Virus Research 155 (2011) 112–122 Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Characterization of Durham virus, a novel rhabdovirus that encodes bothaCand SH protein A.B. Allison a,∗, G. Palacios b, A. Travassos da Rosa c, V.L. Popov c,L.Luc, S.Y. Xiao c,d, K. DeToy b, T. Briese b, W.I. Lipkin b, M.K. Keel a, D.E. Stallknecht a, G.R. Bishop e, R.B. Tesh c a Southeastern Cooperative Wildlife Disease Study, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, United States b Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, NY 10032, United States c Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX 77555, United States d Department of Pathology, University of Chicago Medical Center, Chicago, IL 60637, United States e Mississippi River Field Institute, National Audubon Society, 1208 Washington St., Vicksburg, MS 39183, United States article info abstract Article history: The family Rhabdoviridae is a diverse group of non-segmented, negative-sense RNA viruses that are dis- Received 13 May 2010 tributed worldwide and infect a wide range of hosts including vertebrates, invertebrates, and plants. Of Received in revised form the 114 currently recognized vertebrate rhabdoviruses, relatively few have been well characterized at 10 September 2010 both the antigenic and genetic level; hence, the phylogenetic relationships between many of the verte- Accepted 14 September 2010 brate rhabdoviruses remain unknown. The present report describes a novel rhabdovirus isolated from Available online 21 September 2010 the brain of a moribund American coot (Fulica americana) that exhibited neurological signs when found in Durham County, North Carolina, in 2005. Antigenic characterization of the virus revealed that it was Keywords: Rhabdovirus serologically unrelated to 68 other known vertebrate rhabdoviruses. Genomic sequencing of the virus Durham virus indicated that it shared the highest identity to Tupaia rhabdovirus (TUPV), and as only previously observed Vesiculovirus in TUPV, the genome encoded a putative C protein in an overlapping open reading frame (ORF) of the C protein phosphoprotein gene and a small hydrophobic (SH) protein located in a novel ORF between the matrix Small hydrophobic protein and glycoprotein genes. Phylogenetic analysis of partial amino acid sequences of the nucleoprotein and Overprinting polymerase protein indicated that, in addition to TUPV, the virus was most closely related to avian and small mammal rhabdoviruses from Africa and North America. In this report, we present the morpholog- ical, pathological, antigenic, and genetic characterization of the new virus, tentatively named Durham virus (DURV), and discuss its potential evolutionary relationship to other vertebrate rhabdoviruses. © 2010 Elsevier B.V. All rights reserved. 1. Introduction are spread by direct contact without any apparent arthropod com- ponent. The Rhabdoviridae is one of the four families classified within Of the presently recognized 114 vertebrate rhabdoviruses, 52 the order Mononegavirales (Pringle and Easton, 1997). The rhab- are classified as members (or tentative members) of one of the four dovirus genome, like those of paramyxoviruses, filoviruses, and existing genera (Tordo et al., 2005). Of the 62 remaining unclassi- Borna disease virus (BDV), is a non-segmented, single strand of fied vertebrate rhabdoviruses, 20 are placed within six serogroups negative-sense RNA, although the genome of Orchid fleck virus based on antigenic cross-reactivity, while 42 remain unassigned (OFV) has been proposed to be bipartite (Kondo et al., 2006). Six to any existing serogroup. Although the antigenic relationships genera are currently recognized within the Rhabdoviridae, four for many vertebrate rhabdoviruses have been determined through of which, Vesiculovirus, Lyssavirus, Ephemerovirus, and Novirhab- serological studies (Tesh et al., 1983; Calisher et al., 1989), the dovirus, infect vertebrates, while members of Cytorhabdovirus and phylogenetic relationships between many of these viruses remain Nucleorhabdovirus infect plants (Tordo et al., 2005). Many of the unknown due to the lack of available sequence data. Additionally, vertebrate and plant rhabdoviruses replicate in, and are transmit- as many of the unclassified vertebrate rhabdoviruses are repre- ted by, arthropod vectors, although some viruses (e.g., lyssaviruses) sented by only a single or few isolates, the normal host associations, transmission cycles, and geographical distributions of these viruses remain obscure. ∗ The prototypical rhabdovirus genome consists of five genes Corresponding author. Tel.: +1 706 542 1741; fax: +1 706 542 5865. E-mail address: [email protected] (A.B. Allison). [nucleoprotein (N), phosphoprotein (P), matrix (M), glycoprotein 0168-1702/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2010.09.007 A.B. Allison et al. / Virus Research 155 (2011) 112–122 113 (G), polymerase (L)] that are sequentially transcribed in decreasing 2.2. Virus isolation molar abundance according to their placement in the genome (i.e., 3-N>P>M>G>L-5)(Abraham and Banerjee, 1976). The genomic For virus isolation, samples of brain and liver (∼0.5 cm3) were 3 and 5 ends contain the untranslated leader and trailer sequences, mechanically homogenized in 650 ␮L of medium [1× minimum respectively, which exhibit some degree of terminal complemen- essential medium (MEM), 2.2 g/l NaHCO3, 20% fetal bovine serum tarity (Wertz et al., 1994; Rose and Whitt, 2001). Each of the five (FBS), and 4× antibiotic/antimycotic solution (400 units/mL peni- individual genes is flanked by transcription initiation and termi- cillin, 400 ␮g/mL streptomycin, 1 ␮g/mL amphotericin B) (Sigma, nation/polyadenylation signals, which may be conserved among St. Louis, MO)]. Homogenized tissues were centrifuged (6700 × g members of the same genus (Fu, 2005). Between each transcrip- for 10 min) to pellet debris, and an aliquot (100 ␮L) of clarified tion unit (gene and associated flanking signals) is a nontranscribed supernatant was used to inoculate 2-day-old cultures of Vero cells intergenic region that usually contains a single or dinucleotide in a 12-well plate format. For initial characterization, stock virus sequence [e.g., G or GG in Tupaia rhabdovirus (TUPV)] (Springfeld was inoculated into three 75 cm2 flasks of Vero cells at an m.o.i. et al., 2005). of 0.1 and virus was precipitated on day 4 post-inoculation with In addition to the five structural proteins (N–P–M–G–L) nor- polyethylene glycol (PEG) as described by Killington et al. (1996). mally encoded in the genome, a number of additional proteins, cDNA synthesis from extracted RNA was carried out using random either encoded in the same or overlapping open reading frame decamers, ImProm-II reverse transcriptase (Promega, Madison, (ORF) within an existing gene, or in a novel ORF, have been iden- WI), and GoTaq Flexi DNA polymerase (Promega), according to the tified in numerous rhabdoviruses. For instance, Wongabel virus manufacturer’s instructions. Amplicons were excised and purified (WONV), a putative member of the Hart Park serogroup, has from agarose using a QIAquick Gel Extraction Kit (Qiagen), cloned recently been demonstrated to encode three novel genes (U1, U2, using a PCR CloningPlus Kit (Qiagen), and subsequently purified U3) from three consecutive ORFs located between the P and M using a QIAprep Spin Miniprep Kit (Qiagen) according to the manu- genes, in addition to two genes (U4, U5) in overlapping ORFs facturer’s instructions. Sequencing of clones was performed using of the N and G genes, respectively (Bourhy et al., 2005; Gubala an Applied Biosystems Inc. 3100 Genetic Analyzer (Foster City, CA, et al., 2008). Other gene products in addition to N–P–M–G–L USA). have also been described for vesiculoviruses (Spiropoulou and Nichol, 1993; Springfeld et al., 2005), ephemeroviruses (Walker 2.3. Transmission electron microscopy et al., 1992; Wang et al., 1994), lyssaviruses (Chenik et al., 1995), novirhabdoviruses (Kurath and Leong, 1985; Alonso et al., 2004), Infected monolayers of Vero cells were fixed in a mixture of nucleorhabdoviruses (Scholthof et al., 1994; Huang et al., 2003), 2.5% formaldehyde and 0.1% glutaraldehyde containing 0.03% trini- and cytorhabdoviruses (Tao et al., 2000; Dietzgen et al., 2006). trophenol and 0.03% CaCl2 in 0.05 M cacodylate buffer. Cells were As the genomic sequences of a number of both vertebrate and scraped off the plastic, pelleted in buffer, post-fixed in 1% OsO4 in plant rhabdoviruses have become available in recent years, it 0.1 M cacodylate buffer, stained en bloc with 2% aqueous uranyl appears that many members of the family do not conform to acetate, dehydrated in ethanol, and embedded in Poly/Bed 812 the prototypical N–P–M–G–L genomic organization. Currently, the (Polysciences, Warrington, PA). Ultrathin sections were cut on a function of most of these novel genes remains unknown (Fu, Reichert-Leica Ultracut S ultramicrotome, stained with 0.4% lead 2005). citrate, and examined in a Philips 201 electron microscope at 60 kV. In November 2005, a novel rhabdovirus was isolated from the brain of a moribund American coot (Fulica americana) found in 2.4. Experimental infection of newborn mice Durham County, North Carolina. The virus was provisionally named Durham virus (DURV), after the county in North