牛冠状病毒感染新生犊牛肺上皮细胞模型的建立

Establishment of a Neonatal Calf Lung Epithelial Cell Model Infected with Bovine Coronavirus

[目的]牛冠状病毒(Bovine coronavirus,BCoV)是犊牛腹泻和上呼吸道疾病的主要病原,目前缺少敏感的适合BCoV体外培养的本动物细胞,限制了BCoV致病机制的深入研究。因此,本研究拟从犊牛肺脏组织中分离出牛肺上皮细胞(bovine lung epithelial cell,BLEC)建立BCoV体外感染本动物细胞模型。[方法]采用胰蛋白酶和Ⅰ型胶原蛋白酶两步酶消化法对新生犊牛肺脏组织进行肺上皮细胞初步提取,再利用上皮细胞贴壁时间不同的差异贴壁法纯化BLEC。通过间接免疫荧光试验(immunofluorescence assay,IFA)检测纯化后细胞中标志细胞角蛋白18(cytokeratin 18,CK-18)的表达来鉴定BLEC,并通过PCR方法对细胞进行病原纯净性检测,包括牛轮状病毒(Bovine rotavirus,BRV)、牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)、牛副流感病毒3型(Bovine parainfluenza virus type 3,BPIV3)、BPIV5、牛传染性鼻气管炎(Infectious bovine rhinotracheitis virus,IBRV)、BCoV和支原体(Mycoplasma)。通过PCR和IFA检测BCoV对BLEC的易感情况,并绘制BCoV在BLEC上的一步生长曲线。[结果]分离纯化培养的BLEC形态均匀稳定、呈菱形或砖块状,无常见病原BRV、BVDV、BPIV3、BPIV5、IBRV、BCoV和支原体污染,上皮细胞标志CK-18呈阳性表达。犊牛腹泻来源的BCoV HLJ-325毒株感染BLEC 24 h后可观察到明显的细胞病变效应(cytopathic effect,CPE);PCR能扩增出BCoV N基因;IFA结果显示,BCoV感染BLEC呈现特异性红色荧光。通过实时荧光定量PCR检测BCoV含量建立病毒生长曲线,BCoV在感染BLEC后20 h时病毒载量最高,为4.46×10^(5)拷贝/mL。[结论]本研究成功分离制备了BLEC,证明BCoV易感染BLEC,构建了BCoV体外感染BLEC模型,为研究BCoV引起牛呼吸道疾病的致病机制奠定基础。

[Objective]Bovine coronavirus(BCoV)was the main pathogen of calf diarrhea and upper respiratory tract disease.Currently,there was a lack of sensitive animal cells suitable for in vitro culture of BCoV,which limited in-depth research on the pathogenesis of BCoV.Therefore,it was planned to isolate bovine lung epithelial cells(BLEC)from calf lung tissue to establish a BCoV in vitro infection model of this animal cell.[Method]A two-step enzymatic digestion method of trypsin and typeⅠcollagenase was used to initially extract BLEC from newborn calf lung tissue,and then the differential attachment method of epithelial cells with different attachment time was used to purify BLEC.BLECs were identified by detecting the expression of marker cytokeratin 18(CK-18)in purified cells using indirect immunofluorescence assay(IFA),and the pathogenic purity of the cells was detected by RT-PCR for Bovine rotavirus(BRV),Bovine viral diarrhea virus(BVDV),Bovine parainfluenza virus type 3(BPIV3),BPIV5,Infectious bovine rhinotracheitis virus(IBRV),BCoV and Mycoplasma.The susceptibility of BCoV to BLEC was detected by RT-PCR and IFA,and the one-step growth curve of BCoV on BLEC was drawn.[Result]The BLEC cultured from purification were uniform and stable in shape,diamond-shaped or brick-shaped.There was no contamination by common pathogens such as BRV,BVDV,BPIV3,BPIV5,IBRV,BCoV and Mycoplasma,and the epithelial cell marker CK-18 was positively expressed.Obvious cytopathic effect(CPE)could be observed 24 h after infection of BLEC with the BCoV HLJ-325 strain derived from calf diarrhea.PCR could amplify the BCoV N gene,the IFA results showed that BCoV-infected BLEC showed specific red fluorescence.The BCoV content was detected by Real-time quantitative PCR to establish the virus growth curve.The viral load of BCoV was the highest at 20 h after infection with BLEC,which was 4.46×105 copies/mL.[Conclusion]This study successfully prepared BLEC,proved that BCoV was susceptible to infecting BLEC,and constructed a BCoV in vitro infection model of BLEC,laying the foundation for studying the pathogenic mechanism of bovine respiratory diseases caused by BCoV.