The identification and characterization of animal virus homologs can provide insights into the pathogenesis of human viruses and, in some instances, in vivo
models for investigating methods for the prevention and treatment of human disease (19
). Examples where well-characterized animal viruses have provided such insights include simian immunodeficiency virus, animal poxviruses, herpesviruses, murine norovirus, and woodchuck hepatitis virus (20
). HCV, in contrast, has no satisfactory homolog (6
), and only chimpanzees can be experimentally infected with HCV (22
). Even before the recent U.S. Institute of Medicine recommendations to restrict the use of chimpanzees for biomedical research, limited access to these animals was a challenge for HCV research. NPHV and GBV-B are the most genetically similar to HCV (6
) and could therefore be used as surrogate models of HCV infection. The natural host of NPHV is the horse (6
), in which high frequencies of viremia (from 3 to 8%) have been reported in separate studies (17
). GBV-B was initially detected in a laboratory tamarin (New World monkeys of the family Callitrichidae
). However, subsequent attempts to identify its natural host that concentrated primarily on the screening of New World primates have been unsuccessful. Nonetheless, GBV-B-infected tamarins and marmosets have been used as surrogate models for HCV pathogenesis. The identification of rodent hepaciviruses may finally provide a promising small-animal model for the study of hepaciviruses, with possible relevance to HCV.
Here we identified several lineages of RHV in deer mice that are as highly divergent from each other as are HCV and GBV-B. In light of the recent finding of hepaciviruses infecting horses and dogs (6
), which are considerably more similar to HCV than GBV-B or RHV, it is unlikely that hepaciviruses coevolved with their hosts. The basal radiation of three different lineages of hepaciviruses infecting deer mice () means either that the variants diversified within this host species and subsequently infected another rodent species (Neotoma lepida
) and ultimately a tamarin (GBV-B) or that deer mice became infected with highly genetically distinct hepaciviruses from other host species. Either explanation requires the occurrence of multiple cross-species events that cannot be dated a priori
. Thus, without a chronological anchor, we did not attempt to estimate the evolutionary rates of hepacivirus lineages and their divergence times. The hepaciviruses identified by us in this and previous studies (7
) may have cross-species transmission potential. The high genetic diversity observed among RHV species raises the possibility that hepaciviruses (HCV, NPHV, and GBV-B) may have actually originated in rodents. Serology-enabled approaches, such as the one we recently used to study the host tropism of NPHV (7
), will be very useful in determining the host range and cross-species transmission potential of these novel rodent viruses and in identifying related viruses that infect other animal species.
Viruses genetically related to HPgV include its primate homologs (SPgV), an uncharacterized virus from bats (BPgV) (8
), and a recently identified distinct variant infecting horses (EPgV [Kapoor et al., submitted]). Studies thus far indicate a narrow host range for these viruses, with HPgV being found only in humans and chimpanzees, SPgV being found in New World monkeys, and BPgV and EPgV being found only in bats and horses, respectively (1
). These findings are consistent with the phylogenetic relationships between pegiviruses infecting rodents and other mammalian species (). Indeed, the two lineages of RPgVs infecting deer mice (Peromyscus maniculatus
) and white-throated wood rats (Neotoma albigula
) are more similar to each other than to pegiviruses found in other mammalian species, an observation that is consistent with virus-host cospeciation. However, further investigation of pegiviruses infecting other rodents and mammalian species will be required to solidify or refute the hypothesis that pegiviruses are species specific and have codiverged with the evolution of mammals.
The deduced genome organizations of rodent hepaciviruses and RPgV were similar to those of other members of these genera (1
). The 5′ UTRs of RHV and RPgV are long, consistent with the presence of IRES elements found in other hepaci- and pegiviruses. In the case of RHV, we were able to model an RNA structure based on the structurally conserved domains III found in other hepaciviruses, providing support for this structure’s function as a type IV IRES. Interestingly, the RHV 3′ UTR elements, but not the primary sequence, resembled that of HCV, with a putative variable region immediately downstream of the ORF, followed by a polypyrimidine tract and a 3′ X region. However, a short poly(C) tract replaced the longer poly(U
C) tracts found in HCV isolates. The RPgV 3′ UTR did not have homology to other pegiviruses but, surprisingly, contained repeat sequence elements (RSEs) identical to 5′ UTR sequences from human enterovirus, coxsackievirus, echovirus, and swine vesicular disease virus. It is as yet unclear how RPgV acquired these sequence elements and what function they might have.
Analysis of the RPgV polyprotein sequences revealed both similarities and differences from previously identified pegivirus isolates. Unlike hepaciviruses, pegiviruses typically do not encode a core (nucleocapsid) protein (26
). Nonetheless, biophysical characterization of HPgV particles suggests the presence of a nucleocapsid, although its origin and composition remain a mystery (27
). The RPgV sequence also lacks a convincing capsid protein sequence in either the polyprotein-coding or alternative open reading frames. Rather, the pegivirus polyprotein typically initiates with a signal peptide immediately downstream of the initiation codon that translocates E1 into the ER (position 17 or 21 in human pegiviruses) (28
). For RPgV, this is also the case, but RPgV-cc61 also possessed an additional 223-residue Y protein preceding E1, which may be targeted to the ER and glycosylated. Following the E2 homolog, the RPgV sequence encoded a predicted, 249-residue-long acidic X protein (), potentially homologous to, although highly divergent from, those predicted in EPgV and BPgV (8
). RPgV also possesses an additional predicted signalase site between E2 and NS2 (position 736) that could give rise to yet another glycosylated membrane protein.
Much of our current knowledge of the replication, host interactions, immune responses, and pathogenesis of HCV and pegiviruses comes from experimental infection of primates or cell culture systems. In vitro
models have proven valuable for investigating virus replication (13
), yet these systems fail to mimic the endogenous milieu of the target organ (liver) and may not accurately recapitulate life cycle events, such as polarized cell entry. Finally, cell culture systems cannot reproduce the interaction between virus and immune system, nor do they allow for studies of pathogenesis (12
). The identification and genetic characterization of RHV and RPgV reported here provide a unique opportunity to develop tractable-animal models to study the infection, transmission, immunity, and pathogenesis of hepaciviruses and pegiviruses. Although the current study design precluded direct examination of tissues of infected rodents, it is interesting that the 5′ UTR of RHV contains an miR-122 binding site. These have been previously described in the HCV 5′ UTR (two miR-122 seed sites) as highly conserved among all genotypes and functionally required for replication in hepatocytes (29
). Similarly, we recently reported the presence of one miR-122 site in NPHV (7
), while the GBV-B 5′ UTR contains sites at positions 8 and 23. Tissue-specific expression of miR-122 in the liver of vertebrates (including rodents) is consistent with potential hepatotropism of all hepaciviruses identified to date, including RHV. It will be interesting to define the sites of RHV replication in rodents and NPHV in horses in future investigations. If RHV does indeed resemble HCV in its tissue tropism and pathogenesis, rodents could prove to be a very useful small-animal model. A rodent model for pegivirus infections is also important for studies focused on the viral and host factors underlying virus persistence. Estimates suggest that >20% of the world population has been exposed to GBV-C, with chronic infection established in 1 to 5% of healthy adults (4
). Studying RPgV infections in a natural host amenable to genetic manipulation should provide a powerful approach for unraveling mechanisms favoring resolved infection versus persistence.
Rodent models also provide an opportunity to investigate routes of transmission for RPgV and RHV and how this might relate to HCV transmission, which is due largely to blood-borne routes of exposure. Such studies performed in rodents, including deer mice, have been extremely valuable for understanding hantavirus transmission (31
). Comparative genetic analysis and functional characterization of viral entry may help to unravel the determinants of host specificity and tissue tropism and provide insight into possible routes of cross-species transmission (29
). In addition, defining the natural history of RHV infection, the rate of chronicity, the immune determinants of clearance and protection, and possible disease association holds promise for establishing a highly relevant preclinical model for the development of HCV vaccine strategies and interventions to prevent or reverse virus-associated liver disease.