Development of a fluorescent microbead-based immunoassay for the detection of hepatitis E virus IgG antibodies in pigs and comparison to an enzyme-linked immunoassay

Swine hepatitis E virus (HEV) is a zoonotic virus and pigs are considered as an important reservoir. Swine HEV infection is widespread and most pig herds are infected. Humans can be infected with swine HEV via consumption of undercooked pork or through direct contact with infected pigs. To minimize the risk of zoonotic transmission, sensitive tools to assess the HEV infection status of pigs and pork products are needed. The objective of this study was to develop a fluorescent microbead-based immunoassay (FMIA) for the detection of IgG antibodies against swine HEV and compare it to an in-house enzyme-linked immunoassay (ELISA). Three sets of samples were utilized: (A) samples from pigs infected experimentally with different strains of HEV (positive controls, n = 72), (B) samples from known HEV-negative pigs (negative controls, n = 62) and (C) samples from pigs of unknown HEV infection status (n = 182). All samples were tested by both ELISA and FMIA. The results on the experimental samples with known HEV exposure indicate that both assays have a specificity of 100% while the sensitivity ranges from 84.6% (ELISA) to 92.3% (FMIA). The overall prevalence of HEV IgG antibodies in field samples from pigs with unknown HEV exposure was 21.9% (40/182) for the ELISA and 21.4% (39/182) for the FMIA. The two assays had an almost perfect overall agreement (Kappa = 0.92).     

Cross-species infection of specific-pathogen-free pigs by a genotype 4 strain of human hepatitis E virus

Hepatitis E virus (HEV) is an important pathogen. The animal strain of HEV, swine HEV, is related to human HEV. The genotype 3 swine HEV can infect humans and genotype 3 human HEV can infect pigs. The genotype 4 swine and human HEV strains are genetically related, but it is unknown whether genotype 4 human HEV can infect pigs. A swine bioassay was utilized in this study to determine whether genotype 4 human HEV can infect pigs. Fifteen, 4-week-old, specific-pathogen-free pigs were divided into three groups of five each. Group 1 pigs were each inoculated intravenously with PBS buffer as negative controls, group 2 pigs similarly with genotype 3 human HEV (strain US-2), and group 3 pigs similarly with genotype 4 human HEV (strain TW6196E). Serum and fecal samples were collected at 0, 7, 14, 21, 28, 35, 42, 49, and 56 days postinoculation (dpi) and tested for evidence of HEV infection. All pigs were necropsied at 56 dpi. As expected, the negative control pigs remained negative. The positive control pigs inoculated with genotype 3 human HEV all became infected as evidenced by detection of HEV antibodies, viremia and fecal virus shedding. All five pigs in group 3 inoculated with genotype 4 human HEV also became infected: fecal virus shedding and viremia were detected variably from 7 to 56 dpi, and seroconversion occurred by 28 dpi. The data indicated that genotype 4 human HEV has an expanded host range, and the results have important implications for understanding the natural history and zoonosis of HEV.     

Incidence and seroprevalence of hepatitis E virus infection in pregnant women infected with hepatitis B virus and antibody placental transfer in infants

BackgroundHepatitis E has poor outcomes in pregnant women. Superinfection of hepatitis E virus (HEV) in patients infected with hepatitis B virus (HBV) may worsen liver disease.ObjectivesTo estimate the incidence and seroprevalence of HEV infection among HBV-infected pregnant women, to investigate the transplacental transfer of maternal anti-HEV IgG, and to compare the maternal and neonatal outcomes in anti-HEV positive and negative pregnant women.Study designTotally 391 HBV-infected pregnant women were recruited from April 2012 to October 2014. Paired mothers and infants were followed up at an average 9.8 months postpartum. Anti-HEV IgG and IgM were tested by ELISA.ResultsOf the pregnant women, none was anti-HEV IgM positive and 42 (10.7%) were IgG positive. At the follow-up, 3 seronegative women converted to anti-HEV IgG positive, with an estimated incidence of 17 per 1000 person-years. No significant differences of gestational age, preterm birth rate, Apgar score and birthweight were observed between newborns of anti-HEV IgG positive and negative mothers. Of the 42 neonates born to anti-HEV IgG positive mothers, 38 (90.5%) had anti-HEV IgG in their cord blood. The neonatal and maternal anti-HEV IgG levels were positively correlated (r = 0.827, p < 0.05). All infants were negative for both anti-HEV IgM and IgG at the follow-up.ConclusionsHBV-infected pregnant women rarely have novel HEV infection during late pregnancy in Jiangsu, China. Maternal anti-HEV IgG efficiently transfers into the fetuses, and disappears in infants before 10 months old.     

Molecular and serological survey of hepatitis E virus infection among domestic pigs in Inner Mongolia, China

To evaluate the prevalence and characteristics of swine hepatitis E virus (HEV) infection in Inner Mongolia, China, serum samples obtained from 356 2- to 4-month-old pigs on 14 farms in Inner Mongolia were tested for the presence of anti-HEV antibodies and HEV RNA. Overall, 186 pigs (52%) tested positive for anti-HEV antibodies, while 30 pigs (8%) had detectable HEV RNA levels. The 30 HEV isolates recovered from the viremic pigs were phylogenetically classified into genotype 4 and differed from each other by up to 15.3% in a 412 nt sequence within ORF2. The Inner Mongolian swine HEV strains were most similar to human or swine HEV strains isolated in the other provinces of China but differed by 15.9–18.9% from those in Mongolia (formerly known as Outer Mongolia). These results indicate that farm pigs in Inner Mongolia are frequently infected with markedly divergent genotype 4 HEV strains that may be indigenous to China.     

Hepatitis E virus seroprevalence in three hyperendemic areas: Nepal,Bangladesh and southwest France

BackgroundHepatitis E causes a significant burden of disease in developing countries and has recently been increasingly recognized in developed countries. Comparing population anti-hepatitis E virus (HEV) seroprevalence across populations has been difficult.ObjectivesThe aim of this study was to compare the anti-HEV IgG seroprevalence in both adults and children in three hyper-endemic areas (Nepal, Bangladesh and southwest France) using a sensitive, commercial anti-HEV IgG assay.Study DesignSerum or plasma from adults and children in Nepal (n=498), Bangladesh (n = 1,009) and Southwest France (n = 1031) were tested for anti-HEV IgG using the Wantai assay.ResultsAfter age-standardization, anti-HEV IgG seroprevalence was 47.1%, 49.8% and 34.0% in Nepal, Bangladesh and southwest France, respectively. There was no difference in seroprevalence by gender in any of the countries. A paucity of infections in children 1–10 years-old was consistently observed (less than 15%) at all 3 locations.ConclusionsSurprisingly similar high rates of anti-HEV antibodies were detected using a common, sensitive assay. Despite differences in the epidemiology and circulating genotype of HEV in Nepal, Bangladesh and southwest France, this study found more similarities in population seroprevalence than expected.     

Discrepancy between anti-hepatitis E virus immunoglobulin G prevalence assessed by two assays in kidney and liver transplant recipients

BackgroundHepatitis E virus (HEV) is an emerging clinical threat in Europe among kidney and liver-transplant recipients. The incidence and prevalence of HEV infection in this special population are poorly known. False-negative results have been observed for anti-HEV IgG detection in severely immunocompromized persons. Moreover, large discrepancies have been reported between rates of anti-HEV IgG detection in blood donors and hepatitis E cases.ObjectivesTo compare anti-HEV IgG and IgM prevalence using two different commercial microplate enzyme-immuno assays (MEIAs) (Adaltis and Wantai) in 64 kidney-/liver-transplant recipients.Study designSerum samples tested in our routine clinical practice over the 12/2009–12/2011 period with Adaltis MEIAs were retrospectively tested using Wantai MEIAs. IgG-positive sera were further tested by an immunoblot while those found IgM-positive were further tested with an immunochromatography rapid test and for the presence of HEV RNA.ResultsPositive results on anti-HEV IgG testing were obtained for seven (10.9%) compared to 20 (31.3%) serum samples with Adaltis and Wantai assays, respectively (p = 0.005). Then, 6/7 (86%) of the serum samples positive with Adaltis and 16/20 (80%) of those positive with Wantai were positive with the immunoblot. One patient with chronic HEV infection was IgG-negative with both MEIAs. Regarding anti-HEV IgM, Adaltis and Wantai assays were concordant for 97% of the serum samples, prevalence being 8% with both MEIAs.ConclusionsThe accuracy of currently available commercial or in-house anti-HEV IgG MEIAs should be tested comparatively on a panel of serum samples collected from solid organ-transplant recipients, including some who experienced PCR-documented HEV infection.     

Diagnostic Performance of Five Assays for Anti-Hepatitis E Virus IgG and IgM in a Large Cohort Study

Determination of anti-hepatitis E virus (anti-HEV) antibodies is still enigmatic. There is no gold standard, and results obtained with different assays often diverge. Herein, five assays were compared for detection of anti-HEV IgM and IgG. Serum samples from 500 Swedish blood donors and 316 patients, of whom 136 had suspected HEV infection, were analyzed. Concordant results for IgM and IgG with all assays were obtained only for 71% and 70% of patients with suspected hepatitis E, respectively. The range of sensitivity for anti-HEV detection was broad (42% to 96%); this was reflected in the detection limit, which varied up to 19-fold for IgM and 17-fold for IgG between assays. HEV RNA was analyzed in all patients and in those blood donors reactive for anti-HEV in any assay, and it was found in 26 individuals. Among all of the assays, both anti-HEV IgG and IgM were detected in 10 of those individuals. Twelve had only IgG and, in 7 of those 12, IgG was only detected with the two most sensitive assays. Three of the HEV-RNA-positive samples were negative for anti-HEV IgM and IgG in all assays. With the two most sensitive assays, anti-HEV IgG was identified in 16% of the blood donor samples and in 66% of patients with suspected HEV infection. Because several HEV-RNA-positive samples had only anti-HEV IgG without anti-HEV IgM or lacked anti-HEV antibodies, analysis for HEV RNA may be warranted as a complement in the laboratory diagnosis of ongoing HEV infection.     

Prevalence of hepatitis E virus infection among hemodialysis patients in Japan: evidence for infection with a genotype 3 HEV by blood transfusion

To investigate the prevalence of hepatitis E virus (HEV) infection among patients on maintenance hemodialysis, serum samples collected in January 2003 from 416 patients who had been undergoing hemodialysis for 7.6 +/- 6.3 (mean +/- standard deviation) (range, 0.3-26.0) years in a dialysis unit in Japan and serum samples that had been collected from these patients at the start of hemodialysis were tested for IgG antibodies to HEV (anti-HEV IgG) by an "in-house" enzyme-linked immunosorbent assay (ELISA). Overall, 39 patients (9.4%) had anti-HEV IgG in January 2003, and included 35 patients (8.4%) who had already been positive for anti-HEV IgG at the start of hemodialysis and 4 patients (1%) who seroconverted after initiation of hemodialysis. Periodic serum samples that had been collected from the four seroconverted patients were tested for HEV antibodies and HEV RNA. The four patients became positive for anti-HEV IgG in 1979, 1980, 1988, or 2003, and continued to be seropositive until the end of the observation period. Although anti-HEV IgM was not detectable in the four patients, three were infected transiently with apparently Japanese indigenous HEV strains of genotype 3. The patient who contracted HEV infection in 1979 had been transfused with 2 U of blood 21 days before the transient viremia: one of the two stored pilot serum samples had detectable HEV RNA with 100% identity to that recovered from the patient. Our study provides evidence of transfusion-transmitted HEV infection in Japan in 1979, and that the prevalence of de novo HEV infection during hemodialysis was low (1.1% or 4/374).     

Evaluation of two VIDAS ®prototypes for detecting anti-HEV IgG

BackgroundHigh performance anti-hepatitis E virus (HEV) IgG assays are crucial for epidemiology.ObjectiveTo evaluate the performance of 2 prototypes developed for the VIDAS^® automatic system for detecting anti-HEV IgG, one based on the ORF2 antigen (ORF2 prototype) and the other on the ORF2 and ORF3 antigens (ORF2/3 prototype), with reference to the Wantai anti-HEV IgG assay.Study designThe sensitivity of each assay was determined by testing 113 blood samples, 63 from immunocompetent and 50 from immunocompromised patients, with a proven HEV infection defined by detecting HEV RNA. Their specificity was assessed with 103 blood samples that the Wantai assay indicated was negative for anti-HEV IgM and IgG, and negative for HEV-RNA. Cross reactivity was assessed using samples that were positive for hepatitis A virus IgG (n = 16), hepatitis C virus antibodies (n = 15), hepatitis B virus antigen and anti-HBc antibodies (n = 16), rheumatoid factor (n = 14), and negative for anti-HEV IgG with the Wantai assay.ResultsThe sensitivities in immunocompetent patients were: 95.2% (ORF2), 96.8% (ORF2/3), and 93.6% (Wantai); in immunocompromised patients they were: 66% (ORF2), 72% (ORF2/3), and 68% (Wantai). Both VIDAS prototypes detected low concentrations of anti-HEV IgG. The overall specificity was 100% (ORF2 prototype) and 98.1% (ORF2/3 prototype). Both VIDAS prototypes cross-reacted in five samples (9.6%), mainly those containing HCV antibodies or rheumatoid factor.ConclusionBoth VIDAS^® prototypes performed very well and appear to be suitable for routine detection of anti-HEV IgG.     

Low frequency of acute hepatitis E virus (HEV) infections but high past HEV exposure in subjects from Cambodia with mild liver enzyme elevations,unexplained fever or immunodeficiency due to HIV-1 infection

BackgroundIn Cambodia, previous studies conducted on hepatitis E virus (HEV) infection are scant, sometimes old, and showed inconsistent results. Moreover, there is no data about HEV infection in Cambodian HIV-1-infected patients.ObjectivesTo assess the occurrence of acute HEV infections and the level of past HEV exposure in one Mekong country.Study designUsing anti-HEV IgM and HEV RNA detection, we retrospectively investigated the presence of acute HEV infection in 825 individuals, including 350 subjects with or without fever, 300 subjects with or without liver enzyme elevations (LEE) and 175 antiretroviral treatment (ART)-naïve, severely immunocompromised HIV-1-infected patients. The detection of anti-HEV IgG was also performed to assess ancient HEV exposure.ResultsNine individuals tested positive for anti-HEV IgM yielding an overall rate of 1.1% (95% confidence interval (CI), 0.5–2.0). We did not find significant differences for anti-HEV IgM rates between subjects with unexplained fevers (1.5%) and those with malaria or dengue-associated fever (1.7%) or non-febrile individuals (0%) (P = 0.49), and between subjects with (1.5%) and without (2.0%) LEE (P = 0.87). No HIV-infected patient tested positive for anti-HEV IgM. HEV RNA was not detected in all tested plasma specimens (n = 578). Overall, the anti-HEV IgG prevalence rate was 30.1% (95% CI, 27.0–33.2).ConclusionsThe scarcity of recent HEV infection contrasted with the high level of past HEV exposure. The role of HEV in liver disease is likely minor in Cambodia since no HEV RNA was detected in our studied populations, including HIV-positive patients with severe immunodepression.     

Pathogenesis of highly virulent African swine fever virus in domestic pigs exposed via intraoropharyngeal,intranasopharyngeal, and intramuscular inoculation,and by direct contact with infected pigs

To investigate the pathogenesis of African swine fever virus (ASFV), domestic pigs (n = 18) were challenged with a range (10²–10⁶ 50% hemadsorbing doses (HAD₅₀)) of the highly virulent ASFV-Malawi strain by inoculation via the intraoropharyngeal (IOP), intranasopharyngeal (INP), or intramuscular (IM) routes. A subsequent contact challenge experiment was performed in which six IOP-inoculated donor pigs were allowed to have direct contact (DC) with six naïve pigs for exposure times that varied from 24 to 72 h. All challenge routes resulted in clinical progression and postmortem lesions similar to those previously described in experimental and natural infection. The onset of clinical signs occurred between 1 and 7 days post inoculation (dpi) and included pyrexia with variable progression to obtundation, hematochezia, melena, moribundity and death with a duration of 4–11 days. Viremia was first detected between 4 and 5 dpi in all inoculation groups whereas ASFV shedding from the nasal cavity and tonsil was first detected at 3–9 dpi. IM and DC were the most consistent modes of infection, with 12/12 (100%) of pigs challenged by these routes becoming infected. Several clinical and virological parameters were significantly different between IM and DC groups indicating dissimilarity between these modes of infection. Amongst the simulated natural routes, INP inoculation resulted in the most consistent progression of disease across the widest range of doses whilst preserving simulation of natural exposure and therefore may provide a superior system for pathogenesis and vaccine efficacy investigation.     

Identification of genotype 4 hepatitis E virus strains from a patient with acute hepatitis E and farm pigs in Bali, Indonesia

A previous study revealed that antibodies to hepatitis E virus (HEV) (anti-HEV) are highly prevalent among healthy individuals and farm pigs in Bali, Indonesia, and suggested that HEV infection may occur via zoonosis among Balinese people. However, there were no reports of acute hepatitis E in Bali. To elucidate whether Balinese HEV strains recovered from infected humans and pigs have significant sequence similarity, serum samples obtained from 57 patients (age, mean +/- standard deviation, 31.1 +/- 11.9 years) with sporadic acute hepatitis and from one hundred and one 2- or 3-month-old farm pigs in Bali were tested for anti-HEV and HEV RNA. Among the 57 patients, 2 (3.5%) had high-titer IgM/IgA class anti-HEV antibodies and one of them had detectable HEV RNA (BaliE03-46). Overall, 58 pigs (57.4%) tested positive for anti-HEV, while 5 pigs (5.0%) had detectable HEV RNA. Based on the 412-nucleotide sequence within open reading frame 2, the BaliE03-46 isolate and the 5 swine HEV isolates recovered from the viremic pigs were phylogenetically classified in genotype 4, but were only 77.3-90.8% identical to the genotype 4 HEV isolates reported thus far in China, India, Japan, Taiwan, and Vietnam. The BaliE03-46 isolate of human origin shared high identities of 97.3-98.3% with 4 of the 5 Balinese swine isolates, but differed by 16.1% from the remaining swine isolate. These results suggest that indigenous HEV strains of genotype 4 with marked heterogeneity are circulating in Bali, Indonesia, and that pigs are reservoirs of HEV for Balinese people who have a habit of ingesting uncooked pigs.     

无偿献血人群戊型肝炎病毒感染情况调查

目的调查绍兴市街头无偿献血者戊型肝炎病毒感染情况。方法3701例无偿献血者标本采用ELISA方法检测抗-HEV IgG和抗-HEV IgM,RT-PCR检测抗-HEV IgM阳性血清HEV RNA。结果本市无偿献血者抗-HEV IgG阳性率为29.91%(1107/3701);抗-HEVIgM阳性率为1.35%(50/3701);HEV RNA阳性6例,检出率为0.16%(6/3701),均为HEV基因1型;各季节抗-HEV IgG和抗-HEV IgM的阳性检出率无显著差异。结论加强对献血者感染HEV情况的检测和研究,对保证用血安全,完善安全输血的保障体系有切实的意义。     

Prevalence of antibodies to hepatitis E virus among apparently healthy humans and pigs in Bali, Indonesia: Identification of a pig infected with a genotype 4 hepatitis E virus

In Indonesia where hepatitis E virus (HEV) is believed to be highly endemic, only three outbreaks of HEV transmission have been documented to date in restricted areas (West Kalimantan and East Java). A total of 1,115 serum samples collected from apparently healthy individuals in Bali, Lombok, and Surabaya in Indonesia in 1996 where epidemic HEV transmissions have never been reported, were tested for IgG class antibodies to HEV (anti-HEV). In Bali, anti-HEV was detected in 20% (54/276) of the tested population, in remarkable contrast with 4% (17/446) in Lombok and 0.5% (2/393) in Surabaya. On the other hand, antibodies to hepatitis A virus were highly prevalent in all three regions (95% in Bali, 90% in Lombok, and 89% in Surabaya). Although the majority of the population in Indonesia is Moslem, Balinese people are mostly Hindu and have a habit of consuming pork. Therefore, serum samples were obtained from the 99 farm pigs in Bali and tested for anti-HEV and HEV RNA. The sera from 71 pigs (72%) were positive for anti-HEV and a 2-month-old pig had detectable HEV RNA. The swine HEV isolate recovered from the viremic pig was named SB66-Bali. The SB66-Bali isolate was most closely related to the genotype 4 isolates from China, India, Japan, and Taiwan, but shared only 82.6-90.0% identity in the common 241-412 nucleotides within open reading frame 2 (ORF2). These results indicate that a presumably indigenous HEV strain(s) is circulating in Bali, Indonesia and that HEV infection may occur via zoonosis even in developing countries.     

Hepatitis E virus infection in patients infected with the human immunodeficiency virus

Hepatitis E virus (HEV) is a newly-identified causative agent of acute and chronic hepatitis in severely immunocompromized patients. The present study sought to assess the prevalences of past, recent, on-going, and chronic HEV infections in patients infected with human immunodeficiency virus (HIV) in Marseille, South-eastern France, and to determine if they were correlated with the patients' immunological status or with cirrhosis. Anti-HEV IgG and IgM and HEV RNA testing were concurrently performed on the plasma from 184 patients infected with HIV, including 81 with a CD4+ T-lymphocyte count (CD4 count) <50 cells/mm(3) and 32 with a cirrhosis. Prevalence of anti-HEV IgG and IgM was 4.4% (8/184) and 1.6% (3/184), respectively. Past, recent, and on-going infections were observed in 3.3% (6/184), 1.6% (3/184), and 0.5% (1/184) of the patients, respectively. Anti-HEV antibodies prevalence did not differ significantly according to CD4 count, cirrhosis, sex, age, mode of HIV transmission, and infection with hepatitis B or C virus. Anti-HEV IgG seroreversion was observed in two patients. The patient whose plasma tested positive for HEV RNA had a CD4 count <50 cells/mm(3) ; HEV genotype was 3f. In this patient, longitudinal testing showed HEV RNA positivity during a 10-month period, indicating chronic HEV infection; in contrast, anti-HEV IgG never tested positive. Further studies are needed to evaluate the performance of commercial HEV serological assays in patients infected with HIV and to assess the actual incidence, prevalence, and outcome of HEV infection in this special group of patients. HEV RNA testing is necessary for such purposes.     

Experimental infection of pigs with the newly identified swine hepatitis E virus (swine HEV), but not with human strains of HEV

Summary.  A novel virus of pigs, swine hepatitis E virus (swine HEV), was recently identified and shown to be antigenically and genetically related to human HEV. In the present study, we attempted to infect specific-pathogen-free (SPF) pigs experimentally with swine HEV or with human strains of HEV. Serum samples collected from naturally infected pigs were used as the source of swine HEV. Pigs inoculated intravenously with serum samples containing swine HEV seroconverted to anti-HEV 4 to 8 weeks postinoculation, and the virus spread to an uninoculated pig. Swine HEV was detected in nasal and rectal swab materials as early as 2 weeks postinoculation and for 4 to 8 weeks thereafter. Viremia appeared 4 to 6 weeks postinoculation and lasted 1 to 3 weeks. The inoculated pigs appeared clinically normal and serum liver enzymes were not significantly elevated. In contrast, pigs were not infected when inoculated intravenously with about 10⁵ monkey infectious doses of one of two human strains of HEV (Sar-55 or Mex-14). Received December 9, 1997 Accepted February 18, 1998     

Detection of HEV antigen as a novel marker for the diagnosis of hepatitis E

Infection with hepatitis E virus (HEV) may be diagnosed by the presence of HEV RNA or anti-HEV antibodies. An enzyme immunoassay (EIA) was developed for the detection of antigen. Twenty-four monoclonal antibodies (mAbs) were produced. An indirect sandwich EIA was developed to detect HEV antigen using a combination of three mAbs as coating antibodies. Approximately 44.6% (33/74), 28.6% (50/175), and none (0/27) of sera positive for anti-HEV IgM alone, both anti-HEV IgM and IgG, and anti-HEV IgG alone also were positive for HEV antigen using this EIA. Forty-two HEV antibody-positive sera were tested for HEV RNA and antigen in parallel and the concordance was 81.0% (34/42). All PCR products were found to belong to HEV genotype 4. In order to evaluate the temporal relationship between HEV antigen positivity and HEV RNA, anti-HEV IgG and IgM, and ALT concentrations, macaques were infected with HEV genotypes 1 and 4 and serial samples were collected. The results showed that the antigen EIA can detect the capsid proteins of both genotypes. HEV antigen was detectable prior to ALT elevation and the appearance of anti-HEV antibodies in the infected monkeys and lasted for several weeks in all cases. HEV antigen became detectable in the serum at almost the same time as HEV RNA in feces but persisted for 4 weeks less than HEV RNA. This assay should be valuable for the diagnosis of acute hepatitis E, particularly in the window period prior to seroconversion to anti-HEV.     

Routes of transmission of swine hepatitis E virus in pigs

Hepatitis E virus (HEV) is believed to be transmitted by the fecal-oral route in pigs. To date, in experiments, HEV has been transmitted successfully only by the intravenous or intrahepatic route. To assess the route of HEV transmission, 27 pigs were separated into nine groups of three pigs. Positive-control pigs were inoculated intravenously with swine HEV and served as the source of HEV for the other groups. Uninoculated contact pigs were placed in the positive-control group. On three consecutive days, naive pigs were inoculated using samples collected from the positive-control pigs at 9, 10, and 11 days postinoculation. The tonsils and nasal mucosa of each positive-control pig were swabbed and that swab was used to rub the tonsils and nasal and ocular mucosa of naive pigs. The positive-control pigs were also injected with bacterin, and the same needle was used to immediately inject naive pigs. Feces were collected from positive controls and fed by oral gavage to naive pigs. Weekly fecal and serum samples from each pig were tested for anti-HEV antibodies and HEV RNA. All positive-control pigs shed the virus in feces; two pigs were viremic and seroconverted to anti-HEV. All contact control pigs shed the virus in feces; two seroconverted and one became viremic. One of three pigs in the fecal-oral exposure group shed the virus in feces and seroconverted. Pigs exposed to the contaminated needles or the tonsil and nasal secretion swabs remained negative. This is the first report of experimental fecal-oral transmission of HEV in swine.     

Serological and molecular studies on subclinical hepatitis E virus infection using periodic serum samples obtained from healthy individuals

Subclinical hepatitis E virus (HEV) infection among healthy individuals was studied serologically and molecularly. Serum samples collected at screening between March and April 2004 (or just before retirement) from 266 medical staff members (35 males, 231 females) who had been working for 8.8 +/- 8.5 (mean +/- standard deviation, range, 0.3-35.1) years in a city hospital in Japan and serum samples that had been collected from these staff members at the start of employment were tested for IgA, IgM, and IgG antibodies to HEV (anti-HEV) by in-house enzyme-linked immunosorbent assays. Overall, six subjects (2.3%) tested positive for anti-HEV IgG at the screening; among them, four subjects (1.5%) had already been positive for anti-HEV IgG at the start of employment and two subjects (0.8%) seroconverted after initiation of employment. Periodic serum samples that had been collected from the two seroconverted subjects were tested for HEV antibodies and HEV RNA. The two subjects became positive for anti-HEV IgG in 1978 or 2003, respectively, with no discernible elevation in alanine aminotransferase (ALT) level, and continued to be seropositive up through the screening date. Although anti-HEV IgM was not detectable in the two subjects, one was infected transiently with Japan-indigenous HEV strain of genotype 3 and the other was positive transiently for anti-HEV IgA. The present study indicates that even an individual with subclinical HEV infection had evidence of transient viremia in the absence of ALT elevation and that anti-HEV IgA detection may be useful for serological diagnosis of recent subclinical HEV infection.