Fen Huang

Excretion of infectious hepatitis E virus into milk in cows imposes high risks of zoonosis

Hepatitis E virus (HEV) represents the main cause of acute hepatitis worldwide. HEV infection in immunocompromised patients involves a high risk for the development of chronic hepatitis. Because HEV is recognized as a zoonotic pathogen, it is currently believed that swine is the primary reservoir. However, this is not sufficient to justify the strikingly high seroprevalence of HEV in both developing and Western countries. Thus, this study aimed to identify new zoonotic sources that bear a high risk of transmission to humans. We collected fecal, blood, and milk samples of cows in a typical rural region of Yunnan Province in southwest China, where mixed farming of domestic animals is a common practice. HEV RNA was quantified by quantitative real‐time polymerase chain reaction, and the whole genome was sequenced. HEV infectivity was assessed in rhesus macaques. We found a high prevalence of active HEV infection in cows as determined by viral RNA positivity in fecal samples. Surprisingly, we discovered that HEV is excreted into milk that is produced by infected cows. Phylogenetic analysis revealed that all HEV isolates from cow/milk belong to genotype 4 and subtype 4h. Gavage with HEV‐contaminated raw and even pasteurized milk resulted in active infection in rhesus macaques. Importantly, a short period of boiling, but not pasteurization, could completely inactivate HEV. Conclusion: Infectious HEV‐contaminated cow milk is recognized as a new zoonotic source that bears a high risk of transmission to humans; these results call attention to understanding and establishing proper measurement and control of HEV zoonotic transmission, particularly in the setting of mixed farming of domestic animals. (Hepatology 2016;64:350‐359)

Hepatitis E virus infects neurons and brains.

Hepatitis E virus (HEV), as a hepatotropic virus, is supposed to exclusively infect the liver and only cause hepatitis. However, a broad range of extrahepatic manifestations (in particular, idiopathic neurological disorders) have been recently reported in association with its infection. In this study, we have demonstrated that various human neural cell lines (embryonic stem cell-derived neural lineage cells) induced pluripotent stem cell-derived human neurons and primary mouse neurons are highly susceptible to HEV infection. Treatment with interferon-α or ribavirin, the off-label antiviral drugs for chronic hepatitis E, exerted potent antiviral activities against HEV infection in neural cells. More importantly, in mice and monkey peripherally inoculated with HEV particles, viral RNA and protein were detected in brain tissues. Finally, patients with HEV-associated neurological disorders shed the virus into cerebrospinal fluid, indicating a direct infection of their nervous system. Thus, HEV is neurotropic in vitro, and in mice, monkeys, and possibly humans. These results challenge the dogma of HEV as a pure hepatotropic virus and suggest that HEV infection should be considered in the differential diagnosis of idiopathic neurological disorders.

The global burden of hepatitis E outbreaks: a systematic review.

Hepatitis E virus (HEV) is responsible for repeated water‐borne outbreaks since the past century, representing an emerging issue in public health. However, the global burden of HEV outbreak has not been comprehensively described. We performed a systematic review of confirmed HEV outbreaks based on published literatures. HEV outbreaks have mainly been reported from Asian and African countries, and only a few from European and American countries. India represents a country with the highest number of reported HEV outbreaks. HEV genotypes 1 and 2 were responsible for most of the large outbreaks in developing countries. During the outbreaks in developing countries, a significantly higher case fatality rate was observed in pregnant women. In fact, outbreaks have occurred both in open and closed populations. The control measures mainly depend upon improvement of sanitation and hygiene. This study highlights that HEV outbreak is not new, yet it is a continuous global health problem.

RIG‐I is a key antiviral interferon‐stimulated gene against hepatitis E virus regardless of interferon production

Interferons (IFNs) are broad antiviral cytokines that exert their function by inducing the transcription of hundreds of IFN‐stimulated genes (ISGs). However, little is known about the antiviral potential of these cellular effectors on hepatitis E virus (HEV) infection, the leading cause of acute hepatitis globally. In this study, we profiled the antiviral potential of a panel of important human ISGs on HEV replication in cell culture models by overexpression of an individual ISG. The mechanism of action of the key anti‐HEV ISG was further studied. We identified retinoic acid–inducible gene I (RIG‐I), melanoma differentiation–associated protein 5, and IFN regulatory factor 1 (IRF1) as the key anti‐HEV ISGs. We found that basal expression of RIG‐I restricts HEV infection. Pharmacological activation of the RIG‐I pathway by its natural ligand 5′‐triphosphate RNA potently inhibits HEV replication. Overexpression of RIG‐I activates the transcription of a wide range of ISGs. RIG‐I also mediates but does not overlap with IFN‐α‐initiated ISG transcription. Although it is classically recognized that RIG‐I exerts antiviral activity through the induction of IFN production by IRF3 and IRF7, we reveal an IFN‐independent antiviral mechanism of RIG‐I in combating HEV infection. We found that activation of RIG‐I stimulates an antiviral response independent of IRF3 and IRF7 and regardless of IFN production. However, it is partially through activation of the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) cascade of IFN signaling. RIG‐I activated two distinct categories of ISGs, one JAK‐STAT‐dependent and the other JAK‐STAT‐independent, which coordinately contribute to the anti‐HEV activity. Conclusion: We identified RIG‐I as an important anti‐HEV ISG that can be pharmacologically activated; activation of RIG‐I stimulates the cellular innate immunity against HEV regardless of IFN production but partially through the JAK‐STAT cascade of IFN signaling. (Hepatology 2017;65:1823‐1839).

Unphosphorylated ISGF3 drives constitutive expression of interferon-stimulated genes to protect against viral infections

A tripartite transcription factor complex mediates an interferon-independent antiviral response. An interferon-independent antiviral defense Virally infected cells produce type I interferons (IFNs), which stimulate the phosphorylation and activation of the STAT1 and STAT2 transcription factors. When combined with the transcriptional regulator IRF9, phosphorylated STAT1 and STAT2 form the ISGF3 complex, which drives the expression of IFN-stimulated genes (ISGs) that are important for antiviral immunity. Wang et al. report the formation of an alternative form of ISGF3, U-ISGF3, which drove the expression of ISGs and protected cells from viral infection in the absence of detection of detectable IFN or IFN signaling. In addition to IRF9, U-ISGF3 contained unphosphorylated STAT1 and STAT2. Together, these data suggest that U-ISGF3 drives constitutive expression of ISGs as part of an IFN-independent, antiviral immune response. Interferon (IFN)–stimulated genes (ISGs) are antiviral effectors that are induced by IFNs through the formation of a tripartite transcription factor ISGF3, which is composed of IRF9 and phosphorylated forms of STAT1 and STAT2. However, we found that IFN-independent ISG expression was detectable in immortalized cell lines, primary intestinal and liver organoids, and liver tissues. The constitutive expression of ISGs was mediated by the unphosphorylated ISGF3 (U-ISGF3) complex, consisting of IRF9 together with unphosphorylated STAT1 and STAT2. Under homeostatic conditions, STAT1, STAT2, and IRF9 were found in the nucleus. Analysis of a chromatin immunoprecipitation sequencing data set revealed that STAT1 specifically bound to the promoters of ISGs even in the absence of IFNs. Knockdown of STAT1, STAT2, or IRF9 by RNA interference led to the decreased expression of various ISGs in Huh7.5 human liver cells, which was confirmed in mouse embryonic fibroblasts (MEFs) from STAT1−/−, STAT2−/−, or IRF9−/− mice. Furthermore, decreased ISG expression was accompanied by increased replication of hepatitis C virus and hepatitis E virus. Conversely, simultaneous overexpression of all ISGF3 components, but not any single factor, induced the expression of ISGs and inhibited viral replication; however, no phosphorylated STAT1 and STAT2 were detected. A phosphorylation-deficient STAT1 mutant was comparable to the wild-type protein in mediating the IFN-independent expression of ISGs and antiviral activity, suggesting that ISGF3 works in a phosphorylation-independent manner. These data suggest that the U-ISGF3 complex is both necessary and sufficient for constitutive ISG expression and antiviral immunity under homeostatic conditions.

High prevalence of hepatitis E virus infection in goats

Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide, primarily transmitted by fecal‐oral route. Zoonotic transmission of HEV from HEV‐infected pigs (pork) or cows (milk) to human or non‐human primate has been confirmed, but the risk of HEV in goat is still rarely assessed. In the present study, stool, blood, tissues, and milk of goat were collected for HEV infection investigation from Dali City of Yunnan Province in China, where raw mutton and goat milk are traditionally consumed. Surprisingly, a high prevalence of HEV infection in goat was found. Phylogenetic analysis revealed that all HEV isolates from goat belong to genotype 4 and subtype 4h, and shared a high similarity homology (>99.6%) with HEV isolated from human, swine, and cows in the same area. Results suggested that goats are a previously unrecognized HEV host.

Distinct Antiviral Potency of Sofosbuvir Against Hepatitis C and E Viruses.

Readers may submit letters to the editor concerning articles that appeared in Gastroenterology within one month of publication. Detailed guidelines regarding the content are included in the Instructions to Authors. 65 66 67 68 69 Distinct Antiviral Potency of Sofosbuvir against Hepatitis C and E Viruses 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 Dear Editors: Sofosbuvir (SOF), the direct-acting anti–hepatitis C virus (HCV) drug (targeting HCV RdRp; RNA-dependent RNA polymerase), has been recently reported to be a potential anti-hepatitis E virus (HEV) drug candidate. However, some debates emerged whether SOF is a promising drug candidate for treating hepatitis E. Given the important potential clinical implications, this study has assessed comparatively the antiviral efficacy of SOF in both HCV and HEV models. We believe that the anti-HEV and anti-HCV potency of SOF should be assessed comparatively, before proposing its clinical application for treating HEV-infected patients. In our study, the potential anti-HEV effect of SOF was investigated in HEV replication models with concentrations ranging from 0.01 to 10 mmol/L (Supplementary Figure 1A),

Pregnancy serum facilitates hepatitis E virus replication in vitro

Hepatitis E virus (HEV) infection causes high mortality in pregnant women. However, the pathogenic mechanisms of HEV infection in pregnant women remain unknown. In this study, the roles of pregnancy serum in HEV infection were investigated using an efficient cell culture system. HEV infection was exacerbated by supplementing with pregnancy serum, especially theat in third trimester of pregnancy. Oestrogen receptors (ER-α and ER-β) were activated in cells supplemented with pregnancy serum and were significantly inhibited during HEV infection. Type I IFN, especially IFN-β, showed delayed upregulation in HEV-infected cells supplemented with the serum in the third trimester of pregnancy, which indicated that delayed IFN-β expression may facilitate viral replication. Results suggested that pregnancy serum accelerated HEV replication by suppressing oestrogen receptors and type I IFN in the early stage of infection.

Complete genome sequence of Swine hepatitis e virus prevalent in southwest china.

ABSTRACT Hepatitis E virus (HEV) is an important public health concern in the world, especially in developing countries of Africa and Asia, including China. Hepatitis E is recognized as a zoonotic disease, which is transmitted across species, including between humans and swine. HEV is highly endemic in China, but the complete sequence of HEV in southwestern China is lacking. Swine HEV strain KM01 was isolated from a village in rural Kunming, Yunnan province, China, where swine are housed with humans. Here, we report the complete genome sequence of the swine HEV strain KM01. The sequence and phylogenetic analyses reveal that swine HEV is closely related to the strain isolated from Xinjiang (CHN-XJ-SW13). The genome of the KM01 strain will facilitate further study of HEV molecular epidemiology and genetic diversity in China.

Reply to “No evidence for zoonotic HEV infection through dairy milk in Germany”

detection, the supernatant of disrupted HEV-positive liver material from swine was diluted serially in phosphate-buffered saline (PBS) or in milk samples followed by RNA isolation and quantitative RT-PCR. Liver homogenate dilutions of up to 10, corresponding to 48-92 genome equivalents, were constantly detected out of milk or PBS, respectively. In addition, integrity of extracted RNA was proven by amplification of glyceraldehyde 3-phosphate dehydrogenase messenger RNA. In none of the investigated bulk milk samples could HEVspecific RNA be detected. Pigs represent the main reservoir for zoonotic HEV, but high HEV seroprevalence in the human population might be suggestive of further reservoir hosts. We used broadly binding primers and probe to ensure the detection of both zoonotic genotypes HEV-3 and HEV-4, but our ad-hoc investigation did not point at dairy cows to act as such a virus reservoir. Although infections with HEV-4 have been reported more frequently in the last years, HEV-3 is the predominant zoonotic genotype in Europe. At this time point, it can only be speculated whether among zoonotic genotypes exclusively HEV-4 is able to infect cattle. In their study, Huang et al. analyzed samples originating from a rural area, where cattle are often held in close contact to pigs implying cross-species transmission of HEV. Such mixed farming systems are not very common in Germany. Whether this separation of pork and milk production may prevent the establishment of another HEV reservoir in Europe will have to be elucidated in further studies.