key: cord-0001478-pyic5s73 authors: Woo, Patrick C.Y.; Lau, Susanna K.P.; Teng, Jade L.L.; Tsang, Alan K. L.; Joseph, Marina; Wong, Emily Y.M.; Tang, Ying; Sivakumar, Saritha; Xie, Jun; Bai, Ru; Wernery, Renate; Wernery, Ulrich; Yuen, Kwok-Yung title: New Hepatitis E Virus Genotype in Camels, the Middle East date: 2014-06-03 journal: Emerg Infect Dis DOI: 10.3201/eid2006.140140 sha: 16f9fa5dc35b07c217016a843dff05fa7d9874bf doc_id: 1478 cord_uid: pyic5s73 In a molecular epidemiology study of hepatitis E virus (HEV) in dromedaries in Dubai, United Arab Emirates, HEV was detected in fecal samples from 3 camels. Complete genome sequencing of 2 strains showed >20% overall nucleotide difference to known HEVs. Comparative genomic and phylogenetic analyses revealed a previously unrecognized HEV genotype. As part of a molecular epidemiology study, 203 fecal samples from 203 adult dromedaries (Camelus dromedarius) were submitted to the Central Veterinary Research Laboratory in Dubai, United Arab Emirates, over a 7-month period (January-July 2013). RNA extraction and reverse transcription were performed, as described, to detect other positive-sense single-stranded RNA viruses (5, 6) . Screening for HEV was performed by PCR amplification of a 284-bp fragment of open reading frame (ORF) 2 in HEV; specific primers used were 5′-TTTATTCTC-GTCCAGTCGTTTC-3′ and 5′-GTCAGTGGAG-GACCCATATGT-3′, designed from sequence information from our metagenomic study (P.C.Y. Woo et al., unpub. data) . PCR was performed according to previously described conditions (7); annealing temperature were set at 50°C. DNA sequencing and quantitative real-time reverse transcription PCR were also performed as described (8) . Using strategies we have reported for other positivesense single-stranded RNA viruses, we performed complete-genome sequencing on 2 HEV-positive samples (5, 6) . Comparative genomic analysis was performed as described (9) . Phylogenetic analysis was conducted in MrBayes5D version 3.1.2 (www.fifthdimension.jp/ products/mrbayes5d/) by using an optimal substitution model with 1 million Markov chain Monte Carlo generations; sampling was conducted every 100 generations with a burn-in of 25,000. The substitution model was selected on the basis of the corrected Akaike information criterion by ProtTest version 2.4 (http://darwin.uvigo.es/ software/prottest.html). Reverse transcription PCR for a 284-bp fragment in ORF2 of this HEV, which we named dromedary camel HEV (DcHEV), was positive for 3 fecal samples; viral loads were 3.7 × 10 5 , 4.5 × 10 5 , and 3.2 × 10 7 copies/mL. Complete-genome sequence data for 2 DcHEV strains (GenBank accession nos. KJ496143-KJ496144) revealed that the genome size was 7,220 bases and had a G+C content of 55% (Table) . Overall, the DcHEV genomes differed from all other HEVs by >20% nt (online Technical Appendix Table, wwwnc.cdc.gov/EID/article/20/6/14-0140-Techapp1.pdf). The DcHEV genome contained 3 major ORFs (Table; Figure 1 ). ORF1 polyprotein contained motifs consistent with a methyltransferase, a peptide containing a Y domain, a papain-like cysteine protease, a peptide with a hypervariable region (HVR), a helicase, and an RNA-dependent RNA polymerase. Also present in DcHEV were conserved sequences TLYTRTWS and RRLLXTYPDG, which bound the HVR of HEV1-4 and of 2 recently discovered wild boar HEV strains (10, 11) but not the HVR (11) (12) (13) . As for DcHEV strain 180C, because of the lack of the U insertion as in HEV1, HEV2, and HEV3, ORF2 began at nt 5171 (online Technical Appendix Figure) . For DcHEV strain 178C, similar to HEV4 and the 2 recently discovered wild boar HEV strains (11) , ORF3 began at nt 5161 (online Technical Appendix Figure) and ended at nt 5502, encoding a small phosphoprotein of 113 aa with a multifunctional C-terminal region. As for DcHEV strain 180C, because of the lack of the U insertion as in HEV1, HEV2, and HEV3, ORF3 began at nt 5160. The conserved cis-reactive element (UGAAUAACAUGU) located upstream of ORF2 and ORF3 in both strains might serve as promoter for the synthesis of the subgenomic mRNA for these 2 ORFs. Phylogenetic trees constructed by using ORF1, ORF2, ORF3, and concatenated ORF1/ORF2 excluding the HVR showed that DcHEV was clustered with different HEVs in different phylogenetic trees (Figure 2 ). For ORF1 and concatenated ORF1/ORF2 excluding the HVR, DcHEV was clustered with HEV3; but for ORF2 and ORF3, DcHEV was clustered with HEV1 and HEV2. Recombination analysis performed by using bootscan revealed no obvious and definite site of recombination, similar to what we observed in previous studies for other viruses (14) , although different regions of the DcHEV genome might be more similar to different genotypes of HEV (data not shown). We discovered HEV in dromedaries from the Middle East and named the virus DcHEV. In a recent study conducted in Dubai, HEV accounted for 40% of cases of acute hepatitis in humans (15) . Although HEV is a major pathogen in the Middle East, sequence data for HEVs in the Arabian Peninsula are limited. The study reported here revealed that 1.5% of the adult dromedary fecal samples showed evidence of DcHEV RNA. Because humans come in close contact with dromedaries, our finding of DcHEV in dromedaries indicates a previously unknown potential reservoir and source of HEV infection for humans. Comparative genomic and phylogenetic analyses showed that DcHEV probably represents a previously unrecognized HEV genotype. The conserved motif preceding the HVR in ORF1 resembled those found in HEV3, HEV4, and the 2 recently discovered wild boar HEV strains. Although phylogenetically ORF1 of DcHEV was clustered with HEV3, ORF2 and ORF3 of DcHEV were clustered with HEV1 and HEV2. Of note, ORF2 and ORF3 of the 2 DcHEV strains with complete genomes sequenced in this study resembled those of different HEV genotypes. The presence of a U insertion downstream to the second possible start codon for ORF2 (AUG2) in DcHEV strain 178C resembled the presence of a U insertion in HEV4 and wild boar HEV, leading to 3 possible start codons for its ORF2 but 1 possible start codon for its ORF3; whereas, the lack of this U insertion downstream to AUG2 in DcHEV strain 180C resembled the lack of U insertions in HEV1, HEV2 and HEV3, leading New Hepatitis E Virus Genotype in Camels Dr Woo is professor and head of microbiology at The University of Hong Kong. His research focuses on novel microbe discovery and microbial genomics. Novel hepatitis E virus in ferrets, the Netherlands Hepatitis E virus variant in farmed mink Novel hepatitis E virus genotype in Norway rats Severe hepatitis E virus infection after ingestion of uncooked liver from a wild boar Discovery of seven novel mammalian and avian coronaviruses ) are the optimal substitution models for ORF1, ORF2, ORF3, and concatenated ORF1/ORF2 excluding HVR, respectively. For this analysis we included amino acid positions 1698, 660, 113, and 2282 in ORF1, ORF2, ORF3 and concatenated ORF1/ORF2 excluding HVR, respectively. For ORF2 and concatenated ORF1/ORF2 excluding HVR, the scale bars indicate the estimated number of substitutions per 50 aa. For ORF1 and ORF3, the scale bars indicate the estimated number of substitutions per 20 aa. Boldface indicates the 2 strains of DcHEV with complete genomes sequenced in this study. in the genus Deltacoronavirus supports bat coronaviruses as the gene source of Alphacoronavirus and Betacoronavirus and avian coronaviruses as the gene source of Gammacoronavirus and Deltacoronavirus Natural occurrence and characterization of two internal ribosome entry site elements in a novel virus, canine picodicistrovirus, in the picornavirus-like superfamily Novel betacoronavirus in dromedaries of the Middle East Discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in gammacoronavirus Genetic variability and the classification of hepatitis E virus The hepatitis E virus polyproline region is involved in viral adaptation Analysis of the full-length genome of a hepatitis E virus isolate obtained from a wild boar in Japan that is classifiable into a novel genotype A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo Comparative analysis of 22 coronavirus HKU1 genomes reveals a novel genotype and evidence of natural recombination in coronavirus HKU1 Hepatitis E: a common cause of acute viral hepatitis We thank Wing-Man Ko and Constance Chan for their continuous support. We also thank Shanty Jose and Sweena Liddle for their help with sample preparation.