Beruflich Dokumente
Kultur Dokumente
Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic
Blood Systems Research Institute, San Francisco, San Francisco, CA, USA
Department of Laboratory Medicine, University of California at San Francisco, San Francisco, CA, USA
c
Department of Animal Science, Rural Engineering and Veterinary, Polytechnic Institut of Viseu, Viseu, Portugal
d
Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal
e
Regional Laboratory of Virology, ANTSZ Regional Institute of State Public Health Service, Pecs, Hungary
f
The Pirbright Institute, Woking, Surrey, United Kingdom
g
NCIRD, National Calicivirus Laboratory, Centers for Disease Control and Prevention, Atlanta, GA, USA
h
GRVLB, Rotavirus Surveillance, Centers for Disease Control and Prevention, Atlanta, GA, USA
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 10 January 2014
Received in revised form 29 March 2014
Accepted 1 April 2014
Humans keep more than 80 million cats worldwide, ensuring frequent exposure to their
viruses. Despite such interactions the enteric virome of cats remains poorly understood.
We analyzed a fecal sample from a single healthy cat from Portugal using viral
metagenomics and detected ve eukaryotic viral genomes. These viruses included a novel
picornavirus (proposed genus Sakobuvirus) and bocavirus (feline bocavirus 2), a variant
of feline astrovirus 2 and sequence fragments of a highly divergent feline rotavirus and
picobirnavirus. Feline sakobuvirus A represents the prototype species of a proposed new
genus in the Picornaviridae family, distantly related to human salivirus and kobuvirus.
Feline astroviruses (mamastrovirus 2) are the closest known relatives of the classic human
astroviruses (mamastrovirus 1), suggestive of past cross-species transmission. Presence of
these viruses by PCR among Portuguese cats was detected in 13% (rotavirus), 7%
(astrovirus), 6% (bocavirus), 4% (sakobuvirus), and 4% (picobirnavirus) of 55 feline fecal
samples. Co-infections were frequent with 40% (4/10) of infected cats shedding more than
one of these ve viruses. Our study provides an initial description of the feline fecal virome
indicating a high level of asymptomatic infections. Availability of the genome sequences of
these viruses will facilitate future tropism and feline disease association studies.
2014 Elsevier B.V. All rights reserved.
Keywords:
Metagenomics
Virome
Enteric virus
Felis catus
Sakobuvirus
1. Introduction
Cats are one of the most common pets with worldwide
population estimated at 80400 million. Interactions with
humans presumably accelerated following domestication
* Corresponding author at: Blood Systems Research Institute, University of California, San Francisco, 270 Masonic Ave., San Francisco, CA
94118, USA. Tel.: +1 415 923 5763.
E-mail address: delwarte@medicine.ucsf.edu (E. Delwart).
http://dx.doi.org/10.1016/j.vetmic.2014.04.005
0378-1135/ 2014 Elsevier B.V. All rights reserved.
Picornavirus
Astrovirus
Bocavirus
Rotavirus
Picobirnavirus
Other sequences
48,094
423
348
2
13
479,278
9.11
0.08
0.07
<0.01
<0.01
90.75
Total
528,158
100.00
103
104
Fig. 1. Phylogenetic and genomic analysis of ve feline enteric viruses. The Bayesian inference trees were conducted based on (A) 3D RdRp protein depicting
relationships among representative members of the family Picornaviridae, and a table detailing genomic features of the reference genomes based on
previously published literature (Lau et al., 2012b; Sweeney et al., 2012). Symbols for protease were noted as follows: Y, presence of a protease motif in L; Y/
N, presence of L but L is not protease; N, absence of L. Other motifs are described in the main text (B) the complete ORF2 capsid protein of representative
astroviruses (C) the complete non-structural protein (NS1) of bocaviruses, (D) the partial intermediate capsid protein of rotavirus Segment 6, and (E) the
partial RdRp protein of picobirnavirus Segment 2. Posterior probabilities of the Bayesian analysis (>95%) are shown next to each node. Scale bar indicates
amino acid substitutions per site.
105
Fig. 2. Genome organization, identity plot and pairwise sequence comparison analyses of three feline virus genomes (A) feline sakobuvirus A, (B) feline
astrovirus Viseu, and (C) feline bocavirus 2. Cleavage site predictions of the sakobuvirus are shown below its genome. For each virus, three identity plots are
shown below the genome organization, comparing the coding regions for each virus with three related viruses based on phylogenetic analyses in Fig. 1. The
identity values were color-coded: green, 100%; light green, 30100%, red, <30%. (For interpretation of the references to color in this gure legend, the reader
is referred to the web version of this article.)
106
Fig. 3. Predicted RNA secondary structure of the 50 and 30 UTR of the feline sakobuvirus A (FeAstV-A) and the 30 UTR of the feline astrovirus Viseu. For FeAstVA 50 UTR, the type IV IRES has been annotated according to picornavirus conventions. Domains are labeled II and III; individual helical segments are labeled
II1, II2, III1, and III2, etc.; and individual hairpins are labeled IIIa and IIIb, etc. to maintain the continuity of the current nomenclature. The positions of
conserved domains and the polyprotein AUG start codon are indicated by shaded boxes. For FeAstV-A 30 UTR, detailed structure of the barbell-like
formation between nucleotide 7712 and 7775 was shown. Grey boxes indicate identical nucleotides in members of genera Avihepatovirus, Kobuvirus,
Gallivirus and Passerivirus. The presence of poly(Y) tract characteristic of this barbell-like structure is indicated.
2.1. Astrovirus
Using deep sequencing, PCR and RACE, we obtained a
complete feline astrovirus genome (FeAstV Viseu). Astroviruses are known to infect a wide variety of mammals and
birds, and feline astrovirus has been shown to induce
enteritis by experimental infection of specic-pathogenfree cats (Harbour et al., 1987). Feline astrovirus (FeAstV)
was observed by electron microscopy from cat feces with
or without diarrhea (Hoshino et al., 1981; Marshall et al.,
1987), and FeAstV sequences have been reported in Europe
and in Asia (Lau et al., 2013; Harbour et al., 1987).
The genome of feline astrovirus Viseu was 6780
nucleotides in length, with 49.8% GC content (GenBank
KF374704). Consistent with other astroviruses, three ORFs
were identied, including ORF1a and ORF1b that encode
non-structural proteins, and ORF2 that encodes the
structural capsid proteins. In the ORF1a/1b overlap region
of the FeAstV genome, the conserved slippery ribosomal
frame shift signal (AAAAAAC) was identied. In addition, at
the ORF1ab/ORF2 junction, the highly conserved promoter
sequence UUUGGAGNGGNGGACCNAAN7 AUGNC was
identied, in which the start codon AUG initiating ORF2
is located near 30 end.
Phylogenetically, FeAstV Viseu belongs to the mamastrovirus genogroup 1 (Fig. 1B), and is closely related to a
feline astrovirus (FeAstV2) reported in September 2013 in
cats from Hong Kong (Lau et al., 2013) and more distantly
to another feline astrovirus from the United Kingdom
(FeAstV Bristol, GenBank AF056197). Using pairwise
comparison of the entire capsid protein, FeAstV Viseu
shared 92.6% identity to FeAstV2 from HK, 70.8% to FeAstV
Bristol, and 58.7% to human astrovirus 1. Among feline
astroviruses, FeAstV Viseu and FeAstV2 differ most from
FeAstV Bristol in the C-terminal half of the capsid protein,
sharing %3Flt;40% identity.
According to the ICTV, the ORF2 capsid proteins are
used to distinguish genotypes and species of astroviruses.
Noticeably, the N-terminal half of the feline astrovirus
ORF2s shared high amino acid identities among themselves (FeAstV Viseu and FeAstV 2, 96%, Fig. 2B) and with
human astroviruses (FeAstV Viseu and HAstV1, 89%). The
FeAstVs are therefore most closely related to HAstVs in the
capsid amino terminal half, compared to the next closest
relative, the porcine astroviruses (FeAstV Viseu and
PoAstV2, 65%). In addition, long stretches of near-identical
amino acid residues are observed between feline and
human astroviruses (Fig. 2B, black arrow)
The carboxyl terminal half of the ORF2 capsid protein
showed much weaker conservation (Fig. 2B). A conserved
motif (SRGHAE) was recognized. The 30 UTR was 84
nucleotides long, and shares most nucleotide identities
(94%) to human astrovirus SH1 (GenBank FJ375759). RNA
folding analysis revealed a double stem loop structure in the
3%3Fprime; UTR sequence typical for astroviruses (Fig. 3).
Based on currently available genome information, the
most closely related viruses to the classic human
astroviruses (mamastrovirus 1 genotype 18) are therefore feline astroviruses, with the Bristol strain slightly
closer than Viseu or the Honk Kong FeAstV2 strains (Fig. 1B
and Fig. 2B). Past human-cat cross-species astrovirus
107
108
1 cat with
2 viruses
Sakobuvirus
1 cat with
Sakobuvirus only
Rotavirus
Astrovirus
3 cats with
rotavirus
only
1 cat with
Astrovirus
only
1 cat with
5 viruses
2 cats with
3 viruses
Picobirnavirus
1 cat with
picobirnavirus
only
Bocavirus
109
Table 2
Primers used for the PCR screening.
Primer name
Primer sequence
SakobuAF
SakobuAR
FeAstVAF
FeAstVBF
FBoV2AF
FBoV2AR
FeRVAF
FeRVAR
FePBVAF
FePBVAR
110
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