Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1998-08-04
2001-02-06
Wortman, Donna C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C436S071000
Reexamination Certificate
active
06183950
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the detection of the presence or the likely presence of a virus that has been discharged into the environment.
BACKGROUND OF THE INVENTION
Several nations and terrorist groups have or are believed to have the capability to produce chemical or biological weapons (“CBWs”). Moreover, recent events indicate that certain nations and terrorist groups are willing to use CBWs. For instance, during the war between Iraq and Iran, chemical weapons were deployed by Iraq against both Iranian ground forces and the Kurdish civilian population. An example of terrorist use of chemical weapons against a civilian population is the recent release of a nerve gas in a Tokyo subway station. One type of CBW that is of particular concern are viruses. Characteristics of the types of viruses that are believed to be particularly suitable for use in warfare and terrorist activities are: (1) a relatively short incubation period; (2) debilitating or deadly effects; and/or (3) communicability. Among the types of viruses that exhibit some or all of these characteristics are smallpox, viral encephalitides and viral hemorrhagic fevers. Among the viral hemorrhagic virus is the well-known Ebola virus. The possibility of viral agents being used against military personnel in a warfare situation or against a civilian population in a terrorist attack has created the need for rapid identification of the presence or likely presence of viral agents so that countermeasures can be taken to minimize the effects upon the target population.
SUMMARY OF THE INVENTION
The present invention makes use of the discovery that certain biochemicals (known as biomarkers) associated with viruses are susceptible to rapid detection that permits countermeasures to be taken to reduce the impact of the virus upon the target population.
Briefly, viruses are propagated by infecting host animal cells with a virus. The virus within a host cell uses the resources and environment of the host cell to reproduce. At some point, the viruses produced within a cell rupture the cell wall and move on to infect other cells and repeat the process.
To mass produce a virus, a cell culture is provided that includes host animal cells and certain chemicals that are used to nurture the host cells. The virus is introduced into the cell culture and promptly invades the host cells and begins reproducing. When enough of the virus has been produced, the virus is harvested from the cell culture. Typically, the harvesting collects the virus as well as some or all of the cell culture constituents. The harvested material can be purified. However, purification may degrade the virus and thereby decrease its virulence. Consequently, it is anticipated that any viruses released in a warfare or terrorist situation will be released in an unpurified form that includes components of the cell culture.
The present invention has identified biomarkers associated with the cell culture that can be rapidly detected. More specifically, biomarkers associated with: (1) the animal cells (typically mammalian or bird cells) that are the host cells for the virus and (2) blood serum, which provides the host cells with nutrients and growth factors, are susceptible to rapid identification. While animal cells, such as mammalian and bird cells, are a necessary part of the cell culture, blood serum may or may not be part of the cell culture. A biomarker associated with both mammalian cells and blood serum that is relatively unique to the production of viruses is cholesterol. Consequently, if the virus is dispersed in an unpurified form that includes cell culture materials, cholesterol is likely to be present. Since the cholesterol is associated with the cell culture materials rather than the virus itself, the cholesterol is considered a secondary biomarker. However, in reproducing, the virus acquires cholesterol from the host cells. In this case, cholesterol is considered a primary biomarker because it is part of the virus itself. Since cholesterol is present in the virus itself, rapid detection of the virus is possible even if the virus is dispersed in a purified form in which most or all of the cell culture constituents have been removed.
Other biomarkers that are also indicative of animal cells, including mammalian or bird cells, and blood serum are certain fatty acids. These fatty acids include, among others, palmitic, stearic, oleic and linoleic fatty acids. The detection of fatty acids can be used to further confirm the presence of a virus whose presence is already considered likely based upon the detection of another biomarker, like cholesterol.
Rapid detection of the cholesterol biomarker is possible because the mass spectrum of cholesterol is very distinct relative to the other biomarkers associated with a virus, whether in a purified or unpurified form. Mass spectrometry is a method of chemical analysis that uses the mass of a substance to identify the substance. To elaborate, associated with every type of molecule is a mass spectrum, a kind of “fingerprint”, that is relatively unique to each particular molecule. The chemical analysis of an unknown substance by mass spectrometry involves obtaining a mass spectrum for the substance and comparing the mass spectrum to a library of mass spectra for known substances to identify the chemical components of the unknown substance.
The present invention involves sampling an atmosphere and performing a mass spectrum analysis of the sampled atmosphere to determine if a biomarker indicative of the presence of a virus is present. As previously noted, the present invention utilizes a biomarker that is associated with the cell culture media which is used to produce the virus in quantity, such as cholesterol. If such a biomarker is present, then it is likely that a viral agent is also present and an alarm is issued. The mass spectrum analysis is performed with a few minutes of sampling and, as such, is likely to provide sufficient warning for counter measures to be taken by at least a portion of the target population. While it is expected that viral agents used in warfare and terrorist situations will be dispersed in the atmosphere as aerosols, it is believed that the invention is adaptable to detecting viruses that are dispersed in the water.
In one embodiment, the sampling of the atmosphere is done in a fashion that presents or reduces the possibility that the mass spectrometer's time is used to analyze particles in the atmosphere that are not likely to be viruses. To elaborate, aerosolized viruses in aerosolizing media have an idealized upper limit on their size of approximately 10 microns. Consequently, sampling is done so as to avoid the sampling of particles in the atmosphere that are greater than 10 microns in size. In one embodiment, this is accomplished with a device known as a virtual impactor.
The sampling of the atmosphere is also preferably done so as to heat the sampled atmosphere to distill the biomarkers, such as cholesterol, from the sample and thereby facilitate the mass spectrum analysis. In one embodiment, heating of the sampled atmosphere is accomplished with a pyrolysis device.
To prevent tampering that could reduce the effectiveness of the invention, one embodiment employs a stand-alone power source as either a primary or secondary power source. Relatedly, the intake port for sampling the atmosphere is positioned so as to be difficult to detect and/or to plug.
REFERENCES:
patent: 5469369 (1995-11-01), Rose-Pehrsson et al.
patent: 5550062 (1996-08-01), Wohltjen et al.
Marple, V.A. et al., Aerosol Sample Acquisition for Chemical and Biological Agent Detection, Abstract, Report No.: ARO-25616.1-CHS, Army Research Office, Dec. 1, 1989.
Meuzelaar, Henk L.C., et al., Characterization of Leukemic and Normal White Blood Cells by Curie-Point Pyrolysis-Mass Spectrometry, pp. 111-129, 1981, Journal of Analytical and Applied Pyrolysis.
Tas, A.C., et al., Characterization of Virus Infected Cell Cultures by Pyrolysis/Direct Chemical Ionization Mass Spectrometry, Biomedical and Environm
Madonna Angelo J.
Voorhees Kent J.
Brumback Brenda G.
Colorado School of Mines
Holme Roberts & Owen LLP
Wortman Donna C.
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