key: cord-0903341-45udb96t authors: Moon, Bo‐Youn; Jang, Jiung; Kim, Seong‐Hee; Kim, Yeon‐Hee; Lee, Hyun‐kyoung; So, ByungJae; Park, Choi‐Kyu; Lee, Kyoung‐Ki title: Genetic characterization of canine parvovirus type 2c from domestic dogs in Korea date: 2020-03-01 journal: Transbound Emerg Dis DOI: 10.1111/tbed.13501 sha: 4dc3c2b6a63fb89769cbddd4355dfdc2776ef911 doc_id: 903341 cord_uid: 45udb96t Canine parvovirus type 2 (CPV‐2) is an aetiological agent that causes acute haemorrhagic enteritis and fatal myocarditis in dogs. Since CPV‐2 first emerged in the late 1970s, its rapid evolution has resulted in three antigenic variants: CPV‐2a, CPV‐2b and CPV‐2c. Here, we report, for the first time in Korea, two cases of CPV‐2c infection in two dogs with severe diarrhoea. The complete open reading frame (4,269nt) of CPV‐2, encoding both non‐structural (NS) and structural (VP) proteins, was sequenced. Based on the amino acid Gln present at residue 426 of the VP2 gene, these strains were typed as CPV‐2c, and were named Korea CPV‐2c_1 and Korea CPV‐2c_2. These strains shared 99.48% reciprocal nucleotide sequence identity and had the highest nucleotide identity (99.77%–99.34%) with Asian CPV strains isolated in China, Italy (found in a dog imported from Thailand), and Vietnam from 2013 to 2017. Phylogenetic analysis based on the non‐structural (NS1) and capsid (VP2) genes revealed that Korean CPV‐2c strains clustered closely to Asian CPV strains, and separately from strains isolated in Europe, South America and North America. Amino acid changes never reported before were observed in NS1 (Thr70Pro, Cys287Tyr), VP1 (Lys17Arg, Phe33Leu) and VP2 (Gln365His, Ala516Val). Additional observed mutations, including Phe267Tyr, Tyr324Ile and Gln370Arg, have been previously reported in the recent CPV‐2c strains with Asian origins. These results suggest that the Korean CPV‐2c strains were potentially introduced via neighbouring Asian countries. Since first emerging, mutations of the VP2 gene have generated the antigenic variants CPV-2a, CPV-2b and CPV-2c, which soon replaced the original CPV-2 virus worldwide (Buonavoglia et al., 2001; Miranda & Thompson, 2016; Parrish et al., 1991; Parrish, O'Connell, Evermann, & Carmichael, 1985) . These variants have different biological properties from the original CPV-2 virus and can infect cats and proliferate in feline cell lines (Truyen & Parrish, 1992) . CPV-2c carrying a Glu on residue 426 of VP2 was first identified in Italy in 2000 (Buonavoglia et al., 2001 . A retrospective analysis described circulation of CPV-2c in Germany already in 1996 (Decaro et al., 2007) . Currently, the CPV-2c variants are distributed worldwide, including Asia, Africa, Australia, Europe, and North and South America (Miranda & Thompson, 2016; Woolford, Crocker, Bobrowski, Baker, & Hemmatzadeh, 2017) . In the 2000s, this variant was found with high frequency in Europe and South America, but was relatively rare in Asia, where CPV-2a was predominant (Decaro & Buonavoglia, 2012) . However, the distribution of CPV-2c has since expanded, and it has recently been found in Asia including China (Geng et al., 2015; Wang et al., 2016; Zhao et al., 2017) , Taiwan (Chiang, Wu, Chiou, Chang, & Lin, 2016; Lin et al., 2017) , Vietnam (Hoang et al., 2019) , Laos (Vannamahaxay et al., 2017) , Mongolia (Temuujin et al., 2019) and Thailand (Charoenkul et al., 2019) . Although the VP2 gene of CPV-2 has been relatively well researched, the study of genetic variations in the NS gene has been limited. The NS gene is associated with viral replication, DNA packaging, cytotoxicity and pathogenicity. A recent study has described specific amino acids in NS that are associated with the differentiation of FPV and CPV-2 and CPV-2 NS also harbours more sites that are under selection (Mira, Canuti, et al., 2019) . Recent studies revealed that the migration of CPV-2 might occur across geographical barriers, resulting in dynamic changes in the CPV-2 population at the same location. For example, a CPV-2c strain with the genetic characteristics of Asian-origin CPV-2c was found in dogs imported from Thailand to Italy, and is currently circulating in Italy Mira et al., 2018) . In addition, the genetic divergent properties of CPV-2 in South America resulted from the migration of CPV-2 originating from other continents during the last two decades (Grecco et al., 2018; Miranda & Thompson, 2016; Perez et al., 2014) . Between 2003 and 2006, two CPV-2 variants, CPV-2a and CPV-2b, were detected in Korea, with a higher prevalence of the CPV-2a variant (Jeoung, Ahn, & Kim, 2008; Kang et al., 2008; Yoon, Jeong, Kim, & An, 2009 ). However, CPV-2c was not detected. Here, we report the first identification of CPV-2c variants in Korea, and compare their genetic characteristics and epidemiological relatedness with global CPV-2 strains. were submitted to the Animal and Plant Quarantine Agency for diagnostic purposes. A necropsy was conducted for 17-270, and tissues obtained from the lymphatic gland, lung, spleen, heart, kidney, intestine and brain were used for histopathological examination and molecular assays. The parenchymal organs were removed from 17-270, fixed in 10% neutral buffered formalin and embedded in paraffin wax. The embedded tissues were sectioned and subsequently stained with haematoxylin and eosin. canine coronavirus (CCoV) were screened using Lilif™ CPV PCR kit, Lilif™ CDV Nested-PCR kit, Lilif™ CPIV RT-PCR kit, and Lilif™ CCoV RT-PCR kit (iNtRON Biotechnology), respectively, according to the manufacture's instructions. Canine herpesvirus (CHV) and canine adenovirus type 1 and 2 (CAdV-1 and CAdV-2) were detected using previously described PCR assays (Burr, Campell, Nicolson, & Onion, 1996; Hu et al., 2001) . The 25 μL PCR mixtures contained 100 ng DNA template, primers, 10× HotStarTaq™ Master Mix kit (Qiagen) and distilled water. The PCR products were analysed by electrophoresis on a 2% agarose gel with ethidium bromide. To analyse the complete sequence of the CPV coding regions, five primer pairs were designed based on the genome sequence of the CPV-N strain (NCBI RefSeq NC_001539) ( The obtained novel CPV-2 nucleotide sequences were aligned together with 61 CPV-2 sequences randomly obtained from GenBank using the ClustalW algorithm in Bioedit software 7.2.0 (Ibis Therapeutics). The alignment was used to calculate nucleotide sequence identities using the pairwise distance algorithm. Phylogenetic trees based on the nucleotide sequences of complete NS1 and VP2 gene were analysed with MEGA 6 software using the maximum-likelihood (ML) method according to Hasegawa-Kishino-Yano (HKY) and the Tamura 3-parameter (T92) models with Gamma distribution (five rate categories) (G) and 1,000 bootstrap replicates (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013) . The best-fit model for nucleotide substitution was selected in MEGA 6 and used to determine HKY + G for the NS1 gene and T92 + G for the VP2 gene. The Amino acid Additional mutations detected at Phe267Tyr, Tyr324Ile and Gln370Arg were identical to those reported in recent Asian CPV-2c strains (Chiang et al., 2016; Geng et al., 2015; Hoang et al., 2019) . Phylogenetic analysis of the NS1 and VP2 genes of Korean CPV-2c strains was performed including a data set of 61 CPV-2 genome sequences available in GenBank. This data set includes sequences of CPV-2 strains isolated in Asia (China, Japan and Vietnam), Australia, Europe (Italy, France and Germany), South America (Argentina, Brazil, Ecuador and Uruguay) and the USA. The NS and VP genes of Korean CPV-2c strains closely clustered with those of Asian CPV-2 strains but were separated from those of strains originating in Europe, South America and the USA ( Figure 1 ). The (Simpson et al., 2000) . Taken together, although the role of these residues needs to be further elucidated, these mutated residues may provide a survival advantage, allowing CPV-2c to adapt to a new host or environment. In the present study, unique mutations were found scattered throughout the Korea CPV-2c strain genomes. Analysis of NS1 mutations among global CPV-2 variants has so far been limited. Korea CPV-2c_2 harboured two unique mutations (Thr70Pro and Cys287Tyr) in NS1, which is essential for viral replication and host apoptosis. Apoptosis is typically a host defence mechanism against harmful agents; however, some studies have demonstrated that cell death and cell cycle arrest are beneficial to the virus in parvovirus-infected cells, aiding in viral DNA replication and nuclear egress (Chen & Qiu, 2010) . Two mutations in the VP1 region were observed in Korea CPV-2c_1 (Lys17Arg and Leu33Phe). These mutations are located in unique region of VP1, which is responsible for nuclear transport and phospholipase A2 activity during cell infection (Vihinen-Ranta, Wang, Weichert, & Parrish, 2002) . Unique mutations in VP2, Gln365His in Korea CPV-2c_2 and Ala516Val in Korea CPV-2c_1, were reported in this study for the first time. Residue 365 is also located with residues 370 and 375 in the flexible loop that determines host range. Therefore, these mutations may contribute to host range extension (Simpson et al., 2000) . The substitution at residue 516, Ala516Thr, has been reported as a specific mutation in Hungarian CPV-2 (Csagola, Varga, Lorincz, & Tuboly, 2014) . Continuous monitoring of the genetic characteristics of Korean CPV-2 is therefore necessary for effective disease control. In conclusion, the present study documents the genetic characteristics of the first reported cases of CPV-2c infection in Korea. The Korean CPV-2c strains possess mutations distinct from those observed in CPV-2c strains of other countries. Based on their phylogenetic divergence, the Korean CPV-2c strains clustered with CPV strains of Asian origin. These results suggest that CPV-2c was potentially introduced into South Korea by the transport of dogs and/or fomites contaminated with CPV-2c from neighbouring Asia countries. This work was supported by the grant from Animal and plant quarantine agency (B-1543069-2019-21-04). The authors declare no conflict of interest. The authors confirm that the ethical polices of the journal, as noted on the journal's author guidelines page, have been adhered to. All samples (the gathered faeces and carcasses of dogs) used in this study were those that were submitted Animal and Plant Quarantine Agency for diagnosis of disease. Ethical approval was not required as per institutional guidelines and recommendations. 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