1株犬副流感病毒的分离鉴定及生物学特性研究
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摘要
试验旨在分离鉴定犬副流感病毒(canine parainfluenza virus,CPIV),并对其生物学特性进行研究。用Vero细胞接种感染CPIV阳性犬肺脏组织,盲传4代,收集72 h病毒液进行RT-PCR鉴定、电镜观察、血凝试验、热敏性试验、紫外照射试验及病毒一步生长曲线的测定,同时扩增N基因进行序列分析,并构建系统进化树。结果显示,试验成功从出现咳嗽、流鼻涕等呼吸系统疾病症状的病犬肺脏中分离出1株CPIV,命名为CPIV-BJ01;RT-PCR扩增结果发现,在534 bp处有特异性目的条带。病毒电镜观察发现,其超微结构呈圆形、有囊膜、直径在80～200 nm之间;血凝试验显示,病毒在4和37℃均能凝集1%猪红细胞,与报道的CPIV血凝特性一致;病毒对热敏感,长时间高温下病毒毒价会随之下降;紫外照射可使病毒在短时间内对细胞的感染性急剧下降。病毒一步生长曲线测定结果显示,在12～48 h病毒高速增殖,细胞培养液中病毒滴度急剧上升,之后趋于稳定。CPIV N基因序列与19株有代表性的副流感病毒N基因相比,其核苷酸序列同源性为97.1%～99.8%。遗传进化分析表明,CPIV-BJ01与PIV5 1168-1(登录号:KC237064.1)和PIV5 ZJQ 221(登录号:KX100034.1)位于同一分支上,亲缘关系较近。
The aim of this study was to isolate and identify the canine parainfluenza virus(CPIV),and analyze its biological characteristics.CPIV positive canine lung tissue was inoculated into Vero cells.CPE were observed after four blind passages,then the cell culture fluid after 72 h was assayed for further RT-PCR identification,electron microscopy,HA and biological characteristic experiments.At the same time,N gene of CPIV was amplified,and phylogenetic tree was constructed.The result showed that one CPIV was successfully isolated and named as CPIV-BJ01.The result of RT-PCR showed that there was a specific target fragment of 534 bp.The electron microscopic observation showed that the virus were circular,enveloped and between 80 to 200 nm in diameter.It could agglutinate 1% porcine erythrocytes at 4 or 37 ℃.The virus was sensitive to high temperature and UV irradiation.The one-step growth curve showed that the virus replicated at a high speed between 12 to 48 h.Compared with 19 representative CPIV N genes,the nucleotide sequence homology analysis was 97.1% to 99.8%.The CPIV-BJ01 strain was closely genetically related to strain PIV5 1168-1(GenBank accession No.:KC237064.1) and PIV5 ZJQ 221(GenBank accession No.:KX100034.1).
The aim of this study was to isolate and identify the canine parainfluenza virus(CPIV),and analyze its biological characteristics.CPIV positive canine lung tissue was inoculated into Vero cells.CPE were observed after four blind passages,then the cell culture fluid after 72 h was assayed for further RT-PCR identification,electron microscopy,HA and biological characteristic experiments.At the same time,N gene of CPIV was amplified,and phylogenetic tree was constructed.The result showed that one CPIV was successfully isolated and named as CPIV-BJ01.The result of RT-PCR showed that there was a specific target fragment of 534 bp.The electron microscopic observation showed that the virus were circular,enveloped and between 80 to 200 nm in diameter.It could agglutinate 1% porcine erythrocytes at 4 or 37 ℃.The virus was sensitive to high temperature and UV irradiation.The one-step growth curve showed that the virus replicated at a high speed between 12 to 48 h.Compared with 19 representative CPIV N genes,the nucleotide sequence homology analysis was 97.1% to 99.8%.The CPIV-BJ01 strain was closely genetically related to strain PIV5 1168-1(GenBank accession No.:KC237064.1) and PIV5 ZJQ 221(GenBank accession No.:KX100034.1).
引文
[1] LIU C,LI X,ZHANG J P,et al.Isolation and genomic characterization of a canine parainfluenza virus type 5 strain in China[J].Archives of Virology,2017,162(8):2337-2344.
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[4] 翟俊琼,周霞,邹舒展,等.虎源、小熊猫源副流感病毒5型的鉴定及F基因遗传进化分析[J].中国兽医学报,2018,38(4):700-707. ZHAI J Q,ZHOU X,ZOU S Z,et al.Identification of the parainfluenza virus type 5 from tiger source,red panda source and analysis of F gene genetic evolution in genus[J].Chinese Journal of Veterinary Science,2015,38(4):700-707.(in Chinese)
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[7] 胡晓霞.三种主要犬呼吸道病毒多重PCR方法的建立和应用[D].武汉:华中农业大学,2013. HU X X.Establishment and application of the multiplex PCR for three main canine respiratory tract viruses[D].Wuhan:Huazhong Agricultural University,2013.(in Chinese)
[8] ZHAI J Q,ZHAI S L,LIN T,et al.First complete genome sequence of parainfluenza virus 5 isolated from lesser panda[J].Archives of Virology,2017,162(5):1413-1418.
[9] LEE Y N,LEE C.Complete genome sequence of a novel porcine parainfluenza virus 5 isolate in Korea[J].Archives of Virology,2013,158(8):1765-1772.
[10] BARIK S.Control of nonsegmented negative-strand RNA virus replication by siRNA[J].Virus Research,2004,102(1):27-35.
[11] 仲晓丽.犬副流感病毒单克隆抗体的制备及单链抗体蛋白的表达和活性鉴定[D].武汉:华中农业大学,2015. ZHONG X L.Preparation of monoclonal antibody against CPIV,expression and activity analysis of single chain Fv[D].Wuhan:Huazhong Agricultural University,2015.(in Chinese)
[12] SCHULZ B S,KURZ S,WEBER K,et al.Detection of respiratory viruses and Bordetella bronchiseptica in dogs with acute respiratory tract infections[J].The Veterinary Journal,2014,201(3):365-369.
[13] 闫喜军.犬副流感病毒进化分析及核酸疫苗和犬腺病毒重组疫苗的研究[D].长春:吉林大学,2007. YAN X J.Evolutionary of canine parainfluenza virus and experimental study on CPIV DNA vaccine and recombinant canine adenovirus type-2 vaccine[D].Changchun:Jilin University,2007.(in Chinese)
[14] DAMIAN M,MORALES E,SALAS G,et al.Immunohistochemical detection of antigens of distemper,adenovirus and parainfluenza viruses in domestic dogs with pneumonia[J].Journal Comparative Pathology,2005,133(6):289-293.
[15] MONTEIRO F L,CARQNELUTTI J F,MARTINS M,et al.Detection of respiratory viruses in shelter dogs maintained under varying environmental conditions[J].Virology,2016,47(4):876-881.
[16] 刘腾飞,马素贞,申卫红,等.犬副流感病毒NP基因的克隆与原核表达[J].新疆农业大学学报,2010,32(2):146-150. LIU T F,MA S Z,SHEN W H,et al.Cloning and prokaryotic expression of NP gene of canine parainfluenza virus[J].Journal of Xinjiang Agricultural University,2010,32(2):146-150.(in Chinese)
[17] CANINO B,VITO M.Canine respiratory viruses[J].Veterinary Research,2007,38(2):355-373.
[18] CHATIZANDREOU N,STOCK N,YOUNG N,et al.Relationships and host range of human,canine,simian and porcine isolates of simian virus 5 (parainfluenza virus 5)[J].Journal General Virology,2004,85(10):3007-3016.
[19] SCHULLZ R D.Duration of immunity for canine and feline vaccines:A review[J].Veterinary Microbiology,2006,117(1):75-79.
[20] RUCH G R,MOROFF S,LAPPIN M R.Adenovirus 2,Bordetella bronchiseptica,and parainfluenza molecular diagnostic assay results in puppies after vaccination with modified live vaccines[J].Journal of Veterinary Internal Medicine,2016,30(1):164-166.
[21] 李长磊.犬副流感病毒的分离鉴定及结构蛋白基因的克隆与序列分析[D].长春:吉林农业大学,2013. LI C L.Isolation and identification of canine parainfluenza viruses and cloning and sequence analysis of it’s structural protein genes[D].Changchun:Jilin Agricultural University,2013.(in Chinese)
[22] 刘腾飞.犬副流感病毒的检测和NP基因的克隆、表达及其重组蛋白的免疫学活性的研究[D].乌鲁木齐:新疆农业大学,2010. LIU T F.Detection of canine parainfluenza virus and cloning and expression of CPIV-NP gene and immunocompetence of its recombinant protein[D].Urumqi:Xinjiang Agricultural University,2010.(in Chinese)
[23] 高菽蔓,靳红亮,张守峰,等.犬副流感病毒CC-14株全基因组测序及分析[J].中国预防兽医学报,2015,37(10):802-804. GAO S M,JIN H L,ZHANG S F,et al.Whole genome sequencing and analysis of canine parainfluenza virus CC-14 strain[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(10):802-804.(in Chinese)
[2] ELLIS J,ANSEEUW E,GOW S,et al.Seroepidemiology of respiratory canine coronavirus,canine parainfluenza virus,and bordetella bronchiseptica infections in urban dogs in a humane shelter and in rural dogs in small communities[J].Canadian Veterinary Journal,2011,52(8):861-868.
[3] KRAUS T A,GARAZA L,HORVATH C M,et al.Enabled interferon signaling evasion in an immune-competent transgenic mouse model of parainfluenza virus 5 infection[J].Virology,2007,371(1):196-205.
[4] 翟俊琼,周霞,邹舒展,等.虎源、小熊猫源副流感病毒5型的鉴定及F基因遗传进化分析[J].中国兽医学报,2018,38(4):700-707. ZHAI J Q,ZHOU X,ZOU S Z,et al.Identification of the parainfluenza virus type 5 from tiger source,red panda source and analysis of F gene genetic evolution in genus[J].Chinese Journal of Veterinary Science,2015,38(4):700-707.(in Chinese)
[5] 孙贺廷,夏咸柱,高玉伟,等.虎血清中犬副流感病毒的抗体流行病学调查研究[J].养犬,2003,1(3):7-10. SUN H T,XIA X Z,GAO Y W,et al.Anti-epidemiological investigation of major parainfluenza virus in tiger serum[J].Dog Breeding,2003,1(3):7-10.(in Chinese)
[6] MOCHIZUKI M,YACHI A,OHSHIMA T,et al.Etiologic study of upper respiratory infections of household dogs[J].Virology,2008,70(6):563-569.
[7] 胡晓霞.三种主要犬呼吸道病毒多重PCR方法的建立和应用[D].武汉:华中农业大学,2013. HU X X.Establishment and application of the multiplex PCR for three main canine respiratory tract viruses[D].Wuhan:Huazhong Agricultural University,2013.(in Chinese)
[8] ZHAI J Q,ZHAI S L,LIN T,et al.First complete genome sequence of parainfluenza virus 5 isolated from lesser panda[J].Archives of Virology,2017,162(5):1413-1418.
[9] LEE Y N,LEE C.Complete genome sequence of a novel porcine parainfluenza virus 5 isolate in Korea[J].Archives of Virology,2013,158(8):1765-1772.
[10] BARIK S.Control of nonsegmented negative-strand RNA virus replication by siRNA[J].Virus Research,2004,102(1):27-35.
[11] 仲晓丽.犬副流感病毒单克隆抗体的制备及单链抗体蛋白的表达和活性鉴定[D].武汉:华中农业大学,2015. ZHONG X L.Preparation of monoclonal antibody against CPIV,expression and activity analysis of single chain Fv[D].Wuhan:Huazhong Agricultural University,2015.(in Chinese)
[12] SCHULZ B S,KURZ S,WEBER K,et al.Detection of respiratory viruses and Bordetella bronchiseptica in dogs with acute respiratory tract infections[J].The Veterinary Journal,2014,201(3):365-369.
[13] 闫喜军.犬副流感病毒进化分析及核酸疫苗和犬腺病毒重组疫苗的研究[D].长春:吉林大学,2007. YAN X J.Evolutionary of canine parainfluenza virus and experimental study on CPIV DNA vaccine and recombinant canine adenovirus type-2 vaccine[D].Changchun:Jilin University,2007.(in Chinese)
[14] DAMIAN M,MORALES E,SALAS G,et al.Immunohistochemical detection of antigens of distemper,adenovirus and parainfluenza viruses in domestic dogs with pneumonia[J].Journal Comparative Pathology,2005,133(6):289-293.
[15] MONTEIRO F L,CARQNELUTTI J F,MARTINS M,et al.Detection of respiratory viruses in shelter dogs maintained under varying environmental conditions[J].Virology,2016,47(4):876-881.
[16] 刘腾飞,马素贞,申卫红,等.犬副流感病毒NP基因的克隆与原核表达[J].新疆农业大学学报,2010,32(2):146-150. LIU T F,MA S Z,SHEN W H,et al.Cloning and prokaryotic expression of NP gene of canine parainfluenza virus[J].Journal of Xinjiang Agricultural University,2010,32(2):146-150.(in Chinese)
[17] CANINO B,VITO M.Canine respiratory viruses[J].Veterinary Research,2007,38(2):355-373.
[18] CHATIZANDREOU N,STOCK N,YOUNG N,et al.Relationships and host range of human,canine,simian and porcine isolates of simian virus 5 (parainfluenza virus 5)[J].Journal General Virology,2004,85(10):3007-3016.
[19] SCHULLZ R D.Duration of immunity for canine and feline vaccines:A review[J].Veterinary Microbiology,2006,117(1):75-79.
[20] RUCH G R,MOROFF S,LAPPIN M R.Adenovirus 2,Bordetella bronchiseptica,and parainfluenza molecular diagnostic assay results in puppies after vaccination with modified live vaccines[J].Journal of Veterinary Internal Medicine,2016,30(1):164-166.
[21] 李长磊.犬副流感病毒的分离鉴定及结构蛋白基因的克隆与序列分析[D].长春:吉林农业大学,2013. LI C L.Isolation and identification of canine parainfluenza viruses and cloning and sequence analysis of it’s structural protein genes[D].Changchun:Jilin Agricultural University,2013.(in Chinese)
[22] 刘腾飞.犬副流感病毒的检测和NP基因的克隆、表达及其重组蛋白的免疫学活性的研究[D].乌鲁木齐:新疆农业大学,2010. LIU T F.Detection of canine parainfluenza virus and cloning and expression of CPIV-NP gene and immunocompetence of its recombinant protein[D].Urumqi:Xinjiang Agricultural University,2010.(in Chinese)
[23] 高菽蔓,靳红亮,张守峰,等.犬副流感病毒CC-14株全基因组测序及分析[J].中国预防兽医学报,2015,37(10):802-804. GAO S M,JIN H L,ZHANG S F,et al.Whole genome sequencing and analysis of canine parainfluenza virus CC-14 strain[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(10):802-804.(in Chinese)
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