Full genome analysis of a novel adenovirus from the South Polar skua (Catharacta maccormicki) in Antarctica

Identification of SPSAdV

Initially, the DNA polymerase (pol) gene of a previously unknown adenovirus was amplified by PCR from the kidney of a South Polar skua (SPS T03). Using newly designed primers based on the obtained sequence, the full genome of the novel adenovirus (SPSAdV) was extended from the left-end inverted terminal repeat (ITR) region to the right-end ITR region. The entire viral DNA genome was 26,340 bp and encoded 24 adenoviral genes. The complete nucleotide sequence of SPSAdV was deposited in GenBank (accession number HM585353). The G + C content was found to be 34.2%.

The pol, penton and hexon genes of SPSAdV from four other skuas (SPS T01, SPS T02, SPS T06 and SPS T09) were also sequenced (Table 2 ), while from SPS T08 only the penton gene was sequenced from the liver, suggesting lower virus concentration. Nucleotide sequences of the pol, penton and/or hexon genes of SPSAdV identified in the six skuas were identical, with no evidence of polymorphism. Since separate sterile instruments were used in collecting tissues from each animal under BSL-2 containment, it is unlikely that the identical sequences were due to cross contamination.

Sequence analysis

The positions and coding directions of the 24 genes and open reading frames (ORF) are shown in the schematic genome map (Fig. 2 ). The locations, as well as lengths of each gene (nucleotide and amino acid) and their G + C content, are indicated in Table 3 . The ITR regions were located on left and right ends. IVa2, polymerase, protein precursor (pTP), DNA binding protein (DBP), U exon and ORF8 were transcribed leftward, and sialidase, ORF4, 52K, pIIIa, III (penton), pVII, pX, pVI, hexon, protease, 100K, 22K, 33K, pVIII, E3 region, fiber and ORF7 were transcribed rightward.

The length of the ITR region differed depending on the adenovirus species. For example, the ITR of THEV (AY849321) was 40 bp, whereas that of HAdV-1 (AC000017) was 103 bp. By contrast, the ITR of SPSAdV was 31 bp, whereas the lengths of the 5′ and 3′ terminal ends were the same as those of other species.

The sialidase of SPSAdV, located immediately downstream of the ITR region, was composed of ORF1, ORF2 and ORF3. In SPSAdV, ORF4, located immediately next to the sialidase, was identified as hydrophobic, as in RAdV-1, THEV and FrAdV-1. IVa2, a delayed early gene located downstream of the polymerase gene, was slightly shorter than that in the avirulent turkey enteritis virus (1104 bp) and its G + C content of 29.69% was lower than that of other genes. The E2 region, containing the genes for DNA polymerase, pTP and DBP, consisted of two cleavage sites. The penton base, encoding a major capsid protein of adenovirus, was located between the pIIIa and pVII. And the hexon gene, encoding a capsid protein with a penton base and a fiber knob, had a G + C content of 34.36%.

The protease gene encoded one of the most conserved proteins among all adenovirus genes (Russell, 2009, Weber, 2007). The length of the E3 gene was 891 bp, and the 1389-nucleotide fiber gene encoded a 462-amino acid capsid protein, which was located between the U exon and ORF 7 (22,600–23,988) and transcribed in the rightward direction. ORF7 and ORF8 were genus specific, existing only in Siadenovirus.

Phylogenetic analysis

The viral genome and phylogenetic analysis showed that SPSAdV belonged to genus Siadenovirus in the family Adenoviridae (Fig. 3 ). At the nucleotide level, the SPSAdV pol, penton base and hexon genes exhibited somewhat higher sequence similarity of 73.8%, 79.2% and 77.5% with RAdV-1 than with THEV (68.3%, 75.4% and 74.9%) (Table 4 ).

The fiber gene of SPSAdV revealed only 57.2% and 48.0% nucleotide sequence similarity with that of RAdV-1 and THEV, respectively. Compared with other genera, the pol, penton base and hexon genes of SPSAdV shared < 61% nucleotide sequence similarity with that of Mastadenovirus, Atadenovirus and Aviadenovirus. The nucleotide and amino acid sequences of the pol, penton base, hexon and fiber genes showed nearly equi-distant differences between SPSAdV and other siadenoviruses.

In Fig. 4 , trees were based on the polymerase and hexon genes. Only Siadenovirus and Aviadenovirus sequences were compared. These trees, which also included siadenoviruses from great tit, psittacine and Sulawesi tortoise, showed that SPSAdV was most closely related to RAdV-1.

Samples

Frozen carcasses of nine South Polar skuas (SPS T01-T09), without readily discernable signs of disease, were collected in Lake King Sejong near King Sejong station (latitude 62° 13′ S and longitude 58° 47′ W) in Antarctica (Fig. 1), when ambient temperatures ranged from − 5.6 °C to 2.1 °C. Tissue samples from various organs (heart, trachea, lung, esophagus, intestine, liver, kidney) were obtained using separate sterile instruments from each bird and stored at − 70 °C until used. Autopsy was conducted in a BSL2 laboratory.

PCR and DNA sequencing

Total DNA was extracted from blood and tissue samples using the High Pure PCR Template preparation kit (Roche, Indianapolis, IN), according to the manufacturer's instructions. First and nested PCR were performed in a 50-μL reaction volume containing 1 μL of 10 mM dNTP, 2 μL (10 pM) of each primer, 1 unit of Super-Therm Taq polymerase (JMR Holdings, London, UK) and 2.5 μL (400 ng) of template. Primers used for PCR amplification and sequencing are provided in Table 1.

Initially, adenovirus sequences available in GenBank were aligned using Clustal W, MegAlign program. Regions exhibiting high homology were then selected for designing oligonucleotide primers. For amplification of the full genome, specific primers were designed based on newly acquired sequences. Initial denaturation at 95 °C for 5 min was followed by six cycles each of denaturation at 94 °C for 30 s, annealing at 37 °C for 30 s and elongation at 72 °C for 1 min, then 32 cycles of denaturation at 94 °C for 30 s, annealing at 42 °C for 30 s and elongation at 72 °C for 1 min and finally 72 °C for 5 min in a Peltier PTC-200 thermal cycler (MJ Research, Inc., Watertown, MA). Elongation time was altered between 1 and 3 min depending on the expected product size. PCR products were separated by electrophoresis in 1% agarose gels containing ethidium bromide. The right and left ends of the genome were determined by RACE PCR kit (Takara, Shiga, Japan) and amplified by PCR using enzyme-tagged primer in Table 1 (Kovács and Benkö, 2011).

Amplicons were purified by using a PCR Purification Kit (QIAGEN, Chatsworth, CA) and were sequenced with the Big-Dye® Terminator kit version 3.1 (Applied Biosystems, Foster City, CA) and ABI 3730 automated sequencer (Applied Biosystems) after cloning into the pSTBlue-I vector (Novagen, San Diego, CA).

Phylogenetic analysis

The identity of the sequences was searched by Blast (Altschul et al., 1990). Sequences were edited with EditSeq programs in the Lasergene 6 (DNASTAR) (www.dnastar.com) and aligned using Clustal W (Thompson et al., 1994). Phylogenetic trees were constructed, using maximum-likelihood (ML) and neighbor-joining (NJ) algorithms implemented, rooted at the midpoint, in PAUP (Swofford, 2003) based on the full length of amino acid sequences of polymerase, penton base and hexon. The NJ trees using the partial nucleotide sequences of polymerase and hexon were performed for the analysis of partially characterized siadenoviruses including viruses from parrots, great tit and Sulawest tortoises. An initial ML tree estimation was performed by Modeltest 3.7 (Posada and Crandall, 1998). Topologies were evaluated by bootstrap analysis of 1000 NJ and 100 ML replicates. The genetic distances were computed by the PAUP program.