| BackgroundOn March 31,2013, the first human case of H7N9 avian influenza was reported in China. As of 19 May 2016,there were786 confirmed cases with 307 fatalities. Although there were no definiteevidence suggest whether H7N9 can transmission between human,family cluster cases indicate the possibility of pandemic. A/Anhui/1/2013(H7N9) was one of the candidates for H7N9 vaccines,however, the classical serologic assays for influenza surveillance, diagnosis and vaccine evaluation need to be carried out with live virus,which has risk for biological safety. The pseudovirus infect in a single round, extensive host range,high infectivity and cannot be inactivated by complement,which is much safer than handling the highly virulent subtype H7N9 virus.Thus, the pseudovirus-based neutralization assay is a high-through assay.ObjectiveTo construct the pseudovirus of A/Anhui/1/2013(H7N9) influenza virus, and establish an neutralizing antibodies detection method based on pseudovirus, which can establish foundation for H7N9 pseudovirus-based neutralization assay platform.Methods and results1、Determination of the preferablereassortment of H7N9 pseudovirus.To investigate the influence of different reassortment of hemagglutinin and neuraminidase to the characteristics of pseudovirus expressed HA of A/Anhui/1/2013(H7N9) influenza. Three strains of pseudovirus bearing HA from A/Anhui/l/2013(H7N9) were constructed by co-transfected 293FT cells with backbone plasmid of pNL4-3-Luc.R-E-and membrane plasmid with codon-optimized or wide-type HA and NA derived from A/Anhui/1/2013(H7N9), or codon-optimized HA from A/Anhui/1/2013(H7N9) and NA from A/Brisbane/59/2007(H1N1), named as AHop,-H7N9pp, AHwt-H7N9pp and AHop-H7N1pp, respectively. Western blot assay were used to evaluate the morphology of the pseudovirus particles, luciferase activity assay, immunofluences assay and hemagglution activity assay were used to compare their infectivity, and finally pseudovirus-based neutralization assays were evaluated. Three strains of pseudovirus bearing HA from A/Anhui/1/2013(H7N9) with high infectivity titer were constructed, and AHop-H7N9pp bearing optimized HA and NA from A/Anhui/1/2013(H7N9) demonstrated the most effective infectivity with high virus titer and hemagglution activity.2、Determination of the package parameter to obtain high-infectivity pseudovirus and analyses the biological characteristicsAHop-H7N9pp was packaged at high efficience with optimized conditions, the ratio of transfection backbone plasmid with envelope plasmid was 3:1, plasmid mixture and transfection reagent was at 1:2, the medium was replaced with DMEM containing 2% FBS at 10h after transfection, the time of harvesting AHop-H7N9pp was at 48h after transfection, and purification of pseudovirus was conducted by centrifuge at 2500 rpm for 5min, then 24000rpm for 2h with 20% sucrose cushion. Based on the dateof neutralization assay with AHop-H7N9pp, we found that AHoop-H7N9pp was highly train specificity and could be applied to neutralization assay.3、Validation of a Pseudovirus-based Neutralization Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against H7N9To evaluated the correlation between the pseudovirus particle based neutralization assay and the live virus based microneutralization assay (MN assay),1130 human sera containing 10 H7N9-infected patients convalescence stage sera were detected with pseudovirus.The date show excellent correlation between the two assays.ConclusionTo conclusion, pseudovirus bearing HA and NA from A/Anhui/1/2013(H7N9) withhigh infectivity was constructed with optimized conditions. The pseudovirus has high infectivity, satisfactory hemagglutinin activity and Subtype specificity in neutralizaition assay. Thusit can be used for H7N9 neutralizing antibody detection, laid foundation for H7N9 flu virus neutralzing antibody detection platform for influenza virus centres. |
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