Cultivating viruses involves providing them with a suitable living host environment, as viruses are obligate intracellular parasites, meaning they can only replicate inside living cells. Unlike bacteria or fungi, they cannot grow on artificial nutrient media. Most viruses are successfully grown in cultured cells, embryonated hen's eggs, or laboratory animals.
Primary Methods of Virus Cultivation
The choice of cultivation method depends on the specific virus, its host range, and the purpose of cultivation (e.g., research, vaccine production, diagnosis).
| Cultivation Method | Description | Advantages | Disadvantages |
|---|---|---|---|
| Cell Cultures | Growing viruses in various types of in vitro cell lines (e.g., primary, diploid, continuous) derived from animal or human tissues. | Cost-effective, provides a controlled and uniform environment, allows for observation of cytopathic effects, scalable for research and production. | Requires sterile technique to prevent contamination, some viruses do not grow well or at all in commonly available cell lines, maintaining cell lines can be labor-intensive. |
| Embryonated Eggs | Inoculating viruses into different membranes or cavities of a developing chicken embryo (typically 5-12 days old). | Relatively inexpensive, self-contained and sterile environment, good for propagating large quantities of certain viruses, particularly for vaccine production. | Requires specialized technique for inoculation and harvesting, not all viruses can be cultivated, potential for non-specific reactions from egg components. |
| Laboratory Animals | Infecting live animals with the virus to allow replication in vivo. | Mimics natural infection, useful for studying viral pathogenesis, virulence, immunity, and for testing vaccines and antiviral drugs. | Ethical considerations, high cost, requires specialized animal facilities, genetic variability among animals can affect results, not all viruses cause disease in common lab animals. |
1. Cell Cultures (In Vitro)
Cell cultures are the most widely used method for virus cultivation in laboratories today due to their versatility and ease of manipulation. Cells are grown in sterile containers with specific growth media that provide nutrients and maintain pH.
Types of Cell Cultures:
- Primary Cell Cultures: Directly obtained from fresh tissues (e.g., kidney, embryonic cells) and can be subcultured only a limited number of times before senescence. They most closely resemble the original tissue cells.
- Diploid Cell Strains: These are derived from human embryos or fetal tissues and can undergo a greater number of passages (around 50) before they die. They are often used for vaccine production (e.g., WI-38, MRC-5).
- Continuous Cell Lines: These are immortalized cell lines, often derived from cancerous tissues or transformed through genetic manipulation. They can be propagated indefinitely and are easy to maintain (e.g., HeLa cells, Vero cells, BHK-21). They are extensively used in research and diagnostics.
When viruses replicate within cell cultures, they often cause visible changes known as a cytopathic effect (CPE). These changes can include cell rounding, detachment from the surface, lysis, syncytia (multinucleated giant cell) formation, or inclusion bodies. The presence and type of CPE can help identify the viral infection.
- Example: Poliovirus growth in Vero cells causes characteristic cell rounding and lysis.
2. Embryonated Hen's Eggs
This method is particularly important for the large-scale production of some viral vaccines, most notably the influenza vaccine. Specific sites within the egg are inoculated depending on the virus's tropism.
Common Inoculation Sites:
- Chorioallantoic Membrane (CAM): The membrane lining the shell, used for viruses like vaccinia, herpes simplex virus, and some avian viruses, often resulting in characteristic pocks or lesions.
- Amniotic Cavity: Contains the embryo and amniotic fluid; used for primary isolation of influenza virus.
- Allantoic Cavity: The largest cavity, ideal for high-yield replication of influenza, mumps, and Newcastle disease viruses, crucial for vaccine production.
- Yolk Sac: Primarily used for cultivating chlamydiae, rickettsiae, and some arboviruses.
After incubation, viral growth can be detected by observing lesions on membranes, hemagglutination (clumping of red blood cells by viral particles in allantoic or amniotic fluid), or by examining embryonic death.
3. Laboratory Animals (In Vivo)
Inoculating viruses into living laboratory animals is employed when in vitro methods are unsuitable or when studying the natural course of infection, host immune response, or evaluating vaccine efficacy and antiviral drugs. Animals provide a complete physiological system that mimics human infection more closely.
Considerations:
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Animal Models: Common animal models include mice, guinea pigs, rabbits, ferrets, and non-human primates, chosen based on their susceptibility to the virus.
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Ethical Aspects: Strict ethical guidelines and regulations govern the use of animals in research, ensuring humane treatment and minimizing distress.
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Detection: Viral infection is typically detected by observing disease symptoms (e.g., paralysis, lesions, weight loss), mortality, or by harvesting tissues and organs for viral antigen detection, genomic analysis, or re-isolation of the virus.
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Example: Influenza virus pathogenesis is often studied in ferrets due to their similar respiratory tract physiology to humans.
Challenges in Virus Cultivation
Despite advancements, cultivating all viruses remains a challenge.
- Host Specificity: Some viruses are highly restricted in the types of cells or organisms they can infect and replicate within, making in vitro cultivation difficult or even impossible.
- Uncultivable Viruses: A few viruses, like the hepatitis C virus in its early discovery, could not be readily cultivated under laboratory conditions, requiring alternative molecular methods for study.
- Fastidious Nature: Certain viruses require extremely specific and complex growth conditions that are difficult to replicate in a laboratory setting.
The ability to cultivate viruses is fundamental to virology, enabling the study of viral biology, diagnosis of viral diseases, development of vaccines, and testing of antiviral agents.
