A Novel Adenovirus in Chinstrap Penguins (Pygoscelis antarctica) in Antarctica

Adenoviruses (family Adenoviridae) infect various organ systems and cause diseases in a wide range of host species. In this study, we examined multiple tissues from Chinstrap penguins (Pygoscelis antarctica), collected in Antarctica during 2009 and 2010, for the presence of novel adenoviruses by PCR. Analysis of a 855-bp region of the hexon gene of a newly identified adenovirus, designated Chinstrap penguin adenovirus 1 (CSPAdV-1), showed nucleotide (amino acid) sequence identity of 71.8% (65.5%) with South Polar skua 1 (SPSAdV-1), 71% (70%) with raptor adenovirus 1 (RAdV-1), 71.4% (67.6%) with turkey adenovirus 3 (TAdV-3) and 61% (61.6%) with frog adenovirus 1 (FrAdV-1). Based on the genetic and phylogenetic analyses, CSPAdV-1 was classified as a member of the genus, Siadenovirus. Virus isolation attempts from kidney homogenates in the MDTC-RP19 (ATCC® CRL-8135™) cell line were unsuccessful. In conclusion, this study provides the first evidence of new adenovirus species in Antarctic penguins.

Previously, adenoviruses had been isolated from various vertebrate species on all continents, except Antarctica. Recently, the South Polar skua adenovirus 1 (SPSAdV-1), the single known member of the species, Skua siadenovirus A, was discovered in dead South Polar skua (Catharacta maccormicki) collected near the King Sejong Station in Antarctica [21]. However, there are limited surveillance studies of adenovirus infection in Antarctic wild birds. The South Polar skua can be observed in Antarctica only during the breeding season and feed on the chicks of penguins. Moreover, the South Polar skua shares breeding grounds with the Chinstrap penguin (Pygoscelis antarctica), an endemic species [22]. To ascertain if Chinstrap penguins in Antarctica are also infected with adenoviruses, we conducted an exploratory study on samples collected during 2009 and 2010.
Pair-wise alignment by ClustalW showed a six-amino acid difference between CSPAdVno1 originating from a Chinstrap penguin collected in 2009 and CSPAdVno2, no4 and no8. Twelve amino acid differences were found between CSPAdVno1 and CSPAdVno3, no5, no6 and no7 ( Figure 1). Particularly, CSPAdVno1, no2, no4 and CSPAdVno8 showed a deletion at position 242. As a result of sequence comparison with other types within the genus, Siadenovirus, the insertion of amino acid residue S at position 243 was found in the amino acid sequences of some CSPAdV-1 variants. Further study on the full genome sequence of CSPAdV-1 will be required to support the significance of sequence differences.  Within the genus, Siadenovirus, CSPAdVno1 was shown to have a nucleotide (amino acid) identity of 71.8% (65.5%) with SPSAdV-1, 71% (70%) with raptor adenovirus 1 (RAdV-1), 71.4% (67.6%) with turkey adenovirus 3 (TAdV-3), 69.4% (66.5%) with great tit adenovirus 1 (GTAdV-1) and 61% (61.6%) with frog adenovirus 1 (FrAdV-1). The nucleotide and amino acid sequence identity of each strain of CSPAdV-1 was more than 97% and 96%, respectively (Table 2). However, the nucleotide and amino acid sequences of CSPAdVno3, no5 and no7 and CSPAdVno2, no4 and no8 were identical (Figures 1 and 2). The nucleotide sequence identity of CSPAdV-1 was less (<33%) with other adenovirus genera, such as Atadenovirus, Aviadenovirus and Mastadenovirus. The G+C content of CSPAdVno1 and CSPAdVno2, no4 and no8 was found to be 37.44% and 37.2%, respectively. The partial hexon of CSPAdVno3, no5 and no7 had a G+C content of 37.42%. For phylogenetic analysis, approximately 855-bp of the hexon gene, which contains structural loop regions that encode serotype-specific epitopes, were selected [23]. CSPAdV-1 showed the highest similarity with SPSAdV-1 and RAdV-1 and was classified into the genus, Siadenovirus, based on phylogenetic trees generated by the maximum-parsimony and neighbor-joining methods, implemented in MEGA5.1 (Molecular Evolutionary Genetics Analysis, 5.1) (Figure 2) and PAUP version 4.0b (Phylogenetic Analysis Using Parsimony, 4.0b) [24,25]. A novel adenovirus species is usually defined as one detected in a new host species and having more than a 5%-15% sequence difference at the nucleotide and amino acid levels compared with previously characterized adenovirus species [3]. Based on these criteria, we conclude that CSPAdV-1 (Penguin siadenovirus A) seems to merit the establishment of a new species for it. Isolation of CSPAdV-1 was attempted by inoculating the MDTC-RP19 (ATCC® CRL-8135™) cell line [27] with kidney homogenates, but all such attempts were unsuccessful. Failure of the isolation of RAdV-1 on the chicken embryonic liver cell or MDTC-RP19 cell line has been reported previously [28,29].

Sample Collection
Chinstrap penguin carcasses were gathered at Narębski Point, located on the southeast coast of Barton Peninsula, King George Island, Antarctica (6213'40''S-6214'23''S and 5845'25''W-5847'00''W) during 2009 and early 2010 ( Figure 3). All carcasses were identified, weighed and measured. Internal organs (lung, liver, kidney, heart, intestine, trachea, brain, lymph node and spleen) were dissected using sterile instruments and stored at −70 °C until use for adenovirus identification.  The sequences of PCR primers were designated from the conserved region by the alignment of adenovirus sequences within the genus, Siadenovirus. PCR conditions were performed as follows: 1 cycle of 95 °C for 5 min followed by 14 cycles of denaturation at 95 °C for 40 s, one degree step-down each of 1 cycle annealing from 50 °C to 37 °C for 40 s, extension at 72 °C for 1 min, then 25 cycles of denaturation at 95 °C for 40 s, annealing at 42 °C for 40 s, extension at 72 °C for 1 min and, finally, at 72 °C for 5 min in a Mastercycler (Eppendorf, Germany).

Sequencing and Sequence Analysis
All PCR products were sequenced with the Big Dye terminator v3.1 cycle sequencing kit (ABI) and ABI3730 Automated DNA Sequencer (ABI). Nucleotide sequences were analyzed by ClustalW in MegAlign of DNAstar programs. Phylogenetic trees were generated by maximum-parsimony and neighbor-joining methods, implemented in MEGA5.1 and PAUP version 4.0b [24,25]. Topologies were evaluated by a bootstrap analysis of 1000 iterations by using MEGA5.1 software [26].

Isolation Attempts
For isolation attempts of CSPAdV-1, 5% (w/v) kidney homogenates of virus-infected penguins (CSP10-2 and CSP10-3) were inoculated onto MDTC-RP19 (ATCC® CRL-8135™), a lymphoblastoid cell line of turkey origin (Meleagris gallopavo) with high susceptibility to TAdV-3, a member of the genus, Siadenovirus [27]. Cultures were observed daily for the cytopathic effect, and the cells and supernatant were screened for the adenoviral hexon gene by PCR at each passage.

Conclusions
Previous studies on sub-Antarctic and Antarctic penguins have shown that avian species in this region may become infected with various viruses, including paramyxoviruses, Newcastle disease virus, infectious bursal disease virus and influenza viruses [30][31][32][33][34][35][36][37][38]. The identification of a novel adenovirus species in Chinstrap penguins suggests the possibility of other viruses, including additional previously unrecognized adenoviruses in Antarctic birds.
There is an increasing risk of infectious diseases being introduced into the Antarctic fauna, because of the increased numbers of people travelling to and within Antarctica [38,39]. Consequently, further studies in Antarctic birds may provide new insights into the emergence and dissemination of viral infectious diseases from Antarctica. performed histopathological study and ecological analysis. J.H.K. and H.G.C. provided penguin samples. J.W.S. conceived the project, and provided overall scientific oversight. All authors contributed to the preparation of the final manuscript.

Conflicts of Interest
The authors declare no conflict of interest.