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The viral titre of the harvested egg samples in the first experiment was found to be 9600 HAU/ml for two samples and 13600 HAU/ml in one other. In the Haemagglutination assay, a HI titre of 7680 HIU/ml was recorded. In the second experiment, virus was only found in three of the eggs at dilutions of 10-8 to 10-10, and the infectious dose was recorded as 107.5 ID50/100 μl.
Influenza A, a member of the Orthomyxoviridae family, is one of the most medically important infectious diseases of humans, causing high annual levels of mortality during seasonal epidemics and infecting larger numbers of people globally during occasional pandemics. The virus particles, illustrated in Fig.1, consist of eight single-stranded RNA segments surrounded by an M1 protein capsid and a lipid envelope of cellular origin. The envelope contains two major antigens that are important in characterisation of influenza viruses, haemagglutinin (HA) and neuraminidase (NA), as well as a proton-selective ion channel protein, M2. 1, 2
New Influenza vaccines must be produced annually due to antigenic drift, the accumulation of subtle mutations in HA and NA antigens that occur continually over time. Reassortment of influenza gene segments, referred to as antigenic shift, can result in a novel combination that may be extremely virulent in humans.2, 3 The influenza vaccine is produced by growing virus in embryonated chicken eggs. The influenza virus is injected into the eggs, which are then incubated for several days. The virus replicates in the cells of the chorioallantoic membrane and is released into the allantoic fluid, from which the virus is harvested, purified and chemically inactivated for vaccine production. 4
The target of current influenza vaccine is the HA antigen, and therefore the ability of HA to stimulate production of anti-HA antibodies determines the efficacy of a vaccine. 1 HA initiates entry to the virus by binding to the receptor N-acetylneuraminic acid (sialic acid) on mammalian cells. HA is also able to bind receptors on the surface of red blood cells, and at a certain cell to virus ratio this causes the cells to stick together or agglutinate. The Haemagglutination Assay (HA) allows the quantification of virus particles by their ability to agglutinate red blood cells at a defined concentration. In a positive reaction, sufficient virions are present in the to link the blood cells together, forming a lattice of red blood cells in suspension, illustrated in Fig. 2.A. As the dilution factor increases, the number of particles drops below equivalence with red blood cells and the virus fails to agglutinate the red blood cells fully and the red blood cells form a pellet at the bottom of the well, seen as a round spot from above (Fig.2.B). The reacting virus is diluted until reaction no longer occurs and this dilution is the end-point. One Haemagglutination unit (expressed in HAU/ml) is defined as the minimum concentration of virus that is able to agglutinate a fixed number of blood cells. Since no viral replication is required for agglutination to occur, the ability of virus to agglutinate blood cells does not reflect infectivity of virus. Therefore, an absolute relationship between HA and infectivity cannot be determined using this assay.3, 5
The assay can be modified to include an anti-HA antibody that binds the virus and blocks the interaction that results in Haemagglutination, illustrated in Fig.1.C. This is known as Haemagglutination Inhibition.6 A postitiveis a negative or non-agglutinated reaction, which appears the same as a negative result in the HA assay, where the red blood cells form a pellet at the bottom of the well (Fig.1.C ). By using standard concentrations of virus and blood cells and serially diluting the antiserum, the minimum inhibitory concentration of the antiserum can be determined. In vaccine production, this is used to determine the immunogenicity of the antigen used. 1,7
The aims of the first experiment were: to practice harvesting allantoic fluid from infected eggs and inoculating virus contained in allantoic fluid into fresh eggs, to quantify virus from harvested egg samples using HA assay and quantification of a monoclonal antibody using HI assay. The aims of the second experiment were to: Inoculate embryonated eggs with influenza virus and use the HA assay to determine virus presence in the eggs and infectivity titre.
Organisms vary even when relatively uniform in developmental stage and growth conditions, as the eggs were in this experiment, so some variation between the virus titres of the samples collected from different eggs was to be expected. The reason for the higher titres in samples collected from different eggs could simply be a different number of cells in the chorioallantoic membrane of the eggs harvested, or some other factor that made one particular egg a better environment for virus growth. For example, better overall health or slightly different temperature allowing higher activity of cellular proteins that the virus manipulates for replication. Mistakes made in preparation of the microtiter plates, such as incorrect volume or wrong dilution used, may also cause disparities in the results recorded.
In experiment 1b, the Haemagglutinating units per ml, the reciprocal of the virus dilution that causes 50% dilution in the sample, were determined by Haemagglutination assay. This assay measures the amount of virus in a sample by taking the minimum dilution at which haemagglutination of red blood cells occurs. The titre of sample 2 was higher than that of samples 1 and 3, which may be attributed to biological factors or experimental factors as explained above. It is important to note that different students harvested the three eggs, so the difference may have been due to the harvesting of the eggs by different students (i.e. experimental error), such as collecting less allantoic fluid and instead collecting fluid from other compartments in the egg due to poor technique. The fact that the second replicate of the first virus sample was much higher than the other replicates is most likely due to an error during preparation of the HA plate. The same factors may account for the lack of agglutination in the second replicate in experiment 1c, the Haemagglutination-inhibition assay, in that the virus may have been diluted incorrectly or not added to that row at all. The Haemagglutination-inhibition assay is important for determining the antigenicity of an antigen, as it measures the ability of the antigen to prevent haemagglutination at a defined concentration. The titre of the Monoclonal antibody provided was 7680 HIU/ml.
These reasons may also apply to the apparent lack of virus found in most of the eggs infected with virus stock in experiment 2b, despite an apparently high HAU titre and calculated infectivity titre of 107.5 ID50/100 μl. The fact that virus was found in eggs infected with the most dilute virus stock but not the lowest dilution of 10-7, in which virus was expected to be found, implies that this set of results is due to poor experimental technique. It may be the case that the eggs were defective in some way, that they were not at the right stage of growth or that inadequate sealing with wax lead to bacterial contamination that prevented growth of cells in the egg and therefore replication of virus. However, it is more likely that virus was injected into the wrong part of the egg and therefore could not replicate, or that experimental errors were made in setting up the dilutions or the microtiter plates.