Construction of a replicative expression vector based on the porcine circovirus 2 replicon / 生物工程学报

The antigen gene expression level of a DNA vaccine is the key factor influencing the efficacy of the DNA vaccine. Accordingly, one of the ways to improve the antigen gene expression level of a DNA vaccine is to utilize a plasmid vector that is replicable in eukaryotic cells. A replicative DNA vaccine vector pCMVori was constructed based on the non-replicative pcDNA3.1 and the replicon of porcine circovirus 2 (PCV2) in this study. An EGFP gene was cloned into pCMVori and the control plasmid pcDNA3.1. The two recombinant vectors were transfected into PK-15 cell, and the plasmid DNA and RNA were extracted from the transfected cells. Real-time PCR was used to determine the plasmid replication efficiency of the two plasmids using plasmid before and after Bcl Ⅰ digestion as templates, and the transcription level of the Rep gene in PCV2 replicon was detected by RT-PCR. The average fluorescence intensity of cells transfected with the two plasmids was analyzed with software Image J, and the transcription level of EGFP was determined by means of real-time RT-PCR. The results showed that the replication efficiency of pCMVori in PK-15 cells incubated for 48 h was 136%, and the transcriptions of Rep and Rep' were verified by RT-PCR. The average fluorescence intensity of the cells transfected with pCMVori-EGFP was 39.14% higher than that of pcDNA3.1-EGFP, and the transcription level of EGFP in the former was also 40% higher than that in the latter. In conclusion, the DNA vaccine vector pCMVori constructed in this study can independently replicate in eukaryotic cells. As a result, the expression level of cloned target gene was elevated, providing a basis for developing the pCMVori-based DNA vaccine.

Porcine circovirus 2 and 3 in wild boars in Southern Brazil / Circovírus suíno 2 e 3 em javalis no Sul do Brasil

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.

A molecular survey reveals high occurrence of co-infections in intensive pork production farms with increased rates of mummified swine fetuses in Southern Brazil / [Pesquisa molecular revela alta ocorrência de coinfecções em granjas de produção intensiva de suínos com altas taxas de mumificação fetal no sul do Brasil]

Neste estudo, 308 amostras de fetos mumificados foram testadas para parvovírus suíno (PPV), circovírus suíno tipos 2 e 3 (PCV2 e PCV3) e leptospiras patogênicas. A idade gestacional no momento da perda gestacional e a frequência da mumificação fetal de acordo com a ordem de parto também foram investigadas. As amostras foram coletadas em granjas comerciais de criação de suínos da região sul do Brasil que apresentassem taxas de mumificação fetal igual ou maiores a 2,5%. Fragmentos de pulmão, rim, fígado e coração de fetos suínos mumificados foram coletados para análise molecular. Resultados da PCR foram classificados de acordo com a região de origem das amostras, tendo Santa Catarina, Paraná e Rio Grande do Sul contabilizado 87 (28,25%), 89 (28,90%) e 132 (42,86%) do total de amostras de fetos suínos mumificados, respectivamente. Coinfecções foram observadas na maioria dos casos e PCV3 foi o agente mais prevalente detectado, encontrado em 298 amostras (96,75%). A maioria das perdas gestacionais foi observada entre 50 e 70 dias de gestação (168; 54,5%) e a mumificação fetal não foi associada à ordem de parto das matrizes. Os achados sugerem que as altas taxas de fetos suínos mumificados na região Sul do Brasil podem ser explicadas pela infecção com esses agentes virais.(AU)

cGAS/STING signaling pathways induces the secretion of type Ⅰ interferon in porcine alveolar macrophages infected with porcine circovirus type 2 / 生物工程学报

In order to study the signal pathway secreting type Ⅰ interferon in porcine alveolar macrophages (PAMs) infected with porcine circovirus type 2 (PCV2), the protein and the mRNA expression levels of cGAS/STING pathways were analyzed by ELISA, Western blotting and quantitative reverse transcriptase PCR in PAMs infected with PCV2. In addition, the roles of cGAS, STING, TBK1 and NF-κB/P65 in the generation of type I interferon (IFN-I) from PAMs were analyzed by using the cGAS and STING specific siRNA, inhibitors BX795 and BAY 11-7082. The results showed that the expression levels of IFN-I increased significantly at 48 h after infection with PCV2 (P<0.05), the mRNA expression levels of cGAS increased significantly at 48 h and 72 h after infection (P<0.01), the mRNA expression levels of STING increased significantly at 72 h after infection (P<0.01), and the mRNA expression levels of TBK1 and IRF3 increased at 48 h after infection (P<0.01). The protein expression levels of STING, TBK1 and IRF3 in PAMs infected with PCV2 were increased, the content of NF-κB/p65 was decreased, and the nuclear entry of NF-κB/p65 and IRF3 was promoted. After knocking down cGAS or STING expression by siRNA, the expression level of IFN-I was significantly decreased after PCV2 infection for 48 h (P<0.01). BX795 and BAY 11-7082 inhibitors were used to inhibit the expression of IRF3 and NF-κB, the concentration of IFN-I in BX795-treated group was significantly reduced than that of the PCV2 group (P<0.01), while no significant difference was observed between the BAY 11-7028 group and the PCV2 group. The results showed that PAMs infected with PCV2 induced IFN-I secretion through the cGAS/STING/TBK1/IRF3 signaling pathway.

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)

Infectious diseases dynamics in growing/finishing pigs in Southern Brazil (2005-2016) / Dinâmica de ocorrência das doenças infecciosas em suínos nas fases de crescimento e terminação no sul do Brasil (2005-2016)

This study aimed to determine the frequency and distribution of infectious diseases diagnosed through necropsy examination and histopathological analysis in growing/finishing pigs along 12 years (2005-2016) in Southern Brazil. We evaluated 1906 anatomopathological exams of pigs at growing/finishing phases, of which the infectious diseases corresponded to 75.6% of the cases (1,441/1,906). Porcine circovirus type 2 (PCV2) infections were the most frequent, accounting for 51.3% of the cases (739/1,441) with a higher frequency from 2005 to 2007, characterizing an epidemic distribution, with a gradual decline after 2008. Infectious diseases affecting the respiratory system were the second major cause with 30.1% of the cases. Among these, necrotizing bronchiolitis caused by swine Influenza (15.1%, 218/1,441) and bacterial pneumonia (15%, 216/1,441) were the main conditions. Influenza was mostly diagnosed from 2010 to 2013, accounting for 43.1% (167/387) of the cases. After this period, both respiratory infectious diseases were endemic. Digestive system infectious diseases accounted for 10.5% of the diagnoses (151/1,441), with the following main conditions: Salmonella spp. enterocolitis (43.7%, 66/151), Lawsonia spp. proliferative enteropathy (41.7%, 63/151), and Brachyspira spp. colitis (14.6%, 22/151). The latter had a higher incidence from 2012 to 2014 with all cases detected in this period. Polyserositis and bacterial meningitis represented, respectively, 5.8% (84/1,441) and 2.3% (33/1,441) of the cases diagnosed, with a constant endemic character.(AU)

O objetivo deste estudo consistiu em determinar a frequência e a distribuição das doenças infecciosas diagnosticadas através de exame de necropsia e análise histopatológica em suínos nas fases de crescimento/terminação ao longo de 12 anos (2005-2016) no sul do Brasil. Foram avaliados 1906 laudos anatomopatológicos de suínos nas fases de crescimento/terminação, dos quais as doenças infecciosas corresponderam a 75,6% (1441/1906) do total. As infecções por circovírus suíno tipo 2 (PCV2) foram as mais frequentes, contabilizando 51,3% (739/1441) dos casos, com uma alta frequência de 2005 a 2007 caracterizando uma distribuição epidêmica neste período, e um declínio gradual após o ano de 2008. A segunda principal causa incluiu as doenças infecciosas que afetam o sistema respiratório (30,1% dos casos). Dentre essas, destacaram-se a influenza suína (15,1%; 218/1441) e pneumonias bacterianas (15%; 216/1441). O diagnóstico de influenza apresentou uma frequência elevada de 2010 a 2013, totalizando 43,1% (167/387) dos casos. Após este período, ambas doenças infecciosas respiratórias exibiram caráter endêmico. As doenças infecciosas do sistema digestório totalizaram 10,5% (151/1441) dos diagnósticos, com as seguintes principais condições: enterocolite por Salmonella spp. (43,7%; 66/151), enteropatia proliferativa por Lawsonia spp. (41,7%; 63/151) e colite por Brachyspira spp. (14,6%; 22/151). A colite por Brachyspira spp. apresentou uma alta incidência de 2012 a 2014 com todos os casos detectados no período. As polisserosites e meningites bacterianas representaram 5,8% (84/1441) e 2,3% (33/1441) dos casos diagnosticados, respectivamente, com um caráter endêmico constante.(AU)

Age-specific Prevalence of Porcine Reproductive and Respiratory Syndrome Virus, Porcine Circovirus Type 2, and Mycoplasma hyopneumoniae in Korea Pig Farms

Porcine respiratory disease complex (PRDC) continues to be a significant economic problem to the swine industry. Porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and Mycoplasma hyopneumoniae (MH) are considered to be the most important pathogens that cause PRDC. In this study, we investigated the prevalence of antibodies against PRRSV and MH in the serum of sows and piglets from 89 domestic commercial pig farms by ELISA, and the presence of viral nucleic acids of PRRSV, including North American and European PRRS, and PCV2 was also investigated in the serum of sows and piglets from 89 domestic commercial pig farms by real-time PCR. In case of PRRSV, 78.7% (70/89) of sows were positive for PRRSV antibody, and 96.6% (86/89) of piglets were positive for PRRSV antibody. For MH, 76.4% (68/89) of sows showed positive for MH antibody. In the PRRSV viral nucleic acid detection experiment, 36.0% (32/89) of sows were positive for PRRSV nucleic acids, and virus nucleic acid was detected in 83.1% (74/89) of piglets. In case of virus type, both North American and European types were detected. In case of PCV2, 15.7% (14/89) of sows were positive for PCV2 nucleic acids. Conclusively, PCV2, PRRSV, and MH were widely distributed in pig farms in Korea. These prevalence data related with PRDC provides clinical information for vaccination strategy and development for the control of PRDC.

Porcine circovirus 3 capsid protein induces autophagy in HEK293T cells by inhibiting phosphorylation of the mammalian target of rapamycin / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Porcine circovirus 3 (PCV3) has been detected in major pig-producing countries around the world since its first report in the US in 2016. Most current studies have focused on epidemiological investigations and detection methods of PCV3 because of lack of live virus strains for research on its pathogenesis in porcine cells or even in pigs. We constructed a recombinant plasmid pCMV-Cap carrying the PCV3 orf2 gene to investigate the effects of capsid (Cap) protein expression on autophagic response in human embryonic kidney cell line 293T (HEK293T). We demonstrate that PCV3 Cap protein induced complete autophagy shown as formation of autophagosomes and autophagosome-like vesicles as well as LC3-II conversion from LC3-I via inhibiting phosphorylation of the mammalian target of rapamycin (mTOR) in HEK293T cells. The ubiquitin-proteasome pathway is also involved in the autophagy process. These findings provide insight for further exploration of PCV3 pathogenetic mechanisms in porcine cells.

Porcine circovirus 3 capsid protein induces autophagy in HEK293T cells by inhibiting phosphorylation of the mammalian target of rapamycin / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Porcine circovirus 3 (PCV3) has been detected in major pig-producing countries around the world since its first report in the US in 2016. Most current studies have focused on epidemiological investigations and detection methods of PCV3 because of lack of live virus strains for research on its pathogenesis in porcine cells or even in pigs. We constructed a recombinant plasmid pCMV-Cap carrying the PCV3 orf2 gene to investigate the effects of capsid (Cap) protein expression on autophagic response in human embryonic kidney cell line 293T (HEK293T). We demonstrate that PCV3 Cap protein induced complete autophagy shown as formation of autophagosomes and autophagosome-like vesicles as well as LC3-II conversion from LC3-I via inhibiting phosphorylation of the mammalian target of rapamycin (mTOR) in HEK293T cells. The ubiquitin-proteasome pathway is also involved in the autophagy process. These findings provide insight for further exploration of PCV3 pathogenetic mechanisms in porcine cells.

Combination of mass spectrometry and GST pull-down techniques to study potential interacting protein of PCV2 ORF4 / 生物工程学报

A novel protein encoded by the open reading frame 4 (ORF4) was recently discovered in porcine circovirus type 2 (PCV2). However, little is known about the interaction proteins of ORF4 which hindered better understanding the biological functions of ORF4 in the life cycle of PCV2. In the present study, the ORF4 was inserted into the multiple cloning site of pCMV-N-Flag-GST, yielding recombinant plasmid pCMV-N-Flag-GST-ORF4. The recombinant plasmid was transfected into 293T cells and the intracellular interaction complex of ORF4 were enriched and separated by GST pull-down and SDS-PAGE, sequentially. The potential interacting proteins of PCV2 ORF4 were stained with silver and identified by mass spectrometry (MS). Finally, five candidate ORF4-interacting proteins, including Serine/threonine-protein phosphatase 6 catalytic subunit, alpha cardiac muscle 1, actin, SEC14-like protein 5 and myosin 9 were identified. These results would benefit a better understanding of the biological function of ORF4 in PCV2 infected cells.

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.

Generation and characterization of recombinant classical swine fever virus C-strain expressing the cap protein of porcine circovirus type 2 / 生物工程学报

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a devastating viral disease in swine, leading to significant economic losses to the pig husbandry. C-strain is one of the best modified live vaccines against CSF. The vaccine is highly safe and efficacious and can provide rapid and complete protection against essentially all genotypes of CSFV. Co-infections of pigs with CSFV and porcine circovirus type 2 (PCV2) occur frequently in the field, making it difficult to control the associated diseases. Here, a recombinant C-strain rHCLV-Cap expressing the Cap protein of PCV2 was constructed and evaluated in vitro and in vivo. The recombinant had comparable phenotypes to C-strain in cell cultures and rabbits. At ten days post-immunization, anti-E2, but not anti-Cap, antibodies were detected in the rabbits inoculated with the recombinant virus. Our study warrants further work to construct C-strain-based bivalent vaccines.

Detection and genetic characterization of porcine circovirus 3 from aborted fetuses and pigs with respiratory disease in Korea

A novel porcine circovirus 3 (PCV3) was first detected in pigs showing porcine dermatitis and nephropathy syndrome, reproductive failure, and multisystemic inflammation in the USA. Herein, we report on PCV3 as a potential etiological agent of clinical signs, reproductive failure and respiratory distress on Korean pig farms, based on in situ hybridization, pathological, and molecular findings. Confirmation of the presence of PCV3 may increase co-infection with other causative agents of disease in Korean pig herds, indicating the need for further systemic investigation of pathogenicity and of multiple infections with PCV2 genotypes and bacteria, and the development of an effective PCV3 vaccine.

Porcine transcription factor AP-2δ promotes porcine circovirus type 2 replication through enhancing the activity of the rep gene promoter / 生物工程学报

Several putative transcription factor binding sites (TFBSs) exist in the PCV2 rep gene promoter. To explore if porcine circovirus type 2 (PCV2) could regulate the viral replication by using these TFBSs, we conducted electrophoretic mobility shift assay (EMSA), DNA-pull down and liquid chromatography-tandem mass spectrometric (LC-MS/MS) assays. EMSA confirmed the binding activity of the rep gene promoter with nuclear proteins of host cells. DNA-pull down and LC-MS/MS identified the porcine transcription factor AP-2δ (poTFAP2δ) could bind the PCV2 rep gene promoter. Dual-luciferase reporter assay, quantitative real-time PCR, Western blotting and indirect immunofluorescent assay demonstrated that poTFAP2δ could not only promote the activity of the rep gene promoter, but also enhance the transcription/translation activity of the rep/cap gene and the virus titer of PCV2 during the entire life cycle of PCV2 infection. This study revealed the molecular mechanism of PCV2 using host proteins to enhance the viral replication, provided a new perspective for studying the pathogenic mechanism of PCV2 from virus and host interactions, and provided a theoretical basis for developing highly effective PCV2 vaccines.

Genetic variations in open reading frame 2 gene of porcine circovirus type 2 isolated in Korea during 2016–2017 / 대한수의학회지

The capsid protein of porcine circovirus type 2 (PCV2) encoded by open reading frame 2 (ORF2) is important for neutralizing activity against PCV2 infection. This study investigated the heterogeneity of the ORF2 gene of PCV2 isolated in Korea during 2016–2017. The results revealed that PCV2d is currently the predominant genotype. Moreover, comparison of ORF2 from 17 PCV2 isolates revealed 88.3–100% homology at the nucleotide (deduced amino acid 86.3–100%) level. Interestingly, 61.5% (8/13) of the PCV2d isolates had glycine at position 210. These data provide a useful information for PCV2 epidemiology in Korea.

Porcine circovirus type 2 increases interleukin-1beta and interleukin-10 production via the MyD88–NF-kappa B signaling pathway in porcine alveolar macrophages in vitro

Porcine alveolar macrophages (PAMs) represent the first line of defense in the porcine lung after infection with porcine circovirus type 2 (PCV2) via the respiratory tract. However, PCV2 infection impairs the microbicidal capability of PAMs and alters cytokine production and/or secretion. At present, the reason for the imbalance of cytokines has not been fully elucidated, and the regulatory mechanisms involved are unclear. In this study, we investigated the expression levels and regulation of interleukin-1beta (IL-1β) and IL-10 in PAMs following incubation with PCV2 in vitro. Levels of IL-1β and IL-10 increased in PAM supernatants, and the distribution of nuclear factor kappa B (NF-κB) p65 staining in nucleus, expression of MyD88 and p-IκB in cytoplasm, and DNA-binding activity of NF-κB increased after incubation with PCV2, while p65 expression in PAM cytoplasm decreased. However, when PAMs were co-incubated with PCV2 and small interfering RNA targeting MyD88, those effects were reversed. Additionally, mRNA expression levels of Toll-like receptors (TLR)-2, -3, -4, -7, -8, and -9 increased when PAMs were incubated with PCV2. These results show that PCV2 induces increased IL-1β and IL-10 production in PAMs, and these changes in expression are related to the TLR–MyD88–NF-κB signaling pathway.

Duplex nested reverse transcriptase polymerase chain reaction for simultaneous detection of type 2 porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 from tissue samples

There are high levels of co-incidence of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in porcine tissue. This study established a duplex nested reverse transcriptase polymerase chain reaction (RT-PCR) method that targets the genomic RNA of type 2 PRRSV and the mRNA of PCV2 in infected tissues. The method amplified discriminative bands of 347 bp and 265 bp specific for type 2 PRRSV and PCV2, respectively. The limits of detection of the duplex nested RT-PCR were 10(1.5) TCID₅₀/mL for type 2 PRRSV and 10² infected cells/mL for PCV2. The kappa statistic, which measures agreement between methods, was 0.867, indicating a good level of agreement. This RNA-based duplex RT-PCR approach can be another way to detect type 2 PRRSV and PCV2 simultaneously and with improved convenience.

Analysis of genetic variation diversity of porcine circovirus-2 virus genome isolated from Shanxi area / 中国兽医学报

In order to study genetic variation diversity of porcine circovirus type 2 (PCV2) strains in Shanxi,the genomic sequences of nine PCV2 strains including SXQX,SXCZ,SXTY2,SXJC,SXJX,SXLL,SXPY,SXPG and SXXY recently isolated from some areas of Shanxi from 2013 to 2016,was cloned,sequenced and received by GenBank.The amplified PCV2 genomic sequences,ORF2 sequences and Cap protein amino acid of these nine strains were analysed and compared with those of published 28 PCV2 strains by DNAStar,drawing phylogenetic tree.The results showed that the genomic sequences of SXJX,SXJC and SXXY PCV2 strains were 1 768 bp,and the others were 1 767 bp,which accounted for 33％ and 67％,respectively.The homologies of nucleotide sequences of the nine strains were 94.7％-99.8％,the homologies of nucleotide sequences of the nine strains with the 28 isolates from different regions of the world PCV strain were 93.9％-99.9％,and the homologies of nucleotide sequences of the nine strains with the domestic vaccine strains were 95.1％-99.8％.The phylogenetic analysed that SXJX,SXJC and SXXY belonged to genotype PCV-2D,SXLL,SXPY and SXCZ belonged to genotype PCV-1C,and SXTY14,SXPG and SXQX belonged to genotype PCV-1A/1B.Thus it proved that the epidemic strain of PCV2 was mainly PCV-2b in Shanxi.The homologies of ORF2 nucleotide sequences and Cap amino acid of the nine strains were 90.0％-100.0％ and 87.1 ％-100.0％ respectively,the homologies of ORF2 nucleotide sequences and Cap amino acid of the nine strains with the 28 isolates from different regions of the world PCV strain were 87.6％-100.0％ and 84.1％-100.0％ respectively,and the homologies of ORF2 nucleotide sequences and Cap amino acid of the nine strains with the domestic vaccine strains were 91.0％-100.0％ and 89.3％-100.0％ respectively.The Cap amino acids of SXQX,SXJX,SXTY14,SXPG,SXJC and SXXY PCV2 were 233,ORF2 of SXQX,SXTY14 and SXPG located at 1 033-1 734 bp,ORF2 of SXXY,SXJX and SXJC located at 1 033-1 734 bp,and the Cap amino acids of SXCZ,SXLL and SXPY PCV2 were 234,ORF2 of them located at 1 030-1 734 bp,in addition,the positions of 1 030-1 734 bp were more three bases TCA than other ORF2 genome sequence of 1 767 bp,resulting in increasing a K (Lys) of amino acid sequencein at the 234 position.Also Cap protein of 9 PCV2 strains showed more amino acid variation in addition to the only high-ly conserved glycosylation sites (NYS) (pp.143-145 amino acid).It provided theoretical basis for the PCV2 immune prevention of research in Shanxi,and the data of basic theory of molecular pathogenesis of PCV2.

A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection / Sandwich ELISA com duplo anticorpo baseado no circovirus suíno tipo 2 (PCV2) produzido em cultivo celular para detecção de anticorpos

Few studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm3. Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms.(AU)

Há poucos relatos na literatura de métodos de ELISA (Enzyme-linked immunosorbent assay), para a detecção de anticorpos contra o circovírus suíno tipo 2 (PCV2), baseados em antígenos produzidos em cultivo celular, bem como uma escassez de trabalhos descrevendo técnicas de purificação viral para os membros da família Circoviridae. Isso ocorre, pois os circovírus são de difícil isolamento, não causam efeito citopático e produzem um baixo título viral em cultivo celular. Assim, para superar essas dificuldades encontradas no cultivo do PCV2, este estudo objetivou desenvolver um sandwich ELISA com duplo anticorpo, baseado no antígeno de PCV2 produzido em cultivo celular, para a quantificação de anticorpos anti-PCV2. Um colchão de sacarose descontínuo a 20% e 50% foi utilizado para a purificação viral, o qual possibilitou a separação das proteínas oriundas do cultivo celular no colchão de sacarose a 20% e uma maior concentração viral no colchão de sacarose a 50%. Com a ultracentrifugação isopícnica, o PCV2 ficou mais concentrado na banda com valores de densidade de 1,330 a 1,395g/cm3. A purificação viral foi avaliada pelas técnicas de SDS-PAGE, ELISA indireto e microscopia eletrônica. Assim, o método de ELISA padronizado revelou uma forte correlação linear (r = 0,826, p <0,001) quando comparado com um kit de ELISA comercial. O ensaio demonstrou baixa variabilidade (coeficientes de variação inter-teste de 4,24% e intra-teste de 1,80%) e uma excelente especificidade analítica conferida pelo anticorpo de captura produzido em coelho. Portanto, o método de ELISA demonstrou ser rápido, específico e conveniente para a detecção de anticorpos contra o PCV2 em estudos de infecção natural e experimental, além da monitoria da resposta à vacinação contra o PCV2 em granjas comerciais.(AU)

Recent vaccine technology in industrial animals

Various new technologies have been applied for developing vaccines against various animal diseases. Virus-like particle (VLP) vaccine technology was used for manufacturing the porcine circovirus type 2 and RNA particle vaccines based on an alphavirus vector for porcine epidemic diarrhea (PED). Although VLP is classified as a killed-virus vaccine, because its structure is similar to the original virus, it can induce long-term and cell-mediated immunity. The RNA particle vaccine used a Venezuela equine encephalitis (VEE) virus gene as a vector. The VEE virus partial gene can be substituted with the PED virus spike gene. Recombinant vaccines can be produced by substitution of the target gene in the VEE vector. Both of these new vaccine technologies made it possible to control the infectious disease efficiently in a relatively short time.