Isolation and characterization of a lytic bacteriophage against Pseudomonas aeruginosa.

In recent years, the use of bacteriophages (or 'phages') against multidrug-resistant (MDR) bacteria including Pseudomonas aeruginosa has drawn considerable attention, globally. In this work, we report the isolation and detailed characterization of a highly lytic Pseudomonasphage DRL-P1 isolated from wastewater. Under TEM, DRL-P1 appeared as a member of the phage family Myoviridae. DRL-P1 featured rapid adsorption (~ 5 min), short-latency (~ 30 min), and large burst size (~ 100 PFU per infected cell). DRL-P1 can withstand a wide temperature range (4 °C to 40 °C) and pH (5.0 to 10.0) conditions. The 66,243 bp DRL-P1 genome (MN564818) encodes at least 93 ORFs, of which 36 were functionally annotated based on homology with similar phage proteins available in the databases. Comparative analyses of related genomes suggest an independent evolutionary history and discrete taxonomic position of DRL-P1 within genus Pbunavirus. No toxin or antibiotic resistance genes was identified. DRL-P1 is tolerant to lyophilization and encapsulation techniques and retained lytic activity even after 18 months of storage. We also demonstrated decontaminating potentials of DRL-P1 in vitro, on an artificially contaminated cover-slip model. To the best of our knowledge, this is the first Pbunavirus to be reported from India. Our study suggests DRL-P1 as a potential candidate for various applications.

Rebound of Cotton leaf curl Multan virus and its exclusive detection in cotton leaf curl disease outbreak, Punjab (India), 2015.

Cotton leaf curl disease (CLCuD) outbreaks caused by CLCuD associated begomoviruses (CABs) significantly constrain cotton production in India and Pakistan. In comparison to the CABs circulating in Pakistan, molecular epidemiology, evolution and recombination patterns of CABs circulating in India are less studied. In this work, we characterized CAB complex sequences obtained from the most recent outbreak (Punjab, India, 2015), and rigorously analyzed them with reference to GenBank sequences, submitted from India, Pakistan and other neighbouring countries, using contemporary bioinformatics approaches. In this manuscript, we illustrate the detection of a recombinant, phylogenetically distinct clade of Cotton leaf curl Multan virus (CLCuMuV), suggesting rebound of CLCuMuV in this region. Interestingly, we could not detect Cotton leaf curl Kokhran virus-Burewala strain (CLCuKoV-Bu), which was prevalent in this region, until now. Our study thus indicates substitution of the 'virulent resistance breaking' CLCuKoV-Bu by the re-emerging CLCuMuV recombinants. Our findings corroborate with that of a very recent study from Pakistan and we here discuss epidemiological links between the CAB complexes reported in these two studies. Taken together, these observations signify a shifting epidemiology of CABs, and seem to correlate with the recent prediction of the 'third epidemic' of CLCuD in the Indian subcontinent.

Disappearance of Anopheles minimus and Anopheles dirus from Certain Malaria Endemic Areas of Assam, India.

BACKGROUND: Orang Primary Health Centre (OPHC) and Balipara Primary Health Centre (BPHC) of Assam (India) report mosquito borne diseases annually. Current study was performed to ascertain the prevalence of known malaria and Japanese Encephalitis (JE) vectors and their possible role in disease transmission. METHODS: Malaria epidemiological data for 2006-2010 and JE data for 2008-2013 of Assam, India were obtained from the health authority. Mosquitoes were collected using CDC light traps and identified morpho-taxonomically. RESULTS: Plasmodium falciparum cases (81.5%, 95% CI= 72.0-91.1) were statistically higher in OPHC (P< 0.0001, t= 8.0) during the recent years. There was 4.4 folds rise in the confirmed acute encephalitis syndrome (AES) and 3.2 folds increase in the confirmed JE cases during 2013 as compared to 2008. Altogether 9,218 mosquito specimens (PTND= 153.6), comprising of 44.1% anophelines (PTND= 67.7), 42.3% culicines (PTND= 65.0) and 9.5% mansonia (PTND= 14.6) were recorded. In BPHC, Anopheles vagus was recorded in high density (P< 0.0001), whereas Culex quinquefasciatus was the predominant JE vector (P= 0.04). In OPHC, among the known malaria vectors, the density of Anopheles annularis was significantly high (P< 0.0001). However Culex bitaeniorhynchus was the predominant known JE vector (P< 0.0001) followed by Cx. quinquefasciatus. CONCLUSION: Even in the absence of known efficient vectors, many Anopheles species are still involved in malaria transmission. There was disappearance of Anopheles minimus and Anopheles dirus and establishment of An. annularis, An. vagus and An. philippinensis/nivipes mosquitoes in study area.

Morphometric and morphological appraisal of the eggs of Anopheles stephensi (Diptera: Culicidae) from India.

BACKGROUND & OBJECTIVES: Anopheles stephensi is one of the most important urban malaria vectors in India and contribute about 12% of total malaria cases. An. stephensi has three ecological variants; type, intermediate and mysorensis that can be differentiated on the basis of differences in number of ridges on egg float and on the basis of spiracular indices. Because of its anthropophilic nature the 'type' form is an efficient malaria vector. In the present study, the egg surface morphometry and morphology of An. stephensi 'type' form was studied and detail distinguish- ing characters were recorded for its correct identification. METHODS: Eggs of An. stephensi 'type' form were studied by scanning electron microscopy (SEM) after sputter- coating with gold. In total 23 egg characters were analysed morphologically and morphometrically, which included egg attributes, deck attributes, ventral tubercles, micropyle and float attributes. RESULTS: The dorsal surface of the egg of 'type' form was curved while the ventral surface was concave and both anterior and posterior ends were blunt. The average length and width of egg was 473.94 + 11.18 and 154.69 + 2.66 µm respectively. The number of float ribs observed was 20.33 ± 0.33. The maximum length of float was found to be 246.57 + 15.27 µm, whereas maximum width was 87.16 + 3.83 µm. INTERPRETATION & CONCLUSION: The present study has generated some important data which is specific to An. Stephensi 'type' form and provided significant morphological and morphometric standards for its correct identification. This information could be useful in differentiation of An. stephensi 'type' form from other ecological forms of the same species as well as other species of Anopheles.

Diversity of Cultivable Midgut Microbiota at Different Stages of the Asian Tiger Mosquito, Aedes albopictus from Tezpur, India.

Aedes aegypti and Ae. albopictus are among the most important vectors of arboviral diseases, worldwide. Recent studies indicate that diverse midgut microbiota of mosquitoes significantly affect development, digestion, metabolism, and immunity of their hosts. Midgut microbiota has also been suggested to modulate the competency of mosquitoes to transmit arboviruses, malaria parasites etc. Interestingly, the midgut microbial flora is dynamic and the diversity changes with the development of vectors, in addition to other factors such as species, sex, life-stage, feeding behavior and geographical origin. The aim of the present study was to investigate the midgut bacterial diversity among larva, adult male, sugar fed female and blood fed female Ae. albopictus collected from Tezpur, Northeastern India. Based on colony morphological characteristics, we selected 113 cultivable bacterial isolates for 16S rRNA gene sequence based molecular identification. Of the 113 isolates, we could identify 35 bacterial species belonging to 18 distinct genera under four major phyla, namely Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Phyla Proteobacteria and Firmicutes accounted for majority (80%) of the species, while phylum Actinobacteria constituted 17% of the species. Bacteroidetes was the least represented phylum, characterized by a single species- Chryseobacterium rhizoplanae, isolated from blood fed individuals. Dissection of midgut microbiota diversity at different developmental stages of Ae. albopictus will be helpful in better understanding mosquito-borne diseases, and for designing effective strategies to manage mosquito-borne diseases.

Behavioral Response of Aedes aegypti Mosquito towards Essential Oils Using Olfactometer.

BACKGROUND: Aedes aegypti mosquito is responsible for transmitting human diseases like dengue and chikungunya. Personal or space protection with insect repellents is a practical approach to reducing human mosquito contact, thereby minimizing disease transmission. Essential oils are natural volatile substances from plants used as protective measure against blood-sucking mosquitoes. METHODS: Twenty-three essential oils were evaluated for their repellent effect against Ae. aegypti female mosquito in laboratory conditions using Y-tube olfactometer. RESULTS: The essential oils exhibited varying degree of repellency. Litsea oil showed 50.31%, 60.2 %, and 77.26% effective mean repellency at 1 ppm, 10 ppm and 100 ppm respectively, while DEET exhibited 59.63%, 68.63%, 85.48% and DEPA showed 57.97%, 65.43%, and 80.62% repellency at respective above concentrations. Statistical analysis revealed that among the tested essential oils, litsea oil had effective repellency in comparison with DEET and DEPA against Ae. aegypti mosquito at all concentration. Essential oils, DEET and DEPA showed significant repellence against Ae. aegypti (P< 0.05) at all 3 concentration tested. CONCLUSION: Litsea oil exhibited effective percentage repellency similar to DEET and DEPA. The essential oils are natural plant products that may be useful for developing safer and newer herbal based effective mosquito repellents.

Malaria vector Anopheles culicifacies sibling species differentiation using egg morphometry and morphology.

BACKGROUND: The malaria vector Anopheles culicifacies (sensu lato) is an important malaria vector in Southeast Asia which comprises of five sibling species namely A, B, C, D and E. However, only a few forms have been identified as malaria vectors in various endemic countries. Currently, for the first time egg morphometry and morphology has been used to differentiate the three known vector sibling species of Anopheles culicifacies collected from malaria endemic Madhya Pradesh state of central India. METHODS: The adult An. culicifacies (s.l.) was collected from five districts using standard mosquito collection methods. Adult female mosquitoes were allowed to lay eggs individually. The emerged mosquitoes were identified using allele specific polymerase chain reaction (AS-PCR) to sibling species. Eggs of sibling species A, D and E were studied using scanning electron microscopy (SEM) for morphometric and morphological characteristics. RESULTS: Currently AS-PCR identified four known sibling species (B, C, D and E) of An. culicifacies in the study area. The surface morphology and morphometric attributes of the sibling species A, D and E eggs considerably differed from each other. An. culicifacies E had a narrow deck as compared to A and D, while An. culicifacies A had a bigger micropyle with 6-7 sectors as compared to D and E that had 6 sectors. An. culicifacies D had the smallest float (the structure present on sides of the egg surface in which air is filled that help in floating) and the number of ribs was also fewer than for An. culicifacies A and E. CONCLUSIONS: The present study provides the first evidence that in addition to PCR assay, sibling species of An. culicifacies can also be differentiated using morphological and morphometric characteristics of the egg stage. The results also advocate that the sibling species of An. culicifacies are morphologically dissimilar and can be resolved using advanced microscopy.

Enhancement of PCR Detection Limit by Single-Tube Restriction Endonuclease-PCR (RE-PCR).

BACKGROUND: Polymerase chain reaction (PCR) is widely used in biological research and diagnostics because of its high sensitivity and specificity. However, the sensitivity of PCR is strongly influenced by topological characteristics of the template. Supercoiled templates are known to inhibit PCR, whereas linearized forms of the same supercoiled templates facilitate PCR. OBJECTIVES: This study was conducted to compare the PCR efficiency of circular supercoiled DNA templates to their restriction endonuclease (RE)-mediated linearized forms. Additionally, we also evaluated the possibility of RE digestion of the circular supercoiled templates within the complete PCR buffer. METHODS: Following a systematic approach, we demonstrated that circular supercoiled templates could be efficiently linearized by RE in the complete PCR buffer itself. This allowed linearization of circular supercoiled templates and their subsequent amplification in the PCR buffer in a single-tube format. RESULTS: Using this extremely simple RE-PCR approach, we documented up to tenfold increases in detection efficiency of PCR with two different circular supercoiled templates of clinical origin, including an international calibration standard. CONCLUSIONS: This inexpensive and easy approach to increasing PCR sensitivity can be easily adapted to any standard PCR protocol aimed at amplifying circular supercoiled genomes. Apart from its application in the development of sensitive clinical diagnostic PCR assays for a large number of organisms, this method could also prove to be very useful in simplifying the existing protocols for other applications where pre-PCR restriction digestion is required, such as mutation detection, genotyping, and selective template amplification.

Isolation and characterization of bioactive components from Mirabilis jalapa L. radix.

The present investigation was carried out to isolate and characterize bioactive components from Mirabilis jalapa L. radix ( zǐ mò lì gen). Thin-layer chromatography was used for the separation of spots from fractions of the crude extract. Separated spots were collected for identification of their activities. Free-radical scavenging activity was evaluated by spraying thin-layer chromatography plates (spotted with fractions) with 0.2% of 2,2-diphenyl-1-picrylhydrazyl solution. Activity against human pathogens such as Staphylococcus aureus and Candida albicans were determined using the agar diffusion method. Potential spots were subjected to infrared (IR) analysis and gas chromatography for characterization. Two spots (5F1 and 1F3) showed free-radical scavenging activity. The 1F3 spot was active against both S. aureus and C. albicans, whereas the 5F1 spot was active against S. aureus only. IR spectral analysis indicated that 5F1 spot to be a triterpenoid. Using IR spectral analysis and an IR library search, the 1F3 spot was identified to be a flavone, which may have a hydroxyl group in ring "A" of the flavone nucleus. Our results indicated that the 1F3 and 5F1 spots are potential free-radical scavengers. Both 1F3 and 5F1 exhibited antimicrobial activity. IR spectral analysis coupled with an IR library search indicated 1F3 and 5F1 to be a flavone and a triterpenoid, respectively.

Pharmacokinetic and biodistribution study of eserine and pralidoxime chloride in rabbits following a single application of a transdermal patch.

In the present study, a simple reverse-phase high-performance liquid chromatography method with diode array detection has been developed and validated for the simultaneous determination and quantification of eserine and pralidoxime chloride in rabbit plasma and its application to pharmacokinetic study. The pharmacokinetic study was performed after transdermal application of single patch in rabbits. The plasma levels of both drugs following transdermal application of single patch were maintained for 72 h after removal of the patch. The maximal concentrations (C max) of both drugs were significantly reduced while the mean areas under the plasma concentration vs. time moment curve and mean residence times were evidently increased and extended, respectively. A sustained activity was observed over a period of 3 days. This sustained activity was due to the controlled release of drug into the systemic circulation following transdermal application. Linear correlation was also observed when fraction of drug permeated was correlated with the fraction of drug absorbed at the same time point. Gamma scintigraphy imaging on rabbit following transdermal patch application was performed to ascertain the localization of drugs in rabbit brain.

Morphological and molecular diversity of endophytic Colletotrichum gloeosporioides from tea plant, Camellia sinensis (L.) O. Kuntze of Assam, India.

Thirty isolates of endophytic fungi were isolated from healthy asymptomatic leaves of tea plant (Camellia sinensis) and identified morphologically based on colony morphology, spore shape and size, growth and sporulation rate. Internal transcribed spacer r-DNA sequence analysis supported for molecular identification of all the isolates. Based on morphological and molecular characteristics the isolates were identified as Colletotrichum gloeosporioides. Variations on colony morphology which included the production of conidial masses, led to divide the isolates into different groups. Variations on spore size, growth rate and sporulation rate were exhibited by all the isolates. With RAPD molecular markers, genetic variations among the thirty isolates were observed. Genetic variations and relatedness among the thirty isolates were analyzed with UPGMA phylogram using NTSYS program. Two major groups were obtained among the thirty isolates. Group I comprised of 16 isolates which included three sub groups (Ia, Ib and Ic) and Group II constituted fourteen isolates and it also had three sub groups (IIa, IIb and IIc). A partial co-relationship among the isolates was established on the basis of morphological and molecular based clustering.

Molecular epidemiology of Vibrio cholerae associated with flood in Brahamputra River valley, Assam, India.

Cholera is often caused when drinking water is contaminated through environmental sources. In recent years, the drastic cholera epidemics in Odisha (2007) and Haiti (2010) were associated with natural disasters (flood and Earthquake). Almost every year the state of Assam India witnesses flood in Brahamputra River valley during reversal of wind system (monsoon). This is often followed by outbreak of diarrheal diseases including cholera. Beside the incidence of cholera outbreaks, there is lack of experimental evidence for prevalence of the bacterium in aquatic environment and its association with cholera during/after flood in the state. A molecular surveillance during 2012-14 was carried out to study prevalence, strain differentiation, and clonality of Vibrio cholerae in inland aquatic reservoirs flooded by Brahamputra River in Assam. Water samples were collected, filtered, enriched in alkaline peptone water followed by selective culturing on thiosulfate bile salt sucrose agar. Environmental isolates were identified as V. cholerae, based on biochemical assays followed by sero-grouping and detailed molecular characterization. The incidence of the presence of the bacterium in potable water sources was higher after flood. Except one O1 isolate, all of the strains were broadly grouped under non-O1/non-O139 whereas some of them did have cholera toxin (CT). Surprisingly, we have noticed Haitian ctxB in two non-O1/non-O139 strains. MLST analyses based on pyrH, recA and rpoA genes revealed clonality in the environmental strains. The isolates showed varying degree of antimicrobial resistance including tetracycline and ciprofloxacin. The strains harbored the genetic elements SXT constins and integrons responsible for multidrug resistance. Genetic characterization is useful as phenotypic characters alone have proven to be unsatisfactory for strain discrimination. An assurance to safe drinking water, sanitation and monitoring of the aquatic reservoirs is of utmost importance for combating the impending epidemic threat in the flood affected areas. Further, the management of flood through multi-prong approaches and sustainable utilization of environmental resources would be effective in disease management.

Subchronic dermal exposure to T-2 toxin produces cardiac toxicity in experimental Wistar rats.

Our study aimed to determine the cardiac toxicities of T-2 toxin, a representative mycotoxin that frequently contaminates maize, cereals, and other agricultural products, harvested and stored under damp and cold conditions. Dermal exposure to T-2 toxin caused severe cardiotoxicity in experimental Wistar rats. Electrocardiography studies showed the conduction abnormalities including prolongation of the QT and corrected QT interval, shortening of the PR interval, and tachycardia. Biochemical studies also reported the toxicity of T-2 toxin. T-2 toxin induced acute cardiotoxicity in rats and characterized by significant (p < 0.05) elevation of serum troponin I, creatine kinase (CK) isoenzyme MB, CK isoenzyme NAC, and lactate dehydrogenase as compared to control rats. It is concluded that cardiotoxicity effects of T-2 toxin are thought to be due to direct action on electrocardiac potentials and biochemical changes.

Molecular characterization of midgut microbiota of Aedes albopictus and Aedes aegypti from Arunachal Pradesh, India.

BACKGROUND: Microbiota inhabiting midguts of mosquitoes play a key role in the host - parasite interaction and enhance vectorial capacity of viral diseases like dengue and chikungunya fevers. Mosquito midgut is considered to be an important site for host-pathogen interaction and pathogen survival is thought to be an outcome of this interaction. In the present study we examined the bacterial community in the midgut of Aedes mosquitoes in Arunanchal Pradesh, India, a subtropical zone where dengue fever is reported to be emerging. METHOD: Larvae and pupa of Aedes mosquitoes were collected from a biodiversity hotspot, Bhalukpong, Arunachal Pradesh, India. 16S rRNA gene sequences were used for identification of isolated bacterial population from each species of mosquitoes. We used various diversity indices to assess the diversity and richness of the bacterial isolates in both mosquito species. RESULT: On the basis of 16S rRNA gene sequence analysis a total of 24 bacterial species from 13 genera were identified belonging to 10 families of four major phyla. Phylum Proteobacteria was dominant followed by Firmicutes, Bacteroidetes and Actinobacteria. The midgut bacteria belonging to the phylum Proteobacteria and Firmicutes were isolated from both Ae. albopictus and Ae. aegypti, whereas, bacteria belonging to phylum Bacteroidetes and Actinobacteria were isolated only from Ae. albopictus and Ae. aegypti respectively. Enterobacter cloacae was the dominant bacterial species in both Ae. albopictus (33.65%) and Ae. aegypti (56.45%). Bacillus aryabhattai (22.78%) was the second most common bacterial species in Ae. albopictus whereas, in Ae. aegypti the second most common bacterial species was Stenotrophomonas maltophilia (7.44%). CONCLUSION: The family Enterobacteriaceae of phylum Proteobacteria was dominant in both species of Aedes mosquitoes. To the best of our knowledge, this is the first attempt to study midgut microbiota from a biodiversity hotspot in Northeastern India. Some bacterial genera Enterobacter and Acinetobacter isolated in this study are known to play important roles in parasite-vector interaction. Information on midgut microflora may lead towards the development of novel, safe, and effective strategies to manipulate the vectorial capacity of mosquitoes.

Elemental stoichiometry indicates predominant influence of potassium and phosphorus limitation on arbuscular mycorrhizal symbiosis in acidic soil at high altitude.

The functioning of high-altitude agro-ecosystems is constrained by the harsh environmental conditions, such as low temperatures, acidic soil, and low nutrient supply. It is therefore imperative to investigate the site-specific ecological stoichiometry with respect to AM symbiosis in order to maximize the arbuscular mycorrhizal (AM) benefits for the plants in such ecosystems. Here, we assess the elemental stoichiometry of four Capsicum genotypes grown on acidic soil at high altitude in Arunachal Pradesh, India. Further, we try to identify the predominant resource limitations influencing the symbioses of different Capsicum genotypes with the AM fungi. Foliar and soil elemental stoichiometric relations of Capsicum genotypes were evaluated with arbuscular mycorrhizal (AM) colonization and occurrence under field conditions. AM fungal diversity in rhizosphere, was estimated through PCR-DGGE profiling. Results demonstrated that the symbiotic interaction of various Capsicum genotypes with the AM fungi in acidic soil was not prominent in the study site as evident from the low range of root colonization (21-43.67%). In addition, despite the rich availability of carbon in plant leaves as well as in soil, the carbon-for-phosphorus trade between AMF and plants appeared to be limited. Our results provide strong evidences of predominant influence of the potassium-limitation, in addition to phosphorus-limitation, on AM symbiosis with Capsicum in acidic soil at high altitude. We also conclude that the potassium should be considered in addition to carbon, nitrogen, and phosphorus in further studies investigating the stoichiometric relationships with the AMF symbioses in high altitude agro-ecosystems.

Absence of asymptomatic malaria in a cohort of 133 individuals in a malaria endemic area of Assam, India.

BACKGROUND: Malaria in northeast India affects children and adults annually. The number of malaria cases reported has declined over the past few years. Nevertheless, it is not clear whether there is an actual decline in parasitaemia or whether asymptomatic malaria infections are on the rise, especially in forested and forest-fringed areas. Asymptomatic malaria forms a parasite reservoir that acts as an epicentre for malaria spread during high-transmission season. Therefore it is important to understand the quantum of asymptomatic malaria infections among the vulnerable population. METHOD: Four forest fringed historically malaria endemic villages were selected for the study. A total of 133 individuals without a fever history in the past four weeks were tested for malaria parasite using rapid diagnostic test (RDT), microscopy and polymerase chain reaction (PCR) assay during January - February 2014. Indoor resting Anopheles vectors were collected, identified and tested for sporozoite using VectorTest™ panel assay during October 2013 to March 2014, which is a low transmission season for malaria. Social and demographic data were recorded during the study. RESULTS: Mean age (± SEM) of the participants was 16.1 ± 1.2 years (95 % CI: 13.8-18.4). All participants (100 %) reported to use mosquito nets. Altogether, 43.6 % of participants had education below primary level and only 9 % reported a travel history during the past four weeks. All RDT, microscopy and PCR assays were found negative indicating no asymptomatic malaria parasitaemia. Seven known malaria vector species namely, Anopheles nivipes, An. minimus, An. annularis, An. vagus, An. aconitus, An. philippinensis and An. culicifacies, were recorded in the present study. VectorTest™ sporozoite panel assay conducted on 45 pools (N = 224) of vector mosquitoes were found negative for Plasmodium sporozoite. DISCUSSION: Northeastern states of India report asymptomatic malaria parasitemia along with high malaria transmission. An. minimus and An. dirus are recognised as efficient vectors, but An. culicifacies, An. philippinensis and An. annularis also play role in malaria transmission. Currently all participants were found negative for asymptomatic malaria, however the small sample size may restrict the scope of present results to the population living in more remote areas. CONCLUSION: No cases of asymptomatic malaria infections parasitaemia was found in the present study conducted during a low transmission season indicating that asymptomatic malaria parasitaemia may not be prevalent in the region. Mosquito specimens were tested negative for the malaria sporozoites. Study findings encourage the ongoing malaria intervention efforts and recommends similar investigations in different ecological areas involving large populations.

Antagonistic Potential of Native Trichoderma viride Strain against Potent Tea Fungal Pathogens in North East India.

Indigenous strains of Trichoderma species isolated from rhizosphere soils of Tea gardens of Assam, north eastern state of India were assessed for in vitro antagonism against two important tea fungal pathogens namely Pestalotia theae and Fusarium solani. A potent antagonist against both tea pathogenic fungi, designated as SDRLIN1, was selected and identified as Trichoderma viride. The strain also showed substantial antifungal activity against five standard phytopathogenic fungi. Culture filtrate collected from stationary growth phase of the antagonist demonstrated a significantly higher degree of inhibitory activity against all the test fungi, demonstrating the presence of an optimal blend of extracellular antifungal metabolites. Moreover, quantitative enzyme assay of exponential and stationary culture filtrates revealed that the activity of cellulase, ß-1,3-glucanase, pectinase, and amylase was highest in the exponential phase, whereas the activity of proteases and chitinase was noted highest in the stationary phase. Morphological changes such as hyphal swelling and distortion were also observed in the fungal pathogen grown on potato dextrose agar containing stationary phase culture filtrate. Moreover, the antifungal activity of the filtrate was significantly reduced but not entirely after heat or proteinase K treatment, demonstrating substantial role of certain unknown thermostable antifungal compound(s) in the inhibitory activity.

Next-generation sequencing in clinical virology: Discovery of new viruses.

Viruses are a cause of significant health problem worldwide, especially in the developing nations. Due to different anthropological activities, human populations are exposed to different viral pathogens, many of which emerge as outbreaks. In such situations, discovery of novel viruses is utmost important for deciding prevention and treatment strategies. Since last century, a number of different virus discovery methods, based on cell culture inoculation, sequence-independent PCR have been used for identification of a variety of viruses. However, the recent emergence and commercial availability of next-generation sequencers (NGS) has entirely changed the field of virus discovery. These massively parallel sequencing platforms can sequence a mixture of genetic materials from a very heterogeneous mix, with high sensitivity. Moreover, these platforms work in a sequence-independent manner, making them ideal tools for virus discovery. However, for their application in clinics, sample preparation or enrichment is necessary to detect low abundance virus populations. A number of techniques have also been developed for enrichment or viral nucleic acids. In this manuscript, we review the evolution of sequencing; NGS technologies available today as well as widely used virus enrichment technologies. We also discuss the challenges associated with their applications in the clinical virus discovery.

Knockdown resistance (kdr) mutations in Indian Anopheles culicifacies populations.

BACKGROUND: Anopheles culicifacies s.l. is one of the primary vectors of malaria in India responsible for the highest number of malaria cases. This vector is resistant to DDT in most parts of the country with indication of emerging resistance to pyrethroids. Since knockdown resistance (kdr) is known to confer cross-resistance between DDT and pyrethroids owing to a common target site of action, knowledge of prevalence of knockdown resistance (kdr) alleles is important from insecticide resistance management point of view. METHODS: Nine populations of An. culicifacies belonging to five states of India, representing northern, western and central-east India, were screened for the presence of two alternative kdr mutations L1014F and L1014S using PCR-based assays. Dead and alive mosquitoes, following WHO standard insecticide susceptibility test against deltamethrin and DDT, were tested for allelic association. RESULTS: L1014F mutation was recorded in all populations studied except from Haryana and Rajasthan states in northern India, with low frequencies ranging between 0.012 and 0.076; whereas presence of L1014S mutation was recorded in five populations only belonging to central-east India, with allelic frequencies ranging between 0.010 and 0.046. Both the kdr mutant alleles were found mostly in heterozygous condition without deviating from Hardy-Weinberg equilibrium. Both mutations showed protection against deltamethrin whereas only L1014S mutation showed protection against DDT when tested using additive model. CONCLUSIONS: The two L1014-kdr mutations, L1014F and L1014S, co-occurred in five populations belonging to Chhattisgarh and Odisha states of India whereas L1014F was present in all populations studied except populations from northern states. Both kdr mutations were found with very low allelic frequencies mostly in heterozygous condition and exhibited protection against deltamethrin.

Phylogenetic Characterization of a Novel Insect-Specific Flavivirus Detected in a Culex Pool, Collected from Assam, India.

OBJECTIVE: We report the phylogenetic characterization of a unique flavivirus sequence detected in a wild Culex tritaeniorhynchus mosquito pool, collected from the northeast Indian state of Assam. METHODS: DNA and RNA were extracted from field-collected mosquito pools. Extracts were subjected to PCR and reverse transcriptase PCR amplification using universal and type-specific primers for direct detection of flavivirus-specific viral nucleic acids. An amplified flavivirus nonstructural protein 5 (NS5) genetic region was sequenced and BLAST searched, and phylogenetic analyses performed with reference sequences retrieved from GenBank. RESULTS: Phylogenetic analyses revealed the sequence to be related to insect-specific flaviviruses (ISFs) of the genus Flavivirus, family Flaviviridae. Despite being related to the Palm Creek virus (PCV; an ISF very recently reported from Northern Australia), the present sequence (provisionally named Assam virus) was found to be highly divergent from PCV and other ISF sequences available in GenBank. The partial NS5 sequence analysis demonstrated low nucleotide sequence identity (66-77%) with known ISFs reported from other parts of the globe. CONCLUSION: Findings of this study suggest the presence of a candidate novel ISF - the first to be reported from India.