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1. LABORATORY DIAGNOSIS OF
VIRAL DISEASES
Dr Abhishek Kumar Jain
Assistant Professor
Department of Microbiology
RVRS Govt Medical College Bhilwara
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Sunday, 09 April 2023

2. LABORATORY DIAGNOSIS OF VIRAL
DISEASES
Laboratory diagnosis of viral infections is useful for the following
purposes:
• To start antiviral drugs
• Screening of blood donors for HIV, hepatitis B and hepatitis C.
• Surveillance purpose
• For outbreak or epidemic investigation
• To start post-exposure prophylaxis.
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3. LABORATORY DIAGNOSIS
• Key steps
• Specimen selection and collection
• Specimen transport and storage
• Laboratory examination
• Interpretation and reporting

4. Specimen Collection Guideline
1. Avoid causing harm or discomfort to patient.
2. Collect from appropriate site.
3. Obtain specimen at correct time
4. Use appropriate devices
5. Obtain sufficient quantity of specimen
6. Obtain specimen prior to the start of antimicrobial therapy
7. Label correctly.
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5. SAMPLE SELECTION AND COLLECTION
• General principle
• Selection depends upon the
specific disease syndrome, viral
etiology suspected,
• Each specimen for virus etiology
should be accompanied by a
requisition form
Requisition form
• Patient’s Name.............. Age/Sex...........
• OPD/IPD NO.......................
• Occupation…………..And Address……
• Type Of Specimen.......For...........Test.
• Clinical History............................
• Date And Time Of Collection................
Sign of Clinician

6. Types of Specimen
• Blood for viral culture
• Transported in a sterile tube containing anticoagulant
• Kept at refrigeration temperature (4⁰C) until processing
• Blood for serology
• Serum should be separated from the clot as soon as possible.
• Transported in the sterile tube.
• Serum can be stored for hours or days at 4⁰C.
• For weeks or months at -20⁰C or below before testing.

7. Other common specimens
1. Urine
2. Sputum
3. Stool
4. Pus
5. CSF
6. Swabs
• Throat Swab,
• Nasal swab,
• Oropharyngeal (OP) swab,
• Nasopharyngeal (NP),
• Ear swab,
• Eye swab
• High Vaginal Swab (HVS),
• Cervical swab etc.
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8. LABORATORY EXAMINATION
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Direct Demonstration of Virus
Electron microscopy
Immunoelectron microscopy
Fluorescent microscopy
Light microscopy
Detection of Viral Antigens
By various formats such as ELISA, direct IF, ICT, flow through assays.

9. LABORATORY DIAGNOSIS OF VIRAL
DISEASES
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Detection of Specific Antibodies
Conventional techniques such as HAI, neutralization test and CFT
Newer diagnostic formats such as ELISA, ICT, flow through assays.
Molecular Methods to Detect Viral Genes
Nucleic acid probe—for detection of DNA or RNA by hybridization
PCR—for DNA detection by amplification
Reverse transcriptase-PCR—for RNA detection
Real time PCR—for DNA quantification
Real time RT-PCR—for RNA quantification.

10. LABORATORY DIAGNOSIS OF VIRAL
DISEASES
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Isolation of Virus by
Animal inoculation
Embryonated egg inoculation
Tissue cultures: Organ culture, explant culture, cell line culture (primary, secondary and
continuous cell lines).

11. Direct detection from specimens
• For viruses that are difficult to cultivate, EM can be used as primary
tool for diagnosis.
• As an alternative to tissue culture
• Drawbacks of tissue culture:
Highly expensive
Has low sensitivity with a detection limit of at least 107virions /ml.
The specificity is also low.
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12. Electron Microscope
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13. Electron Microscopy
• Specimens are negatively stained by potassium phosphotungstate and
scanned under EM.
• Shape-Viruses can be identified based on their distinct appearances:
Rabies- bullet shaped
Rotavirus- wheel shaped
Corona virus- petal shaped peplomers
Adenovirus- space vehicle shaped
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14. Immuno-electron Microscopy
• Sensitivity and specificity of EM can be
improved by adding specific antibody to
the specimen to aggregate the virus
particles which can be centrifuged.
• Sediment is negatively stained and
viewed under EM.
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15. Fluorescent Microscopy
• Direct immunofluorescence
technique is employed to
detect viral particles in the
clinical samples.
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16. Clinical applications-
• Clinical applications-
Diagnosis of rabies in skin biopsies, corneal smear of infected
patients.
Syndromic approach- Rapid diagnosis of respiratory
infections caused by influenza, rhinoviruses, respiratory
syncytial virus, adenoviruses and herpesviruses can be carried
out by adding specific antibodies to each of these viruses
Detection of adenovirus from conjunctival smears.
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17. Light Microscopy
Inclusion bodies:
• Histopathological staining of tissue
sections may be useful for detection of
inclusion bodies which helps in the
diagnosis of certain viral infections.
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Basophilic viral inclusion body

18. Light Microscopy
Immunoperoxidase staining:
• Tissue sections or cells coated with viral antigens are stained using
antibodies tagged with horse radish peroxidase (HRP) following
which hydrogen peroxide (H2O2) and a coloring agent (Benzidine
derivative) are added.
• The color complex formed can be viewed under light microscope.
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19. Detection of Viral Antigens
• Various formats are available for detection of viral antigens in
serum and other samples
• Some important antigen detection tests include:
oHBsAg and HBeAg antigen detection for hepatitis B infection
from serum.
oNS1 antigen detection for dengue virus infection from serum
op24 antigen detection for HIV from serum
oRotavirus antigen detection from diarrheic stool
oCMV specific pp65 antigen detection (serum)
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20. Detection of Viral Antibodies
• Antibody detection from serum is one of the most commonly used
method in diagnostic virology.
• Appearance of IgM antibody or a four-fold rise of titer of IgG
antibody indicates recent infection.
• Presence of IgG antibody (without a recent rise) indicates chronic
or past infection.
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21. Detection of Viral Antibodies (Cont..)
• Newer diagnostic formats such as ELISA, ICT, flow through
assays are widely used for antibody detection against most of the
viral infections such as:
Anti-HBc, Anti-HBs and Anti-HBe antibodies for Hepatitis B
infection
Anti-Hepatitis C antibodies
Antibodies against HIV-1 and HIV-2 antigens from serum
Anti-Dengue IgM/IgG antibodies from serum.
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22. Molecular Methods
More sensitive, specific and yield quicker results than culture.
• Polymerase chain reaction (PCR)
• Reverse transcriptase-PCR (RT-PCR)
• Multiplex PCR formats
• BioFire FilmArray
• Real time-PCR (rt-PCR)
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23. Isolation of Virus
• Viruses cannot be grown on artificial culture media.
• Cultivated by animal inoculation, egg inoculation or tissue
cultures.
• Being labor intensive, technically demanding and time consuming,
virus isolation is not routinely used in diagnostic virology.
• The specimen should be collected properly and immediately
transported to the laboratory.
• Refrigeration is essential during transportation as most viruses are
heat labile.
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24. Isolation of Virus
• Because of the ethical issues related to use of animals, animal
inoculation is largely restricted only for research use.
• Research use- To study viral pathogenesis or viral oncogenesis or
for viral vaccine trials
• Diagnostic use- Primary isolation of certain viruses which are
difficult to cultivate otherwise; such as arboviruses and coxsackie
viruses. 25

25. Egg inoculation
• Embryonated eggs - first used
for viral cultivation by Good
pasture in 1931.
• Embryonated hen’s egg has
four sites which are specific for
the growth of certain viruses.
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26. Egg inoculation
• Yolk sac inoculation
• Amniotic sac
• Allantoic sac
• Chorioallantoic Membrane (CAM)
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27. Tissue Culture
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Types Explanation Examples
Organ culture
(obsolete)
Used for certain fastidious
viruses that have affinity
to specific organs.
Tracheal ring culture for
isolation of corona virus
Explant culture
(obsolete)
Fragments of minced
tissue can be grown as
'explants’
Adenoid explants used for
adenoviruses.
Cell line culture This is the only isolation
method which is in use
now.

28. Preparation of the Cell Lines
• Tissues digested – by treatment with proteolytic enzymes (trypsin
or collagenase) followed by mechanical shaking.
• Viral growth medium:
Cells are then washed, counted, and suspended in viral growth
medium containing balanced salt solution added with essential
amino acids and vitamins, salts and glucose supplemented by
5-10% of fetal calf serum and antibiotics.
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29. Preparation of the Cell Lines (Cont..)
• Tissue culture flasks
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30. Preparation of the Cell Lines (Cont..)
• Incubation:
Tissue culture flasks are incubated horizontally in presence of
CO2, either as a stationery culture or as a roller drum culture.
 Rolling of the culture bottle in roller drums provides better
aeration which is useful for isolation of fastidious viruses (e.g.
rotavirus)
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31. Types of Cell Lines
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Type Explanation Examples
Primary cell line  Derived from normal cells.
 Freshly taken from the organs and
cultured
 Capable of very limited growth in
culture, maximum up to 5-10 divisions.
 Maintain a diploid karyosome
 Useful for both primary isolation as well
as growth of the viruses for vaccine
production.
 Monkey kidney cell line-
useful for isolation of
myxoviruses, enteroviruses &
adenoviruses
 Human amnion cell line
 Chick embryo cell line

32. Types of Cell Lines
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Type Explanation Examples
Secondary or diploid
cell lines
 Can divide maximum up to 10-50
divisions before they undergo senescence
(death).
 Also derived from the normal host cells.
 Maintain the diploid karyosome.
 Human fibroblast cell line-
excellent for the recovery of
cytomegalovirus.
 MRC-5 &WI-38 (human
embryonic lung cell strain)

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Type Explanation Examples
Continuous cell lines  Derived from cancerous cell lines, hence
are immortal.
 They also possess altered haploid
chromosome.
 Easy to maintain
 HeLa cell line (Human
carcinoma of cervix cell line)
 Hep-2 cell line (Human
epithelioma of larynx cell
line)- widely used for
respiratory syncytial viruses,
adenoviruses and HSV
 KB cell line (Human
carcinoma of nasopharynx
cell line)
 McCoy cell line (Human
synovial carcinoma cell line)-
useful for isolation of viruses
as well as Chlamydia
 Vero cell line (Vervet monkey
kidney cell line)-used for
rabies vaccine production.

34. Types of Cell Lines (Cont..)
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Human lung fibroblast cell line (Normal)
HeLa cell line (normal, uninfected)
Vero cell line (normal, uninfected)
HEp-2 cell line (normal, uninfect

35. Detection of Viral Growth in Cell Cultures
Two methods are used to detect the growth of the virus in cell
cultures.
• Cytopathic Effect (CPE)
• Shell Vial Technique
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36. Cytopathic Effect (CPE)
▰ Defined as the
morphological change
produced by the virus
in the cell line
detected by light
microscope.
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Type of Cytopathic effect
(CPE)
Virus
Rapid crenation and
degeneration of the entire
cell sheet
Enteroviruses
Syncytium or multinucleated
giant cellformation
Measles,
RSV, HSV
Diffuse roundening and
ballooning of the cell line
HSV
Cytoplasmic vacuolations SV 40 (Simian vacuolating
virus-40)
Large granular clumps
resembling bunches of
grapes
Adenovirus

37. Shell Vial Technique
• Involves centrifugation of cell culture (mixed with the specimen)
to enhance the cell contact and viral replication, followed by
• Detection of early viral antigen in the infected cells by direct
fluorescence technique.
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