Structural and Functional Analysis of Groundnut bud necrosis virus (GBNV) Using Computational and Biochemical Approaches.

Groundnut bud necrosis virus (GBNV) belonging to the genus Orthotospovirus is transmitted by its vector Thrips palmi. It is a tri-segmented RNA virus that consists of L, M, and S RNA segments. We analysed the secondary structure features of GBNV proteins through various software and predicted the transmembrane helix, glycosylation, and signal peptidase sites within the GBNV protein sequences (GN, GC, N, NSm, and NSs). In glycoprotein sequence, extended strands are predominant (52.87%) whereas the N protein sequence mostly contains alpha helices (47.46%). The random coils are present in movement protein (43.97%) and structural protein (39.41%). We generated the 3D structure of GN and N protein using SWISS MODEL software and quality is validated through PROCHECK and PDBsum software. We also expressed the GBNV proteins (GN, GC, N, NSm, and NSs) in bacterial expression system. The recombinant proteins were used to raise polyclonal antibodies in mice. Our study will be useful in understanding GBNV protein structures in further detail by analysing the important domains that interact with the thrips proteins. This will further aid us in understanding virus-vector relationship through the application of protein-protein interaction and other immunodiagnostic techniques.

Polyubiquitin protein of Aedes aegypti as an interacting partner of dengue virus envelope protein.

Dengue virus (DENV) is an arbovirus that comprises four antigenically different serotypes. Aedes aegypti (Diptera: Culicidae) acts as the principal vector for DENV transmission, and vector control is crucial for dengue fever epidemic management. To design effective vector control strategies, a comprehensive understanding of the insect vector and virus interaction is required. Female Ae. aegypti ingests DENV during the acquisition of a blood meal from an infected human. DENV enters the insect midgut, replicates inside it and reaches the salivary gland for transmitting DENV to healthy humans during the subsequent feeding cycles. DENV must interact with the proteins present in the midgut and salivary glands to gain entry and accomplish successful replication and transmission. Ae. aegypti midgut cDNA library was prepared, and yeast two-hybrid screening was performed against the envelope protein domain III (EDIII) protein of DENV-2. The polyubiquitin protein was selected from the various candidate proteins for subsequent analysis. Polyubiquitin gene was amplified, and the protein was purified in a heterologous expression system for in vitro interaction studies. In vitro pull-down assay presented a clear interaction between polyubiquitin protein and EDIII. To further confirm this interaction, a dot blot assay was employed, and polyubiquitin protein was found to interact with DENV particles. Our results enable us to suggest that polyubiquitin plays an important role in DENV infection within mosquitoes.

Combining in silico and in vitro approaches to understand the involvement of methylerythritol 4-phosphate and shikimate pathways in Agrobacterium tumefaciens for enhanced coenzyme Q10 production.

AIMS: To perform an integrated comparative analysis of metabolic pathway to understand coenzyme Q10 (CoQ10) production in Agrobacterium tumefaciens. METHODS AND RESULTS: Comparative analysis of the CoQ10 metabolic pathway in 10 organisms using a genome to KEGG orthology program (G2KO) and the KEGG database elucidated the completeness of the production pathway in A. tumefaciens. The specific roles of the key precursors and the enzymes in the metabolic network were subsequently confirmed using pathway inhibitors and enhancers. While the use of fosmidomycin and glyphosate was found to inhibit CoQ10 production by 54.54% to 99%, the supplementation of polyprenyl pyrophosphate of the methylerythritol 4-phosphate pathway and 4-hydroxybenzoate precursor of the shikimate pathway did increse the production of CoQ10 by 2.3-fold. CONCLUSIONS: The present study provides a comprehensive understanding of the CoQ10 biosynthetic pathway in A. tumefaciens, which would assist rational metabolic engineering strategies for augmenting CoQ10 biosynthesis.

Mucin Protein of Aedes aegypti Interacts with Dengue Virus 2 and Influences Viral Infection.

Dengue, caused by dengue virus (DENV), is the most prevalent vector-borne viral disease, posing a serious health concern to 2.5 billion people worldwide. DENV is primarily transmitted among humans by its mosquito vector Aedes aegypti; hence, the identification of a novel dengue virus receptor in mosquitoes is critical for the development of new anti-mosquito measures. In the current study, we have identified peptides which potentially interact with the surface of the virion particles and facilitate virus infection and movement during their life cycle in the mosquito vector. To identify these candidate proteins, we performed phage-display library screening against domain III of the envelope protein (EDIII), which plays an essential role during host cell receptor binding for viral entry. The mucin protein, which shared sequence similarity with the peptide identified in the screening, was cloned, expressed, and purified for in vitro interaction studies. Using in vitro pulldown and virus overlay protein-binding assay (VOPBA), we confirmed the positive interaction of mucin with purified EDIII and whole virion particles. Finally, blocking of mucin protein with anti-mucin antibodies partially reduced DENV titers in infected mosquitos. Moreover, mucin protein was found to be localized in the midgut of Ae. aegypti. IMPORTANCE Identification of interacting protein partners of DENV in the insect vector Aedes aegypti is crucial for designing vector control-based strategies and for understanding the molecular mechanism DENV uses to modulate the host, gain entry, and survive successfully. Similar proteins can be used in generating transmission-blocking vaccines.

Understanding the role of insects in the acquisition and transmission of antibiotic resistance.

Antibiotic resistance (AR) is a global healthcare threat that requires a comprehensive assessment. Poorly regulated antibiotic stewardship in clinical and non-clinical settings has led to a horizontal dissemination of AR. A variety of often neglected elements facilitate the circulation of AR from antibiotic sinks like concentrated animal feeding operations and healthcare settings to other environments that include healthy human communities. Insects are one of those elements that have received underwhelming attention as vectors of AR, despite their well-known role in transmitting clinically relevant pathogens. We here make an exhaustive attempt to highlight the role of insects as zoonotic reservoirs of AR by discussing the available literature and deriving realistic inferences. We review the AR associated with insects housing various human-relevant environments, namely, animal farm industry, edible-insects enterprise, healthcare institutes, human settlements, agriculture settings and the wild. We also provide evidence-based accounts of the events of the transmission of AR from insects to humans. We evaluate the clinical threats associated with insect-derived AR and propose the adoption of more sophisticated strategies to understand and mitigate future AR concerns facilitated by insects. Future works include a pan-region assessment of insects for AR in the form of AR bacteria (ARB) and AR determinants (ARDs) and the introduction of modern techniques like whole-genome sequencing, metagenomics, and in-silico modelling.

Detection of unprecedented level of antibiotic resistance and identification of antibiotic resistance factors, including QRDR mutations in Escherichia coli isolated from commercial chickens from North India.

AIM: This study aimed to investigate the occurrence of antibiotic resistance phenotype and simultaneously understand its genetic basis in Escherichia coli isolated from the cloacal swabs of commercial chickens from north India. METHODS AND RESULTS: Escherichia coli isolates were assessed for susceptibility to 14 different antibiotics using the disc-diffusion technique and were screened for the presence of 22 antibiotic resistance genes (ARGs) by employing PCR. Isolates were found to be highly resistant to fluoroquinolones (nalidixic acid 91%, norfloxacin 73% and ciprofloxacin 66%), tetracycline (71%), beta-lactams (ampicillin 49% and amoxicillin/clavulanic acid 37%), co-trimoxazole (48%), streptomycin (31%) and chloramphenicol (28%); and comparatively less resistant to cefazolin (13%), amikacin (10%), aztreonam (4%), gentamicin (4%) and ceftriaxone (3%). Sixty-three percent of isolates were resistant to more than four different drugs. Abundance of plasmid-borne ARGs like tetA (83%), sul3 (44%), aadA1 (44%), strA (43%), strB (41%), qnrS (38%), sul2 (28%) and aac(6)-Ib-cr (15%) was observed among the isolates. Forty-five percent of isolates possessed more than five different ARGs. Quinolone resistance-determining region (QRDR) mutations within gyrA and parC genes were found to be the major determiners of quinolone resistance. QRDR mutations included leu83, asn87 and gly87 within gyrase-A polypeptide and ile80 and lys84 within topoisomerase IV (encoded by parC). CONCLUSIONS: Our findings suggest the abuse of antibiotics as feed additives and prophylactic drugs in Indian poultry sector. It also projects this industry as an active hotspot for the replication and selection of ARGs. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings would provide evidence to the authorities for formulating effective strategies for restricting antibiotic usage as non-therapeutic agents in food animals. Occurrence of both plasmid-borne and chromosome-borne resistance towards quinolones can drive movement of resistance phenotype across bacterial species and vertical movement of resistance along the bacterial generations, respectively, which can pose mitigation challenges.

Aedes aegypti lachesin protein binds to the domain III of envelop protein of Dengue virus-2 and inhibits viral replication.

Dengue virus (DENV) comprises of four serotypes (DENV-1 to -4) and is medically one of the most important arboviruses (arthropod-borne virus). DENV infection is a major human health burden and is transmitted between humans by the insect vector, Aedes aegypti. Ae. aegypti ingests DENV while feeding on infected humans, which traverses through its gut, haemolymph and salivary glands of the mosquito before being injected into a healthy human. During this process of transmission, DENV must interact with many proteins of the insect vector, which are important for its successful transmission. Our study focused on the identification and characterisation of interacting protein partners in Ae. aegypti to DENV. Since domain III (DIII) of envelope protein (E) is exposed on the virion surface and is involved in virus entry into various cells, we performed phage display library screening against domain III of the envelope protein (EDIII) of DENV-2. A peptide sequence showing similarity to lachesin protein was found interacting with EDIII. The lachesin protein was cloned, heterologously expressed, purified and used for in vitro interaction studies. Lachesin protein interacted with EDIII and also with DENV. Further, lachesin protein was localised in neuronal cells of different organs of Ae. aegypti by confocal microscopy. Blocking of lachesin protein in Ae. aegypti with anti-lachesin antibody resulted in a significant reduction in DENV replication.

Overexpression of native Musa-miR397 enhances plant biomass without compromising abiotic stress tolerance in banana.

Plant micro RNAs (miRNAs) control growth, development and stress tolerance but are comparatively unexplored in banana, whose cultivation is threatened by abiotic stress and nutrient deficiencies. In this study, a native Musa-miR397 precursor harboring 11 copper-responsive GTAC motifs in its promoter element was identified from banana genome. Musa-miR397 was significantly upregulated (8-10) fold in banana roots and leaves under copper deficiency, correlating with expression of root copper deficiency marker genes such as Musa-COPT and Musa-FRO2. Correspondingly, target laccases were significantly downregulated (>-2 fold), indicating miRNA-mediated silencing for Cu salvaging. No significant expression changes in the miR397-laccase module were observed under iron stress. Musa-miR397 was also significantly upregulated (>2 fold) under ABA, MV and heat treatments but downregulated under NaCl stress, indicating universal stress-responsiveness. Further, Musa-miR397 overexpression in banana significantly increased plant growth by 2-3 fold compared with wild-type but did not compromise tolerance towards Cu deficiency and NaCl stress. RNA-seq of transgenic and wild type plants revealed modulation in expression of 71 genes related to diverse aspects of growth and development, collectively promoting enhanced biomass. Summing up, our results not only portray Musa-miR397 as a candidate for enhancing plant biomass but also highlight it at the crossroads of growth-defense trade-offs.

In-silico Designing and Testing of Primers for Sanger Genome Sequencing of Dengue Virus Types of Asian Origin.

Rarity in reporting whole genome sequence of Dengue virus from dengue endemic countries leaves lacunae in understanding regional pattern of virus mutation and ultimately leading to non-understanding of transmission pattern and clinical outcomes emerging at regional levels. Due to inter-serotype genomic similarity and intra-serotype genomic diversity, appropriate designing of primer pairs appears as an exhaustive exercise. Present paper reports new Dengue virus type-specific primer which may help in characterizing virus specific to Asian origin. Genomes of dengue virus serotypes of Asian region were searched and using advanced bioinformatics tools, serotype specific primers were designed and tested for their targeted amplification efficiency. 19 primers sets for DENV-1, 18 primer sets for DENV-2, 17 for DENV-3 and 18 for DENV-4 were designed. In-silico and experimental testing of the designed primers were performed on virus isolated from both clinical isolates and passaged cultures. While all 17 and 18 primer sets of DENV-3 and DENV-2 respectively yielded good quality sequencing results; in case of DENV-4, 16 out of 18 primer sets and in DENV-1, 16 out of 19 primer sets yielded good results. Average sequencing read length was 382 bases and around 82% nucleotide bases were Phred quality QV20 bases (representing an accuracy of circa one miscall every 100 bases) or higher. Results also highlighted importance of use of primer development algorithm and identified genomic regions which are conservative, yet specific for developing primers to achieve efficiency and specificity during experiments.

Overexpression of native ferritin gene MusaFer1 enhances iron content and oxidative stress tolerance in transgenic banana plants.

Iron is an indispensable element for plant growth and defense and hence it is essential to improve the plant's ability to accumulate iron. Besides, it is also an important aspect for human health. In view of this, we attempted to increase the iron content in banana cultivar Rasthali using MusaFer1 as a candidate gene. Initially, the expression of all five genes of the MusaFer family (MusaFer1-5) was quantified under iron-excess and -deficient conditions. The supplementation of 250 and 350 µM iron enhanced expression of all MusaFer genes; however, MusaFer1 was increased maximally by 2- and 4- fold in leaves and roots respectively. Under iron deficient condition, all five MusaFer genes were downregulated, indicating their iron dependent regulation. In MusaFer1 overexpressing lines, iron content was increased by 2- and 3-fold in leaves and roots respectively, as compared with that of untransformed lines. The increased iron was mainly localized in the epidermal regions of petiole. The analysis of MusaFer1 promoter indicated that it might control the expression of iron metabolism related genes and also other genes of MusaFer family. MusaFer1 overexpression led to downregulated expression of MusaFer3, MusaFer4 and MusaFer5 in transgenic leaves which might be associated with the plant's compensatory mechanism in response to iron flux. Other iron metabolism genes like Ferric reductase (FRO), transporters (IRT, VIT and YSL) and chelators (NAS, DMAS and NAAT) were also differentially expressed in transgenic leaf and root, suggesting the multifaceted impact of MusaFer1 towards iron uptake and organ distribution. Additionally, MusaFer1 overexpression increased plant tolerance against methyl viologen and excess iron which was quantified in terms of photosynthetic efficiency and malondialdehyde content. Thus, the study not only broadens our understanding about iron metabolism but also highlights MusaFer1 as a suitable candidate gene for iron fortification in banana.

Age of initial cohort of dengue patients could explain the origin of disease outbreak in a setting: a case control study in Rajasthan, India.

Dengue fever (DF) and dengue hemorrhagic fever (DHF) is a public health problem with 390 million cases reported in world annually. In Rajasthan, DF with DHF is being reported for about two decades. For undertaking interventions into disease transmission, locating origin of transmission is very important. Present paper reports retrospective analysis of the hospital reported cases of dengue during the year 2013-2014 undertaken in Barmer, Rajasthan. To address task of investigating outbreak, detailed analysis of the data on serological test results (Mac-ELISA assay of NS1, IgG and IgM) performed by local hospital, Balotra was made. The domestic breeding containers were examined for the presence of larvae and adult forms of Aedes aegypti by visiting individual households as well as common places of human aggregation like schools and hospitals. The analysis showed that first dengue cases started from the lot of school going children and then followed by adults and finally during peak period of infection only children around 1-2 years got infected. The subsequent entomological investigations during the outbreak showed school as principal source of mosquito breeding. Present investigations highlight that schools (March to April) play the role of primary sites of disease transmission and should be preferred for undertaking vector control operations to prevent dengue transmission from getting aggravated.

Small and Hungry: MicroRNAs in Micronutrient Homeostasis of Plants.

MicroRNAs are emerging players in plant development and response to stresses, both biotic and abiotic such as micronutrient deficiency. These small RNAs regulate cognate downstream targets either by transcript cleavage or translational inhibition. Micronutrient deficiencies lead to poor quality and yield of crops and impaired human health. Over the years several microRNAs have been identified which regulate expression of genes controlling uptake, mobilization and homeostasis of macronutrients such as nitrogen, phosphorus and sulfur to ensure sufficiency without toxicity. This review attempts to understand the roles played by micro RNAs involved in homeostasis of the micronutrients boron, manganese, zinc, copper, iron, molybdenum and nickel and the cross talk between them upon perception of nutritional stress. Notably and surprisingly, several micro RNAs are not specific for a particular micronutrient stress and the challenge remains to uncover ones (if any) that are directly relevant to the micronutrient. Current findings of this yet infant field could potentiate biotechnological applications towards biofortification, plant innate immunity and remedy heavy metal toxicity.