SHORT REPORTOpen Access
A novel bocavirus in canine liver
Linlin Li1,2, Patricia A Pesavento3, Christian M Leutenegger4, Marko Estrada4, Lark L Coffey1,2, Samia N Naccache2,5,
Erik Samayoa2,5, Charles Chiu2,5, Jianming Qiu6, Chunlin Wang7, Xutao Deng1and Eric Delwart1,2*
Background: Bocaviruses are classified as a genus within the Parvoviridae family of single-stranded DNA viruses and
are pathogenic in some mammalian species. Two species have been previously reported in dogs, minute virus of
canines (MVC), associated with neonatal diseases and fertility disorders; and Canine bocavirus (CBoV), associated
with respiratory disease.
Findings: In this study using deep sequencing of enriched viral particles from the liver of a dog with severe
hemorrhagic gastroenteritis, necrotizing vasculitis, granulomatous lymphadenitis and anuric renal failure, we
identified and characterized a novel bocavirus we named Canine bocavirus 3 (CnBoV3). The three major ORFs of
CnBoV3 (NS1, NP1 and VP1) shared less than 60% aa identity with those of other bocaviruses qualifying it as a
novel species based on ICTV criteria. Inverse PCR showed the presence of concatemerized or circular forms of the
genome in liver.
Conclusions: We genetically characterized a bocavirus in a dog liver that is highly distinct from prior canine
bocaviruses found in respiratory and fecal samples. Its role in this animal’s complex disease remains to be
Keywords: Canine bocavirus 3, Episome, Coinfection
Parvoviruses consist of small non-enveloped, autono-
mously replicating, single-stranded DNA viruses with
genome length between 4.5 - 5.5 kb . Bocavirus, a
genus of the family Parvoviridae, is characterized by the
presence of a third major ORF named NP1. Bocaviruses
are known to infect multiple mammalian species includ-
ing humans , cows , pigs [4-6], gorillas , chim-
panzees , California sea lions , dogs [10-13], cats
, bats , and pine martens . Bocavirus infec-
tions can cause respiratory and gastrointestinal symp-
toms in young animals and humans, but are also often
subclinical in adults [2,16]. While many bocaviruses
were initially identified in feces or respiratory secretion
they can also be found in blood [2,16].
Minute virus of canines (MVC) was the first known
bocavirus infecting dogs. MVC was isolated in 1967 in
the feces of a clinical healthy dog, and later recognized
as causing neonatal diseases and fertility disorders in
dogs . The second species of dog bocavirus (Canine
bocavirus, CBoV) was identified in 2011 in the respira-
tory samples from diseased and healthy dogs . One
genotype of CBoV was associated with respiratory dis-
ease as it showed higher prevalence in diseased animals
than healthy controls . Variants of this CBoV were
also detected in fecal, nasal, urine and blood samples
collected from dogs in Hong Kong .
In this study, an infectious etiology was suspected for
a dog with severe hemorrhagic gastroenteritis, necrotiz-
ing vasculitis, granulomatous lymphadenitis and anuric
renal failure. The clinical and post-mortem workups
for infectious causes in this case included negative
test results for Canine Parvovirus 2, Canine Enteric
Campylobacter, Clostridium perfringens enterotoxin A
gene, Cryptosporidium, and Giardia. Special stains of
histologic specimens revealed no detectable bacteria or
other known infectious agents. Using deep sequencing,
we characterized viral sequences present in the dog’s
liver revealing a third species of canine bocavirus
* Correspondence: firstname.lastname@example.org
1Blood Systems Research Institute, San Francisco, CA, USA
2Department of Laboratory Medicine, University of California, San Francisco,
Full list of author information is available at the end of the article
© 2013 Li et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Li et al. Virology Journal 2013, 10:54
Results and discussion
A sequence of 300 bases showing sequence similarity to
bocaviruses (BLASTx E<10-3) was initially identified by
454 pyrosequencing. The sequence was extended by de-
generate PCR targeting conserved bocavirus regions,
yielding a ~2.5 kb partial genome sequence. The tissue
nucleic acids were further analyzed using the MiSeq Illu-
mina platform generating 16 contigs composed of 133
reads with similarities to bocaviruses (BLASTx E<10-3),
which allowed the amplification of all three ORFs. The
virus was highly divergent from the other two known ca-
nine bocaviruses, MVC and CBoV, and was provisionally
named Canine bocavirus 3 (CnBoV3) [10,11,13].
The nonstructural (NS1) protein encoded by ORF1 was
778 aa long, and contained motifs associated with rolling
circle replication, helicase and ATPase. The NP1 protein
encoded by the middle ORF3 was 194 aa long. The ORF2
encoded capsid proteins VP1 (689 aa) and proteolytically
processed VP2 (560 aa). CnBoV3 shared 51%, 57%, 56% aa
similarity with the NS1, NP1 and VP1 region of CBoV, re-
spectively, and 49%, 52%, 57% aa similarity with the NS1,
NP1 and VP1 regions of MVC. Phylogenetic analysis of
the entire VP1 was performed to determine the relation-
ship between CnBoV3 and other bocaviruses. CnBoV3
was phylogenetically distinct from the known dog boca-
viruses and only loosely related to California sea lion
bocaviruses, CBoV, feline bocavirus and MVC (Figure 1).
Phylogenetic trees constructed by NS1 and NP1 region
yielded similar topology (data not shown).
Bocaviruses are believed to replicate through the parvo-
virus rolling hairpin model, which generate replication
intermediates of concatemers with head-to-head or tail-to
-tail structure . Recent experimental evidence showed
the presence of head-to-tail concatemers or circularized
genomes of human bocavirus (HBoV) 1&3 [18,19] and
porcine bocaviruses , indicating that some bocaviruses
may use a rolling - circle replication model.
Using inverse PCR with primers directed outward from
the 5’ and 3’ extremities of the partial genome sequence we
were able to amplify head-to-tail sequence of the CnBoV3
non-translated regions (NTR) between the VP termination
and the NS1 initiation codons (Figure 2A). The generation
of specific PCR products indicated the presence of conca-
temerized or circular forms of the genome in the liver .
To confirm these results, multiple repeats of the inverse
PCR were generated and directly sequenced as well as
subcloned into a plasmid vector. PCR amplicons and plas-
mid inserts were Sanger sequenced using protocol for
GC-rich/hairpin sequences. The resulting sequences were
aligned and the length of the region between the VP stop
and the NS start codon calculated. This region varied in
length from 392 to 506 nt (Figure 2C). Mfold analyses of
the longest NTR sequence (506 nt) showed that the long
palindromic hairpin terminal repeats (e.g. approximately
150 nt at both ends in MVC or bovine parvovirus, BPV)
were missing, but a typical “rabbit ear” structure was
detected that was nearly identical to a structure in the
MVC 5'NTR sequence (Figure 2B). These results sug-
gested that inverse PCR may have been impeded by strong
secondary DNA structures (Figure 2A &C, between two
red arrows). None of the currently reported NTR regions
of bocavirus obtained by inverse PCR contained both
complete inverted terminal repeats seen in MVC or
Figure 1 Phylogenetic tree based on aligned amino acid sequences of full-length VP proteins of representative bocavirus species.
Li et al. Virology Journal 2013, 10:54
Page 2 of 4
In this study we genetically characterize a third species
of canine bocavirus (CnBoV3), highly divergent from
MVC  and CBoV [10,11], in the liver of a dog with
severe disease. The presence of three distinct canine
bocaviruses, phylogenetically interspersed with viruses
from different mammalian hosts, may reflect their ori-
gins from different cross-species transmissions. The de-
tection of CnBoV3 in liver tissue indicated that the virus
had likely breached the mucosal barrier of the typical
sites of bocavirus replication in the respiratory or enteric
tracts. The detection of episomal forms by PCR indi-
cated that replication may be occurring in hepatocytes
or other liver cell types and that viremia was also likely
present although the lack of available blood sample pre-
vented direct testing. We also detected in that animal
co-infection with a canine circovirus. Circovirus infec-
tion can lead to lymphocytic depletion and immunosup-
pression in the host . Whether CnBoV3, canine
circovirus, or their combination were involved in this
dog’s severe symptoms requires further investigation.
Materials and methods
A dog suffering from vomiting and hemorrhagic gastro-
enteritis was euthanized after a rapid disease course last-
ing seven days. Necropsy showed the presence of
necrotizing vasculitis, granulomatous lymphadenitis and
anuric renal failure. Clinical and post-mortem tests for
multiple infectious agents of enteric disease were all
negative. Liver Tissue was stored at −80?C until further
processing. Tissue samples (~25 mg) were homogenized,
filtered, and nuclease treated as previously described to
enrich for nucleic acids within viral particles . Nu-
cleic acids were then extracted using the QIAamp viral
RNA Mini kit (Qiagen), randomly amplified using ran-
dom RT-PCR with randomized 3’ primers and nucleic
acid libraries prepared as previously described  for
sequencing using the Genome Sequencer FLX Instru-
ment (454 Life Science, Roche). The pyrosequencing
reads were sorted, trimmed, assembled and compared to
the GenBank non-redundant databases as previously
described . Potential viral sequences were identified
with translated protein sequence similarity matches
PCR skips or partially
skips this region
Figure 2 A) Inverse PCR was performed to obtain the NTR sequence between the VP termination and the NS initiation codon of
episomal forms of CnBoV3. Primers (P1 and P2) were situated at VP and NS region respectively. Secondary structures (symbolic in the box) may
have impeded the PCR resulting in several products of different length. B) Highly identical ‘rabbit ear’ structure of CnBoV3 and MVC. C)
Alignments of NTR sequences obtained by sequencing different inverse PCR products directly (PCR1&2) and subcloning products (colony 1–4).
Sequences outside the two arrows were identical. Underlined sequence shows location of “rabbit ear” sequence.
Li et al. Virology Journal 2013, 10:54
Page 3 of 4
(BLASTx to GenBank's NR database with E-value<10-3) Download full-text
to known viral sequences. The presence of virus pro-
tein sequences detected by 454 pyrosequencing was
confirmed by PCR and Sanger sequencing. Genome
walking and degenerate PCR were used to amplify the
viral genome. Enriched viral nucleic acids from the
infected tissue was also handled using the ScriptSeq
sequenced by MiSeq system (illumina). The resulting
near complete genome of CnBoV3 was deposited in
GenBank with accession no. KC580640. Phylogenetic
analyses based on aligned amino acid sequences from
full-length VP proteins were generated by the neigh-
bor joining method in MEGA , using amino acid
p-distances, with 1,000 bootstrap replicates.
The authors declared that they have no competing interests.
LL generated the data, interpreted the data and drafted the manuscript. PP
selected and collected the animal sample and helped interpret results. CL
and ME performed real-time PCR. LC, SN, ES and CC provided help with
Illumina sequencing. CW and XD provided bioinformatics analyses of 454
and Illumina data. JQ assisted in the NTR analysis. ED directed the research
and revised the draft. All authors read and approved the final manuscript.
The work was supported by the Blood Systems Research Institute and NIH
R01 HL105770 to ED. The Bernice Barbour Foundation and the UC Davis
Center for Companion Animal Health supports the research of PP.
1Blood Systems Research Institute, San Francisco, CA, USA.2Department of
Laboratory Medicine, University of California, San Francisco, CA, USA.
3Department of Pathology, Microbiology and Immunology, School of
Veterinary Medicine, University of California, Davis, CA, USA.4IDEXX
Reference Laboratories, CA, USA.5UCSF-Abbott Viral Diagnostics and
Discovery Center, CA, USA.6Department of Microbiology, University of
Kansas, KS, USA.7Stanford Genome Technology Center, Stanford, CA, USA.
Received: 19 December 2012 Accepted: 31 January 2013
Published: 13 February 2013
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Cite this article as: Li et al.: A novel bocavirus in canine liver. Virology
Journal 2013 10:54.
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