Effect of temperature stress on immature stages and susceptibility of Aedes aegypti mosquitoes to chikungunya virus

A high temperature stress of 44.5°C for 10 minutes on the larval stages was found to affect the susceptibility of adult Aedes aegypti mosquitoes to chikungunya virus. At this temperature, the mortality of the mosquito larvae was found to be approximately 95%, whereas a temperature greater than 45°C for 10 minutes was found to be lethal. A temperature tolerant (TT) strain was developed by exposing the larvae to a temperature of 44.5°C for 10 minutes at every generation for five generations. This strain was established to determine whether increase in the susceptibility was due to any selection pressure of higher temperature or to the influence of other intrinsic factors such as expression of immunoresponsive (IR) genes. Other studies on these mosquito strains showed that when maintained at 28 ± 1°C, there was no difference in the larval duration and mortality in the immature stages, but the mean survival of female mosquitoes in the TT strain was 5-6 days longer. Conversely, when mosquitoes were maintained throughout at 37°C the mean survival of the mosquitoes decreased drastically in both strains, but the mean survival of females in the TT strain was 5-6 days longer compared with the unstressed controls. This increases the probability of at least one more blood meal. Fecundity of the TT strain was found to be lower than that of the control mosquitoes. Data suggest that expression of certain IR genes was affected by the heat shock. Some of these genes were up-regulated and down-regulated, which may have affected the susceptibility of mosquitoes to the virus. Although there was some selection in the temperature-tolerant individuals in the TT strain, when stressed by heat they showed expression of IR genes in a pattern similar to that in the normal controls. It appears that an increase in temperature above the average temperature of an area might help increase the proportion of virus-susceptible mosquitoes in the population. Such an increase in temperature in an endemic area would not only enhance the selection of temperature-tolerant individuals in a population having more longevity, but would also affect both intrinsic and extrinsic factors by reducing the extrinsic incubation period and increasing susceptibility of mosquitoes to viruses due to affected expression of IR genes

West Nile virus: the Indian scenario

West Nile virus (WNV) is an important arthropod borne flavivirus; usually causes a mild infection called West Nile fever (WNF) in human and horses. Mosquitoes are the principal vectors of WNV. Various Culex species are found to act as vectors in different geographical regions. The virus is maintained in a bird-mosquito cycle in nature. In India, Culex mosquitoes are tentatively incriminated as vectors of WNV. Experimental studies have shown that Culex tritaeniorhynchus, Cx. vishnui, Cx. bitaeniorhynchus and Cx. univittatus, Culex pipiens fatigans and Aedes albopictus could act as potential vectors of WNV. Transovarial transmission of WNV has been experimentally demonstrated in Culex mosquitoes. Apart from mosquitoes, the role of other arthropods is also considered in the maintenance of WNV during inter-enzootic periods. The possible role of ardeid birds in the maintenance of WNV has been described in India. Though very few clinically overt cases of human encephalitis due to WNV are observed, Japanese encephalitis virus (JEV) is found to dominate in southern India. WNF in horses has not been documented in India. JEV immunized monkeys were protected from WNV challenge and the WNV immunization was found to reduce the disease severity due to JEV. Based on the limited genome sequence analysis, the Indian isolates are grouped together under the genetic lineage-I. WNV infection is diagnosed by IgM antibody capture enzyme linked immunosorbant assay, haemagglutination inhibition test, neutralization test and reverse transcriptase-polymerase chain reaction (RT-PCR). For the effective control of Culex mosquitoes, integrated vector control strategies are recommended. Specific methods are not available for the treatment of WNV infection. However, in patients with encephalitis supportive therapy is recommended. Though a few candidate vaccines are under laboratory trial, no vaccine has been available commercially for the control of WNV infection in human and animals. In view of the global interest on WNV, this paper describes the present status of WNV in India

Electrochemical behaviour and validated determination of the anitcancer drug Tamoxifen

The electrooxidative behaviour of tamoxifen (Tam) and 4-hydroxytamoxifen (TamOH) was investigated by cyclic (CV), differential-pulse adsorptive anodic stripping (DPAdAS) and square-wave adsorptive anodic stripping (SWAdAS) voltammetric techniques. The anodic oxidation peak of Tamoxifen was attributed to the cyclization reaction to form the corresponding phenanthrene derivative and the mechanism of oxidation was postulated on the basis of controlled potential electrolysis and isolation of the oxidative product. Oxidative stripping analysis was successfully applied to the determination of tamoxifen in a bulk pharmaceutical formulation, and sensitivity in human urine and serum was validated. The achieved limits of detection (LOD) of bulk tamoxifen were 1.8 × 10 -6 mol L -1 and 2.4 × 10 -6 mol L -1 for DPAdAS and SWAdAS, respectively. The LOD values for tamoxifen in human urine and serum sample analysis were 4.75 × 10 -7 mol L -1 and 2.63 × 10 -7 mol L -1 and 1.98 × 10 -7 mol L -1 and 3.28 × 10 -7 mol L -1 for DPAdAS and SWAdAS, respectively. 4-hydroxytamoxifen is oxidised at more positive potentials than Tamoxifen, separated from the Tamoxifen stripping peak, and its adsorption to the glassy carbon electrode is less pronounced. This affects the ability to determine this important phase I metabolite in serum and urine sample

Insect cell culture in research: Indian scenario

Insect cell cultures are widely used in viral diagnosis and biotechnology, for the production of recombinant proteins, viral pesticides and vaccines as well as in basic research in genetics, molecular biology, biochemistry, endocrinology and virology. Following KRP Singh's pioneering research in 1967, a large number of cell lines from diptera, hemiptera, and lepidopteran insects were established and characterized in India. With the availability of the modern tools in molecular biology and the advancements made in biotechnology, the indigenous cell lines may prove useful in creating a future without biohazardous chemical pesticides as well as producing life saving pharmaceuticals and vaccines for many diseases. This review summarizes information gathered regarding the insect cell lines established so far in India. It also covers the familiarization of the well characterized continuous cell lines and their potential applications. Special attention is given to virus susceptibility of the cell lines, the yield of virus with a comparative analysis with other conventional systems. The potential applications of dipteran and lepidopteran cell lines in agriculture and biotechnology are also briefly discussed for prospective studies

Evidence of co-infection of chikungunya and densonucleosis viruses in C6/36 cell lines and laboratory infected Aedes aegypti (L.) mosquitoes

<p>Abstract</p> <p>Background</p> <p>Densonucleosis viruses are the etiological agents of insect's disease. We have reported the isolation of densovirus from India and its distribution among the natural populations of <it>Aedes aegypti </it>mosquitoes across the country. Since densonucleosis virus persistently infects mosquito populations, and is demonstrated to negatively affect multiplication of dengue virus in <it>Aedes albopictus</it>, it would be interesting to study if this virus has a role in determining the susceptibility of the vector mosquito <it>Ae. aegypti </it>to chikugunya virus.</p> <p>Methods</p> <p>Mosquito cell lines and adult <it>Ae. aegypti </it>mosquitoes infected with densovirus were superinfected with Chikungunya virus and both the viruses were quantitated by determining their genomic copy number by real time amplification. Comparison was made between the log of genomic copy numbers of the viruses in the presence and absence of each other.</p> <p>Results</p> <p>The log of copy number of the viruses did not vary due to co-infection. Even though the RNA copy number of chikungunya virus increased over the period of time, no change was observed in the RNA copy number between the control and the co-infected group on any given day. Similarly, DNA copy number of densovirus also remained unchanged between the control and the co-infected groups.</p> <p>Conclusion</p> <p>Chikungunya virus neither stimulates the replication of densovirus nor is its own replication suppressed due to co-infection. <it>Ae. aegypti </it>mosquitoes with densovirus infection were as susceptible to infection by chikungunya virus as the uninfected mosquitoes.</p

Generation, annotation, and analysis of ESTs from midgut tissue of adult female Anopheles stephensi mosquitoes

<p>Abstract</p> <p>Background</p> <p>Malaria is a tropical disease caused by protozoan parasite, <it>Plasmodium</it>, which is transmitted to humans by various species of female anopheline mosquitoes. <it>Anopheles stephensi </it>is one such major malaria vector in urban parts of the Indian subcontinent. Unlike <it>Anopheles gambiae</it>, an African malaria vector, transcriptome of <it>A. stephensi </it>midgut tissue is less explored. We have therefore carried out generation, annotation, and analysis of expressed sequence tags from sugar-fed and <it>Plasmodium yoelii </it>infected blood-fed (post 24 h) adult female <it>A. stephensi </it>midgut tissue.</p> <p>Results</p> <p>We obtained 7061 and 8306 ESTs from the sugar-fed and <it>P. yoelii </it>infected mosquito midgut tissue libraries, respectively. ESTs from the combined dataset formed 1319 contigs and 2627 singlets, totaling to 3946 unique transcripts. Putative functions were assigned to 1615 (40.9%) transcripts using BLASTX against UniProtKB database. Amongst unannotated transcripts, we identified 1513 putative novel transcripts and 818 potential untranslated regions (UTRs). Statistical comparison of annotated and unannotated ESTs from the two libraries identified 119 differentially regulated genes. Out of 3946 unique transcripts, only 1387 transcripts were mapped on the <it>A. gambiae </it>genome. These also included 189 novel transcripts, which were mapped to the unannotated regions of the genome. The EST data is available as ESTDB at <url>http://mycompdb.bioinfo-portal.cdac.in/cgi-bin/est/index.cgi</url>.</p> <p>Conclusion</p> <p>3946 unique transcripts were successfully identified from the adult female <it>A. stephensi </it>midgut tissue. These data can be used for microarray development for better understanding of vector-parasite relationship and to study differences or similarities with other malaria vectors. Mapping of putative novel transcripts from <it>A. stephensi </it>on the <it>A. gambiae </it>genome proved fruitful in identification and annotation of several genes. Failure of some novel transcripts to map on the <it>A. gambiae </it>genome indicates existence of substantial genomic dissimilarities between these two potent malaria vectors.</p

Novel transparent nanocomposite films based on chitosan and bacterial cellulose

New nanocomposite films based on different chitosan matrices (two chitosans with different DPs and one water soluble derivative) and bacterial cellulose were prepared by a fully green procedure by casting a water based suspension of chitosan and bacterial cellulose nanofibrils. The films were characterized by several techniques, namely SEM, AFM, X-ray diffraction, TGA, tensile assays and visible spectroscopy. They were highly transparent, flexible and displayed better mechanical properties than the corresponding unfilled chitosan films. These new renewable nanocomposite materials also presented reasonable thermal stability and low O(2) permeability.FCT - SFRH/BD/41388/ 2007FCT - SFRH/BPD/38515/200