The prototype strain of FARV (CT AN 114) was obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) at the University of Texas Medical Branch. As noted previously, CT AN 114 was isolated from an unidentified bird collected during an epidemiologic investigation of an outbreak of eastern equine encephalitis on a commercial pheasant farm in Farmington, CT in 1969. At the time of isolation, it could not be identified and was deposited in WRCEVA virus collection as an unknown. The virus strain used in our studies had been passaged 11 times by intracerebral inoculation of newborn mice and twice in Vero cells. The virus used for sequencing was prepared from a single plaque picked from a monolayer culture of Vero cells.
Vero E6 cells (African green monkey kidney) were used for propagating the virus. The cells were originally obtained from the American Type Culture Collection (Manassas, VA) and were grown at 37°C in minimal essential medium (MEM) with Earle’s salts (Gibco/Invitrogen, Carlsbad, CA) supplemented with 5% heat-inactivated (56°C for 30 min) fetal bovine serum (FBS) and 1% penicillin-streptomycin stock (Sigma, St. Louis, MO).
Outbred ICR mice (Harlan Sprague–Dawley, Indianapolis, IN) were used to prepare antigens and antibodies for FARV and the other rhabdoviruses shown in Figure 3. All animal work was carried out under a protocol approved by the University of Texas Medical Branch Institutional Animal Care and Use Committee.
Antigens and immune reagents
Antigens used in CF tests were prepared from infected newborn mouse brains extracted by the sucrose/acetone method . Specific mouse immune ascitic fluids (MIAF) were also prepared against each of the 24 rhabdoviruses listed in Figure 3. Immunogens were 10% suspensions of homogenized infected mouse brain in phosphate-buffered saline mixed with complete Freund’s adjuvant. The immunization schedule consisted of four intraperitoneal injections given at weekly intervals. Sarcoma 180 cells were given intraperitoneally with the final immunization in order to induce ascites formation.
CF tests were done according to a microtechnique described previously [27, 28], using 2 full units of guinea pig complement. Titers were recorded as the highest dilutions giving 3+ or 4+ fixation of complement on a scale of 0 to 4 + .
Transmission electron microscopy
For ultrastructural analysis, infected Vero cells were fixed for at least 1 h in a mixture of 2.5% formaldehyde prepared from paraformaldehyde powder, and 0.1% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.3, to which 0.03% picric acid and 0.03% CaCl2 were added. The infected cell monolayers were washed in 0.1 M cacodylate buffer, scraped off, and processed further as a pellet. The pellets were post-fixed in 1% OsO4 in 0.1 M cacodylate buffer, pH 7.3, for 1 h, washed with distilled water, and stained en bloc with 2% aqueous uranyl acetate for 20 min at 60°C. The pellets were dehydrated in ethanol, processed through propylene oxide and embedded in Poly/Bed 812 (Polysciences, Warrington, PA). Ultrathin sections were cut on a Leica EM UC7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL), stained with lead citrate and examined in a Phillips 201 transmission electron microscope at 60 kV.
Vero monolayers were grown to 90% confluency in 25 cm2 plastic tissue culture flasks and were infected with FARV or were mock infected. Virus inocula were aspirated following 1 h of absorption at 37°C, washed thrice to remove any unabsorbed virus, replaced with fresh medium (MEM) supplemented with 5% FBS and 1% penicillin/streptomycin, and incubated at 37°C. Four days post infection (p.i.), the cells were harvested into cell lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100). The cell preparations were then treated according to the Biorad instructions for MiniPROTEAN TGX gel analysis of proteins. Briefly, 20 μl samples of loading buffer containing β-mercaptoethanol were added to 20 μl aliquots of the cell lysates. Samples were heated at 95°C for 10 min, and centrifuged for 1 min at 500 × g at 4°C. Proteins were separated under reducing conditions on MiniPROTEAN TGX 4-20% Tris-Glycine SDS-PAGE gels, and were transferred onto Hybond-P PVDF membrane (Amersham) with Transfer Buffer (Biorad, Hercules, CA) according to the manufacturer’s western blotting transfer protocol. Membranes were processed by the WesternBreeze© Chromogenic Immunodetection system (Invitrogen), following the manufacturer’s protocol. The polyclonal MIAF raised to FARV was used at 1:5000 dilution.
Nucleotide sequence accession numbers
The genome sequence of FARV was determined in this study and assigned GenBank accession number KC602379. The GenBank accession numbers for the genome sequences of select rhabdoviruses used in the phylogenetic analyses are listed as follows: ARV, Ade laide River virus (AFR23540); ABLV, Australian bat lyssavirus (NP478343); ARAV, Aravan virus (ABV03822); BEFV, bovine ephemeral fever virus (NP065409); CHPV, Chandipura virus (AED98393); COCV, Cocal virus (ACB47438); CPV, Coastal Plains virus (ADG86364); DMelSV, Drosophila megalomaster sigmavirus (YP003126913); DObSV, Drosophila obscura sigmavirus (ACU65444); DURV, Durham virus (ADB88761); DUVV, Duvenhage virus (AFK93192); EVEX, eel virus European X (AFX58972); EBLV1, European bat lyssavirus 1 (YP001285392); FLAV, Flanders virus (AAN73288); HIRV, Hirame rhabdovirus (NP919035); IKOV, Ikoma lyssavirus (AFQ26098); IHNV, infectious hematopoietic necrosis virus (NP042681); IRKV, Irkut virus (ABV03823); ISFV, Isfahan virus (CAH17548); JURV, Jurona virus (AEG25349); KHUV, Khujand virus (ABV03824); KIMV, Kimberley virus (AFR67096); KOTV, kotonkan virus (YP006202628); LBV, Lagos bat virus (AFW16650); MARV, Maraba virus (AEI52253); MOKV, Mokola virus (YP142354); MOUV, Moussa virus (ACZ81407); NGAV, Ngaingan virus (YP003518294); OVRV, Oak Vale rhabdovirus (AEJ07650); OBOV, Obodhiang virus (YP0062000965); OZEV, Ozernoe virus (ACS70797); PRV, Perinet virus (AEG25355); PFRV, pike fry rhabdovirus (ACP28002); RABV, rabies virus (ABN11300); SHIBV, Shimoni bat virus (ADD84511); SCRV, Siniperca chuatsi rhabdovirus (YP802942); SHRV, snakehead virus (NP050585); SVCV, spring viraemia of carp virus (NP116748); TIBV, Tibrogargan virus (ADG86355); TUPV, tupaia virus (YP238534); VSAV, vesicular stomatitis Alagoas virus (ACB47443); VHSV, viral hemorrhagic septicemia virus (BAM29126); VSIV, vesicular stomatitis Indiana virus (NP041716); VSNJV, vesicular stomatitis New Jersey virus (AAA48442); WCBV, West Caucasian bat virus (ABV03821); and WONV, Wongabel virus (YP002333280). The ICTV has not yet recognized FARV as a member species in the family Rhabdoviridae. As a consequence, its abbreviation ought to be considered tentative and subject to approval.
RNA was extracted from virus stocks (infected Vero cells) using TRIzol LS (Invitrogen) and treated with DNase I (DNA-Free, Ambion, Austin, TX). cDNA was generated using the Superscript II system (Invitrogen) employing random hexamers linked to an arbitrary 17-mer primer sequence , treated with RNase H and then randomly amplified by PCR with a 9:1 mixture of primer corresponding to the 17-mer sequence and the random hexamer linked 17-mer primer. Products greater than 70 base pairs (bp) were selected by column chromatography (MinElute, Qiagen, Hilden, Germany) and ligated to specific adapters for sequencing on the 454 Genome Sequencer FLX (454 Life Sciences, Branford, CT) without fragmentation [30, 31]. After primer removal, redundancy filtering, and sequence assembly, sequence gaps were completed by RT-PCR amplification, using primers based on pyrosequencing data. Amplification products were size-fractioned on 1% agarose gels, prufied (MiniElute, Qiagen) and directly sequenced in both directions with ABI PRISM Big Dye Terminator 1.1 Cycle Sequencing kits on ABI PRISM 3700 DNA Analyzers (Perkin-Elmer Applied Biosystems, Foster City, CA). The terminal sequences for each genome were amplified using the Clontech SMARTer RACE kit (Clontech, Mountain View, CA). Genome sequences were verified by Sanger dideoxy sequencing using primers designed from the draft sequence to create products of 1,000 bp with 500 bp overlaps.
Rapid amplification of cDNA ends (RACE)
Genomic termini were characterized with 5_- and 3_-RACE kits (Invitrogen). Virus-specific primers for FARV were: 5′ – GTC TTG AAG TCG TTT CCC AG, located 934 nt from the 3′ -genomic terminus for reverse transcription, 5′ – TCA GGT TCA TCA GCC ATT TC, located 618 nt from the 3′ – genomic terminus for first PCR with primer UAP (Invitrogen), and 5′ – ACC AGC CGA TGA TGT AAG C, located 556 nt from the 3′ – genomic terminus for second PCR with primer AUAP (Invitrogen). RNA for the second RACE was tailed with poly(A) polymerase (Ambion) and purified using RNeasy® Mini kit (Qiagen). cDNA synthesis was primed with oligo d(T) – adapter primer AP (Invitrogen), and first PCR used primer 5′ – AAC CGT TCC TTC ACT ACA TC, located 1,004 nt from the 3′– antigenomic terminus and primer UAP (Invitrogen), the second PCR used primer 5′ – TCG CTT ACC AGC ATT TTG AG, located 746 nt from the 3′ – antigenomic terminus and primer AUAP (Invitrogen). All primers were at 0.2 M final concentration. PCR products were purified with QIAquick PCR purification kits (Qiagen) and directly dideoxy-sequenced in both directions.
The L protein sequence of FARV was compared with those of 38 other rhabdoviruses downloaded from GenBank. All protein sequences were aligned using MUSCLE  under default settings. Because these sequences were highly divergent, which could negatively impact phylogenetic analysis, all ambiguously aligned regions were removed using the G-blocks program . This resulted in a final sequence alignment of 433 amino acid residues. The phylogenetic relationships among these sequences were determined using the maximum likelihood (ML) method available in PhyML 3.0  employing the WAG-G model of amino acid substitution and subtree pruning and regrafting (SPR) branch-swapping. The robustness of each node was evaluated using the SH and ChiSq minimum statistic. In addition, trees were determined using the neighbor-joining (NJ) and maximum parsimony (MP) methods using the default settings available in PAUP* v. b10, bootstraps were run for both methods.