VIROLOGICA SINICA 2017, 32 (4): 290–297 DOI: 10.1007/s12250-017-3973-z RESEARCH ARTICLE Detection and characterization of three zoonotic viruses in wild rodents and shrews from Shenzhen city, China Bo Wang1#, Chun-Lin Cai2#, Bei Li1, Wei Zhang1, Yan Zhu1, Wei-Hong Chen2, Fei Zhuo2, Zheng-Li Shi1, Xing-Lou Yang1* 1. CAS Key Laboratory of Special Pathogens, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan 430071, China 2. Luohu Center for Disease Control and Prevention, Shenzhen 518020, China Diverse species of rodents and shrews, which are abundant worldwide, harbor a variety of viruses; some of these are closely related to human viruses and possess zoonotic potential. Previously studies have demonstrated that the mammarenavirus and hantavirus carried by rodents or shrews could cause diseases in human population. To determine the distribution of zoonotic viruses in Shenzhen city, the major city in southern China with a high population density, we analyzed 225 rodents (Rattus norvegicus and Rattus flavipectus) and 196 shrews (Suncus murinus) from urban and rural districts for the presence of mammarenavirus, hantavirus, and hepatitis E virus (HEV) by RT-PCR targeting the conserved regions. The infection rates for mammarenavirus, hantaviruses, and HEV in rodents and shrews were 3.56%, 6.89%, and 1.66%, respectively. Partial genome fragment analysis indicated that mammarenavirus and hantavirus strains had more than 90% and 99% nucleic acid identity with Cardamones virus and Seoul virus, respectively, which cause diseases in humans. Although the present HEV strains identified are typically found worldwide, phylogenetic analysis demonstrated a divergence of 16%. To our knowledge, the present work is the first report of the prevalence of mammarenavirus, hantaviruses, and rat HEV strains in rodents and shrews from Shenzhen city, China. Our findings highlight the zoonotic potential of rodent- and shrew-borne mammarenavirus and hantavirus, and the biodiversity of rat HEV isolates in Shenzhen city. The present work suggests that utilization of good hygiene habits is important to minimize the risk of zoonosis. KEYWORDS mammarenavirus; hantavirus; hepatitis E virus (HEV); Shenzhen city INTRODUCTION zoonotic transmission of mammarenavirus and hantavi- ruses has already been demonstrated. Rodents are a harbor of several zoonotic pathogens that Mammarenaviruses (genus Mammarenavirus, family may be transmitted to humans via direct or indirect Arenaviridae) are serious pathogens associated with contact. Some of these viruses are closely related to hemorrhagic fever and diseases of the central nervous system. Rodents are the primary natural reservoir of human viruses, and are therefore suspected to possess zoo- mammarenaviruses (Gonzalez et al., 2007; Charrel and notic potential (Luis et al., 2013). Among these viruses, de Lamballerie, 2010; Radoshitzky et al., 2015). In 2014, Received: 10 March 2017, Accepted: 27 May 2017, the Wenzhou virus (WENV), which is carried by rodents Published online: 17 July 2017 and shrews in Wenzhou city, Zhejiang province, China, # These authors contributed equally to this work. *Correspondence: was isolated and characterized (Li et al., 2015). In 2016, Phone: +86-27-87197311, Fax: +86-27-87197240, the Cardamones variant of WENV, which was found in Email: [email protected] Cambodia and Thailand, was shown to be associated ORCID: 0000-0002-5317-8983 with human infection (Blasdell et al., 2016). 290 AUGUST 2017 VOLUME 32 ISSUE 4 © Wuhan Institute of Virology, CAS and Springer Science+Business Media Singapore 2017 Bo Wang et al. Hantaviruses (genus Orthohantavirus, family Hantavi- were trapped by mouse traps, using fried foods as bait, in ridae, order Bunyavirales) have been shown to cause two Shenzhen city, Guangdong province, southern China. main diseases in humans: hemorrhagic fever with renal Samples of various tissues (liver, lung, and intestine) syndrome (HFRS) in Eurasia, and hantavirus cardiopul- were collected. Rodents and shrews were first identified monary syndrome in North America (Adams et al., 2017). based on morphology; the species of animals from which Rodents are the natural hosts of hantaviruses; humans are virus-positive samples had been collected were further accidental hosts and are infected by aerosols of infected confirmed by sequencing of the mitochondrial cytochrome b rodents (McCaughey and Hart, 2000). HFRS is a severe gene (Ge et al., 2013). The tissue samples were reserved public health issue in China; incidences of HFRS in this at –80 °C until nucleic acid isolation. country have accounted for the highest proportion (ap- proximately 90%) of cases globally in the last few de- Nucleic acid extraction cades (Huang et al., 2012). In China, two specific hant- Tissue samples were suspended at 10% w/v in 1 mL of aviruses cause HFRS: Hantaan virus (HTNV), which is phosphate-buffered saline (pH 7.4), and centrifuged at harbored by the striped field mouse (Aodemus agrarius), 12,000 × g for 10 minutes at 4 °C (Eppendorf, Hamburg, and Seoul virus (SEOV) harbored by the Norway rat (Rattus Germany). Total viral nucleic acids from liver, lung, and norvegicus) (Jiang et al., 2017). intestine samples were extracted from 200 μL of tissue Hepatitis E virus (HEV) is the prototype of the family suspension separately, using the QIAamp Viral RNA Hepeviridae. HEV infections are self-limiting, and the Mini Kit (Qiagen, Hilden, Germany) following the manu- clinical course of symptomatic HEV infections includes facturer’s instructions. Nucleic acids were aliquoted with acute and chronic hepatitis E, fulminant liver failure, and elution buffer and stored at –80 °C until subsequent use. extrahepatic symptoms (Kamar et al., 2012; Smith et al., Detection of viruses 2014). Domestic pigs and wild boars have been recog- nized as the main animal reservoirs of zoonotic HEV, al- First, we mixed RNA extracted from liver, lung, and though deer and camel HEV may also be transmitted to intestine samples from each animal. RT-PCR assays were humans (Meng, 2010; Lee et al., 2016). Currently, mo- conducted for the distinct viruses, using the One-Step lecular detection methods have revealed HEV-related iso- RT-PCR Kit (Invitrogen, Karlsruhe, Germany); sequences lates in wild rats, shrews, and bandicoots from Zhanjiang were amplified to perform phylogenetic analyses. Primer city, Guangdong province, China, which might indicate a combinations targeting specific virus are listed in Table 1. broader host range or spillover infections (Guan et al., For mammarenaviruses, samples were screened by 2013; Li et al., 2013). However, the zoonotic potential of nested RT-PCR with primers amplifying the L segment rodent and shrew HEV remains controversial (Johne et as described previously (Li et al., 2015). For hantavi- al., 2014). ruses, samples were screened by RT-PCR using con- As humans are often in close contact with rodents and sensus primers based on the conserved domain of the L shrews, viruses harbored by these animals represent a fragment (Klempa et al., 2006). For HEV, a hemi-nested threat to human health. Shenzhen is a major city in south- RT-PCR targeting the viral RNA dependent RNA poly- ern China, with an area of 40,000 km2 and a population merase (RdRp)gene was performed, as described previ- of 11 million. The city is located directly north of the ously (Drexler et al., 2012). Hong Kong special administrative region and holds sub- provincial administrative status. Shenzhen city has been Genotyping and phylogenetic analyses of reported to be an important endemic area for HFRS viruses caused by SEOV harbored by Norway rats (Yang et al., RT-PCR products were first examined by agarose gel 2006; Liu et al., 2008). However, little is known about electrophoresis. Bands of the expected length were pu- the presence of mammarenavirus and HEV in rodents rified using the QIAquick Gel Extraction Kit (Qiagen, and shrews from southern China. In this study, three par- Hilden, Germany), and then cloned into the vector ticular viruses with zoonotic potential were molecularly pGEM-T Easy (Promega, Madison, WI, USA). The examined in depth to investigate their relationship with inserts of plasmids were sequenced with primers M13F human isolates. and M13R (Invitrogen, Karlsruhe, Germany), using a 3100 Sequencer (ABI, Waltham, MA, USA). Preliminary MATERIALS AND METHODS sequence data were analyzed using Geneious (Version 10.0.2, Biomatters Limited, Auckland, New Zealand) Sampling (Wang et al., 2017). The derived sequences were sub- Between 2013 and 2016, 225 rodents (Rattus norvegicus mitted to the GenBank database under the subsequent and Rattus flavipectus) and 196 shrews (Suncus murinus) accession numbers: mammarenavirus (KY659332− www.virosin.org AUGUST 2017 VOLUME 32 ISSUE 4 291 Zoonotic viruses in rodents and shrews in Shenzhen city Table 1. Sequences of primers used for RT-PCR and sequencing a Amplicon Virus Primer Sequence (5′–3′) Region Reference size (bp) Mammarenavirus Arena-F1 AYNGGNACNCCRTTNGC L gene First round: Li et al., 2015 Arena-R1 TCHTAYAARGARCARGTDGGDGG 938 Arena-F2 GGNACYTCHTCHCCCCANAC Second round: Arena-R2 AGYAARTGGGGNCCNAYKATG 611 Hantavirus HAN-L-F1 ATGTAYGTBAGTGCWGATGC L gene First round: Klempa et al., 2006 HAN-L-R1 AACCADTCWGTYCCRTCATC 453 HAN-L-F2 TGCWGATGCHACIAARTGGTC Second round: HAN-L-R2 GCRTCRTCWGARTGRTGDGCAA 385 HEV DE-F4228 ACYTTYTGTGCYYTITTTGGTCCITGGTT RdRp gene First round: Drexler et al., 2012 DE-R4598 CCGGGTTCRCCIGAGTGTTTCTTCCA 371 Second round: DE-R4565 GCCATGTTCCAGAYGGTGTTCCA 338 Note: aR: G/A;