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Summary Background The role of subclinical severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in perpetuating the COVID-19 pandemic is unknown because population seroprevalence data are absent. We aimed to establish the sensitivity and specificity of our enzyme immunoassay and microneutralisation assay, and the seroprevalence of SARS-CoV-2 in Hong Kong before and after the pandemic, as well as in Hong Kong residents evacuated from Hubei province, China. Methods We did a multicohort study in a hospital and university in Hong Kong. We evaluated the sensitivity of our enzyme immunoassay and microneutralisation assay with RT-PCR data from patients positive for SARS-CoV-2 and the specificity of our enzyme immunoassay and microneutralisation assay with archived serum samples collected before 2019. We compared the seropositivity of the general population of Hong Kong before and after the pandemic had begun, and determined the seropositivity of Hong Kong residents evacuated from Hubei province, China, in March, 2020. Findings Between Feb 26 and March 18, 2020, we assessed RT-PCR samples from 45 patients who had recovered from COVID-19 to establish the sensitivity of our enzyme immunoassay and microneutralisation assay. To establish the specificity of these assays, we retrieved archived serum. The sensitivity was 91·1% (41 of 45 [95% CI 78·8–97·5]) for the microneutralisation assay, 57·8% (26 of 45 [42·2–72·3]) for anti-nucleoprotein IgG, 66·7% (30 of 45 [51·1–80·0]) for anti-spike protein receptor binding domain (RBD) IgG, and 73·3% (33 of 45 [58·1–85·4]) for enzyme immunoassay (either positive for anti-nucleoprotein or anti-RBD IgG). The specificity was 100% (152 of 152 [95% CI 97·6–100·0]) for both the enzyme immunoassay and microneutralisation assay. Among the Hong Kong general population, 53 (2·7%) of 1938 were enzyme immunoassay positive, but of those who were positive, all 53 were microneutralisation negative, and no significant increase was seen in the seroprevalence between April 12, 2018, and Feb 13, 2020. Among asymptomatic Hubei returnees, 17 (4%) of 452 were seropositive with the enzyme immunoassay or the microneutralisation assay, with 15 (88%) of 17 seropositive with the microneutralisation assay, and two familial clusters were identified. Interpretation Our serological data suggest that SARS-CoV-2 is a new emerging virus. The seropositivity rate in Hubei returnees indicates that RT-PCR-confirmed patients only represent a small proportion of the total number of cases. The low seroprevalence suggests that most of the Hong Kong and Hubei population remain susceptible to COVID-19. Future waves of the outbreak are inevitable without a vaccine or antiviral prophylaxis. The role of age-related cross reactive non-neutralising antibodies in the pathogenesis of COVID-19 warrants further investigation. Funding Richard and Carol Yu, May Tam Mak Mei Yin, Shaw Foundation (Hong Kong), Michael Tong, Marina Lee, and the Government Consultancy Service (see acknowledgments for full list).

Background Hepatitis E virus (HEV) variants belonging to Orthohepevirus species A (HEV-A) are the primary cause of human hepatitis E. However, we previously reported that Orthohepevirus species C genotype 1 (HEV-C1), a divergent HEV variant commonly found in rats, also causes hepatitis in humans. Here, we present a clinical-epidemiological investigation of human HEV-C1 infections detected in Hong Kong, with an emphasis on outcomes in immunocompromised individuals. Methods A surveillance system for detecting human HEV-C1 infections was established in Hong Kong. Epidemiological and clinical characteristics of HEV-C1 cases identified via this system between 1 August 2019 and 31 December 2020 were retrieved. Phylogenetic analysis of HEV-C1 strain sequences was performed. Infection outcomes of immunocompromised individuals with HEV-A and HEV-C1 infections were analyzed. Results HEV-C1 accounted for 8 of 53 (15.1%) reverse-transcription polymerase chain reaction (RT-PCR)–confirmed HEV infections in Hong Kong during the study period, raising the total number of HEV-C1 infections detected in the city to 16. Two distinct HEV-C1 strain groups caused human infections. Patients were elderly and/or immunocompromised; half tested negative for HEV immunoglobulin M. Cumulatively, HEV-C1 accounted for 9 of 21 (42.9%) cases of hepatitis E recorded in immunocompromised patients in Hong Kong. Immunocompromised HEV-C1 patients progressed to persistent hepatitis at similar rates (7/9 [77.8%]) as HEV-A patients (10/12 [75%]). HEV-C1 patients responded to oral ribavirin, although response to first course was sometimes poor or delayed. Conclusions Dedicated RT-PCR–based surveillance detected human HEV-C1 cases that evade conventional hepatitis E diagnostic testing. Immunosuppressed HEV-C1–infected patients frequently progress to persistent HEV-C1 infection, for which ribavirin is a suitable treatment option.

Background Previously, hepatitis E in humans was believed to be caused exclusively by species A variants of the Orthohepevirus genus (HEV-A) of the family Hepeviridae. However, we have previously demonstrated that Orthohepevirus species C (HEV-C), also known as rat hepatitis E virus, also causes hepatitis in humans. Due to high sequence divergence between HEV-A and HEV-C, serological tests based on HEV-A are often insensitive for HEV-C diagnosis. Therefore, we developed an enzymatic immunoassay (EIA) system for differentiating HEV-A and HEV-C antibody signatures in patient sera. Methods HEV-A and HEV-C peptide homologs spanning the entire immunogenic E2s region of HEV ORF2 capsid protein were expressed in E. coli. These peptides, HEV-A4 p239 and HEV-C p241, form virus-like particles (figure 1). Both peptides were coated in separate 96-well plates. Sera obtained from patients with RT-PCR confirmed acute HEV-A infection (n = 54), HEV-C infection (n = 15), and uninfected HEV seronegative controls (n = 126) were tested in parallel in HEV-A4 p239 and HEV-C p241 EIAs for respective IgG antibodies. Sample optical densities (ODs) were divided by mean OD + 3SD of the seronegative controls to generate signal/noise (S/N) ratios. S/Ns marking positivity in either assay were determined by ROC analysis. An algorithm for assay interpretation was developed (table 1) and the performance of this algorithm was measured against the RT-PCR gold standard. HEV-A4 p239 and HEV-C1 p241 virus like particles Transmission electron microscopy images of the two peptides used in this study Diagnostic testing algorithm interpretation Interpretation of results of testing using parallel enzymatic immunoassays Results Using cutoffs determined by ROC analysis (figure 2), HEV-A4 p239 and HEV-C p241 EIAs detected species-specific antibody responses well (sensitivity: 92.6% and 80% respectively) and were specific (92.9% and 98.3% respectively). The DC was 100% congruent with HEV-C RT-PCR and 88.9% congruent with HEV-A RT-PCR in RT-PCR positive samples. Incorporating all three cutoffs into the algorithm, we derived a 3×3 confusion matrix of RT-PCR sample assignation vs EIA algorithm classification (table 2). The Cohen’s κ value was 0.883 indicating excellent inter-rater reliability. ROC analysis for determining S/N cutoffs Curve for A/A represents analysis for HEV-A4 p239 EIA. Curve for C/¬C represents analysis for HEV-C p241 EIA. Curve for r(C/A) represents analysis for the differentiating ratio. 3×3 confusion matrix comparing sample assignations by RT-PCR vs. EIA algorithm Conclusion A parallel EIA system accurately differentiated HEV-A and HEV-C serological signatures in acute patient sera. This method can now be applied to seroprevalence studies to determine seroprevalence of rat hepatitis E in human populations. Disclosures Siddharth Sridhar, FRCPath, Abbott (Other Financial or Material Support, Speaker’s honoraria)

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.

Hepatitis E virus (HEV) infection is an important global public health issue. HEV infections are recognized as a zoonotic disease. Swine are believed to be the main reservoir of HEV. Recently, yaks, cows, and yellow cattle have been reported as new reservoirs of HEV. However, whether other species of cattle and buffaloes are sensitive to HEV infection is unknown. To investigate the prevalence of HEV infection in buffaloes, enzyme-linked immunosorbent assay (ELISA) and reverse transcription-nested polymerase chain reaction (RT-nPCR) were performed. Only one buffalo was positive to anti-HEV IgM antibody (1/106, 0.94%), and none were positive for anti-HEV IgG antibody. To our surprise, five serum (5/106, 4.72%) and three milk samples (3/40, 7.50%) from buffaloes were positive to HEV RNA. All strains of HEV isolated from buffaloes belong to genotype 4. Results indicate that buffaloes may be a new reservoir of HEV. RESUMO Infecção com o vírus Hepatite E (HEV) é uma importante questão de saúde pública global. Infecções HEV são reconhecidas como doença zoológica. Acredita-se que suínos são o principal reservatório de HEV. Recentemente iaques, vacas, e gado amarelo foram reportados como novos reservatórios do HEV. Porém, não se sabe se outras espécies de gado e búfalo são sensíveis a infecção HEV. Para investigar a prevalência de infecção HEV em búfalos, foram realizados prova de imunoabsorção enzimática e polimerização em cadeia inversa ancorada em transcrição. Apenas um búfalo foi positivo para o anticorpo anti-HEV IgM (1/106, 0,94%), e nenhum foi positivo para o anticorpo anti-HEV IgG. Para nossa surpresa cinco (5/106, 4,72%) e três amostras de leite (3/40, 7,50%) de búfalos foram positivos para HEV RNA. Todas as estirpes de HEV isoladas de búfalos pertencem ao genótipo 4. Resultados indicam que búfalos podem ser um reservatório de HEV.

Hepatitis E virus (HEV) is the main pathogen of hepatitis worldwide. However, its infection biology and pathogenesis remain largely unknown. Suitable small animal models are required to advance the study of HEV infection. Although an efficient model of genotype 1 (gt1) and gt3 HEV infection has been established in human liver chimeric mice, the infectivity of gt4 HEV infection in mice has not been comprehensively characterized. In this study, immunocompromised BALB/c nude, immunocompetent BALB/c, and C57BL/6 mice were inoculated with either gt3 or gt4 HEV (19 HEV strains, including human, swine, macaque-adapted, and cow HEV strains). Infectivity was identified by viral RNA and antigen detection, inflammation, and histopathological analysis. Then, HEV-infected BALB/c mice were treated with antiviral drugs. Acute HEV infection was established in BALB/c mice inoculated with eight gt4 HEV strains. However, gt3 HEV strains failed to achieve active HEV infection. HEV infection was established in BALB/c nude and regular mice inoculated with gt4 HEV but not in C57BL/6 mice. Gt4 HEV infection resulted in rapid viremia and high titers in feces, sera, and replication sites. HEV infection in mice showed no gender preference. Furthermore, chronic gt4 HEV infection was well imitated in BALB/c mice for 32 weeks and caused liver fibrosis. Conclusions: BALB/c mice have a great potential for reproducing the process of gt4 HEV infection. The successful establishment of a gt4 HEV small-animal model provides an opportunity to further understand HEV infection biology and zoonotic transmission and develop anti-HEV vaccine.

Hepatitis E virus (HEV) infection causes subclinical diseases, leading to high mortality (>25%) in pregnant women. HEV replication is aggressively escalated in pregnant women, especially in the third trimester of pregnancy. Oestrogen plays an important role in pregnancy. However, the pathogenesis of HEV in pregnant women or immunosuppressive pregnant women (such as HIV-infected or organ-transplanted pregnant women) remains unclear. We investigated the role of oestradiol in HEV infection in a cell culture system. HEV-infected pregnant women had significantly higher oestradiol levels compared with uninfected individuals. HEV infection was significantly increased in cells treated with analogues of oestradiol, diethylstilbestrol (DES) or 17β-oestradiol in a dose-dependent way. However, tamoxifen, an antagonist oestrogen, inhibited HEV replication. HEV infection inhibits oestrogen receptor (ER-α) expression. Immunofluorescence and co-immunoprecipitation assays indicated that ER-α interacted with the helicase of HEV ORF1 indirectly. More importantly, HEV infection was exacerbated in immunosuppressive cells treated with an inhibitor of PI3K-AKT-mTOR signal pathway (LY296004) and supplemented with pregnant women serum with high oestradiol simultaneously. These results strongly suggest that pregnant women with high oestradiol and/or immunosuppression will be vulnerable to HEV infection.

We read with interest the paper by Todt et al 1 concerning the mutagenesis of Hepatitis E virus (HEV) caused by ribavirin treatment. HEV is a major cause of acute hepatitis worldwide and lacks a specific treatment.1 2 It replicates in many extrahepatic sites (spleen, kidney, brain and muscle3–5), but whether the testis is included is unknown. Active HEV has been detected in body fluids (blood,2 urine4 and milk6), but no report exists for semen. Thus, HEV prevalence in the semen of infertile males (n=185) sexually abstinent for at least 2 days was investigated by reverse transcriptase-PCR. Ethical approval was obtained from the Institutional Ethics Committee. Unexpectedly, 28.11% (52/185) of the males were HEV RNA positive (HEV RNA+; detectable HEV ORF1 and ORF2 were considered as HEV RNA+, figure 1A) with comparable viral titers to that from urine (see figure S1 in the online supplementary file 1). The prevalence of HEV in semen was higher in infertile males than in serum of pregnant women (1.82%) or the general population (0.58%) in the same city. ### Supplementary file 1 [SP1.pdf] Figure 1 (A) Positive rate of Hepatitis E virus (HEV) RNA in the semen of …