Virus Cultivation- Purposes and Methods

Viruses are obligate intracellular parasites that depend on living host cells for their survival and replication. Unlike bacteria, they cannot be grown on artificial media or agar plates alone. Therefore, virus cultivation requires special techniques that involve the use of whole organisms, animal tissues, embryonated eggs, or cell cultures.

The primary purposes of virus cultivation are:

• To isolate and identify viruses in clinical samples. Demonstration of virus in appropriate clinical specimens by culture establishes diagnosis of viral diseases.
• To do research on viral structure, replication, genetics, and effects on host cell. Virus cultivation allows the study of various aspects of viral biology and pathogenesis.
• To prepare viruses for vaccine production. Virus cultivation is essential for the development and manufacture of vaccines against viral diseases.

Virus cultivation can be done in vivo (within a living organism) or in vitro (outside a living organism in an artificial environment). In vivo methods involve the inoculation of viruses into susceptible animals or plants and observing the signs of infection or disease. In vitro methods involve the inoculation of viruses into suitable cells or tissues grown in culture media and detecting the viral growth by various methods.

In this article, we will discuss the different methods of virus cultivation in vitro, focusing on animal inoculation, embryonated eggs, and tissue culture techniques. We will also describe the advantages and disadvantages of each method and the types of viruses that can be cultivated by them.

The first step in virus cultivation is to collect suitable clinical specimens that may contain the virus of interest. The choice of specimen depends on the type and location of the viral infection, as well as the availability and invasiveness of the sampling procedure. Some general principles for specimen collection are:

• Collect the specimen as early as possible in the course of infection, when the viral load is likely to be highest.
• Collect the specimen from the site of active viral replication or shedding, such as respiratory secretions, blood, urine, stool, cerebrospinal fluid, skin lesions, or genital swabs.
• Use sterile containers and swabs to avoid contamination with bacteria or fungi.
• Transport the specimen to the laboratory as soon as possible, preferably within 24 hours. If transport is delayed, store the specimen at 4°C or -70°C depending on the stability of the virus.
• Use appropriate transport media to preserve the viability of the virus and prevent its degradation by enzymes or pH changes. Transport media may contain buffers, antibiotics, antifungal agents, and protein stabilizers.

Some examples of clinical specimens and their corresponding viral infections are:

• Respiratory secretions: These include nasal swabs, throat swabs, sputum, bronchoalveolar lavage, or nasopharyngeal aspirates. They are used for the diagnosis of respiratory viruses such as influenza, parainfluenza, respiratory syncytial virus, adenovirus, coronavirus, rhinovirus, and measles.
• Blood: This includes serum, plasma, or whole blood. It is used for the diagnosis of systemic or viremic viruses such as hepatitis B and C, HIV, dengue, yellow fever, Ebola, and cytomegalovirus.
• Urine: This is used for the diagnosis of urinary tract infections caused by viruses such as BK virus, JC virus, adenovirus, and cytomegalovirus.
• Stool: This is used for the diagnosis of enteric viruses such as rotavirus, norovirus, astrovirus, enterovirus, and hepatitis A and E.
• Cerebrospinal fluid: This is obtained by lumbar puncture and used for the diagnosis of central nervous system infections caused by viruses such as herpes simplex virus, varicella-zoster virus, enterovirus, arbovirus, and rabies virus.
• Skin lesions: These include vesicles, pustules, ulcers, or crusts. They are used for the diagnosis of cutaneous viruses such as herpes simplex virus, varicella-zoster virus, poxvirus, papillomavirus, and molluscum contagiosum virus.
• Genital swabs: These are used for the diagnosis of sexually transmitted infections caused by viruses such as herpes simplex virus type 2, human papillomavirus, and HIV.

Another method of virus cultivation is the inoculation of live animals. This technique was first used on human volunteers for the study of yellow fever virus in 1900. However, due to ethical and safety issues, human inoculation is now rarely done and only for relatively harmless viruses. Instead, laboratory animals such as monkeys, rabbits, guinea pigs, rats, hamsters, and mice are used for virus isolation .

The choice of animals and the route of inoculation depend on the type of virus to be isolated. Some common routes of inoculation are intracerebral, intraperitoneal, subcutaneous, intradermal, or intraocular . Handling of animals and inoculation into various routes require special experience and training. The inoculated animals are then observed for signs of disease or death. The infected animals are then sacrificed and infected tissues are examined for the presence of viruses by various tests.

Animal inoculation can also be used to study the pathogenesis, immune response, epidemiology, and oncogenesis of viral infections . For example, mice provide a reliable model for studying viral replication. Some viruses that can be isolated by animal inoculation are coxsackie virus, rabies virus, arboviruses, picornavirus, and some herpes viruses.

However, animal inoculation has some disadvantages as well. Immunity may interfere with viral growth, and the animal may harbor latent viruses that can contaminate the results. Animal inoculation is also more expensive and time-consuming than other methods of virus cultivation. Moreover, animal inoculation raises ethical concerns about animal welfare and rights. Therefore, animal inoculation is not widely used for routine diagnosis of viral diseases, but rather for research purposes.

One of the methods for virus cultivation is the use of embryonated eggs, which are fertilized chicken eggs that contain a developing embryo. Embryonated eggs are used for the cultivation of some viruses that grow in the cells of the embryo and its membranes. Embryonated eggs are also used for manufacturing some viral vaccines, such as influenza, yellow fever, and rabies vaccines.

The advantages of using embryonated eggs for virus cultivation are:

• They are inexpensive and readily available
• They provide a sterile and self-contained environment
• They have different types of cells and tissues that can support the growth of various viruses
• They can be inoculated at different sites depending on the virus affinity and yield

The disadvantages of using embryonated eggs for virus cultivation are:

• They may have low sensitivity and specificity for some viruses
• They may have variations in susceptibility and permissiveness among different batches of eggs
• They may have interference from maternal antibodies or other microorganisms
• They may have ethical issues regarding animal welfare

The procedure of using embryonated eggs for virus cultivation involves the following steps:

• Selection of pathogen-free fertilized eggs of 10-12 days old
• Disinfection of the egg shell with iodine and drilling a small hole with a sterile drill
• Inoculation of the virus suspension or specimen into an appropriate site of the egg
• Sealing the hole with melted paraffin wax or adhesive tape
• Incubation of the inoculated eggs for 2-9 days at 35-37°C
• Observation of the eggs for signs of viral growth, such as death of the embryo, formation of pocks or lesions on the membranes, or hemadsorption
• Harvesting of the virus from the infected site by removing the egg shell and membrane with a pipette

The different sites of inoculation and their examples of viruses are:

• Yolk sac: used for cultivation of Japanese encephalitis, Saint Louis encephalitis, West Nile virus, chlamydia, and rickettsia
• Amniotic cavity: used mainly for primary isolation of influenza virus and mumps virus
• Allantoic cavity: used for serial passages and for obtaining large quantities of virus, such as influenza virus, yellow fever virus, and rabies virus
• Chorioallantoic membrane: used for inoculation of some viruses that produce visible lesions known as pocks, such as herpes simplex virus, poxvirus, and Rous sarcoma virus

The detection and identification of viruses in embryonated eggs can be done by various methods, such as:

• Cytopathic effect: observation of morphological changes in the infected cells under a microscope
• Hemadsorption: adsorption of erythrocytes to the surfaces of infected cells due to viral hemagglutinins
• Heterologous interference: inhibition of infection by a cytopathic challenge virus due to prior infection by a non-cytopathic virus
• Transformation: induction of cell growth and loss of contact inhibition by oncogenic viruses
• Light microscopy: demonstration of viral antigens in infected cells by staining with specific antibodies conjugated with horseradish peroxidase
• Immunofluorescence: detection and identification of viral antigens in infected cells by using specific antibodies labeled with fluorescent dyes
• Electron microscopy: visualization of viral particles in infected cells by using high-resolution imaging techniques

In conclusion, embryonated eggs are a useful tool for virus cultivation and vaccine production. However, they have some limitations and challenges that need to be considered. Therefore, alternative methods such as cell culture and molecular techniques are also widely used in virology.

One of the methods for virus cultivation is the use of embryonated eggs, which are chicken eggs that contain a developing embryo. Embryonated eggs offer various sites for the inoculation of viruses, depending on the type and preference of the virus. The different sites of the egg used for virus isolation are :

• Chorioallantoic membrane (CAM): This is a membrane that surrounds the embryo and the allantoic cavity. It is rich in blood vessels and can support the growth of many viruses. Some viruses, such as poxviruses, produce visible lesions on the CAM called pocks, which can be used for identification and quantification of the virus. Other viruses that can be isolated on the CAM include herpes simplex virus, varicella-zoster virus, and vesicular stomatitis virus.
• Amniotic cavity: This is a fluid-filled space between the embryo and the amnion, which is another membrane that surrounds the embryo. The amniotic cavity is mainly used for primary isolation of influenza virus, which can cause death or malformation of the embryo. Other viruses that can be isolated in the amniotic cavity include mumps virus, rubella virus, and reovirus.
• Allantoic cavity: This is a sac-like structure that collects waste from the embryo. It is connected to the CAM by the allantois, which is a tube-like structure. The allantoic cavity is used for serial passages and for obtaining large quantities of virus, such as influenza virus, yellow fever virus, and rabies virus. These viruses can be harvested from the allantoic fluid and used for vaccine production. Other viruses that can be isolated in the allantoic cavity include Newcastle disease virus, avian adenovirus, and infectious bronchitis virus.
• Yolk sac: This is a sac-like structure that provides nutrients to the embryo. It is connected to the embryo by the yolk stalk. The yolk sac can be used for cultivation of some arboviruses, such as Japanese encephalitis virus, Saint Louis encephalitis virus, and West Nile virus. It can also be used for growth of chlamydia and rickettsia, which are obligate intracellular bacteria that share some features with viruses. Other viruses that can be isolated in the yolk sac include coxsackievirus, echovirus, and poliovirus.
• Embryo: This is the developing organism within the egg. It can be inoculated directly by injecting the virus into its heart or brain. This method can be used for isolation of some neurotropic viruses, such as rabies virus and herpes simplex virus type 1. The infected embryo may show signs of disease or death, which can indicate viral infection. Other viruses that can be isolated in the embryo include measles virus, mumps virus, and vaccinia virus.

These are some of the different sites of the egg used for virus isolation. Each site has its advantages and limitations depending on the type of virus and the purpose of cultivation. Embryonated eggs are a convenient and inexpensive method for virus cultivation, but they have been largely replaced by cell culture techniques in modern virology laboratories.

Tissue culture is a widely used method for growing viruses in the laboratory. It involves the use of animal cells that are grown in flasks or other containers using special media that provide the nutrients and environmental conditions for the cells to survive and multiply. These cells can then be infected with viruses and observed for signs of viral replication and infection .

Tissue culture has several advantages over other methods of virus cultivation, such as animal inoculation or embryonated eggs. Tissue culture allows for the isolation and identification of a wide range of viruses, including those that do not cause visible disease in animals or embryos. Tissue culture also enables the production of large quantities of virus for research, diagnosis, or vaccine development. Furthermore, tissue culture can be used to study the interactions between viruses and host cells, as well as the effects of antiviral drugs or immune responses on viral infection .

There are different types of tissue culture that can be used to grow viruses, depending on the origin, characteristics, and lifespan of the cells. These include:

• Primary cell culture: This is a culture of normal cells that are obtained directly from animal tissues and have not been subcultured (transferred to new flasks). Primary cell cultures retain their normal diploid chromosome number and morphology, but they have a limited capacity for growth and division (usually 5 to 10 times) before they die or undergo changes. Primary cell cultures are often used for the initial isolation of viruses, especially those that are fastidious or require specific receptors or factors for infection. Examples of primary cell cultures are monkey kidney cells, human embryonic kidney cells, and chick embryo cells .
• Diploid cell strains: These are cultures of a single cell type that are derived from primary cell cultures and can be subcultured for a longer period (up to 50 times) before they senesce (stop dividing) or change their characteristics. Diploid cell strains maintain their normal diploid chromosome number and karyotype (arrangement of chromosomes), but they have more defined features and compositions than primary cell cultures. They are usually fibroblasts (connective tissue cells) that can be used to grow some fastidious viruses or to produce vaccines. Examples of diploid cell strains are WI-38 human embryonic lung cells and MRC-5 human fetal lung cells .
• Continuous cell lines: These are cultures of a single cell type that are derived from cancerous tissues or transformed by chemical agents, viruses, or oncogenes (cancer-causing genes). Continuous cell lines have an unlimited capacity for growth and division, but they have abnormal and variable chromosome numbers and karyotypes. They also lose their original morphology and function and become more homogeneous and uniform. Continuous cell lines are useful for the propagation and study of a large number of viruses, as well as for vaccine production. Examples of continuous cell lines are HeLa cells (from human cervical cancer), Hep-2 cells (from human laryngeal cancer), and Vero cells (from African green monkey kidney) .

The type of tissue culture used for virus cultivation depends on the sensitivity and specificity of the cells to the virus. Some viruses can infect multiple types of cells, while others require certain receptors or factors that are only present in specific cell types. Therefore, it is important to select the appropriate tissue culture for each virus.

The growth of viruses in tissue culture can be detected by various methods, such as:

• Cytopathic effect (CPE): This is the most common method for observing viral infection in tissue culture. CPE refers to the morphological changes that occur in infected cells, such as shrinking, swelling, vacuolization, rounding up, detachment, syncytium formation (fusion of multiple cells), or lysis (cell death). Different viruses produce different types of CPE that can help in their identification. For example, adenoviruses cause grape-like clusters of cells, enteroviruses cause crenation (shrinkage) and degeneration of the entire cell sheet, herpesviruses cause focal areas of degeneration, and poxviruses cause pocks (raised lesions) on the cell surface .
• Hemadsorption: This is a method for detecting viruses that express hemagglutinins (proteins that bind red blood cells) on their surface or on the surface of infected cells. Hemagglutinins can cause agglutination (clumping) of red blood cells in the culture medium or adsorption (attachment) of red blood cells to the infected cells. This can be observed under a microscope or by using indicator dyes that change color when hemadsorption occurs. Hemadsorption is useful for detecting viruses such as influenza virus, parainfluenza virus, mumps virus, and togavirus .
• Interference: This is a method for detecting viruses that do not produce CPE or hemadsorption in tissue culture. Interference refers to the phenomenon where one virus inhibits the replication or infection of another virus by competing for cellular resources or inducing antiviral responses. Interference can be used to detect non-CPE-producing viruses by challenging them with a known CPE-producing virus and observing whether the CPE is reduced or absent. For example, rubella virus does not produce CPE but interferes with the replication of picornaviruses .
• Transformation: This is a method for detecting oncogenic viruses that induce malignant changes in infected cells. Transformation refers to the loss of contact inhibition (the ability to stop growing when in contact with other cells), which leads to uncontrolled growth and formation of microtumors or colonies on the cell surface. Transformation can be observed under a microscope or by using staining techniques that differentiate normal and transformed cells. Examples of oncogenic viruses that cause transformation in tissue culture are some herpesviruses, adenoviruses, hepadnaviruses, papillomaviruses, and retroviruses .
• Immunological methods: These are methods that use specific antibodies to detect viral antigens in infected cells or viral particles in the culture medium. Immunological methods include immunofluorescence (using fluorescently labeled antibodies), immunoperoxidase (using enzyme-labeled antibodies), enzyme-linked immunosorbent assay (ELISA), immunochromatography (using lateral flow devices), neutralization test (using antibodies that block viral infectivity), complement fixation test (using antibodies that activate complement proteins), hemagglutination inhibition test (using antibodies that block hemagglutination), and agglutination test (using antibodies that cause agglutination) .
• Molecular methods: These are methods that use nucleic acid probes or amplification techniques to detect viral genomes or transcripts in infected cells or viral particles in the culture medium. Molecular methods include polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), real-time PCR (qPCR), loop-mediated isothermal amplification (LAMP), hybridization assay (using labeled DNA or RNA probes), restriction fragment length polymorphism (RFLP) analysis, nucleic acid sequencing, microarray analysis, and next-generation sequencing .
• Electron microscopy: This is a method that uses high-resolution imaging techniques to visualize viral particles or structures in infected cells or in the culture medium. Electron microscopy can provide information on the size, shape, morphology, symmetry, envelope, capsid, spikes, core, nucleocapsid, inclusion bodies, and assembly sites of viruses.

Tissue culture is the most widely used technique for virus cultivation and identification. It involves the growth of cells derived from tissues in an artificial medium under controlled conditions. Tissue culture can be classified into three different types based on their origin, chromosomal characteristics, and lifespan .

Primary cell culture

Primary cell culture is a culture of normal cells obtained directly from the original tissues that have not been subcultured. These cells retain their normal diploid chromosome number and karyotype, and are capable of only limited growth (5–10 divisions) in vitro. They cannot be maintained in serial culture, but can be subcultured to obtain large numbers of cells. Primary cell cultures are usually sensitive to a wide range of viruses and are useful for primary isolation of viruses .

Some examples of primary cell cultures are:

• Monkey kidney cell culture: used for isolation of myxoviruses, paramyxoviruses, enteroviruses, and some adenoviruses .
• Human embryonic kidney cell culture: used for isolation of adenoviruses, picornaviruses, herpes simplex virus, cytomegalovirus, and varicella-zoster virus .
• Chick embryo cell culture: used for isolation of influenza virus, mumps virus, measles virus, and rubella virus .

Diploid cell strains

Diploid cell strains are cultures of a single cell type that retain their original diploid chromosome number and karyotype. They have specific characteristics and compositions and are usually composed of fibroblasts. They can be cultured for up to 50 serial passages before they undergo senescence (die off) or undergo significant changes in their features. Diploid cells derived from human fibroblasts are useful for isolation of some fastidious viruses. They are also used for production of vaccines .

Some examples of diploid cell strains are:

• WI-38 human embryonic lung cell strain: used for cultivation of fixed rabies virus, measles virus, mumps virus, rubella virus, and varicella-zoster virus .
• MRC-5 human fetal lung cell strain: used for cultivation of adenoviruses, picornaviruses, herpes simplex virus, cytomegalovirus, and varicella-zoster virus .

Continuous cell lines

Continuous or immortal cell lines are cultures of a single cell type that are derived from cancerous tissue or transformed by chemical mutagens, tumorigenic viruses, or oncogenes. They have altered and irregular number of chromosomes and can be maintained in serial culture indefinitely without senescing. They are usually less sensitive to viruses than primary or diploid cells, but they are more convenient and economical to use. They have been used extensively for the growth of a large number of viruses .

Some examples of continuous cell lines are:

• Hep-2 human epithelioma of larynx: used for isolation of respiratory syncytial virus, adenoviruses, and herpes simplex virus .
• HeLa human carcinoma cervix: used for isolation of poliovirus, adenoviruses, measles virus, mumps virus, and rubella virus .
• Vero African green monkey kidney: used for isolation of enteroviruses, arboviruses, herpes simplex virus, cytomegalovirus, and rabies virus .

Cell culture is the most widely used technique for virus cultivation and identification. It involves growing cells derived from tissues or organs in an artificial medium under controlled conditions. Cell culture can be used to isolate and propagate viruses, study their replication and pathogenesis, and produce vaccines.

There are different types of cell cultures based on their origin, characteristics, and lifespan. Primary cell cultures are obtained directly from fresh tissues and have a limited number of divisions before they die. Diploid cell strains are derived from primary cell cultures and retain their normal chromosome number and structure. They can be maintained for up to 50 passages before they undergo senescence or lose their properties. Continuous cell lines are derived from cancerous tissues or transformed by viruses or chemicals. They have abnormal chromosomes and can grow indefinitely in culture.

Depending on the type of virus, different cell cultures may be used for virus cultivation. Some viruses can infect a wide range of cell types, while others are more specific and require certain receptors or conditions to enter and replicate in cells. For example, poliovirus can infect human embryonic kidney cells, monkey kidney cells, and HeLa cells (a continuous cell line derived from human cervical cancer), but not mouse cells. Influenza virus can infect monkey kidney cells, human lung cells, and chicken embryo fibroblasts, but not HeLa cells.

To detect the growth of viruses in cell cultures, various methods can be used. Some of these methods are:

• Cytopathic effect (CPE): This is the visible alteration or damage of the infected cells caused by viral replication. CPE can include cell rounding, shrinking, swelling, vacuolation, syncytia formation (fusion of multiple cells), detachment, or lysis (rupture). Different viruses produce different types of CPE that can help in their identification. For example, herpes simplex virus causes focal degeneration and ballooning of cells, adenovirus causes grape-like clusters of cells, and measles virus causes syncytia formation.
• Hemadsorption: This is the attachment of red blood cells to the surface of infected cells due to the expression of viral hemagglutinins (proteins that bind to sialic acid on red blood cells). Hemadsorption can be used to detect viruses that do not cause CPE or cause CPE at a late stage of infection. Hemadsorption can be observed by adding a suspension of red blood cells to the cell culture and examining it under a microscope. Viruses that cause hemadsorption include influenza virus, parainfluenza virus, mumps virus, and some togaviruses.
• Interference: This is the inhibition of infection by a second virus due to the presence of a first virus in the cell culture. Interference can be used to detect viruses that do not cause CPE or hemadsorption. Interference can be demonstrated by inoculating a cell culture with a non-CPE-producing virus and then challenging it with a CPE-producing virus. If the first virus has infected the cells, it will prevent the second virus from infecting them and producing CPE. For example, rubella virus does not cause CPE but interferes with the infection of picornaviruses that cause CPE.
• Transformation: This is the change in the morphology and behavior of infected cells due to the integration of viral DNA or RNA into their genome. Transformation can result in loss of contact inhibition (the ability to stop growing when in contact with other cells), increased growth rate, altered shape, and formation of tumors. Transformation can be used to detect oncogenic viruses (viruses that cause cancer) such as some herpesviruses, adenoviruses, papillomaviruses, and retroviruses.
• Immunological methods: These are methods that use antibodies (proteins that bind to specific antigens) to detect viral antigens (proteins or other molecules that elicit an immune response) in infected cells. Immunological methods include immunofluorescence (IF), immunoperoxidase (IP), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC). These methods involve staining the infected cells with antibodies labeled with fluorescent dyes, enzymes, or other markers that can be visualized under a microscope or measured by a spectrophotometer. Immunological methods are sensitive and specific for identifying viruses.
• Molecular methods: These are methods that use nucleic acid probes (short segments of DNA or RNA that are complementary to a target sequence) to detect viral nucleic acids (DNA or RNA) in infected cells. Molecular methods include polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), hybridization assays, nucleic acid sequencing, and microarrays. These methods involve amplifying, labeling, hybridizing, or sequencing the viral nucleic acids and detecting them by various techniques such as gel electrophoresis, autoradiography, fluorescence microscopy, or mass spectrometry. Molecular methods are rapid and accurate for identifying viruses.

These are some of the methods for growth and detection of viruses in cell cultures. Each method has its advantages and limitations depending on the type of virus, the availability of reagents and equipment, and the purpose of the study.

Virus cultivation is an essential technique for the isolation, identification, and characterization of viruses. It also has important applications in research, vaccine production, and diagnosis of viral diseases. Virus cultivation can be performed by three main methods: animal inoculation, embryonated egg inoculation, and cell culture. Each method has its advantages and limitations depending on the type of virus, the availability of host cells or organisms, the cost and ethical issues involved, and the purpose of cultivation.

Animal inoculation is one of the primary methods for isolation of certain viruses and for study of pathogenesis of certain viral diseases. However, it is expensive, difficult to maintain, and raises ethical concerns. It is also not suitable for some human viruses that do not cause disease or grow well in animals.

Embryonated egg inoculation is a cheap and convenient method for cultivation of many viruses that can infect different sites of the egg, such as yolk sac, amniotic cavity, allantoic cavity, and chorioallantoic membrane. It can also be used for production of large quantities of virus for vaccine preparation. However, it is not applicable for some viruses that do not infect or grow in eggs, or that cause damage to the embryo.

Cell culture is the most widely used method in diagnostic virology for cultivation and assays of viruses. It involves the growth of viruses in dissociated cells from various tissues or organs that are maintained in artificial media. Cell culture can be classified into three types: primary cell culture, diploid cell strains, and continuous cell lines. Cell culture offers many advantages over animal and egg inoculation, such as higher sensitivity, specificity, speed, safety, and versatility. It can also be used for detection and identification of viruses by various methods, such as cytopathic effect, hemadsorption, heterologous interference, transformation, light microscopy, immunofluorescence, and electron microscopy. However, cell culture also has some drawbacks, such as the requirement of specialized equipment and skills, the risk of contamination and mutation, and the limited availability of some cell types.

In conclusion, virus cultivation is a vital tool in virology that requires careful selection of the appropriate method based on the characteristics of the virus and the objectives of the study. Each method has its strengths and weaknesses that should be considered before choosing the best option for a given situation.

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