Identification of key amino acids of the mouse mammary tumor virus superantigen involved in the specific interaction with T-cell receptor V(beta) domains.

Mouse mammary tumor virus (MMTV) is a retrovirus encoding a superantigen that is recognized in association with major histocompatibility complex class II by the variable region of the beta chain (V(beta)) of the T-cell receptor. The C-terminal 30 to 40 amino acids of the superantigen of different MMTVs display high sequence variability that correlates with the recognition of particular T-cell receptor V(beta) chains. Interestingly, MMTV(SIM) and mtv-8 superantigens are highly homologous but have nonoverlapping T-cell receptor V(beta) specificities. To determine the importance of these few differences for specific V(beta) interaction, we studied superantigen responses in mice to chimeric and mutant MMTV(SIM) and mtv-8 superantigens expressed by recombinant vaccinia viruses. We show that only a few changes (two to six residues) within the C terminus are necessary to modify superantigen recognition by specific V(beta)s. Thus, the introduction of the MMTV(SIM) residues 314-315 into the mtv-8 superantigen greatly decreased its V(beta)12 reactivity without gain of MMTV(SIM)-specific function. The introduction of MMTV(SIM)-specific residues 289 to 295, however, induced a recognition pattern that was a mixture of MMTV(SIM)- and mtv-8-specific V(beta) reactivities: both weak MMTV(SIM)-specific V(beta)4 and full mtv-8-specific V(beta)11 recognition were observed while V(beta)12 interaction was lost. The combination of the two MMTV(SIM)-specific regions in the mtv-8 superantigen established normal MMTV(SIM)-specific V(beta)4 reactivity and completely abolished mtv-8-specific V(beta)5, -11, and -12 interactions. These new functional superantigens with mixed V(beta) recognition patterns allowed us to precisely delineate sites relevant for molecular interactions between the SIM or mtv-8 superantigen and the T-cell receptor V(beta) domain within the 30 C-terminal residues of the viral superantigen.

Suppression of short interfering RNA-mediated gene silencing by the structural proteins of hepatitis C virus.

Viruses have evolved strategies to overcome the antiviral effects of the host at different levels. Besides specific defence mechanisms, the host responds to viral infection via the interferon pathway and also by RNA interference (RNAi). However, several viruses have been identified that suppress RNAi. We addressed the question of whether hepatitis C virus (HCV) suppresses RNAi, using cell lines constitutively expressing green fluorescent protein (GFP) and inducibly expressing HCV proteins. It was found that short interfering RNA-mediated GFP gene silencing was inhibited when the entire HCV polyprotein was expressed. Further studies showed that HCV structural proteins, and in particular envelope protein 2 (E2), were responsible for this inhibition. Co-precipitation assays demonstrated that E2 bound to Argonaute-2 (Ago-2), a member of the RNA-induced silencing complex, RISC. Thus, HCV E2 that interacts with Ago-2 is able to suppress RNAi.

Binding of low concentration of peptide to H-2Kd produced in insect cells requires mouse beta 2-microglobulin co-expression.

A recombinant baculovirus expressing the murine class I MHC heavy chain H-2Kd cDNA under the transcriptional control of Autografa californica nuclear polyhedrosis virus (AcNPV) polyhedrin promoter has been isolated and used to infect Sf9 lepidopteran cells either alone or in association with a previously isolated virus expressing mouse beta 2-microglobulina (beta 2-ma). When infected with the heavy chain-encoding virus alone, H-2Kd was produced in a beta 2-m-free conformation detected on the surface of infected cells by conformation-independent antibodies. When Sf9 cells were co-infected with both viruses, approximately 10% of the heavy chain pool was engaged in the formation of native heterodimeric MHC class I molecules, which were glycosylated and transported to the cell surface as demonstrated by radio-binding experiments and flow cytometry. The assembly of the recombinant class I molecule was dependent on peptide, since heterodimer formation was brought about by H-2Kd-specific peptide ligands both in vivo, upon incubation with dually infected cells, and in vitro, in cell-free detergent extracts. In addition, a change in heavy chain conformation was brought about upon incubation with high concentrations (100 microM) of an H-2Kd-restricted octapeptide epitope from Plasmodium berghei. Furthermore, using low concentrations (3 nM) of a photoaffinity label derivative of this peptide, we show direct binding to cells co-expressing class I heavy chain and mouse beta 2-m but not to cells expressing free heavy chain only.

Hepatitis E virus: a zoonosis adapting to humans.

Hepatitis E virus (HEV) infection is gaining global attention, not only because of the increasing burden of the disease in low endemicity countries, in terms of morbidity and mortality rates, but also due to recent advances in the molecular virology and epidemiology of this emerging pathogen. HEV infection spread can be described as the evolution of a zoonosis towards an established human infection. As known from other viruses, such as the human immunodeficiency virus or the influenza viruses, crossing the species barriers from animals to humans is a recurrent phenomenon. Albeit slow at the beginning, once the virus has adapted to humans, the person-to-person spread can proceed very quickly. Although an optimal cell culture system for HEV is not yet available, outstanding progress has been made with the in vitro expression of HEV-like particles. These new tools have fostered new research to understand the molecular, structural and immunological aspects of human HEV infection. Although some promising data from Phase II vaccine trials are available, recent discoveries will certainly open new avenues for HEV-specific prophylaxis and therapy.

Superantigen-induced immune stimulation amplifies mouse mammary tumor virus infection and allows virus transmission.

Endogenous and infectious mouse mammary tumor viruses (MMTVs) encode in their 3' long terminal repeat a protein that exerts superantigen activity; that is, it is able to interact with T cells via the variable domain of the T cell receptor (TCR) beta chain. We show here that transmission of an infectious MMTV is prevented when superantigen-reactive cells are absent through either clonal deletion due to the expression of an endogenous MTV with identical superantigen specificity or exclusion due to expression of a transgenic TCR beta chain that does not interact with the viral superantigen. A strict requirement for superantigen-reactive T cells is also seen for a local immune response following MMTV infection. This immune response locally amplifies the number of MMTV-infected B cells, most likely owing to their clonal expansion. Collectively, our data indicate that a superantigen-induced immune response is critical for the MMTV life cycle.

Hepatitis B and C virus coinfection: a novel model system reveals the absence of direct viral interference.

Coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) has been associated with severe liver disease and frequent progression to cirrhosis and hepatocellular carcinoma. Clinical evidence suggests reciprocal replicative suppression of the two viruses, or viral interference. However, interactions between HBV and HCV have been difficult to study due to the lack of appropriate model systems. We have established a novel model system to investigate interactions between HBV and HCV. Stable Huh-7 cell lines inducibly replicating HBV were transfected with selectable HCV replicons or infected with cell culture-derived HCV. In this system, both viruses were found to replicate in the same cell without overt interference. Specific inhibition of one virus did not affect the replication and gene expression of the other. Furthermore, cells harboring replicating HBV could be infected with cell culture-derived HCV, arguing against superinfection exclusion. Finally, cells harboring replicating HBV supported efficient production of infectious HCV. Conclusion: HBV and HCV can replicate in the same cell without evidence for direct interference in vitro. Therefore, the viral interference observed in coinfected patients is probably due to indirect mechanisms mediated by innate and/or adaptive host immune responses. These findings provide new insights into the pathogenesis of HBV-HCV coinfection and may contribute to its clinical management in the future.

MHC class II hierarchy of superantigen presentation predicts efficiency of infection with mouse mammary tumor virus.

Superantigens (SAgs) encoded by infectious mouse mammary tumor viruses (MMTVs) play a crucial role in the viral life cycle. Their expression by infected B cells induces a proliferative immune response by SAg-reactive T cells which amplifies MMTV infection. This response most likely ensures stable MMTV infection and transmission to the mammary gland. Since T cell reactivity to SAgs from endogenous Mtv loci depends on MHC class II molecules expressed by B cells, we have determined the ability of MMTV to infect various MHC congenic mice. We show that MHC class II I-E+ compared with I-E- mouse strains show higher levels of MMTV infection, most likely due to their ability to induce a vigorous SAg-dependent immune response following MMTV encounter. Inefficient infection is observed in MHC class II I-E- mice, which have been shown to present endogenous SAgs poorly. Therefore, during MMTV infection the differential ability of MHC class II molecules to form a functional complex with SAg determines the magnitude of the proliferative response of SAg-reactive T cells. This in turn influences the degree of T cell help provided to infected B cells and therefore the efficiency of amplification of MMTV infection.

T cell receptor V beta repertoire in mice lacking endogenous mouse mammary tumor provirus.

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) V beta segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, -7, -8 and -9) and 38CH (Mtv-) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, -7, -8 or -9. The TcR V beta chain (TcR V beta) usage in these mice was analyzed using monoclonal antibodies specific for TcR V beta 2, V beta 3, V beta 4, V beta 5, V beta 6, V beta 7, V beta 8, V beta 11, V beta 12 and V beta 14 segments. Both Mtv-8+ mice and Mtv-9+ mice deleted TcR V beta 5+ and V beta 11+ T cells. Moreover, we also observed the deletion of TcR V beta 12+ cells by Mtv-8 and Mtv-9 products. Mtv-6+ and Mtv-7+ animals deleted TcR V beta 3+ and V beta 5+ cells, and TcR V beta 6+, V beta 7+ and V beta 8.1+ cells, respectively. Unexpectedly, TcR V beta 8.2+ cells were also deleted in some backcross mice expressing Mtv-7. TcR V beta 8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn-->Asp) in position 19 on the TcR V beta 8.2 fragment. Reactivities of BALB.D2 TcR V beta 8.2 and 38CH TcR V beta 8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR V beta 2+, V beta 4+ and V beta 8+ CD4+ T cell subsets in Mtv-8+ and Mtv-9+ mice, and TcR V beta 4+ CD4+ T cells in Mtv-6+ and Mtv-7+ mice, when compared with the T cell repertoire of Mtv- mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.

Acute Hepatitis E Virus infection with coincident reactivation of Epstein-Barr virus infection in an immunosuppressed patient with rheumatoid arthritis: a case report.

BACKGROUND: Hepatitis E virus (HEV) is the most recently discovered of the hepatotropic viruses, and is considered an emerging pathogen in developed countries with the possibility of fulminant hepatitis in immunocompromised patients. Especially in the latter elevated transaminases should be taken as a clue to consider HEV infection, as it can be treated by discontinuation of immunosuppression and/or ribavirin therapy. To our best knowledge, this is a unique case of autochthonous HEV infection with coincident reactivation of Epstein-Barr virus (EBV) infection in an immunosuppressed patient with rheumatoid arthritis (RA). CASE PRESENTATION: A 68-year-old Swiss woman with RA developed hepatitis initially diagnosed as methotrexate-induced liver injury, but later diagnosed as autochthonous HEV infection accompanied by reactivation of her latent EBV infection. She showed confounding serological results pointing to three hepatotropic viruses (HEV, Hepatitis B virus (HBV) and EBV) that could be resolved by detection of HEV and EBV viraemia. The patient recovered by temporary discontinuation of immunosuppressive therapy. CONCLUSIONS: In immunosuppressed patients with RA and signs of liver injury, HEV infection should be considered, as infection can be treated by discontinuation of immunosuppression. Although anti-HEV-IgM antibody assays can be used as first line virological tools, nucleic acid amplification tests (NAAT) for detection of HEV RNA are recommended--as in our case--if confounding serological results from other hepatotropic viruses are obtained. After discontinuation of immunosuppressive therapy, our patient recovered from both HEV infection and reactivation of latent EBV infection without sequelae.

Emerging animal viruses: real threats or simple bystanders?

The list of animal viruses has been frequently added of new members raising permanent concerns to virologists and veterinarians. The pathogenic potential and association with disease have been clearly demonstrated for some, but not for all of these emerging viruses. This review describes recent discoveries of animal viruses and their potential relevance for veterinary practice. Dogs were considered refractory to influenza viruses until 2004, when an influenza A virus subtype H3N8 was transmitted from horses and produced severe respiratory disease in racing greyhounds in Florida/USA. The novel virus, named canine influenza virus (CIV), is considered now a separate virus lineage and has spread among urban canine population in the USA. A new pestivirus (Flaviviridae), tentatively called HoBi-like pestivirus, was identified in 2004 in commercial fetal bovine serum from Brazil. Hobi-like viruses are genetically and antigenically related to bovine viral diarrhea virus (BVDV) and induce similar clinical manifestations. These novel viruses seem to be widespread in Brazilian herds and have also been detected in Southeast Asia and Europe. In 2011, a novel mosquito-borne orthobunyavirus, named Schmallenberg virus (SBV), was associated with fever, drop in milk production, abortion and newborn malformation in cattle and sheep in Germany. Subsequently, the virus disseminated over several European countries and currently represents a real treat for animal health. The origin of SBV is still a matter of debate but it may be a reassortant from previous known bunyaviruses Shamonda and Satuperi. Hepatitis E virus (HEV, family Hepeviridae) is a long known agent of human acute hepatitis and in 1997 was first identified in pigs. Current data indicates that swine HEV is spread worldwide, mainly associated with subclinical infection. Two of the four HEV genotypes are zoonotic and may be transmitted between swine and human by contaminated water and undercooked pork meat. The current distribution and impact of HEV infection in swine production are largely unknown. Avian gyrovirus type 2 (AGV2) is a newly described Gyrovirus, family Circoviridae, which was unexpectedly found in sera of poultry suspected to be infected with chicken anemia virus (CAV). AGV2 is closely related to CAV but displays sufficient genomic differences to be classified as a distinct species. AGV2 seems to be distributed in Brazil and also in other countries but its pathogenic role for chickens is still under investigation. Finally, the long time and intensive search for animal relatives of human hepatitis C virus (HCV) has led to the identification of novel hepaciviruses in dogs (canine hepacivirus [CHV]), horses (non-primate hepaciviruses [NPHV] or Theiler's disease associated virus [TDAV]) and rodents. For these, a clear and definitive association with disease is still lacking and only time and investigation will tell whether they are real disease agents or simple spectators.

Genomic characterization of Brazilian hepatitis C virus genotypes 1a and 1b

Parts of 5' non-coding (5' NC) and of E1 envelope regions of the hepatitis C virus (HCV) genome were amplified from sera of 26 Brazilian anti-HCV antibody-positive patients using the reverse transcription-polymerase chain reaction (RT-PCR). Fourteen samples were PCR positive with primers from the 5' NC region and 8 of them were also positive with primers from the E1 region. A genomic segment of 176 bp from the E1 region of 7 isolates was directly sequenced from PCR products. The sequences were compared with those of HCV strains isolated in other countries and the Brazilian isolates were classified by phylogenetic analysis into genotypes 1a and 1b. This could have a clinical importance since it has been shown that individuals infected with type 1 viruses are less likely to respond to treatment with interferon than individuals infected with types 2 and 3 viruses. Two quasispecies isolated from the same patient with an interval of 13 months differed by two base substitutions (1.1%). The sequence of another isolate presented a three-nucleotide deletion at codon 329

Rotavirus and reovirus interaction with mouse peritoneal resident phagocytic cells

Rotaviruses and reoviruses are involved in human and animal diseases. It is known that both viruses penetrate the gastrointestinal tract but their interaction with phagocytic cells is unknown. To study this interaction, peritoneal resident phagocytic cells were used and rotavirus and reovirus replication in peritoneal phagocytic cells was observed. However, rotavirus replication in these cells led to the production of defective particles since MA-104 cells inoculated with rotavirus phagocytic cell lysate did not show any evidence of virus replication. On the basis of these results, we suggest that, although reovirus dissemination may be helped by these phagocytic cells, these cells may control rotavirus infection and probably contribute to the prevention of its dissemination.

Relationship of cytokines and cytokine signaling to immunodeficiency disorders in the mouse

The contributions of cytokines to the development and progression of disease in a mouse model of retrovirus-induced immunodeficiency (MAIDS) are controversial. Some studies have indicated an etiologic role for type 2 cytokines, while others have emphasized the importance of type 1 cytokines. We have used mice deficient in expression of IL-4, IL-10, IL-4 and IL-10, IFN-g, or ICSBP - a transcriptional protein involved in IFN signaling - to examine their contributions to this disorder. Our results demonstrate that expression of type 2 cytokines is an epiphenomenon of infection and that IFN-g is a driving force in disease progression. In addition, exogenously administered IL-12 prevents many manifestations of disease while blocking retrovirus expression. Interruption of the IFN signaling pathways in ICSBP-/- mice blocks induction of MAIDS. Predictably, ICSBP-deficient mice exhibit impaired responses to challenge with several other viruses. This immunodeficiency is associated with impaired production of IFN-g and IL-12. Unexpectedly, however, the ICSBP-/- mice also develop a syndrome with many similarities to chronic myelogenous leukemia in humans. The chronic phase of this disease is followed by a fatal blast crisis characterized by clonal expansions of undifferentiated cells. ICSBP is thus an important determinant of hematopoietic growth and differentiation as well as a prominent signaling molecule for IFNs

Correlates of protective immunity against hepatitis C virus

Le virus de l’hépatite C (VHC) infecte ~185 millions d’individus dans le monde. Malgré le développement des nouvelles thérapies dirigées contre le VHC, on compte deux millions de nouvelles infections chaque année, en particulier dans les pays sous-développés et les populations marginalisées. Comme pour la plupart des virus à infection chronique, le développement d’un vaccin prophylactique efficace est limité par le manque de caractérisation des déterminants de la mémoire immunitaire protectrice lors des épisodes de réinfection naturelle chez les êtres humains. Le VHC représente un modèle unique au sein des virus à infection chronique pour étudier l’immunité protectrice. En effet ~30% des patients infectés par le VHC peuvent être guéris suite à un premier épisode d’infection spontanément. Dans cette thèse, nous avons étudié l’immunité protectrice contre le VHC dans une cohorte d’utilisateurs de drogues par injection qui sont à risque d’être infectés ou réinfectés. Notre hypothèse est que la majorité des patients qui ont résolu une première infection par le VHC sont protégés contre le développement d’une infection chronique s’ils sont réexposés. Cette immunité protectrice est associée à la présence des cellules T CD4 et CD8 polyfonctionnelles qui possèdent des fréquences, magnitudes et avidités élevées. La capacité protectrice des cellules T mémoire contre les séquences variables du VHC est dépendante de la diversité et flexibilité du répertoire de leurs récepteurs de cellules T (TCR), qui reconnaissent les séquences variables des épitopes ciblés. Notre premier objectif était de définir et détailler les déterminants de l’immunité protectrice conférée par les cellules T spécifiques du VHC. Nos résultats ont montré que la protection pendant l’épisode de réinfection était associée à une augmentation de la magnitude et du spectre des réponses spécifiques par les cellules T CD4 et CD8 polyfonctionnelles, ainsi que par l’apparition d’une population de cellules T tétramère+ CD8+ effectrices qui expriment faiblement le marqueur CD127 (CD127lo) lors du pic de la réponse. Chez les patients qui ont développé une infection chronique pendant l’épisode de réinfection, nous avons observé une expansion très limitée des cellules T CD4 et CD8. Le séquençage des épitopes ciblés par les cellules T CD8 chez ces patients qui sont non-protégés a montré que les séquences de ces épitopes sont différentes des séquences de référence qui étaient utilisées pour tous les essais immunologiques de cette étude. Le deuxième objectif était d’analyser la dynamique du répertoire des TCRs des cellules T CD8 spécifiques chez les patients protégés versus les patients non-protégés. Nos résultats ont montré que le répertoire des cellules T CD8 spécifiques est plus focalisé que chez les patients protégés. En plus, nous avons observé que les clonotypes qui forment le répertoire chez les patients protégés sont distincts de ceux chez les patients non-protégés. Ces clonotypes chez les patients protégés ont montré de plus grandes avidité et polyfonctionnalité que leurs homologues chez les patients non-protégés. En conclusion, nos résultats suggèrent que la protection contre le développement d’une infection chronique pendant l’épisode de réinfection par le VHC est associée à une augmentation de la magnitude, du spectre et de la fonctionnalité des réponses des cellules T spécifiques, ainsi qu’à un répertoire des TCRs plus focalisé composé des clonotypes distincts qui possèdent de plus grandes avidité et polyfonctionnalité que chez les patients non-protégés. L’homologie des séquences des souches virales entre les différents épisodes de l’infection est un déterminant majeur de l’établissement d’une protection efficace. Ces résultats ont donc des implications très importantes pour le développement d’un vaccin prophylactique contre le VHC et d’autres virus à infection chronique.