Download Virology Techniques and more Exercises Virology in PDF only on Docsity!
Chapter 5 - Lesson 4
Virology Techniques
Introduction
Virology is a field within microbiology that encom- passes the study of viruses and the diseases they cause. In the laboratory, viruses have served as useful tools to better understand cellular mechanisms. The purpose of this lesson is to provide a general overview of laboratory techniques used in the identification and study of viruses.
A Brief History
In the late 19th^ century the independent work of Dimitri Ivanofsky and Martinus Beijerinck marked the begin- ning of the field of virology. They showed that the agent responsible for causing a serious disease in tobacco plants, tobacco mosaic virus, was able to pass through filters known to retain bacteria and the filtrate was able to cause disease in new plants. In 1898, Friedrich Loef- fler and Paul Frosch applied the filtration criteria to a disease in cattle known as foot and mouth disease. The filtration criteria remained the standard method used to classify an agent as a virus for nearly 40 years until chemical and physical studies revealed the structural basis of viruses. These attributes have become the ba- sis of many techniques used in the field today.
Background
All organisms are affected by viruses because viruses are capable of infecting and causing disease in all liv- ing species. Viruses affect plants, humans, and ani- mals as well as bacteria. A virus that infects bacteria is known as a bacteriophage and is considered the
This electron micrograph depicts an influenza virus particle or virion. CDC.
Bacteriophage. CDC.
most abundant biological entity on the planet. Many animal disease systems serve well as models for hu- man disease. Many of the techniques used in the study of viruses are the same whether they infect plants or warm- or cold-blooded creatures because the tech- niques are based more on the virus studied rather than the species affected.
Viruses are considered obligate intracellular parasites since they require living host cells to replicate. Virus- es take over or hijack the cellular synthesis machinery in order to reproduce. Viruses are submicroscopic and contain either DNA or RNA as their genome and is enclosed in a protein shell called the capsid. Coded in the DNA or RNA genome of the virus is all the information needed for replication. Some viruses also contain lipids, carbohydrates, and special enzymes that assist in their transmission and replication. Some viruses are enveloped; this envelope is acquired from the host cell either from the cytoplasmic or nuclear membrane. This outer coat is immunogenic, (what the immune system recognizes and what antibody pro- ducing cells respond to), and is necessary for the virus to invade a cell.
Laboratory Techniques
Several different methods are used to study viruses and viral diseases, as the field is constantly changing with the discovery of new methodologies and tech- nologies. This section will provide a cursory overview of the most commonly used techniques in diagnostic virology and will conclude with a brief glimpse of vi- rology in research.
Diagnostic virology is concerned with identifying the virus associated with clinical signs and symptoms. Procedures most commonly used include:
- Detection of a meaningful immune response to the virus (antibody or cell-mediated) by immuno- logic assay(s)
- Identification of the agent by staining of speci- mens or sections of tissue (light and electron mi- croscopy)
- Isolation and identification of the agent (cell cul- ture or fertile eggs)
- Detection of viral nucleic acid (probes or ampli- fication).
Detection of Immune Response
Often, it is difficult to identify a virus in relation to the disease observed, or when conducting a retrospective study of a population to determine exposure to a virus, or when measuring the response of an individual to a vaccine. In these cases, indirect methods of measure are needed, such as measuring antibody response to the virus of interest. Several methods exist for this purpose. A few of the most commonly used methods include:
- Virus neutralization (VN)
- Hemagglutination inhibition (HI)
- Enzyme linked immunosorbent assay (ELISA)
- Indirect fluorescent antibody (IFA)
- Complement fixation (CF)
- Agar-gel immunodiffusion (AGID)
- Agar-gel precipitin (AGP)
- Latex agglutination (LA).
The principles of these assays are fundamentally the same, they depend upon antibody-antigen interactions and consist of a known virus or viral protein, a patient sample (usually serum), and an indicator. If antibod- ies are present in the patient’s serum, they will bind to the virus. If no antibodies are present, no binding will occur. The indicator is observed to determine whether the sample is positive or negative for antibodies.
This illustration provides a 3D graphical represen- tation of a generic influenza viron’s substructure. A portion of the viron’s outer protein coat has been cut away. Dan Higgins, CDC.
Enzyme-Linked Immunosorbent Assay
The enzyme-linked immunosorbent assay ELISA is a very popular technique due to the ease of use and low cost. The ELISA consists of plastic wells coated with either the antigen (virus) of interest or a protein spe- cific to the antigen (virus) of interest. The unknown sample (serum) is allowed to bind to the coated well, an antibody labeled with an enzyme is applied, an in- dicator is added, and then a color change is observed. The presence of color indicates the presence of anti- bodies and the absence of color indicates the absence of antibodies.
Agar-Gel Immunodiffusions (AGID)
The agar-gel immunodiffusion (AGID), also referred to as an agar gel precipitin (AGP) test, involves the diffusion of virus and antibody through an agar (gela- tin-like substance), which will form a line of identity where the antigen-antibody complexes form.
Ab
Ab Ab
Virus
Negative Negative
Positive Line of Idenity
Schematic of an agar gel immunodiffusion (AGID) or agar gel precipitin (AGP) test. “Ab” represents a know antibody to the known virus in the middle
Coat plate with virus or protein
Enzyme-Linked Immunosorbent Assay (ELISA) / Enzyme Immunoassay Assay (EIA)
Add sample
If no antibody present will not bind to coated plate
If antibody is present will bind to coated plate
If no antibody present will not bind
If antibody present will bind
No antibody = no color
Enzyme
Enzyme Enzyme
Enzyme
EnzymeEnzymeEnzymeEnzyme
Indicator added
antibody = color
Light and Electron Microscopy
Light Microscopy
Viruses, unlike bacteria, are too small to be seen us- ing a standard light microscope. Therefore, antibodies labeled with an indicator, most frequently peroxidase or fluorescence, designed to identify the virus of inter- est are used. This label then enables the visualization of the virus cluster (because a single virus is too small to see with a light microscope) with the light micro- scope, in the case of peroxidase, or an ultraviolet (UV) light microscope in the case of fluorescence.
Electron Microscopy
Another way to identify a virus is with the use of the electron microscope. Since viruses are much smaller than bacteria, a regular light microscope does not pro- vide sufficient magnification to see them. The mag- nification of an electron microscope (50,000x magni- fied) provides the ability to see the viral particles. The problem with this method is the lack of sensitivity: a concentration of approximately 10^6 (1,000,000) virus particles per milliliter of fluid is required in order to
see the virus of interest. However techniques can be used to improve this, such as immune electron mi- croscopy. With this technique the sample is incubated with an antibody against the virus of interest and the antibody-antigen reaction results in clumps of the vi- rus, which allows for easier visualization. With the advancements in molecular methodologies, electron microscopy is becoming less widely used.
Rotavirus particles as seen under an electron microscope.
The electron microscope is being used to examine a thin section of the variola virus, revealing some of the structural features displayed by this pathogenic organism. CDC.
Traditional molecular techniques are dot-blot, South- ern blot, and in situ hybridization. These methods are dependent on the use of specific DNA or RNA probes. They are similar in sensitivity to some of the classical methods but are more tedious and expensive, and are not routinely used in diagnostic laboratories.
The technique that has probably had the greatest im- pact on the field of virology is the polymerase chain reaction (PCR, identification of DNA) and the reverse transcriptase polymerase chain reaction (RT-PCR, identification of RNA) along with the development of better, more standardized methods of nucleic acid pu- rification. The rapid advances and decrease in expense associated with nucleotide sequencing, commercial synthesis of oligonucleotides (primers), and the avail- ability of genetic sequences in public databases have contributed to the increased appeal of these methods. The PCR involves the use of two primers designed to identify a target. In the presence of a DNA poly- merase and other components required for DNA am- plification, the target sequence of interest, if present, is amplified. The PCR requires double stranded DNA,
thus it is useful for identifying DNA viruses. The dis- covery of reverse transcriptase, an enzyme capable of making a DNA copy of RNA, enabled the ability to expand PCR to include a reverse transcription step (RT) which generates a double stranded target (RNA- DNA), which is then used in the PCR to identify RNA viruses.
Summary
Virological techniques expand beyond diagnostics into the research laboratory. Many animal disease sys- tems are used as models for human diseases. Using several different types of molecular methods, such as cloning and inserting and deleting genetic informa- tion, viruses are being engineered in a variety of ways to improve human and animal health. Some of the re- sults of these manipulations are improved vaccines, viruses engineered to carry genetic information for use in gene therapy, as well as for use in cancer treat- ment. Virology is a constantly growing and dynamic field with much left to discover.
Reverse Transcriptase (RT) / Plymerase Chain Reaction (PCR)
cDNA
Reverse transcriptase
RNA template 5’^ 3’ 3’ 5’
5’ 3’
3’
5’ Primer Polymerase Primer 5’ 5’
5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’
Reverse Transcription (RT) Polymerase Chain Reaction (PCR) Amplification
Generation of DNA copy (cDNA) of RNA template Primer
References
Castro, A. E., & Heuschele, W. P. (1992). Veterinary diagnostic virology, A practioner’s guide. St. Lou- is, MO: Mosby-Year Book, Inc.
MacLachlan, N., & Dubovi, E. J. (Eds.). (2011). Fenner’s veterinary virology (4th ed.). San Diego, CA: Academic Press, Elsevier Inc.
White, D. E., & Fenner, F. J. (1994). Medical virology (4th ed.). San Diego, CA: Academic Press.
Questions
- Name three assays designed to identify the pres- ence of antibodies in a patient’s serum.
- Name three assays designed to identify the pres- ence of a virus.
- What is a limitation with virus isolation?
- What is an advantage for virus isolation?
- What is a benefit of molecular assays?
