Principles of Virology - Essay Example

Manoo R Name Lecturer Course Date Introduction Influenza may also result from Influenza viruses, which in sequence is regularly caused by having a flu that penetrates and damages sections of or the entire thyroid gland. Influenza viruses are infectious and cause acute diseases which is characterised by coughing, tracheal rales and sneezing, it can be spread from hens to human. Chicken eggs are vulnerable to influenza viruses. It can be transmitted through harvested chicken eggs. Influenza viruses grow in fertile hen’s eggs which are embryonated. As with other viruses, stressful situations may cause latent infections to become active, causing carriers to spread the virus to other susceptible chickens. Chickens with the weak pathogenic form of influenza infections may have signs that are barely distinguishable from other diseases. The only clinical signs may be ocular and nasal discharge, conjunctivitis, swollen infraorbital sinuses, or a decrease in egg production in hens. In order to have good results, for allantoic inoculation of A/Puerto Rco/8/34 (APR/8/34), these eggs will be used to grow influenza viruses. The dilutions of influenza viruses are inoculated and the infected embryonated eggs are then determined by looking for cytopathic effect. Materials and methods This experiment will require various materials which are necessary for detecting virus infection of an embryo. The material include 96-well V-bottom microtitre plate Embryo bin with bench coat Egg rack Needle on handle to pierce eggs 0.5% (v/v) chicken red blood cell suspension (4oC) Single use sterile transfer pipettes 20ml PBS Method The eggs were candled to check whether the there was an embryo developing. Then, a dilution series of A/PR/8/34 from 10-3 to 10-9 was made using PBS with antibiotics, by adding 100 µL to 900 µL at each step. The 4 eggs for each dilution were labelled as 10-5, 10-6, 10-7, 10-8, 10-9 and other 4 eggs as controls. The microtitre plate grid was marked off according as shown in the figure below Then the chilled embryos were arranged upright in rows of four and are grouped according to virus dilution. The air sac and air sac membrane from each embryo was removed to expose the embryo surrounded by allantoic fluid. Then a drop of fluid per egg was transferred to an individual well of a Microtitre V-bottomed plate containing rows of wells that were marked with the particular virus dilution using a separate tip for each egg. Rows of wells are marked with the particular virus dilution. A drop of PBS was added to each control well. Then a drop of 0.5% chicken red blood cells was added to each well containing the allantoic fluid and to the four wells containing 1 drop of PBS. The plates were agitated in both directions to thoroughly mix the contents of the wells and the red cells were allowed to settle over 35 minutes at room temperature on a level bench top. The results was taken and recorded. Results After infecting 4 eggs with virus dilution and having other control eggs and an incubation period had elapsed, the effects were scored as haemagglutinin + or -. The score each well haemagglutinin-positive or -negative, according to the settling pattern of the red cells were recorded as shown below Egg 1 2 3 4 -5 + + + + -6 - + + + -7 - - - - -8 - - - - -9 - - - - Control - - - - From the results it shows that the pattern of the plotted points on the graph suggesting a positive relationship between the concentration and infection rate. This kind of association simply implies that as the concentration increases as the infection increases. This kind of findings simply goes with the general expectation. For a normal infection it is generally hypothesized that it concentration would have adverse effect on demand. The results above can be written in column according to the number of positives per dilution as Dilution + - ∑+ ∑- % Positive 10-5 4 0 7 0 100 10-6 3 1 3 1 75 10-7 0 4 0 5 0 10-8 0 4 0 9 0 10-9 0 4 0 13 0 The table above shows results of mean, EE ratios of control and inoculated eggs with percentage positive dilution. It indicates that dilution for eggs has similar results from 10-7 to 10-9 dilutions for the haemagglutinin. This had haemagglutinin-negative. The proportionate difference was calculated as Proportionate difference (PDF) = = The figure obtained was multiplied by the dilution factor that is the logarithm of dilution to get the negative rock of the TCID50 endpoint and this was done as: PDF x logarithm of the dilution factor = PDF Log= 0.33*1 = 0.33 Negative log TCID50 titre = negative log of next dilution above 50% infected wells + PDF TCID titre =-6+0.33=6.33 This was expressed as 106.33 EID50 / 100 µL, however we always express titre as per ml therefore the endpoint would be 107.33 EID50/ ml The graph above shows how positive Discussion 50% egg infectious dose of influenza virus experiment has proved that the incubation period for virus growth in an egg will depend on the amount of virus dilution injection into an egg. The result showed that the concentration of the virus as well as embryonated cells were good medium for replication. The experiment showed clearly that the positivity increases when dilution series of A/PR/8/34 from 10-9 to 10-5to yield the optimal results. The differences in the dilution series of A/PR/8/34 between 10-7 to 10-9 did not provide a lot of difference positivity but had haemagglutinin-negatives. These results would naturally be different for different dilution series of A/PR/8/34 seeing as they will have different haemagglutinin-negatives. The egg will and the virus dilution concentration of two or more substances reacting to each other will determine the end results. The virus dilution can be set higher if one wants to hasten haemagglutinin-positives if he wants to slow down the infection rate one reduces dilution series of A/PR/8/34. The concentration of the dilution series of A/PR/8/34 within the embryonated would also be taken into consideration in order to determine their initial concentration and then compare it with the conductivity data to determine the level of infection that has taken place. The statistical method of Reed and Muench was used to determine the 50% end point. The results are pooled, and the infection at each dilution is calculated. Determining infection levels is the most valid and sensitive indication of influenza virus. References American Society of Microbiology, 2009. RED BLOOD CELLS, CHICKEN. Available online from (accessed on 19th September, 2013). Flint, J., Enquist, LW., Racaniello, VR & Shalka, A., 2009.Principles of Virology - Virological Methods. New York: Amer Society for Microbiology Raj, G., Kumar, KS, Nainar, A. M. & Nachimuthu, K., 2004. gg:embryo weight ratio as an indicator of dwarfism induced by infectious bronchitis virus RMIT University, 2013. Viral Infections- Practical Manual. Biotechnology and environmental Biology. Sircar, S., 2008. Principles of Medical Physiology. Stuttgart ; New York : Thieme. Read More