G1P[7]型猪轮状病毒的分离鉴定及其全基因组分析

Isolation,identification and whole genome analysis of A G1P[7]porcine rotavirus strain

  • 摘要: 【目的】分离鉴定G1P[7]型猪轮状病毒(PoRV)并进行全基因组分析,为仔猪腹泻病防控及疫苗研发提供理论依据。【方法】腹泻仔猪粪便样本由南京农业大学动物医学院提供,对样本进行猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪德尔塔冠状病毒(PDCoV)、牛病毒性腹泻病毒(BVDV)及PoRV PCR检测。向PCR检测结果为阳性的样本上清液中加入胰蛋白酶,随后接种到MA104细胞进行病毒分离和纯化。通过PCR检测、电镜观察、间接免疫荧光检测和RAN-PAGE检测鉴定毒株。通过绘制病毒增殖曲线和检测病毒对MA104、Vero、IPEC-J2、HT-29、HRT-18和Caco-2细胞的易感性探究毒株生物学特性。利用生物信息学软件分析分离毒株的11个基因与国内外各基因型轮状病毒的同源性并构建病毒全基因组系统发育树。【结果】粪便样本PCR检测结果显示,粪便样本中仅PoRV呈阳性。成功分离出1株PoRV,将该毒株命名为JSNJ2023,病毒在MA104细胞上能稳定增殖和传代,经3次克隆,筛选出适合的克隆株,在第3次克隆中,选择病毒滴度最高的克隆株进行扩大培养,最终成功获得高滴度的单克隆毒株。病毒颗粒呈明显的球形结构,具有清晰的双层衣壳特征,直径约70 nm。间接免疫荧光检测结果显示,JSNJ2023毒株能感染MA104细胞。RNA-PAGE检测结果显示,JSNJ2023毒株具有A群轮状病毒特征性的11条电泳图谱,排列方式为4∶2∶3∶2。病毒增殖曲线显示,接种病毒18 h后JSNJ2023毒株在MA104细胞中的病毒滴度达峰值,随着感染时间的延长,病毒滴度逐渐降低。JSNJ2023毒株对细胞的易感性依次为MA104、IPEC-J2、HRT-18、HT-29、Caco2和Vero细胞。基因同源性分析与系统发育树构建结果显示,JSNJ2023毒株属于猪A群G1P[7]型轮状病毒,其基因型为G1-P[7]-I5。【结论】成功分离获得PoRV JSNJ2023毒株,其属于A群G1P[7]型轮状病毒,基因型为G1-P[7]-I5。JSNJ2023毒株与人、猪及牛轮状病毒存在高度同源性,其进化过程中可能受到人轮状病毒、PoRV与牛轮状病毒的影响,发生了基因重组或交换,形成了独特的基因型(R1-C1-M1-A8-N1-T7-E1-H1)。

     

    Abstract: 【Objective】To isolate and identify G1P[7]porcine rotavirus(PoRV)and conduct a whole genome analysis,which could provide theoretical basis for the prevention and control of piglet diarrhea and vaccine development. 【Method】Excrement samples from diarrheic piglets were provided by college of Veterinary Medicine,Nanjing Agricul【Objective】To isolate and identify G1P[7]porcine rotavirus(PoRV)and conduct a whole genome analysis,which could provide theoretical basis for the prevention and control of piglet diarrhea and vaccine development. 【Method】Excrement samples from diarrheic piglets were provided by college of Veterinary Medicine,Nanjing Agricultural University. The samples were tested for porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus (TGEV),porcine deltacoronavirus(PDCoV),bovine viral diarrhea virus(BVDV)and PoRV by PCR. Trypsin was added to the supernatant of the PCR-positive samples,which were then inoculated into MA104 cells for virus isolation and purification. The strain was identified by PCR,electron microscopy,indirect immunofluorescence assay,and RNAPAGE. The biological characteristics of the strain were explored by drawing the virus proliferation curve and testing the susceptibility of MA104,Vero,IPEC-J2,HT-29,HRT-18,and Caco-2 cells to the virus. Bioinformatics software was used to analyze the homology of the 11 genes of the isolated strain with various genotypes of rotavirus at home and abroad and to construct a phylogenetic tree of whole genome of the virus.【Result】The PCR results of the excrement samples showed that only PoRV was positive. One strain of PoRV was successfully isolated and named JSNJ2023. The virus could stably replicate and passage on MA104 cells. After three rounds of cloning,a suitable clone was selected. In the third round of cloning,the clone with the highest virus titer was expanded,and a high-titer monoclonal strain was finally obtained. The virus particles had a distinct spherical structure with a clear double-layered capsid feature,with a diameter of approximately 70 nm. The indirect immunofluorescence assay results showed that the JSNJ2023 strain could infect MA104 cells. The RNA-PAGE results indicated that strain JSNJ2023 had the characteristic 11-band electrophoretic pattern of group A rotavirus,arranged as 4:2:3:2. The virus proliferation curve showed that the strain JSNJ2023 reached the peak virus titer in MA104 cells 18 h after inoculation,and the virus titer gradually decreased with the extension of the inoculation time. The susceptibility of the strain JSNJ2023 to cell lines was in the order of MA104,IPEC-J2,HRT-18,HT- 29,Caco2 and Vero cells. The results of gene homology analysis and phylogenetic tree construction showed that the strain JSNJ2023 belonged to group A G1P[7]porcine rotavirus,with the genotype of G1-P[7]-I5.【Conclusion】The PoRV strain JSNJ2023 is successfully isolated and identified as a G1P[7]type rotavirus of group A,with the genotype of G1-P[7]-I5. The strain JSNJ2023 has a high degree of homology with human,porcine and bovine rotaviruses,and its evolution may have been influenced by human,porcine,and bovine rotaviruses,resulting in gene recombination or exchange and the formation of a unique genotype R1-C1-M1-A8-N1-T7-E1-H1).

     

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