Red sea bream iridovirus (RSIV) is an important aquatic virus that causes high mortality in marine fish. RSIV infection mainly spreads through horizontal transmission via seawater, and its early detection could help prevent disease outbreaks. Although quantitative PCR (qPCR) is a sensitive and rapid method for detecting RSIV, it cannot differentiate between infectious and inactive viruses. Here, we aimed to develop a viability qPCR assay based on propidium monoazide (PMAxx), which is a photoactive dye that penetrates damaged viral particles and binds to viral DNA to prevent qPCR amplification, to distinguish between infectious and inactive viruses effectively. Our results demonstrated that PMAxx at 75 μM effectively inhibited the amplification of heat-inactivated RSIV in viability qPCR, allowing the discrimination of inactive and infectious RSIV. Furthermore, the PMAxx-based viability qPCR assay selectively detected the infectious RSIV in seawater more efficiently than the conventional qPCR and cell culture methods. The reported viability qPCR method will help prevent the overestimation of red sea bream iridoviral disease caused by RSIV. Furthermore, this non-invasive method will aid in establishing a disease prediction system and in epidemiological analysis using seawater. Megalocytivirus of the family Iridoviridae [Pagrus major) in 1990 [Oplegnathus fasciatus), a highly susceptible fish species [Red sea bream iridovirus (RSIV), which is an enveloped deoxyribonucleic acid (DNA) virus belonging to the genus oviridae , infects in 1990 . Since t in 1990 . RSIV ca in 1990 . RSIVD m species ,4,5. Rec species . The hor species ,7,8—the Several methods, such as cell culture , loop-meThe standard method for assessing the infectivity of RSIV is to use susceptible grunt fin (GF) cells to identify the typical cytopathic effect (CPE), which is characterized by the presence of round and enlarged cells ,15. HoweIn this study, we aimed to develop a viability qPCR for the selective detection of infectious and heat-treated RSIV using PMAxx dye and platinum compounds as survival markers. Furthermore, we optimized viability qPCR procedures for application in seawater through kinetic assays. The results of our study could help national quarantine agencies establish efficient disease control systems through the non-invasive detection of viruses.4 as the survival markers for qPCR assays. The results revealed that PMAxx prevented qPCR amplification of inactivated RSIV better than platinum compounds  and infective (at >1000 μM) RSIV  RSIV c,d.t values negligibly changed from 22.68 ± 0.24 Ct to 23.38 ± 0.24 Ct as the percentage of infectious virus decreased from 100% to 0%  except for that at 100% . Under conditions without the addition of PMAxx, the C0% to 0% a. In con0% to 0% a. Moreovas 12.79 b. These 2.3–107.3 RSIV copies/200 µL), a linear relationship was observed between the Ct value and the concentration of 10-fold serially diluted RSIV  (1.6–108.6 RSIV copies/500 mL) were spiked into seawater and concentrated based on iron flocculation, followed by qPCR and PMAxx-based viability qPCR. As a result, a strong linear relationship was observed between spiked RSIV and the number of recovered RSIV copies  (Upon performing qPCR and PMAxx-based viability qPCR of 200 μL tissue homogenates of different concentrations (10 0.9719) a. Differ 0.9840) b.3.6 RSIV copies/500 mL. Furthermore, the average recovery rate for the number of spikes within all concentration intervals was approximately 78.7%  at both 25 °C and 15 °C on day 0, and 106.1 and 106.3, respectively, on day 14, showing no difference  to 104.2 copies/500 mL on day 1 at 25 °C in environmental seawater. Additionally, on day 7, the copy number was 103.2 RSIV copies/500 mL. However, RSIV was not detected on day 10. Similarly, at 15 °C, the RSIV copy number decreased rapidly from day 1 after RSIV inoculation and was 103.9 copies/500 mL on day 7. However, it was not detected from day 10 onwards  cells was observed at all sampling times in RSIV cultured at 25 °C and 15 °C in the autoclaved seawater group. In environmental seawater, CPE was observed up to day 3 at 25 °C and up to day 7 at 15 °C .The positive control group injected with the RSIV cell culture showed 100% cumulative mortality for 3 weeks a. No morHorizontal transmission through water is one of the most important transmission routes for aquatic viruses ,7,8. HowPropidium monoazide concentration is a key factor influencing the efficiency of viability qPCR to differentiate between infectious and inactive viruses . In this4, as a survival marker for RSIV, showed negative results. A previous study reported that PtCl4 at 2500 μM inhibited the qPCR of the inactivated virus without affecting that of the infectious murine norovirus (MNV) [4 in distinguishing inactivated noroviruses. The inconsistency in these results could be attributed to the differences in experimental conditions, such as inactivation methods and target viruses tested in these studies. It has been reported that PtCl4 penetrates living cells in small amounts and causes a slight decrease in the qPCR fluorescence signal. Furthermore, unlike PMAxx, it does not react with water molecules and becomes inactivated after reacting with viruses [4 treatment in this study could be because the PtCl4 molecules were not eliminated and modified the DNA during the extraction procedure [In our study, PtClus (MNV) , whereinus (MNV) ,31. In cus (MNV)  reported viruses . The suprocedure . Our stut values occurred among the suspensions with different percentages of infectious virus (0–100%). However, Ct and delta Ct values increased significantly as the proportion of infectious RSIV decreased after PMAxx pretreatment. Although viability qPCR did not completely suppress the signal of 100% heat-treated RSIV, it suppressed the signal approximately 16,670-fold from 107.3 RSIV copies/200 μL (infectious RSIV) to 102.9 RSIV copies/200 μL (inactivated RSIV). The RSIV used in this study had a fairly low Ct value, i.e., a high copy number. Therefore, it is possible that not all samples were heat inactivated or that the inactivated virus particles aggregated and prevented PMAxx penetration through the damaged virus [The reliability of the PMAxx-based viability qPCR was confirmed. When RSIV was detected without adding PMAxx, no significant difference in Ced virus . This obed virus ,36,37.The viability qPCR of RSIV in seawater was successfully confirmed. In our study, the oxalic acid-EDTA buffer was used for concentrating seawater. However, this stock solution is highly toxic to cells and fish. The virus suspension was diluted 10-fold with PBS to inoculate the concentrated virus into cells. However, since the virus copy number decreased with dilution, a 10-fold higher RSIV suspension was prepared and introduced into seawater. In experiments evaluating RSIV infectivity in seawater, CPE in cells showed a 10- or 100-fold lower detection limit than that in the viability qPCR. The viability qPCR method reduces the risk of underestimating RSIV present in seawater because samples concentrated in seawater are used directly without further dilution. In addition, viability qPCR is a better method than infectivity assessment through cell inoculation because it is fast and requires no special skills.In this study, we validated the optimized viability qPCR for selective detection of RSIV infectivity in seawater by performing qPCR in various seawater conditions. Furthermore, the cell inoculation and rock bream artificial infection experiments were performed to validate the reliability of the viability qPCR results. Water temperature is often recognized as the most influential factor in virus infectivity; a higher water temperature leads to faster virus inactivation ,39. In tIn environmental seawater, qPCR analysis without the addition of PMAxx confirmed that RSIV was detected up to 14 days at 25 °C and 15 °C. However, viability qPCR and rock bream artificial infection experiments revealed that RSIV stored in environmental seawater at 25 °C and 15 °C showed no virus detection or fish mortality after 7 days. Moreover, no CPE was observed after 3–7 days in the cell inoculation experiments. This observation indicates that RSIV can be inactivated within approximately 7–10 days in seawater, demonstrating that positive results via conventional qPCR in seawater overestimate the disease risk.The three viability experiments we analyzed can be used to assess RSIV infectivity. However, the cell inoculation experiment is limited by its long-time requirement, absence of cell lines, low susceptibility to viruses, and experimental techniques. Rock bream is known to be the most sensitive fish species to RSIV ,44, but 3–106 RSIV copies/L seawater from red sea bream farms. Furthermore, in winter, RSIV was detected in seawater at a low water temperature of 11.7 °C [Our study results demonstrate the potential of viability qPCR as a non-invasive method to assess the risk for RSIV occurrence in actual farm seawater. The findings suggest that the viability qPCR to detect viruses in seawater can help the epidemiological analysis of disease outbreaks. Furthermore, it can help estimate the risk of transmission between farms through disease spread model simulation or by building a disease spread forecasting system for farms around farms with RSIVD outbreaks . Previou 11.7 °C . Howeverg for 5 min. Then, the supernatant was collected and filtered through a 0.22 μm syringe filter. Subsequently, 1 mL of the supernatant was inoculated into the PMF cell line. After 4 h of infection, the culture medium was replaced with fresh L-15 medium and cultured at 25 °C for 7 days. Finally, the harvested supernatant was used as an inoculum in subsequent experiments.Red sea bream iridovirus was obtained from RSIV-infected rock bream spleen tissue and replicated in the PMF cell line  ,46. The ® Genomic DNA Extraction Kit  according to the manufacturer’s instructions. The quantitative analysis of RSIV was performed using a previously reported qPCR assay [t) was employed for quantitative analysis using the same real-time PCR equipment and analysis reagents as described in a previous study [Viral DNA was extracted using the AccuPrepCR assay . The finus study .4; Sigma-Aldrich, St. Louis, MO, USA), were used as viability markers in this study. Propidium monoazide was diluted to a 5 mM concentration in nuclease-free water. Afterward, PtCl4 was diluted to 500 mM using dimethylsulfoxide and further diluted to 50 mM in nuclease-free water. All viability marker stock solutions were stored at −20 °C in the dark. Infectious RSIV (approximately 104 RSIV copies/200 μL) was obtained using the virus suspension obtained via the method described in Photoactivatable dyes, such as PMAxx , and platinum compounds, such as platinum (IV) chloride . Additionally, 0.1% Triton X-100 , which is known to increase virus permeability under all survival marker conditions, was added, and viability assays were performed [For the viability assays, the photoactive PMAxx dye was added to the infectious or inactivated virus suspension at final concentrations of 10, 25, 50, 75, 100, and 200 μM. Photoactivation of the PMAxx dye was performed by incubation in the dark at 20 °C in an orbital shaker (150 rpm) for 30 min, followed by exposure to a light emitting diode (LED) light for 30 min in a photoactivation system . PtClerformed ,27,28. Et values of PMAxx-treated and PMAxx-untreated samples were analyzed according to the infectious virus ratio.Propidium monoazide was selected as the most reliable viability marker and was used in this study to evaluate the performance of survival qPCR. To evaluate the performance of the PMAxx-based viability qPCR assay to selectively detect infectious RSIV in the presence of non-infectious RSIV, the proportion of infectious RSIV in a 200 μL virus suspension was set as 0%, 10%, 30%, 50%, 70%, 90%, or 100% (the remaining proportion was that of the heat-treated RSIV), and then PMAxx-based viability qPCR was performed. Different samples with the above-mentioned concentrations were prepared. Furthermore, qPCR was performed without adding PMAxx. All qPCR experiments were performed in triplicate for all samples. Then, the delta C3∙6H2O) was dissolved in 100 mL distilled water to form a Fe-virus flocculate using iron chloride. Subsequently, 50 µL of the iron chloride solution was added to 500 mL seawater. The seawater was gently stirred (200 rpm) for 1 h at 20 °C using a magnetic stirrer. The resulting Fe-virus flocculates were filtered under reduced pressure through a 0.8 μm pore size polycarbonate filter  held to a filter holder with a receiver . The filter in which the Fe-virus flocculates were captured was transferred to a 5 mL round-bottom tube, and 1 mL oxalate-EDTA buffer was added, as previously reported [A previously reported iron-based flocculation method was used to concentrate the virus in seawater, with slight modifications . Approxireported . Virus rg and 4 °C for 10 min. After centrifugation, the supernatant was filtered through a 0.45 μm syringe filter and sequentially diluted 10-fold to prepare viruses of various concentrations, ranging from 102.3 to 107.3 RSIV copies/200 μL. Afterward, PMAxx-based viability qPCR and qPCR were performed, as mentioned above.Red sea bream iridovirus-infected rock bream spleens were homogenized in PBS and centrifuged at 10,000× 1.6 to 108.6 RSIV copies/500 mL. Subsequently, the seawater sample to which RSIV was added was first filtered through a glass microfiber filter with a 1.6 μm pore size  and then through a 0.45 μm pore MF-Millipore™ membrane filter . Afterward, 50 μL of the previously prepared iron chloride solution was added to the filtered RSIV spiked seawater and gently stirred (200 rpm) for 1 h at 20 °C using a magnetic stirrer. Virus flocculation and resuspension were performed as described above. Three replicates were prepared for each concentration. Next, virus quantification was performed after DNA extraction, as described above, using a resuspended solution of 1 mL concentrated RSIV.Serially diluted RSIV suspensions were spiked into seawater (500 mL) at various concentrations, ranging from 10Owing to the cytotoxicity of the buffer, the virus resuspended in oxalate-EDTA buffer was diluted 10-fold in PBS to determine the intracellular CPE of the concentrated virus. Thus, a concentration of 10 times the viral seawater concentration described above was inoculated. One milliliter of the viral dilution was inoculated onto a PMF cell monolayer cultured in L-15 medium supplemented with 1% FBS and 1% A-A. After 4 h, the inoculum was removed, washed once with Dulbecco’s PBS, and replaced with fresh medium. Cytopathic effects were observed for 7 days.6.2 RSIV copies/mL. After virus inoculation, samples were taken on days 0 (control), 1, 3, 7, 10, and 14. After concentrating the seawater, qPCR and viability qPCR were performed as described in Seawater was collected from the coast of Tongyeong, Gyeongsangnam-do, Republic of Korea. Before analysis, seawater was filtered using a 0.22 μm pore size syringe filter and sterilized in an autoclave. Environmental seawater was used for follow-up research immediately after transporting the collected seawater to the laboratory. Red sea bream iridovirus was inoculated into 1 L capped and clear glass bottles containing 500 mL seawater  and incubated at 25 °C and 15 °C, respectively. Virus concentration at the start of all analyses was 107.2 RSIV copies/mL) 10 times higher than the RSIV inoculation concentration mentioned above was inoculated into autoclaved seawater and environmental seawater. After incubation at 25 °C and 15 °C, respectively, the virus was concentrated for 0, 1, 3, 7, 10, and 14 days. Subsequently, it was diluted 10-fold with PBS. The environmental seawater was filtered through a 0.45 μm pore size syringe filter and inoculated into a PMF cell monolayer.The viability of RSIV in seawater was confirmed by CPE after inoculation of the virus concentrates into PMF cells. A virus suspension with a concentration (102) seawater (500–1000 L/h). For virus inoculation, RSIV was inoculated by 108.2 RSIV copies/mL in 500 mL of environmental and autoclaved seawater, respectively, according to the concentrations obtained through iron flocculation described in To compare RSIV viability in various seawater conditions, rock breams were inoculated with the resuspended virus. All experimental fish were purchased from a rock bream hatchery located in Geoje, Gyeongsangnam-do, Republic of Korea, where RSIVD has not been reported. Approximately 800 rock breams  were acclimatized in a 1600 L tank for 2 weeks. The tank was a flow-through aquaculture system. Furthermore, it was continuously supplied with sand-filtered, 50 μm filter-housed, and ultraviolet (UV)-treated (>30 mW/cmn = 15) after maintaining a 200 L tank at 25 °C. In the RSIV viability infection experiment, 0.1 mL (106.2 RSIV copies/fish) of the resuspended RSIV 10-fold dilution was intraperitoneally injected into each rock bream. In the positive control group, 106.2 RSIV copies/fish (cell culture) was intraperitoneally injected. Furthermore, the oxalate-EDTA control group (virus-free group) was intraperitoneally injected with 0.1 mL oxalate-EDTA buffer diluted 10-fold with PBS. The negative control group was intraperitoneally injected with the same amount of PBS. Rock breams were fed a commercial feed once a day, and they were continuously supplied with sand-filtered, 1 μm filter-housed, and UV-treated (>30 mW/cm2) seawater (100 L/h). Cumulative mortality was observed for 3 weeks.Rock breams were housed in each group .https://www.graphpad.com/scientific-software/prism/; accessed on 7 December 2022). Significant differences according to survival marker concentrations were evaluated using one-way analysis of variance (ANOVA) and Dunnett’s correction based on infectious RSIV. When the Ct values were compared according to infectious RSIV rates, statistical analysis was performed using two-way ANOVA. Furthermore, significant differences in the delta Ct values among groups were analyzed using one-way ANOVA. Significant differences in RSIV viability over time compared to the control in seawater were assessed using Dunnett’s correction based on one-way ANOVA. A p-value of <0.05 was considered statistically significant.Statistical tests were performed using GraphPad Prism 9.5 (


Carica papaya) crops in northwestern (NW) Argentina was sequenced and characterized using next-generation sequencing. The resulting genome is 6667-nt long and encodes five open reading frames in an arrangement typical of other potexviruses. This virus appears to be a novel member within the genus Potexvirus. Blast analysis of RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes showed the highest amino acid sequence identity  with pitaya virus X. Based on nucleotide sequence similarity and phylogenetic analysis, the name papaya virus X is proposed for this newly characterized potexvirus that was mechanically transmitted to papaya plants causing chlorotic patches and severe mosaic symptoms. Papaya virus X (PapVX) was found only in the NW region of Argentina. This prevalence could be associated with a recent emergence or adaptation of this virus to papaya in NW Argentina.An emerging virus isolated from papaya ( Carica papaya L.) is an important fruit crop widely cultivated in tropical and subtropical regions [Papaya  and papaya only by mechanical inoculation [In northwestern (NW) Argentina, papaya plants showing leaf mottling and mosaic symptoms, which differed from those previously reported for PRSV , were deculation .Potexvirus [With the development of next-generation sequencing (NGS) technology, plant virus discovery has increased enormously  and sevetexvirus . This vi2HPO4, pH 7.0 + 0.1% Na2SO3, and 600-mesh silicon carbide as abrasive, and the first three expanded leaves were mechanically inoculated onto five healthy papaya cv. ‘Maradol roja’ seedlings with 60 days after germination (dag) of seeds. In addition, five plants were mock-inoculated (control). The inoculated plants were maintained in an insect-free greenhouse at 26 ± 4 °C and exposed to a photoperiod of 14 h light and 10 h darkness, for symptom development and further description [Papaya plants showing leaf mottling and mosaic were first observed in plantations in Yuto, Jujuy, in 2013. Afterward, papaya orchards in NW  and northeast (NE)  provinces of Argentina were surveyed from 2017 to 2021. Part of the collected symptomatic and asymptomatic leaf samples was stored at −80 °C, and another part was lyophilized and stored at −20 °C until use. For virus maintenance, leaf samples were ground in phosphate buffer 0.01 M Nacription .w/v lead citrate  and 2% w/v uranyl acetate. Tissue preparations were examined with a JEM 1200 EX II electron microscope  and photographed.Fresh symptomatic and asymptomatic leaf samples collected from both papaya field plants, and inoculated plants were analyzed by electron microscopy, using leaf-dip and inclusion in Spurr resin technique . Leaf-diw/v) of cold 0.05 M Tris-citrate (pH 7.4) containing 0.5% Na2SO3 (w/v), 1% polyvinylpyrrolidone  (w/v), and 1% Triton X-100 (w/v). The homogenate was squeezed through muslin and clarified by blending for 20 s; then 25% chloroform (v/v) was added, and centrifugation was performed at 10,000× g for 10 min. The aqueous supernatant was collected, and the virus-like particles were concentrated by centrifugation in a 30% sucrose gradient at 136,000× g at 4 °C for 1 h, in a Beckman SW45 Ti rotor. The pellet was resuspended by stirring in 1 mL of 0.05 M Tris-citrate (pH 7.4) containing 0.5% Na2SO3 (w/v) at 4 °C for 1 h.Leaf tissue from one papaya plant showing leaf mottling and mosaic symptoms was used for the purification of virus-like particles A. PlantsFor NGS, viral RNA was extracted from the suspension (200 μL) of concentrated particles using RNeasy Plant Mini Kit , according to the manufacturer’s protocol. RNA quality and concentration were determined by fluorometric quantitation Qubit 2.0 . RNA preparation (1 µg), subjected to ribosomal RNA depletion with Ribo-Zero rRNA Removal Kit , was used for library construction via TruSeq RNA Library Prep Kit v2 , according to the manufacturer’s protocol. The RNA was fragmented and copied into the first-strand cDNA using 200 units of SuperScrip III reverse transcriptase  and random primers; then, the second-strand cDNA was synthesized using 1 unit of DNA polymerase I  and 2 units of RNase H , according to the manufacturer’s protocol. The cDNA was purified using Agencourt AMPure XP magnetic beads . A total of 3,084,077 paired-end 250-nt long reads were generated using the Illumina MiSeq platform at Instituto de Biotecnología, INTA Castelar, Argentina.http://www.usadellab.org/cms/?page=trimmomatic, accessed on 1 December 2017) with standard parameters [https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 1 December 2017). The putative viral sequences obtained were curated by interactive reading mapping of filtered reads using the Bowtie 2 version 2.3.2 software  [https://www.ncbi.nlm.nih.gov/orffinder/, accessed from January to February 2018). Structural and functional domains of the detected gene products were predicted using the NCBI-CD-search tool available at https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed from January to February 2018), against the CDD v3.19 database with an expected value of 0.01. In addition, the InterPro protein signature database was also used .A trimming step was performed to remove adapters and low-quality reads using the Trimmomatic software version 0.39 (rameters . The prorameters . The asser 2017) . Genome CP and RdRp genes for both nucleotide (nt) and amino acid (aa) sequences. Sequence datasets were refined to conserve non-ambiguous sites using GBlocks with the least stringent conditions [http://iqtree.cibiv.univie.ac.at/) (accessed from August to December 2021) [For phylogenetic analyses, sequences of Alphaflexiviridae members were obtained from the NCBI database . Sequencnditions  in SeaVinditions , accordinditions , availaber 2021) . The SH-er 2021)  and ultrer 2021)  were useer 2021)  was usedHylocereus undatus), Christmas cactus (Schlumbergera truncata), opuntia (Opuntia sp.), Nicotiana glutinosa (60 dag), and Nicotiana rustica (60 dag) (10 plants per species). Plants were maintained in an insect-free greenhouse, as previously described in Mechanical inoculations were done by dusting carborundum (600-mesh) on test leaves followed by rubbing infected tissue homogenized in phosphate buffer as previously described (see Sample Collection and Virus Maintenance). Test plants included papaya (60 dag), pitaya (RdRp gene (ORF1) of the virus sequence obtained by Illumima MiSeq in this work and reference sequences of other related potexviruses, according to Van der Vlugt and Berendsen [https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 15 January 2020) and tested on the samples in which this virus was first detected by NGS.To determine the success of virus inoculation, each plant was RT-PCR tested at 60 days post-inoculation. Sense and antisense primers were designed based on the conserved aa motifs in the C-terminal region of the erendsen . Designe® G2 DNA polymerase enzyme , 200 μM dNTPs and 0.3 μM of each primer in a final reaction volume of 50 μL. The PCR conditions included an initial incubation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 45 s, extension at 72 °C for 60 s, and a final extension at 72 °C for 10 min.Total RNA was extracted using Trizol method , according to the manufacturer’s instructions. The RNA was used for first-strand cDNA synthesis using Moloney murine leukemia virus (MMLV) reverse transcriptase enzyme  and random primers . For virus detection, PCR reactions were performed using 4 μL of cDNA, 1.25 U of GoTaqPCR products were separated on 1.0% agarose gel with TAE buffer . DNA bands were stained with Gel Red , using GelDoc EQ  gel imaging system with the Quantity One analysis software version 4.5.1  and scored by comparison to a 1-kb DNA ladder .polymerase gene containing A, B, and C conserved RdRp motifs [To investigate whether the detected virus might be present in other datasets, we employed the Serratus palmID tool  which exp motifs . The relp motifs . In addihttp://www.diva-gis.org, accessed on 15 April 2022) was used to generate a map showing the virus distribution data. The virus was also tested in Ecuador, using papaya and babaco. A total of 120 papaya samples were collected from four commercial orchards distributed in three provinces, Santa Elena (n = 30), Manabí (n = 60), and Esmeraldas (n = 30), whereas babaco samples were collected from a commercial plant nursery in Azuay province (n = 30) and a commercial production operation in Pichincha province (n = 30).A virus survey was conducted to determine the distribution of this virus in the northern region of Argentina. Samples were collected from papaya orchard and family gardens (sampling sites) located in Jujuy, Salta, Chaco, Corrientes, Formosa, and Misiones provinces. The geographic coordinates for sampling locations were obtained using a handheld global positioning system (GPS) . Virus d® SV Gel and PCR Clean-Up System , and then directly sequenced in both directions by Macrogen Inc. The obtained sequences were assembled with Staden package version 2.0.0b10 , and submitted to the BlastN program [http://blast.ncbi.nlm.nih.gov, accessed on 1 March 2022).The RNA purification, cDNA synthesis, and PCR conditions were the same as previously mentioned. The PCR products corresponding to symptomatic plants, obtained in duplicate, were purified using Wizard program  for homoRdRp gene fragment was used to perform the phylogenetic analyses. The phylogenetic tree was also built using the RdRp partial sequences of isolates of the newly proposed papaya virus and other sequences of closely related species. The most related species were chosen according to the grouping obtained in the phylogenetic tree of RdRp complete sequences  against the RNA-dependent RNA polymerase of pitaya virus X (PiVX). Further inspection of the virus transcript and curation by iterative mapping of filtered reads resulted in a consolidated 6667-nt virus sequence supported by 7564 reads (mean size 108.8-nt), and a mean coverage 131.7X. The virus sequence showing features and identity with several members of the genus The genome sequence of the detected papaya virus consisted of 6667-nt, excluding a polyA tail, and is organized in five ORFs A. SimilaPotexvirus , cactus virus X (CVX), schlumbergera virus X (SchVX), and opuntia virus X (OpVX) revealed nt identities ranging from 66–68% for the RdRp and 64–67% for the CP. At the amino acids level, an average of 66.50% identity was observed for the RdRp and 66% for the CP . Based on the species demarcation criterion for potexviruses, which states that distinct species share <72% (nt) or <80% (aa) identity for the RdRp and CP genes [Sequence alignments with potexviruses indicated the presence of untranslated regions (UTR) of 89-nt and 106-nt at the 5′ and 3′ ends, respectively. The hexanucleotide 5′-GAAAAC-3′, present at the 5′-UTR ends of most potexviruses, was observed B. A putatexvirus D. A Blasr the CP . In turn−61/2.1 × 10−68) corresponded to the NCBI-SRA datasets SRR6846447 of a wastewater metagenome sample from Singapore and SRR11190798 of a pitaya fruit (Hylocereus polyrhizus) sample from Guangdong China, respectively. A rapid NCBI-BlastP of the obtained 95 aa palmprints showed as best hits to the PiVX RdRp  and to ZyVX RdRp , respectively, suggesting that the retrieved virus-like sequences corresponded to strains of these viruses and certainly not to the PapVX reported here. These two SRA datasets were processed and resulted in the assembly of nearly complete virus genomes , with the genomic architecture of members of genus Potexvirus (view SRA datasets). The encoded complete RdRps of both assemblies were subjected to NCBI-BlastP searches, which retrieved as best hit PiVX isolate GZ-ZYP from China  and ZyVX hosted by S. truncata  from Germany for ZyVX. Pairwise identity alignments of PiVX and ZyVX with PapVX showed a genome nt identity of 65.8% and 66.6% respectively and an aa identity of the predicted encoded proteins ranging from 48.7–53.5% for TGB2 protein to the highest of 66.1–70.4% for the CP protein , and genome sequences (nt) (44 species)   showed sspecies) . No recoH. undatus, S. truncata, Opuntia sp., N. glutinosa, and N. rustica. The presence of PapVX was molecularly confirmed only in papaya plants. The developed primer set PapVX5 and PapVX1RC  of the 21 localities sampled from the north of Argentina; only numerical tags were assigned to map locations where the virus was identified . Of the RdRp gene sequences of PapVX obtained  and the MN265368.1 showed nucleotide sequence identity values ranging from 82.99–99.71%, with the lowest identity values being detected between isolates from different locations. The pairwise analysis of the amino acid sequences showed values ranging from 98.56% to 100%. The highest similarities were observed between the sequences of isolates from nearby localities: isolates from Yuto  and Colonia Santa Rosa . In addition, in the phylogenetic tree of the partial RdRp gene sequences of PapVX, two subgroups were observed, one including sequences from Jujuy province  and the other from Salta province (Colonia Santa Rosa and Orán)  .Potexvirus . In this work, we report the genetic and epidemiological studies of a novel potexvirus naturally infecting papaya crop in the NW region of Argentina. The sequencing strategy used in this study (TruSeq RNA Illumina protocol) targeted RNA viruses, whereas additional viral metagenomic analyses could help to understand the diversity of viruses infecting papaya in the subtropical region of Argentina.The ICTV has officially recognized 48 species in the genus In this study, the symptoms observed in papaya plants were different from those attributed to PRSV infection, the only viral disease previously reported in this crop in Argentina . SeveralPapVX exhibits close genetic relationships and shares a common ancestor with PiVX, ZyVX, CVX, SchVX, and OpVX; for this reason, the name papaya virus X is proposed. Recombination and mutation events are major forces attributed to evolution in plant viruses and are associated with emergence of new variants and host adaptation ,53; howeThe presence of PapVX only in papaya crops of NW Argentina suggests that the virus has a restricted geographical distribution or that it has been present in papaya and/or other undetected host plants, which remains to be investigated. In this sense, the fact that this virus was detected exclusively in papaya plantations in Salta and Jujuy, but not in the NE region , suggests a recent emergence in this crop. Even with partial sequences, a geographical clustering was observed, indicating sustained transmission with still limited dispersion.Although the phylogenetic relationships of PapVX with viruses isolated from cactaceous plants were confirmed, it was not experimentally transmitted to the plant species tested in this family . In this sense, Alvarez-Quinto et al.  showed tRdRp gene and will help to monitor the virus distribution and discover potential new hosts. These primers were employed to analyze papaya and babaco samples from Ecuador; results showed that all samples were negative for PapVX. The prospection of PapVX in papaya orchards from Bolivia, which borders the region where this virus was detected in Argentina, could be important to enhance the knowledge of PapVX dispersion. Linear patterns of papaya plants infected with PapVX were observed in the same crop row, which was potentially associated with mechanical labor  performed in the orchards, showing the importance of implementing cultural measures to avoid dispersion of PapVX within the crop. The potential vertical transmission of PapVX (via seeds), which plays a role in the long-distance dispersion of pathogens [The primer set PapVX5 and PapVX1RC resulted in the amplification of a ~737-bp PCR products from the terminal region of the athogens , should The use of high-throughput sequencing to detect known and novel plant viruses or variants has extended considerably , increasThe tropics and subtropics provide many examples of new encounter situations between viruses and wild or cultivated plants ,57. For A virus naturally infecting papaya, tentatively named PapVX, is herewith reported for the first time. Given the rate at which papaya planting materials are being exchanged between farmers, it is likely that this virus, although currently restricted to NW of Argentina, could spread to other papaya-growing areas in the region. Additional surveys in other papaya crops areas, including native plants from hotspot sites and neighboring countries, should be carried out to determine the presence of this virus. Moreover, further epidemiological studies  are needed to understand the risk that PapVX poses to papaya and other crops in the region.


Chili leaf curl virus (ChiLCV) . ChiLCV shows a widespread occurrence in most of the chili (Capsicum spp.) growing regions. ChiLCV has a limited host range and infects tomatoes (Solanum lycopersicum), potatoes (S. tuberosum), and amaranth (Amaranthus tricolor). The virus genome is a monopartite circular single-stranded DNA molecule of 2.7 kb and associated with α and β-satellites of 1.3 and 1.4 kb, respectively. The virus genome is encapsulated in distinct twinned icosahedral particles of around 18–30 nm in size and transmitted by Bemisia tabaci . Recently, bipartite begomovirus has been found to be associated with leaf curl disease. The leaf curl disease has a widespread distribution in the major equatorial regions viz., Australia, Asia, Africa, Europe, and America. Besides the PCR, qPCR, and LAMP-based detection systems, recently, localized surface-plasmon-resonance (LPSR) based optical platform is used for ChiLCV detection in a 20–40 μl of sample volume using aluminum nanoparticles. Management of ChiLCV is more challenging due to the vector-borne nature of the virus, therefore integrated disease management strategies need to be followed to contain the spread and heavy crop loss. CRISPR/Cas-mediated virus resistance has gained importance in disease management of DNA and RNA viruses due to certain advantages over the conventional approaches. Therefore, CRISPR/Cas system-mediated resistance needs to be explored in chili against ChiLCV.Leaf curl disease in a chili plant is caused mainly by  Capsicum annuum L.) is one of the major spice crops belonging to the Solanaceae family. Chili is a native of South America. Portuguese, during the fifteenth century, introduced chili to India, and since then, India is recognized as a secondary center for Capsicum species diversity. C. annuum is the most cultivated species; whereas C. baccatum, C. chinense, and C. frutescens are restricted to homestead gardening in India . In India, during 2019–2020, the production of red chilies was around 1.7 million tons from a cultivated area of around 0.68 million hectares as per the second advance estimate  was demonstrated by Khan et al. (Cotton leaf curl Multan virus (CLCuMuV) and Tomato leaf curl Gujrat virus (ToLCGV) were also reported to occur in chili in India  recognized species and six are tentative species , Begomovirus (445 species), Capulavirus (four species), Citlodavirus (four species), Curtovirus (three species), Eragrovirus (one species), Grablovirus (three species), Maldovirus (three species), Mastrevirus (45 species), Mulcrilevirus (two species), Opunvirus (one species), Topilevirus (two species), Topocuvirus (one species), and Turncurtovirus (three species) as per the recent updates on recognized virus species by ICTV . The genome is encapsulated in a distinctive twinned-icosahedral particle of ~18–30 nm in size and is exclusively transmitted through whitefly  mono-partite begomoviruses having a single DNA genome that is analogous to the DNA-A genome of bipartite begomoviruses, (ii) monopartite begomoviruses with satellite ssDNA designated as β-satellite, and (iii) bipartite begomoviruses with two similar sizes of ssDNA segments designated as DNA-A and DNA-B , Chili leaf curl Palampur virus (ChiLCPaV), ChiLCV, Chili leaf curl Vellanad virus (ChiLCVeV), Chili leaf curl Salem virus (ChiLCSV), Chili leaf curl Gonda virus (ChiLCGV), Chili leaf curl Ahmedabad virus (ChiLCAV), Papaya leaf curl virus (PaLCuV), Chili leaf curl Bijnour virus (ChiLCBV) from India, CLCuMuV and Tomato leaf curl Joydebpur virus (ToLCJoV) from Pakistan, Pepper yellow leaf curl Indonesia virus (PepLCIV) from Indonesia, and Cabbage leaf curl virus (CaLCuV) from Cuba, Pepper leaf curl Bangladesh virus (PepLCVBV) from Bangladesh have been reported , Direct antigen coated ELISA (DAC-ELISA), and double antibody sandwich ELISA (DAS-ELISA) have been routinely used with enhanced sensitivity. Likewise, radioactive probe-based dot-blot hybridization was used to determine the existence or identification of begomovirus in the chili samples , western blots, immunostrip assays, and dot-immuno-binding assays (DIBA) follow the principle of antigen-antibody interaction. Amidst all, an ELISA is the most prominent immunodiagnostic technique for the identification of viruses owing to its high throughput potential. Various formats of ELISA Polymerase chain reaction (PCR) is a more efficient method than ELISA and hence provides a rapid, specific, and accurate detection of known viruses . ChiLCV Isothermal amplification methods such as rolling circle amplification (RCA), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP) have been introduced as an alternative to cost-deprived PCR assays. RCA using φ29 DNA polymerase mediated amplification was carried out in infected genomic DNA samples . In the ChiLCV genome, the IR consists of 279 nucleotides (nts) origin of replication with a 33-nts long potential stem-loop structure containing non-coding non-nucleotide sequence (TAATATTAC), which is required for cleavage and joining of the viral DNA during replication.The IR region is the most prominent and highly conserved region of all the genera of via the rolling-circle (RCR) mode of replication by introducing a nick at the phosphodiester bond of the seventh and eighth residues of non-nucleotide sequence. The cleaved 5' phosphate end of the sequence remains bound to Rep protein while the newly generated 3'-hydroxyl end starts the rolling-circle mode of replication , and tomato , is smaller in size than DNA-A and encodes a single conserved region of ~13–14 kDa protein known as βC1 in complementary orientation . Apart fThe complete genome sequences of 84 isolates of begomoviruses associated with ChiLCD were retrieved from the NCBI GenBank and subjected to phylogenetic dendrogram construction. The phylogenetic tree revealed the 14 distinct clades . The seqRecently, Pandey et al.  ascertaiB. tabaci is polyphagous in nature, and it shows a high frequency of reproduction and quicker dispersal ability thus, facilitating its existence in large groups of populations with different agro-ecological zones. Even single whitefly has the ability to transmit the virus, whereas the presence of eight or more whiteflies on a plant may result in 100% chances of transmission . Moreover, the least immigration frequency of vector was found to be essential in deciding the frequency of infectious host and viruliferous vector. In addition to this, migration of viruliferous vectors, virus acquisition, and transmission frequency were the important traits in deciding the epidemic of ChiLCD. Therefore, by keeping in view the effect of temperature (15–35°C) and transmission phenomenon in the build-up of virus-vector population, spatial and temporal pattern of disease risk has been predicted with ChiLCD distribution in India. This assessment of chili leaf curl disease dynamics would certainly be helpful to the growers in devising effective crop and disease management approaches  incomplete T = 1 icosahedral capsid particles containing viral CP and each CP is bound by seven bases of viral ssDNA  governed by two major genes for two begomoviruses. Further, functional characterization of the genes showed that germin-like proteins gene CchGLP is a vital factor for begomovirus resistance in BG-3821 accession from C. chinense  with 0.1% celite was used to prepare a cocktail of dsRNAs. A single spray of the cocktail of dsRNAs reduced ChiLCD incidence up to 66.7% in N. benthamiana plants for 2 weeks.In a study by Sharma et al. , a demonBacillus thuringiensis (Bt) is the most commonly used commercial biopesticide. The advantages of Bt biopesticides are they highly specific to target insects . Nevertheless, the sap-sucking homopteran whiteflies remain insensitive to Bt toxin. Shukla et al. (Tma12 gene from an edible fern (Tectaria macrodonta) has insecticidal activity against whitefly. Tma12 overexpressing cotton transgenic lines were resistant to whitefly infestation and cotton leaf curl viral disease, with no detectable yield penalty. There is scope to identify such insecticidal proteins that are lethal to whiteflies. These biopesticide molecules need to be expressed in chili to overcome the whitefly-borne ChiLCV.Biopesticides are potent microbial pesticides and biochemicals obtained from microbes and other natural resources. Advancement in plant genetic transformation technology has made it easy to overexpress the potent biomolecules into crop plants thereby conferring resistance against insect pests. Successful application of biopesticides depends on exact information of feeding habits and life cycle of the insect and the mechanism of action of the potent biopesticides. This facilitates determining the precise time and stage of biopesticide application for efficient results. To date, δ-endotoxin from a et al.  reportedviz., to recognize, interfere, and cleave the virus genome so that inhibition of multiplication of invasive viruses and to manage host susceptibility factors essential for virus replication and ultimately improve plant immunity and block the viral attack.In the last decade, the CRISPR/Cas  system has successfully emerged as one of the promising and precise genome-editing methods  and Zinc finger nucleases (ZFN). Therefore, this method has become more dominant and found promising for developing antiviral engineering in plants. In the transgenic model plant system, N. benthamiana CRISPR/Cas9 single guide RNA (sgRNAs) constructs specific to viral Rep, or IR region, showed effective DNA intrusion which thereby conferred resistance against CLCuMuV (Yin et al., N. benthamiana lines (Tashkandi et al., Severely affecting members of the family of CRISPR/Cas-mediated virus resistance in plants can also be established by targeting host factors. For plant virus protein translation, the recruitment of host-specific translation factors to facilitate their infection processes is required. Such host-specific translational factors are recognized and targeted as pro-viral factors. Further, in the recent past, numerous other pro-viral host factors, such as movement, replication, and metabolism-associated regions were characterized and used to limit plant virus diseases. eIF4E, eIF4G, and their isoforms are effective host translation initiation factors, which are exploited to achieve defense against various sub-species of viruses. Chandrasekaran et al.  reportedBemisia tabaci is the only vector of begomoviruses and a serious pest of agricultural and horticultural crop plants. Recently, Heu et al. (B. tabaci. The offsprings were found to have a heritable edited genome. Thus, the development of a gene editing procedure for B. tabaci will helpful for the researchers to use the application of reverse genetics and will lead to the management of this serious insect vector.Like host and virus, vectors can also be tapped for this novel technique, since, u et al.  developeTma12 can be expressed in plants to restrict spread of vectors and ultimately the ChiLCV infection too. For resistance breeding, the reason for the inaccessibility of prominent resistant lines is the lack of a robust screening method for the identification of resistant lines and the evaluation of inheritance patterns. Advance molecular breeding holds the key to the identification of robust resistance sources and further introgression to elite lines. The advanced research needs to be concentrated on the identification of resistance sources using genomics-assisted breeding and developing robust resistance using breeding tools. Besides, RNAi against several transcripts of ChiLCV held the promise of being a fast and effective approach for achieving resistance against various plant viruses. Furthermore, genome editing based on the CRISPR-Cas system has offered a new capable technique for developing resistance against ChiLCV through alteration in host susceptibility factors. For achieving broad-range resistance against plant viruses through the CRISPR-Cas system various non-essential host susceptibility factors may prove to be more potent targets. Transgenic approaches looked to be very promising against ChiLCV, but the major hurdle for the release of transgenic varieties is uncertainties over government norms and public acceptance in India. In the detailed understanding of ChiLCV host–pathogen proteins integration, promotion, and the invention of novel technologies is the responsibility of the scientific community to get rid of ChiLCV.In recent years, there is a substantial increase in understanding of ChiLCD however, certain questions remain unanswered. The devastating and widespread nature of the ChiLCV highlights the urgency to recognize effective control measures for it. Whiteflies serve as a vector for numerous plant viruses and attempts to prevent whitefly population growth will be very beneficial in the field. The geographical distribution of ChiLCD is correlated with the incidence of whitefly in the world. Deciphering the effective environment-friendly biological control agents holds a promising key to control of the whitefly population. Further, whitefly-specific insecticidal proteins such as PS, SH, and DS planned the review, drafted, and finalized. DW and VP prepared the tables, collected the literature, and drafting. SK prepared the figures and reference arrangements. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Yam is an important food staple for millions of people globally, particularly those in the developing countries of West Africa and the Pacific Islands. To sustain the growing population, yam production must be increased amidst the many biotic and abiotic stresses. Plant viruses are among the most detrimental of plant pathogens and have caused great losses of crop yield and quality, including those of yam. Knowledge and understanding of virus biology and ecology are important for the development of diagnostic tools and disease management strategies to combat the spread of yam-infecting viruses. This review aims to highlight current knowledge on key yam-infecting viruses by examining their characteristics, genetic diversity, disease symptoms, diagnostics, and elimination to provide a synopsis for consideration in developing diagnostic strategy and disease management for yam. Dioscorea spp.) is the collective name for a group of multi-species, dioecious, and monocotyledonous vines cultivated primarily for their starchy underground tubers. The Dioscorea genus belongs to the family Dioscoreaceae of the order Dioscoreales and comprises approximately 600 species of domesticated and wild yams. However, only about 10 edible species are widely grown for food in the tropical and sub-tropical regions of the world ..DioscoviD. nummularia samples generated the expected amplicons. The DNUaV positive plants did not show any disease symptoms and it is yet to be determined how it affects yam plants and associated yield.Primer pairs for PCR detection were designed by Sukal et al.  amplifyiBromoviridae contains some of the most important plant RNA viruses, with members distributed worldwide, infecting over 10,000 plant species ..BromovirCucumber mosaic virus) is a member of the genus Cucumovirus. It has an isometric single-stranded, positive-sense tripartite RNA genome consisting of RNA1, RNA2, and RNA3 and two subgenomic regions, RNA4 and RNA4A [Cucumber mosaic virus (CMV) , Betaflexiviridae, Secoviridae (genus Sadwavirus), and Closteroviridae (genus Ampelovirus) have also been reported from yam [In recent years, novel viruses from other plant virus families have been reported from yam. Viruses from the family from yam ,63,101.Yam virus X), genus Potexvirus, family Alphaflexiviridae, isolated from D. trifida in Guadeloupe. The YVX genome is 6158 nt in length, excluding the poly(A) tail, and encodes five ORFs. A large ORF1 encodes the RdRp, whilst ORFs 2, 3, and 4 encode the putative triple gene block proteins, TGBp1, TGBp2, and TGBp3, typical of potexvirus and function in viral movement. ORF5 encodes the CP. A BLAST search of the RdRp and CP amino acid sequences in the NCBI database and phylogenetic analysis confirmed this as a potexvirus but the maximum sequence identity was only 51.9% indicating it was a new member of the genus. Yam plants infected with YVX were symptomless except for a single plant co-infected with a potyvirus that showed mild symptoms. Mechanical transmission of viral isolates to indicator plants N. benthamiana, N. clevelandii, C. quinoa, and C. amaranticolor did not yield local or systemic infection and RT-PCR detection with the potexvirus-specific primers also yielded negative results. A prevalence study carried out by Mambole et al. [D. nummularia from Vanuatu and group two includes sequences from D. bulbifera and D. rotundata from Haiti, and D. trifida and D. rotundata sequences from Guadeloupe.Mambole et al.  reportede et al.  on 383 ySadwavirus, family Secoviridae. Phylogenetic analysis of the protease–polymerase (Pro-Pol) amino acid sequence of DmaV with other members of secovirus indicated that it most closely related to the chocolate lily virus A (CLVA), whilst the amino acid sequence identity indicated it was a putative new member of the family Secoviridae based on ICTV species demarcation criteria.The complete genome sequence of a novel putative secovirus was isolated from yam plants exhibiting mosaic symptoms in Brazil . The genYam asymptomatic virus 1) from an asymptomatic D. alata plant collected from Vanuatu. The genome is 14,855 nt in length, encoding for 10 putative ORFs with a similar organization to that of the little cherry virus 2 (LChV2) of subgroup 1 of genus Ampelovirus. Phylogenetic analysis of the HSP70 and CP amino acid sequences confirmed it to be a novel member of the genus Ampelovirus, family Closteroviridae and distinct from another recently identified ampelovirus, air potato virus 1 (AiPoV1) infecting D. bulbifera from Florida, USA [D. alata plant, confirming its presence in Africa. Sanger sequencing of fifty-five selected amplification products was used for phylogenetic analysis but revealed low variability (93.3 to 100% sequence identity) amongst distinct accessions, suggesting that plant-to-plant transmission through insect vector may be possible.Marais et al.  recentlyida, USA . DiosCloida, USA . Blastn D. rotundata from the Natural Resources Institute , IITA, and Council for Scientific and Industrial Research—Crop Research Institute , were sequenced using high-throughput sequencing (HTS) [Betaflexiviridae and most closely related to sugarcane striate mosaic-associated virus (SCSMaV) (Sugarcane striate mosaic-associated virus). A prevalence study using newly developed YVY-specific PCR primers reported 31 YVY positive samples in a collection of 55 breeding lines and landraces grown in NRI UK, IITA Nigeria, and CSIR-CRI Ghana. Among these, 23 showed mixed infection with YMV. Plants that were singly infected with YVY were generally symptomless except for one plant, whilst plants infected with YMV or mix-infection developed symptoms [The complete genome sequences of two isolates of the tentatively named ‘yam virus Y’ (YVY), obtained from a collection of ng (HTS) . The gensymptoms .Plant viruses are obligate intracellular parasites that can survive only inside living cells . Once thD. opposita plants by cryotherapy of shoot tips. Shoot apices were precultured for 16 h in 0.3 M sucrose, encapsulated in sodium alginate, and dehydrated for 4 h before direct immersion in liquid nitrogen. Regenerated shoot tips were reported to be 90% YMV-free. Similarly, Ita et al. [D. rotundata by cryotherapy of axillary buds of infected stocks. Enlarged axillary buds of infected plants were frozen in liquid nitrogen for one hour, re-warmed at 40 °C, and cultured to regenerate plants. Plantlet regeneration was reported at 76%, whilst YMV elimination was reported at 100%. Similar protocols can be adopted for other yam virusesShin et al.  reporteda et al.  reportedD. alata. Single node vine cuttings were treated at 32 and 36 °C for different time durations and virus elimination was confirmed by RT-PCR using YMMV-specific primers. Treatment at 36 °C for 30 min was reported to be most efficient at 90% elimination [Hot water treatment was successfully used for the elimination of YMMV in mination . Umber emination  reportedD. cayenensis-rotundata [The application of water-dissolved ozone was reported for the sanitation of potyvirus during in vitro propagation of otundata . Potyvirotundata .Efficient virus diagnostic tools and sanitation are essential to facilitating germplasm exchange. The Centre for Pacific Crops and Trees (CePaCT) of the Land Resource Division (LRD) of the Pacific Community (SPC) located in Fiji is the premier genebank of the Pacific supporting the safe conservation and distribution of plant genetic resources of importance to the region. The center (CePaCT) has a unique yam collection from the Pacific region that is invaluable towards mitigating biotic and abiotic stresses in production. However, the yam diversity remains unavailable due to the fact that diagnostic protocols for yam viruses, particularly those endemic to the Pacific region, remain undefined.In the 2000s, virus characterization work carried out in the region, though sporadic, has speculated the existence of known and novel badnavirus and potyvirus diversity in the region ,42,110. In this review, the most prevalent viruses infecting yam globally have been described for their origin, morphology, genome organization, symptoms caused, and diagnostics. As a predominantly vegetatively propagated crop, the use of clean, virus-free planting materials is the most effective method to curtail the spread of yam viruses. A major drawback, however, is the lack of formal seed systems within smallholder farming communities where globally, most yams are cultivated. The use and sharing of virus-infected planting materials by farmers promote the spread of yam viruses to new ecogeographical locations.Yam viruses, such as YMV, YMMV, and badnaviruses, have been reported to be highly prevalent and genetically diverse. In addition, the discovery of integrated viral sequences from badnaviruses within host genomes adds a level of complexity to their diagnostics. As more yam-infecting viruses are being discovered from other virus families, the development of sensitive and specific diagnostic tools for their detection becomes paramount. This leaves the conundrum and predicament of whether a truly ‘virus-free’ plant can be achieved and what level of sanitation is required to meet international standards for germplasm exchange.Serological and PCR-based methods of detection will continue to be the backbone of yam virus diagnostics. Their limitations, however, are that they can only detect known or related viruses. In addition, the high genetic diversity of virus isolates can limit their detection spectrum. The use of HTS technologies for sequencing and discovery of existing and novel yam viruses is gaining traction. Bomer et al.  evaluateDioscorea spp. [Virus elimination through in vitro culture techniques has proven effective for producing virus-free plants in yam. Natural resistance, however, might prove to be more economic and efficient in the long term. Work on genetic resistance and crop improvement has been slow in the past due to a lack of genetic and genomic tools. With the recent publication of several whole-genome sequences of rea spp. ,111,112,rea spp. , researcThis is the first review of global yam viruses and the information contained herein will invaluably facilitate further development in yam virus diagnostics and sanitation for the safe international exchange of yams particularly in the Pacific and those held elsewhere in the world.


Rosa spp.), especially R. hybrida, is one of the most popular ornamental plants in the world and the third largest cut flower crop in Taiwan. Rose mosaic disease (RMD), showing mosaic, line patterns and ringspots on leaves, is a common rose disease caused by the complex infection of various viruses. Due to pests and diseases, the rose planting area in Taiwan has been decreasing since 2008; however, no rose virus disease has been reported in the past five decades. In the spring of 2020, rose samples showing RMD-like symptoms were observed at an organic farm in Chiayi, central Taiwan. The virome in the farm was analyzed by RNA-seq. Rose genomic sequences were filtered from the obtained reads. The remaining reads were de novo assembled to generate 294 contigs, 50 of which were annotated as viral sequences corresponding to 10 viruses. Through reverse transcription-polymerase chain reaction validation, a total of seven viruses were detected, including six known rose viruses, namely apple mosaic virus, prunus necrotic ringspot virus, rose partitivirus, apple stem grooving virus, rose spring dwarf-associated virus and rose cryptic virus 1, and a novel ilarvirus. After completing the whole genome sequencing and sequence analysis, the unknown ilarvirus was demonstrated as a putative new species, tentatively named rose ilarvirus 2. This is the first report of the rose virus disease in Taiwan.Rose ( Rosa spp.), especially R. hybrida, is one of the world’s most popular and important ornamental plants. Due to their elegant and fragrant flowers, rose is widely used in landscaping, cut and potted flowers, food ingredients, cosmetics and for extracting essential oils. The estimated annual production for landscaping was around 15–18 billion stems and 220 million roses in 2003 [Rose  [Ilarvirus and Arabis mosaic virus (ArMV) and strawberry latent ringspot virus (SLRSV) of the genus Nepovirus [A wide range of pathogens, including fungi, bacteria, viruses, nematodes and phytoplasmas, drastically affect the ornamental value of cultivated roses around the world by leaf and flower mosaics, distortion, spotting, discoloration, necrosis, abscission, reduced growth and plant death . The viriphilus) ,8. RMD iepovirus . Symptomepovirus . In addiepovirus .Serological and molecular methods are commonly deployed for virus detection and diagnosis. Enzyme-linked immunosorbent assay (ELISA) is a widely used serological technique for routine virus testing in phytosanitary, quarantine and virus certification. Polymerase chain reaction (PCR), commonly reverse transcription (RT)-PCR used for RNA viruses, is a relatively sensitive method for virus detection. The advanced real-time or quantitative PCR (qPCR) adds further benefits in a routine testing laboratory primarily due to its higher sensitivity and speed, with no need to use agarose gel electrophoresis. Loop-mediated isothermal amplification (LAMP) is suitable for on-site use . The micIlarvirus spp., ApMV, PNRSV, tobacco streak virus (TSV) [Nepovirus spp., ArMV, SLRSV, tobacco ringspot virus and tomato ringspot virus [Orthotospovirus spp., impatiens necrotic spot virus (INSV), iris yellow spot virus (IYSV), tomato spotted wilt virus (TSWV) and tomato yellow fruit ring virus (TYFRV) [Carlavirus spp., rose virus A (RVA) [Partitiviridae, rose cryptic virus 1 (RoCV-1) [Capillovirus sp., apple stem grooving virus (ASGV) [Emaravirus sp., RRV [Luteovirus sp., rose spring dwarf-associated virus (RSDaV) [Roymovirus sp., rose yellow mosaic virus [Closterovirus sp., rose leaf rosette-associated virus (RLRaV) [Cytorhabdovirus sp., rose virus R (RVR) [Begomovirus sp., rose leaf curl virus (RoLCuV) [Rosadnavirus sp., rose yellow vein virus (RYVV) [Through HTS, many rose viruses have been discovered in recent years. Thus far, at least 26 viruses have been reported infecting roses worldwide. They include five us (TSV) , blackbeus (TSV) ,20 and rus (TSV) ; four Neot virus ; four Or (TYFRV) ,23; thre A (RVA) , rose vi A (RVA)  and rose(RoCV-1) ,28 and r(RoCV-1) ; one Caps (ASGV) ; one Ema (RSDaV) ; one Royic virus ; one Clo (RLRaV) ; one Cyt R (RVR) ; one Beg(RoLCuV) ; and ones (RYVV) . Mixed iColletotrichum gloeosporioides), gray mold (Botrytis cinerea), black spot (Diplocarpon rosae), black rot , powdery mildew (Erysiphe simulans var. simulans) and stem canker (Paraconiothyrium fuckelii) [Ilarvirus species, named rose ilarvirus 2 (RIV-2). The occurrence of these rose viruses was also addressed in this study.Rose is the third largest cut flower crop in Taiwan with an annual output value of $20 million, and Nantou is the largest rose planting area with 106.7 ha, followed by Taichung (30.0 ha), Pingtung (22.0 ha) and Changhua (14.4 ha) . Since 2uckelii) . HoweverRose leaves with virus-like symptoms were sampled from the organic rose farm in Fanlu Township, Chiayi County and the campus of Asia University in Wufeng District, Taichung City in central Taiwan in 2020–2022 for virus identification and detection. Rose seedlings were also collected from nurseries in Nantou and Changhua in central Taiwan in 2021–2022 for virus testing. All samples were stored at −80 °C for long-term storage.free™ kit . The cDNA library was constructed using the Universal RNA-Seq Library Preparation kit with NuQuant . The quantity and quality of the cDNA library were analyzed by Qubit® 2.0 Fluorometer with the dsDNA High Sensitivity kit  and Agilent 2100 Bioanalyzer . Paired-end sequencing was performed by Tri-I Biotech Inc.  using the NovaSeq 6000 Sequencing System . Paired-end reads are 151 bp in length. Raw data quality was checked and trimmed with CLC Genomics Workbench v10 [https://www.ncbi.nlm.nih.gov/genome/?term=Rosa+chinensis, accessed on 15 January 2019) to remove rose genome sequences and kept the unmapped reads. The unmapped reads were de novo assembled to generate contigs with SPAdes v3.13.0 [https://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify viruses. BLASTn/BLASTx analyses of the contigs were performed against local and online databases.Foliar total RNA of symptomatic rose samples was extracted using the Total RNA Miniprep Purification kit  following the manufacturer’s instructions. DNA was removed from the total RNA extract using the Invitrogen™ TURBO DNA-ench v10 . The tri v3.13.0 . The con2+ and enzyme stabilizer), 1× Enhancer, enzyme mix of reverse transcriptase (RTase) and Taq DNA polymerase, and RNase block according to the manufacturer’s recommendation. The RT-PCR conditions were set as 30 min for cDNA synthesis at 50 °C, and 2 min for RTase inactivation at 94 °C, followed by 35 cycles of 30 s for denaturation at 94 °C, 30 s for annealing at 56 °C, and 1–1.5 min (depending on the product size) for synthesis at 72 °C and a final cycle of 72 °C for 7 min. The amplified DNA fragments were analyzed by electrophoresis in 1% agarose gels and then eluted from gels using the Micro-Elute DNA Clean/Extraction kit  following the instruction of the manufacturer. The eluted PCR products were ligated to the pCR2.1-TOPO vector using the Invitrogen™ TOPO TA Cloning kit  and the recombinant plasmids were transferred into E. coli DH5α competent cells by Invitrogen™ One Shot™ transformation reaction . The plasmid DNAs of selected clones were isolated using the NautiaZ Plasmid DNA Extraction Mini kit , three clones per PCR product. Nucleotide (nt) sequencing was carried out by Mission Biotech Co., Ltd.  using the Applied Biosystems™ 3730XL automated DNA sequencing system .Primer pairs  designedTaq DNA polymerase (1.25 U), dNTPs (200 μM), forward and reverse primers (200 nM each)   and 1× PRapid amplification of cDNA ends (RACE) was performed to verify both 5′ and 3′ ends of the genome of RIV-2, as described previously . Total RThe complete genome sequences of 24 ilarviruses available in the GenBank databases  were useIn a field survey conducted at an organic rose farm in Chiayi in 2020–2021, rose plants were observed to exhibit RMD-like symptoms, such as mosaic, mottling and yellow spots and flower color-breaking . The sym50 of 834 bp. The contigs were used for virus identification by BLAST analysis. Finally, 50 contigs were annotated as viral sequences, of which six contigs mapped to ApMV, four contigs for tomato necrotic streak virus , six contigs for PNRSV, three contigs for RoCV-1, two contigs for RoPV, three contigs for ASGV, two contigs for RSDaV, 20 contigs for RLRaV, three contigs for RVB and one contig for Plasmopara viticola lesion associated ourmia-like virus 33 (PvLaOV-33). The results of the reads analysis are summarized in Twelve rose samples collected from the rose farm in Chiayi in February 2021 were used for virus detection. Combining the total RNAs of the rose samples to construct a cDNA library for HTS, a total of 42,920,486 reads were obtained. After trimming, 42,862,516 reads were obtained, which were then used to remove rose genome sequences by mapping reference sequences in the NCBI database. The remaining 4,759,744 unmapped reads were used for de novo assembly to generate 294 contigs, with an NPrimers  designedOne rose sample only positive for RIV-2 was used for whole-genome sequencing. Primers  designedIlarvirus species in the GenBank database. The analysis results are shown in Ilarvirus.The genome sequences of RIV-2 were compared with the available genome sequences of During 2020–2022, 92 symptomatic rose samples were tested using the RT-PCR methods described above, of which 82 were from the disease outbreak farm in Chiayi and 10 were from the campus of Asia University in Taichung. The seven viruses, PNRSV, RIV-2, ApMV, RSDaV, RoCV-1, RoPV and ASGV, were detected in Chiayi with detection rates of 19.5%, 13.4%, 7.3%, 7.3%, 3.7%, 3.7% and 2.4%, respectively. In addition, three viruses, RoCV-1, RoPV and RSDaV, were detected in Taichung with detection rates of 40.0%, 20.0% and 10.0%, respectively . On the RMD is a well-known rose virus disease, but it may be overlooked by farmers due to symptoms similar to nutrient deficiencies. In the spring of 2020, the rose plants in the Chiayi organic rose farm suffered severe decline and death, causing the owner’s concern. To our knowledge, as mentioned above, at least 26 viruses have been reported to infect rose plants, making virus detection inconvenient. Therefore, the virome of the farm was analyzed using HTS technology in this study and, surprisingly, 10 viruses were annotated . Of thesIlarvirus in the family Bromoviridae. Based on the genomic characteristics, RIV-2 was further confirmed to be an ilarvirus, including: (1) the critical conserved motifs (S88-H262) of the methyltransferase (MET) domain and the conserved motifs (D764-K1008) of the helicase (HEL) domain in the P1 replicase [361-R605) of the RNA-dependent RNA polymerases (RdRps) of positive-sense RNA viruses in P2 [Ilarvirus genus. However, the threshold for sequence similarity has not yet been defined [RIV-2 was noted in the virome analysis because it has the closest but low nt identity (<90%) to the three genomic RNA segments of TomNSV, a member of the genus eplicase , (2) thees in P2  and (3) es in P2 . Accordi defined . In addi defined ,48. The Ilarvirus [All three genomic RNAs of RIV-2 begin with a consensus ‘GGTATT’ sequence, similar to the previously reported GTATT sequence found in other species of the genus larvirus . The elelarvirus ; whereaslarvirus ,50,51. Tlarvirus ,53. Phyllarvirus .The occurrence of the viruses was investigated for three years from June 2020 to August 2022, at the Chiayi organic rose farm in which the outbreak occurred and two years from July 2021 to September 2022 at the Taichung Asia University campus. Our results showed that ApMV, PNRSV, RIV-2, RoPV, RoCV-1, ASGV and RSDaV were detected in Chiayi. Surprisingly, mixed infections of up to five viruses could be detected in the organic farm in February 2021 . A largeFrankliniella occidentalis) [F. occidentalis does not harbor in Taiwan. It cannot be excluded that PNRSV has other insect vectors. The arthropod vector of ApMV, RoCV-1 and ASGV is still unknown. RSDaV is known to be transmitted by rose-grass aphid (Metopolophium dirhodum) and yellow rose aphid (Rhodobium porosum) [R. porosum is one of the common pests of roses in Taiwan [The main way the rose viruses spread is likely through vegetative propagation in commercial nurseries and subsequent nationwide distribution to gardens through root grafting. However, some viruses may have other transmission mechanisms once in the environment, including seed, pollen and arthropod vectors ,55. ApMVentalis) ,47; howeporosum) . R. poron Taiwan , which mPyrus pyrifolia var. Hengshen) [Ilarvirus. The synergy between these viruses was observed in co-infections of RIV-2 with ApMV, ASGV, RoPV and RSDaV, causing severe damage to roses in Chiayi. The rose varieties cultivated in Taiwan are mainly imported from abroad, such as the Netherlands, the USA and Japan, and there is a risk of invasion by foreign pathogenic microorganisms and pests. Although the HTS technology has been a powerful diagnostic tool, the development of detection techniques, such as RT-PCR and ELISA, is also critical for virus inspection. The RT-PCR methods developed in this study can be adapted to detect rose viruses in samples from nurseries, fields and imported materials. Nepoviruses such as ArMV and SLRSV, which are commonly associated with RMD, were not detected in our HTS analysis. These viruses also need to be tested in the future.To our knowledge, among these rose viruses, ASGV is the only one recorded in Taiwan from pear (engshen) . This is


Dear Editor,Monkeypox is an endemic disease of Central and Western Africa caused by the Monkeypox virus, a member of the Orthopoxvirus genus. The disease was first diagnosed in humans in 1970 in a baby in ZaireA 35-year-old man was referred to the Department of Sexually Transmitted Infections for the evaluation of a painful genital rash that had appeared five days earlier and was followed by fever. His personal history showed that fever and skin lesions appeared 5 days after unprotected anal intercourse with an unknown male partner in Germany. He had genital herpes in his personal history and no other sexually transmitted infections. Physical examination revealed multiple well-circumscribed deep-seated firm papules with central umbilication on the pubic area and the shaft of the penis. Lesions were relatively the same size and same development stage, surrounded by an erythematous halo, followed by swollen lymph nodes in the groin. Physical examination revealed no other skin/mucous lesions. The patient was otherwise healthy. Serological tests for syphilis  and HIV were negative. He was treated with a single oral dose of azithromycin 1 gram and the next day the fever disappeared. By the fourth day, lesions had a central crust  and wereThe incubation period of the Monkeypox virus infection is one to two weeks. Shortly after the prodrome  a rash with centrifugal distribution  appears going through different stages  before healing. Infection can be transmitted through respiratory droplets, close physical contact, including sexual intercourse, and through contact with fomites.The outbreak of monkeypox in the European region is characteristic of males between 31 and 40 years old, predominantly among men who have sex with men.None declared.Milan Bjekic: Approval of the final version of the manuscript; intellectual participation in propaedeutic and/or therapeutic management of studied cases; preparation and writing of the manuscript; study conception and planning.Milica Markovic: Critical literature review; data collection, analysis, and interpretation; intellectual participation in propaedeutic and/or therapeutic management of studied cases; manuscript critical review.Lidija Dejanovic: Effective participation in research orientation; data collection, analysis, and interpretation; manuscript critical review.None declared.


These data highlight the importance of strict adherence of hospital staff to recommended protective measures. If appropriate, pre-exposure or early post-exposure vaccination should be considered for individuals at risk.The extent of monkeypox virus environmental contamination of surfaces is unclear. We examined surfaces in rooms occupied by two monkeypox patients on their fourth hospitalisation day. Contamination with up to 10 Since 4 May 2022, the largest west-African-clade-monkeypox outbreak to date in countries with non-endemic occurrences has been described . The out2 . In the case of a mobile phone, the entire touch screen was wiped off, since the dimensions and thus the total surface area were known. Complex structures, such as door handles, were measured and the swabbed surface area was estimated.Environmental sampling was carried out by carefully swabbing entire surfaces in the patients’ rooms and anterooms, using ESwabs, moistened with medium contained in the swab system  on day 4 of the respective hospital stay. On larger smooth and flat surfaces, as well as on fabrics, defined areas were swabbed using sterile templates for bioburden control with cut-outs of 20 or 100 cmSamples of lesions or from the throat were also obtained from patients by swabbing with ESwabs.2 of surface area.Environmental or patient samples were diluted 1:1 with cobas PCR Medium  and monkeypox virus DNA was detected by automated real-time PCR run on the cobas 6800 system [Virus isolation was attempted using Vero 76 cells (ATCC CRL1587) and standard cultivation conditions for 40/50 of the collected surface specimen  with 1508 and 4.4×108 cp for patients 1 and 2, respectively. In throat swab samples a respective maximum of 1.3×106 and 2.1×107 cp was detected.Both monkeypox patients were men in their thirties. In patient 1, skin lesions – erythema, pustules, excoriations with crustae – were present in the anus, perianal region and scrotum, penis, and a few isolated lesions on the legs, trunk, tongue, and buccal mucosa. In patient 2, lesions were only present in the anus and the perianal region. The highest viral loads in the patients’ specimens were observed in lesional swabs with a maximum of 2.7×10Both patients were hospitalised in isolation rooms with an associated bathroom. The patients’ rooms were separated from the ward corridor by anterooms, which were used for donning and doffing PPE  were detected on toilet seats. Seat surfaces of chairs, which patients reported using most frequently also showed up to 1.4×103 cp/cm2. On the touch display of the mobile phone of patient 2, a total of 1.5×102 cp/cm2 was observed.All surfaces directly touched by the patients’ hands showed viral contamination with the highest loads detected in both bathrooms. A value of 2.4×104 cp/cm2 . Similar3 cp/cm2) was found on upper wall cabinet door handles in the room of patient 1. Viral DNA was observed on all other investigated surfaces in the patients’ rooms, although it was not known at the time of testing whether and to what extent the patients had also touched these surfaces.Monkeypox virus DNA was also found on the patients' room surfaces, presumably touched primarily by medical personnel. The highest level (1.3×105 cp/cm2 (2 and 7.9×103 cp/cm2). Interestingly, we were able to demonstrate infectivity to Vero 76 cells by successful virus isolation for three of the collected samples relative to patient 2, namely the investigator's glove, the soap dispenser operating lever, and a towel on the patient’s bed (6 copies per sample (>103 cp/cm2).Fabrics that were extensively used by the patients also showed viral contamination up to 105 cp/cm2 . Immediant’s bed . All thr2) were detected on the handle of the door leading to the patient's room. Traces of viral DNA were identified on the handle of both anteroom doors located in the ward corridor, outside the anteroom.In the anteroom, all hand-contact points examined yielded positive PCR results. However, only traces of viral DNA  [6 virus copies. Thus, contaminated surfaces with such viral loads or higher, could potentially be infectious and it cannot be ruled out that their contact with especially damaged skin or mucous membranes, could result in transmission.There are no definite data on the required infectious dose with monkeypox virus in humans. However, in contrast to variola virus , a signits (PFU) . Orthopots (PFU) . Dried vts (PFU) . In this6 viral copies on gloves is consistent with the detection of viral DNA on surfaces typically handled only by medical staff such as the door handles of the anteroom. The detection of the virus at very low concentrations even outside the isolation unit indicates that containment protocols may not have been fully adhered to.Detection of up to 1.1×105 cp/cm2 as well as the successful recovery of monkeypox virus from samples with a total of > 106 copies, our findings do not prove that infection can occur from contact with these surfaces. No secondary case in the context of clinical care of the two patients in our study has been observed so far. The study was performed only for two cases and might not be generalised to other cases. In particular, in certain cases, depending on the skin regions mainly affected and the number of lesions, the levels of contamination of different surfaces may vary. The findings in this report are subject to some limitations. As DNA is an environmentally stable molecule, detection of viral DNA by PCR cannot be equated with infectious virus. Despite high contamination with up to 10Overall, these data underscore the importance to remind hospital personnel of the need to follow recommended protection measures for monkeypox. Sufficient time and attention must be given to the careful doffing of PPE and personnel must be properly trained in these procedures. Regular disinfection of frequent hand and skin contact points during the care processes additional to regular room cleaning and surface disinfection using products with at least virucidal activity against enveloped viruses can reduce infectious virus on surfaces and thereby risk of nosocomial transmission . SuitablThose living in the same households of affected individuals should be advised that, in addition to avoiding close physical contact, disinfection of shared skin- and hand-contact surfaces might be useful to prevent transmission ,21. At t


Monkeypox virus has recently emerged from endemic foci in Africa and, since October 20, 2022, more than 73,000 human infections have been reported by the CDC from over 100 countries that historically have not reported monkeypox cases. The detection of virus in skin lesions, blood, semen, and saliva of infected patients with monkeypox infections raises the potential for disease transmission via routes that have not been previously documented, including by blood and plasma transfusions. Methods for protecting the blood supply against the threats of newly emerging disease agents exist and include Pathogen Reduction Technologies (PRT) which utilize photochemical treatment processes to inactivate pathogens in blood while preserving the integrity of plasma and cellular components. Such methods have been employed broadly for over 15 years, but effectiveness of these methods under routine use conditions against monkeypox virus has not been reported.Monkeypox virus (strain USA_2003) was used to inoculate plasma and whole blood units that were then treated with riboflavin and UV light . The infectious titers of monkeypox virus in the samples before and after riboflavin + UV treatment were determined by plaque assay on Vero cells.3 infectious particles per dose, corresponding to greater than 105 DNA copies per mL. Treatment of whole blood and plasma units under standard operating procedures for the Mirasol PRT System resulted in complete inactivation of infectivity to the limits of detection. This is equivalent to a reduction of ≥ 2.86 +/- 0.73 log10 pfu/mL of infectivity in whole blood and ≥ 3.47 +/-0.19 log10 pfu/mL of infectivity in plasma under standard operating conditions for those products.The levels of spiked virus present in whole blood and plasma samples exceeded 10Based on this data and corresponding studies on infectivity in patients with monkeypox infections, use of Mirasol PRT would be expected to significantly reduce the risk of transfusion transmission of monkeypox. Over the past 40 years, the emergence of infectious diseases in human populations has seen a steady increase –3. The rScreening and donor questionnaires have greatly reduced transmission rates of agents that have been identified and classified as significant transfusion risks. These include agents such as HIV, hepatitis B and C viruses, West Nile virus, bacterial contaminants, among others. The safety profile in balance and at times of equilibrium in disease outbreak is high and confidence in these products during such times also remains high. It is during times of outbreak that this equilibrium is thrown out of balance and confidence in the safety and availability of blood products among the public users of these goods and services can be shaken.Not all emerging diseases are blood-borne or transmissible . There iOrthopoxvirus, which include smallpox (Variola) virus and vaccinia virus. Since the eradication of smallpox in 1979, smallpox vaccination was discontinued leading to waning cross-protective immunity in the older population and an increasingly naïve population that threatens human health globally. The most prevalent clinical manifestation of monkeypox virus infections in humans is a smallpox-like rash, often with vesicles, on the skin. Other clinical signs include flu-like symptoms, lymphadenopathy, muscle aches, and headache [Monkeypox virus is a zoonotic, re-emerging virus that is known to have an associated blood viremia in which infectious transfer of disease from donor to host has been demonstrated . The virheadache . The incheadache . It has headache . Yet othheadache . Of mostheadache . Althougheadache –18. SevePathogen reduction of blood products has been routinely practiced for over 15 years in many centers across the world and recently in the United States –22. ThesWe undertook these studies in order to assess the ability and capacity of one such method, Mirasol PRT, to reduce the infectious load of monkeypox virus in spiked whole blood and plasma products under routine operating conditions. The studies were guided by information related to the known titers of virus present in the blood of infected individuals and results are reported in terms of the likely impact that such reduction levels would have on the threat of disease transmission via a transfusion route.All work with monkeypox virus was conducted in a BSL-3 laboratory setting. All human blood products used in this study were obtained via informed consent at an accredited blood banking institution . The protocol for blood collection was approved by the WIRB Institutional Review Board . Informed consent was obtained in writing from all donors prior to donation and was witnessed by blood collection staff at Vitalant. No minors were involved in the study.The use of these blood products at Colorado State University was reviewed by the Colorado State University Institutional Review Board which is part of the Research Integrity and Compliance Review Office (RICRO) at Colorado State University, Office of the Vice President of Research under IRB Tracking Number 19.8726H. The CSU IRB deemed the use of the products under the study protocol “to not meet the requirements of the federal definition of human subject research” due to de-identification of the products and no intended use for diagnosis or treatment of human subjects.Three plasma products were collected at an accredited blood bank and shipped overnight to Colorado State University on dry ice. The products were classified as plasma not for injection, prepared from whole blood products collected in Citrate Phosphate Dextrose (CPD) and frozen prior to shipping to ≤ -20°C.Three non-leukoreduced whole blood (WB) products were collected in Citrate Phosphate Dextrose (CPD) anticoagulant at an accredited blood bank and shipped overnight to Colorado State University at room temperature.Human serum samples for analysis via plaque reduction neutralization test (PRNT) were collected in 2020 prior to the current monkeypox outbreak in the United States. Samples were collected at an accredited blood bank with Institutional Review Board (IRB) approval and all samples were de-identified prior to shipment to CSU. Samples were frozen following collection and shipped to CSU overnight on dry ice.Vero cells (CCL-81) were obtained originally from the International Reagent Resource and frozen stocks prepared. Those stocks screened negative for mycoplasma contamination and were used between total passage level 35 and 41. Virus (strain USA_2003) was amplified in Vero cell culture. Vero cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with glucose, L-glutamine, sodium pyruvate, 10% fetal bovine serum  and penicillin/streptomycin (ATCC). Inoculation of Vero cells with virus was carried out directly in DMEM containing 1% FBS. Medium harvested from infected cells 4 days after inoculation was clarified by centrifugation, supplemented with FBS to 10% and frozen to -80°C in aliquots. All virus titers were measured using a standard agarose overlay plaque assay . ResultsRiboflavin solution  was added to each product, followed by inoculation with 7 mL monkeypox virus, and the bags were placed into the Illuminator  for treatment with UV light per the manufacturer’s instructions as utilized in routine practice in Europe. The device is not approved for use in The United States. The products were treated with 100% of the total recommended light dose, calculated based on the volume of each product (a full treatment consists of exposure to 6.24 J/mL UV light). Average time of treatment for these products was 6 minutes. Samples were removed from each product pre- and post-illumination for viral titer determination via plaque assay.RBC. Average time of treatment for these samples was 53 minutes. Samples were removed from each product pre- and post-illumination for viral titer determination via plaque assay.Each WB product was transferred to an illumination bag per the manufacturer’s instructions for treatment with UV light per the manufacturer’s instructions as utilized in routine practice in Europe. The device is not approved for use in The United States. All solution and disposables were obtained from the manufacturer . Riboflavin solution  was added to each product, followed by inoculation with 10 mL monkeypox virus, and the bags were placed into the illuminator for treatment with UV light -calculated using the measured hematocrit and volume of each product—to a dose of 80 J/mL All pre- and post- illumination samples were serially diluted in sterile phosphate buffered saline (PBS). Plaque assays were performed using Vero cells at confluency in 6-well cell culture plates. Briefly, plates were washed with sterile PBS. All samples were then plated in duplicates at 100 μL per well. Plates were incubated at 37°C for 1 hour with occasional rocking. A 2 mL overlay consisting of 0.5% agarose in minimal essential media  containing 2% FBS and antibiotics was added per well and plates were incubated at 37°C up to 96 hours. The cells were fixed with 10% buffered formalin, followed by the removal of the overlay and staining with 0.2% crystal violet to visualize plaque forming units (pfu). All assays were performed in BSL-3 laboratory setting.The presence of neutralizing antibodies was determined by plaque reduction neutralization test (PRNT). Briefly, serum was first heat-inactivated for 30 minutes at 56°C in a waterbath. Then serum samples were diluted two-fold in DMEM media supplemented with 1% FBS and penicillin/streptomycin starting at a 1:5 dilution on a 96-well plate. An equal volume of monkeypox virus (strain USA_2003) was added to the serum dilutions and the sample-virus mixture was gently mixed. The plates were incubated for 1-hour at 37°C. Following incubation, serum-virus mixtures were plated onto Vero plates as described for viral plaque assays. Antibody titers were recorded as the reciprocal of the highest dilution in which >50% of virus was neutralized.When the post-treatment samples were negative for the presence of virus plaques, the limit of detection (LOD) had been reached. All values at the limit of detection were considered less than or equal to the calculated limit of detection. The theoretical limit of detection and overall log reduction were calculated using the following equations:Descriptive statistics for pathogen reduction are reported, including mean, standard deviation, and number of samples analyzed (N). Sample sizes were determined by sample and product availability and not derived from power calculations.6−4.45 x 106 pfu/mL of virus in the whole blood products and between 2.15 x 106−2.75 x 106 pfu/mL in the plasma products. A slight reduction between expected titer based on dilution of stock titer and the actual titer pre-treatment was observed . Viral titer did decrease after the addition to the products and prior to inactivation. We believe this may have occurred due to blood components that are binding to the virus [The studies performed here utilized whole blood and plasma products spiked with virus at levels of approximately 10he virus . Additiohe virus , 33. HowMultiple factors related to decisions about employing pathogen reduction methods in these cases are at play, including cost-benefit and risk-benefit determinations. Many locations have employed these methods in routine while others have deferred doing so either due to concerns over toxicity, product efficacy following treatment, or overall cost relative to benefit in regions where extensive screening is already taking place for the most common transfusion-transmitted diseases. Nevertheless, diseases are known to jump from animals to humans. The frequency with which this is occurring and the likelihood of further modification of disease behavior through adaptation that comes with highly plastic disease variants that multiply in humans suggests that these occurrences are likely to continue well into our future if not accelerate in pace . How we S1 File(CSV)Click here for additional data file.S2 File(CSV)Click here for additional data file.S3 File(CSV)Click here for additional data file.