Isolation And Identification Of Paramyxovirus From Wild Waterfowl And The Mechanism Of V Protein Antagonizing Innate Immunity Of Host Cell

Newcastle disease (ND), caused by Newcastle disease virus (NDV), is one of the most serious infectious diseases in poultry and even result in 100% morbidity and mortality. ND can cause substantial economic losses in poultry industry because of worldwide distribution. Previous molecular epidemiology studies about Newcastle disease virus focused primarily on poultry and waterfowl, wild birds which are considered reservoirs of NDV, always are ignored in spreading the viruses. As the reservoirs of lots of etiologies, wild birds during the immigration can transmit a lot of infectious diseases, and then makes the diseases erupted and popular. Therefore, it is necessary to process molecular epidemiology of common etiologies. In order to know the status of infection of NDV in wild birds, we collected 1002 samples of wild birds in Xianghai natural reserve in Jilin province from October of 2011 to May of 2013 for NDV molecular epidemiology survey. One side, we investigate the status of NDV existence and conduct the isolation and identifying NDVs. We also selected the representative isolates for sequencing the complete genome and molecular genetic characteristic analysis. On the other hand, we firstly isolated APMV6 in mainland of China, and sequenced the complete genome.1. PCR detection of faece swabs of wild birdsThe swabs were conducted to be sterile according conventional procedures. We extracted the virus RNA from the allantoic fluid as the template of specific PCR detection for NDV and other paramyxoviruses. The results showed that the infection of NDV and APMV6 was existent in wild birds. The rate of NDV positive samples was 4.19%, and that of APMV6 was 0.01%. The rate of NDV positive samples was not seasonal, however, it is obviously different between different years. In 2011, we detected the most number of NDV, the rate is 14.02%.2. Isolation and identifying of NDV from wild birds and phylogenetic analysis of FgeneThe allantoic fluid which was NDV-positive was inoculated in CEF cells for propagation, and then the virus was purified by plaque technique.42 NDV isolates were obtained by specific PCR、HA and HI tests. We got the partial F gene sequences of 42 isolates and alignment and analysis with representative strains from GenBank were conducted. The results showed that all the 42 isolates were lentogenic. Three isolates clustered in genotype 2 class I, while the other 39 isolates clustered in genotype Ⅰ class Ⅱ. The nucleotide sequence identities of the partial F genes of the 3 classⅠ isolates ranging from 97.5% to 98.9%; the phylogenetic analysis showed the 3 classⅠ isolates were closely related to poultry-derived isolates in south of China. The 39 classⅡ isolates had mucleotide sequence identities ranging from 96.4-100%. According to the phylogenetic analysis, NEFU1301, NEFU1302, NEFU1102, NEFU1104 and NEFU1113 had high sequence homology with each other, and they were closely related to the isolates from FarEast region in Russia and Japan, and also to the strains from the south of China. Another 34 classⅡ isolates were clustered into an independent subgroup in genotype Ⅰ. All 42 classⅡ isolates were far from vaccine strains phylogeneticly. The results showed that infection of NDV was existent in wild birds in China, and all the isolates were avirulence showing that virulent-virus infection was not widely existent in wild birds. The NDVs may have exchangs in FarEast region, Japan and the eastern coastline of China. The complete genes of NEFU1106 and NEFU1136 were sequenced and the GenBank numbers were KF361507 and KC894391, respectively. The study not only enriches the epidemiology data of NDV of Northeastern China, but also alerts us to pay more attention to the role of wild birds in NDV spread. It built the basement for the spread, heredity and evolution of NDV.3. Isolation and sequencing of APMV6We identified 2 APMV6 strains by specific PCR and electron microscope. The isolated strains were named JL190 and JL127, respectively. The complete genomes of JL190 and JL127 were obtained by PCR using 16 pairs of specific primers. The APMV6 genome is 16236nt with GenBand accession number were JX522537 and KF267717. The genome construction of APMV6 is 3’leader-NP-P-M-F-SH-HN-L-5’Trailer. The phylogenetic analysis showed that JL127 is more closely related to the TW and FE than HK strain based on the F and HN gene.4. The mechanism of V protein antagonizing innate immune of host cellsThe study constructed the combine plasmids expressing the V protein of NDV and APMV6, respectively. The 293 T cells were cotransfected with combine plasmids and report gene plasmids, after SeV stimulating, the firely luciferase and Renilla luciferase activities were determined. The V protein of NDV remarkably inhibited beta interferon production by decreasing the promotor activity. The V protein of virulent NDV inhibited the PRD III/I promoter much more than that of avirulent NDV. The assay using chimeric protein with N-terminal of V protein of avirulent NDV and C-terminal of V protein of virulent NDV showed that the C-terminal of virulent NDV made important effect on the inhibition. The V protein of APMV6 remarkably inhibited beta interferon production by decreasing the NF-κB activity.