| Newcastle disease(ND)is a serious disease caused by highly virulent Newcastle disease virus(NDV)that poses a significant threat to the poultry industry worldwide.ND was firstly reported in March 1926 on the island of Java,Indonesia,and has caused at least four global pandemics.Currently,the ND has been controlled well in Chinese chicken flocks,but virulent strains of NDV can still be isolated from pigeon and goose flocks.The emergence of new genotypes of NDV in neighboring countries has also put enormous pressure on ND prevention.Therefore,ongoing epidemiological investigations of NDV are of great importance.In this study,the NDVs were isolated and identified from live poultry markets and clinical samples in the East China region from 2020-2022,and a systematic genetic evolution analysis and biological characterization of these NDV strains were conducted in order to provide theoretical data for the scientific prevention and control of ND in China.NDV has a wide host range and can infect most birds,and pigeon-origin NDV is usually considered a variant strain deriving from chicken-origin NDV,posing a significant threat to Chinese pigeon industry.The HN protein is one of the membrane glycoproteins of NDV and plays an important role in mediating virus-receptor recognition,virus budding,and inducing neutralizing antibodies.As previous studies reported,the specific amino acid differences were present at positions 347 and 349 of the HN protein between pigeonand chicken-origin NDVs,but the specific biological significance of the amino acids at these two positions remains unclear.Therefore,in the second chapter of this study,we systematically investigated the effects of mutations at positions 347 and 349 on the pathogenicity,antigenicity,and transmissibility of pigeon-origin NDV.1.Isolation,Identification,and Evolutionary Analysis of NDV in East China Region from 2020 to 2022In order to monitor the trend of NDV(Newcastle Disease Virus)in China,this study collected 2296 samples from live poultry markets and farms for testing between 2020 and 2022.A total of 15 NDV strains were isolated through PCR identification and chicken embryo culture.The host distribution showed that 10 NDV strains were isolated from live poultry market samples,of which 9 were from chickens and 1 was from geese,while 5 NDV strains were isolated from clinical materials,of which 2 were from geese and 3 were from pigeons.Genetic evolution analysis showed that all 10 NDV strains isolated from live poultry markets belonged to Class Ⅰ,including 8 strains of subtype 1b and 2 strains of subtype 1c,with a typical weak cleavage site(112ERQERL117).All 5 NDV strains isolated from clinical materials belonged to ClassⅡ,including 3 strains of subtype Ⅵ.2.1.1.2.2 from pigeons and 2 strains of subtype ⅩⅩfrom geese,with a typical strong cleavage site(112RRQKRF117).The ICPI(intracerebral pathogenicity index)and MDT(mean death time)results showed that all NDV strains isolated from live poultry markets were of weak pathogenicity,while 3 pigeon-derived strains and 2 goose-derived strains isolated from clinical materials were of medium and strong pathogenicity,respectively.The growth kinetics results showed that the replication capacity of goose-derived strains on CEF(chicken embryo fibroblast)was stronger than that of pigeon-derived strains,while the trend was reversed on PEF(pigeon embryo fibroblast).The pigeon-derived strains were grouped with Belgium PPMV-1/Belgium/11-09620/2011,and the goose-derived strains were grouped with quail/South Korea/88-M/514/1988 in Korea.In summary,the results of this study are generally consistent with the current situation of NDV in China,that is,the prevention and control of ND in chickens in China is relatively good,and the prevalent NDV strains are mainly weak strains of Class Ⅰ with a significant decrease in the isolation rate of strong strains of Ⅶ d subtype.However,the prevalence of strong NDV strains in pigeons and geese has increased,with Class Ⅱ Ⅵ and ⅩⅩ subtype strong strains being the predominant strains in pigeons and geese,respectively.2.Study on the Biological Significance of Amino Acids 347 and 349 of HN Protein Previous studies have shown that there are specific amino acid differences at positions 347 and 349 of HN protein between pigeon-origin Newcastle disease virus(NDV)and chicken-origin NDV.Therefore,we performed amino acid site analysis of HN protein for 15 isolates obtained in the first chapter of this study.The results showed that there were specific amino acid differences at positions 347 and 349 of HN protein between pigeon-origin NDV and non-pigeon-origin NDV.To clarify the effect of amino acids 347 and 349 of HN protein on the biological characteristics of pigeon-origin NDV,we first screened a pigeon-origin strain NT-10 with high replication and transmission efficiency in pigeons,and used reverse genetics technology to separately or co-mutate amino acids 347 and 349 of HN protein of NT10 strain into those of non-pigeon-origin NDV.We successfully constructed full-length plasmids and rescued two mutant strains:single mutant strain NT-10 G347E and double mutant strain NT-10 G347E/E349D.The neuraminidase activity and HAd activity of both mutant strains on CEF were higher than that of the parent strain NT-10.Meanwhile,compared with NT-10,the budding ability of progeny virus after infection of CEF was stronger for NT-10 G347E and NT-10 G347E/E349D mutant strains.The MDT of mutant strains was significantly reduced and the ICPI was significantly increased.Cross-neutralization and vaccine protection experiments showed that there were antigenic differences between mutant strains and parent strains.Moreover,the results of vaccine protection experiments showed that only the three viruses were detected in the lungs and trachea,and the virus titers of mutant strains were significantly higher than that of the parent strain.The transmission experiment in chickens showed that the replication and transmission efficiency of mutant strains were higher in chickens,and the replication ability of mutant strains on pulmonary epithelial cells and tracheal epithelial cells was higher than that of the parent strain.In conclusion,this study systematically explored the effect of amino acids 347 and 349 of HN protein on the biological characteristics of pigeon-origin NDV,providing important clues for a deeper understanding of the epidemiology and pathogenic mechanisms of pigeon-origin NDV. |
