David R. McGivern

HIV-1 and HIV-2 share transmission and replication characteristics, but require clinical differentiation. HIV-2 exhibits lower viral loads and slower disease progression, and most patients remain long-term non-progressors. Differentiation is crucial for selecting appropriate viral load monitoring tests and avoiding ineffective antiretroviral treatments, particularly non-nucleoside reverse transcriptase inhibitors, and certain protease inhibitors that do not work against HIV-2. Increased PrEP use may result in new infections testing negative for viral markers. This study aims to identify circulating tRNA expression profiles that could serve as non-viral biomarkers to detect and differentiate HIV-2 from HIV-1 infections. An initial pilot study analyzed 88 tRNAs in plasma samples from HIV-1- ( n = 4) and HIV-2 ( n = 4)-infected individuals, plus four healthy controls, using a tRNA-based PCR array for target identification. Eighteen selected tRNAs were further analyzed in a larger independent group of samples: HIV-1 ( n = 20), HIV-2 ( n = 20), and healthy controls ( n = 20) for independent verification. A plasma tRNA diagnostic model was developed from these results and evaluated in 24 independent samples to detect and differentiate HIV-2 from HIV-1 infection. We identified a tRNA-based model (Model-II) containing six differentially expressed tRNAs (Leu-TAA, mt-Asp-GTC, mt-Ala-TGC, mt-Lys-TTT, mt-LeuT-AA, and Gly-CCC-1) that could detect HIV-2 infection and distinguish it from HIV-1 infection and healthy controls with 100% accuracy, sensitivity, and specificity. We also found that other blood-borne viruses such as HTLV, HBV, and HCV had no cross-reactivity with HIV-2, demonstrating that Model-II is specific for HIV-2 infection. This study establishes proof of concept for tRNA expression profiles as HIV diagnostic biomarkers. The tRNA-based Model-II represents a promising diagnostic tool for identifying HIV-2 infection and differentiating it from HIV-1. IMPORTANCE: HIV-2 infection requires accurate differentiation from HIV-1 to guide appropriate antiretroviral therapy, as HIV-2 exhibits intrinsic resistance to non-nucleoside reverse transcriptase inhibitors and certain protease inhibitors. Current diagnostic challenges include the absence of FDA-approved HIV-2 RNA quantification assays and potential false-negative results in LEVI (low-level viremia with evolving viral infection) syndrome patients on pre-exposure prophylaxis (PrEP), in whom viral markers may be undetectable. We developed a novel diagnostic approach using circulating transfer RNA (tRNA) expression profiles as host-derived, non-viral biomarkers. Our six-tRNA panel (Model-II) achieved 100% accuracy, sensitivity, and specificity in detecting HIV-2 infection while distinguishing it from HIV-1 and other blood-borne viral infections (HTLV, HBV, and HCV). This represents the first tRNA-based diagnostic model for HIV, offering a promising complementary tool for early detection and accurate viral differentiation, particularly in clinical scenarios where traditional viral markers are absent or unreliable.

Objectives: Characterize hepatitis B virus (HBV) isolates from deferred blood donors identified by routine blood screening and confirmatory tests in the US, using virological, serological, and biochemical methods, and evaluate their infectivity in human hepatocytes. Methods: Forty-six (46) hepatitis B surface antigen (HBsAg) -positive plasma samples, including 32 reactive and 14 non-reactive for anti-HBV core (anti-HBc) were identified by blood donation testing and further analyzed for HBeAg/anti-HBe, liver disease markers (ALT and AST), and sequencing to determine virus genotype. To evaluate virus infectivity, plasma was used to inoculate humanized liver chimeric mouse-derived human hepatocytes (HLCM-HH). HBV DNA and HBsAg were monitored in culture media over time. Results: Subjects with confirmed positive screening tests for HBV fell into two groups: 1) older, predominantly Caucasian or African American, anti-HBc non-reactive and genotype A, who had successfully donated blood on a previous occasion when they tested negative for HBV, suggesting recent horizontal transmission, 2) younger, predominantly Asian, anti-HBc reactive with genotypes B or C, attempting to donate for the first time, suggesting distant vertical transmission. HBV from all samples could infect and replicate in HLCM-HH cultures as measured by DNA. Infectivity could be blocked by incubating with anti-HBV immunoglobulin. Kinetics of DNA accumulation suggested more robust replication of viruses from anti-HBc non-reactive plasma. Discussion: Analyses of plasma samples from HBV-infected individuals identified two groups differing in age, race, HBV genotype, and likely transmission route. HBV from anti-HBc non-reactive samples showed greater replicative fitness in HLCM-HH cultures compared to anti-HBc reactive samples.

Background: Pathogen reduction technologies (PRTs) have the potential to reduce the risk of emerging transfusion transmissible infections. Evaluating PRT activity against hepatitis B virus (HBV) presents some unique challenges due to the lack of robust model systems. Surrogate viruses (e.g., duck HBV) can be used, but may differ from the human pathogen in ways that influence susceptibility to a given PRT.Methods: Whole blood (WB) collected from volunteers was spiked with human plasma from deferred HBV‐positive donors. Spiked WB was then treated with the nucleic acid crosslinking compound S‐303 or left untreated. Additional physical and chemical treatments were also assessed. Plasma prepared from the spiked WB was used to inoculate human hepatocyte cultures isolated from chimeric mice with humanized livers. Culture medium was monitored over time for hepatitis B surface antigen (HBsAg) and viral DNA. Cell lysates were analyzed for evidence of covalently closed circular (ccc) DNA.Results: The culture system faithfully measured infectious HBV in blood components. S‐303 treatment of HBV‐spiked WB prevented infection of hepatocyte cultures, as monitored by HBsAg expression, at concentrations as low as 0.125 mM of S‐303 or with incubation times as short as 1 h. Additionally, S‐303 treatment prevented the accumulation of T5 exonuclease‐resistant HBV DNA in inoculated hepatocytes, suggesting that S‐303 prevents establishment of HBV cccDNA.Conclusion: This model mimics transfusion transmission by using donor‐derived HBV and mouse‐passaged human hepatocytes, allowing evaluation of the efficacy of different PRTs against authentic human HBV isolates. We found S‐303 to be a potent inactivator of HBV.

Hepatitis E virus (HEV) causes acute hepatitis in humans, which can progress to chronicity in immunosuppressed individuals. Almost all reported HEV infections are caused by Paslahepevirus balayani genotypes 1–4. The structural ORF2 protein is the major antigen detected in the blood of HEV-infected individuals. ELISA assays to detect IgM antibodies to HEV are the first-line diagnostic tests; however, they showed variable performance with frequently discordant results. A qualitative HEV antigen (ORF2) ELISA is currently available for research use. Here, we report a novel quantitative sandwich ELISA to measure HEV ORF2 protein in 3 matrix types. An optimal pair of capture and detection antibodies was selected among 12 unique combinations tested. A sandwich ELISA protocol was developed using these mAbs and biotin–streptavidin technology. The protocol was further optimized to quantify ORF2 antigen in different matrices by interpolating from a standard curve with a linear range of 3.17 to 50.8 femtomoles/mL. Using this method, ORF2 protein was detected in the cell culture medium of Huh7 cells as early as 2–3 days after transfection with HEV genome RNA and in a medium of human hepatocytes infected with HEV. ORF2 antigen was readily detected in the first 2 weeks post-HEV infection in gerbil sera. In immunosuppressed gerbils, ORF2 was detected up to 6 weeks, and the levels were significantly higher between 3 and 6 weeks post-infection. HEV ORF2 antigen levels showed a strong positive correlation with HEV RNA levels in both cell culture medium and gerbil sera. Our novel sandwich ELISA detected at least 7.3 femtomoles/mL ORF2 protein in human plasma spiked with cell culture propagated HEV and detected ORF2 protein in human plasma samples that tested positive for HEV RNA but negative for anti-HEV antibodies. Further, the assay was nonreactive, with negative human plasma, and HBV or HCV-positive human plasma demonstrating specificity. Overall, our ORF2 antigen ELISA will be useful for quantifying ORF2 antigen in cell culture medium, gerbil serum, and human plasma. Further studies are warranted to evaluate its utility in HEV clinical diagnosis.

Hepatitis E virus (HEV) causes self-limited acute hepatitis in immunocompetent individuals and can establish chronic infection in solid organ transplant recipients taking immunosuppressive drugs. A well characterized small animal model is needed to understand HEV pathogenesis. In this study, we established a robust model to study acute and persistent HEV infection using Mongolian gerbils (Meriones unguiculatus) with or without immunosuppression. Gerbils were implanted subcutaneously with continuous release tacrolimus pellet to induce immunosuppression. Gerbils with or without tacrolimus treatment were inoculated with HEV intraperitoneally. Viremia, fecal virus shedding, serum antibody and ALT levels, liver histopathological lesions, hepatocyte apoptosis, and liver macrophage distribution were assessed. Mild to moderate self-limited hepatitis and IgM and IgG antibody responses against HEV ORF2 were observed in immunocompetent gerbils. Levels of HEV-specific IgM responses were higher and lasted longer in immunocompetent gerbils with higher peak viremia. Persistent viremia and fecal virus shedding with either weak, or absent HEV antibody levels were seen in immunosuppressed gerbils. Following HEV infection, serum ALT levels were increased, with lower and delayed peaks observed in immunosuppressed compared to immunocompetent gerbils. In immunocompetent gerbils, foci of apoptotic hepatocytes were detected that were distributed with inflammatory infiltrates containing CD68⁺ macrophages. However, these foci were absent in immunosuppressed gerbils. The immunosuppressed gerbils showed no inflammation with no increase in CD68⁺ macrophages despite high virus replication in liver. Our findings suggest adaptive immune responses are necessary for inducing hepatocyte apoptosis, CD68⁺ macrophage recruitment, and inflammatory cell infiltration in response to HEV infection. Our studies show that Mongolian gerbils provide a promising model to study pathogenesis during acute and persistent HEV infection.

HAV RNA persists in the liver of infected chimpanzees and interferon receptor-deficientIfnar1^−/−mice for many months after neutralizing antibodies appear, virus has been cleared from the blood, and fecal virus shedding has terminated. Here, we show this viral RNA is located within hepatocytes and that the depletion of macrophages months after the resolution of hepatic inflammation restores fecal virus shedding and circulating viral RNA.

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5′ untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3′ poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3′ poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC ₅₀ 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1 ^−/− mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.

HAV-infected Ifnar1^-/- mice recapitulate many of the cardinal features of hepatitis A in humans, including serum alanine aminotransferase (ALT) elevation, hepatocellular apoptosis, and liver inflammation. Previous studies implicate MAVS-IRF3 signaling in pathogenesis, but leave unresolved the role of IRF3-mediated transcription versus the non-transcriptional, pro-apoptotic activity of ubiquitylated IRF3. Here, we compare the intrahepatic transcriptomes of infected versus naïve Mavs^-/- and Ifnar1^-/- mice using high-throughput sequencing, and identify IRF3-mediated transcriptional responses associated with hepatocyte apoptosis and liver inflammation. Infection was transcriptionally silent in Mavs^-/- mice, in which HAV replicates robustly within the liver without inducing inflammation or hepatocellular apoptosis. By contrast, infection resulted in the upregulation of hundreds of genes in Ifnar1^-/- mice that develop acute hepatitis closely modeling human disease. Upregulated genes included pattern recognition receptors, interferons, chemokines, cytokines and other interferon-stimulated genes. Compared with Ifnar1^-/- mice, HAV-induced inflammation was markedly attenuated and there were few apoptotic hepatocytes in livers of infected Irf3^S1/S1Ifnar1^-/- mice in which IRF3 is transcriptionally-inactive due to alanine substitutions at Ser-388 and Ser-390. Although transcriptome profiling revealed remarkably similar sets of genes induced in Irf3^S1/S1Ifnar1^-/- and Ifnar1^-/- mice, a subset of genes was differentially expressed in relation to the severity of the liver injury. Prominent among these were both type 1 and type III interferons and interferon-responsive genes associated previously with apoptosis, including multiple members of the ISG12 and 2’-5’ oligoadenylate synthetase families. Ifnl3 and Ifnl2 transcript abundance correlated strongly with disease severity, but mice with dual type 1 and type III interferon receptor deficiency remained fully susceptible to liver injury. Collectively, our data show that IRF3-mediated transcription is required for HAV-induced liver injury in mice and identify key IRF3-responsive genes associated with pathogenicity, providing a clear distinction from the transcription-independent role of IRF3 in liver injury following binge exposure to alcohol.