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Laboratory Diagnosis of

Viral Infection

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Learning Objectives

Describe the principles, techniques,standards and recording of results andinterpretation of different methods used in

diagnosis of viral infections

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Difficulties

Can not be seen under light microscope

Can not be cultivated easily

Do not grow on culture media

Treatment was not available

Changed situation

Rapid techniques have emerged

Screening for Blood transfusion

Treatment available

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Techniques used

Microscopy

Detection of Viral Antigen

Growing and detecting viruses in Tissue / Organ / Cell culture

Fertilized hens 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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Microscopy

Electron Microscope /Immune ElectronMicroscopy

Light microscopeInclusion bodies eg NegriBody in Rabies

Fluorescent Microscope -Fluorescent antibodytechnique

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Demonstration of Viral

Antigens

Precipitation on gel eg HBsAg

Immunofluorescence

Counter Immuno Electro Phoresis(CIEP)

Enzyme Linkes Immuno SorbantAssay (ELISA)

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Isolation of Virus

Laboratory animals

Fertilized Hens Egg Chorioallantoic membrane

Allantoic cavity

Amniotic cavity

Yolk sac

Organ/Tissue/Cell Culture Growth identified by serological method

like neutralization.

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Embryonated Egg

Cell Lines/Tissue cultures

Animal inoculation

Chorioallantioc membrane (CAM)

Allantoic cavity

Amniotic cavity

Yolk Sac

Primary

Diploid/ Secondary

Continuous

Suckling mice

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Embryonated Hens Egg

Chorioallantoic membrane (CAM) visible lesionscalled pocks. Each infectious virus particle forms onepock. e.g. Variola, Vaccinia virus

Allantoic cavity Influenza virus (vaccine production)& paramyxoviruses

Amniotic cavity primary isolation of Influenza virus

Yolk sac Chlmyadia, Rickettsiae & some viruses

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Embryonated Hens Egg

Inoculation Harvesting

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Cell Culture

Routinely used for growing viruses

Classified into 3 types: Primary cell culturenormal cells freshly taken from body &

cultured, limited growth1. Rhesus monkey kidney2. Chick embryo fibroblast3. Human amnion cell culture

Diploid cell strains cells of single type (fibroblast cells) that canbe subcultivated for limited number of times, mostly 501. WI-38: human embryonic lung cell2. HL-8: Rhesus embryo cell

Continuous cell linesmalignant cells, indefinite subcultivtion1. HeLa: Human Ca of cervix cell line2. HEP-2: Human epithelioma of larynx3. Vero: Vervet monkey kidney4. McCoy, Detroit-6, BHK-21, Kb

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Cell Culture

Tissues Individual cellstrypsin & mechanical shaking

Cells are washed, counted & suspended in a

growth medium.

Growth mediumMinimum Essential Medium(MEM): essential aminoacids, vitamins, salts,

glucose & bicarbonate in 5% CO2 with 5%fetal calf or calf serum, antibiotics & phenolred indicator

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Detection of virus growth in cell cultures

1. Cytopathic effects (CPE) morphologicalchanges in cultured cells, seen undermicroscope, characteristic CPE for differentgroups of viruses

2. Metabolic Inhibition no acid production inpresence of virus

3. Hemadsorption influenza & parainfluenzaviruses, by adding guinea pig erythrocytes tothe culture

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Detection of virus growth in cell cultures

4. Interference growth of a non cytopathogenicvirus can be tested by inoculating a knowncytopathogenic virus: growth of first virus willinhibit the infection by second

5. Transformation oncogenic viruses inducetransformation & loss of contact inhibitionmicrotumors

6. Immunofluorescence test for viral Ag in cellsfrom viral infected cultures.

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Viral Hemagglutination

Hemagglutination Originally seen with the Influenza virus by

Hirst in 1941.

A convenient method of detection & assay of

Influenza virus.

Due to the presence ofHemagglutinin spikeson the surface.

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Viral Assay

Viral content of a specimen: Total no. of1. Virus particles EM, HA

2. Infectious virions only

Assay of Infectivity: two types1. Quantitative assays actual no. of infectious

particle in an inoculum

2. Quantal assays indicate the presence orabsence of infectious viruses, carried out inanimals, eggs or tissue cultures

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Viral Assay

Assay of Infectivity: Quantitativeassays

Plaque assay in monolayer cellcultures

Pock assay on CAM

*Each plaque/ pock represents one

infectious virus.

Plaquesare clear zones that developon lawns of host cells. The virus plaque is analogous to thebacterial colony.

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Specimens

According to the disease

Respiratory Throat swab

CNS CSFEyes- Conjunctival scrapings

Liver Blood

PUO BloodSkin - Scrapings

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Serological Reactions

Rising titre of antibody in paired sample ofsera 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