Adriano Mondini

Dengue virus type 3 adaptive changes during epidemics in São Jose de Rio Preto, Brazil, 2006-2007.

Global dengue virus spread in tropical and sub-tropical regions has become a major international public health concern. It is evident that DENV genetic diversity plays a significant role in the immunopathology of the disease and that the identification of polymorphisms associated with adaptive responses is important for vaccine development. The investigation of naturally occurring genomic variants may play an important role in the comprehension of different adaptive strategies used by these mutants to evade the human immune system. In order to elucidate this role we sequenced the complete polyprotein-coding region of thirty-three DENV-3 isolates to characterize variants circulating under high endemicity in the city of São José de Rio Preto, Brazil, during the onset of the 2006-07 epidemic. By inferring the evolutionary history on a local-scale and estimating rates of synonymous (dS) and nonsynonimous (dN) substitutions, we have documented at least two different introductions of DENV-3 into the city and detected 10 polymorphic codon sites under significant positive selection (dN/dS > 1) and 8 under significant purifying selection (dN/dS < 1). We found several polymorphic amino acid coding sites in the envelope (15), NS1 (17), NS2A (11), and NS5 (24) genes, which suggests that these genes may be experiencing relatively recent adaptive changes. Furthermore, some polymorphisms correlated with changes in the immunogenicity of several epitopes. Our study highlights the existence of significant and informative DENV variability at the spatio-temporal scale of an urban outbreak.

Population dynamics of DENV-1 genotype V in Brazil is characterized by co-circulation and strain/lineage replacement

Following successive outbreaks of dengue fever caused predominantly by dengue virus (DENV) 2 and 3, DENV-1 is now the primary serotype circulating in Brazil. We sequenced and analyzed Brazilian DENV-1 genomes and found that all isolates belong to genotype V and are subdivided into three lineages, which were introduced during four different events. The first introduction occurred in 1984-85, the second in 1997-99, and the third and fourth occurred from 2004 to 2007. These events were associated with an increase in genetic diversity but not with positive selection. Moreover, a potential new recombinant strain derived from two distinct lineages was detected. We demonstrate that the dynamics of DENV-1 in Brazil is characterized by introduction, movement, local evolution, and lineage replacement. This study strengthens the relevance of genotype surveillance in order to identify, trace, and control virus populations circulating in Brazil and Latin America.

Detection of Mayaro virus infections during a dengue outbreak in Mato Grosso, Brazil

Arboviruses are common agents of human febrile illness worldwide. In dengue-endemic areas illness due to other arboviruses have been misdiagnosed as dengue based only on clinical-epidemiological data. In this study we investigated the presence of Brazilian arboviruses in sera of 200 patients presenting acute febrile illness, during a dengue outbreak in Sinop, MT, Brazil. The results showed that 38 samples were positive to Dengue virus (DENV) type 1, two samples to DENV type 4, and six to Mayaro virus. These results indicate that arboviruses others than DENV are circulating in Sinop and the surrounding region, which are going undiagnosed. In addition, molecular and evolutionary analyses indicate that two MAYV genotypes are co-circulating in Mato Grosso, Brazil. Thus, a strong surveillance program must be implemented to evaluate and monitor the distribution and the true importance of non-dengue arboviruses in the etiology of acute febrile illnesses.

Field-deployable viral diagnostics using CRISPR-Cas13

Taking CRISPR technology further CRISPR techniques are allowing the development of technologies for nucleic acid detection (see the Perspective by Chertow). Taking advantages of the distinctive enzymatic properties of CRISPR enzymes, Gootenberg et al. developed an improved nucleic acid detection technology for multiplexed quantitative and highly sensitive detection, combined with lateral flow for visual readout. Myhrvold et al. added a sample preparation protocol to create a field-deployable viral diagnostic platform for rapid detection of specific strains of pathogens in clinical samples. Cas12a (also known as Cpf1), a type V CRISPR protein, cleaves double-stranded DNA and has been adapted for genome editing. Chen et al. discovered that Cas12a also processes single-stranded DNA threading activity. A technology platform based on this activity detected human papillomavirus in patient samples with high sensitivity. Science, this issue p. 439, p. 444, p. 436; see also p. 381 A nucleic acid detection technology identifies viruses with minimal equipment and sample processing requirements. Mitigating global infectious disease requires diagnostic tools that are sensitive, specific, and rapidly field deployable. In this study, we demonstrate that the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations as low as 1 copy per microliter. We developed HUDSON (heating unextracted diagnostic samples to obliterate nucleases), a protocol that pairs with SHERLOCK for viral detection directly from bodily fluids, enabling instrument-free DENV detection directly from patient samples in <2 hours. We further demonstrate that SHERLOCK can distinguish the four DENV serotypes, as well as region-specific strains of ZIKV from the 2015–2016 pandemic. Finally, we report the rapid (<1 week) design and testing of instrument-free assays to detect clinically relevant viral single-nucleotide polymorphisms.

Serological detection of West Nile virus in horses and chicken from Pantanal, Brazil

In an effort to detect West Nile virus (WNV) in Brazil, we sampled serum from horses and chickens from the Pantanal region of the state of Mato Grosso and tested for flavivirus-reactive antibodies by blocking ELISA. The positive samples were further confirmed for serological evidence of WNV infection in three (8%) of the 38 horses and one (3.2%) of the 31 chickens using an 80% plaque-reduction neutralisation test (PRNT80). These results provide evidence of the circulation of WNV in chickens and horses in Pantanal.

Assessment of the relationship between entomologic indicators of Aedes aegypti and the epidemic occurrence of dengue virus 3 in a susceptible population, São José do Rio Preto, São Paulo, Brazil

The aims of this study were to describe the occurrence of dengue in space and time and to assess the relationships between dengue incidence and entomologic indicators. We selected the dengue autochthonous cases that occurred between September 2005 and August 2007 in São José do Rio Preto to calculate incidence rates by month, year and census tracts. The monthly incidence rates of the city were compared to the monthly Breteau indices (BI) of the São José do Rio Region. Between December 2006 and February 2007, an entomological survey was conducted to collect immature forms of Aedes aegypti in Jaguaré, a São José do Rio Preto neighborhood, and to obtain entomological indices. These indices were represented using statistical interpolation. To represent the occurrence of dengue in the Jaguaré neighborhood in 2006 and 2007, we used the Kernel ratio and to evaluate the relationship between dengue and the entomological indices, we used a generalized additive model in a spatial case-control design. Between September 2005 and August 2007, the occurrence of dengue in São José do Rio Preto was almost entirely caused by DENV3, and the monthly incidence rates presented high correlation coefficients with the monthly BI. In Jaguaré neighborhood, the entomological indices calculated by hectare were better predictors of the spatial distribution of dengue than the indices calculated by properties, but the pupae quantification did not show better prediction qualities than the indices based on the container positivity, in relation to the risk of dengue occurrence. The fact that the municipalitys population had a high susceptibility to the serotype DENV3 before the development of this research, along with the almost total predominance of the occurrence of this serotype between 2005 and 2007, facilitated the analysis of the epidemiological situation of the disease and allowed us to connect it to the entomological indicators.

CO-INFECTION OF DENGUE VIRUS BY SEROTYPES 1 AND 4 IN PATIENT FROM MEDIUM SIZED CITY FROM BRAZIL

SUMMARY The natural co-infection with dengue virus can occur in highly endemic areas where different serotypes have been observed for many years. We report one case of DENV-1/DENV-4 co-infection in human serum detected by molecular tests. Phylogenetic analysis of the sequences obtained indicated the presence of genotype V and II for DENV-1 and DENV-4, respectively.

Dengue in Araraquara, state of São Paulo: epidemiology, climate and Aedes aegypti infestation

ABSTRACT OBJECTIVE To describe the epidemiology of dengue in a medium-sized city in the state of São Paulo. METHODS Data, such as circulating serotypes, severe cases and deaths, age group, sex, among others, were obtained on reported and confirmed dengue cases in Araraquara, state of São Paulo, between 1991 and 2015. Climatic and infestation data were also analyzed. These variables were evaluated descriptively, using statistical measures such as frequencies, averages, minimum and maximum. Dengue incidence rates were calculated according to month, year, age and sex, and time series of dengue cases, infestation, and climatic variables. RESULTS Approximately 16,500 cases of dengue fever were reported between 1991 and 2015. The highest number of reports was recorded in 2015 (7,811 cases). In general, the age group with the highest number of reports is between 20 and 59 years old. The highest incidences, generally between March and May, occurred after the increase in rainfall and infestation in January. CONCLUSIONS Increased levels of infestation due to rainfall are reflected in incidence rates of the disease. It is fundamental to know the epidemiology of dengue in medium-sized cities. Such information can be extended to diseases such as Zika and Chikungunya, which are transmitted by the same vector and were reported in the city. The intensification of surveillance efforts in periods before epidemics could be a strategy to be considered to control the viral spread.

Using adult Aedes aegypti females to predict areas at risk for dengue transmission: A spatial case-control study

INTRODUCTION Traditional indices for measuring dengue fever risk in a given area are based on the immature forms of the vector (larvae and pupae surveys). However, this is inefficient because only adult female mosquitoes actually transmit the virus. Based on these assumptions, our objective was to evaluate the association between an entomological index obtained from adult mosquito traps and the occurrence of dengue in a hyperendemic area. Additionally, we compared its cost to that of the Breteau Index (BI). MATERIAL AND METHODS We performed this study in São José do Rio Preto, SP, Brazil, between the epidemiological weeks of 36/2012 and 19/2013. BG-Sentinel and BG-Mosquitito traps were installed to capture adult mosquitoes. Positive and negative cases of dengue fever were computed and geocoded. We generated biweekly thematic maps of the entomological index, generated by calculating the number of adult Aedes aegypti females (NAF) per 100 households during a week by kriging, and based on the number of mosquitoes captured. The relation between the occurrence of dengue fever and the NAF was tested using a spatial case-control design and a generalized additive model and was controlled by the coordinates of the positive and negative cases of dengue fever. RESULTS Our analyses showed that increases in dengue fever cases occurred in parallel with increases in the number of Ae. aegypti females. The entomological index produced in our study correlates positively with the incidence of dengue, particularly during intervals when vector control measures were applied less intensively. The operational costs of our index were lower than those of the BI: NAF used 71.5% less human resources necessary to measure the BI. CONCLUSIONS Spatial analysis techniques and the number of adult Ae. aegypti females were used to produce an indicator of dengue risk. The index can be applied at various levels of spatial aggregation for an entire study area, as well as for sub-areas, such as city blocks. Even though the index is adequate to predict dengue risk, it should be tested and validated in various scenarios before routine use.