Biomedical subjects

Chris A Whitehouse

Publications and source records attributed to Chris A Whitehouse.

12 recordsLinked to original sources

PubMed2006

Comparison of five commercial DNA extraction kits for the recovery of Francisella tularensis DNA from spiked soil samples.

Francisella tularensis is the etiologic agent of the zoonotic disease tularemia and is thought to be maintained in the environment principally by various terrestrial and aquatic vertebrate animals. The organism is known to persist in water or mud for long periods of time and Francisella-specific DNA has been identified from water and soil. To gain a better understanding of the ecology and epidemiology of F. tularensis, it will be important to further explore its distribution in the environment. Therefore, methods must be established to efficiently extract Francisella-specific DNA from the soil and be able to eliminate potential PCR inhibitors. Thus, we evaluated five commercial DNA extraction kits for their ability to recover F. tularensis-specific DNA from soil samples and eliminate potential PCR inhibitors. The kits evaluated included the Puregene DNA purification kit, QIAamp Stool Mini kit, Epicentre Biotech SoilMaster DNA extraction kit, and the UltraClean and PowerMax soil DNA isolation kits from MoBio. Soil samples were spiked with gamma-irradiated F. tularensis SHU-4 strain (corresponding to a range from 10 to 10(5)CFU). Spiked samples were extracted with each kit and evaluated using a F. tularensis-specific real-time PCR assay and an internal positive control assay that measures the presence of potential PCR inhibitors. DNA extraction using the UltraClean and PowerMax kits resulted in the most consistently positive results at the lowest limit of detection (20 and 100CFU/g soil, respectively) for all soil types tested, suggesting that these kits can provide the most sensitive methods for extracting F. tularensis from environmental soil samples. Processing time and cost were also evaluated.

Costs and Cost Analysis↗

PubMed2005

Reemergence of monkeypox: prevalence, diagnostics, and countermeasures.

Human monkeypox is a viral zoonotic disease that occurs mostly in the rain forests of central and western Africa. However, the disease recently emerged in the United States in imported wild rodents from Africa. Monkeypox has a clinical presentation very similar to that of ordinary forms of smallpox, including flulike symptoms, fever, malaise, back pain, headache, and characteristic rash. Given this clinical spectrum, differential diagnosis to rule out smallpox is very important. There are no licensed therapies for human monkeypox; however, the smallpox vaccine can protect against the disease. The discontinuation of general vaccination in the 1980s has given rise to increasing susceptibility to monkeypox virus infection in the human population. This has led to fears that monkeypox virus could be used as a bioterrorism agent. Effective prevention relies on limiting the contact with infected patients or animals and limiting the respiratory exposure to infected patients.

Animals↗

PubMed2005

Smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus.

Vaccination with live vaccinia virus affords long-lasting protection against variola virus, the agent of smallpox. Its mode of protection in humans, however, has not been clearly defined. Here we report that vaccinia-specific B-cell responses are essential for protection of macaques from monkeypox virus, a variola virus ortholog. Antibody-mediated depletion of B cells, but not CD4+ or CD8+ T cells, abrogated vaccine-induced protection from a lethal intravenous challenge with monkeypox virus. In addition, passive transfer of human vaccinia-neutralizing antibodies protected nonimmunized macaques from severe disease. Thus, vaccines able to induce long-lasting protective antibody responses may constitute realistic alternatives to the currently available smallpox vaccine (Dryvax).

Animals↗

PubMed2005

Smallpox vaccine does not protect macaques with AIDS from a lethal monkeypox virus challenge.

It is unknown whether smallpox vaccination would protect human immunodeficiency virus type 1 (HIV-1)-infected individuals, because helper CD4(+) cells, the targets of HIV-1 infection, are necessary for the induction of both adaptive CD8(+) cell and B cell responses. We have addressed this question in macaques and have demonstrated that, although smallpox vaccination is safe in immunodeficient macaques when it is preceded by immunization with highly attenuated vaccinia strains, the macaques were not protected against lethal monkeypox virus challenge if their CD4(+) cell count was <300 cells/mm(3). The lack of protection appeared to be associated with a defect in vaccinia-specific immunoglobulin (Ig) switching from IgM to IgG. Thus, vaccination strategies that bypass CD4(+) cell help are needed to elicit IgG antibodies with high affinity and adequate tissue distribution and to restore protection against smallpox in severely immunocompromised individuals.

Animals↗

PubMed2005

Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an important human pathogen, which is the cause of a tick-borne illness occurring in many areas of Africa, Asia, and Europe. CCHF is characterized by a sudden onset of high fever, chills, and severe headache. Other symptoms can include gastrointestinal disorders, such as nausea, vomiting, and diarrhea. In severe cases, hemorrhagic manifestations can occur and often present as large areas of ecchymosis, rather than frank bleeding. Exposure to ticks, particularly those in the genus Hyalomma, or direct contact with virus-infected animals or people are considered the major risk factors. Studies on CCHFV are impeded by the biocontainment needed for their manipulation. However, the increasing worldwide medical awareness, the enormous interest of the media in hemorrhagic fever diseases, and their potential to be used as a bioweapon, have greatly spurred on research on this important virus, as evidenced by many new developments including the development of a reverse genetics system which should greatly enhance future research with this virus.

Hemorrhagic Fever Virus, Crimean-Congo↗

PubMed2004

Development of a fluorescent microplate assay for determining cyanovirin-N levels in plasma.

A sensitive immunosorbent competition assay was developed for quantitation of the anti-HIV protein cyanovirin-N (CV-N) in plasma using a 96-well plate format and a fluorescent endpoint. The assay is based on the binding of CV-N in plasma to plate-bound anti-CV-N antibodies, followed by removal of the plasma and addition of europium-labeled CV-N (Eu3+ -CV-N) to compete for the remaining antibody sites. Detection by addition of a dissociative fluorescence enhancement solution and time-resolved fluorescence measurements allowed correlation to the concentration of the native CV-N in plasma. A linear detection range of 1-100 nM (r2>0.99) was obtained for CV-N in mouse plasma. This assay was then utilized for analysis of plasma levels of CV-N samples following subcutaneous injection of CV-N into mice. The results of these studies confirmed the reliability and sensitivity of this assay and the feasibility of its use for pharmacokinetic studies in a variety of species.

Animals↗

PubMed2004

A simple assay for determining antiviral activity against Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that is emerging as a significant human pathogen in many regions of the world, including Africa, Asia, and Europe. In this report, we describe a simple screening method for discovering new antiviral compounds directed against CCHFV. Antiviral activity was determined by assaying infected SW-13 cells (human adrenal gland carcinoma) for protection from cytopathic effect (CPE). By using an in vitro neutral red uptake assay, we were able to quantitatively measure CPE induced by CCHFV. As a proof of concept, we used this method to evaluate the antiviral activity of ribavirin and a series of structural analogs (ribamidine, 6-azauridine, selenazofurin, and tiazofurin) against four geographically diverse strains of CCHFV. Ribavirin inhibited the replication of CCHFV as reported previously using plaque reduction assays. One drug, ribamidine, showed antiviral activity that was 4.5- to 8-fold less than that of ribavirin, and the other three drugs (6-azauridine, selenazofurin, and tiazofurin) did not show significant antiviral activity. There were no significant differences in drug sensitivities among the CCHFV strains. Development of this simple and reliable assay will potentially allow high-throughput screening for discovering additional antiviral drugs to combat this important public health threat.

Animals↗

PubMed2004

Crimean-Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease caused by the arbovirus Crimean-Congo hemorrhagic fever virus (CCHFV), which is a member of the Nairovirus genus (family Bunyaviridae). CCHF was first recognized during a large outbreak among agricultural workers in the mid-1940s in the Crimean peninsula. The disease now occurs sporadically throughout much of Africa, Asia, and Europe and results in an approximately 30% fatality rate. After a short incubation period, CCHF is characterized by a sudden onset of high fever, chills, severe headache, dizziness, back, and abdominal pains. Additional symptoms can include nausea, vomiting, diarrhea, neuropsychiatric, and cardiovascular changes. In severe cases, hemorrhagic manifestations, ranging from petechiae to large areas of ecchymosis, develop. Numerous genera of ixodid ticks serve both as vector and reservoir for CCHFV; however, ticks in the genus Hyalomma are particularly important to the ecology of this virus. In fact, occurrence of CCHF closely approximates the known world distribution of Hyalomma spp. ticks. Therefore, exposure to these ticks represents a major risk factor for contracting disease; however, other important risk factors are known and are discussed in this review. In recent years, major advances in the molecular detection of CCHFV, particularly the use of real-time reverse transcription-polymerase chain reaction (RT-PCR), in clinical and tick samples have allowed for both rapid diagnosis of disease and molecular epidemiology studies. Treatment options for CCHF are limited. Immunotherapy and ribavirin have been tried with varying degrees of success during sporadic outbreaks of disease, but no case-controlled trials have been conducted. Consequently, there is currently no antiviral treatment for CCHF approved by the U.S. Food and Drug Administration (FDA). However, renewed interested in CCHFV, as well as increased knowledge of its basic biology, may lead to improved therapies in the future. This article reviews the history, epidemiology, ecology, clinical features, pathogenesis, diagnosis, and treatment of CCHF. In addition, recent advances in the molecular biology of CCHFV are presented, and issues related to its possible use as a bioterrorism agent are discussed.

Animals↗

PubMed2004

Monkeypox virus detection in rodents using real-time 3'-minor groove binder TaqMan assays on the Roche LightCycler.

During the summer of 2003, an outbreak of human monkeypox occurred in the Midwest region of the United States. In all, 52 rodents suspected of being infected with monkeypox virus were collected from an exotic pet dealer and from private homes. The rodents were euthanized and submitted for testing to the United States Army Medical Research Institute of Infectious Diseases by the Galesburg Animal Disease Laboratory, Illinois Department of Agriculture. The rodent tissue samples were appropriately processed and then tested by using an integrated approach involving real-time polymerase chain reaction (PCR) assays, an antigen-detection immunoassay, and virus culture. We designed and extensively tested two specific real-time PCR assays for rapidly detecting monkeypox virus DNA using the Vaccinia virus F3L and N3R genes as targets. The assays were validated against panels of orthopox viral and miscellaneous bacterial DNAs. A pan-orthopox electrochemiluminescence (ECL) assay was used to further confirm the presence of Orthopoxvirus infection of the rodents. Seven of 12 (58%) animals (seven of 52 (15%) of all animals) tested positive in both monkeypox-specific PCR assays and two additional pan-orthopox PCR assays (in at least one tissue). The ECL results showed varying degrees of agreement with PCR. One hamster and three gerbils were positive by both PCR and ECL for all tissues tested. In addition, we attempted to verify the presence of monkeypox virus by culture on multiple cell lines, by immunohistology, and by electron microscopy, with negative results. Sequencing the PCR products from the samples indicated 100% identity with monkeypox virus strain Zaire-96-I-16 (a human isolate from the Congo). These real-time PCR and ECL assays represent a significant addition to the battery of tests for the detection of various orthopoxviruses. In light of the recent monkeypox virus transmissions, early detection of the virus is crucial for both natural outbreaks and potential acts of bioterrorism.

Animals↗

PubMed2003

Vaccines and animal models for arboviral encephalitides.

Arthropod-borne viruses ("arboviruses") cause significant human illness ranging from mild, asymptomatic infection to fatal encephalitis or hemorrhagic fever. The most significant arboviruses causing human illness belong to genera in three viral families, Togaviridae, Flaviviridae, and Bunyaviridae. These viruses represent a significant public health threat to many parts of the world, and, as evidenced by the recent introduction of the West Nile virus (WNV) to the Western Hemisphere, they can no longer be considered specific to any one country or region of the world. Like most viral diseases, there are no specific therapies for the arboviral encephalitides; therefore, effective vaccines remain the front line of defense for these diseases. With this in mind, the development of new, more effective vaccines and the appropriate animal models in which to test them become paramount. In fact, for many important arboviruses (e.g. California serogroup and St. Louis encephalitis viruses), there are currently no approved vaccines available for human use. For others, such as the alphaviruses, human vaccines are available only as Investigational New Drugs, and thus are not in widespread use. On the other hand, safe and effective vaccines against tick-borne encephalitis virus (TBEV) and Japanese encephalitis virus (JEV) have been in use for decades. New challenges in vaccine development have been met with new technologies in vaccine research. Many of the newer vaccines are now being developed by recombinant DNA technology. For example, chimeric virus vaccines have been developed using infectious clone technology for many of the arboviruses including, WNV, JEV, and TBEV. Other successful approaches have involved the use of naked DNA encoding and subsequently expressing the desired protective epitopes. Naked DNA vaccines have been used for TBEV and JEV and are currently under development for use against WNV. The development of less expensive, more authentic animal models to evaluate new vaccines against arboviral diseases will become increasingly important as these new approaches in vaccine research are realized. This article reviews the current status of vaccines, both approved for use and those in developmental stages, against the major arboviral encephalitides causing human disease. In addition, research on animal models, both past and present, for these diseases are discussed.

Animals↗

PubMed2003

Arbovirus surveillance in Rhode Island: assessing potential ecologic and climatic correlates.

During 1995-2000, mosquitoes were collected from sites throughout Rhode Island and tested for the presence of arboviruses. Mosquito trapping was done weekly from June to October with CO2-baited light traps. In all, 186,537 mosquitoes belonging to 7 different genera were collected, of which Coquillettidia perturbans was most abundant. A total of 6,434 pools were processed for arbovirus isolation, from which 193 arboviral isolations were made. These included 109 Highlands J, 71 eastern equine encephalomyelitis, 1 California encephalitis serogroup, 2 Jamestown Canyon, 3 Cache Valley, and 9 Flanders viruses. Our isolations of Flanders virus represent the 1st reported occurrence of this virus in Rhode Island. After the 1999 sudden occurrence of the West Nile virus (WN) in the New York City area, a dead-bird surveillance program was started to test for this virus. Although no isolations of WN were made from mosquitoes, 87 virus isolations were made from a total of 330 wild birds tested. All the WN-infected birds were either American crows or blue jays. Isolation of WN from dead birds marked the 1st documented appearance of this virus in Rhode Island. Significant interannual variation of arbovirus activity in Rhode Island prompted us to examine if climate-associated factors such as rainfall and temperature correlate with virus activity. Total rainfall amounts from May to June were higher than normal in 1996 and 1998. These years showed significantly higher arbovirus activity. Deviations from normal temperature showed low correlation with arbovirus activity during the 6-year study period. Therefore, precipitation appeared to be more important than temperature in predicting arbovirus activity in Rhode Island.

Animals↗

PubMed2002

Two new rhabdoviruses (Rhabdoviridae) isolated from birds during surveillance for arboviral encephalitis, northeastern United States.

Two novel rhabdoviruses were isolated from birds during surveillance for arboviral encephalitis in the northeastern United States. The first, designated Farmington virus, is a tentative new member of the Vesiculovirus genus. The second, designated Rhode Island virus, is unclassified antigenically, but its ultrastructure and size are more similar to those of some of the plant rhabdoviruses. Both viruses infect birds and mice, as well as monkey kidney cells in culture, but their importance for human health is unknown.

Animals↗