Isolation of Tibet orbivirus from Culicoides and associated infections in livestock in Yunnan, China

Culicoides pools, which included midges that were engorged or not engorged, were removed from liquid nitrogen and immediately homogenized and centrifuged as reported previously [9]. The supernatants were used to inoculate monolayers of baby hamster kidney (BHK-21) cells, Vero cells (from the China National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China), and Madin-Darby bovine kidney (MDBK) cells (From Yunnan biological pharmaceutical factory, Kunming, China) at 37 °C; Aedes albopictus (C6/36) cells (from the National Institute for Viral Disease Control and Prevention) were inoculated at 28 °C. The cells were observed daily (days 1–7 post-inoculation) for cytopathic effects (CPEs).

Aliquots of 30 μL cell culture material (100 plaque-forming units, PFU/mL) were inoculated intracerebrally into 1-day-old suckling mice, and 30 μL of Dulbecco’s modified Eagle’s medium (DMEM) was similarly applied in the control group, with eight suckling mice in each group. Symptoms (or the time of death) in the virus-treated neonatal mice were recorded each day for 14 days post-inoculation.

To reveal the number of dsRNA segment and the genome pattern, viral RNA was extracted using RNAiso Plus (TaKaRa, Dalian, China) according to the manufacturer’s protocols, and then separated on RNA-PAGE as described previously [9‐11].

The virus genome was amplified by full-length amplification of cDNA (FLAC), as described previously [9, 12, 13]. Briefly, viruses were propagated in BHK-21 cells, and total RNA was extracted using RNAiso Plus (TaKaRa) according to the manufacturer’s protocols. Single stranded (ss) RNAs were removed by precipitation with 2 M LiCl (Sigma-Aldrich, St Louis, MO, USA), and dsRNAs were precipitated by the addition of 2.5 volumes of isopropanol and 1 volume of 7.5 M ammonium acetate. The dsRNAs were subjected to 1% agarose gel electrophoresis (AGE) (7 V/cm, for 1 h) in TAE buffer (40 mMTris-acetate, 1 mM EDTA, pH 8.0) and purified from the agarose gel using MinElute gel extraction kits (Qiagen, Hilden, Germany).

An anchor primer, PC3-T7 loop (synthesized by Sangon, Shanghai, China), similar to that described by Maan et al. [13], was ligated to the viral dsRNA. Ligation reactions were carried out in a total volume of 30 μL, as described previously [9, 12]. The ligated dsRNA was purified using MicroElute RNA Cleanup kits (Omega Bio-Tek Inc., Norcross, GA, USA), denatured with dimethyl sulfoxide (DMSO) for a final concentration of 15% (v/v), heated in boiling water for 2 min, and snap-frozen in an ice-water slurry. The full-length complementary DNAs (cDNAs) of 10 viral dsRNA segments were synthesized in a total volume of 50 μL using a PrimeScript II high-fidelity reverse transcription polymerase chain reaction (RT–PCR) kit (TaKaRa) according to the manufacturer’s recommendations. Amplification of the cDNA was carried out with primer PC2 in a total volume of 50 μL, as described previously [9, 12]. The PCR products were viewed after separation on 1% agarose gels in 1× TAE buffer containing a nucleic acid dye, and purified using TaKaRa DNA fragment purification kits (ver. 2.0; TaKaRa). The gel-purified fragments were cloned into the pMD19-T vector (TaKaRa), and introduced into chemically competent Escherichia coli DHα5 cells (TaKaRa). Single colonies were cultured and sequenced by the Shenzhen Huada Genome Institute, Shanghai, China. We performed 2 sequencings from the same RNA extraction and these sequences (that include the differences) were cover 2.5 times.

Initial sequence assembly and analyses were conducted using the DNAStar software package (ver. 4.0; DNASTAR Inc., Madison, WI, USA). Homology and alignment analysis was performed using Clustal X (ver. 2.1) [9] (http://​www.​directoryofshare​ware.​com/​preview/​clustalx/​) and MegAlign (DNASTAR Inc.). Phylogenetic and evolutionary analyses were conducted using MEGA 5.1 (http://​www.​megasoftware.​net/​) based on the neighbor-joining technique and 1,000 bootstrap replications [9].

Serum samples were tested for neutralizing antibodies against TIBOV isolate DH13C120 by 90% plaque-reduction neutralization tests (PRNT 90) using standard methods. Samples were tested with serial two-fold dilutions from 1:10 to 1:1,280. Diluted samples were mixed with equal volumes of culture medium 1× MEM (pH = 7.4) containing DH13C120 (100 PFU) and incubated at 37 °C in a 5% CO₂ for 1 h. Six-well plates of confluent BHK-21 cells were inoculated with the serum–virus mixtures and incubated at 37 °C in a 5% CO ₂ incubator for 1 h. Plates were overlaid with 3 mL of the same medium containing 0.8% agarose, and again with 3 mL of a second overlay medium containing neutral red vital stain (Sigma-Aldrich). The neutralizing antibody titer was identified as the highest serum dilution that reduced the number of virus plaques in the test by ≥ 90%. The samples were considered to be positive when titers were ≥ 20. A ratio of TIBOV PRNT 90 titer between two samples of a same animal ≥ 4 was considered to confirm the presence of a TIBOV infection [14].