Objective In China, 24 cases of human infection with highly pathogenic avian influenza(HPAI) H5 N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5 N6 candi...Objective In China, 24 cases of human infection with highly pathogenic avian influenza(HPAI) H5 N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5 N6 candidate vaccine viruses(CVVs).Methods In accordance with the World Health Organization(WHO) recommendations, we constructed two reassortant viruses using reverse genetics(RG) technology to match the two different epidemic H5 N6 viruses. We performed complete genome sequencing to determine the genetic stability. We assessed the growth ability of the studied viruses in MDCK cells and conducted a hemagglutination inhibition assay to analyze their antigenicity. Pathogenicity attenuation was also evaluated in vitro and in vivo.Results The results showed that no mutations occurred in hemagglutinin or neuraminidase, and both CVVs retained their original antigenicity. The replication capacity of the two CVVs reached a level similar to that of A/Puerto Rico/8/34 in MDCK cells. The two CVVs showed low pathogenicity in vitro and in vivo, which are in line with the WHO requirements for CVVs.Conclusion We obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics,which may aid in our preparedness for a potential H5N6 pandemic.展开更多
[ Objective] This paper aimed to investigate the origin, characteristics and molecular evolution of duck derived H4N6 subtype avian influ- enza virus （DK/SH/Y20/06） and enrich the epidemiologic data of the waterfowl...[ Objective] This paper aimed to investigate the origin, characteristics and molecular evolution of duck derived H4N6 subtype avian influ- enza virus （DK/SH/Y20/06） and enrich the epidemiologic data of the waterfowl origin AIV. [Method] The entire genome of DK/SH/Y20/06 was amplified and subjected to genome sequencing. The molecular software was used for sequence analysis and phylogenetic tree construction of DK/ SH/Y20/06 with some other reference sequences in GenBank. [Result] The results indicated that the amino acid sequence adjacent to HA cleav- age site was PEKASR ↓ GLF, which was the typical characteristics of the LPAIV. The phylogenetic analysis indicated that the HA gene of the isolate was derived from the Eurasian lineage in the eastern hemisphere. The NA gene was at the same branch with A/rnallard/Yan chen/2005（ H4N6）, sharing 98.3% sequence identity. The PB2, PB1, NP and PA gene of this isolate had genetically close relationships with H6 subtype AIV which is epidemic in China at present. The M gene fell into the same branch with A/environment/Korea/CSM05/2004（ H3N1 ）. The NS segment had the highest similarity with A/wild duck/Korea/YS44/2004（H1N2）. The eight genes were not at the same branch and shared a low similarity with other H4N6 subtype avian influenza viruses isolated in North America. [Condusion] These data showed that DK/SH/Y20/06（H4N6） was possibly a re- combinant virus derived from H4N6 subtype, H6N2, H6N5, H3N1 and H1 N2 subtype AIV by complex gene recombination in duck.展开更多
To study the immune effect of recombinant avian influenza virus H5 subtype bivalent inactivated vaccine （ HSN1, Re-6 strain ＋ Re-4 strain） and to provide the basis for formulating reasonable immune procedure of avi...To study the immune effect of recombinant avian influenza virus H5 subtype bivalent inactivated vaccine （ HSN1, Re-6 strain ＋ Re-4 strain） and to provide the basis for formulating reasonable immune procedure of avian influenza vaccine in clinical practice. A total of 12 batches of vaccines from three companies were used for the iannune of SPF chickens and SPF ducks. Each chicken or duck serum was separately collected every 3 weeks until the immunization up to the 24^th week. The serum antibody titers of Re-6 and Re-4 were detected. The results showed that the HI titers of the inoculated SPF chickens and SPF ducks roached the peak when the immune time were the 6^th and 3^rd week after the first immunization respectively; then the titer decreased gradually as time prolonged; the highest titer of SPF chickens was greater than that of SPF ducks; the high titer duration of SPF chickens were longer than that of SPF ducks ; and all the vaccines from the three companies showed a good immune effect.展开更多
A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses;however, there is little data to indicate which of these vectors will b...A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses;however, there is little data to indicate which of these vectors will be the most useful and practical for DNA vaccines in different species. This report examines the use of five expression vectors with different promoters and Kozak sequence to express the same hemagglutinin (HA) protein of an H6N2 avian influenza virus for DNA vaccination in chickens. Although intramuscular vaccination with seven DNA constructs elicited no or limited measurable H6 HA antibody responses in Hy-Line chickens, variable reduction in virus shedding for either oropharyngeal or cloacal swabs post-virus challenge were observed. This indicated that all DNA constructs generated some levels of protective immunity against homologous virus challenge. Interestingly, lower dose (50 or 100 μg) of plasmid DNAs consistently induced better immune response than higher dose (300 or 500 μg). In the transfection experiments there appeared to be a hierarchy in the in vitro expression efficiency in the order of pCAG-optiHAk/ pCAG-HAk > pCI-HAk > VR-HA > pCI-HA > pCI-neo-HA > pVAX-HA. Since the level of in vitro expression correlates with the level of immune response in vivo, in vitro expression levels of the DNA constructs can be used as an indicator for pre-selection of plasmid vaccines prior to in vivo assessment. Moreover, our results suggested that the Kozak sequence could be used as an effective tool for DNA vaccine design.展开更多
基金This study was supported by the National Major Science and Technology Project for Control and Prevention of Major Infectious Diseases in China[No.2018ZX10711001,2018ZX10305409-004-002]Emergency Prevention and Control Project of Ministry of Science and Technology of China[No.10600100000015001206].
文摘Objective In China, 24 cases of human infection with highly pathogenic avian influenza(HPAI) H5 N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5 N6 candidate vaccine viruses(CVVs).Methods In accordance with the World Health Organization(WHO) recommendations, we constructed two reassortant viruses using reverse genetics(RG) technology to match the two different epidemic H5 N6 viruses. We performed complete genome sequencing to determine the genetic stability. We assessed the growth ability of the studied viruses in MDCK cells and conducted a hemagglutination inhibition assay to analyze their antigenicity. Pathogenicity attenuation was also evaluated in vitro and in vivo.Results The results showed that no mutations occurred in hemagglutinin or neuraminidase, and both CVVs retained their original antigenicity. The replication capacity of the two CVVs reached a level similar to that of A/Puerto Rico/8/34 in MDCK cells. The two CVVs showed low pathogenicity in vitro and in vivo, which are in line with the WHO requirements for CVVs.Conclusion We obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics,which may aid in our preparedness for a potential H5N6 pandemic.
基金supported by Science and Technology Commission of Shanghai Municipality Biomedicine Major Projects(09DZ1906602)
文摘[ Objective] This paper aimed to investigate the origin, characteristics and molecular evolution of duck derived H4N6 subtype avian influ- enza virus （DK/SH/Y20/06） and enrich the epidemiologic data of the waterfowl origin AIV. [Method] The entire genome of DK/SH/Y20/06 was amplified and subjected to genome sequencing. The molecular software was used for sequence analysis and phylogenetic tree construction of DK/ SH/Y20/06 with some other reference sequences in GenBank. [Result] The results indicated that the amino acid sequence adjacent to HA cleav- age site was PEKASR ↓ GLF, which was the typical characteristics of the LPAIV. The phylogenetic analysis indicated that the HA gene of the isolate was derived from the Eurasian lineage in the eastern hemisphere. The NA gene was at the same branch with A/rnallard/Yan chen/2005（ H4N6）, sharing 98.3% sequence identity. The PB2, PB1, NP and PA gene of this isolate had genetically close relationships with H6 subtype AIV which is epidemic in China at present. The M gene fell into the same branch with A/environment/Korea/CSM05/2004（ H3N1 ）. The NS segment had the highest similarity with A/wild duck/Korea/YS44/2004（H1N2）. The eight genes were not at the same branch and shared a low similarity with other H4N6 subtype avian influenza viruses isolated in North America. [Condusion] These data showed that DK/SH/Y20/06（H4N6） was possibly a re- combinant virus derived from H4N6 subtype, H6N2, H6N5, H3N1 and H1 N2 subtype AIV by complex gene recombination in duck.
基金Supported by Shandong Winning Project of Quality Monitoring of Avian Influenza Vaccine
文摘To study the immune effect of recombinant avian influenza virus H5 subtype bivalent inactivated vaccine （ HSN1, Re-6 strain ＋ Re-4 strain） and to provide the basis for formulating reasonable immune procedure of avian influenza vaccine in clinical practice. A total of 12 batches of vaccines from three companies were used for the iannune of SPF chickens and SPF ducks. Each chicken or duck serum was separately collected every 3 weeks until the immunization up to the 24^th week. The serum antibody titers of Re-6 and Re-4 were detected. The results showed that the HI titers of the inoculated SPF chickens and SPF ducks roached the peak when the immune time were the 6^th and 3^rd week after the first immunization respectively; then the titer decreased gradually as time prolonged; the highest titer of SPF chickens was greater than that of SPF ducks; the high titer duration of SPF chickens were longer than that of SPF ducks ; and all the vaccines from the three companies showed a good immune effect.
文摘A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses;however, there is little data to indicate which of these vectors will be the most useful and practical for DNA vaccines in different species. This report examines the use of five expression vectors with different promoters and Kozak sequence to express the same hemagglutinin (HA) protein of an H6N2 avian influenza virus for DNA vaccination in chickens. Although intramuscular vaccination with seven DNA constructs elicited no or limited measurable H6 HA antibody responses in Hy-Line chickens, variable reduction in virus shedding for either oropharyngeal or cloacal swabs post-virus challenge were observed. This indicated that all DNA constructs generated some levels of protective immunity against homologous virus challenge. Interestingly, lower dose (50 or 100 μg) of plasmid DNAs consistently induced better immune response than higher dose (300 or 500 μg). In the transfection experiments there appeared to be a hierarchy in the in vitro expression efficiency in the order of pCAG-optiHAk/ pCAG-HAk > pCI-HAk > VR-HA > pCI-HA > pCI-neo-HA > pVAX-HA. Since the level of in vitro expression correlates with the level of immune response in vivo, in vitro expression levels of the DNA constructs can be used as an indicator for pre-selection of plasmid vaccines prior to in vivo assessment. Moreover, our results suggested that the Kozak sequence could be used as an effective tool for DNA vaccine design.
