B亚型禽偏肺病毒的分离鉴定及致病性研究

旨在确定我国部分养鸡场的鸡出现甩头、精神萎靡和肿头综合征是否由禽偏肺病毒（aMPV）引起，本研究从山东、福建、黑龙江等地的发病蛋鸡和肉鸡场采集鼻甲骨、气管和肺等样品，首先利用aMPV特异性的RT-PCR方法对临床样品进行初步检测，将RT-PCR检测阳性样品接种Vero细胞进行病毒分离，然后利用G/F基因序列分析及间接免疫荧光试验（IFA）等鉴定病毒的亚型，最后将分离株感染SPF鸡进行致病性分析。结果显示：在采集的220份样品中，RT-PCR检测结果显示，有3份鼻甲骨样品在228 bp左右出现特异性条带，将阳性病料接种Vero细胞盲传5代后，细胞出现变圆、聚集和融合等aMPV特征性细胞病变（CPE），表明成功分离到3株aMPV，将其分别命名为SD2001、SD2002和HLJ2101。G和F基因同源性分析显示，来自蛋鸡的SD2001、SD2002和来自肉鸡的HLJ2101分离株的G和F基因与其他B亚型aMPV毒株的核苷酸和氨基酸序列相似性均较高，核苷酸的相似性分别为93.4%～98.6%和95.6%～100.0%，氨基酸的相似性分别为88.7%～97.8%和97.6%～100.0%；而与A、C和D亚型的G和F基因同源性较低，核苷酸的相似性分别为27.1%～61.8%和66.8%～74.8%，氨基酸的相似性分别为16.1%～36.7%和72.5%～86.5%，这些结果表明，SD2001、SD2002和HLJ2101分离株属于B亚型aMPV。进一步利用B亚型aMPV特异性的阳性血清进行IFA检测，接种SD2001、SD2002和HLJ2101的Vero细胞均可以观察到特异性的绿色荧光信号，进一步证实3个分离株属于B亚型aMPV。选择SD2001感染3周龄SPF鸡进行了致病性研究，结果发现SPF鸡感染后3～6 d出现精神萎靡、甩头和流鼻涕等症状，鼻甲骨、气管和肺也出现病理性损伤，其发病率为90%（18/20）。3株B亚型aMPV的分离不仅有助于明确我国部分养鸡场出现肿头综合征的发病原因，同时也证实B亚型aMPV流行毒株对鸡有明显的致病性，这些结果为我国家禽疫病的诊断和有效防控提供了重要理论依据。

Isolation,Identification and Pathogenicity of Subtype B Avian Metapneumovirus

The purpose of this study was to identify the avian metapneumovirus (aMPV) as the pathogen of head shaking, mental depression and swollen head syndrome in some poultry farms in China. In this study, nasal turbinate and lung samples were collected from chicken farms in Shandong, Fujian and Heilongjiang. Firstly, the clinical samples were detected by aMPV specific RT-PCR method, and then the positive samples were inoculated into Vero cells for virus isolation. Secondly, the subtypes of the viruses were identified by G and F gene sequence analysis and indirect immunofluorescence assay (IFA). Finally, the pathogenicity of the isolate to SPF chickens was analyzed. Among the 220 samples, the RT-PCR results indicated that three nasal turbinate samples had specific bands at about 228 bp. After the positive samples were inoculated into Vero cells for 5 consecutive generations, the cells showed aMPV characteristic cytopathy (CPE) such as rounding, aggregation and fusion. These results showed that three isolates of aMPV were successfully isolated and named SD2001, SD2002 and HLJ2101, respectively. Phylogenetic analysis showed that the G and F genes of layer-originated SD2001, SD2002 and broiler-originated HLJ2101 isolates had high nucleotide and amino acid sequence homologies with other subtype B aMPV strains, and the nucleotide similarities were 93.4%-98.6% and 95.6%-100.0%, respectively, and the amino acid similarities were 88.7%-97.8% and 97.6%-100.0%, respectively. However, the homologies with the G and F genes of subtypes A, C and D aMPV strains were low, and the nucleotide similarities were 27.1%-61.8% and 66.8%-74.8%, respectively, and the amino acids similarities were 16.1%-36.7% and 72.5%-86.5%, respectively. These results indicated that SD2001, SD2002 and HLJ2101 isolates belonged to subtype B aMPV. Further, the specific positive serum of subtype B aMPV was used for IFA test. The specific green fluorescence signal could be observed in Vero cells inoculated with SD2001, SD2002 and HLJ2101, respectively, which furtherly confirmed that the three isolates belonged to subtype B aMPV. Next, the pathogenicity of the isolate to SPF chickens was tested. SPF chickens infected with SD2001 isolate showed clinical symptoms such as depression, head shaking and sneezing 3-6th day after infection. Histopathological analysis showed that there were pathological lesions in the turbinate, trachea and lung of infected SPF chickens. The incidence rate was 90%(18/20). In this study, three strains of subtype B aMPV were successfully isolated. Our results not only identified the pathogen of swollen head syndrome in some poultry farms in China, but also confirmed that the epidemic strain of subtype B aMPV had obvious pathogenicity to chickens. These results provided an important theoretical basis for the diagnosis and effective prevention of aMPV infection in China.