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Novel partitivirus infection of bat white-nose syndrome (WNS) fungal pathogen pseudogymnoascus destructans links Eurasian and North American isolates

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Novel partitivirus infection of bat white-nose syndrome (WNS) fungal pathogen pseudogymnoascus destructans links Eurasian and North American isolates
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bioRxiv
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Ren, Ping
Rajkumar, Sunanda S.
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English
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White-nose syndrome ( lcsh )
Bats ( lcsh )
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serial ( sobekcm )

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Bat White-nose Syndrome (WNS) fungus Pseudogymnoascus destructans had caused mass mortality in the North American bats. A single clone of the pathogen (Hap_1) was likely introduced in the United States while Eurasian population comprised of several haplotypes. The origin and spread of P. destructans remain enigmatic due in part to a lack of precise population markers. We searched for P. destructans mycoviruses as they are highly host-specific, and their spread could provide a window on the origin of the host fungus. We discovered a P. destructans bipartite virus PdPV-1 with two double-stranded RNA (dsRNA) segments - LS (1,683 bp) and SS (1,524 bp) with motifs similar to viral RNA-dependent RNA polymerase (RdRp) and putative capsid proteins (CPs), respectively. Both LS and SS ORFs were embedded only in the positive strand of each dsRNA segment. Sequence alignments and phylogenetic analysis suggested that both segments constitute the genome of a new virus similar to the mycoviruses in the family Partitiviridae genus Gammapartitivirus. Purified viral particles appeared as isometric virions with approximately 33 nm diameters typical of partitiviruses. A newly developed RT-PCR assay revealed that all US isolates and only a few Eurasian isolates were infected with PdPV-1. PdPV-1 was P. destructans - specific as closely related non-pathogenic fungi P. appendiculatus and P. roses tested negative. Thus, PdPV-1 establishes a link between the Eurasian and North American P. destructans. PdPV-1 could be used as an experimental tool to further investigate fungal biogeography, and the host — pathogen interactions.

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1 Novel Partitivirus Infection of Bat White n ose Syndrome (WNS) Fungal Pathogen Pseudogymnoascus destructans Links Eurasian and North American Isolates Ping Ren 1 a Sunanda S. Rajkumar 1 b Haixin Sui 1, 2 Paul S. Masters 1, 2 Natalia Martinkova 3 Alena Kubto v 4 Jiri Pikula 5 Sudha Chaturvedi 1, 2 Vishnu Chaturvedi 1, 2* Wadsworth Center 1 New York State Department of Health Albany, New York; Department of Biomedical Sciences 2 University of Albany School of Public Health 2 Albany, New York, USA, Institute of Vertebrate Biology 3 Academy of Science of the Czech Republic, Kon!"n, Czech Republic, Department of Botany 4 Faculty of Science, Charles University in Prague, Praha, Czech Republic; University of Veterinary and Pharmaceutical Sciences Brno 5 Brno, Cze ch Republic. a Current address: University of Texas Medical Branch, Galveston, TX, USA b Current address: ICMR Medical Research Institute, Pudicherry, India Corresponding author: vishnu.chaturvedi@he alth.ny.gov ABSTRACT Bat White nose Syndrome (WNS) fungus Pseudogymnoascus destructans had caused mass mortality in the North American bats. A single clone of the pathogen (Hap_1) was likely introduced in the U nited S tates while Eurasian population comp rised of several haplotypes The origin and spread of P. destructans remain enigmatic due in part to a lack of precise population markers. We searched for P. destructans mycoviruses as they are highly host specific, and their spread could p rovide a window on the origin of the host fungus. We discovered a P. destructans bipartite virus PdPV 1 with two double stranded RNA (dsRNA) segments LS ( 1,683 bp ) and SS (1,524 bp ) with motifs similar to viral RNA dependent RNA polymerase (RdRp) and putative capsid pro teins (CPs) respectively Both LS and SS ORFs were embedded only in the positive strand of each dsRNA segment. Sequence alignments an d p hylogenetic analysis suggested that both segments constitute the genome of a new virus similar to the mycoviruses in th e family Partitiviridae genus Gammapartitivirus Purified CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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2 viral particles appeared as isometric virions with approximately 33 nm diameters typical of partitiviruses A newly developed RT PCR assay revealed that all US isolates and only a few Eurasian isola tes were infected with PdPV 1 PdPV 1 was P. destructans specific as closely related non pathogenic fungi P. appendiculatus and P. roseus tested negative. Thus, PdPV 1 establishes a link between the Eurasian and North American P. destructans PdPV 1 coul d be used as an experimental tool to further investigate fungal biogeography, and the host pathogen interactions IMPORTANCE The fungal disease White nose Syndrome threatens many bat species in North America The pathogen Pseudogymnoascus destructans ap peared first in Upstate New York nearly a decade ago The population markers used to date revealed that US population of P. destructans comprised of a single clone (Hap_1) while the Eurasian population was diverse. More precise tools are still needed to pi npoint the exact source of this devastating disease We studied P. destructans for viral infections (mycoviruses ) Fungal viruses are highly host specific and their spread mirrors the distribution of the host fungus. We found a double stranded RNA virus ( PdPV 1 ) with size, shape and sequences similar to fungal partitiviruses. All the US P. destructans and a few Eurasian isolates were positive for PdPV 1. Other closely related Pseudogymnoascus species also found in the bat hibernacula were free of PdPV 1 i nfection These findings provide a new avenue to study P. destructans origin and pathogenic attributes. Pseudogymnoascus destructans is a psychrophilic (cold loving) fungus responsible for bat W hite nose S yndrome (WNS) (1 3) P seudogymnoascus destructans infects at least eleven species of bats in North America of which seven species exhibited disease symptoms. The infected bat species include the endangered Indiana bat ( Myotis sodalist ) and gr a y bat ( M grisesc ens ) ( https://www.whitenosesyndrome.org/about/bats affected wns ). Several species including widely distributed little brown bats ( M. lucifugus ) face local extirpations (4) P seudogymnoascus destructans infects bats during hibernation in the caves and abandoned mines The fungus also persists in the environment in the absence of bats (5) Pseudogymnoascus destructans isolates analyzed to date from the US and Canada represented a clonal population (6, 7) Although European CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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3 bats also suffer from P. destructans infections, the p athogen population comprise of many haplotypes including H a p 1, which is the only haplotype found in fungal isolates from North America (8, 9) Despite apparent genetic homogeneity, p henotypic v ariations were repor ted among North American P. destructans isolates specifically in the mycelial growth rate, exudate production, and pigment formation and diffusion into aga r media (Figure 1A) (7) The underlying cause (s) for the phenotypic variations remain to be discovered. Virus infections in fungi (mycovirus) are rather common and latent mostly (10, 11) The m ajority of m ycoviruses are dsRNA isometric particles although ssRNA, and ssDNA myc oviruses have b een recognized Three well studied mycovirus systems include: i) yeast killer toxins in Saccharomyces cerevisiae and non conventiona l yeasts (12) ii) h ypovirulence in Cryphonectria parasitica and Ophiostoma ulmi the etiologic agents of Chestnut blight and Dutch Elm disease respectively (13, 14) and iii), symbiotic role in fungal endophytes of grasses (15) There are reports linking mycovirus infection to the phenotypic changes in the fungal host (16) Recently ds RNA mycoviruses in human pathogen Aspergillus fumigatus were found to cause aberrant phenotypes and hyp ervirulence (17) Mycoviruses could also provide a phylogenomic s window as their evolution show ed strong a co divergence with their fungal hosts (18) We hypothesized that origin, evolution, and virulence of P. destructans could be investiga ted by focusing on the mycoviruses The current study summarizes the molecular characterization of novel virus (named as PdPV 1) infection in P. destructans All US and some Eurasian isolates tested positive for PdPV 1. PdPV 1 was host specific to P. destr uctans as closely related non pathogenic fungi P. appendiculatus and P. roseus tested negative for the mycovirus (3) The results suggest that PdPV 1 link s the Eurasian and North American P. destructans and provid es an experimental tool for the future investigations of host pathogen interactions (Figure 1) Multiple bands were observed on the agarose gel in t otal nucleic acids extracted from P. destructans ( MYC80251 ) (Fig ure 1 B lane 1). T reatment of nucleic aci ds extracts with various nuclease s and DNase I yielded double bands thereby, suggesting the presence of dsRNA (Fig ure 1 B lane 4). dsRNA bands were estimated to be 1.5 2.0 kb (Fig ure 1B bottom panel ). Two dsRNA segments LS and SS were identified from sequence analysis. The LS segment contain ed an open reading frame (ORF) that encodes 539 amino acids with a molecular mass of approximately 62.7 kDa The small segment SS comprised CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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4 1,524 bp nucleotides with 49% GC content. The SS segment contains an ORF th at encodes 434 amino acids with a molecular mass of approximately 46.9 kDa Both ORFs were identified only on the positive strand of each dsRNA segment. The negative strands did not contain any significant ORFs that are longer than 82 amino acids. The 5' u ntranslated regions of the plus strands of segments LS and SS were 8 and 60 nucleotides, respectively; whereas the 3' untranslated regions of the plus strands of segments LS and SS were 54 and 159 nucleotides, respectively. GenBank search revealed that the ORFs of dsRNA LS and SS have significant similarities to the putative RNA dependent RNA polymerase (RdRp) and the capsid protein (CP), respectively, of viruses from the family Partitiviridae genus Gammapartitivirus The partitivirus was named Pseudogymnoa scus destructans partitivirus (PdPV 1). Phylogenetic trees derived from both RdRp and CP sequences exhibited three major branches and supported earlier conclusion that PdPV 1 is a member of the genus Gammaartitivirus, the family Partitiviridae (Fig ure s 1 a nd supplement ). Also, phylogenetic analyses of the putative RdRp and CP of PdPV 1 showed that PdPV 1 was much closely related to the gammapartitiviruses of PsV S. Tra nsmission electron microsc opy ( TEM ) confirmed the existence of viral particles in P. destr uctans Several isometric PdPV 1 virions with estimated diameter of about 33 nm characteristics of partitiviruses, were observed (Fig ure 1 C ). A specific RT PCR revealed that all P. destructans isolates from the US were positive for PdPV 1 A few Eurasian strains also tested positive for PdPV 1. The closely related fungi P. roseus and P. appendiculatus did not yield any detectable LS or SS segment of PdPV 1 dsRNA (Table S 1). We confirmed the discovery of P. destrucatns mycovirus PdPV 1 by: (i) demonstrat ion of dsRNA by DNA and RNA endonuclease s (ii) nucleotide similarit ies of the cloned dsRNA viral genome, and their phylogenetic alignment s with mycoviruses from the family Partitiviridae genus Gammapartitivirus (iii ) TEM confirmation of isometric viral p articles ( i v) high degree of host specificity for P. destructans and absence from the closely related Pseudogymnoascus species, and (v) exclusive association of PdPV 1 with P. destructans Haplotype 1 (Hpa_1) which is the clonal genotype of all N orth Amer ican isolates and only found in a select number of Eurasian isolates (6, 8, 9) Overall, PdPV 1 showed size, shape and nucleotide sequences typical of the fungal viruses in the family Partitiviridae genus Gammmapar titivirus (19) The discovery of dsRNA mycovirus in P. destructans is consistent with the wide occurrence of m ycoviruses in fungi (10, 20) Earlier observations suggested that partitiviruses caused cryptic infections in CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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5 their hosts without any discernible phenotypic changes (10, 16) A more evolved pattern was discerned in recent studies with viral induced hypovirulence in plant pathogenic fungi without visible phe noty pic effects; virus infections of animal pathogenic fungi and protozoan pathogens show ed hypervirulence (11, 21, 22) Although our limited testing was inconclusive, i n depth experiments are needed to discern the outcome of PdPV 1 infection on P. destructans phenotype and virulence. The absence of PdPV 1 infection in other Pseudog ymnoascus species, collected from the same bat hibernacula in the US suggested a close viral association with P. destructans The observed host specificity of Pd P V 1 was consistent with the narrow host ranges of mycoviruses due to severe bottlenecks in th eir horizontal transmission (11) The PdPV 1 discovery provided an independent and corroborative evidence for the emergence of P. destructans as a novel pathogen (8, 23) The local origin of PdPV 1 in the US was unlikely due to uniform infection of P. destructans isolates tested so far as well as the failure of viral curing experiments ( supplementary information ) A recent Eurasian origin was the most likely explanation based upon the findings of both viral free and viral infe cted P. destructans isolates in the Eurasian populations (Figure 1E) In such a scenario, the likely introduction of PdPV 1 to the US took place by the arrival of a mycovirus infected isolate of P. destructans ( Hap_1 haplotype ) Presently, the influence of different haplotypes of P. destructans on fungal virulence and other attributes remains unknown (9) The association between PdPV 1 and Hap_1 could be come crucial if Hap_1 represent ed a more virulent pathotype of P. destructans (24) PdPV 1 LS and SS nucleotide sequences are available from NCBI GenBank. ( Accession number: KP128044 and KP128045 respectively ) ACKNOWLEDGEMENTS This study was funded in parts with the US Fish & Wildl ife Service ( F15AP00948 ) and the National Science Foundation ( 1203528 ) research awards to VC and SC. DNA sequencing was performed at the Wadsworth Center M olecular Genetics Core. FIGURE LEGEND S Fig. 1 A Phenotypic variation in clonal population of P. destructans There is notable variation of colony color, texture, exudates and growth rates among the US, and Canadian isolates. Fig. 1 B P. destructans nucl eic acid extract analysis. An analytical run on 1% native agarose gel showed bands suggestive of genomic DNA, ribosomal RNA and two additional discrete nucleic acid bands (asterisk). CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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6 These bands were determined to be RNA species since RNase, but not DNase I digestion, led to their disapperance Lane M1: Hind III marker. Lane M2: 2 LOG DNA marker. Lane1: Total nucleic acid. Lane 2: Total nucleic acid + DNase I. Lane 3: Total nucleic acid + RNase III + RNase A. Lane 4: Total nucleic acid + S1 Nuclease + DNase I. Size of the RNA bands. RNA were sized by an analytical run of the purified samples on denaturing polyacrylamide gel The size of the two RNA bands is less than 2 kb. These RNA band s were ma ked by the 18S ribosomal RNA band in total RNA samples Lane M: ssRNA marker (in bp). Lane 1: Total RNA. Lane 2 : Gel purified particle. Fig. 1 C Transmission e lectron microscopy of purified PdPV 1. Several isometric viral particles are visible approximately 33 nm in diameter. The bar represents 100 nm. Fig. 1 D Phylogenetic analyses of PdPV 1. RdRp amino acid seq uences of the representative members of the family Partitiviridae Totiviridae and Chrysoviridae were used to construct Maximum likelihood phylogenetic tree with MEGA 6 (GenBank accession numbers are provided in Table S 3 ) Fig. 1E. Projected origin and tra nsmission of P. destructans The country map highlights areas where PdPV 1 infected fungal isolates were found. Eurasian samples also contained viral negative samples. REFERENCES 1. Blehert DS. 2012. Fungal disease and the developing story of bat white nose syndrome. PLoS pathogens 8: e1002779. 2. Chaturvedi V, Springer DJ, Behr MJ, Ramani R, Li X, Peck MK, Ren P, Bopp DJ, Wood B, Samsonoff WA, Butchkoski CM, Hicks AC, Stone WB, Rudd RJ, Chaturvedi S. 2010. Morphological and molecular c haracterizations of psychrophilic fungus Geomyces destructans from New York bats with White Nose Syndrome (WNS). PloS one 5: e10783. 3. Minnis AM, Lindner DL. 2013. Phylogenetic evaluation of Geomyces and allies reveals no close relatives of Pseudogymnoascu s destructans, comb. nov., in bat hibernacula of eastern North America. Fungal biology 117: 638 649. 4. Thogmartin WE, Sanders Reed CA, Szymanski JA, McKann PC, Pruitt L, King RA, Runge MC, Russell RE. 2013. White nose syndrome is likely to extirpate the en dangered Indiana bat over large parts of its range. Biological Conservation 160: 162 172. 5. Lorch JM, Lindner DL, Gargas A, Muller LK, Minnis AM, Blehert DS. 2012. A culture based survey of fungi in soil from bat hibernacula in the eastern United States an d its implications for detection of Geomyces destructans the causal agent of bat white nose syndrome. Mycologia 105: 237 252. 6. Rajkumar SS, Li X, Rudd RJ, Okoniewski JC, Xu J, Chaturvedi S, Chaturvedi V. 2011. Clonal genotype of Geomyces destructans amon g CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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7 bats with White Nose Syndrome, New York, USA. Emerging infectious diseases 17: 1273 1276. 7. Khankhet J, Vanderwolf KJ, McAlpine DF, McBurney S, Overy DP, Slavic D, Xu J. 2014. Clonal expansion of the Pseudogymnoascus destructans genotype in North America is accompanied by significant variation in phenotypic expression. PloS one 9: e104684. 8. Leopardi S, Blake D, Puechmaille SJ. 2015. White Nose Syndrome fungus introduced from Europe to North America. Current biology : CB 25: R217 219. 9. Zukal J, Bandoucho va H, Brichta J, Cmokova A, Jaron KS, Kolarik M, Kovacova V, Kubtov A, Novkov A, Orlov O, Pikula J, Presetnik P, !uba J, Zahradnkov Jr A, Martnkov N. 2016. White nose syndrome without borders: Pseudogymnoascus destructans infection tolerated in Eur ope and Palearctic Asia but not in North America. Scientific Reports 6: 19829. 10. Ghabrial SA, Castn JR, Jiang D, Nibert ML, Suzuki N. 2015. 50 plus years of fungal viruses. Virology 479: 356 368. 11. Son M, Yu J, Kim K H. 2015. Five Questions about Mycovi ruses. PLoS pathogens 11: e1005172. 12. Schmitt MJ, Breinig F. 2002. The viral killer system in yeast: from molecular biology to application. FEMS microbiology reviews 26: 257 276. 13. Rogers H, Buck K, Brasier C. 1987. A mitochondrial target for double stra nded RNA in diseased isolates of the fungus that causes Dutch elm disease. Nature 329: 558 560. 14. Nuss DL. 2005. Hypovirulence:mycoviruses at the fungal plant interface. Nat. Rev. Microbiol. 3: 632 642. 15. Mrquez LM, Redman RS, Rodriguez RJ, Roossinck MJ 2007. A virus in a fungus in a plant: three way symbiosis required for thermal tolerance. Science 315: 513 515. 16. Pearson MN, Beever RE, Boine B, Arthur K. 2009. Mycoviruses of filamentous fungi and their relevance to plant pathology. Molecular plant pa thology 10: 115 128. 17. Bhatti MF, Jamal A, Petrou MA, Cairns TC, Bignell EM, Coutts RHA. 2011. The effects of dsRNA mycoviruses on growth and murine virulence of Aspergillus fumigatus Fungal Genetics and Biology 48: 1071 1075. 18. Goker M, Scheuner C, Kle nk HP, Stielow JB, Menzel W. 2011. Codivergence of mycoviruses with their hosts. PloS one 6: e22252. 19. Nibert ML, Ghabrial SA, Maiss E, Lesker T, Vainio EJ, Jiang D, Suzuki N. 2014. Taxonomic reorganization of family Partitiviridae and other recent progre ss in partitivirus research. Virus research 188: 128 141. 20. Bozarth RF. 1972. Mycoviruses: a new dimension in microbiology. Environ Health Perspect 2: 23 39. 21. zkan S, Coutts RH. 2015. Aspergillus fumigatus mycovirus causes mild hypervirulent effect on pathogenicity when tested on Galleria mellonella Fungal Genetics and Biology 76: 20 26. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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8 22. Ives A, Ronet C Prevel F, Ruzzante G, Fuertes Marraco S, Schutz F, Zangger H, Revaz Breton M, Lye L F, Hickerson SM, Beverley SM, Acha Orbea H, Launois P, Fasel N, Masina S. 2011. Leishmania RNA Virus Controls the Severity of Mucocutaneous Leishmaniasis. Science 331: 775 778. 23. Warnecke L, Turner JM, Bollinger TK, Lorch JM, Misra V, Cryan PM, Wibbelt G, Blehert DS, Willis CK. 2012. Inoculation of bats with European Geomyces destructans supports the novel pathogen hypothesis for the origin of white nose syndrome. Proceed ings of the National Academy of Sciences of the United States of America 109: 6999 7003. 24. CAIZARES MC, Prez Arts E, GARCA PEDRAJAS NE, GARCA PEDRAJAS MD. 2015. Characterization of a new partitivirus strain in Verticillium dahliae provides further ev idence of the spread of the highly virulent defoliating pathotype through new introductions. Phytopathologia Mediterranea 54: 516 523. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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i Supplementary Information Fungal isolates P. destructans isolate (MYC80251) and other US and Eurasian isolates have been described previously (1 3) The isolates from other Pseudogymnoasc us species were also tested (4) The supplementary table 1 provides additional details of test strain. All fungal isolates were rou tinely maintained on Sabouaraud Dextroxe Agar or Potato Dextrose Agar at 15 C. The cultures were stored in 15% glycerol at 80 C. The methods for the observations of colony morphology and extra cellular enzymes was described previously (1, 5) dsRNA extraction P. destructans was grown in a stationary culture in potato dextrose agar (PDA) at 15C for 2 3 months. About 1 g wet weight mycelium was harvested and grounded to powder in the presence of liquid nitrogen with mortar and pestle. The powder was collected and suspended in 1 ml extraction buffer (150 mM sodium acetate, pH 5.0, 100 mM LiCl, 4% sodium dodecyl sulfate, 10 mM EDTA, pH 8.0, and 20 mM mercaptoethanol) and incubated on ice for 10 min. Total nucleic acids were obtained by traditional phenol chloroform extraction and LiCl, 2 propanol precipitation method. ssRNA and DNA were removed by treatment with S1 nuclease and DNase I (Life Technologies, Carlsbad, CA). The extractions were run on 1% agarose gel, stained with ethidium bromide and visualized with UV transillumin ation. To confirm the dsRNA nature of the mycovirus, the total nucleic acid solutions were treated with RNase III and RNase A (Life Technologies) to see the disappearance of dsRNA. Denaturing polyacrylamide gel was used to run the purified dsRNA extraction s to see the size of two RNA bands (6, 7) cDNA synthesis and sequence analysis Purified dsRNA fractions containing 2 segments were denatured in 90% dimethyl sulfoxide (DMSO) at 65C for 20 min in the presence of random hexadeixinucleotide and quickly chilled on ice. Then first strand cDNA was synthesized using M MLV reverse transcriptase (Life Technologies). The resulting cDNA we re amplified with random primers and the variously sized PCR products were cloned in TOPO TA cloning vector. A series of overlapping cDNA clones were obtained from the sequencing of the positive clones. Determination of the ends of each dsRNA was done usin g FirstChoice RLM RACE Kit (Life Technologies) (8) Nucleotide sequence analysis The nucleotide sequence contigs were assembled by Sequencher 4.8 (Gene Codes Co., Ann Arbor, MI). The conserved sequences were BLAST searched in GenBank by tblastx program. Multiple sequence alignments for two dsRNA segments were CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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ii carried out using ClustalW analysis by M acVector 7.2 (Accelrys Inc., Cary, NC) respectively. The phylogenetic analyses were conducted using maximum likelihood method by MEGA 6.0 (9) Virus purification. About 60 g wet weight fungal mycelium were collected and grounded to powder as described in dsRNA extraction session. The homogenates were mixed with extraction buffer (0.1 M sodium phosphate, pH 7.4, containing 0.1% mercaptoethanol) at a volume of 5 ml/g of wet mycelium. The suspensions were vortex hard after adding equal volume of chloroform and broke the emulsion by centrifuging in a Sorvall GSA rotor at 8,000 rpm for 20 min. The upper aqueous layer was then mixed thoroughly with 0.5 volumes of 30% polyethylene glycol 8000 (PEG) in 0.85% NaCl and hold on ice for 1 hour. PEG precipitates were pelleted in a Sorvall GSA rotor with 16,000x g at 4C for 30 min and resuspended in 0.1 M sodium phosphate, pH 7.4. After centrifuge with 23,000x g at 4C for 20 min to remove unsuspende d debris, the supernatant was u ltra centrifuged in a Beckman SW42 rotor at 76,000x g, 4C for 2 hrs to pellet the virus. The pelleted virus was re suspended in a total of 4 ml 0.1 M sodium phosphate buffer and purified by loading the viral suspension onto pre formed gradients of 10% 50% (w/v) sucrose in 0.1 M sodium phosphate buffer and centrifugin g at 76,000x g, 4C overnight. The collected virus fractions were diluted in 0.1 M sodium phosphate buffer and preserved at 4C for immediate electron microscopy (10 12) Transmission electron micro scopy. Two "l of the virus solution was placed on a glow discharged copper grid covered with a continuous carbon film. After 1 minute of adsorption, the grid was washed with pure water for several seconds and stained with 3"l of 2% (w/v) uranyl acetate sol ution for 1 minute. The staining solution was blotted away by Whatman No. 1 filter paper. The grid was air dried completely before it was examined in a JEOL JEM 1400 electron microscope operating at 120 keV (13) The micrographs were recorded at various magnifications using a 4K x 4K CMOS camera (TVIPS F 416). Reverse transcript PCR (RT PCR) assay. First strand cDNA synthesis was performed as described earlier and followed by PCR using primer pairs V2085/V2090 and V2168/V2164 to amplify SS and LS segments respectively. Then the PCR products were sequenced using the same primers and aligned to confirm the sequences. Viral curing. To cure the viruses from P. destructans isolates, tiny hyphal tips of isolate MYC80251 were inoculated on PDA with 5 "g/ml CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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iii cycloheximide (14) and limited nutrient cAMP rifamycin agar plate (15) After 7 days incubation at 15C, when there was a discernible increase in the colony growth, hyphal tips were cut with sterile blade and transferred on the fresh PDA cycloheximide plates and cAMP rifamycin agar plates. This passage was repeated for five times. For the last passage, the fungal colonies were allowed to grow for about 10 days to obtain sufficient hyphal mass for total RNA extraction and the presence of mycoviruses ex amined by RT PCR. Detection of PdPV 1 dsRNAs or virus particles in broth culture supernatant. P. destructans isolates MY80251, PSEU14, and LBB17 were cultured in a 125 ml flask containing 25 ml Potato dextrose broth medium at 15C without shaking for 4 w eeks. Five ml of culture supernatants were centrifuged at 17,000 g for 5 min to remove conidia and free cells. Then 250 "l resultant supernatants were used for nucleic acids extraction with SDS/phenol and subjected to agarose gel electrophoresis. To detect virus particles, 4 ml resultant supernatants were ultracentrifuged at 148,400 g for 1 hour and the precipitate was suspended in 0.05 M Tris/HCl (pH 7.8) containing 0.15 M NaCl at 4C. The suspension was stained and visualized by transmission electron micr oscope as described earlier. Supplementary figures: Fig. S1 Comparison of the amino acid sequences of putative RdRp of the Pseudogymnoascus destructans virus (PdPV 1), Penicillium stoloniferum virus S (PsV S), Gremmeniella abietina virus MS1 (GaV MS1), Aspergillus ochraceus virus (AoV), Botryotinia fuckeliana partitivirus 1 (BfPV1), Aspergillus fumigatus partitivirus 1 (AfuPV 1), Ustilaginoidea virens partitivirus 1 (UvPV 1), Verticillium dahliae partitivirus 1 (VdPV1), Ophiostoma partitivirus (OPV1), Discula destructiva virus 2 (DdV2), and Fusarium solani virus 1 (FusoV). Red: 100% identity; Blue: consensus match; Green: mismatch. Fig. S 2 Comparison of the amino acid sequences of the capsid protein (CP) of the Pseudogymnoascus destructans virus (PdP V 1), Penicillium stoloniferum virus S (PsV S), Gremmeniella abietina virus MS1 (GaV MS1), Aspergillus ochraceus virus (AoV), Botryotinia fuckeliana partitivirus 1 (BfPV1), Aspergillus fumigatus partitivirus 1 (AfuPV 1), Ustilaginoidea virens partitivirus 1 (UvPV 1), Verticillium dahliae partitivirus 1 (VdPV1), Ophiostoma partitivirus (OPV1), Discula destructiva virus 2 (DdV2), and Fusarium solani virus 1 (FusoV). Red: 100% identity; Blue: consensus match; Green: mismatch. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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iv Fig. S 3 Phylogenetic analyses o f PdPV 1. CP amino acid sequences of representative members of the family Partitiviridae Totiviridae and Chrysoviridae were used to construct Maximum likelihood phylogenetic tree by MEGA 6 (GenBank accession numbers are provided in Table S2). CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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v Supplemen tary Table 1 Fungal isolates used in this study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nternational license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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vi Supplementary Table 2. Details of primers used in this study !"#$%"& '($% & )%*+%',%-& ./&01&2/ & 3-% 4 & 51" & 3:N!V JO22225J25JJ552JO ),W*,($'(L 3:N!0,E 2252J5O2OJ2552OJ ),W*,($'(L .87U: J225OJ255O25OO52OJ552J ),W*,($'(L .87U8 J22JJOOJ25OOJO55O5OJ2J ),W*,($'(L .87UN 5J2OO52J5JOOO5JOO5OOJ5 0"3 T 025B=!-,W*,($'(L .87UD 5OJJOO522JOO5J5J5OJO25 0"3 T 025B=!-,W*,($'(L .87U9 2J55252JO52J2OJJO252JJ ),W*,($'(L .87UU OJO225O5O55JOJJO25JJ2J 0"3 T 025B=!-,W*,($'(L .87UH 2OJJJ22OJO25O2O5OJ5OJJ 0"3 T 025B=!-,W*,($'(L .87U6 O52J52O25OO5OJJOO25J2J 0"3 T 025B=!-,W*,($'(L .8769 2222J22OO5J255J22522OJO25 0O T A50=! -,W*,($'(L .876U 2OOO2O252O2555OJ5JJ255OO 0"3 T 025B=!-,W*,($'(L .8766 25JJ5J22JJ225OJ255O2 0"3 T 025B .876R JOOOJJJ2OJ5O2OJJO5O2OO25OOO ),W*,($'(L .87R7 55O5O55O5OO25JJ2252OO5OO 0O T A50=!-,W*,($'(L .87R8 2O5JJJ2OO52JO2O52O52 ),W*,($'(L .87RD 2J2555J55JJ22O2 5J2 0"3 T 025B .87RU & JOO55J2OO JOO JJ2J2J J2 & 5/#('(L =!-,W*,($'(L .87RH 5O5OOJ2OJ5O5O55522J5 ),W*,($'(L .8:UN 55J2JJ52OOJ5OO5JOOO5 ),W*,($'(L .8:UD 25552JO522JJO5J2JOO5 0O T A50=!-,W*,($'(L .8:U9 5OOOO525J255JO5OOJ5O ),W*,($'(L .8:U6 & OJ25522OJJJ52JOOOJ J2 5/#('(L =!0O T A50=!-,W*,($'(L .8:6U OJ52OJJO52J22JJO2OOJ ),W*,($'(L .88:7 OOJJ2J55255OJ2JO2225 ),W*,($'(L CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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vii Supplementary Table 3 Accession number and other details of amino acid sequences used for phylogenetic analyses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`N:66U 52`N:66H !#*1,1221$)-,8/("&'#*$)E'+*A$. 4A^NR8D68 4A^NR8D6: !#*1,1221,$)-".&2&*13#/$)E'+*-!) A-. T ) 2216U6ND 2216U6N9 !"#$%&'()*&+",$"-%#"./$,.+*"E'+*-!: AM. T : \A:867DD \A:867D9 D&"#221*1+-*#,+./1?Q%+&'&'E'+*-!8 0(A.: G2[R68NH G2[R68N6 D&"#221*1+-*#,+./1?Q%+&'&'E'+*-!8 0(A.8 G23H6U78 G2\9N:R8 E68+#/&6"1"-"+61*#+ 012!E'+*-!8 )-0.8 1A^7DH9U7 1A^7DH99R F".12+'1*&1%#+-51/#*"Q%+&'&'E'+*-! CEA. T : 2JI7DD78 2JI7DD7N F".12+'1*&1%#+-51/#*"012!E'+*-!N CE.N 4A^77R77D:9U 4A^77R77D:99 G#/.1,1221$)-%+821+ # $F+<-#E'+*-!: .M5.: 2[J8:8:N 2[J8:8:D G#/.1,1221$)%+821+#Q%+&'&'E'+*-!: .MA.: 2J_988:7 2J_9887R CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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viii References 1. Chaturvedi V, Springer DJ, Behr MJ, Ramani R, Li X, Peck MK, Ren P, Bopp DJ, Wood B, Samsonoff WA, Butchkoski CM, Hicks AC, Stone WB, Rudd RJ, Chaturvedi S. 2010. Morphological and molecular characterizations of psychrophilic fungus Geomyces destructans from New York bats with White Nose Syndrome (WNS). PloS one 5: e10783. 2. Ren P, Haman KH, Last LA, Rajkumar SS, Keel MK, Chaturvedi V. 2012. Clonal Spread of Geomyces destructans among Bats, Midwestern and Southern United States. Emerg. Infect. Dis. 18: 883 885. 3. Zukal J, Bandouchova H, B richta J, Cmokova A, Jaron KS, Kolarik M, Kovacova V, Kubtov A, Novkov A, Orlov O, Pikula J, Presetnik P, uba J, Zahradnkov Jr A, Martnkov N. 2016. White nose syndrome without borders: Pseudogymnoascus destructans infection tolerated in Europe and Palearctic Asia but not in North America. Scientific Reports 6: 19829. 4. Lorch JM, Muller LK, Russell RE, O'Connor M, Lindner DL, Blehert DS. 2013. Distribution and environmental persistence of the causative agent of white nose syndrome, Geomyces destruct ans in bat hibernacula of the eastern United States. Applied and environmental microbiology 79: 1293 1301. 5. Khankhet J, Vanderwolf KJ, McAlpine DF, McBurney S, Overy DP, Slavic D, Xu J. 2014. Clonal expansion of the Pseudogymnoascus destructans genotype in North America is accompanied by significant variation in phenotypic expression. PloS one 9: e104684. 6. Howitt RL, Beever RE, Pearson MN, Forster RL. 2006. Genome characterization of a flexuous rod shaped mycovirus, Botrytis virus X, reveals high amino a cid identity to genes from plant 'potex like' viruses. Archives of virology 151: 563 579. 7. Lin Y, Zhang H, Zhao C, Liu S, Guo L. 2015. The complete genome sequence of a novel mycovirus from Alternaria longipes strain HN28. Archives of virology 160: 577 580 8. Marvelli RA, Hobbs HA, Li S, McCoppin NK, Domier LL, Hartman GL, Eastburn DM. 2014. Identification of novel double stranded RNA mycoviruses of Fusarium virguliforme and evidence of their effects on virulence. Archives of virology 159: 349 352. 9. Tamur a K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution 30: 2725 2729. 10. Crawford LJ, Osman TA, Booy FP, Coutts RH, Brasier CM, Buck KW. 2006. Molecular characterization of a partitivirus from Ophiostoma himal ulmi Virus Genes 33: 33 39. 11. Lin YH, Chiba S, Tani A, Kondo H, Sasaki A, Kanematsu S, Suzuki N. 2012. A novel quadripartite dsRNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix. Virology 426: 42 50. 12. Ochoa WF, Havens WM, Sinkovits RS, Nibert ML, Ghabrial SA, Baker TS. 2008. Partitivirus structure reveals a 120 subuni t, helix rich capsid with distinctive surface arches formed by quasisymmetric coat protein dimers. Structure 16: 776 786. 13. Kishchenko GP, Danev R, Fisher R, He J, Hsieh C, Marko M, Sui H. 2015. Effect of fringe artifact correction on sub tomogram averagi ng from Zernike phase plate cryo TEM. Journal of structural biology 191: 299 305. 14. Urayama S, Kato S, Suzuki Y, Aoki N, Le MT, Arie T, Teraoka T, Fukuhara T, Moriyama H. 2010. Mycoviruses related to chrysovirus affect vegetative growth in the rice blast fungus Magnaporthe oryzae The Journal of general virology 91: 3085 3094. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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ix 15. Kwon SJ, Lim WS, Park SH, Park MR, Kim KH. 2007. Molecular characterization of a dsRNA mycovirus, Fusarium graminearum virus DK21, which is phylogenetically related to hypoviruses but has a genome organization and gene expression strategy resembling those of plant potex like viruses. Mol Cells 23: 304 315. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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'()% !"#$%& !"#$% &'#$(%) *+,# -.!# *./!#$)01!#$) #2!#) 3!#) 42#5 6/"+#!"#$%& !"#$% &'#$(%) *+,# -.!#) *./!#$)01!#$) #2!#) 3!#) 42#56/"+# !"#$%& !"#$% &'#$(%) *+,# -.!#) *./!#$)01!#$) #2!#) 3!#) 42#56/"+# !"#$%& !"#$% &'#$(%) *+,# -.!#) *./!#$)01!#$) #2!#) 3!#) 42#56/"+# CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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'R)% !"#$%& !"#$% &'#$(%)*+,# -.!#) *./!#$) 01!#$)#2!#) 3!#) 42#5 6/"+# !"#$%& !"#$% &'#$(%)*+,# -.!#) *./!#$) 01!#$) #2!#)3!#) 42#5 6/"+# !"#$%& !"#$% &'#$(%)*+,# -.!#) *./!#$) 01!#$) #2!#)3!#) 42#5 6/"+# !"#$%& !"#$% &'#$(%)*+,# -.!#) *./!#$) 01!#$) #2!#)3!#) 42#5 6/"+# CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;

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. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not http://dx.doi.org/10.1101/059709 doi: bioRxiv preprint first posted online Jun. 19, 2016;