Evolution And The Molecular Mechanism Of Host Preference Of Pigeon-origin Newcastle Disease Virus

Newcastle disease(ND),caused by virulent Newcastle disease virus(NDV),is one of the major diseases that seriously endangers the global poultry industry.Pigeon-origin NDV,also known as pigeon paramyxovirus type 1(PPMV-1),is a host variant of chicken-origin NDV that has been adapted to pigeons and has caused huge economic losses to the pigeon industry worldwide.Since its introduction to China in the 1980s,PPMV-1 has long been prevalent in several provinces and regions of China,seriously hindering the development of the pigeon industry.To understand the prevalence of PPMV-1 in pigeon flocks,we characterized 19 PPMV-1 strains from some regions of China during 2016 to 2020 and further determined the biological characteristics of these isolates.Moreover,to elucidate the evolution and transmission dynamics of PPMV-1 in China and provide a theoretical basis for the development of PPMV-1 prevention and control measures,an evolutionary analysis of dynamics of PPMV-1 in China was conducted.It has been shown that pigeon-origin NDV has a high host preference to pigeons,but the molecular mechanisms involved remain unclear.To this end,we systematically compared the differences in the biological characteristics,pathogenicity and transmission between PPMV-1 and chicken-origin NDV,and then further investigated the molecular mechanism of host preference of PPMV-1 by using a reverse genetic technique.1.Molecular epidemiological investigation of pigeon-origin Newcastle disease virus in some regions of China from 2016 to 2020A total of 19 PPMV-1 strains were isolated from diseased pigeons in some regions of China from 2016 to 2020.Phylogenetic analysis of the F gene showed that all isolates belonged to sub-genotype VI.2.1.1.2.2.The nucleotide sequence identities of F and HN genes among the 19 PPMV-1 isolates was 96-99.9%and 96.4-99.9%,respectively.The amino acid sequence identities of F and HN genes among the 19 PPMV-1 isolates was 96.4-99.8%and 96.5-99.8%,respectively.All 19 isolates were located in the same branch with the European strain PPMV-1/Belgium/11-09620/2011,but they were genetically distant from the vaccine strain La Sota.The nucleotide sequence identities of F and HN genes between all isolates and La Sota were 83.4-84.2%and 81.8-84.2%,respectively.The amino acid sequence identities of F and HN genes between all isolates and La Sota were 87.5-88.8%and 86.7-87.4%,respectively.Compared with sub-genotype VI.2.1.1.2.1 viruses,sub-genotype VI.2.1.1.2.2 viruses had specific amino acid changes in the F and HN proteins.The amino acid cleavage site in F proteins of all PPMV-1 isolates contained multiple basic amino acids(112RRQKRF117),which is typical of virulent viruses.However,the ICPI values showed that all PPMV-1 isolates were mesogenic in nature,indicating that in addition to the F protein cleavage site,other genes or structural domains might influence the viral virulence.Notably,this study found that although some PPMV-1 isolates showed the comparable replication ability in cells derived from pigeon and pigeons,there was a significant difference in replication ability of them in cells derived from chicken and chickens,suggesting that PPMV-1 with high replication ability in cells derived from chicken and chickens has the potential to cause severe disease outbreaks in chicken flocks.In summary,this study revealed the current epidemiological situation of PPMV-1 in some regions of China and also laid the foundation for further studies on the evolutionary dynamics of PPMV-1 in China.2.Evolutionary dynamics of pigeon-origin Newcastle disease virus in ChinaTo elucidate the evolutionary dynamics of PPMV-1 in China,a maximum likelihood tree was constructed based on complete F gene sequences of 427 NDV strains.Phylogenetic analysis showed that all 186 PPMV-1 strains isolated from China belonged to genotype VI,with most of them belonging to sub-genotype VI.2.1.1.2.2.Then,the statistic analysis of the host sources revealed that the majority of PPMV-1 strains(178/186)were isolated from pigeons,which further indicated that PPMV-1 has a high host preference to pigeons.The reconstructed population history showed that the population size change of PPMV-1 in China has gone through six different phases.A Bayesian approach was used to analyze the spatiotemporal dynamics of PPMV-1 in China,and the results indicated that East and South China were the epicenters of the dissemination of PPMV-1,mediating the eight migration routes of PPMV-1 and playing a crucial role in the spread of PPMV-1.Adaptive evolutionary analysis showed that positive selection sites existed in all six genes of PPMV-1 in China,with most of the positive selection sites situated within the NP,P and L genes.Amino acid sequence analysis showed that there were specific amino acid substitutions in all six proteins of PPMV-1 compared with chicken-origin NDV,with 23 substitutions situated at NP,P and L proteins and the remaining 9 substitutions located at M,F and HN proteins.The adaptive evolutionary analysis and amino acid sequence analysis suggested that NP,P and L genes might be associated with the host preference of PPMV-1.Overall,this study systematically analyzed the evolutionary dynamics of PPMV-1 in China,providing a theoretical reference for understanding the evolution of PPMV-1 and formulating reasonable prevention and control measures for PPMV-1,as well as providing clues for further in-depth research on host preference of PPMV-1.3.Comparison of pathogenicity and transmission characteristics of Newcastle disease virus isolates from chicken and pigeon respectivelyIn order to identify key entry points for studying the molecular mechanisms of host preference of PPMV-1,this study provided a comprehensive comparison of the differences in biological characteristics,pathogenicity and transmission between PPMV-1(NT-10 and JS/09/16/Pi)and chicken-origin NDV(Kuwait 256).Firstly,the MDT and ICPI values indicated that Kuwait 256 was a velogenic strain,while NT-10 and JS/09/16/Pi were mesogenic strains.Secondly,the analysis of membrane fusion ability,receptor binding characteristics and neuraminidase activity showed that the membrane fusion ability,receptor binding ability and neuraminidase activity of Kuwait 256 were significantly higher than those of NT-10 and J S/09/16/Pi.In addition,the results of growth curve assay confirmed that Kuwait 256 replicated significantly better than NT-10 and JS/09/16/Pi in cells derived from chicken.However,the replication ability of Kuwait 256 was lower than that of NT-10 and JS/09/16/Pi in cells derived from pigeon.Interestingly,there was no significant difference in the replication levels of these three isolates in mammalian cells.Pathogenicity tests indicated that Kuwait 256 caused 100%morbidity and 100%mortality in SPF chickens,while NT-10 and JS/09/16/Pi did not cause significant clinical signs and had low replication levels in SPF chickens.Notably,Kuwait 256,NT-10 and JS/09/16/Pi caused significant morbidity and mortality in pigeons.Finally,the results of the transmission test showed that both NT-10 and JS/09/16/Pi could be effectively transmitted among pigeons,while Kuwait 256 could not be transmitted among pigeons.In summary,these findings suggested that PPMV-1 showed a poor replication ability in chickens but achieved a transmission advantage over chicken-origin NDV after undergoing some adaptive variation in pigeons,laying the basis for further studies on the molecular mechanism of host preference of PPMV-1.4.The molecular mechanism of host preference of pigeon-origin Newcastle disease virusBased on the existing results of this study,we continued to investigate the molecular mechanism of host preference of PPMV-1.In this study,we first constructed infectious clones of Kuwait 256 and NT-10 strains.And then,two recombinant viruses(rNT-KMFHN and rNT-KNPPL)were successfully rescued by using reverse genetics technique.The differences in biological characteristics,pathogenicity and transmission between the two recombinant viruses and their parental viruses were further evaluated.The results showed that rNT-KNPPL obtained higher virulence and replication ability in cells derived from chicken,which were similar to those of rKuwait 256.In addition,the pathogenicity and replication ability of rNT-KNPPL were also significantly stronger than those of rNT-KMFHN and rNT-10 in chickens.However,rNT-KNPPL could not be transmitted among pigeons compared with rNT-KMFHN and rNT-10.Taken together,these findings suggest that NP,P and L genes play a crucial role in host preference of PPMV-1.