Hepatitis E Virus Genotype 4 in Yak, Northwestern China

al. Identification of rodent homologs of hepatitis C virus and pegiviruses. et al. Serology-enabled discovery of genetically diverse hepaciviruses in a new host. To the Editor: Hepatitis E virus (HEV; family Hepeviridae, ge-nus Hepevirus) is a positive-stranded RNA virus with a genome of ≈7.2 kb that contains 3 open reading frames (1,2). On the basis of sequence analysis , mammalian HEVs are classified into 4 recognized genotypes (3,4). HEV genotypes 1 and 2 are restricted to humans and are often associated with large outbreaks and epidemics in developing countries, especially in Africa and Asia. Genotypes 3 and 4 are zoonotic and have been detected in humans, pigs, and other animal species (1,3–6). Yaks (Bos grunniens) live on the cold highland (altitude >3,000 m, average annual temperature <0°C) surrounding the Qinghai-Tibet Plateau, which includes Qinghai and Gansu Provinces in northwest China. Domestic yaks are usually slaughtered for meat at 3 years of age. Infectious pathogens in yaks have been reported only recently (7,8). On the basis of the high prevalence of HEV in human and pigs in China and close hu-man–yak contact in the Tibet region (4,5), we sought to determine if HEV infects yaks. During March–September 2013, we collected 167 fecal samples from yaks <3 years of age; 92 were from Qinghai Province (56 <1 year of age) and 75 from Gansu Province (48 <1 year of age). Soon after sampling, 10% (wt/vol) fecal suspensions were prepared by using sterile phosphate-buffered saline (0.01 mmol/L phosphate , pH 7.2–7.4; 0.15 mmol/L NaCl, 0.1% diethyl pyrocarbonate). After centrifugation, supernatants were separated, and total RNAs were extracted by using TRIzol reagent (In-vitrogen, Carlsbad, CA, USA). RNAs were used as templates to amplify full-length cDNA by reverse transcription PCR (RT-PCR; SuperScript III Synthesis Kit, Invitrogen), according to the manufacturer's instructions. A positive control sample (GenBank accession no. JU119961) and negative control (water) were included. Briefly, 10 pairs of primers were designed based on HEV genotype 4 (GenBank accession nos. to obtain the HEV genome consisting of all the 3 open reading frames. RT-PCR was then performed in a 25-μL volume containing 2-μL templates and 0.1 μmol/L of each primer; cycles were 94°C for 2 min, followed by 38 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 1 min, with a final extension step of 10 min at 72°C. RT-PCR– amplified DNA fragments of the expected sizes were sequenced in …