Isolation And Identification Of Newcastle Disease Virus In Jiangsu Province And Screening Of Host Cell Proteins Interacting Viral W Protein

Newcastle disease(ND)is a highly contagious disease caused by virulent strains of Newcastle disease virus(NDV)that infects poultry,characterized by damage to the digestive and respiratory systems.The disease has a high incidence and mortality rate,causing significant losses to the global poultry industry.In recent years,with the improvement of ND prevention and control measures,there has been no major outbreak of ND.However,atypical and immune carrier cases are still prevalent in China,and outbreaks may occur in cases of vaccine failure.This study aimed to isolate and identify suspected ND samples from the Jiangsu region from2019 to 2022,perform biological assays such as HA,HI,MDT,and ICPI,analyze the genetic distance,evolutionary trends,and key amino acid mutations of the isolated strains and reference strains from China and abroad,and timely grasp the molecular epidemiological trends of NDV in Jiangsu,providing data reference for ND prevention and control in China.In this study,8 strains were isolated of Newcastle disease virus(NDV)from 36 clinical samples,including 4 chicken-derived strains(JS1838,JS11,A31,MH13),3 pigeon-derived strains(WZ2201,WZ2205,VI-HZ),and 1 wild bird-derived strain(2074).MDT and ICPI values showed that MH13 was a highly virulent strain,VI-HZ,WZ2201,and WZ2205 were moderately virulent strains,and JS11,JS1838,A31,and 2074 were weakly virulent strains.Full genome sequences of the isolates were obtained by PCR amplification,RACE amplification,and sequence assembly,and three different lengths of 15186 nt,15192 nt,and 15198 nt were obtained.Based on the analysis of whole-genome homology,strain 2074 showed 92.70%homology with the Class I reference strain Duck/China/NDV08-004/2004.The homology between the 7 isolates ranged from 76.39% to 99.53%.F protein cleavage site sequence analysis showed that JS11,JS1838,and A31 had weakly virulent molecular characteristics,while MH13,VI-HZ,WZ2201,WZ2205,and 2074 had highly virulent molecular characteristics.Genetic evolution analysis showed that 2074 belonged to the evolutionary branch of the 1.1.2 subtype of Class I strains,while the remaining 7 isolates belonged to the Class II strain group,with JS11 and JS1838 belonging to the 1.2 subtype of genotype I,A31 belonging to genotype II,and MH13 belonging to genotype III.The other 3 isolates(VI-HZ,WZ2201,WZ2205)all belonged to genotype VI,with VI-HZ classified as subtype VI.2.2.2 and WZ2201 and WZ2205 classified as subtype VI.2.1.1.2.2.N-glycosylation site prediction results showed that all isolates contained6 potential glycosylation sites on F protein,and only 2074 isolates had 5 potential glycosylation sites on HN protein.When analyzing amino acid sequence,it was found that some amino acid site mutations appeared in F protein sequence.HN protein also showed partial amino acid site mutationsDuring the analysis of the genome of several Newcastle disease virus(NDV)isolates,it was unexpectedly discovered that the W gene of 8 isolates existed in 4 different lengths(465 bp,540 bp,551 bp,and 684 bp).Further comparison with a large database revealed that there were20 different lengths of the NDV W gene(408 bp,414 bp,429 bp,444 bp,456 bp,468 bp,477 bp,504 bp,534 bp,540 bp,546 bp,552 bp,558 bp,564 bp,591 bp,612 bp,624 bp,666 bp,681 bp,and 684 bp).The 552 bp length was found to be predominant in Class I strains,while the 684 bp length was most common in Class II strains,especially in isolates of genotypes VI and VII,which accounted for 34.2% and 25.2%.In order to investigate the function of W protein,we constructed a recombinant eukaryotic expression plasmid of W gene.After transfecting DF-1 cells with the recombinant plasmid for48 hours and collecting the samples,we used immunoprecipitation,SDS-PAGE gel strip recovery,protein digestion,and tandem mass spectrometry to identify 227 peptide segments and77 host proteins that interact with W protein,excluding non-specific proteins from the blank control group.GO analysis showed that these host proteins were mainly enriched in cellular processing(41)and protein/DNA binding(46).KEGG analysis showed that these host proteins were mainly associated with signal pathways such as ribosomes(8),metabolic pathways(6),and RNA transport(5).PPI network analysis showed that 40 host proteins formed an interaction network centered on actin,ribosome-related proteins,and nuclear import-related proteins,suggesting that W protein may be involved in transcriptional translation,transcriptional regulation,and nuclear transport activities.Based on the above research results and literature reports,this study selected three host proteins(YWHAZ,ANXA2,VIM)for interaction verification,constructed recombinant eukaryotic expression plasmids for the three host proteins,and co-transfected them with the W gene eukaryotic expression plasmid into HEK-293 T cells.After 48 hours,the cells were collected for Co-IP verification,and it was finally determined that the W protein has a direct interaction with the YWHAZ protein,but not with ANXA2 and VIM.It is speculated that the W protein may affect host response and virus replication through YWHAZ activity.