Full genome sequence analysis of a novel adenovirus from a captive polar bear ( Ursus 1 maritimus ) 2

The presence of a novel adenovirus (AdV) was detected by PCR and sequencing, in the internal organs of a 14 captive polar bear that had died in the Budapest zoo. The virus content of the samples proved to be high 15 enough to allow for conventional Sanger sequencing on PCR-amplified genomic fragments. With this 16 approach, the sequence of the entire genome of the putative polar bear adenovirus 1 (PBAdV-1) was 17 obtained. Although the genome was found to be short, consisting of 27,952 base pairs merely, with a 18 relatively balanced G+C content of 46.3%, its organisation corresponded largely to that of a typical 19 mastadenovirus. Every genus-common gene could be identified except that of protein IX. The short E3 region 20 of the PBAdV-1 consisted of two novel, supposedly type-specific ORFs only, whereas no homologue of any of 21 the E3 genes, usually conserved in mastadenoviruses, such as for example that of the 12.5K protein, were 22 present. In the E4 region, only the highly conserved gene of the 34K protein was found besides two novel ORFs showing no homology to any known E4 ORFs. In silico sequence analysis revealed putative splicing 24 donor and acceptor sites in the genes of the E1A, IVa2, DNA-dependent DNA polymerase, pTP, 33K proteins, 25 and also of U exon protein, all being characteristic for mastadenoviruses. Phylogenetic calculations, based on 26 various proteins, further supported that the newly-detected PBAdV is the representative of a new species 27 within the genus Mastadenovirus, and may represent the evolutionary lineage of adenoviruses that 28 coevolved with carnivorans. Molecular characterization of a lizard adenovirus reveals the first atadenovirus with two fiber genes and the first adenovirus with either one short or three long fibers per penton.

ORFs showing no homology to any known E4 ORFs. In silico sequence analysis revealed putative splicing 24 donor and acceptor sites in the genes of the E1A, IVa2, DNA-dependent DNA polymerase, pTP, 33K proteins, 25 and also of U exon protein, all being characteristic for mastadenoviruses. Phylogenetic calculations, based on 26 various proteins, further supported that the newly-detected PBAdV is the representative of a new species 27 within the genus Mastadenovirus, and may represent the evolutionary lineage of adenoviruses that 28 coevolved with carnivorans. 29 another California sea lion with ocular lesions (this virus named otarine AdV-2) (Wright et al., 2015). More 85 recently, pol sequences became available from a cat-associated AdV (Lakatos et al., 2017), as well as from 86 pine martens (Martes martes) and Eurasian otters (Lutra lutra) (Walker et al., 2017). 87

Introduction
In this report, we describe the detection, full genome sequencing and phylogenetic analysis of an AdV 88 found in a captive polar bear (Ursus maritimus) which had died in the Budapest Zoo. Routine PCR screening 89 of the internal organs, namely the liver and the lungs revealed the likely presence of a hitherto unknown 90 AdV. The amount of viral genomic DNA in the samples was found high enough to allow for conventional 91 Sanger sequencing by direct primer walking on PCR-amplified genome fragments. Based on our results we 92 propose this novel polar bear AdV to be a founding member of a new species within the genus 93

Samples 96
A 23-year-old female polar bear, born in captivity in Italy, died suddenly at the Budapest Zoo in 2011. 97 Gross pathology revealed a tumour of about 10 cm in diameter in the liver. Upon histopathology, multifocal 98 glomerulonephritis, chronic fibrosis and amyloidosis were found in the kidney. The tumour was classified as 99 a biliary cystadenoma. Bacteriological examination indicated beta-2 toxin-producing Clostridium perfringens 100 enterotoxaemia as the immediate cause of the death. For routine PCR screening, DNA extraction from liver 101 and lung samples was performed as described earlier (Ballmann and Harrach, 2016). 102

PCR 103
For the initial diagnostic PCR or whenever the expected size of the PCR product did not exceed 1000 bp, 104 Dream Taq Green (ThermoScientific) Master Mix (2×) and for the amplification of larger DNA fragments the 105 PrimeSTAR Max or GXL DNA Polymerase (Takara Bio USA, Inc.) kits were applied. The initial detection of AdV 106 DNA was performed by general nested PCRs with degenerate, consensus primers targeting conserved regions 107 of the pol (Wellehan et al., 2004), IVa2 (Pantó et al., 2015) and hexon genes (the latter performed by single 108 round PCR) (Kiss et al., 1996). 109 The whole genome was sequenced using a primer walking approach. Specific primers were designed 110 based on the sequences gained first with the degenerate primers and later based on the sequences gained 111 by the custom-made primers (specific PCR primers as listed in supplementary Table S1). PCR reaction volume 112 was 25 μl, consisting of 12.5 μl DreamTaq Master Mix (2×) or PrimeSTAR Max or GXL DNA Polymerase 113 (Takara), 0.5 μl (50 pmol/μl) of each primer (outer and inner for the first and second round, respectively) and 114 1 μl of target DNA. Milli-Q water was added up to 25 μl final volume. In the second cycle of nested PCR, 2 μl 115 of the reaction mixture of the first cycle was used as a template. For single round PCR, the same amounts 116 were used, as in the first round of nested PCR. 117 The nested PCR profile comprised an initial denaturation step at 94°C for 5 min, followed by 45 cycles of 118 denaturation at 94°C for 0.5 min, annealing at 46°C for 1 min and elongation at 72°C for 1 min. The final 119 elongation step lasted 3 min at 72°C. For the amplification of the hexon gene, the following thermocycling 120 conditions were used: initial denaturation at 95°C for 5 min; 35 cycles of denaturation, annealing and 121 elongation, 1 cycle consisting of 95°C for 0.5 min, 55°C for 0.5 min and 72°C for 0.5 min, respectively, and a 122 final elongation step at 72°C for 5 min. Using specific primers, the PCR conditions were set according to 123 manufacturer's instruction (Takara Bio USA, Inc.). The PCR products were analysed by electrophoresis in 1% 124 agarose gels containing GelRed™ (Biotium USA, Inc.). 125

DNA sequencing 126
DNA fragments of expected size were cut from the agarose gel and cleaned using NucleoSpin® Gel and PCR 127 Clean-up kit (Macherey-Nagel, Germany). Sequencing was performed on both strands with the BigDye TM 128 Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems/Life Technologies Corporation®, Carlsbad, CA, 129 USA) using the consensus primers. The capillary electrophoresis was performed on the ABI Prism® 3100 130 Genetic Analyzer (Applied Biosystems, USA) by a commercial supplier. 131

Bioinformatics and phylogenetic analysis 132
The identity of the newly-gained sequences was checked by using different BLAST algorithms on the NCBI 133 website. Sequence assembly and editing were performed with the Staden package (Staden et al., 1998).

Genome characteristics 146
The genome of the PBAdV-1 strain BK35 (GenBank accession no. MF773580) was found to be 27,952 bp, with 147 inverted terminal repeats of 80 bp on both ends. The viral DNA has an average G+C content of 46.33%, which 148 can be considered as non-biased. The genome was predicted to contain 27 genes as presented in Table 1 in 149 comparison to other carnivoran AdVs (CAdV-1, CSLAdV-1 and SkAdV-1), bat-WIV12 AdV and human 150 adenovirus 5 (HAdV-5). The number and arrangement of the predicted genes were similar to those of other 151 mastadenoviruses; every genus-specific gene except that of protein IX could be identified. One fiber gene 152 could be determined. The E3 region contains two novel ORFs (E3 ORFA, E3 ORFB) but lacks the 12.5K gene. 153 In the E4 region, two novel ORFs (E4 ORFA, E4 ORFB) were found besides the conserved 34K gene (called 154 ORF6 in HAdVs) but gene ORF6/7 was missing. The three exons of the U exon protein gene could be predicted 155 (Podgorski et al., 2016). The splicing donor and acceptor sites, characteristic for the genus, were predicted in 156 pol, and in the E1A, pTP, IVa2 and 33K genes, too. No evidence of gene duplication was found. A schematic 157 genetic map of the PBAdV-1 is presented in Fig. 1 Here we present the complete coding sequence of a polar bear adenovirus, PBAdV-1. The death of the 177 specimen was caused by beta-2 toxin producing Clostridium perfringens enterotoxaemia. Consequently, the 178 pathogenicity of the novel virus is unclear. 179 Our investigation showed that the PBAdV-1 has a similar genome arrangement to other 180 mastadenoviruses, but it has a non-typical short genome of 27,952 bp. Since seroprevalence of canine AdVs 181 has been described in wild carnivorans, inter alia bears and polar bears (Zarnke and Evans, 1989 The predicted putative splicing sites in the E1A, pTP, pol, IVa2, 33K and U exon protein genes are typical 198 for the genus. Compared to other mastadenoviruses some genes seem to be shorter (52K, pIIIa, V and pVIII) 199 and the similarity is very low; max. 71.2 % in hexon gene (Table 1). The protease cleavage signals in the 200 precursor proteins pTP, pIIIa, pVII, pX, pVI and pVIII could also be determined (Table 2)  identity with our PBAdV-1) has been described from a captive-born polar bear cub that had died in the Berlin 227 Zoo (Dayaram et al., 2018). Compared to that analysis, we determined the genome ends (ITRs), and predicted 228 the splicing sites (for genes E1A, pTP, pol, IVa2, 33K and U exon protein). This is especially important for the 229 pol gene as it results a considerably longer protein, which is more suitable for phylogenetic analysis. Also 230 important that both genome analyses show the lack of protein IX, , which was such unique finding that first 231 we attributed it to some possible technical problem. For this reason, we discussed this topic more in detail.