Genetic diversity and antigenic variations of porcine circovirus type 2 genotypes in Brazilian swine herds: Implications for vaccine efficacy and viral evolutionABSTRACT
Between 2019 and 2021, 106 clinical samples from pigs showing signs compatible with diseases associated with Porcine Circovirus (PCVD) were collected from various regions in Brazil, including the South, Southeast, and Central-West. The animals were sourced from commercial pig farms and had been previously vaccinated against PCV2. The collected samples were genotyped for PCV2, and the sequences were subsequently analyzed. The predominant genotype found was PCV2d (75.47%), followed by PCV2b (22.64%), while PCV2a was not detected. The sequences of PCV2b and PCV2d obtained were highly conserved within their respective genotypes but showed significant differences in antigenic sites, which may affect pathogenicity and/or antigenicity. When comparing PCV2d sequences with ORF2 amino acid sequences from three commercial vaccines used in Brazil (one containing PCV2b and two containing PCV2a), most amino acid residues in epitope regions were conserved, suggesting good cross-reactivity between vaccines and PCV2d. However, mutations were detected in residues essential for viral neutralization, which may impact vaccine efficacy. Further studies are needed to better understand these mutations and strengthen genotype surveillance of PCV2 in Brazil.
Keywords:
porcine circovirus Type 2; Brazil; PCV2b; PCV2d; sequencing
RESUMO
Entre 2019 e 2021, foram coletadas 106 amostras clínicas de suínos com sinais compatíveis com doenças associadas ao Circovírus suíno (PCVD) em diversas regiões do Brasil, incluindo Sul, Sudeste e Centro-Oeste. Os animais eram provenientes de granjas comerciais e haviam sido previamente vacinados contra PCV2. As amostras foram genotipadas, e as sequências analisadas. O genótipo predominante foi o PCV2d (75,47%), seguido pelo PCV2b (22,64%), enquanto o PCV2a não foi detectado. As sequências de PCV2b e PCV2d mostraram alta conservação dentro dos genótipos, mas diferenças significativas nos sítios antigênicos, o que pode afetar a patogenicidade e/ou a antigenicidade. Ao se comparar as sequências de PCV2d com as sequências de aminoácidos ORF2 de três vacinas comerciais usadas no Brasil (uma com PCV2b e duas com PCV2a), a maioria dos resíduos de aminoácidos nas regiões de epítopos foi conservada, sugerindo uma boa reação cruzada entre vacinas e PCV2d. No entanto, foram detectadas mutações em resíduos essenciais para a neutralização viral, o que pode impactar a eficácia da vacina. Estudos adicionais são necessários para compreender melhor essas mutações e reforçar a vigilância dos genótipos de PCV2 no Brasil.
Palavras-chave:
Circovírus suíno tipo 2; Brasil; PCV2b; PCV2d; sequenciamento
INTRODUCTION
Porcine Circovirus type 2 (PCV2) is a globally prevalent pathogen known for its significant role in multifaceted swine diseases (Segalés et al., 2005). PCV2 acts as an immunosuppressive pathogen, rendering pigs more susceptible to other respiratory and enteric tract pathogens (Ciacci-Zanella, 2017).
Despite the clinical disease being controlled after the introduction of vaccines, the virus persists in the pig population and continues to evolve. Global shifts in genotype prevalence have been documented, driven by the virus's rapid rate of evolution. Monitoring PCV2 genotype circulation in swine herds is crucial as mutations may compromise immunity acquired through vaccination, especially against other variants (Dvorak et al., 2016).
In Brazil, PCV2 vaccination rates among swine herds range from 80% to 98% (Ciacci-Zanella, 2017), with the prevailing protocol being the vaccination of three-week-old piglets. Since widespread vaccination adoption, farm health status has shifted towards predominantly self-limited subclinical issues, occasionally punctuated by outbreaks. Deviations from recommended vaccination protocols may enable the virus to persist and continue causing disease (Ciacci-Zanella, 2017).
PCV2 exhibits a notably high mutation rate, estimated at 10³ to 10⁴ substitutions per site per year. This genomic variability has led to the classification of PCV2 into nine distinct genotypes, ranging from PCV2a to PCV2i (Franzo and Segalés, 2018). While PCV2a, PCV2b, and PCV2d are widely identified worldwide, PCV2c has been described in Denmark and Brazil in feral pigs and wild boars (Sato et al., 2017). PCV2e, PCV2f, and PCV2h were described in Mexico, the USA, and China, respectively. In 2020, a new PCV2i genotype was isolated from a pig in the USA (Wang et al., 2020). During the early 2000s, a notable increase in severe clinical diseases affecting pigs was observed, leading to the global presence of Porcine Circovirus-Associated Diseases (PCVD), with PCV2b being the prevailing genotype at that time (Firth et al., 2009; Franzo and Segalés, 2018).
The primary objective of this study is to determine the specific PCV2 genotypes currently circulating within vaccinated swine herds in Brazil, and to conduct a comparative analysis of the most prevalent genotype obtained in this study (PCV2d) with those available in vaccine sequences in the National Center for Biotechnology Information (NCBI) database. We focus particularly on identifying potentially noteworthy alterations in amino acid composition.
MATERIALS AND METHODS
A total of 246 clinical samples, collected between 2019 and 2021, previously tested positive for PCV2, were submitted for genotyping at the Laboratory of Animal Virology Research (LPVA), Department of Preventive Veterinary Medicine (DMVP) at the Universidade Federal de Minas Gerais (UFMG). The samples came from various regions of Brazil, including the southeast, south and midwest regions, with some samples lacking identification of origin. Specimen types included oral fluids, lymphoid organs, and sera from pig herds previously vaccinated against PCV2, exhibiting symptoms resembling PCVD. PCV2 vaccination was conducted using one of seven available vaccines in Brazil.
Total DNA was extracted from collected samples using the mini spin DNA extraction kit (Kasvi), according to the manufacturer’s instruction. Genotyping for PCV2 subtypes (PCV2a, PCV2b and PCV2d) was performed as previously described. (Kwon et al., 2017).
Fifty-one previously positive DNA samples for PCV2 were selected for complete sequencing of the Open Reading Frame 2 (ORF2) gene. Eighteen samples were genotyped in the present study, while 33 PCV2-positive samples were provided by Microvet - Microbiologia Veterinária Especial. All samples were collected from vaccinated pigs exhibiting clinical symptoms consistent with PCVD in 2019. Sequencing was conducted at Simbios Biotecnologia laboratory using the Sanger method.
ORF2 gene sequences from Brazilian samples were aligned with 51 reference sequences comprising PCV2a, PCV2b, PCV2d, and PCV2c sourced from the NCBI GenBank (Franzo and Segales, 2018). To determine the PCV-2d subclade (PCV2d-1 or PCV2d-2) to which the Brazilian samples belong, the PCV2d samples were aligned with reference samples as described by Xiao et al. (2015). Alignment was conducted using MAFFT v7.471, employing the globalpair method, adjusting direction options with 1,0000 iterations. The alignment was refined manually using AliView (https://academic.oup.com/ bioinformatics/article/30/22/3276/2391211).
Phylogeny relationships were inferred using the Neighbor-Joining (NJ) method. The pairwise distance between sequences was estimated using the p-distance method with the MEGA X software under 1,000 bootstrap replications.
The variability within the ORF2 region of PCV2d was assessed by comparing sequenced PCV2 samples with three vaccine sequences available in Brazil (Table 1). Alignment, consensus sequence generation, and visualization were performed using MAFFT v7.471, R software, AliView, and the ggseqlogo library.
RESULTS AND DISCUSSION
A total of 246 field samples collected from various regions were subjected to PCR genotyping, resulting in successful amplification of 106 samples (43.09%). This success rate varied across the study years, with 27.56% of samples genotyped in 2019, 47.25% in 2020, and 48.45% in 2021 (Table 2). The increased diagnostic submissions observed over the years likely reflect a heightened awareness of PCV2-associated diseases and a growing interest in PCV2 diagnostics, possibly linked to the expanded usage of PCV2 vaccines within the swine industry.
The predominant genotype observed in the present study differed from those found in previous studies, with PCV2d identified in the present study as the most prevalent genotype. This accounts for 75.47% of genotyped samples, followed by PCV2b at 22.64% (Gava et al., 2018). Intriguingly, a small subset of samples (1.89%) exhibited coinfections with both PCV2b and PCV2d, while PCV2a was not detected. This shift in genotype prevalence toward PCV2d, surpassing PCV2b, suggests viral dynamics are evolving within Brazilian pig herds (Table 2).
Among the clinical specimens examined, oral fluid emerged as the most effective matrix for PCV2 genotyping, yielding 37.74% of genotyped samples, followed closely by pooled organ samples and sera, each comprising 31.13% of the genotyped samples. This underscores the utility of oral fluid sampling for efficient and cost-effective PCV2 monitoring, with added benefits for animal welfare (Nielsen et al., 2018).
Geographically, Minas Gerais state exhibited the highest number of positive samples (n=51), followed by the states of Mato Grosso (n=20), Paraná (n=14), Rio Grande do Sul (n=8), and Santa Catarina (n=2) (Table 3).
However, 11 samples lacked identification of their origin, highlighting the need for improved sample tracking and metadata collection in future studies. More importantly, regardless of geographical location, PCV2d emerged as the prevailing genotype over PCV2b across all Brazilian states during the evaluated period. This observation aligns with several authors who have proposed that PCV2d has become the predominant genotype globally (Franzo and Segalés, 2018; Qu et al., 2018; Yao et al., 2019). Given the significance of PCV2d as the putatively most common genotype circulating among Brazilian herds, coupled with its association with cases of PCV2 vaccine failures, this genotype has been selected for a through-evaluation at the amino acid level.
Following initial sequencing attempts, a subset of 25 sequences was selected for further phylogenetic analysis. The Brazilian sequences generated in this study have been deposited in GenBank under accession numbers OL614789 to OL614815 (Table 4).
Identification of the PCV2 samples sequenced in the present study and their respective Genbank accession number
The resulting phylogenetic trees revealed distinct sub-clusters within both PCV2b and PCV2d genotypes (Fig. 1).
Notably, all Brazilian PCV2d sequences clustered within the PCV2d-2 subclade, corroborate the prevalence of this genotype observed in the genotyping analysis (Fig. 2). This division of PCV2d genotype into two subclades, PCV2d-1 and PCV2d-2, has already been reported, with most PCV2d-1 samples primarily circulating from 1999 to 2011, and PCV2d-2 samples emerging in 2006 (Xiao et al., 2015). In a phylogenetic study of field samples from pigs with clinical signs of PCVD in Belgium, collected from 2009 to 2018, it was demonstrated that PCV2d-1 samples were found in 2009 and 2010, while in 2018 all PCV2d sequences clustered in the PCVd-2 clade (Wei et al., 2019). Thus, researchers have suggested that PCV2d-1 is possibly an ancestor of PCV2d-2 (Xiao et al., 2015).
The nucleotide sequences of the PCV2 ORF2 gene which encodes the CAP, in all PCV2 Brazilian strains analyzed in the present study, were 695 nucleotides in length, corresponding to 231 amino acids. However, the last two amino acids (position 232 to 233) were excluded from the analysis due to poor sequencing quality.
Phylogenetic tree of 25 sequences from this study, 51 reference strains from PCV2a, PCV2b, PCV2c and PCV2d, according to Franzo & Segalés (2018). Sequences from the present study are indicated by colors. PCV2b are shown in green and PCV2d in red. The phylogenetic tree was constructed based on the nucleotide sequences of the Cap gene (ORF2). The tree was inferred by using the Neighbor-Joining method with 1,000 bootstrap replicates. Only Bootstrap values ≥70% are shown.
Phylogenetic Tree of 22 Sequences from this Study, 3 Brazilian reference sequences from PCV2a, PCV2b, PCV2c, according to Franzo & Segalés (2018) and 19 reference sequences from PCV2d-1 and PCV2d-2, according to Xiao et al. (2015). The PCV-2d sequences in the present study are indicated by a black ball. The Phylogenetic Tree was constructed based on the nucleotide sequences of the Cap gene (ORF2). The tree was inferred using the Maximum Likelihood method with 1,000 bootstrap replicates. Tree was generated on https://Ngphylogeny.Fr/ and displayed on Itol.
The amino acid sequences of the CAP protein from the present study demonstrated conservation within genotypes. When comparing the conserved Brazilian sequences of PCV2b to PCV2d genotypes, 13 positions exhibited mutations in the amino acid sequences, leading to variations at the following positions: Y8F, F53I, I57V, R59K, S64T, R89L/I, S90T, S121T, P134N, S169R/G, N207Y, E210D, V215I (Tab. 5). Notably, 9 of these 13 positions are located within immunogenic regions 53, 57, 59, 64, 121, 134, 169, 207 and 210.
At positions 32, 34 and 68, 24 out of 25 (96%) sequences were identical. The exceptions were in samples ID5297, ID5325, IDI15309 and ID5297, which had mutations at positions P32H, H34N, N68A, 89 and 169, respectively (Table 5). Most of these polymorphisms between genotypes occur in epitope regions, which are regions recognized by the immune system. Such variations can potentially aid the virus to evade the immune response, as the new variants possess different antigenic determinants compared to previously prevalent PCV isolates (Franzo and Segalés, 2020).
Polymorphic sites within PCV2 ORF2 gene sequences from Brazil. Dashes represent an amino acid position similar to GenBank accession number AF201311, according to Mahé et al. (2000). As a reference PCV2d genotype, the Genbank accession number KT819170, described by Gava et al. (2018), was used as a reference PCV2d genotype. Sequences with 100% amino acid identity were excluded
Various techniques have been employed to map linear or conformational epitopes within the PCV2 ORF2, including: generating mutant viruses (PCV1/PCV2 chimeras, recombinant baculoviruses); PEPSCAN analysis for mapping and characterizing epitopes involving the synthesis of overlapping peptides and analysis of the peptides in enzyme-linked immunosorbent assays (ELISAs); developing monoclonal antibodies (mAb) against specific epitope residues from different genotype strains; and, evaluating serological reactivity with experimental and natural infection anti-PCV2 antisera, among others (Mahé et al., 2000; Truong et al., 2001; Lekcharoensuk et al., 2004; Misinzo et al., 2006; Shang et al., 2009; Huang et al., 2011; Saha et al., 2012; Liu et al., 2013; Gava et al., 2018; Khayat et al., 2019; Letunic and Bork, 2021). The recognized epitope regions are often grouped into four general antibody recognition regions labeled A (47 to 84), B (113 to 137), C (156 to 210) and D (228 to 233), along with position 88. These immunogenic regions encompass 54.7% (128/234) of all CAP amino acid residues. In general, most amino acid residues within these antigenic regions are similar, providing cross-protection among vaccines. It's worth noting that, while most amino acid residues within antigenic regions are similar, there are differences among genotypes as well as among strains of the same genotype. Some single residues located on the outer surface of the CAP protein, such as those at positions 59, 89, 134, and 210, play a role in binding strength within the epitope and differ between PCV2b and PCV2d (Saha et al., 2012; Liu et al., 2013; Huang et al., 2020).
According to previous studies, amino acids at positions 59 and 134 are involved not only in binding strength but in the difference of neutralizing antibodies efficacy among monoclonal antibodies (mAb) against PCV2a, PCV2b and PCV2d genotypes (Huang et al., 2020). According to Huang et al. (2020), positions 59 and 134 are moderately conserved among PCV2 strains (approximately 60%). A single mutation at T134 can result in a >50% loss of binding reactivity of mAb, while the mutation at A59 can reduce the binding reactivity of the mAb by approximately 30%. These variations in specific amino acid residues can have a significant impact on the binding strength of antibodies.
When we compared the amino acid sequences of the Brazilian consensus PCV2 (TestD) with the sequences of vaccines used in Brazil and previously deposited in GenBank, we observed significant changes in the epitope regions. Seventeen mutations in antigenic regions occurred between TestD and some or all vaccine strains at positions: (Epitope region A): I53F (all vaccine strains), V57I (cVac2009-2b), K59A (Vac2000-2a)/R (cVac2007-2a, cVac2009-2b), R63T (cVac2007-2a), N68A (all vaccine strains), N75K (cVac2007-2b), N77D (Vac2000-2a, cVac2007-2a), L80V (Vac2000-2a, cVac2007-2a), P88K (Vac2000-2a, cVac2007-2a), and L89I (Vac2000-2a, cVac2007-2a/R (cVac2009-2b; (Epitope region B): N134T (all vaccines). (Epitope region C): R169S (all vaccine strains), L185M (cVac2000-2a), T190S (Vac2000-2a, cVac2007-2a)/A (cVac2009-2b), G191R (Vac2000-2a), T200I (cVac2007-2a), and I206K (cVac2007-2a). Out of 17 mutations in antigenic regions, 13 occurred in cVac2007-2a, 12 in Vac2000-2a, and 10 in cVac2009-2b (Fig. 3).
Location of the immunoreactive regions in the alignment of amino acid from TestD consensus sequences and amino acid sequences available from 3 vaccines licensed in Brazil. Sequences that were not identical in > 90% of the isolates in the group were replaced by dashes. The black squares are four general antibody recognition regions labeled A (47 to 84), B (113 to 137), C (156 to 210) and D (228 to 233), plus at position 88. The D epitope was not demonstrated due to the exclusion of the last 3 amino acids of ORF2 from our Brazilian sequences. Dotted are the amino acid sequences identical to the TestD sequence. Colors indicate amino acid chemistry. Red: Acid Polar, Blue: Basic Polar, Pink: Neutral Polar, Green: Nonpolar.
Of these 17 mutations in antigenic sites of the CAP protein between the Brazilian consensus amino acid sequences of PCV2d genotype (TestD) and vaccine samples, 6 (at positions 53, 57, 80,134, 185 and 210) did not change the physical-chemical property of the amino acids. Therefore, they did not cause significant structural changes in the CAP protein. In 12 positions (59, 63, 68, 75, 77, 88, 89, 169, 190, 191, 200 and 206), mutations were observed that altered the stereo chemical properties of the amino acids (Fig. 3). These are considered the most important mutations for causing significant structural changes in the CAP protein. Among these 12 analyzed mutations, with change on amino acid charge, the vaccine strain with the highest number of mutations was cVac2007-2a with 8 mutations at positions R63T, N68A, N75K, N77D, P88K, R169S, T200I, and I206K; followed by Vac2000-2a with 6 mutations at positions K59A, N68A, N77D, P88K, R169S, and G191R; and, cVac2009-2b with 4 mutations at positions N68A, L89R, R169S, and T190A. All vaccine strains had an amino acid completely different from the TestD consensus at positions 59, 68, 89,134, 169 and 190 (Fig. 3).
Several authors have reported that within an immunogenic region, some single amino acids are essential for the recognition of specific antibody binding and that mutations in these single amino acids can alter, favoring or inhibiting the recognition of antibodies by the antigenic site. Furthermore, mutations in single amino acids can also determine different levels of viral neutralization by a given mAb. This suggests a crucial role for these amino acids in the differential effectiveness of viral neutralization among different PCV2 genotypes, and even among the same genotype strains with different biological characteristics (Shang et al., 2009; Saha et al., 2012; Trible and Rowland, 2012; Liu et al., 2013; Kurtz et al., 2014; Huang et al., 2011, 2020). Studies with universal monoclonal antibodies, which react with eight different PCV2 clusters within PCV2a and PCV2b genotypes, have demonstrated that eight amino acid positions are crucial for binding of the different mAbs at positions 30, 59, 63 (Epitope region A); 89, 130, 133 (Epitope region B); and 206, 210 (Epitope region C) (Saha et al., 2012). These amino acids are located on the outer surface of a capsid protein. Positions 59 and 63 were in loop BC, position 89 in loop CD, positions 130 and 133 in loop EF; position 206 in loop HI and position 210 in β-strand. The flexibility of the loops facilitates the binding between the virion and Fab portion of antibodies (Saha et al., 2012; Huang et al., 2020). Of these positions, two are considered universal, with similar sharing to all cluster strains: 130 and 133 (Fig. 3). As expected, our vaccines were identical to the Brazilian samples (TestD) in these two universal positions, also in 2 other positions: 89 and 210. There was a divergence in three positions, when comparing the TestD samples with the vaccines 59 (all vaccines), 63 (cVac2007-2a) and 206 (cVac2007-2a) (Fig. 3).
Important genotype-specific domains have also been identified using multiple sequence alignment, as six amino acid residues at positions 86 to 91, and four at positions 190, 191, 206 and 210 on the PCV2 CAP (Cheung and Greenlee, 2011). As expected, since the vaccines have different genotypes (PCV2a or PCV2b) from the Brazilian PCV2d (TestD) samples, mutations were observed at positions 190 (all vaccines), 191 (cVac2000-2a) and 206 (cVac2007- 2a) (Fig. 3).
Although PCV2 infected pigs produce high levels of CAP specific antibodies, the onset and severity of PCVD are correlated with the absence or decreased levels of PCV2 neutralizing antibodies, suggesting a crucial role for neutralizing antibodies in the prevention of PCVD (Meerts et al., 2006). Changes in single amino acids can switch the neutralizing phenotype of PCV2 CAP monoclonal antibodies (Saha et al., 2012; Liu et al., 2013; Kurtz et al., 2014; Huang et al., 2020). Studies with mAbs that have different reactivity or neutralization phenotypes have been used to identify these critical amino acids, important for PCV2 neutralization at positions 59, 60, 131, 151, 190, 191, and the C-terminal area including residues at positions 231 to 233 (Saha et al., 2012; Liu et al., 2013; Kurtz et al., 2014). Specific mutations in residues R59A, A60T, A151T/P allow the mutant to be recognized and neutralized by monoclonal antibodies, with complete gain of recognition and neutralization of mAbs against PCV2a or PCV2b. Inverse mutations in these regions result in a complete loss of neutralization. Mutations in other amino acid residues have also shown differences in neutralizing activity, such as T190A which results in complete loss of neutralization. Furthermore, mutations T131P and E191R result in partial but significant loss of neutralization (Saha et al., 2012).
Recently, Huang et al. (2020), using a monoclonal antibody that neutralizes the PCV2a, PCV2b and PCV2d genotypes, detected 18 single amino acids responsible for this neutralization. To date, ten of these amino acids are completely conserved (>99%) among all samples deposited in GenBank (55Y, 56T, 61T, 62V, 128N, 132K, 135A, 137T, 189T and 231L). The rest showed variable levels of conservation with 5 highly conserved (86.89% to 98.33%) at positions 58K, 60T, 131T, 133A and 136L; one moderately conserved (approximately 60%) at position 134; and two highly variable (<25%) at positions 59A and 88K. Most of the amino acids in the epitope regions that neutralize PCV2a, PCV2b and PCV2d genotypes were conserved among different genotypes, which might explain the relatively broad-spectrum reactivity of a monoclonal antibody with PCV2 strains. However, mutations of some amino acids within the epitope regions had significant effects on the neutralizing activity of this specific mAb. Using cryo-electron microscopy single particle analysis in a complex with mAb Fab fragments and alanine scanning mutagenesis, it was observed that the single mutations at positions 128, 131, 132, 134, 135, 136 and 189 in the CAP protein resulted in >50% loss of binding reactivity of specific mAb, while the mutation at position 59 reduced the binding reactivity of the mAb by approximately 30%. The binding levels at positions 56 and 60 mutations were similar to those of the parenteral virus; however, their neutralization activities were significantly lower than that of the parenteral virus.
When comparing the Brazilian amino acid sequences with significant effects on the neutralizing activity described for genotype D (TestD) with vaccines, 15 of the 18 positions were conserved, with differences observed at positions 59, 88 and 134 (Fig. 3). In the Brazilian samples of PCV2d genotype (TestD), when compared to vaccine samples, the mutations were R (cVac2007-2a, cVac2009-2b)/A (cVac2000-2a)59K, K88P (cVac2000-2a,cVac2007-2a) and T134N (all vaccines). These results agree with Huang et al. (2020), which demonstrated that these positions were not conserved among different cluster strains, but they were conserved within specific genotypes, indicating that the positions were involved in the differential neutralization efficacy of mAb against PCV2a, PCV2b and PCV2d.
In summary, the three vaccines analyzed have differences in 3 out of 8 (37.5%) single nucleotides crucial for monoclonal antibody binding; 3 out of 10 (30%) differences among specific genotypes; and 5 out of 15 (33.3%) differences among a single amino acid responsible for viral neutralization, without considering the last 3 nucleotides, which were not analyzed. The vaccines that have the greatest variability in important residues for antibody binding within immunogenic regions and neutralization activity are the PCV2a genotype vaccines (cVac 2007-2a and cVac 2000-2a), followed by the PCV2b genotype vaccine (cVac 2009-2b). The question is how these antigenic differences could be related to vaccine failures observed worldwide, in relation to PCV2d. Studies have demonstrated that a set of single amino acids within epitope regions are responsible for differences in neutralization levels among different genotypes or samples of the same genotype that have biological differences. What has been widely sought after is a vaccine that protects against all strains or genotypes of PCV2.
The existence of a specific amino acid genotype in the crucial binding of mAbs may be the reason for somewhat better results in homologous situations. Cross-protection among PCV2 genotypes has been demonstrated experimentally. However, a recent experimental study indicated that a PCV2 vaccine based on the PCV2b genotype was more effective in protecting pigs against the effects of PCV2b or vaccinated with a bivalent PCV2a/PCV2b vaccine, than with a vaccine based on the PCV2a genotype. However, that protection against PCV2 is known to be dependent on the generation of neutralizing activity (Opriessnig et al., 2013; Bandrick et al., 2020). When PCV2a vaccinated pigs were challenged with PCV2b, then a higher percentage of animals were positive for viral DNA in nasal and fecal swabs 20 days after the challenge than when they were challenged with PCV2a and vice versa (Fort et al., 2007; Beach et al., 2011).
Protection against PCV2 is known to be dependent on the generation of neutralizing activity, and variations in these amino acids can impact significantly the neutralization activity of antibodies. Cross-protection between different PCV2 genotypes has been demonstrated experimentally, but the effectiveness of vaccines against different genotypes may vary due to these antigenic differences.
CONCLUSION
The present study offers valuable insights into the genetic and antigenic variations among PCV2 strains in Brazil, focusing on the PCV2d genotype. The phylogenetic analysis of Brazilian PCV2 strains based on the ORF2 gene revealed a significant prevalence of PCV2d, indicating its dominance in the Brazilian swine population during the study period. This finding aligns with global trends, as PCV2d has been recognized increasingly worldwide.
Amino acid sequence analysis of the Cap protein, a crucial antigenic component of PCV2, identified several mutations in epitope regions when comparing Brazilian PCV2d strains (TestD) to vaccine strains (cVac2000-2a, cVac2007-2a, and cVac2009-2b). These mutations, particularly in essential antibody recognition regions, could potentially impact vaccine effectiveness, contributing to observed vaccine failures against PCV2d.
Furthermore, studies have underscored the importance of specific amino acid residues in determining the neutralization efficacy of monoclonal antibodies against PCV2. Variations in these critical amino acids, especially in regions responsible for antibody binding and neutralization, were observed between Brazilian PCV2d strains and vaccine strains, potentially affecting the cross-protection offered by existing vaccines.
These findings highlight the complexity of PCV2 genetic and antigenic diversity and their potential impact on vaccine efficacy. Developing vaccines that provide broad protection against various PCV2 strains, including PCV2d, remains a significant challenge. Further research is needed to explore strategies to enhance vaccine effectiveness and cross-protection, considering the evolving nature of PCV2 strains.
Overall, the present investigation contributes to our understanding of PCV2 dynamics in Brazil and their implications for swine health. It underscores the importance of ongoing surveillance, vaccine development, and adaptation to emerging PCV2 genotypes to ensure effective control of PCV2-associated diseases in the swine industry.
ACKNOWLEDGMENTS
We would like to thank our colleagues from Microvet- Microbiologia Veterinária especial for kindly providing some of the samples used in the present study, SIMBIOS Biotecnologia for the genetic sequencing work, and Zoetis Animal Health for funding.
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Publication Dates
-
Publication in this collection
14 July 2025 -
Date of issue
Jul-Aug 2025
History
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Received
08 Sept 2024 -
Accepted
03 Dec 2024
