RESUMEN
Bovine alphaherpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses. BoHV-5 causes non-suppurative meningoencephalitis in calves. BoHV-1 is associated with several syndromes and, occasionally, can cause encephalitis. Although both viruses are neurotropic and they share similar biological properties, it is unknown why these alphaherpesviruses differ in their ability to cause neurological disease. Neural tissue samples were collected from BoHV-1- and BoHV-5-intranasally inoculated calves during acute infection, latency and reactivation and the levels of cyclins mRNA expression were analyzed by qRT-PCR. Striking differences in the levels of cyclins mRNA were particularly detected in trigeminal ganglion (TG). The expression levels of cyclins in TG during BoHV-5 latency suggest that these viruses utilize different strategies to persist in the host. It is apparent that a relationship between virus loads and cyclin mRNA levels can be established only during acute infection and other factors might be involved in the regulation of cell cycle components during BoHV latency and reactivation. Bovine alphaherpesviruses neuropathogenicity might be influenced by the differential control of cell cycle components by these herpesviruses. This is the first report on BoHV-5 modulation of cyclins expression in neural tissues from its natural host.
Asunto(s)
Enfermedades de los Bovinos/patología , Ciclinas/biosíntesis , Encefalitis Viral/veterinaria , Infecciones por Herpesviridae/veterinaria , Herpesvirus Bovino 1/crecimiento & desarrollo , Herpesvirus Bovino 5/crecimiento & desarrollo , Meningoencefalitis/veterinaria , ARN Mensajero/biosíntesis , Animales , Encéfalo/patología , Bovinos , Ciclinas/genética , Encefalitis Viral/patología , Perfilación de la Expresión Génica , Infecciones por Herpesviridae/patología , Meningoencefalitis/virología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Bovine herpesvirus type 1 and type 5 (BoHV-1 and BoHV-5) are two alphaherpesviruses that affect cattle with two different syndromes. While BoHV-1 mainly produces respiratory symptoms, BoHV-5 is highly neuropathogenic and responsible for meningoencephalitis in young cattle. The latency-related (LR) gene, which is not conserved between these two herpesviruses, is the only viral gene abundantly expressed in latently infected neurons. The antiapoptotic action of this gene has been demonstrated during acute infection and reactivation from latency and seems to be mainly mediated by a LR protein (ORF-2) which is truncated in amino acid 51 in the case of BoHV-5. In this work, we show that the BoHV-5 LR gene is less efficient at cell survival and apoptosis inhibition in transient as well as in established neuronal cell lines compared to its BoHV-1 homolog. We hypothesize that the BoHV-5 LR gene may have novel functions that are lacking in the BoHV-1 LR gene and that these differences may contribute to its enhanced neuropathogenesis.
Asunto(s)
Infecciones por Herpesviridae/veterinaria , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 5/genética , Rinotraqueítis Infecciosa Bovina/metabolismo , Meningoencefalitis/veterinaria , Proteínas Virales/genética , Latencia del Virus/genética , Animales , Apoptosis/genética , Bovinos , Línea Celular , Expresión Génica , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Herpesvirus Bovino 1/crecimiento & desarrollo , Herpesvirus Bovino 1/metabolismo , Herpesvirus Bovino 5/crecimiento & desarrollo , Herpesvirus Bovino 5/metabolismo , Interacciones Huésped-Patógeno/genética , Rinotraqueítis Infecciosa Bovina/patología , Rinotraqueítis Infecciosa Bovina/virología , Meningoencefalitis/patología , Meningoencefalitis/virología , Neuronas/metabolismo , Neuronas/virología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Virales/metabolismo , Activación ViralRESUMEN
Oncolytic viruses have the ability to infect tumor cells and leave healthy cells intact. In this study, bovine herpesvirus 1 (BHV1; Los Angeles, Cooper, and SV56/90 strains) and bovine herpesvirus 5 (BHV5; SV507/99 and GU9457818 strains) were used to infect two neuronal tumor cell lineages: neuro2a (mouse neuroblastoma cells) and C6 (rat glial cells). BHV1 and BHV5 strains infected both cell lines and positively correlated with viral antigen detection (p < 0.005). When neuro2a cells were infected by Los Angeles, SV507/99, and GU9457818 strains, 40 % of infected cells were under early apoptosis and necroptosis pathways. Infected C6 cells were >40 % in necroptosis phase when infected by BHV5 (GU9457818 strain). Blocking caspase activation did not interfere with cell death. However, when necroptosis was blocked, 60-80 % of both infected cells with either virus switched to early apoptosis pathway with no interference with virus replication. Moreover, reactive oxygen species production and mitochondrial membrane dysfunction were detected at high levels in both infected cell lines. In spite of apoptosis and necroptosis blockage, tumor necrosis factor alpha (TNFA) and virus transcription were positively correlated for all viral strains studied. Thus, these results contribute to the characterization of BHV1 and BHV5 as potential oncolytic viruses for non-human cells. Nonetheless, the mechanisms underlying their oncolytic activity in human cells are still to be determined.
Asunto(s)
Apoptosis/genética , Herpesvirus Bovino 1/crecimiento & desarrollo , Herpesvirus Bovino 5/crecimiento & desarrollo , Necrosis/virología , Neuroglía/virología , Neuronas/virología , Animales , Antígenos Virales/genética , Bovinos , Línea Celular Tumoral , Expresión Génica , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 5/genética , Interacciones Huésped-Patógeno , Humanos , Ratones , Mitocondrias/metabolismo , Mitocondrias/virología , Necrosis/genética , Necrosis/patología , Neuroglía/metabolismo , Neuroglía/patología , Neuronas/metabolismo , Neuronas/patología , Virus Oncolíticos/genética , Virus Oncolíticos/crecimiento & desarrollo , Especificidad de Órganos , Estrés Oxidativo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Replicación ViralRESUMEN
O herpesvírus bovino tipo 1 (BoHV-1), é responsável por manifestações clínicas como a rinotraqueíte infecciosa bovina, abortamentos, conjuntivite, balanopostite e vulvovaginite pustular infecciosa. Esse vírus tem sido responsável por grandes prejuízos produtivos e reprodutivos em diversos rebanhos do país. Deste modo, o objetivo desse trabalho foi estimar a frequência de anticorpos contra o BoHV-1 em fêmeas bovinas de corte, não vacinadas na Microrregião de Imperatriz, Maranhão; além de identificar a faixa etária mais acometida pelo vírus, bem como realizar um estudo dos fatores associados à infecção do vírus e avaliar a técnica ELISA indireto utilizando a soroneutralização (SN) como padrão de referência. O estudo foi realizado em 48 rebanhos de corte, distribuídos em 12 municípios da Microrregião de Imperatriz. As amostras foram coletadas de fêmeas bovinas estratificadas em três faixas etárias, ? 12 meses, entre 12 e 36 meses e ? 36 meses de idade. As amostras foram submetidas a dois testes sorológicos, ELISA indireto e SN. Em cada rebanho, foi aplicado um questionário epidemiológico, com o objetivo de obter informações sobre manejo sanitário e reprodutivo, para o estudo de fatores de risco. A frequência de anticorpos contra o BoHV-1 na Microrregião de Imperatriz foi de 63,23%, os municípios de Açailândia e Buritirana, apresentaram as maiores frequências, ambos com 80,44%, a faixa etária mais acometida, na Microrregião, foi a dos animais com idade ? 36 meses (69,65%). Com base nos resultados obtidos pode-se concluir que a frequência de anticorpos contra o BoHV-1 é alta; entre as faixas etárias mais acometidas foram a dos animais com idade ? 36 meses; foram considerados fatores de risco para a transmissão do vírus, retorno ao cio, reposição de animais oriundos de outros estados/região e a criação de caprinos/ovinos associados com bovinos; a técnica ELISA indireta apresentou concordância moderada quando comparada a técnica de SN, que é a técnica padrão ouro de diagnóstico para BoHV-1
Asunto(s)
Femenino , Animales , Bovinos , Herpesvirus Bovino 1/crecimiento & desarrollo , Herpesvirus Bovino 1/fisiología , Herpesvirus Bovino 1/patogenicidad , Ensayo de Inmunoadsorción Enzimática , Ensayo de Inmunoadsorción Enzimática/veterinariaRESUMEN
The aim of this work was to study the in vitro replication of bovine herpesvirus types 1 and 5 (BoHV-1 and 5) at the beginning and end of the logarithmic growth phase of Madin-Darby Bovine Kidney (MDBK) cells. The replication kinetics and size of lysis and infection plaques of the field isolates 09/210 (BoHV-1) and 97/613 (BoHV-5) and the reference strains BoHV-1.1 Los Angeles 38 (LA38), BoHV-1.1 Cooper, BoHV-5a N569 and BoHV-5b A663 were evaluated. The highest mean virus titre was recorded for N569, followed by LA38 and 97/613. For most of the viruses, the virus titre values increased from 24 h post-infection (hpi) up to 48 hpi and then, they remained unchanged up to 72 hpi. However, the virus titre for 09/210 was significantly lower and a slight, steady increase was observed from 24 to 72 hpi. Furthermore, the largest lysis and infection plaques were recorded for 97/613 and LA38, respectively. According to this work, it is evident that there is a relationship between the replication of BoHV and the multiplication stage of MDBK cells. The results of this study contribute to the understanding of the replication behaviour in cell cultures of several strains of BoHV, which is critical for the rational design of in vitro experiments and vaccine production.
Asunto(s)
Herpesvirus Bovino 1/fisiología , Herpesvirus Bovino 5/fisiología , Replicación Viral , Animales , Bovinos , Línea Celular , Proliferación Celular , Herpesvirus Bovino 1/crecimiento & desarrollo , Herpesvirus Bovino 5/crecimiento & desarrollo , Factores de Tiempo , Carga Viral , Ensayo de Placa Viral , Cultivo de Virus/métodosRESUMEN
Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses infecting cattle. In countries where both viruses circulate, co-infection of cattle is likely. It was shown that recombination occurs at a high frequency in cattle infected dually with two BoHV-1 mutants. In addition, interspecific recombinants are generated in cell culture co-infected with BoHV-1 and BoHV-5. Even if the process of interspecific recombination appears inefficient relative to intraspecific recombination, BoHV-1 and BoHV-5 may give rise to interspecific recombinants in co-infected cattle. Since molecular tools for differentiating BoHV-1 from BoHV-5 are limited and do not allow to localize recombination events between these closely related virus species, 13 PCR sequencing assays were developed to discriminate between BoHV-1 and BoHV-5 at regular intervals throughout the entire respective viral DNA genomes. These assays were used to determine the genetic background of two interspecific BoHV-1/-5 recombinants generated previously. The two crossover points where recombination events occurred between the parental strains were determined. This study provides a detailed analysis of two interspecific recombinant viruses generated in vitro from closely related alphaherpesviruses infecting the same natural host. It demonstrates that recombination can occur within very short fragments of sequence homology. This finding raises questions about the mechanisms involved in the strands exchange and resolution step of the homologous recombination used by herpesviruses. This method will allow monitoring generation of recombinants between closely related herpesvirus species both in vitro and in vivo.
Asunto(s)
ADN Viral/genética , Herpesvirus Bovino 1/crecimiento & desarrollo , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 5/crecimiento & desarrollo , Herpesvirus Bovino 5/genética , Reacción en Cadena de la Polimerasa/métodos , Recombinación Genética , Animales , Secuencia de Bases , Bovinos , Línea Celular , Herpesvirus Bovino 1/clasificación , Herpesvirus Bovino 5/clasificación , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
This report describes an alternative technique to inoculate rabbits and to reproduce infection by Bovine herpesvirus 1 and 5. First, the nostrils are anaesthetized by aspersion with local anaesthetic. A few seconds later, and after proving the insensitivity of the zone, the rabbits are put on their back legs with their nostrils upwards and the inoculum is introduced slowly into each nostril by using disposable droppers. Clinical signs, viral isolation from nasal swabs, histological lesions found, positive polymerase chain reaction and antibodies production confirm the infection. This very simple and bloodless technique, where the animals are exposed to minor distress, may be useful for evaluating the virulence of BoHV-1 and BoHV-5 strains, to study the establishment of latent virus infection and to test the potential of experimental vaccines or properties of antiviral drugs. It may be also suitable for experimental infection with other respiratory viruses in this animal model.
Asunto(s)
Encefalitis Viral/virología , Infecciones por Herpesviridae/virología , Herpesvirus Bovino 1/crecimiento & desarrollo , Herpesvirus Bovino 5/crecimiento & desarrollo , Meningoencefalitis/virología , Virología/métodos , Administración Intranasal , Animales , Anticuerpos Antivirales/sangre , Modelos Animales de Enfermedad , Encefalitis Viral/fisiopatología , Femenino , Infecciones por Herpesviridae/fisiopatología , Herpesvirus Bovino 1/aislamiento & purificación , Herpesvirus Bovino 5/aislamiento & purificación , Hígado/patología , Pulmón/patología , Masculino , Meningoencefalitis/fisiopatología , Nariz/virología , ConejosRESUMEN
Venereal infection of bulls with bovine herpesvirus type 1.2 (BHV-1.2) may result in acute balanoposthitis followed by the establishment of latent infection, presumably in dorsal root nerve ganglia. We herein report the characterization of the acute and latent infection of young bulls with a Brazilian BHV-1.2 isolate and the investigation of neural and non-neural sites in which viral DNA persists during latent infection, i.e. 110 days after inoculation and 50 days after experimental reactivation. Intrapreputial inoculation of BHV-1.2 isolate SV-56/90 (10(6.5)pfu per animal) resulted in severe balanoposthitis, characterized by redness of the penis and preputial mucosa, coalescent vesicles and fibrinous exsudate in all four infected bulls. Virus shedding was detected in preputial secretions and semen up to days 14 and 13 pi, respectively. Dexamethasone administration at day 60 pi led to reactivation of the infection in all animals, resulting in virus shedding in preputial secretions and/or in semen. At day 50 post-reactivation (pr), the animals were euthanized and regional tissues were collected for PCR and virus isolation. Viral DNA was consistently detected in the dorsal root ganglia of nerves genito-femoral (4/4) and obturator (4/4); frequently in the pudendal (3/4), sciatic (3/4) and rectal caudal nerve ganglia (2/3). In addition, viral DNA was detected in the pelvic sympathetic plexus of one bull and in regional lymph nodes (deep inguinal (2/4); sacral (1/4); medial iliac (1/4)) of two bulls. No infectious virus could be recovered from homogenates of DNA positive tissues, indicating the absence of actively replicating virus. These results demonstrate that BHV-1.2 DNA may persist in several sacral nerve ganglia and in regional lymph nodes as well during latent infection, i.e. 50 days after experimental reactivation. These findings may help in understanding the pathogenesis of acute and latent genital infection by BHV-1.2.