Identification and characterization of bocaviruses in cats and dogs reveals a novel feline bocavirus and a novel genetic group of canine bocavirus

Transcription

1 Journal of General Virology (2012), 93, DOI /vir Identification and characterization of bocaviruses in cats and dogs reveals a novel feline bocavirus and a novel genetic group of canine bocavirus Susanna K. P. Lau, 1,2,3,4 3 Patrick C. Y. Woo, 1,2,3,4 3 Hazel C. Yeung, 4 Jade L. L. Teng, 4 Ying Wu, 4 Ru Bai, 4 Rachel Y. Y. Fan, 4 Kwok-Hung Chan 1,2,3,4 and Kwok-Yung Yuen 1,2,3,4 Correspondence Kwok-Yung Yuen kyyuen@hkucc.hku.hk 1 State Key Laboratory of Emerging Infectious Diseases, Hong Kong 2 Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong 3 Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong 4 Department of Microbiology, The University of Hong Kong, Hong Kong Received 29 February 2012 Accepted 1 April 2012 We report the identification and genome characterization of a novel bocavirus, feline bocavirus (FBoV), and novel bocaviruses closely related to canine bocavirus (CBoV) strain Con-161 in stray cats and dogs in Hong Kong, respectively. FBoV was detected by PCR in 7.2, 0.3, 1.6, 2.0 and 0.8 % of faecal, nasal, urine, kidney and blood samples, respectively, from 364 cats, while CBoV was detected in 4.6, 5.1, 6.3 and 0.3 % of faecal, nasal, urine and blood samples, respectively, from 351 dogs. Three FBoV genomes sequenced revealed the presence of three ORFs characteristic of bocaviruses. Phylogenetic analysis showed that FBoVs were related only distantly to other bocaviruses, forming a distinct cluster within the genus, with 59.7 % nucleotide identities to the genome of minute virus of canines. The four CBoV genomes sequenced shared % nucleotide identities with that of CBoV strain Con-161. In addition to the three bocavirus ORFs, they encoded an additional ORF, ORF4, immediately downstream of the ORF for non-structural protein 1 (), which was not found in other bocaviruses including CBoV strain Con-161. They also possessed a putative second exon encoding the C-terminal region of and conserved RNA-splicing signals, previously described in human bocaviruses. Partial sequence analysis of 23 FBoV and 25 CBoV strains demonstrated inter-host genetic diversity, with two potential genetic groups of FBoV and a novel CBoV group, CBoV-HK, distinct from the three groups, CBoV-A to -C, found in the USA. Although the pathogenicity of FBoV and CBoV remains to be determined, their presence in different host tissues suggested wide tissue tropism. INTRODUCTION Parvoviruses are a group of small, non-enveloped, ssdna viruses that cause a wide range of disease in animals. Under the current International Committee on Taxonomy of Viruses (ICTV) classification system, the family Parvoviridae is classified into two subfamilies based on host range: Parvovirinae, infecting vertebrates, and Densovirinae, infecting insects and other arthropods. The subfamily Parvovirinae is further subdivided into five genera, Amdovirus, Bocavirus, Dependovirus, Erythrovirus and Parvovirus (Fauquet et al., 2005). Several novel parvoviruses, potentially 3These authors contributed equally to this paper. The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences of the genomes of FBoVs and CBoVs determined in this study are JQ JQ belonging to a novel genus, have been recently discovered, including human partetravirus (previously known as human parvovirus 4 or PARV4) (Jones et al., 2005; Fryer et al., 2006, 2007), porcine and bovine partetraviruses (previously known as porcine and bovine hokoviruses) (Lau et al., 2008), ovine partetraviruses (Tse et al., 2011), porcine partetravirus-like viruses in wild boar (Adlhoch et al., 2010; Cadar et al., 2011) and human partetravirus-like viruses in primates (Sharp et al., 2010). Bocaviruses are unique among parvoviruses in possessing an additional ORF, ORF3, between the non-structural and structural coding regions, ORF1 and ORF2, in their genomes. The genus was originally named according to the initial two members, bovine parvovirus (BPV) and minute virus of canines (MVC) (Binn et al., 1970; Mochizuki et al., 2002; Spahn et al., 1966; Storz et al., G 2012 SGM Printed in Great Britain 1573

2 S. K. P. Lau and others 1978). The third bocavirus, human bocavirus (HBoV), was discovered in 2005 from respiratory samples and was later also found in faecal samples from children (Allander et al., 2005; Lau et al., 2007; Sloots et al., 2006; Söderlund- Venermo et al., 2009). At least four different HBoV species, HBoV1 4, have now been described, although their pathogenic role in human disease is still not fully understood (Allander et al., 2005; Arthur et al., 2009; Kapoor et al., 2009, 2010b). In the past 2 years, several novel animal bocaviruses have been discovered and their genomes have been characterized. In particular, various groups have reported bocaviruslike sequences in swine samples in China, Sweden and the UK (Blomström et al., 2009; Cheng et al., 2010; Lau et al., 2011; McKillen et al., 2011; Zeng et al., 2011; Zhai et al., 2010). Genome-sequence studies have shown the presence of at least nine potential novel porcine bocavirus species, porcine bocavirus 1 (PBoV1) to PBoV5, PBoV strain WUH1, PBoV1 H18, PBoV2 A6 and PBoV3 22 (Cheng et al., 2010; Lau et al., 2011; Li et al., 2012; Shan et al., 2011a, b; Zeng et al., 2011). We have also described evidence for inter- and intra-host genetic diversity and recombination among porcine bocaviruses (Lau et al., 2011). Apart from swine, another novel bocavirus, gorilla bocavirus species 1 (GBoV1), was discovered in stool samples from western gorillas (Kapoor et al., 2010a). Similar bocavirus-like sequences have also been detected in faecal samples of primates from Cameroon (Sharp et al., 2010). Four novel bocavirus species, California sea lion bocavirus 1 (CslBoV1) to CslBoV4, have been identified in the faecal flora of California sea lions (Li et al., 2011). More recently, a novel canine bocavirus (CBoV), phylogenetically distinct from MVC, was discovered in respiratory samples from dogs (Kapoor et al., 2012). Partial sequences potentially belonging to novel bocaviruses have also been detected in the viral flora of pine marten faeces (van den Brand et al., 2012). Since cats and dogs are closely related animals often sharing similar habitats, and interspecies transmission of viruses between these two animals is not uncommon (Herrewegh et al., 1998; Shackelton et al., 2005; Truyen, 2006), we hypothesized that there are previously unrecognized bocaviruses in cats that may be closely related to their counterparts in dogs. We therefore conducted a study to investigate the presence of bocaviruses in cats and dogs in Hong Kong. A novel bocavirus, feline bocavirus (FBoV), as well as bocaviruses closely related to CBoV, were identified in feline and canine samples, respectively. RESULTS Detection of bocaviruses in feline and canine samples PCR using consensus primers targeted to a 141 bp fragment of the non-structural protein 1 () gene was positive for bocavirus in one faecal sample from a cat, and in one urine sample and one nasopharyngeal swab sample from two dogs. The sequence from the feline sample possessed,77 % nucleotide identity to the corresponding partial sequences of known bocaviruses, suggesting the presence of a potentially novel bocavirus, FBoV. The sequences from canine samples possessed 99 % nucleotide identity to the corresponding sequence of CBoV strain Con-161 (GenBank accession no. HM053672), but only 80 % nucleotide identity to the corresponding sequence of CslBoV (JN420366) and 79 % nucleotide identity to that of PBoV2 A6 (HQ291309), suggesting that they belonged to CBoV. Subsequent PCR using specific primers targeted to a 133 bp fragment of the gene of FBoV was positive in 26 (7.2 %) of 363 faecal samples, one (0.3 %) of 364 nasal samples, six (1.6 %) of 364 urine samples, one (2 %) of 51 kidney samples and three (0.8 %) of 361 blood samples collected from cats (Table 1). PCR using specific primers targeted to a 128 bp fragment of the gene of CBoV was positive in 16 (4.6 %) of 351 faecal samples, 18 (5.1 %) of 351 nasal samples, 22 (6.3 %) of 351 urine samples and one (0.3 %) of 331 blood samples collected from dogs (Table 1). Both FBoV and CBoV were detected in various seasons during the sampling period, although detection rates were higher in some months than others, suggesting a seasonal pattern of infections (Fig. 1). Genome-sequence analysis of FBoV and CBoV Near-complete genome sequences were determined for FBoV detected from three feline samples (797F and 875F from faecal samples and 797U from a urine sample) of two cats, and CBoV from four samples (882F and 831F from faecal samples, 880N from a nasopharyngeal sample and 882U from a urine sample) of three dogs. The sequences obtained for FBoV were bp, with a G+C content of mol%. The sequences obtained for CBoV were bp, with a G+C content of mol%. The two FBoV genomes (797F and 797U) from different Table 1. Detection of bocaviruses in feline and canine samples by PCR Animal samples No. of samples Total Positive samples (%) Feline (2.5) Faecal (7.2) Nasal (0.3) Urine (1.6) Kidney 51 1 (2.0) Blood (0.8) Canine (4.1) Faecal (4.6) Nasal (5.1) Urine (6.3) Blood (0.3) 1574 Journal of General Virology 93

3 Novel feline and canine bocaviruses Positive cats/dogs (%) No. of cats tested No. of dogs tested Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Fig. 1. Seasonal distribution of FBoV (light-grey bars) and CBoV (dark-grey bars) in cats and dogs in Hong Kong. sampling locations of the same cat shared identical genome sequences (except for occasional degenerate bases observed in both sequences), while the other genome (875F) from a different cat possessed 142 nt differences from the former. Three CBoV genomes (880N, 882F and 882U), two from the same dog, also shared identical genome sequences with each other, while the other genome (831F) possessed 214 nt differences from the former. Similar to other bocaviruses, the three FBoV and four CBoV genomes each encode two major non-overlapping ORFs, ORF1 and ORF2, and one additional ORF, ORF3, characteristic of bocaviruses (Allander et al., 2005; Arthur et al., 2009). ORF1 encodes protein and ORF2 encodes the overlapping capsid proteins, while ORF3 encodes (Fig. 2). Within of both FBoV and CBoV, conserved motifs associated with rolling-circle replication, helicase and ATPase were identified. The VP1-unique (VP1u) regions of both viruses also contained phospholipase A 2 motifs required for parvovirus infectivity, with the presence of a calcium-binding loop and catalytic residues. The function of the unique bocavirus protein,, remains unclear, although it has been found to be essential for virus replication in MVC (Sun et al., 2009). In addition to the three ORFs, the three CBoV genomes encode an additional ORF, ORF4, immediately downstream of ORF1, with a predicted signal peptide. This ORF was not found in other known bocaviruses, including CBoV strain Con-161. The three FBoV genome sequences only shared % nucleotide identities to that of the most closely related bocavirus, MVC. The predicted protein of FBoV encodes 804 aa, while encodes 712 aa and encodes 218 aa. Phylogenetic analysis using the genome sequences of FBoV showed that they were related distantly to other bocaviruses, forming a distinct cluster within the genus Bocavirus (Fig. 3). This supported the presence of a novel bocavirus, FBoV, in the genus Bocavirus in the feline samples. The four CBoV genome sequences shared % nucleotide identities with that of CBoV strain Con-161. Similar to HBoV and CBoV strain Con-161, the predicted protein of the four CBoV genomes was relatively short, encoding 644 or 648 aa, compared with aa in other bocaviruses. Recent studies have shown that the four HBoV species possess a second exon encoding the C- terminal region of, and conserved RNA-splicing signals near the end of and the second exon, which may form a spliced transcript encoding aa (Chen et al., 2010; Kapoor et al., 2010b). Similar to CBoV strain Con-161 (Kapoor et al., 2012), the four CBoV genomes also possess a putative second exon and conserved RNAsplicing signals, which may generate a longer of 764 aa (Fig. 2). The of CBoV encodes aa, while encodes 195 aa. ORF4 encodes a 144 aa protein, which overlaps with the predicted second exon of

4 S. K. P. Lau and others bp PBoV1 BPV HBoV1 Exon 2 GBoV Exon 2 CslBoV1 MVC CBoV Con-161 Exon 2 CBoV HK882F FBoV HK797F Exon 2 ORF4 Fig. 2. Comparative genome organization of FBoV, CBoV and other bocaviruses. Boxes labelled Exon 2 represent the second exon of the protein in HBoV1, GBoV and CBoVs. Predicted RNA-splicing sites are indicated by arrows. Phylogenetic analysis of of the present CBoV strains showed that they belonged to CBoV, with % nucleotide identities to CBoV strain Con-161 (Fig. 3). Genetic diversity of FBoV and CBoV As capsid proteins of parvoviruses are responsible for determining cellular tropism and host immune response, the genetic diversity of detected FBoV and CBoV strains was studied by amplifying and sequencing of their respective partial regions. Phylogenetic analysis of the sequences from 23 FBoV strains showed that they possessed up to 6.5 % nucleotide difference from each other, with 20 strains falling into two clusters, FBoV-A (14 strains) and FBoV-B (six strains) (Fig. 4a). FBoV strains from different sampling locations of the same cat were identical, while different strains were observed from different cats. Phylogenetic analysis of the sequences from 25 CBoV strains showed that they possessed up to 10.3 % nucleotide difference from each other. Moreover, they appeared to fall into a separate cluster, CBoV-HK, distinct from the three genetic groups, CBoV-A to -C, described previously among strains from the USA (Fig. 4b). The 6 aa deletion found previously in CBoV-C variants, located at the variable exposed loop of, was not present in the CBoV-HK strains. Interestingly, two different strains, 831F and 831N, were detected in the faecal and nasal samples, respectively, of the same dog (Fig. 4b). Virus culture No cytopathic effect was observed in cell lines inoculated with samples that were positive for FBoV or CBoV by PCR. PCR using the culture supernatants and cell lysates for monitoring the presence of virus replication also showed negative results. DISCUSSION The present study represents the first to describe the existence of bocaviruses in domestic cats. In this study, using consensus primers for PCR of an gene fragment, bocavirus sequences were detected in various tissue samples of cats and dogs. Viral genome sequences were determined from three feline and four canine samples, which confirmed 1576 Journal of General Virology 93

5 Novel feline and canine bocaviruses H1(NC_001358) KRV(AF321230) RMV-2a(EF029111) MPV_3(NC_008185) HaPV(U34255) MPV-1(NC_001630) MPV-2(NC_008186) Lulll(NC_004713) MVM(NC_001510) PARV4 HK1(EU200667) PARV5 BR (DQ873390) BHoV HK1(EU200669) PHoV HK1(EU200671) PPV2(AB076669) BAAV(NC_005889) CslAAV1 1136(JN420371) AAV-1(NC_002077) AAV-2(NC_001401) MDPV(NC_006147) GPV(NC_001701) AAAV DA-1(NC_006263) B19 V9(NC_004295) RmPV(AF221122) SPV(U26342) PmPV(AF221123) ChpPV(U86868) BPV3(AF406967) BPV2(NC_006259) BPV(NC_001540) PBoV2(HM053694) PBoV1(HM053693) PBoV2 A6(HQ291309) PBoV3(JF429834) PBoV3 22(JF713714) PBoV4(JF429835) PBoV1 H18(HQ291308) PBoV SX(HQ223038) FBoV HK797F FBoV HK797U FBoV HK875F MVC(NC ) 93 CBoV HK880N CBoV HK882F CBoV HK882U CBoV HK831F CBoV Con-161(JN648103) HBoV2(NC_012042) HBoV4(NC_012729) HBoV3(NC_012564) HBoV1(NC_007455) GBoV1(NC_014358) PPV(NC_001718) FPV(M38246) MEV(D00765) 84 CPV(NC_001539) RPV-1a(AF036710) AMDV(NC_001662) Silkworm parvo-like virus(s78547) CslBoV1 1136(JN420360) CslBoV2 9822(JN420366) CslBoV (JN420365) Hokovirus Dependovirus Erythrovirus Bocavirus Parvovirus Amdovirus Fig. 3. Phylogenetic analysis of near-complete genome sequences of FBoV and CBoV obtained from two feline faecal (FBoV HK797F and FBoV HK875F), one feline urine (FBoV HK797U), two canine faecal (CBoV HK831F and CBoV HK882F), one canine urine (CBoV HK882U) and one canine nasal (CBoV HK880N) specimens, and those of other parvoviruses with genome sequences available (GenBank accession numbers are in parentheses). A total of 4875 nt positions were included in the analysis. The tree was constructed by the maximum-likelihood (ML) method under the best evolutionary model (GTR+I). Bootstrap values were calculated from trees. Bar, 0.2 nucleotide substitutions per site. Silkworm parvo-like virus was used as an outgroup. AAV-1, Adeno-associated virus 1; AAV-2, adeno-associated virus 2; AMDV, Aleutian mink disease virus; AAAV DA-1, avian adeno-associated virus DA-1; PBoV SX, bocavirus pig/sx/china/2010; BAAV, bovine adeno-associated virus; BHoV HK1, bovine hokovirus HK1; BPV, bovine parvovirus; BPV2, bovine parvovirus 2; BPV3, bovine parvovirus 3; CslAAV1 1136, California sea lion adeno-associated virus ; CslBoV1 1136, California sea lion bocavirus ; CslBoV2 9822, California sea lion bocavirus ; CslBoV3 9805, California sea lion bocavirus ; CBoV Con-161, canine bocavirus Con-161; MVC, minute virus of canines; CPV, canine parvovirus; ChpPV, chipmunk parvovirus; FPV, feline panleukopenia virus; GPV, goose parvovirus; GBoV1, gorilla bocavirus 1; HaPV, hamster parvovirus; HBoV1, human bocavirus 1; HBoV2, human bocavirus 2; HBoV3, human bocavirus 3; HBoV4, human bocavirus 4; PARV4 HK1, human parvovirus 4 HK1; PARV5 BR , human parvovirus 5 BR ; B19 V9, human parvovirus B19 V9; KRV, Kilham rat virus; LuIII, LuIII virus; MVM, minute virus of mice; MEV, mink enteritis virus; MPV-1, mouse parvovirus 1; MPV-2, mouse parvovirus 2; MPV-3, mouse parvovirus 3; MDPV, Muscovy duck parvovirus; H1, parvovirus H1; PmPV, pig-tailed macaque parvovirus; PBoV1, porcine bocavirus 1 pig/zjd/china/2006; PBoV1 H18, porcine bocavirus 1 H18; PBoV2, porcine bocavirus 2 pig/zjd/china/2006; PBoV2 A6, porcine bocavirus 2 A6; PBoV3 22, porcine bocavirus 3 22; PBoV3, porcine bocavirus 3 SH20F; PBoV4, porcine bocavirus 4-1 SH17F-1; PHoV HK1, porcine hokovirus HK1; PPV, porcine parvovirus; PPV2, porcine parvovirus 2; RMV-2a, rat minute virus 2a; RPV-1a, rat parvovirus 1a; RmPV, rhesus macaque parvovirus; SPV, simian parvovirus

6 S. K. P. Lau and others (a) FBoV HK1107U (b) FBoV HK1182U FBoV HK980U FBoV HK1397F FBoV HK1182F FBoV HK980F 62 FBoV HK875F FBoV-A FBoV HK849F FBoV HK828F FBoV HK1167B FBoV HK1017F 55 FBoV HK1376F FBoV HK915F 69 FBoV HK915U FBoV HK1313F 99 FBoV HK797U 53 FBoV HK797F FBoV-B 83 FBoV HK1438F FBoV HK1477F 76 FBoV HK1438B FBoV HK936F FBoV HK1223F 82 FBoV HK1409F 0.05 MVC (NC_004442) 0.1 CBoV Con-ll-208 (JN648106) CBoV Dis-007 (JN648122) CBoV Dis-047 (JN648124) CBoV Con-161 (JN648103) CBoV Con-lll-201 (JN648104) CBoV Con-ll-211 (JN648107) 61 CBoV Con-l-165 (JN648120) CBoV Con-l-166 (JN648121) CBoV Con-l-162 (JN648119) CBoV Con-l-160 (JN648118) CBoV Con-l-159 (JN648117) CBoV Con-l-158 (JN648116) 94 CBoV Con-l-157 (JN648115) CBoV Con-l-153 (JN648113) CBoV Con-l-155 (JN648114) CBoV Con-l-152 (JN648112) CBoV Con-l-149 (JN648110) CBoV Con-l-150 (JN648111) CBoV Con-l-148 (JN648109) CBoV Con-l-147 (JN648108) CBoV Con-ll-206 (JN648105) CBoV Dis-021 (JN648137) CBoV Dis-058 (JN648139) CBoV Dis-026 (JN648138) CBoV HK831F CBoV HK1442U CBoV HK1431U 96 CBoV HK1415U CBoV HK1416U CBoV HK1355U CBoV HK1033U CBoV HK831N CBoV HK1316U CBoV HK930N CBoV HK882B CBoV HK880N CBoV HK882F CBoV HK882U CBoV HK880F CBoV HK881F CBoV HK886F CBoV HK887F 98 CBoV HK882N CBoV HK883N CBoV HK919N CBoV HK880U CBoV HK883U CBoV HK1232U CBoV HK886U CBoV Dis-046 (JN648136) CBoV NY01 (JN648125) CBoV Dis-008 (JN648126) CBoV Dis-009 (JN648127) CBoV Dis-010 (JN648128) 99 CBoV Dis-023 (JN648130) CBoV Dis-011 (JN648129) CBoV Dis-025 (JN648131) CBoV Dis-039 (JN648134) CBoV Dis-038 (JN648133) CBoV Dis-028 (JN648132) CBoV Dis-040 (JN648135) CBoV Dis-017 (JN648123) MVC (NC_004442) CBoV-A CBoV-B CBoV-HK CBoV-C Fig. 4. Genetic diversity of FBoV and CBoV. CBoV strains detected in the present study are shaded. Phylogenetic trees of the partial VP1/2 sequences of 23 FBoV strains (a) and 25 CBoV strains (b) were constructed by the ML method under the best evolutionary model (HKY+G), with bootstrap values calculated from trees. A total of 261 and 270 nt positions were included in the analysis, respectively. Bars, estimated number of substitutions per 20 and 10 nt, respectively. the presence of two different bocaviruses, FBoV and CBoV, respectively. Phylogenetic analysis revealed that the different strains of FBoV and CBoV each formed a distinct cluster among known bocaviruses. While CBoV is phylogenetically closely related to the recently discovered CBoV strain Con- 161, FBoV represents a novel bocavirus identified in cats. The FBoV genomes possessed 59.7 % nucleotide identities to those of other bocaviruses, being only distantly related to CslBoV, CBoV and MVC. Under ICTV criteria for species classification of the genus Bocavirus, members of each species are defined as: (i) probably antigenically distinct; (ii) natural infection is confined to a single host species; and (iii) having,95 % homologous NS gene DNA sequence ( Although the antigenic properties of FBoV and CBoV were not studied, the of FBoV possessed 56.7 % nucleotide identities to those of other known bocaviruses, supporting its classification as a separate bocavirus species. Analysis of partial sequences demonstrated genetic diversity among different strains of FBoV, with most strains belonging to two potential genetic groups. A novel genetic group of CBoVs was identified among the present dogs, suggesting that diverse CBoVs may be distributed in different geographical areas. As the genes of the present CBoV strains exhibited 96.5 % nucleotide identities to CBoV strain Con-161, they should belong to the same bocavirus species. However, a unique ORF4, absent in CBoV strain Con-161 and other bocaviruses, was found in the present CBoV strains. As only one genome sequence of CBoV from strain Con-161 was available among strains detected in a previous study (Kapoor et al., 2012), it remains to be determined whether this ORF is functional and can be found in CBoV strains from other countries. Moreover, phylogenetic analysis of partial sequences revealed a distinct group, CBoV-HK, among the present CBoV 1578 Journal of General Virology 93

7 Novel feline and canine bocaviruses strains, different from the three genetic groups, CBoV-A to C, detected in dogs from the USA (Kapoor et al., 2012). In the present study, we detected only CBoV, but not MVC, in our canine samples, although the consensus primers were theoretically able to detect MVC. This suggests that CBoV may be more prevalent than MVC among our canine population. Further studies are required to better define the epidemiology and genetic diversity of canine bocaviruses in different countries. Despite having DNA genomes with the use of cellular replication machinery, increasing studies have shown that parvoviruses can undergo rapid evolution to generate new genotypes or species and may possess substitution rates close to those of RNA viruses (Duffy et al., 2008; Hokynar et al., 2002; Nguyen et al., 2002; Servant et al., 2002; Shackelton et al., 2005; Shade et al., 1986). Moreover, parvoviruses such as HBoV and PBoV can undergo genetic rearrangement and recombination similar to that which has occurred in RNA viruses (Hoelzer et al., 2008; Hogan & Faust, 1986; Kapoor et al., 2009, 2010b; Lau et al., 2011). We have previously described marked inter- and intra-host genetic diversity and co-infection of two different porcine bocaviruses in the same host (Lau et al., 2011). The presence of within-host genetic diversity has also been described in other parvoviruses including canine parvovirus (CPV) and feline panleukopenia virus (FPV), although their degree of sequence diversity was relatively low compared with that in porcine bocaviruses (Battilani et al., 2006; Hoelzer et al., 2008). In the present study, although no recombination event was detected among the present FBoV and CBoV strains (data not shown), both viruses were found to exhibit inter-host genetic diversity. While intra-host genetic diversity was also observed in of CBoV, the degenerate bases observed in the of FBoV may suggest the presence of variant strains or quasispecies in the same host. Cats and dogs are the commonest domestic pets worldwide that share a close habitat with humans. They are increasingly known to carry a large diversity of viruses, many of which are closely related between these two animals, with interspecies transmission having been described between feline and canine coronaviruses, herpesviruses, papillomaviruses and parvoviruses (Bernard et al., 2012; Herrewegh et al., 1998; Lau et al., 2012; Pellett et al., 2012; Shackelton et al., 2005; Siegl et al., 1985; Truyen, 2006; Woo et al., 2012a, b). In particular, CPV (with the first known strain designated CPV2), belonging to the genus Parvovirus, emerged in 1978 from a feline parvovirus, FPV, differing at several key amino acid residues (Shackelton et al., 2005; Steinel et al., 2000). Although CPV2 can infect feline cells in vitro, it did not infect cats. However, CPV2 was subsequently replaced by a new lineage, CPV2a, and its variants, which can infect both dogs and cats (Truyen et al., 1996). It has been shown that, since its emergence in the 1970s, CPV has undergone an epidemic-like pattern of exponential growth, which was associated with a lineage that acquired a broader host range and greater infectivity (Shackelton et al., 2005). While recombination played no role in the emergence of CPV, its rapid adaptation to dogs was found to be dependent on a high rate of mutation and the positive selection of mutations in the major capsid gene (Shackelton et al., 2005). In the present study, FBoV was also related more closely to MVC, CBoV and CslBoV than to other bocaviruses, although the genetic difference was much larger than that between FPV and CPV. This suggests that feline and canine bocaviruses may share a common ancestor and have co-evolved among cats and dogs, although interspecies transmission is unlikely to have occurred recently. The pathogenicity of the novel FBoVs and CBoVs remains to be determined. The first bocavirus, BPV, is known to be associated with diarrhoea and to cause mild respiratory symptoms in experimentally infected calves (Spahn et al., 1966; Storz et al., 1978). Initially isolated from apparently normal dogs in a canine cell line in 1970, MVC was later found to be a respiratory pathogen of neonatal puppies and to cause fetal deaths in infected dogs (Binn et al., 1970; Mochizuki et al., 2002). However, MVC infections are mostly subclinical in adult animals (Carmichael et al., 1991). Although HBoVs have been detected in respiratory and faecal samples of children worldwide, their pathogenicity in respiratory or enteric disease is still in debate because of the frequent co-detection of other pathogens and their presence in healthy children (Allander et al., 2005; Lau et al., 2007; Sloots et al., 2006; Söderlund- Venermo et al., 2009). The recently discovered novel porcine bocaviruses have been identified in both healthy and diseased pigs, including those with clinical postweaning multisystemic wasting syndrome (Blomström et al., 2009; Cheng et al., 2010; Lau et al., 2011; McKillen et al., 2011; Zeng et al., 2011; Zhai et al., 2010). Although these viruses were most prevalent in porcine faecal samples, their presence in other tissues, including lymph node, serum and nasopharyngeal samples, suggested a wide tissue tropism (Lau et al., 2011). Whilst GBoV1 was associated with acute enteritis in western gorillas (Kapoor et al., 2010a), CBoV group C variants were found to be more prevalent in dogs with respiratory diseases than in healthy animals (Kapoor et al., 2012). However, other bocaviruses from primates and California sea lions detected in faecal samples were not known to cause diseases in these mammals (Li et al., 2011; Sharp et al., 2010). In this study, both FBoV and CBoV were detected in different samples from the respective animals, suggesting a wide tissue tropism. While FBoV was mainly detected in faecal samples in cats, CBoV demonstrated the highest detection rate in urine samples (6.3 %), suggesting that the latter virus may cause more systemic infections in dogs rather than respiratory disease alone. Interestingly, HBoV, still of unresolved clinical significance in humans, has also been occasionally detected in serum and urine samples of infected children (Pozo et al., 2007; Wang et al., 2010). Further studies are required to better understand the epidemiology, evolution, pathogenicity and diversity of bocaviruses in various mammals including humans

8 S. K. P. Lau and others METHODS Collection of animal specimens. In total, 1503 feline and 1384 canine samples from 364 cats and 351 dogs, respectively, collected over a 21 month period (December 2009 August 2011), were provided by the Agriculture, Fisheries and Conservation Department (AFCD) of the Government of Hong Kong Special Administrative Region (HKSAR), as part of a surveillance programme on stray cats and dogs. Nasopharyngeal, faecal, urine, kidney and/or blood samples were collected from the stray cats and dogs by AFCD Animal Management Centres, using procedures described previously (Table 1) (Lau et al., 2005, 2008). To prevent cross-contamination, dissections and collection of tissues were performed using disposable scalpels and from the centre of each tissue after surface decontamination and with protective gloves changed for each tissue sample. All samples were collected immediately after euthanasia as routine policies for disposal of locally captured stray cats and dogs. The study was approved by the Committee on the Use of Live Animals in Teaching and Research at the University of Hong Kong. Detection of bocaviruses. DNA was extracted from all samples using a QIAamp DNA Mini kit (Qiagen), according to the manufacturer s protocol. DNA was subject to PCR for bocaviruses, using forward primer 59-GCCAGCACNGGNAARACMAA-39 and reverse primer 59- CATNAGNCAYTCYTCCCACCA-39 targeted to a 141 bp fragment of the gene, designed by multiple alignments of the nucleotide sequences of regions of known bocaviruses including HBoV, BPV and MVC. As potentially novel bocaviruses were detected, subsequent bocavirus detection was performed using specific primers (forward primer 59-TCTACAAGTGGGACATTGGA-39 and reverse primer 59- GAGCTTGATTGCATTCACGA-39 for FBoV, which targeted a 133 bp fragment of ; forward primer 59-AGGTCGGCCACTGGCTGT-39 and reverse primer 59-CAGCTTAACGGCATTCACTA-39 for CBoV, which targeted a 128 bp fragment of ) designed based on the obtained genome sequences. The PCR mixture (25 ml) contained DNA, PCR buffer (10 mm Tris/HCl ph 8.3, 50 mm KCl, 2 mm MgCl 2 and 0.01 % gelatin), 200 mm of each dntp and U Taq polymerase (Applied Biosystems). The mixtures were amplified by 40 cycles of 94 uc for1min,50uc for 1 min and 72 uc for 1 min and a final extension at 72 uc for 10 min in an automated thermal cycler (Applied Biosystems). Standard precautions were taken to avoid PCR contamination and no false positives were observed in negative controls. PCR products were gel-purified using the QIAquick gel extraction kit (Qiagen). Both strands of the PCR products were sequenced twice with an ABI Prism 3700 DNA Analyser (Applied Biosystems), using the PCR primers. The sequences of the PCR products were compared with known sequences of regions of bocaviruses in GenBank. Genome sequencing and analysis. Near-complete genome sequences were determined for the potentially novel feline bocavirus, FBoV, from three samples (875F, 797F and 797U) of two cats and CBoV from four canine samples (880N, 882U, 882F and 831F) of three dogs, using the strategy described in our previous publications (Lau et al., 2008, 2011; Tse et al., 2011). DNA extracted directly from the specimens was used as template and amplified by degenerate primers designed from multiple alignment of the genomes of HBoV, BPV, MVC and PBoV, and additional primers covering the original degenerate primer sites were designed from the results of the first and subsequent rounds of sequencing. Primer sequences are available on request. The terminal sequences were confirmed by a modified protocol for RACE (Allander et al., 2005; Lau et al., 2011). Sequences were assembled and edited manually to produce final sequences of the viral genome. All assembled sequences were confirmed by independent PCR using specific primers across overlapping regions to ensure accuracy of the assembled sequences. The nucleotide sequences of the genomes and the predicted ORFs were compared with those of other bocaviruses. A maximum-likelihood (ML) phylogenetic tree was constructed using PhyML version 3.0 (Guindon & Gascuel, 2003) under the best evolutionary model (GTR+I) determined by MODELGENERATOR (Keane et al., 2006). Bootstrap values were estimated by using replicates on the ML substitution model. Protein domain and family analysis was performed using InterProScan (Apweiler et al., 2001) and/or multiple sequence alignment. Sequencing of partial sequences. The genetic diversity of FBoV and CBoV strains was studied by amplifying and sequencing of their partial capsid protein sequences, using primers (forward primer 59-AAAATCCTAAACAACAAA-39 and reverse primer 59-TATGGC- AATTCTGGCATT-39 for FBoV; and forward primer 59-GGAGGAGG- TGGAGGACAT-39 and reverse primer 59-CGTCCGTCAGGTCA- GATT-39 for CBoV) targeted to a 562 bp and a 526 bp region of their genes, respectively, designed by multiple alignments of the obtained genome sequences, and the above PCR conditions. Virus culture. Four faecal and three urine samples positive for FBoV were cultured in Vero E6 (African green monkey kidney; ATCC CRL- 1586), CrFK (Crandell feline kidney; ATCC CCL-94), HFL (human embryonic lung fibroblast), CaCO-2 (human colorectal adenocarcinoma; ATCC HTB-37), and primary feline lung and kidney cells. Two faecal, urine and nasal samples positive for CBoV were cultured in MDCK (Madin Darby canine kidney; ATCC CCL-34), and primary canine lung and kidney cells. Nucleotide sequence accession numbers. The nucleotide sequences of the genomes of FBoVs and CBoVs have been deposited in GenBank under accession numbers JQ JQ ACKNOWLEDGEMENTS We thank Director Alan Chi-Kong Wong, Siu-Fai Leung, Thomas Hon-Chung Sit and Howard Kai-Hay Wong (HKSAR AFCD) for facilitation and support; and Veterinary Officers of the AFCD Animal Management Centres for assistance and collection of samples. We are grateful to the generous support of Mrs Carol Yu, Professor Richard Yu, Mr Hui Hoy and Mr Hui Ming in the genomic sequencing platform, and Ms Eunice Lam on emerging infectious disease research. This work is partly supported by the Research Grant Council (grant HKU M), University Grant Council; Committee for Research and Conference Grant, Strategic Research Theme Fund and University Development Fund, the University of Hong Kong; the Shaw Foundation; the Providence Foundation Limited in memory of the late Dr Lui Hac Minh; a donation from Ms Eunice Lam; and the Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Disease for the HKSAR Department of Health. REFERENCES Adlhoch, C., Kaiser, M., Ellerbrok, H. & Pauli, G. (2010). High prevalence of porcine hokovirus in German wild boar populations. Virol J 7, 171. Allander, T., Tammi, M. T., Eriksson, M., Bjerkner, A., Tiveljung- Lindell, A. & Andersson, B. (2005). Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci U SA102, Apweiler, R., Attwood, T. K., Bairoch, A., Bateman, A., Birney, E., Biswas, M., Bucher, P., Cerutti, L., Corpet, F. & other authors (2001). The InterPro database, an integrated documentation resource for protein families, domains and functional sites. Nucleic Acids Res 29, Arthur, J. L., Higgins, G. D., Davidson, G. P., Givney, R. C. & Ratcliff, R. M. (2009). A novel bocavirus associated with acute gastroenteritis in Australian children. PLoS Pathog 5, e Journal of General Virology 93

9 Novel feline and canine bocaviruses Battilani, M., Scagliarini, A., Ciulli, S., Morganti, L. & Prosperi, S. (2006). High genetic diversity of the VP2 gene of a canine parvovirus strain detected in a domestic cat. Virology 352, Bernard, H.-U., Burk, R. D., de Villiers, E.-M. & zur Hausen, H. (2012). Family Papillomaviridae. In Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses, pp Edited by A. M. Q. King, M. J. Adams, E. B. Carstens & E. J. Lefkowitz. San Diego, CA: Elsevier. Binn, L. N., Lazar, E. C., Eddy, G. A. & Kajima, M. (1970). Recovery and characterization of a minute virus of canines. Infect Immun 1, Blomström, A. L., Belák, S., Fossum, C., McKillen, J., Allan, G., Wallgren, P. & Berg, M. (2009). Detection of a novel porcine boca-like virus in the background of porcine circovirus type 2 induced postweaning multisystemic wasting syndrome. Virus Res 146, Cadar, D., Cságola, A., Lőrincz, M., Tombácz, K., SpÎnu, M. & Tuboly, T. (2011). Distribution and genetic diversity of porcine hokovirus in wild boars. Arch Virol 156, Carmichael, L. E., Schlafer, D. H. & Hashimoto, A. (1991). Pathogenicity of minute virus of canines (MVC) for the canine fetus. Cornell Vet 81, Chen, A. Y., Cheng, F., Lou, S., Luo, Y., Liu, Z., Delwart, E., Pintel, D. & Qiu, J. (2010). Characterization of the gene expression profile of human bocavirus. Virology 403, Cheng, W. X., Li, J. S., Huang, C. P., Yao, D. P., Liu, N., Cui, S. X., Jin, Y. & Duan, Z. J. (2010). Identification and nearly full-length genome characterization of novel porcine bocaviruses. PLoS ONE 5, e Duffy, S., Shackelton, L. A. & Holmes, E. C. (2008). Rates of evolutionary change in viruses: patterns and determinants. Nat Rev Genet 9, Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U. & Ball, L. A. (2005). Virus Taxonomy: Classification and Nomenclature of Viruses. Eighth Report of the International Committee on Taxonomy of Viruses. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. San Diego, CA: Elsevier. Fryer, J. F., Kapoor, A., Minor, P. D., Delwart, E. & Baylis, S. A. (2006). Novel parvovirus and related variant in human plasma. Emerg Infect Dis 12, Fryer, J. F., Delwart, E., Hecht, F. M., Bernardin, F., Jones, M. S., Shah, N. & Baylis, S. A. (2007). Frequent detection of the parvoviruses, PARV4 and PARV5, in plasma from blood donors and symptomatic individuals. Transfusion 47, Guindon, S. & Gascuel, O. (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52, Herrewegh, A. A., Smeenk, I., Horzinek, M. C., Rottier, P. J. & de Groot, R. J. (1998). Feline coronavirus type II strains and originate from a double recombination between feline coronavirus type I and canine coronavirus. J Virol 72, Hoelzer, K., Shackelton, L. A., Holmes, E. C. & Parrish, C. R. (2008). Within-host genetic diversity of endemic and emerging parvoviruses of dogs and cats. J Virol 82, Hogan, A. & Faust, E. A. (1986). Nonhomologous recombination in the parvovirus chromosome: role for a CTATTTCT motif. Mol Cell Biol 6, Hokynar, K., Söderlund-Venermo, M., Pesonen, M., Ranki, A., Kiviluoto, O., Partio, E. K. & Hedman, K. (2002). A new parvovirus genotype persistent in human skin. Virology 302, Jones, M. S., Kapoor, A., Lukashov, V. V., Simmonds, P., Hecht, F. & Delwart, E. (2005). New DNA viruses identified in patients with acute viral infection syndrome. J Virol 79, Kapoor, A., Slikas, E., Simmonds, P., Chieochansin, T., Naeem, A., Shaukat, S., Alam, M. M., Sharif, S., Angez, M. & other authors (2009). A newly identified bocavirus species in human stool. J Infect Dis 199, Kapoor, A., Mehta, N., Esper, F., Poljsak-Prijatelj, M., Quan, P. L., Qaisar, N., Delwart, E. & Lipkin, W. I. (2010a). Identification and characterization of a new bocavirus species in gorillas. PLoS ONE 5, e Kapoor, A., Simmonds, P., Slikas, E., Li, L., Bodhidatta, L., Sethabutr, O., Triki, H., Bahri, O., Oderinde, B. S. & other authors (2010b). Human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections. J Infect Dis 201, Kapoor, A., Mehta, N., Dubovi, E. J., Simmonds, P., Govindasamy, L., Medina, J. L., Street, C., Shields, S. & Lipkin, W. I. (2012). Characterization of novel canine bocaviruses and their association with respiratory disease. J Gen Virol 93, Keane, T. M., Creevey, C. J., Pentony, M. M., Naughton, T. J. & Mclnerney, J. O. (2006). Assessment of methods for amino acid matrix selection and their use on empirical data shows that ad hoc assumptions for choice of matrix are not justified. BMC Evol Biol 6, 29. Lau, S. K., Woo, P. C., Li, K. S., Huang, Y., Tsoi, H. W., Wong, B. H., Wong, S. S., Leung, S. Y., Chan, K. H. & Yuen, K. Y. (2005). Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc Natl Acad Sci U S A 102, Lau, S. K. P., Yip, C. C. Y., Que, T. L., Lee, R. A., Au-Yeung, R. K., Zhou, B. P., So, L. Y., Lau, Y. L., Chan, K. H. & other authors (2007). Clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples from children in Hong Kong. J Infect Dis 196, Lau, S. K., Woo, P. C., Tse, H., Fu, C. T., Au, W. K., Chen, X. C., Tsoi, H. W., Tsang, T. H., Chan, J. S. & other authors (2008). Identification of novel porcine and bovine parvoviruses closely related to human parvovirus 4. J Gen Virol 89, Lau, S. K., Woo, P. C., Yip, C. C., Li, K. S., Fu, C. T., Huang, Y., Chan, K. H. & Yuen, K. Y. (2011). Co-existence of multiple strains of two novel porcine bocaviruses in the same pig, a previously undescribed phenomenon in members of the family Parvoviridae, and evidence for inter- and intra-host genetic diversity and recombination. J Gen Virol 92, Lau, S. K., Woo, P. C., Yip, C. C., Choi, G. K., Wu, Y., Bai, R., Fan, R. Y., Lai, K. K., Chan, K. H. & Yuen, K. Y. (2012). Identification of a novel feline picornavirus from the domestic cat. J Virol 86, Li, L., Shan, T., Wang, C., Côté, C., Kolman, J., Onions, D., Gulland, F. M. & Delwart, E. (2011). The fecal viral flora of California sea lions. J Virol 85, Li, B., Ma, J., Xiao, S., Fang, L., Zeng, S., Wen, L., Zhang, X., Ni, Y., Guo, R. & other authors (2012). Complete genome sequence of a novel species of porcine bocavirus, PBoV5. J Virol 86, McKillen, J., McNeilly, F., Duffy, C., McMenamy, M., McNair, I., Hjertner, B., Millar, A., McKay, K., Lagan, P. & other authors (2011). Isolation in cell cultures and initial characterisation of two novel bocavirus species from swine in Northern Ireland. Vet Microbiol 152, Mochizuki, M., Hashimoto, M., Hajima, T., Takiguchi, M., Hashimoto, A., Une, Y., Roerink, F., Ohshima, T., Parrish, C. R. & Carmichael, L. E. (2002). Virologic and serologic identification of minute virus of canines (canine parvovirus type 1) from dogs in Japan. J Clin Microbiol 40, Nguyen, Q. T., Wong, S., Heegaard, E. D. & Brown, K. E. (2002). Identification and characterization of a second novel human erythrovirus variant, A6. Virology 301, Pellett, P. E., Davison, A. J., Eberle, R., Ehlers, B., Hayward, G. S., Lacoste, V., Minson, A. C., Nicholas, J., Roizman, B. & other authors

10 S. K. P. Lau and others (2012). Family Herpesviridae. InVirus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses, pp Edited by A. M. Q. King, M. J. Adams, E. B. Carstens & E. J. Lefkowitz. San Diego, CA: Elsevier. Pozo, F., García-García, M. L., Calvo, C., Cuesta, I., Pérez-Breña, P. & Casas, I. (2007). High incidence of human bocavirus infection in children in Spain. J Clin Virol 40, Servant, A., Laperche, S., Lallemand, F., Marinho, V., De Saint Maur, G., Meritet, J. F. & Garbarg-Chenon, A. (2002). Genetic diversity within human erythroviruses: identification of three genotypes. J Virol 76, Shackelton, L. A., Parrish, C. R., Truyen, U. & Holmes, E. C. (2005). High rate of viral evolution associated with the emergence of carnivore parvovirus. Proc Natl Acad Sci U S A 102, Shade, R. O., Blundell, M. C., Cotmore, S. F., Tattersall, P. & Astell, C. R. (1986). Nucleotide sequence and genome organization of human parvovirus B19 isolated from the serum of a child during aplastic crisis. J Virol 58, Shan, T., Lan, D., Li, L., Wang, C., Cui, L., Zhang, W., Hua, X., Zhu, C., Zhao, W. & Delwart, E. (2011a). Genomic characterization and high prevalence of bocaviruses in swine. PLoS ONE 6, e Shan, T., Li, L., Simmonds, P., Wang, C., Moeser, A. & Delwart, E. (2011b). The fecal virome of pigs on a high-density farm. J Virol 85, Sharp, C. P., LeBreton, M., Kantola, K., Nana, A., Diffo, J. D., Djoko, C. F., Tamoufe, U., Kiyang, J. A., Babila, T. G. & other authors (2010). Widespread infection with homologues of human parvoviruses B19, PARV4, and human bocavirus of chimpanzees and gorillas in the wild. J Virol 84, Siegl, G., Bates, R. C., Berns, K. I., Carter, B. J., Kelly, D. C., Kurstak, E. & Tattersall, P. (1985). Characteristics and taxonomy of Parvoviridae. Intervirology 23, Sloots, T. P., McErlean, P., Speicher, D. J., Arden, K. E., Nissen, M. D. & Mackay, I. M. (2006). Evidence of human coronavirus HKU1 and human bocavirus in Australian children. J Clin Virol 35, Söderlund-Venermo, M., Lahtinen, A., Jartti, T., Hedman, L., Kemppainen, K., Lehtinen, P., Allander, T., Ruuskanen, O. & Hedman, K. (2009). Clinical assessment and improved diagnosis of bocavirus-induced wheezing in children, Finland. Emerg Infect Dis 15, Spahn, G. J., Mohanty, S. B. & Hetrick, F. M. (1966). Experimental infection of calves with hemadsorbing enteric (HADEN) virus. Cornell Vet 56, Steinel, A., Munson, L., van Vuuren, M. & Truyen, U. (2000). Genetic characterization of feline parvovirus sequences from various carnivores. J Gen Virol 81, Storz, J., Leary, J. J., Carlson, J. H. & Bates, R. C. (1978). Parvoviruses associated with diarrhea in calves. J Am Vet Med Assoc 173, Sun, Y., Chen, A. Y., Cheng, F., Guan, W., Johnson, F. B. & Qiu, J. (2009). Molecular characterization of infectious clones of the minute virus of canines reveals unique features of bocaviruses. J Virol 83, Truyen, U. (2006). Evolution of canine parvovirus a need for new vaccines? Vet Microbiol 117, Truyen, U., Evermann, J. F., Vieler, E. & Parrish, C. R. (1996). Evolution of canine parvovirus involved loss and gain of feline host range. Virology 215, Tse, H., Tsoi, H. W., Teng, J. L., Chen, X. C., Liu, H., Zhou, B., Zheng, B. J., Woo, P. C., Lau, S. K. & Yuen, K. Y. (2011). Discovery and genomic characterization of a novel ovine partetravirus and a new genotype of bovine partetravirus. PLoS ONE 6, e van den Brand, J. M., van Leeuwen, M., Schapendonk, C. M., Simon, J. H., Haagmans, B. L., Osterhaus, A. D. & Smits, S. L. (2012). Metagenomic analysis of the viral flora of pine marten and European badger feces. J Virol 86, Wang, K., Wang, W., Yan, H., Ren, P., Zhang, J., Shen, J. & Deubel, V. (2010). Correlation between bocavirus infection and humoral response, and co-infection with other respiratory viruses in children with acute respiratory infection. J Clin Virol 47, Woo, P. C. Y., Lau, S. K. P., Choi, G. K. Y., Yip, C. C. Y., Huang, Y., Tsoi, H. W. & Yuen, K. Y. (2012a). Complete genome sequence of a novel picornavirus, canine picornavirus, discovered in dogs. J Virol 86, Woo, P. C., Lau, S. K., Wong, B. H., Fan, R. Y., Wong, A. Y., Zhang, A. J., Wu, Y., Choi, G. K., Li, K. S. & other authors (2012b). Feline morbillivirus, a previously undescribed paramyxovirus associated with tubulointerstitial nephritis in domestic cats. Proc Natl Acad Sci U SA109, Zeng, S., Wang, D., Fang, L., Ma, J., Song, T., Zhang, R., Chen, H. & Xiao, S. (2011). Complete coding sequences and phylogenetic analysis of porcine bocavirus. J Gen Virol 92, Zhai, S., Yue, C., Wei, Z., Long, J., Ran, D., Lin, T., Deng, Y., Huang, L., Sun, L. & other authors (2010). High prevalence of a novel porcine bocavirus in weanling piglets with respiratory tract symptoms in China. Arch Virol 155, Journal of General Virology 93