1. Laboratory Diagnosis of
Viral Infection
Dr. Sshrutkirti Gupta

2. • Collection of samples
• Transport
• Cultivation
• Methods of diagnosis
Following Points will discussed today

3. Importance of diagnosis of viral
infection
• Rubella diagnosed in 1st trimester-abortion is recommended.
• If baby born to HBV Surface Ag positive mother, immunization at
birth is mandatory.
• Diagnosis of diseases caused by viruses for which antiviral
treatment is available (eg. Herpes)
• Screening of HIV ,HBV helps in preventing transmission of the
disease
• Early detection of epidemics like influenza,
helps to initiate appropriate control measures.

4. Sample collection
• Respiratory tract- Nasal washing, throat
swab, nasal swab, sputum
(adeno, influenza, enterovirus, orthromyxo, paramyxo,
HSV, Rubella)
• Gastrointestinal tract-stool, rectal swab
(Reovirus, Rotavirus, Adenovirus, norwalk calcivirus)
• Maculopapular Rash-Throat swab, rectal
swab
(Adeno, entero, Rubella, measles)

5. • Vesicular rash-Vesicle fluid, scraping or swab
Coxsackie echo, HSV, VZV
• Central Nervous system-
Picorna- Stool ,
Arbovirus- rarely cultured
Rabies- Tissue, saliva, brain biopsy
HSV, CMV, Mumps, Measles- CSF, Biopsy
• Urinary Tract Infection-
Adenovirus, CMV-urine
• Blood born infections
HIV,HTLV,HBV,HCV,HDV-Blood
….Cont Sample collection

6. Methods of diagnosis
• Direct microscopy- virus
• inclusion bodies
• Isolation of virus/cultivation
• Antigen detection
• Antibody detection
• Nucleic acid detection

7. Microscopy
• A-Electron microscopy
• B- Fluorescent microscopy
• C- Light microscopy
Electron Microscopy
• Demonstration of viral particles in clinical samples
• Mainly for those whose cultivation is difficult
• Immunoelectron microscopy – more sensitivity
Rabies virus

9. Direct demonstration-EM
Sample
• Faeces
• Vesicular fluid
• CSF
• Urine
Virus
Rotavirus, HAV, Adeno,
Norwalk, astrovirus.
Herpes simplex, VZ
Enterovirus, VZ
CMV

10. EM structure of Flu virus

11. Microscopy
Fluorescent microscopy
Rabies
Paramyxovirus,
Orthomyxoviruses,
Adenovirus
Herpesvirus

13. Light microscopy
Inclusion bodies
• Rabies- negri bodies
• Vaccinia (Guarnieri bodies)
• Molluscum contagiosum (Molluscum bodies)
• Cowdry TypeA- Herpes, Yellow fever
• Cowdry type B-Adeno, Polio
Stains- Giemsa, Eosin-methylene blue,
Haematoxylin

14. Intracytoplasmic inclusion
bodies

16. Cultivation
• By inoculation in to animals
• By inoculation into embryonated egg
• By inoculation into cell culture
• Sample process to remove bacterial
contamination

17. Animal inoculation
• Suckling mice- Arbovirus, Coxsackie
• Different routes- Intracerebral,
subcutaneous, intraperitoneal, intranasal
• Observe for signs of disease, death
• Tissue tested for presence of virus-
neutralisation test (antisera),
demonstrate inclusion bodies

18. Embryonated Egg inoculation

19. Egg Innoculation
Site
• Chorioallantoic
Membrane
• Allantoic cavity
• Amniotic cavity
• Yolk sac
Virus
• Pox (pocks)-variola or
vaccinia
• Influenza, yellow fever
• Influenza
• Chlamydiae, rickettsiae

20. Tissue culture
• Organ culture
• Explant culture- rarely used
• Cell culture

21. Organ culture
• Bits of organ maintained in tissue
culture growth medium
• Used mainly for highly specified
parasites of certain organ
eg- corona virus- Tracheal ring culture

22. Cell Culture
• Primary cell culture
• Diploid cell culture
• Continuous cell line

23. Primary cell culture
Limited growth up to 5-50 divisions
Use- primary isolation, vaccine production
Examples of cell lines
1. Rhesus monkey kidney cell culture
2. Human amnion cell culture
3.Chick embryo fibroblast cell culture

24. Diploid cell strain
• cells of single type –contains same no. of
chromosomes as in the parent cell
• Limited growth 50 cell division
• Developed from fibroblast
• WI-38-human embryonic lung cell strain
Rabies virus vaccine
HL-8 (Rhesus embryo cell strain)

25. …Cont Cell culture
Continuous cell line
• Single type capable of unlimited growth
eg-
1. HeLa (Human carcinoma of cervix cell line)
2. HEP-2(human epithelioma of larynx cell line)
3. Mckoy (human synovial carcinoma cell line)
4. BHK-21 ( Baby hamster kidney cell line)

26. Detection of growth in cell
culture
• 1 Cytopathic effect
• 2 Haemadsorption
• 3 Interference
• 4 Transformation
• 5 Immunoflurescence
• 6 Electron microscopy
• 7 Detection of enzymes

27. Cytopathic effects
a.Syncytium formation- measles, RSV
neighbouring cell fused together to form
multinucleated giant cell.
b. Cell necrosis & lysis- Enterovirus
c. Roundening of cells- Picornavirus
viral replication leads to neclear pyknosis ,
rounding, refratility, degeneration
d. Discrete focal degneration- Herpes

29. Herpes

30. Cell necrosis by enterovirus

31. Rounding of cells-polio virus

32. Rounding of cells- poliovirus

36. …Cont Cytopathic effects
• Haemadsorption- Influenza,
parainfluenza,Toga –express
Haemagglutinin on infected cells
• RBC suspension layered on cell layer
• RBC adsorb on infected cells

37. Antigen detection
• Immunoflurescence
• Serological tests
Antibody detection
• Serological tests

39. Newer techniques
• Molecular
• DNA PCR
• RNA PCR