INTRODUCTION

Hepatitis E virus (HEV) infection is an important global public health issue. It is usually self-limiting, causes approximately 20 million infections and 70,000 deaths every year, and leads to high mortality in pregnant women (^{Navaneethan et al., 2008}; ^{Rein et al., 2012}). HEV is mainly transmitted through the fecal-oral route, but it has been reported to be transmitted through the consumption of uncooked or undercook pork meat (^{Hoofnagle et al., 2012}; Rein et al., 2012).

HEV infection has been recognized as a zoonotic disease. Swine (^{Hsieh et al., 1999}), camels (^{Lee et al., 2016}), goats (^{Long et al., 2017}), rabbits (^{Cossaboom et al., 2011}) and cows (^{Hu and Ma, 2010}) are natural reservoirs of HEV. The consumption of raw/undercooked pork, liver, or milk has been reported to transmit HEV to humans or non-human primates (Hu and Ma, 2010; ^{Huang et al., 2016}; ^{Park et al., 2016}). In China, HEV has been isolated from Holstein cow (Huang et al., 2016), Yak (^{Xu et al., 2014}), Dairy cow (Hu and Ma, 2010), and Yellow cattle (^{Yan et al., 2016}), but is rarely reported in buffaloes. Thus, it is important to explore the HEV infection in buffaloes in China.

The HEV RNA genome is a 7.2kb positive strand and has at least three open reading frames (ORFs) (^{Li et al., 2008}; ^{Tam et al., 1991}). ORF1 encodes the non-structural polyprotein, ORF2 the capsid protein, and ORF3 a small protein involved in virus secretion (^{Purdy and Khudyakov, 2011}). Eight genotypes of HEV are recognized: genotype 1 and 2 HEV infect humans and are endemic in Asia, Africa, and Mexico (^{Rein et al., 2012}). Genotype 3 and 4 HEV infect humans and animals and are endemic in developing and developed countries (^{Geng et al., 2010}; ^{Xu et al., 2014}). Genotype 4 HEV has become endemic in China since 2000 (^{Zhang et al., 2009}). Recently, genotype 5 and 6 HEV have been reported in wild boars (^{Smith et al., 2014}, 2015; ^{Takahashi et al., 2011}), genotype 7 and 8 HEV has been identified in camel (^{Lee et al., 2016}; ^{Woo et al., 2016}).

In the present study, the prevalence of HEV infection in buffaloes in Guangxi province, South China was investigated. The results showed a high prevalence of HEV infection in buffaloes, which suggests that buffaloes may be a new HEV reservoir.

MATERIAL AND METHODS

To investigate the prevalence of HEV in buffaloes, a total of 106 blood samples and 40 milk samples were collected from January 2016 to June 2017 from Guangxi Province, China, where most buffaloes are raised. All samples were stored at −80°C until use.

HEV RNA was detected by RT-nPCR according to our previous study (^{Huang et al., 2002}). Both HEV ORF1 and HEV ORF2 were positive, which were defined to be HEV RNA positive (Huang et al., 2016; Huang et al., 2002). Total RNA from blood or milk was extracted using the Axygen kit (Axygen, China) according to the manufacturer’s instructions. RT-nPCR was performed using M-MLV reverse transcriptase with specific primers containing the HEV ORF1 and ORF2 genes. The PCR products were detected using electrophoresis on an agarose gel containing 0.5μg/mL ethidium bromide.

The seroprevalence of anti-HEV IgG and IgM were determined using a double-antigen sandwich ELISA kit (KHB, China) in accordance to the manufacturer's instructions. The kit used recombinant HEV fusion proteins derived from putative structural proteins of HEV as a primary antibody. The kit also contained both positive and negative controls.

RESULTS

Five serum (5/106, 4.72%) and three milk samples (3/40, 7.50%) from buffaloes were positive for HEV RNA (Figure. 1). The positivity of HEV RNA in buffaloes in Guangxi Province was consistent with previous reports in other species of cattle: 3.23% (1/31) to 11.67% (7/60) in dairy cow in Xinjiang Province, Northwest China, in 2010 (^{Hu and Ma, 2010}); 3.26% (3/92) in yak in the Qinghai and Gansu provinces of Northwest China, in 2014 (^{Xu et al., 2014}); and 3.00% (8/254) in yellow cattle in Shandong Province, Eastern China, in 2016 (^{Yan et al., 2016}). However, the results were lower than 37.14% (52/140) in Holstein cows in Dali, Yunnan Province, Southwest China, in 2016 (^{Huang et al., 2016}), where mixed farming with swine, goat, and cattle is practiced.

We collected blood samples to investigate seroprevalence of HEV infection in buffaloes. To determine the anti-HEV antibodies in buffaloes, a double-antigen sandwich ELISA kit was used. Only one serum sample from buffaloes was positive for the anti-HEV IgM antibody (1/106, 0.94%), and no sample was positive for the anti-HEV IgG antibody. The seroprevalence of HEV in buffaloes was lower than that in cattle from the provinces of Guizhou (4.00%,1/25), Qinghai (7.40%, 2/27) and Yunnan (44.40%, 12/27) in 2010 (^{Geng et al., 2010}) and in yellow cattle from the province of Shandong (47.00%, 120/254) in 2016 (^{Yan et al., 2016}).

Figure 1 Positive rate of HEV RNA in cattle in China. 

Phylogenetic analysis was performed based on HEV ORF2 (partial) sequences isolated from buffaloes (348 bp). All eight HEV strains isolated from buffaloes in Guangxi Province belong to genotype 4 HEV. These strains share 98.00%-100% similarity with each other. One of them was submitted to the GenBank database (KU974931). The homology analysis indicated that these buffalo HEV strains share 95.10%-95.70% homology (KF736234) to yak HEV isolated from the Qinghai and Gansu provinces of Northwest China (^{Xu et al., 2014}); 98.30%-100% similarity (KU356187) to Holstein cow HEV isolated from Dali, Yunnan Province, Southwest China (^{Huang et al., 2016}); and 84.50%-85.90% similarity (KU904280) to yellow cattle HEV isolated from Shandong, Eastern China (Figure. 2) (^{Yan et al., 2016}).

DISCUSSION

HEV infection has been recognized as a zoonotic disease. A recent report has demonstrated that raw/undercooked milk from cows can lead to active HEV infection in non-human primates (^{Huang et al., 2016}). Moreover, the consumption of camel meat and milk is associated with chronic HEV infection (^{Park et al., 2016}). In the present study, a high prevalence of active HEV infection was found in the serum (4.72%) and milk (7.50%) of buffaloes in Guangxi Province, China. In this area, buffaloes are regarded as an important component of the agricultural economy. Buffalo milk is a new and popular dairy product in Southwest China. Milk is believed to be the best nutriment for people who are undernourished or pregnant. Thus, the consumption of raw/undercooked milk or beef from HEV-infected buffaloes is involved with a high risk of HEV infection from buffaloes to humans.

HEV can be zoonotically transmitted from many domestic animals, including deer, camels, swine, and cows (^{Choi et al., 2013}; ^{Huang et al., 2016}; ^{Lee et al., 2016}; ^{Miyashita et al., 2012}), to humans or non-human primates. Active HEV infection has been found in cattle in many areas of China, such as dairy cow in Xinjiang Province, yak in the Qinghai and Gansu provinces, yellow cattle in Shandong Province, and Holstein cows in Yunnan Province (Hu and Ma, 2010; Huang et al., 2016; ^{Xu et al., 2014}; ^{Yan et al., 2016}). In the present study, a high prevalence of HEV infection was found in buffaloes of Guangxi Province, China. Although the seroprevalence of HEV infection in buffaloes is low, the positive rate of HEV RNA in buffaloes is comparable to that in yak, yellow cattle, or dairy cow.

HEV genotype 3 and 4 HEV have become the predominant HEV genotypes in China (^{Shuai et al., 2017}; ^{Zhang et al., 2009}). All eight HEV strains isolated from buffaloes in Guangxi Province belong to genotype 4 HEV. Homology analysis of HEV isolated from buffaloes showed a high similarity to HEV isolated from Holstein cow (^{Huang et al., 2016}) and yak (^{Xu et al., 2014}). In summary, buffaloes may be a new natural reservoir of HEV. Further research is needed to access the potential for zoonotic transmission from buffaloes to humans.

Figure 2 Phylogenetic tree based on the HEV ORF2 partial sequence (348bp) generated using the Neighbor-Joining method of the MEGA 5.1 program.