Archives of Virology (2021) 166:1183–1191 https://doi.org/10.1007/s00705-021-04975-6

BRIEF REPORT

Discovery of a phylogenetically distinct poxvirus in diseased Crocodilurus amazonicus (family )

Kerstin Seitz1 · Anna Kübber‑Heiss2 · Angelika Auer1 · Nora Dinhopl3 · Annika Posautz2 · Marlene Mötz1 · Alexandra Kiesler1 · Claudia Hochleithner4 · Manfred Hochleithner4 · Gregor Springler5 · Annika Lehmbecker6 · Herbert Weissenböck3 · Till Rümenapf1 · Christiane Riedel1

Received: 17 September 2020 / Accepted: 18 December 2020 / Published online: 12 February 2021 © The Author(s) 2021

Abstract A novel poxvirus was discovered in Crocodilurus amazonicus (Teiidae) presenting with a debilitating skin disease. The generated frst genome sequence of a reptilian poxvirus revealed the closest phylogenetic relationship to avipoxviruses, highlighting potential virus exchanges between avian and reptilian species.

Poxviruses are large enveloped viruses containing a double- [4, 5]. Poxvirus infections of other than crocodil- stranded DNA genome of 130-360 kb. They are important ians have been described in the literature, but no sequence pathogens of high public health and economic impact and information or further characterization of these viruses is are able to infect a wide range of host species, ranging from available [6–11]. insects to mammals. Within the family Poxviridae, two sub- The species afected in this study, Crocodilurus ama- families (Chordopoxvirinae and Entomopoxvirinae) have zonicus (“crocodile tegu”), is part of the family Teiidae. been defned based on their hosts, which are either verte- These are native to the Amazon and Orinoco basins brates or insects [1, 2]. The subfamily Chordopoxvirinae is in South America [12] and belong to one of only two gen- divided into 18 genera, which include a total of 52 species era of living semi-aquatic teiids, and as such, they inhabit [3]. seasonally fooded forested areas near riverbanks or other Poxviruses infecting non-mammalian species belong to watercourses. Their mean body temperature, 31.2°C, is the genera Avipoxvirus and Crocodylidpoxvirus. As also higher than the environmental temperature but relatively low observed in mammalian hosts, poxviruses of avian and croc- compared to that of other teiids, which could be due to their odilian species are primarily associated with skin lesions but association with water habitats. Their variable diet consists can also afect the upper respiratory and gastrointestinal tract of insects, other small reptiles, and frogs [13, 14]. In 2019, fve C. amazonicus, owned by a private collector in Austria, were presented with skin lesions and weight loss. Handling Editor: William G Dundon. Due to the severity of the clinical signs, one had to be * Christiane Riedel euthanized and was sent for further diagnostic evaluation to [email protected] the pathologists of the Research Institute of Wildlife Ecol- ogy, University of Veterinary Medicine, Vienna, Austria. 1 Department of Pathobiology, Institute of Virology, University of Veterinary Medicine, Veterinärplatz 1, The animal was underweight and had multiple elevated, par- 1210 Vienna, Austria tially ulcerated skin lesions with a diameter of up to 4 mm 2 Department of Interdisciplinary Life Sciences, Research on the back and the dorsal areas of head, neck, and tail that Institute of Wildlife Ecology, University of Veterinary did not extend into the underlying muscle (Fig. 1A). Lesions Medicine, Vienna, Austria were subsequently analyzed histologically, revealing multi- 3 Department of Pathobiology, Institute of Pathology, focally and focally extensive epidermal hyperplasia of up to University of Veterinary Medicine, Vienna, Austria 10-15 times the normal thickness with a severely thickened 4 Tierklinik Strebersdorf, Vienna, Austria stratum spinosum forming rete ridges. Keratinocytes showed 5 Labor In Vitro GmbH, Vienna, Austria ballooning degeneration and contained large eosinophilic, intracytoplasmic viral inclusion bodies (Bollinger bodies) up 6 IDEXX Vet Med Labor GmbH, Kornwestheim, Germany

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Fig. 1 Pathologic and histologic examination. (A) Macroscopic pres- displaying severe ballooning of the cells and prominent, eosinophilic entation of skin lesions, represented by multiple, up to lentil-sized, intracytoplasmatic inclusion bodies. (C) Multiple poxvirus-like parti- elevated, partially ulcerated foci. This scale bar is in centimeters. (B) cles in skin lesions detected by transmission electron microscopy of Hematoxylin-eosin-stained tissue section of an afected area of skin, uranyl-acetate-stained tissue sections. to 20 µm in size (Fig. 1B). Large numbers of poxvirus-like a fragment analyzer at the Vienna BioCenter Core Facili- particles were detected within these lesions by transmission ties before being sequenced using Illumina MiSeq chem- electron microscopy (Fig. 1C). istry. The raw reads were quality controlled, and adapter A sample taken from a skin lesion tested positive for sequences were removed before commencing data analysis. the presence of poxvirus DNA when a pan-poxvirus PCR For nanopore sequencing, the DNA was processed accord- for high-GC-content poxviruses developed by Li et al. was ing to the protocol for sequencing of genomic DNA by liga- used [15]. Comparison of this 630-bp amplicon, which cor- tion (SQK-LSK109) provided by ONT, and the library was responded to part of the gene encoding the DNA-dependent loaded onto a Nanpore Flongle Flowcell (ONT). RNA polymerase subunit rpo147 ortholog, to sequences Before contig assembly, the nanopore reads were aligned available in public databases revealed the closest relation- to the genome sequence of penguinpox virus using bow- ship, with 80% sequence identity, to members of the genus tie2-2.2.8 [16]. Nanopore reads aligning to this sequence Avipoxvirus. This isolate was tentatively named "teiidaepox were subsequently used for contig assembly of the Illu- virus 1" (TePV-1). Cultivation in freshly isolated chicken mina reads with SPAdes 3.14.0 [17]. This straightforward embryo fbroblasts was attempted at diferent temperatures approach, combining short reads with reads of up to 30 (22, 28 and 37°C), but this did not result in detectable virus kb length, resulted in the ab initio assembly of two con- replication, either by PCR testing of the supernatant or the tigs with a length of 116,666 and 46,221 bp, respectively. development of a cytopathic efect. In order to characterize These contigs had overlapping ends and could therefore the virus further, we determined the sequence of the virus easily be merged into one genome assembly with a fnal genome, employing a combination of Illumina sequenc- length of 166,425 bp and a GC content of 35.5%. The over- ing technology (150-bp paired-end reads) and nanopore lap between the two contigs was confrmed by site-specifc sequencing (MinION, Oxford Nanopore Technologies PCR and Sanger sequencing. The coverage of the Illumina [ONT]) using DNA extracted from skin. Briefy, an asepti- reads was 124 (138,058 aligned reads), and that of the nano- cally dissected piece of skin containing lesions of approxi- pore sequencing reads was 125 (11,248 aligned reads out mately 10 mg was mechanically homogenized in 60 µl of of 94,061; average length of aligned reads = 1848). The PBS in a TissueLyser II at 30 Hz for 3 min (QIAGEN). length distribution of the aligned reads and a histogram of For nanopore sequencing, 120 µl of lysis bufer was added, the coverage distribution are shown Supplementary File 1. and the rest of the DNA preparation was performed follow- Phylogenetic analysis of the genome sequence of TePV-1 ing the manufacturer’s instructions for preparation of DNA and the amino acid sequences of the putative DNA poly- from tissues using a QIAamp DNA Mini Kit. For Illumina merase (highest amino acid sequence identity, 74.3%, to sequencing, DNA was extracted using an NEB genomic famingopox virus, MF678796.1) and DNA topoisomerase DNA extraction kit according to the manufacturer’s instruc- (highest amino acid sequence identity, 76.0%, to fowlpox tions. The quality of the DNA preparation was checked using virus, NC_002188.1) revealed that TePV-1 is most closely Genomic DNA ScreenTape (Agilent) on a 4200 TapeStation related to members of the genus Avipoxvirus (Fig. 2). (Agilent) at the VetCore genomics facility. The library for Using CLC Workbench, 154 open reading frames Illumina sequencing was prepared using an NEBNext Ultra coding for ≥100 amino acids were detected, and these II DNA Library Prep Kit (New England Biolabs) according sequences were compared to the proteome of other avi- to the manufacturer’s protocol and quality controlled using poxviruses, using BLASTp. The results of this analysis

1 3 Teiidaepox virus 1185

Fig. 2 Phylogenetic relationship of TePV-1 to other members of the pox virus, NC_002188.1; hypsugopox virus, MK860688.1; lumpy subfamily Chordopoxvirinae. (A) Neighbor-joining tree based on skin disease virus, NC_003027.1; magpiepox virus, MK903864.1; the DNA polymerase protein sequence. (B) Neighbor-joining tree myxoma virus, NC_001132.2; Nile crocodilepox virus, DQ356948.1; based on the DNA topoisomerase protein sequence. (C) Neighbor- penguinpox virus, KJ859677.1; pigeonpox virus: KJ801920.1; joining tree of the full genome sequences based on avipoxviruses. pteropox virus, NC_030656.1; saltwater crocodile poxvirus 1, All branches had a bootstrap value of 100. All trees were generated MG450915.1; saltwater crocodile poxvirus 2, MG450916.1; shear- after multiple sequence alignment in CLC Workbench with 1,000 waterpox virus 1, KX857216.1; shearwaterpox virus 2, KX857215.1; replicates. The GenBank accession numbers for sequences employed squirrelpox virus, NC_022563.1; tanapox virus, EF420157.1; turkey- in this analysis are as follows: canarypox virus, NC_005309.1; ept- pox virus, KP728110.2; vaccinia virus, NC_006998.1; variola virus, esipox virus, NC_035460.1; famingopox virus, MF678796.1; fowl- NC_001611.1; Yaba-like disease virus, NC_002642.1. are presented in Table 1. Apart from the presence of arrangement was the same as in other avipoxviruses, ankyrin repeat proteins at the very beginning and the end even though the TePV-1 genome is signifcantly shorter. of the coding region of the TePV-1 genome, the ORF Interestingly, nine reading frames coding for proteins of

1 3 1186 K. Seitz et al. Species SWPV-1 FGPV FGPV FeP2 FeP2 TKPV PEPV FWPV PEPV PEPV FGPV FGPV SWPV-1 FGPV TKPV FGPV FGPV FeP2 FWPV FWPV PEPV FGPV FWPV FWPV TKPV FeP2 PM CNPV FGPV PEPV FWPV FeP2 FGPV e-value 2.43E-87 4.68E-93 2.93E-74 2.61E-122 1.26E-157 2.27E-25 8.05E-45 9.34E-66 6.97E-175 5.22E-85 3.84E-67 0 2.09E-97 1.03E-48 4.06E-44 4.29E-72 2.11E-28 1.91E-111 0 1.06E-133 0 1.57E-157 5.41E-89 1.14E-27 0 1.20E-174 5.00E-20 2.00E-16 0 3.39E-102 3.25E-109 8.20E-98 0 ali 220 600 347 519 612 121 187 261 588 198 296 743 380 134 157 144 179 338 558 353 570 256 154 64 553 289 164 172 635 225 212 161 633 % identity 59.545 33.667 34.582 41.04 42.484 34.711 39.572 42.146 46.259 55.051 36.824 42.665 41.842 52.239 43.312 68.056 34.078 46.154 62.186 50.992 48.772 78.516 76.623 76.562 75.769 79.585 46 35 78.74 62.667 71.226 83.23 90.047 ref loc ref 323041..323703 271364..273295 256018..257064 264529..266415 264529..266415 185548..185937 281304..281861 274191..275423 33882..35669 35800..36411 29544..30545 33068..35515 47782..48978 40555..40962 22080..22559 41945..42382 42434..42961 49969..50982 247565..249259 246462..247523 57639..59369 51121..51906 52429..52893 52647..52856 33712..35373 61089..61958 55108..57021 65721..66398 58126..58821 66549..67034 60145..62046 GenBank no. ARF02867.1 AUD40350.1 AUD40336.1 YP_009046459.1 YP_009046459.1 ALA62542.1 YP_009046231.1 AYO89833.1 YP_009046027.1 YP_009046028.1 AUD40130.1 AUD40134.1 ARF02656.1 AUD40139.1 ALA62391.1 AUD40142.1 AUD40143.1 YP_009046279.1 AYP74252.1 AYP74251.1 YP_009046047.1 AUD40150.1 AXY04490.1 AXY04491.1 ALA62402.1 YP_009046288.1 XP_015684078.2 XP_008122411.1 AUD40154.1 YP_009046053.1 AXY04496.1 YP_009046293.1 AUD40159.1 Protein SWPV1-308, conserved hypothetical protein hypothetical conserved SWPV1-308, Ankyrin repeat family protein Ankyrin family repeat Serpin family protein Serpin family Ankyrin protein repeat Ankyrin protein repeat EFc-like protein EFc-like Ankyrin protein repeat Ankyrin protein repeat No similarityNo found Ankyrin protein repeat Hypothetical protein Hypothetical G-protein-coupled receptor family protein family receptor G-protein-coupled alkaline phosphodiesterase SWPV1-041, DNase II-like protein II-like DNase SWPV1-041, Hypothetical protein Hypothetical Hypothetical protein Hypothetical dUTP pyrophosphatase B-cell lymphoma 2 B-cell lymphoma Serpin family protein Serpin family DNA ligase DNA Putative serpinPutative Semaphorin GNS1/SUR4 Late transcription factor VLTF-2 Late transcription factor Rifampicin resistance protein Rifampicin Rifampicin resistance protein Rifampicin mRNA capping enzyme mRNA Class I histocompatibility antigen, F10 alpha chain-like antigen, Class I histocompatibility Major histocompatibility complex class I-related gene protein gene class I-related complex histocompatibility Major NPH-1 transcription termination factor muT motif expression regulator expression muT motif mutT motif containingmutT motif protein RNA polymerase subunit RPO18 polymerase RNA Early transcription factor small subunit transcription factor Early Length 621 1803 1044 1494 1752 378 558 798 348 1752 603 954 2193 1113 414 474 435 531 1014 1680 1059 1689 771 465 324 1662 870 1134 882 1911 696 636 486 1902 End 1463 3643 4626 6163 7934 8368 8969 9832 9668 11604 12234 13561 15733 16861 17279 17776 18213 18749 19798 21833 22923 24649 25494 26011 25971 27693 28579 29709 30548 32436 33104 33723 34533 36421 Annotation of ORFs in the genome of TePV-1 encoding proteins larger than 100 aa. The start and end of ORFs located on the positive strand are shown in bold. The length of each in bold. The length of each than shown are 100 aa. The start strand larger and end of ORFs located on the positive encoding proteins of TePV-1 Annotation of ORFs in the genome Start 843 1841 3583 4670 6183 7991 8412 9035 9321 9853 11632 12608 13541 15749 16866 17303 17779 18219 18785 20154 21865 22961 24724 25547 25648 26032 27710 28576 29667 30526 32409 33088 34048 34520 1 Table N ORF is given in base pairs. "Protein" indicates the highest-rated BLAST hit for this ORF, which is further loc), the (ref its accession number (GenBank no.), location in the which genome specifed by this ORF, hit for BLAST indicates the highest-rated in base pairs. "Protein" ORF is given virus; canarypox asi - Equus EA, ; CNPV, griseus ; CG, Cricetulus Anolis carolinensis identity (% identity), the and the alignment length species. AC, % amino acid sequence (ali), the e-value virus; penguinpox PM, Protobothrops virus; PEPV, ORF number; magpiepox N., virus; Lingula anatina ; MPPV, LA, fowlpox virus; famingopox FWPV, virus; FGPV, pigeonpox nus ; FePV2, virus turkeypox virus; shearwaterpox TKPV, ; SWPV, mucrosquamatus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

1 3 Teiidaepox virus 1187 Species FGPV FWPV SWPV-1 MPPV CNPV PEPV TKPV FGPV FWPV SWPV-1 FWPV FWPV PEPV TKPV MPPV FGPV MPPV FGPV FGPV FGPV PEPV FWPV FGPV FeP2 FGPV FWPV FeP2 SWPV-1 FWPV FWPV FWPV FWPV AC LA e-value 0 6.82E-127 1.85E-88 1.91E-59 4.02E-29 3.94E-82 9.15E-62 8.93E-102 3.22E-48 0 0 0 0 3.47E-101 0 0 2.17E-47 0 3.94E-141 0 0 1.84E-105 0 0 9.14E-54 3.96E-46 1.22E-88 0 0 0 6.53E-106 6.98E-96 1.00E-07 6.00E-06 ali 791 216 392 119 98 269 125 225 103 633 676 421 390 283 310 659 131 988 268 571 1928 181 717 472 114 209 148 373 630 436 213 183 121 101 % identity 79.267 76.389 41.071 68.908 44.898 47.212 68 64 66.019 65.561 72.485 75.059 63.333 50.883 81.935 48.558 51.145 74.291 71.269 71.979 47.199 79.006 55.23 79.025 70.175 38.756 81.081 75.067 46.825 75.688 69.484 68.306 38 37 ref loc ref 62027..64402 67861..68517 81697..82893 80344..80754 100368..100847 80684..81493 53825..54202 80826..81503 79590..79901 95995..97896 203411..205456 202111..203376 94650..95822 65235..66116 99500..100432 92674..94644 102503..102898 95291..98260 98252..99100 99093..100808 118822..124590 171000..171548 118803..120956 126407..127825 122355..122699 121669..122301 128991..129440 142768..143904 129455..131347 153725..155065 134129..134770 135430..135981 GenBank no. AUD40160.1 AXY04504.1 ARF02682.1 QGM48717.1 NP_955112.1 YP_009046067.1 ALA62425.1 AUD40182.1 AXY04521.1 ARF02699.1 AYP74219.1 AYP74218.1 YP_009046080.1 ALA62437.1 QGM48748.1 AUD40195.1 QGM48751.1 AUD40198.1 AUD40199.1 AUD40200.1 YP_009046096.1 AYP74202.1 AUD40205.1 YP_009046336.1 AUD40207.1 AYO89698.1 YP_009046339.1 ARE67652.1 AXY04551.1 AYP74192.1 AXY04554.1 AXY04556.1 XP_003216736.1 XP_013402139.1 Protein NTPase Uracil DNA glycosylase DNA Uracil SWPV1-075, conserved hypothetical protein hypothetical conserved SWPV1-075, Putative glutathionePutative peroxidase Hypothetical protein Hypothetical Virion protein Virion Glutaredoxin Putative transcriptional factor Putative elongation Hypothetical protein Hypothetical SWPV1-097, putative metalloprotease putative SWPV1-097, RNA helicase NPH-II RNA Virion core proteinase core Virion DNA-binding protein DNA-binding No similarityNo found DNA-binding phosphoprotein DNA-binding DNA-binding virion protein DNA-binding Hypothetical protein Hypothetical Virion core protein core Virion DNA polymerase DNA Hypothetical protein Hypothetical Hypothetical protein Hypothetical B22R family protein B22R family No similarityNo found RNA polymerase subunit polymerase RNA Hypothetical protein Hypothetical PolyA polymerase large subunit PAP-L large polymerase PolyA DNA binding virion phosphoprotein DNA core Hypothetical protein Hypothetical Hypothetical protein Hypothetical SWPV1-124, putative palmitylated EEV envelope lipase EEV envelope palmitylated putative SWPV1-124, EEV maturation protein EEV maturation Ser/Thr kinase Hypothetical protein Hypothetical HAL3 domain containing protein 25-hydroxyvitamin D-1 alpha hydroxylase, mitochondrial D-1 alpha hydroxylase, 25-hydroxyvitamin Glutamate-rich protein 3 isoform X3 3 isoform Glutamate-rich protein Length 2358 657 1140 357 372 795 378 699 312 1854 2037 1260 1170 333 753 936 1944 396 2967 807 1716 5550 459 546 2127 1419 318 600 447 1134 1800 1326 642 540 696 843 End 38759 39444 40600 41215 41540 42653 43218 43859 44164 46041 48064 49338 50508 50708 51828 52968 55055 55380 58654 59452 61169 66739 64939 67372 69501 70891 71202 71880 72375 73865 75696 77052 77671 78655 79822 80702 (continued) Start 36402 38788 39461 40859 41169 41859 42841 43161 43853 44188 46028 48079 49339 50376 51076 52033 53112 54985 55688 58646 59454 61190 64481 66827 67375 69473 70885 71281 71929 72732 73897 75727 77030 78116 79127 79860 1 Table N 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70

1 3 1188 K. Seitz et al. Species CNPV SWPV-1 CNPV FWPV FWPV SWPV-2 PEPV FeP2 FWPV MPPV FGPV FGPV FWPV PEPV FeP2 FWPV FeP2 FGPV FeP2 SWPV-2 SWPV-1 FWPV CNPV CG FWPV CNPV PEPV CNPV TKPV SWPV-1 FWPV FWPV TKPV FeP2 e-value 0 1.15E-98 0 3.61E-177 1.45E-159 4.06E-165 0 1.64E-132 1.32E-54 4.18E-80 4.29E-158 3.22E-102 4.38E-70 0 1.86E-100 1.78E-117 1.64E-121 0 3.66E-34 0 5.47E-61 0 4.89E-50 2.00E-135 5.16E-114 0 2.99E-29 9.90E-36 6.26E-24 1.05E-115 2.66E-149 0 6.65E-30 1.69E-89 ali 433 187 348 260 336 242 303 253 129 143 296 182 121 1288 166 189 324 799 175 316 153 849 139 473 288 450 199 203 163 411 225 648 197 169 % identity 60.277 71.123 77.011 87.308 60.417 86.364 75.908 67.984 62.016 76.224 70.27 75.275 77.686 88.276 79.518 79.894 50.617 79.099 46.286 75.633 58.17 75.972 56.835 47 51.736 65.778 33.166 33.005 34.969 43.796 88.889 80.864 34.01 76.923 ref loc ref 170735..172057 158056..158622 172777..173823 129528..130310 160397..161407 201657..202388 171203..172108 156784..157545 164799..165188 169617..170063 158947..159873 159870..160430 166439..166852 175641..179504 164161..164661 171281..171853 165428..166429 166990..169389 168979..169503 217385..218335 196050..196511 177778..180333 228919..229341 99505..100464 237537..238955 198797..199537 240422..241354 122400..122924 232534..233796 198622..199299 199529..201502 130871..131458 196511..197020 GenBank no. NP_955168.1 ARE67661.1 NP_955171.1 AYP74173.1 AXY04569.1 ARE67397.1 YP_009046124.1 YP_009046362.1 AYO89725.1 QGM48809.1 AUD40238.1 AUD40239.1 AXY04578.1 YP_009046131.1 YP_009046369.1 AXY04581.1 YP_009046371.1 AUD40245.1 YP_009046373.1 ARE67414.1 ARE67699.1 AXY04588.1 NP_955217.1 XP_027254970.1 AYP74141.1 NP_955229.1 YP_009046151.1 NP_955232.1 ALA62492.1 ARF02782.1 AXY04607.1 AXY04609.1 ALA62497.1 YP_009046400.1 Protein No similarityNo found Hypothetical protein Hypothetical SWPV1-137, conserved hypothetical protein hypothetical conserved SWPV1-137, Putative virionPutative protein core Late transcription factor VLTF-1 Late transcription factor Poxvirus myristoylprotein Poxvirus SWPV2-ORF161, putative myristylated IMV envelope protein IMV envelope myristylated putative SWPV2-ORF161, Hypothetical protein Hypothetical DNA-binding virion VP8 core DNA-binding Hypothetical protein Hypothetical Putative IMV membrane protein IMV membrane Putative PolyA polymerase small subunit polymerase PolyA RNA polymerase subunit 22 polymerase RNA Membrane protein Membrane RNA polymerase subunit RPO147 polymerase RNA Protein tyrosine phosphatase tyrosine Protein Putative viral membrane protein membrane viral Putative Virion envelope protein (p35) protein envelope Virion RNA polymerase-associated protein 94 protein polymerase-associated RNA VLTF-4 SWPV2-ORF177, DNA topoisomerase DNA SWPV2-ORF177, SWPV1-166, conserved hypothetical protein hypothetical conserved SWPV1-166, mRNA capping enzyme large subunit capping enzyme large mRNA CNPV194 virion protein Sodium-dependent lysophosphatidylcholine symporter X2 1 isoform Sodium-dependent lysophosphatidylcholine p28-like protein p28-like CNPV206 putative photolyase CNPV206 putative N1R/p28 family protein N1R/p28 family CNPV209 N1R/p28-like protein CNPV209 N1R/p28-like Hypothetical protein Hypothetical SWPV1-208, C4L/C10L-like protein SWPV1-208, No similarityNo found Late transcription factor VLTF-3 Late transcription factor Virion core protein P4b protein core Virion Immunodominant virion protein RNA polymerase subunit RPO19 polymerase RNA Length 330 1305 579 1023 783 1008 732 909 759 381 441 894 561 411 3867 501 570 867 2388 489 951 462 2550 423 1413 861 1356 597 645 513 1209 618 678 1947 495 495 End 80498 82091 82864 83839 84676 85688 86420 87607 88391 88772 89169 90079 90636 91039 94941 95445 96028 96891 99279 99870 100821 101287 104082 104474 105904 107491 108848 109471 110163 110941 112124 111544 112803 114966 115521 116054 (continued) Start 80169 80787 82286 82817 83894 84681 85689 86699 87633 88392 88729 89186 90076 90629 91075 94945 95459 96025 96892 99382 99871 100826 101533 104052 104492 106631 107493 108875 109519 110429 110916 110927 112126 113020 115027 115560 1 Table N 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106

1 3 Teiidaepox virus 1189 Species SWPV-2 FWPV FGPV FWPV FWPV MPPV FGPV FWPV CNPV MPPV PEPV FGPV MPPV FeP2 CNPV PEPV FWPV FWPV FeP2 FWPV PEPV SWPV-1 CNPV FWPV FWPV AC SWPV-2 PEPV TKPV FWPV FeP2 FWPV FeP2 e-value 2.06E-179 0 0 0 3.08E-142 7.46E-54 0 2.15E-100 0 1.09E-62 0 1.83E-75 0 0 0 4.20E-170 2.63E-78 2.60E-138 6.59E-48 1.49E-35 6.67E-161 4.63E-81 4.07E-24 1.63E-75 1.42E-35 1.00E-128 7.90E-11 3.24E-22 7.44E-135 1.65E-102 2.75E-99 0 7.36E-51 ali 373 709 301 893 274 160 369 198 458 112 430 152 383 1157 598 477 140 303 113 118 244 176 147 250 206 486 117 100 292 321 431 737 157 % identity 65.952 83.216 79.07 73.46 74.088 56.875 69.919 72.727 74.891 82.143 59.767 67.105 74.413 90.147 55.017 49.895 72.857 60.726 61.947 45.763 84.836 61.932 38.095 48 37.864 45 35.897 40 59.932 48.287 40.139 41.384 48.408 ref loc ref 268134..269255 203339..205468 203735..204640 207389..210064 210082..210906 229297..229803 210114..211220 213785..214381 286822..288210 234410..234748 227993..229291 215103..215588 236523..237674 213357..216830 295207..297018 236280..237698 57016..57438 226511..227419 221822..222163 55034..55393 240113..241027 267315..267863 302592..303416 232786..233643 49333..50022 302929..303510 251214..251585 164710..165612 243703..244689 240328..241650 248089..250332 247134..248219 GenBank no. ARE67480.1 AYO90282.1 AUD40279.1 AXY04616.1 AXY04617.1 QGM48882.1 AUD40289.1 AXY04624.1 NP_955280.1 QGM48893.1 YP_009046183.1 AUD40295.1 QGM48896.1 YP_009046420.1 NP_955287.1 YP_009046188.1 AYP74103.1 AXY04635.1 YP_009046427.1 AYP74099.1 YP_009046195.1 ARF02806.1 NP_955296.1 AXY04907.1 AYP74093.1 XP_008108335.1 ARE67521.1 YP_009046207.1 ALA62528.1 AXY04659.1 YP_009046446.1 AXY05185.1 YP_009046450.1 Protein SWPV2-ORF229, conserved hypothetical protein hypothetical conserved SWPV2-ORF229, No similarityNo found Early transcription factor large subunit VETF-L large transcription factor Early No similarityNo found Intermediate 3 transcription factor Virion core protein P4a protein core Virion Hypothetical protein Hypothetical Virion protein Virion Putative myristoylated membrane protein membrane myristoylated Putative Phosphorylated virion protein membrane CNPV257 DNA helicase, transcriptional elongation CNPV257 DNA Conserved hypothetical protein hypothetical Conserved Processivity factor Processivity Holliday junction resolvase Holliday Intermediate VITF-3 transcription factor RNA polymerase subunit RPO132 polymerase RNA A-type inclusion like protein inclusion like A-type No similarityNo found A-type inclusion protein A-type Hypothetical protein Hypothetical RNA polymerase subunit RP035 polymerase RNA Hypothetical protein Hypothetical Hypothetical protein Hypothetical Virion assembly protein assembly Virion SWPV1-245, C-type lectin-like EEV protein C-type lectin-like SWPV1-245, Hypothetical protein Hypothetical Hypothetical protein Hypothetical Hypothetical protein Hypothetical Prostacyclin synthase isoform X1 synthase isoform Prostacyclin SWPV2-ORF270, putative interleukin binding protein interleukin putative SWPV2-ORF270, Epidermal growth factor like protein like Epidermal factor growth Ser/Thr protein kinase Ser/Thr protein Hypothetical protein Hypothetical Ankyrin protein repeat Ankyrin protein repeat Ankyrin protein repeat Length 1122 303 2127 318 903 2667 777 450 1092 579 1377 333 1284 453 1152 3474 1569 336 1347 423 912 342 363 744 555 432 777 603 1482 498 360 882 972 1305 2211 576 End 117170 117099 119299 118438 120256 123115 123908 124352 126492 127078 128472 129028 130310 130765 131936 135436 136993 136303 138375 138798 139713 140351 140714 141446 142044 142520 143281 143875 145406 145944 146287 147168 148422 149758 152030 152608 (continued) Start 116049 116797 117173 118121 119354 120449 123132 123903 125401 126500 127096 128696 129027 130313 130785 131963 135425 135968 137029 138376 138802 140010 140352 140703 141490 142089 142505 143273 143925 145447 145928 146287 147451 148454 149820 152033 1 Table N 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142

1 3 1190 K. Seitz et al.

101-206 aa could not be related to any other sequences based on amino acid sequence identity or the presence of Species EA SWPV-1 FGPV FGPV CNPV TKPV TKPV PEPV SWPV-2 SWPV-2 SWPV-1 conserved domains. Seven ORFs were found to encode proteins that are not related to other poxvirus proteins but are related to proteins found in eukaryotes (see Supple- mentary Table 1) and could have been acquired by hori- e-value 9.00E-19 4.78E-96 2.95E-80 2.60E-153 2.49E-85 1.17E-67 4.97E-09 9.22E-117 1.06E-126 2.20E-157 2.43E-87 zontal gene transfer [18]. These sequences, as well as the assembly site of the two contigs and the transitions to the ali 119 400 353 462 329 422 83 560 620 617 220 inverted terminal repeats were confrmed by specifc PCR and Sanger sequencing. Poxvirus-like lesions and infections have been described % identity 48 39.25

39.093 in various reptiles, including crocodilians, tortoises, cha- 50 40.122 33.412 31.325 38.214 38.548 41.977 59.545 meleons, and tegus [6–11, 19]. Despite their description in the literature, they have not yet been characterized at the genetic level, except for poxviruses in Nile and saltwater crocodiles (Nile crocodilepox virus [CRV] and saltwater crocodilepox virus subtypes 1 and 2 [SwCRV1/2], respec- ref loc ref 294643..295941 256018..257064 257748..259259 46721..47704 178533..179828 186082..186339 282988..284919 332667..334556 332667..334556 323041..323703 tively) [10, 11]. This frst report of the genome sequence of a poxvirus causing disease in a reveals it to be most closely related to avipoxviruses. This is surprising, considering the phylogenetic distance between avian and reptilian species and the diferences in homeostasis. The GenBank no. XP_014701435.1 ARF02838.1 AUD40336.1 AUD40338.1 NP_955062.1 ALA62540.1 ALA62543.1 YP_009046233.1 ARE67552.1 ARE67552.1 ARF02867.1 GC content of TePV-1 (35%) is also more similar to that of avipoxviruses than to the known crocodile-infecting poxviruses (62% for CRV, Sw-CRV-1 and -2) [10, 11]. Interestingly, the initial diagnostic PCR only was positive when a primer set for high-GC-content poxviruses was employed. This might indicate that poxviruses of Reptilia are quite variable. Therefore, additional research is war- ranted to examine the diversity of poxviruses of Reptilia and their species specifcity and zoonotic potential.

Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s0070​ 5-021-04975​ -6​ .

Acknowledgments This research was supported using resources of the VetCore Facility (Transcriptomics Unit) of the University of Veterinary Medicine Vienna.

Author contributions CR, KS and TR designed the study; MH and CH examined the ; AKH, AL, AP and GS performed the patho- logical and histopathological examination; HW and ND provided the electron micrographs; and AA, AK, CR, KS and MM performed the diagnostic PCRs, generated the full genome sequence, analyzed the Protein Zinc fnger protein 709-like isoform X3 isoform 709-like protein Zinc fnger SWPV1-279, ankyrin protein repeat SWPV1-279, Serpin family protein Serpin family Ankyrin repeat family protein Ankyrin family repeat CNPV039 G protein-coupled receptor-like protein receptor-like CNPV039 G protein-coupled No similarityNo found Ankyrin repeat family protein Ankyrin family repeat Late transcription factor VLTF-1 Late transcription factor Ankyrin protein repeat SWPV2-ORF300, ankyrin protein repeat SWPV2-ORF300, SWPV2-ORF300, ankyrin protein repeat SWPV2-ORF300, SWPV1-308, conserved hypothetical protein hypothetical conserved SWPV1-308, data and prepared the fgures. CR and KS wrote the manuscript and all authors commented on it. Length 474 1308 1044 1395 978 330 1260 312 1593 1758 1767 621 Funding Open Access funding provided by University of Veterinary Medicine Vienna. It is not applicable for this publication. End 153041 154364 155430 156876 157838 157659 159471 159784 161417 163155 164908 165815 Compliance with ethical standards

Conflict of interest The authors declare no confict of interest. (continued) Start 152568 153057 154387 155482 156861 157330 158212 159473 159825 161398 163142 165195 1 Table N 143 144 145 146 147 148 149 150 151 152 153 154

1 3 Teiidaepox virus 1191

Availability of data and material The sequence of TePV-1 has been 9. Penrith ML, Nesbit JW, Huchzermeyer FW (1991) Pox virus submitted to the GenBank database (accession number MT712273). infection in captive juvenile caimans (Caiman crocodilus fuscus) The raw data are available at SRA (BioProject ID: PRJNA681025). in South Africa. J S Afr Vet Assoc 62:137–139 10. Afonso CL, Tulman ER, Delhon G et al (2006) Genome of Croc- odilepox Virus. J Virol 80:4978–4991. https​://doi.org/10.1128/ Open Access This article is licensed under a Creative Commons Attri- jvi.80.10.4978-4991.2006 bution 4.0 International License, which permits use, sharing, adapta- 11. Sarker S, Isberg SR, Milic NL et al (2018) Molecular characteriza- tion, distribution and reproduction in any medium or format, as long tion of the frst saltwater crocodilepox virus genome sequences as you give appropriate credit to the original author(s) and the source, from the world’s largest living member of the Crocodylia. Sci provide a link to the Creative Commons licence, and indicate if changes Rep. https​://doi.org/10.1038/s4159​8-018-23955​-6 were made. The images or other third party material in this article are 12. Avila-Pires TCS De (1995) Lizards of Brazilian Amazonia, Natio- included in the article’s Creative Commons licence, unless indicated naal Natuurhistorisch Museum, Leiden. otherwise in a credit line to the material. If material is not included in 13. Mesquita DO, Colli GR, Costa GC et al (2006) At the water’s the article’s Creative Commons licence and your intended use is not edge: ecology of semiaquatic teiids in Brazilian Amazon. J Her- permitted by statutory regulation or exceeds the permitted use, you will petol 40:221–229. https​://doi.org/10.1670/123-05a.1 need to obtain permission directly from the copyright holder. To view a 14. Tattersall GJ, Leite CAC, Sanders CE et al (2016) Seasonal repro- copy of this licence, visit http://creativeco​ mmons​ .org/licen​ ses/by/4.0/​ . ductive endothermy in tegu lizards. Sci Adv 2:e1500951. https​:// doi.org/10.1126/sciad​v.15009​51 15. Li Y, Meyer H, Zhao H, Damon IK (2010) GC content-based pan- References pox universal PCR assays for poxvirus detection. J Clin Microbiol 48:268–276. https​://doi.org/10.1128/JCM.01697​-09 16. Langmead B, Salzberg SL (2012) Fast gapped-read alignment 1. Haller SL, Peng C, McFadden G, Rothenburg S (2014) Poxviruses with Bowtie 2. Nat Methods 9:357–359. https​://doi.org/10.1038/ and the evolution of host range and virulence. Infect Genet Evol nmeth​.1923 21:15–40 17. Bankevich A, Nurk S, Antipov D et al (2012) SPAdes: A new 2. Lefkowitz EJ, Wang C, Upton C (2006) Poxviruses: Past, present genome assembly algorithm and its applications to single-cell and future. Virus Res 117:105–118. https://doi.org/10.1016/j.virus​ ​ sequencing. J Comput Biol 19:455–477. https​://doi.org/10.1089/ res.2006.01.016 cmb.2012.0021 3. Lefkowitz EJ, Dempsey DM, Hendrickson RC et al (2018) Virus 18. Bratke KA, McLysaght A (2008) Identifcation of multiple inde- : the database of the International Committee on Tax- pendent horizontal gene transfers into poxviruses using a com- onomy of Viruses (ICTV). Nucleic Acids Res 46:D708–D717. parative genomics approach. BMC Evol Biol 8:67. https​://doi. https​://doi.org/10.1093/nar/gkx93​2 org/10.1186/1471-2148-8-67 4. Weli SC, Tryland M (2011) Avipoxviruses: Infection biology and 19. Marschang RE (2011) Viruses infecting reptiles. Viruses their use as vaccine vectors. Virol J 8:49 3:2087–2126 5. Ariel E (2011) Viruses in reptiles. Vet Res 42:1–12 6. Jacobson ER, Popp JA, Shields RP, Gaskin JM (1979) Poxlike skin Publisher’s Note Springer Nature remains neutral with regard to lesions in captive caimans. J Am Vet Med Assoc 175:937–940 jurisdictional claims in published maps and institutional afliations. 7. Jacobson ER, Telford SR (1990) Chlamydial and poxvirus infections of circulating monocytes of a flap-necked chame- leon (Chamaeleo dilepis). J Wildl Dis 26:572–577. https​://doi. org/10.7589/0090-3558-26.4.572 8. Stauber E, Gogolewski R (1990) Poxvirus dermatitis in a Tegu lizard (Tupinambis teguixin). J Zoo Wildl Med 21(2):228–230

1 3