Co-infection Of NDV And H9N2 Subtype AIV On Viral Proliferation And Pathogenicity

Newcastle disease virus(Newcastlediseasevirus,NDV)is the representative species of paramyxoviridae and paramyxoviridae.Newcastle disease(Newcastledisease,ND)is an acute and highly contagious infectious disease of chickens and many kinds of birds caused by virulent NDV.Birds infected with NDV often show septicemia,which is mainly characterized by dyspnea,dysentery,neurological disorders,mucosal and serous hemorrhage,and its morbidity and mortality are very high.H9N2 subtype avian influenza virus(Avianinfluenzavirus,AIV)belongs to orthomyxoviridae and influenza virus belongs to type An influenza virus,which can cause respiratory symptoms,decrease of laying rate and immunosuppression in poultry,and cause secondary infection of other pathogens in infected birds,resulting in increased morbidity and mortality of poultry.The co-infection of NDV and AIV often occurs in poultry or wild birds,which leads to the spread and pathogenicity of the infected poultry virus.In recent years,studies have only preliminarily discussed the influence of NDV and H9N2 subtype AIV on the mutual interference between the two viruses after co-infection with the host,but the pathogenic mechanism and mutual interference mechanism of the virus have not been clear,and the effect on the pathogenicity of the host has not been reported in depth.In order to understand the effects of NDV and H9N2 subtype AIV on virus proliferation and pathogenicity of SPF chicken embryos,hemagglutination test and hemagglutination inhibition test(HA-HI),RT-PCR,pathogenicity index(ICPI),mean death time of minimum lethal dose(MDT),ELISA,RT-qPCR and tissue sections were used to detect the virus proliferation and the effect of virus on the pathogenicity of chicken embryos.In order to provide new ideas and new directions for clinical prevention and control of AIV co-infection of NDV and H9N2 subtypes.1?Determination of pathogenicity and replication of gene ?d subtype NDV-DQ strainIn order to understand the pathogenicity of gene ?d subtype NDV-DQ strain and the effect on virus proliferation after infection with SPF chicken embryo alone,the virus HA titer and serum HI titer were detected by HA-HI,ICPI,MDT and ELISA methods,the ICPI and MDT of NDV were calculated,and the virus proliferation of chicken embryo allantoic fluid and embryo body collected at 7 periods(3h,6h,12 h,24h,48 h,72h and 96h)was determined at protein level.The results showed that the HA titer of gene ?d subtype NDV-DQ strain was about 8log2,and its hemagglutination ability could be inhibited by NDV positive serum.The ICPI was 1.80 and MDT was 56.6h.According to the identification index of virulent Newcastle disease,the NDV could be judged to be a virulent strain.After NDV infected chicken embryos alone,the increase of NDV virus content in allantoic fluid and embryo body was basically the same,the virus content increased fastest in 24 h?48 h,increased steadily in 24 h?72 h,and reached the highest at 72 h,and the content of NDV virus in allantoic fluid was higher than that in embryo body.2?Determination of pathogenicity and replication of H9N2 subtype AIV-AS strainIn order to understand the pathogenicity of H9N2 subtype AIV-AS strain and the effect on virus proliferation after infection with SPF chicken embryo,the virus HA titer and serum HI titer were determined by HA-HI,MDT and ELISA methods,the MDT of AIV was calculated,and the virus proliferation of chicken embryo allantoic fluid and embryo body at and protein level were collected at 7 periods(3h,6h,12 h,24h,48 h,72h and 96h).The results showed that the average HA titer of H9N2 subtype AIV-AS strain was about 7log2,and its virus hemagglutination could be inhibited by AIV H9 positive serum.The MDT was 74.4 h,which indicated that the AIV strain was attenuated.When AIV was infected with chicken embryo alone,the increase of AIV virus content in allantoic fluid and embryo body was basically the same,the virus content increased fastest in 48 h?72 h,increased steadily in 48h?96 h,and reached the highest at 96 h,and the content of NDV virus in allantoic fluid was higher than that in embryo body.3?Effects of NDV and H9N2 subtype AIV co-infection on virus replication and pathogenicityIn order to understand the effects of gene ?d subtype NDV-DQ virulent strain and H9N2 subtype AIV-AS attenuated strain on virus proliferation and pathogenicity of SPF chicken embryos,NDV(AIV)was designed to infect chicken embryos 12 h,6 h and 3 h in advance,then AIV(NDV),was infected at 0h and set 0h for co-infection group,NDV(AIV)alone infection group and control group.Chicken embryo allantoic fluid and embryo body were collected at 24 h and 48 h after co-infection.The collected samples were tested by RT-PCR,RT-qPCR,ELISA,HA-HI and tissue sections.The results of RT-PCR showed that the specific purpose bands with brightness of 359 b P and 377 bp size could be seen in the collected samples by NDV and AIV nucleic acid RT-PCR amplification.The results of RT-qPCR and ELISA showed that AIV inhibited the proliferation of NDV in chicken embryo allantoic fluid and embryo body,and the degree of inhibition increased with time,NDV promoted the proliferation of AIV in chicken embryo allantoic fluid at 24 h and inhibited the proliferation of AIV at 48 h,while NDV inhibited the proliferation of AIV in embryo body at 24 h and 48 h after co-infection.HA-HI results showed that there was interference between NDV and AIV,and AIV seriously interfered with the proliferation of NDV.After co-infection with NDV and AIV,the whole embryo body was congested and there were bleeding spots in the head,abdomen,back,wings and toes.The observation of pathological tissue section showed that the liver and heart of chicken embryo were injured to varying degrees due to the influence of virus proliferation.To sum up,this study proved that NDV-DQ strain is a virulent strain and H9N2 subtype AIV-AS strain is an attenuated strain.Both viruses can kill chicken embryos and proliferate in chicken embryo allantoic fluid and embryo body,and the virus content in allantoic fluid is higher than that in embryo body.Co-infection of chicken embryos with virulent NDV strain and H9N2 subtype AIV attenuated strain could cause mutual interference of virus proliferation,and the degree of interference was affected by the sequence of infection and infection time.The interference effect of AIV on NDV was more significant than that of NDV on AIV.AIV always inhibited the proliferation of NDV.NDV could promote the proliferation of AIV at 24 hours after co-infection of chicken embryos,and then inhibit the proliferation of AIV.Coinfection of NDV and H9N2 subtype AIV can cause pathogenicity of chicken embryo body,liver and heart.It can cause pathological changes such as liver bleeding,fibrin exudation and cell particle degeneration,and heart tissue hemorrhage and particle degeneration.Co-infection improves the pathogenicity of the virus.The longer the time of co-infection,the more serious the pathological changes of chicken embryo.