Two Cases of Natural Infection of Dengue-2 Virus in Bats in the Colombian Caribbean.

Dengue, a mosquito-borne zoonotic disease, is the most common vector-borne disease in tropical and subtropical areas. In this study, we aim to demonstrate biological evidence of dengue virus infection in bats. A cross-sectional study was carried out in the departments of Cordoba and Sucre, Colombia. A total of 286 bats were captured following the ethical protocols of animal experimentation. The specimens were identified and euthanized using a pharmacological treatment with atropine, acepromazine and sodium pentobarbital. Duplicate samples of brain, heart, lung, spleen, liver, and kidney were collected with one set stored in Trizol and the other stored in 10% buffered formalin for histopathological and immunohistochemical analysis using polyclonal antibodies. Brain samples from lactating mice with an intracranial inoculation of DENV-2 were used as a positive control. As a negative control, lactating mouse brains without inoculation and bats brains negative for RT-PCR were included. Tissue sections from each specimen of bat without conjugate were used as staining control. In a specimen of Carollia perspicillata captured in Ayapel (Cordoba) and Phylostomus discolor captured in San Carlos (Cordoba), dengue virus was detected, and sequences were matched to DENV serotype 2. In bats RT-PCR positive for dengue, lesions compatible with viral infections, and the presence of antigens in tissues were observed. Molecular findings, pathological lesions, and detection of antigens in tissues could demonstrate viral DENV-2 replication and may correspond to natural infection in bats. Additional studies are needed to elucidate the exact role of these species in dengue epidemics.

Molecular and cellular evidence of natural Venezuelan equine encephalitis virus infection in frugivorous bats in Colombia.

BACKGROUND AND AIM: Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis with a high impact on public health in Latin America. However, only in Guatemala, Trinidad and Tobago, and Mexico have found antibodies in VEEV in bats, using immunohistochemistry, the sensitivity and specificity are improved; thus, it is better for demonstrating natural infection in bats as potential hosts. This study aimed to determine the presence of VEEV in tissues of frugivorous bats. MATERIALS AND METHODS: A prospective descriptive cross-sectional study with a non-probabilistic sampling was carried out in 12 localities of Córdoba and Sucre area of the Colombian Caribbean. Two hundred and eighty-six bats were captured using fog nets, and the specimens according to taxonomic keys were classified. According to the Ethics Committee of the University of Córdoba, the bats were treated with analgesics and anesthetics. Blood samples were taken and then euthanized to obtain tissues and organs which were preserved in liquid N2 at -196°C. A portion of each organ was fixed in 10% buffered formalin for the detection of antigens by immunohistochemistry. Several pathological anatomy analyses were performed to determine the histological characteristics of tissue lesions of frugivorous bats naturally infected with the VEEV. RESULTS: Of the 286 bats captured, 23 species were identified. In samples of the brain, spleen, and lung of two frugivorous bats (2/286=0.70%) Artibeus planirostris and Sturnira lilium, the presence of VEEV was confirmed by immunohistochemistry. CONCLUSION: A fragment of the nsP4 non-structural protein gene corresponding to the alphavirus was amplified. Two samples were positive (2/286=0.70%) in frugivorous bats; A. planirostris (code GenBank: MG820274) and S. lilium (code GenBank: MG820275). The present study showed the first molecular evidence and cellular evidence (histopathology and immunohistochemistry) of natural VEEV infection in frugivorous bats in Colombia; these bats could be a host of this zoonosis.

Serological evidence of arboviruses and coccidia infecting horses in the Amazonian region of Brazil.

BACKGROUND: Arboviruses and protozoans can cause neurologic disorders in horses. In Brazilian Amazon, several horses presenting signs compatible with disorders caused by these infectious agents have been observed. OBJECTIVE: To contribute to the knowledge of this epidemiological picture, we sought to construct a serological diagnostic panel for neurotrophic infectious agents in local horses. MATERIAL AND METHODS: A total of 213 blood samples from horses were collected from 29 farms in three municipalities. Samples were evaluated and considered positive when they met the following criteria: titers ≥ 1:80 with the indirect fluorescent antibody test (IFAT) for apicomplexan protozoans; positive recombinant enzyme-linked immunosorbent assay (ELISA) with subsequent titers ≥ 1:10 by the PRNt for viruses; and detection under direct microscopic examination for Trypanosoma evansi. RESULTS: No horses were found to be infected by T. evansi, and only two were infected Toxoplasma gondii and/or Neospora spp. The highest protozoan infection rate was observed for Sarcocystis neurona (40.3%; n = 86/213). Among the positive ELISA samples tested by the plaque reduction neutralization test (PRNT90), 92% (n = 76/83) were positive for St Louis Encephalitis virus, 43% (n = 6/14) were positive for West Nile virus and 33% (n = 16/48) were positive for Mayaro virus. Eighteen percent (n = 39/213) of horses were co-infected by S. neurona and at least one arbovirus, particularly SLEV and/or MAYV. CONCLUSION: Samples positive for SLEV associated with S. neurona, including samples from horses that had recovered from neurological signs were frequent, and must be considered when investigating the possible causes of neurological diseases in South Roraima horses.

A Novel Hepacivirus in Wild Rodents from South America.

The Hepacivirus genus comprises single-stranded positive-sense RNA viruses within the family Flaviviridae. Several hepaciviruses have been identified in different mammals, including multiple rodent species in Africa, Asia, Europe, and North America. To date, no rodent hepacivirus has been identified in the South American continent. Here, we describe an unknown hepacivirus discovered during a metagenomic screen in Akodon montensis, Calomys tener, Oligoryzomys nigripes, Necromys lasiurus, and Mus musculus from São Paulo State, Brazil. Molecular detection of this novel hepacivirus by RT-PCR showed a frequency of 11.11% (2/18) in Oligoryzomys nigripes. This is the first identification of hepavivirus in sigmondonine rodents and in rodents from South America. In sum, our results expand the host range, viral diversity, and geographical distribution of the Hepacivirus genus.

Immunocompetent mice model for dengue virus infection [corrected].

Dengue fever is a noncontagious infectious disease caused by dengue virus (DENV). DENV belongs to the family Flaviviridae, genus Flavivirus, and is classified into four antigenically distinct serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. The number of nations and people affected has increased steadily and today is considered the most widely spread arbovirus (arthropod-borne viral disease) in the world. The absence of an appropriate animal model for studying the disease has hindered the understanding of dengue pathogenesis. In our study, we have found that immunocompetent C57BL/6 mice infected intraperitoneally with DENV-1 presented some signs of dengue disease such as thrombocytopenia, spleen hemorrhage, liver damage, and increase in production of IFNγ and TNFα cytokines. Moreover, the animals became viremic and the virus was detected in several organs by real-time RT-PCR. Thus, this animal model could be used to study mechanism of dengue virus infection, to test antiviral drugs, as well as to evaluate candidate vaccines.

Domain III peptides from flavivirus envelope protein are useful antigens for serologic diagnosis and targets for immunization.

The Flavivirus genus of the Flaviviridae family includes 70 enveloped single-stranded-RNA positive-sense viruses transmitted by arthropods. Among these viruses, there are a relevant number of human pathogens including the mosquito-borne dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), as well as tick-borne viruses such as tick-borne encephalitis virus (TBEV), Langat virus (LGTV) and Omsk hemorrhagic fever (OHFV). The flavivirus envelope (E) protein is a dominant antigen inducing immunologic responses in infected hosts and eliciting virus-neutralizing antibodies. The domain III (DIII) of E protein contains a panel of important epitopes that are recognized by virus-neutralizing monoclonal antibodies. Peptides of the DIII have been used with promising results as antigens for flavivirus serologic diagnosis and as targets for immunization against these viruses. We review here some important aspects of the molecular structure of the DIII as well as its use as antigens for serologic diagnosis and immunization in animal models.

Role of mixed Th1 and Th2 serum cytokines on pathogenesis and prognosis of hantavirus pulmonary syndrome.

The hantavirus pulmonary syndrome (HPS) is an emerging syndrome in the Americas. The disease results from intense immune activation and changes in vascular permeability. The aim of this study was to determine the profile of serum cytokines in HPS patients looking for correlation with the clinical parameters, severity and outcome of illness. Studying 21 HPS patients, we found that IL-6 may have an important role in the pathogenesis of HPS, being associated with fatal outcome. Our results also support a mixed Th1/Th2 immune response during the course of HPS and that the magnitude of Th1 response effector cytokines is correlated to HPS severity. The decreased levels of TGF-beta observed in HPS patients suggest that immunoregulatory activity could be damaged in these patients.

In vitro study of antiviral activity of mycophenolic acid on Brazilian orthobunyaviruses.

OBJECTIVE: Oropouche, Caraparu, Guama, Guaroa and Tacaiuma are ssRNA viruses that belong to the genus Orthobunyavirus and have been associated with human febrile illnesses and/or encephalitis. In this study, we evaluated the antiviral action of mycophenolic acid (MPA) on theseorthobunyaviruses to achieve a therapeutic agent to treat the diseases caused by these viruses. METHODS: The in vitro antiviral evaluation to MPA was done by using plaque assay at different periods of treatment. RESULTS: Results showed that MPA at a concentration of 10 microg/ml has significant antiviral activity on Tacaiuma virus when treatment was initiated either 24 h before or 2 h after viral infection. Moreover, MPA has an inhibitory effect on Guama virus replication, but only when treatment was initiated before cell infection. Addition of guanosine in the culture reverted the inhibitory effect of MPA on Tacaiuma and Guama viruses, suggesting that the antiviral activity of this substance was via depletion of the intracellular guanosine pool. CONCLUSION: Our results suggest that MPA would not be a good therapeutic agent to treat the diseases caused by Oropouche, Caraparu, Guama, Guaroa, and Tacaiuma viruses.

Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses.

A new approach was developed for the rapid detection and identification of Brazilian alphaviruses and flaviviruses. The methodology involves the genus-specific detection of Alphavirus and Flavivirus by a duplex reverse transcription-PCR (D-RT-PCR), followed by multiplex nested PCR (M-N-PCR) or nested PCR (N-PCR) assays for species-specific identification. By this protocol, 25 arboviruses were specifically detected and identified. Detection levels between 10(1.3) and 10(3.5) 50% tissue culture infective doses (TCID(50))/ml of Flavivirus and Alphavirus strains were achieved by D-RT-PCR, and levels of <1 TCID(50)/ml were achieved by M-N-PCR assays. To assess the suitability and clinical application of this methodology, a total of 101 human or animal stored samples were analyzed. Results obtained suggest that this technique could be applied as a rapid diagnostic tool in clinical samples in which arbovirus infection is suspected and differential diagnosis is required, avoiding the need to test specimens by separate PCR methods.

Encuesta serológica sobre el dengue en Ribeirao Preto, Sao Paulo, Brasil / Dengue serologic survey in Ribeirao Preto, Sao Paulo, Brazil

La ciudad de Riberao Preto sufrió una epidemia de dengue 1 que comenzó en noviembre de 1990 y duró hasta marzo de 1991. Durante septiembre y octubre de 1992 se efectuó una encuesta serológica en dicha ciudad con el fin de detectar anticuerpos IgG contra los cuatro serotipos del dengue y otros flavivirus. En 5,4 por ciento de los participantes en la encuesta se detectaron anticuerpos contra el dengue 1. Los residentes del sector noroeste de Riberao Preto tuvieron una seropositividad considereblemente más alta (9,3 por ciento) que los que vivían en los otros sectores de la ciudad. Se observaron también en el sector noroeste cantidades relativamente elevadas de criaderos del vector Aedes aegypti, el número más alto de casos de dengue notificados y condiciones socieconómicas relativamente bajas. El hecho de que la epidemia se limitara principalmente al sector noroeste quizá fué consecuencia de la intensa lucha antivectorial y las actividades educativas iniciadas en respuesta a la epidemia. En el momento de la encuesta de 1992, la mayor parte de la población de Riberao Preto seguía siendo vulnerable a la infección con dengue 1; además aproximadamente 23 000 personas con anticuerpos contra el dengue 1, estaban expuestas a un riesgo relativamente alto de padecer fiebre hemorrágica/síndrome de choque del dengue si se producía una infección con el dengue 2. Por estas razones se concluyó que sería necesario continuar la educación sobre el dengue y las medidas de lucha antivectórial

Encuesta serológica sobre el dengue en Ribeirao Preto, Sao Paulo, Brasil / Dengue serologic survey in Ribeirao Preto, Sao Paulo, Brazil

La ciudad de Riberao Preto sufrió una epidemia de dengue 1 que comenzó en noviembre de 1990 y duró hasta marzo de 1991. Durante septiembre y octubre de 1992 se efectuó una encuesta serológica en dicha ciudad con el fin de detectar anticuerpos IgG contra los cuatro serotipos del dengue y otros flavivirus. En 5,4 por ciento de los participantes en la encuesta se detectaron anticuerpos contra el dengue 1. Los residentes del sector noroeste de Riberao Preto tuvieron una seropositividad considereblemente más alta (9,3 por ciento) que los que vivían en los otros sectores de la ciudad. Se observaron también en el sector noroeste cantidades relativamente elevadas de criaderos del vector Aedes aegypti, el número más alto de casos de dengue notificados y condiciones socieconómicas relativamente bajas. El hecho de que la epidemia se limitara principalmente al sector noroeste quizá fué consecuencia de la intensa lucha antivectorial y las actividades educativas iniciadas en respuesta a la epidemia. En el momento de la encuesta de 1992, la mayor parte de la población de Riberao Preto seguía siendo vulnerable a la infección con dengue 1; además aproximadamente 23 000 personas con anticuerpos contra el dengue 1, estaban expuestas a un riesgo relativamente alto de padecer fiebre hemorrágica/síndrome de choque del dengue si se producía una infección con el dengue 2. Por estas razones se concluyó que sería necesario continuar la educación sobre el dengue y las medidas de lucha antivectórial

Se publica también en inglés en el Bull. PAHO. Vol. 29(1), 1995. Este estudio fue financiado en parte por la Fundación de Apoyo a la Investigación del Estado de Sao Paulo