LETTERS

4. Page LK, Anchor C, Luy E, Kron S, Novel Divergent that the sample was collected from a Larson G, Madsen L, et al. Backyard rac- red fox. coon latrines and risk for Baylisascaris Rhabdovirus in procyonis transmission to humans. Emerg The obtained sequence of RFFRV Infect Dis. 2009;15:1530–1. http://dx.doi. Feces of Red Fox, was partially confirmed by specific org/10.3201/eid1509.090128 Spain primers and Sanger sequencing of 5. Kelly TG, Madhavan VL, Peters JM, PCR amplicons. Five major and 3 mi- Kazacos KR, Silvera VM. Spinal cord involvement in a child with raccoon To the Editor: Rhabdoviruses nor open reading frames (ORFs) were roundworm (Baylisascaris procyonis) (family ) are enveloped identified that had a genome organiza- meningoencephalitis. Pediatr Radiol. single-stranded negative-sense RNA tion similar to that of other rhabdovi- 2012;42:369–73. http://dx.doi.org/10. belonging to the Mononega- ruses (Figure, panel A). No significant 1007/s00247-011-2151-y 6. Wise ME, Sorvillo FJ, Shafir SC, Ash LR, virales order. The International Com- hits were obtained by BLAST analy- Berlin OG. Severe and fatal central ner- mittee on Taxonomy of Viruses rec- sis (http://blast.ncbi.nlm.gov/Blast. vous system disease in humans caused ognizes 11 genera (Cytorhabdovirus, cgi) of N, P, M, and G nucleotide and by Baylisascaris procyonis, the com- Ephemerovirus, Lyssavirus, No- amino acid sequences, which was re- mon roundworm of raccoons: a re- view of current literature. Microbes virhabdovirus, Nucleorhabdovirus, ported previously for novel divergent Infect. 2005;7:317–23. http://dx.doi.org/ Perhabdovirus, Sigmavirus, Sprivivi- rhabdoviruses (4). 10.1016/j.micinf.2004.12.005 rus, Tibrovirus, Tupavirus, Vesiculo- Predicted N, P, and M genes of 7. Blizzard EL, Yabsley MJ, Beck MF, ) (1). In addition, many recently RFFRV consist of 1,629, 2,490, and Harsch S. Geographic expansion of Bay- lisascaris procyonis roundworms, Florida, described rhabdoviruses remain unas- 813 nt, respectively, encoding for 543, USA. Emerg Infect Dis. 2010;16:1803–4. signed. Rhabdoviruses contain 5 ma- 830, and 271 aa (online Technical http://dx.doi.org/10.3201/eid1611.100549 jor genes, encoding for nucleoprotein Appendix Table 1). In addition to the 8. Miyashita M. Prevalence of Baylisascaris (N), phosphoprotein (P), matrix (M), absence of significant hits observed procyonis in raccoons in Japan and experi- mental infections of the worm in labora- glycoprotein (G), and RNA-depen- by BLAST analysis, no significant tory animals. Journal of Urban Living and dent RNA polymerase (L). The Rhab- sequence homology was observed Health Association. 1993;37:137–51. doviridae family includes pathogens with known rhabdovirus proteins in 9. Xie Y, Zhang Z, Niu L, Wang Q, Wang C, of various animal species, humans, pairwise alignments. Furthermore, Lan J, et al. The mitochondrial genome of Baylisascaris procyonis. PLoS ONE. and plants. Viruses of the genus Lys- no conserved motifs were detected in 2011;6:e27066. http://dx.doi.org/10.1371/ savirus are the most relevant to public N, P, and M genes of RFFRV that are journal.pone.0027066 health because they can cause rabies. commonly observed in rhabdoviruses. 10. Sorvillo F, Ash LR, Berlin OGW, Tatabe J, Bats are the driving force within this However, intergenic regions between Degiorgio C, Morse SA. Baylisascaris procyonis: an emerging helminthic zoo- genus; foxes and various other species all major ORFs contained relatively nosis. Emerg Infect Dis. 2002;8:355–9. of wild carnivores also can be infected conserved motifs that could be tran- http://dx.doi.org/10.3201/eid0804.010273 with lyssaviruses and transmit them to scription termination/polyadenylation

humans and dogs (2). sequences (A/U) CU7, similar to other Address for correspondence: Guangyou During a viral metagenomic sur- rhabdoviruses (5). Adjacent to this ter- Yang, Department of Parasitology, College vey, conducted as described previous- mination signal was a stretch of con- of Veterinary Medicine, Sichuan Agricultural ly (3), of fecal samples collected from served nucleotides that might function University, 46 Xinkang Rd, Ya’an, Sichuan, 4 red foxes (Vulpes vulpes) that were as a transcription initiation signal (on- 625014, People’s Republic of China; email: found dead in Álava, Basque Country, line Technical Appendix Table 1). [email protected] Spain, we identified the complete cod- The amino acid sequence of the ing sequence and the partial leader and G protein consisted of 669 aa and trailer sequence of a novel rhabdo- contained an N terminal signal pep- virus, tentatively called red fox fecal tide (1-MYHLIVLLVMLGQRA- rhabdovirus (RFFRV; 15,541 nt, Gen- VA-17), a noncytoplasmic domain Free Online RSS Feed Bank accession no. KF823814; online (aa 18–646), a transmembrane do- Technical Appendix, http://wwwnc. main (647-ITAILMPLLSLAVVVGI- in PubMed Central cdc.gov/EID/article/20/12/14-0236- IMCC-667), and a cytoplasmic tail of Ahead of print Techapp1.pdf) by mapping 8,287 of 2 aa, similar to other rhabdovirus G Peeer-Reviewedr-Revie the 56,519 sequence reads in the sam- proteins as predicted by using Phobius CME ple of a red fox. A proportion of ob- and TMHMM (http://www.cbs.dtu. podcasts tained reads contained sequences that dk/services/TMHMM) (6,7). We pre- were >99% identical to mitochondrial dicted 3 potential glycosylation sites GovDelivery DNA of V. vulpes, which confirmed in the ectodomain at positions 38–40

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(NKT), 554–556 (NAS), and 592–594 (NIS) using NetNGlyc 1.0 (http:// www.cbs.dtu.dk/services/NetNGlyc). Between the G and L genes, a complex intergenic region was pres- ent that contained 3 ORFs of 246 nt (7,413–7,658 aa), 231 nt (7,716–7,946 aa), and 459 nt (7,893–8,355 aa), of which 2 were overlapping frames (U1–3). Additional ORFs between G and L genes were detected previ- ously in other rhabdoviruses (8,9). We detected transmembrane domains in the amino acid sequences of all 3 additional ORFs, suggesting they might act as viroporin (8,9). The L gene of RFFRV contained 6,591 nt (2,197 aa). We detected several conserved domains and mo- tifs, including RNA-dependent RNA polymerase, mRNA-capping region, mRNA capping enzyme, and virus- capping methyltransferase. Align- Figure. Genome organization and phylogenetic analysis of RFFRV. A) Genome organization ment of the deduced amino acid se- of RFFRV. Indicated are the locations of the major ORFs (including the positions of the quence of the L gene with the L gene first and last nucleotide) and 3 theoretical minor ORFs between the G and L genes. B) Phylogenetic maximum-likelihood tree using the WAG+F+I+G model and 100 bootstrap of various other viruses belonging to replicates in MEGA5 (http://www.megasoftware.net) of the deduced amino acid sequence the order by using of the L genes of various viruses of the order Mononegavirales. G, glycoprotein; L, RNA- MAFFT version 7 (http://mafft.cbrc. dependent RNA polymerase; M, matrix; N, nucleoprotein; ORF, open reading frame; P, jp/alignment/software/) and subse- phosphoprotein; RFFRV, RFFRV, red fox fecal rhabdovirus. Only bootstrap values in the quent phylogenetic reconstruction close proximity of the branch of the RFFRV are indicated. Scale bar indicates nucleotide substitutions per site. Viruses and GenBank accession numbers are shown in the by using a maximum-likelihood tree expanded figure legend online (http://wwwnc.cdc.gov/EID/article/20/12/14-0236-F1.htm). (WAG+F+I+G model with 100 boot- strap replicates in MEGA5 [http:// despite a high number of reads in the Disease Entities” European Community www.megasoftware.net]) suggested original sample. The fox might have grant agreement no. 223498; the Virgo that this virus belongs to a novel ge- acquired the virus through spillover Consortium; and the Niedersachsen-Re- nus of the Rhabdoviridiae family. In from a small prey, such as a bat, and search Network on Neuroinfectiology of addition, pairwise identities of the additional studies are required to eluci- the Ministry of Science and Culture of deduced amino acid sequence of the date the prevalence, original host, and Lower Saxony, Germany. In addition, this L gene of RFFRV with that of other pathogenic potential of this novel virus. research was funded partially by the Basque rhabdoviruses of the Rhabdoviridae Government through the research group on family were only <35% (online Tech- Acknowledgments “Systematics, Biogeography and Popula- nical Appendix Table 2). tion Dynamics” (ref. IT317-10; GIC10/76). Because the fox was found dead We thank all researchers and institu- tions for their invaluable help during sam- and no tissue samples were collected, A.R.-G. holds a postdoctoral fellow- pling and for providing the specimens used whether RFFRV played a role in the ship awarded by the Department of Edu- in this study, especially Patricia Lizarraga, animal’s death is unknown. In addi- cation, Universities and Research of the Laura Elorza, Ricardo Gutierrez, and Luis tion, multiple attempts to isolate this Basque Government (ref. DKR-2012-64) Javier Chueca. virus on various cell lines of eukaryotes and was awarded a short-visit research (Vero E6, MDCK, CRFK, N2a, and This work was funded by the Euro- grant from the ConGenOmics Research BHK cells, primary fox kidney cells) pean Community’s Seventh Framework networking program of the European Sci- failed because of the absence of cyto- Program (FP7/2007–2013) under the proj- ence Foundation to visit the Department of pathic effects and viral replication by ect “European Management Platform for Viroscience, Erasmus Medical Centre and quantitative reverse transcription PCR, Emerging and Re-emerging Infectious develop the current research project.

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Rogier Bodewes,1 Rhabdoviridae. Virology. 2008;376:13– In 2010, during an ongoing Aritz Ruiz-Gonzalez,1 23. http://dx.doi.org/10.1016/j.virol.2008. RVFV outbreak in Mauritania, we col- 03.004 Anita C. Schürch, 9. McWilliam SM, Kongsuwan K, lected 163 serum samples (62 from Albert D.M.E. Osterhaus, Cowley JA, Byrne KA, Walker PJ. camels, 8 from cattle, and 93 from and Saskia L. Smits Genome organization and transcription small ruminants) (5). RVFV RNA strategy in the complex GNS-L intergenic Author affiliations: Erasmus Medical was isolated from serum samples as region of bovine ephemeral fever rhabdo- Centre, Rotterdam, the Netherlands virus. J Gen Virol. 1997;78:1309–17. described previously (5). Further mo- (R. Bodewes, A.C. Schürch, A.D.M.E. lecular testing of the samples was Osterhaus, S.L. Smits); University of the Address for correspondence: Rogier Bodewes, conducted by a SYBRGreen–based Basque Country, Vitoria-Gasteiz, Spain Erasmus Medical Centre, Department of real-time reverse transcription PCR (A. Ruiz-Gonzalez); National Institute Viroscience Dr. Molewaterplein 50, 3015GE (RT-PCR) adapted from a conventional for Environmental Protection and Re- Rotterdam, the Netherlands; email: r.bodewes@ RT-PCR and based on generic prim- search, Ozzano dell’Emilia, Italy (A. Ruiz- erasmusmc.nl ers (bun_group_forw 5′-CTGCTAA- Gonzalez); and Viroclinics Biosciences, CACCAGCAGTACTTTTGAC-3′ Rotterdam (A.D.M.E. Osterhaus, S.L. Smits) and bun_group_rev 5′-TGGAGGGTA- AGACCATCGTCAGGAACTG-3′) DOI: http://dx.doi.org/10.3201/eid2012.140236 that target a 250-nt sequence of the S segment of Bunyamwera serogroup References members (6). Real-time RT-PCR was 1. International Committee on Taxonomy performed in a CFX 96 real-time PCR of Viruses. Virus taxonomy: 2013 release Ngari Virus in system (Bio-Rad, Hercules, CA, USA) [cited 2014 Feb 9]. http://www.ictvonline. by using 5 μL RNA with a QuantiTect org/virusTaxonomy.asp Goats during Rift 2. Matha IS, Salunke SR. Immunogenicity SYBR Green RT-PCR Kit (QIAGEN, of purified Vero cell rabies vaccine used Valley Fever Hilden Germany) in a final volume of in the treatment of fox-bite victims in Outbreak, 25 μL. Cycling conditions included RT India. Clin Infect Dis. 2005;40:611–3. at 50°C for 30 min and 95°C for 15 http://dx.doi.org/10.1086/427700 Mauritania, 2010 3. Bodewes R, Ruiz-Gonzalez A, min, followed by amplification with 44 Schapendonk CM, van den Brand JM, To the Editor: Ngari virus cycles of 95°C for 15 s, 55°C for 25 s, Osterhaus AD, Smits SL. Viral metage- (NRIV) is a single-stranded RNA virus 72°C for 30 s, and 77°C for 5 s. A melt- nomic analysis of feces of wild small car- ing curve analysis was then performed nivores. Virol J. 2014;11:89. http://dx.doi. belonging to the family Bunyaviridae, org/10.1186/1743-422X-11-89 genus Orthobunyavirus. The genome starting with 95°C for 60 s, and a tem- 4. Palacios G, Forrester NL, Savji N, comprises 3 segments, the small (S), perature gradient was conducted from Travassos da Rosa AP, Guzman H, medium (M), and large (L) segments, 68°C to 94°C in increments of 0.2°C. Detoy K, et al. Characterization of Farm- Of the 163 serum samples tested, ington virus, a novel virus from birds that is which encode the nucleocapsid (N) distantly related to members of the family protein, the 2 glycoproteins Gn and Gc, 2 samples from goats resulted in a Rhabdoviridae. Virol J. 2013;10:219. http:// and the RNA-dependent RNA-poly- positive signal with cycle thresholds dx.doi.org/10.1186/1743-422X-10-219 merase, respectively. Sequence analy- of 23 (sample 51) and 28 (sample 65), 5. Albertini AA, Ruigrok RW, Blondel D. respectively. Both samples showed Rabies virus transcription and replica- sis showed that NRIV is a reassortant tion. Adv Virus Res. 2011;79:1–22. http:// between Bunyamwera virus (BUNV) similar melting peaks at ≈78.2°C and dx.doi.org/10.1016/B978-0-12-387040- and Batai virus (BATV), both from the shared the identical partial nucleotide 7.00001-9 genus Orthobunyavirus. S and L seg- sequence of the S segment. The se- 6. Coll JM. The glycoprotein G of rhabdo- quence belongs to the Bunyamwera viruses. Arch Virol. 1995;140:827–51. ments derived from BUNV, and the http://dx.doi.org/10.1007/BF01314961 M segment derived from BATV (1,2). serogroup, but the short partial se- 7. Käll L. Krogh A, Sonnhammer EL. A NRIV is more virulent than BUNV and quence was not sufficient for accu- combined transmembrane topology and BATV and is associated with hemor- rate virus determination and identi- signal peptide prediction method. J Mol fication. For this reason, both serum Biol. 2004;338:1027–36. http://dx.doi. rhagic fever. NRIV was first isolated org/10.1016/j.jmb.2004.03.016 from Aedes simpsoni mosquitoes in samples were used to inoculate cell 8. Gubala AJ, Proll DF, Barnard RT, 1979 and from humans in 1993, both monolayers of Vero E6 cells that Cowled CJ, Crameri SG, Hyatt AD, et al. in Senegal (3). During 1997 and 1998, were assayed for virus replication. Genomic characterisation of Wongabel Only sample 51 displayed a cytopath- virus reveals novel genes within the humans were affected with hemorrhag- ic fever diseases in Kenya and Somalia ic effect after 72 h and was further 1These authors contributed equally to this that were caused by Rift Valley fever analyzed. We isolated the viral RNA article. virus (RVFV) and by NRIV (2,4). from cell culture with TRIzol reagent

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