RESUMEN
The aim of this study was to investigate the consequences in calves of two forms of inocula alternative to the use of wild type infectious blood. Two groups of five calves were infected with low cell-passaged virus and infectious blood issued from one animal passage of the same strain. A longitudinal study was implemented and characterised by clinical standardised observations, haematology, BTV RNA detection and viral isolation from blood, detection of serogroup and neutralising antibodies, cytokine expression and post-mortem examination 46 days post-infection (PI). Both tested inocula were able to reproduce clinical expression of the disease, in the bloodstream viral genome was detected until the end of the experiment while virus isolation was possible between days 7 and 31 PI. Humoral immune response developed earlier in calves infected with low cell-passaged virus, while in both groups a massive antibody production was confirmed by the immune balance between IL-4 and IFN-γ expression. Both tested inocula are presented as valid alternative to the use of wild type infectious blood in the study of the pathogenesis of BTV-8 or the efficacy of current and future vaccines.
Asunto(s)
Virus de la Lengua Azul/inmunología , Lengua Azul/virología , Enfermedades de los Bovinos/virología , Viremia/veterinaria , Animales , Formación de Anticuerpos , Lengua Azul/inmunología , Virus de la Lengua Azul/crecimiento & desarrollo , Virus de la Lengua Azul/patogenicidad , Bovinos , Enfermedades de los Bovinos/inmunología , Femenino , Inmunidad Humoral , Interferón gamma/inmunología , Interleucina-4/inmunología , ARN Viral/sangreRESUMEN
Based on sequencing data, norovirus (NoV) recombinants have been described, but no experimental evidence of recombination in NoVs has been documented. Using the murine norovirus (MNV) model, we investigated the occurrence of genetic recombination between two co-infecting wild-type MNV isolates in RAW cells. The design of a PCR-based genotyping tool allowed accurate discrimination between the parental genomes and the detection of a viable recombinant MNV (Rec MNV) in the progeny viruses. Genetic analysis of Rec MNV identified a homologous-recombination event located at the ORF1-ORF2 overlap. Rec MNV exhibited distinct growth curves and produced smaller plaques than the wild-type MNV in RAW cells. Here, we demonstrate experimentally that MNV undergoes homologous recombination at the previously described recombination hot spot for NoVs, suggesting that the MNV model might be suitable for in vitro studies of NoV recombination. Moreover, the results show that exchange of genetic material between NoVs can generate viruses with distinct biological properties from the parental viruses.
Asunto(s)
Norovirus/crecimiento & desarrollo , Norovirus/genética , ARN Viral/genética , Recombinación Genética , Animales , Línea Celular , Análisis por Conglomerados , Genotipo , Macrófagos/virología , Ratones , Viabilidad Microbiana , Datos de Secuencia Molecular , Norovirus/patogenicidad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Análisis de Secuencia de ADN , Homología de Secuencia , Ensayo de Placa Viral , VirulenciaRESUMEN
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
Noroviruses, belonging to the family Caliciviridae, have been identified in human beings and in several animal species including cattle. The distribution of bovine norovirus infections was investigated by both RT-PCR to detect norovirus genomes and a virus-like particles-based ELISA to detect genotype 2 bovine norovirus antibodies. During a 1-year systematic study, a virus prevalence of 7.5% (CI 95%: [3.7; 13.4%]) (10 out of 133 samples) was found in stool samples from diarrhoeic calves screened by RT-PCR. Nucleotide sequencing performed on the polymerase region classified all the norovirus amplicons in the bovine norovirus genotype 2. Rather surprisingly, some rotavirus sequences were also detected. On the basis of the polymerase region, genotype 1 bovine norovirus was not identified. Other enteropathogens were found in all samples. By ELISA, a genotype 2 seroprevalence of 93.2% (CI 95%: [90.4; 95.3%]) was found from calves and adult cattle. Antibody levels against genotype 2 bovine noroviruses rose in the first 6 months of life and were maintained in adults. Together the results of virus prevalence and seroprevalence studies suggest that bovine norovirus infection occurs early in life and that re-infection with serologically related bovine noroviruses strains could occur in adult cattle as reported for rotaviruses. The antibody rise against genotype 2 bovine noroviruses in the adult cattle also suggests a short lived and/or strain specific immunity as already shown in human noroviruses. Genotype 2 bovine noroviruses are endemic in the region investigated.
Asunto(s)
Infecciones por Caliciviridae/veterinaria , Enfermedades de los Bovinos/virología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Gastroenteritis/veterinaria , Norovirus/clasificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Animales , Anticuerpos Antivirales/sangre , Bélgica/epidemiología , Infecciones por Caliciviridae/virología , Bovinos , Enfermedades de los Bovinos/epidemiología , Heces/virología , Gastroenteritis/virología , Norovirus/genética , Filogenia , Estudios SeroepidemiológicosRESUMEN
Porcine noroviruses and sapoviruses belong to the family Caliciviridae and are rarely reported in European countries. In this study, swine stools from a region representative of northern Europe were screened for these viruses by RT-PCR. Both porcine noroviruses and sapoviruses were detected, showing their circulation in this region. The porcine norovirus strains were genetically related to genotype 19 strains in the genogroup II of the genus Norovirus. The porcine sapovirus strains were genetically related to the porcine enteric calicivirus Cowden reference strain and to newly described porcine strains in the genus Sapovirus.
Asunto(s)
Infecciones por Caliciviridae/veterinaria , Norovirus/clasificación , Norovirus/aislamiento & purificación , Sapovirus/clasificación , Sapovirus/aislamiento & purificación , Enfermedades de los Porcinos/virología , Animales , Bélgica , Infecciones por Caliciviridae/virología , Heces/virología , Genotipo , Datos de Secuencia Molecular , Filogenia , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , PorcinosRESUMEN
BACKGROUND: Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and clinical signs and lesions of gastroenteritis were reported in bovines. Due to their genetic proximity, potential zoonotic transmission or animal reservoir can be hypothesized for noroviruses. RT-PCR has become the "gold standard" for the detection of noroviruses in faecal and environmental samples. With such samples, the control for inhibition of the reaction during amplification and detection is crucial to avoid false negative results, which might otherwise not be detected. The aim of the reported method is to detect, with a SYBR Green technology, a broad range of noroviruses with a control for inhibition. RESULTS: A SYBR Green real-time RT-PCR assay was developed making use of a foreign internal RNA control added in the same tube. This assay is able to detect human and bovine noroviruses belonging to genogroups I, II and III and to distinguish between norovirus and internal control amplicons using melting curve analysis. A 10-fold dilution of samples appears to be the method of choice to remove inhibition. This assay was validated with human and bovine stool samples previously tested for norovirus by conventional RT-PCR. CONCLUSION: This SYBR Green real-time RT-PCR assay allows the detection of the most important human and bovine noroviruses in the same assay, and avoids false negative results making use of an internal control. Melting curves allow the discrimination between the internal control and norovirus amplicons. It gives preliminary information about the species of origin. The sensitivity of the developed assay is higher than conventional RT-PCR and a 10-fold dilution of samples showed a better efficiency and reproducibility to remove RT-PCR inhibition than addition of bovine serum albumin.
Asunto(s)
Infecciones por Caliciviridae/diagnóstico , Heces/virología , Gastroenteritis/diagnóstico , Norovirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Infecciones por Caliciviridae/virología , Bovinos , Colorantes Fluorescentes , Gastroenteritis/virología , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and calves. Reverse transcription-polymerase chain reaction (RT-PCR) has become the "gold standard" for detection of noroviruses in faecal and environmental samples. However, false negative results due to co-concentration of RT-PCR inhibitors are a continuous concern. A TaqMan real-time RT-PCR assay making use of a foreign internal RNA control and a RNA standard was developed. Very interestingly, this method is capable of detecting human noroviruses belonging to genogroups I and II, and bovine noroviruses belonging to genogroup III. Inhibitors were removed efficiently by 1/10 dilution of the sample or addition of bovine serum albumin to the RT-PCR mix. This assay was validated with human and bovine stool samples previously tested for norovirus by conventional RT-PCR. The ability to detect norovirus in stool samples that were negative by conventional RT-PCR assay demonstrate the higher sensitivity of the TaqMan assay compared to the conventional RT-PCR assay. This real-time RT-PCR assay allows the detection of both human and bovine noroviruses, avoids false negative results and is able to quantify the level of norovirus contamination.