The “gold standard” filovirus type RefSeq entry should be selected on the basis of experimental importance and accessibility and represent a repository of functional information about a particular filovirus. It is of crucial importance that any functional annotation of a RefSeq entry (e.g., functions of particular genome parts or of genome-encoded proteins), is linked to the actual sequence associated with these experiments. The RefSeq entry should contain the most characterized virus/variant/isolate/sequence, independent of whether this virus, variant, or isolate was the first one discovered or the most widely used experimentally. Importantly, decisions on RefSeq entries do not entail a mandate that future experiments should necessarily be performed with the viruses associated with these entries. However, direct comparisons with RefSeq-associated viruses are highly recommended to further increase the detail associated with the RefSeq entries. These entries should be updated, and, if necessary, corrected on a continuous basis by a filovirus RefSeq subcommittee comprised of filovirus experts, whose composition is currently under consideration.
The authors of this article confirmed or replaced the current taxonomic type virus variants and isolates and the current filovirus RefSeq entries based on the availability of scientific information characterizing a particular virus. If scientific information is scarce for all members belonging to an entire taxon, other criteria such as availability, passaging history, or medical importance were used in decision making. Decisions were reached by consensus or simple majority voting, with the understanding that all authors will apply the final decisions reached by the entire group and enforce them in their functions as authors, peer-reviewers, and/or editors.
3.1. Cuevavirus RefSeq EntriesOnly one cuevavirus, Lloviu virus (LLOV), has been described [18]. At the time of writing, LLOV had not been isolated in culture, and the sequence diversity of LLOV had only been defined in a single study using deep sequencing techniques on samples from deceased Schreibers’s long-fingered bats (Miniopterus schreibersii) [18]. Only one additional study has been published on this virus, characterizing molecular-biological characteristics of the LLOV glycoprotein [20]. The coding-complete genome of one LLOV has been determined (Genbank #JF828358), which therefore automatically became the current RefSeq sequence (#NC_016144) (see [21] for sequencing nomenclature used in this article). In the absence of additional deposited LLOV sequences and characterization data, this RefSeq entry should therefore be upheld but be considered temporary until a complete genome, including all non-coding sequences, is determined.
In line with filovirus strain/variant/isolate definitions outlined previously [19], we propose the variant designation “Asturias” (after the Principality of Asturias in Spain, where Cueva del Lloviu is located in which LLOV was discovered [18]) and the “isolate” name “Bat86” (instead of “MS-Liver-86/2003”) for this virus:
| Full name: | Lloviu virus M.schreibersii-wt/ESP/2003/Asturias-Bat86 |
| Shortened name: | LLOV/M.sch/ESP/03/Ast-Bat86 |
| Abbreviated name: | LLOV/Ast-Bat86 |
Accordingly, in RefSeq #NC_016144 the definition line “Lloviu virus, complete genome” was changed to “Lloviu cuevavirus isolate Lloviu virus M.schreibersii-wt/ESP/2003/Asturias-Bat86, [coding-]complete genome.” The RefSeq <strain> field was cleared; and the RefSeq <isolate> field was filled with “Lloviu virus M.schreibersii-wt/ESP/2003/Asturias-Bat86.” The same changes should be applied to GenBank #JF828358. [Note here and below that the International Nucleotide Sequence Database Collaboration (INSDC) standard currently does not offer options other than “complete” or “partial,” and, in particular, does not provide a possibility for the designation “coding-complete.” Also note here and below that neither RefSeq nor GenBank currently can handle italics or extended Latin characters, which is why the species names are not italicized in the entry’s definition line and <organism> fields and why letters with diacritics revert to their basic Latin letter counterpart].
3.2. Ebolavirus RefSeq EntriesThe genus Ebolavirus includes five species, each of which is represented by one virus.
3.2.1. Bundibugyo VirusBundibugyo virus (BDBV) is the second least characterized ebolavirus. Although at least eight isolates of this virus are available [17,22], all experiments reported to date have been performed with one particular isolate, “811250” (often wrongly referred to as “200706291”). The complete sequence of this isolate is the one found in the current RefSeq entry (NC_014373). This isolate, obtained after two passages of clinical material in Vero E6 cells, came from a male patient who died in 2007 in Uganda [17]. We propose the variant designation “Butalya” (after Butalya Parish, Kikyo Subcounty in Uganda’s Bundibugyo district where BDBV was discovered) and the isolate name “811250” for this virus:
| Full name: | Bundibugyo virus H.sapiens-tc/UGA/2007/Butalya-811250 |
| Shortened name: | BDBV/H.sap/UGA/07/But-811250 |
| Abbreviated name: | BDBV/But-811250 |
Accordingly, in RefSeq #NC_014373, the definition line “Bundibugyo ebolavirus, complete genome” was changed to “Bundibugyo ebolavirus isolate Bundibugyo virus H.sapiens-tc/UGA/2007/Butalya-811250, complete genome.” The RefSeq <isolate> field was filled with “Bundibugyo virus H.sapiens-tc/UGA/2007/Butalya-811250.” The same changes should be applied to GenBank #FJ217161.
3.2.2. Ebola VirusEbola virus (EBOV) is the most thoroughly characterized ebolavirus. Dozens of EBOV isolates are available, but the vast majority of published experiments have been performed with isolates “Mayinga” and “Kikwit” (reviewed in [23]). The “Mayinga” isolate, the first EBOV isolate obtained in 1976, has been used extensively for molecular-biological characterizations. The “Kikwit” variant, obtained during an Ebola virus disease outbreak in 1995, has been used almost exclusively for pathogenesis studies in nonhuman primates in the US (the “Mayinga” isolate is used almost everywhere else) [23]. All available EBOV cDNA clone systems are based on the “Mayinga” isolate (see [24]). The only available mouse- and guinea pig-adapted EBOV strains are derived from the “Mayinga” isolate (see [25]), and all available EBOV protein crystal structures are derived from the “Mayinga” isolate [26,27,28,29,30,31,32,33,34]. The “Mayinga” isolate was therefore chosen as the prototype EBOV for RefSeq (#NC_002549), which lists a complete genome obtained after 3–4 passages in Vero E6 cells. We uphold this decision and propose the variant designation “Yambuku” (after the village in which EBOV first emerged [35,36]) and retain the isolate designation “Mayinga” (the last name of a nurse who succumbed to infection [36]) for this virus:
| Full name: | Ebola virus H.sapiens-tc/COD/1976/Yambuku-Mayinga |
| Shortened name: | EBOV/H.sap/COD/76/Yam-May |
| Abbreviated name: | EBOV/Yam-May |
Accordingly, in RefSeq #NC_002549 the definition line “Zaire ebolavirus, complete genome” was changed to “Zaire ebolavirus isolate Ebola virus H.sapiens-tc/COD/1976/Yambuku-Mayinga, complete genome.” The RefSeq <strain” field was cleared; the RefSeq <isolate> field was filled with “Ebola virus H.sapiens-tc/COD/1976/Yambuku-Mayinga;” and the <organism> field was corrected to “Zaire ebolavirus.” The same changes should be applied to GenBank #AF086833.
3.2.3. Reston VirusReston virus (RESTV) has caused multiple epizootics among captive macaques (1989-1990, 1992, 1996) and domestic pigs in 2008 (reviewed in [37]). At least 10 isolates were obtained during all these outbreaks, and eight complete or coding-complete genomic sequences have been deposited. However, the vast majority of RESTV experiments, in particular those regarding molecular characterization, have been performed with “Pennsylvania” (reviewed in [23]). “Pennsylvania” is the only RESTV variant for which there is a reverse genetics system [38]. In addition, “Pennsylvania” sequences served as the basis for the available RESTV protein crystal structures [39,40,41,42,43]. “Pennsylvania” (NC_004161) was chosen for the current RESTV RefSeq entry, which we propose to maintain. We propose the variant designation “Philippines89” (a reference to the time and place from which this virus was exported to the US in 1989) and the isolate name “Pennsylvania” for this virus:
| Full name: | Reston virus M.fascicularis-tc/USA/1989/Philippines89-Pennsylvania |
| Shortened name: | RESTV/M.fas/USA/89/Phi89-Pen |
| Abbreviated name: | RESTV/Phi89-Pen |
Accordingly, in RefSeq #NC_004161, the definition line “Reston ebolavirus, complete genome” was changed to “Reston ebolavirus isolate Reston virus M.fascicularis-tc/USA/1989/Philippines89-Pennsylvania, complete genome.” The RefSeq <strain> field was cleared; and the RefSeq <isolate> field was filled with “Reston virus M.fascicularis-tc/USA/1989/Philippines89-Pennsylvania”. The same changes should be applied to GenBank #AF522874.
3.2.4. Sudan VirusSudan virus (SUDV) is the second-best characterized ebolavirus. Approximately 15 SUDV isolates have been described, but very few experiments have been performed with any of these isolates. Early experiments focused on isolate “Boneface” (often misspelled “Boniface”). Recently variant “Gulu” isolate “808892” has become a more popular choice, and data from experiments with this virus continue to accumulate (reviewed in [23]). Crystal structures for GP1,2 were determined for both viruses [33,42,44,45]. However, the passaging history of the “Boneface” isolate has not been thoroughly documented and includes passaging in guinea pigs and culturing in various cell types. The “Gulu-808892” isolate, on the other hand, is completely sequenced and is the current virus of choice for nonhuman primate experiments in the US. While the “Boneface” isolate was chosen by the 2010-2011 ICTV Filoviridae Study Group as the type SUDV [12], “Gulu-808892” isolate was chosen as the prototype SUDV for RefSeq (#NC_006432). We propose to support the RefSeq decision and to change the SUDV type virus variant to “Gulu.” As several “Gulu” isolates are available, we propose the variant designation “Gulu” for the virus variant that caused the disease outbreak that started in Gulu District, Uganda, in 2000, and the isolate designation “808892” for the RefSeq entry of this particular virus. (“808892” was obtained after three Vero E6 cell passages of clinical material coming from an infected male who died):
| Full name: | Sudan virus H.sapiens-tc/UGA/2000/Gulu-808892 |
| Shortened name: | SUDV/H.sap/UGA/00/Gul-808892 |
| Abbreviated name: | SUDV/Gul-808892 |
Accordingly, in RefSeq #NC_006432, the definition line “Sudan ebolavirus, complete genome” was changed to “Sudan ebolavirus isolate Sudan virus H.sapiens-tc/UGA/2000/Gulu-808892, complete genome.” The RefSeq <strain> field was cleared; and the RefSeq <isolate> field was filled with “Sudan virus H.sapiens-tc/UGA/2000/Gulu-808892.” The same changes should be applied to GenBank #AY729654.
3.2.5. Taï Forest VirusTaï Forest virus (TAFV) is the least characterized ebolavirus. Only one isolate (“807212” = “CI”) was obtained from a female survivor [46] after seven passages in Vero E6 cells, and the coding-complete genome of this isolate is the only genomic TAFV sequence available [17]. Therefore, this sequence automatically became the current RefSeq sequence (#NC_014372). In the absence of additional deposited TAFV sequences and characterization data, this RefSeq entry should therefore be upheld but be considered temporary.
We propose the variant designation “Pauléoula” (after the village of Pauléoula, Guiglo Department in Moyen-Cavally Region, Côte d’Ivoire, where TAFV was first found [46]) and the isolate name “CI” (for “Côte d’Ivoire”) for this virus:
| Full name: | Taï Forest virus H.sapiens-tc/CIV/1994/Pauléoula-CI |
| Shortened name: | TAFV/H.sap/CIV/94/Pau-CI |
| Abbreviated name: | TAFV/Pau-CI |
Accordingly, in RefSeq # NC_014372, the definition line “Tai Forest ebolavirus, complete genome” was changed to “Taï Forest ebolavirus isolate Taï Forest virus H.sapiens-tc/CIV/1994/Pauléoula-CI, [coding-]complete genome,” and the RefSeq <isolate> field was filled with “Taï Forest virus H.sapiens-tc/CIV/1994/Pauléoula-CI.” The same changes should be applied to GenBank #FJ217162.