First Report of Hepatitis E Virus Infection in Sika Deer in China

Hepatitis E virus (HEV), a single stranded RNA, nonenveloped virus, belongs to the genus Hepevirus, in the family of Hepeviridae. In this study, 46 (5.43%) out of the 847 serum samples from sika deer (Cervus nippon) were detected as seropositive with hepatitis E virus (HEV) by enzyme linked immunosorbent assay (ELISA). These samples were collected from Inner Mongolia and Jilin and Heilongjiang provinces in China, between October 2012 and October 2013. Seroprevalence of HEV infection in male and female deer was 4.82% and 6.52%, respectively. HEV seroprevalence in sika deer from different geographical locations varied from 3.13% to 6.73%. There was no significant difference in HEV seroprevalence between sika deer collected in autumn (5.65%) and winter (4.85%). This is the first report of HEV seroprevalence in sika deer in China, which will provide foundation information for estimating the effectiveness of future measures to control HEV infection in sika deer in China and assessing the potential risk of humans infected with HEV after consumption of undercooked or raw meat from infected sika deer.

Hepatitis E was first recognized in India in 1978 [5]. Avian HEV and mammalian HEV are major species of hepatitis E virus [5]. More than four genotypes (genotypes 1, 2, 3, and 4) have been identified in HEV [6,11,12]; for example, genotypes 1 and 2 of HEV were commonly prevalent in developing countries, and genotype 2 appears to be exclusively anthroponotic, while genotype 1 infects mainly humans but has also been detected in pigs [13,14]. Genotype 3 was distributed around the world and could cause severe HEV infection in humans and animals. Most of HEV genotype 4 infections were in Asian countries, and they have been found in humans and pigs [11,[15][16][17][18]. Recently, genotype 5 has been proposed as the designation for a variant of HEV isolated from wild boars [12]. Haqshenas et al. [19] and Huang et al. [20] identified a novel HEV strain, which was isolated from chicken, but Avian HEV could not infect nonhuman primates [20]. Humans can be infected by six genotypes of mammalian HEV, namely, types 1 to 4 and two additional genotypes from wild boar [5].

Serum Samples.
All the sika deer blood samples (208 from Harbin city, 316 from Changchun city, 163 from Jilin City, and 160 from Chifeng city) were randomly collected from the 4 cities, Northern China, between October 2012 and October 2013. All the sika deer were bred in the semifree range system. Samples were transported to the laboratory in the College of Animal Science and Technology, Jilin Agriculture University, Jilin province, China. They were kept at 37 ∘ C for 2 h and then at 4 ∘ C for 1 h, and then centrifugation was carried out at 1,000 ×g for 10 min, and the serum was separated and kept at −20 ∘ C until further analysis. Information of sika deer about seasons and geographic origin of sampling, gender, and age was recorded.

Serological Examination.
Circulating antibodies (CAb) against HEV were tested by the species-independent doubleantigen sandwich (das) ELISA using a commercially available kit (MP Biomedicals Asia Pacific Pte. Ltd., Singapore) according to the manufacturer's instructions [22]. Positive, negative, and blank controls were supplied in the kit and set in each test.

Statistical
Analysis. The differences of seroprevalence of HEV infection in sika deer from different locations, gender, and seasons were analyzed statistically using SAS software (version 9.1, SAS Institute, Inc., Cary, NC) [36]. Results were considered statistically significant if < 0.05. Odds ratios (OR) and their 95% confidence interval (95% CI) were provided in this study.

Discussion
There are many reports showing that HEV could infect deer all over the world (Table 2). In the present study, we surveyed the HEV seroprevalence in sika deer at 4 cities in Northern China. The overall HEV seroprevalence in sika deer was 5.43%, which was lower than that in red deer (Cervus elaphus) in Spain (10.4%) [27] and Yezo deer in Japan (34.8%) [26] by ELISA and in red deer in Spain (13.6%) [27] and Netherlands (15%) [22] and roe deer in Hungary (34.4%) [28] by RT-PCR but higher than that in red deer in Netherlands (5%) [22] and wild sika deer in Japan (2.6%) [25] and USA (0%) [7] by ELISA and in wild sika deer in Japan by RT-PCR (0%) [25] and Western blot (2.8%) [26] (Table 2). It is well known that due to high discordance between assays in different detection methods, the actual discrepancy is difficult to explain in the prevalence of HEV among different studies. The differences may also be due to the investigated areas that challenged the survival of HEV, the differences in sanitation, animal-welfare for deer, animal husbandry practices, and geographical and ecological factors, for example, rainfall.
Previous studies [29,37] demonstrated the male has the higher positive rates than the female, but the present study indicates the opposite although the difference in prevalence between females and males is not statistically significant (Table 1). In different regions group, HEV seroprevalence in sika deer varied from 3.13% (5/160) to 6.73% (14/208). Sika deer in Harbin has the highest HEV seroprevalence, followed by Changchun, the third was Jilin City, and Chifeng was the last, but the difference was not statistically significant ( = 0.36) (Table 1). Climate, geography, degree of environment contamination with HEV, ecological conditions, feeding conditions, and animal welfares could be the reasons for the differences in HEV seroprevalence in sika deer in different cities.
In terms of the sampling seasons, HEV seroprevalence in sika deer collected in autumn was 5.65%, which was higher than that collected in winter (4.85%), but the difference was not statistically significant ( = 0.65) ( Table 1). This may happen because in autumn the local climate was suitable for the survival of HEV and the resistance of sika deer was improved in winter in four cities. Compared with autumn, sika deer has a small range of activities and the relatively lower exposure of HEV in winter.
Sika deer are popular in China, and they are famous for medicinal value. Velvet antlers, blood, and meat are the main products from sika deer. Deer meat especially was widely recognized by most people with the improvement of living standards in Northern China. A study has shown that HEV could be transmitted from deer to humans [37], so sika deer is considered as the potential source for the spread of HEV to humans, but, in this study, we did not demonstrate that the elder have higher HEV seroprevalence than the younger [38] because the samples were collected in four cities in northern China between October 2012 and October 2013, just including autumn and winter samples, and the animals were adult sika deer, so our results may have not reflected the relationship between HEV seroprevalence and age. Moreover, the study only detected the antibodies against HEV in sika deer, and no sequence information was obtained due to limited volume of serum, so it could not reflect the true infection rate. Further studies should be conducted to get sequence information to confirm the real HEV infection rates and to determine the HEV genotypes.

Conclusion
The present investigation suggests the existence of HEV infection in sika deer, Northern China. The result may provide fundamental information for estimating the effectiveness of future measure to control HEV infection in sika deer in China and assessing the potential risk of humans infected by HEV.