Characterization of a novel orthoreovirus isolated from fruit bat, China

Tingsong Hu 0 Wei Qiu 0 Biao He 2 Yan Zhang 1 Jing Yu 0 Xiu Liang 0 Wendong Zhang 4 Gang Chen 0 Yingguo Zhang 4 Yiyin Wang 0 Ying Zheng 0 Ziliang Feng 0 Yonghe Hu 3 Weiguo Zhou 0 Changchun Tu 2 Quanshui Fan 0 Fuqiang Zhang 0 0 Centre for Disease Control and Prevention, Chengdu Military Region , 168 Daguan Road, Kunming 650032 , China 1 Department of Biochemistry and Molecular Biology, Fudan University Shanghai Medical College , Shanghai 200030 , China 2 Institute of Military Veterinary, Academy of Military Medical Sciences , Changchun 130062 , China 3 General Hospital of Chengdu Military Region of PLA , Chengdu 610083 , China 4 The Animal Epidemic Disease Control Center , Kunming 650032, Yunnan Province , China Background: In recent years novel human respiratory disease agents have been described for Southeast Asia and Australia. The causative pathogens were classified as pteropine orthoreoviruses with a strong phylogenetic relationship to orthoreoviruses of bat origin. Results: In this report, we isolated a novel Melaka-like reovirus (named Cangyuan virus) from intestinal content samples of one fruit bat residing in China's Yunnan province. Phylogenetic analysis of the whole Cangyuan virus genome sequences of segments L, M and S demonstrated the genetic diversity of the Cangyuan virus. In contrast to the L and M segments, the phylogenetic trees for the S segments of Cangyuan virus demonstrated a greater degree of heterogeneity. Conclusions: Phylogenetic analysis indicated that the Cangyuan virus was a novel orthoreovirus and substantially different from currently known members of Pteropine orthoreovirus (PRV) species group. - Background Many emerging infectious diseases are caused by zoonotic transmission, and the consequence is often unpredictable. Zoonoses have been well represented with the 2003 outbreak of severe acute respiratory syndrome (SARS) due to a novel coronavirus [1,2]. Bats are associated with an increasing number of emerging and reemerging viruses, many of which pose major threats to public health, in part because they are mammals which roost together in large populations and can fly over vast geographical distances [3,4]. Many distinct viruses have been isolated or detected (molecular) from bats including representatives from families Rhabdoviridae, Paramyxoviridae, Coronaviridae, Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae, Arenaviridae, Herpesviridae, Picornaviridae, Filoviridae, Hepadnaviridae and Orthomyxoviridae [3-8]. The Reoviridae (respiratory enteric orphan viruses) comprise a large and diverse group of nonenveloped viruses containing a genome of segmented double-stranded RNA, and are taxonomically classified into 10 genera [9-13]. Orthoreoviruses are divided into two subgroups, fusogenic and nonfusogenic, depending on their ability to cause syncytium formation in cell culture, and have been isolated from a broad range of mammalian, avian, and reptilian hosts [10-14]. Members of the genus Orthoreovirus contain a genome with 10 segments of dsRNA; 3 large (L1-L3), 3 medium (M1-M3), and 4 small (S1 to S4) [15]. The discovery of Melaka and Kampar viruses, two novel fusogenic reoviruses of bat origin, marked the emergence of orthoreoviruses capable of causing acute respiratory disease in humans [9,16]. Subsequently, other related strains of bat-associated orthoreoviruses have also been reported, including Xi River virus from China [17,18]. Wong et al. isolated and characterized 3 fusogenic orthoreoviruses from three travelers who had returned from Indonesia to Hong Kong during 2007 2010 [19,20]. In the present study we isolated a novel reovirus from intestinal contents taken from one fruit bat ( Rousettus leschenaultia) in Yunnan province, China. In the absence of targeted sequencing protocols for a novel virus, we applied the VIDISCR (Virus-Discovery-cDNA RAPD) virus discovery strategy to confirm and identify a novel Melaka-like reovirus, the Cangyuan virus. To track virus evolution and to provide evidence of genetic reassortment PCR sequencing was conducted on each of the 10 genome segments, and phylogenetic analysis performed to determine genetic relatedness with other bat-borne fusogenic orthoreoviruses. Results Virus isolation and morphological characterization The Vero-E6 cells showed a syncytial cytopathic effect (CPE) after 24 hours of the first inoculation at 37C (Figure 1). The virus was named Cangyuan virus after the location from which the host bats (Rousettus leschenaultia) were collected (Cangyuan city of Yunnan province, China). After the first passage in Vero E6 cells, Cangyuan virus began to cause syncytial CPE 24 hours post-inoculation; notably earlier than for Melaka virus and other orthoreovirus (Figure 1B) [9,16,17,20]. QPCR analysis demonstrated that the replication of Cangyuan virus began after 12 hours infected the Vero E6 cells (Figure 1C). After the second passage, Virus titrations were performed and the infectious dose of Cangyuan virus was 105.5 TCID50/0.1 ml. QPCR analysis demonstrated that Cangyuan virus is the virus replicating in the cells and responsible for the observed CPE (Table 1). Negative-staining EM of particles in the supernatant recovered from Vero E6 cells infected with Cangyuan virus revealed non-enveloped icosahedral virus-like particles, approximately 7080 nm in diameter, possessing a double capsid with conspicuous spikes or turrets situated on the inner core; features characteristic of the family Reoviridae, genus Orthoreovirus (Figure 1D) [9]. Neutralizing antibody titers Serum samples from 50 fruit bats ( Rousettus leschenaultia) collected from Cangyuan city were screened for antiCangyuan virus neutralizing antibody. According to the Neutralizing Antibody Titers determined in this study, the serum of two bats had a neutralizing antibody titer of 1,280 against Cangyuan virus and the serum of five bats (10%, 5/50) a titer of 640 against Cangyuan virus. Our studies indicated a 26% (13/50) prevalence for antibody titers >1:160 for Cangyuan virus-specific antibodies in fruit bats (Rousettus leschenaultia). The control serum had a neutralizing antibody titer <10. At present, it is not clear whether Cangyuan virus is carried by a Figure 1 Syncytium formation in Cangyuan virus-infected Vero E6 cells. (A) Mock-infected. (B) Cangyuan virus 24 hours post-infection. (C) The viral growth curve of Cangyuan virus infected VeroE6 cells in the 28 hours. (D) Negatively stained electron micrograph of viral particles (arrowheads) recovered from the supernatant of Cangyuan virus-infected Vero E6 cells. Bar = 100 nm. Table 1 QPCRs result of Cangyuan virus infected VeroE6 cells with the L2 segment primers 102.5 22.817 0.358 24 Note: The culture supernatants 0.1 mL (after the second passage,105.5 TCID50/ 0.1 mL titer) was serially diluted until 103 and infected Vero E6 cells. After the 24 hours, the culture supernatants were analyzed by RT-QPCRs. The normal Vero-E6 cells as negative controls for RT-QPCRs (...truncated)