Natural transovarial transmission of dengue virus 4 in Aedes aegypti from Cuiabá , State of Mato Grosso , Brazil

Introduction: Dengue is the most prevalent arboviral disease in tropical areas. In Mato Grosso, outbreaks are reported every year, but studies on dengue in this state are scarce. Methods: Natural transovarial infection of Aedes aegypti by a fl avivirus was investigated in the Jardim Industriário neighborhood of Cuiabá, Mato Grosso. Eggs were collected with ovitraps during the dry, intermediate, and rainy seasons of 2012. After the eggs hatched and the larvae developed to adulthood, mosquitoes (n = 758) were identifi ed and allocated to pools of 1-10 specimens according to the collection location, sex, and climatic period. After RNA extraction, multiplex semi-nested RT-PCR was performed to detect the four dengue virus (DENV) serotypes, yellow fever virus, West Nile virus and Saint Louis encephalitis virus. Results: DENV-4 was the only fl avivirus detected, and it was found in 8/50 pools (16.0%). Three of the positive pools contained females, and fi ve contained males. Their nucleotide sequences presented 96-100% similarity with DENV-4 genotype II strains from Manaus, Amazonas. The minimum infection rate was 10.5 per 1000 specimens, and the maximum likelihood estimator of the infection rate was 11.6 (95% confi dence interval: 4.8; 23.3). Conclusions: This study provides the fi rst evidence of natural transovarial infection by DENV-4 in Ae. Aegypti in Mato Grosso, suggesting that this type of infection might serve as a mechanism of virus maintenance during interepidemic periods in Cuiabá, a city where dengue epidemics are reported every year. These results emphasize the need for effi cient vector population control measures to prevent arbovirus outbreaks in the state.

Dengue poses a signifi cant public health problem in tropical areas worldwide and has been one of the most important reemerging epidemics in recent years (1) (2) .Dengue reemergence is in part attributed to the failure of vector control measures and uncontrolled urbanization combined with certain virus characteristics (1) .Dengue virus (DENV) is a member of the Flaviviridae family and Flavivirus genus.Four antigenically distinct serotypes (DENV-1, 2, 3, and 4) have been described; several intra-serotype genotypes are present, and lineages exhibit up to 6% divergence in the E/NS1 genome sequences (3) (4) .Despite the existence of sylvatic cycles of DENV transmission in Africa and Asia involving primates and sylvatic Aedes species, the viral transmission is essentially urban in the Americas.Here, the environmental conditions associated with the presence of infected vectors and susceptible hosts promote the transmission cycle of the virus (5) .
Brazil is responsible for the majority of annual dengue notifications to the World Health Organization from the Americas (6) .The country has ideal ecological conditions for arbovirus maintenance and dissemination due to the existence of densely populated urban centers infested with the highly anthropophilic vector Aedes aegypti (7) .All four DENV serotypes have been described in Brazil, and the frequencies of these serotypes within the population vary by region.
The reintroduction of DENV-4 into the country was fi rst observed in 2008 in Manaus, Amazonas (AM), followed by reports in 2010 in Boa Vista, Roraima (RR), with subsequent dissemination throughout the country (8) (9) .This serotype spread rapidly through the population, most likely due to a lack of immunity; it had not been observed since 1982 in the country (10) .In 2012, more than 120 thousand cases of dengue were reported in Brazil, with 44,814 in the state of Mato Grosso (MT) (11) .Most of the cases occurring in 2012 were reported in Cuiabá, the state capital (10,742 cases, two fatal).All four DENV serotypes were detected in the city throughout this year, but DENV-4 was responsible for the vast majority of the cases (12) .

METHODS
Aedes Aegypti is also the urban vector of yellow fever virus (YFV).Sporadic human cases in Brazil have been attributed to the sylvatic cycle of transmission since 1942, when the urban cycle was eradicated from the country by massive vaccination of the population.Mato Grosso is considered a risky area for outbreaks, and vaccination is recommended (13) (14) .
The Saint Louis encephalitis virus (SLEV) was described during a dengue outbreak in 20 patients from São José do Rio Preto.Additionally, one human case was reported in Ribeirão Preto, and the virus was detected in three patients from Mato Grosso (12) (15) (16) .Additional evidence of SLEV circulation in Brazil among humans, equines, mosquitoes, birds, and vectors in the Amazon region, the State of São Paulo, and the southern Pantanal has also been reported (17) (18) (19) .
Vertical transmission is a possible mechanism of viral maintenance in nature for various Flavivirus species (24) (25).To explore this phenomenon, several studies have been conducted recently in several regions (26) (27) (28) .Because the country spans much of the continent, dengue epidemiology may vary considerably between regions.This study aimed to estimate the rates of natural transovarial infection by Flavivirus species in an area of Cuiabá, MT that has a high incidence of acute febrile illness suspected to be caused by dengue infection.

Sampling
A prospective study was conducted in the Jardim Industriário II neighborhood of Cuiabá, MT.This area is located in the south of the city (15º 39'10" S and 55º 58'20" W, 211 m), with an estimated 5,300 inhabitants (29) .Ovitraps were placed in 10 peridomiciliary premises (Figure 1 and 2).These traps were constructed and installed according to the methods of Fay and Aliason (30) .Three captures were conducted during each climatic period of 2012 (i.e., the rainy (24/01 to 08/02), intermediate (07/05 to 22/05), and dry (04/07 to 08/08) seasons) following a stratifi ed sampling protocol to obtain a representative sample from the area; 90 pallets were used in total.The eggs were counted, and after emergence and development to adulthood (i.e., approximately seven days), the specifi c size and sex of the living specimens in a dormant state (i.e., at -20°C for four minutes) were identifi ed using a dichotomous key (31) .The specimens were then allocated to pools of 1-10 mosquitoes according to the collection location and stored at -80°C.

RNA extraction and multiplex semi-nested reverse transcriptase (RT)-PCR for fl aviviruses
Mosquito pools were minced in 0.8mL of saline solution (RNAse-free phosphate buffered saline, pH 7.0) and centrifuged for 4 min at 2,500 × g and 4°C.Total RNA extraction was Jardim Industriário II FIGURE 1 -Map of the study area, Jardim Industriário II, in Cuiabá, State of Mato Grosso.performed using TRIzol (Invitrogen) in 0.4mL of supernatant according to the manufacturer's instructions.The total RNA was reverse transcribed using 100U of reverse transcriptase (Superscript III, Invitrogen), a generic flavivirus primer, 10µL of RNA, and 20U of RNAse inhibitor (RNAse OUT, Invitrogen).The mixture was incubated for 50 min at 50°C and 15 min at 70°C (32) (33) (34) .The cDNA (8µL) was subjected to multiplex semi-nested PCR to detect the DENV serotypes, YFV, WNV, and SLEV (34) .Complementary DNA from positive controls was included in the reactions (i.e., DENV-4 strain Boa Vista, SLEV BeH 355964, and YFV 17DD), and a negative control without DNA template (Table 1).An aliquot of 15μL of amplifi cation products was submitted to electrophoresis on a 1.5% agarose gel (Figure 3).

Rev
RNA from the positive pools was subjected independently to single multiplex semi-nested RT-PCR with the primers for DENV-4 used in the multiplex reaction.The products were sequenced (3500 Genetic Analyzer, Applied Biosystems) to confi rm the species identifi cation.Sequences were aligned and analyzed with the software Molecular Evolutionary Genetics Analysis (MEGA) 5.2 and the nucleotide Basic Local Alignment Tool (BLASTn) (NCBI) and then deposited in GenBank (NCBI) under the accession numbers KM191331, KM191332, and KM191333.

Data analysis
Point estimates and confi dence intervals for the prevalence of infection in mosquitoes were calculated with the software Pool Screen, which was kindly provided by the Department of Biostatistics, School of Public Health, University of Alabama (Birmingham, AL).The minimum infection rate (MIR) was calculated as the number of positive pools divided by the number of specimens tested and multiplied by 1,000 (35) (36) .
During the study period, 2,628 eggs were collected from which 758 Ae. aegypti adults emerged: 351 males and 407 females.These adults were allocated to 50 pools (24 pools of females and 26 pools of males) of 1-10 specimens each, with the aim of identifying the frequency of transovarial natural infection by fl aviviruses.Among these pools, eight were positive    for DENV-4: two were obtained in the rainy season, three in the intermediate season, and three in the dry season.Three of the positive pools contained females and fi ve contained males (Table 2).No fl avivirus other than DENV-4 was detected.The MIR was 10.5% (8/758, 95% confi dence interval: 4.7; 23.3).
The MIR was 14.2% among males, 7.4% among females, 14.2% in the dry season, 11% in the intermediate period, and 7% in the rainy season.Three PCR products obtained from DENV-4 positive pools were submitted for nucleotide sequencing; they presented between 96 and 100% similarity with the strain H780563 (GenBank accession number JQ513343), which belongs to genotype II of DENV-4 and was previously isolated from a human sample in Manaus, AM (37).

DISCUSSION
Dengue is endemic in MT.During 2012, the introduction of the DENV-4 serotype was detected in the state and was accompanied by a massive outbreak in the metropolitan area of the capital, Cuiabá.Annually, an unexplained higher incidence of the disease is observed in certain areas of the city, such as the Jardim Industriário neighborhood (38) .This study was conducted to investigate the possible reasons for this higher incidence and consequent higher prevalence of the disease in these areas.Possible causes of the high prevalence of dengue in these neighborhoods primarily include the presence of a critical degree of Ae. aegypti proliferation, the presence of many eggs and the frequency of transovarial DENV-4 infections among mosquitoes.In 2012, the average frequency of Ae.Aegypti estimated using LIRAa (Quick Survey Index Aedes aegypti) in the neighborhoods comprising the Jardim Industriário region was 7.9%.This rate is considered high, as the rate recommended by the Ministry of Health is 1% (39).
In this study, the DENV-4 MIR for Ae.aegypti naturally infected via the transovarial route was 10.5% (8/758).Similar data were obtained in a study conducted on adult females obtained from the pupal and larval stages of Aedes in Fortaleza, Ceará, an area with an elevated incidence of dengue.The MIR was 0.5 and 9.4 for Ae.Aegypti and Ae.albopictus, respectively.During the study period, DENV-2 and DENV-3 were the predominant serotypes in the regions where the immature stages of the vector were sampled (27) .
The nucleotide sequence analysis revealed a high degree of similarity with the DENV-4 genotype II from Manaus, AM.This isolate was described in a spatiotemporal study, which provided evidence of at least three introductions of DENV-4 genotype II to Brazil, two from Venezuela to Roraima and one from Colombia to Amazonas, in 2008-2010.After its introduction, DENV-4 exhibited a rapid geographic spread, most likely due to the lack of immunity in the population; this serotype had not been observed in Brazil since 1982 (37) .
Transovarial transmission of DENV may be passed through generations of Ae. aegypti in the absence of infection of the vector by blood feeding, supporting the maintenance of the virus in nature in the absence of susceptible hosts or during adverse environmental conditions (23) (40) .
In MT, intense efforts are made to achieve vectorial control during the rainy season (December to April).Epidemiological surveillance is conducted throughout the year to monitor and investigate high-priority areas with elevated Ae. aegypti infestation indexes (38) .Positivity for DENV-4 was found during each of the three climatic periods (Table 2), indicating that transovarial transmission is not restricted to the vectorial population peaks observed during the rainy season.These fi ndings indicate that vectors are being born already infected and able to transmit the virus at the beginning of epidemics.This ability might serve as a mechanism of virus maintenance between interepidemic periods, thereby favoring DENV transmission to susceptible hosts and subsequent dengue outbreaks in the studied neighborhood.
During one year of evaluation, elevated levels of natural transovarial infection (MIR = 24.4%)were reported in Thailand four months before a large dengue outbreak (41) .The authors of this study detected all four DENV serotypes, but DENV-4 predominated, followed by DENV-3, DENV-1, and DENV-2.Another study with Ae.Aegypti and Ae.Albopictus in the same location that examined adults obtained from immature stages was not able to fi nd any positivity for DENV (42) .Studies conducted in Bolivia, Taiwan, Southeast Asia, Singapore and French Guiana have reported relatively low rates of natural transovarial infection, with MIR indexes ranging from 0.13% to 0.97% for various DENV serotypes (26) (43) (44) (45) (46) .
In Roraima, Aedes aegypti larvae collected in an urban area tested negative for DENV (47) .The authors of this study concluded that natural transovarial transmission might not be an important mechanism for DENV maintenance in the city, and consequently, the presence of susceptible hosts might be frequent in endemic areas.The life cycle of the mosquito may also indicate that infection rates are diluted between generations.However, these conclusions are not applicable to Cuiabá.
In this study, fi ve pools of Ae.Aegypti containing males and three containing females were positive for DENV-4.In addition to the epidemiological relevance of DENV detection in Ae. aegypti females, which are implicated in the transmission of the virus by their hematophagic and essential anthropophilic habits, DENV infections in male mosquitoes are also relevant; they are considered to be a more precise indicator of natural transovarial transmission because they feed only on plant nectar (48) .Moreover, infected males are able to transmit the virus to females during reproduction, resulting in transmission to their eggs (49) .
None of the pools of Ae.Aegypti was positive for YFV in this study.This result was expected because urban transmission of this virus has not been reported in Brazil since 1942.One study conducted in Senegal reported transovarial infection with a MIR of 0.97% in adults emerging from the eggs of females experimentally infected through the oral route (50) .
Additionally in this study, we did not fi nd any Ae.aegypti pools that were positive for WNV or SLEV.Certain Culicidae species are able to transmit WNV vertically, carrying the virus in their eggs when climatic conditions are unfavorable to their proliferation (51) .In Brazil, serological evidence of WNV circulation in birds and equines has already been reported, demonstrating the importance of surveillance for this virus in host and vector species (17) (20) (21) .SLEV is transmitted vertically in several species of mosquitoes.Although this phenomenon has been most frequently described in the genus Culex, there are some reports involving Aedes species, exhibiting a MIR of 1% (52) (53) .This pathogen was recently detected in three patients with dengue and in Culex spp.from Cuiabá, MT (12).However, the cycle of transmission of this virus in the city is poorly understood.Some studies have demonstrated that the dynamics of fl avivirus transmission is infl uenced by mutations, antigenic variability, ecological conditions, and host factors that affect the susceptibility to infection (54).Although most human arbovirus infections are attributed to DENV in Brazil, it is necessary to extend surveillance to other arboviruses potentially circulating in MT, especially those that have already been reported in neighboring states.Because infection with most of these viruses is clinically characterized by undifferentiated acute febrile illness and asymptomatic infections are common, differential diagnosis through molecular methods is essential to estimate the frequency of infection by various arboviruses in the population (7) .
Moreover, the methods currently available for vector population control have failed to control Ae. aegypti despite the efforts of governmental institutions and intensifi cation of the measures already applied.Studies to elucidate the mechanisms of DENV maintenance in nature are scarce, and the development of new vector population control strategies is necessary (55) .
The fi ndings reported in this study demonstrate that natural transovarial infection by DENV-4 is occurring in Ae.Aegypti in Cuiabá with a relatively high MIR i ndex, which favors the birth of mosquitoes already infected with DENV at the beginning of epidemics.This mechanism may be responsible for virus maintenance during interepidemic periods, especially in critical areas with elevated mosquito density and human disease incidence.Entomological surveillance and data on the detection of infected mosquitoes might be used to estimate DENV serotype frequency, allowing greater understanding of the epidemiological factors that infl uence the occurrence of the disease in the population and further supporting the development of vector population control strategies.

FIGURE 2 -
FIGURE 2 -Map showing the locations of the geo-tagged ovitraps installed in Jardim Industriário II, within the city of Cuiabá, State of Mato Grosso in 2012 to collect eggs from mosquitoes of the genus Aedes during the wet, intermediate, and dry seasons.

TABLE 2 -Specimens of Aedes aegypti larvae hatched from eggs collected in ovitraps; pools were tested by multiplex semi-nested RT-PCR for dengue virus 4. Minimum rate of infection, maximum likelihood estimator and confi dence intervals (95% CI) for the Jardim Industriário II, City of Cuiabá, State of Mato Grosso in 2012.
MRI: minimum infection rate, calculated from the number of positive pools divided by the total number of insects tested × 1,000; EMV: maximum likelihood estimator; CI: confi dence interval; *Cycle I: rainy; Cycle II: intermediate; Cycle III: dry.