Isolation And Identification Of Pigeon Paramyxovirus Type ? Genetic Variation Analysis And Immunogenicity

Pigeon paramyxovirus type ?(PPMV-1)infections,also known as pigeon Newcastle disease(ND)which mainly caused by virulent strains of PPMV-1,variant strains of Newcastle disease virus(NDV),is a highly contagious and infectious diseases.It is still one of the most devastating infectious diseases of pigeon industry throughout the world.In recent years,with the rapid development of pigeon industry,more and more outbreaks of the disease in several provinces which caused great economic losses to pigeon industry in some regions of China were seen.Therefore,it is of great importance to learn the epidemic of pigeon ND,genetic variability of PPMV-1,and prevention and control of the disease by carrying out the isolation and identification,study of molecular epidemiology profile and the main biological characteristics of the pathogen.To survey PPMV-1 among pigeon flocks in Guangxi,isolation and identification of PPMV-1 from clinical samples of dead pigeons suspected to be infected with pigeon NDV from different regions in Guangxi Province between 2011 and 2013 were performed by the hemagglutination(HA),hemagglutination-inhibition(HI)assays and reverse-transcriptase polymerase chain reaction(RT-PCR).The virulence of the isolates were also evaluated by mean death time(MDT)and intracerebral pathogenicity index(ICPI)for NDV and median embryo lethal dose(ELD50)of the viruses.The pathogenicity of the representative isolates of PPMV-1 to pigeons and chickens was also evaluated.The results showed that ten PPMV-1 isolates were identificated and named as GXP110094,GXP120001,GXP120012,GXP120019,GXP120022,GXP120505,GXP120029,GXP120031,GXP120034 and GXP130004 respectively.The MDT,ICPI and ELD50 values of the above isolates were 69 h?102h,0?0.25 and 10-8.68/0.1ml?10-7.63/0.1ml respectively.The results of the challenge tests showed that the PPMV-1 strains GXP120012,GXP120019,GXP120022 and GXP120505 had strong pathogenicity to 35-day-old pigeons with the morbility of 100%and the mortality of 60%?100%,however,the four virus had no pathogenicity to 30-day-old chickens.For know more about the molecular epidemiology profile of PPMV-1 strains of Guangxi isolated in recent years,specific PCR primers were designed to amplify the fusion(F)gene and hemagglutinin-neuraminidase(HN)gene of the PPMV-1 strains based on the sequences of PPMV-1 strains in GenBank.The F and HN gene of the above ten PPMV-1 strains and GXP22 strain isolated in 2000 were amplified by RT-PCR,sequenced and analyzed and their genetic relationships were analyzed by performing the alignment and constructing the phylogenetic tree based on the sequences.The results indicated that the length of open reading frame(ORF)of F protein gene is 1662nt and the protein F were predicted to be 553 aa in length for all eleven PPMV-1 isolates and had the same deduced amino acid sequences at the cleavage site with 112R-R-Q-K/R-R-F117,which is characteristic of velogenic NDVs.The length of ORF of HN protein gene is 1716nt and the protein HN were predicted to be 571 aa in length for all eleven PPMV-1 isolates.The F and HN gene among the ten PPMV-1 isolates shared higher than 99.0%nucleotide and amino acid identities.However,The F and HN gene of them shared 85.6%—85.9%and 85.2%—85.3%nucleotide acid identities with reference virulent strain F48E8,and 83.8%—84.1%and 83.2%—83.4%nucleotide acid identities with attenuated vaccine strain LaSota.The ten PPMV-1s isolated in recent years in Guangxi and the PPMV-1 Pigeon/China/SDLC/2011 strain were located the same branch and shared nucleotide acid identities of 99.3%—99.6%and 99.2%-99.4%of the F and HN gene,respectively.However,The ten new PPMV-1 isolates shared relative lower nucleotide acid identities with 95.2%—95.5%,95.4%—95.7%of F gene,and 93.0%—93.2%,93.5%-93.8%of HN gene with the PPMV-1 strain Pi-Ch-LHLJ-110822 from Heilongjiang and Pi-Ch-LGD110947 from Guangdong isolated in the same period respectively.The phylogenetic tree based on the nucleotide sequences of F gene revealed that PPMV-1 isolate GXP22,the ten isolates and reference strain isolated from the same period in China and the strain IT-227-82 isolated from abroad were classified as class ? genotype ?.To study the immunogenicity of the new isolated PPMV-1 and develop an effective vaccine for the prevention and control of ND in pigeons,the representative PPMV-1 isolate GXP120019 and attenuated NDV LaSota strain were used to create an inactivated oil emulsion(IOE)ND vaccine.Pigeons of different groups were vaccinated with the IOE vaccine GXP120019 and the IOE vaccine LaSota respectively.Five weeks after vaccination,serum was collected from each bird for serological analyses and each treatment group was subsequently challenged with GXP 120019 and a virulent NDV GXC1 strain(subgenotype ?d).The results showed that pigeons vaccinated with IOE vaccines GXP120019 could resist against the challenges of reference strain NDV GXC1 and PPMV-1 isolate GXP 120019,the protection rates were 100%and 100%;pigeons vaccinated with IOE vaccines LaSota could also resist against the challenges of the GXC1 and GXP120019,however the protection rates were 90%and 100%,respectively.This shows that IOE vaccines GXP120019 could completely protect vaccinated pigeons from the infection of the genotype VI PPMV-1 strain and subgenotype ?d virulent NDV strain which is the main genotype of NDV isolated from infected chicken flocks.