Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-06-29
2001-11-13
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069300, C536S023720
Reexamination Certificate
active
06316250
ABSTRACT:
BACKGROUND OF INVENTION
1. Field of Art
The invention relates to the HARDS virus, the etiologic agent of Hantavirus-Associated Respiratory Distress Syndrome.
An epidemic of unexplained adult respiratory distress syndrome, affecting primarily residents of the Four Corners region formed by the borders of New Mexico, Arizona, Utah, and Colorado, was recognized in May, 1993. The disease is characterized by a prodromal illness of fever, myalgias, and, in some cases, conjunctivitis, lasting 1-5 days, followed by a severe and acute illness characterized by pulmonary edema and shock. According to the federal Centers for Disease Control and Prevention (CDC), through Jul. 27, 1993, death from suffocation and/or shock had occurred in 14 (78%) of the 18 patients diagnosed with the illness since the epidemic was recognized. The syndrome was eventually determined to be caused by a viral infection of a newly-identified hantavirus virus subsequently named the HARDS virus (also referred to as Sin Nombre Virus and Four Corner Virus, or FCV). According to the CDC, the predominant vector for this virus is the deer mouse Peromyscus maniculatus, which ranges throughout the southwest U.S. with the potential for spreading HARDS infection within this area.
The infection involves two clinical stages. In the first stage, or prodrome, nonspecific abnormalities such as fever, muscle aches, and cough occur, making the distinction between HARDS virus infection and influenza, “Strep throat”, and other upper respiratory infections difficult. The prodromal infection lasts only briefly, and is followed in 1-5 days by the second stage of HARDS virus infection, characterized by severe pulmonary disease and shock that often proves fatal. By the time pulmonary edema appears, it is often possible to distinguish HARDS infection from infection caused by other microbes, but many authorities consider that stage to be too late for HARDS virus to be effectively treated by antivirals having potential anti-HARDS activity.
Accordingly, the need for a rapid diagnostic test for the presence of the HARDS virus is particularly acute. In addition to assisting clinicians in determining which patients are infected with HARDS virus and in need of hospitalization and treatment, a specific test for HARDS virus would guide public health officials in their efforts to monitor the extent and spread of the disease. A specific test for HARDS vipus would also be helpful for rodent surveillance, studies of the prevalence and transmission of HARDS virus infection, and many other research activities. Such a test would make possible the determination of the range of HARDS virus in rodent populations, thus documenting precisely which human populations are at risk for HARDS infection.
2. Discussion of Related Art
1. Viral Characterization
By early June, it was recognized that the victims of the new syndrome were developing, during their illness, IgM antibodies that reacted with one or both of two members of the Hantavirus genus of Bunyaviruses. Those viruses are known as Puumala virus, a vole virus that causes the human disease nephropathia epidemica (Europe), and Seoul virus, a virus of Norwegian rats first identified in Korea. Scientists at the federal Centers for Disease Control and Prevention (CDC), in Atlanta, Ga., were able to obtain molecular clones and limited sequence information from the new virus. The sequence information supported the notion that the new virus is a hantavirus; and suggested that its closest relatives among the hantaviruses might be Puumala virus, and another rodent virus known as Prospect Hill virus (PHV).
2. Diagnosis of HARDS Virus Infection
The standard method for diagnosis of acute infections caused by other hantaviruses is the detection, via enzyme-linked immunosorbent assay (EIA), of IgM antibodies to the suspected virus. To do this, it is necessary to have the suspected virus growing in tissue culture; such infected cell cultures are the source of viral antigens to which the patient's serum Igm will react. Thus, for example, serum from a patient with suspected infection by Hantaan virus (which occurs in Korea and China) is incubated with lysate of Hantaan virus-infected cells, and the immune complexes are detected by another antibody.
Despite many attempts to grow the HARDS virus in culture by CDC and others, suitable cultures for the production of viral antigens for the assay of immunoreactive serum antibodies have not been achieved, and thus far, infection by HARDS virus has been diagnosed by one of the following routes:
(a) The development, in infected patients, of antibodies to HARDS virus that can be shown to “cross-react” weakly with antigens derived from Puumala virus or Seoul virus. This method is useful for epidemiologic surveillance, but has proved to be insufficiently sensitive to allow diagnosis of HARDS infection before the patient becomes either critically ill and near death, or has recovered from infection. Infection by HARDS virus appears to result in a somewhat less brisk antibody response than that seen in association with infection by other hantaviruses.
(b) Polymerase chain reaction (PCR). CDC scientists developed a PCR method for diagnosing HARDS infection in June, 1993. A small portion of the G2 gene of the viral M segment is copied into DNA using the enzyme reverse transcriptase, and amplified into large amounts of DNA. The amplified DNA (185 bp) is detected on an agarose gel. Application of this technique to patients presenting to the UNM Hospital in Albuquerque, N.Mex. have shown that the HARDS virus can be detected in the peripheral blood cells of ~90% of infected patients, with no known false-positive tests thus far. The method is somewhat slow (36 hours between receipt of blood and detection of the virus) and appears to be subject to false-negative tests, probably attributable to a sufficient difference in viruses between affected patients to prevent the annealing of PCR primers.
An additional concern in use of PCR is false-positive tests. This problem arises when tiny amounts of amplified viral DNA, present in the laboratory on surfaces, centrifuges, gel apparatuses, etc., finds its way into test tubes used in the preparation of a PCR reaction. The contaminating DNA makes an excellent template for the next PCR reaction, making false-positive tests in uninfected patients a significant risk. The risk can be reduced substantially by strict and rigorous physical separation of facilities used for “pre-PCR” activities (for example, areas where RNA is prepared from the blood of patients to be tested for HARDS virus), and “post-PCR” activities, where amplified viral DNA is studied and analyzed. However, such separation may not be sufficient. In many cases, it is helpful to irradiate PCR cocktails—prior to amplification—with ultraviolet light to destroy contaminating template DNAs, before the bona fide target (patient RNA) is added. However, UV irradiation is much more effective in situations where the contaminating DNA is expected to be at least 300 bases long; for this reason, any contamination by the 185 base-pair product produced by the CDC protocol is unlikely to be “sterilized” sufficiently by UV irradiation. This requirement for larger PCR target is shared by many of the most successful schemes for PCR decontamination (
Nature
343:27, 1990).
The expense, slow turnaround time, and labor-intensiveness of PCR (as presently configured) makes it a suboptimal solution for rapid diagnosis of hantavirus infection. Nevertheless, PCR may be the best solution available before a definitive serologic (antibody or antigen) test becomes available.
(c) Immunohistochemistry. It is possible to detect HARDS virus antigens in tissue samples of infected patients by exposing the tissues (lungs or kidneys) to a specific monoclonal antibody, originally raised against Puumala virus. This method has been used by CDC in patient tissues obtained at autopsy, but has not been shown to be an effective means of diagnosis in fresh blood samples from living patients.
It is accordingly desirable to provide a rapi
Hjelle Brian
Jenison Steve
Morgan, Lewis & Bockius, L.L.P.
The University of New Mexico
Wortman Donna C.
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