Abstract: Plasmacytoid dendritic cells (pDCs) play a key role in detecting pathogens by producing large amounts of type I interferon (IFN) by sensing the presence of viral infections through the Toll-Like Receptor (TLR) pathway. TLR9 is a sensor of viral and bacterial DNA motifs and activates the IRF7 transcription factor which leads to type I IFN secretion by pDCs. However, during chronic hepatitis B virus (HBV) infection, pDCs display an impaired ability to secrete IFN-α following ex vivo stimulation with TLR9 ligands. Here we highlight several strategies used by HBV to block IFN-α production through a specific impairment of the TLR9 signaling. Our results show that HBV particle internalisation could inhibit TLR9- but not TLR7-mediated secretion of IFN-α by pDCs. We observed that HBV down-regulated TLR9 transcriptional activity in pDCs and B cells in which TLR9 mRNA and protein levels were reduced. HBV can interfere with TLR9 activity by blocking the MyD88-IRAK4 axis and Sendai virus targeting IRF7 to block IFN-α production. Neutralising CpG motif sequences were identified within HBV DNA genome of genotypes A to H which displayed a suppressive effect on TLR9-immune activation. Moreover, TLR9 mRNA and protein were downregulated in PBMCs from patients with HBV-associated chronic hepatitis and hepatocellular carcinoma. Thus HBV has developed several escape mechanisms to avoid TLR9 activation in both pDCs and B lymphocytes, which may in turn contribute to the establishment and/or persistence of chronic infection.
Abstract: The development of new anti-hepatitis B virus (HBV) therapies, especially immunotherapeutic approaches, has been limited by the lack of a primate model more accessible than chimpanzees. We have previously demonstrated that sylvanus and cynomolgus macaques are susceptible to in vivo HBV infection after intrahepatic HBV DNA inoculation. In this study, we evaluated the susceptibility of primary macaque hepatocytes (PMHs) to HBV infection with a highly efficient HBV genome-mediated transfer system via a recombinant baculovirus (Bac-HBV). Freshly prepared PMHs, isolated from macaque liver tissue by collagenase perfusion, were transduced with Bac-HBV, and intermediates of replication were followed for 9 days post-transduction. Evidence of HBV replication (hepatitis B surface antigen secretion, viral DNA, RNA, and covalently closed circular DNA) was detected from day 1 to day 9 post-transduction. HBV markers were dose-dependent and still detectable at a multiplicity of infection of 10. Importantly, transduced PMHs secreted all typical forms of HBV particles, as evidenced by a cesium chloride gradient as well as transmission electron microscopy. Furthermore, the Toll-like receptor 9 (TLR9) ligand was used to stimulate freshly prepared macaque peripheral blood mononuclear cells to generate TLR9-induced cytokines. We then demonstrated the antiviral effects of both TLR9-induced cytokines and nucleoside analogue (lamivudine) on HBV replication in transduced PMHs. CONCLUSION: Baculovirus-mediated genome transfer initiated a full HBV replication cycle in PMHs; thus highlighted both the baculovirus efficiency in crossing the species barrier and macaque susceptibility to HBV infection. Moreover, our results demonstrate the relevance of thus system for antiviral compound evaluations with either nucleoside analogues or inhibitory cytokines. Cynomolgus macaques are readily available, are immunologically closely related to humans, and may therefore represent a promising model for the development of new immunotherapeutic strategies.
Abstract: Currently approved antiviral monotherapies against chronic hepatitis B fail to eradicate hepatitis B virus (HBV), to overcome the defects in HBV-specific immune responses and to prevent HBV relapse after cessation of therapy. CpG oligodesoxynucleotides (CpG ODN) are synthetic agonists of Toll-like receptor 9 and potent inducers of innate and acquired immunity. Our aim was to establish the proof of concept of the antiviral benefit of combining a nucleoside analogue with CpG-induced cytokines on HBV replication in vitro.
Abstract: Detection and localization of HCV in liver tissue are vital for diagnostic purposes and clinical management of HCV-infected patients, as well as for the elucidation of viropathological mechanisms. The fragility of HCV RNA and the low levels of viral expression in infected tissues are a constant limitation in molecular assays for HCV characterization. HCV antigen detection, by immunochemistry, in liver biopsies is an attractive option for precise localization and quantification of viral proteins with direct access to histological patterns. We describe here a study using a novel immunohistochemical method effective on fixed, archived specimens, including liver biopsies and surgical resection samples. The initial protocol uses a biotin-detection system but can also be used in a polymer-detection system. This protocol offers easy, precise, and strong staining resolution with distinct patterns consistent with the liver pathology, irrespective of the viral HCV genotype examined. This approach provides applications for diagnosis as well as for exploratory pathological studies.
Abstract: One of the major problems in gaining further insight into hepatitis B virus (HBV)/host-cell interactions is to improve the existing cellular models for the study of HBV replication. The first objective of this study was to improve the system based on transduction of HepG2 cells with a recombinant baculovirus to study HBV replication. A new HBV recombinant baculovirus, Bac-HBV-1.1, in which the synthesis of pre-genomic RNA is driven by a strong mammalian promoter, was generated. Transduction with this new recombinant baculovirus led to higher levels of HBV replication in HepG2 cells compared with levels obtained with previously described baculovirus vectors. The initiation of a complete HBV DNA replication cycle in Bac-HBV-1.1-transduced HepG2 cells was shown by the presence of HBV replicative intermediates, including covalently closed circular DNA (cccDNA). Only low levels of cccDNA were detected in the nucleus of infected cells. Data showed that cccDNA resulted from the recycling of newly synthesized nucleocapsids and was bound to acetylated histones in a chromatin-like structure. HBV particles released into the supernatant of transduced HepG2 cells were infectious in differentiated HepaRG cells. Several Bac-HBV-1.1 baculoviruses containing HBV strains carrying mutations conferring resistance to lamivudine and/or adefovir were constructed. Phenotypic analysis of these mutants confirmed the results obtained with the transfection procedures. In conclusion, an improved cell-culture system was established for the transduction of replication-competent HBV genomes. This will be useful for future studies of the fitness of HBV mutants.
Abstract: Porcine circovirus type 2 (PCV2) infection of natural interferon producing cells (NIPCs) impairs the induction of interferon (IFN)-alpha and tumour necrosis factor (TNF)-alpha by cytosine-phosphorothioate-guanine (CpG) oligodeoxynucleotides (ODNs), thereby preventing both their autocrine maturation and the paracrine maturation of myeloid dendritic cells (DCs). The present study shows that the PCV2-mediated inhibition of NIPCs was mediated by viral DNA, although it was independent of virus replication. The inhibitory effect of PCV2 DNA was more diversified than if it had simply targeted CpG-ODN-induced cytokines (IFN-alpha, TNF-alpha, interleukin-6, IL-12). A broad spectrum inhibition was noted, affecting responses induced by toll-like receptor (TLR)-7 and TLR9 agonists, as well as viruses including pseudorabies virus, transmissible gastroenteritis virus and classical swine fever virus. From these results, it would appear that PCV2 DNA can induce a dominant negative signal influencing independent pattern recognition receptor-induced activation cascades. Despite a concomitant internalization of PCV2 DNA and CpG-ODNs, no colocalization was observed, indicating that PCV2 DNA and CPG-ODNs may not target the same receptor. This study describes a novel modulation of the innate immune response, which would render the host more susceptible to secondary or concomitant microbial infections.
Abstract: Cervical cancer development is linked to the persistent infection by high-risk mucosal human papillomaviruses (HPVs) types. The E6 and E7 major oncoproteins from this dsDNA virus play a key role in the deregulation of the cell cycle, apoptosis, and adaptive immune surveillance. In this study, we show for the first time that HPV type 16 (HPV16), the most carcinogenic type among the high-risk subgroup, interferes with innate immunity by affecting the expression of TLRs. Infection of human primary keratinocytes with HPV16 E6 and E7 recombinant retroviruses inhibits TLR9 transcription and hence functional loss of TLR9-regulated pathways. Similar findings were achieved in HPV16-positive cancer-derived cell lines and primary cervical cancers, demonstrating that this event occurs also in an in vivo context. Interestingly, E6 and E7 from the low-risk HPV type 6 are unable to down-regulate the TLR9 promoter. In addition, E6 and E7 from the high-risk HPV type 18, which are known to persist less competently in the host than HPV16, have reduced efficiency compared with HPV16 in inhibiting TLR9 transcription. Furthermore, a CpG motif derived from the HPV16 E6 DNA sequence activated TLR9, indicating this virus is able to initiate innate responses via the receptor it later down-regulates. This study reveals a novel mechanism used by HPV16 to suppress the host immune response by deregulating the TLR9 transcript, providing evidence that abolishing innate responses may be a crucial step involved in the carcinogenic events mediated by HPVs.
Abstract: The purpose of this study was to determine serum profiles of cytokines at a protein level and Creactive protein (CRP) during the development of postweaning multisystemic wasting syndrome (PMWS) in experimentally inoculated pigs. Levels of serum IFN-alpha, IL-6, IL-10, and CRP were examined for a 35-day period in 10 piglets experimentally infected with PCV2 at 3 weeks of age. Four of the infected piglets developed severe PMWS at 14 to 21 days post-infection (d.p.i.) and died prior to termination of the experiment. The remaining six PCV2-infected piglets experienced transient fever, but did not display overt clinical signs of PMWS and were considered as subclinically infected. A bioassay was used to detect IL-6 and ELISAs were used to detect IFN-alpha, IL-10, and CRP. There were no significant differences in cytokine or CRP expression from 0 to 7 d.p.i. between the PMWS-affected and the subclinically infected piglets. Levels of IL-10 and CRP were elevated from 10 and 14 d.p.i. respectively in the PMWS-affected piglets compared to the subclinically infected piglets. There were no significant differences in IFN-alpha and IL-6 expression between the PMWS-affected piglets and the subclinically infected piglets. The present study shows that elevated levels of serum CRP and IL-10 were associated with PCV2-infected piglets that subsequently developed severe PMWS. This may help to provide further insight into the immunoaetiogenesis of this syndrome.
Abstract: Natural interferon-producing cells (NIPC), also called plasmacytoid dendritic cells, are the most potent producers of IFN-alpha in response to viral and bacterial components, serving an important function in innate immune defences. The present work demonstrates that NIPC responsiveness can be primed by immunisation, increasing their capacity to produce IFN-alpha after viral infection. NIPC isolated from pigs immunised against classical swine fever virus (CSFV), a member of the Flaviviridae, were more receptive to viral infection and produced higher levels of IFN-alpha than NIPC from immunologically naive animals. This sensitisation of NIPC was maintained for at least 8 months after immunisation. IFN-alpha production was dependent on live virus and required replication, and the "immune" NIPC responded to lower infectious doses of virus. Co-localisation of the virus with Fc(gamma)RII (CD32) in polarised structures was observed with "immune" NIPC only. This Fc(gamma)RII-dependent virus capture and sensitisation of NIPC, evidently mediated through cytophilic CSFV-specific antibodies, was inhibited by non-specifically aggregated immunoglobulin as well as by pre-formed virus-antibody complexes. In conclusion, these results demonstrate that NIPC not only represent a major player in innate immunity but are also functionally linked to the immunological memory of the adaptive immune system.
Abstract: Viral interactions with dendritic cells (DCs) have important consequences for immune defence function. Certain single-stranded DNA viruses that associate with a number of species, including humans and pigs, exhibit interesting characteristics in this context. Porcine circovirus type 2 (PCV2) can persist within myeloid DCs in the absence of virus replication. Internalization was observed with both conventional blood DCs and plasmacytoid DCs [natural interferon-producing cells (NIPCs)], as well as DC precursors. This PCV2-DC interaction neither induced nor inhibited DC differentiation. The maturation of myeloid DCs induced by a cocktail of interferon-alpha/tumour necrosis factor-alpha (IFN-alpha/TNF-alpha), and the ability to process and present antigen to T lymphocytes, remained intact in the presence of PCV2. The virus was clearly internalized by the DCs, a process noted with both mature and immature cells. This suggested a non-macropinocytic uptake, confirmed by an insensitivity to wortmannin but sensitivity to cytochalasin D, chlorpromazine and bafilomycin. Nevertheless, PCV2 was immunomodulatory, being effected through the reaction of NIPC to danger signals. When NIPCs responded to the CpG-oligonucleotide (CpG-ODN), their costimulatory function which induces myeloid DC maturation was clearly impaired by the presence of PCV2. This was caused by a PCV2-induced inhibition of the IFN-alpha and TNF-alpha normally produced following interaction with CpG-ODN. Thus, the immunomodulatory activity of PCV2 is mediated through the disruption of NIPC function. This would impair the maturation of associated myeloid DC and have major implications for the efficient recognition of viral and bacterial danger signals, favouring the establishment of infections additional to that of PCV2.
Abstract: Functional disruption of dendritic cells (DCs) is an important strategy for viral pathogens to evade host defences. Monocytotropic viruses such as classical swine fever virus (CSFV) could employ such a mechanism, since the virus can suppress immune responses and induce apoptosis without infecting lymphocytes. Here, CSFV was shown to infect and efficiently replicate in monocyte- and in bone marrow-derived DCs. Interestingly, the infected DCs displayed neither modulated MHC nor CD80/86 expression. Stimulation of DCs with IFN-alpha/TNF-alpha or polyinosinic-polycytidylic acid (pIC) induced phenotypic maturation with increased MHC and CD80/86 expression, both with mock-treated and infected DCs. In addition, the T cell stimulatory capacity of CSFV-infected DCs was maintained both in a polyclonal T cell stimulation and in specific antigen-presentation assays, requiring antigen uptake and processing. Interestingly, similar to macrophages, CSFV did not induce IFN-alpha responses in these DCs and even suppressed pIC-induced IFN-alpha induction. Other cytokines including interleukin (IL)-6, IL-10, IL-12 and TNF-alpha were not modulated. Taken together, these results demonstrated that CSFV can replicate in DCs and control IFN type I responses, without interfering with the immune reactivity. These results are interesting considering that DC infection with RNA viruses usually results in DC activation.
Abstract: A panel of 20 porcine sera was distributed to 5 laboratories across Europe and Canada. Each center was requested to test the sera for the presence of porcine circovirus type 2 antibodies using the routine assays, indirect immunofluorescence assay (IFA) and indirect immunoperoxidase monolayer assay (IPMA), and to determine the titer of each serum. Results from all centers were then compiled and correlated. They demonstrate a wide variation in the titers obtained between laboratories. These differences were dependent on the assay used and the choice of fixative. In general, IPMA gave higher titers than did IFA, and paraformaldehyde gave higher titers than did acetone or ethyl alcohol. This report highlights the need for standardized procedures and biologicals for this virus.
Abstract: Dendritic cells (DCs) play crucial roles in innate and adaptive immune responses, rendering them critical targets for virus infections. Porcine circovirus type 2 (PCV2) is associated with the development of postweaning multisystemic wasting syndrome (PMWS) in piglets. We demonstrate here that 80 to 90% of monocyte-derived and bone marrow-derived DCs interact with PCV2 similar to the early stages of an infection. There was no evidence for virus replication, but the virus did persist in DCs without loss of infectivity nor the induction of cell death. This could reflect an abortive infection, but there was no evidence of virus uncoating-the infectivity remained intact for at least 5 days. Alternatively, the results may reflect DC endocytosis of antigenic material. However, there was no modulation of DC surface major histocompatibility complex class I and class II, CD80/86, CD25, CD16, or CD14. Furthermore, infected DC did not transmit virus to syngeneic T lymphocytes, even when the latter were activated. Such coculture did not induce PCV2 replication or death of the lymphocytes or DCs. These results demonstrate that PCV2 can persist in DCs in the absence of virus replication or degradation. Such a silent virus infection presents a novel mechanism of not only immune evasion but also escaping the DC degradation pathway. Because of their migratory capacity, infection of DCs thus provides a potent vehicle for transport of the virus throughout the host without the need for replication. In addition, the lymphopenia seen in PMWS is not a direct effect of the virus on lymphocytes but would require additional events, as proposed by others.
Abstract: The composition of peripheral blood leukocyte populations was studied following experimental PCV2-infection in 3-week-old piglets. Four of 10 PCV2-infected piglets developed clinical and pathological symptoms consistent with postweaning multisystemic wasting syndrome (PMWS) between 14 and 21 days post-inoculation (p.i.), and were characterised as PMWS-affected. Only these four PMWS-affected piglets, but neither the non-symptomatic infected nor control animals, developed a clear leukopenia. Kinetic analysis demonstrated a clear loss of both CD21(+) B and CD3(+) T lymphocytes in the PMWS-affected piglets. By CD3/CD4/CD8 triple labelling, the influence of PCV2 infection on all T cell sub-populations was discernible. A loss of CD3(+)CD4(+)CD8(+) memory/activated Th lymphocytes was particularly notable. However, all T lymphocyte sub-populations-CD3(+)CD4(+)CD8(+) memory Th, CD3(+)CD4(+)CD8(-) nai;ve Th, CD3(+)CD4(-)CD8(+) Tc and CD3(+)CD4(-)CD8(-) gammadelta TCR(+) lymphocytes-were susceptible to PCV2 infection-induced lymphopenia. CD3(-)CD4(-)CD8(+) NK cells were also depleted in the PMWS-affected animals, but granulocytes and monocytes were less affected. In conclusion, PCV2 infection induces primarily a lymphopenia, but only in animals which subsequently develop PMWS. The lymphopenia can be identified early p.i., particularly with the B lymphocytes. Memory/activated Th lymphocytes might be affected more than the other T cell sub-populations, but as time progressed a collapse of both T and B cell populations was clear.
