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.

Detection of coronaviruses in Pteropus & Rousettus species of bats from different States of India.

Background & objectives: Bats are considered to be the natural reservoir for many viruses, of which some are potential human pathogens. In India, an association of Pteropus medius bats with the Nipah virus was reported in the past. It is suspected that the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also has its association with bats. To assess the presence of CoVs in bats, we performed identification and characterization of bat CoV (BtCoV) in P. medius and Rousettus species from representative States in India, collected during 2018 and 2019. Methods: Representative rectal swab (RS) and throat swab specimens of Pteropus and Rousettus spp. bats were screened for CoVs using a pan-CoV reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. A single-step RT-PCR was performed on the RNA extracted from the bat specimens. Next-generation sequencing (NGS) was performed on a few representative bat specimens that were tested positive. Phylogenetic analysis was carried out on the partial sequences of RdRp gene sequences retrieved from both the bat species and complete viral genomes recovered from Rousettus spp. Results: Bat samples from the seven States were screened, and the RS specimens of eight Rousettus spp. and 21 Pteropus spp. were found positive for CoV RdRp gene. Among these, by Sanger sequencing, partial RdRp sequences could be retrieved from three Rousettus and eight Pteropus bat specimens. Phylogenetic analysis of the partial RdRp region demonstrated distinct subclustering of the BtCoV sequences retrieved from these Rousettus and Pteropus spp. bats. NGS led to the recovery of four sequences covering approximately 94.3 per cent of the whole genome of the BtCoVs from Rousettus bats. Three BtCoV sequences had 93.69 per cent identity to CoV BtRt-BetaCoV/GX2018. The fourth BtCoV sequence was 96.8 per cent identical to BtCoV HKU9-1. Interpretation & conclusions: This study was a step towards understanding the CoV circulation in Indian bats. Detection of potentially pathogenic CoVs in Indian bats stresses the need for enhanced screening for novel viruses in them. One Health approach with collaborative activities by the animal health and human health sectors in these surveillance activities shall be of use to public health. This would help in the development of diagnostic assays for novel viruses with outbreak potential and be useful in disease interventions. Proactive surveillance remains crucial for identifying the emerging novel viruses with epidemic potential and measures for risk mitigation.

Involvement of dual serotypes during a severe dengue outbreak in Wadi area, Nagpur district, Maharashtra 2017.

BACKGROUND & OBJECTIVES: An outbreak of dengue-like illness was reported from Wadi area within the Nagpur Municipal Corporation during September-October 2017 with five deaths. Major symptoms reported were high fever (103-106 oF), acute joint pains, myalgia, drowsiness, breathlessness, etc. An investigation was conducted to confirm the etiological agent, its characterization and the vectors involved in the outbreak. METHODS: Serological analysis was conducted to detect dengue (DEN)/chikungunya IgM antibodies in 158 sera samples. Nested-PCR was carried out to serotype eight ELISA positive samples. Adult and larval mosquito collections were conducted in the affected areas to determine species composition and mosquito density. RESULTS: Dengue IgM antibodies were detected in 44 sera samples. Molecular typing revealed involvement of DEN-2 and DEN-3 serotypes. Dengue hemorrhagic fever symptoms were observed in two patients. Aedes aegypti breeding was found rampant with Breteu index and house index ranging from 23 to 70 and 17 to 56, respectively. Major breeding habitats encountered were, used tyres, cement tanks and refrigerator trays. INTERPRETATION & CONCLUSION: Clinical symptoms, detection of anti-DEN IgM antibodies in high number of samples and heavy breeding of Ae. aegypti confirmed it was a dengue outbreak.

Vector competence of certain Culex and Aedes mosquitoes for the Chittoor virus, the Indian variant of the Batai virus.

Chittoor virus (CHITV), a mosquito-borne bunyavirus (Orthobunyavirus: Bunyaviridae) isolated in India, has been found to be antigenically close to the Batai virus (BATV), which has a wide distribution across Asia, Europe, and Africa. The latter virus causes influenza-like illness in humans and mild illness in sheep and goats. BATV has been involved in genetic reassortment with other bunyaviruses, generating novel genome combinations and causing severe clinical manifestations including hemorrhagic fever. Conversely, CHITV has never been associated with any major outbreaks in India, although neutralizing antibodies have been detected in humans and domestic animals. Repeated isolations and seroprevalence have prompted us to determine the vector competence of three important mosquito species, viz., Culex quinquefasciatus, Culex tritaeniorhynchus, and Aedes aegypti, for CHITV. The three mosquito species replicated CHITV to titers of 6.3, 5.0, and 5.2 log10 TCID50/mL, respectively, and maintained the virus for substantial periods. Both of the Culex species demonstrated vector competence, while A. aegypti did not. Horizontal transmission to infant mice was also demonstrated by both Culex species. Active circulation of the virus and the availability of both susceptible hosts and competent vector mosquitoes pose a serious threat to public health should there be a reassortment.

Prevalence and spatial distribution of Ixodid tick populations in the forest fringes of Western Ghats reported with human cases of Kyasanur forest disease and monkey deaths in South India.

Kyasanur forest disease (KFD) is a major tick-borne viral haemorrhagic fever caused by KFD virus (KFDV) (Flaviviridae). The disease was reported to be confined to five districts of Karnataka state India until 2011. During 2012-2016, emergence of KFD has been reported in newer areas of Karnataka and adjoining states. Therefore, survey of tick vectors was carried out in these new areas of Karnataka and adjoining states reported with monkey deaths and human cases of KFD. In all selected sites, ticks from the forest floor were collected by lint clothes using flagging method. Tick samples were tested for KFDV nucleic acid by real-time RT-PCR. A total of 4772 ticks, comprising eight species of genus Haemaphysalis and one species each of genus Amblyomma, Ixodes and Rhipicephalus was collected. Haemaphysalis spinigera, the principal vector of KFDV was the predominant tick species (59.5%) collected followed by H. turturis (8.6%). The abundance of H. spinigera ranged from 9.2 to 33.9 per man-hour in the six districts surveyed. Of 214 (4418 tick samples) pools screened by real-time RT-PCR, two pools of H. spinigera were positive for KFDV. High abundance of Haemaphysalis vectors in the six districts indicated that the districts are receptive for KFD outbreaks. KFDV was detected in the tick vectors in the new foci of the KFD. Data on tick distribution will be useful in creating KFD risk map for strengthening the ongoing preventive measures such as vaccination and supply of insect repellents to the high risk groups and intensive health education.

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 7 [th] and 14 [th] day PI after super-infection was determined by 50 per cent tissue culture infective dose (TCID 50 ) 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.

Vector competence of two Indian populations of Culex quinquefasciatus (Diptera: Culicidae) mosquitoes to three West Nile virus strains.

BACKGROUND & OBJECTIVES: Culex quinquefasciatus is one of the principal vectors of West Nile virus (WNV). The mosquito also acts as a bridge vector as it feeds on both birds and humans. In the background of the recent reports of WNV activity in Kerala and Assam with fatalities, a study was initiated to determine the growth kinetics and transmission mechanisms of three strains of WNV in two populations of Cx. quinquefasciatus. METHODS: Mosquitoes were infected by oral feeding and growth on different post-infection days was determined with the three strains. Horizontal transmission was determined by confirming sickness and mortality in infant mice after infected mosquito bite. F1 generation eggs, larvae, pupae and adults of experimentally infected mosquitoes were screened for WNV to determine vertical (transovarial) transmission. Trans-stadial transmission was determined by detecting WNV in adult mosquitoes emerged from infected larvae. RESULTS: Both the mosquito populations replicated and maintained WNV for a prolonged period with high titers (≥ 5log10 PFU/ml). WNV could be detected in saliva from Days 2 to 32 post-infection. Horizontal transmission by both the populations could be established but no vertical transmission was observed. However, parenterally infected larvae transmitted WNV to adults. INTERPRETATION & CONCLUSION: WNV has been isolated from >10 mosquito species from India, however, vector competence of none of the species has been studied. The present study demonstrates efficient transmission of WNV by Cx. quinquefasciatus mosquitoes. With its country wide prevalence and high vector competence, the mosquitoes could create grave consequences especially when virulent strains with potential to cause acute flaccid paralysis and death are circulating.

Vectorial capacity of Culex gelidus (Theobald) mosquitoes to certain viruses of public health importance in India.

BACKGROUND & OBJECTIVES: Culex gelidus, a widely prevalent mosquito in India and Southeast Asia region, is an important vector of Japanese encephalitis virus (JEV). Experimental studies have shown its potential to transmit West Nile, Kunjin, Murray Valley encephalitis and Ross River viruses. An attempt was therefore made to study its susceptibility and vector competence to some of the arboviruses of public health importance in India. METHODS: Mosquitoes were infected with six viruses, viz. JEV, chikungunya (CHIKV), Chandipura (CHPV), Chittoor (CHITV), Ingwavuma (INGV) and Umbre (UMBV) by intra thoracic inoculation to determine virus susceptibility and vector competence. Growth kinetics of the viruses were studied by determining the titres of inoculated mosquitoes on different days post-infection by titration in Vero E6 cells. Vector competence was studied by detecting the presence of the viruses in saliva of infected mosquitoes. RESULTS: All the six viruses were replicated in Cx. gelidus. JEV, CHPV, CHIKV and CHITV yielded > 5 log10TCID50/ml virus while UMBV and INGV yielded approx 4log10TCID50/ml virus. JEV, CHIKV and CHITV could be detected in the saliva of the infected mosquitoes, while CHPV, INGV and UMBV could not be detected in the saliva of the infected mosquitoes. INTERPRETATION & CONCLUSION: Replication potential and vector competence of Cx. gelidus to some of the viruses of public health importance in India, viz. JEV, CHIKV, CHITV etc, pose a serious threat to general population, especially in the wake of spurt in its population in certain parts of India.

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 s0 tate, 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.

Replication potential and different modes of transmission of West Nile virus in an Indian strain of Culex gelidus Theobald (Diptera: Culicidae) mosquitoes.

BACKGROUND & OBJECTIVES: Culex gelidus mosquito, an important vector of Japanese encephalitis virus, has shown to transmit West Nile virus (WNV), Kunjin and Murray Valley encephalitis viruses experimentally. An attempt was, therefore, made to study the replication kinetics and vector competence of an Indian strain of Cx. gelidus to WNV. METHODS: Mosquitoes were infected by both intrathoracic inoculation and oral feeding and studied the growth kinetics by determining the virus titre on different days post-infection (PI). Vector competence was studied by determining the presence of WNV in saliva on subsequent days PI. Horizontal transmission was determined by demonstrating infection in infant mice by bite of mosquitoes that were fed on viraemic mice previously. Vertical transmission was studied by screening progeny derived from infected mosquitoes. Trans-stadial transmission was determined by screening adult mosquitoes emerged from parenterally inoculated IV instar larvae. RESULTS: The mosquito replicated WNV to 7log10 TCID50/ml on Day 8 PI and maintained the titre for 14 days. Virus dissemination to legs and salivary glands could be detected, but not to ovaries up to Day 10 PI. The mosquitoes picked up infection from viraemic blood and transmitted successfully to infant mice on subsequent feeding. Trans-stadial transmission also could be demonstrated. However, vertical transmission could not be demonstrated. INTERPRETATION & CONCLUSION: The replication potential, maintenance of WNV for prolonged periods and ability to transmit WNV experimentally makes the mosquito a serious threat to public health especially in the wake of active WNV activity in certain parts of India.

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 log10 TCID50/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.

Isolation and characterization of densonucleosis virus from Aedes aegypti mosquitoes and its distribution in India.

OBJECTIVES: Mosquito densonucleosis viruses (DNVs) are known to persistently infect the insect cell line and mosquito population in nature, causing mortality in mosquitoes. Here we report the isolation and characterization of a DNV from Aedes aegypti and its distribution among different Ae. aegypti populations from India. METHODS: We screened Ae. aegypti mosquito populations from different states of India by PCR. Virus isolation and purification was performed using a cesium chloride gradient from a positive mosquito colony. Characterization of this isolate was carried out by electron microscopy, Western blot and sequencing. RESULTS: Electron microscopy showed the presence of parvovirus-like particles, and Western blot showed the presence of 2 viral proteins of 40 and 41 kDa. A total of 3,776 bases of genome were sequenced, which included a 3'UTR of 128 bases, a coding region of 3,507 bases and a 5'UTR of 141 bases. Three open reading frames (ORFs) were identified and characterized. The NIVDNV genome showed 95% similarity with Culex pipiens pallens DNV and 93% similarity with Ae. aegypti DNV. CONCLUSION: Phylogenetic analysis of all 3 ORFs showed that this new isolate falls in the lineage of Brevidensovirus along with other mosquito DNVs.

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.

Inheritance of oral susceptibility of Aedes aegypti to Chikungunya virus.

A colony of rosy eye mutants of Aedes aegypti was established. This strain was refractory to Chikungunya virus by oral route of infection when compared with the wild-type parent strain. The refractoriness of this strain seems to be due to a mesentronal barrier, since both the mosquito strains supported the multiplication of virus after intrathoracic inoculation. The rosy eye strain was also found to be refractory to Sagiyama virus (Alphaviridae: Getah virus subtype) when compared with wild-type parent strain, but no such difference in the oral susceptibility was found with dengue-2 (Flaviviridae) virus. The rosy eye mutant appears to be closely linked to the gene(s) for refractoriness to alpha viruses and may be useful in future studies in understanding the genetic basis of vector competence of Ae. aegypti to arboviruses.

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.

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).

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.

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.

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.