Variations of four Aedes aegypti mosquito strains in their susceptibility to and transmissibility of Tahyna virus

Aedes aegypti mosquitoes originating from different colonies were infected with Tahyna virus by sucking on viraemic newborn mice (peak titre 10(2) X 5 mouse i.c. LD50/0.01 ml). The mosquitoes of laboratory strain "London" proved most susceptible (infection rate 74.2%, transmission rate 60%), while mosquitoes of the laboratory strain "Basel" and strain "Bangkok" (Southeast Asia) were less susceptible (infection rate 23.7 and 17.6%, respectively, transmission rate 6.0 and 10.0%, respectively). Mosquitoes of "Ifakara" strain (East Africa) were found resistant to Tahyna virus infection.     

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.     

Colonization of Aedes aegypti midgut by the endosymbiont-bearing trypanosomatid Blastocrithidia culicis

Monoxenous trypanosomatids inhabit invertebrate hosts throughout their life cycle. However, there have been cases of HIV-positive patients who have presented opportunistic infections caused by these protozoa, offering new perspectives to the study of interactions between monoxenics and hematophagous insect vectors. Some monoxenous trypanosomatids present a symbiotic bacterium in the cytoplasm, which seems to promote biochemical and morphological changes in the host trypanosomatids, such as alterations in plasma membrane carbohydrates and the reduction of the paraxial rod. In this work, we investigated the colonization of Aedes aegypti with Blastocrithidia culicis, an endosymbiont-bearing trypanosomatid. B. culicis remained in the insect digestive tract for 38 days after feeding. Optical microscopy analysis revealed an infection process characterized by a homogenous distribution of the trypanosomatid along the midgut epithelium; no preferential interaction of protozoa with any cell type was observed. Ultrastructural analysis showed that during the colonization process, trypanosomatids interacted mainly with midgut cells through their flagellum, which penetrates the microvilli preferentially near the tight junctions. Prolonged infections promoted insect midgut degradation, culminating with the arrival of protozoa in the hemocel. By demonstrating B. culicis colonization in a bloodsucking insect, we suggest that vector transmission of monoxenous trypanosomatids to vertebrate host may occur in nature.     

Comparative study of dengue 2 virus replication and isoenzymes in two strains of Aedes aegypti

This study, dealing with two strains of Aedes aegypti from Vietnam and French Guiana, shows the variability of the genes coding for 11 isoenzymatic systems and the replication of the dengue 2 virus in parenterally infected mosquitoes. Slight differences are observed in the characteristics of viral replication. No clear correlation is shown with enzymatic patterns which appear widely different from one strain to the other with four of the enzymes studied.     

Aedes aegypti salivary gland extracts modulate anti-viral and TH1/TH2 cytokine responses to sindbis virus infection

Vector-borne viruses are naturally transmitted when a vector salivates during feeding on a vertebrate host. Most laboratory studies of infection disregard the role that the vector plays in the pathogenesis of the virus. In this study, intradermal inoculations of Aedes aegypti salivary gland extract (SGE) and Sindbis virus (SINV) were used to investigate the effect of mosquito feeding on the vertebrate immune response to infection with an arthropod-borne virus. Murine cytokine expression in the skin was quantified by means of real-time RT-PCR. In response to co-inoculation of SINV with SGE, interferon (IFN)-beta expression at 24 and 72 h post inoculation was significantly reduced by 2.2- and 2.3-fold, respectively, when compared to injection of virus alone. IFN-gamma expression in response to SINV infection was significantly decreased by 1.6-fold at 24 h post inoculation when SGE was co-inoculated. In contrast, interleukin (IL)-4 expression was significantly up regulated when SGE was co-inoculated at 24 h post inoculation becoming a 3.3-fold increase by 72 h post inoculation. Compared to expression with SINV alone, IL-10 expression showed a 7.6-fold increase by 72 h post inoculation in mice receiving SGE concurrently with virus. This study suggests that the response to virus is significantly different when an infection is initiated in the presence of mosquito salivary factors, and we identify a possible mechanism for potentiation of viral infections initiated by the natural mosquito vector or in the presence of mosquito saliva.     

The susceptibility of cell lines ofAedes aegypti (Linn.),Aedes albopictus (Skuse) andAedes pseudoscutellaris (Theobald) to infection with Bluetongue virus

Summary Bluetongue virus multiplied in cell lines derived fromAedes albopictus andAedes pseudoscutellaris cells. Virus reached a maximum titre in theAe. pseudoscutellaris cells three days post inoculation, and inAe. albopictus cells six days p. i. Virus growth was demonstrated in both cell lines at 27° C and 37° C. Significant titres of virus were still present in theAe. albopictus cells after five subcultures at 27° C over a period of six weeks. No cytopathic effect was observed in either cell line.A third cell line derived from the mosquitoAe. aegypti did not support the growth of Bluetongue virus.With 1 Figure     

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.     

Dengue-2 virus infection of human mononuclear cell lines and establishment of persistent infections

Summary Twenty three human mononuclear cell lines including ten myelomonocytic cell lines, eight B cell lines and five T cell lines, were examined to determine whether they could be infected with dengue-2 virus. All the cell lines were infected with dengue-2 virus as determined by immunofluorescent staining and by virus titration of culture supernatant fluids. K 562, Jiyoye and Jurkat, respectively, showed the highest percentage of infected cells of these myelomonocytic, B and T cell lines. Antibody to dengue-2 virus at subneutralizing concentrations augmented dengue-2 virus infection of myelomonocytic cell lines, but not of B cell lines or of T cell lines.Persistent dengue-2 virus infection was established using a myelomonocytic cell line (K562), a B cell line (Raji), and a T cell line (HSB-2). These cell lines maintained a high percentage (more than 70%) of dengue-2 virus antigen-positive cells for at least 25 weeks. Very low titers of infectious dengue-2 virus were detected in the culture supernatant fluids of the persistently infected cells. Dengue-2 virus antigen-positive Raji cell clones were established from persistently-infected Raji cells using limiting dilutions and all of the cells in these clones were dengue-2 virus antigen-positive. These findings demonstrate that a variety of human mononuclear cell lines can be infected with dengue-2 virus and may be useful as models for the analysis of dengue virus-human cell interactions in dengue virus infections.     

Toxicity of Brazilian plant seed extracts to two strains of Aedes aegypti (Diptera: Culicidae) and nontarget animals

Seed ethanolic extracts of 21 Brazilian plants were evaluated for ovicidal, larvicidal, and pupicidal activities against insecticide-susceptible (SS) and field-collected (FC) strains of Aedes aegypti (L.) (Diptera: Culicidae), as well as for their effects on nontarget organisms. Myracrodruon urundeuva Fr. Allemao extract was highly toxic to both mosquito strains. Schinopsis brasiliensis Engler extract showed low toxicity and was 38-68 times less toxic to Ae. aegypti larvae than was M. urundeuva extract. The pupicidal activity (LC50) of 14 plant seed extracts ranged between 9 and 433/g/ml, and toxicities were comparable to both mosquito strains. Piptadenia moniliformis Benth. and Luetzelburgia auriculata (Allemao) Ducke extracts showed the highest activities against pupae of FC and SS strains. None of the extracts showed 100% ovicidal activity. In addition, the active extracts did not show high acute toxicity to mice (LD50 > 1.5 g/kg), except that of Enterolobium contortisiliquum (Vell.) Morong. Most of the active extracts exhibited low toxicity against brine shrimp (Artemia sp.) nauplii. The extracts of M. urundeuva, P. moniliformis, and L. auriculata are promising sources of recognized classes of insecticidal compounds with good selectivity against immature stages of Ae. aegypti.     

Classical fowl plague virus reproduction in the body of Aedes aegypti mosquitoes

The results of the studies on fowl plague virus (FPV, Rostok strain) reproduction in Aedes aegypti mosquitoes are presented. The virus-containing allantoic fluid was inoculated intrathoracally in volumes of 0.1 and 0.2 microliter. The virus was isolated in chick embryos and could be detected at 5--14 days after inoculation. After inoculation of 0.1 microliter of virus it could be detected in doses of 0.5, 2.0, 1.75 Ig2 ID50, after inoculation of 0.2 microliter--in doses of 5, 1.5, and 0.5 Ig2 ID50.     

Failure of dengue-2 virus antibody to interfere with the isolation of dengue-2 virus from Aedes aegypti (Diptera: Culicidae)

When isolating dengue virus (DEN) from mosquitoes collected in endemic areas, pools may contain both anti-dengue antibodies from freshly engorged females and virus from DEN infected females. To determine if these antibodies may interfere with virus isolation, we simulated the isolation procedure using Aedes aegypti (L.) that we infected with the 16,681 strain of dengue type 2 virus by intrathoracic inoculation. At 7 d postinfection, we allowed females to engorge on immunized or normal mouse blood. Virus in a mixture of anti-dengue-2 antibodies and dengue-2 virus became inactive after incubation at 37 degrees C for 1 h, but remained infective without incubation. Therefore, at ambient conditions antibodies would not interfere with virus isolation from field-collected Ae. aegypti from endemic areas. In addition, DEN antibodies enhanced virus replication when inoculated into Ae. aegypti, but not C6/36 cells. The mechanism for this in vitro antibody enhancement of infection remains unclear.     

Laboratory evaluation of the response of Aedes aegypti and Aedes albopictus uninfected and infected with dengue virus to deet

Laboratory studies were conducted to compare the response of Aedes aegypti (L.) and Aedes albopictus (Skuse) adults, uninfected and infected with four serotypes of dengue virus to a repellent containing 5% N, N-diethyl-3-methylbenzamide (deet). The results showed that mosquitoes infected with the four serotypes of dengue responded similarly to uninfected mosquitoes.     

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.     

Evaluation of seed extracts from plants found in the Caatinga biome for the control of Aedes aegypti

Dengue fever, currently the most important arbovirus, is transmitted by the bite of the Aedes aegypti mosquito. Given the absence of a prophylactic vaccine, the disease can only be controlled by combating the vector insect. However, increasing reports of resistance and environmental damage caused by insecticides have led to the urgent search for new safer alternatives. In this regard, plants stand out as a source of easy-to-obtain biodegradable insecticide molecules. Twenty (20) plant seed extracts from the Caatinga, an exclusively Brazilian biome, were prepared. Sodium phosphate (50 mM, pH 8.0) was used as extractor. The extracts were used in bioassays and submitted to partial characterisation. A Probit analysis of insecticides was carried out, and intergroup differences were verified by the Student’s t test and ANOVA. All the extracts exhibited larvicidal and ovipositional deterrence activity. The extracts of Amburana cearenses, Piptadenia viridiflora, Erythrina velutina, Myracrodruon urundeuva and Schinopsis brasiliensis were also pupicides, while the extracts of P. viridiflora, E. velutina, A. cearenses, Anadenanthera colubrina, Diocleia grandiflora, Bauhinia cheilantha, Senna spectabilis, Caesalpinia pyramidalis, Mimosa regnelli and Genipa americana displayed adulticidal activity. Egg laying was compromised when females were fed extracts of Ricinus communis, Croton sonderianus and S. brasiliensis. At least two proteins with insecticidal activity were found in all the extracts. Phenol compounds were identified in all the extracts and flavonoids, triterpenes or alkaloids in 14 of them. The results show the potential of plant seed extracts from the Caatinga as a source of active molecules against A. aegypti mosquitos.