Establishment of a Neonatal Calf Lung Epithelial Cell Model Infected with Bovine Coronavirus
[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 type Ⅰ collagenase 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.
Bovine coronavirus(BCoV)bovine lung epithelial cellseparation and purificationinfection model
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