Oral Susceptibility to Yellow Fever Virus of Aedes aegypti from Brazil

The oral susceptibility to yellow fever virus was evaluated in 23 Aedes aegypti samples from Brazil. Six Ae. aegypti samples from Africa, America and Asia were also tested for comparison. Mosquito samples from Asia showed the highest infection rates. Infection rates for the Brazilian Ae. aegypti reached 48.6%, but were under 13% in 60% of sample tested. We concluded that although the low infection rates estimated for some Brazilian mosquito samples may not favor the establishment of urban cycle of yellow fever in some parts of the country, the founding of Ae. aegypti of noteworthy susceptibility to the virus in cities located in endemic and transition areas of sylvatic yellow fever, do pose a threat of the re-emergence of the urban transmission of the disease in Brazil.

Complete nucleotide sequence analysis of a Brazilian dengue virus type 2 strain

In the last decade, dengue fever (DF) in Brazil has been recognized as an important public health problem, and an increasing number of dengue haemorrhagic fever (DHF) cases have been reported since the introduction of dengue virus type 2 (DEN-2) into the country in 1990. In order to analyze the complete genome sequence of a DEN-2 Brazilian strain (BR64022/98), we designed primers to amplify contiguous segments of approximately 500 base pairs across the entire sequence of the viral genome. Twenty fragments amplified by reverse transcriptase-PCR were cloned, and the complete nucleotide and the deduced amino acid sequences were determined. This constitutes the first complete genetic characterization of a DEN-2 strain from Brazil. All amino acid changes differentiating strains related to the Asian/American-Asian genotype were observed in BR64022/98, indicating the Asiatic origin of the strain.

Brazilian Flavivirus phylogeny based on NS5

In this work, a comprehensive phylogenetic study based on 600 base pair nucleotide and on putative 200 amino acid sequences of NS5 was carried out in order to establish genetic relationships among 15 strains of 10 Brazilian flaviviruses: Bussuquara, Cacipacore, dengue type 1, 2 and 4, Iguape, Ilheus, Rocio, Saint Louis encephalitis (SLE), and yellow fever. Phylogenetic trees were created by neighbor-joining and maximum parsimony methods. These trees showed Brazilian flaviviruses grouped into three main branches: yellow fever branch, dengue branch subdivided in types 1, 2 and 4 branches, and Japanese encephalitis virus (JEV) complex branch including SLE virus strains, Cacipacore, Iguape, Rocio, Ilheus and Bussuquara. Viruses transmitted by Aedes mosquitoes, such as dengue and urban yellow fever, that are also the only Flavivirus causing hemorrhagic fevers in Brazil, were grouped in the same cluster. Encephalitis associated viruses, transmitted by Culex mosquitoes such as JEV complex branch including SLE virus strains, Cacipacore, Iguape, Rocio, Ilheus and Bussuquara were also grouped in the same clade.

Larvicidal Activity of essential oils from Brazilian plants against Aedes aegypti L.

Aedes aegypti L. is the major vector of dengue fever, an endemic disease in Brazil. In an effort to find effective and affordable ways to control this mosquito, the larvicidal activities of essential oils from nine plants widely found in the Northeast of Brazil were analyzed by measurement of their LC50. The essential oils were extracted by steam distillation and their chemical composition determined by GL-chromatography coupled to mass spectroscopy. The essential oils from Cymbopogon citratus and Lippia sidoides, reported in the literature to have larvicidal properties against A. aegypti, were used for activity comparison. The results show that Ocimum americanum and Ocimum gratissimum have LC50 of 67 ppm and 60 ppm respectively, compared to 63 ppm for L. sidoides and 69 ppm for C. citratus. These results suggest a potential utilization of the essential oil of these two Ocimum species for the control of A. aegypti.

HLA-A*01 allele: a risk factor for dengue haemorrhagic fever in Brazil's population

Severe forms of dengue, such as dengue haemorrhagic fever (DHF) and dengue shock syndrome, are examples of a complex pathogenic mechanism in which the virus, environment and host immune response interact. The influence of the host's genetic predisposition to susceptibility or resistance to infectious diseases has been evidenced in several studies. The association of the human leukocyte antigen gene (HLA) class I alleles with DHF susceptibility or resistance has been reported in ethnically and geographically distinct populations. Due to these ethnic and viral strain differences, associations occur in each population, independently with a specific allele, which most likely explains the associations of several alleles with DHF. As the potential role of HLA alleles in the progression of DHF in Brazilian patients remains unknown, we then identified HLA-A alleles in 67 patients with dengue fever and 42 with DHF from Rio de Janeiro, Brazil, selected from 2002-2008 by the sequence-based typing technique. Statistical analysis revealed an association between the HLA-A*01 allele and DHF [odds ratio (OR) = 2.7, p = 0.01], while analysis of the HLA-A*31 allele (OR = 0.5, p = 0.11) suggested a potential protective role in DHF that should be further investigated. This study provides evidence that HLA class I alleles might be important risk factors for DHF in Brazilian patients.

Molecular characterisation of dengue virus type 1 reveals lineage replacement during circulation in Brazilian territory

Dengue fever is the most important arbovirus infection found in tropical regions around the world. Dispersal of the vector and an increase in migratory flow between countries have led to large epidemics and severe clinical outcomes, such as dengue haemorrhagic fever and dengue shock syndrome. This study analysed the genetic variability of the dengue virus serotype 1 (DENV-1) in Brazil with regard to the full-length structural genes C/prM/M/E among 34 strains isolated during epidemics that occurred in the country between 1994-2011. Virus phylogeny and time of divergence were also evaluated with only the E gene of the strains isolated from 1994-2008. An analysis of amino acid differences between these strains and the French Guiana strain (FGA/89) revealed the presence of important nonsynonymous substitutions in the amino acid sequences, including residues E297 (Met→Thr) and E338 (Ser→Leu). A phylogenetic analysis of E proteins comparing the studied isolates and other strains selected from the GenBank database showed that the Brazilian DENV-1 strains since 1982 belonged to genotype V. This analysis also showed that different introductions of strains from the 1990s represented lineage replacement, with the identification of three lineages that cluster all isolates from the Americas. An analysis of the divergence time of DENV-1 indicated that the lineage circulating in Brazil emerged from an ancestral lineage that originated approximately 44.35 years ago.

Immune response to the mumps component of the MMR vaccine in the routine of immunisation services in the Brazilian National Immunisation Program

A non-controlled longitudinal study was conducted to evaluate the combined vaccine against measles, mumps and rubella (MMR) immunogenicity in 150 children vaccinated in the routine of three health units in the city of Rio de Janeiro, Brazil, 2008-2009, without other vaccines administered during the period from 30 days before to 30 days after vaccination. A previous study conducted in Brazil in 2007, in 1,769 children ranging from 12-15 months of age vaccinated against yellow fever and MMR simultaneously or at intervals of 30 days or more between doses, had shown low seroconversion for mumps regardless of the interval between administration of the two vaccines. The current study showed 89.5% (95% confidence interval: 83.3; 94.0) seroconversion rate for mumps. All children seroconverted for measles and rubella. After revaccination, high antibody titres and seroconversion rates were achieved against mumps. The results of this study and others suggest that two MMR doses confer optimal immunoresponses for all three antigens and the possible need for additional doses should be studied taking into account not only serological, but also epidemiological data, as there is no serological correlate of protection for mumps.

Spatial and temporal country-wide survey of temephos resistance in Brazilian populations of Aedes aegypti

The organophosphate temephos has been the main insecticide used against larvae of the dengue and yellow fever mosquito (Aedes aegypti) in Brazil since the mid-1980s. Reports of resistance date back to 1995; however, no systematic reports of widespread temephos resistance have occurred to date. As resistance investigation is paramount for strategic decision-making by health officials, our objective here was to investigate the spatial and temporal spread of temephos resistance in Ae. aegypti in Brazil for the last 12 years using discriminating temephos concentrations and the bioassay protocols of the World Health Organization. The mortality results obtained were subjected to spatial analysis for distance interpolation using semi-variance models to generate maps that depict the spread of temephos resistance in Brazil since 1999. The problem has been expanding. Since 2002-2003, approximately half the country has exhibited mosquito populations resistant to temephos. The frequency of temephos resistance and, likely, control failures, which start when the insecticide mortality level drops below 80%, has increased even further since 2004. Few parts of Brazil are able to achieve the target 80% efficacy threshold by 2010/2011, resulting in a significant risk of control failure by temephos in most of the country. The widespread resistance to temephos in Brazilian Ae. aegypti populations greatly compromise effective mosquito control efforts using this insecticide and indicates the urgent need to identify alternative insecticides aided by the preventive elimination of potential mosquito breeding sites.

The invasive spotted-wing Drosophila (Diptera, Drosophilidae) has been found in the city of São Paulo (Brazil)

The invasive spotted-wing Drosophila (Diptera, Drosophilidae) has been found in the city of São Paulo (Brazil). Drosophila suzukii (Matsumura, 1931), the cherry fly or spotted-wing Drosophila, a pest species from the Oriental and southeastern Palaearctic regions belonging to the melanogaster group, invaded the Nearctic and western countries of the Palaearctic regions late last decade (2008) and, more recently (2013), the southern Brazilian states of Rio Grande do Sul and Santa Catarina. Early in 2014 it was reared from blueberries produced in São Joaquim, state of Santa Catarina, that were bought at a São Paulo city grocery store. Despite being a cold-adapted species, after having arrived to the southeastern state of São Paulo, this invasive fly will probably expand its territory to other Brazilian states and South American countries through trade of cultivated soft skin small fruits, such as blueberries and strawberries, as well as naturally through the use of small wild fruits as breeding sites.

Molecular confirmation of ovine herpesvirus 2-induced malignant catarrhal fever lesions in cattle from Rio Grande do Norte, Brazil

Molecular findings that confirmed the participation of ovine herpesvirus 2 (OVH-2) in the lesions that were consistent with those observed in malignant catarrhal fever of cattle are described. Three mixed-breed cattle from Rio Grande do Norte state demonstrated clinical manifestations that included mucopurulent nasal discharge, corneal opacity and motor incoordination. Routine necropsy examination demonstrated ulcerations and hemorrhage of the oral cavity, corneal opacity, and lymph node enlargement. Significant histopathological findings included widespread necrotizing vasculitis, non-suppurative meningoencephalitis, lymphocytic interstitial nephritis and hepatitis, and thrombosis. PCR assay performed on DNA extracted from kidney and mesenteric lymph node of one animal amplified a product of 423 base pairs corresponding to a target sequence within the ovine herpesvirus 2 (OVH-2) tegument protein gene. Direct sequencing of the PCR products, from extracted DNA of the kidney and mesenteric lymph node of one cow, amplified the partial nucleotide sequences (423 base pairs) of OVH-2 tegument protein gene. Blast analysis confirmed that these sequences have 98-100% identity with similar OVH-2 sequences deposited in GenBank. Phylogenetic analyses, based on the deduced amino acid sequences, demonstrated that the strain of OVH-2 circulating in ruminants from the Brazilian states of Rio Grande do Norte and Minas Gerais are similar to that identified in other geographical locations. These findings confirmed the active participation of OVH-2 in the classical manifestations of sheep associated malignant catarrhal fever.

Molecular detection of Neorickettsia risticii in Brazilian free-tailed bats (Tadarida brasiliensis) from Buenos Aires , Argentina

Neorickettsia risticii is the causative agent of Potomac Horse Fever, a severe febrile disease affecting horses, transmitted by trematodes species with a complex life cycle. A total of 30 insectivorous bats (Brazilian free-tailed bat Tadarida brasiliensis) were analyzed by PCR for presence of genus Anaplasma, Ehrlichia, Neorickettsia and Rickettsia. Three samples showed positive reactions for genus Anaplasma, Ehrlichia and Neorickettsia, and the sequences were 99.67% identical to Neorickettsia risticii. The role of bats in the life cycle of N. risticii has yet to be elucidated; however bats may be reservoirs for this bacterium. To our knowledge, this is the first evidence of N. risticii in Argentina.

The yellow fever 17D vaccine virus: molecular basis of viral attenuation and its use as an expression vector

The yellow fever (YF) virus is the prototype flavivirus. The use of molecular techniques has unraveled the basic mechanisms of viral genome structure and expression. Recent trends in flavivirus research include the use of infectious clone technology with which it is possible to recover virus from cloned cDNA. Using this technique, mutations can be introduced at any point of the viral genome and their resulting effect on virus phenotype can be assessed. This approach has opened new possibilities to study several biological viral features with special emphasis on the issue of virulence/attenuation of the YF virus. The feasibility of using YF virus 17D vaccine strain, for which infectious cDNA is available, as a vector for the expression of heterologous antigens is reviewed

Effects of a neuronal nitric oxide synthase inhibitor on lipopolysaccharide-induced fever

It has been demonstrated that nitric oxide (NO) has a thermoregulatory action, but very little is known about the mechanisms involved. In the present study we determined the effect of neuronal nitric oxide synthase (nNOS) inhibition on thermoregulation. We used 7-nitroindazole (7-NI, 1, 10 and 30 mg/kg body weight), a selective nNOS inhibitor, injected intraperitoneally into normothermic Wistar rats (200-250 g) and rats with fever induced by lipopolysaccharide (LPS) (100 µg/kg body weight) administration. It has been demonstrated that the effects of 30 mg/kg of 7-NI given intraperitoneally may inhibit 60% of nNOS activity in rats. In all experiments the colonic temperature of awake unrestrained rats was measured over a period of 5 h at 15-min intervals after intraperitoneal injection of 7-NI. We observed that the injection of 30 mg/kg of 7-NI induced a 1.5oC drop in body temperature, which was statistically significant 1 h after injection (P<0.02). The coinjection of LPS and 7-NI was followed by a significant (P<0.02) hypothermia about 0.5oC below baseline. These findings show that an nNOS isoform is required for thermoregulation and participates in the production of fever in rats.

Fever induction pathways: evidence from responses to systemic or local cytokine formation

The immune and central nervous systems are functionally connected and interacting. The concept that the immune signaling to the brain which induces fever during infection and inflammation is mediated by circulating cytokines has been traditionally accepted. Administration of bacterial lipopolysaccharide (LPS) induces the appearance of a so-termed "cytokine cascade" in the circulation more or less concomitantly to the developing febrile response. Also, LPS-like fever can be induced by systemic administration of key cytokines (IL-1ß, TNF-alpha, and others). However, anti-cytokine strategies against IL-1ß or TNF-alpha along with systemic injections of LPS frequently lead to attenuation of the later stages of the febrile response but not of the initial phase of fever, indicating that cytokines are rather involved in the maintenance than in the early induction of fever. Within the last years experimental evidence has accumulated indicating the existence of neural transport pathways of immune signals to the brain. Because subdiaphragmatic vagotomy prevents or attenuates fever in response to intraperitoneal or intravenous injections of LPS, a role for vagal afferent nerve fibers in fever induction has been proposed. Also other sensory nerves may participate in the manifestation of febrile responses under certain experimental conditions. Thus, injection of a small dose of LPS into an artificial subcutaneous chamber results in fever and formation of cytokines within the inflamed tissue around the site of injection. This febrile response can be blocked in part by injection of a local anesthetic into the subcutaneous chamber, indicating a participation of cutaneous afferent nerve signals in the manifestation of fever in this model. In conclusion, humoral signals and an inflammatory stimulation of afferent sensory nerves can participate in the generation and maintenance of a febrile response.

Attenuation and immunogenicity of recombinant yellow fever 17D-dengue type 2 virus for rhesus monkeys

A chimeric yellow fever (YF)-dengue serotype 2 (dengue 2) virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus. Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.