Validation of a pan-orthopox real-time PCR assay for the detection and quantification of viral genomes from nonhuman primate blood.

BACKGROUND: In 1980, smallpox disease was eradicated from nature and Variola virus, the etiological agent of smallpox, was confined to two laboratories, one located in Russia (Moscow) later moved to VECTOR (Novosibirsk, Siberia) and one in the United States (CDC Atlanta). Vaccinations among the general public ceased shortly after the successful eradication campaign, resulting in an increasingly immunologically susceptible population. Because of the possibility of intentional reintroduction of Variola virus and the emergence of other pathogenic poxviruses, there is a great need for the development of medical countermeasures to treat poxvirus disease. It is highly likely that the U.S. FDA "animal rule" will be necessary for regulatory approval of these interventions. Therefore, relevant animal models and the associated supporting assays will require development to stand up to regulatory scrutiny. METHODS: An optimized real time PCR assay for the detection of orthopoxviruses has been developed by researchers at the United States Army Research Institute of Infectious Diseases (USAMRIID). To support animal studies that will be used to support approval of medical countermeasures by the U.S. FDA, the assay was designed to quantitate poxvirus genomic DNA in a nonhuman primate (cynomolgus macaque) blood matrix as a measurement of viremia. This manuscript describes the validation of the process, including DNA extraction from whole blood anticoagulated with EDTA, for obtaining and quantitating monkeypox genomes by evaluating precision, accuracy, the standard curve, specificity, robustness and stability of the assay and/or components of the assay. RESULTS: The assay had a lower limit of quantitation of 50 genome copies/5 uL sample, upper limit of quantitation of 5 × 107 GC/5uL sample and a limit of detection of 2.5 genome copies /5uL sample. The assay was specific for orthopoxvirus. Matrix effects were detected and suggest the presence of PCR inhibitor(s) that was co-extracted with the target DNA. CONCLUSIONS: The assay has been validated for the purpose of quantitating monkeypox viral load in blood from cynomolgus macaques. This assay has and will continue to support submissions to the FDA for approval of antiviral therapeutics for smallpox.

Postneoliberal Public Health Care Reforms: Neoliberalism, Social Medicine, and Persistent Health Inequalities in Latin America.

Several Latin American countries are implementing a suite of so-called "postneoliberal" social and political economic policies to counter neoliberal models that emerged in the 1980s. This article considers the influence of postneoliberalism on public health discourses, policies, institutions, and practices in Bolivia, Ecuador, and Venezuela. Social medicine and neoliberal public health models are antecedents of postneoliberal public health care models. Postneoliberal public health governance models neither fully incorporate social medicine nor completely reject neoliberal models. Postneoliberal reforms may provide an alternative means of reducing health inequalities and improving population health.

Is there a space for place in family history assessment? Underserved community views on the impact of neighborhood factors on health and prevention.

Family health history tools rarely incorporate environmental and neighborhood factors, although the social and physical environments in which people live are recognized as major contributors to chronic diseases. This paper discusses beliefs about neighborhood influences on chronic disease risk among racially and ethnically diverse individuals in low-income communities in Cleveland, Ohio. We report findings from a qualitative study consisting of 121 interviews with White, African American, and Hispanic participants. Results are organized into four major themes: (1) social and economic environment, (2) physical environment, (3) barriers to healthy behaviors, and (4) participants' views on integrating genetic and non-genetic determinants of health to understand and address disease prevention and management. Findings suggest that integrating environmental factors into family health history assessments would better reflect lay perceptions of disease causation. Results have implications for improving patient-clinician communication and the development of strategies to prevent and manage chronic diseases.

Gene-environment interactions and health inequalities: views of underserved communities.

This article examines the beliefs and experiences of individuals living in underserved ethnically diverse communities in Cleveland, Ohio, regarding the influence of genetic, social, and environmental factors on health and health inequalities. Using a community-engaged methodological approach, 13 focus groups were conducted with African American, Hispanic, and White individuals residing in the Cleveland area to explore attitudes and beliefs about genetics, genetic research, and health disparities and inequalities. Results of this study highlight the range of meanings that individuals attach to genetic variation, genomic research, and gene-environment interactions, and their implications for addressing health inequalities. The majority of participants in all focus groups reported that social and environmental factors were more important than genetics in contributing to health inequalities. Most participants were unfamiliar with genetic research. These data have implications for how genetic information and research might be applied in conjunction with addressing social determinants of health to improve prevention strategies in underserved communities and ultimately reduce health inequalities.

Differentiation of Variola major and Variola minor variants by MGB-Eclipse probe melt curves and genotyping analysis.

Smallpox, caused by the Variola major virus, is considered to be one of the most lethal of all potential biological weapons and has far-reaching consequences. Real-time polymerase chain reaction (PCR) assays are available as a reliable diagnostic tool to detect members of the genus Orthopoxvirus. In addition real-time PCR assays specific for the variola virus have been developed that distinguish it from other orthopoxviruses. However, a positive identification of variola spp. does not classify the virus as the one that causes smallpox (V. major) or as the variant (Variola minor) that causes a much less severe form of the disease. This study reports the development of a real-time PCR minor groove binder (MGB)-Eclipse probe assay utilizing a sequence within the variola B9R/B10R gene complex that reliably differentiates V. major from V. minor by specific probe melting temperatures (T(m)s) and genotyping analysis. The MGB-Eclipse probe assay is an important step beyond the standard TaqMan-MGB assay and we feel this is a significant addition to our current variola species identification algorithm with TaqMan-MGB assays that target the B9R and B10R genes. The probe T(m)s for V. major and V. minor were 62.71 (+/-0.05) and 53.97 (+/-0.44) degrees C, respectively (P=<0.001). We also used the identical sequence to develop a TaqMan((R))-MGB assay that specifically detected V. minor but not V. major variants by qualitative analysis.

Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox.

The potential use of smallpox as a biological weapon has led to the production and stockpiling of smallpox vaccine and the immunization of some healthcare workers. Another public health goal is the licensing of a safer vaccine that could benefit the millions of people advised not to take the current one because they or their contacts have increased susceptibility to severe vaccine side effects. As vaccines can no longer be tested for their ability to prevent smallpox, licensing will necessarily include comparative immunogenicity and protection studies in non-human primates. Here we compare the highly attenuated modified vaccinia virus Ankara (MVA) with the licensed Dryvax vaccine in a monkey model. After two doses of MVA or one dose of MVA followed by Dryvax, antibody binding and neutralizing titres and T-cell responses were equivalent or higher than those induced by Dryvax alone. After challenge with monkeypox virus, unimmunized animals developed more than 500 pustular skin lesions and became gravely ill or died, whereas vaccinated animals were healthy and asymptomatic, except for a small number of transient skin lesions in animals immunized only with MVA.

Beta 2 microglobulin knockout mice are resistant to lethal intraabdominal sepsis.

beta 2 microglobulin knockout (beta2M-/-) mice lack CD8+ T and natural killer T cells. We hypothesized that beta 2M-/- mice are resistant to lethal intraabdominal sepsis. To test this hypothesis, mortality, cytokine production, and physiologic function were assessed in beta 2M-/- mice during sepsis caused by cecal ligation and puncture (CLP). beta 2M-/- mice survived significantly longer than wild-type mice after CLP but ultimately exhibited 100% mortality. Treatment of beta 2M-/- mice with anti-asialoGM1 to deplete natural killer cells conferred greater than 70% long-term survival. Compared with wild-type mice, beta 2M-/- mice treated with anti-asialoGM1 produced decreased amounts of proinflammatory cytokines and did not exhibit hypothermia or metabolic acidosis after CLP. Adoptive transfer of CD8+ T and natural killer cells into beta 2M-/- mice treated with anti-asialoGM1 re-established CLP-induced mortality. CD8 knockout mice treated with anti-asialoGM1, which are specifically deficient in CD8+ T and natural killer cells, exhibited 40% long-term survival after CLP. Furthermore, treatment of wild-type mice with antibodies to CD8 and asialoGM1 conferred a significant survival benefit compared with wild-type mice treated with nonspecific IgG. These findings demonstrate that beta 2M-/- mice treated with anti-asialoGM1 are resistant to CLP-induced mortality and that depletion of CD8+ T and natural killer cells largely accounts for the survival benefit observed in these mice.

Genetic alterations in serrated adenomas: comparison to conventional adenomas and hyperplastic polyps.

Serrated adenoma is a recently described entity characterized by the presence of a hyperplastic (serrated) growth pattern combined with cytologic features of dysplasia. In contrast to conventional (nonserrated) adenomas, the molecular features of serrated adenomas have been poorly studied. Thus, it remains unclear if serrated adenomas are simply a morphologic variant of conventional adenomas or represent a different biologic entity. In this study, 46 serrated adenomas from 39 patients, 32 conventional (nonserrated) adenomas from 31 patients, and 18 hyperplastic polyps from 16 patients were evaluated for loss of heterozygosity (LOH) of APC, p53, p16, and 3p and for K-ras mutations of codons 12, 13, and 61 by polymerase chain reaction (PCR) analysis. Serrated adenomas demonstrated LOH of at least one genetic locus in 32.6% of cases. LOH of the APC gene, 3p, p53, and p16 was seen in 19.4%, 14.2%, 9.3%, and 13.8% of cases, respectively. K-ras mutations were observed in 18% of cases. Similar to serrated adenomas, conventional adenomas demonstrated at least one LOH event in 37.5% of cases and K-ras mutations in another 19% of cases. LOH of APC, 3p, p53, and p16 was observed in 22%, 33%, 5.8%, and 13.4% of cases, respectively. There were no significant differences in either the total number of genetic events or the presence of LOH of any of the individual markers between serrated adenomas and conventional adenomas. However, hyperplastic polyps showed LOH in 22% of cases and a single K-ras mutation (11%). The prevalence of LOH in hyperplastic polyps was lower than both serrated adenomas and conventional adenomas (P < .05). These results support the hypothesis that serrated adenomas represent a biologically similar morphologic variant of conventional adenomas.

Genetic alterations of Carney complex are not present in sporadic cardiac myxomas.

Cardiac myxomas are the most frequent cardiac tumors and cause for significant morbidity and mortality. Recent evidence indicates that cardiac myxomas are, in fact, neoplasms rather than organized thrombi. Cardiac myxomas may present as solitary lesions or in association with the Carney complex. Carney complex has been linked to chromosome 2p16 and the PRKAR1A gene at 17q22-24. In this study, we analyzed sporadic cardiac myxomas to evaluate whether the genetic alterations seen in Carney complex are present in non Carney complex associated cardiac myxomas as well. We analyzed microdissected material from 13 patients with cardiac myxomas for the markers PRKAR1 9CA, D2S2153, D2S2251 and D2S123. None of the cases demonstrated loss of heterozygosity or definite band changes suggestive of microsatellite instability for any of the markers used. We conclude that sporadic cardiac myxomas are genetically not related to Carney complex and most likely do not represent an incomplete form of Carney complex.