Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses.

Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

Ultrasound for the degradation of endocrine disrupting compounds in aqueous solution: A review on mechanisms, influence of operating parameters and cost estimation.

Availability of drinking water is one of the basic humanitarian goals but remains as a grand challenge that the world is facing today. Currently, water bodies are contaminated not only with conventional pollutants but also with numerous recalcitrant pollutants, such as PPCPs, endocrine disrupting compounds, etc. These emerging pollutants require special attention because of their toxicity to living organisms, bio-resistant and can sustain even after primary and secondary treatments of wastewater. Among different treatment technologies, sonolysis is found to be an innovative and promising technique for the treatment of emerging pollutants present in aqueous solution. Sonolysis is the use of ultrasound to enhance or alter chemical reactions by the formation of free radicals and shock waves which ultimately helps in degradation of pollutants. This review summarizes several studies in the sonochemical literature, including mechanisms of sonochemical process, physical and chemical effects of ultrasound, and the influence of several process variables such as ultrasound frequency, power density, temperature and pH of the medium on degradation performance for endocrine disrupting compounds. In addition, this review highlighted techno-economic perspectives focusing on the total cost required for translating the ultrasound-based processes on a large scale. Overall, the objective of this study is to exhibit a critical review of information available in the literature to encourage and promote future research on sonolysis for the degradation of Endocrine Disrupting Compounds (EDCs).

In-Cell Stability Prediction of RNA/DNA Hybrid Duplexes for Designing Oligonucleotides Aimed at Therapeutics.

In cells, the formation of RNA/DNA hybrid duplexes regulates gene expression and modification. The environment inside cellular organelles is heterogeneously crowded with high concentrations of biomolecules that affect the structure and stability of RNA/DNA hybrid duplexes. However, the detailed environmental effects remain unclear. Therefore, the mechanistic details of the effect of such molecular crowding were investigated at the molecular level by using thermodynamic and nuclear magnetic resonance analyses, revealing structure-dependent destabilization of the duplexes under crowded conditions. The transition from B- to A-like hybrid duplexes due to a change in conformation of the DNA strand guided by purine-pyrimidine asymmetry significantly increased the hydration number, which resulted in greater destabilization by the addition of cosolutes. By quantifying the individual contributions of environmental factors and the bulk structure of the duplex, we developed a set of parameters that predict the stability of hybrid duplexes with conformational dissimilarities under diverse crowding conditions. A comparison of the effects of environmental conditions in living cells and in vitro crowded solutions on hybrid duplex formation using the Förster resonance energy transfer technique established the applicability of our parameters to living cells. Moreover, our derived parameters can be used to estimate the efficiency of transcriptional inhibition, genome editing, and silencing techniques in cells. This supports the usefulness of our parameters for the visualization of cellular mechanisms of gene expression and the development of nucleic acid-based therapeutics targeting different cells.

Adsorptive removal of dimethyl phthalate using peanut shell-derived biochar from aqueous solutions: equilibrium, kinetics, and mechanistic studies.

Rise in polymer industry and extensive use of their products leads to leaching of phthalate esters and distributed into the different matrices of the environment. This chemical group has the potential to hamper the life of living organisms and ecosystem. Thus, it is essential to develop cost-effective adsorbents capable of removing these harmful compounds from the environment. In this work, peanut hull-derived biochar was taken as the adsorbent, and DMP was selected as the model pollutant or adsorbates. The biochars of different properties were produced at three pyrolysis temperatures (i.e., 450, 550, and 650 °C) to check how temperature affected the adsorbent properties and adsorption performance. Consequently, the performance of biochars for DMP adsorption was thoroughly studied by the combination of experiments and compared with commercial activated carbon (CAC). All the adsorbents are meticulously characterized using various analytical techniques and used for adsorption DMP from aqueous solutions. The results suggested that adsorption was favoring chemisorption with multi-layered adsorption as adsorption kinetics and isotherm are in good alignment with pseudo-second-order kinetics and Freundlich isotherm, respectively. Further, thermodynamic study revealed DMP adsorption on adsorbent is physically spontaneous and endothermic. The removal efficiency order of four adsorbent was as follows: BC650 > CAC > BC550 > BC450 with maximum efficiency of 98.8% for BC650 followed by 98.6% for CAC at optimum conditions. And as it is a short carbon chain PAE, dominant mechanisms of adsorption for DMP onto porous biochar were H-bonding, π-π EDA interactions, and diffusion within the pore spaces. Therefore, this study can provide strategies for the synthesis of biochar for effectively removing DMP from aqueous solution.

Thickness Mapping and Layer Number Identification of Exfoliated van der Waals Materials by Fourier Imaging Micro-Ellipsometry.

As performance of van der Waals heterostructure devices is governed by the nanoscale thicknesses and homogeneity of their constituent mono- to few-layer flakes, accurate mapping of these properties with high lateral resolution becomes imperative. Spectroscopic ellipsometry is a promising optical technique for such atomically thin-film characterization due to its simplicity, noninvasive nature and high accuracy. However, the effective use of standard ellipsometry methods on exfoliated micron-scale flakes is inhibited by their tens-of-microns lateral resolution or slow data acquisition. In this work, we demonstrate a Fourier imaging spectroscopic micro-ellipsometry method with sub-5 µm lateral resolution and three orders-of-magnitude faster data acquisition than similar-resolution ellipsometers. Simultaneous recording of spectroscopic ellipsometry information at multiple angles results in a highly sensitive system, which is used for performing angstrom-level accurate and consistent thickness mapping on exfoliated mono-, bi- and trilayers of graphene, hexagonal boron nitride (hBN) and transition metal dichalcogenide (MoS2, WS2, MoSe2, WSe2) flakes. The system can successfully identify highly transparent monolayer hBN, a challenging proposition for other characterization tools. The optical microscope integrated ellipsometer can also map minute thickness variations over a micron-scale flake, revealing its lateral inhomogeneity. The prospect of adding standard optical elements to augment generic optical imaging and spectroscopy setups with accurate in situ ellipsometric mapping capability presents potential opportunities for investigation of exfoliated 2D materials.

Nearest-neighbor parameters for the prediction of RNA duplex stability in diverse in vitro and cellular-like crowding conditions.

RNA performs various spatiotemporal functions in living cells. As the solution environments significantly affect the stability of RNA duplexes, a stability prediction of the RNA duplexes in diverse crowded conditions is required to understand and modulate gene expression in heterogeneously crowded intracellular conditions. Herein, we determined the nearest-neighbor (NN) parameters for RNA duplex formation when subjected to crowding conditions with an ionic concentration relevant to that found in cells. Determination of the individual contributions of excluded volume effect and water activity to each of the NN parameters in crowded environments enabled prediction of the thermodynamic parameters and their melting temperatures for plenty of tested RNA duplex formation in vitro and in cell with significant accuracy. The parameters reported herein will help predicting RNA duplex stability in different crowded environments, which will lead to an improved understanding of the stability-function relationship for RNAs in various cellular organelles with different molecular environments.

A C2H2 zinc finger transcription factor of the whitefly Bemisia tabaci interacts with the capsid proteins of begomoviruses and inhibits virus retention.

Begomoviruses are a group of ssDNA viruses exclusively transmitted by the whitefly Bemisia tabaci and constrain vegetable production in the old and new worlds. Although multiple molecular determinants governing the transmission of begomoviruses by whiteflies have been unravelled, factors critical for transmission majorly remain unknown. In this study, a whitefly C2H2 zinc finger (ZF) protein, 100% identical to the vascular endothelial ZF-like gene (vezf) protein was confirmed to interact with the CP of both old- and new-world begomoviruses. This was achieved by a yeast two-hybrid (Y2H) system screening of a whitefly cDNA library using capsid protein (CP) of TYLCV as a bait. In silico annotation of vezf protein revealed that it contains a N-terminal ZF-associated domain (ZAD) alongside multiple C2H2 ZF domains on the C-terminal end. ZAD-ZF proteins form the most abundant class of transcription factors within insects. Herein, we validated the interaction of vezf with four diverse begomoviruses and its functional role in begomovirus transmission. Silencing of the vezf gene of B. tabaci led to increased retention of three diverse begomoviruses tested. Vezf is the first insect transcription factor identified to interact with plant viruses and can be crucial to understand the possible mechanisms by which plant viruses modulate transcription of their insect vectors during transmission.

Accelerating PROTAC drug discovery: Establishing a relationship between ubiquitination and target protein degradation.

PROTACs have emerged as a new class of drugs that can target the "undruggable" proteome by hijacking the ubiquitin proteasome system. Despite PROTACs' success, most current PROTACs interface with a limited number of E3 ligases, hindering their expansion to many challenging therapeutic uses. Currently, PROTAC drug discovery relies heavily on traditional Western blotting and reporter gene assays which are insensitive and prone to artifacts, respectively. New reliable methods to monitor true PROTAC function (i.e., ubiquitination and subsequent degradation of targets at physiological expression levels) without external tags are essential to accelerate the PROTAC discovery process and to address many unmet therapeutic areas. In this study, we developed a new high-throughput screening technology using "TUBEs" as ubiquitin-binding entities to monitor PROTAC-mediated poly-ubiquitination of native target proteins with exceptional sensitivity. As a proof of concept, targets including BRD3, Aurora A Kinase, and KRAS were used to demonstrate that ubiquitination kinetics can reliably establish the rank order potencies of PROTAC with variable ligands and linkers. PROTAC-treated cell lysates with the highest levels of endogenous target protein ubiquitination - termed "UbMax" - display excellent correlations with DC50 values obtained from traditional Western blots with the added benefits of being high throughput, providing improved sensitivity, and reducing technical errors.

Comparative Analysis of Volatiles Emitted from Tomato and Pepper Plants in Response to Infection by Two Whitefly-Transmitted Persistent Viruses.

The whitefly Bemisia tabaci is one of the most important agricultural pests due to its extreme invasiveness, insecticide resistance, and ability to transmit hundreds of plant viruses. Among these, Begomoviruses and recombinant whitefly-borne Poleroviruses are transmitted persistently. Several studies have shown that upon infection, plant viruses manipulate plant-emitted volatile organic compounds (VOCs), which have important roles in communication with insects. In this study, we profiled and compared the VOCs emitted by tomato and pepper plant leaves after infection with the Tomato yellow leaf curl virus (TYLCV) (Bogomoviruses) and the newly discovered Pepper whitefly-borne vein yellows virus (PeWBVYV) (Poleroviruses), respectively. The results identified shared emitted VOCs but also uncovered unique VOC signatures for each virus and for whitefly infestation (i.e., without virus infection) independently. The results suggest that plants have general defense responses; however, they are also able to respond individually to infection with specific viruses or infestation with an insect pest. The results are important to enhance our understanding of virus- and insect vector-induced alteration in the emission of plant VOCs. These volatiles can eventually be used for the management of virus diseases/insect vectors by either monitoring or disrupting insect-plant interactions.

Role of lung ultrasound patterns in monitoring coronavirus disease 2019 pneumonia and acute respiratory distress syndrome in children.

BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, lung ultrasonography (US) has been gaining importance in pediatric intensive care and emergency settings for the screening, diagnosis, and monitoring of pulmonary pathology. PURPOSE: To describe the pattern of lung US changes in patients with COVID-19 pneumonia and its potential role in monitoring ventilated patients. METHODS: This prospective observational study included children aged 1 month to 12 years with a confirmed diagnosis of COVID-19. Lung US was performed using a high-frequency linear probe (5-12 MHz) in all children with moderate/severe respiratory symptoms within 24 hours of admission and then daily until the patient required oxygen therapy. Lung involvement severity was assessed using lung US scores, while lung aeration improvement or deterioration was measured using lung ultrasound reaeration scores (LUSReS). RESULTS: Of 85 children with moderate to severe disease, 54 with pulmonary disease were included. Of them, 50 (92.5%) had an interstitial pattern, followed by pleural line abnormalities in 44 (81.5%), reduced or absent lung sliding in 31 (57.4%), and consolidation in 28 (51.8%). A significantly higher lung US score (median, 18; interquartile range [IQR], 11-22) was observed in ventilated versus nonventilated patients (median, 9; IQR, 6-11). LUSReS improvement after positive end-expiratory pressure titration was positively correlated with improved dynamic lung compliance and oxygenation indices and negatively correlated with the requirement for driving pressure. Successful weaning could be predicted with 100% specificity if loss of LUSReS ≤ 5. CONCLUSION: Interstitial syndrome, fragmented pleural line, and subpleural microconsolidation were the most prevalent lung US findings in children with COVID-19 pneumonia. Thus, lung US may have the ability to monitor changes in lung aeration caused by mechanical ventilation and predict its successful weaning in children with COVID-19.

Potentiating the Anti-Tuberculosis Efficacy of Peptide Nucleic Acids through Combinations with Permeabilizing Drugs.

The emergence of antimicrobial resistance warrants for the development of improved treatment approaches. In this regard, peptide nucleic acids (PNAs) have shown great promise, exhibiting antibiotic properties through the targeting of cellular nucleic acids. We aimed to study the efficacy of PNA as an anti-tuberculosis agent. Since the efficacy of PNA is limited by its low penetration into the cell, we also investigated combinatorial treatments using permeabilizing drugs to improve PNA efficacy. Various concentrations of anti-inhA PNA, permeabilizing drugs, and their combinations were screened against extracellular and intracellular mycobacteria.0.625 to 5 µM anti-inhA PNA was observed to merely inhibit the growth of extracellular M. smegmatis, while low intracellular bacterial load was reduced by 2 or 2.5 log-fold when treated with 2.5 or 5 µM PNA, respectively. Anti-inhA PNA against M. tuberculosis H37Ra exhibited bactericidal properties at 2.5 and 5 µM and enabled a slight reduction in intracellular M. tuberculosis at concentrations from 2.5 to 20 µM. Of the permeabilizing drugs tested, ethambutol showed the most permeabilizing potential and ultimately potentiated anti-inhA PNA to the greatest extent, reducing its efficacious concentration to 1.25 µM against both M. smegmatis and M. tuberculosis. Furthermore, an enhanced clearance of 1.3 log-fold was observed for ethambutol-anti-inhA PNA combinations against intracellular M. tuberculosis. Thus, permeabilizing drug-PNA combinations indeed exhibit improved efficacies. We therefore propose that anti-inhA PNA could improve therapy even when applied in minute doses as an addition to the current anti-tuberculosis drug regimen. IMPORTANCE Peptide nucleic acids have great potential in therapeutics as anti-gene/anti-sense agents. However, their limited uptake in cells has curtailed their widespread application. Through this study, we explore a PNA-drug combinatorial strategy to improve the efficacy of PNAs and reduce their effective concentrations. This work also focuses on improving tuberculosis treatment, which is hindered by the emergence of antimicrobial-resistant strains of Mycobacterium tuberculosis. It is observed that the antibacterial efficacy of anti-inhA PNA is enhanced when it is combined with permeabilizing drugs, particularly ethambutol. This indicates that the addition of even small concentrations of anti-inhA PNA to the current TB regimen could potentiate their therapeutic efficiency. We hypothesize that this system would also overcome isoniazid resistance, since the resistance mutations lie outside the designed anti-inhA PNA target site.

Dielectricity of a molecularly crowded solution accelerates NTP misincorporation during RNA-dependent RNA polymerization by T7 RNA polymerase.

In biological systems, the synthesis of nucleic acids, such as DNA and RNA, is catalyzed by enzymes in various aqueous solutions. However, substrate specificity is derived from the chemical properties of the residues, which implies that perturbations of the solution environment may cause changes in the fidelity of the reaction. Here, we investigated non-promoter-based synthesis of RNA using T7 RNA polymerase (T7 RNAP) directed by an RNA template in the presence of polyethylene glycol (PEG) of various molecular weights, which can affect polymerization fidelity by altering the solution properties. We found that the mismatch extensions of RNA propagated downstream polymerization. Furthermore, PEG promoted the polymerization of non-complementary ribonucleoside triphosphates, mainly due to the decrease in the dielectric constant of the solution. These results indicate that the mismatch extension of RNA-dependent RNA polymerization by T7 RNAP is driven by the stacking interaction of bases of the primer end and the incorporated nucleotide triphosphates (NTP) rather than base pairing between them. Thus, proteinaceous RNA polymerase may display different substrate specificity with changes in dielectricity caused by molecular crowding conditions, which can result in increased genetic diversity without proteinaceous modification.

A repackaged CRISPR platform increases homology-directed repair for yeast engineering.

Inefficient homology-directed repair (HDR) constrains CRISPR-Cas9 genome editing in organisms that preferentially employ nonhomologous end joining (NHEJ) to fix DNA double-strand breaks (DSBs). Current strategies used to alleviate NHEJ proficiency involve NHEJ disruption. To confer precision editing without NHEJ disruption, we identified the shortcomings of the conventional CRISPR platforms and developed a CRISPR platform-lowered indel nuclease system enabling accurate repair (LINEAR)-which enhanced HDR rates (to 67-100%) compared to those in previous reports using conventional platforms in four NHEJ-proficient yeasts. With NHEJ preserved, we demonstrate its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yield favorable expression dynamics for heterologous pathways. We present a case study that deploys LINEAR precision editing and NHEJ-mediated random integration to rapidly engineer and optimize a microbial factory to produce (S)-norcoclaurine. Taken together, this work demonstrates how to leverage an antagonizing pair of DNA DSB repair pathways to expand the current collection of microbial factories.

Utilizing microblogs for optimized real-time resource allocation in post-disaster scenarios.

In the aftermath of a disaster event, it is of utmost important to ensure efficient allocation of emergency resources (e.g. food, water, shelter, medicines) to locations where the resources are needed (need-locations). There are several challenges in this goal, including the identification of resource-needs and resource-availabilities in real time, and deciding a policy for allocating the available resources from where they are available (availability-locations) to the need-locations. In recent years, social media, and especially microblogging sites such as Twitter, have emerged as important sources of real-time information on disasters. There have been some attempts to identify resource-needs and resource-availabilities from microblogging sites. However, there has not been much work on having a policy for optimized and real-time resource allocation based on the information obtained from microblogs. Specifically, the allocation of critical resources must be done in an optimal way by understanding the utility of emergency resources at various need-locations at a given point of time. This paper attempts to develop such a utility-driven model for optimized resource allocation in a post-disaster scenario, based on information extracted from microblogs in real time. Experiments show that the proposed model achieves much better allocation of resources than baseline models-the allocation by the proposed model is not only more efficient in terms of quickly bringing down resource-deficits at various need-locations, but also more fair in distributing the available resources among the various need-locations.

Factors Determining Transmission of Persistent Viruses by Bemisia tabaci and Emergence of New Virus-Vector Relationships.

Many plant viruses depend on insect vectors for their transmission and dissemination. The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is one of the most important virus vectors, transmitting more than four hundred virus species, the majority belonging to begomoviruses (Geminiviridae), with their ssDNA genomes. Begomoviruses are transmitted by B. tabaci in a persistent, circulative manner, during which the virus breaches barriers in the digestive, hemolymph, and salivary systems, and interacts with insect proteins along the transmission pathway. These interactions and the tissue tropism in the vector body determine the efficiency and specificity of the transmission. This review describes the mechanisms involved in circulative begomovirus transmission by B. tabaci, focusing on the most studied virus in this regard, namely the tomato yellow leaf curl virus (TYLCV) and its closely related isolates. Additionally, the review aims at drawing attention to the recent knowhow of unorthodox virus-B. tabaci interactions. The recent knowledge of whitefly-mediated transmission of two recombinant poleroviruses (Luteoviridae), a virus group with an ssRNA genome and known to be strictly transmitted with aphids, is discussed with its broader context in the emergence of new whitefly-driven virus diseases.

Probing the Chiral Domains and Excitonic States in Individual WS2 Tubes by Second-Harmonic Generation.

Distinct from carbon nanotubes, transition-metal dichalcogenide (TMD) nanotubes are noncentrosymmetric and polar and can exhibit some intriguing phenomena such as nonreciprocal superconductivity, chiral shift current, bulk photovoltaic effect, and exciton-polaritons. However, basic characterizations of individual TMD nanotubes are still quite limited, and much remains unclear about their structural chirality and electronic properties. Here we report an optical second-harmonic generation (SHG) study on multiwalled WS2 nanotubes on a single-tube level. As it is highly sensitive to the crystallographic symmetry, SHG microscopy unveiled multiple structural domains within a single WS2 nanotube, which are otherwise hidden under conventional white-light optical microscopy. Moreover, the polarization-resolved SHG anisotropy patterns revealed that different domains on the same tube can be of different chirality. In addition, we observed the excitonic states of individual WS2 nanotubes via SHG excitation spectroscopy, which were otherwise difficult to acquire due to the indirect band gap of the material.

Transmission parameters of pepper whitefly-borne vein yellows virus (PeWBVYV) by Bemisia tabaci and identification of an insect protein with a putative role in polerovirus transmission.

Pepper crops in Israel are infected by poleroviruses, Pepper vein yellows virus 2 (PeVYV-2) and Pepper whitefly-borne vein yellows virus (PeWBVYV). Herein we characterize the transmission of PeWBVYV and the aphid-transmitted PeVYV-2, and show that PeWBVYV is specifically transmitted by MEAM1 species of the whitefly Bemisia tabaci, with a minimum latency period of 120 h, and not by the Mediterranean (MED). PeWBVYV and PeVYV-2 were detected in the hemolymph of MED and MEAM1, respectively, however, amounts of PeWBVYV in the hemolymph of MED or PeVYV-2 in MEAM1 were much lower than PeWBVYV in hemolymph of MEAM1. Moreover, we show that PeWBVYV does not interact with the GroEL protein of the symbiont Hamiltonella and thus does not account for the non-transmissibility by MED. An insect glycoprotein, C1QBP, interacting in vitro with the capsid proteins of both PeWBVYV and PeVYV-2 is reported which suggests a putative functional role in polerovirus transmission.

PNA-mediated efflux inhibition as a therapeutic strategy towards overcoming drug resistance in Mycobacterium smegmatis.

The emergence of antibiotic-resistant strains of Mycobacterium tuberculosis and the decelerating development of new and effective antibiotics has impaired the treatment of tuberculosis (TB). Efflux pump inhibitors (EPIs) have the potential to improve the efficacy of existing anti-TB drugs although with toxicity limitations. Peptide nucleic acids (PNAs), oligonucleotide mimics, by virtue of their high nucleic acid binding specificity have the capability to overcome this drawback. We, therefore, investigated the efflux pump inhibitory properties of a PNA designed against an efflux pump of Mycobacterium smegmatis. LfrA, an efflux pump found in M. smegmatis, is majorly involved in conferring innate drug resistance to this strain and, therefore, was selected as a target for gene silencing via PNA. qRT-PCR and EtBr assays confirmed the EPI activity of the anti-lfrA PNA. On testing the effect of the anti-lfrA PNA on the bactericidal activity of a fluoroquinolone, norfloxacin, we observed that 5 µM of anti-lfrA PNA in combination with norfloxacin led to an enhanced killing of up to 2.5 log-fold against wild-type and a lab-generated multidrug resistant strain, exemplifying its potential in countering resistance. Improved efficacy was also observed against intra-macrophage mycobacteria, where the drug-PNA combination enhanced bacterial clearance by 1.3 log-fold. Further, no toxicity was observed with PNA concentrations up to 4 times higher than the efficacious anti-lfrA PNA concentration. Thus, PNA, as an adjuvant, presents a novel and viable approach to rejuvenate anti-TB therapeutics.

Competitive Interactions Between Whitefly- and Aphid-Transmitted Poleroviruses Within the Plant Host and the Insect Vectors.

Pepper cultivation in Israel has been constrained by two sympatric poleroviruses, Pepper vein yellows virus-2 (PeVYV-2) and Pepper whitefly-borne vein yellows virus (PeWBVYV), which are transmitted specifically by aphids and whiteflies, respectively. This study investigated the interaction between PeVYV-2 and PeWBVYV inside the host plant and the insect vectors. Our results show that PeVYV-2 and PeWBVYV compete against each other inside the host plant and also inside aphids. PeWBVYV was the weaker competitor inside the host plant, with diminished transmission rates when inoculated simultaneously or successively after PeVYV-2 and could only be transmitted efficiently when inoculated first and then challenged by PeVYV-2. Successive inoculations of plants with viruliferous whiteflies with PeWBVYV followed by viruliferous aphids with PeVYV-2 led to a coinfection rate of 60%, but with severely reduced titers of PeWBVYV in the coinfected plants compared with singly infected plants. In contrast, PeVYV-2 was the weaker competitor inside the insect vector, with reduced quantities of the acquired virus and a reduced transmission rate by aphids when given prior acquisition on PeWBVYV. However, we also show that the transmission efficiency of PeVYV-2 and PeWBVYV from coinfected plants by whiteflies and aphids remained comparable to that from singly infected plants. This is likely attributable to the reduced titers of PeWBVYV inside coinfected plants causing lesser impact on transmission of PeVYV-2 by aphids and the stronger competitiveness of PeWBVYV inside the whitefly. Competitive interactions between PeVYV-2 and PeWBVYV inside the host plant and insect vector can thus be beneficial for their coexistence.