Whole Genome Sequencing And Etiological Analysis Of Pigeon Praramyxovirus Type ?

Pigeon paramyxoviruses-1(PPMV-1)is a genotype VI NDV of Newcastle Disease Virus(NDV).It is widely distributed in the world and causes infection in birds and poultry.Untill now,surveillance and research on the virus and its related diseasesis insufficient.And the evolution information and control measures for PPMV-1 need to further develop.The current research will be carried out to analyze the pathogenesis and genetic diversity of PPMV-1,as well as develop diagnostic methods and inactivated vaccine for the disease.The first we investigate the presence of the PPMV-1 in organs and tissues of wild birds and the pathogenesis of the virus.IHC was performed to detect the PPMV-1 in 48 formalin fixed paraffin embedding(FFPE)samplescollected from 14 Eurasian collard-pigeon(ECDO)and 3 rock doves(ROPI)that died from the infection in the United States(US)between 2010 and 2016.At the same time,the pathogenic mechanism of the PPMV-1 on wild bird was studied.The results showed that PPMV-1 was detected in multiple organs and tissues of the 17 infected wildbirds.The highest positive incidence was foundin kidney,spleen and liver,as92.9%,92.9%,83.3% for samples from ECDO,and 66.7%,33.3% and 66.7% forsamples from ROPI,respectively.Acute to subacute interstitial nephritis and necrotizing pancreatitis,50.0% and 66.7% in ECDO and 100% in ROPI,were the most common hispathological changes in wild bird infected with PPMV-1.The widely distribution of the pathogen and hispathological damage to the infected organs are suggested to be the major cause of lethality in wild birds.Secondly,we performed whole genome sequencing PPMV-1 for the 48 American wild bird-origin FFPE samples and 18 Chinese pigeon samples by using the next Generation Sequencing technology(NGS).This would help us to develop a rapid diagnostic technique for PPMV-1in FFPE samples and allantoic fluid collected from infected birds,as well as to provide data for building a new system for NDV classification and facilitate the analysis of genetic of PPMV-1.And at the same time,the sensitivity and accuracy of NGS to detect PPMV-1 was compared to IHC and RT-PCR to demonstrate the feasibility to use NGS to detect the virus in FFPE samples and allantoic fluids samples.The results showed that 79.3% of the IHC-positive samples were found positive in NGS,while 100% of the RT-PCR-positive samples were positive in NGS.Virus from 23 wild FFPE samples and allantoic fluid samples were sequenced yielded a total of 2,624,877 and 60,883,067 NDV reads,representing 99% to 100% coverage of NDV genome.Deduced amino acid analysis of the F protein revealed that the predicted cleavage site of all viruses was at(113RQKR?F117),which is typical for viruses that are virulent to chickens.Phylogenetic analysis showed thatall the 23 NDV from FFPE samples was classified as VI.2.1.1.1 subtype NDV,which was belong to VI.2.1.1.1(VI?a)and VI.2.1.1.1(VI?n)genotype,and the mean genetic distance was 4.2%.The distribution of these viruses from the North American lineages match the distribution of the Eurasian collared-doves and rock pigeons in the U.S.And all the 18 NDV from allantoic fluid were classified as VI.2.1.1.2.1(VI?k)and VI.2.1.1.2.2(VI?j),with the meangenetic distance of 5.3%.The viruses of these sub-genotypes have been independently maintained and continuously evolved for over 20 years,and differ significantly from those causing outbreaks worldwide duringthe 1980 s to 2010 s.The viral reservoir remains unknown and possibilities of the viruses being maintained in both pigeon farms and wild bird populations are viable.The information of 41 F fusion gene sequences obtained in this study has been incorporated into the pilot data for the guidelines of construction of phylogenetic tree and NDV classification system in 2019.Thirdly,we investigate the pathogenicity of the PPMV-1in different hosts as of its genetic diversity and the differences between PPMV-1and current vaccine virus.The viral replication,hispathological changes and transmission patterns PPMV-1was examined in pigeon in Guangxi,China.And the antigenicity and hydrophobicity of F and HN genes in PPMV-1and Class II NDV were also compared for better understanding of the pathogenic characteristic of the virus.The results showed that the mean deathtime(MDT)was and the intracerebral pathogenicity index(ICPI)of 1-day-oldchicks infected with PPMV-1 isolates from Guangxi was 62-114 h and 0.00-0.63,indicating that all of the PPMV-1 isolates belong to mesogenic or lentogenic NDV strains.Significant lesionsin the kidney,pancreas and brain were observed in pigeons died of PPMV-1 infection.Interestingly,we also observed significantly infections in the above organs in chicken,but no clinical symptom was observed.The viral titers and antibody level in pigeons after PPMV-1 infection were significantly higher than those in infected chickens(P < 0.01).The R values of serum cross-inhibition test of all PPMV-1s and La Sotawerelower than 0.8.At the same time,analysis of antigenicity and hydrophobicity of the F and HN genes found mutations in PPMV-1 compared to the La Sota.These results indicate a slightly antigenic difference between La Sota and PPMV-1 isolated from pigeons in Guangxi,which might explain the failure of full protection for pigeons by vaccine derived from standard viral strain,like La Sota.And as subclinical infections usually seen PPMV-1 infected chicken,circulation of this virus might be possible in chicken and pose a risk for the poultry industry.In summary,this study established the NGS method for detection of PPMV-1 in FFPE and allantoic fluid samples based on genome sequencing.Moreover,the phylogenetic and antigenicity of the virus was analyzed and the whole genome sequence of PPMV-1 from wild birds and pigeons was obtained in this study and providing pilot data for construction of new classification system of NDV.These data will facilitate PPMV-1 epidemiology studies,vaccine development,and control of the Newcastle disease in pigeons and poultry.