A novel strain of viral hemorrhagic septicemia virus was discovered in the Great Lakes. This Great Lakes strain of the virus infects a broad range of fish species with outcomes ranging from clinical and subclinical disease, persistent viral shedding, and/or death. Among the most susceptible species to the Great Lakes strain of the virus are juvenile muskellunge. Increased susceptibility to the virus in this regionally and economic important species generated a multitude of research questions to include but not limited to host range, pathogenesis, and diagnostic tools to efficiently detect and minimize viral spread into captive fish stocks. The overarching aim of the current study focuses on assessing the early and latter stages of disease progression in a battery of traditional and non-traditional diagnostically relevant tissues in juvenile muskellunge. Tissue damage from the virus and amount of live virus in each tissue were evaluated in conjunction with advanced diagnostic methods to identify cells targeted by the virus when possible.Esox masquinongy) were challenged with VHSV-IVb at high (1 × 105 PFU mL−1), medium (4 × 103 PFU mL−1), and low (100 PFU mL−1) doses. Samples from spleen, kidneys, heart, liver, gills, pectoral fin, large intestine, and skin/muscle were collected simultaneously from four fish at each predetermined time point and processed for VHSV-IVb reisolaton on Epitheliosum papulosum cyprini cell lines and quantification by plaque assay. The earliest reisolation of VHSV-IVb occurred in one fish from pectoral fin samples at 24 h post-infection. By 6 days post-infection (dpi), all tissue types were positive for VHSV-IVb. Statistical analysis suggested that virus levels were highest in liver, heart, and skin/muscle samples. In contrast, the kidneys and spleen exhibited reduced probability of virus recovery. Virus distribution was further confirmed by an in situ hybridization assay using a VHSV-IVb specific riboprobe. Heart muscle fibers, hepatocytes, endothelia, smooth muscle cells, and fibroblast-like cells of the pectoral fin demonstrated riboprobe labeling, thus highlighting the broad cellular tropism of VHSV-IVb. Histopathologic lesions were observed in areas where the virus was visualized. A novel sublineage of the piscine novirhabdovirus , genotype IVb, emerged in the Laurentian Great Lakes, causing serious losses in resident fish species as early as 2003. Experimentally infected juvenile muskellunge ( Rhabdoviridae [Piscine novirhabdovirus  is a World Organization of Animal Health (OIE) reportable pathogen that is noted for its ability to cause widespread mortality in a number of fish species throughout the northern hemisphere. VHSV belongs to the genus Novirhabdovirus, within the family oviridae , and is oviridae ,3. The voviridae . The viroviridae ,6,7. Theoviridae , amphipooviridae , and pisoviridae . In addioviridae ,11. AlthScophthalmus maximus) experimentally with marine VHSV isolates, reported that infected turbot did not succumb to death in the early stages, nor did the extent of viral distribution in the turbot resemble that of the rainbow trout, (Oncorhynchus mykiss) experimentally infected with VHSV-Ia, as reported earlier by [Presently, direct detection of this OIE reportable virus relies upon protocols detailed in  and the rlier by . Using iOncorhynchus mykiss) and Chinook salmon (Oncorhynchus tshawytscha) to high pathogenicity in lake herring (Coregonus artedi), both of which are members of the family Salmonidae [Esox masquinongy). The detection of viral nucleic acids through in situ hybridization (ISH) assays which utilize a specific nucleic acid probe has been applied to the detection of fish-pathogenic viruses to include other VHSV isolates [The emerging VHSV-IVb sublineage exhibits a number of unique features that range from its fast substitutions in its nucleic acid , wide ralmonidae ,19. To tisolates ,21. The Epithelioma papulosum cyprini (EPC) cell line [12. Plaques were allowed to form for a period of six days, and plaques were visualized by staining with crystal violet (0.5% w/v) following fixation with a 10% formalin solution buffered with phosphate buffered saline (PBS). The virus stock was calculated to have approximately 7.32 × 108 plaque forming units/mL. The Great Lakes VHSV genotype IVb index strain MI03, originally isolated in our laboratory in 2003 from muskellunge , was proell line . Quantifell line . Cryogen2 tissue culture flasks  at 25 °C using a growth formulation of Earle’s salt-based Minimal Essential Medium (MEM)  supplemented with 29.2 mg mL−1 L-glutamine (Invitrogen), Penicillin (100 IU mL−1) and Streptomycin , 10% fetal bovine serum , and sodium bicarbonate .Cell lines were maintained and subcultured in 150 cm−1) and tested for the presence of VHSV. Briefly, kidneys, heart, liver, and spleen were aseptically removed and homogenized using a Biomaster Stomacher  at the high speed setting for 2 min. Homogenates were diluted with Earle’s salt-based minimal essential medium  supplemented with 12 mM tris buffer (Sigma-Aldrich), penicillin (100 IU mL−1), streptomycin (100 µg mL−1) (Invitrogen), and amphotericin B (250 μg mL−1) (Invitrogen) to produce 1:4 dilution (w/v) of original tissues. Samples were centrifuged at 2000× g and the supernatants inoculated into individual wells of a 24-well plate containing EPC cells grown with MEM . Plates were incubated at 15 °C for 7 d and observed for the formation of cytopathic effects (CPE). A second blind passage was performed and assessed for the presence of CPE.Certified VHSV-free juvenile muskellunge (4 months post hatch) were procured from the Rathbun National Fish Hatchery  and approved for use in experimental infection by Michigan State University’s Animal Care Program in accordance with the Institutional Animal Care and Use Committee  protocol 07/07-123-00. Upon arrival, a subsample of five fish were euthanized using an overdose of tricaine methanesulfonate   and transitioned to VHSV-free certified fathead minnows, Pimephales promelas purchased from Robinson Wholesale, Inc .Fish were acclimated to temperatures from 16 °C to 11 °C over a three-week period before the experiment was initiated. All fish were initially held in a 1900 L circular fiberglass tanks in a continuous flow-through system supplied by oxygenated and facility chilled well water at Michigan State University-Research Containment Facility . Fish were sustained at approximate dissolved oxygen levels of 5 mg L5 PFU mL−1, Group 2 (90 fish) at a medium dose of 4 × 103 PFU mL−1, Group 3 (90 fish) at a low dose of 100 PFU mL−1, and Group 4 (90 fish) served as a negative control by immersion in water mixed with 1 mL of sterile maintenance—MEM. All groups were held in static, oxygenated water for 90 min and subsequently divided into two tanks of equal numbers (45 fish). Calculated dose levels were in accordance to those described in [Experimental challenges were performed at the Michigan State University–Research Containment Facility . A group of 360 fish  were randomly assigned into four groups. Groups of fish were challenged by immersion in water containing VHSV MI03 with Group 1 (90 fish) at a high dose 1 × 10ribed in .Samples of tissue were collected in parallel from four fish in each of the groups in fish infected at the medium dose, including the negative control. Moribund and/or apparently healthy fish were euthanized at predetermined hours  and days  and subsequently dissected for collection of pectoral fin, gill, spleen, heart, liver, kidney, posterior intestine, and dorsal musculature with skin. Sampled tissues were divided into two groups, one of which would be stored at −80 °C in 1.5 mL centrifuge tubes  for the viral plaque assay and the second group to be fixed in phosphate buffered formalin (10%) for 24 h and embedded in paraffin wax for histopathology and in situ hybridization.−1), streptomycin (100 µg mL−1) (Invitrogen), and amphotericin B (250 μg mL−1) (Invitrogen) at a range of 1:4 to 1:250 (w/v) and homogenized by mortar and pestle . Homogenates were centrifuged at 2000× g and inoculated onto 24 well plates of confluent monolayers of EPC. Quantification of virus was completed in accordance with the methods described above. Plates were incubated at 15 °C for 6 d and stained to quantify the number of plaques in order to calculate the number of plaques per tissue.Tissues were weighed and diluted with Earle’s salt-based minimal essential medium  supplemented with 12 mM tris buffer (Sigma-Aldrich), penicillin  following the manufacturer’s instruction. The primer set used in this assay is recommended by [® Green Mastermix DNA Polymerase (Promega), and the DNA-ase free water to create a final volume of 50 μL. The reverse transcriptase was inactivated by subjecting the mixture to 94 °C for 2 min, and 30 cycles of PCR  in a Mastercycler Personal Thermal Cycler . The polymerization was finalized by maintaining the mixture for a period of 7 min at 68 °C. The product was visualized by gel electrophoresis in 1.5% agarose gels. Supernatant from tissue homogenates collected from VHSV challenged fish and negative controls were tested for the presence of VHSV using the EPC cell line. For PCR confirmation, total RNA was extracted using QIAamp Viral RNA Mini Kit , according to the manufacturer’s instructions. Reverse transcription was accomplished by a two step protocol using the Affinity Script Multiple Temperature Reverse Transcriptase RT-PCRended by  for deteOnce tissues were fixed in phosphate buffered formalin for 24–36 h, the tissues were trimmed and submitted for embedding into paraffin. Histologic sections were approximately 5 µm in thickness and stained with hematoxylin and eosin as detailed in . www.ncbi.nlm.nih.gov/blast.cgi (accessed on 29 June 2011)) demonstrated no cross reactivity with IHNV, VHSV genotypes I-III, and VHSV-IVa. The probe was purified by high performance liquid chromatography .Using the sequence of VHSV isolate MI03 (Gen Bank Sequence GQ385941.1), a riboprobe was created to anneal to a unique region of the nucleoprotein gene of VHSV-IVb at nucleotide position 279. The oligonucleotide probe sequence was thus 5′-GTC–CCA-TCA-TCT-TCG-CCA-AAG-CCA-CCA-A-3′. The probe was labeled with digoxigenin at the 5′ end (IDT). This specific probe was developed to differentiate VHSV-IVb from the other novirhabdovirus; IHNV, as well as all other VHSV genotypes. The use of the Basic Local Alignment Search Tool  and subsequently pelleted by centrifugation at 200× g for 5 min. The supernatant was removed and immediately replaced with phosphate buffered formalin (10%). Removing EPC cells and fixation of the cell pellet was repeated by the steps described at 48 h post-infection. A negative control of EPC cells was obtained from an additional 150 cm2 flask in which the cells were not exposed to VHSV-IVb. In order to maximize the sensitivity and specificity of this ISH assay, preliminary tests were performed in order to identify the optimal protocol and reagent concentrations as previously described . ISH usi−1 alkaline protease; Ventana Medical Systems) was used for 12 min at 37 °C for a proteolytic treatment followed by a mild cell conditioning step using the citrate buffer-based RiboCC reagent  for 4 min at 95 °C. The slides were then denatured for 4 min at 37 °C, followed by hybridization for 1 h at 37 °C with the antisense oligonucleotide probe for VHSV-IVb suspended in hybridization buffer . The concentration used for the VHSV-IVb probe was 1.59 ng mL−1 . Four stringency washing steps were performed at 42 °C using 0.1× RiboWash  for 4 min for the first three and for 8 min for the fourth washing step. After the stringency washes, the slides were incubated with a rabbit monoclonal antidigoxigenin antibody  at a dilution of 1:10,000 for 32 min at 37 °C. Slides were then incubated in streptavidin-alkaline phosphatase conjugate  for 16 min at 37 °C and the signal was detected automatically using the BlueMap nitroblue tetrazolium-BCIP (5-bromo-4-chloro-3-indolyl phosphate) substrate kit  for 2 h at 37 °C. The final step involved counterstaining the slides with nuclear fast red-equivalent reagent Red Counterstain II  for 4 min before adding a coverslip. Tissues collected from certified VHSV-IVb free naïve muskellunge were used as negative tissue controls whereas experimentally infected muskellunge with RT-PCR confirmed VHSV-IVb -positive tissues were used as positive controls. Additionally, ISH using the developed probe was tried on tissues of infected muskellunge. Briefly, 5 μm thick sections were cut from paraffin-embedded tissues previously collected and placed onto positively charged slides, which were then deparaffinized and fixed using the Discovery XT automated slide-processing system  as programed in the protocol for the RiboMap in situ hybridization reagent system . Protease 3  in order to assess whether the organs/tissue could be differentiated and ranked based on the overall virus load measured. The dependent variable was denoted as the viral load whereas the independent variables were designated as the exposure dose level, sampling time, and organ/tissue sampled. Initially, the virus load obtained from each organ/tissue for individual fish was descriptively analyzed using PROC FREQ. Following a descriptive analysis of the data, logistic regression was applied to assess the probability of observing the virus in each organ/tissue for all fish.Data were analyzed for virus loads greater than “0” followed by the log transformation of each value. Diagnostic evaluation of the data was completed using Tukey’s test of additivity in order to account for instances that may have only one virus load measured for a sampling time. The organs/tissues were compared to one another and ranked based on the paired t-test and through logistic regression, respectively. Subsequently, data analysis was performed using PROC GLM for a repeated measure 3-way ANOVA in instances where the virus load was attained in at least one of the eight sampled organs/tissues. 5 PFU mL−1), VHSV-IVb was not reisolated in any tissues collected between 0 h and 36 h p.i. , initial detection was limited to the pectoral fin of one fish as early as 24 h p.i. By 6 d p.i., VHSV-IVb was distributed to all sampled organs. However, by the following sampling time (8 d p.i.), virus recovery was limited to the skin/muscle of one fish. From 15 to 29 d p.i., VHSV-IVb was reisolated from nearly all tissue types, with a greater number of total isolations occurring at 29 d p.i. In subsequent sampling times, virus isolations decreased with the exception of 43 d p.i. in which a total of 18 virus isolations occurred. However, at 29 d p.i., a total of 22 virus isolations were observed from all tissue samples on that day. Similar to fish exposed at the highest dose, VHSV-IVb was recovered in 94 of the sampled tissues. By the end of the observation period, the total number of VHSV reisolations appeared to be more common in heart, skin/muscle, and gills, whereas it was less common in spleen samples. The titer of VHSV-IVb in the pectoral fin from one fish at 24 h p.i. was 9.75 × 103 PFU g−1 tissue homogenate. From 6 to 29 d p.i., viral loads were consistently higher in all tissues that were confirmed VHSV positive  and gill (5.04 × 108 PFU g −1) of the same fish  with VHSV-IVb, there were overall many fewer virus reisolations which were limited to samples of heart, liver, intestine, gill, and pectoral fin  and turbot, the distribution of each VHSV genotype and sublineage within its susceptible host(s) has not been thoroughly investigated because it was assumed that VHSV pathogenesis in other species resembles that of VHSV genotype Ia in the rainbow trout. This is particularly true for VHSV genotype IVb, the most recent VHSV sublineage to emerge [The piscine novirhabdovirus (VHSV) is known for its wide host range infecting dozens of freshwater and marine fish species in the northern hemisphere . Except o emerge  with itso emerge . Muskello emerge  and is wo emerge  and therIn the current study, after combining the mean infectious viral load in samples from all three dose levels, analyses revealed that the probability of detecting VHSV-IVb was highest in the liver, heart, and skin/muscle, in contrast to the kidneys and spleen in which VHSV-IVb was less likely to be recovered. These findings are surprising given that the kidneys and spleen are traditionally being sampled in most VHSV laboratory studies and field surveillance ,31. AlthIn our study, heart samples also exhibited high loads of infectious virus from as early as 6 dpi and throughout the 64 d observation period for the medium and high dose exposure groups . BrudeseClupea pallasii,) employing immunocytochemistry assay, suggested that early viral tropism is for the fish epidermal cells at 6 dpi followed by the virus spreading to the fibroblasts of the fin soft rays. In our experiment, detection of the virus in the pectoral fin was much earlier than what Lovy et al. [The relatively high virus load in skin and muscle samples and the strong positive signals in a number of cell types suggest a potential involvement of these tissues in the virus entry and shedding. Samples of muscle have previously demonstrated elevated levels of virus in the highly susceptible rainbow trout to VHSV-Ia . Indeed,y et al.  reportedy et al. , who usey et al.  was ableThe broad spectrum of cell types with positive ISH signals expresses the broad predilection of cellular targets by VHSV-IVb in muskellunge. The virus was visualized in cells originating from all three germ cell layers  indicating its pleiotropism. Additionally, histopathologic changes and riboprobe staining confirmed that the virus replicates in the fin, liver, and heart, resulting in damage to affected cells and tissues. Our findings in the muskellunge concur with those described by Kim et al.  for the The results of our study provide evidence for VHSV-IVb distribution in one susceptible host. The dynamics of virus–host interactions may vary from one host to another, a matter that requires additional studies with each of the species at risk. It is imperative that liver and heart be included in sampling for VHSV-IVb in the case of lethal sample testing. Additional processing steps to reduce the inherent cytotoxicity of the liver and environmental contaminants of the gill could enhance virus reisolation. Gills and fins, and the pectoral fin in particular, should be considered for the non-lethal sample scheme for VHSV-IVb screening.


Ecballium elaterium, Malva sylvestris, and Solanum nigrum, sampled at the edges of the crops. Melon and E. elaterium, both belonging to the family Cucurbitaceae, shared three virus species, whereas there was no virus species overlap between melon and the other two weeds. The diversity of cucurbit aphid-borne yellows virus (CABYV) and tomato leaf curl New Delhi virus (ToLCNDV), both in melon and E. elaterium, was further studied by amplicon sequencing. Phylogenetic and population genetics analyses showed that the CABYV population was structured by the host, identifying three sites in the CABYV RNA-dependent RNA polymerase under positive selection, perhaps reflecting host adaptation. The ToLCNDV population was much less diverse than the CABYV one, likely as a consequence of the relatively recent introduction of ToLCNDV in Spain. In spite of its low diversity, we identified geographical but no host differentiation for ToLCNDV. Potential virus migration fluxes between E. elaterium and melon plants were also analyzed. For CABYV, no evidence of migration between the populations of the two hosts was found, whereas important fluxes were identified between geographically distant subpopulations for each host. For ToLCNDV, in contrast, evidence of migration from melon to E. elaterium was found, but not the other way around.Weeds surrounding crops may act as alternative hosts, playing important epidemiological roles as virus reservoirs and impacting virus evolution. We used high-throughput sequencing to identify viruses in Spanish melon crops and plants belonging to three pluriannual weed species, IMPORTANCE It has been reported that about half of the emerging diseases affecting plants are caused by viruses. Alternative hosts often play critical roles in virus emergence as virus reservoirs, bridging host species that are otherwise unconnected and/or favoring virus diversification. In spite of this, the viromes of potential alternative hosts remain largely unexplored. In the case of crops, pluriannual weeds at the crop edges may play these roles. Here, we took advantage of the power of high-throughput sequencing to characterize the viromes of three weed species frequently found at the edges of melon crops. We identified three viruses shared by melon and the cucurbit weed, with two of them being epidemiologically relevant for melon crops. Further genetic analyses showed that these two viruses had contrasting patterns of diversification and migration, providing an interesting example on the role that weeds may play in the ecology and evolution of viruses affecting crops. Solanum nigrum plants using HTS showed a large variability in virome richness, but with little overlap of the viruses found in both species (Datura stramonium in Spain displayed higher levels of within-host genetic diversity when compared to crops (8–Emerging high-throughput sequencing (HTS) technologies are of relatively recent use in phyto-virology and are very powerful, allowing for the characterization of plant viromes, the detection of known viruses, and the discovery of novel ones . These t2– species . In anot species . Also, J species  showed tto crops . There ato crops , 8–11.Cucumis melo L., family Cucurbitaceae) is an important crop cultivated for fresh fruit consumption. The total world production has been estimated at 28.5 million tons in 2020 (www.fao.org), with Spain being one of the main melon producers in the world. Viral infections seriously affect the quality and the yield of agricultural products, and epidemics of emerging viruses represent increasing and significant threats for sustainable crop production. There are more than 35 different viruses reported affecting cucurbits  can infect melon, cucumber, squash, and watermelon plants, where it remains confined to the phloem causing yellowing symptoms , cucurbit chlorotic yellows virus (CCYV), and tobacco mild green mosaic virus (TMGMV) for the first time in melon crops in this country. We also assessed the potential of three important weed species as virus reservoirs for melon infection; one of the species, E. elaterium, S. nigrum, and Malva sylvestris, from eight different sites in the Murcia, Madrid, and Castilla-La Mancha regions of Spain , tomato yellow leaf curl Sardinia virus (TYLCSV), Moroccan pepper virus (MPV), and a sequence related to tomato yellow mottle-associated virus (ToYMaV) (We detected four viruses in (ToYMaV) . The mos(ToYMaV) . MPV and(ToYMaV) . Even wi(ToYMaV) . The seqM. sylvestris, we detected malva vein clearing virus (MVCV), supported by 0.6 M reads, followed by eggplant mottled dwarf virus (EMDV) with 23,700 reads, and chickpea chlorotic dwarf virus (CpCDV) with 5,883 reads  with melon, suggesting its possible role as a virus reservoir for the crop species. Given the potential importance of E. elaterium in the epidemiology of melon viruses, we first resampled E. elaterium during the 2021 season in three sites (Table S4) and sequenced the pooled samples to confirm its virome (Table S1). We detected the same viruses as in 2020 . No disease symptoms were recorded in infected plants during an observation period of 20 days’ postinoculation. Therefore, the detection of TMGMV in the HTS data set may reflect true infections, although the relevance of this virus in disease induction in melon is likely negligible.The relevance of CABYV and ToLCNDV as melon pathogens is well known, but this is not the case for TMGMV, the third virus shared between E. elaterium during the 2020 season. We extracted total RNA from individual samples and screened them for the presence of the two viruses using RT-PCR. We detected CABYV in 32 out of 62 samples (51.6%) from the 7 sites analyzed  (We cloned and Sanger-sequenced a genomic fragment of the CABYV ORF 2 comprising 11.6% of the CABYV genome. This genomic region holds the largest sequence variability for CABYV . We sequM/5.163) . The phyE. elaterium clade shares ancestors with older CABYV isolates in the area, or with isolates from geographically distant areas, we included in our analysis CABYV sequences of isolates collected from cucurbit crops in the same geographical area during 2003–2005 . To further confirm these results, we looked for potential correlations between geographic and genetic distances among pairs of isolates, but no significant correlation was detected .We then sought to test any spatial structuring among CABYV populations, thus considering a population as the isolates from both hosts belonging to the same site. According to our analysis of molecular variance (AMOVA), the entire CABYV population was not structured by site, only 2.8% of the variation was associated to the site. In contrast, 97.2% of the variation was found within populations, suggesting that CABYV populations are possibly structured by the host . We next examined if this difference was maintained across the different sites. To do so, we only considered sites 1, 2, 3, and 4, where CABYV was present in both melon and E. elaterium. For this analysis, we considered a CABYV subpopulation as the isolates belonging to the same host in each of the four sites. Overall, the nucleotide diversity of the CABYV subpopulations from E. elaterium was higher than in melon across the different sites , but lower differences were observed for sites 2 and 3  (E. elaterium (E. elaterium compared to melon (58 sites versus 36 sites) (Tables S6 and S7), and a different distribution pattern of codons under selection . The above results suggest that host adaptation could be contributing to genetic differentiation of the CABYV population.We next estimated the genetic diversity (π) for the CABYV population. The total nucleotide diversity of CABYV was 0.0649 ± 0.0067. The nucleotide diversity of the CABYV isolates from nt sites . In sitectively) . We nextctively) , suggestctively) . In contlaterium . Moreoveelection . An aligE. elaterium and melon sequences within each site. We then examined the CABYV differentiation among geographical subpopulations of the same host, either melon or E. elaterium. In melon, the Fst values ranged from 0 to 0.18 , so the branching of the phylogenetic tree that we built for the isolates in this work had a weak significance (data not shown). Adding isolates from databases strengthened the tree’s significance, and, thus, the analysis illustrated in P < 0.001). To further test this hypothesis, we sought to identify potential correlations between geographic and genetic distances. We found a correlation of 0.8 between the genetic and geographic matrices , suggesting a potential subgrouping according to geographical sites; nevertheless, we found that the nucleotide diversity for the entire population, and the nucleotide diversity within sites, were very low  might provide an indication that barriers to transmission are not absolute and that mechanisms exist for low frequency interspecific transmission   . In thisE. elaterium, D. stramonium, and Sonchus oleraceus can host ToLCNDV; these authors also reported the presence of a monophyletic ToLCNDV population composed of isolates of the ES genotype, with no clustering pattern across hosts, location, or year, although they showed that D. stramonium exhibited larger levels of within-population genetic diversity. In agreement with the results mentioned above, the ToLCNDV isolates reported here showed very low genetic diversity without a specific association to the host. Since ToLCNDV has been recently introduced in Spain (Bemisia tabaci) transmit the virus in a circulative persistent manner  crops in Spain in 2020. We visited eight sites in total , 200 mg of leaf material from each sample was kept in 2-mL tubes and used to build the species-site pools; in a first stage, the pools were prepared for each species and site by mixing these 200 mg . In both cases, tubes were then frozen in liquid N2 and stored at −80°C until total RNA extraction.A survey was conducted in open field melon  following the manufacturer’s protocol. Pool preparations were then treated with RNase-free DNase I , following the manufacturer’s protocol to remove traces of DNA. The pool preparations were then cleaned up by phenol-chloroform extraction  following the manufacturer’s protocol and used for detection and cloning.Leaf tissue from each of the species-site pools prepared as described above was ground with a mortar and pestle in the presence of liquid Ntraction , and thede novo assembly using MEGAHIT v1.2.9. Contigs were then aligned against the RefSeq virus database (last updated: Feb 2021) using tblastx. Viral hits were filtered using the following criteria: contig length 0.3 to 15 kb, e-value lower than 10−4, and length of alignment between the query and the hit >100 amino acids. The alignment between the query and the hit was further checked manually using Geneious Prime v2020.0.3. RefSeq for each virus identified were used as reference to remap the filtered reads using BWA with the mem algorithm. From these alignments, virus genome coverage and average depth were calculated using SAMtools and R basic functions.Raw reads were analyzed using the custom bioinformatics pipeline reported previously  with somin silico by a blastn search against the NCBI database. The KAPA PROBE FAST One-Step qRT-PCR kit  was used. Each 10-μL reaction consisted of 5 μL of 2X Master Mix, 0.2 μL of forward/reverse primers (10 μM) and probe (10 μM), 0.2 μL of 50X RT-Mix, 0.2 μL of 50× ROX high, 2 μL of DEPC-treated water, and 2 μL of RNA (15 ng/μL). The performance of the primers/probe pairs was determined by calculating the PCR amplification efficiency of the reaction from a standard curve of five 1:10 serial dilutions of the pooled total RNA sample (200 ng/μL).Viruses were detected in individual samples by qRT-PCR using primers and TaqMan probes designed in this study or reported in previous studies (Table S2). The specificity of the primers and probes was confirmed Nicotiana benthamiana leaves infected with TMGMV:PV-0124 and maintained frozen at −80°C were used as the inoculum source. The leaves were ground in a sterile mortar together with 30 mM phosphate buffer pH 8.0 (1 g in 5 mL). Carborundum was sprinkled on the expanded cotyledons of 7-day-old melon plants after germination, and the inoculum was applied by rubbing the cotyledons. Plants were checked periodically for symptoms for a period of 20 days. Cotyledons were collected 7 days postinoculation (dpi), and the second leaves were collected at 15 dpi, which were then placed in 1.5-mL tubes. RNA extraction and TMGMV detection in both cotyledons and second leaves were performed as mentioned in the previous sections.Melon  seeds  were germinated in 0.5-L pots with a mixture of peat and coconut fiber (2:1) substrate . The plants were grown in a greenhouse under the following conditions: 16 h:8 h, 25°C:18°C light:dark photoperiod. Eight plants were challenged with TMGMV:PV-0124 , and four were mock inoculated. Mechanical inoculation was carried out according to standard protocols. CTCCTWCCGATATTGGCTCG-3′) and AB-883 (5′-AGCCCATTYHGCGCCRCAGT-3′) were designed based on the nucleotide sequence of isolate CABYV-N (NC003688) as reported previously (ATGTGGGATCCATTATTGCACGA-3′) and AB-885 (5′-TGCCTCGAGTAACATCACTAACA-3′) were designed on the nucleotide sequence of DNA-A of ToLCNDV (NC004611) to amplify the full AV2 gene. Primers were designed in conserved regions flanking variable genomic regions; even then, the conservation of the primer sequences was checked in the HTS data set.Complementary DNAs (cDNAs) to regions of the CABYV and ToLCNDV genomes were synthesized by RT-PCR. The primer pairs AB-882 , respectively, following the recommendations of the manufacturers. RT-PCR products were purified using the GENECLEAN kit . Purified fragments were cloned into a pGEM-T Easy vector  following the manufacturer’s protocol. Stellar competent cells  were transformed and screened by colony PCR using M13 primers. The PCR products with the expected size were Sanger sequenced.RT and PCR were carried out using Expand Reverse Transcriptase  and NXT Nucleic acid sequences were aligned using MUSCLE run in MEGA X  . PhylogOM541599-OM541658) and ToLCNDV (OM475365-OM475424). Raw reads were submitted to the Sequence Read Archive (SRA) under the Bioproject ID no. PRJNA850108.Virus sequences were deposited in the GenBank database under the following accession numbers: CABYV (


Monitoring virus infections can be an important selection tool in honey bee breeding. A recent study pointed towards an association between the virus-free status of eggs and an increased virus resistance to deformed wing virus (DWV) at the colony level. In this study, eggs from both naturally surviving and traditionally managed colonies from across Europe were screened for the prevalence of different viruses. Screenings were performed using the phenotyping protocol of the ‘suppressed in ovo virus infection’ trait but with qPCR instead of end-point PCR and a primer set that covers all DWV genotypes. Of the 213 screened samples, 109 were infected with DWV, 54 were infected with black queen cell virus (BQCV), 3 were infected with the sacbrood virus, and 2 were infected with the acute bee paralyses virus. It was demonstrated that incidences of the vertical transmission of DWV were more frequent in naturally surviving than in traditionally managed colonies, although the virus loads in the eggs remained the same. When comparing virus infections with queen age, older queens showed significantly lower infection loads of DWV in both traditionally managed and naturally surviving colonies, as well as reduced DWV infection frequencies in traditionally managed colonies. We determined that the detection frequencies of DWV and BQCV in honey bee eggs were lower in samples obtained in the spring than in those collected in the summer, indicating that vertical transmission may be lower in spring. Together, these patterns in vertical transmission show that honey bee queens have the potential to reduce the degree of vertical transmission over time. Apis mellifera). With the arrival of the Varroa mite (Varroa destructor), the virus landscape in honey bee colonies was considerably changed by the introduction of a new transmission pathway, thereby influencing virus virulence and evolution [Disease pressure is an inherent driver of the evolution of eusociality ,2 or socvolution ,11,12,13volution ,19,20,21Of the 72 virus species that have been identified in honey bees , the mosIn the first years after managed colonies are left untreated against the Varroa mite, colony mortality increases considerably ,43. ThisEach response to a parasite influences transmission dynamics within and between honey bee colonies. As honey bees live in large groups, the transmission of viruses through trophallaxis, feeding or body contact occurs frequently. This form of transmission between individuals of the same generation is defined as horizontal transmission. However, transmission between generations by either eggs or semen is defined as vertical transmission . Virus iend-point PCR allowed for the quantification of the viral load of the eggs and lowered the detection threshold. Second, as the SOV trait is associated with increased virus resistance across DWV genotypes [The aim of this study was to compare virus infections in eggs collected from naturally surviving colonies (NSCs) and traditionally managed colonies (TMCs) across Europe, along with an analysis of the effects that queen age and sampling season have on infection patterns in these eggs. The previously described SOV phenotyping protocol  was usedenotypes , a shiftend-point PCR to a qPCR approach and to compare the quantification of DWV infections using primers specific to DWV-A or DWV-B and a generic DWV primer (DWV-Fam). All samples were collected following the phenotyping protocol for the SOV trait, as described by de Graaf et al. (2020), and they were screened using qPCR for DWV-A, DWV-B, DWV-Fam, SBV, ABPV and BQCV. To compare the performance of the end-point PCR to that of the qPCR, positive samples covering a 101–108/10 egg range were selected and analyzed using end-point PCR for SBV, ABPV, DWV-A and BQCV.The 187 samples collected as part of the Flemish bee-breeding program in 2020 were used to compare the transition from an Egg samples were collected in 9 countries across Europe during either the spring (the beginning of March to the end of May) or summer (the beginning of June to the end of July) of 2020, depending on the presence of drone brood in each country and in each season . Each coAll samples were first homogenized in the presence of zirconium beads in 0.5 mL QIAzol lysis reagent . RNA was extracted using an RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer’s instructions, including a DNAse step, and it was finally eluted in 30 µL elution buffer. The concentration of the total RNA was measured using Nanodrop . Using random hexamer primers, 200 ng of RNA was retro-transcribed with a RevertAid H Minus First Strand cDNA Synthesis Kit . Honey bee β-actin was used to control RNA integrity.end-point PCR reaction mixtures contained 2 μM of each primer  and 2 μL cDNA product. The end-point PCR assays were performed using the following cycling conditions: 95 °C for 5 min; 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, 35 cycles; final elongation 72 °C for 10 min, hold 4 °C. The end-point PCR amplicons were analyzed by electrophoresis using 1.5% agarose gels stained with ethidium bromide and visualized under UV light. Positive and negative controls were included in each run.All imer see , 1 mM Mg−1). Each plate included a no-template control and a positive control. A standard curve obtained through an 8-fold 5× serial dilution of a known amount of viral plasmid loads (range of 104–1010 copies/µL) was used for absolute quantification. All data were analyzed using CFX Manager™ 3.1 software (Bio-Rad). Baseline correction and threshold setting were performed using the automatic calculation offered by the same software. The maximum accepted quantification cycle (Ct) difference between replicates was set to two Ct. The successful amplification of the β-actin internal reference gene was used to confirm RNA integrity throughout the entire procedure [2 = 0.997 for DWV; Ct = −4.458 × +50.946, R2 = 0.865 for ABPV; Ct = −4.260 × +55.151, R2 = 0.943 for BQCV; and Ct = −4.571 × +45.651, R2 = 0.938 for SBV.The virus load qPCR determination was performed using Platinum™ SYBR™ Green qPCR SuperMix-UDG (Thermo Scientific). Each reaction consisted of 0.4 µM of each primer . Below this threshold, samples cannot be reliably quantified using qPCR . RStudio1–108 copy/reaction range of the starting template) were 102 for DWV-A, 107 for BQCV, 106 for ABPV and 108 for SBV  = 9.4, p < 0.05) and BQCV  = 12.3, p < 0.05) but not for SBV.2  = 8.6, p < 0.05) but not for BQCV  = 0.1, p = 0.75). No significant differences were found between the infection loads of TMCs and NSCs for DWV (t (68) = -0.6, p = 0.52; TMCs = 5.2 ± 0.4, NSCs = 4.9 ± 1.6) and BQCV (t (33) = 1.6, p = 0.11; TMCs = 4.4 ± 0.9, NSCs = 4.8 ± 0.1). On average, most infections hovered around 105 for both DWV and BQCV in this sample cohort.2  = 3.9, p < 0.05). This trend continued with a lower infection frequency in queens aged 2 years (1/10) than in queens aged 1 year (14/42), albeit not significant. No differences in infection frequencies were found between queen ages in NSCs. t (29) = 2.6, p < 0.05) for both TMCs and NSCs. Albeit not significant, the mean DWV infection load for queens aged 2 years (M = 5.3) was lower than the mean of queens aged 1 year (M = 6.2). No significant differences in infection frequencies or infection load were found for BQCV. The infection load did however show a similar general trend, with mean infection loads decreasing with age . Interestingly, the spread in the infection loads of queens aged 0 years largely lacks infection loads lower than 107 for DWV and BQCV. Seasonal differences in sample collection did not influence the infection frequency of DWV in queens aged 0 years  = 1.69, p = 0.16) and older queens  = 2.2, p-value = 0.14). Queen age was unknown for 19 out of 141 samples (13%).end-point PCR. The detection threshold of the qPCR was around 103 (30 Ct) for all viruses, while the end-point PCR showed significantly higher detection thresholds for SBV, BQCV and ABPV . This implies that phenotyping using end-point PCR underestimated the number of virus infections for these viruses. There was no difference between both detection thresholds for DWV. It should be noted that samples negative on qPCR can still be infected below the detection threshold and that positive samples might be infected with viruses in a dormant state. An important advantage of qPCR is that breeding programs can manually set threshold values based on the breeding goal and the virulence of the virus. Each breeding program can thus determine the degree of positive or negative selection desired. Infection loads in eggs are linked with the infection status of the queen [In comparison with the previously described SOV phenotyping protocol , this sthe queen ,54 and hhe queen . Neverth10 lower). In comparison, a previous study in the UK found 40% higher DWV titers when screening with a universal DWV-complex assay than when pooling the results of screening with specific DWV-A and DWV-B assays [The comparison between individual DWV genotypes and the generic DWV showed a large underestimation of DWV infections in this sample cohort when screening for either one of the genotypes or the sum of DWV-A and DWV-B. This can be seen in terms of the underestimation of the number of infected samples (25% of the samples) and the lower infection loads  . The lowIn addition to the high prevalence of DWV, the second most common virus found in this study was BQCV. This virus is the most common cause of queen larval death ,64, but Differences in infection patterns between countries can be caused by climatic conditions, by the seasonality of honey bee viruses ,78,79,80By comparing naturally surviving with traditionally managed populations in the same local context, insights can be gained into which evolutionary adaptations are needed for honey bees to survive without treatment against the Varroa mite. Typical for naturally surviving populations is that they harbor higher mite numbers that serve as an important vector for viruses ,95. HoneHoney bee queens accumulate viral infections and infection loads during queen rearing ,98, duriBy focusing on the role of honey bee queens, this research adds to the growing literature on the relationship between viral infections and honey bee health. Evolutionary patterns of resistance and tolerance can form the theoretical foundation to incorporate virus resilience in breeding programs. This is a promising perspective, as shown by the variability of vertical transmission over time, across queen ages and under different evolutionary conditions.


Plant viruses are the most destructive pathogens which cause devastating losses to crops due to their diversity in the genome, rapid evolution, mutation or recombination in the genome, and lack of management options. It is important to develop a reliable remedy to improve the management of plant viral diseases in economically important crops. Some reports show the efficiency of metal nanoparticles and engineered nanomaterials and their wide range of applications in nanoagriculture. Currently, there are reports for the use of nanoparticles as an antibacterial and antifungal agent in plants and animals too, but few reports as plant antiviral. “Nanophytovirology” has been emerged as a new branch that covers nanobased management approaches to deal with devastating plant viruses. Varied nanoparticles have specific physicochemical properties that help them to interact in various unique and useful ways with viruses and their vectors along with the host plants. To explore the antiviral role of nanoparticles and for the effective management of plant viruses, it is imperative to understand all minute details such as the concentration/dosage of nanoparticles, time of application, application interval, and their mechanism of action. This review focused on different aspects of metal nanoparticles and metal oxides such as their interaction with plant viruses to explore the antiviral role and the multidimensional perspective of nanotechnology in plant viral disease detection, treatment, and management. Food security has always been the priority and important agenda around the globe to feed the large population . Food suFor decades, nanotechnology has proved its potential for the development of effective formulations –28, but Nanoparticles (NPs) are small materials with nanosize ranging from 1 nm to 100 nm , 32 and The nanoparticles could be synthesized by three different methods: biological, physical, and chemical methods. Out of these, biological approaches are considered the best, due to their nontoxic effect, cost-effective, and environmentally friendly nature . The met2, ZnO, MgO, CuO, Cu2O, Al2O3, NiO, and SnO2 , 5044, 5Phytoviruses are always being a challenge for farmers in terms of the production of crops and vegetables. There is a list of experiments that shows the application of different nanoparticles in bacterial and fungal diseases of plants; however, the focused study of nanoparticles on plant virus management is still in its preliminary stages, and the antiviral mechanisms of action of metal nanoparticles are not completely understood. The summary of published work and the available information concerning nanoparticles and plant viruses are gathered in The antiviral mechanism of NPs discussed in different studies and other different possible mechanisms  and the To protect the plants from pathogen invasion, the nanomaterials can be applied directly either into the soil or to seeds or foliage. This direct application is similar to the use of chemical pesticides. However, direct application of nanoparticles to the soil directly affects microorganisms, especially nitrogen-fixing and mineral solubilizing which play a significant role in plant health and nutrition. Silver nanoparticles were the first to be used in plant disease management and showed their antimicrobial activity . The nanThe antiviral mechanisms of metal nanoparticles are not very well understood, but the available studies could provide evidence of the mechanisms involved. The antiviral activity of MeNPs has been observed both in vitro and in vivo on different plants, and it is found to be effective against most of the RNA viruses. Various studies revealed that physical properties like size, shape, and surface area are the key factors to control the biological activity of any nanoparticle , 58. RepBean yellow mosaic virus (BYMV) was studied and reported that the antiviral property of NPs is due to their ability to attach to the envelope glycoprotein of the virus. It binds the disulfide bond regions of the CD4-binding domain present in the envelope glycoprotein gp120 of yellow mosaic virus and prevents entry [The impact of AgNPs on the ts entry . Apart fts entry  and alsots entry .Potato virus Y (PVY) and Tomato mosaic virus (ToMV) and observed the effect. Different concentrations  of AgNPs was sprayed on the plants carrying both diseases and at 50 ppm a concentration of AgNP the striking decrease in disease severity and concentration of both viruses was observed. Furthermore, the transmission electron microscopy (TEM) analysis of the viral sap substantiated the binding of coated protein particles of the virus to AgNPs [Sun-hemp rosette virus (SHRV) indicated complete suppression of the viral disease when spraying with AgNPs at the concentration of 50 mg/L. The detailed result showed the binding of these NPs with virus coat protein and virus inactivation is due to inhibition of virus replication [Sun and his coworkers compared the AgNPs and gold nanoparticles and found AgNPs superior when used for cytoprotective activity towards the virus. It was a general observation that various forms of silver nanoparticles can inactivate viruses by denaturing enzymes through different reactions with self-hydra, amino, carboxyl, phosphate, and imidazole groups –66. Dougto AgNPs . Furtherlication .Tomato spot wilt virus (TSWV) on Chenopodium amaranticolor. Plants sprayed 24 h after inoculation showed weak infection in comparison to plants sprayed before inoculation [Tomato bushy stunt virus (TBSV) [Cyamopsis tetragonoloba, infected with Sun-hemp rosette virus (SHRV), displayed complete suppression of the disease and inactivation of virus replication [2 NPs was studied on tobacco plants against TMV by Cai et al. Both NPs were applied on 3, 7, and 12 days before inoculation of virus. The plant treated 12-days before displayed an extreme antiviral effect by preventing TMV infection and spreading in new leaves [2O3 or TiO2NPs for pretreatment of tobacco plants for 21 days to check the antiviral properties against Turnip mosaic virus (TuMV). The results of the study showed a high decrease in viral proteins, in which the authors suggest could be related to the fact that the NPs interfered with either protein biosynthesis or posttranslational modification processes in the virus, and activated defense mechanisms [bean mild mosaic virus, tobacco plants infected with tobacco necrosis virus and tobacco mosaic virus [The antiviral effect of AgNPs was observed against culation . Similars (TBSV) , while Clication . The antw leaves . Findingchanisms . Variouschanisms –74. Maleic virus .Nicotiana benthamiana plants and given the treatment at different concentrations of titanium dioxide (TiO2) and silver (Ag) nanoparticles, C60 fullerenes, and carbon nanotubes (CNTs) at 100, 200, and 500 mg/L and observed for a 21-day foliar exposure before inoculation of Tobacco mosaic virus (TMV). Plants treated with CNTs and C60 (200 mg/L) exhibited normal phenotype, and viral symptomology was not evident at 5 days postinfection, whereas TiO2 and Ag NP-treated plants show no sign of virus infection suppression [Adeel et al. worked on pression .Many molecular and serological techniques, viz., polymerase chain reaction (PCR), real time PCR, immunological assays such as Enzyme-linked immunosorbent assay (ELISA), and electrochemical immunoassay (ECIA), are being used for diagnostics and identification of plant viral pathogens , 77–80. The device designed to detect the occurrence of any biological analyte, such as a biomolecule, a biological structure, or a microorganism, is known as biosensors. It consists of three parts: (i) a section that identifies the analyte and produces a signal, (ii) a signal transducer, and (iii) a reader device . VariousLettuce mosaic virus, Cowpea mosaic virus, and tobacco mosaic virus with twofold increase of the sensitivity of detection in comparison to traditional methods of ELISA [Cucumber mosaic virus (CMV) by Jiao et al. This method of ELISA consists of three steps: (i) fixation of virus antigen on the surface, (ii) treatment with specific antibodies for the detection of the virus, and (iii) incubation with an enzyme and horse shoe peroxidase- (HRP-) labeled secondary antibody. The reaction was monitored by the mercury electrode. This electrochemical enzyme-linked immunoassay (ECEIA) sensor-based method showed four times higher sensitivity in the detection of CMV in comparison to the standard spectrophotometric ELISA. This was also observed with other plant viruses such as Turnip mosaic virus (TuMV), Tobacco mosaic virus (TMV), Potato virus Y (PVY), Southern bean mosaic virus (SBMV), and Tomato aspermy virus (ToAV).In recent years, various reports have manifested the antibody-based detection of plant viruses –90. Jameof ELISA , 90. IndPlum pox virus (PPV) [Prunus necrotic ringspot virus (PNRSV) and concluded that the biosensor has alike similarity as ELISA [Maize chlorotic mottle virus (MCMV). The sensitivity of the biosensor was found to be similar to ELISA with a detection limit of 250 ng/mL and showed high sensitivity with similar viruses such as Wheat streak mosaic virus (WSMV) [Tobacco mosaic virus (TMV). Later on, this method was employed for the diagnostic of several other viruses, Citrus tristeza virus (CTV) [Potato virus X (PVX) [Potato virus x [Potato virus Y (PVY), Potato virus M (PVM), and Potato virus A (PVA) with a reported sensitivity of 2 ng/mL.In the case of immunosensors, self-assembled monolayers (SAM) were used for diagnostics of plant pathogens. In this method, gold electrodes are the most commonly used substrate for the detection of us (PPV) . Later oas ELISA . Anotheras ELISA . He useds (WSMV) . Laterals (WSMV)  for the us (CTV) , Potato  X (PVX) , Potato  virus x , Potato Chilli vein-banding mottle virus (CVbMV), Watermelon silver mottle virus (WSMoV), and Melon yellow spot virus (MYSV) [Lettuce mosaic virus [Cymbidium mosaic virus (CymMV) or Odontoglossum ringspot virus (ORSV) [An immunoassay is reported to be developed for the detection of multiple substances such as biomarkers and plant pathogens that function based on fluorescence-loaded magnetic microspheres and fluorophore antibodies , 99. A ss (MYSV) . Althougic virus –104 and s (ORSV) .  Table Witches' broom disease of lime (WBDL) caused by Candidatus Phytoplasma aurantifolia [Candidatus Phytoplasma aurantifolia per μL. This technique was applied to detect Rhizoctonia, the disease vector of the Beet necrotic yellow vein virus (BNYVV) [Quantum dots (QD) are small semiconductor nanocrystals that have been used for the construction of biosensors . It has ntifolia . The con (BNYVV) .2, Fe2O3, and ZnO nanoparticles and observed to have increased growth [Turnip mosaic virus was being treated with foliar spray of TiO2 and FeO3 with the concentration of 50 mg/L and observed with enhanced fresh and dry weight, whereas no effect was observed with the treatment of 200 mg/L in comparison to nontreated plants [Potato virus Y-infested tubers were treated with AgNPs, they have shown improved quality parameters in comparison to infected but not treated plants. The reason may be the provocation of resistance or the effect of nanoparticles on virus entry [Biostimulants are substances that enhance the physiological process of plants and promote growth, development, and defense responses. When applied directly to plants or seeds, they cannot be considered pesticides or nutrients . The posd growth , 113. Whd growth . The tobd plants . When thus entry , 113.2 into water and H2O2 [3O4 NPs to tobacco leaves resulted in enhanced production of ROS, which indicates the stimulation of resistance against the virus in tobacco [2 NP, they displayed the expression of pox and pal genes a day after inoculation of PRSV [Under stress conditions (biotic or abiotic), the plant response is observed by increased reactive oxygen species (ROS) that limits the entry of the pathogen and its dissemination and stimulates local and systemic defense responses . When thand H2O2 , 114. Thand H2O2 . The typ tobacco . When cu of PRSV . A simil of PRSV . The AgN of PRSV , 117.Capsicum annum L plants with AgNPs increases cytokinin [2O3 and TiO2 NPs, influence the levels of zeatin, ribose (ZR), abscisic acid, and brassinosteroid (BR) phytohormones [2 [3O4 NPs [Plant hormones play important roles in the defense mechanism of the plant. The phytohormones like salicylic acid (SA), jasmonic acid (JA), and ethylene are the key factors to regulate pathways involved in the defense mechanism and induce appropriate responses. The other phytohormones which can modulate plant defense and responses are auxins, cytokinins, gibberellin, abscisic acid, brassinosteroids, and strigolactone. Different hormonal pathways are up- or downregulated in different types of stress. Nanoparticles have been shown to stimulate hormonal balance in plants . Variousytokinin . Tobaccohormones . When thmones [2  to uninf[3O4 NPs .Nanophytovirology is a very promising field towards sustainable crop protection against viruses. The different nanoparticles and their applications have tremendous potential to deal with plant virus disease-related problems. Among plant viruses, DNA plant viruses specially geminiviruses  are a co


High Plains wheat mosaic virus (HPWMoV) causes a serious disease in major wheat-growing regions worldwide. We report here the complete or partial genomic sequences of five HPWMoV isolates from Australian wheat samples. Phylogenetic analysis of the nucleotide sequences of the eight genomic segments of these five isolates together with others from Genbank found all eight genes formed two lineages, L1 and L2. L1 contained a single isolate from Colorado in the North American Great Plains Region (GPR), and L2 had two unresolved clusters, A and B, of isolates from Australia and the GPR. A quarter of the L2B isolate sequences of the nucleocapsid gene (RNA3) were recombinant, which is unexpected as little evidence of recombination exists in viruses with negative single-stranded RNA genomes. Phylogenies calculated from the amino acid sequences of HPWMoV’s RNA-dependent RNA-polymerase (RNA1), glycoprotein (RNA2), and nucleocapsid protein (RNA3) showed they were closest to those of Palo Verde broom virus. However, its movement protein (RNA4) was closer to those of Ti ringspot-associated and common oak ringspot-associated viruses, indicating the RNA4 segments of their ancestors reassorted to produce the current emaraviruses. To avoid increased yield losses from co-infection, biosecurity measures are advised to avoid HPWMoV introduction to countries where wheat streak mosaic virus already occurs. Aceria tosichella  as their vector: wheat streak mosaic virus , Triticum mosaic virus , and High Plains wheat mosaic virus . To comply with the latest International Committee for Virus Nomenclature recommendations [Emaravirus tritici. WSMD causes the greatest damage to wheat crops when two or more of its causal viruses infect the same plant, which often occurs in wheat crops in the North American Great Plains Region (GPR). This co-infection is frequent because WCM transmits all three wheat viruses, and both their disease cycles and their epidemiology are greatly influenced by its ecology and behavior [Wheat occupies more land than any other staple food crop worldwide and is the second most important global food crop after maize (corn) . Many handations , in 2021behavior ,11,12,13behavior ,14,15,16behavior ,18, but behavior , Ukrainebehavior ,23,24. Ibehavior ,26, whicThe optimum temperature range is 24–27 °C for WCM population increase, as this tiny eriophyid mite vector species requires only 10 days to produce a new generation at these temperatures. Therefore, as climate change advances, global warming is projected to magnify the major global WSMV epidemic in many of the world’s wheat-growing regions by enhancing its spread ,29,30. THPWMoV has an octapartite, negative-sense RNA genome that, in its virions, is coated with thread-like molecules of a 32 kDa nucleocapsid (NC), and these complexes are contained inside 80–200 nm diameter virions that have double membrane envelopes. Its genome’s eight single-stranded RNA components each have a single open reading frame (ORF) encoding a single protein ,34,35,36When Stewart  comparedIn Australia, HPWMoV was first detected in 2003 in preserved leaf samples from wheat-growing regions of New South Wales (NSW), the Australian Capital Territory, Queensland, South Australia, and Victoria ,39. DuriA recent study that included silica gel-preserved survey samples BCWS5, BCHPV1, HP1G, and HP2W from south-west Australia found WSMV was present in each of them . AlthougAfter grinding in liquid nitrogen, a Spectrum Plant Total RNA Kit  was employed to extract total RNA from 20 mg/sample (desiccated) or 50 mg/sample (fresh) leaf material. At the Australian Genome Research Facility , these extracts were assessed using a Nanodrop Spectrophotometer  before library preparation, barcoding (24 samples per lane), and 100 bp single-end sequencing on an Illumina HiSeq2000. In the UK, the Angua pipeline of Fowkes et al.  was used−5 chance probability were analyzed separately. The best substitution models for calculations involving the nt and encoded aa sequences were determined using MEGA11 [The HPWMoV sequences were edited  to extrag MEGA11  and foung MEGA11  for nts g MEGA11  for aa sg MEGA11  and by mg MEGA11  with SH g MEGA11 .The ORFs of RNAs 1–4 from the CoPhil L1 isolate, the Nebraska “Genbank Reference Sequence” isolate, the West Australian Ku-12 isolate, and the Ohio isolate HPVWMoV-NW2 were each used as query sequences in BLASTX searches to identify the most similar 100 proteins in the Genbank protein database. The protein sequences for each RNA were pooled, and both duplicates and incomplete sequences were removed. The remaining 50–100 sequences for each RNA were aligned using MAFFT  and usedThe RNA1 nts phylogeny was calculated from 12 out of 13 nt sequences downloaded, RNA2 from 15 of 17, RNA3 from 22 of 38, RNA4 from 18 of 53, RNA5 from all 14, RNA6 from 18 of 19, RNA7 from all 15 and RNA8 from all 16; those omitted were either incomplete or clearly mislabeled. The initial phylogenies of the RNA1 protein aa’s of different emaravirus species were calculated from 54 sequences selected from 113 downloaded, RNA2 aa’s from 63 of 112 downloaded, RNA3 aa’s from 157 of 168 downloaded, and RNA4 aa’s from 34 of 35 downloaded. Thus, a total of 130 out of 180 sequences for the RNA nt phylogenies, and 308 out of 428 for the protein aa phylogenies were used.Mace and Yitpi were the wheat cultivars from which the sequenced samples were obtained . The HPWEight of the 31 RNA3 ORF sequences were found to be recombinants. Five of these  were among the 10 sequences from Australia, and three  from among the 21 sequences from North America; all the others  were non recombinant (n-rec). The three recombinant sequences from North America came from isolates from Idaho (MN315261), Michigan (MN315262), and Kansas (MW990205), whereas the n-rec sequences came from isolates from Colorado (three), Ohio (nine), and three from unspecified sites in the USA. Tatineni et al.  reported−5.5 to 10−7.2. The other recombinant (MN315261) was an isolate from Idaho; its closest major “parent” was OM302295 from Australia, and its minor parent nts (248 to 456), were detected by six methods with the probability that it occurred by chance of <10−9.0.Seven of the eight recombinant RNA3 sequences had the same two closest “parental” sequences. The major “parent” was closest to the RNA3 of the type Nebraska isolate (NC_029550), and the minor “parent” was the “Nebraska-like” Ohio isolate (MN250339), involving the region from around nts 173 to 583 and detected by six methods with the probability that it occurred by chance ranging from a mean of 10Emaravirus tritici COPhil,” and L2 was of all the other 11–20 ORF sequences (mean 14.75) of each HPWMoV gene. The L2 lineage is subdivided into two clusters, A and B. Cluster L2A consisted of two to six ORFs from wheat isolates all collected in Ohio; in The complete n-rec ORFs for each RNA segment were used to generate ML phylogenies. To check the more distant relationships of HPWMoV, the ORFs of the RNA1–4 genes from four representative HPWMoV isolates were used as query sequences in BLASTX searches of the GenbBank protein database to identify the closest non-HPWMoV proteins; the proteins encoded by RNAs 5–8 were not checked as these have not been identified for all emaraviruses. The phylogenies generated from these sequences  show thaPistacia emaravirus; QOI17315, Maple mottle-associated virus; Outgroup B: UQV97405, Peuraria montana associated virus; QTZ21230, and YP_010088071, Actinidia emaravirus; Outgroup C: YP_009237272, Emaravirus fici; YP_010088072, YP_010088072. Emaravirus kiwii, UQV97406 Pueraria lobata-associated emaravirus, CAG9003604; Ash shoestring-associated emaravirus, QAR18003 Pistacia emaravirus, YP_004327590 Emaravirus rosae, QOI17316 Maple mottle-associated virus, ANQ90730 Emaravirus cajani, YP_009507926 Actinidia chlorotic ringspot-associated virus; YP_009508087 Emaravirus cercidis; QIN85946 Lilac chlorotic ringspot-associated virus; Outlier D: QTW92691 Emaravirus camelliae; Outgroup E: AWS21342 Emaravirus fici; AXI82314 Emaravirus rosae; CAA0079646 Aspen mosaic associated virus, QBM15198 blackberry leaf mottle associated virus, UQV97407 Pueraria lobata associated emaravirus, YP_009268864 Emaravirus toordali; Outlier F: YP_009508085 Emaravirus cercidis; Outgroup G: QGX73506 Emaravirus camelliae, BCO17111 Japanese star anise ringspot-associated virus; Outgroup H: YP_009508084 Emaravirus cercidis, QIN85948 Lilac chlorotic ringspot-associated virus; Outgroup I: CAA0079685 Aspen mosaic-associated virus; QVW29594 Grapevine emaravirus A, YP_009237264; Emaravirus cajani movement protein sequence QRG35061  are mislabeled as RNA5 proteins in Genbank.Large red discs on nodes with SH statistical support of 1.0 and smaller discs with 0.95 SH support. Isolates forming the HPWMoV-containing basal lineage are shown, but those of the other basal lineages/outliers are “collapsed” and consist of: Outgroup A: QAR18002, Emaravirus tritici) of its four other names. Comparisons with previously published analyses may be different or incomplete as the number of emaravirus sequences in Genbank is increasing, and, for this study, we used the complete ORF nt sequences of all HPWMoV genes. By contrast, Pozhylov et al. [Here we report a study of the relationships between HPWMoV isolates from Australia with other isolates from this same species, sometimes recorded under one of three  is more likely to have had a major North American parent, of which the Australian OM302295 is merely the closest known progeny.https://bicon.agriculture.gov.au/BiconWeb4.0/ImportConditions/Questions/EvaluateCase?elementID=0000068254&elementVersionID=294; accessed on 2 November 2022) as a permitted source of certified maize seed for entry to Australia. As mentioned above, HPWMoV has been found to be transmitted in sweet corn seed [One of the USA states supplying the cluster L2B isolates was Idaho MN250353), which is listed in Bicon  occur see . The spr


The start of 2022 was marked by the sudden surge in the detection of the viral disease - monkeypox. The recent and ongoing COVID-19 epidemic makes the re-emerging of viral zoonosis particularly worrisome. The rapid spread of the monkeypox virus has sparked concerns about the start of a new epidemic. In this review, I summarize the epidemiology, clinical signs, and symptoms, transmission, diagnosis, management, and prevention of the monkeypox virus. Clinicians need to have a high index of suspicion for monkeypox in patients with high-risk factors presenting with new onset progressive rash. Patients with confirmed or suspected monkeypox infections need to be isolated until all the lesions have resolved. The start of 2022 was marked re-emerging of the viral zoonosis; the monkeypox virus. The Monkeypox virus cases have increased from one case detected in the UK on May 7, 2022 to 1,285 cases detected in 28 countries by June 8, 2022. . The rapMonkeypox shares many characteristics with the smallpox virus, which also belongs to the Orthopoxvirus family. The clinical symptoms and incubation period of monkeypox are similar to the smallpox virus ,9. One oGenetic mutations and changes have caused the pathology of the virus to change as well. A meta-analysis study concluded that in the 1970s monkeypox virus largely affected children. Due to genetic mutation, the average age of people affected by this has increased and in 2010 the average of infected individuals was found to be 21 . The rapClinical signs and symptomsMonkeypox traditionally presents with a typical picture consisting of lymphadenopathy, fever, headache, body pains, and aches. The rash affects the face and periphery more than the trunk. Mucosal membranes may also be involved including the mouth and genitals. Involvement of the corneal and conjuctival mucosa was seen in a small percentage of patients. The rash in monkeypox patients starts off as macular in appearance and then transitions into papules, vesicular, and pustules and finally drying up to form crusts that fall off . The monA study done in 2004 found similar results. Headache and skin lesions were present in 100% of patients while 82% of patients reported fever, sweating, and chills. Coughing was reported by 73% of patients, lymphadenopathy was reported by 55% of patients and 18% patients reported malaise. Blepharitis, nausea, nasal congestion, back pain, and myalgias were reported in only 9% cases . AnotherReservoirThe monkeypox virus, unlike the smallpox virus, also infects animals. Most of the animals infected appear to be monkeys or small mammals ,17. SeveTransmissionThe route of transmission might also play an important role in the clinical symptom and spread of the virus. Reynolds et al. found that spread of the monkey virus via a bite or scratch resulted in a more severe illness with a shorter incubation period . Many stChen et al. in their study suspected that the D10L, D14L, B10R, B14R, and B19R genes might be responsible for the monkey pox virus virulence . AnotherDiagnosisPolymerase chain reaction (PCR) of samples taken from the patients rash is sensitive . The diaTreatment^tm was used in the 2003 United States monkeypox outbreak. It managed to decrease the symptoms but was ineffective in the prevention of monkeypox disease [Currently, there is no approved treatment for the monkeypox virus infection. A study done in 1988 deduced that the smallpox vaccine was effective in preventing monkeypox . People  disease . The use disease ,24 Tecov disease ,24. Euro disease ,35. BrinPreventionThe current rising number of monkeypox cases might require the use of the smallpox vaccine, especially in high-risk populations. The number of people with a history of smallpox vaccination is decreasing around the world. This means that with each passing year, a greater percentage of people are at risk. World Health Organization (WHO) has approved a newer smallpox vaccine in 2019 to be used for the prevention of the monkeypox virus . The numIsolationPatients with confirmed or suspected monkeypox infections need to be isolated until all the lesions have resolved. Home isolation is recommended for patients who have mild symptoms and who do not require hospitalization. Confirmed or suspected cases of monkeypox virus should be placed in a single-person room with a dedicated bathroom. In case the patient needs to be transported all the exposed skin lesions should be covered with a well-fitted medical mask need to be worn by the patient. Airborne isolation is needed in case of intubation. Gown, gloves, eye protection, and a respirator equipped with N95 filters or higher need to be used by the healthcare provider before entering the patient’s room .ComplicationsThe monkeypox disease can vary significantly in terms of prognosis. It mostly has a benign course yet severe complications can develop. These complications include encephalitis, nausea/vomiting leading to dehydration, and electrolyte imbalance ,15. AnotThe rapid spread of monkeypox has shown the lack of measures in place to prevent epidemics. There is a need to quarantine and check animals being internationally transported for monkeypox virus. A high index of suspicion needs to be in place when examining patients, especially the high-risk population. There is a need to initiate smallpox vaccine in high-risk populations like immunocompromised, those men engaging in homosexual acts with other men and those handling internationally transported animals, especially rodents and other small mammals. Physicians need to be aware of the early signs and symptoms of the monkeypox virus so that early diagnosis and prompt treatment can be initiated. There is a need to do more research in order to come up with more effective preventive and treatment measures.


Faking in a psychological test is often observed whenever an examinee may gain an advantage from it. Although techniques are available to identify a faker, they cannot identify the specific questions distorted by faking. This work evaluates the effectiveness of term frequency-inverse document frequency (TF-IDF)—an information retrieval mathematical tool used in search engines and language representations—in identifying single-item faked responses. We validated the technique on three datasets containing responses to the 10-item Big Five questionnaire  in three faking situations. Each participant responded twice, once faking to achieve an objective in one of three contexts (one to obtain child custody and two to land a job) and once honestly. The proposed TF-IDF model has proven very effective in separating honest from dishonest responses—with the honest ones having low TF-IDF values and the dishonest ones having higher values—and in identifying which of the 10 responses to the questionnaire were distorted in the dishonest condition. We also provide examples of the technique in a single-case evaluation. In this study, we address the problem of interpreting the data collected through questionnaires when responses are distorted by faking. Examinees can easily fake responses to direct questions when they find a direct advantage in doing so, and distortions may take one of two forms: faking bad and faking good. In faking bad, the examinee is likely to exaggerate or make up a psychological disorder  . These sFor this reason, efforts have been made to develop specific tests able to flag the respondent as a faker. Although such procedures may reveal the faker with sufficient accuracy, to the best of our knowledge, no procedure has been proposed to specifically identify faked replies or reconstruct the honest response profile once a faker has been identified and only the faked profile is available. In short, a nondepressed individual who wants to appear depressed may be spotted as a faker; however, no specific procedure is available to identify faked responses.This study aims to develop an efficient technique for spotting faked responses to individual items and not only a faking response style. We will analyze the potential of a standard information-representation model developed within the information retrieval field  , to idenIn both classical test theory and item response theory (IRT), an item’s worth is evaluated with respect to the performance on that item of a sample of participants. In classical test theory, a participant’s overall responding style is not modeled–an example of a responding style is the tendency to use the full range of response alternatives or just the extreme values.Recently, a variant of IRT has been proposed, the IRT tree model . The IRTAs opposed to IRT-tree, the TF-IDF approach offers a purely data-driven and model-free solution for the task of faking-detection that only requires the collection of honest responses. This aspect, in particular, provides some important advantages for a widespread application of the proposed approach compared to IRT. In fact, a TF-IDF based approach does not require a domain expert to design the experiments and can be used following the same process regardless of the domain or type of questionnaire. Additionally, a TF-IDF based approach allows to compare the respondents’ style across different questionnaire items instead of being limited to each single item.Our TF-IDF approach is characterized by combining a response’s relative positioning with respect to the validation sample as well as a specific subject’s response style . From thThe original contributions of this work are:the proposal of a novel approach to precisely identify faking patterns in personality tests;a thorough evaluation of the proposed approach based on TF-IDF, with a comparison to similar techniques based on the distribution of raw response values; andthe creation of three datasets for the development of new item-level faking-detection approaches.TF-IDF is a standard term-weighting method used in information retrieval to compute a numeric representation of textual data based on the distribution of the terms occurring in it. It is one of the mathematical tools search engines use to retrieve web pages relevant to a user query and rank them according to their relevance. It is also used as a text representation strategy in natural language processing .d, is representative of its general meaning proportional to (a) its term frequency (TF) within d and (b) its inverse frequency in the collection  is more representative for the content of d than different ones that are either not frequently used in d or are very frequently used also in other documents.The TF-IDF representation strategy is based on the intuition that a certain term in a document, C, of documents, the TF-IDF value for each term, t, in a document d ∈ C is calculated asd, t) indicates the number of times a target term, t, appears in a document, d. IDF = log(N/n), where N indicates the number of documents in C and n is the number of documents in which t is used.Formally, given a collection, The application of TF-IDF is not only limited to the field of computer science but has also been used, for example, in cognitive neuropsychology to model semantic memory and its disorders  as well q, considering as TF the number of times the subject provides the current response. For example, if a subject responded with a rating of 5 only to one item out of the 10 items, then TF  = 1. Indeed, for each item, the subjects could choose their degree of agreement on a 5-point Likert scale.When applied to item analysis, we compute each response’s TF-IDF in a questionnaire, N/n), where N is the number of participants who responded to the test  and n is the number of times the participant’s response was given also by other participants to the same item. The final score associated with each test item is the product of the TF and IDF values. This adaptation of TF-IDF has high values for responses to single items that are highly atypical. From this perspective, TF-IDF behaves as an anomaly detector, indexing the very atypical responses at the single-item level (not at the subject level).The IDF is computed as log = 9.11. This value will be high for people that provide the same—atypical—response many times in a questionnaire.From this perspective, dishonest responses are expected to have an abnormally high TF-IDF, compared to the TF-IDF distribution of honest respondents.For each response a subject gives on a questionnaire, we compute its corresponding TF and IDF scores as described above. Then, if their product is higher than a certain threshold, we flag the current response as faked.Given a training set, said threshold is selected as the percentile score in the TF-IDF values distribution (for each item) that maximizes the precision metric for faking detection on a separate validation set. It is not calculated on the raw TF-IDF scores but on the corresponding percentile of the distribution of the TF-IDF scores on a certain item. We then evaluated the threshold value following a K-fold cross-validation procedure (with K = 10). This process allowed for an evaluation of the proposed approach’s performance in terms of accuracy, precision, recall, and F1 score.In our study, we first calculated the IDF scores associated with each item in the honest responses. Afterward, we considered a small subset of dishonest responses—as a validation set—and fine-tuned the faking detection threshold for each item based on the distribution of these scores. For example, should we observe that the value associated with the 75th percentile in the distribution of TF-IDF values in our validation set were a reliable threshold to distinguish between honest and dishonest responses for the questionnaire items, we would use said value to detect fakers for each item. We identified the 75th percentile empirically based on our data distribution. More specifically, we first computed the distribution of TF-IDF scores associated with each questionnaire item, then, we evaluated values at different percentiles of each of these distributions as potential threshold candidates to distinguish dishonest from honest responses. Finally, we selected the percentile value that maximises the F1 Score performance metric for the detection of faked response items.When a new questionnaire item is to be evaluated for dishonesty, we rely on the previously computed set of IDF scores for the respective honest responses and on the TF scores computed for the current responses to assign a TF-IDF score to each response. Then, if the obtained TF-IDF value is higher than a certain threshold—which we validated beforehand as explained above—we report the item as faked. In other words, we rely on the TF-IDF score associated with a subject’s responses to a questionnaire as an indicator of their attitude and as a proxy to detect faking behaviors. For this purpose, TF-IDF, as proposed here, acts as an anomaly detector, identifying suspicious differences between the to-be-evaluated case and the distribution of honest respondents’ answers. Furthermore, considering various faking contexts helps us exclude potential TF-IDF approach biases to specific data distributions or situation-specific behaviors , 12.To validate the technique, we conducted three experiments in which we asked participants to respond to the same questionnaire twice, the first time dishonestly according to specific instructions and the second time honestly. We gave the instructions in three specific contexts: (a) a child custody case, (b) a job interview for a sales manager position, and (c) a job interview for a position in a humanitarian organization. We used three faking contexts because faking changes according to the respondent’s objective. Having the same subject responding both dishonestly and honestly allows us to have a ground truth useful for evaluating the TF-IDF method’s accuracy level in identifying faked responses. Indeed, in most practical applications, only one set of responses per subject will be available, and the proposed approach’s goal is to spot faking patterns in the subject’s responses.The experimental procedure was approved by the ethics committee at the Department of General Psychology at the University of Padua (n. 4545). All participants in our experiments were native Italian speakers, and we administered the questionnaires online via social networks in August 2020 to a panel of subjects maintained by our department. We also collected information about the participants’ age, gender, and education level, which are shown in In the three conditions, we asked participants to respond to the same set of 10 items twice, first altering their answers to obtain personal gain in the three contexts described above and then honestly. When a test is taken in a real faking-prone setting, the participant has not responded to the same test honestly before. The order faking first- honest second was adopted to have the faked condition close to the real administration condition.All available responses were in multiple-choice format, and participants could choose only one option for each item. We asked the participants to respond by estimating their agreement using a 5-point Likert scale (where 1 indicates strong disagreement and 5 strong agreement) on the Italian version of the following 10 items from the Big Five Inventory . The Eng“I see myself as someone who …”is outgoing, sociable (extroversion);is reserved (extroversion reversed);tends to find fault with others ;is generally trusting (agreeableness);tends to be lazy ;does a thorough job (conscientiousness);gets nervous easily ;is relaxed, handles stress well ;has an active imagination (openness);has few artistic interests .Before analyzing the collected data, we reversed the responses participants gave for reversed items , so all the items’ final scores were directly comparable.Pretend you and your spouse are separating and fighting in court for the custody of your child (imagine having a child if you don’t have one). A court-appointed psychologist will evaluate your parental skills and require you to respond to the following personality questionnaire. Answer the test in a way that emphasizes your parental skills, hiding behaviors or thoughts that are generally considered negative for a parent. Your goal is to get custody of your child.Pretend you are attending a job interview for the position of sales manager for a very prestigious company. It is your dream job, and you want to give the best possible image of yourself. Your goal is to obtain the job and to be considered better than the other candidates. Answer in a way that makes you look good, hiding behaviors or thoughts that are generally considered negative for the position you aspire to. Try to give a positive image of yourself, even if it means lying.Participants received instructions similar to those we provided to Group 2, with the position being the only difference (a position in a humanitarian organization).The procedure included a question to verify the participants read and fully comprehended the instructions in all three conditions before completing the questionnaire . We did I always had to tell the truth.I received no specific instructions.First I had to tell the truth, and then I had to lie to get custody of the children.I always had to lie to get custody of the children.First I had to lie to get custody of the children, and then I had to tell the truth.Of the 258 participants, 37 did not satisfy this quality check. Therefore, we included only 221 participants in our analysis, discarding the responses of all participants who answered incorrectly.The additional final question for Groups 2 and 3 was “What instructions have you received when filling in the questionnaire?” with the following response choices:I always had to tell the truth.I received no specific instructions.First I had to tell the truth, and then I had to lie to look good in a job interview.I always had to lie to make a good impression.First I had to lie to look good in a job interview, and then I had to tell the truth,This quality check for Group 2 (JIS) resulted in the exclusion of 44 of 287 participants, with 243 participants included in the final analysis, who were selected based on their correct responses to the final questions. From Group 3 (JIHO), we excluded 42 of 272 participants, including 230 participants in the following evaluation.https://github.com/albpurpura/TFIDF-Faking.The proposed approach to compute TF-IDF for questionnaire responses and the data employed for the evaluation are available in our public repository: This section presents the results of the experiments we conducted to validate the proposed approach based on TF-IDF. We begin by giving a few characteristics of the data we collected and providing the analysis we performed with the raw data.As Although the probability of faking changes not only depends on the context but also on the item, we generally expected that the answers given in the faking conditions would score higher than those provided in the honest condition. In order to verify this, we calculated the proportion of responses that increased from the honest to the dishonest condition  or remained unchanged . The oveThis result could have occurred due to different faking strategies adopted by different participants and as well as test-retest issues. In fact, previous studies have shown that the test-retest reliability of the 10-item Big Five Questionnaire is 0.72 . Since tIn addition, we investigated the correlation between honest and dishonest answers. We changed the raw data into TF-IDF indices as described above to verify whether the TF-IDF approach can easily detect faked responses. As a first step, we verified whether the TF-IDF discriminates between honest and dishonest responses more efficiently than raw scores; as it can be seen in the correlation matrices, no linearity exists between the two distributions. For this purpose, we used the KL divergence, which is an entropy-based measure to compare probability distributions. The values of this measure range from 0 to infinity, where 0 indicates that the two considered distributions  completely overlap. Higher values of the KL-divergence indicate a higher degree of separation between honest- and dishonest-response distributions. The results reported in Tables Subsequently, to determine how the TF-IDF representation of honest responses differs from that of the dishonest ones, we calculated the average for each participant over all 10 items see . These rThese results show that fakers can be distinguished from honest respondents based on the average of the TF-IDF over all the responses to the questionnaire items.TF-IDF acts as a novelty detector, with higher values indicating a response that a subject used frequently but all other participants used infrequently. The index is participant specific and item specific. However, TF-IDF can be used not only to spot fakers—as the results in Calculate the TF-IDF score for each response of a target participant.Compare the obtained TF-IDF scores to a threshold specific for each questionnaire item.If the TF-IDF values are outside a certain range, the response is categorized as an anomaly (faked).We applied this procedure to the three datasets , yielding the results summarized in Given that each participant responded to the 10-item test twice, once in the dishonest condition and once in the honest one, we could identify the items with distorted responses for each participant. As indexed by the probability density function, fakers faked between 7 and 9 items more frequently, with few participants faking 1, 2, or 10 items see . We evalWe observed that the accuracy remained relatively stable in the 0.4−0.6 range in all three conditions, indicating that faked items can be identified with the same level of accuracy independently of the specific faking strategy the subject used.ion task .In practice, the TF-IDF can be used to analyze single-subject responses when no honest responses for a suspected faker are available. Figs Psychological tests usually require participants to endorse a statement describing a personality feature or behavior using a Likert scale . Respondents may easily fake responses on such tests by modifying their original, honest responses to achieve an objective .To handle this problem, faking propensity is usually detected using the so-called control scales, which are subsidiary scales added to personality scales . HoweveThe task of identifying faking at the single-item level is challenging for the following reasons:the high variability of responses among individualsthe various degrees of intensity of fakingthe objective of faking modulates where faking takes place and the degree of intensity Currently, no technique is available to identify the specific faked responses. Control scales help identify only the participant’s general propensity to fake and do not pinpoint where faking actually takes place.For these reasons, we propose the use of a new technique inspired by original research in information retrieval. The technique is known as term frequency-inverse document frequency  (TF-IDF)In traditional item analysis, identical responses to the same item from two subjects are evaluated in the same way. In contrast, according to the TF-IDF modeling strategy, two identical responses (from two participants) to the same item may yield different evaluations, depending on other participants’ responses to the same items and the subject’s responses to the questionnaire’s other items. In other words, TF-IDF aggregates in a unique measure the distribution of the group responses to a target item as well as the subject’s response style .TF-IDF, as presented here, can be considered a novelty detector. If the TF-IDF of a subject’s response to an item is abnormally high, it indicates that the participant’s response style does not belong to the sample of honest respondents. To verify the effectiveness of TF-IDF in identifying faked responses, we asked about 700 participants to respond to a short Italian version of the Big Five test . We askThe exploratory analyses, performed on raw scores, indicated thatfaking (good) resulted in an increase in average response values on all but one of the 10 items andfaking in different contexts, as expected, resulted in distortions of responses to different test items.In evaluating the proposed TF-IDF approach, we observed the following:a TF-IDF based representation of item responses leads to a better separation between honest and dishonest responses than do raw scores;the average (of the 10 items of the considered questionnaire) TF-IDF response values of the honest respondents were lower than that of the dishonest ones;lower TF-IDF values characterize honest responses to single items, and larger values are characteristic of dishonest ones;the proportion of dishonest responses associated with large TF-IDF representations  was consistently larger than that of their honest counterparts;when applying the TF-IDF for item-level faking-detection, precision  was between 67% and 71%, indicating that TF-IDF correctly identifies most of the faked single-item responses. The same faking detection procedure, based on raw response values, instead yielded much lower precision values see .All the results indicate that TF-IDF can help catch deceptive responses at the single-item level with unprecedented accuracy. The identification of fakers and faking at the single-item level is achieved without the use of any control scale, only by capitalizing on one distinguishing feature of TF-IDF. It is worth noting that the use of TF-IDF as a novelty detector allows for the spotting of a faker if a group of honest respondents is available. This feature has interesting practical implications given that faking is context-specific and depends primarily on the faker’s strategy, which in turn depends on the objective that faking is expected to achieve .Finally, we have given examples of how the TF-IDF can be used to analyze a single participant’s responses see Figs –7. When Although the present study was validated on the 10 items Big-five test questionnaire, its validity should be evaluated for other tests, as well. Since the role of the presentation order of the honest and dishonest condition has not been investigated here, future studies should balance the sample and provide further information on the possible priming effect.Moreover, in this study, we asked participants to respond to three different scenarios and fake their test responses accordingly. The efficacy of the method should be confirmed also with high-stake test-takers in real settings rather than in low-stake instructed faking conditions as reported in this research.