Three-time Grand Slam champion Stan Wawrinka came up short in the final of the Open Aix Provence Crédit Agricole against Borna Coric on Sunday. The former World No. 3 was beaten by Borna Coric, who continues to dominate the ATP Challenger Tour this year Coric overcame a series of rain delays and a 3 hour 11 minute battle to emerge victorious 6-7(5) 6-3 7-6(4) and capture the trophy The Croatian currently leads the tour with four ATP Challenger titles this season Wawrinka has been going through torrid times in 2025 the Swiss legend beat the likes of Nishesh Basavareddy Borna Gojo and top seed Alexei Popyrin en route to the final Wawrinka lost the match in three tight sets READ ALSO: Jack Draper Proves All-Round Ability By Breaking Huge British Record at 2025 Madrid Open Borna Coric went toe-to-toe with Wawrinka from the beginning of the match who holds a 22-3 record at the Challenger level this season accumulated three straight championships between February and March He had a 16-match winning streak at one point which was coincidentally ended by Wawrinka himself Coric was able to turn the tables on his 40-year-old opponent to win the title Had Stan Wawrinka won the Open Aix Provence trophy he would have become the oldest Challenger champion in the history of the game ‘Stan the man’ pushed Coric to a deciding set tiebreak A series of forced and unforced errors off his magical Yonex wand resulted in Borna Coric getting bragging rights “I’m happy to start to play better tennis in the past couple of months,” laid out an excited Coric after winning the title The former US Open quarter finalist is now up to No that’s the most important thing for me – and also to stay healthy.” Coric now has a 2-4 head to head record against Wawrinka at all levels READ MORE: Jannik Sinner Eyes Grand Comeback Following Return From 3-Month Suspension A passionate sports fan through and through I am currently pursuing my MA in Global Sports Journalism I specialise in tennis and football writing at The PlayOffs and I have prior experience working at EssentiallySports and Sportskeeda sport was my safe space right from my childhood 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provided by betting platforms responsible betting is the key to a safe and enjoyable gaming experience DISCLAIMER: This site is 100% for entertainment purposes only and does not involve real money betting COPYRIGHT © 2025 - THE PLAYOFFS - SHARE THE PASSION FOR SPORTS AND BETTING Borna Coric became the first player to win four ATP Challenger Tour titles this season as he defeated Stan Wawrinka in a legendary final in Aix-en-Provence Alex Michelsen was a surprising clay champion at the other Challenger 175 event in Estoril with four further winners crowned in the lower categories Borna Coric was already leading the ATP Challenger Tour title count this season alongside Emilio Nava (Lugano He secured another run at the 175 category to ensure his return to the Top 100 3 seed Mariano Navone in a straight-set win that was far less comfortable than the scoreline suggested before battling from 4-6 0-2 down (and three break points that game) against Ignacio Buse in the semifinals Stan Wawrinka had already played one Challenger event earlier this season in Naples losing in the quarterfinals to Luciano Darderi 26 and top seed Alexei Popyrin in the second round the 40-year-old pulled through that encounter before defeating Nishesh Basavareddy and Borna Gojo he would need to go through both Croatian Bornas in a row (Gojo and Coric) he secured a chance to become the oldest Challenger Tour champion ever at 40 years and 1 month Wawrinka vs Coric was already a final on the ATP Tour in Chennai 2016 Now it was time for a Challenger championship match between them The Swiss was more clutch in the opening set tie-break the younger opponent dragged him around the court more and more and held his own in the backhand down-the-line battle even when it seemed he was already out of steam and had to save a break point at 2-4 in the third set But Coric still claimed his 7th Challenger title 6-7(5) 6-3 7-6(4) becoming the first player to reach 4 trophies at this level in 2025 while Wawrinka pulled out of Francavilla al Mare Andrea Pellegrino had previously made the semifinals from qualifying in another big clay Challenger this season (CH 125) now producing another run from a similar position at the highest possible category at this level He defeated three Top 100 opponents in a row including the biggest win of his career against World No The toughest matches had him pegged back by Dusan Lajovic or Nicolas Jarry but the Italian quickly regrouped mentally early in both deciding sets Alex Michelsen had played just one match on European clay this season and his start to the campaign in Estoril also wasn’t looking that promising But the American got out of a pickle in the second set against Giovanni Fonio and suddenly began playing as if he were on a hard court he posted impressive straight-set wins over Luca Nardi and Miomir Kecmanovic to make the final Pellegrino’s body language wasn’t the best in the final and it didn’t take long for Michelsen to put him in a state of being too negative But that was how the American played it with a good mix of aggression variety – seemingly all coming at the right time Along with the increased number of free points on serve it was always Michelsen who had the edge and he claimed his 3rd Challenger title (first on clay of any kind) 6-4 6-4 and now has a shot to be seeded for the French Open while Pellegrino chose to take some rest and withdrew from Francavilla al Mare Tomas Barrios Vera has already reached the Punta del Este final and won the Campinas title this season After a couple of rather average appearances in the green clay swing in the United States the Chilean traveled to Europe and played the event in Mauthausen in quicker clay conditions that really suit his game The stacked draw had him start against Jurij Rodionov before surviving a thriller against Martin Landaluce (saved 19/23 break points) before properly getting his game going Cristian Garin had already reached a Challenger semifinal in Merida this season but fell out of the Top 200 after losing points for ATP 250 Estoril and Munich runs from last season He bounced back in Austria with a brutal defeat over Taro Daniel in the opening round before winning three consecutive matches in deciding sets He came very close to defeat against Roman Andres Burruchaga in the semifinals saving four break points at 3-6 0-3 and only losing one further game until the end of the match It was a high-octane start from Barrios Vera and he was in basically every return game in the opening set with Garin’s chances looking quite bleak who did not let his compatriot dominate like that and forced him to dial down that aggression and they weren’t all that one-sided anymore Garin claimed his 5th Challenger title (first since 2018) 3-6 6-1 6-4 and returns to the Top 200 right after falling out Barrios Vera will now play qualifying in Rome while the champion barely missed out (#1 alternate) Tristan Schoolkate won his maiden Challenger title in Guangzhou last year and came into the event after reaching the Gwangju semifinals (which allowed him to bounce back from a surprising defeat in Busan) the opening round turned out to be difficult again with the Australian getting pushed hard by Ryan Peniston Besides another battle with Gwangju runner-up Alibek Kachmazov Terence Atmane won the first tournament of the Asian swing in Busan before retiring to the aforementioned Kachmazov in the opening round in Gwangju that was just the fatigue factor as the Frenchman needed a lot of physical resilience to get through to the final particularly when he fought back from 1-6 0-3 (double-break) down against Marat Sharipov in the quarterfinals he stopped the American from breaking the top 100 it would always come down to who performs better in key moments Atmane had the early lead and even got to 6-3 4-2 before Schoolkate broke for the first time in the match and forced a second-set tie-break But the Frenchman came up with a well-timed forehand pass at 4-all to secure his 4th Challenger title 6-3 7-6(4) winning two trophies in three weeks again after doing it in China in 2023 (Zhangjiagang and Guangzhou) He’s back inside the Top 120 and about 100 points away from a Top 100 debut Hady Habib went on a six-match losing streak after the Australian Open although that was largely caused by his appearing in only high-profile events (including ATP 500 main draws in Doha and Dubai) The Lebanese hit the Challenger Tour again with the start of the European clay season he played much better in Ostrava with quality wins over top-seeded Vit Kopriva or the veteran Daniel Evans He dropped his only set in the first four matches to Oriol Roca Batalla Piros was always going to be on the back foot power-wise but it was his duty to counter-punch actively and ensure Habib doesn’t have a clear upper hand off the ground That’s exactly what he did with some great defensive shotmaking and his domination in baseline rallies is best shown by the fact that he didn’t need to save a break point in the final Piros claimed his 6th Challenger title 6-3 6-2 and while he missed out on the Roland Garros qualifying cut with the champion taking a special exemption Santiago Rodriguez Taverna was the runner-up in San Miguel de Tucuman the week before Porto Alegre and kept up the good form Getting a quick retirement from Valerio Aboian helped conserve some energy but he was clearly doing alright in that department with battles against Ivan Guido Justo or Matheus Pucinelli de Almeida The 25-year-old made multiple Challenger finals in one season for the second time in his career after two appearances in January 2022 Nikolas Sanchez Izquierdo produced some solid results in stacked ITF events recently winning the title at M25 Tarragona and losing in the M25 Reus final The Spaniard had been 0-5 in Challenger semifinals without ever winning a set and locked up another opportunity to reach a milestone in Porto Alegre It was almost a San Miguel de Tucuman rematch in the final as the 26-year-old stopped Barrena 1-6 7-5 6-4 to not allow his opponent to take on Rodriguez Taverna again Rodriguez Taverna was still heavily struggling to figure out his game often overplaying the dropshot or going all-out on the forehand side He almost blew a 4-0 lead in the second set with Sanchez Izquierdo restoring the two breaks Things were looking bleak for him as he went 0-3 (two breaks down in the third) but despite many further obstacles along the way the control over the rallies was in his hands That helped Rodriguez Taverna secure his second Challenger title 4-6 6-4 7-6(6) with both finalists playing Santos next Adam Walton (Wuxi) will be the only Top 100 player in action Main Photo Credit: Susan Mullane – USA TODAY Sports It promises to be an exciting opening day of WTA Rome action ATP Masters 1000 Rome 1/64-Finals Diallo – Giron: Time TBA H2H: first meeting Gabriel Diallo has won four of his last five matches ATP Masters 1000 Rome 1/64-Finals Nishioka – Struff: Time TBA H2H: 2-1 Yoshihito Nishioka has lost three of his last five matches ATP Masters 1000 Rome 1/64-Finals Gaston – Jarry: Time TBA H2H: first meeting Hugo Gaston has lost three of his last five matches Metrics details such as Borna disease virus 1 (BoDV-1) and variegated squirrel bornavirus 1 are zoonotic pathogens that cause fatal encephalitis in humans another mammalian orthobornavirus with high genetic homology to BoDV-1 is believed to share the same geographical distribution as BoDV-1 indicating its potential risk to human health we re-evaluated the whole-genome sequence of BoDV-2 and established a reverse genetics system to investigate its virological properties Compared to the published reference sequence we identified two nonsynonymous nucleotide substitutions in the large (L) gene one of which was critical for restoring polymerase activity enabling the successful recovery of recombinant BoDV-2 (rBoDV-2) we identified two nonsynonymous single-nucleotide polymorphisms (SNPs) in the L gene and one in the phosphoprotein (P) gene Substitution of these SNPs significantly enhanced the growth ability of rBoDV-2 our studies demonstrated that BoDV-2 does not induce superinfection exclusion in cells allowing the persistence of low-fitness genome variants for an extended period of time These findings help to characterize the virological properties of BoDV-2 and shed light on how bornaviruses maintain genetic diversity in infected cells the potential of BoDV-2 to infect and cause disease in humans remains unclear Considering that BoDV-2 isolate No/98 was isolated from a pony that displayed fatal neurological disorders and that BoDV-2 is genetically more closely related to BoDV-1 than VSBV-1 it is likely that BoDV-2 may also possess zoonotic potential we aimed to investigate the virological properties of BoDV-2 using recombinant viral techniques and therefore redetermine the genome sequence of BoDV-2 isolate No/98 which has been maintained in persistently infected cells We found two nonsynonymous nucleotide substitutions in the L gene compared to the published sequence One of these substitutions was crucial for restoring the polymerase activity of L allowing successful recovery of rBoDV-2 through reverse genetics we identified two nonsynonymous single-nucleotide polymorphisms (SNPs) in the L gene and one in the P gene Substituting these SNPs significantly enhanced the growth ability of rBoDV-2 our study demonstrated that BoDV-2 does not induce superinfection exclusion in cells leading to long-term maintenance of low-fitness genome variants in persistently infected cells A Sequence analysis of total RNA extracted from BoDV-2 isolate No/98-infected Vero cells Compared to the reference sequence (RefSeq) of BoDV-2 (accession no substituted nucleotides in the reassessed sequence are represented in bold orange and their percentages of the total are indicated Concomitant amino acid changes are also represented in bold orange The putative RNA-dependent RNA polymerase (RdRp) domain polyribonucleotidyltransferase (PRNTase) domain and C-terminal domain (CTD) are colored blue C RACE analysis of the 5’ and 3’ terminal sequences of both the genome and antigenome of BoDV-2 The 5’ and 3’ ends of total RNA extracted from BoDV-2-infected Vero cells were ligated with each oligoRNA BoDV-2-specific 5’ and 3’ terminal sequences were amplified via RT-PCR and analyzed via direct sequencing The border between the viral terminal sequence and ligated oligoRNA is indicated by the dotted line The analyzed terminal sequences are indicated schematically D Comparison of the reassessed genomic terminal sequences of BoDV-2 with the reference sequence A Schematic representation of the BoDV-2 L gene variants inserted into the expression plasmid and the full-length BoDV-2 cDNA plasmid The positions of the substituted nucleotides compared to the reference sequence are indicated by red bars B Detection of BoDV-2 L variants by western blotting analysis Whole-cell lysates were prepared from 293T cells transfected with the indicated plasmid The primary antibodies used for detection are indicated to the right of the panels Alpha-tubulin was used as the loading control The original scans of these blots are shown as supplementary Figure 1 C BoDV-2 minireplicon assay using the indicated BoDV-2 L variants and BoDV-2 N and P as helper plasmids Data are presented as the means ± standard errors of the means (± SEM) of three biologically independent experiments One-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test were performed for statistical analysis Reverse genetics was performed by transfecting a BoDV-2 cDNA plasmid possessing the indicated substitution in the L gene and five helper plasmids into HEK293T cells transfected HEK293T cells were cocultured with uninfected Vero cells Vero cells were subjected to immunofluorescence assay (IFA) and the infected ratio was measured rBoDV-2 was prepared from cocultured Vero cells and inoculated into uninfected Vero cells Cells were subjected to IFA at 72 h post inoculation and virus titer was calculated F Schematic representation of the growth kinetics assay Vero cells infected with BoDV-2 isolate No/98 or with rBoDV-2 LRG were cocultured with uninfected Vero cells at a ratio of 1 to 19 and passaged every 3 or 4 days G The percentage of infected cells was determined by IFA using an antibody against BoDV N H The copy number of viral RNA was measured via RT-qPCR using the total RNA extracted from each cells indicating that a difference in the nucleotide at position 2762 is detrimental to the polymerase activity of BoDV-2 L These results suggest that the growth ability of rBoDV-2 is not only determined by the L2762 and L3334 substitutions even though both substitutions induce high polymerase activity in the minireplicon assay B BoDV-2 minireplicon assay using the indicated BoDV-2 L variants and BoDV-2 N and P as helper plasmids Values for Gluc activity were normalized to that of the LRG variant as a relative value of 1.0 Data are presented as the means ± SEM of three biologically independent experiments One-way ANOVA and Dunnett’s multiple comparisons test were performed for statistical analysis D Growth kinetics assay of rBoDV-2 variants Vero cells infected with the indicated rBoDV-2 were cocultured with uninfected Vero cells at a ratio of 1 to 19 and passaged every 3 or 4 days C The percentage of infected cells was determined by IFA using an antibody against BoDV N D The copy number of viral RNA was measured via RT-qPCR using total RNA extracted from each cells Two-way ANOVA and Tukey’s multiple comparisons test were performed for statistical analysis c) represent statistically significant differences at p < 0.05; ns These findings suggest that these SNPs influence viral replication through mechanisms other than enhancing polymerase activity in cells B BoDV-2 minireplicon assays using the indicated BoDV-2 P variants and BoDV-2 N and (A) LRG or (B) LRGTR as helper plasmids An unpaired t-test was performed for statistical analysis C–F Growth kinetics assay of rBoDV-2 variants D The percentage of infected cells was determined by IFA using an antibody against BoDV N F The copy number of viral RNA was measured via RT-qPCR using total RNA extracted from each cells The data are presented as the means ± SEM of three biologically independent experiments Two-way ANOVA and Tukey’s or Sidak’s multiple comparisons test were performed for statistical analysis A Schematic representation of rBoDV-2 harboring an artificial expression cassette for the gene of interest (GOI) between the P and M genes B Growth kinetics assay of rBoDV-2 harboring a fluorescent protein Vero cells infected with rBoDV-2 PILRGTR-mCherry or rBoDV-2 PILRGTR-GFP were cocultured with uninfected Vero cells at a ratio of 1 to 19 and passaged every 3 or 4 days The ratio of cells expressing fluorescent proteins was measured via flow cytometry Two-way ANOVA and Sidak’s multiple comparisons test were performed for statistical analysis D Competition assay between rBoDV-2 LRG-mCherry and rBoDV-2 PILRGTR-GFP and uninfected Vero cells were cocultured at a ratio of 1 to 1 to 23 and passaged every 3 or 4 days C The ratio of cells expressing fluorescent proteins was measured via flow cytometry Representative data at 0 and 14 DPC are shown D A nucleotide at position 3743 of the L gene was analyzed via amplicon sequencing which represent rBoDV-2 LRG-mCherry and rBoDV-2 PILRGTR-GFP F Competition assay between rBoDV-2 LRG-mCherry and rBoDV-2 PILRGTR-GFP The rBoDV-2 LRG-mCherry-infected and rBoDV-2 PILRGTR-GFP-infected Vero cells were cocultured at a ratio of 1 to 1 and passaged every 3 or 4 days E The ratio of cells expressing fluorescent proteins was measured via flow cytometry F A nucleotide at position 3743 of the L gene was analyzed via amplicon sequencing G Isolation of Vero cells coinfected with rBoDV-2 LRG-mCherry and rBoDV-2 PILRGTR-GFP Vero cells coinfected with rBoDV-2 LRG-mCherry and rBoDV-2 PILRGTR-GFP were cocultured with uninfected Vero cells at a ratio of 1 to 19 and passaged every 3 or 4 days The ratio of cells expressing fluorescent proteins was measured via flow cytometry and representative data at 0 and 14 DPC are shown and H Data are presented as the means ± SEM of three biologically independent experiments F Data are presented as the means ± SEM of three biologically independent experiments the proportion of cells infected with only the PILRGTR-GFP virus increased; the LRG-mCherry virus could also be transmitted to uninfected Vero cells These observations suggest that BoDV-2 does not exclude superinfection allowing low-fitness variants to persist within infected cells while maintaining their characteristics and thereby maintaining population diversity In 47 sequences of BoDV-1 L registered on the database alanine is completely conserved at position 1112 suggesting that an amino acid residue with a simple side chain is necessary at this position Cysteine may not be suitable at this position because it can disrupt the structure and function of L by creating unrequested sulfate bindings Further research such as structural analysis clarifies the mechanism of how the substitution at position 1112 plays a critical role in the replication of BoDV-2 in a way other than enhancing polymerase activity these SNPs may affect the strength of the interaction of BoDV L with host and/or viral proteins thereby enhancing the efficiency of viral replication through mechanisms other than enhancing polymerase activity Investigating how these SNPs affect viral replication may help to elucidate the previously unknown role of the CTD of BoDV L Although the role of the amino acid residue in BoDV-1 P investigating its potential binding interactions with other viral proteins and its influence on the nuclear translocation capability of P would be of considerable interest it is conceivable that viruses with different sequences may have coexisted before isolation To better understand the characteristics and evolution of BoDV-2 infection it is necessary to examine in detail the competition for growth between viruses with genomic variants not only in vitro but also in vivo These observations suggest that maintenance of genetic diversity through the absence of superinfection exclusion during BoDV-2 infection may play a pivotal role in the establishment of persistent infection This might be achieved by not excluding low-fitness variants that may regulate replication dynamics and cytopathic effects as a viral population in infected cells This hypothesis warrants a long-term in vivo infection experiment with several genotypes or strains of BoDVs the methods used to detect superinfection differed between these studies further studies are necessary to investigate whether the absence of superinfection exclusion is a specific property for BoDV-2 but not for BoDV-1 we successfully established a reverse genetics system for BoDV-2 and revealed lack of superinfection exclusion enables BoDV-2 to maintain the genetic diversity in persistently infected cells Our study provides valuable insights into the biological properties of BoDV-2 and contributes to development of effective vaccines and antiviral drugs for pathogenic mammalian orthobornaviruses Human embryonic kidney (HEK) 293 T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque Japan; #08456-36) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific USA; #10270-106) and 1% penicillin-streptomycin-amphotericin B solution (Fujifilm African green monkey kidney Vero cells were cultured in DMEM supplemented with 5% FBS and 1% penicillin-streptomycin-amphotericin B solution The exact propagation history of the isolate No/98-infected culture is unknown cDNA was synthesized using the SuperScript IV First-Strand cDNA Synthesis System (Thermo Fisher Scientific) and subsequently the complete reaction was used for 2nd strand synthesis with the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs) followed by DNA fragmentation with a Covaris M220 and library preparation with a GeneRead DNA Library L Core kit (QIAGEN) and ION Xpress Barcode adapters (Thermo Fisher Scientific) library size was measured using a 2100 BioAnalyzer system (Agilent Technologies) with Agilent High Sensitivity DNA Chip and reagents and the library was quantified using a QIAseq Library Quant Assay Kit (Qiagen) The library was prepared for sequencing and sequenced together with Ion Torrent Calibration Standard (Thermo Fisher Scientific) using an Ion Torrent S5 XL instrument with Ion 530 chip and reagents (Thermo Fischer Scientific) in 400 bp mode This alignment was visually inspected with Geneious Prime® 2019.2.3 Software (Biomatters) we used the consequence sequence and BAM file generated from the 2nd round of mapping analyses as data input We performed variant calling under the following conditions: minimum coverage of aligned sequences and the minimum variant frequency is set to 50× and 25% The “Find Variations/SNPs” algorithm also calculates P-values representing the probability of sequencing errors as a lower p-value increases the probability that the observed variation in a given position represents a real variant We also excluded observed variants with p-value 10-5 when exceeding 65% strand bias Cultured cells were lysed with 1x sample buffer (50 mM Tris-HCl pH 6.8; 2% sodium dodecyl sulfate (SDS); 5% w/v sucrose; and 5% 2-mercaptoethanol) followed by boiling at 95 °C for 10 min Cell lysates were subjected to SDS-PAGE using an ePAGEL (ATTO Corporation and proteins were subsequently transferred to Trans-Blot® Turbo™ Mini nitrocellulose membranes (Bio-Rad The membranes were blocked with blocking buffer (TBS containing 0.1% Tween-20 with 5% w/v skim milk) and then incubated with the 1:2000 diluted anti-FLAG M2 antibody (Sigma-Aldrich USA; #F1804) or the 1:10000 diluted anti-alpha-tubulin antibody (Sigma-Aldrich followed by incubation with the 1:5000 diluted secondary antibody (horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch The protein bands were detected with Clarity Western ECL substrate reagents (Bio-Rad and chemiluminescence signals were visualized by Fusion Solo S (Vilber HEK293T cells seeded into 48-well plates were transfected with 50 ng of BoDV-2 minigenome plasmid and 5 ng of control plasmid expressing Cypridina luciferase using TransIT293 (Mirus USA; #MIR2700) according to the manufacturer’s instructions Gaussia luciferase and Cypridina luciferase expression levels were measured with a Biolux Gaussia luciferase assay kit (New England Biolabs USA; #E3300) and Biolux Cypridina luciferase assay kit (New England Biolabs according to the manufacturer’s instructions Luminescence signals were detected by a GloMax Discover Microplate Reader (Promega and polymerase activity was quantified by normalizing Gaussia luciferase activity to Cypridina luciferase activity Reverse genetics was performed according to the protocol summarized in our previous review article21 HEK293T cells seeded into 6-well plates were transfected with 2.0 µg of BoDV-2 cDNA plasmid and 0.01 µg of pCAGGS-BoDV-2 G using TransIT293 according to the manufacturer’s instructions the transfected cells were cocultured with uninfected Vero cells which were passaged every 3 or 4 days until the infection spread to almost all the Vero cells The transfected HEK293T cells were removed by adding 1.0 μg/mL puromycin (InvivoGen USA; #ant-pr) to the culture medium at the optimal time point the cells were sonicated using Bioruptor® II (Sonicbio Co. Japan) and centrifuged at 1200 × g for 25 min at 4 °C The supernatant containing rBoDV-2 was used as a viral solution Cultured cells were fixed with 4% paraformaldehyde (Nacalai Tesque Japan; #09154-85) for 10 min and permeabilized with PBS containing 0.5% Triton X-100 (Wako Japan; #169-21105) and 5% bovine serum albumin (Sigma-Aldrich The cells were incubated with the 1:10,000 diluted anti-BoDV N antibody (HB01) followed by incubation with the 1:1,000 diluted Alexa Fluor® 555-conjugated anti-rabbit IgG antibody (Thermo Fisher Scientific USA; #A21429) and 300 nM 4′,6′-diamidino-2-phenylindole (DAPI) (Merck Fluorescence signals were observed by an ECLIPSE TE2000-U fluorescence microscope (Nikon approximately 500 ng of total RNA was reverse transcribed using the Verso cDNA synthesis kit (Thermo Fisher Scientific USA; #AB1453) with anchored oligo dT primer according to the manufacturer’s instructions qPCR was performed using Luna Universal qPCR Master Mix (New England Biolabs USA; #M3003) with forward (5’-ATCTTCTTTTGCGTCGCCAG) and reverse (5’-ACGACCAAATCCGTTGACTCC) primers Each reaction contained 8.0 μl of RNase-free water 1.0 μl of primer mixture (10.0 pmol/μl primer) and 1.0 μl of synthesized cDNA or RNase-free water for the no template control in a total volume of 20.0 μl The thermal program consisted of 1 cycle of 95 °C for 1 min and 40 cycles of 95 °C for 10 s and 60 °C for 30 s All reactions were performed with a CFX Connect real-time system (Bio-Rad and relative expression levels of BoDV-2 RNA were calculated via the relative quantification method with GAPDH mRNA serving as a reference Cultured cells were fixed with 4% paraformaldehyde for 20 min and suspended in PBS containing 2.0% FBS Proportions of cells expressing either or both mCherry and GFP were analyzed by a CytoFLEX S flow cytometer (Beckman Coulter The criterion for assessing the extent of positive detection of each mCherry or GFP signal was set using mock-infected cells as a negative control The number of each read was counted by a proprietary algorithm developed by the Bioengineering Lab the ratio of U to C of the nucleotide at position 3743 representing rBoDV-2 LRG-mCherry and PILRGTR-GFP was analyzed to distinguish two genotypes of rBoDV-2 All statistical analyses were performed with GraphPad Prism 10 software The tests used for each experiment are described in figure legends The sequencing data of total RNA extracted from BoDV-2-infected Vero cells is deposited in the European Nucleotide Archive (ENA) under Sequence Read Archive accession number ERR14722785 Other data used and/or analyzed during the current study is demonstrated in this paper Additional information is available from the corresponding author upon request No custom code and scripts were used during data analysis The versions of the software are described in Materials and Methods Borna disease virus: a mystery as an emerging zoonotic pathogen Epidemiology and host spectrum of Borna disease virus infections Avian Bornavirus Research-A Comprehensive Review Annual (2023) taxonomic update of RNA-directed RNA polymerase-encoding negative-sense RNA viruses (realm Riboviria: kingdom Orthornavirae: phylum Negarnaviricota) Isolation and characterization of a new subtype of Borna disease virus Conservation of coding potential and terminal sequences in four different isolates of Borna disease virus Fatal Encephalitis Associated with Borna Disease Virus 1 Fatal Encephalitic Borna Disease Virus 1 in Solid-Organ Transplant Recipients Severe bornavirus-encephalitis presenting as Guillain-Barre-syndrome The neuropathology of fatal encephalomyelitis in human Borna virus infection Zoonotic spillover infections with Borna disease virus 1 leading to fatal human encephalitis 1999–2019: an epidemiological investigation Active Case Finding of Current Bornavirus Infections in Human Encephalitis Cases of Unknown Etiology Investigation of fatal human Borna disease virus 1 encephalitis outside the previously known area for human cases Human Borna disease virus 1 (BoDV-1) encephalitis cases in the north and east of Germany First detected geographical cluster of BoDV-1 encephalitis from same small village in two children: therapeutic considerations and epidemiological implications A Variegated Squirrel Bornavirus Associated with Fatal Human Encephalitis Genome trimming: a unique strategy for replication control employed by Borna disease virus RNA polymerase II-controlled expression of antigenomic RNA enhances the rescue efficacies of two different members of the Mononegavirales independently of the site of viral genome replication Kanda, T., Sakai, M., Makino, A. & Tomonaga, K. Exogenous expression of both matrix protein and glycoprotein facilitates infectious viral particle production of Borna disease virus 1. J. Gen. Virol. 103 https://doi.org/10.1099/jgv.0.001767 (2022) Reverse Genetics and Artificial Replication Systems of Borna Disease Virus 1 Genomic RNAs of Borna disease virus are elongated on internal template motifs after realignment of the 3’ termini The Borna Disease Virus 2 (BoDV-2) Nucleoprotein Is a Conspecific Protein That Enhances BoDV-1 RNA-Dependent RNA Polymerase Activity Expression and characterization of the Borna disease virus polymerase Overlap of interaction domains indicates a central role of the P protein in assembly and regulation of the Borna disease virus polymerase complex An unconventional pathway of mRNA cap formation by vesiculoviruses Enhanced neurovirulence of borna disease virus variants associated with nucleotide changes in the glycoprotein and L polymerase genes Enhanced polymerase activity confers replication competence of Borna disease virus in mice Structure of the L Protein of Vesicular Stomatitis Virus from Electron Cryomicroscopy BUD23-TRMT112 interacts with the L protein of Borna disease virus and mediates the chromosomal tethering of viral ribonucleoproteins and X proteins and their functional implications A methionine-rich domain mediates CRM1-dependent nuclear export activity of Borna disease virus phosphoprotein Virological and Immunological Outcomes of Coinfections Superinfection Exclusion in Mosquitoes and Its Potential as an Arbovirus Control Strategy Influenza A Virus Superinfection Potential Is Regulated by Viral Genomic Heterogeneity Superinfection exclusion creates spatially distinct influenza virus populations Superinfection exclusion: A viral strategy with short-term benefits and long-term drawbacks Sequence variability of Borna disease virus: resistance to superinfection may contribute to high genome stability in persistently infected cells Selective virus resistance conferred by expression of Borna disease virus nucleocapsid components Versatile Sample Processing Workflow for Metagenomic Pathogen Detection Population- and Variant-Based Genome Analyses of Viruses from Vaccine-Derived Rabies Cases Demonstrate Product Specific Clusters and Unique Patterns Full-Genome Sequences and Phylogenetic Analysis of Archived Danish European Bat Lyssavirus 1 (EBLV-1) Emphasize a Higher Genetic Resolution and Spatial Segregation for Sublineage 1a MUSCLE: multiple sequence alignment with high accuracy and high throughput Divergent Mutational Landscapes of Consensus and Minority Genotypes of West Nile Virus Demonstrate Host and Gene-Specific Evolutionary Pressures Efficient selection for high-expression transfectants with a novel eukaryotic vector Optimal expression of the envelope glycoprotein of orthobornaviruses determines the production of mature virus particles Development of a novel Borna disease virus reverse genetics system using RNA polymerase II promoter and SV40 nuclear import signal A novel borna disease virus vector system that stably expresses foreign proteins from an intercistronic noncoding region Gordon, A. & Hannon, G. J. Fastx-toolkit. FASTQ/A short-reads pre-processing tools. FASTQ/A short-reads preprocessing tools (unpublished) 5, https://github.com/agordon/fastx_toolkit (2010) Joshi, N. & Fass, J. Sickle: a sliding-window, adaptive, quality-based trimming tool for FastQ files (version 1.33). https://github.com/najoshi/sickle (2011) FLASH: fast length adjustment of short reads to improve genome assemblies Download references Germany) for providing BoDV-2-infected Vero cells and to Andrea Aebischer and Kathrin Steffen (Friedrich-Loeffler-Institute Masayuki Horie (Osaka Metropolitan University Japan) for supporting bilateral research.This study was supported in part by JSPS KAKENHI grants JP19J23468 (T.K.) and JP21K19909 (KT); JSPS Overseas Challenge Program for Young Researchers grant 202080194 (T.K.); JSPS Core-to-Core Program JPJSCCA20190008 (K.T.); Kaketsuken Research Grant (K.T.); the Joint Usage/Research Center Program on Institute for Life and Medical Sciences Kyoto University (K.T.); Institute for Life and Medical Sciences Office of Directors’ Research Grants Program Kyoto University (T.K.); and the German Federal Ministry of Education and Research Department of Mammalian Regulatory Network wrote the manuscript.All authors reviewed and approved the manuscript The authors declare no competing interests Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s44298-025-00117-w Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research The first round of the ATP Madrid Open features some exciting matchups and Juan Manuel Cerundolo goes up against Aleksandar Kovacevic With a mix of experience and emerging talents these matches are set to deliver a thrilling start to the tournament Main Photo Credit: Susan Mullane-USA TODAY Sports Metrics details Borna disease virus 1 (BoDV-1) is the causative agent of Borna disease a fatal neurologic disorder of domestic mammals and humans resulting from spill-over infection from its natural reservoir host the bicolored white-toothed shrew (Crocidura leucodon) The known BoDV-1-endemic area is remarkably restricted to parts of Germany To gain comprehensive data on its occurrence we analysed diagnostic material from suspected BoDV-1-induced encephalitis cases based on clinical and/or histopathological diagnosis BoDV-1 infection was confirmed by RT-qPCR in 207 domestic mammals this study markedly raises the number of published laboratory-confirmed human BoDV-1 infections and provides a first comprehensive summary Generation of 136 new BoDV-1 genome sequences from animals and humans facilitated an in-depth phylogeographic analysis allowing for the definition of risk areas for zoonotic BoDV-1 transmission and facilitating the assessment of geographical infection sources Consistent with the low mobility of its reservoir host BoDV-1 sequences showed a remarkable geographic association with individual phylogenetic clades occupying distinct areas The closest genetic relatives of most human-derived BoDV-1 sequences were located at distances of less than 40 km indicating that spill-over transmission from the natural reservoir usually occurs in the patient´s home region prophylactic measures are limited to reducing exposure to the BoDV-1 reservoir we collected material from archived and recent cases of Borna disease in domestic mammals serving as indicators for the presence of the virus Additional diagnostic samples were obtained from BoDV-1-infected human patients and from bicoloured white-toothed shrews collected in BoDV-1-endemic regions in Germany BoDV-1 sequences were generated from this material and used for phylogeographic analysis including also sequence data derived from public databases Grey symbols represent cases confirmed by BoDV-1-specific RT-qPCR in this study without available sequence C Visualisation of endemic regions of BoDV-1 clusters and subclusters by Kernel Density Estimation (KDE) The analysis is based on 214 BoDV-1 sequences with available location Sequences classified as phylogenetic outliers (no additional sequence with at least 98.6% nucleotide sequence identity within a maximal distance of 37.9 km) were excluded from the analysis D Cluster-independent BoDV-1 endemic region visualised by KDE Only sequences meeting the criteria described for panel C) were included ZH Zurich; Austria (AUT): UA Upper Austria The apparently higher sensitivity of Mix-6 RT-qPCR for FFPE-derived RNA is presumably due to its shorter amplicon (75 vs allowing for a more efficient detection of highly degraded RNA Sequencing attempts failed or yielded only short sequence fragments for 18 domestic mammals and three human cases mainly due to low viral loads and/or insufficient RNA quality in FFPE and CSF samples the two sequences of the novel cluster 5 possessed 99.0% nt sequence identity with each other but only 93.8 to 95.1% nt sequence identity with any other BoDV-1 sequence supporting their affiliation to a separate cluster Green asterisks indicate known epidemiologic links into the dispersal area of the respective subclade TH Thuringia; Switzerland (SUI): GR Grisons TG Thurgau; Austria (AUT): UA Upper Austria; Liechtenstein (LIE) The human case Z19_0093 from western BY in 2016 (outlier F; OR468948) was classified as an outlier since its sequence possessed only up to 98.1% nt sequence identity to any other BoDV-1 sequence Two further RT-qPCR-confirmed BoDV-1 infections in horses were detected far from any known BoDV-1-endemic region. These cases had occurred in western Switzerland (canton Geneva [GE]) in 1988 and in eastern BB in 2006 (Fig. 1B) sequencing attempts had failed due to poor RNA quality Epidemiological data were not available for these cases Broken horizontal lines represent the 90th percentile (39.8 km) and the median (15.6 km) of the presented dataset The black line represents the linear regression of genetic and geographic distance Slope and goodness of fit (R2) of the regression line are provided Interestingly, both patients infected with BoDV-1 of cluster 5 had died after developing disease in 2002 and both lived in neighbouring districts in the vicinity of Munich (BY; Fig. 4G and Supplementary Fig. 5F) Cluster 5 has not been detected in shrews or domestic mammals so far We therefore adopted a passive surveillance approach utilising Borna disease in domestic mammals as an indicator of endemic BoDV-1 infection in local shrew populations This approach may provide a potentially less biased fundament for phylogeographic analyses although some variability of the dissemination of susceptible domestic animal populations and of veterinary vigilance within and outside of known endemic areas cannot be excluded Reliable information on the location of infected individuals not only during onset of disease but even more importantly due to the long and possibly highly variable incubation period particularly for cases from earlier decades Despite our extensive efforts to fill these data gaps there are still varying degrees of uncertainty that must be considered when interpreting the results of this study for which we established objectifiable demarcation criteria based on pairwise nt sequence identities of complete coding BoDV-1 genomes The identification of a novel BoDV-1 cluster 5 represented by two human sequences from BY indicates that the genetic variability of BoDV-1 may be higher than currently appreciated and that additional variants may exist within or outside the known endemic areas at least three additional cases were identified in which animals were likely to have been moved to new locations during the incubation period with BoDV-1 sequences suggesting sources of infection at the respective sites of origin the alpaca had been moved no less than eight months prior to death the incubation period may have been shorter as the animal was exhibiting a potentially BoDV-1-associated ataxia already at the time of transfer These findings support the assumption that non-fatal or even asymptomatic human BoDV-1 infections are indeed at least very rare The actual distance to the source of infection is likely to be even lower in many cases as well as unavailability of further sequences representing genetically closer relatives are likely to lead to overestimation rather than underestimation any conclusions regarding the time of infection are complicated by the unknown incubation period and the variable disease progression the time from infection to death of human patients is affected by attempted treatments and life-sustaining measures A larger dataset of human BoDV-1 infections may be required to demonstrate whether their temporal distribution actually differs from the occurrence of BoDV-1 infection in domestic mammals may lead to more extensive data collection allowing for further refinement of phylogeographic analyses in the future In order to facilitate spatio-temporal analyses detailed metadata were requested from the submitters including geographic location (postal code) and date of sampling sex and date of hospital admission were recorded additionally for human cases the accuracy of the geographic location was non-hierarchically categorised as follows: (1) the location of the animal husbandry is known but the location of the husbandry is unknown and may be different (3) only the submitting veterinary practice/clinic is known (4) only the administrative district of origin is known and (5) no information on the accuracy of the location is available Missing data were completed by contacting the authors and/or the initial submitters Fresh-frozen samples were mechanically disrupted in 1 ml TRIzol reagent (Life Technologies Germany) by using the TissueLyser II (Qiagen according to the manufacturers’ instructions After the addition of 200 µl chloroform and a centrifugation step (14,000×g the aqueous phase was collected and added to 250 µl isopropanol Total RNA was extracted using the silica bead-based NucleoMagVet kit (Macherey & Nagel Germany) with the KingFisher™ Flex Purification System (Thermo Fisher Scientific USA) according to the manufacturers’ instructions Additional RNA extraction from fresh-frozen samples selected for HTS was performed according to Wylezich et al.57 the tissue was rapidly frozen in liquid nitrogen and subsequently pulverised using the Covaris cryoPREP (Covaris The resulting powdered tissue was then dissolved in pre-warmed 1 ml lysis buffer AL (Qiagen) RNA was extracted using the RNAdvance tissue kit (Beckman Coulter including a DNase I (Qiagen) digestion step in combination with a KingFisher Flex purification system (Thermo Fisher Scientific Total RNA was eluted in 100 µl nuclease-free water Total RNA from FFPE brain tissues was extracted as described previously58 two FFPE sections of <10 µm thickness underwent deparaffinisation and proteinase K digestion employing the Covaris truXTRAC FFPE total NA kit before RNA extraction To prevent the transfer of paraffin residues formalin de-crosslinking was carried out using 85 µl of the supernatant in a clean 1.5 ml reaction tube (80 °C 175 µl of B1 Buffer from the Covaris kit and 250 µl of 65% isopropanol were added RNA extraction was performed using the Agencourt RNAdvance Tissue Kit For additional 43 BoDV-1-positive fresh-frozen brain samples from domestic mammals, humans or shrews, partial BoDV-1 genome sequences were generated by Sanger sequencing (Table 1) Library quantification was carried out with the QIAseq Library Quant Assay Kit (Qiagen) and fragment size of each library was analysed using Agilent High Sensitivity DNA kit implemented in 2100 BioAnalyzer Instrument (Agilent) Libraries of 500 bpfragment size were sequenced400 bp runs using an Ion Torrent S5 XL instrument (Thermo Fisher Scientific) Libraries of 500 bp DNA fragment size were sequenced in 400 bp runs using Ion 530 chips while libraries of 200 bp DNA fragment size were sequenced in 200 bp runs using Ion 540 or Ion 550 chips on an Ion S5 XL instrument For enrichment of BoDV-1 specific library DNA fragments, an RNA bait set was designed for sequences representing all known members of the family Bornaviridae5 resulting in 17,858 non-redundant specific RNA baits and providing a three-fold genome coverage with a length of 80 nt per probe (myBaits® kit with 1–20 K unique baits; Arbor Bioscience The procedure was performed according to the manufacturer’s instructions with minor modifications 7 µl of each DNA library were combined with the blocking reagent mix of the kit One volume of mineral oil was used to seal the reaction mix before incubation for 24 h at 65 °C and shaking at 550 rpm in a thermomixer The aqueous phase was then transferred to a low-binding tube and purified using the binding beads from the myBaits® kit The enriched target library DNA was finally eluted in 35 μl of 10 mM Tris-HCl 0.05% Tween-20 solution (pH 8.0-8.5) and amplified in duplicates (16 µl DNA each) using the GeneRead DNA Library L amplification Kit (Qiagen) with 10 cycles (denaturation: 2 min at 98 °C; amplification for 10 cycles: 20 s at 98 °C and 30 s at 72 °C; final elongation: 1 min at 72 °C) both duplicates were pooled and purified twice by adding 0.65 or 1.2 volumes of Agencourt AMPure XP Beads (Beckman Coulter) for 500 bp or 200 bp libraries Enriched libraries were eluted in 30 µl buffer EB (Qiagen) The accuracy of the resulting contigs was checked by comparing the consensus sequences generated by both approaches with each other and by sequence annotation as described below Discrepancies at single and polynucleotide level were checked by reviewing the raw data quality and data coverage from mapping and assembly the effects of discrepancies on gene annotation and frameshifting were checked If sequence quality and/or coverage was insufficient Sanger sequencing of RT-PCR amplicons covering the respective positions was performed for confirmation as described below The final consensus sequence was generated by assembly of the overlapping raw sequences after trimming of primer-derived sequence ends and manual quality control Sanger sequencing was also used to fill gaps or confirm not sufficiently reliable positions in sequences generated by HTS BoDV-1-specific primer pairs were selected to generate amplicons of approximately 120 to 180 bp length to cover the respective sequence regions Primer sequences are available upon request Open reading frames (ORFs) were identified by ORF Finder (implemented in Geneious Prime® 2021.0.1) and verified by sequence alignment to the reference sequence All sequences generated in this study are available in the INSDC databases under accession numbers OR203629 As the re-analysis of the original isolates confirmed this suspicion the corresponding GenBank entries have now been corrected (accession numbers AY374523.2 and AY374537.2) Sequences originating from laboratory strains were excluded from the analysis to avoid an impact of adaptive mutations acquired during passaging in cell culture or experimental animals testing the correlation of pairwise patristic distances and geographic distances for all BoDV-1 sequences with available location (n = 238) as well as within the individual BoDV-1 clusters and subclusters IBD matrix correlations were tested in R using the “mantel” function of the “vegan” package (Spearman’s rho statistic and 9999 permutations) Non-parametric KDE was used to visualise spatial distribution patterns of mapped BoDV-1 cases KDE was performed independently for each phylogenetic cluster or subcluster as well as for sequences of all clusters and subclusters combined BoDV-1 sequences identified as phylogeographic outliers by using the outlier definition described in detail in the results section (presence of no other BoDV-1 N-X/P sequence with ≥98.6% nt identity within a distance of ≤37.9 km) were excluded from the KDE The two-dimensional KDE, implemented in ggplot as the “stat_density_2d” function77 was used with a polygon as the bounding box of estimated endemic regions n = 100 grid points were defined in each direction A low bandwidth (h) was set empirically for both approaches in order to minimise the extent of the estimated areas beyond the confirmed cases (subcluster 1A: 1; subcluster 1B: 0.5; cluster 2: 0.6; cluster 3: 0.75; cluster 4: 0.65; combination of all clusters: 1.0) Ethical approval of the analysis of archived human samples was obtained from the local ethical commission of the Faculty for Medicine the Technical University Munich (577/19 S) the Ludwig-Maximilians University Munich (23-0267) and the Medical Board of Hamburg (PV5616) Samples of BoDV-1-positive bicoloured white-toothed shrews were obtained from an ongoing large-scale small mammal screening study (Haring et al. Shrew KS20/0026 originated from a project that was commissioned by the Federal Environment Agency as part of the Environmental Research Plan (Research Code 3718 48 4250; animal ethics permit: 42502-2-1548 Uni Leipzig) and was financed with federal funds All other shrew carcasses included in this study were found dead or preyed by cats Samples from domestic mammals originated from diagnostic necropsies No living animals were handled or killed for the purpose of this study Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Non-parametric two-dimensional kernel density estimation was used to visualise spatial distribution patterns of mapped BoDV-1 cases using “stat_density_2d” function in ggplot Infections of horses and shrews with bornaviruses in Upper Austria: a novel endemic area of Borna disease Fatal encephalitis associated with borna disease virus 1 1999-2019: an epidemiological investigation Fatal encephalitic Borna disease virus 1 in solid-organ transplant recipients Active case finding of current bornavirus infections in human encephalitis cases of unknown etiology [Bornavirus encephalitis as a differential diagnosis to seronegative autoimmune encephalitis] Grosse, L. et al. First detected geographical cluster of BoDV-1 encephalitis from same small village in two children: therapeutic considerations and epidemiological implications. Infection https://doi.org/10.1007/s15010-023-01998-w (2023) and X protein contribute to serological diagnosis of fatal Borna disease virus 1 infections Risk factors for Borna disease virus 1 encephalitis in Germany - a case-control study Hemorrhagic lesion with detection of infected endothelial cells in human bornavirus encephalitis The immunopathogenesis of Borna disease virus infection Fürstenau, J. et al. Borna disease virus 1 infection in alpacas: Comparison of pathological lesions and viral distribution to other dead-end hosts. Vet. Pathol. https://doi.org/10.1177/03009858231185107 (2023) Borna disease in an adult alpaca stallion (Lama pacos) Borna disease virus infection of a horse in Great Britain Pathogenesis of Borna disease in rats: evidence that intra-axonal spread is the major route for virus dissemination and the determinant for disease incubation Borna disease in Switzerland and in the principality of Liechtenstein Borna disease outbreak with high mortality in an alpaca herd in a previously unreported endemic area in Germany Inhibition of Borna disease virus replication by ribavirin Ribavirin inhibits Borna disease virus proliferation and fatal neurological diseases in neonatally infected gerbils Synergistic antiviral activity of ribavirin and interferon-alpha against parrot bornaviruses in avian cells Antiviral activity of favipiravir (T-705) against mammalian and avian bornaviruses Vaccination against borna disease: overview vaccine virus characterization and investigation of live and inactivated vaccines Shrews as reservoir hosts of borna disease virus Distribution of Borna disease virus antigen and RNA in tissues of naturally infected bicolored white-toothed shrews supporting their role as reservoir host species The bicolored white-toothed shrew Crocidura leucodon (HERMANN 1780) is an indigenous host of mammalian Borna disease virus Shedding of infectious Borna disease virus 1 in living bicolored white-toothed shrews In: Handbook of the Mammals of the World: Insectivores Handbuch der Säugetiere Europas [Handbook of European mammals] (eds Niethammer J. Genetic clustering of Borna disease virus natural animal isolates laboratory and vaccine strains strongly reflects their regional geographical origin Meta-analysis of putative human bornavirus sequences fails to provide evidence implicating Borna disease virus in mental illness Epidemiological pattern of classical Borna disease and regional genetic clustering of Borna disease viruses point towards the existence of to-date unknown endemic reservoir host populations Phylogenetic analysis supports horizontal transmission as a driving force of the spread of avian bornaviruses Bicolored white-toothed shrews as reservoir for Borna disease virus Cerebrospinal fluid in Borna disease virus 1 (BoDV-1) encephalitis Human infections with borna disease virus 1 (BoDV-1) primarily lead to severe encephalitis: further evidence from the seroepidemiological BoSOT study in an endemic region in Southern Germany Are human Borna disease virus 1 infections zoonotic and fatal Essentials in bornavirus virology - an epilogue Molecular epidemiology of human Borna disease virus 1 infection revisited Presence of CD4+ and CD8+ T cells and expression of MHC class I and MHC class II antigen in horses with Borna disease virus-induced encephalitis Low prevalence of Borna disease virus 1 (BoDV-1) IgG antibodies in humans from areas endemic for animal Borna disease of Southern Germany Intranasal Borna disease virus (BoDV-1) infection: insights into initial steps and potential contagiosity Axonal transport of Borna disease virus along olfactory pathways in spontaneously and experimentally infected rats Rat model of borna disease virus transmission: epidemiological implications Ocular involvement in BDV-infected rabbits and primates New World camelids are sentinels for the presence of Borna disease virus Molecular identification of small mammal species using novel cytochrome B gene-derived degenerated primers A versatile sample processing workflow for metagenomic pathogen detection Mystery of fatal ‘staggering disease’ unravelled: novel rustrela virus causes severe meningoencephalomyelitis in domestic cats Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses A theoretical and generalized approach for the assessment of the sample-specific limit of detection for clinical metagenomics Full-genome sequencing of German rabbit haemorrhagic disease virus uncovers recombination between RHDV (GI.2) and EBHSV (GII.1) SeqKit: a cross-platform and ultrafast toolkit for FASTA/Q file manipulation SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era ModelFinder: fast model selection for accurate phylogenetic estimates UFBoot2: improving the ultrafast bootstrap approximation New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0 Using ggtree to visualize data on tree-like structures RStudio: Integrated Development for R (2020) R: A Language and Environment for Statistical Computing (2020) Exploring the temporal structure of heterochronous sequences using TempEst (formerly Path-O-Gen) rnaturalearth: World Map Data from Natural Earth (2023) ggplot2: Elegant Graphics for Data Analysis Download references Jessica Geers and Elsbeth Keller-Gautschi for their outstanding technical assistance Austria) and Eva-Maria Wittauer (Bad Kissingen Germany) submitted diagnostic material from confirmed or suspected cases of Borna disease or BoDV-1-infected shrews we like to thank all veterinarians and physicians treating the analysed animals and human patients We are grateful to Sybille Herzog (Giessen Germany) for providing BoDV-1 isolates for re-sequencing Germany) for kindly providing a vial of the bornavirus live vaccine ‘Dessau’ and Christiane Herden (Giessen Germany) for providing the laboratory strain H24 We like to thank Dirk Höper for providing funding technical supervision and advice as well as for critically discussing the data analysis and the manuscript This work was supported by the Federal Ministry of Education and Research within the research consortium “ZooBoCo” (Grant no 01KI1722 and 01KI2005 donated to Martin Beer Ulrich) and the projects “ZooKoInfekt” (01KI1903B; Rainer G Ulrich and Dennis Rubbenstroth) and “Bornavirus - Focal Point Bavaria” (01KI2002; Barbara Schmidt) Friederike Liesche-Starnecker received funding from the German Research Foundation (DFG; no Open Access funding enabled and organized by Projekt DEAL Unit 17 Influenza and Other Respiratory Viruses Center for Neuropathology and Prion Research Friedrich-Alexander Universität Erlangen-Nürnberg (FAU) Section of Clinical & Comparative Neuropathology University of Veterinary Medicine Hannover Florian Hansmann & Wolfgang Baumgärtner Chemical and Veterinary Analysis Agency Stuttgart (CVUAS) Antonie Neubauer-Juric & Marcel Suchowski Institute of Clinical Microbiology and Hygiene Bernhard Nocht-Institute for Tropical Medicine Institute of Novel and Emerging Infectious Diseases Department of Infectious Disease Epidemiology Mohammed Bin Rashid University of Medicine and Health Sciences histopathological and initial laboratory diagnosis and assembly of metadata The manuscript was critically reviewed through the contributions of all authors Nature Communications thanks Andrés Velasco-Villa and the other reviewer(s) for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-024-52192-x Sign up for the Nature Briefing newsletter — what matters in science Miomir Kecmanovic and Marcos Giron are tied at two wins apiece in their head-to-head but this will be their first meeting on clay Kecmanovic has posted solid clay-court numbers recently and has more experience on the surface While Giron has been steady in recent months Kecmanovic’s comfort on clay should give him a slight edge at the ATP Munich Open Ben Shelton and Borna Gojo are set for their first career meeting in the opening round of the 2025 ATP Munich tournament has had limited exposure on clay this season with his sole match resulting in a first-round loss to Alejandro Davidovich Fokina at the Monte Carlo Masters recently defeating Alexander Shevchenko in straight sets during the Munich qualifiers While Shelton has limited clay playing time this season his overall firepower still gives him a slight edge Gojo has looked solid in qualifying and should be well positioned to challenge him on the slower surface Billy Harris and David Goffin will meet for the second time in their careers in the opening round of the 2025 ATP Munich tournament having secured a three-set victory at the 2024 Australian Open While Harris has shown resilience in qualifying matches Goffin’s experience and consistency on clay courts make him the slight favorite in this encounter Main photo credit: Geoff Burke-Imagn Images Montreal’s Gabriel Diallo came up short in his bid to claim a main-draw spot in the Masters-level Madrid Open tennis tournament after a 6-3 7-6 (6) loss to Croatian veteran Borna Coric in Tuesday’s final round of qualifying Coric saved all nine break points he faced while breaking Diallo twice on three chances Diallo was up 6-5 in the second-set tiebreak before surrendering three straight points Diallo stood a good chance of advancing to the main draw as a “lucky loser.” If a player already in the main draw withdraws from the tournament the highest-ranked loser from the final round of qualifying takes that available spot won his match Tuesday with top-seed Daniel Altmaier of Germany yet to play made it into two straight Masters-level main draws last month at Indian Wells losing to France’s Arthur Fils in the second round both times Felix Auger-Aliassime of Montreal (18) and Denis Shapovalov of Richmond Hill (29) both have byes in the first round of the main draw in Madrid as seeded players Open champion Bianca Andreescu of Mississauga continues her comeback from injury when she faces American McCartney Kessler in a first-round match on Wednesday Report an editorial error Report a technical issue Authors and topics you follow will be added to your personal news feed in Following Welcome to The Globe and Mail’s comment community. 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For more information on our commenting policies and how our community-based moderation works, please read our Community Guidelines and our Terms and Conditions Two 14-match win streaks clashed in the final in Zadar as Borna Coric and Valentin Royer battled for the title Meanwhile Thiago Monteiro was the bridesmaid for the second week in a row in South America whereas Carlos Taberner showed some incredible tenacity and stamina to deliver in front of the home crowd in Murcia Read on for a look back at last week’s action on the Challenger Tour Having lost to Daniel Elahi Galan in the Santiago final Monteiro was once again the top seed in Asuncion and had to battle through some fatigue Remy Bertola took a set against him 6-0 but still lost while against Mariano Kestelboim the Brazilian almost got himself in major trouble when he didn’t convert two match points in the second set Despite never really hitting his peak level Monteiro kept fighting to make his second consecutive final with a win over his compatriot Matheus Pucinelli de Almeida Emilio Nava has been playing almost non-stop in South America with a couple of semifinal appearances that both ended at the hands of Galan For a while the American was heading for another clash with the same opponent first turning around another 2025 0-2 head-to-head against Juan Pablo Varillas But Galan was eliminated by hometown hero Adolfo Daniel Vallejo who Nava then defeated comfortably to make his first final since August 2023 Back then Murkel Dellien was his semifinal opponent this time it was the brother Hugo whom he took out in a massive 6-3 5-7 7-5 battle Felipe Meligeni Alves hadn’t competed since qualifying for the ATP 250 in Santiago a few weeks earlier The Brazilian had a pretty comfortable draw in the first two rounds before impressing in a crushing 6-1 6-0 defeat of Juan Carlos Prado Angelo One of the biggest names in the field was former world #17 Cristian Garin but he also couldn’t handle the game of Meligeni Alves with the 27-year-old beginning to dominate after briefly going a set and a break down switching to the altitude hard courts in Morelia Taberner made a semifinal in Punta del Este in February but didn’t achieve much success in the recent altitude clay double in Kigali Conditions in Murcia were so much more to his liking as the Spaniard produced a great win over top-seeded Marton Fucsovics in the quarterfinals which didn’t stop Taberner from taking another marathon against Kimmer Coppejans in the semifinals (2 hours and 44 minutes after looking exhausted in the 2-6 opening set) Jesper de Jong was similarly poor in Kigali not coming close to breaking the top 100 despite only being a few spots away But the Dutchman was also quick to show that in sea-level conditions he’s still a force Despite a tough opening draw in Andrea Pellegrino he handled the Italian comfortably and survived a thriller against Valentin Vacherot 6-3 3-6 7-5 A walkover from Harold Mayot got him into the semifinals and he promptly took advantage with a win over Ivan Gakhov Both finalists are expected in Girona next week with de Jong still a win or two away from securing his top 100 debut Now it was time to switch to clay and while the transition wasn’t 100% smooth at first Coric quickly began dominating to make another final with four straight-set wins When Coric was winning Lugano and Thionville, Royer dominated the Kigali double and also picked up two Challenger Tour titles he didn’t need to switch surfaces before going to Zadar It’s not like the conditions were all that similar with no altitude although both events tend to get quite windy Royer extended his win streak to 14 by the time he made the final Toughest match was his opener against Adrian Andreev with the Bulgarian leading him by a break in the deciding set Zadar tends to be extremely windy with its location on the Adriatic Sea The final was played on the worst day of the week in this regard and the match became a bit of a survival battle for the two players Coric started emerging as the one to handle it better pushing through the wind with some strong backhands down-the-line and nasty dropshots The Croatian picked up his sixth Challenger title 3-6 6-2 6-3 and is the first player to win 3 trophies at this level in 2025 He’s also on the verge of returning to the top 100 and will try to get there in Naples Royer chose to withdraw from Girona this week Main photo credit: David Kirouac-USA TODAY Sports Tennis - Retrouvez sur cette page les infos le suivi LIVE et les résultats du tournoi Challenger ATP 175 prévu à Aix en Provence du 28 avril au 4 mai.. Pierre-Hugues Herbert 🇫🇷 (Alt) 6-2 6-7(7) 6-1 le jeune Péruvien réussit une sacrée semaine contre le Russe Kotov qui a carrément menacé Moutet de mort le Tricolore a pourri son duel avec Opelka en parlant en ralant sans arrêt contre tout et rien face à un arbitre bien trop complaisant le géant Opelka lui a réglé son cas en deux manches mais ce Moutet avec son comportement pitoyable n'a absolument rien à faire sur un court de tennis.  qui sauve deux balles de set en 1ere manche avant de sortir son adversaire de 20 ans qui jouait une wild card pour Roland Garros Ignacio Buse (Q) 🇵🇪 : 6/7 (4) 2/3 abandon même si il a bénéficié de l'abandon du géant US Stan Wawrinka (WC) 🇨🇭 vs. Borna Gojo (PR) 🇭🇷 : 6/4 6/4 Borna Coric 🇭🇷 vs. Ignacio Buse (Q) 🇵🇪 : 4/6 6/2 6/3 Stan Wawrinka (WC) 🇨🇭 vs Borna Coric 🇭🇷 : 7/6 (5) 3/6 6/7 (4) Les organisateurs du tournoi ne pouvaient pas rêver d'une plus belle finale entre un triple vainqueur de Grand Chelem et un vainqueur de Masters 1000 le Suisse et le Croate ont tout donné avec pour chacun de belles périodes La dernière manches sera encore plus intense avec un Coric qui va prendre le plus souvent l'échange à son compte mais Stan ne va jamais rien lâcher avant le jeu décisif C'est encore le Croate qui va faire la différence en premier en se montrant plus percutant assez logique vu la différence d'âge de 12 ans avant de conclure sur une faute en coup droit d'un Stan bien déçu Borna Coric at the 2025 Mutua Madrid Open: Live Stream 104) in the Round of 128 of the Mutua Madrid Open on Thursday Arnaldi's most recent competition was on April 14 Coric heads into this match following a two-set victory over Gabriel Diallo (6-3 7-6) in his last match on Tuesday in the qualifying round Watch Tennis Channel on Fubo! Arnaldi vs. Coric futures oddsTennis odds courtesy of Tipico Sportsbook Tennis odds courtesy of BetMGM Sportsbook. Odds updated Tuesday at 6:57 PM ET. For a full list of sports betting odds, access USA TODAY Sports Betting Scores Odds Hub Borna Gojo at the 2025 Mutua Madrid Open: Live Stream 353 Borna Gojo in the Mutua Madrid Open Round of 128 on Wednesday Gojo's last match on Tuesday was a two-set win against Jerome Kym 7-6 Monfils vs. Gojo futures oddsTennis odds courtesy of Tipico Sportsbook Surreal situation that was experienced this Saturday at the Sioux Falls Challenger tournament is looking to get back to his best level after a tough period due to injuries and has made it to the final despite an impressive incident in the semifinals Gojo went as far as accusing the chair umpire of fixing the match against him as he couldn't believe some of the referee decisions made to his detriment He even called the supervisor and things got tense but he eventually found his composure and defeated Lajal Borna Gojo accused the chair umpire of match fixing because of line calls he’s disagreed with in the first of 2 semifinals here in Sioux Falls, but Gojo keeps his cool in the end and powers through to the final after beating Mark Lajal in straights. pic.twitter.com/IrFID2wu7N This news is an automatic translation. You can read the original news, Borna Gojo pierde los nervios y acusa a un juez de silla de tener amañado el partido Ben Shelton at the 2025 BMW Open: Live Stream 411) in the Round of 32 at the BMW Open on Monday Gojo enters the Round of 32 after his two-set win on Sunday over Alexander Shevchenko (6-4 Shelton lost his most recent match on April 7 2025 in the Round of 64 at the Rolex Monte-Carlo Masters to Alejandro Davidovich Fokina 7-6 Tennis odds courtesy of BetMGM Sportsbook. Odds updated Sunday at 12:56 PM ET. For a full list of sports betting odds, access USA TODAY Sports Betting Scores Odds Hub Christopher Eubanks has lost three of his last five matches Eubanks lost in the opening round of the Challenger tournament in straight sets Chris failed to qualify for the main draw tournament of the Australian Open The former top-30 player has been struggling with his form for a while He is outside the top 100 and hopes for a solid result this week This is his first appearance in Montpellier losing in the second round against Duckworth in straight sets Alibek Kachmazov has won three of his last five matches He lost in the opening round of the Challenger tournament against Kovacevic in straight sets Alibek successfully qualified for the main draw tournament He lost the opening set against Grenier 6-7 but he stepped up his level and successfully turned the match around The young Russian spent most of the last season playing at the Challenger Tour level Chris has never had remarkable results in Europe and is in terrible form Kachmazov is an upcoming talent who thrives under these conditions Borna Coric has lost four of his last five matches The former world number 12 has struggled with his form for a while Coric lost in the opening round against Berankis in straight sets Borna was disappointed at the Australian Open losing against Garin in straight sets in the opening round Borna lost in the final against Bublik in three tight sets This is his fifth appearance in the tournament Corentin Lestienne has won three of his last five matches he lost in the second round of the Challenger tournament against Moller in straight sets Corentin successfully qualified for the main draw tournament He won both of his qualifying matches against his compatriots in straight sets Lestienne defeated Atmane in straight sets today Lestienne lost in the second round against Cobolli in straight sets Coric is the slight favorite in this matchup but so was he last year when he reached the final who has also been struggling with his form Value bet/ the best odds: Borna Coric winning @1.62 @bet365 This article features Stats Insider's prediction and tips for the Monfils vs Gojo match as well as the latest betting odds in Australia Utilising advanced machine learning and data, Stats Insider has simulated the result of Thursday's Monfils-Gojo men's singles match 10,000 times Our independent predictive analytics model currently gives Monfils a 54% chance of winning against Gojo at the ATP Madrid Open tournament The current betting odds in Australia for Thursday's ATP Madrid Open match between Monfils and Gojo are listed here: Odds are correct at the time of publication and subject to change TAB currently has Monfils at $1.57 and Gojo at $2.37 TAB currently has odds for Monfils to win the first set at $1.66 and odds for Gojo to win the first set at $2.20 If you see a 🔥, that means you've found one of our best tips for today across any sport Today's Gael Monfils vs Borna Gojo betting tips are based on world-class modelling and wagering intelligence to help you place smarter bets with your chosen online bookie Even though our predictive analytics model suggests that Monfils is more likely to win the match betting on Gojo to win is the best option due to the 6.6% edge we discovered when comparing our data-led probabilities to the odds that are currently being offered Taking advantage of the edges published on Stats Insider is one of the keys to achieving long-term profitability as a sports bettor And while Monfils is more likely to win the first set on this occassion our suggested bet of Gojo ($2.00) is based on the expectation of that happening Stats Insider provides full coverage of the Gael Monfils vs Borna Gojo match at the ATP Madrid Open tournament including data-driven predictions and expert tips so bookmark this page for the latest betting analysis before the Monfils-Gojo match at the ATP Madrid Open event As always, see our Best Bets to get the best tips for every tennis match plus predictions for a wide range of other sports The 2025 ATP Madrid Open match between Gael Monfils and Borna Gojo is scheduled to begin at 2:10am AEST All dates and times mentioned in this article are in Australian Eastern Standard Time (AEST) Our Gael Monfils vs Borna Gojo predictions are the result of 10,000 data-driven simulations of the game, all curated by our team of skilled data scientists and analysts. We use the latest in predictive analytics technology and machine learning to ensure our tennis tips are trustworthy and reliable giving you the knowledge you need to make informed decisions with confidence If you decide to use our predictions for betting purposes, it's important to gamble responsibly and manage your finances effectively. For free and confidential support, call 1800 858 858 or visit gamblinghelponline.org.au Stats Insider is your go-to source for betting on tennis in Australia, with the latest tennis betting news, tips for every tennis match, and our in-house approach to accurately ranking the world's top 100 men's and women's players. Australia's leading predictive analytics website offers Australian sports fans innovative tools and content to enhance their enjoyment of major sporting events both domestically and internationally Our goal is to transform the sports fan experience by providing readily accessible data-driven content for sports enthusiasts like us © 2015-2025 Hypometer Technologies Pty Ltd (ABN 78 609 507 744) Proudly part of Cipher Sports Technology Group [tgdcta items=”1″ id=”170472″] [tgdcta items=”1″ id=”170476″] ATP Masters 1000 Rome 1/64-Finals Lehecka – Muller: Time TBA H2H: 2-0 Jiri Lehecka has lost four of his last five matches Last Word On Sports is back with more action in our best bets column Add videos to your saved list and come back to them any time This year will be the first edition of the ATP Almaty Open Main draw action begins on Monday at this indoor ATP 250 with the schedule including a fascinating Next Gen clash between Coleman Wong and Justin Engel 7 seed Fabian Marozsan will be the highest-ranked player in action Main Photo Credit: Geoff Burke-USA TODAY Sports This article features Stats Insider's prediction and tips for the Arnaldi vs Coric match along with the latest betting odds in Australia Based on advanced machine learning and data, Stats Insider has simulated the result of Friday's Arnaldi-Coric men's singles match 10,000 times Our independent predictive analytics model is unable to split the two players giving both Arnaldi and Coric an equal 50% chance of winning the match The latest betting odds in Australia for Friday's ATP Madrid Open match between Arnaldi and Coric are listed below: TAB currently has Arnaldi at $1.66 and Coric at $2.20 TAB currently has odds for Arnaldi to win the first set at $1.72 and odds for Coric to win the first set at $2.10 If you see a 🔥, you know it's one of our best tips for today across any sport Stats Insider's Matteo Arnaldi vs Borna Coric betting tips are based on world-class simulations and betting experience to help you place more informed bets with your chosen online bookie Stats Insider provides full betting coverage of the Matteo Arnaldi vs Borna Coric match at the ATP Madrid Open tournament including data-driven predictions and live scorestips so keep checking this article for the latest betting predictions before the Arnaldi-Coric match at the ATP Madrid Open event As always, see our Best Bets for the best tips for every tennis match as well as predictions for a wide range of other sports The 2025 ATP Madrid Open match between Matteo Arnaldi and Borna Coric is scheduled to begin at 12:05am AEST Our Matteo Arnaldi vs Borna Coric predictions have been made thanks to 10,000 data-driven simulations of the game, carefully curated by our team of experienced data scientists and analysts. We use the latest in predictive analytics technology and machine learning to ensure our tennis tips are accurate and reliable giving you the confidence to make informed decisions If you choose to use our predictions for betting purposes, it's vital that you gamble responsibly and know when to stop. For free and confidential support, call 1800 858 858 or visit gamblinghelponline.org.au Stats Insider is your go-to source for betting on tennis in Australia, with the latest tennis betting news, predictions for every tennis match, and our in-house approach to accurately ranking the world's top 100 men's and women's players.