Evaluation Of A Attenuated Live Vaccine Strain HPAIV H7N9/AAca In Mammals Model And Lethal Synergy Between H7N9 Virus And Infected Bacteria

Avian influenza A(H7N9)cause severe disease even death in contracting patients.Since H7N9 virus emerged in China in 2013,it has circulated in our country.And highly pathogenic avain influenza H7N9(HPAI H7N9)virus was isolated in 2017,which obtained an insertion of four amino acids in their HA cleavage site and become highly lethal in chicken.Meanwhile,the human cases of HPAI H7N9 virus were diagnosed in China.Compare with low pathogenic avain influenza H7N9(LPAI H7N9)virus,the mortality of HP AI H7N9 patients is increased Bacterial second infection was diagnosed in part of H7N9 patients,and researchers believed that bacteria might contributed to the adverse clinical outcome.But the research of bacteria-H7N9 virus co-infection in human patients is elimated.Current events have suggested to us that control and eradication spread of H7N9 influenza viruses is necessary.And it is imperative for study pathogenic and lethal synergistic between bacteria and H7N9 influenza viruses and provide reference for the treatment program of H7N9 cases.This thesis contains following parts of studiesFirst,the protection effect of a H7N9/AAca live attenuated vaccine against replication of H7N9 viruses in mammals was evaluated.The H7N9/AAca vaccinie was generated by using reverse genetics,which contains HA and NA genes from H7N9 A/Anhui/1/2013 virus(AH/1),and the backbone of the cold-adapted influenza H2N2 A/AnnArbor/6/60(AAca).In this study,we evaluated the protective efficacy H7N9/AAca against two heterologous HPAI H7N9 viruses(CK/S1220 and CK/SD008-PB2/627K)and one LPAI H7N9 virus(CK/SD057)in mice model The SPF level six-week-old BALB/c mice were anesthetized and vaccinated intranasally with one does or two doses(three weeks apart)of 106 EID50 H7N9/AAca vaccine.On day 21 post-vaccination,sera were collected for antibody detection.At the same time,vaccinated mice and naive mice were anesthetized and inoculated with with 100 X MLD50 CK/S 1220 virus,100 X MLD50 CK/SD008-PB2/627K virus or 100 X MID50 CK/SD057 virus.Nasal turbinates,lungs,spleens,kidneys,and brains were collected form three mice in each group for virus titration in chicken eggs for virus titration;the remaining seven mice in each group were observed daily for body weight change and death for a total of two weeks.As results shown,the mean antibody titers against H7N9/AAca,CK/S1220,CK/SD008-PB2/627K and CK/SD057 were 65,13,20,and 17 respectively after one does vaccination.And one dose of the H7N9/AAca vaccine prevented disease and death in mice challenged with 100 X MLD50 of CK/S1220 and CK/SD008-PB2/627K viruses,but did not prevent replication of the challenge viruses,and prevent the replication of 100 X MID50 of CK/SD057 virus in mice.After two doses of H7N9/AAca vaccinated,the mean antibody titers were increasd to 320,110,160,and 140.And the mice were completely protected from challenge CK/S1220 virus,and very low viral titers were detected in mice challenged with H7N9 virus CK/SD008-PB2/627K.So,one does of H7N9/AAca could efficiently protect the mice challenged by HPAI H7N9 viurses,and two does of H7N9/AAca could completely prevent or dramatically decrease(P<0.001)the replication of HPAI H7N9 viursesSecondly,the protect effect of the H7N9/AAca vaccine on the transmission of CK/SD008-PB2/627K virus via respiratory droplets was evaluated.SPF level guinea pigs were anesthetized and vaccinated intranasally with 106 EID50 H7N9/AAca vaccine.At the day 21 post-vaccination,immunated guinea pigs were used in transmission test.There were two different transmission groups:In one group,vaccinated guinea pigs were inoculated with 106 EID50 CK/SD008-PB2/627K and naive guinea pigs were exposed next to them;in another group,vaccinated guinea pigs were exposed next to CK/SD008-PB2/627K-inculated guinea pigs.Nasal washes form all guinea pigs were collected for virus titration in chicken eggs.If the virus was detected in nasal wash,the animal was judged to be infected.The result shows that high virus titers were detected in nasal washes of CK/SD008-PB2/627K-inoculated guinea pigs with and without vaccination,and exposed animals in both groups were not infected.So,one dose of H7N9/AAca could efficiently prevent transmission of CK/SD008-PB2/627K via respiratory droplet in guinea pigsFinally,the lethal synergistic between bacteria and H7N9 virus in mice model was studied The mice were anesthetized and inoculated with 106 EID50 PG/S1421 or 106 EID50 AH/1 virus PG/S1421 virus can not cause any loss of body weight and death in mice,but AH/1 virus killed all mice within 9 days.The inoculated mice were killed on day 7-8 post inoculation and collected the lower respiratory tract aseptically.The bacteria,invacding in the low respiratory tract,were counted by cultured on solid medium and identified by the sequence 16 SrRNA.The result shows that an abundance of bacteria invaded the lower respiratory tract of AH/1-inoculated mice,but not GP/S1421-inoculated mice,and five different bacteria were isolated from AH/1-inoculated mice,including Staphylococcus,Enter obacter,Cronobacter,Escherichia,Strepotococcus.Then mice were inoculated with sublethal dose of AH/1(104 EID50)and 108 CFU five different bacteriaes respectively.As results shown,in groups of Staphylococcus-AH/1 and Enterobacter-AH/1,there were four of five mice died;and the mice co-inculated with Strepotococcus and AH/1 were all death.So,the co-infection with AH/1 virus and bacteria(Staphylococcus,Enterobacter and Strepotococcus)produced pathogenic and lethal synergistic action,and Strepotococcus presented most severity effect.In addition,when mice challenged with Strepotococcus on 3 days before inoculated with AH/1,two of five mice died and the remaining mice recovered their body weigth;while mice challenged with Strepotococcus on 3 days after inoculated with AH/1,the mice lost body weight fastly and all died.Demonstrated that only when AH/1 virus challenge mice before or together with Strepotococcus it showed lethal synergisticIn summary,our study suggests that H7N9/AAca has the potential to be an effective H7N9 vaccine and should be evaluated in humans,because this vaccine could efficiently protect mammals against HPAI H7N9 visurses and new LPAI H7N9 virus.More than that,the treatment program of H7N9 cases should consider more about nonpathogenic bacteria,especially Strepotococcus species,and the antibiotic therapy should be used as soon as possible once cases are suspected as H7N9 virus infection.