29/249 pooled samples from eight Central and Southern provinces of Vietnam were positive for PCV3 by PCR (Table S1). We found that the positive rates of PCV3 in fetuses and sows in abortion cases were 22.64% and 14.29% respectively, while those of pigs in PRDC cases was 7.14%. The virus prevalence at the herd level in surveyed farms was about 11.65%, which was higher than those of previous study in the North of Vietnam (4.44%) [7]. Investigations in other countries reported higher PCV3-positive rates. PCV3 was detected in 12 of 14 farms (86%) in Poland [12], 53 of 73 farms (73%) in South Korea [4], and 69% (24 of 35 farms) in China [3]. The factors influencing PCV3 prevalence among farms or countries have been attributed to putative factors like biosafety, biosecurity, management of pig flow, environmental conditions, or individual factors, such as existing herd immunity, microorganisms pressure, and infection with immunosuppressive pathogens [12]. We also used tissues as the main sources of specimens, thus, the sample seems not to be the reason for the difference in the ratio of positivity. In addition, the herd size, the geographical location of the farm or the piglet producing unit, PCV2 infection, and vaccination status had no effect on the PCV3 prevalence [3, 12]. PCV3 was detected in the samples collected from all eight provinces examined in this study, together with previous reports, indicating that PCV3 has spread widely in Vietnam.
Phylogenetic tree analysis based on ORF2 sequences of 29 strains and 23 reference sequences showed that the Vietnamese PCV3 isolates were grouped into three different sub-types (a, b, and c) (Fig. 1). More than 79% (23/29) of isolates in this study belong to sub-type b. A previous study identified that 2/3 of strains in three southern provinces belonged to sub-type PCV3b [6]. These results suggested that PCV3b is the most prevalent in the Center and South of Vietnam. Meanwhile, the 5 isolates in group a of this study were clustered into two sub-types (a-1 and a-2), and the remaining strain belonged to sub-type c (Fig. 1). This analysis showed the divergence of the PCV3 in central and southern Vietnam. Another study sequenced 5 strains in the North of Vietnam and found all of them to be sub-type PCV3a-1 [7]. In this study, we first identified PCV3 strains belonging to sub-type c and sub-type a (PCV3a-2) in Vietnam.
The ORF2 nucleotide sequences of the 29 PCV3 isolates in the study were analyzed and compared to the reference strains. The genomic and amino acid identity within PCV3 isolates from Vietnam ranged from 96.90–100% and 96.19–100%, respectively. This seems to be more divergent compared to PCV3 isolates in China [3], but similar to isolates in North Vietnam [7]. Within sub-type PCV3b, the 23 strains examined in this study exhibited 97.21–100% and 96.67–100% similarity at the nucleotide and amino acid levels, respectively. They also shared high genomic and amino acid identity (97.52–99.53% and 97.64–100%, respectively) with reference PCV3 strains isolated in China (MN788140, KY421348, MF460446), Korea (KY996339), Italy (MF162299), and Brazil (MF079254). In addition, PCV3a strains in the study also showed the high homology of nucleotide and amino acid levels (98.29–100% and 97.64–100%, respectively) and with reference isolates (97.67–99.84% and 97.16–100%) (Table S2). A very high identity at nucleotide and amino acid sequences was observed between the PCV3c strain in this study and the three reference isolates (99.22–99.38% and 99.06–99.53%, respectively). These results displayed a similar divergence range at the nucleotide sequence and amino acid levels between PCV3 in the present study and the reference PCV3 strains.
We identified fifteen amino acid substitutions in the Cap of Vietnamese strains in this study (Fig. 2). Three strains of sub-type PCV3a-1 recorded three mutated sites R10K, F104Y, and S156T, which were in the predicted epitopes A, D, E respectively [5]. The specific mutation F104Y was also observed in the amino acid sequences of PCV3a-2 isolates, whereas the strain belonging to sub-type c only had the I94V mutation in Cap. Meanwhile, PCV3b isolates in the study showed 12 amino acid changes, including A5G (1/23), R16K (1/23), and Y23C (1/23) in the predicted epitope A; A75V (1/23) and Y80H (1/23) in the predicted epitope C, I94N (1/23); S95F/C/Y (4/23), L121F/V (2/23), R132G (1/23), S137F (4/23), K139R (7/23), and I150F (15/23) in the predicted epitopes E [5]. Besides, two amino acid substitutions (Y23C and L121F/V) were identified in antibody recognition domains (24–35 and 119–131 amino acids) [15]. These findings showed that PCV3 seems to be gradually accumulating more mutations, which may pose potential challenges in disease control.