Objective: To know the natural history of the first wave of pandemic influenza A(H1N1)pdm09 in the Southern hemisphere, through the detection of antibodies against influenza A(H1N1)pdm09 in a selected community, to es...Objective: To know the natural history of the first wave of pandemic influenza A(H1N1)pdm09 in the Southern hemisphere, through the detection of antibodies against influenza A(H1N1)pdm09 in a selected community, to estimate the population attack rate and its variations, the consultation rates, hospitallization and mortality rates. Methodology: A representative random sample of the population of a commune in Chile (San Felipe) was interviewed and taken blood samples between January and March 2010. A study against the antibodies of the influenza A(H1N1) pdm09 virus was conducted, by the technique of the Hemaglutination Inhibition (HAI) according to standardized methodology. Subjects with antibody titers ≥1:40 were considered positive. Results: 13.5% of the population of San Felipe had antibodies against influenza A(H1N1)pdm09;this percentage reached 30% of the population between 0 and 18 years and 6.1% among those over 19 years. The age variable was the only factor that evidenced significant differences in the prevalence of antibodies. There were no significant differences related to gender, vaccination history against seasonal inluenza, or comorbidity. 51% of people with positive serology showed IN-FLUENZA-LIKE SYMPTOMS. Conclusions: A relevant percentage of subclinical disease was detected in the first pandemic wave in Chile and the proportion of people with SARI and deaths was small. Data from epidemiological surveillance were useful to estimate the trend of TSI but not its magnitude.展开更多
Objective To perform gene expression profiles comparison so that to identify and understand the potential differences in pathogenesis between the pandemic and seasonal A （H1N1） influenza viruses. Methods A549 cells ...Objective To perform gene expression profiles comparison so that to identify and understand the potential differences in pathogenesis between the pandemic and seasonal A （H1N1） influenza viruses. Methods A549 cells were infected with A/California/07/09 （H1N1） and A/GuangdongBaoan/51/08 （H1N1） respectively at the same MOI of 2 and collected at 2, 4, 8, and 24 h post infection （p.i.）. Gene expression profiles of A549 cells were obtained using the 22 K Human Genome Oligo Array, and differentially expressed genes were analyzed at selected time points. Results Microarrays results indicated that both of the viruses suppressed host immune response related pathways including cytokine production while pandemic H1N1 virus displayed weaker suppression of host immune response than seasonal H1N1 virus. Observation on similar anti-apoptotic events such as activation of apoptosis inhibitor and down-regulation of key genes of apoptosis pathways in both infections showed that activities of promoting apoptosis were different in later stage of infection. Conclusion The immuno-suppression and anti-apoptosis events of pandemic H1N1 virus were similar to those seen by seasonal H1N1 virus. The pandemic H1N1 virus had an ability to inhibit biological pathways associated with cytokine responses, NK activation and macrophage recognition .展开更多
In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mu...In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.展开更多
Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of e...Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients.The genetic basis,biological functions,and epitopes of the obtained Bn Abs were assessed and modeled.Results Two Bn Abs,2-12 D5,and 3-37 G7.1,were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset.Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity(ADCC)activity.Albeit derived from distinct Ab lineages,i.e.,V^H1-69-D2-15-JH^4(2-12D5)and V^H1-2-D3-9-JH^5(3-32 G7.1),the Bn Abs were directed toward CR6261-like epitopes in the HA stem,and HA2 I45 in the hydrophobic pocket was the critical residue for their binding.Signature motifs for binding with the HA stem,namely,IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs,were also observed in 2-12D5 and 3-32 G7.1,respectively.Conclusions Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus.The HA stem epitopes targeted by the Bn Abs,and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.展开更多
The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analys...The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analyses of the characteristic of the H1N1 virus infection-related genes,their biological functions,and infection-related reversal drugs were performed.Additionally,we used multi-dimensional bioinformatics analysis to identify the key genes and then used these to construct a diagnostic model for the H1N1 virus infection.There was a total of 169 differently expressed genes in the samples between 21 h before infection and 77 h after infection.They were used during the protein-protein interaction(PPI)analysis,and we obtained a total of 1725 interacting genes.Then,we performed a weighted gene co-expression network analysis(WGCNA)on these genes,and we identified three modules that showed significant potential for the diagnosis of the H1N1 virus infection.These modules contained 60 genes,and they were used to construct this diagnostic model,which showed an effective prediction value.Besides,these 60 genes were involved in the biological functions of this infectious virus,like the cellular response to type I interferon and in the negative regulation of the viral life cycle.However,20 genes showed an upregulated expression as the infection progressed.Other 36 upregulated genes were used to examine the relationship between genes,human influenza A virus,and infection-related reversal drugs.This study revealed numerous important reversal drug molecules on the H1N1 virus.They included rimantadine,interferons,and shikimic acid.Our study provided a novel method to analyze the characteristic of different genes and explore their corresponding biological function during the infection caused by the H1N1 virus.This diagnostic model,which comprises 60 genes,shows that a significant predictive value can be the potential biomarker for the diagnosis of the H1N1 virus infection.展开更多
BACKGROUND:The 2009 H1N1 influenza A virus was first identified in April 2009 and rapidly evolved into a pandemic. Recipients of solid-organ transplants have a higher risk for severe infection because of immunosuppres...BACKGROUND:The 2009 H1N1 influenza A virus was first identified in April 2009 and rapidly evolved into a pandemic. Recipients of solid-organ transplants have a higher risk for severe infection because of immunosuppression.There are limited reports of 2009 H1N1 influenza in liver transplant recipients,especially in China. METHODS:We present a case of a 48-year-old male liver transplant recipient with 2009 H1N1 influenza A virus.He received therapy for acute rejection after transplantation and was confirmed with H1N1 virus infection. RESULTS:The patient was started on oseltamivir(75 mg, orally twice daily)and had a benign hospital course,with defervescence and resolution of symptoms within 72 hours. The follow-up chest radiograph after discharge was normal. CONCLUSIONS:The 2009 H1N1 influenza in this hospitalized transplant recipient was relatively mild,and prolonged viral shedding was not noted.Oseltamivir can be a valid measure in immunocompromised individuals.展开更多
This research focused on the modification of the functional groups of oseltamivir as neuraminidase inhibitor against influenza A virus subtype H1N1.Interactions of three of the best ligands were evaluated in the hydra...This research focused on the modification of the functional groups of oseltamivir as neuraminidase inhibitor against influenza A virus subtype H1N1.Interactions of three of the best ligands were evaluated in the hydrated state using molecular dynamics simulation at two different temperatures.The docking result showed that AD3BF2 D ligand（N-[（1S,6R）-5-amino-5-{[（2R,3S,4S）-3,4-dihydroxy-4-（hydroxymethyl） tetrahydrofuran-2-yl]oxy}-4-formylcyclohex-3-en-1-yl]acetamide-3-（1-ethylpropoxy）-1-cyclohexene-1-carboxylate） had better binding energy values than standard oseltamivir.AD3BF2 D had several interactions,including hydrogen bonds,with the residues in the catalytic site of neuraminidase as identified by molecular dynamics simulation.The results showed that AD3BF2 D ligand can be used as a good candidate for neuraminidase inhibitor to cope with influenza A virus subtype H1N1.展开更多
To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defectiv...To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID<sub>50</sub> of the rAd-H5HA- EGFP was assessed to be 2.26×10<sup>10</sup>/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.展开更多
The rapid spread of the highly pathogenic A/H5N1 avian influenza virus among domestic birds and its transmission to humans has induced world-wide fears of a new influenza pandemic. A/H5N1 has infected over 300 people ...The rapid spread of the highly pathogenic A/H5N1 avian influenza virus among domestic birds and its transmission to humans has induced world-wide fears of a new influenza pandemic. A/H5N1 has infected over 300 people since 1997, and has shown a mortality rate of over 50%. The high mortality in human cases is thought to be enhanced by the excessive secretion of various endogenous factors, including cytokines and interleukins, stimulated by viral infections. Chickens infected with A/H5N1 viruses experience sudden death without showing severe clinical symptoms or inflammation. However, severe hemorrhage and congestion are seen in various tissues in sporadic chicken cases of A/H5N1-infections, especially in the pulmonary tissues, thus indicating that there is ischemia due to vascular abnormalities. Our previous studies have focused on the expression pattern of endothelin-1, which modulates the vascular tone via endothelin receptors. An Indonesian sporadic strain of A/H5N1 virus was intranasally administered to 10-day-old chicks, and the expression of endothelin was examined in the infected birds. All birds died within five days of inoculation, and had moderate inflammation accompanied by severe hemorrhage and congestion in the lungs. Immunohistochemical studies showed enhanced expression of endothelin-1 in the infected lungs. In addition, the real-time PCR analyses revealed that endothelin-1 and endothelin receptor A mRNA were significantly elevated in the birds with A/H5N1 infections. Subsequently, H5N1-infected birds were inoculated with bosentan hydrate, a competitive antagonist of endothelin receptors. Interestingly, the mortality rate of the infected birds was dramatically decreased in a dose-dependent manner by the administration of bosentan hydrate. The pathological lesions, including congestion and hemorrhage in the pulmonary tissues, were clearly inhibited. These findings are promising, and suggest that endothelin receptor antagonists are a potential treatment for the highly pathogenic avian flu.展开更多
Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including tra...Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including trade embargoes and interfering with serologic surveillance in differentiation between infected and vaccinated animals (DIVA strategy). Several distinct DIVA strategies have been presented to conquer these limitations. In this study, the open reading frame of NS1 gene of a H9N2 subtype of AI virus was amplified by polymerase chain reaction. After extraction and purification of NS1 gene from agarose gel, it was inserted into two different pGEX-4T-1 and pMAL-c2X plasmids and transferred in DH5α strain of Escherichia coli by using electroporation procedure. The E. coli colonies possessing recombinant NS1 gene were screened using PCR, restriction mapping and sequencing analysis. The expressed rNS1 protein was purified using affinity chromatography based on MBP (pMAL- c2X) and GST (pGEX-4T-1). The MBP-NS1 and GST- NS1 proteins on SDS-PAGE had bands with molecular weight of 68 and 52 kDa respectively. Western blotting with MBP-NS1 protein showed positive reaction using antisera obtained from chickens challenged with a H9N2 subtype strain. But, the most sera prepared from H9N2 vaccinated chickens were negative in WB. These findings indicated that the MBP-rNS1 protein of 26 kDa expressed by pMAL-c2X plasmid can be used in a DIVA for differentiation of AI infected and vaccinated chickens.展开更多
AIM: To investigate the genetic constitution of an escape mutant H5N1 strain and to screen the presence of possible amino acid signatures that could differentiate it from other Egyptian H5N1 strains.METHODS: Phylogene...AIM: To investigate the genetic constitution of an escape mutant H5N1 strain and to screen the presence of possible amino acid signatures that could differentiate it from other Egyptian H5N1 strains.METHODS: Phylogenetic, evolutionary patterns and amino acid signatures of the genes of an escape mutant H5N1 influenza A virus isolated in Egypt on 2009 were analyzed using direct sequencing and multisequence alignments.RESULTS: All the genes of the escape mutant H5N1 strain showed a genetic pattern potentially related to Eurasian lineages. Evolution of phylogenetic trees of different viral genes revealed the absence of reassortment in the escape mutant strain while confirming close relatedness to other H5N1 Egyptian strains from human and avian species. A variety of amino acid substitutions were recorded in different proteins compared to the available Egyptian H5N1 strains. The strain displayed amino acid substitutions in different viral alleles similar to other Egyptian H5N1 strains without showing amino acid signatures that could differentiate the escape mutant from other Egyptian H5N1.CONCLUSION: The genetic characteristics of avian H5N1 in Egypt revealed evidence of a high possibility of inter-species transmission. No amino acid signatures were found to differentiate the escape mutant H5N1 strain from other Egyptian H5N1 strains.展开更多
文摘Objective: To know the natural history of the first wave of pandemic influenza A(H1N1)pdm09 in the Southern hemisphere, through the detection of antibodies against influenza A(H1N1)pdm09 in a selected community, to estimate the population attack rate and its variations, the consultation rates, hospitallization and mortality rates. Methodology: A representative random sample of the population of a commune in Chile (San Felipe) was interviewed and taken blood samples between January and March 2010. A study against the antibodies of the influenza A(H1N1) pdm09 virus was conducted, by the technique of the Hemaglutination Inhibition (HAI) according to standardized methodology. Subjects with antibody titers ≥1:40 were considered positive. Results: 13.5% of the population of San Felipe had antibodies against influenza A(H1N1)pdm09;this percentage reached 30% of the population between 0 and 18 years and 6.1% among those over 19 years. The age variable was the only factor that evidenced significant differences in the prevalence of antibodies. There were no significant differences related to gender, vaccination history against seasonal inluenza, or comorbidity. 51% of people with positive serology showed IN-FLUENZA-LIKE SYMPTOMS. Conclusions: A relevant percentage of subclinical disease was detected in the first pandemic wave in Chile and the proportion of people with SARI and deaths was small. Data from epidemiological surveillance were useful to estimate the trend of TSI but not its magnitude.
基金supported by the National Basic Research Program of China (973 program: 2010CB534001)
文摘Objective To perform gene expression profiles comparison so that to identify and understand the potential differences in pathogenesis between the pandemic and seasonal A （H1N1） influenza viruses. Methods A549 cells were infected with A/California/07/09 （H1N1） and A/GuangdongBaoan/51/08 （H1N1） respectively at the same MOI of 2 and collected at 2, 4, 8, and 24 h post infection （p.i.）. Gene expression profiles of A549 cells were obtained using the 22 K Human Genome Oligo Array, and differentially expressed genes were analyzed at selected time points. Results Microarrays results indicated that both of the viruses suppressed host immune response related pathways including cytokine production while pandemic H1N1 virus displayed weaker suppression of host immune response than seasonal H1N1 virus. Observation on similar anti-apoptotic events such as activation of apoptosis inhibitor and down-regulation of key genes of apoptosis pathways in both infections showed that activities of promoting apoptosis were different in later stage of infection. Conclusion The immuno-suppression and anti-apoptosis events of pandemic H1N1 virus were similar to those seen by seasonal H1N1 virus. The pandemic H1N1 virus had an ability to inhibit biological pathways associated with cytokine responses, NK activation and macrophage recognition .
文摘In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.
基金supported by the General Program of the National Natural Science Foundation of China[No.31570162]the National Key Research Program[No.2016YFC1200200].
文摘Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients.The genetic basis,biological functions,and epitopes of the obtained Bn Abs were assessed and modeled.Results Two Bn Abs,2-12 D5,and 3-37 G7.1,were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset.Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity(ADCC)activity.Albeit derived from distinct Ab lineages,i.e.,V^H1-69-D2-15-JH^4(2-12D5)and V^H1-2-D3-9-JH^5(3-32 G7.1),the Bn Abs were directed toward CR6261-like epitopes in the HA stem,and HA2 I45 in the hydrophobic pocket was the critical residue for their binding.Signature motifs for binding with the HA stem,namely,IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs,were also observed in 2-12D5 and 3-32 G7.1,respectively.Conclusions Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus.The HA stem epitopes targeted by the Bn Abs,and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.
基金supported by the major national S&T projects for infectious diseases(2018ZX10301401)the Key Research&Development Plan of Zhejiang Province(2019C04005)the National Key Research,and the Development Program of China(2018YFC2000500).
文摘The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analyses of the characteristic of the H1N1 virus infection-related genes,their biological functions,and infection-related reversal drugs were performed.Additionally,we used multi-dimensional bioinformatics analysis to identify the key genes and then used these to construct a diagnostic model for the H1N1 virus infection.There was a total of 169 differently expressed genes in the samples between 21 h before infection and 77 h after infection.They were used during the protein-protein interaction(PPI)analysis,and we obtained a total of 1725 interacting genes.Then,we performed a weighted gene co-expression network analysis(WGCNA)on these genes,and we identified three modules that showed significant potential for the diagnosis of the H1N1 virus infection.These modules contained 60 genes,and they were used to construct this diagnostic model,which showed an effective prediction value.Besides,these 60 genes were involved in the biological functions of this infectious virus,like the cellular response to type I interferon and in the negative regulation of the viral life cycle.However,20 genes showed an upregulated expression as the infection progressed.Other 36 upregulated genes were used to examine the relationship between genes,human influenza A virus,and infection-related reversal drugs.This study revealed numerous important reversal drug molecules on the H1N1 virus.They included rimantadine,interferons,and shikimic acid.Our study provided a novel method to analyze the characteristic of different genes and explore their corresponding biological function during the infection caused by the H1N1 virus.This diagnostic model,which comprises 60 genes,shows that a significant predictive value can be the potential biomarker for the diagnosis of the H1N1 virus infection.
基金supported by a grant from the National Key Technology R&D Program of China(2008ZX10002-26)
文摘BACKGROUND:The 2009 H1N1 influenza A virus was first identified in April 2009 and rapidly evolved into a pandemic. Recipients of solid-organ transplants have a higher risk for severe infection because of immunosuppression.There are limited reports of 2009 H1N1 influenza in liver transplant recipients,especially in China. METHODS:We present a case of a 48-year-old male liver transplant recipient with 2009 H1N1 influenza A virus.He received therapy for acute rejection after transplantation and was confirmed with H1N1 virus infection. RESULTS:The patient was started on oseltamivir(75 mg, orally twice daily)and had a benign hospital course,with defervescence and resolution of symptoms within 72 hours. The follow-up chest radiograph after discharge was normal. CONCLUSIONS:The 2009 H1N1 influenza in this hospitalized transplant recipient was relatively mild,and prolonged viral shedding was not noted.Oseltamivir can be a valid measure in immunocompromised individuals.
文摘This research focused on the modification of the functional groups of oseltamivir as neuraminidase inhibitor against influenza A virus subtype H1N1.Interactions of three of the best ligands were evaluated in the hydrated state using molecular dynamics simulation at two different temperatures.The docking result showed that AD3BF2 D ligand（N-[（1S,6R）-5-amino-5-{[（2R,3S,4S）-3,4-dihydroxy-4-（hydroxymethyl） tetrahydrofuran-2-yl]oxy}-4-formylcyclohex-3-en-1-yl]acetamide-3-（1-ethylpropoxy）-1-cyclohexene-1-carboxylate） had better binding energy values than standard oseltamivir.AD3BF2 D had several interactions,including hydrogen bonds,with the residues in the catalytic site of neuraminidase as identified by molecular dynamics simulation.The results showed that AD3BF2 D ligand can be used as a good candidate for neuraminidase inhibitor to cope with influenza A virus subtype H1N1.
基金supported by the Chinese National S&T Plan(2004BA519A55)Scientific Research Program of State Key Laboratory of Veterinary Biotechnology(NKLVBP200818)
文摘To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID<sub>50</sub> of the rAd-H5HA- EGFP was assessed to be 2.26×10<sup>10</sup>/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.
文摘The rapid spread of the highly pathogenic A/H5N1 avian influenza virus among domestic birds and its transmission to humans has induced world-wide fears of a new influenza pandemic. A/H5N1 has infected over 300 people since 1997, and has shown a mortality rate of over 50%. The high mortality in human cases is thought to be enhanced by the excessive secretion of various endogenous factors, including cytokines and interleukins, stimulated by viral infections. Chickens infected with A/H5N1 viruses experience sudden death without showing severe clinical symptoms or inflammation. However, severe hemorrhage and congestion are seen in various tissues in sporadic chicken cases of A/H5N1-infections, especially in the pulmonary tissues, thus indicating that there is ischemia due to vascular abnormalities. Our previous studies have focused on the expression pattern of endothelin-1, which modulates the vascular tone via endothelin receptors. An Indonesian sporadic strain of A/H5N1 virus was intranasally administered to 10-day-old chicks, and the expression of endothelin was examined in the infected birds. All birds died within five days of inoculation, and had moderate inflammation accompanied by severe hemorrhage and congestion in the lungs. Immunohistochemical studies showed enhanced expression of endothelin-1 in the infected lungs. In addition, the real-time PCR analyses revealed that endothelin-1 and endothelin receptor A mRNA were significantly elevated in the birds with A/H5N1 infections. Subsequently, H5N1-infected birds were inoculated with bosentan hydrate, a competitive antagonist of endothelin receptors. Interestingly, the mortality rate of the infected birds was dramatically decreased in a dose-dependent manner by the administration of bosentan hydrate. The pathological lesions, including congestion and hemorrhage in the pulmonary tissues, were clearly inhibited. These findings are promising, and suggest that endothelin receptor antagonists are a potential treatment for the highly pathogenic avian flu.
文摘Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including trade embargoes and interfering with serologic surveillance in differentiation between infected and vaccinated animals (DIVA strategy). Several distinct DIVA strategies have been presented to conquer these limitations. In this study, the open reading frame of NS1 gene of a H9N2 subtype of AI virus was amplified by polymerase chain reaction. After extraction and purification of NS1 gene from agarose gel, it was inserted into two different pGEX-4T-1 and pMAL-c2X plasmids and transferred in DH5α strain of Escherichia coli by using electroporation procedure. The E. coli colonies possessing recombinant NS1 gene were screened using PCR, restriction mapping and sequencing analysis. The expressed rNS1 protein was purified using affinity chromatography based on MBP (pMAL- c2X) and GST (pGEX-4T-1). The MBP-NS1 and GST- NS1 proteins on SDS-PAGE had bands with molecular weight of 68 and 52 kDa respectively. Western blotting with MBP-NS1 protein showed positive reaction using antisera obtained from chickens challenged with a H9N2 subtype strain. But, the most sera prepared from H9N2 vaccinated chickens were negative in WB. These findings indicated that the MBP-rNS1 protein of 26 kDa expressed by pMAL-c2X plasmid can be used in a DIVA for differentiation of AI infected and vaccinated chickens.
文摘AIM: To investigate the genetic constitution of an escape mutant H5N1 strain and to screen the presence of possible amino acid signatures that could differentiate it from other Egyptian H5N1 strains.METHODS: Phylogenetic, evolutionary patterns and amino acid signatures of the genes of an escape mutant H5N1 influenza A virus isolated in Egypt on 2009 were analyzed using direct sequencing and multisequence alignments.RESULTS: All the genes of the escape mutant H5N1 strain showed a genetic pattern potentially related to Eurasian lineages. Evolution of phylogenetic trees of different viral genes revealed the absence of reassortment in the escape mutant strain while confirming close relatedness to other H5N1 Egyptian strains from human and avian species. A variety of amino acid substitutions were recorded in different proteins compared to the available Egyptian H5N1 strains. The strain displayed amino acid substitutions in different viral alleles similar to other Egyptian H5N1 strains without showing amino acid signatures that could differentiate the escape mutant from other Egyptian H5N1.CONCLUSION: The genetic characteristics of avian H5N1 in Egypt revealed evidence of a high possibility of inter-species transmission. No amino acid signatures were found to differentiate the escape mutant H5N1 strain from other Egyptian H5N1 strains.
