Aphis, Rhopalosiphum, and Sitobion.Aphids are important vectors of numerous plant viruses. Besides plant viruses, a number of insect specific viruses (ISVs), such as nege/nege-like viruses, have been recently discovered in aphids of the genera Indomegoura. INLV1 possessed a single positive-stranded RNA genome with 8945 nucleotides, which was predicted to contain three typical open reading frames (ORFs) of negeviruses , a 44-nt 5′ untranslated region (UTR) and a 98-nt 3′ UTR. Five conserved domains were predicted for INLV1, including an Alphavirus-like methyltransferase domain, a RNA virus helicase core domain, and a RNA-dependent RNA polymerase domain (RdRP) in ORF1, a DISB-ORF2_chro domain in ORF2, and a SP24 domain in ORF3. According to the maximum likelihood phylogenetic tree based on RdRP, INLV1 was grouped with barley aphid RNA virus 1 and Hubei virga-like virus 4, together with another two invertebrate viruses, which formed a distinct clade in the proposed group Centivirus. The alignment of RdRP domains for INLV1 and other nege/kita-like viruses suggested that RdRP of INLV1 contained the permuted C (GDD)- A [DX(4–5)D] –B [GX(2–3)TX(3)N] motifs, which were conserved in the Centivirus and Sandewavirus groups. Furthermore, the high abundance and typical characteristics of INLV1 derived small interfering RNAs clearly showed the active replication of INLV1 in the aphid Indomegoura.In this study, the complete genome sequence of a novel nege-like virus, tentatively named “Indomegoura nege-like virus 1” (INLV1), was identified in aphids of the genus Indomegoura. As far as we know, it is also the first ISV revealed in this aphid genus.INLV1 is the first nege-like virus infecting aphids of the genus The online version contains supplementary material available at 10.1186/s12985-021-01552-w. Aphididae, are one of the most serious pests of agricultural and horticultural crops. Aphids will cause damage through direct feeding or by means of vectors of many important plant viruses [Dicistroviridae and Iflaviridae, and others from various unclassified taxa [Kitaviridae [Aphids, which belong to the order Hemiptera, family  viruses . Apart fied taxa –7. Negevied taxa –10. The aviridae –13.Aphis glycines), which was obtained from Ohio State University using Next Generation Sequencing [Rhopalosiphum and Sitobion). According to phylogenetic analysis, the nege/kita-like viruses infecting these two aphid genera can be classified into two new distinct clades, tentatively designated as Centivirus and Aphiglyvirus, respectively [A few nege/nege-like viruses have hitherto been reported in aphids. The first aphid nege-like virus was discovered in soybean aphid  to extract total RNA from a pool of ten aphids. The Nano Drop spectrophotometer  was used to determine the RNA content. Paired-end (150 bp) sequencing of the RNA library was performed using the Illumina HiSeq 4000 sequencer . Afterwards, the 22,045,205 pairs of raw reads generated were subjected to quality trimming and de novo assembly by adopting Trinity (version 2.8.5) with the default parameters [http://www.boldsystems.org/), and later the potential aphid COI sequence was extracted. The aphid COI sequence was then compared with the nucleotide (nt) database in NCBI, which showed high homology (97% similarities) to the COI of Indomegoura indica (Accession number: NC_045897.1), confidentially indicating that the collected aphid species were highly similar to I. indica and belonged to the genus Indomegoura. The COI sequence of aphids Indomegoura was further confirmed by Sanger sequencing and stored in GenBank under the accession number MW533423 . As a result, a confidently nege-like viral contig was discovered in aphids, which represented almost the complete viral genome with the length of 8876 nt. To investigate the transcript abundance and coverage of the contig, the adaptor- and quality-trimmed reads from the transcriptome were mapped back to this contig using Bowtie2 and Samtools. As a result, high coverage (290X) was confirmed for this nege-like viral contig. Thereafter, the identified viral contig was further compared with the entire NCBI nucleotide (NT) and non-redundant (NR) protein database to avoid false positive results , and its full genome sequence was deposited in GenBank with the accession number MW285725 , a 44-nt 5′ UTR and a 98-nt 3′ UTR (nucleotide position in the genome: 8848–8945 nt)  suggested that the long ORF1 (nucleotide position in the genome: 45-6908 nt) consisted of an Alphavirus-like methyltransferase domain , a RNA virus helicase core domain , and a RNA-dependent RNA polymerase domain . In addition, RNA ribosomal methyltransferase domain (FstJ), which was demonstrated to be present or absent in various negeviruses [Aedes vexans mosquitoes in Finland [http://www.cbs.dtu.dk/services/TMHMM/). As a result, the four transmembrane domains were evidently present in the ORF3 of INLV1 . Furthermore, the complete 5′ and 3′ untranslated region (UTR) were obtained using RACE technology followed by Sanger sequencing, and the full genome sequences of INLV1 was successfully reconstructed. INLV1 had a genome size of 8945 nt (excluding polyA), which was the most homologous to Hubei virga-like virus 4 (HVLV-4) (accession number APG77770.1) and barley aphid RNA virus 1 (BARV-1) (accession number BBV14745.1), with the amino acid (aa) sequence identities of 59.00% and 58.47%, respectively. In terms of the genome organization, INLV1 contained three typical negevirus ORFs  predicted using the Expasy online server  and Cucumber green mottle mosaic virus (NP_044577.1), were used as outgroup. According to recent phylogenetic study on aphid nege/kita-like viruses, it is proposed that the two newly identified groups, Centivirus and Aphiglyvirus, together with the Negevirus subgroups (Nelorpivirus and Sandewavirus), can be classified into a novel viral family or assigned to the family Kitaviridae [Kitaviridae and Virgaviridae, whereas the permuted C-A-B pattern was observed in the groups Centivirus and Sandewavirus , and further trimmed the gaps by Gblock . Besidesaviridae . In thisaviridae , we aligaviridae . In our .450, andIndomegoura.To explore small interfering RNA (siRNA) based anti-viral immunity in aphid host, small RNAs (sRNA) of the aphids were sequenced and virus derived siRNAs (vsiRNAs) were comprehensively characterized. In brief, a sRNA library was prepared using the Illumina TruSeq small RNA sample preparation kit , and sRNA sequencing was performed by Novogene on an Illumina HiSeq 2500 platform. The sRNA reads were pretreated  and sRNAs with the length of 18-nt to 30-nt were extracted. The processed sRNA reads were mapped back to the full viral genome sequence of INLV1 using Bowtie with zero mismatches. vsiRNAs were further analyzed using the custom perl scripts and the Linux bash scripts. As a result, a total of 13,203  vsiRNAs perfectly mapped to INLV1 genome were identified, accounting for 0.06% (0.48% unique) of the sRNA library. The vsiRNAs were mostly 22-nt long , and they were equally derived from the sense and antisense strands of the viral genome  on aphid competence, and to evaluate whether they can be used as the biological agents to control aphid-borne plant viruses in the field.In addition to the recent discoveries of nege/nege-like viruses in various aphid genera including Additional file 1. File S1: COI sequence of the aphid (Genus Indomegoura) used in this study.Additional file 2. File S2: Full genome sequence of Indomegoura nege-like Virus 1.Additional file 3. Table S1: The BLAST results of INLV1 compare to the NCBI NT and NR database.Additional file 4. Table S2: Primers used in this study.Additional file 5. Table S3: Abbreviations of virus names and GenBank accession numbers used in this study.Additional file 6. Figure S1: Prediction of transmembrane domains (TM) in the ORF3 of INLV1. TM 1:68-87 aa; TM 2:107-126 aa; TM 3:138-157 aa; TM 4:177-196 aa.Additional file 7. Figure S2: Alignment of RdRp amino acid sequences of INLV1, previously reported representative nege/kita-like viruses, and plant viruses in the families Kitaviridae and Virgaviridae. Red boxes indicate the position of the motifs - A: DX(4-5)D, B: GX(2-3)TX(3)N, and C: GDD. Virus names and GenBank accessions numbers are listed in Supplementary Table S3.


Swinepox virus (SWPV) is a globally distributed swine pathogen that causes sporadic cases of an acute poxvirus infection in domesticated pigs, characterized by the development of a pathognomonic proliferative dermatitis and secondary ulcerations. More severe disease with higher levels of morbidity and mortality is observed in congenitally SWPV-infected neonatal piglets. In this study, we investigated the evolutionary origins of SWPV strains isolated from domestic pigs and wild boar. Analysis of whole genome sequences of SWPV showed that at least two different virus strains are currently circulating in Germany. These were more closely related to a previously characterized North American SWPV strain than to a more recent Indian SWPV strain and showed a variation in the SWPV-specific genome region. A single nucleotide deletion in the wild boar (wb) SWPV strain leads to the fusion of the SPV019 and SPV020 open reading frames (ORFs) and encodes a new hypothetical 113 aa protein (SPVwb020-019). In addition, the domestic pig (dp) SWPV genome contained a novel ORF downstream of SPVdp020, which encodes a new hypothetical 71aa protein (SPVdp020a). In summary, we show that SWPV strains with altered coding capacity in the SWPV specific genome region are circulating in domestic pig and wild boar populations in Germany. Swinepox virus (SWPV) is the only member of the genus Suipoxvirus, which belongs to the subfamily Chordopoxvirinae, within the family Poxviridae. This virus contains a linear double-stranded DNA genome of 146 kbp and is the etiological agent of an eruptive dermatitis in pigs, known as swinepox. Swinepox was first described as a disease of domestic pigs in Europe in 1842 , AustralClinical studies, experimental infection of laboratory animals and in vitro infection of cell lines originating from different species have shown that SWPV displays a high degree of host specificity with infections restricted to domestic pigs  with a sHaematopinus suis) has been shown to facilitate the spread of the virus between populations [Haematopinus suis in swinepox infections and demonstrated its function as a mechanical vector but not as an intermediate host [In addition to direct contact between infected and susceptible animals, a mechanical route of transmission by insect vectors such as the swine louse  were collected from recurrent sporadic cases of swinepox that occurred in piglets on two conventional pig farms in Westphalia, Germany between July 2019 and January 2020. A complete postmortem examination was performed on three piglets with suspected poxvirus skin lesions and tissue samples  were fixed in 4% buffered formaldehyde and routinely processed for histopathological examination by embedding in paraffin wax according to standard procedures, prior to haematoxylin–eosin (HE) staining of microtome-cut tissue sections. A formalin fixed block containing skin tissue from a previously described swinepox case in a domestic pig in 2008 was also used for retrospective SWPV genome analysis [Sus scrofa) found in Baden-Württemberg with an eruptive dermatitis in October 2019. Ultrastructural examination was performed using a standardized diagnostic electron microscopy protocol with negative staining technique according to the recommendations of the Robert Koch Institute, Germany. Briefly, the unfixed skin lesions were scarified and placed against formvar-filmed electron microscopy copper grids to adsorb virus particles. These were negatively contrasted with 3% (w/v) phosphotungstic acid for 30 s and then ultrastructural investigation performed using an LEO0906 Electron microscope . Samples were initially examined using a 50,000× magnification.Tissue samples from domestic pigs  were observed.Porcine embryonic kidney cell line SPEV , porcine lymphoma cell line 38A1D , porcine1D ,40. Viru® High-Fidelity PCR kit (NEB). For Sanger sequencing of ORF SPV006 and SPV136 coding sequences, two primer pairs were used, 5′-TGAACGGAATCTGAAATACGA-3′ and 5′-AAATATCTCATACAATCATTATACTTAC-3′ for SPV006 and 5′-ATTACAGGAAAGATTGGCGTA-3′ and 5′-TAATTTCCAAGACCTTCGCTT-3′ for SPV136 [A SWPV-specific real-time quantitative PCR (qPCR) was performed to confirm SWPV infection in clinical samples using probe and primer pair sequences targeting the C20L-C1L region . The olir SPV136 .1D lymphoma cell line and porcine epithelia testis cell line Riebe-255 were infected with the two SWPV strains for 2 h at 37 °C. The cell cultures were maintained in EMEM supplemented with 10% FBS and 1% penicillin/streptomycin and monitored for the development of cytopathic effects. Following the infection, supernatant samples were taken at days 0, 3, 6 and 10 post-infection and the amount of viral DNA was quantified by SWPV-specific qPCR with three replicates.The isolated virus strains from domestic pigs and wild boar were used to test the permissivity of porcine-derived cell lines. Monolayers of the kidney-originated cell lines  as well as of the 38ANext generation sequencing (NGS) was used to obtain the complete genome of the SWPV strains isolated from the domestic pig  for further investigations into the etiology of the disease. Two piglets from farm 1  died within 24 h of birth while one piglet from farm 2 (201-200045) was euthanized on day 1 post-birth . Approxi1D) to infection with the German domestic pig and wild boar SWPV strains was investigated together with virus growth kinetics over time. In the absence of a SWPV-specific antibody and with cytopathic effects often absent or subtle in infected cell cultures, a SWPV-specific qPCR was used to assess virus replication. All tested cell lines showed evidence of SWPV replication as evidenced by a decreasing ct value (Virus isolation from confirmed swinepox cases was performed by infection of PK-15 cells with clarified supernatant from pox skin lesions that had been homogenized in PBS from a SWPV-infected domestic pig (Host-ID 201-20046) and wild boar (Host-ID 201-20070). Initially, no cytopathic effects were observed. Therefore, infected cells were blind-passaged until the characteristic CPE of SWPV including cell rounding, ballooning and vacuolization developed at passage four and three for the domestic pig (SWPV/domestic/GER/2019) and wild boar (SWPV/domestic/GER/2019) strains, respectively a–c. The ct value d,e with SWPV is the single member of the genus Suipoxvirus, within the subfamily Chordopoxvirinae and its clade is between that of members of the genus Capripoxvirus such as lumpy skin disease virus and the novel Brazilian porcupinepox virus. However, knowledge about the phylogenetic relationships and sequence diversity within the SWPV species is limited. We therefore compared the two available whole-genome SWPV sequences from domestic pigs in North America  and India  to the new German genome SWPV sequences obtained in this study. Phylogenetic analysis showed that the German domestic pig and wild boar SWPV strains from 2019 form a clade with SWPV/USA/2002 and branch separately from SWPV/India-Assam/16 a. The SWComparison of whole-genome sequences of the two new German SWPV isolates showed 75 synonymous and 37 nonsynonymous changes, which were spread throughout the genome . All preOutbreaks of swinepox are commonly observed in domestic pigs worldwide, but knowledge about the prevalence, strain diversity, wildlife reservoirs and evolutionary origins of SWPV is limited. In this study, SWPV strains isolated from skin samples containing pox lesions obtained from a wild boar piglet and a congenitally infected domestic piglet were sequenced and analyzed to determine the evolutionary origins of these strains. We observed a sequence divergence of only 0.076% (112 nt) between the SWPV strains present in domestic pigs and wild boar, meaning that at least two closely related strains are circulating in German wildlife and livestock pigs. In countries with a comparable, industrial structure of pig farming, the implementation of strict sanitary measures and high hygienic standards serves to shield the livestock population against contact with pathogens present in the environment and especially prevent potential direct or indirect encounters with wild boar. However, given the high nucleotide identity between the two SWPV strains, wild boar may have a role as a potential SWPV wildlife reservoir mediating the spread of SWPV to domestic pig populations. Additional sequencing of SWPV strains in domestic pig and wild boar populations is required to determine the routes of virus transmission.The higher nucleotide sequence identity of the two German SWPV strains (2019) to the reference North American SWPV strain (2002) than to a recent Indian SWPV strain (2016) may reflect the history of domestication and distribution of the domestic pig. Zooarchaeological evidence shows that pigs were domesticated independently in at least two locations, namely Eastern Anatolia , from whGenome analysis of the domestic pig and wild boar SWPV strains showed interesting differences with respect to potential alterations in gene expression in the region of the genome encoding SPV018, SPV019 and SPV020. These genes are unique to SWPV with no homologs within the wider Poxviridae family and are predicted to encode proteins that may be associated with immune evasion, restricted host range and/or virulence. The SWPV strain from wild boar had a one nucleotide deletion at position 13,061 bp resulting in fusion of the SPV019 and SPV020 ORFs. A similar fusion of the SPV019 and SPV020 ORFs has been recently observed in an Indian SWPV sequence from a domestic pig in which a single base pair deletion at position 12,993 bp caused a similar frameshift in SPV020 . This seHaematopinus suis) from the housing units, which indicates that vector-borne mechanical transmission was probably not responsible for the introduction of SWPV into these farms or subsequent virus transmission between animals. The pig louse has previously been shown to be a vector for SWPV transmission as the main lesions observed on infected pigs are on the abdomen and inner surface of the legs, corresponding to the predilection sites of these parasites. However, piglets which have been intravenously infected with SWPV also show lesions in the same positions [The piggeries affected by this outbreak were assessed to be in a good hygienic condition with an absence of pig lice (ositions . The absositions ,54,55, eositions . Poxviruositions ,58,59. HThe in utero infection of piglets with SWPV from clinically unremarkable sows suggests that viremia occurs in immunocompetent animals, together with virus spread to reproductive tissue and vertical virus transmission through the placenta. Although the placentas from these cases were not available for further study, we did observe that the lowest ct values for SWPV apart from skin tissue were present in the umbilical cord. We were unable to consistently identify SWPV infection by qPCR in the blood of sows within the housing units in which swinepox cases occurred. Similar observations have been made previously for other poxvirus infections. Rare cases of generalized poxvirus lesions in the human fetus following in utero virus infection have been reported following both primary vaccinia virus vaccination ,61,62 anEstimating the real prevalence of SWPV in domestic pig populations has been challenging, as swinepox is often neglected by farmers due to the mild clinical signs in noncongenitally infected piglets. Serological testing for antibodies against SWPV is also unreliable and technically challenging, with only limited neutralization activity detected in sera from convalescent animals, which are nevertheless protected against reinfection ,29,67. TWe have characterized two SWPV strains circulating in German wildlife and livestock animals and shown differences with respect to coding capacity within the SWPV-specific terminal region of the genome. The congenital infection of piglets and the detection of SWPV DNA in the umbilical cord suggests that the virus transmission within the domestic pig population is mediated by persistently infected asymptomatic animals. Future studies are required to determine the true prevalence and strain diversity of SWPV in domestic and wild pig species and to understand the role of comorbidities and coinfections in exacerbation of the disease course in infected animals.


Methods: An economic evaluation was conducted alongside a prospective cohort study comparing 608 players from leagues where body checking was allowed in games  with 396 players from leagues where it was not allowed in games . The effectiveness measure was rate of game injuries per 1000 player-hours. Costs were estimated based on associated healthcare use within the publicly funded healthcare system as well as privately paid healthcare costs. Probabilistic sensitivity analysis was conducted using bootstrapping. Results: Disallowing body checking reduced the rate of injuries by 4.32 per 1000 player-hours  and reduced public and total healthcare system costs by $1556  and $1577  per 1000 player-hours, respectively. These finding were robust in over 99% of iterations in sensitivity analyses in the public healthcare and the total healthcare system perspectives. There was no statistically significant difference in privately paid healthcare costs ). Interpretation: Disallowing body checking in non-elite 13–14-year-old ice hockey nationally would prevent injuries and reduce public healthcare costs. Sport-related injuries are the leading cause of injury in youth and are costly to the healthcare system. When body checking is disallowed in non-elite levels of Bantam (ages 13–14 years) ice hockey, the injury rate is reduced, but the impact on costs is unknown. This study compared rates of game injuries and costs among non-elite Bantam ice hockey leagues that disallow body checking to those that did not.  In Canada since 2010, over 600,000 youth participate each year in Hockey Canada youth ice hockey leagues ,4. HowevBody checking has been identified as the most consistent risk factor for all hockey-related injuries and concussions . Body chAn economic evaluation was conducted comparing the rates of game injuries and injury-related costs in a prospective cohort study of 13- to 14-year-old hockey players in British Columbia and Alberta. We used the Consolidated Health Economic Evaluation Reporting Standard (CHEERS) reporting guidelines .Local ice hockey organizations were approached to recruit 13- to 14-year-old hockey players in the lower 60% level of play. Players from both Calgary and Edmonton in 2014–2015 and Edmonton in 2015–2016 comprised the cohort where body checking was allowed. Players from Kelowna and Vancouver in 2014–2015 and from Calgary in 2015–2016 comprised the cohort where body checking was disallowed. Full details of the cohort design, recruitment and data collection have been published previously . The ecoThe measure of effectiveness used for the base case in this economic evaluation was rates of game injuries per 1000 player-hours to standardize the comparison between cohorts.Healthcare system resource use as well as private healthcare costs data were collected and included the frequency of healthcare professional visits, diagnostic imaging, medical treatments, and medication. These data were self-reported by the player or parent and recorded by a team designate on an injury report form (IRF) which also included weekly exposure to hockey sessions and injuries. Details of the treatment of missing data on exposure and injury variables were previously published . For theUnit cost sources from Alberta were used to standardize the costs when comparing policies and remove interprovincial variability. This included physician fee-for-service schedules  for geneThe differences in effectiveness and costs between the policy disallowing body checking and the comparator policy allowing body checking were calculated as the rate of injury or cost per 1000 player-hours, respectively, for the no body checking minus the body checking group. The cost-effectiveness analysis then jointly considered the cost and effectiveness differences. If disallowing body checking resulted in both a reduced rate of injury and a reduced cost, then this policy would be recommended. In scenarios where there were fewer injuries but higher costs or more injuries and lower costs, an incremental cost-effectiveness ratio (ICER) would be calculated . In that case, the ICER quantifies the trade-offs between costs and injury rates to be considered when making policy decisions.The estimates from the cost-effectiveness analysis and data on average player-game hours were used to project the change in total injuries and total costs to all non-elite Bantam players registered in Alberta and also in Canada in the 2016–2017 season. The average game-hours estimate is 38.75 hours per Bantam player [All data analyses were conducted in Stata v14 and R. Injury and cost rates for each group were calculated accounting for player game exposure hours. Non-parametric probabilistic sensitivity analysis was performed using bootstrapping with 10,000 iterations of sampling with replacement while first accounting for stratification by body checking cohort, and then resampling teams and subsequently participants and presented on an incremental cost-effectiveness plane scatterplot. Highest density bootstrap confidence intervals (95%) for the injury and costs rates for each group were calculated. As no specific R package for bootstrapping incorporates all these elements, our statistician adapted commands from several R functions including “draw.bootstrap”, “boot” and “resample”. For the provincial and national projections, confidence intervals were calculated from the injury and cost rate confidence intervals using the average game-hours and relevant population estimates.As previously reported , a totalPublic and private healthcare resource use and costs (unadjusted for the difference in players or exposure hours) are presented in As shown in In probabilistic sensitivity analysis , the no As previously reported , if a poThese study findings support a policy recommendation to disallow body checking in games in 13- to 14-year-old ice hockey leagues as the policy is associated with both reduced rate of game-related injuries and reduced public healthcare costs. The findings were robust in sensitivity analysis. Projecting the results to all Bantam players in Alberta and Canada in the 2015–2016 hockey season, disallowing body checking in non-elite Bantam players could prevent 747 injuries and save $268,983 in public healthcare costs in Alberta. At a national level, this could prevent 6386 injuries and save $2,300,434 in public healthcare costs. In the context of total public healthcare spending in Alberta and in Canada , this iThese findings are consistent with previous economic evaluations of body checking policy in Pee-Wee ice hockey where injuries were lower and costs were lower or remained the same when body checking was disallowed ,25. The Economic evaluation alongside a comparative prospective cohort study provides a unique opportunity to directly identify and measure the healthcare utilization from players as a result of their injuries. The prospective cohort design used in this study capitalized on the variation in body checking policies across Canada to provide an environment for a natural experiment to compare body checking policies and its effect on the rate of injuries and costs. The results may be generalizable to other provinces in Canada since the hockey leagues are similar to the leagues used in this study. This accounts for rules, age categories, and the number of games and practices players are exposed to in a season.This study does have limitations. The non-randomized nature of this study is a limitation, and although the study design capitalizing on the policy variation across Canada was robust, still the lack of randomization could have resulted in differences between the groups. Some out-of-pocket costs that are related to treatment such as travel costs were not collected and may underestimate the true out of pocket costs from this study. There were some injuries for which there was no reported costs due to missing data on the type of visits or treatments used which leads to underestimate of the total costs. This occurred more frequently in the body checking group thus the cost reduction estimates are conservative. This study took place over two seasons of play, and there is variability between seasons in terms of injuries. Additionally, due to the time horizon of this study, the long-term consequences of injuries and concussions were not measured. As a result, the study findings related to the one year time period likely underestimate the overall healthcare costs associated with hockey-related injuries and on long-term health and quality of life outcomes.Players that participate in a league that disallowed body checking had a lower rate of injuries and lower costs to the public healthcare system compared with players in a league that allowed body checking. There is a substantial public health impact both in terms of injuries and costs, and the findings of this study support implementation of policy to disallow body checking in Bantam ice hockey at a national level.


Pea early browning virus (PEBV), Cucumber mosaic virus (CMV), Bean yellow mosaic virus (BYMV) and Pea seedborne mosaic virus (PSbMV). TG-Seq detected all the expected viral amplicons within multiplex PCR (mPCR) reactions. In contrast, the expected PCR amplicons were not detected by gel electrophoresis (GE). For example, for CMV, GE only detected RNA1 and RNA2 while TG-Seq detected all the three RNA components of CMV. In an mPCR to amplify all four viruses, TG-Seq readily detected each virus with more than 732,277 sequence reads mapping to each amplicon. In addition, TG-Seq also detected all four amplicons within a 10−8 serial dilution that were not detectable by GE. Our current findings reveal that the TG-Seq approach offers significant potential and is a highly sensitive targeted approach for detecting multiple plant viruses within a given biological sample. This is the first study describing direct HTS of plant virus mPCR products. These findings have major implications for grain germplasm healthy certification programs and biosecurity management in relation to pathogen entry into Australia and elsewhere.Globally, high-throughput sequencing (HTS) has been used for virus detection in germplasm certification programs. However, sequencing costs have impeded its implementation as a routine diagnostic certification tool. In this study, the targeted genome sequencing (TG-Seq) approach was developed to simultaneously detect multiple (four) viral species of;  In many parts of the world, plant viruses constitute almost 50% of pathogens causing damaging disease in many agricultural and horticultural crops . MinimizCucumber mosaic virus , Pea early browning virus , Bean yellow mosaic virus (BYMV) and Pea seed-borne mosaic virus (PSbMV); . They were used to investigate the capability of TG-Seq to simultaneously detect these multiple plant virus genome targets and compared its utility with gel visualisation of mPCR products and RNA-Seq approaches. A major obstacle to using HTS in a more efficient manner for virus detection is the variable genome structure of RNA viruses  as this 2 and stored at room temperature.Virus-infected leaf material from faba bean infected with CMV, lentil infected with BYMV, field pea infected with PSbMV were collected in Victoria, Australia, and faba bean infected with PEBV ((isolate Lyv66) imported from Libya) was intercepted at the Australian border in post entry quarantine (PEQ). All virus isolates were desiccated on CaClThe total RNA from the four virus-infected plant samples was extracted using the RNeasy Plant Mini Kit  according to manufacturer’s instructions. Crude RNA was treated with RNase-free DNase  and quantified using the Qubit™ RNA BR Assay  then later stored at −80 °C. The isolated total RNA was converted to cDNA using random hexamers and the ImProm-II TM Reverse Transcription System . The cDNA was purified using Qiaquick (Qiagen) purification columns and quantified using a Qubit dsDNA high sensitivity assay kit (Thermo Fisher Scientific).TM Plant  using RNA templates  and each sample was normalized to 1 µg of 10 µL  from nucleotide genome sequences of PEBV, CMV, BYMV and PSbMV  targetinTo determine the percentage of reads that are amplicon specific, a singleplex PCR reaction using specific ORF primers  of the four viruses , that wemPCR was performed using primer pairs targeting either three or four genome regions in each virus, or targeting a genome region from multiple viruses, in a single PCR reaction  was used as a template in a 100-fold serial dilution   . The mPC6, 10−8) . RNase/D6, 10−8) . The TG-4 with the minimum contig length set to 800 bp. All the contigs were subjected to BLASTN search using BLAST version 2.7 [Quality control of the RNA-Seq raw reads obtained was done using Trim Galore . The RNAsion 2.7 . The consion 2.7 . The ORFsion 2.7 , with trThe TG-Seq derived FASTQ files were first inspected using FastQC (version 2.0) to determine any downstream quality control requirement. Quality control of the TG-Seq raw reads was done using Trim Galore  and CLC The local isolates of two monopartite viruses, pea seed-borne mosaic virus (PSbMV) and bean yellow mosaic virus (BYMV); a bipartite virus, pea early browning virus (PEBV); and a tripartite virus; cucumber mosaic virus (CMV) were sequenced  and 16 pBoth gel electrophoresis and TG-Seq detected all four amplicons with each amplicon having more than 967,465 reads mapping to it . An mPCRTo test the sensitivity of TG-Seq for simultaneous detection of multiple genetically diverse plant viruses, a series of mPCR reactions was designed to detect three to four plant viruses in the one assay . When ei−0) for each of the four viruses in nuclease free water was generated  and used as a template for mPCR  for the 10−4, 10−6, 10−8 serial dilutions with the HC-Pro amplicon being nearly invisible in the gel  and RNA-Seq derived raw reads were mapped back to the ORF and the genomes of interest respectively, over 98% mapping reads from the TG-Seq libraries were viral reads, and less than 0.24% of the library reads were nontargets . On the This paper describes the first application of a TG-Seq approach to simultaneously detect multiple plant virus genome targets. The TG-Seq proved to be highly sensitive compared to end point singleplex and mPCR and simultaneously detected four plant viruses: CMVPEBV, BYMV and PSbMV. The TG-Seq enabled targeted sequence analysis of viral genomes and reduced background host sequences to less than 1% of total sequenced reads. TG-Seq proved to be a highly sensitive and unambiguous detection approach in detecting both individual and mixed genomic regions of PSbMV, BYMV, PEBV and CMV when compared to end point mPCR. For example, three genomic regions of BYMV and CMV were successfully detected by TG-Seq, when compared to two BYMV (NIb2 and NIb3) and one CMV (RNA3) amplicons not being detected by mPCR upon gel visualization. These observations are in line with previous studies that have found the sensitivity of mPCR is influenced by the number of different amplicons generated in a given reaction ,32,33 De−2 serial dilution was not detected after gel visualisation. For the 10−4, 10−6 and 10−8 serial dilutions, gel electrophoresis only detected two amplicons  with the HC-Pro amplicon being nearly invisible in the gel. This compares to TG-Seq detecting all four amplicons for each serial dilution, including the 10−8 serial dilution, suggesting an increase in sensitivity of TG-Seq of up to 10−6 when compared to end-point PCR. The limitations of mPCR followed by gel electrophoresis are widely documented and can be as a result of PCR primers competing for amplicons and reagents thus reducing the yield of some of the final amplicons products [Real-time PCR has been widely used for the detection and quantification of the low titre pathogens in a given plant sample ,37,38. Hproducts ,40. The products ,41 and/oproducts  might ha−2, 10−4, 10−6, 10−8). The sequences derived from  transposome beads-based libraries had a good quality precision in virus targets calling and detection of low titre amplicons. These findings corroborate with the previous research of [Although high fidelity DNA polymerase was used in amplifying low concentrated nucleic material , there was variation in the amplicon datasets (number of reads mapping to the virus specific amplicon), as commonly observed in shotgun HTS ,44,45,46earch of  that repMetagenomics approaches such as RNA-Seq are robust diagnostic tools, particularly for virus discovery and whole genome sequencing. This approach has been widely used without requiring prior knowledge of the existing pathogen(s) to identify both viruses and viroids in an infected plant ,49,50,51Taken together, TG-Seq has offered a broad-range capability of detecting multiple plant viruses due to its high sensitivity. Its approach of using targeted primer panels enhances widespread identification of pathogenic plant viruses across multiple plant samples which in turn reduces the cost compared to whole genome sequencing. Whilst our current study focussed on detecting four viruses simultaneously, we recommend future studies to include more target primer panels targeting DNA viruses and additional divergent lineage RNA viruses. We also propose the incorporation of unique dual indices within TG-Seq library reduces the risk of any indexing crossover from multiplexed samples, increasing accurate diagnostics ,59. More


Pneumocystis jirovecii (PJ) pneumonia (PCP), an invasive fungal infection mainly occurring in HIV-negative patients, in-house or commercial PJ-specific real-time quantitative PCR (qPCR) assays are todays’ reliable options. The performance of these assays depends on the type of PJ gene  targeted by the assay. We described the development of a PJ-PCR assay targeting the dihydrofolate reductase (DHFR)-encoding gene. After delineating its analytical performance, the PJ-PCR assay was used to test bronchoalveolar lavage (BAL) fluid samples from 200 patients (only seven were HIV positive) with suspected PCP. Of 211 BAL fluid samples, 18 (8.5%) were positive and 193 (91.5%) were negative by PJ-PCR. Of 18 PJ-PCR-positive samples, 11 (61.1%) tested positive and seven (38.9%) tested negative with the immunofluorescence assay (IFA). All (100%) of the 193 PJ-PCR-negative samples were IFA negative. Based on IFA/PCR results, patients were, respectively, classified as having (n = 18) and not having (n = 182) proven (PJ-PCR+/IFA+) or probable (PJ-PCR+/IFA−) PCP. For 182 patients without PCP, alternative infectious or non-infectious etiologies were identified. Our PJ-PCR assay was at least equivalent to IFA, fostering studies aimed at defining a qPCR-based standard for PCP diagnosis in the future.To support the clinical laboratory diagnosis of  Pneumocystis jirovecii , Aspergillus (aspergillosis), or Mucorales (mucormycosis), account for about 5% of all severe respiratory infections  that occur in immunocompromised patients [PJ organisms [Fungal infections, mainly patients . The nonpatients . Moleculpatients  especialrganisms .PJ [PJ-specific PCR assays, mostly real-time quantitative PCR (qPCR) assays, became reliable options to support today’s clinical laboratory diagnosis of Pneumocystis pneumonia (PCP) [PJ gene—multi-copy mitochondrial  of ribosomal RNA) versus single-copy nuclear ) encoding gene—targeted by the assay. However, the predictably best analytical sensitivity of the mtSSU gene may be detrimental when considering the risk for false-positive results. Notably, quantification of a single-copy gene may better reflect the PJ load—in terms of genomic copies/mL—in an LRT sample [While FDA-cleared molecular panels comprise a variety of detectable bacterial or viral agents from the lower respiratory tract (LRT) bronchoalveolar lavage (BAL) fluid—the preferred sample for PCP diagnosis —or otherPJ . Thus, iia (PCP) . Despiteia (PCP) , which dT sample .PJ DHFR DNA in BAL fluid samples, prospectively collected from patients hospitalized at our institution , using a new in-house qPCR assay (hereafter referred to as PJ-PCR assay).Here, we report on the detection of PJ-microscopy (50 µL) and PCR assay (100 µL), as specified below. All samples allowed one to extract enough human DNA to make PJ detectable in BAL fluid, based on the positive amplification of the human RNase gene (see below for details). This gene was equivalent to the human β-globin gene used elsewhere.Between 1 March 2019 and 1 March 2020, patients hospitalized at the Fondazione Policlinico Universitario A. Gemelli IRCCS  with a sampled BAL fluid for microbiological examination were eligible for the study. We included samples from patients  with PCP- ,11. BefoPneumocystis jirovecii IFA Test kit  to identify PJ asci and trophic forms at microscopy. Using the forward (5′-GGCTGATCAAAGAAGCATGGATA) or reverse (5′-CGGCATAGACATATTCGATACTTGTT) primer pair and the internal probe (5′-TGCGTGAAACAGATACATGGAGCTCTACCC) targeting the PJ DHFR-encoding gene , we developed the PJ-PCR assay following Minimum Information for the publication of real-time Quantitative PCR Experiments (MIQE) guidelines [The immunofluorescence assay (IFA) of BAL fluid samples was performed using the MONOFLUO™ idelines .PJ-PCR assay’s performance, the full-length DHFR gene was amplified, the resulting PCR product was cloned into a pCR 2.1 cloning vector , and the recombinant plasmid DNA concentration was measured with the Qubit™ 4 fluorometer . Using a 1:10 dilution series of DHFR-carrying plasmid (ranging from approximately 10−1 to 109 copies/mL), a standard  curve was generated to calculate the amplification efficiency, which was established to be around 99%. Specifically, using the equation PCR efficiency = 10−1/slope − 1, we plotted the logarithm of the initial DNA concentration on the x-axis and the quantification cycle (Cq)—also termed threshold cycle (Ct)—on the y-axis. Then, we determined the PJ-PCR assay’s analytical sensitivity, expressed as the limit of detection (LoD), which was found to be equivalent to 10 copies/mL. The Ct variation (as assessed at 10X LoD) was 0.6, whereas the PJ-PCR assay had 100% analytical specificity, as shown elsewhere [PJ-PCR assay was stable by testing positive (DHFR-carrying plasmid) samples in qPCRs after the reagents were subjected to several freezing/thawing cycles.To determine the lsewhere . AdditioDHFR gene was expressed as a Ct value; thus, samples with Ct values ≤40 or >40 were scored as PJ-PCR positive or negative, respectively. In each run, the aforementioned human RNase gene was included as an external intrinsic control to monitor PCR inhibition, and no-template (sterile water) controls were included to monitor PCR contamination.As previously described ,15, a di® assay  for 1,3-β-D-glucan (BDG) measurement using a positivity cutoff of 80 pg/mL [In parallel, patients’ serum samples (collected the same day or within ± one day of BAL fluid sampling) were tested with the Fungitell80 pg/mL .PJ-PCR results, the revised/updated EORTC/MSGERC 2008 consensus definitions of invasive fungal disease [PJ-PCR positive) or probable (only PJ-PCR positive) PCP episodes, respectively. In view of conflicting opinions about the role of BDG in the PCP diagnosis [PJ-PCR assay. Furthermore, microbiological standard-of-care testing results allowed one to classify non-PCP episodes as bacterial, viral, or (outside PCP) fungal pneumonia episodes, respectively.According to positive IFA or  disease  allowed iagnosis ,16,17, wn = 211) were investigated for PCP. Based on both PJ-PCR and IFA results, patients were classified as having (n = 18) or not having (n = 182) proven (PCR+/IFA+) or probable (PCR+/IFA−) PCP [As shown in PJ-PCR/IFA results for 211 pneumonia episodes, 191 of which were single and 20 were multiple. One patient had four episodes and eight patients had each two episodes. Of 211 BAL fluid samples studied in total, 18 (8.5%) tested positive  and 193 (91.5%) tested negative  with the PJ-PCR assay.PJ-PCR-positive samples, 11 (61.1%) samples  were IFA positive and seven (38.9%) samples  were IFA negative. A total of 193 (100%) of 193 PJ-PCR negative samples were IFA negative. Seventeen (94.4%) of eighteen patients with PJ-PCR-positive BAL fluid samples had BDG-positive (315 to >500 pg/mL) serum samples, whereas one patient with a PJ-PCR-positive  but IFA-negative BAL fluid sample had a BDG-negative (<80 pg/mL) serum sample. The last patient was a hematological patient for whom a BAL fluid culture yielded positive results for both Pseudomonas aeruginosa and Stenotrophomonas maltophilia.Among 18 PJ-PCR/IFA-negative episodes (Candida (16 samples), Aspergillus (two samples), and/or bacterial species (15 samples). Seven of eighteen patients with PCP were co-infected with bacteria  and two other patients were colonized with Aspergillus or Candida  or non-infectious etiologies were identified (In 41 (21.2%) of 193 episodes , all pat Candida . Among 1entified .PJ-PCR assay allowed one to diagnose PCP in 18 (9.8%) of 183 episodes from patients who, otherwise, were at high risk of developing disease. In addition to meeting clinical and radiological criteria, these patients had at least one of the EORTC/MSGERC designed host factors [PJ DNA in BAL fluid samples. All (100%) of the 193 episodes with a negative PCR result were from patients who had a microbiological  or non-microbiological  reason on which the suspicion of PCP was initially based.The PCP diagnosis remains particularly challenging in HIV-negative immunocompromised or other seriously ill patients, who accounted for 96.5% (193/200) of patients in this study. Only 7 (3.5%) of 200 patients were HIV positive. Excluding 28 episodes from patients with lower suspicion of disease , our PJ- factors . Besides factors , our assPJ microscopically in the BAL fluid (or pulmonary tissue) using conventional (Gomori methenamine silver) or immunofluorescence (specific fluorescent antibody) staining is the current diagnostic criterion for proven PCP [PJ-PCR assay, whereas seven samples with a positive PCR result were negative by the IFA. These findings would be consistent with the lower Ct values observed in these samples, which might reflect a smaller burden of PJ organisms, thus resulting in false-negative results at the microscopic examination. Six of seven patients were patients not living with HIV  that causes coronavirus disease 2019 (COVID-19), because PJ—similar to other fungi —has emerged as a notable coinfection agent in COVID-19 patients [PJ-PCR assay was integrated into our clinical laboratory’s diagnostic respiratory workflow, which includes multiplex PCR-based panels newly commercialized to detect bacterial and viral agents of syndromic infections [Implementing a patients . At the fections . As showfections .PJ qPCR assays, the choice of target genes for amplification remains an open issue [PJ loads in BAL fluids. Very recently, Dellière et al. [PJ in respiratory samples. Therefore, the use of DHFR in our study might be questionable because of the (expected) lower sensitivity of a single-copy nuclear gene compared to multi-copy mitochondrial genes [PJ heat shock protein 70 (HSP70) gene to detect PJ DNA in BAL fluid samples of HIV-positive patients . The analytical sensitivity of their HSP70 real-time assay was ~five copies/reaction, but a cutoff value of ~10 copies/reaction was used to show  a clinical sensitivity of 98% and specificity of 96% for the diagnosis of PCP [PJ-PCR assay developed by us could represent the natural expansion of an existing molecular diagnostic platform in our laboratory [PJ detection [With regard to en issue . Recentlen issue  showed te et al.  found thal genes . Howeveral genes  in 2008.s of PCP . Furthers of PCP  optimizeboratory , which iPJ-PCR assay proved to be a sensitive and specific diagnostic tool for PJ infection in BAL fluid samples, thus emphasizing the increasing role of PJ qPCR assays as a clinical laboratory strategy to diagnose PCP in HIV and non-HIV patients [In conclusion, our patients ,7. We ho


Although the chikungunya virus was discovered more than 60 years ago, it has only really been studied since the outbreak in La Reunion in 2005–2006. Ten years later, between 2014 and 2015, the chikungunya virus spread throughout the Americas, affecting millions of people. The objective of this review is to describe the contributions of research on chikungunya virus infection gained from epidemic in the West Indies and the Guiana Shield.Prevalence data were similar to those found in the Indian Ocean or Asia during epidemics. Clinically, there is now a better understanding of the typical, atypical, and severe forms. Several studies have insisted on the presence of neurological forms of chikungunya infection, such as encephalitis or Guillain–Barré syndrome. Cases of septic shock due to chikungunya virus as well as thrombotic thrombocytopenic purpura were described for the first time. Given the magnitude of the epidemic and the large number of people affected, this has led to a better description and new classifications of chikungunya virus infections in specific populations such as pregnant women, the elderly, and children. Several studies also described the behavior of populations faced with an emerging disease.Aedes albopictus is already established in many parts of northern countries. A better understanding of the disease and its epidemic dynamics will foster better management, the crucial importance of which was demonstrated during the COVID-19 epidemic.Current epidemiological data from tropical regions highlights the risk of spreading emerging diseases at higher latitudes, especially concerning arboviruses, since the vector  Aedes sp. mosquitoes. It was first isolated in 1952 in Tanzania but was never really considered a virus of interest, despite several outbreaks in Asia and Africa, until the Indian Ocean outbreak in 2005–2006 [Chikungunya virus (CHIKV) is an arthropod-borne ARN virus belonging to the Alphavirus genus of the family of Togaviridae that is transmitted by 005–2006 . Knowled005–2006 . Because005–2006 . The objAedes albopictus possible, like for the ECSA and IOL strains, the Asian strain only has one vector, Aedes aegypti. After 15 years of successive CHIKV epidemics in India and Southeast Asia, the Asian strain has propagated through the East until reaching America, as if competing with the ECSA strain , East/Central/South African (ECSA), and Asian genotypes, and since 2005 the Indian Ocean Lineage (IOL) which comes from the ECSA one . During  aegypti .Fig. 1PIn 2005–2006, the island of La Reunion experienced a large-scale CHIKV epidemic, with rapid dissemination and large numbers of daily patients. CVI seroprevalence levels seemed dependent on the virus circulation mode. Seroprevalence was around 40% for epidemic modes  significantly reduced the transmission of CHIKV strains from the Americas, suggesting that colder temperatures may decrease the local transmission of CHIKV by European Ae. Albopictus [. Two studies reinforced the idea that Ae. aegypti was the main vector during CHIKV epidemics by detecting the virus in field-collected females, in Martinique and in French Guiana [Entomological studies related to the 2013–2014 chikungunya epidemic in the Americas focused on 3 main themes: vector competence , the presence or absence of CHIKV in mosquitoes collected in the field, and their resistance to insecticides, which is directly linked to interventions to avoid the spread of the epidemic. In 2011, a first study with it CHIKV . Other eenotypes , 28. Morh Guiana , 29, butAe. aegypti populations from French West Indies and French Guiana exhibit multiple resistances to organophosphates , and pyrethroids (deltamethrin), the effectiveness of vector control is compromised [In the French overseas territories and in Europe, deltamethrin, a pyrethroid compound, is the only allowed molecule to decrease adult mosquito populations. However, in 2014 during the CHIKV epidemic, malathion, an organophosphate compound, was exceptionally authorized for 6 months in French Guiana. However, the lack of evidence of its effectiveness in containing the epidemic, the mistrust from the population against this chemical, and data released on the cancerogenic effects of this molecule pushed local authorities to halt its use . Hence, promised –33.Knowledge acquired during the 2005–2006 period improved our understanding of the disease. Indeed, CVI was hardly known before the Indian Ocean epidemics. The outbreak in La Reunion Island led to the accumulation of knowledge about this infection and especially about new clinical forms. CHIKV was known to cause multiple joint pain and fever, but new forms, besides severe forms, called atypical forms, became increasingly reported. Most of the initially labeled “atypical forms,” for example dermatological forms with a rash and polymorphous eruption, became considered “common forms” during the following epidemics. In 2015, the World Health Organization (WHO) assembled an expert group to develop consensus definitions of the clinical forms of CVI. The resulting definitions described three clinical forms at the acute phase, based on clinical, epidemiological, and laboratory criteria •. (1) A As for several members of the alphavirus family, CHIKV have a strong neurological tropism. Most persons with neurological forms had encephalitis or myelitis. During the outbreak, the incidence of Guillain–Barré syndrome (GBS) was greater than usual, and for the first time, the association between CVI and GBS was shown ••. In MaIn Guadeloupe, 450 patients with confirmed CVI were admitted to the University Hospital of Pointe-à-Pitre. Of these, 25 had severe sepsis or septic shock and 12 died. This finding strongly suggested that CHIKV can, in rare cases, cause severe sepsis and septic shock syndromes, without any bacterial co-infection . It is oIn French Guiana, during the outbreak, most hospitalizations were common forms, driven by painful clinical presentations, and concerns due to the novelty of this infection . As for In tropical areas, a large number of arboviral or infectious diseases coexist, such as dengue virus (DENV), Mayaro virus (MAYV), leptospirosis or malaria, which may be difficult to diagnose, especially without biological tests , 46. BecChronic rheumatic complications after acute CVI were frequent. The term “chronic chikungunya syndrome” covers multiple etiologies, including reactivation of osteoarthritis and posttraumatic degenerative manifestations, fibromyalgia, spondylo-arthritis, and de novo polyarthritis. In Martinique, rheumatologists identified 13% of misdiagnosed patients, which conducted to late treatment initiation. They also noted that 21.1% of patients progressed to a chronic form of de novo seronegative rheumatoid arthritis requiring intensive therapy . SeveralFrom the beginning of the outbreak, physicians and authors reported that the definition of the clinical forms of CVI was not applicable to infants and the elderly . PediatrA study by Godaert et al. suggests that clinical presentations of the acute phase of CVI in the elderly differ from that in younger adults . The eldThe epidemic affected a third of the population in Guadeloupe in just a few months, including children. These studies highlighted a clinical presentation of pediatric CVI combining pain, fever, rash, and edema of the extremities. A study in Guadeloupe, including <24 months old infants, supported these findings; more than 80% of the infants had fever, 69% had a rash, and 48% had hands and/or feet edema . Half ofDuring the epidemic in La Réunion island in 2005/2006, there were frequent and serious neonatal forms of infection: neurosensory, neuropsychological, or neurological disorders such as epilepsy or cerebral palsy (52.6%). To try to prevent these complications, a phase I/II open-label, non-randomized, multicenter therapeutic trial conducted by Guadeloupe’s university hospital was conducted in 4 centers in the Antilles-French Guiana region during the 2014 CHIKV epidemic. The objective was to evaluate the tolerance and safety of intravenous administration of anti-CHIKV human immunoglobulins to newborns at risk of mother-to-child transmission (MTCT) of CHIKV (newborn of a viremic mother at the time of delivery or within 48 h post-delivery). These anti-CHIKV immunoglobulins were developed from CVI-convalescent plasma donors. In French Guiana, only one newborn baby was injected with immunoglobulins; two others were eligible but the parents refused. Unfortunately, the necessary number of inclusions was never reached due to the strict inclusion criteria, and the original question was never answered. A multi-centric trial including more countries in the region could be considered in the event of a new outbreak of CHIKV to increase the sample size and allow concluding on the efficacy of these immunoglobulins, given the potential severity of maternal–fetal infections. Parallel to the CHIKIVIG study, 15 MTCT cases were reported, 5 in Martinique and 10 in Guadeloupe. Seven MTCT cases developed serious clinical manifestations, 4 of which presented cardiovascular failure but no death was reported . A studyDuring the CHIKV outbreak and the 6 following months, of 1003 PLHIV in care in the center of infectious diseases in Martinique, 188 (94 men and 94 women) had confirmed CVI. Clinical presentation was common in 63% of the cases, and severe and atypical forms were scarce. During the acute phase, CD4+ and CD8+ T-cell  absolute numbers dropped significantly, but returned to pre-CHIKV values after the acute phase. Reassuringly, CD4 and CD8 T cell proportions did not decrease during the acute phase. CHIKV infection had no significant impact on this anti-retroviral-treated population .In view of the current importance of neglected tropical diseases, its impact on different populations has been studied, in comparison with dengue. The present results suggest that early in the epidemic, the populations most at risk for CHIKV infection were the most socially vulnerable populations in the poorest neighborhoods, whereas DENV appeared to have affected a richer population and richer areas •. SimilaA clinical trial was conducted at 6 clinical research sites located in Haiti, Dominican Republic, Martinique, Guadeloupe, and Puerto Rico, to evaluate the safety and tolerability of an investigational CHIKV-like particle (VLP) vaccine in endemic regions. This was a randomized, placebo-controlled, double-blind, phase 2 clinical trial to assess the vaccine VRC-CHKVLP059-00-VP (CHIKV VLP). A total of 400 healthy adults aged 18 through 60 years were enrolled between November 2015 and March 6, 2018. Of the 400 participants , 393 (98%) completed the primary safety analysis. All injections were well tolerated. Of the 16 serious adverse events unrelated to the study drugs, 4 (25%) occurred among 4 patients in the vaccine group and 12 (75%) occurred among 11 patients in the placebo group. The durability of the immune response was demonstrated through 72 weeks after vaccination. Phase 3 trials are now needed to assess clinical efficacy .Several million people were affected by CHIKV as it spread in more than 90% of American countries. That led to many behavior and dynamic studies of these populations. An interesting study performed in French Guiana about student perception of CHIKV at the beginning of the outbreak showed that CVI was associated with the highest scores in terms of perceived severity, or fear of its consequences . This score then dropped once the virus was in circulation because of a context of emergence of a new virus. These results also revealed that, unsurprisingly, the adoption of protective behaviors is a multi-factorial process that depends on both sociocultural and cognitive factors , 70. BecAedes albopictus, which is present at higher latitudes all around the world and is already responsible for outbreaks, such as CHIKV in India. Due to important human fluxes between America and Europe, and to the increasing presence of Aedes albopictus in the old continent, there is a non-negligible risk of diffusion of these arboviruses in Western Europe. Although Europe has been, until recently, spared by the phenomenon, it has been reported as suitable for dengue and chikungunya for some years now [Since 2013, the Guiana Shield and the West Indies have seen 4 different arboviral outbreaks: DENV, CHIKV, Zika virus, and yellow fever virus, not counting Covid-19. All of these diseases were public health issues. Several risks should be kept in mind. Mutations led to potential CHIKV transmission by ears now , 74. Tro


Molecular pharming or the technology of application of plants and plant cell culture to manufacture high-value recombinant proteins has progressed a long way over the last three decades. Whether generated in transgenic plants by stable expression or in plant virus-based transient expression systems, biopharmaceuticals have been produced to combat several human viral diseases that have impacted the world in pandemic proportions. Plants have been variously employed in expressing a host of viral antigens as well as monoclonal antibodies. Many of these biopharmaceuticals have shown great promise in animal models and several of them have performed successfully in clinical trials. The current review elaborates the strategies and successes achieved in generating plant-derived vaccines to target several virus-induced health concerns including highly communicable infectious viral diseases. Importantly, plant-made biopharmaceuticals against hepatitis B virus (HBV), hepatitis C virus (HCV), the cancer-causing virus human papillomavirus (HPV), human immunodeficiency virus (HIV), influenza virus, zika virus, and the emerging respiratory virus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have been discussed. The use of plant virus-derived nanoparticles (VNPs) and virus-like particles (VLPs) in generating plant-based vaccines are extensively addressed. The review closes with a critical look at the caveats of plant-based molecular pharming and future prospects towards further advancements in this technology. The use of biopharmed viral vaccines in human medicine and as part of emergency response vaccines and therapeutics in humans looks promising for the near future. Increasingly, plants are being used as vaccine biofactories for expressing antibodies as well as foreign antigens using genetic engineering technologies. Plants are inherently advantageous for the production of vaccines as they inexpensive to grow at a large scale in greenhouses or bioreactors. Orally delivered vaccines preclude the costs and the time involved in the purification of the antigens, by virtue of the plant biomass being directly consumed to confer immunity. Plants can express complex antigens while dispensing with the risk of carrying human pathogens or endotoxins inherent to the bacterial, insect, or mammalian cell systems . Plant mThere are three popular modalities of expressing heterologous protein molecules in plant cells, namely, stable nuclear expression, transient expression using non-replicative or viral vectors, and transplastomic expression within chloroplasts . Of thesThe foreign protein expressed as above could be specifically localized within cellular compartments such as chloroplasts, protein bodies, or endoplasmic reticulum (ER) . Among tA recent comprehensive review of the use of plant-based vaccines for the prevention and cure of human viral diseases is lacking. The current review addresses the use of plant-based vaccines and therapeutic antibodies for prophylaxis and therapy of human viral diseases. In this context are discussed vaccines against viruses such as HBV, HCV, Influenza Virus, HPV, HIV, SARS-CoV-2, and Zika Virus. Specifically, the current global incidence of diseases caused by these viruses, their molecular features relevant to vaccine design, and the latest developments in the generation of plant-derived vaccines against these viruses are addressed. The review closes with a note on the caveats of plant-based vaccines and future perspectives. Through this article, we hope to bring to light the idea that plant-based vaccines show great promise in combating viral diseases and will be the major avenue for low-cost and easy administration of vaccines in developing countries in the future.Hepatitis B Virus (HBV) is the causative agent of severe disease burden globally in the human population. Currently, nearly 257 million people have been affected by HBV infection . HBV cauThe DNA genome of the HBV codes for 3 surface proteins namely, the S , M (medium), and L (large) proteins all generated from the same ORF . The S sHBV vaccine has been one of the most successful, trailblazing vaccines generated in modern times. In 1984, the recombinant HBV S antigen (S-HBsAg) was successfully expressed as a VLP vaccine in yeast  which waNicotiana benthamiana. This venture was highly meritorious as the recombinant antigen exhibited the conformationally correct ‘a’ antigenic determinant and contained the full-length protein with disulfide-linked dimers that assembled into viable VLPs . Interestingly, antibodies against the SARS-CoV-1 cross-react with SARS-CoV-2 and therefore, biologicals and monoclonal antibodies already generated against SARS-CoV-1 could be used to combat Covid-19 infection . TherefoWhen using plant-based edible vaccines, it is important to consider the following factors : the plaThe technology of plant-based vaccines poses several risks to the environment such as gene transfer and undesired exposure to foreign antigens or proteins used as selectable markers . There aRecent research has proved that it is possible to use plants to regularly synthesize complex VLPs and antibodies in their fully assembled state that can stimulate prophylactic immunity or cause therapeutic effects towards disease-amelioration. The successful generation of antigen vaccine candidates for the HBV, HPV, and HIV viruses as well as the production of Influenza virus HA-only VLPs in plants have demonstrated the application of sophisticated transient expression technologies on industrial scales. In 1989, transgenic plants were for the first time used to produce antibodies. These antibodies can be conveyed orally, topically, or parenterally ,308. Pla


Aethina tumida  adults of honey bee pathogens belonging to the following groups: (i) bacteria (Paenibacillus larvae and Melissococcus plutonius), (ii) trypanosomatids (Lotmaria passim and Crithidia mellificae), and (iii) viruses . Specimens were collected from free-flying colonies in Gainesville  in summer 2017. The results of the molecular analysis show the presence of L. passim, C. mellificae, and replicative forms of deformed wing virus (DWV) and Kashmir bee virus (KBV). Replicative forms of KBV have not previously been reported. These results support the hypothesis of pathogen spillover between managed honey bees and the SHB, and these dynamics require further investigation.Knowledge regarding the honey bee pathogens borne by invasive bee pests remains scarce. This investigation aimed to assess the presence in  Aethina tumida (Murray 1867), the small hive beetle (SHB), is a coleopteran species belonging to the Nitidulidae family [Apis mellifera (western honey bee) colonies [a Murray 867, the e family , it is acolonies , and it colonies . Presentcolonies ,5,6,7,8,colonies ,10,11,12colonies  and creacolonies .Honey bees are exposed to pests and pathogens belonging to different groups , some of which are responsible for severe health impairment and colony collapse ,15,16,17Lotmaria passim and Crithidia mellificae are two trypanosomatid species capable of colonizing the digestive system of honey bees [Lotmaria passim has been described only recently [A. mellifera trypanosomatid [C. mellificae infections have been rarely observed [ney bees ,25. The ney bees ,27, and ney bees . Both paney bees ,29,30,31osomatid . Infectiosomatid , whereasobserved ,33,34,35Varroa destructor as it feeds on immature honey bees [Deformed wing virus (DWV) is a positive-sense ssRNA virus belonging to the Picornaviridae family within the Iflavirus genus ,37. Spreney bees . Howeverney bees ,44,45,46ney bees ,48,49.A. tumida infestations [Varroa feeding behavior [Kashmir bee virus (KBV) is a positive-sense ssRNA virus of the Dicistroviridae family within the Cripavirus genus ,51, consstations ,52,60, wstations ,61,62. Istations ,59,63 anbehavior ,65,66 maL. passim, C. mellificae, KBV, and DWV) in addition to pathogenic bacteria (Paenibacillus larvae and Melissococcus plutonius) and other bee viruses (ABPV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), sacbrood virus (SBV), chronic bee paralysis virus (CBPV), and slow paralysis virus ) in SHB specimens collected in Florida, USA in 2017. This is an important first step in determining the role SHBs may plan in the movement of pathogens between honey bee colonies.Herein, we aimed to assess the presence of the abovementioned pathogens . No co-infections with the two were detected.In the SHB individuals, no significant difference was found in the prevalence between p = 0.070). Viral coinfections were found only in one individual SHB, representing a significantly lower proportion of the positives .The frequencies of DWV- and KBV-positive individuals did not significantly differ  to specific virus genome sequences deposited in GenBank. For each virus, the same sequence was recorded in all positive samples. Phylogenetic analysis and pairwise distance analysis indicated the highest homology to DWV type A .A. mellifera and V. destructor from the USA was detected , C. bombi, Ascosphera apis, and Nosema ceranae [N. ceranae [P. larvae [To date, only a few instances of individual SHBs bearing bee pathogens have been reported. This is the case for samples from Mexico (positive for  ceranae ), Florid ceranae ), and ot. larvae ,20,22,24L. passim, C. mellificae, DWV, and KBV in SHB adults collected from free-flying colonies in Florida. Furthermore, all SHB samples that were positive for DWV and/or KBV contained replicative viral forms. Although DWV replication in SHB adults is not a new finding [The present study showed the presence of the honey bee pathogens  finding ,23, replThis is not the first time that DWV and KBV have been reported to infect non-Apis hosts. Replicative DWV was found in hornets (Vespa crabro) , Asian hA. mellifera. The phylogenetic analysis highlighted that the DWV genome detected in the SHB samples belonged to DWV type A, the less virulent genetic variant of this virus [The sequence analysis of DWV and KBV resulted in high identity rates to viral sequences identified in is virus . The KBVL. passim-positive individuals detected in this study  mirrors that reported in a previous survey in which C. bombi was also reported in larval SHBs [C. mellificae. Although C. mellificae is generally considered less spread than L. passim in honey bees [The prevalence of val SHBs . Additioney bees ,34,35, tP. larvae, M. plutonious, ABPV, IAPV, BQCV, SBV, CBPV, SPV major, or SPV minor. This coincides with the results of previous investigations showing low P. larvae [None of the samples was positive for . larvae  and SBV . larvae  loads inThe finding results highlight the need to clarify pathogen transmission between honey bees and SHB adults better. In the case of DWV, horizontal transmission occurs chiefly by the oral route . In thisThe articulate interactions above and active flying behavior  may brinIn summer 2017, one honey bee colony of mixed European origin was selected from an experimental apiary of the University of Florida  based on a conspicuous SHB infestation. No evident signs of other diseases could be detected. Forty SHB adults were randomly sampled alive from the colony combs and hive floor. Once in the laboratory, the collected specimens were randomly separated to compose one pool of thirty adults and ten individual beetle samples.All the SHBs were washed with 95% ethanol to remove possible external microbial contaminants. The ethanol was then allowed to evaporate at room temperature.A TissueLyser II  was used for 3 min at 25 Hz to crush all SHB samples in separate 2 mL Eppendorf tubes filled to the mark with RNase-free water. The resulting suspensions were then split into two equal aliquots from which nucleic acids were extracted (one for DNA and one for RNA).DNA and total RNA were extracted with DNeasy Blood & Tissue Kit (Qiagen) and RNeasy Mini Kit (Qiagen) as previously described ,67. All DNA and RNA extracts were stored at −80 °C until analysis. High pure sterile DNA- and RNA-free water was used as a negative control in all analytical steps.The extracted DNA was analyzed by real-time PCR to detect bacteria and trypanosomatids. The primers that were used are reported in For each target gene, a total reaction volume of 15 μL was prepared as previously described  using 2xAll RNA extracts were retro-transcribed by M-MLV reverse transcriptase  using a blend of oligo-d (T) primers and random hexamers following the manufacturer’s instruction. Five microliters of the obtained cDNAs were used as a template for the PCR reactions, performed using HotStarTaqPlus Polymerase Mix (Qiagen). Primers to amplify the viral genomes of the honey bee viruses investigated herein are reported in To evaluate the replication of the detected viruses, strand-specific RT-PCRs were performed using specific primers, as previously described . All cDNC. mellificae or L. passim and of those showing DWV or KBV infections were statistically compared with a bilateral Fisher’s exact test under the null hypothesis of equality. The same test was also used to compare the frequency of multiple vs. single viral infections. Due to the small number of samples, the test for independence χ2 was not used in this case.The prevalence of the individuals that were positive for L. passim and C. mellificae may colonize, and the viruses DWV and KBV successfully infect A. tumida adults. Additional studies are needed to determine whether these pathogens generate clinical evidence and signs of infection in SHBs. The horizontal and vertical transmission routes of these pathogens in/between SHBs should also be clarified, as well as the potential, if any, of these pathogens to limit SHB populations in the wild.This investigation suggests that the honey bee trypanosomatids Finally, further research is needed to elucidate the epidemiological role that SHBs play in pathogen transmission to honey bees and other insects as a possible dead-end host or vector.


Togaviridae, genus Alphavirus, found in North America and associated with freshwater/hardwood swamps in the Atlantic, Gulf Coast, and Great Lakes regions. EEEV disease in humans is rare but causes substantial illness and death. To investigate the molecular epidemiology and microevolution of EEEV from a fatal case in Alabama, USA, in 2019, we used next-generation sequencing of serum and cerebrospinal fluid (CSF). Phylogenetic inference indicated that the infecting strain may be closely related to isolates from Florida detected during 2010–2014, suggesting potential seeding from Florida. EEEV detected in serum displayed a higher degree of variability with more single-nucleotide variants than that detected in the CSF. These data refine our knowledge of EEEV molecular epidemiologic dynamics in the Gulf Coast region and demonstrate potential quasispecies bottlenecking within the central nervous system of a human host.Eastern equine encephalitis virus (EEEV) is an arbovirus in the family  Culiseta melanura mosquitoes as the principal mosquito vectors  causes disease in equids, domestic birds, and humans  resulting from EEEV infection in humans are seasonal and are typically reported during July–October . All methods followed manufacturer’s recommended protocols unless otherwise noted.https://www.biofiredx.com), were negative; CNS lymphoma also was ruled out. The patient received broad-spectrum antimicrobial drugs and intravenous immunoglobulin, but her condition did not improve. She lapsed into a coma and never regained consciousness. Life support was discontinued, and she died 43 days after initial illness onset.The patient was a woman in her 60s who had lymphoma, for which she was receiving rituximab. She was active and working outdoors until September 2019, when she experienced lethargy and malaise. Approximately 1 week after symptom onset, she was found at home unresponsive and was transferred to the hospital. Her evaluation at the hospital indicated suspected viral encephalitis, but test results for numerous viral and bacterial etiologies, including testing of cerebrospinal fluid (CSF) by BioFire panel . We performed real-time reverse transcription PCR (RT-PCR) to detect viral RNA from the endemic encephalitic arboviruses, West Nile virus (WNV), and EEEV. We performed EEEV real-time RT-PCR as previously described . For whole-genome sequencing, we used the Ion Torrent Personal Genomic Machine system. We prepared libraries by using the Ion Plus Fragment Library Kit barcoded with the Ion Xpress Barcoding Kit and quantified by using the Ion Library TaqMan Quantitation Kit . We prepared sequencing templates by using the Hi-Q View OT2 kit with the Ion One Touch 2 system (both by Thermo Fisher Scientific) and completed sequencing by using a Hi-Q View Sequencing Kit (Thermo Fisher Scientific). We loaded templated ion sphere libraries onto 318 Chips V2 and sequenced them by using the Ion Torrent PGM system (both by Thermo Fisher Scientific). We deposited virus sequences from this study into GenBank (accession nos. MT782294 and MT782295).We generated complementary DNA by using the Ovation RNA-Seq System V2  and completed alignments by using the de novo assembled consensus sequences (GenBank accession nos. MT782294 and MT782295) and Bowtie2 version 2.3.4.1 (https://github.com/BenLangmead/bowtie2) with paired-end, sensitive local parameters. We removed PCR duplicates with MarkDuplicates . We calculated mutational frequency by using custom R scripts over possible nucleotide variables  according to the method described by Matsushita et al.  into the CLC genomic workbench version 12 (QIAGEN) and assembled genomes by using de novo assembly. We identified viral contigs by using BLAST (We inferred phylogenies by using MEGA v7 (https://www.megasoftware.net). We used Bayesian inference with BEAST (https://beast.community) and a Markov chain Monte Carlo approach of 100 million generations to confirm the maximum-likelihood tree topologies. We completed phylogenetic inference by using a maximum-likelihood algorithm with 1,000 bootstrap replicates and the general time-reversible model with gamma distributed rate variation and invariable sites, as determined by the model fit test in MEGA , for further evaluation of potential arboviral etiologies. Serum was negative for IgM against La Crosse virus, Jamestown Canyon virus, Powassan virus, and EEEV; neutralizing antibodies against EEEV were not detected. Test results for WNV and Saint Louis encephalitis virus IgM performed at another laboratory were reportedly negative. CSF was negative for IgM against Powassan virus and EEEV. Because the patient was receiving rituximab therapy, which can suppress antibody production, real-time RT-PCR testing was performed and found to be negative for WNV RNA; however, EEEV RNA was detected in serum and CSF. Quantification cycle (Cq) values were 27.9 (serum) and 20.5 (CSF).Complete genome sequences of EEEV were obtained from each specimen: serum (520× coverage) and CSF . The EEEV consensus sequence from the serum shared 99.79% nt identity with EEEV sequences from Florida isolated in 2010 (GenBank accession no. KU840313) and 2014 (GenBank accession no KU840338). The EEEV consensus sequence from the CSF shared 99.81% nt identity with these same reference sequences. Maximum-likelihood phylogenetic analysis supported these findings and placed the derived sequences from the serum and CSF in a well-supported clade with EEEV isolated from northern Florida in 2013 and 2014 . These dIntrahost variability was measured by detecting single-nucleotide variants (SNVs) in each specimen . We deteThree consensus nucleotides found in the serum were not found in the CSF; however, the corresponding minor SNV populations at positions 775 (NSP1), 5291 (NSP3), and 8728 (E2) in the serum were detected in the CSF with 100% frequency. These consensus level viral populations in the serum resulted in 1 synonymous nucleotide substitution at nt 5921 in NSP3 and 2 nonsynonymous changes at 775 in NSP1-I251T and 8728 in E2-L62S compared with sequences from the CSF and reference EEEV isolates (EEEV causes a severe meningoencephalitis in equids, domestic birds, and humans. In 2019, the number of reported cases in humans increased substantially; 38 cases were confirmed in contrast with the annual average of 8. We confirmed EEEV infection in an immunocompromised person; deep sequencing of the viral RNA directly from the patient’s serum and CSF showed genetic relatedness to recent EEEV isolates in northern Florida and uniquely demonstrated EEEV intrahost variability in a human.Very few sequences of EEEV isolates from Alabama have been described. The sequences from our study cluster within the FL4 (Advances in sequencing have improved our knowledge of intrahost virus variation, or quasispecies, in several arboviruses, including WNV (In addition to intrahost quasispecies diversity, we also observed variation in the consensus sequences derived from each specimen. The consensus sequence derived from serum had 2 nonsynonymous nucleotide changes compared with that of the CSF. One amino acid change, NSP1-I251T, is located in an amphipathic peptide that has been shown to play a role in the membrane association of NSP1 (When evaluating both intrahost virus variants and consensus-level majority variation, we found decreased variation in the CNS is not altogether unexpected because of potential bottlenecks and selection. It is noteworthy that consensus level amino acid changes observed in the serum are not reflected in the CNS. Stochastic generation of virus variants and lack of immune selection cannot explain fixation of 2 nonsynonymous amino acid changes in the peripheral compartment. It is possible that this scenario fits the quasispecies model of cooperative interaction in the virus population as described for poliovirus (Cs. melanura mosquitoes with high EEEV titers (The virus sequences generated in this study were derived from serum and CSF specimens from an immunocompromised person with no detectable serologic antibody response to EEEV, probably because of rituximab therapy for lymphoma. Patients receiving B cell–depleting monoclonal antibody therapy may be predisposed to severe neuroinvasive disease and death after arbovirus infection. Cases have been associated with prolonged RNA detection in serum and CSF or brain tissue and lack of serologic response (


Fighting bulls that participate in bullfighting face energy and metabolic demands due to the high intensity and duration of the exercise performed. Under these conditions, specific corporal mechanisms, such as the acid–base balance, are affected, causing metabolic acidosis. However, fighting bulls also undergo muscular injuries, physiological changes, and high enzyme concentrations that reflect the stress to which they are subjected, and in some bulls, bullfights can trigger electrolytic imbalances that include hypercalcaemia, hypermagnesaemia, and hyperphosphataemia, exacerbated by muscular necrosis and myoglobinuria.During bullfights, bulls undergo physiometabolic responses such as glycolysis, anaerobic reactions, cellular oedema, splenic contraction, and hypovolemic shock. The objective of this review article is to present the current knowledge on the factors that cause stress in fighting bulls during bullfights, including their dying process, by discussing the neurobiology and their physiological responses. The literature shows that biochemical imbalances occur during bullfights, including hypercalcaemia, hypermagnesaemia, hyperphosphataemia, hyperlactataemia, and hyperglycaemia, associated with increased endogenous cortisol and catecholamine levels. Creatine kinase, citrate synthase, and lactate dehydrogenase levels also increase, coupled with decreases in pH, blood bicarbonate levels, excess base, partial oxygen pressure, and oxygen saturation. The intense exercise also causes a marked decrease of glycogen in type I and II muscle fibres that can produce myoglobinuria and muscular necrosis. Other observations suggest the presence of osteochondrosis. The existing information allows us to conclude that during bullfights, bulls face energy and metabolic demands due to the high intensity and duration of the exercise performed, together with muscular injuries, physiological changes, and high enzyme concentrations. In addition, the final stage of the bullfight causes a slow dying process for an animal that is sentient and conscious of its surroundings. Bos taurus, which includes all breeds of bovines involved in various zootechnical practices [tienta in Spanish), while for bulls, it occurs after an outstanding performance where the bull’s life is spared once the bullfight is over. One goal of these practices is to identify bulls that will fight when provoked by a person using some kind of lure [Fighting bulls are considered a specialized breed of cattle that has its origins in the species ractices . As with of lure . During bullfights, the physiometabolic responses correspond mainly to the presence of different types of stressors, such as physical , envirotercios: tercio de varas, tercio de banderillas, and tercio de muleta. In the first stage, a lance is stabbed into the bull’s hump, limiting its mobility [The bullfight is divided in three stages called mobility . The injmobility ,12. The mobility  determinbanderillas) into the bull’s shoulders and/or hump. This action aggravates the muscle damage already inflicted by the lance because every movement the bull makes while charging the matador and his red cape moves the flags inside the wounds. Their sharp points lacerate muscles in different directions, causing additional haemorrhages [In the second stage, the matador stabs six flags (orrhages .puntilla (short knife) to end the fight, which enters near the first and second cervical vertebrae, it will damage the motor nerves, causing the bull to fall with its limbs extended. If the injury is made near the atlantooccipital joint, the bulb is cut the spinal cord and its caudal nerve projections, possibly leading to the same result [In the third stage, the matador uses a sword to kill the bull by causing profuse bleeding in the thoracic cavity, either by piercing the pleura to cause pneumothorax and the consequent respiratory insufficiency, or the lung or right bronchia, allowing blood to leave the lung, enter the bronchia, and reach the trachea, oesophagus, and upper respiratory pathways . In moste result .The death of bulls during bullfights—whether by asphyxia or exsanguination—occurs while the animal is fully conscious because the brainstem and/or brain cortex remain intact ,19.Regarding fighting bulls, studies have determined that aggressiveness  and ferocity  have strong genetic  and enviBos taurus, including fighting bulls) and other species eventually become habituated to novel environmental conditions and reduce their behavioural reactivity [The temperament of the animal could be another factor that affects the quality of its death. However, studies have determined that even bovines with harsh temperaments (activity .Bos taurus brachyceros) under these circumstances, though similar results have been determined in Bos indicus steers [The typical handling practices used with fighting bulls require minimising or eliminating contact with humans. In part for this reason, no scientific studies have yet documented the peculiarities of this breed of bulls  , adaptivOne example of a stressor of physical origin is fatigue due to transport or other causes of strenuous exercise , which rStress responses consist of a series of physiological and behavioural mechanisms designed to promote adaptation and restore homeostasis in the individual , includiBos indicus, excitable Brahman heifers had significantly higher serum cortisol concentrations than docile ones, which negatively affected serum LH concentrations [3, BE, PO2, sO2), others remained within normal ranges , and the rest increased  compared to normal reference values for other bovine species. Clearly, these events could trigger multiple metabolic responses in fighting bulls, including decreases in the acid–base balance and blood pH [In trations . Similartrations  found a trations  and the trations . In relatrations  observedblood pH , as occublood pH . It is wblood pH , so it iblood pH  could beAnimals modify their behavior as a defense mechanism to cope with or avoid stressors . The behCattle can perceive sounds of much higher frequencies than humans, and may perceive the noise in the fighting ring as a threat, which is another stressor that can affect their behavior, producing fear , especiaThe vocalizations of the animals can provide an important source of information about its physical and psychological condition . For thiIn this context, the aim of this review is to present current knowledge on the factors that cause stress in fighting bulls during bullfights, including their dying process, by discussing the neurobiology and physiological responses to which they are subjected. Due to the scarcity of scientific studies of these topics, comparisons to other breeds of cattle are included where appropriate.puya) and flags (banderillas) are stabbed into their bodies, damaging skin, muscles, arteries, veins, and connective tissue, all of which contain physiological sensors called nociceptors. These sensors generate electrical impulses that send signals to the central nervous system, where cattle could detect them as pain [2+, K+, or Na+ ionic channels to create the electrical impulses that travel through neuronal axons to carry the nociceptive signal, successively, to the spinal cord, brainstem, thalamus, and cerebral cortex [Animals’ brains are irrigated through the basioccipital plexus and carotid arteries, which supply blood primarily to the occipital lobe of the cerebral cortex, and the basilar arteries that carry blood rostrally ,61. Duri as pain ,62,63. T as pain . The pro as pain  generate as pain . When acl cortex . In thisl cortex . In addil cortex , first al cortex . This “sl cortex . Transmil cortex . These el cortex ,71 , 0.674 for heifers (p < 0.0001), and 0.696 for steers (p < 0.0001). Thus, they concluded that the percentage of eye white in cattle can be used as a quantitative tool that requires minimal equipment to assess temperament in beef cattle, and that it provides an objective method for temperament selection. These indicators might, therefore, also be used as non-invasive tools for evaluating the degree of excitation in fighting bulls, though under different conditions.Some studies of beef and dairy cattle have used the extension of eye white and ear position as indicators when evaluating the emotional states of animals. Battini et al. , for exaFighting bulls are reared in extensive environments with minimal exposure to humans. Fear is arguably the most investigated emotion in domestic animals. It is a potent stressor. The highly variable results are likely due to different levels of physiological stress such as fear stress, including handling, contact with people, or exposure to novelty. However, we lack scientific studies of this breed that evaluate the degree of expression of positive and negative emotions under different conditions. Since the stimuli that can cause fear in bulls—and other animals—during fights include confronting a closed, unfamiliar environment, isolation, separation from conspecifics, exposure to predators or aggressors, the absence of escape routes or refuge, and the presence of harmful stimuli in conditions that preclude escape . Pain anbanderillas (n = 110 bulls), and at the end of the fight when the bull is killed (estoque) (n = 80 bulls). Their post-event findings showed an increase in the concentration of POMC—a precursor hormone of the β-endorphins and methane cephalins—that was six times higher in the animals after the estoque compared to the concentrations determined when the bulls entered the ring (p < 0.01). With respect to serum ACTH and cortisol levels, that study found higher concentrations in the bulls immediately after leaving the ring, while the lowest values were determined for the samples drawn and analyzed after the final estocada  (p < 0.01).It is possible, however, that the stress provoked could inhibit the transmission of pain stimuli in the brain and spinal cord . To becoAccording to Fernández and Villalón’s  anatomicGomariz et al.  attempteIn addition to the injuries visible at first sight during a bullfight, there are conditions in fighting bulls that could exacerbate muscular and skeletal damage. The study of 120 fighting bulls by Lomillos-Pérez and Alonso de la Varga  detectedbanderillas stage. Dávila et al. [Martínez  and Lomia et al.  point ou+ ions from the intracellular space to the blood. This mechanism aims to self-compensate and cause isotonic dehydration and hyperkalaemia, but the resulting hydroelectrolytic imbalance produces vascular dysfunction. At the same time, other compensating mechanisms are activated to lower arterial pressure. This is detected initially by baroreceptors in the aortic arch and carotid sinus, leading to activation of the sympathetic system that secretes catecholamines, angiotensin II, and the antidiuretic hormone to preserve cardiac output and maintain adequate cerebral and cardiac perfusion [The wound inflicted by the lance causes a loss of blood volume, the first event in a series that ends in hypovolemic shock . Hypovolerfusion .Other essential responses of the fighting bull’s organism during a bullfight include splenic contraction, when erythrocytes are mobilized towards the zones where additional oxygen support is required with increased haematocrit due to the dehydration the animals may present as a consequence of the intense physical activity performed in a short period .Animals are subject to various environmental and behavioural stressors that affect their survival and physical state . To respThe exertion demanded of bulls during the 15–18 min that bullfights usually last can be considered similar to that performed by athletic animals forced to perform enormously intensive exercise ,98. This3−), base excess (BE), oxygen partial pressure (PO2), and oxygen saturation (sO2)—decreased, while others—sodium (Na+), potassium (K+), calcium ion (iCa), and haematocrit (Htc)—remained within normal limits [2, haemoglobin (Hb), and lactate, were above normal values. A study by Muñoz-Juzado et al. [gluteus medius and semitendinosus muscles of bulls aged 1 to 3 years. Those authors found that the highest oxidative muscular potential was manifested in the 2-year-old bulls and that glycolytic capacity increased progressively with age. This contrasts with other bovines, where a reduction in the oxidative potential occurs from the time of birth onwards [Bullfights demand an enormous physical effort by the bulls, so these animals must be in optimal health conditions before participating. The intensity of the fight triggers significant metabolic alterations that are observable after the event, including haematological changes (increased red blood cells and haematocrit), and elevated peroxides and lactic acid production that reduce concentrations of muscle glycogen and pH . Accordil limits . Other ao et al.  evaluate onwards . These f onwards . When co onwards . Likewis onwards . If, in  onwards . FinallyAccording to García-Belenguer et al. , fightinThe existing information allows us to conclude that bulls face energy and metabolic demands during bullfights due to the high intensity and duration of the exercise performed, together with muscular injuries, physiological changes, and high enzyme concentrations. In addition, the final stage of the bullfight causes a slow dying process for an animal that is sentient and conscious of its surroundings. Unfortunately, due to the scant literature on this breed, many gaps exist in the available information; more specific information, such as physiological evaluations, could help verify these effects.