Search Results (14)

Retinitis pigmentosa (RP) is a progressive inherited retinal dystrophy (IRD) that primarily affects rod photoreceptor cells, leading to the degeneration of photoreceptors and the gradual loss of vision. While RP is one of the most studied IRDs, other neurodegenerative diseases affecting the retina and optic nerve, such as glaucoma, also involve common mechanisms of cellular stress and degeneration. Current therapeutic approaches under investigation include gene therapy, retina prosthesis, and neuroprotection. Among these approaches, gene therapy has shown promise, though challenges related to viral vector tropism and transduction efficiency persist. The adeno-associated virus (AAV) vector is commonly employed for gene delivery, but novel serotypes and engineered variants are being explored to improve specificity and efficacy. This study evaluates the gene transfer efficiency of the AAV6.2 vector following intravitreal injection into the murine retina. Male C57BL/6 mice (9 weeks old) were intravitreally injected with 1 µL of AAV2-CMV-EGFP, AAV6-CMV-EGFP, or AAV6.2-CMV-EGFP at a titer of 3.2 × 10¹² vg/mL per eye. Retinal transduction was assessed using in vivo fluorescence imaging, flat-mount imaging, and immunohistochemistry. EGFP expression in retinal ganglion cells, Müller cells, amacrine cells, and bipolar cells was quantitatively analyzed. All three AAV serotypes effectively transduced retinal ganglion cells, but AAV6.2 exhibited enhanced transduction in Müller cells and other neuronal retinal cells, including bipolar and amacrine cells. AAV6.2 demonstrated more localized expression around retinal blood vessels compared to the diffuse expression observed with AAV2. Immunohistochemical analysis revealed that AAV6.2 had significantly higher transduction efficiency in Müller cells (p < 0.001) compared to AAV2 and AAV6. AAV6.2 shows superior transduction efficiency in Müller cells, positioning it as a promising vector for gene therapies targeting retinal degenerative diseases such as RP. Its ability to effectively transduce Müller cells suggests potential applications in neuroprotection and gene replacement therapies. Full article

Congenital cytomegalovirus (CMV) infection is the main cause of non-hereditary sensorineural hearing loss (SNHL). In order to shed light on SNHL pathophysiology, we examined the auditory pathway in CMV-infected fetuses; the temporal lobe, in particular the auditory cortex, and the inner ear. We investigated both inner ears and temporal lobes of 20 human CMV-infected fetuses at 21 weeks of gestation. As a negative group, five fetuses from spontaneous miscarriages without CMV infection were studied. Inner ears and temporal lobes were histologically examined, immunohistochemistry for CMV and CMV-PCR were performed. On the auditory cortex, we evaluated the local microglial reaction to the infection. CMV-positive cells were found in 14/20 brains and the damage was classified as severe, moderate, or mild, according to histological features. Fetuses with severe brain damage had a statistically higher temporal lobe viral load and a higher number of activated microglial cells in the auditory cortex compared to fetuses with mild brain damage (p: 0.01; p: 0.01). In the inner ears, the marginal cells of the stria vascularis were the most CMV positive. In our study, CMV affected the auditory pathway, suggesting a tropism for this route. In addition, in the auditory cortex, microglial activation may favor further tissue damage contributing to hearing loss. Full article

Viral vectors have emerged as powerful tools for delivering and expressing foreign genes, playing a pivotal role in gene therapy. Among these vectors, cytomegalovirus (CMV) stands out as a promising viral vector due to its distinctive attributes including large packaging capacity, ability to achieve superinfection, broad host range, capacity to induce CD8+ T cell responses, lack of integration into the host genome, and other qualities that make it an appealing vector candidate. Engineered attenuated CMV strains such as Towne and AD169 that have a ~15 kb genomic DNA deletion caused by virus passage guarantee human safety. CMV’s large genome enables the efficient incorporation of substantial foreign genes as demonstrated by CMV vector-based therapies for SIV, tuberculosis, cancer, malaria, aging, COVID-19, and more. CMV is capable of reinfecting hosts regardless of prior infection or immunity, making it highly suitable for multiple vector administrations. In addition to its broad cellular tropism and sustained high-level gene expression, CMV triggers robust, virus-specific CD8⁺ T cell responses, offering a significant advantage as a vaccine vector. To date, successful development and testing of murine CMV (MCMV) and rhesus CMV (RhCMV) vectors in animal models have demonstrated the efficacy of CMV-based vectors. These investigations have explored the potential of CMV vectors for vaccines against HIV, cancer, tuberculosis, malaria, and other infectious pathogens, as well as for other gene therapy applications. Moreover, the generation of single-cycle replication CMV vectors, produced by deleting essential genes, ensures robust safety in an immunocompromised population. The results of these studies emphasize CMV’s effectiveness as a gene delivery vehicle and shed light on the future applications of a CMV vector. While challenges such as production complexities and storage limitations need to be addressed, ongoing efforts to bridge the gap between animal models and human translation continue to fuel the optimism surrounding CMV-based vectors. This review will outline the properties of CMV vectors and discuss their future applications as well as possible limitations. Full article

Glaucoma is a multifactorial disease leading to irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form and is associated with the elevation of intraocular pressure (IOP). Reduced aqueous humor (AH) outflow due to trabecular meshwork (TM) dysfunction is responsible for IOP elevation in POAG. Extracellular matrix (ECM) accumulation, actin cytoskeletal reorganization, and stiffening of the TM are associated with increased outflow resistance. Transforming growth factor (TGF) β2, a profibrotic cytokine, is known to play an important role in the development of ocular hypertension (OHT) in POAG. An appropriate mouse model is critical in understanding the underlying molecular mechanism of TGFβ2-induced OHT. To achieve this, TM can be targeted with recombinant viral vectors to express a gene of interest. Lentiviruses (LV) are known for their tropism towards TM with stable transgene expression and low immunogenicity. We, therefore, developed a novel mouse model of IOP elevation using LV gene transfer of active human TGFβ2 in the TM. We developed an LV vector-encoding active hTGFβ2^C226,228S under the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFβ2^C226,228S. We observed a significant increase in IOP 3 weeks post-injection compared to control eyes with an average delta change of 3.3 mmHg. IOP stayed elevated up to 7 weeks post-injection, which correlated with a significant drop in the AH outflow facility (40.36%). Increased expression of active TGFβ2 was observed in both AH and anterior segment samples of injected mice. The morphological assessment of the mouse TM region via hematoxylin and eosin (H&E) staining and direct ophthalmoscopy examination revealed no visible signs of inflammation or other ocular abnormalities in the injected eyes. Furthermore, transduction of primary human TM cells with LV_hTGFβ2^C226,228S exhibited alterations in actin cytoskeleton structures, including the formation of F-actin stress fibers and crossed-linked actin networks (CLANs), which are signature arrangements of actin cytoskeleton observed in the stiffer fibrotic-like TM. Our study demonstrated a mouse model of sustained IOP elevation via lentiviral gene delivery of active hTGFβ2^C226,228S that induces TM dysfunction and outflow resistance. Full article

Congenital cytomegalovirus (CMV) is a leading cause of disease in newborns and a vaccine is a high priority. The guinea pig is the only small animal model for congenital CMV but requires guinea pig cytomegalovirus (GPCMV). Previously, a disabled infectious single cycle (DISC) vaccine strategy demonstrated complete protection against congenital GPCMV (22122 strain) and required neutralizing antibodies to various viral glycoprotein complexes. This included gB, essential for all cell types, and the pentamer complex (PC) for infection of non-fibroblast cells. All GPCMV research has utilized prototype strain 22122 limiting the translational impact, as numerous human CMV strains exist allowing re-infection and congenital CMV despite convalescent immunity. A novel GPCMV strain isolate (designated TAMYC) enabled vaccine cross strain protection studies. A GPCMV DISC (PC+) vaccine (22122 strain) induced a comprehensive immune response in animals, but vaccinated animals challenged with the TAMYC strain virus resulted in sustained viremia and the virus spread to target organs (liver, lung and spleen) with a significant viral load in the salivary glands. Protection was better than natural convalescent immunity, but the results fell short of previous DISC vaccine sterilizing immunity against the homologous 22122 virus challenge, despite a similarity in viral glycoprotein sequences between strains. The outcome suggests a limitation of the current DISC vaccine design against heterologous infection. Full article

Viruses that infect the central nervous system (CNS) are associated with developmental abnormalities as well as neuropsychiatric and degenerative conditions. Many of these viruses such as Zika virus (ZIKV), cytomegalovirus (CMV), and herpes simplex virus (HSV) demonstrate tropism for neural stem cells (NSCs). NSCs are the multipotent progenitor cells of the brain that have the ability to form neurons, astrocytes, and oligodendrocytes. Viral infections often alter the function of NSCs, with profound impacts on the growth and repair of the brain. There are a wide spectrum of effects on NSCs, which differ by the type of virus, the model system, the cell types studied, and the age of the host. Thus, it is a challenge to predict and define the consequences of interactions between viruses and NSCs. The purpose of this review is to dissect the mechanisms by which viruses can affect survival, proliferation, and differentiation of NSCs. This review also sheds light on the contribution of key antiviral cytokines in the impairment of NSC activity during a viral infection, revealing a complex interplay between NSCs, viruses, and the immune system. Full article

Human cytomegalovirus (HCMV) is a highly prevalent herpesvirus that can cause severe disease in immunocompromised individuals and immunologically immature fetuses and newborns. Most infected newborns are able to resolve the infection without developing sequelae. However, in severe cases, congenital HCMV infection can result in life-threatening pathologies and permanent damage of organ systems that possess a low regenerative capacity. Despite the severity of the problem, HCMV infection of the central nervous system (CNS) remains inadequately characterized to date. Cytomegaloviruses (CMVs) show strict species specificity, limiting the use of HCMV in experimental animals. Infection following intraperitoneal administration of mouse cytomegalovirus (MCMV) into newborn mice efficiently recapitulates many aspects of congenital HCMV infection in CNS. Upon entering the CNS, CMV targets all resident brain cells, consequently leading to the development of widespread histopathology and inflammation. Effector functions from both resident cells and infiltrating immune cells efficiently resolve acute MCMV infection in the CNS. However, host-mediated inflammatory factors can also mediate the development of immunopathologies during CMV infection of the brain. Here, we provide an overview of the cytomegalovirus infection in the brain, local immune response to infection, and mechanisms leading to CNS sequelae. Full article

Cytomegalovirus (CMV) establishes persistent, latent infection in hosts, causing diseases in immunocompromised patients, transplant recipients, and neonates. CMV infection modifies the host chemokine axis by modulating chemokine and chemokine receptor expression and by encoding putative chemokine and chemokine receptor homologues. The viral proteins have roles in cellular signaling, migration, and transformation, as well as viral dissemination, tropism, latency and reactivation. Herein, we review the contribution of CMV-encoded chemokines and chemokine receptors to these processes, and further elucidate the viral tropism role of rat CMV (RCMV) R129 and R131. These homologues of the human CMV (HCMV)-encoded chemokines UL128 and UL130 are of particular interest because of their dual role as chemokines and members of the pentameric entry complex, which is required for entry into cell types that are essential for viral transmission and dissemination. The contributions of UL128 and UL130 to acceleration of solid organ transplant chronic rejection are poorly understood, and are in need of an effective in vivo model system to elucidate the phenomenon. We demonstrated similar molecular entry requirements for R129 and R131 in the rat cells, as observed for HCMV, and provided evidence that R129 and R131 are part of the viral entry complex required for entry into macrophages, dendritic cells, and bone marrow cells. Full article

Despite displaying broad tropism in vivo, human cytomegalovirus (CMV) contained in bodily fluids replicates inefficiently in most cultured cell types except fibroblasts. As propagation in fibroblasts leads to the accumulation of genomic changes, a number of strains were generated by serial passaging on endothelial cells. One of these, TB40/E, was shown to contain a mixture of genetically distinct virus variants, and to retain tropism for fibroblasts, endothelial and epithelial cells. Cloning of an endotheliotropic subpopulation produced the TB40-BAC4 variant, extensively used in CMV tropism studies. Because TB40-BAC4 represents only one of the different variants comprising TB40/E, we generated a series of epithelial-cell adapted stocks derived from a TB40/E mixed stock, rather than from TB40-BAC4. Within two passages on ARPE-19 cells, virus populations were produced with the ability to enter and initiate replication with similar efficiencies in both epithelial cells and fibroblasts. Although the ability to release progeny also increased, cell-free virus yields from ARPE-19 cells remained consistently two to three-logs lower than from fibroblasts, hinting at the existence of a post-entry and post-genome synthesis block in epithelial cells. Multinucleated syncytia also rapidly appeared exclusively in ARPE-19 cell cultures, where their numbers and dimensions increased with virus passage. Irrespective of the number of infected nuclei comprising each syncytium, however, only one cytoplasmic virion assembly compartment was consistently observed, leading us to speculate that improvements in entry efficiency associated with ARPE-19 cell adaptation lead to the development of syncytia, which may negatively affect progeny release by limiting the amount of resources available to maturing virions. Full article

Determination of the cellular tropism of viral vectors is imperative for designing precise gene therapy. It has been widely accepted that transduction of hepatocytes using adeno-associated virus serotype 8 (AAV8) is a promising approach to correct inborn errors in neonates, but the type of neonatal hepatic cells transduced by AAV8 has not been thoroughly investigated. To address this question, we used a reporter mouse that carries Cre recombinase (Cre)-inducible yellow fluorescent protein (YFP). Our analysis primarily focused on cholangiocytes, given their pivotal roles in normal liver function and disease. We treated Rosa^YFP/+ mice at postnatal day 2 (P2) with AAV8-cytomegalovirus (CMV) promoter-Cre and analyzed livers at P10 and P56. The vast majority of HNF4α+ hepatocytes were labeled with YFP at both time points, and 11.6% and 24.4% of CK19+ cholangiocytes were marked at P10 and P56, respectively. We also detected YFP+ cells devoid of hepatocyte and cholangiocyte markers, and a subset of these cells expressed the endothelial and fibroblast marker CD34. Next, we used the hepatocyte-specific thyroxine-binding globulin (TBG) promoter. Surprisingly, AAV8-TBG-Cre marked 6.8% and 30.9% of cholangiocytes at P10 and P56, respectively. These results suggest that AAV8 can be a useful tool for targeting cholangiocytes in neonatal livers. Full article

Nipah virus (NiV) is an emergent pathogen capable of causing acute respiratory illness and fatal encephalitis in pigs and humans. A high fatality rate and broad host tropism makes NiV a serious public and animal health concern. There is therefore an urgent need for a NiV vaccines to protect animals and humans. In this study we investigated the immunogenicity of bovine herpesvirus (BoHV-4) vectors expressing either NiV attachment (G) or fusion (F) glycoproteins, BoHV-4-A-CMV-NiV-GΔTK or BoHV-4-A-CMV-NiV-FΔTK, respectively in pigs. The vaccines were benchmarked against a canarypox (ALVAC) vector expressing NiV G, previously demonstrated to induce protective immunity in pigs. Both BoHV-4 vectors induced robust antigen-specific antibody responses. BoHV-4-A-CMV-NiV-GΔTK stimulated NiV-neutralizing antibody titers comparable to ALVAC NiV G and greater than those induced by BoHV-4-A-CMV-NiV-FΔTK. In contrast, only BoHV-4-A-CMV-NiV-FΔTK immunized pigs had antibodies capable of significantly neutralizing NiV G and F-mediated cell fusion. All three vectored vaccines evoked antigen-specific CD4 and CD8 T cell responses, which were particularly strong in BoHV-4-A-CMV-NiV-GΔTK immunized pigs and to a lesser extent BoHV-4-A-CMV-NiV-FΔTK. These findings emphasize the potential of BoHV-4 vectors for inducing antibody and cell-mediated immunity in pigs and provide a solid basis for the further evaluation of these vectored NiV vaccine candidates. Full article

Zika virus (ZIKV) is transmitted by Aedes mosquitoes and exhibits genetic variation with African and Asian lineages. ZIKV Natal RGN strain, an Asian-lineage virus, has been identified in brain tissues from fetal autopsy cases with microcephaly and is suggested to be a neurotropic variant. However, ZIKV Natal RGN strain has not been isolated; its biological features are not yet illustrated. This study rescued and characterized recombinant, single-round infectious particles (SRIPs) of the ZIKV Natal RGN strain using reverse genetic and synthetic biology techniques. The DNA-launched replicon of ZIKV Natal RGN was constructed and contains the EGFP reporter, lacks prM-E genes, and replicates under CMV promoter control. The peak in the ZIKV Natal RGN SRIP titer reached 6.25 × 10⁶ TCID50/mL in the supernatant of prM-E-expressing packaging cells 72 h post-transfection with a ZIKV Natal RGN replicon. The infectivity of ZIKV Natal RGN SRIPs has been demonstrated to correlate with the green florescence intensity of the EGFP reporter, the SRIP-induced cytopathic effect, and ZIKV’s non-structural protein expression. Moreover, ZIKV Natal RGN SRIPs effectively self-replicated in rhabdomyosarcoma/muscle, glioblastoma/astrocytoma, and retinal pigmented epithelial cells, displaying unique cell susceptibility with differential attachment activity. Therefore, the recombinant ZIKV Natal RGN strain was rescued as SRIPs that could be used to elucidate the biological features of a neurotropic strain regarding cell tropism and pathogenic components, apply for antiviral agent screening, and develop vaccine candidates. Full article

Human cytomegalovirus (HCMV) is an opportunistic pathogen causing disease mainly in immunocompromised patients or after congenital infection. HCMV infection of the respiratory tract leads to pneumonitis in the immunocompromised host, which is often associated with a bad clinical course. The related mouse cytomegalovirus (MCMV) likewise exhibits a distinct tropism for the lung and thus provides an elegant model to study host-pathogen interaction. Accordingly, fundamental features of cytomegalovirus (CMV) pneumonitis have been discovered in mice that correlate with clinical data obtained from humans. Recent studies have provided insight into MCMV cell tropism and localized inflammation after infection of the respiratory tract. Accordingly, the nodular inflammatory focus (NIF) has been identified as the anatomical correlate of immune control in lungs. Several hematopoietic cells involved in antiviral immunity reside in NIFs and their key effector molecules have been deciphered. Here, we review what has been learned from the mouse model with focus on the microanatomy of infection sites and antiviral immunity in MCMV pneumonitis. Full article

Propagation of human cytomegalovirus (CMV) in cultured cells results in genetic adaptations that confer improved growth in vitro and significant attenuation in vivo. Mutations in RL13 arise quickly, while mutations in the UL128-131A locus emerge later during fibroblast passage and disrupt formation of a glycoprotein complex that is important for entry into epithelial and endothelial cells. As CMV replicates in the context of host antibodies in vivo, we reasoned that antibodies might mitigate the accumulation of adaptive mutations during cell culture passage. To test this, CMV in infant urine was used to infect replicate fibroblast cultures. One lineage was passaged in the absence of CMV-hyperimmuneglobulin (HIG) while the other was passaged with HIG in the culture medium. The former lost epithelial tropism and acquired mutations disrupting RL13 and UL131A expression, whereas the latter retained epithelial tropism and both gene loci remained intact after 22 passages. Additional mutations resulting in single amino acid changes also occurred in UL100 encoding glycoprotein M, UL102 encoding a subunit of the helicase/primase complex, and UL122 encoding the Immediate Early 2 protein. An epitheliotropic RL13+/UL131A+ virus was isolated by limiting dilution in the presence of HIG and expanded to produce a working stock sufficient to conduct cell tropism experiments. Thus, production of virus stocks by culture in the presence of antibodies may facilitate in vitro experiments using viruses that are genetically more authentic than previously available. Full article