Study On The Differences Of Innate Immune Response Between Two Sub-genotypes Of Newcastle Disease Virus In Both Infected Cells And Pigeons

Pigeon Newcastle disease,also known as pigeon plague is an acute infectious disease caused by the Newcastle disease virus(NDV),a variant of the pigeon paramyxovirus(Pigeon Paramyxovirus type 1,PPMV-1).This is one of the major diseases that endanger pigeon industry,and cause great economic losses to the pigeon industry.At present,the outbreak of Newcastle disease in the pigeon is on the rise,and there are few studies on the inherent immune related factors causing pigeon NDV.Therefore,it is very necessary to study the host specific genes sub-genotype VIb and sub-genotype ?d for pigeon NDV from the main genotypes of pigeons inherent immunity level.In order to further understand the pathogenicity of pigeon NDV,it is important to prevent and control pigeon Newcastle disease virus infection.In this study,chicken specific primers for inducible nitric oxide synthase(iNOS),IFN?,IFNy and pigeon specific primers for IFN?,iNOS,MDA5 and IL-2 were designed and synthesized.The standard plasmid was constructed by cloning and purification,and chicken fluorescent quantitative RT-PCR(qRT-PCR)standard curves for inducible nitric oxide synthase(iNOS),IFN?,IFNy and pigeon fluorescent quantitative RT-PCR(qRT-PCR)standard curves for IFNy,iNOS,MDA5 and IL-2 were constructed.The results showed that the amplification efficiency of the standard curve was between 92%?102%.The linear correlation coefficient was greater than or equal to 0.99.After the repeatability,sensitivity and specificity,the results showed that the real-time fluorescence quantitative RT-PCR method established for the above factors in this study was in line with the requirements of the fluorescence quantitative detection,which could be used for further analysis and detection.In order to detect the expression level of immune related factor,mRNA provided technical support.In addition,two sub-genotypes of pigeon NDV,sub-genotype VIb NDV GXP22 and sub-genotype ?d NDV GXP40 were used in this study to infect single-layered chicken embryo fibroblasts(CEF)and pigeon embryo fibroblasts(PEF)at a dose of 100 TCID50,at the same time,negative control group were set up(equivalent DMEM medium was used to process the corresponding cells).After the lh reactivity of virus with cells,the virus solution was aspirated and a cell maintenance solution of 1%bovine calf serum was added.Cells were collected post infection at Oh,6h,12h,24h,and 36h,respectively.Difference in expressions of these 11 innate immune related factors was detected by the same method.The results showed the most up-regulated expression of TLR2,3 in the CEF cells at 12h that gradually decreased TLR4 and TLR7 were detected up-regulated at 12h,but down regulation occurred after 12h.The sub-genotype ?d NDV induced TLR2,3 expression was stronger than that of sub-genotype VIb NDV,and the up fold was higher;Whereas,in the PEF cells,TLR2,3 were up to up expression at 24h,and then decreased gradually.The sub-genotype VIb NDV induced the expression of TLR2,3 stronger than that of the sub-genotype ?d NDV,and the difference of TLR4 and TLR7 expression was not obvious.The expression of MDA5 caused by two sub-genotypes NDV in the detection cycle was detected.In CEF cells,the expression of MDA5 caused by sub-genotype ?d NDV was more than that caused by sub-genotype VIb NDV.But in PEF cells,the expression of MDA5 caused by sub-genotype VIb NDV was more expressed than that caused by sub-genotype ?d NDV.The expression of interferon was similar to the expression of TLR2 and TLR3.In CEF cells,the expression of IL-6 caused by two sub-genotypes NDV was different.The sub-genotype VIb NDV was found with induced peak expression at 12h after infection and then gradually decreased,but sub-genotype ?d NDV induced expression peak post infection at 36h;In PEF cells,sub-genotype ?d NDV induced a stronger expression in post infection 24h(P<0.05).After infection of two sub-genotypes NDV,the expression of iNOS in CEF and PEF was positively related to expression of IL-6,presumably IL-6 may play a role in promoting iNOS expression.At the same time,the non-immune pigeons of 30 days old were artificially infected two sub-genotypes of pigeon NDV,sub-genotype ?b NDV GXP22 and sub-genotype ?d NDV GXP40 by 1×106ELD50 per 0.2ml ocular and intranasal ways.The changes in serum NDV antibody levels were detected at 3,7,10,and 14 days after infection.Five pigeons were killed 3,7 days post infection,the same method was used to detect the differences of the above 11 inherent immune related factors in the spleen,and detect its viral load.Spleen,pancreas and cecum tonsil of the pigeons were stained with hematoxylin-eosin stain.The results showed that two sub-genotypes of pigeon NDV infected non immune pigeons,both could cause the target organs of the pigeon spleen,pancreas,cecum tonsil and other pathological changes were also seen.The viral load in the spleen increased significantly in 7DPI,and the viral load in group GXP40 was higher than that in GXP22 group.After infection,the level of serum antibody could be raised in 8 days after infection,but the difference of antibody level between infected pigeons was not obvious.After sub-genotype VIb GXP22 post infection 3 days,the TLR3,TLR4,TLR7,MDA5,iNOS could be detected obviously up,and up expression 2.632 folds,2.667 folds,7.840 folds,3.391 folds,3.307 folds,respectively.At the time of 7 days infection,the expressions of cytokines TLR3,TLR7 and iNOS were upregulated 43.520 folds,7.231 folds,3.190 folds respectively;After sub-genotype ?d GXP40 post infection 3days,TLR3,TLR4,TLR7,MDA5,iNOS could be detected up 6.302 folds,6.380 folds,16.069 folds,6.853folds,11.531 folds.At the time of 7 days infection,TLR7,IFNa,MDA5 have obvious up expression,up 12.505 folds,4.873 folds,3.830 folds,respectively.The results showed that in the spleen of 3 days and 7days after both sub-genotypes NDV infection,the expression of TLR3,TLR4,TLR7,MDA5,iNOS and other factors could be detected in the two infection group,and the expression of the inherent immune related factors was similar,but the expression of the inherent immune related factors caused by sub-genotype ?d NDV was stronger than that of sub-genotype VIb NDV.The results of this study demonstrated that the established 7 kinds of fowl intrinsic immunofluorescence quantitative detection methods can be used for clinical detection.It is proved that two kinds of NDV can infect CEF and PEF cells and cause the expression of inherent immune related factors,but different sub-genotypes of NDV in vitro infected cells cause the expression of inherent immune related factors.The sub-genotypes VIb and ?d of pigeon source NDV caused the pathogenesis of non-immune pigeons,and lead to the pathological changes of the main target organs and the up-regulated expression of the innate immune related factors of the non-immune pigeons.Both sub-genotype NDV infection caused the expression changes of the immune related factors in the spleen and the antibody changes in the body.There wrer some differences in the results of PEF infection,indicated that other factors in the animal body participated in immunoregulation in the process of NDV infection.