Virus Growth Protocol Summer 2023

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Arizona State University *

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Feb 20, 2024

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Detection and Quantitation of Viruses LEARNING OBJECTIVES Overall: Examine growth of viruses in tissue culture Detailed: 1. Describe a plaque and compare/contrast it to a bacterial colony 2. Define a virus titer 3. Relate a plaque assay to the steps of virus growth in a cell 4. Predict expected results of an experiment 5. Analyze the results of a simple experiment showing viral plaques 6. Calculate PFU BACKGROUND Viruses are defined as acellular, infectious agents that absolutely require host cells to multiply. In comparison to bacteria, viruses are smaller in size and different in their growth patterns. Unlike bacteria which are capable of independent growth in the presence of nutrients, viruses need a living host cell to replicate within. Bacterial cells multiply exponentially (1 → 2 → 4 → 8) by a process called Binary Fission. This process continues until environmental conditions become limiting (ex. nutrition depletion and accumulation of wastes in the environment of the bacteria). Plotting the growth of bacteria over time will result in a sigmoid shaped growth curve with distinct phases (refer to bacterial growth in Module 2). When a virus attaches and enters a cell, it replicates and assembles new virus particles (virions) within the cell. During this period, the virus cannot be detected in the medium outside the cells. The infected cell will eventually burst and release thousands of virions into the surrounding medium in one single event. Each released virion has the potential to infect other cells and continue the process. Plotting the growth curve of virus (number of virus particles VS time) displays a “step - wise” pattern; each step represents the release of virus from infected cells. While discussing viruses, we often use the terms Tropism and Host range to describe them. Tropism is defined as the ability of virus to infect different cell types. A virus that can infect only few types of cells is defined as “ narrow ” in tropism, while a virus that can infect multiple cell types has broad tropism. Host range refers to the different types of organisms that a virus can infect. Host range may also be defined as broad or narrow depending on the number of different animals that a virus can infect. Both tropism and host range are determined by the ability of the virus to attach to surface receptors for entry into cell. The purpose of this exercise is to understand methods used to cultivate and quantitate viruses in the lab. The study of viruses in a lab involves growing them within host cells. Many different types of cells (eukaryotic and prokaryotic cells) can serve as hosts for viral growth. When grown in uniform layers or “ lawns ” on agar plates, bacteria can serve as hosts for bacteria- specific viruses called bacteriophages . When bacteriophages are introduced into a lawn of

bacteria, they kill bacterial cells and form plaques. These plaques are visualized as clear spaces within the bacterial lawn (FIG. 1). A count of the plaques is used to quantitate how many bacteriophages were present within the original sample used to infect bacteria. When animal cells are used to propagate animal viruses, these cells are incubated in presence of nutrients (dissolved in liquid medium) in flat culture flasks (FIG 2B). This process of growing animal cells is called cell culture. The animal cells divide approximately once in every 24 hours (compared with bacteria like E. coli which can divide every 20 minutes). As they grow, these cells attach to the surface of the culture flask and form monolayers; a single sheet of cells adhering to the bottom of the flask (FIG. 2A). While cultivating animal viruses, the monolayer of animal cells is infected with the virus and will serve as host for viral replication. Eventually, the viruses will destroy cells in the monolayer and be released into the surrounding liquid medium. The liquid medium can be harvested as a source of virus. Virus-infected cells have distinct morphological alterations compared with uninfected cells. These alterations are called cytopathic effect (FIG. 3b) and can be observed under a microscope. Virus-induced cytopathic effects in cells may include a change in shape, shrinkage, detachment from the surface and rounding and cell lysis. These changes can be detected by comparing to a sample of uninfected cells (FIG. 3a). Several viruses, such as HIV and measles, cause specific cytopathic effects which aid in detection of infection. FIG. 1 Bacteriophages grown on an E. coli lawn will form clearing called plaques.

FIG. 2. A) Animal cells form a uniform monolayer when grown in culture flasks. B) Culture flasks. FIG. 3: An intact monolayer of monkey kidney cells seen under a microscope. a) mock infection and b) infection with virus. Cytopathic effect (rounding and detachment) is observed in cells after infection. B) a) b) Viruses 2020 , 12 (2), 180; https://doi.org/10.3390/v12020180

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PROTOCOL One of the most common method for determining the concentration of viruses in a sample is the plaque assay (FIG 4). In this assay, animal cells are grown under appropriate conditions as a single layer of cells attached to a surface like plastic dish. When such a monolayer is exposed to a virus sample, the virus will infect the cells in the monolayer and form zones of clearing called plaques (FIG 4B). A plaque is formed as a result of a single virus particle infecting and multiplying within a host cell. This infected cell subsequently bursts open to release progeny viruses which will infect neighboring cells and lyse them resulting in a clearing or plaque. Each plaque is initiated by a single virus infecting a cell. These plaques can be counted to determine the number of viruses in the original sample that initiated the process. The titer of the virus solution is expressed in terms of a Plaque Forming Unit (PFU)/mL . PFU are analogous to CFU (colony forming units) of bacteria. For example, 5 PFUs represent 5 virus particles in the original sample, each of which have initiated the formation of a plaque on a monolayer of cells. A plaque assay consists of the following steps: 1. Making dilutions of the virus 2. Using the virus dilutions to infect cells in a plaque assay Plaque Assay: Dilutions of the virus: Dilutions are made because there are too many viruses in the sample, and counting them is difficult. You make several dilutions and use them to infect cells. While counting plaques, you select a plate with 30-300 plaques for counting purpose. I. Making dilutions and calculating the dilution factor: A. Make 1:10 dilution or 10 -1 dilution of the original virus sample: 1 ml of original virus sample + 9 ml of broth = 10 ml total volume in tube = 1:10 B. Make 1:100 or 10 -2 dilution of the original virus sample 1 ml of the diluted sample from step A + 9 ml of broth = 10 ml total volume in tube = 1:100 of the original virus sample. ( So, you are not using the original virus sample to make the dilution in B, but rather you are using the previous dilution from step A) C. Make 1:1000 or 10 -3 dilution of the original virus sample 1 ml of the diluted sample from step B + 9 ml of broth = 10 ml total volume in tube = 1:1000 of the original virus sample ( So, you are not using the original virus sample to make the dilution in C, but rather you are using the previous dilution from step B) D. Make 1:10,000 or 10 -4 dilution of the original virus sample 1 ml of the diluted sample from step C + 9 ml of broth = 10 ml total volume in tube = 1:10,000 of the original virus sample ( So, you are not using the original virus sample to make the dilution in D, but rather you are using the previous dilution from step C)

E. In a similar fashion, you can make 1:100,000 and 1:1000,000 dilutions of the original virus by using previous dilutions to make successive dilutions. F. Use 0.1 mL of each dilution to infect cells. So, 0.1 mL becomes the plating factor . Plaque Assay: to calculate the concentration of virus particles in the original sample (PFU/mL) 1. Animal cells are grown in plastic dishes. The plastic dish is incubated at 37 0 C, overnight. The cells will attach to the plate and divide to grow in a monolayer. 2. Next, the media is removed from the dish containing cells and 0.1 mL of virus dilutions (discussed in the section above) is added to the dish for one hour. As a control, another dish of cells is “mock infected” (no virus is added in the mock dish). 3. After one hour, the virus is replaced with the nutrient media in the dishes. The dishes are incubated at 37 0 C overnight to resume cell growth. 4. After 24 hours, the media is removed and the cells are stained with a dye for 5 minutes. The dye is removed and cells are visualized under light to count plaques. A plaque is an area of clearing in a confluent area of cells. It represents the spot where one virus has landed, infected an animal cell and eventually lysed it. Each plaque represents a virus particle or plaque-forming unit (PFU). Plaques appear as a clear unstained zone in an intact monolayer of cells which will be stained with the dye. 5. Plaques are counted on the dish that has approximately between 30-300 plaques. The number is recorded as PFU. Also record what dilution of the virus was used to infect this dish that had 30-300 plaques. 6. The titer or concentration of the virus in the original sample is reported as PFU/mL using the calculations shown below. As an example, if you have diluted the virus 1:100 (the dilution factor is 1/100 or 0.01) and if you have used 0.1 mL of this dilution to infect cells (plating factor is 1/10 or 0.1). Now your equation can be substituted with these values as follows: The plaque count becomes the number of plaques that you have counted in a dish between 30- 300 plaques = Plaque count Dilution factor x plating factor = Plaque count 0.01x 0.1

Fig 4. A dilution scheme used for a plaque assay FIG 5. Monolayer of mock uninfected and virus-infected cells. Plaques can be visualized as clear spots on the dish infected with virus. A) Approximately how many plaques can you count in dish in figure 4? B) If cells in figure 4 were infected with 0.1 mL of 1:10,000 dilution of virus, what is the concentration of the virus sample in PFU/mL? While you do not submit an answer to this question, it will help with assessment and worksheet for Viruses in Module 2 Mock Virus

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Virus Growth Protocol Summer 2023

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