Isolation And Identification Of A Duck-derived Strain Of APMV-4 And Its Pathogenicity To Chickens

APMVs can infect birds worldwide,causing symptoms such as dyspnea,diarrhea and reduced egg production,which seriously affects the poultry industry.APMV-1,also known as NDV,is the most harmful of all the serotypes of avian paramyxoviruses.APMV-4 mainly infects wild birds of Anseriformes,and has been rarely studied due to its low pathogenicity in domestic poultry.However,studies on heterogenous NDV passing through the host-specific barrier and causing pathogenicity in poultry have been reported many times.Therefore,understanding the pathogenic mechanism of wild bird-derived APMV-4 and its potential pathogenicity in chickens is important for the prevention and control of avian paramyxovirus disease.1.The throat swabs of wild ducks in the Yanqi,Xinjiang were collected and amplified through the allantoic cavity of chicken embryo.The strain was identified as APMV-4 and named APMV-4/CH(XJ)/Aythya nyroca/2020 by HA-HI test and PCR.By cloning,sequencing and gene characterization analysis of the F and HN genes of the isolate,the sequence of the F protein cleavage site was ¹¹⁶DIQPR?F¹²¹,which was similar to that of non-toxic NDV;The HN protein encoded 565 amino acids,and the sequence of the sialic acid binding site was ²²⁹NRKSCS²³⁴.In order to understand the proliferation of this isolate on cells,CEF were isolated from 10 d SPF chicken embryos,virus titers in the supernatant of CEF cells were measured by HA assay every 12 hours after infection,draw step growth curve of the virus the results showed that APMV-4 isolates in CEF cells proliferation,but did not form syncytial,virus proliferation in 72 h after the infection peak,hemagglutination titer of about 8.5 log₂.2.The APMV-4/Aythya nyroca/CH(XJ)/2020 isolates of 200?L 2⁸HAU were inoculated into 7d SPF chickens by intranasal eye and air sac inoculation.After 24 h,each group was added to the same age SPF chickens as a cohabitation infection group,and the clinical symptoms,pathological changes,viral shedding,antibody conversion,cohabiting infection ability and tissue affinity of each group were detected.The results showed that the chicks showed clinical symptoms regardless of the infection mode,and the symptoms were especially obvious from 3 to 9 days after infection.Pathological sections and viral load of each tissue showed that the virus was mainly concentrated in lung and trachea,with obvious pathological changes,and there was no significant difference between different groups.The virus was shed through the chicken mouth and cloaca.The amount of virus discharged through the mouth was larger than that in the cloaca,and reached the peak on the 5 dpi,and the antibody potency of the chickens in the air sac infection group reached 4 log₂ on the 5dpi,and reached the peaked at the 14 dpi,and was significantly higher than that of the two groups of co-habiting chickens.3.In this study,the c DNA of APMV-4/CH(XJ)/Aythya nyroca/2020 isolate was used as a template to amplify the full-length ORFs of N,P and L genes,and cloned into the eukaryotic expression vector pc DNA3.1(-).The L gene was amplified by subsection,and the intermediate plasmid pc DNA3.1(-)-L1 was constructed first,and then the final pc DNA3.1(-)-L recombinant plasmid was constructed by using double enzyme digestion,and the three constructed recombinant plasmids were transfected into BHK-21 cells respectively.Finally,the recombinant plasmid that could express APMV-4 N,P proteins and the eukaryotic expression plasmid pc DNA3.1(-)-L was successfully constructed as demonstrated by RT-PCR,double enzyme digestion and Western Blot.In conclusion,a duck-derived APMV-4 strain was successfully isolated in this study and its virulence gene characteristics were analyzed;infected chickens with this isolate by different routes to investigate the potential pathogenicity of duck-derived APMV-4 in chickens;and successfully constructed three recombinant plasmids capable of expressing APMV-4 N,P proteins.This study laid the foundation for the prevention and control of avian paramyxovirus and the construction of a reverse genetic operating system for APMV-4.