Olga A. Maximova

The Transcription Factor IRF8 Activates Integrin-Mediated TGF-β Signaling and Promotes Neuroinflammation

Recent epidemiological studies have identified interferon regulatory factor 8 (IRF8) as a susceptibility factor for multiple sclerosis (MS). However, how IRF8 influences the neuroinflammatory disease has remained unknown. By studying the role of IRF8 in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we found that Irf8(-/-) mice are resistant to EAE. Furthermore, expression of IRF8 in antigen-presenting cells (APCs, such as macrophages, dendritic cells, and microglia), but not in T cells, facilitated disease onset and progression through multiple pathways. IRF8 enhanced αvβ8 integrin expression in APCs and activated TGF-β signaling leading to T helper 17 (Th17) cell differentiation. IRF8 induced a cytokine milieu that favored growth and maintenance of Th1 and Th17 cells, by stimulating interleukin-12 (IL-12) and IL-23 production, but inhibiting IL-27 during EAE. Finally, IRF8 activated microglia and exacerbated neuroinflammation. Together, this work provides mechanistic bases by which IRF8 contributes to the pathogenesis of MS.

Insertion of MicroRNA Targets into the Flavivirus Genome Alters Its Highly Neurovirulent Phenotype

ABSTRACT Flaviviruses such as West Nile, Japanese encephalitis, and tick-borne encephalitis (TBEV) viruses are important neurotropic human pathogens, causing a devastating and often fatal neuroinfection. Here, we demonstrate that incorporation into the viral genome of a target sequence for cellular microRNAs expressed in the central nervous system (CNS) enables alteration of the neurovirulence of the virus and control of the neuropathogenesis of flavivirus infection. As a model virus for this type of modification, we used a neurovirulent chimeric tick-borne encephalitis/dengue virus (TBEV/DEN4) that contained the structural protein genes of a highly pathogenic TBEV. The inclusion of just a single target copy for a brain tissue-expressed mir-9, mir-124a, mir-128a, mir-218, or let-7c microRNA into the TBEV/DEN4 genome was sufficient to prevent the development of otherwise lethal encephalitis in mice infected intracerebrally with a large dose of virus. Viruses bearing a complementary target for mir-9 or mir-124a were highly restricted in replication in primary neuronal cells, had limited access into the CNS of immunodeficient mice, and retained the ability to induce a strong humoral immune response in monkeys. This work suggests that microRNA targeting to control flavivirus tissue tropism and pathogenesis might represent a rational approach for virus attenuation and vaccine development.

MicroRNA-targeting of neurotropic flavivirus: effective control of virus escape and reversion to neurovirulent phenotype.

ABSTRACT Neurotropic flaviviruses can efficiently replicate in the developing and mature central nervous systems (CNS) of mice causing lethal encephalitis. Insertion of a single copy of a target for brain-expressed microRNAs (miRNAs) in the 3′ noncoding region (3′NCR) of the flavivirus genome (chimeric tick-borne encephalitis virus/dengue virus) abolished virus neurovirulence in the mature mouse CNS. However, in the developing CNS of highly permissive suckling mice, the miRNA-targeted viruses can revert to a neurovirulent phenotype by accumulating deletions or mutations within the miRNA target sequence. Virus escape from miRNA-mediated suppression in the developing CNS was markedly diminished by increasing the number of miRNA target sites and by extending the distance between these sites in the virus genome. Insertion of multiple miRNA targets into the 3′NCR altered virus neuroinvasiveness, decreased neurovirulence and neuroinflammatory responses, and prevented neurodegeneration without loss of immunogenicity. Although the onset of encephalitis was delayed, a small number of suckling mice still succumbed to lethal intracerebral infection with the miRNA-targeted viruses. Sequence analysis of brain isolates from moribund mice revealed that the viruses escaped from miRNA-mediated suppression exclusively through the deletion of miRNA targets and viral genome sequence located between the two miRNA targets separated by the greatest distance. These findings offer a general strategy to control the reversion of virus to a virulent phenotype: a simultaneous miRNA targeting of the viral genome at many different functionally important regions could prevent virus escape from miRNA-based attenuation, since a deletion of the targeted genomic sequences located between the inserted miRNA binding sites would be lethal for the virus.

Computerized morphometric analysis of pathological prion protein deposition in scrapie-infected hamster brain.

Transmissible spongiform encephalopathies (TSEs or prion diseases) are characterized by a constellation of typical though variable pathological changes in the brain. Deposition of disease-associated abnormal prion protein (PrPSc) is the pathological feature of TSEs most consistent and accessible for quantification. However, the evaluation of PrPSc deposits detected by immunohistochemical techniques has been traditionally based on arbitrarily assigned semiquantitative scores. This approach is limited by its subjectivity and bias, yielding considerable variability. In this study, we used MetaMorph 6.1 image analysis software for quantitative analysis of immunostained PrPSc deposits in the CNS of hamsters infected with the 263K strain of scrapie agent. Computerized morphometric analysis (CMA) allowed unambiguous detection of even minimal amounts of immunostained PrPSc. CMA values for intensity of staining and area stained correlated well with semiquantitative scores, providing reproducible quantitative data and objective criteria for analyzing PrPSc deposition. CMA provides a simple and reliable method for improved and consistent diagnosis of TSEs that may also be used to quantify other immunostained biomarkers.

Comparative Neuropathogenesis and Neurovirulence of Attenuated Flaviviruses in Nonhuman Primates

ABSTRACT Based on previous preclinical evaluation in mice and monkeys, the chimeric TBEV/DEN4Δ30 virus, carrying the prM and E protein genes from a highly virulent Far Eastern strain of tick-borne encephalitis virus (TBEV) on the backbone of a nonneuroinvasive dengue type 4 virus (DEN4), has been identified as a promising live attenuated virus vaccine candidate against disease caused by TBEV. However, prior to use of this vaccine candidate in humans, its neurovirulence in nonhuman primates needed to be evaluated. In the present study, we compared the neuropathogeneses of the chimeric TBEV/DEN4Δ30 virus; Langat virus (LGTV), a former live TBEV vaccine; and yellow fever 17D virus vaccine (YF 17D) in rhesus monkeys inoculated intracerebrally. TBEV/DEN4Δ30 and YF 17D demonstrated remarkably similar spatiotemporal profiles of virus replication and virus-associated histopathology in the central nervous system (CNS) that were high in cerebral hemispheres but progressively decreased toward the spinal cord. In contrast, the neurovirulence of LGTV exhibited the reverse profile, progressing from the site of inoculation toward the cerebellum and spinal cord. Analysis of the spatiotemporal distribution of viral antigens in the CNS of monkeys revealed a prominent neurotropism associated with all three attenuated viruses. Nevertheless, TBEV/DEN4Δ30 virus exhibited higher neurovirulence in monkeys than either LGTV or YF 17D, suggesting insufficient attenuation. These results provide insight into the neuropathogenesis associated with attenuated flaviviruses that may guide the design of safe vaccines.

Cellular Inflammatory Response to Flaviviruses in the Central Nervous System of a Primate Host

Flaviviruses such as tick-borne encephalitis virus, Japanese encephalitis virus, West Nile virus, and St. Louis encephalitis virus are important neurotropic human pathogens, typically causing a devastating and often fatal neuroinfection. Flaviviruses induce neuroinflammation with typical features of viral encephalitides, including inflammatory cell infiltration, activation of microglia, and neuronal degeneration. Development of safe and effective live-virus vaccines against neurotropic flavivirus infections demands a detailed knowledge of their neuropathogenesis in a primate host that is evolutionarily close to humans. Here, we used computerized morphometric analysis to quantitatively assess the cellular inflammatory responses in the central nervous system (CNS) of rhesus monkeys infected with three antigenically divergent attenuated flaviviruses. The kinetics, spatial pattern, and magnitude of microglial activation, trafficking of T and B cells, and changes in T cell subsets within the CNS define unique phenotypic signatures for each of the three viruses. Our results provide a benchmark for investigation of cellular inflammatory responses induced by attenuated flaviviruses in the CNS of primate hosts and provide insight into the neuropathogenesis of flavivirus encephalitis that might guide the development of safe and effective live-virus vaccines.

Targeted Deletion of the Gene Encoding the La Autoantigen (Sjögren's Syndrome Antigen B) in B Cells or the Frontal Brain Causes Extensive Tissue Loss

ABSTRACT La antigen (Sjögrens syndrome antigen B) is a phosphoprotein associated with nascent precursor tRNAs and other RNAs, and it is targeted by autoantibodies in patients with Sjögrens syndrome, systemic lupus erythematosus, and neonatal lupus. Increased levels of La are associated with leukemias and other cancers, and various viruses usurp La to promote their replication. Yeast cells (Saccharomyces cerevisiae and Schizosaccharomyces pombe) genetically depleted of La grow and proliferate, whereas deletion from mice causes early embryonic lethality, raising the question of whether La is required by mammalian cells generally or only to surpass a developmental stage. We developed a conditional La allele and used it in mice that express Cre recombinase in either B cell progenitors or the forebrain. B cell Mb1Cre La-deleted mice produce no B cells. Consistent with αCamKII Cre, which induces deletion in hippocampal CA1 cells in the third postnatal week and later throughout the neocortex, brains develop normally in La-deleted mice until ∼5 weeks and then lose a large amount of forebrain cells and mass, with evidence of altered pre-tRNA processing. The data indicate that La is required not only in proliferating cells but also in nondividing postmitotic cells. Thus, La is essential in different cell types and required for normal development of various tissue types.

West Nile Virus Spreads Transsynaptically within the Pathways of Motor Control: Anatomical and Ultrastructural Mapping of Neuronal Virus Infection in the Primate Central Nervous System

Background During recent West Nile virus (WNV) outbreaks in the US, half of the reported cases were classified as neuroinvasive disease. WNV neuroinvasion is proposed to follow two major routes: hematogenous and/or axonal transport along the peripheral nerves. How virus spreads once within the central nervous system (CNS) remains unknown. Methodology/Principal Findings Using immunohistochemistry, we examined the expression of viral antigens in the CNS of rhesus monkeys that were intrathalamically inoculated with a wild-type WNV. The localization of WNV within the CNS was mapped to specific neuronal groups and anatomical structures. The neurological functions related to structures containing WNV-labeled neurons were reviewed and summarized. Intraneuronal localization of WNV was investigated by electron microscopy. The known anatomical connectivity of WNV-labeled neurons was used to reconstruct the directionality of WNV spread within the CNS using a connectogram design. Anatomical mapping revealed that all structures identified as containing WNV-labeled neurons belonged to the pathways of motor control. Ultrastructurally, virions were found predominantly within vesicular structures (including autophagosomes) in close vicinity to the axodendritic synapses, either at pre- or post-synaptic positions (axonal terminals and dendritic spines, respectively), strongly indicating transsynaptic spread of the virus between connected neurons. Neuronal connectivity-based reconstruction of the directionality of transsynaptic virus spread suggests that, within the CNS, WNV can utilize both anterograde and retrograde axonal transport to infect connected neurons. Conclusions/Significance This study offers a new insight into the neuropathogenesis of WNV infection in a primate model that closely mimics WNV encephalomyelitis in humans. We show that within the primate CNS, WNV primarily infects the anatomical structures and pathways responsible for the control of movement. Our findings also suggest that WNV most likely propagates within the CNS transsynaptically, by both, anterograde and retrograde axonal transport.

High-throughput automated image analysis of neuroinflammation and neurodegeneration enables quantitative assessment of virus neurovirulence

Historically, the safety of live attenuated vaccine candidates against neurotropic viruses was assessed by semi-quantitative analysis of virus-induced histopathology in the central nervous system of monkeys. We have developed a high-throughput automated image analysis (AIA) for the quantitative assessment of virus-induced neuroinflammation and neurodegeneration. Evaluation of the results generated by AIA showed that quantitative estimates of lymphocytic infiltration, microglial activation, and neurodegeneration strongly and significantly correlated with results of traditional histopathological scoring. In addition, we show that AIA is a targeted, objective, accurate, and time-efficient approach that provides reliable differentiation of virus neurovirulence. As such, it may become a useful tool in establishing consistent analytical standards across research and development laboratories and regulatory agencies, and may improve the safety evaluation of live virus vaccines. The implementation of this high-throughput AIA will markedly advance many fields of research including virology, neuroinflammation, neuroscience, and vaccinology.

Assurance of neuroattenuation of a live vaccine against West Nile virus: A comprehensive study of neuropathogenesis after infection with chimeric WN/DEN4Δ30 vaccine in comparison to two parental viruses and a surrogate flavivirus reference vaccine

The upsurge of West Nile virus (WNV) human infections in 2012 suggests that the US can expect periodic WNV outbreaks in the future. Availability of safe and effective vaccines against WNV in endemic areas, particularly for aging populations that are at high risk of West Nile neuroinvasive disease (WNND), could be beneficial. WN/DEN4Δ30 is a live, attenuated chimeric vaccine against WNV produced by replacement of the genes encoding the pre-membrane and envelope protein genes of the vaccine virus against dengue virus type 4 (DEN4Δ30) with corresponding sequences derived from a wild type WNV. Following intrathalamic inoculation of nonhuman primates (NHPs), a comprehensive neuropathogenesis study was performed and neurovirulence of WN/DEN4Δ30 vaccine candidate was compared to that of two parental viruses (i.e., WNV and DEN4Δ30), as well as to that of an attenuated flavivirus surrogate reference (i.e., yellow fever YF 17D). Clinical and virological data, as well as results of a semi-quantitative histopathological analysis, demonstrated that WN/DEN4Δ30 vaccine is highly attenuated for the central nervous system (CNS) of NHPs in comparison to a wild type WNV. Importantly, based on the virus replicative ability in the CNS of NHPs and the degree of induced histopathological changes, the level of neuroattenuation of WN/DEN4Δ30 vaccine was similar to that of YF 17D, and therefore within an acceptable range. In addition, we show that the DEN4Δ30 vaccine tested in this study also has a low neurovirulence profile. In summary, our results demonstrate a high level of neuroattenuation of two vaccine candidates, WN/DEN4Δ30 and DEN4Δ30. We also show here a remarkable sensitivity of our WNV-NY99 NHP model, as well as striking resemblance of the observed neuropathology to that seen in human WNND. These results support the use of this NHP model for translational studies of WNV neuropathogenesis and/or testing the effectiveness of vaccines and therapeutic approaches.