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Aim To study the inhibitory activities of potential new anti-influenza virus agents,3-O-β-chaco-triosyl pentacyclic triterpenoids against the entry of H5N1influenza viruses.Methods Three target com-pounds were designed and synthesized structurally re-lated to the lead compound 3-O-β-chacotriosyl dioscin derivative (1 )with inhibitory activities against H5N1 influenza viruses.The inhibitory activities of these tar-get compounds were tested at a cellular level pseudo vi-rus system targeting H5N1 influenza viruse entry.Re-sults All the compounds 1 a,1 b and 1 c showed po-tent inhibitory activities against the entry of A/Thai-land/Kan353/2004 pseudo virus into the target cells, of which compound 1 b showed the best inhibitory activ-ity with an IC50 value of (1.25 ±0.22)μmol·L-1. Conclusion The SARs analysis of these compounds indicated that replacement of the aglycone moiety of compound 1 with pentacyclic triterpenoids could in-crease antiviral activity.Different types of pentacyclic triterpen as aglycone residue had the significant influ-ence on the inhibitory activity (1 b >1 c >1 a),sug-gesting ursane type of triterpenes was superior to the two other kinds of triterpenes as aglycone residue.
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Objective To establish neutralization assay based on H5N1 avian influenza pseudotyped virus in vitro and to evaluate neutralizing titer of convalescent serum from 2 patients with H5N1 avian influenza.Methods pHR-Luc,pCMV△8.2 and CMV/R-SH or CMV/R-TH were cotransfected into 293T cell by co-precipitation with calcium phosphate.Pseudotyped virus supernatant was harvested 72 h posttranofection and identified the expression of HA and P24 by Western blot,and then we analyzed infective activity of 200 μL supernatant of pseudotyped virus.293T cell integrated HA was prepared and anti-HA antibodies in convalescent serum were measured with FACS assay.Neutralizing titers of convalescent serums against Shenzhen and Thailand pseudotyped virus were determined based on calculating IC50 with neutralizing assay.Results Pseudotyped virus involved P24 and HA,and precursor protein HA0 could cleavage into HA1 and HA2 with biological activity.Pseudotyped virus possessed better infective activity,and RLA value was about 2 × 104 with 200 μL supernatant.Both convalescent serums contained anti-HA antibodies and had cross-reactivity against different virus clades with FACS assay.Both convalescent serums had neutralizingactivity and could cross-neutralize different virus clades.However,both serums'neutralizing titers against Shenzhen virus were higher than Thailand.Conclusion We successfully constructed infectious pseudotyped virus which integrated HA of Shenzhen or Thailand virus,and it could be used for evaluation of serum neutralizing activity fast,efficiently and safely with broadly application prospect.
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To prepare the RNAse-resistant virus particles containing the partial gene fragments of avian influenza virus H5N1 for use as RNA standard and control in RNA virus detection, the genes coding the coat protein and maturase of E.coli bacteriophage MS2 were amplified by PCR and then cloned into prokaryotic expression vector pET32a to construct the intermediate vector pET32a-MS2. In addition, the gene sequences coding hemagglutinin (HA), neuraminidase(NA) and M protein of the H5N1 virus were also cloned separately to the down-stream of plasmid pET32a-MS2, thus constructing the prokaryotic expression vectors pET32a-NS2-HA, pET32a-MS2-NA and pET32a-MS2-M. These recombinant plasmids were then transformed separately to E.coli BL21(DE3) with induction by IPTG. to express the virus-like particles. The virus-like particles observed under electron microscopy were identified by RT-PCR ,while their stability was confirmed by real-time RT-PCR. In this way, the virus-like particles were successively constructed and identified through PCR amplification, enzymolysis identification and sequencing analysis. These virus-like particles observed under electron microscopy appeared to be circular in shape with a diameter of about 50 nm. Their stability was proved to be rather good. From these observations, it is apparent that these virus-like particles can be used as RNA standard and quality control in the detection of avian influenza virus H5N1.
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Objective To test our hypothesis that sensitivity to avian influenza A(H5N1)virus varies among mouse strain backgrounds, we compared the pathogenicity of H5N1 viral infection in 5 mouse strains. Methods Onehundred-fifty mice from 2 inbred strains(BALB/c and C57BL/6), and 3 outbred stocks(ICR, NIH Swiss, and KM Swiss)were used. Thirty mice of each strain were subjected to an infected group(20 mice), in which mice were inoculated with 0. 1 mL(104.875 TCID50)of A/Goose/Guangdong/NH/2003(H5N1)virus intra-nasally; ten control mice received noninfectious allantoic fluid. Clinical signs were assessed daily for 14 days post-infection. Necropsy was performed on mice that died during the experiment and those euthanized at end of study. Tissue samples were collected for viral isolation and pathological analysis. Results H5N1 virus infection can cause respiratory illness in all 5 strains with severe or minor acute respiratory distress symptoms, but with different mortality rates: 70% in BALB/c; 50% in ICR; 40% in NIH Swiss; 25% in C57BL/6; and 10% in KM Swiss mice. Necrotizing interstitial pneumonia was found in all cases of death. The virus was isolated from the lungs of all infected dead mice. Conclusion A/Goose/Guangdong/NH/2003 (H5N1)virus can infect all mouse strains used in this study, and can cause clinical symptoms and pathological changes similar to those found in humans infected with HSN1 viruses. However, the pathogenicity of H5N1 viral infection varies significantly between the different mouse strains. Thus, in future study of H5N1 virus infections the mouse strain most relevant to their particular research purpose should be selected as animal model.
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Chicken embryo fibroblasts(CEFs)are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus(AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR(QPCR)analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4(RPL4)and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide(YWHAZ)are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene(ACTB)and the ribosomal protein L4(RPL4)gene are the best references.
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Objective To construct pseudotype retrovirus which integrates hemagglutinin(HA)of H5N1 avian influenza virus(AIV)isolated from human in Shenzhen.Methods AIV HA gene was amplified bv RT-PCR,then it was ligated with pGEM-T vector,and identified by restriction enzyme digestion and sequenced.HA gene was cloned into CMV/R vector at the site of Sal Ⅰ and BamH Ⅰ.pHR-Luc,pCMV&8.2 and CMV-HA were co-transfected into 293T cell by co-precipitation with calcium phosphate.The pseudotype virus supernatant was harvested 72 h post-transfection and ultracentrifugation,and the HA and P24 expression on the surface of pseudotype virus was analyzed by western blot.Meanwhile.the infection activity of HIV-HA pseudotype virus was identified in different kinds of cell lines,including MDCK,HeLa,CHO and 293T.Results A/Shenzhen/406H/06 belonged to subclade2.3 with open reading frame(ORF)of HA gene encoded 567 amino acides,whose accession number was EF137706 in GenBank.HA gene was cloned into CMV/R successfully.After co-transfection of above vectors,it revealed that HA protein could integrate pseudotype virus by western blot,and precursor protein HA0 could cleavage into HA1 and HA2 with biological activity.Finally.HIV-HA pseudotype virus could infect 4 kinds of cell lines,which indicated its property of infectivity and catholicity.Conclusion The pseudotype retrnvirns wassuccessfully constructed,which can integrate HA protein of A/Shenzhen/406H/06 and had property of infectivity.It call be used in the further research,including selection of neutralizing antibodies and epitope analysis.
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Objective To study the clinical features and prevention means of the infection caused by the highly dathogenic avian influenza A(H_5N_1).Method The clinical data which were confirmed to be an H_5N_1 infected case were analyzed retrospectively.Results The patient,16-year-old male,had no unambi guous history of direct contact with diseased or dead poultry before the onset of the disease.After the onset of the disease,chest X-ray showed flake shadow of the right lower lung quickly spread to the whole lung,associated with mediastinum,subcutaneous emphysema,acute respiratory distress syndrome(ARDS)was occurred on the fifth day.Mechanical ventilation is the primary measure in the comprehensive treatment.On the sixth day,Chinese Center for Disease Control and Prevention detected the pharyngeal specimen of the patient by RT - PCR,Real- time PCR method and suggested positive for the A/H5/N1 virus nucleic acid andis01ated the avian flu(H_5N_1) virus.The disease course was 10 days from onset of illness to death.Conclusions Human infection by the highly pathogenic avian influenza A(H_5N_1)is a deadly infectious disease.If the lesions is widespread and associated with ARDS,prognosis is poor.