Molecular detection and clinical aspects of porcine circovirus 3 infection in pigs from Brazil / [Detecção molecular e aspectos clínicos da infecção por circovírus suíno 3 em suínos do Brasil]

Porcine circovirus 3 (PCV-3) DNA has been detected in serum samples from apparently healthy pigs as well as pigs with different clinical conditions. Molecular detection of PCV-3 was observed in swine serum samples from Southeastern - Brazil using a nested PCR designed specifically for this study. The epidemiology and clinical aspects of PCV-3 infection were evaluated. The samples originated from 154 pigs of both genders from different production phases and with different clinical presentations, sampled from 31 pig farms visited between 2013 and 2018. In this study, PCV-3 was detected in 26.7% of samples from all populations across varying ages. Statistical association (P=0.0285) was observed only between animals with respiratory signs and PCV-3; no PCV-3-positive animal had diarrhea. No statistical association was observed between PCV-3 and age, or gender of the pigs. Because PCV-3 is a newly discovered virus, there is very little information about its epidemiology. We hope that these data can help in future studies investigating PCV-3 epidemiology.(AU)

O DNA do circovírus suíno 3 (PCV-3) foi detectado em amostras de soro de suínos aparentemente saudáveis, bem como em suínos com diferentes condições clínicas. A detecção molecular do PCV-3 foi observada em amostras de soro de suínos da região Sudeste do Brasil, com uma nested PCR desenhada especificamente para este estudo. A epidemiologia e os aspectos clínicos da infecção por PCV-3 foram avaliados. As amostras foram coletadas de 154 suínos de ambos os sexos, de diferentes fases de produção e com diferentes sinais clínicos. Os animais pertenciam a 31 granjas visitadas entre 2013 e 2018. Neste estudo, o PCV-3 foi detectado em 26,7% das amostras de animais saudáveis e de animais com variados sinais clínicos, de ambos os sexos e de idades variadas. Associação estatística (P=0,0285) foi observada apenas entre animais com sinais respiratórios e PCV-3; nenhum animal positivo para PCV-3 apresentava diarreia. Não foi observada associação estatística entre o PCV-3 e a idade ou o sexo dos suínos. Por se tratar de um vírus recém-descoberto, existem poucas informações sobre sua epidemiologia. Espera-se que os dados deste trabalho possam contribuir para futuros estudos sobre a epidemiologia do PCV-3.(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.

Porcine circovirus 2 (PCV2) increases the expression of endothelial adhesion/junction molecules

Abstract Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus disease, a complex multisystem syndrome in domestic pigs. Despite the significant economic losses caused by porcine circovirus disease, the mechanisms of pathogenesis underlying the clinical findings remain largely unclear. As various reports have highlighted the potential key role of vascular lesions in the pathogenesis of porcine circovirus disease, the aim of this work was to investigate effects of PCV2 infection on vascular endothelial cells, focusing on cell viability and expression of adhesion/junction molecules. PCV2 infection reduced endothelial cell viability, while viral infection did not affected the viability of several other classical cell lines. Also, PCV2 infection in endothelial cells displayed a dual/biphasic effect: initially, infection increased ICAM-1 expression, which can favor leukocyte recruitment and emigration to tissues and possibly inducing characteristic porcine circovirus disease inflammatory lesions; then, secondarily, infection caused an increase in zonula occludens 1 tight junction protein (ZO-1) expression, which in turn can result in difficulties for cell traffic across the endothelium and a potential impairment the immune response in peripheral tissues. These virus-induced endothelial changes could directly impact the inflammatory process of porcine circovirus disease and associated vascular/immune system disturbances. Data suggest that, among the wide range of effects induced by PCV2 on the host, endothelial modulation can be a pivotal process which can help to explain PCV2 pathogenesis in some porcine circovirus disease presentations.

Hallazgos patológicos en cerdos afectados con el síndrome del desmedro multisistémico postdestete de la República Argentina / Pathological findings in pigs affected by the postweaning multisystemic wasting syndrome in Argentina

El síndrome del desmedro multisistémico postdestete (PMWS) fue descrito por primera vez en Canadá en el año 1991 y desde entonces un número creciente de casos han sido diagnosticados en todo el mundo. En la Argentina, el PMWS fue reportado por primera vez recientemente. Se estudiaron 48 cerdos de 5 a 12 semanas de edad con signos característicos de PMWS procedentes de 19 granjas. Si bien se desconoce la distribución real del virus en nuestro país se observó desde el año 2001 un número creciente de granjas con PMWS y distribuidas en las principales provincias productoras. La histopatología fue una herramienta diagnóstica importante en casos sospechosos de PMWS con la observación de diferentes grados de lesión. En los animales estudiados las infecciones secundarias pudieron ser importantes, ya sea por patógenos oportunistas o por complicaciones bacterianas.

Postweaning multisystemic wasting syndrome was first described in Canada in 1991 and at present an increasing number of cases has been diagnosed worldwide. In Argentina the first cases of PMWS were reported recently. Forty eight 5 to 12 week old pigs with signs characteristic of PMWS from 19 farms were studied. Although the real distribution of the virus in our country is not known it was observed an increasing number of farms with PMWS distributed in the major producing provinces. The histopathology was an important tool in diagnosis of suspicious cases of PMWS with the observation of different degrees of lesion. In the studied animals, the secondary infections, either by opportunistic pathogens or secondary bacteria could be important.

Characterization of the Recombinant Proteins of Porcine Circovirus Type2 Field Isolate Expressed in the Baculovirus System

Porcine circovirus (PCV) type2 was isolated using primary porcine kidney cells from lymph node of piglets with typical PMWS. The presence of the virus was identified by PCR using primers specific to PCV type2. The ORFs 1 and 2 were amplified by PCR using primers corresponding to the target genes of the PCV type 2. Cloned genes were inserted into the baculovirus expression vector and PCV recombinant proteins were expressed using baculovirus expression system. Recombinant protein expression was determined by indirect immunofluorescent assay (IFA) and immunoblotting using polyclonal antiserum to PCV. ORF1 gene expressed two proteins with approximately 17 kDa and 31 kDa proteins in the baculovirus system. Recombinant protein of the ORF2 was similar to that of the native virus except minor bands with different molecular weight were detected. Recombinant protein expressed in the baculovirus system showed at least two glycosylation sites based on the tunicamycin treatment. Recombinant protein of the ORF2 assembled virus-like particle in recombinant virus infected insect cells.

Genetic characterization of porcine circovirus-2 field isolates from PMWS pigs

PMWS is a new emerging disease in swine herds worldwide. Field isolates of PCV-2, a putative major causative agent of PMWS, were isolated and genetically characterized. Viral genome of two field isolates (PC201DJ and PC201SS) from pigs showing typical PMWS was sequenced. The nucleotide sequence homology with other PCV-2 isolates was ranging from 95% to 99% in complete viral genomic sequence. The highly conserved nonanucleotide motif of replication origin was identical to that of other PCV-2 isolates. To determine the genetic heterogeneity of PCV-2 isolates, the phylogenetic tree based on the complete genome of PCV-2 isolates were constructed. Two PCV-2 field isolates were closely related to Canadian isolates of PCV-2. PCV-2 isolated from field may have an origin of North America and is possibly originated from importation of breeding stocks. The result indicates that although the genome of PCV-2 is relatively stable in general, minor genetic variations exist among PCV-2 isolates from the different geographic locations. These differences of viral genome might have an important implication for genetic characteristics of PCV-2 infection. Three major immunorelevant epitopes of capsid protein showed variations in amino acid sequences. Also, the variance of amino acid sequence in antigenic epitope existed between two Korean PCV-2 isolates.