Variation in vector competence for dengue 2 virus among 24 collections of Aedes aegypti from Mexico and the United States

Aedes aegypti from 24 collections in Mexico and the United States were challenged orally with dengue 2 virus JAM1409 (DEN-2 JAM1409). The vector competence (VC) of the populations ranged from 24% to 83%. Mosquito populations from the Yucatan exhibited greater VC than those from other areas of Mexico. The presence or absence of a midgut infection barrier (MIB) and a midgut escape barrier (MEB) was determined for mosquitoes in each population. The percentage of mosquitoes exhibiting an MIB ranged from 14% to 59%, and those exhibiting an MEB ranged from 4% to 43% in the collections. The MIB and MEB were not completely independent as determined by regression analysis. Midgut infection rates were dose dependent.     

Low oral receptivity for dengue type 2 viruses of Aedes albopictus from Southeast Asia compared with that of Aedes aegypti

Dengue hemorrhagic fever has been a major health problem in Asia since the 1950s. During this period, the former principal vector of dengue viruses in Asia, Aedes albopictus, was replaced by Aedes aegypti in most major cities of the area. Ae. aegypti is now considered the main vector of dengue viruses in Asia. Surprisingly, however, this mosquito has been described as having a relatively low oral receptivity for dengue viruses compared with Ae. albopictus. In the present study, we compared the relative oral receptivities of Ae. aegypti and Ae. albopictus collected in southeast Asia from both sympatric and allopatric breeding sites. In all instances, the oral receptivity of Ae. aegypti to the dengue type 2 virus used was significantly higher than that of Ae. albopictus. We also compared the relative oral receptivity of Ae. aegypti and Ae. albopictus for two other low-passage strains of dengue 2. In all instances, Ae. aegypti was significantly more receptive than Ae. albopictus. It should be noted, however, that the difference was found only for Ae. albopictus recently collected from the field (Ta Promh strain, Cambodia, 2001) and not for an Ae. albopictus strain that had been colonized for many years (Oahu strain, Hawaii, 1971). We also observed a significant increase in the infection rate of Ae. albopictus of the Ta Promh strain with increasing generations in the laboratory. These observations demonstrate the importance of considering the colonization history of mosquitoes when assessing their susceptibility to infection with dengue viruses and, perhaps, other arboviruses.     

Limited potential for transmission of live dengue virus vaccine candidates by Aedes aegypti and Aedes albopictus

To evaluate the transmission risk of four live dengue (DEN) vaccine candidates developed by the U.S. Army (DEN-1, 45AZ5 PDK 20; DEN-2, S16803 PDK 50; DEN-3, CH53489 PDK 20; and DEN-4, 341750 PDK 20), we tested 3,010 Aedes aegypti and 1,576 Aedes albopictus mosquitoes blood-fed on 21 volunteers who had been administered one of the four vaccine candidates or the licensed yellow fever (YF) vaccine (17D). We used an indirect immunofluorescence assay (IFA) to detect DEN or YF viral antigen in the heads of mosquitoes. Corresponding to the lack of a detectable viremia among volunteers inoculated 8-13 days previously with live DEN-1 or DEN-2 vaccine candidates, only six mosquitoes developed disseminated infections after feeding on these volunteers. These six mosquitoes included 4 of 247 Ae. albopictus fed on volunteers inoculated with the DEN-1 vaccine candidate and 2 of 528 Ae. aegypti fed on volunteers inoculated with the DEN-2 vaccine candidate. Infection was confirmed in each of these IFA-positive mosquitoes by isolating infectious virus from the mosquito's body in Vero-cell culture. None of the 1,252 or the 969 mosquitoes fed on DEN-3 or DEN-4 recipients, respectively, were infected. Overall, dissemination rates in Ae. albopictus and Ae. aegypti were low. Dissemination rates were 0.5%, 0.3%, < 0.1%, and < 0.1% for the DEN-1 through DEN-4 vaccine candidates, respectively. Because of the observed low dissemination rates, it is unlikely that these vaccine viruses would be transmitted under natural conditions.     

Effect of temperature on the vector efficiency of Aedes aegypti for dengue 2 virus

The effect of temperature on the ability of Aedes aegypti to transmit dengue (DEN) 2 virus to rhesus monkeys was assessed as a possible explanation for the seasonal variation in the incidence of dengue hemorrhagic fever in Bangkok, Thailand. In two laboratory experiments, a Bangkok strain of Ae. aegypti was allowed to feed upon viremic monkeys infected with DEN-2 virus. Blood-engorged mosquitoes were separated into two groups and retained at constant temperatures. Virus infection and transmission rates were determined for Ae. aegypti at intervals ranging from 4 to 7 days during a 25-day incubation period. Results of the first experiment for mosquitoes infected with a low dose of DEN-2 virus and maintained at 20, 24, 26, and 30 degrees C, indicated that the infection rate ranged from 25% to 75% depending on the incubation period. However, DEN-2 virus was transmitted to monkeys only by Ae. aegypti retained at 30 degrees C for 25 days. In the second experiment, the infection rate for Ae. aegypti that ingested a higher viral dose, and incubated at 26, 30, 32, and 35 degrees C ranged from 67% to 95%. DEN-2 virus was transmitted to monkeys only by mosquitoes maintained at greater than or equal to 30 degrees C. The extrinsic incubation period was 12 days for mosquitoes at 30 degrees C, and was reduced to 7 days for mosquitoes incubated at 32 degrees C and 35 degrees C. These results imply that temperature-induced variations in the vector efficiency of Ae. aegypti may be a significant determinant in the annual cyclic pattern of dengue hemorrhagic fever epidemics in Bangkok.     

Transovarial transmission of dengue viruses by mosquitoes: Aedes albopictus and Aedes aegypti

Transovarial transmission of all four dengue serotypes was demonstrated in Aedes albopictus mosquitoes. The rates of such transmission varied with the serotype and strain of virus. In general, the highest rates were observed with strains of dengue type 1 and the lowest with dengue type 3. Surprisingly, despite the use of viral strains of the four dengue serotypes which gave the highest rates with Ae. albopictus, transovarial transmission was observed in Aedes aegypti only with dengue type 1, and then only at a relatively low rate. Five different strains of Ae. aegypti were employed, including one that was known to be relatively susceptible to oral infection with dengue viruses. The findings support the view that Ae. aegypti, while of major importance from the point of view of transmission of dengue to man, may be relatively unimportant in the overall natural history of dengue viruses.     

Growth characteristics of ChimeriVax-DEN2 vaccine virus in Aedes aegypti and Aedes albopictus mosquitoes

The chimeric yellow fever (YF) 17D-dengue type 2 (ChimeriVax-DEN2) vaccine virus developed by Acambis, Inc. (Cambridge, MA) contains the prM and E genes of wild-type (wt) dengue 2 (DEN-2) (strain PUO-218) virus in the YF vaccine virus (strain 17D) backbone. The potential of ChimeriVax-DEN2 virus to infect and be transmitted by Aedes aegypti, the principal DEN and YF virus mosquito vector, and Aedes albopictus, a species that occurs in areas of active transmission of YF and DEN viruses, was evaluated. Mosquitoes were intrathoracically (IT) inoculated with virus or were fed a virus-laden blood meal, and the replication kinetics of ChimeriVax-DEN2 were compared with the wt DEN-2 and YF 17D vaccine viruses. Replication of YF 17D virus is attenuated in cultured Ae. albopictus C6/36 mosquito cells and in Ae. aegypti and Ae. albopictus mosquitoes. Growth of ChimeriVax-DEN2 virus similarly was restricted in C6/36 cells and in mosquitoes. ChimeriVax-DEN2 replicated in 56% of IT inoculated Ae. aegypti, and virus disseminated to head tissue in 36%, with a mean viral titer of 1.8 log10 PFU/mosquito. Of mosquitoes, 16% of Ae. aegypti and 24% of Ae. albopictus were infected 14 days after a blood meal containing ChimeriVax-DEN2, but virus did not disseminate to head tissue. In contrast, DEN-2 replicated in all IT inoculated and orally infected Ae. aegypti (mean titer 5.5 log10 PFU/mosquito), and virus disseminated to head tissue in 95%. Of Ae. albopictus, 84% were infected after a blood meal containing DEN-2 virus; dissemination occurred in 36%. Replication of ChimeriVax-DEN2 virus in mosquitoes corresponded to that of YF 17D vaccine virus, which is restricted in its ability to infect and replicate in mosquitoes. Therefore, transmission of ChimeriVax-DEN2 virus by vector mosquitoes is unlikely.     

A mosquito densovirus infecting Aedes aegypti and Aedes albopictus from Thailand.

A previously undescribed mosquito densovirus was detected in colonies of Aedes aegypti and Ae. albopictus from Thailand, using a polymerase chain reaction (PCR)-based assay. Phylogenetic analysis of this virus showed it to be most closely related to ADNV isolated from Russian Ae. aegypti. Both Aedes species were susceptible to oral infection with the Thai-strain virus. Larval mortality for Ae. albopictus was higher (82%) than for Ae. aegypti (51%). Aedes aegypti were able to transmit the virus vertically to a high (58%) proportion of G1 progeny, and the virus was maintained persistently for up to six generations. A PCR survey of adult Ae. aegypti and Ae. albopictus in Thailand indicated that only Ae. aegypti are infected in the field, with an overall prevalence of 44%. Densovirus infection in adult Ae. aegypti showed distinct seasonal variation. The Thai strain densovirus may play a role in structuring Ae. albopictus and Ae. aegypti populations in nature.     

Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti

Mosquitoes (Aedes aegypti) were genetically modified to exhibit impaired vector competence for dengue type 2 viruses (DENV-2). We exploited the natural antiviral RNA interference (RNAi) pathway in the mosquito midgut by constructing an effector gene that expresses an inverted-repeat (IR) RNA derived from the premembrane protein coding region of the DENV-2 RNA genome. The A. aegypti carboxypeptidase A promoter was used to express the IR RNA in midgut epithelial cells after ingestion of a bloodmeal. The promoter and effector gene were inserted into the genome of a white-eye Puerto Rico Rexville D (Higgs' white eye) strain by using the nonautonomous mariner MosI transformation system. A transgenic family, Carb77, expressed IR RNA in the midgut after a bloodmeal. Carb77 mosquitoes ingesting an artificial bloodmeal containing DENV-2 exhibited marked reduction of viral envelope antigen in midguts and salivary glands after infection. DENV-2 titration of individual mosquitoes showed that most Carb77 mosquitoes poorly supported virus replication. Transmission in vitro of virus from the Carb77 line was significantly diminished when compared to control mosquitoes. The presence of DENV-2-derived siRNAs in RNA extracts from midguts of Carb77 and the loss of the resistance phenotype when the RNAi pathway was interrupted proved that DENV-2 resistance was caused by a RNAi response. Engineering of transgenic A. aegypti that show a high level of resistance against DENV-2 provides a powerful tool for developing population replacement strategies to control transmission of dengue viruses.     

Population dynamics of Aedes aegypti from a dengue hyperendemic urban setting in Colombia

This study evaluated if the Aedes aegypti population in the city of Cali, Colombia was composed of genetically distinct local populations with different levels of insecticide resistance and dengue vector competence. Insecticide resistance was assayed biochemically and was associated with varying levels of mixed-function oxidases and non-specific esterases. The genes encoding those enzymes were under selective pressure from insecticides used to suppress Ae. aegypti populations. Vector competence showed heterogeneity among the vector populations ranging from 19% to 60%. Population genetic analysis of random amplified polymorphic DNA-polymerase chain reaction products, expressed as genetic distance, Wright's F(st), and migration rate (Nm), demonstrated moderate genetic differentiation among Ae. aegypti from four sites (F(st) = 0.085). The results from all characteristics evaluated in the study demonstrated spatial and temporal variation between Ae. aegypti populations. At any specific time, the local populations of Ae. aegypti were genetically differentiated and unique with respect to insecticide resistance and vector competence. Both characteristics changed independently.     

The effects of midgut serine proteases on dengue virus type 2 infectivity of Aedes aegypti

Dengue viruses (DENV) cause significant morbidity and mortality worldwide and are transmitted by the mosquito Aedes aegypti. Mosquitoes become infected after ingesting a viremic bloodmeal, and molecular mechanisms involved in bloodmeal digestion may affect the ability of DENV to infect the midgut. We used RNA interference (RNAi) to silence expression of four midgut serine proteases and assessed the effect of each RNAi phenotype on DENV-2 infectivity of Aedes aegypti. Silencing resulted in significant reductions in protease mRNA levels and correlated with a reduction in activity except in the case of late trypsin. RNA silencing of chymotrypsin, early and late trypsin had no effect on DENV-2 infectivity. However, silencing of 5G1 or the addition of soybean trypsin inhibitor to the infectious bloodmeals significantly increased midgut infection rates. These results suggest that some midgut serine proteases may actually limit DENV-2 infectivity of Ae. aegypti.     

Polygamy: the possibly significant behavior of Aedes aegypti and Aedes albopictus in relation to the efficient transmission of dengue virus

The polygamous behavior of male Aedes aegypti (L.) and Ae. albopictus (Skuse) was investigated by co-habiting a newly-emerged male and females in a 30 cm3 cage (1 male: 20 females) for up to 5 consecutive days. As determined by insemination rates, the results indicated that one Ae. aegypti and Ae. albopictus male could successfully mate with 1.10 (0-4), 4.10 (1-8), 5.40 (4-8), 5.10 (2-8), 5.15 (3-9) and 0.20 (0-3), 1.70 (0-3), 2.35 (1-4), 2.30 (0-4), 2.35 (1-4) Ae. aegypti and Ae. albopictus females, respectively on day 1,2,3,4 and 5 consecutively. The possibly significant role of their polygamy in relation to dengue virus transmission is discussed.     

Aedes albolateralis, a potential vector of nocturnally subperiodic Wuchereria bancrofti and dengue type 2 virus

The susceptibility of Aedes albolateralis to nocturnally subperiodic Wuchereria bancrofti and dengue type 2 virus was investigated by using artificial membrane feeding and intrathoracic inoculation techniques, respectively. The results indicated that Ae. albolateralis was susceptible to nocturnally subperiodic W. bancrofti (susceptibility rate = 9.43%) and dengue type 2 virus (susceptibility rate = 100%), suggesting the potential vector of the two pathogens.     

Role of Aedes aegypti and Aedes albopictus during the 2011 dengue fever epidemics in Hanoi,Vietnam

Objective: To record the human cases of dengue fever(DF) and investigate the Aedes mosquito species circulating during the Hanoi 2011 DF epidemics. Methods: 24 different outbreak points were recorded in 8 districts between August and December 2011. Results: 140 patients were hospitalized following dengue diagnostic with a predominance of males(59.3%) and the 15-34 age class. Only DENV-1(11.27%) and DENV-2(88.73%) serotypes were detected in human samples. Mosquito sampling performed in and around patients households revealed the predominance of Aedes aegypti(95.15%) versus Aedes albopictus(4.85%). There is a positive correlation between the population density of Aedes aegypti and the number of human cases and duration of outbreaks. Conclusions: This was not observed for Aedes albopictus. 3 pools of Aedes aegypti were positive with dengue virus, two with DENV-1 and one with DENV-2.     

不同温度对DEN-2型病毒在白纹伊蚊体内外潜伏期的影响

摘要：目的 研究温度对外潜伏期的影响。方法 登革2型病毒人工经口感染白纹伊蚊,冻麻挑出吸饱血的雌蚊放入新的蚊笼,分别置于18℃、２1℃、26℃、31℃和36℃中饲养,第3、4、5、7、10、14、20、25天分别收集并解剖白纹伊蚊,间接免疫荧光法分别检测蚊虫头部、唾液腺和胸腹部的登革2型病毒抗原。结果 置于18、21、26、31和36℃饲养的白纹伊蚊分别在第25、7、5、4、3天时胸腹部中开始检出阳性。21、26、31和36℃饲养的蚊虫唾液腺阳性检出的时间分别为10、7、4、4天,18℃在感染后的25天内,唾液腺均未检出阳性。结论 随着温度的升高,白纹伊蚊的外潜伏期逐渐缩短。     

Variation among strains of Aedes aegypti in susceptibility to oral infection with dengue virus type 2

We compared 18 Aedes aegypti strains for oral susceptibility to dengue virus type 2 (DEN-2) using a feeding protocol in which all parameters remained constant, including the titer of the infectious bloodmeal. For most strains, no significant variation between replicates was observed. Comparisons between pairs of strains showed variation of different degrees, and allowed us to characterize the strains with respect to their oral susceptibility to DEN-2.     

Large genetic differentiation and low variation in vector competence for dengue and yellow fever viruses of Aedes albopictus from Brazil,the United States,and the Cayman Islands

We conducted a population genetic analysis of Aedes albopictus collected from 20 sites in Brazil, the United States (Florida, Georgia, and Illinois), and the Cayman Islands. Using isoenzyme analysis, we examined genetic diversity and patterns of gene flow. High genetic differentiation was found among Brazilian samples, and between them and North American samples. Regression analysis of genetic differentiation according to geographic distances indicated that Ae. albopictus samples from Florida were genetically isolated by distance. Infection rates with dengue and yellow fever viruses showed greater differences between two Brazilian samples than between the two North American samples or between a Brazilian sample and a North American sample. Introductions and establishments of new Ae. albopictus populations in the Americas are still in progress, shaping population genetic composition and potentially modifying both dengue and yellow fever transmission patterns.     

Dispersal of the dengue vector Aedes aegypti within and between rural communities

Knowledge of mosquito dispersal is critical for vector-borne disease control and prevention strategies and for understanding population structure and pathogen dissemination. We determined Aedes aegypti flight range and dispersal patterns from 21 mark-release-recapture experiments conducted over 11 years (1991-2002) in Puerto Rico and Thailand. Dispersal was compared by release location, sex, age, season, and village. For all experiments, the majority of mosquitoes were collected from their release house or adjacent house. Inter-village movement was detected rarely, with a few mosquitoes moving a maximum of 512 meters from one Thai village to the next. Average dispersal distances were similar for males and females and females released indoors versus outdoors. The movement of Ae. aegypti was not influenced by season or age, but differed by village. Results demonstrate that adult Ae. aegypti disperse relatively short distances, suggesting that people rather than mosquitoes are the primary mode of dengue virus dissemination within and among communities.     

Short report: microsatellite sequences as markers for population genetic studies of the mosquito Aedes aegypti, the vector of dengue viruses

We report the isolation of microsatellites from an enriched library of genomic repeated sequences, using a biotin-labeled oligonucleotide bound to streptavidin-coated magnetic particles. Four microsatellites were obtained from a partial library of 120 recombinant clones. This more efficient and rapid method to obtain these specific repeated sequences is preferred to the conventional isolation procedure based on the construction of a genomic library. Microsatellite markers would be promising molecular tools for the study of genetic variability of mosquito populations. Analyses of genetic structure and gene flow would provide information on the distance, direction and rate of dispersal of genes in Aedes aegypti populations. Knowledge on gene dispersal patterns is required to develop vector control strategies.     

Efficiency of dengue serotype 2 virus strains to infect and disseminate in Aedes aegypti

Dengue serotype 2 (DEN-2) viruses with the potential to cause dengue hemorrhagic fever have been shown to belong to the Southeast (SE) Asian genotype. These viruses appear to be rapidly displacing the American genotype of DEN-2 in the Western Hemisphere. To determine whether distinct genotypes of DEN-2 virus are better adapted to mosquito transmission, we classified 15 viral strains of DEN-2 phylogenetically and compared their ability to infect and disseminate in different populations of Aedes aegypti mosquitoes. Envelope gene nucleotide sequence analysis confirmed that six strains belonged to the American genotype and nine strains were of the SE Asian genotype. The overall rate of disseminated infection in mosquitoes from Texas was 27% for the SE Asian genotype versus 9% for the American genotype. This pattern of infection was similar in another population of mosquitoes sampled from southern Mexico (30% versus 13%). Together, these findings suggest that Ae. aegypti tends to be more susceptible to infection by DEN-2 viruses of the SE Asian genotype than to those of the American genotype, and this may have epidemiologic implications.