牛副流感病毒3型山东分离株致病性研究及荧光定量RT-PCR的建立
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摘要
牛副流感病毒3型（Bovine parainfluenza virus type3, BPIV3）是引起犊牛和成年牛上呼吸道感染的重要病原体之一，也是引起牛场牛群呼吸道疾病综合征的重要病原。目前，已从牛体中分离到的BPIV3的基因型主要有A型（BPIV3a）、B型（BPIV3b）和C型（BPIV3c）。2008年本实验室于山东省某牛场的疑似BPIV3感染的牛群中分离得到4株BPIV3。为了进一步明确分离毒株的基因型，对其中一株命名为SD0835进行全基因组测序，通过序列比对及进化树分析发现BPIV3（SD0835）株与国外报道的BPIV3基因A型和基因B型有较大的差异，故而推测本实验室分离的BPIV3可能是一种新的基因型即，暂定为基因C型（BPIV3c）。截止目前，关于BPIV3基因B型和C型对实验动物的致病性研究尚未见报道，而仅有关于BPIV3基因A型的少量报道。
为了明确新分离的基因C型BPIV3（SD0835）的致病性，本研究设计采用动物试验的方式研究该毒株在动物体内的复制、排毒以及组织病理损伤等情况。通过病毒分离培养、滴度检测、实时荧光定量RT-PCR检测、免疫荧光检测、免疫组化检测，证实了BPIV3（SD0835）株对5~6周龄Balb/c小鼠及豚鼠具有一定的致病性。Balb/c小鼠接毒试验结果表明，接毒24小时后病原体即可从肺及气管中检出，且肺组织中病毒RNA的拷贝数明显高于气管组织。免疫组化及免疫荧光结果进一步证明了病毒在肺及气管组织中的复制，而且主要在肺内小支气管周围细胞的胞浆中复制。组织病理学变化主要以肺泡间隔增宽，局灶性纤维素性肺炎为主。血清学检测结果表明小鼠接毒后第6天开始检测到血凝抑制（HI）抗体持续到14天，且HI抗体最高滴度达1:80。
白化豚鼠的致病性研究表明，接毒后豚鼠出现主要以喷嚏、鼻液增多为主的临诊表现，BPIV3主要在肺脏及气管复制，并通过每日采集鼻拭子确定豚鼠接毒后可通过鼻腔排毒直到第6天，实时荧光定量RT-PCR检测表明接毒后第3天肺及气管组织中病毒RNA拷贝数最高，随后持续下降，但在整个接毒期间病毒RNA在肺和气管组织中均可检出。间接免疫荧光及免疫组化试验结果同时也证明了病原体在肺及气管中的复制，同时表明病原体主要于肺组织中的肺泡间隔细胞的胞浆中复制。组织病理学结果表明，接毒后豚鼠肺组织以严重的间质性肺炎为主，随病程发展同时伴有浆液及纤维素渗出，气管病变较为轻微，仅表现为黏膜层萎缩及上皮脱落；肝脏组织空泡变性，脾脏白髓内淋巴细胞减少；颌下淋巴结呈现化脓性淋巴结炎并伴有浆液析出。HI试验表明豚鼠接毒后3天即可检测到特异性抗体，滴度达1:60。
本实验同时建立了BPIV3的实时荧光定量RT-PCR检测方法，所建立方法可以检出下限为17.6拷贝/μl的病毒含量。利用制备的标准品，每个浓度5个重复进行试验并绘制标准曲线，扩增效率为1.942，相关系数r为0.9948，说明相关性较好；同时应用标准品分别进行批内、批间5次重复性试验，变异系数均小于2.5%说明重复性较好，且与其它呼吸道病原体无交叉反应。应用建立的荧光定量RT-PCR检测临床样本的结果与半数组织细胞感染量（TCID50）的测定结果相符，说明本方法适合于临床样品的检测。
综上所述， BPIV3山东分离株（SD0835）可以实验感染5~6周龄Balb/c小鼠及白化豚鼠，病原可以在两者的肺脏及气管中复制，引起各组织脏器不同的病理损伤，同时可刺激机体产生HI抗体，这说明5~6周龄Balb/c小鼠及白化豚鼠对BPIV3基因C型的山东分离株（SD0835）有易感性。本研究所建立的BPIV3基因C型的山东分离株（SD0835）的Balb/c小鼠及白化豚鼠模型，可进一步用于BPIV3基因C型的感染过程和致病机理的研究。同时，本研究首次证实了BPIV3山东分离株（SD0835）实验感染豚鼠后可引起明显的临床症状，为BPIV3基因C型豚鼠实验感染模型的深入研究奠定了基础。
Bovine parainfluenza virus type3(BPIV3) is recognized as one of the most important of the knownviral respiratory pathogens of both young and adult cattle and has also been associated with bovinerespiratory disease complex (BRDC) development in feedlot cattle. To date, genotype A（BPIV3a）,genotype B（BPIV3b）and genotype C（BPIV3c）have been isolated from cattle. During2008there weremany cattle identified with clinical signs considered to be consistent with BPIV3infection. Four BPIV3strains were isolated from four of63bovine nasal swabs collected from Shandong Province. In order tofurther genotyping of the four Shandong isolates, the complete genome for one strain which namedSD0835was determined. The sequence alignment and phylogenetic analysis implicated that the fourShandong BPIV3isolates were distinct from the previously reported genotype A and genotype B ofBPIV3and may represent a new genotype, which was tentatively classified as genotype C (BPIV3c). Todate, the pathogenesis of the genotypes B and C of BPIV3infection in calves and laboratory animalshave not been reported. And only limited studies on the pathogenesis of the genotype A of BPIV3wereconducted and reported.In order to ascertain the pathogenesis of the genotype C of BPIV3SD0835isolate from Shandong Province, animal experimental infections were conducted and the virusreplication, virus excretion period, and pathological changes in animals were evaluated. The studydemonstrated that the BPIV3strain SD0835were pathogenic to Balb/c mice of5to6weeks old andalbino guinea pigs. The pathogenesis of SD0835was characterized by virus isolation and titration,real-time fluorescence quantitative RT-PCR, immunofluorescence staining, and immunohistochemicalstaining. Virus replications in Balb/c mice had occurred in the respiratory tissues as early as24h afterintranasal inoculation and the viral RNA copies of lung were higher than trachea tissues. The results ofimmunofluorescent staining and IHC implicated that the virus replication in lungs and tracheas andlocalization of viral antigens was seen in the cytoplasms of peribronchovascular cells in lungs. Thehistopathologic examinations revealed that alveoli septa thickening and focal cellulose pneumonia wereseen in the lungs of experimentally infected mice. Serologic tests showed the antibodies against BPIV3were detected from6to14days PI and the HI titers reached1:80.
With the same methods, the pathogenesis of SD0835to albino guinea pigs was studied. The notedclinical signs are sneezing, nasal discharges in experimentally infected guinea pigs. The virus isolationresults showed that BPIV3replication occurred mainly in lungs and tracheas and the virus excretionlasted until6days post-infection. The results of real-time fluorescence quantitative RT-PCR suggestedthat the highest copies of lungs and tracheas appeared in3days post-infection and then continued todecline, but the viral RNA could be detected from lungs and trachea of guinea pigs during the14daysof observation. The virus replication in lungs and tracheas and localization of viral antigens in thecytoplasms of alveolar septum cells were observed by using immunofluorescent staining and IHC. Thehistopathologic examinations revealed that interstitial pneumonia was the notable histopathological change along with the disease development, and seriflux and cellulose exudation were also seen.Trachea appeared mucosa atrophy and mucosal epithelium exfoliation. The liver appeared vacuolardegeneration. Lymphocytes were obviously reduced in white pulp of spleen. The submandibular lymphnodes showed suppurative lymphadenitis. HI test revealed that the antibodies against BPIV3weredetected at3days post-infection, and the mean titer of HI antibodies reached1:60.
At the same time, a fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR)was established for BPIV3. It is much more sensitive than conventional RT-PCR and could detect theleast template of17.6copies/μl. Using the plasmid was detected for5times and standard curve wasdrawn. The related coefficient was0.9948and amplification efficiency was1.942. These results showedexcellent correlations. At the same time, the intra-and inter-batch repeatability tests were conductedusing the standard plasmid, respectively and the coefficients of variation were less than2.5%. There wasno cross reaction with the other respiratory pathogens in the fluorescence quantitative RT-PCRdetections. The method had a very good specificity. The results of fluorescence quantitative RT-PCRdetections for clinical samples correlated well with those of median tissue culture infective dose(TCID50) measurement. This indicated that this method was suitable for clinical sample detection forBPIV3.
In sum, the aforementioned results indicated that the SD0835of the genotype C was pathogenic toBalb/c mice of5~6weeks old and albino guinea pigs and could infect them experimentally. Virusreplication occurred mainly in lungs and tracheas and induced the histopathologic changes in theexperimentally infected Balb/c mice and albino guinea pigs with SD0835. At the same time, SD0835infections in Balb/c mice and albino guinea pigs could stimulate the host to generate HI antibodies. Thisindicated that Balb/c mice of5~6weeks old and albino guinea pigs were susceptible to the BPIV3strainSD0835. The Balb/c mice and albino guinea pigs experimental infection models would shed light on thegenotype C of BPIV3infection process and pathogenesis. This research indicated that obvious clinicalsigns were observed in experimentally infected guinea pigs with SD0835for the first time and laid afoundation for further studies of the genotype C of BPIV3in guinea pigs infection model.
为了明确新分离的基因C型BPIV3（SD0835）的致病性，本研究设计采用动物试验的方式研究该毒株在动物体内的复制、排毒以及组织病理损伤等情况。通过病毒分离培养、滴度检测、实时荧光定量RT-PCR检测、免疫荧光检测、免疫组化检测，证实了BPIV3（SD0835）株对5~6周龄Balb/c小鼠及豚鼠具有一定的致病性。Balb/c小鼠接毒试验结果表明，接毒24小时后病原体即可从肺及气管中检出，且肺组织中病毒RNA的拷贝数明显高于气管组织。免疫组化及免疫荧光结果进一步证明了病毒在肺及气管组织中的复制，而且主要在肺内小支气管周围细胞的胞浆中复制。组织病理学变化主要以肺泡间隔增宽，局灶性纤维素性肺炎为主。血清学检测结果表明小鼠接毒后第6天开始检测到血凝抑制（HI）抗体持续到14天，且HI抗体最高滴度达1:80。
白化豚鼠的致病性研究表明，接毒后豚鼠出现主要以喷嚏、鼻液增多为主的临诊表现，BPIV3主要在肺脏及气管复制，并通过每日采集鼻拭子确定豚鼠接毒后可通过鼻腔排毒直到第6天，实时荧光定量RT-PCR检测表明接毒后第3天肺及气管组织中病毒RNA拷贝数最高，随后持续下降，但在整个接毒期间病毒RNA在肺和气管组织中均可检出。间接免疫荧光及免疫组化试验结果同时也证明了病原体在肺及气管中的复制，同时表明病原体主要于肺组织中的肺泡间隔细胞的胞浆中复制。组织病理学结果表明，接毒后豚鼠肺组织以严重的间质性肺炎为主，随病程发展同时伴有浆液及纤维素渗出，气管病变较为轻微，仅表现为黏膜层萎缩及上皮脱落；肝脏组织空泡变性，脾脏白髓内淋巴细胞减少；颌下淋巴结呈现化脓性淋巴结炎并伴有浆液析出。HI试验表明豚鼠接毒后3天即可检测到特异性抗体，滴度达1:60。
本实验同时建立了BPIV3的实时荧光定量RT-PCR检测方法，所建立方法可以检出下限为17.6拷贝/μl的病毒含量。利用制备的标准品，每个浓度5个重复进行试验并绘制标准曲线，扩增效率为1.942，相关系数r为0.9948，说明相关性较好；同时应用标准品分别进行批内、批间5次重复性试验，变异系数均小于2.5%说明重复性较好，且与其它呼吸道病原体无交叉反应。应用建立的荧光定量RT-PCR检测临床样本的结果与半数组织细胞感染量（TCID50）的测定结果相符，说明本方法适合于临床样品的检测。
综上所述， BPIV3山东分离株（SD0835）可以实验感染5~6周龄Balb/c小鼠及白化豚鼠，病原可以在两者的肺脏及气管中复制，引起各组织脏器不同的病理损伤，同时可刺激机体产生HI抗体，这说明5~6周龄Balb/c小鼠及白化豚鼠对BPIV3基因C型的山东分离株（SD0835）有易感性。本研究所建立的BPIV3基因C型的山东分离株（SD0835）的Balb/c小鼠及白化豚鼠模型，可进一步用于BPIV3基因C型的感染过程和致病机理的研究。同时，本研究首次证实了BPIV3山东分离株（SD0835）实验感染豚鼠后可引起明显的临床症状，为BPIV3基因C型豚鼠实验感染模型的深入研究奠定了基础。
Bovine parainfluenza virus type3(BPIV3) is recognized as one of the most important of the knownviral respiratory pathogens of both young and adult cattle and has also been associated with bovinerespiratory disease complex (BRDC) development in feedlot cattle. To date, genotype A（BPIV3a）,genotype B（BPIV3b）and genotype C（BPIV3c）have been isolated from cattle. During2008there weremany cattle identified with clinical signs considered to be consistent with BPIV3infection. Four BPIV3strains were isolated from four of63bovine nasal swabs collected from Shandong Province. In order tofurther genotyping of the four Shandong isolates, the complete genome for one strain which namedSD0835was determined. The sequence alignment and phylogenetic analysis implicated that the fourShandong BPIV3isolates were distinct from the previously reported genotype A and genotype B ofBPIV3and may represent a new genotype, which was tentatively classified as genotype C (BPIV3c). Todate, the pathogenesis of the genotypes B and C of BPIV3infection in calves and laboratory animalshave not been reported. And only limited studies on the pathogenesis of the genotype A of BPIV3wereconducted and reported.In order to ascertain the pathogenesis of the genotype C of BPIV3SD0835isolate from Shandong Province, animal experimental infections were conducted and the virusreplication, virus excretion period, and pathological changes in animals were evaluated. The studydemonstrated that the BPIV3strain SD0835were pathogenic to Balb/c mice of5to6weeks old andalbino guinea pigs. The pathogenesis of SD0835was characterized by virus isolation and titration,real-time fluorescence quantitative RT-PCR, immunofluorescence staining, and immunohistochemicalstaining. Virus replications in Balb/c mice had occurred in the respiratory tissues as early as24h afterintranasal inoculation and the viral RNA copies of lung were higher than trachea tissues. The results ofimmunofluorescent staining and IHC implicated that the virus replication in lungs and tracheas andlocalization of viral antigens was seen in the cytoplasms of peribronchovascular cells in lungs. Thehistopathologic examinations revealed that alveoli septa thickening and focal cellulose pneumonia wereseen in the lungs of experimentally infected mice. Serologic tests showed the antibodies against BPIV3were detected from6to14days PI and the HI titers reached1:80.
With the same methods, the pathogenesis of SD0835to albino guinea pigs was studied. The notedclinical signs are sneezing, nasal discharges in experimentally infected guinea pigs. The virus isolationresults showed that BPIV3replication occurred mainly in lungs and tracheas and the virus excretionlasted until6days post-infection. The results of real-time fluorescence quantitative RT-PCR suggestedthat the highest copies of lungs and tracheas appeared in3days post-infection and then continued todecline, but the viral RNA could be detected from lungs and trachea of guinea pigs during the14daysof observation. The virus replication in lungs and tracheas and localization of viral antigens in thecytoplasms of alveolar septum cells were observed by using immunofluorescent staining and IHC. Thehistopathologic examinations revealed that interstitial pneumonia was the notable histopathological change along with the disease development, and seriflux and cellulose exudation were also seen.Trachea appeared mucosa atrophy and mucosal epithelium exfoliation. The liver appeared vacuolardegeneration. Lymphocytes were obviously reduced in white pulp of spleen. The submandibular lymphnodes showed suppurative lymphadenitis. HI test revealed that the antibodies against BPIV3weredetected at3days post-infection, and the mean titer of HI antibodies reached1:60.
At the same time, a fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR)was established for BPIV3. It is much more sensitive than conventional RT-PCR and could detect theleast template of17.6copies/μl. Using the plasmid was detected for5times and standard curve wasdrawn. The related coefficient was0.9948and amplification efficiency was1.942. These results showedexcellent correlations. At the same time, the intra-and inter-batch repeatability tests were conductedusing the standard plasmid, respectively and the coefficients of variation were less than2.5%. There wasno cross reaction with the other respiratory pathogens in the fluorescence quantitative RT-PCRdetections. The method had a very good specificity. The results of fluorescence quantitative RT-PCRdetections for clinical samples correlated well with those of median tissue culture infective dose(TCID50) measurement. This indicated that this method was suitable for clinical sample detection forBPIV3.
In sum, the aforementioned results indicated that the SD0835of the genotype C was pathogenic toBalb/c mice of5~6weeks old and albino guinea pigs and could infect them experimentally. Virusreplication occurred mainly in lungs and tracheas and induced the histopathologic changes in theexperimentally infected Balb/c mice and albino guinea pigs with SD0835. At the same time, SD0835infections in Balb/c mice and albino guinea pigs could stimulate the host to generate HI antibodies. Thisindicated that Balb/c mice of5~6weeks old and albino guinea pigs were susceptible to the BPIV3strainSD0835. The Balb/c mice and albino guinea pigs experimental infection models would shed light on thegenotype C of BPIV3infection process and pathogenesis. This research indicated that obvious clinicalsigns were observed in experimentally infected guinea pigs with SD0835for the first time and laid afoundation for further studies of the genotype C of BPIV3in guinea pigs infection model.
引文
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