Genomic Analysis And Biological Characterizations Of Three Newcastle Disease Viruses Isolated From Wild Birds

Newcastle disease(ND)has been a threat of aviculture worldwide since 1926,meanwhile it can potentially harm the health of wild birds.Since NDV's wide-spread subclinical infection in wild birds,wild birds are known as the natural reservoirs of NDV,therefore,it is crucial to surveil the status of wild birds infecting with NDV to further benefit the prevention and control of ND.In this study,558 feces samples of wild birds were collected during spring,2014,limiting dilution method was used to isolate and purify NDV from these samples,and isolated NDVs were tested by Haemagglutination test and RT-PCR.The result indicated that 17 NDVs were isolated among 558 samples,with a rate of 3%.And then we cloned F genes of 17 isolates into T vector and Sanger sequenced,using alignment tools to analyze their nucleic acid sequences and amino acid sequences,meanwhile these 17 isolates were compared with other domestic and overseas NDVs,the alignment results showed that all the 17 viruses belong to NSV Class ?,10 of which are gene type ?,7 are gene type ?;F genes of gene type?and?are within the same branch of V4 strain and La Sota strain respectively.Using genome of NDV ZJ-1 as reference,11 pairs of primers were used to amplify genomes of 3 NDVs(Wild bird/HN/150/2014,“HN150” in short;Wild bird/JL/TZ70/2014,“TZ70” in short;Wild bird/JL/M50/2014,“M50” in short),and then we compared similarities of nucleic acids and amino acids of 6 coding regions of 3 selected viruses and ZJ-1,to analyze their protein sequence differences of F protein and HN protein.The results of F protein functional region indicated that HN150 and TZ70 are the same with V4 vaccine strain,M50 is the same with La Sota strain.Then we analyzed antigenic sites of HN protein,the results showed that the M50 and La Sota are the same,but 113 aa and 350 aa of HN150 and 70 changed from R to Q and H to Y respectively.Moreover,the genome of each isolate has the highest similarity with reference virus of the same gene type.The results of ICPI,IVPI and MDT indicated that these 3 isolates are low pathogenic to SPF chickens.To further verify their adaptability and replication capacity in chickens,we infected 4 week-old SPF chickens with 106 EID50/100?L,organs,throat swabs and cloacal swabs were collected at 3 d.p.i and 5 d.p.i to detect viral titers.Titration results revealed that 3 NDVs replicated systemically in chickens,but their replication levels in brains were lower than tracheae and intestines;at 3 d.p.i,replication rates of trachea and intestine were faster than the rate in brain,at 5 d.p.i,the replication rates in small intestines of HN150 and M50 were significant higher than their replication rates in brains at 3 d.p.i.3 viruses could replicate in spleen,pancreas and fabricius bursa from the titration result,at 5 d.p.i,the copy numbers were significantly higher than 3 d.p.i.And then cross hemagglutination inhibition test was conducted by using HN150,TZ70,M50 and La Sota to test their antigenic diversity.The results showed that M50 and La Sota had no antigenic diversity,the R value was 1.0;HN150,TZ70 and La Sota had antigenic diversity,the R values were 0.707 and 0.631 respectively.Wild birds,as the reservoir host of NDVs,more attention need to be paid on their transmitting and spreading function of NDV.As for the different isolate places and hosts in this study,11 genotype?and 7 genotype?wild bird NDVs were isolated and showed their diversity.The genome length of 3 isolates were 15186 bp and no inserts and substitutions were found.The nucleotide similarity between TZ70 and HN150 and V4 are 99.0% and 99.4% respectively,the similarity between M50 and La Sota vaccine strain is 99%.The aa analysis of F protein showed that there is no difference either between TZ70 and HN150 and V4,or between M50 and La Sota.Only the 113 th and 350 th aa of TZ70 and HN150 were substituted by Q and Y.All of the 3 isolates can replicate systematically in chicken body,as for their tissue tropism,they prefer respiratory tract and digestive tract to other organs.Because of the distinguish between genotypes,the tissue tropism and replicate ability of genotype?showed significant difference than genotype?.Antigen diversities were found between La Sota and genotype?isolates,but no Antigen diversity was found between genotype?isolate and La Sota.