Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity

Summary

Here we describe a protocol for pseudovirus packaging and the measurement of antibody neutralizing activity.

Abstract

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. Occasionally, humans also fall victim to it, which results in high mortality. Nonetheless, HPAI virus research is often hindered, considering that it must be handled within biosafety level 3 laboratories. To address this issue, pseudoviruses are adopted as an alternative to wild-type viruses in some experiments of H5 HPAI studies. Pseudoviruses prove to be the ideal tools to study neutralizing antibodies against H5 HPAI viruses. This protocol describes the procedures and critical steps of H5 HPAI pseudovirus preparations and pseudovirus neutralization assays. Also, it discusses the troubleshooting, limitation, and modifications of these assays.

Introduction

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing continual flu outbreaks in poultry and wild birds, accounting for enormous socio-economical losses in the global poultry industry. Sometimes, humans also get infected with it, faced with a high fatality rate^1,2. However, HPAI virus research is often hindered, given that it cannot be handled outside of biosafety level 3 laboratories. To address this issue, pseudoviruses are adopted as an alternative to wild-type viruses in some experiments of H5 HPAI studies. Pseudoviruses are safe enough to practice....

Protocol

All pseudovirus-related experimental operations were performed under ABSL2 condition in the Institut Pasteur of Shanghai Chinese Academy of Sciences (IPS, CAS). Animal experiments were performed based on Institutional Animal Care and Use Committee-approved animal protocols of IPS, CAS.

1. Pseudovirus packaging with Calcium-phosphate transfection

1. Make single-cell suspensions of HEK293FT cells (9 x 10⁵ per mL) in complete DMEM medium (Table 1<.......

Discussion

HEK293FT cells are usually used as packaging cells to produce pseudoviruses. Regular mycoplasma detection is essential during the cell culture. Mycoplasma contamination can drastically decrease the pseudovirus yields and sometimes close to zero. Compared with other contaminations, mycoplasma contaminations do not lead to pH value changes or turbidity of the cell culture medium.Even a high concentration of mycoplasma is not visible with naked eyes or under a microscope. Three popular methods of mycoplasma detection are my.......

Materials

Name	Company	Catalog Number	Comments
1% Chiken Erythrocyte	Bio-channel	BC-RBC-C001	Reagent
96-well cell culture plates (flat-bottom)	Thermo fisher scientific	167008	consumable material
96-well cell culture plates (round-bottom)	Thermo fisher scientific	163320	consumable material
Allegra X-15R	Beckman coulter	--	Equipment/Centrifuge
BD Insulin Syringes	BD	324910	consumable material
Calcium Chloride Anhydrous	AMRESCO	1B1110-500G	Reagent
chloroquine diphosphate	Selleck	S4157	Reagent
Dulbecco’s Modified Eagle Medium (DMEM)	Gibco	12100-046	Reagent
Fetal Bovine Serum	Gibco	16000-044	Reagent
HEK293FT	Gibco	R700-07	Cell line
HEPES FREE ACID	AMRESCO	0511-250G	Reagent
HIV-1 p24 Antigen ELISA	ZeptoMetrix	801111	Reagent kit
Luciferase Assay System Freezer Pack	Promega	E4530	Reagent kit
MDCK.1	ATCC	CRL-2935	Cell line
Microcentrifuge Tubes 1.5 mL	Thermo fisher scientific	509-GRD-Q	consumable material
Nunc Conical Centrifuge Tubes 15 mL	Thermo fisher scientific	339650	consumable material
Nunc Conical Centrifuge Tubes 50 mL	Thermo fisher scientific	339652	consumable material
Nunc EasYFlask 75 cm2	Thermo fisher scientific	156499	consumable material
Penicillin-Streptomycin	Gibco	15140-122	Reagent
Pipette Tips (10 μL)	Thermo fisher scientific	TF102-10-Q	consumable material
Pipette Tips (100 μL)	Thermo fisher scientific	TF113-100-Q	consumable material
Pipette Tips (1000 μL)	Thermo fisher scientific	TF112-1000-Q	consumable material
Serological pipets (5 mL)	Thermo fisher scientific	170355N	consumable material
Serological pipets (10 mL)	Thermo fisher scientific	170356N	consumable material
Trypsin/EDTA	Gibco	25200-072	Reagent
Varioskan Flash	Thermo fisher scientific	--	Equipment/Microplate reader
Water Jacket Incubator	Thermo fisher scientific	3111	Equipment/Cell incubator
Pentobarbital sodium salt	Sigma	57-33-0	Reagent