Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV) or Grapevine leafroll-associated virus 1 (GLRaV-1) and 3 (GLRaV-3). As long-term investigations have shown, GLRaV-1 (2.1%) occurs most frequently in southwestern German wine-growing regions, whereas GLRaV-3 (<0.1%) is almost never found. However, tests conducted over 12 years indicate that there is no general decline in virus-infected planting material. Thus, it can be assumed that a spread of the viruses via corresponding vectors still takes place unhindered. Beyond the examinations regulated within the German Wine Growing Ordinance, one-time tests were carried out on Grapevine Pinot gris virus (GPGV). This analysis showed that GPGV was found in 17.2% of the samples.Viral diseases in viticulture lead to annual losses in the quantity and quality of grape production. Since no direct control measures are available in practice, preventive measures are taken to keep the vines healthy. These include, for example, the testing of propagation material for viruses such as  Grapevine is the perennial crop with the highest number of intracellular pathogens, of which 86 are viruses ,2. ViralArabis mosaic virus (ArMV)Grapevine fanleaf virus (GFLV)Grapevine leafroll-associated virus 1 (GLRaV-1)Grapevine leafroll-associated virus 3 (GLRaV-3)Grapevine fleck virus (GFkV) (rootstock only)Using healthy starting material is a crucial step in practicing integrated plant management in viticulture. The sanitary status of planting material is particularly essential because grapevine is propagated vegetatively. From 1986, that measure has been regulated in Germany by the Wine Growing Ordinance (Rebenpflanzgutverordnung (RebPflV)) . The freNepovirus and are the most typical representatives of this genus in European vineyards [Xiphinema index and Xiphinema diversicaudatum [Ampelovirus. More than ten other viruses are also associated with causing leafroll in grapevine [Maculavirus. No vectors have been found for this virus to date. However, Martelli and Boudon-Padieu [ArMV and GFLV both belong to the genus ineyards . Virus tcaudatum ,7. GLRaVrapevine . Viral vrapevine . GFkV isn-Padieu  listed sn-Padieu .All listed viruses have in common that they are graft transmissible and can cause severe biological, and thus economic, losses ,12,13. TFrom 1986 onwards, the regulation has been amended several times to meet new requirements and current circumstances. Since 2009, the State Institute of Viticulture and Enology (WBI) in Freiburg, Germany, has had the task of carrying out the official virus testing in southwest Germany. The WBI diagnostic laboratory receives wood samples from registered propagation vineyards from the regions concerned. These are tested for viruses utilizing enzyme-linked immunosorbent assay (ELISA).GLRaV-1 was the most abundant virus in Baden-Wuerttemberg followed by GFLV and ArMV. GLRaV-3 was rarely detected.GLRaV-1 infections were more widespread in certified plant material than in pre-basic and basic material.Wuerttemberg showed higher GLRaV-1 and GFLV incidences than Baden. Significant differences exist between pre-basic and basic planting material, but not between certified material.Virus infections are more frequent in scion than in rootstock planting material.This study summarizes twelve years of virus testing at the WBI of the pre-basic, basic, and certified propagating material category, concentrating on scion material. The results showed that:Pre-basicBasicCertifiedStandardAccording to the German Wine Growing Ordinance , grapeviDepending on the category, the phytosanitary requirements for planting material differ significantly. While vines of all categories must be tested for the absence of ArMV, GFLV, GLRaV-1, and GLRaV-3 (GFkV must be monitored only in rootstocks), pre-basic vines are subject to the strictest requirements regarding sampling quantities and retesting intervals. The strictness of the guidelines decreases in the above order. This study will not consider plant material of the standard category, which has only been marketed for commercial purposes in Germany since 2017 following the amendment of the Wine Growing Ordinance .Pre-basic plant material must be derived from a vineyard with plants of a generation prior to the basic plant material of the variety or clone indicated. Planting material must be obtained by the breeder or under his/her supervision and according to his/her instructions in accordance with the principles of conservation breeding. Samples for virus testing are taken from each plant. Five plants are pooled into one mixed sample. Vineyards must be retested in five-year intervals. Virus positive plants must be removed.Basic plant material must be derived from a vineyard planted with approved pre-basic plant material of the variety or clone indicated. Planting material must be obtained by the breeder or under his/her supervision and according to his/her instructions in accordance with the principles of conservation breeding. Samples are taken from each plant. Ten plants are pooled together into one mixed sample for virus testing. Vineyards are first sampled three years after planting and must be retested at least once every six years. Virus-positive plants must be removed.Certified plant material must be derived from a vineyard planted with basic or approved pre-basic plant material of the variety or clone indicated. Samples are taken from every twentieth plant. Ten plants are pooled together into one mixed sample for virus testing. Vineyards are first sampled five years after planting and must be retested at least once every ten years. Virus-positive plants must be removed and must not exceed five out of one hundred plants.The vineyards sampled during this study were selected by the breeding departments of the State Institute of Viticulture and Enology  for the Baden region and the State Education and Research Institute for Viticulture and Pomology  for the Wuerttemberg region. The sampled vine nurseries differ in planting time, grape variety, size, category of propagation material, and geographical position. Wood samples were collected from vineyards by officially trained personnel. Therefore, one-year-old shoots from a stem-near position were cut off and shortened to approximately 15 cm in length and stored at 4 °C until further processing. At the laboratory, thin slices were cut from the lower part of the shoots using a custom-designed machine . Wood from one bundle was pooled into one sample for virus testing and tested immediately as described in w/v) customized “Grapevine” extraction buffer using the homogenizer HOMEX . Scion samples were tested for ArMV, GFLV, GLRaV-1, and GLRaV-3 following the manufacturer’s recommendations. Samples from rootstock plants were also tested for GFkV. In 2018, scion and rootstock samples were additionally tested for GPGV. Positive and negative controls were obtained from the WBI virus collection located in Freiburg, Germany. ELISA plates were evaluated photometrically after 30 and 60 min, using an Infinite F50 reader and Magellan™ software . Samples were considered positive if the absorbance value was twice the value of the negative control sample.Samples were tested by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Assays were performed with the commercially available equipment and products of BioReba  following the protocols provided by the manufacturer. One gram of pooled wood samples was arranged in extraction bags and homogenized in 1:10 . Chi-square tests were conducted to analyze hypotheses that relied only on nominal data. An alpha level of 0.05 was chosen for all statistical tests.Since 2009, the official virus testing for the wine-growing areas Baden and Wuerttemberg has been executed at the WBI following the legislative requirements of the German Wine Growing Ordinance . Until 2X2 = 511.90, p < 0.01. GLRaV-1, the most abundant virus, was detected in 2.1% of the tested samples. GFLV was found in 1.2% and ArMV in 0.4% of the samples. GLRaV-3 was identified in less than 0.1% of the samples and thus was the rarest virus found.Over the last 12 years, 96.3% of the tested samples were free from ArMV, GFLV, GLRaV-1, and GLRaV-3 . ConsequX2  = 430.09. p < 0.001. GFLV and ArMV were both found in 1.3% of the samples. Three pooled samples showed a positive signal for GLRaV-3, which refers to 0.1% of samples  = 165.23. p < 0.001. These differences are caused by higher virus proportions in pre-basic and basic plant material from Wuerttemberg, X2  = 56.14, p < 0.01; X2  = 74.652, p < 0.001. Virus contents in certified material are similar in both regions, X2 = 0.56, p = 0.45).Since the grapevine cultivar Lemberger is a frequent carrier of GLRaV-1 , the virDue to the regulations for virus testing within the German Wine Growing Ordinance , vineyarGrapevine Pinot gris virus (GPGV), a newly emerged Trichovirus, whose symptoms were reported in the sampling area of the WBI [X2  = 665.52, p < 0.01.In 2018, grapevine samples were also tested for  the WBI . The resGrapevine fleck virus (GFkV). In contrast to scion planting material, rootstock samples show fewer virus infections  = 165.23. p < 0.001.Rootstock material is also part of the official virus tests. It is analyzed the same way as scion material but is additionally tested for fections . In totaThis study presents the results of the official virus testing carried out according to the German Wine Growing Ordinance within Germany’s southwestern regions Baden and Wuerttemberg. This official testing allowed the monitoring of virus infestations in vine nurseries of different planting material categories over a time span of twelve years. The results presented here demonstrate that GLRaV-1 was, with few exceptions, the most abundant virus throughout all years and categories, with an occurrence of more than 50% in all virus-positive tested scion samples. Since plant material is screened and selected continuously for the absence of viruses, one should expect the number of virus-positive plants to continuously decrease. In contrast to this, the results presented in this study indicate that virus-positive plants are not reduced over the years but seem to appear in a rather cyclic manner. Since the grapevine cultivar Lemberger is a known carrier of GLRaV-1 and this variety is commonly cultivated, particularly in Wuerttemberg, it might represent a reservoir for this virus . ConsequAmpelovirus and have multiple vector insects. Three soft scale insects and seven mealybug species are known vectors of GLRaV-1 [Parthenolecarnium corni (Bouché) [P. corni is the only widespread vector of leafroll-associated viruses in the sampling areas of this study. A more likely possibility may be a pool of plants infected exclusively with GLRaV-1, acting as a starting point for the distribution by insects. One source may be vineyards planted with already infected plants surrounding the nurseries. In 2009, such a virus repository was detected when a vineyard of pre-basic propagating material had 64 positive GLRaV-1 samples  . However samples . GLRaV-3ectively . NematodFurthermore, what is noticeable about the results of this study is that certified planting material shows by far the highest infection rate of GLRaV-1, with approximately 8% of positive samples. This should not be surprising since those plants are tested only once in ten years. However, it is alarming since only every twentieth plant is sampled in this category and ten plants are pooled into one sample. One can expect that the actual infestation is much higher in those vineyards. Le Maguet et al. ,22 impreThe high proportion of GLRaV-1-infected plants in nurseries for certified material is also alarming since all nurseries are regularly checked visually. Therefore, it can be assumed that many infected grapevines show no symptoms, since otherwise they should have been removed immediately. Furthermore, these assumptions cast doubt on the use of planting material of the standard category whose phytosanitary status is evaluated solely visually . LatentlGrapevine Pinot gris virus (GPGV). Although short RNA readings of symptomatic plants identified the virus, it was rapidly found that GPGV can also be latent in plants [Grapevine red blotch-associated virus (GRBaV), the causative agent of red blotch disease (RBD) [Pathogens that remain latent in their hosts definitely pose a risk, especially if they are still unknown. One prerequisite for PCR and ELISA testing is to know at least part of the pathogen’s genetic code or protein structures. Undetected pathogens in plant material are distributed unintentionally and their occurrence is artificially upregulated . The lonn plants ,24. The n plants . Additiose (RBD) ,27. CaseMaculavirus GFkV and also the Trichovirus GPGV were detected, but in rather low rates. No ampeloviruses were detected. Since the devastating Phylloxera epidemic in the 1860s, American grape varieties have been used almost exclusively as rootstocks because of their tolerance to Daktulusphaira vitifoliae (Fitch) [Contrary to scion material, rootstock plants show relatively low virus incidences. Only 1% of the analyzed, pooled samples were infected. The nepoviruses GFLV and ArMV, the  (Fitch) . Some of (Fitch) . HoweverIn summary, the present study has shown that the monitored viruses are still widely distributed in south-western Germany. Notably, certified planting material had higher GLRaV-1 infestation levels than pre-basic and basic material, suggesting either carryover of unrecognized virus-infected propagation material or viral input from neighboring infected vineyards by its fast moving vectors. However, this result proved the urgency of continuous and close monitoring of the phytosanitary status of planting material. We are aware that within this study only a total of five viruses were investigated, which represent only a small proportion of the viruses from grapevine. In particular, the results from 2018 show that the recently discovered GPGV emerged rather unnoticed to become the most widespread virus in German vineyards, as it is mostly latent in plants. Therefore, it should be considered whether it would not make sense to include viruses like GPGV or GRBaV in the vine planting material regulation, especially since the latter can easily be mistaken for leafroll in optical inspections . In addi


Prunus trees, including almond (P. dulcis), apricot (P. armeniaca), nectarine (P. persica var. nucipersica), peach (P. persica), plum (P. domestica), purple leaf plum (P. cerasifera) and sweet cherry (P. avium), were selected from growing regions Australia-wide and tested for the presence of 34 viruses and three viroids using species-specific reverse transcription-polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) tests. In addition, the samples were tested using some virus family or genus-based RT-PCR tests. The following viruses were detected: Apple chlorotic leaf spot virus (ACLSV) (13/100), Apple mosaic virus (ApMV) (1/100), Cherry green ring mottle virus (CGRMV) (4/100), Cherry necrotic rusty mottle virus (CNRMV) (2/100), Cherry virus A (CVA) (14/100), Little cherry virus 2 (LChV2) (3/100), Plum bark necrosis stem pitting associated virus (PBNSPaV) (4/100), Prune dwarf virus (PDV) (3/100), Prunus necrotic ringspot virus (PNRSV) (52/100), Hop stunt viroid (HSVd) (9/100) and Peach latent mosaic viroid (PLMVd) (6/100). The results showed that PNRSV is widespread in Prunus trees in Australia. Metagenomic high-throughput sequencing (HTS) and bioinformatics analysis were used to characterise the genomes of some viruses that were detected by RT-PCR tests and Apricot latent virus (ApLV), Apricot vein clearing associated virus (AVCaV), Asian Prunus Virus 2 (APV2) and Nectarine stem pitting-associated virus (NSPaV) were also detected. This is the first report of ApLV, APV2, CGRMV, CNRNV, LChV1, LChV2, NSPaV and PBNSPaV occurring in Australia. It is also the first report of ASGV infecting Prunus species in Australia, although it is known to infect other plant species including pome fruit and citrus.One hundred  Prunus species, including P. armeniaca (apricot), P. avium (sweet cherry), P. cerasifera (purple leaf plum), P. domestica (plum), P. dulcis , P. persica (peach) and P. persica var. nucipersica (nectarine), are commercially grown for their nuts, fruits and as ornamental plants [Worldwide l plants ,2,3. Alml plants ,5,6. Curl plants ,8 and thPrunus germplasm imported into Australia requires a minimum of 18 months post-entry quarantine (PEQ) and during that time it is tested using a range of diagnostic techniques for the detection of diseases and pathogens of quarantine significance. Post-border, the biosecurity of these industries is maintained through certification schemes that supply high-health planting material throughout Australia and which, until recently, was tested only for the virus species Apple chlorotic leaf spot virus (ACLSV), genus Trichovirus, and three species of the genus Ilarvirus: Apple mosaic virus (ApMV), Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) [The biosecurity of the Australian almond and stone fruit industries are maintained at the border by the Department of Agriculture.  (PNRSV) ,10.Prunus species. It has helped to clarify pathogens, especially viruses, associated with diseases in Prunus species that had an unknown aetiology. There are now 55 virus species and three viroid species that have been reported to infect almonds, stone fruit and/or ornamental Prunus trees worldwide [Significant advances in molecular and diagnostic technology, such as reverse transcription (RT)-polymerase chain reaction (PCR), PCR and high-throughput sequencing (HTS), have allowed characterisation of new and emerging pathogens and new strains of known pathogens of orldwide ,26,27,28Prunus species overseas and because there was little information about the incidence of viruses and viroids in Australian Prunus species, a national survey of the almond and stone fruit growing regions of Australia was conducted during 20 months in 2013–2015. The survey was used to update the disease status for viruses and viroids of almond and stone fruit industries in Australia and to update the list of viruses and viroids requiring testing during PEQ. The genomes of interesting Australian strains of Prunus-infecting viruses were assembled so that they were represented amongst other international isolates. In undertaking the survey, the molecular diagnostic tests for detection of these viruses were also validated. The results of the survey are reported here.Due to the discovery of new and novel viruses and virus strains in Prunus trees during spring and summer from August 2013 to March 2015 throughout Australia. They were collected from major almond and stone fruit growing regions and some home gardens  was extracted from Prunus tree samples using a modified lysis buffer [For each of the 100 s buffer  and the s buffer , except ®Taq DNA Polymerase (Invitrogen) kit was used for the nested PCR step of the genus or family-based tests  was used for detection of RNA viruses and viroids according to the manufacturer’s instructions except that 0.5 µL of SuperScript™ Taq enzyme mixture and 1 µL of template was used in a total RT-PCR reaction volume of 25 µL. A Platinumed tests , accordindhB gene, NAD) messenger ribonucleic acid (mRNA) by RT-PCR [Primers for the detection of NADH dehydrogenase ND2 subunit . Selected specific RT-PCR and all genus/family-based RT-PCR products were purified using the Qiaquick PCR purification kit , cloned using the pGEM-T Easy Vector system  and sequenced using the ABI BigDye Terminator Version 3.1 kit on an AB3730xl sequencing machine . The resulting sequences were analysed using BLASTn  to deterPrunus trees were tested using virus metagenomic HTS and included 20/100 samples from the survey that were selected based on the Prunus host species, origin and virus species detected by RT-PCR during the survey, along with an additional four LChV2-infected cherry tree samples  from Tasmania that had been submitted to the Tasmanian Plant Biosecurity and Diagnostics Branch to investigate the cause of little cherry symptoms. Five µL aliquots of TNA extract from each of the selected samples were used to prepare 24 individual metagenomic NGS libraries using NEBNext® Ultra™ RNA Library Prep Kit  following the manufacturer’s instructions. The size distribution and concentration of the libraries were determined using the 2200 TapeStation® system  and Qubit® Fluorometer 2.0 (Invitrogen), respectively. The libraries were sequenced using the Illumina MiSeq with a paired read length of 2 × 251 bp. The generated raw sequence reads were quality trimmed, paired, assembled into contigs and BLASTn and BLASTx analysis of the assembled contigs was carried out as previously described [A total of 24 escribed . SpecifiFull-length genome sequences of each virus species assembled from metagenomic HTS were aligned with each other, where multiple sequences were obtained, and with full genome sequences of all corresponding isolates available in GenBank  using MUp values <0.05 [The aligned full genome sequences of virus species from this study and the sequences of corresponding virus species isolates available in GenBank were checked for potential recombination events using RDP , GENECONes <0.05 .Prunus tree samples using species-specific or genus/family-based RT-PCR or nested RT-PCR/PCR tests  of CR tests . The virnd PLMVd . The virnd PLMVd . Using aPrunus trees using species-specific or genus/family-based RT-PCR or nested RT-PCR/PCR tests during the survey: ApLV, ArMV, APV1, APV2, APV3, CLRV, CMLV, CRMaV, CTLaV, LChV1, PPV, PRMV, PcMV, RRSV, SLRSV, TBRV, TBSV, ToRSV and ASSVd  were observed in cherry trees , ApLV (1/24), APV2 (1/24), LChV1 (5/24) and NSPaV (2/24), which were not detected in any of the 24 samples using species-specific RT-PCR or genus/family-based RT-PCR or nested RT-PCR/PCR, were also assembled . HTS alsd RT-PCR .Prunus tree samples in this study have been previously reported [There was evidence of multiple strains of virus species in individual samples: 4/24 samples had full genomes of two CVA strains, 1/24 samples had full genomes of three CVA strains and 1/24 samples had full genomes of two CGRMV strains and two LChV1 strains. . The assreported ,48,49 anACLSV: Three distinct clusters were observed in the phylogenetic analysis of genome sequences of the six Australian ACLSV isolates and genome sequences of 22 ACLSV isolates available from GenBank, which represented diverse hosts and geographic ranges . Five ofAPCLSV: The genomes of the two Australian APCLSV isolates from plum (VIC3) and nectarine (VIC11) obtained in this study had 96% nt identity and they clustered with a South Korean peach isolate (GenBank accession KY310579) and shared 96% nt identity and 98% nt identity, respectively, with this isolate .AVCaV: Two distinct clusters of AVCaV isolates were observed in the phylogenetic analysis of genome sequences from Australia and overseas . The AusCGRMV: Eight CGRMV genomes were obtained by HTS from six Australian cherry samples and one peach sample. Two CGRMV strains, LV27_S1 and LV27_S2, were found in one cherry sample and shared 81% nt identity. All Australian isolates shared 80%–98% nt identity amongst themselves  and 80%–CNRMV: The phylogenetic analysis revealed two distinct clusters of CNRMV isolates and all three Australian cherry isolates occurred in one group . AustralApLV: The phylogenetic analysis revealed two distinct clusters of ApLV isolates. The Australian ApLV apricot isolate from this study clustered with apricot isolates from Italy and France . The AusAPV2: The phylogenetic analysis revealed two distinct clusters of APV2 isolates. The Australian APV2 apricot isolate from this study, VIC18, clustered with two apricot isolates from Japan (GenBank accession KT893295) and the USA (GenBank accession KR998049) and had 96% and 94% nt identity to these two isolates respectively .PBNSPaV: The Australian PBNSPaV isolates from this study had an 82%–98% nt identity amongst themselves and a nt identity range of 71%–99% with GenBank isolates . The phyLChV1: The Australian LChV1 isolates had 76%–87% nt identity amongst themselves and 72%–99% nt identity with GenBank isolates . LChV1 pLChV2: The Australian LChV2 isolates from this study shared 76%–96% nt identity and phylogenetically clustered into two distinct clusters . One groNSPaV: The phylogenetic analysis revealed two distinct clusters of NSPaV and the Australian isolates are represented in both clusters . The VICCVA: The genomes of 20 Australian CVA isolates from apricot, cherry, peach and plum were obtained by HTS, from 13/14 trees that also tested positive by specific or genus/family-based RT-PCR. The 1/14 tree was not used for HTS. Phylogenetic analysis resulted in six distinct clusters of CVA isolates. CVA isolates from this study were distributed amongst all six clusters . CVA isoRecombination analysis of the full genome sequences of Australian isolates and GenBank isolate sequences identified recombination events in ACLSV, CGRMV, CVA, LChV1, LChV2 and PBNSPaV sequences . RecombiAll the recombination events detected in Australian ACLSV and CGRMV isolates, and in 4/6 LChV1, 6/7 LChV2 and 3/4 PBNSPaV Australian isolates had Australian isolates as their major parents while only 2/18 CVA recombination events had Australian major parents . All thePrunus trees tested in this study was not extensive, however, the approach of using a combination of virus species-specific and genus/family-based RT-PCR tests, RT-PCR, or nested RT-PCR/PCR and HTS allowed us to test Australian grown trees for many Prunus infecting viruses and to examine their genetic diversity. This study showed that viruses not previously reported in Australia were present in Prunus species, many of which should be excluded from programs that produce high-quality planting material for the Australian stone fruit and almond industries. This study highlighted the importance of understanding virus genetic diversity to ensure diagnostic tests can detect all known genetic strains of a virus species. It also highlighted the value of metagenomic HTS as a single diagnostic test that can indiscriminately detect all viruses in a single sample. Australia is a large continent and Prunus trees are grown in diverse regions throughout the country, with cool-temperate to Mediterranean climates, sometimes 1000’s of kilometres apart. Some virus species occur in many regions and sometimes closely related virus strains appear in different and distant regions and dissemination of the viruses by propagation material is probably the primary pathway. Prunus material is imported from many countries including Europe and the USA and is also a likely contributor to the diversity of species and genetic diversity within species observed in this study.The number of Prunus species in Australia and are included in certification testing for production of high health planting material, particularly in almonds [Prunus viruses and viroids including APCLSV, APLPV, ASGV, CVA, HSVd and PLMVd, were mainly found in the stone fruit and ornamental Prunus trees, but not in almonds. During the survey ApLV, APV2, CGRMV, CNRMV, LChV1, LChV2, NSPaV and PBNSPaV were detected in stone fruit species and this is the first report of these viruses in Australia. PNRSV was the most commonly detected virus, occurring in 52% of the samples tested. All other viruses that were detected occurred much less frequently (≤14%).One or a combination of viruses were found in 70% of the trees that were tested. PNRSV, PDV, ApMV and ACLSV were detected in this study. These viruses have long been known to occur in many  almonds ,51. OthePrunus infecting viruses that were found in this study. The known mealybug vectors of LChV2 (Phenacoccus aceris and Pseudococcus maritimus) [Prunus infecting viruses in Australia. The high prevalence of PNRSV is likely to be due to a combination of the use of untested planting material, especially as the use of certified material is voluntary, and field transmission via pollen and thrips which occurs in Australia [Prunus limited host range compared to PNRSV, which can also naturally infect other plant hosts [Except for LChV2, vectors are not known for most of the ritimus)  are not ritimus)  and thesustralia ,55. Althustralia  may be dnt hosts ,57,58.Prunus that are reported infrequently [ASPV and CMV were not detected although they are known to occur in other crops in Australia. This result was anticipated as they appear to be minor viruses of equently ,60,61 anPrunus species in other countries [Prunus species (plum) in Australia. Infection could have been through imported infected planting material, but it may also have occurred through mechanical transmission through the sharing of equipment between Prunus and other fruit crops such as citrus or pome fruit, where their growing regions overlap in Australia [Although ASGV is known to infect ountries , this stountries , can alsustralia .Prunus industry’s biosecurity continuum.It is difficult to estimate when ApLV, APV2, CGRMV, CNRMV, LChV1, LChV2, NSPaV and PBNSPaV, which were initially considered exotic, were introduced into Australia, but it is possible they have been present for many years but gone undetected until this study. Indeed, the Victorian plum tree infected with PBNSPaV, ACLSV, ASGV and PNRSV was estimated to be more than 60 years old, which suggests that PBNSPaV may have been present for more than 60 years. These viruses were most likely introduced into Australia before they were known and included as part of the Australian import molecular testing regime. The associated symptoms were probably not observed by biological indexing during Australian PEQ because symptoms were not well described for biological indicator species or the indicators were symptomless. This is especially the case for recently described viruses such as NSPaV, that are only now being discovered through the development and implementation of HTS in association with diseases that previously had an unknown aetiology ,65. TherPrunus germplasm collections may only be tested for endemic viruses upon entry into their collection or tested infrequently, increasing the risk of distributing virus-infected material to nursery and production industries, especially for those viruses like PNRSV, which are also spread by pollen [The lack of detection of some viruses and viroids and low prevalence of others in Australia, especially those that are primarily spread through vegetative propagation, is likely to be a result of certification programs established in the 1970s ,66 in coy pollen ,68.Prunus trees tested. PNRSV occurred in all mixed infections which is not surprising given that PNRSV is easily transmitted by pollen and vegetatively, and was the most frequently detected virus [Prunus industries and trees may not be tested or are tested infrequently instead of annually for viruses. Until this survey was completed in 2015, most Australian Prunus trees were tested only for ApMV, ACLSV, PDV and PNRSV because other viruses were not known to occur or were considered less significant. This infrequent testing, and only for a few viruses, especially before 2015, explains the high incidence of mixed infections observed in stone fruits in this study, especially in cherries, and highlights the need for these Prunus industries to undertake a more proactive virus testing program in which testing is undertaken for all viruses now known to occur in Australia.Like many other studies ,70,71,72ed virus ,68. IntePrunus trees for viruses. RT-PCR and PCR tests were chosen for this survey by comparing primers against available sequences in GenBank to determine their likelihood for detecting targeted virus, however, this process is only as good as the virus sequences available for comparison. Genetic similarity or variability of viruses and co-extracted nucleic acids may influence the reliability of RT-PCR and PCR tests resulting in false-positive or negative results. False-positive results can occur when the PCR primers have a high degree of similarity to other genetic material, including that of the host plant or other organisms within a geographic region [Ilarvirus, Foveavirus, Trichovirus, Capillovirus and Ampelovirus in Prunus trees to overcome the risk of false-negative results, until improved tests can be developed. This study highlighted the need for further improvements in the development, selection and adoption of RT-PCR tests for detection of viruses in Australian grown Prunus species.In this study, the reliability of RT-PCR and PCR diagnostic tests was assessed to ensure they were fit for purpose for routine diagnostic testing of Australian c region . For insc region . Their ac region ,75,76. CPrunus species, which could make RT-PCR a cost-prohibitive diagnostic tool, the reducing cost of HTS [Prunus species in Australia.HTS was used to confirm the first detection of CGRMV, CNRMV, LChV2 and PBNSPaV in Australia by RT-PCR and PCR. It was also useful to confirm the presence of viruses detected by a genus- or family-based RT-PCR test but not by the specific test . Metagenomics HTS detection of additional virus species, specifically ApLV, AVCaV, APV2, NSPaV and LChV1, and strains of CGRMV, CNRMV, LChV2 and PBNSPaV, which were not detected by species-specific or genus- or family-based RT-PCR tests, highlights the high potential for a false-negative diagnosis by RT-PCR due to virus genetic variability. It also highlights the value of metagenomics HTS as a non-discriminative diagnostic tool for virus detection compared to RT-PCR, because it does not rely on knowledge of the virus genome sequences . Given tt of HTS  makes thPrunus tree samples. Similar detection of multiple genetic strains or variants may also be achieved through traditional Sanger sequencing of cloned RT-PCR and PCR amplicons. However, limitations in the sequencing of a few clones due to time, labour and cost constraints may hamper the detections of some strains present in low titre mixed infections. This challenge in the detection of mixed virus genetic strains in a single plant has recently been overcome by HTS for other virus species [In this study, full genomes of Australian ACLSV, APCLSV, AVCaV, ApLV, APV2, NSPaV, PBNSPaV, CNRMV, LChV2, LChV1, CGRMV and CVA isolates were generated by HTS, contributing to the global knowledge of their presence and diversity. Except for ApLV and APV2, multiple genome sequences were obtained for the other viruses. Multiple full-genomes of CVA, CGRMV and LChV1 strains were also detected by HTS in individual  species ,78,79, wPrunus virus isolates of ACLSV, APCLSV, AVCaV, ApLV, APV2, CGRMV, CNRMV, CVA, LChV1, LChV2, NSPaV and PBNSPaV from this study and isolates of each species previously published on GenBank. Where multiple strains of a species were identified, considerable genetic diversity was observed amongst the strains. As reported in other studies [Prunus germplasm collection or an overseas germplasm, although no vector is known for LChV1. It could also be associated with the use of a common infected rootstock.Phylogenetic and sequence identity analysis of full virus genomes was used to determine the genetic relationship between Australian  studies ,80,81,82The multiple genetic strains of CVA, CGRMV or LChV1 within individual trees were highly divergent from each other and occurred in different phylogenetic clusters. For example, the three distinct CVA strains identified in sample TAS12 occurred in the three of the six major CVA clusters reported by . Co-infeBetaflexiviridae and Closteroviridae [Prunus trees that had multiple full-genome genetic strains of CVA, CGRMV and LChV1. This could indicate recent introductions of these viruses into Australia or recent multiple or single infection events.Recombination is a powerful evolutionary driving force that results in the generation of new genotypes in RNA viruses ,86. Recooviridae ,84,87,88oviridae ,81. IntePrunus viruses in Australia conducted by this study provides useful information to support Australian Prunus industry biosecurity. This study has identified 18 viruses and viroids occurring in Australia Prunus species and provides the first report of ApLV, APV2, CGRMV, CNRNV, LChV1, LChV2, NSPaV and PBNSPaV in Australia. This information has been used to update the quarantine status of viruses and viroids in Australian Prunus trees. The low prevalence of some viruses in Australia suggests that quarantine and certification have been useful in limiting their distribution, particularly in almonds and stone fruit species other than cherry. However, the use of certified material is voluntary and the use of untested planting material in combination with field transmission via pollen would explain the higher prevalence of PNRSV and highlights the importance of using pathogen tested material. Since the conclusion of the survey using RT-PCR in 2015, several new viruses of Prunus were reported including Caucasus prunus virus (CPrV), Cherry virus F (CVF), Peach leaf pitting-associated virus (PLPaV), Prunus yellow spot-associated virus (PYSaV), Prunus virus T (PrVT) and Prunus virus F (PrVF) [Prunus industry biosecurity.The comprehensive survey of incidence and genetic diversity of F (PrVF) ,89,90,91F (PrVF) ,92,93,94


Since 2004, we have been developing nanomaterials with antimicrobial properties, the so-called nanoantimicrobials. When the coronavirus disease 2019 (COVID-19) emerged, we started investigating new and challenging routes to nanoantivirals. The two fields have some important points of contact. We would like to share with the readership our vision of the role a (nano)materials scientist can play in the fight against the COVID-19 pandemic. As researchers specifically working on surfaces and nanomaterials, in this letter we underline the importance of nanomaterial-based technological solutions in several aspects of the fight against the virus. While great resources are understandably being dedicated to treatment and diagnosis, more efforts could be dedicated to limit the virus spread. Increasing the efficacy of personal protection equipment, developing synergistic antiviral coatings, are only two of the cases discussed. This is not the first nor the last pandemic: our nanomaterials community may offer several technological solutions to challenge the ongoing and future global health emergencies. Readers’ feedback and suggestions are warmly encouraged. Dear Editor,“Are we ready for pandemic influenza?”With an as-titled article, published in Science in 2003 and heavily cited later, Webby and Webster proposed some interrogatives about the threat of emerging infectious diseases . After tIn late 2019, a novel pneumonia caused by an unknown pathogen emerged in the city of Wuhan, central China .Currently, many different antiviral agents, including re-purposed drugs, are under testing in clinical trials to assess their efficacy against the new virus, but the quest for an effective treatment against COVID-19 is still open ,5,6,7.Different strategies were pursued, to treat coronavirus related to MERS and SARS infections, including the use of inhibitors of viral and host proteases, interferons (IFNs), and host-directed therapies. One of the first antiviral drugs tested, the ribavirin, a nucleoside analog that acts as RNA polymerase inhibitor , was useBased on antiviral treatment against previous coronavirus SARS and MERS, lopinavir/ritonavir combination was tested  too. TheThe vital role of the spike protein (S protein) of coronaviruses makes this glycoprotein an important therapeutic target, because it guides coronavirus entry into host cells, by providing the binding and the fusion of virus on the host cell membrane. S protein is composed of two subunits: S1 recognizes and binds to host receptors, and S2 facilitates fusion between the viral envelope and the host cell membrane . NumerouRemdesivir, an adenosine analogue, is a broad-spectrum antiviral with potent in vitro efficacy against multiple genetically unrelated RNA viruses . It is cIn this scenario of non-univocal medical treatments and vaccine unavailability, it is evident that, besides the population above 75-year-old and people with immunodeficiency, or chronic and oncological diseases, the category that is most exposed to COVID-19 is that of hospital personnel . It is iContagion rates of COVID-19 are much higher than those reported for the well-known SARS . This brSurface contamination has recently been found to be more significant than originally thought in the spread of this viral disease. In fact, despite the use of proper PPE , health-Many resources and biotechnological capabilities are currently directed towards the development of vaccines and treatments against COVID-19. Anyway, given the significance of the surface and air contamination in the spread of the virus, attention should be paid also to the development of antiviral and antibacterial surfaces, along with decontamination equipment and technologies.We hypothesize that using proper air filtering may reduce the viral load in the environment, sufficiently to decrease the probability of health-care worker infection through flaws in PPE, or in confined public transportation . AccordiInverse computational fluid dynamics (CFD) models are available to identify the spread of air particles in passenger vehicles . CFD modHigh-Efficiency Particulate Air (HEPA) filters can limit the spread of airborne fungi, viruses, and bacteria ,43. TheiThe contamination of latex/nitrile gloves, N95 respirators, hospital scrubs, overshoes, and floors in a nosocomial environment is considered a serious issue ,44,45, bBesides nosocomial environments, the contamination of surfaces can be regarded as responsible for many other contagion episodes. As an example, Australian Health Authorities have recently confirmed several cases of COVID-19 among baggage handlers. The circumstances gave rise to concerns about cleaning the luggage and about the virus survival time on hard, smooth materials, such as plastic and metal. The detailed explanation of contamination sources and the current number of contagions among baggage handlers worldwide are still unknown. However, extensive cleaning and disinfecting procedures were implemented in international airports, to reduce the surface contamination of luggage .More generally, many disinfection protocols have been developed so far, which mainly involve the use of sodium hypochlorite , 70–85% Based on these findings, we make a heartfelt appeal to the (Nano)Materials Science community: we can exploit the well-known antimicrobial properties of formulations and nanostructures containing copper, silver, and zinc species ,53 to fiSpecifically, the use of copper salt nanoparticles and/or solutions , which are known for having an antiviral effect ,56,57,58Nanotechnology can offer a great support in the design of contamination-safe equipment in this era of pandemic diseases. Metal-loaded nanocomposites are known to be extremely effective in all those cases in which a controlled and long-lasting ionic release is required ,60. The Metal nanoparticles can act as ion reservoirs for the controlled release of bioactive ions, thus tuning the production of ROS species too. The embedding of metal nanoparticles  in polymer matrices could help in the tuning of metal release properties, and at the same time in minimizing the risk of nanoparticle release into the environment . Moreove2, CdS, MnS2, etc.) [To the best of our knowledge, there are few publications about the treatment of nosocomial PPE with copper nanoparticles or copper oxides and salts. It is evident that inadequate PPE and inappropriate PPE guidelines can be responsible for the death of many health-care workers and for viral nosocomial spreading. Bhattacharjee and co-workers reviewed the topic in 2019, before the spreading of SARS-CoV-2, taking into account other viral pandemic diseases like Ebola, SARS, and MERS . They re2, etc.) . GO graf2, etc.) . Specifi2, etc.) ,67. Anti2, etc.) . However2, etc.) ,69. Thei2, etc.) .2-Ag NPs as active material against MS2 bacteriophage [Recently, polyurethane/CuO nanocomposites were developed, acting as an effective antimicrobial filter for air purification. It is worth noting that microparticles of CuO are a more suitable additive for the modification of polyurethane filters than nanoparticles , thus reriophage .The importance of Nanotechnology in fighting viruses is not enough explored. One of the possible directions of further investigation is related to the use of nanomaterials to fight virus resistance to conventional therapies; this resistance can be due to the accelerated virus adaptation in peripheral protein sequence, thus resulting in the development of a new viral strain .The antiviral action of metal nanoparticles, notably of silver nanoparticles (AgNPs), is well known. They act as viral reproduction inhibitors, and their viricidal activity depends on the target virus. For example, the AgNP ability to inhibit the viral entry in host cells, in the case of the HIV-1 virus, was reported, demonstrating that AgNPs are able to interact with cell receptors . RegardiIt was also demonstrated that gold nanoparticles coated with sulfated ligands, silver nanoparticles, and hybrid silver-copper nanoparticles are able to bind the HIV envelope glycoprotein gp120 and to inhibit in vitro HIV-1 infection in cellular models ,75. In aIt is known that very small metal NPs (<10 nm) are able to go through the cell membrane and inhibit post-attachment virus replication . As an eGiven all these premises, the general question is: can inorganic nanoparticles be useful in affecting the early viral lifecycle stages?The specific question is: is it possible to realize active nanomaterials able to inhibit the binding and fusion of viruses on the host cell?We believe that it is certainly possible to find a nanotechnological solution for these quests.Our idea is to realize hybrid antiviral nanomaterials by functionalizing metal nanoparticles with typical antiviral drugs, with enhanced synergistic efficacy. These synergistic nanoantivirals might offer a great help in increasing the efficacy of PPE and improving the safety of common touch surfaces.They can be used to modify surgical masks and respirators, which are crucial targets in the fight against virus diffusion . Among tHence, we believe that CQ and HCQ could be proficiently used to functionalize metal nanoparticles. Other antiviral/metal nanostructure combinations could be possible, as well.We warmly encourage the readers working in this area to investigate the development of hybrid antiviral metal-CQ/HCQ nanomaterials. The resulting synergistic nanoantivirals could be very useful to prevent the diffusion of the virus. They could be implemented in coatings for hard surfaces and used as PPE-modifiers to increase their protective action.This is of course our perspective viewpoint and needs to be tested. At the present stage, we think that sharing these ideas and triggering our community to develop a deeper level of knowledge about the (unexplored) antiviral properties of nanomaterials could be a good way to fight this pandemic.It is likely that nanotechnology has enormous potential in the prevention, diagnosis, and treatment of COVID-19. In this communication, we do not cover the diagnostics and treatment outcomes, although we certainly quote the considerable perspectives in (i) designing sensors for developing quick-response COVID-19 tests , and  and nanotechnology-enabled highly effective antiviral disinfectants, can be considered the most effective way to prevent viruses from spreading.The readers working in the nanotechnology area are warmly encouraged to contact us with their comments or proposals. Sharing our ideas and setting common actions against this pandemic will be the key to success.The present pandemic is not the first, nor will it be the last one. As researchers working on nanomaterials for the life sciences, we need to ensure that we have tools in place to deal with the COVID-19, as well as with any future pandemics.Nanomaterial-based antiviral and antibacterial textiles, non-woven disposable products, packaging solutions, antiviral coatings, synergistic/multifunctional surfaces, air-conditioning filters, PPE, are just few of the possible examples requiring our prompt technological answer. While many good research papers have been published on metal nanoparticles to be used as antibacterial or antiviral agents, the commercialization of novel functional nanomaterials still appears to be limited by nanotoxicology concerns or by several other practical aspects . This is a good time to ask why and how we can facilitate the turning of these research projects into safe and viable products.In the field of nanoantimicrobials, we have always pursued the use of bioactive nanoparticles as water-insoluble, polymer-confined nano-reservoirs, providing a source of ionic release, without being released as entire nanophases in the contact matrices  ,62.a priori nano-toxicological risks. Other technological solutions need to be envisaged in order to promote nanosafety and support technological knowledge transfer and commercialization. This is becoming more and more a part of our academic mission [Blocking the nanoantivirals in an adequately stable embedding matrix could be, again, the right way to ensure safety, preventing  mission ,90.We encourage our nanomaterials community to actively exploit its impressive nanotechnological background to challenge the ongoing global health emergency.


Dioscorea spp.) is an important crop in tropical and subtropical regions. Many viruses have been recently identified in yam, hampering genetic conservation and safe international exchanges of yam germplasm. We report on the implementation of reliable and cost-effective PCR-based detection tools targeting eight different yam-infecting viruses. Viral indexing of the in vitro yam collection maintained by the Biological Resources Center for Tropical Plants (BRC-TP) in Guadeloupe (French West Indies) unveiled a high prevalence of potyviruses, badnaviruses, Dioscorea mosaic associated virus (DMaV) and yam asymptomatic virus 1 (YaV1) and a high level of coinfections. Infected yam accessions were subjected to a combination of thermotherapy and meristem culture. Sanitation levels were monitored using PCR-based and high-throughput sequencing-based diagnosis, confirming the efficacy and reliability of PCR-based detection tools. Sanitation rates were highly variable depending on viruses. Sixteen accessions were successfully sanitized, paving the way to safe yam germplasm exchanges and the implementation of clean seed production programs worldwide.Yam ( Dioscorea trifida, originating from the Amazonian basin and once widely grown for local consumption, is almost no longer cultivated in Guadeloupe due to its high sensitivity to viruses [D. rotundata, in West Africa, where it is a very popular crop [Yam is a staple food in the tropics and subtropics and the second most important tuber crop in Africa after cassava. Annual worldwide yam production was 73 million tons in 2018, with five African countries accounting for 92% of this production and Nigeria being the largest producer with 48 million tons [Ampelovirus, Aureusvirus, Badnavirus, Carlavirus, Comovirus, Cucumovirus, Fabavirus, Macluravirus, Potexvirus and Potyvirus. Thus, within the last five years alone, the complete genomes of 13 novel yam viral species were sequenced: one potyvirus, Dioscorea mosaic virus ; six ba, Diosco , Diosco, Diosco, Diosco and Dio ; one sas ; two mas  and Yams ; two am and yam Y .D. alata, D. bulbifera, D. cayenensis-rotundata, D. esculenta and D. trifida).With more and more yam-infecting viruses being discovered, determining the viral status of yam germplasm has become increasingly challenging and critical to securing the safe production and exchange of genetic resources and preventing the spread of viral diseases. Indeed, international regulations prohibit the circulation of infected biological resources to avoid pathogenic outbreaks. For this, accurate and sensitive diagnosis tools and methods need to be developed and applied. Such tools and methods are also instrumental for the production of clean (virus-free) planting material produced by sanitation programs such as the one implemented by Guadeloupe’s Biological Resource Center for Tropical Plants (BRC-TP). This program aims at producing virus-free yam plants through sanitation with respect to the viruses identified in Guadeloupe . This geSeveral techniques have proved efficient in eliminating viruses from yams, such as chemotherapy, thermotherapy and cryotherapy associated with cell culture ,22. AlteHere we report on the development of reliable and cost-effective molecular diagnostic tools for indexing all viruses occurring in yams in Guadeloupe and their implementation for virus prevalence studies. We present the first overall assessment of the efficiency of a yam sanitation process combining thermotherapy and meristem excision and the successful production of sanitized virus-free yam plants.in vitro in Guadeloupe and maintained in sterile conditions on solid growth medium  in order to identify conserved regions in the genome of these viruses . PrimersLikewise, the nucleotide sequences of 16 RNA-dependent RNA polymerase (RdRp) domains from isolates of the three potexvirus species reported in yams  were ali6 and 125 nM of oligonucleotide PolyT18. Then, cDNA was synthetized for 1 h at 37 °C with 500 µM of dNTPs, 20 U of RNAse inhibitor and 200 U of the M-MLV reverse transcriptase (Promega).Total nucleic acids (TNAs) were extracted from 100 mg leaf samples collected from yam plants according to the procedure 2 described by Foissac et al. , using a2 concentrations as well as annealing temperatures varied depending on the targeted virus and are detailed in PCR reactions were performed on 3 µL of cDNAs with 200 µM of dNTPs and 1.25 U GoTaq G2 polymerase (Promega). Primers and MgCl2 concentrations as well as annealing temperatures are provided in A first PCR was performed on 3 µL of cDNAs using the generic potexvirus-specific primer pair Potex-2RC/Potex-5 described by van der Vlugt and Berendsen . A nesteBadnavirus indexing was performed according to Le Provost et al.  with thein vitro yam collection. They harbored various combinations of coinfection by yam viruses  filled with wet sterile potting soil 110/60/140 N/P/K . Pots were placed under homemade Plexiglas boxes in order to maintain moisture at as high a level as possible in a room with controlled conditions . After 10 days, boxes were opened slightly, and after another 10 days, completely removed. When yam plantlets had grown approximately 20 leaves (about two months), two young well-developed leaves were collected, placed separately in two grinding bags  and stored at −80 °C until viral indexing.D. trifida plantlets  were collected and processed independently. Double-stranded RNAs (dsRNAs) were purified from leaf samples and used for random amplification as described previously by Marais et al. [Following acclimatization, two leaves from each of four sanitized s et al. . AmplifiSequencing reads were cleaned, demultiplexed  and assein vitro collection, we developed molecular diagnosis tools targeting yam-infecting viruses present in Guadeloupe , yam potexvirus 1 and yam potexvirus 2 sequences , which are widespread in yam genomes .2 and primer concentrations as well as annealing temperatures, which are summarized in All viral detection methods used in this work were optimized in order to increase their sensitivity and reliability, leading to optimal MgClin vitro yam collection conserved by the BRC-TP, using tools and methods described above for the detection of CMV, DMaV, YaV1, YMV, YMMV, badnaviruses, macluraviruses and potexviruses. The majority of sampled plants belonged to the five most cultivated yam species in Guadeloupe, Dioscorea alata (140 sampled plants), D. bulbifera (4), D. cayenensis-rotundata (68), D. esculenta (8) and D. trifida (172). Complete indexing results are provided in D. alata, D. cayenensis-rotundata and D. trifida.Viral indexing was performed on 396 yam accessions of the in vitro BRC-TP collection, except CMV, despite samples being screened for the presence of this virus in separate experiments using two sets of primers that can detect strains and isolates of both CMV subgroups 1 and 2 [All targeted viruses were present in the  1 and 2 ,45. OverMixed infections by up to five distinct viruses were reported in 282 accessions 71.2%; , whereasD. alata, four of D. cayenensis-rotundata and 43 of D. trifida, representing all combinations of co-infection, were subjected to a sanitation cycle: 18 accessions were infected by DMaV, 10 by YaV1, 20 by YMV, 49 by YMMV, 26 by badnaviruses, six by macluraviruses and 12 by potexviruses (D. cayenensis-rotundata accessions showed the highest regeneration level (9.8), D. trifida the lowest (4.1) and D. alata an intermediate level (7.1). Viral detection tools were used to monitor the efficiency of sanitation. A total of 57 infected mother vitroplants from 10 accessions of xviruses . Most acxviruses . On averin vitro collection nor in the fields in Guadeloupe during viral prevalence studies [Each meriplant and its mother plant were indexed twice for DMaV, YaV1, YMV, YMMV, badnaviruses, macluraviruses and potexviruses. No CMV indexing was performed since this virus could not be detected in the BRC-TP  studies . After oTwelve non-sanitized or partially sanitized meriplants were subjected to a second cycle of sanitation . A totalD. alata accessions, one D. cayenensis-rotundata accession and 11 D. trifida accessions  of dsRNA [In order to confirm the results obtained using RT-PCR-based diagnostic, the virome of four fully sanitized of dsRNA . For thiSanitation success rate is defined as the ratio of sanitized meriplants divided by the number of meriplants generated from an infected vitroplant. Sanitation success rates obtained in this work differed between viruses , rangingSexual reproduction provides natural sanitation for most plant viruses, which are not seed transmitted . HoweverA comprehensive diagnostic strategy was designed in order to increase sensitivity and to reduce costs and labor. Extraction of high quality TNAs from yam was achieved using the method developed by Foissac et al. . This meAlphaflexiviridae and Betaflexiviridae, whose members are difficult to detect by RT-PCR [Several potexviruses have been reported in yams . Degenery RT-PCR ,52. Indey RT-PCR .Taking advantage of the above-mentioned comprehensive diagnostic tools, we undertook the first large-scale virus prevalence study in a yam germplasm collection. The indexing of 396 accessions conserved by the BRC-TP showed that 91.6% (363/396) of these accessions were infected by at least one virus and 71.5% of them were co-infected by two to five viruses, demonstrating that viruses could easily coexist in the yam plant .D. alata, 25% in D. bulbifera, 73.5% in D. cayenensis-rotundata, 87.5% in D. esculenta and 79.1% in D. trifida. Potyviruses cause severe damage in a wide range of crops [Potyviruses displayed the highest prevalence in the analyzed yam collection, with 71% of the accessions (281/396) infected by YMV and/or YMMV . Prevaleof crops  includinof crops , therefoSurprisingly, none of the accessions were infected by CMV, although this virus has one of the broadest host spectrum ranges among plant viruses . CMV wasD. alata accessions were mostly infected by YaV1 and/or YMMV, whereas D. cayenensis-rotundata accessions were mostly infected by YMV, D. esculenta by macluraviruses and/or YaV1, and D. trifida by YMMV, badnaviruses and/or YMV. Overall, YMV was mostly detected in D. cayenensis-rotundata and D. trifida accessions, YMMV in D. alata and D. trifida accessions, YaV1 in D. alata and D. esculenta accessions, potexviruses in D. trifida accessions and macluravirus in D. esculenta accessions  and sweet potato leaf curl virus , respectively, were sanitized by the combination of thermotherapy and meristem excision [The sanitation method described in this work involves a combination of thermotherapy and meristem culture. Thermotherapy allows the reduction of the viral replication rate in vascularized tissues and meristem excision favors the regeneration of healthy plantlets from virus-free totipotent cells. This combination proved moderately efficient on the vitroplants used in this work, since only 36 out of 351 generated meriplants (9.7%) were fully sanitized, representing 16 of the 57 accessions subjected to sanitation . Comparaexcision ,59. The Rubus spp [It is important to stress out that the meriplants considered as virus-free in our work are sanitized only for the viruses for which optimized diagnosis tools are currently available, with the exception of the four plants assessed by HTS. Therefore, the sanitation scheme reported in this paper does not yet include recently characterized yam viruses such as AiPoV1, DMV and YVY, for which molecular diagnostic tools are being developed. However, the sanitation process reported in this work  has beenubus spp . Howeverubus spp .Dioscorea rotundata Poir.), this approach led to the regeneration of 70% virus-free plants from YMV-infected plants [Sanitation is a long and time-consuming process: a complete sanitation cycle takes up to nine months, not including the whole plant verification step . Hence, d plants . This sa


Mosquito viruses cause unpredictable outbreaks of disease. Recently, several unassigned viruses isolated from mosquitoes, including the Omono River virus (OmRV), were identified as totivirus-like viruses, with features similar to those of the Totiviridae family. Most reported members of this family infect fungi or protozoans and lack an extracellular life cycle stage. Here, we identified a new strain of OmRV and determined high-resolution structures for this virus using single-particle cryo-electron microscopy. The structures feature an unexpected protrusion at the five-fold vertex of the capsid. Disassociation of the protrusion could result in several conformational changes in the major capsid. All these structures, together with some biological results, suggest the protrusions’ associations with the extracellular transmission of OmRV. Culex mosquito and is closely related to the family Totiviridae. However, current researches have reported the extracellular transmission ability of OmRV, which is lacked in most members of Totiviridae. In the current study, we isolated a new strain, OmRV-LZ, and obtained its high-resolution cryo-electron microscopy (cryo-EM) structure. Unexpectedly, a protrusion structure has been found located at the five-fold vertex, which is unrecognized in the previous studies. Structural and molecular biological experiments were applied to try to investigate its functions. The results may be helpful to understand the extracellular transmission ability of OmRV-LZ and similar double-stranded RNA (dsRNA) viruses.Mosquito is a reservoir of viruses, with a large amount of them perform significant research value. Omono River virus (OmRV) has been isolated from  Some e virus) , while se virus) ,5 and dee virus) . Therefoe virus) , or mosqs (AsTV) , were ids (AsTV) –13, and s (AsTV) –13. The llularly ,14. Howellularly ,8,15–17.Infectious myonecrosis virus (IMNV), the first isolated totivirus-like virus, has been identified as the infectious agent that led to the collapse of shrimp yields in Brazil ,16. NotaHere, we report cryo-EM structures of a newly isolated OmRV strain (named OmRV-LZ). The results show that OmRV-LZ harbours unexpected pentamer protrusions and reveal that the protrusion is composed of a small protein cleaved from the polypeptide precursor encoded by ORF1. We further demonstrated that the protrusion might associate with the extracellular transmission of OmRV-LZ.Culex samples were collected from Leizhou, Guangdong Province (PR of China). After homogenization and centrifugation, the supernatants of the samples were added to C6/36 cells and could have serious cytopathic effects (CPEs) on these cells. Sample was then amplified and purified by gradient ultra-centrifugation for subsequent analyses.de novo contig assembly revealed a genome sequence of 7613 nucleotides (nt). A BLASTn search revealed that the sequence most closely matched that of OmRV-AK4  [Next-generation sequencing and It has been demonstrated that the MCPs of OmRV and IMNV were generated from a precursor peptide ,15. The Two fractions were obtained after CsCl density gradient ultra-centrifugation, and both of the fragments were subjected to further structure determination. The cryo-EM micrographs and two-dimensional (2-D) class averaging revealed that the lower and upper fractions from ultra-centrifugation contained virus particles with and without the genome, respectively . The finThe diameter of the OmRV-LZ without the protrusions is 450 Å, and that with protrusions is 540 Å. Similar to that of other members of Totiviridae, OmRV-LZ has a major capsid with icosahedral symmetry and a T = 1 lattice comprising 60 dimers composed of MCP-A and MCP-B. The central cross-sectional view of the map with the genome shows at least five visible layers of viral genome RNA packaged inside the capsid . A pore ab initio atomic models of the MCPs , another member of Totiviridae, has been determined by X-ray crystallography [The structure of lography . To escalography . To invelography ,25. HoweTo date, only two structures of totivirus-like viruses, OmRV-AK4 and IMNV, have been determined, and only IMNV has been reported to have a fibre-like protrusion ,21,22. HTo obtain better resolution and avoid the artificial features resulting from the imposed icosahedral symmetry, we applied sub-particle refinement with C1 symmetry to the protrusion. The result shows that the protrusion of OmRV-LZ is exactly pentameric; C5 symmetry was then applied to the final reconstruction of the protrusion. The final resolution of the protrusion is 4.10 Å . OverallThe resolution of the protrusion is good enough to build a rough poly-alanine backbone model containing 126 alanines . The bacOn the other hand, the quality of the stem structure is good enough to clearly identify several large sidechains of the residues . A signi3 Å3  to compare the relative expression of viral RNA , which forms the protrusion based on the structure and model of protrusion, was expressed and purified iral RNA . One groiral RNA . Then, tiral RNA . These rThe newly isolated mosquito-specific virus strain OmRV-LZ shares high homology in both sequence and structure with the previously reported OmRV-AK4 ,22, exceHelminthosporium victoriae virus 190S, Trichomonas vaginalis virus 1, Giardia lamblia virus and Leishmania RNA viruses 1) have been determined, showing that these viruses lack a fibre-like protrusion on the capsid [Undoubtedly, extracellular transmission primarily requires the ability to recognize, bind and enter a cell. The protrusion occupies the outermost capsid in the OmRV-LZ . Therefoe capsid ,26–29. Oe capsid . CompareThe head part of the protrusion is mainly composed of a β-jelly roll structure . The β-jHowever, the mechanism of extracellular transmission of OmRV-LZ needs to be further studied.The inner core of the protrusion is negatively charged  and it mThe structure of the particles without protrusions reveals that disassociation of the protrusion leads to several conformational changes and slight expansion of capsid , which iOn the other hand, the disassociation of the protrusions causes the pore at five-fold vertex expanding slightly, which would benefit the release of nascent single-stranded RNA, and therefore might trigger the transcription. However, more evidences and further investigations are needed.Leishmania RNA viruses 1 (Totiviridae) [Penicillium chrysogenum virus (Chrysoviridae) [Rosellinia necatrix quadrivirus 1 (Quadriviridae) [The capsid protein subunit of OmRV-LZ shares a conserved core structure with that of ScV-L-A, containing five pairs of well-aligned α-helices . Moreoveviridae) , Penicilviridae)  and Roseviridae)  indicativiridae) .Commonly, MCP dimers form a capsid enwrapping the genome in many dsRNA viruses. In several members of Reoviridae, the N-terminus of subunit B of the inner capsid often forms a long arm interacting with other capsid subunits to enhance the stability of the capsid shell ,39,40. HAedes albopictus mosquito cells  were provided and maintained by the Guangdong Provincial Center for Disease Control and Prevention. The cells were cultured in complete medium containing 60% Dulbecco’s modified Eagle’s medium , 30% RPMI 1640 medium (Gibco), 10% foetal bovine serum (Gibco), and 100 U/mL penicillin/100 μg/mL streptomycin (Gibco) at 28°C with 5% CO2.C6/36 Culex samples were collected from Leizhou, Guangdong Province (PR of China). Samples were mixed with complete medium, homogenized on Precellys 24 , and centrifuged at 10,000 × g for 30 min to remove the debris. The supernatants were added to C6/36 cells (1:100 v/v) in 24-well culture plates for 7 days. After three blind passages, the culture medium was used as a virus stock.2 cell culture flasks at 72 h p.i. The fluid was centrifuged at 10,000 × g for 30 min to remove the cell debris. Then, the supernatant was loaded onto a 30% sucrose cushion and centrifuged for 3 h at ~80,000 × g in an SW28 rotor  to aggregate the viruses. The pellets were resuspended in PBS and further centrifuged on a 30–50% cesium chloride (CsCl) gradient . Two bands (upper and lower bands) were collected separately. To remove CsCl, each fraction was diluted in excess PBS and pelleted by centrifugation at ~130,000 × g for 3 h. The final pellets were recovered in ~100 μL of PBS.For virus purification, cell fluid was collected from at least four 175 cmThe total genome was extracted from a purified virus suspension (full particles) using the QIAamp MinElute Virus Spin Kit  according to the manufacturer’s instructions. cDNA was synthesized using the SuperScript IV First-Strand Synthesis System  with random hexamers, according to the manufacturer’s instructions.Fastp [Megahit [For next-generation sequencing of the viral genome, the TruSeq Stranded Total RNA Library Prep Kit  was used for library preparation, and sequencing was performed on the HiSeq X Ten platform (Illumina). In total, 1 Gb of clean paired-end (150 bp) reads were quality trimmed by Fastp , and con[Megahit . FinallyCryo-EM samples were prepared as previously described . BrieflyEPU software (Thermo Fisher Scientific) with defocus ranging from 1.0 μm to 3.0 μm. Movies were recorded with 39 frames under a dose rate of 1e-/Å2/s, giving a total dose of ~39 e-/Å2. The nominal magnification was 75,000× giving a calibrated pixel size of 1.09 Å.Cryo-EM data of the full viruses were collected under a Titan Krios 300 kV electron microscope (Thermo Fisher Scientific) equipped with a Falcon III camera (Thermo Fisher Scientific) working in linear mode. Automatic data collection was performed with SerialEM software [-/Å2/s, giving a total dose of ~60 e-/Å2. The nominal magnification was 54,000×, giving a calibrated pixel size of 0.9 Å. Statistics for data collection are summarized in Cryo-EM data of the empty viruses were collected under a Talos Arctica 200 kV electron microscope (Thermo Fisher Scientific) equipped with a K3 camera  working in super-resolution mode. Automatic data collection was performed with software  with defMotionCor2 [Gctf [Ethan [Relion3.1 [Both sets of cryo-EM data were processed in the same way as in the initial step. Beam-induced motion correction and dose weighting were performed using tionCor2 . Contrasr2 [Gctf . Virus pf [Ethan . Severalelion3.1  to cleanRelion3.1 [JSPR [3-D classification was performed using elion3.1  to disti.1 [JSPR , generatRelion3.1 [Relion3.1 [After the refinement of the whole virus, the head part of protrusion was further classified and refined. The coordinators of protrusions were estimated using shift and rotation parameters  of the whole virus, which was obtained during previous refinement, by a modified script from a "block-based" reconstruction method . Then, telion3.1 . The inielion3.1 . The shiRelion3.1 and displayed and segmented using UCSF Chimera [The resolution of all the maps was estimated using the “gold-standard” FSC with the 0.143 criterion ,52. The  Chimera .Some of the small proteins were found in the micrographs from the data of the empty viruses and were boxed manually. A total of 10,749 particles were boxed, and reference-free 2-D classification was performed. Due to the limited orientations, we did not conduct further refinement and 3-D reconstruction.COOT [Phenix [The atomic models of MCP-A, MCP-B and the protrusion were built in COOT  based on [Phenix .Phenix [APBS [The qualities of all the atomic models were validated by using Phenix . The surix [APBS . StatistA pair of primers was designed to clone the gene of the ORF1 P3 N-terminal 126-aa fragment (P3N126):Forward: ATATATGGATCCATCGACTGCGACTCATCA;Reverse: ATATATCTCGAGAGCTGGTGCGTAAGGAGT.E. coli DH5α and E. coli BL21 (DE3) for amplification and expression, respectively. Purification of recombinant proteins with His-tags was performed by using a Ni-NTA affinity column. Purified recombinant proteins were used to immunize New Zealand rabbits to produce serum and anti-P3N126 antibodies, which were made by GenScript Company . The specificity and the range of reactivity of the antibodies have been validated by the manufacturer.Amplified PCR products were ligated into the pET28a plasmid, and the recombinant plasmids were transformed into -ΔΔCt method [A. albopictus β-actin gene used in qPCR are listed below [To test the effect of the anti-P3N126 fragment antibody on the infectivity of OmRV-LZ, aliquots of OmRV-LZ viruses were incubated with antibodies and an equal volume of PBS (negative control) at 4°C overnight. After incubation, the viruses were added with monolayer C6/36 cells in 12-well culture plates. Infection was stopped by adding TRIzol reagent (Invitrogen) onto the cells at 0, 6, 12 and 24 h p.i.. Total RNA was extracted according to the manufacturer’s instructions. qPCR was performed using the StarScript II Green Fast One-Step qRT-PCR Kit  in a Lightcycler 480 system . Relative gene expression was analysed according to the Livak 2t method . Statisted below :OmRV-LZ-Forward: AACGTCTGTGGCCATCTCTGOmRV-LZ-Reverse: CAGCAGCTCTTTGCGTGTTTAedes albopictus β-actin-Forward: TGACTGAACGTGGCTACTCGAedes albopictus β-actin-Reverse: ACTTCTCGAGGGAGGAGGACS1 Fig(A) Fourier shell curves of the OmRV-LZ full particle with the protrusion (2.79 Å), full particle without the protrusion (2.95 Å), empty particle with the protrusion (3.40 Å) and the protrusion structure obtained from C5 reconstruction (4.10 Å). (B) Representative atomic models and the corresponding cryo-EM maps of the α-helix and several residues with large sidechains.(TIF)Click here for additional data file.S2 Fig(A) Superposition of MCP-A in OmRV-LZ (blue) and ScV-L-A (pink). (B) Zoomed-in view of the region framed by the yellow dotted box in panel B, showing five pairs of α-helices (labelled α1–5) with conserved folds between OmRV-LZ and ScV-L-A. Other mismatched structures are set to translucent for clarity.  Zoomed-in view of the region framed by the green dotted box in panel B. (C) Atomic model of the cap-snatching reaction centre in ScV-L-A. The sidechain of the active site His154 (coloured pink) and nearby key residues Tyr150, His151, Asp152, Tyr452, Tyr538 and Asp540 that may be involved in the cap-snatching reaction (coloured grey) are displayed. (D) Atomic model of the region in OmRV-LZ MCP-A corresponding to the cap-snatching reaction centre of ScV-L-A MCP-A. All sidechains in this region are displayed.(TIF)Click here for additional data file.S3 Fig(A) Protrusion structure surface rendered with electrostatic potentials. (B) The sidechain of Tyr367 of the protrusion is inserted into the positively charged canyon area composed of two neighbouring MCP-As that are superposed with the ribbon model and rendered with electrostatic potentials.(TIF)Click here for additional data file.S4 FigReference-free 2-D classification of the OmRV-LZ full particles. Two classes without the protrusion are highlighted by red boxes.(TIF)Click here for additional data file.S1 MovieModel morph of capsid decamer and MCP-A monomer between OmRV-LZ structure with protrusion (blue) and without protrusion (red), showing the global tilt and local conformational changes.(MP4)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


We found that, although sporadic single transmission events could occur in most US counties, outbreaks will likely be restricted to the Gulf Coast region and to late spring through autumn. Seasonal fluctuations in birth rates will confer natural population-level protection against early-trimester infections. Overall, outbreak control will be more effective and efficient than prevention, and vaccination will be most effective at >70% coverage. Our county-level risk profiles should serve as a critical resource for resource allocation.Zika virus is transitioning to become a long-term public health challenge, and countries should remain informed of the risk for emergence. We developed a stochastic epidemiologic model to profile risk for Zika virus emergence, including trimester-specific fetal risk across time, in all 3,208 counties in the United States, including Puerto Rico. Validation against known transmission in North America demonstrated accuracy to predict epidemic dynamics and absolute case counts across scales (R Aedes mosquitoes that for >60 years remained only an esoteric threat to human health , Zika virus emergence and transmission continues globally, and WHO warns that Zika virus is set to remain as a long-term public health challenge ; depleting adult vectors ; and vaccination, which, should a successful candidate vaccine come to market, might reduce individual and community risk for infection once herd-immunity thresholds are achieved  into each county across time and under varying control measures. We simulated stochastic trajectories by using an adaptation of the fundamental Gillespie stochastic simulation algorithm, an adaptive tau leaping procedure .For each county and each scenario, we conducted We selected parameters from ecologic and epidemiologic literature  Figure 1Infection with Zika virus is most concerning during pregnancy, where maternal infection has been linked to congenital birth defects, most notably microcephaly  based on US Census data. For each county, we fit generalized additive models to the monthly data to estimate the fraction of annual births per month for each county. We then coupled the county-level generalized additive model output, indicating the expected proportion of annual births in each calendar month, to annual birth numbers for each individual county to calculate monthly county-specific expected pregnancies.From the monthly birth data for each county, we back-calculated the time of conception, assuming a 40-week gestation and a constant rate within a given month. On the basis of this calculation, we derived pregnancy cohorts, defined as the number of women becoming pregnant per month of the simulation, which allowed us to follow each cohort throughout their pregnancies and evaluate the number of pregnancies in their first, second, or third trimester during each month for each county or municipality. To calculate infections during pregnancy during the simulations, we derived the number of fetal exposures per week per trimester by drawing simultaneously from 3 binomial distributions each week, each with the size equal to the number of first-, second-, or third-trimester pregnancies in the county during the week of interest, and with a probability equal to the proportion of the population infected during that week . By drawWe compared models by comparing reported or published estimated incidence and case counts for known Zika that have arisen from local transmission in the United States including Puerto Rico against the respective simulated data. In addition, given similarities between Zika virus and dengue virus, such as common transmission vectors and dynamics  provided information on the monthly dynamics  of the epidemic for each health region, namely the proportion of cases per month in each of the 8 health regions. Separately, to obtain best estimates for total cumulative incidence, we used a recent and thorough report from CDC by Chevalier et al.  in 86% of US counties  had median incidence (among simulations with transmission) of <1% of the population , panel BAlong the Gulf Coast, outbreaks were more sustained. In Harris County, Texas, home to the Houston metropolitan area (population ≈4.8 million), the model predicted the largest epidemics in the 50 states, with a median epidemic size of 6,538 infections  from an import during peak vector abundance. Although the entire Gulf Coast region is at risk for outbreaks, only 3 states contributed 97% of the top 100 counties with the largest simulated outbreaks: Florida (40%), Texas (35%), and Louisiana (22%). Mississippi contributed the other 3%.According to our model, no counties within the 50 states sustained transmission beyond the first winter  Figure 8Our model showed final epidemic size was particularly sensitive to time of introduction  after introduction of index cases during peak vector abundance  required to achieve herd immunity: V = 1–1 / R0, where R0 is the basic reproductive number), in agreement with previous estimates , Brownsville County (Texas), and Puerto Rico, with separate tests across each of Puerto Rico’s 8 health regions. Overall, the model accurately predicted the dynamics and absolute case counts for each site tested  , panel COverall, our model predicts interventions would be more effective at preventing additional transmission initiated by the index case than reducing the probability of an outbreak taking place. Therefore, given limited resources, a reactive approach focused on infection control rather than complete prevention might prove most beneficial. However, for those counties with highest overall invasion potential  is a viable intervention to pursue while maternal Zika virus vaccine and risk profiles, as they relate to gestational age, are being developed (Some limitations to the proposed model might influence county level risk profiles for Zika virus transmission. For areas with a high level of travel-associated imports of Zika virus cases, such as cities with cruise ship ports and large airports, the associated risk might be an underestimate because our model does not consider multiple imports of infectious persons (Zika virus transmission is expected to persist as a long-term public health challenge, and the United States remains an entirely susceptible population, with risk for transmission. As long as Zika virus circulates anywhere, the continued importation into the United States remains a potential risk. Our comprehensive profiling efforts should serve a critical need for decision making across all levels of government regarding efficient use of local, state, and national resources aimed at preventing and controlling Zika virus transmission and should provide critical information to inform future vaccination efforts.Additional information about comprehensive profiling of Zika virus risk with natural and artificial mitigating strategies, United States.


P = 0.024), and that Dead SO+ and live hydrogen positive cells (live H2O2+) were positively correlated with freezability, respectively  and . Related to sperm functional integrity, sperm from PF bulls had greater dead intact acrosome (DIAC) than those from GF bulls  whereas sperm from GF bulls tended to have greater live intact acrosome (LIAC) than those from PF bulls . Sperm with dead reacted acrosome (DRAC) in PF bulls were greater compared to those in GF . While DIAC  and DRAC  were negatively correlated with freezability phenotype, LIAC  was positively correlated. Protamine deficiency (PRM) was similar between sperm from GF and PF bulls  and  was correlated with freezability. Sperm characteristics associated with cryotolerance are important for advancing both fundamental andrology and assisted reproductive technologies across mammals.The objective of this study was to ascertain the cellular and functional parameters as well as ROS related changes in sperm from bulls with varied sperm freezability phenotypes. Using principal component analysis (PCA), the variables were reduced to two principal components, of which PC1 explained 48% of the variance, and PC2 explained 24% of the variance, and clustered animals into two distinct groups of good freezability (GF) and poor freezability (PF). In ROS associated pathophysiology, there were more dead superoxide anion positive (Dead SO+) sperm in GF bulls than those in PF (15.72 and 12.00%;  Bull fertility, regarded as the ability of viable sperm to fertilize the egg and then support early embryonic development, is essential for the propagation of species, and it is an important economic trait for animal breeding programs. There is a global need for modern and sustainable food farming of animals such as cattle for the production of quality milk and meat to feed the world. The use of fresh or frozen sperm through artificial insemination (AI) combined with genomic selection has accelerated the genetic improvement of livestock , 2. CryoHarsh procedures of cooling, freezing, and thawing cause dramatic changes in the cell which induce injuries to the sperm membrane, thereby reducing sperm quality . More spOxidative stress is a potential factor for poor fertility status with abnormal sperm parameters and indicates the imbalance between the overproduction of ROS and total antioxidant capacity in the cell. Oxidative stress gives rise to impaired sperm function by causing DNA damage, thus remains a challenging factor for male infertility and potential pregnancy losses –23. At tDespite the significance of sperm freezability affecting bull fertility and its economic impact on cattle farming, there exists no optimal extenders and no conventional methods to accurately evaluate sperm freezability. These gaps in the knowledge and technology bases are important because they are preventing advances both in cryobiology of mammalian male gamete and in the ART. Causes underlying differing freezability remain to be elusive, although physical damage to the membranes and biochemical changes have been associated with cryo-induced oxidative stress –34. The The specific objective was to ascertain the cellular and functional parameters as well as ROS related changes in sperm from bulls with varied sperm freezability phenotypes. The results of this study advance our understanding of sperm cryopreservation and the development of more integrative methods for precision reproduction through applied biology.r = 0.91; P = 0.0002), live H2O2− , dead H2O2− , dead H2O2+ , live SO+ , dead SO– , DIAC , and DRAC , LIAC , LRAC , and MitoSOX– . From the PCA patterns, PC2 was the component that separated the GF and PF groups. It was correlated with LPO , live H2O2+ , dead SO+ , and PRM .Using PCA, we reduced the variables to two principal components, PC1 explaining 48% of the variances and PC2 explaining 24% of the variances, and clustered animals into two distinct groups of GF and PF, with an outlier for the GF . Based oP = 0.462). Lipid peroxidation (LPO), as a second messenger of oxidative stress, in bull sperm was measured by the reaction of thiobarbituric acid (TBA). The difference between the GF and PF bulls was similar for lipid peroxidation . The percentages of live hydrogen peroxide negative (Live H2O2−) and live hydrogen peroxide positive (Live H2O2+) sperm cells did not differ between the GF and PF bulls, respectively  and . Likewise, percentages of dead H2O2 negative (Dead H2O2−) and dead H2O2 positive (Dead H2O2+) sperm were similar GF and PF bulls, respectively  and . As for the assessment of superoxide anion (SO) in semen from GF and PF bulls, the percentages of live superoxide anion (Live SO–) and live superoxide anion positive (Live SO+) were similar between the freezability groups, respectively , . The percentage of dead SO– (Dead SO–) were similar between the GF and PF groups , while the dead superoxide anion positive (Dead SO+) sperm in the GF bulls were greater than in the PF . The levels of mitochondria-specific reactive oxygen species were measured in sperm from bulls with different sperm freezability phenotypes. The percentages of mitochondrial negative ROS (MitoSOX–) and positive ROS (MitoSOX+) were similar between the GF and PF, respectively  and  as depicted in By assessing the total antioxidant capacity (TAC) in the GF and PF bulls, the current study revealed that the FRAP values of GF and PF were similar . The results generated by the pattern of forward and side scatter (FSC-SSC) light revealed the acrosome status of sperm from different freezability bulls. Sperm samples from PF bulls had greater dead intact acrosome (DIAC) than those from the GF bulls  whereas GF bulls had almost greater live intact acrosome (LIAC) than the PF bulls . There was also greater dead reacted acrosome (DRAC) in PF bulls compared to those from the GF . However, live reacted acrosome (LRAC) percentages were similar between the GF and PF bulls . We also demonstrated through protamine deficiency assay that the levels of PRM were similar between the GF and PF bulls  as depicted in Analyses of data on sperm functional parameters revealed that GF had greater PTV than PF  and dead reacted acrosome (DRAC)  were negatively correlated with freezability phenotype. The number of cells with live intact acrosome (LIAC)  and Dead SO+  were positively correlated with freezability  and PF (n = 5) were performed for freezability bulls. The PTV was significantly correlated with live H2O2+ , and PRM  whereas dead H2O2+  and live SO–  have no significant association with PTV.Pearson correlation was performed for the sperm parameters between the GF and PF groups. Using scatter plots, we demonstrated the distributions of bulls for sperm functional parameters in relation to freezability status. The number of cells with dead intact acrosome (DIAC)  in male fertility remains elusive. In bovine sperm cells, Gürler et al.  to 66.19% (n = 5) (population average 57.9 ± 6.5%) (P = 0.0001)  was collected using an artificial vagina and processed immediately. Bull semen was preserved in a commercial Egg-Yolk-Tris based extender and frozen at Alta Genetics' laboratories using standard protocols ; therefore, the experiments conducted in our laboratories did not involve live animals. Five bulls were assigned to a good freezability group (GF); whereas the other five were assigned to poor freezability group (PF) according to the post-thaw viability (PTV) data provided by Alta Genetics Inc. Post-thaw viability of sperm was assessed using a fluorescent stain combination of SYBR-14 with propidium iodide  as described previously  16, 77), 7716, 7 0.0001) . The bul 0.0001) . The bulrotocols . Cryopreg for 10 min. Sperm were washed twice with PBS and diluted to a concentration of 5 × 106 cells in 100 μL of PBS solution. Thiobarbituric acid reactive substances assay was employed to evaluate lipid peroxidation by measuring malondialdehyde (MDA) produced from the oxidation of polyunsaturated fatty acids in the sperm according to the previously described method (g in 4°C for 10 min to pellet the precipitated proteins. The clear supernatant (200 μL) was collected and transferred into a 96-well plate , together with an MDA calibration curve. The absorbance was measured at 532 nm in a microplate reader . The TBARS value was expressed as μM of MDA.For thiobarbituric acid reactive substances (TBARS) assay, sperm were thawed at 37°C for 30 s and then the extender was removed by centrifugation at 800 × d method . Brieflyg for 6 min to remove the extender. The pellet was washed twice with PBS and suspended in modified Tyrode Hepes-buffered medium (sp-TALPH) at a concentration of 5 × 106 cells in 200 μL. Antioxidants in sperm were extracted by sonication . The total antioxidant capacity (TAC) was assessed by the method of the ferric reducing antioxidant power (FRAP) (3.6H2O in the ratio of 10:1:1] was mixed with 200 μL sperm cell extract and vortexed. The absorbance was measured at 593 nm against a reagent blank and absorbance of a standard solution (FeSO4.7H2O). The FRAP value was expressed as μM of FeSO4.7H2O.Cryopreserved sperm in straws were thawed the same way as above and centrifuged at 500 × r (FRAP) . The FRAg for 10 min. Also, 2 × 106 sperm cells were suspended in 300 μL of PBS for the flow cytometry assays, including mitochondrial ROS, acrosome reaction, and protamine deficiency assay. Sperm ROS were measured by following a previously reported protocol , 10 μL of 40 μM dihydroethidium , and 10 μL of 40 μM Hoechst 33258  were added to the resuspended sperm cells (2 × 106 sperm/mL) and incubated at 37°C in the water bath in the dark for 25 min. Following the incubation, the excitation of Hoechst 33258 was performed with a laser-excited fluorescence channel (VL1) at 405 nm and detected using a 445/45 nm pass filter. The excitation of DHE was performed with laser-excited fluorescence channel (YL2) at 561 nm and detected using a 615 nm pass filter whereas excitation of H2DCFDA was performed with laser-excited fluorescence channel (BL1) at 488 nm and detected using a 530 nm filter. A total of 10,000 events was evaluated for each bull sample and cell populations were gated as percentages of live-dead H2O2+, and live-dead superoxide anion positive and negative . Sperm stained with H2DCFDA and Hoechst indicating ROS and live cells are depicted in Sperm pellet was washed twice in 1 mL of PBS at 800 × protocol , with so6 cells was used for each analysis. Sperm were incubated in 3 mM of MitoSOX™ Red and 0.05 μM of SYTOX® Green  at 37°C in the dark for 15 min. Following the incubation, samples were then washed using PBS and analyzed using flow cytometer; the excitation of Mito-SOX™ Red was performed with laser-excited fluorescence channel (YL2) at 561 nm and detected using a 620 nm pass filter whereas excitation of SYTOX was performed with laser-excited fluorescence channel (BL1) at 488 nm and detected using a 530 nm filter. A total of 10,000 events was counted after gating as percentages of live MitoSOX– and MitoSOX+. Sperm stained with MitoSOX red for mitochondrial superoxide generation, and SYTOX (green) for dead cells are depicted in Mitochondrial ROS (mROS) levels in sperm cells were determined using a modified method of the fluorescent probe of Mito-SOX™ Red  . Briefly6 cells and then incubated at 37°C in a water bath in the dark for 20 min. The excitation of FITC-PNA was performed with laser-excited fluorescence channel (BL1) at 488 nm and detected using a 530 nm filter, and PI was excited at (BL3) at 488 nm and detected using a 620 nm filter. A total of 10,000 events using a flow cytometer was evaluated for each sample. The cell populations were distinguished as live reacted acrosome (LRAC), live intact acrosome (LIAC), dead reacted acrosome (DRAC), and dead intact acrosome (DIAC). Sperm stained with FITC-PNA and PI indicating acrosome and dead cells were viewed using fluorescence microscopy  according to the methods described by , with mocroscopy .6 sperm cells were resuspended in TNE buffer  after thawing and centrifugation twice in PBS at 5,000 × g at room temperature for 15 min. The CMA3 solution (0.25 mg/mL) was prepared by dissolving CMA3 (Sigma-Aldrich) in McIlvaine's buffer ; 200 μL of which were added to spermatozoa. The resuspended sperm was incubated at 37°C in a water bath in the dark for 20 min and washed twice in PBS by centrifugation (500 × g at room temperature for 10 min). Sperm cells were gated using FSC and SSC and identified by PI-positive signal excited using the fluorescence channel (BL3) at 488 nm. The signals were detected using a 620 nm filter whereas CMA3 was excited using a laser-excited fluorescence channel (VL2) at 405 nm and detected with a 530 nm pass filter. For each bull sample, a minimum 10,000 of events was assessed. The percentage of CMA3 positivity was expressed as median fluorescence intensity (MFI) read from the histogram, and measured by the ratio between the MFI of each sample and MFI of negative cells.Protamine deficiency assay was performed to assess the extent of replacement of histones by protamines using chromomycin A3 (CMA3) according to a previously reported assay . Sperm pt-test procedure of SAS 9.4 was used to conduct a two-sample t-test. For non-normally distributed outcomes, Wilcoxon rank-sum tests were performed using Proc NPAR1WAY. The actual probability values of statistical significance were reported.Principal component analysis (PCA) of standardized data (mean-centered and divided by the standard deviation of each variable) was used to reduce sperm functional parameters and oxidative stress parameters to two principal components (PC) while maintaining total variance in data. The loadings, representing correlation coefficients of variables with principal component 1  and principal component 2 , were used to map these variables in a biplot. The PC scores or rankings were used to map bulls having different freezability phenotype on the same biplot. The CORR procedure of SAS for Windows v9.4  was used to determine Pearson and Spearman's correlation coefficients (in case of non-parametric data) between post-thaw viability with sperm functional and oxidative stress parameters. The The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MH, TD, EM, and AK: conception and design of the study. AK, ET, MH, DS, and TD: sample acquisition and processing. MH, MU, TD, DS, EM, AK, and WT: acquisition, analysis, and interpretation of data. MH, MU, TD, AK, and EM: drafting the article or revising it critically for important intellectual content. All authors contributed to the article and approved the submitted version.ET employed by the company URUS Group LP. 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.