Virology 485 (2015) 16–24

Contents lists available at ScienceDirect

Virology journal homepage: www.elsevier.com/locate/yviro

Characterization of a novel adenovirus isolated from a skunk$ Robert A. Kozak a, James G. Ackford a, Patrick Slaine a, Aimin Li b, Susy Carman c, Doug Campbell a, M. Katherine Welch c, Andrew M. Kropinski a, Éva Nagy a,n a

Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada Public Health Ontario, Toronto, Ontario, Canada c Animal Health Laboratory, Laboratory Services Division, University of Guelph, Guelph, Ontario, Canada b

art ic l e i nf o

a b s t r a c t

Article history: Received 5 February 2015 Returned to author for revisions 17 May 2015 Accepted 19 June 2015

Adenoviruses are a ubiquitous group of viruses that have been found in a wide range of hosts. A novel adenovirus from a skunk suffering from acute hepatitis was isolated and its DNA genome sequenced. The analysis revealed this virus to be a new member of the genus Mastadenovirus, with a genome of 31,848 bp in length containing 30 genes predicted to encode proteins, and with a G þC content of 49.0%. Global genomic organization indicated SkAdV-1 was similar in organization to bat and canine adenoviruses, and phylogenetic comparison suggested these viruses shared a common ancestor. SkAdV-1 demonstrated an ability to replicate in several mammalian liver cell lines suggesting a potential tropism for this virus. & 2015 Elsevier Inc. All rights reserved.

Keywords: Mastadenovirus Skunk adenovirus Whole genome sequence Liver tropism

Introduction Members of the diverse family Adenoviridae are classified into five genera, Mastadenovirus, Aviadenovirus, Atadenovirus, Siadenovirus, and Ichtadenovirus (Harrach et al., 2011). Adenoviruses are non-enveloped, icosahedral viruses that range in diameter from 70 to 100 nm. Their double-stranded DNA genomes are linear, nonsegmented, ranging in size from 26 to 48 kb, and typically contain between 22 and 40 genes (Davison et al., 2003). To date, over 44 species have been identified in the family Adenoviridae, and there is a wide range of host species capable of sustaining these viruses (Hall et al., 2012; Marek et al., 2014; Park et al., 2012; Robinson et al., 2013). Due to the relative speed and low cost of sequencing technologies, new isolates will continue to be added to each of these genera, and it is likely that the number of novel species will continue to grow. Adenoviruses have been isolated from human and a wide range of animal species, including lizards (Wellehan et al., 2004), fowl (Griffin and Nagy, 2011; Ojkic and Nagy, 2000), dogs (Morrison et al., 1997), bats (Li et al., 2010), horses (Cavanagh et al., 2012), titi monkeys (genus Callicebus) (Chen et al., 2011), and sea lions (Cortes-Hinojosa et al., 2015). While numerous mammalian species harbor adenoviruses, no novel viruses from this family have yet been reported in skunks (family Mephitidae), although these animals may serve as a spillover

host for canine adenoviruses (Alexander et al., 1972; Karstad et al., 1975). Skunks have traditionally been the focus of rabies virus surveillance, although they may also serve as hosts for parvoviruses (Allison et al., 2013), orthoreoviruses (Victoria et al., 2008), West Nile virus (Anderson et al., 2001) and Powassan virus (El Khoury et al., 2013). Therefore it is evident that skunks may serve as an underappreciated reservoir of viruses capable of infecting other mammals (Chen et al., 2011). We have characterized a novel adenovirus isolated from a skunk, hereafter referred to as skunk adenovirus 1 (SkAdV-1), and sequenced its genome using Illumina MiSeq next-generation sequencing technology. Genomic analysis reveals similarities between SkAdV-1 and both bat adenoviruses (BtAdV) and canine adenoviruses (CAdV). Phylogenetic analysis suggests that SkAdV-1 may have evolved in skunks from the common ancestor of both bat and canine adenoviruses. This study is the first in depth investigation into double-stranded DNA viruses, and more specifically adenoviruses, found in skunks. Based on our results we propose that this novel skunk adenovirus is a founding member of a new species, to be termed Skunk mastadenovirus A, within the genus Mastadenovirus.

Results Gross pathology and virus isolation

☆ The GenBank accession number for the skunk adenovirus sequence is KP238322. n Corresponding author. Tel.: þ 1 519 824 4120x54783. E-mail address: [email protected] (É. Nagy).

http://dx.doi.org/10.1016/j.virol.2015.06.026 0042-6822/& 2015 Elsevier Inc. All rights reserved.

A dead skunk (Mephitis mephitis) was originally presented to the Canadian Wildlife Health Cooperative, Department of Pathobiology, University of Guelph, for surveillance of wildlife rabies. However, the brain tissue was negative for rabies virus by indirect fluorescent

R.A. Kozak et al. / Virology 485 (2015) 16–24

17

Fig. 1. Virus replication and cytopathic effect (CPE) in MDCK cells infected with SkAdV-1. Cells were infected with a multiplicity of infection (MOI) of 5 (A) or were left uninfected (B), and were evaluated for CPE at 24 h post-infection (h.p.i.) and imaged using brightfield microscopy. (C) Cells were infected with SkAdV-1 at an MOI of 5, and at various h.p.i. the titers were determined in MDCK cells in order to establish a one-step growth curve. All experiments were performed in duplicate, and error bars represent the standard deviation.

antibody test. This juvenile female skunk was in good body condition, weighing 283 g. On post-mortem and histological evaluation, there were lesions of acute hepatitis, and mild icterus was observed in a wide range of tissues. The lungs were edematous and a clear pericardial effusion was present. Spleen and mesenteric lymph nodes were enlarged. The liver was bronze-colored, but otherwise unremarkable. Tissues were fixed in 10% formalin, sectioned in routine fashion and stained with hematoxylin and eosin for histological examination. No significant changes were observed in brain, heart, kidney, adrenal glands, esophagus and trachea. Lymphoid tissue in spleen and lymph node were depleted. There was congestion, edema and interstitial inflammation in the lung. In the liver, there was diffuse hepatic necrosis, with large, amphophilic intranuclear inclusions present in affected areas. Collectively, these changes were interpreted to be typical of adenovirus infection. Frozen liver tissue was subsequently used for virus isolation and analysis by transmission electron microscopy. Images displayed virions that were icosahedral in shape (data not shown), with a diameter of approximately 80–100 nm, both of which are morphological characteristics of adenoviruses (Pantelic et al., 2008). Virus isolation was attempted in different cell lines, and the most significant cytopathic effect (CPE) was observed in Madin-Darby canine kidney (MDCK) cells. The virus was plaque purified in MDCK cells and used for subsequent work. The CPE, manifested as cell rounding and detachment was seen as early as 24 h post-infection (h.p.i.) (Fig. 1A and B). Replication of the virus in MDCK cells was evaluated through a single step growth curve (Fig. 1C). Productive infection in these cells yielded viral titers of 108 PFU/ml after three days. Electron microscopic examination of the purified virus preparation showed typical adenovirus particles (Fig. 2). Characteristics of SkAdV-1 genome The genome of SkAdV-1 was sequenced using an Illumina MiSeq, with paired-end libraries at 2  150 bp cycle run. Any reads

Fig. 2. Electron micrograph of purified SkAdV-1 particles after negative staining. The bar represents 20 nm.

with a Q score below 30 were discarded, and 2857 times coverage of the complete viral genome was achieved. The genome length of SkAdV-1 is 31,848 bp, with left and right inverted terminal repeats each being 181 bp in length. The virus has a base composition of 26.2% A, 24.8% C, 24.2% G, 24.8% T, and the G þC content was 49.0%. These and related data are presented in Table 1 and in comparison to canine and bat adenoviruses. A large number of potential genes were identified by in silico analysis using Kodon and comparative genomics with other sequenced adenoviral genomes. An open reading frame (ORF) was taken as a potential coding sequence if it contained more than 75 nucleotides after an ATG as possible start codon. The initial list was reduced to 30 genes based on significant homology to known adenovirus genes, 11 of these genes were on the complementary DNA strand. The overall

18

R.A. Kozak et al. / Virology 485 (2015) 16–24

number of predicted genes and their arrangement were similar to other adenoviruses, notably BtAdVs and CAdVs (Li et al., 2010; Morrison et al., 1997). Furthermore, SkAdV-1 showed significant nucleotide sequence similarity to BtAdV-2 (species Bat mastadenovirus B) and CAdV-1 (species Canine mastadenovirus A) with 64% and 64.2% pairwise identity, respectively. Global genomic analysis revealed the region of greatest difference between SkAdV-1 and canine and bat adenoviruses was at the left and right end regions. The global organization of the SkAdV-1 genome is illustrated in Fig. 3. Genes predicted to encode proteins included a number of core genes common to adenoviruses including: DNA polymerase, pTP, 52K, penton base, hexon, protease and DNA binding protein. No evidence of gene duplication was found. Additionally, we did not detect any unique ORFs at either end of the SkAdV-1 genome, a region traditionally associated with variation in adenovirus genomic content (Davison et al., 2003). Collectively, analysis of the genes of SkAdV-1 indicated that the majority of them were similar to those of canine and

Table 1 Characteristics of SkAdV-1 genome and comparison to canine and bat mastadenoviruses. Designation

Genome size (bp)

Percent GC

CG dinucleotides

% identitya

SkAdV-1 CAdV-1 CAdV-2 BtAdV-3 BtAdV-2

31,848 30,536 31,323 31,681 31,161

49.0 47.0 50.3 56.8 53.5

984 989 1064 1895 1432

100 62.6 64.2 63.5 64

a

Percent nucleotide identity compared to SkAdV-1.

bat adenoviruses, although the small T-antigen showed greatest similarity to one found in equine adenovirus 1 (EAdV-1). The complete list of predicted genes, including homologs with the greatest percent identity is summarized in Table 2. Interestingly, similar to bat adenoviruses, no gene predicted to code for VA RNA was detected in SkAdV-1 (Li et al., 2010). Phylogenetic analysis of SkAdV-1 Phylogenetic analysis of sequences of four genes of the SkAdV1 genome indicated that SkAdV-1 should be classified within the genus Mastadenovirus. SkAdV-1 formed a separate branch, distinct from CAdVs and BtAdVs, although they likely share a common ancestor. Specifically, the amino acid sequences of the DNA polymerase, penton base, hexon and fiber were examined. Molecular typing and classification of adenoviruses are often done by analysis of the hexon amino acid sequence (Lu and Erdman, 2006). The hexon of SkAdV-1 was similar in length to those of CAdVs and BtAdVs, and was shorter than the HAdV sequences examined. The assessment of the DNA polymerase sequence found highest identity (69%) to CAdV-2; although SkAdV-1 was arranged in its own branch, separate from both the canine and bat adenoviruses (Fig. 4A). Phylogeny indicated the hexon of SkAdV-1 to be most similar to those of CAdVs and BtAdVs, with highest identity (86%) to that of CAdV-2 (Fig. 4B). We next examined the predicted sequences of the fiber and penton base proteins. These proteins interact with cellular receptors, and act in concert to facilitate viral entry into the host cell (Russell, 2009). Both predicted proteins showed divergence from other adenoviruses, with highest identity (43% and 82%, respectively) to CAdV-1 (Fig. 4C and D). This

Fig. 3. Organization and global comparison of SkAdV-1 genome. The genome is represented by a thick black line, and predicted genes and ORFs and their directions are denoted in purple arrows. The predicted organization of the SkAdV-1 indicates 30 predicted genes distributed over both strands. Below the genome map, global pairwise comparison of homology to canine and bat adenoviruses is shown. Analysis was performed using mVISTA and regions of greater than 75% homology are colored red.

R.A. Kozak et al. / Virology 485 (2015) 16–24

19

Table 2 Predicted gene products of SkAdV-1. Gene

Location (bp)

Strand

Length (aa)

Description

Closest viral homolog (GenBank accession no.)

Identity (%)

E1A 28.6K E1B 20.8K E1B 50.5K IX IVa2 pol pTP 52K pIIIa III pVII V pX pVI hexon protease DBP 100K 22K pVIII E3 12.5K E3 ORFA U exon fiber E4 ORF6/7 E4 34K E4 ORFD E4 ORFC E4 ORFB E4 ORFA

462–1305 1413–1961 1775–3136 3213–3512 3531–5149 4622–12599 7900–12599 9775–10965 10832–12577 12625–14061 14091–14525 14620–15924 15953–16165 16235–17056 17132–19855 19868–20488 20533–21846 21859–23952 23804–24310 24452–25120 25107–25454 25521–26726 26743–26910 26909–28729 28759–29763 28990–29763 29771–30160 30160–30534 30567–30926 31008–31403

þ þ þ þ – – – þ þ þ þ þ þ þ þ þ – þ þ þ þ þ – þ – – – – – –

260 182 453 99 449 1143 610 396 581 478 144 434 70 273 907 206 437 697 168 222 115 401 55 606 89 257 129 124 119 131

Early E1A protein E1B protein, small T-antigen E1B protein, large T-antigen protein XI/hexon associated IVa2 multifunctional protein DNA polymerase precursor terminal protein late encapsidation protein IIIa protein precursor penton base protein core protein protein V, minor core protein Mu peptide precursor minor capsid protein precursor capsid protein 23K endopeptidase DNA binding protein 100 kDa hexon assembly protein 22 kDa encapsidation protein hexon associated protein E3 12.5K control protein E3 ORFA U exon fiber protein E4 ORF6/7 control protein E4 34 kDa early protein E4 ORFD E4 ORFC E4 ORFB E4 ORFA

BtAdV-2 (YP_004782095.1) EAdV-1 (AEP16406.1) BtAdV-2 (YP_004782097.1) CAdV-2 (AP_000611.1) CAdV-2 (AP_000612.1) CAdV-2 (AP_000613.1) BtAdV-3 (YP_005271183.1) BtAdV-2 (YP_004782102.1) CAdV-1 (NP_044192.1) CAdV-1 (NP_044193.1) BtAdV-2 (YP_004782105.1) CAdV-2 (AP_000619.1) BtAdV-2 (YP_004782107.1) CAdV-2 (NP_044197.1) CAdV-2 (ACF19805.1) BtAdV-2 (YP_004782110.1) BtAdV-3 (YP_005271193.1) CAdV-2 (AP_000625.1) BtAdV-3 (YP_005271196.1) BtAdV-3 (YP_005271197.1) CAdV-1 (NP_044204.1) BtAdV-3 (YP_005271198.1) BtAdV-3 (YP_005271199.1) CAdV-1 (ACT22747.1) BtAdV-3 (YP_005271201.1) CAdV-2 (AP_000634.1) EAdV-1 (AEP16435.1) BtAdV-3 (YP_005271204.1) BtAdV-3 (YP_005271205.1) CAdV-2 (AP_000638.1)

53 54 48 63 70 69 79 69 79 82 72 62 83 66 86 84 60 73 56 80 41 33 74 43 48 52 30 31 61 45

BtAdV-2¼bat adenovirus 2; EAdV-1¼ equine adenovirus 1; CAdV-2¼canine adenovirus 2; BtAdV-3¼ bat adenovirus 3; CAdV-1¼ canine adenovirus 1.

phylogenetic analysis reinforced the hypothesis that SkAdV-1 is a member of a novel mastadenovirus species. Additionally, we performed multiple alignments of the predicted fiber and penton base proteins using ClustalX2 (Larkin et al., 2007). Comparison of the fiber sequences indicated that the SkAdV-1 fiber was approximately 50 amino acids longer than that of BtAdV-3 or CAdV-2. However, there was significant similarity between the knob regions of SkAdV-1 fiber and those of CAdV-1, CAdV-2 and BtAdV-3 (Fig. 5B). Interestingly, examination of the penton base sequence revealed that it did not contain the RGD motif (Fig. 5A), which is a known cell adhesion motif, suggesting that SkAdV-1 may enter cells through an RGD independent mechanism, similar to that observed with CAdV-2 (Soudais et al., 2000).

Virus replication in mammalian liver cell lines The fact that the virus was isolated from an acute case of hepatitis in a skunk, suggested that SkAdV-1 would replicate in other mammalian liver cells. Moreover, the liver tropism of some mammalian AdVs is well established (Einfeld et al., 2001; Nakamura et al., 2003). Therefore, we examined CPE and virus titers, at 72 h.p.i., in three mammalian (Huh-7, Huh-7.5 and HepG2) and one non-mammalian (avian CH-SAH) liver cell lines infected with SkAdV-1. The cells were infected at a multiplicity of infection (MOI) of 3 and extensive CPE was seen in all cells except for CH-SAH (Fig. 7). Moreover, the virus titers were 1  107 PFU/ml in Huh-7 cells, 1  108 PFU/ml in Huh-7.5 cells and 2  107 PFU/ml in HepG2 cells. By contrast, in the chicken derived CH-SAH cells the virus titer was 9  104 PFU/ml.

Virus replication in bat and canine cell lines Our sequence analysis indicated a high probability that SkAdV1 evolved from the common ancestor of BtAdVs. To investigate potential cell tropisms, both bat (Tb-1 Lu) and canine (MDCK) cell lines were infected with SkAdV-1 and virus titers were determined at 72 h.p.i. (Fig. 6). Titers were three logs higher in MDCK cells (p ¼0.01), and there was greater CPE, compared with Tb-1 Lu cells. Recent work by Li et al. (2010) demonstrated that while Tb-1 Lu cells were not susceptible to BtAdV-3, the virus could infect MDCK cells (Li et al., 2010). This similar phenotype was noted with SkAdV-1, although when the number of CG nucleotides was analyzed using EMBOSS 6.3.1 geecee (〈http://mobyle.pasteur.fr〉), SkAdV-1 had fewer GC dinucleotides than any BtAdV species (Table 1). It has been noted that reduction in the number of CpG dinucleotides is often linked to viral adaptation and stability within a particular host species, suggesting SkAdV-1 may be adapting to the skunk host (Karlin et al., 1994; Upadhyay et al., 2013).

Discussion Massive parallel sequencing technology has expanded our knowledge of the viruses present in both animals and humans (Ge et al., 2012; Lysholm et al., 2012; Ng et al., 2012; Wevers et al., 2010). Here we have isolated a novel adenovirus from a skunk exhibiting acute hepatitis, and sequenced and characterized its genome. Our investigation showed that the SkAdV-1 genome is similar in size and arrangement to the genomes of four types of three species within the genus, notably bat and canine adenoviruses (Kohl et al., 2012; Li et al., 2010; Morrison et al., 1997). However, phylogenetic placement of this virus indicates that SkAdV-1 is a member of its own distinct species though within the genus Mastadenovirus. We failed to find any unique ORFs, nor any gene duplications. The analyses of the DNA polymerase, penton base, hexon, and fiber predicted amino acid sequences suggest that SkAdV-1 and both CAdVs and BtAdVs may have

20

R.A. Kozak et al. / Virology 485 (2015) 16–24

Fig. 4. Phylogenetic comparison of SkAdV-1 genes. Phylogenetic trees were based on amino acid sequences of the (A) DNA polymerase, (B) hexon, (C) fiber, and (D) penton base. Sequences were aligned using ClustalW (BLOSUM cost matrix), and phylogenic analysis was in Geneious Version 8.05 using the PhyML function with the unweighted pair group method with arithmetic mean (UPGMA) under default settings (Bootstrap resampling method, 1000 replicates).

evolved from a common ancestor. It is also possible that a bat adenovirus may have made a species jump into a skunk, and evolved within that host. This theory is supported by the observation that SkAdV-1 showed reduced titers in the Tb-1 Lu fruit bat cell line. However, unlike BtAdV-3, SkAdV-1 did not replicate well in Vero cells (data not shown), potentially indicating further viral adaptation. Moreover, the report of adenovirus transmission between bats, coupled with the detection of high virus titers in the intestines of infected bats suggests a gastrointestinal disease that could also spread to other animals (Sonntag et al., 2009).

Studies examining the epidemiology of CAdV-2 have demonstrated a wide distribution across a number of mammals, and postulate that this virus may have evolved from bat adenovirus (Buonavoglia and Martella, 2007; Kohl et al., 2012). Notably, there have been reports of disease similar to infectious canine hepatitis, suggesting SkAdV-1 infections may be widespread but under reported (Alexander et al., 1972; Karstad et al., 1975). However, these studies failed to determine if the viral infection was in fact due to SkAdV-1 or due to CAdV-1, the etiological agent of the similar disease in dogs. Therefore, it remains to be determined

R.A. Kozak et al. / Virology 485 (2015) 16–24

21

Fig. 5. Multiple alignments of the penton base and fiber knob region sequences of selected mammalian adenoviruses. (A) The sequence alignment of the penton base from CAdV-1, CAdV-2, BtAdV-3, SkAdV-1, and HAdV-3 was performed using ClustalX2. The RGD motif in HAdV-3 is highlighted (\widehat\widehat\widehat), and absent from the remaining adenovirus sequences. (B) Multiple alignments of the knob region of the fiber sequences of selected mammalian adenoviruses. The sequence alignments were performed using ClustalX2.

whether skunks act as a reservoir, an amplifier host, a spillover event for SkAdV-1, or if in fact the virus evolved from a predecessor in this animal. It is possible that this virus has a broader host range, similar to other adenoviruses, and further investigation is necessary (Buonavoglia and Martella, 2007; Chen et al., 2011; Yu et al., 2013). Furthermore, it must be noted that although SkAdV-1 was isolated from a diseased animal, Koch's postulates have not been fulfilled to link this virus to the documented pathology. Therefore, additional work with animal models will be required to formally demonstrate it. The pathology documented in this case is in agreement with that associated with adenoviruses (Morrison et al., 1997). Hepatitis is a known sequela of adenoviral infection, as evidenced by a recent report showing TMAdV causes hepatitis in new world monkeys (Chen et al., 2011). Furthermore, this new skunk adenovirus is capable of infecting and causing CPE in a number of mammalian liver cell lines, specifically Huh-7, Huh-7.5 and HepG2, suggesting SkAdV-1 may display a liver tropism commonly found among other adenoviruses (Einfeld et al., 2001; Nakamura et al., 2003). The higher titers obtained in Huh-7, Huh-7.5 and HepG2 cell lines compared to CH-SAH could suggest the virus replicates or is internalized more efficiently in mammalian liver cells. It has been reported that CAR and integrin are highly expressed in liver cells (Fechner et al., 1999), however expression levels could be significantly lower in avian cells, thereby reducing viral uptake. Nevertheless, it is also possible that virus internalization or the

later steps of viral replication are inferior in avian cells, and further studies are required to elucidate on these. The absence of other receptors, presence of mitigating factors (eg. Homologs of mouse CAR-like soluble protein) (Kawabata et al., 2007), or differences in receptor structure must also be considered. Adenovirus attachment is facilitated by fiber protein interacting with CAR, followed by an interaction of the RGD motif on the penton base with αVβ3 or αVβ5 integrins to allow internalization (Bergelson et al., 1997; Zhang and Bergelson, 2005). Assessment of the SkAdV-1 penton base sequence did not reveal an RGD, RGE or other known integrin binding motif, suggesting that viral entry into cells may be through an unknown pathway (Belin and Boulanger, 1993; Bergelson et al., 1997; Soudais et al., 2000). CAdV-2 enters the cell through a CAR-independent pathway, and as this virus likely evolved from the same common ancestor as SkAdV-1, it is likely they share similar mechanisms of infection (Soudais et al., 2000). Furthermore, other mammalian adenoviruses have been isolated that lack this motif, a recent example is a novel adenovirus in species Human mastadenovirus D, which was found to contain YGD on the penton base (Robinson et al., 2013). The varied levels of virus replication may also be due to differences in host immune factors and the ability of SkAdV-1 to modulate the innate immune response. The E3 region is involved in suppression of host cytokines, chemokines, MHC-I, and apoptosis (Friedman and Horwitz, 2002; Lesokhin et al., 2002; Wold and Gooding, 1991). This region is dispensable for replication, and

22

R.A. Kozak et al. / Virology 485 (2015) 16–24

variations in it may alter viral pathogenesis (Marinheiro et al., 2011). Interestingly our elucidation of the SkAdV-1 genome predicted the presence of the E3 12.5K followed by E3 ORFA, which is

a similar organization to canine adenoviruses (Morrison et al., 1997).

Conclusions Currently, there is a paucity of studies on the virome of skunks, and the main focus of many skunk studies is on rabies. The characterization of a novel skunk adenovirus not only broadens our understanding of the evolution of adenoviruses, but also suggests that these animals may be an underappreciated reservoir of viruses. The data here present a strong case for proposing SkAdV-1 as the first representative of a novel species in genus Mastadenovirus, as it meets multiple criteria laid out by the International Committee on the Taxonomy of Viruses; importantly the phylogenetic distance of the DNA polymerase is more than 15%. Further studies are required not only to characterize the epidemiology of this adenovirus in the skunk population, but to also determine its role in causing disease, and its host range.

Materials and methods Virus isolation and propagation

Fig. 6. Viral titers and CPE in Tb-1 Lu and MDCK cells. Cells were infected at an MOI of 3, and (A) the virus titers were determined at 72 h.p.i. by titration in MDCK cells. Experiments were performed in triplicate, and error bars represent the standard error of the mean. Samples were compared using Students t-test with significant differences indicated by * (p r 0.05). (B) Less CPE was seen in Tb-1 Lu cells compared to MDCK cells at this time point. Experiments were performed in triplicate and one representative sample is shown.

To isolate virus, tissue homogenate of a skunk liver was filtered through a 0.45 mm syringe top filter (BD Biosciences, Mississauga, Ontario) before being added to MDCK, Vero, A-72, Crandall-Rees feline kidney and mink lung epithelial cell cultures. The cell lines were evaluated daily for CPE, and based on the extent of CPE the MDCK cell line was selected for further work. Plaque purification, virus propagation and titrations were performed in MDCK cells as described previously (Griffin and Nagy, 2011; Ojkic and Nagy, 2000).

Fig. 7. SkAdV-1 replication in mammalian and non-mammalian liver cell lines. Mammalian (Huh-7, Huh-7.5 and HepG2) and avian (CH-SAH) liver cell lines were infected at an MOI of 3 and CPE was evaluated at 72 h.p.i. by brightfield microscopy. Experiments were performed in triplicate, and one representative example is shown.

R.A. Kozak et al. / Virology 485 (2015) 16–24

23

DNA extraction and sequencing

Nucleotide accession numbers

SkAdV-1 was concentrated and the genomic DNA was extracted as described previously (Ojkic and Nagy, 2001). Concentration and quality of the genomic DNA were determined using Qubit dsDNA Assay kit (Life Technology Inc., Oakville, Canada). Adenoviral genomic DNA was acoustically fragmented using the Covaris M220 ultrasonicator and viral libraries were prepared using Illumina Truseq DNA Library Preparation kit (Illumina, California, USA). Indexed libraries were evaluated using Agilent High Sensitivity DNA chip on an Agilent 2100 Bioanalyser system (Agilent Technologies Canada Ltd, Mississauga, Canada) according to manufacturer's specifications.

Dilutions of the concentrated and purified virus sample were adhered to a copper grid for 10 s, stained with 1% uranyl acetate for 7 s, then washed once with double distilled H2O, and allowed to air dry. Grids were imaged using the FEI Tecnai G2 F20 operated at 120 kV. The transmission electron microscopy was performed at the University of Guelph.

The GenBank accession number of the skunk adenovirus 1 (SkAdV-1) genome sequence reported in this paper is KP238322. In order to perform phylogenetic comparisons, the following adenovirus genomes were retrieved from GenBank: bovine adenovirus 3 (BAdV-3, AF030154), bovine adenovirus 1 (BAdV-1, AC_000191.1), bovine adenovirus 2 (BAdV-2 AF252854), bovine adenovirus 10 (BAdV-10, AF027599), canine adenovirus 1 (CAdV-1, Y07760), canine adenovirus 2 (CAdV-2, U77082), equine adenovirus 1 (EAdV-1, L79955), equine adenovirus 2 (EAdV-2, L80007), human adenovirus 12 (HAdV-12, X73487), human adenovirus 1 (HAdV-1, AF534906), human adenovirus 8 (HAdV-8, AB448767), human adenovirus 4 (HAdV-4, AY487947), human adenovirus 3 (HAdV-3, NC_011203), human adenovirus 12 (HAdV-12, AB330093), simian adenovirus 22 (SAdV-22, AY530876), human adenovirus 40 (HAdV-40, L19443), human adenovirus 52 (HAdV52, DQ923122), murine adenovirus 1 (MAdV-1, NC_000942), murine adenovirus 2 (MAdV-2, NC_014899), murine adenovirus 3 (MAdV-3, EU835513), porcine adenovirus 5 (PAdV-5, AF289262), tree shrew adenovirus 1 (TSAdV-1, AF258784), titi monkey adenovirus (TMAdV, NC_020487), bat adenovirus 2 (BtAdV-2, FJ983127), bat adenovirus 3 (BtAdV-3, GU226970).

Cell lines and virus replication

Statistical analyses

The mammalian cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fischer Scientific, Whitby, Ontario) supplemented with 10% fetal bovine serum (FBS) (Sigma, Oakville, Ontario), 2 mM L-glutamine, and penicillin (100 IU/ml)/streptomycin (100 ug/ml) (Sigma, Oakville, Ontario). The cell lines used include MDCK, HepG2, Huh-7, Huh-7.5, and Tb-1 Lu. The chicken hepatoma cells (CH-SAH) were maintained in DMEM/F12 (Sigma) supplemented with 10% FBS (Sigma, Oakville, Ontario), 2 mM L-glutamine and penicillin (100 IU/ml)/streptomycin (100 mg/ml) (Sigma, Oakville, Ontario) (Griffin and Nagy, 2011). To assess viral replication cell monolayers were grown to 80% confluency prior to infection, and were infected as described previously (Ojkic and Nagy, 2001).

Figures were generated using GraphPad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA). Statistical analyses were also performed using this software. Differences between means were evaluated using the Student's t-test and were deemed significant at p-values r0.05.

Electron microscopy

Genomic and phylogenetic analysis The primary deep sequence data, representing 2857-fold coverage of the viral genome, was de novo assembled using SeqMan NGen12 (DNASTAR, Madison, WI). The ORFs were identified in Kodon (Applied Maths, Austin, TX) with respect to other adenoviruses, with homologs identified on the basis of BLASTP analyses. Splice sites were calculated using alternative splice site predictor (ASSP) (Wang and Silverman, 2006), and cross-referenced with the annotated genomes of BtAdV-2 and BtAdV-3 (Accession no. JN252129, GU226970). Amino acid sequences for the DNA polymerase, penton base, hexon and fiber were downloaded from GenBank, and aligned using ClustalW (BLOSUM cost matrix). Phylogenetic analysis was performed in Geneious Version 8.05 (Biomatters, Auckland, New Zealand) using the PhyML function with the unweighted pair group method with arithmetic mean (UPGMA) under default settings (Bootstrap resampling method with 1000 replicates). Multiple alignments of the penton base and fiber sequences of selected mammalian adenoviruses were performed using ClustalX2 (Larkin et al., 2007). The number of CG dinucleotides were enumerated using EMBOSS 6.3.1 geecee (〈http://genome.lbl.gov/vista/index.shtml〉). Evaluation of global pairwise identity of the SkAdV-1 whole genome to other adenoviral sequences was by using mVISTA (〈http://genome.lbl.gov/ vista/mvista〉) (Brudno et al., 2003).

Acknowledgments The authors would like to acknowledge Susan Emery, Betty-Anne McBey and David Leishman for their excellent technical assistance. This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), RGPIN 41625-2013. The authors have no conflicts of interest to declare. References Alexander, A.D., Flyger, V., Herman, Y.F., McConnell, S.J., Rothstein, N., Yager, R.H., 1972. Survey of wild mammals in a Chesapeake Bay area for selected zoonoses. J. Wildl. Dis. 8, 119–126. Allison, A.B., Kohler, D.J., Fox, K.A., Brown, J.D., Gerhold, R.W., Shearn-Bochsler, V.I., Dubovi, E.J., Parrish, C.R., Holmes, E.C., 2013. Frequent cross-species transmission of parvoviruses among diverse carnivore hosts. J. Virol. 87, 2342–2347. Anderson, J.F., Vossbrinck, C.R., Andreadis, T.G., Iton, A., Beckwith 3rd, W.H., Mayo, D.R., 2001. Characterization of West Nile virus from five species of mosquitoes, nine species of birds, and one mammal. Ann. N.Y. Acad. Sci. 951, 328–331. Belin, M.T., Boulanger, P., 1993. Involvement of cellular adhesion sequences in the attachment of adenovirus to the HeLa cell surface. J. Gen. Virol. 74, 1485–1497. Bergelson, J.M., Cunningham, J.A., Droguett, G., Kurt-Jones, E.A., Krithivas, A., Hong, J.S., Horwitz, M.S., Crowell, R.L., Finberg, R.W., 1997. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275, 1320–1323. Brudno, M., Do, C.B., Cooper, G.M., Kim, M.F., Davydov, E., Program, N.C.S., Green, E.D., Sidow, A., Batzoglou, S., 2003. LAGAN and Multi-LAGAN: efficient tools for largescale multiple alignment of genomic DNA. Genome Res. 13, 721–731. Buonavoglia, C., Martella, V., 2007. Canine respiratory viruses. Vet. Res. 38, 355–373. Cavanagh, H.M., Mahony, T.J., Vanniasinkam, T., 2012. Genetic characterization of equine adenovirus type 1. Vet. Microbiol. 155, 33–37. Chen, E.C., Yagi, S., Kelly, K.R., Mendoza, S.P., Tarara, R.P., Canfield, D.R., Maninger, N., Rosenthal, A., Spinner, A., Bales, K.L., Schnurr, D.P., Lerche, N.W., Chiu, C.Y., 2011. Cross-species transmission of a novel adenovirus associated with a fulminant pneumonia outbreak in a new world monkey colony. PLoS Pathog. 7, e1002155. Cortes-Hinojosa, G., Gulland, F.M.D., Goldstein, T., Venn-Watson, S., Rivera, R., Waltzek, T.B., Salemi, M., Wellehan Jr., J.F.X., 2015. Phylogenomic characterization of California sea lion adenovirus-1. Infect. Genet. Evol. 31, 270–276.

24

R.A. Kozak et al. / Virology 485 (2015) 16–24

Davison, A.J., Benko, M., Harrach, B., 2003. Genetic content and evolution of adenoviruses. J. Gen. Virol. 84, 2895–2908. Einfeld, D.A., Schroeder, R., Roelvink, P.W., Lizonova, A., King, C.R., Kovesdi, I., Wickham, T.J., 2001. Reducing the native tropism of adenovirus vectors requires removal of both CAR and integrin interactions. J. Virol. 75, 11284–11291. El Khoury, M.Y., Camargo, J.F., White, J.L., Backenson, B.P., Dupuis 2nd, A.P., Escuyer, K.L., Kramer St, L., George, K., Chatterjee, D., Prusinski, M., Wormser, G.P., Wong, S.J., 2013. Potential role of deer tick virus in Powassan encephalitis cases in Lyme disease-endemic areas of New York, USA. Emerg. Infect. Dis. 19, 1926–1933. Fechner, H., Haack, A., Wang, H., Wang, X., Eizema, K., Pauschinger, M., Schoemaker, R., Veghel, R., Houtsmuller, A., Schultheiss, H.P., Lamers, J., Poller, W., 1999. Expression of coxsackie adenovirus receptor and alphav-integrin does not correlate with adenovector targeting in vivo indicating anatomical vector barriers. Gene Ther. 6, 1520–1535. Friedman, J.M., Horwitz, M.S., 2002. Inhibition of tumor necrosis factor alphainduced NF-kappa B activation by the adenovirus E3-10.4/14.5K complex. J. Virol. 76, 5515–5521. Ge, X., Li, Y., Yang, X., Zhang, H., Zhou, P., Zhang, Y., Shi, Z., 2012. Metagenomic analysis of viruses from bat fecal samples reveals many novel viruses in insectivorous bats in China. J. Virol. 86, 4620–4630. Griffin, B.D., Nagy, E., 2011. Coding potential and transcript analysis of fowl adenovirus 4: insight into upstream ORFs as common sequence features in adenoviral transcripts. J. Gen. Virol. 92, 1260–1272. Hall, N.H., Archer, L.L., Childress, A.L., Wellehan Jr., J.F., 2012. Identification of a novel adenovirus in a cotton-top tamarin (Saguinus oedipus). J. Vet. Diagn. Investig. 24, 359–363. Harrach, B., Benkő, M., Both, G.W., Brown, M., Davison, A.J., Echavarría, M., Hess, M., Jones, M.S., Kajon, A., 2011. Virus Taxonomy: Classification and Nomenclature of Viruses: Ninth Report of the International Committee on Taxonomy of Viruses. In: King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J. (Eds.), Elsevier, San Diego, pp. 125–141. Karlin, S., Doerfler, W., Cardon, L.R., 1994. Why is CpG suppressed in the genomes of virtually all small eukaryotic viruses but not in those of large eukaryotic viruses? J. Virol. 68, 2889–2897. Karstad, L., Ramsden, R., Berry, T.J., Binn, L.N., 1975. Hepatitis in skunks caused by the virus of infectious canine hepatitis. J. Wildl. Dis. 11, 494–496. Kawabata, K., Tashiro, K., Sakurai, F., Osada, N., Kusuda, J., Hayakawa, T., Yamanishi, K., Mizuguchi, H., 2007. Positive and negative regulation of adenovirus infection by CAR-like soluble protein, CLSP. Gene Ther. 14, 1199–1207. Kohl, C., Vidovszky, M.Z., Muhldorfer, K., Dabrowski, P.W., Radonic, A., Nitsche, A., Wibbelt, G., Kurth, A., Harrach, B., 2012. Genome analysis of bat adenovirus 2: indications of interspecies transmission. J. Virol. 86, 1888–1892. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948. Lesokhin, A.M., Delgado-Lopez, F., Horwitz, M.S., 2002. Inhibition of chemokine expression by adenovirus early region three (E3) genes. J. Virol. 76, 8236–8243. Li, Y., Ge, X., Zhang, H., Zhou, P., Zhu, Y., Zhang, Y., Yuan, J., Wang, L.F., Shi, Z., 2010. Host range, prevalence, and genetic diversity of adenoviruses in bats. J. Virol. 84, 3889–3897. Lu, X., Erdman, D.D., 2006. Molecular typing of human adenoviruses by PCR and sequencing of a partial region of the hexon gene. Arch. Virol. 151, 1587–1602. Lysholm, F., Wetterbom, A., Lindau, C., Darban, H., Bjerkner, A., Fahlander, K., Lindberg, A.M., Persson, B., Allander, T., Andersson, B., 2012. Characterization of the viral microbiome in patients with severe lower respiratory tract infections, using metagenomic sequencing. PloS ONE 7, e30875. Marek, A., Ballmann, M.Z., Kosiol, C., Harrach, B., Schlotterer, C., Hess, M., 2014. Whole-genome sequences of two turkey adenovirus types reveal the existence

of two unknown lineages that merit the establishment of novel species within the genus Aviadenovirus. J. Gen. Virol. 95, 156–170. Marinheiro, J.C., Dos Santos, T.G., Siqueira-Silva, J., Lu, X., Carvalho, D., da Camara, A.A., Arruda, E., Arruda, K., Erdman, D.D., Harsi, C.M., 2011. A naturally occurring human adenovirus type 7 variant with a 1743 bp deletion in the E3 cassette. J. Gen. Virol. 92, 2399–2404. Morrison, M.D., Onions, D.E., Nicolson, L., 1997. Complete DNA sequence of canine adenovirus type 1. J. Gen. Virol. 78, 873–878. Nakamura, T., Sato, K., Hamada, H., 2003. Reduction of natural adenovirus tropism to the liver by both ablation of fiber-coxsackievirus and adenovirus receptor interaction and use of replaceable short fiber. J. Virol. 77, 2512–2521. Ng, T.F., Marine, R., Wang, C., Simmonds, P., Kapusinszky, B., Bodhidatta, L., Oderinde, B.S., Wommack, K.E., Delwart, E., 2012. High variety of known and new RNA and DNA viruses of diverse origins in untreated sewage. J. Virol. 86, 12161–12175. Ojkic, D., Nagy, E., 2000. The complete nucleotide sequence of fowl adenovirus type 8. J. Gen. Virol. 81, 1833–1837. Ojkic, D., Nagy, E., 2001. The long repeat region is dispensable for fowl adenovirus replication in vitro. Virology 283, 197–206. Pantelic, R.S., Lockett, L.J., Rothnagel, R., Hankamer, B., Both, G.W., 2008. Cryoelectron microscopy map of Atadenovirus reveals cross-genus structural differences from human adenovirus. J. Virol. 82, 7346–7356. Park, Y.M., Kim, J.H., Gu, S.H., Lee, S.Y., Lee, M.G., Kang, Y.K., Kang, S.H., Kim, H.J., Song, J.W., 2012. Full genome analysis of a novel adenovirus from the South Polar skua (Catharacta maccormicki) in Antarctica. Virology 422, 144–150. Robinson, C.M., Zhou, X., Rajaiya, J., Yousuf, M.A., Singh, G., DeSerres, J.J., Walsh, M.P., Wong, S., Seto, D., Dyer, D.W., Chodosh, J., Jones, M.S., 2013. Predicting the next eye pathogen: analysis of a novel adenovirus. mBio 4 (2) e00595-12. Russell, W.C., 2009. Adenoviruses: update on structure and function. J. Gen. Virol. 90, 1–20. Sonntag, M., Muhldorfer, K., Speck, S., Wibbelt, G., Kurth, A., 2009. New adenovirus in bats, Germany. Emerg. Infect. Dis. 15, 2052–2055. Soudais, C., Boutin, S., Hong, S.S., Chillon, M., Danos, O., Bergelson, J.M., Boulanger, P., Kremer, E.J., 2000. Canine adenovirus type 2 attachment and internalization: coxsackievirus-adenovirus receptor, alternative receptors, and an RGDindependent pathway. J. Virol. 74, 10639–10649. Upadhyay, M., Samal, J., Kandpal, M., Vasaikar, S., Biswas, B., Gomes, J., Vivekanandan, P., 2013. CpG dinucleotide frequencies reveal the role of host methylation capabilities in parvovirus evolution. J. Virol. 87, 13816–13824. Victoria, J.G., Kapoor, A., Dupuis, K., Schnurr, D.P., Delwart, E.L., 2008. Rapid identification of known and new RNA viruses from animal tissues. PLoS Pathog. 4, e1000163. Wang, Y., Silverman, S.K., 2006. Experimental tests of two proofreading mechanisms for 50 -splice site selection. ACS Chem. Biol. 1, 316–324. Wellehan, J.F., Johnson, A.J., Harrach, B., Benko, M., Pessier, A.P., Johnson, C.M., Garner, M.M., Childress, A., Jacobson, E.R., 2004. Detection and analysis of six lizard adenoviruses by consensus primer PCR provides further evidence of a reptilian origin for the atadenoviruses. J. Virol. 78, 13366–13369. Wevers, D., Leendertz, F.H., Scuda, N., Boesch, C., Robbins, M.M., Head, J., Ludwig, C., Kuhn, J., Ehlers, B., 2010. A novel adenovirus of Western lowland gorillas (Gorilla gorilla gorilla). Virol. J. 7, 303. Wold, W.S., Gooding, L.R., 1991. Region E3 of adenovirus: a cassette of genes involved in host immunosurveillance and virus-cell interactions. Virology 184, 1–8. Yu, G., Yagi, S., Carrion Jr., R., Chen, E.C., Liu, M., Brasky, K.M., Lanford, R.E., Kelly, K.R., Bales, K.L., Schnurr, D.P., Canfield, D.R., Patterson, J.L., Chiu, C.Y., 2013. Experimental cross-species infection of common marmosets by titi monkey adenovirus. PloS ONE 8, e68558. Zhang, Y., Bergelson, J.M., 2005. Adenovirus receptors. J. Virol. 79, 12125–12131.