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Background In June 2019, Nipah virus (NiV) infection was detected in a 21-year-old male (index case) of Ernakulum, Kerala, India. This study was undertaken to determine if NiV was in circulation in Pteropus species (spp) in those areas where the index case had visit history in 1 month. Methods Specialized techniques were used to trap the Pteropus medius bats (random sampling) in the vicinity of the index case area. Throat and rectal swabs samples of 141 bats along with visceral organs of 92 bats were collected to detect the presence of NiV by real-time reverse transcriptase-polymerase chain reaction (qRTPCR). Serum samples of 52 bats were tested for anti-NiV Immunoglobulin (Ig) G antibodies by Enzyme-Linked Immunosorbent Assay (ELISA). The complete genome of NiV was sequenced by next-generation sequencing (NGS) from the tissues and swab samples of bats. Results One rectal swab sample and three bats visceral organs were found positive for the NiV. Interestingly, 20.68% (12/58) of Pteropus were positive for anti-NiV IgG antibodies. NiV sequences of 18,172; 17,200 and 15,100 nucleotide bps could be retrieved from three Pteropus bats. Conclusion A distinct cluster of NiV sequences, with significant net-evolutionary nucleotide divergence, was obtained, suggesting the circulation of new genotype (I-India) in South India. NiV Positivity in Pteropus spp. of bats revealed that NiV is circulating in many districts of Kerala state, and active surveillance of NiV should be immediately set up to know the hotspot area for NiV infection.

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.

The COVID-19 pandemic is a global health crisis that poses a great challenge to the public health system of affected countries. Safe and effective vaccines are needed to overcome this crisis. Here, we develop and assess the protective efficacy and immunogenicity of an inactivated SARS-CoV-2 vaccine in rhesus macaques. Twenty macaques were divided into four groups of five animals each. One group was administered a placebo, while three groups were immunized with three different vaccine candidates of BBV152 at 0 and 14 days. All the macaques were challenged with SARS-CoV-2 fourteen days after the second dose. The protective response was observed with increasing SARS-CoV-2 specific IgG and neutralizing antibody titers from 3rd-week post-immunization. Viral clearance was observed from bronchoalveolar lavage fluid, nasal swab, throat swab and lung tissues at 7 days post-infection in the vaccinated groups. No evidence of pneumonia was observed by histopathological examination in vaccinated groups, unlike the placebo group which exhibited interstitial pneumonia and localization of viral antigen in the alveolar epithelium and macrophages by immunohistochemistry. This vaccine candidate BBV152 has completed Phase I/II (NCT04471519) clinical trials in India and is presently in phase III, data of this study substantiates the immunogenicity and protective efficacy of the vaccine candidates., SARS-CoV-2 vaccines are needed to fight the pandemic. Here, authors show protective efficacy and immunogenicity of the inactivated SARS-CoV-2 vaccine BBV152 in rhesus macaques. This vaccine is currently in clinical development.

The COVID-19 pandemic is a global health crisis that has severely affected mankind and posed a great challenge to the public health system of affected countries. The availability of a safe and effective vaccine is the need of the hour to overcome this crisis. Here, we have developed and assessed the protective efficacy and immunogenicity of an inactivated SARS-CoV-2 vaccine (BBV152) in rhesus macaques (Macaca mulata). Twenty macaques were divided into four groups of five animals each. One group was administered a placebo while three groups were immunized with three different vaccine candidates at 0 and 14 days. All the macaques were challenged with SARS-CoV-2 fourteen days after the second dose. The protective response was observed with increasing SARS-CoV-2 specific IgG and neutralizing antibody titers from 3rd-week post-immunization. Viral clearance was observed from bronchoalveolar lavage fluid, nasal swab, throat swab, and lung tissues at 7 days post-infection in the vaccinated groups. No evidence of pneumonia was observed by histopathological examination in vaccinated groups, unlike the placebo group which showed features of interstitial pneumonia and localization of viral antigen in the alveolar epithelium and macrophages by immunohistochemistry. Data from this study substantiate the immunogenicity of the vaccine candidates and BBV152 is being evaluated in Phase I clinical trials in India (NCT04471519).

Arboviruses cause the most devastating diseases in humans and animals worldwide. Several hundred arbovirus are transmitted by mosquitoes, sand flies or ticks and are responsible for more than million deaths annually. Development of a model system is essential to extrapolate the molecular events occurring during infection in the human and mosquito host. Virus overlay protein binding assay (VOPBA) combined with MALDI TOF/TOF MS revealed that Dengue-2 virus (DENV-2) exploits similar protein molecules in Drosophila melanogaster and Aedes aegypti for its infection. Furthermore, the virus susceptibility studies revealed that DENV-2 could propagate in D. melanogaster, and DENV-2 produced in fruit fly is equally infectious to D. melanogaster and Ae. aegypti. Additionally, real time PCR analysis revealed that RNAi coupled with JAK-STAT and Toll pathway constitutes an effector mechanism to control the DENV-2 infection in flies. These observations point out that D. melanogaster harbors all necessary machineries to support the growth of arboviruses. With the availability of well-established techniques for genetic and developmental manipulations, D. melanogaster, offers itself as the potential model system for the study of arbovirus-vector interactions.

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.

Kyasanur forest disease (KFD) outbreak was confirmed in Dodamarg Taluka, Sindhudurga district (Maharashtra) in India during the year 2016. The rise in suspected KFD cases was reported in January 2016, peaked during March, and then declined gradually from April 2016. The outbreak was thoroughly investigated considering different socio-clinical parameters.Total, 488 suspected KFD cases were investigated using KFD specific real-time RT-PCR and anti-KFDV IgM enzyme-linked immunosorbent assay (ELISA). Sero-epidemiological survey was carried out in the affected area using anti-KFDV IgG ELISA.Among suspected KFD cases, high age-specific attack rate (105.1 per 1000 persons) was observed in adults (aged 40-59 years). Out of 488 suspected KFD cases, 130 were laboratory confirmed. Of these, 54 cases were KFDV real-time RT-PCR positive, 66 cases were anti-KFDV IgM ELISA positive and 10 cases were positive by both the assays. Case fatality ratio among laboratory-confirmed KFD cases were 2.3% (3/130). Majority of laboratory-confirmed KFD cases (93.1%) had visited Western Ghats forest in Dodamarg for activities like working in cashew nut farms (79.8%), cashew nut fruit collection (76.6%), collection of firewood (68.5%) and dry leaves/grass (40.3%), etc., before the start of symptoms. Common clinical features included fever (100%), headache (93.1%), weakness (84.6%), and myalgia (83.1%). Hemorrhagic manifestations were observed in nearly one-third of the laboratory-confirmed KFD cases (28.5%). A seroprevalence of (9.7%, 72/745) was recorded in KFD-affected area and two neighboring villages (9.1%, 15/165). Serosurvey conducted in Ker village showed clinical to subclinical ratio of 6:1 in KFD-affected areas.This study confirms the outbreak of KFD Sindhudurg district with 130 cases. Detection of anti-KFDV IgG antibodies among the healthy population in KFD-affected area during the KFD outbreak suggested the past exposure of KFD infection. This outbreak investigation has helped health authorities in adopting KFD vaccination strategy for the population at risk.

Objectives: The objective of this study was to understand natural disease progression in infant CD1 mice after the bite of Aedes aegypti mosquitoes infected by the Zika virus (ZIKV, MR-766 strain). Methods: A. aegypti mosquitoes were experimentally infected with ZIKV MR-766 strain via the oral feeding route. Infected mosquitoes were allowed to feed on infant CD1 mice. Sick mice were euthanized, and their organs were collected and subjected to real-time RT-PCR, histo­pathology, and immunohistochemistry. Results: Clinical symptoms appeared in mice after 4–5 days of being bitten by mosquitoes, following which they were euthanized. Real-time RT-PCR analysis showed the presence of viral RNA in various organs such as the brain, liver, kidney, spleen, lungs, and intestines of the mice. The brain tissue specimens showed higher viral loads as determined by threshold values (Ct value) in the real-time RT-PCR assay. Histopathological and immunohistochemistry studies also revealed the presence of the virus and associated lesions in the brain, indicating that ZIKV shows tropism for neuronal tissue. Conclusions: This study demonstrates ZIKV pathogenesis in infant CD1 mice and that these mice are highly susceptible to natural infection with this ZIKV strain.

The spread and establishment of Chandipura virus (CHPV) infection in India has raised serious epidemiological concerns. The virus interface with the vertebrate hosts (including humans) and vector competence are the important parameters of disease prevalence. Interestingly, in the present study, a highly zoophilic species of the sandfly Sergentomyia was found to be a potential vector of CHPV in Gujarat. This is probably the first report from India of male sandflies testing positive for CHPV in RT-PCR analysis. These findings signify vertical transmission of the virus among sandflies and have epidemiological significance. Health Officers from Gujarat referred 9 pools comprising 277 adult sandflies from disease-affected and unaffected areas to the National Institute of Virology, Pune. The pools were subjected to RT-PCR analysis and sequencing. Of the 9, 2 female and one male pool tested positive for CHPV. Phylogenetic analysis showed similarity of the new sandfly-borne CHPV strains with the human strain from Andhra Pradesh (AP) 2003. The present study highlights the possible role of Sergentomyia spp. in the transmission of CHPV in India.

Bat-borne viral diseases are a major public health concern among newly emerging infectious diseases which includes severe acute respiratory syndrome, Nipah, Marburg and Ebola virus disease. During the survey for Nipah virus among bats at North-East region of India; Tioman virus (TioV), a new member of the Paramyxoviridae family was isolated from tissues of Pteropus giganteus bats for the first time in India. This isolate was identified and confirmed by RT-PCR, sequence analysis and electron microscopy. A range of vertebrate cell lines were shown to be susceptible to Tioman virus. Negative electron microscopy study revealed the “herringbone” morphology of the nucleocapsid filaments and enveloped particles with distinct envelope projections a characteristic of the Paramyxoviridae family. Sequence analysis of Nucleocapsid gene of TioV demonstrated sequence identity of 99.87% and 99.99% nucleotide and amino acid respectively with of TioV strain isolated in Malaysia, 2001. This report demonstrates the first isolation of Tioman virus from a region where Nipah virus activity has been noticed in the past and recent years. Bat-borne viruses have become serious concern world-wide. A Survey of bats for novel viruses in this region would help in recognizing emerging viruses and combating diseases caused by them., Highlights • First isolation of Tioman virus from Pteropus bat in North East region of India. • Isolate was identified by RT-PCR, sequencing and electron microscopy. • TioV isolate showed highest sequence identity with Malaysia strain of TioV. • Bat-borne viruses are concern for human and animal health. • Survey of bats for novel viruses is needed to recognize emerging viruses.

Background & objectives: The presence of Cat Que virus (CQV) in Culex mosquitoes and pigs has been reported in China and Vietnam. Due to the spread of similar species of the Culex mosquitoes in India, there is a need to understand the replication kinetics of this virus in mosquito models. As a part of preparedness and to identify the presence of this CQV in humans and swine, this study was carried out to develop diagnostic tests. Methods: Serological and molecular diagnostic assays were developed for testing the mosquito population, human and swine serum samples. In this line, RNA-dependent RNA polymerase (L), glycoprotein (M) and nucleocapsid (S) genes-based reverse transcription-polymerase chain reaction (RT-PCR) assays were developed for CQV. Real-time RT-PCR was used for screening of retrospectively collected human serum samples (n=1020) with acute febrile illness during 2014-2017. Simultaneously, an in-house anti-CQV swine and human IgG ELISAs were also developed to detect anti-CQV IgG antibody. Human serum samples (n=883) with post-onset of disease (POD) >4 days and swine serum samples (n=459) were tested for the presence of anti-CQV IgG antibodies. CQV NIV 612,045 isolate was used for susceptibility and replication kinetics experiment using three different species of mosquitoes to understand its behaviour in Indian mosquitoes. Results: All human serum samples (n=1020) screened for the presence of CQV using real-time RT-PCR were found to be negative. Anti-CQV IgG antibody positivity was recorded in two of 883 human serum samples tested. Virus susceptibility experiments indicated that three species of mosquito, namely Aedes aegypti, Culex quinquefasciatus and Cx. tritaeniorhynchus supported multiplication of CQV by intrathoracic as well as artificial membrane/oral feeding routes. Interpretation & conclusions: Anti-CQV IgG antibody positivity in human serum samples tested and the replication capability of CQV in mosquitoes indicated a possible disease causing potential of CQV in Indian scenario. Screening of more human and swine serum samples using these assays is required as a proactive measure for understanding the prevalence of this neglected tropical 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.

Understanding the population dynamics of mosquito populations through life table analysis and insecticide susceptibility is important to assess the likely impact of vector control strategies as well as to aid the design of novel interventions. Variation in the life tables and other biological data was compared for two populations ofCulex quinquefasciatusSay 1823 from geographically isolated regions, Gorakhpur and Pune from India. Under a standardized rearing regime and constant laboratory conditions, mosquitoes were reared and biological attributes of these populations were compared. Development and survival of immature and adult stages ofCulex quinquefasciatuswere found significantly different in Gorakhpur and Pune populations. Principal component analysis of morphological data revealed that the two populations form significantly different clusters which can be differentiated from each other based on siphon, saddle, anal gills, and pecten teeth related variables. Insecticide susceptibility results suggest that the larvae from both areas were more susceptible to deltamethrin as compared to DDT and malathion. The current study provides baseline information on survivorship, morphological variation and insecticide susceptibility ofCulex quinquefasciatus. The results obtained in this study suggest that different geographical areas with contrasting habitats have significant influence on survival and reproductive strategies ofCulex quinquefasciatus.

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 log 10 and 4.67 log 10 TCID 50 /ml of virus on day 3 post-infection (PI), respectively while Cx. quinquefasciatus yielded a titre of 4 log 10 TCID 50 /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.

For the design of effective antiviral strategies, understanding the fundamental steps of the virus life cycle, including virus-host interactions, is essential. We performed a virus overlay protein binding assay followed by proteomics for identification of proteins from membrane fractions of A7 (Aedes aegypti) cells, C6/36 (Aedes albopictus) cells and the midgut brush border membrane fraction of Ae. aegypti mosquito that bind to dengue-2 virus. Actin, ATP synthase β subunit, HSc 70, orisis, prohibitin, tubulin β chain, and vav-1 were identified as dengue-2-virus-binding proteins. Our results suggest that dengue-2 virus exploits an array of housekeeping proteins for its entry in mosquito cells.

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.

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.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.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.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.

Experiments were conducted to determine the persistence of chikungunya viral (CHIKV) RNA in experimentally infected Aedes aegypti mosquitoes stored for prolonged periods at 28°C. Intra-thoracically inoculated mosquitoes with confirmed positivity were killed by quick freezing at -80°C, applied to sticky tape, and stored at 28°C with 80 ± 5% relative humidity (RH). At weekly intervals, five mosquitoes were removed from the tape randomly and assayed individually for detection of viral RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). CHIKV RNA was detected up to 12 weeks in dry mosquitoes by RT-PCR. Virus could not be isolated either in cell culture or in the suckling Swiss-albino mouse system at any stage. This study demonstrated the persistence of CHIKV viral RNA up to 12 weeks when stored at 28°C with RH 80 ± 5%. This finding will have significance in CHIKV surveillance programs in mosquito populations or field-based studies in countries where maintenance of a cold chain is a concern.

Experiments were conducted to demonstrate the role of male Aedes aegypti mosquitoes in the maintenance and transmission of chikungunya virus (CHIKV) to female mosquitoes. We demonstrated that infected male mosquitoes are capable of infecting females during mating. The infection rate in female mosquitoes was 11% when virgin female mosquitoes were allowed to coinhabit with infected males. The body suspension of venereally infected female mosquitoes induced illness in infant Swiss albino mice, which demonstrated the infectivity of the venereally transmitted virus. The presence of CHIKV in the brains of the ill mice was confirmed by a reverse transcription–polymerase chain reaction specific for partial sequences of nonstructural protein 4 and envelope 1 genes. In the light of the recent report of transovarial transmission of CHIKV in mosquitoes, although at a lower level, this finding has significance because it may help in transmission of the virus to females venereally to start a new infection cycle.

Background Chikungunya virus (CHIKV) has reemerged as a life threatening pathogen and caused large epidemics in several countries. So far, no licensed vaccine or effective antivirals are available and the treatment remains symptomatic. In this context, development of effective and safe prophylactics and therapeutics assumes priority. Methods We evaluated the efficacy of the siRNAs against ns1 and E2 genes of CHIKV both in vitro and in vivo. Four siRNAs each, targeting the E2 (Chik-1 to Chik-4) and ns1 (Chik-5 to Chik-8) genes were designed and evaluated for efficiency in inhibiting CHIKV growth in vitro and in vivo. Chik-1 and Chik-5 siRNAs were effective in controlling CHIKV replication in vitro as assessed by real time PCR, IFA and plaque assay. Conclusions CHIKV replication was completely inhibited in the virus-infected mice when administered 72 hours post infection. The combination of Chik-1 and Chik-5 siRNAs exhibited additive effect leading to early and complete inhibition of virus replication. These findings suggest that RNAi capable of inhibiting CHIKV growth might constitute a new therapeutic strategy for controlling CHIKV infection and transmission., Author Summary Despite having immense medical importance, still vaccine, chemoprophylactic, or effective therapeutic measures are not commercially available for chikungunya. Only strict attention to good infection control practices may prevent CHIKV infection. The pathogenic properties of CHIKV necessitate the development of an efficient antiviral therapies. Four siRNAs each, targeting the E2 and ns1 genes of chikungunya were designed and evaluated for their efficiency in inhibiting CHIKV growth in in vitro and in vivo model systems. Efficiency of these siRNAs in controlling CHIKV replication in vitro and in vivo was assessed by the real time PCR, IFA and plaque assay. Chik-1 and Chik-5 siRNA ids efficiently inhibited CHIKV replication in the virus-infected Vero-E6 cells and mice. CHIKV replication was completely inhibited in the virus-infected mice when administered 72 hours post infection (p.i.). The combination of Chik-1 and Chik-5 siRNAs exhibited additive effect leading to early and potent inhibition of virus replication. Taken together, these findings suggest the promising efficacy of RNAi ids in silencing sequence-specific genes of CHIKV and might constitute a new therapeutic strategy for controlling the CHIKV infection and transmission.

Mosquito midgut plays a crucial role in its vector susceptibility and pathogen interaction. Identification of the sustainable microflora of the midgut environment can therefore help in evaluating its contribution in mosquito-pathogen interaction and in turn vector competence. To understand the bacterial diversity in the midgut of Aedes aegypti mosquitoes, we conducted a screening study of the gut microbes of these mosquitoes which were either collected from fields or reared in the laboratory “culture-dependent” approach. This work demonstrated that the microbial flora of larvae and adult Ae. aegypti midgut is complex and is dominated by Gram negative proteobacteria. Serratia odorifera was found to be stably associated in the midguts of field collected and laboratory reared larvae and adult females. The potential influence of this sustainable gut microbe on DENV-2 susceptibility of this vector was evaluated by co-feeding S. odorifera with DENV-2 to adult Ae. aegypti females (free of gut flora). The observations revealed that the viral susceptibility of these Aedes females enhanced significantly as compared to solely dengue-2 fed and another gut inhabitant, Microbacterium oxydans co-fed females. Based on the results of this study we proposed that the enhancement in the DENV-2 susceptibility of Ae. aegypti females was due to blocking of prohibitin molecule present on the midgut surface of these females by the polypeptide of gut inhabitant S. odorifera.

Four populations of Culex tritaeniorhynchus (Giles) (Diptera: Culicidae), collected from Bellary, Cuddalore, Pune, and the Microbial Containment Complex laboratory culture in India were analyzed for morphological and allozyme variation. Multivariate analysis based on eight morphological characteristics and three morphometric indices was used to investigate the morphological variations among the four populations. Principal component analysis of the data suggested that siphon, saddle, and anal gills related variables were most important. Discriminant factor analysis of morphological data revealed that the four populations form significantly different clusters which can be differentiated from each other based on siphon, saddle, and pectin teeth related variables. Allozyme electrophoresis of the four populations revealed that the mean heterozygosity per locus value had high variation, ranging from 0.0879 to 1.794. Fst values between 0 and 0.519 suggested genetic differentiation within these populations. Fis values ranged from 0 to 1 with most of the values closer to 1. The allelic frequencies and Nei's genetic identity values showed that genetic differences between populations were small, but significant. Some of the morphological and allozyme variations in the Cx. tritaeniorhynchus populations could be partly attributed to the environmental conditions. The findings suggested that transition of morphological characters and allozyme variations in Cx. tritaeniorhynchus populations seem to be consequences of influence and selection by the environmental conditions. These results indicated that populations of Cx. tritaeniorhynchus in non-endemic areas of Japanese encephalitis (JE) virus infection have higher adaptability as compared to endemic areas of JE infection.

Background 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 Aedes aegypti mosquitoes across the country. Since densonucleosis virus persistently infects mosquito populations, and is demonstrated to negatively affect multiplication of dengue virus in Aedes albopictus, it would be interesting to study if this virus has a role in determining the susceptibility of the vector mosquito Ae. aegypti to chikugunya virus. Methods Mosquito cell lines and adult Ae. aegypti 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. Results 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. Conclusion Chikungunya virus neither stimulates the replication of densovirus nor is its own replication suppressed due to co-infection. Ae. aegypti mosquitoes with densovirus infection were as susceptible to infection by chikungunya virus as the uninfected mosquitoes.

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.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.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.Phylogenetic analysis of all 3 ORFs showed that this new isolate falls in the lineage of Brevidensovirus along with other mosquito DNVs.

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.

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.

An epizootic of febrile illness among the Madras red breed of sheep had occurred in 1994 in Verrapuram, Chennai, India. The epizootic was suspected as Rift Valley fever (RVF)-like sickness based on clinical features. However, its etiological agent could neither be isolated nor implicated conclusively. During the post-epizootic period a male lamb died of similar clinical features and the spleen was immediately collected. Inoculation of spleen suspension in infant mouse brain yielded a virus that was serially passaged in infant mice and rhabdomyosarcoma (RD) cells. Electron microscopic observations revealed virus particles resembling flaviviruses. RT-PCR performed on extracted total RNA from infected cells and mouse brains with flavivirus-specific or RVF-specific primers gave negative results. However, an amplicon of 280 bp was obtained with pestivirus-specific primers from the 5'-UTR. Further, a nested PCR yielded a product of 157 bp. Nucleotide sequencing of the 157 bp product showed 100% homology to BVDV-1. Western blot analysis with a flavivirus envelope protein-specific MAb revealed three proteins of 33 K, 45 K and 55 K. Further studies suggested that the 33 K and 55 K proteins were glycosylated. This is the first report of isolation of BVDV-1 from a lamb in India.

Hemagglutinin activity (HA) was studied in the midgut extracts from highly (h) and lowly susceptible strains of Aedes aegypti mosquitoes to Dengue-2 virus (DEN-2). HA in the midgut extracts from these two isofemale strains of mosquitoes was high in as compared to (h) mosquitoes. HA was found to be higher with chicken red blood cells (RBCs) than with rabbit and human RBCs of O group. Larval midgut extracts showed higher activity than those from adult female mosquitoes. Exposure of midgut extracts to 100 degrees C for 10 mins destroyed the activity. The activity was observed between pH 6 and pH 10. HA in midgut extracts was also studied using twenty different carbohydrates; five of them showed an inhibition of HA. The inhibitory carbohydrates, when incorporated into DEN-2-infected bloodmeal, showed a reduction in the susceptibility of mosquitoes to the virus as compared to the control ones fed on the virus alone. Similarly, when these carbohydrates were incorporated in the DEN-2-infected inoculum, the inoculated mosquitoes showed a reduction in the susceptibility to the virus. HA in the virus-infected midgut extracts was higher than that in the uninfected controls. These results suggest that the presence of HA in the midgut may be one of the factors that affect the susceptibility of Ae. aegypti mosquitoes to DEN-2.

The Ades aegypti mosquito has been considered the principal vector of Chikungunya (CHIK) virus. As CHIK epidemics usually occur in urban regions and Anopheles stephensi is another highly endophilic and anthropophilic mosquito, there is a very high probability of this mosquito to feed on CHIK virus-infected patients, to pick up and transmit the virus. Therefore the present study was conducted to test the CHIK virus transmission capability for the A. stephensi mosquito. The obtained results showed that this mosquito species is capable of transmitting CHIK virus. It is surmised that during any epidemic of febrile illness CHIK virus isolation attempts should also be made from this mosquito species.

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.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.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.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.

Chikungunya (CHIK) virus is prevalent throughout Southeast Asia and Africa. It has caused numerous large outbreaks in India. No active or passive surveillance has been carried out since the last epidemic occurring in 1971. During a recent outbreak of Dengue (DEN)-like illness in eastern India, Aedes aegypti mosquitoes collected from the affected area were positive for CHIK virus. Evidence of dual infection with CHIK and DEN typel virus was also obtained. A widely circulating low-virulent CHIK virus is a possible explanation for the epidemiological pattern of the CHIK virus disease in this region.

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.

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.

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 (0.5p0.1) after the treatment with deltamethrin.

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.

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).

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.

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.