Full Genome Sequence of Bluetongue Virus Serotype 4 from China

Abstract

The complete genomic sequence of a bluetongue virus serotype 4 (BTV-4) strain (strain YTS-4), isolated from sentinel cattle in Yunnan Province, China, is reported here. This work is the first to document the complete genomic sequence of a BTV-4 strain from China. The sequence information will help determine the geographic origin of Chinese BTV-4 and provide data to facilitate future analyses of the genetic diversity and phylogenetic relationships of BTV strains.

GENOME ANNOUNCEMENT

Bluetongue virus (BTV) is the type species of the genus Orbivirus, within the family Reoviridae (1). The genome of BTV consists of 10 linear double-stranded RNA segments encoding the seven structural proteins (VP1 to VP7) and four nonstructural proteins (NS1, NS2, NS3/NS3a, and NS4) (2, 9, 10, 11). To date, 26 BTV serotypes (4, 7) have been described worldwide, and seven BTV serotypes (BTV-1, BTV-2, BTV-3, BTV-4, BTV-12, BTV-15, and BTV-16) have been isolated in China (6, 13, 14). Complete genome sequences are available for three BTV-4 isolates in GenBank, one from South Africa and two from Italy (3). There have been no reports, however, describing the full genomic sequence of BTV-4 strains isolated from China or other locations. It is necessary to acquire and analyze additional complete genomic sequences of BTV-4 strains to study the molecular features of BTV-4 strains and establish phylogenic relationships among BTV serotypes. We report here the complete genomic sequence of BTV-4 strain YTS-4, isolated from the blood of sentinel cattle in Mangshi, Dehong District, Yunnan Province, China, in 1996 (5).

Virus-infected BHK-21 cells culture medium was filtered through 0.22-μm Millipore filters and treated with Cryonase cold-active nuclease (TaKaRa). Viral double-stranded RNA (dsRNA) was isolated using RNAiso Plus (TaKaRa) and ligated to an “anchor” primer as described by Maan et al. (8). The ligation product was purified with MicroElute RNA Cleanup (Omega), and full-length cDNA copies of viral 10 dsRNA segments were synthesized using a PrimeScriptII high-fidelity RT-PCR kit (TaKaRa); PCR was subsequently performed with PHX-B-F/R primers as described previously (8). The purified PCR products were subject to library construction (total length, 400 bp) using a TruSeq DNA sample preparation kit, version 2 (Illumina), with multiple indexes. Paired-end sequencing was performed on an Illumina MiSeq system. In order to obtain high-quality data for de novo assembly, multiple filtering steps were used to remove potential sequences with low quality from PCR or sequencing adapters, including reads that contained more than 50% low-quality bases (Q20) and trimming of the adaptor sequences. After filtration, we performed de novo assembly using Newbler 2.6 and Velvet (12) to construct a consensus sequence for each segment of the YTS-4 genome.

The sizes (in base pairs) of YTS-4 segments 1 to 10 are 3,944, 2,926, 2,772, 1,981, 1,736, 1,638, 1,154, 1,125, 1,052, and 822, respectively. They encode proteins with amino acid lengths as follows: VP1, 1,302; VP2, 956; VP3, 901; VP4, 644; VP5, 526; VP6/NS4, 330/77; VP7, 349; NS1, 552; NS2, 354; and NS3/NS3a, 229/216. This is the first report of a complete sequence of a BTV-4 strain isolated in China. It will facilitate future investigations of the molecular characteristics and geographic origins of BTV-4 strains from China as well as other countries.

Nucleotide sequence accession numbers.

The full genomic sequence of the BTV-4 strain YTS-4 was deposited in GenBank. The accession numbers JX560413 to JX560422 correspond to YTS-4 segments 1 through 10.