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At present, these methods depend on developing antibody reagents that bind specifically to variola proteins that are distinct from those made by other orthopoxviruses. Polyclonal antibody. One monoclonal antibody has been described that is specific for variola virus and can be used to distinguish variola from other poxviruses Damon, However, monoclonal antibodies detect a single epitope in a single viral protein, and most are conformation dependent.

Specificity for geographically unrelated variola isolates would depend on defining a fully conserved and stable epitope or using a mix of monoclonals that would recognize epitopes in several unique variola virus proteins having no homologues or differing substantially from the related proteins in the other poxviruses. Even when well-characterized reagents are available, designing antigen detection methods that demonstrate the presence of viral proteins in patient materials has been challenging for many human pathogens. Most successes are achieved when the clinical material is a cutaneous lesion specimen, which would be the case for variola at the symptomatic stage of infection.

In one example of a poxvirus detection method applied to respiratory secre- tions, a biosensor technique using cyan-5 dye labeled antivaccinia antibody was used to detect vaccina proteins in human throat swab specimens that had been spiked with vaccinia virus from tissue culture Donaldson et al.

One would expect such approaches to be feasible for variola detection, but their development currently depends on generating panels of antibodies that are highly specific for variola proteins. Pilot experiments were conducted in which ELISAs were used to detect monkeypox virus during the recent outbreaks in Africa and variola virus in specimens from nonhuman primates Karem et al. Nevertheless, although inhibi- tors may be encountered, nucleotide detection methods are generally pre- ferred for viral detection because nucleotides can be extracted from patient materials and concentrated for PCR testing, whereas similar processes to enhance sensitivity are difficult for protein detection in respiratory secre- tions or other clinical specimens that would be available from patients in the pre-eruptive phase of smallpox.

Proteomics methods may emerge that can identify a specific sequence of amino acid residues by direct analysis of a sample using mass spectroscopy or other methods that do not require antibody reagents, but these tools are not yet applicable for clinical use. With the exception of measuring antibody titers by plaque reduc- tion neutralization assay, serologic assays for IgG and IgM antibodies to variola and other poxviruses are also protein-based detection techniques.

ELISA methods detect antibodies in serum samples through their binding to immobilized viral antigens. The development of such an assay for detecting variola virus IgG and IgM antibodies is feasible, but specificity requires the identification of unique proteins that do not elicit cross-reactive antibodies as a result of exposure to other poxviruses, such as by vaccination with vac- cinia. It is anticipated that most variola infections would be symptomatic;.

These immunodiagnostic tests will rely on hybridoma technology and technology for producing recombinant anti- bodies to major neutralizing and protective antigens of variola virus and those of other orthopoxviruses pathogenic for humans Russian federation Patent ; Razumov et al. Detection in the Environment The technical capacity for environmental detection of variola virus would be important in the event of an intentional release. Widespread distribution of the virus could be achieved because poxviruses are stable in aerosol form and can be lyophilized. The molecular methods for variola virus detection that have been developed since use PCR and in situ hybridization assays that have proven valuable for the clinical detection of many viral pathogens in patient specimens, and a few of these methods have been validated using archived tissues from variola cases.

These methods could be applied to the identification of variola virus in such specimens with certain modifications in the way the materials are prepared for testing. For example, it would be necessary to take into account the inhibitory effects of detergents and other materials on PCR sensitivity, as shown in experiments with vaccinia virus Kurth et al. The specificity of PCR for variola virus detection should be preserved, but sensitivity in such samples is difficult to predict. Ideally, tools for detecting the presence of variola virus in the environ- ment would need to be rapid, portable, and easily deployable.

Author Summary

Because pox- virus genome detection methods require relatively complex equipment and reagents, it would be necessary at present to bring materials suspected of containing variola virus to a laboratory facility. A more practical variation of the method for field use would be the use of dried reagents in a dual-. Even if PCR or ELISA methods were used that could differentiate variola from other poxviruses in environmental samples, their sensitivity in field testing would need to be established.

Criteria for speci- ficity might need to be lowered to ensure that a positive sample was not missed under field conditions, with the assumption that all specimens would need to be retested and results validated in a reference laboratory. Nanotechnology-based tools may be developed that can discriminate viruses based on their particle size and other properties; if so, it would be necessary to have at least inactivated variola particles to assess their sensitivity for environmental detection.

Whereas most Class I devices are exempt from premarket notification, most Class II devices do require such notification [ k ], and most Class III devices require premarket approval PMA , including submission of clinical data to support marketing claims.

Rapid Identification of Known and New RNA Viruses from Animal Tissues

The potential classification of IVDs for variola virus detection has not been established, although it appears likely, given the critical impor- tance of accurate detection methods, that premarket notification including both general and special controls Class II designation would be required. Methods that detect viral proteins have been pursued to a lesser extent but could also be expanded without the need for live virus.

Although not essential, better characterization of the sensitivity and specificity of both nucleic acid and protein methods for variola virus detection in relevant samples could be achieved by additional testing of tissues from nonhuman primates infected with the virus. Preservation of tissues for this purpose should be included in antiviral, vaccine, or patho- genesis studies done in animals infected with variola. Since methods devel- oped using only variola proteins could prove inadequate for their detection in clinical materials from infected individuals, archived clinical specimens could be tested to confirm the sensitivity and specificity of such tests, if possible.

Further work on protein-based detection would benefit particu- larly from access to proteins made in variola virus-infected cells instead of proteins made using expression vectors to ensure the reliability of the test and to standardize reagents. High-throughput assays, including serologic methods to identify recently infected individuals, would be needed to test large numbers of samples in a possible outbreak situation. However, the development of most new methods would not require live virus as this research could build on work with other validated methods and be scaled up.

Some future approaches that might prove valuable, such as those that detect viral particles, could require access to variola virions made in culture cells for their validation. One caveat related to variola detection and smallpox diagnosis is that genomic sequencing of enough geographically diverse isolates is necessary to ensure that PCR tests have adequate specificity. PCR and sequencing of the amplicons would be the first step in a forensic analysis of the source of a variola isolate should a reintroduction of the virus occur, and would also be accomplished most effectively if background information were available on the complete genome sequence of as many variola isolates as possible.

It is expected that use of the live virus would not be necessary for this purpose, assuming that sufficient DNA is still available in stored specimens in the U. Finally, it is not yet clear whether the FDA will require the use of live variola virus in the evaluation of new diagnostic methods. Saleh, P. Kyusung, J. Huggins, M. Jahrling, and S. Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents.

Biel, S. Diagnostic electron microscopy is still a timely and rewarding method. Damon, I. Fields, D. Knipe, P. Howley, and D. Donaldson, K.


Kramer, and D. A rapid detection method for Vaccinia virus, the surrogate for smallpox virus. Espy, M. Cockerill, R. Meyer, M. Bowen, G. Poland, T. Hadfield, and T.

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Fedele, C. Negredo, F. Molero, M. Sanchez-Seco, and A. Fitzgibbon, J. Simultaneous identification of orthopoxviruses and alphaviruses by oligonucleotide macroarray with special emphasis on detection of variola and Venezuelan equine encephalitis viruses. Ibrahim, S. Kulesh, S. Saleh, I. Damon, J. Esposito, A. Schmaljohn, and P. Real-time PCR assay to detect smallpox virus.

Karem, K. Reynolds, C. Hughes, Z. Braden, P. Nigam, S. Crotty, J. Glidewell, R.

Isolation of Viruses

Ahmed, R. Amara, and I. Monkeypox-induced immunity and failure of childhood smallpox vaccination to provide complete protection. Komarova, E. Aldissi, and A. Direct electrochemical sensor for fast reagent-free DNA detection.

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Kulesh, D. Baker, B. Loveless, D. Norwood, S. Zwiers, E. Mucker, C. Herrera, D. Miller, D. Christensen, L. Wasieloski, Jr. Huggins, and P. Kurth, A. Achenbach, L. Miller, I. Mackay, G. Pauli, and A. Orthopox- virus detection in environmental specimens during suspected bioterror attacks: Inhibitory influences of common household products. Laassri, M. Chizhikov, M. Mikheev, S. Shchelkunov, and K. Lapa, S. Shchelkunov, V. Mikhailovich, A. Sobolev, V. Blinov, I. Babkin, A.

Guskov, E. Sokunova, A. Zasedatelev, L. Sandakhchiev, and A. Carroll, S. Gardner, M. Walsh, E. Vitalis, and I. On the origin of smallpox: Correlating variola phylogenics with historical smallpox records. Loparev, V. Massung, J. Esposito, and H. Detection and differentia- tion of Old World orthopoxviruses: Restriction fragment length polymorphism of the crmB gene region. Nitsche, A. Ellerbrok, and G. Nuovo, G. Plaza, and C. Rapid diagnosis of smallpox infection and differentiation from its mimics.

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Babkin, C. Drosten, S. Shchelkunov, M. From time to time, data on the same sequence of bases can be reanalysed, enabling the identification of new viruses and viroids as information on their genomes is accrued in databases. Subsequently, even viruses with a sequence of bases notably different from known viruses are easier to detect.

Potentially great crop losses make identifying viruses in crops important. Most viruses are also able to migrate between plant species, which makes the prevention of the spread of viruses and the use of uncontaminated propagating materials essential. Due to the increasingly active import of plants, the importance of virus identification has grown. Finland wishes to avoid such harmful organisms, which could, for example, halve horticultural crops and which have even forced the discontinuation of farming elsewhere.

For a couple of years already, the Finnish Food Authority, located on Viikki Campus , has been using deep sequencing equipment with which to determine the sequence of bases in small RNA sequences.

Last year the equipment was deployed in earnest in a research project investigating viral infections in potatoes. In the future, the method will be utilised, among other things, to ensure the purity of vegetable and berry plant propagation material. Similar deep sequencing methods can be used to test for viruses also in insects, eelworms and warm-blooded animals, in addition to which the technology enables the identification of bacteria and fungi. At the Finnish Food Authority, deep sequencing is in fact used, in addition to identifying plant viruses, for typing bacterial strains in connection with food poisoning epidemics.

A sweetpotato strain cultivated in cell culture was supposed to be free from viruses, yet three previously unknown viruses were identified in it. More viruses were found, for example, in plant cell cultures and core plant materials. So far, the technique has been successfully used to identify viruses in dozens of plant species, including potato, tomato, pepino, vine, raspberry and cocksfoot, as well as many wild plants. In Finland, the method has uncovered the black raspberry necrosis virus BRNV in wild raspberries, the raspberry leaf blotch virus RLBV in raspberries grown in plastic tunnels and outdoors, as well as the Lily virus X and the woolly burdock yellow vein virus WBYVV in woolly burdocks, previously not known to occur in Finland.

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Doctoral Programme in Plant Sciences. More than researchers from 39 different research groups are part of the centre. University of Helsinki. Small photo: raspberry leaf blotch virus RLBV was identified for the first time in raspberries in Finland thanks to the new method. In a decade, all plant viruses will be easy and quick to identify. Life science news. Sustainability news. Thanks to novel techniques, identifying plant viruses is no longer based on guessing the identity of the virus and choosing the methods of identification accordingly. Now, a sample of the plant will suffice.