A novel avian isolate of hepatitis E virus from Pakistan.

BACKGROUND: Avian hepatitis E virus (aHEV) has been associated with hepatitis-splenomegaly syndrome (HSS) in chickens along with asymptomatic subclinical infection in many cases. So far, four genotypes have been described, which cause infection in chickens, specifically in broiler breeders and layer chickens. In the present study, we isolated and identified two novel aHEV strains from the bile of layer chickens in Pakistan evincing clinical symptoms related to HSS. METHODOLOGY: Histology of liver and spleen tissues was carried out to observe histopathological changes in these tissues. Bile fluid and fecal suspensions were used for viral RNA isolation through MegNA pure and Trizol method which was further used for viral genome detection and characterization by cDNA synthesis and amplification of partial open reading frame (ORF) 1, ORF2 and complete ORF3. The bioinformatics tools; Molecular Evolutionary Genetics Analysis version 6.0 (MEGA 6), Mfold and ProtScale were used for phylogenic analysis, RNA secondary structure prediction and protein hydropathy analysis, respectively. RESULTS: Sequencing and phylogenetic analysis on the basis of partial methyltranferase (MeT), helicase (Hel) domain, ORF2 and complete ORF3 sequence suggests these Pakistani aHEV (Pak aHEV) isolates may belong to a Pakistani specific clade. The overall sequence similarity between the Pak aHEV sequences was 98-100%. The ORF1/ORF3 intergenic region contains a conserved cis-reactive element (CRE) and stem-loop structure (SLS). Analysis of the amino acid sequence of ORF3 indicated two hydrophobic domains (HD) and single conserved proline-rich domain (PRD) PREPSAPP (PXXPXXPP) with a single PSAP motif found in C-terminal. Amino acid changes S15 T, A31T, Q35H and G46D unique to the Pak aHEV sequences were found in the N-terminal region of ORF3. CONCLUSIONS: Our data suggests that Pak aHEV isolates may represent a novel Pakistani clade and high sequence homology to each other support the supposition they may belong to a monophyletic clade circulating in the region around Pakistan. The data presented in this study provide further information for aHEV genetic diversity, genotype mapping, global distribution and epidemiology.

Prevalence of human herpesviruses in biliary fluid and their association with biliary complications after liver transplantation.

BACKGROUND: Beta-herpesviruses are common opportunistic pathogens that cause morbidity after liver transplantation (LT). METHODS: Objective of the study was to evaluate the prevalence and correlation of herpesviruses in bile, blood and liver tissue and to investigate their association with biliary complications and retransplantation (re-LT) free survival after LT. The study design is a single-center case-control study. We performed quantative polymerase chain reaction (qPCR) for herpesvirus 1-8 DNA in bile, blood and liver tissue of 73 patients after first LT and analyzed their clinical courses retrospectively. RESULTS: The median follow-up was 48 months (range 2-102), during which a total of 16 patients underwent re-LT and 11 patients died. Of the patients, 46.5% received valganciclovir prophylaxis at the time of bile sample acquisition. Cytomegalovirus (CMV) (18.3%), human herpesvirus 6 (HHV-6) (34.2%), human herpesvirus 7 (HHV-7) (20.5%) and Epstein-Barr virus (EBV) (16.4%) were highly prevalent in bile after LT, while herpes simpex virus 1 and 2 (HSV-1, HSV-2), varicella-zoster virus (VZV) and human herpesvirus 8 (HHV-8) were not or rarely detected in bile. Valganciclovir prophylaxis did not reduce the prevalence of HHV-6 and HHV-7 in bile, but it did reduce the presence of CMV and EBV. The presence of HHV-6 in bile was associated with non-anastomotic biliary strictures (NAS) and acute cellular rejection (ACR). CONCLUSIONS: CMV, EBV, HHV-6 and HHV-7 are more prevalent in biliary fluid than in liver biopsy or blood serum after LT. HHV-6 and HHV-7 might be associated with biliary complications after LT. Biliary fluids might be an attractive target for routine herpesvirus detection.

Experimental infection of rabbit with swine-derived hepatitis E virus genotype 4.

Hepatitis E virus (HEV) is a zoonotic virus that is capable of causing cross-species infection. Rabbits can be experimentally infected with human- and swine-derived HEV-4 in the species Orthohepevirus A, and avian-derived HEV-3 in the species Orthohepevirus B suggesting rabbits can serve as an animal model for zoonotic HEV infection study. However, these studies show that the infectivity of swine HEV isolates in rabbits is not consistent. In this study, the animal study was conducted by the experimental infection of rabbit with a swine-derived HEV-4 isolated in China (designated CHN-SD-sHEV) for 28 weeks post-inoculation (wpi) in compassion to that infected with a rabbit-derived HEV-3 (designated HEV-SX-rHEV). Two rabbits were euthanized every 2 wpi for pathological examinations. The results showed that rabbits infected with CHN-SD-sHEV had the viremia and virus fecal shedding from 1 wpi to 22 wpi and seroconverted from 10 to 28 wpi. Meanwhile, elevated ALT levels were detected at 2 wpi. Moreover, virus replication was confirmed by the detection of both positive- and negative-strand HEV RNAs in the livers and spleens. Diarrhea and hepatocellular lesions were also observed in some animals. In contrast, rabbits experimentally infected with CHN-SX-rHEV exhibited earlier seroconversion, viremia and virus fecal shedding and hepatocellular lesions. Taken together, our data demonstrate that in comparison to the previously reported cases, the swine-derived HEV-4 isolated in China could cross-species infect rabbit accompanied with prolonged virus fecal shedding and liver lesions.

Phylogenetic Analysis and Characterization of the Complete Hepatitis E Virus Genome (Zoonotic Genotype 3) in Swine Samples from Mexico.

Hepatitis E virus (HEV) is an emerging public health problem with an estimated 20 million infections each year. In Mexico, Orthohepevirus A, genotype 2, has been reported in humans, but genotype 3 has only been reported in swine (zoonotic). No diagnostic tests are publicly available in Mexico, and only partial sequences have been reported from swine samples. Hence, research is necessary to determine circulating strains, understand the features and dynamics of infection on pig farms, determine how to implement surveillance programs, and to assess public health risks. In this study, a next-generation sequencing (NGS) approach was applied to obtain a complete genome of swine HEV. Liver, feces, and bile samples were taken at slaughterhouses and a farm in Mexico. RT-PCR was used to determine positive samples and confirmed by Sanger sequencing. Of the 64 slaughterhouse samples, one bile sample was positive (B1r) (1.56%). Of 21 sample pools from farm animals, 14 were positive (66.66%), representing all stages of production. A complete sequence strain MXCDg3_B1c|_2016 was obtained from the bile of a domestic swine in the fattening stage. In addition, two partial sequences-MXCDg3_H2cons|_2016 (1473 nt) and MXCDg3_C3Acons|_2016 (4777 nt)-were obtained from sampled farm animals. Comparison with all reported genome HEV sequences showed similarity to genotype 3 subgenotype a (G3a), which has been previously reported in acute cases of human hepatitis in the US, Colombia, China, and Japan.

Molecular Detection of Indigenous Hepatitis E Virus (HEV) from Tibetan Pigs in Tibet, China.

Hepatitis E is an important public health concern throughout the world. Many molecular and serological surveys have reported the prevalence and genotypic characteristics of HEV in humans and animals worldwide. However, the genotypic characterization of this virus is very limited in Tibetan pigs. Hence, we aimed to explore the genotype of HEV, prevailing among Tibetan pigs in China. For this purpose, 253 bile samples of Tibetan pigs (free-range animals) were collected from different slaughterhouses during 2017-2018 and subsequently tested for HEV RNA by RT-nPCR. A total of 11 out of 253 (4.35%) samples tested were positive for HEV RNA. Based on the sequence alignment and phylogenetic analysis, all the isolated HEV strains belonged to genotype 4 and clustered into subtype 4b by sharing more than 84.8-95.2% identities with other reported strains. Our results concluded that HEV genotype 4 is prevailing among Tibetan pigs in Tibet, China.

A comprehensive study of hepatitis E virus infection in pigs entering a slaughterhouse in Slovenia.

Hepatitis E is a zoonotic viral disease of pigs with increasing public health concern in industrialized countries. Presented broad study of hepatitis E virus (HEV) presence in pigs in Slovenia is the first attempt to overview the HEV situation in pigs entering a slaughterhouse and, further, to analyse the possibility of HEV entering into the food supply chain. 2433 samples from 811 clinically healthy pigs were collected at four slaughterhouses in Slovenia. Sampling covered three different age groups of pigs and three different types of samples (faeces, bile and liver) important for tracing HEV in a pig population. In addition, 63 swab samples were collected systematically from three different sites on the slaughter line, as well as 22 samples of minced meat and 30 bratwurst samples. All the samples were screened for the presence of HEV nucleic acids by specific real-time RT-PCR assay. In the group of three month old pigs 13.7% of faeces, 13.0% of bile and 2.1% of liver samples were HEV positive. In the group of six months old pigs only 0.25% of liver and 0.25% of bile samples were positive. In the category of sows, no positive samples were found. Two out of 63 swab samples collected on the slaughter line were HEV positive. All tested samples of minced meat and bratwurst were negative. The phylogenetic analysis of 50 HEV positive samples, with comparison of 366 nucleotides in ORF1 region, revealed high diversity of identified strains of HEV in pigs, belonging into subtypes 3a, 3b, 3c and 3e.

Hepatitis E Virus (HEV) egress: Role of BST2 (Tetherin) and interferon induced long non- coding RNA (lncRNA) BISPR.

BACKGROUND: The biology of Hepatitis E Virus (HEV), a common cause of epidemic and sporadic hepatitis, is still being explored. HEV exits liver through bile, a process which is essential for its natural transmission by feco-oral route. Though the process of this polarised HEV egress is not known in detail, HEV pORF3 and hepatocyte actin cytoskeleton have been shown to play a role. METHODS: Our transcriptome analysis in Hepatitis E virus (HEV) replicon transfected Huh7 cells at 24 and 72 hrs indicated that at 24hrs, both LncBISPR and BST2, expressed by a bidirectional promoter were highly upregulated whereas at 72 hrs, BST2 expression was comparatively reduced accompanied by normal levels of BISPR. These findings were confirmed by qPCR analysis. Co-localisation of BST2 and HEV pORF2 was confirmed in HEV transfected Huh7 by confocal microscopy. To investigate the role of BISPR/BST2 in HEV life cycle, particularly virus egress, we generated Huh7 cells with ~8kb deletion in BISPR gene using Crispr-Cas9 system. The deletion was confirmed by PCR screening, Sanger sequencing and Real time PCR. Virus egress in ΔBISPR Huh7 and Huh7 cells was compared by measuring HEV positive strand RNA copy numbers in cell lysates and culture supernatants at 24 and 72 hrs post HEV replicon transfection and further validated by western blot for HEV pORF2 capsid protein. RESULTS: ΔBISPR Huh7 cells showed ~8 fold increase in virus egress at 24 hrs compared to Huh7 cells. No significant difference in virus egress was observed at 72hrs. Immunohistochemistry in histologically normal liver and HEV associated acute liver failure revealed BST2 overexpression in HEV infected hepatocytes and a dominant canalicular BST2 distribution in normal liver in addition to the cytoplasmic localisation reported in literature. CONCLUSIONS: These findings lead us to believe that BISPR and BST2 may regulate egress of HEV virions into bile in vivo. This system may also be used to scale up virus production in vitro.

Detection of high biliary and fecal viral loads in patients with chronic hepatitis C virus infection / Detección de concentraciones víricas elevadas en la bilis y en las heces de pacientes con infección crónica por el virus de la hepatitis C

BACKGROUND: The life cycle of the hepatitis C virus (HCV) is closely associated with lipid metabolism. Recently, NPC1L1 (a cholesterol transporter) has been reported to function as an HCV receptor. This receptor is expressed in the hepatocyte canalicular membrane and in the intestine; serving as a key transporter for the cholesterol enterohepatic cycle. OBJECTIVES: We hypothesized that HCV might have a similar cycle, so we aimed to study the presence of HCV in bile and stools of infected patients. MATERIALS AND METHODS: Blood, feces, and duodenal bile samples were collected from patients infected with HCV. The biliary viral load was normalized to the bile salt concentration of each sample and the presence of HCV core protein was also evaluated. A total of 12 patients were recruited. HCV RNA was detected in the bile from ten patients. RESULTS: The mean viral load was 2.5log10IU/60mg bile salt. In the stool samples, HCV RNA was detected in ten patients (mean concentration 2.7log10IU/g of feces). CONCLUSIONS: HCV RNA is readily detectable and is present at relatively high concentrations in the bile and stool samples of infected patients. This may be relevant as a source of infection in men who have sex with men. Biliary HCV secretion may perhaps play a role in the persistence of viral infection via an enterohepatic cycle of the virus or intrahepatic spread

INTRODUCCIÓN: El ciclo de vida del virus de la hepatitis C (VHC) está estrechamente ligado al metabolismo lipídico. Recientemente, se ha descrito que el NPC1L1 (un transportador del colesterol) actúa como un receptor del VHC. Este receptor se expresa en la membrana canalicular de los hepatocitos y en el intestino, y actúa como uno de los principales transportadores durante la circulación enterohepática del colesterol. OBJETIVOS: Planteamos la hipótesis de que el VHC podría tener un ciclo similar, por lo que nuestro objetivo fue estudiar la presencia del VHC en la bilis y en las heces de pacientes infectados. MATERIALES Y MÉTODOS: Se obtuvieron muestras de sangre, heces y bilis duodenal de pacientes infectados por el VHC. La concentración vírica en la bilis se normalizó respecto a la concentración de sales biliares de cada muestra y también se evaluó la presencia de la proteína central del VHC. Se reclutaron un total de 12 pacientes. Se detectó el ARN del VHC en la bilis de 10 pacientes. RESULTADOS: La media de la concentración vírica fue 2,5log10UI/60mg de sales biliares. En las muestras de heces, se detectó el ARN del VHC en 10 pacientes (media de la concentración 2,7log10UI/g de heces). CONCLUSIONES: El ARN del VHC es fácilmente detectable y está presente en concentraciones relativamente elevadas en las muestras de bilis y heces de pacientes infectados. Esto puede tener importancia como foco de infección en varones que mantienen relaciones sexuales con otros varones. Es posible que la secreción biliar del VHC pueda desempeñar un papel en la persistencia de la virosis a través de la circulación enterohepática del virus o la propagación intrahepática

Detection of high biliary and fecal viral loads in patients with chronic hepatitis C virus infection.

BACKGROUND: The life cycle of the hepatitis C virus (HCV) is closely associated with lipid metabolism. Recently, NPC1L1 (a cholesterol transporter) has been reported to function as an HCV receptor. This receptor is expressed in the hepatocyte canalicular membrane and in the intestine; serving as a key transporter for the cholesterol enterohepatic cycle. OBJECTIVES: We hypothesized that HCV might have a similar cycle, so we aimed to study the presence of HCV in bile and stools of infected patients. MATERIALS AND METHODS: Blood, feces, and duodenal bile samples were collected from patients infected with HCV. The biliary viral load was normalized to the bile salt concentration of each sample and the presence of HCV core protein was also evaluated. A total of 12 patients were recruited. HCV RNA was detected in the bile from ten patients. RESULTS: The mean viral load was 2.5log10IU/60mg bile salt. In the stool samples, HCV RNA was detected in ten patients (mean concentration 2.7log10IU/g of feces). CONCLUSIONS: HCV RNA is readily detectable and is present at relatively high concentrations in the bile and stool samples of infected patients. This may be relevant as a source of infection in men who have sex with men. Biliary HCV secretion may perhaps play a role in the persistence of viral infection via an enterohepatic cycle of the virus or intrahepatic spread.

First detection and complete genome sequence of a phylogenetically distinct human polyomavirus 6 highly prevalent in human bile samples.

Oncovirus-associated malignancies are potentially preventable diseases with major public health significance. Human polyomaviruses (HPyVs) may be associated with oncogenesis or symptomatic illnesses in immunocompromised patients, but the site of viral shedding of most recently discovered HPyVs remains obscure. Using real-time PCR assay using specific primers targeting the HPyV6 large tumor antigen gene, we detected a phylogenetically distinct HPyV6 which was highly prevalent in the bile samples of patients with malignant biliary obstruction (18.8%) and acute gallstone cholangitis (5.5%). The prevalence rate and mean viral load of this HPyV6 were highest in the cholangiocarcinoma subgroup (27.6% and 2.41 × 104copies/ml). These findings were confirmed with another real-time PCR assay using specific primers targeting the HPyV6 viral capsid protein 2 gene. These bile HPyV6 strains may represent a novel clade of HPyV6 as they formed a distinct cluster from the other HPyV6s and exhibited >2% differences in amino acid sequences in their major proteins. While HPyV6 was unlikely the cause of the patients' acute symptoms and liver dysfunction, the virus may be related to immunosuppression in patients with malignancy and/or important in the oncogenesis of cholangiocarcinoma in patients without coinfection with other oncogenic microbes. Further studies to ascertain a causative role of HPyV6 in cholangiocarcinoma should be conducted.

Detection of Torque Teno Sus Virus in Pork Bile and Liver Sausages.

Torque teno viruses (TTV) are small DNA viruses widespread among humans and pigs. The clinical significance of TTV infections in either humans or pigs is uncertain. In fact, TTV viremia is highly prevalent in patients with different pathologies, but it can also be frequently observed in healthy subjects. Virus infection in pigs is considered a putative cofactor in several diseases; despite being detected frequently in healthy animals, its role still remains unknown. The present study aimed to investigate the presence of Torque teno sus virus (TTSuV) in 62 bile samples collected from pigs at slaughterhouse and in 36 fresh pork liver sausages bought at point of sale. Quantitative Real-Time PCR, confirmed that 19.4 and 58.3 % of bile and sausage samples tested positive for TTSuV, respectively. The mean viral load was established as 5.6 × 104 GE/µl for bile and 7.16 × 103 GE/g for sausages. TTSuV nucleotide sequence analysis confirmed a wide heterogeneity among the circulating TTSuV strains, which included both TTSuV1 and TTSuV2.

Hepadnavirus detected in bile and liver samples from domestic pigs of commercial abattoirs.

BACKGROUND: Preliminary studies showed the prevalence of a virus similar to human hepatitis B virus (HBV-like) in swine from farms in China and the molecular evidence of Hepadnavirus infection in domestic pigs herds in Brazil. In this study, we genetically characterize the swine Hepadnavirus strains in swine from slaughterhouses located in certified abattoirs from Rio de Janeiro State, Brazil and evaluate its hepatotropic potential. RESULTS: Bile and liver samples from swine were positive for partial genome amplification (ORF S and ORF C), direct sequencing and viral load quantification. Sequencing of the gene encoding the surface antigen allowed classification of Hepadnavirus into genotypes, similar to HBV genotype classification. Indirect immunofluorescence confirmed the presence of HBsAg antigen in liver tissue sections. CONCLUSIONS: So far our data suggest that commercial swine house an HBV-like virus and this relevant finding should be considered in studies on the origin and viral evolution.

Seroprevalence and evolutionary dynamics of genotype 4 hepatitis E virus in Shandong Province, China.

AIM: To investigate the seroprevalence and evolutionary dynamics of hepatitis E virus (HEV) and assess the ancestor of HEVs in China's Shandong Province. METHODS: A total of 2028 serum, 60 fecal and 82 bile samples were collected from the general human population, patients and swine, respectively. This seroepidemiological study was conducted using an immunnosorbent assay and HEV RNA was detected by the reverse transcription-nested polymerase chain reaction (RT-nPCR) method. Complete genome sequences of the prevalent strains (CH-YT-HEV01, CH-YT-HEV02 and CH-YT-sHEV01) were determined, and the sequences were analyzed phylogenetically. In addition, the evolutionary dynamics of three HEV isolates were determined using the framework of coalescent analysis in the program package BEAST, and the time of the most recent common ancestors (TMRCAs) of China-indigenous genotype 4 HEV isolates was calculated. RESULTS: The overall viral burden in the general human population was 0.1%, and the positive rates of anti-HEV IgG and IgM in the serum specimens were 25.1% (509/2028) and 2.3% (51/2028), respectively. In addition, IgG positivity increased with age. The phylogenetic analysis based on the full-length nucleotide sequences showed that the strain CH-YT-HEV02 was directly related to CH-YT-sHEV01 with a 94% identity, suggesting that they were involved in cross-species transmission. The isolate CH-YT-HEV01 was close to HB-3 and CHN-SD-sHEV with a bootstrap value of 100%, sharing a 96.1%-96.4% identity with each other. Surprisingly, the HB-3 strain was a representative strain prevalent in swine in Hubei, and the isolate CHN-SD-sHEV was obtained from swine in Shandong in a previous report. TMRCA for the clade of CH-YT-HEV01 and HB-3 was 2003, which was consistent with the TMRCA for the clade of CHN-SD-sHEV and HB-3, and they were both earlier than the TMRCA for the clade of CH-YT-HEV01 and CHN-SD-sHEV (2004). CONCLUSION: The strains CH-YT-HEV01, CHN-SD-sHEV and HB-3 are involved in trans-regional transmission, and the ancestors of HEVs in Shandong come from Hubei Province.

The prevalence of hepatitis E virus infections among swine, swine farmers and the general population in Guangdong Province, China.

Hepatitis E virus (HEV) infection is widespread in China, but few studies have been carried out in Guangdong Province. This study aimed to characterize the prevalence of HEV infections among swine, swine farmers and the general population in Guangdong Province. We conducted an epidemiological study that included swine, swine farmers and health examination attendees in Guangdong from 2011 to 2013. The overall seroprevalence of anti-HEV antibodies in swine was 64.7%. The results revealed that growing pigs, sows and boars (OR ranges from 3.5 to 21.5) have a higher risk than nursery pigs. HEV RNA in swine bile showed that HEV is epidemic in swine in the Pearl River Delta, with the highest prevalence of 22.73% in Foshan. Some genomes of HEV strains from each district were sequenced. Phylogenetic analysis of partial open reading frame 2 (ORF2) shows that they belong to genotype IV and are most closely related to isolates from China. In total, 307 participants were enrolled in the study, including 114 swine farmers and 193 attendees from hospitals. IgG anti-HEV was detected in 48.25% of swine farmers and in 38.34% of the general population. Seroprevalence rates were almost stratified by age, with a higher positive rate for males compared to females across all age groups. Women on swine farms appeared to have a lower risk of infection compared to the general population, revealing that the risk factors for HEV infection are not unique. The results suggested that there were other risk factors for HEV infection. HEV infection is prevalent in Guangdong, but due to the small sample sizes, more investigations are needed to assess the potential impact of HEV infection, and many additional risk factors should be considered.

Occult cytomegalovirus cholangitis as a potential cause of cholestatic complications after orthotopic liver transplantation? A study of cytomegalovirus DNA in bile.

Cholestatic complications, important causes of morbidity and mortality after orthotopic liver transplantation (OLT), often have an unclear etiology. Human cytomegalovirus (CMV) infections occur in immunosuppressed patients and can be detected in blood samples. However, CMV analyses of body fluids and biopsies are more sensitive. Here we evaluated whether a CMV analysis of bile could reveal occult CMV cholangitis. We evaluated OLT patients undergoing endoscopic retrograde cholangiography (ERC) for suspected biliary complications after OLT at a tertiary care center. Biliary CMV DNA levels were measured with real-time polymerase chain reaction. A nonanastomotic biliary lesion (NABL) group consisted of patients with nonanastomotic strictures (NASs) at the time of ERC (n = 59) and patients with normal ERC findings but microscopic biliary lesions in biopsy samples (n = 12). The anastomotic stricture (AS) group comprised patients with ASs only (n = 53). In all, 124 OLT patients underwent 240 ERC procedures. Biliary CMV DNA was detected in 14 of the 124 patients and was more frequently found in the NABL group (12/71 for the NABL group versus 2/53 for the AS group, P = 0.02). Concurrent sampling of CMV DNA in blood yielded negative results. Biliary CMV was more frequently detected in patients with a positive recipient status (13/73 or 17.8% versus 1/44 or 2.3%, P < 0.05). There was no significant difference in the incidence of biliary CMV between patients with a high-risk CMV status and patients with a low-risk CMV status. The median interval between OLT and biliary CMV detection was 8.4 months (range = 0.4-212.8 months). In conclusion, biliary CMV was detected in a substantial number of patients after OLT and was significantly associated with NASs or microscopic biliary lesions. A potential occult CMV infection could, therefore, be considered as a contributory etiological factor in the development of biliary complications.

Hepatitis E virus in liver and bile samples from slaughtered pigs of Brazil.

The objective of this study was to detect and identify hepatitis E virus (HEV) strains in liver and bile samples from slaughtered pigs in the state of Paraná, Brazil. Liver and bile samples were collected from 118 asymptomatic adult pigs at a slaughterhouse in a major Brazilian pork production area. The samples were assayed using a nested reverse transcription-polymerase chain reaction protocol with primer sets targeting open reading frames (ORF)1 and 2 of the HEV genome. HEV RNA was detected in two (1.7%) liver samples and one (0.84%) bile sample using both primers sets. The HEV strains were classified as genotype 3b on the basis of their nucleotide sequences. These data suggest that healthy pigs may be a source of HEV infection for consumers of pig liver and slaughterhouse workers in Brazil.

Hepatitis E virus in liver and bile samples from slaughtered pigs of Brazil

The objective of this study was to detect and identify hepatitis E virus (HEV) strains in liver and bile samples from slaughtered pigs in the state of Paraná, Brazil. Liver and bile samples were collected from 118 asymptomatic adult pigs at a slaughterhouse in a major Brazilian pork production area. The samples were assayed using a nested reverse transcription-polymerase chain reaction protocol with primer sets targeting open reading frames (ORF)1 and 2 of the HEV genome. HEV RNA was detected in two (1.7%) liver samples and one (0.84%) bile sample using both primers sets. The HEV strains were classified as genotype 3b on the basis of their nucleotide sequences. These data suggest that healthy pigs may be a source of HEV infection for consumers of pig liver and slaughterhouse workers in Brazil.

Simple and rapid detection of swine hepatitis E virus by reverse transcription loop-mediated isothermal amplification.

Hepatitis E virus (HEV) is an enteric pathogen of humans and animals, and pigs have been considered an important reservoir of this virus. Recent evidence has indicated the cross-species transmission of hepatitis E virus (HEV) from pigs to humans, causing zoonosis, mostly via consumption of uncooked or undercooked animal meat/viscera. In this study, we have developed a one-step RT-LAMP assay for rapid detection of swine HEV. Specific primer sets targeting the ORF3 gene were designed. The sensitivity of the RT-LAMP assay was 10(1) copies/µl of RNA template, which was tenfold higher than that of RT-nPCR. The specificity of this assay was demonstrated by the lack of amplification of DNA/RNA from other swine viruses. Furthermore, a total of 41 bile samples were subjected to RT-LAMP and RT-nPCR. Eighteen positive samples were detected by RT-nPCR, while 36 positive samples were detected by RT-LAMP, indicating that the sensitivity of the RT-LAMP assay was higher than that of the conventional RT-nPCR assay. The RT-LAMP assay reported here may be used for diagnosis of swine HEV, not only in laboratories but also under field conditions.