Viral Diseases How To Diagnose By: Dr. Amr

Special characters of viruses Viruses are prokaryotes; Not cellular, No ribosomes, No membrane-bound organells. Can reproduce ONLY inside host cells “Obligate intracellular”. Reproduction is the only characteristic of life. Very small size: 20-300 nm ‘diameter’. Only 1 kind of nucleic acids (DNA or RNA)

Virus Laboratory diagnosis 1- Direct detection C- Antigens A- Particles B- Inclusion bodies C- Antigens D- Nucleic acid Laboratory diagnosis 1- Direct detection

By Electron Microscope 1- Direct detection A- Viral particles

By Inverted Microscope Site of viral replication 1- Direct detection B- Inclusion bodies = Site of viral replication In the” nucleus or - cytoplasm of infected cells

a- ELISA 1- Direct detection C- Viral antigens

ELISA plate Color change is detected by Spectrophotometer

b- RIA ‘Radio Immune Assay’ 1- Direct detection C- Viral antigens Gamma counter

c- IF ‘Immunofluorescence’ 1- Direct detection C- Viral antigens Solid phase Immunofluorescence slide

Seen with Fluorescent microscope ‘UV’

PCR & RT-PCR, probes 1- Direct detection D- Viral nucleic acid

2- Virus isolation A- Tissue culture Viruses are ‘Obligate intra-cellular’ 2- Virus isolation A- Tissue culture

Cell culture: 1- Primary cell line: Somatic cells from animal ‘monkey kidney cells’ or human. - Maintained for short period in culture. 2- Semi-continuous cell line: Human embryo lung ‘fibroblasts’ Limited passage number (30) Susceptible to many viruses 3- Continuous cell line: Tumor cells ‘HELA’ ( human cervical cancer cells) Indefinite passage number (300) but susceptible to few viruses

Passage: dilution of cells to keep them growing (e.g: twice / week). Culture media FCS ‘foetal calf serum’  Growth factors Amino acids Vitamins Antibiotics & Antifungals Passage: dilution of cells to keep them growing (e.g: twice / week). Detection: CPE ‘Cytopathic effect’ (Cell changes that can be seen by the microscope) Cell death & detachment from surface (polio v.) Rounding & grape-like cluster formation (adeno v.) Syncytium ‘giant cell formation’ (measles, mumps)

If the virus doesn’t produce a CPE, its presence can be detected by: 1- Haemadsorption: attachment of erythocytes to the surface of virus infected cells e.g. in mumps, parainfluenza and influenza viruses 2-Haemagglutination: The HA test can be used to detect and quantitate the virus in vitro. Using sheep RBCs.

3- Hemagglutination inhibition: It is a test used for detection of specific antibodies that could prevent haemagglutination by the virus. 4- Detection of the virus antigens or its genome in infected cells. 5- Inclusion bodies in some infected cells.

2- Virus isolation B- Embryonated egg Some viruses will replicate in the living tissues and membranes of developing embryonated hen’s eggs, such as influenza virus. Egg-adapted strains of influenza virus replicate well in eggs and very high virus titers can be obtained.

Serologic detection of ‘antiviral antibodies’ Detection of IgM / or at least (4-fold increase) of IgG. 3- Indirect detection - By IF, ELISA, RIA patient serum ? Solid phase IF Serologic detection of ‘antiviral antibodies’ = ‘Host response’

4- Animal pathogenicity One of the earliest ways of detecting a virus. Animals with actively replicating cells give more observed response as suckling mice. limited by virus ‘species specificity’, human viruses may need primates for replication.

5- Viral quantitation 2- Biochemical 1- Physical ‘EM’ Enzymes (RT) Ag (p24 of retrovirus) 1- Physical ‘EM’ - Does not differentiate bet. ‘infective, non-inf.’ 3- Biological “plaque assay” ‘infectious only’ 4- Molecular “Q. PCR”

5- Viral quantitation “Plaque assay” Dilutions of the virus are used to infect a cultured cell monolayer, which is then covered with soft agar to restrict diffusion of the virus, resulting in localized cell killing and the appearance of plaques after the monolayer is stained. Counting the number of plaques directly determines the number of infectious virus particles applied to the plate. 5- Viral quantitation “Plaque assay”

*Number of plaques = number of infectious v. particles

5- Viral quantitation “Q PCR”