Laboratory Diagnosis of Viral Infections

Methods for Virus Cultivation

For laboratory diagnosis of viral diseases the indication and identification of certain virus present in clinical specimen is elaborated.

Indication of viruses means the discovery of virus presence in the clinical material, whereas virus identification assumes the exact determination of virus species or type (primarily, viral serotype and viral genetic variant or genotype).

Three basic groups of laboratory tests are used in clinical practice for indication and identification of viruses:

rapid (or express) tests allowing direct detection of viruses in clinical specimens;

isolation of viral culture;

serological tests that confirm the diagnosis of viral infection by detection of specific antibodies against viral antigens in patient’s serum.

As current viral taxonomy is primarily based on genetic ground, the precise determination of viral genetic variant (genotype) is pivotal for correct identification of virus. For this purpose versatile molecular genetic tests like methods of nucleic acid sequencing, PCR and nucleic acid hybridization are commonly used.

PCR is the most convenient and reliable as well as sensitive and specific genetic test with possibility of full automation that is available now for clinical practice.

On the other hand, DNA microarray technologies based on parallel hybridization of multitude of nucleic acid probes placed in DNA biochip allow simultaneous testing of hundreds of clinical specimens for specific viral DNA or RNA. This creates excellent opportunities for mass screening of population for viral infections.

Finally, highly sophisticated methods of nucleic acid sequencing play the role of the reference tests for precise identification of viruses.

Serotype of virus is determined by reactions with specific antiviral antibodies, e.g. by neutralization tests, ELISA, immunofluorescence assay, complement fixation test, inhibition of hemagglutination, etc.

PCR and molecular hybridization of nucleic acids as well as ELISA and immunofluorescence assay are the most commonly used methods for rapid detection of virus directly in clinical specimen.

Genetic tests made possible the identification of fastidious viruses that can’t be cultured in laboratory cell lines – PCR or nucleic acid probing reveals nucleic acids of these viruses directly in the host tissues. For instance, noncultivatable sarcoma Kaposi virus was detected in tissues of AIDS patients and was proven to be the herpes family representative (herpesvirus type 8) by methods of molecular genetic analyses.

For isolation of virus cultures (as the viruses can propagate only inside the living cells) three main models are commonly used: laboratory cell cultures, chicken embryos and susceptible laboratory animals.

The main aims of virus cultivation presume the efficient laboratory diagnosis of viral infections; investigation of pathogenesis of viral diseases; and laboratory design and trials of antiviral drugs and vaccines.

The most simple and cheap model of virus propagation is their cultivation in embryonated chicken eggs under strictly controlled conditions. Virus-containing material can be inoculated in any compartment of the embryonated egg.

Indication of viral growth in the fertile chick eggs is performed by estimation of embryo death, vessels impairment, production of pocks or plaques on the chorioallantoic membrane (e.g., it is characteristic for herpes, smallpox, or vaccinia viruses).

Viruses, expressing hemagglutinins in their external coat, are shown to induce hemagglutination and hemadsorption phenomenons, clumping erythrocytes of different animal species. This is essential for ortho- and paramyxoviruses, certain types picornaviruses, and others.

As the result, after cultivation in chicken embryo the indication of hemagglutinating virus is performed by hemagglutination test. In this case the twofold dilutions of the allantoic fluid are mixed with sensitive red blood cell suspension. During the incubation the virions interact with erythrocytes, mediating their aggregation. The surface viral hemagglutinins of virion can attach simultaneously to two and more separate red blood cells that promote erythrocyte clumping.

The endpoint of hemagglutination test (or virus titer) is determined as the greatest dilution of the virus-containing material, which causes a clearly marked hemagglutination.

Similarly virus indication by hemadsorption is performed using the microscopy of the mixture of virus-infected embryo cells with erythocytes. In latter case the eukaryotic infected cells, surrounded by adsorbed erythrocytes, are readily discernible in the smear.

Identification of hemagglutinating virus in the chicken embryo is carried out by virus hemagglutination inhibition test. After primary incubation of the virus-containing allantoic fluid with the dilutions of antiviral type-specific antibodies, the erythrocyte suspension is added. As antibodies block the viral hemagglutinins, the hemagglutination is inhibited, and the virus serotype is determined.

Nevertheless, the most suitable and widespread model of virus investigation is the viral cultivation in cell (or tissue) cultures.

All cell cultures are prepared from animal or human source. The tissue is minced into small pieces and homogenized. Then it is treated by proteolytic enzyme (mainly, by trypsin or collagenase) to disintegrate intercellular matrix. The cell suspension is placed into culture multiwell plate or flask with sterile nutrient complex medium, containing all necessary growth factors, such as amino acids, carbohydrates, vitamins, salts and sometimes fetal serum components and antibiotics to prevent bacterial growth. The cells propagate in the medium and finally form a single layer of the cells (cell monolayer), attached to the plastic surface.

There are three main types of cell cultures: primary, secondary or diploid, and continuous.

Primary cell cultures are obtained from different human and animal tissues (human skin fibroblasts, monkey kidney cells, etc.) They grow for 1-3 weeks with final autolysis and culture death. In most of cases primary cultures undergo only 1-2 passages, thus they are not suitable for long-term cultivation.

Cell passage (or subculture) means reinoculation of a small portion of basic cell culture grown in the medium into another well or flask with fresh nutrient medium for further propagation. Cell passaging substantially expands the survival time of laboratory cell lines.

Diploid (or semi-continuous) cell cultures are designed for longer cultivation. The sources for diploid cell lines are fetal tissues (e.g., human diploid lung fibroblasts, human breast epithelial cells, etc.) They are also of limited survival, but can propagate for 40-50 or more passages. The nuclei of diploid cells retain their normal chromosome pattern. They can be used for cultivation of most viruses.

And continuous cell cultures are originated from the almost immortal tumor cell lines (HeLa cells from human cervical cancer, HEp-2 from larynx carcinoma, McCoy cells, and many others.) Tumor cells can divide indefinitely long in vitro. These lines are appropriate for many viruses, but their genome might be not completely stable, rendering rare but cumulating chromosome aberrations. Also they can be occasionally contaminated by bacterial intracellular parasites (e.g., mycoplasmas) in course of long-time cultivation.

Viral indication and identification in the cell cultures is carried out by several methods.

Viruses are indicated by their cytopathic effect. During cultivation the viruses impact on the cell life cycle resulting in changes of cell morphology and viability. This viral action is known as cytopathic effect.

Different types of cytopathic effects exist – degeneration and destruction of monolayer, cell lysis or necrosis, plaque appearance, inclusion formation, cytoplasmic vacuolization, symplast, syncytium and giant cell formation. Many viruses develop a special cytopathic effect, which is used for viral indication.

Inclusion formation is the characteristics of many viruses. RNA viruses (e.g. rhabdoviruses) usually form inclusions in the cell cytoplasm, while DNA-containing agents (e.g. herpesviruses) – in the nuclei of infected cells. However, the cells, infected with DNA-containing poxviruses, produce the specific inclusions (Guarniery bodies) in cytoplasm. Some viruses are shown to form inclusions both in the cell nucleus and cytoplasm (measles virus). Inclusions are the sites of virus intracellular replication.

Different viruses can induce symplast and syncytium formation in the cell cultures because of virus-induced cell fusion activity. Giant polynuclear epithelial cells (symplasts) are formed after measles virus action. Respiratory syncytial virus and HIV exert the damage of infected cells by syncytium formation.

Plaques appear after cell monolayer destruction and lysis, caused by virus. Plaques look like clear zones within the cell culture. This method is used for the determination of virus quantity, because each plaque is the result of single virus particle propagation within susceptible cells. Total virus count is calculated by multiplication of plaque quantity by the dilution of the sample.

Indirect evaluation of viral cytopathic activity is performed by color reactions in the cell cultures. Test tubes, containing cell cultures, virus sample, and nutrient medium with indicator dye, are incubated for several days. During incubation the virus replicates within susceptible cells and destruct them. Control test tubes are free of virus, and the abundant growth of the cell culture is observed. In the control tubes the color of the indicator medium is changed due to the accumulation of products of cellular metabolism, resulting in the medium acidification and pH lowering. And vice versa, viral cytopathic activity blocks cell metabolism retaining the initial color of the medium.

Hemagglutination and hemadsorption are also used for hemagglutinin-containing virus indication in the infected cell cultures. Influenza and parainfluenza viruses are determined by these reactions.

Viral identification in the cell and tissue cultures is based on virus neutralization reactions, promoted by specific antiviral antibodies, which block viral activity. The inhibition of virus hemagglutination and hemadsorbtion, neutralization of plaque and inclusion formation, neutralization of color reactions can be applied.

And obviously, for precise indication and identification of viral isolates grown in cell culture lines, all genetic methods (like PCR or nucleic acid hybridization) or serological tests (immunofluorescence or ELISA test) are commonly used in clinical virology laboratories.

Tissue and cell cultivation makes possible the rapid detection and diagnosis of viral infections. Also it is the suitable technology necessary to obtain the pure viral culture from cell lysate. It can be used for virus diagnosticum production or vaccine design.

Sometimes the viruses are inoculated to propagate in the susceptible laboratory animals. For instance, Coxsackie viruses infect newborn “suckling” mice with characteristic disease development. Indication and identification of viruses in this model is similar with the above described.

Serological testing allows to confirm the diagnosis of viral infections by detection of specific viral antibodies arisen in patient’s serum. A broad group of serological methods are currently used in laboratory practice – ELISA (the most common test), hemagglutination inhibition assay, radioimmunoassay, complement fixation test, neutralization test and some others.

Identification of specific antibodies of IgM class indicates acute primary viral infection

In many clinical situations serological paired sera test is elaborated, where patient’s antiviral antibodies are tested at least twice – early after the disease onset and next closer to patient’s recovery. The fourfold increase of the titers of specific antibodies gives the confirmation of current viral infection.