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ROCKVILLE, MD, United States

Daum L.T.,Longhorn Vaccines and Diagnostics | Worthy S.A.,Longhorn Vaccines and Diagnostics | Yim K.C.,Virion Systems, Inc. | Nogueras M.,BioReliance Corporation | And 3 more authors.
Epidemiology and Infection | Year: 2011

Pathogen detection and genetic characterization has dramatically changed in recent years. Clinical laboratories are transitioning from traditional culture and primer-specific sequencing to more robust and rapid nucleic acid testing such as real-time PCR and meta-genomic characterization, respectively. Specimen collection is the first step in any downstream molecular diagnostic procedure. PrimeStore Molecular Transport Medium (MTM) is an optimized blend of nucleic acid stabilizing reagents that includes a non-specific internal positive control that can be amplified using real-time RT - PCR for tracking the integrity of a specimen from the point of collection to detection. PrimeStore MTM is shown here to effectively kill pathogens, including highly pathogenic H5 influenza virus, inactivate nucleases and to protect and preserve released RNA at ambient temperature for up to 30 days for downstream real-time and traditional RT - PCR detection and genetic characterization. PrimeStore MTM is also compatible with a variety of commercial extraction kits. PrimeStore is suited for routine clinical specimens and has added utility for field collection in remote areas, triage centres, border crossings and during pandemics where cold-chain, transport, and dissemination of potentially infectious pathogens are a concern. © Copyright Cambridge University Press 2010. Source


Boukhvalova M.S.,Virion Systems, Inc.
Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] | Year: 2010

Viral infection is normally detected either by viral culture or by PCR methods. Rarely is a combination of the two techniques used in the same study. Yet, when applied simultaneously, viral culture and PCR may reveal important features of viral biology, such as an abortive replication, as in the case of respiratory syncytial virus (RSV) infection. In this unit, we describe methods for detecting abortive RSV replication in a cotton rat model by using the plaque-forming unit assay and the real-time reverse-transcription PCR (qRT-PCR) assay. All steps of the process of monitoring viral replication in vivo are described, starting from the design of animal infection protocols. We continue on to the methods for extracting and processing lung samples for viral culture and RNA extraction, and finish with the actual methods of viral titration by the qRT-PCR and the plaque-forming unit assays. 2010 by John Wiley & Sons, Inc. Source


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 404.56K | Year: 2007

DESCRIPTION (provided by applicant): The development of a small animal model to study human immunodeficiency virus type-1 (HIV-1) infection would significantly facilitate studies of disease pathogenesis, as well as vaccine and anti-viral drug development and testing. However, HIV-1 replication is subjected to a number of species-specific restrictions at the level of cellular entry and/or post-entry. To date, no satisfactory small animal model for HIV-1 infection has been identified. The cotton rat has been a superb model for human infectious diseases. This animal is susceptible to an extraordinary spectrum of human pathogens, particularly viruses. Inspired by these observations, we have cloned and characterized a battery of more than 270 cotton rat genes of immunological and inflammatory importance, and reagents for their detection have been developed. Human immunodeficiency virus (HIV-1) was shown to infect cotton rats and infectious virus was transmitted from animal to animal by blood with low efficiency. In new studies developed during the Phase I of this SBIR, we have demonstrated that cotton rat cells expressing human co-receptors for HIV-1 (hCD4 with hCXCR4 or hCD4 with hCCR5) support HIV-1 infection (by kinetic experiments that measured the production of p24gag in cotton rat infected cells) and DNA integration (by cloning experiments that isolated chimeric DNA containing HIV-1 DNA sequences linked to cotton rat DNA sequences from infected cotton rat cells). Furthermore, we have shown that cotton rat cells are able to produce infective particles that can infect fresh human PBMCs. The data presented in our Progress Report indicate that the main blockage for HIV replication in cotton rat cells occurs during viral entrance and that it could be circumvented by the expression of HIV-1 co-receptors. Due to our success in demonstrating the feasibility of the model in vitro during phase I, our goal in phase II is to produce line(s) of transgenic cotton rats expressing hCD4 with hCXCR4 and hCD4 with hCCR5 molecules and test them for their infectivity to HIV-1. An HIV-1-permissive cotton rat could be widely used by the research community. The hypothesis to be tested is that cotton rats (S. hispidus) after engineered to produce HIV-1 co-receptors will be permissive to productive HIV-1 infection. We will test our hypothesis by producing lines of transgenic animals expressing hCD4 with hCCR5 and hCD4 with hCXCR4. Finaly, we will Test these transgenic lines of cotton rats in infection protocols using HIV-1. At the completion of the work proposed for phase II of this SBIR, we will have produced several lines of transgenic cotton rat expressing human co-receptors for HIV-1. Most importantly, we will have characterized these lines of animals in ex vivo and in vivo experiments An estimated 5 million people became infected with HIV worldwide in 2003, and as many as 3 million died from AIDS, according to the Joint United Nations Program on HIV/AIDS (UNAIDS). The total number of infected people worldwide is estimated at 40 million (and 600 new infections per hour). Preventive vaccine and prophylactic therapies against HIV infection and more efficient drugs for AIDS are not only urgently needed but they are also scientifically possible. However, one of the major obstacles in translational HIV research rests in the absence of inexpensive and efficient pre-clinical trial models. This is reflected in the fact that the 30 vaccine candidates that are currently being tested in clinical trials in 21 countries (only 2 have advanced to clinical phase III efficacy trials) are very similar to each other with nearly all based on only one hypothesis (protection by eliciting a cell mediated immune response), while other strong alternative hypotheses have been largely neglected (vaccines that induce neutralizing antibodies and live attenuated vaccines) due to monetary and logistic impediment to test a greater number of candidates in preclinical settings. Thus, several potential vaccines and therapeutics turn out to be buried in laboratory notebooks because the institutions where they were developed cannot afford to continue with non-human primate trials. The lack of a small animal model for HIV infection that can be used for screening a larger variety of candidates in preclinical studies is one of the most evident obstacles to speed up the process of developing HIV vaccines and AIDS therapies.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 438.79K | Year: 2006

DESCRIPTION (provided by applicant): Respiratory Syncytial virus (RSV) is the leading viral cause of death in children under 1 year and is an increasing cause of morbidity and mortality in transplant patients and the elderly. RSV causes upper and lower respiratory tract infections, occasionally leading to severe bronchiolitis and pneumonia. There is no safe and effective vaccine against RSV. Anti-RSV immunotherapy, although effective in prophylactic settings, does not provide any clinically beneficial outcome when applied therapeutically, indicating that RSV-induced pathology is mostly the result of the inflammatory response to infection rather than a direct viral effect. A combined antiviral and anti-inflammatory therapy might represent the most safe and efficient treatment against RSV infection. The expression of COX-2 and its products, prostaglandins and thromboxanes, has been correlated with the development of many inflammatory processes. Our recent studies in the cotton rat, the animal model of choice for previous studies of RSV immunoprophylaxis, strongly support our hypothesis that induction of COX-2 during RSV infection plays a pivotal role during RSV-induced inflammation and pathology and, conversely, that inhibition of COX-2 activity is a beneficial treatment for RSV-induced bronchiolitis. This application is designed to determine efficacy and safety profiles for COX-2 specific, non-steroidal anti-inflammatory drug (NSAID) treatment of RSV-induced lung pathology. Our hypothesis is that inhibition of COX-2 activity generated during RSV infection will be of therapeutic benefit during acute RSV disease, preventing the development of inflammation and pathology. The experiments will focus on primary RSV disease, with two goals in mind. The first will be to determine if COX-2 inhibition can by itself be an effective and safe treatment regimen during the acute phase of primary RSV infection. The second will be to determine whether treatment with COX-2-specific inhibitors can be complemented with antiviral therapy to improve the final outcome of RSV disease. These studies will involve correlating treatment of RSV-infected animals with pulmonary histopathology and inflammation.


Boukhvalova M.S.,Virion Systems, Inc. | Sotomayor T.B.,Virion Systems, Inc. | Sotomayor T.B.,West Virginia University | Point R.C.,Virion Systems, Inc. | And 3 more authors.
Journal of Interferon and Cytokine Research | Year: 2010

Interferon (IFN) therapy in humans often causes flu-like symptoms by an unknown mechanism. Poly ICLC is a synthetic dsRNA and a Toll-like receptor 3(TLR3)agonist with a strong IFN-inducing ability. In this work, we analyzed the effect of poly ICLC on pulmonary responses to influenza and respiratory syncytial virus (RSV) infections in the cotton rat (Sigmodon hispidus) model. Viral replication, pulmonary inflammation, and expression of IFN, TLR, and chemokines were monitored and compared. Antiviral effect of poly ICLC against influenza virus and RSV was best achieved at high poly ICLC concentrations that, in the absence of virus infection, induced a strong IFN response. The antiviral doses of poly ICLC, however, also increased lung inflammation, an unexpected finding because of the reported poly ICLC safety in BALB/c mice. Similarly, in contrast to murine model, pathology of RSV infection was increased in cotton rats treated with poly ICLC. Augmented lung inflammation was accompanied by an earlier induction of IFN and TLR responses and a stronger chemokine expression. Overall, these findings indicate significant association between antiviral IFN action and pulmonary inflammation and highlight important animal model-specific variations in the potential of IFN to cause pathology. © 2009 Mary Ann Liebert, Inc. Source

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