Epidemic Investigation And Genetic Evolutionary Analysis Of Newcastle Disease Virus In Wild Birds In China From 2013 To 2022

Newcastle disease（ND）is a highly contagious infectious disease in birds caused by the Newcastle disease virus（NDV）and is classified as a notifiable infectious disease by the World Organisation for Animal Health（WOAH）.At least 241 species of birds in 27 orders have been reported to be infected with NDV,and the disease has become the main pathogen in wild birds worldwide.The dynamic migration of migratory birds provides conditions for the spread of NDV.In order to investigate the prevalence and spread of ND in wild birds in China,this study carried out continuous monitoring of NDV in wild birds in 17 provinces in China,analyzed the epidemic characteristics,genetic diversity and evolution of Newcastle disease in migratory birds passing through China and wild birds residing in important habitats,and established a technology to rapidly obtain the full sequence of NDV.The specific research is as follow:1.Molecular epidemiological investigation of NDV in wild birds in China.In order to study the distribution and genotypic diversity of NDV in wild birds in China,active early warning monitoring and epidemic surveillance of migratory birds in the eastern,central and western parts of China were carried out for up to 10 years.From 2013 to 2022,a total of 113017 wild bird samples were collected and 67 NDV strains were isolated in active early warning monitoring,with rates of isolation of 2013（0.21%）,2014（0.23%）,2015（0.04%）,2016（0.05%）,2017（0.03%）,2018（0.03%）,2019（0.00%）,2020（0.06%）,2021（0.01%）,2022（0.00%）.The host species included 15 species belonging to 5 orders,Stork,Anseriformes,Steroidales,Ploveriformes and crane.It was the first time that NDV was detected in Great Bittern,Red-crowned,Pelagic Cormorant and Terek Sandpiper.In the passive monitoring of dead wild birds,a total of 2838 dead wild bird tissues were collected,which excluded the death of wild birds caused by NDV infection,and confirmed that there has been no outbreak of wild birds caused by NDV in wild birds in China in recent years.2.The establishment of NDV sequence determination and analysis method by high-throughput sequencing technologyIn recent years,high-throughput sequencing based on metagenomics has become more and more widely used in the detection and identification of pathogens/new pathogens,and this study applied high-throughput sequencing technology to the determination and rapid confirmation of NDV whole sequence in wild birds,and successfully established a rapid determination of NDV whole gene sequence and rapid diagnosis method of Newcastle disease virus in wild birds through optimization.At the same time,in order to achieve the rapid analysis of NDV sequences,a local database of 400 NDV whole gene sequences containing all epidemic strains and 42 branches was established,and the data analysis library can meet the rapid evolutionary comparison analysis of the currently circulating strong/weak strains and vaccine strains without relying on the network.We selected 4 NDV isolates to verify the method,and the results showed that22G of data could be obtained in 36 hours through nucleic acid extraction,library establishment,and Next Seq1000/2000 machine sequencing.After removing the host information,the STD data were spliced according to the group,and the proportion of s1,s2,s3 and s4 obtaining the full length of the NDV genome was 98.30%,99.99%,99.99%and 99.99%,respectively,of which s2,s3 and s4 obtained all the coding region sequences of the 6 internal fragments of NDV,and s1 obtained most of the coding regions of the 6 internal fragments of NDV.The high-throughput sequencing technology method for detecting NDV established by this institute can be used to quickly and efficiently confirm the diagnosis of NDV,accelerate the determination of whole gene sequences and the comparison speed of evolutionary analysis,and greatly shorten the detection and analysis time.This method can be used for rapid diagnosis of Newcastle disease,gene sequencing,and identification of multiple genotypes of mixed infection.3.Genetic evolution analysis of NDV in wild birdsA combination of first-generation sequencing and second-generation sequencing was used to determine and analyze the whole gene sequence of the obtained Newcastle disease isolates.67 Newcastle disease isolates were grouped according to collection time,location and host type,and 26 were selected as representative strains for analysis,and the motif analysis of the lysis site of fusion protein of all strains was¹¹²G/E-K/R-Q-G/E-R↓L¹¹⁷,all of which showed the molecular characteristics of NDV weakening.Genetic distance analysis and genetic evolution analysis showed that among the 26 virus strains,14 NDV strains belonged to Class I genotype1.1.2,6 NDV strains belonged to Class I genotype1.2,1 NDV strain belonged to Class II genotype I.1.1,1 NDV strain belonged to Class II genotype I.1.2.1,and 4 NDV strains belonged to Class II genotype I.2.The 14 plants are Class I genotype1.1.2 and form two branches,of which J70,J71,S229 and SH548 are common evolutionary ancestors,and JL31,ZH1626,ZH1647,ZH1603,ZH1648,ZH1935,ZH1610,ZH1605,ZH1665,ZH16181 are common evolutionary ancestors.The six Class I genotype1.2 subgenotypes formed two branches,of which J97,J100 and J192 were common evolutionary ancestors,and A159,CZ418 and SH136 were common evolutionary ancestors.One strain was a Class II genotype I.1.2.1 subgenotype.The four strains were Class II genotype I.1.2 subgenotypes,forming two branches,of which S264 and S267 had a common ancestor,and I48 and I51 had a common ancestor.It was found that Class I genotype1.1.2 NDV may be a subgenotype unique to wild bird populations in China,and no studies have found the occurrence of this subtype NDV in wild birds in other regions.Class I genotype1.2,Class II genotype I.1.2.1,Class II genotype I.2 NDV found in the survey have high homology with NDV prevalent in wild bird flocks in Russia,and it is speculated that NDV was introduced into China from Russia through migratory bird migration,which further confirmed that wild bird migration promoted the transcontinental transmission of Newcastle disease virus.In summary,this study carried out a detailed survey of the prevalence of NDV in wild birds in China,analyzed the epidemic trend and subtype diversity of Newcastle disease in wild birds in China,and found that multiple genotypes of NDV Class I and Class II weakly virulent types continued to circulate in wild birds in China,and there were two epidemic modes of foreign introduction and domestic self-circulation. This study is of great significance for assessing the pathogenic ecology and public health risk of NDV in migratory birds.