Exploratory Study of the Frequency of Detection and Tissue Distribution of Porcine Circovirus 3 (PCV-3) in Pig Fetuses at Different Gestational Ages.

Porcine circovirus 3 (PCV-3) has been associated with several pig diseases. Despite the pathogenicity of this virus has not been completely clarified, reproductive disorders are consistently associated with its infection. The aim of the present work was to analyze the presence of PCV-3 DNA in tissues from pig fetuses from different gestational timepoints. The fetuses were obtained either from farms with no reproductive problems (NRP, n = 249; all of them from the last third of gestation) or from a slaughterhouse (S, n = 51; 49 of the second-third of gestation and 2 from the third one). Tissues collected included brain, heart, lung, kidney, and/or spleen. Overall, the frequency of detection of PCV-3 was significantly higher in fetuses from the last third of the gestation (69/251, 27.5%) when compared to those from the second-third (5/49, 10.2%), although the viral loads were not significantly different. Moreover, the frequency of detection in NRP fetuses (69/249, 27.7%) was significantly higher than in S ones (5/51, 9.8%). Furthermore, PCV-3 DNA was detected in all tissue types analyzed. In conclusion, the present study demonstrates a higher frequency of PCV-3 DNA detection in fetuses from late periods of the gestation and highlights wide organ distributions of the virus in pig fetuses.

Multisystemic lymphoplasmacytic inflammation associated with PCV-3 in wasting pigs.

Porcine circovirus 3 (PCV-3) has been detected in diseased and healthy pigs of different ages. Several reports have associated the agent with reproductive failure and mummified and stillborn piglets. One report from North America has proposed a consistent potential association with postweaning disorders. Thus, the present case report aimed to describe the histopathological lesions and their association with the presence of PCV-3 genome in postweaning pigs showing growth-retardation and thrown-back ears. All affected animals displayed multi-organic lymphoplasmacytic periarteritis, lymphocytic myocarditis and/or lymphoplasmacytic meningoencephalitis. PCV-3 genetic material was detected by in situ hybridization within the lesions and confirmed by PCV-3 real-time quantitative PCR detection in tissues. This study represents the first report of PCV-3 associated with clinical disease in postweaning pigs in Europe.

Porcine circovirus 3 (PCV-3) as a causal agent of disease in swine and a proposal of PCV-3 associated disease case definition.

Porcine circovirus 3 (PCV-3) was discovered in 2015 using next-generation sequencing (NGS) methods. Since then, the virus has been detected worldwide in pigs displaying several clinical-pathological outcomes as well as in healthy animals. The objective of this review is to critically discuss the evidence existing so far regarding PCV-3 as a swine pathogen. In fact, a significant number of publications claim PCV-3 as a disease causal infectious agent, but very few of them have shown strong evidence of such potential causality. The most convincing proofs of disease association are those that demonstrate a clinical picture linked to multisystemic lymphoplasmacytic to lymphohistiocytic perivascular inflammation and presence of viral nucleic acid within these lesions. Based on these evidence, individual case definitions for PCV-3-reproductive disease and PCV-3-systemic disease are proposed to standardize diagnostic criteria for PCV-3-associated diseases. However, the real frequency of these clinical-pathological conditions linked to the novel virus is unknown, and the most frequent outcome of PCV-3 infection is likely subclinical based on its worlwide distribution.

Porcine Circovirus 3 Detection in Aborted Fetuses and Stillborn Piglets from Swine Reproductive Failure Cases.

Porcine circovirus 3 (PCV-3) has been widely detected in healthy and diseased pigs; among different pathologic conditions, the strongest evidence of association comes from reproductive disease cases. However, simple viral detection does not imply the causality of the clinical conditions. Detection of PCV-3 within lesions may provide stronger evidence of causality. Thus, this study aimed to assess the frequency of PCV-3 detection in tissues from fetuses/stillborn piglets in cases of reproductive problems in domestic swine, as well as the histopathologic assessment of fetal tissues. Fetuses or stillborn piglets from 53 cases of reproductive failure were collected and analyzed by PCV-3 qPCR. The presence of porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV-2), and porcine parvovirus 1 (PPV1) was also checked. PCV-3 qPCR positive samples with a high viral load were tested by PCV-3 in situ hybridization (ISH), sequenced, and phylogenetically analyzed. PCV-3 DNA was detected in 18/53 (33.9%) reproductive failure cases and in 16 of them PCV-3 was the only pathogen found. PCV-2 DNA was found in 5/53 (9.4%), PRRSV RNA in 4/53 (7.5%) and PPV1 was not detected. Four out of the six PCV-3 qPCR-positive cases with Ct value <30 were positive when tested by ISH. In these samples, PCV-3 was detected within mild histopathologic lesions, such as arteritis and periarteritis in multiple tissues. The present work emphasizes the need to include PCV-3 as a potential causative agent of reproductive failure in swine.

Porcine lymphotropic herpesvirus DNA detection in multiple organs of pigs in Brazil.

We investigated the porcine lymphotropic herpesvirus (PLHV) DNA presence in multiple organs of pigs. Biological samples (n = 136) included tissue fragments of the central nervous system, heart, kidney, liver, lungs, spleen, urinary bladder, and urine. Sixty-eight (50%) organs were PLHV DNA-positive. None of the urine samples were detected with the virus genome. Although the presence of the PLHV DNA in the urinary bladder and kidney has been detected, it was not possible to show whether urine can be considered an effective route of virus shedding. This study warns to the risk of PLHV zoonotic transmission by xenotransplantation of tissues of porcine origin.

Frequency of Detection and Phylogenetic Analysis of Porcine circovirus 3 (PCV-3) in Healthy Primiparous and Multiparous Sows and Their Mummified Fetuses and Stillborn.

Porcine circovirus 3 (PCV-3) has been suggested as a putative causal agent of swine reproductive disease. A number of different studies have pointed out this association, but there is still a lack of information regarding the normal rates of PCV-3 infection in farms with normal reproductive parameters. The objective of the present study was to assess the frequency of PCV-3 detection in primiparous and multiparous sows and in tissues from their respective fetuses from farms with average reproductive parameters. Sera from 57 primiparous and 64 multiparous sows from 3 different farms were collected at two time points. Brain and lung tissues from 49 mummies and 206 stillborn were collected at farrowing. Samples were tested by PCR, and when positive, quantified by quantitative PCR. Thirty-nine complete genomes were obtained and phylogenetically analyzed. All sera from multiparous sows were negative, while 19/57 (33.3%) primiparous sows were PCV-3 PCR positive. From the 255 tested fetuses, 86 (33.7%) had at least one tissue positive to PCV-3. The frequency of detection in fetuses from primiparous sows (73/91, 80.2%) was significantly higher than those from multiparous ones (13/164, 7.9%). It can be concluded that PCV-3 is able to cause intrauterine infections in absence of overt reproductive disorders.

Detection and genotyping of Porcine circovirus 2 (PCV-2) and detection of Porcine circovirus 3 (PCV-3) in sera from fattening pigs of different European countries.

PCV-2 is considered one of the most economically important viral agents in swine worldwide. Recently, PCV-3 has been found in pigs affected by different disorders and in healthy animals. The objective of this epidemiological work was to describe the frequency of detection of PCV-2 and PCV-3 in pig farms of 9 European countries. Moreover, a second aim was to assess the most frequent PCV-2 genotypes found in the studied farms. Sera from 5 to 10 pigs per farm were collected from 2 to 11 farms per studied country. A total of 624 sera of fattening pigs (10-25 week old) from 64 farms from Spain (n = 11), Belgium (n = 10), France (n = 8), Germany (n = 8), Italy (n = 7), Denmark (n = 8), the Netherlands (n = 5), Ireland (n = 5) and Sweden (n = 2) were analysed by conventional PCR. In addition, one or two PCV-2-positive samples per farm were genotyped by sequencing the ORF2 gene. PCV-3 PCR-positive samples with relatively low Ct values were also sequenced and phylogenetically analysed. PCV-2 DNA was detected in pig sera from all European tested countries, but Sweden. A total of 132 out of 624 (21%) sera were positive for PCV-2 PCR, corresponding to 30 out of the 64 (47%) tested farms. PCV-3 DNA was detected in 52 out of 624 (8%) sera, corresponding also to 30 out of the 64 (47%) studied farms from all tested countries. A total of 48 PCV-2 PCR-positive samples were successfully sequenced and genotyped, being PCV-2d the most frequently genotype found (n = 28), followed by PCV-2b (n = 11) and PCV-2a (n = 9). These results pointed out PCV-2d as the most prevalent genotype currently in Europe. The PCV-3 phylogenetic analysis showed high identity (>98%) among sequences from all the analysed countries. The relatively low co-infection (3%), likely suggest an independent circulation patterns of PCV-2 and PCV-3.

Detection of Porcine Circovirus 3 in Wildlife Species in Spain.

Porcine circovirus 3 (PCV-3) is the third member of the family Circoviridae, genus Circovirus, able to infect swine. A high prevalence of viral DNA has been recorded in wild boars. Recently, PCV-3 DNA was identified in Italian wild ruminants. Based on these previous results, this study assessed the frequency of PCV-3 DNA detection in free-ranging ruminants and Lagomorpha species in Spain. In addition, the genetic characterization of the PCV-3 PCR-positive samples was performed. A total of 801 serum samples, including red deer (Cervus elaphus, [CE]; n = 108), roe deer (Capreolus capreolus, [CC]; n = 87), Pyrenean chamois (Rupicapra pyrenaica, [RP]; n = 133), Iberian ibex (Capra pyrenaica, [CP]; n = 92), mouflon (Ovis aries, [OA]; n = 91), fallow deer (Dama dama, [DD]; n = 104), European rabbit (Oryctolagus cuniculus, [OC]; n = 101), and European hare (Lepus europaeus, [LE]; n = 85) from Catalonia (northeast Spain) were tested by conventional polymerase chain reaction (PCR) and, when positive, sequenced. Overall, PCV-3 DNA was found in three out of 801 analyzed sera (0.37%) corresponding to one red deer (1/108, 0.9%), one mouflon (1/91, 1.1%), and one fallow deer (1/104, 0.96%). None of the samples collected from Lagomorpha species resulted PCR positive. The partial genome sequences detected in positive samples displayed high identity with some PCV-3 sequences detected in wild boars and domestic pigs (99.7% and 100%, respectively). In conclusion, the present study indicated that free-ranging ruminant and Lagomorpha species are not relevant in the epidemiology of PCV-3 in Spain.

Similar frequency of Porcine circovirus 3 (PCV-3) detection in serum samples of pigs affected by digestive or respiratory disorders and age-matched clinically healthy pigs.

Porcine circovirus 3 (PCV-3) has been identified in pigs affected by different disease conditions, although its pathogenicity remains unclear. The objective of the present study was to assess the frequency of PCV-3 infection in serum samples from animals suffering from post-weaning respiratory or digestive disorders as well as in healthy animals. A total of 315 swine serum samples were analysed for PCV-3 DNA detection by conventional PCR; positive samples were further assayed with a quantitative PCR and partially sequenced. Sera were obtained from 4 week- to 4 month-old pigs clinically diagnosed with respiratory (n = 129) or digestive (n = 126) disorders. Serum samples of age-matched healthy animals (n = 60) served as negative control. Pigs with clinical respiratory signs had a wide variety of pulmonary lesions including suppurative bronchopneumonia, interstitial pneumonia, fibrinous-necrotizing pneumonia and/or pleuritis. Animals with enteric signs displayed histopathological findings like villus atrophy and fusion, catarrhal enteritis and/or catarrhal colitis. Overall, PCV-3 DNA was detected in 19 out of 315 analysed samples (6.0%). Among the diseased animals, PCV-3 was found in 6.2% (8 out of 129) and 5.6% (7 out of 126) of pigs with respiratory and digestive disorders, respectively. The frequency of PCV-3 PCR positive samples among healthy pigs was 6.7% (4 out of 60). No apparent association was observed between PCR positive cases and any type of histopathological lesion. The phylogenetic analysis of the partial genome sequences obtained showed high identity among viruses from the three groups of animals studied. In conclusion, PCV-3 was present in the serum of diseased and healthy pigs to similar percentages, suggesting that this virus does not seem to be causally associated with respiratory or enteric disorders.

Detection of Equid gammaherpesvirus 2 and 5 DNA in the upper respiratory tract of asymptomatic horses from Southern Brazil.

Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) are members of the Herpesviridae family and have been reported in horse populations worldwide. This study aimed to evaluate the presence of herpesvirus DNA in the upper respiratory tract of horses. Twenty-six nasal swabs were collected from asymptomatic adult horses of two different horse farms (A, n = 18; B, n = 8), both located in Southern Brazil. The EHV-1, EHV-2, EHV-4, and EHV-5 DNA analyses were performed using nested PCR assays targeting the glycoprotein B gene. Four (15.3%) and 12 (46.1%) of the 26 nasal swab samples were positive for the EHV-2 and EHV-5, respectively. Four (15.3%) horses were detected with both viruses simultaneously. DNA of EHV-2 and EHV-5 in both single and mixed infections was identified in horses from both herds. All swab samples were negative for EHV-1 and EHV-4. This study reports the first detection of EHV-2 and EHV-5 in the upper respiratory tracts of horses in Brazil. The high detection rate of EHV-2 and EHV-5 in asymptomatic adult horses demonstrates that these gammaherpesviruses are circulating in Brazil.

Molecular analysis of the full-length F gene of Brazilian strains of canine distemper virus shows lineage co-circulation and variability between field and vaccine strains.

Canine distemper is a highly contagious systemic viral disease, with worldwide distribution that affects a wide variety of terrestrial carnivores. This study characterized full-length fusion (F) genes from 15 Brazilian wild-type canine distemper virus (CDV) strains collected between 2003-2004 (n = 6) and 2013-2016 (n = 9). Using deduced amino acid (aa) sequence analysis, 14 strains were classified into Europe 1/South America 1 (EU1/SA1) lineage, with a temporal clustering into past (2003-2004) and contemporary (2013-2016) strains. One strain clustered to Rockborn-like lineage, showing high similarity (98.5%) with the Rockborn vaccine strain. In analyzed strains, the fusion protein signal-peptide (Fsp) coding region was highly variable at the aa level (67.4%-96.2%). The Brazilian strains were more Fsp-divergent from the North America 1 (NA1) strains (24.5%-36.3%) than from the Rockborn (11.2%-14.9%) vaccine strain. Seventeen cysteine residues in the full-length F gene and four non-conserved glycosylation sites in the Fsp region were detected. The results reveal that past and contemporary CDV strains are currently co-circulating. This first analysis of full-length F genes from Brazilian wild-type CDV strains contributes to knowledge of molecular epidemiology of CDV viral infection and evolution.

A ten years (2007-2016) retrospective serological survey for Seneca Valley virus infection in major pig producing states of Brazil.

Seneca Valley virus (SVV) is the etiological agent of vesicular disease in pigs, clinically indistinguishable of classical viral vesicular infections, including foot-and-mouth disease. The first outbreaks of SVV infection in Brazil were reported in 2014. However, it was not known whether the virus was circulating in Brazilian pig herds before this year. This study is a retrospective serological investigation of porcine health status to SVV in Brazil. Serum samples (n = 594) were grouped in before (2007-2013, n = 347) and after (2014-2016, n = 247) SVV outbreaks in Brazil. Twenty-three pig herds were analyzed, of which 19 and 4 were sampled before and after the beginning of SVV outbreaks, respectively. Two herds sampled after 2014 presented animals with SVV-associated clinical manifestations, while the other two housed asymptomatic pigs. Anti-SVV antibodies were evaluated by virus neutralization test. The results demonstrated that pig herds of different Brazilian geographical regions and distinct pig categories were negative to anti-SVV antibodies in sera obtained before 2014. Antibodies to SVV were detected only in serum samples obtained after 2014, particularly in herds with the presence of pigs with SVV-clinical signs. These results present robust serological evidence that the SVV was not present in the major Brazilian pig producing regions prior to 2014.