Varying abilities of recombinant polypeptides from different regions of hepatitis E virus ORF2 and ORF3 to detect anti‐HEV immunoglobulin M

Following infection with hepatitis E virus (HEV), anti‐HEV immunoglobulin (Ig) M is thought to develop before anti‐HEV IgG and to be a better marker for differentiating between the acute and convalescent phases of infection. In order to select polypeptides for improved detection of anti‐HEV IgM, six and three overlapping polypeptides from open reading frames (ORFs) 2 and 3, respectively, of HEV genotypes 1 and 4 were expressed as fusion proteins in Escherichia coli. The reactivities of the polypeptides with anti‐HEV IgM were evaluated using immunoblotting and enzyme immunoassays (EIAs). The data indicated that polypeptides from the N‐terminus of ORF3 and middle region of ORF2 were weakly or not reactive with anti‐HEV IgM, while those from the remaining regions of ORF2 and ORF3 contained reactive epitopes. Anti‐HEV IgM against the N‐ or C‐terminus of ORF2 appeared earlier and disappeared faster than that against polypeptides from the C‐terminus of ORF3, based on serum samples from rhesus monkeys infected experimentally, and from patients infected naturally, with HEV. The N‐ and C‐terminal polypeptides from ORF2 complemented one another in detecting anti‐HEV IgM and EIA sensitivity was improved significantly with a combination of these polypeptides. The reactivities of ORF2 polypeptides from genotypes 1 and 4 were similar but that of ORF3 differed with sera from monkeys infected by the two genotypes. Thus, a combination of N‐ and C‐terminal polypeptides of ORF2 from one genotype may be effective in EIAs to detect anti‐HEV IgM. J. Med. Virol. 81:1052–1061, 2009. © 2009 Wiley‐Liss, Inc.     

Valuable antibody detection method for classifying hepatitis E virus genotypes

Nucleotide‐based methods are conventionally used to classify the hepatitis E virus (HEV) genotypes. A serological enzyme immunoassay (EIA) using open reading frame 3 (ORF3) C‐terminal peptides was developed to conveniently and accurately classify and evaluate the genotypes of HEV. The sera of mice immunized with HEV genotype 1, 3, and 4 reacted highly specifically to the peptides of the corresponding genotypes. Most (84.2%) clinical sera infected with HEV genotype 4 were positive for anti‐HEV antibodies when tested with the ORF3 peptides of genotype 4, but were negative for genotypes 1 and 3. Monkey and clinical serial sera infected with HEV reacted strongly to the homologous genotype ORF3 peptides. The indirect EIAs were more sensitive, with stronger reactivity, than commercial anti‐HEV immunoglobulin G assays when serial sera from monkeys infected with HEV genotype 1 or 4 were tested. All our results indicate that the serological typing EIA assays described in this study are more effective and convenient for the classification of HEV genotypes than molecular approaches, and can be used to screen large numbers of serum samples and differentiate genotypes for the diagnosis of HEV infections.     

Hepatitis E virus antigen detection as an early diagnostic marker: Report from India

Hepatitis E virus (HEV) is implicated in many outbreaks of viral hepatitis in the Indian subcontinent. The conventional diagnosis of such outbreaks rests on the detection of anti‐HEV IgM antibodies. However, IgM antibodies develop after 4–5 days of infection. An early‐diagnostic marker is imperative for timely diagnosis of the outbreak and also initiation of control measures. This study aimed to determine the use of hepatitis E virus antigen detection as an early diagnostic marker in an outbreak in comparison to anti‐HEV IgM and RT‐PCR analyses. Forty samples were collected during a suspected outbreak of viral hepatitis due to HEV. A total of 36 samples were positive for one or more HEV markers. The positivity for anti‐HEV IgM, HEV antigen, and RT‐PCR was 91.6%, 69.4%, and 47.2% respectively. RT‐PCR and HEV antigen detection gave the highest positive results (100%) in the first 3 days of illness. Positive HEV PCR declined to 54% by Days 4–7, whereas HEV antigen and IgM detection were 88% and 100%, respectively. Sequencing of representative HEV samples indicated that the strains responsible for this outbreak belonged to genotype I, subtype 1a. HEV antigen was found to be an early diagnostic marker of acute infection. HEV antigen was detected in three additional cases in the early phase (1–3 days), and they had no detectable anti‐HEV IgM antibodies. These three samples were also positive for HEV RNA. After Day 7, anti‐HEV IgM was the main diagnostic indicator of infection. J. Med. Virol. 85:823–827, 2013. © 2013 Wiley Periodicals, Inc.     

Immunohistochemistry for hepatitis E virus capsid protein cross-reacts with cytomegalovirus-infected cells: a potential diagnostic pitfall

Immunohistochemistry for hepatitis E virus (HEV) ORF2 (capsid) protein is a powerful tool for tissue-based diagnosis of hepatitis E, particularly useful in evaluating abnormal liver values in immunocompromised patients. We report here a previously unobserved reactivity of the HEV ORF2 antibody to human cytomegalovirus (CMV) proteins and contrast the staining patterns encountered in HEV and CMV infection, respectively. As part of a routine diagnostic work-up, the liver biopsy of an immunocompromised patient with elevated liver values was examined histologically for infection with viruses including CMV and HEV. Cytopathic changes were found, suggestive of CMV infection, which was confirmed by immunohistochemistry. Surprisingly, reactivity of a portion of CMV-infected cells with a mouse monoclonal antibody (clone 1E6) against HEV ORF2 protein was also detected. This observation prompted a screening of 22 further specimens (including liver, gastrointestinal, lung, brain and placental biopsies) with confirmed CMV infection/reactivation. Immunoreactivity of CMV-infected cells with HEV ORF2 antibody was observed in 18 of 23 specimens. While the HEV ORF2 antibody showed cytoplasmic, nuclear and canalicular positivity in hepatitis E cases, positivity in CMV-infected cells was limited to the nucleus. In conclusion, the HEV ORF2 antibody (clone 1E6) shows unexpected immunoreactivity against CMV proteins. In contrast to the hepatitis E staining pattern with cytoplasmic, nuclear and occasional canalicular positivity, reactivity in CMV-infected cells is restricted to the nucleus. Awareness of this cross-reactivity and knowledge of the differences in staining patterns will prevent pathologists from misinterpreting positive HEV ORF2 immunohistochemistry in liver specimens.     

Acute hepatitis E virus infection in Taiwan 2002–2006 revisited: PCR shows frequent co‐infection with multiple hepatitis viruses

Sporadic cases of acute hepatitis E virus (HEV) infection with production of anti‐HEV IgM have been reported occasionally in Taiwan despite no reported outbreaks in the past. This study was undertaken to determine whether serological markers correlated with virus detection. From 2002 to 2006, 72 reported cases of acute hepatitis E seropositive for anti‐HEV IgM in Taiwan were enrolled for investigation. Acute phase serum samples were collected for detection of HEV RNA, HBV DNA, HCV RNA, and GBV‐C RNA by PCR. The results showed that viral sequences of HEV, HBV, HCV and GBV‐C were detected in 54 (75%), 21 (29.2%), 9 (12.5%), and 22 (30.6%) of cases, respectively. Acute hepatitis A co‐infection was excluded in all patients because none were seropositive for anti‐HAV IgM and, nine patients (12.5%) did not seroconvert to anti‐HEV IgG. These results suggest that serum markers did not correlate completely with viremia in the diagnosis of acute HEV infection. Multiple viruses may co‐infect with acute hepatitis E virus in Taiwan. Detection of hepatitis E viremia together with seropositivity for anti‐HEV IgM and followed by seroconversion to anti‐HEV IgG should be included in the diagnostic criteria for HEV infection. J. Med. Virol. 81:1734–1742, 2009. © 2009 Wiley‐Liss, Inc.     

Molecular virology of hepatitis E virus

This review details the molecular virology of the hepatitis E virus (HEV). While replicons and in vitro infection systems have recently become available, a lot of information on HEV has been generated through comparisons with better-studied positive-strand RNA viruses and through subgenomic expression of viral open reading frames. These models are now being verified with replicon and infection systems. We provide here the current knowledge on the HEV genome and its constituent proteins - ORF1, ORF2 and ORF3. Based on the available information, we also modify the existing model of the HEV life cycle.     

Cross-protection of hepatitis E virus genotypes 1 and 4 in rhesus macaques

The purpose of this study was to determine cross-protection between HEV genotypes 1 and 4, which are prevalent in China. Fecal suspensions of genotypes 1 and 4 from patients, as well as genotype 4 from swine, were inoculated intravenously into rhesus macaques. Each inoculum contained 5 x 10(4) genome equivalents of HEV. After infection, serum and fecal samples were collected serially and the levels of alanine aminotransferase (ALT) and anti-HEV IgG and IgM in sera, and HEV RNA in fecal samples, were measured. Liver biopsies were carried out. All the infected monkeys (12/12) developed anti-HEV IgG and exhibited fecal shedding of virus. IgM was detected in 11 of 12, and ALT elevation occurred about 2-6 weeks post-inoculation in 10 of 12, infected monkeys. Hepatic histopathology was consistent with acute viral hepatitis and the ORF2 antigen of HEV was detected in the granular cytoplasm of hepatocytes by immunohistochemistry. After recovery from their initial HEV infection, the monkeys were challenged with a heterologous genotype or heterologous source of HEV and monitored for hepatitis and fecal shedding. Previous infection with HEV completely or partially protected against subsequent challenge with a heterologous virus, because 7 of 11 monkeys did not develop HEV infection or shed virus in the feces, and none of them developed hepatitis or exhibited ALT elevation or liver biopsy findings of hepatitis. In conclusion, previous HEV infection may give rise to cross-genotype and cross-host-species protection.     

Genotypic characterization of symptomatic hepatitis E virus (HEV) infections in Egypt

BackgroundHepatitis E virus (HEV) is a common cause of acute viral hepatitis (AVH) in many developing countries. In Egypt, HEV seroprevalence is among the highest in the world; however, only a very limited number of Egyptian HEV sequences are currently available.ObjectivesThe objectives were to determine the HEV genotype(s) currently circulating in Egypt.Study designAVH patients without serologic evidence of hepatitis A, B, and C viruses were evaluated for possible HEV infection using serologic assays for anti-HEV IgM and anti-HEV IgG and real-time PCR for HEV RNA. Stool suspensions from suspected cases were inoculated into rhesus macaques to confirm the presence of HEV. Sequence analysis was utilized to determine HEV genotype.ResultsOf 287 subjects with AVH enrolled, 58 had serologic evidence of acute HEV infection. Stool samples for two of these patients were repeatedly positive for HEV RNA by real-time PCR. Macaques experimentally inoculated with these human stools also developed viremia. Sequence analysis of open reading frame (ORF) 1 demonstrated that these isolates belonged to HEV genotype 1 and were 3.9–9.5% divergent from other genotype 1 isolates. ORF2 was 5.3–8.7% divergent from previously reported Egyptian isolates.ConclusionsThis study strongly suggests that genotype 1 HEV related to other North African isolates is circulating in acute symptomatic patients in Egypt. Further evaluation of genotypic variability is underway in this highly endemic cohort and is considered an important component of our increased understanding of HEV pathogenesis.     

Occurrence of hepatitis E virus IgM, low avidity IgG serum antibodies, and viremia in sporadic cases of non-A, -B, and -C acute hepatitis.

Serum samples were taken from 57 patients with sporadic non-A, -B, and -C (Non A, B, C) acute hepatitis at different times after onset of the disease and tested for the presence of the hepatitis E virus (HEV) RNA, IgM, and low avidity IgG antibodies. The viral antibodies were detected using two ELISA. One assay (GL) was produced using a mixture of recombinant peptides specified by ORF2 and ORF3 of the viral genome. The other was produced with an ORF2 specified peptide, pE2. The latter occurs naturally as homodimer, it is recognized strongly in its dimeric form by human sera and, in the primate model, it confers protection against experimental HEV infection. Nineteen samples were positive for one or more of these acute markers of HEV infection, 14 of which were acute sera with elevated ALT levels and 5 were convalescent sera with normal ALT level. The results showed that icteric phase of sporadic hepatitis lasts for about 17 days and it coincides with a period when viremia is subsiding as HEV antibodies are developing. Viremia was intermittent and all but one of the 5 instances were confined to the icteric phase with elevated ALT levels. On two of these occasions, viremia preceded detection of HEV antibody, on another 2 occasions it was concurrent with the detection of pE2 specific IgM and/or low avidity IgG and only in one case of protracted viremia was the viral genome detected concurrently with avid pE2 IgG antibody. Ten (71%) of the 14 acute sera were reactive for pE2 IgM, eight (57%) were reactive for low avidity pE2 IgG, and six (43%) for the GL IgM. The sensitivity for the diagnosis of acute hepatitis E may be increased to 87% by combining pE2 IgM and viremia. GL IgM was detected later, but persisted for a longer period of time than the pE2 antibodies, and it was the only acute antibody detected in the convalescent sera.     

Full‐length sequence of genotype 3 hepatitis E virus derived from a pig in Thailand

Hepatitis E virus (HEV) infection in pigs was investigated in two principal swine farming areas in Thailand. Anti‐HEV antibodies and HEV RNA in sera were examined in 258 pigs reared on five commercial farms from age 1 to 6.5 months and sows. Overall, 167 of 258 (64.7%) pigs were positive for anti‐HEV IgG, while 20 of 258 (7.75%) had detectable HEV RNA. Sequence analysis of 20 HEV isolates obtained from viremic pigs revealed that they were 92.3–100% identical to each other and had 82.2–88.2% nucleotide similarity to other reported genotype 3 isolates in 415 nucleotide sequences within ORF2 region. Further characterization by sequencing the complete genome of the Thai swine HEV isolate (named Thai‐swHEV07) and phylogenetic analysis showed that Thai‐swHEV07 segregated into a cluster consisting of swine isolates from Japan, Mongolia, and Kyrgyzstan within the HEV genotype 3. The Thai‐swHEV07 had a genomic length of 7,229 nt excluding the polyadenylated region at 3′ terminus of the genome. Comparison of Thai‐swHEV07 and 27 reported strains of genotype 3 revealed 80.4–85.9% nucleotide identity, with the highest identity of 85.9% to the novel swHEV strain from Mongolia. These findings suggest that genotype 3 HEV isolates are markedly heterogeneous. J. Med. Virol. 81:657–664, 2009 © 2009 Wiley‐Liss, Inc.     

Roles of the genomic sequence surrounding the stem‐loop structure in the junction region including the 3′ terminus of open reading frame 1 in hepatitis E virus replication

Hepatitis E virus (HEV), a member of the family Hepeviridae, causes both acute and chronic viral hepatitis. We have previously demonstrated that the stem‐loop structure in the junction region (JR) of HEV genome plays a critical role in HEV replication. However, the function of the sequence bordering the JR, including the 3′ terminus of open reading frame (ORF1), in HEV replication is unknown. In this study, a panel of HEV Renilla luciferase (Rluc) replicons containing various deletions at 5′ or 3′ termini of the JR was constructed to determine the effect of the deletions on HEV replication in Huh7 human liver cells. We showed that even a single nucleotide deletion at the 5′ terminus of the JR abolished HEV replication, whereas deletions at the 3′ terminus of the JR also decreased virus replication efficiency. Furthermore, we also constructed firefly luciferase and Rluc dual‐reporter HEV replicons containing the 3′ terminal ORF1 of various lengths and the JR inserted upstream of the Rluc reporter. A higher level of HEV replication was observed in cells transfected with replicons containing the 3′ terminal ORF1 than that of the JR only replicon. We also showed that the ORF3 noncoding sequence along with the JR promoted a higher level of translation activity than that promoted by JR and the ORF2 noncoding sequence.     

CD8+ lymphocytes but not B lymphocytes are required for protection against chronic hepatitis E virus infection in chickens

Hepatitis E is an important global disease, causing outbreaks of acute hepatitis in many developing countries and sporadic cases in industrialized countries. Hepatitis E virus (HEV) infection typically causes self-limiting acute hepatitis but can also progress to chronic disease in immunocompromised individuals. The immune response necessary for the prevention of chronic infection is T cell-dependent; however, the arm of cellular immunity responsible for this protection is not currently known. To investigate the contribution of humoral immunity in control of HEV infection and prevention of chronicity, we experimentally infected 20 wild-type (WT) and 18 immunoglobulin knockout (JH-KO) chickens with a chicken strain of HEV (avian HEV). Four weeks postinfection (wpi) with avian HEV, JH-KO chickens were unable to elicit anti-HEV antibody but had statistically significantly lower liver lesion scores than the WT chickens. At 16 wpi, viral RNA in fecal material and liver, and severe liver lesions were undetectable in both groups. To determine the role of cytotoxic lymphocytes in the prevention of chronicity, we infected 20 WT and 20 cyclosporine and CD8⁺ antibody-treated chickens with the same strain of avian HEV. The CD8 ⁺ lymphocyte-depleted, HEV-infected chickens had higher incidences of prolonged fecal viral shedding and statistically significantly higher liver lesion scores than the untreated, HEV-infected birds at 16 wpi. The results indicate that CD8 ⁺ lymphocytes are required for viral clearance and reduction of liver lesions in HEV infection while antibodies are not necessary for viral clearance but may contribute to the development of liver lesions in acute HEV infection.     

Identification of the first strain of swine hepatitis E virus in South America and prevalence of anti-HEV antibodies in swine in Argentina

In Argentina, a country considered non-endemic for hepatitis E virus (HEV) infection, serologic evidence of HEV infection has been observed in different human population groups. In other countries, a high degree of genetic relatedness has been observed between human and swine HEV genotype 3 sequences, suggesting zoonosis as one probable route of infection. This is the first identification of swine HEV in South America. HEV RNA was detected and sequenced in the ORF 1 and ORF 2 regions from swine fecal samples from a herd located in Pergamino, in the province of Buenos Aires. These strains all group into genotype 3 and exhibit a close relationship to two novel HEV variants previously identified in Argentina from sporadic acute cases of non-A to -C hepatitis in humans. In addition, using a modified commercial ELISA, the presence of anti-HEV antibodies was surveyed in five provinces across the country and all five showed a prevalence of HEV antibodies, ranging from 4% to 58%. The results suggest that swine could be an important reservoir for virus transmission in Argentina as has been suggested for other non-endemic areas. The Argentine human strains and swine strain described in this article seem to be closely related to a human Austrian strain, suggesting a potential European origin of HEV infection in these cases.     

Quasispecies dynamics of a hepatitis E virus 4 from the feces and liver biopsy of an acute hepatitis E patient during virus clearance

The presence of a quasispecies in hepatitis E virus (HEV) infection has been documented, however, the implications of a quasispecies in HEV-host interaction are poorly understood. Here, we analyzed the whole genome sequences of a HEV 4d from the feces and liver biopsy of a patient during the icteric and convalescent phases in an acute hepatitis E infection. Viral RNAs were extracted, reversely transcribed and seven fragments encompassing the entire viral genome were amplified and cloned. By sequencing multiple colonies of each cloned viral genome amplicon with Sanger sequencing, we verified the existence of the HEV quasispecies or intra-host genetic variations within the fecal and liver biopsy samples. There were broader genetic variations in the HEV ORF1 region including the PCP, HPX, and RdRp regions during the convalescent phase whereas more genetic variations in the ORF2 P domain during the icteric phase. The quasispecies dynamics might reflect host immune pressure during viral clearance.     

JNK2 modulates the CD1d‐dependent and ‐independent activation of iNKT cells

Invariant natural killer T (iNKT) cells play critical roles in autoimmune, anti‐tumor, and anti‐microbial immune responses, and are activated by glycolipids presented by the MHC class I‐like molecule, CD1d. How the activation of signaling pathways impacts antigen (Ag)‐dependent iNKT cell activation is not well‐known. In the current study, we found that the MAPK JNK2 not only negatively regulates CD1d‐mediated Ag presentation in APCs, but also contributes to CD1d‐independent iNKT cell activation. A deficiency in the JNK2 (but not JNK1) isoform enhanced Ag presentation by CD1d. Using a vaccinia virus (VV) infection model known to cause a loss in iNKT cells in a CD1d‐independent, but IL‐12‐dependent manner, we found the virus‐induced loss of iNKT cells in JNK2 KO mice was substantially lower than that observed in JNK1 KO or wild‐type (WT) mice. Importantly, compared to WT mice, JNK2 KO mouse iNKT cells were found to express less surface IL‐12 receptors. As with a VV infection, an IL‐12 injection also resulted in a smaller decrease in JNK2 KO iNKT cells as compared to WT mice. Overall, our work strongly suggests JNK2 is a negative regulator of CD1d‐mediated Ag presentation and contributes to IL‐12‐induced iNKT cell activation and loss during viral infections.     

Hepatitis E virus ORF3 antigens derived from genotype 1 and 4 viruses are detected with varying efficiencies by an anti-HEV enzyme immunoassay

The function of the hepatitis E virus (HEV) open reading frame 3 (ORF3) protein product remains unclear but it is able to induce a strong antibody response following HEV infection. Therefore, it has been used in some enzyme immunoassays (EIAs) for detecting anti-HEV antibody. In order to evaluate the difference in antigenicity of HEV ORF3 polypeptides derived from genotypes 1 and 4, two EIAs were developed, based on ORF3 polypeptides from genotypes 1 and 4 HEV. Serial weekly serum samples from two rhesus monkeys vaccinated with ORF3 antigens derived from the genotype 4 ORF3 protein and nine rhesus monkeys experimentally infected with genotypes 1 and 4 HEV were tested for anti-HEV using the assays. HEV ORF3 antigens derived from viruses of genotypes 1 and 4 showed different patterns of reactivity with sera obtained from monkeys immunized with ORF3 antigens or infected experimentally with HEV. The genotype 1 ORF3 polypeptide exhibited stronger reactivity with the sera from monkeys infected with genotype 1 than the genotype 4 ORF3 polypeptide. The genotype 4 ORF3 polypeptide demonstrated stronger reactivity with the sera from monkeys infected with genotype 4 than did the genotype 1 ORF3 polypeptide. The HEV ORF3 polypeptide contains genotype-specific antigens and the antigen-antibody reactions between the same genotypes were stronger than those between different genotypes.     

Humanized Rag2γc (RAG-hu) mice can sustain long-term chronic HIV-1 infection lasting more than a year

HIV-1 infection is characterized by life-long viral persistence and continued decline of helper CD4 T cells. The new generation of humanized mouse models that encompass RAG-hu, hNOG and BLT mice have been shown to be susceptible to HIV-1 infection and display CD4 T cell loss. Productive infection has been demonstrated with both R5 and X4 tropic strains of HIV-1 via direct injection as well as mucosal exposure. However the duration of infection in these mice was evaluated for a limited time lasting only weeks post infection, and it is not established how long the viremia can be sustained, and if the CD4 T cell loss persists throughout the life of the infected humanized mice. In the present study we followed the HIV-1 infected RAG-hu mice to determine the long-term viral persistence and CD4 T cell levels. Our results showed that viremia persists life-long lasting for more than a year, and that CD4 T cell levels display a continuous declining trend as seen in the human. These studies provide a chronic HIV-1 infection humanized mouse model that can be used to dissect viral latency, long-term drug evaluation and immune-based therapies.     

Immunity against hepatitis E virus infection: Implications for therapy and vaccine development

Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis worldwide and an emerging cause of chronic infection in immunocompromised patients. As with viral infections in general, immune responses are critical to determine the outcome of HEV infection. Accumulating studies in cell culture, animal models and patients have improved our understanding of HEV immunopathogenesis and informed the development of new antiviral therapies and effective vaccines. In this review, we discuss the recent progress on innate and adaptive immunity in HEV infection, and the implications for the devolopment of effective vaccines and immune‐based therapies.