Virology practice Dr. Y. Hamadt Allah Elhaj Ass. Prof. medical microbiology

Cultivation of viruses Since viruses are obligate intracellular parasites they have to be grown in living cells. There are three systems for their cultivation: 1- tissue culture. 2- embryonated eggs. 3- intact animals. 1- tissue cultures: Pieces of animal or human tissues are trypsinized to get separate cells. Thses are grown in presentceof growth medium containing serum, on glass or plastic tubes, bottles or plates with a flat side.

A monolayer or sheet of cells is formed on the flat side of container within few days. Viruses are inoculated on the monolayer. There are three types of tissue cultures: 1.Primary cell lines: these are prepared from organ fragments e.g. monkey kidney. such cells can only divide for several passages (4 - 6) and then degenerate. 2. Continuous cell lines: these are derived from tumour cells and they can divide indefinitely e.g. hela cells which are derived from carcinoma of the cervix.

3. Human diploid cell lines: these are usually fibroblasts derived from human embryo tissues. They have diploid number of chromosomes. They grow rapidly and can be subcultured up to about 50 passages in culture e.g. human embryo lung tissue.

Detection of virus replication in cell culture: 1- cytopathgenic effects (CPE): these are changes in cells that can be observed microscopically: a.Cell death and detachment from the glass surface is produced by many viruses e. g. poliovirus. b.Rounding and grape like cluster formation is produced by adenovrus. c.Cyncytium or multinucleated giant cell formation are characteristic of measles or mumps.

d. Cell transformation: the cells lose the property of contact inhibition present in normal cells and pile up to form foci of malignantly transformed cells e.g. tumour viruses.

2- Plaque formation: plaques are virally infected areas in tissue culture monolayer. They can be seen by the naked eye as unstained areas when using vital stains. 3- Inclusion bodies: these are intranuclear or intracytoplasmic structures which may appear in virus infected cells and can be seen by the light microscope. They are often the site of virus replication. Their presence is of diagnostic value e.g. the (Negri bodies) in nerve cells of rabid animals.

4- Haemadsoption: When RBCs are added to infected cells they will appear as rosettes or clumps on the areas where the virus is growing. This is useful in haemagglutinating viruses such as influenza virus. 5- Fluorescent antibody staining: infected cell sheets on cover slips or microtiter plates may be treated with fluorescein labelled specific antibody and examined for positive fluorescence.

6- Interference: in some viruses which do not produce CPE their growth can be proved by their ability to interfere with the growth of another CPE producing virus. 7- detection of viral antigens by serology: including complement fixation test, haemagglutination test,…..

8- neutralization tests: neutralization of effects of virus on tissue culture by specific antisera can be used to identify and type the virus isolated.

Plaque

CPE: inclusion bodies Negri bodies Negri bodies 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)

Hemadsorbtion add red blood cells

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2- embryonated eggs: Different viruses can grow in various cavities of embryonated eggs or in the developing embryo itself. The age of the embryo used and the site of inoculation vary according to the virus inoculated. Chorioallantoic membrane inoculation is used in pox and herpes viruses. The influenza virus can readily grow in the amniotic sac and in the respiratory cells of the embryo.

3- intact animal: Animal inoculation was mainly used in the past when tissue culture methods were not known, however, animal inoculation is still used for studying viral oncogenesis, pathogenesis of viral disese, immune response to viruses and for primary isolation of some viruses. The white sucking mouse is the most widely used; it is susceptible to encephalitis viruses by the intracerebral route.

Diagnosis of virus infection the laboratory procedure used in diagnosis of viral disease include: a. Direct detection of viruses or their antigens in clinical specimens. b. Isolation of viruses. c. Serologic detection of antiviral antibodies.

a. Direct detection of viruses or their antigens in the specimens: Can be achieved by different techniques: 1- Light microscope: this can be used to visualize some large viruses e.g. poxviruses in which elementary bodies can be seen in skin lesions (papules and vesicles). Inclusion bodies can also seen under the light microscope in several viral infections. In rabies, intracytoplasmic inclusions called (negri bodies) can be detected in nerve cells.

2- Electron microscope: is used to demonstrate virus particles in vesicular fluid or tissue extracts treated with special stains. It is only successful if large numbers of particles (10 9 /ml) are present. 3- Immunoelectron microscope (IEM): addition of specific antisera to the clinical material leads to aggregation of virus so it can be detected more readily than separate virus particles e.g. diagnosis of HAV and rota virus in stool.

4- Immunofluorescence microscopy: detection of virus in smears from lesions using fluorescein labeled specific antisera. E.g diagnosis of rabies virus in brain smears. 5- Solide – phase immunoassay: both radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) can be used for detection of viral antigens in different clinical specimens such as detect of HCV, HBV, rotaviruses,….

6- Nucleic acid hybridization: using DNA probes, it is possible to detect virus nucleic acid in pathologic specimens or in tissue samples. The probe which is a single strand of the nucleic acid of the virus in question will hybridize with its complementary strand in the specimen. Probes are labeled and can be easily detected. 7- Polymerase chain reaction (PCR): is a recent technique which involves amplification of a short sequence of a target DNA or RNA ( which may be in low concentration e.g one copy). Leading to accumulation of large amount of that sequence, so it can be easily detected.

b. Isolation of viruses: Isolation of viruses from clinical specimens by inoculation on tissue culture, chick embryo or laboratory animals according to the virus in question.

c. Serologic detection of antiviral antibodies: Serologic diagnosis of virus infections can be established by detecting a rising antibody titre to the virus. the first sample should be collected early after onset, the second 1 -2 weeks later. If paired sera are not available or rapid diagnosis is needed, as in diagnosis of rubella in early pregnancy; detection of IgM antibodies to the virus is resorted to. The detection of IgM in a single serum sample, indicates recent infection. c. Serologic detection of antiviral antibodies: Serologic diagnosis of virus infections can be established by detecting a rising antibody titre to the virus. the first sample should be collected early after onset, the second 1 -2 weeks later. If paired sera are not available or rapid diagnosis is needed, as in diagnosis of rubella in early pregnancy; detection of IgM antibodies to the virus is resorted to. The detection of IgM in a single serum sample, indicates recent infection.

Neutralization test This is based on the neutralization of the virus infectivity by mixing it with specific antibody before inoculation into cultures.

123 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 2. 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.

231 Principle of Neutralization test

Viral Hemagglutination Inhibition Test Useful for viruses that aren’t cytopathic Test based on viral hemagglutination, the ability of some viral surface proteins to clump red blood cells Serum from an individual will stop viral hemagglutination if the serum contains antibodies against the specific virus Commonly used to detect antibodies against influenza, measles, and mumps

Complement Fixation Test Based on the generation of membrane attack complexes during complement activation that disrupt cytoplasmic membranes Used to detect the presence of specific antibodies in an individual’s serum Can detect antibody amounts too small to be detected by agglutination

Fluorescent Antibody Test Uses fluorescent dyes as labels Fluorescein is the most important dye used in these test – Chemically linked to an antibody without affecting antibody’s ability to bind antigen – Glows bright green when exposed to fluorescent light Fluorescein-labeled antibodies used in two types of tests – Direct fluorescent antibody test – Indirect fluorescent antibody tests

Direct Fluorescent Antibody Tests Identifies the presence of antigen in tissue – Tissue sample flooded with labeled antibody – Antibody and antigen are allowed to bind for a short period – Unbound antibody washed from the preparation – Results observed under a fluorescent microscope Used to identify small numbers of bacteria in patient tissues Not a quantitative test – the amount of fluorescence observed is not directly related to the amount of antigen present

Indirect Fluorescent Antibody Tests Can be used to detect antigens in cells or patient tissues Also used to detect specific antibodies in serum via a two-step process

Indirect Fluorescent Antibody Test Figure 17.14

ELISA Stands for enzyme-linked immunosorbent assay 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

Antibody Sandwich ELISA Modification of the ELISA technique Commonly used to detect antigen Antigen being tested for is “sandwiched” between two antibody molecules

Antibody Sandwich ELISA

Advantages of the ELISA Can detect either antibody or antigen Can quantify amounts of antigen or antibody Easy to perform, inexpensive, and can test many samples quickly Plates coated with antigen and gelatin can be stored for later testing