Wenhai Yu

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)

Seroepidemiology and Molecular Characterization of Hepatitis E Virus in Macaca Mulatta from a Village in Yunnan, China, where Infection with this Virus Is Endemic

Background Hepatitis E virus (HEV) infection is a significant public health concern and has been identified as a zoonotic infection. Objectives Since no reports have characterized the epidemiological and genotypic features of HEV infections in Macaca mulatta (rhesus macaques) from Yunnan, China, where swine HEV infections are endemic, we aimed to investigate these characteristics. Materials and Methods Seroepidemiological and molecular characterization of HEV in both Macaca mulatta and pigs from the Yunnan province of China were conducted using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-nested PCR (RT-nPCR). Four hundred and eighty-two stool samples (320 from Macaca mulatta and 162 from pigs) and 92 serum samples (all from Macaca mulatta) were collected for the detection of HEV RNA and anti-HEV antibodies (IgG/IgM). Results Thirty-three rhesus macaques (35.87%) were positive for HEV IgG. Of these, 3 were also positive for HEV IgM. Four different strains of swine HEV RNA were detected in pigs; however, we failed to detect any in Macaca mulatta. Conclusions Results indicate that Macaca mulatta may not be a natural reservoir of HEV.

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.

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.

Increased oestradiol in hepatitis E virus-infected pregnant women promotes viral replication

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.