Mice lacking alpha/beta and gamma interferon receptors are susceptible to junin virus infection.

Junin virus (JUNV) causes a highly lethal human disease, Argentine hemorrhagic fever. Previous work has demonstrated the requirement for human transferrin receptor 1 for virus entry, and the absence of the receptor was proposed to be a major cause for the resistance of laboratory mice to JUNV infection. In this study, we present for the first time in vivo evidence that the disruption of interferon signaling is sufficient to generate a disease-susceptible mouse model for JUNV infection. After peripheral inoculation with virulent JUNV, adult mice lacking alpha/beta and gamma interferon receptors developed disseminated infection and severe disease.

Human rabies and rabies in vampire and nonvampire bat species, Southeastern Peru, 2007.

After a human rabies outbreak in southeastern Peru, we collected bats to estimate the prevalence of rabies in various species. Among 165 bats from 6 genera and 10 species, 10.3% were antibody positive; antibody prevalence was similar in vampire and nonvampire bats. Thus, nonvampire bats may also be a source for human rabies in Peru.

Hantavirus pulmonary syndrome in Central Bolivia: relationships between reservoir hosts, habitats, and viral genotypes.

In August 2002, two cases of hantavirus pulmonary syndrome (HPS) were confirmed in Mineros and Concepcion, within the Santa Cruz Department of Bolivia. Extensive alteration of the native ecosystem, from dense forest to pasture or sugarcane, had occurred in both regions. An ecologic assessment of reservoir species associated with the human disease identified a single hantavirus antibody-positive Oligoryzomys microtis from Mineros and three hantavirus antibody-positive Calomys callosus from Concepcion. In Mineros, the virus from the O. microtis was 90% similar to sequences published for Rio Mamore virus. Viral nucleotide sequences from two C. callosus were 87-88% similar to the sequence of Laguna Negra virus. The viral sequence from the C. callosus was 99% identical to viral sequences obtained from the HPS patient in this area, implicating C. callosus as the host and Laguna Negra virus as the agent responsible for the HPS case near Concepcion.

In vivo imaging systems (IVIS) detection of a neuro-invasive encephalitic virus.

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

Hantavirus pulmonary syndrome in Santa Cruz, Bolivia: outbreak investigation and antibody prevalence study.

We report the results of an investigation of a small outbreak of hantavirus pulmonary syndrome in 2002 in the Department of Santa Cruz, Bolivia, where the disease had not previously been reported. Two cases were initially reported. The first case was a physician infected with Laguna Negra virus during a weekend visit to his ranch. Four other persons living on the ranch were IgM antibody-positive, two of whom were symptomatic for mild hantavirus pulmonary syndrome. The second case was a migrant sugarcane worker. Although no sample remained to determine the specific infecting hantavirus, a virus 90% homologous with Río Mamoré virus was previously found in small-eared pygmy rice rats (Oligoryzomys microtis) trapped in the area. An antibody prevalence study conducted in the region as part of the outbreak investigation showed 45 (9.1%) of 494 persons to be IgG positive, illustrating that hantavirus infection is common in Santa Cruz Department. Precipitation in the months preceding the outbreak was particularly heavy in comparison to other years, suggesting a possible climatic or ecological influence on rodent populations and risk of hantavirus transmission to humans. Hantavirus infection appears to be common in the Santa Cruz Department, but more comprehensive surveillance and field studies are needed to fully understand the epidemiology and risk to humans.

Rapid, non-invasive imaging of alphaviral brain infection: reducing animal numbers and morbidity to identify efficacy of potential vaccines and antivirals.

Rapid and accurate identification of disease progression are key factors in testing novel vaccines and antivirals against encephalitic alphaviruses. Typical efficacy studies utilize a large number of animals and severe morbidity or mortality as an endpoint. New technologies provide a means to reduce and refine the animal use as proposed in Hume's 3Rs (replacement, reduction, refinement) described by Russel and Burch. In vivo imaging systems (IVIS) and bioluminescent enzyme technologies accomplish the reduction of animal requirements while shortening the experimental time and improving the accuracy in localizing active virus replication. In the case of murine models of viral encephalitis in which central nervous system (CNS) viral invasion occurs rapidly but the disease development is relatively slow, we visualized the initial brain infection and enhance the data collection process required for efficacy studies on antivirals or vaccines that are aimed at preventing brain infection. Accordingly, we infected mice through intranasal inoculation with the genetically modified pathogen, Venezuelan equine encephalitis, which expresses a luciferase gene. In this study, we were able to identify the invasion of the CNS at least 3 days before any clinical signs of disease, allowing for reduction of animal morbidity providing a humane means of disease and vaccine research while obtaining scientific data accurately and more rapidly. Based on our data from the imaging model, we confirmed the usefulness of this technology in preclinical research by demonstrating the efficacy of Ampligen, a TLR-3 agonist, in preventing CNS invasion.

TC83 replicon vectored vaccine provides protection against Junin virus in guinea pigs.

Junin virus (JUNV) is the etiological agent of the potentially lethal, reemerging human disease, Argentine hemorrhagic fever (AHF). The mechanism of the disease development is not well understood and no antiviral therapy is available. Candid 1, a live-attenuated vaccine, has been developed by the US Army and is being used in the endemic area to prevent AHF. This vaccine is only approved for use in Argentina. In this study we have used the alphavirus-based approach to engineer a replicon system based on a human (United States Food and Drug Administration Investigational New Drug status) vaccine TC83 that express heterologous viral antigens, such as glycoproteins (GPC) of Junin virus (JUNV). Preclinical studies testing the immunogenicity and efficacy of TC83/GPC were performed in guinea pigs. A single dose of the live-attenuated alphavirus based vaccine expressing only GPC was immunogenic and provided partial protection, while a double dose of the same vaccine provided a complete protection against JUNV. This is the first scientific report to our knowledge that the immune response against GPC alone is sufficient to prevent lethal disease against JUNV in an animal model.

Susceptibility of the Aotus nancymaae owl monkey to eastern equine encephalitis.

Eastern equine encephalitis virus (EEEV) is an arthropod-borne virus associated with life-threatening encephalitis in humans, equines, birds and many other domestic animals. To investigate the suitability of the Aotus nancymaae New World owl monkey as a viable animal model for EEE candidate vaccine testing we used clinical presentation, serology, viral isolation and PCR to evaluate pathogenesis and immunity in infected animals. Monkeys were inoculated subcutaneously (SQ) or intranasally (IN) with 10(4)pfu of virulent EEEV and were initially followed for 45 days. While none of the animals displayed clinical signs of disease, all of the SC inoculated animals (n=6) manifested a viremia averaging 3.2 days (+/-0.8 days). Likewise, serologic responses (IgM, IgG and PRNT) were observed in all SC infected animals. Interestingly, none of the IN inoculated animals (n=6) became viremic or mounted an antibody response and no pathological abnormalities were observed in two animals that were necropsied on day 6 post-infection (p.i.) from each group. To determine if the antibodies produced by the SC inoculated animals were protective against homologous challenge, three animals from the SC group were serologically evaluated on day 253 p.i. and were administered an inoculum identical to initial challenge on day 270 p.i. A positive control group of four naïve animals was also infected as before. All of the naïve positive control animals manifested a similar viremia as observed initially, averaging 2.75 days (+/-0.5 days) while none of the previously challenged animals became viremic. On days 45 and 253 p.i. geometric mean PRNT titers in the SC group were 453 and 101, respectively. This study demonstrates that the Aotus nancymaae can be reproducibly infected with EEE virus and can serve as a suitable model for infection and immunogenicity for the evaluation of candidate vaccines against EEEV.

CD4+ T cells provide protection against acute lethal encephalitis caused by Venezuelan equine encephalitis virus.

Studying the mechanisms of host survival resulting from viral encephalitis is critical to the development of vaccines. Here we have shown in several independent studies that high dose treatment with neutralizing antibody prior to intranasal infection with Venezuelan equine encephalitis virus had an antiviral effect in the visceral organs and prolonged survival time of infected mice, even in the absence of alphabeta T cells. Nevertheless, antibody treatment did not prevent the development of lethal encephalitis. On the contrary, the adoptive transfer of primed CD4(+) T cells was necessary to prevent lethal encephalitis in mice lacking alphabeta T cell receptor.

Evaluation of immunity and protective efficacy of a dengue-3 pre-membrane and envelope DNA vaccine in Aotus nancymae monkeys.

A dengue (DEN) virus type 3 DNA vaccine expressing pre-membrane and envelope genes was tested for immunogenicity and protective efficacy in Aotus monkeys. Five of six vaccinated animals demonstrated moderate DEN-specific antibody responses as measured by ELISA and virus neutralization in vitro. By contrast, none of the six control animals developed detectable anti-DEN antibodies. When five vaccinated animals were challenged with live DEN-3 virus and viremia determined by PCR amplification of viral RNA in serum samples, one animal was completely protected and two were partially protected as indicated by a decrease in mean days of viremia. The results demonstrate the ability of the DEN-3 DNA vaccine to elicit a neutralizing antibody response and to partially protect against live virus challenge. These findings support the inclusion of this construct in a tetravalent DNA vaccine.

The New World primate, Aotus nancymae, as a model for examining the immunogenicity of a prototype enterotoxigenic Escherichia coli subunit vaccine.

The colonization factors (CF) of enterotoxigenic Escherichia coli (ETEC) are being targeted for inclusion in a multi-subunit ETEC vaccine. This study was designed to examine the preclinical safety and immunogenicity of CF CS6, encapsulated in a biodegradable poly(DL-lactide-co-glycolide) (meCS6), and administered in the presence or absence of a mutated heat-labile enterotoxin, LT(R192G), in the non-human primate, Aotus nancymae. A. nancymae were inoculated intranasally (IN) with meCS6 (200 microg; positive control), or intragastrically (IG) with meCS6 (200 or 1000 microg) with or without 2 microg LT(R192G) in three doses given at 2-week intervals. In a second experiment, A. nancymae were inoculated IG with 950 microg of meCS6 with or without 2 microg LT(R192G) in four doses given every 48 h. Blood was collected to assess anti-CS6 and -LT serum immunoglobulin G (IgG) and IgA responses and safety variables (complete blood count and chemistry). Safety parameters were unchanged from baseline following all vaccinations. In Experiment 1, a dose-related serologic response to CS6 was observed; 78.6 and 57.1% of monkeys given 1000 microg meCS6 (n = 14) had a serum IgG and IgA response, respectively, compared to only 28.6% of monkeys given 200 microg meCS6 (n = 14) with a serum IgG and IgA response. No significant effect on the number of responders or the magnitude of responses was observed with the addition of LT(R192G). The three-dose, 2-week regimen with 1000 microg meCS6 was more effective at eliciting an immune response than the four-dose, 48-h regimen with 950 microg meCS6. Results from this study indicate that A. nancymae provide a useful ETEC preclinical safety and immunogenicity model.

New World monkey Aotus nancymae as a model for Campylobacter jejuni infection and immunity.

Three groups of six monkeys (Aotus nancymae) each were inoculated intragastrically with increasing doses of Campylobacter jejuni. Infection resulted in fecal colonization (100% of monkeys), dose-related diarrhea, and robust immune responses. Colonization duration and diarrhea rate were reduced upon secondary challenge. A. nancymae may be useful for studying anti-Campylobacter vaccine efficacy.

Saturación sensorial y lactancia materna como métodos analgésicos no farmacológicos: estudio randomizado controlado / Sensory overload and breastfeeding as analgesic method no pharmacologic: control randomized study

OBJETIVO: Verificar el efecto analgésico de la saturación sensorial (Sat) y compararlo con la lactancia materna (LM) y un grupo control. MATERIAL Y MÉTODOS: Ensayo clínico, randomizado con 167 recién nacidos a término sanos, en quiénes se cuantificó la intensidad de dolor agudo al recibir una vacuna (hepatitis B) a las 48 horas de vida. Se formaron tres grupos de manera aleatoria: grupo 1 (control, sin método analgésico), grupo 2 (analgesia con LM) y grupo 3 (analgesia con Sat); los estímulos sensoriales fueron: táctil (caricias de la madre sobre el rostro y espalda), olfatorio (colonia sin alcohol), auditivo (voz de la madre), gustativo (lactancia materna), visual (rostro de la madre frente a su bebé). Se utilizó la Escala para Dolor Agudo Neonatal (DAN: Douleur Aiguë du Nouveau-né), con un score de dolor del 1 al 10 y 7 categorías de dolor. RESULTADOS: El grupo con saturación sensorial tuvo menos dolor (3.02 en la escala de DAN); el grupo dos (lactancia materna): 4.15, y control: 9.02. Hubo diferencia significativa entre grupos. Dolor extremo, se presentó en 60% del grupo control, 3,8% en el grupo lactancia materna y 0% en el grupo con saturación sensorial. La categoría NO DOLOR fue más frecuente en el grupo tres (Sat): 33,3% versus 16,9% del grupo con LM y 0% grupo control, p<0.0001. CONCLUSIONES: La saturación sensorial tuvo mayor efecto analgésico que la lactancia materna. Ambas demostraron buen efecto analgésico, comparadas con el grupo control.

OBJECTIVES: To verify the analgesic effect of sensory overload (SO) and compared with breastfeeding (BF) and a control group. MATERIAL AND METHODS: We conducted a randomized clinical trial in 167 healthy term infants, in which the intensity acute pain was quantified to receive hepatitis B vaccine within 48 hours of life. Three groups were formed at random: one group (control, no method of analgesia), group two (analgesia with BF) and group three (analgesia with SO), sensory stimuli were: touch (petting of the mother on baby face and back), olfactory (colony nonalcoholic), auditory (motherÆs voice), gustatory (breastfeeding), visual (mother and baby - face to face). We used the Neonatal Acute Pain Scale (DAN: Douleur Aigue du Nouveau-né), which gives a pain score of 1 to 10, giving 7 categories of pain. RESULTS: In group three (sensory overload) expressed less pain with an average score of 3.02 on the scale of DAN, the group two (breastfeeding): 4.15, and one group (control): 9.02, with significant difference between groups. The category of extreme pain occurred in 60% of the control group, 3.8% of the breastfed group and there was not extreme pain in the sensory overload group. NO PAIN category was more frequent in all three group (Sat): 33.3% versus 16.9% (group LM) and 0% (control group), p <0.0001. CONCLUSIONS: The sensory overload has greater analgesic effect of breastfeeding. Both demonstrate good analgesic effect compared with the control group.

Aplicabilidad de la hoja de mantenimiento de salud en medicina familiar / Applicability of the leaf maintenance of health in family medicine

Para determinar la aplicabilidad (elaboración y seguimiento) de la Hoja de Mantenimiento y Salud (HMS) se revisó una muestra de 481 historias clínicas de un centro ambulatorio de Medicina Familiar, y se encuestó el 50 por ciento de los médicos familiares en ejercicio en Maracaibo (Venezuela). Las historias correspondían a pacientes inscritos 4 ó más años antes de la revisión. El 52 por ciento tenía elaborada la HMS (56,8 por ciento en el sexo femenino, 80,5 por ciento en mayores de 40 años, y 97,6 por ciento en casos de hipertensión arterial). En el 63 por ciento de las historias la razón entre el número de consultas y el seguimiento de la HMS fue superior a 6. El 90 por ciento de las historias con HMS elaborado no tenían actualizado ninguno de los parámetros programados en ella. Estos resultados, aceptables respecto a la elaboración del HMS, son deficientes en cuanto a la utilización a travéz del tiempo. Los médicos encuestados informaron haber sido entrenados en el uso del instrumento, tenían una opinión favorable acerca de su utilidad, y mencionaron dificultades en su aplicación, principalmente relacionados con la falta de tiempo y la complejidad de su uso. Se propone una revisión del instrumento para mejorar su aplicabilidad