Measuring parameters of Bovine Enterovirus infection

1Lab report 2- Bovine Enterovirus

Measuring parameters of Bovine Enterovirus infection

AbstractThe purpose of this experiment is to measure the parameters of bovine enterovirus infections. This was accomplished by performing four assays: a plaque assay, a TCID50 assay, a viral protein assay and finally a intracellular viral RNA assay followed by a SDS polyacrylamide gel electrophoresis.

IntroductionThe aim of this experiment is to demonstrate the many ways to monitor the growth and infectious cycle of viruses. In this experiment bovine enterovirus (BEV) is used to infect cultures of baby Hamster Kidney cells (BHK21) which are then harvested at different times and undergo a number of assays.

BHK21 are commonly used for vaccine production and came to light in 1961 from 5 Syrian hamsters at 1 day old. They are fibroblasts meaning they play an important role in the structure of animal tissues. They synthesise collagen, a protein abundant in the connective tissue and flesh of mammals, reticular, glycosaminoglycans, glycoproteins and elastic fibres.

The BEV causes respiratory, enteric and reproductive diseases in cattle despite its fairly non-pathogenic characteristics. It has an icosahedral protein capsid composed of 4 proteins, VP1-VP4 that holds a single strand positive RNA genome of 7500 bases. For it to replicate the BEV has to attach to a surface receptor on the host and penetrate into the cytoplasm then uncoat its genome. It is spread through oro-fecal and respiratory routes but is not zoonosis so does not infect humans.

Plaque assays are used to determine the concentration of viruses needed for an infectious dose. This is done by counting the number of plaque forming units (PFU), with each one representing one virus. The cells are then covered in a layer of agar to insure the virus only spreads to neighbouring cells, after a number of infectious cycle’s pfu’s developed due to the cells dying. These are then exposed during staining and counted by eye revealing the number of infectious units of virus of each diluted sample.

The 50% tissue culture infective dose (TCID50) assay, as its name suggests, measures the amount of virus required to kill or cause cytopathic effects in 50% of infected host cells. This assay is used to determine the virus’ lethal dose, mostly studied in clinical research.

A protein assay determines the viral protein in a sample. To study viral proteins, a cell culture is infected and incubated allowing virus growth and infectious cycles. The starting amount of protein in the virus is usually known and the cells are lysed after incubation to expose the proteins and observed using fluorescence detection.

Matthew Dower - 1021492


The purpose of sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis is to separate proteins according to their size. This is achieved by making each protein linear so they have no secondary, tertiary or quaternary structure using SDS, as it is a detergent and dissolves the hydrophobic bonds in the protein. The sulphate present in SDS negatively charges the protein overwhelming any positive charge from R-groups. Therefore the proteins are all the same shape and all have the same resistance when passing through the gel from the negative end to the positive, due to an electric field put in place. The polyacrylamide gel allows the different sized proteins to move at different rates, and therefore be separated due to size. (Figure 4)

MethodThe method of this experiment can be seen in close detail on page 26-38 in the lab manual. This section will go into detail on the purpose of these methods and how they aid the experiment.

A BEV growth curve samples series was prepared prior to the labs and we set up cell cultures and performed a number of assays on the next day, these results where taken the next week and can be seen in the result section below.

The first assay we performed was a plaque assay. A series of diluted virus samples were created and used to infect the BHK cell monolayers causing an infectious centre in a susceptible cell. This formed PFU’s which were then counted after they had been revealed by staining. From this number and with the knowledge of volume and dilution factor employed the concentration of infectious virus in the original sample and the number of plaque forming units per sample unit volume (pfu/ml) can be estimated. (Figure 1)

The next assay used was the TCID50 assay. Multiple replicates of cell monolayer culture were created and again, infected with a series of virus sample dilutions, incubated and the pfu’s observed after staining. (Figure 2)

A viral protein synthesis assay was conducted. To distinguish between the host and viral protein, the viral protein was pulse-labelled. The cells are lysed and proteins classifying due to size using SDS-polyacrylamide gel, then observed under x-rays to illuminate the radioactivity. (Figure 3)Lastly a gel electrophoresis was done, however this failed to show any RNA. (Figure 4)

Equipment listFor a fully detailed list of the equipment used in this experiment refer to pages 23-25 of the lab manual. This list consists of the essentials used throughout the experiment.

GMEM-BHK 90mm dishes Versene Trypsin



96 well tissue culture plates 12 well tissue culture plates Inverted microscopes Bovine enterovirus SDS gel sample buffer Microfuge tubes Water baths Agarose gel electrophoresis mini tank Haemocytometer

Results

Figure 1 shows the plaque assay of both 12 well plates. The wells circled in red are the ones we used to count to plaques: 2 hours 10-3 to -4,5 hours 10-3, 8 hours 10-5 to -6, 14 hours 10-5 (Appendix A)



Figure 1b shows the log titre of the plaque assay against time. Calculations can be seen in appendix A.

Figure 2 shows the 96 well plate TCID50 assay along with a map above showing the different time samples and dilutions.



Figure 3 shows our viral protein assay for the mock, 2 hours, 5 hours and 8 hour samples (labelled). The ladder referred to in this report is in the red box; the other ladders seen are from another group and are not included in this report.

Figure 4 shows the intracellular viral RNA assay, however no RNA was present on our sample or any sample from the class.

DiscussionFigure 1 shows the plaque assay of both 12 well plates. The wells circled in red are the ones we used to count to plaques: 2 hours 10-3 to -4,5 hours 10-3, 8 hours 10-5 to -6, 14 hours 10-5 (Appendix A). The other samples didn’t contain any cells and are therefore clear as the stain could not bind, some wells didn’t have any plaques as the virus didn’t infect any cells, this may be due to the higher dilutions as there wasn’t enough virus present to cause infection. The samples with a less diluted virus and more time formed more plaques as there was more of the virus present to infect and kill the cells.



Figure 1b shows the log10 titre of each sample against time, showing positive correlation so as the time of each sample increases so does the titre from 1.85 at 2 hours 10-3.5 to 2.83 at 14 hours-5, this is a difference of 0.98 in 12 hours. This suggests that the concentration of the virus increases with time as the virus undergoes more replication cycles and therefore infects more cells.

Figure 2 shows the different virus dilutions of the TCID50 assay at 2,5,8 and 10 hours, as well as a mock and controls (+ and -). This method provides insight into the the cytopathic effects of the Bovine enterovirus at different stages of its life cycle.At the lower dilutions of the bovine enterovirus the wells should be completely blue as no cells should die as there aren’t enough viruses present to kill them. However this can only be seen in the control ‘-‘wells (H10 and H12) that contain no virus, this may be because the dilutions weren’t big enough and the virus was still capable of killing the cells. The majority of the wells contained no cells as they have been killed off by the virus, therefore are clear, this should not be the case. The wells that contain both blue stain and clear patches shows that some cells are alive and some dead, we referred to this dilution as the end-point of the assay and used it to calculate the 1 TCID50 unit of virus that causes cytopathic effects in 50% of the cultures.(Appendix B)

The end point and titre show us the concentration of virus needed of each time sample to cause infection. (Appendix B) We can see that the bovine enterovirus had the highest titre at 10 hours with x10-1.7 and the least at both 5 and 8 hours with 103.7. This suggests that the bovine enterovirus is more pathogenic at 10 hours and less at 5 and 8 hours. This may be due to a larger amount of viruses present in the 10 hour sample as it had more time to replicate before being harvested. However the end points are inaccurate as there is no clear identification of when the cells are alive and others are dead, as the majority of the cells had been killed. This again may be due to a dilution too high and the viruses killing all the cells or due to a contaminant; the cells could also have been damaged and washed away during staining.

Figure 3 show the ladder of the viral protein assay, it should be noted that some proteins share the same weight and are therefore present on the same location on the ladder, but may not be the same. It should also be noted that the darker lines indicate a higher amount of that protein.It can be seen that there is a larger variety and abundant of proteins in the 5 hour sample than the 2 hour sample (36 kDa); however there seems to be less variety and abundance of protein in the 8 hour sample. This may be because the samples are at different stages of the replication cycle. The 2 hour sample could be near the start of the cycle and the genome was being replicated, therefore there isn’t as much polyprotein. The 5 hour sample may have synthesised the polyprotein so there is more protein and the 8 hour sample may be on the verge of lysing therefore the polyprotein has been cleaved and there is less protein. The mock has more protein suggesting that in the samples the virus has taken over the cell and stop the production of host proteins.



Figure 4 shows no signs of protein on the SDS-polyacrylamide gel; the same results were seen by all partners in the lab so the reason must be a communal one. This reason could have been a variety of things, such as reverse transcription didn’t take place due to the superscript 2 being inaccurate and therefore no protein was synthesised. Another theory could be the 100,000 dilution was too high and synthesis couldn’t occur or it was so diluted it could not be seen on the film. There is also the fact that RNA is degraded by ribonucleases which could have contaminated the sample as it is naturally present on our hands. There could have also been a problem with the illuminator that exposed the film to x-rays as the ladder didn’t show up which was prepared prior to the labs, unless it too was prepared using the same batch of possible contaminated materials.

Conclusion To conclude the bovine enterovirus caused infection and underwent a number of infectious cycles in the BHK cells. It also produced protein in the viral protein assay outlining its life cycle and the effects on the host cell. However more insight could have been gained if the viral RNA sample had worked. Overall the bovine enterovirus had a significant impact on the BHK cell cultures and the three assays that worked did well to measure the parameters of the bovine enterovirus.

Appendix

Appendix A

Number of plaques Titre Non diluted titre

2 hours 10^-3.5 7.21111 72.1111 228035.32065 Hours 10^-3 16 160 160000

8 Hours 10^-5.5 23.664319 236.64319 74833147.3214 Hours 10^-5 68 680 68000000

Sample Titre Log102 hours 10^-3.5 72.1111 1.85800212

5 Hours 10^-3 160 2.204119983

8 Hours 10^-5.5 236.64319 2.374094011

14 Hours 10^-5 680 2.832508913

Appendix B

96 well end points



Sample End point 0.05ml Titre in 1mlMock x10-3.5 x10-2.2

2 hours x10-4 x10-2.7

5 hours x10-5 x10-3.7

8 hours x10-5 x10-3.7

10 hours x10-3 x10-1.7

The titre of 1ml was calculated by multiplying the 0.05ml by 20 as 0.05mlx20=1ml


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Measuring parameters of Bovine Enterovirus infection