ABSTRACT Vesicular stomatitis virus is the causative agent of the vesicular stomatitis disease, which mainly affects cattle, swine, and horses causing significant economic losses. Many other animal species, including humans, can be also affected by the disease. Up to now, the epidemiology of vesicular stomatitis disease is not well understood. Previous epidemiological studies in Central America described the existence of an association between the presence of vesicular stomatitis New Jersey virus antibodies and environmental risk factors such as mean annual rainfall, temperature, and elevation. Additionally, molecular epidemiology studies revealed that the phylogenetic relationship between vesicular stomatitis New Jersey virus isolates is more related to ecological factors than temporal factors. We have performed a genetic analysis of vesicular stomatitis virus samples isolated from animals in different geographical regions in Venezuela. We sequenced the hypervariable region of the phosphoprotein gene of 51 samples collected over eight years. The virus was found in a wide range of environmental conditions, at high and low altitudes and with variable levels of precipitation and temperatures. In contrast with previous reports, our phylogenetic analysis showed that viruses from different ecological regions did not present significant genetic differences. cattle swine losses species humans now understood rainfall temperature elevation Additionally Venezuela 5 years conditions temperatures reports differences

RESUMO: Pseudorraiva (PR) é uma doença viral altamente contagiosa de grande importância sanitária e econômica na indústria suína. O objetivo deste estudo foi avaliar diferentes regiões genômicas, químicas de PCR em tempo real e equipamentos para o diagnóstico molecular de PR. Foram avaliados quatro genes (GB, GC, GE, gD), três diferentes produtos químicos (SybrGreen, sondas de hidrólise e plexor) e dois equipamentos (ABI7500, Rotorgene 3000). Oligonucleotídeos com alvo para gB baseados na química de sondas de hidrólise apresentaram o melhor desempenho nos testes de eficiência (99%), de limite de detecção (10-1.5 TCID50 mL-1) e sensibilidade diagnóstica. Portanto, estes foram selecionados para fatores de verificação de desempenho, tais como a repetibilidade, reprodutibilidade e robustez (1,39% de variância entre dias, 24% variância entre analistas e 4,07% de variância por erro de análise). A qPCR, padronizada e validada neste trabalho, mostrou-se confiável para o diagnóstico de PR e pode ser utilizada em laboratórios de diagnóstico que se seguem normas internacionais como ISO 17025 e ISO 16140.

ABSTRACT: Pseudorabies (PR) is a highly contagious viral disease of great animal health and economic importance in swine industry. The aim of this study was to evaluate different genomic regions, real-time PCR chemistries and equipment for the molecular diagnosis of PR. Eight primer pairs targeting four genes (gB, gC, gE, gD), three different qPCR chemistries (SybrGreen, hydrolysis probes and plexor) and two equipment (ABI7500, Rotorgene 3000) were evaluated. Oligonucleotides targeting gB using hydrolysis probes showed the best performance after evaluating efficiency (99%), the detection limit (10-1.5 TCID50 mL-1) and diagnostic sensitivity and; therefore, those primers were selected for performance verification factors such as repeatability, reproducibility and robustness (1.39% variance between days, 24% variance between analysts and 4.07% variance in analysis error). The qPCR standardized and validated in this research proved to be reliable for the diagnosis of PR and may be used in diagnostic laboratories that follow ISO 17025 and ISO 16140.

Complexo de Doenças Respiratórias de Suínos (CDRS ) é um grupo de doenças que causam grandes perdas na indústria suína. Vários agentes infecciosos estão relacionados com a CDRS , entre eles o circovírus suíno 2 (PCV -2), vírus da pseudo-raiva (SuHV -1) , Haemophilus parasuis (HP) ,Mycoplasma hypneumoniae (MH ) e Pasteurela multocida (PM). O objetivo com este estudo foi desenvolver PCR em tempo real (qPCR) para a detecção destes agentes infecciosos. Os oligonucleotídeos foram concebidos para cada agente infeccioso específico e marcado com fluoróforos diferentes para amplificar partes específicas do genoma em dois grupos de reacções , uma qPCR dúplex em SuHV -1 e PCV- 2 e uma qPCR multiplex para detectar as três bactérias simultaneamente. As reações foram testadas em 142 amostras de pools de linfonodos e pulmões de suínos com sinais clínicos de CDRS. Foram detectadas 135 amostras positivas para PCV- 2 , 61 para a HP, 29 para PM e 30 para MH e zero para SuHV -1, dentre esses foram registrados 76 casos de co-infecção . As qPCRs desenvolvidas neste estudo são ferramentas úteis no diagnóstico da CDRS.

Porcine Respiratory Disease Complex (PRDC) is a group of diseases that cause high losses in the swine industry. Several infectious agents are related to PRDC including porcine circovirus 2 (PCV-2), pseudorabies virus (SuHV-1),Haemophilus parasuis (HP), Mycoplasma hypneumoniae (MH) and Pasteurela multocida (PM). The aim of this study was to develop real-time PCRs (qPCR) for the detection of these infectious agents. Oligonucleotides were designed for each specific infectious agent and labeled with different fluorophores to amplify specific parts of the genome. This was done in two groups of reactions—a duplex qPCR for SuHV-1 and PCV-2 and a multiplex qPCR to detect the three bacteria simultaneously. The reactions were tested in 142 pooled samples of swine lymph nodes and lungs with clinical signs of PRDC. There were 135 samples that tested positive for PCV-2, 61 for HP, 29 for PM, 30 for MH and zero for SuHV-1. We recorded 76 cases of co-infection. The qPCRs developed in this study are useful tools in the diagnosis of PRDC.