key: cord-282062-h9smg0w9
authors: Takano, Tomomi; Yanai, Yoshitomo; Hiramatsu, Kanae; Doki, Tomoyoshi; Hohdatsu, Tsutomu
title: Novel single-stranded, circular DNA virus identified in cats in Japan
date: 2018-09-14
journal: Arch Virol
DOI: 10.1007/s00705-018-4020-6
sha: 
doc_id: 282062
cord_uid: h9smg0w9

We detected a novel feline stool-associated circular DNA virus (FeSCV) in fecal samples from cats with diarrhea using consensus primers matching those of circovirus and cyclovirus. FeSCV is a circular DNA virus containing a genome with a total length of 2,046 nt encoding 2 open reading frames. Phylogenetic analyses indicated that FeSCV is classified into a clade different from that of circovirus and cyclovirus. Since the FeSCVs detected in several cats in the same household had genetically similar genomes, these viruses are most likely derived from the same origin. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00705-018-4020-6) contains supplementary material, which is available to authorized users.

RCR III) are commonly present [7] . Circoviruses include porcine circovirus 2, which causes post weaning multisystemic wasting syndrome in pigs [8] , beak and feather disease (BFD) virus, which causes BFD in psittacines [9] , and canine circovirus (CaCV), which causes vasculitis and gastroenteritis in dogs [10] . Cycloviruses include human cyclovirus, which is associated with human acute flaccid paralysis [11] , and bat cyclovirus isolated from bat feces [12] . In cats, no Circoviridae family other than feline cyclovirus has been reported. Feline cyclovirus was identified by next-generation sequencing analysis in which the viral gene was detected in a pooled fecal sample collected from 4-5 healthy cats. Its pathogenicity in cats has not been clarified [13] . In this study, we performed nested PCR using Circoviridae family consensus primers and detected novel CRESS DNA viruses in several cats with diarrhea symptoms. The full genomic sequences of these viruses were clarified, and a phylogenetic analysis was performed.

Fecal samples were collected from 20 cats at a private cattery in Japan. These cats were maintained under conditions in which they had contact with each other. Fourteen of the 20 cats developed diarrhea and did not respond to anthelmintics or antibiotics. The remaining 6 cats were healthy. The cats with diarrhea were separated from the other cats, and newly-collected feces were subjected to experiments. Feline coronavirus was detected in fecal samples from 5 healthy cats and 11 cats with diarrhea. In addition, feline bocavirus was detected in fecal samples from 3 cats with diarrhea ( We named the virus detected in this study feline stoolassociated circular virus (FeSCV). To determine the complete genomic sequence of FeSCV, inverse primers (CV841F 5'-TTC TCC CGA CCT GGA CAT AG and CV664R 5'-ACA GAG ATG ATA GCG TCC GG) were prepared based on a section of the base sequence of the acquired Rep gene, and inverse PCR was performed. Fig. S1 shows the schematic position of the inverse PCR primers and other primers used for the FeSCV genome. Prior to inverse PCR, circular DNA was multiplied using TempliPhi TM 100 Amplification Kit (GE Healthcare, USA). Viral DNA was amplified by inverse PCR, which was performed in a total volume of 50 μL using the following mixture: 3 μL of multiplied circular DNA mixed with 10 μL of 5X PrimeSTAR buffer (TaKaRa, Japan), 4 μL of dNTP mixture (TaKaRa, Japan) containing 2.5 mM of each dNTP, 1 μL of 20 μM primer mix (FC1F), 0.5 μL of PrimeSTAR HS DNA polymerase (2.5 U/mL; TaKaRa, Japan), and 31.5 μL of distilled water. Using a thermal cycler, the DNA was amplified at 98°C for 1 min followed by 30 cycles of denaturation at 98°C for 10 sec, primer annealing at 55°C for 15 sec, and synthesis at 72°C for 1 min with a final extension at 72°C for 5 min. From all samples, approximately 1,800-bp amplicons were acquired. The sequences of the viral genomes were obtained by sequencing the overlapping PCR products (amplicons of 400 and 1,800 bp in size). Phylogenetic trees based on Rep were analyzed using MEGA software (version 6). Phylogenetic relationships were evaluated using the neighbor-joining algorithm, and branching order reliability was analyzed by 1,000 replications of bootstrap resampling. All 4 strains of FeSCV were circular DNA viruses containing a genome (Fig. 1B) .

Of the viruses that infect eukaryotic organisms, those most similar to FeSCV were rodent stool-associated circular viruses (RodSCV). Of note, the putative Rep and putative Cap of FeSCV were approximately 50% homologous with RNA helicase and the hypothetical protein of Giardia intestinalis, respectively. The FeSCV genomes were submitted to GenBank under 3 accession numbers LC389584 (KU14), LC406404 (KU7), LC406405 (KU8), and LC406406 (KU9), respectively. Based on the base sequence of FeSCV clarified in this study, specific primers to detect the FeSCV gene (FeSCV1F 5'-GCT AAG GTC TGC CTC AGG TG and FeSCV1R 5'-CTA TGT CCA GGT CGG GAG AA) were prepared. PCR was performed as described above except the reaction temperature and time were modified as follows: DNA was amplified at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 30 sec, primer annealing at 55°C for 45 sec, and synthesis at 72°C for 45 sec with a final extension at 72°C for 7 min. When an amplicon with the expected size of 303 bp was detected, the sample was regarded as FeSCV-positive. Of the 14 cats with diarrhea, 10 were FeSCV-positive (71.4%). Three of the 6 healthy cats were FeSCV-positive (50.0%) ( Table 1 ; see FeSCV-specific primer).

To detect feline circovirus or feline cyclovirus in feline fecal samples, nested PCR using Circoviridae consensus primers was performed. The degenerate primers used in Table 2 Highly conserved motifs of Rep detected in FeSCV based on the report by Rosario et al. [7] x: any amino acid u: hydrophobic amino acid (F, I, L, V, or M) this nested PCR assay were prepared based on the consensus sequence of the Rep gene in 12 strains of circoviruses and one strain of cyclovirus [14] since we expected only circoviruses and cycloviruses to be detected. However, a novel CRESS DNA virus, FeSCV, was detected in the fecal samples. Phylogenetic analysis based on Rep revealed that FeSCV belongs in a clade different from that of circoviruses and cycloviruses for which the reason is unclear. Very few known CRESS DNA viruses, including circoviruses, have been confirmed to have pathogenicity in the host. Moreover, many studies on novel CRESS DNA viruses analyzed only viral genes, that is, only a few studies analyzed whether the viral infection is manifested in the host. The FeSCV found in this study was detected in 13 of the 20 cats. Based on this result, cats may be the natural host of FeSCV. Furthermore, the 4 FeSCVs (KU7 strain, KU8 strain, KU9 strain, and KU14 strain) are genetically similar and share approximately 100% of their genomes. This data suggests that these FeSCVs have the same origin. The FeSCV infection rates in healthy cats and cats with diarrhea were 50.0 and 71.4%, respectively and demonstrate that although the infection rate was slightly higher in cats with diarrhea, half of the healthy cats were also infected with FeSCV. Thus, it is unclear whether FeSCV is pathogenic. Furthermore, the rate of co-infection with FeSCV and other viruses was approximately 2-fold higher in cats with diarrhea (64.2%) than in healthy cats (33.3%). In dogs, a relationship between CaCV and gastroenteritis has been suggested. Dowgier et al. [15] reported that CaCV infection and the development of acute gastroenteritis were correlated in dogs co-infected with other enteric viruses. The incidence of enteritis may increase when FeSCV-infected cats are co-infected with other viruses. Further investigation of FeSCV pathogenicity in cats is necessary.

The putative Rep of FeSCV was 50% homologous with RNA helicase of Giardia intestinalis in Blastx analysis, and the putative Cap of FeSCV was approximately 45% homologous with a cysteine protease (hypothetical protein) of Giardia intestinalis. We first suspected that the FeSCV gene identified in this study was derived from Giardia intestinalis. However, we concluded that FeSCV is a circular DNA virus based on the following: 1) No Giardia intestinalis was detected in the fecal test, and 2) the complete genome of FeSCV was amplified using the rolling-circle amplification and inverse PCR assays. Similar with FeSCV, RodSCV is reported to have Rep with <52% homology with RNA helicase of Giardia intestinalis [16] . Moreover, both the putative Rep and putative Cap of FeSCV are similar to proteins derived from Giardia intestinalis. To our knowledge, no study has reported a gene encoding Cap or putative Cap of CRESS DNA virus generated by recombination with parasitic or bacterial genes. Further investigation is necessary for the origin of the putative Cap of FeSCV.

A region presumed to be a classical NLS was detected in the putative Cap of FeSCV. As circoviruses lack DNA polymerase, virus particles have to transfer into the nucleus for viral DNA replication [17] . In BFDV and duck circoviruses, viral DNA may by transported into the host cell nucleus via NLS-containing viral Cap [18, 19] . It is unclear whether the putative Cap of FeSCV functions similar to the Cap of BFDV and duck circoviruses. Further studies regarding the intracellular localization of FeSCV putative Cap is required.

We detected a novel CRESS DNA virus, FeSCV, in fecal samples from cats. Although it was detected using consensus primers of circovirus and cyclovirus, FeSCV was phylogenetically positioned in a clade different from that of these viruses. FeSCV was detected in several cats that were housed together, suggesting that FeSCVs are derived from the same origin. In addition, FeSCV is considered to cause diarrhea in cats via co-infection with other enteric viruses. Therefore, it is necessary to perform a large-scale epidemiological survey and clarify whether diarrhea is associated with FeSCV infection.

Consensus statement: virus taxonomy in the age of metagenomics

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Characterization of the genomic sequence of a novel CRESS DNA virus identified in Eurasian jay (Garrulus glandarius)

Identification and genetic characterization of a novel circular singlestranded DNA virus in a human upper respiratory tract sample

Plasma virome of cattle from forest region revealed diverse small circular ssDNA viral genomes

Pervasive chimerism in the replication-associated proteins of uncultured singlestranded DNA viruses

Revisiting the taxonomy of the family Circoviridae: establishment of the genus Cyclovirus and removal of the genus Gyrovirus

Molecular evolution of porcine circovirus type 2 genomes: phylogeny and clonality

Evidence for specificity of psittacine beak and feather disease viruses among avian hosts

Circovirus in tissues of dogs with vasculitis and hemorrhage

Novel cyclovirus in human cerebrospinal fluid

Bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses

Faecal virome of cats in an animal shelter

Multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee feces

A molecular survey for selected viral enteropathogens revealed a limited role of Canine circovirus in the development of canine acute gastroenteritis

The fecal viral flora of wild rodents

Replication of porcine circoviruses

Structural determination of importin alpha in complex with beak and feather disease virus capsid nuclear localization signal

Identification of two functional nuclear localization signals in the capsid protein of duck circovirus

Acknowledgements This work was in part supported by MEXT/JSPS KAKENHI Grant number JP16K08027.

Conflict of interest All authors declare that have no conflict of interest.Ethical approval All applicable international, national, and institutional guidelines for the care and use of animals were followed by the authors. Samples were obtained from an animal hospital, and this study does not include animal experiments.