Carlavirus, Idaeovirus and Capillovirus and are tentatively named birch carlavirus, birch idaeovirus, and birch capillovirus, respectively. The recently discovered birch leafroll-associated virus was systematically detected by HTS in symptomatic seedlings but not in symptomless ones. The new carlavirus was detected only in one of the three symptomatic seedlings. The novel putative Capillovirus was detected in all seedlings—irrespective of their BLRD status—while the Idaeovirus was identified in a plant without leaf symptoms at the time of sampling. Further efforts are needed to complete Koch’s postulates and to clarify the possible association of the detected viruses with the BLR disease. Our study elucidates the viral population in single birch seedlings and provides a comprehensive overview for the diversities of the viral communities they harbor, to date.To unravel the virome in birch trees of German and Finnish origin exhibiting symptoms of birch leaf-roll disease (BRLD), high-throughput sequencing (HTS) was employed. In total five viruses, among which three were so far unknown, were detected by RNAseq. One to five virus variants were identified in the transcriptome of individual trees. The novel viruses were genetically—fully or partially—characterized, belonging to the genera  The wide application of HTS technologies has significantly facilitated the discovery and characterization of viral agents in woody hosts, including trees, surpassing the limitations of more traditional approaches –3. This Betula sp.), the use of HTS approaches has led to the discovery of a novel virus, birch leafroll-associated virus , in trees from Germany and Finland affected by the birch leaf-roll disease (BLRD) [In birch (e (BLRD) . This die (BLRD) –15 and ce (BLRD) . Due to Carlavirus, Idaeovirus and Capillovirus were identified and novel viruses were—fully or partially—genetically characterized. However, HTS is a qualitative method. Therefore, it is not adequate for attributing causal roles to some of the pathogens identified. To study the etiology of the disease and possible synergistic effects between the identified viral agents, further studies involving infectivity tests with infectious clones in greenhouse experiments will be necessary.In the present study, we aimed to provide additional knowledge on viruses in birches, as well as to investigate the composition of the virome of single trees. The exhaustive collection of nucleic acid sequences deriving from viral agents from five birch seedlings of German and Finnish origin are described. A complex of known and novel viruses and of diverse variants of those agents was found to infect the tested samples. Apart from the already described viruses, novel viral sequences from the genera Betula pubescens donor tree (Bpub3) with severe BLRD leaf symptoms  from Rovaniemi (Finland) were grafted on two non-symptomatic B. pubescens rootstocks, generating grafted seedlings BpubFinn407501_3A and BpubFinn407507_3I. One twig originating from a B. pendula tree (Bpen 5) from Berlin, Germany, which was also exhibiting BLRD symptoms, was grafted on a non-symptomatic B. pendula rootstock, generating grafted seedling BpenGer407526_5M. One twig originating from the B. pendula tree MO197 from Berlin, Germany, not exhibiting symptoms at the time of the grafting, was grafted on a non-symptomatic B. pendula rootstock, generating the grafted seedling BpenGerM0197542. One CLRV-negative and symptomless birch seedling, the B. pubescens (BpubGer4), obtained from the same German nursery , was used as a negative control. The following growing season, symptoms similar to the ones exhibited by the donor trees could be observed on three grafted birches already at the beginning of May and developed further until the end of September  long for the carlavirus, 533 nt for the idaeovirus and 420 nt for the capillovirus. PCR products were directly submitted for Sanger sequencing (Macrogen) without previous cloning.Pooled samples of three to five leaves from different twigs of each seedling were used. The first-strand cDNAs were synthesized from 1 μg of total RNA in a 20 μl reaction volume of 1 x RT buffer (Thermo Scientific) containing 1 μM dNTPs mix, 200 U RevertAid Premium reverse transcriptase (Thermo Scientific), 20 U Ribolock RNase inhibitor (Thermo Scientific) and 100 pmol of random hexamer-oligonucleotides (Biomers.net GmbH). Subsequent PCR amplifications were conducted in a 25 μl volume of 1 x DreamTaq Buffer (Thermo Scientific) containing 0.2 μM dNTP mix, 0.625 U of DreamTaq DNA polymerase and 1 μM of each forward and reverse primer . The theAssuming a dsRNA stage of the tentative carlavirus, 5’ and 3’ ends of the genome were determined using a 5’ Rapid amplification of cDNA-ends (5’ RACE) strategy, and a polyA-anchored Long Distance-RT-PCR, respectively. The 5’ RACE reaction was performed according to the kit manufacturer’s instructions   and Opisthokonta (Fungi and Vertebrata)  and specifically, to two variants of this virus , while 2.835 reads are analyzed as representing agent(s) in the family Betaflexiviridae, with affinities to helleborus net necrosis virus (2.826 reads) and apple stem grooving virus (9 reads). The presence of 426 reads from Human mastadenovirus E (dsDNA viruses) is attributed to possible contamination of the sample during sample handlings or sequencing .The results obtained by MEGAN analysis regarding the taxonomic content of contigs assembled from the RNA-Seq reads are shown in tebrata) . From thsee ref.  for detaB. pubescens samples BpubFinn407501_3A and BpubFinn407507_3I are both found to be infected by the badnavirus BLRaV, respresented by a high number of reads . RNA2 is 6,459 bp-long and exhibits a lower level of identity with the birch CLRV isolate , and is similar to the identity level observed with the cherry CLRV isolate . The genomic sequences of this new CLRV variant have been deposited in GenBank under accession numbers MK402281  and MK402282 .CLRV was detected by HTS in only one of the tested symptomatic birches, the B. pendula pollen RNA-Seq dataset (SRX2768860).In the dataset from the seedling BpenGerMO197542, two long contigs of an uncharacterized virus with affinities to idaeoviruses were assembled see . The firIn August 2015, virus-like symptoms were detected on seedling BpenGerMO197542 . White-yBetaflexiviridae, Capillovirus, From the RNA-seq dataset of the BpenGer407526_5M seedling, an 821 bp-long contig was assembled, which exhibits identities with apple stem grooving virus .As the encoded proteins of the new viral sequence show less than 80% aa identity with CP sequences from other capilloviruses, they are suggested to belong to a novel species of the genus es clade . In concThe presence of the capillovirus sequence was confirmed by RT-PCR not only in the seedling Bpen5MGer407526_5M from which it originated, but also, in all four other seedlings analyzed here (symptomatic and not symptomatic) and in other trees from Berlin and Rovaniemi .Carlavirus (Betaflexiviridae) -PCR and 5’ RACE enabled the completion of the genome by generating sequences that perfectly matched the contig in the overlap regions. The presence of the virus was confirmed by specific RT-PCR performed in the seedling in which it was first detected in tree BpenGer407526_5M and in other trees in Berlin . The fulThe genome of this virus is 8,896 base pairs (bp) long, which is similar to the genome size of typical carlaviruses  . It showCarlavirus and it is tentatively named birch carlavirus (BiCV).Phylogenetic relationships between the birch carlavirus and the sequences of carlaviruses known to date were estimated based on amino acid sequences comparisons. The topology of the trees was similar, irrespective of whether the ML or NJ algorithms were used. B. pubescens seedlings, the virome is less complex; in BpubFinn407501_3A, the presence of a BLRaV and a BCV variant was shown , an isolate of the newly discovered birch carlavirus (BiCV), as well as a capillovirus sequence (BCV) . In the as shown , while ias shown . The virCherry leaf roll virus (CLRV) as the only BLRD causal agent [Based on these findings, new scenarios for the disease etiology can be proposed. The initial hypothesis implicating al agent , 15 is nApple mosaic virus) and BLRaV in birches was observed in urban landscapes in Berlin, with an incidence in symptomatic leaves of around 29% within three years of investigation. These data indicate that mixed infections in birch are widespread. It is not easy to establish a correlation between such viral complexes and the appearance of symptoms or to differentiate symptomatology in cases of infection by a single virus or by two virus species [The use of HTS has revealed that mixed infections are frequent in birch, involving different viruses. The complex virome observed in the tested birch seedlings has been repeatedly observed in more birches in Berlin in other studies. In an investigation of viruses’ distribution in urban parks in Berlin for two consecutive years, BiCV was present in 16% of the tested birches . In five species . In cont species —or to otIn light of a holistic understanding of the disease pathogenesis, the “pathobiome” concept has been developed, which represents the pathogenic agents integrated within its biotic environment . UnderstProteobacteria, Terrabacteria, FCB Group bacteria), unclassified Totiviruses, as well as thousands of unassigned reads, are part of the birch metagenome. Our findings should be examined under the holistic view of the “hologenome theory” [Apart from the virome that is described in detail here, attention should also be given to the remaining microorganisms detected in the samples. Bacterial species  are reported. In comparison to cultivated plants, few measures have been taken regarding knowledge on viruses present in forest ecosystems. Missing data or unawareness concerning viral incidence in forests may lead to unjustified disease diagnosis and determination of the causal agent. It is not only birch that suffers from viral diseases, as virus-like symptoms are commonly observed in Fraxinus sp. , in Quercus sp. , in Ulmus sp. , in Acer sp. (Germany), in Populus sp. , and in Sorbus sp.  [To our knowledge, it is the first time that metagenome data of a forest tree species ( Europe) . Based o Europe)  and futuS1 FigThe trees were reconstructed using the Maximum Likelihood method and the statistical significance of branches was evaluated by bootstrap analysis . Only bootstrap values above 70% are indicated. The scale bar represents 10% amino acid divergence.(TIF)Click here for additional data file.


We screened nonequine animals with unexplained neurologic signs or death in South Africa during 2010–2018 for Shuni virus (SHUV). SHUV was detected in 3.3% of wildlife, 1.1% of domestic, and 2.0% of avian species. Seropositivity was also demonstrated in wildlife. These results suggest a range of possible SHUV hosts in Africa. Peribunyaviridae: Orthobunyavirus) was isolated in the 1960s from livestock, Culicoides midges, and a febrile child in Nigeria (Shuni virus (SHUV) (https://www.qiagen.com), according to the manufacturer’s recommendations. We screened all samples by a SHUV real-time reverse transcription PCR (rRT-PCR) (https://www.inqababiotec.co.za) and phylogenetic analyses  with unexplained neurologic or febrile disease or sudden unexplained death from across South Africa were submitted to the Zoonotic Arbo and Respiratory Virus Program, Centre for Viral Zoonoses, University of Pretoria  as part of a passive zoonotic arbovirus surveillance program. We extracted RNA under Biosafety Level (BSL) 3 conditions using the QIAamp viral RNA mini kit (blood) or the RNeasy mini kit (tissue)  . We confanalyses . We alsoSyncerus caffer) (n = 45) and white rhinoceros (Ceratotherium simum) (n = 48) from Kruger National Park were collected in March 2014 and June 2016, respectively, by South African National Parks and from wild Nile crocodiles (Crocodylus niloticus) (n = 34) from northern KwaZulu-Natal collected during 2009–2012 by the Faculty of Veterinary Sciences, University of Pretoria, as part of surveillance studies. We examined tissue samples from a SHUV PCR-confirmed positive buffalo (MVA43/10) microscopically under a light microscope using routinely prepared hematoxylin and eosin stained . We excluded animals that were found dead, aborted, or stillborn from OR analysis.We calculated odds ratios (OR) and 95% CI in EpiInfo version 7.2.0.1 (Theileria sp. sable and Theileria separate (We detected SHUV RNA in 15/608 (2.5%) animals tested from 10 different animal species: 12/361 (3.3%) wildlife, 2/196 (1.0%) nonequine domestic animals, and 1/51 avian species (2.0%) . We deteseparate .In 9/15  positive infections, we detected SHUV in the central nervous system (CNS) , indicatPositivity of infection was highest in the North West , followed by Limpopo Province   Figure 1Necropsy examination on the buffalo showed no specific macroscopic lesions on histopathology examination of brain tissue . PatholoWe used phylogenetic analyses on the small segment of the Simbu serogroup to verify the molecular results. All novel sequences from this study, with 1 exception, were closely related to SHUV strains identified in horses in South Africa . An isolWe detected antibodies to SHUV by an eb-ELISA in 3/44 (6.8%) African buffalo and 2/48 (4.2%) white rhinoceroses but none in crocodiles. SHUV-specific IgG was confirmed, using microtiter virus neutralization assay, in 1 buffalo and 1 rhinoceros. Two of 3 buffalo and 1 rhinoceros positive for SHUV epitope antibodies were negative by microtiter virus neutralization assay, suggesting that these antibodies may have been elicited in response to closely related orthobunyavirus. Confirmation for the third buffalo was not possible because of depleted serum.Our findings suggest that SHUV may have a wide host range, including several wildlife and domestic species, and should be included in the differential diagnosis of neurologic disease in animals. This study highlights the role of this virus as a potential emerging zoonotic pathogen in Africa that warrants increased surveillance and further investigation. Future epidemiologic studies would benefit from an increased sample size and more extensive serosurveys. Investigation of human infections may define SHUV’s importance as a zoonosis. The causative link between clinical manifestations in the various species and the evidence of SHUV infection must be regarded with caution because other possible infectious and noninfectious etiologies were not excluded by comprehensive investigations in all cases.Additional information about the study of Shuni virus in wildlife and nonequine domestic animals, South Africa.


Rapid urbanization, global trade, and the exceptionally great numbers of worldwide visitors during Hajj and Umrah have all placed the Kingdom of Saudi Arabia at a significant risk of introducing several vector-borne tropical diseases, such as dengue fever virus (DENV) infection. In this study we estimated DENV infection cost of illness (COI) in Saudi Arabia in the period 2013–2017, by processing national data including all declared cases recorded in referral centers in the western region, being the endemic region of the country. Using a statistically validated predictive model that was built on a representative sample of 717 laboratory-confirmed cases of DENV infection, direct costs, due to care-related expenditures, were estimated by applying the predictive equation to national data. However, indirect costs, which are due to productivity loss, were estimated using the human capital model based on gross domestic product adjusted for invalidity duration. Further, under-reporting was adjusted by using an expansion factor EF = 3. We observed highest estimated costs in 2016 with over US$168.5 Million total costs, including direct (US$29.0 Million) and indirect (US$139.5 Million) costs, for a total 4415 confirmed cases. The total DENV COI for the five years was estimated as US$551.0 Million for a total 15,369 patients (59.7%) out of 25,745 declared cases, resulting in an average cost of US$11 947.6 by patient. Depending on the year, productivity years loss costs accounted for 63.3% to 83.8% of the estimated total costs. Dengue has a substantial local economic burden that costs US$110.2 Million per year, stressing the urgent need for an effective national prevention strategy to perform considerable cost-savings besides reducing morbidity. The global incidence of DENV infection has evidenced a dramatic increase in the recent two decades with a great number of cases that are misclassified or underreported. These epidemiological characteristics generate high economic costs, especially in endemic regions and countries such as Saudi Arabia. This two-phase study aimed at providing economic data that helps political efficiency and resource prioritization for dengue prevention programs, by assessing the economic burden of disease over the last five years 2013–2017. A double-method used to estimate direct costs due to care expenses and indirect costs due to productivity loss, by using a predictive and an economic model, respectively. Assuming an expansion factor of 3 to correct under-reporting, the average estimated costs of dengue illness per year in the current study was US$117.87 million. Comparison of these findings with international reports emphasized the substantial disease burden of dengue fever in Saudi Arabia. Despite some limitations, this study provided the first economic data of dengue fever infection burden in Saudi Arabia. Vector-borne diseases constinue to be a major global problem, causing a significant health economic burden notably in the Eastern Mediterranean Refgions (EMR) of the World Health Organization (WHO) . The gloAedes aegypti, the locally identified vector of the disease ), Bakhsh Hospital (private), Al Ahli Hospital (private). All these centers had a significant flow of patients who were registered as confirmed cases of DF during the study period.The initial design consisted of a two-phase method for data collection and analysis, namely:Phase I: calculation of actual direct and indirect costs on the representative sample of patients (Source Database);Phase II: estimation of direct and indirect costs on national data for the years 2013–2017 (5 datasets), which was provided by the Public Health Department, at Jeddah, and is available at the Dengue Fever Operation Room Database, covering all governmental and private hospitals in Jeddah district (Target Database).Fig 1) and described separately below.However, estimation of indirect costs could not be done following this method due to substantial absence of relevant data in both Source and Target Databases. Thus, only direct costs were estimated using the two-phase Bottom-Up method; while the estimation of indirect costs used an alternative method, namely the Human Capital Model. The two methods are illustrated summarily in a flowchart , who were diagnosed and treated in 2016 in one of the selected centers (N = 717). Cases with no laboratory confirmation were excluded.Collected data included four types of variables: 1) all health interventions  provided for the patient: these data were collected by reviewing the medical records in the participating hospitals; 2) the number of units or episodes for each intervention  for each patient over the management period; 3) tariffs applied for each intervention, with respect to the prices applied in each center: these data were provided by the economic officers from each hospital and completed by reviewing the Saudi Food and Drug Authority (SFDA) list of medications costs ; 4) sociDirect costs of the sample (N = 717), which represent the outcome of Phase I, were calculated for each patient by summing up the unitary costs of each intervention multiplied by the number of episodes . Specific assumptions were considered for the calculation of emergency room consultations costs ; fluid costs ; antipyretics ; discharge medication (assumed to be 2-day prescription of paracetamol and omeprazole). According to the type of intervention, direct costs were divided into 4 sub-categories: a) Admissions costs ; b) Investigations costs (imaging and laboratory workups); c) Care costs  Past Direct Costs, corresponding to any care costs incurred prior to admission and diagnosis. Total direct costs were calculated as the sum of all intervention costs and was analyzed as the dependent variable in Phase II. Data management, processing and calculations in Phase I were done using Microsoft Excel 2013 for Windows.This Phase consisted in building the predictive model for direct costs of dengue, from the representative sample (N = 717), as a function of the relevant demographic and clinical explanatory variables including gender, age, marital status, nationality, hospital type, and severity level. This approach is based on the assumption that direct costs are independently associated with various clinical and demographic factors.Using R Package, version 3.6.1 , univariate and multivariate Ordinary Least Squares (OLS) regression models were carried out to predict Direct Costs, the dependent variable, as a function of the abovementioned demographic and clinical factors. The level of statistical significance was fixed as p<0.05. The dependent variable, Direct Costs, was not normally distributed; therefore, it underwent Box-Cox transformation using a lambda = 0.1, as it was the optimal lambda value for the equation, and one that enabled the greatest Log-Likelihood. The normality of the Box-Cox transformed variable was tested by the creation of a histogram for the residuals, which indicated that the residuals are normally distributed. The OLS model using Box-Cox transformed Direct Costs was tested for other basic assumptions including linearity, homoskedasticity and inter-independence of the explanatory variables. All assumptions were fulfilled, except homoskedasticity that was violated, with Breusch-Pagan test  and NCV test , indicating heteroskedasticity of the model. Consequently, robust regression was carried out using Box-Cox transformed Direct Costs as the dependent variable and the aforementioned demographic and clinical factors as the explanatory variables. Two robust regression models were built. The first model (Robust Model 1) included all six explanatory variables and showed significance for only disease severity and hospital type. The second model (Robust Model 2) included only disease severity and hospital type, and both were significant. Both Robust Models 1 and 2 were tested on the Source Database and their respective mean absolute errors, mean square errors (MSEs), and Root MSEs (RMSEs) were analyzed. These showed that Robust Model 1 had a lower MAE (10466.4) compared with Robust Model 2 (10477.2), indicating better performance of Robust Model 1 in terms of absolute error. However, Robust Model 2 showed lower MSE (271991845.5 versus 278610358.6) and RMSE (16492.2 versus 16691.6), indicating higher accuracy, in terms of error metrics, of Robust Model 2 than Robust Model 1 respectively. Consequently, Robust Model 2 including severity level and hospital type as predictors, was used to predict Box-Cox transformed Direct Costs. The robust regression equation formula with the estimated parameters are presented in the results section. The predictive model was applied on Target database  for each patient, and Estimated Direct Costs (EDC) were calculated by back-transforming Box-Cox Direct Costs using a specific formula see  section.Table 1). Further, the model considers caregiver DALS, assuming the participation of a third person  to provide care, assistance, and/or hospital visits to the patient, which results in absenteeism that is estimated as half the DALS of the patient. Specific assumptions were used for estimating DALS of unemployed patients including housewives, jobseekers, retired individuals and under school-age children. Subsequently, indirect costs were calculated per patient by adjusting the GDP per capita for the number of DALS (both patient’s and caregivers if applicable); and in case of death, as the sum of per capita GDP per PYLS. Total estimated indirect costs (EIC) of dengue were calculated for each year (2013–2017) using the equation: EIC = patient DALS cost + caregiver DALS cost + PYL cost. Data management, processing and calculations were done using Microsoft Excel 2013 for Windows and SPSS version 21.0 for Windows .This model is based on the estimation, on the Target Databases (2013–2017), of the disability days costs, which depend on both productivity days lost and GDP per capita. Productivity days loss is divided into days of activity loss (DALS) and productivity years loss (PYLS). Both DALS and PYLS were estimated using a systematic model by Shepard et al.  as descrEstimated total costs (ETC) were computed as: ETC = EDC+EICWhere,EDC: are the direct costs as estimated using the predictive modelEIC: are the indirect costs as estimated using the Human Capital ModelResults were presented as the yearly (2013–2017) and total EDC, EIC and ETC. The average cost by patient (ACP) was calculated as: ACP = ETC/N, where N is the number of confirmed cases in the given year. Costs were expressed in the local currency  as well as in US$.Both Source and Target databases were initially managed for missing data by using appropriate imputation methods available on the Statistical Package for Social Sciences version 21.0 for Windows . Missing data were of two types: missing observations for a given variable, or missing variable (unobserved variable).Missing observations concerned both Source and Target databases and included age, gender, professional status, nationality, severity level and hospital type. These were imputed using multiple regression imputations to predict the missing values for each variable as a function of the other relevant variables , and with respect of the local demographic figures . Multiple imputations were ran and analyzed, and those with figures that were inconsistent with the local figures were excluded. For numerical variables like age, where <5% data was missing, mean substitution was used after adjusting for other variables; e.g. for a single non-Saudi male with missing age data, the mean age of single Saudi males in the sample was attributed.Unobserved variables concerned only Target Databases. These included treatment modality and mortality; both variables were unoserved in the national data (Target Databases). Imputation was conducted in two steps. First, each Target Database (2013–2017) was merged individually with the Source Database (N = 717), which was used as a reference dataset for the missing variables. This step was based on the assumption that populations from source (N = 717) and target datasets (2013–2017) are comparable, and thus can be analyzed in a combined dataset. Second, unobserved variables from Target Databases were treated as missing observations and imputed by using multiple or logistic regression imputation, as appropriate, as a function of relevant independent variables from the combined database. Five imputations were performed for each variable and for each combined dataset, resulting in five imputed datasets for each Target Database (2013–2017). Imputed datasets of each year were carefully analyzed, and those which showed the best consistency with the actual epidemiological figures, by reference to source database and clinical sense, were considered valid. Imputations were rejected if they were clearly inconsistent with the Source dataset with respect of the following figures: total percentage of hospitalized patients (64.0%); mortality rate ; hospitalization and mortality rates by severity level . Valid datasets (3 for each year) were used to estimate Indirect Costs by applying the Human Capital Model, and the final Indirect Costs were calculated as the average of the 3 valid estimates for each year. As to Direct Costs, they were not affected by mortality and treatment modality, as none of the two variables was included in the predictive model for Direct Costs.Based on literature, an expansion factor of 3 was assumed to correct under-reporting, which corresponds to the lowest borderline of the EF range in studies from Southeast Asia and Latin America –39.Table 2).Demographic data of the confirmed cases of DF were characterized by remarkable male predominance  and relatively young age (mean [SD] age = 24.70 [15.86] years), and 55.5% were national citizens. Majority of cases were D.WS-; while 16.7% were D.WS+ and 1.1% were severe dengue entailing 63.9% of hospitalizations. Mortality rate was 0.7% . Management of the 717 cases occasioned 506 outpatient visits, 1875 days of hospitalization, and 472 days of emergency room occupation , 1875 days of hospitalization , and 472 days of ER ; thus, the total costs related to admissions were 1,749,688 SAR with an average 2,440.3 SAR by patient. Total cost of investigations was 5,321,530.5 SAR , of which 636,899.9 SAR for imaging and 4,684,630.6 for blood testing. The total care costs were 6,064,008.5 SAR , of which 4,917,430.7 were fluids and electrolytes . Consequently, direct costs were calculated as 13,214,717.00 SAR . Health consumption and the related costs are detailed in Table 4.The first robust regression model, Robust Model 1, which included all six demographic and clinical factors, showed only hospital type and severity level as significant predictors of Box-Cox transformed Direct Costs. The second model, Robust Model 2, was carried out including the two significant variables. Both models are presented in According to the predictive model above, the Box-Cox transformed Direct Costs was estimated using the equation:Where,13.62: model intercept value of Box-Cox transformed Direct Costsai: effect of hospital type on Box-Cox transformed direct costs bj: effect of severity level on Log-transformed direct costs The Estimated Direct Costs (EDC) was obtained by back transformation of Box-Cox transformed Direct Costs using the following formula:Where Lambda is the Box-Cox transformation factor = 0.1.Table 5. These include total percentage of hospitalizations, mortality rate, and distributions of both hospitalization and mortality rates by severity level, by reference to source dataset (N = 717). These two variables are relevant for the estimation of EIC using the Human Capital Model. In general, all imputations provided total percentage of hospitalizations that is consistent with Source Database and acceptable ranges of mortality rates by respect of the 95% confidence interval of mortality  in source dataset. Inconsistencies lied principally in the distribution of hospitalizations and or mortality rate by severity level, upon which the decision of retention or rejection of the given imputation was based. Note that the high variability of mortality rates in severe dengue in some imputations is due to a small number of cases; thus, imputations with high mortality rates among severe dengue cases  were considered valid.Key figures of the 5 imputations of mortality and treatment modality by severity level for each Target Database (2013–2017) are depicted in Table 6. Additionally, the 3 imputation-based calculations of indirect costs using the economic model based on GDP per capita are depicted by year and by imputation in S1 Fig in US$; whereas the average values of the three imputations are presented in Table 6. Fig 2 depicts: (a) the EDC and EIC by year; and (b) the EIC broken up by DALS, care giver DALS (CG-DALS) and PYLS. Note that, unlike EIC, EDC do not vary by the presented imputation, as EDC did not depend on the imputed variables as per the predictive model.By processing imputed variables on 2013–2017 national databases, Direct Costs were calculated using the predictive model developed in Phase II analysis and Indirect Costs were calculated using the GDP-based model ; final results of EDC and EIC with ETC in Saudi Riyal and US$ are presented in Fig 2A), and, outstandingly, PYLS represented the major proportion of EIC . Thus, PYLS accounted for 63.3% to 83.8% of the estimated total costs, depending on the year.We observed highest ETC in 2016 (210 669 789.1 SAR = 56 178 610.4 US$) for a total 4415 confirmed cases (61.2%) out of a total 7219 suspected ones; while the lowest ETC was observed in 2014 (61 726 236.5 SAR = 16 460 329.7 US$) corresponding to 1524 confirmed cases (34.9% of the 4361 suspected one). The total dengue costs for the five-year period was estimated as 688 582 292.7SAR (US$183 621 944.7), for a total 15,369 patients out of 25,745 suspected cases, resulting in an average cost of 44 803.3 SAR (US$ 11 947.6) by patient. Remarkably, EIC were substantially higher than EDC .Under-reporting was assumed as follows: for every reported dengue case, there are 3 actual dengue cases ; thus, underreporting was corrected by multiplying by expansion factors (EF3) to scale reported cases. Results of costs after under-reporting correction are presented in This is the first study investigating the economic burden of dengue fever infection in Saudi Arabia, based on local data. Its findings emphasized the substantial economic burden of dengue fever in Jeddah and surrounding areas, representing the endemic center of the country and weighing heavily on the national economy. Dengue fever costs were divided into direct and indirect costs, each analyzed using a specific method. For direct costs, we employed a bottom-up approach based on data collected from a representative sample of laboratory-confirmed cases of dengue fever from the highest endemic region in Saudi Arabia during 2016. Direct costs of the sample were modeled as a function of predictors and the predictive model was applied on national data to estimate the respective direct costs. For indirect costs we employed the Human Capital Method, which is an economic model that is used in most of COI studies and is based on the estimation of productivity lost costs by adjusting the GDP per capita ,40..In general, three major methods are used to estimate indirect costs in the literature, including the willingness to pay method (WTP), friction cost method (FCM), and human capital method (HCM). In the WTP, the amount which an individual is eager to pay to alleviate the burden of an illness or mortality is quantified. The FCM relies on the productivity lost due to time required to restore the original production level. In such a method, the productivity losses are probably replaceable and thus the main losses are limited to a short period, during which a sick employee could be replaced. FCM is a conservative method because job absenteeism or death are the major determinants of productivity loss. In HCM, the person is viewed as a capital investment and can be valued by his/her societal economic contribution. Therefore, in cases of dengue fever, the temporary productivity loss is estimated as the discounted present value of future earnings expected over the course of the infection, considering the assumption that future earnings are used as a proxy for future productivity Although both methods used in this study are statistically and methodologically valid, it is plausible that relying on multiple sources of data would provide more accurate national estimates. Furthermore, besides the results of estimated costs that are discussed in the following sections, data processing sheds light on the considerable lack of economic data with a questionable quality of the available data, which made the cost analysis laborious and yielded some methodological limitations. This denotes the need to integrate effective data collection and management procedures as part of disease surveillance and control strategies.For the total 15,369 patients included over the 5 years in the current study, the estimated yearly cost of dengue illness was US$110.17 million after assuming an EF of 3. Considering US dollar inflation rates in 2018 , our annThe productivity loss contributes to the largest proportion of total costs in several other COI studies, accounting for 50–60% of the total costs ,30,43, wTherefore, it is acceptable that our ETC estimate and EF are conservative, probably considering three major factors. First, disease estimates in the current study reflect the patients with a symptomatic dengue in its acute phase, which potentially affects the quality of life (QOL) of patients; however, chronic fatigue can occur post-dengue infection, especially in females, elders and patients having acute phase chills and may persist from 2 months to 2 years in 8.5%-57% of the patients –52. Thes. The degrees and magnitudes of variation of COI should be evaluated in multiple facilities of the same type as suggested previously [The variations in cost estimation may be affected by several factors. First, the discrepancy of costs between hospital types as indicated in the current study. More specifically, hospital type had a significant effect on the cumulative changes in EDC over the study period and was a significant predictor of direct costs of dengue. Similarly, in Thailand, there was a significant difference in the COI between large referral centers and community or provisional hospitals eviously . Second,eviously ,57. Howeeviously , while reviously .Dengue vaccination and dengue control programs may offer a cost-effective approach, with a significant disparity among developing and developed countries. In the early 1990s, Shepard and Halstead  found thIn light of these findings, it is imperative to assess the efficacy and cost-effectiveness of dengue vaccination campaigns in Saudi Arabia, besides other preventive and surveillance programs, using local epidemiological and economic indicators, with the aim of reducing the substantial health and economic burden of the disease –65.In this study, we evidenced some limitations that might affect the interpretation of the outcomes. Inclusion of patients in a hospital setting indicates that they experienced relatively moderate to severe symptoms and, therefore, a proportion of mildly symptomatic patients may have remained undiagnosed and escaped inclusion. Thus, despite the correction factor (EF = 3), the included sample might be unrepresentative of the whole population. Furthermore, the used EF was based on existing literature rather than real evidence from Saudi Arabia. Applying a constant EF from non-local studies would possibly be problematic given that EF may differ according to the intensity of transmission , transmiThe method used for indirect costs estimation is limited by the assumptions regarding DALS due to absenteeism, which may be over-estimated especially in non-severe cases. On the other hand, this method was limited by the multiple imputations required to correct missing variables. This urges the implementation of reliable data collection and management systems to enable more accurate estimations.Drivers of higher costs, such as prolonged disabling symptoms and effects on tourism and other economic sectors that might be associated with dengue epidemics were not considered in our statistical analysis. In addition, the inability to include costs of vaccination and vector control strategies (as they were beyond the objectives of the study) would eventually add to the limiting factors.Evidently, there is a need to conduct future local studies, on a larger scale, that entail a multiregional methodological approach to provide robust national evidence. This is to overcome the potential bias that may emerge from estimating the economic burden in an endemic region like Jeddah, along with the existence of substantial spatial differences across the Kingdom.In general, future studies should be based on standardized monetary units to facilitate the comparisons between countries of different purchasing powers and the outcomes should be analyzed considering the financial structure of local healthcare systems. It is also recommended to combine medical costs with costs related to reduced QOL, tourism estimates, and vector control expenses. A comprehensive analysis of the economic aspects in the private sector is needed to help guide decision-making and policy-making, particularly in terms of employing vaccine campaigns and the development of antiviral drugs. Estimates of productivity loss could be derived via the human capital approach rather than self-reported information that can be subject to recall bias. However, more accurate assumptions are needed for cost estimations for those working in both the formal and informal sectors. Another crucial element that should be taken in mind is quantifying the cyclical variations to capture the common patterns of disease transmission to reveal accurate cost estimates during the endemic and epidemic times. This could be partially done via collecting data over multiple years.Importantly, intensified vector control should be a critical component of health sector planning in Saudi Arabia, particularly in light of mass gatherings during Hajj and Umrah, primarily constituted from populations from Southeast and South Asia, and the possible transmission of dengue and other vector-borne neglected tropical diseases –71. The Finally, it is essential to establish future comparative analyses of the cost-effectiveness of a dengue vaccine. This should include avoiding the inherent difficulties implied by such studies as per experts’ recommendations, including the application of well-defined and cited assumptions, considering the vetted costs of vaccination and its possible side effects as well as surveillance programs aiming to monitor the safety of new vaccines, utilizing natural units to report cost-effectiveness , and the estimated effects on mortality since policymakers may wrongfully expect a high mortality.Rapid urbanization, global trade, and the exceptionally great numbers of worldwide visitors during Hajj and Umrah have all placed the Kingdom of Saudi Arabia at a significant risk of introduction of several vector-borne tropical diseases. Of them, dengue fever has been established as an endemic disease for two decades and this could be emphasized in the current study. Dengue has a substantial local economic burden that costs US$110.17 million per year resulting in an average cost of US$11 947.6 per patient. Costs were highly disparate across the different types of healthcare institutions and productivity years loss accounted for approximately 80% of the total costs.These estimates, as with other global estimates, are subject to underestimation given several factors, including the use of unstandardized expansion factors, likelihood of exclusion of the mildly-affected cases, underestimation of other symptoms and chronic complications that have a prolonged economic impact, and exclusion of vector control and other preventive activities from the analysis. In light of study limitations, there is a need to conduct future studies employing a multiregional approach, using standardized monetary units¸ quantifying cyclical patterns of the vector and health burden, and investigating the cost-effectiveness of a dengue vaccine.S1 Fig(TIF)Click here for additional data file.


The World Health Organization (WHO) recommends that governments scale up testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.Costs of molecular tests, including costs of diagnostic kits, analysis equipment and human resources, may be regarded as an important barrier for scaling up SARS-CoV-2 testing. However, large-scale testing may result in earlier detection of cases, including asymptomatic ones, and thus prevent many new infections and avert hospitalizations. Therefore, financial costs associated with an increased number of tests may in some scenarios be offset by savings in future hospitalization costs. The number of prevented hospitalizations, however, does not simply depend on the number of performed tests. If testing is scaled up, a decrease in the percentage of positive results will ensue, although we cannot predict to which extent, as we are unable to know the number of undetected cases. a(1+r)t where a corresponds to the additional cases detected by the increased frequency of testing, r to the infection growth rate and t to the number of days over which the user wishes to project results. Additional required inputs include the number of tested patients, the number of confirmed infection cases, the frequency of hospitalized patients, and the costs of hospitalization and tests. As an example, the tool displays predefined scenarios for different countries in the WHO European Region and the Region of the Americas, along with the corresponding data sources. To address this challenge, we developed an online tool,This tool only considers direct costs of testing and hospitalizations, which vary between countries. Prevention of new infections, after testing is scaled up, is expected to prompt additional savings in indirect costs, including decreased productivity losses subsequent to hospital admissions or quarantines. Importantly, less stress on national health systems would allow patients with other diseases to be adequately treated. Since hospitals are being directed to treat patients with COVID-19, some diagnoses and treatments are being cancelled or postponed.Costs should not be the sole or main reason behind decisions in health care. Nevertheless, data showing that increasing SARS-CoV-2 testing may prompt savings for health-care services, may also persuade decision-makers to scale up testing in a fast and sustained way, and thus meet the recommendation of WHO to increase testing.


Plums are a kind of stone fruit, a category that includes peaches, cherries, apricots, and almonds. In Korea, Japanese plum trees are usually cultivated as they best suit the climate. To date, there have been few studies in Korea on viruses infecting plum trees compared to those infecting peach trees.To identify viruses and viroids infecting plum trees, we collected leaf samples from six different plum cultivars and subjected them to RNA-sequencing (RNA-seq). Six different plum transcriptomes were de novo assembled using the Trinity assembler followed by BLAST searching against a viral reference database.We identified hop stunt viroid (HSVd) and six viruses, including apple chlorotic leaf spot virus (ACLSV), little cherry virus-1 (LChV-1), peach virus D (PeVD), peach leaf pitting-associated virus (PLPaV), plum bark necrosis stem pitting-associated virus (PBNSPaV), and prunus necrotic ringspot virus (PNRSV), from six plum cultivars by RNA-seq. RT-PCR confirmed the infection of HSVd and three viruses—ACLSV, PBNSPaV, and PNRSV—in plum trees. However, RT-PCR demonstrated that plum trees in this study were not infected by LChV-1, PeVD, or PLPaV. It is likely that the three viruses LChV-1, PeVD, and PLPaV as identified by RNA-seq were contaminants from other peach libraries caused by index misassignment, which suggests that careful confirmation by other methods should be carried out in next-generation sequencing (NGS)-based virus identification. Taken together, we identified a viroid and three viruses infecting plum trees in Korea. Prunus domestica) , are widely grown   and the ly grown .Plum pox virus (PPV) in the genus Potyvirus causing Sharka disease is the most destructive (Plum bark necrosis stem pitting-associated virus (PBNSPaV) in the genus Ampelovirus (Hop stunt viroid (HSVd) in the genus Hostuviroid are known to infect plum trees -based approaches are widely used in the identification of viruses infecting plants . NGS tecFor NGS, plant leaf samples with obvious symptoms are harvested and subjected to nucleic acid extraction and libraries are prepared from extracted nucleic acids. The most frequently used methods for library preparation are double-stranded (ds) RNA library, mRNA library, ribosome-depleted RNA library, and small RNA library. Two recent studies compared different viral sequence enrichment methods, suggesting that the yield of viral sequences by NGS was dependent on viral genome organizations and the number of viral reads in the data .goya or oyat are grown in Korea, their fruits are relatively smaller with diverse fruit colors. It seems that the native plum species in Korea might be propagated by seedlings.Japanese plum trees are usually cultivated in Korea as they best suit the climate. Most plum cultivars in Korea have been derived from Japan or other countries. Although several native plum species called Prunus necrotic ringspot virus (PNRSV) in the genus Ilarvirus and Apple chlorotic leaf spot virus (ACLSV) in the genus Trichovirus, and HSVd infecting Japanese plum trees have been reported in Korea (Prunus species by RNA-sequencing (RNA-seq).To date, there have been few studies in Korea on viruses infecting plum trees compared to those infecting peach trees. Only two viruses, in Korea . In thisLeaf samples from five plum cultivars—Akihime, Formosa, Hollywood, Gadam, and Yeonsik—grown in an orchard in Hoengseong, Korea were harvested in May 2013 and leaf samples from a European plum (unknown cultivar) were harvested in September 2012. The plum trees had been properly sprayed with agricultural pesticides such as insecticides and fungicides. We collected five leaves from each plant representing a cultivar. Leaf samples were combined and subjected to total RNA extraction using Fruit-mate for RNA Purification  and the RNeasy Plant Mini Kit for RNA extraction , following the manufacturers’ instructions. The quality and quantity of extracted RNA were measured by gel electrophoresis and NanoDrop Spectrophotometers .PRJNA295439. Using the PRINSEQ program 25 Magnetic Beads  according to the manufacturer’s instructions. The mRNA libraries were prepared using the TruSeq RNA Library Preparation Kit v2  following the manufacturer’s instructions. The quality control of the generated libraries was carried out using a 2100 Bioanalyzer . The average insert size was 350 bp. The six plum libraries were paired-end (2 × 100 bp) sequenced by Macrogen Co.  using the HiSeq 2000 platform. In order to reduce the cost for NGS, six plum libraries in this study and six peach libraries in the previous studies , 2018b w program , we obtaFor de novo transcriptome assembly, we used Trinity   with def−10. We only extracted the genome sequences of plant viruses and viroids from the NCBI’s reference viral genome database (http://www.ncbi.nlm.nih.gov/genome/viruses/) and we established an in-house plant viral genome database. The assembled contigs from each plum transcriptome were blasted against a database composed of plant viruses and viroids. In addition, we conducted BLASTX with a cutoff E-value of 1e–3 using the assembled transcriptome against the viral protein database. The identified contigs associated with viral sequences were used for a BLASTX search against an NCBI non-redundant proteins (NR) database to remove sequences derived from the plum hosts. Finally, we obtained clean virus-associated contigs from each plum transcriptome.To identify viruses and viroids in the assembled plum transcriptomes, we carried out the MEGABLAST algorithm with a cutoff E-value of 1eTo confirm the presence of identified viruses in the six plum samples, RT-PCR was carried out using virus-specific primers . We perfhttp://trimal.cgenomics.org/). The best-fitting substitution model for each aligned protein sequence was selected using IQ-TREE (http://tree.bio.ed.ac.uk/software/figtree/).Full-length amino acid sequences for the coat protein (CP) and movement protein (MP) of ACLSV and the CP and MP of PNRSV were subjected to BLASTP search against the NR database. Homologous viral protein sequences were retrieved and subjected to sequence alignment using MAFFT ver. 7.310  with the G-INS-I (accurate) strategy . Aligned IQ-TREE . The phy IQ-TREE . FigTreeP. salicina) (AH), Gadam  (PG), Yeonsik  (PY), Hollywood (P. cerasifera) (HW), an unknown European plum (P. domestica) (EP), and Formosa  (FM)  (FM) –1E. We cna) (FM)  and 1D. na) (FM) , 2018b wTo identify viruses and viroids infecting plum in detail, we used the Trinity assembler. The clean reads obtained from each library were further subjected to de novo transcriptome assembly . The numNext, we examined the number of identified viruses and viroids in each plum cultivar . We idenAfter combining all virus- and viroid-associated reads, we had identified six viruses and a viroid (HSVd) . Among tAs the plum trees were also co-infected by diverse viruses and a viroid, we decided to examine viral populations based on viral reads and reads per kilobase million (RPKM) values. To calculate viral reads, raw sequence reads were mapped on the individual reference viral genome using the Burrows–Wheeler Aligner (BWA) program. The mapped SAM file was subjected to the BBMap program to calculate viral reads. Based on viral RNAs, the dominant virus and viroid in AH were PLPaV and HSVd, respectively . PLPaV wIt is important to calculate viral abundance according to the viral genome size. For that, we calculated the RPKM values for each virus and viroid using BBMap program. Based on the RPKM values, HSVd (96%) was overwhelmingly the major viral pathogen infecting AH and FM . In PG aWe carried out RT-PCR to confirm the presence of viruses identified by RNA-seq. We have previously confirmed that HSVd infects plum trees by RT-PCR . For RT-P. salicina in Brazil, four isolates from peach in Korea, and two isolates from peach in China.We revealed the phylogenetic relationships of ACLSV and PNRSV by generating phylogenetic trees using the available sequences of CP and MP. Based on the CP protein sequences, the ACLSV isolate FM was closely related to ACLSV isolate SQ6 (AEZ03868.1) and SQ3 (AEZ03866.1) from peach in China . The phyThe phylogenetic tree generated using CPs of known PNRSV isolates showed that most PNRSV isolates were closely related to three possible clades . The PNRPrunus species by RNA-seq.Many viruses and viroids infecting stone fruits—including peach, plum, apricot, and cherry—are being identified . Of the Initially, RNA-seq revealed six viruses and a viroid from plum transcriptomes. PCR-based approaches are frequently used to support NGS results; similarly, we conducted RT-PCR for the identified viruses using virus- and viroid specific primers. In many cases, the RT-PCR results were consistent with those of RNA-seq. However, RT-PCR confirmed infection of three viruses, namely ACLSV, PNRSV, and PBNSPaV, and one viroid (HSVd) in plum trees. There are several possible reasons for the discrepancy between the RNA-seq and RT-PCR. The first is primer sequences; due to the low sequence coverage of the identified viruses, we used known primer sequences instead of newly designed primers. If the genome of an infected virus shows strong sequence diversity, the binding affinity of the known primers can be very low. For example, the strong sequence diversity among Asian prunus viruses as well as ACLSV and APCLSV has been previously reported . The secP. domestica), not the other P. salicina cultivars or P. cerasifera. By contrast, infection of PBNSPaV in P. cerasifera and P. serrulata has been previously reported in China  with a hexaploid genome (2n = 6× = 48) than from P. salicina with a diploid genome (2n = 16)  tails . Compare2n = 16) . Based o2n = 16) , it seemTaken together, we identified three viruses ACLSV, PBNSPaV, and PNRSV and one viroid (HSVd) infecting plum trees by RNA-seq and RT-PCR. In addition, we confirmed that the three viruses identified by RNA-seq could have been contaminants caused by index misassignment. Therefore, we suggest that careful confirmation by other methods be carried out in NGS-based virus identification.10.7717/peerj.9588/supp-1Supplemental Information 1The accession number of individual RNA-seq and the number of total reads and clean reads (trimmed reads). Clean reads were analyzed by PRINSEQ program.Click here for additional data file.10.7717/peerj.9588/supp-2Supplemental Information 2Virus-associated contigs were assembled by Trinity (v.2.8.6) assembler and viral reads were mapped on the reference genome using BBMap to calculate mapped bases, coverage, number of reads, and RPKM.Prunus salicina) (AH), Gadam  (PG), Yeonsik  (PY), Hollywood (Prunus cerasifera) (HW), an unknown European plum (Prunus domestica) (EP), and Formosa  (FM).The names of plum samples were abbreviated as follows: Akihime  was used as outgroup. Ultrafast bootstrap with 1,000 iterations was indicated. The scale bar represents 0.5 substitutions/amino acid position.Click here for additional data file.10.7717/peerj.9588/supp-5Supplemental Information 5The PNRSV isolate HW is indicated by red color. The MPs of lilac leaf chlorosis virus (LLCV), ApMV, and blueberry shock virus (BIShV) were used as outgroups. Ultrafast bootstrap with 1,000 iterations was indicated. The scale bar represents 0.5 substitutions/amino acid position.Click here for additional data file.


Plum pox virus, Soybean mosaic virus, Turnip mosaic virus, and Potato virus Y. Potyviruses adopt polyprotein processing as their genome expression strategy. Among the 11 known viral proteins, the nuclear inclusion protein b (NIb) is the RNA-dependent RNA polymerase responsible for viral genome replication. Beyond its principal role as an RNA replicase, NIb has been shown to play key roles in diverse virus–host interactions. NIb recruits several host proteins into the viral replication complexes (VRCs), which are essential for the formation of functional VRCs for virus multiplication, and interacts with the sumoylation pathway proteins to suppress NPR1-mediated immunity response. On the other hand, NIb serves as a target of selective autophagy as well as an elicitor of effector-triggered immunity, resulting in attenuated virus infection. These contrasting roles of NIb provide an excellent example of the complex co-evolutionary arms race between plant hosts and potyviruses. This review highlights the current knowledge about the multifunctional roles of NIb in potyvirus infection, and discusses future research directions.Potyviruses represent the largest group of known plant RNA viruses and include many agriculturally important viruses, such as  Potyviridae is the largest family of known plant RNA viruses and is comprised of 10 definitive and 3 proposed genera [Potyvirus is the largest genus, including many agriculturally important viruses that cause serious diseases in many crops worldwide [Potyvirus include Potato virus Y (PVY), Plum pox virus (PPV), Soybean mosaic virus (SMV), Turnip mosaic virus (TuMV), Lettuce mosaic virus (LMV), Sweet potato feathery mottle virus (SPFMV), and Sugarcane mosaic virus (SCMV), 4 of which have been ranked in the 10 most economically or scientifically important plant viruses [d genera . Among torldwide ,3. Examp viruses . Becausecis on its own C-terminal end), and nuclear inclusion a protease (NIa-Pro), responsible for the remaining cleavage sites, to produce 10 functional proteins: P1, HC-Pro, P3, 6K1, cylindrical inclusion (CI) protein, 6K2, viral genome-linked protein (VPg), NIa-Pro, nuclear inclusion b (NIb), and capsid protein (CP) [trans and may need other viral-replication-associated proteins and/or host factors to deliver its RdRp activity in the VRC [Potyviruses have a positive-sense, single-stranded RNA genome of approximately 10,000 nucleotides that encodes a long open reading frame (ORF) ,3. Durinein (CP) ,9,10,11.ein (CP) . All RNAein (CP) ,14,15. Hein (CP) ,16,17,18ein (CP) . Thus, N the VRC . Like ot the VRC ,23,24,25Euphorbia ringspot virus (the genus Potyvirus) and Ugandan/cassava brown streak virus (the genus Ipomovirus) in which the NIb is flanked by NIa-Pro and a Maf/HAM1 pyrophosphatase-like sequence [Tobacco vein mottling virus is four in the carboxy region and six in the amino region around the scissile bond [cis or in trans by the NIa protein but cleavage between NIa-Pro and NIb occurs in cis only [Tobacco etch virus (TEV) also prefers cleavage in trans at the site between NIb and CP and in cis at the site between NIa-Pro and NIb [cis processing of the polyprotein at the site between NIa-Pro and NIb is forced by the polyprotein conformation in vivo. The potyviral NIbs have a molecular mass (MM) of nearly 60 kDa. The TuMV NIb contains 517 amino acids with an estimated MM of 59.6 kDa  [Arabidopsis (AtRH8) also interact with the potyviral NIb [A. thaliana and an α-expansin (NbEXPA1) from Nicotiana benthamiana are two crucial host factors of TuMV [AtRH9 knockout or NbEXPA1 knockdown plants. Since both AtRH9 and NbEXPA1 interact with NIb and are present in the VRC, it is possible that such interactions promote viral replication by stimulating NIb’s RdRp activity. In addition, AtRH9, as an RNA helicase, may participate in the separation of RNA duplexes during viral genome replication [NIb is probably the most “sticky” among potyviral proteins, as it is a very active recruiter that interacts with many pro-viral host proteins and co-opts them to promote viral infection. Through the interactions with NIb, many of them are recruited to the VRC. A poly(A)-binding protein (PABP) of cucumber was the first host factor identified to interact with the NIb of a potyvirus (c virus) . Subsequiral NIb ,74,75,76iral NIb ,78. Sinciral NIb ,54, it hiral NIb ,75,79. R of TuMV ,81. The lication . NbEXPA1lication . The NbEIn virally infected cells, viral protein function may be modulated by post-translational modifications (PTMs), including phosphorylation, ubiquitination, methylation, and sumoylation ,84,85. SCucumber green mottle mosaic virus of the genus Tobamovirus, and Pepino mosaic virus of the genus Potexvirus) [Autophagy is an evolutionarily conserved regulated mechanism of all eukaryotic cells that degrades and recycles defective organelles, toxic proteins and macromolecules, and various other aggregates to maintain cellular homeostasis during many biological processes such as development and abiotic and biotic stresses ,94. Accuexvirus) . Besides NIb, another important viral protein, HC-Pro, which is the major potyviral VSR, is also a target of autophagy. In this case, it has been shown that both host proteins rgs-CaM, a calmodulin-like protein, and NBR1, an autophagy cargo receptor, can mediate the degradation of HC-Pro via autophagy to restrict virus infection ,98. MoreR)-conferred genetic resistance is among the most effective strategies to control viral pathogens for crop production. This type of resistance works based on a well-known gene-for-gene interaction model [R gene product recognizes a specific viral effector, known as avirulence factor (Avr), to activate effector-triggered immunity (ETI), which confers strong resistance and is often associated with the hypersensitive response (HR) [Pvr4 from Criollo de Morelos 334 (CM334) variety confers dominant resistance to six potyviruses, including Pepper mottle virus (PepMoV), Pepper severe mosaic virus (PepSMV), and PVY [Pvr4 gene [N. benthamiana, but its activity is suppressed by the nucleotide-binding-Apaf1-R protein-CED4 (NBARC) and LRR domain in the absence of NIb [Rk gene, which provides resistance to a root knot nematode [Among a number of antiviral defense mechanisms deployed by plants to restrict viral infection ,102,103,on model ,105. In nse (HR) ,107. R pnse (HR) . In pepp and PVY . The pro and PVY ,111. NIbvr4 gene . Pvr4 isvr4 gene . The CC e of NIb . When Pve of NIb . Therefonematode . Pvr4 not only confers broad-spectrum resistance to potyviruses but also shows durability under field conditions [Pvr4-conferred resistance in the field have been documented. The evolutionary constraint acting on NIb is considered a major factor influencing the durability of Pvr4 [nditions ,114. So  of Pvr4 . As NIb  of Pvr4 . This as of Pvr4 . In cont of Pvr4 . Thus, t of Pvr4 . Taken tIn summary, potyviral NIb is a multifunctional protein, and functions as much more than just an RdRp in the viral infection process. It is a recruiter that co-opts proviral host proteins involved in the assembly and activation of the VRC, and a suppressor of host defense response that specifically tackles the NPR1-mediated immunity signaling pathway. Meanwhile, it is also a target of host antiviral defense, e.g., as a substrate of selective autophagy, and furthermore it is an elicitor that activates ETI. NIb’s multifunctionality is attributed to the complex interaction network of NIb, and establishes the significance of NIb in the co-evolutionary arms race between plants and potyviruses. We summarize the diverse functions of NIb in the potyviral infection in Although extensive studies have been devoted to the characterization of NIb’s roles in potyvirus infection in the past 30 years, much more remains to be explored. To date, we still do not understand clearly how NIb joins other viral proteins and host factors to form the VRC. We still cannot establish an active in vitro viral replicase complex to study potyvirus replication. The availability of this cell-free system would significantly accelerate research on the potyvirus replication mechanism and characterization of the components essential for virus replication. Since RNA viral replication is always associated with cellular membranes ,62, it w


Virus disease spreads effortlessly mechanically or through minute insect vectors that are extremely challenging to avoid. Emergence and reemergence of new viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), H1N1 influenza virus, avian influenza virus, dengue virus, Citrus tristeza virus, and Tomato yellow leaf curl virus have paralyzed the economy of many countries. The cure for major viral diseases is not feasible; however, early detection and surveillance of the disease can obstruct their spread. Therefore, advances in the field of virus diagnosis and the development of new point-of-care testing kits become necessary globally. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is an emerging technology for gene editing and diagnostics development. Several rapid nucleic acid diagnostic kits have been developed and validated using Cas9, Cas12, and Cas13 proteins. This review summarizes the CRISPR/Cas-based next-generation molecular diagnostic techniques and portability of devices for field-based utilization. Similarly, the Tobacco mosaic virus, Tomato spotted wilt virus, Tomato yellow leaf curl virus, Cucumber mosaic virus, Potato virus Y, African cassava mosaic virus, and Potato virus X are major plant pathogens that adversely affect economies each year  caused three major epidemics of respiratory distress syndrome, which comprise severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 in Guangdong, China; the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 in Saudi Arabia; and the respiratory coronavirus, the novel coronavirus disease 2019 (COVID-19), which started from Wuhan province, China, and later extended its infection in South Korea, Japan, Italy, Iran, and USA and has further spread to India, Bangladesh, and Pakistan  kits, which can be used at the site of infection. The recent outbreak of SARS-CoV-2 accelerates research in the field of diagnostics to develop rapid, accurate, and ultrasensitive kits. Nucleic acid-based testing (NAT) tools proved ultrasensitive and accurate diagnostics for multiplexed virus infection, thus major attempts are being performed to convert typical diagnostics into nucleic acid POCT tools. Popular NAT kits are based on quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), which requires expensive infrastructure and trained manpower, therefore not feasible to utilize at the point of infection. Nowadays, recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR)-based tools are popular among NAT tools. These tools may replace costly qRT-PCR-based testing systems. The CRISPR/CRISPR-associated protein (Cas) system is rapidly becoming well known for its powerful potential not only in therapeutics but also in getting a unique identity in the field of molecular diagnostics. Intense research efforts are being conducted around the globe to identify efficiently CRISPR's search function to be utilized in diagnostics. Recently, an improved understanding of other diverse CRISPR/Cas systems has expanded CRISPR's applications in disease diagnosis. The recent discovery of different classes of RNA-guided nuclease, Cas protein has unlocked the door to new tools that offer cost-effective and portable diagnostics through nucleic acid screening.The modern NAT kits are reliable and highly sensitive such as quantitative PCR kits for SARS-CoV-2  is a 2–6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas nuclease in the CRISPR bacterial adaptive immune system. The most commonly studied endonuclease belongs to the type II CRISPR system, i.e., Cas9 that targets phage's DNA, while the type V CRISPR system, i.e., Cas12 also targets DNA, and type III and type VI groups exhibit RNA targeting activity, which includes Cas13 Ford, .in vitro  recognizes 5' NGG 3' as PAM, while Francisella novicida Cas9 (FnCas9) recognizes 5' NGG 3' as PAM sequence on target site  and guided by single-guide RNA (sgRNA), which facilitates its specific binding to target double-stranded DNA (dsDNA). Binding of sgRNA activates nuclease activity of Cas9, which makes a blunt double-stranded DNA break in vitro  nuclease, is a unique protein that has specific recognition and cleavage activity for complementary RNA and known for trans cleavage or collateral cleavage of nearby RNA , was also developed, which utilizes the target-specific nicking activity of Cas9 and nicking endonuclease (NEase). Unlike CRISDA, this technique does not require any external primers and hence proved more specific for the mutated targets  was developed for the diagnosis of African swine fever virus (ASFV). In this diagnostic kit, CRISPR/Cas9-based ultrasensitive lateral flow nucleic acid diagnosis of ASFV was demonstrated , are also introduced, which successfully diagnose both SARS-CoV-2 and HIV  and DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) are two major CRISPR/Cas12-based diagnostic systems, which have been applied worldwide   kits are the most popular POCT kits, which are generally based on the molecular affinity of various molecules such as antigen–antibodies, streptavidin-biotin, etc. It is the simplest device for POCT for various pathogens, diseases, or hormonal changes in humans, animals, or plants. These colorimetric devices are developed on paper material such as nitrocellulose paper and generally contain one or two test lines and a control line and do not require any specialized electronic device to visualize the results. CRISPR/Cas9-based CASLFA  are an advanced form of LFA, emerged as cost-effective and portable platforms for POCT, and are primarily made up of fibers and direct fluid from an inlet to the outlet through imbibition and could be run by applying a voltage into a triangular sheet of wet paper. English et al.  have preThe setup for paper-based LFA and microfluidic devices are very tricky to perform in the laboratory and required good experimental skills. Therefore, an easy and POCT alternative for this kit is the fluorescence-based diagnosis of CRISPR/Cas-cleaved fluorescent molecules. These days, many battery-operated and inexpensive fluorescence detectors are available, which can be used in non-laboratory conditions. Such devices can be developed by the use of LED and filter foils, which can detect up to a lower limit of detection of approximately 6.8 nM fluorescein (Katzmeier et al., Toehold switches are synthetic RNAs that act as mRNAs whose role is to switch between the start and stop of the translation. In a normal situation, a toehold forms a hairpin loop structure that blocks the translation of the reporter gene by checking the ribosome movement. Pardee et al.  first utThe recent development in the field of molecular diagnostics presented the positive side of the COVID-19 outbreak. This incidence suggests that the only way to treat a virus disease is its early diagnosis in the mass population and containment of the pathogen. CRISPR/Cas-based diagnostics may prove to be a better option to develop ultra-sensitive, inexpensive, and rapid non-laboratory-based detection kits for several viruses and other pathogens. In this review, we have summarized the several kits such as CASLFA, FELUDA, DETECTR, HOLMES, SHERLOCK, etc., which are developed by the CRISPR-associated nucleases Cas9, Cas12, or Cas13. We also focus on techniques used for the portability of the devices to deploy them into field conditions.AS has developed the concept. SS and AS has written manuscript. DU evaluated and corrected the text. 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.


The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Wuhan City, China, late in December 2019 is an example of an emerging zoonotic virus that threatens public health and international travel and commerce. When such a virus emerges, there is often insufficient specific information available on mechanisms of virus dissemination from animal-to-human or from person-to-person, on the level or route of infection transmissibility or of viral release in body secretions/excretions, and on the survival of virus in aerosols or on surfaces. The effectiveness of available virucidal agents and hygiene practices as interventions for disrupting the spread of infection and the associated diseases may not be clear for the emerging virus. In the present review, we suggest that approaches for infection prevention and control (IPAC) for SARS-CoV-2 and future emerging/re-emerging viruses can be invoked based on pre-existing data on microbicidal and hygiene effectiveness for related and unrelated enveloped viruses. Coronaviridae family was subsequently named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)  . This meS-CoV-2) . As of AS-CoV-2) , there hS-CoV-2)  (Zhu et S-CoV-2) .Coronaviridae is just one of several families of enveloped viruses that have emerged/re-emerged in recent years (Coronaviridae), Crimean Congo hemorrhagic fever (Nairoviridae), Rift Valley fever (Phenuiviridae), Ebola virus disease and Marburg virus disease (Filoviridae), Nipah and Hendra virus disease (Paramyxoviridae), and Lassa fever (Arenaviridae)).The nt years   has shown person-to-person transmission , yet thiAs mentioned in The animal-to-human and person-to-person transmission of SARS-CoV-2 and associated COVID-19 disease appears to occur in a manner similar to that described for MERS-CoV and SARS-CoV. That is, the transmission of SARS-CoV-2  primaril50 (ID50) is the smallest number of infectious virus particles that will lead to infection of 50% of an exposed population (50) values that reflect the doses capable of infecting half of the subjects exposed. As prospective studies in humans of highly pathogenic viruses with potentially fatal outcomes (such as SARS-CoV-2) cannot ethically be performed, very limited data exist on the infectivity of the emerging/re-emerging viruses in The infectivity of a virus refers to its ability to initiate infection of a host cell with production of viral progeny. The infectious dosepulation , and is pulation . Those wpulation . IPAC mapulation . Typical50 values.The estimates that have been reported for viruses listed in Once infected with one of these emerging/re-emerging viruses, during the prodromal period before actual appearance of symptoms, as well as once symptoms appear, the infected individual may become a shedder of infectious particles, as mentioned above. The extent to which virus shedding might lead to dissemination of the associated disease depends upon a number of factors, including the amount of virus released (shed), the infectivity of the virus within the released matrix , and the survival of the released viruses within such matrices once dried on HITES. Extent of virus shedding, unfortunately, is commonly measured through detection of genomic material e.g., , 2020b, As displayed in Knowledge of the transmissibility and infectivity of emerging/re-emerging viruses enables one to assess the risk of spread of a viral disease in the case that infectious virus is shed from an infected individual and is deposited on environmental surfaces/fomites or in droplets/aerosols. Another important factor to consider when assessing risk is the survival  of these viruses on the environmental surfaces/fomites or in air in the form of droplets/aerosols.10 reduction in infectivity per unit time, infectivity half-life, infectious titer after a measured duration, etc.). For There is much more information addressing survival of infectious viruses on environmental surfaces than in aerosols. The data that are available address a number of environmental factors of relevance , includiInfectious virus survival (persistence) of SARS-CoV-2 on experimentally contaminated prototypic HITES and in air has recently been reported by Infectious virus surviving in aerosols/droplets or on HITES represents a source for dissemination of emerging/re-emerging viruses, including SARS-CoV-2. The enveloped viruses listed in p-chloro-m-xylenol (PCMX)) (10) within the 1–5 min contact times tested.Among pathogens, prions are considered to be the least sensitive to microbicides, requiring highly caustic solutions for inactivation. Bacterial spores and protozoan cysts/oocysts are next on the microbicidal susceptibility spectrum. Small, non-enveloped viruses are considered to be less susceptible to microbicides, although these viruses display increased susceptibility to high pH, oxidizers such as sodium hypochlorite, activated hydrogen peroxide, alcohols, and a variety of microbicidal actives, relative to spores and protozoan cysts/oocysts. Mycobacteria, fungi, vegetative bacteria, and enveloped viruses appear to be more susceptible to certain formulated microbicides, such as alcohols, oxidizers, quaternary ammonium compounds (QAC), and phenolics ) . A numbe (PCMX)) , and as It is of interest that the enveloped viruses are considered to be the most susceptible to a variety of formulated microbicidal actives, even more so than fungi and vegetative bacteria, yeast, and mycobacteria . Viral eCoronaviridae family, reviewed recently by It can be assumed as a starting point, therefore, that the enveloped emerging/re-emerging viruses listed in 10 of infectious Ebola virus–Makona variant (EBOV/Mak) suspended in an organic load and evaluated in liquid virucidal efficacy studies  by aqueous solutions of 70% ethanol or 0.5% or 1% NaOCl (≥0.5%) after contact times ≥2.5 min (10) for EBOV/Mak and vesicular stomatitis virus following as little as 5 s contact time  have been recommended for decontaminating environmental surfaces or materials used for food preparation . The effIn the case of highly pathogenic emerging/re-emerging viruses, such as SARS-CoV-2, effective and frequent targeted hygiene using appropriate microbicides is essential for prevention of infectious virus dissemination. Practicing hygiene inappropriately and only once daily may not be sufficient, as recontamination of HITES could potentially occur, particularly under healthcare settings where SARS-CoV-2 infected patients are treated. For instance, infectious coronavirus 229E was detected on HITES  in a university classroom in which samples were collected daily over a 1-week period . VigilanCoronaviridae, Orthomxyoviridae, Paramyxoviridae, Phenuiviridae, indicate that UV fluencies of 3–14 mJ/cm2 should inactivate 4 log10 of the enveloped viruses in 2 resulted in 3 log10 inactivation. These fluency values are relatively low, compared to those needed to inactivate 4 log10 of the least UV-susceptible viruses, such as those of the Adenoviridae (98–222 mJ/cm2) and Polyomaviridae (235–364 mJ/cm2) families of non-enveloped viruses (The enveloped emerging/re-emerging viruses listed in  viruses .The WHO has posted a webpage entitled “Coronavirus Disease (COVID-19) advice for the public” . Basic pThe U.S. CDC has posted a webpage entitled “Coronavirus 2019 (COVID-19) How to protect yourself and others” . This inWhile handwashing practices practically can be applied in developed countries, there are still some three billion people in developing countries without access to basic handwashing facilities in the home and where proper hand hygiene may not be practiced in the majority of healthcare facilities . Basic IAs Dr. Anthony Fauci eloquently stated in 2005 , “PublicCoronaviridae), African swine fever virus (Asfarviridae), Schmallenberg virus (Peribunyaviridae), Crimean-Congo hemorrhagic fever virus (Nairoviridae), Rift Valley fever virus (Phenuiviridae), West Nile virus and Zika virus (Flaviviridae), Hantaviruses (Hantaviridae), and Lassa viruses (Arenaviridae).It is fortunate that so many of the emerging/re-emerging viruses  has primarily been described on the basis of measurement of SARS-CoV-2 RNA, and not on the basis of recovery of infectious virus using cell-based assays. The measurement of RNA does not inform as to the infection potential of the surface contamination. Similarly, articles describing the contamination of waste-water streams by SARS-CoV-2 have been based on measurement of viral RNA, not infectious virus. As a result, it is not presently known whether infectious virus is present in such waste streams and, therefore, whether the finding of viral RNA in waste water represents real risk in terms of onward dissemination of the virus. Another knowledge gap, that has already been mentioned above, is the true mortality rates for the various emerging/re-emerging viruses addressed in this review. As these viral infections are relatively deadly, empirical data on true mortality rates are lacking. For the same reason, accurate data on human MID are not generally available for these viruses. The risk of acquiring an infection of one of these viruses from a contaminated environmental surface, over a period of time following an initial contamination event, will remain difficult to assess until such knowledge gaps have been resolved.The likelihood of experiencing future emergent zoonotic viruses is high , and def


The COVID 19 pandemic caused from SARS CoV 2 was declared on March 11, 2020 . HydrogeThe use of HI also against virus infection is well documented in the scientific literature. Goyal et al. studied the effectiveness of a vapour of HI versus different viruses: feline calicivirus; human adenovirus type 1; transmissible gastroenteritis coronavirus of pigs (very similar to SARS-CoV); avian influenza virus (AIV); and swine influenza virus (SwIV). Authors demonstrated that adenovirus, TGEV and AIV died at the lowest vaporized HI volume tested 25 ml). They evaluated that the total exposure time, including injection and aeration, was 2-3 hours, varying with the amount of hydrogen peroxide being vaporized  ml. They. RudnickAn hydrogen peroxide spray for decontamination of surfaces in hospitals was successfully tested by Cadnum et al. They observed that a 1.4% solution of hydrogen peroxide (IHP) spray disinfectant could be very effective against Staphilococcus aureus and strains of vancomycin-resistant enterococci . In a veThe authors declare no competing interests.


Tomato leaf curl Guangdong virus (ToLCGdV) is a begomovirus associated with a Tomato yellow leaf curl disease (TYLCD) epidemic in Guangdong province, China. Being the least conserved protein among geminivirus proteins, the function of C4 during ToLCGdV infection has not been elucidated. In this study, the infectious clones of ToLCGdV and a ToLCGdV mutant (ToLCGdVmC4) with disrupted C4 ORF were constructed. Although ToLCGdV and ToLCGdVmC4 could infect Nicotiana benthamiana and tomato plants, ToLCGdVmC4 elicited much milder symptoms compared with ToLCGdV. To further verify the role of C4 in viral pathogenesis, C4 was expressed in N. benthamiana from Potato virus X (PVX) vector. The results showed that ToLCGdV C4 enhanced the pathogenicity of PVX and induced more severe developmental abnormalities in plants compared with PVX alone or PVX-mC4. In addition, ToLCGdV C4 suppresses systemic gene silencing in the transgenic N. benthamiana line 16c, but not local gene silencing induced by sense GFP in wild-type N. benthamiana plants. Moreover, C4 suppresses transcriptional gene silencing (TGS) by reducing the DNA methylation level of 35S promoter in 16c-TGS N. benthamiana plants. Furthermore, C4 could also interact with the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1), suggesting that C4 may suppress gene silencing by interfering with the function of BAM1 in the cell-to-cell spread of RNAi. All these results suggest that C4 is a pathogenic determinant of ToLCGdV, and C4 may suppress post-transcriptional gene silencing (PTGS) by interacting with BAM1. Geminiviridae constitutes a large family of plant viruses with small, single-stranded and circular DNA (sscDNA) genomes encompassed within characteristic twinned icosahedral particles  and movement protein (AV2), which may both be involved in virus movement. The complementary-sense strand of DNA A component encodes the replication-associated protein , transcriptional activator protein , replication enhancer protein  and C4 protein (ORF AC4). The DNA B component of bipartite begomovirus encodes two proteins, the nuclear shuttle protein  on the virion-sense strand and the movement protein  on the complementary-sense strand  C4 or Tomato leaf curl Yunnan virus (TLCYnV) C4 contributes to PVX infection, and induces severe developmental abnormalities similar with phenotypes induced by virus infection in both N. benthamiana and Arabidopsis (Tomato golden mosaic virus (TGMV) has no obvious effect on symptom development (Arabidopsis SHAGGY-like protein kinase (AtSK) family  family , b.African cassava mosaic virus (ACMV) and Sri Lankan cassava mosaic virus (SLCMV), but not East African cassava mosaic Cameroon virus (EACMCV) nor Indian cassava mosaic virus (ICMV), have been shown to suppress RNA silencing by specifically binding to the single-stranded miRNAs and siRNAs to block the miRNA-mediated cleavage of target mRNAs (Tomato yellow leaf curl virus (TYLCV) C4 interacts with the receptor-like kinases (RLKs) BARELY ANY MERISTEM 1 (BAM1) and its homolog BAM2, interfering with the function of BAM1 and BAM2 in the intercellular spread of RNAi (Mungbean yellow mosaic virus (MYMV) AC4 also interacts with BAM1 at plasma membrane (PM), and the PM location of MYMV AC4 depends on S-palmitoylation (Cotton leaf curl Multan virus (CLCuMuV) C4 have been shown to repress both PTGS and TGS by interacting with S-adenosyl methionine synthetase (SAMS) and inhibiting SAMS activity  to suppress both post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) . AC4 encet mRNAs . A recen of RNAi . Moreoveoylation . AC4/C4 activity .Tomato leaf curl Guangdong virus (ToLCGdV)  which is a major limiting factor for tomato cultivation worldwide . A new bToLCGdV) . ToLCGdVToLCGdV) . As the mC4 were constructed. ToLCGdV C4 is a pathogenic determinant by contributing to ToLCGdV and PVX infection. ToLCGdV C4 is also a VSR by suppressing systemic gene silencing but not local gene silencing. Moreover, ToLCGdV C4 could reverse methylation-mediated TGS. In addition, ToLCGdV C4 suppresses PTGS maybe by interacting with BAM1. These results demonstrate that ToLCGdV C4 plays an important role in virus infection by functioning as both pathogenic determinant and VSR.In this study, the infectious clones of ToLCGdV and ToLCGdVSmaI-digested pGreenII (mC4 was generated by QuickChange® site-directed mutagenesis (Agilent Technologies) with pGreenII-1.3A-ToLCGdV as templates.To construct ToLCGdV infectious clones pGreenII-1.3A-ToLCGdV which contains a 1.3-mer tandem repeat of ToLCGdV, the full-length sequence and a 0.8-kb fragment of ToLCGdV were amplified with primer pairs ToLCGDV-F1/R1 and ToLCGDV-F2/R2 , respectpGreenII  by SeamlmC4 and introduced into pGR107 (C4 and the mutated C4 nucleotide sequences were amplified from pGreenII-1.3A-ToLCGdV or pGreenII-1.3A-ToLCGdVo pGR107  to obtaio pGR107 . SubsequFor bimolecular fluorescence complementation (BiFC) assays, C4 was amplified and cloned into pSPYNE-35S and pSPYCE-35S split YFP destination vectors . For subPrimers used for plasmid construction in this study are listed in Nicotiana benthamiana and tomato plants used in this study were grown in a climate chamber with a 13h/11h light/dark photoperiod at 24°C. N. benthamiana were agroinfiltrated at 5–6 leaf stage, and tomato plants were inoculated at 6–7 leaf stage.For western blot detection, samples were taken and weighed, followed by ground in liquid nitrogen. Two volumes of 1 × SDS loading buffer was added and boiled for 10 min. After centrifugation for 10 min at 12 000 rpm, 20 μl supernatant was loaded per lane on the SDS-PAGE gel. Then proteins were transferred to the nitrocellulose membranes. The secondary antibody was purchased from Sigma-Aldrich, Inc., Alkaline phosphatase was visualized with NBT/BCIP .AC4/C4 protein sequences were obtained by searching National Center for Biotechnology Information (NCBI) GenBank using the protein sequence of ToLCGdV C4. Phylogenetic trees were constructed with MEGA7  softwareTotal RNA was extracted with Trizol reagent (Takara) and treated with RNase-free rDNase I (Takara) before reverse transcription reaction. Equal amount of treated RNA was subjected to reverse transcription using PrimeScript RT Reagent Kit (Takara). Real-time RT-PCR was performed with SYBR Premix Ex Taq II (Takara) using CFX96 Real-Time System .N. benthamiana plants (provided by Dawei Li lab) and 16c-TGS plants were used. 16c-TGS plants were generated as described (Agrobacterium infiltration was performed as described previously (Tomato bushy stunt virus (TBSV) p19 and an empty vector (pGD) were used as positive and negative controls, respectively. GFP fluorescence was observed under a long-wave UV lamp  and photographed using a Nikon D70 digital camera with a Y48 yellow filter.To identify the suppressor activity of C4, 16c transgenic escribed . Agrobaceviously . Tomato Agrobacterium strains harboring the corresponding plasmids were co-infiltrated into 5–6 leaf stage N. benthamiana plants. Samples were taken at 3 dpi and visualized under Zeiss LSM710 confocal microscope . Excitation wavelengths were as follows: EYFP, 514 nm; RFP, 561 nm.Genomic DNA was extracted with a cetyltrimethylammonium bromide (CTAB) method . CytosinN. benthamiana leaves were collected at 3 dpi to perform Co-IP assays with anti-c-Myc agarose affinity gel .Co-immunoprecipitation assays were performed according to the previously published protocols . InoculaTomato leaf curl Guangdong virus was firstly reported and named by Euphorbia leaf curl virus (EuLCuV) (Euphorbia pulcherrima-infecting monopartite begomovirus firstly identified in China ((EuLCuV) , an Euphin China . ToLCGdVin China . In contin China . Thus, imC4 which contains an unexpressed C4 were constructed. pGreenII-1.3A-ToLCGdV   , while pl 15 dpi . Westernl 15 dpi . In summAgrobacterium strain containing pGreenII-1.3A-ToLCGdV or pGreenII-1.3A-ToLCGdVmC4 was infiltrated into tomato plants. Tomato plants agroinfiltrated with pGreenII-1.3A-ToLCGdV developed obvious symptoms including leaf curling and yellowing at 30 dpi  vector pGR107   and sGFP was used as negative control. Three days after agroinfiltration, strong GFP fluorescence was observed in regions co-infiltrated with sGFP and p19 (AC4/C4 encoded by some begomoviruses have been reported to function as a VSR . To explutilized . AgrobacP (sGFP) , 2018 we and p19 . However and p19 . The res and p19 .N. benthamiana  vector which contains a portion of the 35S promoter sequence  was performed to assess the cytosine methylation level of 35S promoter. Cytosine methylation level of ten CG, four CNG, and forty-eight CHH sites in the 35S promoter region were analyzed. C4 expressed from the PVX vector reduced cytosine methylation remarkably at CG (48%), CNG (51%), and CHH (5%) sites . These rTomato yellow leaf curl virus C4 and MYMV AC4 have been reported to interact with BAM1 in PM and plasmodesmata (PD) to inhibit the intercellular spread of RNAi , Asystasia begomovirus 2 (ABgV2), Allamanda leaf curl virus (AllLCV), and Pouzolzia mosaic Guangdong virus (PouMGDV). All these begomoviruses are firstly isolated from wild plant hosts (Begomovirus contains over 320 species and is one of the largest plant virus genera (Tomato leaf curl Yunnan virus (TLCYnV), a begomovirus firstly isolated from Malvastrum coromandelianum, evolved from Tomato yellow leaf curl China virus (TYLCCNV) by recombination and is highly infectious to a range of host plants by acquiring a more virulent C4 (Tomato yellow leaf curl Sardinia virus (TYLCSV) and TYLCV  C4 induces the expression of a RING finger E3 ligase, RKP, which antagonizes with an inhibitor of cyclin-dependent kinase (CDK) to induce cell cycle  (Tomato leaf curl Yunnan virus (TYLCCNB) , C4 of CTYLCCNB) , and C4 an virus . TYLCCNBan virus .Tomato leaf curl Guangdong virus C4 inhibits systemic gene silencing but not local gene silencing, this is also the case for TYLCV C4 (TYLCV C4 . AC4/C4 TYLCV C4 . We founTYLCV C4 , implyinAll datasets generated for this study are included in the article/ZL, ZD, and ZH conceived and designed the experiments. ZL, ZD, YT, and LY conducted the experiments. XS, XW, GL, and YZ analyzed the data. ZL and ZD wrote the manuscript. All authors read and approved the manuscript.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.