Analysis Of Genetic Variation Of IBV In Laying Hens In Hebei Province And Preliminary Study On Anti-IBV Of Chinese Herbs

This study conducted genetic evolution analysis of the S1 gene in IBV isolates from Hebei region,as well as in vitro resistance tests of Chinese herbal medicine to IBV,in order to determine the genotype,genetic evolution,and recombination of IBV prevalent in Hebei region,and to explore the mechanism of Chinese herbal medicine’s resistance to IBV in vitro.The research content and results include the following four parts:1.Isolation and identification of IBV in Hebei region118 suspected IBV infected samples from livestock farms in Hebei region were inoculated into the allantoic cavity of chicken embryos,PCR identification after 5repetitions of subculture.And inoculate the positive diseased materials into chicken embryos and chicks,observe the pathological changes in the chick embryos and the incidence of disease in the chicks.11 local strains of IBV were obtained based on comprehensive PCR identification,chicken embryo lesion status,and chick pathogenicity test results.2.Genetic Evolution Analysis of IBV in Hebei RegionPurify the target band of S1 gene and construct a plasmid.The plasmid that is confirmed to be positive will be sent for sequencing.53 reference strains were selected from Gen Bank,and DNAStar software was used to analyze the nucleotide and amino acid sequence homology of the S1 gene of the Hebei strain.The Neighbor Joining method in MEGA software was used to construct a genetic evolution tree of the S1 gene.RDP4 and Sim Plot software were used to analyze the recombination events of the Hebei strain.Online software Yin OYang-1.2 and Net NGlyc-1.0 were used to analyze the O-and N-glycosylation sites,respectively,DNAstar software was used to analyze the S1 protein cleavage sites of IBV isolates.The results showed that the S1 gene has a total length of 1611 bp to 1621 bp,encoding approximately 540 amino acids.The nucleotide sequence homology of the S1 gene in 11 isolated strains ranged from 92.8% to 99.9%;The homology between 11 isolated strains and 53 reference strains at home and abroad is 73.0%～96.7%,and the homology with commonly used vaccine strains M41,H120,W93,Ma5,and QX in China is74.7%～96.1%.The homology between the S1 gene amino acid sequences of 11 isolated strains is 92.4%～99.6%,and the homology with the S1 gene amino acid sequences of 53 domestic and foreign IBV reference strains is 68.9%～94.9%.Among them,the homology with the S1 gene sequence of the domestic GI-19 genotype reference strain is 88.0%～94.9%,and the homology with commonly used vaccine strains in China is 70.9%～92.4%.Genetic evolution analysis shows that 11 isolated strains belong to the same branch as the GI-19 genotype(QX type)IBV.Gene recombination showed that the isolated strains HB.SJZ.21 and HB.XT.1 were two recombinant viruses,and the HB.SJZ.21 strain may be composed of HB.XT.9strain and HB.SJZ.8 strains were recombined,while HB.XT.1 strain may have been recombined from HB.SJZ.8 strain and a Chilean isolate.The results showed that the IBV epidemic strain in Hebei region is the GI-19 genotype,with varying degrees of variation compared to the vaccine strain,and there is recombination phenomenon between isolated strains.The S1 protein cleavage sites of 11 IBV isolates are consistent,and the N-linked and O-linked glycosylation sites of most isolates exhibit some conservatism.3.In vitro screening experiment of anti IBV Chinese herbal medicineUsing the traditional water decoction method to extract 18 Chinese herbal medicines,the maximum safe concentration of 18 Chinese herbal medicines and ribavirin on CEK cells was determined using CPE observation and CCK-8 detection methods,and effective Chinese herbal medicines against IBV in vitro were preliminarily screened.The results showed that the maximum safe concentration range of 18 traditional Chinese medicines was between 0.0781mg/m L and 10mg/m L;The TCID50 of IBV is 10-4.67/0.1m L.Astragalus membranaceus,Radix Isatidis,licorice,dandelion,and honeysuckle have the best antiviral effect,with an effective inhibition rate of over 80%.Ribavirin,scallion whiskers,houttuynia cordata,cauliflower,pomegranate peel,and Rhizoma menispermi have an effective inhibition rate of over 75%.Three types of Chinese herbal medicines are used to act on viruses in order to determine the best antiviral pathway for Chinese herbal medicine.Among the direct killing methods,dandelion,licorice,astragalus membranaceus,and soybean root have better killing effects on the virus,with effective inhibition rates of 81.53%,75.85%,75.14%,and 75.12%,respectively;Among the modes of adsorption blocking,licorice,isatis root,and forsythia suspensa have stronger adsorption blocking effects on viruses,with effective inhibition rates for IBV being 83.60%,83.30%,and 77.92%,respectively.Compared with ribavirin,the effective inhibition rates have increased by7.86%,7.56%,and 2.18%,respectively;In the mode of replication blocking effect,the inhibitory rates of Astragalus membranaceus,Houttuynia cordata,Stephania japonica,Lonicera japonica,and Ribavirin on IBV were 83.59%,79.91%,75.47%,75.11%,and 63.45%,respectively.The effective inhibitory rates of Ribavirin on IBV were lower than those of 15 other Chinese herbal medicines except for Echinacea purpurea.4.Preliminary Study on the Mechanism of Chinese Herbal Medicine Against IBV in VitroChinese herbal medicine was selected to replicate and block the action of IBV on CEK cells,and fluorescence quantitative PCR technology was used to detect the relative m RNA expression of related factors in CEK cells.Compared with the virus control group,Forsythia suspensa,Allium multiflorum,and Lonicera japonica can significantly increase IFN-α and IFN-β m RNA expression level in CEK after IBV infection through the MDA5 signaling pathway.Other drugs except Astragalus membranaceus significantly upregulated the m RNA level of MAVS.Houttuynia cordata,Pomegranate peel,Cyrtomium fortune,Stephania,Radix isatidis,Astragalus membranaceus,Rhodiola,Scutellaria baicalensis,Beidou root,Licorice,throughing NF-κB signaling pathway significantly reduces the m RNA expression level of IL-6and TNF-α.In summary,11 strains of GI-19 type IBV were successfully isolated from the allantoic cavity of chicken embryos.Among 18 Chinese herbal medicines,16 were selected to have a certain inhibitory effect on IBV.The best way to detect the anti IBV effect of most Chinese herbal medicines through three modes of action is through replication blockade.Chinese herbal medicine can significantly increase IFN-α and IFN-β m RNA expression level in CEK after IBV infection through the MDA5 signaling pathway.Except for Astragalus membranaceus,all other drug test groups significantly upregulated the m RNA level of MAVS.Chinese herbal medicine through NF-κB signaling pathway significantly reduces the m RNA expression level of IL-6 and TNF-α.