First identification and characterization of ovine gammaherpesvirus type 2 in horses and artiodactyla from an outbreak of malignant catarrhal fever in Mexico.

Ovine gammaherpesvirus 2 (OvHV-2), a member of the genus Macavirus, causes sheep-associated malignant catarrhal fever (SA-MCF), a fatal lymphoproliferative disease affecting a wide variety of ungulates in addition to horses. This study described an outbreak of SA-MCF in Mexico and the identification of the OvHV-2 virus in primary rabbit testis cultures through the generation of intranuclear inclusion bodies, syncytia, immunofluorescence (IF), immunocytochemistry (ICC), immunohistochemistry (IHC), endpoint polymerase chain reaction (PCR), and partial sequencing of the ORF75 gene. The animals involved in this outbreak showed mucogingival ulcers in the vestibule of the mouth and tongue, hypersalivation, corneal opacity, reduced food consumption, and weight loss of variable severity. These clinical signs and the histopathological findings suggested the diagnosis of SA-MCF. Buffy coat fractions from the anticoagulated blood samples of ill animals were collected and analyzed by PCR. Positive buffy coats were used to inoculate the primary cell cultures of rabbit testis to identify the virus. Small clusters of refractile cytomegalic cells, characteristic of viral cytopathic effects, were observed between 48 and 72 h post-infection. Furthermore, intranuclear acidophilic inclusion bodies (IBs) were identified in the inoculated primary culture cells, and the cytoplasm showed immunoreactivity with hyperimmune rabbit serum against OvHV-2. Moreover, in the liver histological sections from sick deer, immunoreactive juxtanuclear IBs were identified with the same rabbit hyperimmune serum. The obtained sequences were aligned with the OvHV-2 sequences reported in GenBank and revealed a nucleotide identity higher than 98%. Based on the evidence provided in this study, we conclude that the outbreak of SA-MCF in the municipality of Tequisquiapan in the state of Queretaro, Mexico, was caused by OvHV-2. This is the second study reporting that horses are susceptible to OvHV-2 infection and can develop SA-MCF. We identified for the first time in Mexico, the presence of OvHV-2 in buffy coats from horses and Artiodactyla.

Infection dynamics of porcine circovirus type 3 in longitudinally sampled pigs from four Spanish farms.

Porcine circovirus type 3 (PCV-3) is a recently discovered virus in domestic pigs and wild boar. The virus has been described in pigs with different clinical/pathological presentations and healthy animals, but the dynamics of infection is currently unknown. The aim of this study was to longitudinally monitor PCV-3 infection in 152 pigs from four different healthy farms (A, B, C and D) by means of PCR in serum. The selected animals were sampled five (farm A) or six (farms B-D) times from weaning until the end of the fattening period. PCV-3 genome was found in pigs from all tested ages and farms; few animals had an apparent long-term infection (4-23 weeks). PCV-3 frequency of detection remained fairly uniform along tested ages within farms A and C, but was more variable among sampling times in farms B and D. Eight partial genome sequences were obtained from six different animals. Phylogenetic tree and pairwise distance analysis showed high similarity among sequences and with available genomes from different countries. This is the first study on PCV-3 infection dynamics in longitudinally sampled pigs. Most pigs got infection during their life, although PCV-3 did not appear to be linked with any specific age.

Porcine circovirus 3 is highly prevalent in serum and tissues and may persistently infect wild boar (Sus scrofa scrofa).

Porcine circovirus 3 (PCV-3) prevalence has been minimally investigated in wild boar; dynamics of infection and viral tissue distribution are currently unknown. In this study, serum samples from 518 wild boar (from years 2004 to 2018) were used to study frequency of infection. Also, serum samples from 19 boar captured and recaptured at least two times for a period of time from 1 month to 1 year were collected to determine PCV-3 infection dynamics. Finally, to elucidate PCV-3 DNA organic distribution, sera, different tissues and faeces were obtained from 35 additional wild boar. PCV-3 DNA was extracted and amplified with a conventional PCR. For the PCV-3 PCR-positive sera from the longitudinally sampled and different tissue types, a quantitative PCR was performed. Genome sequence was obtained from a number of PCV-3 PCR-positive samples from different years, different time-points of infection and tissues. Obtained results confirmed the susceptibility of wild boar to the virus, showing high frequency of PCV-3 detection (221 out of 518, 42.66%) and demonstrating circulation at least since 2004. Compiled data indicate the possibility of long-term infections, since 5 out of 10 PCV-3 PCR-positive boars longitudinally sampled showed positivity in samplings separated for more than 5 months. All tested tissue types' harboured PCV-3 genome, with the highest percentage of PCR positivity in submandibular lymph node, tonsil, lung, liver, spleen and kidney. The amount of DNA in all tested PCV-3 PCR-positive samples was moderate to low. All partial and complete PCV-3 sequences obtained from wild boar displayed high nucleotide identity, higher than 98%. In conclusion, this study further confirms that wild boar is susceptible to PCV-3 infection, showing high frequency of detection in this animal species. Furthermore, PCV-3 can be found in different tissues of wild boar and is apparently able to cause persistent infection.

Current Knowledge on Porcine circovirus 3 (PCV-3): A Novel Virus With a Yet Unknown Impact on the Swine Industry.

Porcine circovirus 3 (PCV-3) is a recently described virus belonging to the family Circoviridae. It represents the third member of genus Circovirus able to infect swine, together with PCV-1, considered non-pathogenic, and PCV-2, one of the most economically relevant viruses for the swine worldwide industry. PCV-3 was originally found by metagenomics analyses in 2015 in tissues of pigs suffering from porcine dermatitis and nephropathy syndrome, reproductive failure, myocarditis and multisystemic inflammation. The lack of other common pathogens as potential infectious agents of these conditions prompted the suspicion that PCV-3 might etiologically be involved in disease occurrence. Subsequently, viral genome was detected in apparently healthy pigs, and retrospective studies indicated that PCV-3 was already present in pigs by early 1990s. In fact, current evidence suggests that PCV-3 is a rather widespread virus worldwide. Recently, the virus DNA has also been found in wild boar, expanding the scope of infection susceptibility among the Suidae family; also, the potential reservoir role of this species for the domestic pig has been proposed. Phylogenetic studies with available PCV-3 partial and complete sequences from around the world have revealed high nucleotide identity (>96%), although two main groups and several subclusters have been described as well. Moreover, it has been proposed the existence of a most common ancestor dated around 50 years ago. Taking into account the economic importance and the well-known effects of PCV-2 on the swine industry, a new member of the same family like PCV-3 should not be neglected. Studies on epidemiology, pathogenesis, immunity and diagnosis are guaranteed in the next few years. Therefore, the present review will update the current knowledge and future trends of research on PCV-3.

Genetic characterisation of Porcine circovirus type 2 (PCV2) strains from feral pigs in the Brazilian Pantanal: An opportunity to reconstruct the history of PCV2 evolution.

Since its discovery, Porcine circovirus type 2 has emerged as one of the most relevant swine infectious diseases, causing relevant economic losses for the pig industry. While four genotypes were identified, only three (PCV2a, PCV2b and PCV2d) are currently circulating and display a worldwide distribution. Another genotype, PCV2c, has been described only once in Danish archive samples collected between 1980 and 1990. In addition to commercial pigs, PCV2 has been demonstrated to infect wild boars and other wild species, which can potentially serve as a reservoir for domestic populations. In this study, eight sequences obtained from feral pigs in the Pantanal region (Mato Grosso do Sul State, Brazil) were compared with reference sequences and other Brazilian sequences, and the results revealed remarkable genetic diversity, with all four genotypes currently recognised being detected (PCV2a, PCV2b, PCV2c and PCV2d). This finding represents a remarkable discovery, as it is the first detection of PCV2c since 1990 and the first-ever detection of PCV2c in live animals. The peculiar population history and ecological scenario of feral pigs in the Pantanal coupled with the complex, and still only partially known relationship of feral pigs with other PCV2 susceptible species (i.e., domestic pigs, wild boars and peccaries), open exciting questions concerning PCV2 origin and evolution. Overall, the results of the present study led us to form the following hypothesis: the PCV2 strains found in feral pigs may be the last descent of the strains that circulated among European pigs in the past, or they may have infected these feral pigs more recently through a bridge species.

Expresión de citocinas en cerdos co-infectados con circovirus porcino tipo 2 y el virus del síndrome respiratorio y reproductivo porcino / Cytokine expression in growing pigs co-infected with porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus

The aim of this work was to quantify porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), as well as cytokine mRNA expression (IL-6, IL-12, TNF-α, and IFN-γ) in superficial inguinal lymph nodes (SILN) of pigs from postweaning multisystemic wasting syndrome (PMWS)-affected and healthy farms. Genetic characterization of detected PCV2 sequences was also carried out. Based on the clinical outcome and the quantification of PCV2 nucleic acid by in situ hybridization and real time PCR, pigs were grouped into three categories: 1) PMWS, pigs with signs of wasting and high viral load in SILN (n = 4); 2) wasted-non-PMWS, pigs with signs of wasting and low to intermediate viral loads in SILN (n = 3); and 3) healthy, pigs with no clinical signs and low viral loads (n = 3). PRRSV was detected in three PMWS affected and two-wasted-non-PMWS pigs. The genetic analysis of PCV2 sequences revealed the presence of two genotypes PCV2a and PCV2b in PMWS-affected farms. Cytokine mRNA expression revealed that PMWS affected pigs had low expression of IFN-γ, whereas wasted-non-PMWS pigs showed higher amounts of IFN-γ. These results suggest that an imbalance in cytokines could be involved in the pathogenesis of PMWS.

Los objetivos de este trabajo fueron la cuantificación de circovirus porcino tipo 2 (PCV2) de virus del síndrome respiratorio y reproductivo porcino (PRRSV), así como la expresión de citocinas (IL-6, IL-12, TNF-α, e IFN-γ) en nódulos linfáticos inguinales superficiales (NLIS) de cerdos afectados por el síndrome de desmedro posdestete (postweaning multisystemic wasting syndrome, PMWS) y en cerdos sanos. Con base en el diagnóstico clínico y a la cuantificación de PCV2 por hibridación in situ y qRT-PCR, los cerdos se distribuyeron en tres grupos: 1) PMWS, cerdos con signos de desmedro y con cargas virales altas en NLIS (n = 4); 2) con desmedro sin PMWS, cerdos con signos de desmedro y cargas virales de intermedias a bajas en NLIS (n = 3); y 3) cerdos sanos, sin signos clínicos y con cargas virales bajas (n = 3). El PRRSV fue detectado en tres de los cerdos afectados por el PMWS y en dos cerdos con desmedro, pero sin el síndrome. Se caracterizaron las secuencias nucleotídicas del ORF2 de los PCV2 encontrados y los análisis genéticos revelaron la presencia de 2 genotipos PCV2a y PCV2b en las granjas afectadas con el PMWS. El perfil de expresión de citocinas mostró una baja expresión de IFN-γ en los cerdos con PMWS, mientras que los cerdos con desmedro sin PMWS mostraron valores elevados de esta citocina. Estos resultados sugieren que existe un desbalance en la producción de citocinas que puede estar implicado en la patogénesis de la enfermedad.

Phylogenetic networks to study the origin and evolution of porcine circovirus type 2 (PCV2) in Cuba.

Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of postweaning multisystemic wasting syndrome (PMWS), which is considered one of the most economically important swine diseases worldwide. In this study, a comparison between methodologies based on classical phylogenetic trees and networks to infer the origin of PCV2 in Cuba was performed. In addition, the mechanisms supporting the genetic variability of Cuban PCV2 populations were investigated. A retrospective study, using pig sera collected in Cuba from 1993 to 2004, to evaluate the presence of PCV2 genome and PCV2-specific antibodies was also conducted and revealed a lack of evidence of PCV2 infection in Cuban swine from years 1993 to 2004. A total of 24 complete Cuban PCV2 sequences collected between 2005 and 2009 from different regions of the country were analyzed. Three classical methods of phylogenetic analysis, namely Neighbour-Joining, Maximum Parsimony and Bayesian Inference, as well as haplotype network construction, were used. Whereas the classical phylogenetic trees suggested different origins for the Cuban PCV2 strains, the haplotype network revealed a direct connection between all the Cuban sequences in agreement with the obtained epidemiological and viral sequence data. Moreover, the importation of pigs carried out in 2005 from the Quebec-Ontario region, Canada, seems to be the most likely origin of PCV2 in Cuba. Likewise, the genetic variability of Cuban PCV2 sequences was supported by geographic segregation and positive selection pressure with estimated rates of nucleotide substitution on the order of 3.12×10(-3) and 6.57×10(-3) substitutions/site/year, which are closer to those reported for RNA viruses.

Hallazgos histopatológicos, hibridización in situ y estudio ultraestructural del Síndrome de Emaciación Multisistémico Porcino (PMWS) en granjas en Venezuela / Histopathological findings, in situ hybridization and ultrastructural study of Porcine Multisystemic Wasting Syndrome (PMWS) in pig farms in Venezuela

El síndrome multisistémico de emaciación post destete porcino (PMWS, por sus siglas en Inglés) ha tenido en años recientes extraordinaria importancia en la industria porcina en muchos países, particularmente en Canadá, Estados Unidos de América, Comunidad Europea, Asia y Sur América. El síndrome afecta a cerdos jóvenes, en edad comprendida generalmente entre las 8 y 14 semanas. El PMWS se caracteriza por emaciación, diarrea, ictericia y aumento de volumen de los linfonódulos. El circovirus porcino tipo 2 (PCV2, por sus siglas en inglés) ha sido descrito como el principal agente infeccioso en el PMWS y simultáneamente puede concurrir con otras enfermedades virales tales como: parvovirus porcino, virus del síndrome respiratorio y reproductivo porcino, además de otras entidades bacterianas. El diagnóstico del PMWS se basa en la edad de los cerdos, aspectos clínicos, emaciación, palidez de mucosas, hallazgos de necropsia, estudios histopatológicos, exámenes de hibridización in situ (HIS) en tejidos que revelan la presenciadel PCV2, además de estudios ultraestructurales. El objetivo principal de la presente investigación fue mostrar una mayor y actualizada casuística del PMWS en nuestro país. El presente estudio se realizó en 120 lechones de 8 a 16 semanas de edad, ubicados en 10 granjas porcinas de los estados Aragua y Carabobo, Venezuela. Los lechones presentaban pobre condición corporal, severa palidez, disnea, diarrea, dificultad respiratoria e hipertrofia de los linfonodos inguinales superficiales. Al realizar las necropsias se evidenció neumonía en grado variable e incremento del tamaño de los nódulos linfáticos inguinales, mesentéricos y traqueobronquiales, mientras que la histopatología revelaba depleción linfoide, con pérdida de la arquitectura de los linfonódulos con histiocitosis, células sincitiales e histiocitos con corpúsculos basófilos intensos intracitoplasmáticos, hallazgos altamente sugestivos del PMWS. Adicionalmente, se procesaron muestras de estos mismos tejidos mediante la técnica HIS, lo que permitió demostrar la presencia de PCV2 en todos los tejidos linfoides afectados y confirmar el diagnóstico de PMWS. El estudio ultraestrutural permitió observar las características del ente viral involucrado. Este es el primer trabajo que incluye métodos ultraestructurales para describir este síndrome en Venezuela.

Nowadays, the postweaning multisystemic wasting syndrome (PMWS) is an important disease in the swine industry, particularly in Canada, United States of America, Europe, Asia, and South America. The PMWS mainly affects pigs between 8 and 14 weeks of age. The PMWS is characterized by emaciation, diarrhea, icterus, and an enlargement of lymph nodes. The porcine circovirus type 2 (PCV2) has been described as the main infectious agent responsible for PMWS. It can concurrently occur with other viral diseases such as porcine parvovirus (PPV), porcine respiratory and reproductive viral syndrome, as well as other bacterial entities. The PMWS diagnosis is based on the pig’s age, clinical aspects, weight loss, macro and microscopic findings, presence of nucleic acids in tissues which reveal the existence of the PCV2, detected by an in situ hybridization (ISH) technique, and ultrastructural studies. This investigation studied the histological findings, in situ hybridization, and ultrastructural assessment of PMWS in pig farms in Venezuela. The main purpose of this study was to show a greater and updated cases of PMWS in our country. A total of 120 piglets ages 8 to 16 weeks, from 10 pig farms of the States of Aragua and Carabobo, Venezuela, were evaluated. The piglets showed poor body condition, pale appearance of mucous membranes, dyspnea, diarrhea, respiratory distress, and hypertrophy of superficial inguinal lymph nodes. At necropsy, diverse degrees of pneumonia, enlargement of superficial inguinal, mesenteric, and tracheobronquial lymph nodes were found, while the histological findings showed lymphoid depletion, loss of lymph nodes architecture, hystiocytosis, syncitial cells, and the presence of cytoplasmic inclusions in hystiocitic cells. All these findings were suggestive of PMWS. Furthermore, samples of the same tissues were processed through the ISH, which allowed showing the presence of PCV2 in all affected lymphoid tissues, confirming the diagnosis of PMWS. The ultrastructural study showed the characteristics of the virus involved. This is the first study in Venezuela that includes ultrastructural methods describing the PMWS.

Retrospective serological survey of Porcine circovirus-2 infection in Mexico.

Postweaning multisystemic wasting syndrome (PMWS) is considered a multifactorial emerging disease of which Porcine circovirus-2 (PCV-2) is the necessary infectious cause. However, retrospective studies have shown that PMWS is not a new disease and that PCV-2 has been circulating in pig farms for years. Most of these studies were performed in Europe and Asia; only a few were performed in North or South America. A PCV-2 retrospective serological survey was carried out with 659 serum samples collected from pigs in Mexico between 1972 and 2000. Serological analyses were performed with an immunoperoxidase monolayer assay (IPMA). The overall prevalence of PCV-2 antibodies was 59% (387/659); the prevalence was 27% (24/90) for the period from 1972-1979; 44% (74/169) from 1980-1989, and 72% (289/400) from 1990-2000. Antibodies to PCV-2 were detected in at least 1 pig from all tested years since 1973. This study shows evidence of enzootic PCV-2 infection in Mexico for many years before the first description of PMWS in the country (in 2001), further supporting results obtained in other parts of the world. To date, this study provides the earliest evidence of PCV-2 infection in the North and South American continents.

Coinfección de circovirus porcino tipo 2 con el virus del síndrome respiratorio y reproductivo porcino en Venezuela / Coinfection of porcine circovirus type 2 with porcine reproductive and respiratory syndrome virus in Venezuela

El circovirus porcino tipo 2 (PCV2) que ha sido asociado al síndrome de emaciación multisistémico postdestete (PMWS), generalmente es diagnosticado conjuntamente con otros agentes infecciosos en cerdos. El virus del síndrome respiratorio y reproductivo porcino (PRRSV) es probablemente el más frecuentemente asociado a estos casos. Aunado a ello, se ha sugerido una potencial relación entre ambos agentes, basándose en infecciones experimentales. Tomando en cuenta que ambos virus, PRRSV y PCV2, han sido detectados en Venezuela, es posible que la coinfección por estos agentes ocurra de forma natural bajo condiciones de campo en el país; en razón de lo cual, el objetivo del presente trabajo fue describir los signos clínicos, las lesiones macroscópicas y microscópicas, así como la detección de ambos virus en cerdos naturalmente coinfectados en Venezuela. Entre noviembre del 2001 y noviembre del 2002, fueron estudiados 27 cerdos entre 6 y 12 semanas de edad, provenientes de 11 granjas, las cuales presentaron capacidades entre 300 y 1.500 madres, estando ubicadas en los estados Aragua, Carabobo, Cojedes, Mérida, Yaracuy y Zulia. Los signos clínicos y las lesiones macro y microscópicas de los cerdos seleccionados fueron registradas. Muestras de tonsila, pulmón, nódulos linfáticos, bazo, hígado, intestino y riñón, fueron fijadas en formalina bufferada al 10 por ciento para la realización del examen histopatológico. Los casos compatibles con PMWS fueron procesados según la técnica de hibridación in situ (HIS) para detectar PCV2, realizándose inmunohistoquímica (IHQ) para PRRSV a las muestras positivas a PCV2, de manera de detectar aquellos casos de co-infección viral. De los 27 cerdos con signos clínicos como retraso en el crecimiento y disnea, 10 evidenciaron lesiones microscópicas consistentes con PMWS, de estos últimos, cinco resultaron positivos a la prueba de HIS, tres de ellos (dos con PMWS y uno con infección subclínica de PCV2).

Primer reporte en Venezuela del síndrome de emaciación multisistémico post destete porcino / Postweaning multisystemic wasting swine syndrome first report in Venezuela

En años recientes el Síndrome Multisistémico de Emaciación Post Destete Porcino (PMWS) ha tenido importancia en la industria porcina en muchos países, particularmente en Canadá, Europa, Estados Unidos de América, Asia y Sur América. El síndrome afecta cerdos jóvenes, principalmente entre 6 y 14 semanas de edad. El PMWS está caracterizado por emaciación, neumonía, diarrea, ictericia y aumento de volumen de los linfonódulos. El síndrome ha sido asociado con un circovirus porcino tipo 2 (PCV2) y, simultáneamente pueden ocurrir otras enfermedades: parvovirus porcino, virus del síndrome respiratorio y reproductivo porcino (PRRS), etc. El diagnóstico del PMWS se basa en la edad de los cerdos afectados, la típica apariencia de emaciación, hallazgos de necropsia, histopatológicos, y exámenes de Hibridación in situ (HIS) en tejidos que revelan la presencia de PCV2. El presente estudio se realizó en 12 lechones de 8 a 12 semanas de edad, ubicados en 3 granjas porcinas de estados centrales de Venezuela (Aragua/Carabobo). Los lechones presentaban signos de consunción, palidez generalizada, disnea, diarrea e hipertrofia de linfonódulos superficiales inguinales. A la necropsia se evidenció neumonía con pulmones no colapsables e incremento de tamaño de linfonódulos inguinales, mesentéricos y traqueobronquiales; mientras en la histopatología se evidenció depleción linfoide con pérdida de la arquitectura folicular en linfonódulos, bazo y placas de Peyer, con presencia de infiltración histiocitaria, células sincitiales y corpúsculos intensamente basófilos intracitoplasmáticos en histiocitos, lo cual es altamente sugestivo de PMWS. Adicionalmente se procesaron muestras de estos mismos tejidos mediante una técnica de HIS, que permitió demostrar la presencia de PCV2 en los tejidos linfoides afectados y confirmar el diagnóstico de PMWS. Esta es la primera descripción de esta entidad patológica en Venezuela.