Genomic studies of envelope gene sequences from mosquito and human samples from Bangkok, Thailand.

Dengue virus (DENV) is an RNA virus showing a high degree of genetic variation as a consequence of its proofreading inability. This variation plays an important role in virus evolution and pathogenesis. Although levels of within-host genetic variation are similar following equilibrium, variation among different hosts is frequently different. To identify dengue quasispecies present among two hosts, we collected patient samples from six acute DENV cases and two pools of Aedes aegypti mosquitoes and analyzed the genetic variation of regions of the viral envelope gene. Among human and mosquito samples, we found three major clusters originating from two subpopulations. Although several shared lineages were observed in the two hosts, only one lineage showing evidence of neutral selection was observed among two hosts. Taken together, our data provide evidence for the existence of a DENV quasispecies, with less genetic variation observed in mosquitoes than humans and with circulating lineages found in both host types.

Genetic and evolutionary analysis of cell-fusing agent virus based on Thai strains isolated in 2008 and 2012.

Increasing attention is being devoted to ecological and evolutionary relationships between insect-specific flaviviruses and globally important human-pathogenic flaviviruses such as dengue viruses. One such insect flavivirus, cell-fusing agent virus (CFAV), remains poorly investigated. In this study, we isolated 13 and 16 CFAV strains from Aedes aegypti mosquitoes collected in Thailand in 2008 and 2012, respectively, and performed genetic and evolutionary analyses based on gene regions encoding the envelope protein (E) and nonstructural proteins 3 (NS3) and 5 (NS5). Consistent with previously reported CFAV strains, E, NS3 and NS5 regions comprised 1,290, 1,761 and 2,664 nucleotides, respectively. Nucleotide and amino acid identities of these three regions were >98% among the 29 isolates, and approximately 95-96% and 96-99%, respectively, between the isolates and previously reported CFAV strains. When amino acid sequences from representative strains of six insect-specific and seven mosquito-borne flaviviruses were compared, average identities of 14.9%, 31.8% and 44.3% were calculated for E, NS3 and NS5 regions, respectively. Phylogenetic analysis based on nucleotide and amino acid data indicated that the Thai CFAV isolates of the current study were distinct from previously reported CFAV strains from Indonesia and Puerto Rico. Analysis of each gene region consistently uncovered a clade made up of nearly the same subset of Thai CFAV isolates; this result, and the isolation of CFAV from mosquitoes reared from larvae, suggest that the virus is maintained by vertical transmission and conserved in a particular environment without considerable evolutionary alteration. The most recent common ancestor of the Thai CFAV isolates in this study was dated to 11-27 years ago, and is estimated to have diverged 46-86 years ago from previously reported CFAV strains. Superinfection with CFAV of Aedes mosquitoes carrying dengue viruses present in Thailand for over 50 years has most likely taken place.

Isolation of infectious chikungunya virus and dengue virus using anionic polymer-coated magnetic beads.

Mosquitoes-borne viruses are a major threat for human populations. Among them, chikungunya virus (CHIKV) and dengue virus (DENV) cause thousands of cases worldwide. The recent propagation of mosquito vectors competent to transmit these viruses to temperate areas increases their potential impact on susceptible human populations. The development of sensitive methods allowing the detection and isolation of infectious viruses is of crucial interest for determination of virus contamination in humans and in competent mosquito vectors. However, simple and rapid method allowing the capture of infectious CHIKV and DENV from samples with low viral titers useful for further genetic and functional characterization of circulating strains is lacking. The present study reports a fast and sensitive isolation technique based on viral particles adsorption on magnetic beads coated with anionic polymer, poly(methyl vinyl ether-maleic anhydrate) and suitable for isolation of infectious CHIKV and DENV from the four serotypes. Starting from quite reduced biological material, this method was accurate to combine with conventional detection techniques, including qRT-PCR and immunoblotting and allowed isolation of infectious particles without resorting to a step of cultivation. The use of polymer-coated magnetic beads is therefore of high interest for rapid detection and isolation of CHIKV and DENV from samples with reduced viral loads and represents an accurate approach for the surveillance of mosquito vector in area at risk for arbovirus outbreaks.

Dengue-2 virus carrying capacity of Thai Aedes aegypti strains with different susceptibility to deltamethrin.

Deltamethrin-resistant Aedes aegypti currently threatens the effectiveness of dengue hemorrhagic fever control operations in Thailand. Although a previous study has suggested that insecticide resistance may increase Ae. aegypti susceptibility to dengue-2 virus infection, our experimental data showed no significant association between laboratory-induced deltamethrin-resistance in a Thai Ae. aegypti isolate and its susceptibility to dengue -2 infection.

Update on the proteomics of major arthropod vectors of human and animal pathogens.

Vector-borne diseases (VBDs) are defined as infectious diseases of humans and animals caused by pathogenic agents such as viruses, protists, bacteria, and helminths transmitted by the bite of blood-feeding arthropod (BFA) vectors. VBDs represent a major public health threat in endemic areas, generally subtropical zones, and many are considered to be neglected diseases. Genome sequencing of some arthropod vectors as well as modern proteomic and genomic technologies are expanding our knowledge of arthropod-pathogen interactions. This review describes the proteomic approaches that have been used to investigate diverse biological questions about arthropod vectors, including the interplay between vectors and pathogens. Proteomic studies have identified proteins and biochemical pathways that may be involved in molecular crosstalk in BFA-pathogen associations. Future work can build upon this promising start and functional analyses coupled with interactome bioassays will be carried out to investigate the role of candidate peptides and proteins in BFA-human pathogen associations. Dissection of the host-pathogen interactome will be key to understanding the strategies and biochemical pathways used by BFAs to cope with pathogens.

Natural transovarial dengue virus infection rate in both sexes of dark and pale forms of Aedes aegypti from an urban area of Bangkok, Thailand.

Transovarial dengue virus infection status of two forms of adult Aedes aegypti (dark or Ae. aegypti type form and pale or form queenslandensis), reared from field-collected larval and pupal stages, was determined by one-step RT-PCR and dengue viral serotype by nested-PCR. Natural transovarial transmission (TOT) of dengue virus was detected in the two Ae. aegypti forms, and in both adult males and females. Male Ae. aegypti had a higher rate of TOT dengue virus infection than female. The overall minimum infection rate among the male and female populations was 19.5 and 12.3 per 1,000 mosquitoes, respectively. All four dengue serotypes were detected in mosquito samples, with DEN-4 being the predominant serotype. Thus, both male and female Ae. aegypti have influences on the epidemiology of dengue virus transmission.

Protein expression in the salivary glands of dengue-infected Aedes aegypti mosquitoes and blood-feeding success.

Mosquito salivary glands (SG) play an essential role in food digestion and pathogen transmission. The function of the salivary components during infection is poorly understood. In this study, female Aedes aegypti mosquitoes were infected with dengue virus serotype 2 (DENV-2) via an artificial membrane feeding apparatus. The mosquito SGs were examined for DENV-2 infection for 14 days post-infection (dpi). The amount of dengue virus increased throughout the 14 dpi. Three different meals were provided for the Ae. aegypti mosquitoes. SG protein expression was compared among sugar-fed (SF), blood-fed (BF), and dengue-infected blood-fed (DF) mosquitoes using SDS-PAGE coupled with densitometric analysis. The SG of SF mosquitoes had fewer protein bands than those of BF and DF mosquitoes. The major SG proteins seen among BF and DF mosquitoes had molecular weights of 12-15, 25-30, 35-40, 45-50, 55-60 kDa and 61-67 kDa. We compared the SG protein band expression profiles in BF and DF mosquitoes. Two bands (35-40 and 61-67 kDa) were expressed more by DF mosquitoes and 3 different bands (25-30, 45-50, and 55-60 kDa) were expressed more by BF mosquitoes. These SG proteins may have some role in facilitating blood-feeding and dengue infection. We speculate these specific SG proteins in dengue-infected mosquitoes may increase the chance of blood-feeding and virus transmission by infected mosquitoes. These results may be useful for designing additional tools to investigate the interaction between Ae. aegypti SG and the dengue virus.

Prospective field study of transovarial dengue-virus transmission by two different forms of Aedes aegypti in an urban area of Bangkok, Thailand.

A prospective field study was conducted to determine transovarial dengue-virus transmission in two forms of Aedes aegypti mosquitoes in an urban district of Bangkok, Thailand. Immature Aedes mosquitoes were collected monthly for one year and reared continuously until adulthood in the laboratory. Mosquitoes assayed for dengue virus were processed in pools and their dengue virus infection status was determined by one-step RT-PCR and nested-PCR methods. Of a total 15,457 newly emerged adult Ae. aegypti, 98.2% were dark and 1.8% of the pale form. The results showed that the minimum infection rate (MIR) by transovarial transmission (TOT) of dengue virus during the one-year study ranged between 0 to 24.4/1,000 mosquitoes. Dengue virus TOT increased gradually during the hot summer months, reaching a peak in April-June, while dengue cases peaked in September, a rainy month near the end of the rainy season. Therefore, mosquito infections due to TOT were prevalent four months before a high incidence of human infections. TOT dengue virus infections occurred in both forms of Ae. aegypti. All four dengue serotypes were detected, with DEN-4 predominant, followed by DEN-3, DEN-1, and DEN-2, respectively.

Control of mosquito vectors of tropical infectious diseases: (3) susceptibility of Aedes aegypti to pyrethroid and mosquito coils.

We collected 11 groups of Aedes aegypti mosquitoes from various locations of Thailand. After rearing in the laboratory, the colonies were tested for KT50 values to dl,d-T80-allethrin 0.5% mosquito coils in a 25 m3 room semi-field test and KD50 and LD50 values to dl,d-T80-allethrin by a topical application method. Two groups of mosquitoes were susceptible to allethrin similar to a SS (known allethrin sensitive) group, and other 9 groups showed various levels of lower susceptibility to allethrin; of these 6 had susceptibilities similar to a BS (known allethrin resistant) group with extremely low susceptibility, while the remaining 3 groups had susceptibilities to allethrin between the SS and BS groups. The KD50 values with the topical application were found to correlate highly with the KT50 values in the 25 m3 room semi-field test, providing a useful test method for insect susceptibility evaluation. The allethrin mosquito coils, even at higher concentrations, had no activity against the 6 decreased susceptibility groups, similar to the BS group. With the 25 m3 room semi-field test, mosquito coils with d,d-T-prallethrin at concentrations of 0.1 to 0.15% plus a synergist and those with methoxymethyl-tetrafluorobenzyl tetramethylcyclopropane carboxylate (K-3050) at a concentration of 0.1% plus a synergist were found to be highly effective against these mosquito groups. These two pyrethroids had smaller KD50 and LD50 values for topical application, and were more effective than dl,d-T80-allethrin, having the potential to control Ae. aegypti mosquitoes with low allethrin susceptibility.

Suppression of Plasmodium falciparum by serum collected from a case of Plasmodium vivax infection.

BACKGROUND: It has frequently been reported that Plasmodium vivax suppressed Plasmodium falciparum and ameliorated disease severity in patients infected with these two species simultaneously. The authors investigate the hypothesis that immunological responses stimulated by P. vivax may play a role in suppressing co-infecting P. falciparum. METHODS: Sera, taken sequentially from one of the authors (YN) during experimental infection with P. vivax, were added to in vitro cultures of P. falciparum. Cross-reactive antibodies against P. falciparum antigens, and cytokines were measured in the sera. RESULTS: Significant growth inhibitory effects upon P. falciparum cultures (maximally 68% inhibition as compared to pre-illness average) were observed in the sera collected during an acute episode. Such inhibitory effects showed a strong positive temporal correlation with cross-reactive antibodies, especially IgM against P. falciparum schizont extract and, to a lesser degree, IgM against Merozoite Surface Protein (MSP)-119. Interleukin (IL)-12 showed the highest temporal correlation with P. vivax parasitaemia and with body temperatures in the volunteer. CONCLUSION: These results suggest the involvement by cross-reactive antibodies, especially IgM, in the interplay between plasmodial species. IL-12 may be one of direct mediators of fever induction by rupturing P. vivax schizonts, at least in some subjects. Future studies, preferably of epidemiological design, to reveal the association between cross-reactive IgM and cross-plasmodial interaction, are warranted.

Control of mosquito vectors of tropical infectious diseases: (1) bioefficacy of mosquito coils containing several pyrethroids and a synergist.

The bioefficacy of mosquito coils containing several pyrethroids were tested in a 25 m3 room against Culex pipiens quinquefasciatus, Aedes aegypti and Anopheles dirus. The test results were compared with tests against Culex pipiens pallens in Japan. Based on the KT50 values (the 50% knockdown time) of mosquito coils containing dl, d-T80-allethrin, d, d-T-prallethrin and methoxymethyl-tetrafluorobenzyl tetramethyl-cyclopropanecarboxylate (K-3050) at doses of 0.05-0.5% (w/w) with or without a synergist, the pyrethroid susceptibility of the four mosquito species was as follows: Cx. p. quinquefasciatus was several times more tolerant to pyrethroids than Cx. p. pallens, Ae. aegypti was a further several times more tolerant than Cx. p. quinquefasciatus, and An. dirus was more susceptible than Cx. p. pallens (KT50 value: about half of Cx. p. pallens). The order of their susceptibilities is common for pyrethroids. Mosquito coils containing d, d-T-prallethrin and K-3050 at doses of 0.05-0.2% (w/w) and N-(2-ethylhexyl)bicycle-[2,2,1]-hept-5-ene-2,3-dicarboxyimide as a synergist at a ratio of 2 times the active ingredient were highly effective against Ae. aegypti, the most important mosquito vector for dengue fever.

Control of mosquito vectors of tropical infectious diseases: (2) pyrethroid susceptibility of Aedes aegypti ( l) collected from different sites in Thailand.

Four strains (SS, BS, A and B) of Aedes aegypti collected from different sites in Bangkok and at different times were examined for their pyrethroid susceptibility. Mosquito coils containing dl, d-T80-allethrin, d, d-T-prallethrin and methoxymethyl-tetrafluorobenzyl tetramethyl-cyclopropanecarboxylate (K-3050) with or without a synergist were tested by the 25 m3 semi-field test method. One strain (SS) was the most susceptible with KT50 values of about < 30 minutes for all mosquito coils, while the other three strains (BS, A and B) were found to be around 10 to 20 times more tolerant to pyrethroids than the SS strain. A similar tendency for the pyrethroid susceptibility of the four strains was obtained with tests by topical application method. In field efficacy tests, mosquito coils with d, d-T-prallethrin 0.20% plus N-(2-ethylhexyl)bicycle-[2,2,1]- hept-5- ene-2,3-dicarboxyimide as a synergist exhibited a repellent effect of about 85%, while those with K-3050 0.10% plus the synergist exhibited a greater repellent effect of about 90%. In contrast, the repellent effect of commercial dl, d-T80-allethrin 0.20% coils was as low as about 50%. The d, d-T-prallethrin and K-3050 coils with the synergist were confirmed to be highly effective in repelling Ae. aegypti.

A new densovirus isolated from the mosquito Toxorhynchites splendens (Wiedemann) (Diptera: Culicidae).

A new densovirus was isolated and characterized in laboratory strains of Toxorhynchites splendens. The virus was detected by polymerase chain reaction (PCR) from mosquitoes reared in our laboratory. PCR fragments from each mosquito were compared by single strand conformation polymorphism (SSCP) assay and found to be indistinguishable. Thus, it is likely the densoviruses from these mosquitoes contain homologous nucleotide sequences. The PCR fragment corresponding to a 451 bp densovirus structural gene segment from each of 5 mosquitoes had 100% identical nucleotide sequences. Phylogenetic analysis of the structural gene sequence suggests the newly isolated densovirus is more closely related to Aedes aegypti densovirus (AaeDNV) than to Aedes albopictus densovirus (AalDNV). Analysis of offspring and predated larvae suggests that vertical and horizontal transmission are responsible for chronic infections in this laboratory strain of Toxorhynchites splendens. The virion DNA is 4.2 kb in size, is closely related to, but distinct from, known densoviruses in the genera Brevidensovirus and Contravirus. Thevirus is tentatively named Toxorhynchites splendens densovirus (TsDNV).

Protozoal contamination of water used in Thai frozen food industry.

This study evaluated the prevalence of contamination of water that was used for food preparation. Since protozoal cysts can be found in small numbers in water, 1,000 liters of either untreated or treated water were filtered through activated carbon block filters (1 microm nominal porosity). Identification of protozoa was performed using specific monoclonal antibodies against Giardia and Cryptosporidium parasites followed by fluorescence microscopy. Twelve of 20 untreated water samples (60%) were found to be contaminated by Giardia cysts, with an average of 53.33 cysts/1,000 liters (geometric mean 39.43), whilst 7 samples (35%) were contaminated by Cryptosporidium oocysts, with an average of 28.57 oocysts/1,000 liters (geometric mean 26.92). Three samples of untreated water (15%) were positive for both organisms. In contrast, none of the treated water samples were contaminated.

Markedly reduced severity of Dengue virus infection in mosquito cell cultures persistently infected with Aedes albopictus densovirus (AalDNV).

AalDNV-infected C6/36 cells serially passaged for over 10 weeks showed a decline in percentage of anti-AalDNV-positive cells (APC) from an initial 92% to approximately 20%. Cultures of persistent APC were indistinguishable from uninfected cultures by direct microscopy but most stained cells from early APC passages had enlarged nuclei with eosinophilic inclusions, while late APC passages had few and naive cells none. Super challenge of persistent APC cultures did not increase percentage APC and supernatants from persistent APC cultures gave low APC (40%) in naive C6/36 cell cultures. When challenged with dengue virus serotype 2 (DEN-2), naive C6/36 cells showed severe cytopathic effects (CPE) and high mortality within 4 days, as did early passage APC cultures. Remarkably, DEN-2 infections in persistent APC cultures were much less severe, being characterized by reduced DEN-2 infection percentage, retarded DEN-2 virion production, no CPE and no significant mortality. Reasons for rapid reduction in APC and resistance to superinfection upon serial passage remain unproven but may relate to production of AalDNV-defective interfering particles (DIP) by molecular mechanisms still open to speculation. More difficult to explain is cross-protection against DEN-2-induced mortality seen in persistent APC cultures. However, by comparison to work on shrimp viruses, we speculate that this may involve blockage of viral-triggered apoptosis. The phenomena described raise questions regarding the potential for persistent infections by unknown viruses to confound experimental results with insect cell lines.

Susceptibility and transovarial transmission of dengue virus in Aedes aegypti: a preliminary study of morphological variations.

Two types of morphological variants, the dark form and the pale form of Aedes aegypti were selected from wild-caught mosquitos. Ascertaining any differences between the two forms for susceptibility to dengue type 2 virus was performed by oral feeding. Transovarial transmission was further determined from the progenies of infected mosquitos by tracing them to the third generation. Significant differences in oral infection were not observed between these two forms of mosquitos. Transovarial transmission was found in the progenies of infected females of both forms, and the filial infection rates (FIRs) were also similar. However, there was a trend of declining FIR in the later generation. In order to achieve an accurate result, more tests are currently underway to obtain a larger number of progeny. Although the FIR was low in the present study under laboratory conditions, higher rates might occur under field conditions, which could have a significant impact on the maintenance of dengue viruses in nature.

Potential development of temephos resistance in Aedes aegypti related to its mechanism and susceptibility to dengue virus.

The addition of temephos to water containers as a larvicide against Aedes aegypti was commonly used as a part of DHF control programs. The widespread, or long-term, application of insecticides can lead to the development of mosquito resistance to the insecticides through selection pressure. This presents a problem for disease control. Therefore, this study was conducted in the laboratory to observe the potential development of resistance to temephos and the mechanism involved in Ae. aegypti, and to study the significance for dengue infection. The larvae were selected in consecutive generations. The level of resistance to temephos was detected by WHO assay technique. After 19 generations of selection, a low level of resistance was found. The resistance ratio at LC50 was 4.64 when compared with the non-selected group. The assay for major enzyme-based resistance mechanisms was done in a microtiter plate to detect elevated non-specific esterases, monooxygenase, and insensitive acetylcholinesterase in the temephos-selected and non-selected groups. It revealed a significant increase in esterase activity when compared with the non-selected group. There was no elevation of monooxygenase or insensitive acetylcholinesterase activities. However, when an esterase inhibitor (S, S, S-tributyl phosphorotrithioate, or DEF) was added to temephos and the susceptibility in the selected group was studied, the resistance ratio was reduced from 16.92 to 3.57 when compared with a standard susceptible strain (Bora Bora). This indicates that the esterases play an important role in temephos resistance. Dengue-2 virus susceptibility was studied by oral feeding to females of the temephos-selected (S19) and the non-selected groups. The dissemination rates, when the titer of virus in the blood meal was 7.30 MID50/ml, were 11.11% and 9.38% for the selected and non-selected groups, respectively. When the titer of virus in the blood meal was 8.15 MID50/ml, the dissemination rates increased to 24.24% and 33.33%, respectively. A statistical difference in viral susceptibility was not found between the two groups. This suggested that the low level of temephos resistance might not affect oral susceptibility. However, this needs further study.

Temephos resistance in two forms of Aedes aegypti and its significance for the resistance mechanism.

Aedes aegypti, at the larval stage, has been subjected to the temephos selection in laboratory. The level of temephos resistance was detected in a microplate by biochemical assay using WHO bioassay technique. The major enzyme-based resistance mechanisms involved in temephos resistance include elevated nonspecific esterase, oxidase and insensitive acetylcholinesterase. After 19 generations of temephos selection, the selected group showed resistance ratios of 4.64 and 16.92, when compared with a non-selected group and the WHO susceptible strain, respectively. The two seperated forms, type form and the pale form of Ae. aegypti showed low levels of resistance to temephos after 19 generations of selection, with resistance ratios of 4.82 and 4.07 for the type form and the pale form, respectively; when compared with the non-selected strain, 17.58 and 14.84, when compared with the WHO susceptible strain. This showed that the type form could develop higher level resistance than the pale form. The esterase inhibitor (S,S,S-tributyl phosphorotrithioate, DEF) or synergist implicated detoxifying esterase in all the temephos selected groups and the presence of elevated esterase were confirmed by biochemical assay. There were significant differences in elevated esterase activity between the temephos selected groups and the non-selected group. However no significant difference between the type form and the pale form was found. Besides the elevated esterase, there was no change in monooxygenase activity and no evidence of insensitive acetylcholinesterease for all temephos selected groups. These results suggest that temephos resistance could be developed in Ae. aegypti under selection pressure and that the main mechanism is based only on esterase detoxification.

Comparative susceptibility to oral infection with dengue viruses among local strains of Aedes aegypti (Diptera: Culicidae) collected at different seasons of the year.

The vector competence of Aedes aegypti (L.) mosquitoes, collected during the hot, rainy and cool seasons from different localities in Thailand, was tested for a correlation with a seasonal cyclic pattern of dengue incidence. Under laboratory conditions, some groups of mosquitoes exhibited differences in susceptibility to oral infection but showed no correlation to dengue cases that peak during the rainy season. Thus, the environmental conditions of each season, although they might affect a temporal change of mosquito vector competence, they might not have any direct effects on virus transmission patterns. Mosquito populations from different parts of Thailand were also likely to be homogeneous in their susceptibility to dengue virus during the study period. Other factors, such as characteristics of the virus, vector density and frequency of host-vector contact, should be considered for seasonal pattern of dengue diseases.