禽呼肠孤病毒C-98、T-98分离株S2基因的克隆及序列分析

Cloning and Sequence Analysis of S2 Gene of Avian Reovirus C-98 and T-98 Strains

参考GenBank中禽呼肠孤病毒176株(ARV 176)S2基因设计引物,对禽呼肠孤病毒内蒙古地区分离株C-98和禽呼肠孤病毒天津地区分离株T-98进行总RNA的提取,以其为模板,应用RT-PCR方法扩增病毒S2基因的cDNA片段,将纯化的cDNA片段克隆到pEASY-T1载体后进行序列测定。结果表明,获得的ARV C-98和ARV T-98的S2基因全长为1324bp,包括15bp的5′非编码区和58bp的3′非编码区,阅读框位于5′端第16位核苷酸和第1266位核苷酸之间。ARV C-98和ARV T-98分离株S2基因的核苷酸同源性很高,达到了99.8%,将C-98、T-98株的S2基因核苷酸序列和参考毒株的S2基因序列进行同源性分析,结果表明,ARV C-98和ARV T-98分离株的S2基因与参考毒株ARV 176、ARV 918、ARV 919、ARV 1733、ARV GX2010、ARV S1133和DRV YJLS2基因的核苷酸同源性最高,在99.2%～99.8%之间;与参考毒株ARV 138和ARV AVS-BS2基因的核苷酸同源性在92.1%～94.1%之间;与参考毒株ARV601SI、ARV 916和ARV 1017-1S2基因的核苷酸同源性在87.3%～87.8%之间;与参考毒株DRV 091、DRV S12和DRV S14 S2基因的核苷酸同源性在77.3%～78.2%之间;与参考毒株MRV 3、MRV 3-jin-1和MRV 3-T3D S2基因的核苷酸同源性最低,在2.4%～5.5%之间。遗传进化树分析显示:ARV C-98和ARV T-98分离株与参考毒株共分为4个遗传进化分支。

According to the $2 gene sequence of Avian reovirus strain 176 （ARV 176） published in GenBank, two pairs of primers were synthesized. The total RNA of ARV isolate T-98 and C-98 isolated in Tianjin and Inner Mongolia were extracted. The $2 gene of virus was amplified by RT-PCR and then cloned into pESAY-T1 plasmid and sequenced. The results showed that the $2 genes of ARV C-98 and ARV T-98 isolates were 1 324 bp, included 15 bp 5＇ non-coding region and 58 bp 3＇ non-coding region, and contained one ORF（ 16-1 266）. Compared nucleotide sequence showed that high homology was 99.8% between ARV T-98 and ARV C-98. The homology of $2 genes of ARV C-98 and ARV T-98 isolates was 99.2%-99.8% comparing with S2 genes of reference strains ARV 176, ARV 918, ARV 919, ARV 1733, ARV GX2010, ARV S1133 and DRV YJL, and was 92.1% -94.1% comparing with $2 genes of reference strains ARV 138 and ARV AVS-B, and was 87.3%-87.8% comparing with $2 genes of reference strains ARV 601SI, ARV 916 and ARV 1017-1, and was 77.3%-78.2% comparing with S2 genes of reference strains DRV 091,DRV S!2 and DRV S14, and was 2.4%-5.5% comparing with S2 genes of reference strains MRV 3, MRV 3-jin- 1 and MRV 3-T3D. Phylogenetic analysis indicated that S2 genes of ARV T-98 and C-98 and 20 ARV strains were separated into four lineages.