Genetic diversity of bovine viral diarrhoea virus in Beijing region , China from 2009 to 2010

The genetic diversity of bovine viral diarrhea viruses (BVDV) from Beijing region, China between 2009-2010, was investigated using a phylogenetic analysis of partial 5’untranslated region (5’UTR) nucleotide sequences from 61 BVDV positive samples, and for 13 selected samples, the N pro region was also investigated. All the sixty-one blood samples were collected from cattle that were BVDV persistently infected (PI) within a BVD eradication program on Beijing farms during 2009 and 2010. Phylogenetic analysis based on 5’UTR sequences indicated that all BVDV positive samples were of the BVDV-1 genotype, but were clustered into four different subtypes: sub-genotype 1b (n = 31), 1m (n = 24), 1c (n = 4) and 1d (n = 2). BVDV-1b and 1 m are the most prevalent sub-genotypes in Beijing. The results also demonstrate that in most herds, certain BVDV-1 sub-genotype predominates. It is the first time that BVDV-1d is reported in China with unclear origin. The result of this study will be useful in constructing an effective vaccination plan to further control BVDV in Beijing region.


INTRODUCTION
Bovine viral diarrhea virus (BVDV), a member of the genus Pestivirus, family Flaviviridae, causes significant losses in cattle farming worldwide (Vilcek et al., 2001).BVDV has a positive single-stranded RNA genome, approximately 12.3 kb in length, and encodes an open reading frame (ORF) that is translated into a single polyprotein of approximately 4000 amino acids.The ORF is flanked by untranslated regions (5'UTR, 3'UTR) (Meyers and Thiel, 1996).The 5'UTR highly conserved segments provide sufficient sequence data for reliable genetic classification of new BVDV isolates (Toplak et al., 2004).
Several BVDV-1c were identified in Xinjiang (Huang et al., 2008), and several isolates were clustered into BVDV-1m (Xue et al., 2010).Xue et al. (2010) also reported that several isolates from China were clustered into a new BVDV sub-genotype 1p.
Pestiviruses are known to be highly variable antigenically and genetically (Paton, 1995).When designing and constructing effective vaccination strategies to control BVD, the genetic diversity of circulating BVDV needs to be considered.After screening persistently infected cows in Beijing dairy herds, we investigated the genetic diversity of BVDV obtained in positive serum samples by performing phylogenetic analysis of partial 5'UTR genome part, and for selected samples the N pro region to check the results was obtained.

Animals and samples
Animals investigated in this study were from 11 dairy farms in Beijing, which had an area of 16,808 km 2 and about 150,000 cattle.Before this study, BVDV PI cattle screening under a BVDV eradication program were enforced on 15 dairy farms in Beijing during 2009 and 2010, including about 30,000 cattle (Zhang et al., 2012).Sixty one (61) sera samples were collected from all the PI cattle which had been tested positive in an antigen capture ELISA screening test (HerdCheck BVDV Antigen Test Kit/Serum Plus, IDEXX, Sweden) (Shannon et al., 1991) within the program.These positive samples belong to 11 farms, which are located at 7 districts of Beijing, including Fangshan, Tongzhou, Daxing, Haidian, Changping, Shunyi and Miyun District, and most of Beijing's cattle are kept in these areas.The samples were stored at -70°C.All the sera samples examined in this study are listed in Table 1.

RNA isolation
Total RNA was extracted from sera samples using TIANamp Virus RNA Kit (Tiangen Biotech, China) according to the manufacturer's instructions.The extraction was accomplished with 140 µl of sera and 560 µl of TRIZOL, and the RNA was resuspended in 40 µl of DEPC-treated water.The RNA isolation was performed directly on sera, which excluded the possibility of BVDV contamination during cell culture and the authenticity of the results was upheld (Vilcek et al., 2001;Xue et al., 2010).

cDNA synthesis and PCR
cDNA synthesis was performed using Quantscript cDNA First-strand Synthesis Kit (Tiangen Biotech, China) with random primers.Subsequently, a 288 bp DNA product was amplified from the 5'UTR using GoTaq Green Master Mix (Promega Corporation, USA) for 35 cycles.Utilized primer, 324 and 326, were widely used to amplify a cDNA fragment for genetic diversity analysis of BVDV isolates on 5'UTR (Vilcek et al., 1994(Vilcek et al., , 2001(Vilcek et al., , 2003)).The conditions for amplification were 30 s at 94°C, 30 s at 56°C, and 30 s at 72°C.For 13 selected samples, a 428 bp DNA product was amplified from N pro region employing the same method with primers BD1 and BD3 (Vilcek et al., 2001).The conditions for amplification were 30 s at 94°C, 30 s at 56°C, and 1 min at 72°C.

Sequencing and phylogenetic analysis
Amplified products were separated by electrophoresis in 1.5% agarose gel in Tris -Acetate EDTA buffer , and purified using a QIAquick Gel Extraction Kit (QIAGEN, Germany).The resulting DNA were then sequenced in both directions by using primers 324/326 or BD1/BD3 and an ABI 3730XL sequencing device utilizing fluorescent labeled dideoxynucleotide terminators.All the sequences were confirmed as BVDV by blasting the specified sequences against existing sequences in the NCBI Genbank.Nucleotides sequences were aligned using the Clustal W V.12.10.Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4.0, employing the Neighbor-Joining method with 1000 Bootstrap replications (Tamura et al., 2007).

RESULTS AND DISCUSSION
The genetic diversity of BVDV field positive samples was surveyed by determining the nucleotide sequences of the 5'UTR of 61 positive samples and the N pro region of 13 selected samples, obtained in Beijing between 2009 and 2010.The 5'UTR phylogenetic tree was constructed with the nucleotide sequences of the 61 positive samples, and 36 representative strains retrieved from GenBank.The N pro phylogenetic tree was created using the nucleotide sequences of the 13 selected samples and 24 representative strains retrieved from GenBank.The 13 selected samples are clustered into the four sub-genotypes by the 5'UTR phylogenetic tree.We selected these samples, and constructed the N pro tree to independently test the results obtained from the 5'UTR tree.
As expected from previous analyses (Vilcek, 2001;Mahony et al., 2005) both phylogenetic trees produced consistent results (Mahony et al., 2005;Vilcek et al., 2001).All the 61 BVDV field positive samples investigated in this study were of the BVDV-1 genotype (Figures 1 and 2).Of the total, 31 positive samples were of the BVDV-1b sub-genotype, 24 were of the BVDV-1m sub-genotype, 4 were of the BVDV-1c sub-genotype, and two were of the BVDV-1d sub-genotype.To the best of our knowledge, this is the first time BVDV-1d has been reported in China, and the first time BVDV-1m, BVDV-1b and BVDV-1c has been reported in Beijing.Based on these results, the BVDV-2 genotype was absent in Beijing during this period.From the distribution of 61 positive samples obtained (Table 1), we can see that in most herds (8 out of 11) there is one predominant sub-genotype.The alignment of these sequences reveals that mutations were usually located in two variable regions, 208-228 and 298-328 of the NADL strain, which is consistent with the results obtained by Vilcek et al. (1997).Xue et al. (2010) identified BVDV-1p from cattle in Beijing, however, in the current study no samples clustered into the BVDV-1p sub-genotype (Xue et al., 2010).Two most prevalent sub-genotypes in Beijing are BVDV-1b and 1m.A Blast search of the NCBI Genbank, indicated that these BVDV-1b and 1m sequences are similar with the strains reported in China previous to this study (Vilcek et al., 1997).
Four positive samples were clustered into the BVDV-1c sub-genotype, which is the predominant sub-genotype in Australia.A Blast analysis of the BVDV-1c positive samples against NCBI Genbank non-redundant database identifies VR924 and VR1000 (Mahony et al., 2005) as the most similar sequences which were both collected from Australia.The two herds exhibiting BVDV-1c as the prevalent sub-genotype, the Jinxing and Jinyindao herds had imported cows from Australia in 2005 and 2006.However, when importing cattle, a BVDV antigen capture ELISA was conducted to confirm that no BVDV PI animal was introduced.So it is an interesting question: how was BVDV-1c introduced?Interestingly, two farms, Zhongyi and Lvhe Ermu, imported cows from Australia without developing BVDV-1c as the prevalent sub-genotype (Li et al., 1983).The self-clearance action of BVDV infection should be considered.In order for BVDV infection to persist in a herd for an extended period of time (without re-introduction), one or more sero-negative animals need to be in early pregnancy while there are PI animals in the herd.Otherwise, self-clearance will occur (Lindberg and Alenius, 1999).Consequently, we should acknowledge that PI animals may not be identified if they are aborted, stillborn, experience an early death or are traded before a testable age.
After blasting, the most similar sequence to the two BVDV-1d positive samples was isolate 2900/83 (Tajima et al., 2001), initially reported in Germany.So far as we know, frozen semens imported from Germany were used in Beijing dairy farms.However, how BVDV-1d was introduced is still unknown and worth further investigation.
is indispensable for a better understanding of the pathogenesis and epidemiology of BVDV infections.On the basis of this study, the BVDV-1b and BVDV-1m sub-genotypes should be considered first when constructing a vaccine to control the BVDV infections in Beijing.Consequently, further studies needs evaluation of the antigenicity of Beijing isolates belonging to different sub-genotypes.

Table 1 .
BVDV positive samples used in this study.