Genetic Characterization of Canine Parvovirus Type 2 Detected in Wild Raccoon Dogs (Nyctereutes procyonoides) in the Republic of Korea Open Access

Canine parvovirus type 2 (CPV-2) emerged in 1978 and is the major cause of hemorrhagic gastroenteritis and myocarditis in puppies (Appel et al. 1979). The virus is nonenveloped and single-stranded DNA with two nonstructural proteins (NS1 and NS2) and two structural proteins (VP1 and VP2). Protein VP2 is a major determinant of antigenic type and host range. From 1979 to 1981, the original antigenic type (CPV-2) had been replaced by an antigenic variant CPV-2a. The original strain mutated into three antigenic subtypes, CPV-2a, CPV-2b, and CPV-2c, by the change of several amino acids in the VP2 gene. Five amino acid substitutions in the VP2 capsid protein differentiate CPV-2 to CPV-2a (Met-87 to Leu; Ile-101 to Thr; Ala-300 to Gly; Asp-305 to Tyr; and Val-555 to Ile; Parrish et al. 1991). A second antigenic variant CPV-2b appeared around 1984, and the only significant difference from CPV-2a was the substitution of Asn-426 to Asp in the VP2 protein. Those two antigenic variants have now replaced the original strain CPV-2 throughout the canine population worldwide (Pratelli et al. 2001). Recently, a new variant, CPV-2c, has been reported in Italy, Vietnam, and the American continent (Decaro et al. 2005). This variant is distinguishable from CPV-2b by the substitution of Asp-426 to Glu of VP2 (Buonavoglia et al. 2001). All wild members of the Canidae, domestic dogs, and some of species of Felidae probably are susceptible to CPV. Despite many global reports isolating CPV from wild animals such as bobcat (Lynx rufus), coyote (Canis latrans), and raccoon dog (Nyctereutes procyonoides; Barker and Parrish 2001), the research about CPV in the Republic of Korea (South Korea) has previously been limited to domestic dogs (Kang et al. 2008; Moon et al. 2008; Yoon et al. 2009), except one serologic survey in raccoon dogs (Cha et al. 2013). In this study, we describe the identification of CPV-2 in raccoon dog tissue samples in South Korea.

During 2016–17, 136 raccoon dog carcasses were collected from 12 wildlife rescue centers in South Korea. The raccoon dog carcasses collected died by a car accident, poaching, dehydration, or during hospitalization in the wildlife rescue center. Carcasses were stored at –20 C before autopsy. Brain, trachea, lung, liver, kidney, and intestine were sampled. Total nucleic acid was extracted from 100 mg of tissue samples by using Maxwell^® RSC Instrument and Maxwell^® RSC Viral Total Nucleic Acid Purification Kit (Promega, Madison, Wisconsin, USA) following the manufacturer's instructions. Extracted total nucleic acid was stored at –80 C before processing. Initial canine parvovirus screening was performed by a VeTeK^™ CPV Detection Kit (iNtRoN Biotechnology, Seongnam, South Korea). The targeted VP2 gene primers were used to amplify and sequence the full VP2 gene (1,755 base pairs) from one CPV-positive sample (Yoon et al. 2009). Cycle sequencing reactions were performed by using the BigDye^® Terminator Cycle Sequencing Kit version 1.1 (Applied Biosystems, Foster City, California, USA). Reactions were purified by using SigmaSpin^™ Post-Reaction Clean-Up Columns (Sigma, St. Louis, Missouri, USA) and sequenced on an ABI PRISM 3130 Automated Capillary DNA Sequencer (Applied Biosystems) according to the manufacturer's instructions. The nucleotide sequences were aligned and compared to 19 selected canine and feline parvovirus sequences available from the GenBank database by using ClustalW software implemented in BioEdit version 7.0.9.0 (Hall 1999). The phylogenetic trees were drawn by using the neighbor-joining method by the maximum composite likelihood model with MEGA 7 software (Kumar et al. 2016). Deduced amino acid sequences of the complete VP2 gene of these sequences were also aligned and compared.

In this study, CPV-2 was detected from three raccoon dogs exhibiting diarrhea and dehydration: 16M130 (GenBank accession no. MH643886), 17Ra5 (GenBank no. MH213135), and 17Ra171 (GenBank no. MH213140). The VP2 genes of 16M130 and 17Ra5 were clustered with the original antigenic type (CPV-2) and displayed the highest nucleotide identity (99.7%) with the CPV-2 isolate RDPV-DP3 from a raccoon dog in China (GenBank no. MF996334). Moreover, the VP2 gene of 17Ra171 showed 99.8% identity at the nucleotide level with CPV-2 isolate HB3 from a raccoon dog in China (GenBank no. GU392240; Fig. 1). Deduced amino acids were aligned, and the main substitution site was checked. Amino acid substitution sites in the VP2 capsid protein that differentiated CPV-2 to other variants were aligned, and 16M130, 17Ra5, and 17Ra171 had no substitutions from CPV-2 (Table 1).

Two antigenic variants (CPV-2a and CPV-2b) have replaced the original strain of CPV-2 throughout the canine population worldwide (Pratelli et al. 2001), including South Korea. Recently isolated CPV from domestic dogs in South Korea were variants CPV-2a and CPV-2b (Kang et al. 2008; Moon et al. 2008; Yoon et al. 2009). In 2011, 21 CPVs were detected in samples from domestic dogs and wild animals in China, but none of them were CPV-2 (Chen et al. 2011). The original antigenic type (CPV-2) has already been replaced with other variants in South Korean domestic dog populations, but CPV-2 still remains in wild animal populations. This result suggested that CPV evolved separately in wild animals and domestic dogs.

The CPV vaccines used in South Korea are mostly derived from the original strain, and one of them is made of a CPV-2b variant (Yoon et al. 2009). Groups of domestic dogs inoculated with either the CPV-2 vaccine or CPV-2b vaccine had high neutralizing antibody titers to the CPV-2, but the CPV-2 vaccine–inoculated group had lower neutralizing antibody titers to the CPV-2b than did the CPV-2b vaccine–inoculated group (Pratelli et al. 2001). For this reason, domestic dogs can be protected by the CPV-2 from infections from raccoon dogs because CPV-2 vaccines are broadly used in animal hospitals in South Korea.

Raccoon dogs usually live in farmlands, amid streams, houses, and forests (National Institute of Biological Resources 2016), despite their decreasing habitat density in South Korea since 2006. With increasing contact between raccoon dogs and domestic dogs, the emergence of a novel CPV-2 variant from raccoon dogs in China was reported (Yu et al. 2018). Moreover, new CPV-2a and CPV-2b variants were isolated from domestic dogs in China (Wang et al. 2016). These reports suggested that CPVs evolved in raccoon dogs or domestic dogs may be spread between hosts. Therefore, further active and epidemiologic surveys are needed to understand the circulation of CPV variants between raccoon dogs and domestic dogs.

This research was supported by grant 2017-01-01-007 from the National Institute of Environmental Research. The funder had no role in the study design, data collection and interpretation, or the decision to submit the work for publication. The subject was partially supported by Korea Ministry of Environment as the Public Technology Program Based on Environmental Policy (2016000210002).