Lab Diagnosis Of Viral Diseases

Prabin ShahBScMLT, MSc(Biochemistry)

17/04/2023 Dept of Oral Pathology, TNGDC, Chennai

Introduction

Types of Viral Lab Diagnosis

CONTENTS

What is a virus?

It is a segment of either RNA or DNA protected by a protein coat and in some families of viruses a host derived envelope with attached viral proteins

It lacks: - Protein synthesizing machinery- Energy producing system- No mitochondria- No stores of amino acids, nucleotides

energy rich molecules It is a compulsory intra cellular parasite

Diagnostic Methods in Virology

1. Direct Examination

2. Indirect Examination (Virus Isolation)

3. Serology

Direct Examination

1. Antigen Detection immunofluorescence, ELISA etc.

2. Electron Microscopy morphology of virus particles

immune electron microscopy

3. Light Microscopy histological appearance

inclusion bodies

4. Viral Genome Detection hybridization with specific nucleic acid probes

polymerase chain reaction (PCR)

Indirect Examination

1. Cell Culture cytopathic effect (CPE) haemadsorption

immunofluorescence

2. Eggs pocks on CAM

haemagglutination

inclusion bodies

3. Animals disease or death

Serology

Detection of rising titres of antibody between acute and convalescent stages of infection, or the detection of IgM in primary infection.

Classical Techniques Newer Techniques

1. Complement fixation tests (CFT) 1. Radioimmunoassay (RIA)2. Haemagglutination inhibition tests 2. Enzyme linked immunosorbent assay (EIA)3. Immunofluorescence techniques (IF) 3. Particle agglutination4. Neutralization tests 4. Western Blot (WB)5. Counter-immunoelectrophoresis 5. RIBA, Line immunoassay

Specimens for Routine Tests

Clinical Category Blood Throatswab

Faeces CSF Other

1. Meningitis + + + +2. Encephalitis + + + + Brain biopsy3. Paralytic disease + + + +4. Respiratory illness + + Nasopharyngeal aspirate5. Hepatitis +6. Gastroenteritis +7. Congenital diseases + Urine, saliva8. Skin lesions + + Lesion sample e.g. vesicle

fluid, skin scrapping9. Eye lesions Eye swab10. Myocarditis + Pericardial fluid11. Myositis + +12. Glandular fever +13. Post Mortem + Autopsy

After use, swabs should be broken into a small bottle containing 2 ml of virus transport medium.Swabs should be sent to the laboratory as soon as possible without freezing. Faeces, CSF, biopsy orautopsy specimens should be put into a dry sterile container.

Electron Microscopy

106 virus particles per ml required for visualization, 50,000 - 60,000 magnification normally used. Viruses may be detected in the following specimens.

Faeces Rotavirus, Adenovirus

Norwalk like viruses

Astrovirus, Calicivirus

Vesicle Fluid HSV

VZV

Skin scrapings papillomavirus

Electronmicrographs

Adenovirus Rotavirus

(courtesy of Linda Stannard, University of Cape Town, S.A.)

Inclusion bodies

Negribodies

Negribodies

neuron

Immunohistochemical staining of intra-cytoplasmic viral inclusions in the neuron of a human rabies patient. (Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 39.9)

Immune Electron Microscopy

The sensitivity and specificity of EM may be enhanced by immune electron microscopy. There are two variants:-

Classical Immune electron microscopy (IEM) - the sample is treated with specific anti-sera before being put up for EM. Viral particles present will be agglutinated and thus congregate together by the antibody.

Solid phase immune electron microscopy (SPIEM) - the grid is coated with specific anti-sera. Virus particles present in the sample will be absorbed onto the grid by the antibody.

Immunofluorescence

• Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes.

• Immunofluorescent labeled tissue sections are studied using a

fluorescence microscope.

• Fluorescein is a dye which emits greenish fluorescence under UV light. It can be tagged to immunoglobulin molecules.

Direct IF Indirect IF

                                                               

            

                                                               

            

ELISA

Embryonated Egg

Cell Lines/ Tissue cultures

Animal inoculation

Chorioallantioc membrane (CAM)

Allantoic cavity

Amniotic cavity

Yolk Sac

Primary

Diploid/ Secondary

Continuous

Suckling mice

Virus Culture

Embryonated Hen’s Egg

• Chorioallantoic membrane (CAM) – visible lesions called pocks. Each infectious virus particle forms one pock. e.g. Variola, Vaccinia virus

• Allantoic cavity – Influenza virus (vaccine production) & paramyxoviruses

• Amniotic cavity – primary isolation of Influenza virus

• Yolk sac – Chlamyadia, Rickettsiae & some viruses

Growth of virus on embryonated eggs

Davis, Duylbecco, Eisen, Ginsberg “Microbiology” 4th ed, J.B. Lippincott 1990, Fig. 48-1

Embryonated Hen’s Egg

Types of Tissue Culture

Organ culture

Explant culture

Cell culture

Cell Culture

• Tissues Individual cells trypsin & mechanical shaking

• Cells are washed, counted & suspended in a growth medium.

• Growth medium – Minimum Essential Medium (MEM): essential aminoacids, vitamins, salts, glucose & bicarbonate in 5% CO2 with 5% fetal calf or calf serum, antibiotics & phenol red indicator

Cell Culture• Routinely used for growing viruses• Classified into 3 types:

– Primary cell culture – normal 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 can be subcultivated for limited number of times, mostly 501. WI-38: human embryonic lung cell2. HL-8: Rhesus embryo cell

– Continuous cell lines – malignant cells, indefinite subcultivation1. HeLa: Human Ca of cervix cell line2. HEP-2: Human epithelioma of larynx3. Vero: Vervet monkey kidney4. McCoy, Detroit-6, BHK-21, KB

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, erythocytes are absorbed on surface of cells

Cytopathic Effect

Cytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning of cells . (Virology Laboratory, Yale-New Haven Hospital, Linda Stannard, University of Cape Town)

Hemadsorbtion

add red blood cells

Haemadsorption

Syncytial formation caused by mumps virus and haemadsorption of erythrocytes onto the surface of the cell sheet. (courtesy of Linda Stannard, University of Cape Town, S.A.)

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 – growth appear in a pile-up fashion producing microtumors

6. Immunofluorescence – test for viral Ag in cells from viral infected cultures.

Transformationloss of contact inhibition

Problems with cell culture

• Long period (up to 4 weeks) required for result.• Often very poor sensitivity, sensitivity depends on a

large extent on the condition of the specimen.• Susceptible to bacterial contamination.• Susceptible to toxic substances which may be present in

the specimen.• Many viruses will not grow in cell culture e.g. Hepatitis

B, Diarrhoeal viruses, parvovirus, papillomavirus.

Rapid Culture Techniques

Rapid culture techniques are available whereby viral antigens are detected 2 to 4 days after inoculation. The CMV DEAFF test is the best example, whereby

• The cell sheet is grown on individual cover slips in a plastic bottle.

• Following inoculation, the bottle then is spun at a low speed for one hour (to speed up the adsorption of the virus) and then incubated for 2 to 4 days.

• The cover slip is then taken out and examined for the presence of CMV early antigens by immunofluorescence.

DEAFF test for CMV

(Virology Laboratory, Yale-New Haven Hospital)

Animal inoculation

• Useful for study of pathogenesis, immune response, epidemilogy and oncogenesis

• Earlier human volunteers and monkey were used• Suckling mice are used (arbo, coxsackie)• Mice is inoculated (intracerebral, subcutaneous,

intraperitoneal, intranasal)• Growth of virus is indicated by death, disease or

viable lesion

SEROLOGY

Serology

Criteria for diagnosing Primary Infection

• 4 fold or more increase in titre of IgG or total antibody between acute and convalescent sera

• Presence of IgM• Seroconversion• A single high titre of IgG (or total antibody) - very

unreliable

Criteria for diagnosing Reinfection

• fold or more increase in titre of IgG or total antibody between acute and convalescent sera

• Absence or slight increase in IgM

– Primary and secondary responses to viral infections• IgM (1st exposure)• IgG (2nd exposure)

Figure 5.18: Primary (1 degree) and secondary (2 degree) antibody responses toward a viral pathogen.

Serological Test

Solid-Phase ImmunoassaysEnzyme immunoassay Passive latex agglutinationImmunofluorescence immunoassay Passive hemagglutinationChemiluminescence immunoassay

ImmunoflourescenceIndirect immunofluorescence NeutralizationAnticomplement immunofluorescence

Agglutination AssaysPassive latex agglutinationPassive hemagglutinationHemagglutination inhibition

Complement fixation

Neutralization

Immunoblotting

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 of Hemagglutinin spikes on the

surface.

• Reversal of hemagglutination – Elution– Due to the presence of Neuraminidase enzyme, Receptor

Destroying Enzyme (RDE)– Destruction of receptor – reversal of hemagglutination –

release of virus from the red cell surface– Found only in Myxoviuses.

Hemagglutination (HA) Test

+

RBC Suspension HA Virus Settling Pattern

Hemagglutination-Inhibition (HI) Test

+

RBC Suspension HA Virus Settling Pattern

+

Antibody

ELISA (ELISA) can detect unknown Ag or Ab by

direct or indirect means. A positive result is visualized when a

colored product is released by an enzyme-substrate reaction.

• Uses an enzyme as the label

– Reaction of the enzyme with its substrate produces a colored product indicative of a positive test

• Most common form of ELISA is used to detect the presence of antibodies in serum

ELISA Procedures

Figure 5-19a

Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third Edition . ASM Press, 2000.

Figure 5-19b

© Hank Morgan/Science Photo Library/Photo Researchers, Inc.

HIV ELISA test.

Western Blot

HIV-1 Western Blot• Lane1: Positive Control• Lane 2: Negative Control• Sample A: Negative• Sample B: Indeterminate• Sample C: Positive

Dr.T.V.Rao MD 48

Figure 5.21b: The structure of HIV-1.

Image courtesy of Bio-Rad Laboratories

Figure 5.21c: The typical results of a Western blot testing patient serum for HIV-1 antibodies.

(c)(b)

Figure 5.21a: The basic principles behind the Western blotting procedure. Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third Edition. ASM Press, 2000.

Neutralization Test

1 2 3

B

Following virus isolation:

1.Divide culture yield into small volume in a set of test tubes

2. Prepare the panel of antisera against which the virus isolate is to be challenged

3. To each test tube add one antisera and leave one as a virus control and one as serum control

• Incubate for one hour then inoculate each into cell culture tubes, incubate and observe daily.

2 31

Principle of Neutralization test

54

Complement fixation: Principle

Complement fixation tests detect lysins- Ab that fix complement and can lyse target cells. Involves mixing test Ag and Ab with complement and then with sensitized sheep RBCs. If complement is fixed by the Ag-Ab, the RBCs remain intact and the test is positive. If RBCs are hemolyzed, specific Ab are lacking and the test is negative.

Results and Interpretations:• No haemolysis is considered as a positive test. • haemolysis of erythrocytes indicative of a negative test.

1 2 3 4

A

B

• Microtiter plate showing Haemolysis (Well A2, A3, A4 and B4) and No Haemolysis (Well A1, B1, B2 and B3)

MOLECULAR TECHNICS

Polymerase Chain Reaction • PCR allows the in vitro amplification of specific target

DNA sequences by a factor of 106 and is thus an extremely sensitive technique.

• It is based on an enzymatic reaction involving the use of

synthetic oligonucleotides flanking the target nucleic sequence of interest.

• These oligonucleotides act as primers for the thermostable Taq polymerase. Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC), annealing of primers to their complementary sequences (50oC), and primer extension (72oC) result in the exponential production of the specific target fragment.

• Further sensitivity and specificity may be obtained by the nested PCR

• Detection and identification of the PCR product is usually carried out by

agarose gel electrophoresis,

hybridization with a specific oligonucleotide probe,

restriction enzyme analysis, or DNA sequencing.

• Advantages of PCR:

– Extremely high sensitivity, may detect down to one viral genome per sample volume

– Easy to set up

– Fast turnaround time

• Disadvantages of PCR

– Extremely liable to contamination

– High degree of operator skill required

– Not easy to set up a quantitative assay.

Schematic of PCR

Each cycle doubles the copy number of the target

Other Newer Molecular Techniques

• Commercial proprietary techniques such as LCR, NASBA, TMA depend on exponential amplification of the signal or the target.

• PCR and related techniques are bound to play an increasingly important role in the diagnosis of viral infections.

• DNA chip is another promising technology where it would be possible to detect a large number of viruses, their pathogenic potential, and their drug sensitivity at the same time.

References

• BAILEY & SCOTT`S DIAGNOSTIC MICROBILOGY-12th Edition

• TOPLEY & WILSION`S Textbook of Microbiology

• Ananthanarayan and Paniker`s Textbook of Microbiology- 8th Edition

• Koneman`s Color Atlas and Textbook of Diagnostic Microbiology- 6th Edition