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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