Characterisation of Mhc class I and class II DRB polymorphism in red-bellied tamarins (Saguinus labiatus).

The infection of red-bellied tamarins (Saguinus labiatus) with GB virus B (GBV-B) is an important surrogate model of hepatitis C virus infection in man. To fully exploit the value of this model, we have characterised MHC class I G and class II DRB alleles in eight tamarins representing a cross-section of a UK breeding colony. The results indicated a high degree of classes I and II DRB allele sharing. Each animal transcribed three to four putative surface-expressed class I alleles and two to four class II DRB alleles. Most animals also transcribed at least one class I allele predicted to result in a C-terminal truncated protein. These results represent the first description of MHC polymorphism in this species and provide a foundation for characterisation of MHC diversity in breeding populations of red-bellied tamarins. The data will facilitate the identification of associations between MHC polymorphism and control of viral infections, and detailed dissection of cellular immune responses against GBV-B.

In Vivo Characterization of a Bank Vole-Derived Cowpox Virus Isolate in Natural Hosts and the Rat Model.

Cowpox virus (CPXV) belongs to the genus Orthopoxvirus in the Poxviridae family and is endemic in western Eurasia. Based on seroprevalence studies in different voles from continental Europe and UK, voles are suspected to be the major reservoir host. Recently, a CPXV was isolated from a bank vole (Myodes glareolus) in Germany that showed a high genetic similarity to another isolate originating from a Cotton-top tamarin (Saguinus oedipus). Here we characterize this first bank vole-derived CPXV isolate in comparison to the related tamarin-derived isolate. Both isolates grouped genetically within the provisionally called CPXV-like 3 clade. Previous phylogenetic analysis indicated that CPXV is polyphyletic and CPXV-like 3 clade represents probably a different species if categorized by the rules used for other orthopoxviruses. Experimental infection studies with bank voles, common voles (Microtusarvalis) and Wistar rats showed very clear differences. The bank vole isolate was avirulent in both common voles and Wistar rats with seroconversion seen only in the rats. In contrast, inoculated bank voles exhibited viral shedding and seroconversion for both tested CPXV isolates. In addition, bank voles infected with the tamarin-derived isolate experienced a marked weight loss. Our findings allow for the conclusion that CPXV isolates might differ in their replication capacity in different vole species and rats depending on their original host. Moreover, the results indicate host-specific differences concerning CPXV-specific virulence. Further experiments are needed to identify individual virulence and host factors involved in the susceptibility and outcome of CPXV-infections in the different reservoir hosts.

Cell culture models and animal models of viral hepatitis. Part II: hepatitis C.

The lack of a preventive vaccine, coupled with common unresponsiveness to treatment and coinfection with HIV, has made HCV a major threat to public health. The authors review in vitro and in vivo models that are being used to study HCV and to develop new treatments and preventive measures.

GB virus B as a model for hepatitis C virus.

GB viruses A and B (GBV-A and GBV-B) are members of the Flaviviridae family and are isolated from tamarins injected with serum from a human hepatitis patient. Along with a related human virus, GB virus C, or alternatively, hepatitis G virus (GBV-C/HGV), the three viruses represent the GB agents. Of the three viruses, GBV-B has been proposed as a potential surrogate model for the study of hepatitis C virus (HCV) infections of humans. GBV-B is phylogenetically most closely related to HCV and causes an acute, self-resolving hepatitis in tamarins as indicated by an increase in alanine aminotransferase and changes in liver histology. Similarities between GBV-B and HCV are found at the nucleotide sequence level with the two viruses sharing 28% amino acid homology over the lengths of their open reading frames. Short regions have even higher levels of homology that are functionally significant as shown by the ability of the GBV-B NS3 protease to cleave recombinant HCV polyprotein substrates. The shared protease substrate specificities suggest that GBV-B may be useful in testing antiviral compounds for activity against HCV. Although there are numerous similarities between GBV-B and HCV, there are important differences in that HCV frequently causes chronic infections in people, whereas GBV-B appears to cause only acute infections. The acute versus chronic course of infection may point to important differences between the two viruses that, along with the numerous similarities, will make GBV-B in tamarins a good surrogate model for HCV.

Changes in immune cell populations in the periphery and liver of GBV-B-infected and convalescent tamarins (Saguinus labiatus).

Flaviviruses related to hepatitis C virus (HCV) in suitable animal models may provide further insight into the role that cellular immunity contributes to spontaneous clearance of HCV. We characterised changes in lymphocyte populations in tamarins with an acute GBV-B infection, a hepatitis virus of the flaviviridae. Major immune cell populations were monitored in peripheral and intra-hepatic lymphocytes at high viraemia or following a period when peripheral virus was no longer detected. Limited changes in major lymphocyte populations were apparent during high viraemia; however, the proportions of CD3(+) lymphocytes decreased and CD20(+) lymphocytes increased once peripheral viraemia became undetectable. Intrahepatic lymphocyte populations increased at both time points post-infection. Distinct expression patterns of PD-1, a marker of T-cell activation, were observed on peripheral and hepatic lymphocytes; notably there was elevated PD-1 expression on hepatic CD4(+) T-cells during high viraemia, suggesting an activated phenotype, which decreased following clearance of peripheral viraemia. At times when peripheral vRNA was not detected, suggesting viral clearance, we were able to readily detect GBV-B RNA in the liver, indicative of long-term virus replication. This study is the first description of changes in lymphocyte populations during GBV-B infection of tamarins and provides a foundation for more detailed investigations of the responses that contribute to the control of GBV-B infection.

Fatal cowpox virus infection in cotton-top tamarins (Saguinus oedipus) in Germany.

Cowpox virus (CPXV) was isolated from a fatal outbreak among cotton-top tamarins. Samples from healthy common marmosets in contact were also CPXV genome positive. The CPXV isolated from the cotton-top tamarins exhibited a unique hemagglutinin sequence. Pathogenicity investigations using a Wistar rat model characterized the isolate as low pathogenic.

A cooperative interaction between nontranslated RNA sequences and NS5A protein promotes in vivo fitness of a chimeric hepatitis C/GB virus B.

GB virus B (GBV-B) is closely related to hepatitis C virus (HCV), infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5' nontranslated RNAs (NTRs) of these viruses fold into four similar structured domains (I-IV), with domains II-III-IV comprising the viral internal ribosomal entry site (IRES). We previously reported the in vivo rescue of a chimeric GBV-B (vGB/III(HC)) containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/III(HC) genome (within the 3'NTR, upstream of the poly(U) tract, and NS5A coding sequence) are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s) underlying these compensatory mutations, and to determine whether 5'NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5'NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5'NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3'NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/III(HC) RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5'NTR, 3'NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses.

Immunity against the GBV-B hepatitis virus in tamarins can prevent productive infection following rechallenge and is long-lived.

GB virus-B (GBV-B) is the virus most closely related to hepatitis C virus (HCV). Thus, we have used GBV-B infection of tamarins, which develop acute hepatitis following experimental infection, as a surrogate model to study protective immunity. As challenge virus, we first produced a GBV-B pool from an infected tamarin, which was not infected with the related GBV-A viruses. Its infectivity titer was 10(6.6) tamarin 50% infectious doses per ml. Next, two tamarins that were convalescent from recombinant GBV-B infection were re-challenged. In the original infection viremia persisted for 8 and 12 weeks, respectively, and both animals developed moderately severe hepatitis. Each tamarin was re-challenged four times with 10(4.3) tamarin 50% infectious doses of the GBV-B challenge virus. In one animal, each re-challenge produced 1-2 weeks of viremia; hepatitis was observed following the first re-challenge. In the other animal, however, only the first re-challenge produced viremia, lasting 1 week. During the primary infection, peak GBV-B titers were about 10(8) genome equivalents/ml in both animals; following re-challenges, peak titers ranged from 10(3) to 10(6) genome equivalents/ml. Analysis of the polyprotein sequence of viruses recovered from both animals following the first re-challenge demonstrated that these did not represent immune escape variants since mutations were not detected. Neutralization studies suggested that the immunity was not humoral in nature. We also demonstrated that the immunity was long-lived: 1 year after the fourth challenge, the animal with sterilizing immunity had low titer viremia for only 1 week following an additional challenge.

A chimeric GB virus B with 5' nontranslated RNA sequence from hepatitis C virus causes hepatitis in tamarins.

Only humans and chimpanzees are fully permissive for replication of hepatitis C virus (HCV), an important cause of liver cirrhosis and cancer worldwide. The absence of suitable animal models limits opportunities for in vivo evaluation of candidate hepatitis C therapeutics and slows progress in the field. Here, we describe a chimeric virus derived from GB virus B (GBV-B), an unclassified hepatotropic member of the family Flaviviridae that is closely related to HCV and infects tamarins (Saguinus sp.), in which a functionally important HCV regulatory sequence replaced an analogous sequence in the 5' nontranslated region (5'NTR) of the GBV-B genome. The transplanted sequence comprised domain III of the internal ribosome entry site (IRES), which directly binds the 40S ribosome subunit and is a target for candidate therapeutics. The chimeric 5'NTR retained ribosome binding activity and was competent in directing protein translation both in cell-free translation reactions and in transfected primary tamarin hepatocyte cultures. Virus rescued from the chimeric RNA replicated in the liver of tamarins, causing biochemical and histopathological changes typical of viral hepatitis. However, adaptive mutations were required elsewhere in the genome for efficient replication. Virus was not rescued from other, translationally competent, chimeric RNAs in which domain II of the IRES was exchanged. Thus, the 5'NTR appears to contain virus-specific replication signals that interact with other sites within the viral genome or with viral proteins. In conclusion, such novel chimeric flaviviruses offer opportunities for new insights into HCV replication mechanisms, while potentially facilitating the evaluation of candidate therapeutics in vivo.

Experimental norovirus infections in non-human primates.

Noroviruses, with Norwalk virus as the prototype strain, are the most common cause of viral gastroenteritis in people of all ages. Limited information on the immunology of Norovirus infections has been obtained by studies both in the natural setting and in experimentally infected volunteers. Interpretation of these studies is difficult due to the lack of information on the history of Norovirus exposure and the cross-reactivity of antibodies. An animal model for Norovirus infections would be important to study the immune response, e.g., for vaccine assessment. In the present study the susceptibility of common marmosets, cotton top tamarins, cynomolgus, and rhesus macaques to Norovirus infection was tested. Following oral inoculation, low level replication may have occurred in common marmosets and cotton top tamarins but not in cynomolgus macaques, based on short-term viral shedding; neither clinical symptoms nor antibody responses were observed in these species. In contrast, rhesus macaques were found susceptible to Norwalk virus infection as one animal shed virus for a longer period of time and developed Norwalk virus specific IgM and IgG responses. Further research on Norovirus susceptibility in rhesus macaques may yield an animal model to study the immune response and pathogenesis after Norovirus infection.

Rabies in tamarins (Callithrix jacchus) in the state of Ceará, Brazil, a distinct viral variant?

Presently, the State of Ceará reports the largest percentage of human rabies cases originated from wild animals in Brazil, transmitted by the principal simian species, the tamarin (Callithrix jacchus), found in various locations throughout the State, but concentrated along the coast. Epidemiological studies indicated that possibly the same virus caused the deaths in humans and non-human primates. This rabies virus seem to be different from all other identified so far.

Novel gamma-1 herpesviruses identified in free-ranging new world monkeys (golden-handed tamarin [Saguinus midas], squirrel monkey [Saimiri sciureus], and white-faced saki [Pithecia pithecia]) in French Guiana.

The recent finding of a novel Epstein-Barr virus-related lymphocryptovirus (CalHV-3) in a captive colony of common marmoset (Callithrix jacchus) in the United States modifies the view that the host range of lymphocryptovirus is restricted to humans and Old World primates. We investigated the presence of Epstein-Barr virus-related viruses in 79 samples of New World monkeys caught in the wild, including six species of the Cebidae family and one of the Callitrichidae, living in the rain forest of French Guiana. Using a degenerate consensus PCR method for the herpesvirus DNA polymerase gene, we identified three novel lymphocryptoviruses from golden-handed tamarin (Saguinus midas) of the Callitrichidae family and squirrel monkey (Saimiri sciureus) and white-faced saki (Pithecia pithecia) of the Cebidae family. With the CalHV-3 strain, these three novel viruses constitute a well-supported phylogenetic clade in the Lymphocryptovirus genus, which is clearly distinct from the lineage of Old World lymphocryptovirus, hosted by catarrhine monkeys and humans. In tamarins, the prevalence of the novel lymphocryptovirus was more than 50%, indicating that it circulates well in the wild population, perhaps due to specific ecoethological patterns such as confrontations and intergroup migration. The detection and partial molecular characterization of the polymerase gene of three novel Gamma-1-Herpesvirinae from New World monkeys caught in the wild clearly indicate that free-ranging populations of platyrrhine are natural hosts of lymphocryptoviruses. Further characterization of these novel viruses will provide new insight not only into the origin and evolution of Gammaherpesvirinae but also into their pathogenicity.

Five new or recently discovered (GBV-A) virus species are indigenous to New World monkeys and may constitute a separate genus of the Flaviviridae.

In previous studies, human hepatitis viruses have been experimentally transmitted to New World monkeys of the genus Saguinus (tamarins). Recently, two Flaviviridae-like agents (GBV-A and GBV-B) were identified in tamarins that developed hepatitis following inoculation with serum of the 11th tamarin passage of a potentially new human hepatitis agent. However, it was not shown that these viruses originated from the initial inoculum. We here report the discovery of indigenous species-specific viruses related to GBV-A in several species of New World monkeys and suggest that GBV-A virus was fortuitously acquired during passage in tamarins. Sera or plasma from 98 wild-caught New World monkeys representing 10 different species was tested by RT-PCR with conserved degenerate primers to the 5' noncoding region of the genome. Viral sequences were identified in 33 animals and sequence analysis was performed on the amplicons. In addition, the genomic region corresponding to the putative NS3 RNA helicase of GBV-A was amplified from most positive animals and sequenced. We detected GBV-A-like viruses in 13 (35%) of 37 S. mystax, 7 (78%) of 9 S. nigricollis, 3 (25%) of 12 S. labiatus, 2 (50%) of 4 S. oedipus, 2 (100%) of 2 Callithrix jacchus, and 6 (50%) of 12 Aotus trivirgatus monkeys. Each positive animal was infected with a unique strain of the GBV-A-like viruses. Analysis of the 5' NC and NS3 helicase sequences revealed that these viruses could be classified into 5 major genetic groups with genetic distances equivalent to or greater than those found among major genetic groups of hepatitis C virus. Species-specific GBV-A-like viruses were found in S. mystax, S. nigricollis, S. oedipus, C. jacchus, and A. trivirgatus species. The viruses specific for S. nigricollis were closely related to GBV-A, suggesting that GBV-A was acquired by passage through this species during the initial transmission studies. The natural history of the GBV-A-like viruses was studied in serial serum samples from 9 S. mystax and 2 A. trivirgatus monkeys. Each animal was chronically infected and the viral strain did not vary during 9-27 months of follow-up. Finally, we demonstrated that four S. mystax were positive upon arrival to the United States from the country of origin. No apparent disease was associated with chronic infection of the GBV-A-like viruses. In conclusion, many New World monkeys are persistently infected with indigenous species-specific viruses that may represent a new genus within the virus family Flaviviridae.

Importance of amino acid 216 in nonstructural protein 2B for replication of hepatitis A virus in cell culture and in vivo.

Clinical isolates of hepatitis A virus (HAV) replicate inefficiently in cell culture unless mutations are acquired throughout the genome. An Ala-to-Val substitution in the nonstructural protein 2B (2B-216) was known to have a major impact on replication in cell culture. Analysis of chimeric viruses confirmed that the 2B-A[216]V change was critical for efficient replication and that Leu or Ile could substitute for Val. Viruses containing Val, Ile, or Leu at 2B-216 all replicated with similar kinetics in cell culture, whereas the virus containing Ala at this position grew 10- to 20-fold less efficiently. In contrast, in vivo, virus with either Ala or Val at 2B-216 replicated equally efficiently when tested in a chimpanzee and in tamarins, and each amino acid was stably maintained. Attempts to complement wild-type 2B in trans with adapted 2B provided by co-infection with a second viable HAV mutant failed to enhance replication of the virus containing the wild-type 2B sequence.

Species-specific variants of GB virus A in captive monkeys .

Sequences from the putative 5' nontranslated region of GB virus A were isolated from mystax, owl monkeys, and tamarins. Though sequences of isolates from each animal species are virtually identical at the nucleotide level (95%), isolates from different species are dramatically different (52 to 79% identical) and genetically cluster on this basis.

In vivo analysis of the 3' untranslated region of GB virus B after in vitro mutagenesis of an infectious cDNA clone: persistent infection in a transfected tamarin.

GB virus B (GBV-B), the virus most closely related to hepatitis C virus (HCV), infects tamarins and causes acute hepatitis. The 3' untranslated region (UTR) of an infectious GBV-B clone (pGBB) has a proximal short sequence followed by a poly(U) tract and a 3' terminal sequence. Our investigators previously demonstrated that the 3' terminal sequence was critical for in vivo infectivity. Here, we tested the effect of deleting the short sequence and/or the poly(U) tract from pGBB; infectivity of each mutant was tested by intrahepatic transfection of two tamarins with transcribed RNA. A mutant lacking both regions was not viable. However, mutants lacking either the short sequence or the poly(U) tract were viable. All four tamarins had a wild-type-like acute infection and developed acute hepatitis. Whereas we found that five tamarins transfected with the wild-type clone pGBB had acute resolving infection, one tamarin transfected with the poly(U) deletion mutant became persistently infected. This animal had viremia and hepatitis until its death at week 90. The genomes recovered at weeks 2, 7, 15, 20, 60, and 90 lacked the poly(U) stretch. Eight amino acid changes were identified at week 90. One change, in the putative p7 protein, was dominant at week 15. Thus, persistence of GBV-B, like persistence of HCV, was associated with the emergence of virus variants. Four tamarins inoculated with serum collected at weeks 2 and 90 from the tamarin with persistent infection had an acute resolving infection. Nonetheless, the demonstration that GBV-B can persist in tamarins strengthens its relevance as a surrogate model for the study of HCV.

Hepatitis A virus infection in tamarins: experimental transmission via contaminated factor VIII concentrates.

An experimental hepatitis A virus (HAV) transmission study was performed in 3 tamarins, using a factor VIII concentrate linked to a recent outbreak of HAV infections in German hemophiliacs. The typical indicators for HAV infection were investigated in feces and serum samples. One tamarin showed a classical HAV infection with seroconversion. HAV antigen and HAV RNA were detected in feces of a second animal, but no seroconversion was observed until 19 weeks after inoculation. The HAV sequences from the reverse-transcription- and polymerase chain reaction-positive samples of the 2 animals were identical to the deduced HAV sequences of the chain of infection (from plasma pool to final product to patients). The study results provide conclusive evidence of the presence of infectious HAV in coagulation factor concentrate. Because a number of HAV transmission episodes have been described for solely solvent/detergent-treated factor VIII preparations, continued use of these agents seems questionable.

Virus-specific cytotoxic T cell responses are associated with immunity of the cottontop tamarin to Epstein-Barr virus (EBV).

The cytotoxic responses of peripheral blood lymphocytes from cottontop tamarins to in vitro restimulation with autologous lymphoblastoid cell lines (LCL) were assayed. Lymphocytes from immune tamarins that had recovered from EBV challenge developed potent cytotoxicity for natural killer (NK) cell targets and for autologous LCL. The cytotoxicity for LCL targets was EBV-specific, as B cell blasts uninfected with EBV were not killed. The cell lines could be maintained by repeated stimulation with LCL and the addition of IL-2. Flow cytometry showed that they were T cell lines expressing CD2, CD3, CD4, CD8 and CD25. Dual-colour flow cytometry revealed two subpopulations, one CD4+ CD8+ population and the other CD4- CD8+. After separation by magnetic cell sorting both subpopulations were shown to be cytotoxic and the CD4+ CD8+ fraction was also shown to be MHC class II-restricted; the MHC restriction of the CD8+ subpopulation could not be determined. The unseparated T cells and both the subpopulations were able to inhibit LCL outgrowth in vitro. In contrast, PBL from naive tamarins stimulated by autologous LCL developed less NK cell cytotoxicity and little cytotoxicity for LCL. The cytotoxic response was enhanced at higher levels of LCL stimulation, but the cells were unable to inhibit LCL outgrowth in vitro. We conclude that cytotoxic responses capable of inhibiting LCL growth in vitro correlate with in vivo immunity in the tamarin model and provide a basis for understanding the mechanism of vaccine-induced immune protection.

TT viruses (TTV) of non-human primates and their relationship to the human TTV genotypes.

Sera from eight different non-human primate species, in total 216 samples, were analysed for the presence of TT virus (TTV) sequences. A very high incidence of TTV infection was found in sera from both common chimpanzees and pygmy chimpanzees, 48.8% and 66.7%, respectively. Sequence analysis of PCR fragments from two pygmy chimpanzees and seven common chimpanzees resulted in a total of 14 different TTV sequences. Phylogenetic analysis, including human TTV of all known genotypes, revealed that: (i) TTV from pygmy chimpanzees are closely related to viruses from human genotypes 2 and 3; (ii) TTV sequences obtained from common chimpanzees cluster together with human TTV genotypes 5 and 6, the latter only at the protein level; (iii) TTV from the common chimpanzee subspecies Pan troglodytes verus and Pan troglodytes schweinfurthii cluster together, suggesting an ancient host-pathogen relationship before subspeciation 1.6 million years ago; and (iv) TTV of common and pygmy chimpanzees may have been acquired by these animals in different zoonotic events not longer than 2.5 million years ago.

The sequence and genomic organization of a GB virus A variant isolated from captive tamarins.

Recently, gene fragments of several novel variants of GB virus A were isolated from the serum of distinct monkey species that had not been experimentally inoculated with an infectious agent. These variants appeared to be species-specific in that sequences isolated within a species were virtually identical, though sequences were strikingly different when compared between each species. In the present study, the nucleotide sequence of one of these variants, GBV-Alab, was extended to near-genome length. Similar to the other GB viruses, GBV-Alab appears to encode a single large polyprotein of 2967 amino acids that is post-translationally cleaved by cellular and viral proteases into the individual viral proteins. The structural proteins are found at the N-terminal end of the polyprotein, while the nonstructural proteins are found at the C teminus. Amino acid sequence comparisons of the large polyprotein demonstrate that GBV-Alab is 74% identical to GBV-A and 48% identical to GBV-C, sharing only marginal identity with GBV-B and HCV-1 at 27%. Examination of the GBV-Alab polyprotein reveals that structural motifs are conserved for a protease, a helicase and a replicase. Phylogenetic analysis of the polyprotein confirms previous results that GBV-Alab is a member of the Flaviviridae, distinct from GBV-B and HCV, though more closely related to GBV-A and GBV-C.