LAB DIAGNOSIS OF VIRAL DISEASES

24TH DECEMBER, 2018

What is the purpose of Lab Diagnosis ?

1. To start antiviral drugs for those viral infections for which specific drugs are available
such as herpes, CMY, influenza and respiratory syncytial virus (RSV)
2. Screening of blood donors for HIV, hepatitis B and hepatitis C
3. Surveillance purpose: To assess the disease burden in the community.
4. For outbreak or epidemic investigation, to initiate appropriate control measures
5. To start post-exposure prophylaxis of antiretroviral drugs to the health care workers
following needle stick injury
6. To initiate certain measures: For example,
 If rubella is diagnosed in the first trimester of pregnancy, termination of pregnancy
is recommended
 If newborn is diagnosed to have hepatitis B infection, then immunoglobulins (HBIG)
should be started within 12 hours of birth.

Lab Diagnosis of Viral Diseases includes

 Collection of Samples and Transport
 Microscopy
 Serological tests for detection of antigens and antibodies
 Molecular methods
 Viral isolation

Collection of Samples
 Sterile precautions must be followed
 No blood must be present as it hinders PCR.
 Cotton swabs are also avoided due to the same reason
Eg – Nylon Flocked Swabs are used for throat swabs.
  Samples like – CSF, Serum, urine, scrapings of wound, etc
 Transported at 40 C

 Viral Transport Media – balanced salt solution, essential amino acids and vitamins, salts
and glucose supplemented by 5-10% of fetal calf serum and antibiotics. Medium is
buffered with bicarbonate to maintain a pH of 7.2 - 7.4 and phenol red is added as pH
indicator.

Microscopy
A. Light Microscopy –
Viruses cannot be visualized under light microscope as they are extremely small (except
pox virus)
Light microscopy is useful in the following situations.
 Inclusion bodies
 Immunoperoxidase staining

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B. Electron Microscopy – (Negative staining by potassium phosphotungstate)
i. Shape:
 Rabies virus - bullet-shaped
 Rotavirus - wheel-shaped
 Coronavirus - petal-shaped peplomers
 Adenovirus - space vehicle-shaped
 Astrovirus - star-shaped peplomers
ii. Direct detection of viruses by EM is preferred as primary tool for diagnosis in the
following situations:
 For viruses that are difficult to cultivate - Hepatitis A and E viruses
 When there is a need for faster results - Herpes simplex virus and varicella-zoster
virus
iii. Virus detection from tissue cultures
Demerits - Highly expensive
Low sensitivity and specificity

Electron Microscopy
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Serology - Detection of Viral Ag and Ab
A. Detection of Viral Ag
 Enzyme-linked immunosorbent assay (ELISA)
 Immuno - chromatographic test (ICT) – rapid, colloidal gold base, anti human Ab as
control, no need of skills and less sensitive
 IF – can detect seversl virus at once, sensitive and costly.
Eg - HBsAg antigen detection for hepatitis B virus infection from serum
CMV specific pp65 antigen detection in peripheral blood leukocyte.

B. Detection of Viral Ab
 Most commonly used method
 Appearance of IgM Ab or a four-fold rise of titer of IgG Ab indicates recent infection.
 Presence of IgG antibody (without a recent rise) indicates chronic or past infection.
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 Eg - Paul -Bunnell test for EBV
HAI for Influenza virus
CFT for Polio virus and arbovirus
Neutralization test for Polio virus and arbovirus

Molecular Methods
 More sensitive, specific and yield quicker results than culture.
o Nuclear Probes
o PCR
o Real time PCR
o Reverse transcriptase PCR

Viral isolation
A. Animal Inoculation
 Used mostly for research purposes
 Limited diagnostic purposes
 Suckling mice
 Route of administration – Intra Cerebral / Intra peritoneal
 Observed for – death / disease
a. Coxsackie –A – Flaccid paralysis
b. Coxsackie –B – Spastic paralysis

B. Egg Inoculation
- Embryonated hen's eggs are used for cultivation of viruses.
- Specimens can be inoculated by four different routes into embryonated 7 to 12
days old hen's eggs and then incubated for 2-9 days.

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  Allantoic Sac
 It is a larger cavity, hence is used for better yield of viral vaccines.
 Eg - Influenza vaccine
Yellow fever vaccine
Rabies vaccine
 Yolk Sac Inoculation
 Amniotic Sac
 Chorio - allantoic Membrane
 It is preferred for poxviruses and other viruses such as HSV. Viruses produce
visible lesions called as pocks on chorioallantoic membrane (CAM).
 Pock counting: Each pock is derived from a single virion. So, the number pocks
would represent the number of viral particles present in the inoculum.

C. Tissue Culture - Types

 Organ culture: It was previously used for certain fastidious viruses that have affinity to
specific organs
o Eg - Tracheal ring culture for isolation of corona virus.
 Explant culture: Fragments of minced tissue can be grown as 'explants
o Eg - Adenoid explants used for adenoviruses.
This method is obsolete now.
 Cell line culture: This is the only isolation method which is in use now.

Preparation for Cell line Culture

Tissues are completely digested by treatment with proteolytic enzymes (trypsin or collagenase),
followed by mechanical shaking so that the components are completely dissociated into
individual cells.
 Viral growth medium: The cells are then washed, counted, and suspended in viral
growth medium.

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  Tissue culture flasks: The viral growth medium containing cells is dispensed in tissue
culture flasks.
 Monolayer sheet formation: On incubation, the cells adhere to the glass surfaces of the
flask and then they divide to form a confluent monolayer sheet of cells within a week
covering the floor of tissue culture flask
 Incubation: Tissue culture flasks are incubated horizontally in presence of CO2, either
as a stationary culture or as a roller drum culture.

Primary cell lines Diploid cell lines Continuous cell lines

Normal cells Normal cells Cancerous cells

Very limited growth 10-50 divisions Indefinite divisons

Diploid Diploid haploid

Isolation and vaccine Fastidious virus isolation and Commonly used cell line
preparation vaccine preparation(MMR,
Rabies, Chicken pox)

1. Monkey kidney cell 4. Human fibroblast cell 6. HeLa (human ca cervix

line lines- CMV cell line)
2. Human amnion cell 5. MRC-5, WI-138 7. Hep-2 (Human
line epithelioma of larynx cell
3. Chick embryo cell line)
line 8. Vero (vervet monkey
kidney cell line)

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Detection of Viral Growth in Cell Cultures
A. Cytopathic Effects
 morphological change produced by the virus in the cell line detected by light microscope
 unique for each virus and that helps for their presumptive identification

VIRUS CPE

Adenovirus Clumps-bunch of grapes

HSV Diffuse rounding and ballooning

Measles, Syncitium and multi-nucleate

RSV
Enterovirus Rapid crenation and degeneration

B. Viral Interference
C. Haemadsorption
D. Direct-IF
E. Electron microscopy

Viral Assays - used for quantification of viral particles

 Physical - Real time PCR
RIA/ELISA
Haemagglutination assays
Electron microscopy
 Biological - End point assays
 LD50
 TC ID 50
Plaque assays
Pock assays

Reference –
1. Class Notes
2. Essentials of Medical Microbiology, Apurba Sastry, 2nd ed.

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