As a first step to addressthis challenge, we generated transgenic rice plants expressing NifBfrom the Archaea Methanocaldococcus infernus and Methanothermobacter thermautotrophicus. The recombinant proteins were targeted to the mitochondria to limitexposure to O2 and to have access to essential [4Fe–4S]clusters required for NifB-co biosynthesis. M. infernus and M. thermautotrophicus NifB accumulatedas soluble proteins in planta, and the purified proteinswere functional in the in vitro FeMo-co synthesis assay. We thus reportNifB protein expression and purification from an engineered staplecrop, representing a first step in the biosynthesis of a functionalNifDK complex, as required for independent biological nitrogen fixationin cereals.The engineering of nitrogen fixation in plants requiresassemblyof an active prokaryotic nitrogenase complex, which is yet to be achieved.Nitrogenase biogenesis relies on NifB, which catalyzes the formationof the [8Fe–9S–C] metal cluster NifB-co. This is thefirst committed step in the biosynthesis of the iron–molybdenumcofactor (FeMo-co) found at the nitrogenase active site. The productionof NifB in plants is challenging because this protein is often insolublein eukaryotic cells, and its [Fe–S] clusters are extremelyunstable and sensitive to O The MoFe protein isa dinitrogenase encoded by nifDK that contains thecatalytic site which binds and reduces N2. The Fe proteinis a dinitrogenase reductase encoded by nifH, whichprovides electrons to the MoFe protein.9 Mo-nitrogenase has three essential metal clusters. The first isthe [4Fe–4S] cluster located in the Fe protein, and the othersare the P-cluster [8Fe–7S] and FeMo-co [7Fe–9S–C–Mo-R-homocitrate] located in the MoFe protein.10There arethree types of nitrogenases: molybdenum- (Mo), vanadium-(V), and iron-only (Fe) nitrogenases.12 The P-cluster is formedby the reductive coupling of two [4Fe–4S] cluster pairs atthe MoFe protein in the presence of the Fe protein, MgATP, and a reductant.14 FeMo-co is one of the most complex [Fe–S] clusters discoveredin nature thus far and is synthesized in a regulated and coordinatedprocess, depending on a multitude of proteins.The [4Fe–4S] clusters are synthesized by NifUand NifS,where NifU provides the scaffold for the assembly of [Fe–S]clusters, and NifS mobilizes sulfur (S) from cysteine for [Fe–S]cluster synthesis on NifU.15 Theprocess of FeMo-co biosynthesis is initiated by NifU and NifS withthe assembly of [4Fe–4S] cluster units that are transferredto NifB.18 NifB is an S-adenosyl-l-methionine (SAM) radical enzyme that carries a catalytic [4Fe–4S]cluster  and two additional [4Fe–4S] clusters used as substrates to generate the [8Fe–9S–C]product called NifB-co.22 It has been shown that NifB-co production is 80%lower in Azotobacter vinelandiifdxN mutants, suggesting that FdxN is required for NifB-cobiosynthesis.23 Although the precise functionof FdxN remains unclear, it is thought to provide NifB with electronsneeded for NifB-co formation.10 NifB-cosynthesized by NifB is matured into FeMo-co on NifEN/NifH complexes,25 which is then inserted into the active site of the apo-MoFe proteinto reconstitute active Mo-nitrogenase.26 As the function of NifB is to catalyze the first committed stepin the biosynthesis of FeMo-co (the production of NifB-co), nifB mutants lack FeMo-co.28 NifB-co isalso the precursor cluster for the biosynthesis of FeV-co and FeFe-cofound at the active sites of the alternative V-nitrogenase and Fe-onlynitrogenase29 and therefore required for allBNF.The proteinsinvolved in FeMo-co biosynthesis can be functionallydivided into four classes: molecular scaffolds ,metallocluster carriers , substrate providers, and NifH.A. vinelandii and Klebsiella oxytoca comprise anN-terminal SAM-radical domain containing the CxxxCxxC SAM-bindingmotif and a C-terminal NifX-like domain.31 However, the simplest NifB architecture is a standalone SAM-radicaldomain because the NifX-like sequence is not essential for NifB activity.For example, the single-domain NifB proteins from Methanosarcinaacetivorans, Methanobacterium thermoautotrophicum, Methanocaldococcus infernus, and Methanothrix thermoacetophila are functionally equivalentto A. vinelandii NifB.32 This single-domain architecturefacilitates the heterologous expression of stable NifB in Escherichia coli.33 When a libraryof 28 NifB proteins was screened for expression in Saccharomyces cerevisiae, only six accumulated aspredominantly soluble proteins targeted to mitochondria, and fourof these had a single-domain architecture.34 When the same NifB variants were expressed transiently in Nicotiana benthamiana and targeted to the mitochondriaor chloroplasts, only three accumulated as soluble proteins. All threewere single-domain proteins, corroborating their superior expressionand performance in eukaryotic hosts.35The NifB proteins of M.infernus NifB or Methanothermobacterthermautotrophicus NifB targeted to the mitochondria,in each case, together with A. vinelandii FdxN. Both NifB proteins accumulated in a soluble form, and theirfunctionality was confirmed by FeMo-co synthesis in vitro.M. thermautotrophicus NifB exhibited higher NifB-coconversion activity in vitro, compared withM. infernus NifB. The production of functional NifB in rice represents an importantstep toward the expression of active nitrogenase to achieve BNF incereal crops.Here, we generated rice plants expressing stably S. cerevisiae codon-optimized nifB from M. infernus (nifBMi) and M. thermautotrophicus (nifBMt) and fdxN from A. vinelandii (fdxNAv) were used for rice transformationbecause there are no rare codons in these three gene sequences inthe context of rice codon usage.36 The nifBMi gene (hereafter OsnifBMi to indicate expressionin rice), nifBMt (hereafter OsnifBMt), and the fdxNAv gene (hereafter OsfdxNAv) were introduced into separate vectorsfor rice transformation. Expression was driven by the strong constitutive ZmUbi1 + 1st i promoter. An N-terminalmitochondrial leader sequence from the S. cerevisiae cytochrome c oxidase subunit IV (Cox4) was added to direct the proteinsto mitochondria because this sequence was previously shown to targetrecombinant eGFP to the rice mitochondria effectively.37 An N-terminal Twin-Strep (TS) tag was addedbetween the Cox4 signal and the OsNifBMi or OsNifBMt proteins for detection and purification of OsNifB. A C-terminal hemagglutinin (HA) tag was added to OsFdxNAv to enable immunodetection. The OsnifBMi and OsnifBMt constructs were used separately, ineach case, combined with OsfdxNAv and a third construct carrying the hygromycin phosphotransferase(hpt) gene for selection. Transgenic rice callusexpressing nif transgenes were produced by directDNA transfer, as described.39 Plantlets were regeneratedfrom the corresponding callus lines under hygromycin selection andgrown to maturity, as described.39Figure S1). Four lines each from MiB and MtBwere selected for immunoblot analysis. We identified three lines,MiB32, MiB115, and MtB35 that accumulated the recombinant proteinsat the highest levels. Accumulation of OsNifBMi, OsNifBMt, and OsFdxNAv in these callus lines and the corresponding regenerated plants wasdetermined by immunoblot analysis using antibodies specific for NifBMi, and the TS and HA tags . Based on their SDS-gel migration patterns, the recombinantproteins were correctly processed, resulting in the expected molecularweights of 38 kDa (OsNifBMi), 35 kDa (OsNifBMt), and 11 kDa (OsFdxNAv). As seen previously when expressed in S. cerevisiae,40 the migration of the HA-tagged FdxNAv protein was less distinct, probably dueto its smaller size. We thus confirmed that OsNifBMi, OsNifBMt, and OsFdxNAv proteins accumulated in the soluble form in rice callus  by strep-tag affinitychromatography (STAC), and the purification process was monitoredby sampling the total extract, cell-free extract, flow-through, wash,and elution fractions for analysis by SDS-PAGE and immunoblotting(S4). No significant amount of proteinwas lost during centrifugation and filteration of the cell extract,confirming that both OsNifBMi and OsNifBMt proteinswere soluble in the mitochondria. The elution fractions featured aband matching the anticipated size of correctly processed OsNifBMi or OsNifBMt. Isolated NifB yields were 44and 87 μg per 100 g fresh weight callus for OsNifBMi and OsNifBMt, respectively . Side by side comparison of NifBMi and NifBMt proteins isolated fromyeast and rice suggested the correct targeting and the specific processingof Cox4 signals from both OsNifB proteins , A. vinelandii NifEN with the permanent [4Fe–4S] clusters but lacking FeMo-coprecursor cluster (apo-NifENAv), A. vinelandii NifH protein (NifHAv), and A. vinelandii NifDK withthe P-cluster but lacking FeMo-co (apo-NifDKAv). Molybdate, R-homocitrate, and SAM wereadded as they are the required substrates for NifB-dependent in vitroFeMo-co synthesis.The minimal protein components for FeMo-co synthesisin vitro are NifB, NifEN, and NifH.OsNifBMi and OsNifBMt proteins were colorless and inactive in FeMo-cosynthesis. However, when loaded with [4Fe–4S] clusters fromNifU, the OsNifBMi and OsNifBMt proteins were functionalin the FeMo-co synthesis assay, in which the OsNifB-dependentactivation of apo-NifDK was measured by in vitro acetylene reductionactivity of the reconstituted enzyme  (The as-isolated d enzyme 3. This s1 NifDK) 3.OsNifBMt activity was affecting cell growthand developmentand precluding the generation of plants expressing OsNifBMt (OsNifBMt. The MtB15 line expressed OsNifBMt at high levels (inaddition to OsFdxNAv)not only in callus (S6a) but also in leaves ofthe corresponding regenerated plants (S6b), whichindicates that expression of NifBMt islikely not detrimental to the plants. Similarly, NifBMi expression was shown to be stable in T1 plantsof the MiB115 line (S7).To rule out that this higher OsNifBMt 1, we gen115 line 5 and S7.10 Many of these components are extremely sensitiveto O2, which is an additional challenge when transferringthe trait to plants.42 One solution isto express the nitrogenase and its accessory proteins in the plantmitochondria, a strategy that would reduce O2 exposureand supply energy for nitrogenase activity and a ready source of [Fe–S]clusters generated by proteins similar to the bacterial NifUS system.7The engineering of staple crops to fix nitrogenhas been an importantgoal of plant biotechnology for several decades. If successful, thisapproach offers the potential to reduce or even abolish our dependenceon nitrogen fertilizers, while maintaining the nitrogen content ofsoils. Natural BNF occurs only in some prokaryotes and is catalyzedby a nitrogenase complex with assistance from various accessory proteinsrequired to assemble and incorporate metal cofactors into nitrogenase.nif genesare involved in the assemblyand activity of nitrogenase and its metal cofactors in bacteria, notall are expected to be required to reconstitute nitrogenase activityin plants because some of the accessory functions can be fulfilledby endogenous proteins.43 The minimal geneset that must be transferred to plants includes NifD, NifK, and NifHwhich form the nitrogenase enzyme and NifE, NifN, and NifB which catalyzeessential reactions in the biosynthesis of FeMo-co, the active-sitecofactor of nitrogenase.10 These six geneshave been expressed in the mitochondria of S. cerevisiae and in some cases, also transiently in N. benthamiana shows that secondary structureelements and relevant residues in both structures match, with onlytwo differences: (1) the NifBMi H22 residue, which appears to stabilize the K-cluster,21 has been modeled by AlphaFold in the rotatedposition compared to its equivalent H24 residue in NifBMt and (2) the C-terminal stretch of NifBproteins. This region was well resolved in the NifBMt structure containing the K-cluster, but it was shown to bedisordered before K-cluster formation (in the absence of K2-cluster)in the crystal structure of another Archaeal NifB homolog.19 It was proposed that the short C-terminal stretchacted as a strap closing the side of the NifB β-barrel structureand stabilizing the K2-cluster.21 It shouldalso be noted that the confidence of the AlphaFold model for thisregion of NifBMi was low.Likely reasons to explain the differentoutcomes when expressingNifBS. cerevisiae.40 Analysis of the ScNifBMi protein isolated from different S. cerevisiae strains showed that while NifUS was important for providing [4Fe–4S]clusters, FdxN was more important for NifB activity.34 Earlier studies had shown that FdxN was required for efficientNifB-co biosynthesis,23 but its exact roleis still unknown. Several non-exclusive roles in NifB Fe–Scluster acquisition or maturation or as a participant of the NifBreaction have been proposed.10 For example,NifB produced recombinantly in S. cerevisiae required FdxN to acquire the EPR signatures of its three clusters.34 FdxN could also promote the reductive couplingof K1- and K2-clusters to form the [8Fe–8S] K-cluster, a reactionintermediate of NifB-co synthesis.21 Finally,FdxN could serve as an electron donor to the NifB RS-cluster for thereductive cleavage of SAM and release of the dA• radical.Earlierstudies involved the co-expression of NifB with NifU, NifS,and FdxN in 2H4 min–1 mg–1 NifDK) when co-expressed with either NifM alone or with NifM, NifS,and NifU in S. cerevisiae.49 This may reflect the distinct mechanisms usedto incorporate [4Fe–4S] clusters into the NifB and NifH proteinsor different requirements for these clusters. While NifH containsa permanent [4Fe–4S] cluster only required for catalysis, NifBrequires a [4Fe–4S] cluster for catalysis and two additional[4Fe–4S] clusters as substrates for NifB-co biosynthesis.In contrast to NifB, NifH showed similar activity , M. thermautotrophicusnifB (nifBMt), and A. vinelandiifdxN (fdxNAv), the mitochondrial targeting peptide Cox4, and the TS and HA tagswere codon-optimized for S. cerevisiae using the GeneOptimizer tool  and synthesized by Thermo Fisher Scientific, as described.40 The empty vector pUC57 was digested with Acc65I and SalI, allowing the insertion of the ZmUbi1 + 1st i promoter. The Cox4-TS-nifBMi-nos cassette was generated by PCRusing pN2XJ2140 as the template and wasintroduced into the intermediate vector pUC57-ZmUbi1 + 1st i at the SalI and SphI sites to produce pMiNifB. The pMiNifB vector was digestedwith BamHI and BstEII, allowingthe insertion of the nifBMt sequence generated by PCR using pN2SB10334 as the template, to produce pMtNifB. The pMiNifB vectorwas digested with SalI and BstEII,allowing insertion of the synthetic Cox4-fdxNAv-HA cassette, to generatepAvFdxN. All restriction enzymes and T4 DNA ligase were obtained fromPromega  or New England Biolabs .The plasmids were amplified in E. coli DH5α cells grown at 37 °C in lysogeny broth medium supplementedwith 100 μg/mL ampicillin. The fidelity of DNA constructs wasverified by Sanger sequencing . Thesequences of cloning primers and DNA constructs for rice expressionare listed in Tables S1 and S2.The sequences of Oryza sativa cv. Nipponbare) were isolated as explantsfor particle bombardment. The embryos were transferred to Murashige& Skoog (MS) osmoticum (MSO) medium for 4 h in the dark beforetransformation with 10 mg gold particles coated with the transgeneconstructs (pMiNifB and pAvFdxN or pMtNifB and pAvFdxN) and the hygromycinphosphotransferase (hpt) selectable marker at a molarratio of 3:3:1. The bombarded embryos were maintained on MSO mediumfor 16 h in the dark and then transferred to MS selection medium for4 weeks in the dark, with one subculture after 2 weeks. Half of theresistant callus was kept under selection, and the other half wastransferred to MS regeneration medium with a 12 h photoperiod for3–4 weeks to regenerate transgenic plantlets. The transgenicplantlets were transferred to rooting medium (HMS) with a 12 h photoperiodfor 2 weeks and planted to soil in the greenhouse with a 12 h photoperiodand 80% relative humidity. Media compositions are listed in Table S3.Seven-day-old mature rice embryos (2) in 2 mL Eppendorf tubesusing 3 mm BeadBug steel balls and a microtube homogenizer  operating at 400 rpm for 20 s. Leaf powderwas resuspended in 7 volumes (v/w) of extraction buffer comprising100 mM Tris-HCl (pH 8.6), 200 mM NaCl, 10% glycerol, 1 mM PMSF, 1μg/mL leupeptin, and 5 mM EDTA and homogenized twice. Cell debriswas removed by centrifugation , and the supernatant was collected and stored at −80°C. Soluble rice callus extracts were prepared using a blender(see STAC purification section).Solublerice leaf protein extracts were prepared by grinding ca. 50 mg ricetissue (snap-frozen in liquid NMi (generated in-house), and monoclonal antibodiesagainst the strep-tag II  and the HA tag or Rubisco  were diluted at 1:2,000–1:5,000 in TBS-Tsupplemented with 5% bovine serum albumin (BSA). Secondary antibodies(Thermo Fisher Scientific) were diluted at 1:20,000 in TBS-T supplementedwith 2% non-fat milk and incubated for 2 h at room temperature. Membraneswere developed on medical X-ray films  usingenhanced chemiluminescence.Rice proteins were separatedby SDS-PAGE and then immunoblotted to Protran Premium 0.45 μmnitrocellulose membranes  using asemi-dry transfer apparatus at 20 V for 45 min. Loading equivalence was confirmed by stainingpolyacrylamide gels with Coomassie brilliant blue or nitrocellulosemembranes with Ponceau S. The membranes were blocked with 5% non-fatmilk in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.02% Tween-20 (TBS-T)for 1 h at room temperature, before incubation with primary antibodiesovernight at 4 °C. Primary polyclonal antibodies against NifBOsNifBMi and OsNifBMt were prepared for STAC purification at O2 levels below 1 ppm in an anaerobic chamber . Callus was disruptedin lysis buffer comprising 100 mM Tris-HCl (pH 8.5), 300 mM NaCl,10% glycerol, 3 mM sodium dithionite (DTH), 5 mM 2-mercaptoethanol,1 mM PMSF, 1 μg/mL leupeptin, 10 μg/mL DNAse I, and 1:200(v/v) BioLock solution at a ratio of 1:3 (w/v). Total extracts were prepared by lysing thecell suspensions under anaerobic conditions using the Oster 4655 blender modified with a water-cooling systemoperating at full speed in 4 cycles of 2 min at 4 °C. Extractswere transferred to centrifuge tubes equipped with sealing closures and centrifuged  using the Beckman Coulter Avanti J-26 XP device.The supernatant was passed through Nalgene 0.2 μm filter cups(Thermo Fisher Scientific) to yield a cell-free extract of solubleproteins. This was loaded at 2.5 mL/min onto a 5 mL Strep-Tactin XPcolumn (IBA Lifesciences) attached to an ÄKTA FPLC system . The column was washed with 150 mL of 100 mM Tris-HCl(pH 8.0), 300 mM NaCl, 10% glycerol, 2 mM DTH, and 5 mM 2-mercaptoethanolat 16 °C, and bound proteins were eluted with 15–20 mLof the same wash buffer supplemented with 50 mM biotin (IBA LifeSciences).The elution fraction was concentrated using the Amicon Ultra centrifugalfilter  with a cut-off sizeof 10 kDa. Biotin was removed by passing the protein through PD-10desalting columns  equilibrated with wash buffer. Thedesalted eluate was concentrated and snap-frozen in Nalgene cryovialsand stored in liquid nitrogen.OsNifBMi and OsNifBMt was determined by Coomassie geltitration against standards of the purified S. cerevisiae NifB (ScNifBMi and ScNifBMt) protein, as shownin Figure S5. Amino terminal amino acidsequencing was performed by Edman degradation . 25 pmol OsNifBprotein was separated by SDS-PAGE, transferred to 0.2 μm Sequi-BlotPVDF membranes (Thermo Fisher Scientific) in 50 mM borate buffer (pH9.0), stained with freshly prepared 0.1% Coomassie R-250 (Sigma-Aldrich)in 40% methanol and 10% acetic acid, and then destained using 50%methanol.The yield of purified 34 For the in vitro synthesis of FeMo-co, each100 μL reaction contained 3 μM NifHAv, 1 μM OsNifB, 1.5 μM apo-NifENAv, 0.6 μM apo-NifDKAv, 17.5 μM Na2MoO4, 175 μM R-homocitrate, 9 μM [Fe4–S4]cluster-loaded NifUEc (holo-NifUEc), 125 μM SAM, 1 mg/mL BSA, and theATP-regenerating mixture .For the positive control FeMo-co synthesis assay, holo-NifUEc was omitted, and OsNifB was replacedwith 2.5 μM NifB-co. The reactants were incubated for 60 minat 30 °C. For the acetylene reduction assays, 500 μL ofthe ATP-regenerating mixture and 2.0 μM NifHAv were added to the reaction tube. The reaction mixture wasthen transferred to 9 mL serum vials under an argon/acetylene (94%/6%)atmosphere. The reaction was incubated for 20 min at 30 °C. Tomeasure ethylene formation, 50 μL of the gas phase was takenfrom the reaction vials and injected in the Shimadzu GC-2014 gas chromatographerequipped with the Porapak N 80/100 column .FeMo-co synthesis and apo-NifDK reconstitution assays were carriedout in vitro in an anaerobic chamber, as previously described.Standard deviation (SD) of invitro activity data was calculated based on two biological replicates.


FEBS Open Bio focused on Virology. The section contains four review articles, each focused on a particular aspect of virus–host interaction, including the antiviral response, subversion of the host translational machinery, repurposing of cellular factors, and the development of antiviral drugs.The spread of pathogenic viruses implies host infection, replication, and virus dissemination. In each step, viruses have to overcome the host defenses designed to neutralize the threat they pose. The host–virus relationship represents a constant multistage battle for power as the host/cell does not voluntarily give in to the viral enemy. Upon infection, cells recognize viral pathogen‐associated molecular patterns, activating the innate antiviral defenses. As such, during most of the replication cycle, the virus has to deal with the cellular antiviral response. At this point, it should not be forgotten that viruses are obligate intracellular parasites and thus are entirely dependent on the host cell for their replication. This dependency has pushed viruses to evolve unorthodox strategies to subvert and repurpose cellular factors and processes required for efficient replication. Even if a virus has the potential to be successful at each step necessary for its spread, this does not mean it has won the war against the host. Another threat to viruses is represented by antiviral drugs designed to diminish their survival and promote the host's wellbeing. This editorial outlines the contents of this special ‘In the Limelight’ issue of  HSF1heat shock factor 1PAMPspathogen‐associated molecular patternsRBPRNA‐binding proteinsvRNAsviral messenger RNAsViruses are obligate intracellular parasites, which depend on cells for their replication . In consA successful infection implies that a virus has managed to overcome all host antiviral responses and has been capable of taking control of the cell processes required for its efficient replication . SuccessAs the Editor of this issue, I thank all authors and reviewers for the time devoted and their excellent contributions to this ‘In the Limelight’ section. I hope the provided insights into the world of host–virus relationships will be of interest to all of our readership.The authors declare no conflict of interest.MLL wrote the editorial.


Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2), Grapevine leafroll-associated virus 3 (GLRaV-3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine fanleaf virus (GFLV) and its satellite RNA (satGFLV), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine Pinot gris virus (GPGV), Grapevine satellite virus (GV-Sat), Hop stunt viroid (HSVd), and Grapevine yellow speckle viroid 1 (GYSVd-1). Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for validation of sRNA-seq predicted infections, including various combinations of viruses or viroids and satellite RNA. mRT-PCR could further be used for rapid and cost-effective routine molecular diagnosis, including widespread, emerging, and seemingly rare viruses, as well as viroids which testing is usually overlooked.Metagenomic approaches used for virus diagnostics allow for rapid and accurate detection of all viral pathogens in the plants. In order to investigate the occurrence of viruses and virus-like organisms infecting grapevine from the Ampelographic collection Kromberk in Slovenia, we used Ion Torrent small RNA sequencing (sRNA-seq) and the VirusDetect pipeline to analyze the sRNA-seq data. The used method revealed the presence of:  Vitivirus have been identified in South Africa [Grapevine is one of the most susceptible plants to viral infections. More than 86 viruses belonging to different families and genera have been reported to infect grapevine , and theh Africa .Most grapevine viruses have an RNA genome, including viruses associated with four major and widespread disease complexes  . VirusesGrapevine leafroll-associated virus 3 (GLRaV-3) in California are more than USD 90 million annually [Viral pathogens are spread over long distances by infected material (nursery productions), whereas infections within a vineyard or an area are transmitted mechanically and by insects, mites, or nematodes . Virusesannually . TherefoHigh-throughput sequencing technology (HTS), which targets all nucleic acid types, enables rapid and accurate detection, including previously described and novel viruses and virus-like organisms ,15,16,17The aim of the presented work was to perform sRNA-seq for the diagnosis of grapevine viral pathogens in six grapevine varieties from the Ampelographic collection Kromberk, Slovenia, and to develop an mRT-PCR assay for the validation of sRNA-seq data that could be further used for rapid and cost-effective routine molecular diagnosis in large-scale surveys.A total of 13 cuttings from six grapevine varieties, two red, ‘Cipro’ (‘Rosenmuscateller’) and ‘Pokalca’ (‘Schioppettino’), and four white, ‘Malvazija’ , ‘Volovnik’ (‘Vela pergola’), ‘Rebula’ , and ‘Poljšakica’, were collected from the Ampelographic collection Kromberk near Nova Gorica, Slovenia, in 2017 . CuttingThe selected samples were pooled together into four pools representing either samples of the same variety  or of different varieties (L4). Small RNAs (sRNAs) were isolated using mirVana™ miRNA Isolation Kit  according to the manufacturer’s instructions for the enrichment of sRNAs. The quantity and quality of sRNAs were assessed using the Agilent 2100 Bioanalyzer  according to the manufacturer’s instructions. Libraries of sRNAs were constructed using the Ion Total RNA-Seq Kit v2  and were barcoded using the Xpress™ RNA-Seq Barcode 1–16 Kit  according to the manufacturer’s instructions. The yield and size distribution of the amplified cDNA libraries were determined using the Agilent 2100 Bioanalyzer . Libraries were pooled at equimolar concentrations and prepared for sequencing using the Ion PI™ Hi-Q™ OT2 200 Kit and Ion PI™ Hi-Q™ Sequencing 200 Kit  according to the manufacturer’s instructions. Sequencing was performed on Ion PI™ chips v3 using an Ion Proton™ System , according to the manufacturer’s instructions. Raw sequencing data were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under BioProject number PRJNA667593, BioSamples: SAMN16378719-SAMN16378722. The sRNA-seq data were analyzed using the VirusDetect pipeline with default parameters . The pip2, and stabilizers), 0.2 µL (0.1 µL for GRVFV) of each 10 µM forward and reverse primer , 1 µL of pooled cDNA, and nuclease-free water up to 25 µL. Primers are listed in Confirmation of sRNA-seq-predicted infections was performed by mRT-PCR. Total RNA was extracted from 100 mg of frozen leaves using the RNeasy Plant Mini Kit . First strand cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit  according to the manufacturer’s instructions. mRT-PCR was performed using the KAPA2G Fast Multiplex PCR Kit . The reaction mixture was prepared using 12.5 µL of KAPA2G Fast Multiplex Mix , Grapevine leafroll-associated virus 2 (GLRaV-2), Grapevine leafroll-associated virus 3 (GLRaV-3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine fanleaf virus (GFLV) and its satellite RNA (satGFLV), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine Pinot gris virus (GPGV), Grapevine satellite virus (GV-Sat), Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd-1). The highest number of viral entities (nine) was found in the library that was a mixture of three different varieties (L4). Eight viral pathogens were detected in the library of variety ‘Cipro’ (L1), while in the other two libraries (L2 and L3), the number of identified viral pathogens was seven  were mapped to viral reference sequences, while 42,342,716 (59.72%) originated from grapevine. The BLASTN search of the unique viral-assembled contigs revealed the presence of widespread viruses associated with four major disease complexes, emerging virus, GV-Sat (first report in Slovenia) , as well2 and/or annealing temperature lower than 60 °C . In this study, the lowest GC content had again primers for GLRaV-3 amplification (30%), while all other primers for amplification of predicted viruses in L2, L3, and L4 had GC content in the range of 40–60%. In L1, all primers for virus amplification had a GC content of 40–60%, except for the reverse primer of GLRaV-2 (66.67%)  [Additionally, in this study, we described the application of the mRT-PCR approach for validation of the sRNA-seq data. Simultaneous amplifications of different combinations of nine viruses or two viroids and satGFLV were performed. According to the KAPA2G Fast Multiplex Kit protocol, employed primers should have a similar temperature melting (Tm) and GC content of 40–60%. In our study, the Tm of primers used for virus amplification was not similar; the lowest Tm had a primer pair for GLRaV-3 amplification (51.81 °C for forward and 48.91 °C for reverse primer) . Conside(66.67%) . Althoug(66.67%) . Thus fa. (2007) . Hajizad. (2007)  develope. (2007) . In this. (2007) ,62. Lowe. (2007) .However, in this study, we showed that mRT-PCR is highly effective, reliable, and sensitive, enabling validation of all viral pathogens predicted with sRNA-seq.High-throughput screening and high-throughput validation of viral entities for some important old grapevine varieties from the Ampelographic collection Kromberk, Slovenia, was performed. The mRT-PCR protocol described herein provides a simple, time-saving, cost-efficient method for the rapid and reliable validation of sRNA-seq data and successful detection of viral pathogens belonging to different families and genera.


Japanese encephalitis is a serious disease transmitted by mosquitoes. With its recent spread beyond the traditional territory of endemicity in Asia, the magnitude of global threat has increased sharply. While much of the current research are largely focused on changing epidemiology, molecular genetics of virus, and vaccination, little attention has been paid to the early history of virus isolation and phenotypic characterization of this virus. In this review, using this piece of history as an example, I review the transition of the concept and practice of virus isolation and characterization from the early period of history to modern times. The spectacular development of molecular techniques in modern times has brought many changes in practices as well as enormous amount of new knowledge. However, many aspects of virus characterization, in particular, transmission mechanism and host relationship, remain unsolved. As molecular techniques are not perfect in all respects, beneficial accommodation of molecular and biologic data is critically important in many branches of research. Accordingly, I emphasize exercising caution in applying only these modern techniques, point out unrecognized communication problems, and stress that JE research history is a rich source of interesting works still valuable even today and waiting to be discovered. Japanese encephalitis virus (JEV) is the most serious cause of encephalitis in humans among mosquito-borne viruses ,2. CurreCulex tritaeniorhynchus, endowed with a capacity of long distance dispersal  (a flavivirus) discovered accidentally in 1975 in the Joseph Peleg’s  culture . Because in Cuba , occurrerto Rico . With the advent of metagenomic techniques, more commensalistic viruses were discovered, and some new viruses were found to be even beneficial to the hosts. Thus, establishing a causal relationship as a requirement for the newly discovered viruses partly became unimportant or irrelevant; and further revision of already-modified Koch’s postulates became again necessary in the eyes of modern virologists, since many new viruses cannot be propagated . The scientists who discovered viruses or isolated viruses of notable significance have received a special accolade, and their contributions have been celebrated in virology and/or in medical history books ,20,21. WAccordingly, dispute over who isolated a particular virus for the first time in history has erupted occasionally, such as in the discovery of human immunodeficiency virus in a form of highly open public argument or in the discovery of Ebola virus in a form of more subtle discussion. As shown in the following paragraphs, the “first” isolation of JEV superficially resembles the famous retrospective debate over who was first to discover virus as a new disease entity distinct from microorganisms, Ivanovskii, Beijerinck, Loeffler and Frosch or someone else? . One comThe understanding of the requirements for “first” virus isolation evolved by trials and errors over years, since little was known in the early part of history about the characteristics of viruses. Further, techniques used at first were rudimentary when even syringes had not been invented. The following requirements were a retrospective compilation, which means that at any given stage of JEV isolation period researchers recognized these criteria only partially. It should be noted that in historical accounts, sometimes “virus isolation” has been used for the events of “first recognition of viral etiology” or “first virus detection”; while “virus discovery” has been loosely used for virus isolation, virus detection and/or recognition of viral etiology. These ambiguous usages of words also contributed to controversies. With respect to the history of virus isolation and techniques used, an ebook available free online  is very Development of unglazed porcelain filter by Charles E. Chamberland in 1884 for proof of viral etiology was unquestionably a major landmark. It was in 1924 when the etiology of JE was confirmed by this method. However, the conclusion was not without a controversy because some claimed evidence of bacterial etiology. This problem was not uncommon at the time because it was a time of transition from the age of bacterial to newly emerging viral etiology of infectious diseases. In modern times, the requirement of submicroscopic size was weakened by the discoveries of giant viruses (such as mimiviruses) some of which are microscopically visible; and requirement of filterability was violated by the discoveries of non-viral, filterable, and infectious entities . This was found early as a good indication of viral agent, but debate persisted for some time because a small number of microorganisms that grow in media are filterable. Nonetheless, for JEV isolation, this was an important criterion. The absence of replication in culture media may suggest automatically that early virologists recognized obligatory need of living cells for viral replication. However, that interpretation was not necessarily uniform among early workers. Most early workers heavily relied on trials and errors and careful observation of diseased animal or human for this information. For JE, initially sampling sources for virus isolation were broad and included most major organs, the brain, cerebrospinal fluid, blood, saliva, nasopharyngeal secretion, urine, and feces. Though it was claimed by some that JEV was isolated from blood, it was not considered an optimal source, because of too many negative results. Ultimately, central nervous system, in particular, the brain, was chosen for isolation by most workers. For other sources of the virus, direct transmission was considered, as described in In the 1920s and early 1930s, Asian monkeys, guinea pigs, and rabbits were used by many. Some used even dogs. The low rate of success in producing a neurologic symptom consistently over passages was a major problem, as the rate of claimed success varied considerably among workers. Cell/tissue culture technique was developed extensively since the early research in the 1910s by Alexis Carrel and many others. By the time of nationwide JE outbreak of 1935, use of chick embryo developed by Ernest W. Goodpasture in 1931 was added to the list of choices available for JEV isolation . More imIn the 1920s and 1930s, when little was known about the factors determining the development of a sign, symptom or syndrome of JE, variations in animal species chosen, its age, dose, route of inoculation, and other factors enormously complicated the establishment of cause-effect relationship and delayed discovery of JEV. Appearance of a symptom or syndrome in experimental animals quite different from that in humans was other complication. Nevertheless, most investigators recognized the importance of establishing a cause-effect relationship with the agent isolated. Initially, reproducibility of the development of a neurologic symptom in experimental animals was thought to be sufficient to fulfill this requirement for JEV. However, the results were variable. To improve reliability, workers emphasized in vivo neutralization test (NT) using mice and homologous antiserum-virus mixture  because Causal relationship was also determined by immunologic (later serologic) tests. Among arboviruses, it was determined first for yellow fever virus with complement fixation (CF) test , followeThough this is one of the most important requirements today, in the early part of history (before 1930), it was rarely performed simply because of the paucity of similar viruses isolated. As virus isolation activities accelerated in early 1930s, by the time JEV was isolated in 1935 this was a requirement for the confirmation of new virus isolation. However, the kinds of viruses to compare with varied depending on the inventory of viruses available in each laboratory. Thus, in Japan St. Louis encephalitis virus (SLEV) and Rift Valley fever virus were used for JEV, while in the U.S., SLEV was compared not only with JEV but with western equine encephalitis virus, Russian spring-summer encephalitis virus, and other neurotropic viruses ,35. Preservation of infectivity of isolated virus was a major concern in the early period. A stock of well-preserved virus was indispensable for the confirmation of reproducibility by other competing researchers. If infectivity was lost, it negatively affected the claim of “first” virus isolation.Preservation of infectivity was a serious challenge in the early period of JE research, because at first little was known about the optimal conditions for a longer preservation. Although preservation of vaccinia virus used for vaccination at a low temperature was known before 1900 , the ideIn the early part of the 1930s, lyophilization was found to improve virus preservation. However, invention of a practical freeze dryer for a prolonged preservation of virus was developed only in 1935 . FurtherThis was an added requirement uniquely for vector-borne viruses, such as JEV. At the time, nearly all isolates were made of the specimens collected from patients, and causal relationship was established with these isolates. When JEV was isolated from mosquitoes in 1938 for the first time, the validity was seriously questioned by physicians because few of them could understand how a virus in mosquitoes could cause JE in humans. Thus, Tokushiro Mitamura and his colleagues  conducteIt has been speculated that relaxation of refrain from meat consumption among Buddhists in Japan at the end of feudal period in mid-19th century coincided with emergence of encephalitic outbreak. While it is generally agreeable, strictly speaking, swine breeding and popularity of the consumption of pork were recorded in Okinawa and southwestern parts of Kyushu for nearly a few centuries even during this period of refrain. In Okinawa, in particular, because of heavy influence of Chinese culture (including cuisine), consumption of pig meat and swine breeding had been a long tradition. Similarly in Kyushu, because of the influence of European visitors (including missionaries), pig breeding and meat consumption persisted even during the “refrain period,” albeit at a level below Okinawa’s. Because these are the regions of the country where earliest cases of JE typically begin to emerge at the beginning the season to start a dispersal to the northeast, any historical record of encephalitis outbreak there would suggest possible occurrence of JE much earlier. The fact that such a record was not found then favors the theory that JE first appeared only around 1871, although a possibility of lack of individuals interested in documenting diseases or loss of documents as an alternative explanation cannot be entirely ruled out. The earliest record of encephalitis outbreak with a considerable number of fatality was recorded in 1871 through 1873. An outbreak of equine encephalitis which occurred in 1897 is strongly believed to be a JE outbreak. The peak of epidemic occurred usually in August through September. Because of this seasonality, often the epidemic was called “summer encephalitis.” Subsequently, outbreak occurred in 1901, 1903, 1907, 1909, 1912, 1916, 1917, and 1919. Among those outbreaks, the 1912 outbreak was notable for high incidence in the elderly people and the case fatality rate (CFR) approximately around 60%. In the 1919 outbreak, more than 7000 cases were reported, again heavily among the elderly people. This is puzzling because JE is known to be primarily a pediatric disease today. An early age-stratified epidemiologic study of the encephalitic cases in the western region of the country considered endemic focus showed that the proportions in 1–10, 11–20, 51–60, and >60 yr groups were 8.5, 5.8, 21.1, and 40.6%, respectively . The othThese reports contrasted to the predominantly pediatric pattern in Korea, where the seasonality and other aspects of JE were very similar to those in Japan. After WWII, even in Japan JE became predominantly a pediatric problem. It was suspected that the most likely reason for the unusually high ratio for the elderly people in Japan was because in public health reports, the statistics was then based on clinical diagnosis but not on laboratory confirmation . HoweverIn the explosive 1924 outbreak, out of four main islands of Japan  only Hokkaido was spared, as cases were recorded as north as Aomori of Honshu. After the 1924 outbreak, epidemic continued to occur. In the outbreaks of 1926, 1925, 1927, 1931, 1936, and 1937, CFR consistently exceeded 50%. Even in inter-epidemic periods, case number per annum often exceeded 600. It is interesting that in most of those years of outbreak in Japan, JE epidemic also occurred simultaneously in Korea with a very similar pattern of seasonality . In muchMultiple theories were proposed on the etiology . They raAs described earlier, the amount of historical documents made available to the scientists outside Japan (primarily to the scientists in the USA) in English represented only a small portion of the vast volume of publications in Japan. This is exemplified by the large numbers of articles covered in monographs published after the 1924 and 1935 JE outbreaks ,48. I prBetween 1924 and 1938, three major outbreaks of JE occurred in Japan, though smaller numbers of clinical cases were continuously reported in inter-epidemic periods. In the current literature, a confusion is found, because of three different dates assigned to “first” isolation of JEV among authors. In In the investigation of the 1924 outbreak, many workers representing multiple universities  were involved in isolating the etiological agent . Among mThe tradition of quoting 1924 as the date of the “first” JEV isolation most likely originated in the publication of Thomas M. Rivers, then the world leading authority of newly emerged virology independent of the traditional bacteriology or microbiology. It should be noted that in his publication , he mereIn the case of “first” isolation in 1933, Michitomo Hayashi inoculated multiple brain specimens from the deceased victims into the brain of Asian monkeys . Among tIn the “first” isolation during the outbreak that affected multiple regions of the country in 1935, a large number of researchers representing governmental laboratories, universities, research institutions, and hospitals located in multiple locations participated in isolation. The word “virus” was officially adopted for the suspected agent during an emergency conference held that year. As shown in Because many strains were “first” isolated simultaneously during one outbreak, when the prototype of JEV was selected in the U.S. for a compilation of arboviruses and related zoonotic viruses , selectiOnly the records of shipment or transfer for the 1935–1950 period which were documented in publications are used. Other known transfers which were documented but deficient in data recording and other transactions without any record are excluded. Green: Shipment/transfer of Nakayama strain; Red: Shipment/transfer of all other strains. As a virus exchange, SLEV was shipped from L. T. Webster of the Rockefeller Institute of Medical Research (RIMR) to Univ. Tokyo and St. Luke’s International Hospital in 1936 and later to Niigata University. For many transactions across the Pacific both virus and antiserums from patients were shipped. The names in double quotation marks refer to strains. www.cdc.gov/arbocat/virusbrowser.aspx, accessed on 9 October 2022) has served for many years as an invaluable source of voucher specimens for arbovirologists around the world. Regarding the information on the 1935 strain submitted to the Catalogue at the time of virus deposition, I found it difficult to identify the strain partly because of the lack of specific strain designation in the references submitted at the time of virus deposition. The International Catalogue of Arboviruses published by the American Society of Tropical Medicine and Hygiene   had been isolated only two years earlier. To compare the properties of these encephalitic viruses, the investigators of two countries arranged virus exchange. As a result, Nakayama and other strains of JEV (as well as homologous antiserums from patients) were shipped to some laboratories in the U.S., while SLEV (and the antiserums) were shipped to laboratories in Japan. In each country, imported virus was further shared with other laboratories. Other shipments from Japan to the U.S. are known, but due to the troubling style of recording, only those transactions documented in publications between 1935 and 1950 ,91,92,93In the meanwhile, unbeknownst among most JE researchers on both sides of the Pacific, during the difficult time of devastation during WWII, all JEV isolates in Japan, including the original Nakayama strain, were lost there ,64,90. ANakayama strain of JEV occupies a special place in the history of JE research not only as the “first” strain but also for serving as source for the only JE vaccine and material for research worldwide for nearly half a century. Obviously, for many researchers, the following history rarely told may be of interest. After returning home to Yobo Kenkyusho , Nakayama strain was shared with another governmental public health institution dedicated to veterinary product quality inspection and belonging to the Ministry of Agriculture and Fisheries  and Kitasato Institute, the birth place. In the subsequent JEV research, researchers studying any difference among Nakayama strain derivatives in different laboratories designated the derivative held at Yoken as “Nakayama-NIH,” another held at Yakken as “Nakayama-Yakken,” and the stock held at the origin of the return trip (RIMR-NY) as “Nakayama-RFVI” .It has been long thought that “NIH” affixed to Nakayama strain meant Yoken of Japan, because some leading JE researchers in charge of vaccine project in that institution preferred their professional affiliation be called “National Institute of Health” in publications, following the namesake designation in the U.S. However, it turns out that this “NIH” attached to the vaccine actually meant NIH of the U.S. because of the following background. During the post-war period, quality certification of JEV vaccine produced in the U.S. Army was the responsibility of the National Institute (still singular in 1947) of Health. The agency then issued procedural instructions and quality standards which must be met by the lyophilized vaccine produced by using chick embryo . As U.S.When virus strain is passaged many times, increased mutation and possible phenotypic change are the major concerns. By the end of 1940s, the passage level of Nakayama strain exceeded well over 100. In fact, antigenic difference among the Nakayama strain derivatives was reported ,104. FurIn the early period of research, virus transfer to another institution meant shipping infected tissue or even organs. Naturally, the quantity of infectious virus shipped varied greatly. Furthermore, shipping blocks of tissues was cumbersome and dangerous. To standardize the amount of infectious virus per shipment, to make storage of virus simpler, efficient and/or to make it much less dangerous, adaptation of virus to tissue or cell culture was explored. As a result of this effort, pooled supernatant of infected in vitro culture was dispensed in a fixed quantity to a large number of vials for an easy storage and shipment. JEV was one of the beneficiaries of this technologic improvement. Another benefit of this improvement was minimal need of passage ,33. In this section, early attempts to characterize JEV and its mechanisms of transmission and persistence in nature are briefly summarized. These contributions are later compared with the applications of molecular techniques for respective subject in In the early period, the relationship of JEV among known animal viruses was initially determined on the basis of antigenic relationship. Additionally for JEV, the relationship with other neurotropic viruses  was investigated. Jordi Casals, initially using complement fixation technique and hyperimmune serums, clearly distinguished JEV from eastern equine encephalitis, western equine encephalitis, and louping ill virus but recognized a close relationship with SLEV . He thenTraditionally, regarding JEV persistence in temperate regions, four mechanisms have been proposed: overwintering of JEV in vectors, overwintering in vertebrate hosts, repeated  introduction of the virus carried by vectors blown by atmospheric current, and repeated  introduction of the virus by infected birds. JEV in overwintering mosquitoes was recorded in Korea as well as in the semitropical Amami-Oshima and Okinawa Islands further to the south of Kyushu of Japan ,110,111.JEV was once isolated from pigs early in the season (presumably infected between April and June) in Hokkaido in 1986, which raised a possibility of viral overwintering in northern part of Japan . ActuallJEV overwintering probably occurs sporadically in other regions as well, but the persistence of virus overwintering is most likely only for a short period of time. Additionally, in northern temperate regions, isolation of JEV in overwintering mosquitoes has been rare. This is not surprising because overwintering female mosquitoes generally do not blood-feed before hibernation. Accordingly, in the subsequent absence of any report of newly infected pigs in early spring in Hokkaido and elsewhere in northern temperate regions, it has been difficult to interpret the mode of transmission except to recognize occurrence of infrequent (but short) viral overwintering. On the other hand, repeated  introduction of virus-infected mosquitoes blown by atmospheric air current has been more strongly supported by the sequence similarity of newly introduced genotype I JEV between the strains in the continental parts of Asia and those isolated in Japan, since genotype I had not existed there before .As for vertical transmission of JEV in mosquitoes, it was discovered for the first time in 1938 ,40. It wThough almost totally overlooked for years, JEV transmission between larvae in aquatic conditions also occurs . This moShortly after the first JEV isolation in 1935, an interest in other vertebrates as possible hosts of this virus grew. As a result, JEV was isolated from horses in 1937 , pigs inThe importance of birds in JEV transmission was more clearly recognized during an extensive research by U.S. investigators  in Japan in the 1950s. They found (i) that ardeid birds are regularly and silently infected with JEV in the summer; and multiple strains of JEV were isolated from those birds. Because the viremia levels are sufficient for vector transmission, they concluded that bird-mosquito-bird cycle of transmission cycle in the summer was certain. They further speculated that viremic birds disseminated virus to mosquitoes in distant locations; (ii) that epidemic ceased with the arrival of cold season, resulting in concurrent cessation of mosquito biting activity; (iii) that they had no idea if and how JEV persists in temperate regions after summer and speculated the possibilities of overwintering in mosquitoes, latently infected hosts, or repeated introduction of virus by migratory birds at the beginning of each JE season, but without ever reaching a conclusion; and (iv) that, because of the complexity of involvement of multiple hosts, control of one amplifying host (such as pigs) would not be sufficient to suppress JE epidemic. However, they neither used the word “reservoir” nor speculated its identity. Furthermore, they did not state that mosquito-host-mosquito cycle was the only mechanism of JEV transmission ,120. EssAlthough the role of pigs in JEV transmission has been a subject of strong interest recently, important first reports in history, such as virus isolation from pigs , discoveMore recently, a report of nasopharyngeal contact transmission of JEV between pigs drew attention . Though The aforementioned confusion over the “first” date of JEV isolation in the literature also derived from a language barrier in international scientific communication at the time. Because most early articles on JEV isolation were published in Japanese and because global bibliographic database on biomedical literature did not exist, few in other countries were aware of these publications. Accordingly, the publications in the early JE research found in nearly all bibliographic databases popularly used today are limited to a very small number of articles in English. Accordingly, the number of early JE papers retrievable from these databases is hardly commensurate with the actual number . FurtherEven in a more recent compilation of the past discoveries of arboviruses and zoonotic viruses around the world, which is otherwise considered most comprehensive thus far, all pertinent records including “first” isolations of JEV, DEN-1, Apoi virus, Yokose virus, Sagiyama virus, Akabane virus, Chuzan virus, and other viruses of veterinary importance from Japan and other arbovirus isolations in China and Korea are conspicuously and totally missing. For that matter, JEV research in whole Asia is not even mentioned .The lack of interest in examining the documents written in languages other than English then led to excessive dependence on secondary or tertiary reading even when the originals were written in English. This increased the probability of the errors in quotation made by the earlier authors being perpetuated in JE literature. One example of frequent equivocal quotation relates to isolation of Nakayama strain from a 19 yr-old male. Actually, the strain was isolated from a 6 yr-old male, and the article was published in English . Incorrect interpretation of published documents or reporting of incorrect information is another source of communication problem. One such example concerns the widespread belief in ardeid birds as reservoirs of JEV. As described in In another examples of communication problem, the source of the problems was often simple incorrect information. In one of such examples, it was reported that JE is “air or water borne, the results of mosquito bite or spread by ticks” . While tDevelopment of metagenomics, phylogenetics, and phylogeography contributed to the enormous advancement in virologic research; and their advantages over traditional techniques in many aspects of investigation are numerous. Nevertheless, like in natural transmission studies of most vector-borne viruses still with multiple unresolved basic questions, differences in the results or conclusions among reports are also frequently found in these new molecular fields despite the fact that every author strives to present the best results and conclusion. Rather than frustrating over a lack of consensus, one can seize these differences in results or conclusions as excellent sources of innovative ideas and concepts to consider for further advancement. Thus, they serve as sources of research questions. They are also sources of cautions to be exercised. In this review, I emphasize accommodation of both traditional and modern techniques, since they complement each other. The three fields  discussed below partially overlap because they share genome sequencing as a necessary source of information. The topics selected below provide sources for comparison in JEV characterization between the early part of history and more recent age of molecular virology. In the past few decades, using a combination of metagenomics and bioinformatics, virus discovery has been revolutionized, resulting in the discoveries of a huge number of new viruses. One of the reasons for promoting metagenomic technique is that too many new viruses cannot be cultivated easily, which justifies use of culture-independent and sequence-independent detection/isolation method ,137. As Despite the popularity of metagenomic virus isolation without a physical isolation of infectious entity, the application is considered problematic  because The fifth concern is that this new technique does not necessarily provide information regarding host range, tissue specificity, mode of transmission, virulence, and other biologic traits. Accordingly, even the proponents of metagenomics agree that the traditional and newer methods play complementary roles in virus isolation . As in the aforementioned cases of metagenomically discovered viruses, new viruses are determined mostly by the position in phylogenetic tree supported by an assumed mathematical reliability of selected algorithm. Recently, International Committee on Taxonomy of Viruses (ICTV) endorsed a comparative genomics as a sole acceptable basis for the recognition of virus taxa . PhylogeA byproduct of phylogenetic studies is molecular clock. The complexity and problem of understanding phylogenetic data are shown in the following comparison of JEV molecular clock date determined with 95% posterior probability by three groups. It is stressed that comparing results of molecular clock dates among reports is not simple because of differences in software, assumptions used in parameter setting, and other analytical tools used. Nevertheless, it is generally agreed that genotype V is considered the oldest in JEV because it is located closest to the root of the phylogenetic tree. The dates determined for the genotype V were circa 1550s A.D. , circa 1For animals and plants, paleontological records , historical floral and faunal patterns of geographic distribution, and others can be used as collaborating supports of the accuracy of sequence-based phylogenies. For the taxonomy of bacteria, archaea, and eukaryotes, universally conserved genes serve as reliable guides for taxonomic classification. However, for nearly all phylogenies of naturally occurring RNA viruses, there are few or no such supports. For RNA viruses, RNA-dependent RNA polymerase gene (RdRp) is most conserved, which is why many RNA virus family trees are built on RdRp sequences. Still, the diversity of sequences in RdRp for many RNA virus groups is too great to be reliably used for the entire family. That is the fundamental weakness of RNA viral phylogenies. For vector-borne viruses, such as flaviviruses, it has been proposed to use endogenous viral elements (EVEs) integrated in the genomes of vectors, to offset the weakness. However, these vestigial signals are not found for all flaviviruses.In the phylogeny of flaviviruses, the topologies based on NS3 and open reading frame (ORF) have been found to be very similar; while that of NS5 (including RdRp) was different ,148. ForIn phylogeographic studies, accurate information regarding the date and location of each virus isolate is important. Furthermore, we need to recognize that inventory of available strains sequenced for a given study also changes the result considerably. Genotype V of JEV is a good example. Traditionally, the publication by a British physician, James H. Hale and two colleagues at the University of Malaya in Singapore  in 1952  has beenBecause the Muar strain had long been the sole strain of genotype V, it was once speculated that it represented “a transitional lineage between Murray Valley encephalitis virus and JEV, which subsequently became extinct” . UnexpecAnyone accessing sequence data from GenBank notices that the person who isolated a strain earlier and the person who submitted its sequence many years later are different. Here lies a potential problem. As sequence data are customarily retrieved from GenBank or other databanks, associated information  deposited at the time of sequence submission is automatically used by most users of the GenBank on an assumption that it accurately corresponds to the isolate. However, when the submitters of sequences rely only on secondary reading, there is no guarantee of eliminating the possibility of misquotation. As an example of Muar strain of JEV, in one article Singapore was identified the geographic isolation site . This isAnother interesting subject that emerges from the issue surrounding Muar strain is the fact that Muar was the site of heavy fighting in 1942. Since JE outbreak began to become noticeable in Malaysia in 1942 , it is pIn another example of phylogeographic issue that generated contradictory results, based on E-gene sequences, it was earlier concluded that tick-borne flaviviruses dispersed westward from the east along the northern region of Eurasia . When thTheoretically, molecular data, if enhanced with biologic data, are more reliable. Actually, molecular data and phenotypic (biologic) data complement each other in the full characterization of a virus. Thus, if current molecular definition of viruses is considered two-dimensional, its combination with biologic traits makes it tridimensional. The following examples of flaviviruses are useful to understand it. Among multiple mosquito-borne flaviviruses , Zika virus and dengue virus serotypes) in tropical Asia, New Guinea and northern Australia, serologic cross reaction has been known. Existence of such non-specific antibodies in the human populations is not only a source of serologic complication in diagnostics but a factor that distorts efficacy evaluation of vaccines (for such diseases as JE and dengue). A recent report revealed a wide-spread interspecific proteomic recombination among those flaviviruses. Since this is not detectable in nucleotide or amino acid sequence-based phylogenies, it requires a different software . This maIdentification of host (or host range) is an important subject not only for studying the evolutionary history of viruses but epidemiology caused by pathogenic viruses. From the days of poliovirus research in the 1950s, numerous discoveries of virus-specific receptors in hosts have been reported; and they established the foundation of the concept of RNA virus-specific host range. Most of these studies were performed in the laboratories, in particular for important human viruses. More recently, it has become popular to apply phylogenetics of viruses and bioinformatics data on host genomes to identify the hosts more precisely for known flaviviruses or to predict the hosts for newly discovered viruses based on sequence characteristics ,158,159.First, when one attempts to correlate a viral genomic trait (or traits) with the corresponding host (or hosts), those viruses with the narrowest host range are most ideal. The examples include human-specific viruses, such as polio and measles viruses. For these viruses the virus–host relationship is direct with human the only host. On the other hand, for vector-borne arboviruses (such as JEV), it is indirect. Actually, on the parts of most vector-borne viruses, human is only one of the incidental hosts and unimportant for their survival. In host studies, many of the crucially important sources of information are found in voucher specimens collected in the field but not in the laboratories . For mosSecond, recent host studies are all based on the traditional notion that vertebrates are the reservoirs. As revealed earlier, there exists no RNA arbovirus with a scientifically validated vertebrate reservoir. The only arbovirus with validated vertebrate reservoirs is African swine fever virus, the only DNA arbovirus. Thus far, the only validated reservoirs of RNA arboviruses are vectors themselves, based on the current definition of “reservoir” for arboviruses . From thNodaviridae; not arboviruses), for example, replicate in mosquitoes, yeasts, vertebrates, and plants. Totiviruses replicate in fishes, mosquitoes, fungi, and protozoa. Mokola virus (not an arbovirus but a rhabdovirus affiliated to lyssavirus group) replicates in mosquito and vertebrate and has been often isolated from mosquitoes in nature. Similarly, members of the Genus Flavivirus are not only vector-borne and insect-specific viruses but viruses of nematodes, crustaceans, squids, sharks, and salmons. The vertebrate flaviviruses  readily replicate in mosquito cells, even though they are not vector-borne; but, to make the matter further complicated, other group of vertebrate flaviviruses (known by No Known Vector viruses) do not replicate in arthropod cells. Accordingly, for those groups of viruses that have such a broad range of hosts, the accuracy of linking genetic signals found in sequence data of one host or one group of hosts to a particular virus or a closely-related group of viruses becomes less certain. Third, many viruses are known to replicate in multiple phyla of organisms, some even in multiple kingdoms. A few Nodaviruses . Some of the examples are infection of killer whale by SLEV , of alliRecently, a molecular marker, TMEM41B (a transmembrane protein), was identified as a pan-flavivirus host factor to explain difference in flavivirus infection among human populations in Asia . HoweverConsidering the complexities of host selection described above, it is necessary to understand host selection of vector-borne flaviviruses holistically in the context of the outcome of complicated interactions among viral, vectorial, host, and other ecologic factors. Actually, even the authors of one of those reports readily admit the difficulty of identifying hosts .In recent decades, often viral assay has been performed by measuring viral RNA as a substitute for infectious unit assay. It is based on an assumption that copy number of viral RNA correlates with infectious unit . This practice has been adopted in many branches of molecular studies including the analysis of the dynamics of viral replication. Although this correlation is valid when infectious unit assay is also performed as reference within the same set of experiments, when no such calibration of the quantity of viral RNA to infectious unit is conducted, the significance of the quantitative value of viral RNA alone is far less clear.Prolonged RNA shedding has been suspected to be a possible evidence of direct transmission of JEV between hosts, JEV persistence or overwintering in temperate regions . ActuallAs for the significance of arboviral RNA persistence on overwintering, in all past reports of persistent detection of viral RNA in hosts through winter, when spring arrived, infectious agent has never been isolated from those hosts . This inIn a more recent study, JEV RNA shedding into the vaginal mucosa of pig for as long as 28 days was reported; and the result was discussed in the context of possible sexual transmission of JEV in pigs . ActuallThis re-examination of the early history of “first” JEV isolation illustrates how the concepts and requirements for virus isolation and characterization evolved by trials and errors over time. While technological advancement has been phenomenal and the depth of our knowledge about viruses has increased considerably over years, some fundamental issues on natural JEV transmission that troubled the workers several decades ago still remain without a definitive conclusion. Additionally, this review revealed an enigma as to why such invaluable contributions to JE studies accomplished in the early part of history have remained untold for so long.Among several troubling issues commented on, language barrier as a cause of international communication problem is unique to JE research. Though the exact background or reason is unknown, it is possible that before WWII, JE was primarily considered either a regional or domestic issue in Japan, even though JE was known in Korea, China, and Far Eastern Soviet Union. Thus, despite the fact that a large number of research reports were published in their native language, only a very small number of the workers published their reports in English. The traditional emphasis on German rather than English in science in Japan prior to WWII was possibly another reason. After WWII, however, JE became an international medical issue and English the global currency of scientific communication. Accordingly, had early historical JE research documents been systematically translated, ambiguities clarified and comprehensively published in English for the benefits to the researchers around the world, some of the communication problems, including the difference in “first” virus isolation date, could have been averted. In the future, it is highly desirable that each JE-endemic country (where English is not a mother tongue) post online a collection of selected, important research documents and publications which can be translated with a more advanced language software uniquely crafted for each language, to make the documents available worldwide.As for the issues related to the strategies adopted to solve research questions, one common denominator among many proponents of molecular techniques is to analyze complex topics (ranging from virus taxonomy to host group determinants) by means of genomic and bioinformatics data alone. The advantage is that most parameters are quantifiable; and the support and popularity of this approach are considerable. In fact, a popular method for phylogenetic studies was once maximum parsimony, which was subsequently replaced by more improved methods. On the other hand, the proponents of holistic understanding of these complex issues regard such strategies too simplistic. Though they too prefer a simpler explanation if possible, for complex issues, they recognize complex explanations as rational and necessary choices. For the sake of healthy science, it is desirable that both sides of argument be kept alive. The history of international JEV-SLEV exchange (both virus and information) shown in this review may appear to some readers nothing but a fond memory now that, on top of the above problems, multiple reasons for restricting international (and even domestic) shipment of viruses have been erected as barriers due to a variety of necessities and concerns, such as increasing threat of bioterrorism, increased awareness of intellectual property right protection and even politics. Thus, cross examination of new viruses by other groups is far more difficult. When virus shipment is increasingly difficult, as it is today, at least sharing information can be improved. As an example, since 1960 through 1990th, American Committee on Arthropod-borne Viruses (ACAV) within the American Society of Tropical Medicine and Hygiene collected voluntarily-submitted research activity news and other developments from many laboratories around the world, printed them in an informal newsletter (“Arthropod-borne Virus Information Exchange”), and distributed it twice a year free of charge. Amazingly, many laboratories submitted preliminary results and other data still unpublished. A similar newsletter was also launched in Australia. What is sorely needed today is revival of such a collaborative and more open spirit in international scientific communication, this time online.


Viruses are a major constraint for yam production worldwide. They hamper the conservation, movement, and exchange of yam germplasm and are a threat to food security in tropical and subtropical areas of Africa and the Pacific where yam is a staple food and a source of income. However, the biology and impact of yam viruses remains largely unknown. This review summarizes current knowledge on yam viruses and emphasizes gaps that exist in the knowledge of the biology of these viruses, their diagnosis, and their impact on production. It provides essential information to inform the implementation of more effective virus control strategies. Dioscorea (family Dioscoreaceae), which includes more than 600 species worldwide [D. pentaphylla, D. hamiltonii, D. oppositifolia, and D. polystchya have interesting medicinal properties [Yam is a vernacular name used for various species of the genus orldwide . Some oforldwide ,2,3. Othoperties ,4. Approoperties ,6. The moperties . The conoperties ,8.Virus infections have been reported in all yam growing areas, although geographical distributions vary between yam viruses . The phyBetween 2012 and 2022, 14 new viruses have been found to infect yams ,30,35,54This review addresses these gaps to help identify where additional efforts are needed for the design and implementation of effective control strategies, including more comprehensive sanitation programs. It is also intended to inform decision-makers with regard to the international exchange of yam germplasm.Aureusvirus, Ampelovirus, Badnavirus, Carlavirus, Cucumovirus, Dioscovirus, Fabavirus, Macluravirus, Potexvirus, Potyvirus, Sadwavirus, Velarivirus) and eight families   RNA1 and RNA2 using a relatively low number of yam samples originating from different varieties and geographical areas conserved during a germplasm collection in Guadeloupe and Cote d’Ivoire. This approach enabled authors to demonstrate that both the host range and geographic distribution of DMaV were larger than initially thought. It also revealed that DMaV displays a high nucleotide variability in both its RNA1 (18.3%) and RNA2 (16.6%) and that intra-plant nucleotide variability was similarly very high in both genomic RNAs . Authors hypothesized that high genetic diversity could help DMaV escape sequence-specific host defense mechanisms.The International Committee for the Taxonomy of Viruses (ICTV) currently recognizes 25 viruses that infect yams, in 12 genera (viridae) . Co-infe viruses ,30,55. FD. cayenensis-rotundata complex. The spread of YMV to D. trifida in French Guiana was estimated to have occurred about 80 years ago, and more recently to D. alata in Burkina Faso and Puerto Rico about 53 and 30 years ago, respectively. Interestingly, Mendoza et al. [D. alata species, followed by a geographical dispersion of this virus probably through the long-distance movement of infected germplasm [Accessing yam samples from diverse geographical origins will also assist in studying the extent to which the diversity of specific yam viruses is determined by their geographical origin. Recent studies by Mendoza et al.  built ona et al.  reportedermplasm . Likewisermplasm ,28,30,55Mixed infections of multiple virus species and strains in vegetatively propagated crops such as yams provide conditions that favor the emergence of new strains via recombination . PhylogeD. trifida in Guadeloupe in 1977 [D. alata and D. rotundata genotypes collected in Nigeria has not revealed any contig mapping to CMV [Virus diagnosis is key to efficient disease management, in particular during sanitation programs, which are the backbone of any clean seed production program. Inaccurate diagnosis leads to either false negatives or false positives that can have minor  to major impacts . For example, based on electron microscope observations, CMV was reported on  in 1977 . Still,  in 1977 . Additio in 1977 ,16, but g to CMV ,58, whicg to CMV  proved ug to CMV ,58. Low g to CMV ,43,58,61g to CMV ,48,62,63In the last four decades, a wide range of serology-based tests, including immuno-electron microscopy (IEM), dot blot immunoassay (DBIA) and direct tissue blot immunoassays (DTBI), immunosorbent electron microscopy (ISEM), and variations of the enzyme-linked immunosorbent assay (ELISA) have been developed for the detection of yam viruses ,64,65,66The use of PCR for virus diagnostics has greatly improved the sensitivity, specificity, and speed of yam virus indexing compared to that achieved by serological tests . PCR hasImmunocapture-PCR (IC-PCR) is a sensitive hybrid assay that combines the general specificity of antibodies for trapping viral particles with the increased specificity and sensitivity of PCR amplification of viral DNA sequences . This teQuantitative PCR (qPCR) is less commonly used for the routine diagnosis of yam viruses, although it was reported to be more sensitive than conventional RT-PCR for the detection of YMV . FurtherThe detection of badnaviruses in yams is hampered by the presence of endogenous Dioscorea bacilliform virus sequences (eDBVs) and the high genomic and serological variability of badnaviruses ,26,62. PPCR-denaturing gradient gel electrophoresis (PCR-DGGE) ,73,74 haEpisomal DBVs are circular DNAs, whereas endogenous DBVs are linear DNAs . RollingIsothermal amplification methods use a single temperature for DNA amplification and require only a simple, low-cost heat source rather than more expensive thermal cycling equipment . To dateAmpelovirus, Badnavirus, Macluravirus, Potyvirus, and Sadwavirus  are often observed on infected yam leaves . HoweverInfectious virus clones have helped unravel virus–host and virus–virus interactions for numerous plant viruses ,97,98, iD. rotundata plants and assessed the impact of infection on a range of physiological traits and yield. For this, they performed virus indexing targeting YMV, YMMV, DaBV (BDALV), and CMV using TAS-ELISA, whose sensitivity and specificity is not optimal, as discussed above. Symptoms and impact of YMV infection on plant growth and yield losses were registered and compared between infected and mock-inoculated plants. However, the presence and impacts of other viruses  that have since been identified and shown to be widespread in West Africa [Assessing losses due to plant pathogens is a difficult task due to the number of parameters that must be considered to draw valid statistical analyses ,101. Vert Africa ,17,23,24t Africa . HTS of For a better estimation of the impact of viruses on yam production, the sole evaluation of yield losses based on statistical comparisons between yields of infected and healthy plants is not sufficient. Several other criteria are impacted by viral infections and must be considered. For example, decreased tuber size and quality reduces the seed pool available for the next planting and makes harvesting more difficult and time consuming, incurring additional labor costs. Likewise, decreased tuber food palatability negatively impacts consumer preferences and marketing channels. In addition, estimation of yield losses should be based on a comprehensive approach using a conceptual framework involving virologists, statisticians, and economists, similar to what has been achieved for other vegetatively propagated crops ,107,108.In this regard, the use of severity scores based on leaf symptoms as a proxy of infection status or resistance to viruses ,94,109, Planococcus citri [Vegetative propagation plays a predominant role in the persistence and spread of viruses in many important crops . In yamsus citri , whereasus citri ,109,111.us citri ,34,35,54The possible role of weeds as reservoirs and the impact of farming practices on the epidemiology of yam viruses has not been extensively addressed either , althougVery little is currently known about how yam viral diseases are spread through the informal trade or exchange of infected plant material. Social epidemiology approaches  could prThis review complements the efforts undertaken in recent years by the scientific community and stakeholders to mitigate the impact of viral diseases on yam production . ParticuVegetative propagation promotes the accumulation and spread of viruses. It results in the presence of diverse variants in plants and/or in co-infections by distinct viruses. In yams, this situation challenges accurate viral diagnostics. The ability of molecular techniques such as PCR, HTS, and isothermal amplification to overcome this problem makes them particularly appealing. HTS can also inform the host range and epidemiology of yam viruses—in particular, the role of reservoir plants such as weeds—in their spread and help unravel antiviral defense mechanisms through the profiling of yam virus-specific small interfering RNAs, as has been achieved for other crops ,122,123.Most yam viruses have an international distribution , in partAureusvirus, are known to be transmitted by insects. However, vector competence studies are complex to perform, time consuming, and provide a lower scientific “return on investment” than most other fields of virology, hence why the current wide gap in knowledge on vector-borne transmission of yam viruses, which also applies to the role of weeds in the epidemiology of yam viruses and to the biology of these viruses for similar reasons, including their symptomatology. In potato, vertical transmission is known to be only partial for potato virus X, Andean potato mottle virus, potato virus Y, and potato leafroll virus, which means that only a proportion of daughter tubers are infected in turn [Elucidation on the epidemiology of yam viruses is critical for developing comprehensive control strategies and relies primarily on the characterization of biological vectors such as insects because members of all viral genera reported in yam, except the genus  in turn ,125. WheDioscorea spp., in particular to distinguish D. cayenensis and D. rotundata species, in a similar manner to studies on eggplant [Yams host a wide range of endogenous viral elements (EVEs) originating from badnaviruses ,62,77 aneggplant .The production of clean (virus-free) yam seed is based on sanitization by thermotherapy and meristem culture ,58,133. Dioscorea” and “Potato AND Solanum” for all publication categories revealed an average of 48 and 258 articles per year, respectively, during the last 20-year period (August 2002 to August 2022), reflecting a strong disparity in the research efforts made on these staple food crops and their pathogens. In the case of potato, the impact of certain viruses on yield has been successfully measured [Research efforts on the epidemiology, symptomatology, impact, and transmission of yam viruses have been scarce, probably due to the fact that yams remain understudied and underfunded compared to many other tuber crops . For exameasured ,120, sevmeasured ,119, themeasured . Similar