Isolation of Tibet Orbivirus from Culicoides jacobsoni (Diptera, Ceratopogonidae) in China

Abstract

Background Tibet Orbivirus (TIBOV) is a recently discovered Orbivirus known to infect cattle, Asian buffalo and goats in south-western China. It was first isolated from mosquitoes and subsequently from biting midges (Culicoides spp.) in Yunnan, China, indicating that it is an arbovirus. Little is known of its potential to cause disease, but the economic importance of related viruses promoted an investigation of potential Culicoides spp. vectors of TIBOV. Methods Biting midges were collected approximately once per week between May and December 2020, at a cattle farm in Wulong village, Shizong County, Yunnan Province, China. Approximately 3000 specimens of nine species were subsequently used in attempts to isolate virus, and a further 2000 specimens of six species were tested for the presence of bluetongue virus (BTV) and TIBOV using a RT-qPCR test. Results Virus isolation attempts resulted in the isolation of three viruses. One isolate from a pool of Culicoidesjacobsoni was identified as TIBOV, while the other two viruses from C.orientalis and C.tainanus remain unidentified but are not BTV or TIBOV. RT-qPCR analysis did not detect BTV in any specimens, but a single pool containing five specimens of C. jacobsoni and another containing five specimens of C. tainanus produced PCR quantification cycle (Cq) values of around 28 that may indicate infection with TIBOV. Conclusions The isolation of TIBOV from C. jacobsoni satisfies one criterion required to prove its status as a vector of this virus. This isolation is supported by a low Cq value produced from a different pool of this species in the RT-qPCR test. The low Cq value obtained from a pool of C. tainanus suggests that this species may also be able to satisfy this criterion. Both of these species are widespread throughout Asia, with C. jacobsoni extending into the Pacific region, which raises the possibility that TIBOV may be more widespread than is currently known. Graphical abstract

Full Genome Sequencing of Three Sedoreoviridae Viruses Isolated from Culicoides spp. (Diptera, Ceratopogonidae) in China

“…Three viruses isolated from Culicoides without blood meals collected from Shizong County of Yunnan Province, China, were identified by full genome sequencing in this study. Viral strain YNV/KM3, isolated from unsorted Culicoides species was identified as EHDV, while strains YNV/01-1 and YNV/03-2 isolated from C. tainanus and C. orientalis , respectively [ 35 ], were identified as BAV (genotype A) and EHDV-7, respectively, in this study. The latter two results confirm that BAV and EHDV can infect C. tainanus and C. orientalis , respectively, and provide the first evidence that these species are potential vectors of these respective viruses.…”

“…Insects were collected from cattle farms at Wulong Village (24.64° N, 104.29° E, 975 m a.s.l.) in Shizong County, then identified, sorted and processed for virus isolation as described previously [ 35 ]. Details of two isolates, YNV/01-1 and YNV/03-2, were reported previously [ 35 ], while the same methods were used to isolate a third virus from a pool of approximately 100 unidentified parous female Culicoides free from visible blood.…”

“…Cell pellets were collected after scraping cells and centrifugation at 360× g for 5 min. Viral genomes were extracted and prepared by methods modified from references [ 35 , 44 , 45 ]. Briefly, RNA was extracted from cell pellets using RNAiso-plus kit (Takara) according to the manufacturer’s instructions.…”

“…These factors identify Yunnan as one of the most active provinces in China for insect-borne diseases, particularly if the threat of transboundary diseases from neighboring countries is considered. This high level of arboviral activity is evidenced by the initial discovery in Yunnan of BAV and Tibet Orbivirus (TIBOV) [ 12 , 34 , 35 ], the presence of a large number of strains of BTV and EHDV [ 28 , 36 , 37 , 38 ], and the high prevalence of dengue virus in southern Yunnan [ 39 , 40 , 41 ].…”

“…Shizong County of Yunnan Province was the site of the first domestic case of bluetongue disease in China, prompting a number of longitudinal studies into the ecology of this virus in the area [ 36 , 42 , 43 ]. Duan et al [ 35 ] reported the isolation of three viruses from C. tainanus Kieffer and C. jacobsoni Macfie in one such study; however, they only identified one of these viruses. This paper reports full genome sequencing of the remaining two viruses and from a third virus isolated from a pool of unsorted Culicoides collected in Shizong in 2019 and 2020.…”