4. DISCUSSION
Porcine circovirus type 2 is one of the major swine viruses causing enormous economic losses to the swine industry globally. Although commercially available vaccines have been widely used in the last two decades, vaccine failures remain questionable. PCV2 has a high nucleotide substitution rate that varies between 10-3and 10-4 substitution/site/year, similar to RNA viruses (Franzo et al., 2016). In addition, both point mutation and recombination contribute to the evolution of PCV2 (Firth, Charleston, Duffy, Shapiro, & Holmes, 2009), which could facilitate the evasion of the host immune responses. To date, at least eight PCV2 genotypes (A to H) were classified based on the phylogenetic analysis (Franzo & Segales, 2018).
In Thailand, data on the prevalence and genetic diversity of PCV2 in the past five years are limited. In this study, the prevalence and genetic diversity of PCV2 in Thailand were investigated, and the high prevalence of PCV2 (53.95% (396/734)) was observed in Thai pig populations from wide geographical regions during 2019-2020. In the current study, PCV2d was a dominant genotype, which is in agreement with the previous report (Thangthamniyom et al., 2017). Not only that, the proportion of PCV2d in Thailand seems to be increasing, indicating that the predominant PCV2d was gradually taking over other genotypes in the country. During 2009-2015, 54.81% of Thai PCV2 were PCV2d, while 40% were PCV2b (Thangthamniyom et al., 2017). The proportion of PCV2d in this study was increased to 86.27% in 2019-2020, and PCV2b was dropped to 13.73%. This increasing number of PCV2d infections could raise awareness regarding the PCV2 vaccine efficacy, mainly derived from PCV2a or PCV2b.
Other than point mutation, natural recombination can also lead to the emergence of novel PCV2 variant strains. Various studies demonstrated that both intra- and inter-genotypic recombination were found in many PCV2 strains (Cai et al., 2011; Cheung, 2009; Hesse, Kerrigan, & Rowland, 2008; Huang et al., 2013; Jang et al., 2021; Kim et al., 2009; Kleymann et al., 2020; Ma et al., 2007; Ramos et al., 2013). However, the evidence for genetic recombination of PCV2 is scant in Thailand due to the lack of complete genome sequences datasets. In this study, the presence of both PCV2d and PCV2b in the same farm (but at different time points) was occasionally found. Coincidental infection with different PCV2 genotypes might provide an opportunity for natural inter-genotypic recombination.
One recombinant PCV2 strain was identified in this study, 19NPT29. The results showed that the recombinant virus might be generated from a 19RBR10-like strain (PCV2d) and a KU-1605-like strain (PCV2b). Evidently, all Thai PCV2b isolates shared 99.6-99.7% nucleotide sequence identity with the South Korean KU-1605 (data not shown), which was suspected to be a parental strain of most Thai PCV2b isolates. Therefore, it is possible that PCV2b from South Korea (e.g., KU-1605) might be introduced into Thailand via non-pig transmission sources before spreading in the Thai pig populations and then being recombined with the local Thai PCV2d strains (e.g., 19RBR10).
The time and place where the recombination events occurred in 19NPT29 are unknown, mainly due to the limited number of samples from the farm of origin and the short duration of the study. The virus was found in a pig farm in Nakornpathom in 2019. During the study, there was only one sample submission from this farm, in which 19NPT29 was identified. Co-circulation of PCV2b and PCV2d in this farm was not observed. In fact, 19NPT29 was the only PCV2 strain found in this farm. It is possible that co-circulation of PCV2b and PCV2d took place in this farm prior to the investigation and gave rise to the emergence of 19NPT29. Alternatively, 19NPT29 might emerge in the other area and then spread to this farm.
Recombination breakpoints can be found in both ORF1 and ORF2 of recombinant PCV2 strains. ORF1 was predicted to be a primary target for inter-genotypic recombination (Hesse et al., 2008; Kim et al., 2009), possibly from the high conservation of nucleotide sequences in this region between the two genotypes that facilitating the recombination events. On the other hand, ORF2 encoded for the capsid gene that plays a significant role in immunogenicity, the virulence of the virus, and grouping the virus genotypes (Nawagitgul et al., 2000; Olvera et al., 2007). Hence, recombination events within ORF2 may contribute to both antigenicity and virulence of the novel virus (Franzo et al., 2016). Two recombination breakpoints were identified in ORF1 and ORF2 genes at positions 508 and 1356 of 19NPT29. Therefore, the antigenicity and pathogenesis of this recombinant virus should be further studied. Although the virus was detected in pigs that showed respiratory signs, the clinical outcomes can be affected by many factors.
For deduced amino acid analysis, the ORF2 gene encoded for capsid protein is recognized for phylogenetic analysis and still plays a vital role in both antigenic and immunogenic properties. Besides, antibody recognition sites were described from four regions at positions A (51-84), B (113-139), C (161-207), and D (228-233) in the Capsid protein (Lekcharoensuk et al., 2004; Mahe et al., 2000). In the present study, 31 deduced PCV2d ORF2 amino acid sequences presented four unique amino acid mutations corresponding to antibody recognition regions compared to different PCV2 genotypes. Specific mutations of PCV2d mainly occurred at positions 133, 134, 136 and 232 located in immunoreactive domains B and D. Notably, mutational events within the critical epitopes, especially at amino acid 133-135, may be responsible for the virulence of PCV2 (Krakowka et al., 2012). Hence, the mutations in the epitope regions of Thai PCV2d isolates may enhance the virus’s ability to escape the host immune responses and contribute to the disease severity. The effects of amino acid variation found in this study need further investigation to clarify the pathogenicity and virus properties.
To date, both intra-genotypic and inter-genotypic recombination of PCV2 have been reported worldwide, including Chile (Neira et al., 2017), China (Cai et al., 2011; Huang et al., 2013; Ma et al., 2007), Uruguay (Ramos et al., 2013), USA (Cheung, 2009; Hesse et al., 2008), South Korea (Jang et al., 2021; Kim et al., 2009), the Lesser Antilles in the Caribbean Sea (Kleymann et al., 2020), and lastly, Thailand. Therefore, recombination analysis should not be neglected in PCV2 genetic studies (e.g., virus evolution, phylogenetic analysis).
In conclusion, this study reveals PCV2d as a dominant PCV2 genotype in Thailand and an emergence of a recombinant virus between PCV2b and PCV2d through inter-genotypic recombination. Regular surveillance and monitoring in the farms may help gain a comprehensive view of PCV2 genetic evolution to implement early interventions against the emergence of PCV2 variants. Further investigation is needed to increase our understanding of the pathogenicity and virulence of the recombinant virus.