Detection of bluetongue virus in Culicoides spp. in southern Yunnan Province, China

Abstract

Background Culicoides (Diptera: Ceratopogonidae) are vectors for many arboviruses. At least 20 species are considered as vectors or potential vectors of bluetongue virus (BTV) which cause bluetongue disease in ruminants. A BTV prevalence of 30–50% among cattle and goats in tropical southern Yunnan Province, China, prompted an investigation of the potential BTV vectors in this area. Methods Culicoides were collected by light trapping at three sites in the tropical region of Yunnan Province. Species were identified based on morphology and DNA sequences of cytochrome c oxidase subunit 1 (cox1). PCR and quantitative PCR following reverse transcription were used to test for the presence of BTV RNA in these specimens. Phylogenetic analysis was used to analyze the cox1 sequences of Culicoides specimens infected with BTV. Results Approximately 67,000 specimens of Culicoides were collected, of which 748 were tested for the presence of BTV. Five specimens, including two of Culicoides jacobsoni, one of C. tainanus and two of C. imicola, were identified as infected with BTV. No specimens of C. (subgenus Trithecoides) or C. oxystoma tested were positive for BTV infection. Conclusions To our knowledge this is the first report of C. jacobsoni as a potential BTV vector and the fourth report of an association between C. tainanus and BTV, as well as the first direct evidence of an association between BTV and C. imicola in Asia. A fourth potential cryptic species within C. tainanus was identified in this study. Further analysis is required to confirm the importance of C. jacobsoni and C. tainanus in BTV epidemiology in Asia.

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

“…tainanus and C . jacobsoni as they have been recently reported as potential vectors of BTV in China and Asia [ 14 , 15 , 19 ].…”

“…Prepared Culicoides specimens were submitted for non-destructive nucleic acid extraction using a procedure described by Duan et al [ 14 , 15 ]. Briefly, midges were incubated in 50 μl of lysis buffer from a Genomic DNA Extraction kit (TIANGEN, Beijing, China) at 30 °C for 16 h. The DNA and RNA were extracted together using a MagMAX™-96 Viral RNA Isolation kit following the manufacturer’s directions and a MagMAX™ Express-96 magnetic particle processor (both Ambion Inc., Thermo Fisher Scientific).…”

“…The presence of BTV RNA was tested in the same pools of insect lysate using a pan BTV serotype RT-qPCR method [ 15 ] with primers BTVF-MH and BTVR-MH and probe BTVP-MH [ 23 ]. Properly diluted positive controls with a Cq of approximately 25 and a negative control (water) without signal were run with each batch tested.…”

“…All specimens submitted for cox 1 amplification were processed individually. A 5.5-µl aliquot of nucleic acid from each specimen was added to a 30-µl reaction volume containing PrimeSTAR HS DNA Polymerase, a high-fidelity DNA polymerase (Takara Bio), and amplified according to Duan et al [ 14 , 15 ].…”

“…Recently, Culicoides tainanus and C . jacobsoni , both of which are common and widespread species of biting midges in Asia, were reported as satisfying these two criteria with respect to BTV in Yunnan Province, China [ 14 , 15 ], suggesting the potential of these species to be involved with other Orbivirus , particularly viruses associated to bovids. In this study, the potential Culicoides vectors of TIBOV were studied in Shizong, Yunnan Province, China.…”