Hepatitis E virus infection of slaughtered healthy pigs in Brazil

Hepatitis E virus (HEV) is an emerging pathogen that can be transmitted through contaminated raw or undercooked meat derived from domestic pigs. HEV infections have been documented among pig herds, pig products and environmental samples raising concern about the spread of the virus. HEV genotypes 3 and 4 are considered zoonotic and have been linked to human cases. HEV was detected in 51 of 335 bile samples (15.2%) from healthy pigs in Minas Gerais, Brazil. Phylogenetic analysis of partial sequences from ORF1 and ORF2 regions yielded discordant results, assigning isolates to subtypes 3c and 3i, respectively, suggesting intragenotypic HEV recombination.     

Seroepidemiology and molecular characterization of hepatitis E virus infection in swine and occupationally exposed workers in Punjab,India

Hepatitis E virus (HEV) has two discrete epidemiological patterns: waterborne epidemics in developing countries only, caused by HEV genotype I, and sporadic zoonotic outbreaks in developing and developed countries caused by genotypes III and IV. This study was designed to investigate seroprevalence, molecular detection and the characterization of HEV by nested RT‐PCR in swine as well as the occupational risk to exposed human population in Punjab state of north‐western India. The occupational risk‐exposed group comprised of swine farmers (organized – mixed feed feeders and unorganized – swill feeders), slaughterhouse workers, sewage workers and veterinary internes. During the study period, blood and faecal samples were collected from 320 swine and 360 humans with both high and low occupational exposure risks. The overall seroprevalence of swine HEV was 65.00%, with a significantly higher seropositivity in growing pigs (2–8 months of age). The prevalence of HEV RNA in swine faecal samples by nRT‐PCR was 8.75% with a significantly higher detection in swill‐fed pigs. With humans in the high occupational exposure risk population, significantly higher anti‐HEV IgG seropositivity was observed (60.48%) as compared to control population (10.71%). Strong evidence of association between human anti‐HEV IgG seropositivity and certain occupational exposure risk groups was observed (p < 0.05). This indicates that unorganized swine farmers, slaughterhouse workers and sewage workers have higher odds of HEV infection in this study region. Percentage of nucleotide similarity between swine and human HEV isolates was less than that found in countries with zoonotic HEV outbreaks. Molecular characterization revealed the circulation of G IV and G I genotypes among swine and human population in Punjab state, respectively.     

A Cross‐Sectional Study of Hepatitis E Virus Infection in Healthy People Directly Exposed and Unexposed to Pigs in a Rural Community in Northern Thailand

A cross‐sectional study of the association between occupational pig exposure and hepatitis E virus (HEV) infection in adult pig farmers and the general population who were not directly exposed to pigs was conducted in Nan Province, Thailand, from November 2010 to April 2011. All participants were interviewed to provide information on their job history, eating habits and other potential confounders. The prevalence of anti‐HEV immunoglobulin G antibodies (IgG) among 513 subjects was 23.0%. Hand washing with water and soap was associated with a lower seroprevalence of HEV infection, whereas living in an area with frequent flooding (OR 1.64, 95% CI: 1.00–2.68) and consuming internal pig organs more than twice per week (OR 3.23, 95%CI: 1.15–9.01) were both associated with a higher seroprevalence of anti‐HEV IgG. There was no association between HEV seroprevalence and frequent, direct occupational pig contact.     

屠宰猪肝和血清中乙型肝炎病毒及戊型肝炎病毒的检测

应用1对乙型肝炎病毒（HBV）S基因保守区的引物，采用PCR方法从屠宰猪肝、血清中检测到了HBV，序列分析表明，扩增片段与已发表的HBVS基因的同源性高达98％～100％。电镜负染色样品观察结果表明，在HBV表面抗原ELISA检测强阳性反应的血清样品中存在有形态、大小与人HBV Dane颗粒和小球状颗粒相似的病毒粒子。针对戊型肝炎病毒（HEV）（）RF2／ORF3重叠区设计了简并引物，采用巢式RT-PCR对屠宰猪肝和血清样品进行了检测。结果表明，部分屠宰猪肝中存在HEV。     

猪戊型肝炎病毒大庆株DQ1全基因序列分析

本试验采用RT．-PCR的方法对戊型肝炎病毒大庆株（DQ1HEV株）的全基因进行分片段扩增，并对其两个末端采用末端快速扩增法（RACE）进行扩增、克隆，测序。与已报道的人的14株HEV的4个基因型的核苷酸和氨基酸进行比较，与IV型HEV的同源性最高。ORF1区与IV型HEV核苷酸的同源性为82．6％～83．6％．氨基酸同源性为93．5％，ORF2区与IV型HEV核苷酸的同源性为87．0％～88.4％，氨基酸同源性为95．8％～97．4％。ORF3区与IV型HEV核苷酸的同源性为94．4％～96．5％，氨基酸同源性为90.3％～96．5％。其结果表明DQ1 HEV株为IV型HEv。     

Hepatitis E virus infection in swine workers: A meta‐analysis

Hepatitis E virus (HEV) infects both humans and animals. Swine has been confirmed to be the principal natural reservoir, which raises a concern that HEV infection would be substantially increasing among swine workers. The present study calculated the pooled prevalence of IgG antibodies against HEV among swine workers and the general population in previous cross‐sectional studies. We conducted a meta‐analysis comparing the prevalence of HEV infection between swine workers and the general population, including local residents, blood donors and non‐swine workers. Through searches in three databases (PubMed and OVID in English, and CNKI in Chinese) and after study selection, a total of 32 studies from 16 countries (from 1999 through 2018) were included in the meta‐analysis. A random‐effect model was employed in the study; an I ² statistic assessed heterogeneity, and the Egger's test detected publication bias. The comparative prevalence of anti‐HEV IgG was pooled from the studies. Compared to the general population, the prevalence ratio (PR) for swine workers was estimated to be 1.52 (95% CI 1.38–1.76) with the I ² being 71%. No publication bias was detected (p = 0.40). A subgroup analysis further indicated increased prevalence of anti‐HEV IgG in the swine workers in Asia (PR = 1.49, 95% CI: 1.35–1.64), in Europe (PR = 1.93, 95% CI: 1.49–2.50) and in all five swine‐related occupations, including swine farmers, butchers, meat processors, pork retailers and veterinarians (PR ranged between 1.19 and 1.75). In summary, swine workers have a relatively higher prevalence of past HEV infection, and this finding is true across swine‐related occupations, which confirms zoonotic transmission between swine and swine workers.     

Development of a novel enzyme-linked immunosorbent assay to detect anti-IgG against swine hepatitis E virus

Swine hepatitis E virus (HEV) is widespread throughout pigs in both developing and industrialized countries. This virus is an important zoonotic agent and a public concern worldwide. Infected pigs are asymptomatic, so diagnosing swine HEV relies on detection of the virus or antibodies against the virus. However, several obstacles need to be overcome for effective and practical serological diagnosis. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) that used a purified recombinant capsid protein of swine HEV. The potential clinical use of this assay was evaluated by comparing it with a commercial kit (Genelabs Technologies, Diagnostics, Singapore). Results of the ELISA were highly correlated with those of the commercial kit with a sensitivity of 97% and specificity of 95%. ROC (receiving operator characteristic) analysis of the ELISA data produced a value of 0.987 (95% CI, 0.977~0.998, p < 0.01). The cut-off value for the ELISA was also determined using negative pig sera. In summary, the HEV-specific ELISA developed in the present study appears to be both practical and economical.     

A systematic review and meta‐analysis of predictors of human hepatitis E virus exposure in non‐endemic countries

The reported incidence of clinical hepatitis E cases is rising in some non‐endemic countries, with concurrent concerns regarding potential hepatitis E virus (HEV) contamination of the blood supply. Therefore, the characterization of major potential sources of human HEV exposure is important to inform risk assessment and public health policy. A systematic review was conducted, including a comprehensive search in six electronic bibliographic databases, verified by hand‐searching reference lists of HEV reviews, and a grey literature search, of the broad research question ‘what is the evidence of the association between predictors of human HEV exposure, and HEV IgG seropositivity, in non‐endemic countries?’ Using forms designed a priori, captured studies were appraised at first‐level screening, second‐level characterization, and third‐level data extraction and risk of bias assessment. Meta‐analysis yielded summary estimates of association between potential predictors and odds of HEV seropositivity. Meta‐analysis and meta‐regression of the odds of HEV seroprevalence in specific groups characterized potential sources of HEV exposure. From 4,163 captured citations, 245 relevant studies underwent data extraction, investigating HEV seroprevalence or predictors in both healthy subjects and targeted patient groups. Across these groups, increasing age was a predictor of HEV IgG seropositivity. Both human immunodeficiency virus patients and haemodialysis patients had significantly increased odds of HEV seropositivity relative to the general population. Working with pigs, in forestry, or in hospitals, was significantly associated with increased odds of HEV seropositivity, as were consumption of meat, pork or game meat, or hunting. Chronological time was not associated with HEV seropositivity within our data sets. Further study of the distribution of potential dietary or behavioural predictors between high and lower prevalence areas within non‐endemic countries could improve our understanding of the relative importance of specific HEV transmission pathways.     

Hepatitis E in southern Vietnam: Seroepidemiology in humans and molecular epidemiology in pigs

Viral pathogens account for a significant proportion of the burden of emerging infectious diseases in humans. The Wellcome Trust‐Vietnamese Initiative on Zoonotic Infections (WT‐VIZIONS) is aiming to understand the circulation of viral zoonotic pathogens in animals that pose a potential risk to human health. Evidence suggests that human exposure and infections with hepatitis E virus (HEV) genotypes (GT) 3 and 4 results from zoonotic transmission. Hypothesising that HEV GT3 and GT4 are circulating in the Vietnamese pig population and can be transmitted to humans, we aimed to estimate the seroprevalence of HEV exposure in a population of farmers and the general population. We additionally performed sequence analysis of HEV in pig populations in the same region to address knowledge gaps regarding HEV circulation and to evaluate if pigs were a potential source of HEV exposure. We found a high prevalence of HEV GT3 viral RNA in pigs (19.1% in faecal samples and 8.2% in rectal swabs) and a high HEV seroprevalence in pig farmers (16.0%) and a hospital‐attending population (31.7%) in southern Vietnam. The hospital population was recruited as a general‐population proxy even though this particular population subgroup may introduce bias. The detection of HEV RNA in pigs indicates that HEV may be a zoonotic disease risk in this location, although a larger sample size is required to infer an association between HEV positivity in pigs and seroprevalence in humans.     

Hepatitis E virus and related viruses in wild,domestic and zoo animals: A review

Hepatitis E is a human disease mainly characterized by acute liver illness, which is caused by infection with the hepatitis E virus (HEV). Large hepatitis E outbreaks have been described in developing countries; however, the disease is also increasingly recognized in industrialized countries. Mortality rates up to 25% have been described for pregnant women during outbreaks in developing countries. In addition, chronic disease courses could be observed in immunocompromised transplant patients. Whereas the HEV genotypes 1 and 2 are mainly confined to humans, genotypes 3 and 4 are also found in animals and can be zoonotically transmitted to humans. Domestic pig and wild boar represent the most important reservoirs for these genotypes. A distinct subtype of genotype 3 has been repeatedly detected in rabbits and a few human patients. Recently, HEV genotype 7 has been identified in dromedary camels and in an immunocompromised transplant patient. The reservoir animals get infected with HEV without showing any clinical symptoms. Besides these well‐known animal reservoirs, HEV‐specific antibodies and/or the genome of HEV or HEV‐related viruses have also been detected in many other animal species, including primates, other mammals and birds. In particular, genotypes 3 and 4 infections are documented in many domestic, wildlife and zoo animal species. In most cases, the presence of HEV in these animals can be explained by spillover infections, but a risk of virus transmission through contact with humans cannot be excluded. This review gives a general overview on the transmission pathways of HEV to humans. It particularly focuses on reported serological and molecular evidence of infections in wild, domestic and zoo animals with HEV or HEV‐related viruses. The role of these animals for transmission of HEV to humans and other animals is discussed.     

北京郊区猪戊型肝炎流行初步调查

用间接ELISA试剂盒对北京市郊区所采集的174份猪血清样品进行了抗体检测,结果显示,其抗体总阳性率达62.1%。针对戊型肝炎病毒(HEV)ORF2/ORF3重叠区设计了简并引物,采用巢式RT-PCR对随即采取的少量猪粪便样品进行了抗原检测。结果表明,部分粪便样品中存在HEV。     

Prevalence of hepatitis E virus infection among people and swine in mainland China: A systematic review and meta‐analysis

Hepatitis E virus (HEV) infection remains an important public health problem, and it is endemic primarily in developing countries. This study aimed to evaluate the seroprevalence of HEV among the general population, occupational population and swine in mainland China and its risk factors based on a systematic review and meta‐analysis. Systematic search from EMBASE, PubMed, Web of Science, Cochrane Library and several Chinese databases, such as Wanfang (WF) Data, China National Knowledge Infrastructure (CNKI), Chinese Science and Technology Periodical Database (VIP) and SINOMED, was searched from inception up to 25 April 2018. The overall seroprevalence of HEV and its corresponding 95% confidence interval (CI) as well as the correlation coefficients between different groups were estimated using stata 12.0 and r ‐3.4.1 software. Potential sources of heterogeneity were explored using subgroup and sensitivity analyses. Twenty‐eight studies with 57,274 participants (including human and swine) were included. The seroprevalence of anti‐HEV immunoglobulin G (IgG) among the general population, occupational population and swine was 27.3% (95% CI: 22.4–32.2), 47.4% (95% CI: 40.1–54.8) and 66.4% (95% CI: 61.7–71.1), respectively. The overall prevalence of IgM among the general population was 1.8% (95% CI: 0.7–2.9). The odds ratio for the occupational population, as compared to the general population, was 2.63. The highest anti‐HEV IgG prevalence (59%) was observed in East China, whereas the lowest (34.8%) was noted in Northeast and North China. In the occupational population, the highest prevalence (77.0%) was observed among swine vendors. Seven studies included 30,392 participants (humans and swine); the correlation coefficient for the prevalence of anti‐HEV IgG between the professional population and adult pigs was 0.88. Sensitivity analyses showed that the stability of results was not considered significant. This research found that HEV is common in China, and contact with pork or other pig products may be an important mode of HEV transmission.     

A serological and virological survey for evidence of infection with Newcastle disease virus in Australian chicken farms

OBJECTIVE: To determine the prevalence and distribution of antibodies to Newcastle disease virus on Australian chicken farms and to determine the pathotype and relationships of the Newcastle disease viruses present on those farms. DESIGN: A cross-sectional survey of 753 commercial chicken farms. PROCEDURE: The survey comprised a detailed questionnaire and collection of venous blood samples. The titre of antibodies to Newcastle disease virus was determined by haemagglutination inhibition. Virus isolation was conducted from cloacal and tracheal swabs taken from chickens in serologically positive flocks. Virus isolates were pathotyped on the basis of the deduced Fusion protein cleavage site determined by nucleotide sequencing of a 265 bp region of the genome in the region of the cleavage site. RESULTS: Antibody evidence of Newcastle disease virus infection was found on 300 of the 753 surveyed farms throughout all 11 geographic regions of the survey. The highest prevalence occurred in the Sydney basin, New South Wales and Victoria east regions. Antibody titres were also highest in the regions where serologically positive flocks were most prevalent. The 259 virus isolates revealed nine different RNA sequences. Of the nine virus groups isolated, the most common group W was identical in sequence to the V4 vaccine strain. Five of the other groups had novel RNA sequences in the region of the F protein cleavage site. CONCLUSIONS: Antibodies to Newcastle disease virus are highly prevalent in the Australian chicken flock but all identified strains were avirulent in nature.     

Seroepidemiology of Hepatitis E in Selected Population Groups in Croatia: A Prospective Pilot Study

Hepatitis E has become an emerging infection in many European countries. We analysed the prevalence of hepatitis E virus (HEV) infection in selected population groups in Croatia. Overall HEV IgG seropositivity was 5.6%, while 1.9% participants showed IgM antibodies suggestive of recent infection. No IgM‐positive sample was positive for HEV RNA. HEV IgG antibodies were most prevalent in alcohol abusers (8.9%) and war veterans (8.6%), compared with 6.1% among injecting drug users and 2.7% in healthcare professionals. No individual with high‐risk sexual behaviour tested HEV seropositive. HEV IgG positivity increased significantly with age from 1.8% to 2.3% in individuals younger than 40 years to 11.3% in individuals older than 50 years (P = 0.023). The mean age of HEV‐positive participants was significantly higher than that of HEV‐negative participants (50.9 ± 11.8 years versus 41.2 ± 11.8 years, P = 0.008). Seroprevalence rates were significantly higher in residents of suburban and rural areas compared with residents of urban areas (14.5% versus 2.5%, P = 0.003). Additionally, an increasing prevalence of HEV IgG antibodies was observed from 1.8% in participants living in families with two household members to 12.1% in those living with more than four members (P = 0.046). Gender, marital status, educational level, sexual orientation, source of drinking water, history of blood transfusions, surgical procedures, tattooing and travelling were not associated with HEV seroprevalence. Logistic regression showed that living in suburban/rural areas was the main risk factor for HEV seropositivity (OR = 6.67; 95%CI = 1.89–25.0; AOR = 7.14, 95%CI = 1.89–25.0).     

东北边境地区野猪及放养杂交野猪戊型肝炎流行病学调查

为了解东北边境地区野猪及放养杂交野猪群体猪戊型肝炎病毒(HEV)感染情况,于2015—2018年在吉林省、黑龙江省的中朝、中俄边境和内蒙古自治区境内加格达奇周边地区采集6月龄以上杂交野猪血清、粪便或肛拭子样品共520份,采集野猪血清和粪便样品共248份。ELISA检测、RT-nPCR检测、全基因组测序、同源性及进化分析结果显示,杂交野猪和野猪感染HEV的血清抗体总阳性率为34.1%(136/399);核酸总阳性率为1.56%(12/771),12份核酸阳性样品均来自杂交野猪,病毒基因组ORF2部分核苷酸序列同源性为85.4%～100.0%,属于基因4型,4a、4b亚型。对4a亚型的1份阳性样品(LJG-18)进行病毒全基因组扩增测序,其核苷酸序列与日本的人源毒株JKO-ChiSai98C同源性最高,为94.9%,与吉林省猪源毒株Ch-S-1同源性为90.2%。结果表明:东北边境地区放养杂交野猪群具有较高的HEV血清抗体阳性率,HEV流行毒株以4a亚型为主。本试验针对我国野猪及放养杂交野猪群体开展猪戊型肝炎流行病学调查,为该病的流行情况提供了新的科学数据,对我国养猪业健康发展和公共卫生安全具有重要意义。     

Risk for interspecies transmission of zoonotic pathogens during poultry processing and pork production in Peru: A qualitative study

Interspecies transmission of pathogens is an unfrequent but naturally occurring event and human activities may favour opportunities not previously reported. Reassortment of zoonotic pathogens like influenza A virus can result from these activities. Recently, swine and birds have played a central role as “mixing vessels” for epidemic and pandemic events related to strains like H1N1 and H5N1. Unsafe practices in poultry markets and swine farms can lead to interspecies transmission, favouring the emergence of novel strains. Thus, understanding practices that lead to interspecies interactions is crucial. This qualitative study aimed to evaluate poultry processing practices in formal and informal markets and the use of leftovers by swine farmers in three Peruvian cities: Lima (capital), Tumbes (coastal) and Tarapoto (jungle). We conducted 80 direct observations at formal and informal markets and interviewed 15 swine farmers. Processors slaughter and pluck chickens and vendors and/or processors eviscerate chickens. Food safety and hygiene practices were suboptimal or absent, although some heterogeneity was observed between cities and chicken vendors versus processors. Both vendors (76%) and processors (100%) sold the chicken viscera leftovers to swine farmers, representing the main source of chicken viscera for swine farms (53%). Swine farmers fed the chicken viscera to their swine. Chicken viscera cooking times varied widely and were insufficient in some cases. Non‐abattoired poultry leads to the sale of poultry leftovers to small‐scale swine farms, resulting in indirect but frequent interspecies contacts that can lead to interspecies transmission of bacterial pathogens or the reassortment of influenza A viruses. These interactions are exacerbated by suboptimal safety and hygiene conditions. People involved in these activities constitute an at‐risk population who could play a central role in preventing the transmission of pathogens between species. Educational interventions on hygiene and food safety practices will be important for reducing the risk of interspecies influenza transmission.     

Serological surveillance and factors associated with influenza A virus in backyard pigs in Southern Brazil

Backyard pig populations are not monitored for influenza A virus (IAV) in Brazil and there are limited data about seroprevalence and risk factors in these populations. Our goal was to assess possible factors associated with IAV seroprevalence in backyard pig populations using an indirect ELISA protocol based on a recombinant nucleoprotein. Following the IAV screening using NP‐ELISA, subtype‐specific serology based on hemagglutination inhibition (HI) assay of the ELISA‐positive pigs was conducted. The survey comprised a total of 1,667 sera samples collected in 2012 and 2014 in 479 holdings and the estimated seroprevalence was 5.3% (3.84%–7.33%) and 2.3% (1.34%–3.71%) in the respective years. In both years, H1N1pdm09 was the most prevalent subtype. The multivariable analysis showed main factors such as “age,” “sex,” “number of suckling pigs” and “neighbours raising pigs” that presented the greatest effect on IAV seroprevalence in these pig populations. These factors may be associated with the low biosecurity measures and management of backyard holdings. In addition, the low IAV seroprevalences found in these backyard pig populations could be related to a low number of animals in each pig holding and low animal movement/replacement that do not favour IAV transmission dynamics. This low frequency of H1N1pdm09 seropositive pigs could also be due to sporadic human‐to‐pig transmission of what is now a human seasonal influenza A virus; however, these factors should be explored in future studies. Herein, these results highlight the importance of IAV continued surveillance in backyard pig holdings, since it is poorly known which IAVs are circulating in these populations and the risk they could pose to public health and virus transmission to commercial farms.     

The study of seroprevalence of hepatitis E virus and an investigation into the lifestyle behaviours of the aborigines in Malaysia

Malaysia is a non‐endemic country for hepatitis E virus (HEV) infection. However, seroprevalence as high as 50% among samples of aboriginal people were reported over two decades ago. A total of 207 samples collected from seven aboriginal villages in rural settlements across two states in Malaysia were analysed for anti‐HEV IgG and IgM by an enzyme‐linked immunoassay. Following the detection of anti‐HEV seroprevalence, we organized health outreach to inform and educate the community. Qualitative interviews were conducted with individuals tested positive for anti‐HEV antibodies. Data derived from interviews and observations were used to investigate possible lifestyle behaviours associated with HEV infection. Anti‐HEV IgG was detected in six samples (5.9%) from the village of Dusun Kubur. Qualitative inquiry and observation study revealed poor dietary and household hygiene, contaminated food and water, contact with animal faeces, unsanitary and domestic waste disposal, and wildlife reservoirs could be the contributing factors for transmission and acquisition of HEV infection. Investigation during health outreach is important to provide insights for future empirical research and implementation for improvement of lifestyle behaviours among the aborigines. Managing the risk of HEV infection in the aborigines may reduce the risk of HEV transmission to the local communities.