Validation of a real-time PCR assay for detection of swinepox virus.

Swine are the only known hosts of swinepox virus (SWPV), the sole member of the genus Suipoxvirus, family Poxviridae. Rapid diagnosis is recommended for appropriate interventions because of the high morbidity associated with this virus. This study describes a real-time quantitative PCR (qPCR) assay for rapid detection and quantification of SWPV. The detection limit, repeatability, reproducibility, and specificity of this assay were determined. The efficiency was 96%, and the R2 value was 0.996. The detection limit was 1 fg or 10-0.5 TCID50/50 µL. Tests showed that the greatest source of error in the SWPV qPCR assay was variation between analysts rather than different qPCR kits or equipment. All nucleic acids from other viruses or samples collected from swine were negative in the specificity test. qPCR for SWPV is a new method with tested variables that allows main sources of error in laboratory diagnosis and viral quantification to be identified.

Phylodynamics of classical swine fever virus in Brazil.

The classical swine fever virus is the etiologic agent of one of the diseases with the greatest impact on swine farming worldwide. An extensive area of Brazil is considered free of the disease, but some states in Northeast Brazil have registered outbreaks since 2001. The objective of this study was to analyze the genetic variations of the virus and its spread over time and space. Partial sequences of the viral E2 protein obtained from samples collected during the Brazilian outbreaks were compared with sequences from the GenBank database (NCBI). The results demonstrated the continuous presence of the virus in the state of Ceará, with diffusion to at least two other states. The Brazilian Northeast virus presents specific polymorphisms that separate it from viruses isolated in other countries.

Prostaglandin A1 inhibits the replication of bovine viral diarrhea virus.

Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10µg/mL).

Prostaglandin A1 triggers Mayaro virus inhibition and heat shock protein 70 expression in an epithelial cell model.

INTRODUCTION: The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment. METHODS: Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6µg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively. RESULTS: PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography. CONCLUSIONS: PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.

Prostaglandin A1 triggers Mayaro virus inhibition and heat shock protein 70 expression in an epithelial cell model

Abstract INTRODUCTION: The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment. METHODS: Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6μg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively. RESULTS: PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography. CONCLUSIONS: PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.

Prostaglandin A1 inhibits the replication of bovine viral diarrhea virus

ABSTRACT Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5 µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10 µg/mL).

Effect of prostaglandin A1 in porcine cells persistently infected with classical swine fever virus.

Cyclopentenone prostaglandins are potent inhibitors of a wide variety of RNA and DNA viruses. In this report we describe that prostaglandin A1 (PGA1) potently inhibited the replication of classical swine fever virus in cultures of PK-15 cells. The highest non-toxic dose (5 microg/ml) inhibited virus yield in 99% at the initial phase of infection and in 77% in persistent infected cells. However when PGA1 was removed from persistently infected cells, the inhibition of virus replication was partially reverted.

Implementação da técnica de imuno-histoquímica (IHQ) para o diagnóstico do circovírus suíno tipo-2 (CVS-2) / Implementation of immune-histochemical (IHC) technique applied to diagnostic of porcine circovirus type-2 antigen in swine tissues

Circovírus Suíno Tipo-2 (CVS-2) é um virus não-envelopado, DNA fita única circular, classificado na família Circoviridae, associado à Síndrome Multissistêmica do Definhamento dos Suínos (SMDS). A SMDS é considerada uma doença emergente, multifatorial e de distribuição cosmopolita. Os sinais clínicos são inespecíficos e o diagnóstico, além dos achados de necropsia e histopatologia compatíveis com SMDS, deve ser corroborado pela demonstração do ácido nucléico e (ou) do antígeno viral nas lesões. A técnica de Imuno-Histoquímica (IHQ) permite a demonstração de antígenos virais pela aplicação de anticorpos específicos e conjugados de anticorpos. A implementação da IHQ foi realizada aplicando-se anticorpo policlonal anti – CVS -2 em 23 blocos de cortes histológicos, fixados com formalina e embebidos em parafina, sabidamente positivos ou negativos para o CVS-2. A análise histológica prévia demonstrou que os tecidos positivos continham lesões características da SMDS. O genoma do CVS-2 foi demonstrado pela Hibridização “in situ” (HIS). A IHQ implementada foi testada em 16 blocos com de três a quatro amostras de tecidos de animais, totalizando, respectivamente, 48 a 64 amostras de tecido de suínos com suspeita clínica da SMDS. Em dez blocos (40 amostras de tecidos) foram confirmadas pela detecção do genoma viral. Os resultados obtidos pela aplicação da IHQ padronizada foram correlacionados com as lesões histopatológicas compatíveis com a SMDS. A IHQ vem sendo aplicada com êxito no rastreamento do vírus em suídeos de diversas granjas brasileiras onde se estabelece a suspeita de ocorrência de SMDS.

Classical Swine Fever in Brazil: study for the survey of classical swine fever outbreaks in Brazil from 1978 to 2004 / Peste Suína Clássica no Brasil: estudo para a avaliação dos surtos depeste suína clássica no Brasil de 1978 a 2004

The programs developed in Brazil with the aim to control and eradicate swine fever provided an opportunityfor the survey of Classical Swine Fever (CSF) outbreaks. Were concerned CSF official programs, strategies and results, during 26 years. Based in epizootic official data we showed that the number of CSF outbreaks from 1978 to 2004 drastically decreased in all country, although different eradicating strategies were applied in those official programs, especially in fourteen States of "CSF Free Zone". Were evaluated both CSF official programs: Swine Pests Combat Program (SPCP) from 1984 to 1991 and CSF Eradication and Control Program (CSFECP) from 1992 to 2004 by the decreasing of CSF outbreaks number. Considering the technical evolution in swine production systems, statistical analysis to compare the ranking of CSFoutbreaks in each program was performed by Mann-Whitney test, that showed at 95% confidence level(Table T) a significant difference (p< 0.05) between programs, as suggested in CSF outbreaks profileplotted diagram. The number of CSF outbreaks occurred from 2000­2004 in "CSF-infected" and "CSF-free" zones, was analyzed. Also, we regarded with most important recent CSF outbreak in Brazil occurred in 1997, during CSFECP, that was figured out by stamping out measures without appealing to preventive vaccination regimen. Those results suggest that the efficacy of implemented CSF eradication programs depends on the continuity of defined strategies as rigorous vigilance, notification, virus diagnostic screening and sanitary police measures in order to enable quick and adequate action upon CSFV detection

Os programas oficiais para o controle e erradicação de pestes suínas forneceram uma oportunidade de levantar o perfil de ocorrência da Peste Suína Clássica (PSC). Independente das estratégias aplicadasdurante 26 anos foi demonstrado que o número de surtos de PSC de 1978 até 2004 caiu drasticamente emtodo país, especialmente nos quatorze Estados inclusos na "Zona Livre de PSC". O estudo comparou o número de surtos de PSC durante a vigência do Programa de Combate às Pestes Suínas (PCPS) de 1984a 1991 e o Programa de Controle e Erradicação da PSC (PCEPSC) de 1992 a 2004. Considerando aevolução tecnológica nos sistemas de produção de suínos, a diferença nos resultados obtidos após aimplementação de cada programa foi avaliada pelo teste estatístico Mann Whitney por meio da ordenaçãodo número de surtos ocorridos. Essa análise demonstrou uma diferença significativa (p< 0,05) entre osprogramas no nível de confiança de 95% (Tabela T) com havia sido sugerido pelo diagrama do perfil deocorrência da PSC. A eficácia do PCEPSC para debelar o mais importante surto de PSC ocorridorecentemente no Brasil, em 1997, também foi considerada. Paralelamente, o número de surtos ocorridosde 2000 a 2004 nas áreas infectadas com a PSC e na zona livre de PSC foi avaliado. Os resultados sugeremque a eficácia dos programas de erradicação depende da continuidade das estratégias definidas como avigilância rigorosa, notificação, rastreamento do vírus e medidas sanitárias que agilizem a ação nomomento de detecção de vírus da PSC