Study On The Pathogenicity Of Different Genotypes Of Newcastle Disease Virus To Quail And Differencial Expressions Of TLRs Signaling Pathway Related Factors

Newcastle disease(ND)is a kind of acute and severe infectious disease caused by Newcastle diseases virus(NDV)of the virulent strains.In recent years,ND has become one of the major diseases which cause serious damage to the quail industry.There are reports of disease outbreaks in several provinces in China,which has led to the huge economic losses to the quail industry.According to nucleotide sequences of Toll-like receptor signaling pathways of innate immunity factor-related genes(TLR3,TLR7,IL-6,IL-8,IFN?,AvBD-9)and housekeeping gene(P-actin)of different poultry species published in GenBank,and related reports and literature,7 pairs of specific primers were designed and synthesized.The target fragments of above-mentioned genes were amplified by PCR using the primers,and then were cloned to construct a plasmid standard.Fluorescence quantitative real-time PCR(qRT-PCR)methods were developed and the specificity and reproducibility tests were done.The results showed that the linear correlation coefficients of the established the standard curves of 7 qRT-PCR methods(TLR3,TLR7,IL-6,IL-8,IFNa,AvBD-9 and ?-actin)are in 0.99?1.00;and the amplification efficiency are in 1.83?2.13 and the solubility curves have a single melting peak within 40 cycles.In the repeatability test,the intra-assay and inter-assay coefficient of variation of each target gene is less than 4.0%,and the standard deviation is less than 0.5.It is indicated that the established detection methods have the characteristics of high specificity,stability and repeatability,which provides certain technical support for quantitative detection of the transcription level of mRNA of the innate immune related factors in quail.To study the differences of pathogenicity of different genotypes of NDV,the GXQ53 strain of genotype ?,GXGF12001 strain of genotype ? and Chinese reference strain of genotype IX F48E8 were used to artificially infect 35-day-old non-vaccinated quails.The clinical symptoms and signs score of the experimental animals were observed and recorded.The gross lesions and scores of the spleen,brain,and cecal tonsils of each group were observed and recorded at 5 days post-infection(dpi),8 dpi,and 11 dpi.Tissue samples were collected for histology observation,and detection of viral load and the gene expression of the innate immunne related factors.The results were as follows:(1)A11 the infected groups showed a typical clinical symptoms and postmortem lesions of ND.The scores of clinical symptoms in the F48E8 group,the GXGF 12001 group,and the GXQ53 group were 12.0 points,6.0 points and 6.0 points respectively.The mean scores of the postmortem lesions were 3.0 points,1.67 points,and 1.83 points respectively.The results of histopathology showed that F48E8,GXGF 12001 and GXQ53 strains can lead to different degrees of damage to the spleen,cecum tonsil and brain.The spleen and cecum tonsil all showed the reduction of lymphocytes and the necrotic foci.The lesions of F48E8 and GXGF12001 groups worsened with the increase of infected time,while the lesions of GXQ53 group was gradually reduced.The lesions of brain were mainly presented as microglial hyperplasia,and those of the three groups all had the trend to worsen with the increase of the infected time.In terms of the results of the postmortem tissue sections,F48E8 showed more severe damage to the immune and nervous systems of infected quails than GXGF12001,while the damage of GXQ53 infected group was weaker than the fonner two groups.(2)The results of viral load of the infected groups in the spleen,cecum tonsil and brain indicated that F48E8 and GXGF 12001 strains replicated not only in the tonsils of the spleen and cecal tonsil but also in the brain across the blood-brain barrier,while GXQ53 strain only replicated in the cecal tonsil.The results of the number of virus copies showed that the replication ability of the GXGF12001 strain in the spleen was stronger than that of the F48E8 strain,whereas the ability of the F48E8 strain to replicate in the brain was stronger than that of the GXGF 12001 strain.The viral replication ability to replicate in the cecal tonsils was:GXGF 12001 strain>F48E8 strain>GXQ53 strain.(3)The relative expression levels of innate immune related factors in different tissues after infecting NDV showed that TLR3,TLR7,IL-6,IL-8,IFN?,and AvBD-9 were all expressed in the spleen,brain,and cecal tonsils.Among them,the expression of TLR3,IL-6,and IL-8 in the spleen,TLR7,IL-8,AvBD-9,and IL-6 in cecal tonsils,IFNa and AvBD-9 in brain were all significantly increased(P<0.05),and the relative expression of cytokines in these tissues were positively correlated with the number of virus copies.In summary,we speculated that TLR3,TLR7,IL-6,IL-8,IFNa,AvBD-9 may be involved in the immune response of quail to NDV infection.This study has successfully established qRT-PCR methods for detecting the genes of innate immune-related factors TLR3,TLR7,IL-6,IL-8,IFN? and AvBD-9 and housekeeping gene ?-actin;The results of artificial infection tests showed that the GXQ53 strain of genotype ?,GXGF12001 strain of genotype? and Chinese reference strain of genotype IX F48E8 can replicated in the spleen,cecal tonsil and brain of quails and cause damage to these tissues and organs.Moreover,the pathogenicity of F48E8 strain was higher than that of GXGF 12001 strain,and GXQ53 strain has the lowest pathogenicity.They subsequently can result in quails death.From the results of the cytokine mRNA detection,we speculated that TLR3,TLR7,IL-8 and IFN? may be involved in the immune response of NDV infection in spleed,brain and cecal tonsils.The significantly up-regulated expression level of IL-6 in spleen and cecal tonsils and the significantly up-regulated expression level of AvBD-9 in the brain may be related to TLR7 mediated MyD88 dependent pathway and TLR3 mediated TRIF dependent pathway.