Arch Virol (2014) 159:3453–3456 DOI 10.1007/s00705-014-2212-2
ANNOTATED SEQUENCE RECORD
Novel dicistrovirus from bat guano Ga´bor Reuter • Pe´ter Pankovics • Zolta´n Gyo¨ngyi ´ kos Boros Eric Delwart • A
•
Received: 20 May 2014 / Accepted: 24 August 2014 / Published online: 29 August 2014 Ó Springer-Verlag Wien 2014
Abstract A novel dicistrovirus (strain NB-1/2011/HUN, KJ802403) genome was detected from guano collected from an insectivorous bat (species Pipistrellus pipistrellus) in Hungary, using viral metagenomics. The complete genome of NB-1 is 9136 nt in length, excluding the poly(A) tail. NB-1 has a genome organization typical of a dicistrovirus with multiple 3BVPg and a cripavirus-like intergenic region (IGR)-IRES. NB-1 shares only 41 % average amino acid sequence identity with capsid proteins of Himetobi P virus, indicating a potential novel species in the genus Cripavirus, family Dicistroviridae.
Dicistroviruses are small, non-enveloped viruses with a positive-sense, single-stranded RNA genome of *850010,000 nucleotides (nt) in size [3]. Dicistrovirus genomes contain two non-overlapping open reading frames (ORF1
Nucleotide sequence data reported are available in the GenBank database under accession number KJ802403. ´ . Boros G. Reuter (&) P. Pankovics A Regional Laboratory of Virology, National Reference ´ NTSZ Regional Institute Laboratory of Gastroenteric Viruses, A of State Public Health Service, Szabadsa´g u´t 7., 7623 Pe´cs, Hungary e-mail: [email protected] G. Reuter E. Delwart Blood Systems Research Institute, San Francisco, CA, USA G. Reuter E. Delwart University of California, San Francisco, CA, USA Z. Gyo¨ngyi Department of Public Health, Faculty of Medicine, University of Pe´cs, Pe´cs, Hungary
and ORF2) with the structural (capsid) proteins encoded by the 30 -proximal ORF. A defining characteristic of dicistroviruses is that they have two internal ribosome entry sites (IRES): one for translation of ORF1 and the second – the IGR (intergenic region) IRES – for translation of the capsid proteins (VP2-VP4-VP3 and VP1) [11]. ORF1 encodes the 2C-like helicase (Hel), genome-linked viral protein (VPg), 3C-like protease (Pro), and 3D-like RNA-dependent RNA polymerase (Pol) domains. To our current knowledge, all members of the family Dicistroviridae infect invertebrates and have been identified in aphids, leafhoppers, flies, bees, ants, silkworms and shrimps. Some dicistroviruses are pathogenic to arthropods such as honey bees and shrimp or to insect pests of medical and agricultural importance [1]. Here, we report the detection and complete genome characterization of a novel dicistrovirus identified in bat guano using viral metagenomics. Forty to fifty peppercorn-sized bat fecal samples (used as bat guano pool ‘‘NB-1’’) were collected from a 0.5-m2 sampling area in the backyard of a family house located in a garden suburb territory 5 km from the town of Pe´cs (Southwest Hungary) in April 2011. The host origin of ‘‘NB-1’’ was determined by bat species identification methods [7] using PCR and sequencing. ‘‘NB-1’’ was diluted with PBS, passed through a 0.45-lm sterile filter and centrifuged at 6,000 9 g for 5 min. The pellet was mixed with nucleases to enrich for particle-protected nucleic acids [16], and nucleic acids were extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). Viral nucleic acid libraries were constructed by sequenceindependent random RT-PCR amplification [5, 17]. Pyrosequencing reads (using 454 GS FLX technology) and assembled sequence contigs were compared to GenBank nucleotide and protein databases using BLASTn/BLASTx.
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Fig. 1 Predicted genome organization of dicistrovirus strain NB-1/ 2011/HUN (KJ802403). Numbers for nucleotide (nt; upper number) and amino acid (lower letters and number) sequences indicate the first sequence position of the next region. Amino-terminal cleavage sites are indicated at the borders of the regions. Amino acid sequence alignments of the multiple 3BVPg proteins are shown. The predicted
RNA secondary structure of IGR-IRES has been annotated as proposed previously for cripaviruses [3, 11]. The conserved IGRIRES motifs (in PSIV, DCV, RhPV and strain NB-1/2011/HUN) and the predicted CUU start codon (AUG-unrelated initiation codon) are indicated by shaded boxes. ORF, open reading frame; UTR, untranslated region; IGR, intergenic region; PK, pseudoknot
Fig. 2 Phylogenetic analysis of NB-1/2011/HUN (KJ802403, bold letters) and representative prototype strains of the genera Cripavirus and Aparavirus, in the family Dicistroviridae. Phylogenetic trees of the amino acid sequence alignment of 3D RNA polymerase (ORF1) and the complete capsid proteins (ORF2) were created. Abbreviations: ALPV, aphid lethal paralysis virus, BQCV, black queen cell virus; CrPV, cricket paralysis virus; DCV, drosophila C virus; HiPV,
Himetobi P virus; HoCV-1, Homalodisca coagulata virus-1; PSIV, Plautia stali intestine virus, RhPV, Rhopalosiphum padi virus; TrV, triatoma virus; ABPV, acute bee paralysis virus; IAPV, Israeli acute paralysis virus; KBV, Kashmir bee virus; SINV-1, Solenopsis invicta virus-1; TSV, Taura syndrome virus; MCDV, mud crab dicistrovirus; ’’BatDV’’, ’’bat dicistrovirus’’
The partial genome of dicistrovirus from ‘‘NB-1’’ was assembled from pyrosequencing reads and completed by primer-walking methods using RT-PCR and 50 /30 RACE [4]. The complete dicistrovirus genome was re-amplified by RT-PCR. PCR products were sequenced directly using on automated sequencer (ABI PRISM 310 Genetic Analyzer, Stafford, USA). Representative dicistrovirus sequences were aligned using ClustalX [15], and phylogenetic trees based on an amino acid (aa) alignment of 3D (ORF1) or the capsid polyprotein were created using the neighbor-joining
method employing the Jones-Taylor-Thornton matrixbased model of MEGA5 [14]. The RNA secondary structure of the IGR-IRES was predicted using Mfold [17], with manual corrections for dicistrovirus IGR-IRES structures published previously [11, 12]. Polyprotein cleavage sites were predicted from alignments with Himetobi P and Plautia stali intestine virus (PSIV), for which cleavage sites have been determined experimentally [8–10, 13]. The sequence of NB-1/2011/HUN was submitted to GenBank with the accession number KJ802403.
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From the bat (Pipistrellus pipistrellus) fecal sample ‘‘NB-1’’, a total of 244 viral sequence reads (singletons and contigs) were obtained (BLASTx cutoff E score B10-10) after de novo assembly from 5,710 viral reads from viruses of the families Dicistroviridae, Circoviridae, Parvoviridae and Iflaviridae. A total of 84 sequence reads (designated as NB-1) assembled into six contigs covering *88 % of the partial replicase (ORF1) and capsid proteins (ORF2) of dicistroviruses. The partial genome sequence shared closest aa sequence identity to the homologous proteins of Himetobi P virus (genus Cripavirus, family Dicistroviridae). The complete genome of NB-1 (KJ802403) is 9136 nt in length, excluding the poly(A) tail (Fig. 1). The coding region (ORF1) at the 50 end of the NB-1 genome encodes the nonstructural proteins; the coding region (ORF2) at the 30 end of NB-1 genome encodes the structural/capsid proteins. ORF1 encodes an 1820-aa (5460-nt) polyprotein with putative protease-mediated cleavage sites Q/VDVG (2B/ 2C), Q/VLIP (2C/3A), E/S (3A/3BVPg1), E/A (3BVPg5/3C) and Q/GHIL (3C/3D) (Fig. 1). The cleavage site between 2A and 2B could not be identified, as the ‘‘aphthovirus/ picornavirus-like’’ 2A with the NPG;P ‘‘ribosome skipping’’ motif was not found. The NB-1 genome appears to encode five 3BVPg proteins in tandem, with the 30 -proximal copy (3BVPg5) distinct in sequence from the other VPgs (Fig. 1). The 2C, 3C and 3D proteins share 52 %, 42 %, and 50 % aa sequence identity, respectively, with the corresponding Hel-Pro-Pol proteins of Himetobi P virus (AB183472) as the closest match in GenBank. Upstream of ORF2 was a highly structured sequence similar to the intergenic region (IGR)-IRES of other dicistroviruses including PSIV, Drosophila C virus (DCV) and Rhopalosiphum padi virus (RhPV) in the genus Cripavirus (Fig. 1). In addition, highly conserved IGR-IRES nucleotide motifs (e.g., bulge sequence G6100AUCA and U6149GC) [10, 11] and a C6276UU start codon (IGR-IRES-mediated translation initiation at an AUG-unrelated initiation codon like in PSIV) [12] also were found in NB-1 dicistrovirus (Fig. 1). Dicistrovirus-like capsid protein sequences were identified in ORF2 using a conserved domain search [6]. NB-1 capsid proteins have a maximum of 41 % average aa sequence identity to Himetobi P virus (AB183472) capsid proteins. The putative 3C-like-protease-mediated cleavage sites of the ORF2 polyprotein are Q/IKLN (VP2/VP4), L/SKPE (VP4/VP3) and Q/IYAT (VP3/VP1) (Fig. 1). The NB-1 capsid proteins share 51 % (VP2), 37 % (VP4), 50 % (VP3) and 37 % (VP1) aa sequence identity to the corresponding VPs of Himetobi P virus (AB183472). The 50 UTR and the 30 UTR of NB-1 are 623 nt and 170 nt long, and similar nt sequences were not found in GenBank. The phylogenetic analysis confirms that NB-1 is most closely related to Himetobi P virus (Fig. 2). During manuscript preparation, a partial (*1800-nt-long) dicistroviral
3455
sequence (‘‘bat dicistrovirus’’, KF170223) covering the partial non-structural region 3C/3D (found in a lung specimen of bat species Pipistrellus pipistrellus collected in Paris, France) became available in GenBank [2]. This sequence has 97 % aa sequence identity to the corresponding 3C/3D protein of NB-1 (Fig. 2). While the primary origin of the dicistrovirus NB-1 remains unknown, the virus is present in and shed from insectivorous bats in Europe, based on two independent studies. According to the current species demarcation criteria for the Dicistroviridae [3], sequence identity between the capsid proteins of isolates and strains of a species is above 90 %. Based on this rule, NB-1 potentially represents a novel species in the genus Cripavirus, family Dicistroviridae. Acknowledgments This work was supported by grants from the Hungarian Scientific Research Fund (OTKA, K83013 and OTKA, K111615) and NHLBI Grant R01-HL105770. Conflict of interest of interest.
The authors declare that they have no conflict
References 1. Bonning BC, Miller WA (2010) Dicistroviruses. An Rev Entomol 55:129–150 2. Dacheux L, Cervantes-Gonzales M, Guigon G, Thiberge JM, Vandenbogaert M, Maufrais C, Caro V, Bourhy H (2014) A preliminary study of viral metagenomics of French bat species in contact with humans: identification of new mammalian viruses. PLoS One 9:e87194 3. Culley AI, Lang AS, Suttle CA (2012) Family Dicistroviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus taxonomy, classification and nomenclature of viruses, 9th report of the ICTV. Elsevier Academic Press, Amsterdam, pp 840–854 4. Boros A, Pankovics P, Simmonds P, Reuter G (2011) Novel positive-sense, single-stranded RNA (?ssRNA) virus with dicistronic genome from intestinal content of freshwater carp (Cyprinus carpio). PLoS One 6:e29145 5. Kapoor A, Victoria J, Simmonds P, Slikas E, Chieochansin T, Naeem A, Shaukat S, Sharif S, Alam MM, Angez M, Wang C, Shafer RW, Zaidi S, Delwart E (2008) A highly prevalent and genetically diversified Picornaviridae genus in South Asian children. Proc Natl Acad Sci USA 105:20482–20487 6. Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY et al (2011) CDD: a conserved domain database for the functional annotation of proteins. Nucleic Acids Res 39:D225–D229 7. Mayer F, von Helversen O (2001) Cryptic diversity in European bats. Proc Biol Sci 268:1825–1832 8. Nakashima N, Ishibashi J (2010) Identification of the 3C-protease-mediated 2A/2B and 2B/2C cleavage sites in the non-structural polyprotein precursor of a dicistrovirus lacking the NPGP motif. Arch Virol 155:1477–1482 9. Nakashima N, Nakamura Y (2008) Cleavage sites of the ‘‘P3 region’’ in the nonstructural polyprotein precursor of a dicistrovirus. Arch Virol 153:1955–1960
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3456 10. Nakashima N, Sasaki J, Toriyama S (1999) Determining the nucleotide sequence and capsid-coding region of Himetobi P virus: a member of a novel group of RNA viruses that infect insects. Arch Virol 144:2051–2058 11. Nakashima N, Uchiumi T (2009) Functional analysis of structural motifs in dicistroviruses. Virus Res 139:137–147 12. Sasaki J, Nakashima N (1999) Translation initiation at the CUU codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro. J Virol 73:1219–1226 13. Sasaki J, Nakashima N, Saito H, Noda H (1998) An insect picorna-like virus, plautia stali intestine virus, has genes of capsid proteins in the 30 part of the genome. Virology 244:50–58 14. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using
123
G. Reuter et al. maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 15. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 16. Victoria JG, Kapoor A, Li L, Blinkova O, Slikas B, Wang C, Naeem A, Zaidi S, Delwart E (2009) Metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis. J Virol 83:4642–4651 17. Zucker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415
ANNOTATED SEQUENCE RECORD
Novel dicistrovirus from bat guano Ga´bor Reuter • Pe´ter Pankovics • Zolta´n Gyo¨ngyi ´ kos Boros Eric Delwart • A
•
Received: 20 May 2014 / Accepted: 24 August 2014 / Published online: 29 August 2014 Ó Springer-Verlag Wien 2014
Abstract A novel dicistrovirus (strain NB-1/2011/HUN, KJ802403) genome was detected from guano collected from an insectivorous bat (species Pipistrellus pipistrellus) in Hungary, using viral metagenomics. The complete genome of NB-1 is 9136 nt in length, excluding the poly(A) tail. NB-1 has a genome organization typical of a dicistrovirus with multiple 3BVPg and a cripavirus-like intergenic region (IGR)-IRES. NB-1 shares only 41 % average amino acid sequence identity with capsid proteins of Himetobi P virus, indicating a potential novel species in the genus Cripavirus, family Dicistroviridae.
Dicistroviruses are small, non-enveloped viruses with a positive-sense, single-stranded RNA genome of *850010,000 nucleotides (nt) in size [3]. Dicistrovirus genomes contain two non-overlapping open reading frames (ORF1
Nucleotide sequence data reported are available in the GenBank database under accession number KJ802403. ´ . Boros G. Reuter (&) P. Pankovics A Regional Laboratory of Virology, National Reference ´ NTSZ Regional Institute Laboratory of Gastroenteric Viruses, A of State Public Health Service, Szabadsa´g u´t 7., 7623 Pe´cs, Hungary e-mail: [email protected] G. Reuter E. Delwart Blood Systems Research Institute, San Francisco, CA, USA G. Reuter E. Delwart University of California, San Francisco, CA, USA Z. Gyo¨ngyi Department of Public Health, Faculty of Medicine, University of Pe´cs, Pe´cs, Hungary
and ORF2) with the structural (capsid) proteins encoded by the 30 -proximal ORF. A defining characteristic of dicistroviruses is that they have two internal ribosome entry sites (IRES): one for translation of ORF1 and the second – the IGR (intergenic region) IRES – for translation of the capsid proteins (VP2-VP4-VP3 and VP1) [11]. ORF1 encodes the 2C-like helicase (Hel), genome-linked viral protein (VPg), 3C-like protease (Pro), and 3D-like RNA-dependent RNA polymerase (Pol) domains. To our current knowledge, all members of the family Dicistroviridae infect invertebrates and have been identified in aphids, leafhoppers, flies, bees, ants, silkworms and shrimps. Some dicistroviruses are pathogenic to arthropods such as honey bees and shrimp or to insect pests of medical and agricultural importance [1]. Here, we report the detection and complete genome characterization of a novel dicistrovirus identified in bat guano using viral metagenomics. Forty to fifty peppercorn-sized bat fecal samples (used as bat guano pool ‘‘NB-1’’) were collected from a 0.5-m2 sampling area in the backyard of a family house located in a garden suburb territory 5 km from the town of Pe´cs (Southwest Hungary) in April 2011. The host origin of ‘‘NB-1’’ was determined by bat species identification methods [7] using PCR and sequencing. ‘‘NB-1’’ was diluted with PBS, passed through a 0.45-lm sterile filter and centrifuged at 6,000 9 g for 5 min. The pellet was mixed with nucleases to enrich for particle-protected nucleic acids [16], and nucleic acids were extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). Viral nucleic acid libraries were constructed by sequenceindependent random RT-PCR amplification [5, 17]. Pyrosequencing reads (using 454 GS FLX technology) and assembled sequence contigs were compared to GenBank nucleotide and protein databases using BLASTn/BLASTx.
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Fig. 1 Predicted genome organization of dicistrovirus strain NB-1/ 2011/HUN (KJ802403). Numbers for nucleotide (nt; upper number) and amino acid (lower letters and number) sequences indicate the first sequence position of the next region. Amino-terminal cleavage sites are indicated at the borders of the regions. Amino acid sequence alignments of the multiple 3BVPg proteins are shown. The predicted
RNA secondary structure of IGR-IRES has been annotated as proposed previously for cripaviruses [3, 11]. The conserved IGRIRES motifs (in PSIV, DCV, RhPV and strain NB-1/2011/HUN) and the predicted CUU start codon (AUG-unrelated initiation codon) are indicated by shaded boxes. ORF, open reading frame; UTR, untranslated region; IGR, intergenic region; PK, pseudoknot
Fig. 2 Phylogenetic analysis of NB-1/2011/HUN (KJ802403, bold letters) and representative prototype strains of the genera Cripavirus and Aparavirus, in the family Dicistroviridae. Phylogenetic trees of the amino acid sequence alignment of 3D RNA polymerase (ORF1) and the complete capsid proteins (ORF2) were created. Abbreviations: ALPV, aphid lethal paralysis virus, BQCV, black queen cell virus; CrPV, cricket paralysis virus; DCV, drosophila C virus; HiPV,
Himetobi P virus; HoCV-1, Homalodisca coagulata virus-1; PSIV, Plautia stali intestine virus, RhPV, Rhopalosiphum padi virus; TrV, triatoma virus; ABPV, acute bee paralysis virus; IAPV, Israeli acute paralysis virus; KBV, Kashmir bee virus; SINV-1, Solenopsis invicta virus-1; TSV, Taura syndrome virus; MCDV, mud crab dicistrovirus; ’’BatDV’’, ’’bat dicistrovirus’’
The partial genome of dicistrovirus from ‘‘NB-1’’ was assembled from pyrosequencing reads and completed by primer-walking methods using RT-PCR and 50 /30 RACE [4]. The complete dicistrovirus genome was re-amplified by RT-PCR. PCR products were sequenced directly using on automated sequencer (ABI PRISM 310 Genetic Analyzer, Stafford, USA). Representative dicistrovirus sequences were aligned using ClustalX [15], and phylogenetic trees based on an amino acid (aa) alignment of 3D (ORF1) or the capsid polyprotein were created using the neighbor-joining
method employing the Jones-Taylor-Thornton matrixbased model of MEGA5 [14]. The RNA secondary structure of the IGR-IRES was predicted using Mfold [17], with manual corrections for dicistrovirus IGR-IRES structures published previously [11, 12]. Polyprotein cleavage sites were predicted from alignments with Himetobi P and Plautia stali intestine virus (PSIV), for which cleavage sites have been determined experimentally [8–10, 13]. The sequence of NB-1/2011/HUN was submitted to GenBank with the accession number KJ802403.
123
Novel dicistrovirus from bat guano
From the bat (Pipistrellus pipistrellus) fecal sample ‘‘NB-1’’, a total of 244 viral sequence reads (singletons and contigs) were obtained (BLASTx cutoff E score B10-10) after de novo assembly from 5,710 viral reads from viruses of the families Dicistroviridae, Circoviridae, Parvoviridae and Iflaviridae. A total of 84 sequence reads (designated as NB-1) assembled into six contigs covering *88 % of the partial replicase (ORF1) and capsid proteins (ORF2) of dicistroviruses. The partial genome sequence shared closest aa sequence identity to the homologous proteins of Himetobi P virus (genus Cripavirus, family Dicistroviridae). The complete genome of NB-1 (KJ802403) is 9136 nt in length, excluding the poly(A) tail (Fig. 1). The coding region (ORF1) at the 50 end of the NB-1 genome encodes the nonstructural proteins; the coding region (ORF2) at the 30 end of NB-1 genome encodes the structural/capsid proteins. ORF1 encodes an 1820-aa (5460-nt) polyprotein with putative protease-mediated cleavage sites Q/VDVG (2B/ 2C), Q/VLIP (2C/3A), E/S (3A/3BVPg1), E/A (3BVPg5/3C) and Q/GHIL (3C/3D) (Fig. 1). The cleavage site between 2A and 2B could not be identified, as the ‘‘aphthovirus/ picornavirus-like’’ 2A with the NPG;P ‘‘ribosome skipping’’ motif was not found. The NB-1 genome appears to encode five 3BVPg proteins in tandem, with the 30 -proximal copy (3BVPg5) distinct in sequence from the other VPgs (Fig. 1). The 2C, 3C and 3D proteins share 52 %, 42 %, and 50 % aa sequence identity, respectively, with the corresponding Hel-Pro-Pol proteins of Himetobi P virus (AB183472) as the closest match in GenBank. Upstream of ORF2 was a highly structured sequence similar to the intergenic region (IGR)-IRES of other dicistroviruses including PSIV, Drosophila C virus (DCV) and Rhopalosiphum padi virus (RhPV) in the genus Cripavirus (Fig. 1). In addition, highly conserved IGR-IRES nucleotide motifs (e.g., bulge sequence G6100AUCA and U6149GC) [10, 11] and a C6276UU start codon (IGR-IRES-mediated translation initiation at an AUG-unrelated initiation codon like in PSIV) [12] also were found in NB-1 dicistrovirus (Fig. 1). Dicistrovirus-like capsid protein sequences were identified in ORF2 using a conserved domain search [6]. NB-1 capsid proteins have a maximum of 41 % average aa sequence identity to Himetobi P virus (AB183472) capsid proteins. The putative 3C-like-protease-mediated cleavage sites of the ORF2 polyprotein are Q/IKLN (VP2/VP4), L/SKPE (VP4/VP3) and Q/IYAT (VP3/VP1) (Fig. 1). The NB-1 capsid proteins share 51 % (VP2), 37 % (VP4), 50 % (VP3) and 37 % (VP1) aa sequence identity to the corresponding VPs of Himetobi P virus (AB183472). The 50 UTR and the 30 UTR of NB-1 are 623 nt and 170 nt long, and similar nt sequences were not found in GenBank. The phylogenetic analysis confirms that NB-1 is most closely related to Himetobi P virus (Fig. 2). During manuscript preparation, a partial (*1800-nt-long) dicistroviral
3455
sequence (‘‘bat dicistrovirus’’, KF170223) covering the partial non-structural region 3C/3D (found in a lung specimen of bat species Pipistrellus pipistrellus collected in Paris, France) became available in GenBank [2]. This sequence has 97 % aa sequence identity to the corresponding 3C/3D protein of NB-1 (Fig. 2). While the primary origin of the dicistrovirus NB-1 remains unknown, the virus is present in and shed from insectivorous bats in Europe, based on two independent studies. According to the current species demarcation criteria for the Dicistroviridae [3], sequence identity between the capsid proteins of isolates and strains of a species is above 90 %. Based on this rule, NB-1 potentially represents a novel species in the genus Cripavirus, family Dicistroviridae. Acknowledgments This work was supported by grants from the Hungarian Scientific Research Fund (OTKA, K83013 and OTKA, K111615) and NHLBI Grant R01-HL105770. Conflict of interest of interest.
The authors declare that they have no conflict
References 1. Bonning BC, Miller WA (2010) Dicistroviruses. An Rev Entomol 55:129–150 2. Dacheux L, Cervantes-Gonzales M, Guigon G, Thiberge JM, Vandenbogaert M, Maufrais C, Caro V, Bourhy H (2014) A preliminary study of viral metagenomics of French bat species in contact with humans: identification of new mammalian viruses. PLoS One 9:e87194 3. Culley AI, Lang AS, Suttle CA (2012) Family Dicistroviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus taxonomy, classification and nomenclature of viruses, 9th report of the ICTV. Elsevier Academic Press, Amsterdam, pp 840–854 4. Boros A, Pankovics P, Simmonds P, Reuter G (2011) Novel positive-sense, single-stranded RNA (?ssRNA) virus with dicistronic genome from intestinal content of freshwater carp (Cyprinus carpio). PLoS One 6:e29145 5. Kapoor A, Victoria J, Simmonds P, Slikas E, Chieochansin T, Naeem A, Shaukat S, Sharif S, Alam MM, Angez M, Wang C, Shafer RW, Zaidi S, Delwart E (2008) A highly prevalent and genetically diversified Picornaviridae genus in South Asian children. Proc Natl Acad Sci USA 105:20482–20487 6. Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY et al (2011) CDD: a conserved domain database for the functional annotation of proteins. Nucleic Acids Res 39:D225–D229 7. Mayer F, von Helversen O (2001) Cryptic diversity in European bats. Proc Biol Sci 268:1825–1832 8. Nakashima N, Ishibashi J (2010) Identification of the 3C-protease-mediated 2A/2B and 2B/2C cleavage sites in the non-structural polyprotein precursor of a dicistrovirus lacking the NPGP motif. Arch Virol 155:1477–1482 9. Nakashima N, Nakamura Y (2008) Cleavage sites of the ‘‘P3 region’’ in the nonstructural polyprotein precursor of a dicistrovirus. Arch Virol 153:1955–1960
123
3456 10. Nakashima N, Sasaki J, Toriyama S (1999) Determining the nucleotide sequence and capsid-coding region of Himetobi P virus: a member of a novel group of RNA viruses that infect insects. Arch Virol 144:2051–2058 11. Nakashima N, Uchiumi T (2009) Functional analysis of structural motifs in dicistroviruses. Virus Res 139:137–147 12. Sasaki J, Nakashima N (1999) Translation initiation at the CUU codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro. J Virol 73:1219–1226 13. Sasaki J, Nakashima N, Saito H, Noda H (1998) An insect picorna-like virus, plautia stali intestine virus, has genes of capsid proteins in the 30 part of the genome. Virology 244:50–58 14. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using
123
G. Reuter et al. maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 15. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 16. Victoria JG, Kapoor A, Li L, Blinkova O, Slikas B, Wang C, Naeem A, Zaidi S, Delwart E (2009) Metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis. J Virol 83:4642–4651 17. Zucker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415
