METHODS FOR DECTECTION OF VIRUSES
The term virus has originated from a Latin word which means poison. In a biological sense, the word has been in use for about 100 years. Scientists first applied the word to describe such agents which they thought to be responsible for causing certain diseases. The term gradually came to signify a special type of entity whose properties were found in many aspects to be completely different from those of living organisms. Information about these substances was gradually gathered and a whole branch of study of developed. This branch of study is referred to as Virology and is today one of the most important field of science.
Many numerous laboratories all over the world, busy in gathering newer information about viruses and in developing fresh ideas regarding them. Virology, the discipline dealing with the viruses, is of comparatively of recent origin, it is roughly a century old. However, several incidences linked directly to viruses have been known to mankind from the very dawn of civilization, as for example, such diseases as smallpox, Rabies or the mosaic disease of tobacco or such incidences as tulip break. In fact the most outstanding example of applied virology, vaccination, introduction by Jenner in 1796, antedates the actual discovery of viruses by almost one hundred years. Credit for the discovery of viruses goes to Iwanowski (1892). However, contribution of pioneers like Pauster, Mayer and Beijerinck can never be over emphasized. Iwanowski demonstrated that agents responsible for causing the mosaic disease of tobacco were filterable indicating the existence of the disease causing agents smaller than bacteria their distinct nature was established later by Beijerinck who described them as contagium vivum fluidum or living infection fluid.
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The filterable nature of the casual agents of many unestablisehed diseases both plants and animals were gradually discovered; ‘filterable' agents destroying bacteria was discovered too (bacteriophages). Moreover, it is also become clear that these mysterious agents were always found in association with some living organisms or other but never alone. The real breakthrough in the progress of virology was achieved in the frontiers with the development and utilization of sophisticated techniques like electron microscopy, X-ray crystallography and ultra centrifugation. At about the same time another development went a long way in establishing the true physicochemical and biological nature of viruses. This was the successful crystallization of tobacco mosaic virus (TMV) particles by Stanley (1935).
Utilizing these techniques the information concerning the chemistry and physics of viruses was obtained at a rapid rate. New viruses were discovered. Ultra structure of well known viruses like TMV and bacteriophages were elucidated. Viruses could be successfully cultivated in the laboratory thus rendering the task of getting viruses in the pure form comparatively easier.
Several aspects of the physiology of viruses, particularly their ability to reconstitute and replicate were also established. That they could replicate only at the expense of the metabolites and energy of the host, while guiding the entire operation through their own genomes, added an extra dimension to their characteristic.
A duality in their character, involving traits, purely inanimate and also purely animate came to be recognized. Their capacity to mutate and recombine genetically was established. Their unique biology was accepted. All this happen during the course of last few decades.
In recent years the emphasis has been on the proper assessment of the role played by viruses in causing diseases and to develop ways and means to fight them. With the establishment of a viral connection to many a disease, particularly of animals, proper immunization procedures have been developed; chemotherapeutic agents have been discovered and prepared. Control of viral diseases by checking their modes of transmission has been achieved. The role of viruses play in causing cancer in mammals and their organisms is being investigated extensively and intensively all over the world. Epidemiology of many viral diseases are becoming better and better understood, enabling health authorities to devise effective fighting strategies against them.
Though young, the science of virology has already assumed a significance which is second to none. Many ideas concerning them are getting clarified. One hopes the future will bring in a better understanding and a better adaptation to these mysterious agents which defy full understanding as yet.
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The data so far collected give a favorable and valuable approaches on the recent trends in prediction of latest diseases caused by their and eradicating the future infections.
Some of these are listed for consideration. Viruses have an important position in the biological system of this planet, strictly speaking not to be considered members of the living world. Attempts have been made from time to time to define a virus according to Bawden (1964) consider viruses as submicroscopic, infectious entities that multiply only intracellular and are potentially pathogenic
Thus viruses strictly speaking, could not be defined they could surely be set apart from other organisms on the basis of certain discriminative characters like presences of a single nucleic acid absence of enzymes etc. Viruses and the evolution of viruses are still remains a matter of speculation.
Phylogeny of viruses thus remained obscure, as yet. But one thing has become quit clear. We may now say that whatever be their mode of appearance on this planet, whether as an end to a process of gradual retrogression or as independent entities originating denovo, there exists a unique and exclusive relationship between a virion and its host .In the proper understanding of this relationship lies the answer to the riddle of the biological status of these particles.
Recent reports and analysis on viruses have thrown some light on detection methods
Methods for detection of viruses
1. SEROLOGICAL CHARACTERIZATION:
Serological techniques are also in wide range though restrictive, use in the study of viruses. It is common knowledge that when an animal (vertebrate) is infected with a foreign material, be it a virus or bacterium or a macromolecule like proteins, there are produced in its blood stream proteins which combine specifically with the substance entered. This occurred of specific combination can be demonstrated in vitro and forms the basis of serological characterization. It is of great help particularly for detection of viruses and or their; products.
The proteins specifically produced in the blood stream as a result of external stimulus are called antibodies. Any substance which will stimulate the production of antibodies in vitro is called an antigen. Blood serum containing antibodies is termed antiserum. The serum obtained from an animal not injected with a specific antigen is called normal serum. Antigen-antibody interactions form the basis of serological studies.
2. NEUTRALISATION REACTION
These tests are used to determine the level of infectivity of a virus or detect new serotypes of viruses. Normally the infectivity of a known type of virus would be neutralized by specific antibody effective against it. In general procedure serum containing the antibody is serially diluted and added to containers such as Petri plates or plastic cups containing tissue monolayers. A standard amount of virus is then added to each cup and the mixture incubated after adding the host cells. The highest dilution that inhibits cytopathic effect on host cell is taken to be a measure of infectivity. In a modification of this procedure called plaque reduction method, the virus serum mixture and the cell monolayers are overlaid with agar and incubated till plaques develop. The end point is usually taken to be the highest dilution of the serum that reduces number of plaques by at least 50%. Different serotypes of the same virus often have different dilution end points. If a new viral isolate is not neutralized by antisera against all the known serotypes, it may be regarded as a new serotype. .
3. COMPLEMENT FIXATION TEST
This is a useful method for preliminary screening of a viral isolate, and for placing it correctly with in family or genus. Complement is a specific group of heat labile proteins present in normal blood serum which are vitally necessary for antigen-antibody reaction to occur. Whenever an antigen-antibody association is formed, the complement is said to get fixed. Complement proteins present freely help normal serum to destroy red blood cells. However fixed complement, usually with specific antibody, removes the power of normal serum to neutralize red cells altogether. This test is therefore used more for detecting antibody in sera.
4. PRECIPITIN TEST (IMMUNODIFFUSION TEST)
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To proceed with this reaction purified antigen (virus) has to be prepared first. When virus antigen is being prepared from tissues extreme care has to be taken to eliminate any possible protein or similar contaminant which themselves might act as antigen and confuse the results. There are instances when erroneous conclusions have been drawn only because the antigen preparations were contaminated (see Van RegenmorteI1966). Usual procedures adopted for preparation of purified samples of virus have been outlined earlier.
Antiserum is obtained by injecting the purified antigen (virus) into a suitable animal like rabbit. Domestic fowl and also the horse are sometimes employed. The antigen is usually injected into the vein which runs along the upper surface of the ear. About two weeks after injection (the incubation period) the rabbit is bled from the other ear. A small cut is made near the base of the ear. The blood is collected in a tube and is allowed to clot. The serum is decanted off and centrifuged to remove any remaining blood cells. Antiserum prepared in this way is not sterile and has to be stored with proper precaution to prevent bacterial growth.
In the earlier days of serology, the precipitin test was carried out in liquid medium. A convenient method was to use 1 ml samples of antiserum at constant dilution in a series of test tubes to each of which 1 ml of antigen solution at different dilutions was added. The tubes were then set after a brief shaking in a water bath with temperature at 50°C. The tubes were then observed for the appearance of precipitate often after a period of hours.
In recent years the precipitin test in liquid (usually normal saline) medium has been largely replaced by precipitin reactions in agargel, Devised by Ouchterlony (1950), this method is known as the Plate test. In this method agargel is prepared in a petri plate with a number of wells in it. Usually one well is bored in the centre and six or seven in the peripheral regions. The known antigen is placed in the central well. The unknown and a suitable control are placed in the peripheral ones. The preparation is. Kept moist and within a few hours the protein solutions diffuse through gel. A clear line of precipitate is produced where antigen and antibody meet. The precipitate can be made clearly visible by staining with a dye.
In this type of test, the combination between antigen and antibody is detected by reaction offered by animal tissues. It is an in vivo system of detection of antigen-antibody reaction. Anaplylaxis tests are used in assessing artificial immunization procedures.
The precipitin test, the complement fixation test (CFf) and 2- anaphylaxis find extensive application in animal virology, particularly medical virology. The role of CFf in elucidation of the problem is possible viral oncogenis cannot be overemphasized.
Serological data have also been utilized for finding out relatedness or otherwise amongst viruses. As early as in 1937 Bawden and Pirie reported that the viruses then known as Cucumber viruses 3 and 4 were in fact related to tobacco mosaic virus. On the other hand, they reported that tobacco necrosis virus was in fact comprised of a number of serologically distinct virus types. Since then serological' relationships between otherwise distinct virus types have been reported from time to time. For instance, the serological relationship between pea mosaic and bean yellow mosaic viruses.
6. Direct Detection of Viral Antigen:
In recent years more and more sophisticated and better techniques have been developed to detect viral antigen in a given sample. With these techniques it is possible to detect the presence of a Virus or viral products with rapidity and high degree of specify. These methods are based on direct interaction between the virion or viral antigen. Present in situ in tissues or in secretions and specific antibodies which are pre labeled or tagged in some way as to permit the ready recognition of the interaction. Depending upon the mode of labeling used the methods are (i) immunofluorscence (ii) immunoperoxidase staining and iii)Enzyme lincked immune sorbent assay(ELISA) of these techniques ELISA has found wide acceptance and usage for its high degree of sensitivity and optional simplicity. Owing to this factors ELISA had found wide applications in diagnostic medicine.
7. Direct detection of viral nucleic acids
With the development of the technique of in situ DNA hybridization, it has been possible to detect viral DNA in host cell. It is found to be particularly useful when the DNA is present in cell but is not expressed as in the case of integrated retroviral DNA or episomal DNA of papovirus infected cell. Specific probes from a collection of cloned fragments of whole viral genome are selected and used for the purpose. These probes are initially labeled and then used. Earlier viral nucleic acid from a source (which is to be identified) is separated by electrophoresis on agarose gel and then transferred to nitrocellulose filter papers by Southern blotting technique. The nitrocellulose paper containing different band of nucleic acid is then made interact with the labeled probes. Specific binding of a known probe with a specific band indicates presence of viral nucleic acid. Source nucleic acid is often fragmented before electrophoresis by restriction endonucleases.
8. Molecular biological detection
Real-time (Reverse transcription-) PCR detection of a specific genome segment of a virus Any soil (or) biological samples are diluted in a test tube and viruses are eluted at high pH. The viral RNA/DNA can be extracted. In a real-time PCR cycle, the extracted nucleic acid can be identified using specific fluorescence-marked DNA probes.
9.Detection of virus growth in cell cultures:
i) Cytopatic effect: Many viruses cause morphological changes in cultured cells in which they grow. These changes can be readily observed by microscopic examination of the cultures. These changes are known as 'cytopathic effects' (CPE) and the viruses causing CPE are called 'cytopathogenic viruses'. The CPE produced by different groups of viruses are characteristic and help in the presumptive identification of virus isolates. For example, enteroviruses produce rapid CPE with crenation of cells and degeneration of the entire cell sheet; measles virus produces syncytium formation; herpes virus causes discrete focal degeneration; adenovirus produces large granular clumps resembling bunches of grapes: SV 40 produces, prominent cytoplasmic vacuolation.
ii) Metabolic inhibition:In normal cell cultures, the 'medium turn's acid due to cellular metabolism. When viruses grow in cell cultures, cell metabolism is inhibited and there is no acid production. This can be made out by the color of the indicator (phenol red) incorporated in the medium.
iii) Hemadsorption:When hemagglutinating viruses (such as influenza and parainfluenza viruses) grow in cell cultures, their presence can be indicated by the addition of guinea pig erythrocytes to the cultures. If the viruses are multiplying in the culture, the erythrocytes will adsorb onto the surface of cells. This is known as ‘hemadsorption'.
iv) Interference:The growth of a noncytopathogenic virus in cell culture can be tested by the following challenge with a known cytopathogenic virus. The growth of the first will inhibit infection by the second virus by interference.
v) Transformation:Tumours forming viruses induce cell transformation and loss of contact inhibition, so that growth appears in a piled up manner producing microtumours.
vi) Immunofluorescence:Cells from virus infected cultures can be stained by fluorescent conjugated antiserum and examined under the UV microscope for the presence of virus antigen. This gives positive results than other methods and, therefore, finds wide application in diagnostic virology.
We conclude that new human viruses will continue to be discovered in the immediate future; Current analysis say that at least 38 undiscovered species that will be reported on an average of approximately one per year to 2020.Approximately 1400 pathogen species are been recognized . Of them 200 are viruses, but novel virus species are being reported in humans at a rate of two per year. Viruses are a major public health concern, whether it may cause disease on large scale like HIV/AIDS, more novel events such as the SARS epidemic or potential future threats such as pandemic influenza. An analysis patterns on virus discovery is therefore of considerable very interesting and some times very dangerous. Some older biological detection techniques still prove reliable in virology analysis labs. Clinical virologists are engaged in the field of diagnostic virology to determine whether pathogenic viruses are present in clinical specimens collected from patients with suspected infections. During the past twenty years in the field of virology, technical advances took place and have made constant and enormous progress in various areas, including, mycology, mycobacteriology, immunology and parasitology. The diagnostic capabilities of modern clinical virology laboratories have improved rapidly and expanded greatly due to a technological revolution in the field of microbiology and immunology. The rapid techniques for nucleic acid amplification and characterization combined with automation and user-friendly software have significantly broadened the diagnostic areas for the clinical virologist. The conventional model for clinical virology has been labor-intensive and frequently required days or weeks before test results to be available. The physical structure of laboratories, staffing patterns and turn-around time all have been influenced greatly on these technical advances. Such changes will undoubtedly continue and lead the field of diagnostic virology inevitably to a truly modern discipline in feature. Now the world around is developing a computational methods that are very much useful in identifying the conserved viral sequences at the genus level for all viral genomes available in Gene Bank and to established a virus probe library. The virus probes are used not only to recognize known viruses but also for discerning the genera of emerging or uncharacterized ones. Our virus identification strategy has great potential in the diagnosis of viral infections and promise to do better in coming tomorrow