| Avian influenza virus(AIV)and newcastle disease virus(NDV)are two important viruses that seriously threaten the poultry industry.Although many cases of wild poultries co-infected with AIV and NDV have been reported,the effect and mechanism of these two viruses co-infection on their replication is not yet clear.Using indirect immune fluorescent confocal laser technology(IFA),RT-qPCR,western blotting technique(WB)and ELISA test,the expression of virus nucleoprotein(NP)in protein level and virus replication in cellular level were analyzed when AIV and NDV co-infected in chicken embryo fibroblast(DF-1),human non-small cell lung cancer cells(A549)and the african green monkey kidney cells(Vero)in the early infection group(EIG,-6 h),concurrent group(CG,0 h)and post infection group(PIG,+6 h).1.NDV strong strain Herts/33 and AIV attenuated strain H9N2 was used to infect DF-1 cells individually.Obvious cell lesions were observed 12 h after NDV infection,and cells began to shed after 24 h.AIV infections have similar results.Western blot and RT-qPCR showed that NDV and AIV replication were both in the logarithmic phase in 8 h after infection.NDV replication was in the stable period after infection of 16-24 h,while AIV was still in the logarithmic replication stage in 24 h after infection.Combined with the results of cytopathic effect,12 h and 24 h after NDV and AIV infection were selected as the co-infection time.2.WB and IFA analysis in DF-1 cells showed that NDV significantly inhibits AIV replication in EIG(p<0.05),most significantly inhibits AIV replication in PIG(p<0.01)and CG(p<0.01)in 12 h,while NDV most significantly inhibits(p<0.001)AIV replication in the three groups in 24 h.AIV only significantly inhibits NDV replication in EIG both in 12 h and 24 h(p<0.05),but significantly promotes the NDV replication in CG and PIG(p<0.05).In A549 cells,NDV significantly inhibits AIV replication in the three groups in 12 h(p<0.05)and most significantly inhibits AIV replication in 24 h(p<0.001).AIV most significantly inhibits NDV replication in the three groups in 12 h(p<0.001)and in EIG and CG in 24 h(p<0.001)except in PIG.However,there was no significant inhibition in Vero cells(p>0.05).These findings indicates that AIV and NDV co-infection in DF-1 and A549 cells reciprocal inhibits its replication.3.ELISA test indicated that IFN-? in AIV infected A549 cells expressed stably in 600-400 pg/mL from 3 h to 24 h.IFN-? in NDV infected A549 cells expressed highly from 12 h to 24 h with maximum 7758 pg/mL.In co-infection NDV influencing AIV replication the expression of INF-P in the three groups most significantly increased than AIV individual infection group both in 12 h and 24 h(p<0.001).In return,in AIV influencing NDV replication the expression of INF/? in EIG and PIG was significantly inhibited(p<0.05),but significantly increased in CG(p<0.05).These data manifested that IFN-? may be involved in the reciprocal inhibitions of virus replication in co-infection of Aiv and NDV.4-Using JAK signal pathway inhibitor Ruxolitinib(RUX)and Tofacitinib(TOF)block IFN-? production,WB test showed that the expression of AIV NP in CG significantly increased in RUX or TOF and RUX+TOF treatment A549 cells(p<0.05),significantly increased in the TOF treatment(p<0.05),most significantly increased in the RUX and RUX+TOF treatment(p<0.001)in EIG.In PIG the NP expression significantly decreased in RUX and TOF treatment(p<0.05),but significantly increased in RUX +TOF treatment(p<0.05).In return the NDV NP expression in EIG significantly increased in RUX or TOF treatment(p<0.05),most significantly increased in RUX+TOF treatment(p<0.001).In PIG and CG,NDV NP expression significantly inhibited in RUX or TOF treatment(p<0.05),but significantly increased in RUX+TOF treatment(p<0.05).These results further proved that JAK signal pathway play a role in the reciprocal inhibitions of virus replication in co-infection of AIV and NDV. |
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