Community-Based Screening for Hepatitis B and C Infectivity Using Two Quantitative Antigens to Identify Endemic Townships.

Screening and linkage to care are essential to achieve viral hepatitis elimination before 2030. The accurate identification of endemic areas is important for controlling diseases with geographic aggregation. Viral activity drives prognosis of chronic hepatitis B and hepatitis C virus infection. This screening was conducted in Chiayi County from 2018-2019. All residents aged 30 years or older were invited to participate in quantitative HBsAg (qHBsAg) and HCV Ag screening. Among the 4010 participants (male:female = 1630:2380), the prevalence of qHBsAg and HCV Ag was 9.9% (396/4010) and 4.1% (163/4010), respectively. High-prevalence townships were identified, three for qHBsAg > 15% and two for HCV Ag > 10%. The age-specific prevalence of qHBsAg was distributed in an inverse U-shape with a peak (16.0%, 68/424) for subjects in their 40 s; for HCV, prevalence increased with age. Concentrations of qHBsAg < 200 IU/mL were found in 54% (214/396) of carriers. The rate of oral antiviral treatment for HCV was 75.5% (114/151), with subjects younger than 75 years tending to undergo treatment (85.6% vs. 57.4%, p < 0.001). QHBsAg and HCV Ag core antigens can reflect the concentration of the viral load, which serves as a feasible screening tool. Using quantitative antigen screening for hepatitis B and C in community-based screening, two hyperendemic townships were identified from an endemic county.

Prevalence of hepatitis B, C, and D virus infection in Haiti: A systematic review and meta-analysis.

Background: Viral hepatitis causes an important global health burden. In 2016, the World Health Assembly adopted an objective to globally eliminate this as a public health threat by 2030. However, significant gaps exist between countries in their progress. Haiti is the last country that has introduced infant hepatitis B vaccines into the routine immunization program in the Region of the Americas, and its schedule still does not incorporate birth dose vaccines. As the first step to raise awareness of viral hepatitis in this country, we conducted a systematic review and meta-analysis to estimate the prevalence of hepatitis B (HBV), C (HCV), and D (HDV) viruses in Haiti. Methods: We searched PubMed, EMBASE, Web of Science and Scopus for studies reporting the prevalence of HBV, HCV and HDV among Haitian, with no language restriction, published until November 30th, 2021. Prevalence was pooled via a random-effects meta-analysis using a generalized linear mixed model with the logit link. Results: Of 453 articles retrieved, 25 studies were included: 16 reported the prevalence of hepatitis B surface antigen (HBsAg), three for anti-HCV antibody, and six for both HBsAg and anti-HCV. No study was found for HDV prevalence. The pooled prevalence of HBsAg was 0.7% [95% confidence interval (CI): 0.3-1.4, I 2 = 77.7%] among children, 3.5% (95% CI: 2.8-4.4, I 2 = 93.2%) in the general adult population and 7.4% (95% CI: 4.0-13.3, I 2 = 83.9%) in high-risk adult population. The pooled prevalence of anti-HCV antibody was 0.9% (95% CI: 0.6-1.4, I 2 = 93.5%) among the general population and 1.4% (95% CI: 0.4-4.2, I 2 = 0.0%) in high-risk adult population. No study reported the prevalence of anti-HCV antibody exclusively in children. Interpretation: The prevalence of blood-borne hepatitis, particularly that of HBV, is substantial in Haiti. The introduction of birth dose hepatitis B vaccines and improving access to testing and treatment services should be urgently considered to meet the elimination goal. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022298081, identifier: PROSPERO (CRD42022298081).

[Sero-epidemiological characteristics of the hepatitis D virus infection among hepatitis B virus infected-patients at a single center in Xinjiang region].

Objective: To investigate the sero-epidemiological characteristics of the hepatitis D virus (HDV) infection among hepatitis B virus (HBV)-infected patients in Xinjiang region. Methods: A single-center cross-sectional analysis method was used to select 264 cases of hepatitis B virus infection who were hospitalized in the Center for Infectious Diseases and Liver Diseases of the First Affiliated Hospital of Xinjiang Medical University from August 2021 to January 2022. All patients were tested for HDV Ag, HDV IgM, HDV IgG, and HDV RNA. The infection status of hepatitis D virus was analyzed by grouping according to their clinical type, HBV viral load, and HBsAg level. A paired t-test was used for data with measurement data conforming to normal distribution. A paired rank sum test was used for data that did not conform to normal distribution before and after treatment. Results: A total of 36 cases (13.64%) and 26 cases (9.85%) were positive for HDV serological markers and HDV RNA. According to clinical type grouping, the positive rates of HDV serum markers in patients with chronic hepatitis B, hepatitis B-related cirrhosis, liver cancer, and liver failure were 13.46%, 12.43%, and 20.83%, respectively, and there was no statistically significant difference among the three groups (χ2=0.86, P=0.649). The positive rates of HDV RNA were 11.54%, 8.11%, and 20.83%, respectively, and there was no statistically significant difference among the three groups (χ2=4.015, P=0.134). According to HBV viral load grouping, the positive rates of HDV serum markers among patients with viral loads <20, 20-2 000, and >2 000 IU/ml were 17.15%, 7.81%, and 6.67%, respectively, and the difference was not statistically significant among the three groups (χ2=4.846, P=0.089). The positive rates of HDV RNA were 9.47%, 10.94%, and 10%, respectively, and the difference was not statistically significant among the three groups (χ2=0.113, P=0.945). According to HBsAg level grouping, the positive rates of HDV serum markers in HBsAg<0.05, 0.05~250, and >250 IU/ml were 14.29%, 16.67%, and 10.85%, respectively, and there was no statistically significance between the three groups (χ2=1.745, P=0.418). The positive rates of HDV RNA were 4.76%, 8.77%, and 11.63%, respectively, and there was no statistically significant difference among the three groups (χ2=1.221, P=0.543). Clinical outcome, disease course, HBV DNA, serological markers of viral hepatitis, routine blood test, biochemical indicators, coagulation function, and other laboratory indicators were compared between HDV serum marker and/or nucleic acid positive and negative patients, and there was no statistically significant difference (P>0.05). Conclusion: The positive rate of HDV serological markers and HDV RNA is 13.64% and 9.85%, respectively, at a single center in the Xinjiang region, and there is still a high HDV infection rate among the HBV-infected patients with low levels of viral load and HBsAg.

Multimodal investigation of rat hepatitis E virus antigenicity: Implications for infection, diagnostics, and vaccine efficacy.

BACKGROUND & AIMS: Rat hepatitis E virus (Orthohepevirus species C; HEV-C1) is an emerging cause of viral hepatitis in humans. HEV-C1 is divergent from other HEV variants infecting humans that belong to Orthohepevirus species A (HEV-A). This study assessed HEV-C1 antigenic divergence from HEV-A and investigated the impact of this divergence on infection susceptibility, serological test sensitivity, and vaccine efficacy. METHODS: Immunodominant E2s peptide sequences of HEV-A and HEV-C1 were aligned. Interactions of HEV-C1 E2s and anti-HEV-A monoclonal antibodies (mAbs) were modeled. Recombinant peptides incorporating E2s of HEV-A (HEV-A4 p239) and HEV-C1 (HEV-C1 p241) were expressed. HEV-A and HEV-C1 patient sera were tested using antibody enzymatic immunoassays (EIA), antigen EIAs, and HEV-A4 p239/HEV-C1 p241 immunoblots. Rats immunized with HEV-A1 p239 vaccine (Hecolin), HEV-A4 p239 or HEV-C1 p241 peptides were challenged with a HEV-C1 strain. RESULTS: E2s sequence identity between HEV-A and HEV-C1 was only 48%. There was low conservation at E2s residues (23/53; 43.4%) involved in mAb binding. Anti-HEV-A mAbs bound HEV-C1 poorly in homology modeling and antigen EIAs. Divergence resulted in low sensitivity of commercial antigen (0%) and antibody EIAs (10-70%) for HEV-C1 diagnosis. Species-specific HEV-A4 p239/HEV-C1 p241 immunoblots accurately differentiated HEV-A and HEV-C1 serological profiles in immunized rats (18/18; 100%) and infected-patient sera (32/36; 88.9%). Immunization with Hecolin and HEV-A4 p239 was partially protective while HEV-C1 p241 was fully protective against HEV-C1 infection in rats. CONCLUSIONS: Antigenic divergence significantly decreases sensitivity of hepatitis E serodiagnostic assays for HEV-C1 infection. Species-specific immunoblots are useful for diagnosing HEV-C1 and for differentiating the serological profiles of HEV-A and HEV-C1. Prior HEV-A exposure is not protective against HEV-C1. HEV-C1 p241 is an immunogenic vaccine candidate against HEV-C1. LAY SUMMARY: Rat hepatitis E virus (HEV-C1) is a new cause of hepatitis in humans. Using a combination of methods, we showed that HEV-C1 is highly divergent from the usual cause of human hepatitis (HEV-A). This divergence reduces the capacity of existing tests to diagnose HEV-C1 and also indicates that prior exposure to HEV-A (via infection or vaccination) is not protective against HEV-C1.

Role of Hepatitis E Virus Infection in North American Patients With Severe Acute Liver Injury.

INTRODUCTION: The aim of this study was to determine the role of hepatitis E virus (HEV) infection in a large cohort of prospectively enrolled patients with severe acute liver injury (ALI). METHODS: Serum samples from 594 consecutive adults enrolled between 2008 and 2018 in the US Acute Liver Failure Study Group ALI registry were tested for anti-HEV IgM and anti-HEV IgG levels. Those with detectable anti-HEV IgM underwent further testing for HEV RNA using real-time polymerase chain reaction. RESULTS: The median age of patients was 38 years; 41% were men and 72% Caucasian. Etiologies of ALI included acetaminophen hepatotoxicity (50%), autoimmune hepatitis (8.9%), hepatitis B virus (8.9%), and idiosyncratic drug-induced liver injury (7.9%). Overall, 62 patients (10.4%) were negative for anti-HEV IgM but positive for IgG, whereas only 3 men (0.5%) were positive for both anti-HEV IgM and IgG. These 3 cases were initially diagnosed as having indeterminate, HEV, and hepatitis B virus-related ALI. One of these patients had detectable HEV RNA genotype 3, and another anti-HEV IgM+ patient had detectable HEV antigens by immunohistochemistry on liver biopsy. On multivariate modeling, older (odds ratio: 1.99) and non-Caucasian subjects (odds ratio: 2.92) were significantly more likely to have detectable anti-HEV IgG (P < 0.0001). DISCUSSION: Acute HEV infection is an infrequent cause of ALI in hospitalized North American adults. The anti-HEV IgG+ patients were significantly older and more likely to be non-Caucasian. These data are consistent with other population-based studies that indicate exposure to HEV in the general US population is declining over time and might reflect a cohort effect.

Enhancing the antigenicity and immunogenicity of monomeric forms of hepatitis C virus E2 for use as a preventive vaccine.

The E2 glycoprotein of hepatitis C virus (HCV) is the major target of broadly neutralizing antibodies (bNAbs) that are critical for the efficacy of a prophylactic HCV vaccine. We previously showed that a cell culture-derived, disulfide-linked high-molecular-weight (HMW) form of the E2 receptor-binding domain lacking three variable regions, Δ123-HMW, elicits broad neutralizing activity against the seven major genotypes of HCV. A limitation to the use of this antigen is that it is produced only at low yields and does not have a homogeneous composition. Here, we employed a sequential reduction and oxidation strategy to efficiently refold two high-yielding monomeric E2 species, D123 and a disulfide-minimized version (D123A7), into disulfide-linked HMW-like species (Δ123r and Δ123A7r). These proteins exhibited normal reactivity to bNAbs with continuous epitopes on the neutralizing face of E2, but reduced reactivity to conformation-dependent bNAbs and nonneutralizing antibodies (non-NAbs) compared with the corresponding monomeric species. Δ123r and Δ123A7r recapitulated the immunogenic properties of cell culture-derived D123-HMW in guinea pigs. The refolded antigens elicited antibodies that neutralized homologous and heterologous HCV genotypes, blocked the interaction between E2 and its cellular receptor CD81, and targeted the AS412, AS434, and AR3 domains. Of note, antibodies directed to epitopes overlapping with those of non-NAbs were absent. The approach to E2 antigen engineering outlined here provides an avenue for the development of preventive HCV vaccine candidates that induce bNAbs at higher yield and lower cost.

Demonstration of real-time and accelerated stability of hepatitis E vaccine with a combination of different physicochemical and immunochemical methods.

Hepatitis E, which is caused by infection with hepatitis E virus (HEV), is a global health problem in both developed and developing countries. An efficacious hepatitis E vaccine was licensed (by China) in 2011 with a trade name of Hecolin®. The antigen contained in this vaccine is a truncated version of the sole capsid protein encoded by open reading frame 2, which is designated p239. In this study, the real-time and real-condition stability and accelerated stability of five lots of hepatitis E vaccine products at the end of the designated shelf life, were assessed by a well-established quality analysis platform. The protein integrity of p239 that was recovered from the vaccine lots was demonstrated using CE-SDS, LC-MS and MALDI-TOF MS. The particle characteristics of the recovered vaccine antigen were assessed by TEM and HPSEC. The immunogenicity of hepatitis E vaccines was assessed by a mouse potency assay, which is part of product release and stability testing. Several methods were employed to assess the antigenicity of vaccines with or without adjuvant dissolution. Specifically, the well-established methods of sandwich ELISA and surface plasma resonance (SPR)-based BIAcore were used with unique murine monoclonal antibodies. Most interesting, two 'dissolution-free' immunoassays were also used for in situ antigenicity assessment of the vaccines. In addition to the confirmation of vaccine stability at the end of expiry dating, i.e., after storage in recommended conditions (2-8 °C) for 36 months, the mouse potency assay and sandwich ELISA were used to assess the accelerated stability of prefilled syringes to demonstrate the feasibility of out-of-cold-chain storage. In summary, molecular and functional characterization confirmed the shelf life stability of the vaccine at the end of expiry dating and the feasibility of transporting the hepatitis E vaccine for a given period of time out of cold chains.

Establishment of a Highly Sensitive Assay for Detection of Hepatitis E Virus-Specific Immunoglobulins.

Hepatitis E, a liver disease caused by infection with the hepatitis E virus (HEV), is a worldwide emerging disease. The diagnosis is based on the detection of viral RNA and of HEV-specific immunoglobulins (Ig). For the latter, various assays are commercially available but still lack harmonization. In this study, a Luminex-based multiplex serological assay was established that measures the presence of total IgG, IgA, and IgM antibodies, targeting a short peptide derived from the viral E2 protein. For the validation, 160 serum samples with a known HEV serostatus were used to determine the assay cutoff and accuracy. Thereby, HEV IgG- and RNA-positive sera were identified with a sensitivity of 100% and a specificity of 98% (95% confidence interval [CI], 94% to 100%). Application of the assay by retesting 514 serum samples previously characterized with different HEV-IgG or total antibody tests revealed a high level of agreement between the assays (Cohen's kappa, 0.58 to 0.99). The established method is highly sensitive and specific and can be easily implemented in a multiplex format to facilitate rapid differential diagnostics with a few microliters of sample input.

Hepatitis E Virus (HEV) Open Reading Frame 2 Antigen Kinetics in Human-Liver Chimeric Mice and Its Impact on HEV Diagnosis.

BACKGROUND: Hepatitis E virus infection (HEV) is an emerging problem in developed countries. Diagnosis of HEV infection is based on the detection of HEV-specific antibodies, viral RNA, and/or antigen (Ag). Humanized mice were previously reported as a model for the study of HEV infection, but published data were focused on the quantification of viral RNA. However, the kinetics of HEV Ag expression during infection remains poorly understood. METHODS: Plasma specimens and suspensions of fecal specimens from HEV-infected and ribavirin-treated humanized mice were analyzed using HEV antigen-specific enzyme-linked immunosorbent assay, reverse transcription-quantitative polymerase chain reaction analysis, density gradient analysis, and Western blotting. RESULT: Open reading frame 2 (ORF2) Ag was detected in both plasma and stool from HEV-infected mice, and levels increased over time. Contrary to HEV RNA, ORF2 Ag levels were higher in mouse plasma than in stool. Interestingly, ORF2 was detected in plasma from mice that tested negative for HEV RNA in plasma but positive for HEV RNA in stool and was detected after viral clearance in mice that were treated with ribavirin. Plasma density gradient analysis revealed the presence of the noninfectious glycosylated form of ORF2. CONCLUSION: ORF2 Ag can be used as a marker of active HEV infection and for assessment of the effect of antiviral therapy, especially when fecal samples are not available or molecular diagnostic tests are not accessible.

A Territorywide Prevalence Study on Blood-Borne and Enteric Viral Hepatitis in Hong Kong.

BACKGROUND: Viral hepatitis epidemiological data are important for the World Health Organization plan of eliminating viral hepatitis. We aimed to document the prevalence of viral hepatitis A to E in Hong Kong. METHODS: This community-based study was open to all Hong Kong Chinese citizens aged ≥18 years. Baseline data and risk factors were collected. Hepatitis A-E serology was measured, including hepatitis B e antigen, antibodies to hepatitis B e antigen, antibodies to hepatitis D, hepatitis B virus (HBV) DNA for hepatitis B surface antigen (HBsAg)-positive participants, and antibodies to hepatitis B surface antigen and antibodies to hepatitis B core antigen (anti-HBc) in HBsAg-negative participants. Hepatitis C virus (HCV) RNA and genotypes were determined in anti-HCV-positive participants. RESULTS: A total of 10 256 participants were recruited from February 2015 to July 2016. Overall HBsAg seroprevalence was 7.8% (95% confidence interval [CI], 7.3%-8.3%), which was reduced significantly with HBV vaccination (odds ratio, 0.15 [95% CI, .11-.21]). Among HBsAg-negative participants, anti-HBc seroprevalence increased from 5.4% (<26 years) to 60.1% (>65 years). No hepatitis D virus (HDV) cases were detected. Anti-HCV positivity was 0.5% (95% CI, .3%-.6%). Prevalence of antibodies to hepatitis A virus (anti-HAV) and hepatitis E virus (anti-HEV) was 65.2% (95% CI, 64.2%-66.1%) and 33.3% (95% CI, 32.4%-34.2%), respectively, and were influenced by age, family income, and being born in mainland China. CONCLUSIONS: HBV seroprevalence remained high despite universal vaccination. High anti-HBc seroprevalence underlines the potential issue of HBV reactivation during profound immunosuppression. HCV and HDV remained uncommon. Anti-HAV seroprevalence had decreased whereas anti-HEV seroprevalence had risen.

Origin, antigenicity, and function of a secreted form of ORF2 in hepatitis E virus infection.

The enterically transmitted hepatitis E virus (HEV) adopts a unique strategy to exit cells by cloaking its capsid (encoded by the viral ORF2 gene) and circulating in the blood as "quasi-enveloped" particles. However, recent evidence suggests that the majority of the ORF2 protein present in the patient serum and supernatants of HEV-infected cell culture exists in a free form and is not associated with virus particles. The origin and biological functions of this secreted form of ORF2 (ORF2S) are unknown. Here we show that production of ORF2S results from translation initiated at the previously presumed AUG start codon for the capsid protein, whereas translation of the actual capsid protein (ORF2C) is initiated at a previously unrecognized internal AUG codon (15 codons downstream of the first AUG). The addition of 15 amino acids to the N terminus of the capsid protein creates a signal sequence that drives ORF2S secretion via the secretory pathway. Unlike ORF2C, ORF2S is glycosylated and exists as a dimer. Nonetheless, ORF2S exhibits substantial antigenic overlap with the capsid, but the epitopes predicted to bind the putative cell receptor are lost. Consistent with this, ORF2S does not block HEV cell entry but inhibits antibody-mediated neutralization. These results reveal a previously unrecognized aspect in HEV biology and shed new light on the immune evasion mechanisms and pathogenesis of this virus.

Development and evaluation of a rapid point-of-care test for detecting the hepatitis E virus antigen.

BACKGROUND: Hepatitis E virus (HEV)-caused acute viral hepatitis is a major threat to public health worldwide. Recently, an enzyme linked immunosorbent assay (ELISA) kit detecting the HEV antigen was reported to have good concordance with the HEV RNA load and showed good clinical performance. But the ELISA kits can barely satisfy the needs of community clinics. In this study, a fluorescent microbead-based immunoassay (FMIA) for detecting the HEV antigen was developed and evaluated. METHODS: A mouse anti-HEV monoclonal antibody (mAb) conjugated with fluorescent microbeads was used as capturing antibody and another mouse mAb was used as detection antibody. Overall, 150 serum samples were collected from HEV-infected patients (n = 50) and non-HEV cases (n = 100) to evaluate the performance of the FMIA. RESULTS: The FMIA results showed a strong linear correlation with the viral RNA load. The diagnostic sensitivity and specificity of the HEV antigen FMIA were 92.0% (46/50) and 100.0% (100/100), respectively, and the test was consistent (kappa = 0.937, p = 0.627) with the commercial HEV antigen ELISA. The FMIA also showed good consistency with the PCR results (kappa = 0.939, p = 0.134). CONCLUSIONS: As a rapid point-of-care (POC) test, a FMIA that is developed with acceptable performance is suitable for acute hepatitis E diagnosis, especially in developing countries and regions, because of its reduced time and simplified operation.

Rational design and evaluation of HBsAg polymeric nanoparticles as antigen delivery carriers.

The present work is focused on the development and evaluation of single dose sustained-release Hepatitis B surface antigen (HBsAg) loaded nanovaccine for Hepatitis B. The conventional treatment suffers from repeated administration and hence requires a booster dose. Therefore, polymeric nanovaccine of HBsAg was developed by double emulsion solvent evaporation technique, utilizing central composite design for formulation optimization. The effects of independent variables (like polymer amount, stabilizer concentration, aqueous/organic phase ratio and homogenizer speed) were also studied on critical quality attributes like particle size and entrapment efficiency. Nanovaccine was characterized in terms of physicochemical parameters, release, internalization and in vivo immunological evaluation in BALB/c mice after administration by different routes such as oral, sub-cutaneous, nasal and intramuscular. The designed nanovaccine demonstrated nanometric size with smooth surface, negative zeta potential, maximum entrapment, sustained release and better internalization in macrophage and MRC-5 cell line. The immune-stimulating activity of nanovaccine administered by different routes was evaluated by measuring anti-HBsAg titre like specific immunoglobulin IgG and IgA response and cytokine level (interleukin-2, interferon-Y) measurement. The results indicated that the nanovaccine administered by intramuscular route produced better humoral as well as cellular responses and potential carriers for antigen delivery at single dose administration via intramuscular route.

Targeting naturally occurring epitope variants of hepatitis C virus with high-affinity T-cell receptors.

Hepatitis C virus (HCV) readily establishes chronic infection, which is characterized by failure of virus-specific CD8+ T cells. HCV uses epitope mutation and T-cell exhaustion to escape from the host immune response. Previously, we engineered high-affinity T-cell receptors (HATs) targeting human immunodeficiency virus escape mutants. In this study, the affinity of a T-cell receptor specific for the HLA-A2-restricted HCV immunodominant epitope NS3 1406-1415 (KLVALGINAV) was improved from a KD of 6.6 µM to 40 pM. These HATs could also target HCV NS3 naturally occurring variants, including an escape variant vrt1 (KLVVLGINAV), with high affinities. The HATs can be used as high-affinity targeting molecules at the centre of the immune synapse for the HLA-restricted NS3 antigen. By fusing the HAT with a T-cell activation molecule, an anti-CD3 single-chain variable fragment, we constructed a molecule called high-affinity T-cell activation core (HATac), which can redirect functional CTLs possessing any specificity to recognize and kill cells presenting HCV NS3 antigens. This capability was verified with T2 cells loaded with prototype or variant peptides and HepG2 cells expressing the truncated NS3 prototype or variant proteins. The results indicate that HATac targeting the HLA-restricted NS3 antigen may provide a useful tool for circumventing immune escape mutants and T-cell exhaustion caused by HCV infection.

TMEM2 inhibits hepatitis B virus infection in HepG2 and HepG2.2.15 cells by activating the JAK-STAT signaling pathway.

We have previously observed the downregulation of TMEM2 in the liver tissue of patients with chronic hepatitis B virus (HBV) infection and in HepG2.2.15 cells with HBV genomic DNA. In the present study, we investigated the role and mechanism of TMEM2 in HepG2 and HepG2.2.15 during HBV infection HepG2 and HepG2.2.15. HepG2 shTMEM2 cells with stable TMEM2 knockdown and HepG2 TMEM2 and HepG2.2.15 TMEM2 cells with stable TMEM2 overexpression were established using lentivirus vectors. We observed reduced expression of TMEM2 in HBV-infected liver tissues and HepG2.2.15 cells. HBsAg, HBcAg, HBV DNA, and HBV cccDNA levels were significantly increased in HepG2 shTMEM2 cells but decreased in HepG2 TMEM2 and HepG2.2.15 TMEM2 cells compared with naive HepG2 cells. On the basis of the western blotting results, the JAK-STAT signaling pathway was inhibited in HepG2 shTMEM2 cells but activated in HepG2 TMEM2 and HepG2.2.15 TMEM2 cells. In addition, reduced and increased expression of the antiviral proteins MxA and OAS1 was observed in TMEM2-silenced cells (HepG2 shTMEM2 cells) and TMEM2-overexpressing cells (HepG2 TMEM2 and HepG2.2.15 TMEM2 cells), respectively. The expression of Interferon regulatory factor 9 (IRF9) was not affected by TMEM2. However, we found that overexpression and knockdown of TMEM2, respectively, promoted and inhibited importation of IRF9 into nuclei. The luciferase reporter assay showed that IRF9 nuclear translocation affected interferon-stimulated response element activities. In addition, the inhibitory effects of TMEM2 on HBV infection in HepG2 shTMEM2 cells was significantly enhanced by pre-treatment with interferon but significantly inhibited in HepG2.2.15 TMEM2 cells by pre-treatment with JAK1 inhibitor. TMEM2 inhibits HBV infection in HepG2 and HepG2.2.15 by activating the JAK-STAT signaling pathway.

Transmitted/Founder Viruses Rapidly Escape from CD8+ T Cell Responses in Acute Hepatitis C Virus Infection.

UNLABELLED: The interaction between hepatitis C virus (HCV) and cellular immune responses during very early infection is critical for disease outcome. To date, the impact of antigen-specific cellular immune responses on the evolution of the viral population establishing infection and on potential escape has not been studied. Understanding these early host-virus dynamics is important for the development of a preventative vaccine. Three subjects who were followed longitudinally from the detection of viremia preseroconversion until disease outcome were analyzed. The evolution of transmitted/founder (T/F) viruses was undertaken using deep sequencing. CD8(+) T cell responses were measured via enzyme-linked immunosorbent spot (ELISpot) assay using HLA class I-restricted T/F epitopes. T/F viruses were rapidly extinguished in all subjects associated with either viral clearance (n = 1) or replacement with viral variants leading to establishment of chronic infection (n = 2). CD8(+) T cell responses against 11 T/F epitopes were detectable by 33 to 44 days postinfection, and 5 of these epitopes had not previously been reported. These responses declined rapidly in those who became chronically infected and were maintained in the subject who cleared infection. Higher-magnitude CD8(+) T cell responses were associated with rapid development of immune escape variants at a rate of up to 0.1 per day. Rapid escape from CD8(+) T cell responses has been quantified for the first time in the early phase of primary HCV infection. These rapid escape dynamics were associated with higher-magnitude CD8(+) T cell responses. These findings raise questions regarding optimal selection of immunogens for HCV vaccine development and suggest that detailed analysis of individual epitopes may be required. IMPORTANCE: A major limitation in our detailed understanding of the role of immune response in HCV clearance has been the lack of data on very early primary infection when the transmitted viral variants successfully establish the acute infection. This study was made possible through the availability of specimens from a unique cohort of asymptomatic primary infection cases in whom the first available viremic samples were collected approximately 3 weeks postinfection and at regular intervals thereafter. The study included detailed examination of both the evolution of the viral population and the host cellular immune responses against the T/F viruses. The findings here provide the first evidence of host cellular responses targeting T/F variants and imposing a strong selective force toward viral escape. The results of this study provide useful insight on how virus escapes the host response and consequently on future analysis of vaccine-induced immunity.

Characterization of Two Novel Linear B-Cell Epitopes in the Capsid Protein of Avian Hepatitis E Virus (HEV) That Are Common to Avian, Swine, and Human HEVs.

UNLABELLED: Antisera raised against the avian hepatitis E virus (HEV) capsid protein are cross-reactive with human and swine HEV capsid proteins. In this study, two monoclonal antibodies (MAbs) against the avian HEV capsid protein, namely, 3E8 and 1B5, were shown to cross-react with the swine HEV capsid protein. The motifs involved in binding both MAbs were identified and characterized using phage display biopanning, peptide synthesis, and truncated or mutated protein expression, along with indirect enzyme-linked immunosorbent assay (ELISA) and Western blotting. The results showed that the I/VPHD motif is a necessary core sequence and that P and H are two key amino acids for recognition by MAb 3E8. The VKLYM/TS motif is the minimal amino acid sequence necessary for recognition by MAb 1B5. Cross-reactivity between the two epitopes and antibodies against avian, swine, and human HEVs in sera showed that both epitopes are common to avian, swine, and human HEVs. In addition, amino acid sequence alignment of the capsid proteins revealed that the key motifs of both novel epitopes are the same in HEVs from different animal species, predicting that they may be common to HEV isolates from boars, rabbits, rats, ferrets, mongooses, deer, and camels as well. Protein modeling analysis showed that both epitopes are at least partially exposed on the surface of the HEV capsid protein. Protective capacity analysis demonstrated that the two epitopes are nonprotective against avian HEV infection in chickens. Collectively, these studies characterize two novel linear B-cell epitopes common to avian, swine, and human HEVs, which furthers the understanding of HEV capsid protein antigenic structure. IMPORTANCE: More and more evidence indicates that the host range diversity of hepatitis E virus (HEV) is a global public health concern. A better understanding of the antigenic structure of the HEV capsid protein may improve disease diagnosis and prevention. In this study, binding site mapping and localization as well as the antigenic biology of two novel linear B-cell epitopes common to several different species of HEV were characterized. These findings partially reveal the antigenic structure of the HEV capsid protein and provide potential applications for the development of diagnostics and interventions for HEV infection.

Detection and localization of rabbit hepatitis e virus and antigen in systemic tissues from experimentally intraperitoneally infected rabbits.

Rabbit hepatitis E virus (HEV) is a novel genotype of HEV, and is considered to pose a risk of zoonotic transmission. Research into the systemic distribution of rabbit HEV in rabbits during different periods of infection has rarely been reported. To better understand this virus, we infected rabbits with second-passage rabbit HEV via an intraperitoneal route. After inoculation, the infection showed two types, temporary and constant infection. The detection of HEV RNA in the feces varied with time, and serum antigen correlated with fecal HEV RNA. Viremia only appeared 72 days after inoculation. The rabbits remained antibody negative throughout the experimental period. When HEV was localized, several organs besides the liver were HEV RNA positive. Tissue antigen was observed immunohistochemically in the different cells of various organs, especially in parts of the small intestine and the characteristic rabbit gut-associated lymphoid tissue. These data provide valuable information for future research into the pathogenesis of HEV.