Notable mutations of porcine parvovirus 1 and 4 circulating in commercial pig farms in South Korea

Article information

Korean J Vet Res. 2024;64.e4

Publication date (electronic) : 2024 March 29

doi : https://doi.org/10.14405/kjvr.20230036

Beomsu Park¹, Jihyeon Hong¹, Jongsu Jun¹, An Kook Choi¹, Choi Kyu Park², Young Soo Lyoo^1,

¹College of Veterinary Medicine, Konkuk University, Seoul 05029, Korea

²College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea

^*Corresponding author: Young Soo Lyoo, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea Tel: +82-2-450-3719 E-mail: lyoo@konkuk.ac.kr

Received 2023 September 5; Revised 2024 February 20; Accepted 2024 February 23.

Parvoviridae is a family of animal viruses that infect and cause various diseases in many mammals, including pigs, dogs, cats, and humans [1]. Parvovirus is a single-stranded DNA virus, approximately 5 kb in genomic length. A complex palindromic hairpin structure of approximately 120 to 200 bases is necessary for DNA replication in both terminal sequences of the virus [2].

The most widely known porcine parvovirus is porcine parvovirus (PPV)1, a major cause of reproductive failure syndrome in pigs. This reproductive failure syndrome is referred to as SMEDI and includes stillbirths, mummified fetuses, early embryonic death, and infertility [3]. Recently, a new dominant strain of PPV1 called the 27a-like strain has emerged in Europe, which shows a lack of neutralization against the existing vaccine strains [4]. Identifying whether the 27a-like strain exists in Korean pig populations is necessary. PPV4 was first discovered in the United States in 2005 and belongs to copiparvovirus, such as Bovine parvovirus 2 [5]. Unlike other parvoviruses, it has Bocaviruses like ORF3. PPV4 infections may persist in pigs, but its pathogenicity has not been revealed.

The transfer of viruses between different hosts to produce a new self-sustaining epidemic is rare. A rare example is the canine parvovirus, which adapted to dogs as the host range changes to the feline parvovirus [6]. The canine adaptation of the parvoviruses involved capsid protein changes, altering the recognition of the host transferrin receptors, allowing canine transferrin receptor binding, and its use as a receptor for cell infection [7]. Hence, the parvovirus must be monitored for these changes because its host may change due to protein mutations. As some viruses have spread from pigs to other species, including humans [6,8], genetic alterations in PPVs should be monitored, and signs of changes in host specificity should be closely scrutinized. From 2016 to 2022, 120 oral fluid samples for each breeding stage were submitted for laboratory diagnosis from domestic pig farms in South Korea.

Viral DNA was extracted from the oral fluid and identified by polymerase chain reaction (PCR) using a known multiplex primer set [9]. The multiplex PCR conditions were as follows: pre-denaturation at 95°C for 10 minutes; 35 cycles of denaturation at 95°C for 30 seconds, varied annealing temperature for 30 seconds, and extension at 72°C for 1 minute; and a final extension at 72°C for 5 minutes. The PCR products were evaluated by 2% agarose gel electrophoresis. Positive samples were sequenced using sequencing primers. The new primer was designed using the Primer3 tool of the Geneious Prime v2021.2.2 (Biomatters, New Zealand) according to the sequence data obtained from each sample. The amplified DNA was run on a 2% agarose gel, and the appropriate DNA band was extracted using a QIAquick Gel Extraction kit (QIAGEN, USA) and subjected to sequence analysis (Bionics, Korea). Table 1 lists the oligonucleotide sequence of each primer set used in this study. The obtained sequences were evaluated and assembled with Geneious Prime v2021.2.2. The complete nucleotide sequences obtained were compared with the sequence data available in GenBank, and sequence alignment was carried out using the MUSCLE included in Geneious Prime [10]. Translation of the nucleotide sequences into amino acid sequences and the alignment of the amino acid sequences were also carried out using the MUSCLE included in Geneious Prime. Phylogenetic analysis was performed using the PHYML in Geneious prime software in Hasegawa Kishino Yano (HKY) method mode with 500 bootstrap replicates [11]. The maximum likelihood tree was analyzed based on VP1 of PPV1 to determine the presence of a strain genetically and antigenically different from the existing vaccine strain, such as the 27a-like strain [2,12]. The VP1 sequence of canine parvoviruses (M19296.1) was used as an outgroup.

Table 1.

Sets of primers used to diagnose and obtain almost complete genome sequences

Five full genes and one VP1 gene of PPV1 from different geographical regions have been sequenced. PPV4 from different geographical regions has succeeded in sequencing 5 full genes and one ORF3 gene. The completed 12 sequences were called PPV1KUIP22-1 to 6 and PPV4KUIP22-1 to 6. All sequence information is registered in the GenBank and has received accession numbers (ON982164, OP377030-OP377035, and OP377054- OP377058).

In the case of PPV1, a premature termination codon was formed by a mutation in one of the sequences. The nucleotide at 482 of the NS2 gene of PPV1KUIP22-1 was mutated from G to A, resulting in a stop codon, and the amino acid sequence length was shortened from 162 to 161 (Fig. 1A). PPV1 shows a significant difference in pathogenicity with only one change in the amino acid [4,13]. Therefore, a premature stop codon mutation of PPV1 NS2 may show significant differences in the pathogenicity and infectivity of the virus, even though only one amino acid is shortened. In addition, a premature stop codon mutation of porcine bocavirus, which is phylogenetically close to porcine parvovirus, was observed in the previous study, and these mutations may cause a change in host specificity [14].

Fig. 1.

Notable mutations of porcine parvovirus (PPV)1 and PPV4. Premature stop codon of NS2 (A) and phylogenetic tree based on VP1 (B) of PPV1 included. The yellow mark on the tree indicates a 27a-like strain. Canine parvovirus was used as an outgroup. NADL-2 (NC_001718), Kresse (PPU44978), and 27a (AY684871) were also used to make a tree and nodes mean bootstrap value. Frameshift mutation (C) of ORF3 of PPV4 is also indicated.

Phylogenetic analysis confirmed that PPV1KUIP22-1 is a 27a-like strain (Fig. 1B). The 27a strain in Germany, which has changed its neutralizing epitope because of mutation of aa 228, lacks cross-neutralization against the NADL-2 vaccine strain [4,15]. The same mutation as above was observed in PPV1KUIP22-1, and the commercial vaccine currently used in South Korea is a virus like NADL-2, such as PVK-1. No clear evidence has shown that Korean 27a-like strains are difficult to defend with existing vaccines. Nevertheless, the emergence of new PPV1 strains with different antigenicity is possible. The existence of the 27a-like PPV1 must be considered, and continuous variation monitoring through additional sampling, sequencing, antigenicity, and cross-neutralization assay with existing vaccine strain is required. Because PPV1KUIP22-1 is a 27a-like strain and has a premature stop codon at the same time, further research will be needed to determine the changes in antigenicity.

For PPV4, one nucleotide is inserted into nucleotide at 545 of ORF3 in all collected sample sequences, resulting in a frameshift mutaction (Fig. 1C). The stop codon of ORF3 disappears by this mutation, and the new stop codon is the same as that of the VP gene. This must be confirmed by virus isolation and further in vitro analysis. In most cases, frameshift mutations cause lower fitness of viruses, but some major changes have also been observed, such as host changes, immune avoidance, pathogenicity, and transmission [16]. Although the role of ORF3 in PPV4 is unknown, it may be involved in the replication of viruses [17]. The role of the protein will change significantly if a frameshift occurs and a longer protein is translated. Nevertheless, there are no successful cases of PPV4 isolation in cell culture. Therefore, further study will be needed to determine how this frameshift affects the virus after successful isolation in vitro. The possibility that ORF3 protein is a virtual protein that does not exist cannot be excluded owing to the lack of such data with the isolated virus strain.

Through the premature stop codon of PPV1 and frameshift of PPV4, an important mutation described elsewhere can be a precursor to host range change. In addition, 27a-like strains of PPV1, which show a lack of cross-neutralization against the existing vaccine strains, have been confirmed in Korea. Therefore, it is necessary to check whether the host range of PPVs and antigenicity are changed through continuous monitoring.