Comparison of 4 laboratory tests for the detection of bovine rabies viral infection in Paraguay: fluorescent antibody test, rapid detection test, histologic lesions, and RT-PCR.

Rabies virus (RABV; Lyssavirus rabies) is a neurotropic virus that can be transmitted to mammals by the hematophagous bat Desmodus rotundus. An accurate, accessible method for the detection of RABV in cattle is necessary in Paraguay; thus, we evaluated the detection of RABV using 4 techniques: fluorescent antibody test (FAT), immunochromatography rapid detection test (RDT; Anigen Rapid Rabies Ag test kit; Bionote), a reverse-transcription PCR (RT-PCR) assay, and histologic lesions in different portions of the CNS of 49 Paraguayan cattle to determine the most sensitive and specific technique. By FAT and RDT, 15 of 49 (31%) samples were positive. By RT-PCR amplification of N and G genes, 13 of 49 (27%) and 12 of 49 (25%) were positive, respectively. RDT had high agreement with FAT (kappa = 1); sensitivity was 100% (95% CI: 97-100%) and specificity was 100% (95% CI: 99-100%). The amplification of the N and G genes resulted in substantial agreement (kappa of 0.9 and 0.8, respectively) compared with FAT, and the sensitivity and specificity of the N gene were 87% (95% CI: 66-100%) and 100% (95% CI: 98-100%), respectively, and those of the G gene were 80% (95% CI: 56-100%) and 100% (95% CI: 98-100%), respectively. Histologic lesions observed were lymphoplasmacytic meningoencephalitis, gliosis, and neuronophagia. The agreement observed between the FAT and RDT tests suggests that RDT is an accurate tool for the detection of RABV. Histopathology can be used to confirm lesions caused by RABV and to rule out other conditions; the RT-PCR assay is useful for molecular epidemiology studies.

A recombinant fusion protein containing the domain III of the dengue-2 envelope protein is immunogenic and protective in nonhuman primates.

We have previously reported the construction and evaluation in mice of recombinant fusion proteins formed by a fragment (aa 286-426) of the dengue envelope protein and the P64k protein from Neisseria meningitidis. In this work we describe the immunization of Macaca fascicularis monkeys with two variants of these proteins [PD3 (insertion variant) and PD5 (fusion variant)] corresponding to serotype 2. Four doses of the proteins adjuvated in Freund's adjuvant were administered and the kinetics of antibody induction was monitored by ELISA and neutralization tests. Monkeys receiving PD3 or PD5 developed functional antibodies (Abs) in a dose-dependent manner. Following challenge with 5 log PFU of wild type dengue-2 virus (DEN2), animals immunized with PD5 were protected from developing viremia. These results constitute a proof-of-concept demonstrating that a fragment of the dengue envelope protein, containing the domain III and produced as a recombinant fusion protein in Escherichia coli, induces functional and protective immunity in a nonhuman primate model.

IL-10 levels in Dengue patients: some findings from the exceptional epidemiological conditions in Cuba.

The pathogenesis associated with Dengue haemorrhagic fever, has yet to be fully elucidated, with no definitive in vivo evidence. The exceptional epidemiological circumstances in Cuba allow the evaluation of different mediators in a well-defined situation. In the present study, we describe the determination of levels of IL-12, IL-10 and RANTES in the sera of Cuban patients hospitalised with Dengue fever or Dengue haemorrhagic fever. The results showed that levels of serum IL-10 were higher in patients than controls, and those patients with secondary infections had consistently higher levels. All the Dengue haemorrhagic fever patients had increased levels of IL-10. In contrast, levels of IL-12 did not differ between patients and controls. Finally, RANTES serum levels detected in patients were lower than those observed in the controls. The association of increased levels of IL-10 in Dengue patients with a sequential infection suggests a possible role of this cytokine in the pathogenesis of Dengue disease.

[Detection of lymphoproliferative response in monkeys innoculated with dengue 4 virus]. / Detección de respuesta linfoproliferativa en monos inoculados con virus dengue 4.

A group of 6 Cynomolgus monkeys was immunized with dengue 4 virus (H247 standard strain). 60 days after innoculation the cellular proliferation against the same serotype as well as against hetereologous serotypes 1 and 2 was measured. A proliferative response predominantly of the specific serotype and some cross reactivity were detected. These results showed a behavior of primates similar in some extent to that of human beings and mice regarding the specific serotype and cross serotype response to dengue virus.

Detección de respuesta linfoproliferativa en monos inoculados con virus dengue 4 / Detection of lymphoproliferative response in monkeys innoculated with dengue 4 virus

Se inmunizó con virus dengue 4 (cepa estándar H247), un grupo de 6 monos Cynomolgus y, 60 d después de la inoculación, se midió la proliferación celular frente al mismo serotipo, así como frente a los serotipos heterólogos 1 y 2. Se detectó una respuesta proliferativa predominantemente serotipo específica y alguna reactividad cruzada. Estos resultados evidencian un comportamiento de los primates en alguna medida similar al de humanos y ratones, en la respuesta serotipo específica y serotipo cruzada al virus dengue

Immune response to synthetic peptides of dengue prM protein.

The immunological activities of five synthetic peptides of the prM protein of dengue-2 (DEN-2) virus containing B cell epitopes were evaluated in BALB/c mice. Two peptides elicited neutralizing antibodies against all four DEN serotypes. Virus-specific proliferative responses were demonstrated in mice immunized with four of the five peptides, demonstrating the presence of T cell epitopes. Mice immunized with three of the five peptides conjugated with bovine albumin showed statistically significant levels (P<0.05) of protection when challenged with DEN-2 virus. These results could constitute the basis for the establishment of the role of DEN virus pre and M antigens in the development of anti-flaviviral vaccines.

Dengue in Nicaragua, 1994: reintroduction of serotype 3 in the Americas / Dengue en Nicaragua, 1994: reintroducción del serotipo 3 en las Américas

El objetivo principal de este informe fue describir la reaparición del serotipo 3 del dengue en las Américas después de 17 años de ausencia, tal como se observó recientemente en Nicaragua. Se examinaron en total 356 muestras de suero obtenidas por medio del sistema nicaragüense de vigilancia del dengue durante una epidemia en octubre de 1994. En 43% de las muestras se detectaron anticuerpos IgM contra el dengue y los sueros de 12 de las 18 áreas atendidas por los sistemas locales integrales de salud dieron resultados positivos. Además, se aislaron virus de dengue en 5 de 24 sueros de pacientes con síntomas hemorrágicos: en 3 se aisló el serotipo 3 y en 2, el serotipo 1. Mediante pruebas de laboratorio, en 26 de 39 pacientes hospitalizados en León y Managua se consideró probable o se confirmó el diagnóstico de dengue con manifestaciones hemorrágicas o dengue hemorrágico. En 18 pacientes diagnosticados de dengue con manifestaciones hemorrágicas, los síntomas más comunes fueron fiebre, cefalea, vómito, mialgia, artralgia y epistaxis. Los ocho pacientes restantes, en los que se diagnosticó dengue hemorrágico probable, tuvieron fiebre, malestar general, hemorragias, trombocitopenia y hemoconcentración, y los títulos de anticuerpos a la prueba de inhibición de la hemaglutinación oscilaron de 640 a 20 480. Se confirmó así la reintroducción del serotipo 3 del dengue en la Región y su capacidad para producir casos de dengue hemorrágico. En Nicaragua, por lo menos, es evidente que la reintroducción del serotipo 3 del dengue ha producido un aumento de los casos de dengue clásico y dengue hemorrágico. Si no se presta atención urgente al control del mosquito vector de la enfermedad, la experiencia descrita podría constituir el preludio lúgubre de futuros acontecimientos similares en las Américas

The principal aim of the report presented here is to describe the reappearance of dengue serotype 3 in the Americas, following a 17-year absence, through the recent experience of Nicaragua. In all, 356 serum samples obtained through Nicaragua's dengue monitoring system in October 1994 during an epidemic were examined. Anti-dengue IgM antibodies were detected in 43% of these, with sera from 12 of the 18 areas covered by Nicaragua's local integrated health care systems yielding positive results. In addition, dengue virus was isolated from 5 of 24 sera obtained from patients with hemorrhagic symptoms, dengue 3 being isolated from 3 of these samples and dengue 1 from the other 2. A diagnosis of dengue with hemorrhagic manifestations or of hemorrhagic dengue was supported or confirmed by laboratory findings obtained from 26 of 39 patients hospitalized in León or Managua. The most frequent symptoms of 18 patients diagnosed as having dengue with hemorrhagic manifestations were fever, headache, vomiting, myalgia, arthralgia, and epistaxis. The remaining eight patients, diagnosed as having probable hemorrhagic dengue, exhibited fever, general malaise, hemorrhaging, thrombocytopenia, hemoconcentration, and hemagglutination-inhibition antibody titers ranging from 640 to 20 480. Overall, the reappearance of dengue serotype 3 in the Region was confirmed, together with its ability to produce cases of hemorrhagic dengue. At least in Nicaragua, it is apparent that the introduction of dengue serotype 3 has prompted an increase in the number of classical dengue and hemorrhagic dengue cases, a scenario that might constitute the grim prelude to future developments in the Americas if urgent attention is not given to controlling the disease's mosquito vector