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.

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.

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.

Genetic characterization of porcine circovirus type 2 in captive wild boars in southern Brazil.

Porcine circovirus type 2 (PCV2) has been identified in pig population in Brazil since 2000, but scarce studies involving wild boars with PCV2 infection are reported in the country. This study aimed to perform the genetic characterization of PCV2 detected in clinically healthy captive wild boars from farms located in Southern Brazil. Bronchial and mesenteric lymph nodes from 129 clinically healthy captive wild boars were tested by nested PCR for PCV2 detection. Six out of 38 positive samples (29.5%) were submitted to a quantitative real time PCR (qPCR) and genetic sequencing. Viral load up to 1.19 × 109 viral DNA copies/uL was detected in lymph nodes samples by qPCR. According to the ORF2 gene sequence analysis, all PCV2 samples were classified into PCV2b genotype. Comparisons based on a 702 nt region of the ORF2 of all six isolates revealed a high degree of similarity between these isolates. The ORF2 sequences characterized here share 97.1-100% of nucleotide identity and 95.7-100% of amino acid identity with other PCV2b isolated in Brazil from wild boars and feral pigs. This study reports the first detection and genetic characterization of PCV2b in captive wild boars in Brazil and provides important information on PCV2 infection in this domesticated species.

Influenza A virus infection in Brazilian swine herds following the introduction of pandemic 2009 H1N1.

Influenza A virus (FLUAV) infections are endemic in pork producing countries worldwide but in Brazil it was not considered an important pathogen in pigs. Since the emergence of 2009 pandemic H1N1 (H1N1pdm) FLUAV, many outbreaks of respiratory disease were observed in pig herds. The aim of this study was to evaluate FLUAV infection in swine in 48 pig farms located in seven Brazilian states with previous reports of influenza-like signs by clinical, serological and virological cross-sectional studies. Serological results showed that pigs from all farms had anti-influenza antibodies by NP-ELISA. Antibodies to H3N2, H1N2 and H1N1pdm were detected by HI in pigs from 24 farms. Co-infection with two or more FLUAV subtypes was detected in pigs in seven of those 24 farms. Detection of FLUAV in nasal swabs and oral fluids by RT-qPCR indicated a global concordance >81% for the two biological samples. Moreover, our results show that H1N1pdm, H1N2 and H3N2 viruses are widespread in Brazilian pig herds. The monitoring of FLUAV emergence and evolution in pigs is urgent, as well the study of the pathogenesis of Brazilian isolates, aiming to control influenza in pigs.

Unravelling the genetic components involved in the immune response of pigs vaccinated against influenza virus.

A genome-wide association study for immune response to influenza vaccination in a crossbred swine population was conducted. Swine influenza is caused by influenza A virus (FLUAV) which is considered one of the most prevalent respiratory pathogens in swine worldwide. The main strategy used to control influenza in swine herds is through vaccination. However, the currently circulating FLUAV subtypes in swine are genetically and antigenically diverse and their interaction with the host genetics poses a challenge for the production of efficacious and cross-protective vaccines. In this study, 103 pigs vaccinated with an inactivated H1N1 pandemic virus were genotyped with the Illumina PorcineSNP60V2 BeadChip for the identification of genetic markers associated with immune response efficacy to influenza A virus vaccination. Immune response was measured based on the presence or absence of HA (hemagglutinin) and NP (nucleoprotein) antibodies induced by vaccination and detected in swine sera by the hemagglutination inhibition (HI) and ELISA assays, respectively. The ELISA test was also used as a measurement of antibody levels produced following the FLUAV vaccination. Associations were tested with x(2) test for a case and control data and using maximum likelihood method for the quantitative data, where a moderate association was considered if p<5×10(-5). When testing the association using the HI results, three markers with unknown location and three located on chromosomes SSCX, SSC14 and SSC18 were identified as associated with the immune response. Using the response to vaccination measured by ELISA as a qualitative and quantitative phenotype, four genomic regions were associated with immune response: one on SSC12 and three on chromosomes SSC1, SSC7, and SSC15, respectively. Those regions harbor important functional candidate genes possibly involved with the degree of immune response to vaccination. These results show an important role of host genetics in the immune response to influenza vaccination. Genetic selection for pigs with better response to FLUAV vaccination might be an alternative to reduce the impact of influenza virus infection in the swine industry. However, these results should to be validated in additional populations before its use.

Influenza A Viruses of Human Origin in Swine, Brazil.

The evolutionary origins of the influenza A(H1N1)pdm09 virus that caused the first outbreak of the 2009 pandemic in Mexico remain unclear, highlighting the lack of swine surveillance in Latin American countries. Although Brazil has one of the largest swine populations in the world, influenza was not thought to be endemic in Brazil's swine until the major outbreaks of influenza A(H1N1)pdm09 in 2009. Through phylogenetic analysis of whole-genome sequences of influenza viruses of the H1N1, H1N2, and H3N2 subtypes collected in swine in Brazil during 2009-2012, we identified multiple previously uncharacterized influenza viruses of human seasonal H1N2 and H3N2 virus origin that have circulated undetected in swine for more than a decade. Viral diversity has further increased in Brazil through reassortment between co-circulating viruses, including A(H1N1)pdm09. The circulation of multiple divergent hemagglutinin lineages challenges the design of effective cross-protective vaccines and highlights the need for additional surveillance.

Detection of porcine Circovirus type 2 (PCV2) variants PCV2-1 and PCV2-2 in Brazilian pig population.

In the present study whole genome of six Brazilian isolates of PCV2 were sequenced, analyzed and compared with 35 other sequences (24 from other countries and 17 from Brazil). The phylogenetic analysis showed that mostly Brazilian variants of PCV2 were grouped as PCV2-1. Two isolates among the six analyzed here could not be grouped with any other PCV2-2 analyzed in this study. One of these isolates was from an aborted fetus with myocarditis and the other from a PMWS affected pig. The results pointed here showed that both groups of PCV2 are present in Brazilian pig population without any clear geographical correlation.

Erradicação da doença de Aujeszky em Santa Catarina: importância da condição sanitária das leitoas de reposição / Aujeszky's diasease eradication in Santa Catarina State: relevance of sanitary status of replacement gilts

A doença de Aujeszky (DA) é uma infecção causada por um herpesvírus, o vírus da DA (VDA), primariamente em suínos. Esta doença está presente no Estado de Santa Catarina (SC) desde 1984. Devido ao impacto da DA no mercado exportador de carne suína, no comércio de reprodutores e nas perdas de produtividade, um programa de erradicação, financiado por uma parceria entre a indústria e a associação de produtores tem tido sucesso em eliminar gradualmente a DA de rebanhos suínos de SC. O último caso de DA no estado foi identificado em julho de 2004. Durante o processo de despovoamento/repovoamento, foi detectado um rebanho suíno positivo para o VDA localizado no oeste de SC. Estudos de rastreabilidade da origem daqueles animais indicaram que a fonte era uma granja que distribuía reprodutores ilegalmente sem certificação sanitária. Este suinocultor mantinha um sistema de integração que incluía 40 diferentes produtores para quem eram comercializados reprodutores e/ou suínos para terminação. Testes de soroprevalência detectaram anticorpos anti-VDA em 12 daqueles rebanhos. Devido a sua localização dentro do raio de 2,5km do foco inicial, uma outra granja, onde havia uma central de inseminação artificial que distribuía sêmen suíno para outras 5 granjas do mesmo proprietário, foi testada e também resultou positiva. Os objetivos deste artigo são descrever as condições sanitárias frente ao VDA naquelas granjas que receberam suínos ou sêmen suíno daqueles suinocultores, as medidas para controlar e eliminar o VDA dos rebanhos positivos e a situação atual decorrente deste trabalho. Além disso, o artigo busca alertar que medidas de vigilância ativa e normas sanitárias para comércio e distribuição de material genético devem ser seguidas, caso contrário, a DA pode recrudescer e tornar-se fora de controle como ocorria antes do início deste programa.

Aujeszky's disease (AD) is an herpesvirus infection, caused by the pseudorabies virus (PRV), primarily in swine and present in Santa Catarina State (SC) since 1984. Due to the impacts of AD in the pork export, breeder's trade and productivity losses, an eradication program, financed by industry and swine producers association, has successfully eradicated the AD of swine herds in SC State. The last case of AD in the State was identified in July of 2004. During the depopulation/repopulation process, a PRV positive swine herd located in the west region of SC State was detected. Traceability studies of the origin of those animals indicated that the source was a swine farm which illegally distributed breeders without sanitary certification. This swine producer maintained an integration system which included 40 different producers, to whom were commercialized breeders and/or finishers. Serum-prevalence tests detected PRV antibodies in 12 of those herds. Due to the location into the 2.5km of radius from the initial outbreak, another swine farm, which had an artificial insemination center that distributed swine semen to another 5 herds of the same owner was tested positive as well. The objectives of this paper are to describe the sanitary status related to AD on the farms which have received pigs or swine semen from these swine producers, the measures to control and eliminate ADV from positive herds and the outcome of this work. Beyond that, to alert that measures of active surveillance and sanitary rules for commerce and distribution of genetic materials must be properly fulfilled, otherwise, AD can reactivate and become out of control as occurred before the eradication program.

Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system

Aujeszky' s disease (AD) is an infectious disease causing important economic losses to the swine industry worldwide. The disease is caused by an alpha-herpesvirus, Aujeszky' s disease virus (ADV), an enveloped virus with a double stranded linear DNA genome. The ADV genome encodes 11 glycoproteins, which are major targets for the immune system of the host in response to the infection. The glycoprotein E (gE) is a non-essential protein and deletion of the gE gene has been used for the production of marker vaccines. Aiming to develop molecular reagents for the production of a gE specific ELISA test, the gE gene was amplified by PCR, cloned and expressed into a baculovirus expression system. The recombinant DNA vector pFastBac-gE-ADV was used for site-specific transposition into the recombinant baculovirus (bacmid). Colonies with recombinant bacmid-pFastBac-gE-ADV were selected by antibiotic and color selection and the presence of the gE gene was confirmed by PCR. The recombinant bacmid-pFastBac-gE-ADV was cotransfected in insect Trichoplusia ni and the presence of the recombinant DNA and gE protein were detected by PCR, SDS-PAGE and Western blotting, respectively.

A doença de Aujeszky (DA) é uma enfermidade infecto-contagiosa que causa grandes perdas econômicas ao produtor e à agroindústria suinícola em todo o mundo. É causada pelo vírus da doença de Aujeszky (VDA), um alfaherpesvírus envelopado com genoma DNA de fita dupla e linear. O genoma do VDA codifica 11 glicoproteínas, as quais são os maiores alvos do sistema imune do hospedeiro em resposta a infecção. A glicoproteína E (gE) é uma proteína não essencial e a deleção do gene da gE é muito utilizada para a produção de vacinas com marcadores. Com o objetivo de desenvolver insumos moleculares para a produção de um teste de ELISA específico para gE do VDA, a seqüência do gene da gE foi amplificada, clonada e expressa no sistema de expressão em baculovírus. O produto da amplificação foi clonado no vetor pGem®-T Easy e subclonado no plasmídeo de expressão pFastBac™1. O DNA recombinante pFastBac-gE-VDA foi usado para a transposição sítio-específica no baculovírus recombinante (bacmid). Após seleção por antibióticos e cor, as colônias com o recombinante bacmid-pFastBac-gE-VDA foram selecionadas e a presença do gene da gE foi confirmada por PCR. O DNA recombinante viral, bacmid-pFastBac-gE-VDA, foi usado para cotransfecção de células de inseto Trichoplusia ni e a presença do recombinante e a proteína gE foi determinada por PCR, por SDS-PAGE e Western blotting, respectivamente.

Identificação do circovírus suíno tipo 2 por reação em cadeia da polimerase e por imunoistoquímica em tecidos suínos arquivados desde 1988 no Brasil / Identification of porcine circovirus type 2 by polymerase chain reaction and immunohistochemistry on archived porcine tissues since 1988 in Brazil

A síndrome multissistêmica do definhamento dos suínos (SMDS) é uma doença de importância econômica causada pelo circovírus suíno tipo 2 (PCV2). Uma pesquisa retrospectiva foi realizada em amostras de órgãos de suínos fixados em blocos de parafina arquivados, que haviam sido submetidos à Embrapa Suínos e Aves entre 1985 e 1998 para o diagnóstico histopatológico. Vinte e cinco casos foram selecionados com base nas lesões histológicas características da SMDS, tais como linfoadenopatia, pneumonia intersticial, hepatite e nefrite intersticial. A presença de PCV2 nos cortes histológicos foi pesquisada por reação em cadeia da polimerase interna (nested-PCR), na qual utilizou-se primers específicos para a seqüência da ORF2 do PCV2 e também por imunoistoquímica, utilizando um anticorpo monoclonal específico para o capsídeo do PCV2. O DNA viral e os antígenos específicos do PCV2 foram detectados em amostras de tecidos de dois dos 25 casos analisados, sendo um desses datado de 1988. Esses resultados indicam que o PCV2 já estava presente no Brasil desde 1988.

Postweaning multisystemic wasting syndrome (PMWS) is of economical importance a disease caused by porcine circovirus type 2 (PCV2). A retrospective investigation was performed on paraffin-embedded organs samples from swine submitted to Embrapa Swine and Poultry Research Center between 1985 and 1998 for histopathologic diagnosis. A total of 25 cases were chosen from the archival collection of the Animal Health Laboratory at Embrapa Swine and Poultry based on characteristic pathological lesions of PMWS, such as lymphadenopathy, interstitial pneumonia, hepatitis and interstitial nephritis. The sections were investigated by nested-PCR (polymerase chain reaction) which used specific primers for the ORF2 sequence of the PCV2 and by immunohistochemistry using a monoclonal antibody specific for PCV2 capsid antigen. Virus specific DNA and antigen were detected in tissue samples of two out of 25 analyzed cases. The earliest positive sample originated from 1988. These results indicate that PCV2 is present in Brazil since 1988.

Lack of evidence of porcine reproductive and respiratory syndrome virus (PRRSV) infection in domestic swine in Brazil

Este artigo descreve a primeira prevalência de anticorpos e inoculação experimental de amostras suspeitas do vírus da síndrome reprodutiva e respiratória dos suínos (PRRSV) de suínos de rebanhos do Brasil positivos pelo teste de ELISA. Com base na hipótese de que este agente está presente em plantéis de suínos mundialmente, o objetivo deste trabalho foi estabelecer uma metodologia de diagnóstico e também investigar a ocorrência de PRRSV em rebanhos suínos brasileiros. Cinqüenta e quatro granjas, número total de granjas que importaram material genético (suínos vivos e sêmen suíno) de países onde a PRRS era endêmica no período de 1990 a dezembro de 2000, de oito estados brasileiros foram todas incluídas neste estudo. O tamanho da amostragem de soros coletados foi para que se detectasse uma prevalência de 5 por cento, com um nível de confiança de 95 por cento. Um total de 3785 amostras de soro foram testadas para detecção de anticorpos para PRRSV por ELISA. Após a realização dos testes de ELISA, os quais foram realizados utilizando dois kits comerciais, todos os suínos positivos foram retestados sorologicamente, examinados e depois, material adicional foi coletado para detecção viral. O material para isolamento viral foi inoculado em células de cultivo permissíveis e também em suínos (bio-ensaio suíno). Além disso, reação de transcriptase reversa e reação da polimerase em cadeia (RT-PCR) e nested RT-PCR também foram realizadas. Não foi demonstrada a presença de PRRSV ou de RNA de PRRSV por isolamento viral ou RT-PCR (ou nested RT-PCR), respectivamente, em todas as amostras testadas. Além disso, os suínos inoculados com amostras suspeitas de PRRSV não soro-converteram ou produziram lesões características de PRRS no bio-ensaio suíno. Portanto, nossos resultados não indicam evidências da presença de PRRSV nas amostras analisadas dos rebanhos suínos deste estudo.