1株B亚型禽偏肺病毒的分离与鉴定

Isolation and identification of subtype B avian metapneumovirus

从山东省多个地区的商品肉鸡养殖场采集64份疑似病料，通过RT—PCR方法检测出37份禽偏肺病毒阳性病料，以此作为病毒分离材料接种SPF鸡胚，盲传至第7代时鸡胚生长明显迟滞。取阳性尿囊液在CEF中培养，呈现禽偏肺病毒典型细胞病变（CPE）：细胞变圆、悬浮，有合胞体形成。将分离株接种于Veto细胞及DF-1细胞均出现类似病变。对分离株F基因进行测序，并与GenBank中发表的部分代表序列比较。结果显示，与B亚型禽偏肺病毒的核苷酸同源性最高，为97．4％～99．3％；与A亚型禽偏肺病毒核苷酸同源性较低，为77．4％～78．1％；而与C亚型禽偏肺病毒核苷酸同源性最低，为69．5％～69．7％。基因分型显示分离株为B亚型禽偏肺病毒，将该毒株命名为禽偏肺病毒SDWF株。

In 2010,64 shares of suspected avian metapneumovirus （aMPV） cases were collected from Shandong province. The samples were detected by RT-PCR method and 37 shares were posi- tive of subtype B aMPV. Six to eight day-old specific pathogen free embryonating chicken eggs were used to isolate the virus following inoculation by the yolk-sac route. After seven passages, embryo haemorrhages could be observed and the embryo grewth delayed. Then the egg allantoic fluid was inoculated onto chicken embryo fibroblast. After a few passages, scattered focal areas of cell rounding and syncytial formation could be seen,which were regarded as characteristic CPE of aMPV. The isolated virus was named as SDWF strain. The CPE also could be seen in a range of avian and mammalian cells like Veto cells and DF-lcells. The virus was identified by RT-PCR using specific primers. The PCR products were cloned and sequenced. The phylogenetic tree analysis indicated that there was a close genetic relationship between SDWF strain and subtype B aMPV strains isolated from Europe. The genotyping research showed that SDWF strain belonged to subtype B aMPV.