Virology and Viral Diseases, Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liege, Boulevard de Colonster, 20, B43b B-4000 Liege BELGIUM
etienne.thiry@ulg.ac.be
Etienne Thiry is born on the 4th April 1957 in Brussels. He was graduated as doctor in veterinary medicine in 1980 at the university of Liège (ULg). He got a master of sciences in molecular biology in 1982 at the university of Brussels. He got the doctorate in veterinary sciences in 1985 with a thesis on the conditions of the re-excretion of bovine herpesvirus 1. In 1992, he was graduated in medical and epidemiology from the university Pierre and Marie Curie in Paris. He was recognized in 2001 as diplomate of the European college for veterinary public health.
Since 1980, Etienne Thiry has occupied several positions in the laboratory of veterinary virology headed by Prof. Paul-Pierre Pastoret at ULg. He is professor of virology and viral diseases since 1993. He is also part time professor of veterinary virology at the university of Brussels since 2000. He was the head of the department of infectious and parasitic diseases of the veterinary faculty at ULg from 2001 to 2006. He won the International Pfizer award by the international committee of the World Buiatrics (bovine medicine) Society in 1996.
His research interests cover several aspects of animal virology, especially herpesviruses, caliciviruses and orbiviruses. Recombination within alphaherpesvirus genomes was the main research topic over the last 7 years. The study of the cluster of ruminant alphaherpesviruses related to bovine herpesvirus 1, the molecular epidemiology of the foodborne noroviruses and the genetic variation of feline calicivirus are also part of his research activities. The recent outbreak of bluetongue in northern Europe orientated the activities of his laboratory to the orbivirus causing this disease.
Etienne Thiry is member of the National Committee for Microbiology of the Belgian Royal Academy since 1999. He was the treasurer of the Belgian Society for Microbiology for ten years. He was the president of the Belgian Association of animal health and epidemiology from 1999 to 2006. From 1994 to 2000, he was member of the board of the European Society for Veterinary Virology. He takes part to several scientific committees. Among others, he is member of the scientific committee of the Belgian Federal Food Safety Agency, the Belgian scientific committee for influenza, the Belgian chamber of veterinary medicinal products, the expert board for notifiable diseases in the Grand-Duchy of Luxembourg and the expert committee for animal health at the French Food Safety Agency. He was recently appointed as vice-chairman of the European Advisory Board on Cat Diseases.
Abstract: Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and calves. Reverse transcription-polymerase chain reaction (RT-PCR) has become the "gold standard" for detection of noroviruses in faecal and environmental samples. However, false negative results due to co-concentration of RT-PCR inhibitors are a continuous concern. A TaqMan real-time RT-PCR assay making use of a foreign internal RNA control and a RNA standard was developed. Very interestingly, this method is capable of detecting human noroviruses belonging to genogroups I and II, and bovine noroviruses belonging to genogroup III. Inhibitors were removed efficiently by 1/10 dilution of the sample or addition of bovine serum albumin to the RT-PCR mix. This assay was validated with human and bovine stool samples previously tested for norovirus by conventional RT-PCR. The ability to detect norovirus in stool samples that were negative by conventional RT-PCR assay demonstrate the higher sensitivity of the TaqMan assay compared to the conventional RT-PCR assay. This real-time RT-PCR assay allows the detection of both human and bovine noroviruses, avoids false negative results and is able to quantify the level of norovirus contamination.
Abstract: Among enteric caliciviruses, noroviruses belong to the genus Norovirus, one of the four accepted genera in the family Caliciviridae. These single-stranded, positive-sense RNA viruses are highly variable both genetically and antigenically. Several animal enteric caliciviruses that are morphologically indistinguishable and genetically closely related to human noroviruses have been identified. The first bovine enteric noroviruses were described in Great Britain and are known as Newbury Agent 2. At least three genetic clusters of porcine noroviruses join together within genogroup II noroviruses. Human noroviruses are the most important cause of acute gastroenteritis illness in people of all ages. In the USA, they are associated with approximately 30-50% of all food-borne outbreaks. Until now, noroviruses have not been associated with gastroenteritis outbreaks in immunocompetent animals. Neither bovine nor porcine noroviruses can replicate in cell culture, although human norovirus can grow in a complex 3D culture system. However, the recently discovered murine noroviruses can replicate in cell culture and are therefore used as model viruses to study human noroviruses. This review focusses on virus classification, virion structure, pathogenesis, epidemiology, immune response and diagnosis of animal noroviruses in comparison with human noroviruses. The classification of animal enteric caliciviruses within the Norovirus genus raises the question of whether transmission from an animal reservoir to humans could occur. Answering this question is important in determining the risk of cross-species infections affecting the epidemiology and evolution of these viruses and so complicating the control of human norovirus infections.
Abstract: Over the last decade, atypical myopathy (AM) in grazing horses has emerged in several European countries. An exploratory analysis was conducted to determine horse- and pasture-level indicators or factors associated with AM in Belgium. Belgian cases of AM confirmed by histology (n=57) were compared to their healthy co-grazing horses (n=77) and to pastured horses not involved with AM as controls (n=386). The pastures where confirmed cases were grazing (42 pastures; 38 sites; 44 incidences of AM) were compared with those of the controls (216 pastures; 96 sites; no incidence of AM). Statistically significant (P0.05) exploratory variables, identified by means of adjusted odds ratios, suggested that indicators or factors associated with individual horses (young age, inactivity, body condition poor to normal), management practices (permanent pasturing, spreading of manure) and pasture characteristics (humid, sloping pastures, accumulated dead leaves, presence of waterway) may increase the risk of AM. Specific interventions based on these factors might help to reduce the incidence of AM.
Abstract: To the Editor: In August 2006, several northern European countries including Belgium reported cases of bluetongue (BT). This noncontagious, arthropod-borne animal disease is caused by Bluetongue virus (BTV), genus Orbivirus, family Reoviridae. The genome of BTV consists of 10 segments of double-stranded RNA; 24 serotypes have been reported. Serotype 8 (BTV-8) was implicated in the emergence in Belgium. All ruminant species are thought to be susceptible to BT. We report laboratory-confirmed clinical cases of BT in yaks (Bos grunniens grunniens).
Abstract: Caprine herpesvirus 1 (CpHV-1) is a virus able to cause genital infection leading to vulvovaginitis or balanoposthitis in adult goats. CpHV-1 shares several biological similarities with herpes simplex type 2 (HSV-2) infection in man, such as genital tropism, type and site of typical lesions and it might provide an animal model for studies on antiviral drugs for HSV-2 infection in man. In this view the efficacy of cidofovir (CDV) drug was tested in six goats intravaginally infected with BA.1 strain of CpHV-1. Three goats received an intravaginal application of 3 ml of a 1% CDV preparation at 4h post infection and then every 12 h for five consecutive days. Three goats were kept as untreated controls. The goats were daily examined for clinical evidence of the infection and viral shedding. CDV was able to protect against disease progression and inhibited the onset of the local lesions due to the CpHV-1 replication. Treated animals shed virus for a shorter period (3 days less) and at lower titres than the control animals. CpHV-1 infection in goats may represent an excellent animal model for the study of novel strategies for the treatment of primary genital HSV-2 infection in man.
Abstract: BACKGROUND: The emergent nature of atypical myopathy or atypical myoglobinuria (AM) necessitates precise description of its clinical and epidemiologic features. PURPOSE: To define key features of AM to help practitioners recognize the disease and to advise owners to take preventive measures. ANIMALS: Belgian cases of AM confirmed by histology (CC horses; n = 57) from autumn 2000 to spring 2005 were included in the study. Co-grazing horses (Co-G horses; n = 77) that remained free of any abnormal clinical signs constituted a control group. METHODS: History, environmental characteristics, clinical signs, and laboratory results associated with AM were determined by a retrospective case series study. RESULTS: Young horses in poor or normal body condition were found to be at risk for AM. Pastures were characterized by poor natural drainage and vegetation of low nutritional value. Features of AM were seasonal occurrence, apparent link with weather conditions (ie, lack of solar radiation with no heavy frost and an excess of precipitation or relative humidity), sudden onset of clinical signs, and rapid death. Evaluation of serum creatine kinase activity indicated severe muscle destruction in CC horses and subclinical disease in a few Co-G horses. CONCLUSIONS: The association of AM with specific environmental conditions and individual animals suggests that young horses should not be pastured on bare premises subject to humidity when the weather has been very wet and cold for several days. Management of AM outbreaks should include control of Co-G horses who are apparently healthy.
Abstract: The Asian lineage highly pathogenic avian influenza (HPAI) H5N1 virus is a known pathogen of birds. Only recently, the virus has been reported to cause sporadic fatal disease in carnivores, and its zoonotic potential has been dominating the popular media. Attention to felids was drawn by two outbreaks with high mortality in tigers, leopards and other exotic felids in Thailand. Subsequently, domestic cats were found naturally infected and experimentally susceptible to H5N1 virus. A high susceptibility of the dog to H3N8 equine influenza A virus had been reported earlier, and recently also HPAI H5N1 virus has been identified as a canine pathogen. The ferret, hamster and mouse are suitable as experimental animals; importantly, these species are also kept as pets. Experimental intratracheal and oral infection of cats with an HPAI H5N1 virus isolate from a human case resulted in lethal disease; furthermore, cats have been infected by the feeding of infected chickens. Spread of the infection from experimentally infected to in-contact cats has been reported. The epidemiological role of the cat and other pet animal species in transmitting HPAI H5N1 virus to humans needs continuous consideration and attention.
Abstract: Bovine herpesvirus 1 (BoHV-1), classified as an alphaherpesvirus, is a major pathogen of cattle. Primary infection is accompanied by various clinical manifestations such as infectious bovine rhinotracheitis, abortion, infectious pustular vulvovaginitis, and systemic infection in neonates. When animals survive, a life-long latent infection is established in nervous sensory ganglia. Several reactivation stimuli can lead to viral re-excretion, which is responsible for the maintenance of BoHV-1 within a cattle herd. This paper focuses on an updated pathogenesis based on a molecular characterization of BoHV-1 and the description of the virus cycle. Special emphasis is accorded to the impact of the latency and reactivation cycle on the epidemiology and the control of BoHV-1. Several European countries have initiated BoHV-1 eradication schemes because of the significant losses incurred by disease and trading restrictions. The vaccines used against BoHV-1 are described in this context where the differentiation of infected from vaccinated animals is of critical importance to achieve BoHV-1 eradication.
Abstract: BACKGROUND: Like human herpesvirus 2 (HHV-2) in humans, infection by caprine herpesvirus 1 in goats is associated with genital lesions, and this provides a unique model to study the efficacy and effects of anti-herpetic drugs. METHODS: The antiviral activity of cidofovir was assessed in goats infected experimentally, using various therapeutic protocols. RESULTS: Topic administration of cidofovir 1% cream prevented the onset of virus-induced genital lesions and drastically reduced virus shedding. CONCLUSION: Cidofovir appears to be a very efficient drug for the prevention of genital lesions caused by an alphaherpesvirus.
Abstract: Canine parvovirus (CPV), which causes hemorrhagic enteritis in dogs, has 3 antigenic variants: types 2a, 2b, and 2c. Molecular method assessment of the distribution of the CPV variants in Europe showed that the new variant CPV-2c is widespread in Europe and that the viruses are distributed in different countries.
Abstract: BACKGROUND: Caprine herpesvirus 1 (CpHV-1) is responsible of systemic diseases in kids and genital diseases leading to abortions in goats. CpHV-1 is widespread and especially in Mediterranean countries as Greece, Italy and Spain. CpHV-1 is antigenically and genetically closely related to bovine herpesvirus 1 (BoHV-1). Taking into account the biological properties shared by these two viruses, we decided in the current study to assess the protection of a live attenuated glycoprotein E (gE) negative BoHV-1 vaccine against a genital CpHV-1 infection in goats. RESULTS: The vaccine was inoculated intranasally twice three weeks apart followed by a subsequent CpHV-1 intravaginal challenge which is the natural route of infection in three goats. To analyse the safety and the efficacy of this marker vaccine, two groups of three goats served as controls: one immunised with a virulent CpHV-1 and one uninoculated until the challenge. Goats were clinically monitored and all sampling procedures were carried out in a blind manner. The vaccine did not induce any undesirable local or systemic reaction and goats did not excrete gE-negative BoHV-1. After challenge, a significant reduction in disease severity was observed in immunised goats. Moreover, goats immunised with either gE-negative BoHV-1 or CpHV-1 exhibited a significant reduction in the length and the peak of viral excretion. Antibodies neutralising both BoHV-1 and CpHV-1 were raised in immunised goats. CONCLUSION: Intranasal application of a live attenuated gE-negative BoHV-1 vaccine is able to afford a clinical protection and a reduction of virus excretion in goats challenged by a CpHV-1 genital infection.
Abstract: BACKGROUND: The genus Varicellovirus of the Herpesviridae subfamily Alphaherpesvirinae includes a cluster of viruses antigenically and genetically related to bovine herpesvirus 1 (BoHV-1): namely bovine herpesvirus 5 (BoHV-5), bubaline herpesvirus 1 (BuHV-1), caprine herpesvirus 1 (CpHV-1), cervid herpesviruses 1 (CvHV-1) and 2 (CvHV-2) and elk herpesvirus 1 (ElkHV-1). Considering the serological relationship between these ruminant alphaherpesviruses, several surveys have studied the occurrence of BoHV-1 related virus infection in wild and domestic ruminant species. In this way, a recent investigation has indicated, in Belgium, a high increase in the serological prevalence of BoHV-1 related virus infection in free-ranging red deer population. In this context, it has been decided to investigate the presence of an alphaherpesvirus spreading in the Belgian free-ranging red deer population. RESULTS: The current study reports the first isolation in a free-ranging red deer of a BoHV-1 closely related virus. The isolate was antigenically, genomically and genetically characterised by comparison with several ruminant alphaherpesvirus. Immunofluorescence assays revealed the isolate was antigenically distinct from bovine and caprine alphaherpesviruses. Similarly, BamHI and BstEII restriction analyses demonstrated the genomic difference between the isolate and the other ruminant alphaherpesviruses. Next, the sequencing of selected parts of UL27 and US8 genes showed a high degree of homologies between each BoHV-1 related ruminant alphaherpesvirus and the isolate. Besides the close relationship between all ruminant alphaherpesviruses, the phylogenetic analysis revealed that the isolate clustered with CvHV-1. CONCLUSION: The first isolation of a virus closely related to BoHV-1 in a free-ranging red deer is reported. Data demonstrate that a CvHV-1 strain, named Anlier, circulates in wild red deer in continental Europe. Anlier strain show consistent differences with the virus isolated from Scottish farmed red deer. All together, these results improve our understanding of ruminant alphaherpesviruses.
Abstract: Vaccination guidelines are non-compulsory recommendations which assist the veterinary practitioner to use vaccines efficiently. They complement the official information contained in the shortened form of the summary of product characteristics that is included in the package insert of the product. The aim of this article is to clarify the role of guidelines and examine how they can improve the use of vaccines in practical conditions. The development of vaccination guidelines is explained. Several issues are discussed: primary vaccination schedule; interference with maternally derived antibodies; duration of immunity; vaccination and ageing. Three guidelines dealing with the vaccination of cats against upper respiratory tract disease are compared, as an example. In conclusion, vaccination guidelines are essential tools to assist veterinarians in good vaccination practices. They fill the gap that exists between the official recommendations included in the regulations and the licensing dossiers and the daily use of the vaccines.
Abstract: Feline herpesvirus (FHV-1; felid herpesvirus 1 (FeHV-1)) is an alphaherpesvirus of cats closely related to canine herpesvirus-1 and phocine herpesvirus-1. There is only one serotype of the virus and it is relatively homogenous genetically. FeHV-1 is an important cause of acute upper respiratory tract and ocular disease in cats. In addition, its role in more chronic ocular disease and skin lesions is increasingly being recognised. Epidemiologically, FeHV-1 behaves as a typical alphaherpesvirus whereby clinically recovered cats become latently infected carriers which undergo periodic episodes of virus reactivation, particularly after a stress. The primary site of latency is the trigeminal ganglion. Conventional inactivated and modified-live vaccines are available and protect reasonably well against disease but not infection, although viral shedding may be reduced. Genetically engineered vaccines have also been developed, both for FeHV-1 and as vector vaccines for other pathogens, but none is as yet marketed.
Abstract: In the family Herpesviridae, the subfamily Alphaherpesvirinae contains numerous pathogenic viruses, i.e. herpes simplex and varicella-zoster viruses. These double-stranded DNA viruses exhibit a complex cycle combining lytic and latent infections. Moreover, both intranuclear replication and a sophisticated DNA replication machinery allow an efficient proof-reading mechanism of correction. A low mutation rate is therefore encountered by these viruses. Recombination can be identified as a key element of the genetic biodiversity of alphaherpesviruses, together with mutations. The experimental data recently obtained in the bovine herpesvirus 1 homologous model support the importance of recombination in alphaherpesvirus evolution and its role in the mechanisms involved by the virus to escape from medical tools of prevention and treatment.
Abstract: Embryo transfer is a globally executed technique which, when properly done, has both economic and sanitary advantages. International guidelines are available to prevent infection of the embryo with pathogens, both originating from the donor animals as from the environment. This manuscript describes the bacteria, viruses, protozoa, fungi and prions that are of major concern in the context of embryo transfer in cattle. In addition, the actual scientific knowledge on these pathogens is evaluated in terms of the current international and national guidelines and legislation.
Abstract: Molecular biology and technical advances in DNA recombination have ushered in a new era in vaccinology. This article examines the recent development of specific marker vaccines and examines the impact of their use on the diagnosis and prevention of major infectious diseases. Gene-deleted vaccines, DIVA strategies (differentiating infected from vaccinated animals) and similar methods have been successfully applied in the control and eradication of Aujeszky's disease, infectious bovine rhinotracheitis, classical swine fever, foot and mouth disease and, recently, avian influenza. The efficacy and performance of existing marker vaccines and their companion diagnostic tools (which should be assesed by an independent body) are discussed, as are the ways in which these tools are deployed by competent authorities. The limits and the advantages of the use of marker vaccines are carefully analysed in the light of practical experiences. Although these vaccines can limit the speed and the extent of virus dissemination and thus reduce the number of animals slaughtered, marker vaccines are no substitute for sanitary measures. Early detection and warning systems and the quick implementation of sanitary measures, including stamping out, remain key issues in the control of highly contagious diseases.
Abstract: Caprine herpesvirus 1 (CpHV-1) is responsible of systemic infection in neonatal kids as well as abortion and fertility disorders in adult goats. This virus is closely related to bovine herpesvirus 1 (BoHV-1) which causes infectious bovine rhinotracheitis. Glycoprotein D (gD) mediates important functions in alphaherpesviruses and is also a main immunogen. The sequence of CpHV-1 gD gene and the biochemical properties of its translation product were analyzed and compared to those of BoHV-1 and other alphaherpesviruses. A relatively high homology was found between CpHV-1 and BoHV-1 glycoproteins D amino acid sequences (similarity of 68.8%). Moreover, six cysteine residues are conserved by CpHV-1 gD and the other studied alphaherpesviruses. CpHV-1 gD has a molecular mass similar to BoHV-1 gD and contains complex N-linked oligosaccharides. In contrast to the BoHV-1 gD, CpHV-1 gD is expressed as a late protein. In spite of the observed differences which could influence its biological functions, CpHV-1 gD shares most characteristics with other alphaherpesviruses and especially BoHV-1.
Abstract: Rift valley fever (RVF) is an arboviral disease produced by a bunyavirus belonging to the genus Phlebovirus. Several species of Aedes and Culex are the vectors of this virus that affects sheep, goats, buffalos, cattle, camels and human beings. The human disease is well known, especially during periods of intense epizootic activity. The initial description of the disease dates back to 1930, when animals and human outbreaks appeared on a farm in Lake Naivasha, in the Great Rift Valley of Kenya. Until 2000, this disease was only described in Africa, and then outbreaks were also declared in the Kingdom of Saudi Arabia (2000-2001 and 2004) and in Yemen (2000-2001). Animal and human cases were recorded. This work presents a retrospective summary of the data collected on animal RVF cases during this epidemic in Yemen. Results from several RVF surveys were gathered from the Yemeni vet services and FAO experts. Geographical data (topographic maps and data freely available on internet) were used for the location of outbreaks. After cleaning and standardization of location names, all the data were introduced into a GIS database. The spatial distribution of outbreaks was then studied at two scales: at the national level and at a local scale in the particular area of Wadi Mawr in the Tihama plain, Western coast of Yemen.
Abstract: Vaccines used in control programmes of Bovine herpesvirus 1 (BoHV-1) utilize highly attenuated BoHV-1 strains marked by a deletion of the glycoprotein E (gE) gene. Since BoHV-1 recombinants are obtained at high frequency in experimentally coinfected cattle, the consequences of recombination on the virulence of gE-negative BoHV-1 were investigated. Thus, gE-negative BoHV-1 recombinants were generated in vitro from several virulent BoHV-1 and one mutant BoHV-1 deleted in the gC and gE genes. Four gE-negative recombinants were tested in the natural host. All the recombinants were more virulent than the gE-negative BoHV-1 vaccine and the gC- and gE-negative parental BoHV-1. The gE-negative recombinant isolated from a BoHV-1 field strain induced the highest severe clinical score. Latency and reactivation studies showed that three of the recombinants were reexcreted. Recombination can therefore restore virulence of gE-negative BoHV-1 by introducing the gE deletion into a different virulence background.
Abstract: Herpesviruses have mainly co-evolved with their hosts for millions of years. Consequently, different related host species may have been infected by various genetically related herpesviruses. Illustrating this concept, several ruminant alphaherpesviruses have been shown to form a cluster of viruses closely related to bovine herpesvirus 1 (BoHV-1): namely bovine herpesvirus 5, bubaline herpesvirus 1, caprine herpesvirus 1, cervid herpesviruses 1 and 2 and elk herpesvirus 1. These viruses share common antigenic properties and the serological relationships between them can be considered as a threat to BoHV-1 eradication programmes. BoHV-1 is a herpesvirus responsible for infectious bovine rhinotracheitis, which is a disease of major economic concern. In this article, the genetic properties of these ruminant alphaherpesviruses are reviewed on a comparative basis and the issue of interspecific recombination is assessed. The pathogenesis of these infections is described with emphasis on the host range and crossing of the host species barrier. Indeed, the non bovine ruminant species susceptible to these ruminant alphaherpesviruses may be potential BoHV-1 reservoirs. The differential diagnosis of these related infections is also discussed. In addition, available epidemiological data are used to assess the potential of cross-infection in ruminant populations. A better knowledge of these ruminant alphaherpesvirus infections is essential to successfully control infectious bovine rhinotracheitis.
Abstract: Herpesviruses are DNA viruses characterized by a low rate of nucleotide substitution. Therefore, other mechanisms must be involved to their evolution, like recombination that can be seen as an essential evolutionary driving force of these viruses. Recombination contributes to the long-term evolution of alphaherpesviruses. It acts also to continuously create new alphaherpesvirus strains. We have used bovine herpesvirus 1 to investigate recombination both within DNA concatemers in infected cells and in vitro and in vivo at the end of the lytic cycle. The following results have been obtained: (i) intramolecular recombination occurs at the level of concatemers and gives rise to genomic segment inversions; (ii) intraspecific recombination occurs frequently both in vitro and in vivo; (iii) interspecific recombination is possible and requires two highly genetically related viruses; (iv) only simultaneous or closely separated infections lead to the production of recombinant viruses; (v) recombination between wild-type and glycoprotein defective vaccine virus can produce a glycoprotein defective virus keeping part of the virulence of parental wild-type virus. Recombination, by exchanging genomic segments, may modify the virulence of alphaherpesviruses. It must be carefully assessed for the biosafety of antiviral therapy, alphaherpesvirus-based vectors and live attenuated vaccines.
Abstract: Taking into account the close antigenic relationship between bovine herpesvirus 1 (BoHV-1) and caprine herpesvirus 1 (CpHV-1), a live attenuated glycoprotein E (gE) negative BoHV-1 vaccine was assessed in goats with the aim to protect against CpHV-1 infection. Vaccine safety was evaluated by intranasal inoculation of two groups of goats with either a gE-negative BoHV-1 vaccine or a virulent BoHV-1. The length of viral excretion and the peak viral titre were reduced with the gE-negative vaccine. To assess the efficacy, two goats were inoculated intranasally twice 2 weeks apart with a gE-negative BoHV-1 vaccine. Four weeks later, immunised and control goats were challenged with CpHV-1. A 2 log(10) reduction in the peak viral titre was observed and the challenge virus excretion lasted 2 days more in immunised than in control goats. These data indicate the safety and the partial efficacy of a live attenuated gE-negative BoHV-1 vaccine intranasally administrated in goats.
Abstract: Intramolecular recombination is a frequent event during the replication cycle of bovine herpesvirus 1 (BoHV-1). Recombinant viruses frequently arise and survive in cattle after concomitant nasal infections with two BoHV-1 mutants. The consequences of this process, related to herpesvirus evolution, have to be assessed in the context of large use of live marker vaccines based on glycoprotein E (gE) gene deletion. In natural conditions, double nasal infections by vaccine and wild-type strains are likely to occur. This situation might generate virulent recombinant viruses inducing a serological response indistinguishable from the vaccine one. This question was addressed by generating in vitro BoHV-1 recombinants deleted in the gE gene from seven wild-type BoHV-1 strains and one mutant strain deleted in the genes encoding gC and gE. In vitro growth properties were assessed by virus production, one step growth kinetics and plaque size assay. Heterogeneity in the biological properties was shown among the investigated recombinant viruses. The results demonstrated that some recombinants, in spite of their gE minus phenotype, have biological characteristics close to wild-type BoHV-1.
Abstract: Bovine herpesvirus 4 (BoHV-4) has been isolated from cattle throughout the world. Interestingly, a survey of wild African buffaloes mainly from the Maasai Mara Game Reserve in Kenya revealed that 94% of the animals tested had anti-BoHV-4 antibodies [Rossiter, P.B., Gumm, I.D., Stagg, D.A., Conrad, P.A., Mukolwe, S., Davies, F.G., White, H., 1989. Isolation of bovine herpesvirus-3 from African buffaloes (Syncerus caffer). Res. Vet. Sci. 46, 337-343]. These authors also proposed that the serological antigenic relationship existing between BoHV-4 and alcelaphine herpesvirus 1 (AlHV-1) could confer to BoHV-4 infected buffaloes a protective immune response against lethal AlHV-1 infection. In the present study, we addressed two questions related to Rossiter et al. paper. Firstly, to investigate the role of the African buffalo as a natural host species of BoHV-4, the seroprevalence of anti-BoHV-4 antibodies was analysed in wild African buffaloes throughout eastern and southern Africa. A total of 400 sera was analysed using two complementary immunofluorescent assays. These analyses revealed that independently of their geographical origin, wild African buffaloes exhibit a seroprevalence of anti-BoHV-4 antibodies higher than 68%. This result is by far above the seroprevalence generally observed in cattle. Our data are discussed in the light of our recent phylogenetic study demonstrating that the BoHV-4 Bo17 gene has been acquired from a recent ancestor of the African buffalo. Secondly, we investigated the humoral antigenic relationship existing between BoHV-4 and AlHV-1. Our results demonstrate that among the antigens expressed in AlHV-1 infected cells, epitope(s) recognised by anti-BoHV-4 antibodies are exclusively nuclear, suggesting that the putative property of BoHV-4 to confer an immune protection against AlHV-1 relies on a cellular rather than on a humoral immune response.
Abstract: A retrospective epidemiological study (n = 7,875) of neurologically expressed disorders (NED) in ruminants before the onset of the bovine spongiform encephalopathy epidemic (years studied, 1980 to 1997) was carried out in Belgium. The archives of all veterinary laboratories and rabies and transmissible spongiform encephalopathy (TSE) epidemiosurveillance networks were consulted. For all species, a significantly higher number of NED with virological causes (rabies) was reported south of the Sambre-Meuse Valley. During the period 1992 to 1997, for which the data were complete, (i) the predicted annual incidence of NED varied significantly as a function of species and area (higher numbers in areas where rabies was present) but was always above 100 cases per million, and (ii) the mean incidence of suspected TSE cases and, among them, those investigated by histopathological examination varied significantly as a function of species and area. The positive predictive value of a presumptive clinical diagnosis of NED ranged from 0.13 (game) to 0.63 (sheep). Knowledge of the positive predictive value permits the definition of a reference point before certain actions (e.g., awareness and training campaigns) are undertaken. It also shows the usefulness of a systematic necropsy or complementary laboratory tests to establish an etiological diagnosis. TSE analysis of a small, targeted historical sampling (n = 48) permitted the confirmation of one case and uncovered another case of scrapie. The results of the present study help to develop and maintain the quality of the worldwide clinical epidemiological networks for TSE, especially in countries that in the past imported live animals, animal products, and feedstuffs from countries with TSE cases.
Abstract: Within the Herpesviridae family, Alphaherpesvirinae is an extensive subfamily which contains numerous mammalian and avian viruses. Given the low rate of herpesvirus nucleotide substitution, recombination can be seen as an essential evolutionary driving force although it is likely underestimated. Recombination in alphaherpesviruses is intimately linked to DNA replication. Both viral and cellular proteins participate in this recombination-dependent replication. The presence of inverted repeats in the alphaherpesvirus genomes allows segment inversion as a consequence of specific recombination between repeated sequences during DNA replication. High molecular weight intermediates of replication, called concatemers, are the site of early recombination events. The analysis of concatemers from cells coinfected by two distinguishable alphaherpesviruses provides an efficient tool to study recombination without the bias introduced by invisible or non-viable recombinants, and by dominance of a virus over recombinants.Intraspecific recombination frequently occurs between strains of the same alphaherpesvirus species. Interspecific recombination depends on enough sequence similarity to enable recombination between distinct alphaherpesvirus species. The most important prerequisite for successful recombination is coinfection of the individual host by different virus strains or species. Consequently the following factors affecting the distribution of different viruses to shared target cells need to be considered: dose of inoculated virus, time interval between inoculation of the first and the second virus, distance between the marker mutations, genetic homology, virulence and latency. Recombination, by exchanging genomic segments, may modify the virulence of alphaherpesviruses. It must be carefully assessed for the biosafety of antiviral therapy, alphaherpesvirus-based vectors and live attenuated vaccines.
Abstract: Canine herpesvirus-1 (CHV-1) is presumed to be enzootic in the dog population and is associated with reproductive disorders and neonatal mortality. To advise dog breeders towards an effective management of CHV-1 infected colonies, 27 breeding bitches were studied during one reproductive cycle in field conditions: the effect of cycle stage, kennel size, initial antibody titre, mating and gestation on serologic and viral excretion patterns was evaluated, while the association between reproductive disorders and CHV-1 antibody titres and viral excretion was also analysed. All initially seronegative bitches seroconverted, while 40% of the initially seropositive bitches became seronegative at one or two occasions. No difference in antibody patterns was observed between mated and unmated bitches. Of the mated bitches, 46% experienced infertility, foetal resorption or mummification. No difference in antibody patterns was observed depending on the occurrence of reproductive disorders even if a decrease in antibody titres during early or late-di-oestrus was often present. Significantly higher titres were observed at all cycle stages in large kennels. None of the vaginal and nasal samples or buffy coats tested positive for CHV-1 DNA. The mixed image of clinical and sub-clinical carriage in this study demonstrated CHV-1 has a complex and difficult to predict clinical behavior. Preventive management with vaccination of reproducing bitches in kennels with reproductive disorders should therefore be advised.
Abstract: An infectious cDNA clone of the hypervirulent bovine viral diarrhoea virus (BVDV) strain 890 (isolate 256) was produced by a streamlined PCR procedure. As compared to the published sequence of strain 890, the nucleotide sequencing of cloned cDNA corresponding to isolate 256 revealed several mutations seven of which were attributed to the cloning procedure. The infectious transcript was transfected into permissive cells and led to viral multiplication (AvrII+ strain). In vitro, viral titres reached by the parental strain exceed those of the AvrII+ strain by more than one order of magnitude. The latter was clearly less virulent to young calves as indicated by clinical, haematological and virological parameters. Thirty-four days after inoculation with AvrII+ strain, calves were challenged with the virulent parental strain. The animals were protected as compared to unvaccinated controls. Therefore, our approach led to the production of an attenuated strain with potential use as a vaccine strain and will be useful for studies of virulence determinants in BVDV-2.
Abstract: Serum samples of goats experiencing natural and experimental infections and/or reactivation of caprine herpesvirus 1 (CpHV.1) were analysed with neutralization and Western blotting (WB) tests. WB immunological patterns resulted differently and related to neutralizing titers. In serum samples having neutralizing titer 1:2-1:4, antibodies to two proteins of Mw of 150 and 34 kDa were present. Antibodies against several proteins, two of those being characterized by monoclonal antibodies as gB and gC, were visualized by WB in sera having titer > or = 1:8. The neutralizing antibody titers and the pattern of antibody reactivity were hypothesized to modulate the reactivation and re-excretion process of CpHV.1.
Abstract: The purpose of the present study was to identify a potential interference of bovine herpesvirus-1 (BoHV-1) with sperm-oocyte interactions during bovine in vitro fertilization. An inhibition of almost 70% of sperm-zona binding was observed when bovine cumulus-denuded oocytes were inseminated in the presence of 10(7) 50% tissue culture infective dose/ml BoHV-1. The inhibitory effect of BoHV-1 on sperm-zona binding was mediated by an interaction of the virus with spermatozoa, but not with oocytes. Treatment of spermatozoa with BoHV-1, however, did not affect sperm motility and acrosomal status. Antiserum against BoHV-1 prevented the virus-induced inhibition of sperm-zona binding, indicating that BoHV-1 itself affects the fertilization process. In order to investigate which BoHV-1 glycoprotein(s) are responsible for the virus-sperm interaction, BoHV-1 was treated with monoclonal antibodies against the viral glycoproteins gB, gC, gD and gH prior to insemination. Anti-gC completely prevented the inhibitory effect of BoHV-1 on sperm-zona binding, while anti-gD caused a reduction of this inhibition. Further evidence for the involvement of gC and gD in the virus-sperm interaction was provided by the fact that purified gC and gD decreased sperm-zona binding in a dose-dependent way with gC being more effective than gD. These results indicated that BoHV-1 inhibits bovine sperm-zona binding by interacting with spermatozoa. The binding of BoHV-1 to a spermatozoon is mediated by the viral glycoproteins gC and gD, and therefore seems to be comparable with the mechanisms of BoHV-1 attachment to its natural host cell.
Abstract: Bovine herpesvirus 1 glycoprotein D (gD) gene expression by recombinant replication defective human adenovirus type 5 (HAdV-5) was investigated in calves using indirect immunofluorescence microscopy (IIFM), confocal laser scanning microscopy (CLSM) and RT-PCR. One fold intranasal instillation of HAdV-5-expressing gD in the cattle upper respiratory tract showed a short term expression of at least 5 days, but not 10 days, limited only to epithelial cells localised in the epithelium of the nasal mucosa in one out of six calves. Observed limited gene transfer into well differentiated cattle airway epithelial cells must be taken into consideration in order to enhance transfection efficiency, and consequently the vaccine potential of this vector.
Abstract: Reporting of clinically suspected cattle is currently the most common method for detecting cases of bovine spongiform encephalopathy (BSE). Improvement of clinical diagnosis and decision-making remains crucial. A comparison of clinical patterns, consisting of 25 signs, was made between all 30 BSE cases, confirmed in Belgium before October 2002, and 272 suspected cases that were subsequently determined to be histologically, immunohistochemically, and scrapie-associated-fiber negative. Seasonality in reporting suspected cases was observed, with more cases being reported during wintertime when animals were kept indoors. The median duration of illness was 30 days. The 10 most relevant signs of BSE were kicking in the milking parlor, hypersensitivity to touch and/or sound, head shyness, panic-stricken response, reluctance to enter in the milking parlor, abnormal ear movement or carriage, increased alertness behavior, reduced milk yield, teeth grinding, and temperament change. Ataxia did not appear to be a specific sign of BSE. A classification and regression tree was constructed by using the following four features: age of the animal, year of birth, number of relevant BSE signs noted, and number of clinical signs, typical for listeriosis, noted. The model had a sensitivity of 100% and a specificity of 85%. This approach allows the use of an interactive decision-support tool, based entirely on odds ratios, a statistic independent of disease prevalence.
Abstract: Two strains of caprine herpesvirus type 1 (CpHV-1) were isolated after the experimental reactivation of two seropositive goats in Spain. Viral DNA from these isolates was compared with DNA from bovine herpesvirus type 1 and CpHV-1 reference strains by restriction endonuclease analysis. The two Spanish isolates were closely related but could easily be distinguished from each other and from the reference strains.
Abstract: The ability of two soluble formulations, namely chitosan and glycol chitosan, when used as an intranasal adjuvant, to improve the immunogenicity of an intranasal human adenovirus type 5 replication defective expressing bovine herpesvirus 1 (BoHV-1) glycoprotein D based vaccine, was investigated in cattle. Their adjuvant effects on immune response by increasing clinical and especially virological protection against an intranasal BoHV-1 challenge were then evaluated. The best virological protection was obtained in calves immunized with the vaccine vector adjuvanted with glycol chitosan which decreased the challenge BoHV-1 virus excretion titres by 0.5-1.5 log when compared to those obtained in calves immunized with the vaccine vector alone or adjuvanted with chitosan. A slight difference in clinical scores was observed in calves immunized with the adjuvanted vaccine vector compared to calves immunized with the vaccine vector alone. The obtained data suggest that the tested soluble formulation of glycol chitosan has promising potential use as an intranasal adjuvant for recombinant viral vector vaccines in cattle.
Abstract: We report an outbreak of gastroenteritis due to Norovirus in a care unit in a Belgian hospital involving thirty-three people. The origin of the outbreak was traced to one nursing assistant. The virus strain identified by reverse transcription polymerase chain reaction and electron microscopy belonged to the genogroup II.
Abstract: Homologous recombination between strains of the same alphaherpesvirus species occurs frequently both in vitro and in vivo. This process has been described between strains of herpes simplex virus type 1, herpes simplex virus type 2, pseudorabies virus, feline herpesvirus 1, varicella-zoster virus, and bovine herpesvirus 1 (BoHV-1). In vivo, the rise of recombinant viruses can be modulated by different factors, such as the dose of the inoculated viruses, the distance between inoculation sites, the time interval between inoculation of the first and the second virus, and the genes in which the mutations are located. The effect of the time interval between infections with two distinguishable BoHV-1 on recombination was studied in three ways: (i) recombination at the level of progeny viruses, (ii) interference induced by the first virus infection on beta-galactosidase gene expression of a superinfecting virus, and (iii) recombination at the level of concatemeric DNA. A time interval of 2 to 8 h between two successive infections allows the establishment of a barrier, which reduces or prevents any successful superinfection needed to generate recombinant viruses. The dramatic effect of the time interval on the rise of recombinant viruses is particularly important for the risk assessment of recombination between glycoprotein E-negative marker vaccine and field strains that could threaten BoHV-1 control and eradication programs.
Abstract: Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that has a worldwide distribution in the population of cattle. Many factors make human contamination by BoHV-4 likely to occur. In this study, we performed in vitro experiments to assess the risk and the consequences of human infection by BoHV-4. First, by using a recombinant BoHV-4 strain expressing enhanced green fluorescent protein under the control of the human cytomegalovirus immediate-early gene promoter, we tested 21 human cell lines for their sensitivity and their permissiveness to BoHV-4 infection. These experiments revealed that human cell lines from lymphoid and myeloid origins were resistant to infection, whereas epithelial cells, carcinoma cells, or adenocarcinoma cells isolated from various organs were sensitive but poorly permissive to BoHV-4 infection. Second, by using the HeLa cell line as a model of human cells sensitive but not permissive to BoHV-4 infection, we investigated the resistance of infected cells to apoptosis and the persistence of the infection through cellular divisions. The results obtained can be summarized as follows. (i) BoHV-4 nonpermissive infection of HeLa cells protects them against tumor necrosis factor alpha-induced apoptosis. (ii) BoHV-4 infection of HeLa cells persists in cell culture; however, the percentage of infected cells decreases with time due to erratic transmission of the viral genome through cell division. (iii) BoHV-4 infection has no effect on the rate of HeLa cell division. Altogether, these data suggest that BoHV-4 could infect humans. This study also stresses the importance of considering the insidious effects of nonpermissive infection when the biosafety of animal gammaherpesviruses for humans is being considered.
Abstract: The control of infectious bovine rhinotracheitis induced by bovine herpesvirus 1 (BoHV-1) requires sensitive and specific diagnostic assays. As BoHV-1 is antigenically and genetically related to four other alphaherpesviruses of ruminants-namely, BoHV-5, caprine herpesvirus 1 (CpHV-1), cervine herpesvirus 1 (CvHV-1) and CvHV-2-diagnostic tests able to discriminate BoHV-1 from these related viruses are needed to avoid misdiagnosis, especially because some of these viruses are able to cross the species barrier. In this study, murine monoclonal antibodies (MAbs) specific for BoHV-1, BoHV-5, CpHV-1, CvHV-1, and CvHV-2 were produced with the aim of setting up an immunofluorescence assay able to discriminate between these related herpesviruses. Produced MAbs were selected for their viral specificity by enzyme-linked immunosorbent assay and indirect immunofluorescence staining of virus-infected cells. Radioimmunoprecipitation characterization of the selected MAbs revealed that four of them are directed against glycoprotein C (gC) and one of them is directed against gD of these related viruses. The obtained results demonstrate that the antibodies produced allow an unambiguous discrimination of each of the four alphaherpesviruses related to BoHV-1.
Abstract: Homologous recombination between different species of alphaherpesviruses has been described between herpes simplex viruses 1 and 2 but has not yet been observed between other alphaherpesviruses. In the present study we chose to assess to what extent in vitro recombination can occur between members of a well-defined group of closely related viruses such as ruminant alphaherpesviruses. At 24 h after infection of epithelial bovine kidney cells with a double-deleted mutant of bovine herpesvirus 1 (BoHV-1) (containing green fluorescent protein and red fluorescent protein genes) and different ruminant alphaherpesviruses, four types of progeny viruses were detected and distinguished according to their phenotype. Frequent recombination events between identical or different strains of BoHV-1 were observed (up to 30%), whereas only two BoHV-1/BoHV-5 recombinants were identified, and no recombinants between BoHV-1 and less closely related caprine and cervine herpesviruses were detected. Restriction analysis of the genomes of the two BoHV-1/BoHV-5 recombinants showed different genetic backgrounds. One possessed a restriction pattern close to BoHV-1, whereas the other one was close to BoHV-5. This exhaustive analysis of each combination of coinfection in a unique situation of five closely related alphaherpesviruses revealed the importance of a high degree of genetic relatedness and similar parental virus growth kinetics for successful interspecific recombination.
Abstract: Herpesviruses are large DNA viruses, which possess a number of advantages as gene delivery vectors. These relate to an ability to package large DNA insertions and establish lifelong latent infections in which the viral genome exists as a stable episome in the nucleus. For gene therapy to become a potential future treatment option, biosafe therapeutically efficient gene transfer is a central, but more and more stringent requirement. This review highlights the progress in development of herpesvirus based vectors, describes their properties as wall as discusses the biosafety concerns that are associated with their use in gene therapy. Thought was also given to biosafety issues pertaining to design and production of herpesvirus vector systems in therapeutic gene delivery.
Abstract: During a field trial to evaluate the efficacy of repeated vaccinations with bovine herpesvirus type 1 (BHV-1) marker vaccines, a glycoprotein E (gE)-negative BHV-1 strain was isolated from the nasal secretions of two cows, eight months after vaccination with a gE-negative live-attenuated vaccine, initially given intranasally, then intramuscularly. The strain isolated was characterised using immunofluorescence, restriction analysis and PCR. All the techniques used identified the isolated virus as a gE-negative BHV-1 phenotypically and genotypically identical to the Za strain used as a control.
Abstract: A classification of neurological or neurologically expressed disorders that occur in Western European cattle aged 12 month and over has been established on the basis of aetiology, frequency and conditions of appearance, age and type of animals concerned and the main clinical signs observed. Neurologically expressed disorders have been classified according to different groups of causes: biological, non-biological and non-specific or unknown. Differential diagnosis of neurologically expressed disorders is an essential element in the clinical epidemiological surveillance of bovine spongiform encephalopathy. A growing number of aetiologies are described in the scientific literature. The identification and centralised management of neurological disorders will make it possible, one the one hand, to take account of the inherent variability in the clinical forms encountered and in the diagnostic approaches of the observers and, on the other hand, to identify new risk factors in order to control them.
Abstract: Recombination is thought to be an important source of genetic variation in herpesviruses. Several studies, performed in vitro or in vivo, detected recombinant viruses after the coinoculation of two distinguishable strains of the same herpesvirus species. However, none of these studies investigated the evolution of the relative proportions of parental versus recombinant progeny populations after coinoculation of the natural host, both during the excretion and the reexcretion period. In the present study, we address this by studying the infection of cattle with bovine herpesvirus 1 (BoHV-1). The recombination of two BoHV-1 mutants lacking either glycoprotein C (gC(-)/gE(+)) or E (gC(+)/gE(-)) was investigated after inoculation of cattle by the natural route of infection. The results demonstrated that (i) recombination is a frequent event in vivo since recombinants (gC(+)/gE(+) and gC(-)/gE(-)) were detected in all coinoculated calves, (ii) relative proportions of progeny populations evolved during the excretion period toward a situation where two populations (gC(+)/gE(+) and gC(-)/gE(+)) predominated without fully outcompeting the presence of the two other detected populations (gC(+)/gE(-) and gC(-)/gE(-)), and (iii) after reactivation from latency, no gC(+)/gE(-) and gC(-)/gE(-) progeny viruses were detected, although gC(+)/gE(-) mutants, when inoculated alone, were detected after reactivation treatment. In view of these data, the importance of gE in the biology of BoHV-1 infection and the role of recombination in herpesvirus evolution are discussed.
Abstract: Herpesvirus genomes are often characterized by the presence of direct and inverted repeats that delineate their grouping into six structural classes. Class D genomes consist of a long (L) segment and a short (S) segment. The latter is flanked by large inverted repeats. DNA replication produces concatemers of head-to-tail linked genomes that are cleaved into unit genomes during the process of packaging DNA into capsids. Packaged class D genomes are an equimolar mixture of two isomers in which S is in either of two orientations, presumably a consequence of homologous recombination between the inverted repeats. The L segment remains predominantly fixed in a prototype (P) orientation; however, low levels of genomes having inverted L (I(L)) segments have been reported for some class D herpesviruses. Inefficient formation of class D I(L) genomes has been attributed to infrequent L segment inversion, but recent detection of frequent inverted L segments in equine herpesvirus 1 concatemers [Virology 229 (1997) 415-420] suggests that the defect may be at the level of cleavage and packaging rather than inversion. In this study, the structures of virion and concatemeric DNA of another class D herpesvirus, bovine herpesvirus 1, were determined. Virion DNA contained low levels of I(L) genomes, whereas concatemeric DNA contained significant amounts of L segments in both P and I(L) orientations. However, concatemeric termini exhibited a preponderance of L termini derived from P isomers which was comparable to the preponderance of P genomes found in virion DNA. Thus, the defect in formation of I(L) genomes appears to lie at the level of concatemer cleavage. These results have important implications for the mechanisms by which herpesvirus DNA cleavage and packaging occur.
Abstract: Replication-defective human adenoviruses type 5 (HAd5) expressing the bovine herpesvirus type 1 (BHV-1) glycoprotein gC or gD under the control of the human cytomegalovirus immediate-early promoter/enhancer (AdCMVgC or AdCMVgD) or the 5' regulatory region of the human desmin gene (AdDESMgC or AdDESMgD) were generated. A preliminary experiment performed on rabbits showed that the intranasal administration of AdCMV elicited higher levels of BHV-1 neutralizing antibodies than the intramuscular administration of AdDESM. The obtained results allowed to select the replication-defective AdCMVgC and AdCMVgD for further assessment of their potential as a recombinant vaccine in cattle. Calves were injected intranasally twice 3 weeks apart with either AdCMVgC or AdCMVgD or a combination of these two recombinants or a commercially available live vaccine for comparison. The highest BHV-1 neutralizing antibody titres were obtained with AdCMVgD followed by the live vaccine and to a lower extent with the combination of the two recombinants (AdCMVgC+AdCMVgD). Calves were protected against intranasal BHV-1 challenge performed 3 weeks after the second immunization. In view of the obtained results, recombinant HAd5 may be developed as an intranasal vaccine vector in cattle administrated either alone or sequentially with non-human adenovirus-based vectors.
Abstract: The effects of the vaccination of neonatal calves with a glycoprotein E (gE)-negative bovine herpesvirus type 1 (BHV-1) were investigated in naïve and passively immunised calves either with the recommended dose or a 5-fold concentrated one. After inoculation (PI), all calves excreted the virus vaccine except three passively immunised calves inoculated with the lower titre. No antibody response could be detected in passively immunised calves, whatever the dose used, and they all became BHV-1 seronegative and remained so after dexamethasone treatment (PDT). Nevertheless, as shown by a gamma-interferon assay, all calves that excreted the vaccine PI developed a cell-mediated immune response and a booster response was observed PDT, suggesting viral reactivation. The vaccine virus was recovered PDT from nasal secretions in two calves and BHV-1 DNA were detected in trigeminal ganglia from five calves belonging to all inoculated groups. The results show that the BHV-1 gE-negative vaccine can establish latency not only in naïve but also in passively immunised neonatal calves after a single intranasal inoculation. Moreover, this study shows for the first time that the gE-negative vaccine, when used in passively immunised calves, can lead to seronegative vaccine virus carriers.
Abstract: This study was conducted to investigate the glycoprotein E (gE) antibody response raised after inoculation with a low infectious dose of bovine herpesvirus 1 (BHV-1) in six calves possessing high levels of passive immunity from cows repeatedly vaccinated with gE deleted marker vaccine. Four out of the six calves developed gE antibodies 3-5 weeks after infection, whereas the two other ones remained seronegative to gE. After 5 months of infection, the six calves were treated with dexamethasone. Virus was only re-excreted by the four calves which previously seroconverted against gE. The two other calves became seronegative against BHV-1, 30-32 weeks after infection. A second dexamethasone treatment performed 11 months after infection failed to demonstrate a latent infection in these two calves. Moreover, the lack of identification of a cell-mediated immune response, after the two dexamethasone treatments, and the failure to detect BHV-1 DNA sequences in trigeminal ganglia strongly suggest that these two calves were not latently infected. In conclusion, the presence of high levels of maternal immunity lacking gE antibodies does not prevent latency after infection with a low titre of BHV-1. Moreover, latency is associated with a serological response to gE. These results confirm that the gE deletion is a good marker to identify young calves latently infected with a field virus.
Abstract: Homologous recombination occurs frequently between strains of the same alphaherpesvirus species. Studies of this phenomenon require techniques that can differentiate parental strains from putative recombinant progeny viruses. Usually, progeny viruses generated by co-infection of two distinguishable parental strains are first cloned by selection of a single plaque and then characterised by PCR. An assay designed to investigate recombination between two bovine herpesvirus 1 (BHV-1) strains lacking either the glycoprotein gC or gE ORF is described. A PCR assay was developed in which a single step co-amplifies both BHV-1 glycoprotein-encoding sequences. Because the usual procedure for virus isolation, viral plaque picking, can lead to polyclonal virus preparations, a PCR protocol alone does not differentiate between samples containing recombinant viruses (gC+/gE+) and those containing a mixture of both single deleted parental strains (gC-/gE+ and gC+/gE-), and false positives resulting from recombination could occur. To reduce this possibility, double-label immunofluorescence staining of isolated plaques was developed, which coupled with PCR, allows straightforward discrimination between parental strains and progeny recombinant viruses. This assay will be useful for further studies of recombination, especially those evaluating the potential emergence of recombinants between BHV-1 marker vaccine and wildtype strains.
Abstract: Myxomatosis is a major viral disease of the European rabbit (Oryctolagus cuniculus). Two forms of the disease (nodular and amyxomatous) exist. The clinical diagnosis of the nodular form is easily performed on the basis of typical skin lesions whereas that of amyxomatous forms must be based on virus isolation or detection of specific antibodies to myxoma virus (MV). The seroprevalence of MV was studied between March 1998 and February 1999 in 16 farms from three European countries considered free of myxomatosis on the basis of the absence of typical clinical signs. MV antibodies were detected by enzyme-linked immunosorbent assay (ELISA) (sensitivity 100%, specificity 90%) in all 16 farms; the seroprevalences corrected for test inaccuracy (95% confidence interval) were 55+/-7.7% and 37+/-6.1% for does and broilers, respectively. The association between herd sizes, types of rabbitries, and presence of recurrent respiratory or digestive troubles and seroprevalence of MV antibodies was tested in logistic multiple regressions. In all models, the seroprevalence of MV antibodies was significantly higher in herds (does and broilers) with recurrent respiratory or digestive troubles than in herds without these problems. The seroprevalence was also higher in herds (does and broilers) where animals were housed totally or partially in outdoors rabbitries than in totally enclosed rabbitries. The effect of herd sizes on the presence of MV antibodies was the same in does and broilers; intermediate sizes were at lower risk than the smaller and larger ones.
Abstract: This study was conducted to compare the pathogenesis of acute and latent infections with closely related bovine herpesvirus types 1 (BHV-1) and 5 (BHV-5) in their natural host. Two groups of eight calves were inoculated intranasally with BHV-1 or BHV-5. Although BHV-1 and BHV-5 similarly replicate in the nasal mucosa after inoculation, both viruses differ markedly in their ability to cause disease, BHV-5 being responsible of some fatal encephalitis while BHV-1 inducing rhinotracheitis. Virus isolation and immunohistochemistry demonstrated that BHV-5 replicates extensively in neurons of the central nervous system (CNS) and in respiratory cells of lungs, tracheal and nasal mucosae. Invasion of the CNS likely occurs through the trigeminal and olfactory pathways. Both groups developed cross-neutralising antibodies during this experiment suggesting partial clinical cross-protection afforded by the two infections. Three months after primary infection, experimental reactivation showed that BHV-5 was able to establish latency in the trigeminal ganglia but also the CNS of surviving calves. Moreover, laboratory findings suggested that BHV-5 could also persist in the tracheal and nasal mucosae. These results indicate that, after primary infection, BHV-1 and BHV-5 displayed similar biological features and consequently need to be considered together for the control of BHV-1 infection.
Abstract: The beta-1,6-N-acetylglucosaminyltransferase (beta1,6GnT) gene family encodes enzymes playing crucial roles in glycan synthesis. Important changes in beta1,6GnT expression are observed during development, oncogenesis, and immunodeficiency. The most characterized beta1,6GnTs in this gene family are the human (h) C2GnT-L and h-IGnT, which have core 2 [Galbeta1-->3(GlcNAcbeta1-->6)GalNAc] and I branching [GlcNAcbeta1-->3(GlcNAcbeta1-->6)Gal] activities, respectively. Recently, h-C2GnT-M was shown to be unique in forming core 2, core 4 [GlcNAcbeta1-->3(GlcNAcbeta1-->6)GalNAc], and I structures. To date, the beta1,6GnT gene family has been characterized only in mammals. Here, we describe that bovine herpesvirus type 4 (BHV-4) encodes a beta1,6GnT expressed during viral replication and exhibiting all of the core 2, core 4, and I branching activities. Sequencing of the BHV-4 genome revealed an ORF, hereafter called BORFF3-4, encoding a protein (pBORFF3-4) exhibiting 81.1%, 50.7%, and 36.6% amino acid identity with h-C2GnT-M, h-C2GnT-L, and h-IGnT, respectively. Reverse transcriptase-PCR analysis revealed that BORFF3-4 is expressed during BHV-4 replication. Expression of BORFF3-4 in Chinese hamster ovary cells directed the expression of core 2 branched oligosaccharides and I antigenic structures on the cell surface. Moreover, a soluble form of pBORFF3-4 had core 4 branching activity in addition to core 2 and I branching activities. Finally, infection of a C2GnT-negative cell line with BHV-4 induced expression of core 2 branched oligosaccharides. This study extends the beta1,6GnT gene family to a viral gene and provides a model to study the biological functions of a beta1,6GnT in the context of viral infection.
Abstract: We investigated the excretion of either a glycoprotein E (gE)-negative bovine herpesvirus type 1 (BHV1) vaccine strain or a conventional modified-live vaccine strain in both naïve and passively immunised calves. The replication of gE-negative strain was considerably reduced in the maternally immunised calves, in comparison with the non-immune calves. On the other hand, the excretion of the gE-positive conventional vaccine strain was not reduced and even seemed to be prolonged in the presence of maternal antibodies. These results suggest that BHV1 gE may play a role in virus survival in the presence of antibodies.
Abstract: The consequences of the vaccination of neonatal calves with the widely used live-attenuated temperature-sensitive (ts) bovine herpesvirus type 1 (BHV-1) were investigated. The ts strain established acute and latent infections in all vaccinated calves either with or without passive immunity. Four of seven calves vaccinated under passive immunity became clearly BHV-1 seronegative by different serological tests, as did uninfected control calves after the disappearance of maternal antibodies, and they remained so for long periods. A cell-mediated immune response was detected by a BHV-1 gamma interferon assay, but this test failed to detect the seronegative latent carriers (SNLCs). While they are not detected, SNLCs represent a threat for BHV-1-free herds or countries. This study demonstrates that SNLCs can be easily obtained by inoculation with a live-attenuated BHV-1 under passive immunity and that latent carrier animals without any antibody do exist. Consequently, this situation could represent a good model to experimentally produce SNLCs.
Abstract: The presence of maternally derived antibodies can interfere with the development of an active antibody response to antigen. Infection of seven passively immunized young calves with a virulent strain of bovine herpesvirus type 1 (BHV-1) was performed to determine whether they could become seronegative after the disappearance of maternal antibodies while latently infected with BHV-1. Four uninfected calves were controls. All calves were monitored serologically for 13 to 18 months. In addition, the development of a cell-mediated immune response was assessed by an in vitro antigen-specific gamma interferon (IFN-gamma) production assay. All calves had positive IFN-gamma responses as early as 7 days until at least 10 weeks after infection. However, no antibody rise was observed after infection in the three calves with the highest titers of maternal antibodies. One of the three became seronegative by virus neutralization test at 7 months of age like the control animals. This calf presented negative IFN-gamma results at the same time and was classified seronegative by enzyme-linked immunosorbent assay at around 10 months of age. This calf was latently infected, as proven by virus reexcretion after dexamethasone treatment at the end of the experiment. In conclusion, this study demonstrated that BHV-1-seronegative latent carriers can be obtained experimentally. In addition, the IFN-gamma assay was able to discriminate calves possessing only passively acquired antibodies from those latently infected by BHV-1, but it could not detect seronegative latent carriers. The failure to easily detect such animals presents an epidemiological threat for the control of BHV-1 infection.
Abstract: Bovine herpesvirus type 5 (BHV-5) is the causative agent of a fatal meningo-encephalitis in calves and is closely related to BHV-1 which causes infectious bovine rhinotracheitis. The gene encoding BHV-5 glycoprotein gH was sequenced. A high degree of conservation was found between BHV-1 and BHV-5 deduced gH amino acid sequences (86. 4%), which is also observed for all alphaherpesvirus gH sequences. Transcriptional analysis revealed a 3.1 kb mRNA as the specific gH transcript which was detected 2 h post-infection (p.i.). Twelve out of twenty-one MAbs directed against BHV-1 gH immunoprecipitated a 108-110 kDa glycoprotein, which was then designated BHV-5 gH. Synthesis and intracellular processing of BHV- 5 gH was analysed in infected MDBK cells using gH cross-reacting MAbs. Glycoprotein gH was expressed as a beta-gamma protein, detected by radioimmunoprecipitation as early as 3 h p.i. Glycosylation studies indicated that BHV-5 gH contains N-linked carbohydrates which are essential for the recognition of the protein by the MAbs. This suggests that N-linked glycans are involved in protein folding or are targets for the gH cross-reacting MAbs. Plaque- reduction neutralization assays showed that at least one BHV-1 gH antigenic domain is lacking in BHV-5 which may possibly relate to in vivo differences in virus tropism.
Abstract: In the context of infectious bovine rhinotracheitis (IBR) control programmes using glycoprotein E (gE) deleted marker vaccines, a PCR assay was developed to allow the genotypic differentiation between wildtype bovine herpesvirus type 1 (BoHV-1) and gE negative strains. This assay is based on the PCR amplification of a 281 bp DNA fragment within the gE gene. The specificity of the amplification was confirmed by restriction endonuclease analysis and nucleotide sequencing of the PCR product. Its ability to determine the gE genotype of BoHV-1 strains was demonstrated on isolates coming from 20 experimental calves infected with four different BoHV-1 strains. This PCR assay may be a useful tool for monitoring the spread of live marker vaccine and the gE genotype of viral field isolates.
Abstract: Cells infected with the wild-type (WT) strain of channel catfish virus (CCV) secreted a glycoprotein with an apparent molecular mass (MM) superior to 200 kDa into the culture medium. This protein, designated gp250, was the sole viral glycoprotein detected in the culture medium after [3H]mannose labeling of the infected cells. When cells were infected with the attenuated V60 strain, a glycoprotein of 135 kDa (designated gp135) was detected instead of gp250. Because WT gene 50 is predicted to encode a secreted, mucin-type glycoprotein, we expressed this gene transiently and detected a glycoprotein of the same apparent MM as gp250 in the culture medium of transfected catfish cells. The increased mobility in SDS-PAGE of the secreted V60 glycoprotein correlated with the presence of a major deletion in V60 gene 50. Therefore, we concluded that gp250 in the WT and gp135 in the V60 strains are both likely encoded by gene 50. An important shift in the relative mobility of gp250 in SDS-PAGE was observed after tunicamycin treatment of infected cells labeled with [3H]glucosamine, confirming the presence of N-linked sugars on gp250. We observed variations in the size of PCR products derived from gene 50 amplification in three different field isolates. Such genetic variations are a characteristic feature of mucin genes and are linked to crossing-over events between internal repeated sequences, such as those present in gene 50.
Abstract: This study was conducted to determine whether young calves with maternal antibodies against bovine herpesvirus type 1 (BHV-1) but without antibodies against glycoprotein E (gE) can produce an active antibody response to gE after a BHV-1 infection. Five calves received at birth colostrum from gE-seronegative cows which had been vaccinated two or three times with an inactivated BHV-1, gE-deleted marker vaccine. After inoculation with a wild-type virulent strain of BHV-1, all the passively immunised gE-negative calves shed virus in large amounts in their nasal secretions. All the calves seroconverted to gE within two to four weeks after inoculation and then had high levels of gE antibodies for at least four months. The development of an active cell-mediated immune response was also detected by in vitro BHV-1-specific interferon-gamma assays. All the calves were latently infected, because one of them re-excreted the virus spontaneously and the other four did so after being treated with dexamethasone. The results showed that under the conditions of this work the gE-negative marker could also distinguish between passively immunised and latently infected calves.
Abstract: Bovine herpesvirus type 1 (BHV-1) glycoprotein H (gH) is a structural component of the virion which forms a complex with glycoprotein gL. To study the role of BHV-1 gH in the virus infectious cycle, a gH null mutant was constructed in which the gH coding sequences were deleted and replaced by the Escherichia coli lacZ cassette. The BHV-1 gH null mutant was propagated in trans-complementing MDBK cells, stably transfected with plasmid pMEP4 containing the BHV-1 gH gene under the control of the inducible mouse metallothionein promoter. Experiments with the BHV-1 gH null mutant showed that gH is essential in the infectious cycle of the virus and is specifically involved in virus entry and cell-to-cell spread. The lack of infectivity of virions devoid of gH is not due to a defect in attachment. Moreover, PEG-induced fusion of virions to target cells provides evidence that BHV-1 gH is required for virion penetration.
Abstract: OBJECTIVE: To compare cellular and humoral immune responses of beef (Belgian White and Blue [BWB]) and dairy (Friesian-Holstein [FH]) cattle to Psoroptes ovis infestation and to determine whether P ovis infestation impaired immune responses to infectious bovine rhinotracheitis virus (IBR) vaccine or an immunogenic protein (keyhole-limpet hemocyanin [KLH]). ANIMALS: 19 BWB and 6 FH 1-year-old calves. PROCEDURE: 2 trials were performed. In each trial, 7 (trial 1) or 6 (trial 2) BWB calves and 3 FH calves were experimentally infested with P ovis and 3 BWB calves were maintained as uninfested controls. Animals were inoculated with KLH and IBR virus vaccine twice; 3 BWB calves in each trial were treated with ivermectin. Serum antibody responses to KLH, IBR virus, and P ovis were measured by use of ELISA. A lymphocyte transformation assay was used to determine nonspecific responses to 3 mitogens and specific lymphocyte reactivity to P ovis antigen. RESULTS: In each trial, 3 BWB and 3 FH calves developed clinical signs of psoroptic mange and mites could be recovered. Infested and control animals developed similar antibody titers to KLH and IBR virus. Antibodies to P ovis were detected early in some infested calves, and this was correlated with a marked cell-mediated immune response. Lymphocyte responsiveness to the 3 mitogens was not significantly different among groups. CONCLUSIONS: In these calves, infestation with P ovis induced a marked humoral and cell-mediated immune response. Immunosuppression was not evident.
Abstract: Within the framework of developing a marker vaccine against bovine herpesvirus 1 (BHV1), several mutants with deletions in non-essential glycoprotein genes were constructed. Glycoprotein gC, gG, gI and gE single deletion mutants, a gI/gE double deletion mutant and a gE frame-shift mutant were made. The virulence and immunogenicity of these mutants were evaluated in specific-pathogen-free calves. Except for the gC deletion mutant, all mutants were significantly less virulent than the parental wild-type (wt) BHV1 strain Lam. The virulence of the gI and the gI-/gE- mutants was almost completely reduced. Upon challenge infection, the calves of the control group became severely ill, whereas all other calves remained healthy. The reduction of the virus shedding after challenge infection was related to the virulence of the strain of primary inoculation. Virus shedding was almost completely reduced in calves first inoculated with Lam-wt or with gC- and the least reduced in calves inoculated with gI- or gI-/gE-. Six weeks after challenge, all calves were treated with dexamethasone to study whether mutant or challenge virus or both could be reactivated. The gC- and the gG- mutants were reactivated, whereas none of the other mutants were reisolated. Reactivation of challenge virus was reduced in all calves inoculated with mutant viruses. The gC deletion mutant was too virulent and the gI and the gI/gE deletion mutants were the least immunogenic, but based on residual virulence and immunogenicity, both the gG and the gE deletion mutants are candidates for incorporation in live BHV1 vaccines. However, it also depends on the kinetics of the anti-gG and anti-gE antibody response after wild-type virus infection, whether these deletion mutants are really suitable to be incorporated in a marker vaccine.
Abstract: Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in bovine peripheral blood mononuclear cells and B-lymphoma cells. Using a BHV-1 glycoprotein H null mutant, we have demonstrated that although penetration of BHV-1 is not required, attachment of BHV-1 viral particles is essential for the induction of apoptosis.
Abstract: The prevalence of cattle seropositive to bovine herpesvirus-4 (BHV-4) is high in Belgium. In Belgian farms, clinical signs associated with BHV-4 infection essentially involve the genital tract and consist mainly of postpartum metritis or metroperitonitis. The role of BHV-4 in abortion has not been definitively demonstrated but epidemiological and experimental facts suggest its involvement. A seroepidemiological investigation was therefore conducted as a case-control study to compare the seroprevalences of BHV-4 infections in the aborted-cow population and in a randomly selected control group in the province of Liège (Belgium). The seroprevalence (17.2%) in aborted cows was significantly higher than that of the control group (10.0%). The odds ratio (OR) was 1.87 (1.06 < 3.30). BHV-4 infection is therefore considered as a risk factor for abortion in cows.
Abstract: This study reports that in bovine herpesvirus 4, glycoprotein B (gB) is a heterodimer and a major component of the virion, unlike gBs of Epstein-Barr virus (gp110) and murine gammaherpesvirus 68, two other gammaherpesviruses. These are new characteristics with regard to the general features of gB in the Gammaherpesvirinae subfamily.
Abstract: Genes encoding glycoprotein gH and gL homologues were localized in the genome of the gamma-herpesvirus bovine herpesvirus-4 (BHV-4). Both genes were sequenced and glutathione S-transferase fusion proteins were produced and used to immunize rabbits against the translation products of the two genes. The anti-gH serum recognized a protein with an apparent molecular mass (MM) of 110 kDa both in infected cells and in virions. This protein was sensitive to endo-beta-N-acetylglucosaminase-H (endoH) and endoglycosidase F-N-glycosidase F (endoF-PNGaseF) digestion. A protein with the same relative mobility was immunoprecipitated from infected cells radiolabelled with [3H]glucosamine which confirmed that this product (gp110), now designated BHV-4 gH, was glycosylated. Western blotting with the anti-gL serum detected in infected cells a product with an apparent MM ranging from 31-35 kDa and diffusely migrating protein species ranging from 45-65 kDa. Tunicamycin, monensin, endoH or endoF-PNGaseF treatments showed that both the 31-35 kDa and the 45-65 kDa proteins were glycosylated, gp31-35 being a precursor of the 45-65 kDa glycoprotein species. In radioimmunoprecipitation assays, the anti-gL serum immunoprecipitated from infected cells two glycosylated proteins with apparent MMs of 31-35 kDa (gp31-35) and 45-55 kDa (gp45-55). However a third glycoprotein, gp110, was also immunoprecipitated together with gp31-35 and gp45-55. gp110 and gp45-55 were subsequently confirmed to be virion glycoproteins corresponding to mature forms of BHV-4 gH and gL respectively. In addition, the present study clearly demonstrated complex formation between BHV-4 gH and gL both in virions and in infected cells.
Abstract: The nucleotide sequence of a 10.5 kb region (map position 0.332 to 0.410) of bovine herpesvirus type 1 (BHV-1) was determined. This region contained three open reading frames (ORFs) homologous to herpes simplex virus DNA polymerase catalytic subunit (DNApol, UL30), major DNA-binding protein (MDBP, UL29) and ICP18.5 assembly protein (ICP18.5, UL28). The BHV-1 DNApol. MDBP and ICP18.5 ORFs were 1246, 1203 and 826 amino acids long with a calculated molecular mass of 134.2 kDa, 124.4 kDa and 86.9 kDa, respectively. They showed a high homology with alphaherpesvirus homologs despite large differences in the G + C content of the UL30-UL28 segment ranging from 44.4% for varicella zoster virus to 71.5% for BHV-1. Particularly well conserved among Alphaherpesvirinae are the putative functional domains of the DNApol and MDBP proteins which are discussed. Phylogenetic analysis revealed that BHV-1 clustered in the Varicellovirus genus with the animal D-type viruses. In this group, the BHV-1 position was shown to vary according to the investigated genes. Indeed, pseudorabies virus clustered with BHV-1 in the DNApol tree but with equine herpesvirus 1 in the ICP18.5 tree.
Abstract: Mutant viruses with deletions in genes encoding non-essential glycoproteins are considered as promising bovine herpesvirus 1 (BHV1) vaccine candidates. The present study compared the influence of various gene deletions (gC, gE, gI, gG) on the induction of cell-mediated immune responses against the virus. The highest BHV1 specific lymphoproliferative response was observed in the group of calves inoculated with the gC- mutant. However, in all groups of inoculated calves, limiting dilution analysis showed marked individual variability in the number of BHV1 specific T lymphocytes that were stimulated. The same animals were then challenged with wild-type BHV1. In these animals, limiting dilution analysis did not reveal gE, gI nor gG as a major T lymphocyte antigen. However, further analysis suggested the T cell antigenicity of gE in a low number of BHV1 hyperimmunized calves. Stimulation of MHC unrestricted cytotoxicity was also evaluated after inoculation with the various deletion mutants. Cytotoxicity in gC- inoculated calves was as high as in BHV1 inoculated calves. In conclusion, among the BHV1 deletion mutants that were tested, the gC- mutant stimulated the best cell-mediated immune responses.
Abstract: Bovine herpesvirus 4 (BHV-4) belongs to the gammaherpesvirinae subfamily. Although the whole sequence of BHV-4 genome is not known it was possible, based on random sequencing, to assume that its genomic organization consists of genes clustered in blocks whose orientation and location in the genome are conserved within a herpesvirus subfamily. Between these blocks lie genes which are specific to either a particular virus or a virus subfamily. BHV-4 genome consists of 5 gene blocks conserved among the gammaherpesviruses and particularly within the Epstein-Barr virus (EBV) and the herpesvirus saimiri (HVS) genomes. Analysis of the regions located outside the gene blocks showed the presence of 12 open reading frames (ORFs). Protein database comparisons showed that no ORF translation products were similar to proteins encoded by alpha- or beta-herpesviruses. Nevertheless, 5 ORFs were homologous in amino acid sequences to proteins encoded by HVS and one was similar to a protein encoded by both HVS and EBV. On the basis of the molecular data BHV-4 is more closely related to HVS than to EBV. Genes homologous to cellular genes have been described in both HVS and EBV genomes. No genes homologous to presently sequenced cellular genes were found among those found in the BHV-4 genome to date.
Abstract: This study was conducted to evaluate the suitability of the rabbit as a model for bovine, herpesvirus 5 (BHV-5) acute infection. In a preliminary experiment, a total of 24 one-month old New Zealand white rabbits were inoculated with BHV-5 or bovine herpesvirus 1 (BHV-1) by the intraconjunctival, intracerebral or intranasal routes. BHV-5 or BHV-1 inoculated in the conjunctiva induced virus proliferation in the eye mucosae and the nasal cavity of rabbits without meningo-encephalitis. On the other hand, only BHV-5 infection by intranasal or intracerebral routes produced a fatal meningo-encephalitis. The intranasal route was used in a further experiment for the establishment of a rabbit model for BHV-5 infection. A total of 45 rabbits were inoculated intranasally with BHV-5 or BHV-1. The results showed that intranasal inoculation of BHV-5 strain N569 in rabbits was followed by the development of a lethal meningo-encephalitis for 66% of rabbits while all BHV-1 infected rabbits remained healthy throughout this experiment (28 days). Analysis between the mortalities of rabbits infected with BHV-5 and BHV-1 were highly significant (p < 0.001). The presence of BHV-5 in the central nervous system (CNS) was confirmed by virus isolation (essentially the cerebrum, midbrain and pons) and by immunohistochemical staining of BHV-5 antigen (essentially in the neurons of the cerebrum) only in BHV-5 infected rabbits showing clinical signs of meningo-encephalitis. The findings obtained confirmed the suitability of a rabbit model for the establishment of BHV-5 neurological acute infection and also as a valuable tool for the comparative study of BHV-5 and BHV-1 neuropathogenicity.
Abstract: The glycoproteins of bovine herpesvirus 1 (BHV-1) play important roles in the interactions between virions and target cells. A 108 kDa glycoprotein, designated gII or gp 108, has been identified by two different panels of monoclonal antibodies. The gII- and gp 108-specific monoclonal antibodies were shown to react with the same protein, which was identified by N-terminal sequencing as the homologue of herpes simplex virus type 1 (HSV-1) gH. When BHV-1 gH was purified by immunoadsorbent chromatography, gL was co-purified. The gH-gL complex induced the production of antibodies that neutralized virus infectivity and inhibited virus penetration. Affinity-purified gH-gL did prevent penetration, but not attachment of BHV-1, which suggests that the gH-gL complex is essential for penetration of BHV-1 into susceptible cells.
Abstract: The cell-mediated immunity (CMI) following bovine herpesvirus 4 (BHV4) infection has been poorly investigated in cattle. The in vivo response measured by a delayed type of hypersensitivity (DTH) assay has been reported to be positive in only few animals showing serological evidences of BHV4 infection. We have investigated the CMI following BHV4 infection by an in vitro antigen-specific interferon gamma (IFN-gamma) release assay, as an indicator of an actively acquired immunity to BHV4. Our preliminary results using a partially purified antigen suggest that there was a measurable CMI in 75 out of 168 animals (44.4%) originating from a farm with a clinical history and serological evidences (76.3% seropositivity) of BHV4 infection. If the results of serological tests and BHV4 IFN-gamma test are interpreted in parallel, 81.5% of the animals are classified positive, demonstrating the complementarity of these tests. The specificity of the BHV4 IFN-gamma test was supported by the absence of a measurable CMI in 41 animals originating from a farm with no clinical history or serological evidence of BHV4 infection. In an allied study, we developed a bovine herpesvirus 1 (BHV1) IFN-gamma test. This allowed us to measure the antigen specific IFN-gamma release after stimulation with a mixture of BHV1 and BHV4 antigens. Animals that were classified negative by the BHV4 IFN-gamma test and by the BHV1 IFN-gamma test, were classified negative after stimulation with a mixture of both antigens. Animals that were classified positive by the BHV4 IFN-gamma test or the BHV1 IFN-gamma test, were classified positive after stimulation with a mixture of both antigens. Taken together these results suggest that the in vitro assessment of the CMI after BHV4 infection should be further investigated as a specific and valuable alternative to the DTH assay.
Abstract: This paper focuses on the structure and functions of bovine herpesvirus 1 minor glycoproteins gH, gE, gG and gp42. It reviews the progress which has been made in their identification and characterization, in the study of their temporal expression and processing in infected cells, and finally in the understanding of their biological activities. In addition, aspects discussed include a comparison with two other alphaherpesviruses, namely herpes simplex virus and pseudorabies virus.
Abstract: Bovine herpesvirus 4 (BHV-4) DNA sequences located outside the gene blocks conserved among the gammaherpesviruses BHV-4, herpesvirus saimiri (HVS) and Epstein-Barr virus (EBV) were analysed. Twelve potential open reading frames (ORFs) were found. Protein database comparisons showed that no ORF translation products were similar to proteins encoded by alpha- or betaherpesviruses. Nevertheless, six of the ORFs were homologous in amino acid sequences to proteins encoded by HVS but apparently not to those encoded by EBV. Furthermore, the location and orientation of these six ORFs in the BHV-4 genome were similar to the corresponding ORFs in the HVS genome. No genes homologous to known cellular genes were found in the BHV-4 genome; this feature is the major difference between the BHV-4 and HVS genomes with regards to the overall gene content.
Abstract: Because several observations have suggested that replication of the gammaherpesvirus bovine herpesvirus 4 (BHV-4) is influenced by the physiological state of the host cell, a study was carried out to determine the relationship between BHV-4 infection and the cell cycle. The temporal expression of BHV-4 late (L) proteins in unsynchronized cell cultures was first investigated by flow cytometry. Interestingly, L protein expression occurred in a limited number of cells infected with a high multiplicity of infection, and a reciprocal correlation between the percentage of positive cells and the cell density at the time of infection was demonstrated. Moreover, the finding that a BHV-4 early-late protein was expressed in nearly all the cells suggested that a blockage in the viral replication cycle occurred in some infected cells at the stage of viral DNA synthesis or L protein expression. Because this blockage could be the consequence of the dependence of one or both of these events on the cell cycle, they were investigated after infection of synchronized cell cultures. The following findings were made. (i) Cell transition through the S phase quantitatively increased the rate of BHV-4 DNA replication. (ii) BHV-4 DNA synthesis could not be detected in cells arrested in G0. (iii) Synchronization of MDBK cells with Lovastatin before infection increased the percentage of cells expressing L proteins. (iv) In contrast, infection of cells arrested in G0 led to few positive cells. Taken together these results showed that BHV-4 DNA replication and consequently the expression of L proteins are dependent on the S phase of the cell cycle. This dependence could be of importance for several biological properties of BHV-4 infection in vitro and might have implications for the biology of the virus in vivo.
Abstract: The translation product of the bovine herpesvirus-1 (BHV-1) gH gene was identified and characterized. Synthetic peptides were used to generate specific antisera and a glycoprotein of 108K was precipitated by one of the antisera. Cross-immunoprecipitations with monoclonal antibodies to BHV-1 glycoprotein gp108 and the anti-gH peptide antiserum demonstrated that gp108 is the translation product of the gH open reading frame. Glycoprotein gH synthesis and intracellular processing was analyzed in infected Madin-Darby bovine kidney cells using anti-gp 108 monoclonal antibodies. Glycoprotein gH is expressed as a beta-gamma protein and could be detected by radioimmunoprecipitation as early as 2 hr postinfection. Cotranslational N-glycosylation of gH is essential for the recognition by monoclonal antibodies, suggesting that N-linked glycans are involved in protein folding or that they are targets for most of monoclonal antibodies used in this study.
Abstract: Sensitivity, specificity and predictive values of an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against bovine leukaemia virus (BLV) were evaluated using a representative sample of 145 serum pools, comprising from 3 to 48 individual sera. The sample was constituted according to the frequency distribution of the negative and positive pools analysed during a screening involving the whole cattle population of Belgium. Sensitivity and specificity were estimated to 88.9% and 100% and the predicted negative and positive values were 99.9% and 100%, respectively. These results indicate the use of serum pools is suitable for the detection of BLV infected herds in eradication campaigns.
Abstract: We quantified the CD4+ T cell proliferation specific for bovine herpesvirus 1 (BHV-1) in peripheral blood mononuclear cells from cattle. The stimulation index as detected in proliferative assays performed in the presence of BHV-1 antigen is highly variable in immune cattle. By using proliferative assays performed after negative selection we showed that, as expected, CD4+ T cells were the limiting cell type for antigen-induced proliferation. Neither B, gamma delta T nor CD8+ cells seemed to be involved. The limiting dilution method was established to obtain quantitative estimations, namely frequencies of specific T cells. When limiting dilution cultures were supplemented with interleukin-2 (IL-2), an IL-2 induced unspecific cell proliferation masked the specific T cell proliferation. Natural killer cells were not the major cell type involved, but CD4+ lymphocytes themselves seemed to respond to IL-2 irrespective of the presence of antigen. When cultures were performed without addition of IL-2, the frequency of BHV-1 specific proliferative T cells could be obtained by the difference between the frequency of proliferating cells calculated in the presence and absence of antigen. The method provides a sensitive and quantitative means to measure the T cell immune response to BHV-1 vaccine candidates.
Abstract: Three glycoproteins of bovine herpesvirus-1 (BHV-1) other than glycoproteins gI, gIII, and gIV were identified by monoclonal antibody (MAb) analyses. Monoclonal antibodies were obtained by immunization of mice with either BHV-1 envelope or virus infected cells, from which the glycoproteins gI, gIII, and gIV were removed by immunoaffinity. In the latter immunization procedure mice were tolerized either against normal cellular antigens with or without glycoproteins gI, gIII, gIV, and nucleocapsid. From 154 anti-BHV-1 hybridomas isolated, 39 MAbs precipitated a 108K glycoprotein. Two other glycoproteins of respectively 42K and 93K were precipitated each by one MAb. These three glycoproteins were detected in infected cell lysate. Nine anti-108K glycoprotein MAbs neutralized BHV-1 infectivity and three non-neutralizing MAbs were able to reduce plaque development when virus was grown in the presence of these MAbs. It is therefore suggested that this glycoprotein is involved in viral entry into the cell and in cell-to-cell spread of the virus.
Abstract: Cell surface heparan sulfate serves as the initial receptor for several alphaherpesviruses and at least one betaherpesvirus. This study shows that during the process of adsorption of the gammaherpesvirus bovine herpesvirus 4 (BHV-4), the viral glycoprotein gp8 interacts with heparinlike moieties of cell surface. This conclusion is based on the following findings. (i) Soluble heparin was capable of blocking BHV-4 infection of Georgia bovine kidney cells by inhibition of viral attachment. (ii) Nevertheless, after virus adsorption to Georgia bovine kidney cells, heparin was partially capable of removing adsorbed virus. (iii) Enzymatic digestion of cell surface heparan sulfate but not of chondroitin sulfates A, B, and C reduced the binding of the virus to the cells, and rendered the cells partially resistant to infection. (iv) Radiolabeled purified BHV-4 bound to wild-type Chinese hamster ovary cells, whereas binding of the virus to mutant Chinese hamster ovary cell lines that where deficient in either all glycosaminoglycans or only heparan sulfate was significantly impaired. (v) Using heparin-affinity chromatography, gp8 glycoprotein was shown to bind specifically to immobilized heparin and to elute in the presence of soluble heparin. These data together showed that the gammaherpesvirus BHV-4, like alphaherpesviruses and one betaherpesvirus, adsorbs to cells by binding to cell surface heparin-like moieties. Therefore, this study extends the group of herpesviruses interacting with heparinlike moieties at the cell surface to a member of the gammaherpesvirinae subfamily.
Abstract: Vaccinia virus recombinants expressing the three major bovine herpes virus-1 (BHV-1) glycoproteins gI, gIII and gIV were used to identify the major target antigens for BHV-1-specific CTL isolated from immune cattle. Peripheral blood mononuclear cells (PBMC) expanded in vitro in the presence of interleukin-2 (IL-2) and lysed both gIII- and gIV-infected target cells. Secondary in vitro stimulation of PBMC was also performed in the presence of either fixed BHV-1-infected autologous fibroblasts or ultraviolet (UV)-inactivated virus. Both methods of antigen presentation allowed the proliferation of BHV-1-specific CTL but the target glycoprotein for these CTL differed depending on the method of stimulation. Vaccinia-gIV-infected targets were lysed predominantly when PBMC were stimulated by fixed infected fibroblasts, whilst PBMC stimulated by UV-inactivated virus lysed mostly vaccinia-gIII-infected targets. This observation could be explained by a different processing pathway of BHV-1 antigens in each cell type involved.
Abstract: Restriction maps of cervid herpesviruses 1 and 2 which are antigenetically related to bovine herpesvirus 1, were deduced from Southern blot hybridization with HindIII restriction fragments of BHV-1 DNA as probes.
Abstract: Three major bovine herpesvirus type 4 (BHV-4) glycoproteins have been described previously. By using monoclonal antibodies produced against BHV-4 envelope proteins from which the three major antigens had been removed by immunoaffinity, a fourth glycoprotein was identified. This protein (gp1) has a high Mr (greater than 300K), is detected about 8 h post-inoculation of infected cells and is strictly expressed as a gamma protein. Moreover, gp1 was identified by a polyclonal antiserum from an infected animal, indicating that this glycoprotein is an antigen recognized by the immune system of infected animals.
Abstract: The overall arrangement of genes in the unique central part of the bovine herpesvirus type 4 (BHV-4) genome has been deduced by analysis of short DNA sequences. Twenty-three genes conserved in at least one of the completely sequenced herpesviruses have been identified and localized. All of these genes encoded amino acid sequences with higher similarity to proteins of the gammaherpesviruses Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS) than to the homologous products of the alphaherpesviruses varicella-zoster virus and herpes simplex virus type 1 or the betaherpesvirus human cytomegalovirus. The genome organization of BHV-4 had also an overall colinearity with that of the gammaherpesviruses EBV and HVS. Furthermore, the BHV-4 genes content and arrangement were more similar to those of HVS than to those of EBV, suggesting that BHV-4 and HVS are evolutionarily more closely related to each other than either are to EBV. BHV-4 DNA sequences were generally deficient in CpG dinucleotide. This CpG deficiency is characteristic of gammaherpesvirus genomes and suggests that the BHV-4 latent genome is extensively methylated. Despite several biological features similar to those of betaherpesviruses, BHV-4 displays the molecular characteristics of the representative members of the gammaherpesvirinae subfamily.
Abstract: An enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies for capture and detection, was developed for detecting bovine viral diarrhoea virus (BVDV) antigens in blood samples. The test was evaluated using 761 field samples of known status (viraemic or not). When an appropriate cut-off value was chosen, the sensitivity, specificity, and predictive values of the assay were 100%, higher than the values obtained by classical virus isolation. Correlation with the latter technique exceeded 90%. The ELISA is a good candidate for replacing virus isolation as a reference method for BVDV antigen detection in persistently infected carriers. A method based on the mean of the standard deviation ratio can be used to choose the cut-off value in order to optimise reproducibility.
Abstract: The location and nucleotide sequence of the bovine herpesvirus type 4 (BHV-4) thymidine kinase (TK) gene was determined. The coding region of the TK gene is 1335 nucleotides long and corresponds to a polypeptide of 445 amino acids. Comparison of TK amino acid sequences of BHV-4 and 16 herpesvirus TKs reveals a greater homology to those of the gammaherpesviruses EBV and specially HVS, than to those of alphaherpesviruses. The open reading frames detected in the vicinity of TK gene were homologous to the corresponding ones in other herpesviruses.
Abstract: Forty-three cattle seronegative to bovine herpesvirus-1 (BHV-1) were given from one to five intradermal injections of BHV-1 inactivated antigen at four-week intervals. This delayed hypersensitivity test was assessed by the increase in skin thickness. The activity of the antigen was assessed in five animals which had a previous natural BHV-1 infection with clinical signs and seroconversion. Anti-BHV-1 antibodies were detected by seroneutralisation and an enzyme-linked immunoassay. Only one animal showed a significant but slight increase in skin thickness after the first test, but it was negative after a second test. The animals remained seronegative after the first test. Seroconversion was identified in 11 of the 43 animals (25 per cent) submitted to repeated delayed hypersensitivity tests. Five of 37 animals seroconverted after only two tests. The serological response was transient in seven of 11 seroconverted calves. Repeated hypersensitivity tests were therefore able to induce a serological response in seronegative calves but the response was weak and often transient. The test must therefore be applied cautiously to seronegative animals.
Abstract: Three bovine herpesvirus type 4 (BHV-4) proteins released into the culture medium of infected cells were identified, with Mr values of 135K, 16K and 14.5K. Among these three proteins, two were precipitated by the monoclonal antibodies characterized in this work. One is a glycoprotein of 135K (gp8) which does not seem to be involved in BHV-4 neutralization. Moreover, this 135K glycoprotein adsorbed onto uninfected susceptible cells. The attachment of gp8 to cells was totally inhibited by the prior adsorption of unlabelled viral proteins. Moreover, anti-gp8 monoclonal antibodies were effective in inhibiting the adsorption of gp8. These results indicate that gp8 could be involved in BHV-4 attachment.
Abstract: Bovine herpesvirus type 4 (BHV-4) is a ubiquitous virus of cattle. Its genome is a 144 +/- 6 kb double-stranded DNA consisting of a unique central part (L-DNA) flanked at both ends by tandem repeats called polyrepetitive DNA (prDNA or H-DNA). The overall arrangement of genes has been obtained by the analysis of homologies between short BHV-4 DNA sequences and corresponding genes of Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS). The gene expression is temporally regulated. Glycoprotein precursor p (gp10/gp17) is expressed as gamma 1 polypeptide. Glycoproteins gp1, gp8, gp11 and their precursors are gamma 2 proteins. The analysis of strain variations allows the definition of two types of strains, based on the DNA patterns: the Movar 33/63-like and the DN 599-like strains. Only the M40 strain, isolated in India, fails to fit this classification. The genomic variations have been compiled to build a dendrogram showing three levels of divergence between BHV-4 strains or isolates. The available molecular data indicate that the BHV-4 genome shares much similarity with the DNA of EBV and HVS, two representative members of the gammaherpesvirinae. BHV-4 may therefore be classified in the subfamily gammaherpesvirinae.
Abstract: Sera from cattle of the Accra Plains in Ghana were screened for bovine type-4 herpesvirus (BHV-4) antibodies by an indirect immunofluorescence test. Among the 176 screened serums, 14% were found positive at a 1/100 dilution. Further studies are necessary for a better understanding of the relationships between BHV-4 or some other agents and necrotic vaginitis associated with poor fertility observed in cattle in this area.
Abstract: Genomes of herpesvirus aotus type 2 (HVA-2) and bovine herpesvirus type 4 (BHV-4) have previously been shown to be closely similar. Moreover, preliminary serological data indicated that HVA-2 is antigenically related to BHV-4. To extend this study, structural components of four BHV-4 strains and HVA-2 were compared by SDS-PAGE, radioimmunoprecipitation and Western blotting. The overall pattern of structural proteins was the same for HVA-2 and BHV-4 but variations were observed in electrophoretic profiles of glycoproteins, mainly of the two major ones (gp6/gp10/gp17 and gp11/VP24). Variations between HVA-2 and BHV-4 glycoproteins were greater than those observed among BHV-4 strains.
Abstract: Twenty-one monoclonal antibodies (MAbs) directed against the NY-1 and the Osloss-c strains of bovine viral diarrhoea virus were produced and characterized by indirect immunofluorescence assay, radioimmunoprecipitation and neutralization tests. Fourteen MAbs directed against the NY-1 strain recognized the gp48 and showed a weak neutralizing activity in the presence of goat anti-mouse immunoglobulin serum.
Abstract: The glycoprotein gp6/gp10/gp17, gp11/VP24 and gp8 are major bovine herpesvirus type 4 antigens. The temporal expression of these glycoproteins was studied and it was shown that gp6/gp10/gp17 appeared as early as 6 h post-inoculation (p.i.), and gp11/VP24 and gp8 were detected 8 h p.i. Moreover, a precursor of two components of gp6/gp10/gp17 glycoprotein [p(gp10/gp17)] was a beta-gamma protein, whereas the gp11/VP24 and gp8 glycoproteins were gamma proteins.
Abstract: A mathematical model for the epidemiology of rinderpest was developed, starting from a simplified descriptive analysis of the disease. A formula for the calculation of the probability of infection of a susceptible animal was first established. A deterministic failure threshold of the infection was then deduced. Deterministic and stochastic approaches were adopted using iterative methods on a computer. These allowed a description of the spread and the variability of an infection process in a population to be made. An illustration of the use of this model showed that, in some cases, variability effects due to stochastic factors were very important. In these particular conditions, the use of the deterministic model alone was not adequate for a good description of the infection. Consequently, improvements of the model were proposed in order to make it more realistic and to allow its use for the evaluation of the efficiency of field operations.
Abstract: Twenty-four Belgian field isolates of bovine herpesvirus 4 (BHV-4), together with four reference strains were compared by radio-immunoprecipitation and western blotting using a polyvalent antiserum and monoclonal antibodies raised against major glycoproteins. Most of these strains showed the same protein profile as the European reference strain Movar 33/63. For two strains the molecular weight of gp 6, p (gp 10/gp 17) and gp 10 were the same as those of the American reference strain DN 599. No relationship could be established between the protein profiles and origin of the isolates or with the restriction patterns. This study provides a view of the molecular weight variations of the major BHV-4 glycoproteins among field isolates.
Abstract: Herpesvirus aotus type 2 (HVA-2) was isolated from a culture of kidney cells from a healthy owl monkey (Aotus trivirgatus). Bovine herpesvirus type 4 (BHV-4) is frequently isolated from diseased and even healthy cattle and occasionally from sheep, wild ruminants and cats. The two viruses are related antigenically, as was revealed by an indirect fluorescent antibody test using polyclonal antisera from experimentally infected rabbits or monoclonal antibodies raised against six BHV-4 proteins, three of which were glycosylated. The genome structures of the two viruses consist of a unique central sequence flanked at both ends by G + C-rich tandem repeats. Restriction maps (produced using EcoRI, BamHI and HindIII) of these two viruses were nearly identical but the unique sequence of the HVA-2 genome possessed two additional BamHI sites. Four genomic regions of variable size were detected, two located in the unique part, one in the repetitive part and one in the left junction between the unique and the repeated part of the genome; these slight variations were similar to those observed between various BHV-4 isolates. These results suggest that HVA-2 and BHV-4 belong to the same virus species; HVA-2 could be either a BHV-4 contaminant of owl monkey kidney cell cultures or an isolate from an owl monkey accidentally infected with BHV-4.
Abstract: Twenty-eight Belgian field isolates of bovine herpesvirus 4 (BHV-4) coming from a variety of clinical diseases have been studied by restriction analysis and Southern blot hybridization. The unique central part of the genome was very well conserved among strains; only one variation in a restriction site was detected in 3 isolates which contain an additional EcoRI site also present in the LVR 140 strain; three regions in the unique part of the genome varied in size, one of these was highly variable. The polyrepetitive fragments (prDNAs) situated in tandem at both genomic ends were also variable in size; most of the isolates exhibited prDNA units of one size (major prDNA) and some of them also contained prDNA units having a different size and present in a lower amount (minor prDNA) than the major prDNA. Other isolates possessed two major prDNAs of different sizes which were both present in the same genome. The left junction fragment between the unique and the repeated sequences was also highly variable. No relationship could be established between the restriction pattern and the origin of the isolates; patterns of isolates coming from the same herd were similar except in one case. This study provides a view of the genome variability existing between BHV-4 field isolates.
Abstract: In infected cattle, bovine herpesvirus type 4 (BHV-4) induces an immune response with low neutralizing antibody levels or in the absence of such antibodies. For the study of this phenomenon, monoclonal antibodies (MAbs) raised against two BHV-4 glycoproteins identified previously (150K/120K/51K and 120K/16.5K) were used in neutralization tests. None of the MAbs except for MAb 16 could neutralize alone; pairs of MAbs against the 150K/120K/51K and 120K/16.5K glycoproteins were able to neutralize BHV-4 infectivity. MAbs involved in neutralization were used in competitive binding assays to identify epitopes relevant for BHV-4 neutralization. These MAbs showed a low avidity and a weak neutralizing activity, and they partially decreased BHV-4 attachment to cells. These results suggest that the BHV-4 glycoprotein domains involved in viral infectivity are poorly exposed to the immune system.
Abstract: The biology of bovine herpesvirus-4 (BHV-4) infection of cattle is reviewed. The infection is distributed worldwide. Most of isolated viruses are non-pathogenic in cattle; some of them are able to produce a genital disease. Twenty-nine structural polypeptides were described; ten of them are glycosylated. Two major glycoproteins were characterized by monoclonal antibodies. Restriction maps of BHV-4 DNA are available for the enzymes EcoRI, BamHi and HindIII. The strain variations studied by restriction analysis are very weak. The virus is able to persist in a latent state after primary infection. The identified sites of latency are nervous ganglia and mononuclear blood cells. The immune response of cattle after BHV-4 infection is characterized by low or undetectable levels of neutralizing antibodies. Four envelope proteins are recognized by convalescent sera and are the main antigenic components. Skin test remains negative in immunized cattle. Bovine herpesvirus-4 is not strictly species-specific: infection was proved in American bison (Bison bison), African buffalo (Syncerus caffer), sheep and probably cat, because feline herpesvirus-2 is in fact a BHV-4 strain. Finally BHV-4 shares antigenic and genomic relationships with alcelaphine herpesvirus-1, the causal agent of the African form of malignant catarrhal fever.
Abstract: The restriction map of the bovine herpesvirus 4 (BHV-4) genome (V. Test strain) was established for the restriction enzymes EcoRI, BamHI and HindIII by analysis of clones from a lambda library (Sau3AI partial digestion) and from a plasmid library (EcoRI fragments). One genome unit was defined as the length of the unique central part, flanked at both ends by one of the terminal tandem repeats called polyrepetitive DNA (prDNA) and was estimated to be 113 +/- 2 kbp. A restriction map of the prDNA of the V. Test strain showed internal 200 bp tandem repeats of different sequences. This region in the prDNA was highly polymorphic between BHV-4 strains, even in a viral DNA preparation from a plaque-purified strain. The right junction between the repeated and the unique sequence of the genome occurred at an almost constant site, but the left junction contained a modified prDNA and was variable between BHV-4 strains. The unique central part of the genome was very similar in the four strains under consideration, with a few variations due to the presence or absence of a restriction site and four length variations were observed, located at positions 0.006 to 0.034 (left end), 0.211 to 0.225, 0.864 to 0.881 and 0.962 to 0.984 (right end). The total length variation of 1 genome unit does not exceed 1 kbp.
Abstract: More than 400 small ruminant sera from Zaïre were screened for antibodies to IBR, CHV2, BVD, bovine and ovine PI3, BRS and rinderpest viruses. Sera from local animals were negative for BVD, PI3 and rinderpest viruses: 8% of sera were positive for IBR virus, all with higher titers to CHV2; 31% of sera were positive to BRS virus.
Abstract: The current situation of infectious bovine rhinotracheitis/infectious pustular vulvovaginitis infection in various European countries is reviewed. Whilst some have a high serological prevalence and use live virus vaccines to control the disease, others have a low prevalence and two countries (Denmark and Switzerland) have national eradication schemes which are almost complete. Serology remains important for diagnosis although other tests such as delayed cutaneous hypersensitivity may have a role to play. New tests such as polymerase chain reaction may find increasing application where high sensitivity is required, such as the detection of virus in semen.
Abstract: Thirty-five hybridoma cell lines secreting monoclonal antibodies (Mabs) against bovid herpesvirus-4 (BHV-4) strain V. Test were produced. These hybrid cells resulted from the fusion of SP2/0 myeloma cells with splenocytes of BALB/c mice previously immunized with purified BHV-4. A modified indirect fluorescent antibody test (IFAT) was applied as a screening procedure and was compared with an indirect enzyme-linked immunosorbent assay. The selected Mabs were tested by the same IFAT against a panel of BHV-4 field isolates and against bovine herpesvirus-1, bovine herpesvirus-2 and alcelaphine herpesvirus-1 (AHV-1). Comparison of BHV-4 field isolates with Mabs confirmed their close antigenic relationships, but slight antigenic differences were observed between different isolates. One of the Mabs also reacted against AHV-1, indicating an antigenic relationship between BHV-4 and AHV-1. None of the Mabs reacting with BHV-4 possessed neutralizing activity against the strain used for immunization.
Abstract: Two-hundred bovine sera from western Zaire were screened for antibodies to 8 viruses: BHV-1, BHV-2, BHV-4, BVD-MD virus, bovine adenovirus A and B, bovine rotavirus and bovine coronavirus. Positive sera were found to all these viruses. For animals whose origin was undoubted, the main features were the high prevalence of infections by rotavirus and BHV-4 and the low prevalence of infections by coronavirus and BVD-MD virus.
Abstract: Five 13- to 18-month old Belgian Blue bulls were used in this experiment. Four bulls (Nos. 2, 3, 4 and 5) were inoculated intratesticularly with 10(5) plaque-forming units of bovine herpesvirus-4 (BHV-4) in each testicle (Day 0). The challenge BHV-4 strain was previously isolated from testicle cells of a bull exhibiting orchitis and azoospermia. The fifth bull (No. 1) was used as a control and received the same volume of uninfected cell culture supernatant. For 5 days, beginning on Day 51 post-infection, two bulls (Nos. 4 and 5) and the control bull (No. 1) received 0.1 mg kg-1 of dexamethasone. Unilateral castrations were then performed at regular intervals for viral examination. Treatment with dexamethasone reactivated latent BHV-4, but no clinical signs were observed in treated bulls until the end of the experiment (Day 93). Only Bull 3 showed conjunctivitis and temporary azoospermia. The virus was recovered from various samples showing that: (i) BHV-4 can be present in a latent state in the testicles and mononuclear blood cells; (ii) dexamethasone reactivates the virus; (iii) the virus is excreted by nasal and ocular routes. Each infected bull seroconverted and a booster antibody response appeared after dexamethasone treatment as shown by immunofluorescence. Neutralizing antibodies were detected in each bull by complement-dependent neutralization test with titres higher than those obtained by a classical neutralization test. No booster response of neutralizing antibodies was observed after dexamethasone treatment. The antigenically relevant envelope BHV-4 proteins were identified by Western blotting using sera samples from the animals. DNA restriction endonuclease profiles of viruses reisolated after primary infection and reactivation showed only small differences.
Abstract: Bovine herpesvirus type 4 proteins were identified by PAGE of [35S]methionine- or [3H]glucosamine-labelled purified virions. Thirty-one monoclonal antibodies (MAbs) raised against the V. Test strain were used to identify 29 proteins, ten of which were glycosylated. All of these glycoproteins belonged to the viral envelope and a 140K non-glycosylated protein appeared to be the major nucleocapsid protein. The MAbs were classified into two groups. The first group precipitated three glycoproteins of Mr 150K, 120K and 51K. The 120K and 51K glycoproteins were linked by disulphide bonds and the 150K glycoprotein was linked to the others by non-covalent bonds. The second group precipitated a different 120K glycoprotein.
Abstract: The effect of prostaglandins PGE2 and PGF alpha 2 on infectious bovine rhinotracheitis (IBR) virus (bovine herpesvirus 1; BHV-1) was studied by using the measurement of the mean size of plaques produced in cell culture by IBR/Cu5 strain. Three concentrations (0.1, 1 and 10 micrograms/ml) of prostaglandins, PGE2 and PGF alpha 2 were used. Each prostaglandin provoked an increase in the mean plaque size when compared to control plaques, at the concentrations 1 and 10 micrograms/ml for PGE2 and 10 micrograms/ml for PGF alpha 2.
Abstract: Two experimental porcine models of Aujeszky's disease (AD) were compared for assessing the efficacy of potential antiviral compounds. While signs were the same following intranasal inoculation and in-contact transmission of the causal herpesvirus, SHV-1 (Suid herpesvirus), the time-course of the disease was different. There was less variation in clinical signs between pigs following artificial infection, but the disease was more severe, making this a consistent but also more stringent test system. A nucleoside analogue, BW B759U (9-[1,3-dihydroxy-2-propoxy-methyl] guanine), was administered intramuscularly in divided twice daily doses at 50 mg kg-1 to three-week-old piglets in these two SHV-1 disease models. In artificially infected animals, in which treatment was begun 1.5 hours preinfection and continued for six days, there was a delay in the onset of clinical signs (4.8 compared with 3.2 days), a 1 log10 reduction in virus shedding, and a 1 to 2 log10 reduction in virus recovered from tissues, but all the treated piglets died from AD. In contrast, none of the BW B759U-treated piglets in the in-contact model system died during the eight-day medication period, although two piglets died subsequently. Mean serum concentrations of BW B759U two hours after intramuscular dosing were about 45 microM, at nine hours 15 to 20 microM and at 17 hours 3.5 microM. The in vitro IC50 of BW B759U against SHV-1 varies from 1.5 to 80 microM depending on the cell line in which the assay is carried out. There were no overt signs of BW B759U toxicity in the treated piglets.
Abstract: Bovid herpesvirus-4 (BHV-4) isolates V.Test and LVR140, isolated from genital disease, respectively, in bull and in cow, and the reference strains Movar 33/63 and DN599 were compared by several methods: cross-serological relationship studied by indirect immunofluorescence; kinetics of intracellular and extracellular viral production; comparison of the mean plaque size; restriction analysis of viral DNA with restriction enzymes EcoRI, BamHI and HindIII. BHV-4 strains were serologically identical and the kinetics of viral production were very similar. Comparison of the mean plaque size allowed classification into 3 classes (Class I, Movar33/63; Class II, LVR140; Class III, V.Test and DN599) and restriction analysis of viral DNA revealed clear differences between the electrophoretic patterns of the four BHV-4 strains. The differentiation between BHV-4 strains can therefore be achieved by a biological method (mean plaque size) and by restriction analysis. The two genital isolates are easily differentiated by the two methods.
Abstract: The presence of antibodies against bovine herpesvirus 1 (BHV-1), bovid herpesvirus 6 (BHV-6), herpesvirus of Cervidae type 1 (HVC-1), reindeer herpesvirus, bovine herpesvirus 2 (BHV-2) and bovid herpesvirus 4 (BHV-4) was investigated in wild ruminants of France and Belgium between 1981 and 1986. There were no animals serologically positive for BHV-4. Antibodies against BHV-2 were demonstrated in roe deer (Cervus capreolus) (less than 1%) and chamois (Rupicapra rupicapra) (1%) in France. Animals seropositive to the four related viruses (BHV-1, BHV-6, HVC-1, reindeer herpesvirus) were detected in red deer (Cervus elaphus) in France and Belgium (1% and 11%, respectively), in roe deer (less than 1%) from France, in chamois (4%) in France and in ibex (Capra ibex) (4%) from France. The presence of antibodies against HVC-1, especially in red deer from Belgium, may suggest that wild ruminants in continental Europe are now infected with this virus, which previously has been isolated only in Scotland.
Abstract: Transport was studied as a cause of reactivation of infectious bovine rhinotracheitis virus (Bovine herpesvirus-1; BHV-1) in heifers vaccinated 2-6 months before transport, using a double dose of the thermosensitive (ts) vaccine strain (Tracherine). Eight out of 19 animals showed ts strain re-excretion over a period of 1-3 days, beginning, in 5 out of the 8 heifers, the day after transport. In 14 other heifers, only sera were examined by sero-neutralisation: only 1 out of these 14 animals showed a rise in BHV-1 neutralising antibodies. Transport can therefore be considered as a stimulus of BHV-1 reactivation.
Abstract: The various mechanisms of virus persistence are described. Four kinds of virus persistence are presented, depending on their epidemiological significance, persistence associated with continuous multiplication and transmission (feline calicivirus, calf rotavirus); persistence associated with continuous multiplication but discontinuous transmission (equine infectious anemia retrovirus); persistence with continuous multiplication associated with immunotolerance (feline leukaemia retrovirus; mucosal disease pestivirus); latent carrier state with discontinuous transmission (feline and pigeon herpesvirus; infectious bovine rhinotracheitis herpesvirus). The classification of these kinds of virus persistence is based upon their epidemiological significance, even if the mechanisms allowing the persistence are completely different.
Abstract: Description of the interactions between bovine herpesvirus type 1 (BHV-1) and cattle was performed by the method known as kinetic logic. This logical formalization uses variables with two possible values, 1 and 0, which tell whether an element is present or not at a significant level. To each variable is associated a function which tells if the element is being produced at a significant rate. The temporal relation between a variable and its associated function is given by specific time delays. The BHV-1 infection system is described by a set of five logical equations which tell in what conditions each function is on or off. The five functions are: V, development of viral multiplication; R, development of reactivation of the latent virus; A, development of an immune response; G, establishment of the viral genome; M, development of a memory of a first immune response. Several examples are detailed in a dynamic analysis, in connection with known experimental data.
Abstract: Evolution of sensitizing antibodies involved in antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-complement cell lysis and complement-facilitated ADCC was followed in bulls after primary infection by a wild strain of infectious bovine rhinotracheitis virus (bovine herpesvirus 1; BHV-1) and after experimental reactivation of the virus. These antibodies were detected between the 4th and the 7th day after primary infection, reached a maximum level after 2 weeks and rose slightly after reactivation of the virus following dexamethasone treatment. The presence of endogenous complement slightly enhanced the ADCC reaction.
Abstract: Twelve cattle were divided into 2 groups. The first was intranasally co-infected with 2 strains of infectious bovine rhinotracheitis virus (Bovine herpesvirus 1; BHV 1): the thermosensitive vaccine strain IBR/ts RLB106 and a Belgian field isolate IBR/Cu5. Reactivation of BHV 1 was induced by dexamethasone treatment 2 months later and again 5 months later for 3 animals that only reexcreted small quantities of virus during the first dexamethasone treatment. The second group was intranasally infected with IBR/Cu5. Two months later, an attempt to reinfect this group with IBR/ts RLB106 failed. Four months after the primary infection, these cattle were treated with dexamethasone. Except after reinfection and at the beginning or the end of the (re)excretion periods, excreted and reexcreted viruses replicated at 35, 37 and 40 degrees C, indicating the presence of the wild-type virus. Only one isolate, out of 116 cloned from the nasal exudates collected during the excretion and reexcretion periods, expressed the thermosensitive phenotype. This isolate was characterized by its mean plaque size as the IBR/ts RLB106 strain. The epizootiological significance of these findings is discussed, with emphasis on the weak spreading capacity of the ts vaccine strain and the possibility of emergence of recombinant viruses.
Abstract: Efficient methods of diagnosis and prophylaxis of infectious bovine rhinotracheitis must consider the concept of latency of the etiological agent, infectious bovine rhinotracheitis virus (Bovine herpesvirus 1; BHV 1). The identification of BHV 1 in nasal mucus samples or a rise in specific antibodies have to be cautiously interpreted, because they can signify either a primary infection or a reexcretion of the virus after reactivation. The isolated virus can also either be a vaccine or a virulent strain. Another aspect of BHV 1 infection diagnosis is the detection of latent carriers, which are able to transmit the virus to uninfected animals; delayed hypersensitivity test seems to be a good candidate. The classical methods of prophylaxis protect the animal against the disease, but they should also impede the reexcretion of virulent strains by latent carriers. Since, in several countries, attenuated viruses are used as vaccines, a special emphasis has to be laid on the persistence of these vaccine viruses in a latent form in the bovine population.
Abstract: The effect of dexamethasone on infectious bovine rhinotracheitis virus (Bovine herpesvirus 1: BHV 1) was studied by measuring the mean plaque size produced by eight virus strains under two concentrations of dexamethasone phosphate (0.1 and 1 mM). Dexamethasone induced a significant reduction of the mean plaque size, whatever the strain used. Some differences were noted between the BHV 1 strains studied, depending on the dexamethasone concentration.
Abstract: The biochemical characteristics of infectious bovine rhinotracheitis virus (Bovine herpes-virus 1, BHV 1) are reviewed: description of the virion particle and virus purification, analysis of structural and non structural proteins, nucleic acid properties, restriction endonuclease analysis of the viral DNA. The authors emphasize the biochemical methods able to insure a better understanding of the molecular epidemiology of BHV 1 and of its latency; also to develop biochemical weapons against the infection.
Abstract: Two cattle latently infected with infectious bovine rhinotracheitis virus (Bovine herpesvirus 1, BHV 1) were intradermally injected with inactivated BHV 1 antigen (delayed hypersensitivity test, DHT, skin test). Another animal, free of BHV 1 infection was similarly treated. Two latent carriers of the virus, intradermally injected with Phosphate-Buffered Saline solution were used as control. The evolution of spontaneous multiplication of lymphocytes and lymphoblastic transformation in vitro induced by three phytomitogens (con A, PHA, PWM) and BHV 1 antigen was followed in all animals. The delayed hypersensitivity test provoked an increase in spontaneous lymphocyte multiplication in latent carriers as well as an increase in mitogen- and antigen-induced blastogenesis on the 2nd and the 9th day following the treatment. A similar increase occurred on the 9th day in the BHV 1-free animal. Therefore, in latent carriers of BHV 1, delayed hypersensitivity testing induces an anamnestic lymphocyte reaction corresponding to the cutaneous one. This first lymphocyte activity is followed, one week later, by a new reaction which occurs both in infected and uninfected animals.
Abstract: The effect of various concentrations of acyclovir on the mean plaque size of pseudorabies virus (SHV), infectious bovine rhinotracheitis virus (IBR virus) and pigeon herpesvirus (PHV) has been studied. Acyclovir significantly reduced the mean plaque size of SHV and PHV, whereas IBR virus was less affected and did only show a reduction of the mean plaque size at the highest concentration of acyclovir used (1000 microM). In vivo effect of acyclovir was tested using pigeons and budgerigars experimentally infected with PHV and rabbits experimentally infected with a very low dose of SHV. Intramuscular injections of acyclovir (100 mg/kg/day; three injections/day) did not prevent the appearance of clinical disease in infected pigeons nor did reduce the level of viral excretion. The same treatment applied, as for the pigeons, before infection protected most of the budgerigars as long as they were treated, but most of them died soon after the end of the treatment. Only one rabbit was protected by the treatment. SHV was recovered in the lung of only one of the treated animals, whereas it was isolated in the lungs of each control animal.
