Improvement of a RT-PCR assay for Yellow Fever virus genome detection

The aim of the present study was to describe an improved protocol of reverse transcription polymerase chain reaction (RT-PCR) for Yellow Fever virus genome detection. A strain of ribonucleic acid of Yellow Fever virus was submitted to the improved protocol of RT-PCR and the amplicons were visualized under ultraviolet transilluminator, purifed and sequenced. The nucleotide sequence obtained was compared with sequences available in GenBank using the tblastx tool. The amplicons produced by the strain of ribonucleic acid of Yellow Fever virus exhibited fragments of 400 and 800 base pairs and the consensus sequence exhibited a similarity of 100% with Yellow Fever virus sequences recorded in GenBank. The improved protocol described in this study allowed Yellow Fever virus genome detection and enabled the elimination of the nested-PCR step, which has been frequently associated with contamination. In addition, it reduced the time of reaction, the cost of reagents and the possibility of sample contamination. New methods of investigating these infections must be elaborated and a continuous vigilance of these viruses in their different vectors and hosts is required to avoid negative impacts on human health, tourism and trade.(AU)

Larval application of sodium channel homologous dsRNA restores pyrethroid insecticide susceptibility in a resistant adult mosquito population.

BACKGROUND: Mosquitoes host and pass on to humans a variety of disease-causing pathogens such as infectious viruses and other parasitic microorganisms. The emergence and spread of insecticide resistance is threatening the effectiveness of current control measures for common mosquito vector borne diseases, such as malaria, dengue and Zika. Therefore, the emerging resistance to the widely used pyrethroid insecticides is an alarming problem for public health. Herein we demonstrated the use of RNA interference (RNAi) to increase susceptibility of adult mosquitoes to a widely used pyrethroid insecticide. METHODS: Experiments were performed on a field-collected pyrethroid resistant strain of Ae. aegypti (Rio de Janeiro; RJ). Larvae from the resistant Ae. aegypti population were soaked with double-stranded RNAs (dsRNAs) that correspond either to voltage-gate sodium channel (VGSC), P-glycoprotein, or P450 detoxification genes and reared to adulthood. Adult mortality rates in the presence of various Deltamethrin pyrethroid concentrations were used to assess mosquito insecticide susceptibility. RESULTS: We characterized the RJ Ae. aegypti strain with regard to its level of resistance to a pyrethroid insecticide and found that it was approximately 6 times more resistant to Deltamethrin compared to the laboratory Rockefeller strain. The RJ strain displayed a higher frequency of Val1016Ile and Phe1534Cys substitutions of the VGSC gene. The resistant strain also displayed a higher basal expression level of VGSC compared to the Rockefeller strain. When dsRNA-treated mosquitoes were subjected to a standard pyrethroid contact bioassay, only dsRNA targeting VGSC increased the adult mortality of the pyrethroid resistant strain. The dsRNA treatment proved effective in increasing adult mosquito susceptibility over a range of pyrethroid concentrations and these results were associated with dsRNA-specific small interfering RNAs in treated adults, and the corresponding specific down regulation of VGSC gene expression level. Finally, we demonstrated that the efficiency of our approach was further improved by 'tiling' along the VGSC gene in order to identify the most potent dsRNA sequences. CONCLUSIONS: These results demonstrate that dsRNA applied to mosquito larvae retains its biological activity into adulthood. Thus, the RNAi system reported here could be a useful approach to control the widespread insecticide resistance in mosquitoes and other insect vectors of human diseases.

Contrasting patterns of insecticide resistance and knockdown resistance (kdr) in Aedes aegypti populations from Jacarezinho (Brazil) after a Dengue Outbreak

ABSTRACT After a dengue outbreak, the knowledge on the extent, distribution and mechanisms of insecticide resistance is essential for successful insecticide-based dengue control interventions. Therefore, we evaluated the potential changes to insecticide resistance in natural Aedes aegypti populations to Organophosphates (OP) and Pyrethroids (PY) after chemical vector control interventions. After a Dengue outbreak in 2010, A. aegypti mosquitoes from the urban area of Jacarezinho (Paraná, Brazil) were collected in 2011 and 2012. Insecticide resistance to OP Temephos was assessed in 2011 and 2012 by dose–response bioassays adopting WHO-based protocols. Additionally, in both sampling, PY resistance was also investigated by the Val1016Ile mutation genotyping. In 2011, a random collection of mosquitoes was carried out; while in 2012, the urban area was divided into four regions where mosquitoes were sampled randomly. Bioassays conducted with larvae in 2011 (82 ± 10%; RR95 = 3.6) and 2012 (95 ± 3%; RR95 = 2.5) indicated an incipient altered susceptibility to Temephos. On the other hand, the Val1016IIe mutation analysis in 2011, presented frequencies of the 1016Ilekdr allele equal to 80%. Nevertheless, in 2012, when the urban area of Jacarezinho was analyzed as a single unit, the frequency of the mutant allele was 70%. Additionally, the distribution analysis of the Val1016Ile mutation in 2012 showed the mutant allele frequencies ≥60% in all regions. These outcomes indicated the necessity of developing alternative strategies such as insecticide rotations for delaying the evolution of resistance.

Evaluation of arboviruses of public health interest in free-living non-human primates (Alouatta spp., Callithrix spp., Sapajus spp.) in Brazil.

INTRODUCTION: The aim of the present study was to evaluate the presence of arboviruses from the Flavivirus genus in asymptomatic free-living non-human primates (NHPs) living in close contact with humans and vectors in the States of Paraná and Mato Grosso do Sul, Brazil. METHODS: NHP sera samples (total n = 80, Alouatta spp. n = 07, Callithrix spp. n = 29 and Sapajus spp. n = 44) were screened for the presence of viral genomes using reverse transcription polymerase chain reaction and 10% polyacrylamide gel electrophoresis techniques. RESULTS: All of the samples were negative for the Flavivirus genome following the 10% polyacrylamide gel electrophoresis analysis. CONCLUSIONS: These negative results indicate that the analyzed animals were not infected with arboviruses from the Flavivirus genus and did not represent a risk for viral transmission through vectors during the period in which the samples were collected.

Evaluation of arboviruses of public health interest in free-living non-human primates (Alouatta spp., Callithrix spp., Sapajus spp.) in Brazil

INTRODUCTION: The aim of the present study was to evaluate the presence of arboviruses from the Flavivirus genus in asymptomatic free-living non-human primates (NHPs) living in close contact with humans and vectors in the States of Paraná and Mato Grosso do Sul, Brazil. METHODS: NHP sera samples (total n = 80, Alouatta spp. n = 07, Callithrix spp. n = 29 and Sapajus spp. n = 44) were screened for the presence of viral genomes using reverse transcription polymerase chain reaction and 10% polyacrylamide gel electrophoresis techniques. RESULTS: All of the samples were negative for the Flavivirus genome following the 10% polyacrylamide gel electrophoresis analysis. CONCLUSIONS: These negative results indicate that the analyzed animals were not infected with arboviruses from the Flavivirus genus and did not represent a risk for viral transmission through vectors during the period in which the samples were collected. .

Genetic diversity of dengue virus serotypes 1 and 2 in the State of Paraná, Brazil, based on a fragment of the capsid/premembrane junction region.

INTRODUCTION: The precise identification of the genetic variants of the dengue virus is important to understand its dispersion and virulence patterns and to identify the strains responsible for epidemic outbreaks. This study investigated the genetic variants of the capsid-premembrane junction region fragment in the dengue virus serotypes 1 and 2 (DENV1-2). METHODS: Samples from 11 municipalities in the State of Paraná, Brazil, were provided by the Central Laboratory of Paraná. They were isolated from the cell culture line C6/36 (Aedes albopictus) and were positive for indirect immunofluorescence. Ribonucleic acid (RNA) extracted from these samples was submitted to the reverse transcription polymerase chain reaction (RT-PCR) and nested PCR. RESULTS: RT-PCR revealed that 4 of the samples were co-infected with both serotypes. The isolated DENV-1 sequences were 95-100% similar to the sequences of other serotype 1 strains deposited in GenBank. Similarly, the isolated DENV-2 sequences were 98-100% similar to other serotype 2 sequences in GenBank. According to our neighbor-joining tree, all strains obtained in this study belonged to genotype V of DENV-1. The DENV-2 strains, by contrast, belonged to the American/Asian genotypes. CONCLUSIONS: The monitoring of circulating strains is an important tool to detect the migration of virus subtypes involved in dengue epidemics.

Genetic diversity of dengue virus serotypes 1 and 2 in the State of Paraná, Brazil, based on a fragment of the capsid/premembrane junction region / Diversidade genética dos vírus dengue sorotipos 1 e 2, no Estado do Paraná, Brasil, baseada no fragmento da junção do gene capsídeo/pré-membrana

INTRODUCTION: The precise identification of the genetic variants of the dengue virus is important to understand its dispersion and virulence patterns and to identify the strains responsible for epidemic outbreaks. This study investigated the genetic variants of the capsid-premembrane junction region fragment in the dengue virus serotypes 1 and 2 (DENV1-2). METHODS: Samples from 11 municipalities in the State of Paraná, Brazil, were provided by the Central Laboratory of Paraná. They were isolated from the cell culture line C6/36 (Aedes albopictus) and were positive for indirect immunofluorescence. Ribonucleic acid (RNA) extracted from these samples was submitted to the reverse transcription polymerase chain reaction (RT-PCR) and nested PCR. RESULTS: RT-PCR revealed that 4 of the samples were co-infected with both serotypes. The isolated DENV-1 sequences were 95-100% similar to the sequences of other serotype 1 strains deposited in GenBank. Similarly, the isolated DENV-2 sequences were 98-100% similar to other serotype 2 sequences in GenBank. According to our neighbor-joining tree, all strains obtained in this study belonged to genotype V of DENV-1. The DENV-2 strains, by contrast, belonged to the American/Asian genotypes. CONCLUSIONS: The monitoring of circulating strains is an important tool to detect the migration of virus subtypes involved in dengue epidemics.

INTRODUÇÃO:A identificação precisa da variante genética do vírus da dengue é importante para compreender a dispersão, virulência e identificação das cepas responsáveis pelas epidemias. O objetivo da pesquisa foi investigar a variação genética do fragmento da junção do gene capsídeo/pré-membrana dos sorotipos 1 e 2. MÉTODOS: Amostras de onze municípios do Estado Paraná, Brasil, foram cedidas pelo Laboratório Central do Paraná e consistiam em isolados de cultura de células da linhagem C6/36 (Aedes albopictus), positivos para técnica de imunofluorescência indireta. O Ribonucleic acid (RNA) dessas amostras foi extraído, seguido da transcrição reversa, reação em cadeia da polimerase (PCR) e nested PCR. RESULTADOS: Co-infecção por DENV-1 e 2 (virus da dengue 1 e 2) foi observada em quatro pacientes, através da técnica Reverse transcriptase-polymerase chain reaction (RT-PCR). Para o DENV-1 a porcentagem de similaridade variou de 95 a 100% comparando com cepas do Genbank. Para o DENV-2 a porcentagem de similaridade variou de 98 a 100%. De acordo com o cladograma gerado, todas as cepas deste estudo se agruparam no genótipo V para DENV-1. Para o DENV-2 foi encontrada a cepa referente ao genótipo asiático/americano. CONCLUSÕES: O monitoramento das cepas circulantes torna-se uma ferramenta importante na detecção da migração dos subtipos do vírus da dengue envolvidos em epidemias.

Detecção do vírus da dengue em populações naturais de mosquitos / Detection of dengue virus in natural populations of mosquitoes

A dengue é causada por um Flavivirus que apresenta elevada diversidade genética, com quatro sorotipos e vários genótipos. A enfermidade é endêmica na maioria dos países das Américas, e as fêmeas de Aedes (Stegomyia) aegypti (Linnaeus, 1762) são as únicas transmissoras, com importância epidemiológica. Um único mosquito infectado permanece assim pelo resto de sua vida, podendo infectar múltiplos hospedeiros humanos. Com a detecção do vírus da dengue em mosquitos, podemos revelar o sorotipo circulante ou a entrada de um novo sorotipo em uma determinada região, sem quaisquer implicações éticas, além de apresentar reprodutibilidade dos resultados. Esta revisão aborda as técnicas para detecção viral em mosquitos, suas vantagens e limitações, bem como as pesquisas já realizadas com populações naturais de Aedes aegypti.

Dengue is caused by Flavivirus which exhibits high genetic diversity, with four serotypes and various genotypes. The disease is endemic in most countries in the Americas, and the female Aedes (Stegomyia) aegypti (Linnaeus, 1762) are the only transmitters of epidemiological importance. A single infected mosquito remains so for the rest of his life and can infect multiple human hosts. For dengue virus detection in mosquitoes, the circulating serotype can be revealed or detect an entry of a new serotype in the region, without any ethical implications, and reproducible results. This review covers the techniques for detecting virus in mosquitoes, their advantages and limitations, as well as previous studies with natural populations of Aedes aegypti.

El dengue es causado por un Flavivirus que presenta una alta diversidad genética, cuatro serotipos y varios genotipos. Esta enfermedad es endémica en la mayoría de los países del continente Americano y sólo las hembras de Aedes (Stegomyia) aegypti (Linnaeus, 1762) son las transmisoras de esta enfermedad de importancia epidemiológica. Un único mosquito infectado permanece así por el resto de su vida pudiendo infectar múltiples hospederos humanos. El detectar el serotipo de virus de dengue presente en los mosquitos permite conocer, ya sea, el serotipo circulante o el ingreso de un nuevo serotipo a una determinada región sin ningún tipo de implicaciones éticas presentando así resultados reproducibles. Ésta revisión aborda las técnicas de detección viral en mosquitos, sus ventajas y limitaciones, así como estudios previos con poblaciones naturales de Aedes aegypti.

[Anopheles cruzii parity in dense rain forest in Southern Brazil]. / Paridade de Anopheles cruzii em Floresta Ombrófila Densa no Sul do Brasil.

OBJECTIVE: To determine the parity and ovarian development of Anopheles cruzii species during the seasons. METHODS: Collections were carried out fortnightly in the morning in the Palmito State Park in the municipality of Paranaguá, Southern Brazil, between April 2004 and April 2005. Adult mosquitoes were captured using human landing rate. Dissections were performed using Detinova's and Polovodova's methods and follicular development was assessed following Christophers and Mer's criteria. RESULTS: A total of 208 specimens of Anopheles cruzii were dissected. Most females dissected were nulliparous in the seasons; 14.4% of them were found to be nulliparous above Christophers and Mer's stage II, which shows previous blood meal prior to the first oviposition. It was observed that Anopheles cruzii populations comprised young mosquitoes, probably due to high mortality among parous females. CONCLUSIONS: The likely gonotrophic discordance is epidemiologically relevant because female mosquitoes can search for more than one host to complete the maturation of their eggs.

Paridade de Anopheles cruzii em Floresta Ombrófila Densa no Sul do Brasil / Anopheles cruzii parity in dense rain forest in Southern Brazil

OBJETIVO: Conhecer a paridade e desenvolvimento ovariano da espécie Anopheles cruzii, durante os períodos estacionais. MÉTODOS: As capturas foram realizadas quinzenalmente, no período matutino, de abril/2004 a abril/2005, no Parque Estadual do Palmito, município de Paranaguá litoral do Estado do Paraná. Mosquitos adultos foram capturados usando a técnica pouso homem. As dissecções foram feitas utilizando-se a técnica de Detinova e de Polovodova e a avaliação do desenvolvimento folicular, segundo os critérios de Christophers e Mer. RESULTADOS: Foram dissecadas 208 fêmeas de Anopheles cruzii. A maioria das fêmeas dissecadas nas estações eram nulíparas. Sendo que 14,4 por cento eram nulíparas com folículo além do estádio II de Christophers & Mer, o que evidencia o exercício da hematofagia previamente à primeira oviposição. Observou-se que as populações de Anopheles cruzii são constituídas de indivíduos jovens, talvez em razão da alta mortalidade de fêmeas paridas. CONCLUSÕES: A provável discordância gonotrófica das fêmeas dissecadas é importante do ponto de vista epidemiológico, considerando que a fêmea pode procurar mais de um hospedeiro para completar a maturação dos seus ovos.

OBJECTIVE: To determine the parity and ovarian development of Anopheles cruzii species during the seasons. METHODS: Collections were carried out fortnightly in the morning in the Palmito State Park in the municipality of Paranaguá, Southern Brazil, between April 2004 and April 2005. Adult mosquitoes were captured using human landing rate. Dissections were performed using Detinova's and Polovodova's methods and follicular development was assessed following Christophers and Mer's criteria. RESULTS: A total of 208 specimens of Anopheles cruzii were dissected. Most females dissected were nulliparous in the seasons; 14.4 percent of them were found to be nulliparous above Christophers and Mer's stage II, which shows previous blood meal prior to the first oviposition. It was observed that Anopheles cruzii populations comprised young mosquitoes, probably due to high mortality among parous females. CONCLUSIONS: The likely gonotrophic discordance is epidemiologically relevant because female mosquitoes can search for more than one host to complete the maturation of their eggs.