Diversity of sandflies in Vidarbha region of Maharashtra, India, a region endemic to Chandipura virus encephalitis

Background & objectives: Sandflies are implicated as vectors of Chandipura virus (CHPV) (Vesiculovirus: Rhabdoviridae). The virus is prevalent in central India including Vidarbha region of Maharashtra. CHPV causes encephalitis in children below 15 yr of age with case fatality rates ranging from 56 to 78 per cent. The present study was undertaken to determine the sandfly fauna in the CHPV endemic Vidharba region. Methods: A year round survey of sandflies was conducted at 25 sites in three districts of Vidarbha region. Sandflies were collected from their resting sites using handheld aspirators and identified using taxonomical keys. Results: A total of 6568 sandflies were collected during the study. Approximately 99 per cent of the collection belonged to genus Sergentomyia, which was represented by Ser. babu, Ser. bailyi and Ser. punjabensis. Genus Phlebotomus was represented by Ph. argentipes and Ph. papatasi. Ser. babu was the predominant species (70.7%) collected during the study. Ph. argentipes was detected in four villages with 0.89 per cent, whereas Ph. papatasi was detected in only one village with 0.32 per cent of the total collection. CHPV could not be isolated despite processing all the sandflies for virus isolation in cell culture. Interpretation & conclusions: The present study showed influence of higher temperature and relative humidity on sandfly population dynamics. An important observation during the study was the absence or decline in the population of Ph. papatasi and Ph. argentipes in the study area. Surge in Sergentomyia population and their breeding/resting in close vicinity to humans pose a concern as they are known to harbour CHPV and other viruses of public health importance

Bagaza virus inhibits Japanese encephalitis & West Nile virus replication in Culex tritaeniorhynchus & Cx. quinquefasciatus mosquitoes.

Background & objectives: Studies have shown that certain flaviviruses influence susceptibility of mosquitoes by inhibiting/enhancing replication of important flaviviruses. Hence, a study was designed to determine whether Bagaza virus (BAGV), a flavivirus isolated from Culex tritaeniorhynchus mosquitoes in India, alters susceptibility of Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes to Japanese encephalitis (JEV) and West Nile viruses (WNV). Methods: JEV and WNV infection in Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes in the presence of BAGV was carried out by intrathoracic (IT) inoculation and oral feeding methods. Mosquitoes were infected with BAGV and WNV/JEV either simultaneously or in a phased manner, in which mosquitoes were infected with BAGV by IT inoculation followed by super-infection with JEV/WNV after eight days post-infection (PI). JEV and WNV yield on 7th and 14th day PI after super-infection was determined by 50 per cent tissue culture infective dose (TCID50) method. Results: In Cx. tritaeniorhynchus mosquitoes, prior infection with BAGV significantly reduced JEV and WNV replication while in Cx. quinquefasciatus, BAGV influence was only seen with WNV. Reduction in virus titre was observed in IT inoculated and oral fed mosquitoes irrespective of the infection mode. JEV replication was also found reduced in Cx. tritaeniorhynchus mosquitoes persistently infected with BAGV at passage four. Interpretation & conclusions: BAGV infection in Cx. tritaeniorhynchus and Cx. quinquefasciatus mosquitoes altered their susceptibility to JEV and WNV producing low virus yield. However, the role of BAGV in inhibiting JEV/WNV replication in field mosquitoes needs further investigations

Isolation of Chandipura virus (Vesiculovirus: Rhabdoviridae) from Sergentomyia species of sandflies from Nagpur, Maharashtra, India.

Background & objectives: An outbreak of acute encephalitis syndrome was reported from Vidarbha region of Maharashtra state, India, during July 2012. Anti-IgM antibodies against Chandipura virus (CHPV) were detected in clinical samples. Sandfly collections were done to determine their role in CHPV transmission. Methods: Twenty nine pools of Sergentomyia spp. comprising 625 specimens were processed for virus isolation in Vero E6 cell line. Diagnostic RT-PCR targeting N-gene was carried out with the sample that showed cytopathic effects (CPE). The PCR product was sequenced, analysed and the sequences were deposited in Genbank database. Results: CPE in Vero E6 cell line infected with three pools was detected at 48 h post infection. However, virus could be isolated only from one pool. RT-PCR studies demonstrated 527 nucleotide product that confirmed the agent as CHPV. Sequence analysis of the new isolate showed difference in 10-12 nucleotides in comparison to earlier isolates. Interpretation & conclusions: This is perhaps the first isolation of CHPV from Sergentomyia spp. in India and virus isolation during transmission season suggests their probable role in CHPV transmission. Further studies need to be done to confirm the precise role of Sargentomyia spp. in CHPV transmission.

Preliminary findings on Bagaza virus (Flavivirus: Flaviviridae) growth kinetics, transmission potential & transovarial transmission in three species of mosquitoes.

Background & objectives: Bagaza virus (BAGV), a flavivirus synonymous with Israel turkey meningoencephalitis virus, has been found to circulate in India. BAGV has recently been held responsible for inducing febrile illness in humans and causing unusually high mortality to wild birds in Spain. A study was therefore, undertaken to determine its replication kinetics in certain mosquitoes and to determine vector competence and potential of the mosquitoes to transmit BAGV experimentally. Methods: Aedes aegypti, Culex tritaeniorhynchus and Cx quinquefasciatus mosquitoes were inoculated with BAGV; samples were harvested every day and titrated in BHK-21 cell line. Vector competence and experimental transmission were determined by examining the saliva of infected mosquitoes for virus and induction of sickness in suckling mice, respectively. Results: Cx. tritaeniorhynchus and Ae. aegypti mosquitoes yielded 5 log10 and 4.67 log10 TCID50/ml of virus on day 3 post-infection (PI), respectively while Cx. quinquefasciatus yielded a titre of 4 log10TCID50/ml on day 4 PI. BAGV was detected in saliva of all the infected mosquitoes demonstrating their vector competence. Experimental transmission of BAGV to infant mice as well as transovarial transmission was demonstrated by Cx. tritaeniorhynchus but not by Ae. aegypti and Cx. quinquefasciatus mosquitoes. Interpretation & conclusions: Replication of BAGV to high titres and dissemination to saliva in three most prevalent mosquitoes in India is of immense public health importance. Though no major outbreak involving man has been reported yet, BAGV has a potential to cause outbreaks in future.

Xenodiagnosis: use of mosquitoes for the diagnosis of arboviral infections.

The arboviruses have a worldwide distribution and, mosquitoes and ticks contribute principally in their transmission. In the last two decades, arboviral diseases have been recognised due to their resurgence and spread in newer geographic areas. Surveys to determine the prevalence of arboviruses in any region largely depend on the isolation attempts from the arthropods along with the serosurveys. Xenodiagnosis means use of insects for the diagnosis of infectious diseases affecting human being. The present communication discusses the application of mosquitoes for propagation and assays of arboviruses, the technique of mosquito inoculation and importance of xenodiagnosis.

Effect of temperature and insecticide stresses on Aedes aegypti larvae and their influence on the susceptibility of mosquitoes to dengue-2 virus.

Two major factors, higher temperatures and the application of insecticides, can drastically alter the genetic structure of a vector mosquito population. Due to these two stresses, the majority of the population gets wiped out, but the ones that withstand the stress and survive are likely to pass on survivability, and have an altered physiology. Our study shows that exposures to higher temperatures and DDT during the larval stage affects their susceptibility as adult mosquitoes to the DEN-2 virus. The overall transcription and translation status of heat shock protein (Hsp70) in virus high- and low-susceptible was the same as that in other batches. In the case of a DDT-resistant (R-7) strain two bands were obtained during RT-PCRs after heat shock. These two alleles were obtained only with HY-1 in which R-7 males were used for the crosses, suggesting that the second allele is probably male sex linked. The higher expression of Hsp70 may provide DDT-resistant strains a better chance of survival high temperature environments, particularly in homozygotes and hybrids. It was also interesting to note that these strains have a significantly lower susceptibility to the virus. Wide-spread DDT-resistance and a rise in temperature above the average temperature during summer may result in a population with a low susceptibility to the virus. Several families of heat shock proteins are known to be expressed in mosquitoes, and may have a cumulative role in determining susceptibility to the virus, which itself is governed by several genes.

Development of monoclonal antibody based antigen capture ELISA to detect chikungunya virus antigen in mosquitoes.

BACKGROUND & OBJECTIVES: Chikungunya (CHIK) virus has caused numerous large outbreaks in India. No active or passive surveillance has been carried out since the last epidemic which occurred in 1971. For active surveillance, it is necessary to have a test, which can detect the virus from a large number of field-collected mosquitoes. METHODS: The present study describes the standardization of monoclonal antibody (MAb) based antigen capture ELISA to detect chikungunya virus antigen from the mosquitoes. CHIK virus antigen from suspension of experimentally infected mosquitoes and their progeny was captured on mouse polyclonal antibody, while biotinylated CHIK Mab was used as a probing antibody. CHIK virus antigen in the head squashes of virus inoculated mosquitoes was detected using indirect immunofluorescence antibody (IFA) test for confirmation of ELISA results. RESULTS: The ELISA test was sensitive enough to detect antigen even if a small fraction of a single infected mosquito homogenate was incorporated in the test. The IFA test failed to detect CHIK antigen in 10 and 25 microliters of suspension whereas with ELISA it was detected in all the samples. Progeny of Aedes aegypti and Ae. albopictus mosquitoes infected with chikungunya virus did not show the possibility of existence of transovarial transmission. INTERPRETATION & CONCLUSION: This test is rapid and simple since it can be completed in two days as compared to the conventional mosquito inoculation and IFA techniques, which require at least 10 days. There is an additional advantage with this test that a large number of samples can be processed, and the remaining homogenate of the mosquitoes can be used for screening other viruses. Experimental data raised using this test showed that transovarial transmission of this virus does not occur in these vector species.

Highly-substrate active isoenzyme acetylcholinesterase-II, in rosy eye mutant of Aedes aegypti mosquito.

Insecticide bioassays were carried out on larvae and adults of rosy eye mutant and wildtype strains of A. aegypti. Both the strains were equally susceptible to DDT, malathion and deltamethrin. Biochemical assays showed an increase in acetylcholinesterase enzyme (AChE) activity in all the stages of mutant strain with both the substrates i.e. acetylthiocholine iodide and S-butyrylthiocholine iodide. However, there was no difference in the percent inhibition of enzyme activity with propoxur in these two strains. Polyacrylamide gel electrophoresis performed in native conditions on the homogenates of adults of rosy eye mosquitoes showed that AChE-II allele was highly active with the substrate acetylthiocholine iodide as compared to wildtype strain. Frequency of the highly active AChE-II allele in the mutant strain was about 68%, whereas it was about 5% in the wildtype strain.

Dengue virus and insecticide susceptibility status of Aedes aegypti mosquitoes from Belagola village, Mandya District, Karnataka state: during and post-epidemic investigations.

Aedes aegypti mosquitoes collected during and after dengue virus epidemic season were exposed to diagnostic dosages of insecticides. Mosquitoes collected during the epidemic showed resistance to DDT only, while mosquitoes collected after the control measures showed tolerance to malathion and low resistance to DDT. Since there was no indication of malathion resistance in the mosquitoes collected during the epidemic, the second batch was subjected to malathion selection pressure for 8 generations to find out the mechanism of resistance in this strain. There was 2-4 fold increase in resistance to malathion. Polyacrylamide electrophoresis showed increase in intensity of three esterase bands after selection. Quantitative enzyme assays showed an increase in esterase activity by 3-4 folds. Mosquitoes collected during the epidemic showed higher susceptibility to dengue-2 virus as compared to the laboratory strain, while the mosquitoes collected after malathion fogging showed further increase in the susceptibility to dengue virus. Virus susceptibility of the mosquitoes after eight generations of selection with malathion did not show any further significant increase.

Putative chikungunya virus-specific receptor proteins on the midgut brush border membrane of Aedes aegypti mosquito.

Two proteins (putative receptors) of 60 and 38 kDa, for chikungunya (CHIK) virus were detected in the brush border membrane fraction (BBMF) of the normal population of Aedes aegypti mosquitoes. Mosquitoes were infected orally with CHIK virus and infectivity checked by testing the head squashes. BBMF was prepared from proved positive and negative mosquitoes. The receptor proteins were found to be present in both the proved genotypes. However, dot-b'ot assays showed that the CHIK virus binding activity of BBMF/mg protein was noticeably low in the proved negative mosquitoes as compared to the positives. BBMF from the larvae of the normal populations also showed the presence of the receptor proteins, binding to CHIK virus. Receptor proteins from larvae as well as the adults were found glycosylated. CHIK virus receptor proteins of 24, 45, 58, 60 and 62 kDa were also seen in the membrane fraction of the C6/36 cells.

Effect of sublethal dosages of insecticides on chikungunya virus susceptible and refractory strains of Aedes aegypti.

Three strains of Aedes aegypti mosquitos viz (i) CRS, refractory to Chikungunya (CHIK) virus by oral route of infection but susceptible to DDT (2) CSS, susceptible to CHIK virus and also susceptible to DDT (3) CSS-DDTR, susceptible to CHIK virus but resistant to DDT, were examined for the effect of sublethal dosages of DDT and deltamethrin on their fecundity. Biochemical analysis showed that there was an increase in glutathione s-transferase activity in the CSS-DDTR strain which was associated with DDT resistance. There was an increase in acetylcholinesterase activity in the CRS strain, however it was not associated with resistance to all the three insecticides tested. No significant differences in the fecundity of these three strains were observed, though there was some increase in the number of non layers in CSS-DDTR strain after the treatment of DDT and mean number of eggs laid by CSS and CRS strains was slightly reduced (0.5 > p < 0.1) after the treatment with deltamethrin.

Biochemical basis of DDT-resistance in Aedes aegypti population from a dengue affected area in Shahjahanpur city.

Entomological studies conducted in Jalalnagar, Shahjahanpur city, Uttar Pradesh, India, during an outbreak of dengue in 1992, showed that Ae. aegypti mosquitoes were resistant to DDT and had some tolerance to malathion in the adults and the larvae. Biochemical analysis suggested that DDT resistance was related to elevated glutathione s-transferase and tolerance to malathion was due to a little increase in esterase activity. Crosses of DDT-resistant and susceptible strain suggested that resistance was codominant (metabolic type).

Zinc, copper & iron contents in cooked foods & estimates of their daily intakes in young hostel residents.

Dietary zinc, copper and iron intakes of 19 men and 11 women (aged 18-25 yr) were determined during three periods of five consecutive days each, using, weighment method and atomic absorption analysis of food samples. Daily zinc, copper and iron intakes of men ranged from 13.1 +/- 1.2 to 15.3 +/- 1.4 mg/d, 1.62 +/- 0.15 to 1.84 +/- 0.17 mg/d and 20.5 +/- 2.8 to 29.1 +/- 4.5 mg/d and of women 8.8 +/- 2.0 to 9.0 +/- 1.6, 1.42 +/- 0.23 to 1.69 +/- 0.62, and 10.0 +/- 2.4 to 14.7 +/- 2.6 mg/d respectively. Cereals provided 67.7 per cent of dietary zinc, 47.7 per cent copper and 62 per cent iron in men and 60 per cent of Zn, 44 per cent of Cu and 59 per cent of Fe in women, suggesting lower contribution of cereals to Cu intakes than Zn and Fe. Men had mineral intakes close to the recommended allowances for Indians, whereas the intakes of women were 30 to 60 per cent lower. When expressed as mg/1000 kcal, lower calorie intakes of women were responsible for their low mineral intakes. Intra-individual variation in mineral intakes was higher (18.39%) than that of energy intakes (14-17%) in both men and women. Phytate: Zn molar ratios were > 15 indicating risk of zinc deficiency in these students.

Insecticide susceptibility status of certain populations of Aedes aegypti mosquito from rural areas of Maharashtra state.

Ae. aegypti mosquitoes from rural areas of Maharashtra state were subjected to insecticide bio-assays. All the populations showed resistance to DDT at larval and adult stages. No resistance to deltamithrin and malathion was detected at any stage. Biochemical analysis of these mosquito populations showed that resistance to DDT was probably due to increase in the kinetics of glutathione S-transferase.

Insecticide susceptibility status & enzyme profile of Aedes albopictus populations from different localities of Maharashtra state.

Susceptibility status of five populations of Ae. albopictus mosquitoes from Maharashtra state, to DDT, malathion, fenitrothion, bromophos, propoxur and deltamethrin was studied and compared with the laboratory population. Four populations survived when adults were exposed to 4 per cent DDT impregnated paper for 2 h; though three of these populations had lower DDT-LC50 values at larval stages in comparison with the laboratory population. Results of topical application of DDT on these four populations supported these findings by showing comparatively higher LD50 values at adult stages in comparison with the laboratory population. All the populations were highly susceptible to other pesticides tested i.e., malathion, fenitrothion, bromophos, propoxur and deltamethrin. These populations were distinguished from each other by esterase isoenzyme patterns.