Background: The present study compared corticospinal excitability to the biceps brachii muscle during arm cycling at a self-selected and a fixed cadence . We hypothesized that corticospinal excitability would not be different between the two conditions. Methods: The SSC was initially performed and the cycling cadence was recorded every 5 s for one minute. The average cadence of the SSC cycling trial was then used as a target for the FC of cycling that the participants were instructed to maintain. The motor evoked potentials (MEPs) elicited via transcranial magnetic stimulation (TMS) of the motor cortex were recorded from the biceps brachii during each trial of SSC and FC arm cycling. Results: Corticospinal excitability, as assessed via normalized MEP amplitudes , was not different between groups. Conclusions: Focusing on maintaining a fixed cadence during arm cycling does not influence corticospinal excitability, as assessed via TMS-evoked MEPs. It is well established that rhythmic locomotor outputs in non-human animals  are partially controlled by neural circuits located in the spinal cord, referred to as central pattern generators (CPGs) ,2. EvideArm cycling has been introduced as a model of locomotor output for examining changes in neural excitability during rhythmic movement, with the vast majority of these studies using a set cadence and power output for each participant ,5. WhileWhen humans engage in a novel motor task, they typically focus on how to perform the said task, placing them in what is known as the “cognitive stage” of motor learning, according to the Fitts and Posner model . This suArm cycling is a motor task that may be considered novel and a number of studies have been published that have examined corticospinal excitability during cycling in humans ,12,13,14Several studies have examined the influence of cycling cadence on neuromuscular activation. Marias et al. (2004) examined the effects of a spontaneous chosen crank rate (SCCR) and crank rates 20% higher and lower than the SCCR during arm cycling on integrated electromyography (iEMG) levels in the biceps brachii muscles in humans. The researchers concluded that there were no significant differences in the iEMG between the crank rate conditions of the biceps brachii, suggesting that the SCCR was not chosen to minimize the level of muscle activity and that the degree of muscle activation was similar between the two groups . This fiThe purpose of the current study was thus to determine if corticospinal excitability between SSC and FC arm cycling was different. It was hypothesized that corticospinal excitability, as assessed via the amplitude of motor evoked potentials (MEPs) elicited via TMS of the motor cortex, would not be different between SSC and FC arm cycling.Prior to the experiment all participants were informed of the experimental protocol and written informed consent was obtained. This study was in accordance with the Helsinki declaration, and experimental procedures were approved by the Interdisciplinary Committee on Ethics in Human Research at the Memorial University of Newfoundland (ICEHR #20171250). All experimental procedures were in accordance with the Tri-Council guidelines in Canada, and potential risks of participation were disclosed to all participants.Eleven participants  were recruited from the School of Human Kinetics and Recreation (HKR) at Memorial University using a convenience sampling technique. Prior to testing, each participant completed a magnetic stimulation safety-checklist to screen for existing contraindications to magnetic stimulation . To determine hand dominance, participants completed an Edinburgh Handedness Inventory questionnaire to ensure that all evoked responses were recorded from the dominant arm . AdditioA one-group within-subjects design was used. Participants attended two lab sessions with at least 24 h in between visits. The first visit was for a half-hour familiarization session and the second was the testing session, lasting approximately 1 h. The experiment was completed on an arm cycle ergometer  with the arm cranks set at 180 degrees out of phase see . Each paAll measurements were taken at a single position—6 o’clock relative to a clock face. This position was relative to the participants’ dominant hand, such that the TMS would be triggered when the right or left hand was at the 6 o’clock position for a right- or left-handed dominant individual, respectively. We have examined this position previously ,12,13,15max) in the biceps brachii muscle in each condition. The MEP amplitude made relative to Mmax and bEMG , as a measure of corticospinal excitability, served as the dependent variable. The SSC trial was completed first, followed by the FC trial, and responses were triggered as the arm crank of the dominant arm passed the 6 o’clock position.The study required participants to cycle at two different cadences, both at a constant workload of 25 W. The cadences (FC and SSC) served as the independent variables in the study. The TMS and Erb’s point stimulation were delivered at the 6 o’clock position to elicit MEPs and maximal M-wave . The EMG was recorded using a bi-polar configuration with an interelectrode distance of 2 cm. Electrodes were placed in the middle of the muscle belly of the biceps brachii. A ground electrode was placed over the lateral epicondyle on the dominant arm. Prior to electrode placement, the skin at the recording site was shaved to remove hair, abraded using an abrasive pad to remove dead epithelial cells, and cleaned with an isopropyl alcohol swab to reduce impedance for the EMG recordings. Signals were sampled online at 5 kHz using a CED 1401 interface and Signal 5.11 software (Cambridge Electronic Design (CED) Ltd., Cambridge, UK). The EMG signals were amplified (gain of 300) and filtered using a 3-pole Butterworth band-pass filter (10–1000 Hz) using a CED 1902 amplifier.EMG activity was recorded from the biceps brachii and lateral head of the triceps brachii of the dominant arm using pairs of surface electrodes  . The anode was placed on the acromion process and the cathode was placed over the skin in the supraclavicular fossa. A pulse duration of 200 μs was used and the stimulation intensity was gradually increased until the M-wave amplitude of the biceps brachii reached a plateau, referred to as Mmax. This stimulation intensity was increased by 10% and used for the remainder of the experiment to ensure maximal M-waves were elicited during each trial [Electrical stimulation of the brachial plexus at Erb’s point was used to measure Mch trial .Motor evoked potentials (MEPs) were measured during both cycling trials from the biceps brachii and served as the dependent variable in the study. TMS  was used to elicit MEPs in the biceps brachii by placing a circular coil (13.5 cm outside diameter) over the vertex. TMS is a valid and reliable technique for eliciting MEPs, which are recorded from the muscle as a measure of the excitability of the corticospinal tract . The vertex was located by measuring the mid-point between the nasion and the inion and between the participant’s tragi, and marks were placed for both measurements directly on the scalp. The intersection of the measurements was defined as the vertex ,23,24. TAfter the stimulation intensities were set for the TMS and Erb’s point stimulation, the cycling trials were completed. The participant was first instructed to cycle forward at a comfortable pace, and the monitor displaying the cycling cadence was moved out of the participant’s sight, such that the participant was blinded to their cycling cadence. When the participant reached a steady cadence, as observed by the researcher, the trial was started. A steady cadence was defined as a cadence that fluctuated no more than ±1 rpm over a 5 s period. While the participant was cycling, the researcher recorded the cadence every 5 s and calculated the average cadence over the duration of the trial. After a 1 min break the participant was instructed to cycle forward maintaining a target cadence, as specified by the researcher, by observing their cadence on the monitor. This target cadence (FC) was equal to the average of the cadence over the duration of the SSC trial. During both trials the arm ergometer was set to a fixed power output of 25 W. While cycling, each participant received 12 MEPs and 2 M-waves per trial, which were delivered when the dominant hand passed the 6 o’clock position. The order of the stimulations was randomized during the trial, and the stimulations were evoked every 7–8 s. To prevent anticipation of the stimulation, 2 frames without stimulation were added. The total length of cycling was approximately 2 min per trial.max, thus MEPs were normalized to Mmax evoked during the same trial to account for potential changes in peripheral excitability. All measurements were taken from the averaged files of all 12 MEPs and 2 M-waves. All measurements were made from the dominant arm.Data were analyzed off-line using Signal 5.11 software . To determine if the central motor drive projecting to the biceps brachii was similar between the two arm cycling conditions, the mean rectified EMG 50 ms prior to the TMS stimulus artifact was measured . The peat-tests were used. Additionally, paired-samples t-tests were used to assess whether statistically significant differences in MEP amplitudes normalized to Mmax occurred between the SSC and FC conditions. All statistics were completed on group data with a significance level of p < 0.05. All data are reported as mean ± SE (standard error) in the figures.To compare the pre-stimulus EMG between the conditions (SSC and FC), paired-samples Cycling cadence. p = 0.118).MEP amplitude. max of the biceps brachii during the SSC and FC arm cycling trials. The average MEP amplitude  when cycling at a SSC and a FC was 16.2% (SD (standard deviation) = 12.25) and 14.1% (SD = 11.75), respectively, with a mean difference of 2.1%. This difference was not statistically significant (p = 0.146).Pre-stimulus EMG of the biceps brachii for MEPs. The group mean (n = 11) pre-stimulus EMG of the biceps brachii prior to the TMS stimulus artifact during the SSC and FC arm cycling can be seen in p = 0.061).Pre-stimulus EMG of the triceps brachii for MEPs. The group mean (n = 11) pre-stimulus EMG of the triceps brachii prior to the TMS stimulus artifact during the SSC and FC arm cycling can be seen in p = 0.58).This is the first study to compare corticospinal excitability projecting to the biceps brachii between self-selected (SSC) and fixed cadence (FC) arm cycling. There were no significant differences in corticospinal excitability, as assessed via TMS-evoked MEP amplitudes recorded from the biceps brachii, between the two arm cycling conditions. Maintaining a pre-determined cadence (FC) during arm cycling does not increase corticospinal excitability, when compared to cycling at a voluntarily chosen cadence (SSC).A prior concern in studies from our lab and also in the work of others was that the attentional demands of maintaining a set cadence could inadvertently alter (likely increase) the measures of corticospinal excitability. The current finding that corticospinal excitability is not different between SSC and FC arm cycling lends support to our previous finding that corticospinal excitability is task-dependent and is higher during arm cycling than an intensity- and position-matched tonic contraction . In thatPrior work has shown that visual attention modulates corticospinal excitability and directing visual attention toward the specific features of an observed action facilitates corticospinal excitability more than passive observation ,26. AtteAdditional factors should be considered when interpreting the present results. This study assessed MEP amplitudes and therefore conclusions can only be made regarding the overall excitability of the corticospinal tract. In future, research assessing spinal excitability, with TMES  for example, to the target muscle to determine if changes in corticospinal excitability are occurring at the spinal and/or supraspinal level may be of interest . For insThe novel finding in this study is that corticospinal excitability, as assessed by changes in MEP amplitude, projecting to the biceps brachii is not different between SSC and FC arm cycling. We can indirectly (because attention was not directly measured) conclude that corticospinal excitability during arm cycling is independent of attentional demands, as corticospinal excitability is not different when focusing attention on maintaining a set cadence compared to cycling at a voluntarily chosen cadence.


Pneumocystis jirovecii (PJ) also causes pneumonia in immunocompromised hosts. Although both cause opportunistic infections, it is rare to have a co-infection in a non-human immunodeficiency virus carrier.Varicella-zoster virus (VZV) causes herpes zoster. An 84-year-old woman with hemolytic anemia referred because of acute respiratory failure. She had received prednisolone without PJ pneumonia prevention. She developed dyspnea and desaturation while eating, and thus was treated based on a presumptive diagnosis of aspiration pneumonia. Physical examination revealed a vesicular rash on the left side of her neck suggesting herpes zoster infection. Polymerase chain reaction of her sputum for PJ and VZV was positive, which confirmed a diagnosis of pneumonia due to PJ and VZV co-infection. Despite acyclovir and sulfamethoxazole and trimethoprim administration, she died on hospital day 19.Clinicians should suspect PJP when patients on systemic corticosteroids develop pneumonia and they have not received prophylactic treatment for PJP in non-HIV carriers. When such patients have a VZV rash, clinicians should aggressively seek signs of opportunistic infections. Our case hereby highlights the importance of recognizing the possibility of a VZV and PJ co-infection. Pneumocystis jirovecii (PJ) causes life-threatening pneumonia, known as Pneumocystis jirovecii pneumonia (PJP), in immunocompromised hosts. Both pathogens cause known opportunistic infections, but co-infection with both entities in a non-human-immunodeficiency-virus (HIV) carrier is uncommon. We describe a case of VZV and PJ co-infection in a non-HIV carrier on chronic systemic corticosteroids.Varicella-zoster virus (VZV) rarely causes pneumonia in immunocompromised or immunocompetent hosts. An 84-year-old woman with a recent diagnosis of hemolytic anemia referred to our hospital because of acute respiratory failure. During the 64 days before this referral, the patient received prednisolone titrated between 10 mg and 80 mg daily. As of the referral, she received 40 mg/day of prednisolone. A few days prior, she developed a fever without any accompanying symptoms. On the day of the referral, she developed dyspnea, desaturation, and altered mental status while eating, and was transported to our hospital. We supposed that the patient inspired food while eating and that inspiration-induced hypoxia caused deterioration of her mental status. Her vital signs on arrival were a body temperature of 37.0 °C, heart rate of 150 beats/min, blood pressure of 170/70 mmHg, respiratory rate of 24/min, and 88% oxygen saturation on a reservoir mask of 10 L/min. Her Glasgow Coma Scale (GCS) was 7 (E2V1M4). She had hypoxemia refractory to full mask oxygenation and was eventually intubated in the emergency room. The white blood cell (WBC) count was 9200 cells/mcL . Computed tomography (CT) identified patchy infiltrates along the bronchi in her right lung, with consolidation and ground-glass opacity in her left lung  and vancomycin (500 mg to 1 g at a time). However, both respiratory status and pneumonia were refractory to the current antibiotic regimen and her condition worsened. Specifically, the worst PaO2/FiO2 ratio was 120. The β-D-glucan value measured on hospital day 6 was elevated at 285.6 pg/mL . We started sulfamethoxazole and trimethoprim (7.5 mg/kg/day) therapy, and increased her prednisolone to 80 mg/day to treat the presumptive PJP. Polymerase chain reaction (PCR) of her sputum for PJ and VZV was positive, which confirmed a diagnosis of pneumonia due to PJ and VZV co-infection. We added intravenous acyclovir (500 mg every 12 h). At that time, we observed eschars on her left neck and external ear canal, which suggested systemic VZV infection. The patient remained comatose and her respiratory status and renal function continued to decline despite the treatment. The patient’s family opted for palliative care and she died on hospital day 19.Sputum cultures on admission and on hospital day 4 were positive for This was a case of PJ and VZV co-infection. Systemic corticosteroid use can suppress the immune system, thereby rending the patient susceptible to opportunistic infections.PJ colonizes in more than 50% of immunocompetent adults . In immuVZV causes a characteristic vesicular rash called varicella (chickenpox) or herpes zoster (shingles). VZV is occasionally associated with serious complications such as central nervous system disorders, pneumonia, arthritis, osteomyelitis, necrotizing fasciitis, and secondary bacterial infections, including sepsis . VaricelThere are a limited number of reports in the literature of co-infections of PJ with members of the Herpesviridae family, such as cytomegalovirus and human herpesvirus type 6, in immunocompromised HIV and non-HIV hosts Table  6–8]. K. K8]. KoWe might have missed opportunities to diagnose this patient with VZV or PJ co-infection. First, our patient used prednisolone for 2 months without administration of sulfamethoxazole/trimethoprim as preventative treatment for PJP. This rendered the patient susceptible to PJP. Second, when her pneumonia was refractory to antibiotics, we could have considered other pathogens, including PJ and VZV. Because she had herpes zoster on admission, systemic VZV infection could have been considered. In HIV carriers, CD4+ cell counts are usually measured to estimate the risk of opportunistic infections. Herpesviridae pneumonia can develop later than PJP in the course of acquired immunodeficiency syndrome . In contClinicians should suspect PJP when patients on systemic corticosteroids develop pneumonia and they have not received prophylactic treatment for PJP in non-HIV carriers. When such patients have a VZV rash, clinicians should aggressively seek signs of opportunistic infections. Our case hereby highlights the importance of recognizing the possibility of a VZV and PJ co-infection.


Henbane mosaic virus , which was first discovered at the beginning of 20th century in the United Kingdom in cultivated henbane. A field tomato plant with severe necrotic symptoms of unknown etiology was sampled in Slovenia and high-throughput sequencing analysis using small RNA and ribosomal RNA depleted total RNA approaches revealed a mixed infection with Potato virus M , Southern tomato virus  and henbane mosaic virus in the sample. The complete genomic sequence of henbane mosaic virus was assembled from the sequencing reads. By re-inoculation of the infected material on selected test plants, henbane mosaic virus was isolated and a host range analysis was performed, demonstrating the virus was pathogenic on several plant species. Due to limited metadata in public repositories, the taxonomic identification of the virus isolate was initially putative. Thus, in the next step, we used small RNA sequencing to determine genomic sequences of four historic isolates of the virus, obtained from different virus collections. Phylogenetic analyses performed using this new sequence information enabled us to taxonomically position Henbane mosaic virus as a member of the Potyvirus genus within the chili veinal mottle virus phylogenetic cluster and define the relationship of the new tomato isolate with the historic ones, indicating the existence of at least four putative strains of the virus. The first detection of henbane mosaic virus in tomato and demonstration of its pathogenicity on this host is important for plant protection and commercial tomato production. Since the virus was initially present in a mixed infection, and its whole genome was not sequenced, it has probably been overlooked in routine diagnostics. This study confirms the applicability of a combination of high-throughput sequencing and classic plant virus characterization methods for identification and phylogenetic classification of obscure viruses and historical viral isolates, for which no or limited genome sequence data is available.High-throughput sequencing has dramatically broadened the possibilities for plant virus research and diagnostics, enabling discovery of new or obscure viruses, and virus strains and rapid sequencing of their genomes. In this research, we employed high-throughput sequencing to discover a new virus infecting tomato,  Potyvirus, family Potyviridae), which had a poorly characterized genome, despite being the subject of several biological studies in the past.The immense sequence data generating potential of high-throughput sequencing (HTS) has enabled an accelerated discovery of new virus species in recent years  and incrHyoscyamus niger) in 1932 in the United Kingdom    and lateStation) . Howeverlladonna  and in Dramonium . HMV wasas HMV-A . The viras HMV-A  in D. stSolanum lycopersicum L.) is one of the most widely grown vegetable crops worldwide  and Southern tomato virus . Prior to this study only a short fragment of the HMV genome sequence was available in databases with very limited metadata, thus, the initial species identification was only putative. To address this gap, we have obtained four other historic isolates of the virus from two different virus collections and used small RNA high-throughput sequencing to obtain complete genome sequences. Besides the first detection of the HMV in an important vegetable crop (tomato), we also report the first detection of the virus in Slovenia and the first complete genome sequence. Moreover, we demonstrate the utility of HTS for the rapid characterization of known plant virus species with no or little sequence information. We discuss implications of the approach for other viral species and discuss the power of the method for the revision of historical virus isolates and viral collections in general.In all the previous reported cases HMV was found in different solanaceous plants, which were not crop or vegetable plants, important from the agricultural perspective. However, in this study, we report for the first time natural HMV infection in field-grown tomato, found in Slovenia in 2015 (Ankaran) showing severe necrotic symptoms. Tomato (orldwide . Tomato orldwide  which caorldwide . HMV wasS. lycopersicum L.) sample (with laboratory diagnostic identification number D159/15) with necrotic disease symptoms was collected in Slovenia (Ankaran) in the summer of 2015 and stored at -80°C for further analyses. The sample contained a mixed infection with HMV, PVM, and STV. The HMV virus isolate found in this sample will be designated throughout the manuscript as isolate HMV-SI/L representing henbane mosaic virus (HMV) from Slovenia (SI) from S. lycopersicum (L).A tomato  and will be from this point on labeled as HMV-PV-76 and HMV-PV-79, respectively. The original host of isolates HMV-PV-76 and HMV-PV-79 is H. niger (henbane), and their origin is United States  and England, respectively. Detailed information for the isolates is shown in Supplementary Table Four other isolates were sourced from virus collections as lyophilized leaf material and stored at -20°C for further analyses. Isolates HMV-146 and HMV-R were obtained from the Institute for Sustainable Plant Protection (IPSP), Italy. The original host of isolate HMV-146 is Orthotospovirus, family Tospoviridae), tomato spotted wilt virus , potato virus S  and potato virus M . ELISA was performed using kits containing virus specific antibodies as follows: INSV , TSWV , PVS , and PVM . Additionally, selected test plants  were mechanically inoculated with a (1:10) dilution of the original sample in phosphate buffer (0.02 M with 2% PVP) and applied to the first two to three completely expanded leaves dusted with carborundum. Test plants were visually inspected for symptoms and tested by electron microscopy and ELISA for PVM 4 weeks after mechanical inoculation. The original plant sample and symptomatic test plants were also tested for STV using RT-PCR  in the testing and confirmation steps, RNA was isolated from leaf samples using RNeasy Plant Mini Kit  following the manufacturer’s protocol, with some minor modifications as follows. RLT buffer without β-mercaptoethanol was added to plant material and RNA was eluted from the RNeasy Mini Spin columns using 50 μl of RNase-free warm water (65°C). All of the samples were stored at -80°C between sampling and extraction. Isolated RNA was stored at -80°C, when not in use.Ribosomal RNA depleted total RNA sequencing was performed for the sample HMV-SI/L. The RNeasy Plant Mini Kit  was again used for RNA isolation, including the optional DNase treatment . Ribosomal RNA was then depleted from the total RNA and libraries for sequencing were prepared using the ScriptSeqTM Complete Kit (plant leaf) . The libraries were sequenced using MiSeq  in 2 × 300 bp (V3) mode.Small (s)RNA sequencing was performed for samples HMV-SI/L; HMV-146; HMV-R; HMV-PV-76; HMV-PV-79. In this case, total RNA was isolated using TRIzol reagent  following the manufacturer’s protocols. Total RNA from the four samples was sent to Seqmatic LLC (United States) for sRNA library preparation and sequencing using a HiSeq 2000  in 1 × 50 bp mode.In the first stage, HTS of sample HMV-SI/L was performed for both, rRNA depleted total RNA and sRNA. Two different detection pipelines were used to detect viral sequences in the HTS data for both approaches as previously described . In bothFor samples HMV-146, HMV-R, HMV-PV-76 and HMV-PV-79, sRNA sequencing was performed and the resulting datasets were analyzed to identify viral sequences using the pipeline described previously . CompletFurthermore, in order to validate the assemblies within the repeated region (TATATA) around position 9980 nt, we designed universal PCR primers for all HMV isolates: HMV-UNI-F: 5′-TTAGCCCGATATGCTTTC-3′ and HMV-UNI-R: 5′-CTATCTTCCACTTCAGGT-3′. The RT-PCR reaction was performed using OneStep RT-PCR Kit . The reaction conditions were 50°C for 30 min, 95°C for 15 min, followed by 35 cycles of 94°C for 30 s, 48°C for 60 s, and 72°C for 60 s. The amplicons obtained by RT-PCR were purified using MinElute PCR Purification Kit  prior to sequencing . The sequences obtained were aligned against the HMV genomes and the repeated region was validated.1 and the individual putative cleavage sites for each gene product were manually determined based on known cleavage sites for other potyviruses reported in the literature  out of the six primer pairs designed for genome assembly was used. Negative controls for RNA isolation and no template controls were also tested. RT-PCR was done as described above using the OneStep RT-PCR Kit  in a 10 μl reaction volume.AM184113). The results were visualized as heatmaps. In addition, nucleotide identities of the HMV-SI/L polyprotein sequence were compared with four other isolates  and visualized using SimPlot 3.5.1 . The comparison was also performed using partial genome sequences (1600 nt) to enable comparison with the published sequence of isolate HMV PHYS/H-Hungary  resources3, including 130 viral species. Sequences of the five HMV isolates sequenced in this study were added to the alignment. Additionally, we performed blastn searches using HMV-SI/L against the NCBI nt database and added also two viral species with sequenced complete genomes (KY623506 and MF997470) with relatively high similarity to HMV (detected within first 100 blastn hits), which were not present in the initial ICTV alignment. All sequences were codon aligned applying MUSCLE in MEGA7  and with relatively high similarity to HMV (detected within first 100 blastn hits), which was not present in the initial ICTV alignment. Several Rymovirus species  were used as an outgroup. As described above, the partial genome sequences were codon aligned applying MUSCLE in MEGA7 and phylogenetic tree was constructed in MEGA7 on the basis of this alignment using maximum likelihood algorithm with GTR + G + I substitution model. Bootstrap replication (1000 pseudoreplicates) were used to assess the statistical support of the groups within the tree.We aligned also partial genome sequences (∼1600 nt) of the six HMV isolates  and other N. clevelandii plant material, to which PVM and HMV were successfully transferred, was then used to inoculate several test plants of S. lycopersicum cv. Moneymaker. The leaf material of S. lycopersicum with a single infection of HMV (confirmed by ELISA and RT-PCR) was collected and further used as an inoculum for the host range analysis.HMV was detected in the field-grown tomato sample in a mixed infection with two other viruses. The isolation of HMV from the mixed infection was achieved by several re-inoculation steps. A tomato sample, positive for HMV, PVM, and STV, was used for inoculation of several test plants (see section “First Test on Sample HMV-SI/L”). The A total of 20 different plant species/varieties from several botanical families were selected Table  and inclN. benthamiana and S. lycopersicum cv. Moneymaker using the same method as described in section “First Test on Sample HMV-SI/L.”Out of four isolates obtained from the virus collections, HMV-PV-76 was the only isolate present in a single infection. Thus, additionally we also checked the pathogenicity of the isolate HMV-PV-76 and compared it to the HMV-SI/L isolate by inoculating S. lycopersicum and N. benthamiana  and leaves from a Figure  infectedS. lycopersicum L.) with severe necrotic symptoms  did not show any symptoms, the ELISA assay was negative for PVM and no virus particles were observed in those plant samples. Four of the test plants  were PVM positive by ELISA . Since the disease symptoms observed on the original sample  and HMV-PV-79 contained Potato virus Y .Before this study only a partial genome sequence of a single isolate of HMV was present in the public database, with no affiliated publication and little metadata (NCBI GenBank Acc. No. AM184113). The overlapping part of HMV-SI/L isolate sequenced in this study was 88% identical to this sequence, leading to a putative identification of the virus as HMV. To validate this putative identification, we obtained four other isolates designated as HMV from virus collections  and performed sRNA sequencing to obtain their complete genome sequences. Using the sRNA virus detection pipeline  we detecPotyvirus with a single open reading frame (ORF) encoding a polyprotein of 3248–3249 amino acids and an estimated molecular weight of 367 kDa. The ORF encoded all typical potyviral protein products, with an unusually long P1 protein (476 amino acid residues)  Figure . Additio) Figure  and was ) Figure  and seve) Figure . All HMVPotyviridae family , Gloriosa stripe mosaic virus (EF427894), Yam mild mosaic virus (JX470965), Tobacco vein banding mosaic virus (EF219408), Chili ringspot virus (JN008909), Wild tomato mosaic virus (DQ851495), Chili veinal mottle virus (AJ237843), African eggplant mosaic virus (MF997470), and Pepper veinal mottle virus (DQ645484). Phylogenetic analysis based on the alignment of the smaller subset of partial genome sequences  clustered together with high bootstrap support. HMV-146 and HMV-SI/L were significantly different from the HMV-R, HMV-PV-76, HMV-PV-79 group, but also from each other, thus they represented separate groups on the tree Figure . HMV PHYnr) and blastn (against NCBI nt) similarity searches did not show significant similarity  of HMV P1 product to any known sequence.SimPlot analysis Figure  demonstrH. niger, S. lycopersicum  of the eight inoculated plants expressed disease symptoms 3 weeks after mechanical inoculation. Smaller number of D. stramonium (4/8) and P. floridana (2/8) plants expressed disease symptoms. Most severe disease symptoms were observed in the following two cases: in D. stramonium, where 4 of 8 plants died 6 weeks post inoculation and in P. floridana, where plants showed very strong dwarf growth. In Solanum melongena, the HMV did not move from the inoculated leaves and local lesions developed  than plants infected with HMV-SI/L, and died 4 weeks after inoculation. Similarly, in the case of S. lycopersicum cv. Moneymaker, the symptoms induced by HMV-PV-76 were more severe , in comparison to HMV-SI/L  in High-throughput sequencing has in the recent decade facilitated discovery of many new viruses and viral strains and largely improved the possibilities for broad range virus detection and screening. The increased rate of virus discovery, also brings new challenges, especially considering the biological characterization of the newly detected viral sequences. The latter is crucial to establish their importance, i.e., their impact on agriculture and trade  or theirHowever, for henbane mosaic virus described here, the perspective was reversed; very limited genomic information was available for a virus first described in 1932  and subsPotyvirus genus. Interestingly, HMV is placed within the chili veinal mottle virus cluster and is not closely related to any of the virus species , for which a distant serological relationship with HMV has been previously speculated . Similar results were obtained by S. melongena. On the other hand, HMV-R showed no disease symptoms on S. melongena  is the largest genus of plant viruses, including 168 virus species, which cause diseases in a wide range of plant species. Among them, according to the latest ICTV report, 114 have complete genome sequences available, for 48 there are just partial genomic sequences available, and for 6 of them there is no sequence data in public repositories (The study of HMV presented in this paper demonstrated the benefit of using HTS for characterization of known viral species with limited or absent genomic information. The sitories ; the latsitories . As a reAP, MR, DK, and NM designed the experiments. PP discovered new type of disease symptoms on tomato, started further investigation, and provided the field tomato sample infected with HMV-SI/L. NM performed first screening analysis on the field tomato sample. AP performed laboratory part of the experiments and analyzed the data with the assistance of IA and DK. MŽ recorded TEM photos. AP wrote the draft of the manuscript. All authors significantly contributed with reviewing and editing the manuscript.IA was employed by company Fera Science Ltd., Sand Hutton, York, United Kingdom and PP was employed by company KZ Agraria Koper z.o.o., Koper, Slovenia. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dasheen mosaic virus, Taro bacilliform virus, Colocasia bobone disease virus, and Taro vein chlorosis virus) and a relatively new one (Taro bacilliform CH virus), this paper explores the details of each virus by examining their characteristics and highlighting information that could be used to mitigate taro infections and disease management.Taro is an important crop in parts of the world, especially in the Pacific Islands. Like all plants, it is also susceptible to virus infections that could result in diseases, which negatively affects the source of food and trade revenue. Understanding the biology of taro viruses could improve current knowledge regarding the relationship between viruses and taro, thus allowing for a better approach towards the management of the diseases that are associated with them. By compiling and discussing the research on taro and its four major viruses ( Colocasia esculenta (L.) Schott) is a perennial root crop from the family Araceae (common name: Aroids) with substantial socio-economic importance in tropical and sub-tropical regions, especially Southeast Asia and the Pacific Island countries .,34.33,34Taro plants that were infected with DsMV have the typical feather mottle symptoms with conspicuous mosaic patterns—yellow, whitish patterns against the green; pale green against dark green—and sometimes malformation and dwarfing of the leaves  6,31]. ,31. 6,31Taro bacilliform virus is an aroid-specific pararetrovirus from the family Caulimoviridae and it has been classified in the genus Badnavirus. This classification is based on the examination of bacilliform-shaped virions that were found in the infected plants and the mealybug transmission studies . The. The4]. Taro bacilliform CH Virus (TaBCHV). The first complete nucleotide sequence was recorded in China, before others in the United States in America (USA) and East Africa -based assay being the most used in studies, which is potentially due to it being more efficient ,53,54. IDetection assays are essential in the screening of taro germplasms, ensuring that they are virus free and suitable for movement across the border ,5,54. A successful viral infection usually occurs due to the viral genome containing ORFs that could encode the proteins that are necessary for host infections . The genProteins that are encoded by the viral genome could be directly responsible for host infections. As such, understanding the functions of ORFs and the proteins could illuminate the molecular interaction of taro and the viruses. However, potentially due to its complexity, studies regarding this interaction remains elusive. Previous work speculates that the genome of TaBV could integrate into the taro genome ,46,59. AAs mentioned before, Alomae and Bobone are the major diseases infecting taro ,28. It hDue to its nature, Alomae is considered to be the most lethal taro disease that significantly affects the crop ,29. A stIf left uncontrolled, the taro diseases could potentially be widely distributed and the outcomes would be devastating to the taro crop industry. Hence, a certain degree of disease management and control is essential in maintaining a considerable amount of taro safe for consumption. One of the most important approaches is to ensure the better supervision of the movement of germplasms by authorities (and researchers) across the border, especially with regards to symptoms identification and diagnostic assays . As suchA relatively common method of management is roguing, which is the periodic physical removal of infected plant parts. It is an established viral disease control strategy and used in various plant species to protect against respective viruses, although it can be quite tedious ,68. At tApart from that, there is an approach to introduce the intercropping of non-host plants among taro cultivars . This meCyrtorhinus fulvus, could effectively control the Tarophagus population, although no research has been done to further explore this method in the last decade [Another approach is to use biological control of the vector population. Using predators to control the vectors spreading viruses on taro showed potential, considering the population of said vectors is still relatively small . Few stut decade ,69.Observation from another study found the number of plants with Alomae symptoms was reduced in the colder months when compared to warmer ones, highlighting a possible factor of the timing and temperature of the soil and surroundings, although this observation could be simply due to the virus inactivity rather than its complete removal . HoweverThis review has outlined the characteristics of viruses infecting taro plants. It is essential for the taro planting materials to be virus-free, which is best achieved through the development of suitable diagnostic assays and the careful screening of such materials across the border. Although traditional methods of cultivation may prove to be a challenge in the management of taro viruses, continuous exposure to current information regarding taro could help local farmers, especially the next generation of modern farmers, to better fortify their crops against diseases. The bridge that was formed between current research, farmers, and local authorities could improve overall the understanding of taro viruses and diseases that are associated with them, thus providing an insight regarding the best way to mediate the problem. This would not only be beneficial to the local consumers, but could help to sustain the crop-based economy of taro growing regions.


Cydia pomonella granulovirus isolates, the Mexican isolate CpGV-M and the CpGV-R5, in ratios of 99% M + 1% R, 95% M + 5% R, 90% M + 10% R, 50% M + 50% R, and 10% M + 90% R. CpGV-M and CpGV-R5 differ in their ability to replicate in codling moth larvae carrying the type I resistance. This ability is associated with a genetic marker located in the virus pe38 gene. Six successive cycles of replication were carried out with each virus population on a fully-permissive codling moth colony (CpNPP), as well as on a host colony (RGV) that carries the type I resistance, and thus blocks CpGV-M replication. The infectivity of offspring viruses was tested on both hosts. Replication on the CpNPP leads to virus lineages preserving the pe38 markers characteristic of both isolates, while replication on the RGV colony drastically reduces the frequency of the CpGV-M pe38 marker. Virus progeny obtained after replication on CpNPP show consistently higher pathogenicity than that of progeny viruses obtained by replication on RGV, independently of the host used for testing.To test the importance of the host genotype in maintaining virus genetic diversity, five experimental populations were constructed by mixing two  Codling moth, the main insect pest for apple and pear production , is wideBaculoviruses are authorized as biological control agents in field conditions because they are specific to one or few insect species and harmless for beneficial insects .Cydia pomonella granulovirus (CpGV) isolate originates from Mexico (CpGV-M) [The first (CpGV-M) . This is(CpGV-M) . In Euro(CpGV-M) .Betabaculovirus”, one of the four genera of the Baculoviridae family. In their life cycle, baculoviruses alternate between two types of particles, the budded virus (BV), a virus particle where a nucleocapsid that contains the genome acquires a membrane of cellular origin when budding out of the cell, and an occluded form, where virus particles are protected within a proteinaceous paracrystalline structure, the occlusion body (OB). BVs are responsible for within-host cell infections, while OBs are responsible for infection from one host to another, via the external environment. In the alpha-, gamma-, and delta-baculoviruses, usually called nucleopolyhedroviruses (NPV), OBs can contain many independent virions, whose genomes might not be identical [Since 2004, resistance to CpGV-M has been reported in orchards, first in Germany  and Frandentical . In the dentical . These OAutographa californica multiple nucleopolyhedrovirus, AcMNPV) are characterized by an important genetic diversity, both within a single isolate or between isolates. Analysis by restriction fragment length polymorphism (RFLP) allowed the characterization of nine genotypes from a population of Spodoptera frugiperda multiple nucleopolyhedrovirus SfMNPV [Panolis flammea larval cadaver [Alpha-baculoviruses  found in the region of Saint-Andiol , followed by selection for resistance to CpGV-M as previously described [GV colony harbored the sex-linked resistance now called type I resistance [Two escribed . Brieflysistance . Both cosistance .50 = 13.10 OB/µL (6.55–23.20)), but not on RGV ((LC50 = 2.22 × 106 OB/µL (1.19 × 106–5.67 × 106). The CpGV-R5 isolate is able to overcome the resistance of the RGV colony (LC50 = 22.43 OB/µL (13.73–34.36)) and also replicates on CpNPP (LC50 = 6.76 OB/µL (2.6–13.37)) [CpGV-M (laboratory stock 2020-s1) and CpGV-R5 (laboratory stock 2016-r16), the two virus isolates used in this work, have been described in previous studies ,31. The –13.37)) .Five experimental virus populations were constructed by mixing in various proportions OBs of the two isolates. The proportions of each isolate in the mixed virus populations were 99% CpGV-M + 1% CpGV-R5, 95% CpGV-M + 5% CpGV-R5, 90% CpGV-M + 10% CpGV-R5, 50% CpGV-M + 50% CpGV-R5, and 10% CpGV-M + 90% CpGV-R5. Pure CpGV-M and CpGV-R5 were used as reference virus populations. These OBs mixtures are referred to as Passage 0 OBs (P0) .GV) insects   or resistant (R number) .GV) larvae were individually deposited in 24-well plates. Each well contained 1 mL formaldehyde-free diet  inoculated with 50 µL of each P0 viral suspension at 800 OBs/µL per well, that is a concentration of 40 OBs/µL of diet. At this concentration, mortality higher than 90% was expected. Plates were then incubated at 25 °C (±1 °C) with a 16:8-h (light/dark) photoperiod and a relative humidity of 60% (±10%). Four days later, all larvae presenting signs of viral infection were extracted from the rearing diet, stored at 25 °C for one more day, then frozen (−20 °C). OBs were then extracted and purified as previously described [Forty-eight 3rd–4th instar (7 days old) susceptible (CpNPP) or resistant  in TBE buffer and visualized on a UV transilluminator after ethidium bromide staining. No attempt at quantification of the relative frequencies of each genotype was done.GV on OBs at passages P1, P3, and P6. The bioassays of the original mixes (P0) both on susceptible and resistant larvae have been previously published [Bioassays of the 10 virus lineages (5 different frequencies × 2 hosts) have been performed both on CpNPP and Rublished . They arCpNPP, there was a reduction of the pathogenicity in all virus populations for CpNPP larvae. From P1CpNPP–P6CpNPP, the general trend was a progressive increase of the pathogenicity in the five virus lineages, roughly reaching the efficiency of the pure genotype virus populations, CpGV-M or CpGV-R5, on this permissive host . In addition, the efficacy on both hosts was not correlated with the original frequency of the parental virus genotypes: M95-R05 P6RGV showed the lowest LC50 on both RGV and CpNPP, while M99-R01 P6RGV had the highest LC50 on CpNPP, but not on RGV.The results of the bioassays performed with the P1pe38 gene. The analysis of virus lineages after six passages on the susceptible (P6CpNPP) and the resistant host (P6RGV) revealed the retaining of genetic diversity in all populations of P6CpNPP, but such diversity was clearly reduced or absent in P6RGV  often implies an evolutionary tradeoff. In the presence of the selection factor, resistant individuals have a selective advantage compared to susceptible ones. Conversely, in the absence of the selection factor, susceptible individuals are favored due to their higher fitness. Under this hypothesis, when both genotypes are present in a population, a progressive increase of susceptible genotypes should be observed in the absence of the selection trait . Two appUndorf-Spahn et al.  studied 50s . The two virus isolates replicated in the permissive host (CpNPP) at approximately the same level, in terms of OB production, and showed a slight difference in their LC76 OB/µL ). To comBy mixing OBs from the two virus isolates (P0), experimental virus populations were constructed covering all the range of proportions for each virus isolate. These experimental virus populations were then allowed to evolve on the two hosts for six successive passages, constituting 10 different lineages, five on each host. Their pathogenicity was evaluated on both hosts at P0, P1, P3, and P6.4 OBs. However, the probability of successful infection with a second virus might be related to the delay between the first infection and the second one, as has been shown for SfMNPV [As CpGV OBs contain one genome , we have chosen conditions in which each larvae would eat more than one OB, thus, increasing the probability of the presence of multiple genotypes in the larvae. In the four-day infection, period a L3 larva ate approximately 500 µL of diet; at 40 OB/µL, that means in the range of 10r SfMNPV .For a given number of OBs ingested, the probability of multiple genotype infection depends on the relative frequency of the genotypes. It is maximal for the even distribution of the genotypes (50% of each in our experiments with only two genotypes) and decreases when increasing the differences in the relative frequencies. Accordingly, in the experimental virus population containing 99% CpGV-M + 1% CpGV-R5, the probability of having larvae infected by only one genotype will be higher than for the 50% CpGV-M + 50% CpGV-R5 population, for the same number of OBs ingested.GV host, the presence of CpGV-M genomes in the offspring will rely on the replication of at least one genome of CpGV-R5, as the LC50 for pure CpGV-M is far above the dose use for the test. It would be expected that if both viruses were almost equally fit in the permissive host, the virus lineages would maintain their genotypic diversity, while lineages replicating in the non-permissive host would eliminate the CpGV-M genotype.We hypothesized that the permissive CpNPP host would not influence the outcome of viral infections, while in the resistant RGV when in co-infection with CpGV-R5; thus, P1RGV contained CpGV-M genomes.We have previously demonstrated  that theCpNPP and P1CpNPP . This decrease was statistically significant for all but the M99 + 1R P1CpNPP, and this can be explained by the higher frequency of infections with only one genotype under this condition.Surprisingly, there was an overall reduction in the efficacy against CpNPP between P0x could be the consequence of diversity generation via recombination, leading to some less adapted gene combinations that would be counter selected in the following passages. Recombination of NPV has been demonstrated in cell cultures [pe38 gene. Further work will be required to explore this point.In plant or animal selection, it is common to observe an increase in the fitness of hybrids and a decrease when reproducing these hybrids on successive generations. The results we obtained resemble this effect, if we consider the whole inoculum ingested as the selection unit. The reduction of the efficacy of the control at P1cultures  and in vCpNPP, the LC50s of all lineages recovered to the level of the parent viruses on CpNPP. When tested on the RGV, the host in which these lineages did not replicate previously, there was no such reduction in efficacy, but a continuous improvement. The efficacy of P6CpNPP lineages approached the level of CpGV-R5 on this host (LC50 for CpGV-R5 was 22.43 (13.73–34.36); LC50s for P6CpNPP ranged between 28.24 and 40.77). This trend was independent of the original frequency of the parent genotypes. The hypothesis of progressive replacement of one genotype by the second in the experimental populations could explain the observed behavior. Over passages, CpGV-M would be expected to reduce progressively and disappear. PCR analysis of the virus lineages revealed that virus lineages replicating in a fully-permissive host retained the markers of both genotypes, while improving their ability to control the resistant insects; conversely, in virus lineages submitted to strong selection, the CpGV-M-specific marker was absent or highly reduced, and the efficacy was not as good , the LC50s did not reach the level of the P0RGV virus populations, neither on CpNPP nor on RGV, after six passages. Increasing the number of passages and reducing the multiplicity of infection would eventually lead to a highly-pathogenic isolate similar to CpGV-R5, following a process similar to that used to select this isolate.It would also be expected that the PiAccording to our results, allowing the virus isolates to adapt to the specific host populations resulted in similar levels of control, while preserving the virus genetic diversity.In CM populations in orchards, there was a variable proportion of resistant individuals, mixed with susceptible individuals. This was reflected by a two-plateau dose mortality line when challenged with a virus.Using a single virus genotype against host populations with variable genetic background can result in failures of the control due to the variable susceptibility of the host to that virus genotype. Mixed virus populations will be able to colonize each individual of the host population. Providing genetic diversity is supposed to facilitate adaptation of populations to a new environment. For a virus, this new environment can be a host harboring a new resistance trait or the presence in the host of an inhibition molecule.12 and 1013 OB/Ha [13 OB/Ha and calculated 2404 ± 1608 OBs/mm2 of leaves or fruit. Accordingly, the estimate concentration obtained with commercial products would be at least 271 OB/mm2 in the orchards. Taking into account the observations on the size of the feeding holes excavated by neonate larvae [In apple orchards, the success of control of the codling moth with CpGV requires massive release of the virus. The usual dose in the field is between 3 × 1013 OB/Ha ,40. Glen13 OB/Ha  applied e larvae  and assuA recent analysis of the commercial virus preparations developed to fight field resistance showed that they were genotypically diverse, contrary to the products commercialized before 2005, when pure CpGV-M was the active ingredient. The two products analyzed contained at least three genotypes each , among t


The aim of the study was to evaluate costs associated with colonic endoscopic submucosal dissection (ESD) for treatment of colorectal cancer. The study is a retrospective analysis of data on 395 patients treated by colonic ESD. The operation, consumable items, and medication accounted for 71% of the total costs for colonic ESD treatment. Medication and consumable items' costs were higher if lesions occurred in the transverse colon and right hemicolon compared to the left hemicolon. Medication, consumable items, and total costs were higher for larger lesions. Lesion numbers and carcinoma were associated with higher medication, consumable items, operation, and total costs. Positive surgical margins and complications of hemorrhage or perforation were positively correlated with higher costs for medication, consumable items, and total costs. Labor costs for doctors and nurses remain low in China. Costs for medication and consumable items were higher for treatment involving the transverse colon or right hemicolon (vs. the left hemicolon), larger lesions, carcinoma, and a positive surgical margin. A benchmark cost estimate for ESD treatment including 4 days of postoperative hospitalization was determined to be approximately 5400 USD. Based ul cancer , 6. At tThe study is based on retrospective analysis of data from 395 patients undergoing colonic ESD between January 2015 and December 2017 at the Department of Gastroenterology, Renmin Hospital of Wuhan University, China.Tumor size  was included. Pathologies of the neoplasm were summarized as polyp , adenoma, carcinoid, and carcinoma. Moreover, macroscopic morphology of the lesions  was presented as well . The operators consisted of junior operators and senior professors. On one hand, there were three well-trained junior doctors (trained for more than three years and operated upon more than one hundred pig stomachs and colons) who participated in the beginning steps for 72 cases that were supervised by senior professors. On the other hand, four senior professors who practiced colonic ESD for more than 500 cases and more than ten years were enrolled. The indications strictly followed the colonic ESD guidelines including polys/LST bigger than 2 cm, a submucosal tumor, carcinoid or rectal carcinoid smaller than 2 cm, and early carcinoma. The operations (techniques and procedures) were conducted according to colonic ESD guidelines, such as lesion observation, marking, submucosal injection, incision, dissection, and wound management. Complications  were involved as well.The analyzed colonic ESD patient medical costs spanned hospital admission to discharge. Medical costs were categorized as direct, indirect, and intangible. Direct costs were related to treatment and patient care including admission, medical procedures, consumable items, medications, and nursing. Indirect costs were those ascribed to disease-related morbidity and mortality. Intangible costs encompassed nonfinancial outcomes such as pain and mental suffering. Direct medical charges were obtained from hospitalized patient charge lists . t-test was used for comparison between groups. P<0.05 was considered statistically significant.Statistical analysis was performed using SPSS version 17.0 . Numerical data are presented as mean ± SD. Student'sWe divided costs into several categories: hospitalization, laboratory, imaging, nursing, medication, anesthesia, consumable items, and operation. The hospital financial system precluded more specific cost stratification in this study. Total costs were divided into those covered by National Health Insurance (NHI) and those paid by the patients themselves. Operation, medication, and consumable items' costs accounted for 71% of the total costs for colonic ESD .We examined relationships between cost and lesion location. Medication and consumable items' costs were higher for lesions in the transverse colon and right hemicolon (>2300 USD) than for lesions in the left hemicolon (<1900 USD). Total costs showed the same trend, with costs exceeding 6700 USD for lesions in the transverse colon and right hemicolon but less than 5800 USD for lesions in the left hemicolon. No major differences in operation costs were observed for lesions in different locations .P>0.05). However, abdominal pain occurred significantly more frequently following procedures for lesions of the ascending and transverse colon than for other locations (P<0.05)  .P<0.05). Operation costs, however, did not significantly differ according to lesion size (P> 0.05). The presence of multiple lesions (>1) was significantly correlated with higher costs for medication, consumable items, and operation and total costs compared to patients with a single lesion (P< 0.05). Carcinoma was associated with significantly (P< 0.05) higher costs for medication, consumable items, and operation and total costs compared to other pathologies . With respect to complications, hemorrhage and perforation were associated with significantly higher medication, consumable items, and total costs compared to complications of infection and abdominal pain . Hospitalization of >10 d was correlated with significantly higher operation costs , perforation , infection , and abdominal pain . The incidence of complications was significantly higher within 4 d after the procedure (87.7%) than at 4 d (12.3%) . We examon costs .Lastly, in order to clarify the relationship between costs and outcomes, the costs have been analyzed for defined outcomes of colorectal ESD. More detailedly, the costs of uncomplicated curative ESD of cancer (and HGIN), uncomplicated noncurative ESD of cancer, and complicated ESD of cancer (and of other benign lesions) were analyzed, respectively .Minimally invasive ESD is a promising technique for treatment of digestive tract diseases, especially polyps, adenoma, and early stage carcinoma . Most weNearly 90% of complications occurred within 4 d after the procedure. With respect to lesion location, abdominal pain occurred more often when lesions were located in the ascending and transverse colon. This might be due to poor gas release from high positions of the colon, with the pneumogastric nerve only exiting above the splenic flexure , 12. Whe


Generally, viral pathogens remain under-diagnosed under the limited health services, lack of awareness and poorly coordinated health efforts, especially in the developing and under-developed countries. The research advances made during the last two decades are paving novel and effective ways to tackle such infectious agents in a better way. Additionally, enhanced disease surveillance and monitoring, rapid diagnostics, inventing effective prophylactics, and adapting appropriate public health measures would certainly help in combating the emerging viral threats.The word “Emerging Diseases” encompasses the potential threat posed by infectious pathogens to humans and animals. In the recent times, several epidemics and few pandemics of viral origin have haunted the global population. Majorly, the transboundary and zoonotic pathogens are of concerns to the public health. Most recently, Zika virus has been found with confounding situation in Asian countries. Correspondingly, other infectious viruses like Ebola virus, SARS, MERS, Dengue, Chicken Guinea, West Nile and Japanese encephalitis have created panic owing to their severe life-threatening consequences, affecting larger population globally with a high toll of human deaths, gaining potential of rapid spread across borders of many countries, and creating economic impacts and high sufferings to the mankind. Of note, nearly two-thirds of these viral pathogens have been of zoonotic nature having significant public health concerns. Predisposing factors such as deforestation, urbanization, ecotourism, fast population movement, changing climatic issues , and ecological alterations with the evolution of newer viral pathogens /strains and jumping the host species barriers, i.e., to maintain epidemiological study on humans, animals, virus strains from various sources including environment, which is relevant to “One Health” approach advocated recently. In the recent years, Chandipura Virus (CHPV) has emerged as an encephalitic pathogen and found associated with a number of outbreaks in different parts of India. The article by Sapkal et al. narrate the overview on CHPV focusing on prevalence and seasonal activity of this virus in India through outbreak investigations, serosurvey, and diagnosis of the referred clinical specimens.In this special issue on “Emerging and Zoonotic Virus Challenges of Developing Nations”, a review article by N. Kobayashi from Japan has highlighted various factors which may synergistically increase risks for the emergence of pathogens, transmission of pathogens, and opportunity of infections. Added to this, he pointed out that the spread of emerging viruses might be indirectly related to socioeconomic problems including civil wars, increase of refugees, and natural disasters. These human factors always compromise the human health and increase the risk for emerging infectious diseases. Despite such situations, scientists have the significant role to reduce the risks for the emerging viral diseases. This article emphasizes the first priority to ensure adequate surveillance of viral diseases, et al. highlights the adaptation of Newcastle Disease Virus (NDV) in feral birds and their potential role in interspecies transmission. The review details the pertinent features of several historical and contemporary aspects of NDV and the vital role of feral birds in its molecular epidemiology and ecology. Reports are very less on the existence of Bovine Viral Diarrhea Virus (BVDV) in pigs. The North Eastern (NE) part of India is having the maximum pig population in India and the article by Chakraborty et al. emphasized on the BVDV in pigs from this region of India with genetic profiling of virus strains. Several semi-captive/free-ranging wild avian species have been noted playing a vital role in the dissemination of viruses, which is an important consideration to control the disease outbreaks. In this series, Newcastle disease (ND), caused by Avian Paramyxovirus serotype 1 (APMV-1), is a notifiable disease throughout the world due to the economic impact on trading restrictions and its embargoes placed in endemic regions. The article by Rahman et al. has given a comprehensive Indian perspectives over highlighting the recent progress, focusing challenges with diagnosis and prophylactic interventions. Lately, Antimicrobial Resistance (AMR) or Multidrug Resistance (MDR) has become a serious health issue, globally. Even resitance in virus has been noted. The review article on “The Quest for Materials-based Hydrogels with Antimicrobial and Antiviral Potentialities” by Iqbal highlights the current concepts in this area. The review has pin-pointed various methodological approaches including biomaterials-based therapeutic hydrogels aiming to resolve these issues. The information is also given on the potential research activities, and possible mechanisms of actions of hydrogels are discussed with a closeup look at the future recommendations. In recent years, the two porcine viral diseases have gained the attraction of researchers due to the losses associated with them and influence of concurrent infections. In this area, the research group of Mukherjee et al. investigated the expression of various cytokines including IL2, INF_G, IL-10, TNF_Beta, IL12 and TNF Alpha by quantitative reverse transcription-polymerase chain reaction in growing piglets based on the viral load of two main porcine viruses namely porcine circovirus 2 and classical swine fever virus to define the influence of co-infection on host immunity. Another article on “Zoonotic viral diseases of equines” by Kumar et al. provided an overview of the infectious zoonotic diseases and their impact on human and animal health. Among the emerging viral infections, a new addition is the Picobirnavirus, which has been documented in enteric and respiratory infections in several mammalian and avian host species. It is an opportunistic infection seen more commonly with immune-compromised individual with concurrent infection. The review article by Malik et al. emphasized on its relevance to Indian human and animal population. This viral infection of wide host range needs the attention of researchers globally to find out its control measures. One of the viral diseases of zoonotic concern, Japanese Encephalitis (JE) has recently been declared as a notifiable disease in India due to its expanding geographical distribution, on which, the article by Kulkarni st century. The compilation of articles included in this issue would be helpful for the public health professionals, clinicians, animal owners, epidemiologists, academicians, researchers and policymakers in formulating the strategies for adopting appropriate preventive and control measures for the prevailing and upcoming viral diseases.This thematic issue will serve as a scientific platform to share the knowledge on emerging infectious viral diseases which are showing added prominence in the 21


C. gattii, presented lesions on the nasal region and respiratory signs. Strains were typed as molecular type VGII, mating type alpha, MLST subtypes ST442 and ST185. Since Rio de Janeiro is known as an endemic area for C. neoformans VNI, these cases might be a warning for a possible emergence of C. gattii VGII in southeast Brazil.Two cats infected by  However, in the southern regions of the country, C. neoformans predominates, and infections by C. gattii VGII are rare and usually considered as imported cases [Cryptococcosis is a worldwide-distributed fungal disease of humans and animals caused by members of the omplexes . Both spomplexes . Since 1, Canada . In the ed cases .C. neoformans than dogs in studies conducted in Western Australia [Cryptococcosis is the most common systemic mycosis in cats. These animals were found to be particularly more susceptible to infections by ustralia . Feline ustralia .C. neoformans from C. gattii. We describe herein two cases of feline cryptococcosis due to C. gattii in the state of Rio de Janeiro, southeast Brazil, focusing on the differential diagnosis, treatment and clinical outcome, along with the phenotypic identification, molecular typing and in vitro antifungal susceptibility of the agents. Moreover, a role for these animals in cryptococcosis within the concept of One Health is discussed.In Brazil, very few animal cases of cryptococcosis have been documented ,8 and mo2Cryptococcus spp. were observed in Panotic-stained smears. Exudate from the same lesion and secretion from the nasal cavities obtained with a swab were seeded on to Sabouraud-dextrose agar. The physiologic characterization was performed as follows: the isolated yeasts were tested for thermotolerance at 37 °C, melanin production on Niger seed agar, and assimilation of carbon and nitrogen compounds. The species complexes C. neoformans and C. gattii were differentiated via incubation on canavanine-glycine-bromothymol blue (CGB) medium. The mating type was determined applying polymerase chain reaction using specific primers for the pheromone genes. Subtyping was performed according to the ISHAM consensus multi-locus sequence typing scheme for C. neoformans and C. gattii [http://mlst.mycologylab.org/). The results revealed the presence of C. gattii VGII, mat alfa, sequence type ST442. Antifungal susceptibility testing was performed according to the CLSI M27-A3 guideline. The drugs tested were fluconazole, voriconazole, itraconazole, flucytosine, and amphotericin B. The minimal inhibitory concentrations (MICs) were evaluated by means of the epidemiologic cutoff values (ECVs) suggested for the specific molecular types of C. gattii [−1 for itraconazole was consistent with the non-WT population  was prescribed once a day (day 0), and the cat was followed up every 30 days for clinical evaluation. After the disappearance of all the clinical signs initially presented, which occurred after six months of therapy, the cat was discharged b.C. gattii. The isolates were further characterized as VGII, mat alfa, sequence type ST185, which were identified according to the techniques described for the first case (see above). The antifungal susceptibility testing and the ECVs classification of the strain were performed as described for case 1 (see above). The MIC's (μg/ml) were as follows: 16 for fluconazole, 4.0 for flucytosine, and 0.12 for amphotericin B, values consistent with WT population, as well as 1.0 for both itraconazole and voriconazole, which were consistent with the non-WT population , weighing 5.2 kg. The cat had been previously treated at a private veterinary clinic with itraconazole  for one month, with no clinical response. At clinical examination, a soft subcutaneous swelling over the left periocular medial region, a lesion on the conjunctiva of the left eye, polyp-like mass in the nostrils and sneezing were observed a. Exudatpulation .Fig. 2CaItraconazole was replaced by fluconazole  (day 0). The cat was followed up every 30 days for clinical evaluation. The clinical cure occurred after five months of therapy, when the cat was discharged b.3C. gattii was properly identified in three cases, with one case due to the molecular type VGII. Thus, animal cryptococcosis is probably underdiagnosed and under-reported in Brazil, with the real incidence being severely biased. Herein, we report two cases of feline cryptococcosis due to C. gattii VGII in an urban area of the Southeast Brazil, largely known to be endemic for human cryptococcosis caused by C. neoformans VNI [Since the first description in Brazil in 1971, only 27 other cases of feline cryptococcosis have been reported . Of thesmans VNI .C. neoformans VNI and C. gattii VGI, while C. gattii VGII has rarely been isolated [C. gattii VGII was identified [Environmental studies in Rio de Janeiro have isolated mainly isolated . Authorsentified . Consideentified , reinforC. gattii VGII and VGIII infections in North America fail to respond to this azole, presenting persistent high antigen titers and frequent relapses. In these cases, itraconazole is a better option [C. gattii, a cat presenting a nasal granuloma achieved clinical cure after six months therapy of itraconazole/5-flucytosine combination [Cats with localized cutaneous lesions can be treated successfully with fluconazole, which is the drug of choice for feline cryptococcosis. However, many cats with r option . In prevbination  and the bination . Case 2 bination , as was C. gattii [in vitro against Sporothrix spp [Cryptococcosis and sporotrichosis infections are thought to be acquired from the environment. However, in the Rio de Janeiro sporotrichosis epizootic, feline cases are mainly related to be acquired by scratches or bites from other infected cats , whereas. gattii . This phhrix spp . These dC. gattii in urban environments is a challenge because it comes with a great potential to generate outbreaks of cryptococcosis among immunocompetent individuals and animals. Future studies on the etiological agents of cryptococcosis in domestic animals in urban environments are necessary to understand the dynamics of this mycosis in these animals.The emergence of The authors declare that no conflict of interests exist.The procedures were approved by the Animal Ethics Committee under the license number LW 37/12.


This paper presents standards and best practices for reporting genome sequences of uncultivated viruses.The online version of this article (doi:10.1038/nbt.4306) contains supplementary material, which is available to authorized users. in silico host prediction. Community-wide adoption of MIUViG standards, which complement the Minimum Information about a Single Amplified Genome (MISAG) and Metagenome-Assembled Genome (MIMAG) standards for uncultivated bacteria and archaea, will improve the reporting of uncultivated virus genomes in public databases. In turn, this should enable more robust comparative studies and a systematic exploration of the global virosphere.We present an extension of the Minimum Information about any (x) Sequence (MIxS) standard for reporting sequences of uncultivated virus genomes. Minimum Information about an Uncultivated Virus Genome (MIUViG) standards were developed within the Genomic Standards Consortium framework and include virus origin, genome quality, genome annotation, taxonomic classification, biogeographic distribution and The online version of this article (doi:10.1038/nbt.4306) contains supplementary material, which is available to authorized users. An unprecedented diversity of viruses are being discovered through culture-independent sequencing3. Progress has been made in reconstructing genomes of uncultivated viruses de novo, from biotic and abiotic environments, without laboratory isolation of the virus–host system. For example, in the past 2 years, more than 750,000 uncultivated virus genomes (UViGs) have been identified in metagenome and metatranscriptome datasets9, five times the total number of genomes sequenced from virus isolates (11. Although double-stranded DNA (dsDNA) genomes are over-represented in UViGs because most metagenomic protocols exclusively target dsDNA, UViGs nonetheless enable an assessment of global virus diversity and an evaluation of structure and drivers of viral communities. UViGs also contribute to improving our understanding of the evolutionary history of viruses and virus–host interactions.Current estimates are that virus particles massively outnumber live cells in most habitatsisolates , and UVi12. Although some aspects of MISAG and MIMAG can be applied to UViGs, the extraordinary diversity of viral genome composition and content, replication strategies, and hosts means that the completeness, quality, taxonomy and ecology of UViGs need to be evaluated via virus-specific metrics.Analysis and interpretation of standalone genomes present substantial challenges, whether the genomes are eukaryotic, bacterial, archaeal or viral. To address these challenges, MISAG and MIMAG standards were drafted to improve the quality of reporting of microbial genomes derived from single cell or metagenome sequences, which are often incompletehttp://gensc.org) maintains metadata checklists for MIxS, encompassing genome and metagenome sequences13, marker gene sequences14 and single amplified and metagenome-assembled bacterial and archaeal genomes12. Here we present a set of standards that extend the MIxS checklists to include identification, quality assessment, analysis and reporting of UViGs . Although MIUViG standards and best practices were designed for genomes of viruses infecting microorganisms, they can also be applied to viruses infecting animals, fungi and plants, and are compatible with standards that are already in place for epidemiological analysis of these viruses16 23 23 . Viral mde novo assembly of both abundant and rare viruses, increased confidence that the sequence is of viral origin, and the ability to sequence both active and 'inactive' or 'cryptic' viruses . However, virus-enriched datasets can have over-representation of virulent viruses with high burst size (high number of virus particles released from each infected cell) and under-representation of larger viruses with capsids ≥0.2 μm, such as giant viruses, as a result of the selective filtration steps used25. Furthermore, in silico approaches are often the only option available to determine the host range of UViGs obtained from virus-enriched samples.The main advantages of datasets produced after enrichment for viruses are good 28. These sequences could originate from viruses that are replicating in cells, from temperate viruses (proviruses or prophages) that are either integrated into host genomes or present as episomal elements in the host cell, or from free virus particles present in samples.Virus sequences are also present in non-virus-enriched datasets, including sorted cells, tissues, or environmental samples collected on 0.2 μm filters29  or from the capture of extracellular DNA in the virus fraction. Cellular sequences can also derive from cell genome fragments that are encased in virus capsids or comparable particles , DNA-containing membrane vesicles, or gene transfer agents34.Regardless of the type of dataset, the viral origin of UViGs must be validated because even samples enriched for virus particles still contain a substantial amount of cellular DNA38; eukaryotic viruses39; or combinations of bacteriophages, archaeal viruses and large eukaryotic viruses40 , which can results in inclusion of host gene(s) in a virus genome. Manual annotation of genes on the edge of a provirus prediction is recommended.• Partial genomes assembled as circular contigs. Partial genomes are sometimes misassembled as circular contigs owing to repeats47. These circularized fragments could be incorrectly identified as complete genomes. The size and gene content of circular contigs should be manually validated as consistent or at least plausible in comparison with known reference genomes.• Errors in gene prediction. For novel viruses with little or no similarity to known references, gene prediction can be challenging in the absence of accompanying transcriptomics or proteomics data. Outputs of automatic gene predictors applied to novel viruses should be checked for gene density (most viruses do not include large noncoding regions), as well as typical gene prediction errors, such as internal stop codons causing artificially shortened genes.• Inaccurate functional annotation. The annotation of open reading frames predicted from novel viruses often requires sensitive profile similarity approaches. Although such sensitive searches are necessary to detect homology in the face of high rates of virus sequence evolution, the inferred function should be cautiously interpreted and remain general .• Clustering of partial genomes. Incomplete genomes can be difficult to classify using genome-based taxonomic classification methods. For example, the estimation of whole-genome average nucleotide identity from partial genomes could vary by up to 50% from the complete genome value , high-quality draft genomes and finished genomes . These cllance16 . UViG qullance16 . This asllance16 , or base46. Similarly, UViG fragments can be analyzed to assess the functional gene complement of unknown viruses or link them to potential hosts. Importantly, current methods for automatic virus sequence identification40 cannot reliably identify short (<10 kb) viral sequences, which should be interpreted with utmost caution.Contigs or genome bins representing <90% of the expected genome length, or for which no expected genome length can be determined, would be considered genome fragments. This category might include UViG fragments large enough to be assigned to known virus groups on the basis of gene content and average nucleotide identity. However, high-quality draft or finished genomes are required to establish new taxa . Sequenc12. Repeat regions may lead to erroneous assembly of partial genomes as circular contigs47. Thus, the length of the assembled circular contig should be considered when assessing UViG completeness  on the basis of a comparison of sequences currently available in NCBI RefSeq10 and IMG/VR11 .For higher taxonomic ranks than species, no consensus has been reached on which approach should be used, although several have been proposed67) should be included in the UViG description. Similarly, the predicted segmentation state of the genome (segmented or nonsegmented) should be reported, typically derived from taxonomic classification and comparison with the closest references  encoding only a couple of proteins, double-stranded RNA viruses with up to 12 segments, or large and complex dsDNA viruses with genome sizes that are as large as those of some bacteria. Viruses are very diverse and tend to evolve faster than cellular organisms, in terms of both their genetic sequence and genome content. For all these reasons, viruses are not incorporated into the universal tree of life and a 'one size fits all' virus taxonomy has not been reported. Instead, there are different classification rules for different groups of viruses.91, and another operational classification scheme relying on nucleic acid type and method of genome expression was proposed by David Baltimore in 1971  in 1975  and interim update articles (“Virology Division news”) in Archives of Virology. Using some of the stable properties of viruses that were previously highlighted, experts in the ICTV developed a universal virus taxonomy similar to the classical Linnaean hierarchical system, in which virus groups were assigned to familiar taxonomic ranks including order, family, genus and species.The need for a specific set of rules to name and classify viruses led to the establishment of the International Committee on Nomenclature of Viruses (ICNV)975 ref. . The ICT93. Importantly, with large sections of the virosphere still to be explored, virus taxonomy represents only the current best attempt at recapitulating virus evolutionary history on the basis of available data. Virus classification will need to remain dynamic, expanding as we discover new viruses and being refined as our understanding of virus evolution improves.In the postgenomic era, virus classification is increasingly based on the comparison of genome and protein sequences, which provides a unique opportunity to evaluate phylogenetic and evolutionary relationships between viruses and reconcile the taxonomy of viruses with their reconstructed evolutionary trajectory. The ICTV has undertaken the immense task of re-evaluating virus classification in light of sequence-based informationIn silico approaches are often the only option for UViGs , which include CRISPR spacers71, to longer (>100 bp) nucleotide sequence matches, including proviruses integrated into a larger host contig73 . contig73 . Host-raf UViGs7 . Finallyin silico, in vitro and in vivo studies, including experimental validation to unequivocally demonstrate a viral infection of a given microbial host. Host predictions should be reported along with details regarding the specific tool(s) used and, importantly, their estimated accuracy as derived either from published benchmarks or from tests conducted in the study  through one of the member databases, at DDBJ (https://www.ddbj.nig.ac.jp/index-e.html), EMBL-EBI's European Nucleotide Archive  or NCBI . If needed, INSDC database curators can be contacted directly for large-scale batch dataset submissions. Where new datasets are generated as part of a UViG study, sequenced samples should be described according to the environment-relevant MIxS checklists and raw read data should be submitted. High-quality and finished UViGs should be submitted as assemblies, the former reported as “draft” accompanied by the required metadata  and data centers , will provide a framework for a systematic exploration of viral genome sequence space and enable the research community to better utilize and report UViGs.MIUViG standards and best practices for UViG analysis are the virus-specific counterparts to MISAG and MIMAGPublisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figure 11,2. SRF: surface, DCM: deep chlorophyll maximum.Top panel represents the number of distinct virus contigs ≥ 10kb identified in each dataset. The bottom panel depicts the ratio of “shared”, i.e., detected in both viral and microbial fraction of the sample, and “unique”, i.e., detected only in one fraction, contigs in each microbial and viral fraction. Datasets were originally analyzed in refs. Supplementary Figure 2Genome length of virus genomes from NCBI RefSeq were compared at different taxonomic ranks and are presented separately for four main types of viruses . Genome length variation was calculated as a coefficient of variation, i.e. standard deviation of genome length in the group divided by average genome length in the grouop (for groups with >1 genome). Underlying data are available in Supplementary Figure 3Only genome pairs with ANI >60% and AF >20% were considered. ANI and AF were binned in 1% intervals, and are represented here as a heatmap . On the top right corner , three main groups of genome pairs are delineated with black dashed circles, and the proposed standard cutoff is highlighted in dark red. Note that for this clustering, the cutoff was applied as follows: pairs of genomes with ≥ 85% AF were first selected, and whole genome (wg) ANI was then calculated by multiplying the observed ANI by the observed AF. This wgANI was then compared to the corresponding whole genome ANI cutoff (i.e. 95% ANI * 85% AF = 80.75% wgANI). This allows for hits with ≤ 95% ANI but ≥ 85 % AF to be considered as well, i.e. a pair of genomes with 90% ANI on 100% AF would be considered as “passing” the cutoff. Examples of genome comparisons for each group are presented in Supplementary Figure 4For each example, nucleotide similarity (blastn) and amino acid similarity (tblastx) are displayed, alongside the ANI, AF, and wgANI (i.e. ANI over the whole length of the shorter genome). AF, alignment fraction; ANI, average nucleotide identity; wgANI, whole-genome average nucleotide identity.Supplementary Figure 5To evaluate the impact of genome fragmentation on whole-genome average nucleotide identity (wgANI) estimation, pairs of genomes from NCBI RefSeq with wgANI ≥ 70% and ≥ 20kb were selected, random fragments were generated (from 1 to 45kb) from one of the two genomes, and then compared to the other complete genome. The resulting estimated wgANI between the fragment and complete genome was then compared with the original values estimated from the two complete genomes (y-axis). Boxplots lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles), while whisker extend from the nearest hinge to the smallest/largest value no further than 1.5 * IQR from the hinge .Supplementary Figures 1–5 (PDF 1122 kb)Supplementary Notes 1–4 (PDF 196 kb)List of mandatory and optional metadata for UViGs (XLSX 9 kb)21 (XLSX 17 kb)List of metadata from previous standards relevant for UViGset al.22 (XLSX 5 kb)Comparison between UViGs categories and the quality categories proposed for small DNA/RNA virus whole-genome sequencing for epidemiology and surveillance by Ladner 23–31 (XLSX 6 kb)List and characteristics of tools used to identify virus sequences in mixed datasets published or updated since 2012Variation in genome length for virus families and genera with two or more genomes, from NCBI RefSeq v83. (XLSX 25 kb)http://vogdb.org/) (XLSX 85 kb)List of potential marker genes for virus orders, families or genera, based on the VOGdb v83 List of UViGs from the GOV dataset32–35 (XLSX 6 kb)List of databases providing collections of HMM profiles for virus protein familiesCurrent species demarcation criteria from ICTV ninth and tenth reports. (XLSX 46 kb)in silico host prediction18,37–42 (XLSX 6 kb)Approaches available for