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Molecular diagnosis of Variola and other high-risk viruses

2007; Future Medicine; Volume: 2; Issue: 4 Linguagem: Inglês

10.2217/17460794.2.4.315

1746-0808

Antônio Tenório, María Paz Sánchez‐Seco, Giovanni Fedele, Cristina Domingo, Anabel Negredo,

Viral gastroenteritis research and epidemiology

Future VirologyVol. 2, No. 4 EditorialFree AccessMolecular diagnosis of Variola and other high-risk virusesAntonio Tenorio, Maria Paz Sánchez-Seco, Giovanni Fedele, Cristina Domingo & Ana Isabel NegredoAntonio Tenorio† Author for correspondenceArbovirus & Imported Viral Diseases Unit, Instituto de Salud Carlos III, Madrid, Spain. , Maria Paz Sánchez-SecoArbovirus & Imported Viral Diseases Unit, Instituto de Salud Carlos III, Madrid, Spain, and, Alert & Emergency Unit, Instituto de Salud Carlos III, Madrid, Spain. , Giovanni FedeleArbovirus & Imported Viral Diseases Unit, Instituto de Salud Carlos III, Madrid, Spain, and, Alert & Emergency Unit, Instituto de Salud Carlos III, Madrid, Spain. , Cristina DomingoArbovirus & Imported Viral Diseases Unit, Instituto de Salud Carlos III, Madrid, Spain. & Ana Isabel NegredoArbovirus & Imported Viral Diseases Unit, Instituto de Salud Carlos III, Madrid, Spain. Published Online:29 Jun 2007https://doi.org/10.2217/17460794.2.4.315AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Recently developed diagnostic methods based on genome amplification of highly pathogenic viruses allow for the rapid attainment of more sensitive results. Furthermore, the risk of accidental infection is much lower compared with traditional methods, which are often based on growing the virus on cell cultures or in experimental mice. This changing methodology, which should be permanently actualized to include the whole known or predicted viral variability, is demanding the re-evaluation of the standard operational procedures for the front-line diagnosis of high-risk viruses.A combined action will improve the efficiency of the diagnosis, increasing the speed of delivery, sensitivity and specificity of the first results.OverviewWhich viruses are we talking about & how can they appear?A small number of viruses, including Variola, are commonly considered high-risk pathogens and are included in the Bio-Safety Level 4 (BSL-4) category of microorganisms: • Variola, an eradicated virus, is known to be stored in Russian and US laboratories, and could potentially reappear as a result of accidental or deliberate release• Ebolavirus and Marburgvirus, two Filoviridaefrom the Old World hosted by as yet unknown natural reservoirs• Lassa virus in the Old World, along with Junin, Guanarito, Oliveros and Sabia viruses in South America, all Arenaviridae that are primarily transmitted by contact with wild rodents or their excreta• Crimean-Congo hemorrhagic fever virus, a tick-born Nairovirus, which is widespread throughout the Old World• Hendra virus and Nipah virus, two viruses recently discovered in Australia and South Asia, respectively, and maintained in batsWith the exception of Variola virus, the agent that caused smallpox, the other cited viruses are primarily transmitted to humans via a wild animal or arthropod, and then secondarily through human-to-human infection. Fortunately, these barrier-crossing viruses demonstrate a very low efficacy in terms of human-to-human transmission, with a low risk of local outbreaks and virtually no risk of causing pandemics.Natural outbreaks & imported viral infectionsAlthough Ebolavirus is well known as a lethal agent causing viral hemorrhagic fever, other viruses more frequently cause this deadly syndrome. In fact, infection by Ebola or Marburg viruses are easily recognized by epidemiologists due to their high lethality.Other viruses, such as Lassa, its related American hemorrhagic viruses, and the Crimean-Congo hemorrhagic virus, although also transmissible via direct human-to-human infection, cause fewer deaths. This fact, along with insufficient healthcare resources in the majority of affected countries, leads to a high level of undetected, and therefore unreported, outbreaks.Increasingly, such natural infections appear unexpectedly in nonendemic areas throughout the world, imported by travellers infected hours or days previously while in endemic areas.Accidental releaseRecently, two laboratory accidents with Ebolavirus have been declared in the USA and Russia. Moreover, after its apparent disappearance, SARS coronavirus has suddenly reappeared in laboratory workers accidentally infected in different countries. Luckily, even minimal outbreaks were avoided and the risk of epidemics was controlled.Deliberate releaseAlthough Variola is an eradicated virus, the increased activity of international terrorist groups raises the risk of its deliberate release. In fact, this virus is, potentially, an ideal biological weapon because of its easy aerosolization, high transmissibility and lethality in unvaccinated populations (individuals born after 1980).All the other high-risk pathogens have also been included as potential biological weapons. Although they are not as lethal and transmissible as the Variola virus infection, their potential as a terror weapon is considerable owing to the widespread panic they would cause. This risk is compounded by the relative ease with which these viruses could be acquired.Current procedures to detect the first suspected case of infectionNowadays, different guidelines for the management and control of suspected high-risk viral infections are applicable to different countries and regions around the world. Each guideline usually defines the category of the 'suspected case' following more or less strict clinical and epidemiological criteria. When a case fits these criteria, the patient must be isolated and attended to by health workers with personal protection equipment. Samples also need to be sent immediately to a specialized laboratory to exclude or confirm the suspicion.Classical diagnosis was based on the viral culture, a dangerous and time-consuming procedure lasting several days and strictly limited to a few highly specialized laboratories in the world, operating in facilities with the maximum containment BSL-4. As a result, most of the guidelines maintain that viral diagnosis must be carried out in BSL-4 facilities.In recent years, molecular methods that detect and identify these viruses within a few hours have been published, at least with the use of well-characterized laboratory viral strains. Moreover, it is now possible to work on clinical samples inactivated by adding a solution that preserves the genomes and destroys their infectivity, with a much lower risk of accidental infection.Two key issuesRecent improvements in molecular diagnosis will improve the future efficiency of current operational procedures for the front-line diagnosis of high-risk viral infections. Until then, however, virologists and public health authorities should consider two key questions: the availability of efficient methods for molecular diagnosis and the possibility of opening the first-line diagnosis to laboratories lacking BSL-4 facilities.Methods for the molecular diagnosis of high-risk virusesAn adequate method for molecular diagnosis must include: • A design that considers the natural variability of the target virus• Contrasted specificity, measured as the probability of generating false-positive results with clinical samples taken from similar syndromes• High sensitivity, characterized not only using the common laboratory strains, but also with positive controls representing the whole spectrum of wild viral strains• Controls to avoid false-negative results due to inhibitions or other unsuspected problems occurring during the processing of each sampleClearly, the main problems in fulfilling these prerequisites stem from the low level of knowledge regarding these viruses' natural variability and from the availability of natural strains representing the whole spectrum of wild strains.Unfortunately, the majority of published methods for the molecular diagnosis of high-risk viruses have not been designed considering natural variability as a key factor in developing a highly sensitive and specific method.Natural variability of the high-risk virusesVariola, the only high-risk virus using a DNA genome, exhibits a very low level of natural variability, frequently sharing highly conserved genomic regions with other related viruses. This fact is not surprising since DNA viruses have enzymes that are able to correct, with a high degree of efficiency, the introduction of errors in the descending genomes.By contrast, it is common knowledge that the enzymes responsible for the replication of RNA genomes are inefficient at correcting these mistakes. This property ensures a high plasticity for the RNA viruses, which produce new variants after each replication, enhancing their capacity to adapt themselves to new bottlenecks, such as immune response, antiviral molecules and species barriers.This fact has important implications for molecular diagnosis, as the design of detection methods must take into account the higher level of natural variability for RNA viruses.Moreover, the variability of RNA in viruses is qualitatively enhanced in those viruses associated with reservoir species that exist in isolated populations. In these cases, the viruses will evolve independently into each different ecological niche, generating unpredictable levels of viral diversity, with up to 20% of differences for the same virus found in different locations.Covering the known & suspected viral diversitySuch tremendous variability makes it increasingly difficult to select potentially conserved target regions in as yet undescribed evolutionary lineages. As a rule, the more variability that is known and considered, the safer the design achieved.One of the best possibilities is to design the primers of the amplification in conserved coding regions, preferably ending at their 3´ ends on methionine or tryptophan, two amino acids encoded by a single codon, or alternatively in other amino acids with a low level of degeneracy in their genetic code.After selecting the regions for the primers, the potential variability of the amino acids coded in the region should also be considered, including the degeneracy at these positions, or the polyvalent nucleoside inosine in their sequences. Unfortunately, no available software is capable of properly considering such factors and a good design still depends on the previous experience and know-how of the molecular virologist.Alternatively, the design of a diagnostic method in noncoding regions, showing highly conserved secondary structures for some RNA viruses, could be considered.Once a suitable method has been designed, it should be properly validated. Unfortunately, for most laboratories, it would be impossible to use specimens with Variolavirus and very difficult to use samples representing the known spectrum of variability of each highly pathogenic virus.Only cooperation between specialized laboratories could increase knowledge on the natural variability and diversity of these viruses, and promote the elaboration and distribution of standard collections of samples or genomes needed to validate and control the methods, generating the tools required for the permanent revision of the methods, and ensuring the best capacity for properly detecting high-risk viruses.The example of collaboration in the SARS epidemics should be followed in order to obtain better conditions to control the impact of highly pathogenic viruses all over the world.In brief, molecular diagnostic methods are biologically safe but will require continuous revision of their design and should be properly controlled to avoid the generation of false-positive and -negative results.Where to carry out front-line diagnosisAs stated previously, the standard operational procedure for high-risk virus suspicion involves sending samples to one of the few BSL-4 facilities available in the world. Hospital workers must follow strict operational procedures to obtain, pack and send the samples to the designated laboratory. Undoubtedly, this recommendation must be maintained, and the specialized laboratory should provide the preliminary and definitive confirmed results as soon as possible.However, the use of the recently described molecular methods allows inactivated, noninfective biological specimens to be worked with. If this is accepted, why not approve the parallel sending of samples to another designated laboratory that could act as the first-line in diagnosis? The ease of sending the specimen and laboratory procedures will permit a quicker preliminary result that could have a great impact on patient management, and on the control of a potential outbreak.In a nutshellHighly pathogenic viruses are exotic in most first world countries, while causing many deaths in endemic countries when an outbreak occurs. Several cases of travellers returning from endemic areas after being infected by highly pathogenic viruses are detected each year in different countries, and other threats, such as their accidental or deliberate release should be considered. Rapid diagnosis is needed not only for the management of the patient, but also to control a potential outbreak.Efficient methods for obtaining and assaying the clinical samples are therefore critical. Even though the specimens potentially contain high-risk viruses, they will likely be obtained at hospitals without BSL-4 facilities, and should be inactivated as soon as possible, even in facilities with a lower category of biological contention. Regional authorities should consider this critical point when establishing recommendations for the early detection and control of such potentially epidemic viruses.At the same time, the methods need to be both sensitive and specific and not only use the available control strains, but also consider the broad variability of such RNA viruses evolving independently in different ecological niches.Taking the best care of the patient and avoiding new infections and resulting panic among the public are the responsibility of public health authorities, who should provide suitable tools for specialized hospitals and laboratories so they can be ready for a quick response.Future perspectiveMolecular diagnosis of high-risk viruses is now replacing traditional methods, based on growing the virus, as a front-line method. As a consequence of this: • Better methods need to be developed, as well as a qualitative increase in the knowledge of viral diversity and variability in these wild-type-1 viruses.• National and regional guidelines for the management and control of high-risk viral infections must recognize the potential utility and safety of these new techniques and should consider present advantages and disadvantages in sending specimens to specialized laboratories other than those with few BSL-4 facilities.FiguresReferencesRelatedDetails Vol. 2, No. 4 STAY CONNECTED Metrics History Published online 29 June 2007 Published in print July 2007 Information© Future Medicine LtdPDF download