Diagnosis of viral infections

The laboratory diagnosis of viral infections based on three general approaches 1-Direct detection of viral components either in cells derived from infected tissues or free in fluid specimens. 2-Isolation of viruses, usually in cell cultures, followed by identification. 3-Demonstration of a significant increase in serum levels of antibodies to the etiological virus during the course of the illness or the presence of antibodies of class [IgM] specific to the etiological virus.

Specimens in viral infection Timing Specimens for virus isolation & direct detection as well as blood samples must be collected within the first few days of the illness . Choice of the specimens This done by knowing the pathogenesis of the virus . e.g. Polio virus infection, the neurological samples is not important in the diagnosis, but throat wash is important, because the virus start replication in the oropharynx. Clinical data Because each type of virus need special type of tissue culture for replication, so clinical data is important. Specimen transport & storage Specimens should delivered without delay, kept at neutral pH to avoid affection of viral infectivity. Keep the specimens at 4 °C if delay is expected for four days. (refrigeration). If the delay is > four days the specimens should be kept at -70 °C (freezing). If a virus suspected to be present is labile to freezing, the sample should be diluted with an equal volume of 2SP(sucrose in phosphate buffer pH 7.2).

Culture & isolation Is the gold standard in viral diagnosis. Viral Culture Viruses don't grow on artificial media, they must be cultured on cell cultures or other type of living system. 1- Emboryonated eggs Inoculation of embryonated eggs used for growing of some viruses,( mumps & influenza viruses). Fertilized egg is incubated for (5-14) days after that inoculated. The site of inoculation & the period of incubation is depend on the type of virus. Then embryonated examined for the evidence of viral replication Death of the yolk sac. Pocks formation as cytopathic effect on the chorion. Examination of the embryonated egg fluid for the viral antigens.

2- Laboratory animals In the past, mice & chimpanzees were inoculated with viruses, but now this has been largely replaced by the use of cell cultures. 3- Cell cultures Cell culture originated from viable host cells & grow into a monolayer on the sides of glass or plastic test tubes. Cells are kept moist & supplied with nutrients by keeping them immersed in cell culture medium & maintenance medium. Cell culture incubated 1-4 weeks, depending on the type of the virus. Periodically the cell are inspected for the presence of virus, indicated by areas of dead or dying cells called cytopathic effect (CPE). Cell culture help in 1-Isolation & growth of a variety of viruses. 2-Studying viral replication. 3-Development of attenuated mutant strains, & vaccine production.

A cell culture becomes a cell line once it has been passed (sub-cultured) in vitro. Cell lines are classified as Primary cell lines. Low passage cell lines. Continuous cell lines. Primary cell lines Those cell lines that have been passed only once or twice since harvesting. Further passage of primary cells results in decreased the receptivity of the cell lines to viral infection. Examples of the primary cell lines 1-Human embryonic kidney. 2-Guinea pig embryo. 3-Chick embryo. 4-Primary monkey kidney.

Low passage cell lines Those cell lines that remain virus sensitive through 20 to 50 passages. Human dipliod fibroblast cells such as lung fibroblast, are a commonly used low passage cell line. Examples 1-Human diploid fibroblast cell line. 2-MRC5 institution of the medical research. 3-WI 38. Continuous cell lines Those cell lines that can be passed & remain sensitive to virus infections indefinitely. 1-HEp-2 human epidermiod cancer of the larynx. 2-HELLA cervical carcinoma.

Identification of viruses detected in cell culture Infected cell in cell culture change their morphology, & eventually lyse or detach from the glass surface while dying. These changes in the infected cell called cytopathic effects of the virus (CPE). Viruses are detected in cell culture by the recognition of these (CPE). Viruses have distinct CPEs, just as colonies of bacteria on agar plates have unique morphologies. CPE may be quantitated as in the following QUANTITATION INTERPRETATION Negative uninfected monolayer Equivocal(+/-) atypical alteration of monolayer involving few cells. 1+ 1-25% of monolayer exhibit CPE 2+ 25-50%of monolayer exhibit CPE 3+ 50-75% of monolayer exhibit CPE 4+ 75-100% of monolayer exhibit CPE Viruses detected in cell culture identified by 1-Cell type that support viral replication. 2-Time need to detect CPE. 3-Morphology of the CPE.

Syncytia formed by membrane fusion of infected & non infected cells. Morphology of CPE These CPE suggestive of infection by a family, but don't allow determination of the species of the virus. Examples of CPE (a) Herpesviruses: loss of architecture & rounding of cells in 24-48 hours. (b) Paramyxoviruses: (measles virus, respiratory syncytial virus) cells coalesce into large multinucleated syncytia in 3-5 days. Syncytia formed by membrane fusion of infected & non infected cells. (c) Cytomegalovirus (CMV) appearance of foci of cell lysis in 2-3 weeks. (plaques) (d) Viruses may cause the appearance of inclusion bodies in infected cells. Viral inclusions They are intracellular structures formed by: a- aggregates of viruses within an infected cell. b- abnormal accumulation of viral components from inefficient viral maturation. Inclusion bodies are visible by conventional microscopy. Location & appearance of inclusion bodies provide information about infecting virus. E.g. stained smear from based of skin vesicle used to detect VZV or HSV inclusions is called a Tzank test.

Human embryo skin muscle cells were infected with human CMV & stained at selected times to demonstrate: (A) uninfected cells, (B) late CPE (nuclear inclusions, cell enlargement), (C) cell degeneration (D) a focus of cell lysis in a cell monolayer ( plaque).

For definitive identification of the virus 1- Fluorescent-labeled antisera available for most viruses, & used for culture conformation. 2 - Viral neutralization used to identify viruses with many serotype for which fluorescent labeled antisera are not available. 3 - Acid liability test used to differentiate enteroviruses from rhinoviruses. 4 - Some viruses, such as influenza, parainfluenza, & mumps, can be detected by hemadsorption, since infected cells contain viral hem-adsorbing glycoprotein in their outer membranes. 5 - Other methods EM, or immune electron microscopy to detect the virus inside the infected cells.

Shell vial method A rapid modification of a conventional cell culture. Virus is detected more quickly, by staining host cell for viral antigens before the development of CPE. Here the monolayer of the cell culture is grow on cover slip (shell vial) & specimens are inoculated onto the shell vial cell layer by low speed centrifugation, which enhances viral infectivity. So the shell vial method (a) Shorten the time of virus to get in the cell. (b) Enhance replication of the virus. (c) Decrease the time of CPE of the virus to occurs. Used for viruses required long incubation before producing CPE. e.g. CMV need four weeks to shown CPE in conventional cell culture, by shell vial method few days required for CPE to appear.

Rapid diagnostic tests These tests in comparison to the cell culture (required days), few hours required after receiving the specimens to detect the virus. These tests includes 1-Direct EM examination of the specimens. e.g. examination of the stool for Rotavirus & Hepatitis A. 2-Immune electron microscopy. Allows visualization of virus particles present in numbers too small for easy direct detection 3-Antigen detection method The antigen of the virus is detected by immunofluorescent technique. e.g. detection of the RSV antigen in the respiratory secretions. 4-Agglutination method Here antibody is added to the specimen to detect the presence of the viral antigen. 5-Molecular technique (Nucleic acid hybridization) Use to detect viral genome & genome sequence in clinical specimens.

Examples of the molecular methods A- Insitu hybirdization technique. use single stranded, complementary nucleic acid probes. It is highly sensitive & specific This technique used to identify a- Adenovirus in nasopharyngeal washings. b- CMV in urine. c- HIV in blood of seronegatine individuals B-PCR. Increases the sensitivity of nucleic acid hybridization by increases the amount of viral genome obtained from the patient. This approach is useful for rapid detection of the virus in patient specimens when the amount of viral nucleic acid in patient sample is small.

6-Serology Serological test used to detect antibodies produced against viral antigens. From these serological tests we have 1-Radioimmunoassay RIA. 2-Enzyme immune assay. 3-Indirect immunofluorescent tests. 4-Hemagglutination inhibition test, measure antibodies directed against hemagglutinating viruses, such as influenza viruses. 5-Complement fixation test. The aim of the serological tests 1- Detect a significant increase in the titer of antibodies to the etiological virus. 2- The presence of IgM specific antibodies to the etiological virus.