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.

A thioredoxin-like protein of Bemisia tabaci interacts with coat protein of begomoviruses.

Bemisia tabaci (whitefly) is the sole vector of begomoviruses, which transmits them in a persistent and circulative manner from infected to healthy plants. During this process, begomoviruses interact with various proteins in the insect vector B. tabaci that would play a specific role in the virus transmission. Identification and characterization of such proteins are important to understand the complete process of virus transmission. Coat protein (CP) of begomoviruses is the only protein which is reported to interact with proteins of the insect vector B. tabaci. In this study, we performed yeast two-hybrid assay using CP of cotton leaf curl Rajasthan virus (CLCuV) and Tomato leaf curl New Delhi virus (ToLCNDV) as bait in separate experiments and cDNA prepared from total RNA of B. tabaci was used as prey. Yeast two-hybrid assay resulted in identification of a thioredoxin-like protein (TLP) from CLCuV yeast two-hybrid library. Later TLP was also found to interact with CP of ToLCNDV. In vitro pull-down assay showed TLP interaction with CP of both CLCuV and ToLCNDV. TLP was found to interact with ToLCNDV virus particles isolated from tomato leaves.

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.

A Bemisia tabaci midgut protein interacts with begomoviruses and plays a role in virus transmission.

Begomoviruses are a major group of plant viruses, transmitted exclusively by Bemisia tabaci (Gennadius) in a persistent circulative non-propagative manner. The information regarding molecular and cellular basis underlying Begomovirus - whitefly interaction is very scarce. Evidences have suggested that the insect gut possesses some crucial protein receptors that allow specific entry of virus into the insect haemolymph. We have performed yeast two hybrid gut cDNA expression library screening against coat protein of Tomato leaf curl New Delhi virus (ToLCV) and Cotton leaf curl Rajasthan virus (CLCuV) as bait. Midgut protein (MGP) was the common protein found interacting with both ToLCV and CLCuV. MGP was localized in whole mount B. tabaci as well as in dissected guts through confocal microscopy. Pull down and dot blot assays confirmed in vitro interaction between ToLCV/CLCuV coat protein and MGP. Immunolocalization analysis also showed colocalization of ToLCV/CLCuV particles and MGP within insect's gut. Finally, anti-MGP antibody fed B. tabaci, exhibited 70% reduction in ToLCV transmission, suggesting a supportive role for MGP in virus transmission.

Influence of Groundnut bud necrosis virus on the Life History Traits and Feeding Preference of Its Vector, Thrips palmi.

The effect of Groundnut bud necrosis virus (GBNV) infection on the life history traits of its vector, Thrips palmi, and its feeding preference on GBNV-infected plants were studied. A significant difference was observed in the developmental period (first instar to adult) between the GBNV-infected and healthy thrips, wherein the developmental period of GBNV-infected thrips was decreased. However, there was no effect on the other parameters such as preadult mortality, adult longevity, and fecundity. Further investigation on a settling and feeding choice assay of T. palmi to GBNV-infected and healthy plants showed that T. palmi preferred GBNV-infected cowpea plants more than the healthy cowpea plants. This preference was also noticed for leaf disks from GBNV-infected cowpea, groundnut, and tomato plants.

Detection and Localization of Wolbachia in Thrips palmi Karny (Thysanoptera: Thripidae).

Thrips palmi Karny is a globally distributed polyphagous agricultural pest. It causes huge economic loss by its biological behaviors like feeding, reproduction and transmission of tospoviruses. Since T. palmi shows close morphological similarities with other thrips species, we employed mitochondrial cytochrome oxidase 1 (mtCO1) gene as a molecular marker. BLAST analysis of this sequence helped us to identify the collected specimen as T. palmi. We observed the female to male ratio of about 3:1 from collected samples and suspected the presence of Wolbachia. The presence of Wolbachia was detected by PCR using genus specific primers of 16S rRNA gene. Further confirmation of Wolbachia strain was achieved by conducting PCR amplification of three ubiquitous genes ftsZ, gatB and groEL. A phylogenetic tree was constructed with concatenated sequences of ftsZ and gatB gene to assign supergroup to Wolbachia. Finally, we localized Wolbachia in abdominal region of the insect using fluorescent in situ hybridization with the help of confocal microscope. Our result confirmed the presence of Wolbachia supergroup B strain for the first time in T. palmi.

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.

Regulation and safety measures for nanotechnology-based agri-products.

There is a wide range of application for nanotechnology in agriculture, including fertilizers, aquaculture, irrigation, water filtration, animal feed, animal vaccines, food processing, and packaging. In recent decades, nanotechnology emerged as a prospective and promising approach for the advancement of Agri-sector such as pest/disease prevention, fertilizers, agrochemicals, biofertilizers, bio-stimulants, post-harvest storage, pheromones-, and nutrient-delivery, and genetic manipulation in plants for crop improvement by using nanomaterial as a carrier system. Exponential increase in global population has enhanced food demand, so to fulfil the demand markets already included nano-based product likewise nano-encapsulated nutrients/agrochemicals, antimicrobial and packaging of food. For the approval of nano-based product, applicants for a marketing approval must show that such novel items can be used safely without endangering the consumer and environment. Several nations throughout the world have been actively looking at whether their regulatory frameworks are suitable for handling nanotechnologies. As a result, many techniques to regulate nano-based products in agriculture, feed, and food have been used. Here, we have contextualized different regulatory measures of several countries for nano-based products in agriculture, from feed to food, including guidance and legislation for safety assessment worldwide.

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.

Aeromonas hydrophila-induced alterations in cytosolic calcium activate pro-apoptotic cPKC-MEK1/2-TNFα axis in infected headkidney macrophages of Clarias gariepinus.

Alterations in intracellular-calcium (Ca2+)i homeostasis is critical to Aeromonas hydrophila-induced headkidney macrophages (HKM) apoptosis of Clarias gariepinus, though the implications are poorly understood. Here, we describe the role of intermediate molecules of Ca2+-signaling pathway that are involved in HKM apoptosis. We observed phosphoinositide-3-kinase/phospholipase C is critical for (Ca2+)i release in infected HKM. Heightened protein kinase-C (PKC) activity and phosphorylation of MEK1/2-ERK1/2 was noted which declined in presence of 2-APB, Go6976 and PD98059, inhibitors to IP3-receptor, conventional PKC isoforms (cPKC) and MEK1/2 respectively implicating Ca2+/cPKC/MEK-ERK1/2 axis imperative in A. hydrophila-induced HKM apoptosis. Significant tumor necrosis factor-α (TNFα) production and its subsequent reduction in presence of MEK-ERK1/2 inhibitor U0126 suggested TNFα production downstream to cPKC-mediated signaling via MEK1/2-ERK1/2 pathway. RNAi and inhibitor studies established the role of TNFα in inducing caspase-8-mediated apoptosis of infected HKM. We conclude, alterations in A. hydrophila-induced (Ca2+)i alterations activate cPKC-MEK1/2-ERK1/2-TNFα signaling cascade triggering HKM apoptosis.