Samir Mansour Moraes Casseb

Oropouche fever epidemic in Northern Brazil: epidemiology and molecular characterization of isolates.

BACKGROUND Oropouche fever virus is an important arbovirus associated with febrile disease that re-emerged in 2006 in several municipalities of Pará State, Bragantina region, Amazon, Brazil, 26 years after the last epidemic. OBJECTIVE To investigate an Oropouche fever outbreak in this region. STUDY DESIGN A serologic survey and prospective study of acute febrile cases were performed in Magalhães Barata (urban and rural areas) and Maracanã (rural area) municipalities. Serology (IgM-ELISA and hemagglutination-inhibition [HI]), virus isolation, RT-PCR and real-time-PCR were used to confirm Oropouche virus (OROV) as responsible for the febrile outbreaks. RESULTS Real-time-PCR showed high titers of OROV in acute-phase serum samples from febrile patients. From 113 of 119 acutely febrile patients with paired serum samples, OROV infections was confirmed by serologic conversion (n=76) or high titers (n=37) for both HI and IgM-ELISA. Patients had a febrile disease characterized by headache, chills, dizziness, photophobia, myalgia, nausea, and vomiting. Females and children under 15 years of age were most affected. Nucleotide sequencing of six OROV isolates identified that genotype II was associated with the human disease epidemic. CONCLUSIONS Oropouche fever, which has re-emerged in the Bragantina region in eastern Amazon 26 years after the last epidemic, is caused by genotype II, a lineage previously found only in Peru and western Brazil.

A simple one-step real-time RT-PCR for diagnosis of dengue virus infection

Dengue is the most important arbovirus disease in tropical and sub‐tropical countries, and can be caused by infection with any of the four‐dengue virus (DENV) serotypes. Infection with DENV can lead to a broad clinical spectrum, ranging from sub‐clinical infection or an influenza‐like disease known as dengue fever (DF) to a severe, sometimes fatal, disease characterized by hemorrhage and plasma leakage that can lead to shock, known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The diagnosis of dengue is routinely accomplished by serologic assays, such as IgM and IgG ELISAs, as well as HI tests, analyzing serum samples obtained from patients with at least 7 days of symptoms onset. These tests cannot be used for diagnosis during the early symptomatic phase. In addition, antibodies against dengue are broad reactive with other flaviviruses. Therefore, a specific diagnostic method for acute DENV infection is of great interest. In that sense, the real‐time RT‐PCR has become an important tool that can be used for early and specific detection of dengue virus genome in human serum samples. This study describes a simple, specific, and sensitive real‐time RT‐PCR for early diagnosis of dengue virus infection. J. Med. Virol. 80:1426–1433, 2008.

Molecular epidemiology of Oropouche virus, Brazil.

Oropouche virus (OROV) is the causative agent of Oropouche fever, an urban febrile arboviral disease widespread in South America, with >30 epidemics reported in Brazil and other Latin American countries during 1960–2009. To describe the molecular epidemiology of OROV, we analyzed the entire N gene sequences (small RNA) of 66 strains and 35 partial Gn (medium RNA) and large RNA gene sequences. Distinct patterns of OROV strain clustered according to N, Gn, and large gene sequences, which suggests that each RNA segment had a different evolutionary history and that the classification in genotypes must consider the genetic information for all genetic segments. Finally, time-scale analysis based on the N gene showed that OROV emerged in Brazil ≈223 years ago and that genotype I (based on N gene data) was responsible for the emergence of all other genotypes and for virus dispersal.

Molecular epidemiology of Saint Louis encephalitis virus in the Brazilian Amazon: genetic divergence and dispersal.

Saint Louis encephalitis virus (SLEV), a member of the genus Flavivirus (family Flaviviridae), is an encephalitogenic arbovirus broadly distributed in the Americas. Phylogenetic analysis based on the full-length E gene sequences obtained for 30 Brazilian SLEV strains was performed using different methods including Bayesian and relaxed molecular clock approaches. A new genetic lineage was suggested, hereafter named genotype VIII, which co-circulates with the previously described genotype V in the Brazilian Amazon region. Genotypes II and III were restricted to São Paulo state (South-east Atlantic rainforest ecosystem). The analysis also suggested the emergence of an SLEV common ancestor between 1875 and 1973 (mean of 107 years ago), giving rise to two major genetic groups: genotype II, more prevalent in the North America, and a second group comprising the other genotypes (I and III-VIII), broadly dispersed throughout the Americas, suggesting that SLEV initially emerged in South America and spread to North America. In conclusion, the current study demonstrates the high genetic variability of SLEV and its geographical dispersion in Brazil and other New World countries.

Evaluation of two molecular methods for the detection of Yellow fever virus genome

Yellow fever virus (YFV), a member of the family Flaviviridae, genus Flavivirus is endemic to tropical areas of Africa and South America and is among the arboviruses that pose a threat to public health. Recent outbreaks in Brazil, Bolivia, and Paraguay and the observation that vectors capable of transmitting YFV are presenting in urban areas underscore the urgency of improving surveillance and diagnostic methods. Two novel methods (RT-hemi-nested-PCR and SYBR(®) Green qRT-PCR) for efficient detection of YFV strains circulating in South America have been developed. The methods were validated using samples obtained from golden hamsters infected experimentally with wild-type YFV strains as well as human serum and tissue samples with acute disease.

Arbovirus eco-epidemiology in the area affected by the Cuiabá-Santarém Highway (BR-163), Pará State, Brazil

The current study describes the eco-epidemiological aspects of arbovirus diseases in the municipalities (counties) of Novo Progresso and Trairao, Para State, Brazil, in the area affected by highway BR-163. Hemagglutination inhibition (HI) antibodies to different arboviruses were detected, with monotypic reactions to MAYV and OROV, two important arboviruses associated with epidemics in the Amazon. IgM antibodies to OROV and MAYV were found in human sera, suggesting recent infections by these viruses. Two DENV-3 strains were isolated from febrile patients in Novo Progresso and identified as genotype III strains. In general, the data suggest that the area displays ideal conditions for maintenance and circulation of arboviruses, plus a population with low immunization levels. Dynamic surveillance of local immigrants and wild animals is thus important, focusing on antibody prevalence and isolation of arboviruses, thereby allowing effective control of infections by these viral agents in the resident population along highway BR-163 in Para State.

Full-length sequencing and genetic characterization of Breu Branco virus (Reoviridae, Orbivirus) and two related strains isolated from Anopheles mosquitoes.

Breu Branco virus (BE AR 492347) was isolated from Anopheles (Nyssorhynchus) triannulatus mosquitoes captured in Tucuruí, Pará State, northern Brazil, in 1988. No cross-reactivity by complement-fixation tests was observed between Breu Branco virus and other known arboviruses. Results of electron microscopy and physicochemical tests suggested that Breu Branco virus may be a member of the family Reoviridae. In order to elucidate its taxonomic status, a comprehensive genetic characterization was conducted for Breu Branco virus and related strains (BE AR 494475 and BE AR 486204) that were also isolated from Anopheles mosquitoes in the same area. This included full-length genome sequencing, determination of genetic traits and phylogenetic analysis. Breu Branco virus showed a similar genome organization to members of the genus Orbivirus, family Reoviridae. Genetically, Breu Branco virus was indistinguishable from strains BE AR 494475 and BE AR 486204. Phylogenetic analysis suggested that Breu Branco virus BE AR 492347 and its related strains constitute a novel species of the genus Orbivirus. Breu Branco virus is the first Brazilian orbivirus and the fifth orbivirus in the world to be sequenced fully.

Bovine lactoferrin activity against Chikungunya and Zika viruses

Chikungunya (CHIKV) and Zika (ZIKV) viruses are arboviruses which have recently broken their sylvatic isolation and gone on to spread rampantly among humans in some urban areas of the world, especially in Latin America. Given the lack of effective interventions against such viruses, the aim of this work was to evaluate the antiviral potential of bovine lactoferrin (bLf) in their infections. Through viability, plaque, immunofluorescence and nucleic acid quantification assays, our data show that bLf exerts a dose-dependent strong inhibitory effect on the infection of Vero cells by the aforementioned arboviruses, reducing their infection efficiency by up to nearly 80 %, with no expressive cytotoxicity, and that such antiviral activity occurs at the levels of input and output of virus particles. These findings reveal that bLf antimicrobial properties are extendable to CHIKV and ZIKV, underlining a generic inhibition mechanism that can be explored to develop a potential strategy against their infections.

Environmental influences on antibody-enhanced dengue disease outcomes

Because an enriched environment (EE) enhances T-cell activity and T-lymphocytes contribute to immunopathogenesis during heterologous dengue virus (DENV) infections, we hypothesised that an EE increases dengue severity. To compare single serotype (SS) and antibody-enhanced disease (AED) infections regimens, serial intraperitoneal were performed with DENV3 (genotype III) infected brain homogenate or anti-DENV2 hyperimmune serum followed 24 h later by DENV3 (genotype III) infected brain homogenate. Compared AED for which significant differences were detected between the EE and impoverished environmental (IE) groups (Kaplan-Meyer log-rank test, p = 0.0025), no significant differences were detected between the SS experimental groups (Kaplan-Meyer log-rank test, p = 0.089). Survival curves from EE and IE animals infected with the AED regimen were extended after corticoid injection and this effect was greater in the EE than in the IE group (Kaplan-Meyer log-rank test, p = 0.0162). Under the AED regimen the EE group showed more intense clinical signs than the IE group. Dyspnoea, tremor, hunched posture, ruffled fur, immobility, pre-terminal paralysis, shock and death were associated with dominant T-lymphocytic hyperplasia and presence of viral antigens in the liver and lungs. We propose that the increased expansion of these memory T-cells and serotype cross-reactive antibodies facilitates the infection of these cells by DENV and that these events correlate with disease severity in an EE.

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.