Introduction
The Rhabdoviridae is a large and diverse family of negative-sense (−) single-stranded RNA (ssRNA) viruses infecting both animals and plants.1,2 Many rhabdoviruses replicate in arthropods, some of which (e.g., mosquitoes, sand flies, biting midges, and ticks) may serve as vectors of transmission of infection among humans and other vertebrate hosts.3 Other rhabdoviruses, such as the drosophila sigmaviruses, are known to be transmitted entirely within arthropod populations.4 Indeed, a recent metagenomics study of a wide range of terrestrial and marine arthropods has revealed that the genomes of rhabdoviruses and other (−) ssRNA viruses are abundant and diverse, suggesting that arthropods are reservoirs of genetic diversity for these viruses and have played a key role in their long history of evolution.5 These data also suggest that adaptation of some rhabdoviruses to transmission to vertebrates by hematophagous vectors may be a relatively recent and specialized aspect of their evolution. Distinguishing arthropod-specific viruses from those that may present a serious risk of human infection will become increasingly important as the pace of novel virus discovery escalates.
Herein, we describe the isolation of several novel rhabdoviruses from mosquitoes collected in Colombia, Panama, and the Florida Everglades. We show that these viruses are closely related phylogenetically, and in genome organization, to Arboretum virus (ABTV) and Puerto Almendras virus (PTAMV) that were isolated recently from mosquitoes in Peru.6 Like ABTV and PTAMV, the novel rhabdoviruses replicated effectively in mosquito cells but failed to show evidence of replication in vertebrate cell cultures or newborn mice, suggesting they may not be well adapted to replication in vertebrates. Our data support the formation of a new genus Almendravirus within the family Rhabdoviridae to accommodate these novel viruses.
Materials and Methods
Description of viruses.
Coot Bay virus (CBV; strain EVG 5-53) was isolated from a pool of Anopheles quadrimaculatus mosquitoes collected on July 25, 2013, from a mangrove swamp near Coot Bay (25°11′N, 80°55′W) in the Everglades National Park, FL. Rio Chico virus (RCHV; strain GAM-195) was isolated from a pool of unidentified mosquitoes collected in December 2012 in largely old-growth forest near Gamboa (9°07′N, 79°42′W), Central Panama. Balsa virus (BALV; strains CoB 76 and CoB 84) was isolated from two pools of Culex erraticus mosquitoes collected between June 9 and June 15, 2013, in an open secondary forest near the beach (La Balsa) in San Bernardo (9°21′N, 75°57′W), Cordoba Department, Colombia. Details of the sources of isolation of ABTV and PTAMV have been described.6 ABTV (strain LO-121) was isolated on February 22, 2009, from a pool of Aedes (Ochlerotattus) fulvus mosquitoes and PTAMV (strain LO-39) was isolated on March 25, 2009, from Psorophora albigenu mosquitoes, each at Puerto Almendras (3°50′S, 73°22′W), Loreto Department, Peru.
Processing of field samples and virus isolation.
Pools of field-collected female mosquitoes were homogenized in 1.0 mL of phosphate-buffered saline, pH 7.4, with 20% fetal bovine serum, using a TissueLyser (Qiagen, Valencia, CA) and 3-mm stainless steel beads. After centrifugation at 10,000 rpm in a microcentrifuge for 5 minutes, 100 μL of the supernatant was inoculated into single 12.5-cm2 flasks with monolayer cultures of C6/36 cells. The cultures were maintained at 28°C for 6–7 days and were examined every 2 days for evidence of viral cytopathic effect (CPE). Cultures showing CPE were harvested for transmission electron microscopy and next-generation sequencing as described below.
Evaluation of growth of viruses in vertebrate cell cultures.
Vertebrate cell lines were originally obtained from the American Type Culture Collection (ATCC). Baby hamster kidney cells (BHK-21, ATCC CCL-10), African green monkey kidney cells (Vero E6, ATCC CRL-1586), and duck embryo fibroblasts (ATCC CCL-141) were cultured at 37°C, and toad (Xenopus laevis) epithelial cells (XLK-WG, ATCC CRL-2527) were cultured at 28°C. Cells were grown in 25-cm2 flasks in 5 mL of culture medium as recommended in the ATCC specification sheets. Confluent monolayers were inoculated with 200 μL of virus supernatant obtained following culture in C6/36 mosquito cells and adsorbed for 2 hours. Each flask was then rinsed three times with 5 mL of maintenance medium with aspiration to remove all remaining medium between washes. The medium was then replaced and the cultures were then examined daily for CPE for 6–7 days. Assays for BALV (strain CoB 76) and CBV (strain EVG 5-53) RNA were conducted on cell culture supernatant fluids that were collected daily and stored at −80°C. After each daily collection, all remaining cell culture fluid was removed by aspiration and fresh medium was added. The samples were assayed by polymerase chain reaction (PCR) using primers targeting the respective L genes (primer sequences and PCR conditions are available upon request).
Evaluation of growth in suckling mice.
One litter of 2-day old ICR mouse pups (N = 10) was inoculated intracranially with approximately 15 μL of culture fluid from C6/36 cultures infected with BALV (strain CoB 76) or CBV (strain EVG 5-53). After inoculation, the pups were returned to their dams and were examined daily for 14 days for signs of illness or death. The mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN); animal work at University of Texas Medical Branch was carried out under an Institutional Animal Care and Use Committee-approved protocol (no. 9505045).
Transmission electron microscopy.
For ultrastructural analysis, infected C6/36 cells were fixed for at least 1 hour 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 monolayers were washed in 0.1 M cacodylate buffer, and cells were scraped off and processed further as a pellet. The pellets were postfixed in 1% OsO4 in 0.1 M cacodylate buffer (pH 7.3) for 1 hour, washed with distilled water, and stained en block with 2% aqueous uranyl acetate for 20 minutes 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 μL tramicrotome (Leica Microsystems, Buffalo Grove, IL), stained with lead citrate, and examined in a Phillips 201 transmission electron microscope (FEI Phillips, Hillsboro, OR) at 60 kV.
Extraction of viral RNA.
C6/36 cells grown to 90% confluence in 25-cm2 culture flasks were infected with respective viruses. The virus harvest and isolation of viral RNA for next-generation genome sequencing was processed as described previously.7
Next-generation sequencing.
Viral RNA (∼0.9 μg) was fragmented by incubation at 94°C for 8 minutes in 19.5 μL of fragmentation buffer (Illumina 15016648, Illumina, San Diego, CA). Sequencing libraries were prepared from the sample RNAs using an Illumina TruSeq RNA v2 kit following the manufacturer's protocol. The samples were sequenced on a HiSeq 1500 using the Rapid-Run 2 × 50 paired-end protocol. Reads in FASTQ format were quality filtered, and any adapter sequences were removed, using Trimmomatic software.8 The de novo assembly program ABySS9 (Michael Smith Genome Sciences Centre, Vancouver, Canada) was used to assemble the reads into contigs, using several different sets of reads, and k values from 20 to 40. Contigs covering nearly the full length of the viruses in each sample were obtained from 150,000 reads (BALV strains CoB 76 and CoB 84) and 1,000,000 reads for CBV. Reads were mapped back to the contigs using bowtie2,10 and visualized with the Integrated Genomics Viewer11 to verify that the assembled contigs were correct. There were 11.3, 13.6, and 26 million read-pairs in samples of BALV CoB 76, BALV CoB 84, and CBV, respectively. Read-pairs mapping to the virus in each sample were ∼288,000 (2.1%), 403,000 (3.6%), and 468,000 (1.8%), respectively.
Bioinformatic analysis.
Translation of genome sequences and structural analysis of proteins was conducted using bioinformatics resources accessed through the ExPASy portal (http://www.expasy.org/proteomics), including Translate (nucleotide sequence translation), Compute PI/MW (pI and molecular weight prediction), TMHMM and TMPred (transmembrane protein prediction), SignalP (signal peptidase cleavage sites), and NetNGlyc (N-linked glycosylation sites).
Phylogenetic analysis.
Sequence alignments of complete L protein sequences were created using MUSCLE (MEGA 6.0)12,13 and ambiguously aligned regions were removed using Gblocks.14 The resulting alignment comprising 921 amino acids was used to infer phylogenetic relationships using the maximum-likelihood (ML) method in MEGA 6.0 employing the WAG model of amino acid substitution, earest neighbor interchange (NNI) and subtree pruning and regrafting (SPR) branch-swapping. The phylogenetic robustness of each node was determined using 100 bootstrap replicates and NNI branch-swapping. Trees were annotated using Figtree version 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree).
Results and Discussion
Each of the four virus isolates was initially isolated in cultures of C6/36 cells. On the first passage, CBV (EVG 5-53) produced marked CPE in the mosquito cells within 48 hours. The two BALV virus strains (CoB 76 and CoB 84) also caused massive CPE within 2 days. The original mosquito homogenate yielding RCHV (GAM195) produced CPE in the C6/36 cell culture 4 days after inoculation, but on the second C6/36 passage, CPE was observed after 2 days. However, subsequent next generation sequencing of the GAM195 isolate indicated that it contained two distinct viruses, RCHV and Wallerfield virus, a negevirus, so we are uncertain which virus caused the CPE.
After one or two passages in C6/36 cells, the four rhabdovirus isolates were inoculated into cultures of Vero E6 and BHK-21 cells maintained at 37°C. No CPE was observed in the mammalian cells after 14 days. Samples (0.15 μL) of each virus suspension were also inoculated intracranially into single litters of 1- to 2-day-old, outbred (ICR) mice. Pups were examined daily for 14 days, but no illness or deaths were observed in the animals. To further assess the potential for virus replication in vertebrate cells, one isolate of BALV (CoB 76) and the CBV (EVG 5-53) isolate were each inoculated onto confluent monolayers of Vero cells, duck embryo cells, and toad (X. laevis) cells, and the culture supernatants were tested daily by PCR for the presence of viral RNA. In each cell line, BALV RNA was detected as a strong band in the inoculum and as weaker bands of decreasing intensity in supernatants collected on day 0 (triple-washed cells) and from day 1 to day 4, but was not detected in supernatants collected on day 5 or day 6. CBV was also detected as a moderately intense band in the inoculum, but was not detected in the supernatants collected from any of the cell lines from day 0 (postinoculation triple-washed cells) to day 6. There was no evidence of CPE in any of the cell lines inoculated with BALV or CBV. Taken together, these results suggested that these viruses, as proposed previously for ABTV and PTAMV, do not replicate in vertebrate cells and may be insect-specific viruses.6
Three of the virus isolates were characterized initially by electron microscopy. Ultrathin sections of infected C6/36 cells were processed as described previously15 and examined by transmission electron microscopy. BALV strain CoB 76 virions (50–55 nm in diameter and 180–310 nm in length) were observed budding into intracytoplasmic vacuoles or from the plasmalemma into extracellular space (Figure 1A and B). A virion was also observed in the process of internalization into a clathrin-coated vesicle (Figure 1B). BALV strain CoB 84 virions (∼50 nm in diameter and 140–290 nm in length) were found mostly inside intracellular vacuoles (Figure 1C). CBV virions (∼50 nm in diameter and 130–320 nm in length) were forming either from the cell surface or into expanded cisterns of granular endoplasmic reticulum (Figure 1D). In each case, the variations in the length of virions resulted in morphology that varied from the bullet-like shapes to long rod-like shapes, sometimes with two rounded ends. These variations were also observed previously for ABTV and PTAMV,6 and may be indicative of the packaging of full-length genomes and smaller defective-interfering particles.
Ultrastructure of Balsa virus (BALV) and Coot Bay virus (CBV) virions in C6/36 mosquito cells. (A) BALV strain CoB 76 virions forming into a spacious intracellular vacuole (VAC). (B) BALV strain CoB 76 virions forming at the cell surface; cross-sections of the virions. One virion is seen in the process of internalization into a clathrin-coated vesicle (VES). (C) CBV strain EVG 5-53 virions forming into expanded cisterns of granular endoplasmic reticulum (RER). (D) BALV strain CoB 84 virions forming at the cell surface; cross-sections of the virions. Virions are indicated with arrows. Bars = 100 nm.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
Ultrastructure of Balsa virus (BALV) and Coot Bay virus (CBV) virions in C6/36 mosquito cells. (A) BALV strain CoB 76 virions forming into a spacious intracellular vacuole (VAC). (B) BALV strain CoB 76 virions forming at the cell surface; cross-sections of the virions. One virion is seen in the process of internalization into a clathrin-coated vesicle (VES). (C) CBV strain EVG 5-53 virions forming into expanded cisterns of granular endoplasmic reticulum (RER). (D) BALV strain CoB 84 virions forming at the cell surface; cross-sections of the virions. Virions are indicated with arrows. Bars = 100 nm.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
Ultrastructure of Balsa virus (BALV) and Coot Bay virus (CBV) virions in C6/36 mosquito cells. (A) BALV strain CoB 76 virions forming into a spacious intracellular vacuole (VAC). (B) BALV strain CoB 76 virions forming at the cell surface; cross-sections of the virions. One virion is seen in the process of internalization into a clathrin-coated vesicle (VES). (C) CBV strain EVG 5-53 virions forming into expanded cisterns of granular endoplasmic reticulum (RER). (D) BALV strain CoB 84 virions forming at the cell surface; cross-sections of the virions. Virions are indicated with arrows. Bars = 100 nm.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
Complete genome sequences were determined for BALV strain CoB 76 and BALV CoB 84 (11,287 nucleotides and 11,286 nucleotides, respectively). For CBV and RCHV, complete coding regions were sequenced (10,869 nucleotides and 10,735 nucleotides, respectively) with only the extreme genome termini not determined. BLAST searches (blastx)16 of the National Center for Biotechnology Information nonredundant protein sequences (nr) database indicated that each of the genomes was most closely related to ABTV (GenBank KC994644) and PTAMV (GenBank KF534749) which had been isolated from mosquito pools collected in Brazil by passage in C6/36 cells and which had also failed to grow in mammalian cell cultures or in newborn mice.6 Translation of the genome sequences indicated that BALV, CBV, and RCHV each have similar organizations to ABTV and PTAMV, comprising the five canonical rhabdovirus structural protein genes (N, P, M, G, and L) and a gene encoding a small hydrophobic protein (U1 or SH) located between the G and L genes (Figure 2). Each gene is flanked by transcription regulatory sequences which conform to the consensus sequences 5′-UUGUUUGGAUU (transcription initiation) and [U]7GAGUU-3′ (transcription termination), except for CBV in which transcription initiation sequences appear to be highly variable and somewhat cryptic (Table 1). The novel genome sequences have been deposited in GenBank under accession numbers KX228196–KX228199.
Comparisons of the genome organizations of Arboretum virus (ABTV), Puerto Almendras virus (PTAMV), Coot Bay virus (CBV), Rio Chico virus (RCHV), and Balsa virus (BALV) strains CoB 76 and CoB 84. Schematic representation of the genome organizations with the locations of each open reading frame shown approximately to scale.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
Comparisons of the genome organizations of Arboretum virus (ABTV), Puerto Almendras virus (PTAMV), Coot Bay virus (CBV), Rio Chico virus (RCHV), and Balsa virus (BALV) strains CoB 76 and CoB 84. Schematic representation of the genome organizations with the locations of each open reading frame shown approximately to scale.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
Comparisons of the genome organizations of Arboretum virus (ABTV), Puerto Almendras virus (PTAMV), Coot Bay virus (CBV), Rio Chico virus (RCHV), and Balsa virus (BALV) strains CoB 76 and CoB 84. Schematic representation of the genome organizations with the locations of each open reading frame shown approximately to scale.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
Putative transcriptional regulatory sequences of almendraviruses
| Gene | Location | ABTV | PTAMV | CBV | RCHV | BALV |
|---|---|---|---|---|---|---|
| N | TI* | AACAATCCTAA | AACAACCCTAA | AACAAACAAAA | AACAAACCTAA | AACAAACCTAA |
| AACAAGCAAAG | ||||||
| AACAAACTAAA | ||||||
| TTP† | TATTAAAAAAACTCAA | AATGAAAAAAACTCAA | ATATAAAAAACTCAA | ATATAAAAAACTCAA | TTATAAAAAACTCAA | |
| P | TI | AACAAACCTAA | AACAACCCTAA | AACGATTAATT | AACAAACCTAA | AACAAACCTAA |
| TTP | TTCGAAAAAAACTCAA | TATCAAAAAAACTCAA | ATATAAAAAACTCAA | TTATAAAAAACTCAA | ATATAAAAAACTCAA | |
| M | TI | AACAAACCTAC | AACAAGCCTAA | AACATATAAAT | AACAAACTTAA | AACAAACCTAA |
| TTP | GTTGAAAAAAACTCAA | TTAGAAAAAAACTCAA | ATATAAAAAACTCAA | ATTTAAAAAACACAA | ATATAAAAAACTCAG | |
| G | TI | AACAAACCTAG | AACAACCCTAA | AACATAAAAGA | AACAATCCTAA | AACAAACCTAA |
| TTP | TATCAAAAAAACTCAA | TATCAAAAAAACTCAG | ATATAAAAAACTCAA | ATATAAAAAACTCAA | TTATAAAAAACTCAA | |
| U1 | TI | AACAATCCTAA | AACAAACCTAC | AAGAAATAAAA | AATAATCCTAC | TAAAAACCTAA |
| TTP | TATGAAAAAAACTCAA | TATGAAAAAAACTCAA | ATATAAAAAACTCAA | ATTTAAAAAACTCAA | TTTTAAAAAACTCAA | |
| L | TI | AACAAACCTAG | AACAAACCTAG | AAGAAATACAA | AACAAACCTAA | AACAAACCTAA |
| TTP | TATGAAAAAAACTCAT | GATGAAAAAAACTCAA | ATATAAAAAACTCAC | ATATAAAAAACTCAT | TTATAAAAAACTCAA | |
| Con | TI | AACAAACCTAA | AACAAACCTAA | Cryptic | AACAAACCTAA | AACAAACCTAA |
| TTP | TATGAAAAAAACTCAA | TATGAAAAAAACTCAA | ATATAAAAAACTCAA | ATATAAAAAACTCAA | TTATAAAAAACTCAA |
ABTV = Arboretum virus; BALV = Balsa virus; CBV = Coot Bay virus; PTAMV = Puerto Almendras virus; RCHV = Rio Chico virus.
Transcription initiation sequence.
Transcription termination-polyadenylation sequence.
The structural characteristics of polypeptides encoded in each of the genes of BALV, CBV, and RCHV were compared with those of ABTV and PTAMV. The N gene of each virus contains a single long open reading frame (ORF) encoding a polypeptide with the structural characteristics of rhabdovirus nucleoproteins. The N proteins range in size from 429 to 438 amino acids (predicted molecular weights 48.2–49.8 kDa) and share amino acid sequence identity (p-distance; MEGA 6.0) ranging from 28.4% (ABTV and CBV) to 61.9% (ABTV and PTAMV). Alignment of the sequences with the vesicular stomatitis Indiana virus (VSIV) N protein indicated conservation of eight basic residues that are located in the RNA-binding cavity, six of which coordinate with phosphate groups in the ribonucleoprotein (RNP) with VSIV genomic RNA, and conservation of two large aromatic residues (F, W, Y) that form a hydrophobic patch on the C-terminal lobe of the VSIV RNA-binding cavity (Supplemental Figure 1).17,18 Interestingly, one of the six RNA-coordinating residues in VSIV N (R-146) is not conserved in the CBV N protein, aligning to a polar residue (S-155) in stretch of amino acids that lacks basic residues. Variations in phosphate-coordinating residues have also been reported between VSIV and rabies virus RNPs.18
The P genes of each virus contain long ORFs encoding polypeptides that vary significantly in size (210–320 amino acids; predicted 24.4–36.5 kDa) and display little overall sequence identity (> 25%). However, the proteins are similar in net charge (pI = 5.07–5.89) and, like the P proteins of other rhabdoviruses, they are rich in charged amino acids (D, E, K, R, H) which, in VSIV, form extensive acidic and basic domains on the protein surface.19 Structural variability is typical of rhabdovirus P proteins which are intrinsically disordered.20 Uniquely among this set of viruses, the P genes of each strain of BALV contains an alternative ORF (Px) commencing 35 nucleotides downstream of the P ORF initiation codon. The putative Px proteins (81 amino acids; 9.8 kDa) are highly basic (pI = 10.66) and share a high level of sequence identity (97.5%) (Figure 3A). Small basic proteins are commonly encoded in alternative ORFs in the P genes of rhabdoviruses21 including VSIV for which two small basic carboxy-coterminal proteins (C and C′) have been shown to be expressed in infected cells.22,23
(A) ClustalX alignment of the amino acid sequences of the Px proteins of Balsa virus (BALV) strains CoB 76 and CoB 84 showing the high level of sequence identity and the high proportion of basic amino acid residues. Basic amino acids (K, R) are shaded in black and acidic amino acids (D, E) are shaded in grey. (B) Comparison of the amino acid sequences of the U1 proteins of each virus illustrating the similar structures including the predicted transmembrane domains (shaded grey) and relatively high proportions of basic amino acid residues (K, R, H) in the C-terminal domains.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
(A) ClustalX alignment of the amino acid sequences of the Px proteins of Balsa virus (BALV) strains CoB 76 and CoB 84 showing the high level of sequence identity and the high proportion of basic amino acid residues. Basic amino acids (K, R) are shaded in black and acidic amino acids (D, E) are shaded in grey. (B) Comparison of the amino acid sequences of the U1 proteins of each virus illustrating the similar structures including the predicted transmembrane domains (shaded grey) and relatively high proportions of basic amino acid residues (K, R, H) in the C-terminal domains.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
(A) ClustalX alignment of the amino acid sequences of the Px proteins of Balsa virus (BALV) strains CoB 76 and CoB 84 showing the high level of sequence identity and the high proportion of basic amino acid residues. Basic amino acids (K, R) are shaded in black and acidic amino acids (D, E) are shaded in grey. (B) Comparison of the amino acid sequences of the U1 proteins of each virus illustrating the similar structures including the predicted transmembrane domains (shaded grey) and relatively high proportions of basic amino acid residues (K, R, H) in the C-terminal domains.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
The M genes contain ORFs encoding mildly basic proteins (pI = 8.61–9.40) of similar size (159–182 amino acids; predicted 19.1–21.4 kDa). They share identifiable levels of sequence identity across the data set, but poor homology with the M proteins of VSIV and other rhabdoviruses (Supplemental Figure 2). Rhabdovirus M proteins are a structural component of virions and play roles in maturation and modulating intracellular processes during virus replication.24 The N-terminal domain of the VSIV M protein contains a basic region that is important in membrane localization and late budding domain motifs (PPPY and PSAP) that are required for membrane fusion and release of virions from infected cells.25 Similar clusters of basic residues are evident in the N-terminal domains of the M proteins of each of the viruses in this data set (Supplemental Figure 2), and sequences similar to recognized late budding motifs have been identified previously in ABTV and PTAMV.6 However, we could find no evidence of recognized late budding motifs in the CBV, RCHV, or BALV M proteins.
The G genes each contain a single long ORF encoding a class 1 transmembrane glycoprotein with an N-terminal signal domain and a near-C-terminal transmembrane domain (Figure 4). The unprocessed G proteins vary in size (442–476 amino acids; predicted 51.5–55.8 kDa) and in the number (2–6) of predicted N-glycosylation sites (NetNGlyc 1.0 server), the locations of which are not generally conserved. Each virus contains the 12 highly conserved cysteine residues (CI–CXII) that are characteristic of rhabdovirus G proteins,26 except for CBV which lacks cysteines CVI and CVII. These 12 cysteine residues form six disulfide bridges that stabilize the folded secondary structures of the trimerization, fusion and plextrin homology domains of the G protein.27 In most animal rhabdoviruses, cysteines CVI and CVII form a disulfide bridge that stabilizes an exposed loop in the fusion domain.27 Interestingly, this disulfide bridge is also absent from the G proteins of sigmaviruses which are known to be insect specific.4,26 As reported previously for ABTV and PTAMV,6 each virus also contains two additional conserved cysteine residues that are likely to form a unique seventh disulfide bridge in the lateral domain.
ClustalX alignment of the amino acid sequences of the G proteins of each virus. The signal domains and transmembrane domains are shaded grey. Cysteine residues in the ectodomain are shaded black. Cysteine residues that form conserved disulfide bridges in animal rhabdoviruses (CI–CXII) are numbered according to the scheme of Walker and Kongsuwan.26 Predicted N-glycosylation sites are underlined. Fully conserved (*), strongly conserved (:), or weakly conserved (.) amino acids are indicated below the alignment.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
ClustalX alignment of the amino acid sequences of the G proteins of each virus. The signal domains and transmembrane domains are shaded grey. Cysteine residues in the ectodomain are shaded black. Cysteine residues that form conserved disulfide bridges in animal rhabdoviruses (CI–CXII) are numbered according to the scheme of Walker and Kongsuwan.26 Predicted N-glycosylation sites are underlined. Fully conserved (*), strongly conserved (:), or weakly conserved (.) amino acids are indicated below the alignment.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
ClustalX alignment of the amino acid sequences of the G proteins of each virus. The signal domains and transmembrane domains are shaded grey. Cysteine residues in the ectodomain are shaded black. Cysteine residues that form conserved disulfide bridges in animal rhabdoviruses (CI–CXII) are numbered according to the scheme of Walker and Kongsuwan.26 Predicted N-glycosylation sites are underlined. Fully conserved (*), strongly conserved (:), or weakly conserved (.) amino acids are indicated below the alignment.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
The U1 genes each encode small hydrophobic proteins (51–80 amino acids; predicted 6.1–9.5 kDa) which are predicted to feature a short N-terminal luminal domain, a central transmembrane domain, and a longer C-terminal cytoplasmic domain that is rich in basic residues (TMpred; http://embnet.vital-it.ch/software/TMPRED_form.html) (Figure 3B). This structure is characteristic of class 1A viroporins.28 ORFs encoding viroporin-like proteins have been reported in a wide range of animal rhabdoviruses, including ABTV and PTAMV, and are usually located immediately following the G gene.6,21,29 The α1 viroporin of the rhabdovirus, bovine ephemeral fever virus, has been shown to localize in the Golgi complex, increase cellular permeability, and interact with importins to disrupt nuclear trafficking.30
The L genes of each virus encode the large multifunctional RNA-dependent RNA polymerase (2,035–2,062 amino acids; predicted 234.5–241.1 kDa) that contains each of the conserved regions (CRI–CRVI) and functional domains (RdRp, capping, connector, methyltransferase, and CTD) that are characteristic of the L proteins of all nonsegmented (−) ssRNA viruses. Amino acid sequence identity between the L proteins of viruses in the data set ranges from 42.4% (ABTV and CBV) to 65.1% (ABTV and PTAMV).
An ML phylogenetic tree was inferred using the complete L protein sequences of CBV, RCHV, BALV, and 134 other animal rhabdoviruses, including all members of existing genera, proposed new genera, and many rhabdoviruses that are currently unassigned (Figure 5). Novirhabdoviruses (infecting fish) and several rhabdovirus genomes detected by metagenomics analysis of arthropods were excluded as the L protein sequences are too divergent, limiting the length of the Gblocks-pruned alignment and thus reducing phylogenetic resolution. Fecal fox rhabdovirus, which was detected by metagenomics analysis of fecal samples from red foxes (Vulpes vulpes) in Spain,31 was the most divergent sequence and was used as the outgroup in the tree. In this analysis, CBV, RCHV, and BALV clustered with ABTV and PTAMV as a distinct monophyletic group with strong bootstrap support (bootstrap proportion = 100%). Although several of the deeply rooted nodes in the tree were unresolved, the almendravirus clade was more deeply rooted than clades representing all other genera except lyssaviruses.
A maximum-likelihood phylogenetic tree inferred from a MUSCLE alignment of the L protein sequences of 137 animal rhabdoviruses. Clades containing viruses assigned to eight existing genera (Lyssavirus, Tupavirus, Sigmavirus, Perhabdovirus, Sprivivirus, Vesiculovirus, Tibrovirus, and Ephemerovirus) and two proposed new genera (Hapavirus, Ledantevirus) have been collapsed to reduce complexity. Asterisks (*) indicate well-supported nodes in the tree (bootstrap proportion ≥ 75%). Horizontal branch lengths are drawn to a scale of amino acid substitutions/site, the scale bar indicating a value of 0.5.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
A maximum-likelihood phylogenetic tree inferred from a MUSCLE alignment of the L protein sequences of 137 animal rhabdoviruses. Clades containing viruses assigned to eight existing genera (Lyssavirus, Tupavirus, Sigmavirus, Perhabdovirus, Sprivivirus, Vesiculovirus, Tibrovirus, and Ephemerovirus) and two proposed new genera (Hapavirus, Ledantevirus) have been collapsed to reduce complexity. Asterisks (*) indicate well-supported nodes in the tree (bootstrap proportion ≥ 75%). Horizontal branch lengths are drawn to a scale of amino acid substitutions/site, the scale bar indicating a value of 0.5.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
A maximum-likelihood phylogenetic tree inferred from a MUSCLE alignment of the L protein sequences of 137 animal rhabdoviruses. Clades containing viruses assigned to eight existing genera (Lyssavirus, Tupavirus, Sigmavirus, Perhabdovirus, Sprivivirus, Vesiculovirus, Tibrovirus, and Ephemerovirus) and two proposed new genera (Hapavirus, Ledantevirus) have been collapsed to reduce complexity. Asterisks (*) indicate well-supported nodes in the tree (bootstrap proportion ≥ 75%). Horizontal branch lengths are drawn to a scale of amino acid substitutions/site, the scale bar indicating a value of 0.5.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0403
Previous studies have identified ABTV and PTAMV as novel and phylogenetically distinct rhabdoviruses and proposed the creation of a new genus Almedravirus to which the viruses should be assigned.6,21 Our data support this proposal and indicate that the genus should include five new species (Arboretum almendravirus, Puerto Almendras almendravirus, Coot Bay almendravirus, Rio Chico almendravirus, and Balsa almendravirus). Each of these viruses has been isolated from mosquitoes, they failed to grow in mammalian cell cultures or in suckling mice, and they have a genome organization featuring a gene between the G and L genes that encodes a small hydrophobic class 1A viroporin-like protein. They also have a similar a similar arrangement of cysteine residues in the G proteins, supporting previous observations of the genus-specificity of disulfide bridges.26
Failure of these viruses to grow in newborn mice or in vertebrate cells in vitro may be indicative of poor adaptation to replication in vertebrates. An increasing number of RNA viruses isolated from mosquitoes in recent years have been characterized as insect specific, replicating only in mosquitoes with no apparent vertebrate host. These include positive-sense ssRNA viruses (flaviviruses, togaviruses, negeviruses, mesoniviruses, nodaviruses, tymoviruses), double-stranded RNA viruses (reoviruses), and (−) ssRNA viruses (rhabdoviruses).32 Indeed, recent metagenomics surveys of various arthropods have revealed a vast and deeply rooted diversity of both positive-sense and negative-sense RNA viruses, suggesting that they may be the ancestral hosts from which vertebrate-infecting viruses have evolved.5,33 Our analysis indicated that, although several deep nodes remained unresolved, the almendravirus clade is clearly basal to most recognized families of arthropod-borne rhabdoviruses infecting vertebrates. However, it was also evident that the clade is not more deeply rooted than lyssaviruses (infecting only mammals) or several other viruses that were isolated from mosquitoes that have a broad host range in mammals and reptiles (Bahia Grande virus, Reed Ranch virus, and Muir Springs virus).34 We suggest, therefore, that further experimental infections and epidemiological studies are required to determine whether the almendraviruses are truly insect specific.
Poor resolution of deeper roots in the phylogeny also suggests that, unless major ancestral lineages are now extinct, there may be many as yet undiscovered rhabdoviruses that would help clarify the phylogenetic position of almendraviruses and add further insights into the evolutionary history of the family.
ACKNOWLEDGMENTS
We thank Richard Hoyos and Juan David Suaza for help for fieldwork and entomological processing of the samples in Colombia. We thank Matt Aliota for his assistance in screening samples at NYSDOH.
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