Wang et al. Virology Journal (2017) 14:105 DOI 10.1186/s12985-017-0774-9 RESEARCH Open Access Isolation of Tibet orbivirus from Culicoides and associated infections in livestock in Yunnan, China Jinglin Wang*, Huachun Li*, Yuwen He, Yang Zhou, Aiguo Xin, Defang Liao and Jinxin Meng Abstract Background: Culicoides-borne orbiviruses, such as bluetongue virus (BTV) and African horse sickness virus (AHSV), are important pathogens that cause animal epidemic diseases leading to significant loss of domestic animals. This study was conducted to identify Culicoides-borne arboviruses and to investigate the associated infections in local livestock in Yunnan, China. Methods: Culicoides were collected overnight in Mangshi City using light traps during August 2013. A virus was isolated from the collected Culicoides and grown using baby hamster kidney (BHK-21), Vero, Madin-Darby bovine kidney (MDBK) and Aedes albopictus (C6/36) cells. Preliminary identification of the virus was performed by polyacrylamide gel (PAGE) analysis. A full-length cDNA copy of the genome was amplified and sequenced. Serological investigations were conducted in local cattle, buffalo and goat using plaque-reduction neutralization tests. Results: We isolated a viral strain (DH13C120) that caused cytopathogenic effects in BHK-21, Vero, MDBK and C6/36 cells. Suckling mice inoculated intracerebrally with DH13C120 showed signs of fatal neurovirulence. PAGE analysis indicated a genome consisting of 10 segments of double-stranded RNA that demonstrated a 3–3–3–1 pattern, similar to the migrating bands of Tibet orbivirus (TIBOV). Phylogenetic analysis of the viral RNA-dependent RNA polymerase (Pol), sub-core-shell (T2, and outer core (T13) proteins revealed that DH13C120 clustered with TIBOV, and the amino acid sequences of DH13C120 virus shared more than 98% identity with TIBOV XZ0906. However, outer capsid protein VP2 and outer capsid protein VP5 shared only 43.1 and 79.3% identity, respectively, indicating that the DH13C120 virus belongs to TIBOV, and it may represent different serotypes with XZ0906. A serosurvey revealed the presence of neutralizing antibodies with 90% plaque-reduction neutralization against TIBOV DH13C120 in local cattle (44%), buffalo (20%), and goat (4%). Four-fold or higher levels of TIBOV-2-neutralizing antibody titers were detected between the convalescent and acute phases of infection in local livestock. Conclusions: A new strain of TIBOV was isolated from Culicoides. This study provides the first evidence of TIBOV infection in livestock in Yunnan, China, and suggests that TIBOV could be a potential pathogen in livestock. Keywords: Tibet orbivirus, Isolation, Culicoides, Whole genome analysis, Infection in animals * Correspondence: [email protected]; [email protected] Yunnan Animal Science and Veterinary Institute, Qinglongshan Jindian PanLong District Kunming, Kunming, Yunnan province 650224, People’s Republic of China © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. Virology Journal (2017) 14:105 Page 2 of 9 Background Assay of neurovirulence in suckling mice The viral genus Orbivirus within the family Reoviridae Aliquots of 30 μL cell culture material (100 plaque- has a genome consisting of 10 segments of double- forming units, PFU/mL) were inoculated intracerebrally stranded (ds) RNA. It contains 22 recognized virus spe- into 1-day-old suckling mice, and 30 μL of Dulbecco’s cies and 10 unclassified viruses [1]. BTV and AHSV modified Eagle’s medium (DMEM) was similarly applied negatively impact animal husbandry and veterinary health. in the control group, with eight suckling mice in each In addition, Equine encephalosis virus (EEV), Epizootic group. Symptoms (or the time of death) in the virus- hemorrhagic disease virus (EHDV), Palyam virus (PALV) treated neonatal mice were recorded each day for 14 days and Peruvian horse sickness virus (PHSV) are important post-inoculation. pathogens causing epidemic diseases in animals. In these orbiviruses, although PHSV is mosquito-borne, most of Polyacrylamide gel electrophoresis (PAGE) these viruses are carried by Culicoides [2, 3]. Therefore, To reveal the number of dsRNA segment and the ge- Culicoides is considered a potent vector of important nome pattern, viral RNA was extracted using RNAiso animal arbovirus diseases, which cause major economic Plus (TaKaRa, Dalian, China) according to the manufac- losses in domestic animals [2, 4–6]. turer’s protocols, and then separated on RNA-PAGE as The TIBOV was first isolated from Anopheles macula- described previously [9–11]. tus mosquitoes collected in 2009 from Motuo County, Tibet, China [7]. Subsequently, TIBOV was isolated from Full genome sequencing Culex fatigan mosquitoes and Culicoides specimens col- The virus genome was amplified by full-length amplifica- lected from Guangdong and Yunnan, respectively [8]. tion of cDNA (FLAC), as described previously [9, 12, 13]. Here, we report a new TIBOV named DH13C120, iso- Briefly, viruses were propagated in BHK-21 cells, and total lated from Culicoides. Sequence analysis of its entire RNA was extracted using RNAiso Plus (TaKaRa) accor- genome and evidence of DH13C120 infection in local ding to the manufacturer’s protocols. Single stranded (ss) livestock were obtained from samples collected in the RNAs were removed by precipitation with 2 M LiCl southwest border area of Yunnan Province in 2013. (Sigma-Aldrich, St Louis, MO, USA), and dsRNAs were precipitated by the addition of 2.5 volumes of isopropanol Methods and 1 volume of 7.5 M ammonium acetate. The dsRNAs Collection of Culicoides specimens were subjected to 1% agarose gel electrophoresis (AGE) Specimens were collected during August 2013 from the (7 V/cm, for 1 h) in TAE buffer (40 mMTris-acetate, suburb of Manbing Town in Mangshi City, Dehong 1 mM EDTA, pH 8.0) and purified from the agarose Prefecture, Yunnan Province. Samples were collected gel using MinElute gel extraction kits (Qiagen, Hilden, overnight using light traps (12 V, 300 mA; Wuhan Lucky Germany). Star Environmental Protection Tech. Co., Ltd., Hubei, An anchor primer, PC3-T7 loop (synthesized by China). Captured Culicoides, killed by freezing at −20 °C Sangon, Shanghai, China), similar to that described by for 20 min and identified by using morphologic charac- Maan et al. [13], was ligated to the viral dsRNA. Ligation teristics, were pooled into groups of approximately 100 reactions were carried out in a total volume of 30 μL, as insects and stored in liquid nitrogen until viral isolation. described previously [9, 12]. The ligated dsRNA was purified using MicroElute RNA Cleanup kits (Omega Isolation of viruses Bio-Tek Inc., Norcross, GA, USA), denatured with di- Culicoides pools, which included midges that were methyl sulfoxide (DMSO) for a final concentration of engorged or not engorged, were removed from liquid 15% (v/v), heated in boiling water for 2 min, and snap- nitrogen and immediately homogenized and centri- frozen in an ice-water slurry. The full-length comple- fuged as reported previously [9]. The supernatants mentary DNAs (cDNAs) of 10 viral dsRNA segments were used to inoculate monolayers of baby hamster kid- were synthesized in a total volume of 50 μL using a ney (BHK-21) cells, Vero cells (from the China National PrimeScript II high-fidelity reverse transcription poly- Institute for Viral Disease Control and Prevention, merase chain reaction (RT–PCR) kit (TaKaRa) according Chinese Center for Disease Control and Prevention, to the manufacturer’s recommendations. Amplification Beijing, China), and Madin-Darby bovine kidney (MDBK) of the cDNA was carried out with primer PC2 in a total cells (From Yunnan biological pharmaceutical factory, volume of 50 μL, as described previously [9, 12]. The Kunming, China) at 37 °C; Aedes albopictus (C6/36) cells PCR products were viewed after separation on 1% (from the National Institute for Viral Disease Control and agarose gels in 1× TAE buffer containing a nucleic acid Prevention) were inoculated at 28 °C. The cells were dye, and purified using TaKaRa DNA fragment purifica- observed daily (days 1–7 post-inoculation) for cytopathic tion kits (ver. 2.0; TaKaRa). The gel-purified fragments effects (CPEs). were cloned into the pMD19-T vector (TaKaRa), and Wang et al. Virology Journal (2017) 14:105 Page 3 of 9 introduced into chemically competent Escherichia coli Results DHα5 cells (TaKaRa). Single colonies were cultured and Isolation of viruses sequenced by the Shenzhen Huada Genome Institute, Culicoides were processed to isolate viruses in BHK-21, Shanghai, China. We performed 2 sequencings from the Vero and C6/36 cells. A CPE was observed with one same RNA extraction and these sequences (that include pool (DH13C120) collected from Dehong. Isolate the differences) were cover 2.5 times. DH13C120 caused CPEs in BHK-21, Vero, MDBK and C6/36 cells at 48, 48, 144, and 120 h post-infection, re- Sequence analysis and phylogenetic comparisons spectively. The characteristic CPE in BHK-21,