Influenza virus growth in eggs

10 December 2009

candle-eggsBefore the development of cell culture, many viruses were propagated in embryonated chicken eggs. Today this method is most commonly used for growth of influenza virus. The excellent yield of virus from chicken eggs has led to their widespread use in research laboratories and for vaccine production. In fact the vast majority of influenza vaccines – both inactivated and infectious – are produced in chicken eggs. How is influenza virus propagated in eggs?

The illustration below shows a cutaway view of an embryonated chicken egg. The different routes of inoculation into the egg are shown, as well as the different compartments in which viruses replicate.

viruses_in_eggs

For propagation of influenza virus, pathogen-free eggs are used 11-12 days after fertilization. The egg is placed in front of a light source to locate a non-veined area of the allantoic cavity just below the air sac. This is marked with a pencil. After all the eggs have been ‘candled’ in this way, a small nick is made in the shell at this position using a jeweler’s scribe. Next, a hole is drilled at the top of the egg with a Dremel motorized tool. If this is not done, when virus is injected, the pressure in the air sac will simply force out the inoculum.

After all the eggs have been nicked and drilled, they are inoculated with virus using a tuberculin syringe – a 1 ml syringe fitted with a 1/2 inch, 27 gauge needle. The needle passes through the hole in the shell, through the chorioallantoic membrane, and the virus is placed in the allantoic cavity, which is filled with allantoic fluid. The two holes in the shell are sealed with melted paraffin, and the eggs are placed at 37 degrees C for 48 hours.

During the incubation period, the virus replicates in the cells that make up the chorioallantoic membrane. As new virus particles are produced by budding, they are released into the allantoic fluid. To harvest the virus, the top of the egg shell – the part covering the air sac – is removed. We used to have a special tool to do this, which was placed over the egg. When the handle of this tool is squeezed, it makes a neat crack around the top of the egg. It was then easy to remove the flap of shell with tweezers. The shell membrane and chorioallantoic membrane are pierced with a pipette which is then used to remove the allantoic fluid – about 10 ml per egg. Sufficient virus may be produced in one or two eggs (depending on the viral strain) to produce one 15 microgram dose of vaccine.

We used to grow so much influenza virus that a large walk-in warm room was used as an egg incubator. When you opened the door of the incubator and heard peeping, it meant that someone had left unused eggs too long and they had hatched. Then you were left with the task of catching the evasive chicks.

  • Diane

    so each single flu vaccine is, in some ways, made by hand? I had no idea. How unbelievably time-consuming. How many eggs can one innoculate in a day?

  • http://twitter.com/lbwhcn Bo Liang

    I am surprised that chicken egg cells are permissive for some viruses that do not seem to naturally infect avian! Also, it seems that different type of viruses should be inoculated at different locations.Why is that? Is it because of different tropism of these viruses?

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  • Teeny

    The last paragraph made me laugh. Chasing hatchlings sounds more fun than catching drosophila though!

    Out of curiousity, would the chicks have flu and would that dangerously transmissable to the staff?

  • http://www.virology.ws profvrr

    I would put one in my lab coat and walk around surprising people with
    the sound. To answer your question: yes, depending on the viral
    strain, it could replicate in chicks. But the chicks that hatched were
    always from eggs that had not been inoculated with virus – unused
    eggs. But even if the chicks had been infected, they were no dangerous
    in terms of transmission than working with large volumes of virus in
    the laboratory – where aerosol generation often occurs as the virus is
    pipetted, centrifuged, etc.

  • http://www.virology.ws profvrr

    I could do hundreds in a day. Probably someone well trained could do
    at least a thousand a day. For up to a billion eggs worldwide, that is
    a lot of time.

  • http://www.virology.ws profvrr

    Yes, the cells in different parts of the embryonated egg are
    different, and serve as a host for only certain viruses. How this is
    controlled is largely unknown.

  • PP

    That makes a mind-boggling number of eggs, too. That's crazy.

  • PP

    How come influenza vaccines haven't moved to cell-culture based growing? I had the idea it was in order anyway?

  • o.jeff

    Re: Egg inoculation

    Sanofi-Aventis appears to have developed a very high volume automatic inoculation system that is shown running in this video at over 32,000 eggs per hour according to the monitoring panel shown in the video.

    http://www.cbsnews.com/video/watch/?id=5450993n

  • http://www.virology.ws profvrr

    Yes, I've seen that video as well. Not surprising that the process is
    automated. But I agree with PP, there has to be a better way. Cell
    culture grown vaccines are in the pipeline, but even better, in my
    opinion, are influenza virus-like particles grown in plants. Twenty
    thousand doses per square meter of plants, twenty cents a dose.

  • http://www.facebook.com/people/Janarthan-Rama-Murti/100000313951980 Janarthan Rama Murti

    Dear sir,
    I have a few questions. Firstly, NDV is a highly virulent avian pathogen (depending on strain), how is it possible to obtain significant yield if the virus is propagated by inoculation into chicken embryo. Logically speaking, the chicken embryo suffers from the infection & will die before significant amount of virus is produced.
    Secondly, are all strains of influenza virus capable of growing well in chicken embryo? What determines whether a field strain of influenza adapts to conditions in chicken embryo, in terms of viral genetic changes & techniques used to bring about this adaptation in field strains.
    Thirdly, can strains of avian influenza (AI), especially HPAIs adapt to condition in chicken embryo? We expect the same situation as the NDV described above to occur. Then, how is propagation of these virus in large amount are possible for vaccine production?

  • http://www.virology.ws profvrr

    Not all strains of NDV kill the embryo, hence the virus yield can
    vary. As for influenza, different strains vary in their ability to
    multiply in the egg. Some field isolates replicate poorly or not at
    all, some better. The yield of H5N1 strains is poor because of the
    pathogenicity for the embryo. To produce a vaccine, reassortants with
    the PR8 strain are produced which have only the HA and NA genes from
    H5N1, and the other 6 from PR8, which grows very well in eggs.

  • M. A. Ramakrishnan,

    Nice description Dr Vincent. When I was in my PhD , we used lot of eggs for virus isolation (Bluetongue) through intra-venous route and we followed few simple procedure to save time and avoid some hazard.
    1. insert a small pin in to the rubber cork and use it for piercing egg shell and memrane. This is very simple and we can avoid egg shell powder due to Dremel motorized tool or similar tool.
    2. Use cellophane tape (Scotch tape) instead of paraffin wax.

    M. A. Ramakrishnan, University of Minnesota (rama@umn.edu)

  • swapnil

    Can we cultivate entero viruses in egg embryo

  • scucic

    Are chicken embryos of different age used for cultivation of different viruses? For example, 10-day old chicken embryos used for, say, NDV and FPV? If so, why?

  • maramakrishnan

    Yes. Different ages of embryonated eggs were used for cultivation of various viruses. Based on the route of inoculation (for effective viral growth/receptors in different location) different ages of embryonated eggs are used. For example, for the grow of Bluetongue virus (Reoviridae; causing disease in sheep), the route of inoculation is intravenous (IV) and 13 days eggs used for inoculation (in 13 days we can see a clear vein and easy to inoculate)

  • scucic

    Are chicken embryos of different age used for cultivation of different viruses? For example, 10-day old chicken embryos used for, say, NDV and FPV? If so, why?

  • maramakrishnan

    Yes. Different ages of embryonated eggs were used for cultivation of various viruses. Based on the route of inoculation (for effective viral growth/receptors in different location) different ages of embryonated eggs are used. For example, for the grow of Bluetongue virus (Reoviridae; causing disease in sheep), the route of inoculation is intravenous (IV) and 13 days eggs used for inoculation (in 13 days we can see a clear vein and easy to inoculate)

  • ErikCarter

    Is it possible to grow the cells that line the chorioallentoic membrane in a large “bioreactor” vat (I don't know the correct term). It seems like it would be easier than harvesting all the eggs and dealing with the chicks if eggs are left for too long as you mentioned. All you would need is some nutrient broth (?bovine serum albumin?) to maintain the cells.

    (The fact that this method isn't routinely employed implies that there is an issue with it. So really what I'm asking is what makes this method insufficient to produce virus?)

  • Kamalanadhan Ravivarma

    This is for good article for all reserchers, Thank you for virology blog

  • Eflesch

    Can you inoculate an embryonated egg with 2 different viruses at one time?

  • Cyanpku

    what happened to those unexpected chicks after being caught?

  • nermeen

    what result of inoculation on egg by avian flu?

  • http://www.virology.ws profvrr

    Avian influenza viruses replicate in embryonated chicken eggs. Highly
    pathogenic strains (eg H5N1) kill the embryos before viral yield is
    high enough for vaccine production; this problem was overcome by
    deleting the polybasic HA cleavage site and reassorting with genes
    from the A/PR/8/34 strain.

  • Doan Thuy

    please give me the name of the lights used for propagation of influenza viruses in chicken eggs

  • James_laten26

    are there any method in laboratories where lab scientist could do instead of using those egg…?? using hundred of egg in a day its crazy actually.. instead your going to eat that or give to those people who need more good cholesterol’s….

  • http://www.virology.ws profvrr

    Yes, there are other methods for growing viruses, but for influenza
    vaccine, eggs is the most efficient. However, cell-based influenza
    vaccines are moving forward, and it is likely that these will replace
    egg produced vaccines in the near future.

  • Makne

    Hi, do you know if rhadovirus strains can growth in eggs?

  • http://www.virology.ws profvrr

    Yes, some rhabdoviruses will replicate in eggs. Depends on the strain.

  • Ash

    well… you may hear several “cracks” as the chicks necks are broken. As the chicks get older, the defects of inbreeding the bloodlines becomes quite apparent. Nothing too major, a manky foot or turned beaks, but with companies using upwards of 400 000 eggs per day, it’s difficult to re-home these chicks

  • http://pulse.yahoo.com/_EWWNHSZD5U4JU6SKPANJW2XIQA Chris

    Yes, but one will mask /out growth the other one

  • http://pulse.yahoo.com/_EWWNHSZD5U4JU6SKPANJW2XIQA Chris

    Can we do cell based influenza diagnostic test for avian influenza?

  • ema

    How do you make the (purified?) virus to inject into the eggs?

  • http://www.virology.ws profvrr

    Virus injected into eggs doesn’t have to be purified – it can be from
    another egg, or also produced in cell culture.

  • Byiringiro christophe

    why u use embryonic egg not normal egg

  • http://www.virology.ws profvrr

    Look at the image of the embryonated egg in this post – only that form
    contains the various cell sheets (such as the chorioallantoic membrane) in
    which the virus replicates. Such cells are not present in the unfertilized
    egg.

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  • Rohannarayanm

    i think he mentionedUNUSED eggs?

  • Rohannarayanm

     they say the ostrich egg is the worlds largest cell. But them wen there are so many cells which make up the CAM, how can it be called a single cell?
    even if its an unfertilized egg, the whole egg is nothin but one huge cell?

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  • Dmack5347

    How is sterilityof the eggs achived?

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  • Wael99992001

    i work in a vaccine production center in jordan  i and i rebeat i  inoculate 3800 eggs today with ndv -b1 virus is that a big number ? . with some training the proceses comes easier .

  • Roshni

    Influenza B is one of the viruses in the trivalent vaccine and it does not infect birds. So then how does it infect chicken eggs?

  • http://www.virology.ws profvrr

    Many viruses replicate in eggs that do not replicate in birds. Influenza B is one of them.  Viral replication in the animal from which cells are derived is not necessary for the cells to be susceptible to infection. For example, human influenza A viruses replicate in dog kidney cells but not in dog kidney. Poliovirus replicates in monkey cell kidney culture but not in monkey kidney.

  • Roshni

    Thank you! Also, is there a limit to how much virus a cell can produce? In the sense, by prolonging apoptosis or by knocking down certain genes (IFN related) that play a role in antiviral pathways, would they be able to increase the yield? Depending on the host and the strain of virus, the infectivity and virus production will be different, but say for instance a cell produces 1000 virions, is that the limit of the cell or can it roduce upto 2000 virions ?  

  • shobha

    thank u for ur infermation

  • Himu907

    What about the infection period of different virus?and on which factor incubation time depends?

  • http://pulse.yahoo.com/_WLSQN3HLPA2V6U2NEH3JJE23BM me

    I think she figured that out 2 years ago dimwad.