• View in gallery

    Transmission electron microscopy of Vero cells infected with Sierra Nevada (BA-T) virus. (A) Virions forming at the cell surface. N = nuclei of adjacent cells. Bar = 1 μm. (B) High power of a portion of an infected Vero cell with budding (thick arrowheads) virions (v). Thin arrows indicate cell membranes of adjacent cells. Bar = 100 nm.

  • View in gallery

    Coverage plot in log scale showing the number of mapped reads across the genomic assembly. Low coverage regions were polymerase chain reaction (PCR) amplified and sequenced by Sanger.

  • View in gallery

    Schematic of the annotated Sierra Nevada virus (SNVV) genome, in comparison to Nyamanini virus (NYMV), and Midway virus (MIDWV). Coordinates of interpro domains are annotated on each genome. i = site of transcription initiation motif; t = site of transcription termination motif; CC = putative coiled-coil domains (see Figure 4C).

  • View in gallery

    (A) Phylogenetic tree of RdRP sequences of the order Mononegavirales. The order Mononegavirales comprises five families, within which the family Paramyxoviridae is split into two subfamilies: Pneumovirinae and Paramyxovirinae. The Sierra Nevada virus (SNVV) branches within a clade of Nyamiviridae and branches closely, but as sister to Nyamanini virus (NYWV) and Midway virus (MIDWV). In comparison, the soybean cyst nematode virus 1 (SbCNV) is a distant, deep branching member of the Nyamiviridae. The accession numbers and full names of all the viruses in the tree are listed in Supplemental Table 1. (B) Coiled-coil domain prediction for open-reading frame (ORF) III in SNVV, NYMV, and MIDWV. The coiled-coil domain prediction was done based on a 28-residue window using the program of COILS15; there are two peaks shown along the curves, and both of them partially overlap with each other across the three viruses, suggesting a similar pattern of the coiled-coil domain distribution in their genomes. (C) Sequence alignment for the putative coiled-coil regions of ORF III in SNVV, NYMV, and MIDWV. Alignment of the sequences of N- and C- terminal coiled-coil domain across SNVV, NYMV, and MIDWV shows that 39% of the residues are conserved in the N-terminal domains, whereas 63% of the residues are conserved in the C-terminal domains.

  • 1.

    Pringle CR, Alexander DJ, Billeter MA, Collins PL, Kingsbury DW, Lipkind MA, Nagai Y, Orvell C, Rima B, Rott R, ter Meulen V, 1991. The order Mononegavirales. Arch Virol 117: 137140.

    • Search Google Scholar
    • Export Citation
  • 2.

    Kuhn JH, Bekal S, Caì Y, Clawson AN, Domier LL, Herrel M, Jahrling PB, Kondo H, Lambert KN, Mihindukulasuriya KA, 2013. Nyamiviridae: proposal for a new family in the order Mononegavirales. Arch Virol 158: 118.

    • Search Google Scholar
    • Export Citation
  • 3.

    Mihindukulasuriya KA, Nguyen NL, Wu G, Huang HV, da Rosa AP, Popov VL, Tesh RB, Wang D, 2009. Nyamanini and midway viruses define a novel taxon of RNA viruses in the order Mononegavirales. J Virol 83: 51095116.

    • Search Google Scholar
    • Export Citation
  • 4.

    McKercher DG, Wada EM, Straub OC, 1963. Distribution and persistence of infectious bovine rhinotracheitis virus in experimentally infected cattle. Am J Vet Res 24: 510514.

    • Search Google Scholar
    • Export Citation
  • 5.

    McKercher DG, Wada EM, Ault SK, Theis JH, 1980. Viral agents recovered from Ornithodoros coriaceus, a vector of epizootic bovine abortion. Am J Vet Res 41: 803805.

    • Search Google Scholar
    • Export Citation
  • 6.

    Wada EM, McKercher DG, Castrucci G, Theis JH, 1976. Preliminary characterization and pathogenicity studies of a virus isolated from ticks (Ornithodoros coriaceus) and from tick-exposed cattle. Am J Vet Res 37: 12011206.

    • Search Google Scholar
    • Export Citation
  • 7.

    King DP, Chen C-I, Blanchard MT, Aldridge BM, Anderson M, Walker R, Maas J, Hanks D, Hall M, Stott JL, 2005. Molecular identification of a novel deltaproteobacterium as the etiologic agent of epizootic bovine abortion (foothill abortion). J Clin Microbiol 43: 604609.

    • Search Google Scholar
    • Export Citation
  • 8.

    Anderson ML, Kennedy PC, Blanchard MT, Barbano L, Chiu P, Walker RL, Manzer M, Hall MR, King DP, Stott JL, 2006. Histochemical and immunohistochemical evidence of a bacterium associated with lesions of epizootic bovine abortion. J Vet Diagn Invest 18: 7680.

    • Search Google Scholar
    • Export Citation
  • 9.

    Djikeng A, Halpin R, Kuzmickas R, DePasse J, Feldblyum J, Sengamalay N, Afonso C, Zhang X, Anderson N, Ghedin E, 2008. Viral genome sequencing by random priming methods. BMC Genomics 9: 5.

    • Search Google Scholar
    • Export Citation
  • 10.

    Zerbino DR, Birney E, 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18: 821829.

  • 11.

    Assefa S, Keane TM, Otto TD, Newbold C, Berriman M, 2009. ABACAS: algorithm-based automatic contiguation of assembled sequences. Bioinformatics 25: 19681969.

    • Search Google Scholar
    • Export Citation
  • 12.

    Edgar RC, 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 17921797.

  • 13.

    Gouy M, Guindon S, Gascuel O, 2010. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 27: 221224.

    • Search Google Scholar
    • Export Citation
  • 14.

    Talavera G, Castresana J, 2007. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol 56: 564577.

    • Search Google Scholar
    • Export Citation
  • 15.

    Lupas A, Van Dyke M, Stock J, 1991. Predicting coiled coils from protein sequences. Science 252: 11621164.

  • 16.

    Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS, 2009. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res 37: W202W208.

    • Search Google Scholar
    • Export Citation
  • 17.

    Bekal S, Domier LL, Niblack TL, Lambert KN, 2011. Discovery and initial analysis of novel viral genomes in the soybean cyst nematode. J Gen Virol 92: 18701879.

    • Search Google Scholar
    • Export Citation
  • 18.

    Rassa JC, Wilson GM, Brewer GA, Parks GD, 2000. Spacing constraints on reinitiation of paramyxovirus transcription: the gene end U tract acts as a spacer to separate gene end from gene start sites. Virology 274: 438449.

    • Search Google Scholar
    • Export Citation
  • 19.

    Zdobnov EM, Apweiler R, 2001. InterProScan–an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17: 847848.

    • Search Google Scholar
    • Export Citation
  • 20.

    Mason JM, Arndt KM, 2004. Coiled coil domains: stability, specificity, and biological implications. ChemBioChem 5: 170176.

  • 21.

    Castagné N, Barbier A, Bernard J, Rezaei H, Huet J-C, Henry C, Da Costa B, Eléouët J-F, 2004. Biochemical characterization of the respiratory syncytial virus P–P and P–N protein complexes and localization of the P protein oligomerization domain. J Gen Virol 85: 16431653.

    • Search Google Scholar
    • Export Citation
  • 22.

    Shaji D, Shaila MS, 1999. Domains of rinderpest virus phosphoprotein involved in interaction with itself and the nucleocapsid protein. Virology 258: 415424.

    • Search Google Scholar
    • Export Citation
  • 23.

    Curran J, Boeck R, Lin-Marq N, Lupas A, Kolakofsky D, 1995. Paramyxovirus phosphoproteins form homotrimers as determined by an epitope dilution assay, via predicted coiled coils. Virology 214: 139149.

    • Search Google Scholar
    • Export Citation
  • 24.

    Taylor R, Hurlbut H, Work T, Kingston J, Hoogstraal H, 1966. Arboviruses isolated from argas ticks in Egypt: Quaranfil, Chenuda, and Nyamanini. Am J Trop Med Hyg 15: 7686.

    • Search Google Scholar
    • Export Citation
  • 25.

    Takahashi M, Yunker C, Clifford C, Nakano W, Fujino N, Tanifuji K, Thomas L, 1982. Isolation and characterization of midway virus: a new tick-borne virus related to nyamanini. J Med Virol 10: 181193.

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  • 26.

    Kemp GE, Lee VH, Moore DL, 1975. Isolation of Nyamanini and Quaranfil viruses from Argas (Persicargas) arboreus ticks in Nigeria. J Med Entomol 12: 535537.

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  • 27.

    Kaiser MN, 1966. Viruses in ticks. I. Natural infections of Argas (Persicargas) arboreus by Quaranfil and Nyamanini viruses and absence of infections in A.(p.) persicus in Egypt. Am J Trop Med Hyg 15: 964975.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

Whole Genome Analysis of Sierra Nevada Virus, a Novel Mononegavirus in the Family Nyamiviridae

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  • Center for Vaccine Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Tsinghua University School of Medicine, Beijing, China; Center for Biodefense and Emerging Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas; Center for Tropical Diseases, University of Texas Medical Branch, Galveston, Texas; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas

A novel mononegavirus was isolated in 1975 from ticks (Ornithodoros coriaceus) collected during investigation of an outbreak of epizootic bovine abortion (EBA) in northern California. It was originally designated “bovine abortion-tick virus” (BA-T virus). The EBA is now known to be associated with a deltaproteobacterium infection, and not a virus. The BA-T virus had remained uncharacterized until now. We have determined by electron microscopy, serology, and genome sequencing that the BA-T virus is a fourth member of the newly proposed family Nyamiviridae, and we have renamed it Sierra Nevada virus (SNVV). Although antigenically distinct, phylogenetically SNVV is basal to Nyamanini virus (NYMV) and Midway virus (MIDWV), two other tick-borne agents. Although NYMV was found to infect land birds, and MIDWV seabirds, it is presently unknown whether SNVV naturally infects birds or mammals.

Members in the order Mononegavirales are non-segmented, negative-strand RNA viruses encoding 5–10 open-reading frames (ORF).1 Ebola, rabies, and measles are just a few members of this order, and causative agents of serious human infectious diseases. In addition to the four well-established families—Bornaviridae, Filoviridae, Paramyxoviridae, and Rhabdoviridae—the order Mononegavirales also includes Nyamiviridae, a novel and recently proposed family.2 Nyamiviridae presently has only three members: Nyamanini virus (NYMV), Midway virus (MIDWV), and soybean cyst nematode virus 1 (SbCNV); the first two comprise a novel genus, Nyavirus.3

Here, we report the characterization of a novel virus, originally designated as bovine abortion-tick (BA-T) virus, that was isolated in 1975 from ticks (Ornithodoros coriaceus) collected during investigation of an outbreak of epizootic bovine abortion (EBA) in northern California. The EBA is an infectious disease associated with abortion and/or premature birth in cattle grazing foothills and montane pastures in the Sierra Nevada mountain range of California, Nevada, and Oregon.4 Subsequent experimental studies in heifers have indicated that BA-T virus was not the causative agent of EBA.5,6 The disease has now been associated with a deltaproteobacterium transmitted by the same tick species,7,8 but the BA-T virus has remained uncharacterized. Because the original designation, bovine abortion-tick (BA-T) virus, is misleading, we propose instead the name “Sierra Nevada virus” (SNVV) for the new virus.

The SNVV was originally isolated at the University of California/Davis in Vero cell cultures inoculated with a homogenate of O. coriaceus ticks collected in northern California (exact site unknown). The isolate was initially sent to the Rocky Mountain Laboratory in Hamilton, MT and was subsequently sent to the University of Texas Medical Branch (UTMB) for further study. Initial studies at UTMB indicated that the virus produced a viral cytopathic effect in both Vero and BHK cells within 48 hours after inoculation. Likewise, intracranial inoculation of newborn mice with SNVV produced illness and death within 2–3 days. Animal use was done under protocol no. 9505045, approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Medical Branch.

In a cross-box complement fixation assay comparing NYMV and MIDWV mouse brain antigens and a SNVV Vero cell antigen with specific hyperimmune mouse ascitic fluids (immune sera) prepared to the three viruses, NYMV and MIDWV were distinct but showed a close antigenic relationship (Table 1). In contrast, SNVV immune serum only reacted with the homologous antigen, indicating that it is antigenically distinct (Table 1). In ultrathin sections of infected Vero cells, transmission electron microscopy showed virion formation at the cell surface (Figure 1). Virions of variable sizes from 200 to 400 nm were observed budding from the host cell plasmalemma. Although the morphology of SNVV is similar to NYMV and MIDWV, the virion size of the former is about twice the size of the latter two viruses.3

Table 1

Results of complement fixation tests comparing Nyamanini, Midway, and Sierra Nevada viruses

AntigenComplement Fixation Test
Immune serum
Nyamanini (Tick-39)MidwaySierra NevadaControl
Nyamanini512*/12832/3200
Midway128/5121024/≥ 51200
Sierra Nevada0064/≥ 20
Control0000

Reciprocal of the highest antibody titer/reciprocal of highest antigen titer. 0 = < 8.

Figure 1.
Figure 1.

Transmission electron microscopy of Vero cells infected with Sierra Nevada (BA-T) virus. (A) Virions forming at the cell surface. N = nuclei of adjacent cells. Bar = 1 μm. (B) High power of a portion of an infected Vero cell with budding (thick arrowheads) virions (v). Thin arrows indicate cell membranes of adjacent cells. Bar = 100 nm.

Citation: The American Society of Tropical Medicine and Hygiene 91, 1; 10.4269/ajtmh.14-0076

Whole-genome sequencing and phylogenetic analysis were carried out to further characterize SNVV. Virus genomic RNA was isolated by processing cell supernatants through a 0.45 μM filter to remove eukaryotic cells, enrichment for intact virions with a Vivaspin 20 column (Sartorius, Goettingen, Germany) followed by treatment with 2 U Turbo DNase (Ambion, Austin, TX), 1 U Benzonase (Novagen, Madison, WI), and 2 U Rnase One (Promega, Madison, WI) for 1 hour at 37°C. Viruses were then pelleted through a 10%/15%/20% sucrose gradient by spinning at 39,000 rpm for 1.5 hours. Viral pellets were resuspended in nuclease free water and the presence of virus was confirmed by electron microscopy. Pelleted virus was lysed and genomic material purified using Trizol (Invitrogen, Carlsbad, CA) or the Viral RNA Mini Kit (Qiagen, Valencia, CA). Extracted Viral RNAs were reverse transcribed and barcoded using the SISPA methodology.9 This virus was sequenced as part of a multiplex of other samples. A multiplex sequencing pool was built using equimolar amounts of each barcoded DNA; this pool was purified with a MinElute PCR kit (Qiagen, Valencia, CA). The virus pool was sequenced on the HiSeq2000 (Illumina, San Diego, CA) with 100 basepair (bp) paired-ends. Demultiplexed reads were quality trimmed using the trim.pl script (http://wiki.bioinformatics.ucdavis.edu/index.php/Trim.pl), and de-novo assembly was performed using Velvet10 with a kmer of 51, and an expected coverage of 300. Contigs were ordered by ABACAS.pl11 and two gaps in the assembly were closed by polymerase chain reaction (PCR) and Sanger sequencing. To validate the assembly, reads were mapped back to the assembled genome using SMALT (http://www.sanger.ac.uk/resources/software/smalt/). A coverage plot was generated in R using a pileup file of the mapped reads (Figure 2). The SNVV genome was deposited in GenBank, under accession no. KF530058.

Figure 2.
Figure 2.

Coverage plot in log scale showing the number of mapped reads across the genomic assembly. Low coverage regions were polymerase chain reaction (PCR) amplified and sequenced by Sanger.

Citation: The American Society of Tropical Medicine and Hygiene 91, 1; 10.4269/ajtmh.14-0076

The 11,840 nt SNVV segment sequence may still be missing close to 190 nucleotides at its 3′ end, based on the alignment to the NYMV genome. It nevertheless potentially encodes six protein-coding genes: ORFI (353 aa), ORFII (274 aa), ORFIII (399 aa), ORFIV (145 aa), ORFV (599 aa), and ORFVI (1918 aa). Genomic organization of SNVV is highly syntenic to that of NYMV and MIDWV (Figure 3). A BLASTN analysis indicates that the SNVV genome has a 73% identity with NYMV and MIDWV. Because of the high similarity to NYMV and MIDWV, we have named the six ORFs according to the nomenclature for NYMV and MIDWV: ORFI, nucleocapsid protein (N); ORFII, X protein; ORFIII, phosphoprotein (P); ORFIV, matrix protein (M); ORFV, glycoprotein (G); ORFVI, RNA-dependent RNA polymerase (RdRP).2

Figure 3.
Figure 3.

Schematic of the annotated Sierra Nevada virus (SNVV) genome, in comparison to Nyamanini virus (NYMV), and Midway virus (MIDWV). Coordinates of interpro domains are annotated on each genome. i = site of transcription initiation motif; t = site of transcription termination motif; CC = putative coiled-coil domains (see Figure 4C).

Citation: The American Society of Tropical Medicine and Hygiene 91, 1; 10.4269/ajtmh.14-0076

To place SNVV in a phylogenetic context, BLASTX queries were performed using the RdRP gene against the GenBank non-redundant (nr) database. The first 1,000 protein matches were downloaded from GenBank, and the type species of each genus in the order Mononegavirales was retained (see Supplemental Table 1 for accessions). These protein sequences were aligned with those of SNVV, NYMV, MIDWV, and SbCNV using MUSCLE12 and the resulting multiple sequence alignment was inspected and manually adjusted in Seaview Version 4.4.0.13 Character sets for phylogenetic analysis were selected using Gblocks14 and a maximum likelihood phylogenetic tree was generated with RAxML-HPC, using the GTR substitution model, rates across sites modeled on a gamma distribution and 1,000 bootstrap replicates. Our analysis places SNVV within the Nyamiviridae family, and indicates it is basal to the Nyavirus genus containing NYMV and MIDWV (Figure 4A).

Figure 4.
Figure 4.

(A) Phylogenetic tree of RdRP sequences of the order Mononegavirales. The order Mononegavirales comprises five families, within which the family Paramyxoviridae is split into two subfamilies: Pneumovirinae and Paramyxovirinae. The Sierra Nevada virus (SNVV) branches within a clade of Nyamiviridae and branches closely, but as sister to Nyamanini virus (NYWV) and Midway virus (MIDWV). In comparison, the soybean cyst nematode virus 1 (SbCNV) is a distant, deep branching member of the Nyamiviridae. The accession numbers and full names of all the viruses in the tree are listed in Supplemental Table 1. (B) Coiled-coil domain prediction for open-reading frame (ORF) III in SNVV, NYMV, and MIDWV. The coiled-coil domain prediction was done based on a 28-residue window using the program of COILS15; there are two peaks shown along the curves, and both of them partially overlap with each other across the three viruses, suggesting a similar pattern of the coiled-coil domain distribution in their genomes. (C) Sequence alignment for the putative coiled-coil regions of ORF III in SNVV, NYMV, and MIDWV. Alignment of the sequences of N- and C- terminal coiled-coil domain across SNVV, NYMV, and MIDWV shows that 39% of the residues are conserved in the N-terminal domains, whereas 63% of the residues are conserved in the C-terminal domains.

Citation: The American Society of Tropical Medicine and Hygiene 91, 1; 10.4269/ajtmh.14-0076

Using MEME, a motif-based sequence analysis tool,16 we identified conserved transcription termination motifs (3′-AG(G/A)AA(U/A)GUUUUU-5′) downstream of every SNVV virus ORF (Table 2). Similar motifs have also been reported for other viruses in the family Nyamiviridae, indicating further conservation across this family.3,17 A study of the paramyxovirus simian virus 5 (SV5), also a mononegavirus, suggests that the poly-U tract in every termination motif may function as a template for polyadenylation, and a genome spacer separating each gene end from the next gene start.18 It still remains to be determined whether the same function applies for the transcription termination motifs seen in the Nyamiviridae.

Table 2

Conserved transcription initiation and termination motifs upstream and downstream of every SNVV ORF, and comparison of motifs across NYMV, MIDWV, and SbCNV3,6

Table 2

Conserved transcription initiation motifs 3′-CGUUGGNNGG(G/U)-5′ were also found upstream of all the SNVV ORFs (Table 2). Compared with the initiation motifs found in NYMV, MIDWV, and SbCNV,3,17 SNVV shows high similarity with NYMV and MIDWV, but not with SbCNV. Despite that difference, all four viruses share a common characteristic where the initiation motif is located directly after the termination motif for the upstream gene, which is different from the paramyxovirus SV5 where there is always a 1 to 22 nucleic acid long spacer between the termination motif and the initiation motif for the following gene.18

To further characterize the function of the six ORFs encoded by SNVV, we have performed protein domain searches against each of these using the InterProScan webserver.19 As expected, we find protein domains associated with RNA polymerases (IPR014023, IPR025786, IPR026890) for ORFVI (RdRP), and a Borna disease virus P40 nucleoprotein subdomain 2 (IPR015970) for ORFI (nucleoprotein) (Figure 3). Using COILS,15 we found two putative coiled-coil domains in the P protein (ORF III) of SNVV. Similar domains were also predicted for the P proteins of NYMV and MIDWV, but not SbCNV. Interestingly, the specific location of this domain is approximately the same in SNVV, NYMW, and MIDWV. As shown in (Figure 4B), two peaks in the probability curves are identified, indicating the existence of two putative coiled-coil domains in this protein. A coiled-coil domain is a type of structural motif in the amphipathic α-helix, and is formed by heptad repeats denoted (a-b-c-d-e-f-g)n;15 “a” and “d” often refer to hydrophobic amino acids at the interface of two helices, whereas “e” and “g” are solvent and polar residues.20 The putative coiled-coil domains found in the N- and C-termini of SNVV, NYMV, and MIDWV show different characteristics in sequence composition. As shown in the sequence alignment in (Figure 4C), the N-terminal coiled-coil domains are of low conservation across the three viruses, whereas the C-terminal domains are better conserved where the “a” and “d” amino acid sites are always hydrophobic residues (i.e., isoleucine, leucine, or valine). Most members of the Paramyxoviridae family, such as the respiratory syncytial virus,21 rinderpest virus,22 Sendai virus, parainfluenza virus, measles virus, Newcastle disease virus, and mumps virus,23 have been reported to contain a coiled-coil domain in the P protein. None of these proteins, however, contain two domains, as predicted for the nyaviruses. Without a functional assay we cannot determine whether these predicted domains are real.

In summary, based on its genomic structure and phylogeny SNVV appears to be closely related to NYMV and MIDWV, pointing toward a third variant of the Nyavirus genus. However, antigen reactivity and virion size indicate that it may represent a closely related but distinct group. Although NYMV and MIDWV have been isolated from bird species and their ticks,3,2427 it is unclear whether SNVV can also infect birds. Based on its phylogenetic location it is likely that birds are also natural vertebrate hosts. This, however, will require confirmation by surveillance efforts.

ACKNOWLEDGMENTS

We thank Conrad E. Yunker for providing information on the origin and history of BA-T (SNVV) virus.

  • 1.

    Pringle CR, Alexander DJ, Billeter MA, Collins PL, Kingsbury DW, Lipkind MA, Nagai Y, Orvell C, Rima B, Rott R, ter Meulen V, 1991. The order Mononegavirales. Arch Virol 117: 137140.

    • Search Google Scholar
    • Export Citation
  • 2.

    Kuhn JH, Bekal S, Caì Y, Clawson AN, Domier LL, Herrel M, Jahrling PB, Kondo H, Lambert KN, Mihindukulasuriya KA, 2013. Nyamiviridae: proposal for a new family in the order Mononegavirales. Arch Virol 158: 118.

    • Search Google Scholar
    • Export Citation
  • 3.

    Mihindukulasuriya KA, Nguyen NL, Wu G, Huang HV, da Rosa AP, Popov VL, Tesh RB, Wang D, 2009. Nyamanini and midway viruses define a novel taxon of RNA viruses in the order Mononegavirales. J Virol 83: 51095116.

    • Search Google Scholar
    • Export Citation
  • 4.

    McKercher DG, Wada EM, Straub OC, 1963. Distribution and persistence of infectious bovine rhinotracheitis virus in experimentally infected cattle. Am J Vet Res 24: 510514.

    • Search Google Scholar
    • Export Citation
  • 5.

    McKercher DG, Wada EM, Ault SK, Theis JH, 1980. Viral agents recovered from Ornithodoros coriaceus, a vector of epizootic bovine abortion. Am J Vet Res 41: 803805.

    • Search Google Scholar
    • Export Citation
  • 6.

    Wada EM, McKercher DG, Castrucci G, Theis JH, 1976. Preliminary characterization and pathogenicity studies of a virus isolated from ticks (Ornithodoros coriaceus) and from tick-exposed cattle. Am J Vet Res 37: 12011206.

    • Search Google Scholar
    • Export Citation
  • 7.

    King DP, Chen C-I, Blanchard MT, Aldridge BM, Anderson M, Walker R, Maas J, Hanks D, Hall M, Stott JL, 2005. Molecular identification of a novel deltaproteobacterium as the etiologic agent of epizootic bovine abortion (foothill abortion). J Clin Microbiol 43: 604609.

    • Search Google Scholar
    • Export Citation
  • 8.

    Anderson ML, Kennedy PC, Blanchard MT, Barbano L, Chiu P, Walker RL, Manzer M, Hall MR, King DP, Stott JL, 2006. Histochemical and immunohistochemical evidence of a bacterium associated with lesions of epizootic bovine abortion. J Vet Diagn Invest 18: 7680.

    • Search Google Scholar
    • Export Citation
  • 9.

    Djikeng A, Halpin R, Kuzmickas R, DePasse J, Feldblyum J, Sengamalay N, Afonso C, Zhang X, Anderson N, Ghedin E, 2008. Viral genome sequencing by random priming methods. BMC Genomics 9: 5.

    • Search Google Scholar
    • Export Citation
  • 10.

    Zerbino DR, Birney E, 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18: 821829.

  • 11.

    Assefa S, Keane TM, Otto TD, Newbold C, Berriman M, 2009. ABACAS: algorithm-based automatic contiguation of assembled sequences. Bioinformatics 25: 19681969.

    • Search Google Scholar
    • Export Citation
  • 12.

    Edgar RC, 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 17921797.

  • 13.

    Gouy M, Guindon S, Gascuel O, 2010. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 27: 221224.

    • Search Google Scholar
    • Export Citation
  • 14.

    Talavera G, Castresana J, 2007. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol 56: 564577.

    • Search Google Scholar
    • Export Citation
  • 15.

    Lupas A, Van Dyke M, Stock J, 1991. Predicting coiled coils from protein sequences. Science 252: 11621164.

  • 16.

    Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS, 2009. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res 37: W202W208.

    • Search Google Scholar
    • Export Citation
  • 17.

    Bekal S, Domier LL, Niblack TL, Lambert KN, 2011. Discovery and initial analysis of novel viral genomes in the soybean cyst nematode. J Gen Virol 92: 18701879.

    • Search Google Scholar
    • Export Citation
  • 18.

    Rassa JC, Wilson GM, Brewer GA, Parks GD, 2000. Spacing constraints on reinitiation of paramyxovirus transcription: the gene end U tract acts as a spacer to separate gene end from gene start sites. Virology 274: 438449.

    • Search Google Scholar
    • Export Citation
  • 19.

    Zdobnov EM, Apweiler R, 2001. InterProScan–an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17: 847848.

    • Search Google Scholar
    • Export Citation
  • 20.

    Mason JM, Arndt KM, 2004. Coiled coil domains: stability, specificity, and biological implications. ChemBioChem 5: 170176.

  • 21.

    Castagné N, Barbier A, Bernard J, Rezaei H, Huet J-C, Henry C, Da Costa B, Eléouët J-F, 2004. Biochemical characterization of the respiratory syncytial virus P–P and P–N protein complexes and localization of the P protein oligomerization domain. J Gen Virol 85: 16431653.

    • Search Google Scholar
    • Export Citation
  • 22.

    Shaji D, Shaila MS, 1999. Domains of rinderpest virus phosphoprotein involved in interaction with itself and the nucleocapsid protein. Virology 258: 415424.

    • Search Google Scholar
    • Export Citation
  • 23.

    Curran J, Boeck R, Lin-Marq N, Lupas A, Kolakofsky D, 1995. Paramyxovirus phosphoproteins form homotrimers as determined by an epitope dilution assay, via predicted coiled coils. Virology 214: 139149.

    • Search Google Scholar
    • Export Citation
  • 24.

    Taylor R, Hurlbut H, Work T, Kingston J, Hoogstraal H, 1966. Arboviruses isolated from argas ticks in Egypt: Quaranfil, Chenuda, and Nyamanini. Am J Trop Med Hyg 15: 7686.

    • Search Google Scholar
    • Export Citation
  • 25.

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Author Notes

* Address correspondence to Elodie Ghedin, NYU-Biology, 100 Washington Square East, 1009 Silver Center New York, NY 10003. E-mail: elodie.ghedin@nyu.edu

Financial support: LC is a visiting scholar from Tsinghua University and supported in part by the China Scholarship Council. This work was also supported in part by NIH contract HHSN272201000040I/HHSN270004 (RBT), the Department of Pathology start-up funds, and a grant from the Institute for Human Infections and Immunity, University of Texas Medical Branch (NV).

Disclosure: The GenBank accession no. for the Sierra Nevada virus is KF530058. Animal use reported in this work was done under protocol no. 9505045, approved by the IACUC at the University of Texas Medical Branch.

Authors' addresses: Matthew B. Rogers, Center for Vaccine Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, and Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, E-mail: rogersm@pitt.edu. Lijia Cui, Center for Vaccine Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, and Tsinghua University School of Medicine, Beijing, China, E-mail: lic80@pitt.edu. Adam Fitch, Center for Vaccine Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, E-mail: fitcha@pitt.edu. Vsevolod Popov, Nikos Vasilakis, and Robert B. Tesh, Center for Biodefense and Emerging Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, TX, Center for Tropical Diseases, University of Texas Medical Branch, Galveston, TX, and Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas, E-mails: vpopov@utmb.edu, nivasila@utmb.edu, and rtesh@utmb.edu. Amelia P. A. Travassos da Rosa, Center for Biodefense and Emerging Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, TX, E-mail: aptravas@utmb.edu. Elodie Ghedin, Department of Biology, Center for Genomics and Systems Biology, New York University, New York, NY, E-mail: elodie.ghedin@nyu.edu.

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