RESUMEN
Murine hepatitis virus (MHV) infection is one of the most prevalent types of mice infection in laboratory. MHV could cause death in mice and even interfere with the results in animal experiments. Herein, we developed two isothermal approaches based on the Multienzyme Isothermal Rapid Amplification (MIRA), for rapid detection of MHV in conserved M gene. We designed and screened several pairs of primers and probes and the isothermal fluorescence detector was applied for the exonuclease â ¢ reverse transcription MIRA (exo-RT-MIRA) assay. To further simplify the workflow, the portable fluorescence visualization instrument, also as a palm-sized handheld system, was used for the naked-eye exo-RT-MIRA assay. The amplification temperature and time were optimized. The assay could be processed well at 42 °C 20 min for the exo-RT-MIRA and the naked-eye exo-RT-MIRA assay. The limit of detection (LoD) of the exo-RT-MIRA assay was 43.4 copies/µL. The LoD of the naked-eye exo-RT-MIRA assay was 68.2 copies/µL. No nonspecific amplifications were observed in the two assays. A total of 107 specimens were examined by qPCR and two assays developed. The experimental results statistical analysis demonstrated that the exo-RT-MIRA assay with the qPCR yielded sufficient agreement with a kappa value of 1.000 (p < 0.0001). The results also exhibited a good agreement (kappa value, 0.961) (p < 0.0001) between the naked-eye exo-RT-MIRA assay and the qPCR assay. In our study, the exo-RT-MIRA assay and the naked-eye exo-RT-MIRA assay presented the possibility of new methods in MHV point-of-testing diagnosis.
Asunto(s)
Límite de Detección , Técnicas de Diagnóstico Molecular , Virus de la Hepatitis Murina , Técnicas de Amplificación de Ácido Nucleico , Sensibilidad y Especificidad , Animales , Técnicas de Amplificación de Ácido Nucleico/métodos , Ratones , Virus de la Hepatitis Murina/genética , Virus de la Hepatitis Murina/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Cartilla de ADN/genética , Temperatura , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Hepatitis Viral Animal/diagnóstico , Hepatitis Viral Animal/virología , Fluorescencia , ARN Viral/genéticaAsunto(s)
Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/transmisión , Electrocirugia/métodos , Transmisión de Enfermedad Infecciosa de Paciente a Profesional/prevención & control , Laparoscopía/métodos , Virus de la Hepatitis Murina/fisiología , Humo , Animales , Electrocirugia/efectos adversos , Electrocirugia/instrumentación , Humanos , Laparoscopía/efectos adversos , Laparoscopía/instrumentación , Ratones , Viabilidad Microbiana , Virus de la Hepatitis Murina/aislamiento & purificación , Humo/efectos adversos , Humo/análisis , Humo/prevención & controlRESUMEN
Gap junctions (GJs) are important for maintenance of CNS homeostasis. GJ proteins, connexin 43 (Cx43) and connexin 47 (Cx47), play a crucial role in production and maintenance of CNS myelin. Cx43 is mainly expressed by astrocytes in the CNS and forms gap junction intercellular communications between astrocytes-astrocytes (Cx43-Cx43) and between astrocytes-oligodendrocytes (Cx43-Cx47). Mutations of these connexin (Cx) proteins cause dysmyelinating diseases in humans. Previously, it has been shown that Cx43 localization and expression is altered due to mouse hepatitis virus (MHV)-A59 infection both in vivo and in vitro; however, its mechanism and association with loss of myelin protein was not elaborated. Thus, we explored potential mechanisms by which MHV-A59 infection alters Cx43 localization and examined the effects of viral infection on Cx47 expression and its association with loss of the myelin marker proteolipid protein. Immunofluorescence and total internal reflection fluorescence microscopy confirmed that MHV-A59 used microtubules (MTs) as a conduit to reach the cell surface and restricted MT-mediated Cx43 delivery to the cell membrane. Co-immunoprecipitation experiments demonstrated that Cx43-ß-tubulin molecular interaction was depleted due to protein-protein interaction between viral particles and MTs. During acute MHV-A59 infection, oligodendrocytic Cx47, which is mainly stabilized by Cx43 in vivo, was down-regulated, and its characteristic staining remained disrupted even at chronic phase. The loss of Cx47 was associated with loss of proteolipid protein at the chronic stage of MHV-A59 infection.
Asunto(s)
Astrocitos/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Hepatitis/metabolismo , Microtúbulos/metabolismo , Virus de la Hepatitis Murina/fisiología , Animales , Astrocitos/citología , Conexinas/deficiencia , Hepatitis/virología , Ratones , Ratones Endogámicos C57BL , Virus de la Hepatitis Murina/aislamiento & purificaciónRESUMEN
The oral drug FTY720 affects sphingosine-1-phosphate (S1P) signaling on targeted cells that bear the S1P receptors S1P1, S1P3, S1P4, and S1P5. We examined the effect of FTY720 treatment on the biology of mouse neural progenitor cells (NPCs) after transplantation in a viral model of demyelination. Intracerebral infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in an acute encephalomyelitis, followed by demyelination similar in pathology to the human demyelinating disease, multiple sclerosis. We have previously reported that intraspinal transplantation of mouse NPCs into JHMV-infected animals resulted in selective colonization of demyelinated lesions, preferential differentiation into oligodendroglia accompanied by axonal preservation, and increased remyelination. Cultured NPCs expressed transcripts for S1P receptors S1P1, S1P2, S1P3, S1P4, and S1P5. FTY720 treatment of cultured NPCs resulted in increased mitogen-activated protein kinase phosphorylation and migration after exposure to the chemokine CXCL12. Administration of FTY720 to JHMV-infected mice resulted in enhanced migration and increased proliferation of transplanted NPCs after spinal cord engraftment. FTY720 treatment did not improve clinical disease, diminish neuroinflammation or the severity of demyelination, nor increase remyelination. These findings argue that FTY720 treatment selectively increases NPC proliferation and migration but does not either improve clinical outcome or enhance remyelination after transplantation into animals in which immune-mediated demyelination is initiated by the viral infection of the central nervous system.
Asunto(s)
Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Enfermedades Desmielinizantes/patología , Virus de la Hepatitis Murina/aislamiento & purificación , Células-Madre Neurales/citología , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Animales , Axones/patología , Células Cultivadas , Sistema Nervioso Central/patología , Enfermedades Desmielinizantes/virología , Clorhidrato de Fingolimod/farmacología , Inmunosupresores/farmacología , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Esclerosis Múltiple/patología , Esclerosis Múltiple/virología , Células-Madre Neurales/efectos de los fármacos , Oligodendroglía/citologíaRESUMEN
During attempts to clone retroviral determinants associated with a mouse model of Langerhans cell histiocytosis (LCH), suppression subtractive hybridization (SSH) was used to identify unique viruses in the liver of severe combined immunodeficiency (SCID) mice transplanted with LCH tissues. A partial genomic sequence of a murine coronavirus was identified, and the whole genome (31428 bp) of the coronavirus was subsequently sequenced using PCR cloning techniques. Nucleotide sequence comparisons revealed that the genome sequence of the new virus was 91-93% identical to those of known murine hepatitis viruses (MHVs). The predicted open reading frame from the nucleotide sequence encoded all known proteins of MHVs. Analysis at the protein level showed that the virus was closely related to the highly virulent MHV-JHM strain. The virus strain was named MHV-MI. No type D retroviruses were found. Degenerate PCR targeting of type D retrovirus and 5'-RACE targeting of other types of retroviruses confirmed the absence of any retroviral association with the LCH xenografted SCID mice.
Asunto(s)
Infecciones por Coronavirus/virología , Histiocitosis de Células de Langerhans/virología , Virus de la Hepatitis Murina/genética , Virus de la Hepatitis Murina/aislamiento & purificación , Técnicas de Hibridación Sustractiva/métodos , Adolescente , Animales , Secuencia de Bases , Modelos Animales de Enfermedad , Femenino , Genoma Viral , Humanos , Ratones , Ratones SCID , Datos de Secuencia Molecular , Virus de la Hepatitis Murina/clasificación , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Carbohydrate structures, including Lewis X (Le(x)), which is not synthesized in mutant mice that lack α1,3-fucosyltransferase 9 (Fut9(-/-)), are involved in cell-cell recognition and inflammation. However, immunological alteration in Fut9(-/-) mice has not been studied. Thus, the inflammatory response of Fut9(-/-) mice was examined using the highly neurovirulent mouse hepatitis virus (MHV) JHMV srr7 strain. Pathological study revealed that inflammation induced in the brains of Fut9(-/-) mice after infection was more extensive compared with that of wild-type mice, although viral titers obtained from the brains of mutant mice were lower than those of wild-type mice. Furthermore, the reduction in cell numbers in the spleens of wild-type mice after infection was not observed in the infected Fut9(-/-) mice. Although there were no clear differences in the levels of cytokines examined in the brains between Fut9(-/-) and wild-type mice except for interferon-ß expression, some of those in the spleens, including interferon-γ, interleukin-6, and monocyte chemoattractant protein 1, showed higher levels in Fut9(-/-) than in wild-type mice. Furthermore, Fut9(-/-) mice were refractory to the in vivo inoculation of endotoxin (LPS) compared with wild-type mice. These results indicate that Le(x) structures are involved in host responses against viral or bacterial challenges.
Asunto(s)
Fucosiltransferasas/deficiencia , Inmunidad Innata/inmunología , Lipopolisacáridos/inmunología , Virus de la Hepatitis Murina/inmunología , Animales , Encéfalo/patología , Encéfalo/virología , Recuento de Células , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/prevención & control , Citocinas/metabolismo , Modelos Animales de Enfermedad , Fucosiltransferasas/genética , Fucosiltransferasas/fisiología , Inmunidad Innata/genética , Inmunidad Innata/fisiología , Ratones , Ratones Noqueados , Ratones Mutantes , Virus de la Hepatitis Murina/aislamiento & purificación , Bazo/patología , Bazo/virologíaRESUMEN
The mouse hepatitis virus (MHV) has a high mutation rate, leading to various neuropathologies after infection. The srr7 mutant was isolated from the MHV strain cl-2, which induces characteristic spongiform degeneration in the brain. To investigate outcomes of srr7 infection, we re-cloned srr7(H2) from the viral stock srr7(Mix). During this re-cloning, we obtained the mutant viruses, Mu-1, Mu-2, and Br-1 which was isolated from the mice brain infected with srr7(Mix). We examined mutant viruses for infectivity independent of the major MHV receptor (MHVR), because these mutants exhibited high virulence similar to cl-2, which is MHVR-independent. To confirm MHVR-independence in vitro, we used a combination of spinoculation and ultraviolet radiation to detect distinct plaque formation (SpinoPlaque(UV+)) afrer infection of BHK cells, which do not express MHVR. Using this technique, we found that the unique neuropathologies caused by infection with the mutant viruses result from infecting neurons, which do not express MHVR. Infection with the mutant viruses was 100% correlated with SpinoPlaque(UV+) formation. This is in contrast to infection with srr7, which does not from SpinoPlaque(UV+).
Asunto(s)
Infecciones del Sistema Nervioso Central/patología , Infecciones del Sistema Nervioso Central/virología , Virus de la Hepatitis Murina/patogenicidad , Virus de la Hepatitis Murina/efectos de la radiación , Mutación , Rayos Ultravioleta , Animales , Encéfalo/patología , Encéfalo/virología , Histocitoquímica , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Microscopía , Virus de la Hepatitis Murina/aislamiento & purificación , VirulenciaRESUMEN
The focus of this protocol is mouse hepatitis virus (MHV), with occasional references to other coronaviruses. Many of these protocols can be easily adapted to other coronaviruses. Protocols for propagating MHV in DBT and 17CL-1 cells; the storage and titration of viral stocks; purification of MHV on sucrose gradients; and the generation of recombinant viruses by a cDNA assembly method and by targeted recombination will be presented. Protocols are also included for the propagation of DBT, 17CL-1, and L2 cells used for growing and titrating MHV, and for the growth of BHK-R cells and FCWF cells. The latter two cell lines are used for regenerating infectious MHV by an in vitro cDNA assembly protocol and by a targeted recombination protocol, respectively, allowing reverse genetic manipulation of these viruses. An additional protocol for the maintenance of the large plasmids used for generating recombinant MHVs will also be presented.
Asunto(s)
Virus de la Hepatitis Murina/crecimiento & desarrollo , Virus de la Hepatitis Murina/genética , Virología/métodos , Animales , Línea Celular , Clonación Molecular/métodos , Ratones , Virus de la Hepatitis Murina/aislamiento & purificación , Preservación Biológica/métodos , Carga Viral/métodos , Cultivo de Virus/métodosRESUMEN
The highly neuropathogenic cl-2 and less virulent srr7 viruses isolated from the neurotropic JHM strain of the mouse hepatitis virus exhibit super acute spread of virus (SAS), a term applied when rapid viral spread from an organ or part of the initially infected site to another non-adjacent organ or part is detected within 12 h after infection. Herein, we used a cytospin procedure to confirm SAS in splenic cells derived from mice whose brains were infected with these viruses. The cytospin procedure enabled effective preservation of the cells on glass slides. With this method, we could characterize extremely low populations of infected cells in the spleen (less than 0.1%) at 12 h post-inoculation with srr7. We observed that all kinds of splenic cells examined were infected, including B220(+)Ly-6C(+) plasmacytoid dendritic cells. The population of viral antigen-positive splenic cells was only slightly higher in cl-2 infection than in srr7 infection, but the cells showing viral production were present in numbers significantly higher in cl-2 infection compared with srr7 infection.
Asunto(s)
Hepatitis Viral Animal/virología , Virus de la Hepatitis Murina/patogenicidad , Enfermedades de los Roedores/inmunología , Enfermedades de los Roedores/virología , Bazo/virología , Animales , Modelos Animales de Enfermedad , Hepatitis Viral Animal/patología , Ratones , Ratones Endogámicos BALB C , Virus de la Hepatitis Murina/aislamiento & purificación , Bazo/patología , Factores de TiempoRESUMEN
MHV-Wüts18 is an RNA-negative, temperature-sensitive mutant of mouse coronavirus, strain murine hepatitis virus (MHV)-A59. We have previously identified the putative causal mutation of MHV-Wüts18 as a C to U transition at codon 2446 in ORF1b, which results in a substitution of proline 12 with serine in non-structural protein 16. Here, we have used a vaccinia virus-based reverse genetic system to produce a recombinant virus, inf-MHV-Wüts18((AGC)) that encodes nsp16 serine 12 with AGC rather than UCU; a difference that facilitates the isolation of second-site revertants. Sequence analysis of nine inf-MHV-Wüts18((AGC)) revertant viruses suggests that their phenotype is most probably due to the intra-molecular substitution of amino acids in nsp16. However, the revertant viruses displayed different plaque sizes and whole genome sequencing of two revertants showed that they were isogenic apart from a mutation in nsp13. These results are discussed in the context of a model of coronavirus MHV nsp16 structure.
Asunto(s)
Virus de la Hepatitis Murina/crecimiento & desarrollo , Virus de la Hepatitis Murina/genética , Mutación Missense , Mutación Puntual , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Animales , ADN Complementario/genética , ADN Viral/genética , Vectores Genéticos , Ratones , Modelos Moleculares , Virus de la Hepatitis Murina/aislamiento & purificación , Estructura Terciaria de Proteína , ARN Viral/genética , Análisis de Secuencia de ADN , Virus Vaccinia/genética , Ensayo de Placa Viral , Replicación ViralRESUMEN
The emergence of a previously unknown coronavirus infection, Severe Acute Respiratory Syndrome (SARS), demonstrated that fecally contaminated liquid droplets are a potential vehicle for the spread of a respiratory virus to large numbers of people. To assess potential risks from this pathway, there is a need for surrogates for SARS coronavirus to provide representative data on viral survival in contaminated water. This study evaluated survival of two surrogate coronaviruses, transmissible gastroenteritis (TGEV) and mouse hepatitis (MHV). These viruses remained infectious in water and sewage for days to weeks. At 25 degrees C, time required for 99% reduction in reagent-grade water was 22 days for TGEV and 17 days for MHV. In pasteurized settled sewage, times for 99% reduction were 9 days for TGEV and 7 days for MHV. At 4 degrees C, there was <1 log(10) infectivity decrease for both viruses after four weeks. Coronaviruses can remain infectious for long periods in water and pasteurized settled sewage, suggesting contaminated water is a potential vehicle for human exposure if aerosols are generated.
Asunto(s)
Virus de la Hepatitis Murina/fisiología , Virus de la Gastroenteritis Transmisible/fisiología , Microbiología del Agua , Virus de la Hepatitis Murina/aislamiento & purificación , Aguas del Alcantarillado/virología , Virus de la Gastroenteritis Transmisible/aislamiento & purificaciónRESUMEN
The aim of this study was to determine the susceptibility of murine embryonic stem (mESCs) to mouse hepatitis virus (MHV-A59) and mouse minute virus (MMVp) and the effect of these viruses on germline transmission (GLT) and the serological status of recipients and pups. When recipients received 10 blastocysts, each injected with 10(0) TCID(50) MHV-A59, three out of five recipients and four out of 14 pups from three litters became seropositive. When blastocysts were injected with 10(-5) TCID(50) MMVp, all four recipients and 14 pups from four litters remained seronegative. The mESCs replicated MHV-A59 but not MMVp, MHV-A59 being cytolytic for mESCs. Exposure of mESCs to the viruses over four to five passages but not for 6 h affected GLT. Recipients were seropositive for MHV-A59 but not for MMVp when mESCs were cultured with the virus over four or five passages. The data show that GLT is affected by virus-contaminated mESCs.
Asunto(s)
Blastocisto/virología , Infecciones por Coronavirus/virología , Células Madre Embrionarias/virología , Células Germinativas/fisiología , Hepatitis Viral Animal/transmisión , Virus Diminuto del Ratón/patogenicidad , Virus de la Hepatitis Murina/patogenicidad , Infecciones por Parvoviridae/virología , Animales , Transferencia de Embrión , Femenino , Fertilización In Vitro , Citometría de Flujo , Hepatitis Viral Animal/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Virus Diminuto del Ratón/aislamiento & purificación , Virus Diminuto del Ratón/metabolismo , Virus de la Hepatitis Murina/aislamiento & purificación , Virus de la Hepatitis Murina/metabolismo , Receptores Virales/metabolismo , Estudios Seroepidemiológicos , Replicación ViralRESUMEN
Although some previously common infections, such as Sendai virus and Mycoplasma pulmonis, have become rare in laboratory rodents in North American research facilities, others continue to plague researchers and those responsible for providing biomedical scientists with animals free of adventitious disease. Long-recognized agents that remain in research facilities in the 21st century include parvoviruses of rats and mice, mouse rotavirus, Theilers murine encephalomyelitis virus (TMEV), mouse hepatitis virus (MHV), and pinworms. The reasons for their persistence vary with the agent. The resilience of parvoviruses, for example, is due to their resistance to inactivation, their prolonged shedding, and difficulties with detection, especially in C57BL/6 mice. Rotavirus also has marked environmental resistance, but periodic reintroduction into facilities, possibly on bags of feed, bedding, or other supplies or equipment, also seems likely. TMEV is characterized by resistance to inactivation, periodic reintroduction, and relatively long shedding periods. Although MHV remains active in the environment at most a few days, currently prevalent strains are shed in massive quantities and likely transmitted by fomites. Pinworm infestations continue because of prolonged infections, inefficient diagnosis, and the survivability of eggs of some species in the environment. For all of these agents, increases in both interinstitutional shipping and the use of immunodeficient or genetically modified rodents of unknown immune status may contribute to the problem, as might incursions by wild or feral rodents. Elimination of these old enemies will require improved detection, strict adherence to protocols designed to limit the spread of infections, and comprehensive eradication programs.
Asunto(s)
Enfermedades de los Roedores/parasitología , Enfermedades de los Roedores/virología , Animales , Enterobius/aislamiento & purificación , Ratones , Virus de la Hepatitis Murina/aislamiento & purificación , Parvovirus/aislamiento & purificación , Rotavirus/aislamiento & purificación , Theilovirus/aislamiento & purificaciónRESUMEN
Immunocompetent, but not RAG1(-/-) mice infected with MHV-JHM develop demyelination. Transferred CD8 T cell-enriched splenocytes reconstitute demyelination, and this ability is dependent on donor IFN-gamma. We used IFN-gammaR1(-/-) mice to examine the target of IFN-gamma in CD8 T cell-mediated demyelination. In IFN-gammaR1(-/-)RAG1(-/-) recipients, demyelination is decreased, but not eliminated, while viral titers are significantly increased when compared to IFN-gammaR1(+/+)RAG1(-/-) recipients. IFN-gammaR1(-/-) CD8 T cells retain virus-specific effector function regardless of IFN-gammaR1 expression. Although IFN-gammaR1 responsiveness is critical for maximal demyelination, increased levels of infectious virus coupled with adoptive transfer of CD8 T cells may result in myelin destruction independent of IFN-gammaR1 expression.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Enfermedades Virales del Sistema Nervioso Central/inmunología , Infecciones por Coronavirus/inmunología , Enfermedades Autoinmunes Desmielinizantes SNC/inmunología , Interferón gamma/fisiología , Virus de la Hepatitis Murina/fisiología , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/fisiología , Enfermedades Virales del Sistema Nervioso Central/patología , Enfermedades Virales del Sistema Nervioso Central/virología , Quimiotaxis , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Cruzamientos Genéticos , Enfermedades Autoinmunes Desmielinizantes SNC/etiología , Enfermedades Autoinmunes Desmielinizantes SNC/patología , Enfermedades Autoinmunes Desmielinizantes SNC/virología , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Interferón gamma/análisis , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Virus de la Hepatitis Murina/aislamiento & purificación , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Organismos Libres de Patógenos Específicos , Linfocitos T/trasplante , Receptor de Interferón gammaRESUMEN
Serologic monitoring of sentinel mice exposed to soiled bedding is a common method of detecting viral infections in mice. Because bedding transfer protocols vary, the sensitivity of this method has not been documented sufficiently. We examined the reliability of bedding transfer during various stages of infection with mouse parvovirus (MPV) and mouse hepatitis virus (MHV). Most mice exposed to bedding contaminated with MPV 0, 3, or 7 d previously seroconverted, whereas only mice exposed to bedding contaminated with MHV 4 h previously seroconverted, thus confirming the differing stabilities of these viruses. Index mice were inoculated with 30 times the infectious dose 50 (ID50) of MPV or 300 ID50 of MHV. At 3 d, 1 wk, and 2 wk postinoculation (PI), we transferred 25, 50, or 100 ml of bedding to cages of sentinel mice. Viral infection and shedding by index mice was confirmed by serology and fecal polymerase chain reaction assay. Transfer of soiled bedding between mice in static cages induced seroconversion of sentinel mice most reliably during peak viral shedding (1 wk PI for MPV and 3 d PI for MHV). Soiled bedding transfer between mice in individually ventilated cages induced a higher prevalence of sentinel seroconversion to MPV and MHV than that after transfer between mice in static cages. Our findings indicate that although soiled bedding transfer is an effective method for detecting MHV and MPV under optimal conditions, the method is less than 100% reliable under many conditions in contemporary mouse facilities.
Asunto(s)
Infecciones por Coronavirus/veterinaria , Vivienda para Animales , Ratones , Virus de la Hepatitis Murina/aislamiento & purificación , Infecciones por Parvoviridae/veterinaria , Parvovirus/aislamiento & purificación , Enfermedades de los Roedores/virología , Vigilancia de Guardia/veterinaria , Animales , Anticuerpos Antivirales/sangre , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/transmisión , Transmisión de Enfermedad Infecciosa/veterinaria , Femenino , Técnica del Anticuerpo Fluorescente , Infecciones por Parvoviridae/diagnóstico , Infecciones por Parvoviridae/transmisión , Enfermedades de los Roedores/transmisión , Estadísticas no ParamétricasRESUMEN
We developed a microfabricated electrochemical DNA chip for detection of polymerase chain reaction (PCR) products from 16S rRNA sequences of Clostridium piliforme (Cp), Helicobacter bilis (Hb) and Helicobacter hepaticus (Hh), and the nucleocapsid protein gene of mouse hepatitis virus (MHV). This chip does not require DNA labeling, and the hybridization signal can be detected as an anodic current. The average anodic currents of 9 (Cp), 5 (Hb), 8 (Hh) and 7 (MHV) PCR positive samples derived from feces of spontaneously infected mice (Cp, Hb and Hh) and MHV-contaminated tumor cells were 27.9+/-7.2, 31.9+/-8.1, 29.3+/-10.1, and 27.6+/-3.0 nA, respectively. On the other hand, the average anodic currents of 19 (Cp), 27 (Hb), 18 (Hh), and 13 (MHV) PCR negative samples were 0.3+/-2.9, 3.7+/-2.4, -1.0+/-1.7, and -2.3+/-2.7 nA, respectively. The anodic current increased with increasing concentrations of pathogens. For experimentally infected samples, the results of PCR/electrophoresis were in complete accord with those of this system when anodic currents of 6.1 (Cp), 8.5 (Hb), 2.4 (Hh), and 3.1 nA (MHV) were taken as the cut-off value. The results suggested that the electrochemical DNA chip system is useful for specific and quantitative detection of PCR products.
Asunto(s)
Clostridium/aislamiento & purificación , Helicobacter hepaticus/aislamiento & purificación , Helicobacter/aislamiento & purificación , Virus de la Hepatitis Murina/aislamiento & purificación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Animales , Clostridium/genética , Cartilla de ADN , Electroquímica , Helicobacter/genética , Infecciones por Helicobacter/diagnóstico , Infecciones por Helicobacter/veterinaria , Helicobacter hepaticus/genética , Ratones , Modelos Biológicos , Virus de la Hepatitis Murina/genética , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/metabolismo , Enfermedades de los Roedores/diagnóstico , Sensibilidad y EspecificidadRESUMEN
The present study contains information about proper microbiological monitoring of laboratory animals' health and the standardization of microbiological monitoring methods in Korea. Microbiological quality control for laboratory animals, composed of biosecurity and health surveillance, is essential to guard against research complications and public health dangers that have been associated with adventitious infections. In this study, one hundred and twenty-two mice and ninety rats from laboratory animal breeding companies and one animal facility of the national universities in Korea were monitored in 2000-2003. Histopathologically, thickening of the alveolar walls and lymphocytic infiltration around the bronchioles were observed in mice and rats from microbiologically contaminated facilities. Cryptosporidial oocysts were observed in the gastric pits of only conventionally-housed mice and rats. Helicobacter spp. infection was also detected in 1 of 24 feces DNA samples in mice and 9 of 40 feces DNA samples in rats by PCR in 2003, but they were not Helicobacter hepaticus. This paper describes bacteriological, parasitological, and virological examinations of the animals.
Asunto(s)
Animales de Laboratorio/microbiología , Ratones Endogámicos/microbiología , Ratas Endogámicas/microbiología , Organismos Libres de Patógenos Específicos , Animales , Animales de Laboratorio/parasitología , Animales de Laboratorio/virología , Cryptosporidium/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Helicobacter/aislamiento & purificación , Vivienda para Animales , Corea (Geográfico) , Ratones , Ratones Endogámicos/parasitología , Ratones Endogámicos/virología , Virus de la Hepatitis Murina/aislamiento & purificación , Mycoplasma/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Cuarentena/normas , Ratas , Ratas Endogámicas/parasitología , Ratas Endogámicas/virología , Virus Sendai/aislamiento & purificaciónRESUMEN
The CC chemokine ligand 2 (CCL2, monocyte chemoattractant protein-1) is important in coordinating the immune response following microbial infection by regulating T cell polarization as well as leukocyte migration and accumulation within infected tissues. The present study examines the consequences of mouse hepatitis virus (MHV) infection in mice lacking CCL2 (CCL2(-/-)) in order to determine if signaling by this chemokine is relevant in host defense. Intracerebral infection of CCL2(-/-) mice with MHV did not result in increased morbidity or mortality as compared to either wild type or CCR2(-/-) mice and CCL2(-/-) mice cleared replicating virus from the brain. In contrast, CCR2(-/-) mice displayed an impaired ability to clear virus from the brain that was accompanied by a reduction in the numbers of antigen-specific T cells as compared to both CCL2(-/-) and wild-type mice. The paucity in T cell accumulation within the central nervous system (CNS) of MHV-infected CCR2(-/-) mice was not the result of either a deficiency in antigen-presenting cell (APC) accumulation within draining cervical lymph nodes (CLN) or the generation of virus-specific T cells within this compartment. A similar reduction in macrophage infiltration into the CNS was observed in both CCL2(-/-) and CCR2(-/-) mice when compared to wild-type mice, indicating that both CCL2 and CC chemokine receptor 2 (CCR2) contribute to macrophage migration and accumulation within the CNS following MHV infection. Together, these data demonstrate that CCR2, but not CCL2, is important in host defense following viral infection of the CNS, and CCR2 ligand(s), other than CCL2, participates in generating a protective response.
Asunto(s)
Quimiocina CCL2/inmunología , Infecciones por Coronavirus/inmunología , Virus de la Hepatitis Murina/inmunología , Receptores de Quimiocina/inmunología , Animales , Encéfalo/inmunología , Encéfalo/virología , Recuento de Células , Quimiocina CCL2/deficiencia , Quimiocina CCL2/genética , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/virología , Modelos Animales de Enfermedad , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Virus de la Hepatitis Murina/aislamiento & purificación , Receptores CCR2 , Receptores de Quimiocina/deficiencia , Receptores de Quimiocina/genética , Linfocitos T/inmunologíaRESUMEN
Mouse hepatitis virus (MHV) is the most prevalent virus that infects mice, and most MHV strains are enterotropic. Experiments were performed to elucidate the duration of enterotropic MHV-Y shedding by immunocompetent BALB/ c and C57BL/6 mice and immunocompromised B and T cell-deficient mice. Although the use of molecular diagnostics to detect MHV infection is increasing, it is unclear whether the viral RNA detected is always infectious. The ability to detect MHV-Y transmission to sentinel mice exposed directly to infected mice or to soil bedding from infected mice was compared with reverse transcriptase-polymerase chain reaction-based detection of viral RNA in the feces. The BALB/c mice developed subclinical intestinal infection, and transmitted MHV-Y for four weeks. The C57BL/6 mice also developed subclinical intestinal infection, but only transmitted virus for two weeks. The T cell-deficient mice developed severe disseminated disease by two weeks and transmitted virus for four weeks. The B cell-deficient mice developed subclinical intestinal infection and transmitted virus for longer than three months, although virus RNA was not detected in feces late in the infection. Viral RNA detected in the feces of infected mice was almost always infectious. Non-infectious RNA was detected in a few mice for several days after transmission had ceased. In addition, constant exposure of naive mice to infected mice, via the use of serial sentinels, prolonged viral transmission.
Asunto(s)
Hepatitis Viral Animal/transmisión , Transmisión Vertical de Enfermedad Infecciosa , Virus de la Hepatitis Murina , Enfermedades de los Roedores/transmisión , Animales , Animales no Consanguíneos , Heces/virología , Femenino , Hepatitis Viral Animal/diagnóstico , Hepatitis Viral Animal/inmunología , Inmunocompetencia/inmunología , Huésped Inmunocomprometido/inmunología , Ratones , Ratones Endogámicos , Virus de la Hepatitis Murina/genética , Virus de la Hepatitis Murina/inmunología , Virus de la Hepatitis Murina/aislamiento & purificación , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Enfermedades de los Roedores/inmunología , Vigilancia de Guardia , Especificidad de la Especie , Esparcimiento de Virus/inmunologíaRESUMEN
Mouse hepatitis virus (MHV) is one of the most prevalent viruses infecting laboratory mice. Most natural infections are caused by enterotropic strains. Experiments were done to compare the pathogenesis of enterotropic strain MHV-Y in immunocompetent BALB/c and C57BL/6 mice with that in B and T cell-deficient mice. In situ hybridization was used to identify sites of virus replication, and reverse transcriptase-polymerase chain reaction analysis was used to detect viral RNA in feces and blood. MHV-Y caused acute subclinical infections restricted to the gastrointestinal tract in BALB/c and C57BL/6 mice. Viral RNA was detected in small intestine and associated lymphoid tissues of immunocompetent mice for 1 week and in cecum and colon for 2 weeks. Infected B cell-deficient mice developed chronic subclinical infection also restricted to the gastrointestinal tract. Viral RNA was detected in the small intestine, cecum, colon, and feces for 7 to 8 weeks. In contrast, infected T cell-deficient mice developed multisystemic lethal infection. During the first week, viral RNA was restricted to the gastrointestinal tract. However, by 2 weeks, mice developed peritonitis, and viral RNA was detected in mesentery and visceral peritoneum. Three to four weeks after virus inoculation, T cell-deficient mice became moribund and viral RNA was detected in multiple organ systems. These results suggest that B cells promote clearance of MHV-Y from intestinal mucosa and that T cells are required to prevent dissemination of MHV-Y from the gastrointestinal tract and associated lymphoid tissues.