RESUMO
Bovine viral diarrhea virus (BVDV) and bovine gammaherpesvirus 4 (BoHV-4) infect the uterus of cattle, being responsible for huge economic losses. Most of the pathogenesis of BoHV-4 in the bovine reproductive tract has been elucidated by conducting tests on primary cultures. Thus, it is important to have optimal in vitro conditions, avoiding the presence of other pathogens that can alter the results. BVDV is one of the most frequent viral contaminants of cell cultures. Considering that non-cytopathic (NCP) BVDV biotype can generate persistently infected (PI) cattle, which are the major source for virus transmission in susceptible herds, it is important to check products derived from cattle that are intended to be used in research laboratories. The aim of this work was to evaluate how the natural infection of bovine endometrial cells (BEC) with a NCP BVDV strain (BEC + BVDV) affects BoHV-4 replication. We have demonstrated a delay in BoHV-4 gene expression and a decrease in viral load in the extracellular environment in BEC + BDVD cells compared to BEC (BVDV-free) cells. These results confirm that replication of BoHV-4 in BEC primary cultures is affected by previous infection with BVDV. This finding highlights the importance of ruling out BVDV infection in bovine primary cell cultures to avoid biological interference or misinterpretation of results at the time of performing in vitro studies with BoHV-4.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina , Coinfecção , Vírus da Diarreia Viral Bovina Tipo 1 , Vírus da Diarreia Viral Bovina , Animais , Bovinos , Coinfecção/veterinária , Diarreia , FemininoRESUMO
Neurons of the dorsal root ganglia (DRG) express a diversity of voltage-gated sodium channels. From rat DRG we have cloned and functionally expressed a tetrodotoxin-sensitive sodium channel alpha subunit, NaCh6/Scn8a/rPN4, and a splice variant, rPN4a. Primary structure analysis shows NaCh6/Scn8a/rPN4 to be highly homologous (99%) to NaCh6 and most likely represents the same transcript. The splice variation in rPN4a is homologous in sequence and location to that of rat brain I. Tissue distribution analyzed by RT-PCR showed NaCh6/Scn8a/rPN4 to be expressed at its highest levels in rat brain, at moderate levels in spinal cord, and at lower levels in DRG, nodose ganglia, and superior cervical ganglia and to be absent from sciatic nerve, heart, and skeletal muscle. In contrast, rPN4a shows no expression in brain and low-level expression in spinal cord, whereas in DRG its expression is comparable to that of NaCh6/Scn8a/rPN4. Functional analysis of these channels expressed in Xenopus oocytes showed that NaCh6/Scn8a/rPN4 and rPN4a exhibited similar properties, with V(1/2) approximately -100 mV for steady-state inactivation and V(1/2) approximately -40 mV for activation. rPN4a recovered from inactivation significantly faster than NaCh6/Scn8a/rPN4. NaCh6/Scn8a/rPN4 was inhibited by tetrodotoxin with an IC50 approximately 1 nM. Coexpression of the beta1 subunit accelerated inactivation kinetics, but the beta2 subunit was without effect.
Assuntos
Processamento Alternativo , Gânglios Espinais/metabolismo , Ativação do Canal Iônico , Canais de Sódio/fisiologia , Sequência de Aminoácidos , Animais , Masculino , Dados de Sequência Molecular , Oócitos , Especificidade de Órgãos , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Canais de Sódio/biossíntese , Canais de Sódio/genética , Canais de Sódio/isolamento & purificação , Xenopus laevisRESUMO
Dorsal root ganglion neurons express a wide repertoire of sodium channels with different properties. Here, we report the cloning from rat, dorsal root ganglia (DRG), cellular expression, and functional analysis of a novel tetrodotoxin-sensitive peripheral sodium channel (PN), PN1. PN1 mRNA is expressed in many different tissues. Within the rat DRG, both the mRNA and PN1-like immunoreactivity are present in small and large neurons. The abundance of sodium channel mRNAs in rat DRG is rBI > PN1 >/= PN3 >>> rBIII by quantitative reverse transcription-polymerase chain reaction analysis. Data from reverse transcription-polymerase chain reaction and sequence analyses of human DRG and other human tissues suggest that rat PN1 is an ortholog of the human neuroendocrine channel. In Xenopus oocytes, PN1 exhibits kinetics that are similar to rBIIa sodium currents and is inhibited by tetrodotoxin with an IC50 of 4.3 +/- 0.92 nM. Unlike rBIIa, the inactivation kinetics of PN1 are not accelerated by the coexpression of the beta-subunits.
Assuntos
Gânglios Espinais/metabolismo , Neurônios/metabolismo , Neuropeptídeos , Canais de Sódio/biossíntese , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar , Humanos , Substâncias Macromoleculares , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.7 , Oócitos/fisiologia , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos , Canais de Sódio/química , Canais de Sódio/fisiologia , Tetrodotoxina/farmacologia , Transcrição Gênica , Xenopus laevisRESUMO
Small neurons of the dorsal root ganglia (DRG) are known to play an important role in nociceptive mechanisms. These neurons express two types of sodium current, which differ in their inactivation kinetics and sensitivity to tetrodotoxin. Here, we report the cloning of the alpha-subunit of a novel, voltage-gated sodium channel (PN3) from rat DRG. Functional expression in Xenopus oocytes showed that PN3 is a voltage-gated sodium channel with a depolarized activation potential, slow inactivation kinetics, and resistance to high concentrations of tetrodotoxin. In situ hybridization to rat DRG indicated that PN3 is expressed primarily in small sensory neurons of the peripheral nervous system.
