Virus Cultivation in the Cell lines: Introduction, Principle, Procedure, Result- Interpretation, and Keynotes

Virus Cultivation in the Cell lines: Introduction, Principle, Procedure, Result- Interpretation, and Keynotes

Introduction of Virus Cultivation in the Cell lines

Viruses are intercellular pathogens that are dependent on host cell machinery for their multiplication and growth. Nowadays monolayer cell cultures are mostly used in diagnostic and research work in viral diseases.

Cell culture is the most widely used system for the cultivation of viruses. It is a more convenient method compared to other methods like egg inoculation and animal inoculation. Candling of an egg is done prior to specimen inoculation for the cultivation of the virus as shown above picture.

The cell cultures are used for the isolation of viruses from clinical specimens for diagnosis of viral disease. It is also useful for biochemical studies of viral replication, and the production of viral antigens and vaccines. Depending upon the number of divisions that a cell line undergoes in vitro before dying. The cell lines have been classified as primary cell lines, diploid and continuous cell lines. Primary cell lines are often from fetal organs or tissues and have 5 or 10 divisions whereas diploid cell lines are a number of divisions in a culture that is roughly related to the life span of the species of animal.  e.g.  50 for fetal human cells while 10 for fetal cells from horses and cows. Continuous cell lines are cells of a single type capable of indefinite propagation in vitro.

The most commonly used cell culture system in most laboratories includes chick embryo fibroblast cells, human amnion cells, Rhesus monkey kidney cells,  HeLa (Human carcinoma of cervix cell line), Hep 2 (Human epithelioma of larynx cell line), McCoy (Human synovial carcinoma cell line), Vero (Vervet monkey kidney cell line), and W1-38 (Human embryonic lung cell line).

Principle of Virus Cultivation in the Cell lines 

Viruses infect healthy cells that grow in the laboratory. When susceptible cells are used for the inoculation of viruses, they show pathological change,s and viruses, they show pathological changes, and viruses can be harvested from the cells for further tests. The growth of viruses in the cell line can be known by a) cytopathic effects, b) immunofluorescence, c) haemagglutination and haemadsorption, and haemadsorption, and d) interference.

Many viruses kill the infected viral cells in which they grow and bring about detectable changes in the morphology of the cells. These changes are collectively known as cytopathic effects. Some viruses however do not express any cytopathic effect (e.g., rubella virus)

The most important precaution to be taken during the maintenance of cell lines is sterility. Contamination of cell lines should be prevented and even cross-contamination among cell lines should be avoided.

Requirements for Virus Cultivation in the Cell lines 

The following are the requirements for Virus Cultivation in the Cell lines –

  •  Inverted microscope
  • Incubator
  • Hemocytometer
  • Biosafety cabinet (BSC)
  • Sterile glassware
  • Pre-sterilized tissue culture plasticware
  • Pasteur pipettes and measuring pipettes
  • Membrane filter
  • Syringes
  • Vials
  • Discard jar
  • Eagle’s minimum essential medium (MEM)
  • sodium bicarbonate (NaHCO3)
  • EDTA trypsin mixture
  • Fetal calf serum (FCS)
  • Sterile double distilled water
  • Virus inoculum,
  • 70% Ethanol/ spirit and sodium hypochlorite
  •  A monolayer of cell culture in a culture flask is treated with trypsin or versene to disperse cells.
  • Specimen: Suspected virus-infected specimen like the cerebrospinal fluid (CSF), stool, rectal swab, throat swab, etc.

Procedure of Virus Cultivation in the Cell lines 

  1. Discard the trypsin versene mixture and add a small amount of MEM with 10% FCS to the monolayer of cells.
  2. Count the cells with the medium in a hemocytometer for appropriate splitting.
  3. Inoculate the cells into sterile flasks or tubs for viral inoculation.
  4. Fill the new flask with MEM and incubate in a horizontal position.
  5. Select a healthy monolayer, which is also confluent, for viral inoculation.
  6. Inoculate the monolayer of cells with the virus using a sterile Pasture pipette, and incubate at 37°C.
  7. Observe for the cytopathic effect (CPE) 7 days after inoculation.

Quality Control

  1. Sterility precautions should be taken perfectly.
  2. Susceptible cells are to be selected for the appropriate virus.

Observations

  • After incubation, the flasks are observed for confluency, and healthy monolayer cells; and virus-infected cells are classified.
  • Viruses are known to produce cytopathic effects are identified by observing the same in the infected cell lines.
  • Non- cytopathogenic viruses are identified by other methods like immunofluorescence, haemagglutination and haemadsorption, and interference.

Results and Interpretation of Virus Cultivation in the Cell lines 

The cell lines are observed for any cytological alterations that are diagnostic of viral infections.

Keynotes on Virus Cultivation in the Cell lines

  1. The main purpose of virus cultivation is to isolate and identify viruses in clinical samples, to do research on the viral structure, replication, genetics, and effects on the host cells, and to prepare viruses for vaccine production.
  2. The most important aspect to be taken care of in cell cultures is sterility. Hence precautions for sterility should be meticulously followed.
  3. Susceptible cells should be selected for appropriate viral inoculations.

Further Readings on Virus Cultivation in the Cell lines 

  1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173454/
  2. https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&ved=2ahUKEwiOhbiu4_PxAhUx7HMBHfX2AFAQFjAMegQIDBAD&url=https%3A%2F%2Fapps.who.int%2Firis%2Frest%2Fbitstreams%2F1088173%2Fretrieve&usg=AOvVaw2RBaX3pO2PZ3JyW0s7U3Ol
  3. https://www.sigmaaldrich.com/NP/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/microbial-cell-culture/virus-cultivation
  4. https://www.intechopen.com/chapters/40221
  5. https://courses.lumenlearning.com/microbiology/chapter/isolation-culture-and-identification-of-viruses/
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