THE LABORATORY DIAGNOSIS OF VIRAL INFECTIONS

Principles of viral diagnosis Detection techniques in virology can be thought of simply as two types: Direct diagnosis Those which detect the virus or parts of the virus Indirect diagnosis Those which detect the body’s response to a viral infection

Direct diagnosis Through detection of the infecting virus itself Visualization of virus particles: Electron microscopy In vitro propagation: Virus isolation on cell culture, embryonated eggs or in experimental animals Visualization of the effects of viral infection on infected tissue or cell cultures: light microscopy

Direct diagnosis Detection of viral antigens: Immunostaining of infected cells or tissues; Detection of viral antigen in body fluids or excretions Detection of viral nucleic acid (viral genome): Qualitative (‘yes or no’ answer); Quantitative (‘how much?’); Genotyping (‘what type?’)

Indirect diagnosis Through detection of the infected host’s immune response Humoral immunity: Antibodies of different classes (IgG, IgM, IgA) Cellular immunity: Cytotoxic T-cells

Laboratory Diagnosis of Viral Infections The procedure Includes: Collection of specimen Transport of specimen Specimen processing and inoculation Virus identification

Specimen collection The specimens sent to the laboratory must be representative Taken at the right time, From the correct site Stored and transported in the right way Viral transport medium is used for transportation Preserve viral infectivity within the specimen Prevent specimen from drying Stop the growth of bacteria and fungi

Specimen collection In addition to the above, 5-10 ml of clotted blood for serological tests is always required.

Immunofluorescence (IF) Tests Immunofluorescence techniques rely on the use of antibodies to label a specific target antigen with a fluorescent dye (also called fluorophores or fluorochromes) such as fluorescein isothiocyanate (FITC). Two IF methods are distinguished depending on whether the fluorophore is conjugated to the primary or the secondary antibody: Direct IF Indirect IF

A. Direct IF Virus or virus antigen is detected by a specific antiserum coupled with a fluorescent dye (e.g., fluorescein isothiocyanate). The dye becomes visible as a green fluorescence viewed by ultraviolet microscopy.

B. Indirect IF Virus or virus antigen is first reacted with an “unlabeled” specific antibody. The reaction is then detected by the use of labeled secondary antibody prepared against the specific primary antibody.

Cell culture of CMV grown in fbroblasts. A, Cytopathic effect (CPE) Cell culture of CMV grown in fbroblasts. A, Cytopathic effect (CPE). B, Cell culture monolayer of uninfected fbroblasts. C, DEAFF test – Immunofluorescent staining of fbroblasts infected with cytomegalovirus.

Enzyme-Linked Immunosorbent Assay (ELISA) and Radioimmunoassay (RIA) These techniques are used for the detection of viral antigens and antibodies developed against them, such as “p24” antigen of HIV, hepatitis antigens, rotavirus, and their corresponding antibodies. Virus or virus antigen is reacted with a specific antibody immobilized to a plastic surface. In ELISA, an enzyme labeled specific antibody is then added to the reaction. The whole thing is then detected by addition of a substrate that lead to the development of color change.

ELISA E 1. Anti-analyte 3. Anti-analyte-enzyme 2. Sample 4. Substrate

ELISA

Enzyme-Linked Immunosorbent Assay (ELISA) and Radioimmunoassay (RIA) In RIA, the label is radioactive iodine, and the detection depends on measuring the radioactive emissions.

Electron Microscopy (EM) and Immunoelectron Microscopy (IEM) Samples are negatively stained with phosphotungistic acid, i.e., the virions, which are not penetrated by the stain, stand out as white particles on a dark background. At least 106 particles must be present on the EM grid to stand a chance of being identified. In IEM, the specimen is reacted with specific antibody that agglutinates a particular virus, thus making the virions easier to find An antibody specific for virus antigen which is labeled with gold particles can also be used, in this case the virus itself can be visualized

Electron Microscopy (EM) and Immunoelectron Microscopy (IEM) These techniques can be used for the identification of viruses that can not be grown in cell culture, e.g., rotavirus, adenoviruses, and small round viruses in feces and HBV in blood. Electron micrograph of a negatively stained Papilloma virus specimen

Detection of viral nucleic acid Nucleic acid hybridization It is used for the detection of specific DNA fragments (test DNA) by the use of labeled DNA sequences (reference DNA). Only those fragments of test DNA possessing sequences complementary to the reference DNA will reanneal to form double stranded DNA.

Detection of viral nucleic acid Polymerase Chain Reaction (PCR) PCR is used for the amplification and detection of nucleic acids (e.g., a viral genome). PCR consists of three major steps, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time

Detection of viral nucleic acid Denaturation at 94°C: During the denaturation, the double strand (template) melts open to single stranded DNA. Annealing at 55°C: The single stranded primers anneal to the single stranded template, and on that little piece of double stranded DNA (template and primer), the polymerase can attach and start copying the template. Extension (polymerization) at 72°C: This is the ideal working temperature for the DNA polymerase.

Complement Fixation Test

Radial Hemolysis Test Virus-linked to sheep or human RBCs by chromium chloride Molten agarose is poured into a petri dish Cool, punch small wells, fill with a serum sample Incubate overnight Antibodies diffuse and combine with antigen on RBCs Complement is added Lysis of RBCs on which both antigen and antibody are present This technique is used for Influenza and Rubella viruses.

Radial Hemolysis Test

Western Blot Virus proteins are separated by electrophoresis Protein bands are transferred onto nitrocellulose paper Test serum is added, any specific antibody attaches to the viral proteins Antihuman antibody labelled with an enzyme is added Enzyme substrate is added The blot is examined for the presence of stained bands which indicate the presence of complexes of specific antibody with antigen.