Elevation of cell-associated HIV-1 transcripts in CSF CD4+ T cells, despite effective antiretroviral therapy, is linked to brain injury.

Antiretroviral therapy (ART) can attain prolonged undetectable HIV-1 in plasma and cerebrospinal fluid (CSF), but brain injury remains prevalent in people living with HIV-1 infection (PLHIV). We investigated cell-associated (CA)-HIV-1 RNA transcripts in cells in CSF and blood, using the highly sensitive Double-R assay, together with proton Magnetic Resonance Spectroscopy (1H MRS) of major brain metabolites, in sixteen PLHIV. 14/16 CSF cell samples had quantifiable CA-HIV-1 RNA, at levels significantly higher than in their PBMCs (median 9,266 vs 185 copies /106 CD4+ T-cells; p<0.0001). In individual PLHIV, higher levels of HIV-1 transcripts in CSF cells were associated with greater brain injury in the frontal white matter (Std ß=-0.73; p=0.007) and posterior cingulate (Std ß=-0.61; p=0.03). 18-colour flow cytometry revealed that the CSF cells were 91% memory T-cells, equally CD4+ and CD8+ T-cells, but fewer B cells (0.4 %), and monocytes (3.1%). CXCR3+CD49d+integrin ß7-, CCR5+CD4+ T-cells were highly enriched in CSF, compared with PBMC (p <0.001). However, CA-HIV-1 RNA could not be detected in 10/16 preparations of highly purified monocytes from PBMC, and was extremely low in the other six. Our data show that elevated HIV-1 transcripts in CSF cells were associated with brain injury, despite suppressive ART. The cellular source is most likely memory CD4+ T cells from blood, rather than trafficking monocytes. Future research should focus on inhibitors of this transcription to reduce local production of potentially neurotoxic and inflammatory viral products.

A pilot investigation of the association between HIV-1 Vpr amino acid sequence diversity and the tryptophan-kynurenine pathway as a potential mechanism for neurocognitive impairment.

HIV infection compromises both the peripheral and central immune systems due to its pathogenic and neuropathogenic features. The mechanisms driving HIV-1 pathogenesis and neuropathogenesis involve a series of events, including metabolic dysregulation. Furthermore, HIV-subtype-specific variations, particularly alterations in the amino acid sequences of key viral proteins, are known to influence the severity of clinical outcomes in people living with HIV. However, the impact of amino acid sequence variations in specific viral proteins, such as Viral protein R (Vpr), on metabolites within the Tryptophan (Trp)-kynurenine (Kyn) pathway in people living with HIV remains unclear. Our research aimed to explore the relationship between variations in the Vpr amino acid sequence (specifically at positions 22, 41, 45, and 55, as these have been previously linked to neurocognitive function) and peripheral Trp-Kyn metabolites. Additionally, we sought to clarify the systems biology of Vpr sequence variation by examining the link between Trp-Kyn metabolism and peripheral inflammation, as a neuropathogenic mechanism. In this preliminary study, we analyzed a unique cohort of thirty-two (n = 32) South African cART naïve people living with HIV. We employed Sanger sequencing to ascertain blood-derived Vpr amino acid sequence variations and a targeted LC-MS/MS metabolomics platform to assess Trp-Kyn metabolites, such as Trp, Kyn, kynurenic acid (KA), and quinolinic acid (QUIN). Particle-enhanced turbidimetric assay and Enzyme-linked immunosorbent assays were used to measure immune markers, hsCRP, IL-6, suPAR, NGAL and sCD163. After applying Bonferroni corrections (p =.05/3) and adjusting for covariates (age and sex), only the Vpr G41 and A55 groups was nearing significance for higher levels of QUIN compared to the Vpr S41 and T55 groups, respectively (all p =.023). Multiple regression results revealed that Vpr amino acid variations at position 41 (adj R2 = 0.049, ß = 0.505; p =.023), and 55 (adj R2 = 0.126, ß = 0.444; p =.023) displayed significant associations with QUIN after adjusting for age and sex. Lastly, the higher QUIN levels observed in the Vpr G41 group were found to be correlated with suPAR (r =.588, p =.005). These results collectively underscore the importance of specific Vpr amino acid substitutions in influencing QUIN and inflammation (specifically suPAR levels), potentially contributing to our understanding of their roles in the pathogenesis and neuropathogenesis of HIV-1.

Neuropathogenesis of SARS-CoV-2 in human neuronal, microglial and glial cells.

Neurological complications, both acute and chronic, are reported commonly in COVID-19 affected individuals. In this context, the understanding of pathogenesis of SARS-CoV-2 in specific cells of central nervous system (CNS) origin is relevant. The present study explores infection biology of a clinical isolate of SARS-CoV-2 in human cell lines of neural origin such as the glioblastoma (U87-MG), neuroblastoma (SHSY5Y) and microglia (C20). Despite showing clear evidence of infection by immunofluorescence with an anti-spike protein antibody, all the three neural cell lines were observed to be highly restrictive to the replication of the infecting virus. While the U87-MG glioblastoma cells demonstrated no cytopathic effects and a low viral titre with no signs of replication, the SHSY5Y neuroblastoma cells exhibited cytopathic effects with bleb formation but no evidence of viable virus. The C20 microglial cells showed neither signs of cytopathic effects nor viable virus. Ultrastructural studies demonstrated intracellular virions in infected neural cells. The presence of lipid droplets in infected SHSY5Y cells suggested an impact on host cell metabolism. The decrease in viral RNA levels over time in all the neural cell lines suggested restricted viral replication. In conclusion, this study highlights the limited susceptibility of neural cells to SARS-CoV-2 infection. This reduced permissibility of neural cell lines to SARS-CoV-2 may point to their inherent lower expression of receptors that support viral entry in addition to the intracellular factors that potently inhibit viral replication. The study findings prompt further investigation into the mechanisms of SARS-CoV-2 infection of neural cells.

Human Immunodeficiency Virus (HIV-1) Targets Astrocytes via Cell-Free and Cell-Associated Infection.

BACKGROUND: Infection of astrocytes by Human Immunodeficiency Virus (HIV-1) remains a topic of debate, with conflicting data, yet instances of astrocytes containing viral DNA have been observed in vivo. In this study, we aimed to elucidate potential routes through which astrocytes could be infected and their ability to produce infectious particles using primary human astrocytes. METHODS: We infected primary astrocytes derived from either neuroprogenitor cells (NPCs) or induced pluripotent stem cells (iPSCs) that express both C-X-C chemokine receptor type 4 (CXCR4) and the C-C chemokine receptor type 5 (CCR5) coreceptors, using either cell-free HIV-1 virus directly or cell-associated virus indirectly through infected macrophages and microglia. RESULTS: Low-level infectivity by cell-free viruses was primarily attributed to a defect in the entry process. Bypassing HIV-specific receptor-mediated entry using pseudotyped viruses resulted in productive infection and the release of infectious particles. CONCLUSIONS: These findings suggest that astrocytes may be one of the potential sources of neurotoxicity in HIV-associated neurocognitive disorders (HAND) and could possibly act as reservoirs for HIV in the central nervous system (CNS).

The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis.

Engagement of host receptors is essential for viruses to enter target cells and initiate infection. Expression patterns of receptors in turn dictate host range, tissue tropism, and disease pathogenesis during infection. Mammalian orthoreovirus (reovirus) displays serotype-dependent patterns of tropism in the murine central nervous system (CNS) that are dictated by the viral attachment protein σ1. However, the receptor that mediates reovirus CNS tropism is unknown. Two proteinaceous receptors have been identified for reovirus, junctional adhesion molecule A (JAM-A) and Nogo-66 receptor 1 (NgR1). Engagement of JAM-A is required for reovirus hematogenous dissemination but is dispensable for neural spread and infection of the CNS. To determine whether NgR1 functions in reovirus neuropathogenesis, we compared virus replication and disease in wild-type (WT) and NgR1-/- mice. Genetic ablation of NgR1 did not alter reovirus replication in the intestine or transmission to the brain following peroral inoculation. Viral titers in neural tissues following intramuscular inoculation, which provides access to neural dissemination routes, also were comparable in WT and NgR1-/- mice, suggesting that NgR1 is dispensable for reovirus neural spread to the CNS. The absence of NgR1 also did not alter reovirus replication, neural tropism, and virulence following direct intracranial inoculation. In agreement with these findings, we found that the human but not the murine homolog of NgR1 functions as a receptor and confers efficient reovirus binding and infection of nonsusceptible cells in vitro. Thus, neither JAM-A nor NgR1 is required for reovirus CNS tropism in mice, suggesting that other unidentified receptors support this function. IMPORTANCE Viruses engage diverse molecules on host cell surfaces to navigate barriers, gain cell entry, and establish infection. Despite discovery of several reovirus receptors, host factors responsible for reovirus neurotropism are unknown. Human NgR1 functions as a reovirus receptor in vitro and is expressed in CNS neurons in a pattern overlapping reovirus tropism. We used mice lacking NgR1 to test whether NgR1 functions as a reovirus neural receptor. Following different routes of inoculation, we found that murine NgR1 is dispensable for reovirus dissemination to the CNS, tropism and replication in the brain, and resultant disease. Concordantly, expression of human but not murine NgR1 confers reovirus binding and infection of nonsusceptible cells in vitro. These results highlight species-specific use of alternate receptors by reovirus. A detailed understanding of species- and tissue-specific factors that dictate viral tropism will inform development of antiviral interventions and targeted gene delivery and therapeutic viral vectors.

Human cytomegalovirus infection perturbs neural progenitor cell fate via the expression of viral microRNAs.

Human cytomegalovirus (HCMV) preferentially targets neural progenitor cells (NPCs) in congenitally infected fetal brains, inducing neurodevelopmental disorders. While HCMV expresses several microRNAs (miRNAs) during infection, their roles in NPC infection are unclear. Here, we characterized expression of cellular and viral miRNAs in HCMV-infected NPCs during early infection by microarray and identified seven differentially expressed cellular miRNAs and six significantly upregulated HCMV miRNAs. Deep learning approaches were used to identify potential targets of significantly upregulated HCMV miRNAs against differentially expressed cellular messenger RNA (mRNAs), and the associations with miRNA-mRNA expression changes were observed. Gene ontology enrichment analysis indicated cellular gene targets were significantly enriched in pathways involved in neurodevelopment and cell-cycle processes. Viral modulation of selected miRNAs and cellular gene targets involved in neurodevelopmental processes were further validated by real-time quantitative reverse transcription polymerase chain reaction. Finally, a predicted 3' untranslated region target site of hcmv-miR-US25-1 in Jag1, a factor important for neurogenesis, was confirmed by mutagenesis. Reduction of Jag1 RNA and protein levels in NPCs was observed in response to transient expression of hcmv-miR-US25-1. A hcmv-miR-US25-1 mutant virus (ΔmiR-US25) displayed limited ability to downregulate Jag1 mRNA levels and protein levels during the early infection stage compared with the wild type virus. Our collective experimental and computational investigation of miRNAs and cellular mRNAs expression in HCMV-infected NPCs yields new insights into the roles of viral miRNAs in regulating NPC fate and their contributions to HCMV neuropathogenesis.

Opioid abuse and SIV infection in non-human primates.

Human immunodeficiency virus (HIV) and drug abuse are intertwined epidemics, leading to compromised adherence to combined antiretroviral therapy (cART) and exacerbation of NeuroHIV. As opioid abuse causes increased viral replication and load, leading to a further compromised immune system in people living with HIV (PLWH), it is paramount to address this comorbidity to reduce the NeuroHIV pathogenesis. Non-human primates are well-suited models to study mechanisms involved in HIV neuropathogenesis and provide a better understanding of the underlying mechanisms involved in the comorbidity of HIV and drug abuse, leading to the development of more effective treatments for PLWH. Additionally, using broader behavioral tests in these models can mimic mild NeuroHIV and aid in studying other neurocognitive diseases without encephalitis. The simian immunodeficiency virus (SIV)-infected rhesus macaque model is instrumental in studying the effects of opioid abuse on PLWH due to its similarity to HIV infection. The review highlights the importance of using non-human primate models to study the comorbidity of opioid abuse and HIV infection. It also emphasizes the need to consider modifiable risk factors such as gut homeostasis and pulmonary pathogenesis associated with SIV infection and opioid abuse in this model. Moreover, the review suggests that these non-human primate models can also be used in developing effective treatment strategies for NeuroHIV and opioid addiction. Therefore, non-human primate models can significantly contribute to understanding the complex interplay between HIV infection, opioid abuse, and associated comorbidities.

Duck Tembusu virus induces incomplete autophagy via the ERK/mTOR and AMPK/mTOR signalling pathways to promote viral replication in neuronal cells.

Duck Tembusu virus (DTMUV) is a neurotropic virus in the genus Flavivirus that causes massive economic losses to the poultry industry in China and neighbouring countries. Autophagy is pivotal in cellular responses to pathogens and in viral pathogenesis. However, little is known about the roles of autophagy in DTMUV replication and viral pathogenesis, especially in neuropathogenesis. In this study, mouse neuroblastoma cells (Neuro-2a) were used to establish a cell model of DTMUV infection. Our experiments indicated that DTMUV infection induced incomplete autophagy in Neuro-2a cells. Then, we used different autophagy regulators to alter the autophagy induced by DTMUV and found that incomplete autophagy promoted DTMUV replication. Furthermore, we showed that DTMUV infection activated the ERK and AMPK pathways, resulting in decreased phosphorylation of the autophagy repressor mTOR, subsequently leading to autophagic induction. In addition, we utilized ICR mice in an animal model of DTMUV infection to evaluate the autophagic responses in brain tissues and investigate the effects of autophagy on viral replication and tissue lesions. Our results confirmed that DTMUV induced incomplete autophagy in mouse brain tissues and that autophagy inducer treatment promoted DTMUV replication and aggravated DTMUV-induced lesions, whereas autophagy inhibitor treatment had the opposite effects. In summary, DTMUV infection induced incomplete autophagy through the ERK/mTOR and AMPK/mTOR signalling pathways to promote viral replication in mouse neuronal cells, and DTMUV-induced incomplete autophagy contributed to the neuropathogenesis of DTMUV.

Human microglial models to study HIV infection and neuropathogenesis: a literature overview and comparative analyses.

HIV persistence in the CNS despite antiretroviral therapy may cause neurological disorders and poses a critical challenge for HIV cure. Understanding the pathobiology of HIV-infected microglia, the main viral CNS reservoir, is imperative. Here, we provide a comprehensive comparison of human microglial culture models: cultured primary microglia (pMG), microglial cell lines, monocyte-derived microglia (MDMi), stem cell-derived microglia (iPSC-MG), and microglia grown in 3D cerebral organoids (oMG) as potential model systems to advance HIV research on microglia. Functional characterization revealed phagocytic capabilities and responsiveness to LPS across all models. Microglial transcriptome profiles of uncultured pMG showed the highest similarity to cultured pMG and oMG, followed by iPSC-MG and then MDMi. Direct comparison of HIV infection showed a striking difference, with high levels of viral replication in cultured pMG and MDMi and relatively low levels in oMG resembling HIV infection observed in post-mortem biopsies, while the SV40 and HMC3 cell lines did not support HIV infection. Altogether, based on transcriptional similarities to uncultured pMG and susceptibility to HIV infection, MDMi may serve as a first screening tool, whereas oMG, cultured pMG, and iPSC-MG provide more representative microglial culture models for HIV research. The use of current human microglial cell lines (SV40, HMC3) is not recommended.

HIV-1C and HIV-1B Tat protein polymorphism in Southern Brazil.

The transactivator of transcription (Tat) is a key HIV regulatory protein. We aimed to identify the frequency of key polymorphisms in HIV-1C compared with HIV-1B Tat protein, chiefly in the cysteine-, arginine-, and glutamine-rich domains and identify novel point mutations in HIV-1B and C sequences from Southern Brazil. This study was the first to investigate the genetic diversity and point mutations within HIV-1 Tat C in a Brazilian cohort. This was an observational, cross-sectional study, which included sequences of HIV-1B (n = 20) and HIV-1C (n = 21) from Southern Brazil. Additionally, 344 HIV-1C sequences were obtained from the Los Alamos database: 29 from Brazil and 315 from Africa, Asia, and Europe. The frequency of C31S substitution on HIV-1 Tat C in Brazil was 82% vs. 10% in the HIV-1B group (p < 0.0001). The frequency of the R57S substitution among the HIV-1C sequences from Brazil was 74% vs. 20% in HIV-1B (p = 0.004), and that of substitution Q63E in HIV-1C was 80% and 20% in HIV-1B (p < 0.0001). The mutation P60Q was more frequent in HIV-1B than in HIV-1C (55% and 6.12%, respectively, p < 0.0001)). Novel point mutations in the HIV-1C and B Tat functional domains were described. The frequency of C31S and other key point mutations in HIV-1 Tat C in Brazil were similar to those described in Africa, although lower than those in India. The Tat-B and C sequences found in Southern Brazil are consistent with biological differences and have potential implications for HIV-1 subtype pathogenesis.

Altered expression of fractalkine in HIV-1-infected astrocytes and consequences for the virus-related neurotoxicity.

HIV-1 infection in the central nervous system (CNS) causes the release of neurotoxic products from infected cells which trigger extensive neuronal loss. Clinically, this results in HIV-1-associated neurocognitive disorders (HAND). However, the effects on neuroprotective factors in the brain remain poorly understood and understudied in this situation. HAND is a multifactorial process involving several players, and the complex cellular mechanisms have not been fully elucidated yet. In this study, we reported that HIV-1 infection of astrocytes limits their potential to express the protective chemokine fractalkine in response to an inflammatory environment. We next confirmed that this effect was not due to a default in its shedding from the cell surface. We then investigated the biological mechanism responsible for this reduced fractalkine expression and found that HIV-1 infection specifically blocks the interaction of transcription factor NF-κB on its promoter with no effect on other cytokines. Moreover, we demonstrated that fractalkine production in astrocytes is regulated in response to immune factors secreted by infected/activated microglia and macrophages. In contrast, we observed that conditioned media from these infected cells also trigger neuronal apoptosis. At last, we demonstrated a strong neuroprotective action of fractalkine on human neurons by reducing neuronal damages. Taken together, our results indicate new relevant interactions between HIV-1 and fractalkine signaling in the CNS. This study provides new information to broaden the understanding of HAND and possibly foresee new therapeutic strategies. Considering its neuro-protective functions, reducing its production from astrocytes could have important outcomes in chronic neuroinflammation and in HIV-1 neuropathogenesis.

Dissecting the localization of Tilapia tilapinevirus in the brain of the experimentally infected Nile tilapia, Oreochromis niloticus (L.).

Tilapia tilapinevirus or tilapia lake virus (TiLV) is an emerging virus that inflicts significant mortality on farmed tilapia globally. Previous studies reported detection of the virus in multiple organs of the infected fish; however, little is known about the in-depth localization of the virus in the central nervous system. Herein, we determined the distribution of TiLV in the entire brain of experimentally infected Nile tilapia. In situ hybridization (ISH) using TiLV-specific probes revealed that the virus was broadly distributed throughout the brain. The strongest positive signals were dominantly detected in the forebrain (responsible for learning, appetitive behaviour and attention) and the hindbrain (involved in controlling locomotion and basal physiology). The permissive cell zones for viral infection were observed mostly to be along the blood vessels and the ventricles. This indicates that the virus may productively enter into the brain through the circulatory system and widen broad regions, possibly through the cerebrospinal fluid along the ventricles, and subsequently induce the brain dysfunction. Understanding the pattern of viral localization in the brain may help elucidate the neurological disorders of the diseased fish. This study revealed the distribution of TiLV in the whole infected brain, providing new insights into fish-virus interactions and neuropathogenesis.

Neuropathophysiology of coronavirus disease 2019: neuroinflammation and blood brain barrier disruption are critical pathophysiological processes that contribute to the clinical symptoms of SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19) is caused by the novel SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) first discovered in Wuhan, Hubei province, China in December 2019. SARS-CoV-2 has infected several millions of people, resulting in a huge socioeconomic cost and over 2.5 million deaths worldwide. Though the pathogenesis of COVID-19 is not fully understood, data have consistently shown that SARS-CoV-2 mainly affects the respiratory and gastrointestinal tracts. Nevertheless, accumulating evidence has implicated the central nervous system in the pathogenesis of SARS-CoV-2 infection. Unfortunately, however, the mechanisms of SARS-CoV-2 induced impairment of the central nervous system are not completely known. Here, we review the literature on possible neuropathogenic mechanisms of SARS-CoV-2 induced cerebral damage. The results suggest that downregulation of angiotensin converting enzyme 2 (ACE2) with increased activity of the transmembrane protease serine 2 (TMPRSS2) and cathepsin L in SARS-CoV-2 neuroinvasion may result in upregulation of proinflammatory mediators and reactive species that trigger neuroinflammatory response and blood brain barrier disruption. Furthermore, dysregulation of hormone and neurotransmitter signalling may constitute a fundamental mechanism involved in the neuropathogenic sequelae of SARS-CoV-2 infection. The viral RNA or antigenic peptides also activate or interact with molecular signalling pathways mediated by pattern recognition receptors (e.g., toll-like receptors), nuclear factor kappa B, Janus kinase/signal transducer and activator of transcription, complement cascades, and cell suicide molecules. Potential molecular targets and therapeutics of SARS-CoV-2 induced neurologic damage are also discussed.

Pathological modeling of TBEV infection reveals differential innate immune responses in human neurons and astrocytes that correlate with their susceptibility to infection.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is a member of the Flaviviridae family, Flavivirus genus, which includes several important human pathogens. It is responsible for neurological symptoms that may cause permanent disability or death, and, from a medical point of view, is the major arbovirus in Central/Northern Europe and North-Eastern Asia. TBEV tropism is critical for neuropathogenesis, yet little is known about the molecular mechanisms that govern the susceptibility of human brain cells to the virus. In this study, we sought to establish and characterize a new in vitro model of TBEV infection in the human brain and to decipher cell type-specific innate immunity and its relation to TBEV tropism and neuropathogenesis. METHOD: Human neuronal/glial cells were differentiated from neural progenitor cells and infected with the TBEV-Hypr strain. Kinetics of infection, cellular tropism, and cellular responses, including innate immune responses, were characterized by measuring viral genome and viral titer, performing immunofluorescence, enumerating the different cellular types, and determining their rate of infection and by performing PCR array and qRT-PCR. The specific response of neurons and astrocytes was analyzed using the same approaches after enrichment of the neuronal/glial cultures for each cellular subtype. RESULTS: We showed that infection of human neuronal/glial cells mimicked three major hallmarks of TBEV infection in the human brain, namely, preferential neuronal tropism, neuronal death, and astrogliosis. We further showed that these cells conserved their capacity to mount an antiviral response against TBEV. TBEV-infected neuronal/glial cells, therefore, represented a highly relevant pathological model. By enriching the cultures for either neurons or astrocytes, we further demonstrated qualitative and quantitative differential innate immune responses in the two cell types that correlated with their particular susceptibility to TBEV. CONCLUSION: Our results thus reveal that cell type-specific innate immunity is likely to contribute to shaping TBEV tropism for human brain cells. They describe a new in vitro model for in-depth study of TBEV-induced neuropathogenesis and improve our understanding of the mechanisms by which neurotropic viruses target and damage human brain cells.

Differences in Neuropathogenesis of Encephalitic California Serogroup Viruses.

The California serogroup of orthobunyaviruses comprises a group of mosquitoborne viruses, including La Crosse (LACV), snowshoe hare (SSHV), Tahyna (TAHV), Jamestown Canyon (JCV), and Inkoo (INKV) viruses, that cause neurologic disease in humans of differing ages with varying incidences. To determine how the pathogenesis of these viruses differs, we compared their ability to induce disease in mice and replicate and induce cell death in vitro. In mice, LACV, TAHV, and SSHV induced neurologic disease after intraperitoneal and intranasal inoculation, and JCV induced disease only after intranasal inoculation. INKV rarely induced disease, which correlated with less viral antigen in the brain than the other viruses. In vitro, all viruses replicated to high titers; however, LACV, SSHV, and TAHV induced high cell death, whereas JCV and INKV did not. Results demonstrated that CSG viruses differ in neuropathogenesis in vitro and in vivo, which correlates with the differences in pathogenesis reported in humans.

A Mutation in the UL24 Gene Abolishes Expression of the Newly Identified UL24.5 Protein of Herpes Simplex Virus 1 and Leads to an Increase in Pathogenicity in Mice.

Herpes simplex virus 1 (HSV-1) infects the host via epithelia and establishes latency in sensory neurons. The UL24 gene is conserved throughout the Herpesviridae family, and the UL24 protein is important for efficient viral replication and pathogenesis. Multiple transcripts are expressed from the UL24 gene. The presence of a transcription initiation site inside the open reading frame of UL24 and an ATG start codon in the same open reading frame led us to suspect that another protein was expressed from the UL24 locus. To test our hypothesis, we constructed a recombinant virus that expresses a hemagglutinin tag at the C terminus of UL24. Western blot analysis revealed the expression of an 18-kDa protein that is not a degradation product of the full-length UL24, which we refer to as UL24.5. Ectopically expressed UL24.5 did not induce the dispersal of nucleolar proteins, as seen for UL24. In order to characterize the role of UL24.5, we constructed a mutant virus encoding a substitution of the predicted initiation methionine to a valine. This substitution eliminated the expression of the 18-kDa polypeptide. Unlike the UL24-null mutant (UL24X), which exhibits reduced viral yields, the UL24.5-null mutant exhibited the same replication phenotype in cell culture as the parental strain. However, in a murine ocular infection model, we observed an increase in the incidence of neurological disorders with the UL24.5 mutant. Alignment of amino acid sequences for various herpesviruses revealed that the initiation site of UL24.5 is conserved among HSV-1 strains and is present in many herpesviruses.IMPORTANCE We discovered a new HSV-1 protein, UL24.5, which corresponds to the C-terminal portion of UL24. In contrast to the replication defects observed with HSV-1 strains that do not express full-length UL24, the absence of UL24.5 did not affect viral replication in cell culture. Moreover, in mice, the absence of UL24.5 did not affect viral titers in epithelia or trigeminal ganglia during acute infection; however, it was associated with a prolonged persistence of signs of inflammation. Strikingly, the absence of UL24.5 also led to an increase in the incidence of severe neurological impairment compared to results for wild-type control viruses. This increase in pathogenicity is in stark contrast to the reduction in clinical signs associated with the absence of full-length UL24. Bioinformatic analyses suggest that UL24.5 is conserved among all human alphaherpesviruses and in some nonhuman alphaherpesviruses. Thus, we have identified UL24.5 as a new HSV-1 determinant of pathogenesis.

Axonal Transport Enables Neuron-to-Neuron Propagation of Human Coronavirus OC43.

Human coronaviruses (HCoVs) are recognized respiratory pathogens for which accumulating evidence indicates that in vulnerable patients the infection can cause more severe pathologies. HCoVs are not always confined to the upper respiratory tract and can invade the central nervous system (CNS) under still unclear circumstances. HCoV-induced neuropathologies in humans are difficult to diagnose early enough to allow therapeutic interventions. Making use of our already described animal model of HCoV neuropathogenesis, we describe the route of neuropropagation from the nasal cavity to the olfactory bulb and piriform cortex and then the brain stem. We identified neuron-to-neuron propagation as one underlying mode of virus spreading in cell culture. Our data demonstrate that both passive diffusion of released viral particles and axonal transport are valid propagation strategies used by the virus. We describe for the first time the presence along axons of viral platforms whose static dynamism is reminiscent of viral assembly sites. We further reveal that HCoV OC43 modes of propagation can be modulated by selected HCoV OC43 proteins and axonal transport. Our work, therefore, identifies processes that may govern the severity and nature of HCoV OC43 neuropathogenesis and will make possible the development of therapeutic strategies to prevent occurrences.IMPORTANCE Coronaviruses may invade the CNS, disseminate, and participate in the induction of neurological diseases. Their neuropathogenicity is being increasingly recognized in humans, and the presence and persistence of human coronaviruses (HCoV) in human brains have been proposed to cause long-term sequelae. Using our mouse model relying on natural susceptibility to HCoV OC43 and neuronal cell cultures, we have defined the most relevant path taken by HCoV OC43 to access and spread to and within the CNS toward the brain stem and spinal cord and studied in cell culture the underlying modes of intercellular propagation to better understand its neuropathogenesis. Our data suggest that axonal transport governs HCoV OC43 egress in the CNS, leading to the exacerbation of neuropathogenesis. Exploiting knowledge on neuroinvasion and dissemination will enhance our ability to control viral infection within the CNS, as it will shed light on underlying mechanisms of neuropathogenesis and uncover potential druggable molecular virus-host interfaces.

Tick-borne encephalitis virus: molecular determinants of neuropathogenesis of an emerging pathogen.

Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis. The transmission cycle involves the virus, the Ixodes tick vector, and a vertebrate reservoir, such as small mammals (rodents, or shrews). Humans are accidentally involved in this transmission cycle. Tick-borne encephalitis (TBE) has been a growing public health problem in Europe and Asia over the past 30 years. The mechanisms involved in the development of TBE are very complex and likely multifactorial, involving both host and viral factors. The purpose of this review is to provide an overview of the current literature on TBE neuropathogenesis in the human host and to demonstrate the emergence of common themes in the molecular pathogenesis of TBE in humans. We discuss and review data on experimental study models and on both viral (molecular genetics of TBEV) and host (immune response, and genetic background) factors involved in TBE neuropathogenesis in the context of human infection.

Functional analysis of the latency-related gene of bovine herpesvirus types 1 and 5.

Bovine herpesvirus type 1 and type 5 (BoHV-1 and BoHV-5) are two alphaherpesviruses that affect cattle with two different syndromes. While BoHV-1 mainly produces respiratory symptoms, BoHV-5 is highly neuropathogenic and responsible for meningoencephalitis in young cattle. The latency-related (LR) gene, which is not conserved between these two herpesviruses, is the only viral gene abundantly expressed in latently infected neurons. The antiapoptotic action of this gene has been demonstrated during acute infection and reactivation from latency and seems to be mainly mediated by a LR protein (ORF-2) which is truncated in amino acid 51 in the case of BoHV-5. In this work, we show that the BoHV-5 LR gene is less efficient at cell survival and apoptosis inhibition in transient as well as in established neuronal cell lines compared to its BoHV-1 homolog. We hypothesize that the BoHV-5 LR gene may have novel functions that are lacking in the BoHV-1 LR gene and that these differences may contribute to its enhanced neuropathogenesis.

Differential expression of cyclins mRNA in neural tissues of BoHV-1- and BoHV-5- infected cattle.

Bovine alphaherpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses. BoHV-5 causes non-suppurative meningoencephalitis in calves. BoHV-1 is associated with several syndromes and, occasionally, can cause encephalitis. Although both viruses are neurotropic and they share similar biological properties, it is unknown why these alphaherpesviruses differ in their ability to cause neurological disease. Neural tissue samples were collected from BoHV-1- and BoHV-5-intranasally inoculated calves during acute infection, latency and reactivation and the levels of cyclins mRNA expression were analyzed by qRT-PCR. Striking differences in the levels of cyclins mRNA were particularly detected in trigeminal ganglion (TG). The expression levels of cyclins in TG during BoHV-5 latency suggest that these viruses utilize different strategies to persist in the host. It is apparent that a relationship between virus loads and cyclin mRNA levels can be established only during acute infection and other factors might be involved in the regulation of cell cycle components during BoHV latency and reactivation. Bovine alphaherpesviruses neuropathogenicity might be influenced by the differential control of cell cycle components by these herpesviruses. This is the first report on BoHV-5 modulation of cyclins expression in neural tissues from its natural host.