【摘要】：犬冠狀病毒病是由犬冠狀病毒(Canine coronavirus,CCoV)引起的一種急性、高度接觸性胃腸道傳染病。在德國1971年首次報(bào)道后,該病毒在亞洲、歐洲、南美洲、北美洲等多個(gè)國家和地區(qū)相繼被報(bào)道并呈現(xiàn)全球流行趨勢,給養(yǎng)犬業(yè)帶來極大的危害。近年來,隨著CCoV高致病性變異毒株的發(fā)現(xiàn)及病毒致病性增強(qiáng)在歐洲國家陸續(xù)報(bào)道,該病再一次引起了廣泛的關(guān)注。在過去五年,我國關(guān)于CCoV的流行情況及遺傳進(jìn)化性等報(bào)道較少。鑒于此,本試驗(yàn)通過對(duì)2014年5月~2015年4月,在黑龍江三個(gè)地區(qū)采集的腹瀉犬糞便樣品,進(jìn)行CCoV的檢測、遺傳進(jìn)化性分析、病毒的初步分離。明確黑龍江三個(gè)地區(qū)CCoV的基本流行情況,為CCoV的防控以及后續(xù)相關(guān)研究提供理論基礎(chǔ)。為了明確黑龍江三個(gè)地區(qū)CCoV的流行現(xiàn)狀及遺傳進(jìn)化性,本試驗(yàn)采集201份腹瀉犬糞便拭子樣品,采用RT-PCR方法針對(duì)CCoVM和S基因片段進(jìn)行擴(kuò)增,對(duì)擴(kuò)增序列進(jìn)行測序及分子進(jìn)化性分析。M基因RT-PCR結(jié)果顯示,201份樣品中57份樣品為CCoV陽性(28.36%,57/201),其中CCoV-Ⅰ型和CCoV-Ⅱ型分別占15.79%(9/57)和84.21%(48/57)。序列分析顯示,57個(gè)CCoV毒株的M基因序列核苷酸同源性為88.4%~100%,推導(dǎo)氨基酸序列同源性為93%~100%;57個(gè)CCoV毒株與中國參考株HF3比較,核苷酸同源性為88.7%~96.2%,推導(dǎo)氨基酸序列同源性為92.2%~97.4%;CCoV-Ⅰ型和CCoV-Ⅱ型毒株與中國參考毒株和其他國家參考毒株相比較呈現(xiàn)遺傳多樣性。57個(gè)CCoV陽性樣品與CPV-2、CaKV和CBo V混合感染率分別為31.58%(18/57)、33.33%(19/57)、5.26%(3/57)。犬感染CCoV的年齡、免疫情況、地區(qū)與季節(jié)性都有較大的差異。57個(gè)CCoV陽性樣品中S基因擴(kuò)增出28份,其中26個(gè)CCoV-Ⅱa亞型毒株,1個(gè)CCoV-Ⅱb亞型毒株和1個(gè)CCoV-Ⅰ型毒株。S基因序列分析顯示,26個(gè)CCoV-Ⅱa亞型毒株之間的核苷酸同源性為86.3%~100%,推導(dǎo)氨基酸序列同源性為87.6%~100%;26個(gè)CCoV-Ⅱa亞型毒株與中國參考株V1之間核苷酸同源性為89.6%~92.2%,推導(dǎo)氨基酸序列同源性為91.1%~97.5%。26個(gè)CCoV-Ⅱa亞型毒株與中國參考株和歐洲、北美、南美洲、亞洲參考株相比較呈現(xiàn)出不同的進(jìn)化史。本試驗(yàn)選取單純感染的23株CCoV陽性樣品,利用MDCK進(jìn)行病毒分離,其中9株樣品盲傳至第4代出現(xiàn)明顯CPE,通過RT-PCR鑒定為陽性,繼續(xù)盲傳第7代眼觀CPE消失,RT-PCR鑒定為陰性;將9株樣品重新接種到MDCK細(xì)胞,盲傳至第5代眼觀4株出現(xiàn)明顯CPE,通過巢式RT-PCR鑒定為陽性,繼續(xù)盲傳,第6代無眼觀CPE存在,巢式RT-PCR鑒定為陰性。本研究揭示,黑龍江三個(gè)地區(qū)腹瀉犬中同時(shí)存在CCoV-Ⅰ型、CCoV-Ⅱa亞型和CCoV-Ⅱb亞型,且CCoV-Ⅱ型毒株具有較高的感染率及遺傳多樣性。在黑龍江三個(gè)地區(qū)CCoV與CPV-2、CaKV有著很高的混合感染率。CCoV在MDCK細(xì)胞上能出現(xiàn)CPE,但是病毒增殖極其不穩(wěn)定。
[Abstract]:Canine coronavirus (canine coronavirus) is an acute, highly contagious gastrointestinal infection caused by canine coronavirus (Canine coronavirus,CCoV). After the first report in Germany in 1971, the virus has been reported in Asia, Europe, South America, North America and many other countries and regions. In recent years, with the discovery of highly pathogenic variant strains of CCoV and the increasing of viral pathogenicity in European countries, the disease has once again attracted wide attention. In the past five years, there have been few reports on the prevalence and genetic evolution of CCoV in China. In view of this, from May 2014 to April 2015, the fecal samples of diarrhea dogs collected in three areas of Heilongjiang Province were tested for CCoV, genetic evolution analysis and preliminary isolation of the virus. In order to provide theoretical basis for the prevention and control of CCoV and related research, the basic epidemic situation of CCoV in the three regions of Heilongjiang Province is clarified. In order to identify the prevalence and genetic evolution of CCoV in three regions of Heilongjiang Province, 201 fecal swabs from diarrhea dogs were collected and amplified by RT-PCR for CCoVM and S gene fragments. The results of sequencing and molecular evolution analysis showed that 57 of the 201 samples were CCoV positive (28.36 / 57), and CCoV- 鈪，

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