High Prevalence of Hepatitis E Virus in Swedish Moose – A Phylogenetic Characterization and Comparison of the Virus from Different Regions

Background

Hepatitis E virus (HEV) infects a range of species, including humans, pigs, wild boars and deer. Zoonotic transmission may contribute to the high HEV seroprevalence in the human population of many countries. A novel divergent HEV from moose (Alces alces) in Sweden was recently identified by partial genome sequencing. Since only one strain was found, its classification within the HEV family, prevalence in moose and zoonotic potential was unclear. We therefore investigated samples from 231 moose in seven Swedish counties for HEV, and sequenced a near complete moose HEV genome. Phylogenetic analysis to classify this virus within the family Hepeviridae and to explore potential host specific determinants was performed.

Methods and Findings

The HEV prevalence of moose was determined by PCR (marker for active infection) and serological assays (marker of past infection) of sera and 51 fecal samples from 231 Swedish moose. Markers of active and past infection were found in 67 (29%) animals, while 34 (15%) were positive for HEV RNA, 43 (19%) were seropositive for anti-HEV antibodies, and 10 (4%) had both markers. The number of young individuals positive for HEV RNA was larger than for older individuals, and the number of anti-HEV antibody positive individuals increased with age. The high throughput sequenced moose HEV genome was 35-60% identical to existing HEVs. Partial ORF1 sequences from 13 moose strains showed high similarity among them, forming a distinct monophyletic clade with a common ancestor to HEV genotype 1-6 group, which includes members known for zoonotic transmission.

Conclusions

This study demonstrates a high frequency of HEV in moose in Sweden, with markers of current and past infection demonstrated in 30% of the animals. Moose is thus an important animal reservoir of HEV. The phylogenetic relationship demonstrated that the moose HEV belonged to the genotype 1-6 group, which includes strains that also infect humans, and therefore may signify a potential for zoonotic transmission of this HEV.     

上海部分地区戊型肝炎病毒（HEV）基因型的分析

为了解戊型肝炎病毒 (HEV)在上海部分地区流行的基因型 ,采用RT nPCR的方法检验 35例急性散发性戊型肝炎患者中HEVRNA ,并对阳性产物进行克隆测序 ,然后对其基因型进行分析。结果显示在 35例急性散发性戊型肝炎患者中PCR阳性为 9例 ,测序证实 8例为HEV的基因序列 ;其中 1例为HEV 1型 ,7例为HEV 4型。提示在上海部分地区的急性散发性戊型肝炎中以HEV 4型感染为主     

Detection of Hepatitis E Virus Antibodies in Dogs in the United Kingdom

Hepatitis E virus (HEV) genotypes 3 and 4 are zoonotic pathogens, with pigs predominantly implicated in disease transmission. The rapid rise in human cases in developed countries over the past decade indicates a change in epidemiology of HEV, and it has been suggested that additional animal species may be involved in transmission of infection. Multiple studies have identified contact with dogs as a risk factor for HEV infection in industrialised nations, and a low seroprevalence to HEV has previously been reported in dogs in low-income countries. In this study we aimed to evaluate the possibility that dogs are susceptible to HEV, and determine the frequency with which this occurs. Serum samples from UK dogs with and without hepatitis were screened for HEV-specific antibodies, and canine liver and stool samples were analysed by qPCR for the presence of HEV RNA. We describe evidence to show HEV infection occurs at low levels in dogs in the UK, but the strain of origin is undetermined. The low seroprevalence level of HEV in dogs implies the risk of zoonotic disease transmission is likely to be limited, but further investigations will be required to determine if HEV-infected dogs can transmit HEV to man.     

戊型肝炎病毒遗传多样性和进化机制的研究进展

戊型肝炎病毒(hepatitis E virus, HEV)是一种引发全球急性病毒性肝炎的人兽共患病病原。HEV具有丰富的遗传多样性,不同基因型或基因亚型的流行与地理位置、宿主物种以及防控策略等密切相关。欧洲和美洲HEV流行株为HEV-3,包括3a-i亚型,而亚洲流行株含HEV-3和HEV-4;我国的流行毒株已从HEV-1进化到HEV-4。近年来,研究发现HEV进化的影响机制,包括同义密码子使用模式、氨基酸突变和基因重组等,其中氨基酸突变是病毒持续流行的主要驱动力。因此,本文就HEV的分类、全球流行特征、进化机制等进行综述,以期为戊型肝炎的有效防控以及疫苗开发提供参考。     

Identical consensus sequence and conserved genomic polymorphism of hepatitis E virus during controlled interspecies transmission

High-throughput sequencing of bile and feces from two pigs experimentally infected with human hepatitis E virus (HEV) of genotype 3f revealed the same full-length consensus sequence as in the human sample. Twenty-nine percent of polymorphic sites found in HEV from the human sample were conserved throughout the infection of the heterologous host. The interspecies transmission of HEV quasispecies is the result of a genomic negative-selection pressure on random mutations which can be deleterious to the viral population. HEV intrahost nucleotide diversity was found to be in the lower range of other human RNA viruses but correlated with values found for zoonotic viruses. HEV transmission between humans and pigs does not seem to be modulated by host-specific mutations, suggesting that adaptation is mainly regulated by ecological drivers.     

寡核苷酸芯片法、实时PCR 和测序法进行乙肝病毒 基因分型的比较

目的:比较寡核苷酸芯片法、实时荧光PCR和测序法在对慢性乙肝患者病毒基因分型的比较和方法学评价。方法:对126例不同基因型的慢性乙肝患者的血清样本分别用寡核苷酸芯片法、实时荧光PCR法和测序法进行基因分型,并评价各种方法的临床表现、所需时间和检测成本。结果:寡核苷酸芯片法、实时荧光PCR分别能检测到1%和0.1%比例的基因型。在126例慢性乙肝患者的临床样本中,寡核苷酸芯片法、实时荧光PCR和测序法分别检测出41(33%)、41(33%)和45(36%)例为B型,76(60%)、76(60%)、81(64%)例为C型。寡核苷酸芯片法、实时荧光PCR均检出9例B、C混合基因型。在三种检测方法中实时荧光PCR是最快速和廉价的。结论:寡核苷酸芯片法、实时荧光PCR能检出B、C混合基因型,而测序法只能检测出样本的主导基因型。     

Bats Worldwide Carry Hepatitis E Virus-Related Viruses That Form a Putative Novel Genus within the Family Hepeviridae

Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis in tropical and temperate climates. Tropical genotypes 1 and 2 are associated with food-borne and waterborne transmission. Zoonotic reservoirs (mainly pigs, wild boar, and deer) are considered for genotypes 3 and 4, which exist in temperate climates. In view of the association of several zoonotic viruses with bats, we analyzed 3,869 bat specimens from 85 different species and from five continents for hepevirus RNA. HEVs were detected in African, Central American, and European bats, forming a novel phylogenetic clade in the family Hepeviridae. Bat hepeviruses were highly diversified and comparable to human HEV in sequence variation. No evidence for the transmission of bat hepeviruses to humans was found in over 90,000 human blood donations and individual patient sera. Full-genome analysis of one representative virus confirmed formal classification within the family Hepeviridae. Sequence- and distance-based taxonomic evaluations suggested that bat hepeviruses constitute a distinct genus within the family Hepeviridae and that at least three other genera comprising human, rodent, and avian hepeviruses can be designated. This may imply that hepeviruses invaded mammalian hosts nonrecently and underwent speciation according to their host restrictions. Human HEV-related viruses in farmed and peridomestic animals might represent secondary acquisitions of human viruses, rather than animal precursors causally involved in the evolution of human HEV.     

西北农林科技大学动物医学院，兽医免疫学研究所，杨凌712100

禽戊型肝炎病毒(Hepatitis E virus,HEV)与人、猪HEV同属于肝炎病毒属,它们在遗传性和抗原性上有一定的相关性。自禽HEV被分离鉴定以来,许多国家从血清学或分子流行病学方面证实了该病毒的存在和流行。目前,GenBank上共有5个禽HEV的全基因组或接近全基因组的序列,分为3个基因型,并且其全基因组包含3个ORFs,其中ORF2基因编码病毒的衣壳蛋白,包含病毒主要的抗原表位,是血清学检测和疫苗设计的主要靶蛋白。禽HEV由于其对家禽养殖业的危害以及人畜共患的可能性,正被引起越来越多的关注。本文结合国内禽HEV的分离鉴定从禽HEV病原学、致病性以及衣壳蛋白抗原性等方面进行了总结概述。     

Evolutionary history and population dynamics of hepatitis E virus

Background

Hepatitis E virus (HEV) is an enterically transmitted hepatropic virus. It segregates as four genotypes. All genotypes infect humans while only genotypes 3 and 4 also infect several animal species. It has been suggested that hepatitis E is zoonotic, but no study has analyzed the evolutionary history of HEV. We present here an analysis of the evolutionary history of HEV.

Methods and Findings

The times to the most recent common ancestors for all four genotypes of HEV were calculated using BEAST to conduct a Bayesian analysis of HEV. The population dynamics for genotypes 1, 3 and 4 were analyzed using skyline plots. Bayesian analysis showed that the most recent common ancestor for modern HEV existed between 536 and 1344 years ago. The progenitor of HEV appears to have given rise to anthropotropic and enzootic forms of HEV, which evolved into genotypes 1 and 2 and genotypes 3 and 4, respectively. Population dynamics suggest that genotypes 1, 3 and 4 experienced a population expansion during the 20^th century. Genotype 1 has increased in infected population size ∼30–35 years ago. Genotype 3 and 4 have experienced an increase in population size starting late in the 19^th century until ca.1940-45, with genotype 3 having undergone additional rapid expansion until ca.1960. The effective population size for both genotype 3 and 4 rapidly declined to pre-expansion levels starting in ca.1990. Genotype 4 was further examined as Chinese and Japanese sequences, which exhibited different population dynamics, suggesting that this genotype experienced different evolutionary history in these two countries.

Conclusions

HEV appears to have evolved through a series of steps, in which the ancestors of HEV may have adapted to a succession of animal hosts leading to humans. Analysis of the population dynamics of HEV suggests a substantial temporal variation in the rate of transmission among HEV genotypes in different geographic regions late in the 20^th Century.     

Complete Genome Sequence of Hepatitis E Virus from Rabbits in the United States

Hepatitis E virus (HEV) is a single-strand positive-sense RNA virus in the family Hepeviridae. The disease caused by HEV, hepatitis E, is an important public health problem in developing countries of Asia and Africa and is also endemic in many industrialized countries, including the United States. HEV has been identified from several other animal species in addition to humans, including the pig, chicken, mongoose, deer, rabbit, ferret, bat, and fish. Here we report the complete genome sequence of the first strain of HEV from rabbits in the United States. Sequence and phylogenetic analyses revealed that the U.S. rabbit HEV is a distant member of the zoonotic genotype 3 HEV, thus raising a concern for potential zoonotic human infection. A unique 90-nucleotide insertion within the X domain of the ORF1 was identified in the rabbit HEV, and this insertion may play a role in the species tropism of HEV.     

Experimental Infection of Rabbits with Rabbit and Genotypes 1 and 4 Hepatitis E Viruses

Background

A recent study provided evidence that farmed rabbits in China harbor a novel hepatitis E virus (HEV) genotype. Although the rabbit HEV isolate had 77–79% nucleotide identity to the mammalian HEV genotypes 1 to 4, their genomic organization is very similar. Since rabbits are used widely experimentally, including as models of infection, we investigated whether they constitute an appropriate animal model for human HEV infection.

Methods

Forty-two SPF rabbits were divided randomly into eleven groups and inoculated with six different isolates of rabbit HEV, two different doses of a second-passage rabbit HEV, and with genotype 1 and 4 HEV. Sera and feces were collected weekly after inoculation. HEV antigen, RNA, antibody and alanine aminotransferase in sera and HEV RNA in feces were detected. The liver samples were collected during necropsy subject to histopathological examination.

Findings

Rabbits inoculated with rabbit HEV became infected with HEV, with viremia, fecal virus shedding and high serum levels of viral antigens, and developed hepatitis, with elevation of the liver enzyme, ALT. The severity of disease corresponded to the infectious dose (genome equivalents), with the most severe hepatic disease caused by strain GDC54-18. However, only two of nine rabbits infected with HEV genotype 4, and none infected with genotype 1, developed hepatitis although six of nine rabbits inoculated with the genotype 1 HEV and in all rabbits inoculated with the genotype 4 HEV seroconverted to be positive for anti-HEV IgG antibody by 14 weeks post-inoculation.

Conclusions

These data indicate that rabbits are an appropriate model for rabbit HEV infection but are not likely to be useful for the study of human HEV. The rabbit HEV infection of rabbits may provide an appropriate parallel animal model to study HEV pathogenesis.     

The prevalence and genomic characteristics of hepatitis E virus in murine rodents and house shrews from several regions in China

Background

Urban rodents and house shrews are closely correlated in terms of location with humans and can transmit many pathogens to them. Hepatitis E has been confirmed to be a zoonotic disease. However, the zoonotic potential of rat HEV is still unclear. The aim of this study was to determine the prevalence and genomic characteristics of hepatitis E virus (HEV) in rodents and house shrews.

Results

We collected a total of 788 animals from four provinces in China. From the 614 collected murine rodents, 20.19% of the liver tissue samples and 45.76% of the fecal samples were positive for HEV. From the 174 house shrews (Suncus murinus), 5.17% fecal samples and 0.57% liver tissue samples were positive for HEV. All of the HEV sequences obtained in this study belonged to Orthohepevirus C1. However, we observed a lower percentage of identity in the ORF3 region upon comparing the amino acid sequences between Rattus norvegicus and Rattus losea. HEV derived from house shrews shared a high percentage of identity with rat HEV. Notably, the first near full-length of the HEV genome from Rattus losea is described in our study, and we also report the first near full-length rat HEV genomes in Rattus norvegicus from China.

Conclusion

HEV is prevalent among the three common species of murine rodents (Rattus. norvegicus, Rattus. tanezumi, and Rattus. losea) in China. HEV sequences detected from house shrews were similar to rat HEV sequences. The high identity of HEV from murine rodents and house shrews suggested that HEV can spread among different animal species.

     

First Serological Evidence on Endemicity of HEV Infection in Wild Boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>) Populations from Portugal

Hunting is a common and popular pastime in Portugal. Hunted animals are, generally, for human consumption as meat or local products that are consumed without cooking, increasing the risk of zoonotic transmission of several infectious agents. The present study intended to characterize HEV infection in hunted wild boars (species Sus scrofa) from two regions of Portugal in order to estimate its importance as reservoir for zoonotic spread of HEV to humans, and its possible implication in public health. Markers for both past and/or ongoing HEV infection were evaluated in serum, bile and stool samples of 29 wild boars. The presence of specific HEV antibodies as marker of past infection was evaluated in serum samples, while active HEV infection was evaluated through the detection of HEV genome in bile and stool samples. HEV specific antibodies were detected in 14% of the studied animals, while none of the tested bile or stool samples revealed detectable HEV genome. Despite no active HEV infection was demonstrated in the hunted animals included in the present study, serological analysis revealed the endemicity of HEV infection in Portuguese wild boars from the studied regions, corroborating its possible role as zoonotic reservoir of such virus. The proved endemicity of HEV infection among wild boars further support the importance of including HEV in national and regional surveillance programs for wild animal diseases, as well as to the awareness for thorough cook all wild boar products and to the education of occupationally exposed people in order to prevent HEV infection.     

Immunoenzymatic analysis and genetic detection of Cryptosporidium parvum in lambs from Italy

Cryptosporidiosis is a worldwide-diffused protozoan disease causing important economic losses to animal husbandry and livestock production. Additionally, several species/genotypes of Cryptosporidium have a relevant zoonotic potential and ruminants may be important sources of infection for human beings. Nonetheless, in Europe, little is known of the presence of Cryptosporidium in sheep nor on the species/genotypes involved. To obtain information on the occurrence of cryptosporidiosis in lambs and the potential zoonotic role of the Cryptosporidium isolates, one hundred and forty-nine faecal samples individually collected from lambs in central Italy have been examined for the presence of Cryptosporidium. All faecal specimens were processed with a commercial ELISA kit immunoassay and all ELISA-positive samples were further analyzed genetically. Twenty-six ELISA-positive samples scored positive at the PCR and the sequences obtained displayed 100% identity with the zoonotic Cryptosporidum parvum. This work suggests for the first time that lambs in Italy may shed C. parvum, thus representing a potential public health hazard.     

First isolation of hepatitis E virus genotype 4 in Europe through swine surveillance in the Netherlands and Belgium

Hepatitis E virus (HEV) genotypes 3 and 4 are a cause of human hepatitis and swine are considered the main reservoir. To study the HEV prevalence and characterize circulating HEV strains, fecal samples from swine in the Netherlands and Belgium were tested by RT-PCR. HEV prevalence in swine was 7-15%. The Dutch strains were characterized as genotype 3, subgroups 3a, 3c and 3f, closely related to sequences found in humans and swine earlier. The HEV strains found in Belgium belonged to genotypes 3f and 4b. The HEV genotype 4 strain was the first ever reported in swine in Europe and an experimental infection in pigs was performed to isolate the virus. The genotype 4 strain readily infected piglets and caused fever and virus shedding. Since HEV4 infections have been reported to run a more severe clinical course in humans this observation may have public health implications.     

Detection and characterization of three zoonotic viruses in wild rodents and shrews from Shenzhen city,China

Diverse species of rodents and shrews, which are abundant worldwide, harbor a variety of viruses;some of these are closely related to human viruses and possess zoonotic potential. Previously studies have demonstrated that the mammarenavirus and hantavirus carried by rodents or shrews could cause diseases in human population. To determine the distribution of zoonotic viruses in Shenzhen city, the major city in southern China with a high population density, we analyzed 225 rodents(Rattus norvegicus and Rattus flavipectus) and 196 shrews(Suncus murinus) from urban and rural districts for the presence of mammarenavirus, hantavirus, and hepatitis E virus(HEV) by RT-PCR targeting the conserved regions. The infection rates for mammarenavirus, hantaviruses,and HEV in rodents and shrews were 3.56%, 6.89%, and 1.66%, respectively. Partial genome fragment analysis indicated that mammarenavirus and hantavirus strains had more than 90% and 99% nucleic acid identity with Cardamones virus and Seoul virus, respectively, which cause diseases in humans. Although the present HEV strains identified are typically found worldwide,phylogenetic analysis demonstrated a divergence of 16%. To our knowledge, the present work is the first report of the prevalence of mammarenavirus, hantaviruses, and rat HEV strains in rodents and shrews from Shenzhen city, China. Our findings highlight the zoonotic potential of rodent-and shrew-borne mammarenavirus and hantavirus, and the biodiversity of rat HEV isolates in Shenzhen city. The present work suggests that utilization of good hygiene habits is important to minimize the risk of zoonosis.     

戊型肝炎病毒研究新进展

戊型肝炎病毒(hepatitis E virus,HEV)是引起全球多地区大规模流行和散发病例的急性病毒性肝炎的病原体,发展中国家尤为突出;我国是戊型肝炎(hepatitis E,HE)流行的高发区.HE是一种人畜共患病,猪是人HEV病毒的主要储库,因而HE已成为一个全球性的的公共卫生问题.对HEV的病毒学和基因组特征、临床表征、流行病学、主要抗原表位以及HEV诊断试剂和疫苗研制的进展进行了综述.     

基因Ⅰ、Ⅳ型戊型肝炎病毒高灵敏度通用引物的设计和初步应用

设计针对国内流行的戊型肝炎病毒(HEV)基因Ⅰ、Ⅳ型,在这两型序列的保守区域设计了一套RT-PCR引物——E引物,并将E引物与目前较常用的3对通用引物(Meng、ConORF1和ConORF2引物)比较了检测基因Ⅰ、Ⅳ型HEV的灵敏度。对基因Ⅰ型HEV,E引物能检出的稀释度为105,参考引物能检出的稀释度为10^2～10^4;对基因Ⅳ型HEV,E引物能检出的稀释度为10^2,参考引物能检出的稀释度为10^1～10^2。在17份HEV-IgM阳性血清中,E引物检出5份,检出率为29．4％;参考引物只能检出1份或2份,检出率最高为11．8％。E引物在33份HEV-IgM阳性的隐性感染血清中检出6份,阳性率18．2％;在79份HEV-IgM阳性的临床肝炎血清中检出36份,阳性率45．6％。以上结果初步表明,对于在国内流行的基因Ⅰ、Ⅳ型HEV,E引物的检测灵敏度要高于目前常用的通用引物。     

Understanding animal welfare

Background

Hepatitis E virus (HEV) is a zoonotic pathogen of which swine was reported as major reservoirs. HEV has been divided into 4 different genotypes according to phylogenetic analysis. Recent reports showed that genotype 4 HEV is freely transmitted between humans and swine in eastern China, including Shanghai area. This paper investigated the recent infection status of HEV among swine population of Shanghai area in China.

Methods

480 swine faecal specimens were collected from 23 farms which distribute all over Shanghai from September to November, 2007 and tested for the presence of HEV RNA by the polymerase chain reaction (PCR).

Results

Our results showed that 26.1% (6/23) of the swine farms were positive for HEV RNA and the positive rate of the six farms were ranged from 9.1% to 33.3%. The HEV RNA positive rate for total samples were 5% (24/480). The resulted positive band specific for HEV was sequenced and sequence analysis indicated that all of these isolates belonged to genotype 4 HEV. Phylogenetic analysis showed that the 24 isolates clustered into 4 distinct subgroups, sharing 83.3–89.7% inter-subgroup and 97–99% intra-subgroup identities. More over, isolates in three of the four subgroups closely clustered with previous identified strains, sharing up high to 97% identity with them.

Conclusion

These results suggested that there were 4 different subgenotypes of HEV prevalent in Shanghai, and some of them may not be indigenous to Shanghai but introduced from other geographic regions.