Isolation and Identification of a Natural Reassortant Mammalian Orthoreovirus from Least Horseshoe Bat in China

March Isolation and Identification of a Natural Reassortant Mammalian Orthoreovirus from Least Horseshoe Bat in China Data Availability Statement: All relevant data are within the paper. 0 1 2 Lihua Wang 0 1 2 Shihong Fu 0 1 2 Lei Cao 0 1 2 Wenwen Lei 0 1 2 Yuxi Cao 0 1 2 Jingdong Song 0 1 2 Qing Tang 0 1 2 Hailin Zhang 0 1 2 Yun Feng 0 1 2 Weihong Yang 0 1 2 Guodong Liang 0 1 2 0 1 State Key Laboratory for Infectious Disease Prevention and Control, Key Laboratory for Medical Virology, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention , Beijing , China , 2 Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases , Hangzhou , China , 3 Yunnan Institute of Endemic Diseases Control and Prevention , Yunnan , China 1 Funding: Funding provided by National Natural Science Foundation of China (81290342); Ministry of Science and Technology , China (2011CB504702); National Key Technology R & D Program of the Ministry of Science and Technology (2014BAI13B04) and State Key Laboratory for Infectious Disease Prevention and Control (2014SKLID03). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 Academic Editor: Jonas Waldenstrom, Linneaus University , SWEDEN - Mammalian orthoreoviruses (MRVs) have a wide geographic distribution and can infect virtually all mammals. Infections in humans may be either symptomatic or asymptomatic. This study describes the isolation and identification of a natural reassortant MRV from least horseshoe bats (Rhinolophus pusillu) in China, referred to as RpMRV-YN2012. Methods and Results The RpMRV-YN2012 was obtained from urine samples of Rhinolophus pusillus by cell culture. Negative-staining electron microscopy revealed that RpMRV-YN2012 was a non-enveloped icosahedral virus with *75 nm in diameter. Polyacrylamide gel electrophoresis (PAGE) migration patterns of the genome segments showed that RpMRVYN2012 contained 10 segments in a 3:3:4 arrangement. The whole genome sequence of RpMRV2012 was determined. The consensus terminal sequences of all segments of 5GCUAh. . .yUCAUC-3 (h = A, U or C; y = C or U) were similar to the MRV species within the genus Orthoreovirus. Its evolution and evidence of genetic reassortment were analyzed by sequence comparison and phylogenetic analysis. The results showed that RpMRV-YN2012 is a novel serotype 2 MRV that may have originated from reassortment among bat, human, and/or pig MRV strains which associated with diarrhea, acute gastroenteritis and necrotizing encephalopathy in animals and humans. RpMRV-YN2012 is a novel bat reassortant MRV, which may have resulted from a reassortment involving MRVs known to infect humans and animals. It is necessary to identify whether RpMRV-YN2012 is associated with diarrhea, acute gastroenteritis and necrotizing Competing Interests: The authors have declared that no competing interests exist. encephalopathy in clinical patients. In addition, we should carefully monitor its evolution and virulence in real time. Mammalian orthoreoviruses (MRVs), prototypes of the genus Orthoreovirus (family Reoviridae), are non-enveloped viruses with a segmented double-stranded RNA (dsRNA) genome (*23,500 bp) [1]. MRVs have four major serotypes (type 1 Lang, type 2 Jones, type 3 Dearing, and type 4 Ndelle) [1, 2]. Each MRV particle contains 10 genome segments divided into three size classes based upon their characteristic mobility during gel electrophoresis: three large (L1, L2 and L3) segments, three medium segments (M1, M2 and M3), and four small segments (S1, S2, S3 and S4) [13]. The virions have an average diameter of 7080 nm with a typical icosahedral, double-layered protein capsid structure [13]. Although MRVs had been assumed to cause mild respiratory or gastrointestinal diseases, recent studies have shown that they can cause severe illnesses in humans and other mammals, including upper respiratory tract infections, encephalitis, and diarrhea [4, 5]. MRVs have been isolated from many mammalian species, including humans and bats [4, 5]; however, the natural reservoirs or direct progenitors remain unclear. The significance of bats as a source of emerging infectious diseases has been recognized. Bats also are being increasingly recognized as reservoir hosts for viruses which can cross species to infect humans and other domestic and wild mammals [68]. Indeed, many recent outbreaks of emerging viruses, such as the Hendra virus, Nipah virus, Ebola virus and severe acute respiratory syndrome coronavirus (SARS-like CoVs), have been associated with bat transmission events [913]. Here, we describe the first isolation of a novel natural reassortant MRV strain, named RpMRV-YN2012, from the least horseshoe bat (Rhinolophus pusillus) in China. The whole genome sequence of RpMRV2012 was determined. Its evolution and evidence of genetic reassortment were analyzed by sequence comparison and phylogenetic analysis. Materials and Methods Ethics Statement Bats were treated according to the guidelines of Regulations for the Administration of Laboratory Animals (Decree No. 2 of the State Science and Technology Commission of the People's Republic of China, 1988). The sampling was approved by the Ethics Committee of Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention. Sample collection Clean plastic sheets measuring 2.0 by 2.0m were placed under bat roosting sites at approximately 17:00. The fresh urine and fecal samples were collected at the following morning (approximately 7:30 to 8:00). Pharyngeal swab and anal swab samples from captured bats were immersed into maintenance media in a virus sampling tube (Yocon, China). After collecting the swab samples, all bats were released at their capture site. The samples were transported to the laboratory under chilled conditions and stored at 80C until being processed. Cell culture and virus isolation Cell lines used in this study were BHK-21(ATCC CCL-10) and Tb1Lu (ATCC CCL88). Cells were grown in Dulbecos Modified Eagle Medium (DMEM) containing high glucose (Invitrogen, Breda, The Netherlands), supplemented with penicillin, streptomycin and 10% fetal bovine serum (FBS) (Invitrogen, Breda, The Netherlands) at 37C in the presence of 5% CO2. The urine and fecal samples were thawed at 4C and centrifuged at 16,000xg for 5 min to pellet debris. Supernatant was filtered through a 0.45-m filter (Millipore) to remove bacterium-sized particles, and then was diluted 1:10 in cell culture media. Two aliquots of 200 l diluted supernatant were added to monolayer BHK-21and Tb1Lu cells in a 24-well plate separately. After rocked for 2 h at 37C, 1ml of fresh cell culture media was added and then incubated for 7 days at 37C. The flasks were observed daily for toxicity, contamination, or viral cytopathic effect (CPE). By CPE occurrence in the third subcultivation, the supern (...truncated)