Molecular epidemiology of canine picornavirus in Hong Kong and Dubai and proposal of a novel genus in Picornaviridae

Infection, Genetics and Evolution 41 (2016) 191–200

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Research paper

Molecular epidemiology of canine picornavirus in Hong Kong and Dubai and proposal of a novel genus in Picornaviridae Patrick C.Y. Woo a,b,c,d,e,⁎,1, Susanna K.P. Lau a,b,c,d,e,1, Garnet K.Y. Choi b,1, Yi Huang b, Saritha Sivakumar f, Hoi-Wah Tsoi b, Cyril C.Y. Yip b, Shanty V. Jose f, Ru Bai b, Emily Y.M. Wong b, Marina Joseph f, Tong Li b, Ulrich Wernery f,⁎, Kwok-Yung Yuen a,b,c,d,e a

State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China Department of Microbiology, The University of Hong Kong, Hong Kong, China c Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong, China d Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong, China e Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou 310006, China f Central Veterinary Research Laboratory, Dubai, United Arab Emirates b

a r t i c l e

i n f o

Article history: Received 19 February 2016 Received in revised form 25 March 2016 Accepted 29 March 2016 Available online 3 April 2016 Keywords: animal RNA viruses picornavirus dog

a b s t r a c t Previously, we reported the discovery of a novel canine picornavirus (CanPV) in the fecal sample of a dog. In this molecular epidemiology study, CanPV was detected in 15 (1.11%) of 1347 canine fecal samples from Hong Kong and one (0.76%) of 131 canine fecal samples from Dubai, with viral loads 1.06×103 to 6.64×106 copies/ml. Complete genome sequencing and phylogenetic analysis showed that CanPV was clustered with feline picornavirus (FePV), bat picornavirus (BatPV) 1 to 3, Ia io picornavirus 1 (IaioPV1) and bovine picornavirus (BoPV), and this cluster was most closely related to the genera Enterovirus and Sapelovirus. The Ka/Ks ratios of all the coding regions were b 0.1. According to the definition of the Picornavirus Study Group of ICTV, CanPV, FePV, BatPV 1 to 3, IaioPV1 and BoPV should constitute a novel genus in Picornaviridae. BEAST analysis showed that this genus diverged from its most closely related genus, Sapelovirus, about 49 years ago. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Picornaviruses are positive-sense, single-stranded RNA viruses with icosahedral capsids. They are widely distributed in human and various animals in which they can cause respiratory, cardiac, hepatic, neurological, mucocutaneous and systemic diseases of varying severity (Tracy et al. 2006). Based on genotypic and serological characterization, the family Picornaviridae is currently divided into 29 genera. Since the SARS epidemic, there has been a boost in the interest in studying novel zoonotic viruses including picornaviruses (Chiu et al. 2008; Kapoor et al. 2008; Lau et al. 2012b; Li et al. 2009; Woo et al. 2012a; Woo et al. 2010). Novel human picornaviruses including the novel rhinovirus species, human rhinovirus C, have also been discovered in the past few years (Drexler et al. 2008; Jones et al. 2007; Lau et al. 2009; Lau et al. 2007b; McErlean et al. 2008). We have also described the discovery of novel picornaviruses from wild dead birds as well as bats

⁎ Corresponding authors at: State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, University Pathology Building, Queen Mary Hospital, Hong Kong. E-mail addresses: [email protected] (P.C.Y. Woo), [email protected] (U. Wernery). 1 PCY Woo, SKP Lau and GKY Choi contributed the same to the manuscript.

http://dx.doi.org/10.1016/j.meegid.2016.03.033 1567-1348/© 2016 Elsevier B.V. All rights reserved.

of diverse species in Hong Kong (Lau et al. 2011a; Woo et al. 2010). Recently, we have also reported the discovery of a novel picornavirus from dromedary camels (Woo et al. 2015). The identification of novel picornaviruses and previously unknown animal hosts for these viruses are crucial for better understanding of their genetic diversity, evolution, biology and potential for cross species transmission and emergence. Although dogs might have been acquainted with humans for over 30,000 years, no picornavirus was discovered until 2011. In 2011, a novel canine kobuvirus was discovered in fecal samples of dogs (Kapoor et al. 2011; Li et al. 2011). In 2012, we discovered a novel canine picodicistrovirus, which was renamed as Cadicivirus A and constituted a new genus Dicipivirus (Woo et al. 2012a). In the same year, we described another novel canine picornavirus (CanPV) (strain 325F), which is still unclassified to any genus (Woo et al. 2012b). The epidemiology of CanPV and its host specificity are unknown. In this article, we report the molecular epidemiology of CanPV in Hong Kong and Dubai. Two complete genomes of CanPV found in dogs from Hong Kong and Dubai respectively were sequenced and analyzed. Based on the results, we propose a novel genus in Picornaviridae, which comprises CanPV, our previously discovered bat picornaviruses (BatPV) 1, 2 and 3 and feline picornavirus (FePV), IaioPV1 discovered in Ia io bats in mainland China and BoPV discovered in cattle in Japan (Lau et al. 2011a; Lau et al. 2012b; Nagai et al. 2015; Wu et al. 2012).

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2. Materials and methods 2.1. Animal surveillance and sample collection For the fecal samples collected in Hong Kong, 1347 dogs were captured from 46 different locations in Hong Kong Special Administrative Region (HKSAR) for 86 months (June 2007 to Aug 2014) by the Department of Agriculture, Fisheries and Conservation (AFCD), HKSAR. Fecal samples were collected from these dogs by the Kowloon Animal Management Centre, AFCD using procedures described previously (Lau et al. 2010a; Lau et al. 2010b; Lau et al. 2005; Lau et al. 2007a; Lau et al. 2010c; Woo et al. 2005a; Woo et al. 2009; Woo et al. 2005b). For the 131 canine fecal samples from Dubai, they were left-over specimens submitted for pathogens screening at the Central Veterinary Research Laboratory in Dubai, United Arab Emirates (UAE) from January 2013 to July 2014. For the other animal fecal samples from bats, cats, cattle, pigs and rodents in Hong Kong and cats and dromedary camels in Dubai were collected as described in our previous publications (Table 1) (Lau et al. 2010a; Lau et al. 2013a; Lau et al. 2012a; Lau et al. 2011a; Lau et al. 2005; Lau et al. 2007a; Lau et al. 2008; Lau et al. 2013b; Lau et al. 2012b; Lau et al. 2011b; Tse et al. 2012; Tse et al. 2011a; Tse et al. 2011b; Woo et al. 2014a; Woo et al. 2014b; Woo et al. 2014c; Woo et al. 2012c).

72°C for 10 min in an automated thermal cycler (Applied Biosystem, Foster City, CA, USA). Standard precautions were taken to avoid PCR contamination and no false-positive was observed in negative controls. All PCR products were gel-purified using the QIAquick gel extraction kit (QIAgen, Hilden, Germany). Both strands of the PCR products were sequenced twice with an ABI Prism 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA), using the two PCR primers. The sequences of the PCR products were compared with known sequences of the 5’-UTR of picornaviruses in the GenBank database.

2.4. Quantitative real-time RT-PCR Quantitative real-time RT-PCR to detect the 3’ UTR of CanPV was performed on the 15 positive fecal samples by the use of Premix Ex Taq™ (Probe qPCR) (TaKaRa, China) with primers 5’-GGTAGAGTTAAGTTGTCC GA-3’ and 5’-AACCACTACTGATTCTAATGATTG-3’ and probe 5’-6FAMCCACGCCGAGTAGGATCGAGGGTACAGAC-IBFQ-3’ and a LightCycler 96 System (Roche Applied Science, Mannheim, Germany). The reaction mixture contained 1 × Premix Ex Taq (Probe qPCR), 0.3 μM of each primer, 0.1 μM of probe, 6.4 μl of nuclease free water, and 2 μl of cDNA template or standard. The cDNA template was generated as aforementioned. The reaction was subjected to thermal cycling at 95°C for 30 s followed by 50 cycles of 95°C for 5 s and 56°C for 30 s.

2.2. RNA extraction Viral RNA was extracted from the fecal samples using EZ1 Virus Mini Kit v2.0 (Qiagen, Hilden, Germany). The RNA was eluted in 60 μl of AVE buffer (Qiagen, Hilden, Germany) and was used as the template for RTPCR. 2.3. RT-PCR of 5’-untranslated region (UTR) of picornaviruses and DNA sequencing Picornavirus screening in the canine fecal samples was performed by amplifying a 112-bp fragment of the 5’-UTR of picornaviruses using conserved primers (5’- CGGCCCCYGAATGYGGCTAA-3’ and 5’- ACACGGAC ACCCAAAGTAGT -3’) designed by multiple alignment of the nucleotide sequences of the 5’-UTR of various picornaviruses using previously described protocols (Lau et al. 2011a; Woo et al. 2010; Yip et al. 2010). Reverse transcription was performed using the SuperScript III kit (Invitrogen, San Diego, CA, USA) and the reaction mixture (10 μl) contained RNA, first-strand buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2), 5 mM of DTT, 50 ng of random hexamers, 500 μM of each dNTPs and 100 U Superscript III reverse transcriptase. The mixtures were incubated at 25°C for 5 min, followed by 50°C for 60 min and 70°C for 15 min. The PCR mixture (25 μl) contained cDNA, PCR buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 2 mM MgCl2 and 0.01% gelatin), 200 μM of each dNTPs and 1.0 U Taq polymerase (Applied Biosystem, Foster City, CA, USA). The mixtures were amplified in 60 cycles of 94°C for 1 min, 60°C for 1.5 min and 72°C for 2 min and a final extension at Table 1 Animals screened for CanPV in the present study. Animals

Sampling period

No. of animals

Hong Kong Bats Cats Cattles Dogs Pigs Rodents

Jul 2008 - Aug 2014 Jun 2007 - Aug 2014 Oct 2008 - Aug 2014 Jun 2007 - Aug 2014 Sep 2008 - Aug 2014 Sep 2008 - Aug 2014

2224 1254 620 1347 670 601

Dubai Dromedaries Cats Dogs

Jan 2013 - July 2014 Jan 2013 - July 2014 Jan 2013 - July 2014

751 51 131

2.5. Genome sequencing Two complete genomes of CanPV, one from Hong Kong (strain 244F) and another one from Dubai (strain 6D), including the full 5’-UTR regions, were amplified and sequenced using strategies we previously used for complete genome sequencing of other picornaviruses, with the RNA extracted from the fecal samples as templates (Lau et al. 2011a; Woo et al. 2010; Yip et al. 2010). The RNA was converted to cDNA by a combined random-priming and oligo (dT) priming strategy. The cDNA was amplified by primers designed from the complete genome sequence of CanPV strain 325F, and additional primers designed from the results of the first and subsequent rounds of sequencing. The 5’ ends of the viral genomes were confirmed by rapid amplification of cDNA ends using the SMARTer RACE cDNA Amplification Kit (Clontech, USA). Sequences were checked manually and assembled to produce final sequences of the full viral genomes.

2.6. Genome analysis The nucleotide sequences of the genomes and the deduced amino acid sequences of the open reading frame were compared to those of CanPV strain 325F and other picornaviruses. The unrooted phylogenetic tree of 5’-UTR was constructed using neighbor-joining method for aligned nucleotide sequences in ClustalX 2.1. The maximumlikelihood phylogenetic trees of P1, P2 (excluding 2A) and P3 (excluding 3A) were constructed using the PhyML 3.0 program (Guindon and Gascuel 2003) and the Approximate Likelihood-Ratio Test (aLRT) method (Anisimova and Gascuel 2006). Secondary structure prediction in the 5’-UTR was performed using RNAstructure Web Server on three strains (Reuter and Mathews 2010).

2.7. Estimation of synonymous and non-synonymous substitution rates The number of synonymous substitutions per synonymous site, Ks, and the number of non-synonymous substitutions per nonsynonymous site, Ka, for each coding region among the two strains of CanPV were calculated using KaKs_Calculator Toolbox 2.0 (Wang et al. 2010).

P.C.Y. Woo et al. / Infection, Genetics and Evolution 41 (2016) 191–200 Fig. 1. (a) Phylogenetic analysis of nucleotide sequences of the 72-bp fragment (excluding primer sequences) of the partial 5’ UTR of CanPV detected from 16 fecal samples of 16 dogs in the present study. The three strains with genomes completely sequenced are shaded in gray. The tree was constructed by the neighbor-joining method, and bootstrap values calculated from 1,000 trees. Bootstrap values expressed as percentages are shown at nodes and only those N75% are shown. The scale bar indicates the estimated number of substitutions per 20 nucleotides. Phylogenetic analyses of the (b) P1, (c) P2 (excluding 2A), and (d) P3 (excluding 3A) regions of CanPV (shown in boldface type). The scale bars indicate the estimated number of substitutions per 5 (P1), 5 (P2) and 10 (P3) amino acids, respectively. Virus abbreviations (GenBank accession numbers shown in parentheses): AEV, avian encephalomyelitis virus (NC_003990); AiV, Aichi virus A (NC_001918); BatPV1, bat picornavirus 1 (HQ595340); BatPV2, bat picornavirus 2 (HQ595342); BatPV3, bat picornavirus 3 (HQ595344); BHUV1, Hunnivirus A (NC_018668); BoPV, bovine picornavirus (LC006971, LC036580); CPDV-209, Cadicivirus A (JN819202); DcEV19CC dromedary picornaviruses (KP345887); DHAV-1, duck hepatitis A virus 1 (NC_008250); DPV, avian sapelovirus (NC_006553); EMCV, encephalomyocarditis virus (NC_001479); ERBV, equine rhinitis B virus 1 (NC_003983); EV-A, Enterovirus A (NC_001612); EV-B, Enterovirus B (NC_001472); EV-C, Enterovirus C (NC_002058); EV-D, Enterovirus D (NC_001430); EV-E, Enterovirus E (NC_001859); EV-F, Enterovirus F (KC748420); EV-G, Enterovirus G (NC_004441); EV-H, Enterovirus H (NC_003988); EV-J, Enterovirus J (NC_013695); FePV, feline picornavirus (NC_016156); FMDV-O, foot-and-mouth disease virus type O (NC_004004); HAV, hepatitis A virus (NC_001489); HCoSV-A1, Cosavirus A (NC_012800); HPeV, human parechovirus (NC_001897); HRV-A, Rhinovirus A (FJ445142); HRV-B, Rhinovirus B (NC_001490); HRV-C, Rhinovirus C (NC_009996); IaioPV1, Ia io picornavirus 1 (JQ814852); MsPV1, Mischivirus A (JQ814851); MoV, Mosavirus A (NC_023987); PoSpV1, porcine sapelovirus (NC_003987); PTV1, porcine teschovirus (NC_003985); RosaV, Rosavirus A (JF973686); SaV, Salivirus A (NC_012957); SePV1, Aquamavirus A (NC_009891); SwPV1, Swine pasivirus 1 (NC_018226); SVV, Seneca Valley virus (NC_011349); SpV1, simian sapelovirus (NC_004451); TV1, Passerivirus A (NC_014411); TV2, Oscivirus A (NC_014412); TuASV, Avisivirus A (KC465954); TuGV, Gallivirus A (NC_018400); THV-2993D, Megrivirus A (HM751199).

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2.8. Estimation of divergence dates The tMRCA was estimated based on an alignment of 3Dpol sequences, using the uncorrelated lognormal distributed relaxed clock model (UCLD) in BEAST version 1.8 (http://evolve.zoo.ox.ac.uk/beast/) (Drummond et al. 2012). The sampling dates of all strains were collected from the literature or from the present study, and were used as calibration points. Sequences were aligned according to the codon positions. Depending on the data set, Markov chain Monte Carlo (MCMC) sample chains were run for 1 × 108 states, sampling every 1,000 generations under the HKY/SRD06 model of substitution. A constant population coalescent prior was assumed for all data sets. The median and HPD were calculated for each of these parameters from two identical but independent MCMC chains using TRACER 1.3 (http://beast.bio.ed.ac.uk). The tree was annotated by TreeAnnotator, a program of BEAST and displayed by FigTree (http://tree.bio.ed.ac.uk/ software/figtree/). 2.9. Viral culture The three fecal samples positive for CanPV with their genomes completely sequenced were cultured in MDCK (Madin-Darby canine kidney) and DH82 (canine macrophagemonocyte) cells respectively. After centrifugation, the fecal samples were diluted fivefold with viral transport medium and filtered. Two hundred microliters of the filtrate was inoculated to 200 μl of MEM. Four hundred microliters of the mixture was added to 24-well tissue culture plates, with each of the cell lines, by adsorption inoculation. After 1 h of adsorption, the wells were washed twice with PBS solution, and the medium was replaced by 1 ml of serum-free MEM. Cultures were incubated at 37°C with 5% CO2 and inspected daily by inverted microscopy for cytopathic effects. After one week of incubation, subculturing to fresh cell line was performed even if there were no cytopathic effects, and culture lysates were collected for RT-PCR for CanPV. Three blind passages were carried out for each sample. 3. Results 3.1. Animal surveillance for CanPV RT-PCR for a 112-bp fragment in the 5’ UTR of CanPV and sequencing showed that CanPV was positive in 15 (1.11%) of the 1347 canine fecal samples from Hong Kong and one (0.76%) of the 131 canine fecal samples from Dubai. Multiple alignments and phylogenetic analysis showed that there were 0 to 11 base differences among the sequences of the 112 bp in the 5’ UTR of these positive samples (Fig. 1a). All the others fecal samples were negative for CanPV (Table 1). 3.2. Quantitative real-time RT-PCR Quantitative RT-PCR showed that the amount of CanPV RNA ranged from 1.06×103 to 6.64×106 copies per ml of fecal sample (Table 2). 3.3. Genome organization and coding potential of CanPV The size of the three genomes of CanPV [strain 325F reported previously (Woo et al. 2012b) and strains 244F and 6D from the present study] were from 7918 to 7948 bases, after excluding the polyadenylated tract, and the G +C content was 40.3% to 40.7%. Their genome organizations are similar to other picornaviruses, with the characteristic gene order 5’-L, VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3Cpro, 3Dpol-3’. Both 5’ (665 to 668 bases) and 3’ (143 bases) ends of the genomes contain UTRs. Downstream to the 5’-UTR, each genome contains a large open reading frame of 7110 to 7137 bases, which encodes potential polyprotein precursors of 2369 to 2378 amino acids. The hypothetical protease cleavage sites of the polyproteins, as

determined by multiple alignments with other picornaviruses, are shown in Fig. 2. 3.4. Phylogenetic analyses The phylogenetic trees constructed using the amino acid sequences of P1, P2 (excluding 2A) and P3 (excluding 3A) of CanPV and other picornaviruses are shown in Fig. 1b, c and d and the corresponding pairwise amino acid identities are shown in Table 3. In all three phylogenetic trees, CanPV was clustered with FePV, BatPV 1 to 3, IaioPV1 and BoPV. Furthermore, this cluster of CanPV, FePV, BatPV 1 to 3, IaioPV1 and BoPV was most closely related to the genera Enterovirus and Sapelovirus, with high aLRT supports. 3.5. 3.4 Genome analyses The 5’ UTR of CanPV contained six domains, I to VI. Domain I contained 121 nucleotides and formed a cloverleaf structure, whereas domains II, III, IV, V and VI are the main domains of the internal ribosome entry site (IRES) element, responsible for directing the initiation of translation in a cap-independent manner, which requires both canonical translation initiation and IRES trans-acting factors (Shih et al. 2011). Similar to enteroviruses, BatPV 3 and BoPV but different from sapeloviruses, BatPV 1, BatPV 2 and FePV (IRES type of IaioPV1 is unknown), CanPV possessed a putative type I IRES element (Fig. 3). The conserved GNRA motif was found in domain IV. Upstream to the AUG start codon, the conserved Yn-Xm-AUG motif was present at domain VI. Similar to sapeloviruses, BatPV 1 to 3, FePV, IaioPV1 and BoPV but different from enteroviruses, a leader protein (L) without the characteristic catalytic amino acid residues with proteolytic activity is present downstream to the 5’ UTR. The P1 (capsid-coding) regions in the genomes of CanPV encode the capsid genes VP4, VP2, VP3 and VP1. Similar to BatPV 1 to 3, FePV and BoPV, but different from IaioPV1 and members of Enterovirus and Sapelovirus, the cleavage sites at the junctions of VP4/VP2, VP2/VP3 and VP3/VP1 in the genome of CanPV were Lys/Ser, Gln/Gly and Gln/ Gly respectively (Fig. 2). Similar to FePV but different members of Enterovirus and Sapelovirus, BatPV 1 to 3, IaioPV1 and BoPV, the VP1 of CanPV possessed the AALXAXETG motif (Table 4). Similar to BatPV 1, BatPV 2, IaioPV1 and BoPV (strain Bo-12-7) but different from members of Enterovirus and Sapelovirus and FePV, the cleavage site at the VP1/2A junction was Asp/Gly (Fig. 2). The P2 regions in the genomes of CanPV encode non-structural proteins 2A, 2B and 2C. Similar to members of Sapelovirus, BatPV 1 to 3, FePV, IaioPV1 and BoPV, but different from members of Enterovirus, all the cleavage junctions in P2 of CanPV were Glu/Gly (Fig. 2). The 2A protein of picornaviruses is a highly variable region (9 to 305 amino Table 2 Viral loads of CanPV in fecal samples of dogs by qRT-PCR. Sample number

Origin of sample

Time of specimen collection

Viral load of CanPV (copies/ml)

169F 208F 210F 216F 244F 325F 353F 490F 491F 492F 1204F 1228F 1627F 1932F 2443F 6D

Hong Kong Hong Kong Hong Kong Hong Kong Hong Kong Hong Kong Hong Kong Hong Kong Hong Kong Hong Kong Hong Kong Hong Kong Hong Kong Hong Kong Hong Kong Dubai

Dec 2007 Jan 2008 Jan 2008 Jan 2008 Feb 2008 Oct 2008 Oct 2008 Apr 2009 Apr 2009 Apr 2009 Dec 2010 Jan 2011 Feb 2012 Nov 2012 Feb 2014 Jan 2013

1.02×104 3.24×105 3.67×106 3.21×103 1.06×103 1.35×105 8.32×103 1.13×103 6.64×106 6.82×105 2.80×104 6.85×103 1.53×104 3.53×104 1.39×104 6.02×104

P.C.Y. Woo et al. / Infection, Genetics and Evolution 41 (2016) 191–200

acids). The 2A protein of CanPV (257 to 265 amino acids in length with 89.8-92.8% amino acid identities among the three strains of CanPV) shared the highest amino acid identity (30%) to that of BatPV 2. An eight amino acid deletion was observed in the 2A of strain 244F. Similar to other members of Enterovirus and Sapelovirus, BatPV 1 to 3, FePV, IaioPV1 and BoPV, the 2A of CanPV possessed the characteristic chymotrypsin-like structures with cysteine-reactive catalytic sites (Table 4). Similar to other members of Enterovirus and Sapelovirus,

195

BatPV 1 to 3, FePV, IaioPV1 and BoPV, the Asn-Pro-Gly-Pro (NPGP) motif observed in the 2A and 2B of avihepatoviruses and avisiviruses required for co-translational cleavage (Ryan and Flint 1997), was absent in CanPV. Furthermore, the conserved H-box/NC motif involved in cell proliferation control was also absent in CanPV, members of Enterovirus and Sapelovirus, BatPV 1 to 3, FePV, IaioPV1 and BoPV (Hughes and Stanway 2000; Tseng et al. 2007; Woo et al. 2010). Similar to other picornaviruses, 2C of CanPV possessed the GXXGXGKS motif for NTP-

Fig. 2. Multiple-sequence alignment of the polyproteins of CanPV with those of closely related picornaviruses, including bat picornavirus 1 to 3, feline picornavirus, Ia io picornavirus 1, bovine picornavirus, enteroviruses, rhinoviruses and sapeloviruses. Gaps introduced to maximize alignment are indicated by dashes. Conserved amino acids are indicated by an asterisk below the sequence alignment.

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Table 3 Comparison of genomic features of CanPV and representative species of other picornavirus genera and amino acid identities between the predicted proteins P1, P2, P3, 3Cpro, and 3Dpol proteins of CanPV and the corresponding proteins of representative species of other picornavirus genera. Genera

Enterovirus Enterovirus Sapelovirus Sapelovirus Sapelovirus Unclassified Unclassified Unclassified Unclassified Unclassified Unclassified Unclassified Unclassified Unclassified Unclassified a b

Species

Enterovirus C Human rhinovirus 58 Simian sapelovirus 1 Avian sapelovirus Porcine sapelovirus 1 Bat picornavirus 1 Bat picornavirus 2 Bat picornavirus 3 Feline picornavirus Ia io picornavirus 1 Bovine picornavirus 11-39 Bovine picornavirus 12-7 Canine picornavirus 244F Canine picornavirus 325F Canine picornavirus 6D

Accession no.

Genome features

Pairwise amino acid identity (%)

Size

Canine picornavirus 244F

G+C

a

P3

b

3C

pro

(bases) Content P1 (%)

P2

NC_002058.3 FJ445142.1 NC_004451.1 NC_006553.1 NC_003987.1 NC_015940.1 NC_015941.1 NC_015934.1 NC_016156.1 JQ814852.1 LC006971.1

7440 7140 8126 8289 7491 7753 7693 7749 7391 7543 7570

46.3 38.9 40.4 42.7 41.0 44.8 43.1 50.2 51.2 45.0 41.2

36.3 36.7 44.1 43.2 43.5 57.9 56.5 55.8 55.3 63.8 53.3

39.7 37.7 49.6 39.7 38.7 59.9 57.4 60.9 55.5 61.4 52.5

52.4 52.9 59.9 54.2 55.4 66.7 66.1 64.4 61.7 64.8 59.0

37.5 43.1 51.6 40.6 48.1 65.0 63.9 61.7 56.5 69.9 53.0

LC036580.1 KU871313 NC_016964.1 KU871312.1

7611 7918 7948 7944

40.1 40.3 40.7 40.3

51.3 89.4 97.0

53.2 99.5 99.1

57.4 98.7 98.2

47.5 99.5 98.4

Canine picornavirus 325F 3D

pol

a

P1

P2

58.5 57.3 63.7 60.1 59.3 68.1 66.7 65.6 64.2 63.2 62.5

36.2 37.3 44.1 42.2 44.5 57.5 54.5 54.2 53.7 63.0 52.1

39.7 37.9 44.1 39.7 38.7 60.1 57.6 60.6 55.3 61.6 52.5

62.6 98.7 98.5

50.8 89.4 89.4

53.5 99.5 99.1

P3

b

pro

Canine picornavirus 6D P1

P2a

P3b

3Cpro 3Dpol

58.9 57.3 64.4 60.3 59.3 68.5 67.2 65.6 64.2 63.4 62.9

36.2 37.0 44.1 43.0 43.4 57.6 55.9 55.5 54.7 63.7 52.6

39.7 37.7 48.2 39.7 38.9 60.1 57.8 60.6 55.3 61.6 52.9

52.5 53.0 60.0 54.7 55.5 67.0 66.4 64.2 61.8 65.1 58.9

37.0 44.1 52.2 41.2 48.6 65.6 64.5 61.2 55.2 70.5 51.9

58.9 57.1 63.9 60.6 59.3 68.3 66.9 65.4 64.6 63.4 62.7

62.6 98.7 98.5

51.1 97.0 89.4 -

53.2 99.1 99.1 -

57.2 98.2 98.7 -

46.4 98.4 98.9 -

62.8 98.5 98.5 -

3C

3D

52.5 52.9 60.3 54.5 55.5 67.2 66.5 64.4 61.7 65.1 59.2

37.0 43.1 52.2 41.2 48.6 65.6 64.5 61.2 56.0 70.5 52.5

57.2 98.7 98.7

47.0 99.5 98.9

pol

P2 region excluding 2A. P3 region excluding 3A.

Fig. 3. Predicted type I IRES structure of CanPV strain 244F. The Yn-Xm-AUG motif and the AUG start codon are underlined. The AUG in the Yn-Xm-AUG motif is shown in boldface.

Table 4 Comparison of genomic features of CanPV, BatPV 1 to 3, FePV, IaioPV1, BoPV and the genera Enterovirus and Sapelovirus in the Picornaviridae family. Poliovirus (Enterovirus)

Simian sapelovirus 1 (Sapelovirus)

BatPV 1, BatPV 2

BatPV 3

CanPV

FePV

IaioPV1

BoPV

5’UTR

Pattern: Yn-Xm-AUG IRES (Belsham et al., 2008; Fernandez-Miragall et al., 2009; Hellen and de Breyne, 2007)

Y9X18 Type I

Y16-17 X17-18 Type IV

No Type IV

Y8X19 Type I

Y7-11X9-18 Type I

No Type IV

Y14X10 (Bo-11-39) Type I

L

Protease (Gorbalenya et al., 1991; Roberts and Belsham, 1995) Cleaved into VP4 and VP2 Myristylation site (Chow et al., 1987): GXXX[ST] Motif: [PS]ALXAXETG Functions (Hughes and Stanway 2000; Ryan and Flint 1997): NTPase motif (Gorbalenya et al. 1989b): GXXGXGKS Helicase (Gorbalenya et al., 1989a, b): DDLXQ Catalytic triad (Gorbalenya et al. 1989a): H-D/E-C RNA-binding domain motif (Hammerle et al. 1992): KFRDI Motif (Gorbalenya et al. 1989a): GXCG, GXH Motif KDE[LI]R (Kamer and Argos 1984): Motif: GG[LMN]PSG (Kamer and Argos 1984) Motif: YGDD (Kamer and Argos 1984) Motif: FLKR(Kamer and Argos 1984)

N†

Y/N†

Y/N†

Y/N†

Y/N†

Y/N†

Unknown Unknown (Incomplete 5' UTR) Y/N†

Y Y Y Chymotrypsin-like protease Y

Y Y Y Chymotrypsin-like protease or unknowne Y

Y Y Y Chymotrypsin-like protease Y

Y Y Y Chymotrypsin-like protease Y

Y Y AALTAPETG Chymotrypsin-like protease Y

Y Y AALXAXETG Chymotrypsin-like protease Y

Y Y Y Chymotrypsin-like protease Y

Y Y Y Chymotrypsin-like protease Y

Y§ H-E-C Y

Y§ H-E-C Y

Y§ H-E-C NFRDI

DDVGQ H-E-C KYRDI

Y§ H-E-C Y

DDVGQ H-E-C K[FY]RDI

Y§ H-E-C DFRDI

Y§ H-D-C NFRD[IV]

Y Y Y Y Y

Y Y Y Y Y

AMH Y Y Y Y

Y Y Y Y Y

AMH Y Y Y Y

A[MI]H Y Y Y Y

AMH Y Y Y Y

Y Y Y Y Y

VP0 VP1 2A 2C

3Cpro

3Dpol

Y/N†

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Regions Function, conserved motif or feature

Y†, presence of L and L is protease; Y/N†, presence of L but L is not protease; N†, absence of L Y§, motif DDLXQ is present.

197

198

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binding (Gorbalenya et al. 1989b). Similar to members of Enterovirus and Sapelovirus, BatPV 1, BatPV 2, FePV, IaioPV1 and BoPV, the DDLXQ motif important for putative helicase activity was present in CanPV (Gorbalenya et al., 1989a, b). The P3 regions in the genomes of CanPV encode 3A, 3B (VPg, small genome-linked protein), 3Cpro (protease) and 3Dpol (RNA-dependent RNA polymerase). Similar to the 3Cpro of members of Enterovirus and Sapelovirus, BatPV 1 to 3, FePV and IaioPV1, the catalytic triad of the 3Cpro of CanPV is His-Glu-Cys (Table 4). This is different from the 3Cpro of picornaviruses of BoPV and some other genera, such as Avihepatovirus, Hepatovirus, Megrivirus, Parechovirus, Tremovirus and Avisivirus, which have catalytic triads of His-Asp-Cys. Similar to all other picornaviruses, CanPV also possessed the conserved GXCG motif that forms part of the active site of the protease. Instead of the GXH motif in members of Enterovirus and Sapelovirus, BatPV 3 and BoPV, CanPV contained the AMH motif as in BatPV 1, BatPV 2, FePV and IaioPV1. Similar to members of Enterovirus and Sapelovirus and FePV, but different from BatPV 1 to 3, IaioPV1 and BoPV, CanPV also contained the conserved RNA-binding motif, KFRDI (Gorbalenya et al. 1989a; Hammerle et al. 1992). Similar to other picornaviruses, the 3Dpol of CanPV contained the conserved KDE[LI]R, GG[LMN]PSG, YGDD and FLKR motifs (Kamer and Argos 1984). 3.6. 3.5 rates

Estimation of synonymous and non-synonymous substitution

Using the three CanPV genome sequences for analysis, the Ka/Ks ratios for the various coding regions were calculated (Table 5). All Ka/Ks ratios were low (b0.1), suggesting that CanPV was stably evolving in dogs. 3.7. 3.6 Estimation of divergence dates Using the uncorrelated lognormal distributed relaxed clock model (UCLD) on 3Dpol, the date of the most recent common ancestor (MRCA) of CanPV, BatPV 1, BatPV 2 and IaioPV1 was estimated to be 1977 (HPDs, 1926 to 1995), approximately 39 years before the present (Fig. 4). Moreover, the MRCA date of CanPV, BatPV 1 to 3, FePV, IaioPV1 and BoPV was estimated to be 1967 (HPDs, 1913 to 1987), approximately 49 years before the present (Fig. 4). The estimated mean substitution rate of the 3Dpol data set under the UCLD model was 1.6× 10-2 substitution per site per year. 3.8. 3.7 Viral culture No cytopathic effect was observed in any of the cell lines inoculated with the samples that were positive for CanPV by RT-PCR. RT-PCR using the culture supernatants and cell lysates for monitoring the presence of viral replication also showed negative results.

definition of the Picornavirus Study Group of the International Committee for Taxonomy of Viruses (http://www.picornastudygroup.com/ definitions/genus_definition.htm), picornaviruses that belong to the same genus should have the following properties: (1) the Leader, 2A, 2B and 3A polypeptides would normally be expected to be homologous between members of a genus; (2) members of a genus should normally share a structurally homologous IRES (this rule may not apply if rule 1 is true); and (3) members of a genus should normally share phylogenetically related P1, P2 and P3 genome regions, each sharing N 40%, N40% and N 50% amino acid identity, respectively. The P1, P2 and P3 regions of CanPV, FePV, BatPV 1, BatPV 2, BatPV 3, IaioPV1 and BoPV possess only 36.2-60.3%, 36.3-58.6%, 36.0-60.8%, 35.6-59.1%, 36.7-62.2%, 35.361.4% and 38.0-52.6% amino acid identities respectively to the corresponding regions of Enterovirus and Sapelovirus (Table 3), the two genera most closely related to these viruses. Therefore, these viruses should not be classified under either Enterovirus or Sapelovirus. On the other hand, the Leader, 2A, 2B and 3A polypeptides of CanPV, FePV, BatPV 1 to 3, IaioPV1 and BoPV are homologous, although the IRES elements of CanPV, BatPV 3 and BoPV belong to type I while those of FePV, BatPV 1 and BatPV 2 belong to type IV and that of IaioPV1 is still unknown as the 5’ UTR of its genome is incomplete (Wu et al. 2012). Moreover, these viruses are also clustered in the phylogenetic trees constructed using the P1, P2 and P3 regions, with 47.7-63.8%, 51.9-72.2% and 54.4-86.2% amino acid identities respectively among them (Fig. 1 and Table 3). Therefore, these viruses should be classified under the same genus in the Picornaviridae family. BEAST analysis showed that this genus diverged from its most closely related genus, Sapelovirus, about 49 years ago (Fig. 4). Within this novel genus, at least six picornavirus species exist. Phylogenetically, BatPV 1 and BatPV 2 are always clustered with high aLRT supports; and therefore may belong to the same species (Fig. 1). For IaioPV1, it is clustered with CanPV in the P1 tree but clustered with BatPV 1 and BatPV 2 in the P2 and P3 trees (Fig. 1). For CanPV, it is clustered with IaioPV1 in the P1 tree but clustered with IaioPV1 and BatPV 1 and BatPV 2 in the P2 and P3 trees (Fig. 1). Similarly, FePV, BatPV 3 and BoPV are also not clustered with the same virus in all three trees (Fig. 1). In addition to their distinct phylogenetic positions, BatPV 1 and BatPV 2, BatPV 3, CanPV, FePV, IaioPV1 and BoPV all show unique genomic features, including the IRES elements in their 5’ UTR, as well as the amino acid sequences of [PS]ALXAXETG in their VP1, DDLXQ motif in their helicase, RNA-binding domain motif and CXCG, GXH motif in their 3Cpro, in comparative genome analysis (Table 44). All these show that BatPV 1 and BatPV 2, BatPV 3, CanPV, FePV, IaioPV1 and BoPV probably belong to six distinct species in this genus. The pathogenicity of the various picornaviruses discovered in dogs remains to be determined. In 2011, a novel canine kobuvirus, which was also the first picornavirus reported, was discovered independently by two groups in diarrheic dogs of the United States of America using the deep sequencing approach (Kapoor et al. 2011; Li et al. 2011).

4. Discussion CanPV is detected in dogs from both Hong Kong and Dubai. Three years ago, we reported the discovery of the novel CanPV from a dog in Hong Kong (Woo et al. 2012b). In the present molecular epidemiology study, we observed the presence of this virus in around 1% of fecal samples from dogs in Hong Kong and Dubai, with a median viral load of 2.17 × 104 copies per ml (Table 2). From the data in the present study, it seems that CanPV is found more commonly during the winter months (Table 2). As for host specificity, CanPV is not present in all the other mammals tested in the present study (Table 1). In addition, the Ka/Ks ratios of all coding regions in the CanPV genome are b0.1 (Table 5). These show that the virus is stably evolving in dogs and support that dogs are the natural reservoir of CanPV. The present CanPV as well as FePV, BatPV 1 to 3, IaioPV1 and BoPV probably constitute a novel genus in Picornaviridae. According to the

Table 5 Synonymous and non-synonymous substitution rates of each coding region in the three genomes of CanPV. Putative proteins

No. of amino acids

Ka

Ks

Ka/Ks

L VP4 VP2 VP3 VP1 2A 2B 2C 3A 3B 3C 3D

49 65 243 226 311 257-265 111 331 108 23 183 461

0.0423 0.0184 0.0537 0.0427 0.0685 0.0364 0.0184 0.0056 0.0052 0.0386 0.0047 0.0095

0.9510 1.3363 2.0745 1.7629 1.1518 1.2175 1.3143 1.0348 0.9444 0.6482 0.7621 0.7407

0.0445 0.0115 0.0244 0.0231 0.0540 0.0299 0.0137 0.0052 0.0060 0.0918 0.0061 0.0128

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199

Fig. 4. Estimation of tMRCA of CanPV and those of closely related picornaviruses, including bat picornavirus 1 to 3, feline picornavirus, Ia io picornavirus 1, bovine picornavirus, enteroviruses, rhinoviruses and sapeloviruses based on 3Dpol gene. The mean estimated dates are labeled. The taxa are labeled with their sampling years.

Subsequently, this canine kobuvirus was also detected in dogs in the United Kingdom, Italy and Korea, implying that this virus is present globally (Carmona-Vicente et al. 2013; Di Martino et al. 2013; Oem et al. 2014). However, none of the studies performed on both diarrheic and non-diarrheic dogs found that this canine kobuvirus was associated with diarrhea (Carmona-Vicente et al. 2013; Di Martino et al. 2013; Oem et al. 2014). In 2012, we reported the discovery of another phylogenetically distinct picornavirus in fecal samples of apparently healthy dogs (Woo et al. 2012b). This picornavirus possesses a unique feature of having two functional IRES elements. No pathogenicity study has been performed on this virus so far. As for the CanPV in the present study, all the CanPV positive fecal samples collected in Hong Kong were from apparently healthy dogs but the one collected in Dubai was from a dog with diarrhea. From the available data, it seems that all these three picornaviruses in dogs are unlikely to be associated with diarrhea. One of the obstacles in further studying the pathogenicity of these picornaviruses in dogs is the difficulty in isolating the viruses. So far, these three picornaviruses were only detected in fecal samples of dogs by RT-PCR or deep sequencing. When these picornaviruses can be cultured, animal challenge experiments should be performed to further study their possible pathogenicity in various organ/systems.

Acknowledgements This work is partly supported by the HKSAR Health and Medical Research Fund; Strategic Research Theme Fund, The University of Hong Kong; Research Grant Council Grant, University Grant Council; and Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Disease for the HKSAR Department of Health.

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