Search

Swift and early diagnosis of dengue is important for case management and epidemiological purpose. We developed a dengue NS1 antigen specific capture enzyme-linked immunosorbent assay (ELISA) for early diagnosis using a panel of monoclonal antibodies (mAbs), which are generated against monomeric non-structural (NS1) antigen expressed in E. coli. The mAbs were tested for its usefulness in dengue diagnosis during dengue outbreak in Tamil Nadu, India and compared with routinely used methods like RT-PCR, MAC-ELISA and commercially available NS1 antigen detection kit. The results of the comparative analysis suggest that the raised mAb may be used for the routine screening of suspected sera and also for the detection of dengue virus antigen in field collected vectors (mosquitoes).

Mosquitoes are vectors of devastating pathogens and parasites, causing millions of deaths every year. Dengue is a mosquito-borne viral infection found in tropical and subtropical regions around the world. Recently, dengue transmission has strongly increased in urban and semiurban areas, becoming a major international public health concern. Aedes aegypti (Diptera: Culicidae) is a primary vector of dengue. Shedding light on genetic deviation in A. aegypti populations is of crucial importance to fully understand their molecular ecology and evolution. In this research, haplotype and genetic analyses were conducted using individuals of A. aegypti from 31 localities in the north, southeast, northeast and central regions of Tamil Nadu (South India). The mitochondrial DNA region of cytochrome c oxidase 1 (CO1) gene was used as marker for the analyses. Thirty-one haplotypes sequences were submitted to GenBank and authenticated. The complete haplotype set included 64 haplotypes from various geographical regions clustered into three groups (lineages) separated by three fixed mutational steps, suggesting that the South Indian Ae. aegypti populations were pooled and are linked with West Africa, Columbian and Southeast Asian lineages. The genetic and haplotype diversity was low, indicating reduced gene flow among close populations of the vector, due to geographical barriers such as water bodies. Lastly, the negative values for neutrality tests indicated a bottle-neck effect and supported for low frequency of polymorphism among the haplotypes. Overall, our results add basic knowledge to molecular ecology of the dengue vector A. aegypti, providing the first evidence for multiple introductions of Ae. aegypti populations from Columbia and West Africa in South India.

Mosquitoes (Diptera: Culicidae) are a key threat for millions of people worldwide, since they act as vectors for devastating pathogens and parasites. The standard method of utilisation of morphological characters becomes challenging due to various factors such as phenotypical variations. We explored the complementary approach of CO1 gene-based identification, analysing ten species of mosquito vectors belonging to three genera, Aedes, Culex and Anopheles from India. Analysed nucleotide sequences were found without pseudo genes and indels; they match with high similarity in nucleotide Basic Local Alignment Search Tool (BLASTn) search. The partial CO1 sequence of Anopheles niligricus was the first time record submitted to National Center for Biotechnology Information (NCBI). Mean intra- and interspecies divergence was found to be 1.30 and 3.83 %, respectively. The congeneric divergence was three times higher than the conspecifics. Deep intraspecific divergence was noted in three of the species, and the reason could be explained more accurately in the future by improving the sample size across different locations. The transitional and transversional substitutions were tested individually. Ts and Tv substitutions in all the 1st, 2nd and 3rd codons were estimated to be (0.44, 99.51), (40.35, 59.66) and (59.16, 40.84), respectively. Saturation of the sequences was resolved, since both the Ts and Tv exhibited a linear relationship suggesting that the sequences were not saturated. NJ and ML tree analysis showed that the individuals of the same species clustered together based on the CO1 sequence similarity, regardless of their collection site and geographic location. Overall, this study adds basic knowledge to molecular evolution of mosquito vectors of medical and veterinary importance and may be useful to improve biotechnological tools employed in Culicidae control programmes.

Accurate and early diagnosis of dengue infection is essential for dengue case management. In outbreak conditions, it is essential to include two different tests to diagnose dengue and the choice depends on the number of days after the onset of illness in which the sample is collected. During the laboratory diagnosis of dengue in late acute and convalescent phase by MAC-ELISA, it is necessary to rule out possible cross reactions of closely related flavivirus, such as Japanese encephalitis virus which is commonly co-circulating. In the present investigation, the usefulness of dengue virus NS1 and prM antibodies in diagnosing and differentiating dengue from Japanese encephalitis infection was assessed using samples collected during out-breaks. It was shown here that, detection of antibodies against dengue NS1 and prM proteins increases the sensitivity of dengue diagnosis until 15 days. Moreover, detection of antibodies against both proteins was able to differentiate dengue from Japanese encephalitis infection.

Detection of nonstructural protein (NS1) is an important diagnostic marker during acute phase of dengue infection. Not only for diagnostic purpose, the protein had important role in vaccine design as well, as a candidate for studying virus assembly and maturation. Various researchers employed different expression systems and strategies for recombinant NS1 protein production. Attempts to express NS1 protein in prokaryotic and yeast expression system result in formation of insoluble protein which needs to undergo refolding to attain native structural and functional forms. Here, we report the production of soluble NS1 protein inE. coliby using appropriate vector and employing suitable culture conditions to maximize protein production. Proteins were purified using metal affinity chromatography. SDS-PAGE and western blot analysis reveal the native structure of NS1 protein. Solid phase ELISA using the recombinantly expressed antigen with positive and negative dengue samples showed that the expressed protein retains its antigenic and immunological properties. To our knowledge, this is the first report on the successful production of functionally active recombinant dengue-2 NS1 protein production without undergoing anyin vitroposttranslational modification process.

As original tribal ways of living have morphed from a forest dweller existence, dengue is no longer an urban infection but is now also found in rural hilly areas. The spread of dengue is enhanced by the frequent movement of people to endemic areas where there is a vector mosquito presence. The impact of the virus is known to be great in the immunologically naive population. Our study reports on the threat of the dengue virus in these hilly areas.

Background & objectives: Japanese encephalitis (JE) is a mosquito-borne zoonotic disease. The JE virus (JEV) does not cause any disease among its natural hosts and transmission continues through mosquitoes belonging to Culex vishnui subgroup. This study was aimed to investigate the prevalence of JEV in mosquitoes and humans in the Thanjavur district, a non-endemic region for JE, in Tamil Nadu, by using standard available assays. Methods : A sero-surveillance study was conducted in Thanjavur district among the normal rural school children in the 5–12 yr age group, during the JE season (October) and post-JE season (February) from 2011 to 2013 for the detection of JEV infection. Vector abundance studies were carried out from 2011 to 2014. JE seropositivity and its association between the seasons were analysed statistically. Results : The occurrence of JE infection among children aged 5–12 yr was very high in the study area. The infection rates for JE in two consecutive seasons for 2011–12 and 2012–13 were 32.2 and 65.2%, respectively. The Cx. tritaeniorhynchus sp. dominated the catch, and was majorly responsible for the transmission. There was a significant difference in the human infection rate compared to the years 1991–92 and 1992–93; and a marked decrease in the cattle to pigs ratio (123 : 1) compared to the studies in 1991–93. Interpretation & conclusion : The study unearthed the prevailing situation of JE among children, who are at higher risk of developing the disease during the transmission season. The decrease in the cattle to pigs ratio might be the one of the reasons for increase in the JEV infection among the children population compared to 20 years before. This trend requires urgent attention as it could be prevented with effective surveillance systems and vaccines.

Specimens of Anopheles pseudosundaicus, new species, belonging to the Pyretophorus Series of Anopheles (Cellia), were collected from the coastal areas of Alleppey and Kollam districts in Krerala, southwest India. Female (holotype), male (allotype), pupa and fourth-stage larva of this species are described and illustrated to distinguish it from An. subpictus Grassi and An. sundaicus. (Rodenwaldt). It differs from An. sundaicus by the absence of speckling on legs and from An. subpictus in having a complete prehumeral dark spot on costa in wings. A partial sequence of Cytochrome oxidase C subunit 1 (CO1) of mt DNA places An. pseudosundaicus phylogenetically distant from An. sundaicus and closer to An. subpictus.

A 4-yr (2002-2006) entomological study was carried out in Kurnool district, Andhra Pradesh state, south India, to identify the mosquito vectors of Japanese encephalitis virus (family Flaviviridae, genus Flavivirus, JEV). In total, 37,139 female mosquitoes belonging five genera and 18 species resting on vegetation were collected in villages and periurban areas at dusk. Mosquito species composition and pattern of JEV infection in mosquitoes varied in periurban and rural areas. In periurban area, Culex gelidus Theobald was abundant, making up 49.7% of total catch followed by Culex tritaeniorhynchus Giles (44.5%). In rural area, Cx. tritaeniorhynchus was predominant, making up 78.9% of total catch followed by Culex quinquefasciatus Say (10.8%), Anopheles subpictus Grassi (7.1%), and Cx. gelidus (1.1%). In light trap collections, Cx. gelidus and Cx. tritaeniorhynchus predominated in periurban and rural areas, respectively. Of 50,145 mosquitoes screened JEV isolations were made only from Cx. gelidus and Cx. tritaeniorhynchus. Based on high abundance and frequent JEV isolation, Cx. tritaeniorhynchus was found to be the principal vector in both areas, whereas Cx. gelidus plays a secondary vector role in periurban areas only.

Following a report of dengue outbreak from January 2010 to 2012 in the Tirunelveli, Theni, Dharmapuri and Thiruvallur districts of Tamil Nadu state, India, an investigation was carried out. The study was to demonstrate the probable presence of Chikungunya viral antibodies in patients clinically suspected of dengue fever. Out of 331 samples analysed, dengue viral antibodies were observed in 14.8% ( n = 49) of patients, while 16.6% ( n = 55) were positive for Chikungunya viral specific IgM antibodies. In the four districts surveyed, patients found positive for Chikungunya were found to be higher than dengue. The clinician should consider Chikungunya in the differential diagnosis of dengue-like infection appearing in the community.

Dengue fever is a rapidly spreading mosquito-borne virus infection, which remains a serious global public health problem. As there is no specific treatment or commercial vaccine available for effective control of the disease, the attempts on developing novel control strategies are underway. Viruses utilize the surface receptor proteins of host to enter into the cells. Though various proteins were said to be receptors of Dengue virus (DENV) using Virus Overlay Protein Binding Assay, the precise interaction between DENV and host is not explored. Understanding the structural features of domain III envelope glycoprotein would help in developing efficient antiviral inhibitors. Therefore, an attempt was made to identify the sequence motifs present in domain III envelope glycoprotein of Dengue virus. Computational analysis revealed that the NGR motif is present in the domain III envelope glycoprotein of DENV-1 and DENV-3. Similarly, DENV-1, DENV-2 and DENV-4 were found to contain Yxxphi motif which is a tyrosine-based sorting signal responsible for the interaction with a mu subunit of adaptor protein complex. High-throughput virtual screening resulted in five compounds as lead molecules based on glide score, which ranges from -4.664 to -6.52 kcal/Mol. This computational prediction provides an additional tool for understanding the virus-host interactions and helps to identify potential targets in the host. Further, experimental evidence is warranted to confirm the virus-host interactions and also inhibitory activity of reported lead compounds.

Chikungunya fever is one of the reemerging vector-borne diseases. It has become a major global health problem especially in the developing countries. There are no vaccines or specific antiviral drugs available to date. This study reports small molecule inhibitors of envelope glycoprotein 2 (E2 glycoprotein) which are predicted based on Chikungunya virus-host interactions. E2 glycoprotein of Chikungunya virus interacts at 216 residue of the host receptor protein which plays a vital role in initiating infection. Understanding the structural aspects of E2 glycoprotein is crucial to develop specific inhibitors to prevent the virus binding from host receptors. In silico method was adopted to predict the sequence motifs of envelope protein, as the method like yeast two hybrid system is laborious, time consuming, and costly. The E2 glycoprotein structure of the Indian isolate was modeled using two templates (2XFC and 3JOC) and then validated. The class III PDZ domain binding motif was found to be identified at 213-216 amino acids. The corresponding peptide structures which recognize the PDZ domain binding motif were identified by the literature search and were used for generating five point pharmacophore model (ADDDR) containing acceptor, donor and aromatic ring features. Databases such as Asinex, TosLab and Maybridge were searched for the matches for the predicted pharmacophore model. Two compounds were identified as lead molecules as their glide score is 5 kcal/mol. Since the pharmacophore model is developed based on Chikungunya virus-host interaction, it can be used for designing promising antiviral lead compounds for the treatment of Chikungunya fever.An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:21.

In India, the eastern state of Bihar is particularly badly affected by visceral leishmaniasis (VL). It was in Bihar in the 1980s that the first clear signs of resistance to pentavalent antimonials, which had then been the standard antileishmanial treatment for several decades, were observed. New drugs and new formulations of old drugs have since been developed for the treatment of VL. However, despite some initial signs of benefit after each major revision in the method of treatment of VL in India, the VL-related case fatality rates recorded in India since the 1970s show no clear evidence of long-term success. In fact, the most recent data indicate that such rates have stabilised or even increased, probably because of the continued usage of sodium stibogluconate in northern Bihar.

Dengue is a serious public health problem in tropical and subtropical countries. It is caused by any of the four serologically distinct dengue viruses, namely DENV1-4. The viruses are transmitted by Aedes mosquitoes. Understanding various defence mechanisms of insects has become a prime area of research worldwide. In insects, the first line of defence against invading pathogens includes cellular mechanisms and a battery of antimicrobial peptides such as defensins, cecropins etc. Defensins--cationic, cysteine-rich peptides consisting of approximately 40 amino acids with broad-spectrum activity against Gram-positive bacteria--have been reported from a wide range of organisms. In the dengue vector mosquito, Aedes aegypti, three isoforms of defensins are reported to be expressed in a spatial and temporal fashion. This report presents the three-dimensional structures of the three isoforms of Ae. aegypti defensins predicted by comparative modeling. Prediction was done with Modeller 9v1 and the structures validated through a series of tests. The best results of the prediction study are presented, and may help lead to the discovery of new synthetic peptides or derivatives of defensins that could be useful in the control of vector-borne diseases.

The use of cotton plug as closure of a bacterial culture flask had been reported to have many disadvantages such as inhibitory nature of cotton to certain microbes, chances of contamination during handling and accumulation of used cotton as biological waste. To overcome the disadvantages of cotton plugs, we have developed a new method of capping bacterial culture flasks. In the present study, three sets of experiments were conducted, one was to find out the efficiency of bacterial growth in culture flasks closed by either glass caps or cotton plugs and the second set was to find out the chances of getting contamination of sterile broth closed by either glass caps or cotton plugs and the third set was to find out the evaporation of water in conical flasks closed by glass caps or cotton plug. The results showed that the bacterial cultures closed by glass caps showed better growth with less chance of contamination and evaporation of the culture media. By this method, the bacterial culture work is made very simpler than using cotton plug.