犬副流感病毒的分离鉴定及部分基因序列分析

摘要

目的：分离并鉴定犬副流感病毒（canine parainfluenza virus，CPIV），并对该病毒N、HN和F基因进行序列分析，研究其遗传变异情况；为CPIV诊断、治疗以及预防奠定分子生物学基础。方法用Vero细胞接种感染CPIV阳性犬肺组织，盲传3代，观察病变情况，收集72 h培养液进行PCR鉴定、血凝特性以及形态学观察。同时，用3对特异性引物，扩增N、HN和F全基因，进行序列测定和分析，并制作系统进化树。结果从犬肺中成功分离出一株犬副流感病毒，并命名为QF20100726。该病毒能凝集豚鼠、猪、鸡和人O型血，电镜观察病毒呈圆形、长丝状等形态多样、大小不等的颗粒状；序列分析表明，与人源、犬源等10株有代表性的副流感病毒基因相比，N基因核苷酸同源性为95.7％～99.8％，氨基酸同源性为97.4％～99.6％，其中有两处氨基酸发生新的变异，分别是第257位由V变成了A，第301位由A变成了T；HN基因核苷酸同源性为95.5～99.8％，氨基酸同源性为96.3～99.6％，F基因核苷酸同源性为94.7％～99.6％，氨基酸同源性为95.6％～99.3％。有两处发生特异变异，分别是56位由T变成了S，89位由T变成了M。结论成功分离犬副流感病毒QF20100726分离株，其血凝特性及超微形态特征均符合CPIV特性，N、HN和F全基因生物进化树显示，该分离株与犬副流感病毒分离株08－1990（登录号：KC237063）亲缘关系最近；N基因氨基酸在第257位（由V变成了A）和第301位（由A变成了T），F基因氨基酸在第56位（由T变成了S）和89位（由T变成了M）发生了特异性变异。这些数据将为CPIV的防控奠定基础。%Objective To isolate and identify canine parainfluenza virus ( CPIV) from the a dog suspected of CPIV infection, then to analyze the gene sequence variation of N,HN and F genes and provide a molecular biology basis for the diagnosis, treatment, prevention and control of CPIV infection.Methods Samples of the lung tissue was taken from a dead dog suspected of CPIV infection, and were inoculated into Vero cells.CPE were observed after three blind passages, then the cell culture fluid after 72-hour-cultuere was assayed for further PCR identification, blood coagulation characteristics and morphological observation.Moreover, 3 pairs of oligonucleotide primers were designed, and N, HN and F complete genes of CPIV were amplified by RT-PCR from positive CPIV culture fluid.The genetic variation of the three genes were further analyzed by biological software, and phylogenetic trees were produced.Results One strain of CPIV was isolated and named QF20100726.The results showed that the CPIV strain could agglutinate Guinea pig, pig,chicken and human o type blood cells, and had the basic ultrastructural chatacteristics of parainfluenza virus.Compared with 10 representative CPIV genes, N, HN and F gene phylogenetic analysis of the QF20100726 CPIV gene showed 95.7% to 99.8%, 94.7% to 99.6% and 94.7% to 99.6% nucleotide identity, respectively, and 97.4% to 99.6%, 96.3 to 99.6%, 95.6%to 99.3%amino acid identity, respectively.Of these aa substitutions, 2 substitutions ( V208A, A301T) occurred in the N open reading frame (ORF), and 2 substitutions (T56S, T89M) occurred in the F open reading frame ( ORF) .Conclusions One strain QF20100726 of CPIv is successfully isolated, and the phylogenetic trees of N, HN and F genes from the QF20100726 CPIV strain show close phylogenetic relationship with other CPIV isolates.The data provide a valuable molecular biology basis for the studies on prevention and control of CPIV infection.