Viruses are among the most infectious pathogens, responsible for the highest death toll around the world. Lack of effective clinical drugs for most viral diseases emphasizes the need for speedy and accurate diagnosis at early stages of infection to prevent rapid spread of the pathogens. Glycans are important molecules which are involved in different biological recognition processes, especially in the spread of infection by mediating virus interaction with endothelial cells. Thus, novel strategies based on nanotechnology have been developed for identifying and inhibiting viruses in a fast, selective, and precise way. The nanosized nature of nanomaterials and their exclusive optical, electronic, magnetic, and mechanical features can improve patient care through using sensors with minimal invasiveness and extreme sensitivity. This review provides an overview of the latest advances of functionalized glyconanomaterials, for rapid and selective biosensing detection of molecules as biomarkers or specific glycoproteins and as novel promising antiviral agents for different kinds of serious viruses, such as the Dengue virus, Ebola virus, influenza virus, human immunodeficiency virus (HIV), influenza virus, Zika virus, or coronavirus SARS-CoV-2 (COVID-19). Viruses are among the most infectious pathogens, responsible for the highest number of deaths worldwide. Although the pathogenic mechanisms of viruses are diverse, all existing viruses need a host to maintain their existence . The comViral infections result in millions of deaths and huge economic losses annually. In recent years, important examples are the viruses Ebola, Zika, SARS, MERS or recently the actual pandemic SARS-CoV-2 coronavirus (which have infected more than 140 million and killed more than 3 million people so far (data from 21 April 2021) .This means that novel systems for rapid and efficient detection of viruses are critical to control the infection spread. From the last decade, nanotechnology has signified an important advance in the development of nanomaterials for detection devices ,10,11,12Glycan molecules have been demonstrated to have a very important role in many biological recognition processes . Indeed,Therefore, in this review we summarized the recent advances in the fabrication of novel functionalized glyconanomaterials, evaluating the effect of different glycomolecules as biomarkers in the recognition of human viruses and the important role in the detection of viral glycoproteins in relevant cases such as the Dengue virus, Ebola virus, human immunodeficiency virus (HIV), influenza virus, Zika virus, and coronavirus SARS-CoV-2 as well as their novel application as potential antiviral agents. Different sections will be focused on proteins, oligosaccharide-functionalized nanomaterials, and glyconanoparticles as biosensors for virus detection and glyconanoconjugates for virus inhibition . AnotherOne of the most promising strategies for biosensor devices to detect viruses is based on protein-based biosensors ,18,19,20In such cases, some of the main examples using nanomaterials involve the functionalization with lectins, proteins which specifically interact with glycans from glycoproteins. In particular, Concanavalin A (ConA) is a well-known lectin, which binds specifically α-D-mannosyl and α-D-glucosyl residues , which aThus, Oliveira and coworkers  developeDifferences in viral envelope are an essential feature of immune response differentiation after arbovirus infection. The structural components of the viral envelope can be used to identify and differentiate arboviruses . TherefoThe degree of affinity of the sensor to the virus decreased from ZIKV > DENV2 > CHIKV > YFV. The sensor shows the highest response to ZIKV due to the interaction of ConA with specific carbohydrate residues present in the structural glycoprotein E of ZIKV b. Also, Another approach in the design of protein-based biosensors focuses on the use of proteins mimicking the carbohydrate-binding receptor of the virus.Fetuin-A  is a 64-kDa glycoprotein that is found in relatively high concentrations in human serum and recently has been used for the new selective nanobiosensor developed for influenza virus . The strTherefore, this could be a potential diagnostic sensor for clinical applications, because of the advantages of speed, the use of very small sample volumes, and its suitability for the selective determination of the virus from complex real samples.Another strategy for detection of viral infection involves the development of an imm unosensor capable of monitoring cytokines . CytokinA graphene-nitrocellulose ink was printed on a flexible polyimide film (Kapton) in an IDE pattern by aerosolizing graphene and depositing the aerosol mist in highly focused lines. The electrode was chemically modified to generate carboxylic groups as functional groups on the surface for covalent immobilization of anti-bovine antibody (IFN-γ or IL-10) which was then used for detecting the corresponding IFN-γ and IL-10 cytokines a. The syMost viruses recognize glycans on glycoproteins and glycolipids of the mammalian cells as receptors , especiaThus, Yates and coworkers  developeCarbon nanotubes were functionalized with pyrenemethylamine by π–π stacking interaction of the pyrene moiety and the SWNT. Then chemical modification was performed to incorporate heparin to the surface. This oligosaccharide is highly sulfated, having the highest negative charge density of any known biological macromolecule, whereas DENV envelope protein has positively charged β-strands located within a well-conserved binding pocket for electrostatic interaction with receptor sulfate groups. Therefore, interaction between the glycan and virus is detected by the resistance on p-type SWNTs. This method was highly selective since a functionalized sensor responded to the presence of DENV but not to influenza H1N1 . A limitHowever, actually the most important developments in glycan-sensors for virus detection have been focused on the coronavirus SARS-CoV-2, responsible for the pandemic respiratory disease COVID-19 (starting in December 2019) , which tIn this context, sialic acids were determined as a receptor (or recognition motif) for the glycoproteins from the coronavirus, especially the spike protein . In SARSThus, recently Gibson and coworkers  describeN-hydroxyethyl acrylamide), PHEA, was chosen as the polymer to give colloidally stable particles , by the displacement of an ω-terminal pentafluorophenyl (PFP) group, and immobilization onto gold particles via an α-terminal thiol. In the next reaction step, poly  to ensure glycosylation. The assays revealed that in this system sialic acid gave the strongest response and consequently was taken forward. With the successful identification of sialic acid as a target ligand, its application as the capture unit in lateral flow was examined. It is important to highlight that the performance does not only depend on the affinity of the capture ligand but also on the flow of the particles. For this reason, “half” lateral flow assays were established to optimize the particles. The negative test line was  2,3-silyllactose-BSA, which the nanoparticle should not bind to . The positive control was immobilized SARS-CoV-2 and the sialic acid-AuNPs 35 nm nanoparticles flowed over them. The lateral flow devices could clearly detect the virus-like particles at a concentration of 5 µg/mL protein. Sialic acid particle system had a clear preference for SARS-CoV-2, demonstrating selectivity in this glyconanoparticle system. This data supports the notion that the terminal sialic acid is the key binding motif. Therefore, this system can detect the spike glycoprotein from SARS-CoV-2 virus under 30 min, thus being interesting for the creation of rapid point-of-care diagnostics in a format which requires no infrastructure and limited training.Other glycans with a relevant role in cell communication, signaling and adhesion, also acting as immuno-modulators during infectious diseases are phosphoglycans such as glycosylphosphatidylinositol (GPI) . This moThus, recently a glycobiosensor based on synthetic GPI was developed by Orozco and coworkers  Figure . The sysT. gondii) was attached to screen-printed gold electrodes (SPAuEs) through a linear alkane thiol phosphodiester and the interaction with antibodies was detected and quantified by electrochemical impedance spectroscopy (EIS). No significant increase in the Rct (charge transfer resistance) value was observed when the GPI-electrode was incubated with the non-reactive serum whereas an increase was clearly observed with the reactive serum.This phosphoglycan -3-trimethyl chitosan (HTCC) cationic nano/microspheres were prepared by crosslinking of chitosan with genipin, followed by reaction with glycidyltrimethyl-ammonium chloride (GTMAC). After cytopathic and PCR analyses, these glycoparticles adsorbed in a strong and selective way human coronavirus NL63 (HCoV-NL63).Therefore, another strategy associated with detection of immunoresponses involves the fabrication of oligosaccharide nanoparticles in antitumor therapy ,41. DextGlycomolecules conjugated with nanostructures, such as nanoparticles have been also investigated for multiple applications, in particular for novel antiviral therapy.In this sense, sulfated glycopolymers such as heparin and carrageenin have been reported to interact with several types of viruses. However, inherent heterogeneity and toxicity of these naturally occurring glycopolymers may hamper clinical applications . To overThe recognition between cell surface glycans and carbohydrate binding proteins in pathogens and host cells is often the first stage of many infectious processes. Blocking these interactions provides a strategy to inhibit pathogen adhesion and subsequent infection. Importantly, this approach can prevent the virus from mutating and developing resistance . DC-SIGNIn this sense, recently Martin and coworkers  synthesi50 values as low as 0.37 μg/mL.The results showed that both the nanocarbon platforms  and the multivalent glycan display (glycodendrimers or glycofullerens) strongly influenced the glyconanomaterials’ ability to interact with DC-SIGN and prevent viral infection: glycoconjugates based on MWCNTs functionalized with glycofullerenes were found to be the most potent inhibitors, with ICGold nanoparticles (GNPs) displaying glycans have been extensively studied as multivalent glycomaterials for sensing and biomedical applications . Zhou anThis review emphasized the most recent advances in the design and application of different glyconanomaterials for the detection and inhibition of human viruses. This nanotechnological approach, in which it combines the power of nanomaterials, for example nanoparticles—which have been demonstrated in the last decade to be an important tool for biomedical applications—with glycan molecules—which have a key role in biological recognition systems—opens up timely a future way forward in biomedicine. Advantages of this methodology are for example high sensitivity and efficiency, whereas a potential disadvantage in the case of therapy application is the possible cytotoxicity of these nanomaterials.Novel systems using tailor-made oligosaccharides or particular proteins were emphasized with their highly sensitive, rapid, and economic features for potential industrial diagnosis kits, in particular of high relevance in the case of the actual pandemic (SARS-CoV-2 virus detection).


Cucumis species were reported as resistant. Germplasm accessions of Cucumis melo, as well as C. anguria, C. ficifolius, C. myriocarpus and C. metuliferus, were mechanically infected with isolates belonging to the European and Asian strain of CGMMV and screened for resistance by scoring symptom severity and comparing the accumulation of virus by qRT-PCR. The wild species C. anguria and C. ficifolius showed no symptoms and did not accumulate CGGMV following inoculation, while C. metuliferus was highly susceptible to the isolates of both strains of CGMMV. The virus accumulated also in C. myriocarpus and the European isolate produced symptoms, but the Asian isolate did not. Thirty C. melo accessions were susceptible to CGMMV. An isolate-dependent expression of symptoms was observed in 16 melon accessions: they showed mild and severe symptoms at 14 and 21 days after inoculation with the European and Asian isolate, respectively. Freeman’s Cucumber showed few or no symptoms following inoculation with the isolate of either CGMMV strain. This particular accession also showed reduced virus accumulation, whereas most other tested germplasm accessions showed significantly higher viral loads and, therefore, may well be a candidate for breeding programs aiming to reduce the losses produced by CGMMV with resistant commercial melon cultivars.Cucumber green mottle mosaic virus (CGMMV) is a severe threat to melon production worldwide. At present, there are no cultivars available on the market which show an effective resistance or tolerance to CGMMV infection; only wild  Tobamovirus, family Virgaviridae [Diseases produced by viruses cause economic losses in commercial cucurbit production around the world . Among taviridae , and cauaviridae . In cucuaviridae . In meloaviridae . It is maviridae .CGMMV was first described infecting cucumbers in England in 1935 by Ainsworth . Its incChenopodium amaranticolor, on which local lesions appear only when isolates of the Asian strain are inoculated [Worldwide, CGMMV isolates are grouped in two major clusters based on biological differences and genome sequences: a first cluster constitutes the European strain and includes most isolates from France, the Netherlands and Uzbekistan. A second cluster is formed by isolates from Asian countries, such as Japan and South Korea. Spain is currently the first country where both these strains have been described co-infecting the same crops and in the same region . Althougoculated .Commercial tomato and pepper hybrids that carry virus-resistant genes successfully control tobamoviruses in solanaceous crops. Resistance is an important factor that determines concentrations of virus in several virus-cucurbits host plant pathosystems. This happens, for example, with cucurbit yellow stunting disorder virus and Watermelon mosaic virus in melon ,12,13. HC. melo and eight wild Cucumis species. Six wild species were found to be resistant  but only a few accessions of the Indian momordica and Kachri melon groups displayed a partially resistant response, with recessive and polygenic control [C. figarei and C. zeyheri [makuwa group [Rajamony et al., screened 187 accessions of  control ,17,18. O zeyheri . Apart fwa group . Resistawa group . These pC. melo, selected to represent the variability of the species, and four wild Cucumis species: C. anguria, C. ficifolius, C. metuliferus and C. myriocarpus. We report on the evolution of symptom expression for both virus isolates in all accessions, and that of the viral loads on a representative number of accessions.In the present paper we inoculated isolates belonging to the European and the Asian strain of CGMMV in a collection of 47 accessions of Single inoculations in melon accessions with CGMMV isolates CGMMV-SP and CG-SPCu16 that represent the European and the Asian strains, respectively, produced symptoms that were scored according to the scales from 0 to 3, as defined in the Materials and Methods .C. melo accessions which belonged to the Asian ameri and the Spanish ibericus types, such as Birjucekutskaja and Piñoncillo, but also to the Asian Dudaim types, Indian Kachri and momordica and African and Indian wild agrestis, as well as to oriental conomon melons.The time of appearance of these symptoms in individual plants was variable, but most of the replicate plants of each accession showed similar symptoms at 21 dpi . The plaibericus Rochet accession BGV004884 and the makuwa accession PI 420176 from Japan) while two others showed intermediate symptoms with CG-SPCu16 and severe symptoms with the European isolate . Two additional melon accessions were also susceptible, but the Indian momordica PI 381789 displayed intermediate symptoms with both virus isolates, while the Khira melon from India PI 271332 had a variable response to the virus isolate CG-SPCu16. A virus isolate-dependent expression of symptoms was observed in 16 melon accessions: they showed mild and severe symptoms at 14 and 21 dpi when infected with the European and Asian isolate, respectively . The only accession resistant to both isolates of the virus was the conomon Japanese accession Freeman’s Cucumber. Regarding wild types, C. metuliferus expressed severe symptoms with the isolates of both CGMMV strains, and C. myriocarpus displayed intermediate symptoms following the inoculation with CGMMV-SP and none with CG-SPCu16. The inoculation with CGMMV produced no symptoms in C. anguria, and C. ficifolius   .Cucumis species, C. anguria and C. ficifolius tested negative for CGMMV, whereas CGMMV concentrations in C. metuliferus and C. myriocarpus ranged between the orders of e5 to e7 with the CGMMV-SP, and e3 to e4 with CG-SPCu16. Melon accessions that showed severe symptoms with both virus isolates had similar viral loads at 14 and 21 dpi (p < 0.05) (2 at 14 dpi with CGMMV-SP), and CGMMV was not detected at 21 dpi in plants inoculated with CG-SPCu16 (Among the wild d 21 dpi . However < 0.05) . FreemanG-SPCu16 . The aveG-SPCu16 .We tested a collection of 47 melon accessions for resistance to CGMMV. The majority of genotypes were very susceptible after mechanical inoculation, as was made clear by the expression of typical CGMMV symptoms and the accumulation of virus in apical leaves. The high susceptibility observed along the whole range of diversity of this species confirmed that this virus represents a major threat to melon cultivation. The majority of the accessions tested were susceptible to the European (CGMMV-SP) and the Asian (CG-SPCu16) isolates of CGMMV, which is relevant to countries such as Spain, where both strains occur, sometimes even in the same greenhouse  Among thagrestis accessions PI 164797, PI 185111 or PI 536476) and Kachri (PI 614521), groups, where resistance had been previously described. Other melon accessions that were susceptible to both main virus isolates were some Indian accessions of momordica and conomon that otherwise are multi-resistant to other viruses: Shiro Uri Okayama, which is resistant to cucumber mosaic virus (CMV) [Equally susceptible as most Spanish landraces were also most of the accessions of Asian origin that belong to many melon groups such as ameri, Dudaim, even certain wild types (wild us (CMV) , accessius (CMV)  and momous (CMV) ,24,25. Rus (CMV) .chinensis Korean accession PI 161375 “Sonwang Charmi” and the flexuosus melon BGV004853 showed the opposite behavior, expressing strong symptoms with the European isolate after 21 dpi, and intermediate symptoms with the Asian isolate after 14 dpi. PI 420176 and PI 161375 are resistant to CMV and multiresistant, respectively [The expression of symptoms following inoculation with CGMMV was variable in other melon accessions. The makuwa melon PI 420176 already expressed severe symptoms with the Asian isolate after 14 dpi, and intermediate symptoms with the European isolate after 21 dpi, similarly to the Rochet type. The ectively . The resectively . Accessiectively , and whecantalupensis and reticulatus (Europe) and ameri, chandalak and casaba (Asia and North-Africa) melon types  is shown here, for the first time, to be resistant to CGMMV, and this entry is of enormous interest as it crosses perfectly with melons. With respect to wild species, we confirm the resistance of escribed ,35. ThisC. melo groups, including accessions of the momordica, Kachri, and makuwa, the melon groups in which CGMMV has been previously reported, and additional inodorus-ibericus, cantalupensis-reticulatus, casaba, Asian ameri-chandalack, flexuosus, Dudaim, conomon, chinensis and wild agrestis types not screened previously [Cucurbits breeding group at COMAV-UPV. The type and origin of these accessions is shown in anguria, C. ficifolius, C. myriocarpus and C. metuliferus coming from the COMAVs germplasm collection were also included.Forty-seven accessions were selected from a previously established melon core collection to represent some of the most important eviously ,31,32,36Cucumis accessions, the isolates were propagated after mechanical inoculation in cucumber (cv. Cumlaude) in a plant growth chamber. Infected plants were grown in an insect-proof greenhouse where temperature was partially controlled (25–30 °C). Approximately 3 weeks after inoculation, the leaves of plants that showed typical symptoms of virus infection were used as the virus source [In order to investigate differences in resistance, we mechanically inoculated plants with each one of two isolates that represent the two different strains of CGMMV: the strain of European origin (CGMMV-SP) (GenBank GQ411361) and that of Asian origin (CG-SPCu16), as described by . Before s source .For each virus isolate, 0.5 g of leaf tissue was taken at 5 weeks after sowing and from the second leaf down from the plant apex which displayed CGMMV symptoms. The tissue was homogenized in 1.5 mL of 50 mM sodium phosphate buffer (pH 7.0) and inoculated mechanically by rubbing 150 µL of the extract onto leaves dusted with carborundum powder. At least ten plants of each accession were inoculated with each virus isolate, and two plants with sodium phosphate buffer as controls on 7th of October 2019. All inoculated plants were maintained in an insect-proof greenhouse under controlled conditions.. Cycling conditions consisted of incubation at 95 °C for 5 min, 45 cycles of 95 °C for 5 s and 60 °C for 30 s. Relative CGMMV levels were calculated using the 2−ΔCt expression, a variation of the Livak method [−ΔCt = 2−−Ct (reference gen)). Three technical replications were performed per sample and the tests were run on an ABI Prism 7000 DNA sequence detection system  as followed: 10 min at 50 °C, 1 min at 95 °C, 40 cycles of 15 s at 95 °C and 1 min at 60 °C.Inoculated plants were evaluated for the expression of CGMMV symptoms at 14 and 21 days post inoculation (dpi) using the following scale: 0 (symptomless), 1 , 2  and 3  . The prek method ,39, wherResistance was evaluated as the response of the host plant to virus infection, estimated from symptom severity and the viral titers in all inoculated plants. The interaction effects from inoculated isolates of virus, and dpi on viral loads, were investigated using the Kruskal–Wallis test. All effects were tested at the 5% significance level. Analyses were performed using Statistics 9.1 statistical software.


Cyclops vicinus, which is vulnerable to fish predation. Long-term data (January 2014 to February 2019) showed that C. vicinus was abundant in winter when the foraging activity of fish was lower. This pattern was reversed in spring, summer, and autumn. C. vicinus is consumed frequently by fish because it has a body size larger than that of other cyclopoid copepods (Mescyclops leuckarti and Thermocyclop sp.). In this respect, winter formed a seasonal refuge when C. vicinus populations could grow efficiently. In addition, there was an abundant phytoplankton presence (Cyclotella sp. and Rhodomonas sp.) in winter. These species formed a food source that supported the population growth of C. vicinus. The evolution of the predator avoidance mechanisms of prey contributes significantly to the security of local biodiversity and the stability of the freshwater food web.Predator avoidance mechanisms play a critical role in the survival and stable population growth of prey. Here, we describe a new defense strategy for Cyclops vicinus in the Upo Wetlands, South Korea, is an evolved temporal defense mechanism to avoid fish predation. Long-term data (2014–2019) showed that fish consumed the most cyclopoid copepods from spring to autumn. Lepomis macrochirus preferentially consumed C. vicinus; thus, C. vicinus density was lower from spring to autumn. However, C. vicinus was abundant in winter when fish consumed fewer copepods. Nauplii density began to increase in late autumn (October–November), and their population growth was fueled through consumption of Cyclotella sp. and Rhodomonas sp. Culture experiments showed that Cyclotella sp. contributed more to the growth stage (copepodite or subadult) after nauplii than Rhodomonas sp. C. vicinus density was lower in the winters of 2013 and 2016 when the densities of these phytoplankton prey species were lower. In summary, although winter conditions were suitable for copepod survival and population growth, C. vicinus relied heavily on the diversity and species composition of its food sources. The winter dominance of C. vicinus could increase regional biodiversity and contribute significantly to the stability of the freshwater food web.Frequent predation induces various defense strategies in prey, including morphological changes or migration patterns in zooplankton. We hypothesized that the winter dominance of  Predation is an important factor that affects local population size, species diversity, and biological interactions ,2. ImbalMoina micrura is a behavioral mechanism for avoiding predation [Daphnia spp.; [In freshwater ecosystems, foraging activity by predator has led to the development of different defense strategies by prey species. In terms of the evolutionary arms race ,14, the redation . Littoraia spp.; ,23,24). ia spp.; ,26.Lepomis macrochirus), an exotic species, established itself in the wetlands of South Korea by occupying mainly vegetated areas where other predatory fish are restricted [L. macrochirus is capable of efficient foraging activity, even with a moderate aquatic macrophyte abundance [Micropterus salmoides) was introduced around the same period as L. macrochirus and interfered with their establishment by consuming L. macrochirus; however, the high quantity of aquatic macrophytes that is supported by Korean wetlands greatly hindered the predatory activities of M. salmoides and contributed to the stable establishment of L. macrochirus [L. macrochirus populations only have a minor refuge effect [The refuges for zooplankton introduced in previous studies are mainly created by spatial differences in their environmental characteristics. These spaces provide an advantage for zooplankton because chemical factors (such as low dissolved oxygen) or physical factors  strongly restrict predator distributions ,28. Howestricted . L. macrbundance . Largemorochirus . Althougrochirus ,32,33, ve effect . The pree effect .L. macrochirus has been reported in various studies [In this study, we report on a new defense strategy by which copepod communities avoided predators. Copepods have the position as primary consumers in freshwater food webs, and since they are a food source for fish and invertebrates, various defense strategies are needed to avoid predators. Previous studies suggest that behavioral reactions or distribution changes such as jumps or accelerated movement , migrati studies ,41, whicCyclotella sp. and Rhodomonas sp.).The aims of this study were to elucidate (1) the seasonal changes in cyclopoid copepod communities in relation to environmental variations, (2) the consumption pattern of copepods by fish predators, and (3) the influence of winter growth on copepods in relation to their food sources . To address these objectives, we surveyed the environmental variables, cyclopoid copepods, fish, and phytoplankton in the Upo Wetlands in southeastern South Korea. During this long-term study (January 2013‒February 2019), we analyzed the seasonal responses of copepods to fish predation and food sources . We found high copepod consumption by fish during all seasons except winter, in addition to a close relationship between the winter distribution of cyclopoid copepods and the seasonality of some phytoplankton species (2) is larger than the combined area of the other three wetlands (1.05 km2). In the past, the Upo Wetlands were intermittent wetlands with very large water level changes depending on flooding in Topyeong Stream; however, it has maintained its current form and water depth since the construction of an embankment [Phragmites australis, Paspalum distichum, Zizania latifolia, Spirodela polyrhiza, Salvinia natans, Trapa japonica, Ceratophyllum demersum, and Hydrilla verticillata, which cover the water surface from spring to autumn. The four distinct seasons in the middle and lower reaches of the Nakdong River lead to the seasonal growth and dynamic succession of various biological communities, including aquatic macrophytes. Water temperatures range from 10 to 28 °C from spring to autumn, providing suitable conditions for the growth of various aquatic organisms; however, in winter, a low water temperature of 1.6 °C hinders the growth of most aquatic organisms.The Upo Wetlands are riverine wetlands located in the middle and lower reaches of the Nakdong River . The majbankment . The watC. vicinus.Biweekly monitoring was conducted for 7 years (January 2013‒February 2019). Prior to the investigation, we selected six sampling points (1 m × 1 m quadrats) within the wetland characterized by similar environmental variations . Three quadrats were used to investigate environmental variables and cyclopoid copepods. The remaining three quadrats were used to collect biological items for stable isotope analysis, which was performed to identify the potential food source for http://www.kma.go.kr access on 8 January 2021), which was collected from Hapcheon Station .In order to explain the seasonal and yearly distribution of cyclopoid copepods in relation to environmental variables in the Upo Wetlands, we used rainfall data during field survey time (January 2013‒February 2019) obtained from the Korea Meteorological Administration  were measured at the three sampling points. Water depth was measured using a 1.5 m steel ruler. Water temperature and DO were recorded using a DO meter . A conductivity meter  and an Orion 250A pH meter  were used to determine the conductivity and pH, respectively. Turbidity was measured using a turbidimeter . For cyclopoid copepod enumeration, we collected 10 L water samples using a 10 L column water sampler  from each quadrat. The sampler was placed vertically into the water to collect copepods from the entire water column of the quadrat. Water samples were filtered through a 70 µm mesh plankton net and the filtrate was preserved in formalin . CopepoL. macrochirus) collected in each season using a methanol–formaldehyde solution (3:1). We identified and counted all food items  in the gut contents of L. macrochirus, and the abundance of each food item was calculated as the abundance per weight of the gut. We used 30 L. macrochirus individuals in each season to identify food consumption patterns.In order to understand the influence of the winter distribution of copepods on fish predation and food sources, we investigated the seasonality of fish and phytoplankton. Fish were collected using a cast net (7 mm × 7 mm) and scoop net (5 mm × 5 mm) along 200 m transects in each season  from January 2014 to February 2019. Fish and copepods were collected on different days. The cast net and scoop net were used for 30 min and 20 min, respectively. Fish samples were identified to the species level according to Kim and Park  and the For phytoplankton enumeration, we collected 1 L surface water samples from January 2013 to February 2019 and fixed the samples with neutral Lugol’s solution. All phytoplankton samples were kept in a laboratory refrigerator at 4 °C and analyzed as soon as possible after collection. Phytoplankton identification and enumeration were carried out using a microscope , and 1000× magnification was used for species identification. After allowing 48 h for phytoplankton samples to deposit, the samples were concentrated to 30 mL using a siphonage method and stored at 4 °C. Then, each sample was added to a 0.1 mL counting chamber (20 × 20 mm) and phytoplankton species were identified and counted according to Hu et al. [C. vicinus groups . Suspended particulate organic matter , epiphytic particulate organic matter , benthic particulate organic matter , and C. vicinus groups  were sampled in each winter month  from 2014 to 2019. These samples were collected three times per month in addition to the regular monitoring program. We collected 5 L of surface water per sample (n = 4). To process the SPOM samples, micro- or macroinvertebrates were initially removed using a 32 µm mesh size plankton net, and the water samples were then filtered through GF/F glass fibers . The surfaces of the submerged parts  of the macrophytes present at the sampling points were gently brushed in a tank filled with distilled water to retain the EPOM. The BPOM was obtained by carefully floating the bottom substrate. Care was taken not to mix organic matter other than the bottom surface in this process. Similar to the SPOM processing step, plant debris and zooplankton were removed using a 32 µm mesh size plankton net. To obtain C. vicinus groups, a total of 10 L water was filtered through a plankton net (70 µm mesh size) using a 10 L column sampler . To prevent injury in dense samples of C. vicinus groups, filtration using a plankton net was conducted before the detailed separation of the C. vicinus group in the laboratory. Copepod groups were sorted into nauplii, copepodites, and adults using fine tweezers and spoids. In January and February, when the nauplii and copepodites densities were low, only adults were used for stable isotope analysis.Stable isotope analysis was implemented to identify the potential food items of −1 HCl to remove inorganic carbon. The samples were then rinsed with deionized distilled water to remove the acid. All samples were freeze dried and then ground with a mortar and pestle. Powdered samples were frozen at −70 °C until analysis. Carbon and nitrogen isotope ratios were determined using continuous-flow isotope mass spectrometry . Prior to analysis, the samples were placed overnight in a sealed CF-IRMS through which 99.999% He was flowing at a few mL/min. Instrument linearity  was tested daily and confirmed to be <0.03‰/nA over the 1–10 nA range. 100 (±10) μg silver-encapsulated cellulose samples , producing approximately 4–6 nA signal at the collectors, were loaded in a 99-position zero-blank CF-IRMS and converted to a mixture of carbon monoxide, carbon dioxide, water, and hydrogen gases over glassy carbon chips in a quartz tube at 1080 °C, within a stream of 99.999% carrier He flowing at 110 mL/min. Data are expressed as the relative (‰) difference per mL between the sample and the conventional standards of Pee Dee Belemnite (PDB) carbonate for carbon and atmospheric N2 for nitrogen, according to the following equation:13C or 15N, and R is the 13C:12C or 15N:14N ratio. A secondary standard of known relationships to the international standard was used as the reference material. The standard deviations of δ13C and δ15N for 20 replicate analyses of the peptone  standard were ±0.1 and ±0.2‰, respectively.The POM samples  were treated with 1 mol LC. vicinus growth experiments, C. vicinus and two food algae species (Rhodomonas sp. and Cyclotella sp.) were collected from the sampling point in the Upo Wetlands, where field investigations were conducted. We selected the collection period considering the seasonal variability of each target species in the field. C. vicinus was obtained using 150 µm mesh plankton nets during late autumn (late October‒early November 2016). In the laboratory, adult females (length: 1.2–1.8 mm) with eggs were sorted and anesthetized with carbonated water. Eggs were removed with a dissecting pin and maintained in small dishes containing 25 mL of aged tap water at room temperature (18–20 °C). Nauplii that hatched after approximately 2 days were transferred within 12 h of hatching into 500 mL sterilized beakers filled with 500 mL distilled water.For the Rhodomonas sp. and Cyclotella sp. were separated from phytoplankton samples collected in November 2016 and January 2017, respectively. To increase the abundance of the target species, the water samples collected from the Upo Wetlands were filtered using a 32 µm mesh plankton net. Rhodomonas sp. and Cyclotella sp. were extracted from phytoplankton samples using a microscope . To provide stable food for C. vicinus growth, Rhodomonas sp. and Cyclotella sp. were maintained using a plant growth chamber , with 50 photon flux density (µmol·m−2·sec−1), and a 12 L:12 D light: dark cycle in Bold’s Basal Medium [Rhodomonas sp. and Cyclotella sp. were cultured at 10 °C and 5 °C, respectively, considering their seasonal distribution patterns within the Upo Wetlands.l Medium . Rhodomo4 cells mL−1, 1 × 105 cells mL−1, and 4.5 × 105 cells mL−1) were established according to Hopp and Maier [C. vicinus growth. A concentration of 4.5 × 105 cells mL−1 was considered sufficient for C. vicinus survival and population growth (22.5 mg CL−1), and 1 × 104 cells mL−1 was insufficient (0.5 CL−1). The required quantity of algae was calculated from the relationship between algal density and carbon content [C. vicinus growth experiment was prepared such that water samples obtained from the sampling points where C. vicinus was collected were filtered twice using 0.45 µm mesh filter paper. We determined that there were no algae or organic matter available as food for C. vicinus in the filtered water using a microscope . To avoid pH changes owing to photosynthesis, we stored filtered water in the refrigerator (2 °C) and removed it just prior to the experiment. Growth experiments were conducted using a plant growth chamber , with a 50-photon flux density (µmol·m−2·sec−1), and a 12 L:12 D light–dark cycle. We placed 20 beakers containing each food concentration, with 50 individual nauplii placed in each 500 mL beaker. Each day during the experiment, we transferred the nauplii and copepodites to a fresh culture medium (filtered field water) that met each food condition, before providing food algae, to maintain the supply of algae under each condition. Growth experiments were conducted for approximately 80 days, and the surviving individuals in five randomly selected beakers were counted every 5 days. We calculated the survival rate (%) as the number of surviving individuals on each day/total number of individual nauplii  in each beaker.Three different food concentration conditions for each algal species  for final analysis. The stress value for the two-dimensional solution was 0.156, which was lower than the generally accepted maximum stress value of <0.2 [p < 0.05 considered significant. NMDS ordination was conducted using the R package “vegan”  to examine the distribution patterns of the cyclopoid copepod groups due to environmental variations. The NMDS ordination plots were generated based on the Euclidean distance, and the goodness of fit was assessed in terms of the loss of stress. Each variation was log transformed after being assessed for normality using the Shapiro–Wilk test. Rare copepod species with observed densities of <50 ind.L of <0.2 . The sigon 2.5–3 ).For statistical analyses of the growth experiment, we applied a one-way nested ANOVA  to explain the survival and growth patterns under different food conditions. Although we prepared 20 replicates (beakers) for each experimental group (three food conditions), pseudoreplication required careful consideration  . TherefoMost of the environmental variables  showed clear seasonal fluctuations . RainfalCyclops vicinus, Mesocyclops leuckarti, and Thermocyclops sp. were the most dominant (96%), followed by M. fuscus and Eucyclops sp. (4%). Long-term monitoring data (January 2013‒February 2019) showed different seasonal variability of the three dominant species (Mesocyclops leuckarti and Thermocyclops sp. were abundant in summer‒autumn (June‒October), while a high C. vicinus density occurred in winter (January‒February). While M. leuckarti and Thermocyclops sp. growth were sustained in summer and autumn in each year, the winter peak of C. vicinus differed each year. C. vicinus was abundant in the winters of 2014, 2015, 2017, and 2018 (mean 220 ind./L); however, densities were relatively low in the winters of 2013, 2016, and 2019 (mean 52 ind./L). Adult C. vicinus were dominated by a high density of females with eggs in late winter (February) and were mainly dominated by females without eggs in early and midwinter  and Thermocyclops sp. (Th) were abundant in summer and were associated with higher water temperature, rainfall, and turbidity. In contrast, a high C. vicinus (Cv) density was found in winter, associated with lower water temperature, rainfall, and turbidity. Nauplii and copepodites distributions were mainly related to autumn.We fitted the five cyclopoid copepod groups to NMDS ordination sites and selected four environmental variables that were significantly correlated with those locations  from the Upo Wetlands during the study period (C. carassius (14.9%). The relative richness of the remaining fish species  was approximately 7%, although this varied over time. The fish community was abundant in summer and autumn and fluctuated every year in spring. In contrast, few fish species were present during most winter periods . The seasonal patterns of these fish communities were similar every year.We collected a total of six fish species , followed by branchiopods (27%). Predation by L. macrochirus on the remaining four food communities  was relatively low. The consumption patterns of copepods and branchiopods for L. macrochirus showed distinct seasonal variability. In summer and autumn, copepod consumption by L. macrochirus was approximately twice as high as that of the branchiopods, whereas, in spring, branchiopod consumption was higher. L. macrochirus consumed more C. vicinus (49.5%) than M. leucarti (31.5%) and Thermocyclops sp. (18.9%)  .C. vicinus in winter  and indicated a shift in the food source composition. However, the close relationship between C. vicinus and SPOM was rare in January and February 2017 and February 2019. During these periods, the δ13C value of SPOM was heavier than that of other winter periods, whereas the δ13C value of C. vicinus was similar to that of other winter periods. Nauplii and copepodites, which declined in December, also relied on SPOM. The δ13C and δ15N values of EPOM and BPOM were always higher than those of SPOM, and their relevance to C. vicinus was very low. The consistent contribution of SPOM to C. vicinus during winter indicated that C. vicinus mainly consumed phytoplankton.The stable isotope analysis results showed the annual consumption patterns of three potential food sources  of n winter . C. viciCyclotella sp. During late autumn and winter (October‒February), Cyclotella sp. abundance was approximately four times greater than that in other seasons (spring to mid-autumn). However, during winter, Cyclotella sp. showed a different distribution pattern every year. The Cyclotella sp. abundance in the winters of 2014, 2015, and 2017  accounted for the majority of the total Bacillariophyceae; however, abundances were lower in the winters of 2013, 2016, and 2019 (January‒February). Chlorophyceae and Cyanophyceae, which had the second-highest abundances after Bacillariophyceae, were mainly abundant in summer. Although the Cryptophyceae abundance was only 0.9% of the total phytoplankton, the seasonal distribution of the dominant species, Rhodomonas sp., was an important factor in explaining the winter dominance of C. vicinus. However, their abundance was lower in autumn  of 2015 and 2018 than in autumn of other years, similar to Cyclotella sp.Phytoplankton in the Upo Wetlands were dominated by Bacillariophyceae (84.5%), followed by Chlorophyceae (11%) and Cyanophyceae (3.6%) . Thus, tC. vicinus growth patterns with the various concentrations of food algae differed significantly depending on the algae species (Cyclotella sp. or Rhodomonas sp.) , while the survival rate gradually decreased over time in treatments with lower food concentrations (1 × 105 cells mL−1 and 1 × 104 cells mL−1). The number of nauplii decreased to less than half within 45 days of the onset of the experiment under the intermediate Cyclotella sp. concentration (1 × 105 cells mL−1), and at the lower concentration (1 × 104 cells mL−1), a less than 50% survival rate was observed 20 days earlier than the intermediate concentration. In contrast, the survival rate of nauplii that had Rhodomonas sp. injected as a food source gradually decreased at all food concentrations. Although the C. vicinus survival rate was greater at the higher concentration of Rhodomonas sp., it was <20% under all food concentrations after 80 days. Interestingly, C. vicinus survival did not decrease for 10 days under high or intermediate Rhodomonas sp. concentrations; however, it began to decrease suddenly after 15 days. At low food concentrations, the survival rate decreased significantly from the beginning of the experiment, and all individuals died within 40 days. C. vicinus survival rates in the three concentrations of both food algae species were significantly different (The nas sp.) . Most naifferent .C. vicinus, M. leuckarti, and Thermocyclops sp.) were identified as dominant species. These species are widely distributed internationally, and their seasonal distribution and food preferences have been reported in various empirical studies [M. leuckarti and Thermocyclops sp. were most abundant during summer‒autumn in the Upo Wetlands. Summer and autumn are beneficial for obtaining energy for spawning because water temperatures are high, and the water is also rich in food sources such as phytoplankton, which increase metabolic activity. Therefore, summer and autumn are the optimal seasons for copepod population growth and development since adequate supplies of food are available, and predators can be avoided.Cyclopoid copepods are frequently found in freshwater ecosystems . They are primary consumers of phytoplankton or bacteria in freshwater food webs and are food sources for fish and invertebrates . Thus, i studies ,57,58. T studies . We founC. vicinus was more abundant during winter than during the general growth seasons (spring‒autumn) in the Upo Wetlands. The main reason why population growth in aquatic animals tends to be rarer in winter is that low water temperatures slow the metabolic rate and also make it difficult to acquire food, which greatly restricts growth and development [C. vicinus in the Upo Wetlands had a relatively long life cycle in winter. This can be attributed to a decrease in metabolic activity at low water temperatures. Nauplii and copepodites were mainly present from late autumn to early winter (late October‒ early December) and were supported by a high density of C. vicinus adults during most winter periods (December‒February). Given that nauplii and copepodites stages are bottlenecks in copepod development, the low nauplii and copepodite densities in most winter periods indicated that minimal C. vicinus reproduction occurs in winter. We speculate that in winter, C. vicinus adults come from the growth of nauplii and copepodites in autumn, which perform minimal reproductive activity during the winter and die off after laying resting eggs in February when water temperatures begin to increase. Based on the seasonal distribution pattern of C. vicinus domination every winter, the resting eggs appeared to remain dormant from spring to autumn and then hatched in late autumn. Although nauplii and copepodites were observed in spring (March‒May), these were considered to be the prior stages of M. leuckarti and Thermocyclops sp., which are mainly distributed in summer and autumn.Interestingly, we found that elopment . In partelopment ,61. HoweC. vicinus would be an inefficient defense strategy if it were used solely to avoid relatively suitable environmental conditions  from spring to autumn. However, the spring‒autumn period in many locations has relatively high densities of other zooplankton communities, which consume similar food sources, as well as being the preferred growth season for predators such as fish which preferentially feed on C. vicinus. This makes spring‒autumn a difficult period for C. vicinus growth and indicates that winter may be an efficient period to avoid excessive interactions (competition or predation). In this location in South Korea, it is clear that the winter distribution of C. vicinus provides an evolutionary advantage to C. vicinus over other seasons. Changes in their seasonal distribution are closely related to the efficiency and persistence of population growth, which is the inherent nature of organisms. This is consistent with the evolutionary direction of almost all life on Earth.This winter distribution of L. macrochirus, a dominant fish species in the Upo Wetlands, consumed more copepods and cladocerans than any other food source. Previous studies have also suggested that L. macrochirus is a representative planktivorous fish, which mainly consumes zooplankton [L. macrochirus consumed more cladocerans every spring than copepods; however, their choice changed to concentrated consumption of copepods in summer and autumn. Choi et al. [L. macrochirus in wetlands in this area may lead to the near extinction of pelagic cladocerans. Pelagic cladocerans have a relatively large body size and frequent movements, making them relatively easy for fish such as L. macrochirus to locate and capture. We also found pelagic cladoceran species such as Simocephalus vetulus and Daphnia sp. in the gut contents of L. macrochirus during spring. Owing to the selective consumption of pelagic cladocerans, high densities of epiphytic cladocerans  are found in the summer and autumn in most wetlands in South Korea. These species are difficult for L. macrochirus to consume.In this study, plankton ,63. Cladplankton . Althougplankton . This foL. macrochirus in summer and autumn was mainly focused on C. vicinus. This selective consumption explains the low C. vicinus densities in summer and autumn. We assume that the higher consumption levels of C. vicinus by L. macrochirus, compared with other cyclopoid copepods (M. leuckarti and Thermocyclops sp.), is due to their ease of discovery. Cyclops vicinus has a larger body size relative to M. leuckarti and Thermocyclops sp., and hence, it can be prioritized during L. macrochirus foraging activities. The size of Cyclops spp. females ranges from 1.2 to 1.8 mm, while female M. leuckarti and Thermocyclops sp. range in size from 0.9 to 1.0 mm and from 0.7 to 0.8 mm, respectively [M. leuckarti and Thermocyclops sp. [C. vicinus is vulnerable to predators even in areas where aquatic macrophytes are abundant. This is likely to contribute to selective food consumption by L. macrochirus in the Upo Wetlands, where aquatic macrophytes are dominant.The consumption of cyclopoid copepods by ectively . Althougectively ,67. The lops sp. ; howeverC. vicinus by L. macrochirus may have induced the greater winter population growth of C. vicinus. Given that consumption of C. vicinus by L. macrochirus was observed to be constant from spring to autumn, it is evident that C. vicinus is also present in spring‒autumn in the Upo Wetlands. However, following the decline in cladoceran populations in summer and autumn, the concentrated consumption of copepods by L. macrochirus interrupts the stable population growth of C. vicinus. In this regard, winter is the only season that C. vicinus is able to avoid predation by fish. Fish, including L. macrochirus, have a low metabolic rate under low water temperatures, making it difficult to carry out efficient feeding activities during winter. Choi and Kim [L. macrochirus is usually concentrated in withered vegetation during winter and that foraging activity and movement are minimal. Thus, the absence of predators in winter is advantageous for C. vicinus, which is strongly influenced by fish predation from spring to autumn. From this finding, we suggest that winter serves as a temporal refuge for stable population growth of C. vicinus.Based on this information, it is logical to assume that the high consumption of  and Kim  and Spee and Kim  suggesteIn the Upo wetlands, phytoplankton were mainly abundant in winter because of the seasonal variability of Bacillariophyceae, i.e., a dominant species of phytoplankton. Most wetlands located in Korea, including the Upo Wetlands, are almost completely covered by aquatic macrophytes from spring to autumn, which reduces light entering the water and strongly restricts phytoplankton growth because of nutrient competition with aquatic macrophytes . Winter C. vicinus distribution in winter. In years when the winter peak of C. vicinus was observed , Cyclotella sp. accounted for >80% of total Bacillariophyceae, while the C. vicinus density was lower in years in which there was a lower Cyclotella sp. abundance . Winter water temperature is an important factor in determining the winter distribution of Cyclotella sp., and we found that the Cyclotella sp. abundance and C. vicinus density were low in years when water temperatures remained relatively high in winter . The matching temporal distributions of Cyclotella sp. and C. vicinus indicate that Cyclotella sp. is closely related to the winter distribution of C. vicinus. Santer [Eudiaplomus gracilis, and M. leucarti prefer food algae with soft shells, while Cyclops abyssorum may consume the dinoflagellate Ceratium furcoides, which has a hard shell. C.vicinus, which is able to feed on prey with hard shells, can grow in winter, unlike M. leuckarti and Thermocyclops sp., because Cyclotella sp. is available as a food source. The results of stable isotope analysis provide a physical basis for the use of Cyclotella sp. as a winter food source for C. vicinus. The SPOM, supported primarily by phytoplankton, clearly contributed as a food source for C. vicinus in winter , when Cyclotella sp. was abundant, but had a weaker association with SPOM in the winters of 2013, 2016, and 2019, when Cyclotella sp. was not dominant in the phytoplankton community. Moreover, the drastic decrease in C. vicinus in February 2018 can also be attributed to the low Cyclotella sp. abundance. The rapid increase in water temperature during this period caused the depletion of Cyclotella sp.; this association was also found in Rhodomonas spp. belonging to Cryptophyceae. Although the Rhodomonas sp. abundance was low during winter, high density was observed in late autumn (October–November), just before winter began. We assumed that the late autumn growth of Rhodomonas sp. influenced the germination and growth of nauplii and copepodites. We found that Rhodomonas sp. was abundant in the late autumn of the previous year when the winter dominance of C. vicinus was observed in January‒February in 2014, 2015, 2017, and 2018.We focused on the fact that the Bacillariophyceae distribution was supported by different species composition in the winter of every year. Interestingly, the difference in the species composition of Bacillariophyceae was closely related to the . Santer  suggesteRhodomonas sp. did not play an important role in the winter distribution of C. vicinus, it contributed as a food source in the early growth stages of C. vicinus . Growth experiments using microcosms showed that the Rhodomonas sp. concentration did not significantly affect C. vicinus growth. Nauplii survived for nearly 10 days at concentrations of 4.5 × 105 cells mL−1 and 1 × 104 cells mL−1 of Rhodomonas sp. ; however, their survival rate declined sharply from 15 days onward. Under the low food concentration, survival rates dropped sharply from the beginning of the experiment, and all individuals were dead within 40 days. This means that Rhodomonas sp. is rarely used as a suitable food source in the growth stage of C. vicinus after 10 days. This result was clearly different in the case of Cyclotella sp., in which survival rates rarely decreased under high food concentrations. From these results, we concluded that the concentration and type of food algae strongly influence C. vicinus growth. Hopp and Maier [C. vicinus nauplii had a very high mortality rate under low food algae concentration conditions, whereas M. leuckarti and Thermocyclops crassus were not significantly affected by the food concentration. The summer diapause of Cyclops spp., which is frequently observed in Europe, is interpreted as a strategy caused by competition or food shortages among the nauplii stages [Although we surmised that nd Maier  also sugi stages . In genei stages ,78. For i stages  explaineC. vicinus. Cyclopoid copepods, which are distributed in spring in various regions, frequently consume rotifers as a food source. Devetter and Seďa [Synchaeta sp. and Polyartra sp.), while hard-bodied rotifer species (K. cochlearis and K. longispina) were consumed more slowly. Choi et al. [C. vicinus is mainly distributed at the nauplii or copepodites stages in late autumn, the possibility of consumption cannot be ruled out. The effects of rotifers as a food source on C. vicinus require further investigation.Although the results are not presented here, we suggest an additional possibility based on previous studies. The rotifers distributed in late autumn may have affected the winter distribution of and Seďa  suggestei et al.  found thC. vicinus populations diverged from the community distribution from spring to autumn. High foraging activities during spring‒autumn, owing to the establishment of exotic fish species such as L. macrochirus, required a new survival strategy for stable C. vicinus population growth. We assumed that the winter distribution of copepods, such as the hypolimnion and vegetated areas, is an evolved form of refuge utilization. The zooplankton refuges reported in previous studies include moving to a specific space or concentrating in one region, based on predators’ habitat preferences. Thus, the presence of predator-avoidance spaces is important for prey such as zooplankton. However, these previous refuges do not have adequate conditions as habitats for zooplankton. It is difficult for phytoplankton, which are food sources for zooplankton, to grow in the hypolimnion and vegetated areas because of the low light, and these places offer inconsistent refuge efficiency owing to seasonal or regional differences in growth. Although these spaces are somewhat suitable for avoiding predators, they have long-term environmental conditions that are adverse for the stable growth of prey populations. From this point of view, winter is an efficient temporal refuge for the copepod C. vicinus, providing adequate food sources and effective predator avoidance. The use of this temporal refuge by C. vicinus is an evolutionary defense strategy to resist the predatory activities of L. macrochirus, which has developed to enable efficient food acquisition in areas with a high aquatic macrophyte cover. L. macrochirus has also evolved to use aquatic macrophytes as refuge to avoid predation by M. salmoides [C. vicinus is positioned at a higher trophic level than its main predator through an evolved defense strategy—winter distribution. In terms of the evolutionary arms race, a predator’s foraging efficiency will need to adapt to the evolution of its prey. However, provided other prey species are present from spring to autumn, L. macrochirus survival will not be threatened, and there may not be a reason to choose the poor conditions of winter distribution. It is clear that C. vicinus will increase its chances of survival by actively utilizing this temporal refuge and also produce more offspring through stable population growth. This evolution of prey defense strategies against predators increases local biodiversity and affects the stable interaction between aquatic organisms in freshwater food webs. Based on the discovery of this new form of refuge, it is necessary to investigate the presence of evolutionary strategies of predators or/and prey in areas where prey–predator interactions are frequent.We surmised that the winter almoides ,63. CurrC. vicinus, which are significantly larger species than other cyclopoid copepods, results in higher predation pressure due to a higher chance of being detected by the main fish predators in the shared summer habitat, which has dense vegetation and low visibility. The fish predator, L. macrochirus, is well adapted to the summer habitat and detects, encounters, and ingests prey most frequently. However, fish predators are seriously impaired in winter temperatures and thus are unable to compete in the predator‒prey arms race. Furthermore, winter offers a stable uncontested food opportunity—phytoplankton abundance that is different from the warm season and specific to the cold season. Culture experiments showed that Cyclotella sp. contributed more to the growth stage (copepodite or subadult) after nauplii than Rhodomonas sp. From this finding, we assume that the timing of peak reproduction and abundance of C. vicinus has changed from the warm to the cold season.A habitat-related disadvantage of


Fragaria × ananassa Duchesne) production given their negative impact on plant vigor and growth. Strawberry accessions from the National Clonal Germplasm Repository were screened for viruses using high throughput sequencing (HTS). Analyses of sequence information from 45 plants identified multiple variants of 14 known viruses, comprising strawberry mottle virus (SMoV), beet pseudo yellows virus (BPYV), strawberry pallidosis-associated virus (SPaV), tomato ringspot virus (ToRSV), strawberry mild yellow edge virus (SMYEV), strawberry vein banding virus (SVBV), strawberry crinkle virus (SCV), strawberry polerovirus 1 (SPV-1), apple mosaic virus (ApMV), strawberry chlorotic fleck virus (SCFaV), strawberry crinivirus 4 (SCrV-4), strawberry crinivirus 3 (SCrV-3), Fragaria chiloensis latent virus (FClLV) and Fragaria chiloensis cryptic virus (FCCV). Genetic diversity of sequenced virus isolates was investigated via sequence homology analysis, and partial-genome sequences were deposited into GenBank. To confirm the HTS results and expand the detection of strawberry viruses, new reverse transcription quantitative PCR (RT-qPCR) assays were designed for the above-listed viruses. Further in silico and in vitro validation of the new diagnostic assays indicated high efficiency and reliability. Thus, the occurrence of different viruses, including divergent variants, among the strawberries was verified. This is the first viral metagenomic survey in strawberry, additionally, this study describes the design and validation of multiple RT-qPCR assays for strawberry viruses, which represent important detection tools for clean plant programs.Viruses are considered of major importance in strawberry ( Fragaria × ananassa Duchesne) is a widely grown hybrid species of the genus Fragaria (family Rosaceae), which is cultivated as a source of food in many parts of the world [F. virginiana from North America and F. chiloensis from South America [http://www.fao.org/faostat/en/#data/QC, accessed on 26 October 2020). California strawberries are available year-round because of the mild climate, which allows harvesting fruits over a long period of time [The garden or commercial strawberry  located in Corvallis . The NCGR contains 2013 active strawberry accessions, representing 46 taxa including species and subspecies . Besides having foundational material for horticultural distribution, the NCGR also separately houses pathogen-positive accessions for virology research.An important resource of genetic material from Various viral diseases have been reported in strawberry and are associated with plant decline and yield loss . The Reliable and efficient diagnostic tests are critical in determining viral infection in strawberry and consequently the control of disease. Multiple laboratory-based tests are available for the diagnosis of viruses in strawberry, mainly involving ELISA and reverse transcription PCR (RT-PCR), previously reviewed in . In contCrinivirus, strawberry criniviruses 3 and 4 (SCrV-3 and 4), were identified in strawberry plants displaying virus-like symptoms [Recently, next generation sequencing or high throughput sequencing (HTS) has been used to reveal the etiology of several diseases in strawberry. For example, a novel virus  was associated with the strawberry decline disease in Canada . Two newsymptoms ,12. In 2symptoms  sequenceTo further investigate the virome of strawberry, we analyzed 45 plants collected at the NCGR by HTS. Multiple variants of viruses known to infect strawberry were annotated and characterized, including near-complete genomes. To complement these HTS results, new RT-qPCR assays were developed and validated for 14 different viruses.w/w) tissue of Phaseolus vulgaris cultivar Black Turtle Soup (BTS). BTS is naturally infected by two different endornaviruses and is used as an internal control in HTS virus screening [In the summer of 2020, 45 accessions of strawberry were received as propagative material from the NCGR, under a USDA Animal and Plant Health Inspection Service (APHIS) movement permit, for HTS analysis at Foundation Plant Services . These 45 plants were previously determined to be positive for viruses or virus-like agents using RT-PCR and bio-indexing. Stolons were propagated under mist and later transferred to single 1-gal pots within a greenhouse. Four months after bud break, 0.7 g of leaf tissue from each strawberry plant was collected and spiked with 5% . Subsequently, cDNA libraries were end-repaired, adapter-ligated by unique dual-indexes, and PCR enriched. Finally, the amplicons were sequenced in an Illumina NextSeq 500 platform using a single-end 75-bp format.Sequenced reads were demultiplexed and adapter trimmed using Illumina bcl2fastq2 v2.20.0.422. To obtain the highest quality contig for an annotated virus, three de novo assemblies were performed: (1) a de novo assembly using SPAdes v3.14.1 ; (2) a dE-value < 1 × 10−5) in this database to a virus known to infect land plants went through a final manual annotation check.To annotate de novo assemblies, all contigs greater than 200 bp were aligned to the June 2020 version of the GenBank non-redundant database of nucleotide sequences using BLASTn with a reduced word size of 7 to identify known viruses. Additionally, to screen for potential unknown viruses, contigs greater than 200 bp were aligned to the June 2020 version of the GenBank non-redundant database of nucleotide sequences using BLASTx. Then, sequences with significant hits  and proteins encoded by the HTS-detected viruses were annotated by ORFfinder and BLASTp analysis. Conserved domains present in the putative proteins were searched in the Pfam database  using HMFollowing the protocol described in , new RT-After initial assay design was completed, in silico analysis facilitated by purpose-built scripts implementing the procedures described below was used to incorporate additional viral genetic diversity into assay design. For each of the viruses, the RT-qPCR assay was first evaluated against all virus sequences in the July 2020 version of GenBank as well as all virus isolates identified during this study. First, a BLAST database search is used to identify and obtain all sequences overlapping the current assay region. To maximize sensitivity, a tBLASTn translated alignment exploiting codon redundancy was used. Once target sequences were collected and their species identification confirmed, all existing primers and probes were aligned to all target sequences covering the assay region. This alignment was accomplished using a script that used an end-gap-free nucleotide alignment to identify the best matching probe, forward and reverse primer sequences to each variant. In each case, the variant sequences corresponding to the matching oligos were collected and analyzed for divergence. Thus, all unique candidate sequence variants were inspected for total or partial divergence to an existing primer/probe sequence. The location and quantity of nucleotide differences and the frequency of the differences were also determined, and the assays were updated with extra primers or probes as needed. A probe or primer was added when more than two nucleotide mismatches or a single mismatch near the end were detected during the sequence comparison. Lastly, in order to reduce the effective number of primers in each reaction, degenerate bases were not used in the oligos.Once assay design was completed, their efficiency was calculated via serial dilutions of 1:1 to 1:10,000,000 and replicated in triplicate; standard curves were generated by the QuantStudio 6 Flex Real-Time PCR System software .Leaf tissue was collected from all the propagated strawberry plants and TNA was extracted as described above but reducing the input material to 0.2 g and omitting the addition of BTS. RT-qPCR reactions were completed in the QuantStudio 6 Flex Real-Time PCR System using the TaqMan Fast Virus 1-Step Master Mix  as per manufacturer’s protocol. Each reaction  included 2 µL of TNA and final primer and probe concentrations of 900 and 250 ῃM, respectively. In addition, the new assays were multiplexed with an 18S rRNA assay to confirm the presence of RNA .https://fps.ucdavis.edu/index.cfm, accessed on 1 July 2021). All 12 plants were previously analyzed by RT-PCR and HTS, revealing the presence of several viruses included in this study.The new RT-qPCR assays were used to test 12 virus-positive strawberry plants located at FPS. Given the nature of FPS as a center for distribution of virus-tested propagation material, occasionally plants infected by viruses are identified and adopted as positive controls in routine screening  among the 45 samples we analyzed. For each distinct infection , we selected the longest sequence as representative of the infecting isolate. Thus, a total of 65 partial sequences, including several near complete genomes, were annotated for their protein coding regions and deposited in GenBank.We used the nucleotide identity to the closest homolog in GenBank as an estimate of the amount of additional nucleotide diversity each of these sequences provide. As indicated in New RT-qPCR assays were developed for SMoV, BPYV, SPaV, ToRSV, SMYEV, SVBV, SCV, SPV-1, ApMV, SCFaV, SCrV-4, SCrV-3, FClLV and FCCV. According to the in silico analysis, most assays needed multiple forward and reverse primers and/or probes  to coverTo confirm the presence of viruses identified by HTS, source plants were analyzed by newly designed RT-qPCR assays. Virus detection was then validated by comparing HTS and RT-qPCR results for each virus-infected sample. In all cases, RT-qPCR and HTS results agreed . LikewisTo further validate the new assays, twelve FPS positive control plants were analyzed by both HTS and RT-qPCR . The folThe primary objectives of this study were to increase available genetic diversity of strawberry viruses by characterizing a diverse set of infected plants and to develop a comprehensive set of RT-qPCR assays for detecting viruses infecting strawberries. The new sequence resources were utilized in the design of the detection assays.The NCGR provided us with a vast collection of accessions of geographically and genetically diverse provenance. From that collection, all strawberry samples identified as pathogen-infected were included in a viral metagenomic survey using HTS. This approach allowed us to efficiently obtain sequences for all viruses infecting the sample. It is the first such survey of this type in strawberry, templated on work done in previous crops ,22,23. TConsiderable additional genetic diversity was observed in several cases of strawberry viruses. In total, 65 partial genome sequences from 14 strawberry-infecting viruses were deposited in GenBank. These represent a substantial contribution to sequence resources for strawberry viruses, increasing the number of sequences deposited in GenBank by approximately 10%. Moreover, these sequences extend the genetic diversity of strawberry infecting viruses characterized in previous amplicon-based studies ,25,26,27Four different criniviruses infecting strawberry were found during the metagenomics survey . Most of these viruses displayed considerable genetic diversity, with the exception of SCrV-3, which lacked divergent variants. In that sense, an outstanding question we believe needs to be addressed is whether SCrV-3 and SCrV-4 represent new virus species or whether they are strains of a known virus. Novel data, like those generated here, will help to understand the biology and molecular biology of criniviruses, which remains to be fully understood .Utilizing these new genetic resources, a comprehensive set of new RT-qPCR detection assays were designed for strawberry viruses. New assays were validated in vitro to confirm their reliability. For example, twelve strawberry samples previously screened for viruses and located at FPS were retested using the newly designed RT-qPCR assays. While we did not compare the sensitivity of our new assays with other PCR-based methods such as end-point RT-PCR, it is generally accepted that RT-qPCR is a highly sensitive method. Multiple primers and probes were employed to detect diverse virus isolates, as demonstrated with a similar assay for grapevine leafroll-associated virus 3, a highly divergent virus infecting grapevine .The high throughput nature of RT-qPCR is desirable for clean plant certification facilities that process large sample numbers. Except for previous work describing RT-qPCR assays for strawberry necrotic shock virus and SCV, there is a scarcity of published assays of this kind for detecting strawberry viruses ,33. The


Bunyavirales order, which is the largest group of RNA viruses, comprising 12 families, including a large group of emerging and re-emerging viruses. These viruses can infect a wide variety of species worldwide, such as arthropods, protozoans, plants, animals, and humans, and pose substantial threats to the public. In view of the fact that a better understanding of the life cycle of a highly pathogenic virus is often a precondition for developing vaccines and antivirals, it is urgent to develop powerful tools to unravel the molecular basis of the pathogenesis. However, biosafety level −3 or even −4 containment laboratory is considered as a necessary condition for working with a number of bunyaviruses, which has hampered various studies. Reverse genetics systems, including minigenome (MG), infectious virus-like particle (iVLP), and infectious full-length clone (IFLC) systems, are capable of recapitulating some or all steps of the viral replication cycle; among these, the MG and iVLP systems have been very convenient and effective tools, allowing researchers to manipulate the genome segments of pathogenic viruses at lower biocontainment to investigate the viral genome transcription, replication, virus entry, and budding. The IFLC system is generally developed based on the MG or iVLP systems, which have facilitated the generation of recombinant infectious viruses. The MG, iVLP, and IFLC systems have been successfully developed for some important bunyaviruses and have been widely employed as powerful tools to investigate the viral replication cycle, virus–host interactions, virus pathogenesis, and virus evolutionary process. The majority of bunyaviruses is generally enveloped negative-strand RNA viruses with two to six genome segments, of which the viruses with bipartite and tripartite genome segments have mostly been characterized. This review aimed to summarize current knowledge on reverse genetic studies of representative bunyaviruses causing severe diseases in humans and animals, which will contribute to the better understanding of the bunyavirus replication cycle and provide some hints for developing designed antivirals.Bunyaviruses are members of the  As the handling of many infectious and highly pathogenic members of the order Bunyavirales is restricted to occurring in a laboratory with a biosafety level −3, or even −4, many studies focused on different aspects of the viral life cycle and pathogenesis have been hampered. Reverse genetics systems established up to now, such as the minigenome (MG), infectious virus-like particles (iVLP), and infectious full-length clone (IFLC) systems, have been efficient tools for researchers to conduct some important experiments at lower biocontainment than at level 3 or 4. Among these experimental systems constructed for bunyaviruses, MG and iVLP systems are used to model their partial life cycle, which enables the dissection of virus invasion (a process the virus enter the host cells), viral genome replication, transcription, ribonucleoprotein assembly, virion packaging, and budding processes. The IFLC system is used to rescue recombinant viruses, which has made it possible to manipulate viral genomes at the RNA/DNA molecular level. These established reverse genetics systems have exerted great potential to help us to better understand the bunyaviruses and to develop antivirals and vaccines.Arenaviridae, Hantaviridae, Nairoviridae, Peribunyaviridae, and Phenuiviridae , medium (M), and small (S), while the viral genome of viruses in Arenaviridae is mainly composed of two segments, i.e., L and S segments. These established systems suggest that the reverse genetics technology has been widely developed for bunyaviruses and widely used by researchers to dissect all aspects of the viral life cycle.Over the past few decades, reverse genetics technology has revolutionized the field of RNA virology, which has made it possible to manipulate RNA viral genomes and rescue genome analogs , infectious iVLP, and recombinant viruses from cDNA clones. Here, we summarize the already established MG, iVLP, and IFLC systems for bunyaviruses causing severe diseases in humans and animals. These bunyaviruses are mostly distributed in the families This review provides an overview of the current status of reverse genetic studies on important bi- and trisegmented emerging and re-emerging bunyaviruses, recent advances in the applications of these systems. Furthermore, we also discuss the limitations of the currently established systems for bunyaviruses and unsolved problems that urgently need to be addressed.Bunyavirales is a newly proposed order consisting of related viruses distributed among 12 families: Arenaviridae, Cruliviridae, Filoviridae, Hantaviridae, Leishbuviridae, Mypoviridae, Nairoviridae, Peribunyaviridae, Phasmaviridae, Phenuiviridae, Tospoviridae, and Wupedeviridae proposed by the International Committee on Taxonomy of Viruses, of which the viral genome is composed of linear, segmented, single-stranded, negative-sense, or ambisense RNA genomes; this order is now comprised of 48 genera and over 383 species , which is endemic across large regions within Western Africa and is estimated to infect hundreds of thousands of individuals annually (Hantaviridae family) is found predominantly in Europe and Asia (Nairoviridae family) can infect human host primarily through infected ticks or by contact with infected hosts or their body fluids and tissues (Peribunyaviridae family), which causes disease in livestock animals, avian species, and humans, is considered the prototype of the bunyavirus as it is the most characterized to date (Phenuiviridae family), which is found in Sub-Saharan Africa, is associated with disease in camels, cattle, goats, and sheep but also causes severe morbidity and mortality in humans  and the nucleocapsid protein (N) in an ambisense arrangement, and L codes for the RNA-dependent RNA polymerase (RdRp) and a zinc-binding protein (Z) for some members in the genera Mammarenavirus and Reptarenavirus  and Phlebovirus (RVFV for instance) encode the NSm protein as part of a precursor with the glycoproteins, whereas NSm of virus from the genus Orthotospovirus (tomato spotted wilt virus for instance) is translated from a subgenomic messenger RNA (mRNA) in an ambisense manner  on both the 5′ and 3′ ends that are denoted as 5′ UTR and 3′ UTR, which play important roles in the virus life cycle by promoting transcription, replication, and encapsidation of the viral genome. Bunyaviruses replicate exclusively in the host cell cytoplasm, with maturation and budding occurring in the Golgi apparatus, and are released pathways . The virpathways . The bunpathways . Initiatpathways . The mRNAccording to the viral replication cycle and the initial transcripts of the cDNA clones, two categories of reverse genetics systems can be constructed for negative-strand RNA bunyaviruses, including sense (−) and antisense (+) MG, iVLP, and IFLC systems, of which the initial transcripts are genomic RNA (vRNA) and antigenomic RNA (cRNA), respectively. It is theoretically possible that both (+) and (−) systems can successfully model the partial or entire virus replication cycle. It has been demonstrated that the sense (+) and antisense (−) reverse genetics systems of Uukuniemi virus (UUKV) show no obviously different efficiencies in rescuing MG RNAs and the recombinant virus .via the intracellular RNA receptor RIG-I  can efficiently substitute for each other to form functional RNP complex , chloramphenicol acetyltransferase, or luciferase. Viral proteins, such as RdRp and N, required for the replication and transcription of MG, are provided in trans form to drive expression of the encoded reporter protein, and viral proteins can be provided via either a co-infecting helper virus or by expression plasmids. MG systems are generally used as model systems for virus genome transcription and replication, and are frequently, although not always, generated as precursors to the development of IFLC systems.MGs or minireplicons are virus-like genome segments  that contain the It is incontestable that a breakthrough came when Palese et al. established the first MG system for the modification of a negative-sense segmented RNA virus, influenza A virus, in 1989 . Howevercis- and trans-acting factors involved in bunyavirus replication and transcription  was pioneered by Dunn et al. in 1995, and they performed a wide-scale mutagenic analysis of viral N to examine the effects of different N mutants on viral RNA transcription and replication using the T7-driven MG system , AKAV, LACV, SBV, CVV, KRIV, and OROV  system, where it was proven that viral N and L are absolutely required and sufficient to reconstitute the transcriptase activity  by Phlebovirus) was also constructed in 2006, and the authors reported that the RNP complexes are incorporated into iVLPs but are not required for the generation of particles. Morphological analysis of these particles by electron microscopy revealed that iVLPs, either with or without MGs, display a surface morphology indistinguishable from that of the authentic UUKV and that they bud into Golgi vesicles in the same way as UUKV does (Phlebovirus), Habjan et al. developed an iVLP system and revealed that a human interferon-induced protein, MxA, inhibits both primary and secondary transcriptions of RVFV (Orthonairovirus), a so-called transcription and entry-competent virus-like particle (tc-VLP) system first developed in 2014, the authors revealed that the endonuclease domain of CCHFV L protein is located around amino acid D693 using this system (Bandavirus) based on the M segment was developed by Rezelj et al. in 2019 and revealed the reassortment potential of SFTSV and the closely related bandaviruses and phleboviruses  and Phlebovirus (RVFV and UUKV) genome reassortment potential , the classical live-attenuated vaccine candidates, including recombinant virus with genome segment rearrangement , the National Natural Science Foundation of China (U20A20135), the National Program on Key Research Project of China (2018YFE0200402), the Innovation Team Project of Hubei Provincial Health Commission (WJ2019C003), the Special Project of Hubei Science and Technology Innovation Platform (2020DFE018), and the Biological Resources Programme, Chinese Academy of Sciences (KFJ-BRP-017-74).The authors declare that the review 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.


If each of the four nucleotides were represented equally in the genomes of viruses and the hosts they infect, each base would occur at a frequency of 25%. However, this is not observed in nature. Similarly, the order of nucleotides is not random . Codon usage and codon order are also nonrandom. Furthermore, nucleotide and codon biases vary between species. Such biases have various drivers, including cellular proteins that recognize specific patterns in nucleic acids, that once triggered, induce mutations or invoke intrinsic or innate immune responses. In this review we examine the types of compositional biases identified in viral genomes and current understanding of the evolutionary mechanisms underpinning these trends. Finally, we consider the potential for large scale synonymous recoding strategies to engineer RNA virus vaccines, including those with pandemic potential, such as influenza A virus and Severe Acute Respiratory Syndrome Coronavirus Virus 2.RNA in Disease and Development > RNA in DiseaseRNA Evolution and Genomics > Computational Analyses of RNARNA Interactions with Proteins and Other Molecules > Protein‐RNA RecognitionThis article is categorized under: Nucleotide sequences in the genomes of viruses and the hosts they infect are highly patterned; this review discusses the causes, consequences and applications of these genomic compositional biases. As examples, internal ribosome entry sites (IRESs), first discovered in the Picornaviridae . Other types of nucleotide biases are also described, such as the 70% GC content of the rubella virus genome ; nine by two codons ; Ile is encoded by three codons; five amino acids are encoded by four codons  and three are encoded by six codons . Representation of each of the degenerate codons can be highly skewed. For example, across the HIV‐1 genome, ~37% of bases are adenine, and adenines are heavily selected for at degenerate positions  metric. Key to genome composition variation is how long a virus has been adapting to its host; for example, a virus that has recently switched host may change its genome composition profile as it adapts to a new host  underlying codon preferences are somewhat speculative, successive codons encoding the same amino acid are more likely to use the same degenerate base and so the same tRNA, possibly allowing for faster recycling of tRNAs, if tRNA diffusion away from the ribosome happens slower than the rate of translation . Given the four bases of RNA—adenine (A), cytosine (C), guanine (G) and uracil (U)—all possible combinations give rise to 16 possible dinucleotides  ratio, represented by the formula:2.4Vertebrate genomic dinucleotide composition has been studied since the 1960s, when the striking observation was made that CpG motifs are under‐represented in vertebrate genomes , which is proposed to have resulted in a loss of CpG motifs from vertebrate genomes over evolutionary time  were translated more slowly  may also regulate translation  varies depending on the tRNA pool available , or by copying errors that then become fixed in the viral genome (selection). We have already considered the A‐rich genome of HIV‐1, and understand that this has arisen due to the mutational activities of the cellular protein APOBEC3G. Similarly, the cellular proteins of the adenosine deaminase acting on RNA (ADAR) family convert adenosine to inosine; evidence for ADAR acting on virally derived nucleic acids was first reported in the genome of vesicular stomatitis virus . In general, recognition of a viral PAMP by a host cell protein  triggers type I interferon signaling; these PRRs may themselves be upregulated by interferon, and in this case are known as interferon stimulated genes (ISGs) . Viral mimicry of genomic CpG suppression was hypothesized to be due to aberrant CpG frequency sensing by an as yet unidentified PRR  and short (ZAPS) forms G(n)CG . Since ZAP recognizes CpG motifs in ssRNA, it is possible that secondary structure of RNA—i.e., CpG context, is an important factor in determining whether CpG motifs can be recognized by ZAP, and there is some evidence indicating this. First, in the crystallography paper characterizing ZAP‐RNA binding, the optimal binding motif for ZAP on RNA was found to be CCG(C/G), (A/U)CG(A/U), (A/U)CG(C/G) and (C/G)CG(A/U) in H1N1 genomes over time  but add AACGAA or UUCGUU motifs  that UpA dinucleotides are cleaved by the cellular ISG RNaseL  to draw significant attention to the subject of large scale genome recoding examined the effects of modifying codon pair bias in the poliovirus genome , displaying a 50% lethal dose (LD50))/PD50 ratio 1000‐fold higher than that of wildtype PR8 virus. This result, and others from the same lab  was recoded to increase disfavoured codon pair usage. Three viral genome segments  were modified and tested separately or in combination for their effects on viral growth characteristics in cells and vaccine potential in mice. Single or combinatorial segment modifications all displayed around 10‐fold defect in multicycle replication assays in vitro. However, in BALB/c mice, the triple reassortant had a 3000‐fold reduction in virus titre at 24 h post‐infection. The triple reassortant virus was further tested for its 50% protective dose . Publications on this so far report mixed results  are avoided.Let us consider the example of IAV as a candidate virus for which a large scale recoded virus could be developed for vaccination. IAV live attenuated vaccines are most commonly produced in embryonated hen's eggs, although there is a motion to switch production to cell culture‐based systems  for any other deoptimization strategy. However, further work must be undertaken to fully understand the mechanisms impacted by this recoding before we can consider using this approach commercially.Figures The authors have declared no conflicts of interest for this article.Eleanor Gaunt: Conceptualization; data curation; formal analysis; writing‐original draft; writing‐review & editing. Paul Digard: Conceptualization; writing‐original draft; writing‐review & editing.lncRNAs regulate the innate immune response to viral infection