Genomic characterization of Canine circovirus detected in a dog with intermittent hemorrhagic gastroenteritis in Brazil

ABSTRACT: Because Canine circovirus (CanineCV) is a new species of the genus Circovirus, several issues related to its epidemiology, pathogenesis and clinical disease remain unknown. Thus, this study aimed to perform the characterization of the first complete genome sequence of CanineCV detected in a dog with diarrhea in Brazil. A stool sample was collected of a ten-month-old female German Shepherd dog which had signs of intermittent hemorrhagic gastroenteritis, vomiting, and a history of eating raw pork. The complete CanineCV genome was sequenced by Next-Generation Sequencing. The sequence had 2,063 nucleotides, showed a typical genomic organization for circovirus, and was grouped with strain 214 described in the United States by phylogenetic analysis. One amino acid change was found in the replicase protein, and because of that it was considered unique to CanineCV. Therefore, the characterization of the complete genome of Brazilian CanineCV can be used in future studies of molecular epidemiology, pathogenesis and development of diagnostic tools for the prevention and control of this disease.


MATERIALS AND METHODS
This study was approved by the Committee on Ethics in the Use of Animals (CEUA) from Regional University of Blumenau (FURB) under protocol nº 006/13. In January 2013, a female ten-month-old German Shepherd dog, un-spayed, vaccinated against giardiasis, canine adenovirus type 1 and 2, canine distemper virus, canine parvovirus, parainfluenza virus, coronavirus, and Leptospira spp., presented intermittent hemorrhagic gastroenteritis associated with melena, some emetic episodes, 38.9 °C body temperature, and absence of abdominal pain. In addition, the dog was constantly fed with raw pork. Initially, giardiasis was suspected, but a fecal examination performed by zinc sulphate flotation and salt flotation techniques was negative for helminth eggs and protozoan oocysts; nevertheless, symptomatic treatment was conducted with fluid therapy and antibiotics (metronidazole) for giardiasis, with no adequate resolution of the clinical signs. The infection time course was three months with intermittent diarrhea, and there was no evidence of transmission to other dogs. Therefore, to establish an etiological diagnosis, a stool sample was molecularly analyzed for the presence of Cryptosporidium spp., Giardia duodenalis, canine parvovirus (CPV), CanineCV, and porcine circovirus (PCV) DNA.
The stool sample (0.2 g) was subjected to a freezing cycle (-196 °C/2 min) and thawing (60 °C/2 min). Then, the DNA was extracted using a QIAamp ® DNA Stool Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations.
PCR and nested-PCR were used as described by XIAO et al. (2001) for the DNA detection of Cryptosporidium spp. PCR and semi-nested PCR for Giardia duodenalis were also prepared as reported by READ et al. (2004).
The complete genome of CanineCV was amplified by rolling circle amplification (RCA) using the enzyme phi29 DNA polymerase (Thermo Fisher Scientific, Carlsbad, CA), following the manufacturer's recommendations. The digestion reaction with the enzyme NcoI (New England Biolabs, Ipswich, England) was performed to confirm the presence of expected product size of the complete CanineCV genome, according to the manufacturer's instructions. After visualizing of 1% agarose gel stained with Brilliant Green Plus (Neotaq, Botucatu, Brazil) by UV light exposure (Figure 1), DNA band of expected size was purified with a GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, United Kingdom) and quantified by Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Carlsbad, CA), following the manufacturer's instructions.
The sequencing library was prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA), and the sequencing was performed on the Illumina NextSeq platform (2 x 150 cycles) (Illumina, San Diego, CA) following the manufacturer's recommendations. The reads generated were analyzed and assembled with Geneious v.6.1.8 (KEARSE et al., 2012) to obtain the complete CanineCV D1056 genome. The complete genome sequence, Open Reading Frame 1 (ORF1) and Open Reading Frame 2 (ORF2) of CanineCV D1056 were aligned with 48 CanineCV sequences deposited on GenBank using ClustalW (LARKIN et al., 2007) implemented in MEGA v.6 (TAMURA et al., 2013). Afterwards, a phylogenetic tree was constructed based on the nucleotide sequence for the complete CanineCV D1056 genome by the Maximum Likelihood method (MEGA v.6) using the GTR+G as a substitution model and 1,000 bootstrap replicates. The complete CanineCV D1056 genome was deposited in GenBank under accession number MK424788.
The attempt to isolate CanineCV D1056 was performed in MDCK (Madin-Darby canine kidney) cell culture according to the procedure described for PCV2 (CRUZ & ARAUJO JR, 2014), with some modifications. A 6.25% stool suspension was prepared with minimum essential media (MEM) (Gibco, Grand Island, NE) and 0.5X Antibiotic-Antimycotic 100X (Gibco, Grand Island, NE). The suspension was centrifuged at 3,220 x g for 15 min at 4 ºC, and the supernatant was filtered through a 0.22 µm sterile membrane. The inoculation was performed after the trypsinization of the control and infected MDCK cells. Five passages were performed. Between each subculture, the cells were treated with 100 mM D-glucosamine pH 7.0. Aliquots of the control and infected cell suspensions of each passage were separated for DNA extraction using an Illustra Blood GenomicPrep Mini Spin Kit (GE Healthcare, Buckinghamshire, United Kingdom) according to the manufacturer's instructions. Finally, qPCR for CanineCV was performed as reported above.

RESULTS AND DISCUSSION
In this study, the stool sample was positive for CanineCV (Ct value 32) by qPCR. Analysis of the complete CanineCV D1056 sequence showed that it was 2,063 nt in length, 51% GC, and a genomic organization typical of the circovirus, as previously reported for CanineCV (KAPOOR et al., 2012;LI et al., 2013). Thus, two major ORFs, 1 and 2 in opposite orientation, were reported in CanineCV D1056 genome, which encode the viral replicase (Rep, 303 amino acids, aa) and capsid protein (Cap, 270 aa), respectively. In addition, two noncoding intergenic regions were reported in the viral genome. The 5'-intergenic region (135 nt in length) between the start codons of the two major ORFs contains a typical stem-loop structure for the initiation of rolling-circle replication and the conserved 9-nt sequence (TAGTATTAC) between the circoviruses. The 3'-intergenic region (203 nt in length) is located between the stop codons of the two major ORFs. Sequences of amino acids highly conserved among circoviruses associated with rolling-circle replication (FTINN, TPHLQ and CSK) and the dNTP binding (GCGKS) were identified in the replicase protein. For the capsid protein, the N-terminus region was rich in arginine residues, as usually observed in circoviruses. The nucleotide identity shared among CanineCV D1056 with the other 48 CanineCV sequences for complete genome, replicase and capsid nucleotide sequences was 83.5% -98.0%, 81.1% -98.5% and 80.3% -98.3%, respectively. The phylogenetic tree showed that CanineCV D1056 was grouped with strain 214 (GenBank JQ821392.1) (Figure 2). Strain 214 was described in the United States in 2012 from asymptomatic dog sera and was the first complete CanineCV sequence reported (KAPOOR et al., 2012). The nucleotide identities found for the two sequences with respect to the complete genome, replicase and capsid were 98%, 97.3% and 98.3%, respectively. For amino acid identities, the values found were 96.7% and 98.9% for replicase and capsid proteins, respectively. In the protein residue 16 of the replicase protein, one amino acid change was considered unique to CanineCV D1056 (Cysteine) compared with other CanineCV (Glycine) sequences used in this study. In Brazil, a CanineCV contig of 200 bases in length was obtained after the dog virome characterization in 2018 (WEBER et al., 2018). However, the authors did not determine the complete CanineCV genome, and there was no description of the clinical signs associated with this sequence. Consequently, the first complete CanineCV genome sequencing in Brazil associated with intermittent hemorrhagic gastroenteritis in a dog in January 2013 was reported in this study. The animal was negative for Cryptosporidium spp., Giardia duodenalis, and CPV. For CanineCV, it is suggested that the occurrence of coinfections may increase the severity of the clinical disease (LI et al., 2013;THAIWONG et al., 2016;ANDERSON et al., 2017;DOWGIER et al., 2017). Moreover, a higher frequency of CanineCV infections had been previously described in younger animals (< 1 year old) (HSU et al., 2016;ZACCARIA et al., 2016;DOWGIER et al., 2017). Such characteristic was also observed in the present case since the dog was ten months old.
As formerly described by the frequent intake of raw pork by the animal, qPCR for PCV was performed and resulted negative. However, since domestic dog infection with PCV2a (HERBST & WILLEMS, 2017) and PCV3 (ZHANG et al., 2018) has already been reported, it is important not to provide domestic dogs with raw meat because of the high risk of transmission of various pathogens, including zoonotic agents.
The attempt to isolate CanineCV D1056 in MDCK cells was unsuccessful, since viral DNA was detected by qPCR only during the inoculation phase. As the amount of viral DNA found by qPCR for CanineCV was low, this may have hindered viral isolation. Moreover, the presence of viral DNA in the inoculum does not indicate the existence of an integral viral particle, which is necessary for the virus to adsorb in the cells.

CONCLUSION
It is known that the pathogenesis and development of clinical diseases caused by CanineCV remain uncertain, so several studies are essential to obtain a better understanding of the factors involved in the interaction of this viral agent with its hosts. Hence, the present study was a pioneer in performing the genetic characterization of Brazilian CanineCV D1056 detected in a female dog with intermittent hemorrhagic gastroenteritis, which can assist in future research related to the molecular epidemiology, pathogenesis, and development of serological and molecular diagnostic methods for CanineCV detection.