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.

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.

Implication of the Whitefly, Bemisia tabaci, Collagen Protein in Begomoviruses Acquisition and Transmission.

Begomoviruses are the largest group of plant viruses transmitted exclusively by the whitefly, Bemisia tabaci (Gennadius), in a persistent, circulative, and nonpropagative manner. Begomoviruses in association with B. tabaci cause enormous loss to world agricultural crops. Transmission, retention, and circulation of begomovirus in B. tabaci are facilitated by its interaction with several proteins of the insect and its endosymbionts. However, very few such proteins have been identified from B. tabaci that are involved in this specific interaction. Here, we have performed yeast two-hybrid assay between B. tabaci complementary DNA expression library and the coat protein (CP) of tomato leaf curl New Delhi virus (ToLCNDV) and cotton leaf curl Rajasthan virus (CLCuV). Collagen was the common protein found to be interacting with both of the viruses. The collagen protein was found to be localized in gut layers of B. tabaci. Additionally, pull-down and dot-blot assays confirmed the association of endogenous collagen with ToLCNDV CP. Immunolocalization analysis also showed colocalization of ToLCNDV particles and collagen within insect gut. Finally, B. tabaci fed on anticollagen antibody and exhibited ∼46% reduction in ToLCNDV transmission, suggesting a supportive role for collagen in virus transmission.

Comparison of sagittal anchorage conservation of mini-implants and modified Nance palatal buttons.

OBJECTIVE: To compare the anchorage potential of mini-implants with modified Nance palatal buttons during simultaneous first and second maxillary molar distalization. METHODS: Mini-implants (1.4 x 10 mm) placed to obtain indirect anchorage for maxillary molar distalization using a superelastic Ni-Ti open coil spring were compared with anchorage derived from a modified Nance palatal button incorporated in a distal jet appliance. Appliances were placed bilaterally in 19 adolescent patients. Lateral cephalograms with guide wires to differentiate the right from left sides were used for evaluation. All measurements (angular and linear) were obtained from these guide wires. RESULTS: Anchorage loss at the first premolar was 13% with mini-implant-supported Ni-Ti coil spring appliances and 24.75% with the Nance palatal button (distal jet appliance) on the right side. On the left side, anchorage loss was 15.4% with mini-implant-supported Ni-Ti coil spring appliances and 23.9% with the Nance palatal button (distal jet appliance). CONCLUSION: Mini-implants do not provide absolute anchorage when used indirectly. However, anchorage conservation is more efficient than modified Nance palatal buttons.

Distalization of molars using two stage mini-implants -- a case report.

This article describes a two stage mini-implant approach to distalize the maxillary molars and then retract the anterior teeth into class I, providing a non-compliant, non-extraction treatment.

Late-developing supernumerary mandibular premolar: a case report.

This report presents a case of a patient who developed a supernumerary mandibular premolar during the course of orthodontic treatment. Evidence of this tooth comes from consecutive panoramic radiographs. It is not routine practice to screen for developing supernumerary teeth during orthodontic treatment. Therefore, the possibility of supernumerary tooth interference with occlusal development or orthodontic treatment should always be kept in mind. A brief review of literature concerning the late formation of supernumerary teeth is included.