Aedes mosquito bites. Although the mortality rate associated with CHIKV infection is not very high, CHIKVF has been confirmed in more than 40 countries, not only in tropical but also in temperate areas. Therefore, CHIKV is a growing major threat to the public health of the world. However, a specific drug is not available for CHIKV infection. As demonstrated by many studies, the processes completing the replication of CHIKV are assisted by many host factors, whereas it has become clear that the host cell possesses some factors limiting the virus replication. This evidence will provide us with an important clue for the development of pharmacological treatment against CHIKVF. In this review, we briefly summarize cellular molecules participating in the CHIKV infection, particularly focusing on introducing recent genome-wide screen studies that enabled illuminating the virus-host interactions.Chikungunya virus (CHIKV), the causative agent of Chikungunya fever (CHIKVF) that is often characterized by fever, headache, rash, and arthralgia, is transmitted to humans by  Alphavirus genus in the Togaviridae family, which is composed of more than 30 recognized viruses (arthropod-borne viruses) (Chikungunya virus (CHIKV) is an enveloped RNA virus that causes Chikungunya fever (CHIKF) in humans. CHIKV is classified into the  viruses . Most alviruses) . A CHIKVviruses) .in vitro studies revealed that the A226V substitution in E1 enhanced the replication fitness of CHIKV in the Aedes albopictus mosquito, which thrives in both tropical and temperate regions , and West African (WA) lineages based on the E1 gene sequence . Althoug regions . Howevertus cell .9 copies, and the high level of viremia was shown to be often correlated with the severity of the medical condition  are often involved in critical biological activities of the host, the application of candidate inhibitors to the treatment of CHIKV-infected individuals remains an obstacle  bind to various bioactive proteins such as cell growth factors, cytokines, chemokines, enzymes, and protease inhibitors to regulate their activities . In addigenicity . In the genicity . Our pregenicity . We showgenicity . In addigenicity . Interesn humans .via bafilomycin A1 treatment, suggesting that part of the conformational changes in E2 occurs before endosome acidification  identified fuzzy homolog (FUZ) and TSPAN9 as cellular proteins that promoted the entry process of CHIKV . Depletivia budding  encoding nsP1–4 is first translated from the viral RNA released into the cytoplasm, which yields precursors of the non-structural protein. It has been well demonstrated that the majority of CHIKV isolates possess an opal stop codon (UGA) between the nsP3 and nsP4 genes that produces an nsP123 precursor; on the other hand, a full-length nsP1234 polyprotein is generated by the readthrough of the opal stop codon . The nsP budding .nsP3 is an accessory protein necessary for the nsP4’s RNA polymerase activity and has been shown to possess ADP-ribosylhydrolase activity in its N-terminal domain, whereas the C-terminal domain is hypervariable . In a reA study using a set of siRNA libraries targeting cellular factors involved in membrane trafficking revealed critical roles of endosomal sorting complexes required for transport (ESCRT) proteins in the intracellular replication of CHIKV . ESCRT, in vitro. Furthermore, three drugs targeting the fatty acid synthesis pathway, calmodulin signaling pathway, or fms-related tyrosine kinase 4, all of which were identified as cellular enhancers for CHIKV infection using an siRNA library screen, significantly reduced virus replication in C57BL/6 mice -expressing CHIKV, and identified 156 enhancing and 41 inhibitory genes for virus replication . Then, bL/6 mice . TherefoL/6 mice .Generally, the loss-of-function genetic screen using the siRNA and CRISPR-Cas9 system provides insights into the dependencies of the virus on host factors and machinery, whereas the gain-of-function screen, which ectopically expresses a certain set of functional genes, is able to identify cellular factors that limit virus replication. Particularly, it has been demonstrated that the gain-of-function screen using a cDNA library is a powerful approach in a comprehensive study of host antiviral mechanisms such as interferon (IFN)-stimulated genes . RecentlAs seen in many human pathogenic viruses, CHIKV hijacks the host machinery to create a favorable environment for virus replication . On the AT and YS contributed to the conceptualization, writing, review, and editing of this manuscript. Both authors have read and agreed to the submission of 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Advances in testicular cancer screening and therapy increased 10-year survival to 97% despite a rising incidence; eventually expanding the population of survivors requiring follow-up. We analyzed 10-year follow-up costs after testicular cancer treatment in Germany during 2000, 2008, and 2015.Testicular cancer follow-up guidelines were extracted from the European Association of Urology. Per patient costs were estimated with a micro-costing approach considering direct and indirect medical expenses derived from expert interviews, literature research, and official scales of tariffs. Three perspectives covering costs for patients, providers, and insurers were included to estimate societal costs. Cost progression was compared across cancer histology, stage, stakeholders, resource use, and follow-up years.p < 0.001). Advanced SGCT spending shrank from EUR 13,866 to EUR 9,724 (p < 0.001). In contrast, expenditure for stage II SGCT increased from EUR 7,159 to EUR 9,724 (p < 0.001). While insurers covered 32% of costs in 2000, only 13% of costs were reimbursed in 2015 (p < 0.001). 70% of SGCT follow-up resources were consumed by medical imaging . Spending was unevenly distributed across follow-up years .Mean 10-year follow-up costs per patient for stage I seminomatous germ-cell tumors (SGCT) on surveillance declined from EUR 11,995 in 2000 to EUR 4,430 in 2015 (The increasing prevalence of testicular cancer survivors caused German statutory insurers to cut per patient cost by up to 80% by budgeting services and decreasing reimbursement rates. The economic burden was gradually redistributed to patients and providers. Germany spent 6.8% of the gross domestic product on cancer in 2014, representing a 1% increase from 1995 , stage I (surveillance), stage II, and stage III. As EAU guidelines focus on the first 10 years post-treatment, a 10-year time horizon was assumed appropriate. Cost data were derived with a micro-costing approach based on patient level data. The developed costing approach estimates follow-up costs from all three perspectives—payers, providers, and insurers—to then estimate the overall societal impact in 2000, 2008, and 2015.The payer perspective assesses opportunity costs relevant for testicular cancer follow-ups. Time consumption was derived from literature and expert opinions Table . ForgoneThe provider perspective estimates opportunity costs for all relevant health care providers involved in testicular cancer follow-ups. In Germany, the provider perspective is relevant since budgeting and reimbursement restrictions lead to a discrepancy between the provider’s possibility to bill medical services and actual performed medical activities. Time consumption were again based on previous literature and expert interviews Table . The forThe insurer perspective considers the bill that the provider hands to the insurer. Due to Germany’s dual insurance system, a separate costing approach was considered for both the social health insurance (SHI) and the private health insurance (PHI). SHI and PHI funds are charged according to reimbursement rates published in a regularly updated catalogs Brück , 2015. RThe frequency of recommended consultations, examinations, and diagnostic tests was extracted from EAU guidelines. Thereafter, the opportunity costs for physicians and patients, resulting from forgone time consumption for follow-ups, were calculated alongside the resulting expenditure bill for the insurance Table . The hosWe conducted a probabilistic sensitivity analysis to account for variations in the length of physician–patient consultations, examinations, and diagnostic tests. Therefore, provider’s and payer’s time consumption parameters were drawn by random sampling from their defined distribution Table . The anaEstimated 10-year prostate cancer follow-up costs ranged from EUR 4430 to EUR 36,304 per person . The decline was even sharper for follow-up schedules for stage I SGCT after surveillance  and with SGCT/NSGCT in stage III with metastasis . In contrast, follow-up cost for stage II SGCT surged from EUR 1493 in 2000 to EUR 1855 in 2015 (p < 0.001). Regarding NSGCT, follow-up costs for stage I after surgery/chemotherapy increased from EUR 1466 in 2000 to EUR 1680 in 2015 (p < 0.001).10-year follow-up costs for payers decreased throughout the examined period . Follow-up costs for SGCT in stage I on surveillance steeply declined , while SGCT in stage II surged . There was a decline in follow-up costs for advanced SGCT/NSGST in stage III from EUR 7177 in 2000 to EUR 4996 (p < 0.001), followed by an increase to EUR 6630 in 2015 (p < 0.01).The calculated 10-year follow-up costs for providers showed varying patterns depending on cancer stage and histology . The decline in 2008 can mainly be attributed to the decreased costs for insurance funds  compared to the following three (EUR 4059) and five EUR 3119) years (Fig. 119 yearsCancer follow-up costs are relevant to assess the entire financial cancer burden on society. Therefore, costs incurred by all stakeholders—patients, providers, and insurers—were analyzed. Results underline that all three stakeholders face a significant economic barrier to testicular cancer follow-up. Consequently, the consideration of cancer follow-up costs is relevant and must be considered as an essential component of future clinical and health economic research. Precisely, cancer survivorship is an emerging financial and non-financial cost driver for the overall healthcare system, especially for patients. However, the overall financial burden depends on the cancer stage and the post-treatment “survivorship trajectory” (Lorgelly and Neri From a societal perspective follow-up costs decreased from 2000 to 2015 across most cancer stages—yet, costs were redistributed among stakeholders. In 2000, health insurances paid for one third of all follow-up costs. In 2015, insurances covered less than 13%. German statutory health insurers limited their cost exposure by the stepwise budgeting of medical services and lowering of reimbursement rates. Consequently, the financial burden of follow-up costs was gradually shifted to providers and patients.Our results demonstrate that costs were particularly reduced during follow-up years five to ten and transferred to providers and patients. The increased economic burden to providers could especially disincentivize registered doctors in rural areas, with enhanced patient flows, to perform cancer follow-ups. Similarly, medical services and travel expenses not covered by health insurers discourage patient adherence to follow-ups. Research unveiled a low adherence to recommended follow-ups (Nielsen et al. Recently, health policy concentrated on primary and tertiary prevention strategies. As a results, early detection and improved treatment options significantly reduced mortality and drove 10-year survival rates to 97% Tarver . The comConsequently, follow-up schedules should be adjusted to consider the growing number of survivors. There is an unmet need for more sophisticated follow-up patterns with a detailed match between evidence-based schedules and suitable patients to account for differential risk factors (Alfano et al. Shared patient-centered survivorship trajectories between GPs, urologists and oncologist are necessary (De Padova et al. Strengths of this study entail the comprehensive micro-costing method, wholistic approach to include all three perspectives, and assessment of costs for various treatment alternatives. First, follow-up spending was estimated by combining patient level data with EAU guidelines and reimbursement quotes. Second, direct and indirect medical costs were analyzed for insurers, providers, and patients. Third, relevant follow-up costs for different treatment options and cancer stages allow cost-effectiveness evaluations of testicular cancer treatment options. Results permit a detailed cost analysis across stakeholders, resource use, and follow-up year. Based on literature review, to the best of our knowledge, this is the first study assessing testicular cancer follow-up costs in Germany from 2000 to 2015.However, the study is prone to some limitations. Firstly, follow-up costs are empirically determined and consequently not confirmed in clinical studies. Secondly, this health economic evaluation does not permit conclusions about the efficacy and cost-effectiveness of advances in diagnostic options and improved EAU guidelines. Thirdly, the underlying 100% “uptake” of guideline recommendations might not model reality. Further research is necessary to examine the follow-up costs of non-testicular cancer patients in and beyond Germany.Testicular cancer follow-ups encompass a considerable economic burden to all stakeholders. Follow-up costs differ depending on cancer histology, stage, and treatment strategy. Consequently, we proposed to consider follow-up expenses in cost-effectiveness evaluations of cancer treatments. Our results educate health care decision makers on the emerging economic burden shifted to patients and providers by statutory health insurers in Germany. The redistribution of the financial burden from insurers to patients and providers likely disincentivizes adherence to follow-up guidelines and consequently negatively influences health outcome—especially for the unprivileged population not covered by private insurance. Improved long-term care for cancer survivors, who are at risk for cardiovascular diseases, is urgently needed.


The welfare of farmed rabbits is a growing concern for consumers, who are demanding animal-friendly housing systems. At the same time, it is important to understand the impact of such a system on the economic sustainability of this sector. Through a face-to-face interview, we collected data of structures, productivity, and costs from six farms using conventional or structurally enriched cages. Looking at the results, the enriched cage, in comparison with conventional housing systems, is both non-penalizing and economically sustainable. In addition, the adoption of a more animal-friendly housing system leads to a reduction of drugs cost. This result is opening the discussion on the opportunity to contribute to the reduction of antibiotic use, therefore pursuing an improvement of animal welfare. Due to the complexity of the topic, we believe that further studies on the economic sustainability of this sector are needed to confirm our results.2 and on eight cost indices expressed in EUR/kg of produced live weight. The results showed that WRSA significantly reduced the productivity index per walkable cage area in buildings and cages, thanks to the longer platform area included in the cage compared to the other systems. Concerning cost indexes, total variable costs were not different among housing systems, whereas significant differences were observed within costs items. As for the feed costs, DP underperforms compared to BI or WRSA (1.15 vs. 1.02 and 0.99 EUR/kg produced live weight); for drugs costs, BI was less competitive compared to DP and WRSA (0.12 vs. 0.06 and 0.05 EUR /kg). In conclusion, under the conditions of the present study, the economic results of farms that adopted housing systems designed to improve rabbit welfare, such as WRSA enriched systems, were economically sustainable and, comparable to conventional housing systems based on BI or DP cages, also provided a significant reduction in drug use in the tested farms. A comprehensive collection of data from more farms at a European level would be necessary to confirm these results on the economics of farms adopting alternative housing systems for rabbits.This research evaluates the economic sustainability of rabbit farms using different housing systems—bicellular (BI), conventional dual-purpose (DP) and enriched cages designed according to the World Rabbit Science Association guidelines (WRSA)—through a field-based study involving six farms over the course of five years. The cages were compared based on three productivity indices expressed in kg of produced live weight/m During the last 15 years, consumers’ concerns about rabbit breeding practices have grown . That’s No specific EU rules yet exist for farmed rabbits, but the European Parliament resolution of 14 March 2017 .Currently, as for housing systems, conventional farms use conventional cages  or structurally enriched cages designed according to guidelines of the World Rabbit Science Association (WRSA) . Both haFarms equipped with bicellular cages (BI) house reproducing does with the offspring until weaning (30–35 days of age) when kits are moved to bicellular cages  for fattening and until slaughter. In the case of dual-purpose cage (DP), at the time of weaning, the doe is moved to a clean and disinfected enclosure and the kits remain in their cage until the end of fattening . The cage is equipped with a removable nest box for offspring and lactation, which is taken away for the fattening period. Structurally enriched cages, also called welfare-cages or WRSA, are of recent use. The management of animals in WRSA cages works the same as in dual-purpose cages, but cages are higher and with enrichments (a raised platform and a plastic footrest) providing more surface area for the fattening rabbits and an opportunity for movement . For morOne of the greatest limitations for farmers in changing the housing system and adopting the best technology depends on individual choices and preferences, often linked to economic advantages. Farmers can choose to adopt higher standards in animal welfare if they also gain advantages in terms of a higher price paid by the market  or in terms of a cost reduction .However, market dynamics influence the economic performance of the sector, as farmers are also subjected to high price volatility over the past 10 years, due to a cyclical market crisis within a trend of decline in consumption ,20,21,22The possibility of farmers to adopt any animal welfare innovation following EU regulations largely depends on the impact of such innovation on the economics of the business. This is an urgent issue in Europe and Italy, the third largest EU producer, which, together with Spain and France, supplied 83% of the total EU production of professional rabbit farming in 2018 ,24. WithThe literature cited above has addressed the issue of cost analysis by adopting the approach of the typical farm. This method does not require the collection of data at the farm-level for a significant number of farmers. As such, they are a considerably lower cost option relative to farm surveys, but they do not generally ensure the representativeness of the results .To contribute to the debate on the economic advantages and disadvantages related to the adoption of animal-friendly housing systems in rabbit farms, this study aimed at comparing the economic performance of farms adopting three housing systems for reproducing does and growing rabbits, i.e., conventional cages for does and bicellular cages for growing rabbits (BI), and dual-purpose cages (DP) or structurally enriched cages (WRSA) for both categories, based on farm-level survey. To the best of our knowledge, this is the first attempt at performing a statistical analysis that compares, on the basis of the main cost elements observable at farm-level, these three housing systems for rabbit farms.Face to face interviews were conducted on 6 farms between February and April 2019 and information was collected over the 5-year period between 2014–2018, regarding the type of rabbit housing, productivity and costs. Farms were located in the Regions of Veneto and Friuli Venezia Giulia, north-east Italy. They were selected based on the housing systems adopted for growing rabbits: bicellular (BI), conventional dual-purpose (DP), and structurally enriched cages (WRSA). A total number of 19 observations  were collected from six farms . Data relating to periods of less than one year were excluded from the analysis. One of the farms (farm C) changed the housing system in 2017, from bicellular to WRSA. A different number of observations was available per farm, for which an unbalanced dataset was used with 9 observations from farms with bicellular cages (BI) (9 observations × 3 farms); 7 observations from farms with conventional dual-purpose cages (DP) (7 observations × 2 farms); and 3 observations from farms with structurally enriched cages (WRSA) (3 observations × 2 farms). Regarding the evaluation of the age of the cages, this was possible for DP, installed on the 2 farms in 2010 and 2015, and for WRSA, installed on the 2 farms in 2017. BI cages were installed before 2014. The production systems were consistent with the description provided by EFSA [2, including masonry works, automatic feeding system (with silos), cleaning system (scrapers), air conditioning system, and electrical and water system. The cost of the cages changed according to the housing system. The price of WRSA and DP cages was EUR 78.08 and EUR 70.15 and included 1 fattening/doe cage plus half a replacement/recovery cage. For BI, the price was EUR 18.30 for the fattening cage and EUR 47.77 for the doe cage. Fixed costs were determined as depreciation costs considering a depreciation time of 20 years for the structures (shed plus services) and 10 years for the cages  and fixed costs (FC) were attributed to the final product, i.e., kilogram of produced live weight. Among VC, the analysis considered the following: feed, drugs (including veterinary service costs), reproduction , and energy costs . Feed costs included all the quantity of feeds (medicated or not) consumed in one year on the farm for all categories of rabbits . All coshe cages .To allow a comparison among different years, all the monetary values were discounted to 2018 based on the price index of the goods purchased by farmers . As VAT 2). In detail:2): this was obtained by dividing the produced live weight in one year by the area (m2) of cages available to rabbits. This is equal to the cage floor for BI and DP cages, and the cage floor plus platform for WRSA cages. This index includes the areas of both fattening and reproduction cages.Yearly productivity of the walkable cage area (kg/m2): this was obtained by dividing the slaughtered live weight in one year by the area (m2) of the cage, which excludes the platform area in WRSA. This index includes the areas of both fattening and reproduction cages.Productivity of the base cage area (kg/m2): this was obtained by dividing the slaughtered live weight in the year by the area (m2) of the shed declared by farmers. In the all-in/all-out system, all shed areas are considered in the calculation.Productivity of the total shed area  (kg/mCost indexes (EUR/kg): these were obtained by dividing the yearly expenditure for each cost items by the produced live weight in one year, expressed in kg. “Total variable costs” (VC) includes feed, drugs, energy, and reproduction. The economic information comes from the farm accountancy, the data recorded by the breeders, or both. “Total fixed costs” (FC) includes the depreciation costs of the shed and cage.The economic indexes give the average monetary costs of different cost items per kilogram of live weight produced in one year (EUR/kg) , calculaThe analysis of differences in productivity and economic indexes among housing systems started verifying the ANOVA assumptions: the normality distribution of the residuals (Shapiro-Wilk test) and homoscedasticity . All the variables have a normal distribution and were analysed using a two-way ANOVA, taking the fixed effect of the housing system and management. When heteroscedastic distribution was detected (costs for feed and reproduction), the Welch ANOVA was performed. The Tukey-Kramer test and Games-Howell post-hoc tests were performed.To assess the existence of a farm effect, the one-way ANOVA between-subjects was performed, looking for statistically significant effects associated with the management (random effects).p-value < 0.05 were accepted as statistically significant. Significance levels of p-value < 0.10 were also discussed. The analysis was performed using the IBM SPSS (version 26.0).The differences between the means with a 2) compared to those with DP cages (196.82 kg/m2) . There is also a trend towards a significant effect  between WRSA and BI, with a higher value in the latter.The yearly productivity of the walkable cage area was lower in farms with WRSA cages (158.08 kg produced live weight/m2) did not differ among housing systems, despite the fact that the productivity of the total shed area tended to be higher in farms using BI cages (115.94 kg/m2) compared to those using DP cages (100.26 kg/m2) , which could be attributed to the second floor of BI cages, compared to the flat-deck DP cages.The productivity of the base cage area . The drug cost was significantly higher in farms adopting BI housing (EUR 0.11/kg produced live weight) compared to those with DP (EUR 0.06/kg) and WRSA cages (EUR 0.05/kg) . The energy costs were higher in farms with BI and DP cages  than in those with WRSA cages (EUR 0.03/kg) . Concerning reproduction, costs results were statistically higher in farms with WRSA cages (0.19 €/kg) than in those with BI (0.08 €/kg), and tended to be higher than DP cages (EUR 0.08/kg) .As for differences in the cost indexes , VC, as p-value = 0.08). Finally, the cage cost was significantly higher in farms with WRSA cages (EUR 0.10/kg) compared to those with BI and DP  .Regarding FC, despite the significance of the F-test, no difference among farms with different housing systems was recorded. Nevertheless, the shed cost tended to be lower for the BI (EUR 0.20/kg slaughtered live weight) compared to DP (EUR 0.23/kg)  to animals compared to BI and DP cages, without affecting production per cage area.We reported that the changes of the housing system, which addressed animal welfare through the use of WRSA cages, did not reduce the yearly productivity of farms in terms of produced live weight per mAs for costs, the results of the present study do not identify significant differences for aggregated costs (variable and fixed), which could be due to the small sample size, the variability of the production factors other than housing among the farms, or a combination thereof. The total costs ranged from 1.54 to 1.66 to 1.54 EUR /kg of live weight produced in farms with BI, D and WRSA housing systems. On average, variable costs accounted for 81.6% of the total, with feed costs being the main item at 66.7% of the total, drugs at 4.6%, energy at 3.4% and reproduction at 7.4%. Fixed costs were 18.4%, with shed costs accounting for 13.1% and cages costs for 5.3% of the total. Field data dating back to 2014 gave a production cost ranging from 2.06 to 1.91 EUR/kg of meat  .2 of the cage.Under our conditions, farms using WRSA cages were competitive for costs related to feeding, drugs and energy, whereas the worst performance was recorded in farms with BI cages for drug costs and in those with DP cages for feed costs. The analysis of the indexes related to structures (cages) shows that BI and DP appear to have lower depreciation costs than WRSA, mainly due to the lower cost for mIndeed, feed costs can vary largely, according to all factors affecting feed intakes, such as diet composition  and feeding regime (restricted or ad libitum feeding), besides the health of the animals on a farm  and, lasAs for farms with WRSA cages, the advantages in terms of lower feed, drugs and energy costs are cancelled due to higher costs for reproduction and the depreciation of cages. The former reason was mainly linked to a farm effect within the surveyed farms, whereas the latter depends on the higher purchasing price of WRSA cages compared to the conventional cages, which is a general condition for all farms.In conclusion, based on these preliminary results, since total FC or VC did not change among housing systems, whereas drug costs were lower in WRSA cages compared to BI cages, we can state that in the perspective of evolution towards good welfare practices in rabbit farming, the replacement of traditional cages with structurally enriched cages, potentially associated with better hygiene conditions, could be economically sustainable, provided that market prices remain acceptable. These results should be confirmed on a larger number of observations. Furthermore, an exhaustive assessment of production costs for the different housing systems should also include labour costs. To reduce the farmers’ effect on performance indexes and enlarge perspectives about alternative housing systems for rabbits, further research should increase the number of farms and include other housing systems, such as elevated parks for conventional farms and organic systems per niche farms.


Culex (Latin for “gnat”) quinquefasciatus, which he collected along the Mississippi River. Originally written as “C. 5-fasciatus,” the name refers to 5 (“quinque”) black, broad, transverse bands (“fasciatus” or “fasciae”) on the mosquito’s dorsal abdomen (quinquefasciatus is the official scientific name, there are at least 5 synonymous names for this species.In 1823, the American entomologist Thomas Say described  abdomen . The namCx. quinquefasciatus is found throughout subtropical and tropical areas worldwide, except for exceedingly dry or cold regions. This mosquito is a principal vector of many pathogens, transmitting the phlebovirus Rift Valley fever virus and the 2 flaviviruses St. Louis encephalitis virus and West Nile virus, in addition to filarial worms and avian malarial parasites.Say described this species as “exceedingly numerous and troublesome.” “Quinx” are among the world’s most abundant peridomestic mosquitoes, earning the nickname “southern house mosquito.”


Mentha piperita, Mentha longifolia, Salvia officinalis, and Salvia rosmarinus, grown at high altitudes in the Taif region, KSA, on Aphis punicae. Furthermore, the toxicity of these EOs against the predator Coccinella undecimpunctata was estimated. A total of 17, 14, 16, and 26 compounds were identified in the EOs of M. piperita, M. longifolia, S. officinalis, and S. rosmarinus, respectively. They showed a variation in the major compounds: M. piperita , M. longifolia , S. officinalis , and S. rosmarinus . A contact toxicity test on A. punicae imago and C. undecimpunctata larvae showed that LC50 were approximately four-fold greater for all tested EOs towards aphids compared to towards the predator, while the two species of Salvia sp. were more effective than the other two species of Mentha sp. The LC50 values on A. punicae ranged from 1.57 to 2.97 µg/mL, while on C. undecimpunctata larvae, they ranged from 5.96 to 10.33 µg/mL. Furthermore, the EOs of two species of Salvia sp. showed excellent repellence and deterrence against A. punicae. In conclusion, the tested EOs, especially those from Salvia sp., have been shown to be promising natural aphicides, repellent, and deterrent against A. punicae, and they are safe for important insect predators.Chemical insecticides have many harmful effects, including as foodborne residues and environmental contaminants, as well as side effects on natural enemies and serious risks for human health. The use of plant-derived essential oils (EOs) as effective bio-agents has become an essential component of integrated pest management. In this study, the contact toxicity, deterrent, and repellent activities were evaluated for essential oils obtained from  The widespread utilization of synthetic pesticides poses hazards for both the environment and human health due to their toxicity and poor biodegradability ,2. In adEssential oils (EOs) derived from medicinal and aromatic plants are considered safe substances for the environment and human health. Thus, they can be used as active substances for pest control . In thisAphis punicae Passerini (Hemiptera: Aphididae) is the main sucking–piercing insect pest affecting pomegranate. Both nymphs and imagos infest young leaves, vegetative and flower buds, flowers, and young fruits, resulting in the discoloration and drying of these affected parts ×100  , where N50) was estimated using Probit analysis. Then, significant differences among the LC50 values were determined using the confidence intervals of the relative median potency (RMP). Differences between each pair of LC50 values were considered statistically significant if 1.0 was not present in the 95% confidence interval of RMP. Analysis of variance (One-Way ANOVA) and Tukey’s test were conducted to assess repellent and deterrent effects. Moreover, Two-Way ANOVA was performed to estimate the interaction between EOs and concentrations on aphid deterrence. Statistical analysis was determined using the SPSS software program, version 20, Armonk, NY: IBM Corp, USA.The mortality (%) in each treatment was corrected with that in the control depending on Abbott’s formula. The selectivity ratios (LC50 for the predator/LC50 for aphid) were estimated . Each leM. x piperita, M. longifolia, S. officinalis, and S. rosmarinus, respectively. The chemical compositions of the tested essential oils are presented in M. piperita, M. longifolia, S. officinalis, and S. rosmarinus, respectively. The major component of M. piperita EO was Carvone (61.16%), followed by α -Cubebene (10.99%) and D-Limonene (4.08%). In M. longifolia, Pulegone (48.6%), l-Menthone (34.49%), and Eucalyptol (4.5%) were the major components. In Salvia sp., Eucalyptol (33.52%), α-pinene (22.68%), and Camphene (14.44%) were the major components in S. officinalis, while α-pinene (36.65%) was the major component in S. rosmarinus, followed by p-Cymene (7.08%), Eucalyptol (6.91%), and Camphene (6.8%).The obtained yields of EOs were 0.72 ± 0.03, 0.94 ± 0.09, 1.35 ± 0.08, and 0.89 ± 0.05 (w/w) for 50 values of the four tested EOs on the aphid A. punicae indicated that the LC50 values for both S. officinalis (1.574 µg/mL) and S. rosmarinus (1.653 µg/mL) EOs were lower than those of both M. piperita (2.971 µg/mL) and M. longifolia (2.4 µg/mL)  and S. officinalis versus S. rosmarinus . Meanwhile, these comparisons for C. undecimpunctata larvae showed that that all comparisons among the four tested EOs were significant, except for M. longifolia versus M. piperita  and S. rosmarinus versus S. officinalis  (The LC4 µg/mL) . The samectively . It was , 0.475) .10, LC20, and LC30) on the deterrence of A. punicae is presented in 10 of M. piperita (4.3%), while the highest values were achieved with LC30 of S. officinalis (80.4%) and S. rosmarinus (75.2%). There were significant differences among the deterrent effect of the three tested LCs for all EOs on A. punicae . Meanwhile, LC10 indicated that there was no significant difference among the tested EOs , while LC20  and LC30  indicated a significant difference between both Menth sp. and both Salvia sp. In general, Two-Way ANOVA analyses . Moreover, there was a significant difference in the deterrent effect among the tested concentrations . Meanwhile, the interaction effect between the tested concentrations and the tested EOs showed no significant difference .The effect of the tested EOs with three less lethal concentrations  and M. longifolia (5.2%), while the highest repellence was achieved with the highest concentration of 20 µg/mL for S. officinalis (63.4%) and S. rosmarinus (71.4%). There were significant differences in the repellent effect of the five tested concentrations for all EOs on A. punicae .The repellence of the tested EOs at five different concentrations compared with the control is shown in Salvia sp. collected from the same region had different yields and chemical compositions [Mentha sp., the same results were obtained [S. officinalis (1.72–2.06%) was higher than that of S. rosmarinus (0.99–1.08%), which were collected from the same region and at the same altitude [Mentha sp. varied in their yield of EOs: Mentha pulegium (2.93%), M. piperita (1.23%) and Mentha spicata (0.9%) [The EO yields for the tested plants ranged from 0.72 to 1.35 (w/w). Moreover, there were variations in the major EO constituents’ content for all tested plant species. In general, the environmental conditions  and collection site affected the EO yields and chemical compositions, where altitude is considered an indirect control affecting plant metabolism ,27. Moreositions ,29. Addiobtained . In a pra (0.9%) . Bioratia (0.9%) ,33,34.Many investigations demonstrate that EOs repel the insects and also act on them as neurotoxic compounds ,36 whereS. officinalis and S. rosmarinus EOs were more toxic than both M. piperita and M. longifolia for aphids and the coccinellid predator. In this regard, the effects of the EO compounds, rosacide (from S. rosemarinus), sagix , and cura (from Curcuma longa), on Aphis craccivora were very promising, especially at higher concentrations [Rodolia cardinalis larvae. However, S. officinalis achieved 100% mortality for Acyrthosiphon pisum, but caused 45% mortality for Myzus persicae with the same concentration (2 µL/L) through the fumigation method [Trichoplusia ni due to the increased penetration of the tested toxicants through the integument rather than through the inhibition of detoxicative enzymes [Our findings revealed that both the trations , and then method . On othe enzymes .A. punicae and C. undecimpunctata. This finding is in agreement with many previous investigations [A. punicae compared to C. undecimpunctata. In general, the EOs were more effective towards target insect pests than natural enemies. Regarding the contact toxicity, Satureja intermedia EO was more toxic for imago females of Aphis nerii than Coccinella septempunctata imago [Myzus persicae but not to ladybeetle-related TRP channels. Interestingly, the exposure to the Negramina essential oil did not affect the predatory abilities of Coleomegilla maculata but increased the abilities of Eriopis connexa to prey upon M. persicae. These findings provided a physiological basis for the insecticidal and selectivity potential of this EO. Such differential susceptibility results from the differences in the life history traits and in differential receptor expressions (quantities and/or types) in aphids and ladybeetles [In the present study, the effects of the EOs varied according to the plant species and concentration, where the mortality rates increased with an increase in the EO concentration for all EOs on both igations ,45,46. Mta imago ,47,48. Ata imago . Computaybeetles . S. officinalis (63.4%) and S. rosmarinus (71.4%). This result is in accordance with previous findings [Neotoxoptera formosana and may play a role in plant defense against attacks by insect pests. The repellent activity of S. rosmarinus EOs against female imago of Planococcus citri under laboratory conditions amounted to 57.1 and 45.2% in choice and no-choice tests, respectively [Acanthoscelides obtectus and Leptinotarsa decemlineata [The repellent activity of EOs from various plants has been clearly confirmed through their major active constituents . In thisfindings , where rectively . Also, imlineata . The repmlineata .30 of S. officinalis (80.4%) and S. rosmarinus (75.2%), and there were significant differences among the tested EOs as well as the tested concentrations; however, the interaction effect between the tested concentrations and the tested EOs showed no significant difference. The same findings were obtained previously, where the Two-Way ANOVA revealed that there was a significant difference in the deterrent effect for the cabbage aphid Brevicoryne brassicae among different five EOs and also two different concentrations, while the interaction effect of different essential oils at different concentrations showed no significant difference [S. rosmarinus EOs against female imago of Planococcus citri under laboratory conditions amounted to 57.1 and 45.2% in choice and no-choice tests, respectively [S. rosmarinus on the cabbage aphid B. brassicae, showed that they reduced reproductivity and led to an increased mortality rate in the aphid population [10 and LC25 against Brevicoryne brassicae L. and the black aphid Aphis fabae [In the present study, deterrence also increased with LC increase, where the highest values were achieved with LCfference . The detectively . Evaluatpulation . The detis fabae . In geneis fabae .S. officinalis and S. rosmarinus, had strong negative effects on the pomegranate aphid A. punicae due to the low impact of these EOs for the insect predator. Moreover, an appropriate composition of EOs can be used to control aphids in integrated pest management programs. Therefore, the results suggest that these EOs may help in the population reduction of A. punicae through their toxicity, repellent, and nymph production deterrence effects. Moreover, these findings are promising for future research as they have the potential to be launched on a commercial scale. Other future investigations could be performed on these EOs to evaluate their compatibility with other biocontrol agents such as entomopathogenic fungi and bacteria.The findings of the current study show that the studied EOs, especially


C. liriopes. ClPV1 genome has two double stranded RNAs (dsRNAs), named as dsRNA 1 and dsRNA 2, which in the lengths of 1,807 and 1,706 bp, respectively. The dsRNA 1 and dsRNA 2 encoded proteins showing significant amino acid (aa) sequence identity to the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) of partitiviruses, respectively. Phylogenetic analysis using the aa sequences of RdRp and CP indicated that ClPV1 was grouped to members of the putative Epsilonpartitivirus genus in the Partitiviridae family. Spherical viral particles in approximately 35 nm in diameter and packaging the ClPV1 genome were isolated. Virus elimination and virus transfection with purified viral particles, and biological comparison revealed that ClPV1 could reduce the virulence and conidia production of C. liriopes. To the best of our knowledge, this is the first report of mycovirus in C. liriopes fungus.Here, we report a novel double-stranded RNA virus designated Colletotrichum liriopes partitivirus 1 (ClPV1) from the plant pathogenic fungus  Totiviridae, Partitiviridae, Chysoviridae, Quadriviridae, Endornaviridae, Megabirnaviridae, Reoviridae, Amalgaviridae, Curvulaviridae, Polymycoviridae, and a kingdom, Orthornavirae  in plant pathogenic fungi or oomycetes , the enhColletotrichum spp. are considered as an economically significant groups of plant pathogens that cause anthracnose disease in a variety of plant species worldwide , from a Colletotrichum liriopes strains Cl-B-2 and SJM3-2 were originally isolated from the Paris polyphylla and Pachysandra terminalis plants that were infected by anthracnose disease, respectively, in Hunan province of China. The Cl-B-2-P1 was a derivative strain of the Cl-B-2 obtained by protoplast regeneration. The SJM3-2-T5 was a derivative strain transfected with viral particles of ClPV1. All strains were grown on potato dextrose agar  at 27°C. For dsRNA or total RNA extraction, mycelial plugs were cultured on potato dextrose (PD) broth  liquid medium at 27°C, with an orbital shaker at 110 rpm for 4 to 7 days.Double stranded RNAs were extracted from fungal mycelium using CF-11 cellulose  column chromatography with the methods described by The dsRNAs were purified and used as a template for cDNA synthesis. The cDNA library was constructed using random hexadeoxynucleotide primers  based on the methods described previously . InternaPotential ORFs in each full-length cDNA sequence were deduced and their homologous amino acid (aa) sequences were searched in NCBI by ORF Finder and BLASTp programs, respectively. Multiple sequence alignment was carried out using the CLUSTALX 1.8 program . PhylogeWe performed the protoplast regeneration method to eliminate ClPV1 from its host fungus. Protoplast preparation was made using the method as described by Single-spore isolation and virus detection were carried out for evaluating the transmission efficiency of virus ClPV1. Conidia were produced on PDA plate and single-spore isolates were obtained by picking the conidia individually into fresh PDA plates. The presence of virus in the single-conidian isolates was confirmed by RT-PCR detection.C. liriopes strain Cl-B-2 according to the procedure described previously by C. liriopes was grown on PD for 7 days. Approximately 50 g of mycelia were harvested and ground to fine powder with liquid nitrogen. The powder was mixed with 150 mL phosphate buffer . Then, the suspension was centrifuged at 10,000 × g for 30 min to remove the hyphal cell debris. The supernatant was transferred to centrifugation at 100,000 × g under 4∘C for 2 h. The resultant pellet was resuspended in 0.1M phosphate buffer and subsequently fractioned with 20–50% (w/w) sucrose gradients for 2.5 h at 100,000 × g in a Beckman SW55 rotor. The fractions containing particles were further precipitated by ultracentrifugation at 100,000 × g. The pellet was resuspended in 100 μl of 0.01M sodium phosphate buffer, pH 7.0.Virus particles were purified from mycelial of A drop of purified virus suspension was stained negatively with 1% uranyl acetate and observed under a transmission electron microscope (TEM). The dsRNAs from the viral particles were extracted with phenol chloroform isoamyl alcohol and detected using 1% agarose gel electrophoresis. The purified virus particles were subjected for sodium dodecyl sulfate (SDS)-polyacrylamide (12%) gel electrophoresis and stained with Coomassie brilliant blue R250.C. liriopes strain SJM3-2 was used as a virus recipient in the transfection experiment. Protoplasts of the recipient were prepared using the method described previously  plates for colony regeneration at 27°C. The putative transfected regenerated isolates were selected and sub-cultured on new PDA plates. The presence of virus infection was tested by dsRNA extraction and RT-PCR amplification using viral specific primer pairs.The virus-free eviously , and subeviously . About 2Morphology and growth rates were assessed by culturing mycelial plugs, collected from 4-day-old actively growing plates, on PDA for 3–7 days at 27°C in the dark. Mycelial growth rates were tested by measuring the diameters of each colony, with each strain has three replicates.Malus pumila Mill.) as described previously  test, C. liriopes strain Cl-B-2 was found to be dsRNA-positive. After being treated with DNase I and S1 nuclease, a dsRNA band, in the size of approximately 2 kbp was observed on a 1% agarose gel  were 64 and 27 bp in the dsRNA1, and 176 and 33 bp in the dsRNA 2, respectively. The 5′-terminus between the two dsRNAs were conserved containing the conserved stretch “CCCATTATA.” Conserved terminal sequences might be involved in recognition and replication of the viral dsRNAs. The 3′ UTRs between the dsRNA 1 and dsRNA 2 were shorter and less conserved . In viewQHB49873.1, Query cover: 99%; E value: 0; identity: 65.21%), Plasmopara viticola lesion associated Partiti-like 2 , Colletotrichum eremochloae partitivirus 1 , Penicillium aurantiogriseum partiti-like virus , Penicillium aurantiogriseum partitivirus 1-cp , et al. When searched by conserved domain database search and multiple protein alignment, conserved motifs (Motif III to Motif VIII) which were also present in and characteristic of RdRp sequences of other members of the Partitiviridae family, were detected in the ClPV1 encoded RdRp , followed by PvlaPL2 and CePV1.The ORF2 in dsRNA 2 potentially encoded a protein of 498 aa, with a molecular mass of 55.51 kDa. BLASTp search revealed that the ClPV1 ORF2 had significant aa sequence identity, ranging from 30.36 to 58.37%, to the CP of the family Partitiviridae . Virulence assays on detached apple fruits were conducted. After 14 days, the average lesion areas caused by Cl-B-2-P1 (41.3 mm) were larger than those caused by Cl-B-2 (21.2 mm), indicating that ClPV1 might confer hypovirulence to its fungal host C. liriopes strain Cl-B-2 . The lesion diameter caused by SJM 3-2-T5 on apple fruits was significantly smaller than that caused by SJM3-2  can be found below: JZ and XL conceived and designed the experiments. JZZ, JG, and ZH performed the experiments. XZ analyzed the data. JZ, JZZ, JG, and ZH wrote the manuscript. 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.


Plant viruses cause yield losses to crops of agronomic and economic significance and are a challenge to the achievement of global food security. Although conventional plant breeding has played an important role in managing plant viral diseases, it will unlikely meet the challenges posed by the frequent emergence of novel and more virulent viral species or viral strains. Hence there is an urgent need to seek alternative strategies of virus control that can be more readily deployed to contain viral diseases. The discovery in the late 1980s that viral genes can be introduced into plants to engineer resistance to the cognate virus provided a new avenue for virus disease control. Subsequent advances in genomics and biotechnology have led to the refinement and expansion of genetic engineering (GE) strategies in crop improvement. Importantly, many of the drawbacks of conventional breeding, such as long lead times, inability or difficulty to cross fertilize, loss of desirable plant traits, are overcome by GE. Unfortunately, public skepticism towards genetically modified (GM) crops and other factors have dampened the early promise of GE efforts. These concerns are principally about the possible negative effects of transgenes to humans and animals, as well as to the environment. However, with regards to engineering for virus resistance, these risks are overstated given that most virus resistance engineering strategies involve transfer of viral genes or genomic segments to plants. These viral genomes are found in infected plant cells and have not been associated with any adverse effects in humans or animals. Thus, integrating antiviral genes of virus origin into plant genomes is hardly unnatural as suggested by GM crop skeptics. Moreover, advances in deep sequencing have resulted in the sequencing of large numbers of plant genomes and the revelation of widespread endogenization of viral genomes into plant genomes. This has raised the possibility that viral genome endogenization is part of an antiviral defense mechanism deployed by the plant during its evolutionary past. Thus, GM crops engineered for viral resistance would likely be acceptable to the public if regulatory policies were product-based (the North America regulatory model), as opposed to process-based. This review discusses some of the benefits to be gained from adopting GE for virus resistance, as well as the challenges that must be overcome to leverage this technology. Furthermore, regulatory policies impacting virus-resistant GM crops and some success cases of virus-resistant GM crops approved so far for cultivation are discussed. To ensure food security as the global population increases in a changing climate, there is a need to improve crop production, which continues to face multiple constraints. Viral diseases constitute one of the major threats to crop production worldwide , and theThe use of recombinant DNA techniques to genetically engineer plants was first reported in 1983 ,5,6. TomEngineering plants for virus resistance has grown considerably since evidence emerged in the 1980s showing that viral genes can be used to engineer plants for antiviral resistance , as elucMany genetic modification strategies have been successfully used to control plant viruses in laboratories, greenhouses, and to a limited extent, in field trials. Since its elucidation in 1990, many RNA and DNA viruses have been successfully controlled using RNA silencing approaches, which suppress gene expression in a sequence-specific manner. Many RNA silencing precursors, including: sense/antisense, small interfering RNA (siRNA), microRNA (miRNA), and hairpin RNA (hpRNA) have been harnessed to generate virus resistant transgenic crops ,22. SomeNon-viral antiviral genes, including especially R genes and ribosome-interacting proteins, as well as protease inhibitors have also been incorporated into crop genomes to generate resistance against viruses. Mechanistically, most of these non-viral genes silence host genes that are involved in virus replication ,29,30,31Application of GE in virus management has also been facilitated by the advancements and breakthroughs in genome editing. This has included the use of site-specific nucleases to target the viral genome or host plant factors that are involved in the viral infection cycle. Four major classes of site specific nucleases (SSNs) that have successfully been employed to engineer virus resistance are meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently, the clustered regularly interspaced palindromic repeats/ CRISPR-associated 9 (CRISPR/Cas9) technology ,35. In eIn plants, the CRISPR/Cas system was first used successfully to generate geminivirus resistance in both model and crop plants, including beet curly top virus (BCTV) and merremia mosaic virus (MeMV). This system was shown to simultaneously target multiple begomoviruses, including wheat dwarf virus (WDV), tomato yellow leaf curl virus (TYLCV), tobacco curly shoot virus (TbCSV), bean yellow dwarf virus (BeYDV), where the guide RNA (gRNA) was designed from the intergenic region (IR), or CP and Rep genes, ,41,42. CAs discussed above, GE has provided new opportunities in plant virus control efforts by addressing most of the shortcomings of conventional breeding and agronomic practices. As presented here, some of the advantages of GE for virus resistance include short lead time, relatively low production cost, high efficacy, ease of interspecies gene transfers, no major effect on the genetic background of the cultivar and can be used in vegetatively propagated crops.One of the major limitations of plant breeding, which so far is the most effective plant virus management approach, is the length of time required to produce a resistant plant. This contrasts with the relatively short plant transformation lead time, even for very recalcitrant crops such as cassava. Today, the transformation process involves designing the gene construct and its transfer in the regenerative tissue of the target plant. The transgene can be synthesized and used for transformation within a week or two after being designed. Moreover, there are institutional core facilities that are specialized in transforming specific crops, which depending on the crop, can take weeks to several months in generating the transformed lines. This contrasts with conventional breeding, which can take up to 20 generations of selection and may be complicated if there is a tight genetic linkage between the desired resistance gene(s) and the genes that confer undesirable traits ,47.The relatively low cost of production of virus resistant transgenic plants is partly due to the short turnaround time of the plant transformation process, as well as low manpower input. Furthermore, as discussed above, the advent of core facilities specialized in plant transformation has reduced duplication of purchases of expensive instruments used in plant transformation . These core facilities also have specialized technical knowledge for specific crops; this significantly reduces the time and effort required to construct transgenes and transform plants with different levels of amenability to transformation in modest labs. In the USA, the cost of transformation and transgenic development in potato and tomato, for example, are currently ~$1700 per construct and ~$1500, respectively.The need for backcrossing to introduce the transgene into the most desirable plant genotypes, which are typically not amenable to transformation, has been suggested . HoweverAdvances in genomics have provided new pathways that can be harnessed to produce transgenes that specifically target viruses with RNA or DNA genomes. The early generation of transgenic constructs were produced using whole genes or large segments of the viral genome . These tThese drawbacks have been overcome with the recent elucidation of tasiRNAs, and to an extent, artificial microRNAs (amiRNAs), both of which can be multiplexed to target multiple viruses from the same construct ,28,51. IThe advent of the CRISPR/Cas system  has alsoThe obvious limitation with transgenes containing ~21-mers is the apparent ease with which the target virus can mutate leading to a breakdown of the resistance. This can be mitigated by designing 21-mers from conserved regions and/or invariable motifs of viral genes.Vasconcellea species (family: Caricaceae) are resistant to PRSV, but are not sexually compatible with C. papaya [In conventional plant breeding, transfer of genes from distantly related species may be quite challenging because of sexual incompatibility. Given that virus resistance genes are frequently found in wild species that may not be sexually compatible with cultivated species, this becomes a serious drawback. This is the case with papaya ringspot virus (PRSV) where several wild . papaya . Because. papaya ,54.Although both conventional breeding and GE involve changes in the genetic makeup of an organism with respect to DNA sequences and the order of genes, the level of genetic changes brought about by GE is much less and is well defined. This contrasts with conventional breeding where thousands of uncharacterized genes are transferred during genetic crosses. Furthermore, in GE, the gene to be introduced is selected based on the function against the target pathogen and the type of resistance mechanism to be used. Moreover, with GE, the end products, such as proteins, metabolites, as well as the phenotype are well characterized prior to release. Because in conventional breeding genomes of both parents are mixed together and randomly re-assorted into the genome of the offspring, undesirable genes are transferred along with target genes while some desirable genes are lost in the offspring.To be improved using conventional breeding, the plant species must be amenable to cross-fertilization with the species containing the gene(s) for virus resistance. One of the main challenges of breeding for crop improvement is the inability or difficulty to cross fertilize. This is especially true for cultivated polyploids, for instance potato and cassava that are propagated using the vegetative mode of reproduction, as allopolyploidy combined with heterozygosity makes breeding challenging ,57,58. TFrom the epidemiological perspective, managing viral diseases of clonally propagated crops is more challenging due to the buildup over time of viral diseases in vegetative propagules. This partly explains why two of the most damaging plant virus diseases, caused by cassava mosaic viruses and potato virus Y, are found in clonally propagated cassava and potato. This adds to the need of adopting GE in the management of viruses of clonally propagated crops.In spite of the benefits offered by GE for virus resistance, there are potential risks if safeguards are not followed prior to deploying this technology. These potential risks include horizontal gene transfer, genetic erosion, recombination of transgene with infecting viruses, and deleterious effect of the transgene to humans and/or animals. As addressed below, it has been scientifically documented so far that GE crops, including virus-resistant ones, do not pose undue, new, or additional risks to humans, animals, or the environment relative to crops produced from conventional breeding. Moreover, these concerns are based unfortunately on the assumption that transgenes are unnatural and inherently harmful by their very nature. Here, we discuss these risks and the way forward.Concerns have been expressed about the potential of transgenes and/or selectable markers to transfer to other crops or to wild species through cross-pollination. The selectable marker gene can also be transferred to bacteria, especially in the root system, where a high rate of gene transfer with plants is known to occur . Such trAgrobacterium selectable markers, which are often antibiotic resistance genes that are included in virus-resistance transgenes, it is possible that the selectable marker can be transferred to microorganisms, thereby increasing the development of microorganisms with antibiotic resistance in the environment. However, there appears to be little indication that there is documented evidence of such transfers. In fact, in a three-year analysis of kanamycin and neomycin resistant bacterial populations collected from GM maize and non-GM crop fields, no differences in bacterial genotypes were observed. Interestingly, bacterial populations from the GM maize field tested negative for the presence of neomycin phosphotransferase II (nptII) marker gene [As for ker gene . Thus, hOne of the concerns about large scale cultivation of transgenic crops is the possibility of a progressive depletion and loss of biodiversity as landraces are replaced by more potent GM crops. This is obviously an important concern and GE crop improvement programs must take it into consideration. Regarding engineering for virus resistance, a case can be made that introduction of virus resistance genes may preserve biodiversity by introducing resistance to highly susceptible cultivars, thereby rescuing them from virulent viruses. A classic example is the high-quality tomato variety named “San Marzano”, which is popular with the canning industry but is highly susceptible to viruses, especially cucumber mosaic virus (CMV). The absence of natural resistance to CMV almost resulted in the abandonment of this variety in the Mediterranean, especially in southern Italy . ExtinctThe possibility of recombination occurring between the transgene and infecting viral genomes, giving rise to new and more devastating strains, is another risk that has been expressed regarding virus resistant transgenics. Recombination and reassortment (for segmented genome viruses) between viruses in mixed infected plants are frequent occurrences and drive virus evolution , as has Curiously, integration of viral genomes into plant genomes is not limited to DNA viruses and retroviruses. In fact, non-retroviral integrated RNA virus sequences (NIRVs) are widespread in plant genomes . While tnptll, which is routinely included in transgenes, is unlikely to cause a negative effect to humans and animals as reported in two reviews [The possible adverse effects of transgenes to humans and animals, as well as to the plants have been expressed. However, an investigation of GM maize in rats either showed no negative effect  or the t reviews ,84.Toxicity of transgenes to the transgenic plant is increasingly unlikely since constructs are now routinely screened for the ability to target host genes, thanks to the availability of plant genome sequencing data. Furthermore, transgenic plants are rigorously screened for any morphological abnormalities. Taken together, the risks of transgene causing negative effects to humans and animals are overstated, especially for GM crops containing transgenes of viral origin.Fundamentally, there are two GMO regulatory models: preventive (USA/Canada model) and precautionary (European Union model). Most other countries employ a combination of the two approaches, depending on the product being regulated and the socio-political pressures on the regulators by policy makers. In the precautionary approach the priority is to anticipate and guard against environmental contamination . This apIn the USA, three federal agencies enact regulations on both conventional and GM crops  Figure . The FooGM crop regulation in Canada is product-based as in the USA, thus, GMO crops undergo the same review process as other new agricultural products . CorrespIn the EU, The European Food Safety Authority (EFSA) assesses and provides scientific advice to risk managers on any possible risks that the deployment  of GM crops may pose to humans, animals, and the environment  Figure . EFSA’s As for South Africa, GM crops are regulated under the GMO Act, which oversees research and development, import/export, production, consumption and other uses of GMOs and their products . The GMOAs shown in Carica papaya) was one of the first virus-resistant transgenic plants to be approved in response to the PRSV epidemic in Hawaii (USA) in 1990s. This is because of the inability to develop resistance through conventional breeding to contain the epidemic. Transgenic papaya lines developed under the trade names “Rainbow” and “SunUp” and expressing the coat protein gene of PRSV were first introduced in Hawaii in 1998 [Papaya ringspot virus (PRSV)-resistant papaya  worldwide [TMY” and “NewLeaf™ Y”, respectively [Potato leaf roll virus (PLRV) and potato virus Y (PVY) are arguably the two most important viruses of potato  production worldwide [Bemisia tabaci) and it causes characteristic symptoms of yellow-green mosaic leaves, distorted pods and stunted growth [Bean golden mosaic virus (BGMV) and related viruses are considered the most destructive viruses of bean  was one of the first crops that was genetically engineered in the early 1990s in an effort to contain epidemics of zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and cucumber mosaic virus (CMV) [Squash (us (CMV) . Like otus (CMV) . Subsequus (CMV) .Cassava is a staple food crop that is resilient to climate change and cultivated in several tropical regions of Africa, Latin America, and South Asia. However, it is affected by at least 20 different viral diseases, of which cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are the most damaging ,122,123.The need to achieve food sustainability and food security in an ever-increasing world population in a changing climate is central to current crop improvement efforts. Viral diseases constitute a major constraint to crop production across all geographic regions. Although conventional breeding has been key to the management of numerous viral diseases, it has its limits. Some of the disadvantages of antiviral plant breeding include absence of natural resistance, complexity, and difficulties in introgressing available resistance genes to cultivars by crossing, amount of time required to produce a cultivar, and the cost of sustaining an effective breeding program. Many of these drawbacks are overcome by the use of GE. However, the overarching challenge regarding GM crops is acceptance of the technology by the public. Yet, there has been no evidence of adverse consequences GM crops and products to human health from its consumption, as confirmed by credible international organizations ,128.Unfortunately, policies regulating GM crop cultivation vary considerably from country to country. The USA approach that is product-based appears to be more forward thinking, as it assesses the risks of the product and not the process. Thus, GM foods are considered not to be fundamentally different from other foods in terms of overall composition, and therefore there is no need for legislation specifically dealing with GM foods. In this case, regulation focuses on the nature of the final food product rather than the process by which the food product is made . Thus, pWhile it must be stressed that some of the concerns raised about GM crops are founded and should be addressed prior to release, a lot of the opposition is either out of ignorance or it is outright manipulative. For example, as discussed in this review, there are claims that GM crops adversely affect humans and animals , yet theIt must be emphasized that much of the hostility towards GM crops has been from Europe, and that this attitude is usually relayed to developing countries, especially sub-Saharan Africa. To leverage new discoveries in genomics and biotechnology and improve crop production, the research community will need to properly communicate new technologies to the public. Incidentally, attitudes towards GM crops in Europe appear to be changing. For example, a recent survey of EU citizens by Eurobarometers shows an increasing acceptance of GM crops  and this


Phytophthora pluvialis has been characterized and designated as Phytophthora pluvialis RNA virus 1 (PplRV1). The genome of the PplRV1 reference genome is 6742 bp that encodes two predicted open reading frames (ORFs). ORF1 and ORF2 overlap by a 47 nt “slippery” frameshift sequence. ORF1 encodes a putative protein of unknown function. ORF2 shows high similarity to the RNA-dependent RNA polymerase (RdRp) of other dsRNA viruses. Phylogenetic analysis of the putative PplRV1 RdRp and its most closely related viruses showed PplRV1 is distinct from other known viruses (below 33% amino acid similarity), which indicates this virus may belong to a new virus family. Analyses of the geographical distribution of PplRV1 in relation to two genetically distinct classes of its host revealed two corresponding genotypes of the PplRV1 (termed a and b), which share 92.3% nt identity. The reference genome for the second genotype is 6760 bp long and a prediction of its genetic organization shows three ORFs, with ORF2 being split into two ORFs, ORF2a and ORF2b, that is conserved in seven of eleven genotype b isolates. Additionally, a quick and simple diagnostic method using qPCR has been developed, which is suitable for large scale screens to identify PplRV1 in Phytophthora.A new dsRNA virus from the oomycete  Phytophthora is a genus in the family Oomycota, which was previously classified within the fungi domain and separated owing to their highly divergent genome sequence [Phytophthora species are plant pathogens and cause enormous economic losses in crops worldwide. The most notorious plant pathogen, Phytophthora infestans, causes potato late blight which in the 1840s triggered a series of events that led to several million people dying from starvation [Phytophthora agathidicida, the causal agent of the devastating disease kauri dieback which threatens kauri tree  populations of high ecological and cultural relevance in New Zealand [sequence . Most Pharvation . Another Zealand ,4,5.Phytophthora pluvialis is a pathogen causing red needle cast disease. In 2008, a severe needle disease of Pinus radiata caused by a new and undescribed Phytophthora was reported in New Zealand [P. pluvialis was isolated from stream, soil and canopy drip in tanoak-Douglas fir forest in Oregon (USA) and described as a new pathogen species within the Phytophthora clade III [P. pluvialis was first recognized in the North Island of New Zealand when an outbreak of red needle cast disease on pine trees (Pinus radiata) occurred [ Zealand . In 2013lade III . A year occurred ,8.P. pluvialis isolates from New Zealand, which suggested P. pluvialis was potentially introduced from the USA into New Zealand on two separate occasions [A study using single nucleotide polymorphism (SNP) and a minimum spanning network identified two clusters of ccasions . A subseccasions .Phytophthora since the first virus was identified in 2005. Originally, an endornavirus was isolated from a Phytophthora isolate from Douglas fir [Phytophthora isolate from Douglas fir but also in Phytophthora ramorum [Phytophthora cactorum and designated Phytophthora cactorum RNA virus 1 (PcRV1) [Totiviridae [Phytophthora [Increasing numbers of viruses have been reported in glas fir  and nameglas fir . PEV1 ha ramorum . In 2020 (PcRV1) . Phylogeiviridae . To dateophthora ,16,17,18Phytophthora species. For example, within Phytophthora infestans alone four viruses have been described. An RNA virus was reported in P. infestans and designated Phytophthora infestans RNA virus 1 (PiRV-1) [P. infestans in 2018 [Several RNA viruses have been found in single (PiRV-1) . PiRV-1 (PiRV-1) . Another in 2018 . In 2012 in 2018 . The gen in 2018 .Phytophthora species. In 2011, Liu et al. found gemini-like sequences integrated into the mitochondrial genomes of P. infestans and Phytophthora sojae [P. infestans mitogenome shares approximately 70% similarity with the one found in P. sojae. It has been assumed that the replication-initiation sequences of some circular DNA viruses, such as geminiviruses, are frequently transferred into a wide variety of eukaryotic hosts [Beside RNA viruses, DNA viruses have also been reported in ra sojae . The seqic hosts . Furtheric hosts .Dichanthelium lanuginosum, a tropical panic grass, colonized by the fungus Curvularia protuberata which itself is infected with the C. protuberata thermal tolerance virus, is more heat-tolerant than plants harboring virus-free C. protuberata [Cryphonectria parasitica, which causes chestnut blight [P. infestans [P. infestans isolates that contained PiRV-2 produced a larger number sporangia. Further surveys found PiRV-2 was present in most strains of a predominant clonal lineage of P. infestans in North America, which suggested PiRV-2 could be the cause of the resurgence of potato late blight in the 1980s–1990s [Viruses or viral-like sequences can contribute to host-adaptive phenotypes, including thermal tolerance and virulence. For example, tuberata . A groupt blight ,22. Becat blight . Additionfestans . Compare0s–1990s . Mycovir0s–1990s .Phytophthora viruses on Phytophthora, either hypervirulence or hypovirulence, may lead to alternative options to control pathogenic Phytophthora. This study discovered and characterized an RNA virus associated with New Zealand P. pluvialis, which provides fundamental knowledge and may identify leads for developing a biocontrol method to manage pathogenic P. pluvialis.Understanding of the potential impacts of An initial screen for double-stranded RNA elements was performed with isolates NZFS 4014 to 4019 collected in Gisborne, New Zealand, using the CF-11 cellulose chromatography protocol , with suP. pluvialis isolates collected in the New Zealand regions of Auckland (n = 1), Bay of Plenty/Wanganui (n = 1), Coromandel (n = 2), Gisborne (n = 7), Hawke’s Bay (n = 1), Nelson (n = 3), Northland (n = 5), Taranaki (n = 1), Taupo (n = 1) and Wairarapa (n = 1) were obtained from the New Zealand Forest Research Institute (Scion) . Among these P. pluvialis, two are isolates whose cluster is unknown, 12 isolates belong to New Zealand cluster 1 (NZ1), and nine isolates belong to New Zealand cluster 2 (NZ2) [Pseudotsuga menziesii), and 22 isolates were sourced from radiata pine (Pinus radiata)  and 250 mg/L rifampicin  at room temperature in the dark. 2 (NZ2) . Moreoveradiata) . IsolatePhytophthora mycelia were collected for dsRNA extraction. Mycelia were ground in liquid nitrogen using a sterilized pestle and mortar. The dsRNA was extracted using Double-RNA Viral dsRNA Extraction Mini Kit  according to the manufacturer’s instructions for plant tissue.After 12–14 days, 100–400 mg P. pluvialis clusters NZ1 and NZ2, respectively [Virus dsRNA was reverse-transcribed into complementary DNA (cDNA) with primer SEQ31 designed based on the viral sequences initially isolated using SuperScript™ IV First-Strand cDNA Synthesis Reaction Kit . Subsequently, it was amplified via Polymerase chain reaction (PCR) with primers SEQ31 and SEQ6663 . The fulectively . To obtaP. pluvialis isolates were fully/near completely sequenced.The purified PCR products were sequenced via a commercial Sanger sequencing service . Subsequently a sequencing strategy using eight distinct overlapping PCR was established . The sequence assembly were aligned to the preliminary contigs 1 and 2 assembled from the initial screen. The ORFs in the virus genome were predicted by Geneious 10.0.9.Low-quality ends of the Sanger sequences amplified were trimmed and P. pluvialis isolates. Virus genomes from P. pluvialis isolates belonging to NZ1 and NZ2 [Virus genomes from NZFS 3052 and NZFS 4018 were used as the references for virus sequence assembly from additional  and NZ2  were ali and NZ2 ,28 via G and NZ2 ,30 via GA conserved region within the virus genome was found through NCBI Conserved Domain Database  searchesPhytophthora/Nothophytophthora isolates was used for the virus screen, including 25 P. pluvialis isolates, nine other Phytophthora species/isolates and one Nothophytophthora species. The 25 P. pluvialis isolates were collected in Bay of Plenty (n = 2), Gisborne (n = 1), Hawke’s Bay (n = 3), Nelson (n = 5), Rangitikei (n = 3), Taranaki (n = 1), Taupo (n = 3), Waikato (n = 1), Wairarapa (n = 2), Wanganui (n = 1), Wellington (n = 3) in New Zealand. Two P. cactorum isolates were collected in the Bay of Plenty in New Zealand. Two Phytophthora cryptogea isolates were collected in the New Zealand regions of South Canterbury and Auckland. Two Phytophthora multivora isolates were collected in Auckland and the Bay of Plenty, and three Phytophthora hybrid isolates were collected in Auckland, the Bay of Plenty and Northland regions in New Zealand.A panel of Phytophthora total RNA was extracted using RNeasy Mini Kit  following the manufacturer’s instructions. The RT- quantitative PCR (RT-qPCR) primers and probes were designed by Primer3 [Phytophthora total RNA was used in each 20-µL reaction. The optimized reactions are summarized in Phytophthora/Nothophytophthora strains  and contig 2 (2865 nt) shared approximately 30% amino acid identity with PiRV-3 hypothetical proteins and RdRp, respectively [Initial screen for ectively  while coectively .P. pluvialis NZFS 3052. The PplRV1_3052 genome is 6742 nt with a GC content of 55.4%, and two predicted ORFs. ORF1 is 3549 nt in length, starting at nt position 455 and terminating at 4003. A BLASTx analysis of the predicted ORF1 amino acid sequence showed similarity to hypothetical proteins found in other RNA viruses, including PiRV-3. The ORF2 is 2631 nt in length, starting at position 4039 and terminating at position 6669. The ORF2 is predicted to encode an RdRp, and contains six predicted motif regions. A 47 nt slippery sequence was predicted to span ORF1 and ORF2 from nt 3955 to nt 4001 , was confirmed by RT-PCR. The complete PplRV1 reference genome sequence was obtained through Sanger sequencing using primer walking and 5′ RACE  from RNA nt 4001 A. A sign nt 4001 A. TogethP. pluvialis population, we isolated PplRV1 from a range of P. pluvialis isolates spanning both New Zealand clusters. Two distinct genotypes of PplRV1 (PplRV1a and b) were identified from the two host clusters  with complete correspondence, respectively. PplRV1 (genotype a) from isolate NZFS 3052  was chosen as the reference genome for the first genotype of the virus, and we propose to designate the virus genotype associated with NZFS 4018  as the reference genome for PplRV1b.To investigate the genetic diversity of PplRV1 in New Zealand PplRV1_4018 has a genome size of 6760 nt with a GC content of 55.2%. Three ORFs including two sub-ORFs of ORF2 were identified in PplRV1_4018. ORF1 has a length of 3549 nt, starting at position 455 and terminating at 4003. ORF2a has a length of 1041 nt, which begins at position 4039 and ends at 5079. ORF2b is 1671 nt in length, from position 5051 to position 6721. Both ORF2a and ORF2b show similarity to parts of RdRp albeit encoded in two ORFs B. ORF2a PplRV1a and PplRV1b share 92.3% nt identity. Although PplRV1b has a different genome size and GC content from those of PplRV1a, it contains a perfectly conserved slippery sequence of the same size and in the same position and therefore identical stem-loops A,B.virus 10 (HTV10), Phytophthora infestans RNA virus 3 (PiRV-3), and Bremia lactucae associated fusagravirus 1 (BlaFV1). Within these related viruses, CvTV belongs to Totiviridae, and CtV1, SfV1 and BlaFV1 are dsRNA viruses, while WiV28, and SV, KV HTV10 and PiRV3 are unclassified RNA viruses. PplRV1a RdRp has very low residue/base similarity percentage with these other viruses. The highest amino acid similarity is 32.64% with dsRNA virus, BLaFV1, and the lowest amino acid similarity is 24.94% with unclassified RNA virus, SV (BLASTx searches using the protein sequence of the putative RdRp encoded by PplRV1a ORF2 found similarities with the RdRp regions of Wuhan insect virus 28 (WiV28), Circulifer tenellus virus 1 (CtV1), Spissistilus festinus virus 1 (SfV1), Culex vishnui subgroup totivirus (CvTV1), Stinn virus (SV), Koroku virus (KV), Hubei toti-like irus, SV . Alignmeirus, SV . Fully cirus, SV . As typiirus, SV , while iirus, SV . Except P. pluvialis isolates were fully/near completely sequenced. Except PplRV1_3052 and PplRV1_4018, PplRV1 from NZFS 4015, NZFS 4016 and NZFS 4019 were also fully sequenced. The genome size of PplRV1_4015, PplRV1_4016 and PplRV1_4019 is 6758, 6758 and 6756 nt, respectively. The near-complete genomes of other PplRV1 genotypes are about 6500 nt  rather than the NZ2 PplRV1-derived reference genome PplRV1b (92.0–93.2%). Conversely, NZ2-derived PplRV1 sequences are most similarity to those of PplRV1b (99.2–99.5%) rather than PplRV1a (91.2–92.5%) . The intra-variation and inter-variation between PplRV1 genotypes validate the link between the host clusters and virus genotypes.The data indicate two distinct PplRV1 variants associated with the two 2–92.5%) . The datP. pluvialis in New Zealand.A phylogenetic analysis also supports that NZ1-associated PplRV1a sequences and NZ2-associated PplRV1b sequences are clustered in two different groups, which has a 100% support for the grouping . In the Phytophthora trnM-trnP-trnM region was developed [Phytophthora and allows the simultaneous detection and quantification of the host.To facilitate further studies of PplRV1, a qPCR diagnostic for the rapid detection and quantification of the virus was developed. Based on the genome comparisons, the ORF1 region of PplRV1_4018 was chosen as a target as it is the common part of the virus genome, and generic for both PplRV1a and PplRV1b genotype viruses. Furthermore, a multiplex assay that targets the eveloped . This hoPhytophthora/Nothophytophthora isolates  has the putative RdRp split between ORF2a and ORF2b that is conserved in seven of eleven genotype b isolates. The qPCR multiplex diagnostic developed for the rapid detection and quantification of both the virus and host revealed that all P. pluvialis isolates tested (n = 25) were infected with PplRV1.A novel RNA virus designated as PplRV1 was isolated from P. pluvialis population shows that P. pluvialis cluster 1 (carrying PplRV1a) was the initial introduction into New Zealand [P. pluvialis cluster 2 is diverse from that of cluster 1, the phylogenetic analysis revealed that all P. pluvialis cluster 1 and cluster 2 isolates were grouped in a single terminal clade, which indicates there was only a single incursion of P. pluvialis into New Zealand [As reported previously, the genetic diversity of  Zealand . Althoug Zealand . Since R Zealand . PplRV1 A −1 frameshifting potentially occurs in a “slippery” overlap sequence between ORF1 and ORF2 in PplRV1. Two stem-loop structures were also predicted downstream of this slippery sequence, which supports the possibility of ribosomal frameshifting in the progress of PplRV1 polyprotein translation. Stem-loops relevant to slippery sequences are also known as “stimulatory” RNA secondary structures, and are able to modulate the −1 frameshift efficiency . TherefoA BLAST search using the PplRV1 ORF2 sequence revealed that it has similarity to PiRV-3, totiviruses and the RdRps of unclassified RNA viruses. However, phylogenetic analysis of PplRV1 RdRps and these related viral RdRps found PplRV1 does not cluster with any of those viruses . MoreovePhytophthora using a single sample. Compared with the traditional diagnostic method of using standard PCR, our diagnostics can be used to determine the titers of PplRV1 by using an appropriate standard. For that purpose, the target regions of the assay were placed on a plasmid, and appropriate dilutions were created to represent specific copy numbers.In this study, a diagnostic method using qPCR technique has been developed, which can accurately and quantitatively detect PplRV1 and P. pluvialis and not in other Phytophthora/Nothophytophthora species pure cultures so far, despite ten isolates being from other species. It would be beneficial to investigate a broader variety of Phytophthora species and other oomycete species as well as environmental samples, i.e., infected pine needles, and water and soil samples from pine woods. A comprehensive survey of PplRV1 in isolates from North America could shed light on the biology of PplRV1, and potentially on the history of its introduction to New Zealand. With the help of PplRV1 diagnostics described in this work, we can obtain a better understanding of the prevalence and host range of PplRV1.We detected PplRV1 exclusively in P. pluvialis cluster , indicating co-evolution of PplRV1 with its host P. pluvialis cluster since its introduction to New Zealand [P. pluvialis isolates. The lack of PplRV1 movement between host clusters could be caused by exclusion , a lack of a vector for PplRV1 in New Zealand, or the inability for PplRV1 to move by anastomosis between the two P. pluvialis clusters present in New Zealand. It is unlikely that PplRV1 forms a virion and the unencapsulated RNA may be unstable in the environment. Whether the initial introduction of P. pluvialis carried PplRV1, or if this association occurred shortly after entry into New Zealand, will require further research to confirm. Importantly, research is needed to survey the distribution of PplRV1 in North America, and to determine its genomic features. In addition, determining whether PplRV1 is present in other Phytophthora species within New Zealand, the USA and other countries, especially those growing Pseudotsuga menziesii and Pinus radiata, would provide a clearer picture of its worldwide distribution and introduction history into New Zealand.Investigation of PplRV1 (a and b) distribution correlated entirely with the  Zealand . The resThe unique genetic structure of some PplRV1b isolates leads to additional questions, for example, how are ORF2a and ORF2b expressed to form a functional RdRp enzyme? A thorough biochemical investigation of the RdRp enzymes from isolates with the different genome structures could illuminate the function and potential evolutionary relevance of the different genome organizations described in this work.P. pluvialis strains isolated in North America, and the comparison of their genomes with the ones described here, will provide further insight into the function of the virus.An analysis of the presence of PplRV1 in P. pluvialis isolate without one of the PplRV1 virus variants has been observed. The generation of such a PplRV1-free isolate will be crucial for future work to understand the ecological and evolutionary roles of the virus.The significance of PplRV1a and b for its host remains unclear. To date, no C. parasitica which was used as a biocontrol agent of chestnut blight in Europe [P. infestans and could impact the ecological fitness of P. infestans [P. pluvialis results in severe red needle cast disease in New Zealand, future research on the virus transmission and whether PplRV1 affects host virulence  will be important for the development of a potential biocontrol for red needle cast disease.Some mycoviruses affect the host virulence and may play an important role in the development of the controlling methods for plant diseases. For example, CHV1 decreases the pathogenicity of its host n Europe ; Conversnfestans . Since P


Marigold plants with symptoms of mosaic, crinkle, leaf curl and necrosis were observed and small RNA and ribo-depleted total RNA deep sequencing were conducted to identify the associated viruses. Broad bean wilt virus 2, cucumber mosaic virus, turnip mosaic virus, a new potyvirus tentatively named marigold mosaic virus (MMV) and a new partitivirus named as marigold cryptic virus (MCV) were finally identified. Complete genome sequence analysis showed MMV was 9811 nt in length, encoding a large polyprotein with highest aa sequence identity (57%) with the putative potyvirus polygonatumkingianum virus 1. Phylogenetic analysis with the definite potyviruses based on the polyprotein sequence showed MMV clustered closest to plum pox virus. The complete genome of MCV comprised of dsRNA1 (1583 bp) and dsRNA2 (1459 bp), encoding the RNA-dependent RNA polymerase (RdRp), and coat protein (CP), respectively. MCV RdRp shared the highest (75.7%) aa sequence identity with the unclassified partitivirus ambrosia cryptic virus 2, and 59.0%, 57.1%, 56.1%, 54.5% and 33.7% with the corresponding region of the definite delta-partitiviruses, pepper cryptic virus 2, beet cryptic virus 3, beet cryptic virus 2, pepper cryptic virus 1 and fig cryptic virus, respectively. Phylogenetic analysis based on the RdRp aa sequence showed MCV clustered into the delta-partitivirus group. These findings enriched our knowledge of viruses infecting marigold, but the association of the observed symptom and the identified viruses and the biological characterization of the new viruses should be further investigated. Recently in China BBWV-2 has been reported in many new host plants and here BBWV-2 is firstly reported in marigold, which enriches our knowledge of its host range.CMV is the type member of the genus B and II . In thisRaphanus . FurtherRaphanus . SequencPotyvirus is the largest group of the plant positive-strand RNA viruses, containing 183 currently approved species [The genus  species . In this species . Compare species , and thePartitiviridae have been shown to infect plants, fungi, and protozoa, and phylogenetic analysis based on amino acid sequences of RdRPs shows all the members cluster into five clades corresponding to the five partitivirus genera, which could be also characterized with the hosts and genome segment and protein lengths [Deltapartitivirus. Conserved genomic features in delta-partitiviruses are also found in MCV. Partitiviruses consistently mediate persistent infections of their hosts and are classically considered to have few, if any, deleterious effects on host cells and are usually asymptomatic in plants. Moreover, some plant partitivirus genes appear to be associated with mutualistic host effects [Homona magnanima population [Viruses in the family  lengths . With th lengths ,57. In t effects ,59, but  effects , but theSmall RNA analysis shows that vsiRNAs derived from the MMV genome are mainly 21- and 22-nt in length A, suggesPotyvirus and marigold cryptic virus in the Deltapartitivirus. This work expanded the current knowledgeof the virome of marigold. However, the association between the observed symptoms and these viruses should be further investigated.In summary, mixed infection of five viruses infecting marigold was identified by deep sequencing, including TuMV and BBWV-2, first reported in marigold, and two new viruses, marigold mosaic virus in the


To improve the rough texture and hypoglycemic ability of sea buckthorn insoluble dietary fiber (IDF), a novel combined modification method was developed in this study. The IDF was treated with ball milling and cellulase treatment to obtain co-modified insoluble dietary fiber (CIDF). The physicochemical and functional properties of IDF, milled insoluble dietary fiber (MIDF), and CIDF were studied. After treatments, MIDF had smaller particle sizes and a looser structure, and CIDF exhibited a wrinkled surface and sparse porous structure according to scanning electron microscopy (SEM) and X-ray diffraction. Compared to IDF, MIDF and CIDF showed improved water-holding, oil-binding, and swelling capacities, improved by 16.13, 14.29, and 15.38%, and 38.5, 22.2, and 25.0%, for MIDF and CIDF, respectively. The cation exchange ability of modified samples showed improvement as well. Treatments also changed the fluidity of MIDF and CIDF. Due to the smaller particles and increased stacking, the bulk density (BD) and angle of repose of MIDF improved by 33.3% and 4.1° compared to IDF, whereas CIDF had a looser structure and thus decreased by 7.1% and 13.3° with increased fluidity. Moreover, the modification also enhanced the effects of CIDF on glucose adsorption, glucose diffusion inhibition, starch digestion inhibition, starch pasting interference, and α-amylase activity inhibition. In summary, IDF modified by ball milling combined with cellulose treatment could be developed as a functional ingredient for regulating glucose content. Diabetes is a major public health problem and a substantial global economic burden . RelevanHippophae rhamnoides) belongs to the Elaeagnaceae family, and is widely cultivated in China, Central Asia, and Mongolia . The cellulase  was obtained from Beijing Sunon Co. Ltd. The heat-stable α-amylase  was purchased from Sigma-Aldrich . The glucosidase was bought from Nanjing Duly Biotech Co., Ltd. . Other chemicals used in this study were all of the analytical grade.The IDF of sea buckthorn seed meal was prepared according to the previous method  with somThe IDF was processed with a planetary ball mill  and mixed two times with the quantity of zirconia balls (3–5 mm diameter) in a 250-ml vessel at 600 rpm/min for 6 h. An intermittent method (run for 60 min and then cool it for 10 min) was carried out to maintain the milled insoluble dietary fiber (MIDF).The cellulase treatment was based on the method of the previous study  with somA scanning electron microscopy (SEM)  was used to analyze the surface morphology of the samples. Referring to a previous study , 2 mg ofThe X-ray diffraction (XRD) patterns of samples were obtained from X-ray diffraction , at the operating voltage and current at 40 kV and 40 mA, scanning from 5 to 70° with the angle step of 0.02°.−1.The Fourier transfer-infrared spectrometry (FT-IR) spectrum of samples was detected using an FT-IR instrument  according to the previous study  with KBr0), mixed with 15 ml distilled water, and then incubated at room temperature for 2 h. After the treatment, the mixture was centrifugated at 4,000 rpm for 15 min. The supernatant was then removed. The wet weight with the centrifugal tube was recorded as M1. The supernatant was dried to a constant weight and recorded as M2. The waterholding capacity (WHC) was calculated by the following equation:Referring to the previous method , 1 g (M)0) and mixed with 25 ml corn oil, and then, the mixture was shaken in a 36°C water bath for 2 h. After the treatment, the oil was removed by centrifuging the mixture at 4,000 rpm for 30 min. The wet weight of the tube and sample was recorded as M1. The oil-binding capacity (OBC) was calculated by the following equation:According to the previous study , 1 g (M)0. The volume of the cylinder was recorded again as V1 after being kept for 24 h at room temperature. The swelling capacity (SC) was calculated by the following equation:Following the reported method , 1 g (M)1, then filled with IDF, and then weighed and recorded again as W2. The BD was calculated by the following equation:The bulk density (BD) of samples was calculated according to the method reported in the previous study . A volumReferred to the reported method , a funne− was detected, and then dried at 60°C. After that, 0.1 g of acidic sample was weighed and dispersed into 50 ml 5% NaCl solution, titrated with 0.01 M NaOH, and the pH of the mixture was recorded.Based on the previous method  with somAccording to the previous method , 1 g of The pasting property of corn starch (CS), the mixture of CS with different concentrations of CIDF , and a mixture of IDF/CS, MIDF/CS, and CIDF/CS were determined by a rapid viscosity analyzer (RVA) . The test procedure of the RVA assay was performed according to a previous study  test was performed using statistical software . All trials were done in triplicate, and the statistical means and standard deviations were calculated and shown.A schematic showing the preparation method of IDFs is shown in The microstructure of IDF is known to change depending on the modifications. This also influences the physicochemical properties and special functional groups including -OH and -COOH . ConsequScanning electron microscopy images of IDF, MIDF, and CIDF were taken and shown in −1 were mainly caused by O-H in hydroxyl groups. It was apparent that the modifications enhanced it, which might be related to the increase in specific surface and exposed hydrogen bonds . When the addition exceeded 10%, the viscosity of the fiber was not sufficient to compensate for the reduction in starch pasting and the viscosity decreased significantly (p < 0.05).Starch is one of the most widely used ingredients in food, and the addition of IDF affects its physicochemical properties, such as water retention, thermodynamic properties, rheology and texture, and in particular the pasting property . The stuSubsequently, IDF, MIDF, and CIDF were added at 10% of starch mass to evaluate the effects of different fibers on the starch pasting properties. The results are shown in The previous study has reported that IDF from wheat bran with modification by several methods also had great capacity in absorbing glucose in solution due to the changes in structure . Differein vitro , Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University (BTBU), Yulin City Science and Technology Plan Project (No. CXY-2020-074), and Science and Technology Planning Project of Xining (No. 2021-Y-15).MY, TY, and HY were employed by Puredia Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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