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Laboratory diagnosis of Viral infections

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River’s Postulates (Modified from Koch’s postulates)

1. Isolate virus from diseased hosts.2. Cultivation of virus in host cells.3. Proof of filterability.4. Production of a comparable disease when

the cultivated virus is used to infect experimental animals.

5. Reisolation of the same virus from the infected experimental animal.

6. Detection of a specific immune response to the virus.

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Indications for lab diagnosis of viral infection

•If rubella is diagnosed in the first trimester of pregnancy, abortion is recommended

•If a baby is borne of an HbsAg positive mother ,abortion is recommended

For proper management of certain diseases

•for which antiviral chemotherapy is available (herpes viruses)

Diagnosis of diseases caused by viruses

•For hepatitis B & HIV virus helps to prevent spread of these viruses

Screening of blood donors

•To initiate appropriate control measures

Early detection of epidemics

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3 General approaches for Laboratory diagnosis of Viral infections

Direct demonstration of virus & its components

Isolation of virus

Detection of specific antibodies

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Microscopy

•Examine specimen for viruses

Electron Microscope

•Labeled antibody

Immuno-electron microscopy

•Fluorescent tag bound to Fc region of Ab

Immunofluorescence

•Histological appearance of affected cells

•Inclusion bodies

Light microscope

Electronmicrographs

Adenovirus Rotavirus

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

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Inclusion bodies

•Inclusion bodies are virus-specific intracellular globular masses which are produced during replication of virus in host cells.

•They can be demonstrated in virus infected cells under light microscope after fixation & staining

•Eg: Negri bodies in rabies .

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Negribodies

Negribodies

neuron

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Isolation of virus

• Laboratory animals• Fertilized Hen’s EggChorioallantoic membraneAllantoic cavityAmniotic cavityYolk sac• Organ/Tissue/Cell Culture• Growth identified by serological method like

neutralization.

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Regular Methods in Use

• Egg inoculation Pox virus, Influenza

• Into tissue culture

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Egg inoculation

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Cell culture

Cell Cultures are most widely used for virus isolation, there are 3 types of cell cultures:

1. Primary cells - Monkey Kidney

2. Semi-continuous cells - Human embryonic kidney and skin fibroblasts

3. Continuous cells - HeLa, Vero, Hep2, LLC-MK2, MDCK

Primary cell culture are widely acknowledged as the best cell culture systems available since they support the widest range of viruses. However, they are very expensive and it is often difficult to obtain a reliable supply. Continuous cells are the most easy to handle but the range of viruses supported is often limited.

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Cytopathic Effect (1)

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)

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Cytopathic Effect (2)

Syncytium formation in cell culture caused by RSV (top), and measles virus (bottom). (courtesy of Linda Stannard, University of Cape Town, S.A.)

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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.)

Tissue culture

Tissue culture / Explant culture

Fragments of minced tissue can be used as “explants”

This method is rarely used nowadays

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Serology 

• The development of antibodies to different components of the virus is used in staging the disease. For example in hepatitis B and HIV infections this approach is used.

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Viral Serology

– 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.

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Serological Diagnosis

• Detection of Immunologlublins Ig G. Ig M Ig A

• Raise of titers Ist sample later sample (convalescent sample) tested after 10 – 14 days Raise of titer is diagnostic

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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

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PCR

Each cycle doubles the copy number of the target

A target DNA sequence can be amplified to the point where it can be readily identified using labelled probes in a hybridisation assay

• The technique is used for the diagnosis of infections caused by HIV , HPV , Herpes simplex virus, Hepatitis B & C,Enterovirus,EBV ,Rubella & Rotavirus

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Polymerase Chain Reaction

• 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.

– A positive result may be difficult to interpret, especially with latent viruses such as CMV, where any seropositive person will have virus present in their blood irrespective whether they have disease or not.

• .

Detection of specific antobodies

Micro plate ELISA for HIV antibody: colored wells indicate reactivity

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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

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ELISA• Enzyme-Linked Immuno-Sorbant

Assays (ELISAs)– Enzyme reacts with substrate to produce

colored product– Very sensitive

• HIV test– If positive twice, Western Blotting is performed next

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Neutralization, hemagglutination, and hemagglutination inhibition assays. In the assay shown, tenfold dilutions of serum were incubated with virus. Aliquots of the mixture were then added to cell cultures or erythrocytes. In the absence of antibody, the virus infected the monolayer (indicated by CPE) and caused hemagglutination (i.e., formed a gel-like suspension of erythrocytes). In the presence of the antibody, infection was blocked (neutralization), and hemagglutination was inhibited, allowing the erythrocytes to pellet. The titer of antibody in the serum was 100. pfu, Plaque-forming units.From Medical Microbiology, 5th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 51-6.

Antibody detection

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Diagnostic methods for common viruses

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