Influenza Virus Propagation in Embryonated Chicken Eggs

Summary

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Abstract

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 °C and 55-60% humidity for 10 – 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 °C, the eggs are chilled for at least 4 hr at 4 °C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 °C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Introduction

Influenza A continues to be a major threat to human health. It is a potentially devastating respiratory disease with a large global burden causing up to 500,000 deaths worldwide annually ¹. Influenza viruses are in the family Orthomyxoviridae and carry 8 negative-sense single-stranded RNA in their genome ^1,2. The high mutability (i.e., antigenic “drift”) of the viral genome prevents long-term immunity. Moreover, influenza is increasingly resistant to anti-viral drugs ³.

The 2009 H1N1 influenza pandemic highlighted all the challenging issues associated with influenza disease (pandemic str....

Protocol

NOTE: General remarks: Perform all procedures involving manipulation of the egg under sterile conditions, and sterile technique should be used accordingly. Pre-clean all equipment with 70% ethanol before use. In general, influenza virus inoculation and harvest should be conducted in a BSL-2 laboratory. However, if this protocol is used to propagate much more pathogenic influenza viruses (e.g., pandemic and pre-pandemic strains, strains that pose a danger to poultry and livestock, highly pathogenic avian influenz.......

Discussion

Influenza causes a large global burden of disease, and work continues into understanding the pathogenesis of lung injury ⁷. To facilitated research in this deadly disease, various methods have been developed to propagate the influenza virus ⁶. Here, we describe a technique to produce influenza virus in chicken eggs. The advantage of this method is that it is highly reproducible and results in large quantities of high titer influenza virus stocks, which is often necessary for in vitro studie.......

Materials

Name	Company	Catalog Number	Comments
Name of Reagent/ Equipment	Company	Catalog Number	Comments/Description
Phosphate buffered saline (PBS)	Cellgro	21-040-CV
serum pathogen-free (SPF) fertilized chicken eggs	VALO BioMedia	n/a
humidified egg incubator	FARM iNNOVATORS	Model 2100
automatic egg turner	FARM iNNOVATORS	Model 3200
egg candler	FARM iNNOVATORS	Model 3300
1 ml syringe	BD Bioscience	309659
18G needle	BD Bioscience	305196
20G / 22G needle	BD Bioscience	305176 / 305156
HEPES	Sigma-Aldrich	H3375
Ethanol, >99.5%	Sigma-Aldrich	459844	diluted to 70% using water
glue gun "Ad tech Hi Temp Project Pro"	Ad tech, Adhesive Technologies, Inc.	Model 1105
glue "Ad tech MINI SIZE, Multi Temp"	Ad tech, Adhesive Technologies, Inc.	220-34ZIP30
MDCK cells	ATCC	CRL-2935
Washed Pooled Turkey Red Blood Cells, 10%	Lampire Biological Laboratories	724908