Cultivation tasks and models
Cultivation of viral agents is a key step in transitioning to laboratory diagnostic methods. Virus propagation is necessary to solve three fundamental tasks:
- Laboratory diagnostics of infectious diseases in patients.
- Scientific purposes, including detailed study of infection pathogenesis and mechanisms of immunity development.
- Production tasks aimed at obtaining specific diagnostic preparations and vaccines.
For successful virus propagation in modern virology, three main biological models (cultivation media) are used:
- Laboratory animals (in vivo).
- Developing avian embryos (most commonly chicken).
- Cell or tissue cultures (in vitro).
Stages of cultured virus analysis
When working with viral cultures, there is an important methodological distinction between pathogen detection and its precise recognition. This process is strictly divided into two sequential stages.
Virus indication The essence of indication is detecting the very fact of virus replication (reproduction) in the provided biological model. The method is based on identifying the biological properties of the pathogen and the features of its interaction with living cells (e.g., recording the cytopathic effect that the virus exerts on the infected cell).
Virus identification The essence of identification is the precise determination of the taxonomic affiliation of the detected virus (its species and type). This stage is implemented using immunological reactions. They are based on the specific interaction of antigens of the cultured virus with pre-known laboratory antibodies.
Cultivation in laboratory animal organisms
This is historically the first biological model (in vivo). Adult or newborn white mice are most frequently used as objects for infection. Hamsters, rabbits, monkeys, and other animals may also be used.
Features of infection and indication Methods of animal infection strictly depend on the virus tropism — its natural affinity for specific body tissues. Depending on this, routes of administration are chosen: subcutaneous, intramuscular, intranasal, or intracerebral (directly into the brain).
Indication of replication (evaluation of results) is carried out according to the following criteria:
- Appearance of visible clinical symptoms of disease in the animal.
- Pathomorphological changes in tissues and organs detected during autopsy.
- Performance of the hemagglutination assay (HA). It is carried out with a suspension prepared from the organs of the infected animal and allows the detection of viruses capable of agglutinating erythrocytes.
Method limitations Currently, this model is rarely used. The main reasons are: pronounced species refractoriness of animals to many human viruses, high risk of contamination of the experiment by extraneous microbes, significant economic costs for maintaining the vivarium, and serious ethical considerations.
Cultivation in chicken embryos
This method is historically significant and is still widely used in industry, despite certain limitations. Developing chicken embryos aged 5 to 12 days are used as the object.
Technique and spectrum of viruses The infection technique involves injecting the test material into various cavities of the embryo (allantoic or amniotic) or directly into its tissues. This method has high efficacy for cultivating influenza, herpes, smallpox, and several other pathogens.
Advantages and disadvantages Undeniable advantages of the model include:
- The ability to accumulate viral mass in very large quantities.
- Complete absence of latent viral infections in the embryo itself, making the medium "clean".
- High economic accessibility and simplicity of implementation for any laboratory.
The key disadvantage of the method is that many viruses are physically incapable of multiplying in avian embryo tissues.
How to know that the virus has replicated (Indication)? The fact of virus replication in the embryo is judged by the death of the embryo, the appearance of specific lesions on its body or membranes (pocks, local hemorrhages), as well as a positive hemagglutination assay (HA) performed with fluid extracted from the cavities of the infected embryo.
Briefly: answers to topical questions
What types of cell cultures are used to grow viruses in vitro?
To grow viruses in vitro, several types of cell cultures are used.
- Primary cultures — human tissue cell cultures.
- Continuous cell lines — human tissue cell cultures.
- Diploid fibroblasts — human fibroblasts requiring prolonged incubation.
- Pig embryo cell cultures — used to isolate certain viruses (e.g., flaviviruses).
- Fast-growing cell cultures — used in the virological method to isolate DNA viruses.
Which specific immunological reactions are used for virus identification?
Immunological reactions based on the interaction of virus antigens with known antibodies are used for virus identification.
- NT — neutralization test (in cell culture or mice, using type-specific sera).
- DFA — direct fluorescent antibody assay.
- ELISA — enzyme-linked immunosorbent assay.
- CFT — complement fixation test.
- HI — hemagglutination inhibition test (e.g., with goose erythrocytes).
- PHA — passive hemagglutination assay.
What virus indication methods are used in cell cultures, besides evaluating the cytopathic effect?
In addition to evaluating the cytopathic effect, the following methods are used to indicate viruses in cell cultures:
- Formation of intracellular inclusion bodies — detection of clusters of viral particles or their components; inclusions can be intranuclear or perinuclear cytoplasmic.
- Plaque formation phenomenon — detection of negative virus colonies ("sterile" spots in the cell layer under an agar overlay).
- Hemadsorption and hemagglutination reactions — detection of virus interaction with erythrocytes; during hemadsorption, erythrocytes adhere to infected cells.
- Color test — recording changes in medium pH.
To remember the stages of working with a virus, use the association: "First the INDIcator lights up (Indication — is there a virus at all?), and then we look at the ID card (Identification — exactly who is this virus?)".
What else to study on this topic
- Cytopathic effect (CPE) of viruses on cells
- Mechanism of the hemagglutination reaction (HAR)
- Immunological reactions for virus identification
- Viral tropism: mechanisms of tissue affinity
- Cell cultures (in vitro) as the third cultivation model
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