6.3 Isolation, Culture, and Identification of Viruses
3 min read•june 18, 2024
Viruses, once mysterious filterable agents, revolutionized microbiology. Their discovery in the late 1800s led to virology's birth, challenging the idea that all pathogens were cellular. This revelation sparked intense research into virus-host interactions and infection mechanisms.
Cultivating viruses involves careful specimen collection, isolation, and identification. Methods range from animal studies to cell cultures, each with pros and cons. Quantification and purification techniques are crucial for research, disease monitoring, and developing for therapies.
Historical Context and Virus Cultivation
Historical context of filterable agents
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- Viruses initially discovered as filterable agents that could pass through filters retaining bacteria
- Experiments by Dmitri Ivanovsky and Martinus Beijerinck in late 1800s demonstrated tobacco mosaic disease caused by filterable agent smaller than bacteria ()
- Findings challenged prevailing notion that all infectious agents were cellular microorganisms (bacteria, protozoa)
- Discovery of viruses as filterable agents led to development of virology as distinct field of study
- Researchers began investigating unique properties and behavior of viruses, differing from bacteria and other microorganisms
- Realization that viruses required living host cells for replication sparked further research into virus-host interactions and mechanisms of viral infection (cell entry, replication, assembly, release)
Steps in virus cultivation
- Specimen collection and handling
- Collect appropriate clinical specimens based on suspected viral infection and site of infection
- Nasopharyngeal swabs, blood, urine, stool, or tissue biopsies
- Transport specimens to laboratory promptly under appropriate conditions to maintain virus viability
- Use viral transport media to prevent desiccation and maintain pH
- Keep specimens cold (4°C) or frozen (-70°C) depending on virus and expected processing time
- Collect appropriate clinical specimens based on suspected viral infection and site of infection
- and cultivation
- Inoculate specimens onto suitable host systems
- Cell cultures, , or laboratory animals (mice, guinea pigs)
- Monitor host systems for signs of viral infection
- (CPE) in cell cultures, disease symptoms in animals
- Confirm presence of virus using specific diagnostic tests
- Immunofluorescence assays, PCR
- Determine to quantify the amount of infectious virus in a sample
- Inoculate specimens onto suitable host systems
- Virus identification and characterization
- Perform serological tests to identify virus based on antigenic properties
- Neutralization assays, ELISA
- Conduct molecular tests to detect and characterize virus at genetic level
- PCR, genome sequencing
- Examine virus morphology using electron microscopy to observe distinctive structural features (capsid symmetry, envelope presence)
- Perform serological tests to identify virus based on antigenic properties
Virus Cultivation Methods
In vivo vs in vitro cultivation methods
- In vivo methods involve use of living host systems to cultivate viruses
- Laboratory animals (mice, ferrets) or embryonated eggs
- Advantages:
- Provide more natural environment for virus replication, allowing study of virus-host interactions and pathogenesis
- Useful for viruses that do not replicate well in cell cultures (hepatitis viruses)
- Limitations:
- Ethical concerns associated with animal use
- Higher costs and longer time required for animal studies
- Potential for host adaptation and changes in viral properties
- In vitro methods involve use of cell cultures derived from various tissues or organisms to cultivate viruses
- Primary cell cultures, continuous cell lines, or organ cultures
- Advantages:
- Provide more controlled and standardized environment for virus replication
- Allow study of virus-cell interactions at cellular and molecular level
- Generally less expensive and faster than in vivo methods
- Easier to manipulate and observe viral growth (plaque assays, focus-forming assays)
- Limitations:
- Not all viruses can be cultivated in cell cultures (norovirus, hepatitis viruses)
- Cell cultures may not fully recapitulate complexity of in vivo host systems
- Risk of contamination and artifacts
Virus Isolation and Quantification
- Virus isolation techniques are used to separate and grow specific viruses from complex samples
- measurements determine the amount of virus in a patient's body fluids, crucial for disease monitoring
- methods are employed to obtain highly pure viral preparations for research and vaccine production
- Viral vectors, modified viruses used to deliver genetic material to cells, are important tools in gene therapy and vaccine development
Key Terms to Review (42)
Anchorage dependency: Anchorage dependency is a characteristic of cells that require attachment to a solid or semi-solid surface to grow and proliferate. This phenomenon is crucial in the study of cell cultures, especially for understanding cellular behaviors and virus-host interactions.
Bacterial lawn: A bacterial lawn is a uniform and continuous layer of bacteria grown on an agar plate. This technique is often used to study the effects of bacteriophages and antibiotics.
Bacteriophages: Bacteriophages are viruses that infect and replicate within bacteria. They play a crucial role in bacterial genetics and microbial diversity.
Cell Culture: Cell culture is the process of growing and maintaining cells in a controlled, artificial environment outside of their natural organism. It is a fundamental technique used in various fields, including microbiology, to study the properties and behavior of cells in isolation from the complex in vivo environment.
Contact inhibition: Contact inhibition is a regulatory mechanism that functions to keep cells from proliferating excessively by halting cell division when they come into contact with each other. This process is crucial for maintaining tissue architecture and preventing tumor formation.
Continuous cell line: A continuous cell line is a population of cells from a single origin that can be maintained in vitro for an indefinite number of generations. These cells are often used in research to study viruses and other pathogens.
Cytopathic Effects: Cytopathic effects refer to the structural and functional changes that occur in host cells due to viral infection. These effects are a key indicator of successful viral replication and can be used to detect and identify viruses during the isolation, culture, and identification processes.
Cytopathic effects (CPEs): Cytopathic effects (CPEs) are observable changes in host cells caused by viral invasion. These changes can include cell lysis, syncytia formation, and inclusion bodies.
Cytopathogenesis: Cytopathogenesis refers to the process by which viruses induce pathological changes in the host cells they infect, leading to cellular damage or death. This term is particularly relevant in the context of virus isolation, culture, and identification, as the cytopathic effects observed in infected cells are often used as a diagnostic tool to detect and characterize viral infections.
Embryonated bird’s egg: An embryonated bird's egg is a fertilized bird egg containing a developing embryo, often used as a biological system to culture and propagate viruses. This method allows for the growth and multiplication of viruses in a controlled environment.
Embryonated Eggs: Embryonated eggs refer to chicken eggs that have been incubated and contain a developing embryo. These eggs are commonly used in the isolation, culture, and identification of viruses, as the embryo provides a suitable environment for viral growth and replication.
Enzyme immunoassays (EIAs): Enzyme immunoassays (EIAs) are a biochemical technique used to detect the presence of an antigen or antibody in a sample. The method utilizes an enzyme linked to an antibody or antigen as a marker for detecting a specific target molecule.
Focus-Forming Assay: A focus-forming assay is a laboratory technique used in the field of virology to quantify the number of infectious viral particles present in a sample. It is a crucial method for isolating, culturing, and identifying viruses, as it allows researchers to determine the titer or concentration of viable virus particles that can infect and replicate within host cells.
HeLa cell line: HeLa cells are an immortal cell line derived from cervical cancer cells taken from Henrietta Lacks in 1951. They are widely used in scientific research due to their ability to divide indefinitely.
HeLa cells: HeLa cells are a type of immortalized human cervical cancer cells that have been widely used in biomedical research since the 1950s. These cells are notable for their ability to continuously divide and proliferate, making them a valuable tool for studying various aspects of virology, including virus isolation, culture, and identification.
Hemagglutination: Hemagglutination is the agglutination or clumping of red blood cells, which can occur due to the interaction between certain molecules, such as viruses or antibodies, and receptors on the surface of red blood cells. This process is an important tool in the isolation, culture, and identification of viruses, as well as in various agglutination assays.
Hemagglutination assay: A hemagglutination assay is a laboratory technique used to detect the presence of viruses or antibodies that cause red blood cells to agglutinate, or clump together. It is commonly used in virology for virus quantification and vaccine efficacy testing.
Hemagglutination inhibition (HAI) assays: Hemagglutination inhibition (HAI) assays are laboratory techniques used to detect the presence of specific antibodies against viruses that cause agglutination of red blood cells. It is commonly used for identifying and quantifying viral infections.
Herpes Simplex Virus: Herpes simplex virus (HSV) is a highly contagious virus that can cause a variety of infections, primarily affecting the skin, mucous membranes, and nervous system. It is a member of the Herpesviridae family and is a common cause of viral infections in humans, with two main types: HSV-1 and HSV-2.
Immunofluorescence Assay: The immunofluorescence assay is a powerful analytical technique that uses fluorescent-labeled antibodies to detect and visualize specific target molecules, such as proteins or pathogens, within cells or tissues. This method is particularly useful in the context of virus isolation, culture, and identification.
In vitro: In vitro refers to experiments conducted outside of a living organism, typically in a controlled laboratory environment such as a petri dish or test tube. This method is used to study cellular and molecular processes without the complexity of whole organisms.
In vivo: In vivo refers to experiments or processes performed within a living organism. It contrasts with in vitro, which involves studies outside of a living organism, often in a controlled laboratory environment.
Influenza vaccine: The influenza vaccine is an immunization designed to protect against the influenza virus, commonly known as the flu. It stimulates the immune system to produce antibodies specific to the virus, reducing the risk of infection.
Influenza Virus: The influenza virus is a type of RNA virus that causes the contagious respiratory illness known as the flu. It is a member of the Orthomyxoviridae family and is a significant public health concern due to its ability to cause seasonal epidemics and occasional global pandemics.
Lacks: Lacks refers to cells or biological samples that do not contain a certain element, such as a specific protein, receptor, or genetic material. In microbiology, this term is often used to describe host cells that do not possess particular viral components necessary for infection.
Lysis: Lysis is the process of cell rupture or disintegration, resulting in the release of the cell's contents. This term is particularly relevant in the context of viruses and their interaction with host cells during the viral infection cycle.
NAAT: NAAT (Nucleic Acid Amplification Test) is a molecular technique used to detect the genetic material of pathogens. It is highly sensitive and specific, making it ideal for diagnosing viral infections.
Neutralization Assay: A neutralization assay is a laboratory technique used to measure the ability of antibodies or other neutralizing agents to inhibit the infectivity of a virus. It is a key method in the isolation, culture, and identification of viruses.
Pap smear: A Pap smear is a medical test that collects cells from the cervix to detect cervical cancer and viral infections, such as human papillomavirus (HPV). It involves microscopic examination of the collected cells to identify abnormal changes.
PCR (Polymerase Chain Reaction): PCR, or Polymerase Chain Reaction, is a powerful molecular biology technique used to amplify specific DNA sequences exponentially. It is a fundamental tool employed across various fields, including microbiology, genetics, forensics, and diagnostics, to study and detect the presence of genetic material from a wide range of organisms, including prokaryotes, viruses, fungi, and protozoa.
Plaque Assay: A plaque assay is a technique used to quantify the number of infectious viral particles in a sample by counting the number of plaques, or clear areas, formed on a lawn of host cells. It is a crucial method for isolating, culturing, and identifying viruses in the context of 6.3 Isolation, Culture, and Identification of Viruses.
Polymerase chain reaction: Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify and detect DNA sequences. It allows for the rapid and specific identification of genetic material from various sources, including viruses.
Primary cell culture: Primary cell culture involves the isolation of cells directly from tissues or organs and their subsequent maintenance in an artificial environment. These cultures provide a more accurate representation of cellular physiology compared to established cell lines.
Reverse transcriptase-PCR (RT-PCR): Reverse transcriptase-PCR (RT-PCR) is a laboratory technique used to amplify and quantify RNA sequences by converting them into complementary DNA (cDNA). It is crucial for detecting and studying RNA viruses.
Tobacco Mosaic Virus: Tobacco mosaic virus (TMV) is a plant virus that infects a wide range of plant species, particularly tobacco plants, and is one of the most well-studied and significant viruses in the field of virology. It is an icosahedral, positive-sense, single-stranded RNA virus that serves as a model organism for understanding viral structure, replication, and host-pathogen interactions.
Vero Cells: Vero cells are a continuous cell line derived from the kidney epithelial cells of the African green monkey (Cercopithecus aethiops). These cells are widely used in virology research, particularly for the isolation, culture, and identification of various viruses.
Viral Load: Viral load refers to the amount or concentration of virus particles present in an infected individual's body fluids, typically measured in the blood. It is an important indicator of the severity of a viral infection and is widely used in the diagnosis, monitoring, and treatment of various viral diseases.
Viral Titer: Viral titer refers to the concentration or amount of virus particles present in a sample, typically measured in terms of the number of infectious virus particles per unit volume. It is a crucial metric in the isolation, culture, and identification of viruses, as it provides information about the viral load and helps in the quantification and standardization of viral samples.
Viral Vectors: Viral vectors are modified viruses that are used as vehicles to deliver genetic material, such as DNA or RNA, into target cells. These engineered viruses are designed to infect cells and introduce the desired genetic information without causing the original viral disease.
Virus Isolation: Virus isolation is the process of separating and identifying a specific virus from a sample, such as a clinical specimen or environmental sample. This technique is a crucial step in the identification and characterization of viruses, and it is an essential part of the broader field of 6.3 Isolation, Culture, and Identification of Viruses.
Virus Purification: Virus purification is the process of isolating and concentrating viruses from a complex mixture, such as a biological sample, to obtain a highly pure and concentrated viral preparation. This is a crucial step in the study, identification, and characterization of viruses within the context of 6.3 Isolation, Culture, and Identification of Viruses.
Western blot: Western blot is a lab technique used to detect specific proteins in a sample through gel electrophoresis and antibodies. It is widely utilized for confirming the presence of viral proteins and immune responses.