Selection of D2S3, an Aedes aegypti (Diptera: Culicidae) strain with high oral susceptibility to Dengue 2 virus and D2MEB, a strain with a midgut barrier to Dengue 2 escape

Family-based phenotypic selection was used to breed two genetic strains of Aedes aegypti L. that differ in susceptibility to infection with dengue serotype 2 virus (DEN-2) strain JAM1409. A Dengue 2 Susceptible on 3 chromosomes (D2S3) strain was bred from Ae. aegypti aegypti and Ae. aegyptiformosus P1 parents to have a high midgut infection rate (MIR) and a high disseminated infection rate (DIR). A Dengue 2 Midgut Escape Barrier (D2MEB) strain was bred from D2S3 and Houston P1 parents to have a high MIR and a low DIR. After selection in the F2 generation, single strand conformation polymorphism genotypes were determined at cDNA marker loci throughout the genome to test for Mendelian ratios and thereby identify regions containing deleterious or lethal alleles. Both strains were orally challenged with two other DEN-2 genotypes, two DEN-1 genotypes, one DEN-3 genotype, and two DEN-4 genotypes. There were significant differences in MIR and DIR for the different virus strains in both the D2S3 and the D2MEB mosquito lines.     

Enhanced vector competence of Aedes aegypti (Diptera: Culicidae) from the Torres Strait compared with mainland Australia for dengue 2 and 4 viruses

Australian Aedes aegypti (L.) mosquitoes colonized from the Torres Strait and three mainland localities (Charters Towers, Townsville, and Cairns) were fed on blood suspensions containing dengue virus type 2 (DEN-2) or dengue virus type 4 (DEN-4). Variation was found in oral susceptibility to DEN-2 (59 -99% infection) and DEN-4 (28-79% infection) among Ae. aegypti assayed for virus at 8, 12, 16, or 20 d after ingestion of infected blood. Torres Strait Ae. aegypti were the most susceptible to DEN-2 and were significantly more efficient in transmission to capillary tube at 16 d (76% transmission) than mainland Ae. aegypti populations (20-28% transmission). Torres Strait Ae. aegypti were also the most susceptible to DEN-4, although transmission did not vary significantly from mainland populations at 16 d (12% compared with 0-4%) or 20 d (16% compared with 4-16%). Disseminated infection (i.e., leg infection) with either DEN-2 or DEN-4 was not an accurate predictor of transmission potential. This study demonstrates differences among Australian Ae. aegypti populations in vector competence for DEN-2 and DEN-4. Torres Strait Ae. aegypti were more frequently infected and able to transmit DEN-2 at higher rates than mainland populations. These data indicate that the Torres Strait region is potentially more receptive to dengue transmission than mainland localities, a finding discussed with respect to past outbreaks.     

Horizontal and vertical transmission of dengue virus type 2 in highly and lowly susceptible strains of Aedes aegypti mosquitoes.

Isofemale lines of Aedes aegypti mosquitoes highly and lowly susceptible to dengue type 2 (DEN-2) virus (DEN(h) and DEN(l), respectively) were established by oral feeding and individual rearing. The susceptibility at F13 generation was found to be 61% and 25% for the DEN(h) and DEN(l) line, respectively. The virus-infected mosquito females were allowed to probe on bovine albumin phosphate saline pH 7.2 (BAPS) through membrane feeders. The presence of virus in the probed BAPS was determined either by ELISA or by intrathoracic (i.t.) inoculation of mosquitoes or by both methods. The rate of oral transmission of virus was found to be 2 times higher in the DEN(h) isofemale line than in the DEN(l) one. Similarly, vertical transmission rate of the virus was found to be 7 times higher in the DEN(h) line. When batches of eggs obtained from infected female mosquitoes were allowed to hatch after two months the vertical transmission rate of the virus was very high. It is possible that, at room temperature, the virus gets an opportunity to multiply and increase its copy number in the quiescent embryos. The progeny obtained from the infected mosquitoes was found to be capable of transmitting the virus horizontally when allowed to probe on BAPS through the membrane feeder. This is the first report demonstrating horizontal transmission of DEN-2 virus by mosquitoes infected through vertical transmission. The higher vertical transmission rate of the virus in the progeny obtained from the eggs dessicated for a longer time and the horizontal transmission of the virus from the progeny is of very high epidemiological significance.     

Susceptibility of selected strains of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) to chikungunya virus.

The relative susceptibility of selected strains of Aedes aegypti (L.) and Aedes albopictus (Skuse) fed on a viremic monkey to infection with chikungunya virus was determined. Infection rates were consistently higher in 10 strains of Ae. albopictus tested than in 7 strains of Ae. aegypti tested, regardless of the geographic location from which the strains originated or the dose of virus ingested. Similarly, virus dissemination rates were higher in the Ae. albopictus strains compared with the Ae. aegypti strains. For nearly all (11 of 12) strains tested of both species, groups of mosquitoes with one or more females with a disseminated infection transmitted virus by bite to weanling mice. Based on these studies, Ae. albopictus appears to be a more competent laboratory vector of chikungunya virus than does Ae. aegypti.     

Analysis of inheritance of oral susceptibility of Aedes aegypti (Diptera: Culicidae) to dengue-2 virus using isofemale lines

Isofemale lines were compared to determine possible genetic variation in oral susceptibility within the Fare strain of Aedes aegypti (L.) to dengue-2 (DEN-2) virus. Three groups of 12 isofemale lines each were tested statistically using the SAS CATMOD procedure of analysis of variance. The "isofemale line" effect was highly significant, demonstrating genetic variability in oral susceptibility among females. The length of time eggs were stored before hatching influenced the oral susceptibility of the adult mosquitoes for DEN-2 review.     

Comparative analysis of midgut bacterial communities of Aedes aegypti mosquito strains varying in vector competence to dengue virus

Differences in midgut bacterial communities of Aedes aegypti, the primary mosquito vector of dengue viruses (DENV), might influence the susceptibility of these mosquitoes to infection by DENV. As a first step toward addressing this hypothesis, comparative analysis of bacterial communities from midguts of mosquito strains with differential genetic susceptibility to DENV was performed. 16S rRNA gene libraries and real-time PCR approaches were used to characterize midgut bacterial community composition and abundance in three Aedes aegypti strains: MOYO, MOYO-R, and MOYO-S. Although Pseudomonas spp.-related clones were predominant across all libraries, some interesting and potentially significant differences were found in midgut bacterial communities among the three strains. Pedobacter sp.- and Janthinobacterium sp.-related phylotypes were identified only in the MOYO-R strain libraries, while Bacillus sp. was detected only in the MOYO-S strain. Rahnella sp. was found in MOYO-R and MOYO strains libraries but was absent in MOYO-S libraries. Both 16S rRNA gene library and real-time PCR approaches confirmed the presence of Pedobacter sp. only in the MOYO-R strain. Further, real-time PCR-based quantification of 16S rRNA gene copies showed bacterial abundance in midguts of the MOYO-R strain mosquitoes to be at least 10–100-folds higher than in the MOYO-S and MOYO strain mosquitoes. Our study identified some putative bacteria with characteristic physiological properties that could affect the infectivity of dengue virus. This analysis represents the first report of comparisons of midgut bacterial communities with respect to refractoriness and susceptibility of Aedes aegypti mosquitoes to DENV and will guide future efforts to address the potential interactive role of midgut bacteria of Aedes aegypti mosquitoes in determining vectorial capacity for DENV.     

Passage of ingested Mansonella ozzardi (Spirurida: Onchocercidae) microfilariae through the midgut of Aedes aegypti (Diptera: Culicidae)

When virus and microfilariae are ingested concurrently by a mosquito, microfilariae (mf) may penetrate the mosquito midgut and introduce virus directly into the mosquito hemocoel, allowing mosquitoes to become infectious much sooner than normal and enhancing transmission of viruses by mosquitoes. Mansonella ozzardi (Manson) is a benign filarial nematode parasite of humans in Latin America and is transmitted by black flies (Diptera: Simuliidae) and biting midges (Diptera: Ceratopogonidae). Because M. ozzardi and dengue are sympatric, we wanted to know whether M. ozzardi mf had the ability to penetrate the midgut of Aedes aegypti (L.) (Diptera: Culicidae) and thus play a potential role in the enhancement of dengue transmission. To test this, the F1 progeny from locally collected Ae. aegypti were fed on M. ozzardi-infected human males in an endemic village in northern Trinidad. Mosquitoes were dissected at various times after feeding and examined for mf in the midguts and thoraces. Microfilariae penetrated the midguts of 43% of 63 mosquitoes that ingested mf. Overall, 11% of mf penetrated the midgut by 17 h after being ingested. The intensity of midgut penetration was positively correlated to the numbers of mf ingested. Because midgut penetration is a key requirement for mf enhancement to occur, the potential exists that M. ozzardi could be involved in the enhancement of dengue virus transmission.     

Effect of urea on the hemagglutinating and complement-fixing antigens of type 2 Dengue virus

Sephadex G-200 filtration was used for fractionating dengue type 2 (DEN-2)-infected suckling mouse brain (SMB) supernatant fluids. The high-molecular-weight fraction, which was eluted in the void volume, showed no increase in type specificity when tested against immune ascitic fluid to the four prototype dengue viruses. A void-volume fraction obtained after the infected SMB supernatant fluids were treated with urea displayed significant increases in complement fixation (CF) type specificity. Immune ascitic fluid prepared against the more typespecific DEN-2 antigen demonstrated neutralizing ability and greater CF type specificity. When DEN-2 sucrose-acetone-extracted hemagglutinating (HA) antigens were treated with 6 m urea at 37 C for various time intervals, all HA antigen was destroyed in 15 min. Urea treatment of infected SMB supernatant fluids indicated that the CF antigens were more stable to the effect of urea than were the HA antigens. After urea treatment of the SMB supernatant fluids, the CF type specificity increased as the hemagglutination inhibition titer decreased.     

RNA interference, arthropod-borne viruses, and mosquitoes

RNA interference (RNAi) probably functions as an antiviral mechanism in most eukaryotic organisms. Variations in the activity of this antiviral pathway in mosquitoes could explain, in part, why some mosquitoes are competent vectors of medically important, arthropod-borne viruses (arboviruses) and others are not. There are three lines of evidence that show the RNAi pathway exists in Aedes species that transmit arboviruses. The first is that recombinant Sindbis viruses expressing a RNA fragment from a genetically unrelated dengue-2 virus (DENV-2) interfere with DENV-2 replication in Aedes aegypti mosquitoes by a mechanism similar to virus-induced gene silencing described in plants. The second is that transfection of C6/36 (Aedes albopictus) cells with either double-stranded RNA or synthetic small interfering RNAs derived from an arbovirus genome interferes with replication of the homologous virus. The third is that a hairpin DENV-2-specific RNA transcribed from a plasmid can generate virus-resistant C6/36 cells. We hypothesize that genetically modified mosquitoes can be generated that transcribe a flavivirus-specific dsRNA, triggering the RNAi response soon after ingestion of a blood meal. This could induce the RNAi pathway in the midgut prior to establishment of virus infection and profoundly change vector competence. Towards this goal, we are developing transgenic A. aegypti lines that are refractory to DENV by exploiting the RNAi pathway.     

Comparison of the transmission potential of two genetically distinct Sindbis viruses after oral infection of Aedes aegypti (Diptera: Culicidae)

Within mosquitoes, arboviruses encounter barriers to infection and dissemination that are critical determinants of vector competence. The molecular mechanisms responsible for these barriers have yet to be elucidated. The prototype Sindbis (SIN) strain, AR339, and viruses derived from this strain, such as TR339 virus, have limited infection and transmission potential in the medically important arthropod vector, Aedes aegypti (L.). However, the Malaysian SIN virus strain, MRE16, disseminates in nearly 100% of Ae. aegypti 14 d after oral infection. Here, we compare the spatial and temporal infection patterns of MRE16 and TR339 viruses in Ae. aegypti. The results indicate that a midgut escape barrier is primarily responsible for the significantly lower dissemination and transmission potentials observed after oral infection with TR339 virus. MRE16 and TR339 viruses now represent a well-characterized model system for the further study of virus determinants of vector infection, particularly determinants affecting the midgut escape barrier in Ae. aegypti.     

Oral receptivity of Aedes aegypti formosus from Franceville (Gabon, central Africa) for type 2 dengue virus

Dengue is widely distributed in the tropics but epidemic activity was rarely reported in Africa before the 1980's. In the past 15 years, increased epidemic dengue fever has been reported both in East and West Africa, raising concern about the ability of local populations of Aedes aegypti to transmit dengue viruses. Ae. aegypti is present in two forms in Africa: Ae. aegypti aegypti and Ae. aegypti formosus. This latter form, much darker, was not originally a local species but is now colonizing artificial breeding sites within cities. We have been able to demonstrate the oral susceptibility for dengue type 2 virus of Ae. aegypti formosus collected in Franceville, Gabon (Central Africa). However, these mosquitoes sampled exhibited lower infection rates than those of a control colony of Ae. aegypti aegypti originating from French Polynesia.     

贵州白纹伊蚊对登革病毒易感性的研究

目的 用细胞、分子生物学技术进行贵州省白纹伊蚊不同地理株对登革病毒(DEN)易感性的研究。方法 采集贵州省9个地(州)市共计15个县(区)白纹伊蚊幼虫标本，饲养为成蚊；取羽化后3～5日龄期的贵州不同地理株白纹伊蚊，用不同型别的DEN分别经口连续感染3d，于首次感染后的4、7、10、14d收集感染成蚊标本；制备蚊悬液，碘化钠法提取RNA，用DENNS1基因区通用引物经逆转录．聚合酶链反应(RT-PCR)检测DEN核酸；蚊悬液接种C6／36细胞进行病毒分离，制作细胞抗原片，经间接免疫荧光法检测DEN抗原；同时感染白纹伊蚊海南株作为对照。结果 DEN1-4型国际参考株感染白纹伊蚊贵州省不同地方株，其感染比率分别为12／15、12／15、8／15和13／15。结论 白纹伊蚊贵州省不同地方株对DEN1-4型国际参考株普遍易感，表明贵州省具备引起登革热流行的条件。     

Maturation of influenza A virus hemagglutinin--estimates of the pH encountered during transport and its regulation by the M2 protein.

The susceptibility of H7 influenza A viruses to the M2-mediated alteration in HA resulting from treatment with amantadine or rimantadine depends both on the pH stability of HA and the pH encountered during transport to the plasma membrane of the particular virus-infected cell. pH stabilities of a range of virus mutant HAs exhibited an inverse correlation with drug sensitivity and the proportion of low-pH HA expressed on the surface of infected cells in the absence of drug. The lowest pH encountered during transport was thus estimated from the proportion of HA expressed in its native conformation and its pH stability profile. Lower drug sensitivity and improved HA maturation of MDCK cells compared to that in CEF reflect the higher pH within the appropriate compartment of the former. Differences in apparent pH resulting from infection with two closely related virus strains, Rostock or Weybridge, e.g., approximately 6.0 and 5.4, respectively, in CEF, were abrogated by rimantadine treatment (pH approximately 5.2 in CEF) and were attributable to intrinsic properties of their respective M2 proteins. The greater activity of the Rostock M2, which was estimated to be capable of increasing vesicular pH by as much as 0.8 pH units, correlates with the lower pH stability of the HA. This emphasizes the essential relationship between the characteristics of the two virus proteins as well as the subtle role of M2 in regulating the pH of the transport pathway to protect the structural integrity of the hemagglutinin glycoprotein.     

Synthesis of dengue virus RNA in vitro: initiation and the involvement of proteins NS3 and NS5

Summary An assay for flavivirus RNA-dependent RNA polymerase activity in vitro was established using extracts of Vero cells infected with dengue virus type 2 (DEN-2) or Kunjin virus (KUN). RNA synthesis was initiated on a template of viral replicative form (RF) and RF was converted to the replicative intermediate (RI). The RNA-dependent RNA polymerase complex of DEN-2 utilised either DEN-2 or KUN RF as template, and similarly the KUN polymerase complex utilised either DEN-2 or KUN RF template. In addition, antibodies against the nonstructural proteins NS3 and NS5 inhibited the conversion of RF to RI, indicating that NS3 and NS5 are involved in viral RNA replication.     

Production of viral antigens in culture fluid of C6/36 mosquito cell line infected with dengue type 4 virus strains isolated from patients with different clinical severities

Viral antigen production was examined in the culture fluid of Aedes albopictus clone C6/36 cell line incubated at 28 degrees C and 37 degrees C after infection with four strains of dengue type 4 (DEN-4) virus which were isolated from patients with different clinical severities. During the observation period from day 1 to day 18, the number of infected cells at each day for all four strains did not show any significant difference (P >0.05). Antigen production as determined by the hemagglutination (HA) test and sandwich enzyme linked immunosorbent assay (ELISA) was higher at 28 degrees C than at 37 degrees C for all four DEN-4 virus strains examined. The amount of viral antigen produced was highest for CT93-74 strain (dengue hemorrhagic fever syndrome (DHF) grade II) and was significantly different in comparison to other strains (P <0.001). This strain was followed by CT93-158 and CT93-129 strains (both of DHF grade I), and CT93-77 strain (dengue fever (DF)). The viral antigen production was apparently proportional to the clinical severity of the patient from whom the virus was isolated. These results show that CT93-74 strain could be used to produce DEN-4 virus antigen of sufficiently high titer in the C6/36 cell culture instead of classical extraction of this antigen from suckling mouse brains.     

Susceptibility of Aedes aegypti, Culex quinquefasciatus Say, and Anopheles quadrimaculatus Say to 19 pesticides with different modes of action

To access the relative potency of pesticides to control adult mosquitoes, 19 pesticides with various modes of action were evaluated against Aedes aegypti, Culex quinquefasciatus Say, and Anopheles quadrimaculatus Say. On the basis of 24-h LD50 values after topical application, the only pesticide that had higher activity than permethrin was fipronil, with LD50 values lower than permethrin for 107-, 4,849-, and 2-fold against Ae. aegypti, Cx. quinquefasciatus Say, and An. quadrimaculatus Say, respectively. Abamectin, imidacloprid, spinosad, diazinon, and carbaryl showed slightly lower activity than permethrin (<20-fold). However, bifenazate showed very low activity against the three mosquito species tested, with LD50 values higher than permthrin for >1000-fold. On the basis of 24-h LD50 values, Cx. quinquefasciatus was the least susceptible species to nine pesticides tested (DNOC, azocyclotin, chlorfenapyr, carbaryl, spinosad, imidaclorid, diazinon, abamectin, and permethrin) , whereas Ae. aegypti was the least susceptible species to six pesticides tested (dicofol, amitraz, propargite, hydramethylnon, cyhexatin, and diafenthiuron), and An. quadrimaculatus was the least susceptible species to four pesticides tested (bifenazate, pyridaben, indoxacarb, and fipronil). Our results revealed that different species of mosquitoes had different susceptibility to pesticides, showing the need to select the most efficacious compounds for the least susceptible mosquito species to achieve successful mosquito control.     

Rates of infection in, and transmission of, African horse-sickness virus by Aedes aegypti mosquitoes.

Very low infection rates (less than 3%) were obtained when Aedes aegypti mosquitoes ingested blood contained 5.8--6.5 log10 MLD50/0.02 ml African horse sickness virus (AHSV). When A. aegypti mosquitoes were inoculated intrathoracically with virus, however, high infection rates were achieved. Mosquitoes infected by inoculum failed to transmit virus to embryonated hens eggs by bite, and virus could not be detected in membrane or blood when inoculated mosquitoes were allowed to engorge on uninfected blood through a chick skin membrane. It was concluded that the mosquito A. aegypti is unlikely to be an effective vector of AHSV.