Chikungunya Virus Interacts with Heat Shock Cognate 70 Protein to Facilitate Its Entry into Mosquito Cell Line.

AIM: The study was designed to identify putative Chikungunya virus (CHIKV) receptor/s on C6/36 cells that facilitate viral entry. METHODS: The virus overlay protein binding assay (VOPBA) was adopted to identify CHIKV-interacting bands present in C6/36 cell membrane and identity of the protein was established by mass spectrometry. The role of this protein as a putative CHIKV receptor on C6/36 cells was confirmed by infection inhibition assay. Cell surface localization of the identified protein was studied by indirect immunofluorescence assay (IFA) on nonpermeabilized cells and by flow cytometry. Interaction between this protein and CHIKV was confirmed by co-immunoprecipation (Co-IP) and Western blotting. The effect of depletion of the identified protein by quercetin was demonstrated by infection inhibition assay. RESULTS: A 70-kDa protein was identified as a CHIKV-interacting protein by VOPBA. MALDI-TOF analysis followed by homology search revealed that this protein could be heat shock cognate 70 (HSC 70). Anti-HSC 70 antibodies blocked CHIKV entry into C6/36 cells in a dose-dependent manner. IFA and flow cytometry analysis demonstrated HSC 70 localization on C6/36 cell surface. Co-IP experiments confirmed the interaction between HSC 70 and CHIKV envelope. Quercetin- and YM-01-treated C6/36 cells exhibited dose-dependent infection inhibition. CONCLUSION: HSC 70 serves as a putative CHIKV receptor on C6/36 cells.

The impact of transgenesis on mosquito fitness: A review.

Transgenic mosquitoes developed by genetic manipulation, offer a promising strategy for the sustainable and effective control of mosquito-borne diseases. This strategy relies on the mass release of transgenic mosquitoes into the wild, where their transgene is expected to persist in the natural environment, either permanently or transiently, within the mosquito population. In such circumstances, the fitness of transgenic mosquitoes is an important factor in determining their survival in the wild. The impact of transgene expression, insertional mutagenesis, inbreeding depression related to laboratory adaptation, and the hitchhiking effect involved in developing homozygous mosquito lines can all have an effect on the fitness of transgenic mosquitoes. Therefore, real-time estimation of transgene-associated fitness cost is imperative for modeling and planning transgenic mosquito release programs. This can be achieved by directly comparing fitness parameters in individuals homozygous or hemizygous for the transgene and their wild-type counterparts, or by cage invasion experiments to monitor the frequency of the transgenic allele over multiple generations. Recent advancements such as site-specific integration systems and gene drives, provide platforms to address fitness issues in transgenic mosquitoes. More research on the fitness of transgenic individuals is required to develop transgenic mosquitoes with a low fitness cost.

Eco-bio-social determinants of dengue vector breeding: a multicountry study in urban and periurban Asia.

OBJECTIVE: To study dengue vector breeding patterns under a variety of conditions in public and private spaces; to explore the ecological, biological and social (eco-bio-social) factors involved in vector breeding and viral transmission, and to define the main implications for vector control. METHODS: In each of six Asian cities or periurban areas, a team randomly selected urban clusters for conducting standardized household surveys, neighbourhood background surveys and entomological surveys. They collected information on vector breeding sites, people's knowledge, attitudes and practices surrounding dengue, and the characteristics of the study areas. All premises were inspected; larval indices were used to quantify vector breeding sites, and pupal counts were used to identify productive water container types and as a proxy measure for adult vector abundance. FINDINGS: The most productive vector breeding sites were outdoor water containers, particularly if uncovered, beneath shrubbery and unused for at least one week. Peridomestic and intradomestic areas were much more important for pupal production than commercial and public spaces other than schools and religious facilities. A complex but non-significant association was found between water supply and pupal counts, and lack of waste disposal services was associated with higher vector abundance in only one site. Greater knowledge about dengue and its transmission was associated with lower mosquito breeding and production. Vector control measures (mainly larviciding in one site) substantially reduced larval and pupal counts and "pushed" mosquito breeding to alternative containers. CONCLUSION: Vector breeding and the production of adult Aedes aegypti are influenced by a complex interplay of factors. Thus, to achieve effective vector management, a public health response beyond routine larviciding or focal spraying is essential.

Chikungunya virus isolated in Lakshadweep islands in the Indian Ocean: evidence of the Central/East African genotype.

Virological investigation was carried out to determine the etiology of suspected Chikungunya fever among humans reported in the Lakshadweep islands in the Indian Ocean. Three out of 23 acute sera samples showed cytopathological changes in Vero cell lines. Further, indirect immunofluorescence antibody test and reverse transcriptase-polymerase chain reaction studies demonstrated the Chikungunya virus etiology during the episode. E1 gene sequence analysis has confirmed the involvement of the Central/East African genotype of the Chikungunya virus.

Biological nanopesticides: a greener approach towards the mosquito vector control.

Mosquitoes, being a vector for some potentially dreadful diseases, pose a considerable threat to people all around the world. The control over the growth and propagation of mosquitoes comprises conventional pesticides, insect growth regulators and other microbial control agents. However, the usage of these common chemicals and conventional pesticides eventually has a negative impact on human health as well as the environment, which therefore becomes a major concern. The lacuna allows nanotechnology to come into action and exploit nanopesticides. Nanopesticides are majorly divided into two categories-synthetic and biological. Several nanoformulations serve as a promising nanopesticide viz. nanoparticles, e.g. biologically synthesised nanoparticles through plant extracts, nanoemulsions prepared using the essential oils like neem oil and citronella oil and nanoemulsion of conventional pesticides like pyrethroids. These green approaches of synthesising nanopesticides make use of non-toxic and biologically derived compounds and hence are eco-friendly with a better target specificity. Even though there are numerous evidences to show the effectiveness of these nanopesticides, very few efforts have been made to study the possible non-target effects on other organisms prevalent in the aquatic ecosystem. This study focuses on the role of these nanopesticides towards the vector control and its eco-safe property against the other non-target species.

Environmentally benign nanometric neem-laced urea emulsion for controlling mosquito population in environment.

The increasing risk of vector-borne diseases and the environmental pollution in the day-to-day life due to the usage of the conventional pesticides makes the role of nanotechnology to come into the action. The current study deals with one of the applications of nanotechnology through the formulation of neem urea nanoemulsion (NUNE). NUNE was formulated using neem oil, Tween 20, and urea using the microfluidization method. Prior to the development of nanoemulsion, the ratio of oil/surfactant/urea was optimized using the response surface modeling method. The mean droplet size of the nanoemulsion was found to be 19.3 ± 1.34 nm. The nanoemulsion was found to be stable for the period of 4 days in the field conditions which aids to its mosquitocidal activity. The nanoemulsion exhibited a potent ovicidal and larvicidal activity against A. aegypti and C. tritaeniorhynchus vectors. This result was corroborated with the histopathological analysis of the NUNE-treated larvae. Further, the effect of NUNE on the biochemical profile of the target host was assessed and was found to be efficacious compared to the bulk counterpart. The nanoemulsion was then checked for its biosafety towards the non-target species like plant beneficial bacterium (E. ludwigii), and phytotoxicity was assessed towards the paddy plant (O. sativa). Nanometric emulsion at the concentration used for the mosquitocidal application was found to be potentially safe towards the environment. Therefore, the nanometric neem-laced urea emulsion tends to be an efficient mosquito control agent with an environmentally benign property.

Natural vertical transmission of dengue virus in Aedes albopictus (Diptera: Culicidae) in Kerala, a southern Indian state.

The natural occurrence of vertical transmission of dengue viruses in Aedes albopictus (Skuse) mosquitoes was examined in the state of Kerala in southern India. Adults and larvae of Ae. albopictus collected from Kerala were screened for dengue viruses by using enzyme-linked immunosorbent assay with dengue-specific monoclonal antibodies. The possibility of the vertical transmission of dengue virus in Ae. albopictus was further evidenced by the detection of the virus in field-collected adult males as well as females emerged from field-collected larvae. Two pools, one pool from the adult males and one pool from the emerged females derived from field-collected larvae, were collected in the relatively hot months of June and March, respectively, and found to be positive for dengue virus antigen. Dengue serotype 2 virus was isolated from field-collected male adults in Kerala. These findings suggest that dengue virus is maintained in Ae. albopictus mosquitoes during the dry season by vertical transmission in nature.

A Preliminary Study to Forecast Japanese Encephalitis Vector Abundance in Paddy Growing Area, with the Aid of Radar Satellite Images.

Vector mosquitoes of Japanese encephalitis (JE) breed mostly in rice fields, and human cases occur scattered over extended rural rice-growing areas. From this, one may surmise an ecological connection with the irrigation facilities and paddy cultivation. Furthermore, it has been hypothesized that a particular stage of paddy growth is a premonitory sign that can lead to a markedly increased population of the vector mosquitoes. The present study aimed to forecast the vector abundance by monitoring the paddy growth using remote sensing and geographical information systems. The abundance of the JE vector Culex tritaeniorhynchus peaked when the paddy crop was at its heading stage and dipped when the crop reached the maturing stage. A significant positive correlation was observed between paddy growth and adult density (r = 0.73, p < 0.008). The sigma naught values (σ0) derived from satellite images of paddy fields ranged from -18.3 (during transplantation stage) to approximately -10 (during the noncultivation period). A significant positive correlation was observed between σ0 and paddy growth stages (r = 0.87, p < 0.05) and adult vector density (r = 0.74, p = 0.04). The σ0 value observed during the vegetative and flowering stages of paddy growth ranged from -17.6 to -17.16, at which period the vector density started building up. This could be the spectral signature that denotes the "risk," following which a high vector abundance is expected during heading stage of the paddy.

Economic and disease burden of dengue illness in India.

Between 2006 and 2012 India reported an annual average of 20,474 dengue cases. Although dengue has been notifiable since 1996, regional comparisons suggest that reported numbers substantially underrepresent the full impact of the disease. Adjustment for underreporting from a case study in Madurai district and an expert Delphi panel yielded an annual average of 5,778,406 clinically diagnosed dengue cases between 2006 and 2012, or 282 times the reported number per year. The total direct annual medical cost was US$548 million. Ambulatory settings treated 67% of cases representing 18% of costs, whereas 33% of cases were hospitalized, comprising 82% of costs. Eighty percent of expenditures went to private facilities. Including non-medical and indirect costs based on other dengue-endemic countries raises the economic cost to $1.11 billion, or $0.88 per capita. The economic and disease burden of dengue in India is substantially more than captured by officially reported cases, and increased control measures merit serious consideration.

Community-based control of Aedes aegypti by adoption of eco-health methods in Chennai City, India.

BACKGROUND: Dengue is highly endemic in Chennai city, South India, in spite of continuous vector control efforts. This intervention study was aimed at establishing the efficacy as well as the favouring and limiting factors relating to a community-based environmental intervention package to control the dengue vector Aedes aegypti. METHODS: A cluster randomized controlled trial was designed to measure the outcome of a new vector control package and process analysis; different data collection tools were used to determine the performance. Ten randomly selected intervention clusters (neighbourhoods with 100 houses each) were paired with ten control clusters on the basis of ecological/entomological indices and sociological parameters collected during baseline studies. In the intervention clusters, Aedes control was carried out using a community-based environmental management approach like provision of water container covers through community actors, clean-up campaigns, and dissemination of dengue information through schoolchildren. The main outcome measure was reduction in pupal indices (pupae per person index), used as a proxy measure of adult vectors, in the intervention clusters compared to the control clusters. RESULTS: At baseline, almost half the respondents did not know that dengue is serious but preventable, or that it is transmitted by mosquitoes. The stakeholder analysis showed that dengue vector control is carried out by vertically structured programmes of national, state, and local administrative bodies through fogging and larval control with temephos, without any involvement of community-based organizations, and that vector control efforts were conducted in an isolated and irregular way. The most productive container types for Aedes pupae were cement tanks, drums, and discarded containers. All ten intervention clusters with a total of 1000 houses and 4639 inhabitants received the intervention while the ten control clusters with a total of 1000 houses and 4439 inhabitants received only the routine government services and some of the information education and communication project materials. The follow-up studies showed that there was a substantial increase in dengue understanding in the intervention group with only minor knowledge changes in the control group. Community involvement and the partnership among stakeholders (particularly women's self-help groups) worked well. After 10 months of intervention, the pupae per person index was significantly reduced to 0·004 pupae per person from 1·075 (P = 0·020) in the intervention clusters compared to control clusters. There were also significant reductions in the Stegomyia indices: the house index was reduced to 4·2%, the container index to 1·05%, and the Breteau index to 4·3 from the baseline values of 19·6, 8·91, and 30·8 in the intervention arm. CONCLUSION: A community-based approach together with other stakeholders that promoted interventions to prevent dengue vector breeding led to a substantial reduction in dengue vector density.

Estimating dengue vector abundance in the wet and dry season: implications for targeted vector control in urban and peri-urban Asia.

BACKGROUND: Research has shown that the classical Stegomyia indices (or "larval indices") of the dengue vector Aedes aegypti reflect the absence or presence of the vector but do not provide accurate measures of adult mosquito density. In contrast, pupal indices as collected in pupal productivity surveys are a much better proxy indicator for adult vector abundance. However, it is unknown when it is most optimal to conduct pupal productivity surveys, in the wet or in the dry season or in both, to inform control services about the most productive water container types and if this pattern varies among different ecological settings. METHODS: A multi-country study in randomly selected twelve to twenty urban and peri-urban neighborhoods ("clusters") of six Asian countries, in which all water holding containers were examined for larvae and pupae of Aedes aegypti during the dry season and the wet season and their productivity was characterized by water container types. In addition, meteorological data and information on reported dengue cases were collected. FINDINGS: The study reconfirmed the association between rainfall and dengue cases ("dengue season") and underlined the importance of determining through pupal productivity surveys the "most productive containers types", responsible for the majority (>70%) of adult dengue vectors. The variety of productive container types was greater during the wet than during the dry season, but included practically all container types productive in the dry season. Container types producing pupae were usually different from those infested by larvae indicating that containers with larval infestations do not necessarily foster pupal development and thus the production of adult Aedes mosquitoes. CONCLUSION: Pupal productivity surveys conducted during the wet season will identify almost all of the most productive container types for both the dry and wet seasons and will therefore facilitate cost-effective targeted interventions.

Temporal variation in the susceptibility of Culex tritaeniorhynchus (Diptera: Culicidae) to Japanese encephalitis virus in an endemic area of Tamil Nadu, South India.

The study area, Cuddalore, is one of the endemic districts for Japanese encephalitis (JE) in southern India and there is a strong seasonality in JE case incidence, as well as JE virus (JEV) infection in the principal vector Culex (Culex) tritaeniorhynchus Giles. In a longitudinal 3-year study (July 2003 to June 2006), we determined the susceptibility of wild-caught female Cx. tritaeniorhynchus for JEV infection over several seasons from several villages. The susceptibility varied in all four seasons with the lowest value (4.82 geometric mean [GM]) in hot and wet seasons and highest (13.22 GM) in cool and wet seasons. Infection rate was significant between seasons (7.08-11.85 GM) and years (4.82-13.22 GM). Although the vector was abundant throughout the year, with an average per man-hour density ranging from 58 to 652, the JEV infection rates showed no correlation with vector abundance during different seasons in the index villages. The temporal and spatial changes in the competency of the vector appeared to influence the JEV infection rate in vector, which may at least partially explain the seasonality in JEV human cases in the study area.

Impact of ivermectin drug combinations on Pediculus humanus capitis infestation in primary schoolchildren of south Indian rural villages.

BACKGROUND: Antifilarial drug combinations including ivermectin provide antifilarial activity with ancillary benefits on intestinal helminths and ectoparasites, such as chiggers and lice. The impact of single oral dose of antifilarial drugs, viz; (1) diethylcarbamazine (DEC) alone, (ii) DEC + albendazole (ALB), (iii) ivermectin (IVR) + DEC and (iv) IVR + ALB, was determined, on the head louse (Pediculus humanus capitis) in primary school children in a rural community in south India. METHODS: Primary school children (n = 534) of age 6-10 years from four villages of South India were examined for the presence of head lice before and after single dose of DEC + ivermectin drug combination. The effectiveness and the duration of cure sustained by these drugs were quantified. The head louse was examined by "combing method" during post-treatment periods at 15, 45, 60 and 75 days interval. RESULTS: The antifilarial drug consumption rate was similar (96-98%) in all treatment arms. In pre-treatment survey the prevalence of head lice in children administered with DEC, DEC + ALB, IVR + DEC and IVR + ALB arm was 86%, 80%, 87% and 80%, respectively, with the latter two arms demonstrating significant reduction in louse infestation (P < 0.05) for 60 days. CONCLUSION: Single dose with IVR combination demonstrates a greater impact in reducing head louse infestation in the endemic rural communities for nearly 60 days. Therefore, in regions such as Africa where ivermectin is part of the antifilariasis campaign, this drug will have an additional benefit in reducing head lice infestation.

Prediction of 3-dimensional structure of salivary odorant-binding protein-2 of the mosquito Culex quinquefasciatus, the vector of human lymphatic filariasis.

Olfaction of insects is currently recognized as the major area of research for developing novel control strategies to prevent mosquito-borne infections. A 3-dimensional model (3D) was developed for the salivary gland odorant-binding protein-2 of the mosquito Culex quinquefasciatus, a major vector of human lymphatic filariasis. A homology modeling method was used for the prediction of the structure. For the modeling, two template proteins were obtained by mGenTHERADER, namely the high-resolution X-ray crystallography structure of a pheromone-binding protein (ASP1) of Apis mellifera L., [1R5R:A] and the aristolochene synthase from Penicillium roqueforti [1DI1:B]. By comparing the template protein a rough model was constructed for the target protein using MODELLER, a program for comparative modelling. The structure of OBP of the mosquito Culex quinquefasciatus resembles the structure of pheromone-binding protein ASP1 of Apis mellifera L., [1R5R:A]. From Ramachandran plot analysis it was found that the portion of residues falling into the most favoured regions was 86.0%. The predicted 3-D model may be further used in characterizing the protein in wet laboratory.