Evidence of vertical transmission of Senecavirus A in naturally infected sows.

Senecavirus A (SVA) is a Picornaviridae RNA virus that causes vesicular disease (VD) and transitory neonatal losses in pigs. The major ways SVA is spread are by oral, nasal, and feces. Vertical transmission of SVA was investigated during a VD epidemic in a farrow-to-finish herd in Brazil. Vesicular lesions were observed on sows before farrowing and on piglets within 24 h of birth. Analyses included RT-qPCR, viral isolation, sequencing, and virus-neutralization assays on serum, vesicular fluid, colostrum, and milk. Five out of ten sows were viremic before farrowing, and 46.7% of tested piglets had high viral loads in the first 24 h after birth. Infectious virus was detected in colostrum and milk from one postnatal sow. Despite high levels of neutralizing antibodies (nAbs) in piglet serum, colostrum, and milk, piglets were not protected from viremia and clinical illness. These findings support the vertical and congenital transmission of SVA.

Immunogenicity of an Inactivated Senecavirus A Vaccine with a Contemporary Brazilian Strain in Mice.

Senecavirus A (SVA) is a picornavirus that is endemic in swine, causing a vesicular disease clinically indistinguishable from other vesicular diseases, like foot-and-mouth disease. The widespread viral circulation, constant evolution, and economic losses caused to the swine industry emphasize the need for measures to control the agent. In this study, we evaluated the immunogenicity of a whole-virus-inactivated vaccine using a representative contemporary Brazilian SVA strain in Balb/ByJ mice. The animals were vaccinated with two doses by an intramuscular route. The humoral response induced by the vaccination was evaluated by an in-house ELISA assay for IgG detection. The cellular response was assessed by flow cytometry after in vitro SVA stimulation in splenocyte cultures from vaccinated and non-vaccinated groups. Protection against SVA was assessed in the experimental groups following an oral challenge with the homologous virus. The vaccination induced high levels of IgG antibodies and the proliferation of CD45R/B220+sIgM+, CD3e+CD69+, and CD3e+CD4+CD44+CD62L- cells. These results indicate the immunogenicity and safety of the vaccine formulation in a murine model and the induction of humoral and cellular response against SVA.

A polyvalent virosomal influenza vaccine induces broad cellular and humoral immunity in pigs.

BACKGROUND: Influenza A virus (IAV) is endemic in pigs globally and co-circulation of genetically and antigenically diverse virus lineages of subtypes H1N1, H1N2 and H3N2 is a challenge for the development of effective vaccines. Virosomes are virus-like particles that mimic virus infection and have proven to be a successful vaccine platform against several animal and human viruses. METHODS: This study evaluated the immunogenicity of a virosome-based influenza vaccine containing the surface glycoproteins of H1N1 pandemic, H1N2 and H3N2 in pigs. RESULTS: A robust humoral and cellular immune response was induced against the three IAV subtypes in pigs after two vaccine doses. The influenza virosome vaccine elicited hemagglutinin-specific antibodies and virus-neutralizing activity. Furthermore, it induced a significant maturation of macrophages, and proliferation of B lymphocytes, effector and central memory CD4+ and CD8+ T cells, and CD8+ T lymphocytes producing interferon-γ. Also, the vaccine demonstrated potential to confer long-lasting immunity until the market age of pigs and proved to be safe and non-cytotoxic to pigs. CONCLUSIONS: This virosome platform allows flexibility to adjust the vaccine content to reflect the diversity of circulating IAVs in swine in Brazil. The vaccination of pigs may reduce the impact of the disease on swine production and the risk of swine-to-human transmission.

Immunological profile of mice immunized with a polyvalent virosome-based influenza vaccine.

BACKGROUND: Influenza A virus (IAV) causes respiratory disease in pigs and is a major concern for public health. Vaccination of pigs is the most successful measure to mitigate the impact of the disease in the herds. Influenza-based virosome is an effective immunomodulating carrier that replicates the natural antigen presentation pathway and has tolerability profile due to their purity and biocompatibility. METHODS: This study aimed to develop a polyvalent virosome influenza vaccine containing the hemagglutinin and neuraminidase proteins derived from the swine IAVs (swIAVs) H1N1, H1N2 and H3N2 subtypes, and to investigate its effectiveness in mice as a potential vaccine for swine. Mice were immunized with two vaccine doses (1 and 15 days), intramuscularly and intranasally. At 21 days and eight months later after the second vaccine dose, mice were euthanized. The humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with a polyvalent influenza virosomal vaccine were investigated. RESULTS: Only intramuscular vaccination induced high hemagglutination inhibition (HI) titers. Seroconversion and seroprotection (> 4-fold rise in HI antibody titers, reaching a titer of ≥ 1:40) were achieved in 80% of mice (intramuscularly vaccinated group) at 21 days after booster immunization. Virus-neutralizing antibody titers against IAV were detected at 8 months after vaccination, indicating long-lasting immunity. Overall, mice immunized with the virosome displayed greater ability for B, effector-T and memory-T cells from the spleen to respond to H1N1, H1N2 and H3N2 antigens. CONCLUSIONS: All findings showed an efficient immune response against IAVs in mice vaccinated with a polyvalent virosome-based influenza vaccine.

Human-to-swine introductions and onward transmission of 2009 H1N1 pandemic influenza viruses in Brazil.

Introduction: Once established in the human population, the 2009 H1N1 pandemic virus (H1N1pdm09) was repeatedly introduced into swine populations globally with subsequent onward transmission among pigs. Methods: To identify and characterize human-to-swine H1N1pdm09 introductions in Brazil, we conducted a large-scale phylogenetic analysis of 4,141 H1pdm09 hemagglutinin (HA) and 3,227 N1pdm09 neuraminidase (NA) gene sequences isolated globally from humans and swine between 2009 and 2022. Results: Phylodynamic analysis revealed that during the period between 2009 and 2011, there was a rapid transmission of the H1N1pdm09 virus from humans to swine in Brazil. Multiple introductions of the virus were observed, but most of them resulted in self-limited infections in swine, with limited onward transmission. Only a few sustained transmission clusters were identified during this period. After 2012, there was a reduction in the number of human-to-swine H1N1pdm09 transmissions in Brazil. Discussion: The virus underwent continuous antigenic drift, and a balance was established between swine-to-swine transmission and extinction, with minimal sustained onward transmission from humans to swine. These results emphasize the dynamic interplay between human-to-swine transmission, antigenic drift, and the establishment of swine-to-swine transmission in shaping the evolution and persistence of H1N1pdm09 in swine populations.

Introductions of Human-Origin Seasonal H3N2, H1N2 and Pre-2009 H1N1 Influenza Viruses to Swine in Brazil.

In South America, the evolutionary history of influenza A virus (IAV) in swine has been obscured by historically low levels of surveillance, and this has hampered the assessment of the zoonotic risk of emerging viruses. The extensive genetic diversity of IAV in swine observed globally has been attributed mainly to bidirectional transmission between humans and pigs. We conducted surveillance in swine in Brazil during 2011-2020 and characterized 107 H1N1, H1N2, and H3N2 IAVs. Phylogenetic analysis based on HA and NA segments revealed that human seasonal IAVs were introduced at least eight times into swine in Brazil since the mid-late 1980s. Our analyses revealed three genetic clades of H1 within the 1B lineage originated from three distinct spillover events, and an H3 lineage that has diversified into three genetic clades. The N2 segment from human seasonal H1N2 and H3N2 viruses was introduced into swine six times and a single introduction of an N1 segment from the human H1N1 virus was identified. Additional analysis revealed further reassortment with H1N1pdm09 viruses. All these introductions resulted in IAVs that apparently circulate only in Brazilian herds. These results reinforce the significant contributions of human IAVs to the genetic diversity of IAV in swine and reiterate the importance of surveillance of IAV in pigs.

A retrospective study of porcine reproductive and respiratory syndrome virus infection in Brazilian pigs from 2008 to 2020.

Porcine reproductive and respiratory syndrome (PRRS) is a viral disease characterized by reproductive impairment or failure in breeding animals, and a respiratory disease in pigs of any age. Brazil is the fourth largest pork producer and exporter globally, and PRRS virus (PRRSV) infection has never been reported in the country. This study aimed to investigate the status of porcine biological samples from commercial swine herds, quarantined imported boars, wild boars and feral pigs to update PRRS information in Brazil. A total of 14,382 samples were collected from 2008 to 2020, including sera (n = 12,841), plasma (n = 1,000) and oral fluids (n = 541), comprehending 137 herds and free-living pigs in eight Brazilian states. One out of 1,000 (0.1%) plasma and 15 out of 12,841 (0.11%) serum samples tested positive for PRRSV antibodies through ELISA. Upon ELISA retesting, only the plasma sample, from one 8-day-old piglet remained positive. All sixteen previously PRRSV antibody-positive samples were tested through RT-PCR and found to be negative. The presence of false-positive or singleton reactors are quite expected. Thus, the use of different/alternative diagnostic tests is indicated for an efficient PRRSV detection. Taken together, our findings demonstrated no conclusive evidence of PRRSV infection in the tested pigs, highlighting the importance to reinforce the surveillance program to prevent the introduction and eventual dissemination of PRRSV in Brazil.

Porcine Circovirus 3a Field Strains in Free-Living Wild Boars in Paraná State, Brazil.

Porcine circovirus 3 (PCV-3) was identified in domestic pigs worldwide. Although PCV-3 has also been detected in wild boars, information regarding its circulation in this free-living animal species is scarce. To investigate PCV-3 occurrence in free-living wild boars in Brazil, 70 serum samples collected between January 2017 and June 2019 in Paraná state, Brazil were analyzed by PCR assay. Amplicons measuring 330 bp in length were amplified in seven (10.0%) of the serum samples and confirmed to be PCV3-specific by nucleotide (nt) sequencing. As the amplified products from the serum samples yielded only intermediate levels of viral DNA, lung samples from the seven PCR-positive wild boars were also evaluated by PCR. Of these samples, five lung samples were positive and provided high levels of viral DNA. The three lung samples that presented the highest levels of viral DNA were selected for amplification and sequencing of the whole PCV-3 genome. The three full-length sequences obtained were grouped in PCV-3 clade "a", and the sequences exhibited 100% nucleotide similarity among them. The PCV-3 field strains of this study showed nucleotide and amino acid similarities of 98.5-99.8% and 98.8-100%, respectively, with whole-genome PCV-3 sequences from around the world.

Distribution difference of colostrum-derived B and T cells subsets in gilts and sows.

Piglets are highly vulnerable to infections, but colostrum provides them with some protection. The function of colostrum components is unknown, as is if the amount and subsets of leukocytes in colostrum differ between gilts and sows. This study serially characterized leukocyte populations in colostrum for differential leukocyte counts. Differences in humoral and cellular composition of colostrum between 40 gilts and 40 sows (parities orders 3-4) from a commercial herd were examined. Flow cytometry is a useful tool to identify and quantify leukocyte subsets in sow colostrum. Overall, there were no (p ≥ 0.05) parity differences in total macrophages, granulocytes, and T and B cells. However, the sows' colostrum presented significantly higher (p ≤ 0.05) T lymphocyte subsets than gilts, such as central memory CD4+T cells, effector memory CD4+T cells, and central memory CD8+T cells. Among B-lymphocytes, percentages of SWC7+CD5+ cells were significantly higher in sow colostrum than in that of gilts. As expected, IgG concentrations were significantly higher in sows than in gilts. Colostrum from sows had significantly greater mitogenic activity than colostrum from gilts and this fact can be associated with the potential to accelerate the maturation of a newborn's gastrointestinal tract. Our findings suggest that parity order may be one among other factors influencing the cell population and, consequently, the immune adaptive response in piglets that induces neutralizing antibodies and cellular immune responses to antigens.

Investigation of hemotropic Mycoplasmas in fetuses and sows with reproductive failure.

Swine eperythrozoonosis or porcine hemoplasmosis is an infectious disease caused mainly by Mycoplasma suis and is distributed worldwide. This study investigated the occurrence of porcine hemothropic mycoplasmas (PHMs) in fetuses and sows with reproductive failure. Two hundred and seventy-six samples (80 sows' blood and 196 fetal tissue samples) from 27 farms with reproductive disorders were evaluated. The PHMs DNA was detected in 15 out of 80 (18.7%) sows but it was not detected in the fetuses. The bacterial load ranged from 1.32 × 102 to 2.61 × 105 copies/µL. From the 27 tested herds, 11 (40.7%) showed at least one positive sow per farm. The majority of the reproductive problems observed in PMHs positive sows were stillborn fetuses (46.7%) and stillborn associated with fetal mummification (26.7%). So, we evidenced that porcine hemoplasmas circulate among sows in Brazilian herds, however, its real impact on reproductive problems remains unknown.

Evaluation of two multiplex RT-PCR assays for detection and subtype differentiation of Brazilian swine influenza viruses.

Influenza A virus (IAV) subtypes H1N1, H1N2, and H3N2 are endemic in swine herds in most pork producing countries; however, the viruses circulating in different geographic regions are antigenically and genetically distinct. In this sense, the availability of a rapid diagnostic assay to detect locally adapted IAVs and discriminate the virus subtype in clinical samples from swine is extremely important for monitoring and control of the disease. This study describes the development and validation of a multiplex RT-PCR assay for detection and subtyping of IAV from pigs. The analytical and diagnostic specificity of the assays was 100% (94.3-100.0, CI 95%), and the limit of detection was 10-3 TCID50/mL. A total of 100 samples (IAV isolates and clinical specimens) were tested, and the virus subtype was determined for 80 samples (80%; 71.1-86.7, CI 95%). From these, 50% were H1N1, 22.5% were H1N2, and 7.5% were H3N2. Partial subtyping was determined for 8.75% samples (H1pdmNx and HxN2). Additionally, mixed infections with two virus subtypes (H1N2 + H3N2 and H1N1pdm + H1pdmN2; 2.5%) and reassortant viruses (H1pdmN2, 6.25%; and H1N1hu, 2.5%) were detected by the assay. A rapid detection of the most prevalent IAV subtypes and lineages in swine is provided by the assays developed here, improving the IAV diagnosis in Brazilian laboratories, and contributing to the IAV monitoring.

Genetic diversity and evolution of the emerging picornavirus Senecavirus A.

Senecavirus A (SVA) is an emerging picornavirus that causes vesicular disease (VD) in swine. The virus has been circulating in swine in the United Stated (USA) since at least 1988, however, since 2014 a marked increase in the number of SVA outbreaks has been observed in swine worldwide. The factors that led to the emergence of SVA remain unknown. Evolutionary changes that accumulated in the SVA genome over the years may have contributed to the recent increase in disease incidence. Here we compared full-genome sequences of historical SVA strains (identified before 2010) from the USA and global contemporary SVA strains (identified after 2011). The results from the genetic analysis revealed 6.32 % genetic divergence between historical and contemporary SVA isolates. Selection pressure analysis revealed that the SVA polyprotein is undergoing selection, with four amino acid (aa) residues located in the VP1 (aa 735), 2A (aa 941), 3C (aa 1547) and 3D (aa 1850) coding regions being under positive/diversifying selection. Several aa substitutions were observed in the structural proteins (VP1, VP2 and VP3) of contemporary SVA isolates when compared to historical SVA strains. Some of these aa substitutions led to changes in the surface electrostatic potential of the structural proteins. This work provides important insights into the molecular evolution and epidemiology of SVA.

Porcine parvovirus VP1/VP2 on a time series epitope mapping: exploring the effects of high hydrostatic pressure on the immune recognition of antigens.

Porcine parvovirus (PPV) is a DNA virus that causes reproductive failure in gilts and sows, resulting in embryonic and fetal losses worldwide. Epitope mapping of PPV is important for developing new vaccines. In this study, we used spot synthesis analysis for epitope mapping of the capsid proteins of PPV (NADL-2 strain) and correlated the findings with predictive data from immunoinformatics. The virus was exposed to three conditions prior to inoculation in pigs: native (untreated), high hydrostatic pressure (350 MPa for 1 h) at room temperature and high hydrostatic pressure (350 MPa for 1 h) at - 18 °C, and was compared with a commercial vaccine produced using inactivated PPV. The screening of serum samples detected 44 positive spots corresponding to 20 antigenic sites. Each type of inoculated antigen elicited a distinct epitope set. In silico prediction located linear and discontinuous epitopes in B cells that coincided with several epitopes detected in spot synthesis of sera from pigs that received different preparations of inoculum. The conditions tested elicited antibodies against the VP1/VP2 antigen that differed in relation to the response time and the profile of structurally available regions that were recognized.

One-step multiplex RT-qPCR for the detection and subtyping of influenza A virus in swine in Brazil.

Pandemic H1N1, human-like H1N2 and H3N2 influenza A (IAV) viruses are co-circulating in swine herds in Brazil. The genetic analysis of the Brazilian IAVs has shown that they are genetically distinct from viruses found in swine in other countries; therefore, an update of the diagnostic assays for IAV detection and subtyping is needed. This study describes the development and validation of a TaqMan based - one-step multiplex RT-qPCR to discriminate the hemagglutinin and neuraminidase genes of the three major IAV subtypes circulating in pigs in Brazil. The RT-qPCR assays presented 100% (95.7-100, CI 95%) of diagnostic sensitivity in the analysis of 85 IAVs, previously characterized by sequencing. The limits of detection ranged from 5.09 × 101 to 5.09 × 103 viral RNA copies/µL. For the analytical specificity, 73 pig samples collected during 2017 and 2018 were analyzed, resulting in the identification of the subtype in 74.0% (62.9-82.7, CI 95%) of samples. From these, 46.3% were H3N2, 33.3% were H1N1, 11.1% were H1N2 and 3.7% were HxN1. Mixed viral infections (3.7%) and reassortant viruses (1.9%) were also detected by the test. This multiplex RT-qPCR assay provides a fast and specific diagnostic tool for identification of different subtypes and lineages of IAV in pigs, contributing to the monitoring of influenza in swine.

Serological surveillance and factors associated with influenza A virus in backyard pigs in Southern Brazil.

Backyard pig populations are not monitored for influenza A virus (IAV) in Brazil and there are limited data about seroprevalence and risk factors in these populations. Our goal was to assess possible factors associated with IAV seroprevalence in backyard pig populations using an indirect ELISA protocol based on a recombinant nucleoprotein. Following the IAV screening using NP-ELISA, subtype-specific serology based on hemagglutination inhibition (HI) assay of the ELISA-positive pigs was conducted. The survey comprised a total of 1,667 sera samples collected in 2012 and 2014 in 479 holdings and the estimated seroprevalence was 5.3% (3.84%-7.33%) and 2.3% (1.34%-3.71%) in the respective years. In both years, H1N1pdm09 was the most prevalent subtype. The multivariable analysis showed main factors such as "age," "sex," "number of suckling pigs" and "neighbours raising pigs" that presented the greatest effect on IAV seroprevalence in these pig populations. These factors may be associated with the low biosecurity measures and management of backyard holdings. In addition, the low IAV seroprevalences found in these backyard pig populations could be related to a low number of animals in each pig holding and low animal movement/replacement that do not favour IAV transmission dynamics. This low frequency of H1N1pdm09 seropositive pigs could also be due to sporadic human-to-pig transmission of what is now a human seasonal influenza A virus; however, these factors should be explored in future studies. Herein, these results highlight the importance of IAV continued surveillance in backyard pig holdings, since it is poorly known which IAVs are circulating in these populations and the risk they could pose to public health and virus transmission to commercial farms.

Production and application of anti-nucleoprotein IgY antibodies for influenza A virus detection in swine.

Influenza A virus (IAV) causes an important respiratory disease in mammals and birds leading to concerns in animal production industry and public health. Usually, antibodies produced in mammals are employed in diagnostic tests. However, due to animal welfare concerns, technical advantages and the high cost of production, alternatives to the production of antibodies in mammals have been investigated. The aim of this study was to produce egg yolk immunoglobulin (IgY) in laying hens against a highly conserved protein (nucleoprotein- NP) of IAV and to evaluate the application of anti-NP IgY antibodies in virus detection by immunocytochemistry (ICC) and immunohistochemistry (IHC). Three laying hens of the White Leghorn line were inoculated seven times with a recombinant NP protein and their eggs collected seven days after the 3rd, 5th and 7th inoculations. Immunoglobulin Y antibodies were purified from egg yolk through precipitation with ammonium sulfate. The titers and specificity of the purified antibodies were determined by ELISA, western blotting, ICC and IHC. High levels of specific anti-NP antibodies were detected by ELISA after the 5th inoculation, reaching a peak after the 7th inoculation. The mean yield of total protein in yolk after the 7th inoculation was 13.5 mg/mL. The use of western blotting and ICC demonstrated that anti-NP IgY binds specifically to NP protein. Moreover, the use of anti-NP IgY antibody in ICC test revealed positive staining of MDCK cells infected with IAV of the three subtypes circulating in swine (H1N1, H1N2, and H3N2). However, no staining was observed in lung tissues through the IHC test. The data obtained showed that anti-NP IgY antibodies bound specifically to influenza virus NP protein, detecting the main virus subtypes circulating in swine, reinforcing their usefulness in the influenza diagnosis.

Structure analysis of capsid protein of Porcine circovirus type 2 from pigs with systemic disease

Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.(AU)

Structure analysis of capsid protein of Porcine circovirus type 2 from pigs with systemic disease

Abstract Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.

Low occurrence of pathogenic Yersinia enterocolitica in pig tonsils at slaughter in Southern Brazil.

Yersinia enterocolitica is a foodborne pathogen and pigs are the main reservoir of it in their tonsils. As Brazil is a large producer and exporter of pork meat and information regarding this pathogen is still quite scarce, this study aimed at evaluating the direct detection of Y. enterocolitica followed by pathogenic Y. enterocolitica (PYE) determination in tonsils of slaughtered pigs. For this purpose, 400 pig tonsils were collected from 15 farms in four federally certified slaughterhouses in Southern Brazil. Initially, samples were screened using conventional PCR targeting of the 16sRNA gene, followed by multiplex PCR (mPCR) in order to detect three virulence genes (ail, yadA, and virF) and quantitative real-time PCR (qPCR) for the detection of the ail gene. One hundred and one (25.2%) of the samples tested positive for the 16sRNA gene. However, a PYE was detected in one out of the 101 Y. enterocolitica positive samples. The three virulence genes were determined by mPCR and confirmed by partial DNA sequencing. Thus, a significant occurrence of Y. enterocolitica was observed in pig tonsils from federally inspected slaughterhouses in Brazil, although the presence of pathogenic strains was quite low.

Structure analysis of capsid protein of Porcine circovirus type 2 from pigs with systemic disease.

Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.