746 Huang F et al. man primates are housed. Therefore, investigation of the epidemiology of HEV in Macaca mulatta in this region is necessary. Macaca mulatta is a commonly used animal model in the evaluation of the eﬃcacy of HEV vaccines, the patho- genesis of HEV infections, and other studies to inves- tigate HEV, such as xenotransplantation (11, 12). Organ transplant recipients have been reported to be at risk of HEV infection, and thus, the study of xenotransplan- tation in Macaca mulatta may lead to the development of beneﬁcial therapeutics to avoid HEV infection during organ transplantation (12). Although the Yunnan prov- ince has the most diverse population of wild animals in China, epidemiological and genotypic data for HEV are lacking. 2. Objectives We sought to immediately investigate the epidemiol- ogy of HEV in Macaca mulatta following the detection of genotype 4 swine HEV RNA in the rhesus macaques in a village in Yunnan. 3. Materials and Methods 3.1. Stool and SerumSamples Fresh stool samples (162 from pigs and 320 from Macaca mulatta) and serum samples (from 92 rhesus macaques) were separately collected between 2008 and 2011. The samples were stored at -70°C until use. 3.2. Detection of HEV RNA Stool specimens were suspended at 10% w/v in phos- phate-buﬀered saline (PBS; pH 7.4), containing 0.01% di- ethyl pyrocarbonate (DEPC), and centrifuged at 12000 × g for 10 min. Total RNA was extracted from the supernatant of each stool sample and serum sample with TRIzol® rea- gent (Invitrogen, USA) according to the manufacturer’s instructions. Reverse transcription was performed using a reverse transcriptase kit (AMV XL for RT-PCR; Takara, Japan) according to the manufacturer’s directions. Pre- viously described HEV-speciﬁc primers were used (10); these included the forward primer (P1) 5′-AAT TAT GCY CAG TAY CGR GTT G-3′ and the reverse primer (P2) 5′-CCC TTR TCY TGC TGM GCA TTC TC-3′, and internal primers, which included the forward primer (P3) 5′-GTW ATG CTY TGC ATW CAT GGC T-3′ and the reverse primer (P4) 5′-AGC CGA CGA AAT CAA TTC TGT C-3′. These primers had been previously conﬁrmed to detect all 4 known mammalian HEV genotypes. The expected RT-nPCR product was 348 Table 1. Serological Investigation of HEV in Macaca Mulatta Hepatitis E Virus in Macaca Mulatta bp. The RT-PCR protocol was carried out by incubation at 42°C for 30 min, followed by 85°C for 5 min. The resulting cDNA was ampliﬁed by nested PCR at 94°C for 2 min, fol- lowed by 39 cycles of 94°C for 1 min, 42°C for 1 min, and 72°C for 1 min, with a ﬁnal incubation at 72°C for 7 min. The PCR products were detected both by electrophoresis on agarose gel containing 0.5 µg/mL ethidium bromide and by sequencing on a DNA analyzer (Applied Biosys- tems 3730 DNA Analyzer; Invitrogen, USA). 3.3. Detection of Anti-HEV IgG and IgM Antibodies Serum samples were tested for the presence of HEV-spe- ciﬁc IgG and IgM by using commercial ELISA kits (Wantai, China) containing recombinant ORF2 peptides from the HEV genome as well as both positive and negative con- trols. The sensitivity and speciﬁcity of the kits have been previously reported (13, 14). Sera were tested in duplicate according to the manufacturer’s directions, with cutoﬀ values for IgG and IgM assays set at 0.22 and 0.26, respec- tively and also determined based on the mean OD450 val- ues from the negative controls (± standard deviation). 3.4. Sequence and Phylogenetic Analysis The nucleotide sequences of the ampliﬁed PCR products and of prototypes of diﬀerent genotypes of HEV strains were aligned using MEGA 3.0 software (version 3.0, http:// www.megasoftware.net). The genomic sequences of pro- totype HEV strains were obtained from GenBank. Phylo- genetic trees were generated by the minimum evolution and interior branch methods. Bootstrapping with 1,000 resamplings of the data was performed to calculate branch percentages. The identity between nucleotide sequences was calculated using the MegAlign program (DNAstar package version 5.03; Lasergene, DNAstar Inc., Madison, WI, USA). 3.5. Statistical Analysis Statistical analysis was performed using SPSS statistical software. The descriptive statistics have been reported. A P value of < 0.05 was considered statistically signiﬁcant. 4. Results 4.1. Detection of Anti-HEV IgG and IgM Antibodies in Sera of Macaca Mulatta Thirty-three (35.87%) serum samples from Macaca mu- latta were positive for anti-HEV IgG antibodies, while only 0.44% was positive for HEV IgM (3/92). Fifty percent (21/42) 1–4, y (Adolescent) 5–19, y (Adult) ≥ 20, y (Elder) Number HEV-positive Positive rate (%) 39 16 41.3 48 15 31.25 5 2 40 Male 50 12 24 Female 42 21 50 Hepat Mon. 2011;11(9):745-749