Methods of derection and identification of viruses

How to test if you have a viruses usually

PCR

PCR

Used to amplify genetics information to large enough quantities that it can be readily observed

Steps of identification

1. Sequence its genetic material, or genetic material, and identify regions that are unique to that specific virus

2. PCR then targets these particular segments by collecting samples

3. The sample is taken to a central laboratory

4. Genetics information can be encoded via DNA or RNA

5. RNA must be reverse transcribed to make a strand of complementary DNA

6. The unique regions of genetic code will be identified by complementary primers primer and copied by enzymes

7. One strand of DNA becomes hundreds of millions

8. Fluorescent dye are able to detected them using probes

9. A sample with fluorescent dye will be marked positive

   
   

1

DNA From cell

2

PCR product

3

PCR anaylsis

4

PCR Amplification

5

Addition of probe

Gel electrophoresis of DNA

What type of genetic material is corona virus made up of

ss-RNA

Methods of detection of viral antigen

1. Immunofluorescence

2. Imunoelectrophoresis

3. Radio immunoassay

4. ELISA

Serology

The development of antibodies to different antigens of the virus is used in staging the disease. For example in HBV and HIV

Serology means

antigen / antibody reaction

Serology forms

the mainstay of viral diagnosis

Immunoassays

ap into the immune system’s memory of the virus, Showing if someone has previously been infected. They work by targeting virus-specific antibodies generated by the immune system during infection. may detect IgG antibodies, the most abundant class , and IgM antibodies, the type that’s first produced in response to a new infection

The presence of IgM antibodies suggests

a recent infection but they are unreliable in a diagnosis

The presence of IgG antibodies suggests

extended period after infection; their presence usually indicates that someone was exposed and recovered.

Digital PCR

quantifies individual pieces of target DNA, shows promise in further boosting accuracy

Results (uninfected)

1. Negative - Take the vaccine

2. Was injured and recovered - Plasma

Results (infected)

1. Plasma

2. Antiviruses

3. Interferon

4. Comfort

Problems associated with Serology

1. long length of time required for diagnosis.

2. mild local infections may not produce a detectable humoral immune response.

3. Extensive antigenic cross-reactivity between related viruses may lead to false positive results.

4. Patients given blood give a false positive result due to the transfer of antibody.

Cultivation of animal virus

1. In living animal

2. In embryoted egg

3. By cell culture

Cultivation and Isolation in a living animals

The animal is exposed to the virus by injection of a viral preparation or specimen into the brain, blood, muscle, body cavity, skin, or footpads. Used in research to study the immune system's response to viral infections.

Example of viruses that are cultivated in living animals

HIV: immunodeficient mice grafted to produce human T cells and human gamma globulin.

Identification in living animals

1. Death

2. Defects

The infected animal tissue can be prepared for

examination with an electron microscopе.

Which virus

Paramyxovirus

What are the cytopathic effects of said virus

Syncytium and faint basophillic cytoplasmic inclusion bodies

Which Virus is shown

Proxvirus

What are the cytopathic effect of said virus

Pink eosinophilic cytoplasmic inclusion bodies (arrows) and cell swelling

Which virus is shown

Herpesvirus

What cytopathic effect said virus has

Cytoplasmic stranding (arrow) and nuclear inclusion bodies (dashed arrow)

Which virus

Adenovirus

What is the cytopathic effect of said virus

Cell enlargement, rounding and distinctive “grape-like” clusters

Clutivation in embryoted egg

Use embryonated chicken, duck or turkey for inoculation of viral suspension.

Identification in embryoted egg

1. Death of the embryo

2. Defects in embryonic development.

3. Localized areas of damage in the membranes, resulting in discrete, opaque spots called pocks (a variant of pox).

The virus injected site in an embryoted egg

1. Target the embryo

2. Target the Amniotic cavity

3. Target Chorioallantois membrane

4. Target Yolk sac

Example of virus in egg

influenza viruses,

Cultivation using cell culture

Preferred type of growth medium for virus, more convenient than the previous two methods. They use isolated cell from animal that are cultured in vitro. Normal cells will form monolayer.

Identification of cell culture

If viruses are present, the cells of monolayer will deteriorate as they multiply.

CPE

Cell deterioration

CPE can be detected by

1. Plaques by phage

2. Microscopic observation via electron microscope (histopathology)

Problems with cell culture

1. Long period (up to 4 weeks) required for result.

2. Often very poor sensitivity because it depends on the condition of the specimen.

3. Susceptible to bacterial contamination.

4. Susceptible to toxic substances which may be present in the specimen.

5. Many viruses don’t grow in cell culture e.g. Hepatitis B, Diarrhoeal viruses, parvovirus, papillomavirus.

Problems with electron microscopy

1. Expensive equipment

2. Expensive maintenance

3. Require experienced

Indirect Examination

1. Cell culture

2. Eggs

3. Animals

Direct Examination

1. Antigen Detection

2. Electron Microscopy

3. Light Microscopy

4. Viral Genome Detection