The Mechanism Of The Distinct Pathogenicity Between Genotype ?d And Genotype ? NDV In Geese

Newcastle Disease(ND),caused by Newcastle disease virus(NDV),is a highly contagious infectious disease for poultry.Traditionally,geese are usually considered to be resistant even to the most virulent strains of NDV for chickens.However,NDV outbreaks in goose flocks have been frequently reported in China since 1997,which have caused serious loss for waterfowl industry.After several years,it has been confirmed that genotype ?d NDV is responsible for the ND outbreaks in goose flocks,which indicates that compared with the early classical virulent NDVs,genotype VII NDV is able to cross the host barrier to infect waterfowl and shows strong pathogenicity.However,most pathogenesis studies of NDV are focused on chickens,and the studies of NDV pathogenicity in waterfowl are relatively limited.In addition,the mechanism of strong pathogenicity to waterfowl of genotype ?d NDV compared to to strains of early genotype is unclear.Previously,our team has identified that the M,F and HN genes of the virus are crucial to the pathogenicity of genotype VII NDV to chickens.In this study,to elucidate the potential mechanism of the high pathogenicity of genotype ?d NDV in geese,four strains,including Herts/33 of early genotype(IV),JS5/05 of late genotype(VII)and two recombinant viruses based on M+F+HN exchange between Herts/33 and JS5/05(JS-MFHNH and Her-MFHNJ),were chosen.Their biological characteristics(virus titer,syncytia formation,virulence),pathogenicity to goose,virus replication and cytokine response,inflammatory mediator production in infected lymphocytes were compared.1.Comparison of biological characteristics of genotype ?d and IV NDV in vitroIn this study,Herts/33,JS5/05 and two recombinant viruses(JS-MFHNH and Her-MFHNJ)were chosen as model viruses.First of all,the biological characteristics and virulence index(ICPI)of the four strains were determined.The results showed that the titers(HA,EID50 and TCID50)of the four strains were similar and all of them were virulent based in ICPI values.Secondly,infection experiment in goose embryo fibroblast(GEF)and goose spleen lymphocytes were conducted,and virus replication and syncytia formation in GEF,growth curve,apoptosis and inflammatory mediators in lymphocytes were tested.The results showed that the replication of each strain was similar in GEF but different in lymphocytes.The virus titer of the recombinant virus Her-MFHNJ expressing M+F+HN of JS5/05 was about 10 times higher than its parental virus Herts/33.The titer of the recombinant virus JS-MFHNH harboring M+F+HN of Herts/33 was about 3 times lower than its parental virus JS5/05.In addition,apoptosis induced by JS5/05 is higher than that induced by Herts/33 in splenocytes.As for the recombinant viruses,apoptosis induced by JS-MFHNH was lower than that of JS5/05,and the proportion of apoptotic cells induced by Her-MFHNJ was significantly higher than that induced by Herts/33.In addition,Her-MFHNJ also induced a significant increase in the level of nitric oxide and reactive oxygen in lymphocytes compared to Herts/33,similar with JS5/05.These results indicated that there was no significant difference in virulence,virus titer,replication ability in GEF between NDV of genotype VII and IV and two recombinant viruses,whereas virus replication,levels of apoptosis and inflammatory mediators in lymphocytes infected with JS5/05 and Her-MFHNJ were stronger than Herts/33 and JS-MFHNH.These findings suggest that the pathogenicity of genotype ?d NDV in geese may be related to virus replication efficiency in lymphocytes and the extent of virus-induced apoptosis and inflammatory mediators.2.Comparison of pathogenicity of genotype ?d and IV NDV in geeseFor geese challenge study,Herts/33,JS5/05,JS-MFHNH and Her-MFHNJ were chosen as the model viruses.The clinical symptoms and gross lesions of geese infected by different strains were compared.The results showed that when the challenge dose was 108 ELD50,geese infected with JS5/05 and Her-MFHNJ exhibited onset of symptoms from day 3 post infection(pi),whereas geese infected by Herts/33 and JS-MFHNH started to show disease symptoms from day 5 pi.The main symptoms of geese were similar,including nose mucus,dyspnea,diarrhea,twisted head and neck and paralysis in the late stage.Geese infected with JS5/05 and Her-MFHNJ showed more serious disease signs than those infected with Herts/33 and JS-MFHNH,but only one of Her-MFHNJ-infected geese died at day 7 pi.Pathological examination showed that among geese infected with JS5/05 and Her-MFHNJ,from day 3 pi,necrosis appeared in the spleen and pancreas,hyperaemia and hemorrhage in the throat,small intestine,spleen and thymus.The lesions of geese infected with Herts/33 and JS-MFHNH appeared late,and only hemorrhage and swelling in small intestinal mucosa and thymus were observed.Histologic examination demonstrated atht genotype VII NDV and the recombinant virus harboring JS5/05 M+F+HN genes caused more severe lymphocyte necrosis in the spleen,thymus and bursa in geese compared to genotype IV NDV.These pathological findings suggest that genotype VII NDV produed more severe tissue damage in the immune system of geese.Moreover,viral load of JS5/05 and Her-MFHNJ in the immune organs(spleen,thymus,bursa of Fabricius)was higher than that of geese infected with Herts/33 and JS-MFHNH.The recombinant virus Her-MFHNJ expressing M+F+HN of JS5/05 significantly up-regulated the nitric oxide level compared with the parental virus Herts/33.The expression of representative cytokine genes in the immune organs of infected geese was measured using qRT-PCR,and the results showed that JS5/05 significantly up-regulated expression of most tested cytokine genes in the spleen and thymus compared to Herts/33.As for the recombinant viruses,Her-MFHNJ expressing M+F+HN of JS5/05 significantly up-regulated the expression of IL-1?,IL-2,TNF-a,IFN-? and IL-8 in the spleen and thymus.In contrast,the recombinant virus JS-MFHNH expressing M+F+HN of JS5/05 significantly down-regulated the expression of IL-1?,IL-2,TNF-a,IL-6,IFN-p,IL-8 and IL-18 in the spleen and thymus.In conclusion,these results suggest that replication efficiency,the level of inflammatory mediators and cytokine response in the immune organs infected with genotype VII strain and recombinant virus strain expressing M+F+HN of genotype? were significantly higher than those of genotype IV strain and recombinant virus strain carrying M+F+HN of genotype ?.Summary1.In vitro infection experiments showed that the syncytium formation in GEF,virus replication,induction of apoptosis and inflammatory mediators in goose lymphocytes induced by JS5/05 were higher compared to Herts/33,which are associated with the M,F,HN genes of NDV.2.Goose infection experiment showed that compared with the early genotype IV strain,genotype VII virus is more pathogenic to geese and causes more severe tissue lesions in the immune system,which are related to the significantly higher replication and the intense inflammatory response induced by the virus.These characteristics are determined by the synergy of the M,F,HN genes.3.Taken together,high level of virus replication and the strong inflammatory response contribute to the immunopathology in the immune system of goose caused by genotype VII NDV.The M,F and HN genes act as molecular determinant for these viral phenotypes.