Genotype 3f Research Articles

Longitudinal survey of hepatitis E virus in extensively raised pigs in Spain

Hepatitis E virus (HEV) is an emerging zoonotic virus of public health concern, of which pigs, wild boar and red deer are the main reservoirs. The European Food Safety Authority (EFSA) has recently prioritized the development of monitoring programs of HEV at different stages of the pig food chain, including outdoor pig farming. Pigs managed under these extensive production systems frequently share habitat and natural resources with wild boar and red deer during fattening stages and cross-species transmission of HEV among these species has previously been suggested. In this context, we aimed to (I) to evaluate the risk of HEV circulation within the production phases of extensively raised pigs and at the domestic-wildlife interface, and (II) to identify the genotypes circulating within these hosts. A total of 1452 pigs from seven different pig farms were longitudinally sampled during the breeding, rearing, and fattening production phases. In addition, 138 and 252 sympatric wild boar and red deer, respectively, were analysed. Anti-HEV antibodies were found in 1245 (85.7 %) out of the 1452 Iberian pigs sampled. The seroprevalence was 30.4 % in the breeding phase, 95.4 % in the rearing phase and 97.0 % in the fattening phase. Statistically significant differences (P < 0.05) were found among the three production phases. The seroprevalence was significantly higher (P < 0.001) in fattening pigs compared to those found in sympatric wild boar (31.9 %) and red deer (2.0 %). Three (1.0 %) out of the 293 serum pools analysed were positive for viral RNA. One of them was identified in pigs at the rearing phase (genotype 3 f) and two in wild boar (genotypes 3 f and 3 m). The high seroprevalence detected in extensively raised pigs, together with the detection of the zoonotic HEV-3 f and HEV-3 m subtypes in sympatric domestic and wild swine, highlights the risk of zoonotic transmission and the need to establish surveillance programs and control measures, particularly in breeding and rearing phase, in these epidemiological scenarios.

Large-scale HEV genotype 3 outbreak on New Caledonia Island.

Several symptomatic cases of HEV infections were reported to the New Caledonia Island Public Health Service between August and December 2023. This prompted epidemiological and virological investigations to identify the source of infection. HEV RNA was assessed in symptomatic patients, various food items, and pig farms on the Island. HEV strains were characterized by sequencing. A seroprevalence study was also conducted on asymptomatic blood donors before and after the outbreak. One hundred twenty-seven symptomatic cases were reported. Hospitalization was required for 29/127 patients (22.8%). Hospitalized patients presented more frequently with comorbidities, including liver and cardiovascular diseases (80.7% vs. 27%, p < 0.01), and 3 persons died (2.3%). Among the 100 HEV RNA-positive samples received at the French National Reference Centre for HEV, viral sequencing was possible for 76 samples. All strains were identified as HEV genotype 3, and 74/76 strains were grouped together (nucleotide identity: 98%-100%). Full-length sequencing indicated a new HEV-3 subtype within HEV-3 subclade abk. Only genotype 3f strains were detected on the Island's pig farms. No food items tested positive for HEV RNA. The seroprevalence of HEV IgG and IgM in blood donors was 9.2% (9/98) and 0%, respectively, in 2020, rising to 17.3% (17/98) and 2% (2/98) in 2024. Although all previous large-scale epidemics in Asia and Africa were associated with HEV-1 or 2, the New Caledonia outbreak was linked to HEV-3. A high number of symptomatic cases were admitted to the hospital, with a case-fatality rate of 2.3%.

Low prevalence of hepatitis E virus in the liver of Corsican pigs slaughtered after 12 months despite high antibody seroprevalence.

Hepatitis E virus (HEV) infection can be acute and benign or evolve to chronic hepatitis with rapid progression toward cirrhosis or liver failure in humans. Hence, hepatitis E (HE) disease is a major public health concern. In countries where pig populations are highly contaminated with HEV, human cases of HE are mainly foodborne, occurring frequently after consumption of raw or undercooked pork products or liver. Among factors associated to the presence of HEV in pork livers from intensive rearing systems, early slaughter (≤6 months) seems to be major. In Corsica, local pigs are raised in extensive farming systems and slaughtered after 12 months. To evaluate if slaughter of pigs over 12 months reduces the risk of HEV presence in livers, 1197 liver samples were randomly collected in 2 Corsican slaughterhouses. Presence of HEV RNA was detected in liver and HEV seroprevalence was determined in paired serum. The sampling included 1083 livers from animals between 12 and 48 months and 114 livers from animals<12 months. The samples were predominantly from semi-extensive and extensive farms (n=1154). Estimated HEV seroprevalence was high, that is, >88%, and HEV RNA prevalence in adult pig livers (>12 months old) was low, that is, 0.18%. However, in livers from younger animals(<12 months), including piglets below 6 months old, 5.3% (6/114) of the samples were positive for HEV RNA. Sequences recovered from positive livers belonged to HEV genotype 3c and 3f. The presence of infectious HEV was confirmed in two livers by the detection of HEV replication in HepaRG cell cultures. Thus, this study demonstrates the low prevalence of HEV in livers of pigs over 12 months, even in farms with high HEV circulation. This observation may open new perspectives on the preferential use of livers from animals older than 12 months in raw pork liver products.

Detection of Hepatitis E Virus in Hyalomma lusitanicum Ticks Feeding on Wild Boars.

Little is known about the role of ticks in maintaining highly prevalent zoonotic viruses in wildlife, such as hepatitis E virus (HEV), which do not require ticks for transmission between animals and humans. In this cross-sectional study, adult female ticks were collected from Eurasian wild boar (Sus scrofa) in autumn 2015 in Spain. HEV RNA in both ticks and wild boar was evaluated by RT-qPCR. Twenty-nine adult Hyalomma lusitanicum ticks were collected from 29 wild boars. HEV RNA was detected in a total of 10 tick (34.4%) and 11 wild boar serum samples (37.9%). In two cases, detectable HEV RNA was found in a wild boar but not in the tick collected from them. In contrast, one HEV-positive tick was collected from an HEV-negative wild boar. All viral sequences were consistent with genotype 3f. We describe for the first time the presence of HEV RNA in adult Hyalomma lusitanicum ticks.

Hepatitis E virus genotype 1f outbreak in Bangladesh, 2018.

Hepatitis E virus (HEV) infection is a significant public health issue in many developing countries, causing waterborne outbreaks as well as sporadic hepatitis. We report here an outbreak of HEV genotype 1f infection during April-May 2018 among peopleliving at Halisohor, a low land in thesouthern part of Chottogram District of Bangladesh. A total of 933 patients were admitted toCombined Military Hospital (CMH), Chottogram, with symptoms of acute hepatitis. Among them, 550 patients were tested by ELISA for HEV-specific (IgM) and all were positive. Genotyping, sequencing, and phylogenetic analysis based on ORF2 region revealed that the outbreak was caused by genotype 1f and the strains were closely related to the previously reported HEV strains that caused the outbreak in Bangladesh in 2010. The current outbreak was most likely linked with water supplyas fecal contamination in water was evident and could be prevented by ensuring access to safe drinking water.

Parsonage-Turner syndrome associated with hepatitis E infection in immunocompetent patients

Introduction The hepatitis E virus (HEV) is the leading cause of acute hepatitis around the world. In recent years, knowledge has increased concerning extrahepatic manifestations caused by HEV, including neurological manifestations such as Parsonage-Turner syndrome (PTS). PTS is characterized by severe shoulder or arm pain and patchy paresis with muscle weakness. The aim of the present study was to assess the association between HEV and PTS. Materials and Methods We reported two cases of PTS associated with HEV, which were diagnosed in a short period of time in the same village. PTS was diagnosed by physical examination and electrophysiological studies, and serology testing for IgM, low-avidity IgG, and RNA of HEV established the diagnosis of acute HEV infection. Results A 44-year-old man who presented cervicobrachial pain accompanied by paresthesia, dyspnea, and isolated derangement of liver enzymes and 57-year-old women with cervical pain radiated to upper limbs, paresthesia, and liver cytolysis, although, this patient was initially diagnosed as having drug-induced hepatitis. Finally, the diagnosis was Parsonage- Turner syndrome associated with hepatitis e virus. In both patients, symptoms were bilateral and they required hospital admission. Both consumed vegetables are grown in a local patch and the phylogenetic analysis showed genotype 3f. Then, we reviewed the literature on PTS and HEV and we found 62 previously described cases that were more likely to be men (86.20 %) with more frequent bilateral symptoms (85.71 %). Genotype 3 is the most commonly associated. Three of those cases were diagnosed in Spain. Conclusions According to our findings, HEV should be considered in patients with neuralgic amyotrophy, including those with the absence of liver cytolysis.

Hepatitis E virus genotype 1f outbreak in Bangladesh, 2018

Hepatitis E virus (HEV) infection is a significant public health issue in many developing countries, causing waterborne outbreaks as well as sporadic hepatitis. We report here an outbreak of HEV genotype 1f infection during Apr-May 2018 among persons living at Halisohor, a low land of southern part of Chottogram District of Bangladesh. A total of 933 patients were admitted in Combined Military Hospital (CMH) Chottogram with symptoms of acute hepatitis. Among them 550 patients were tested by ELISA for HEV specific IgM and all were positive. Genotyping, sequencing and phylogenetic analysis based on ORF 2 region revealed that the outbreak was caused by genotype 1f and the strains were closely related to the previously reported HEV strains that caused outbreak in Bangladesh in 2010. The current outbreak was most likely linked with water supply as fecal contamination in water was evident and could be prevented by ensuring access to safe drinking water.&#x0D; Bangladesh J Microbiol, Volume 37 Number 1 June 2020, pp 35-37

Viral load and genotype of recent hepatitis E virus outbreak in Chittagong, Bangladesh

Introduction: There is an outbreak of hepatitis E virus (HEV) infection in Chittagong, Bangladesh in the year 2018. Due to unavailability of data of HEV infections in this region, objectives of the present study are to find the viral load and genotype the HEV infections. Methods: It was an observational study done in a tertiary care hospital during one year. A total of 125 Ig-M HEV positive cases were recruited tested by ELISA. After collection blood samples those were sent to Sliea Gen L.L.C, Texas, USA for detection of viral load and genotype maintaining proper procedure. Ethical clearance was taken from the hospital authority and informed written consent was taken from all patients and funding was provided by Sliea Gen L.L.C. USA. Results: Among the total 125 study patients, male was 90(72') and female was 35(29') where 7(5.6') were pregnant. Among them most 45(36.0') were at age group 30-40 years. Mean ± SD of age was 33.78 ± 14.29 years. All (100') cases were Ig-M Anti HEV positive and 9(7.2') cases were also positive for HBsAg, 1(0.8') cases were Anti HCV positive and none was Anti HAV positive. Regarding viral load Log 4-6 were found in 55(44.0') cases and >Log 6 were found in 23(18.4') cases and again 30(24.0') cases had 1000-10,000 IU/ml, 25(20.0') cases had 1,0,000-1,00,000 IU/ml and 23(18.4') cases had 1.00,000- 10,00,000 IU/ml viral load. Genotype 1F was found in 68(54.4') cases as the only genotype and no other genotype was found in the sample. Conclusion: So, in this region Chittagong, Bangladesh genotype 1F is the main genotype found and also they showed variable viral load.

Detection of HEV RNA amounts and genotypes in raw milks obtained from different animals

Hepatitis E virus (HEV) is one of the major foodborne viral pathogens transmitted through the fecal-oral route. Four genotypes of HEV are known to infect humans and it is reported that different types of HEV are active in zoonotic transitions. It is known that the HEV genotype 1 and HEV genotype 2 infections are generally acute and the HEV genotype 3 infections are chronic. Therefore, in the studies related to HEV infections, it is important to determine the genotypes to monitortreatment regimens. Although raw milk is often used in communities due to its low cost, there are limited data on the rates and the genotypes of HEV in our country and in the world. In light of this information, we aimed to investigate epidemiologically the quantity and genotypes of HEV RNA in 231 raw milk (48 cow milk, 65 goat milk, 65 sheep milk, and 53 donkey milk) samples. Viral RNAs were isolated from raw milk samples and the ORF2 region of HEV was investigated by the qRt-PCR method to determine quantitatively the presence of HEV RNA. In addition, among HEV RNA positive samples, the ORF2 region of HEV was amplified by nested PCR and the amplicons were sequenced. HEV RNA was detected in 47 (20.34%) raw milk samples, Positivity was detected in 14 (29.16%) of cow milk, 12 (18.46%) of goat milk, 8 of sheep milk (12.3) and 13 of donkey milk (24.5%). The amount of HEV RNA in cow milk found as the highest in both proportion and quantity. When the distribution of the HEV genotypes in the 47 positive samples was examined, 27 (57.44%) HEV genotype 1a, 10 (21.27%) HEV genotype 1b, 4 (8.5%) HEV genotype 4c, 2 (4.2%) HEV genotype 3a, (2.13) HEV genotype 1c, 1 (2.13%) HEV genotype 3e, 1 (2.13%) HEV genotype 3f and 1 (2.13%) HEV genotype 3g were determined. Although genotype 1a is more frequent, it has been revealed that different genotypes encountered in our country. In conclusion, it has been determined that HEV, one of the major foodborne viral agents, may be encountered in raw milk, and the genotypes that can cause infections in human are found especially in raw milk from animal sources. For the prevention of foodborne outbreaks, the presence of HEV in raw milk should not be ignored.

Complete genome sequencing of a genotype 3 hepatitis E virus strain identified in a swine farm in Italy

In this study, we investigated hepatitis E virus (HEV) infection in piglets sampled in two farms in southern Italy. The virus was detected in 11 out of 15 animals tested. Based on sequence analysis, the 6 Italian strains examined belonged to two clusters containing both swine and human strains of either genotype 3 subtype e or f from Europe and Japan. The two Italian strain clusters shared nucleotide identity of 81.8% and 87.5% in the ORF2 (capsid protein) and ORF1 (RdRp) diagnostic fragments, respectively, confirming the heterogeneity of genotype 3 viruses circulating in pigs in Italy. The complete genome of one genotype 3 subtype e strain and the full ORF2 and ORF3 coding regions of one of the genotype 3f strains, obtained in this study, were compared to other HEV sequences available on line (NCBI database). The results of analysis showed that porcine strains clustered together with human and swine strains detected in Europe. Most changes in the coding region corresponded to synonymous mutations, and only the ORF3 showed a positive selection. Further, analyses are needed to understand the clinical significance of HEV genotypes and subtypes.

Genotype 3fのE型肝炎：東京例（2012年）と埼玉例（2014年）の分子疫学的検討

Recently we saw two patients with acute hepatitis, infected with hepatitis E virus (HEV) of a rare genotype 3f. One patient (Tokyo/2012) had traveled Spain 1 month before developing hepatitis, while the other patient (Saitama/2014) did not have history of traveling abroad. Nearly entire genomic sequence of HEV was recovered from both patients; 7,336 nucleotides (nt) from the Tokyo case in 2012 (isolate name=JAO-SpaTok12, accession=LC055972) and 7,306 nt from the Saitama case in 2014 (JMH-Sai14, LC055973). Phylogenetic analysis revealed that JMH-Sai14 from the Saitama patient, who denied having traveled abroad, could be classified into a subgroup of genotype 3f, together with European isolates as well as an already reported Japanese isolate HE-JA12-0725 (AB850879) from a patient living in Gunma and having never traveled abroad when he/she developed hepatitis in 2012. On the other hand, the Tokyo patient's sequence segregated to a separate branch together with European isolates. Based on these findings it is possible that HEV genotype 3f has already made inroad into Japan as in the case of 3e.

Seroprevalence of hepatitis E virus (HEV) and detection of HEV RNA with a transcription-mediated amplification assay in blood donors from Catalonia (Spain).

Hepatitis E virus (HEV) is an emerging threat to the safety of blood transfusion. The aim of this study was to determine HEV immunoglobulin (Ig)G and RNA prevalence in Catalan blood donors. Nearly 10,000 samples were collected from anonymized, unpaid donors at the Banc de Sang i Teixits (Barcelona, Spain) from June to December 2013. For the serology study, a subset of 1082 donations was tested in parallel for HEV IgG using Wantai and Mikrogen enzyme-linked immunosorbent assay tests. Samples were tested individually (individual-donation nucleic acid test [ID-NAT]) for HEV RNA using the Procleix HEV assay (95% limit of detection 7.9 IU/mL). Procleix repeat-reactive donations were confirmed by an in-house real-time polymerase chain reaction (PCR) test. The prevalences of IgG anti-HEV in Catalan blood donors were 19.96% (Wantai assay) and 10.72% (Mikrogen assay). Screening of 9998 samples with the Procleix HEV assay yielded three real-time PCR-confirmed and IgM and IgG anti-HEV-positive donations with viral loads of 250, 564, and 2755 IU/mL. The donation with highest viral load was genotype 3f. HEV RNA positivity rate was one per 3333 donations (0.03%; 95% confidence interval, 0.01%-0.09%). The Procleix HEV ID-NAT screening system has provided evidence of HEV RNA presence in Catalan blood donors. Further data are needed to assess the impact of HEV infection in at-risk patients to design the best strategy to increase blood safety.

Risk factors and molecular characterization of acute sporadic symptomatic hepatitis E virus infection in Thailand

ObjectiveTo report clinical outcomes and viral genotypes of acute symptomatic hepatitis E virus (HEV) infection in Thailand. MethodsForty patients with acute symptomatic HEV infection were recruited during 2009-2013. Clinical, demographic and laboratory data were collected. Diagnosis was accomplished by detection of anti-HEV IgM and/or HEV RNA in the serum or stool. HEV genotypes were classified by direct sequencing of RT-PCR products and phylogenetic analysis. ResultsThe high risk group, comprising immune-compromised, liver cirrhosis and very elderly (>80 years) patients (17 cases), had higher levels of serum alkaline phosphatase at presentation compared with the low risk group. Two fatal cases resulted from acute hepatitis E in the high risk group. Initial clinical presentation did not show statistically significant differences. In six cases (6/40), the virus could be detected in serum or stool by RT-PCR and sequencing. Upon molecular characterization, the viruses were classified as HEV genotype 3f and were in the same cluster as Thai swine HEV. ConclusionsOur data showed that acute HEV infection has various clinical presentations and outcomes. Higher levels of serum alkaline phosphatase were observed in high risk patients. All isolated viruses were identified as HEV genotype 3f possibly originating from swine.

Genomic analysis of the Chinese genotype 1F rubella virus that disappeared after 2002 in China

Genotype 1F was likely localized geographically to China as it has not been reported elsewhere. In this study, whole genome sequences of two rubella 1F virus isolates were completed. Both viruses contained 9,761 nt with a single nucleotide deletion in the intergenic region, compared to the NCBI rubella reference sequence (NC 001545). No evidence of recombination was found between 1F and other rubella viruses. The genetic distance between 1F viruses and 10 other rubella virus genotypes (1a, 1B, 1C, 1D, 1E, 1G, 1J 2A, 2B, and 2C) ranged from 3.9% to 8.6% by pairwise comparison. A region known to be hypervariable in other rubella genotypes was also the most variable region in the 1F genomes. Comparisons to all available rubella virus sequences from GenBank identified 22 nucleotide variations exclusively in 1F viruses. Among these unique variations, C9306U is located within the recommended molecular window for rubella virus genotyping assignment, could be useful to confirm 1F viruses. Using the Bayesian Markov Chain Monte Carlo (MCMC) method, the time of the most recent common ancestor for the genotype 1F was estimated between 1976 and 1995. Recent rubella molecular surveillance suggests that this indigenous strain may have circulated for less than three decades, as it has not been detected since 2002.

Case Report: Severe bilateral amyotrophic neuralgia associated with major dysphagia secondary to acute hepatitis E

Introduction:Several acute neurological syndromes can be triggered by immune events. Hepatitis E virus (HEV), an emerging infectious disease, can be one of these triggers.Case report:We report the case of a 36-year-old man that presented nausea and a dull abdominal pain for a week and then felt an acute neuralgic pain involving both shoulders that lasted for 8 to 10 hours. Immediately after, the patient presented a severe bilateral muscular weakness of the proximal part of both upper limbs, corresponding to an amyotrophic neuralgia. Two days after the shoulder pain, the patient presented a dysphagia necessitating tube feeding. A blood sample confirmed hepatitis caused by hepatitis E virus (HEV; genotype 3F). Oral feeding resumed progressively after five months. The patient was fully independent for the activities of daily living but was still unable to work after six months.Conclusion:Amyotrophic neuralgia and hepatitis E are both under-diagnosed. It is noteworthy that HEV can trigger amyotrophic neuralgia. Antiviral drugs, oral steroids and intravenous immunoglobulins can be proposed, but the optimal treatment has not yet been determined.

Severe bilateral amyotrophic neuralgia associated with major dysphagia secondary to acute hepatitis E

Several acute neurological syndromes can be triggered by immune events. Hepatitis E virus (HEV), an emerging infectious disease, can be one of these triggers. We report the case of a 36-year-old man that presented nausea and a dull abdominal pain for a week and then felt an acute neuralgic pain involving both shoulders that lasted for 8 to 10 hours. Immediately after, the patient presented a severe bilateral muscular weakness of the proximal part of both upper limbs, corresponding to an amyotrophic neuralgia. Two days after the shoulder pain, the patient presented a dysphagia necessitating tube feeding. A blood sample confirmed hepatitis caused by hepatitis E virus (HEV; genotype 3F). Oral feeding resumed progressively after five months. The patient was fully independent for the activities of daily living but was still unable to work after six months. Amyotrophic neuralgia and hepatitis E are both under-diagnosed. It is noteworthy that HEV can trigger amyotrophic neuralgia. Antiviral drugs, oral steroids and intravenous immunoglobulins can be proposed, but the optimal treatment has not yet been determined.

Hepatitis E virus genotype 3f sequences from pigs in Thailand, 2011–2012

Phylogenetic analysis of partial ORF1 and ORF2 genes of Hepatitis E virus (HEV) strains from pigs in Thailand during 2011-2012 was performed. The result indicated that the current Thai strains belonged to the genotype 3 subgroup 3f, which were similar to the previous HEVs circulating in humans in Thailand.

Rubella Epidemics and Genotypic Distribution of the Rubella Virus in Shandong Province, China, in 1999–2010

BackgroundThe rubella vaccine was introduced into the immunization program in 1995 in the Shandong province, China. A series of different rubella vaccination strategies were implemented at different stages of measles control in Shandong province.Methodology/Principal FindingsThe average reported incidence rate of rubella cases remained at a low level in Shandong province after 1999. However, rubella epidemics occurred repeatedly in 2001/2002, 2006, and 2008/2009. The age of the onset of rubella cases gradually increased during 1999–2010, which showed that most cases were found among the 10 years old in 1999 and among the 17 years old in 2010. Phylogenetic analysis was performed and a phylogenetic tree was constructed based on the World Health Organization standard sequence window for rubella virus isolates. All rubella viruses isolated in Shandong province were divided into 4 genotypes: 1E, 1F, 2A, and 2B. Genotype 1E viruses accounted for the majority (79%) of all these viruses. The similarity of nucleotide and amino acid sequences among genotype 1E viruses was 98.2–100% and 99.1–100%, respectively. All Shandong genotype 1E strains, differed from international genotype 1E strains, belonged to cluster 1 and interdigitated with the viruses from other provinces in mainland China. The effective number of infections indicated by a Bayesian skyline plot remained constant from 2001 to 2009.Conclusions/SignificanceThe gradual shift of disease burden to an older age group occurred after a rubella-containing vaccine was introduced into the childhood immunization schedule in 1995 in Shandong province. Four genotypes, including 1E, 1F, 2A, and 2B, were found in Shandong province during 2000–2009. Genotype 1E, rather than genotype 1F, became the predominant genotype circulating in Shandong province from 2001. All Shandong genotype 1E viruses belong to the genotype 1E/cluster 1; they have constantly circulated, and co-evolved and co-circulated, with those from other provinces.

Identical Consensus Sequence and Conserved Genomic Polymorphism of Hepatitis E Virus during Controlled Interspecies Transmission

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

Genotype 3 Diversity and Quantification of Hepatitis E Virus RNA

Genotype 3 hepatitis E viruses (HEVs) are distributed across the world and are now considered to be an emerging public health concern in industrialized countries. At least 10 genotype 3 subtypes have been identified in humans and animals worldwide. It was recently reported that the sensitivities of HEV RNA assays differ greatly. We have assessed the influence of genotype 3 diversity on the performances of two HEV RNA assays: one targeting the ORF3 gene and the other targeting the ORF2 gene. We tested a panel of 5 HEV-positive reference samples of genotypes 3a, 3b, 3c, 3e, and 3f at 10-fold serial dilutions. The HEV RNA concentrations obtained with both reverse transcription (RT)-PCRs were correlated, but the RT-PCR based on ORF2 underestimated the HEV RNA concentrations. The mean [ORF3 - ORF2] difference was 1.41 log copies/ml. We also tested 34 clinical specimens of genotypes 3c (n = 15), 3e (n = 4), and 3f (n = 15), representing the most prevalent subtypes in Europe. The mean [ORF3 - ORF2] differences were 1.41 log copies/ml for genotype 3c, 0.96 log copies/ml for genotype 3e, and 0.70 log copies/ml for genotype 3f. The bias between the 2 RT-PCR assays was significantly greater for genotype 3c than for genotype 3f (P = 0.007). We therefore recommend the use of an RT-PCR protocol based on ORF3 to quantify HEV RNA of genotype 3 strains.