Construction And Application Of Pseudotyped Retrovirus Expressing Hemagglutinin And Neuraminidase From H5N1 Avian Influenza Virus

Background and Objective:By February 9th 2011, according to the statistical data from World Health Organization (WHO), there were accumulatively 520 cases with highly pathogenic H5N1 avian influenza, including 307 death cases, and the mortality rate is up to 59.0%.40 patients had been reported in China,14 survived. There is no sufficient evidence to reveal H5N1 AIV mutual person-to-person transmission, but H5N1 AIV could occur mutation through the antigen drift or antigen shift, which bring great challenges for H5N1 AIV vaccine. If H5N1 AIV recombinated with human influenza virus and got the ability of person-to-person transmission, and the human was lack of immune protection, it will bring into worldwide pandemic. In 2006, we used the H5N1 AIV convalescent plasma to successfully cure one severe H5N1 influenza patient, indicating the importance of passive immune therapy for H5N1 influenza. For wild type H5N1 influenza virus, there were lots of difficulties for researchers because of high pathogenicity, such as lower safety, time consuming, and strict standard for experimental condition. Fortunately, pseudotyped virus provided an effective tool to deal with these difficulties. Therefore, we first separated the A/Shenzhen/406H/06 H5N1 strains, and HA, NA and NS gene were amplified with RT-PCR assay, and the gene characterization and molecular genetic evolution features were analyzed; H5N1 pseudotyped virus was constructed which integrated HA and NA from A/Shenzhen/406H/ 06 and A/Thailand/KAN-1/04 strains, and it was indentified with electron microscope and Western-blot. Neutralizing assay in vitro was established based on pseudotyped virus, which provided strong technical tools for screening human monoclonal neutralizing antibody.Methods:1. The Virus solution isolated from Tracheal secretions of the patients infected with H5N1 avian influenza virus in Shenzhen. After filtration with 0.22μm membrane, the virus solution was used to infect 4×106 MDCK cells in 10cm Petri dish. The cell supernatant was collected after 48 hours. The virus was concentrated by 70000×g ultracentrifugation with 20% sucrose in the bottom. Virus RNA was extracted by Trizol reagent, sedimentaried by isopropanol, then it was dissolved in Rnase-free water. The concentration (A260) and purity (A260/A280) were measured by UV spectrophotometer.2. The Uni12 RT-primer (5'-AGCAAAAGCAGG-3') was designed according to the 12 conservative bases in the 3'End of the 8 gene sequences in A influenza virus. Then the genome c-DNA library was constructed with RT reaction. We designed 3 pairs of Specific primers for HA, NA and NS genes respectively for PCR amplification. The PCR conditions was 94℃2 min, (94℃30 s,55℃45s,72℃2min,30cycles), and 72℃7min. The PCR products were detected by 1%(W/V) agarose gel electrophoresis.3. The PCR products were recovered by agarose gel recovered kit and inserted into pGEM-T Easy vector by T4-Ligase. The vector was used to transform JM109 competent cells. The positive clones were identified with blue-white selection,37℃overnight, recombinant plasmid was identified with NotⅠdigestion and sequenced by Invitrogen con.4. The sequences of HA,NA and NS genes were got from GenBank, and the amino acids sequences were inferred. The sequence specificity and homology of HA, NA and NS genes from Shenzhen AIV isolations were analyzed by Cluster and Mega3.1, and the polygenetic trees were drawn.5. HA and NA gene specific primers were designed and involved SalⅠand BamHⅠdigestion sites. The recombinant plasmid pGEM-T-HA and pGEM-T-NA PCR were considered as templates, the products were recovered and then digested by SalⅠand BamHⅠ.The plasmid CMV/R was done the same digestion. After ligation, the recombinant plasmid were used to transform E. Coli JM109 competent cells and then the positive clones were identified and obtained.6. The plasmid pHR-Luc (14μg), pCMV△8.2 (14μg), CMV/R-HA (2μg) and CMV/R-NA (0.5μg) were co-transfected into 293T cell by calcium phosphate co-precipitation method. Co-transfection of plasmid pHR-Luc, pCMV△8.2 and pVSV-G was positive control, co-transfection of plasmid pHR-Luc, pCMV△8.2 was negative control. The cell supernatant was collected after 48 hours.7. The concentrated H5N1 pseudotyped virus was fixed with 2% glutaraldehyde, then adsorpted into a clean copper net,2% Sodium phosphotungstate was used for negative staining, finally observed with Electron microscopy. HA and P24 expression were identified with Western-blot, the HA was incubated with H5N1 AIV immunized mouse serum (1: 1000 dilution), and P24 was incubated with anti-P24 polyclonal antibody(1:1000 dilution). The second antibody was AP labeled rabbit anti-mouse IgG (1:3000 dilution)8. MDCK cell was seeded into 24-well plate, after 24 hours incubation, it was infected with different doses of pseudotyped virus including 1000μL,200μL, and 40μL supernatant. Meanwhile, the polybrene was added with final concentration 8μg/mL in order to promote virus adsorption. Each dose repeated 3 times. RLA value was measured after 48 hours to evaluate pseudotyped virus infectivity.9. MDCK cell was seeded into 24-well plate and incubated overnight. Serum was serially 2-fold diluted from 1:10 start point, then diluted serum was incubated with 200μL pseudotyped virus supernatant for 2 hours at 37℃, mixture was added into MDCK cell. After incubation 2 hours, mixture was discarded and RLA values were measured for another 48 hours incubation. Inhibition rate was calculated as followed:inhibition%= (MOCK-Serum Inhibition)/MOCK×100%.Results:1. H5N1 AIV RNA purification and RT-PCR amplification for HA, NA, and NS gene:RNA purified from H5N1 AIV was measured using ultraviolet spectrophotometer with A260 122.3μg/mL and A260/A280 1.82. The RNA was considered as template for RT-PCR amplification of HA, NA, and NS gene, the size of fragment was about 1.7kb,1.4kb, and 800bp respectively as expected.2. Gene clone and identification of HA, NA, and NS gene:PCR fragments were purified and ligated with pGEM-T Easy vector, recombinant plasmid was digested with NotⅠ, there were two fragments with size of 3.0kb and target gene after digestion. Then the recombinant plasmid was send to sequence, and virus strain was named after A/Shenzhen/406H/2006, the accession number of HA, NA, and NS gene in Genebank were EF137706, EF137708, EF137710 respectively.3. Gene characterization of HA, NA, and NS gene: HA gene encoded 567aa, with special sequence of SPLRERRRKR↓GLF in cleavage site. Compared to other HA gene from different strains, the receptor binding site was relative conserved, including Tyr91, Trp149, Ile151, His179,Asn182,Leu190,Gln192,130～134(GVSSA), and 220-224(NGQSG). HA involved 7 conserved glycosylation sites, there was another potential glycosylation site incurred Ala156Thr mutation. NA gene encoded 449aa and include 4 domains: N-terminal, non-polarity transmembrane domain, stalk, and head. Compared to H1N1 strains, there was 49-68 deletion in the stalk which lead to lack of 4 potential glycosylation sites (NQS, NNT,NQT,NIS). NS gene had two open reading frame (ORF) which encoded NS1 and NS2, NS1 had 225aa and possessed 80～84 deletion compared with A/Hongkong/156/97.4. Construction of poly genetic tree based on HA, NA, and NS gene:H5N1 AIV could be classified into North-America lineage and Euro-Asian lineage based on HA, and Euro-Asian lineage involved China mainland clade, Qianghai clade,02/03 Hongkong clade and 97 Hongkong clade. A/Shenzhen/406H/06 belonged to China mainland clade. NA gene involved swine lineage, human lineage, and avian lineage, A/Shenzhen/406H/06 belonged to avian lineage. NS gene involved allele A and allele B, A/Shenzhen/406H/06 belonged to allele B and had homology of 98.3% and 94.8% with A/Dk/Fujian/1734/05 and A/Dk/Hunan/191/05 strains respectively.5. Construction and identification of CMV/R-HA, CMV/R-NA vector:HA and NA gene were amplified with specific primer involved SalⅠand BamHⅠrestriction endonuclease site, and the size of fragments was expected. After ligation with CMV/R vector, the recombinant positive plasmid was digested into expected fragments with SalⅠand BamHⅠdigestion.6. Electron microscope analysis for H5N1 pseudotyped virus:The pseudotyped virus derived from lentiviral virus vector, the capsid protein was HIV-1 gag and envelope was HA, NA instead of gp120, the diameter is about 100nm similar to HIV-1. Meanwhile, pseudotyped virus located in budding, release stage similar to wild type virus.7. Western-blot identification for HA, P24 from H5N1 pseudotyped virus:After incubation with H5N1 AIV immunized mouse polyclonal serum, there were 3 fragments for SZ and TH pseudotyped virus with molecular weight 80×103,55×103, and 25×103, which corresponded to HAO, HA1, and HA2. Meanwhile, there were no fragments for negative control co-transfected pHR-Luc and pCMV△8.2 vector. However, after incubation with anti-P24 polyclonal antibody, there were 3 fragments corresponding to P55, P41, and P24, indicating it existed HIV virus-like particle.8. Infectivity of H5N1 pseudotyped virus:SZ and TH pseudotyped virus could infect target cell, with lower titre compared with VSV-G positive pseudotyped virus, the titre for 200μL supernatant was about 2×104, which was much higher than negative control pHR-Luc and pCMV△8.2 vector (HIV virus-like particle)9. Neutralizing assay in vitro based on pseudotyped virus:The SZ and AH serum was serially diluted and incubated with SZ and TH pesudotyped virus, and the VSV-G pseudotyped virus was considered to be negative control. IC50 of SZ serum against SZ pseudotyped virus was about 1:10240, which was much higher than that against TH pseudotyped virus (IC50 about 1:160-320). SZ serum couldn't neutralize VSV-G pseudotyped virus. AH serum could neutralize SZ and TH virus, with IC50 about 1: 5120-10240 against SZ pseudotyped virus and 1:2650-5120 against TH pseudotyped virus.Conclusion:1. There are many consequent alkaline amino acids in HA cleavage sites for A/Shenzhen/406H/06. The strain has highly pathogenic characterization, In addition, Ala156Thr mutation lead to a potential glycosylation site at Asn154, which maybe influence the virulence of AIV. The NA stalk 49-68aa deletion may reduce of the virulence of H5N1 AIV, and NS1 80～84aa deletion reduced the ability to anti-interferon. but the potential glycosylation site at HA 156 site could offset the negative influence, finally its replication, amplification, pathogenicity and transmission could Not be influenced.2. Although Shenzhen is near to Hongkong, A/Shenzhen/406H/06 and 97/02/03 Hong Kong isolation strains were at different evolutionary branching. It suggested A/Shenzhen/406H/06 did not derive from Hongkong. In addition, although A/Shenzhen/ 406H/06 is at the same lineage with South-east Asian isolated strains, they did Not belong to the same clade. Meanwhile, it was also different from 05 Qinghai isolated strains. On the contrary, it had high Homology with A/Anhui/1/05 and A/Dk/Fujian/1734/05 strain, indicating they had the same ancestors.3. H5N1 pseudotyped virus was successfully constructed to integrate HA and NA envelope based on lentiviral vector. Whose capsid protein derived from HIV-1 gag and envelope from H5N1 AIV. The pseudotyped virus has one-cycle infective activity with better safety, and is similar to wild type virus in cell tropism, adsorption and budding. Finally it possessed reporter gene and could be used in many assays and fields.4. H5N1 AIV induced human serum could neutralize many different strains, but the neutralization titres were different. For SZ pseudotyped virus, neutralizing titre of SZ serum was higher than AH serum. For TH pseudotyped virus, neutralizing titre of AH serum was higher than SZ serum. It suggested that human serum induced by subclade2.3 strain had cross reaction to different stains, so it was hoped broadly neutralizing antibody could be developed form peripheral blood.