Serological evidence of hepatitis E virus infection in different animal species from the Southeast of Brazil

Serological evidence of hepatitis E virus infection (HEV) has been observed in both humans and different animal species living in non-endemic areas, suggesting that animals could be important reservoir for virus transmission to man. Antibodies to HEV have been detected in some Brazilian population groups. Nevertheless, sporadic cases of acute HEV infection have never been reported. We collected 271 serum samples from several domestic animals and also from pig handlers from Southeast of Brazil in order to investigate the seroprevalence of HEV infection. Anti-HEV IgG was detected in cows (1.42%), dogs (6.97%), chickens (20%), swines (24.3%), and rodents (50%), as well as in pig handlers (6.3%). The recognition of swine HEV infections in pigs in many countries of the world led us to investigate a larger sample of pigs (n = 357) from the same Brazilian region with ages ranging from 1 to > 25 weeks. IgG anti-HEV was detected in 100% of 7-day old pigs. Following a gradual decline between weeks 2 and 8 (probably due to loss of maternal IgG), the prevalence then steady increased until it reached 97.3% of animals older than 25 weeks. Besides the detection of anti-HEV antibodies in different animal species, the results showed that swine HEV infection seems to be almost universal within this Brazilian pig population. This is the first report that shows evidences of HEV circulation in Brazilian animal species and pig handlers.     

以合成多肽为抗原的戊肝抗体检测试剂的研制

用戊肝病毒（ＨＥＶ）基因组编码氨基酸序列１－９０１－９１４／２－５１５－５３０、３－９１－１２３、２－６１３－６５４相应的三段合成多肽为抗原、研制出一种检测抗－ＨＥＶＩｇＧ的ＥＬＩＳＡ试剂。以该试剂检测中国、缅甸、印度和前苏联肠道传播非乙型肝炎（ＥＴ－ＮＡＮＢＨ）病人血清１０５份,仅３份中国病人血清阴性,阳性率为９７．１％;检查实验感染ＨＥＶＬ赤猩猩血清,感染前阴性,感染后阳性;检查正常人血清９９     

Comparative studies on the diagnosis of hepatitis E virus antibodies with ORF-2 and ORF-3 proteins expressed in insect cells

Hepatitis E is a worldwide health problem, especially in developing countries. The virus genome contains three different open reading frames (ORFs): ORF-1, which is believed to encode nonstructural proteins, and ORF-2 and ORF-3, which are believed to encode structural proteins. Presently, serologic tests for the detection of human antibodies to hepatitis E virus (HEV) infection are primarily based on the ORF-2 structural protein expressed inEscherichia coli, insect cells or synthetic peptides. We report here the comparative studies on the diagnosis of HEV infection with full-length ORF-2 and ORF-3 proteins expressed in insect cells. We found that 31 of 74 (42%) sera were positive for IgM antibody to HEV (anti-HEV) using the ORF-2 protein as an antigen, as compared to 6 of 74 sera (8%) using the ORF-3 protein as an antigen (p<0.001). Similarly, 49 of 74 sera (66%) were positive for IgG anti-HEV utilizing the ORF-2 protein versus 12 of 74 sera (16%) when the ORF-3 protein was used (p<0.001). These results suggest that the recombinant ORF-2 protein is more sensitive as a diagnostic antigen for detecting antibodies to HEV in both acute-phase and convalescent-phase sera than ORF-3 protein.     

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.     

No transmission of hepatitis E virus in pigs fed diets containing commercial spray-dried porcine plasma: a retrospective study of samples from several swine trials

Background

Hepatitis E virus (HEV) has been reported in the human population and pigs are a recognized reservoir for HEV and a possible source of HEV transmission to humans. Spray-dried porcine plasma (SDPP) is an ingredient commonly used in feed for pigs around the world. Even though processing conditions used to produce SDPP should be adequate to inactivate HEV, it was of interest to analyze commercial SDPP samples for presence of genome and antibodies (AB) against HEV and to retrospectively analyze serum samples collected from pigs used in past experiments that had been fed diets containing either 0% or 8% SDPP to detect potential transmission of HEV as determined by seroconversion.

Results

Eighty-five commercial SDPP samples were analyzed by ELISA and 100% of them contained AB against HEV, while 22.4% (11 of 49 samples analyzed) were positive for HEV RNA.Frozen sera samples (n?=?140) collected from 70 pigs used in past experiments that had been fed diets containing either 0% or 8% commercial SDPP was analyzed by ELISA for AB against HEV. Age of pigs at sera sampling ranged from 3 to 15 weeks and feeding duration of diets ranged from approximately 4 to 9 weeks. One lot of SDPP used in one experiment was analyzed and confirmed to contain HEV RNA. Regardless of the diet fed, some sera samples collected at the beginning of an experiment contained AB titer against HEV. These sera samples were collected from weaned pigs prior to feeding of the experimental diets and the HEV titer was probably from maternal origin. However, by the end of the experiments, HEV titer was not detected or had declined by more than 50% of the initial titer concentration.

Conclusions

To our knowledge, this is the first study reporting presence of HEV AB titer and RNA in SDPP. Retrospective analysis of serum collected from pigs fed diets with SDPP revealed no indication of seroconversion to HEV. The results indicate that feeding SDPP in diets for pigs does not represent a risk of transmitting HEV, even though HEV genome may be detected in SDPP.

     

Serological evidence for hepatitis e virus infection in laboratory monkeys and pigs in animal facilities in Japan

In laboratory animal facilities, monkeys and pigs are used for animal experiments, but the details of hepatitis E virus (HEV) infection in these animals are unknown. The risk of infection from laboratory animals to humans has become a concern; therefore, much attention should be paid to the handling of these animals during their care and use, including surgical procedures performed on infected animals. In this connection, serum samples collected from 916 monkeys and 77 pigs kept in 23 animal facilities belonging to the Japanese Association of Laboratory Animal Facilities of National University Corporations (JALAN) and the Japanese Association of Laboratory Animal Facilities of Public and Private Universities (JALAP) in Japan were examined for the purpose of detecting antibodies to HEV and HEV RNA by using ELISA and RT-PCR, respectively. One hundred and seven serum samples of 916 (11.7%) monkeys were positive for anti-HEV IgG, and 7 and 17 serum samples of 916 (0.8% and 5.3%) monkeys were positive for anti-HEV IgM and IgA, respectively. Thirty-six samples from 62 (58.1%) farm pigs were positive for anti-HEV IgG, whereas all samples tested from miniature pigs were negative (0/15, 0%). Seven samples from 62 (9.1%) farm pigs and 7 samples from 916 (0.8%) monkeys were positive for IgM antibody, but these HEV-IgM antibody positive serum samples were HEV-RNA negative by RT-PCR. The IgM antibody positive rate (9.1%) of farm pigs was much higher than that of monkeys (0.8%). These results suggest the relative levels of risk of HEV infection from these animals to animal handlers and researchers who work with them in laboratory animal facilities.     

Mapping of linear B cell epitopes on open reading frames 2- and 3-encoded proteins of hepatitis E virus using synthetic peptides

Abstract Hepatitis E virus (HEV) is the causative agent of non-A, non-B hepatitis which is transmitted by the fecal-oral route and occurs principally in the form of large epidemics and outbreaks in developing countries. Two overlapping synthetic peptides corresponding to overlapping DNA sequences of the ORF 3 of HEV genome were found to be immunoreactive with sera from patients involved in two epidemics of enterically transmitted non-A, non-B hepatitis. The results suggested the existence of two distinct epitopes. The four synthetic peptides representing these two epitopes from Burma and Mexico strains of hepatitis E virus, were used to investigate anti-HEV reactivities. HEV antibodies were detected in 84–88% of HEV-infected individuals according to the peptide used. The results suggest that a peptide-based ELISA can provide an accurate tool for the diagnosis of acute hepatitis type E.     

中国西藏部分地区猪戊型肝炎病毒流行病学调查

戊型肝炎病毒(Hepatitis E Virus,HEV)感染是一个重要的全球公共卫生问题,而猪被认为是HEV的天然宿主。HEV可以跨种间传播,且已经证实生吃感染的猪肉会导致人感染。在中国西藏许多地区仍然有生吃猪肉、猪肝等的习惯,且不同种家畜混合饲养,极易造成HEV感染和传播。然而中国西藏地区猪HEV流行情况报道甚少。文中对中国西藏5个地区市(拉萨、日喀则、山南、那曲和昌都)猪血清进行HEV Immunoglobulin-M(Ig M)和Ig G抗体检测,并通过逆转录巢氏PCR(RT-n PCR)进行HEV RNA检测和定量RT-PCR(q RT-PCR)进行病毒拷贝计算,首次报道了藏猪血清HEV RNA阳性率。结果显示,在西藏猪中HEV有较高的流行趋势。猪血清HEV Ig M抗体阳性率高达7.6%(26/340),HEV Ig G抗体阳性率为1.8%(6/340),HEV RNA阳性率高达7.6%(26/340),血清中病毒拷贝高达1.7×107 copies/m L,而且5个地区有不同的流行趋势。结果表明西藏猪HEV感染情况严重。有关部门应加强管理,以避免人与动物之间的交叉感染和暴发。     

Cirrhosis,Liver Transplantation and HIV Infection Are Risk Factors Associated with Hepatitis E Virus Infection

Background

Acute and chronic hepatitis E have been associated with high mortality and development of cirrhosis, particularly in solid-organ recipients and patients infected by human immunodeficiency virus. However, data regarding the epidemiology of hepatitis E in special populations is still limited.

Aims

Investigate seroprevalence and possible factors associated with HEV infection in a large cohort of immunosuppressed patients.

Methods

Cross-sectional study testing IgG anti-HEV in serum samples from 1373 consecutive individuals: 332 liver-transplant, 296 kidney-transplant, 6 dual organ recipients, 301 non-transplanted patients with chronic liver disease, 238 HIV-infected patients and 200 healthy controls.

Results

IgG anti-HEV was detected in 3.5% controls, 3.7% kidney recipients, 7.4% liver transplant without cirrhosis and 32.1% patients who developed post-transplant cirrhosis (p<0.01). In patients with chronic liver disease, IgG anti-HEV was also statistically higher in those with liver cirrhosis (2% vs 17.5%, p<0.01). HIV-infected patients showed an IgG anti-HEV rate of 9.2%, higher than those patients without HIV infection (p<0.03). Multivariate analysis showed that the factors independently associated with anti-HEV detection were liver cirrhosis, liver transplantation and HIV infection (OR: 7.6, 3.1 and 2.4). HCV infection was a protective factor for HEV infection (OR: 0.4).

Conclusions

HEV seroprevalence was high in liver transplant recipients, particularly those with liver cirrhosis. The difference in anti-HEV prevalence between Liver and Kidney transplanted cases suggests an association with advanced liver disease. Further research is needed to ascertain whether cirrhosis is a predisposing factor for HEV infection or whether HEV infection may play a role in the pathogeneses of cirrhosis.     

不同戊肝抗原检测抗-HEV IgM反应性研究

目的比较不同戊肝抗原检测抗-HEVIgM反应性。方法用HEVE30、E42、E33合成肽和HEVORF-2重组抗原建立酶免疫试验（EIA）检测肝病患者和健康人群中抗-HEVIgM。结果60份抗-HEV阳性血清中,用E30、E42、E33及重组抗原包被检测抗-HEVIgM,阳性率分别为76.6%,26.6%,18.3%,66.7%。用E30抗原进一步检测戊肝急性期及恢复期血清,抗HEVIgM阳性率为90%及3.3%。结论以HEVE30为抗原的EIA特异性强、灵敏度高,是戊型肝炎早期诊断实用可靠的方法。     

基因重组戊肝病毒多价复合抗原的表达及初步应用

目的：利用基因工程技术体外表达高效HEV多价复合抗原,建立一套敏感和特异的抗HEV检测体系,方法：将前期构建的基因重组戊肝病毒多价复合抗原表达质粒转化大肠埃希菌JM109。筛选鉴定阳性克隆并进行诱导表达,表达产物经分子筛层析纯化后,利用western blot作抗原特异性鉴定,以此抗原建立ELISA检测卫生部生物药品检定所HEV血清参比品及临床血清标本,同时以Genelab公司抗HEVELISA试剂盒作为对照进行对比研究。结果：重组质粒转化大肠埃希菌JM109株后的阳性克隆,经诱导在SDS-PAGE中出现一条分子量大小约为31.5kD的蛋白条带,该蛋白纯化后经Western Blotting检测证实为HEV特异性抗原,以此抗原包被酶联板建立ELISA检测53份国家卫生部标准HEV参比血清,灵敏度达100%（38/38）,特异度达93.3%(14/15)。用此方法检测68份临床标本,并与Gene4labELISA试剂盒结果比较,两者阴阳性相符的有64份,总符合率为94.1%。结论：本实验克隆表达的基因重组戊肝病毒多价复合抗原具有良好抗原性,以此抗原建立的抗HEVELISA具有灵敏度高,特异性强的特点,可望为戊型肝炎的血清学诊断和流行病学调查提供一种更为有效的检测手段。     

Rapid detection of porcine circovirus type 2 using a TaqMan-based real-time PCR

Background

In assays for anti-hepatitis E virus (HEV) immunoglobulin M (IgM), large volumes of the patient's sera cannot be easily obtained for use as a positive control. In this study, we investigated an alternative chemical method in which rabbit anti-HEV IgG was conjugated with human IgM and was used as a positive control in the anti-HEV IgM assay. Rabbit anti-HEV IgG was isolated from immune sera by chromatography on protein A-Sepharose and was conjugated with human IgM by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as a crosslinker.

Results

The specific anti-HEV IgG antibody titer was 100,000 times that of the negative control, i.e., prebleed rabbit serum. The results of anti-HEV IgM enzyme-linked immunosobent assay showed that the antibody conjugate was similar to anti-HEV IgM antibodies produced in humans. The results of a stability experiment showed that the antibody conjugate was stable for use in external quality assessment or internal quality control trials.

Conclusions

We concluded that the chemically conjugated rabbit-human antibody could be used instead of the traditional serum control as a positive control in the anti-HEV IgM assay.     

Development and validation of a recombinant nucleocapsid protein-based ELISA for detection of the antibody to porcine reproductive and respiratory syndrome virus

Three indirect enzyme-linked immunosorbent assays (iELISA) based on the North American like (NA-like), European like (EU-like) and co-expressed NA- and EU-like recombinant nucleocapsid proteins (N-protein) of porcine reproductive and respiratory syndrome virus (PRRSV) were validated for the detection of the antibodies in porcine sera. A total of 422 serum samples from unvaccinated pigs were tested. The cut-off value was optimized by a two-graph receiver operating characteristics analysis at a 95% confidence level. This assay was validated with Western blot analysis and IDEXX HerdChek™ ELISA. Cross-reactivity results showed that iELISA was PRRSV-specific. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were less than 10%. The results indicate that iELISA is simpler to produce and perform, time-saving and suitable for large scale surveys of PRRSV infection at low cost, and is potentially useful to evaluate the efficiency of various vaccines against PRRSV.     

不同基因型戊型肝炎病毒存在多种类型抗原表位

以戊型肝炎病毒(HEV)ORF2重组蛋白p166Us为免疫原制备单克隆抗体(McAbs),采用间接ELISA和免疫印迹法,检测McAbs与不同基因型和亚型HEV重组蛋白p166Bur(Ⅰa型)、p166Pak(Ⅰb型)、p166Mor(Ⅰc型)、p166Mex(Ⅱ型)、p166Us(Ⅲ型)、p166Nz(猪HEV,Ⅲ型)和p166Chn(Ⅳ型)的反应性,采用抗原或抗体竞争ELISA分析p166蛋白与天然HEV颗粒之间抗原表位的关系。结果获得4D3、2E3、11E11、12H5、3A3和1F16株稳定分泌McAbs的杂交瘤细胞株。4D3分泌的McAb与7种p166均发生反应,其与免疫原p166Us的结合可被Ⅰ、Ⅱ、Ⅲ或Ⅳ型天然HEV颗粒或病人血清竞争抑制。2E3、11E11和12H5分泌的McAbs只与p166Us、p166Nz和p166Chn发生反应,它们与p166Us的结合仅能被Ⅲ和IV型病毒或血清所抑制。3A3分泌的McAb只与p166Us及p166Nz结合,1F1分泌的McAb只与p166Us结合,两者均能被Ⅲ型美国株竞争抑制,而Ⅰ、Ⅱ、Ⅳ型不能抑制它们与p166Us的结合。由此可见,不同基因型和亚型HEV ORF2编码蛋白p166上存在多种类型抗原表位,其中包括Ⅰ、Ⅱ、Ⅲ、Ⅳ基因型共同的,Ⅲ、Ⅳ基因型共有的和第Ⅲ基因型特异的等,这些表位与天然HEV颗粒上的抗原表位具有相同的免疫学特征。     

Occurrence of Hepatitis C Virus infection in type 2 diabetic patients attending Plateau state specialist hospital Jos Nigeria

Background

Hepatitis E virus (HEV) is highly endemic in several African countries with high mortality rate among pregnant women. The prevalence of antibodies to HEV in Ghana is not known. Therefore we evaluated the prevalence of anti-HEV IgG and anti-HEV IgM among pregnant women seen between the months of January and May, 2008 at the Obstetrics and Gynaecology Department, Korle-Bu Teaching Hospital, Accra, Ghana.

Results

One hundred and fifty-seven women provided blood samples for unlinked anonymous testing for the presence of antibodies to HEV. The median age of participants was 28.89 ± 5.76 years (range 13–42 years). Of the 157 women tested, HEV seroprevelance was 28.66% (45/157). Among the seropositive women, 64.40% (29/45) tested positive for anti-HEV IgM while 35.60% (16/45) tested positive to HEV IgG antibodies. HEV seroprevalence was highest (46.15%) among women 21–25 years of age, followed by 42.82% in = 20 year group, then 36.84% in = 36 year group. Of the 157 women, 75.79% and 22.92% were in their third and second trimesters of pregnancy, respectively. Anti-HEV antibodies detected in women in their third trimester of pregnancy (30.25%) was significantly higher, P < 0.05, than in women in their second trimester of pregnancy (25.0%).

Conclusion

Consistent with similar studies worldwide, the results of our studies revealed a high prevalence of HEV infection in pregnant women.     

Seroprevalence and Incidence of hepatitis E in Blood Donors in Upper Austria

Background

In recent years various studies showed, that hepatitis E virus (HEV) is a growing public health problem in many developed countries. Therefore, HEV infections might bear a transmission risk by blood transfusions. The clinical relevance still requires further investigations. The aim of this study was to provide an overview of acute HEV infections in Upper Austrian blood donors as well as a risk estimation of this transfusion-related infection.

Methods and Findings

A total of 58,915 blood donors were tested for HEV RNA using a commercial HEV RT-PCR Kit. 7 of these donors (0.01%) were PCR-positive with normal laboratory parameters in absence of clinical signs of hepatitis. Viral load determined by quantitative real-time PCR showed a HEV nucleic acid concentration of 2,217 293,635 IU/ml. At follow-up testing (2–11 weeks after donation) all blood donors had negative HEV RNA results. Additionally, genotyping was performed by amplification and sequencing of the ORF1 or ORF2 region of the HEV genome. All HEV RNA positive donor samples revealed a genotype 3 isolate. For the antibody screening, anti-HEV IgM and IgG were detected by ELISA. Follow up serological testing revealed that no donor was seropositive for HEV IgM or IgG antibodies at time of donation. Moreover, we verified the prevalence of anti-HEV IgG in 1,203 of the HEV RNA negative tested blood donors. Overall 13.55% showed positive results for anti-HEV IgG.

Conclusions

In the presented study, we investigated HEV infections in blood donations of Upper Austria over 1 year. We concluded that 1 out of 8,416 blood donations is HEV RNA positive. Seroprevalence of anti HEV IgG results in an age-related increase of 13.55%. Therefore, based on this data, we recommend HEV-PCR screening to prevent transmission of hepatitis E virus by transfusion.     

Seroprevalence of antibodies to hepatitis A and E viruses in pediatric age groups in Turkey

Hepatitis A and hepatitis E are enteric transmitted viral diseases occurring in epidemic and sporadic forms especially in developing countries. Previous studies in Turkey showed that most residents are infected with HAV by the second decade of life. Since HEV is generally transmitted by the same route as HAV we conducted a community-based seroprevalence study for HAV and HEV infection in Ahatli area in Antalya, Turkey where socioeconomic conditions are low. Anti-HAV total immunoglobulin was tested by using a microparticle EIA (Axsym-Abbott Lab). Anti-HEV IgG was assayed by a micro ELISA method (Genelabs-Singapore). Of the 338 sera tested, 112 (33.1%) were positive for anti-HAV total antibody. Anti-HEV IgG was detected in three (0.89%) of the serum samples. Seropositivity rates of HAV in preschool and school children were 19.9% and 43.9% respectively (p < 0.001). No antibody to HEV was detected in preschool children, while the prevalence of anti-HEV IgG was 1.6% in children attending school. Our data showed that seroprevalence of anti-HAV is high among children samples but HEV infection appears to be relatively rare in pediatric age groups.     

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。