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Techniques that directly detect or characterize virus genomes will play a dominant role in  the virus diagnostic laboratory at the beginning of the 21st century. Discuss

 

Presently, diagnosis of virus infections is usually made by conventional diagnostic techniques. Serology is the mainstay of virus diagnosis. An acute infection can be made by the detection of virus-specific IgM or rising titres of IgG or total antibody. However, in the latter case, the diagnosis is usually retrospective because paired acute and convalescent sera are required. In addition, many viruses such as diarrhoeal and respiratory viruses, cause clinical disease before the development of antibodies and thus a serological diagnosis would be retrospective. Many virus infections can be diagnosed by virus isolation in cell culture. However, it may take up to several weeks before viral induced changes such as cytopathic effect to become apparent and therefore, again the diagnosis is usually retrospective. Moreover, virus isolation requires a large amount of expertise and cost in setting a laboratory, not withstanding the safety issues that may be involved.

Tests based on the direct detection of virus particles (electron microscopy), viral antigens, or viral genomes from the clinical specimen are becoming more and more popular, as these methods can give a result within a few hours to two days after the collection of the specimen. This is an important consideration for viral diseases which are potentially treatable by specific antiviral therapy. As more and more effective antiviral compounds become available, there will be increasingly demands on the virus diagnostic laboratory to offer rapid diagnostic methods. Methods that characterize viral genomes, whether directly from specimens, or indirectly from isolates, are playing an increasingly important role in virology laboratory, especially reference laboratories. Characterization of the genome is playing an important role for the epidemiological investigation, antiviral resistance, and prognostic purposes.

A. Direct Detection of Viral Genome

Viral genome can be detected directly from specimens without having been grown first in cell culture. This may be done by hybridization with oligonucleotide probe, or by a genome amplification method such as the polymerase chain reaction (PCR). Besides rapid turnover, genome amplification methods offer a very high sensitivity rate, often surpassing greatly that of cell culture and other direct detection methods.

1. Hybridization

Specific viral genomes in a clinical specimen may be detected by the use of a DNA or RNA probe with a sequence homologous to that of the target sequence to be detected. The hybridization may be carried out on a solid phase eg. dot or slot-blot, sandwich., in liquid, or even in situ in the infected cells. The probe is radioactively or chemically labeled and thus allowing the detection of the hybrids afterwards by techniques such as autoradiography or chemoluminescence. Hepatitis B DNA had been detected in the past by various hybridization techniques. In general, hybridization techniques are not difficult to perform. The dot or slot-blot techniques may be used to screen a large number of clinical specimens. However, hybridization is not particularly sensitive and the sensitivity may not be greater than conventional techniques which are far easier to perform. Therefore, hybridization had now all but replaced by amplification methods. However, hybridization is still used for the verification of amplified products, especially in commercial assays.

2. Amplification Methods

In the case of amplification methods. specific viral target sequences are amplified to the order of several million fold so that the amplified product could be detected or characterized easily. The most popular amplification method is PCR The technique is based on the use of synthetic oligonucleotides flanking the target nucleic sequence of interest. These oligonucleotides act as primers for the thermostable Taq polymerase. Repeated temperature (usually 30 to 40) cycles of denaturation of the template DNA ( 94oC), annealing of the primers to their complementary sequences (50oC), and primer extension (70oC) result in the exponential production of the target sequence. The amplified product of the expected molecular weight may be detected by gel electrophoresis and the identity of the product could be further confirmed by hybridization with a complementary nucleic acid probe. The amplified product may be characterized by restriction enzyme analysis or by nucleic acid sequencing. PCR is a comparatively rapid and simple molecular biological technique. However, its extreme sensitivity may cause problems, especially since it is easy for false positive results to arise due to contamination. The interpretation of a positive result may also be very difficult especially in the case of viruses which may become latent, such as CMV. However, this can be overcome by the use of quantitative assays that can determine the viral load. Other amplification methods have been developed commercially, such as the ligase chain reaction (LCR), isothermal amplification (NASBA - nucleic acid based amplification), and the branched DNA technique. However PCR remains the most flexible technique it is relatively easy to design primers and set up a new assay. Genome amplification methods are now commonly used in the detection and determining of HIV, HBV, and HCV viral loads, diagnosis of norovirus infections, and also those of emerging viruses such as influenza H5N1 and SARS.

B. Characterization of Viral Genome

Increasingly viral genomes are being characterized by laboratories in addition to detection. A number of methods are available, the gold standard being sequencing. However, this requires expensive equipment, a high degree of expertise, and is relatively also. Other more rapid and easier methods that are commonly used include hybridization by type-specific oligonucleotide probes and a number of get based methods such as RFLP, SSCP, heteroduplex analysis, and dHPLC. There are a number of situations where characterization of the genome is useful.

Identification of isolates - molecular methods may be used in lieu of conventional methods using type-specific antisera for the identification of viral isolates e.g. entertoviruses, adenoviruses, influenza and parainfluenza viruses. It is particularly useful in situations where it is increasingly difficult and expensive to obtain high quality type-specific antisera for identification, such as enteroviruses and adenoviruses.

Epidemiological Research. - molecular epidemiology is particularly useful in outbreak situations such as those involving respiratory and diarrhoeal viruses, and also HIV, HBV and HCV. The usual method is to amplify a certain gene region of the virus and then sequence the amplicon. Phylogenetic analysis is then carried out on the nucleic acid of the various isolates obtained in the outbreak. Establishing an early epidemiology link may help in controlling the outbreak. Besides outbreak situations, molecular epidemiological techniques may be used to study the transmission of a virus worldwide, such as the transmission of the various HIV-1 subtypes throughout the world.

Antiviral Resistance - as more effective antiviral agents become available, there will be more demand for antiviral susceptibility tests. Genotypic testing for HIV anti-viral resistance is now routinely used for the management of HIV infection. Antiviral testing is also increasingly used in other virus infections such as hepatitis B, CMV, and influenza A viruses.

Prognosis - infection with certain genotypes of particular viruses may carry a poorer prognosis. For example, infection with HCV genotype 1 carries a poorer prognosis than genotypes 2 and 3. Patients infected with genotype 1 are offered 48 weeks of interferon therapy instead of 24 weeks for other types.

Future potential usage

Molecular techniques are likely to play an increasing role in the detection and characterization of viruses that are normally diagnosed by virus isolation, such as respiratory viruses, enteroviruses and herpesviruses. Virus isolation techniques require a large amount of resources and expertise and it is conceivable that molecular techniques may replace isolation for the diagnosis of these viruses in future. Molecular techniques have also proved to be important in the diagnosis of emerging virus infections when they are quite often the first diagnostic assays available e.g. SARS and influenza H5N1. However, molecular biology techniques are exceedingly unlikely to supplant serology in the diagnosis of certain virus infections such as hepatitis A and B, rubella, parvovirus and HIV. Serological techniques for these viruses are well developed and a rapid diagnosis can usually be made as antibody would be detectable by time time of onset of symptoms. However, there may be certain areas where molecular biology techniques have a possible role, such as to assess the response of a hepatitis B carrier to interferon, to monitor for viraemia in the case of hepatitis C, or for the diagnosis of congenital HIV infection. Molecular biological techniques are not likely either to supplant other rapid methods of diagnosis which are much simpler to perform, such as immunofluorescence or EIA of nasopharyngeal aspirate for RSV and other respiratory viruses.

Current molecular bioliogical techniques need to be further simplified, automated, and the cost reduced before they will gain widespread acceptance by routine diagnostic laboratories. Virus isolation offer the advantage of being able to detect a number of viruses at any one time whereas techniques such as hybridization and PCR can only look for only one virus at a time. However this may change with the development of multiplex PCR assays and microarray assays. DNA microarray or DNA chip involves of coating of specific labelled oligonculeotide probes onto individual fields on a chip. Each microarray can hold hundreds of thousands of fields and DNA bound to individual probes will be visualised. At present microarray technology is mainly used for gene expression experiments. Looking further ahead, microarray technology may one day be used to detect viruses from clinical specimens. Besides detecting the presence of the virus, it may be able to determine the genotype and antiviral sensitivity at the same time, as well as providing important molecular epidemiological data. It is quite possible that molecular biology techniques may become the method of choice for the diagnosis of many if not most viral infections in the near future.

 

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