Murine CMV Research Articles

Hearing loss caused by CMV infection is correlated with reduced endocochlear potentials caused by strial damage in murine models

Congenital cytomegalovirus (CMV) infection is a significant cause of neonatal hearing loss. However, at the cochlear level, the anatomical lesions and pathophysiological mechanisms that underlie hearing loss are still not clearly understood. In murine models of CMV infection, we have observed early damage to the capillary networks in stria vascularis, as well as hearing loss manifested in ABR threshold elevations. Our experimental hypothesis is that strial damage causes a reduced endocochlear potential (EP) resulting in impaired haircell activation and consequent hearing loss. We have studied strial damage, EP, and ABR threshold elevations in two mouse models (BALB/c and C57BL6 strains) infected with murine CMV. Neonatal (P3) pups were inoculated with murine CMV (2µl of 200pfu) by intra cerebral injection. Control mice were saline injected. At 6 weeks, ABR thresholds to tonal stimuli at 8, 16 and 32 kHz were determined for each ear. At 8 weeks a sub-group of treated and control animals was prepared for study of cochlear capillary networks using scanning electron microscopy of corrosion cast specimens. In a second group, at 8 weeks, EP measurements from both cochleas were made. We report that in both mouse strains, CMV infection caused capillary loss in the stria vascularis, initially at the cochlear apex, and extending to lower cochlear turns in some subjects. After CMV infection, in both BALB/c and C57BL6 mice, reduced EPs and ABR threshold elevations were observed, and there was a within-animal correlation between loss of EP and ABR threshold elevations across the sound frequencies tested. These results suggest that CMV induced damage to stria vascularis results in EP reduction that is correlated with ABR threshold elevations. Extrapolating to the human condition, we suggest that strial damage and its physiological consequences may contribute to the initial hearing loss in congenital CMV infection. The early involvement of cochlear capillary damage may encourage a focus on therapeutic interventions that can prevent vascular damage, or subsequently promote vascular healing or angiogenesis.

Prolonged activation of cytomegalovirus early gene e1-promoter exclusively in neurons during infection of the developing cerebrum

The brain is the major target of congenital cytomegalovirus (CMV) infection. It is possible that neuron disorder in the developing brain is a critical factor in the development of neuropsychiatric diseases in later life. Previous studies using mouse model of murine CMV (MCMV) infection demonstrated that the viral early antigen (E1 as a product of e1 gene) persists in the postnatal neurons of the hippocampus (HP) and cerebral cortex (CX) after the disappearance of lytic infection from non-neuronal cells in the periventricular (PV) region. Furthermore, neuron-specific activation of the MCMV-e1-promoter (e1-pro) was found in the cerebrum of transgenic mice carrying the e1-pro-lacZ reporter construct. In this study, in order to elucidate the mechanisms of e1-pro activation in cerebral neurons during actual MCMV infection, we have generated the recombinant MCMV (rMCMV) carrying long e1-pro1373- or short e1-pro448-EGFP reporter constructs. The length of the former, 1373 nucleotides (nt), is similar to that of transgenic mice. rMCMVs and wild type MCMV did not significantly differed in terms of viral replication or E1 expression. rMCMV-infected mouse embryonic fibroblasts showed lytic infection and activation of both promoters, while virus-infected cerebral neurons in primary neuronal cultures demonstrated the non-lytic and persistent infection as well as the activation of e1-pro-1373, but not -448. In the rMCMV-infected postnatal cerebrum, lytic infection and the activation of both promoters were found in non-neuronal cells of the PV region until postnatal 8 days (P8), but these disappeared at P12, while the activation of e1-pro-1373, but not -448 appeared in HP and CX neurons at P8 and were prolonged exclusively in these neurons at P12, with preservation of the neuronal morphology. Therefore, e1-pro-448 is sufficient to activate E1 expression in non-neuronal cells, however, the upstream sequence from nt -449 to -1373 in e1-pro-1373 is supposed to work as an enhancer necessary for the neuron-specific activation of e1-pro, particularly around the second postnatal week. This unique activation of e1-pro in developing cerebral neurons may be an important factor in the neurodevelopmental disorders induced by congenital CMV infection.

MCMV triggers ROS/NLRP3-associated inflammasome activation in the inner ear of mice and cultured spiral ganglion neurons, contributing to sensorineural hearing loss.

Congenital cytomegalovirus (CMV) infection is the most common infectious cause of sensorineural hearing loss in children. While the importance of CMV-induced SNHL has been described, the mechanisms underlying its pathogenesis and the role of inflammatory responses remain elusive. The present study established an experimental model of hearing loss after systemic infection with murine CMV (MCMV) in newborn mice. Auditory brainstem responses were tested to evaluate hearing at 3 weeks, expression of inflammasome-associated factors was assessed by immunofluorescence, western blot analysis, reverse transcription-quantitative polymerase chain reaction and ELISA. MCMV sequentially induced inflammasome-associated factors. Furthermore, the inflammasome-associated factors were also increased in cultured spiral ganglion neurons infected with MCMV for 24 h. In addition, MCMV increased the content of reactive oxygen species (ROS). These results suggest that hearing loss caused by MCMV infection may be associated with ROS-induced inflammation.

Natural killer cells attenuate cytomegalovirus-induced hearing loss in mice.

Congenital cytomegalovirus (CMV) infection is the most common non-hereditary cause of sensorineural hearing loss (SNHL) yet the mechanisms of hearing loss remain obscure. Natural Killer (NK) cells play a critical role in regulating murine CMV infection via NK cell recognition of the Ly49H cell surface receptor of the viral-encoded m157 ligand expressed at the infected cell surface. This Ly49H NK receptor/m157 ligand interaction has been found to mediate host resistance to CMV in the spleen, and lung, but is much less effective in the liver, so it is not known if this interaction is important in the context of SNHL. Using a murine model for CMV-induced labyrinthitis, we have demonstrated that the Ly49H/m157 interaction mediates host resistance in the temporal bone. BALB/c mice, which lack functional Ly49H, inoculated with mCMV at post-natal day 3 developed profound hearing loss and significant outer hair cell loss by 28 days of life. In contrast, C57BL/6 mice, competent for the Ly49H/m157 interaction, had minimal hearing loss and attenuated outer hair cell loss with the same mCMV dose. Administration of Ly49H blocking antibody or inoculation with a mCMV viral strain deleted for the m157 gene rendered the previously resistant C57BL/6 mouse strain susceptible to hearing loss to a similar extent as the BALB/c mouse strain indicating a direct role of the Ly49H/m157 interaction in mCMV-dependent hearing loss. Additionally, NK cell recruitment to sites of infection was evident in the temporal bone of inoculated susceptible mouse strains. These results demonstrate participation of NK cells in protection from CMV-induced labyrinthitis and SNHL in mice.

Cochlear Spiral Ganglion Neuron Apoptosis in Neonatal Mice with Murine Cytomegalovirus–Induced Sensorineural Hearing Loss

Sensorineural hearing loss (SNHL) is the most common consequence of congenital cytomegalovirus (CMV) infection, and could result in neurological abnormalities and intellectual and developmental disabilities. To explore the mechanism of murine CMV (MCMV)-induced SNHL in neonatal mice model. A repeated measures design was used. Total 72 neonatal BALB/C mice (36 males and 36 females) were randomly divided into two groups. MCMV suspension (50% tissue culture infective dose = 10(4.15) IU/0.1 ml, 15 μl) or physiological saline was intracranially injected into neonatal mice in the experimental or control group, respectively. Auditory brainstem response (ABR) was measured at three weeks postinjection. At 1, 3, 5, 7, 14, and 21 days postinjection, MCMV-DNA polymerase chain reaction analysis was performed to detect MCMV infection in cochlea, followed by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling analysis and immunohistochemistry staining. Extended latency, decreased amplitude, and increased threshold of ABR wave I were observed in the experimental group. Polymerase chain reaction test was positive from 3 to 21 days postinjection in the experimental group and negative at each time point in the control group. The average apoptosis index was higher in the experimental group than that in the control group from 3 to 21 days postinjection (p < 0.01). In addition, compared with the control group, B-cell lymphoma 2 and B-cell lymphoma 2-associated protein ratio was decreased in the experimental group (p < 0.01). Spiral ganglion neuron apoptosis was an important component of the mechanism of SNHL in MCMV-infected mice.

Pathological changes of the inner ear cochlea in different time windows of murine cytomegalovirus-induced hearing loss in a mouse model

Conclusions: Murine congenital cytomegalovirus (CMV) (MCMV) infection of the inner ear cochlea, which caused continuous pathological change, occurred on the third day after intracerebral injection and persisted for a very long time. Objective: Our study used the MCMV-induced hearing loss neonatal mouse model to investigate the pathological changes of the cochlea in different time windows. Methods: Sixty newborn BALB/c mice were randomly and equally divided into the experimental group (MCMV intracerebral injection) and control group (normal saline intracerebral injection). At 1, 3, 5, 7, and 21 days after intracerebral injection, cochleas were extracted and evaluated by MCMV-DNA PCR analysis and histopathological examination. Results: Two mice died before the end of the experiment in the experimental group, while there were no deaths in the control group. In the experimental group, the MCMV-DNA PCR samples did not show positive results in the gel electrophoresis until the 3–21 days after intracerebral injection, while no positive result was found in the control group. Pathological changes including hemorrhage and inflammatory cell infiltration of the scala vestibule and scala tympani began on the third day, spiral ganglion cell gap widening and cell thinning began on the seventh day, and scala tympani fibrosis began on the 14th day.

Aberrant fetal macrophage/microglial reactions to cytomegalovirus infection.

ObjectiveCongenital cytomegalovirus (CMV) infection is the leading viral cause of neurodevelopmental disorders in humans, with the most severe and permanent sequelae being those affecting the cerebrum. As the fetal immune reactions to congenital CMV infection in the brain and their effects on cerebral development remain elusive, our aim was to investigate primitive innate immunity to CMV infection and its effects on cerebral corticogenesis in a mouse model for congenital CMV infection using a precise intraplacental inoculation method.MethodsAt 13.5 embryonic days (E13.5), pregnant C57BL/6 mice were intraplacentally infected with murine CMV (MCMV). Placentas and fetal organs were collected at 1, 3, and 5 days postinfection and analyzed.ResultsMCMV antigens were found frequently in perivascular macrophages, and subsequently in neural stem/progenitor cells (NSPCs). With increased expression of inducible nitric oxide synthase and proinflammatory cytokines, activated macrophages infiltrated into the infectious foci. In addition to the infected area, the numbers of both meningeal macrophages and parenchymal microglia increased even in the uninfected areas of MCMV-infected brain due to recruitment of their precursors from other sites. A bromodeoxyuridine (BrdU) incorporation experiment demonstrated that MCMV infection globally disrupted the self-renewal of NSPCs. Furthermore, BrdU-labeled neurons, particularly Brn2+ neurons of upper layers II/III in the cortical plate, decreased in number significantly in the MCMV-infected E18.5 cerebrum.InterpretationBrain macrophages are crucial for innate immunity during MCMV infection in the fetal brain, while their aberrant recruitment and activation may adversely impact on the stemness of NSPCs, resulting in neurodevelopmental disorders.

Effects of cytomegalovirus infection on embryogenesis and brain development

Congenital cytomegalovirus (CMV) infection is a significant cause of brain disorders, such as microcephaly, mental retardation, hearing loss and visual disorders in humans. The type and severity of brain disorder may be dependent on the stage of embryonic development when the congenital infection occurs. Developmental disorders may be associated with the type of embryonic cells to which CMV is susceptible and the effects of the infection on the cellular functions of these cells. Early murine embryos, including embryonic stem (ES) cells, are not susceptible to CMV infection. A part of the embryonic cells acquire susceptibility during early development. Mesenchymal cells are the targets of infection at midgestation, affecting organogenesis of the brain, eyes and oral-facial regions. In contrast to ES cells, neural stem progenitor cells (NSPC) from fetal brains are susceptible to murine CMV (MCMV) infection. The viral infection inhibits proliferation and differentiation of the NSPC to neuronal and glial cells in addition to induction of neuronal cell loss. These cellular events may cause brain malformations, such as microcephaly and polymicrogyria. Furthermore, MCMV persists in neuronal cells in developing brains, presumably resulting in neuronal dysfunction.

Induction of cytomegalovirus-infected labyrinthitis in newborn mice by lipopolysaccharide: a model for hearing loss in congenital CMV infection

Congenital cytomegalovirus (CMV) infection is the most common infectious cause of sensorineural hearing loss in children. Here, we established an experimental model of hearing loss after systemic infection with murine CMV (MCMV) in newborn mice. Although almost no viral infection was observed in the inner ears and brains by intraperitoneal (i.p.) infection with MCMV in newborn mice, infection in these regions was induced in combination with intracerebral (i.c.) injection of bacterial lipopolysaccharide (LPS). The susceptibility of the inner ears was higher than that of the brains in terms of viral titer per unit weight. In the labyrinths, the viral infection was associated with the mesenchymal vessels and accompanied by inflammatory cells induced by LPS, causing hematogenous targets of infection in the labyrinths. Viral infection also spread in the perilymph regions such as the scala tympani and scala vestibuli, probably from infected brains via meningogenic and cochlear nerve routes. Viral infection was not observed in the scala media in the endolymph, including the Corti organ. However, viral infection was observed in the spiral limbus, including the stria vascularis. These results suggest that hearing loss caused by labyrinthitis after congenital CMV infection may be enhanced by inflammation caused by systemic bacterial infection in the neonatal period.

Growth retardation and microcephaly induced in mice by placental infection with murine cytomegalovirus.

The placenta is regarded as a site of congenital cytomegalovirus (CMV) infection. The placental infection of fetuses with murine CMV (MCMV) was investigated in a mouse model. The placentas and fetuses were examined using the polymerase chain reaction (PCR) and Southern blotting for viral DNA and immunostaining for viral antigen. Since the transplacental infection rarely occurs, the placentas were directly injected with MCMV at day 12.5 of gestation; the embryos were then allowed to develop until day 18.5 of gestation. Formation of infected foci at day 18. 5 of gestation was found in more than 60% of the injected placentas. Infection of about 50% of the embryos occurred from the infected placentas. The frequency of infection in the brain was 27%, which was the same as that in the liver and higher than that in the lungs. In the brains, infected cells were often observed in the ventricular zone of the cerebrum and sometimes in the cortical plate and the hippocampus. Developmental retardation with microcephaly was observed in about 25% of offspring exposed to infection in utero. These results suggest that formation of infected foci in the placenta is important for embryonic congenital infection, and that the cerebral ventricular zone is one of the most susceptible sites for CMV infection in the embryonic stage.

Disordered migration and loss of virus-infected neuronal cells in developing mouse brains infected with murine cytomegalovirus.

Microcephaly is the most prominent symptom of the developmental brain abnormalities induced by congenital cytomegalovirus (CMV) infection. To investigate the effect of CMV infection on neuronal migration in developing brains, mouse embryos on one side of uteri received, on day 15.5 of gestation (E15.5), an injection of murine CMV (MCMV) into the cerebral ventricles, and the embryos on the other side of the uteri were injected with minimum essential medium (MEM). Labeling with 5-bromo-2-deoxyuridine (BrdU) was accomplished by intraperitoneal injection of BrdU 6 h later. Disturbance of the neuronal migration and loss of neurons were observed postnatally in the brains of MCMV-infected mice, which were identified by immunohistochemical staining of viral antigen. Double staining of BrdU-labeled and viral antigen-positive cells in brains on the 7th postnatal day showed that the migration of BrdU-single-labeled cells, mainly localized in cerebral layers II-III, mostly preceded that of the viral antigen-positive cells. However, about 7.5% of the cells observed were double-labeled, especially in the layers III-IV, and a few double-stained cells were markedly disturbed in migration. In the brains of offspring labeled with BrdU 72 h after infection with MCMV on E15.5, most of the double-stained cells were seen around the ventricular and subventricular zones. These findings suggest that a disturbance of neuronal migration in addition to neuronal loss may play a crucial role in the development of microcephaly in congenital CMV infection in humans.

Developmental disorders of the mouse brain induced by murine cytomegalovirus: Animal models for congenital cytomegalovirus infection

Developmental disorders induced by congenital cytomegalovirus (CMV) infection mainly involve the central nervous system. The type and degree of the brain disorders seems to depend on infection time during gestation, virulence, route of infection and viral susceptible cells in each embryonal stage. Since transplacental transmission has been reported not to occur with murine CMV (MCMV), we developed mouse models for congenital CMV infection by surgical injection of MCMV into the mouse conceptus or embryo at different gestational stages. For the early stage, the mouse embryos were not infected with MCMV even after injecting the virus into the blastocysts, which were developed in the pseudo-pregnant mothers or cultured in vitro. Isolated whole mouse embryos of day 7.5 of gestation (E7.5), adsorbed with a high titer of MCMV and cultured for 3 days, were susceptible to MCMV infection. Therefore, the mouse embryo acquires the susceptibility around this period. Microphthalmia and cerebral atrophy were induced in mouse embryos after injection of MCMV into the conceptus on E8.5. Viral antigen-positive cells were widely distributed in the mesenchyme around the oral and nasal cavities and in the mesenchyme around the brain, especially the endothelial cells of vessels and the perivascular mesodermal cells, then infection extends to the eyes, brain or choroid plexus. This finding suggests that mesenchymal infection may be the critical step in disrupting organogenesis, resulting in brain disorders. For the late stage, mouse embryos were infected with MCMV by injecting the virus into the cerebral ventricles on E15.5. Brains of the offspring showed massive necrosis with gliomesodermal proliferation in the cerebral cortex. Viral antigen-positive cells were observed in laminar array in the lesion-free cortex and the hippocampus, suggesting that the infected cells migrate in association with the lamina formation. Immunohistochemical double-staining showed that brain cells susceptible to MCMV infection may be mainly neuronal and endothelial cells, resulting in cerebral atrophy with reduction of neuronal cells and cystic lesions, presumably due to ischemic vascular changes.