Human Pegivirus (HPgV) – formerly known as GB virus C and hepatitis G virus – is a poorly characterized RNA virus that infects approximately one-sixth of the global human population and is transmitted frequently in the blood supply. Here, we create an animal model of HPgV infection by infecting macaque monkeys with a new simian pegivirus (SPgV) discovered in wild baboons. Using this model, we provide a high-resolution, longitudinal picture of SPgV viremia where the dose, route, and timing of infection are known. We detail the highly-variable acute-phase of SPgV infection, showing that the viral load trajectory early in infection is dependent upon the infecting dose, whereas the chronic-phase viremic set-point is not. We also show that SPgV has an extremely low propensity for accumulating sequence variation, with no consensus-level variants detected during the acute phase of infection and an average of only 1.5 variants generated per 100 infection days. Finally, we show that SPgV RNA is highly concentrated in only two tissues: spleen and bone marrow, with bone marrow likely producing the majority of virus detected in plasma. Together, these results reconcile several paradoxical observations from cross-sectional analyses of HPgV in humans and provide an animal model for studying pegivirus biology.

Killer cell Immunoglobulin-like Receptors (KIRs) modulate disease progression of pathogens including HIV, malaria, and hepatitis C. Cynomolgus and rhesus macaques are widely used as nonhuman primate models to study human pathogens and so considerable effort has been put into characterizing their KIR genetics. However, previous studies have relied on cDNA cloning and Sanger sequencing that lacks the throughput of current sequencing platforms. In this study, we present a high throughput, full-length allele discovery method utilizing PacBio circular consensus sequencing (CCS). We also describe a new approach to Macaque Exome Sequencing (MES) and the development of the Rhexome1.0, an adapted target capture reagent that includes macaque-specific capture probesets. By using sequence reads generated by whole genome sequencing (WGS) and MES to inform primer design, we were able to increase the sensitivity of KIR allele discovery. We demonstrate this increased sensitivity by defining nine novel alleles within a cohort of Mauritian cynomolgus macaques (MCM), a geographically isolated population with restricted KIR genetics that was thought to be completely characterized. Finally, we describe an approach to genotyping KIRs directly from sequence reads generated using WGS/MES reads. The findings presented here expand our understanding of KIR genetics in MCM by associating new genes with all eight KIR haplotypes and demonstrating the existence of at least one KIR3DS gene associated with every haplotype.

Anti-HIV CD8 T cells included in therapeutic treatments will need to target epitopes that do not accumulate escape mutations.Identifying the epitopes that do not accumulate variants but retain immunogenicity depends on both host major histocompatibility complex (MHC) genetics and the likelihood for an epitope to tolerate variation. We previously found that immune escape during acute SIV infection is conditional; the accumulation of mutations in T cell epitopes is limited, and the rate of accumulation depends on the number of epitopes being targeted. We have now tested the hypothesis that conditional immune escape extends into chronic SIV infection and that epitopes with a preserved wild-type sequence have the potential to elicit epitope-specific CD8 T cells. We deep sequenced simian immunodeficiency virus (SIV) from Mauritian cynomolgus macaques (MCMs) that were homozygous and heterozygous for the M3 MHC haplotype and had been infected with SIV for about 1 year. When interrogating variation within individual epitopes restricted by M3 MHC alleles, we found three categories of epitopes, which we called categories A, B, and C. Category B epitopes readily accumulated variants in M3-homozygous MCMs, but this was less common in M3-heterozygous MCMs. We then determined that chronic CD8 T cells specific for these epitopes were more likely preserved in the M3-heterozygous MCMs than M3-homozygous MCMs. We provide evidence that epitopes known to escape from chronic CD8 T cell responses in animals that are homozygous for a set of MHC alleles are preserved and retain immunogenicity in a host that is heterozygous for the same MHC alleles. IMPORTANCEAnti-HIV CD8 T cells that are part of therapeutic treatments will need to target epitopes that do not accumulate escape mutations. Defining these epitope sequences is a necessary precursor to designing approaches that enhance the functionality of CD8 T cells with the potential to control virus replication during chronic infection or after reactivation of latent virus. Using MHChomozygous and -heterozygous Mauritian cynomolgus macaques, we have now obtained evidence that epitopes known to escape from chronic CD8 T cell responses in animals that are MHC homozygous are preserved and retain immunogenicity in a host that is heterozygous for the same MHC alleles. Importantly, our findings support the conditional immune escape hypothesis, such that the potential to present a greater number of CD8 T cell epitopes within a single animal can delay immune escape in targeted epitopes. As a result, certain epitope sequences can retain immunogenicity into chronic infection.

Killer cell Immunoglobulin-like Receptors (KIRs) modulate disease progression of pathogens including HIV, malaria, and hepatitis C. Cynomolgus and rhesus macaques are widely used as nonhuman primate models to study human pathogens and so considerable effort has been put into characterizing their KIR genetics. However, previous studies have relied on cDNA cloning and Sanger sequencing that lacks the throughput of current sequencing platforms. In this study, we present a high throughput, full-length allele discovery method utilizing PacBio circular consensus sequencing (CCS). We also describe a new approach to Macaque Exome Sequencing (MES) and the development of the Rhexome1.0, an adapted target capture reagent that includes macaque-specific capture probesets. By using sequence reads generated by whole genome sequencing (WGS) and MES to inform primer design, we were able to increase the sensitivity of KIR allele discovery. We demonstrate this increased sensitivity by defining nine novel alleles within a cohort of Mauritian cynomolgus macaques (MCM), a geographically isolated population with restricted KIR genetics that was thought to be completely characterized. Finally, we describe an approach to genotyping KIRs directly from sequence reads generated using WGS/MES reads. The findings presented here expand our understanding of KIR genetics in MCM by associating new genes with all eight KIR haplotypes and demonstrating the existence of at least one KIR3DS gene associated with every haplotype.

CD8+ T Lymphocytes (CTL) can control AIDS virus replication. However, natural selection favoring viral variants that escape CTL recognition is a common feature of both simian immunodeficiency virus (SIV) infection of macaques and HIV infection of humans. Emerging data indicate that CTL directed against alternate reading frame (ARF)-derived epitopes (a.k.a. cryptic epitopes) are important components of the total virus-specific response in SIV and HIV infection but the contributions of these responses during the critical first several weeks of infection have not been determined. We used a focused deep sequencing approach to examine acute phase viral evolution in response to CTL targeting two polypeptides encoded by ARFs of SIVmac239 in SIV-infected rhesus macaques. We report high magnitude CTL responses as early as three weeks post-infection against epitopes within both ARFs, which both overlap the 5′ end of the env gene. Further, mutations accumulated in the epitopes by three to four weeks post infection consistent with viral escape. Interestingly, these mutations largely maintained the primary amino acid sequence of the overlapping Envelope protein. Our data show that high frequency CTL target cryptic epitopes and exert selective pressure on SIV during the acute phase, underscoring the importance of these unique immune responses.