ROCKVILLE, MD, United States
ROCKVILLE, MD, United States
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Shirey K.A.,University of Maryland, Baltimore | Pletneva L.M.,Virion Systems, Inc. | Puche A.C.,University of Maryland, Baltimore | Keegan A.D.,University of Maryland, Baltimore | And 3 more authors.
Mucosal Immunology | Year: 2010

Severe respiratory syncytial virus (RSV)-induced bronchiolitis has been associated with a mixed Th1 and Th2 cytokine storm. We hypothesized that differentiation of alternatively activated macrophages (AA-Mφ) would mediate the resolution of RSV-induced lung injury. RSV induced interleukin (IL)-4 and IL-13 by murine lung and peritoneal macrophages, IL-4Rα/STAT6-dependent AA-Mφ differentiation, and significantly enhanced inflammation in the lungs of IL-4Rα-/- mice. Adoptive transfer of wildtype macrophages to IL-4Rα-/- mice restored RSV-inducible AA-Mφ phenotype and diminished lung pathology. RSV-infected Toll-like receptor (TLR)4-/- and interferon (IFN)-Β-/- macrophages and mice also failed to express AA-Mφ markers, but exhibited sustained proinflammatory cytokine production (e.g., IL-12) in vitro and in vivo and epithelial damage in vivo. TLR4 signaling is required for peroxisome proliferator-activated receptorγ expression, a DNA-binding protein that induces AA-Mφ genes, whereas IFN-Β regulates IL-4, IL-13, IL-4Rα, and IL-10 expression in response to RSV. RSV-infected cotton rats treated with a cyclooxygenase-2 inhibitor increased expression of lung AA-Mφ. These data suggest new treatment strategies for RSV that promote AA-Mφ differentiation. © 2010 Society for Mucosal Immunology.


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.


Hubner M.P.,Uniformed Services University of the Health Sciences | Hubner M.P.,University of Bonn | Killoran K.E.,Uniformed Services University of the Health Sciences | Rajnik M.,Uniformed Services University of the Health Sciences | And 11 more authors.
PLoS Neglected Tropical Diseases | Year: 2012

Background: Chronic helminth infections induce a Th2 immune shift and establish an immunoregulatory milieu. As both of these responses can suppress Th1 immunity, which is necessary for control of Mycobacterium tuberculosis (MTB) infection, we hypothesized that chronic helminth infections may exacerbate the course of MTB. Methodology/Principal Findings: Co-infection studies were conducted in cotton rats as they are the natural host for the filarial nematode Litomosoides sigmodontis and are an excellent model for human MTB. Immunogical responses, histological studies, and quantitative mycobacterial cultures were assessed two months after MTB challenge in cotton rats with and without chronic L. sigmodontis infection. Spleen cell proliferation and interferon gamma production in response to purified protein derivative were similar between co-infected and MTB-only infected animals. In contrast to our hypothesis, MTB loads and occurrence and size of lung granulomas were not increased in co-infected animals. Conclusions/Significance: These findings suggest that chronic filaria infections do not exacerbate MTB infection in the cotton rat model. While these results suggest that filaria eradication programs may not facilitate MTB control, they indicate that it may be possible to develop worm-derived therapies for autoimmune diseases that do not substantially increase the risk for infections.


Jensen K.,Uniformed Services University of the Health Sciences | Patel A.,Uniformed Services University of the Health Sciences | Larin A.,Hospital for Endocrine Surgery | Hoperia V.,Hospital for Endocrine Surgery | And 5 more authors.
Journal of Pathology | Year: 2010

To test the hypothesis that herpes viruses may have a role in thyroid neoplasia, we analysed thyroid tissues from patients with benign (44) and malignant (65) lesions for HSV1 and HSV2 DNA. Confirmatory studies included direct sequencing, analysis of viral gene expression, and activation of viral-inducible signalling pathways. Expression of viral entry receptor nectin-1 was examined in human samples and in cancer cell lines. In vitro experiments were performed to explore the molecular mechanisms underlying thyroid cancer cell susceptibility to HSV. HSV DNA was detected in 43/109 (39.4%) examined samples. HSV capsid protein expression correlated with HSV DNA status. HSV-positive tumours were characterized by activation of virus-inducible signalling such as interferon-beta expression and nuclear NFκB expression. Lymphocyte infiltration and oncocytic cellular features were common in HSV-positive tumours. HSV1 was detected with the same frequency in benign and malignant thyroid tumours. HSV2 was significantly associated with papillary thyroid cancer and the presence of lymph node metastases. The expression of HSV entry receptor nectin-1 was increased in thyroid tumours compared to normal thyroid tissue and further increased in papillary thyroid cancer. Nectin-1 expression was detected in all examined thyroid cancer cell lines. Nectin-1 expression in cancer cells correlated with their susceptibility to HSV. Inhibition of PI3K/AKT or MAPK/ERK signalling did not affect the level of nectin-1 expression but decreased thyroid cancer cell susceptibility to HSV. These findings showed that HSV is frequently detected in thyroid cancer. During tumour progression, thyroid cells acquire increased susceptibility to HSV due to increased expression of viral entry mediator nectin-1 and activation of mitogenic signalling in cancer cells. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Dambrosi S.,Laval University | Martin M.,Laval University | Yim K.,Virion Systems, Inc. | Miles B.,Virion Systems, Inc. | And 3 more authors.
Journal of Medical Virology | Year: 2010

Herpes simplex virus (HSV) resistance to acyclovir or foscarnet results from mutations in viral thymidine kinase (TK) and/or DNA polymerase (pol) genes. Replication kinetics and virulence of TK and/or DNA pol clinical mutants were assessed using models of mouse encephalitis and cotton rat genital infection. Replication capacities in Vero cells of a DNA pol altered strain (L850I) and a TK/DNA pol mutant (C467deletion/A912V) were significantly lower than those of unrelated wild-type (WT) strains, while a double DNA pol mutant (S724N/P920S) demonstrated replication kinetics similar to the WT. The replication of a TK-deficient mutant (G439.5addition) was impaired (low m.o.i.) or unaltered (high m.o.i.) compared to that of a WT virus depending on the viral inoculum. Compared to a survival rate of 6%for mice infected intranasally with WTHSV-1 or -2 viruses, G439.5add, C467deletion/A912V and L850I strains were associated with survival rates of 100% (P<0.05) whereas mice infected with the S724N/P920S mutant had a survival rate of 33% (P=0.08). Brain viral titers were higher in mice infected with WTHSV-1 or -2 strains and the double DNA pol mutant. All strains except the DNA pol mutant L850I were able to establish latency in the dorsal root ganglia of cotton rats. A good correlation was generally found between replication kinetics of DNA pol mutants and their neurovirulence potential in mice whereas such correlation was not straightforward for TK mutants. © 2010 Wiley-Liss, Inc.


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.


Yim K.,Sigmovir Biosystems, Inc. | Yim K.,Virion Systems, Inc. | Miles B.,Virion Systems, Inc. | Zinsou R.,Virion Systems, Inc. | And 3 more authors.
Vaccine | Year: 2012

Annually adjusted inactivated influenza vaccines can prevent infection and limit the spread of seasonal influenza when vaccine strain closely matches circulating strain. For the years when the match is difficult to achieve, a rapid screening of a larger repertoire of vaccines may be required but is difficult to accomplish due to the lack of a convenient small animal model of seasonal influenza vaccines. The goal of this work was to determine whether the cotton rat Sigmodon hispidus, a small laboratory animal susceptible to infection with unadapted influenza viruses, may become such a model. Cotton rats were immunized with a trivalent inactivated vaccine (TIV) FluLaval (2006/2007) and vaccine immunogenicity and antiviral efficacy was evaluated against the homologous H1N1 and a heterologous H3N2 challenge. FluLaval induced a strong virus-specific IgG and neutralizing antibody response against homologous virus, elicited sterilizing immunity in the lungs and significantly reduced viral replication in the nose of infected animals. FluLaval was efficacious in cotton rats as either a single-time or a double immunization, although higher level of protection of the upper respiratory tract was achieved following two doses of vaccine. Antibodies against a heterologous influenza strain were induced in FluLaval-vaccinated animals, but vaccine lacked antiviral efficacy and did not reduce replication of a heterologous virus. Similarity of these findings to human TIV data suggests that the cotton rat may prove to be a reliable small animal model of human influenza vaccines. © 2011 Elsevier Ltd.


Boukhvalova M.S.,Virion Systems, Inc. | Prince G.A.,Virion Systems, Inc. | Blanco J.C.,Virion Systems, Inc.
Virology Journal | Year: 2010

Background. Infectivity of retroviruses such as HIV-1 and MuLV can be abrogated by compounds targeting zinc finger motif in viral nucleocapsid protein (NC), involved in controlling the processivity of reverse transcription and virus infectivity. Although a member of a different viral family (Pneumoviridae), respiratory syncytial virus (RSV) contains a zinc finger protein M2-1 also involved in control of viral polymerase processivity. Given the functional similarity between the two proteins, it was possible that zinc finger-reactive compounds inactivating retroviruses would have a similar effect against RSV by targeting RSV M2-1 protein. Moreover, inactivation of RSV through modification of an internal protein could yield a safer whole virus vaccine than that produced by RSV inactivation with formalin which modifies surface proteins. Results. Three compounds were evaluated for their ability to reduce RSV infectivity: 2,2'-dithiodipyridine (AT-2), tetraethylthiuram disulfide and tetramethylthiuram disulfide. All three were capable of inactivating RSV, with AT-2 being the most potent. The mechanism of action of AT-2 was analyzed and it was found that AT-2 treatment indeed results in the modification of RSV M2-1. Altered intramolecular disulfide bond formation in M2-1 protein of AT-2-treated RSV virions might have been responsible for abrogation of RSV infectivity. AT-2-inactivated RSV was found to be moderately immunogenic in the cotton rats S.hispidus and did not cause a vaccine-enhancement seen in animals vaccinated with formalin-inactivated RSV. Increasing immunogenicity of AT-2-inactivated RSV by adjuvant (Ribi), however, led to vaccine-enhanced disease. Conclusions. This work presents evidence that compounds that inactivate retroviruses by targeting the zinc finger motif in their nucleocapsid proteins are also effective against RSV. AT-2-inactivated RSV vaccine is not strongly immunogenic in the absence of adjuvants. In the adjuvanted form, however, vaccine induces immunopathologic response. The mere preservation of surface antigens of RSV, therefore may not be sufficient to produce a highly-efficacious inactivated virus vaccine that does not lead to an atypical disease. © 2010 Boukhvalova et al; licensee BioMed Central Ltd.


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.


Boukhvalova M.S.,Virion Systems, Inc. | Yim K.C.,Virion Systems, Inc. | Prince G.A.,Virion Systems, Inc. | Blanco J.C.G.,Virion Systems, Inc.
Current Protocols in Cell Biology | 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. Curr. Protoc. Cell Biol. 46:26.6.1-26.6.19. © 2010 by John Wiley & Sons, Inc.

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