How to Diagnose Viral Infections and Cultivate Viruses: A Step-by-Step Overview

How to Diagnose Viral Infections and Cultivate Viruses. Master the diagnosis of viral infections and the cultivation of viruses. Critically examine the complete guide covering key lab methods and step-by-step procedures to use.

DIAGNOSIS OF VIRAL INFECTION & CULTIVATION OF VIRUSES

BY MRS. M. I. OKON

THREE (3) GENERAL APPROACHES TO THE DIAGNOSIS OF VIRAL INFECTIONS

1. Direct detection of the viral components (genome, antigens): In cells derived from infected tissues or in fluid specimens

2. Isolation of viruses: In cell cultures, tissue cultures followed by identification of the isolate

3. Detection & Measurement of antibody titer produced against the viral agent in patient’s serum or the presence of IgM antibodies specific to the etiologic virus

DIRECT DETECTION OF VIRAL COMPONENTS

Rapid diagnostic tests

Only few hours required after receiving the specimens to detect the virus.

These tests include;

1. Direct Electron Microscopy (EM) examination of the specimens. e.g. examination of the stool for Rotavirus & Hepatitis A. Mainly for detecting viral gastroenteritis; adenovirus, calicivirus, Norwalk-like viruses etc. Occasionally used to detect viruses in skin lesions.

2. Immune electron microscopy: Allows visualization of virus particles present in numbers too small for easy direct detection. For enhanced sensitivity and specificity of detection with EM

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Disadvantages of EM: expensive; requires a highly skilled observer, poor sensitivity in sample required for visualization.

3. Antigen detection method: The antigen of the virus is detected by immunofluorescent technique. e.g. detection of the RSV antigen in the respiratory secretions; detection of rotavirus antigen in faeces; detection of HSV and VZV in skin scrapping.

Advantages: rapid (hours)

Disadvantages: often tedious, poor sensitivity and specificity, result is difficult to read and interpret

4. Agglutination method: Here antibody is added to the specimen to detect the presence of the viral antigen. E.g detection of HBsAg in serum (this is usually considered as a serological test)

5. Molecular technique: Use to detect viral genome & genome sequence in clinical specimens. Examples of test include; Southern-blot, Nucleic acid hybridization, PCR.

Tests are very sensitive.

ISOLATION OF VIRUSES/CULTIVATION OF VIRUSES

Why Culture Viruses

• To isolate and identify viruses in clinical samples

• To carry out research on viral structure, replication, genetics and effects on the host cell

• To prepare viruses for vaccine production

Viruses can only grow on living cells

1. In vitro- cell or tissue culture methods

2. In vivo- laboratory-bred animals and embryonic bird tissues

Methods for the Cultivation of Viruses

1. Inoculation of virus into animals

2. Tissue culture

3. Inoculation of virus into embryonated eggs

ANIMAL INNOCULATION

Viruses can be cultivated in laboratory animals such as mice, guinea pig, hamster and rabbits. The selected animals should be healthy and free from any communicable diseases. Mice (less than 48 hours old) are most commonly used. Mice are susceptible to togavirus and coxsackie viruses, which are inoculated by intracerebral and intranasal route. After inoculation, virus multiply in host and develops disease. The animals are observed for symptoms of disease and death. Then the virus is isolated and purified from the tissue of these animals. Live inoculation was first used on human volunteers for the study of yellow fever virus.

Advantages of Animal Inoculation

• Diagnosis, Pathogenesis and clinical symptoms are determined.

• Production of antibodies can be identified.

• Primary isolation of certain viruses.

• Mice provide a reliable model for studying viral replication.

• Used for the study of immune responses, epidemiology and oncogenesis.

Disadvantages of Animal Inoculation

• Expensive and difficulties in maintenance of animals.

• Difficulty in choosing of animals for particular virus

• Some human viruses cannot be grown in animals, or can be grown but do not cause disease.

• Mice do not provide models for vaccine development.

• Issues related to animal welfare systems.

INOCULATION INTO EMBRYONATED EGG

Goodpasture in 1931 first used the embryonated hen’s egg for the cultivation of virus. The process of cultivation of viruses in embryonated eggs depends on the type of egg which is used. Viruses are inoculated into chick embryo of 7-12 days old. For inoculation, eggs are first prepared for cultivation, the shell surface is first disinfected with iodine and penetrated with a small sterile drill.

After inoculation, the opening is sealed with gelatin or paraffin and incubated at 36°c for 2-3 days. After incubation, the egg is broken and virus is isolated from tissue of egg. Viral growth and multiplication in the egg embryo is indicated by the death of the embryo, by embryo cell damage, or by the formation of typical pocks or lesions on the egg membranes.

Viruses can be cultivated in various parts of egg like chorioallantoic membrane, allantoic cavity, amniotic sac and yolk sac.

Chorioallantoic Membrane (CAM): Inoculation is mainly for growing poxvirus, Herpes simplex virus. After incubation, visible lesions called pocks are observed, which is grey white area in transparent CAM. This method is suitable for plaque studies.

Allantoic cavity: Inoculation is mainly done for production of vaccine of influenza virus, yellow fever, rabies. Most of avian viruses can be isolated using this method.

Amniotic sac: Inoculation is mainly done for primary isolation of influenza virus and the mumps virus. Growth and replication of virus in egg embryo can be detected by haemagglutination assay.

Yolk sac inoculation: It is also a simplest method for growth and multiplication of virus.

• It is inoculated for cultivation of some viruses and some bacteria (Chlamydia, Rickettsiae)

Advantages of Inoculation into embryonated egg

• Widely used method for the isolation of virus and growth.

• Ideal substrate for the viral growth and replication.

• Isolation and cultivation of many avian and few mammalian viruses.

• Cost effective and maintenance is much easier.

• Less labor is needed.

• The embryonated eggs are readily available.

• They are free from contaminating bacteria and many viruses.

• Widely used method to grow virus for some vaccine production.

Disadvantages of Inoculation into embryonated egg

The site of inoculation varies with different virus. That is, each virus has different sites for their growth and replication.

TISSUE CULTURE

They include;

1. Organ cultures – are mainly done for highly specialized parasites of certain organs

e.g. tracheal ring culture is done for isolation of coronavirus.

2. Cell culture – it is mostly used for cultivation of viruses.

Cell culture is mostly used for identification and cultivation of viruses. Cell culture is the process by which cells are grown under controlled conditions. Cells are grown in vitro on glass or a treated plastic surface in a suitable growth medium.

At first growth medium, usually balanced salt solution containing 13 amino acids, sugar, proteins, salts, calf serum, buffer, antibiotics and phenol red are taken and the host tissue or cell is inoculated. On incubation the cell divides and spreads out on the glass surface to form a confluent monolayer.

Types of cell culture based on the origin and the chromosome property of the tissue culture are classified into 3 types.

1. Primary cell culture: These are normal cells freshly taken from animal or human body. They are able to grow only for limited time and cannot be maintained in serial culture. They are used for the primary isolation of viruses and production of vaccine.

Examples: Monkey kidney cell culture, Human embryonic kidney, chick embryo cell culture.

2. Diploid cell culture (Semi-continuous cell lines): They are diploid and contain the same number of chromosomes as the parent cells. • They can be subcultured up to 50 times by serial transfer. They are used for the isolation of some fastidious viruses and production of viral vaccines. Examples: Human embryonic lung strain, Rhesus embryo cell strain.

3. Continuous cell lines: They are derived from cancer cells. They can be serially cultured and are so named as continuous cell lines. They can be maintained either by serial subculture or by storing in deep freeze at -70°c. Due to derivation from cancer cells they are not useful for vaccine production. Examples: HeLa (Human Carcinoma of cervix cell line), HEP- 2 (Human Epithelioma of larynx cell line), BHK-21 (Baby Hamster Kidney cell line).

DETECTION AND MEASUREMENT OF ANTIBODY TITRE (SEROLOGY)

Serological test used to detect antibodies produced against viral antigens. IgM is the first antibody to appear followed by IgG with much higher titer.

From these serological tests we have;

1) Radioimmunoassay (RIA)

2) Enzyme immune assay (EIA)/ELISA: Better sensitivity, specificity and reproducibility than classical techniques.

3) Indirect immunofluorescent tests.

4) Hemagglutination inhibition test, measure antibodies directed against hemagglutinating viruses, such as influenza viruses.

5) Complement fixation test.

The aim of the serological tests;

1- Detect a significant increase in the titer of antibodies to the etiological virus.

2- The presence of IgM specific antibodies to the etiological virus.

 

 

CONTROL OF VIRAL INFECTIONS/VACCINES

Control of viral infections and diseases involves the use of immunoprophylaxis, antiviral agents and interferons.

IMMUNOPROPHYLAXIS

Immunoprophylaxis against viral illnesses includes the use of vaccines or antibodycontaining preparations to provide a susceptible individual with immunologic protection against a specific disease. Immunization against viral illnesses can be either active or passive.

Active Prophylaxis

Active immunization involves administering a virus preparation that stimulates the body’s immune system to produce its own specific immunity. Viral vaccines now available for use include the following types: (1) attenuated live viruses; (2) killed viruses; (3) recombinant produced antigens. A vaccinee is a person who has been vaccinated.

Immune Response to Vaccines: Vaccination evokes an antibody response and stimulates T lymphocytes. Vaccine effectiveness is assessed in terms of percentage of recipients protected and the duration and degree of protection. Most effective viral vaccines protect more than 90 percent of recipients and produce fairly durable immunity.

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