Preparation and verification of highly pathogenic H5N8 subtype avian influenza pseudovirus
Objective In order to study the structure of the highly pathogenic H5N8 subtype(A/Astrakhan/3212/2020)a-vian influenza virus,the corresponding pseudovirus was prepared and its infective ability at the cellular level,as well as the neutralizing ability of serum antibodies,were preliminarily verified.Methods By analyzing the amino acid sequences of the H5N8 subtype(A/Astrakhan/3212/2020)virus hemagglutinin(HA)and neuraminidase(NA),the target HA and NA se-quences were optimized and the corresponding plasmid was extracted.Moreover,H5N8 subtype pseudovirus was prepared by a lentivirus packaging system.The fabricated pseudovirus was verified by luciferase readings,hemagglutination inhibition tests,transmission electron microscopy,and serum neutralization tests after infection with 293T-ST6GALl cells containing α-2,6 sialic acid receptors.Results H5N8 subtype pseudovirus was successfully prepared,and its infectivity to 293T-ST6GAL1 cells was 103 TCID50/mL.After being 50 times-concentrated,the hemagglutination activity of the pseudovirus in-creased from 16 to 128.Typical influenza virus particles could be observed by transmission electron microscopy.The neutral-ization test results showed that the neutralization activity of 3 H5N8 positive sera was significantly higher than that of other positive sera and negative sera.Conclusion This study successfully prepared the highly pathgenic H5N8 subtype avian in-fluenza pseudovirus,which provides an important experimental platform for future vaccine development and screening.
H5N8 subtype avian influenza virusPseudovirus preparationHemagglutininNeuraminidaseNeutralization test
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