Abstract: More than 120 variants of rotavirus strains with different VP7 (G type) and VP4 (P type) combinations are reported thus far. Among them Wa-like G1P[8] rotaviruses are the most common human strains worldwide. However, characterization of their entire genome complement is limited to a few old prototype strains, and no complete genome data for any G1P[8] strain isolated in the last decade are available. Both the currently licensed rotavirus vaccines Rotarix and RotaTeq possess the G1 and P[8] specificities. Therefore, comprehensive genetic information of the currently circulating G1P[8] strain is important to assess the impact of rotavirus vaccines on the circulating rotavirus strains. Here we report the complete genome sequence of a G1P[8] rotavirus strain Dhaka16-03 isolated in 2003 from a Bangladeshi child hospitalized with severe diarrhea. Based on a full-genome classification system, Dhaka16-03 was shown to posses the typical Wa-like genotype constellation: G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-E1-H1. The strain was phylogenetically more closely related to contemporary human rotavirus strains (isolated in the 2000s) with a range of G and P-genotypes than to those of the prototype G1P[8] strains. Since the vaccine strains are developed based on strains isolated several decades ago, it is important to know how much the vaccine strains differ from the currently circulating G1P[8] and other Wa-like strains. Our complete genome characterization of a recent G1P[8] strain will be helpful to assess the ongoing rotavirus vaccine trials and their implementation programs in the forthcoming years.
Abstract: From January 2004 to December 2005, a subset of stool specimens (n = 189) from patients who attended an urban hospital in Bangladesh, in which no pathogen was detected, was tested for the presence of noroviruses by conventional reverse transcription-polymerase chain reaction (RT-PCR). Norovirus RNA was detected in 37 samples (19.6%) in the no-pathogen-detected samples and the estimated overall norovirus detection rate was 8.5%. Diarrhea was generally moderate in the norovirus-infected patients and vomiting was the most common feature among them. Genetic analysis indicated that the GII genogroup was the most predominant norovirus strain (82.4%). The GI strain was found in 17.6% of samples and no cases of GIV were detected. This study indicates that a remarkable proportion of the diarrhea patients is hospitalized due to norovirus infection. Therefore, routine diagnosis of this virus in hospitalized patients is required. Since our study was based on hospitalized patients, community surveillance would be helpful to estimate the true burden of the virus in the country. The data regarding the genetic information of the circulating norovirus strains would be very useful for the norovirus vaccine development programs.
Abstract: The full-length genome of a rare human G8P[14] rotavirus strain, BP1062/04, identified during a surveillance study in Hungary was determined and analyzed. This strain showed a G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3 genomic constellation. Phylogenetic analysis of each genome segment revealed common origins with selected animal and zoonotic human strains. The closest relatedness was seen with suspect zoonotic Hungarian G6P[14] strains in the NSP1 and NSP3 gene phylogeny, with ovine strains in the VP1, VP2, NSP4 gene phylogeny, and with bovine strains in the NSP5 gene phylogeny. The outer capsid VP7 and VP4 genes could not be derived from cognate genes of any known human or animal G8P[14] strains. The remaining genes, NSP2, VP3 and VP6, gave no definite clues to the host origin, although each was clearly different from true human strains. Altogether, our findings suggest that strain BP1062/04 represents an example of a direct zoonotic transmission event.
Abstract: BACKGROUND: Genome diversity of hepatitis B virus (HBV) is prominent among DNA viruses; which, allowed the virus to be genetically classified into eight genotypes and several subgenotypes. OBJECTIVE: To introduce and to characterize a novel subgenotype HBV, classified as A6. STUDY DESIGN: HBV full-length genomes were isolated and sequenced from three African-Belgian patients chronically infected with the virus. Using phylogenetic reconstruction and genetic distance calculation, the evolutionary relationships of the novel strains were investigated. RESULTS: Phylogenetic analysis based on complete genome sequences of genotype A strains revealed distinct clusters supported by high bootstrap values. The three African-Belgian strains clustered separately from the other known A subgenotypes (A1-A5) with maximal bootstrap support (100%). The mean inter-subgenotypic nucleotide divergence over the complete genome sequence between the novel A6 strains and A1-A5 was higher than 4%. CONCLUSION: Phylogenetic analysis of the complete genome sequences yielded maximal bootstrap value support for nodes that establish the new lineage as a novel subgenotype. In addition, nucleotide divergence more than 4% based on full-length genome of the virus, clearly demonstrated that the three African-Belgian strains belonged to a novel subgenotype of HBV, which was assigned as "A6". Noteworthy, the phylogeny of genotype A demonstrated that the A6 is a basal lineage that diverged earlier from the other African subgenotypes of genotype A.
Abstract: The use of whole genome amplification and analysis of viruses is of increasing importance, as data generated using these methods are currently used for clinical diagnostics, epidemiological studies, phylogenetic analyses, and studies of genome organization and evolution. The best known amplification method for DNA is the polymerase chain reaction (PCR). This technique, however, has drawbacks: PCR produces relatively small amplicons and also requires prior knowledge of sequence data for the construction of the required consensus or degenerate primers. For circular DNA templates, it is possible to overcome these drawbacks by using the multiply primed rolling-circle amplification (RCA) technique, which mimics the rolling-circle mechanism that occurs in nature for replication of circular DNA molecules, e.g., plasmids. The RCA protocol described here is optimized for the amplification of circular DNA virus genomes.
Abstract: The first fully sequenced papillomavirus (PV) of marsupials, tentatively named Bettongia penicillata papillomavirus type 1 (BpPV1), was detected in papillomas from a woylie (Bettongia penicillata ogilbyi). The circular, double-stranded DNA genome contains 7,737 bp and encodes 7 open reading frames (ORFs), E6, E7, E1, E2, E4, L2, and L1, in typical PV conformation. BpPV1 is a close-to-root PV with L1 and L2 ORFs most similar to European hedgehog PV and bandicoot papillomatosis carcinomatosis virus types 1 and 2 (BPCV1 and -2). It appears that the BPCVs arose by recombination between an ancient PV and an ancient polyomavirus more than 10 million years ago.
Abstract: G11 rotaviruses are believed to be of porcine origin. However, a limited number of G11 rotaviruses have been recently isolated from humans in combination with P[25], P[8], P[6], and P[4]. To investigate the evolutionary relationships of these strains, we analyzed the complete genomes of 2 human G11P[25] strains, 2 human G11P[8] strains, and 3 porcine reference strains. Most of the 11 gene segments of these 7 strains belonged to genotype 1 (Wa-like). However, phylogenetic clustering patterns suggested that an unknown G11P[25] strain with a new I12 VP6 genotype was transmitted to the human population, in which it acquired human genotype 1 gene segments through reassortment, resulting in a human G11P[8] rotavirus strain with an entire human Wa-genogroup backbone. This Wa-like backbone is believed to have caused the worldwide spread of human G9 and G12 rotaviruses. G11 human rotavirus strains should be monitored because they may also become major human pathogens.
Abstract: Rotaviruses are responsible for more than 600.000 child deaths each year. The world wide introduction of two life oral vaccines RotaTeq and Rotarix is believed to reduce this number significantly. Before the licensing of both vaccines, two new genotypes, G9 and G12, emerged in the human population and were able to spread across the entire globe in a very short time span. To quantify the VP7 mutation rates of these G9 and G12 genotypes, and to estimate their most recent common ancestors we used a Bayesian Markov Chain Monte Carlo (MCMC) framework. Based on 356 sequences for G9 and 140 sequences for G12 we estimated mutation rates (nt substitutions/site/year) of 1.87x10(-3) [1.45 - 2.27x10(-3)] for G9 and 1.66x10(-3) [1.13 - 2.32x10(-3)] for G12. For both the G9 and G12 strains, one particular (sub) lineage was able to disseminate and cause disease across the world. The most recent common ancestors of these particular lineages were dated back to 1989 [1986-1992] and 1995 [1992-1998] for the G9 and G12 genotypes respectively. These estimates suggest that a single novel rotavirus (for example a vaccine escape mutant) can spread worldwide in little more than a decade. These results re-emphasize the need for thorough and continued rotavirus surveillance, in order to detect such potential spreading events at an early stage.
Abstract: In the early phase of an emerging pandemic such as A/H1N1v 2009, it is essential to have a good understanding of its transmissibility, which is often summarized by the reproductive number. Before a country is affected, its government may want to make their own assessment of what is going on in areas of the world that have previously been affected by the disease. However, having access to detailed data is problematic. The only publicly available international dataset with information for a large number of countries was the WHO cumulated case counts per country. In this paper, we show how and in which situations the recorded history of cumulated case counts provides valuable information to estimate the effective reproductive number in an early phase and for a large number of countries.
Abstract: Molecular evolutionary patterns of 62 HBV full-length genomes obtained from Belgian patients were characterized. Phylogenetic analysis revealed diverse HBV subgenotypes including A2 and A6 (46.8%), D1-D4 (38.8%), E (9.7%), C1 (1.6%), and B2 (1.6%). The study population consisted of patients with different ethnic origin (Caucasian, Turkish, Asian, Arab, and African). One HBV D/C recombinant isolate was identified, which encoded subtype adw2. An HBV subgenotype D4 with an aberrant subtype ayw4 was detected. Although none of the genotypes was associated with a specific disease outcome, several nucleotide substitutions, deletions and insertions were observed within the HBV preS1/S and X genes, particularly among patients with active chronic hepatitis B infection and patients with cirrhosis. Within the immunological domain of the HBsAg gene, the most frequent substitutions were sT125M and sT118A. High rates of precore and basal core promoter mutations were detected in patients infected with genotype D of HBV. Almost half of the patients who received lamivudine therapy for at least 1 year had HBV variants associated with lamivudine drug resistance. In conclusion, the most common HBV genotypes in West Europe (A and D) also prevail in Belgium. The highest degree of genetic diversity was detected in HBV genotype D. In addition, this study reveals the circulation of exotic HBV genotypes B, C, and E in Belgium. J. Med. Virol. 82:379-389, 2010. (c) 2010 Wiley-Liss, Inc.
Abstract: Although few simian rotaviruses (RVs) have been isolated, such strains have been important for basic research and vaccine development. To explore the origins of simian RVs, the complete genome sequences of strains PTRV (G8P[1]), RRV (G3P[3]), and TUCH (G3P[24]) were determined. These data allowed the genotype constellations of each virus to be determined and the phylogenetic relationships of the simian strains with each other and with nonsimian RVs to be elucidated. The results indicate that PTRV was likely transmitted from a bovine or other ruminant into pig-tailed macaques (its host of origin), since its genes have genotypes and encode outer-capsid proteins similar to those of bovine RVs. In contrast, most of the genes of rhesus-macaque strains, RRV and TUCH, have genotypes more typical of canine-feline RVs. However, the sequences of the canine and/or feline (canine/feline)-like genes of RRV and TUCH are only distantly related to those of modern canine/feline RVs, indicating that any potential transmission of a progenitor of these viruses from a canine/feline host to a simian host was not recent. The remaining genes of RRV and TUCH appear to have originated through reassortment with bovine, human, or other RV strains. Finally, comparison of PTRV, RRV, and TUCH genes with those of the vervet-monkey RV SA11-H96 (G3P[2]) indicates that SA11-H96 shares little genetic similarity to other simian strains and likely has evolved independently. Collectively, our data indicate that simian RVs are of diverse ancestry with genome constellations that originated largely by interspecies transmission and reassortment with nonhuman animal RVs.
Abstract: SUMMARYEuroRotaNet, a laboratory network, was established in order to determine the diversity of co-circulating rotavirus strains in Europe over three or more rotavirus seasons from 2006/2007 and currently includes 16 countries. This report highlights the tremendous diversity of rotavirus strains co-circulating in the European population during three years of surveillance since 2006/2007 and points to the possible origins of these strains including genetic reassortment and interspecies transmission. Furthermore, the ability of the network to identify strains circulating with an incidence of 1% allowed the identification of possible emerging strains such as G8 and G12 since the beginning of the study; analysis of recent data indicates their increased incidence. The introduction of universal rotavirus vaccination in at least two of the participating countries, and partial vaccine coverage in some others may provide data on diversity driven by vaccine introduction and possible strain replacement in Europe.
Abstract: Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major co-circulating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence was dated back to 1854 (1807-1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates, in terms of receptor preferences, plaque phenotype, growth characteristics and cell tropism. This is the first report describing resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy in the development of vaccines.
Abstract: Patients with immune-mediated inflammatory diseases (IMID) such as RA, IBD or psoriasis, are at increased risk of infection, partially because of the disease itself, but mostly because of treatment with immunomodulatory or immunosuppressive drugs. In spite of their elevated risk for vaccine-preventable disease, vaccination coverage in IMID patients is surprisingly low. This review summarizes current literature data on vaccine safety and efficacy in IMID patients treated with immunosuppressive or immunomodulatory drugs and formulates best-practice recommendations on vaccination in this population. Especially in the current era of biological therapies, including TNF-blocking agents, special consideration should be given to vaccination strategies in IMID patients. Clinical evidence indicates that immunization of IMID patients does not increase clinical or laboratory parameters of disease activity. Live vaccines are contraindicated in immunocompromized individuals, but non-live vaccines can safely be given. Although the reduced quality of the immune response in patients under immunotherapy may have a negative impact on vaccination efficacy in this population, adequate humoral response to vaccination in IMID patients has been demonstrated for hepatitis B, influenza and pneumococcal vaccination. Vaccination status is best checked and updated before the start of immunomodulatory therapy: live vaccines are not contraindicated at that time and inactivated vaccines elicit an optimal immune response in immunocompetent individuals.
Abstract: The connection between nephropathia epidemica (NE) and vegetation dynamics has been emphasized in recent studies. Changing climate has been suggested as a triggering factor of recently observed epidemiologic peaks in reported NE cases. We have investigated whether there is a connection between the NE occurrence pattern in Belgium and specific trends in remotely sensed phenology parameters of broad-leaved forests. The analysis of time series of the MODIS Enhanced Vegetation Index revealed that changes in forest phenology, considered in literature as an effect of climate change, may affect the mechanics of NE transmission.
Abstract: BACKGROUND: Recently, several novel hepatitis B virus (HBV) subgenotypes have been introduced that do not meet proper definition of "subgenotypes". In particular for HBV genotype A, such novel subgenotypes have been reported. OBJECTIVE: To comprehensively reanalyse all HBV subgenotypes A, and to propose a novel, consistent alternative for HBV classification. STUDY DESIGN: All HBV full-length genome subgenotypes A1-A6 were reanalysed using phylogenetic reconstruction and genetic distance calculation in order to study their evolutionary relationships. RESULTS: Phylogenetic analysis based on the complete genome sequence of subgenotype A strains revealed four distinct clusters supported by high bootstrap values, whereas only the three groups A1, A2 and A6 could be assigned as subgenotypes. Previously introduced subgenotype A3, "tentative A4" and A5 clustered together in one main branch and were designated as "quasi-subgenotypes". Also genetic distances failed to classify these three groups as definite subgenotypes. These results advocate for a new classification of HBV genotype A into subgenotype A1, A2, "quasi-subgenotype A3" and A4. CONCLUSION: Detailed phylogenetic analysis of the complete genome sequences demonstrates that some of available HBV genotype A strains may not be considered as definite "subgenotypes". These strains, which are mainly of African origin, could be considered as "quasi-subgenotypes" which puts them in between the "clade" and "subgenotype" definition. Geographical origin may have a key role in further classification of HBV subgenotypes.
Abstract: Summary. Hepatitis B virus (HBV) viral load and its genotype play important roles in clinical outcome, management of disease and response to antiviral therapy. In many parts of the world such as Europe or the Middle East, distinguishing HBV genotype D from non-D is most relevant for treatment decisions, because genotype D-infected patients respond poorly to interferon-based therapeutic regimens. Here, we developed an in-house real-time PCR to concordantly assess HBV genotype (D vs non-D) based on melt curve analysis and quantify the viral load. Genotype distinction was established with control plasmids of all HBV genotypes and validated with 57 clinical samples from patients infected with six different HBV genotypes. Our in-house real-time PCR assay could discriminate HBV genotype D from non-D using single-step melt curve analysis with a 2 degrees C difference in the melt curve temperature in all samples tested. Viral load quantification was calibrated with the WHO HBV international standard, demonstrating an excellent correlation with a commercial kit (r = 0.852; P < 0.0001) in a linear range from 3.2 x 10(2) to 3.2 x 10(10) IU/mL. In conclusion, we developed a rapid, simple and cost-effective method to simultaneously quantify and distinguish HBV genotypes D from non-D with a single-step PCR run and melt curve analysis. This assay should be a useful diagnostic alternative to aid clinical decisions about initiation and choice of antiviral therapy, especially in geographical regions with a high prevalence of HBV genotype D.
Abstract: BACKGROUND: Nephropathia epidemica (NE), an emerging rodent-borne viral disease, has become the most important cause of infectious acute renal failure in Belgium, with sharp increases in incidence occurring for more than a decade. Bank voles are the rodent reservoir of the responsible hantavirus and are known to display cyclic population peaks. We tried to relate these peaks to the cyclic NE outbreaks observed since 1993. Our hypothesis was that the ecological causal connection was the staple food source for voles, being seeds of deciduous broad-leaf trees, commonly called "mast". We also examined whether past temperature and precipitation preceding "mast years" were statistically linked to these NE outbreaks. RESULTS: Since 1993, each NE peak is immediately preceded by a mast year, resulting in significantly higher NE case numbers during these peaks (Spearman R = -0.82; P = 0.034). NE peaks are significantly related to warmer autumns the year before (R = 0.51; P < 0.001), hotter summers two years before (R = 0.32; P < 0.001), but also to colder (R = -0.25; P < 0.01) and more moist summers (R = 0.39; P < 0.001) three years before. Summer correlations were even more pronounced, when only July was singled out as the most representative summer month. CONCLUSION: NE peaks in year 0 are induced by abundant mast formation in year-1, facilitating bank vole survival during winter, thus putting the local human population at risk from the spring onwards of year 0. This bank vole survival is further promoted by higher autumn temperatures in year-1, whereas mast formation itself is primed by higher summer temperatures in year-2. Both summer and autumn temperatures have been rising to significantly higher levels during recent years, explaining the virtually continuous epidemic state since 2005 of a zoonosis, considered rare until recently. Moreover, in 2007 a NE peak and an abundant mast formation occurred for the first time within the same year, thus forecasting yet another record NE incidence for 2008. We therefore predict that with the anticipated climate changes due to global warming, NE might become a highly endemic disease in Belgium and surrounding countries.
Abstract: Group A human rotaviruses (RVs) are a major cause of severe gastroenteritis in infants and young children. Yet, aside from the genes encoding serotype antigens (VP7; G-type and VP4; P-type), little is known about the genetic make-up of emerging and endemic human RV strains. To gain insight into the diversity and evolution of RVs circulating at a single location over a period of time, we sequenced the eleven-segmented, double-stranded RNA genomes of fifty-one G3P[8] strains collected from 1974 to 1991 at Children's Hospital National Medical Center, Washington, D. C. During this period, G1P[8] strains typically dominated, comprising on average 56% of RV infections each year in hospitalized children. A notable exception was in the 1976 and 1991 winter seasons when the incidence of G1P[8] infections decreased dramatically, a trend that correlated with a significant increase in G3P[8] infections. Our sequence analysis indicates that the 1976 season was characterized by the presence of several genetically distinct, co-circulating clades of G3P[8] viruses, which contained minor but significant differences in their encoded proteins. These 1976 lineages did not readily exchange gene segments with each other, but instead remained stable over the course of the season. In contrast, the 1991 season contained a single major clade, whose genome constellation was similar to one of the 1976 clades. The 1991 clade may have gained a fitness advantage after reassorting with as of yet unidentified RV strain(s). This study reveals for the first time that genetically distinct RV clades of the same G/P-type can co-circulate and cause disease. The findings from this study also suggest that, although gene segment exchange occurs, most reassortant strains are replaced over time by lineages with preferred genome constellations. Elucidation of the selective pressures that favor maintenance of RVs with certain sets of genes may be necessary to anticipate future vaccine needs.
Abstract: Consistent evidence underlines the utility of human papillomavirus (HPV) DNA testing in the management of women with equivocal cervical cytological abnormalities, but not in case of low-grade lesions. We performed a meta-analysis including studies where the high-risk probe of the Hybrid Capture-II is used to triage these two cytological categories. The triage test-positivity rate reflects the colposcopy referral workload.Data were pooled on the HPV test positivity rate in women with atypical squamous cells of undetermined significance (ASCUS/ASC-US) or low-grade squamous intraepithelial lesions (LSIL), derived from different cytological classification systems. The meta-analysis was restricted to studies, published between 1991 and 2007. A random-effect model was applied for meta-analytical pooling and the influence of covariates on the HPV positivity rate was analyzed by meta-regression. The variation by age was assessed within individual studies since age strata were not defined uniformly. On an average, 43% (95% CI: 40-46%) of women with ASCUS/ASC-US were high-risk HPV positive (range 23-74%). In women with LSIL, the pooled positivity rate was 76% (95% CI: 71-81%; range 55-89%). In spite of considerable inter-study heterogeneity, the difference in HPV positivity between the two triage groups was large and highly significant: 32% (95% CI: 27-38%). HPV rates dropped tremendously as age and cutoffs of test positivity increased. Other factors (cytological classification system, country, continent, collection method and year of publication) had no statistically significant impact, except in LSIL triage where HPV positivity was significantly lower in European compared to American studies. Women with LSIL, especially younger women, have high HPV positivity rates suggesting limited utility of reflex HPV triaging these cases. Research is needed to identify more specific methods to triage women with low-grade squamous cervical lesions.
Abstract: HCV replicates in liver via an intermediate negative strand RNA. To study the relevance of HCV genome replication, quantitative strand-specific HCV real-time RT-PCR assays were developed and applied to livers explanted because of end-stage cirrhosis. The assays have broad ranges of determination and a high reproducibility and accuracy. Analysis of five different samples showed an even distribution of HCV genomes in four livers. Hepatic concentrations of positive (PS)- and negative (NS)-strand RNA did correlate with each other, with PS/NS ratios ranging between 3 and 340. Hepatic concentrations of HCV-PS or -NS RNA did not correlate with serum HCV-RNA levels or with genotypes. A high HCV envelope-2 protein expression correlated with a low NS concentration. HCV-PS and -NS levels, E2 protein expression and genotype did not correlate with biochemical tests or with histological changes in the explanted liver, but the ratio NS/PS, a marker of viral replication, correlated with the severity of the recurrent post-transplant hepatitis caused by HCV. This suggests the existence of an extra-hepatic location of HCV with comparable viral replication rate being responsible for the infection of the newly transplanted liver.
Abstract: BACKGROUND: We conducted for the first time a systematic review, including a meta-analysis, of the incidence of symptomatic rotavirus (RV) infections, because (1) it was shown to be an influential factor in estimating the cost-effectiveness of RV vaccination, (2) multiple community-based studies assessed it prospectively, (3) previous studies indicated, inconclusively, it might be similar around the world. METHODOLOGY: Pubmed (which includes Medline) was searched for surveys assessing prospectively symptomatic (diarrheal) episodes in a general population and situation, which also reported on the number of the episodes being tested RV+ and on the persons and the time period observed. A bias assessment tool was developed and used according to Cochrane guidelines by 4 researchers with different backgrounds. Heterogeneity was explored graphically and by comparing fits of study-homogenous 'fixed effects' and -heterogeneous 'random effects' models. Data were synthesized using these models. Sensitivity analysis for uncertainty regarding data abstraction, bias assessment and included studies was performed. PRINCIPAL FINDINGS: Variability between the incidences obtained from 20 studies is unlikely to be due to study groups living in different environments (tropical versus temperate climate, slums versus middle-class suburban populations), nor due to the year the study was conducted (from 1967 to 2003). A random effects model was used to incorporate unexplained heterogeneity and resulted in a global incidence estimate of 0.31 [0.19; 0.50] symptomatic RV infections per personyear of observation for children below 2 years of age, and of 0.24 [0.17; 0.34] when excluding the extreme high value of 0.84 reported for Mayan Indians in Guatemala. Apart from the inclusion/exclusion of the latter study, results were robust. CONCLUSIONS/SIGNIFICANCE: Rather than assumptions based on an ad-hoc selection of one or two studies, these pooled estimates (together with the measure for variability between populations) should be used as an input in future cost-effectiveness analyses of RV vaccination.
Abstract: BACKGROUND: Hepatitis B virus (HBV) is primarily transmitted from mother to child, by sexual contact, intravenous drug abuse, or unsafe health care-related injection practices. Despite increased safety efforts, nosocomial acquired hepatitis B infection remains problematic. OBJECTIVES: A large HBV outbreak was investigated comprising 36 patients with acute HBV infection in a primary care physician's practice. STUDY DESIGN: In a retrospective study (2003-2008), 36 serum samples from patients with acute HBV infection were collected. They had received several injections by the same physician at least 3 months before the onset of clinical symptoms. As a control group, sera were collected from HBV patients from other physicians from the same province. Full-length HBV genomes were amplified and were phylogenetically analysed. RESULTS: HBV complete genomes of 32 patients were successfully amplified and sequenced, and clustered together with the reference genotype A, subgenotype A2 strains. We also analysed 26 control HBV genotype A samples. All 32 HBV strains from the patient group clustered in a monophyletic branch with a bootstrap value of 100, whereas the control samples branched separately in another clade. The genetic distance value showed small differences within the patients group, whereas the rate within the control group was seven times higher. These observations confirm that the source of transmission was clearly different in both groups. CONCLUSION: Maximum likelihood analysis and genetic distance calculations based on the full-length genomes of HBV strains isolated from patients and controls provided strong evidence for a common nosocomial source of infection for all 32 patient cases.
Abstract: A limited number of human G6P[14] rotavirus strains that cause gastroenteritis in humans have been isolated in Europe and Australia. The complete genome sequences were determined for five of these human strains--B10925-97 (isolated in Belgium in 1997), 111/05-27 (Italy, 2005), PA169 (Italy, 1987), MG6 (Australia, 1993), and Hun5 (Hungary, 1997)--and their genetic relatedness to animal rotavirus strains was evaluated by sequencing the complete genome of the sheep rotavirus OVR762 (G8P[14]; Spain, 2002), the guanaco (Lama guanicoe) rotavirus strains Arg/Chubut/99 and Arg/RÃo Negro/98 (G8P[14] and G8P[1], respectively; Argentina, 1999 and 1998), the sable antelope strain RC-18/08 (G6P[14]; South Africa, 2008), and the bovine rotavirus strain Arg/B383/98 (G15P[11]; Argentina, 1998). These analyses revealed an overall consensus genomic constellation (G6/G8)-P[14]-I2-(R2/R5)-C2-M2-(A3/A11)-N2-T6-(E2/E12)-H3, together with a few gene reassortments, and the phylogenetic analyses confirmed that the P[14] human strains evaluated in this study were closely related to rotavirus strains isolated from sheep, cattle, goats, guanacos, and antelopes and to rabbits (albeit to a lesser extent), suggesting that one (or more) of these animal species might be the source of the human G6P[14] strains. The main feature of the genotype and phylogenetic analyses was the close overall genomic relatedness between the five human G6P[14] rotavirus strains and the ovine and antelope rotavirus strains. Taken together, these data strongly suggest a common origin for the human P[14] strains and those of the even-toed ungulates belonging to the mammalian order Artiodactyla, with sheep probably playing a key role in the interspecies transmission responsible for the introduction of P[14] rotavirus strains into the human population.
Abstract: BACKGROUND: In July 2004, a sharp increase of hepatitis A, a notifiable disease in Belgium, was detected. OBJECTIVES: We investigated the outbreak in order to identify the source and take appropriate action. STUDY DESIGN: We conducted an outbreak investigation which included a matched case-control study to analyse the association with a range of food items and food providers. A phylogenetic analysis was used to study the relation between the outbreak cases and the identified source. RESULTS: We registered 269 cases of hepatitis A. Consumption of raw beef (OR 16.0; 95% CI 2.1-120.7) was the most probable way of infection. A food handler working at an epidemiologically linked meat distribution plant had contracted hepatitis A 1 month before the start of the outbreak. HAV strains from the food handler and the patients involved in the outbreak were monophyletically related. CONCLUSIONS: Since serological immunity in Belgium is decreasing over time, foodborne outbreaks of hepatitis A are a substantial risk. In this outbreak, a single food handler, at the level of the distribution chain, has been identified as the most likely source, through cross-contamination of raw beef. This outbreak investigation suggests the need to consider vaccination against hepatitis A in food handlers.
Abstract: BACKGROUND: Elderly infected with Human Respiratory Syncytial Virus (RSV) often bear low viral loads that stay below the detection limits of commercial assays. A more sensitive detection of RSV infections can improve patient management, guide containment strategies, and possibly prevent morbidity and mortality among populations most severely affected by RSV. OBJECTIVE: To test the sensitivity for RSV detection by using an alternative extraction method in combination with a new amplification procedure. STUDY DESIGN: Nasopharyngeal washes and sputum samples (n=78) form clinical cases, and broncheo-alveolar lavages (n=27) from an experimental RSV rat model were obtained. An ultrasone-based RNA extraction method was combined with a multi-component Nucleic Acid enzymes (MNAzyme) amplification procedure for simultaneous detection of RSV-A, RSV-B, and an Internal Extraction control IEC. RESULTS: Compared to standard real-time PCR technology, this method resulted in an increased detection sensitivity, ranging from 0.9 to 4.93 log (average 2.05+/-1.01) for RSV-A and 0.76 to 4.28 log (average 1.30+/-0.92) for RSV-B. CONCLUSIONS: An ultrasone-based extraction method with MNAzyme amplification resulted in improved detection of RSV in different respiratory samples, including sputum. This generic method for nucleic acid extraction should be readily applicable for any other respiratory pathogen.
Abstract: In rotaviruses, intragenic recombination or gene rearrangement occurs almost exclusively in the genome segments encoding for non-structural proteins. Rearranged RNA originates by mechanisms of partial sequence duplications and deletions or insertions of non-templated nucleotides. Of interest, epidemiological investigations have pointed out an unusual bias to rearrangements in genome segment 11, notably in rotavirus strains of lapine origin, as evidenced by the detection of numerous lapine strains with super-short genomic electropherotype. The sequence of the full-length genome segment 11 of two lapine strains with super-short electropherotype, LRV-4 and 3489/3, was determined and compared with rearranged and normal cognate genome segments of lapine rotaviruses. The rearranged genome segments contained head-to-tail partial duplications at the 3' end of the main ORF encoding NSP5. Unlike the strains Alabama and B4106, intermingled stretches of non-templated sequences were not present in the accessory RNA of LRV-4 and 3489/3, while multiple deletions were mapped, suggesting the lack of functional constraints. Altogether, these findings suggest that independent rearrangement events have given origin to the various lapine strains that have super-short genome pattern.
Abstract: BACKGROUND: The first European rotavirus surveillance network, EuroRotaNet, comprising 16 laboratories in 15 European countries, has been established. METHODS: Fecal samples from gastroenteritis cases positive for group A rotavirus antigen were collected from multiple European countries from 2005 to mid-2008 and were subjected to G and P genotyping. Epidemiological data collected included age, sex, geographical location, setting, dates of onset and sample collection, and clinical symptoms. RESULTS: A total of 8879 rotavirus-positive samples were characterized: 2129 cases were from the 2005-2006 season, 4030 from the 2006-2007 season, and 2720 from the ongoing 2007-2008 season. A total of 30 different G and P type combinations of strains circulated in the region from 2005 through 2008. Of these strains, 90% had genotypes commonly associated with human infections-G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]-and 1.37% represented potential zoonotic introductions. G1P[8] remained the most prevalent genotype in Europe as a whole, but the incidence of infection with G1P[8] rotavirus strains was <50% overall, and all 3 seasons were characterized by a significant diversity of cocirculating strains. The peak incidence of rotavirus infection occurred from January through May, and 81% of case patients were aged <2.5 years. Conclusions. Data gathered through EuroRotaNet will provide valuable background information on the rotavirus strain diversity in Europe before the introduction of rotavirus vaccines, and the network will provide a robust method for surveillance during vaccine implementation.
Abstract: Rodent-borne hantaviruses are associated with two main clinical disorders in humans: hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Although hantavirus diseases can be life threatening and numerous research efforts are focused on the development of hantavirus prevention, no specific antiviral therapy is yet available and, at this time, no WHO-approved vaccine has gained widespread acceptance. This review will summarize the current knowledge and recent progress as well as new speculative approaches in the development of hantavirus vaccines.
Abstract: The aim of this study was to evaluate the diagnostic reliability and prognostic significance of the quantification of cytomegalovirus (CMV) DNA in amniotic fluid (AF). We retrospectively reviewed the results for 282 amniotic fluid samples that had been tested for CMV by a quantitative real-time PCR. We observed three cases in which no CMV genomes were detected in the AF but in which the children were nevertheless congenitally infected. Hence, we conclude that a negative result by PCR for CMV in AF cannot rule out the possibility of congenital infection. No false-positive PCR results were observed. A correlation between the CMV viral load in AF and the fetal and neonatal outcomes could not be demonstrated in our study. Instead, a correlation was found between the CMV viral load and the gestational age at the time of amniocentesis.
Abstract: Papillomaviruses (PVs) are a large family of small DNA viruses infecting mammals, reptiles, and birds. PV infection induces cell proliferation that may lead to the formation of orogenital or skin tumors. PV-induced cell proliferation has been related mainly to the expression of two small oncoproteins, E6 and E7. In mammalian PVs, E6 contains two 70-residue zinc-binding repeats, whereas E7 consists of a natively unfolded N-terminal region followed by a zinc-binding domain which folds as an obligate homodimer. Here, we show that both the novel francolin bird PV Francolinus leucoscepus PV type 1 (FlPV-1) and the chaffinch bird PV Fringilla coelebs PV contain unusual E6 and E7 proteins. The avian E7 proteins contain an extended unfolded N terminus and a zinc-binding domain of reduced size, whereas the avian E6 proteins consist of a single zinc-binding domain. A comparable single-domain E6 protein may have existed in a common ancestor of mammalian and avian PVs. Mammalian E6 C-terminal domains are phylogenetically related to those of single-domain avian E6, whereas mammalian E6 N-terminal domains seem to have emerged by duplication and subsequently diverged from the original ancestral domain. In avian and mammalian cells, both FlPV-1 E6 and FlPV-1 E7 were evenly expressed in the cytoplasm and the nucleus. Finally, samples of full-length FlPV-1 E6 and the FlPV-1 E7 C-terminal zinc-binding domain were prepared for biophysical analysis. Both constructs were highly soluble and well folded, according to nuclear magnetic resonance spectroscopy measurements.
Abstract: BACKGROUND: End-stage liver disease due to hepatitis C viral (HCV) infection is the most common reason for liver transplantation. One of the major risk factors for infection with HCV is intravenous drug use (IVDU). The pretransplantation characteristics and outcome of liver transplantation in patients with chronic hepatitis C (CHC) infected after IVDU are poorly known. METHODS: We performed a retrospective cohort study in patients with CHC who underwent liver transplantation between 1998 and 2002 in Belgium. Seven patients with and 60 patients without a history of IVDU were compared. RESULTS: Patients with CHC infected after IVDU were primarily men, significantly younger, and affected more by genotype 2 or 3. There was no relapse in substance use. No patients required a second transplantation or developed surgical complications. Progression to fibrosis in the posttransplantation period seemed to be slower. Graft and patient survival, and compliance were similar in both groups. CONCLUSIONS: Compared with patients in the non-IVDU group, patients with CHC infected after IVDU in complete remission have the same compliance, and patient and graft survival after liver transplantation. Therefore, patients with IVDU should not be excluded for liver transplantation because of HCV-induced cirrhosis.
Abstract: We describe the design and characterization of a potent human respiratory syncytial virus (RSV) nucleocapsid gene-specific small interfering RNA (siRNA), ALN-RSV01. In in vitro RSV plaque assays, ALN-RSV01 showed a 50% inhibitory concentration of 0.7 nM. Sequence analysis of primary isolates of RSV showed that the siRNA target site was absolutely conserved in 89/95 isolates, and ALN-RSV01 demonstrated activity against all isolates, including those with single-mismatch mutations. In vivo, intranasal dosing of ALN-RSV01 in a BALB/c mouse model resulted in potent antiviral efficacy, with 2.5- to 3.0-log-unit reductions in RSV lung concentrations being achieved when ALN-RSV01 was administered prophylactically or therapeutically in both single-dose and multidose regimens. The specificity of ALN-RSV01 was demonstrated in vivo by using mismatch controls; and the absence of an immune stimulatory mechanism was demonstrated by showing that nonspecific siRNAs that induce alpha interferon and tumor necrosis factor alpha lack antiviral efficacy, while a chemically modified form of ALN-RSV01 lacking measurable immunostimulatory capacity retained full activity in vivo. Furthermore, an RNA interference mechanism of action was demonstrated by the capture of the site-specific cleavage product of the RSV mRNA via rapid amplification of cDNA ends both in vitro and in vivo. These studies lay a solid foundation for the further investigation of ALN-RSV01 as a novel therapeutic antiviral agent for clinical use by humans.
Abstract: Temporal and spatial fluctuations in the genotype distribution of human rotaviruses are continuously observed in surveillance studies. New genotypes, such as G9 and G12, have emerged and spread worldwide in a very short time span. In addition, reassortment events have the potential to contribute substantially to genetic diversity among human and animal rotaviruses. With the recent introduction of the two rotavirus vaccines, RotaTeq and Rotarix, in many countries, it appears that the total number of hospitalizations due to rotavirus infections is being reduced, at least in developed countries that implemented a universal immunization program. However, continued surveillance is warranted, especially regarding the long-term effects of the vaccines. No data analyses are available to clarify whether rotavirus vaccine introduction would allow other rotavirus P and G genotypes, which are not covered by the current vaccines, to emerge into the human population and fill the apparent gap. This kind of data analysis is essential, but its interpretation is hampered by natural and cyclical genotype fluctuations.
Abstract: BACKGROUND: Group A rotaviruses are the most common cause of severe diarrhea in infants and children worldwide and continue to have a major global impact on childhood morbidity and mortality. In recent years, considerable research efforts have been devoted to the development of two new live, orally administered vaccines. Although both vaccines have proven to confer a good protection against severe rotavirus gastroenteritis, these vaccines will have to be screened and may have to be updated regularly to reflect temporal and spatial genotype fluctuations. In this matter, the genetic characterization of circulating and new emerging rotavirus strains will need to be compulsory and accurate. An extended classification system for rotaviruses in which all the 11 genomic RNA segments are used, has been proposed recently. The use of this classification system will help to elucidate the role of gene reassortments in the generation of genetic diversity, host range restriction, co-segregation of certain gene segments, and in adaptation to a new host species. RESULTS: Here we present a web-based tool that can be used for fast rotavirus genotype differentiation of all 11 group A rotavirus gene segments according to the new guidelines proposed by the Rotavirus Classification Working Group (RCWG). CONCLUSION: With the increasing sequencing efforts that are being conducted around the world to unravel complete rotavirus genomes of human and animal origin, this tool will be of great help to analyze and correctly classify the large amount of new data. The web-based tool is freely available at http://rotac.regatools.be.
Abstract: Papillomaviruses (PVs) have been shown as being the etiologic agents of various benign and malignant tumours in many vertebrate species. In dolphins and porpoises, a high prevalence of orogenital tumours has recently been documented with at least four distinct novel species-specific PV types detected in such lesions. Therefore, we generated the immunological reagents to establish a serological screening test to determine the prevalence of PV infection in Atlantic bottlenose dolphins [(Tursiops truncatus (Tt)]. Using the baculovirus expression system, virus-like particles (VLPs) derived from the L1 proteins of two TtPV types, TtPV1 and TtPV2, were generated. Polyclonal antibodies against TtPV VLPs were produced in rabbits and their specificity for the VLPs was confirmed. Electron microscopy and enzyme-linked immunosorbent assay (ELISA) studies revealed that the generated VLPs self-assembled into particles presenting conformational immunodominant epitopes. As such, these particles are potential antigen candidates for a TtPV vaccine. Subsequently, the VLPs served as antigens in initial ELISA tests using sera from six bottlenose dolphins to investigate PV antibody presence. Three of these sera were derived from dolphins with genital tumour history and showed positive PV ELISA reactivity, while the remaining sera from lesion-free dolphins were PV antibody-negative. The results suggest that the developed screening test may serve as a potential tool for determining PV prevalence and thus for observing transmission rates in dolphin populations as the significance of PV infection in cetaceans starts to unfold.
Abstract: Techniques for the single-step amplification of whole genomes have been developed into powerful tools for phylogenetic analyses, epidemiological studies and studies on genome organization. Recently, the bacteriophage phi29 DNA polymerase has been used for the efficient amplification of circular DNA viral genomes without the need of specific primers by a rolling-circle amplification (RCA) mechanism. Various protocols have been applied for detection of novel viruses, for differentiation between circular and linear forms of viral genomes and for generation of infectious genomic clones directly from specimens. Here, we summarize the broad application of the RCA technique to DNA viruses infecting humans, animals and plants.
Abstract: Hantaviruses, members of the family Bunyaviridae, are the causative agents of hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Hantaviruses are currently demarcated into species based on the guidelines provided by the International Committee on Taxonomy of Viruses (ICTV). These guidelines however, are often ignored by the descriptors of novel hantaviruses. With this study we attempted to refine the second ICTV guideline for hantavirus species demarcation by phylogenetically analyzing all in Genbank available complete sequences derived from the S, M or L segments of hantaviruses. S and M segment amino acid sequence comparison allowed clear and unequivocal distinction between different hantavirus species, and lead us to propose additional criteria for the demarcation of hantavirus species (S segment amino acid distance >10% or M segment amino acid distance >12%) and hantavirus groups (S segment amino distance >24% or M segment amino acid distance >32%). With this study, we propose to adjust the second rule of the ICTV classification guidelines ("a 7% difference in amino acid identity when comparing the complete S segment and M segment sequences") to a more appropriate rule, "a 10% difference in S segment similarity and a 12% difference in M segment similarity based on complete amino acid sequences" in accordance with the current situation in the hantavirus field.
Abstract: Until recently, human coronaviruses (HCoVs), such as HCoV strain OC43 (HCoV-OC43), were mainly known to cause 15 to 30% of mild upper respiratory tract infections. In recent years, the identification of new HCoVs, including severe acute respiratory syndrome coronavirus, revealed that HCoVs can be highly pathogenic and can cause more severe upper and lower respiratory tract infections, including bronchiolitis and pneumonia. To date, no specific antiviral drugs to prevent or treat HCoV infections are available. We demonstrate that chloroquine, a widely used drug with well-known antimalarial effects, inhibits HCoV-OC43 replication in HRT-18 cells, with a 50% effective concentration (+/- standard deviation) of 0.306 +/- 0.0091 microM and a 50% cytotoxic concentration (+/- standard deviation) of 419 +/- 192.5 microM, resulting in a selectivity index of 1,369. Further, we investigated whether chloroquine could prevent HCoV-OC43-induced death in newborn mice. Our results show that a lethal HCoV-OC43 infection in newborn C57BL/6 mice can be treated with chloroquine acquired transplacentally or via maternal milk. The highest survival rate (98.6%) of the pups was found when mother mice were treated daily with a concentration of 15 mg of chloroquine per kg of body weight. Survival rates declined in a dose-dependent manner, with 88% survival when treated with 5 mg/kg chloroquine and 13% survival when treated with 1 mg/kg chloroquine. Our results show that chloroquine can be highly effective against HCoV-OC43 infection in newborn mice and may be considered as a future drug against HCoVs.
Abstract: Hantaviruses, which are mainly rodent-borne viruses, cause hemorrhagic fever with renal syndrome in the Old World, and hantavirus pulmonary syndrome in the New World. A neutralization test based on quantitative RT-PCR, the replication reduction neutralization test (RRNT), was developed for efficient detection of hantavirus-neutralizing antibodies. The effectiveness of the RRNT was evaluated by examining several hantaviruses and hantavirus-specific convalescent human serum samples. All convalescent serum samples tested by RRNT caused significant decreases in hantavirus genomes with only one specific hantavirus species, which allowed a straightforward identification of the related hantavirus. The results obtained by RRNT were completely comparable with the results obtained by focus reduction neutralization test (FRNT). The RRNT approach is a reliable and rapid alternative for FRNT, hitherto considered as the gold standard for hantavirus serology.
Abstract: Human immunodeficiency virus type-1 (HIV-1), hepatitis B and C and other rapidly evolving viruses are characterized by extremely high levels of genetic diversity. To facilitate diagnosis and the development of prevention and treatment strategies that efficiently target the diversity of these viruses, and other pathogens such as human T-lymphotropic virus type-1 (HTLV-1), human herpes virus type-8 (HHV8) and human papillomavirus (HPV), we developed a rapid high-throughput-genotyping system. The method involves the alignment of a query sequence with a carefully selected set of pre-defined reference strains, followed by phylogenetic analysis of multiple overlapping segments of the alignment using a sliding window. Each segment of the query sequence is assigned the genotype and sub-genotype of the reference strain with the highest bootstrap (>70%) and bootscanning (>90%) scores. Results from all windows are combined and displayed graphically using color-coded genotypes. The new Virus-Genotyping Tools provide accurate classification of recombinant and non-recombinant viruses and are currently being assessed for their diagnostic utility. They have incorporated into several HIV drug resistance algorithms including the Stanford (http://hivdb.stanford.edu) and two European databases (http://www.umcutrecht.nl/subsite/spread-programme/ and http://www.hivrdb.org.uk/) and have been successfully used to genotype a large number of sequences in these and other databases. The tools are a PHP/JAVA web application and are freely accessible on a number of servers including: http://bioafrica.mrc.ac.za/rega-genotype/html/, http://lasp.cpqgm.fiocruz.br/virus-genotype/html/, http://jose.med.kuleuven.be/genotypetool/html/.
Abstract: In 2003, we described the first human G6P[6] rotavirus strain (B1711). To investigate further the molecular origin of this strain and to determine the possible reassortments leading to this new gene constellation, the complete genome of strain B1711 was sequenced. SimPlot analyses were conducted to compare strain B1711 with other known rotavirus gene segments, and phylogenetic dendrograms were constructed to analyse the origin of the eleven genome segments of strain B1711. Our analysis indicated that strain B1711 acquired its VP1-, VP2-, VP4-, VP6- and NSP1-5-encoding gene segments from human DS-1-like P[6] rotavirus strains, and its VP3 and VP7 gene segments from a bovine rotavirus strain through reassortment. The introduction of animal-human reassortant strains, which might arise in either of the hosts, into the human rotavirus population is an important mechanism for the generation of rotavirus diversity, and might be a challenge for the current rotavirus vaccines and vaccines under development.
Abstract: BACKGROUND: Biometric fingerprint identity verification is currently introduced in visa application and entry screening at border control. The system implies physical contact between the skin and the surface of the fingerprint-capturing and reading devices. AIM: To assess the risk of infection transmission through fingerprinting. METHODS: The medical literature was reviewed for the potential of microorganisms to be carried on the skin of hands in the community, to be transferred from hands to inanimate surfaces, to survive on surfaces, and to be transferred in doses exceeding the infectious dose. The fingerprinting procedures as currently applied were reviewed. RESULTS: Factors that favor transfer of microorganisms are large skin-surface contact between flat fingers (2 x 20 cm(2)) and fingerprint-capturing device, nonporous contact surface, large overlap of contact surface and short turnaround time between successive applicants, high contact pressure, and difficulties to disinfect devices. Transmission risk exists for enteric viruses (rotavirus, norovirus, and hepatitis A virus), respiratory viruses (respiratory syncytial virus, rhinovirus, influenza virus, etc.), and enteropathogenic bacteria with low infectious doses (Shigella dysenteriae, Enterohemorrhagic Escherichia coli, etc.). Using Monte Carlo risk analysis on US data, transmission of human rotavirus is estimated at 191 [95% credible intervals (CI) 0-289] per million fingerprint-capturing procedures. Application of 70% isopropyl hand rub and 85% ethanol hand gel reduces the risk to 77 (95% CI 0-118) and 0.3 (95% CI 0-0.3) transmissions per million procedures, respectively. CONCLUSIONS: The fingerprinting procedure as currently used is associated with a risk of infection transmission. Simple hygienic measures can considerably reduce this transmission risk.
Abstract: Recently, a classification system was proposed for rotaviruses in which all the 11 genomic RNA segments are used (Matthijnssens et al. in J Virol 82:3204-3219, 2008). Based on nucleotide identity cut-off percentages, different genotypes were defined for each genome segment. A nomenclature for the comparison of complete rotavirus genomes was considered in which the notations Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx are used for the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 encoding genes, respectively. This classification system is an extension of the previously applied genotype-based system which made use of the rotavirus gene segments encoding VP4, VP7, VP6, and NSP4. In order to assign rotavirus strains to one of the established genotypes or a new genotype, a standard procedure is proposed in this report. As more human and animal rotavirus genomes will be completely sequenced, new genotypes for each of the 11 gene segments may be identified. A Rotavirus Classification Working Group (RCWG) including specialists in molecular virology, infectious diseases, epidemiology, and public health was formed, which can assist in the appropriate delineation of new genotypes, thus avoiding duplications and helping minimize errors. Scientists discovering a potentially new rotavirus genotype for any of the 11 gene segments are invited to send the novel sequence to the RCWG, where the sequence will be analyzed, and a new nomenclature will be advised as appropriate. The RCWG will update the list of classified strains regularly and make this accessible on a website. Close collaboration with the Study Group Reoviridae of the International Committee on the Taxonomy of Viruses will be maintained.
Abstract: The healthy skin of two female domestic pigs (Sus scrofa domestica) was sampled with cotton-tipped swabs. Total genomic DNA was extracted from the samples and subjected to PCR with degenerate papillomavirus (PV)-specific primers. Similarity searches performed with blastn showed that partial E1 and L1 sequences of two novel PVs were amplified. Subsequently, the complete genomes of these Sus scrofa papillomaviruses (SsPVs) were amplified by long-template PCR, cloned and sequenced using a transposon insertion method. They contained the typical PV open reading frames (ORFs) E1, E2, E4, E6, L1 and L2, but the E7 ORF was absent in both viruses. Pairwise nucleotide sequence alignment of the L1 ORFs of the SsPVs showed 98.5 % similarity, classifying these viruses as SsPV type 1 'variants' (SsPV-1a and -1b). Based on a concatenated alignment of the E1, E2, L1 and L2 ORFs of SsPV-1 variants a and b, and 81 other human and animal PV type species, a neighbour-joining phylogenetic tree was constructed. This phylogenetic analysis showed that the SsPV-1a and -1b variants did not cluster with the other PVs of artiodactyls (cloven-hoofed) host species, but clustered on the edge of the genus Alphapapillomavirus, very near to the root of this genus.
Abstract: Since auditory disability causes serious problems in the development of speech and in the total development of a child, it is crucial to diagnose possible hearing impairment as soon as possible after birth. This study evaluates the neonatal hearing screening program in Flanders, Belgium. The auditory ability of 118,438 babies was tested using the automated auditory brainstem response. We selected 194 babies with indicative hearing impairment and 332 matched controls to investigate the association between the presence of human cytomegalovirus (HCMV) in urine samples and sensorineural hearing loss and to analyze the sensibility and specificity of a cell culture assay and a quantitative PCR detection method. Our results indicate that significantly more babies with confirmed hearing impairment were HCMV positive after birth. Further, based on the results of our study, babies with HCMV viral loads above 4.5 log copies/ml urine seem to be 1.4 times more likely to have confirmed hearing impairment. Our follow-up study suggests that the hearing impairment of children infected with HCMV after birth is less likely to improve than that of HCMV-negative infants. Our results confirm that the presence of HCMV before or shortly after birth influences the outcome of hearing impairment.
Abstract: The genome of the hepatitis C virus (HCV) exhibits a high genetic variability. This remarkable heterogeneity is mainly attributed to the gradual accumulation of mutational changes, whereas the contribution of recombination events to the evolution of HCV remains controversial so far. While performing phylogenetic analyses including a large number of sequences deposited in the GenBank, we encountered a full-length HCV sequence (AY651061) that showed evidence for inter-subtype recombination and was, therefore, subjected to a detailed analysis of its molecular structure. The obtained results indicated that AY651061 does not represent a "simple" HCV 1c isolate, but a complex 1a/1c mosaic genome, showing five putative breakpoints in the core to NS3 regions. To our knowledge, this is the first report on a mosaic HCV full-length sequence with multiple breakpoints. The molecular structure of AY651061 is reminiscent of complex homologous recombinant variants occurring among other members of the flaviviridae family, e.g. GB virus C, dengue virus, and Japanese encephalitis virus. Our finding of a mosaic HCV sequence may have important implications for many fields of current HCV research which merit careful consideration.
Abstract: Although G9 rotaviruses have become one of the important rotavirus genotypes worldwide, they have been uncommon in China. Recently, we reported G9 rotaviruses as a highly prevalent genotype in Xinjiang, the northwest part of China [Yang, X., Matthijnssens, J., Sun, H., Muhamaiti, J., Zhang, B., Nahar, S., Van Ranst, M., Rahman, M., 2008. Temporal changes of rotavirus strain distribution in a northwest city of China, 1996-2005. Int. J. Infect. Dis., June (Epub ahead of print)]. Here we report the genetic variations of the Xinjiang-G9 rotaviruses isolated between 1999 and 2005. Sequence analysis of the VP7 genes of Xinjiang-G9 strains indicated that they were more closely related to the contemporary global G9 strains than to the prototype Chinese G9 strains. However, their VP4 genes were most similar to those from the locally circulating G1P[8], G2P[4], G3P[6] and G3P[8] strains. This indicates that reassortment rather than antigenic drift might be the preferred evolutionary mechanism for the emergence of the G9 rotaviruses in Xinjiang. These findings will be of major significance for understanding the emergence of newly introduced rotavirus strains.
Abstract: The recent discoveries of novel human coronaviruses, including the coronavirus causing SARS, and the previously unrecognized human coronaviruses HCoV-NL63 and HCoV-HKU1, indicate that the family Coronaviridae harbors more members than was previously assumed. All human coronaviruses characterized at present are associated with respiratory illnesses, ranging from mild common colds to more severe lower respiratory tract infections. Since the etiology of a relatively large percentage of respiratory tract diseases remains unidentified, it is possible that for a certain number of these illnesses, a yet unknown viral causative agent may be found. Screening for the presence of novel coronaviruses requires the use of a method that can detect all coronaviruses known at present. In this chapter, we describe a pancoronavirus degenerate primer-based method that allows the detection of all known and possibly unknown coronaviruses by RT-PCR amplification and sequencing of a 251-bp fragment of the coronavirus polymerase gene.
Abstract: Phylogenetic analysis of novel dolphin (Tursiops truncatus) papillomavirus sequences, TtPV1, -2, and -3, indicates that the early and late protein coding regions of their genomes differ in evolutionary history. Sliding window bootscan analysis showed a significant a change in phylogenetic clustering, in which the grouped sequences of TtPV1 and -3 move from a cluster with the Phocoena spinipinnis PsPV1 in the early region to a cluster with TtPV2 in the late region. This provides indications for a possible recombination event near the end of E2/beginning of L2. A second possible recombination site could be located near the end of L1, in the upstream regulatory region. Selection analysis by using maximum likelihood models of codon substitutions ruled out the possibility of intense selective pressure, acting asymmetrically on the viral genomes, as an alternative explanation for the observed difference in evolutionary history between the early and late genomic regions of these cetacean papillomaviruses.
Abstract: For health economic evaluations of rotavirus vaccination, estimates of the health and cost burden of rotavirus are required. Due to differences in health care systems and surveillance organisations, this is difficult to achieve by imputing estimates from one country to others. This study aimed to estimate the burden of rotavirus disease in Belgium. In children younger than 7 years of age, rotavirus is predicted to account annually for about 5,600 hospitalisations (676:100,000 children); 26,800 outpatient, general practitioner and paediatrician visits; and about 44,600 episodes for which no medical care is sought. This burden is estimated to represent direct costs of 7.7 million Euro and indirect costs of 12.8 million Euro. Rotavirus disease causes a substantial health and economic burden in Belgium.
Abstract: Recently, the Food and Drug Administration (FDA) of the USA approved the first integrase inhibitor for inclusion in treatment regimens of HIV-1 patients failing their current regimens with multi-drug resistant strains. However, treatment failure has been observed during integrase inhibitor-containing therapy. Several mutational pathways have been described with signature mutations at integrase positions 66, 92, 148 and 155. Therefore, a genotypic assay for the amplification and sequencing of HIV-1 integrase was developed. The assay displayed a detection limit of 10 HIV-1 III(B) RNA copies/ml plasma. As the HIV-1 pandemic is characterised by a large genetic diversity, the new assay was evaluated on a panel of 74 genetically divergent samples belonging to the following genetic forms A, B, C, D, F, G, J, CRF01-AE, CRF02-AG, CRFF03-AB, CRF12-BF and CRF13-cpx. Their viral load ranged from 178 until >500,000 RNA copies/ml. The amplification and sequencing was successful for 70 samples (a success rate of 95%). The four failures were most probably due to low viral load or poor quality of RNA and not to subtype issues. Some of the sequences obtained from integrase inhibitor-naïve patients displayed polymorphisms at integrase positions associated with resistance: 74IV, 138D, 151I, 157Q and 163AE. The relevance of these polymorphisms in the absence of the signature mutations remains unclear.
Abstract: A fatal case of Coxsackie B3 myocarditis in a neonate is described. The clinical features became evident in the 3rd week of life, but enteroviral RNA was detected in the dried blood spot of the baby collected on day 4 after birth. This is the first report on the detection of enteroviral RNA in Guthrie card dried blood using reverse-transcriptase PCR. Materials and methods used are described in detail.
Abstract: Group A rotavirus classification is currently based on the molecular properties of the two outer layer proteins, VP7 and VP4, and the middle layer protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.
Abstract: Papillomatosis has been documented in several carnivores, and papillomavirus (PV) types have been characterized from lesions in a number of carnivore species: the canine oral PV (COPV), the Felis domesticus PV type 1 (FdPV-1) isolated from a Persian cat, the Procyon lotor PV type 1 (PlPV-1) isolated from a raccoon, the canine PV type 2 (CPV-2) from a dog's foot pad lesion and the canine PV type 3 (CPV-3) associated with a canine epidermodysplasia verruciformis - like disease. A tissue sample was taken from a papillomatous lesion on the oral mucosa of a polar bear (Ursus maritimus). Extracted DNA was used as a template for multiply primed rolling-circle amplification (RCA), and restriction enzyme analysis of the RCA product indicated the presence of papillomaviral DNA. The genome of this PV was cloned and the complete genomic sequence was determined. The Ursus maritimus PV type 1 (UmPV-1) genome counts 7582 basepairs and is smaller than that of other papillomaviruses from carnivore species. UmPV-1 contains the typical noncoding region NCR1, but unlike the carnivore PVs of the Lambda genus, UmPV-1 does not possess a second noncoding region NCR2. Phylogenetic analysis based on a nucleotide sequence alignment of the L1 ORF of UmPV-1 and 51 other PV types indicates that UmPV-1 does not cluster with any of the other carnivore PVs, but branches off near the root of the common branch of the genus Alphapapillomavirus.
Abstract: Epstein-Barr virus (EBV) has been implicated in the pathogenesis of different types of malignancies. While nonmelanoma skin cancers, lymphomas and Kaposi sarcomas are the most frequently reported malignancies after solid organ transplantation, EBV-associated smooth muscle tumors (EBV-SMT) after transplantation are rare and thus far only 18 cases in kidney recipients have been reported. A case of a 51-year-old kidney transplant recipient diagnosed with EBV-SMT is reported together with a review of the literature.
Abstract: In Europe, Puumala virus and Dobrava virus are the major hantaviruses that cause hemorrhagic fever with renal syndrome in humans. As hantaviruses can cause diseases with high morbidity and mortality rates, and as to date there is no specific treatment, efforts are concentrated on the development of vaccines. In this study we characterized the immunogenicity of recombinant nucleocapsid proteins of Puumala virus (PUUV) linked to a carrier protein corresponding to the outer membrane protein A from Klebsiella pneumoniae (rP40). The rP40 molecule is a novel carrier protein that facilitates exogenous antigen uptake by dendritic cells. We cloned and expressed the recombinant PUUV proteins in the E. coli mutant ICONE 200 using the tryptophan promoter-controlled pTEXmp18 expression vector. All recombinant PUUV proteins were found to be highly immunogenic in NMRI mice after three immunizations of 10 microg each of the protein. Only the truncated construct, P40-Puu118, gave high antibody titers after two vaccinations of 0.2 microg each. Likewise in the challenge experiments in NMRI mice, only the truncated construct P40-Puu118 resulted in 100% protection after three immunizations of 10 microg each. The results suggest that P40-Puu118 in particular is a good candidate for a recombinant vaccine against Puumala virus. All recombinant proteins linked to rP40 induced high antibody responses, indicating that rP40 is a carrier protein with potential for use in other vaccines.
Abstract: This study is the first prospective study to assess the prevalence, epidemiology, and risk factors of HIV-1 drug resistance in newly diagnosed HIV-infected patients in Belgium. In January 2003 it was initiated as part of the pan-European SPREAD program, and continued thereafter for four inclusion rounds until December 2006. Epidemiological, clinical, and behavioral data were collected using a standardized questionnaire and genotypic resistance testing was done on a sample taken within 6 months of diagnosis. Two hundred and eighty-five patients were included. The overall prevalence of transmitted HIV-1 drug resistance in Belgium was 9.5% (27/285, 95% CI: 6.6-13.4). Being infected in Belgium, which largely coincided with harboring a subtype B virus, was found to be significantly associated with transmission of drug resistance. The relatively high rate of baseline resistance might jeopardize the success of first line treatment as more than 1 out of 10 (30/285, 10.5%) viruses did not score as fully susceptible to one of the recommended first-line regimens, i.e., zidovudine, lamivudine, and efavirenz. Our results support the implementation of genotypic resistance testing as a standard of care in all treatment-naive patients in Belgium.
Abstract: A 52-year-old woman with no previous history of major health problems presented with an acute abdomen and symptoms of shock. Three days earlier she had been diagnosed as having acute laryngitis which was treated with steroids. On admission she was suffering from hypotension, renal failure, liver failure and coagulopathy. Emergency laparotomy revealed purulent fluid spread diffusely throughout the abdominal cavity. Streptococcus pyogenes was grown in culture from this fluid, enabling a diagnosis of streptococcal toxic shock syndrome (STSS) with primary peritonitis to be made. This combination is rare, and has been described only a few times. Only one other patient is known in whom this combination was preceded by respiratory symptoms. The treatment consists of abdominal lavage, intravenous administration of antibiotics and immunoglobulins, and support for renal function, liver function, respiration and coagulation.
Abstract: Hepatitis C virus (HCV) genotyping is a tool used to optimize antiviral treatment regimens. The newly developed Versant HCV genotype assay (LiPA) 2.0 uses sequence information from both the 5' untranslated region and the core region, allowing distinction between HCV genotype 1 and subtypes c to l of genotype 6 and between subtypes a and b of genotype 1. HCV-positive samples were genotyped manually using the Versant HCV genotype assay (LiPA) 2.0 system according to the manufacturer's instructions. For the comparison study, Versant HCV genotype assay (LiPA) 1.0 was used. In this study, 99.7% of the samples could be amplified, the genotype of 96.0% of samples could be determined, and the agreement with the reference method was 99.4% when a genotype was determined. The reproducibility study showed no significant differences in performance across sites (P = 0.43) or across lots (P = 0.88). In the comparison study, 13 samples that were uninterpretable or incorrectly genotyped with Versant HCV genotype assay (LiPA) 1.0 were correctly genotyped by Versant HCV genotype assay (LiPA) 2.0. Versant HCV genotype assay (LiPA) 2.0 is a sensitive, accurate, and reliable assay for HCV genotyping. The inclusion of the core region probes in Versant HCV genotype assay (LiPA) 2.0 results in a genotyping success rate higher than that of the current Versant HCV genotype assay (LiPA) 1.0.
Abstract: BACKGROUND: Centers for Disease Control (CDC) guidelines require confirmation of hepatitis C virus (HCV) screening-test-positive sera with a low signal/cut-off (S/CO) ratio by recombinant immunoblot or PCR. The UK Health Protection Agency has suggested that a second enzyme immunoassay (EIA) could be used as an alternative for confirmation in non-immunocompromised patients. OBJECTIVE: To evaluate the UK HPA approach in 17,936 consecutive in-house sera submitted for HCV testing. STUDY DESIGN: AxSYM-positive sera (S/CO> or =1.0) were tested with Monolisa Plus. AxSYM-positive sera of patients that were confirmed PCR-positive were considered HCV+. All other AxSYM-positive sera were confirmed with immunoblot according to CDC guidelines. RESULTS: 17,299 sera were negative with AxSYM. Of the 637 AxSYM-positive sera, 384 were from patients confirmed as PCR-positive. Of other 250 sera, 120 were negative with immunoblot, 103 were positive and 30 were indeterminate. All 30 immunoblot-indeterminate sera were PCR-negative. Two patients were Monolisa Plus+ and immunoblot- and PCR-. One patient was known as immunoblot-, while the other patient was diagnosed with non-A non-B hepatitis in 1980s. Nine sera from HCV-positive patients were Monolisa Plus-. Two PCR- sera were from immunocompetent patients who were PCR- for > or =8 years and six PCR- sera and one PCR+ serum were from immunocompromised patients. Sensitivity and specificity of confirmation with Monolisa Plus were 98.15% and 98.33% and the positive and negative predictive values were 99.58% and 92.91% in AxSYM-positive sera (excluding immunoblot-indeterminate/PCR-negative sera). If immunocompromised patients that were false-negative were excluded, sensitivity was 99.58%. CONCLUSION: Monolisa Plus can be used as an alternative to immunoblot for the confirmation of AxSYM-positive sera.
Abstract: BACKGROUND: Very little is known about human rotaviruses in the northwest of China. To investigate the genomic diversity, we evaluated the distribution of rotavirus genotypes in this region covering a 10-year period (1996-2005). METHODS: Rotavirus antigen was detected in stool specimens by enzyme immunoassay (EIA), and G and P genotyping was performed by reverse transcription-polymerase chain reaction and nucleotide sequencing methods. RESULTS: A total of 783 stool specimens collected from children with diarrhea, under 5 years of age, attending an urban hospital in Xinjiang were tested for rotavirus antigen, and 398 (50.8%) were positive. Overall, the most prevalent rotavirus genotype was G1P[8] (40.0%), followed by G3P[8] (17.5%), G2P[4] (8.3%), and G2P[6] (6.5%). G1 rotavirus was the most prevalent genotype until 2004. However, in 2005, G3 rotavirus (51.9%) became a dominating strain. Only one G9 strain was isolated in this region (isolated for the first time in 1999) and it became a more prevalent strain (21.2%) in 2005. CONCLUSIONS: The results of this study are of importance to the decision makers in the evaluation of rotavirus vaccines in China.
Abstract: BACKGROUND AND AIMS: Anti-alpha4 integrin therapy with natalizumab is efficacious in refractory Crohn's disease and in multiple sclerosis, but carries an estimated 1/1000 risk of progressive multifocal leukoencephalopathy (PML) caused by reactivation of latent JC virus infection. Although anti-alpha4 integrin therapies are likely to be introduced in the clinic, screening for the risk of PML has not been developed. METHODS: We prospectively collected urine, serum, plasma and buffy coats from 125 patients with Crohn's disease, 100 control subjects with gastrointestinal (GI) disease, and 106 healthy volunteers. Four to eight weeks after this first sample collection, we additionally collected a set of urine, serum, plasma and buffy coat samples from the 125 patients with Crohn's disease, and a next set of samples was collected 12-16 weeks after the first collection. JC viral loads were determined with quantitative real-time polymerase chain reaction (PCR), and JC virus seroprevalence with a specific enzyme-linked immunosorbant assay (ELISA). RESULTS: The overall JC virus seroprevalence was 65%. JC virus DNA copies were detected in the urine from 29-44% of subjects, both those with Crohn's disease and controls. Median viral loads were significantly higher in patients with Crohn's disease who were immunosuppressed (7.36x10(6) copies/ml) compared to healthy volunteers (2.77x10(5) copies/ml) and compared to GI controls (1.8x10(6) copies/ml). Clearance at any time point occurred in 4/107 (3.7%) subjects only. JC viraemia was found in two patients with Crohn's disease. CONCLUSIONS: The natural history of JC virus in patients with Crohn's disease is still unknown. Our study results show that JC virus latency and urine viral shedding is frequent in immunosuppressed patients with Crohn's disease. More prospective studies are needed in order to agree on possible recommendations concerning the exclusion of patients with JCV viraemia from anti-alpha4 integrin treatment.
Abstract: The genome of a novel virus, tentatively named bandicoot papillomatosis carcinomatosis virus type 2 (BPCV2), obtained from multicentric papillomatous lesions from an adult male southern brown bandicoot (Isoodon obesulus) was sequenced in its entirety. BPCV2 had a circular double-stranded DNA genome consisting of 7277 bp and open reading frames encoding putative L1 and L2 structural proteins and putative large T antigen and small t antigen transforming proteins. These genomic features, intermediate between Papillomaviridae and Polyomaviridae are most similar to BPCV1, recently described from papillomas and carcinomas in the endangered western barred bandicoot (Perameles bougainville). This study also employed in situ hybridization to definitively demonstrate BPCV2 DNA within lesion biopsies. The discovery of BPCV2 provides evidence of virus-host co-speciation between BPCVs and marsupial bandicoots and has important implications for the phylogeny and taxonomy of circular double-stranded DNA viruses infecting vertebrates.
Abstract: The immobilization of DNA strands is an essential step in the development of any DNA biosensor. Self-assembled mixed DNA/alkanethiol films are often used for coupling DNA probes covalently to the sensor surface. Although this strategy is well accepted, the effect of introducing a spacer molecule to increase the distance between the specific DNA sequence and the surface has rarely been assessed. The major goal of this work was to evaluate a number of such spacers and to assess their impact on for example the sensitivity and the reproducibility. Besides the commonly used mercaptohexyl (C(6)) spacer, a longer mercapto-undecyl (C(11)) spacer was selected. The combination of both spacers with tri(ethylene)glycol (TEG) and hexa(ethylene)glycol (HEG) was studied as well. The effect of the different spacers on the immobilization degree as well as on the consecutive hybridization was studied using surface plasmon resonance (SPR). When using the longer C(11) spacer the mixed DNA/alkanethiol films were found to be more densely packed. Further hybridization studies have indicated that C(11) modified probes improve the sensitivity, the corresponding detection limit as well as the reproducibility. In addition two different immobilization pathways, i.e. flow vs. diffusion controlled, were compared with respect to the hybridization efficiency. These data suggest that a flow-assisted approach is beneficial for DNA immobilization and hybridization events. In conclusion, this work demonstrates the considerable impact of spacers on the biosensor performance but also shows the importance of a flow-assisted immobilization approach.
Abstract: We identified sequences from two distantly related papillomaviruses in genital warts from two Burmeister's porpoises, including a PV antigen-positive specimen, and characterized Phocoena spinipinnis papillomavirus type 1 (PsPV-1). The PsPV-1 genome comprises 7879 nt and presents unusual features. It lacks an E7, an E8 and a bona fide E5 open reading frame (ORF) and has a large E6 ORF. PsPV-1 L1 ORF showed the highest percentage of nucleotide identity (54-55 %) with human papillomavirus type 5, bovine papillomavirus type 3 (BPV-3) and Tursiops truncatus papillomavirus type 2 (TtPV-2). This warrants the classification of PsPV-1 as the prototype of the genus Omikronpapillomavirus. PsPV-1 clustered with TtPV-2 in the E6 and E1E2 phylogenetic trees and with TtPV-2 and BPV-3 in the L2L1 tree. This supports the hypothesis that PV evolution may not be monophyletic across all genes.
Abstract: During treatment of chronic hepatitis C patients with interferon and ribavirin, a lot of side effects are described. Twenty-three percent to 44% of patients develop depression. A minority of patients evolve to psychosis. To the best of our knowledge, no cases of psychogenic parasitosis occurring during interferon therapy have been described in the literature. We present a 49-year-old woman who developed a delusional parasitosis during treatment with pegylated interferon alpha-2b weekly and ribavirin. She complained of seeing parasites and the larvae of fleas in her stools. This could not be confirmed by any technical examination. All the complaints disappeared after stopping pegylated interferon alpha-2b and reappeared after restarting it. She had a complete sustained viral response.
Abstract: Conservation efforts to prevent the extinction of the endangered western barred bandicoot (Perameles bougainville) are currently hindered by a progressively debilitating cutaneous and mucocutaneous papillomatosis and carcinomatosis syndrome observed in captive and wild populations. In this study, we detected a novel virus, designated the bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1), in lesional tissue from affected western barred bandicoots using multiply primed rolling-circle amplification and PCR with the cutaneotropic papillomavirus primer pairs FAP59/FAP64 and AR-L1F8/AR-L1R9. Sequencing of the BPCV1 genome revealed a novel prototype virus exhibiting genomic properties of both the Papillomaviridae and the Polyomaviridae. Papillomaviral properties included a large genome size ( approximately 7.3 kb) and the presence of open reading frames (ORFs) encoding canonical L1 and L2 structural proteins. The genomic organization in which structural and nonstructural proteins were encoded on different strands of the double-stranded genome and the presence of ORFs encoding the nonstructural proteins large T and small t antigens were, on the other hand, typical polyomaviral features. BPCV1 may represent the first member of a novel virus family, descended from a common ancestor of the papillomaviruses and polyomaviruses recognized today. Alternatively, it may represent the product of ancient recombination between members of these two virus families. The discovery of this virus could have implications for the current taxonomic classification of Papillomaviridae and Polyomaviridae and can provide further insight into the evolution of these ancient virus families.
Abstract: BACKGROUND: Estimating evolutionary rates for slowly evolving viruses such as papillomaviruses (PVs) is not possible using fossil calibrations directly or sequences sampled over a time-scale of decades. An ability to correlate their divergence with a host species, however, can provide a means to estimate evolutionary rates for these viruses accurately. To determine whether such an approach is feasible, we sequenced complete feline PV genomes, previously available only for the domestic cat (Felis domesticus, FdPV1), from four additional, globally distributed feline species: Lynx rufus PV type 1, Puma concolor PV type 1, Panthera leo persica PV type 1, and Uncia uncia PV type 1. RESULTS: The feline PVs all belong to the Lambdapapillomavirus genus, and contain an unusual second noncoding region between the early and late protein region, which is only present in members of this genus. Our maximum likelihood and Bayesian phylogenetic analyses demonstrate that the evolutionary relationships between feline PVs perfectly mirror those of their feline hosts, despite a complex and dynamic phylogeographic history. By applying host species divergence times, we provide the first precise estimates for the rate of evolution for each PV gene, with an overall evolutionary rate of 1.95 x 10(-8) (95% confidence interval 1.32 x 10(-8) to 2.47 x 10(-8)) nucleotide substitutions per site per year for the viral coding genome. CONCLUSION: Our work provides evidence for long-term virus-host co-speciation of feline PVs, indicating that viral diversity in slowly evolving viruses can be used to investigate host species evolution. These findings, however, should not be extrapolated to other viral lineages without prior confirmation of virus-host co-divergence.
Abstract: AIM: To study the predictive value of the vegetative-depressive symptoms of the Zung Depression Rating Scale for the occurrence of depression during treatment with peg-interferon alpha-2b of chronic hepatitis C (CHC) patients. METHODS: The predictive value of vegetative-depressive symptoms at 4 wk of treatment for the occurrence of a subsequent diagnosis of major depressive disorder (MDD) was studied in CHC patients infected after substance use in a prospective, multi-center treatment trial in Belgium. The presence of vegetative-depressive symptoms was assessed using the Zung Scale before and 4 wk after the start of antiviral treatment. RESULTS: Out of 49 eligible patients, 19 (39%) developed MDD. The area under the ROC curve of the vegetative Zung subscale was 0.73, P = 0.004. The sensitivity at a cut-point of > 15/35 was 95% (95% CI: 74-100). The positive predictive value equalled 44% (95% CI: 29-60). CONCLUSION: In this group of Belgian CHC patients infected after substance use, antiviral treatment caused a considerable risk of depression. Seven vegetative-depressive symptoms of the Zung scale at wk 4 of treatment predicted 95% of all emerging depressions, at a price of 56% false positive test results.
Abstract: Human respiratory syncytial virus (HRSV) is the leading viral cause of severe respiratory illness for infants and young children worldwide. Two major antigenic groups (A and B) of HRSV exist, and viruses from both subgroups can cocirculate during epidemics; however, their frequencies might differ between seasons. The subgroup prevalence and genotype distribution patterns of HRSV strains were investigated in a community in Belgium during 10 successive epidemic seasons (1996 to 2006). A regular 3-year cyclic pattern of subgroup dominance was observed, consisting of two predominant HRSV-A seasons, followed by a single HRSV-B-dominant year. HRSV infections with both subgroups were more prevalent among children younger than 6 months and had a peak incidence in December. The most frequently detected genotypes were GA5 and GB13, the latter including strains with the 60-nucleotide duplication in the G gene. Furthermore, GA5 remained the dominant HRSV genotype in two consecutive epidemic seasons twice during the study period. Additional variability was detected among the GB13 isolates, due to the usage of a novel termination codon in the G gene. Dual infections with both HRSV subgroups were detected for 9 patients, and subsequent infections with the heterologous HRSV subgroup were documented for 15 patients. Among five patients with homologous reinfections, only one was caused by HRSV-B. Our results support the hypothesis that the overall prevalence of HRSV-A over HRSV-B could be due to a more-transient subgroup A-specific immune protection.
Abstract: G12 rotaviruses were first detected in diarrheic children in the Philippines in 1987, but no further cases were reported until 1998. However, G12 rotaviruses have been detected all over the world in recent years. Here, we report the worldwide variations of G12 rotaviruses to investigate the evolutionary mechanisms by which they managed to spread globally in a short period of time. We sequenced the complete genomes (11 segments) of nine G12 rotaviruses isolated in Bangladesh, Belgium, Thailand, and the Philippines and compared them with the genomes of other rotavirus strains. Our genetic analyses revealed that after introduction of the VP7 gene of the rare G12 genotype into more common local strains through reassortment, a vast genetic diversity was generated and several new variants with distinct gene constellations emerged. These reassortment events most likely took place in Southeast Asian countries and spread to other parts of the world. The acquirement of gene segments from human-adapted rotaviruses might allow G12 to better propagate in humans and hence to develop into an important emerging human pathogen.
Abstract: Adherence of Actinobacillus actinomycetemcomitans to epithelial cells is an important step in periodontal disease pathogenesis. Recent publications describe the subgingival presence of a wide array of viruses [e.g., human cytomegalo-virus (hCMV)]. Since viruses can increase cellular susceptibility for bacterial adherence, we investigated whether hCMV renders epithelial cells more prone to adherence by Actinobacillus actinomycetemcomitans. Cultivated HeLa and primary epithelial cells were shown to be semi-permissive for hCMV infection, which resulted in increased bacterial adherence. This increase correlated with viral concentrations, was evident in all Actinobacillus actinomycetemcomitans strains examined, and increased during the first 24 hrs, followed by a slight decrease. Immediate early antigen expression was not correlated with the increased adherence of Actinobacillus actinomycetemcomitans. The results confirmed our hypothesis that the adherence of Actinobacillus actinomycetemcomitans is influenced by hCMV in vitro.
Abstract: Puumala virus, a hantavirus belonging to the Bunyaviridae family, causes a human disease known as nephropathia epidemica, a mild form of hemorrhagic fever with renal syndrome. The implementation of effective decontamination procedures is critical in hantavirus research to minimize the risk of personnel exposure. This study investigated the efficacy of Clidox((R)), Dettol((R)), ethanol, Halamid-d((R)), peracetic acid, sodium hypochloride and Virkon((R))S for inactivating Puumala virus. A real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to quantify Puumala virus before and after treatment with these products. Inactivation of Puumala virus was effective after 10min with all products except ethanol. Inactivation with absolute ethanol was effective only after 30min. Using the qRT-PCR method, this study has shown that the commercially available products Clidox((R)), Halamid-d((R)) and Virkon((R))S in particular represent a rapid and safe way to decontaminate surfaces with possible Puumala virus contamination. These products can be used in solutions of 1-2%, with contact times greater than 10min, for inactivating effectively Puumala virus.
Abstract: Human group B rotaviruses were isolated from hospitalized patients in Bangladesh between July 2003 and December 2004. Phylogenetic analyses of the gene segments encoding the hemagglutinin (VP4), glycoprotein (VP7) and RNA-binding protein (NSP2) of group B rotaviruses showed that Bangladeshi strains were more similar to the Indian strains than to the prototype Chinese strains. Moreover, all human strains were clustered together and were distantly related to the animal strains. With limited sequence data, the evolutionary rate of the glycoproteins (VP7) of human group B rotaviruses was estimated to be 1.57x10(-3) nucleotide substitutions/(siteyear), which was comparable to other rapidly evolving RNA viruses. The most recent common ancestor (MRCA) of the extant human group B rotaviruses was calculated to date to around 1976.
Abstract: BACKGROUND: Resistance testing has been implemented into clinical guidelines as it has shown some beneficial effect on subsequent therapy response. CASE REPORT: Routine population-based genotypic resistance testing for a newly diagnosed HIV-1 patient revealed the presence of resistance mutations M41L, V179D and T215E within reverse transcriptase and no mutations within protease. Four weeks after initiation of the combination tenofovir+lamivudine+efavirenz, no response was observed despite good adherence to therapy and efavirenz drug levels in the therapeutic range. Retrospective single genome sequencing of the baseline sample revealed the presence of minority viral variants with additional mutations: a mutation conferring resistance to lamivudine (M184IV), a thymidine associated mutation (K219R) and mutations possibly associated with non-nucleoside reverse transcriptase resistance (F227S, M230IV). CONCLUSIONS: This case illustrates that undetected drug-resistant minority variants can reduce the efficacy of a normally very potent first-line regimen tenofovir+lamivudine+efavirenz. The presence of drug-resistance mutations at diagnosis should be considered as a warning sign against the use of low genetic barrier drugs in first-line regimens, even when these drugs are considered to be active according to routine resistance testing.
Abstract: The papillomaviruses form a large group of species-specific pathogens that cause epithelial proliferations in a wide spectrum of animal hosts. Previous reports demonstrated a relatively high frequency of a variety of skin lesions in captive European harvest mice. The Micromys minutus papillomavirus (MmPV) was isolated from one of these lesions found on a captive European harvest mouse in a regional zoo in Chicago. In this study we present the entire genomic sequence of MmPV. The MmPV genome is organized into the seven classical papillomaviral open reading frames. Phylogenetic analysis places MmPV together with a papillomavirus (PV) isolated from a Syrian golden Hamster (HaOPV) in the genus Pipapillomavirus. The similar clustering pattern of the MmPV-HaOPV pair and their rodent hosts support the hypothesis of papillomaviral and host co-phylogenetic descent. The availability of the complete genomic sequence of a mouse PV should allow researchers to use MmPV as a model for PV carcinogenesis.
Abstract: Approximately 20,000 stool specimens from patients with diarrhea visiting 1 urban and 1 rural hospital in Bangladesh during January 2001-May 2006 were tested for group A rotavirus antigen, and 4,712 (24.0%) were positive. G and P genotyping was performed on a subset of 10% of the positive samples (n = 471). During the 2001-2005 rotavirus seasons, G1P[8] (36.4%) and G9P[8] (27.7%) were the dominant strains, but G2[4] and G12P[6] were present in 15.4% and 3.1% of the rotavirus-positive patients, respectively. During the 2005-06 rotavirus season, G2P[4] (43.2%) appeared as the most prevalent strain, and G12P[6] became a more prevalent strain (11.1%) during this season. Because recently licensed rotavirus vaccines include only the P[8] specificity, it is unknown how the vaccines will perform in settings where non-P[8] types are prevalent.
Abstract: We describe the antiviral activity of plant lectins with specificity for different glycan structures against the severe acute respiratory syndrome coronavirus (SARS-CoV) and the feline infectious peritonitis virus (FIPV) in vitro. The SARS-CoV emerged in 2002 as an important cause of severe lower respiratory tract infection in humans, and FIPV infection causes a chronic and often fatal peritonitis in cats. A unique collection of 33 plant lectins with different specificities were evaluated. The plant lectins possessed marked antiviral properties against both coronaviruses with EC(50) values in the lower microgram/ml range (middle nanomolar range), being non-toxic (CC(50)) at 50-100 microg/ml. The strongest anti-coronavirus activity was found predominantly among the mannose-binding lectins. In addition, a number of galactose-, N-acetylgalactosamine-, glucose-, and N-acetylglucosamine-specific plant agglutinines exhibited anti-coronaviral activity. A significant correlation (with an r-value of 0.70) between the EC(50) values of the 10 mannose-specific plant lectins effective against the two coronaviruses was found. In contrast, little correlation was seen between the activity of other types of lectins. Two targets of possible antiviral intervention were identified in the replication cycle of SARS-CoV. The first target is located early in the replication cycle, most probably viral attachment, and the second target is located at the end of the infectious virus cycle.
Abstract: OBJECTIVES: Evaluation of a wide variety of pyridine N-oxide derivatives on their inhibitory activity against feline coronavirus (FIPV strain) and human SARS-CoV (Frankfurt strain-1) in cell culture. METHODS: FIPV and SARS-CoV were exposed to confluent Crandel feline kidney (CRFK) and simian kidney (Vero) cell cultures in the presence of serial concentrations of the test compounds. The anti-cytopathic activity of the pyridine N-oxide derivatives was monitored by spectrophotometric analysis. RESULTS AND CONCLUSIONS: A wide variety of pyridine N-oxide derivatives have been found to be inhibitory against feline coronavirus (FIPV strain) and human SARS-CoV (Frankfurt strain-1) in CRFK and simian kidney (Vero) cell cultures, respectively. The oxide part on the pyridine moiety proved indispensable for anti-coronavirus activity. The potency and virus specificity of the pyridine N-oxide derivatives varied depending the nature and specific location of substituents (i.e. alkyl, halogeno, nitro, etc.) on the different parts of the molecule. The most selective compounds were active in the higher microgram per litre range, being non-toxic at 50-100 mg/L. There was a poor structure-antiviral activity relationship (SAR) for the pyridine N-oxide derivatives against Fe-CoV and SARS-CoV. One of the most active and selective compounds was shown to inhibit Fe-CoV replication at the transcriptional level.
Abstract: The close genetic and antigenic relatedness among the group 2 coronaviruses human coronavirus OC43 (HCoV-OC43), bovine coronavirus (BCoV), and porcine hemagglutinating encephalomyelitis virus (PHEV) suggests that these three viruses with different host specificities diverged fairly recently. In this study, we determined the complete genomic sequence of PHEV (strain PHEV-VW572), revealing the presence of a truncated group 2-specific ns2 gene in PHEV in comparison to other group 2 coronaviruses. Using a relaxed molecular clock approach, we reconstructed the evolutionary relationships between PHEV, BCoV, and HCoV-OC43 in real-time units, which indicated relatively recent common ancestors for these species-specific coronaviruses.
Abstract: We describe the cloning and expression of Dobrava hantavirus (DOBV) nucleocapsid proteins and a truncated form consisting of the first 118 N-terminal amino acids, and the capacity of these E. coli ICONE 200-expressed recombinant proteins (rNp) to induce a protective immune response against DOBV in mice. As an alternative carrier protein, the outer membrane protein A derived from Klebsiella pneumoniae (rP40) has been coupled to different rNp constructs. All recombinant proteins were found to be highly immunogenic after three immunizations of rNp. The immunizations resulted in the induction of a strong Np-specific IgG response with a predominance of IgG1 over IgG2b and IgG2a, suggesting a mixed Th1/Th2 cell involvement. A specific IgG3 response could not be detected. Mice immunized with recombinant DOBV rNp without rP40 showed lower nucleocapsid-specific antibody responses in comparison with the rP40-conjugated constructs, but all mice were found to be protected against DOBV challenge. Our results indicate that the rNp constructs coupled to rP40, represent promising vaccine candidates.
Abstract: Various semisynthetic derivatives of glycopeptide antibiotics including vancomycin, eremomycin, teicoplanin, ristocetin A and DA-40926 have been evaluated for their inhibitory activity against feline infectious peritonitis virus (FIPV) and human (SARS-CoV, Frankfurt-1 strain) coronavirus in cell culture in comparison with their activity against human immunodeficiency virus (HIV). Several glycopeptide derivatives modified with hydrophobic substituents showed selective antiviral activity. For the most active compounds, the 50% effective concentrations (EC(50)) were in the lower micromolar range. In general, removal of the carbohydrate parts of the molecules did not affect the antiviral activity of the compounds. Some compounds showed inhibitory activity against both, whereas other compounds proved inhibitory to either, FIPV or SARS-CoV. There was no close correlation between the EC(50) values of the glycopeptide derivatives for FIPV or SARS-CoV.
Abstract: The complete genomic DNA of a novel papillomavirus (PV) was isolated from a basosquamous carcinoma on the wing of an Egyptian fruit bat (Rousettus aegyptiacus). Initial short sequences of the E1 and L1 genes of this virus were retrieved by PCR with degenerate papillomavirus-specific primers, and the entire R. aegyptiacus papillomavirus type 1 (RaPV-1) DNA was then amplified by long template PCR, cloned and sequenced with a transposon insertion method. The RaPV-1 genome counts 7970 basepairs and contains the typical papillomavirus open reading frames (ORF) (E1, E2, E4, E6, E7, L1 and L2). Based on a concatenated alignment of the E1, E2, L1 and L2 open reading frames of RaPV-1 and 46 other human and animal papillomavirus type species, a neighbor-joining phylogenetic tree was constructed. This phylogenetic analysis shows that RaPV-1 has a close-to-root position in the papillomavirus evolutionary tree. Since RaPV-1 is only distantly related to other papillomaviruses (with maximally 50% nucleotide sequence identity across the L1 open reading frame), it cannot be assigned to one of the existing papillomavirus genera and therefore represents the first member of a novel, as yet unnamed, close-to-root papillomavirus genus. This is the first time a papillomavirus has been isolated and characterized from a member of the Chiroptera order.
Abstract: Severe human infection with Hantavirus is characterized by high fever, cold chills, thrombocytopenia, arterial hypotension, acute renal failure, and/or adult respiratory distress syndrome (ARDS)-like pulmonary involvement, but the clinical course varies greatly between individuals. We investigated whether genetically determined differences in tumor necrosis factor (TNF)-alpha production can influence the severity of Hantavirus disease. We studied a TNF-alpha single-nucleotide promoter polymorphism (SNP) at position -238 (a guanine [G]-to-adenine [A] transition) and ex vivo TNF-alpha production in a recall study of 36 Belgian patients who had a serologically proven form of Puumala virus-induced Hantavirus infection with the kidney as main target organ. In our study, the highest creatinine levels were found in patients with the lowest ex vivo TNF-alpha production. Creatinine levels correlated inversely with TNF-alpha production (R = -0.35, p < 0.05). The number of thrombocytes was significantly lower in patients with the GA-238 genotype (low TNF-alpha producers) compared with patients with the GG-238 genotype. In our study, genetically determined low production of TNF-alpha was associated with some parameters indicating a more severe clinical course of Puumala Hantavirus infection in humans, possibly by impaired activation of TNF-alpha-dependent antiviral mechanisms, which could in turn result in decreased clearance of Hantavirus.
Abstract: A 5-yr-old female Egyptian fruit bat (Rousettus aegyptiacus) had a small raised pigmented mass removed from the lateral canthus of the left eye. Six additional variably sized, raised, smooth to cauliflower-like skin masses were observed randomly distributed throughout the left wing membranes. Four masses were removed and diagnosed microscopically as basosquamous carcinomas and papillomas. Additional masses, removed 6 mo and 1 yr later, showed bony invasion and squamous differentiation. Immunohistochemistry detected positive intranuclear staining for bovine papillomavirus antibody in all samples. Polymerase chain reaction done on DNA extracts from formalin-fixed, paraffin-embedded tumor tissue amplified a 450 base-pair segment analogous to the L1 region of human papillomavirus types 96 and 5. Basic Local Alignment Search Tool analysis of sequenced amplicons suggests a novel chiropteran papillomavirus. To our knowledge, this is the first report of papillomavirus-associated carcinoma in a chiropteran species.
Abstract: Coinfection with other hepatitis viruses modifies the viral profile in serum and leads to more liver damage and more rapid progression during the course of hepatitis C virus infection. The viral interference is not only carried out by virus-virus or by virus-cell interactions but also by an enhanced immune response. A superinfecting viral infection does not crossactivate protective immune responses to the pre-existing virus albeit the latter can become undetectable. The induced cytokine stimulation might enhance the hepatic inflammation. Moreover, hepatitis B virus coinfection increases the risk of development of hepatocellular carcinoma in hepatitis C virus patients through common necro-inflammatory pathways or by direct oncogenic activity of hepatitis B virus. Viral interaction also complicates the management of the coinfection because hepatitis C virus impairs the humoral response to hepatitis A virus and hepatitis B virus vaccines, and because pharmacological suppression of hepatitis C virus endangers dually infected patients with reactivation of coinfected hepatitis B virus. Optimized strategies and follow-up are thus necessary in the treatment of infection with multiple viruses. It seems thus necessary to look for markers of hepatitis B virus and/or hepatitis D virus infection in chronic hepatitis patients positive for hepatitis C virus antibodies but negative for hepatitis C virus RNA, and equally well to search for hepatitis C virus RNA in HBsAg-negative/anti-HBc-positive patients with a low level of serum hepatitis B virus DNA.
Abstract: A novel papillomavirus (PV) was isolated from a genital condyloma of a free-ranging bottlenose dolphin inhabiting the coastal waters of Charleston Harbor, SC, USA: Tursiops truncatus papillomavirus type 2 (TtPV2). This novel virus represents the first isolated North American cetacean PV and the first American bottlenose dolphin PV. After the viral genome was cloned, sequenced and characterized genetically, phylogenetic analyses revealed that TtPV2 is most similar to the only published cetacean PV isolated and characterized thus far, Phocoena spinipinnis PV type 1 (PsPV1). A striking feature of the genome of TtPV2, as well as that of PsPV1, is the lack of an E7 open reading frame, which typically encodes one of the oncogenic proteins believed to be responsible for malignant transformation in the high-risk mucosotropic human papillomaviruses (HPVs). TtPV2 E6 contains a PDZ-binding motif that has been shown to be involved in transformation in the case of high-risk genital HPVs.
Abstract: Several G8P[6] and G8P[8] rotavirus strains were isolated from hospitalized patients in the Democratic Republic of Congo in 2003. To investigate their overall genomic relatedness and to determine to which genogroup they belonged, the complete genomes of strains DRC88 (G8P[8]) and DRC86 (G8P[6]) were determined. Genomic comparison of these two African G8 strains revealed that 10 out of their 11 gene segments, except for VP4, were nearly identical (>98.9% identical at the nucleotide level), suggesting that this rare G8P[8] rotavirus strain originated recently from a reassortment between a common G8P[6] strain and a strain with a P[8] specificity. A very close evolutionary relationship between 9 out of the 11 gene segments of DRC88 and DRC86 and rotavirus strains belonging to the DS-1-like (G2P[4]) "genogroup" was found, and several possible reassortment events preceding the occurrence of G8P[8] and G8P[6] human rotaviruses were hypothesized. Since the genes of G2P[4] rotavirus strains are very well adapted to infect humans, the acquirement of a new VP7 (G8) gene, and especially the replacement of P[6] (believed to be of animal origin) by P[8] (most common in human rotaviruses), might make DRC88-like rotaviruses very well equipped to become a predominant human rotavirus strain and an important pathogen on the African continent and the rest of the world. These findings have important implications for rotavirus vaccine development and highlight that typing of new rotavirus strains by merely sequencing their VP7 and VP4 genes provides us with only the tip of the iceberg regarding rotavirus diversity.
Abstract: Epidemiological and phylogenetic studies of hepatitis C virus (HCV) have identified six major HCV genotypes and have attempted to characterize their origin and spread worldwide. Putative regions of endemic infection have been identified for all HCV genotypes except HCV genotype 5a. Although HCV genotype 5a was previously thought to be largely restricted to the northern part of South Africa, this study reports an unexpected cluster of the genotype in West Flanders Province in Belgium. To investigate the molecular epidemiology of this cluster and of HCV genotype 5a in general, a rigorous phylogenetic analysis of Belgian and South African HCV genotype 5a samples was performed. Remarkably, the Belgian and South African strains form two distinct clusters of similar diversity. We used a Bayesian coalescent method to estimate the rate of virus spread through time for HCV genotype 5a in both regions. Our results indicate that HCV genotype 5a strains have been spreading independently in Belgium and South Africa for more than 100 years, with a rate of spread characteristic of an epidemic genotype. These findings have major implications for tracing the origin of HCV genotype 5a. Here, we speculate about the possible origins of these clusters.
Abstract: Gene rearrangements in a head-to-tail fashion have been described several times for gene segments of the rota-, phytoreo-, and orbiviruses. Several mechanisms have been proposed to explain the occurrence of partial duplications, however, none of these models has been fully satisfactory to explain the occurrence of all the observed duplicated genes. Based on recently available structural data about the lambda3 RNA-dependent-RNA-polymerase of reoviruses, we propose the 'loop model' as a plausible explanation for the occurrence of partial gene duplications in dsRNA viruses.
Abstract: In human immunodeficiency virus type 1 (HIV-1), an interaction exists between the in vivo evolution of Gag protein and protease to escape from antiretroviral drug selective pressure. Therefore, it was decided to develop a genotypic assay for the amplification and sequencing of HIV-1 gag and protease. As the HIV-1 pandemic is characterised by a large genetic diversity, the assay developed was evaluated on a panel of 28 genetically divergent samples belonging to the following subtypes A1, B, C, D, F1, F2, G, H, J, CRF01-AE, CRF02-AG and CRF13-cpx. The assay displayed a detection limit ranging between 500 RNA copies/ml and 5000 RNA copies/ml plasma. Full-length sequences could be obtained for 25 samples. The population sequences of the three other samples lacked a part of the sequence because of heterogeneous signal, probably due to the presence of quasi-species with insertions/deletions of a different length.
Abstract: OBJECTIVES: There is some reluctance to treat intravenous drug users (IVDUs) with chronic hepatitis C (CHC) because of presumed lower compliance and response to antiviral therapy. We intended to evaluate the compliance and response to antiviral treatment for CHC in IVDUs compared with non-IVDUs. METHODS: A retrospective cohort study--secondary analysis of the results of a treatment trial--was performed in Belgium and The Netherlands. A total of 406 previously untreated CHC patients, including 98 (24%) IVDUs, were studied for compliance (presentation at the end of treatment), complete response (alanine aminotransferase within normal limits and serum hepatitis C virus polymerase chain reaction negative) at the end of therapy and sustained virological response (SVR). RESULTS: Non-compliance (8.2%) in IVDUs was not different from non-IVDUs (6.8%) (relative risk=1.20; 95% confidence interval=0.55-2.62). Complete response after controlling for hepatitis C virus was similar (relative risk=1.19; 95% confidence interval=0.89-1.60). Controlling for treatment arm, age, sex, presence of cirrhosis or hepatitis C virus viral load before treatment did not change these results. There was a marginally significant difference in the sustained virological response between IVDUs (46.6%) and non-IVDUs (34.6%) (relative risk=1.35; 95% confidence interval=1.00-1.81), also disappearing after adjusting for genotype. No difference in compliance or sustained virological response was found between active and non-active IVDUs or between IVDU patients in or without a methadone maintenance program. CONCLUSIONS: In this group of Benelux patients, IVDUs showed similar compliance and response to treatment with interferon and ribavirin compared with other patients with CHC infection. Therefore, it is no longer justifiable to withhold treatment to chronic hepatitis C patients who use intravenous drugs.
Abstract: The healthy skin of a seven-year-old female goat (Capra hircus) was sampled with a cotton tip swab. Total genomic DNA was extracted from the sample and subjected to multiply primed rolling-circle amplification (RCA). Restriction analysis showed that the complete genome of a papillomavirus was amplified, measuring approximately 7,600 bp in length. The Capra hircus papillomavirus (ChPV-1) genome was cloned from this rolling-circle amplification product, and the complete nucleotide sequence was determined. The ChPV-1 genome counts 7,542 bp, and contains the typical papillomaviral open reading frames (ORFs). Based on a concatenated alignment of the E1, E2, L1 and L2 open reading frames of ChPV-1 and 54 other animal and human papillomavirus types, a neighbor-joining phylogenetic tree was constructed. In this tree ChPV-1 clusters with BPV-3, -4 and -6. Pairwise nucleotide sequence alignments of the L1 open reading frame of ChPV-1 with its closest relatives showed less than 60% similarity, placing the ChPV-1 in a novel genus.
Abstract: The Belgian rotavirus strain B4106, isolated from a child with gastroenteritis, was previously found to have VP7 (G3), VP4 (P[14]), and NSP4 (A genotype) genes closely related to those of lapine rotaviruses, suggesting a possible lapine origin or natural reassortment of strain B4106. To investigate the origin of this unusual strain, the gene sequences encoding VP1, VP2, VP3, VP6, NSP1, NSP2, NSP3, and NSP5/6 were also determined. To allow comparison to a lapine strain, the 11 double-stranded RNA segments of a European G3P[14] rabbit rotavirus strain 30/96 were also determined. The complete genome similarity between strains B4106 and 30/96 was 93.4% at the nucleotide level and 96.9% at the amino acid level. All 11 genome segments of strain B4106 were closely related to those of lapine rotaviruses and clustered with the lapine strains in phylogenetic analyses. In addition, sequence analyses of the NSP5 gene of strain B4106 revealed that the altered electrophoretic mobility of NSP5, resulting in a super-short pattern, was due to a gene rearrangement (head-to-tail partial duplication, combined with two short insertions and a deletion). Altogether, these findings confirm that a rotavirus strain with an entirely lapine genome complement was able to infect and cause severe disease in a human child.
Abstract: INTRODUCTION: Severe acute respiratory syndrome (SARS) is an emerging infectious disease that first occurred in humans in the People's Republic of China in November 2002 and has subsequently spread worldwide. A novel virus belonging to the Coronaviridae family has been identified as the cause of this pulmonary disease. The severity of the disease combined with its rapid spread requires the development of fast and sensitive diagnostic assays. RESULTS: A real-time quantitative RT-PCR was designed in the nsp11 region of the replicase 1B domain of the SARS-coronavirus (SARS-CoV) genome. To evaluate this quantitative RT-PCR, cRNA standards were constructed by in vitro transcription of SARS-CoV Frankfurt 1 RNA using T7 RNA polymerase, followed by real-time RT-PCR. The assay allowed quantitation over a range of 10(2) to 10(8) RNA copies per reaction. CONCLUSIONS: Extrapolated to clinical samples, this novel assay has a detection range of 10(4) to 10(10) copies of viral genome equivalents per millilitre. In comparison to the current de facto cRNA Artus Biotech standard, the in-house cRNA standard gives a 100-fold higher absolute quantity, suggesting a possible underestimation of the viral load when using the Artus Biotech standard.
Abstract: BACKGROUND: The enteric subgroup F adenoviruses type 40 (Ad40) and 41 (Ad41) are the second most important cause of acute infantile gastroenteritis after rotaviruses. Repeated community outbreaks have been associated with antigenic changes among the Ad40 and Ad41 strains due to host immune pressure. Therefore large field epidemiological surveys and studies on the genetic variations in different isolates of Ad40 and Ad41 are important for disease control programs, the design of efficient diagnostic kits and vaccines against subgroup F adenoviruses. A novel method using sodium dodecyl sulphate SDS/EDTA-pretreated chromatography paper strips was evaluated for the collection, storage and shipping of Ad40/41 contaminated stool samples. RESULTS: This study shows that adenoviral DNA can be successfully detected in the filter strips by PCR after four months storage at -20 degrees C, 4 degrees C, room temperature (20-25 degrees C) and 37 degrees C. Furthermore no adenoviral infectivity was observed upon contact with the SDS/EDTA-pretreated strips. CONCLUSIONS: Collecting, storing and transporting adenovirus type 40 and 41 positive stool samples on SDS/EDTA-pretreated chromatography filter strips is a convenient, biosafe and cost effective method for studying new genome variants and monitoring spread of enteric adenovirus strains during outbreaks.
Abstract: Substance use is emerging in Belgium. The 'typical user' starts at a young age. Polydrug use has become very common. Cannabis and alcohol are the most frequently used substances among the school population. Heroin and cocaine are the most frequently injected drugs. Sharing of injecting material and paraphernalia is reported to happen in half of the subjects injecting drugs. Substance use causes a lot of adverse organic, social and psychiatric events. Management of substance use consists of information, drug-free treatment and harm reduction, including substitution and maintenance programs. The management of care in Belgium differs between the regions.
Abstract: In industrialised countries, injecting drug use is currently the most important risk factor for infection with hepatitis C, resulting in high prevalence rates of hepatitis C among injecting drug users. To contain the hepatitis C epidemic major efforts should be done to prevent new infection among injecting drug users. Monitoring infection rates are crucial as it may provide feedback on the effectiveness of interventions. In this article the epidemiology of hepatitis C among injecting drug users in Belgium is briefly reviewed. More specifically the prevalence of anti-HCV antibodies, the prevalence of co-infections, the proportion of chronic HCV carriers, the distribution of genotypes and preventive measures among injecting drug users in Belgium are discussed and compared to the situation elsewhere in Western Europe.
Abstract: BACKGROUND: Environmental factors trigger the onset of inflammatory bowel disease (IBD) in genetically predisposed individuals. Exposure to seasonal external factors during the maturation of the immune system is suspected to be an inducing factor for IBD. Some studies suggested an association between the month of birth and the later development of IBD. We studied this putative relationship in a large cohort of Belgian patients with Crohn's disease (CD). METHODS: Data from 1025 patients born between 1935 and 1990 were collected. Diagnosis of CD was based on generally accepted clinical, endoscopic, and histologic criteria. As a control group, a cohort of 5125 non-IBD patients seen at the same hospital and matched for birth year and sex was used. Odds ratios were calculated using multivariate unconditional logistic regression including the matching variables and allowing for cyclic variation in risk with month of birth. RESULTS: A cyclic pattern described by a 4-month periodic function was observed with peaks in April and August. Moreover, being born in June significantly reduced the risk of developing CD later in life (P = 0.012). CONCLUSION: In this Belgian cohort, a significant association was found between the month of birth and later development of IBD; a significant reduced risk to develop CD was observed for people born in June. Moreover, environmental yearly reoccurring factors during pregnancy or postpartum might be associated with the occurrence of CD later in life.
Abstract: In this study, we report the complete genome sequence of two contemporary human coronavirus OC43 (HCoV-OC43) strains detected in 2003 and 2004, respectively. Comparative genetic analyses of the circulating strains and the prototype HCoV-OC43 strain (ATCC VR759) were performed. Remarkably, a lower than expected similarity is found between the complete genomes and more in particular between the spike genes of the BE03 and BE04 strains. This finding suggests the existence of two genetically distinct HCoV-OC43 strains, circulating in Belgium in 2003 and 2004. Spike gene sequencing of three additional 2003 and two additional 2004 HCoV-OC43 strains, and subsequent phylogenetic analysis confirm this assumption. Compared to the ATCC prototype HCoV-OC43 strain, an important amino acid substitution is present in the potential cleavage site sequence of the spike protein of all contemporary strains, restoring the N-RRXRR-C motif, associated with increased spike protein cleavability in bovine coronaviruses. We here describe specific characteristics associated with circulating HCoV-OC43 strains, and we provide substantial evidence for the genetic variability of HCoV-OC43.
Abstract: A 32-base pair deletion in the CC-chemokine receptor 5 gene (CCR5), associated with resistance to human immunodeficiency virus type 1 (HIV-1) infection, has recently been suggested to act as an adverse host factor in hepatitis C virus (HCV) infection. To examine this hypothesis, we determined the CCR5-Delta32 allele frequency by polymerase chain reaction in a Belgian cohort of 163 HCV-infected patients and 310 healthy control subjects. The resulting CCR5-Delta32 allele frequencies were 0.080 and 0.119 for the patient group and control group, respectively. In contrast with a previous study, we could not show a statistically significant difference between the CCR5-Delta32 allele frequencies in HCV patients and controls. Moreover, genotype distributions in both populations were in agreement with Hardy-Weinberg equilibrium. Our results do not support the hypothesis that the CCR5-Delta32 mutant allele is a risk factor for hepatitis C virus infection.
Abstract: BACKGROUND: Group A rotavirus genotypes G1, G2, G3 and G4 are the main etiological agents of infantile diarrhea. The G9 rotavirus has recently emerged as a fifth important genotype all over the world. OBJECTIVE: To characterize the VP7 gene of group A rotaviruses from gastroenteritis patients admitted to the Gasthuisberg University Hospital, Leuven, Belgium, during 1999-2003. STUDY DESIGN: Rotavirus antigen was detected in stool specimens using an enzyme immunoassay. G-typing was performed by reverse transcriptase polymerase chain reaction (RT-PCR) amplification and sequencing of the complete VP7 gene. RESULTS: The genotype distribution varied markedly over the four rotavirus years in Belgium. In the 1999-2000 rotavirus year, G1 was the predominating type (72%), and G9 was present in 5% of the rotavirus-positive patients. In the 2000-2001 and 2002-2003 years, G9 appeared as the dominating strain (45% and 53%, respectively). In the 2001-2002 year, between two G9 epidemic years, G1 was dominating (66%) but G9 was still present in 24%. All the G9 isolates were combined with P[8] and shared a high gene sequence similarity (<3% sequence divergence on the nucleotide and amino acid level). Phylogenetic analysis of the VP7 genes revealed that our Belgian G9 strains clustered together with recent G9 strains from all over the world, distinct from the prototype G9 strains isolated in the 1980s. CONCLUSION: Our study indicates that although the first introduction of G9 isolates in the Belgian population was recorded in 1997, G9 strains were able to establish themselves quickly as the predominant genotype. The emergence of G9 as an important pathogen in both developing as industrialized countries necessitates the urgent consideration of the G9 moiety in rotavirus vaccines.
Abstract: BACKGROUND: Four human coronaviruses are currently known to infect the respiratory tract: human coronaviruses OC43 (HCoV-OC43) and 229E (HCoV-229E), SARS associated coronavirus (SARS-CoV) and the recently identified human coronavirus NL63 (HCoV-NL63). In this study we explored the incidence of HCoV-NL63 infection in children diagnosed with respiratory tract infections in Belgium. METHODS: Samples from children hospitalized with respiratory diseases during the winter seasons of 2003 and 2004 were evaluated for the presence of HCoV-NL63 using a optimized pancoronavirus RT-PCR assay. RESULTS: Seven HCoV-NL63 positive samples were identified, six were collected during January/February 2003 and one at the end of February 2004. CONCLUSIONS: Our results support the notation that HCoV-NL63 can cause serious respiratory symptoms in children. Sequence analysis of the S gene showed that our isolates could be classified into two subtypes corresponding to the two prototype HCoV-NL63 sequences isolated in The Netherlands in 1988 and 2003, indicating that these two subtypes may currently be cocirculating.
Abstract: Coronaviruses are enveloped, positive-stranded RNA viruses with a genome of approximately 30 kb. Based on genetic similarities, coronaviruses are classified into three groups. Two group 2 coronaviruses, human coronavirus OC43 (HCoV-OC43) and bovine coronavirus (BCoV), show remarkable antigenic and genetic similarities. In this study, we report the first complete genome sequence (30,738 nucleotides) of the prototype HCoV-OC43 strain (ATCC VR759). Complete genome and open reading frame (ORF) analyses were performed in comparison to the BCoV genome. In the region between the spike and membrane protein genes, a 290-nucleotide deletion is present, corresponding to the absence of BCoV ORFs ns4.9 and ns4.8. Nucleotide and amino acid similarity percentages were determined for the major HCoV-OC43 ORFs and for those of other group 2 coronaviruses. The highest degree of similarity is demonstrated between HCoV-OC43 and BCoV in all ORFs with the exception of the E gene. Molecular clock analysis of the spike gene sequences of BCoV and HCoV-OC43 suggests a relatively recent zoonotic transmission event and dates their most recent common ancestor to around 1890. An evolutionary rate in the order of 4 x 10(-4) nucleotide changes per site per year was estimated. This is the first animal-human zoonotic pair of coronaviruses that can be analyzed in order to gain insights into the processes of adaptation of a nonhuman coronavirus to a human host, which is important for understanding the interspecies transmission events that led to the origin of the severe acute respiratory syndrome outbreak.
Abstract: Vero E6 cells are commonly used for in vitro studies of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and for antiviral evaluation purposes. A better understanding of the SARS-CoV growth kinetics in Vero E6 cells is crucial to help elucidate the mechanism of antiviral activity of selective antiviral agents. In this study, the growth kinetics of SARS-CoV in Vero E6 cells were studied by quantitation of intra- and extracellular viral RNA load as well as extracellular virus yield at different time points post-infection. At 12h post-infection, the intracellular viral RNA load was 3x10(2)-fold higher than at the time of infection, and the extracellular viral RNA load was increased with a factor of 2 x 10(3). Intracellular viral RNA levels started to rise at 6h post-infection. One hour later (at 7h post-infection), the levels of extracellular SARS-CoV RNA also began to rise. This was corroborated by the fact that infectious progeny SARS-CoV also first appeared in the supernatant between 6 and 7h post-infection. At 12h post-infection, SARS-CoV reached titers in the supernatant of 5.2 x 10(3) CCID(50)/ml.
Abstract: BACKGROUND: Rotavirus genotyping is performed by using reverse transcription PCR with type-specific-primers. Because the high rotavirus mutation rate generates an extensive genomic variation, different G-type-specific primer sets are applied in different geographical locations. In Bangladesh, a significant proportion (36.9%) of the rotavirus strains isolated in 2002 could not be G-typed using the routinely used primer set. To investigate the reason why the strains were untypeable, nucleotide sequencing of the VP7 genes was performed. RESULTS: Four nucleotide substitutions at the G1 primer-binding site of the VP7 gene of Bangladeshi G1 rotaviruses rendered a major proportion of circulating strains untypeable using the routine primer set. Using an alternative primer set, we could identify G1 rotaviruses as the most prevalent genotype (44.8%), followed by G9 (21.7%), G2 (15.0%) and G4 (13.8%). CONCLUSION: Because of the natural variation in the rotaviral gene sequences, close monitoring of rotavirus genotyping methods is important.
Abstract: The prior diagnosis of fatal astrocytoma in a 60-year-old man with Crohn's disease treated with natalizumab, a monoclonal antibody against alpha4 integrins, was reclassified as JC virus-related progressive multifocal leukoencephalopathy (PML). Analysis of frozen serum samples showed that JC virus DNA had appeared in the serum three months after the initiation of open-label natalizumab monotherapy and two months before the appearance of symptomatic PML. There was staining of the brain lesion for polyomavirus. This case report, along with two others, suggests that anti-alpha4-integrin therapy can result in JC virus-induced PML.
Abstract: Partial sequences of a novel papillomavirus were amplified from a cutaneous lesion biopsy of a raccoon (Procyon lotor), by using PCR with degenerate papillomavirus-specific primers. The Procyon lotor papillomavirus type 1 (PlPV-1) DNA was amplified with long template PCR in two overlapping fragments, together encompassing the entire genome, and the complete PlPV-1 genomic sequence was determined. The PlPV-1 genome consists of 8170 bp, and contains the typical papillomaviral open reading frames, encoding five early proteins and two late capsid proteins. Besides the classical non-coding region (NCR1) between the end of L1 and the start of E6, PlPV-1 contains an additional non-coding region (NCR2) of 1065 bp between the early and late protein region, which has previously also been described for the canine oral papillomavirus (COPV) and the Felis domesticus papillomavirus (FdPV-1). Phylogenetic analysis places PlPV-1 together with COPV and FdPV-1 in a monophyletic branch which encompasses the Lambda papillomavirus genus.
Abstract: The clinical significance of human coronaviruses in more severe respiratory illnesses has recently been shown to be higher than was previously assumed. Rapid and reliable diagnosis of human coronavirus infections therefore becomes indispensable in a routine clinical setting. In this study, we present a very sensitive and specific TaqMan-based, real-time quantitative reverse transcriptase PCR (qRT-PCR) for the rapid detection and quantitation of human coronaviruses (HCoVs) OC43 and 229E. Absolute viral load measurement in clinical samples was achieved through the construction of in-house HCoV OC43 and 229E cRNA standards for the generation of a standard curve. The HCoV OC43 assay allows quantitation over a range from 20 to 2 x 10(8) RNA copies per reaction mixture (5 microl RNA extract). When this is extrapolated to clinical samples, this corresponds to a detection range of 10(3) to 10(10) viral genome equivalents per ml. By using the HCoV 229E qRT-PCR assay, viral RNA copies ranging from 200 to 2 x 10(9) per reaction mixture can be detected, which corresponds to 10(4) to 10(11) viral genome equivalents per ml sample. A total of 100 respiratory samples screened for the presence of HCoVs OC43 and 229E by using conventional RT-PCR were assessed in parallel by the qRT-PCR assays. By use of the real-time qRT-PCR techniques, the detection rate of HCoVs OC43 and 229E increased from 2.0% to 3.1% and from 0.3% to 2.5%, respectively. The real-time qRT-PCR assays described here allow the rapid, specific, and sensitive laboratory detection and quantitation of human coronaviruses OC43 and 229E.
Abstract: The performance of the m1000 system (Abbott Laboratories, Illinois) as a front-end extraction system for high-throughput "in-house" quantitative real-time PCR assays was analyzed and compared to that of manual extraction of plasma and serum samples (hepatitis C virus [HCV] and hepatitis B virus [HBV]) and EDTA-blood samples (cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). Linearity of extraction was tested on dilution series of HCV and HBV reference materials. The correlation coefficient for standard curves based on repeated extraction runs was 0.97 +/- 0.06 for HCV and 0.97 +/- 0.03 for HBV, indicating a linear extraction from 100 to 1.0 x 10(5) HCV IU/ml and from 100 to 1.0 x 10(6) HBV IU/ml. Intra- and interrun variability was below 0.23 log(10) IU/ml for 2.98 to 5.28 log(10) HCV IU/ml and 2.70 to 5.20 log(10) HBV IU/ml. Correlation between automated and manual extraction was very good. For HCV, the correlation coefficient was 0.91 and the mean difference in viral load was 0.13 log(10) HCV IU/ml. For HBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.61 log(10) HBV IU/ml. For CMV and EBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.33 log(10) copies/ml. Accuracy was confirmed with a reference panel (QCMD, Glasgow, Scotland) for all four assays. No cross-contamination was observed when extracting strongly positive polyomavirus samples (8.10 log(10) copies/ml) interspersed with polyomavirus-negative samples. Automated extraction via the m1000 system offers a high reliability of extraction and resulted in a strong reduction of the required extraction hands-on time for high-throughput PCR compared to manual extraction protocols.
Abstract: To validate a commercially available line probe assay for samples containing the infrequently found hepatitis C virus (HCV) genotype 5a, we sequenced a 511-nucleotide fragment of the NS4b region of Belgian and South African HCV genotype 5a samples. Phylogenetic analysis of the sequence data was performed. For the 77 HCV genotype 5a samples collected, there was 100% concordance between the genotype assignment by the line probe assay and the genotyping based on nucleotide sequencing, despite sequence heterogeneity in the probe binding sites of some samples.
Abstract: Acute retinal necrosis (ARN) is a severe ocular syndrome consisting of a moderate-to-severe anterior uveitis, vasculitis, and vaso-occlusive retinal necrosis. It can occur in healthy individuals at any age, but reports of this condition in children are rare.
Abstract: Group C rotaviruses were detected by reverse transcription-PCR in 14 (2.3%) of 611 group A rotavirus-negative stool specimens from the patients admitted to the International Centre for Diarrhoeal Disease Research, Bangladesh hospital, Dhaka, Bangladesh, during July to December 2003. The low rate of detection suggested that infection with group C rotaviruses was an uncommon cause of hospitalization due to gastroenteritis. In addition, coinfections with pathogenic enteric bacteria were frequently observed in group C rotavirus-infected patients. Nucleotide sequence comparison of the VP4, VP6, and VP7 genes revealed that the Bangladeshi group C rotaviruses were most similar to Nigerian group C rotavirus strains. Phylogenetic analysis suggested that all human group C rotaviruses, including the strains isolated in our study, clustered in a monophyletic branch, which was distantly related to the branch comprised of animal group C rotaviruses.
Abstract: Human respiratory syncytial virus (HRSV) is the most important cause of acute respiratory disease in infants. Two major subgroups (A and B) have been identified based on antigenic differences in the attachment G protein. Antigenic variation between and within the subgroups may contribute to reinfections with these viruses by evading the host immune responses. To investigate the circulation patterns and mechanisms by which HRSV-B viruses evolve, we analyzed the G protein genetic variability of subgroup B sequences isolated over a 45-year period, including 196 Belgian strains obtained over 22 epidemic seasons (1982 to 2004). Our study revealed that the HRSV-B evolutionary rate (1.95 x 10(-3) nucleotide substitutions/site/year) is similar to that previously estimated for HRSV-A (1.83 x 10(-3) nucleotide substitutions/site/year). However, natural HRSV-B isolates appear to accommodate more drastic changes in their attachment G proteins. The most recent common ancestor of the currently circulating subgroup B strains was estimated to date back to around the year 1949. The divergence between the two major subgroups was calculated to have occurred approximately 350 years ago. Furthermore, we have identified 12 positively selected sites in the G protein ectodomain, suggesting that immune-driven selective pressure operates in certain codon positions. HRSV-A and -B strains have similar phylodynamic patterns: both subgroups are characterized by global spatiotemporal strain dynamics, where the high infectiousness of HRSV permits the rapid geographic spread of novel strain variants.
Abstract: A novel rotavirus strain (Dhaka6) isolated from a 21-year-old Bangladeshi male patient was characterized by sequence analysis of its VP7 and VP4 gene segments. Phylogenetic analysis of the VP7 gene of the Dhaka6 strain revealed a common evolutionary lineage with porcine G11 rotavirus strains. This isolate is the first reported G11 rotavirus strain infecting a human host. Comparison of the VP4 gene sequences with all currently recognized 24 different P genotypes revealed only low nucleotide (54 to 71%) and amino acid (52 to 76%) sequence identities. This lack of high sequence similarity in the VP4 gene indicates that the Dhaka6 isolate represents a new group A rotavirus P genotype, to which we propose assignment of the designation P[25].
Abstract: After notification of a case of fulminant hepatitis B virus (HBV) infection in a 83-year-old female resident of a nursing home to the Flemish Health Inspectorate, a seroepidemiological study and a retrospective cohort study were conducted among the 94 residents and 47 nursing staff to assess the extent of HBV transmission and to identify risk factors. Susceptible residents were vaccinated against HBV and their serological response to hepatitis B vaccination determined. From December 2002 to April 2003, five residents with acute hepatitis B infection were identified with an attack rate of 5.5% and a case fatality rate of 40%. Three other residents were identified as HBV surface antigen and e-antigen positive carrier. None of the nursing staff tested positive for acute HBV infection or HBV carriage. Diabetic patients who were exposed to a shared finger-stick device for blood sampling were 10.7 times more likely to contract an acute HBV infection (RR 10.7; 95% CI 1.3-91.3). Other potential risk factors were undergoing podiatric care and being exposed to the shared razor blade of the hairdresser. The transmission of the infection could be controlled by restricting the use of finger-stick capillary sampling devices to individual patients, assigning separate glucometers to individual patients and, implementation of standard infection-control recommendations like wearing gloves and performing hand hygiene by the nursing staff, assigning separate podiatric sets to individual patients. HBV vaccination of the residents with three doses of HBV vaccine has not induced a sufficient degree of protection. The outbreak stresses the need for appropriate and generally applied standard procedures in nursing homes to prevent bloodborne pathogens.
Abstract: The aim of this study was to determine the genotypic variation of hepatitis C among drug users in Flanders and to relate the distribution of genotypes to the characteristics of the population. Hepatitis C virus RNA (HCV-RNA) quantification and genotyping was performed on stored samples from 161 anti-HCV-positive injecting and non-injecting drug users. Information on sociodemographic status, drug-related risk behaviour and sexual risk behaviour was available for each drug user. HCV-RNA was present in 152 of 161 samples (94.4%). Genotype 1 was predominant (48.7%), followed by genotype 3 (41.2%), genotype 4 (8.8%) and genotype 2 (1.4%). In the multivariate analysis, lack of a history of injecting drug use was confirmed as a statistically significant predictor for infection with genotype 1. Predictors for infection with genotype 3 were the presence of anti-HBc antibodies and a history of injecting drug use. Being tattooed emerged as a statistically significant predictor for infection with genotype 4. The 94.4% prevalence of HCV-RNA among anti-HCV-positive drug users was considerably higher than the 54-86% chronicity rate found globally among HCV-infected patients. The results of this study suggest the existence of separate transmission networks for injecting drug users and non-injecting drug users. Finally, the results suggest that tattooing practices play a role in the spread of HCV among drug users.
Abstract: The complete genome of a novel papillomavirus was isolated from a cutaneous papillomatous lesion of a North American porcupine (Erethizon dorsatum) using multiply primed rolling-circle amplification (RCA). The nucleotide sequence, genome organization, and phylogenetic position of the Erethizon dorsatum papillomavirus type 1 (EdPV-1) were determined. EdPV-1 is only distantly related to other benign cutaneous papillomavirus sequences and is the first member of the novel Sigma papillomavirus genus.
Abstract: Hemorrhagic cystitis that occurs late after bone marrow transplantation (BMT) in BMT recipients is often associated with adenovirus or polyomavirus BK infections. Intravesical instillation of cidofovir in a BMT recipient with intractable hemorrhagic cystitis resulted in clinical improvement. Local cidofovir therapy for viral hemorrhagic cystitis could be an alternative to intravenous administration of cidofovir.
Abstract: Since it is not clear yet whether enfuvirtide resistance is restricted to gp41, it was decided to develop a genotypic assay for the detection of drug resistance in the entire human immunodeficiency virus type 1 (HIV-1) env gene. Given the increasing prevalence of HIV-1 non-B subtypes in Europe, it is important to evaluate the performance of the assay on a panel of genetically divergent samples. A panel of 1 laboratory and 10 clinical isolates from 10 patients was tested, all enfuvirtide naive and chosen according to the subtype as determined in the pol region (A, B, C, H, CRF01-AE, CRF02-AG, CRF05-DF, CRF11-cpx and U), while their env sequences belonged to subtypes A, B, C, H, A/G recombinant, B/H recombinant, CRF01-AE, CRF02-AG, CRF05-DF and CRF11-cpx. The detection limits of the gp120 and the gp41 PCRs ranged between 500 and 5000 RNA copies/ml plasma. The highest sensitivity was obtained for the laboratory strain, whereas the detection limit for all patient samples, except for the subtype C sample, was 1000 RNA copies/ml. The numerous insertions and deletions in the gp120 gene, that were often present as quasi-species, necessitated the sequencing of cloned PCR products. The gp41 gene displayed less diversity and less insertions/deletions. Especially, the heptad repeat 1 was highly conserved and none of the enfuvirtide naive samples contained any of the already known enfuvirtide resistance mutations at amino acid positions 36-45. This study demonstrates that the assay is able to genotype genetically diverse HIV-1 strains with a good sensitivity.
Abstract: A 37-year-old Belgian patient presented with acute nephropathia epidemica (NE) shortly after a camping holiday in southern France. Unusual symptoms were initial noncardiogenic lung involvement, followed by severe acute renal failure, acute acalculous cholecystitis, presence of immunoblasts in the bone marrow, and hemolytic anemia, presenting as hemolytic uremic syndrome. Positive immunoglobulin (Ig) A and rising IgG titers against Puumala hantavirus (PUUV) were detected, but IgM remained negative on days 8 and 20. The results of reverse-transcriptase-polymerase chain reaction performed on day 8 were positive for PUUV. This is the first report of an iatrogenically IgM-negative hantavirus case due to the selective removal of heavy-weight molecules during plasma exchange via the centrifugation technique. This is also the first report of proven NE from the Mediterranean part of France.
Abstract: Group A rotaviruses are the main cause of severe diarrhoea in humans and animals throughout the world. We report the first description of a clinically apparent infection with a P[14], G3 rotavirus (strain B4106) in a hospitalized 6-year-old child. The VP7 gene of the B4106 strain had the closest sequence similarity (94% and 97% on the nucleotide and amino acid level, respectively) with strain 30/96 (P[14], G3), a lapine rotavirus isolated in an Italian rabbit in 1996 while the VP4 gene had the closest similarity with strain 30/96 on the nucleotide level (96%), and with lapine strains C-11 (P[14], G3) and Alabama (P[14], G3), isolated in the United States in the 1980s on the amino acid level (99%). The host restriction determinant gene NSP4 of B4106 was also most similar to lapine strain Alabama (95% nt identity and 97% aa identity). Phylogenetic analysis showed that the VP4, VP7, and NSP4 genes of the B4106 strain share a common evolutionary lineage with those of lapine rotavirus strains. We therefore hypothesize that a lapine rotavirus was able to cross the host species barrier and caused disease in a new host. The increasing detection of strains in humans that were previously believed to be restricted to animals raises questions whether interspecies transmission of rotaviruses is a common event in nature.
Abstract: OBJECTIVE: Human aminopeptidase N (APN/CD13/ANPEP) has been identified as the receptor for human coronavirus (HCoV) 229E. In this study, we analyzed the region of the APN gene that encodes a stretch of amino acid residues, essential for its HCoV-229E receptor function (amino acids 260-353). METHODS: Full-length APN exon 3, intron 3 and exon 4, was PCR-amplified and sequenced in DNA samples from 100 unrelated Caucasian Belgian healthy volunteers. RESULTS: We identified seven polymorphisms, including four intron 3 and three exon 4 variations. Apart from the already known C956T exon 4 mutation, the six other polymorphisms have not yet been described. The most prevalent APN variations in this population (C956T leading to an alanine to valine substitution, G978T, G987A and intron3-C389T) always occurred together at an allele frequency of 8.5%. Haploid DNA sequencing demonstrated the presence of these four variations on the same allele. Three polymorphisms in intron 3, intron3-G395C, intron3-C86T, and intron3-C429T, were identified with an allele frequency of 3.5%, 1% and 0.5% respectively. Five haplotypes were identified in the population of 100 individuals. CONCLUSION: These results demonstrate that there is a relatively broad spectrum of variations in the APN domain critical for coronavirus binding.
Abstract: INTRODUCTION: The recent outbreak of severe acute respiratory syndrome (SARS) warrants the search for effective antiviral agents to treat the disease. This study describes the assessment of the antiviral potential of nitric oxide (NO) against SARS coronavirus (SARS-CoV) strain Frankfurt-1 replicating in African Green Monkey (Vero E6) cells. RESULTS: Two organic NO donor compounds, S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP), were tested in a broad range of concentrations. The non-nitrosylated form of SNAP, N-acetylpenicillamine (NAP), was included as a control compound in the assay. Antiviral activity was estimated by the inhibition of the SARS-CoV cytopathic effect in Vero E6 cells, determined by a tetrazolium-based colorimetric method. Cytotoxicity of the compounds was tested in parallel. CONCLUSION: The survival rate of SARS-CoV infected cells was greatly increased by the treatment with SNAP, and the concentration of this compound needed to inhibit the viral cytopathic effect to 50% was 222 microM, with a selectivity index of 3. No anti-SARS-CoV effect could be detected for SNP and NAP.
Abstract: The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with phi29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 x 10(4)-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information.
Abstract: Structural point mutations in exon 1 at codons 52, 54 and 57 and a promotor polymorphism at -221 bp of the mannose-binding lectin (MBL) gene are associated with increased susceptibility to various infectious diseases. We developed a genotyping method based on the 5' nuclease (TaqMan) assay in combination with the use of minor-groove-binder (MGB) probes in screening for these mutations/polymorphisms. In contrast to conventional probes, MGB probes have a short length and can be used for detection of mutations that are in close proximity to each other, as is the case for the structural mutations in exon 1 of the MBL gene. Results obtained with the 5' nuclease assay using MGB probes were identical with results obtained with classical techniques such as restriction fragment length polymorphism, allele-specific PCR, and sequencing. In conclusion, the 5' nuclease assay using MGB probes is useful for large-scale screening of point mutations/polymorphisms, even when they are in close proximity.
Abstract: BACKGROUND: The appropriate management of women with minor cytologic lesions in their cervix is unclear. We performed a meta-analysis to assess the accuracy of human papillomavirus (HPV) DNA testing as an alternative to repeat cytology in women who had equivocal results on a previous Pap smear. METHODS: Data were extracted from articles published between 1992 and 2002 that contained results of virologic and cytologic testing followed by colposcopically directed biopsy in women with an index smear showing atypical cells of undetermined significance (ASCUS). Fifteen studies were identified in which HPV triage and the histologic outcome (presence or absence of a cervical intraepithelial neoplasia of grade II or worse [CIN2+]) was documented. Nine, seven, and two studies also documented the accuracy of repeat cytology when the cutoff for abnormal cytology was set at a threshold of ASCUS or worse, low-grade squamous intraepithelial lesion (LSIL) or worse, or high-grade squamous intraepithelial lesion (HSIL) or worse, respectively. Random-effects models were used for pooling of accuracy parameters in case of interstudy heterogeneity. Differences in accuracy were assessed by pooling the ratio of the sensitivity (or specificity) of HPV testing to that of repeat cytology. RESULTS: The sensitivity and specificity were 84.4% (95% confidence interval [CI] = 77.6% to 91.1%) and 72.9% (95% CI = 62.5% to 83.3%), respectively, for HPV testing overall and 94.8% (95% CI = 92.7% to 96.9%) and 67.3% (95% CI = 58.2% to 76.4%), respectively, for HPV testing in the eight studies that used the Hybrid Capture II assay. Sensitivity and specificity of repeat cytology at a threshold for abnormal cytology of ASCUS or worse was 81.8% (95% CI = 73.5% to 84.3%) and 57.6% (95% CI = 49.5% to 65.7%), respectively. Repeat cytology that used higher cytologic thresholds yielded substantially lower sensitivity but higher specificity than triage with the Hybrid Capture II assay. The ratio of the sensitivity of the Hybrid Capture II assay to that of repeat cytology at a threshold of ASCUS or worse pooled from the four studies that used both triage tests was 1.16 (95% CI = 1.04 to 1.29). The specificity ratio was not statistically different from unity. CONCLUSION: The published literature indicates that the Hybrid Capture II assay has improved accuracy (higher sensitivity, similar specificity) than the repeat Pap smear using the threshold of ASCUS for an outcome of CIN2+ among women with equivocal cytologic results. The sensitivity of triage at higher cytologic cutoffs is poor.
Abstract: The sequence identity of the enterovirus VP1 gene has been shown to correlate with the serotype concept. Enterovirus molecular typing methods are therefore often based on sequencing of the VP1 genomic region and monophyletic clustering of VP1 sequences of a homologous serotype. For epidemiological surveillance, 342 enterovirus samples obtained from patients with aseptic meningitis in Belgium from 1999 to 2002 were first diagnosed as being enterovirus positive by amplification of the 5' noncoding region (5'NCR) by reverse transcription (RT)-PCR. Subsequently, samples were molecularly typed by RT-nested PCR amplification and sequencing of a portion of the VP1 gene. Phylogenetic analyses were performed to investigate enteroviral evolution and to examine the serotype and genotype correlation of the two genomic regions. Our typing results demonstrated echovirus 30, echovirus 13, echovirus 18, and echovirus 6 to be the most predominant types. Echoviruses 13 and 18 were considered to be emerging human serotypes since 2000 and 2001, respectively, as they had been rarely reported before. Several serotypes existed as multiple genotypes (subtypes) from 1999 to 2002, but genomic differences mainly resided at synonymous sites; these results strongly suggest that the subtypes exhibit similar antigenic properties. Phylogenetic analyses confirmed that VP1 is an adequate region for molecular typing. Serotype-specific clusters are not observed commonly in phylogenetic trees based on the 5'NCR, and the phylogenetic signal in the 5'NCR was found to be particularly low. However, some substructure in the 5'NCR tree made a tentative prediction of the enterovirus type possible and was therefore helpful in PCR strategies for VP1 (e.g., primer choice), provided some background knowledge on the local spectrum of enteroviruses already exists.
Abstract: We developed a novel method that uses sodium dodecyl sulfate-EDTA-treated chromatography paper strips to collect unconcentrated fresh stool samples. After the paper strips were stored for 4 months at room temperature, rotavirus RNA could be successfully amplified by using reverse transcriptase PCR. The use of filter paper strips as a specimen support allows (self-)collection of stool samples by untrained persons. Diarrheal stool samples from remote areas can be stored and transported to a central diagnostic laboratory without the need for freezers or special shipping conditions. This convenient and inexpensive rotavirus sample collection system can be of use in epidemiological surveillance studies and vaccine trials.
Abstract: Human respiratory syncytial virus (HRSV) is the most common etiological agent of acute lower respiratory tract disease in infants and can cause repeated infections throughout life. In this study, we have analyzed nucleotide sequences encompassing 629 bp at the carboxy terminus of the G glycoprotein gene for HRSV subgroup A strains isolated over 47 years, including 112 Belgian strains isolated over 19 consecutive years (1984 to 2002). By using a maximum likelihood method, we have tested the presence of diversifying selection and identified 13 positively selected sites with a posterior probability above 0.5. The sites under positive selection correspond to sites of O glycosylation or to amino acids that were previously described as monoclonal antibody-induced in vitro escape mutants. Our findings suggest that the evolution of subgroup A HRSV G glycoprotein is driven by immune pressure operating in certain codon positions located mainly in the second hypervariable region of the ectodomain. Phylogenetic analysis revealed the prolonged cocirculation of two subgroup A lineages among the Belgian population and the possible extinction of three other lineages. The evolutionary rate of HRSV subgroup A isolates was estimated to be 1.83 x 10(-3) nucleotide substitutions/site/year, projecting the most recent common ancestor back to the early 1940s.
Abstract: A truncated recombinant nucleocapsid protein (rNp118) consisting of the first 118 amino-terminal amino acids (AA) of the Puumala hantavirus (PUUV) nucleocapsid protein expressed in Escherichia coli, was evaluated for its antigenicity and reliability as serodiagnostic antigen in an enzyme-linked immunosorbent assay (ELISA) for detection of PUUV antibodies in human sera. The PUUV nucleocapsid protein has been shown to contain several B-cell epitopes, mapped within the first 118 amino-terminal AA. This finding makes the rNp118 an interesting recombinant protein to use as serodiagnostic antigen. The sensitivity of this new PUUV-rNp118 ELISA, was compared with those of a commercially available PUUV ELISA assay and an home-made ELISA based on a recombinant whole nucleocapsid protein of PUUV. Eighty-six human serum samples clinically suspected for PUUV-induced nephropathia epidemica, and previously screened with the reference assays, were tested. The sensitivity of the new assay was compared with that of the reference assays and an excellent correlation between the assays was found. Sera found to be negative by other methods were also negative in our assay. The ELISA based on rNp118 represents an alternative and valid test for detection of antibodies to PUUV in human sera.
Abstract: Since there are indications of an increasing amount of non-B subtypes in Western Europe it was decided to assess the performance of the ViroSeq HIV-1 Genotyping System on a set of samples from the AIDS Reference Laboratory at the University Hospitals Leuven, a hospital with an increasing number of patients infected with non-B subtypes. The set consisted of 383 samples comprising 12 different subtypes and the genotyping kit was assessed for its amplification capabilities as well as its sequencing capabilities. Amplification failed in 32 samples (8.4%) and there was a tendency of a lower performance of the kit when it concerned the amplification of non-B subtypes. Regarding the sequencing performance of the HIV-1 Genotyping System, three different results could be considered. The performance of the entire set of primers (A, B, C, F, G and H) on the different subtypes showed a significant decrease of positive results for subtypes A, G and the recombinants whereas a tendency to less positive results could be detected for subtypes CRF12_BF, D, H and J. When looking at the performance of the individual primers for the different subtypes, only one result differed significantly: there were less positive results by applying primer F on subtype A. A tendency to less positive results was found for other combinations of primer and subtype, most of which comprised combinations with primers B, C, F and H. A final result was obtained by comparing the overall sequencing results of a certain primer on all the non-B subtypes with the results of the same primer on subtype B. Primer F showed significant less positive results and a tendency to less positive results was found for primer H. The other primers showed comparable results. All of the above results regarding the sequencing primers did not include primer D since this is a back-up primer for primer A. Analysis of the results for primer D showed that less positive results were found for all the non-B subtypes, most of which were significant. The overall performance of primer D on all non-B subtypes was only 15.7%. The use of primer D as a back-up primer was also investigated: it generated a positive result in only 17.3% of the cases where primer A failed. Most of these positive results were subtype B (74%). As a result of sequencing problems 65 out of 351 (18.5%) samples had to be processed with "in-house" procedures.
Abstract: To achieve the goal of poliovirus eradication, surveillance of endemic areas is a crucial step in the poliovirus eradication program. Currently, six countries still have endemic poliovirus. We have tested a novel method which uses SDS/EDTA-treated chromatography paper strips to collect and transport poliovirus-containing stool samples. The SDS/EDTA-treated paper strips were soaked with different dilutions of poliovirus-containing feces and stored at different temperatures. After storing the SDS/EDTA paper strips for 5 months at 37 degrees C, poliovirus RNA could be successfully amplified using RT-PCR. Infectivity of wild-type poliovirus type 1, 2, and 3 was lost upon contact with the SDS/EDTA-treated strips. This easy, inexpensive, and biosafe chromatography paper strip method for the collection and transportation of poliovirus samples can be of use in poliovirus surveillance and polio vaccination programs.
Abstract: By using an isothermal multiply primed rolling-circle amplification protocol, the complete genomic DNA of a novel papillomavirus was amplified from a skin lesion biopsy of a Florida manatee (Trichechus manatus latirostris), one of the most endangered marine mammals in United States coastal waters. The nucleotide sequence, genome organization, and phylogenetic position of the Trichechus manatus latirostris papillomavirus type 1 (TmPV-1) were determined. TmPV-1 is the first virus isolated from the order of Sirenia. A phylogenetic analysis shows that TmPV-1 is only distantly related to other papillomavirus sequences, and it appears in our phylogenetic tree as a novel close-to-root papillomavirus genus.
Abstract: We report detection of subgroup B respiratory syncytial virus in cerebrospinal fluid from a 4-month-old infant, using a 1-step reverse transcription-polymerase chain reaction assay. The infant was diagnosed with bilateral pneumonia and had a febrile seizure. The patient recovered uneventfully after 9 days in the hospital.
Abstract: Hantaviruses are rodent-borne bunyaviruses that are associated with two main clinical diseases in humans: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. It has been suggested that host-related immune mechanisms rather than direct viral cytopathology may be responsible for the principal abnormality (vascular dysfunction) in these syndromes. This review summarizes the current knowledge on hantaviral host immune responses, immune abnormalities, laboratory diagnosis, and antiviral therapy as well as the current approaches in vaccine development.
Abstract: Noroviruses are the most common cause of nonbacterial gastroenteritis outbreaks. A novel method was tested that utilizes sodium dodecyl sulfate (SDS)/EDTA-pretreated chromatography paper strips to collect and transport norovirus-containing fresh stool samples. As confirmation, the strips were infected with different dilutions of human norovirus-containing faeces and stored at different temperatures (-80, -20, 4 degrees C, room temperature and 37 degrees C). Reverse transcription (RT)-PCR analysis was carried out after storage of the strips for 1, 7, 14, 60, and 120 days. The presence of amplifiable norovirus RNA on the strips was inversely correlated with the duration of storage and the storage temperature. Norovirus RNA remained detectable for a sufficient amount of time at room temperature or even in warmer climatic conditions to allow transportation of the samples from a field site to a reference laboratory using regular postal mail. To prove that viral infectivity was lost upon contact with the SDS/EDTA-treated strips, feline calicivirus F9 (10(7) TCID(50)/ml) was loaded onto the strips. After elution and dialysis, no cytopatic effect could be observed upon incubation on Crandell-Reese feline kidney cells. The SDS/EDTA-pretreated chromatography filter paper strips are an inexpensive, biosafe and adequate method to collect, transport and store norovirus samples.
Abstract: Equus caballus papillomavirus type 1 (EcPV-1) was isolated from a cutaneous papilloma, the most common neoplasm in horses. The complete EcPV-1 nucleotide sequence and genomic organization were determined. Phylogenetic analysis showed that EcPV-1 is a close-to-root papillomavirus, with only distant relationships to the fibropapillomaviruses and the benign cutaneous papillomaviruses. To produce EcPV-1 virus-like particles (VLPs), the EcPV-1 L1 major capsid protein was expressed in insect cells using a recombinant baculovirus vector. The self-assembled EcPV-1 VLPs were morphologically indistinguishable from wild type papillomavirus virions. Monoclonal antibodies were developed against intact and denatured EcPV-1 VLPs. When tested by ELISA, all monoclonal antibodies produced against intact (#18) and some against denatured EcPV-1 VLPs (#16) reacted with intact EcPV-1 VLPs only, demonstrating that the VLPs carry type-specific conformational as well as linear epitopes on their surface. Recombinant EcPV-1 VLPs offer the potential of a noninfectious vaccine to prevent and eradicate equine cutaneous papillomatosis.
Abstract: We report on chloroquine, a 4-amino-quinoline, as an effective inhibitor of the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro. Chloroquine is a clinically approved drug effective against malaria. We tested chloroquine phosphate for its antiviral potential against SARS-CoV-induced cytopathicity in Vero E6 cell culture. Results indicate that the IC50 of chloroquine for antiviral activity (8.8 +/- 1.2 microM) was significantly lower than its cytostatic activity; CC50 (261.3 +/- 14.5 microM), yielding a selectivity index of 30. The IC50 of chloroquine for inhibition of SARS-CoV in vitro approximates the plasma concentrations of chloroquine reached during treatment of acute malaria. Addition of chloroquine to infected cultures could be delayed for up to 5h postinfection, without an important drop in antiviral activity. Chloroquine, an old antimalarial drug, may be considered for immediate use in the prevention and treatment of SARS-CoV infections.
Abstract: Polyomavirus infection and related nephropathy is being increasingly recognized as an important cause of allograft dysfunction in adult renal transplant recipients. We prospectively monitored pediatric renal transplant recipients for the presence of BK and JC polyomavirus in urine and blood using a quantitative PCR assay to evaluate the prevalence and clinical relevance of polyomavirus infection in the pediatric renal transplant population. Of 46 pediatric renal recipients who were evaluated, nine (20%) demonstrated isolated BKV viruria, while five (11%) had concomitant BKV viremia and viruria. JCV viruria was found in eight (17%) patients. BKV viremia was associated with a significantly higher urinary BKV viral load: median urinary viral load 1.9 x 10(9) copies/mL (range 6.7 x 10(2)-1.8 x 10(11)) for the group with concomitant viremia and viruria vs. 1.8 x 10(3) copies/mL (range 2.5 x 10(2)-4.5 x 10(6)) for the group with isolated viruria (p < 0.0001). In children that were followed prospectively since their transplantation, the BKV urinary viral load increased markedly before viremia became detectable a few weeks later. None of the patients with JCV viruria or isolated BKV viruria had renal dysfunction. Among the five patients with BKV in both urine and blood, two developed biopsy-proven BKV nephropathy associated with deterioration of the renal function. Management of the BKV nephropathy consisted of reduction of immunosuppression alone or in combination with antiviral treatment with cidofovir. This study shows that polyomavirus infection and related interstitial nephritis is a relevant clinical issue in the pediatric renal transplant population. Monitoring the polyomaviral load in the urine and the blood of the patients using a quantitative PCR technique is a useful tool in the diagnosis and subsequent management of this infection. Even before viremia is present, an important rise in the urinary viral load should draw the attention of the transplant clinician and raise the issue of adapting the immunosuppression.
Abstract: The origin of the severe acute respiratory syndrome-coronavirus (SARS-CoV) remains unclear. Evidence based on Bayesian scanning plots and phylogenetic analysis using maximum likelihood (ML) and Bayesian methods indicates that SARS-CoV, for the largest part of the genome ( approximately 80%), is more closely related to Group II coronaviruses sequences, whereas in three regions in the ORF1ab gene it shows no apparent similarity to any of the previously characterized groups of coronaviruses. There is discordant phylogenetic clustering of SARS-CoV and coronaviruses sequences, throughout the genome, compatible with either ancient recombination events or altered evolutionary rates in different lineages, or a combination of both.
Abstract: Rupture of the fetal membranes is a common, but potentially serious complication of invasive fetal procedures. Quintero described a technique to seal the fetal membrane defect by means of a bloodpatch, usually called 'amniopatch' in this application. The successful use in two consecutive patients with ruptured membranes after a fetoscopic intervention at respectively 17 and 22 weeks' gestational age is described, together with a literature review of published experience.
Abstract: A binary classification system has been established for group A rotaviruses, with the viral capsid protein VP7 defining G types and VP4 defining P types. At least 15 G types and 21 P types have been isolated globally with various G and P combinations. Most of the currently circulating human rotaviruses belong to G1P[8], G2P[4], G3P[8], and G4P[8]. We report a human rotavirus strain (B1711) with a novel genotypic VP7/VP4 combination of G6P[6]. This unique rotavirus was isolated from a 13-month-old human immunodeficiency virus (HIV)- negative child of an HIV-seropositive Malian mother that was hospitalized with severe diarrhea in Belgium after returning from a trip to Mali. The VP7 and VP4 genes of the rotavirus strain were sequenced, and phylogenetic trees were constructed. Nucleotide and amino acid sequence comparisons with 15 known G genotypes indicated that the VP7 sequence of strain B1711 was most closely related to an American (Se584) and an Italian (PA151) human G6 strain (95 to 96% nucleotide and 98% amino acid identity). Comparison of the VP4 sequence with 21 P types showed the closest similarity to P[6] genotypes, with greatest similarity to a G8P[6] Malawi strain (mw131) (97% nucleotide and 98% amino acid identity). The B1711 strain is the first reported rotavirus isolate with a G6P[6] genotypic combination. The discovery and surveillance of novel human and nonhuman rotavirus G or P types or of novel G/P combinations is essential for the design of future rotavirus vaccines and for our understanding of rotavirus diversity and evolution.
Abstract: Non-polio enteroviruses are the most common cause of aseptic meningitis worldwide. From May to September 2000, a major outbreak of aseptic meningitis occurred in Belgium. Cerebrospinal fluid samples (CSF) of 122 patients were found to contain enterovirus RNA using diagnostic RT-PCR that targeted a 231-bp gene fragment in the 5' noncoding region. In addition, a molecular typing method was developed based on RT-nested PCR and sequencing directly from CSF(a) 358-bp fragment in the aminoterminal part of the VP1 capsid protein. To identify the enterovirus type, nucleotide sequences of the VP1 amplicons were compared to all the enterovirus VP1 sequences available in GenBank. Echovirus 30 (31.2%), echovirus 13 (23.8%), and echovirus 6 (20.5%) were identified most frequently during the epidemic. Coxsackievirus B5 was present in 15.6% of the samples, and could be subdivided in two distinct epidemic clusters, coxsackievirus B5a (10.7%) and B5b (4.9%). Other enteroviruses encountered were echovirus 16 (5.7%), echovirus 18 (1.6%), coxsackievirus B4 (0.8%) and echovirus 7 (0.8%). The high prevalence of echovirus 13, considered previously a rare serotype, indicates it is an emerging epidemic type. To verify the typing results and to explore further the intratypical genetic variation, phylogenetic analysis was carried out. Geographical clustering of most of the strains within each type and subtype could be observed. The RT-nested PCR strategy, carried out directly on clinical samples, is a simple and rapid method for adequate molecular typing of the Group B enteroviruses causing aseptic meningitis.
Abstract: OBJECTIVES: We evaluated the role of intracoronary, intrapulmonary and macrophage-mediated delivery of C. pneumoniae (Cp) on coronary lesion formation. METHODS: Pigs were allocated to one of three coronary protocols (intracoronary, macrophage or control groups) or to a fourth-a pulmonary group. In the intracoronary group Cp was injected into the wall of the left anterior descending (LAD) and right coronary arteries (RCA) and vehicle into the circumflex (CX). In the macrophage group autologous macrophages preincubated with Cp or not were injected into the LAD and CX wall, respectively. Animals in the control group received vehicle in LAD and CX. In the pulmonary group aerosolised Cp was given intrabronchially, after a single injection of vehicle into the LAD wall. Delivery into the coronary artery wall was performed with a balloon catheter with low-profile injector ports. RESULTS: Seroconversion occurred in the following proportions: 5/6 (intracoronary group), 4/5 (macrophage group), 0/6 (control group), and 1/6 (intrapulmonary group). Significantly higher maximal intimal thickness (MIT) was observed in LADs of intracoronary and pulmonary groups when compared to corresponding CXs. The presence of Cp antigen was associated with higher MIT (r=0.73; P<0.0001). Injection of macrophages into the coronary artery wall did not induce proliferation. Arteries without coronary interventions were morphologically normal. CONCLUSIONS: Intracoronary and intrapulmonary but not macrophage-mediated Cp inoculation were associated with moderate intimal proliferation in the absence of a lipid-rich diet. Pre-existing coronary lesions seem a prerequisite for Cp-induced proliferation.
Abstract: Human rhinoviruses (HRV), responsible for approximately 60% of the common colds, are divided into two groups, according to their receptor specificity. The major group of HRVs gains access to human cells by binding to the intercellular adhesion molecule-1 (ICAM-1), whereas HRVs of the minor group use members of the low-density lipoprotein receptor (LDLR) family for cell entry. Previous studies confirmed that the HRV-binding region of ICAM-1 is located in the amino-terminal immunoglobulin-like (Ig) domain 1, which is encoded by exon 2 of the ICAM-1 gene. An A --> T transversion in codon 29 of ICAM-1 exon 2 causes a lysine to methionine substitution (K29M), and was found at a high frequency (33.2%) in Kilifi (Kenya), as well as in other African populations. In this study we examined whether polymorphisms in exon 2 of ICAM-1 could be detected in a Caucasian population, assuming that these could be of importance in HRV binding. DNA from 100 healthy, unrelated, Belgian volunteers was obtained through a noninvasive swish-and-spit method. Using a primer set in the adjacent intron sequences, the full-length ICAM-1 exon 2 was amplified by polymerase chain reaction (PCR), followed by direct sequencing of the PCR product. No polymorphisms could be demonstrated in exon 2 of the ICAM-1 gene among all 100 tested individuals. The rhinovirus-binding Ig domain 1 of ICAM-1 seems to be a highly conserved region in the Caucasian population.
Abstract: Rotavirus infections are a major cause of severe diarrhea in children younger than 2 years. In Belgium they cause many hospitalizations because of dehydration. A study of the laboratory diagnosis of rotavirus infections in 28.251 stool samples at a university teaching hospital in Belgium during a twenty-year period (1981-2002) showed a marked seasonality. The virus was most often diagnosed during the winter months: 54% of the rotavirus isolates were found in the first three months of the year, with 21% of the positive samples occurring in February. Recently, rotaviruses can be genotyped based on differences in the viral outer capsid protein VP7. Vaccines are currently being developed against the four most prevalent genotypes G1, G2, G3 and G4. During the last three epidemic seasons (1999-2002) in Belgium, G1 was the most prevalent genotype and accounted for 62% of the rotavirus isolates recovered. G2, G3 and G4 were also isolated, and other emerging types need to be carefully monitored too, since G9 (45%) was co-dominant with G1 (42%) in the 2000-2001 rotavirus season in Belgium. The future development of an efficient rotavirus vaccine will need to take the diversity of the rotavirus genotypes into account.
Abstract: The objective of this prospective study was to evaluate the clinical and laboratory parameters distinguishing viral from nonviral lower respiratory tract infection in elderly patients and to determine the yield of virological diagnostics in elderly patients with lower respiratory tract infection. The study was conducted in a 184-bed geriatric department in a university hospital during 4 winter months. All consecutive elderly persons admitted with a lower respiratory tract infection were included in the study. Clinical and laboratory parameters, a nasopharyngeal swab, and serological results for respiratory viruses were obtained for all participants. Available blood and sputum cultures were analysed. A total of 165 elderly persons (mean age, 82+/-6.8 years) were hospitalised with a lower respiratory tract infection. Familial flu-like illness (OR, 4.25; 95%CI, 1.4-13), better functionality (OR, 4; 95%CI, 1.3-14.15), and leucocyte count <10(10)/l (OR, 3; 95%CI, 1.3-7.1) were predictive for viral lower respiratory tract infection. Sixty (36.5%) definite diagnoses (positive blood culture, viral culture, or serological test) and seven (4.2%) probable diagnoses (positive sputum culture) were obtained. An early diagnosis (within 72 h) was possible in 38 (23%) and a late diagnosis in 29 (17.6%) participants. A nasopharyngeal swab contributed in 60.5% of the cases to an early diagnosis. Viral culture identified half (22/43) of the lower respiratory tract infections caused by influenza but only one of six lower respiratory tract infections caused by respiratory syncytial virus. In conclusion, a history of flu-like illness in family members and a total leucocyte count within normal limits makes a viral cause more likely in elderly people hospitalised with a lower respiratory tract infection during winter. Viral culture and rapid antigen detection are insensitive in elderly patients hospitalised with a lower respiratory tract infection.
Abstract: In this study we evaluated the performance of the VERSANT HIV-1 Resistance Assays (LiPA) in detecting drug resistance in therapy-naive HIV-infected patients diagnosed in Belgium in 2000. We compared the results with population sequencing and found concordance to be in line with previous studies in treatment-experienced patients (86.87% for reverse transcriptase (RT); 92.77% for protease (PRO)). Discordance was mainly due to indeterminate reactions on LiPA (8.45% for RT; 6.85% for PRO) and minor discordances (4.13% for RT; 0.25% for PRO). Major discordances were rare (0.46% for RT; 0.12% for PRO). Indeterminate reactions were significantly associated with strains belonging to non-B subtypes.
Abstract: An internal RNA standard proved less suitable in bacterial gene expression experiments. We therefore developed a method for simultaneous RNA and gDNA (genomic DNA) isolation from in vitro and in vivo samples containing staphylococci and combined it with quantitative PCR. The reliability of gDNA for bacterial quantification and for standardisation in gene expression experiments was evaluated. Quantitative PCR proves equivalent to quantitative culture for in vitro samples, and superior for in vivo samples. In gene expression experiments, gDNA permits a good standardisation for the initial amount of bacteria. The average interassay variability of the in vitro expression is 20.1%. The in vivo intersample variability was 73.3%. This higher variability can be attributed to the biological variation of gene expression in vivo. This method permits exact quantification of the number of bacteria and the expression of genes in staphylococci in vivo (e.g., in biofilms, evolution in time) and in vitro.
Abstract: The introduction of potent combinations of antiviral drugs is a major breakthrough in the treatment of HIV. We investigated the long-term virologic outcome and the development of resistance after initiating highly active antiretroviral therapy (HAART) in drug-naive patients in daily clinical practice. Twenty-five treatment-naive HIV-1 patients were started on HAART. Fifteen patients responded with a drop in viral load below the limit of detection during 35.5 (interquartile range: 7) months of therapy. In 6 of 10 patients with virologic failure, virus with resistance-related mutations against the received drugs emerged. Compared with responders (R), nonresponding (NR) patients were in a later disease stage at therapy start (p = 0.0089) with lower CD4 cell counts at baseline (p = 0.040), and a lower proportion of nonresponders showed protease inhibitor (PI) levels above C(min) (p = 0.049). More NR patients showed secondary PI mutations at baseline (p = 0.079), and the CCR2-64I coreceptor polymorphism was absent among NR patients, compared with 38.5% of R patients displaying CCR2-64I (p = 0.053), although the differences were not significant. In conclusion, starting HAART in antiretroviral drug-naive HIV-infected patients followed in daily clinical practice prevented viral breakthrough for up to 44 months in 60% of the patients. Virologic failure was associated with the development of resistance-related mutations, a later stage of disease at start of therapy and lower PI drug levels.
Abstract: PURPOSE: The purpose of our study was to characterize a human papillomavirus (HPV) 16 DNA integration in the genome of a rapidly progressive, lethal cervical cancer in a 39-year-old woman. EXPERIMENTAL DESIGN: An HPV 16 integration site from cervical cancer tissue was cloned and analyzed using Southern blot hybridization, nucleotide sequencing, fluorescence in situ hybridization analysis for chromosomal localization and comparison with the draft human genome sequence. RESULTS: HPV 16 DNA (3826 bp) was integrated into the genome of the tumor sample and contained an intact upstream regulatory region and E6 and E7 open reading frames. Both 5' and 3' viral-cell junction regions contained direct repeat and palindrome sequences. The chromosomal location of the viral integration and cellular deletion was mapped to chromosome 14q32.3 using both a somatic cell hybrid panel and fluorescence in situ hybridization. Search of the draft human genome sequence confirmed the chromosomal location and revealed a disruption of the TNFAIP2 cytokine/retinoic acid-inducible gene. CONCLUSIONS: On the basis of the lack of sequence homology between the viral and cellular site of integration and the structure of the viral-cell junctions, it seems that HPV 16 DNA integrates into the host genome by a mechanism of nonhomologous recombination. We suggest that, taken together, maintenance of E6 and E7 expression, loss of the E2 gene and disruption of the TNFAIP2 gene through viral integration contributed to the rapid progression of cervical cancer in this patient. Availability of the human genome sequence will facilitate identification of cellular genes involved in cervical cancer by high-throughput analysis of viral integration sites.
Abstract: Human papillomavirus (HPV) induced epithelial cell proliferation is responsible for a broad range of lesions. Treatment of such lesions is characterized by a high relapse rate. Four patients are described who were treated locally with cidofovir (cream 1% or injection of 2.5 mg/ml solution) for multi-treated relapsing HPV-associated lesions. Three of the four patients had a complete response. The fourth patient showed a clinical response in the first cycle of treatment, but new lesions appeared during four successive cycles of cidofovir.
Abstract: BACKGROUND: Polymorphisms in the gene that encodes the human cellular receptor for group B coxsackieviruses and adenoviruses (HCAR) could be responsible for differences in susceptibility to infections with these pathogens. Moreover, adenovirus subgroup C-mediated gene therapy could be influenced by mutations in the coding exons for the aminoterminal immunoglobulin-like 1 (Ig1) domain, which is the essential component for adenovirus fiber knob binding. RESULTS: Using two primersets in the adjacent intron sequences, HCAR exons 2 and 3, which comprise the full-length Ig1 domain, were amplified by polymerase chain reactions in 108 unselected and unrelated healthy Belgian volunteers. After nucleotide sequencing, no polymorphisms could be demonstrated in the adenovirus-binding Ig1 exons 2 and 3 of the HCAR gene. CONCLUSIONS: The adenovirus-binding Ig1 domain seems to be a highly conserved region in the Caucasian population which is a reassuring finding regarding adenovector-based gene therapy.
Abstract: A number of reports suggest that the sexually transmitted pathogen of cattle, Tritrichomonasfoetus, and a gastrointestinal commensal of pigs, Tritrichomonas suis, are very similar and may be co-specific. A conclusive review of the taxonomic and nomenclatural status of these species has not been presented so far. Toward this end, we reexamined and compared porcine and bovine trichomonads with regard to their morphology, pathogenic potential, and DNA polymorphism. Using light and electron microscopy, no distinguishing features between T. foetus and T. suis strains were found in size, general morphology, and karyomastigont structure. Both bovine and porcine trichomonads showed pathogenic potential in the subcutaneous mouse assays and did not separate into distinct groups according to strain virulence. Three DNA fingerprinting methods (i.e. RFLP, RAPD, and PCR-based analysis of variable-length DNA repeats) that produce species-specific DNA fragment patterns did not distinguish between the bovine and porcine strains. Sequencing of a variable 502-bp DNA fragment as well as comparison of 16S rRNA gene sequences did not reveal species-specific differences between the cattle and porcine strains. Therefore, we conclude that T. foetus and T. suis belong to the same species. To prevent confusion that may arise from T. foetus-T. suis synonymy, we propose to suppress the older name suis and maintain its accustomed junior synonym foetus as a nomen protectum for both cattle and porcine trichomonads. The case has been submitted to the International Commission on Zoological Nomenclature for ruling under its plenary power.
Abstract: Recently, individuals have been identified who possess T cell responses to herpes simplex virus (HSV) antigens despite the absence of detectable anti-HSV antibodies in their serum. The significance of this immune seronegative status is unclear, but it could indicate resistance to overt HSV infection. The aims of the present study were to investigate whether genetic differences in receptors used by HSV for cell entry (herpesvirus entry mediator [HVEM], nectin-1, and nectin-2) could be detected in immune seronegative individuals. Coding polymorphisms were identified in the HVEM and nectin-1 genes. The variant receptor proteins were expressed, and their ability to bind the viral ligand glycoprotein D and to mediate HSV entry after transient transfection into normally resistant cells was compared with that of their wild-type counterparts. HSV entry activity in wild-type and variant forms of the receptors was indistinguishable, which indicates that the polymorphisms observed are unlikely to explain the possible restrictions on HSV replication or spread in immune seronegative individuals.
Abstract: BACKGROUND: An avian papillomavirus genome has been cloned from a cutaneous exophytic papilloma from an African grey parrot (Psittacus erithacus). The nucleotide sequence, genome organization, and phylogenetic position of the Psittacus erithacus papillomavirus (PePV) were determined. This PePV sequence represents the first complete avian papillomavirus genome defined. RESULTS: The PePV genome (7304 basepairs) differs from other papillomaviruses, in that it has a unique organization of the early protein region lacking classical E6 and E7 open reading frames. Phylogenetic comparison of the PePV sequence with partial E1 and L1 sequences of the chaffinch (Fringilla coelebs) papillomavirus (FPV) reveals that these two avian papillomaviruses form a monophyletic cluster with a common branch that originates near the unresolved center of the papillomavirus evolutionary tree. CONCLUSIONS: The PePV genome has a unique layout of the early protein region which represents a novel prototypic genomic organization for avian papillomaviruses. The close relationship between PePV and FPV, and between their Psittaciformes and Passeriformes hosts, supports the hypothesis that papillomaviruses have co-evolved and speciated together with their host species throughout evolution.
Abstract: Both genetic and environmental factors have been implicated in the etiology of inflammatory bowel diseases (IBD) i.e., Crohn's disease (CD) and ulcerative colitis (UC). Polymorphisms in cytokine genes are likely to influence an individual's predisposition to IBD. In intron 2 of the interleukin-1 receptor antagonist (IL-1ra) gene, a variable number of an 86-bp tandem repeat (VNTR) polymorphism leads to the existence of five different alleles. In order to analyze the association between certain IL-1ra VNTR-alleles and IBD, we investigated the IL-1ra genotype and allele frequencies in 342 unrelated IBD patients and in 401 healthy control individuals. CD patients were also genotyped for the three main associated variants in the NOD2/CARD15 gene. In the IBD group, a significant decrease in the frequency of IL-1ra allele 1 (P=0.048) compared to controls was observed. The frequency of IL-1ra genotype 1/1 was significantly lower in the IBD population vs the control group (P=0.018). Analysis of the CD population without NOD2 homozygotes and compound heterozygotes revealed a more significant decrease in IL-1ra genotype 1/1 compared to controls (P=0.038). These results support the hypothesis that the IL-1ra VNTR-polymorphism could be among the genetic factors that are of importance in IBD susceptibility.
Abstract: The initial step during hepatitis B virus (HBV) infection is the specific attachment of the virus to the hepatocyte. Here we studied whether the binding of HBV to hepatocytes can, as is the case with most other enveloped viruses, be blocked by polyanionic compounds. Viral particles produced by HepAD38 cells were used as inoculum and HBV-negative HepG2 cells, as well as primary human hepatocytes, as target cells. Three sulphated polymers, that is, PAVAS (a co-polymer of acrylic acid with vinyl alcohol sulphate), heparin and dextran sulphate (DS) (MW 5000), and the sulphonated polymer PAMPS [poly(2-acryl-amido-2-methyl-1-propanesulfonic acid] (MW approximately 7000-12000), proved strong inhibitors of the binding of HBV to HepG2 cells and primary hepatocytes. The 50% effective concentration (EC50) for inhibition of HBV binding to HepG2 cells by PAVAS, heparin, DS and PAMPS was 1.3 microg/ml, 1.6 microg/ml, 1.8 microg/ml and 3.3 microg/ml, respectively, and to primary hepatocytes 1.6 microg/ml (PAVAS), 1.6 microg/ml (heparin), 2.6 microg/ml (DS) and 4.1 microg/ml (PAMPS). These values are in the same range as the concentrations required for these compounds to prevent such viruses as herpesviruses and HIV from binding to cells. These findings may be helpful in elucidating the mechanism of the initial interaction of HBV with hepatocytes.
Abstract: The relationship between adherence, virological response to highly active antiretroviral therapy (HAART) and the presence and development of genotypic resistance was assessed in 41 HIV-infected patients on HAART. Four adherence parameters (drug taking adherence, dosing adherence, timing adherence and drug holidays) were scored prospectively using electronic event monitoring. Genotypic resistance at baseline and after therapy failure was scored retrospectively and a genotype-based susceptibility score was calculated. Overall median adherence rates were high. All adherence parameters were better in virological responders (n=31) compared to non-responders (n=10), drug taking adherence and number of drug holidays being significantly different. Responders had a significantly higher susceptibility score. Stepwise logistic regression showed that the number of drug holidays and a low susceptibility score were highly predictive for therapy failure. Despite the presence of a limited number of baseline resistance mutations, perfectly adherent patients can control virus replication for a prolonged period.
Abstract: OBJECTIVES: To analyze the practical use of the anticytomegalovirus IgG avidity and its impact on the follow-up of pregnancy. To evaluate the performance of IgG avidity to exclude the risk of congenital infection. METHODS: 409 IgM-positive women without a documented seroconversion were prospectively followed. Data concerning the follow-up of the pregnancies were collected (amniotic fluid puncture and samples from the offspring). These observations were compared to those of 76 seroconversions during the same period. RESULTS: High avidity excluding a primary infection within the past 3 months was observed in 270 women. As the gestational age was less than 3 months for 121 women, exclusion of a primary infection was achieved in 30% of the cases. The rate of amniotic fluid puncture was influenced by the serological result: high avidity (9%), low avidity (42%) and seroconversion (65%). CONCLUSIONS: A high avidity index during the first trimester of pregnancy could reasonably be considered as a good indicator of past infection and invasive prenatal diagnosis is not necessary. Nearly 70% of the IgM-positive women could be reassured if the first serology was systematically performed before 12 weeks of gestation.
Abstract: We report a fatal case of disseminated adenovirus infection with fulminant hepatic failure in an 8-month-old child after peripheral blood stem cell transplantation. The virus was identified in blood, urine, respiratory aspirate and stool samples and was typed as adenovirus type 2 through PCR and sequencing of part of the hexon gene, with the use of degenerated consensus primers.
Abstract: A novel papillomavirus was cloned from hyperkeratotic cutaneous lesions of a Persian domestic cat. The Felis domesticus papillomavirus (FdPV-1) genome counts 8300 bp and has a typical genome structure with an early region (E1, E2, E4, E6, E7), a late region (L1, L2), and a noncoding upstream regulatory region (URR or NCR1) between the end of L1 and the beginning of E6. The FdPV-1 also shows an unusual second noncoding region (NCR2) of 1.3 kb, situated between the end of E2 and the beginning of L2. This NCR2 is uniquely related to a similar region in the canine oral papillomavirus (COPV). Phylogenetic analysis places FdPV-1 together with COPV, the cottontail rabbit papillomavirus, human papillomavirus type 1 (HPV-1), and HPV-63 in the group of the benign cutaneous papillomaviruses. The position of FdPV-1 in the phylogenetic tree allows us to hypothesize that already in an early phase of the papillomavirus molecular evolution, a split occurred into viruses with a dual tropism primarily for cutaneous epithelia but also secondarily for mucosal surfaces, and viruses with a specific monotropism for mucosal surfaces. The close relationship between FdPV-1 and COPV, and between their Canidae and Felidae hosts, supports the hypothesis that papillomaviruses have speciated and coevolved together with their hosts throughout vertebrate evolution. A papillomavirus mutation rate of 0.73 to 0.96 x 10(-8) nucleotide substitutions per base per year was calculated.
Abstract: Intravenous drug use is a major route of hepatitis C virus (HCV) transmission. In Belgium, more than 70% of the intravenous drug users (IVDUs) are HCV seropositive. In the past, medical treatment of HCV-positive IVDUs has been controversial. However, current studies support that the anti-HCV therapy of IVDUs should be the same as in other HCV-infected patients. In prison populations, HCV screening and therapy has to be performed. Patients should be counseled about the benefits of alcohol abstinence, should be educated about safer injection techniques to avoid reinfection, and should be vaccinated to avoid hepatitis A or B co-infections. Treatment of HCV infections should not be withheld from patient populations with complicated social problems. Physicians should rather develop individual treatment and follow-up plans in order to optimize compliance in IVDUs.
Abstract: The human cellular receptor for group B coxsackieviruses and adenoviruses (HCAR) is a transmembrane glycoprotein which belongs to the immunoglobulin superfamily. We describe alternative splicing of the HCAR-gene and the existence of three exon-skipping splice variants in addition to the originally identified seven exon-encompassing mRNA transcript. Expression of the splice variants theoretically results in truncated proteins, possibly leading to impaired viral binding and/or the occurrence of soluble viral receptors due to the absence of the transmembranous region. Consequently, this could markedly influence the efficacy of an adenovirus subgroup C-mediated gene therapy.
Abstract: Genital condylomata acuminata are nonmalignant human papillomavirus (HPV)-induced tumors in which HPV types 6 and 11 are most commonly found. Usual treatments for condylomata acuminata are nonspecific and are based on the destruction or removal of infected tissue. These procedures are often painful and are characterized by a high relapse rate. We report here what is to our knowledge the first double-blind, placebo-controlled study of the use of cidofovir, a nucleotide analogue, for the treatment of genital papillomavirus infections. Thirty patients were enrolled in the study; 19 received cidofovir, and 11 received placebo. The median number of warts and the median baseline wart area were comparable for both groups. Nine (47%) of 19 patients in the cidofovir group had a complete response (total healing), compared with 0 of the patients in the placebo group (P=.006). None of the patients in the cidofovir group experienced progression of the disease, compared with 5 (45%) of 11 patients in the placebo group. The side effects recorded for both groups were comparable.
Abstract: The inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis (UC) are complex multifactorial traits involving both environmental and genetic factors. Recent studies have shown the important role of pro-inflammatory cytokines and chemokines, including RANTES, in IBD. RANTES is the natural ligand for the CC-chemokine receptor 5 (CCR5). The chromosomal location of the CCR5 gene on 3p21 coincides with an IBD-susceptibility locus identified by genome-wide scanning. A 32-bp deletion (A32) in the CCR5 gene results in a nonfunctional receptor and is found with high frequency in Caucasians. In this study, we investigated the presence of the CCR5delta32 allele in a large cohort of IBD patients and in a healthy control population. Blood samples were obtained from 538 unselected IBD cases (433 unrelated IBD patients: 289 CD, 142 UC, 2 indeterminate colitis; 105 affected first-degree relatives) and 135 unaffected first-degree family members. Of the IBD patients, 36% had familial IBD with at least two members being affected. There were no significant differences in the CCR5delta32 mutation frequency between IBD patients and healthy controls, nor between CD and UC patients. There was no correlation between the CCR5delta32 genotype and the age at IBD-diagnosis, the frequency of surgical intervention, or disease localization. Only the association between CCR5delta32 homozygosity and the presence of anal lesions in CD patients was statistically significant (P=0.007). Analysis by the transmission/disequilibrium test showed no significant transmission distortion to the probands or their clinically silent siblings. Based on these results, it is unlikely that the CCR5delta32 allele is an important marker for predisposition to IBD.
Abstract: OBJECTIVES: To estimate the prevalence and the evolution over time (1995-1998) of genotypic resistance to antiviral drugs in antiretroviral drug-naive HIV-1-infected patients in Belgium. DESIGN: Belgian Aids Reference Laboratories provided retrospective samples and clinical data from antiretroviral drug-naive HIV-1-infected patients who visited the hospital for the first time in 1995 (n=45), 1997 (n=75) and 1998 (n=111). Genotypic resistance to the three available classes of drugs was monitored using the Line Probe Assay (Innogenetics, Gent, Belgium). Additionally, ARMS-151 was performed for scoring multinucleoside resistance. RESULTS: The prevalence of genotypic resistance at baseline to nucleoside analogue reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were each between 10% and 20% for 1995, 1997 and 1998 without an increasing trend over time. For NRTIs, resistance mutations were mainly related to zidovudine in 1995, whereas in 1997 and 1998 baseline resistance was scored for zidovudine, lamivudine or for both drugs simultaneously. No patients displayed the multi-nucleoside resistance Q151M mutation. Baseline resistance mutations to protease inhibitors (PIs) did not rise significantly: 4.4% in 1995, 8% in 1997 and 9.9% in 1998. When scoring any resistance-related mutation, 26.6% displayed genotypic baseline resistance in 1995, 26.6% in 1997 and 31.5% in 1998. DISCUSSION: The prevalence of genotypic baseline resistance to any drug, as scored with LiPA, in naive HIV-1 patients in Belgium is 29%, with baseline resistance mutations to one or several drugs from all available classes of antiviral drugs. The ability of LiPA to pick up minor variants could be an explanation for the higher overall prevalence we observe, when compared to recent estimates in other countries of 16.3% and 22%, which were based on sequencing methods. According to the European guidelines for resistance testing, resistance testing in Belgium before starting antiviral therapy should be considered.
Abstract: Non-human adenoviruses such as bovine adenovirus type 3 (BAV-3) that do not replicate in human cells but can infect human cells in culture could provide an attractive alternative to human adenoviral vectors for gene therapy. In addition, a large-animal model for genetic diseases can be very useful for the assessment of the efficacy of adenovector-mediated gene delivery in man. Recombinant human subgroup C adenovectors use the coxsackie and adenovirus receptor (CAR) to enter their target cells. Through RT-PCR and sequencing we determined the complete coding sequence of bovine CAR which serves as the primary adenoviral attachment site on bovine cells. A multiple sequence alignment, involving all the previously identified CAR species (man, mouse, rat, pig, and dog) showed that bovine CAR was most related to porcine CAR (92% nucleotide similarity) and demonstrated a highly conserved adenovirus binding Ig1 domain.
Abstract: OBJECTIVES: To document the prevalence of reproductive tract infections (RTI) and sexually transmitted infections (STI) among women attending a basic healthcare clinic in Dhaka, Bangladesh, to identify risk factors associated with the diseases and to estimate the incidence of syphilis, hepatitis C (HCV), hepatitis B (HBV), and herpes simplex type 2 (HSV-2) infection. METHODS: A cross sectional sample of 2335 consecutive women was examined during 1996-8. Women were interviewed about risk factors for RTI/STI and tested for Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Treponema pallidum, HIV, HCV, HBV, HSV-1 and HSV-2 infection as well as vaginal candidosis and bacterial vaginosis. Women with antibodies to T pallidum were retested at regular intervals. One year after ending the study seroconversion for syphilis, HBV, HCV, and HSV-2 infection was detected among women initially negative for the respective diseases. RESULTS: The overall prevalence rate of N gonorrhoeae, C trachomatis, T vaginalis, and T pallidum infection was 0.5%, 1.9%, 2.0%, and 2.9% respectively. Overall, 35% of the women had antibodies to hepatitis B core antigen, 0.9% had HCV, and 12% HSV-2 infection. Risk factors for gonorrhoea/C trachomatis infection were a husband not living at home or suspected of being unfaithful. HSV-2 infection was associated with the same risk factors and with a polygamous marriage. The prevalence of HSV-2 infection among women "at risk" was 23%. HIV infection was not diagnosed. Repeated serological examination indicated that only 32% of women with serological evidence of syphilis had active disease. The seroincidences of HBV, HCV, and HSV-2 were 0.03, 0.007, and 0.009 per person year. Seroconversion for syphilis was not observed.
Abstract: We compared the two commercially available sequencing kits for HIV-1 drug resistance testing, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA, U.S.A.) and the TRUGENE HIV-1 Genotyping Kit (Visible Genetics, Inc., Toronto, Ontario, Canada), with our in-house genotyping system. Fifteen viral isolates from African patients (6 treated and 9 untreated) covering a panel of HIV-1 subtypes A through J and 7 plasma samples from Belgian and African patients (2 treated and 5 untreated) were tested. All the samples could be amplified and sequenced by the three systems; however, for all systems, alternative amplification/sequencing primers had to be used for some samples belonging to subtype B as well as to other subtypes. The consensus sequence was partially derived from only one strand for the in-house system and for the ViroSeq Genotyping System. The TRUGENE HIV-1 Genotyping Kit scored the highest number of ambiguities, followed by the ViroSeq Genotyping System and the in-house system. For 11 samples, these differences in reporting mixtures affected 14 resistance-related positions, which altered the interpretation toward protease inhibitors for 2 samples when using version 1.2 RetroGram software (Virology Networks, Utrecht, The Netherlands). All three systems were able to sequence diluted samples with a viral load down to 10 3 or 10 4 RNA copies/ml. Our data therefore suggest that the performance of amplification and sequencing primers must be improved to allow fast and reliable resistance testing for all HIV-1 subtypes.
Abstract: The inflammatory bowel diseases (IBD), Crohn's disease (CD), and ulcerative colitis (UC), are complex multifactorial traits involving both environmental and genetic factors. Mannan-binding lectin (MBL) plays an important role in non-specific immunity and complement activation. Point mutations in codons 52, 54 and 57 of exon 1 of the MBL gene are associated with decreased MBL plasma concentrations and increased susceptibility to various infectious diseases. If these MBL mutations could lead to susceptibility to putative IBD-etiological microbial agents, or could temper the complement-mediated mucosal damage in IBD, MBL could function as the link between certain microbial, immunological and genetic factors in IBD. In this study, we investigated the presence of the codon 52, 54 and 57 mutations of the MBL gene in 431 unrelated IBD patients, 112 affected and 141 unaffected first-degree relatives, and 308 healthy control individuals. In the group of sporadic IBD patients (n = 340), the frequency of the investigated MBL variants was significantly lower in UC patients when compared with CD patients (P = 0.01) and with controls (P = 0.02). These results suggest that MBL mutations which decrease the formation of functional MBL could protect against the clinical development of sporadic UC, but not of CD. This could be explained by the differential T-helper response in both diseases.
Abstract: The measurement of the avidity of cytomegalovirus (CMV) immunoglobulin G (IgG) antibodies has been shown by several investigators to be useful in identifying and excluding primary CMV infections in pregnant women. In this work, we examined the diagnostic utility of reflex testing of CMV IgM-positive specimens from pregnant women by using a CMV IgG avidity assay. The utility of this approach was directly dependent on the sensitivity of the CMV IgM assay employed during the initial screen. The higher initial reactivity rate of the AxSYM CMV IgM assay was necessary in order to detect CMV IgM in specimens containing low-avidity CMV IgG antibodies, indicative of a primary CMV infection, which other CMV IgM assays (Behring, Vidas, Captia, and Eurogenetics) fail to detect in some cases. The use of the AxSYM CMV IgM assay, followed by an avidity test, should result in more accurate diagnosis of CMV infection in pregnant women.
Abstract: HIV-1 samples from six patients undergoing diverse anti-HIV therapies possessed the E138A mutation in their reverse transcriptase (RT) genome. Patients were receiving the following therapies: TIBO monotherapy (one patient); zidovudine plus didanosine combination therapy (one); zidovudine monotherapy (one); sequential therapy with zidovudine, then stavudine and finally zalcitabine plus didanosine (one); and two were drug naive. E138K, not E138A, is a known TSAO-specific resistance mutation, emerging under selective pressure in vitro. Our phenotypic data on the patient isolates, confirmed by data on an E138A mutant acquired through in vitro mutagenesis, indicated that an alanine substitution for glutamate at codon 138 of the HIV-1 RT renders the virus TSAO resistant, confirming the importance of this amino acid residue in the activity of TSAO derivatives. In addition, we have demonstrated through phenotypic analysis of the E138A and A98S mutants (after in vitro mutagenesis) that the mutation A98S, found in one of these patients, could be partially responsible for the phenotypic reversal of TSAO resistance. This reversal could be explained by the restoration of a hydrogen bond between 98S and the main-chain residue L349, which compensates for the loss of the E138-G99 main-chain hydrogen bond. As TSAO derivatives have not been used in the clinical setting, the presence of the E138A mutation at a frequency of 6.7% in our study of 90 TSAO-inexperienced HIV-seropositive individuals implies that 138A of the RT must be a natural variant and that the mutant virus is replication competent. Our observations suggest that the E138A mutation may likely arise in patients under the selective pressure of TSAO or related compounds that show a decreased antiviral potency toward the E138A variant.
Abstract: We examined the cause of death during a 12-month period (1995/96) in all consecutive patients admitted to hospital with leptospiral infection in Seychelles (Indian Ocean), where the disease is endemic. Leptospirosis was diagnosed by use of the microscopic agglutination test and a specific polymerase chain reaction assay on serum samples. Seventy-five cases were diagnosed and 6 patients died, a case fatality of 8%. All 6 patients died within 9 days of onset of symptoms and within 2 days of admission for 5 of them (5 days for the 6th). On autopsy, diffuse bilateral pulmonary haemorrhage (PH) was found in all fatalities. Renal, cardiac, digestive and cerebral haemorrhages were also found in 5, 3, 3 and 1 case(s), respectively. Incidentally, haemoptysis and lung infiltrate on chest radiographs, which suggest PH, were found in 8 of the 69 non-fatal cases. Dengue and hantavirus infections were ruled out. In conclusion, PH appeared to be a main cause of death in leptospirosis in this population, although haemorrhage in other organs may also have contributed to fatal outcomes. This cause of death contrasts with the findings generally reported in endemic settings.
Abstract: Papillomaviruses (PVs) are highly species- and site-specific pathogens of stratified squamous epithelium. Although PV infections in the various Felidae are rarely reported, we identified productive infections in six cat species. PV-induced proliferative skin or mucous membrane lesions were confirmed by immunohistochemical screening for papillomavirus-specific capsid antigens. Seven monoclonal antibodies, each of which reacts with an immunodominant antigenic determinant of the bovine papillomavirus L1 gene product, revealed that feline PV capsid epitopes were conserved to various degrees. This battery of monoclonal antibodies established differential expression patterns among cutaneous and oral PVs of snow leopards and domestic cats, suggesting that they represent distinct viruses. Clinically, the lesions in all species and anatomic sites were locally extensive and frequently multiple. Histologically, the areas of epidermal hyperplasia were flat with a similarity to benign tumors induced by cutaneotropic, carcinogenic PVs in immunosuppressed human patients. Limited restriction endonuclease analyses of viral genomic DNA confirmed the variability among three viral genomes recovered from available frozen tissue. Because most previous PV isolates have been species specific, these studies suggest that at least eight different cat papillomaviruses infect the oral cavity (tentative designations: Asian lion, Panthera leo, P1PV; snow leopard, Panthera uncia, PuPV-1; bobcat, Felis rufus, FrPV; Florida panther, Felis concolor, FcPV; clouded leopard, Neofelis nebulosa, NnPV; and domestic cat, Felis domesticus, FdPV-2) or skin (domestic cat, F. domesticus, FdPV-1; and snow leopard, P. uncia, PuPV-2).
Abstract: Polymorphisms of the chemokine receptor genes CCR5 and CCR2 are associated with resistance to HIV-1 infection or delayed progression to AIDS. Few data are available on their combined prevalence in healthy subjects; we therefore examined the occurrence of CCR5-Delta32 and CCR2-64I polymorphisms in a sample of 310 healthy Belgians. Allele frequencies were 0.119 and 0.074 for CCR5-Delta32 and CCR2-64I, respectively. Genotype distributions for both polymorphisms were found to be in accordance with Hardy-Weinberg equilibrium, but a significant (p = 0.002) linkage disequilibrium between CCR5-Delta32 and CCR2-64I was observed. The high prevalence of CCR5-Delta32 and CCR2-64I in Belgians may need to be taken into account in the design of studies of antiretroviral treatments.
Abstract: Cytomegalovirus (CMV) is a frequent cause of congenital infections in humans occurring in 0.4 to 2.3% of life births. Although preexisting maternal antibodies are generally protective, transplacental transmission of CMV during pregnancy may occur after recurrent maternal infection. We report severe bilateral hearing loss in an infant affected as a result of a CMV reactivation in pregnancy.
Abstract: High-risk mucosal human papillomaviruses encode an E6 oncoprotein, which binds the cellular p53 tumor suppressor protein, thereby marking it for degradation through the ubiquitin-mediated pathway. A common p53 polymorphism at codon-72 of exon 4 results in translation to either arginine or proline. Recently reported data suggested an increased susceptibility to E6/ubiquitin-mediated degradation of the Arg72-p53 isoform and an over-representation of the homozygous Arg72-p53 genotype in cervical cancer patients. We have analyzed this polymorphism in a larger series of patients with cervical cancer and in controls in the Czech Republic. We found no statistically significant differences between the codon-72 p53 genotypes of cervical cancer patients and the control women. Based on these results, it is unlikely that Arg72-p53 is associated with an increased risk for human papillomavirus-associated cervical tumor development in Czech women.
Abstract: BACKGROUND: We intended to evaluate the stability of human immunodeficiency virus (HIV) type 1 virions in whole blood and in culture medium. MATERIALS AND METHOD: EDTA whole-blood samples taken from 12 patients were left at room temperature for up to 7 days, and aliquots of a laboratory virus stock spiked in EDTA, in heparinized or in citrated whole blood, with or without the addition of Triton X-100, or spiked in culture medium were left at room temperature for up to 120 days before plasma was separated and frozen at -80 degrees C. Viral load was measured for all frozen plasma samples using different viral load assays. p24 antigen and infectivity were also measured in the spiked samples. RESULTS: The patient whole-blood samples did not show any decrease in viral load during this 7-day period. The spiked samples decayed by not more than 1 log after 120 days (about 4 months), with the fastest decay in medium. Virus infectivity decayed very slowly from 20,000 units mL-1 to undetectable amounts after 56 days. CONCLUSIONS: These results indicate that HIV-1 virions in whole blood possess a long-term stability in terms of viral load, p24 antigen level and infectivity, which is not sufficiently recognized by laboratory and health care workers.
Abstract: A novel immunochromatographic membrane-based assay for the detection of specific IgG antibodies to Mycobacterium tuberculosis was evaluated in patients with active tuberculosis in a low-prevalence population. The sensitivity of the test for detecting active tuberculosis was 41.5% (17/41 patients positive); its specificity in a group of patients with other lung diseases was 91.4% (3/35 false positive), while in a group of 47 healthy controls it was 100%. The sensitivity of the immunochromatographic test equaled that of auramine staining, but different subsets of tuberculosis patients were detected by the two tests. The suboptimal sensitivity of this immunochromatographic test implies that, even though it could be a useful adjunct, it cannot be a replacement for the diagnosis of tuberculosis by other microbiological methods along with clinical and radiological data.
Abstract: Because the biological spectrum of human papillomavirus (HPV) genotypes present in cervical cancer lesions varies according to the geographical region studied, and because little genotype information is available for Central and Eastern European countries, we studied the endemic HPV-genotype spectrum in cervical samples collected from women visiting gynaecological departments of selected hospitals in the Czech Republic. In a series of 389 samples, 171 (44.0%) were positive for HPV DNA using a consensus-primer polymerase chain reaction (PCR). Genotyping of the HPV PCR products was done using dot-blot hybridisation with type-specific oligonucleotide probes and thermocycle DNA sequencing. Twenty-two different HPV types were detected, HPV-16 being the most prevalent type irrespective of severity of the lesions (55.0%). Multiple HPV types were found in 16.4% of our HPV-DNA-positive samples. The prevalence of HPV infection was 23.0% in women with normal findings and 59.4% in patients with cervical neoplasia, and increased significantly with the severity of the disease: 52.9% in low-grade lesions, 58.0% in high-grade lesions, and 73.5% in cervical carcinomas (P for trend < .00001). In the sera of 191 subjects, 89 with normal findings and 102 with different forms of cervical neoplasia, the prevalence of HPV-specific IgG antibodies was tested by an enzyme-linked immunosorbent assay (ELISA) using virus-like particles (VLPs) of HPV-16, -18, and -33. Antibodies were significantly more prevalent in HPV-DNA-positive than in HPV-DNA-negative women and there was no association with age. In agreement with the results of HPV genotyping, antibodies reactive with HPV-16 VLPs were the most frequent and, moreover, their prevalence increased with the cervical lesion severity. About half of the subjects with smears in which either HPV-16 or HPV-33 DNA had been detected possessed antibodies reactive with homotypic VLPs. With HPV-18-DNA-positive subjects, however, fewer than 25% displayed homotypic antibodies. In general, subjects older than 30 years of age had antibodies reactive to HPV-specific VLPs more often than subjects younger than 30 years of age. In women with benign findings, the seropositivity to HPV-16, -18, and -33 VLPs increased with age, whereas in women with cervical neoplasia the seropositivity decreased with age.
Abstract: Hantavirus disease is a recently recognised zoonosis. The main vectors are infected but healthy wild rodents or laboratory rats. Transmission to man occurs via contact with, or inhalation of aerosolised excretions. The target organs in man are the kidney (Old World) or the lung (New World), probably via a local hyperproduction of pro-inflammatory cytokines. To date, more than 33 different hantaviruses have been characterised, at least 14 of them being of clinical importance. Each serotype has its own rodent vector and its own geographical spread. In Europe, the red bank vole (Clethrionomys glareolus) is the main rodent vector, carrying the Puumala (PUU) serotype, which is the etiologic agent of a viral affection known as Nephropathia epidemica (NE). PUU infection has been recognised for the first time in Belgium in 1983. From this date on, approximately 500 cases have been diagnosed. In our regions, a diagnosis of hantavirus disease is mandatory when a febrile patient presents with lumbaches, headache, and an acute renal failure with proteinuria, and particularly with thrombocytopenia.
Abstract: The sensitivity and discriminatory power of the 151 and 215 amplification refractory mutation system (ARMS) were evaluated, and their performance for the detection of drug resistance in mixed genotypic populations of the reverse transcription (RT) gene of HIV-1 were compared with T7 sequencing, cycle sequencing, the line probe assay (LiPA) HIV-1 RT test, and the recombinant virus assay (RVA). ARMS and the LiPA HIV-1 RT test were shown to be able to detect minor variants that in particular cases comprised only 1%. T7 sequencing on an ALF semiautomated sequencer could correctly score mixtures only when variants were present at 50%. Cycle sequencing on an ABI PRISM 310 improved the sensitivity for mixtures to about 25%. Using RVA, it was shown that at least 50% of the virus population needed to carry the resistance mutation at codon 184 to afford phenotypic resistance against lamivudine. The two point mutation assays therefore proved to be more sensitive methods than sequencing and RVA to reliably determine a gradual shift in HIV-1 drug resistance mutations in follow-up of patients infected with HIV-1. In 4 of 5 treated patients who were followed by ARMS, a gradual shift in resistant genotypic populations was observed during a period of 6 to 19 months. For 1 patient, a shift from wild to mutant type at position 151 occurred within 2 months, without mixed genotypic intermediate type's being detected.
Abstract: Respiratory papillomatosis is a rare and often severe disease, usually localized in the larynx. It may cause respiratory distress and even life-threatening obstruction of the airways. Treatment is generally based on the evaporation of the lesions with a CO2 laser, but microsurgery, cytotoxic and/or cytostatic drugs, interferons, and vaccines are also used. Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] (HPMPC) was shown to suppress the growth of tumors induced by rabbit papillomavirus as well as human papillomavirus (HPV). The efficacy of cidofovir was assessed in 17 patients with severe respiratory papillomatosis. Cidofovir at a concentration of 2.5 mg/ml was injected directly in the different laryngeal papillomatous lesions during microlaryngoscopy under general anesthesia. Biopsies were taken before the treatment was started both for anatomopathology and viral typing. HPMPC kinetics in serum was monitored in three patients, the drug levels being determined by high-performance liquid chromatography. Complete disappearance of the papillomatosis was observed in 14 patients. Four patients relapsed and were successfully treated again with cidofovir. Of the three remaining patients, one progressed while under treatment with cidofovir, after an initial marked response. One patient had a partial remission and remained stable for more than 1 year after the last injection. He had a very aggressive and extensive disease originally. Finally, one patient was lost to follow-up after four injections. Intratumoral injections of cidofovir for the treatment of severe laryngeal papillomatosis is a powerful new therapeutic approach for this disease. Treatment was well tolerated, and no significant side effects were noted.
Abstract: We identified a potential new subtype within human T-cell lymphotropic virus type 2 (HTLV-2), HTLV-2d, present in members of an isolated Efe Bambuti Pygmy tribe. Two of 23 Efe Pygmies were HTLV-2 seropositive, with HTLV-2 Western blot and enzyme-linked immunosorbent assay reactivities. From one of them the entire genome of the HTLV-2 strain Efe2 could be amplified and sequenced. In all gene regions analyzed, this strain was the most divergent HTLV-2 strain, differing by 2.4% (tax/rex) to 10.7% (long terminal repeat) from both subtypes HTLV-2a and HTLV-2b, yet major functional elements are conserved. The similarity between the HTLV-2 Efe2 Gag and Env proteins and the corresponding HTLV-2a and -2b proteins is consistent with the observed serological reactivity. In the proximal pX region, one of the two alternative splice acceptor sites is abolished in HTLV-2 Efe2. Another interesting feature of this potential new subtype is that it has a Tax protein of 344 amino acids (aa), which is intermediate in length between the HTLV-2a Tax protein (331 aa) and the HTLV-2b and -2c Tax proteins (356 aa) and similar to the simian T-cell lymphotropic virus type 2 (STLV-2) PP1664 Tax protein. Together these two findings suggest a different phenotype for the HTLV-2 Efe2 strain. Phylogenetic analyses confirmed that the Pygmy Efe2 strain potentially belonged to a new and quite divergent subtype, HTLV-2d. When the STLV-2 bonobo viruses PP1664 and PanP were used as an outgroup, it was clear that the Pygmy HTLV-2 Efe2 strain had the longest independent evolution and that HTLV-2 evolution is consistent with an African origin.
Abstract: Although acute liver failure due to the varicella-zoster virus is rare, it is frequently fatal. Immunologic impairment is a significant predisposing factor. Classic symptoms at presentation are rash, abdominal pain, and fever. After some days patients go on to develop full-blown liver failure. The diagnosis can be confirmed by histological examination and electron microscopy with fluorescent staining, immunohistochemistry, and in situ hybridization of the liver. In cases of high suspicion, acyclovir therapy should not be delayed.
Abstract: Two isoforms of NAD(P)(+)-dependent malic enzyme (EC 1.1.1.39) were isolated from hydrogenosomes of Trichomonas vaginalis. A positively charged isoform at pH 7 was obtained in a single purification step using cation-exchange chromatography. The second isoform, negatively charged at pH 7.5, was partially purified using a combination of anion-exchange and affinity chromatography. Both isoforms displayed similar physical and kinetic properties. Molecular weight determination of the native enzyme suggested a homotetrameric arrangement of the 60 kDa subunits. The enzyme utilized NAD+ (Km, 6-6.3 microM) preferentially to NADP+ (Km, 125-145 microM). The NAD(+)-dependent activity showed a broad pH optimum with maximum under alkaline conditions (pH 9) likely to be present inside hydrogenosomes. Immunocytochemical studies using a polyclonal rabbit antibody raised against purified T. vaginalis malic enzyme proved hydrogenosomal localization of the enzyme. Subfractionation of hydrogenosomes suggested an association of the malic enzyme with the hydrogenosomal membranes. The 60 kDa malic enzyme subunit was highly sensitive to non-enzymatic cleavage by an iron-ascorbate system resulting in two enzymatically inactive fragments of about 31 kDa. Microsequencing of the fragments revealed that the 60 kDa subunit was cleaved at the metal-binding site between Asp279-Ile280. The enzyme inactivation was inhibited by an excess of manganese. Iron-dependent posttranslational modification might contribute to the regulation of malic enzyme activity in vivo.
Abstract: Human papillomaviruses (HPV) are associated with benign lesions and show specificity towards the location or tissues that they infect. HPVs are responsible for warts. Among more than 60 different HPV types known to occur in humans, a strong association has been found between types 16 and 18 and cervical cancer, and such an association is also suspected for types 31, 33, 35, 45, 51, 52, and 56. We describe the effects of (S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine (HPMPC), following local intratumoral injection, in a 69-year-old woman with hypopharyngeal and esophageal papillomatous lesions, polymerase chain reaction (PCR) positive for HPV types 16 and 18, that relapsed after surgery and that also failed to respond to Nd-Yag laser photocoagulation and alpha-interferon treatment (6 x 10(6) U five times a week for 4 weeks followed by three times a week for 2 months). HPMPC was given at 1.25 mg/kg, with a sclerosing needle, through the biopsy channel of a video-endoscope, directly into the tumor, from March until July 1993 at seven different occasions. The first four injections were given at an interval of 1 week at the level of the hypopharynx. The next three injections were given at an interval of 3 to 5 weeks. During the fourth to the seventh session, half of the dose was injected into the hypopharyngeal and the other half into the esophageal tumor. Three further injections of HPMPC were administered at the level of the esophageal tumor in September 1993 with 2-week intervals. After HPMPC treatment, the lesions became smaller and flat until they completely disappeared.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Replication of vif-negative human immunodeficiency virus type 1 (HIV-1) is attenuated in certain cell lines and highly impaired in peripheral blood lymphocytes in vitro. To determine whether intact vif is positively selected during natural HIV-1 infection and to determine vif sequence variability, we employed PCR amplification, cloning, and sequencing to investigate the vif region of replicating virus in short-term-passage HIV-1 primary isolates from five asymptomatic individuals and from five persons with AIDS. A total of 46 vif clones were obtained and analyzed. Recombinant proviruses were constructed from selected vif clones from one patient and found to be fully infectious. We found that 38 of the 46 clones sequenced carried open vif reading frames and that there was a low degree of heterogeneity of vif genes within isolates from the same individual and among isolates from different donors. The cysteines previously found to be essential for vif protein function were conserved in all clones. A phylogenetic tree constructed from all available vif nucleotide sequences resulted in a virus grouping similar to those of gag and env. Direct sequencing of vif amplified by PCR from uncultured lymphocytes of 15 individuals at various stages of progression toward AIDS demonstrated vif open reading frames in 13 of 15 samples tested. There was no obvious correlation between disease status and the presence of an intact vif within this sample group at the time of sample procurement. The conservation of the vif open reading frame in vitro and in vivo and its limited variability following virus transmission in vitro are consistent with a role for vif in natural HIV-1 infection.
Abstract: Consensus primer mediated PCR protocols have the potential to amplify previously uncharacterized human papillomavirus (HPV) genotypes. In a study on 500 cervicovaginal samples, we amplified four sequences (L1AE 1 to L1AE 4) that failed to hybridize to any of the available HPV type-specific oligonucleotide probes. Nucleotide sequencing revealed that the sequences were derived from the L1 region of hitherto unsequenced genotypes. Comparison of phylogenetic trees based on the amplified L1 sequences with E6-derived phylogenetic trees resulted in the identification of L1AE 1 and L1AE 2 as putative novel HPV PCR genotypes. L1AE 1 was related to HPV 39, whereas L1AE 2 was related to HPV 51. The L1AE 3 and L1AE 4 sequences occupied L1-phylogenetic branches equivalent to the positions of HPV 66 and HPV 61, respectively, in an E6-phylogenetic tree.
Abstract: Forty-two women attending a colposcopy clinic for evaluation of abnormal cervical cytology and 13 normal controls were studied for the presence of lymphocyte proliferation (LP) cell-mediated immune (CMI) responses and serological reactivity to E7 peptides of human papillomavirus type 16 (HPV-16). HPV was typed by Southern blot hybridization of exfoliated cervicovaginal cell DNA. Positive LP responses (stimulation index > or = 5.0) to one or more E7 peptides were observed in 28.6% (12 of 42) of patients and 23.1% (three of 13) of controls. Of patients infected with HPV-16, -31 or -33, 63.6% (seven of 11) showed a positive LP response compared with 14.3% (two of 14) of women infected with other HPV types (P = 0.02), 17.6% (three of 17) negative for HPV (P = 0.02) and 23.1% (three of 13) of controls (HPV status unknown) (P = 0.05). C-terminal peptide 109 (amino acids 72 to 97) elicited positive LP responses in 45.4% (five of 11) of patients infected with HPV -16, -31 or -33 compared with 7.1% (one of 14) patients infected with other HPVs (P = 0.04), 5.9% (one of 17) of women negative for HPV (P = 0.02) and 7.7% (one of 13) of controls (P = 0.05). HPV-16 group-specific LP responses of borderline significance were also observed against E7 peptides 103, 105 and 108 (17-37, 37-54 and 62-80) (P = 0.07). ELISA reactivity (IgG) to E7 peptide 109 (72-97) was present in 7.7% (one of 13) of controls, 35.3% (six of 17) of HPV-negative patients, 42.9% (six of 14) of patients infected with other HPVs, and only 9.1% (one of 11) of patients infected with HPV-16, -31 or -33. CMI responses to C-terminal HPV-16 E7 peptide 109 (72-97) were thus significantly related to ongoing cervical infection with HPV-16 and closely related types, whereas serological reactivity to E7 peptides was not HPV type-specific.
Abstract: Recurrent respiratory papillomatosis (RRP) is the most common benign tumour of the larynx, affecting both children and adults. We present a series of 25 patients, including 10 cases of juvenile multiple, 8 cases of adult solitary, and 7 cases of adult multiple RRP. Biopsy tissue from each patient was screened by Southern blot hybridization and polymerase chain reaction for the presence of human papillomavirus (HPV) DNA. Sera from patients and age- and sex-matched controls were tested for the presence of HPV-specific antibodies using a synthetic peptide derived from the minor capsid protein (L2) of HPV 6/11. By Southern blot hybridization and/or polymerase chain reaction, biopsies from all patients were positive for HPV 6/11 DNA. There was no difference in antibody response between cases and controls. Female cases and controls had significantly higher antibody titers than male subjects. A correlation was observed between the HPV-specific antibody level and the number of surgery-necessitating recurrences.
Abstract: Mastomys natalensis is a rodent of African origin afflicted with a very high incidence of skin tumors (keratoacanthomas and squamous carcinomas), which are associated with a papillomavirus, M. natalensis papillomavirus (MnPV). We have determined the genomic sequence of MnPV, which has a size of 7687 bp. The genomic organization is similar to that of other papillomaviruses, with open reading frames E6, E7, E1, E2, and E4 in the early and L2 and L1 in the late region. Due to an unusually large hinge region, the transcriptional activator E2 has a size of 542 amino acids rather than 400 to 460 amino acids, as in other papillomaviruses. An open reading frame E5 coding for a small hydrophobic membrane protein is missing, as is the case for some cutaneous human papillomaviruses (HPV). This fact, together with the composition of cis-responsive elements in its long control region and phylogenetic evaluation of segments of its E6, E1, and L1 genes, indicates a relationship of MnPV to the cottontail rabbit papillomavirus and several HPV types found in lesions of cutaneous epithelia, in particular to those that are associated with epidermodysplasia verruciformis. MnPV may be a useful model system for tumorigenesis of cutaneous epithelia in humans.
Abstract: Severe papillomatosis developed in the oral cavity and spread throughout the haired skin of the trunk and limbs of an 8-month-old female Chinese Shar Pei dog. The dog had received corticosteroids prior to referral, which was associated with the onset of demodecosis and papillomatosis. Papillomavirus structural antigens were detected in biopsies by immunohistochemistry using a panel of monoclonal and polyclonal antibodies. An 8.2-kilobase papillomavirus-specific DNA molecule was detected in the cutaneous lesions by high stringency Southern blot hybridization using a cloned canine oral papillomavirus DNA probe. Restriction enzyme analysis revealed that the virus in the cutaneous lesions was identical to the canine oral papillomavirus. Discontinuation of the steroids combined with the use of a mitocide, antibiotics, and an autogenous vaccine resolved the demodecosis and papillomatosis. This case report suggests that corticosteroid-induced immunosuppression can expand the tissue tropism of papillomaviruses.
Abstract: The canine oral papillomavirus (COPV) is associated with oropharyngeal papillomatosis in dogs, coyotes, and wolves. We have determined the complete nucleotide sequence of COPV, the largest of all known PV genomes (8607 bp). The genomic architecture of the COPV genome is similar to that of other PVs except for a unique and large noncoding region of 1.5 kb between the end of the early region (E2) and the beginning of the late region (L2) and a small (345 bp) upstream regulatory region between the end of L1 and the beginning of E6. Although COPV displays a primarily mucosal tropism, the COPV nucleotide sequence showed the highest overall similarity to cutaneous papillomaviruses such as HPV-1, HPV-63, CRPV (cottontail rabbit PV), FdPV (Felis domesticus PV), and MnPV (Mastomys natalensis PV).
Abstract: Human papillomaviruses (HPVs) are a heterogeneous group of small dsDNA viruses which cause a variety of proliferative epithelial lesions at specific anatomical sites. Although more than 65 different virus types have been cloned and characterized, no uniform classification system exists. In order to classify HPV DNA types, phylogenetic trees were constructed based on nucleotide sequence alignments using parsimony and distance matrix algorithms. The resulting phylogenetic trees provide a classification of the HPVs into specific groups encompassing the known tissue tropism and oncogenic potential of each HPV type. The implications of a phylogenetic taxonomy on the diagnostic detection of HPVs and the concept of different HPV species are discussed.
Abstract: Six cell lines, that were cloned from murine C127 cells infected by bovine papillomavirus type 1 (BPV1), were found to differ in the degree of transformation in vitro and of tumorigenicity in vivo. In these cell lines the degree of tumorigenicity was inversely correlated with IL-6 induction by IL-1 beta. Whereas the parental C127 cell line produced 15-30 U/ml of IL-6 spontaneously, none of the transformed cell lines produced significant levels of IL-6 constitutively. On induction by human IL-1 beta the parental C127 cell line produced up to 300 U/ml of IL-6, whereas the fully transformed ID14 cell line failed to produce any. The less transformed cell lines produced lower yields of IL-1 beta-induced IL-6, dependent on their degrees of transformation and tumorigenicity. Gelatinase B (96 kDa), a matrix metalloproteinase inducible by IL-1 beta, was dose-dependently regulated in the parental C127 cell line and in the weakly transformed cell line Tlc. These data suggest that transformation processes by BPV1 generally impair IL-1-regulated gene transcription. This impairment seems not to be located at the IL-1 beta receptor level, since in all the cell lines studied the numbers and affinities of the IL-1 beta binding sites were found to be comparable. This impairment seems not to be mediated by transformation-induced inactivation of the protein kinase C pathway since phorbol 12-myristate 13-acetate (PMA) induced IL-6 production equally well in all C127 cell-derived clones. It is suggested that BPV1 transformation can change the expression of host genes that might play a functional role in tumor immune surveillance and tumorigenicity in vivo.
Abstract: Human papillomavirus type 13(HPV-13) is associated with oral focal epithelial hyperplasia (FEH) in humans. A recent epidemic of a FEH-like disease in a pygmy chimpanzee (Pan paniscus) colony allowed us to clone a novel papillomavirus genome. To assess the homology between HPV-13 and the pygmy chimpanzee papillomavirus type 1 (PCPV-1), the complete nucleotide sequences of both FEH-related viruses were determined. In both viruses, all eight major open reading frames were located on one strand and the genomic organization was similar to that of other mucosal papillomaviruses. The genomes of PCPV-1 and HPV-13 showed extensive overall sequence homology (85%). They could be classified, using phylogenetic analysis, together with HPV types 6, 11, 43, and 44 in a group associated with benign orogenital lesions. These data indicate that two phylogenetically related papillomaviruses can elicit similar pathology in different primate host species, reflecting viral genomic similarities.
Abstract: The induction of proteolytic enzymes is an important mechanism in the migration of monocytes into tissues and body fluids. The monocytic cell line THP-1 was used as a model system to study the production of a particular gelatinase. Upon stimulation with phorbol myristate acetate (PMA) the cells differentiated to the adherent phenotype and produced significant amounts of a 96-kD gelatinase in a dose-dependent way. The secretion rate was maximal between 12 and 24 h after induction. Study of gelatinase mRNA steady state levels showed that the synthesis of THP-1 gelatinase is regulated by PMA at transcriptional or posttranscriptional levels. Stimulation of signal transduction pathways with other substances, including calcium ionophore A 23187, dibutyryl cyclic AMP, and dexamethasone, were ineffective in inducing gelatinase mRNA or enzyme activity. However, THP-1 cells were responsive to the cytokine interleukin (IL)-1 beta, to bacterial lipopolysaccharide (LPS), and the lectin concanavalin A (Con A), the kinetics of gelatinase induction being similar to those of induction by PMA. The THP-1 cells did not synthesize and/or secrete detectable levels of IL-6 after stimulation with PMA, Con A, LPS, or IL-1 beta. The 96-kD monocytic THP-1 gelatinase was shown to be a neutral metalloproteinase that cross-reacted with hepatoma-derived and neutrophil gelatinases in immunoprecipitation experiments. The active enzyme produced by THP-1 cells consistently showed, however, a molecular mass different from that of normal granulocyte-, monocyte-, and tumor cell-derived gelatinases.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Bradykinin was found to induce production of IL-6 in human diploid fibroblasts, as well as in a hepatoma-derived cell line, but not in a human melanoma or an osteosarcoma cell line. With the exception of the melanoma cell line, these cells were also found to be responsive to IL-1 beta. The response to bradykinin was faster but less high than that induced by IL-1. Experiments in which IL-1 (-alpha or -beta) and bradykinin were applied simultaneously revealed a synergistic interaction. Of the other cytokines tested, TNF-alpha and IFN-gamma weakly induced IL-6. Neither IL-2, IFN-alpha, nor IFN-beta was able to induce IL-6, either in the absence or the presence of bradykinin. These observations constitute further evidence for the existence of interactions between cytokine and noncytokine peptides, thus linking the neuroendocrine and immune systems.
Abstract: An epizootic of focal epithelial hyperplasia (FEH) or Morbus Heck in a pygmy chimpanzee (Pan paniscus) colony is described. Papovavirus-like particles were observed in the nuclei of epithelial cells. Analysis of the DNA of the lesions revealed an episomal papillomavirus-specific band with a molecular size of approximately 8,000 bp. In situ hybridization under high stringency conditions showed cross-hybridization between the chimpanzee papillomavirus DNA and human papillomavirus (HPV) type 13. The latter virus is uniquely associated with oral disease in man. This is the first demonstration of the association of a HPV 13-related pygmy chimpanzee papillomavirus (PCPV) and oral epithelial hyperplasia in a nonhuman primate.
