Objectives and Models of Cultivation
Cultivating viral agents is a key step in diagnostic virology. Propagating viruses is necessary to achieve three fundamental goals:
- Laboratory diagnosis of infectious diseases in patients.
- Research purposes, including the detailed study of infection pathogenesis and mechanisms of immunity.
- Production purposes, aimed at obtaining specific diagnostic reagents and vaccines.
To successfully propagate viruses in modern virology, three primary biological models (culture systems) are utilized:
- Laboratory animals (in vivo).
- Developing avian embryos (most commonly chicken eggs).
- Cell or tissue cultures (in vitro).
Stages of Analysis of Cultured Viruses
When working with viral cultures, there is an important methodological distinction between detecting the pathogen and precisely determining its identity. This process is strictly divided into two consecutive stages.
Viral Indication The essence of indication is proving the actual fact of viral replication in the provided biological model. The method is based on identifying the biological properties of the pathogen and its interaction features with living cells (e.g., recording the cytopathic effect that the virus exerts on the infected cell).
Viral Identification The essence of identification is the precise determination of the taxonomic identity of the detected virus (its species and type). This stage is implemented using immunological assays based on the specific interaction of viral antigens with pre-characterized laboratory antibodies.
Cultivation in Laboratory Animals
This is the historically first biological model (in vivo). Adult or newborn white mice are most frequently used as subjects for inoculation. Hamsters, guinea pigs, rabbits, monkeys, and other animals may also be used.
Inoculation and Indication Features Methods of animal inoculation strictly depend on viral tropism — the virus's natural affinity for specific body tissues. Depending on this, the route of administration is chosen: subcutaneous, intramuscular, intranasal, or intracerebral (directly into the brain).
Indication of replication (evaluating results) is performed using the following criteria:
- Appearance of visible clinical symptoms of disease in the animal.
- Pathomorphological changes in tissues and organs detected during necropsy.
- Performing the hemagglutination assay (HA). This is conducted using a suspension prepared from the organs of the infected animal to detect viruses capable of agglutinating erythrocytes.
Limitations of the Method Currently, this model is rarely used. The main reasons include pronounced species refractoriness of animals to many human viruses, a high risk of experimental contamination with extraneous microbes, significant economic costs for housing animal facilities, and serious ethical considerations.
Cultivation in Embryonated Chicken Eggs
This method is historically significant and is still widely applied in industry despite certain limitations. Developing chicken embryos aged 5 to 12 days are used as the object.
Technique and Spectrum of Viruses Inoculation involves introducing the test material into various cavities of the embryo (allantoic or amniotic) or directly into its tissues. This method is highly effective for propagating influenza viruses, herpesviruses, variola virus, and a number of other pathogens.
Advantages and Disadvantages Undeniable advantages of this model include:
- The ability to accumulate high yields of viral mass.
- The complete absence of latent viral infections in the embryo itself, making the medium "clean".
- High economic accessibility and simple implementation for any laboratory.
A key drawback is that many viruses are physically incapable of multiplying in avian embryonic tissues.
How to Determine Viral Replication (Indication)? The fact of viral replication in the embryo is determined by embryo death, the appearance of specific lesions on its body or membranes (pocks, local hemorrhages), and a positive hemagglutination assay (HA) performed with fluid extracted from the cavities of the infected embryo.