Detection of DENV-4 genotype I from mosquitoes collected in the city of Manaus, Brazil
© de Figueiredo et al; licensee BioMed Central Ltd. 2013
Received: 17 September 2012
Accepted: 11 February 2013
Published: 19 February 2013
Dengue epidemics have been reported in Brazil since 1981. In Manaus, a large city in the Amazon region, dengue is endemic with all four-virus serotypes (DENV-1, -2, -3, and -4) simultaneously causing human disease. In 2008, during a surveillance of dengue virus in mosquitoes in the district of Tancredo Neves in Manaus, 260 mosquitoes of Aedes genus were captured, identified and grouped into pools of 10 mosquitoes.
RNA extracts of mosquito pools were tested by a RT-Hemi-Nested-PCR for detection of flaviviruses. One amplicon of 222 bp, compatible with dengue virus serotype 4, was obtained from a pool of Aedes aegypti. The nucleotide sequence of the amplicon indicated that the mosquitoes were infected with DENV-4 of genotype I. This virus of Asian origin has been described in Manaus in 2008 infecting acute febrile illness patients.
This is the first report of dengue virus serotype 4 genotype I infecting Aedes aegypti in the Americas.
KeywordsEmerging flavivirus Dengue serotype 4 in Brazil RT-Hemi-Nested-PCR for Flavivirus
Dengue virus serotypes 1 through 4 (DENV-1, 2, 3 and 4) are the most important arboviruses worldwide based on number of cases and mortality. DENV infects approximately 50 million of people per year in 100 countries . In Brazil, the incidence of dengue has been on an upward trend, and in the last decade 700,000 cases have been reported per year. Most Brazilian states are infested by Aedes aegypti and endure dengue transmission. The average age of dengue hemorrhagic fever patients has decreased in the last years, affecting a rising proportion of children. In recent years dengue outbreaks have included many atypical cases, including myocarditis, hepatitis, meningoencephalitis and acute kidney failure, and fatality rates have also increased [2, 3].
DENV is transmitted to humans mainly by Aedes aegypti mosquitoes, which acquire the infection throught blood feeding on infected individuals or by transovarial transmission . The first isolation of a DENV (DENV-1 and -4) in Brazil occurred in 1981, in the northern state of Roraima, from acute febrile patients . Then, DENV-4 had disappeared from the country until 2008, when it was found infecting acute febrile patients in the city of Manaus, the capital of Amazonas state, which is located in the middle of the rain forest . A phylogenetic analysis showed that the DENV-4 isolated in Manaus belonged to the genotype I, which has never been described in the American Continent . In 2010, another DENV-4 genotype was detected in Roraima State; the genotype II, which has been circulating in Central America, the northern of South America and the Caribbean . This DENV-4 genotype II rapidly spread through Brazil, producing outbreaks in the most populated areas of the northeastern and southeastern of the country (Ministry of Health of Brazil, 2012). Recently, the origin and evolution of DENV-4 genotypes (I and II) from northern and northeastern Brazil were analyzed . We report here the first detection of DENV-4 genotype I infecting Aedes aegypti in the city of Manaus.
In 2008, as a part of a vector surveillance program for dengue virus detection, 203 Aedes aegypti and 57 Aedes albopictus were captured using adult mosquito traps in Tancredo Neves, a poor district of Manaus city. The mosquitoes were identified based on morphological characteristics in CO2 atmosphere and those from the same specie or genus, captured in the same place, were pooled (~10 adults/pool) based on day of collection. A volume of 1.5 ml PBS (pH7.8) containing 4% bovine albumin was added to each pool of mosquito. The insects were crushed using plastic pistils, and centrifuged at 2500x g for 30 minutes, at 4°C, to pellet the carcasses. The supernatant was split in 2 aliquots and stored at -80°C until use.
Vector surveillance is at the root of dengue control and prevention, with many methods available for detecting the presence of the mosquito vectors, Aedes aegypti and Ae. albopictus, in or around human residences. We show here an important result from a vector surveillance work performed in the city of Manaus where dengue is hyperendemic and has an almost continuous circulation of all 4 dengue virus serotypes .
Interestingly, the topology of phylogenetic tree showed that BHI_3681(JQ513345) strain is most closely related to Thailand isolates. The introduction of DENV-4 genotype I might be related to trading among oriental companies that have facilities in the city of Manaus and receive company worker visits and imported containers from Asia. DENV-4 of genotype II has been also introduced in 2010 and presently, genotypes (I and II) of the virus are circulating in Manaus. This work also showed that the RT-PCR and Hemi-nested-PCR allowed the diagnosis of dengue virus infecting mosquitoes [4, 10]. Therefore, it is a helpful tool for surveillance of dengue and other flaviviruses in vectors.
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq.
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