Methods of Identification and Isolation of Virus

Introduction

Specimen processing

In addition to patients, identification and demographic as well as other information should be also included such as,

• Source of specimen
• Clinical history or
• Date and time of specimen collection

If this information is not available a call for additional details should be made to the requesting physicians or to the person caring for the patient.

Processing of viral specimens should occur in a biological safety cabinet whenever possible. This products specimen from contaminated by the processing technologies and protect the lab from infectious aerosols created when specimens are manipulated. Latex gloves and lab coats should be worn during manipulated of all patients specimens vortexing, pipetting and centrifugal can all create dangerous aerosols. Vortexing should be done in a tightly capped tube behind a shield. After vortexing, the tube should be opened in a biological safety cabinet. Pipetting also should be performed behind a protective shield and pipette must be discarded into a disinfectant fluid so that the disinfectant reaches the inside of the pipette.

More than one patient's specimens or series of cell culture tubes for that specimens should not be opened for inoculation at one time. Aerosols and microsplashes may contribute to cross contamination of the cultures. Containing specimens can be re-processed with antibiotics containing virus transport media (VTM) if they were not originally handled in this manner or they can be filtered using a disposable membrane filter of size 22µm.

Transport of specimen for virus isolation

The diagnosis of viral infections by culture relies on the collection of proper specimens, proper care to protect the virus in the specimens from environmental damage, and use of an adequate transport system to maintain virus activity. A variety of transport media has been formulated, beginning with early bacteriological transport media. Certain swab-tube combinations have proven to be both effective and convenient of the liquid transport media, sucrose -based and broth-based media appear to be the most widely accepted and used. Studies on virus stability show that most viruses tested are sufficiently stable in transport media to withstand a transport time of 1 to 3 days. Some viruses may withstand longer transport times. In many cases, it is not necessary to store virus specimens in a freeze or send them to the laboratory on wet ice or frozen on dry ice. However, the specimen should not be exposed to environmental extremes. Modern viral transport media allow for the most effective use of viral culture and culture enhancement techniques for the diagnosis of human viral infections.

The constituent of suitable viral transport media is designed to provide an isotonic solution containing protective proteins , antibiotics to control microbial contamination and one or more buffers to control the PH. Liquid transport media are used primarily for transporting swabs or materials released into the medium from a collection swab.

Isolation of virus

We have seen how viruses are activated; however, in order to study them thoroughly, one must have them free from an unnecessary contaminating material such as a component of tissue cells. It is essential that the viruses are available in the reasonably pure from. To achieve this, they must be isolated first and then suitably purified. The principle techniques used for this purpose are:

• Ultrafiltration
• Ultracentrifugation
• Chemical precipitation
• Adsorption chromatography
• Solvent Extraction

1.Ultrafiltration

The viruses which have already in the living cells must have more or less similar condition of PH and temperature, before proceeding to any of the techniques. So, selection of proper pH of the extracting medium is quite important, the infected tissue first should be homogenized in a cold condition with a suitable buffer. On proper homogenization, the tissue gets completely disrupted and a uniform suspension is obtained. The suspension is subjected to the isolation of virus through any of the mentioned techniques.

Ultrafiltration technique is based upon the fact that virus particles have a smaller size than any other microorganism. Therefore, separate viruses from a mixture of variously sized particles, one have simply to use a filtration device capable of retaining the larger particles while allowing the smaller particles to pass through it.

In order to retain these viral particles, the viral suspension may further be passed through ultrafilters made up of cellulose acetate.

In recent years, the diatomaceous earth has been increasing use as a filtrating aid; charcoal and bentonite have also been successfully used. More sophisticated gel filtration technique using agar-agar or cephalexin allow selective filtration of virus particles.

Demerits

There are few other types of microorganism which have size range comparable to those of larger viruses. The rickettsiae: a pathogenic group of microorganism and mycoplasma, the cell wall-less bacteria are two examples of such organisms. Therefore, the technique of ultrafiltration is likely to be useless in case of virus suspension that contains such types of microorganism.

2.Ultracentrifugation

This is based on the principle that, out of the particle in a suspension, the heavier ones have the tendency to settle down first. This is because of the attraction of gravitational force. Usually, the process would be a slow one. In a case of extremely small particles, the process of settling down or sedimentation may take an almost indefinite time period. The rate of sedimentation could be increased by increasing the gravitational pull itself. This can be done by applying centrifugal force on the suspension. This is commonly achieved by rotating centrifuge tube containing the suspension in a centrifuge machine. Such rotation or centrifugation at a very high speed produces force equivalent to many thousand times the gravitational force. Depending upon the mass of the particles of the particles to segregated, suitable gravitational force should be applied.

Centrifugation at extremely high speed, generating thousands of times of normal gravitational pull is known as ultracentrifugation. Virus particles present in a suspension containing cell residue and other materials may be purified by a two-step centrifugation process. In the first step, the residual, cell materials or bacterial cells are separated out by centrifuging the suspension at low speed; the semi-purified suspension could be further purified by the filtration. The filtrates containing the virus particle are centrifuged at high speed. The virus particle sediment and form a small heap or pellet at the bottom. This technique is also called differential centrifugation. The speed of ultracentrifugation for viral suspension may reach 40000g-150000g.

Methods of Virus Identification

For the successful detection and identification of the viruses different methods have been used such as:

• Virus isolation in cell cultures
• Immunofluorescence-based assay
• Molecular techniques to determine nucleic acid.

In addition to this, the most commonly used methods in order to quantify viruses can be subdivided into three major categories:

• Techniques measuring viral infectivity such as viral plaque assay, immunofluorescence foci assay, etc.
• Technique examining the viral nucleic acid and protein such as hemagglutination assay, ELISA, immunoblotting, immunoprecipitation, etc.
• A technique that depends on the on direct counting of physical viral particles such as transmission electron microscopy and viral flow cytometry.

Although the above-mentioned techniques have several limitations, the improved techniques of virus quantification and identification are constantly being explored to overcome their limitations. Here is some of the most common virus identification and quantification protocol which is currently being used both in diagnostic virology and experimental lab are mentioned below:

1. Microscopy in Cell Culture
2. Conventional Methods of Virus Quantification
3. Measurement of Viral Proteins and Nucleic Acid
4. Direct Counting of Viral Particles.

1. Microscopy cell culture

Microscopic cell culture is based on system to classify animal viruses into four distinct division consisting-

1. Viruses that caused cell degeneration,
2. Viruses that caused formation of inclusion bodies and cell degeneration,
3. Viruses that caused the formation of multinucleated cells (syncytia),
4. Viruses that caused no cytopathic effect (CPE).

Since then, cell cultures have been successfully followed routinely for Vitro isolation of viruses. Presumptive identification of virus types can be made by observing morphological changes produced in a host cell that is a cytopathic effect, caused by cytopathogenic viruses.

• Immunofluorescence (IF) Assay

Immunofluorescence technique is widely used for rapid detection of virus infections by identifying virus antigens in a clinical sample. This assay technique has been successfully used for better management of influenza virus infection and surveillance of influenza virus activity. In addition to this IF assay is also well-accepted for laboratory diagnostics test, however, sometimes this technique could be quite expensive, due to a cost of antibodies used in this assay.

• Molecular Methods

The establishment of molecular methods for the direct identification of specific viral genome from a clinical specimen is one of the greatest achievements of the 21st century.

2. Conventional methods for virus quantification

• Viral Plaque Assay

Viral plaque assay is one of the most widely used methods in virology to purify a clonal population of a virus as well as to determine viral titer. A plaque usually is a result of infection of the cell by a single virion on the host cell monolayer. As such, to perform a plaque assay, susceptible host cell monolayer is infected with 10-fold serial dilutions of virus.

• Immunofluorescence Foci Assay (IFA)

The IFA is also known as fluorescent foci assay FFA which is a rapid method of virus titration that allows the quantification of virus in cell lines. This technique does not support plaque formation or do not exhibit the detectable cytopathic effect.IFA is not dependent on the ability of induction of cell death, rather it utilizes antibody-based staining methods in order to detect virally infected cells.

3. Measurement of viral proteins and nucleic acid

• Polymerase Chain Reaction (PCR)

PCR is one of the most widely used laboratory methods for detection of viral nucleic acids. This technique can also be use to determine viral RNA, by adding an initial step in which the RNA is converted into DNA; know as reverse transcriptase PCR that is RT-PCR.

• Immunoblotting

Immunoblotting also knew to be western blot; WB analysis technique that detects specific viral proteins isolated from a cell, tissue, organ, or body fluid.

• Immunoprecipitation

Immunoprecipitation method utilizes specific antigen-antibody interaction and binding of a protein to a fragment of an antibody.

• Enzyme-Linked Immunosorbent Assays (ELISA)

Indirect ELISA can be used for screening of blood samples collected at blood bank as well as presumptive diagnosis of HIV infection by detecting antibodies.

• Hemagglutination Assay (HA)

4. Direct counting of viral particles

• Flow Cytometry (FCM)

• Transmission Electron Microscopy (TEM)

REFERENCE

Cheesbrough, M.Medical Laboratory Manual for Tropical Countries.Vol. Vol 2. ELBS London, 2007.

Tille, P.Diagnostic Microbiology.13th. Elsevier, 2014.

D, Grenwood, Slack RCB and Peutherer J. Medical Microbiology. Dunclude Livingstone: ELBS, 2001.