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Describe the principles, techniques, standards and recording of results and interpretation of different methods used in diagnosis of viral infections

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Can not be seen under light microscope
Can not be cultivated easily
Do not grow on culture media
Treatment was not available
Changed situation
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Rapid techniques have emerged
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Screening for Blood transfusion
Treatment available

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Microscopy
Detection of Viral Antigen
Growing and detecting viruses in
– Tissue / Organ / Cell culture
– Fertilized hen’s egg
– Laboratory animal inoculation eg mice
Detection of antobody in serum
– IgG – Rising titre in paired sample
– IgM – Indicates current / recent infection

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Electron Microscope /
Immune Electron
Microscopy
Light microscope –
Inclusion bodies eg Negri
Body in Rabies
Fluorescent Microscope -
Fluorescent antibody technique

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Laboratory animals
Fertilized Hen’s Egg
– Chorioallantoic membrane
– Allantoic cavity
– Amniotic cavity
– Yolk sac
Organ/Tissue/Cell Culture
Growth identified by serological method like neutralization.

Virus Culture
Embryonated Egg Chorioallantioc membrane (CAM)
Allantoic cavity
Amniotic cavity
Yolk Sac
Cell Lines/
Tissue cultures
Primary
Diploid/ Secondary
Continuous
Animal inoculation Suckling mice

Embryonated Hen’s Egg
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Chorioallantoic membrane (CAM) – visible lesions called pocks. Each infectious virus particle forms one pock. e.g. Variola, Vaccinia virus
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Allantoic cavity – Influenza virus (vaccine production) & paramyxoviruses
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Amniotic cavity – primary isolation of Influenza virus
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Yolk sac – Chlmyadia, Rickettsiae & some viruses

Embryonated Hen’s Egg
Inoculation
Harvesting

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Routinely used for growing viruses
Classified into 3 types:
– Primary cell culture – normal cells freshly taken from body & cultured, limited growth
1.
Rhesus monkey kidney
2.
Chick embryo fibroblast
3.
Human amnion cell culture
– Diploid cell strains – cells of single type ( fibroblast cells ) that can be subcultivated for limited number of times, mostly 50
1.
WI-38: human embryonic lung cell
2.
HL-8: Rhesus embryo cell
– Continuous cell lines – malignant cells , indefinite subcultivtion
1.
HeLa: Human Ca of cervix cell line
2.
HEP-2: Human epithelioma of larynx
3.
Vero: Vervet monkey kidney
4.
McCoy, Detroit-6, BHK-21, Kb

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Tissues trypsin & mechanical shaking
Individual cells
Cells are washed, counted & suspended in a growth medium.
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Growth medium – Minimum Essential Medium
(MEM): essential aminoacids, vitamins, salts, glucose & bicarbonate in 5% CO
2 with 5% fetal calf or calf serum, antibiotics & phenol red indicator

Cell Culture Bottles / Tubes

Detection of virus growth in cell cultures
1.
Cytopathic effects (CPE) – morphological changes in cultured cells, seen under microscope, characteristic CPE for different groups of viruses
2.
Metabolic Inhibition – no acid production in presence of virus
3.
Hemadsorption – influenza & parainfluenza viruses, by adding guinea pig erythrocytes to the culture

Detection of virus growth in cell cultures
4.
Interference – growth of a non cytopathogenic virus can be tested by inoculating a known cytopathogenic virus: growth of first virus will inhibit the infection by second
5.
Transformation – oncogenic viruses induce transformation & loss of contact inhibition – microtumors
6.
Immunofluorescence – test for viral Ag in cells from viral infected cultures.

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Hemagglutination
– Originally seen with the Influenza virus by
Hirst in 1941.
– A convenient method of detection & assay of
Influenza virus.
– Due to the presence of Hemagglutinin spikes on the surface.

Viral Assay
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Viral content of a specimen : Total no. of
1.
Virus particles – EM, HA
2.
Infectious virions only
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Assay of Infectivity : two types
1.
Quantitative assays – actual no. of infectious particle in an inoculum
2.
Quantal assays – indicate the presence or absence of infectious viruses, carried out in animals, eggs or tissue cultures

Viral Assay
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Assay of Infectivity: Quantitative assays
– Plaque assay in monolayer cell cultures
– Pock assay on CAM
*Each plaque/ pock represents one infectious virus.
– Plaques are clear zones that develop on lawns of host cells.
– The virus plaque is analogous to the bacterial colony.

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– Respiratory – Throat swab
– CNS – CSF
– Eyes- Conjunctival scrapings
– Liver – Blood
– PUO – Blood
– Skin - Scrapings

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Rising titre of antibody in paired sample of sera is diagnostic
– First sample – At the earliest
– Second sample – After 2 weeks
Single sample - IgM type of antibody detection. Indicates recent / current infection.
Techniques – Neutralization, ELISA, CFT,
Haemagglutination Inhibition (HAI)Test