Among 264 samples collected from June 2022 to January 2023, 95 samples tested positive for PCV2 (35.98%). Following this, we selected 47 samples exhibiting a specific, bright PCR band to amplify and sequence the entire genome of PCV2. Analysis of the PCV2 genome revealed that 47 Vietnamese PCV2 strains collected between 2022 and 2023 fell into two main genotypes PCV2b and PCV2d, accounting for 19.15% (9/47) and 80.85% (38/47), respectively (Figure S1). These findings indicate that PCV2d was the high prevalent genotype in swine farming across various provinces in Vietnam during the specified period.
To investigate the molecular evolution of PCV2 in Vietnam, 222 ORF2 Vietnamese sequences sourced from NCBI, alongside 47 sequences from this study, were utilized to reconstruct a phylogenetic tree (Fig. 1). Overall, PCV2 displayed considerable genetic diversity in Vietnam, with five genotypes observed: PCV2a, PCV2b, PCV2d, PCV2g, and PCV2h. Notably, Vietnam's PCV2 history included just two PCV2g strains documented in 2008, along with two PCV2a isolates identified in both 2008 and 2018. Furthermore, PCV2h genotypes were predominantly present from 2008 to 2015 but have since ceased to be detected in recent genetic studies in Vietnam. In 2019, four PCV2h sequences were deposited into GenBank, but they were derived from local PCV2 vaccines (Doan et al. 2022). Genotypes PCV2b and PCV2d have consistently dominated in Vietnam over time. Specifically, during 2022–2023, only these two genotypes were observed, with PCV2d being much more prevalent than PCV2b. Overall, it appears that changes in PCV2 genotypes from PCV2b to PCV2h and subsequently to PCV2d occurred in Vietnam in 2008 and 2014, respectively.
To delve deeper into the distribution of PCV2 genotypes across two primary regions of Vietnam, we determined the frequencies of all genotypes over time in these areas (Fig. 2). In southern Vietnam, only three genotypes PCV2b, PCV2d, and PCV2h were detected, yet only PCV2d remained present in the pig population of this area. Conversely, all five PCV2 genotypes were identified between 2008 and 2023 in northern Vietnam. This trait shares numerous similarities with the PCV2 epidemic in China (Lv et al. 2020). However, as of now, only PCV2b and PCV2d are present in this area. These results show clear epidemiological differences in PCV2 between the two primary pig production regions of Vietnam.
The discovery of the PCV2 strain KhanhHoa/2007 and CaMau/2007 marked the first identification of ORF2 sequences in Vietnam. To assess the changes in amino acid levels within the Capsid protein among Vietnamese strains, we compared the Capsid amino acid sequences of these strains with the original Vietnamese strains (Fig. 3). We identified 17 significant amino acid substitutions in the Capsid protein sequences of Vietnamese PCV2 strains, occurring at a very high frequency. These substitutions include F8Y, I58F, V57I, K/A59R, K63R, N68A, R/I/H89L, T90S, T121S, P/S131T, N134T, R/G169S, S/T190A, A191G, D210E, I115V, and L/F234P. Importantly, many of these changes are situated within crucial antigenic regions of the Capsid protein. Specifically, K63R, N68A, R/I/H89L, T90S, T121S, S/T190A, and A191G were located at B-cell epitope positions (Shang et al. 2009; Guo et al. 2011; Ge et al. 2013), while K/A59R was within neutralizing epitopes (Mahe et al. 2000)d G169S was found within decoy epitopes (Trible et al. 2012).
In order to identify positively selected sites during molecular evolution of Capsid proteins of Vietnamese PCV2 strains, the predicted Capsid protein sequences of all Vietnamese strains were tested with HyPhy FUBAR, FEL, SLAC and MEME methods (Tables S1). Fourteen sites were recognized as positive selected sites within the capsid protein by four methods. Among these, positions 89 and 191 were flagged for adaptive selection by all four methods, while position 30 was pinpointed by three of them. Additionally, positions 88 and 190 were identified to be under adaptive selection by two methods. Furthermore, nine positions were identified by either FUBAR or MEME to be under positive selective pressure.