key: cord-343949-zmuvq6e3
authors: Lu, Gang; Zhang, Xin; Luo, Jie; Sun, Yankuo; Xu, Haibin; Huang, Ji; Ou, Jiajun; Li, Shoujun
title: First report and genetic characterization of feline kobuvirus in diarrhoeic cats in China
date: 2018-06-06
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.12916
sha: 
doc_id: 343949
cord_uid: zmuvq6e3

Feline kobuvirus (FeKoV) is a newly discovered organism, classified under the species Aichivirus A of the genus Kobuvirus. Since it was first reported in 2013, molecular evidence for FeKoV in the feline population has been restricted to two countries: Korea and Italy. In this study, we collected faecal samples from cats in southern China and detected the FeKoV RNA in these samples. A prevalence rate of 9.9% (8/81) was identified by RT‐PCR, and all positive samples were obtained from diarrhoeic animals. In addition, FeKoV was shown positive associated with diarrhoea in cats, with a correlation coefficient of 0.25. Next, we designed three primer pairs with degenerate bases, which targeted the conservative overlapping region of the entire published FeKoV genome, and sequenced the near‐complete genome of the first Chinese field FeKoV strain, WHJ‐1, using long‐fragment PCR. Finally, we analysed WHJ‐1's homology and phylogeny using the polyprotein gene. The results indicated that FeKoV has rapidly mutated since it was first discovered. This study will help to better understand FeKoV's epidemiology, evolutionary pattern and genetic diversity.

1), Aichivirus E (rabbit picornavirus) and Aichivirus F (bat kobuvirus) (Adams, King, & Carstens, 2013; Adams et al., 2017; Pringle, 1999) . Other members of the Kobuvirus genus have been detected in sheep, goats, rodents and wild boar (Lee et al., 2012; Phan et al., 2011; Reuter, Boros, Pankovics, & Egyed, 2010; Reuter et al., 2013) . The species Aichivirus A includes six types: Aichi virus, canine kobuvirus, murine kobuvirus, Kathmandu sewage kobuvirus, roller kobuvirus and feline kobuvirus (FeKoV) (Adams et al., 2017) .

Kobuviruses are small, nonenveloped, icosahedral particles with linear, positive-sense ssRNA genomes of 8.2-8.4 kb (Lee et al., 2012; Reuter, Boldizsar, & Pankovics, 2009; Yamashita et al., 1998; Yu et al., 2011) . Their genome is composed of a 5 0 untranslated region (UTR), a large open reading frame (ORF), and a 3 0 UTR. The ORF encodes a single polyprotein precursor, which is cleaved into three structural viral proteins (VP0, VP1 and VP3) and eight nonstructural proteins (L, 2A, 2B, 2C, 3A, 3B, 3C and 3D) . Kobuviruses have similar genome organizations to those described in other picornaviruses, 5 0 -L-VP0-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3 0 (Choi, Lee, Lee, & Oem, 2015; Kapoor et al., 2011; Pankovics et al., 2016; Reuter et al., 2009 ).

The first serological evidence supporting kobuviral infections in cats was reported in the United Kingdom by N. Carmona-Vicente et al. in 2013 (Carmona-Vicente et al., 2013 . Using the Aichi virus as an antigen, IgG antibody to this organism was detected in 67 of 97 cat serum samples, indicating an Aichi virus crossreactive kobuviral infection is common in cats. The first FeKoV molecular evidence was reported in Korea by Joon-Yee Chung et al. in 2013 (Chung et al., 2013 . Using reverse transcription polymerase chain reaction (RT-PCR), six of 39 collected faecal samples from Korean cats were confirmed positive for kobuvirus RNA. Phylogenetic analysis revealed that FeKoV (12D240, FK-13) is most closely related to the canine kobuvirus (Choi et al., 2015) . In 2015, Barbara Di Martino et al. screened faecal samples obtained from asymptomatic and diarrhoeic cats for FeKoV RNA in Italy (Di Martino, Profio, Melegari, Marsilio, & Martella, 2015) . FeKoV RNA was found in five of 37 diarrhoeic cats but was undetected in asymptomatic cats.

The full-length genome sequence of one Italian FeKoV strain (FeKoV/ TE/52/IT/13) was sequenced, and it displayed a high nucleotide identity (96.0%) to the Korean strain, FK-13.

Until now, molecular reports of the kobuvirus in cats have been restricted to Korea and Italy, and only three FeKoV strain genomes have been sequenced (Cho et al., 2014; Choi et al., 2015; Di Martino et al., 2015) . Therefore, it is important to investigate and genetically characterize FeKoV infections in other countries to assess the global epidemiology of this emerging virus. Our study determined that FeKoV is present in China. We evaluated its molecular prevalence in cats in China, sequenced the near-complete genome of one Chinese FeKoV strain and analysed its homology and phylogeny based on the sequence. 

The FeKoV screening RT-polymerase chain reaction (RT-PCR) was performed using one published primer pair with broad reactivity with various kobuvirus species (Reuter et al., 2009) . The primer pair, UNIV-kobu-F and UNIV-kobu-R, was designed targeting a 216-bp fragment of the 3D RdRp region of the three prototype kobuviruses (Aichi virus, bovine kobuvirus and porcine kobuvirus). FeKoV RNA was detected by PCR using PrimeSTAR â HS (premix) (Takara, Dalian, China). The PCR conditions were 35 cycles at 98°C for 10 s, 50°C

for 15 s and 72°C for 30 s, followed by 1 cycle at 72°C for 5 min.

The PCR product was electrophoresed in a 1% ethidium-bromidestained agarose gel, and the faecal samples yielding PCR products of 

Thirty-eight kobuvirus polyprotein ORF sequences were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/). Their strain names and accession numbers are shown in Figure 2 . The WHJ-1 polyprotein ORF sequence was then aligned with the kobuvirus polyprotein gene sequences using Bioedit (version 7.0.9.0). The nucleotide and amino acid homologies between these sequences were calculated by the Megalign module of the DNAStar package (version 7.1.0). Finally, a neighbour-joining tree based on maximum composite likelihood was constructed using a 1,000-bootstrap value in Mega (version 5.05).

Faecal samples that were only positive for FeKoV were processed for viral isolation, which was performed by inoculating 0.5 ml of the filtrate onto confluent cell layers grown in 25-cm 2 flasks at 37°C. Six cell types (CRFK, MDCK, A549, PK-15, DF-1, Vero) were used and grown in Dulbecco's Modified Eagle's Medium (Gibco, Shanghai, China) supplemented with 100 lg/ml of streptomycin, 100 units/ml of penicillin and 10% foetal calf serum. After inoculating for 1 hr, the inocula were removed and fresh medium was added. Negative noninoculated controls were also cultivated. Cultures were inspected daily by inverted microscopy for cytopathic effects (CPE) until 4 days postinoculation.

The cultures were frozen and used for further passage. Culture lysates and supernatant were also collected for FeKoV RT-PCR.

To detect the presence of FeKoV, RNA was extracted from the col- One field FeKoV strain demonstrated here was named WHJ-1.

To elucidate the possibility of a correlation between FeKoV and feline diarrhoea, the data were processed for calculating the Phi coefficient of association. The obtained correlation coefficient was 0.25. In addition, the p value of one-tailed and one-tailed Fisher exact probability test was 0.023 (p < 0.05) and 0.046 (p < 0.05), respectively. The result suggested a positive association between FeKoV and feline diarrhoea.

To acquire the WHJ-1 genome, all FeKoV genome sequences available in the NCBI database were retrieved and aligned, and three primer pairs were designed based on the FeKoV genome conservative region (Figure 1a,b) . After agarose gel electrophoresis, the PCR products amplified by the three primer pairs had bands of %3,000, 950, and 4,400 bp, respectively (Figure 1c) . After sequencing and assembly, the near-complete genome of WHJ-1 was obtained, including a 647-nucleotide 5 0 UTR, a 7,311-nucleotide complete polyprotein 

The nucleotide and amino acid sequences of the WHJ-1 polyprotein and cleaved viral proteins were obtained and compared with those of three FeKoV strains and four representative kobuviruses (Table 1) .

WHJ-1 had a higher homology with FeKoV than with kobuviruses of other origins when analysing each gene at both the nucleotide and the amino acid levels. In addition, except the 3A gene homology between WHJ-1 and the canine and mouse kobuviruses, higher nucleotide and amino acid identities were found in each gene between WHJ-1 and the canine kobuvirus compared with mouse, human and bovine kobuviruses. Among the polyprotein genes of the three FeKoV strains, WHJ-1 had a higher nucleotide identity with the Italian strain, FeKoV/ TE/52/IT/13 (93.2%), and a higher amino acid homology with the Korean strain, FK-13 (98.6%). The highest nucleotide identity was found between the FK-13 3B gene and the 12D240 2A gene, with a value of 95.1%, and a highest amino acid identity was found between the FeKoV/TE/52/IT/13 2B protein, the FK-13 3A and 3B proteins, and the 12D240 VP0 and 2B proteins, with a value of 100%. The highest nucleotide divergence was found in the FeKoV/TE/52/IT/13 3B gene, with a value of 88.9%, and the highest amino acid divergence was found in the 12D240 L protein, with a value of 83.9%.

Phylogenetic analysis of the kobuvirus polyprotein ORF gene indicated the Chinese FeKoV strain, WHJ-1, was clustered with the Italian and Korean FeKoV strains (Figure 2 ). In addition, the phylogenetic tree showed that FeKoV was most closely related to the canine kobuvirus, as indicated by the nucleotide and amino acid homology analyses. Both FeKoV and the canine kobuvirus were grouped in Aichivirus A, the species that comprise the genus Kobuvirus together with Aichivirus B-F.

After serial passages, no CPE were observed in the cultured cells, and no kobuvirus RNA was detected in the culture supernatant or cells by RT-PCR.

In this study, we provided the first evidence to support FeKoV circulation in the Chinese feline population, demonstrating that FeKoV is not geographically restricted to Korea and Italy (Cho et al., 2014 (Cho et al., , 2015 Choi et al., 2015; Chung et al., 2013; Di Martino et al., 2015) .

All FeKoV-positive faecal samples were collected from diarrhoeic cats, with a prevalence rate of 8/52, similar to those identified in Korea (6/39) and Italy (5/37) (Chung et al., 2013; Di Martino et al., 2015) . No FeKoV was found in asymptomatic cats in this study, nor in another study in Italy. However, a study conducted by Yoon- (Cho et al., 2015) . FeKoV prevalence studies in China, Korea and Italy indicate FeKoV is commonly detected in diarrhoeic cats (Di Martino et al., 2015) . In addition, our field study To isolate FeKoV, we used six cell types originating from cats, dogs, humans, pigs, chickens and monkeys but detected no FeKoV replication in any of these cell lines after serial passages.

To date, kobuvirus culturing remains an important problem to be solved. To our knowledge, except for the Aichi virus strain, A846/88, and the Bovine kobuvirus strain, U-1, other attempts to isolate kobuviruses have failed (Reuter, Kecskemeti, & Pankovics, 2010; Yamashita et al., 1991 Yamashita et al., , 2003 . The lack of pure viral stocks after culturing makes it difficult to study kobuviral pathogenicity.

In conclusion, in this study, we first determined FeKoV presence in faecal samples from diarrhoeic cats in southern China. We also sequenced the near-complete genome of one field FeKoV strain using long-fragment PCR. Homology analysis indicated FeKoV's genetic variation. Our study indicated FeKoV was associated with feline diarrhoea. Further study is required to isolate this virus and study its pathogenicity in cats.

This work was supported by The National Natural Science Foundation of China (31672563) 

The authors declare no conflict of interest.

Lu http://orcid.org/0000-0002-4969-1768

Yankuo Sun http://orcid.org/0000-0001-6283-4172

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