Laboratory Diagnosis of viral diseases

Preface:

Laboratory diagnosis of viral infection need a separate concern because,

- Virus is a very tiny particle to be visualized under routine laboratory microscope.

- Virus are obligate intracellular organism and cannot be cultured in artificial culture media.

           

Learning objectives:

Methods for diagnosis of viral infections are:

A. Microscopic methods:

1. Demonstration of inclusion bodies

2. Immuno-fluorescence, Fluorescent microscopic examination

3. Electron microscope

 

B. Molecular techniques for detection of viral genome

1. Polymerase chain reaction

2.Other molecular techniques

 

C. Immunological techniques

1. Detection of viral antigens

2. Detection of viral specific antibodies

 

D. Virus isolation by culture methods:

1. Egg inoculation

2. Animal inoculation

3. Tissue culture / cell culture

 

A. Microscopic methods:

1. Demonstration of Inclusion bodies:

Definition:Structure with distinct size ,shape, location & staining properties seen under light microscope within virus infected cells are called inclusion bodies.

Methods to demonstrate inclusion bodies:

1. The inclusion bodies are large enough to be visualized by routine microscope. The inclusion bodies can be stained with Giemsa stain, eosin methylene blue stain, H & E stain.

2. The inclusion bodies can also be stained with fluorescence dyes & visualized under fluorescent microscope.

Examples of inclusion bodies:

Intracytoplasmic inclusion bodies:

            Negri bodies: Rabies

            Molluscum bodies: Molluscumcontagiosum

Intra-nuclear bodies:

            Herpes virus, Yellow fever virus, Adeno virus, Polio virus

2. Florescent microscope:

This method can detect viral particles / viral antigens / inclusion bodies in the speciemen

Principle: Speciemen is smeared on the slide and fixed. A fluorescent dyes tagged on antibodies which are specific against viral antigens is used to stain the smear. The stained smear is examined under fluorescent microscope.

Examples:

Anti rabies antibodies tagged with auramin dye is used to detect rabies viral particle / inclusion bodies in the smear from brain speciemen or impression smear from cornea.

3. Electron microscope:

This type of microscope can demonstrate as small as 20 nm sized viral particles. However this method cannot be employed for routine diagnostic laboratory and its use is limited up to research level.

Principle:

The speciemen is stained by potassium phosphotungstate. An electron beam instead of light is allowed to pass through the stained object. Instead of glass lens, electromagnetic lenses are used to capture the electrons getting scattered from the object. These produce a magnified image on the screen.

Types of electron microscopes:

 

i.                    Transmission electron microscope

ii.                  Scanning electron microscope

Clinical application:

Rarely it can be applied for detection of rota virus or hepatitis A virus from stool speciemen.

 

B. Molecular techniques for detection of viral genome

1. Polymerase chain reaction

- Conventional PCR: The virus specific genetic sequence (template) is defined by a set of the primers. With use of these primers, the multiple copies of the template are developed in the laboratory which is detected by electrophoresis.

- Reverse trancriptase - PCR : used for detection of RNA viruses. In this method, the viral RNA is first reversely transcribed to its complementary DNA strand; This DNA sequence is further identified by PCR. e.g Dengue virus, Influenza viruses

- Real time PCR: During each cycle of amplification, the number of sequences multiplied are measured; This will reflect the initial number of viral nucleic acids. So, the viral quantity in the speciemen can be detected by this method: e.g HIV viral load.

 

2.Other molecular techniques

- Gene probe

- Southern blot analyses: for DNA virus

- Northern blot analyses: for RNA virus

- Restricted fragment polymorphism (RFLP)

C. Immunological techniques

1. Detection of viral antigens

Various serological techniques used for detection of antigens:

            ELISA, immunocromatography (ICT), Reverse passive haemagglutination (RPHA)

These technologies are useful for diagnosis of various viral infections:

- Hepatitis B surface antigen in Hepatitis B viral infection

- NS1 antigen in Dengue viral infection

- p24 antigen detection in HIV infection

 

2. Detection of viral specific antibodies

Various serological techniques used for detection of virus specific IgG or IgM antibodies in patient's serum:

- Enzyme Linked Immuno-sorbent Assay (ELISA)

- Immunochromatography

- Passive agglutination

- Immuno-fluroscence

These technologies are useful for diagnosis of various viral infections:

- Human Immuno deficiency Virus

- Arboviruses i.e. Dengue, Chikunguniya, Japanese encephalitis, crimean - congo haemorrhagic fever

- Hepatitis viruses: Hepatitis A, Hepatitis C, Hepatitis D & Hepatitis E, Antibodies against hepatitis B surface antigens and core antigens

- Herpes viruses: i.e. Herpes, Cytomegalo virus

- Measles, mumps, rubella

 

D. Virus culture:

Speciemen collection & transport:

Appropriate speciemen which most likely contains aetiological viral agent should be collected and transported to the laboratory without delay.

In case of delay in transportation, the speciemen must be transported in viral transport media (A buffered solution containing antibiotics for bacterial growth inhibition and protein for maintenance of viability of viruses.)

Viruses are obligate intracellular organism. So, they need cells for their growth. There are 3 different methods for virus culture

1. Animal inoculation:

Laboratory animals used for study are:

Mice ( infant mice is preferred ), Guinea pig, Rabbit etc.

The route of administrations:

Intra-cerebral, subcutaneous, intra-peritoneal, intra-nasal etc.

The growth of virus in inoculated animals may be evident by:

Death, disease or development of visible lesion

Uses:

- Diagnosis of viral disease (these method is not used nowadays).

- To study pathogenesis, immune response and epidemiology of agents

 

2. Egg inoculation:

The speciemen can be inoculated at four different sites in embryonated egg depend upon type of viruses

i. Chorioallantoic membrane (CAM): e.g. for Variola or vaccinia virus forms pocks on CAM

ii. Allantoic cavity: Used to have large population of viruses particularly for vaccine production e.g. Influenza viruses, paramyxoviruses, rabies viruses

iii. Amniotic sac: Used for isolation of influenza viruses.

iv. Yolk sac: For isolation of some viruses including arboviruses; rickettsiae, chlamydiae.

 

3. Tissue culture / cell culture:

Tissue culture:Speciemen may be inoculated in tissue or a small piece of organ maintained in laboratory to give good yield of viruses. However this method is rarely used.

Cell culture:

Principle: The cells are dissociated from the tissue or organ in the laboratory with the help of proteolytic enzyme like trypsin. These cells are washed and suspended in the medium (Minimum Essential Medium) which support viability and growth of cells. The suspended cells are then dispensed in bottles, tubes or petri dishes. The cells adhere to glass surface of these containers. On further incubation, they will grow, multiply and form a sheet of cells on the surface of container. Speciemen is inoculated over it; after proper incubation, virus present in speciemen infect the cells and produce its growth effect (Cytopathic effect). This is evidence for presence of virus in the inoculated speciemen.

Minimum Essential Medium (MEM)

            This medium is composed of essential amino acids, vitamins, salts, glucose and buffering system. Phenol red is added as an indicator.

Development of cell line:

- The cells suspended in MEM.

- This is supplemented with 5% fetal calf serum.

- Antibiotic is added for prevention of bacterial growth.

- This suspension is dispensed in bottle, tube or petri dishes & incubated at 37* C with 5 % carbon dioxide.

- Cells will adhere to the glass surface & divide to form a monolayer sheet over entire glass surface which is covered with the medium.

Examples of cell culture:

A. Primary cells culture: These are formed by normal cells of the body; They have limited growth potentials;  They cannot be maintained by serial passage. e.g. Monkey kidney cell culture

B. Diploid cell strain: These cells can be maintained for maximum up to 50 serial passage. e.g. human fibroblast.

C. Continuous cell lines: These usually developed from cancer cells, which are capable of continuous serial passage for indefinite times e.g. HeLa, HEp-2, Vero cell lines.

Detection of virus growth in cell culture:

Speciemen inoculated in cell culture is incubated to allow the growth of virus present in it. The viral growth in cells can be evident by,

1. Cytopathic effect: Virus can change the morphology of cells in which they grow. These are known as cytopathic effects (CPE). e.g. Enterovirus will lead degeneration of cells & make them retractile and crenated. CPE in cells can be detected by inverted microscope. 

2. Metabolic inhibition: The metabolism of infected cells will be inhibited by intracellular growth of virus. This can be detected by pH indicator present in cell culture medium (MEM). The virus infected cells would not produce acid.

3. Hemadsorption: The wash erythrocytes will be adsorbed over virus infected cells. e.g. in case of influenza and parainfluenza viruses.

4. Immuno-fluorescence: Viral antigens will be expressed over infected cells. The fluorescence conjugated antiserum against these viral antigens will bind to infected cells and stain them. These fluorescence stained cells can be demonstrated by fluorescent microscope.