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.

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.