The present study was supported by the NIH grants HL120947 (P.C.), HL103868 (P.C.), and the American Heart Association Grant-in-Aid (P.C.)

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...

<ol>
	<li>Centers for Disease Control and Prevention (CDC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimates+of+deaths+associated+with+seasonal+influenza+---+United+States,+1976-2007.">Estimates of deaths associated with seasonal influenza --- United States, 1976-2007.</a> MMWR. Morbidity and mortality weekly report. 59 (33), 1057-1062 (2010).</li><li>Neumann, G., Noda, T., Kawaoka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+and+pandemic+potential+of+swine-origin+H1N1+influenza+virus.">Emergence and pandemic potential of swine-origin H1N1 influenza virus.</a> Nature. 459 (7249), 931-939 (2009).</li><li>Tumpey, T. M., Garcia-Sastre, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenicity+of+Influenza+Viruses+with+Genes+from+the+1918+Pandemic+Virus:+Functional+Roles+of+Alveolar+Macrophages+and+Neutrophils+in+Limiting+Virus+Replication+and+Mortality+in+Mice.">Pathogenicity of Influenza Viruses with Genes from the 1918 Pandemic Virus: Functional Roles of Alveolar Macrophages and Neutrophils in Limiting Virus Replication and Mortality in Mice.</a> J Virol. 79 (23), 14933-14944 (2005).</li><li>Doherty, P. C., Turner, S. J., Webby, R. G., Thomas, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+and+the+challenge+for+immunology.">Influenza and the challenge for immunology.</a> Nat Immu. 7 (5), 449-455 (2006).</li><li>Webster, R. G., Govorkova, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuing+challenges+in+influenza.">Continuing challenges in influenza.</a> Ann N Y Acad Sci. 1323 (1), 115-139 (2014).</li><li>Szretter, K. J., Balish, A. L., Katz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unit+15G.1+Influenza:+propagation,+quantification,+and+storage.+.">Unit 15G.1 Influenza: propagation, quantification, and storage. .</a> Current protocols in microbiology. , (2006).</li><li>Short, K. R., Kroeze, E. J. B. V., Fouchier, R. A. M., Kuiken, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+influenza-induced+acute+respiratory+distress+syndrome.">Pathogenesis of influenza-induced acute respiratory distress syndrome.</a> Lancet Infect Dis. 14 (4), 57-69 (2014).</li><li>Ito, T., Suzuki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+sialic+acid-galactose+linkages+in+the+chicken+egg+amnion+and+allantois+influence+human+influenza+virus+receptor+specificity+and+variant+selection.">Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection.</a> J Virol. 71 (4), 3357-3362 (1997).</li><li>Robertson, J. S., Nicolson, C., Major, D., Robertson, E. W., Wood, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+amniotic+passage+in+the+egg-adaptation+of+human+influenza+virus+is+revealed+by+haemagglutinin+sequence+analyses.">The role of amniotic passage in the egg-adaptation of human influenza virus is revealed by haemagglutinin sequence analyses.</a> J Gen Virol. 74 (Pt 10), 2047-2051 (1993).</li><li>Gaush, C. R., Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+and+plaque+assay+of+influenza+virus+in+an+established+line+of+canine+kidney+cells).">Replication and plaque assay of influenza virus in an established line of canine kidney cells).</a> Appl Microbiol. 16 (4), 588-594 (1968).</li></ol>

The authors have nothing to disclose.

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...

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 &#8220;drift&#8221;) 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 strain, anti-viral resistance, delayed vaccine production). The vaccine formulation is determined annually by the World Health Organization for the most likely pathogenic influenza strains (one each for H1N1, H3N2 and influenza B) 4. Because this method is based on predicted influenza strains, pathogenic strains are occasionally incorrectly identified and vaccine efficacy drops dramatically. Moreover, the appearance of novel influenza strains can circumvent these preventative programs causing pandemic disease 2.
The creation of a universal influenza vaccine has been elusive 5. Therefore, research must continue into understanding the pathogenesis of lung injury. To facility laboratory research, several methods have been developed for viral isolation and propagation 6. Human influenza viruses can be amplified in a variety of mammalian cell substrates. However, generating high titer viruses in large quantities are best achieved in embryonated eggs. The following protocol describes a technique for influenza A virus propagation and storage from existing virus stocks.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Reagent/ Equipment</td>
			<td style="width:225px;">Company</td>
			<td style="width:128px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Phosphate buffered saline (PBS)</td>
			<td style="width:225px;">Cellgro&#160;</td>
			<td style="width:128px;">21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serum pathogen-free (SPF) fertilized chicken eggs&#160;</td>
			<td>VALO BioMedia</td>
			<td style="width:128px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">humidified egg incubator&#160;</td>
			<td>FARM iNNOVATORS</td>
			<td>Model 2100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">automatic egg turner</td>
			<td>FARM iNNOVATORS</td>
			<td>Model 3200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">egg candler</td>
			<td>FARM iNNOVATORS</td>
			<td>Model 3300&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1 ml syringe</td>
			<td style="width:225px;">BD Bioscience</td>
			<td style="width:128px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">18G needle</td>
			<td style="width:225px;">BD Bioscience</td>
			<td style="width:128px;">305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">20G / 22G needle</td>
			<td style="width:225px;">BD Bioscience</td>
			<td style="width:128px;">305176 / 305156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">HEPES</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:128px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Ethanol, &#62;99.5%</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:128px;">459844</td>
			<td>diluted to 70% using water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">glue gun &#34;Ad tech Hi Temp Project Pro&#34;</td>
			<td style="width:225px;">Ad tech, Adhesive Technologies, Inc.</td>
			<td>Model 1105</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">glue &#34;Ad tech MINI SIZE, Multi Temp&#34;</td>
			<td style="width:225px;">Ad tech, Adhesive Technologies, Inc.</td>
			<td>220-34ZIP30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">MDCK cells</td>
			<td style="width:225px;">ATCC</td>
			<td>CRL-2935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Washed Pooled Turkey Red Blood Cells, 10%</td>
			<td style="width:225px;">Lampire Biological Laboratories</td>
			<td>724908</td>
			<td></td>
		</tr>
	</tbody>
</table>

influenza virus propagation in embryonated chicken eggs

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 &#176;C and 55-60% humidity for 10 &#8211; 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 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;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 &#176;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.

Multiple methods have been developed to titer influenza virus. One consideration when choosing an appropriate method is that some determine total particles regardless of viability (e.g., hemagglutinin assay) whereas others are based on infectivity, which will assess viable virions (e.g., plaque assay) 6. Here, the viral titer of allantoic fluid collected from chicken eggs inoculated with a mouse-adapted influenza virus (A/PR/8/34) was determined by plaque assay (Figure&#160;1...

Watch this Scientific Journal Video about Influenza Virus Propagation in Embryonated Chicken Eggs at JoVE.com

Influenza Virus Propagation in Embryonated Chicken Eggs

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.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

influenza-virus-propagation-in-embryonated-chicken-eggs

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JoVE Journal

Immunology and Infection

42.3K Görüntüleme.  Cedars-Sinai Medical Center. 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.