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Porcine circovirus disease (PCV) causes substantial economic losses in the pig industry, primarily from porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3). Novel vaccines are necessary to prevent and control PCV infections. PCV coat proteins are crucial for eliciting immunogenic proteins that induce the production of antibodies and immune responses. A vaccine platform utilizing Semliki Forest virus RNA replicons expressing vesicular stomatitis virus glycoprotein (VSV-G), was recently developed. This platform generates virus-like vesicles (VLVs) containing VSV-G exclusively, excluding other viral structural proteins. In our study, we developed a novel virus-like vesicle vaccine by constructing recombinant virus-like vesicles (rVLVs) that also express EGFP. These rVLVs were created using the RNA replicon of Venezuelan equine encephalomyelitis (VEEV) and New Jersey serotype VSV-G. The rVLVs underwent characterization and safety evaluation in vitro. Subsequently, rVLVs expressing PCV2d-Cap and PCV3-Cap proteins were constructed. Immunization of C57 mice with these rVLVs led to a significant increase in anti-porcine circovirus type 2 and type 3 capsid protein antibodies in mouse serum. Additionally, a cellular immune response was induced, as evidenced by high production of IFN-γ and IL-4 cytokines. Overall, this study demonstrates the feasibility of developing a novel porcine circovirus disease vaccine based on rVLVs.

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Seven novel porcine parvoviruses (nPPVs) (PPV2 through PPV8) have been described, although their pathogenicity and possible effects on porcine reproductive failure (PRF) are undefined. In this study, these nPPVs were assessed in gilts from Colombia; their coinfections with PPV1, PCV2, PCV3, PCV4, and PRRSV and an association between the nPPVs and the reproductive performance parameters (RPPs) in sows were determined. For this, 234 serum samples were collected from healthy gilts from 40 herds in five Colombian regions, and the viruses were detected via real-time PCR. The results confirmed the circulation of PPV2 through PPV7 in Colombia, with PPV3 (40%), PPV5 (20%), and PPV6 (17%) being the most frequent. Additionally, no PCV4 or PPV8 was detected. PPV2 to PPV7 were detected in concurrence with each other and with the primary PRF viruses, and these coinfections varied from double to sextuple coinfections. Additionally, the association between nPPVs and PRF primary viruses was statistically significant for the presence of PPV6 in PCV3-positive (p < 0.01) and PPV5 in PPRSV-positive (p < 0.05) gilts; conversely, there was a significant presence of PPV3 in both PCV2-negative (p < 0.01) and PRRSV-negative (p < 0.05) gilts. Regarding the RPPs, the crude association between virus detection (positive or negative) and a high or low RPP was only statistically significant for PCV3 and the farrowing rate (FR), indicating that the crude odds of a low FR were 94% lower in herds with PCV3-positive gilts. This finding means that the detection of PCV3 in gilts (PCV3-positive by PCR) is associated with a higher FR in the farm or that these farms (with positive gilts) have lower odds (OR 0.06, p-value 0.0043) of a low FR. Additionally, a low FR tended to be associated with the detection of PPV4 and PPV5 (p-value < 0.20). This study is important for establishing the possible participation of nPPVs in PRF.

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Porcine circovirus type 3 (PCV3) is a nonenveloped virus of the Circoviridae family. This virus has been identified in pigs of different ages and pigs with several clinical manifestations of the disease or even in apparently healthy pigs. While PCV3 was first reported in 2015, several retrospective studies have reported the virus before that year. The earliest report indicates that PCV3 has been circulated in swine farms since 1996. In this study, we evaluated the presence of PCV3 in samples collected in Mexico in 2008, 2015, 2020, and 2021. This study assessed PCV3 DNA by qPCR and antibodies against CAP protein by indirect ELISA. The results showed that PCV3 (DNA and anti-CAP antibodies) was detected in the samples collected from 2008 to 2021. The highest prevalence was in 2008 (100%), and the lowest was in 2015 (negative). Genetic analysis of ORF2 showed that the virus identified belonged to genotype a, as most of the viruses identified thus far. PCV3 was detected in samples from piglets with respiratory signs and growth retardation, sows with reproductive failure, or asymptomatic piglets and sows. Pigs with respiratory signs, growth retardation, or reproductive failure had a higher prevalence of antibodies and qPCR-positive samples. In conclusion, this study showed that PCV3 has been circulating in Mexico since 2008 and that PCV3 DNA and antibodies were more prevalent in samples from pigs with clinical manifestations of diseases.

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[This corrects the article DOI: 10.3389/fvets.2023.1174718.].

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This study aimed to evaluate the relationship between porcine circovirus type 3 (PCV3) viral load and histopathological findings in perinatal piglet tissues and to develop an immunohistochemical method for detecting the virus in lesions. The quantitative polymerase chain reaction (qPCR) cycle threshold (Ct) when amplifying PCV3 DNA and the area of perivascular inflammatory infiltrates in different organs [central nervous system (CNS), lung, heart, liver, spleen, and lymph nodes] were compared. To develop an immunohistochemistry technique, rabbit sera were produced against PCV3-capsid protein peptides selected using bioinformatic analyses. The assay was initially implemented using a tissue sample previously tested using qPCR and in situ hybridization to optimize the procedure and reagent dilutions. To evaluate immunohistochemistry performance, tissue samples from another 17 cases were analyzed using standardized parameters. The most common microscopic lesion was multisystemic periarteritis, with associated vasculitis, as the mesenteric vascular plexus is one of the most affected organs. Other tissues, such as the heart, lung, CNS, and skeletal muscle, were also affected. Comparison of the Ct values for different tissues showed no significant difference, except in lymphoid organs (spleen and lymph nodes), which had significantly higher viral loads than the CNS tissues. There was no correlation between Ct values and perivascular inflammatory infiltrates. PCV3 immunohistochemistry revealed granular immunolabeling, mainly in the cytoplasm of cells in the vascular mesenteric plexus, heart, lung, kidney, and spleen.

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Porcine circovirus type 3 (PCV3) has been widely detected worldwide in healthy and sick pigs. Recently its association with clinical disease and reproductive failure has been proven through the detection of intralesional viral mRNA in affected pigs. This study aims to describe the occurrence of PCV3-associated reproductive failure (abortions) in sow herds in southern Brazil. Eleven fetuses from five different litters from two herds were analyzed. These herds reported an increase in the rate of late-gestation abortions, stillbirths, and the percentage of mummified piglets. At gross examination, six of the fetuses had large caudally rotated ears and one fetus was mummified. Microscopically, multisystemic vasculitis, lymphocytic interstitial pneumonia, myocarditis, and encephalitis were observed. These six fetuses with gross and histological lesions were positive in qPCR analysis for PCV3, and PCV3 transcription was shown through in situ hybridization (ISH-RNA) within the histologic lesions. Samples from all 11 fetuses tested negative in PCR exam for Porcine Circovirus type 1 and 2, Porcine Reproductive and Respiratory Syndrome, Porcine Parvovirus, and Atypical Porcine Pestivirus. Furthermore, based on the ORF2 analysis, the PCV3a clade was identified. This is the first report of PCV3a-associated reproductive failure in pig herds in South America.

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Four genotypes of circovirus have been recognized in swine, with PCV2 and PCV3 being the most associated with clinical manifestations, while PCV4 does not have a defined disease. In addition, PCV2 is associated with different syndromes grouped as diseases associated with porcine circovirus (PCVAD), while PCV3 causes systemic and reproductive diseases. In the present study, we retrospectively detected PCV2, PCV3, and PCV4 in Colombia during two periods: A (2015-2016) and B (2018-2019). During period A, we evaluated stool pools from the 32 Colombian provinces, finding a higher prevalence of PCV3 compared to PCV2 as well as PCV2/PCV3 co-infection. Furthermore, we determined that PCV3 had been circulating since 2015 in Colombia. Regarding period B, we evaluated sera pools and tissues from abortions and stillborn piglets from the five provinces with the highest pig production. The highest prevalence found was for PCV3 in tissues followed by sera pools, while PCV2 was lower and only in sera pools. In addition, PCV2/PCV3 co-infection in sera pools was also found for this period. The complete genome sequences of PCV3 and PCV3-ORF2 placed the Colombian isolates within clade 1 as the majority in the world. For PCV2, the predominant genotype currently in Colombia is PCV2d. Likewise, in some PCV3-ORF2 sequences, a mutation (A24V) was found at the level of the Cap protein, which could be involved in PCV3 immunogenic recognition. Regarding PCV4, retrospective surveillance showed that there is no evidence of the presence of this virus in Colombia.

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Porcine circovirus type 3 (PCV3) is widely distributed worldwide, and its association with clinical disease in pigs has been studied in recent years. This study describes a novel PCV3-associated clinical disease in piglets from Brazil. Since September 2020, we received 48 piglets with large caudally rotated ears, weakness, and dyspnea. Most piglets were from gilts and died 1-5 days after birth. Two piglets that presented similar clinical signs and survived until 35-60 days had a marked decrease in growth rate. At post-mortem examination, the lungs did not collapse due to marked interlobular edema. Microscopically, the main feature was multisystemic vasculitis characterized by lymphocytes and plasma cells infiltrating and disrupting the wall of vessels, lymphohistiocytic interstitial pneumonia, myocarditis, and encephalitis. Viral replication was confirmed in these lesions through in situ hybridization (ISH-RNA). Seventeen cases were positive for PCV3 in PCR analysis, and all samples tested negative for porcine circovirus (PCV1, and PCV2); porcine parvovirus (PPV1, 2, 5, and 6); atypical porcine pestivirus (APPV); porcine reproductive and respiratory syndrome (PRRSV); and ovine herpesvirus-2 (OvHV-2). Phylogenetic analysis of the ORF2 sequence from five different pig farms showed that the PCV3a clade is circulating among Brazil's swineherds and causing neonatal piglet losses. This is the first report of PCV3a-associated disease in neonatal pigs from farms in Brazil.

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Porcine circovirus 2 (PCV2) has a considerable economic impact on the pork industry worldwide for more than two decades. In 2016, a new circovirus, porcine circovirus 3 (PCV3), was described; since then, it has been reported to be associated with diseased or even in clinically healthy swine in several countries. Considering the importance of wild boars as reservoirs of swine pathogens and the extensive distribution of these animals in Rio Grande do Sul and throughout the national territory, we searched for PCV2 and PCV3 in twenty-six wild boars coupled with necropsy and histologic examination of the sampled animals. Using PCR, 182 tissue samples were analyzed, including the heart, kidneys, liver, lung, lymph nodes, spleen, and tonsils. PCV2 and PCV3 were detected in 57.7% (15/26) and 15.4% (4/26) of wild boars, respectively. Furthermore, co-infection with PCV2 and PCV3 was detected in one of these animals, with PCV2 or PCV3 DNA detection in multiple organs. Histological examination showed mild to moderate and multifocal lymphoplasmacytic interstitial nephritis distributed randomly throughout the renal cortex, apparently unrelated to PCV2 or PCV3 detection. The wild boar population in Brazil is extensive, indicating the presence of a larger number of swine pathogen hosts. In the present study, more than half of the wild boars harbored PCV2; and although less frequently, PCV3 was also detected. Therefore, free-living wild boars can serve as reservoirs of swine circoviruses in southern Brazil.

O circovírus suíno 2 (PCV2) tem causado impacto econômico na indústria suína em todo o mundo por mais de duas décadas. Em 2016, um novo circovírus foi descrito - circovírus suíno 3 (PCV3) - e desde então tem sido relatado em vários países associado a doenças ou mesmo suínos saudáveis. Diante da importância dos javalis como reservatórios de patógenos suínos, e da ampla distribuição desses animais no Rio Grande do Sul e em todo o território nacional, foi realizada pesquisa de PCV2 e PCV3 em vinte e seis javalis (10 fêmeas e 16 machos). Necropsia e exame histológico foram realizados. Utilizando PCR, foram analisadas 182 amostras de tecidos incluindo: coração, rins, fígado, pulmão, linfonodos, baço e tonsila. PCV2 e PCV3 foram detectados por PCR em 57,7% (15/26) e 15,4% (4/26) dos javalis, respectivamente. Um destes animais estava co-infectado por PCV2 e PCV3. O DNA do PCV2 ou PCV3 foi detectado em multiplos órgãos. No exame histológico foi observada nefrite intersticial linfoplasmocitária multifocal leve a moderada, distribuída aleatoriamente pelo córtex renal, aparentemente sem relação com a detecção de DNA viral. A população de javalis no Brasil é extensa, resultando em maior número de hospedeiros para patógenos de suínos. No presente estudo, mais da metade dos javalis capturados abrigavam PCV2 e, embora menos frequente, PCV3 também foi detectado. Os javalis de vida livre podem servir como reservatórios de circovírus suínos no sul do Brasil.

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Porcine circovirus type 3 (PCV3) has been recently described as a potential cause of abortions and systemic vasculitis in pigs. Although the virus has been detected by real-time PCR in several porcine tissues from countries worldwide, PCV3-associated diseases have not been satisfactorily clarified. The objective of this study was to investigate the association between the presence of PCV3 mRNA detected by in situ hybridization (ISH) within histological lesions and PCV3 DNA detected by real-time PCR in naturally infected pigs. A total of 25 PCV3 PCR-positive cases were analyzed. Formalin-fixed tissues from these cases were evaluated for histologic lesions and for ISH-RNA positive signals for PCV3. The most frequent tissue type with histopathologic lesions was heart, 76.2%, with lymphoplasmacytic myocarditis and epicarditis as the most frequent lesions observed. Lymphoplasmacytic interstitial pneumonia was also a frequent finding, 47.6%. There were also lesions in kidney, liver, spleen and lymph nodes. PCV3-ISH-RNA positive signals were mostly observed in association with lymphoplasmacytic inflammatory infiltrate in various tissues, including arteries. Based on our results, the minimum set of specimens to be submitted for histopathology and mRNA in situ hybridization to confirm or exclude a diagnosis of PCV3 are heart, lung and lymphoid tissues (i.e., spleen and lymph nodes), especially for differential diagnosis related with PCV2-associated diseases.

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BACKGROUND: PCV3 is a member of the Circovirus family, associated with disease and mortality in pigs. It is not clear whether PCV3 putatively causes clinical symptoms and disease. In the present case, we reported a gilt infected with PCV3 associated with reproductive failures, vertical transmission, tissue lesions, viral replication by in situ hybridization, and the hypothesis that some strains of PCV3 clade one are associated with reproductive failures at the field level. CASE PRESENTATION: In May 2019, a pig farm in Colombia reported increased reproductive failures, and the presence of PCV3 in gilts and sows was established in a single form or coinfections, mainly with PCV2 and PPV7. Ten sows with a single infection with PCV3 were found, and one gilt with a pre-farrowing serum viral load above 103 was studied. This gilt was followed up during the pre-farrowing, farrowing period and on her litter for 6 weeks. During dystocic farrowing, a mummy and ten piglets were released, including two weak-born piglets. The highest viral loads for PCV3 were found in the mummy and the placenta. In the weak-born piglets, there were viral loads both in serum and in tissues, mainly in the mesenteric ganglia and lung. Replication of PCV3 in these tissues was demonstrated by in situ hybridizations. PCV3 was also found in the precolostrum sera of piglets and colostrum, showing vertical transmission. The viral load in piglets decreased gradually until week six of life. The viral genome's complete sequencing was made from the mummy, and its analysis classified it as PCV3 clade one. CONCLUSIONS: This report confirms that PCV3 can cause disease at the field level, and putatively, in this case, we find the generation of reproductive failures. The ability of PCV3 to cause disease as a putative pathogen may be associated with the viral load present in the pig and the strain that is affecting the farm. For this case, we found that viral loads above 103 (4.93 log genomic copies / mL) in the gilt were associated with clinical manifestation and that some PCV3 strains belonging to clade one are more associated with the reproductive presentation.

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The clinical implications of recently discovered porcine circovirus 3 (PCV3) infections are still unknown. The potential role of this emerging virus in reproductive loss in swine has been described. Herein, we report a high prevalence of PCV3 in mummified fetuses from sows maintained in modern farms in Rio Grande do Sul, Santa Catarina, Paraná, Goiás, and Mato Grosso do Sul states, Brazil. For this analysis, 276 mummified fetuses from 11 commercial swine farms were included. The presence of PCV3 DNA was confirmed using PCR, and the complete sequence of five different viral strains was obtained. Sequences of PCV3 genomes available on GenBank were then used for phylogenetic tree construction. Of the 276 mummified fetuses examined, 270 (nearly 97%) were positive for PCV3. In 93.1% of the fetuses, co-infections with at least one of the following agents were identified: porcine parvovirus (PPV), porcine circovirus 2 (PCV2) and Leptospira spp. Twelve fetuses were positive for PCV3 alone. The amino acid sequence of the capsid gene for the five viral strains shared 98-100% homology among them. Analysis of the DNA sequence indicates that the viruses identified in this study belong to the PCV3a1 subgroup. In summary, PCV3 DNA was detected in mummified fetuses at a surprisingly high rate. The role of PCV3 in porcine circovirus-associated disease (PCVAD) is still uncertain. However, considering that PCV3 has been detected in a variety of conditions, even in healthy animals, the present results confirm the need to investigate PCV3 as a causative agent of fetal mummification in swine.

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Porcine circovirus 3 (PCV3) is an emerging virus that was first identified in the United States in 2016. Since its first detection, PCV3 has already been found in America, Asia, and Europe. Although PCV3 has already been described in Brazil, knowledge of its detection and sequence variation before 2016 is limited, as well as its distribution in the main swine producing regions of Brazil. In this study, 67 porcine clinical samples collected from nine states in Brazil between 2006 and 2007 were analyzed for PCV3 infection by PCR. Results showed that 47.8% of the samples were PCV3 positive, across all nine states. Of the PCV3-positive samples, 37.5% were also positive for PCV2. Interestingly, no clinical signs were associated with samples that were detected singularly with PCV3 infection. Moreover, the positive PCV3 rate in healthy pigs was higher (29.8%) than that found in unhealthy pigs (17.9%), suggesting that most pigs could live with PCV3 infection without any clinical sign in the analyzed samples. Nucleotide sequence analysis showed that PCV3 strains obtained in this study shared 94.44% to 99.83% sequence identity at the open reading frame 2 (ORF2) gene level with available strains from different countries. PCV3 Brazilian sequences collected in 2006 and 2007 shared 97.94% to 99.62% identity with the strains obtained in 2016. The results of neutrality and selective pressure tests indicated that the PCV3 Cap protein seems unable to tolerate high levels of variation on its sequence. Phylogenetic analysis grouped the Brazilian strains in PCV3a and PCV3b genotypes clusters, both including strains collected in America, Asia, and Europe. Taking the results together, multiple events of introduction of PCV3 may have occurred in Brazil, and Brazilian PCV3 strains may show genetic stability over the past 10 years.

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Porcine circovirus 3 (PCV3) was recently discovered and is a new species of the genus circovirus. Clinically, it is associated with absence of symptoms or with different clinical syndromes. It has been reported in different countries of America, Europe and Asia. Last year, in Colombia, some farms have reported symptoms similar to those caused by PCV2. Samples were taken from two farms located in the centre of the country, and the presence of PCV3 was determined by PCR in two samples, one from a pool of sera and another from mesenteric lymph node. The strains were fully sequenced (GenBank accession numbers MH327784 and MH327785) and classified into subgroups a1 and a2. According to this classification and its analysis, strain a2 is located within the group called "Linker" that may be evolving towards group "b". In addition to the above, the two Colombian strains were compared with 104 strains reported in the GenBank database. The phylogenetic tree obtained grouped according to the classification of subgroups a1, a2, b1 and b2. It was found that subgroups a1 and a2 were well grouped when comparing whole genomes, but the same was not observed with the strains of group "b". In the latter, no subgroups were evidenced when comparing complete genomes. It is suggested that a new classification of PCV3 subgroups should be proposed, based on whole genome sequences. This is the first report of PCV3 in Colombia and its complete genome sequence.

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Porcine circovirus 3 (PCV3) is an emerging virus that was identified in the United States in 2016. Since its first identification, PCV3 has been identified in Brazil, China, United States, Poland, and Republic of Korea. In this study, we used molecular phylogenetic analysis of available sequences to address questions surrounding the emergence of PCV3 in porcine world industry. Our data indicate that PCV3 did not emerge through recombination events among currently known circoviruses and that its speciation is not a recent evolutionary event. The most common recent ancestor analysis suggests that PCV3 lineages have emerged over the past 50 years. PCV3 is not genetically closely related with other Porcine circovirus and it has been evolving undetected for some time in swine and probably in bovine population. We also found groups of genetically related isolates of PCV3 originated from different countries that may be associated with dispersal routes, suggesting that PCV3 has already been circulating in pig-producing countries for some time before its first detection.

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