Vector competence experiments with Rocio virus and three mosquito species from the epidemic zone in Brazil

First-generation progeny of field-collected Psorophora ferox, Aedes scapularis, and Aedes serratus from the Rocio encephalitis epidemic zone in S.Paulo State, Brazil, were tested for vector competency in the laboratory. Psorophora ferox and Ae. scapularis are susceptible to per os infection with Rocio virus and can transmit the virus by bite following a suitable incubation period. Oral ID50S for the two species (10(4.1) and 10(4.3) Vero cell plaque forming units, respectively) did not differ significantly. Infection rates in Ae. serratus never exceeded 36%, and, consequently, an ID50 could not be calculated for this species. It is unlikely that Ae. serratus is an epidemiologically important vector of Rocio virus. The utility of an in vitro feeding technique for demonstrating virus transmission by infected mosquitoes and difficulties encountered in working with uncolonized progeny of field-collected mosquitoes are discussed.

A Saint Louis encephalitis and Rocio virus serosurvey in Brazilian horses

Introduction Arboviruses are an important public health problem in Brazil, in especially flaviviruses, including the Saint Louis encephalitis virus (SLEV) and the Rocio virus (ROCV), are especially problematic. These viruses are transmitted to humans or other vertebrates through arthropod bites and may cause diseases with clinical manifestations that range from asymptomatic infection, viral hemorrhagic fever to encephalitis. Methods A serological survey of horses from various regions of Brazil using an enzyme-linked immunosorbent assay (ELISA) with recombinant SLEV domain III peptides and ROCV E protein as antigens. Results Overall, 415 (55.1%) of the 753 horses that were screened were seropositive for flavivirus and, among them, monotypic reactions were observed to SLEV in 93 (12.3%) and to ROCV in 46 (6.1%). These results suggested that these viruses, or other closely related viruses, are infecting horses in Brazil. However, none of the studied horses presented central nervous system infection symptoms. Conclusions Our results suggest that SLEV and ROCV previously circulated among horses in northeast, west-central and southeast Brazil.

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus)

Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus)

ABSTRACT: Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.

A Saint Louis encephalitis and Rocio virus serosurvey in Brazilian horses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

An Experimental Model of Meningoencephalomyelitis by Rocio Flavivirus in Balb/C Mice: Inflammatory Response, Cytokine Production, and Histopathology

Rocio virus (ROCV) is a flavivirus, probably transmitted by Culex mosquitoes and maintained in nature as a zoonosis of wild birds. Rocio virus caused a human epidemic of severe encephalitis that lasted from 1973 to 1980 in the Ribeira valley, in the southeastern coast of Brazil. After this outbreak, serologic evidence of ROCV circulation has been reported and public health authorities are concerned about a return of ROCV outbreaks in Brazil. We show here a study on the pathogenesis and the physiopathology of ROCV disease in the central nervous system of a Balb/C young adult mice experimental model. The animals were intraperitoneally infected by ROCV and followed from 0 to 9 days after infection, when all of them died. Nervous tissue samples were collected from infected animals for immunohistochemistry and molecular biology analysis. We observed the virus in the central nervous system, the inflammatory changes induced by Th1 and Th2 cytokines, and the final irreversible damage of nervous tissues by neuronal degeneration and apoptosis. These findings can help to better understand the pathogenesis and physiopathology of the human meningoencephalomyelitis by ROCV and other flaviviruses.

Molecular characterization of two rocio flavivirus strains isolated during the encephalitis epidemic in são paulo state, brazil and the development of a one-step rt-pcr assay for diagnosis

Rocio virus (ROCV) was responsible for an explosive encephalitis epidemic in the 1970s affecting about 1,000 residents of 20 coastland counties in São Paulo State, Brazil. ROCV was first isolated in 1975 from the cerebellum of a fatal human case of encephalitis. Clinical manifestations of the illness are similar to those described for St. Louis encephalitis. ROCV shows intense antigenic cross-reactivity with Japanese encephalitis complex (JEC) viruses, particularly with Ilheus (ILHV), St. Louis encephalitis, Murray Valley and West Nile viruses. In this study, we report a specific RT-PCR assay for ROCV diagnosis and the molecular characterization of the SPAn37630 and SPH37623 strains. Partial nucleotide sequences of NS5 and E genes determined from both strains were used in phylogenetic analysis. The results indicated that these strains are closely related to JEC viruses, but forming a distinct subclade together with ILHV, in accordance with results recently reported by Medeiros et al. (2007).

Neutralising antibodies for West Nile virus in horses from Brazilian Pantanal

Despite evidence of West Nile virus (WNV) activity in Colombia, Venezuela and Argentina, this virus has not been reported in most South American countries. In February 2009, we commenced an investigation for WNV in mosquitoes, horses and caimans from the Pantanal, Central-West Brazil. The sera of 168 horses and 30 caimans were initially tested using a flaviviruses-specific epitope-blocking enzyme-linked immunosorbent assay (blocking ELISA) for the detection of flavivirus-reactive antibodies. The seropositive samples were further tested using a plaque-reduction neutralisation test (PRNT90) for WNV and its most closely-related flaviviruses that circulate in Brazil to confirm the detection of specific virus-neutralising antibodies. Of the 93 (55.4%) blocking ELISA-seropositive horse serum samples, five (3%) were seropositive for WNV, nine (5.4%) were seropositive for St. Louis encephalitis virus, 18 (10.7%) were seropositive for Ilheus virus, three (1.8%) were seropositive for Cacipacore virus and none were seropositive for Rocio virus using PRNT90, with a criteria of > four-fold antibody titre difference. All caimans were negative for flaviviruses-specific antibodies using the blocking ELISA. No virus genome was detected from caiman blood or mosquito samples. The present study is the first report of confirmed serological evidence of WNV activity in Brazil.

Human disease in ribeira valley, brazil caused by caraparu, a group c arbovirus - Report of a case

The clinical and laboratory data of a disease in a resident of Ribeira Valley, São Paulo State, southeastern Brazil, caused by an agent close or identical to Caraparu, a Group C arbovirus, was described. Although there is evidence of an intensive circulation of several arboviruses in the area, no diagnosis of human disease by these agents has been made, except the encephalitis cases caused by Rocio virus during an epidemic in 1975-1977. An antigenic difference between Caraparu strains isolated in São Paulo and in Pará States and a close antigenic relationship between Caraparu strain from São Paulo and Bruconha virus were suggested by the serological tests.

Cytokine and nitric oxide production by mouse macrophages infected with brazilian flaviviruses

The Flaviviridae family, Flavivirus genus includes viruses that are transmitted to vertebrates by infected mosquitoes or ticks. The genus Flavivirus includes a variety of viruses that cause diseases such as acute febrile illness, encephalitis, and hemorrhagic fever. Flaviviruses primarily infect blood monocytes and tissue macrophages, which have been shown to be permissive, supporting viral replication and serving as virus reservoirs. On the other hand, these cells may have an important antiviral activity related to modulation by cytokine production and by the capacity of these cells to synthesize reactive free radicals such as nitric oxide (NO) which can have a microbicidal effect. The present study was performed in order to determine the production of cytokines interleukin-1beta (IL-1β), tumor necrosis factor -alpha (TNF-α), transforming growth factor- beta (TGF-β) and interferon -alpha (IFN-α) and NO by macrophages infected with one of four Brazilian flaviviruses, Bussuquara virus (BUSV), Yellow Fever virus (YFV), Rocio virus (ROCV) and Encephalitis Saint Louis virus (SLEV), and to verify the possible antiviral effect of NO during macrophage infection with ROCV. Moreover, we asked if the different viruses were able to regulate bacterial lipopolysaccharide (LPS) induced cytokine production. Our results showed that YFV and SLEV reduced the production of IL-1β and TGF-β by LPS-stimulated macrophages, while ROCV only diminished LPS-stimulated TGF-β synthesis. On the other hand, BUSV more likely favored an enhancement of the LPS-induced production of IL-1β by macrophages. Additionally, while most of the viruses stimulated the production of IFN-α, none of them altered the production of TNF-α by murine macrophages. Interestingly, all viruses induced synthesis of NO that was not correlated with antiviral activity for ROCV.

Mosquito (Diptera: Culicidae) Diversity of a Forest-Fragment Mosaic in the Amazon Rain Forest

To study the impact of Amazonian forest fragmentation on the mosquito fauna, an inventory of Culicidae was conducted in the upland forest research areas of the Biological Dynamics of Forest Fragments Project located 60 km north of Manaus, Amazonas, Brazil. The culicid community was sampled monthly between February 2002 and May 2003. CDC light traps, flight interception traps, manual aspiration, and net sweeping were used to capture adult specimens along the edges and within forest fragments of different sizes (1, 10, and 100 ha), in second-growth areas surrounding the fragments and around camps. We collected 5,204 specimens, distributed in 18 genera and 160 species level taxa. A list of mosquito taxa is presented with 145 species found in the survey, including seven new records for Brazil, 16 new records for the state of Amazonas, along with the 15 morphotypes that probably represent undescribed species. No exotic species [Aedes aegypti (L.) and Aedes albopictus (Skuse)] were found within the sampled areas. Several species collected are potential vectors of Plasmodium causing human malaria and of various arboviruses. The epidemiological and ecological implications of mosquito species found are discussed, and the results are compared with other mosquito inventories from the Amazon region.

Diagnóstico molecular de arboviroses brasileiras em soros de pacientes com doença febril aguda

Pós-graduação em Microbiologia - IBILCE

Avaliação da diversidade de insetos hematófagos da subordem Nematocera e de vertebrados silvestres: transmissão de arbovírus na área de influência do Projeto Salobo, Carajás, Pará

Com o objetivo de avaliar a diversidade de insetos hematófagos e de vertebrados silvestres, bem como, a fauna de arbovírus circulante antes das ações de exploração mineral na jazida polimetálica do Salobo, Província Mineral de Carajás, Pará, Brasil, no período de dezembro de 2005 a junho de 2007, um estudo longitudinal foi realizado (sete viagens) sendo capturados e identificados insetos hematófagos (famílias Ceratopogonidae, Culicidae, Psychodidae e Simulidae) capturados em armadilhas luminosas CDC e Shannon, e atração humana; e também foram capturados e identificados vertebrados silvestres das classes das aves (redes de nylon), dos mamíferos e dos répteis (armadilhas Shermann e Tommahwak); foi feita pesquisa e determinação da prevalência de anticorpos nos soros e/ou plasmas desses vertebrados contra arbovírus e tentativas de isolamento viral. Foram capturados 44.795 (1.220 lotes) insetos hematófagos, sendo a família Psychodidae a mais prevalente. As espécies mais abundantes de culicídeos foram Haemagogus leucocelaenus e Haemagogus janthinomys. Foram também capturados 1.288 vertebrados silvestres, e os roedores Proechimys guyannensis e Oryzomys capito, e as aves Turdus albicollis e Phlegopsis nigromaculata foram as espécies mais prevalentes. Foram isoladas em camundongos recém-nascidos, três cepas do Virus Tucunduba, obtidas a partir de lotes de Anopheles (Nys.) species, Culex coronator e Wyeomyia species; foram detectados anticorpos para os seguintes arbovírus: encefalite Saint Louis (VSLE), Ilhéus, encefalite eqüina Oeste, Cacipacoré, Icoaraci, Rocio, Bussuquara e Mucambo, sendo a maior prevalência de anticorpos obtida para o VSLE.

Spatial distribution of arboviral mosquito vectors (Diptera, Culicidae) in Vale do Ribeira in the South-eastern Brazilian Atlantic Forest

Mosquitoes are vectors of arboviruses that can cause encephalitis and hemorrhagic fevers in humans. Aedes serratus (Theobald), Aedes scapularis (Rondani) and Psorophora ferox (Von Humboldt) are potential vectors of arboviruses and are abundant in Vale do Ribeira, located in the Atlantic Forest in the southeast of the State of Sao Paulo, Brazil. The objective of this study was to predict the spatial distribution of these mosquitoes and estimate the risk of human exposure to mosquito bites. Results of the analyses show that humans are highly exposed to bites in the municipalities of Cananeia, Iguape and Ilha Comprida. In these localities the incidence of Rocio encephalitis was 2% in the 1970s. Furthermore, Ae. serratus, a recently implicated vector of yellow fever virus in the State of Rio Grande do Sul, should be a target for the entomological surveillance in the southeastern Atlantic Forest. Considering the continental dimensions of Brazil and the inherent difficulties in sampling its vast area, the habitat suitability method used in the study can be an important tool for predicting the distribution of vectors of pathogens.

Mosquitoes in degraded and preserved areas of the Atlantic Forest and potential for vector-borne disease risk in the municipality of Sao Paulo, Brazil

In order to assess the epidemiological potential of the Culicidae species in remaining areas of the Brazilian Atlantic Forest, specimens of this family were collected in wild and anthropic environments. A total of 9,403 adult mosquitoes was collected from May, 2009 to June, 2010. The most prevalent among species collected in the wild environment were Anopheles (Kerteszia) cruzii, the Melanoconion section of Culex (Melanoconion), and Aedes serratus, while the most common in the anthropic site were Coquillettidia chrysonotum/albifera, Culex (Culex) Coronator group, and An. (Ker.) cruzii. Mosquito richness was similar between environments, although the abundance of individuals from different species varied. When comparing diversity patterns between environments, anthropic sites exhibited higher richness and evenness, suggesting that environmental stress increased the number of favorable niches for culicids, promoting diversity. Increased abundance of opportunistic species in the anthropic environment enhances contact with culicids that transmit vector-borne diseases.