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. 2021 May 9;10(5):575. doi: 10.3390/pathogens10050575

Genomic Characterisation of a Novel Avipoxvirus Isolated from an Endangered Northern Royal Albatross (Diomedea sanfordi)

Subir Sarker 1,*, Ajani Athukorala 1, Tadiwa Nyandowe 1, Timothy R Bowden 2,†, David B Boyle 2
Editor: Anna Honko
PMCID: PMC8151833  PMID: 34065100

Abstract

Marine bird populations have been declining globally with the factors driving this decline not fully understood. Viral diseases, including those caused by poxviruses, are a concern for endangered seabird species. In this study we have characterised a novel avipoxvirus, tentatively designated albatrosspox virus (ALPV), isolated from a skin lesion of an endangered New Zealand northern royal albatross (Diomedea sanfordi). The ALPV genome was 351.9 kbp in length and contained 336 predicted genes, seven of which were determined to be unique. The highest number of genes (313) in the ALPV genome were homologs of those in shearwaterpox virus 2 (SWPV2), while a further 10 were homologs to canarypox virus (CNPV) and an additional six to shearwaterpox virus 1 (SWPV1). Phylogenetic analyses positioned the ALPV genome within a distinct subclade comprising recently isolated avipoxvirus genome sequences from shearwater, penguin and passerine bird species. This is the first reported genome sequence of ALPV from a northern royal albatross and will help to track the evolution of avipoxvirus infections in this endangered species.

Keywords: avipoxvirus, complete genome, endangered, evolution, northern royal albatross

1. Introduction

Marine bird populations have been declining globally [1] with the sustainability of the albatrosses (family Diomedeidae) and large petrels (Macronectes and Procellaria spp.) being of particular concern [2,3,4]. This group includes some of the world’s most endangered bird species, with rapidly decreasing populations and their conservation status markedly deteriorating in recent years [5,6]. The northern royal albatross (Diomedea sanfordi), which is one of the largest seabirds in the world, is categorised as an “endangered” species under the International Union for Conservation of Nature (IUCN) Red List and is ranked as Category B for conservation priority [7]. The northern royal albatrosses range widely throughout the Southern Ocean, though rarely into Antarctic waters. The breeding range is restricted to the Chatham Islands and Taiaroa Head on the Otago Peninsula, Dunedin, New Zealand. The total breeding population in the Chatham Islands colonies (99% of the total) is estimated at approximately 6500–7000 pairs, which equates to a total population of 17,000 mature individuals [8]. Northern royal albatrosses are normally solitary foragers, but they may congregate at food sources at sea. Most of their food is thought to be obtained by seizing dead or dying prey from the surface and also by scavenging discards and offal from fishing boats. Breeding birds forage over the continental shelves to shelf edges in New Zealand waters. Non-breeding and young birds can be found anywhere in the Southern Ocean throughout the year, with the main wintering areas off the coasts of southern South America [8].

Human activities such as fisheries and pollution have been documented as threats for incidental mortality of these species [7,9,10,11,12,13]. Invasive alien species, degradation or loss of nesting habitats, storms and flooding, and marine pollution or plastic ingestion are also significant factors in population declines [6,7]. Infectious diseases, including those caused by avipoxviruses, have been identified as an important risk factor in the conservation of small and endangered bird populations, including albatrosses [14,15,16,17,18,19]. The impact of the introduction of avipoxviruses has been severe for the avifauna of various archipelagos [20]. For example, the emergence of an avipoxvirus with a high prevalence (88%) in Hawaiian Laysan albatrosses (Phoebastria immutabilis) enabled one of the first detailed studies of the epidemiology and population-level impact of the disease in these seabirds [21].

Avipoxviruses are large, double-stranded DNA (dsDNA) viruses comprising the genus Avipoxvirus. They occur worldwide and are known to infect a large number of wild and domestic avian species across 76 families and 20 orders [22,23,24]. The behaviour of wild birds allows avian poxviruses to reach new hosts through bird migration, species introductions, and habitat change. Avipoxviruses have been identified as an important risk factor in the conservation of endangered bird populations [19,25]. In affected birds, avipoxvirus infection can cause two different forms of disease, defined as cutaneous or diphtheritic. The cutaneous form is characterised by proliferative ‘wart-like’ lesions that commonly develop on unfeathered body areas, including the eyes, feet, legs, face and around the beak. The less common diphtheritic form is characterised by soft and yellowish cankers and proliferative lesions on the mucous membranes of the upper alimentary and respiratory tracts [23,26,27].

Little is known about the effects of poxviruses on some bird taxa, particularly for seabird species including the northern royal albatross (D. sanfordi). The aim of the present study was to characterise the genome sequence of a novel poxvirus, which was isolated from a skin lesion that was collected in 1997 from an endangered northern royal albatross on the Otago Peninsula, near Dunedin, on the South Island of New Zealand.

2. Results

2.1. Genome of ALPV

The complete genome of ALPV was assembled into a contiguous sequence of linear double-stranded DNA 351,909 bp in length (the second-largest avipoxvirus genome so far characterised) and submitted to GenBank under accession number MW365933. Like many other avipoxviruses [25,28,29], the ALPV genome contained a well-conserved central coding region surrounded by two identical inverted terminal repeat (ITR) regions, comprising 4069 bp each (coordinates 1–4069 sense and 347,841–351,909 antisense orientation). The nucleotide composition of the ALPV genome was A + T rich (69.9%), which was in agreement with other avipoxviruses isolated from yellow-eyed penguin [19], shearwater [25] and passerine bird species [30,31] (Table 1). The ALPV genome showed the highest nucleotide identities with penguinpox virus 2 (PEPV2, GenBank accession no. MW296038) (98.92%), followed by shearwaterpox virus 2 (SWPV2, GenBank accession no. KX857216) (95.75%), canarypox virus (CNPV, GenBank accession no. AY318871) (92.71%) and mudlarkpox virus (MLPV, GenBank accession no. MK903864) (88.47%) (Table 1).

Table 1.

Comparative analysis of representative avipoxviruses and ALPV based on complete genome nucleotide sequences.

Avipoxviruses (Abbreviation) GenBank Accession Numbers Genome Identity (%) Genome Length (kbp) A + T Content (%) Number of ORFs References
Albatrosspox virus (ALPV) MW365933 352 69.9 336 This study
Penguinpox virus 2 (PEPV2) MW296038 98.92 350 69.9 327 [19]
Shearwaterpox virus 2 (SWPV2) KX857215 95.75 351 69.8 312 [25]
Canarypox virus (CNPV) AY318871 92.71 360 69.6 328 [32]
Mudlarkpox virus (MLPV) MT978051 88.47 343 70.2 352 [30]
Magpiepox virus (MPPV) MK903864 78.75 293 70.4 301 [31]
Shearwaterpox virus 1 (SWPV1) KX857216 61.44 327 72.4 310 [25]
Penguinpox virus (PEPV) KJ859677 49.83 307 70.5 285 [33]
Fowlpox virus (FWPV) AF198100 48.89 289 69.1 260 [29]
Pigeonpox virus (FeP2) KJ801920 47.54 282 70.5 271 [33]
Flamingopox virus (FGPV) MF678796 46.51 293 70.5 285 [24]
Turkeypox virus (TKPV) KP728110 33.39 189 70.2 171 [34]
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2.2. Genome Annotation and Comparative Analyses of ALPV

The ALPV genome encoded 336 putative genes, 45 to 1936 amino acids in length, that have been numbered from left to right (Figure 1 and Table 2). Among them, four ORFs were located within the inverted terminal repeats (ITRs) and were therefore present as diploid copies. Comparative analysis of the predicted ORF sequences was performed, and a significant number of ORFs (329) were found to be homologs with other chordopoxvirus (ChPV) gene products (Table 2). Among these conserved ChPV gene products, the highest number of protein-coding genes (313) in ALPV were homologs to the recently isolated SWPV2 [25]. The remaining ten gene products (ALPV-079, -155, -163, -165, -166, -167, -168, -175, -233 and -236) were homologous to ORFs of CNPV, and a further six gene products (ALPV-003, -009, -090, -127, -229 and -334) were homologs to SWPV1 (Table 2). All conserved genes of ALPV showed the highest sequence similarity to homologs of avipoxviruses isolated from Pacific shearwater, canary and yellow-eyed penguin bird species, implying a common evolutionary history [19,25,32]. In comparison to SWPV2, two gene products (SWPV2-121 and -213) were absent from the ALPV genome, and a further nineteen genes were predicted to be truncated/fragmented (Figure 1 and Table 2). In comparison to vaccinia virus strain Copenhagen (VACV-Cop), 138 ORFs of ALPV showed homology to VACV-Cop and the sequence identities ranging from 20.9–76.7% (Table 2).

Figure 1.

Figure 1

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Comparative genomic illustration of the novel ALPV. A sequence alignment using MAFFT in Geneious (version 10.2.2) was performed to compare ORFs between albatrosspox virus (ALPV, GenBank accession no. MW365933) and shearwaterpox virus 2 (SWPV2, GenBank accession no. KX857215). The arrows symbolise genes and open reading frames (ORFs), with orientation indicating their direction of transcription. Each gene or ORF is colour coded, as indicated by the key in the legend. The top graph represents the mean pairwise sequence identity over all pairs in the column between ALPV and SWPV2 (green: 100% identity; mustard: ≥30% and <100% identity; red: <30% identity).

Table 2.

Albatrosspox virus (ALPV) genome annotations and comparative analysis of ORFs.

ALPV Synteny ALPV Genome Coordinates SWPV2 Synteny ALPV AA Size SWPV2 AA Size SWPV2 BLAST Hits ALPV AA Identity (%) Compared to Avipoxviruses ALPV AA Identity (%) Compared to VACV-Cop VACV BLAST Hits Notes
ALPV-001 827-1342 SWPV2-001 171 171 SWPV2-001 hypothetical protein 100 identical to ALPV-336
ALPV-002 2271-1645 SWPV2-002 208 208 SWPV2-002 C-type lectin-like protein 100 identical to ALPV-335
ALPV-003 2553-2326 75 56.1 SWPV1-002 C-type lectin-like protein, identical to ALPV-334
ALPV-004 2679-3347 SWPV2-003 222 222 SWPV2-003 conserved hypothetical protein 100 identical to ALPV-333
ALPV-005 3464-3925 SWPV2-004 153 134 SWPV2-004 conserved hypothetical protein 86.9 identical to ALPV-332
ALPV-006 4899-4042 SWPV2-005 285 169 SWPV2-005 C-type lectin-like protein 46.9
ALPV-007 5455-4946 SWPV2-005 169 169 SWPV2-005 C-type lectin-like protein 100
ALPV-008 7809-5743 SWPV2-006 688 688 SWPV2-006 ankyrin repeat protein 100 23.9 B4R
ALPV-009 8828-8184 214 43.1 SWPV1-006 ankyrin repeat protein
ALPV-010 11218-9458 SWPV2-007 586 586 SWPV2-007 ankyrin repeat protein 100 28.8 M1L
ALPV-011 11483-12052 SWPV2-008 189 189 SWPV2-008 conserved hypothetical protein 100
ALPV-012 12765-12259 SWPV2-009 168 168 SWPV2-009 conserved hypothetical protein 100
ALPV-013 14555-13083 SWPV2-010 490 490 SWPV2-010 Ig-like domain protein 100
ALPV-014 14719-16368 SWPV2-011 549 528 SWPV2-011 ankyrin repeat protein 96.2 28.7 M1L
ALPV-015 16429-16935 SWPV2-012 168 168 SWPV2-012 C-type lectin-like protein 100 24.0 A40R
ALPV-016 17039-18478 SWPV2-013 479 479 SWPV2-013 ankyrin repeat protein 100 26.7 B4R
ALPV-017 19149-18577 SWPV2-014 190 190 SWPV2-014 IL-10-like protein 100
ALPV-018 20576-19266 SWPV2-015 436 436 SWPV2-015 ankyrin repeat protein 100 27.5 M1L
ALPV-019 20767-22026 SWPV2-016 419 419 SWPV2-016 ankyrin repeat protein 100 38.2 M1L
ALPV-020 23769-22162 SWPV2-017 535 535 SWPV2-017 ankyrin repeat protein 100 21.5 B4R
ALPV-021 24886-23810 SWPV2-018 358 358 SWPV2-018 putative serpin 100 27.9 C12L
ALPV-022 26242-24968 SWPV2-019 424 424 SWPV2-019 vaccinia C4L/C10L-like protein 100-
ALPV-023 26520-27056 SWPV2-020 178 178 SWPV2-020 hypothetical protein 100
ALPV-024 28172-27270 SWPV2-021 300 300 SWPV2-021 alpha-SNAP-like protein 100
ALPV-025 29443-28295 SWPV2-022 382 382 SWPV2-022 ankyrin repeat protein 100 22.0 C9L
ALPV-026 31392-29512 SWPV2-023 626 626 SWPV2-023 ankyrin repeat protein 100
ALPV-027 32608-31511 SWPV2-024 365 365 SWPV2-024 ankyrin repeat protein 100
ALPV-028 33146-32718 SWPV2-025 142 142 SWPV2-025 C-type lectin-like protein 100
ALPV-029 34224-33202 SWPV2-026 340 340 SWPV2-026 ankyrin repeat protein 100 26.8 B4R
ALPV-030 34487-34287 66 hypothetical protein, unique to ALPV, containing a transmembrane helix
ALPV-031 34467-34826 SWPV2-027 119 119 SWPV2-027 hypothetical protein 100
ALPV-032 35782-35054 SWPV2-028 242 242 SWPV2-028 Ig-like domain putative IFN-gamma binding protein 100
ALPV-033 36598-35858 SWPV2-029 246 246 SWPV2-029 Ig-like domain protein 100
ALPV-034 38673-36694 SWPV2-030 659 659 SWPV2-030 ankyrin repeat protein 100 23.5 K1L
ALPV-035 39392-38991 SWPV2-031 133 133 SWPV2-031 C-type lectin-like protein 100
ALPV-036 39776-39489 SWPV2-032 95 95 SWPV2-032 conserved hypothetical protein 100
ALPV-037 39840-40379 SWPV2-033 179 179 SWPV2-033 conserved hypothetical protein 100
ALPV-038 41625-40384 SWPV2-034 413 413 SWPV2-034 vaccinia C4L/C10L-like protein 99.8 24.3 C10L
ALPV-039 41743-42726 SWPV2-035 327 327 SWPV2-035 G protein-coupled receptor-like protein 100
ALPV-040 44520-42745 SWPV2-036 591 591 SWPV2-036 ankyrin repeat protein 99.7 23.2 B4R
ALPV-041 45885-44593 SWPV2-037 430 430 SWPV2-037 ankyrin repeat protein 100 27.7 B18R
ALPV-042 47751-45934 SWPV2-038 605 605 SWPV2-038 ankyrin repeat protein 100 21.6 B4R
ALPV-043 48462-47857 SWPV2-039 201 201 SWPV2-039 conserved hypothetical protein 100
ALPV-044 49946-48504 SWPV2-040 480 480 SWPV2-040 ankyrin repeat protein 100 25.2 B18R
ALPV-045 50212-51210 SWPV2-041 332 332 SWPV2-041 G protein-coupled receptor-like protein 100-
ALPV-046 52589-51237 SWPV2-042 450 450 SWPV2-042 ankyrin repeat protein 100 29.7 VACV-Cop-B4R
ALPV-047 53030-52656 SWPV2-043 124 124 SWPV2-043 conserved hypothetical protein 100
ALPV-048 55603-53198 SWPV2-044 801 801 SWPV2-044 alkaline phosphodiesterase-like protein 100
ALPV-049 56143-55691 SWPV2-045 150 150 SWPV2-045 hypothetical protein 100
ALPV-050 57255-56197 SWPV2-046 352 352 SWPV2-046 ankyrin repeat protein 100
ALPV-051 58528-57302 SWPV2-047 408 408 SWPV2-047 DNase II-like protein 100
ALPV-052 59069-58554 SWPV2-048 171 171 SWPV2-048 C-type lectin-like protein 100
ALPV-053 59689-59249 SWPV2-049 146 146 SWPV2-049 conserved hypothetical protein 100
ALPV-054 60104-59682 SWPV2-050 140 140 SWPV2-050 conserved hypothetical protein 100
ALPV-055 60647-60156 SWPV2-051 163 163 SWPV2-051 conserved hypothetical protein 100
ALPV-056 61081-60644 SWPV2-052 145 145 SWPV2-052 CNLV056 dUTPase 100 52.8 F2L
ALPV-057 62028-61108 SWPV2-053 306 306 SWPV2-053 putative serpin 100
ALPV-058 62601-62059 SWPV2-054 180 180 SWPV2-054 bcl-2 like protein 100
ALPV-059 63674-62658 SWPV2-055 338 338 SWPV2-055 putative serpin 100
ALPV-060 64555-63743 SWPV2-056 270 206 SWPV2-056 conserved hypothetical protein 99.5
ALPV-061 66342-64645 SWPV2-057 565 565 SWPV2-057 DNA ligase 100 46.9 A50R
ALPV-062 67433-66381 SWPV2-058 350 350 SWPV2-058 putative serpin 100 25.0 K2L
ALPV-063 68580-67504 SWPV2-059 358 358 SWPV2-059 hydroxysteroid dehydrogenase-like protein 100 38.2 A44L
ALPV-064 69493-68642 SWPV2-060 283 283 SWPV2-060 TGF-beta-like protein 100
ALPV-065 71324-69573 SWPV2-061 583 583 SWPV2-061 semaphorin-like protein 100 31.7 A39R
ALPV-066 71606-71424 SWPV2-062 60 399 SWPV2-062 hypothetical protein 100
ALPV-067 71608-71784 59 hypothetical protein, unique to ALPV
ALPV-068 72000-71728 SWPV2-062 90 399 SWPV2-062 hypothetical protein 90.8
ALPV-069 72255-72082 SWPV2-063 57 57 SWPV2-063 hypothetical protein 100
ALPV-070 72415-73188 SWPV2-064 257 257 SWPV2-064 GNS1/SUR4-like protein 100
ALPV-071 73281-73748 SWPV2-065 155 155 SWPV2-065 late transcription factor VLTF-2 100 46.6 A1L
ALPV-072 73765-75420 SWPV2-066 551 551 SWPV2-066 putative rifampicin resistance protein, IMV assembly 100 56.2 D13L
ALPV-073 75452-76321 SWPV2-067 289 289 SWPV2-067 mRNA capping enzyme small subunit 100 57.5 D12L
ALPV-074 76342-77283 SWPV2-068 313 132 SWPV2-068 CC chemokine-like protein 100
ALPV-075 77690-77361 SWPV2-069 109 109 SWPV2-069 hypothetical protein 100
ALPV-076 77761-79668 SWPV2-070 635 635 SWPV2-070 NPH-I, transcription termination factor 100 60.8 D11L
ALPV-077 80351-79665 SWPV2-071 228 228 SWPV2-071 mutT motif putative gene expression regulator 100 38.0 D10R
ALPV-078 81048-80335 SWPV2-072 237 232 SWPV2-072 mutT motif 97.9 46.3 D9R
ALPV-079 81601-81287 104 100 CNPV-077 hypothetical protein
ALPV-080 81796-82065 89 hypothetical protein, unique to ALPV
ALPV-081 82693-82448 81 hypothetical protein, unique to ALPV
ALPV-082 83172-82690 SWPV2-073 160 160 SWPV2-073 RNA polymerase subunit RPO18 100 57.5 D7R
ALPV-083 84332-83508 SWPV2-074 274 274 SWPV2-074 Ig-like domain protein 100
ALPV-084 86356-84455 SWPV2-075 633 633 SWPV2-075 early transcription factor small subunit VETFS 100 72.2 D6R
ALPV-085 87561-86560 SWPV2-076 333 334 SWPV2-076 Ig-like domain protein 98.8
ALPV-086 90271-87887 SWPV2-077 794 794 SWPV2-077 NTPase, DNA replication 100 54.3 D5R
ALPV-087 91091-90426 SWPV2-078 221 221 SWPV2-078 CC chemokine-like protein 100
ALPV-088 91830-91174 SWPV2-079 218 218 SWPV2-079 uracil DNA glycosylase 99.5 54.2 D4R
ALPV-089 92626-91871 SWPV2-080 251 303 SWPV2-080 putative RNA phosphatase 94.7 34.1 H1L
ALPV-090 93861-92674 395 71.9 SWPV1-075 conserved hypothetical protein
ALPV-091 93946-94311 SWPV2-081 121 112 SWPV2-081 TNFR-like protein 80.8 33.9 B28R
ALPV-092 96471-96866 SWPV2-082 131 131 SWPV2-082 putative glutathione peroxidase 100
ALPV-093 96891-97193 SWPV2-083 100 100 SWPV2-083 conserved hypothetical protein 100
ALPV-094 97677-97198 SWPV2-084 159 159 SWPV2-084 conserved hypothetical protein 100
ALPV-095 98047-97664 SWPV2-085 127 127 SWPV2-085 conserved hypothetical protein 100
ALPV-096 98384-98133 SWPV2-086 83 83 SWPV2-086 HT motif protein 100
ALPV-097 99122-98736 SWPV2-087 128 146 SWPV2-087 conserved hypothetical protein 87.7
ALPV-098 100027-99224 SWPV2-088 267 267 SWPV2-088 virion protein 100
ALPV-099 100102-100929 SWPV2-089 275 275 SWPV2-089 T10-like protein 100
ALPV-100 101074-100937 SWPV2-090 45 45 SWPV2-090 conserved hypothetical protein 100
ALPV-101 101313-101056 SWPV2-091 85 85 SWPV2-091 ubiquitin 100
ALPV-102 102422-101445 SWPV2-092 325 339 SWPV2-092 conserved hypothetical protein 95.9
ALPV-103 102692-102450 SWPV2-093 80 80 SWPV2-093 hypothetical protein 100
ALPV-104 103285-102698 SWPV2-094 195 195 SWPV2-094 beta-NGF-like protein 100
ALPV-105 103815-103309 SWPV2-095 168 168 SWPV2-095 putative interleukin binding protein 100
ALPV-106 104127-103870 SWPV2-096 85 85 SWPV2-096 hypothetical protein 100
ALPV-107 104455-104138 SWPV2-097 105 105 SWPV2-097 conserved hypothetical protein 100
ALPV-108 105044-104472 SWPV2-098 190 190 SWPV2-098 N1R/p28-like protein 100
ALPV-109 105246-105623 SWPV2-099 125 125 SWPV2-099 putative glutaredoxin 2, virion morphogenesis 100 32.2 G4L
ALPV-110 106270-105566 SWPV2-100 234 234 SWPV2-100 putative elongation factor 100
ALPV-111 106264-106572 SWPV2-101 102 102 SWPV2-101 conserved hypothetical protein 100
ALPV-112 106708-106941 SWPV2-102 77 77 SWPV2-102 hypothetical protein 100
ALPV-113 107186-109084 SWPV2-103 632 632 SWPV2-103 putative metalloprotease, virion morphogenesis 100 43.9 G1L
ALPV-114 111113-109068 SWPV2-104 681 681 SWPV2-104 NPH-II, RNA helicase 100 43.8 I8R
ALPV-115 111148-112416 SWPV2-105 422 422 SWPV2-105 virion core proteinase 100 54.4 I7L
ALPV-116 112421-113596 SWPV2-106 391 391 SWPV2-106 DNA-binding protein 100 34.8 I6L
ALPV-117 113597-113842 SWPV2-107 81 81 SWPV2-107 putative IMV membrane protein 100
ALPV-118 113864-114403 SWPV2-108 179 179 SWPV2-108 thymidine kinase 100 52.0 J2R
ALPV-119 114524-114772 SWPV2-109 82 82 SWPV2-109 HT motif protein 100
ALPV-120 114842-115711 SWPV2-110 289 289 SWPV2-110 DNA-binding phosphoprotein 99.7 33.5 I3L
ALPV-121 115712-115921 SWPV2-111 69 69 SWPV2-111 conserved hypothetical protein 100
ALPV-122 115928-116860 SWPV2-112 310 310 SWPV2-112 DNA-binding virion protein 100 58.0 I1L
ALPV-123 117040-118998 SWPV2-113 652 652 SWPV2-113 conserved hypothetical protein 100 20.9 O1L
ALPV-124 118928-119323 SWPV2-114 131 131 SWPV2-114 virion core protein 100 34.98 E11L
ALPV-125 119601-119320 SWPV2-115 93 93 SWPV2-115 putative IMV redox protein, virus assembly 100 51.6 E10R
ALPV-126 119628-122594 SWPV2-116 988 988 SWPV2-116 DNA polymerase 100 50.3 E9L
ALPV-127 123413-122586 275 80.4 48.2 E8R SWPV1-111 putative membrane protein
ALPV-128 125130-123415 SWPV2-117 571 502 SWPV2-117 conserved hypothetical protein 87.2 49.9 E6R
ALPV-129 130930-125192 SWPV2-118 1912 1916 SWPV2-118 variola B22R-like protein 99.8
ALPV-130 136264-130997 SWPV2-119 1755 1767 SWPV2-119 variola B22R-like protein 99.3
ALPV-131 142243-136544 SWPV2-120 1899 1839 SWPV2-120 variola B22R-like protein 95.5
ALPV-132 142444-142992 SWPV2-122 182 182 SWPV2-122 RNA polymerase subunit RPO30 100 57.0 E4L
ALPV-133 143024-145189 SWPV2-123 721 721 SWPV2-123 conserved hypothetical protein 100 28.0 E2L
ALPV-134 145182-146600 SWPV2-124 472 472 SWPV2-124 poly(A) polymerase large subunit PAPL 100 50.5 E1L
ALPV-135 146953-146594 SWPV2-125 119 119 SWPV2-125 DNA-binding virion core protein 100 38.8 F17R
ALPV-136 147029-147652 SWPV2-126 207 207 SWPV2-126 conserved hypothetical protein 100
ALPV-137 147746-148192 SWPV2-127 148 148 SWPV2-127 conserved hypothetical protein 100 40.4 F15L
ALPV-138 148426-148725 SWPV2-128 99 99 SWPV2-128 conserved hypothetical protein 100
ALPV-139 154234-148796 SWPV2-129 1812 1801 SWPV2-129 variola B22R-like protein 99.4
ALPV-140 154384-155520 SWPV2-130 378 378 SWPV2-130 putative palmitoylated EEV envelope lipase 100 38.0 F13L
ALPV-141 155598-157475 SWPV2-131 625 625 SWPV2-131 putative EEV maturation protein 100 26.3 F12L
ALPV-142 157518-158906 SWPV2-132 462 462 SWPV2-132 conserved hypothetical protein 100 27.0 F11L
ALPV-143 158997-160331 SWPV2-133 444 444 SWPV2-133 putative serine/threonine protein kinase, virus assembly 100 53.6 F10L
ALPV-144 160306-160947 SWPV2-134 213 213 SWPV2-134 conserved hypothetical protein 100 31.5 F9L
ALPV-145 161030-161230 SWPV2-135 66 66 SWPV2-135 conserved hypothetical protein 100
ALPV-146 161556-162110 SWPV2-136 184 184 SWPV2-136 HAL3-like domain protein 100
ALPV-147 162371-163336 SWPV2-137 321 321 SWPV2-137 N1R/p28-like protein 100
ALPV-148 163448-165463 SWPV2-138 671 671 SWPV2-138 ankyrin repeat protein 100 26.0 M1L
ALPV-149 165489-167159 SWPV2-139 556 556 SWPV2-139 ankyrin repeat protein 100 26.1 B4R
ALPV-150 167380-168702 SWPV2-140 440 440 SWPV2-140 conserved hypothetical protein 100 33.3 G5R
ALPV-151 168710-168898 SWPV2-141 62 62 SWPV2-141 RNA polymerase subunit RPO7 98.4 55.2 G5.5R
ALPV-152 168891-169457 SWPV2-142 188 188 SWPV2-142 conserved hypothetical protein 100 31.8 G6R
ALPV-153 170468-169422 SWPV2-143 348 348 SWPV2-143 virion core protein 100 34.6 G7L
ALPV-154 171554-170634 SWPV2-144 306 306 SWPV2-144 putative thioredoxin binding protein 100
ALPV-155 171682-171915 77 97.1 CNPV-150 ankyrin repeat protein
ALPV-156 173269-172031 SWPV2-145 412 412 SWPV2-145 ankyrin repeat protein 100 42.9 M1L
ALPV-157 173945-173496 SWPV2-146 149 149 SWPV2-146 hypothetical protein 100
ALPV-158 175111-174173 SWPV2-147 312 312 SWPV2-147 Rep-like protein 100
ALPV-159 181355-175545 SWPV2-148 1936 875 SWPV2-148 variola B22R-like protein 98.2
ALPV-160 186885-181408 SWPV2-149 1825 1831 SWPV2-149 variola B22R-like protein 99.7
ALPV-161 187202-189685 SWPV2-150 827 834 SWPV2-150 hypothetical protein 90.5
ALPV-162 190813-189782 SWPV2-151 343 343 SWPV2-151 TGF-beta-like protein 100
ALPV-163 190815-191294 159 64.7 CNPV-157 TGF-beta-like protein
ALPV-164 191763-192263 SWPV2-150 166 834 SWPV2-150 hypothetical protein 47.8
ALPV-165 192733-193446 237 97.2 CNPV-159 N1R/p28-like protein
ALPV-166 193490-194494 334 91.3 CNPV-169 N1R/p28-like protein
ALPV-167 194549-195412 287 78.7 CNPV-169 N1R/p28-like protein
ALPV-168 195417-195821 134 98.5 CNPV-160 N1R/p28-like protein
ALPV-169 197008-195920 SWPV2-152 362 358 SWPV2-152 TGF-beta-like protein 98.9
ALPV-170 197058-197507 SWPV2-153 149 149 SWPV2-153 TGF-beta-like protein 100
ALPV-171 197843-198883 SWPV2-154 346 320 SWPV2-154 N1R/p28-like protein 95.3
ALPV-172 199118-200155 SWPV2-155 345 345 SWPV2-155 Ig-like domain protein 99.7
ALPV-173 200427-200933 SWPV2-156 168 168 SWPV2-156 Ig-like domain protein 97
ALPV-174 201028-202059 SWPV2-157 343 350 SWPV2-157 N1R/p28-like protein 93.6
ALPV-175 202011-202415 134 92.5 CNPV-226 N1R/p28-like protein
ALPV-176 202488-203126 SWPV2-158 212 212 SWPV2-158 thymidylate kinase 100 45.2 A48R
ALPV-177 203179-203961 SWPV2-159 260 260 SWPV2-159 late transcription factor VLTF-1 100 66.2 G8R
ALPV-178 203975-204982 SWPV2-160 335 335 SWPV2-160 putative myristylated protein 100 38.0 G9R
ALPV-179 204983-205714 SWPV2-161 243 243 SWPV2-161 putative myristylated IMV envelope protein 100 54.7 L1R
ALPV-180 205774-206064 SWPV2-162 96 96 SWPV2-162 conserved hypothetical protein 100
ALPV-181 206965-206054 SWPV2-163 303 303 SWPV2-163 conserved hypothetical protein 100 42.0 L3L
ALPV-182 206991-207749 SWPV2-164 252 252 SWPV2-164 DNA-binding virion core protein 100 36.3 L4R
ALPV-183 207750-208142 SWPV2-165 130 130 SWPV2-165 conserved hypothetical protein 100 41.6 L5R
ALPV-184 208096-208542 SWPV2-166 148 148 SWPV2-166 putative IMV membrane protein 100 42.5 J1R
ALPV-185 208576-209484 SWPV2-167 302 302 SWPV2-167 poly(A) polymerase small subunit PAPS 100 56.0 J3R
ALPV-186 209481-210041 SWPV2-168 186 186 SWPV2-168 RNA polymerase subunit RPO22 100 55.0 J4R
ALPV-187 210444-210034 SWPV2-169 136 136 SWPV2-169 conserved hypothetical protein 100 47.9 J5L
ALPV-188 210487-214353 SWPV2-170 1288 1288 SWPV2-170 RNA polymerase subunit RPO147 100 70.6 J6R
ALPV-189 214856-214356 SWPV2-171 166 166 SWPV2-171 putative protein-tyrosine phosphatase, virus assembly 100 47.6 H1L
ALPV-190 214872-215441 SWPV2-172 189 189 SWPV2-172 conserved hypothetical protein 100 48.9 H2R
ALPV-191 216503-215517 SWPV2-173 328 328 SWPV2-173 ankyrin repeat protein 100
ALPV-192 217538-216546 SWPV2-174 330 330 SWPV2-174 putative IMV envelope protein 100 32.2 H3L
ALPV-193 220029-217630 SWPV2-175 799 799 SWPV2-175 RNA polymerase associated protein RAP94 100 55.2 H4L
ALPV-194 220198-220710 SWPV2-176 170 170 SWPV2-176 late transcription factor VLTF-4 100
ALPV-195 220711-221661 SWPV2-177 316 316 SWPV2-177 DNA topoisomerase 100 58.0 H6R
ALPV-196 221666-222127 SWPV2-178 153 153 SWPV2-178 conserved hypothetical protein 100 33.8 H7R
ALPV-197 222401-222090 SWPV2-179 103 103 SWPV2-179 conserved hypothetical protein 100
ALPV-198 222409-224949 SWPV2-180 846 846 SWPV2-180 mRNA capping enzyme large subunit 100 53.9 D1R
ALPV-199 225020-225340 SWPV2-181 106 106 SWPV2-181 HT motif protein 100
ALPV-200 225759-225337 SWPV2-182 140 140 SWPV2-182 virion protein 100
ALPV-201 225813-226247 SWPV2-183 144 144 SWPV2-183 hypothetical protein 100
ALPV-202 226312-226884 SWPV2-184 190 190 SWPV2-184 conserved hypothetical protein 100
ALPV-203 226950-227777 SWPV2-185 275 275 SWPV2-185 N1R/p28-like protein 100
ALPV-204 228314-227844 SWPV2-186 156 156 SWPV2-186 C-type lectin-like protein 100 30.8 A34R
ALPV-205 228622-229299 SWPV2-187 225 225 SWPV2-187 deoxycytidine kinase-like protein 100
ALPV-206 229305-229805 SWPV2-188 166 166 SWPV2-188 Rep-like protein 99.4
ALPV-207 229864-230367 SWPV2-189 167 167 SWPV2-189 conserved hypothetical protein 100
ALPV-208 230421-231251 SWPV2-190 276 276 SWPV2-190 N1R/p28-like protein 100
ALPV-209 231324-232472 SWPV2-191 382 382 SWPV2-191 N1R/p28-like protein 100
ALPV-210 232528-232713 SWPV2-192 61 61 SWPV2-192 conserved hypothetical protein 100
ALPV-211 232932-233888 SWPV2-193 318 318 SWPV2-193 N1R/p28-like protein 100
ALPV-212 233949-235367 SWPV2-194 472 472 SWPV2-194 putative photolyase 100
ALPV-213 235424-235579 51 hypothetical protein, unique to ALPV, containing a transmembrane helix
ALPV-214 235557-236078 SWPV2-195 173 173 SWPV2-195 N1R/p28-like protein 100
ALPV-215 236184-236723 SWPV2-196 179 200 SWPV2-196 conserved hypothetical protein 89
ALPV-216 236767-237699 SWPV2-197 310 310 SWPV2-197 N1R/p28-like protein 100
ALPV-217 237747-238142 SWPV2-198 131 131 SWPV2-198 N1R/p28-like protein 100
ALPV-218 238197-238361 SWPV2-199 54 54 SWPV2-199 conserved hypothetical protein 100
ALPV-219 238421-238951 SWPV2-200 176 176 SWPV2-200 N1R/p28-like protein 100
ALPV-220 239662-239012 SWPV2-201 216 216 SWPV2-201 deoxycytidine kinase-like protein 100
ALPV-221 239836-240906 SWPV2-202 356 356 SWPV2-202 vaccinia C4L/C10L-like protein 100 28.1 C10L
ALPV-222 241181-241795 SWPV2-203 204 204 SWPV2-203 CC chemokine-like protein 100
ALPV-223 241885-243090 SWPV2-204 401 401 SWPV2-204 conserved hypothetical protein 100
ALPV-224 243204-244196 SWPV2-205 330 330 SWPV2-205 N1R/p28-like protein 100
ALPV-225 244284-245555 SWPV2-206 423 223 SWPV2-206 N1R/p28-like protein 99.5
ALPV-226 245795-245619 58 hypothetical protein, unique to ALPV, containing a transmembrane helix
ALPV-227 246013-245858 51 hypothetical protein, unique to ALPV, containing a transmembrane helix
ALPV-228 246363-247412 SWPV2-207 349 349 SWPV2-207 N1R/p28-like protein 100
ALPV-229 247466-248308 280 80.9 SWPV1-198 N1R/p28-like protein
ALPV-230 248847-248644 SWPV2-208 67 85 SWPV2-208 N1R/p28-like protein 62.7
ALPV-231 249295-249483 SWPV2-209 62 213 SWPV2-209 N1R/p28-like protein 100
ALPV-232 250029-250292 SWPV2-210 87 285 SWPV2-210 N1R/p28-like protein 100
ALPV-233 250283-250615 110 98.2 CNPV-227 N1R/p28-like protein
ALPV-234 253677-251134 SWPV2-211 847 847 SWPV2-211 ankyrin repeat protein 99.9 31.6 B4R
ALPV-235 253931-254650 SWPV2-212 239 239 SWPV2-212 hypothetical protein 100
ALPV-236 254650-255027 125 73.3 CNPV-227 N1R/p28-like protein
ALPV-237 255062-255346 SWPV2-214 94 126 SWPV2-214 N1R/p28-like protein 96.4
ALPV-238 257103-255799 SWPV2-215 434 434 SWPV2-215 ankyrin repeat protein 100 27.3 B4R
ALPV-239 257301-257498 SWPV2-216 65 65 SWPV2-216 hypothetical protein 100
ALPV-240 257446-257922 SWPV2-217 158 158 SWPV2-217 MyD116-like domain protein 100
ALPV-241 257952-258566 SWPV2-218 204 204 SWPV2-218 CC chemokine-like protein 98
ALPV-242 258748-260163 SWPV2-219 471 471 SWPV2-219 ankyrin repeat protein 100 31.4 M1L
ALPV-243 260183-261709 SWPV2-220 508 508 SWPV2-220 ankyrin repeat protein 100 37.0 M1L
ALPV-244 261780-263084 SWPV2-221 434 432 SWPV2-221 conserved hypothetical protein 99.5
ALPV-245 263129-264100 SWPV2-222 323 323 SWPV2-222 ribonucleotide reductase small subunit 100 70.9 F4L
ALPV-246 264281-265606 SWPV2-223 441 441 SWPV2-223 ankyrin repeat protein 99.8 33.3 B4R
ALPV-247 266342-265665 SWPV2-224 225 225 SWPV2-224 late transcription factor VLTF-3 100 76.7 A2L
ALPV-248 266557-266330 SWPV2-225 75 75 SWPV2-225 virion redox protein 100
ALPV-249 268550-266571 SWPV2-226 659 659 SWPV2-226 virion core protein P4b 100 54.1 A3L
ALPV-250 269284-268637 SWPV2-227 215 215 SWPV2-227 immunodominant virion protein 100
ALPV-251 269323-269832 SWPV2-228 169 169 SWPV2-228 RNA polymerase subunit RPO19 100 55.2 A5R
ALPV-252 270948-269827 SWPV2-229 373 373 SWPV2-229 conserved hypothetical protein 100 39.3 A6L
ALPV-253 273084-270955 SWPV2-230 709 709 SWPV2-230 early transcription factor large subunit VETFL 100 59.6 A7L
ALPV-254 273148-274050 SWPV2-231 300 300 SWPV2-231 intermediate transcription factor VITF-3 100 38.7 A8R
ALPV-255 274242-274015 SWPV2-232 75 75 SWPV2-232 putative IMV membrane protein 100
ALPV-256 276924-274243 SWPV2-233 893 893 SWPV2-233 virion core protein P4a 100 39.6 A10L
ALPV-257 276942-277781 SWPV2-234 279 279 SWPV2-234 conserved hypothetical protein 100 39.5 A11R
ALPV-258 278284-277778 SWPV2-235 168 168 SWPV2-235 virion protein 99.4 32.9 A12L
ALPV-259 278299-278556 SWPV2-236 85 56 SWPV2-236 conserved hypothetical protein 87.0
ALPV-260 278754-278545 SWPV2-237 69 69 SWPV2-237 putative IMV membrane protein 100
ALPV-261 279080-278802 SWPV2-238 92 92 SWPV2-238 putative IMV membrane protein 100 27.5 A14L
ALPV-262 279258-279097 SWPV2-239 53 53 SWPV2-239 putative IMV membrane virulence factor 100 35.3 A 14.5L
ALPV-263 279564-279274 SWPV2-240 96 96 SWPV2-240 conserved hypothetical protein 100
ALPV-264 280654-279548 SWPV2-241 368 368 SWPV2-241 predicted myristylated protein 100 43.0 A16L
ALPV-265 281248-280670 SWPV2-242 192 192 SWPV2-242 putative phosphorylated IMV membrane protein 100 33.2 A17L
ALPV-266 281266-282654 SWPV2-243 462 462 SWPV2-243 DNA helicase, transcriptional elongation 100 50.2 A18R
ALPV-267 282891-282622 SWPV2-244 89 89 SWPV2-244 conserved hypothetical protein 100 42.9 A19L
ALPV-268 283237-282899 SWPV2-245 112 112 SWPV2-245 conserved hypothetical protein 100 45.3 A21L
ALPV-269 283236-284540 SWPV2-246 434 434 SWPV2-246 DNA polymerase processivity factor 100 25.9 A20R
ALPV-270 284537-284995 SWPV2-247 152 152 SWPV2-247 Holliday junction resolvase protein 100 45.0 A22R
ALPV-271 285012-286163 SWPV2-248 383 383 SWPV2-248 intermediate transcription factor VITF-3 100 51.3 A23R
ALPV-272 286189-289662 SWPV2-249 1157 1157 SWPV2-249 RNA polymerase subunit RPO132 100 74.6 A24R
ALPV-273 291456-289651 SWPV2-250 601 601 SWPV2-250 A type inclusion-like protein 100
ALPV-274 292918-291491 SWPV2-251 475 475 SWPV2-251 A type inclusion-like/fusion protein 100 56.4 A27L
ALPV-275 293341-292919 SWPV2-252 140 140 SWPV2-252 conserved hypothetical protein 100 41.8 A28L
ALPV-276 294263-293346 SWPV2-253 305 305 SWPV2-253 RNA polymerase subunit RPO35 100 42.6 A29L
ALPV-277 294465-294238 SWPV2-254 75 75 SWPV2-254 conserved hypothetical protein 100
ALPV-278 294590-294931 SWPV2-255 113 113 SWPV2-255 conserved hypothetical protein 100 31.6 A31R
ALPV-279 294940-295302 SWPV2-256 120 120 SWPV2-256 conserved hypothetical protein 100
ALPV-280 296145-295291 SWPV2-257 284 284 SWPV2-257 DNA packaging protein 100 47.1 A32L
ALPV-281 296260-296805 SWPV2-258 181 181 SWPV2-258 C-type lectin-like EEV protein 100
ALPV-282 297030-297854 SWPV2-259 274 274 SWPV2-259 conserved hypothetical protein 100
ALPV-283 297914-298723 SWPV2-260 269 269 SWPV2-260 putative tyrosine protein kinase 100
ALPV-284 298766-299782 SWPV2-261 338 338 SWPV2-261 putative serpin 100
ALPV-285 300562-299804 SWPV2-262 252 252 SWPV2-262 conserved hypothetical protein 100
ALPV-286 300672-301604 SWPV2-263 310 310 SWPV2-263 G protein-coupled receptor-like protein 100
ALPV-287 301615-301905 SWPV2-264 96 96 SWPV2-264 conserved hypothetical protein 100
ALPV-288 301971-302501 SWPV2-265 176 169 SWPV2-265 beta-NGF-like protein 96
ALPV-289 302911-302519 SWPV2-266 130 130 SWPV2-266 HT motif protein 100
ALPV-290 303015-303659 SWPV2-267 214 214 SWPV2-267 conserved hypothetical protein 100
ALPV-291 304032-303670 SWPV2-268 120 120 SWPV2-268 HT motif protein 100
ALPV-292 304198-304533 SWPV2-269 111 111 SWPV2-269 CC chemokine-like protein 100
ALPV-293 304612-305193 SWPV2-270 193 193 SWPV2-270 putative interleukin binding protein 100
ALPV-294 305303-305683 SWPV2-271 126 126 SWPV2-271 EGF-like protein 100 34.8 C11R
ALPV-295 305685-306602 SWPV2-272 305 305 SWPV2-272 putative serine/threonine protein kinase 100 41.1 B1R
ALPV-296 306645-307127 SWPV2-273 160 160 SWPV2-273 conserved hypothetical protein 100
ALPV-297 307210-307677 SWPV2-274 155 147 SWPV2-274 C-type lectin-like protein 99.3 22.6 A34R
ALPV-298 307720-308139 SWPV2-275 139 139 SWPV2-275 putative interleukin binding protein 100
ALPV-299 308208-308435 SWPV2-276 75 75 SWPV2-276 conserved hypothetical protein 100
ALPV-300 308637-310421 SWPV2-277 594 594 SWPV2-277 ankyrin repeat protein 99.8 22.3 B4R
ALPV-301 310445-310669 SWPV2-278 74 74 SWPV2-278 hypothetical protein 100
ALPV-302 310712-311566 SWPV2-279 284 284 SWPV2-279 ankyrin repeat protein 99.6 33.9 B24R
ALPV-303 311621-312913 SWPV2-280 430 430 SWPV2-280 ankyrin repeat protein 99.8 C18L
ALPV-304 313106-314296 SWPV2-281 396 396 SWPV2-281 ankyrin repeat protein 100 28.6 M1L
ALPV-305 314299-315675 SWPV2-282 458 458 SWPV2-282 ankyrin repeat protein 100 32.5 B4R
ALPV-306 315784-317997 SWPV2-283 737 737 SWPV2-283 ankyrin repeat protein 100 25.8 M1L
ALPV-307 318053-319768 SWPV2-284 571 571 SWPV2-284 ankyrin repeat protein 100 28.8 B4R
ALPV-308 319772-320674 SWPV2-285 300 300 SWPV2-285 putative serine/threonine protein kinase 100 24.6 M1L
ALPV-309 320747-321481 SWPV2-286 244 244 SWPV2-286 ankyrin repeat protein 100 32.8 B1R
ALPV-310 322082-323665 SWPV2-287 527 527 SWPV2-287 ankyrin repeat protein 100 23.7 M1L
ALPV-311 324261-323680 SWPV2-288 193 193 SWPV2-288 conserved hypothetical protein 100 26.1 B18R
ALPV-312 324329-325831 SWPV2-289 500 500 SWPV2-289 ankyrin repeat protein 100
ALPV-313 326047-327447 SWPV2-290 466 466 SWPV2-290 ankyrin repeat protein 100 28.3 M1L
ALPV-314 327518-328306 SWPV2-291 262 262 SWPV2-291 N1R/p28-like protein 100 28.2 B4R
ALPV-315 328368-328586 SWPV2-292 72 72 SWPV2-292 hypothetical protein 100
ALPV-316 329054-328590 SWPV2-293 154 154 SWPV2-293 C-type lectin-like protein 100
ALPV-317 329231-330304 SWPV2-294 357 357 SWPV2-294 ankyrin repeat protein 100 34.4 A40R
ALPV-318 330452-331042 SWPV2-295 196 196 SWPV2-295 ankyrin repeat protein 100
ALPV-319 331147-332760 SWPV2-296 537 537 SWPV2-296 ankyrin repeat protein 100 37.4 M1L
ALPV-320 332794-333168 SWPV2-297 124 124 SWPV2-297 EFc-like protein 100
ALPV-321 333178-333678 SWPV2-298 166 166 SWPV2-298 conserved hypothetical protein 100
ALPV-322 333750-334406 SWPV2-299 218 218 SWPV2-299 Ig-like domain protein 100
ALPV-323 334433-336322 SWPV2-300 629 629 SWPV2-300 ankyrin repeat protein 99.8
ALPV-324 336421-337368 SWPV2-301 315 315 SWPV2-301 G protein-coupled receptor-like protein 100 40.8 B4R
ALPV-325 337435-339069 SWPV2-302 544 544 SWPV2-302 ankyrin repeat protein 99.8
ALPV-326 339250-339417 SWPV2-303 55 55 SWPV2-303 hypothetical protein 100 33.9 B4R
ALPV-327 339592-341124 SWPV2-304 510 514 SWPV2-304 ankyrin repeat protein 99.2
ALPV-328 341457-343424 SWPV2-305 655 637 SWPV2-305 ankyrin repeat protein 96.6 36.8 M1L
ALPV-329 343616-345025 SWPV2-306 469 469 SWPV2-306 Ig-like domain protein 100 25.8 M1L
ALPV-330 345157-345531 SWPV2-307 124 124 SWPV2-307 EFc-like protein 100
ALPV-331 345865-347868 SWPV2-308 667 689 SWPV2-308 ankyrin repeat protein 96.5
ALPV-332 348446-347985 SWPV2-309 153 186 SWPV2-309 conserved hypothetical protein 86.9 identical to ALPV-005
ALPV-333 349231-348563 SWPV2-310 222 222 SWPV2-310 conserved hypothetical protein 100 identical to ALPV-004
ALPV-334 349357-349584 75 56.1 SWPV1-002 C-type lectin-like protein, identical to ALPV-003
ALPV-335 349639-350265 SWPV2-311 208 208 SWPV2-311 C-type lectin-like protein 100 identical to ALPV-002
ALPV-336 351083-350568 SWPV2-312 171 171 SWPV2-312 hypothetical protein 100 identical to ALPV-001
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Note: ALPV, albatrosspox virus (GenBank accession no. MW365933); SWPV1, shearwaterpox virus 1 (GenBank accession no. KX857216); SWPV2, shearwaterpox virus 2 (GenBank accession no. KX857215); CNPV, canarypox virus (GenBank accession no. AY318871). Avipoxviruses as being identity to SWPV2 unless indicated in the note column. Truncated or fragmented ORFs of ALPV compared to SWPV2 are highlighted in italic font.

Interestingly, ALPV contained seven predicted protein-coding genes (ORF030, -067, -080, -081, -213, -226 and -227) that were not present in any other characterised poxvirus genomes, nor did they match any sequences in the NR protein database using BLASTX and BLASTP; these unique ORFs encoded proteins of 51 to 89 amino acids in length (Table 2). Furthermore, four of these unique protein-coding genes (ALPV-ORF030, -213, -226 and -227) were predicted to contain a single transmembrane helix (TMH) using the software packages employed in this study (Table 2). However, we did not find any known motif, nor significant homology with known proteins, for the unique ORFs encoded in the ALPV genome when using the Phyre2, HHpred and SWISS-MODEL, which might be due to the lack of closely related structures in the database.

Comparison of the ALPV genome to that of other avipoxviruses was performed using dot plot analyses. The ALPV genome was shown to be highly syntenic with SWPV2, PEPV2, CNPV and MLPV (Figure 2A–D) and demonstrated significant differences compared to SWPV1, PEPV, FGPV and TKPV (black and orange arrows, Figure 2E–H).

Figure 2.

Figure 2

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Dot plots of the ALPV genome (x-axis) vs. other poxvirus genomes (y-axis). (A) ALPV vs SWPV2, (B) ALPV vs PEPV2, (C) ALPV vs CNPV, (D) ALPV vs MLPV, (E) ALPV vs SWPV1, (F) ALPV vs PEPV, (G) ALPV vs FGPV and (H) ALPV vs TKPV (refer to Table 2 for virus details and GenBank accession numbers). The Classic colour scheme was chosen in Geneious (version 10.2.2) for the dot plot lines according to the length of the match, from blue for short matches to red for matches over 100 bp long. Window size = 12.

2.3. Evolutionary Relationships of ALPV

Phylogenetic analysis using concatenated amino acid sequences of the selected nine core poxvirus proteins supported the inclusion of the newly assembled ALPV in the genus Avipoxvirus. In the maximum likelihood (ML) tree, ALPV was located within a sub-clade comprising SWPV2, PEPV2 and CNPV with strong bootstrap support (100%) (Figure 3), suggesting that it may represent an ancient evolutionary lineage within the genus. Using the same set of concatenated protein sequences, we found that the maximum inter-lineage sequence identity values were 100% among ALPV, SWPV2 and PEPV2, which mirrored the phylogenetic position of this novel avipoxvirus sequenced from an endangered northern royal albatross. A large number of poxviruses were positioned in the phylogenetic tree when we used partial nucleotide sequences of the DNA polymerase gene (Supplementary Figure S1) and p4b gene (Supplementary Figure S2). We discovered that several other avipoxviruses were represented within the ALPV, PEPV2, CNPV, SWPV2, MPPV and MLPV clade. This included a poxvirus isolated from a common bullfinch (Pyrrhula pyrrhula) in Belgium [35] and a northern harrier (Circus cyaneus) in Spain [35], which is almost identical to ALPV within this relatively small fragment of the genome.

Figure 3.

Figure 3

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Phylogenetic relationships between ALPV and other chordopoxviruses. A maximum likelihood (ML) tree was constructed from multiple alignments of the concatenated amino acid sequences of the selected nine poxvirus core proteins using CLC Genomic Workbench (version 9.5.4, CLC bio, a QIAGEN Company, Prismet, Aarhus C, Denmark). The numbers on the left show bootstrap values as percentages. The ML tree is displayed as a phylogram. The labels at branch tips refer to original ChPV GenBank accession numbers followed by abbreviated species names. Saltwater crocodilepox virus (SwCRV1) [36] was used as an outgroup. The position of the novel ALPV is highlighted using a purple box and the subclade relevant to ALPV is shown with pink shading.

3. Discussion

This study presents the characterisation of the complete genome sequence of a novel avipoxvirus, ALPV, isolated from cutaneous pox lesions in a juvenile northern royal albatross. In addition to the highest number of genes being homologous to SWPV2, a further ten were homologs to CNPV and six to SWPV1. An additional seven genes were not present in any other known poxvirus, nor did they match any sequences in the NR protein database. Given this genome structure, gene content, genome nucleotide similarities and phylogenetic relationships, the authors postulate that the ALPV genome is most closely related to avipoxviruses isolated from shearwater, penguin and canary bird species.

It has been reported that an avipoxvirus may have caused the death of some northern royal albatrosses in 1997 [37]. However, as far as we are aware, there have been no further scientific studies investigating the epidemiology and characteristics of avipoxviruses circulating in this population. Moreover, avipoxviruses have been reported to be a significant cause of chick mortality in several other albatross species. For instance, an avipoxvirus spread by bird fleas has caused high chick mortalities in some seasons within colonies of shy albatrosses (Thalassarche cauta) in Tasmania [38] and may act as a potential threat to adults and chicks of Buller’s albatross (Thalassarche bulleri) [37]. An avipoxvirus has also been described as a key threat for the rapid population decline of a critically endangered seabird, the waved Albatross (Phoebastria irrorata), in the Galápagos Islands, Ecuador [39]. Given the conservation status of the northern royal albatross, it would be important to improve understanding of the epidemiology, transmission and genetic diversity of circulating avipoxviruses, including ALPV, and the threat that they pose to this and other albatross species.

Identifying the transmission mode is essential to characterise incidence, ecology, and effective control of disease in wild populations. However, it is not yet known how this avipoxvirus is transmitted. Mechanical transmission by biting arthropods is thought to play a role in the transmission of avipoxviruses within wild bird populations. Ticks, fleas [40], hippoboscid flies [41], and mosquitos [20,42] are all potential mechanical vectors. It would therefore seem likely that, as for other avipoxviruses, transmission of ALPV in the northern royal albatross is also mediated by insect vectors. Moreover, poxvirus infection can also occur through ingestion, parenteral inoculation, or droplet or aerosol exposure to mucous membranes or broken skin. Some poxviruses can be transmitted by fomites (inanimate objects) [43]. For example, studies have revealed that sheeppox and goatpox viruses are predominantly transmitted via aerosols [44]; whereas poxviruses from the genus Parapoxvirus can pass from one animal to another through direct or indirect contact. However, unfortunately, there are no available studies addressing whether closely related avipoxviruses employ similar or different routes of spread.

There are a number of factors that threaten the populations of large sea birds, particularly albatrosses. Amongst these are longline fishing, climate change and diseases such as those caused by avipoxviruses. It is well-established in the literature that avipoxviruses are mechanically transmitted by biting insects. Although yet to be confirmed, it is therefore expected that this will also be the case for ALPV.

4. Materials and Methods

4.1. Sampling and Virus Isolation

Cutaneous pox lesions were collected from an endangered juvenile northern royal albatross (Diomedea sanfordi), located on the Otago Peninsula, near Dunedin, on the South Island of New Zealand. Sampling was conducted in March 1997 by Wallaceville Animal Research Centre, New Zealand, and lesions sent to the Australian Animal Health Laboratory, Geelong, Victoria, Australia (sample ID: SL 08/05/1997). Virus isolation was undertaken by homogenisation of the tissue samples (~10% w/v) in the presence of antibiotics. This material was then inoculated onto the chorioallantoic membranes (CAMs) of 10 to 12 day old embryonated chicken eggs. The CAMs were harvested 3 to 5 days later and examined for the presence of pock lesions. The infected CAMs were similarly homogenised and passaged onto fresh CAMs. Similarly, the homogenised tissue samples were inoculated onto monolayers of chicken embryo skin cells and examined for 7 to 10 days for the development of cytopathic effect. Additional passages were undertaken with frozen and thawed tissue culture cells inoculated onto fresh chicken embryo skin cells. All passages were stored frozen at −80 °C.

4.2. DNA Extraction and Sequencing

Infected cell culture pellets were digested with DNAase and RNAase, and then with trypsin. Released virus was pelleted through a 36% sucrose cushion for 80 min at 20,000 rpm. Poxvirus cores were released from the pelleted virus with 1% Triton X100 and mercaptoethanol by incubation for 10 min on ice. The released cores were pelleted through a 36% sucrose cushion and the viral DNA released by Proteinase K/RNAase digestion followed by phenol/chloroform extraction and ethanol precipitation, as reported previously [45,46]. Sequencing was undertaken using TruSeq (Illumina) protocols and standard multiplex adaptors available in March 2011. A paired-end 100-base-read protocol was used for sequencing on an Illumina GAIIx instrument using a previously established protocol [47].

4.3. Genome Assembly and Annotation

The resulting 3,343,202 paired-end raw sequence reads were used to assemble the complete genome of ALPV as described previously [25,31,48,49] using CLC Genomics Workbench (version 9.5.4, CLC bio, a QIAGEN Company, Prismet, Aarhus C, Denmark) and Geneious (version 10.2.2, Biomatters, New Zealand). Briefly, the sequences were processed to remove Illumina adapters, low quality reads and ambiguous bases. Trimmed sequence reads were mapped against the chicken genome (Gallus gallus, GenBank accession number NC_006088) to remove likely host DNA contamination. In addition, reads were further mapped to Escherichia coli bacterial genomic sequence (GenBank accession no. U00096) to remove possible bacterial contamination. Unmapped reads were used as input data for de novo assembly using CLC Genomics Workbench (version 9.5.4). This resulted in the generation of a 351,909 bp genome. Clean raw reads (1.15 million) were mapped back to the assembled ALPV genome and resulted in an average coverage of 136.33x. The genome was annotated according to the previously published protocol [19] using Geneious software (version 10.2.2, Biomatters, Auckland, New Zealand). Open reading frames (ORFs) longer than 50 amino acids, with a methionine start codon (ATG) and minimal overlap with other ORFs (not exceeding 50% of one of the genes), were selected and annotated. ORFs shorter than 50 amino acids that had been previously annotated in other poxvirus genomes were also included. Similarity BLAST searches were performed on the predicted ORFs and were annotated as potential genes if predicted ORFs showed significant sequence similarity to known viral or cellular genes (BLAST E value ≤ e−5) [50]. Additional BLAST searches were performed on the predicted ORFs of ALPV against VACV-Cop [51].

To predict the function of unique ORFs tentatively identified in this study, the derived protein sequence of each ORF was searched by multiple applications to identify conserved domains or motifs. Transmembrane helices were searched using the TMHMM package (version 2.0) [52] and TMpred [53]. Additionally, searches for conserved secondary structure (HHpred) [54] and protein homologs using Phyre2 [55] were used to predict the function of unique ORFs identified in this study. To identify the likely promoter sequences of predicted unique ORFs of ALPV, a promoter motif search analysis was conducted using CLC Genomic Workbench (version 9.5.4), where vaccinia virus unique promoter sequences were used [51,56,57,58].

4.4. Comparative Genomics

Genomic features of the newly sequenced ALPV were visualised using Geneious (version 10.2.2). Sequence similarity percentages between representative ChPV and ALPV complete genome sequences were determined using tools available in Geneious (version 10.2.2). Dot plots were created based on the EMBOSS dottup program in Geneious software, with word size = 12 [59].

4.5. Phylogenetic Analyses

Phylogenetic analyses were performed using the novel ALPV genome sequence determined in this study, together with other selected ChPV genome sequences available in GenBank (Table 3). Nucleotide sequences of the partial DNA polymerase and partial p4b genes, as well as concatenated amino acid sequences of the selected nine poxvirus core proteins, were aligned as described previously [30] using the MAFTT L-INS-I algorithm implemented in Geneious (version 7.388) [60]. To determine the best-fit model to construct phylogenetic analyses, a model test was performed using CLC Genomics Workbench (version 9.5.4), which favoured a general-time-reversible model with gamma distribution rate variation and a proportion of invariable sites (GTR+G+I). Phylogenetic analyses for nucleotide sequences were performed using the GTR substitution model with 1000 bootstrap support in CLC Genomics Workbench (version 9.5.4), but the LG substitution model was chosen for concatenated amino acid sequences in Geneious (version 10.2.2).

Table 3.

Related poxvirus genome sequences used in further analysis of ALPV.

Virus Abbreviation GenBank Accession Number Reference
Albatrosspox virus ALPV MW365933 This study
Canarypox virus CNPV AY318871 [32]
Cheloniidpox virus 1 ChePV-1 MT799800 [61]
Fowlpox virus FWPV AF198100, MF766430-32,
MH709124-25,
MH719203, MH734528,
AJ581527, MW142017 		[28,29,62,63]
Flamingopox virus FGPV MF678796 [24]
Magpiepox virus MPPV MK903864 [31]
Mudlarkpox virus MLPV MT978051 [30]
Nile crocodilepox virus CRV DQ356948 [64]
Penguinpox virus PEPV KJ859677 [33]
Penguinpox virus 2 PEPV2 MW296038 [19]
Pigeonpox virus FeP2 KJ801920 [33]
Saltwater crocodilepox virus 1 SwCRV1 MG450915 [36,65]
Shearwaterpox virus 1 SWPV1 KX857216 [25]
Shearwaterpox virus 2 SWPV2 KX857215 [25]
Turkeypox virus TKPV NC_028238 [34]
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5. Conclusions

This study reports the genomic characterisation of a novel avipoxvirus, ALPV, isolated from an endangered northern royal albatross. The ALPV genome sequence was sufficiently divergent from other known avipoxviruses to be considered a novel species within the genus Avipoxvirus, family Poxviridae. This discovery has enhanced our understanding of the pathogen landscape relevant to northern royal albatrosses in New Zealand. Obtaining and sequencing additional poxvirus isolates will also be important to further investigate the epidemiology, transmission, pathogenesis and host specificity of ALPV infections in this endangered bird species.

Acknowledgments

Subir Sarker is the recipient of an Australian Research Council Discovery Early Career Researcher Award (grant number DE200100367) funded by the Australian Government. We also gratefully acknowledge the funding contributed by the Australian Biosecurity CRC for Emerging Infectious Disease in support of this work. The authors are thankful to the New Zealand Ministry for Primary Industries, Animal Health Laboratory, for providing the skin lesion from which ALPV was isolated.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/pathogens10050575/s1, Figure S1: Maximum likelihood (ML) phylogenetic tree from partial nucleotide sequences of the DNA polymerase gene of selected avipoxviruses, Figure S2: Maximum likelihood phylogenetic tree from partial nucleotide sequences of the p4b gene of selected avipoxviruses.

Click here for additional data file. (940.5KB, zip)

Author Contributions

Conceptualization, S.S., T.R.B. and D.B.B.; Formal analysis, S.S., A.A., T.N. and D.B.B.; Funding acquisition, S.S. and D.B.B.; Investigation, S.S. and D.B.B.; Methodology, S.S. and D.B.B.; Writing—original draft, S.S.; Writing—review and editing, S.S., A.A., T.N., T.R.B. and D.B.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The complete genome sequence and associated datasets generated during this study were deposited in GenBank under the accession number MW365933.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Footnotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Click here for additional data file. (940.5KB, zip)

Data Availability Statement

The complete genome sequence and associated datasets generated during this study were deposited in GenBank under the accession number MW365933.

Articles from Pathogens are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)

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