Similar levels of dead J2s were observed after immersion of J2s in oxamyl at concentrations higher than 8 μL L−1 (24 and 48 h) or 4 μL L−1 (96 h). An evident decrease of egg differentiation was observed when fluopyram concentration was increased to 8 μL L−1, while no significant decrease in egg differentiation was recorded at any concentration of oxamyl. Egg hatching was decreased at concentrations of fluopyram higher than 4 μL L−1, while no reduction was observed even when the concentration of oxamyl was increased to 64 μL L−1. The efficacy of fluopyram in soil was superior compared to that of oxamyl. For the first time, the systemic action of fluopyram is recorded in trials with tomato plants. On the other hand, compared to oxamyl, fluopyram seems to be more toxic to non-target organisms such as the entomopathogenic nematodes Steinernema feltiae and Heterorhabditis bacteriophora.Root-knot nematodes can cause tremendous losses in vegetable crops. Farmers usually rely on synthetic nematicides to protect their crops. Recently, newly released nematicides are giving farmers an alternative in chemical control for nematodes. In the present study, the efficacy of vintage nematicide was compared to that of a relatively new nematicide, fluopyram. The latter was always more effective in substantially lower concentrations than oxamyl. Fluopyram paralyzed more than 80% J2s after 24 h immersion at the concentration of 0.25 μL L Greenhouse cultivated vegetable crops are suffering from root-knot nematodes (RKN) ,2. RKN cAfter the removal of effective fumigant nematicides, farmers struggle to fill the management gap continuously using the same non-fumigant nematicides. Oxamyl, a non-fumigant nematicide, is still in use, registered with different commercial names and different granulated and liquid formulations. Oxamyl, under different commercial names, is probably the most known nematicide all over the world. It is being used either in protected cultivating vegetables such as solanaceous and cucurbitaceous species or in open field cultivations. Oxamyl could be considered as the exception of the group of organophosphates and carbamates which have been withdrawn from the market in recent years. The fact that this chemical is one of the few nematicides still in the market, and because farmers use this active ingredient every year, could create biodegradation problems and efficacy decrease . There a® prime, Vellum® one, ILevo, which are registered in many countries all over the world. It is a succinate dehydrogenase inhibitor (SDHI) fungicide belonging to the pyridinyl-ethyl-benzamide group. The main advantage of this compound is its ecotoxicological profile. It has low toxicity for rats (LD50 > 2000 mg kg−1) and bees (LD50 > 100 μg per bee). Another advantage of this nematicide is its low recommended dosage preventing an adverse effect to the agroenvironment. Fluopyram reduced root galling on lima bean, but a 22% reduction in emergence was recorded [M. javanica exposed to doses of fluopyram higher than 1.0 mg/L were immobilized after 24 h (>85.9%) [This situation has enforced chemical scientists to search for new molecules to find new nematicides or to test already registered substances such as fungicides, insecticides, and acaricides against plant parasitic nematodes ,16,17. Mrecorded  which corecorded . Second-(>85.9%) ,22,23, nM. javanica, (2) to study the effect of fluopyram and oxamyl in egg differentiation and hatching of M. javanica, (3) to study either the direct effect of fluopyram and oxamyl in dare soil infested with M. javanica or its systemic effect using tomato plants, (4) to ascertain the effect of fluopyram and oxamyl on two entomopathogenic nematodes.The objectives of the present study were: (1) to ascertain the effects of fluopyram and oxamyl in a series of concentrations in three different incubation periods against juveniles of M. javanica in the motility bioassays using either fluopyram or oxamyl in a series of concentrations. The percentage of dead J2s increased by increasing the incubation time or the concentration. Constantly, fluopyram has shown 100% paralysis for all concentrations higher than 2 mg/L. The effect of fluopyram at the lowest concentration of 0.25 mg/L resulted in more than 80% dead juveniles after 24 h of incubation. The percentage of dead J2s was increased when the exposure time was increased to 48 and 96 h. On the other hand, similar efficacy in juveniles’ motility was recorded when oxamyl was used in higher concentrations. Specifically, oxamyl concentration had to increase to 8 mg/L to record a percentage of dead juveniles higher than 95%. After 96 h of incubation, only concentrations higher than 4 mg/L showed 100% dead juveniles. Based on the results, it is clear that fluopyram is very effective even at the lowest concentration tested.Egg differentiation was not inhibited by low concentration of fluopyram . The conSignificantly fewer juveniles were hatched from eggs treated with 4 mg/L of fluopyram or higher . No furtThere was a gradual decrease in the number of nematodes per gram of root as the concentration of fluopyram was increased to 1 mg/L . Any fur−1. By increasing oxamyl concentration from 8 to 64 mg/L, there was a constant decrease of nematode numbers in tomato roots.A gradual decrease in the total number of nematodes inside the roots was recorded by increasing the concentration of fluopyram . The numNo significant differences were recorded between plants transplanted into fluopyram- and oxamyl-treated soil at concentrations of 0.25–8 mg/L. However, significant differences between the two nematicides were recorded as the concentration was increased to 16, 32, and 64 mg/L .Steinernema feltiae were dead at a percentage of 64–69% when they were incubated for 24 h in concentrations from 1 to 32 mg/L . Fluopyram was also toxic to third-stage instars of Heterorhabditis becteriophora. There was an increase of dead instars as the concentration increased from 2 to 64 mg/L after 24 h of incubation. The toxic effect of fluopyram on H. becteriophora was 100% at the concentration of 8 mg/L (48 h of incubation) or 2 mg/L (96 h of incubation). Oxamyl was less toxic to third-stage instars of both entomopathogenic nematodes tested. Specifically, the highest percentage (12%) of dead instars of S. feltiae was recorded at the concentration of 64 mg/L after 24 h of incubation, which was increased to 17% at the same concentration after 48 h of incubation (S. feltiae as the concentration increased. The highest percentage of dead instars (50%) was recorded at the concentration of 64 mg/L. Much lower toxicity was recorded when H. becteriophora was used. The highest toxicity (7%) was recorded at the concentration of 64 mg/L after 96 h of incubation. However, it seems that there was no clear effect of oxamyl to H. becteriophora third-stage instars irrespective of concentration and incubation time.There was a clear toxic effect of fluopyram on third-stage instars of entomopathogenic nematodes as this was recorded in the motility tests. Instars of  32 mg/L . A furthcubation . When neMeloidogyne javanica. We also investigated the possible negative impact of these nematicides against two entomopathogenic nematodes.In this study, we conducted a comparison between an old (oxamyl) and a recently released nematicide (fluopyram). Specifically, we investigated the concentration- and time-dependent effect of fluopyram and oxamyl against M. incognita juveniles which resulted in tomato root protection after exposure to fluopyram for 2 h. A similar level of immobilization was also reported by Oka and Saroya [−1 of fluopyram after 24-h exposure of M. javanica juveniles, although this was evident only in one of the two trials conducted. The percentage of dead J2s increased as the concentration of fluopyram increased. When the incubation time increased to 48 and 96 h, a complete kill of J2s (100% dead J2s) was recorded at the concentration of 2 mg/L (48 h of incubation) or at concentration of 0.25 mg/L (96 h of incubation). On the other hand, it is clear that a higher concentration of oxamyl was always required to gain the same percentage of kill effect at the same time, compared to fluopyram. This was expected, since the nematicides tested have different modes of action. Oxamyl is a carbamate acting as an inhibitor of acetyloholynesterase, whereas flyopyram is an SDHI inhibitor [We showed that a concentration as low as 0.25 or 0.5 mg/L was effective in J2s paralysis after exposure to fluopyram for 24 h. Our results are in agreement with those reported from Faske and Hurd  showing d Saroya  at the cnhibitor ,24.To the best of our knowledge, there are no published data in the literature for the effect of either fluopyram or oxamyl on egg differentiation. It was not affected when oxamyl was used at concentrations from 0.25 to 16 mg/L. A slight decrease of egg differentiation, compared to the untreated eggs, was recorded when the concentration was increased to 32 and 64 mg/L. Fluopyram ceased egg differentiation at a higher percentage as the concentration was increased to 8 and 16 mg/L (30 and 40% less differentiated eggs). This percentage was even higher at concentrations of 32 and 64 mg/L (70 and 80% less differentiated eggs). Depending on the gap between the uprooting of the previous crop and the transplanting of the next, eggs might be either developed  or undifferentiated (cells multiplication stage).−1, since the hatching rate was similar to that observed in the untreated control after the removal of the eggs into clean water [Our results show that no inhibition on egg hatching was recorded after immersion of egg masses in all tested concentrations of oxamyl. In contrast, a hatching inhibition was evident as the concentration of fluopyram was increased from 2 to 64 mg/L. Three-day incubation of eggs at a concentration of 5 mg/L of fluopyram resulted in 96% hatching inhibition . Howeveran water . In our A drastic reduction of J2s survival was recorded as the concentration of fluopyram in soil was increased to 1 mg/L. Complete protection against RKN was recorded in soil treated with fluopyram at a concentration of 2 mg/L or higher. On the other hand, slight efficacy on J2s was recorded at concentrations of oxamyl up to 8 mg/L. Further oxamyl concentration increase resulted in J2s survival decrease that was inferior to that observed in fluopyram treatments. Except from preliminary experiments testing the action of chemical compounds against juveniles and eggs, in vitro experiments using soil are always necessary to conduct to clarify the efficacy of molecules in conditions very close to real.M. javanica. On the other hand, once J2s entered the roots, it was difficult to reduce nematode population when plants were transplanted into soil treated with either oxamyl or fluopyram.Both nematicides tested have been reported to have acropetally systemic activity ,25. AlthIt is clear from the results reported that fluopyram is very toxic to entomopathogenic nematodes. This could be undesirable in case that the farmer uses an EPN formulation to control soil insects. It has been reported that fluopyram has shown high toxicity to the nematode fauna of the soil . It affeAlthough fluopyram is very effective, even at very low concentrations killing second-stage juveniles, this is not the case as far as egg differentiation and egg hatching is concerned. However, the farmer could keep irrigating the soil for some days after the end of the previous crop to enhance the eggs into hatching and maximize the efficacy of the nematicide. Modern agriculture will rely on new molecules with innovative modes of action. Commonly, these new molecules are superior to the vintage ones, considering that they are active in lower doses, cost less, and show lower toxicity . We haveMeloidogyne javanica originally obtained from tomato roots in Crete, Greece was reared on tomato (Solanum lycopersicum) Mill. cv. Belladonna and maintained in a temperature-controlled glasshouse at 23–28 °C. Eggs were extracted with 1% sodium hypochlorite solution [A population of solution . Second-® 24-well plates which were exposed to fluopyram and oxamyl solutions. All treated and control plates were covered with a lid, wrapped with aluminum foil, and incubated at 26 ± 1 °C. Juveniles were observed with the aid of an inverted microscope  at 100× magnification after 12, 24, 48, and 96 h and were ranked as either alive or dead. Dead juveniles were certified by disturbing them with a needle. Those that remained immotile and had a straight shape were considered as dead. The experiment was conducted twice, while every treatment was replicated 5 times.Solutions of fluopyram and oxamyl were tested for J2s motility at concentrations of 0.25, 0.5, 1, 2, 4, 8, 16, 32, and 64 mg/L. The chemicals were serially diluted in distilled water to produce test solutions of the abovementioned concentrations. Distilled water served as control. Approximately 50 J2s were used per treatment well in CellstarM. javanica eggs were isolated from infected tomato roots using hypochlorite solution and following the procedure described by [ribed by . A cleanM. javanica originally obtained from Crete and reared on tomato (Solanum lycopersicum) Mill. cv. Belladonna and maintained in a temperature-controlled glasshouse at 23–25 °C were handpicked and placed in small extracting trays made by 6 cm Petri dishes. Solutions of either fluopyram or oxamyl  were added to each extracting tray to cover the egg mass. Egg masses submerged in clean water served as the control. Extracting trays were maintained for 7 days in an incubator at 25 ± 1 °C, and then test solutions were removed by gently washing them with tap water. Each egg mass was returned to the same extracting tray and filled with clean water, covered to avoid loss of water, and placed in the incubator. Hatched J2s were collected and counted every 7 days, and all extracting trays were filled with clean water. The experiment was terminated after 30 days when no more J2s were collected from the extracting trays. The experiment was performed twice, and each treatment was replicated five times.Mature egg masses (dark yellow) from a population of M. javanica was evaluated using tomato seedlings, cv. Belladonna. Seedlings at the four-leaf stage were grown in soil collected from a greenhouse  were drenched with solutions of fluopyram or oxamyl . The same procedure was repeated 4 days later. Pots under control treatment were drenched with water. Plants were maintained for another 3 days in a growth room at 25 ± 1 °C, and roots were carefully washed free of soil. Plants were transplanted into pots containing 200 g of soil inoculated with 500 J2s of ribed in , and allM. javanica each. Infected plants were maintained for 3 days in a growth room at 25 ± 1 °C, and the roots were carefully washed free of soil. Then, infected plants were transplanted into pots containing soil treated with fluopyram. Pots with soil irrigated with water served as the control treatment. One kilogram of sandy soil collected from a greenhouse was divided into plastic bags after being autoclaved. Water solutions of fluopyram at concentrations of 0.25, 0.5, 1, 2, 4, 8, 16, 32, and 64 mg/L were used to treat 1 kg of soil in each bag. Moisture level was standardized to 40% of the WHC. Soil was thoroughly mixed and maintained at 22 ± 1 °C for 24 h. Five plastic pots were filled with the treated soil, and one seedling was transplanted into the center of each pot. Aluminum foil was used to cover the soil around the plant stem to avoid water evaporation. All plants were placed in a growth room at 25 ± 1 °C and uprooted 28 days later, and the roots were gently washed free of soil. The roots were stained, and all nematode stages were counted as described previously.J2s were collected using the hypochlorite method as described previously. Four-weeks-old tomato plants in small plastic pots (6 cm deep and 3 cm in diameter) were inoculated with 500 J2s of M. javanica, two species of entomopathogenic nematodes were used. Specifically, Steinernema feltiae and Heterorhabditis becteriophora were used as non-target organisms to monitor the action of fluopyram and oxamyl. Both species were supplied by Koppert B.V . The chemicals were serially diluted in distilled water to produce test solutions . Distilled water served as control. Approximately 50 third-stage infective instars were used per treatment well in Cellstar® 24-well plates. Larvae were observed with the aid of an inverted microscope  at 100× magnification after 24, 48, and 96 h and ranked either as alive or dead.The procedure described previously (“Nematode motility bioassays”) was repeated, but instead of Data were subjected to one-way analysis of variance (ANOVA) using the General Linear Model (GLM). Treatment means were compared using the LSD test. Statistical analysis in all cases was conducted using the SAS statistical package. Whenever appropriate, two experiments were combined and analyzed together, if no variation was revealed between the data.M. javanica. However, fluopyram was more effective than oxamyl against J2s of M. javanica in substantially lower concentrations. Decrease of egg differentiation along with the inhibition of egg hatching were observed only with fluopyram. The efficacy of fluopyram in soil was superior compared to that of oxamyl. On the other hand, compared to oxamyl, fluopyram was more toxic to the entomopathogenic nematodes S. feltiae and H. bacteriophora.In conclusion, our work showed that oxamyl and fluopyram can control


There is only limited knowledge of the presence and incidence of viruses in peas within the United Kingdom, therefore high-throughput sequencing (HTS) in combination with a bulk sampling strategy and targeted testing was used to determine the virome in cultivated pea crops. Bulks of 120 leaves collected from twenty fields from around the UK were initially tested by HTS, and presence and incidence of virus was then determined using specific real-time reverse-transcription PCR assays by testing smaller mixed-bulk size samples. This study presents the first finding of turnip yellows virus (TuYV) in peas in the UK and the first finding of soybean dwarf virus (SbDV) in the UK. While TuYV was not previously known to be present in UK peas, it was found in 13 of the 20 sites tested and was present at incidences up to 100%. Pea enation mosaic virus-1, pea enation mosaic virus-2, pea seed-borne mosaic virus, bean yellow mosaic virus, pea enation mosaic virus satellite RNA and turnip yellows virus associated RNA were also identified by HTS. Additionally, a subset of bulked samples were re-sequenced at greater depth to ascertain whether the relatively low depth of sequencing had missed any infections. In each case the same viruses were identified as had been identified using the lower sequencing depth. Sequencing of an isolate of pea seed-borne mosaic virus from 2007 also revealed the presence of TuYV and SbDV, showing that both viruses have been present in the UK for at least a decade, and represents the earliest whole genome of SbDV from Europe. This study demonstrates the potential of HTS to be used as a surveillance tool, or for crop-specific field survey, using a bulk sampling strategy combined with HTS and targeted diagnostics to indicate both presence and incidence of viruses in a crop. Peas are a key horticultural crop with produce for human consumption as fresh or frozen and combinable peas for the production of pea protein e.g., cattle feed. Peas are also a key element in crop rotations for nitrogen fixation . In the Viruses reported from pea crops in the United Kingdom (UK) largely originate from pre-1960s research [Conventional approaches to conducting virus surveys tend to focus on collecting multiple symptomatic samples which get tested for a set number of target viruses, using targeted approaches such as ELISA or PCR, the suite of viruses would likely comprise those viruses expected to be present and those viruses known to infect the host which have not yet been detected in a given geographic region. Where there is limited knowledge of the presence of a disease in a crop, a diagnostic focus on the viruses expected to be present, may lead to the exclusion of potentially important pathogens which may not previously have been reported in that region or host, or may be as yet undescribed ,4. Due tHigh-throughput sequencing (HTS) is now being widely applied in plant pathology to identify putative viral sequences which are then subsequently confirmed using conventional targeted methods. Currently, most applications of HTS have focussed on resolving the causal agents of diseases of unknown aetiology e.g., ,6,7 and Therefore, the aim of this study was both to identify what viruses are present in UK peas and at what incidence. The approach detailed below uses a standardised random sampling strategy to take a sample to represent the field, these leaves were then sub-sampled into both a ‘bulked field sample’ (BFS) and smaller bulks of mixed sizes. The whole crop sample was tested by HTS and resulting sequence data was then used to infer the presence of candidate virus targets. Subsequent testing used mixed-bulk size sub-samples and real-time reverse-transcription PCR , to both confirm the presence of inferred viruses and to estimate the prevalence of those viruses in plants in field samples. Due to the relatively low depth of sequencing used to generate candidate virus sequences, a sub-set of ‘bulk field’ pooled samples were reanalysed by sequencing at increased depth to investigate the validity of the pooled sequencing approach. Additionally, this approach was accompanied by testing a limited number of symptomatic samples to compare the two approaches for efficacy of approach.Between May and July 2019, twenty pea crops from around UK were sampled by staff from Processors and Growers Research Organisation (PGRO) . Sample 2 square was randomly selected. From this eleven parallel transect-lines, at 10 m intervals were made and a single plant sampled at random at each 10 m interval along each transect, making a total of 121 sample points [At each field, a 100 me points . Each inAdditionally, 28 individual plants exhibiting symptoms consistent with viral infections, such as yellowing, mosaic and thickening of leaves See , were scThe process used to analyse the pea leaf samples collected at the twenty sites is shown in (a)HTS analysis–Bulk field sample (BFS): A representative sample of the whole field was obtained by using a cork borer to take a piece of every leaf. This bulk field sample was tested by HTS.(b)Confirmation and incidence–Individual leaves and bulked samples: Subsamples were also taken from the initial field sample for both confirmation of HTS detections and estimating the incidence of viruses. (i) Fifteen individual leaves were randomly chosen, and (ii) fifteen bulked samples of seven leaves were randomly chosen. A cork-borer was used to obtain a piece of each leaf within the bulked sample.From the bulked field sample 120 leaves were selected and then sampled in two ways:The additional symptomatic samples from the BFS-sample sites were analysed as four bulks, representing symptomatic samples from sites Market Weighton, Wainfleet, Eye and Langton, and these bulks were run on the MiSeq. The nine general surveillance samples were run individually on the MiSeq. Where possible, symptomatic material was taken. If no symptoms were present, material was taken from around the plant.A pea sample held in the Fera Freeze-Dried Virus isolate collection was tested by HTS as part of ongoing work to characterise the viruses in the collection. The sample originated from East Anglia, United Kingdom and was sent in to Fera Science Ltd. for testing in 2007. The sample was originally screened for pea enation mosaic virus-1 (PEMV-1), pea early browning virus (PEBV) and pea seed-borne mosaic virus (PSbMV) by ELISA. The sample tested positive for PSbMV and was freeze-dried as a reference isolate of the virus. In 2019 the sample was tested by HTS and real-time RT-PCR and RT-PCR as described below.The BFS, bulked symptomatic samples and individual symptomatic samples were extracted by Qiagen RNeasy mini kit with a DNase step, following manufacturers’ instructions. The RNA extract was ribosome depleted using the Ribozero plant leaf kit  and dual unique indexed libraries produced using the TruSeq stranded RNA library prep kit (Illumina) as per the manufacturer’s instruction. The resulting BFS libraries were pooled with other indexed libraries, diluted to 10 pM, mixed with 5% PhiX library (Illumina) and sequenced on an Illumina MiSeq using a 600 cycle V3 kit. The resulting sequences were analysed using the “ANGUA” pipeline briefly as follows: Sequences were trimmed to remove low-quality nucleotides from the 3′ end, using a Phred score threshold of 20 using Sickle , and asshttps://fred.fera.co.uk/smcgreig/angua (accessed on 17 November 2021).The described pipeline is maintained within a Conda environment, allowing for easy software version control, environment portability and program management. From within this environment, the Angua2 script can be run, which automates directory structure, trimming, assembling, contig filtering, BLASTx/BLASTn execution and MEGAN file creation. An experimental version of this pipeline, along with the Conda environment setup file, is publicly available and can be found here: Phylogenetic trees were produced using the Maximum likelihood algorithm and 500 bootstraps in MEGA 7 . PairwisFor virus confirmation and incidence testing, individual leaves, bulked samples and symptomatic plants were extracted by a magnetic bead extraction, using Invimag Virus RNA/DNA mini-kit . The samples were then tested for the internal control Cox , and subThe detection of soybean dwarf virus (SbDV) was confirmed by first testing by real time RT-PCR, samples which tested positive were then tested by reverse-transcriptase PCR and sent for Sanger sequencing  ,32, assaReal-time RT-PCR assay were carried out using 10 µL 2× iTaq universal probes reaction mix , 0.05 µL iScript reverse transcriptase (Biorad), 1 µL of 7.5 µM forward and reverse primers, 0.5 µL of 5 µM probe, 1 µL sample and molecular grade water to make a total volume of 20 µL. Samples were cycled at 50 °C for 10 min, 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Assays were carried out on either 7900HT Fast Real-Time PCR System, 7500 Real-Time PCR System, ViiA 7 Real-Time PCR System, QuantStudio 6 Real-Time PCR System or QuantStudio 12K Real-Time PCR System . Preliminary validation of assays indicated a cut off of Ct 35 was suitable for determining a positive detection (data not presented). Any Ct higher than this was recorded as negative.RT-PCR was carried out using 12.5 µL 2× 1-Step PCR Reddymix , 1 µL Verso enzyme mix, 9 µL molecular grade water, 1 µL of 10 µM forward and reverse primers and 1 µL sample. Samples were cycled at 50 °C for 15 min, 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and final elongation of 72 °C for 5 min. RT-PCR products were analysed by electrophoresis using 1% agarose gel stained with ethidium bromide.Hepworth  describeSamples from three of the whole crop pooled samples were re-sequenced at greater depth from library preparations previously produced for the initial runs. These were samples from sites Market Weighton, Perth and Chirnside. Sites Market Weighton and Perth were selected due to the presence of mixed infections including soybean dwarf virus partial sequences in the initial analysis, likely due to low concentration of the virus within the whole crop pooled sample possibly due to low incidence in the field . The samPolerovirus), pea enation mosaic virus-1 , pea enation mosaic virus-2 , pea enation mosaic virus-satellite RNA (PEMV satRNA) and soybean dwarf virus . No virus was detected at six of the sites and no further testing was carried out on these samples. By HTS, TuYV was most commonly detected, being present at thirteen sites, PEMV-1 at four sites, PEMV-2 at nine sites, PEMV satRNA at three sites and SbDV at two sites. Results are presented in Of the twenty BFS samples tested, fourteen were identified as having at least one virus present. The viruses identified were turnip yellows virus  values were calculated . RepreseWhere HTS indicated the presence of virus from a whole crop sample, real-time RT-PCR was used to both confirm infection and determine incidence of the virus from the corresponding mixed-bulk size samples. The calculated incidence of TuYV ranged from 2–93%, with seven sites having an incidence under 25% and six sites over 50%. PEMV-1 was present at about 40% incidence in two sites, and 16% and 5% incidence in the other two sites. The highest incidence of PEMV-2 found was 86%, five sites had an incidence between 20 and 40% and three had an incidence below 10%. SbDV was present at two sites with less than 5% incidence at both sites. Results are presented in At sites Ulceby, Langtoft, Louth, Market Weighton and Langton HTS identified PEMV-2 but not PEMV-1. Due to the association of these two viruses, the sites were subjected to further testing by real-time RT-PCR. From further testing on four of these sites, only one bulked sample from Langtoft tested positive for PEMV-1. Results for Langtoft and Louth are shown in As SbDV is a new record for the UK, the samples which tested positive by real-time RT-PCR were then tested by RT-PCR and submitted for Sanger sequencing (MWG GmbH). From Market Weighton one individual leaf sample and one bulked sample tested positive by both methods  and from Perth one individual leaf sample tested positive by both PCR methods (GenBank accession: OK492198).Symptomatic samples from Market Weighton, Wainfleet, Eye and Langton were tested as bulks. HTS testing identified TuYV, PEMV-1, PEMV-2 and PEMV satellite RNA, The samples were then individually tested by real-time RT-PCR for TuYV, PEMV-1 and PEMV-2, There was a difference in the identity of viruses indicated by HTS in the bulk field sample and the viruses detected in the symptomatic samples taken at that site. For example, at Market Weighton, SbDV was detected in the bulked field sample but not in the eight symptomatic samples.In the nine general surveillance samples HTS inferred the presence of TuYV, PEMV-1, PEMV-2, PEMV satRNA, Pea seed-borne mosaic virus (PSbMV), Bean yellow mosaic virus (BYMV) and four contigs ranging from 200 to 400 nt of turnip yellows virus associated RNA (TuYVaRNA), The samples were tested by real-time RT-PCR for TuYV, PEMV-1 and PEMV-2, three samples were also tested for PSbMV to confirm presence. Results of testing of the symptomatic samples by real-time RT-PCR in most cases were concordant with the viruses inferred by HTS. Exceptions to this are the Market Rasen sample, which tested positive for TuYV (Ct 24), PEMV-1 (Ct 32) and PEMV-2 (Ct 32). Further, both Ramsey-2 and Ramsey-4 tested positive for TuYV , PEMV-1  and PEMV-2 , respectively.As a quality control step to check whether any viruses had been missed on the initial testing, Bulked Field Samples (BFS) from three sites  were re-analysed on the MiSeq at an increased sequencing depth. This increased depth equated to approximately a six-fold increase in depth of sequencing over the initial analysis. HTS inferred the presence of the same plant infecting viruses as on the initial run. Running the samples at greater depth did mean that more sequence data was obtained for each virus species at the three sites shown in Testing by HTS inferred the presence of PSbMV, TuYV and SbDV in this sample, whole genomes were obtained for the three viruses, GenBank accession numbers and reads obtained by HTS can be found in Crops can be host to a wide range of viruses, and with the movement of plants and changing climate it is important to determine a baseline for virus presence in a crop . The aimEnamovirus), pea enation mosaic virus-2 , Pea seed-borne mosaic virus, bean yellow mosaic virus  and pea enation mosaic virus satellite RNA (PEMV satRNA). Two more viruses were also detected, turnip yellows virus  and soybean dwarf virus , and finally turnip yellows virus-associated RNA. TuYV represents a new host record for the UK, and the finding of SbDV is a new record for the UK.In this study, six viruses, one satellite RNA and an associated RNA were detected by HTS across all 20 sites, and 28 symptomatic samples. This included viruses that were previously reported as present in UK peas; pea enation mosaic virus-1 , and has a wide host range including 13 plant families [TuYV is known to be present and widespread in the UK and is commonly found in oilseed rape  which form on the underside of leaves, but can also cause dwarfing, yellow mosaic and leaf curl ,46,47. PIn this study HTS and subsequent testing detected PEMV-1 in only five of twenty sites and at incidence less than 40%. From Hagedorn’s work PEMV-1 may have been expected in more of the fields  describeThere were instances where PEMV-2 was identified without the PEMV-1 helper virus. As these two viruses are always thought to be present together, some samples from sites where only PEMV-2 was found were tested for PEMV-1, of these only one bulked sample tested positive. Further, in-depth HTS testing of Market Weighton did not identify PEMV-1 in the sample. Gaafar et al.  also fouSbDV was detected at a low incidence at two sites and it was also found in the historic isolate. The historic isolate was sequenced as part of ongoing work to sequence isolates from the Fera Freeze-Dried Virus Collection. This pea isolate was originally diagnosed with PSbMV by ELISA. Testing by HTS inferred the presence of PSbMV, TuYV and SbDV. This virus has not been reported previously in the UK, but is known to be present in Germany  and FinlPSbMV and BYMV were only observed in the single symptomatic samples. Both viruses are known to be present in the UK. PSbMV is a seed-transmitted virus, plants grown from infected seed become a source of inoculum, PSbMV can then be transmitted to surrounding plants by aphid vectors and wind-mediated contact transmission . In the Nicotiana benthamiana but no effect was seen on peas [Similar to Gaafar et al.  TuYVaRNA on peas ,55. The  on peas . As pea  on peas ,43.Solemoviridae (formerly Luteoviridae) are phloem-limited [Overall, the results from the real-time RT-PCR testing correlated with the viruses inferred from HTS data with a few minor discrepancies. These findings could be explained by sensitivity or sub-sampling strategy. Viruses of the -limited , if littIn some cases viruses were not seen in both the whole crop sample and the symptomatic field samples, there was no trend of more viruses being seen in the symptomatic plants rather than the BFS. Gaafar et al.  tested bBFS were initially run as single libraries by HTS. Three of the twenty sites were then chosen for in-depth sequencing, in which they were the only samples on the run. All three sites were chosen because they were later in the season and had a greater chance of virus infection, additionally short contigs of SbDV were found in the Market Weighton and Perth sites. In all three cases, the same plant viruses were detected by both the initial HTS and the in-depth run, suggesting that running the BFS as a single library is sufficient to detect the presence of viruses. In all three cases running in-depth increased the depth and the genome coverage of the virus sequences recovered . After tAs the study was to focus on viruses, an rRNA depleted total RNA HTS method was chosen because the majority of plant viruses have an RNA genome or intermediary RNA step, it has also been previously shown to be able to detect viruses with either an RNA or DNA genome ,57.Testing the BFS by HTS identified prominent viruses at the site, which could then be confirmed using real-time RT-PCR. This process reduced further testing, as samples were only tested for viruses indicated to be present. Whereas in a traditional sample, every sample or multiple small bulks would have to be tested for the full suite of viruses selected for the study.For the incidence testing, mixed bulk sample sizes were chosen to find a balance between cost and confidence intervals for analysing both high and low virus incidence. 2 area can be used for monoculture field crops but wouldn’t be as useful when mixed species are present or for sampling trees which take up a lot more space. Pea leaves are very easy to extract and a high yield of RNA was recovered after extraction, but if the material has inhibitors present, such as phenolics or polysaccharides, this could make the extraction process more difficult. Peas have a very small window for testing prior to being harvested which puts pressure on a fast turn-around for results and there is no option for re-testing, this also means the results obtained are true for that point in time. Where plants spend longer in the ground, e.g., trees, it would be possible to re-sample from the same site at different times points or sample different areas. In this study, the aim was to identify which viruses are present in UK peas to inform a baseline so re-testing was not necessary. Another limitation of this method is that it could not be used to demonstrate that an area is free from a particular virus as, if the virus is present at a very low incidence it could be missed by this testing. For further testing a whole genome was not required, as with the confirmation of SbDV, whereas if the aim of a study was to investigate genetic variability, testing the samples as BFS would not be recommended due to the relative limited coverage of individual genomes as demonstrated by the relative differences in depth of genome coverage between BFS, smaller bulks and individual samples  and pea necrotic yellow dwarf virus (PNYDV) a virus which has spread through pea crops in Europe. PSbMV was only found in three symptomatic samples and PNYDV was not found in this initial work. Sequencing of historic isolates can also give greater understanding of the plant health impact of more recent virus discoveries . The finThis work provides a comprehensive snapshot of the incidence and prevalence of viruses in UK pea crops. The virus baseline being established here can help inform effective crop management in the future. Within this study twenty BFS, each comprising of 120 leaves, were tested by HTS allowing for identification of unexpected viruses. Despite pooling a large number of leaves into a single BFS, viruses present at a low incidence were still detected by HTS. Three of the BFS were re-tested in-depth by HTS and no further plant viruses were identified, suggesting that the initial run was sufficient for the intended purpose of identifying candidate viruses for subsequent confirmation testing. By using HTS to identify candidate viruses, the testing of smaller mixed-size bulks could be focused for viruses inferred from sequencing, thus reducing confirmation testing and consequently incurred costs by comparison to running a similar exercise using targeted diagnostic approaches. The added advantage of such an approach is that the initial screening method is generic and could therefore be applied to other crops. Whilst the method here is applied as a general survey of pea crops, this testing could be used within routine surveys e.g., certification or in regulatory systems. For application in other crops considerations would need to be given to the specific traits of that crop  and those of the target pathogens  in the scope of the study. The value of a generic workflow approach such as this is that it can give information on the presence, distribution and incidence of viruses in a single sampling exercise. Within regulatory testing the sample size is governed by the detection threshold of a given pathogen and the efficacy of the test method being applied, as recommended in International Standards of Phytosanitary management (ISPM) standard 31 . Here 12The reliability of this testing approach at the limit of detection is currently unknown, but within this study there are instances of virus being detected by HTS at less than 4% calculated incidence at the site. Three of the Bulked Field Samples were re-tested by HTS to give a greater depth of sequencing and no additional viruses were found suggesting the initial comparatively low sequence depth was adequate. If using this approach in routine surveillance, re-running a sub-set of BFS could be used as a quality assurance measure, however this would be limited by cost implications. The workflow described here is cost-effective as, by comparison to traditional targeted approaches, only candidate viruses identified by HTS are the focus of confirmation testing. Whereas, in a traditional survey samples would be tested by a pre-determined suite of specific tests for known viruses and/or emerging threats. While, this could give evidence that the surveyed area is free from a particular virus, it would not give the added benefit of detecting viruses which would not be included in a fixed suite of targeted tests, such as the TuYV and SbDV in this study.


Paramyxoviridae are poorly understood. We screened pooled feces samples from three Pteropus vampyrus colonies and assayed tissues, rectal swabs, and oral swabs from 95 individuals of 23 pteropodid species sampled at 17 sites across the Indonesian archipelago with a conventional paramyxovirus PCR; all tested negative. Samples from 43 individuals were screened with next generation sequencing (NGS), and a single Pteropus vampyrus collected near Flores had Tioman virus sequencing reads. Tioman virus is a bat-borne virus in the genus Pararubulavirus with prior evidence of spillover to humans. This work expands the known range of Tioman virus, and it is likely that this isolated colony likely has sustained intergenerational transmission over a long period.Diverse paramyxoviruses have coevolved with their bat hosts, including fruit bats such as flying foxes (Chiroptera: Pteropodidae). Several of these viruses are zoonotic, but the diversity and distribution of  Bats  are unique animals with ecological, immunological, and behavioral attributes that set them apart from other mammalian orders . Bats coParamyxoviridae, and proactive discovery of novel paramyxoviruses and their natural reservoir host species benefits public health . We. We19]. Acerodon celebensis, A. mackloti, Pteropus alecto, P. chrysoproctus, P. hypomelanus, P. lombocensis, P. ocularis, P. temminckii, and P. vampyrus) and 14 smaller fruit bats   were recognized as belonging to multiple subspecies or found across a broad geographic range, and representative samples were taken from each established day roost. Habitats ranged from tracts of pristine mangrove forest to fragments of lightly disturbed secondary forest near human settlement. In addition, eight P. alecto fecal samples were collected in Central Sulawesi during 2012, and 91 P. vampyrus fecal samples were collected from two sites in West Java during 2012–2013  . Members012–2013 .later  and kept on wet ice until transfer to a −20 °C freezer. Voucher specimens were preserved in the collections at the Museum Zoologicum Bogoriense at the Indonesian Institute of Sciences in Cibinong, Indonesia . The selected clone was validated by restriction digestion and PCR amplification, followed by expansion in maxi culture and purification  to produce a stock control.Nucleic acids were extracted from bat tissues at the Australian Animal Health Laboratory in Geelong, Victoria. Carbide beads and 300–500 uL of AVL Buffer  were added to each sample and these were homogenized in a Mini-Beadbeater . The samples were centrifuged for 5 min at 13,000 rpm, and RNA was extracted from 180 µL of the supernatant using a MagMax-96 Viral RNA Isolation Kit  per the manufacturer’s instructions. RNA was eluted into 50 µL buffer and quantitated with a NanoDrop Spectrophotometer  to confirm successful RNA extraction. RNA was stored at −80 °C until shipment to Duke-NUS Medical School for conventional PCR and Illumina sequencing. cDNA was synthesized from extracted tissue and feces RNA using Superscript III Reverse Transcriptase  and screened for paramyxoviruses with a hemi-nested conventional PCR . A param® RNA Exome kit  following the manufacturer’s protocol. Library quality was assessed with an Agilent Bioanalyzer High Sensitivity DNA Assay and sequenced on an Illumina HiSeq2000 platform using a 76 bp paired-end run.Liver, lung, and kidney tissue samples from 43 bats were pooled based on species and location . A totalRubulavirinae subfamily.FASTQ files were examined with FastQC to determine quality . TrimminPteropus vampyrus individual from a small island northwest of Flores (n = 5048) of all reads . However, the Indonesian sequence was most closely related to viral sequences from a P. hypomelanus specimen in Malaysia. Additionally, the L gene sequence of Tioman viruses clustered most closely with other Pararubulavirus sequences collected from Pteropus species across multiple continents  [Menangle pararubulavirus and Sosuga pararubulavirus, which are both pathogenic to humans [Tioman virus was first detected in Malaysia on Tioman Island in 2001 during surveillance efforts to detect Nipah virus . This viositive) . Experimositive) . This geo humans .Pteropus, the natural reservoir, range from Oceania, through Australia and tropical/subtropical Asia, to Madagascar [Pteropus rufus tested (0.57%) and in one additional pteropodid bat (Rousettus madagascariensis) [P. giganteus individual (1.5% of bats screened) captured in Assam, India [P. conspicillatus bats were tested for Tioman virus-specific antibodies, and 38% were seropositive [P. poliocephalus [Pteropus across the genus’ distribution, though questions remain about its endemicity to Australia and the islands of the Pacific.Evidence of Tioman virus infection in bats has now been detected in six countries. Species in the genus dagascar . A studyriensis) . Tioman m, India . In New positive . A recencephalus . HenipavPteropus vampyrus, thus expanding the bat’s known range [P. vampyrus that founded the island’s population. The day roost is within a conservation area on an offshore island with a dense mangrove swamp forest and few beaches. The rugged terrain and the presence of multiple, large (~3 m) Komodo dragons (Varanus komodoensis) likely act as a deterrent to hunting or long-term human settlement. These factors may have helped promote population growth and stability, with over 5000 individuals located at a single site. This situation stands in stark contrast to western Indonesian roosts that contend with human settlements and occupy any suitable tree in disturbed landscapes, resulting in spatial fragmentation of large colonies [The remote island northeast of Flores on which the Tioman virus-infected individual was found is a new locality record for wn range . The phywn range . Presenccolonies .The relative isolation and apparent stability of the Flores colony combined with its large population size are both likely key contributing factors that promote sustained bat-to-bat transmission of Tioman virus. Isolation on islands often results in ecological feedback that leads to decreased influence from interspecific interactions, but increased influence from intraspecific interactions . Tioman


Solemoviridae includes viruses with icosahedral particles (26–34 nm in diameter) assembled on T=3 symmetry with a 4–6 kb positive-sense, monopartite, polycistronic RNA genome. Transmission of members of the genera Sobemovirus and Polemovirus occurs via mechanical wounding, vegetative propagation, insect vectors or abiotically through soil; members of the genera Polerovirus and Enamovirus are transmitted by specific aphids. Most solemoviruses have a narrow host range. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Solemoviridae, which is available at ictv.global/report/solemoviridae.The family  CP, capsid protein; RdRP, RNA-directed RNA polymerase; sgRNA, subgenomic mRNA; VPg, virus protein genome-linked.T=3 lattice symmetry  on a symmetry . CP monosymmetry  [1]. SobThe genome comprises a polycistronic, positive-sense RNA molecule of 4–6 kb without a 3′-poly(A) tail. The genome 5′-end has a covalently attached virus protein genome-linked (VPg). The genome contains 4–10 open reading frames (ORFs) . The 5′-Replication occurs after synthesis of RdRP from genomic RNA. A conserved 5′-end sequence, 5′-ACAA(AA)−3′, and a stable 3′-end stem–loop are essential for template recognition. The VPg of cereal yellow dwarf virus RPV acts as a primer for full-length negative-sense strand RNA synthesis . SobemovInfections can be symptomless or cause severe diseases. The family includes important plant pathogens with high economic impact, of which rice yellow mottle virus, potato leafroll virus, sugarcane yellow leaf virus and beet-infecting poleroviruses are the most devastating. Virulent infections induce chlorosis, phloem necrosis, stunting and sometimes sterility. Sobemoviruses infect different tissue types, whereas poleroviruses and enamoviruses are phloem-specific. Whereas sobemoviruses are mainly transmitted via mechanical wounds , transmiictv.global/taxonomy. The genera Sobemovirus and Polerovirus each include 20 or more species and the genus Enamovirus includes 5 species. The genus Polemovirus includes the species Poinsettia latent virus.Current taxonomy: Solemoviridae: ictv.global/report/solemoviridae.Full ICTV Report on the family


Despite many recent efforts to predict and control emerging infectious disease threats to humans, we failed to anticipate the zoonotic viruses which led to pandemics in 2009 and 2020. The morbidity, mortality, and economic costs of these pandemics have been staggering. We desperately need a more targeted, cost-efficient, and sustainable strategy to detect and mitigate future zoonotic respiratory virus threats. Evidence suggests that the transition from an animal virus to a human pathogen is incremental and requires a considerable number of spillover events and considerable time before a pandemic variant emerges. This evolutionary view argues for the refocusing of public health resources on novel respiratory virus surveillance at human–animal interfaces in geographical hotspots for emerging infectious diseases. Where human–animal interface surveillance is not possible, a secondary high-yield, cost-efficient strategy is to conduct novel respiratory virus surveillance among pneumonia patients in these same hotspots. When novel pathogens are discovered, they must be quickly assessed for their human risk and, if indicated, mitigation strategies initiated. In this review, we discuss the most common respiratory virus threats, current efforts at early emerging pathogen detection, and propose and defend new molecular pathogen discovery strategies with the goal of preempting future pandemics. In this report, we seek to review the impact that respiratory viruses have had upon mankind, discuss current efforts regarding their control, and propose new strategies to detect emerging respiratory virus threats and mitigate them before they become widespread. The goal in proposing new strategies is to identify methods that will be widely embraced, preemptive, and sustainable, rather than our current practice of being surprised by new threats. The latter forces countries to invest intensive and expensive response efforts towards respiratory virus control, while enduring significant morbidity.Adenoviridae, Coronaviridae, Orthomyxoviridae, Picornaviridae, and Pneumoviridae), they have two things in common: many are RNA viruses, and most are zoonotic. A review of these epidemics is necessary for planning better future mitigation strategies.In recent decades, the world has experienced numerous epidemics of emerging or reemerging respiratory viruses. These viruses have been responsible for much morbidity and mortality , in addiPneumoviridae family .hMPV was first discovered in 2001 in the Netherlands, yet it is thought to have been in circulation for at least 50 years beforehand . RetrospPicornaviridae family.HRV-C was identified in 2004, following an outbreak of influenza-like illness in New York State . In limiPicornaviridae family.EV-A71 infections are recognized as the cause of hand, foot, and mouth disease and various neurological conditions, especially among children under five years. The virus was first isolated from an encephalitic patient in California in 1969, and between 1972 and 1990 was associated with additional outbreaks across six countries . EV-A71 Coronaviridae family.SARS-CoV emerged in China in 2003, and rapidly spread to 29 countries before it was halted, with heroic public health and hospital infection control measures. Though no cases of SARS-CoV infection have been identified since 2004, it is recognized to have infected more than 8000 people and caused 774 deaths during the short time the virus was circulating . This RNAdenoviridae family.HAdV14 reemerged in the United States in 2006, causing at least 750 illnesses and 13 deaths , this adOrthomyxoviridae family.The H1N1pdm09 virus emerged in Mexico in 2009, and quickly spread throughout the world, causing an estimated 60.8 million illnesses and at least 12,469 deaths from 2009–2010 in the United States alone . This swCoronaviridae family.Middle East Respiratory Syndrome Coronavirus (MERS-CoV) emerged in Saudi Arabia in 2012, causing alarming respiratory illnesses. As of September 2020, MERS-CoV has been confirmed as a cause of 2494 illnesses and 858 deaths in 27 countries. It is recognized to be enzootic in camels ) an) anParamCR assay .Molecular detection algorithms for adenoviruses and enteroviruses incorporate both qPCR/qRT-PCR to identify human strains of these viruses and conventional PCR in a pan-species RT-PCR approach, with sequencing to characterize novel human and animal virus strains  97,98,99,100,101As part of our pathogen discovery pathway, field specimens with evidence of a novel virus are transported to one of our dedicated research laboratories for further study. This may involve work to determine if a novel virus meets Bradford Hill or other related criteria to be considered as a human pathogen. Often, characterization involves assessing viral infectivity in various cell lines to support hypotheses for host range and pathogenicity. From the original specimen, 250 µL of the sample is inoculated in an appropriate cell line. This procedure requires previous knowledge relating to characteristics of the viral family to determine which cell culture line would provide the optimal conditions for growing the virus. Cytopathic effects (CPE) are monitored in the inoculated cells to assess successful viral amplification, and virus in the supernatant is harvested when 80% CPE is observed. We next develop a real-time molecular assay for that pathogen. The resultant virus is confirmed to grow in cell culture using qPCR/qRT-PCR, and the virus concentration is titered. In the case of novel viruses, the DNA/RNA may be extracted and sent for whole-genome sequencing. Isolated viruses are stored for future use by our team or apportioned to other researchers through our online biorepository. If the virus is especially threatening it may be necessary to determine the virus’ geographical and host prevalence. This may involve the development of a serum neutralization assay and seroepidemiological studies of animal workers and their animals. Further concerns may lead to pathogenesis studies in animal models.In this manuscript, we endeavored to review the most common respiratory virus threats and detail their morbidity. We discussed current efforts at early emerging pathogen detection and noted their recent failure in providing early warning for the 2009 influenza A virus and 2020 coronavirus pandemics. We also proposed an alternate One Health strategy for novel respiratory virus detection and argued that it is likely to be more efficient and more sustainable than current efforts, especially when integrated with research perspectives from ecology and evolution. Finally, we provided considerable detail regarding laboratory methods in employing this One Health strategy. We proposed establishing networks for novel respiratory virus surveillance in known geographical hotspots for emerging infectious diseases. We proposed targeting sites where humans and animals interface, such as live-animal markets, large industrial farms, or alternatively, when that is not possible, large hospital sites with high rates of pneumonia admissions where the etiology of these patients might be studied for novel respiratory virus pathogens. By employing targeted, minimally invasive surveillance studies, future respiratory virus threats may be more effectively detected mitigated prior to causing tremendous human and economic costs to the affected nations.


Perca fluviatilis) are increasingly farmed as a human food source. Viral infections of European perch remain largely unexplored, thereby putting farm populations at incalculable risk for devastating fish epizootics and presenting a potential hazard to consumers. To address these concerns, we applied metatranscriptomics to identify disease-associated viruses in European perch farmed in Switzerland. Unexpectedly, in clinically diseased fish we detected novel freshwater fish filoviruses, a novel freshwater fish hantavirus, and a previously unknown rhabdovirus. Hantavirus titers were high, and we demonstrated virus in macrophages and gill endothelial cells by using in situ hybridization. Rhabdovirus titers in organ samples were low, but virus could be isolated on cell culture. Our data add to the hypothesis that filoviruses, hantaviruses, and rhabdoviruses are globally distributed common fish commensals, pathogens, or both. Our findings shed new light on negative-sense RNA virus diversity and evolution.European perch ( Perca fluviatilis; order Perciformes, family Percidae) to farming and the growing consumer interest in this fish , leading to the central nervous system (CNS) signs of loss of equilibrium and aberrant swimming behavior and to higher mortality  and 33 g . Eleven live juvenile European perch were sent to the Centre for Fish and Wildlife Health (FIWI), University of Bern , where they were euthanized and subjected to microbiological  and pathologic (including histopathologic) examination. Because the remaining fish at the farm exhibited clinical signs of disease, they were kept in quarantine for an additional 2 months and subsequently euthanized.Lepomis macrochirus) fry cells (BF-2) and fathead minnow  epithelioma papulosum cyprini (EPC) cells. These cells were originally obtained from the Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health .We used bluegill (G) gene of PRV isolate 9574.1 (GenBank accession no. JF502613), according to a previously published protocol, by using the primer pair oPVP116/118 and oPVP126/Rha2  and incubated at 15°C. We monitored the cell cultures for CPE daily for 7 days by using light microscopy and then monitored subcultures for another 7 days. We harvested supernatants from cell cultures showing CPE and tested by reverse transcription PCR (RT-PCR) for the common European perch pathogen PRV, targeting the glycoprotein , according to the manufacturer’s instructions. We then prepared a high-throughput sequencing (HTS) library with the TruSeq Stranded Total RNA kit  and performed HTS on a HiSeq 3000 machine (Illumina), generating paired-end reads of 2 × 150 bp. We performed bioinformatic analysis as described previously . We performed 3′ and 5′ rapid amplification of cDNA ends (RACE), as described previously, on RNA extracted from pooled organs and CNS tissue as well as cell culture supernatants (Filoviridae (Hantaviridae (Rhabdoviridae (L) sequence using maximum-likelihood phylogenetics. We applied the maximum-likelihood method in MEGA X  https://acdbio.com) (Appendix).During a complete necropsy of the 11 fish, we used 3 whole perch for histologic examination. We fixed these fish in 10% buffered formalin for 24 hours and embedded cut sections of the gills, longitudinal head sections, and longitudinal cut sections of the body cavity in paraffin. We prepared 3-μm sections and stained them with hematoxylin and eosin according to standard protocols. We conducted chromogenic in situ hybridization on all formalin-fixed paraffin-embedded (FFPE) tissues used for histopathology. We performed staining with the RNAscope system  infection , 11 randomly selected live fish were sent to FIWI for microbiological and pathologic examination. Clinical signs included anorexia, lethargy, skin ulcerations, multifocal hemorrhages, and eroded tail fins. Culture and PCR indicated that skin ulcerations were caused by oomycete  to suspensions of pooled perch CNS and pooled visceral organs. We selected these cell lines because of their high susceptibility to diverse fish viruses (7) signals with inverse complementarity of 13 nt of the 3′ and 5′ terminal genome sequences.To further characterize the putative novel perhabdovirus, we performed metatranscriptomics by using HTS and bioinformatic analysis of pooled CNS tissue and visceral organ RNA extracts of 5 fish. We found 16 sequence scaffolds 247–1,454-nt long with mean k-mer coverages of 1.0–5.9 and nucleotide sequence identities of 62%–98% to perhabdovirus genomes (Appendix Table 1). We mapped these scaffolds to LTRV (GenBank accession no. AF434991) and PRV (GenBank accession no. JX679246) as references and closed sequence gaps by RT-PCR and RACE followed by Sanger sequencing. The resulting complete genome of the novel virus was 11,595-nt long and had the characteristic genomic organization of (pe)rhabdoviruses , panel AL gene confirmed a close relationship of the new virus to the 2 members of the species Sea trout perhabdovirus  (N), phosphoprotein (P), matrix protein (M), and G genes . The current perhabdovirus sequence-based species demarcation criterion is a minimum divergence of 15% in the L gene (L gene sequence of the novel virus is most closely related to that of the isolate 18/203 (divergence 10%); L genes of both viruses are ≈19% divergent from the next closest related perhabdovirus, sea trout perhabdovirus isolate R6146 (GenBank accession no. MN963999), thus indicating that we discovered a novel perhabdovirus that should be assigned to a new species along with isolate 18/203. We named the new virus Egli (EGLV) virus after a local Swiss-German word for European perch and deposited the complete viral genome sequence into GenBank (accession no. MN510774). Detection of EGLV in European perch FFPE tissue sections by RNA in situ hybridization was unsuccessful.Phylogenetic comparison of the F434992) , panel BThamnaconus septentrionalis) captured in the East China Sea . The deduced amino acid sequences were 28%–30% identical to proteins of Huángjiāo virus (HUJV), a recently identified virus of marine greenfin horse‐faced filefish , transcriptional activator (VP30), and large protein (L) containing an RNA-directed RNA polymerase (RdRp) domain, as well as 1–2 novel proteins (L gene sequences with those of representative classified viruses of the family Filoviridae (>55% and >23% sequence divergence over complete genome sequences determined by using PASC (Thamnovirus percae”). The available KNDV genome sequence is 49% divergent from HUJV and 37% divergent from FIWIV (Thamnovirus kanderense”). In contrast, OBLV was >62% divergent from HUJV, FIWIV, and KNDV viruses and thus represents a new species (“Oblavirus percae”) within a new genus (“Oblavirus”). All attempts to detect FIWIV or OBLV viruses in 3 European perch FFPE tissue sections were unsuccessful.We named these viruses Fiwi virus , after FIWI; Oberland virus , after Bernese Oberland; and Kander virus , after the Kander River, which flows through Bernese Oberland. Whereas the FIWIV genome appears to be coding complete, the sequences of OBLV are coding incomplete at the 5′ terminus and of KNDV at both the 5′ and the 3′ termini. All attempts to determine the authentic 3′ and 5′ termini by RACE were unsuccessful, most likely because of low viral RNA loads. However, all 3 sequences have the genomic features of HUJV, encoding the filovirus-typical proteins nucleoprotein (NP), polymerase cofactor (VP35), glycoprotein  similar to various HUJV genes, but attempts to demonstrate a physical linkage of these sequences by RT-PCR were not successful. Still, the presence of these scaffolds is indicative of at least 1 additional novel filovirus, which we were not able to further characterize.Halieutaea stellate) and WERSV in red spikefish  captured in the East China Sea (Appendix Table 1) , M (GenBank accession no. MN510770), and S (GenBank accession no. MN510771) segments of a novel virus, here named Bern perch virus. Similar to WEMBV and WERSV, the Bern perch virus (BPV) L segment  was deduced to encode the L protein including an RdRp domain, the M segment  was deduced to encode the glycoprotein precursor, and the S segment  was deduced to encode the nucleocapsid protein . Phylogenetic analysis of the L protein confirmed the close relationship of Bern perch virus to all currently classified actinoviruses , medium (M), and small (S) segments of Wēnlǐng minipizza batfish virus (WEMBV) and Wēnlǐng red spikefish virus (WERSV). WEMBV was recently identified in apparently healthy minipizza batfish , and 1 novel hantavirus in morbid farmed European perch. The discovery of a novel rhabdovirus was not surprising and adds to the role of rhabdoviruses in fish health; rhabdoviruses, in particular those of the genera Striavirus) in striated frogfish (Antennarius striatus ) and HUJV in greenfin horse‐faced filefish captured in the East China Sea (Zeus faber) and blue spotted goatfish filovirus in blue spotted goatfish (Upeneichthys lineatus) purchased at a fish market in Sydney, New South Wales, Australia  and detected a likely fourth filovirus in the diseased perch. Until recently, filoviruses, notorious for causing disease in humans with extremely high lethality  and fish hantaviruses  (Lophius litulon] captured in China [Eviota zebrina) purchased at a fish market in Sydney (Eptatretus burgeri) captured in China (We also identified a novel hantavirus, Bern perch virus. Hantaviruses are best known as rodent-borne viruses of the mammantavirin genus Additional methods and results for study of filoviruses and hantaviruses in freshwater fish, Switzerland, 2017.


Environmentally-based archaeological research at Zaraa Uul, including zooarchaeology, phytolith analysis, and radiocarbon dating, is the first of its kind in Mongolia and presents critical new insight on the relationship between periods of occupational intensity and climatic amelioration from the earliest anatomically modern humans to the adoption of pastoralism. The palaeoenvironmental and faunal record of Zaraa Uul show that Early-Middle Holocene hydrology and species distributions were distinct from all other periods of human occupation. Holocene hunter-gatherers inhabited an ecosystem characterized by extensive marshes, riparian shrub and arboreal vegetation along the hill slopes and drainages. The exploitation of species associated with riparian and wetland settings supports the hypothesis of, but suggests an earlier timing for, oasis-based logistical foraging during the Early-Middle Holocene of arid Northeast Asia. The onset of wetter conditions at 8500 cal BP agrees with other regional studies, but multiple lines of evidence present the first integrated field- and laboratory-based record of human-environment relationships in arid East Asia during the Holocene Climatic Optimum. We compare it to Late Pleistocene climatic amelioration, and highlight specific responses of the hydrological, vegetative and faunal communities to climate change in arid Northeast Asia. Her proposed chronology for the region is summarized in Wetlands are a critical resource for hunter-gatherers in many environmental contexts  and evidThis chronology, beginning with the earliest verified sites post-dating the Last Glacial Maximum (LGM) and continuing into the early stages of herding and bronze metallurgy, is the only date-based chronology for the region and is grounded on site assemblages collected by major interdisciplinary scientific expeditions during the 1920s and 1930s –13. WhilHaloxylon ammodendron)  metatarsal, Coelondonta antiquitatus [woolly rhinoceros] scapula and juvenile Hyaenidae [hyaena] skull) on the north-east wall and extended the unit to recover these . Each grid was laid out on a north-east to south-west axis, set parallel to the orientation of the local basin-range landforms; the x-axis was labelled at 1 m intervals with numbers and the y-axis was labelled at 1 m intervals with letters. Small bone and seemingly undiagnostic fragments were collected, along with bone from the sieve, in a common grid bag with the catalogue number and the name “bone” rather than an individual number . Artifacts and diagnostic or larger bone fragments were mapped in three dimensions within the grid using a string, line level and datum (10 cm above ground surface). In order to track changes in elevation within the excavation, numerous elevation points were collected within the grid using the datum. Primary levels  were determined based on matrix characteristics , beginning with Level 1 uppermost. Within deeper matrices, sublevels were set at approximately 10 cm intervals within the main level , with the exception of 2018 excavations at Locus 3 where sublevels were placed at 5 cm intervals in Level 1. The above procedure was followed for all units.Geoarchaeological research at this site was led by Rosen and included walking observations, excavation and sediment collection from three geological test pits  around t14C were taken from multiple levels of loci 1–3. Pottery and bone collagen were processed at the University Georgia Center for Applied Isotope Studies and the A. E. Lalonde AMS Laboratory. Collagen for all samples sent to the A. E. Lalonde AMS Laboratory was processed using ultra filtration by Janz at the Trent Environmental Archaeology Laboratory (TEAL) according to the UCI-KCCAMS procedure for ultrafiltration ). All were derived from Locus 2 (ZU2.T2/3) with the exception of the one likely intrusive from Locus 3 (Rodentia femur), and one possible intrusive from each Zuun Shovkh (Cricetidae tibia) and Otson Tsokhio (Rodentia tooth) . The calcium-rich context of these burials has caused extensive mineralization on the surfaces of most artifacts and bones. This is especially true of the Holocene layers. There are no studies relating to the timeline for surface mineralization in these sites, especially as it might relate differently to large ungulate versus less porous bone such as that of small avian fauna. Comparison between level of surface mineralization on Early-Middle Holocene fauna and actual dates was inclusive: dates did not match expectations based on relative degree of mineralization. However, bones with no evidence of surface mineralization visible to the naked eye were categorized as intrusive (N = 6) and although they are reported in oth) see .The highest of the observed beach terraces was recorded at an approximate elevation of 1044 m a.s.l. (Garmin GPS). A large lithic scatter eroding from the whitish/yellow silty sand deposits of this ridge, Zuun Shovkh, are typologically consistent with the Initial to Early Upper Palaeolithic in Mongolia , during which time the East Asian Summer Monsoon System (EASM) brought heightened precipitation far into Northeast Asia –51. At Zally dry . One lumally dry , 48, 52.Level 1 of Zaraa Uul Locus 2 (ZU2.T2/3) and Locus 3 (ZU3.T1) extends ~5–10 cm below the surface and is characterized by recently re-deposited sediments–a poorly consolidated mix of aeolian deposits and slope wash. Redeposition of artifacts from slightly farther upslope in Locus 2, Level 1 would explain both the lack of spatial patterning  and the Level 2 extends ~11–50 cm below surface and is characterized by a darker A-horizon which represents several thousand years of Holocene soil development under stable landscape conditions. A potsherd (T2-3m:1a) from Locus 2 at the Level 2/3 boundary was dated to 6794–6665 cal BP (5910 ± 30) , but rec3 derived from the older Pleistocene marls. Levels 2 and 3 were heavily disturbed and intermixed in Locus 2, but can be characterized by distinct changes in colour and texture. Level 3 was less well-developed in Locus 3. Artifacts in Locus 3 were primarily distributed in Level 2 at 30–50 cm below the surface  which provided a hard, sparsely vegetated surface for camping and working. Site activity after 8500 cal BP contributed active anthropogenic disturbance of these sediments during the formation of Holocene soils and was responsible for the intermixing of levels 2 and 3 at Locus 2 (ZU2.T2/3). Site occupation over the next two millennia resulted in the mixing of occupation debris, as did rodent activity and processes of erosion and redeposition during the more arid conditions of the Late Holocene. Radiocarbon dates on bone and ceramics consistently, with only two exceptions, predate 6500 cal BP. One date from Locus 1 shows continued human activity at 6276–6003 cal BP. Regular use of the site appears to end Following what appears to be a hiatus of about three thousand years, the locality was used in Bronze Age mortuary rituals. There are several later periods of discontinuous activity represented: 3555–3382 cal BP (3230 ± 25) in Locus 1, 2753–2353 cal BP  in loci 1 and 3, 1520–1350 cal BP (1523 + 20) in Locus 2, and 1863–1717 cal BP (1850 ± 20) in Locus 1. The first two phases correspond with periods of known Bronze Age burial activity (Ulaanzuukh and Slab Grave periods ) upslopePhytoliths were analyzed at Zaraa Uul to investigate a) the diversity of micro- environments around the site which were available to be exploited for different types of resources on the part of the Early and Middle Holocene populations who visited this locality, b) to address questions of seasonality of occupation, and c) to help contextualize the environmental background for the faunal remains at the site. All samples came from the Early-Middle Holocene contexts in loci 1 and 2 (see below).Marmota sibirica) and badger (Meles leucurus) in Locus 2 (see below) support a summer or fall occupation. Some of the phytolith forms are from the leaves and wood  of woody shrubs or trees (Here we report on the counts of phytolith samples from ten samples. The results are listed in or trees . These por trees .Stipa sp. (c.f. Stipa capillata) which is typical of the natural undegraded steppe vegetation found widely across Central Asia. The phytoliths from this genus occur in both Locus 1 and Locus 2 samples  were identifiable to family, order, genera, species, or body size class. Excavations at Otson Tsokhio were limited in scope (2 x 2 m unit in 2018). Preservation was excellent for all assemblages.All classes of elements  were represented in all assemblages for both large and small species. There was some variation in relative frequency of elements represented across loci and body size of prey: cranial elements were most common across the site for large ungulates (66.2% of MNI), especially at Otson Tsokhio (79.1% of MNI); large ungulate vertebral elements were most common at Locus 1 (52.9% compared to 12.2% for entire assemblage); and frequencies of both medium and small ungulate feet were higher at Locus 3. This variation is likely due less to transport and more to peculiarities of fragmentation/preservations  and the use of bone for raw materials, rather than transport. Issues related to transport will be discussed in other publications.p <0.001). Otson Tsokhio fauna was least heavily fragmented, which may relate to both the size/density of the bones and the degree of dietary or non-dietary processing. Spiral fractures are not present in the Otson Tsokhio assemblage and limited at Zaraa Uul , but there is evidence of cut marks on fauna from both Zaraa Uul  and Otson Tsokhio (N = 2). Carnivore gnawing is present at Zaraa Uul  and very rare at Otson Tsokhio (N = 2). Cut marks on a femoral head and the distal shaft of an ulna are related to carcass processing. Wide, irregular grooves associated with hacking were also found at Otson Tsokhio on both the spine of the rhinoceros scapula and the giant elk metatarsal. Primary processing (butchery) is therefore the main type represented at Otson Tsokhio.Holocene and Pleistocene assemblages clearly related to human activity but reveal very different patterns of use and deposition. Degrees of fragmentation between sites were assessed by measuring maximum length of all bone fragments without any fresh breaks . A KruskLepus tolai) bone from Locus 2 (N = 2) and Locus 3 (N = 1). Calcination was present on only two faunal specimens, one each from Locus 2 and Locus 3, suggesting that burning was incidental to cooking rather than their use fuel. Burning was not present on bone from Palaeolithic assemblages. Taphonomic variation between Zaraa Uul and Otson Tsokhio assemblages may be related to the nature of these deposits . Rodent gnawing is rare (N = 3 for Locus 3 and N = 1 for Otson Tsokhio).Bone at Zaraa Uul shows greater evidence, not only of carnivore gnawing, but also secondary processing (post-butchery bone modification): long bones from Locus 2 are often split along the long axis (N = 107) and percussive flaking is present in both loci 2 and 3 . Although no hearths or ash pits were found, burning was present across Locus 1 (N = 8), Locus2 (N = 101), and Locus 3 (N = 53), including burning on hare . The emphasis on small to micro-sized fauna at Zuun Shovkh suggests that the faunal deposit may not be anthropogenic and etching on many of the bones indicate they may have been deposited as predator pellets or feces. This sample was therefore not included in our consideration of dietary changes.X2 = 11.81, df = 1, p < 0.01) increase in both avian  and carnivore  species compared to the Pleistocene layers. There is a statistically significant  shift across all body sizes of prey between 8500–7500 cal BP (Locus 2) and 7200–6500 cal BP (Locus 3) components, with an increase in large-bodied (45.5% to 58.8%) prey and corresponding decline in medium-bodied (20.0% to 9.0%), as well as small- and micro-bodied, prey  or carnivores (1.4%). Fauna from Zaraa Uul show a primary reliance on ungulates  with a statistically significant  as well as several other species that are prefer forested or shrubby habitats and are regionally extinct or rare  (Panthera tigris (tiger) bone dates to 6640–6494 cal BP (5765 ± 25). This find is ecologically consistent with the presence of key prey species such as deer and boar that were also being hunted by humans. The bone is from an animal’s paw  and as such may be a remnant of skin used for clothing or furnishing. Equids represent the primary category of identifiable large-bodied prey from the Upper Palaeolithic through the early Oasis 2 phase of occupation, whereas identified specimens in the later phase of Oasis 2 are dominated by bovines (Equus hemionus (khulan or wild ass), where equid species are identifiable to species (based on size), while both Equus hemionus and Equus ferus (wild horse) are present in Pleistocene layers.Otson Tsokhio fauna includes easant]) . The Pan bovines . Middle Struthio asiaticus/anderssoni] eggshell was recovered at Otson Tsokhio and both of the two fragments we dated pre-date the fauna by several thousand years. Mother-of-pearl (nacre) was recovered in both Locus 2 and 3 (Uno sp.). Such artifacts have been found in burial contexts to the north, in the far eastern steppe region of Dornod province. Uno spp. may have inhabited the basin at Zaraa Uul or been imported from the north, where the shells are better represented archaeologically at sites like Tamsagbulag and Hag (Inner Mongolia) , Coelodonta antiquitatis [woolly rhinoceros], Megaloceros sp. [giant elk]) and Hyaenids  prior to or during the early Holocene; 2) increased diversity of smaller-bodied and more specialized herbivores during the Middle Holocene Climatic Optimum; and 3) late Holocene aridification and the rise of domesticated herd animals, which corresponds with the local retreat of species such as cervids, wild boar, and various small-bodied species . Divergent faunal communities during Pleistocene and Holocene wet phases underscores epochal changes in global ecology. Locally, in the Gobi Desert, the Holocene is characterized by the late Pleistocene/early Holocene evaporation and infilling of deep saline lake basins, resulting in the widespread development of freshwater marshes, enhanced vegetative biomass, and increasing species diversity during the Holocene.Zooarchaeological data highlights substantive changes in the nature of faunal communities: 1) local extinctions of numerous large-bodied herbivores and carnivores Click here for additional data file.S2 TableAll measurements for (XLSX)Click here for additional data file.


Amyotrophic lateral sclerosis (ALS) is a progressive neuromotor disease characterized by the loss of upper and lower motor neurons (MNs), resulting in muscle paralysis and death. Early cortical hyper-excitability is a common pathological process observed clinically and in animal disease models. Although the mechanisms that underlie cortical hyper-excitability are not completely understood, the molecular and cellular mechanisms that cause enhanced neuronal intrinsic excitability and changes in excitatory and inhibitory synaptic activity are starting to emerge. Here, we review the evidence for an anterograde glutamatergic excitotoxic process, leading to cortical hyper-excitability via intrinsic cellular and synaptic mechanisms and for the role of interneurons in establishing disinhibition in clinical and experimental settings. Understanding the mechanisms that lead to these complex pathological processes will likely produce key insights towards developing novel therapeutic strategies to rescue upper MNs, thus alleviating the impact of this fatal disease. First described by Jean-Martin Charcot in 1869, amyotrophic lateral sclerosis (ALS), one of the most common neuromotor diseases, is characterized by an inexorable loss of upper and lower motor neurons (MNs). The upper motor neurons of the primary motor cortex are large pyramidal neurons in layer V (LVPNs), whose axonal projections form the corticospinal tracts, which descend to directly or indirectly excite the lower motor neurons (LMNs) of brainstem and the spinal cord . AlthougThe clinical outcome in ALS is largely based on lower MN degeneration, presenting mostly as the onset of weakness in limb 80%) or bulbar 20%) muscles . A relen0% or bul0% muscleIn an attempt to identify the origin of neurodegeneration and the correlation between upper MN and lower MN dysfunction, the “dying forward”  hypothes+, K+) present, which shapes the neuronal output [Excitability of a neuron is a measure of its electrophysiological properties. This relates to the neuron’s ability to produce an output or action potential (AP) by depolarizing its membrane potential to a threshold level in response to an input. This intrinsic ability of a neuron is referred to as its “intrinsic excitability” and is defined by factors including the types and number of excitatory receptors and voltage gated ion channels  [+ current and depleted K+ conductance [2+ influx and MN firing frequency [+ channel activity, further reinforces the correlation between increased PIC and cortical dysfunction [One prime factor affecting the neuronal excitability is the persistent inward current (PIC) generated by a inactivation-resistant population of Nat (INaP) . In ALS,ductance  and prodductance ,53. Seveductance  and in mductance . More reductance , to the ductance , clearlyductance . A depolductance , and henductance  by direcrequency ,58. Furtrequency ,59,60, arequency  by riluzfunction ,54,61.+ conductance, documented in clinical studies showing peripheral motor axonal excitability [+ currents [+ conductance remains to be explored. Activating Kv7.2/3 potassium channels  by retigabine, an activator of Kv7.2/3 potassium channels drives K+ efflux, counterbalancing the Na+ influx and thus dampening MN hyper-excitability as observed in ALS patient-derived MNs [+ and decreased K+ conductances, providing additional avenues for therapeutic exploration.Similarly, another contributor to MN hyper-excitability in ALS is reduced Ktability ,63 and icurrents . Howeverived MNs  and LVPNived MNs . This ev2+ and Na+ ions resulting in a synaptic hyper-active state [2+ buffering capacity of MNs in ALS is reported to be impaired, with defective Ca2+ ATPase and Ca2+/Na+ exchangers exacerbating the increased intracellular Ca2+ burden [2+ overabundance and sustained depolarization [Cortical hyper-excitability in ALS is also associated with both functional and morphological synaptic changes ,15,31,66ve state . Additio+ burden  leading rization ,76,77. Grization ,15. Thisrization .Clinical observation of functional and structural cortical excitatory measurements in ALS patients serve as a marker of altered cortical output function, i.e., the cortical state of excitability. Enhanced cortical excitability prior to symptom onset due to significant lower MN degeneration has been observed in ALS patients by utilizing advanced clinical neurophysiological and neuroimaging techniques , suggestCorticospinal neurons, namely LVPNs in the deep motor cortical layer, are the source of corticospinal projections to spinal cord controlling voluntary movement execution. Besides direct corticospinal projections, LVPNs also make direct corticofugal connections to the thalamus, cortico-cortical connections to layer 4 and layer 2/3 motor cortex, and corticobulbar connections to motor nuclei in the brainstem . EvidencA receptors [A receptor ligand, in sporadic ALS patients [The maintenance of normal neuronal activity in the cortical neural circuit is an interaction between the excitatory pyramidal neurons and abundant interneurons that mainly cause inhibition via γ-aminobutyric acid (GABA) neurotransmission. GABAergic interneurons in the motor cortex can be activated via a sub-threshold stimulus . In ALS eceptors  and funcpatients  provide patients  and animpatients ,103, andpatients . A more patients . Additiopatients  and somapatients  have alspatients ,106 or hpatients , it remaHyper-excitability is a prevalent feature observed clinically in ALS patients and in animal models for ALS. What causes cortical hyper-excitability and how hyper-excitability drives upper MN degeneration and death is just beginning to be unraveled. Evidence from electrophysiological and histological studies in humans and in animal models provides insight into an early cortical involvement in the disease process that precedes lower MN degeneration. Cortical hyper-excitability is clearly a result of increased excitation and impaired inhibition, and the evidence so far points to a number of molecular and cellular mechanisms that contribute to cortical hyper-excitability and that may drive the degeneration of upper MNs. It will be critically important to further characterize these mechanisms and understand their consequences, in order to develop rational therapeutic strategies that normalize hyper-excitation and alleviate the disease.