| Yaks are necessary and important breeds for local residents in high-altitude environments. Diarrhoea in newborn yaks is a common disease that reduces productivity and increace loss by death, thereby resulting in major economic losses in the yak industry. However, diarrhea-associated virus etiology and epidemiological data in yaks have been largely under-investigated due to the harsh local natural and geographical conditions, poor traffic access and fragmentary veterinary services, which greatly restricted the scientific prevention and control of the disease. Thus, the aim of this study was to do a thorough survey of diarrhea virues in yaks, to identify the association between viruses and diarrhoea, to study the molecular characteristics, detection assay and epidemiology of yak Ast V. The following results were obtained: 1?Virus identification of yak stool samples and the relationship with yak diarrheaTo investigate the diversity of viral species, we collected 20 diarrheal faecal specimens of yaks between 30 and 60 days of age from four farms of the grassland in northwest of Sichuan province from May to July 2013. The stool suspensions were pooled by choosing every 5 samples of 20 diarrheal faecal specimens. Then we did viral nucleic acid extraction and reverse transcription. Finally, we mixed 4 c DNA to construct a library and rloaded on a Hi Seq 2000(Illumina) for sequencing.The nine viruses found in the pooled diarrhoeic samples, in order of abundance of nucleic acid sequence, were influenza A virus, bovine viraldiarrhea virus(BVDV), rotavirus, ungulate tetraparvovirus 1(hokovirus), astrovirus(Ast V), bovine enterovirus, hepatitis E virus, kobuvirus and woodchuck hepatitis virus. BRV and BVDV are known to cause bovine diarrhea. However, the remaining seven viruses were first discovered in yak, and 5 of which were also first discovered in bovine industry in China. To determine which viruse was associated with diarrhea in yaks, an additional 138 faecal samples from 63 diarrheal and 75 healthy calves investigated for the presence of the prevalence of the nine viruses. The results show that BEV, kobuvirus, BRV, BVDV, ungulate tetraparvovirus 1 and influenza A virus were detected, and the detection rate in diarrheal and healthy faecal samples were Ast V(60.3%and 2.7%), BVDV(3.2%and 1.3%), influenza A(3.2%and 0), BEV(79.4%and 68%), kobuvirus(43.9%and 34.7%), BRV(28.6%and 21.3%), ungulate tetraparvovirus 1(4.7%and 1.3%). But none of the healthy or diarrhoeic yaks was found to be positive for HEV and WHV. This may be related to the sensitivity of the PCR method. Thus, Ast V, BEV, kobu and BEV virus were prevalent in Yak population, and compared with healthy yaks, only Ast V had a significantly higher prevalence rate in diarrheal samples, indicating a correlation with the clinical symptoms of diarrhea in yaks. 2?Amplification of yak Ast V genome sequenceTo further investigate the genomic information, 12 different pairs of primers were designed to amplify the genome sequence of diarrhoeic faecal sample 8(S8) based on the gene fragments obtained from viral metagenome technology and genome sequence of Bo Ast V in Gen Bank. A near-full genome of yak Ast V S8 was obtained from diarrhoeic sample S8, excluding the 5' UTR. The near-full genome of yak Ast V S8 was 6243 bp in length, with a 51.8% G+C content, which shared 41.2%~76.6% similarity with Bo Ast V isolated from faeces. As typical for mamastroviruses, yak Ast V S8 had three putative ORFs, encoding the protease with ORF1 a, RNA-dependent RNA polymerase(Rd Rp) with ORF1 b and capsid protein with ORF2. The complete ORF1 a was 2454 bp long. The nucleotide and amino acid sequences of ORF1 a shared 72.9%~84.3% and 75.4%~92.5% identity, respectively, with cogent ORFs of other Bo Ast V. The complete ORF1 b was 1509 bp in length, compared with Bo Ast V, the nucleotide and amino acid sequences of ORF1 b had 75.7%~87.5% and 87.1%~96.8% similarity, respectively. In yak Ast V S8, ORF1 b appeared to be the least divergent compared with other ORFs. ORF2 was 2310 bp long. In comparison with other Bo Ast V isolated from faeces, the ORF2 nucleotide and amino acid sequence identity showed the most divergence,with 50.7% ~65.1 % and 54.0 %~69.2% homology. Sequence alignment showed that the variation of ORF1 a and ORF1 b of yak Ast V was mainly caused by the insertion and deletion and the mutation of the base. The variation of ORF2 gene sequence of the strain is very large, which is mainly related to the recombination of the gene.the recombination breakpoint was most strongly supported at position 268. In the first partition(positions 1–268), yak Ast V S8 is most closely related to Bo Ast V. In the second partition(positions 269–769), however, yak Ast V S8 had a closer genetic distance to Cc Ast V-2. Therefore, based on the genetic diversity and evolution, yak Ast V S8 was proposed as a novel virus. The results of this study provide a reference for further understanding of the genetic variation and molecular detection of yak. 3?Development and Application of an RT-PCR Assay for Yak Ast VIn this study, we can not detect the positive samples of yak Ast V positive samples which have been verified by 2 PCR methods of detection of by Bo Ast V method. Thus, it was necessary develop an RT-PCR assay for detecting the novel yak Ast V. As a fact that, the anterior segment of the ORF2 Ast V gene sequence is highly conserved within species. Firstly we obtained 13 1~630 bp ORF2 gene fragments from 20 fecal samples of diarrheic yak, which shared a homology of 99.3%~100% within fragments, but only a homology of 59.5%~80.8%with Bo Ast V. Then, the 1~630 bp ORF2 gene fragment of yak Ast V was selected as the target gene, we redesigned detection primers and developed an RT-PCR assay for yak Ast V successfully. The results show that this RT-PCR assay developed in this study was specific to yak Ast V, no amplifying for Bo Ast V and other pathogens tested in this study. The detection limit of viral nucleic acid of the assay was 57.3 fg/?L. The assay was significantly better in detecting yak Ast V compared to the other two reported assays for detecting bovine astrovirus. Therefore, this study established an RT-PCR assay for yak Ast V with good specificity, high sensitivity, good stability, providing a powerful tool for the detection and epidemiological investigation of yak Ast V.Then we use the RT-PCR assay to detect 331 yak diarrheal samples collected from 4 provinces of the Tibetan Plateau. The detecting results of clinical samples showed that the positive rates of yak Ast V were 55.5%(76/137) in diarrheic fecal samples in Sichuan province, 10%(6/60)in diarrheic fecal samples in Qinghai province, 8.2%(4/49)in diarrheic fecal samples in Yunnan province, 31.8%(27/85)in diarrheic fecal samples in Xizang province, indicating that Bo Ast V was prevalent in the Qinghai Tibet Plateau, the detection rate of diarrhea in different regions is different. A random selection of Sichuan(8), Qinghai(6), Yunnan(4), Tibet(7) a total of 25 yak Ast V positive PCR products established phylogenetic tree can be divided into 2 branches, and there was no relationship between the classification and geographical distribution. |
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