Korean Journal of
Veterinary Service

Noroviruses (NoVs) are a group of single-stranded positive-sense RNA viruses within the Caliciviridae family, which can infect a wide range of animal species, including humans, causing acute gastroenteritis, and are a significant public health concern worldwide (Chan et al, 2017). Canine norovirus (CNoV) can cause acute gastroenteritis with symptoms such as vomiting, diarrhea, and lethargy, leading to significant morbidity and mortality in affected dogs. Most noroviruses have a genome organization consisting of three open reading frames (ORFs), except for murine norovirus, which possesses an additional the fourth ORF (Chhabra et al, 2019). Noroviruses are classified into ten genogroups (GI∼GX) based on the diversity of their VP1 amino acid sequences. Additionally, they can be further divided into 49 confirmed capsid genotypes based on VP1 amino acid sequences and 60 confirmed P-types based on partial RdRp sequences (Chhabra et al, 2019). Significant sequence variation has been identified among various strains of canine and feline noroviruses that have been reported to date, leading to the classification of a minimum of four genotypes belonging to three distinct genogroups, including GIV.2, GVI.1, GVI.2, and GVII (Bodnar et al, 2017).

The first report of CNoV occurred in Italy in 2007, when a 60-day-old mixed breed puppy with diarrhea and vomiting was found to be co-infected with canine parvovirus type-2a (Martella et al, 2008). Since their first report, detection of noroviruses in dogs and cats has been reported in various countries, including Europe, the Americas, and Asia. Several studies have been conducted in different regions, including South Korea, to investigate the prevalence, molecular characteristics, genetic diversity, and potential public health threat of NoVs (Mesquita et al, 2010 & 2014; Ntafis et al, 2010; Pinto et al, 2012; Tse et al, 2012; Soma et al, 2015; Di Martino et al, 2016 & 2017; Bodnar et al, 2017; Lyoo et al, 2018; Ma et al, 2021).

This report outlines the development of precise primers intended for the identification of CNoV, along with an analysis of the molecular characteristics of CNoV. This analysis is based on a diarrhea case observed in a seven-month-old dog that was treated at an animal clinic located in the Gwangju Region of South Korea, during December 2021. The dog showed gastroenteric disease with vomiting and hemorrhagic diarrhea and recovered after receiving antibiotic treatment. A clinical specimen (rectal swab) was collected from the diarrheic dog and subjected to real-time RT-PCR assays for POBGEN™ canine circovirus, rotavirus type A and norovirus detection kit (Postbio Inc., Namyangju, Korea) as well as culture methods for diarrhea-causing bacterial pathogens. The test results were positive for CNoV and enterotoxigenic E. coli (ETEC). The STp gene was detected in E. coli isolates by PCR using a pathogenic E. coli detection kit (GeNet Bio, Daejeon, Korea).

The oligonucleotide primers were designed based on the nucleotide sequences of the RNA-dependent RNA polymerase (RdRp) region of CNoVs obtained from previously published norovirus 5 sequences in GenBank (accession no. EU224456, MK067289, JF930689, NC_045762, and LC389583) (Fig. 1). To perform the design, sequence alignment was conducted using the BioEdit Sequence Alignment Editor Version 7.0.9.0 software, and BLAST was performed using NCBI nucleotide (http://www.ncbi.nlm.nih.gov/BLAST) for validation.

Figure 1. Sequence alignment of the RdRp regions of five norovirus sequences to select primers for semi-nested PCR to detect genotype GIV.2. Dots indicate sequence identities, and dashes indicate lack of nucleotide, and slashes indicate the skip of nucleotide.

The nucleotide sequences of three primers used in this study were as follows: NorG4_346F sense: 5’-TGGAATTYGACCCRGAGGT-3’ (nt 346∼364) and NorG4_875R antisense: 5’-TGACTCTCTGGRACGAGGT-3’ (nt 857∼875) for the RT-PCR reaction; NorG4_346F sense and NorG4_823R antisense: 5’-TAGACGCCATCTTCATTCAC-3’ (nt 804∼823) for the semi-nested PCR (SN-PCR) reaction. The predicted sizes of RT-PCR and SN-PCR products are 530 and 478 bp, respectively. The nucleotide positions are relative to the CNoVs (GenBank accession no. EU224456).

RT-PCR reaction was conducted in a total volume of 20 μL using the Maxime™ RT-PCR PreMix kit (iNtRon Biotechnology, Seongnam, Korea). For the RT-PCR assay, 1 μL of extracted RNA, 10 pmol each of the NorG4_346F sense and NorG4_875R antisense primers, and RNase-free water to a final volume of 20 μL were added. The reaction was performed in a thermocycler (ProFlex PCR System, Applied Biosystems by Thermo Fisher Scientific, Singapore). The cycling conditions included reverse transcription at 45℃ for 30 min, followed by inactivation of the RTase at 95℃ for 5 min. The PCR amplification consisted of 40 cycles at 94℃ for 30 s, 55℃ for 35 s, and 72℃ for 45 s. The final step was held at 72℃ for 7 min, followed by cooling down to 4℃. By the way, after cDNA synthesis using reverse transcriptase, PCR amplification was successful with the primer NorG4_875R antisense, whereas it was not possible with NorG4_823R antisense.

The second PCR reactions (SN-PCR) were performed using Maxime™ PCR PreMix Kit (i-StarTaq) kit (iNtRon Biotechnology, Seongnam, Korea). The SN-PCR mixtures were composed of 1 μL of the first amplicons, 1 μL of the primer NorG4_346F sense (10 pmol) and NorG4_823R antisense (10 pmol), and RNase-free water to a final volume of 20 μL. The PCR amplification steps were performed with the following temperature and time conditions: one step of 5 min/94℃, followed by 30 cycles of 30 s/94℃, 35 s/55℃, 45 s/72℃, and a final step of 7 min/72℃, followed by cooling down to 4℃. Electrophoresis of PCR amplicons was performed on a 1.2% agarose gel in TBE buffer using SYBR^Ⓡ Safe DNA gel stain (Invitrogen, CA, USA) and visualized under UV light using a Quantum CX5 gel documentation system (Vilber Lourmat, France).

The sequencing was outsourced to Cosmo Genetech (Daejeon, Korea) and was performed using an ABI 3730XL DNA Analyzer. The phylogenetic relationships of the nucleotide sequences were analyzed by constructing a maximum likelihood tree using MEGA software (version 11.0). The reliability of the tree was evaluated by bootstrap analysis with 1,000 replicates (Tamura et al, 2021).

The RT-PCR assay conducted on a rectal swab of a dog with hemorrhagic diarrhea did not yield the expected 530 bp amplification product, while the SN-PCR assay produced a 478 bp product (Fig. 2). We sequenced a 478 bp fragment of the RdRp gene of the confirmed CNoV strain Dog/GIV.2/gw/s377/2021/KOR, and the nucleotide sequence has been submitted to GenBank under accession number OR294045.

Figure 2. The detection of the canine norovirus genotype GIV using RT-PCR and SN-PCR assays with the newly designed primers in this study. Lane 1 and 2 did not show amplification of the 530 bp fragment in the RT-PCR assay. The SN-PCR assay resulted in the amplification of a 478 bp fragment in lanes 3 and 4. Lane 1∼2, retal swab samples from the same dog with bloody stool symptoms; lane 3∼4, RT-PCR products of lane 1 and 2; lane M, 100 bp DNA ladder (invitrogen); lane NC, negative control (nuclease-free water).

We compared the obtained sequence with the sequences available in GenBank. This strain showed a similarity of 91.9∼95.1% to the cat norovirus genotype GIV (accession no. NC_045762, ON595846, LC389583, EF450827, KT245136, and LC011951) (Fig. 3). We found the highest similarity (95.1% nt and 98.7% aa) with the feline norovirus strain CU081210E/USA/2010 (accession no. NC_045762) reported by Pinto et al. in 2012 (Fig. 4). The RdRp region of the CNoV strain (Dog/GIV.2/gw/s377/2021/KOR) indentified in this study exhibited high nucleotide identity with feline norovirus strains. Therefore, it is speculated that the CNoV detected in this study originated from cats. And the CNoV detected exhibited nucleotide sequence identity of 94.5% and amino acid sequence identity of 98.1% with the pistoia/387/06/ITA norovirus (accession no. EF450827) identified in a captive lion cub in 2007 (Martella et al, 2007). The CNoV detected in this study showed 85.8% nucleotide identity and 96.8% amino acid identity with the CNoV strain 170/07 initially reported in Italy in 2008 (accession no. E224456) (Martella et al, 2008). Di Martino et al. (2016) reported the circulation of genetically related NoVs in different host species, indicating that cats and dogs can carry NoVs of the same genotypes, GIV.2, GVI.1, and GVI.2.

Figure 3. Phylogenetic tree constructed using a 471-nucleotide (nt) alignment of the partial RdRp region, including CNoV strain detected in this study (denoted by black circle), as well as reference sequences of CNoV (accession numbers indicated). The tree was reconstructed using the maximum likelihood method with bootstrap test (1000 replicates) based on the Tamura 3-parameter model in MEGA software version 11.0.

Figure 4. The nucleotide sequences alignment of the partial RdRp region of the norovirus using the BioEdit software. The nucleotide sequence (Dog/GIV.2/gw/s377/2021/KOR) of the identified canine norovirus in this study was compared to four reference nucleotide sequences (NC_045762, JF939046, MW662289, and FN663929). Dots indicate sequence identities, while dashes indicate the absence of nucleotides.

In this study, utilizing the previously reported sequences of CNoVs, we developed novel diagnostic SN-PCR method. The newly designed primers, NorG4_346F, NorG4_875R, and NorG4_823R, were used to test an additional 53 diarrheal samples from dogs submitted to our laboratory from animal clinics in Gwangju area in 2022. The results of the diagnostic tests revealed no positive reactions. Lyoo et al. (2018) reported a detection rate of 3.1% for CNoV in dog feces from small animal clinics and animal shelters in Korea, classifying them as genogroup GIV. Previously, the detection rates of CNoV were reported as 4.1% in Japan, 4.6% in Italy, 7.6% in Spain, 7.8% in China and 9.7% in the United States (Soma et al, 2015; Bodnar et al, 2017; Ford-Siltz et al, 2019; Ma et al, 2021). The detection rate of canine norovirus in Greece was reported to be 8.3%, and all cases were co-infected with the same CNoVs strain and canine coronavirus, indicating the easy spread of CNoVs among dogs (Ntafis et al, 2010). In this study, the CNoV-infected dog was co-infected with ETEC. CNoV was not detected in the sample using the JV102 and JV103 primers reported by Mesquita et al. (2010), so novel primers were designed and tested in this study.

Noroviruses, which exhibit a high degree of genetic diversity, are the leading viral cause of acute gastroenteritis in humans across all age groups. These viruses have the ability to infect various mammalian species, but their potential for zoonotic transmission, particularly within genogroup IV that includes viruses infecting humans, canines, and felines, remains poorly understood. Although human-infective noroviruses have been detected in the feces of swine, dogs, and cattle, the risk of zoonotic transmission has been considered very low (Stals et al, 2012). Human noroviruses are classified into GI, GII, and GIV. Canine noroviruses are classified into GIV, GVI, and GVII; and feline noroviruses are classified into GIV and GVI. It showed a nucleotide sequence identity of 72.5% and an amino acid sequence identity of 80.1% with the GIV.1 norovirus infect humans (accession no. KC196342) detected in sewage in Seoul in 2010 (Martella et al, 2007; Han et al, 2014).

Previously, the positive antibody response to CNoV GIV.2 in the human population of Italy indicated the possibility of zoonotic transmission from animals to humans, likely due to social interactions between humans and companion animals (Di Martino et al, 2014). However, based on genomic, phylogenetic, and structural analyses, there is also a report suggesting that the differences in the major capsid protein and non-structural proteins of GIV and GVI noroviruses may limit cross-species transmission between humans, canines, and felines (Ford-Siltz et al, 2019).

We conclude that the SN-PCR described here is a useful tool for the diagnosis of CNoV in faeces from naturally infected dogs. Furthermore, our study revealed that the noroviruses detected in dogs are genetically closely related to those found in cats, suggesting the potential for inter-species transmission. For a more comprehensive assessment of such inter-species viral infections, large-scale virological and serological investigations would be necessary.

This work was supported by Gwangju Metropolitan City Institute of Health & Environment’s Research Capacity Enhancement Project in 2022.

No potential conflict of interest relevant to this article was reported.