Identification of rodent homologs of hepatitis C virus and pegiviruses

Abstract

Hepatitis C virus (HCV) and human pegivirus (HPgV or GB virus C) are globally distributed and infect 2 to 5% of the human population. The lack of tractable-animal models for these viruses, in particular for HCV, has hampered the study of infection, transmission, virulence, immunity, and pathogenesis. To address this challenge, we searched for homologous viruses in small mammals, including wild rodents. Here we report the discovery of several new hepaciviruses (HCV-like viruses) and pegiviruses (GB virus-like viruses) that infect wild rodents. Complete genome sequences were acquired for a rodent hepacivirus (RHV) found in Peromyscus maniculatus and a rodent pegivirus (RPgV) found in Neotoma albigula. Unique genomic features and phylogenetic analyses confirmed that these RHV and RPgV variants represent several novel virus species in the Hepacivirus and Pegivirus genera within the family Flaviviridae. The genetic diversity of the rodent hepaciviruses exceeded that observed for hepaciviruses infecting either humans or non-primates, leading to new insights into the origin, evolution, and host range of hepaciviruses. The presence of genes, encoded proteins, and translation elements homologous to those found in human hepaciviruses and pegiviruses suggests the potential for the development of new animal systems with which to model HCV pathogenesis, vaccine design, and treatment.

Importance: The genetic and biological characterization of animal homologs of human viruses provides insights into the origins of human infections and enhances our ability to study their pathogenesis and explore preventive and therapeutic interventions. Horses are the only reported host of nonprimate homologs of hepatitis C virus (HCV). Here, we report the discovery of HCV-like viruses in wild rodents. The majority of HCV-like viruses were found in deer mice (Peromyscus maniculatus), a small rodent used in laboratories to study viruses, including hantaviruses. We also identified pegiviruses in rodents that are distinct from the pegiviruses found in primates, bats, and horses. These novel viruses may enable the development of small-animal models for HCV, the most common infectious cause of liver failure and hepatocellular carcinoma after hepatitis B virus, and help to explore the health relevance of the highly prevalent human pegiviruses.

FIG 1

Phylogenetic analysis of new rodent viruses using the partial helicase sequences generated by generic PCR. Bootstrap resampling was used to determine the robustness of branches; values of ≥70% (from 1,000 replicates) are shown. Host origins of newly reported viral sequences are indicated by red and yellow circles (see the key) and for previously described hepaciviruses (blue circles) and pegiviruses (green circles).

FIG 2

Phylogenetic analysis of conserved regions in the helicase (motifs I to VI) (A) and RdRp (B) genes of rodent hepaciviruses and pegiviruses aligned with representative members of the Hepacivirus, Pegivirus, Pestivirus, and Flavivirus genera. Trees were constructed by neighbor joining of pairwise amino acid distances with the program MEGA5 (according to the distance scale provided). Bootstrap resampling was used to determine the robustness of branches; values of ≥70% (from 1,000 replicates) are shown. Regions compared corresponded to positions 3667 to 4470 (helicase domain of NS3) and 7711 to 8550 (RdRp in NS5B; numbered according to the AF011751 HCV genotype 1a reference sequence).

FIG 3

Predicted structures in the RHV-339 and RPgV-cc61 UTRs. (A) RHV-339 5′ UTR structure predictions were made by homology searching and structural alignment within domain III. Bases conserved in all hepaciviruses (HCV, NPHV, and GBV-B) are highlighted with blue circles. (B) Structure prediction of the RHV 3′ UTR using Mfold. The ORF stop codon is boxed, and a putative variable region equivalent to that of HCV is indicated. A poly(C) tract of around 10 nt separates this from the downstream 3′ X region, which was predicted to fold into four stem-loops (3′SL1 to -4). (C) Predicted structure of the RPgV 3′ UTR. The black bar cartoon of the 3′ UTR (475 nt) was drawn to scale, with poly(C) tracts indicated in blue. Strong stem-loop structures immediately downstream of the ORF stop codon (boxed) and at the very end of the genome are indicated. RSEs, potentially folding into similar stem-loop structures, are outlined in red. The structure prediction of the RPgV 3′ UTR was done using Mfold.

FIG 4

(A) Amino acid sequence divergence between RHV and human virus (HCV), equine virus (NPHV), and GBV-B using 300-nt fragments by 15-nt increments across the sequence alignment (the midpoint is plotted on the y axis). Within-species distances for human and equine hepaciviruses are included for comparison. (B, C) Genome diagram of RHV showing predicted N-linked glycosylation sites (downward-pointing arrows) and proposed cleavage sites of cellular peptidase (black triangles), NS2-3 protease (white triangle), and NS3-4A protease (gray triangles) (sequence positions were numbered using the RHV sequence).

FIG 5

(A) Amino acid sequence divergence between RPgV and HPgV, New World primate PgV (SPgV), equine PgV (EPgV), and bat PgV (BPgV) using 300-base fragments by 15-base increments across the sequence alignment (the midpoint is plotted on the y axis). Within-species distances for human and simian pegiviruses (from New World primates [GBV-A]) are included for comparison. The divergence scan commenced at the predicted signalase cleavage site at the start of the E1-encoding genes (position 1016). (B, C) Genome diagram of RPgV showing predicted N-linked glycosylation sites (downward-pointing arrows) and proposed cleavage sites of cellular peptidase (black triangles), NS2-3 protease (white triangle), and NS3-4A protease (gray triangles) (sequence positions were numbered using the RPgV sequence).