Characterization of virus‒host recombinant variants of the hepatitis E virus.

Hepatitis E virus is a single-strand, positive-sense RNA virus that can lead to chronic infection in immunocompromised patients. Virus-host recombinant variants (VHRVs) have been described in such patients. These variants integrate part of human genes into the polyproline-rich region that could introduce new post-translational modifications (PTMs), such as ubiquitination. The aim of this study was to characterize the replication capacity of different VHRVs, namely, RNF19A, ZNF787, KIF1B, EEF1A1, RNA18, RPS17, and RPL6. We used a plasmid encoding the Kernow strain, in which the fragment encoding the S17 insertion was deleted (Kernow p6 delS17) or replaced by fragments encoding the different insertions. The HEV RNA concentrations in the supernatants and the HepG2/C3A cell lysates were determined via RT-qPCR. The capsid protein ORF2 was immunostained. The effect of ribavirin was also assessed. The HEV RNA concentrations in the supernatants and the cell lysates were higher for the variants harboring the RNF19A, ZNF787, KIF1B, RPS17, and EEF1A1 insertions than for the Kernow p6 del S17, while it was not with RNA18 or RPL6 fragments. The number of ORF2 foci was higher for RNF19A, ZNF787, KIF1B, and RPS17 than for Kernow p6 del S17. VHRVs with replicative advantages were less sensitive to the antiviral effect of ribavirin. No difference in PTMs was found between VHRVs with a replicative advantage and those without. In conclusion, our study showed that insertions did not systematically confer a replicative advantage in vitro. Further studies are needed to determine the mechanisms underlying the differences in replicative capacity. IMPORTANCE: Hepatitis E virus (HEV) is a major cause of viral hepatitis. HEV can lead to chronic infection in immunocompromised patients. Ribavirin treatment is currently used to treat such chronic infections. Recently, seven virus-host recombinant viruses were characterized in immunocompromised patients. These viruses have incorporated a portion of a human gene fragment into their genome. We studied the consequences of these insertions on the replication capacity. We found that these inserted fragments could enhance virus replication for five of the seven recombinant variants. We also showed that the recombinant variants with replicative advantages were less sensitive to ribavirin in vitro. Finally, we found that the mechanisms leading to such a replicative advantage do not seem to rely on the post-translational modifications introduced by the human gene fragment that could have modified the function of the viral protein. The mechanisms involved in improving the replication of such recombinant viruses remain to be explored.

Influence of Nasopharyngeal Viral Load on the Spread of the Omicron BA.2 Variant.

We used variant typing polymerase chain reaction to describe the evolution of severe acute respiratory syndrome coronavirus 2 Omicron sublineages between December 2021 and mid-March 2022. The selective advantage of the BA.2 variant over BA.1 is not due to greater nasopharyngeal viral loads.

Effector memory CD8 T cell response elicits Hepatitis E Virus genotype 3 pathogenesis in the elderly.

Genotype 3 Hepatitis E virus (HEV-3) is an emerging threat for aging population. More than one third of older infected patients develops clinical symptoms with severe liver damage, while others remain asymptomatic. The origin of this discrepancy is still elusive although HEV-3 pathogenesis appears to be immune-mediated. Therefore, we investigated the role of CD8 T cells in the outcome of the infection in immunocompetent elderly subjects. We enrolled twenty two HEV-3-infected patients displaying similar viral determinants and fifteen healthy donors. Among the infected group, sixteen patients experienced clinical symptoms related to liver disease while six remained asymptomatic. Here we report that symptomatic infection is characterized by an expansion of highly activated effector memory CD8 T (EM) cells, regardless of antigen specificity. This robust activation is associated with key features of early T cell exhaustion including a loss in polyfunctional type-1 cytokine production and partial commitment to type-2 cells. In addition, we show that bystander activation of EM cells seems to be dependent on the inflammatory cytokines IL-15 and IL-18, and is supported by an upregulation of the activating receptor NKG2D and an exuberant expression of T-Bet and T-Bet-regulated genes including granzyme B and CXCR3. We also show that the inflammatory chemokines CXCL9-10 are increased in symptomatic patients thereby fostering the recruitment of highly cytotoxic EM cells into the liver in a CXCR3-dependent manner. Finally, we find that the EM-biased immune response returns to homeostasis following viral clearance and disease resolution, further linking the EM cells response to viral burden. Conversely, asymptomatic patients are endowed with low-to-moderate EM cell response. In summary, our findings define immune correlates that contribute to HEV-3 pathogenesis and emphasize the central role of EM cells in governing the outcome of the infection.

Mobilization of γδ T Cells and IL-10 Production at the Acute Phase of Hepatitis E Virus Infection in Cytomegalovirus Carriers.

Alterations in the γδ T cell compartment have been reported in immunocompromised individuals infected with hepatitis E virus (HEV)-g3. We now report the analysis of blood γδ T cells from acutely HEV-infected individuals in the absence of immunosuppression. In these patients, non-Vδ2 (ND2) γδ T cells outnumbered otherwise predominant Vδ2 cells selectively in human CMV (HCMV)-seropositive patients and were higher than in HCMVpos controls, mimicking HCMV reactivation, whereas their serum was PCR-negative for HCMV. Stimulation of their lymphocytes with HEV-infected hepatocarcinoma cells led to an HEV-specific response in γδ subsets of HCMVpos individuals. HEV infection was associated with a lowered expression of TIGIT, LAG-3, and CD160 immune checkpoint markers on ND2 effector memory cells in HCMVneg but not in HCMVpos HEV patients. γδ cell lines, predominantly ND2, were generated from patients after coculture with hepatocarcinoma cells permissive to HEV and IL-2/12/18. Upon restimulation with HEV-infected or uninfected cells and selected cytokines, these cell lines produced IFN-γ and IL-10, the latter being induced by IL-12 in IFN-γ-producing cells and upregulated by HEV and IL-18. They were also capable of suppressing the proliferation of CD3/CD28-activated CD4 cells in transwell experiments. Importantly, IL-10 was detected in the plasma of 10 of 10 HCMVpos HEV patients but rarely in controls or HCMVneg HEV patients, implying that γδ cells are probably involved in IL-10 production at the acute phase of infection. Our data indicate that HEV mobilizes a pool of ND2 memory cells in HCMV carriers, promoting the development of an immunoregulatory environment.

Anti-SARS-CoV-2 spike protein and neutralizing antibodies at 1 and 3 months after three doses of SARS-CoV-2 vaccine in a large cohort of solid organ transplant patients.

The immunogenicity of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) vaccine was improved by the administration of a third dose. The aim of our retrospective study was to assess the evolution of binding and neutralizing antibody concentration until 3 months after the third dose in a large cohort of solid organ transplant (SOT) patients (n = 872). At 1 month after the third dose, anti-SARS-CoV-2 antibodies were detected by means of enzyme-linked immunosorbent assay tests in 578 patients (66.3%). In a subgroup of patients, 70% (180 out of 257) had anti-SARS-CoV-2 antibody concentrations ranging from 1.2 to 18 411 binding antibody units (BAU)/ml and 48.5% (115 out of 239) had a neutralizing antibodies titer that can confer clinical protection against SARS-CoV-2. Three-hundred ninety-three patients out of the 416 (94.5%) who were seropositive at month 1 and were tested at 3 months after vaccination remained seropositive. Between months 1 and 3 after vaccination, binding and neutralizing antibodies concentrations decreased significantly. The proportion of protected patients against the SARS-CoV-2 also slightly decreased. In conclusion, this study shows that although two-third of SOT develop anti-SARS-CoV-2 antibodies after three doses, one-third of them remain weak or non-protected. It is important to measure anti-SARS-CoV-2 antibodies to define the strategy that can optimize SOT protection against SARS-CoV-2.

Novel T cell interferon gamma release assay (IGRA) using spike recombinant protein for COVID19 vaccine response and Nucleocapsid for SARS-Cov2 response.

We explored the performance of a whole blood interferon gamma release assay (IGRA) based on the stimulation of SARS-Cov2-specific T cells by purified recombinant proteins. Twenty volunteers vaccinated with BNT162b2 were selected first for T cell response evaluation using an in-house IGRA, a commercial IGRA, and ELISpot showing a S2 > S1 poly-epitopic response. Next, 64 vaccinated and 103 non-vaccinated individuals were tested for humoral and T cell response (IGRA-Spike/-nucleocapsid recombinant proteins). Following the second vaccine injection, humoral (100%) and IGRA-Spike T cell (95.3%) responses took place irrespective of sex, age, and vaccine type. The humoral response declined first, followed by IGRA-Spike T cell response after the second vaccine injection. Altogether, this study confirms the utility of the IGRA-Spike/-nucleocapsid assay to complement serology in COVID19 vaccinated individuals and those who have recovered from SARS-Cov2.

Glucocorticoid use as a cause of non-cellular immune response to SARS-Cov2 Spike in patients with immune system diseases.

Disease modifying therapies compromise immune response to SARS-Cov2 or its vaccine in patients with immune system diseases (ISD). Therefore, analysis of the humoral and cellular responses against Spike is of utmost importance to manage ISD patients. A single-center retrospective study was conducted to evaluate the impact of COVID-19 immunization in 87 ISD patients and 81 healthy controls. We performed a whole blood interferon gamma release assay using SARS-Cov2 Spike and Nucleocapsid recombinant proteins in order to evaluate T-cell memory response, and an IgG anti-Spike ELISA to evaluate humoral response. Cellular (26.4%) and humoral (44.8%) responses were negative against Spike in ISD patients following COVID-19 immunization. In univariate analysis, an anti-Spike T cell defective response was associated with the use of glucocorticoids (Odds ratio [OR] = 10.0; p < 10-4), serum albumin level ≤40 g/L (OR = 18.9; p < 10-4), age over 55 years old (OR = 3.9, p = 0.009) and ≤2 vaccine injections (OR = 4.9; p = 0.001). The impact of glucocorticoids persisted after adjustment for age and number of vaccine injections (OR = 8.38, p < 0.001). In contrast, the humoral response was impacted by the use of anti-CD20 mAb (OR = 24.8, p < 10-4), and an extended time since immunization (≥75 days; OR = 4.3, p = 0.002). Double defective cellular/humoral responses (6.9%) were typically encountered in glucocorticoids and/or anti-CD20 mAb treated ISD with a serum albumin level ≤40 g/L (OR = 17.5; p = 0.002). Glucocorticoid usage, B cell depleting therapies, and a low serum albumin level were the main factors associated with a non-response to COVID-19 immunization in ISD patients. These results need further confirmation in larger studies.

Adaptive lymphocyte profile analysis discriminates mild and severe forms of COVID-19 after solid organ transplantation.

Solid organ transplant recipients are at high risk for the development of severe forms of COVID-19. However, the role of immunosuppression in the morbidity and mortality of the immune phenotype during COVID-19 in transplant recipients remains unknown. In this retrospective study, we compared peripheral blood T and B cell functional and surface markers, as well as serum antibody development during 29 cases of mild (World Health Organization 9-point Ordinal Scale (WOS) of 3-4) and 22 cases of severe COVID-19 (WOS 5-8) in solid organ transplant (72% kidney transplant) recipients hospitalized in our center. Patients who developed severe forms of COVID-19 presented significantly lower CD3+ (median 344/mm3 (inter quartile range 197; 564) vs. 643/mm3 (397; 1251)) and CD8+ T cell counts (124/mm3 (76; 229) vs. 240/mm3 (119; 435)). However, activated CD4+ T cells were significantly more frequent in severe forms (2.9% (1.37; 5.72) vs. 1.4% (0.68; 2.35)), counterbalanced by a significantly higher proportion of Tregs (3.9% (2.35; 5.87) vs. 2.7% (1.9; 3.45)). A marked decrease in the proportion of NK cells was noted only in severe forms. In the B cell compartment, transitional B cells were significantly lower in severe forms (1.2% (0.7; 4.2) vs. 3.6% (2.1; 6.2)). Nonetheless, a majority of transplant recipients developed antibodies against SARS-CoV-2 (77% and 83% in mild and severe forms, respectively). Thus, our data revealed immunological differences between mild and severe forms of COVID-19 in solid organ transplant recipients, similar to previous reports in the immunocompetent population.

Hepatitis E, what is the real issue?

Hepatitis E virus (HEV) infection is a worldwide disease and the primary cause of acute viral hepatitis with an estimated 3.3 million symptomatic cases every year and 44,000 related deaths. It is a waterborne infection in the developing countries. In these countries, HEV genotypes 1 and 2 cause large outbreaks and affect young subjects resulting in significant mortality in pregnant women and patients with cirrhosis. In developed countries, HEV genotypes 3 and 4 are responsible for autochthonous, sporadic hepatitis and transmission is zoonotic. Parenteral transmission by the transfusion of blood products has been identified as a potential new mode of transmission. HEV can also cause neurological disorders and chronic infections in immunocompromised patients. The progression of acute hepatitis E is usually asymptomatic and resolves spontaneously. Diagnosis is based on both anti-HEV IgM antibodies in serum and viral RNA detection in blood or stools by PCR in immunocompetent patients, while only PCR is validated in immunocompromised individuals. Ribavirin is the only validated treatment in chronic infection. A vaccine has been developed in China.

Hepatitis E virus replication in human intestinal cells.

OBJECTIVE: Hepatitis E virus (HEV), one of the most common agent of acute hepatitis worldwide, is mainly transmitted enterically, via contaminated water for HEV genotypes 1 (HEV1) and HEV2, or by eating raw or undercooked infected meat for HEV genotype 3 (HEV3) and HEV4. However, little is known about how the ingested HEV reaches the liver or its ability to replicate in intestinal cells. DESIGN: We developed human primary cultures of small intestine epithelial cells and intestinal explants obtained from small bowel resections. The epithelial cells were also polarised on transwells. Cells were infected with Kernow-p6 strain or clinically derived virions. RESULTS: Primary intestinal cells supported the growth of Kernow-p6 strain and HEV1 and HEV3 clinically derived virions. Polarised enterocytes infected with HEV1 and HEV3 strains released HEV particles vectorially: mostly into the apical compartment with a little basally. Iodixanol density gradient centrifugation of enterocyte-derived HEV virions gave bands at a density of 1.06-1.08 g/cm3, corresponding to that of quasi-enveloped HEV particles. Ribavirin therapy inhibited HEV excretion from the basal surface but not from the apical side of infected human enterocytes. HEV virions also infected intestinal tissue explants. Lastly, HEV RNA and antigen were detected in the intestinal crypts of a chronically infected patient. CONCLUSION: HEV can replicate in intestinal cells and reaches the liver as quasi-enveloped virions.

Ribavirin for Hepatitis E Virus Infection After Organ Transplantation: A Large European Retrospective Multicenter Study.

BACKGROUND: Ribavirin is currently recommended for treating chronic hepatitis E virus (HEV) infection. This retrospective European multicenter study aimed to assess the sustained virological response (SVR) in a large cohort of solid organ transplant (SOT) recipients with chronic HEV infection treated with ribavirin monotherapy (N = 255), to identify the predictive factors for SVR, and to evaluate the impact of HEV RNA mutations on virological response. METHODS: Data from 255 SOT recipients with chronic HEV infection from 30 European centers were analyzed. Ribavirin was given at the median dose of 600 (range, 29-1200) mg/day (mean, 8.6 ± 3.6 mg/kg/day) for a median duration of 3 (range, 0.25-18) months. RESULTS: After a first course of ribavirin, the SVR rate was 81.2%. It increased to 89.8% when some patients were offered a second course of ribavirin. An increased lymphocyte count at the initiation of therapy was a predictive factor for SVR, while poor hematological tolerance of ribavirin requiring its dose reduction (28%) and blood transfusion (15.7%) were associated with more relapse after ribavirin cessation. Pretreatment HEV polymerase mutations and de novo mutations under ribavirin did not have a negative impact on HEV clearance. Anemia was the main adverse event. CONCLUSIONS: This large-scale retrospective study confirms that ribavirin is highly efficient for treating chronic HEV infection in SOT recipients and shows that the predominant HEV RNA polymerase mutations found in this study do not affect the rate of HEV clearance.This large-scale retrospective study that included 255 solid organ transplant recipients confirms that ribavirin is highly efficient for treating chronic hepatitis E virus (HEV) infection and shows that HEV RNA polymerase mutations do not play a role in HEV clearance.

Risk for Hepatitis E Virus Transmission by Solvent/Detergent-Treated Plasma.

Hepatitis E has emerged as a major transfusion-transmitted infectious risk. Two recipients of plasma from 2 lots (A and B) of pooled solvent/detergent-treated plasma were found to be infected by hepatitis E virus (HEV) that was determined to have been transmitted by the solvent/detergent-treated plasma. HEV RNA viral loads were 433 IU in lot A and 55 IU in lot B. Retrospective studies found that 100% (13/13) of evaluable lot A recipients versus 18% (3/17) of evaluable lot B recipients had been infected by HEV (p<0.001), albeit not necessarily at time of transfusion. Among evaluable recipients, 86% with a transfused HEV RNA load >50,000 IU were infected, most likely by the HEV-containing solvent/detergent-treated plasma, versus only 7% with a transfused HEV RNA load <50,000 IU (p<0.001). Overall, solvent/detergent-treated plasma might harbor HEV. Such an occurrence might result in a dose-dependent risk for transfusion-transmitted hepatitis E.

Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes.

Hepatitis E virus (HEV) is a common cause of acute viral hepatitis worldwide. Most HEV infections are asymptomatic, but immunocompromised patients infected with HEV genotype 3 (HEV3), HEV4, or HEV7 may develop chronic infections. The HEV particles in stools are naked (nHEV), while those in the serum and culture supernatants (eHEV) are associated with lipids. Hepatocytes are polarized epithelial cells that have basolateral (oriented toward the blood) and apical (oriented toward the bile) exosomal pathways. We isolated a subclone, F2, from the human hepatocarcinoma cell line HepG2/C3A that grew as a polarized monolayer culture and had better HEV production than HepG2/C3A cells. F2 cells cultured on semipermeable collagen inserts and infected basolaterally with nHEV3 released 94.6% of virus particles apically, those infected with eHEV3 released 96.8% apically, and eHEV1-infected cells released 99.3% apically. Transcytosis was not involved. Density gradient centrifugation and NP-40 treatment showed that HEV particles released both apically and basolaterally were lipid associated. The apically released HEV3 and HEV1 particles were six and nine times more infectious than those released basolaterally, respectively. Confocal microscopy indicated that the open reading frame 2 (ORF2) capsid protein colocalized apically with ORF3 virus protein, the apical marker DPP4, and the recycling endosome GTPase Rab27a. The amounts of soluble glycosylated ORF2 secreted apically and basolaterally were similar. These polarized-hepatocyte data suggest that infectious HEV particles are mainly released into bile, while the small fraction released into blood could spread HEV throughout the host.IMPORTANCE Hepatitis E virus (HEV) in stools is naked, while that in culture supernatants and patients' blood is lipid associated. Its life cycle in hepatocytes, polarized cells with a basolateral side communicating with blood and an apical side connected with bile, is incompletely understood. We have developed a polarized hepatocyte model and used the cells to analyze the supernatants bathing the apical and basolateral sides and HEV subcellular distribution. HEV particles from both sides were lipid associated, and most infectious HEV particles left the cell via its apical side. Similar amounts of the open reading frame 2 (ORF2) soluble capsid protein were secreted from both sides of the hepatocytes. This model mimicking physiological conditions should help clarify the HEV cell cycle in polarized hepatocytes.

No evidence of sexual transmission of HEV among individuals using HIV pre-exposure prophylaxis.

We investigated the seroprevalence and incidence of hepatitis E virus (HEV) infection in men who have sex with men (MSM) who have been exposed to pre-exposure prophylaxis (PrEP) against HIV as sexual transmission of HEV has been suggested. A total of 147 PrEP-using MSM and 147 blood donors matched for sex, age and geographical area were tested for anti-HEV IgG and IgM. Among them, 135 have been followed for 1 year, at the end of which serological tests for HEV were performed retrospectively on stored samples. Laboratory data on sexual transmitted infections (STIs) and viral hepatitis, including hepatitis A virus (HAV), were collected. Baseline seroprevalence rates in PrEP users were 42.2% (anti-HEV IgG) and 3.4% (anti-HEV IgM). Those of the control blood donors were similar (anti-HEV IgG 43.5% and anti-HEV IgM 4.1%). There was no incident of HEV infection despite the rates of bacterial STIs (incidence rate (IR) = 46.6%) and HAV infection (IR = 15.8%). Age was the only risk factor associated with anti-HEV IgG seropositivity at baseline and at the end of follow-up. Sexual transmission does not seem to be a major route of HEV infection in MSM, unlike HAV.

Hepatitis E, what's the real issue?

Hepatitis E Virus (HEV) infection is a worldwide disease and the primary cause of acute viral hepatitis in the world with an estimated 20 million cases every year and 70 000 deaths. Hepatitis E is a waterborne infection in the developing countries. In these countries, HEV genotypes 1 and 2 cause large outbreaks and affect young subjects, resulting in significant mortality in pregnant women and patients with cirrhosis. In the developed countries, HEV genotypes 3 and 4 are responsible for autochthonous, sporadic hepatitis and transmission is zoonotic. Parenteral transmission by the transfusion of blood products has been identified as a potential new mode of transmission. The prevalence of positive HEV viraemia in blood donors in Europe ranges from 1/600 to 1/2500 in highly endemic European countries. HEV can cause neurological disorders and chronic infections in immunocompromised patients. The progression of acute hepatitis E is usually asymptomatic and resolves spontaneously. Diagnostic tools include anti-HEV IgM antibodies in serum and/or viral RNA detection in the blood or the stools by PCR. Ribavirin is used to treat chronic infection. A vaccine has been developed in China.

Does HEV-3 subtype play a role in the severity of acute hepatitis E?

Hepatitis E virus genotype 3 (HEV-3) is a major aetiologic agent of acute hepatitis in industrialized countries. Two main HEV-3 subtypes are found in Europe: subtypes 3c and 3f. We have analysed the clinical and biological parameters from 100 French immunocompetent patients with an HEV subtype 3f or subtype 3c infection, included in a prospective multicentre study. Stepwise regression analysis found that infections with HEV subtype 3f were associated with fever (OR: 6.1 95%CI: 1.4-26.1), have a greater virus load (OR: 7.4; 95%CI: 1.3-42.2) and require more frequent hospitalization (OR: 7.6; 95%CI: 1.1-51.4) than those infected with subtype 3c. The directed acyclic graph strengthens the multivariate analyses indicating a direct link between the HEV subtype, HEV RNA concentration, fever and hospitalization. Further studies on patients in other European countries are needed to confirm this relationship and determine the underlying mechanism.

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.