Understanding neurotropic enteric viruses: routes of infection and mechanisms of attenuation.

The intricate connection between the gut and the brain involves multiple routes. Several viral families begin their infection cycle in the intestinal tract. However, amongst the long list of viral intestinal pathogens, picornaviruses, and astroviruses stand out for their ability to transition from the intestinal epithelia to central or peripheral nervous system cells. In immunocompromised, neonates and young children, these viral infections can manifest as severe diseases, such as encephalitis, meningitis, and acute flaccid paralysis. What confers this remarkable plasticity and makes them efficient in infecting cells of the gut and the brain axes? Here, we review the current understanding of the virus infection along the gut-brain axis for some enteric viruses and discuss the molecular mechanisms of their attenuation.

Preclinical Safety Assessment of the EBS-LASV Vaccine Candidate against Lassa Fever Virus.

There are currently no prophylactic vaccines licensed to protect against Lassa fever caused by Lassa virus (LASV) infection. The Emergent BioSolutions (EBS) vaccine candidate, EBS-LASV, is being developed for the prevention of Lassa fever. EBS-LASV is a live-attenuated recombinant Vesicular Stomatitis Virus (rVSV)-vectored vaccine encoding the surface glycoprotein complex (GPC) from LASV and has two attenuating vector modifications: a gene shuffle of the VSV N gene and a deletion of the VSV G gene. Preclinical studies were performed to evaluate EBS-LASV's neurovirulence potential following intracranial (IC) injection and to determine the biodistribution and vector replication following intramuscular (IM) inoculation in mice. In addition, the potential EBS-LASV toxicity was assessed using repeated-dose IM EBS-LASV administration to rabbits. All mice receiving the IC injection of EBS-LASV survived, while mice administered the unattenuated control vector did not. The vaccine was only detected in the muscle at the injection site, draining lymph nodes, and the spleen over the first week following IM EBS-LASV injection in mice, with no detectable plasma viremia. No toxicity was observed in rabbits receiving a three-dose regimen of EBS-LASV. These studies demonstrate that EBS-LASV is safe when administered to animals and supported a first-in-human dose-escalation, safety, and immunogenicity clinical study.

Impact of equine herpesvirus-1 ORF15 (EUL45) on viral replication and neurovirulence.

Equine herpesvirus 1 (EHV-1) causes respiratory illness, fetal loss, perinatal mortality, and myeloencephalopathy. This study investigated ORF15's impact on virus infectivity and neurovirulence. The Ab4p neurovirulent strain of EHV1 was used as a backbone to create Ab4p attB, Ab4p∆ORF15, and Ab4p∆ORF15R chimeras via BAC DNA transfection into RK-13 cells. Viral growth kinetics, plaque size, transcription, and growth were assessed in MDBK cells, mouse neurons, and fetal equine brain cells. Neurovirulence was evaluated post-intranasal inoculation into male CBA/N1 SPF mice, measuring signs, virus titers, and histopathological changes. Deletion of EUL45 (Ab4p-∆EUL45) reduced viral replication efficiency, resulting in decreased release and smaller plaques. EUL45 deletion also upregulated neighbouring genes (EUL46 and EUL44). Ab4p-∆EUL45 exhibited reduced virulence and poor growth in neural cells compared to wild-type viruses. This study sheds light on EUL45's role in EHV-1, viral replication, and regulation of EUL46 and EUL44 expression, suggesting potential as a vaccine candidate.

Age-dependent Powassan virus lethality is linked to glial cell activation and divergent neuroinflammatory cytokine responses in a murine model.

Powassan virus (POWV) is an emergent tick-borne flavivirus that causes fatal encephalitis in the elderly and long-term neurologic sequelae in survivors. How age contributes to severe POWV encephalitis remains an enigma, and no animal models have assessed age-dependent POWV neuropathology. Inoculating C57BL/6 mice with a POWV strain (LI9) currently circulating in Ixodes ticks resulted in age-dependent POWV lethality 10-20 dpi. POWV infection of 50-week-old mice was 82% fatal with lethality sequentially reduced by age to 7.1% in 10-week-old mice. POWV LI9 was neuroinvasive in mice of all ages, causing acute spongiform CNS pathology and reactive gliosis 5-15 dpi that persisted in survivors 30 dpi. High CNS viral loads were found in all mice 10 dpi. However, by 15 dpi, viral loads decreased by 2-4 logs in 10- to 40-week-old mice, while remaining at high levels in 50-week-old mice. Age-dependent differences in CNS viral loads 15 dpi occurred concomitantly with striking changes in CNS cytokine responses. In the CNS of 50-week-old mice, POWV induced Th1-type cytokines (IFNγ, IL-2, IL-12, IL-4, TNFα, IL-6), suggesting a neurodegenerative pro-inflammatory M1 microglial program. By contrast, in 10-week-old mice, POWV-induced Th2-type cytokines (IL-10, TGFß, IL-4) were consistent with a neuroprotective M2 microglial phenotype. These findings correlate age-dependent CNS cytokine responses and viral loads with POWV lethality and suggest potential neuroinflammatory therapeutic targets. Our results establish the age-dependent lethality of POWV in a murine model that mirrors human POWV severity and long-term CNS pathology in the elderly. IMPORTANCE: Powassan virus is an emerging tick-borne flavivirus causing lethal encephalitis in aged individuals. We reveal an age-dependent POWV murine model that mirrors human POWV encephalitis and long-term CNS damage in the elderly. We found that POWV is neuroinvasive and directs reactive gliosis in all age mice, but at acute stages selectively induces pro-inflammatory Th1 cytokine responses in 50-week-old mice and neuroprotective Th2 cytokine responses in 10-week-old mice. Our findings associate CNS viral loads and divergent cytokine responses with age-dependent POWV lethality and survival outcomes. Responses of young mice suggest potential therapeutic targets and approaches for preventing severe POWV encephalitis that may be broadly applicable to other neurodegenerative diseases. Our age-dependent murine POWV model permits analysis of vaccines that prevent POWV lethality, and therapeutics that resolve severe POWV encephalitis.

Alpha-synuclein and RNA viruses: Exploring the neuronal nexus.

Alpha-synuclein (α-syn), known for its pivotal role in Parkinson's disease, has recently emerged as a significant player in neurotropic RNA virus infections. Upregulation of α-syn in various viral infections has been found to impact neuroprotective functions by regulating neurotransmitter synthesis, vesicle trafficking, and synaptic vesicle recycling. This review focuses on the multifaceted role of α-syn in controlling viral replication by modulating chemoattractant properties towards microglial cells, virus-induced ER stress signaling, anti-oxidative proteins expression. Furthermore, the text underlines the α-syn-mediated regulation of interferon-stimulated genes. The review may help suggest potential therapeutic avenues for mitigating the impact of RNA viruses on the central nervous system by exploiting α-syn neuroprotective biology.

VP1 is the primary determinant of neuropathogenesis in a mouse model of enterovirus D68 acute flaccid myelitis.

Enterovirus D68 (EV-D68) is an emerging pathogen that can cause severe respiratory and neurologic disease [acute flaccid myelitis (AFM)]. Intramuscular (IM) injection of neonatal Swiss Webster (SW) mice with US/IL/14-18952 (IL52), a clinical isolate from the 2014 EV-D68 epidemic, results in many of the pathogenic features of human AFM, including viral infection of the spinal cord, death of motor neurons, and resultant progressive paralysis. In distinction, CA/14-4231 (CA4231), another clinical isolate from the 2014 EV-D68 outbreak, does not cause paralysis in mice, does not grow in the spinal cord, and does not cause motor neuron loss following IM injection. A panel of chimeric viruses containing sequences from IL52 and CA4231 was used to demonstrate that VP1 is the main determinant of EV-D68 neurovirulence following IM injection of neonatal SW mice. VP1 contains four amino acid differences between IL52 and CA4231. Mutations resulting in substituting these four amino acids (CA4231 residues into the IL52 polyprotein) completely abolished neurovirulence. Conversely, mutations resulting in substituting VP1 IL52 amino acid residues into the CA4231 polyprotein created a virus that induced paralysis to the same degree as IL52. Neurovirulence following infection of neonatal SW mice with parental and chimeric viruses was associated with viral growth in the spinal cord. IMPORTANCE: Emerging viruses allow us to investigate mutations leading to increased disease severity. Enterovirus D68 (EV-D68), once the cause of rare cases of respiratory illness, recently acquired the ability to cause severe respiratory and neurologic disease. Chimeric viruses were used to demonstrate that viral structural protein VP1 determines growth in the spinal cord, motor neuron loss, and paralysis following intramuscular (IM) injection of neonatal Swiss Webster (SW) mice with EV-D68. These results have relevance for predicting the clinical outcome of future EV-D68 epidemics as well as targeting retrograde transport as a potential strategy for treating virus-induced neurologic disease.

Identification of a novel neurovirulence factor encoded by the cryptic orphan gene UL31.6 of herpes simplex virus 1.

Although the herpes simplex virus type 1 (HSV-1) genome was thought to contain approximately 80 different protein coding sequences (CDSs), recent multi-omics analyses reported HSV-1 encodes more than 200 potential CDSs. However, few of the newly identified CDSs were confirmed to be expressed at the peptide or protein level in HSV-1-infected cells. Furthermore, the impact of the proteins they encode on HSV-1 infection is largely unknown. This study focused on a newly identified CDS, UL31.6. Re-analyzation of our previous chemical proteomics data verified that UL31.6 was expressed at the peptide level in HSV-1-infected cells. Antisera raised against a viral protein encoded by UL31.6 (pUL31.6) reacted with a protein with an approximate molecular mass of 37 kDa in lysates of Vero cells infected with each of three HSV-1 strains. pUL31.6 was efficiently dissociated from virions in high-salt solution. A UL31.6-null mutation had a minimal effect on HSV-1 gene expression, replication, cell-to-cell spread, and morphogenesis in Vero cells; in contrast, it significantly reduced HSV-1 cell-to-cell spread in three neural cells but not in four non-neural cells including Vero cells. The UL31.6-null mutation also significantly reduced the mortality and viral replication in the brains of mice after intracranial infection, but had minimal effects on pathogenic manifestations in and around the eyes, and viral replication detected in the tear films of mice after ocular infection. These results indicated that pUL31.6 was a tegument protein and specifically acted as a neurovirulence factor by potentially promoting viral transmission between neuronal cells in the central nervous system.IMPORTANCERecent multi-omics analyses reported the herpes simplex virus type 1 (HSV-1) genome encodes an additional number of potential coding sequences (CDSs). However, the expressions of these CDSs at the peptide or protein levels and the biological effects of these CDSs on HSV-1 infection remain largely unknown. This study annotated a cryptic orphan CDS, termed UL31.6, an HSV-1 gene that encodes a tegument protein with an approximate molecular mass of 37 kDa, which specifically acts as a neurovirulence factor. Our study indicates that HSV-1 proteins important for viral pathogenesis remain to be identified and a comprehensive understanding of the pathogenesis of HSV-1 will require not only the identification of cryptic orphan CDSs using emerging technologies but also step-by-step and in-depth analyses of each of the cryptic orphan CDSs.

Hyperglycemia facilitates EV71 replication: Insights into miR-206-mediated regulation of G3BP2 promoting EV71 IRES activity.

Background: Neurotropic virus infections actively manipulate host cell metabolism to enhance virus neurovirulence. Although hyperglycemia is common during severe infections, its specific role remains unclear. This study investigates the impact of hyperglycemia on the neurovirulence of enterovirus 71 (EV71), a neurovirulent virus relying on internal ribosome entry site (IRES)-mediated translation for replication. Methods: Utilizing hSCARB2-transgenic mice, we explore the effects of hyperglycemia in EV71 infection and elucidate the underlying mechanisms. Results: Remarkably, administering insulin alone to reduce hyperglycemia in hSCARB2-transgenic mice results in a decrease in brainstem encephalitis and viral load. Conversely, induced hyperglycemia exacerbates neuropathogenesis, highlighting the pivotal role of hyperglycemia in neurovirulence. Notably, miR-206 emerges as a crucial mediator induced by viral infection, with its expression further heightened by hyperglycemia and concurrently repressed by insulin. The use of antagomiR-206 effectively mitigates EV71-induced brainstem encephalitis and reduces viral load. Mechanistically, miR-206 facilitates IRES-driven virus replication by repressing the stress granule protein G3BP2. Conclusions: Novel therapeutic approaches against severe EV71 infections involve managing hyperglycemia and targeting the miR-206-stress granule pathway to modulate virus IRES activity.

Herpes simplex virus infection induces necroptosis of neurons and astrocytes in human fetal organotypic brain slice cultures.

BACKGROUND: Herpes simplex virus (HSV) encephalitis (HSE) is a serious and potentially life-threatening disease, affecting both adults and newborns. Progress in understanding the virus and host factors involved in neonatal HSE has been hampered by the limitations of current brain models that do not fully recapitulate the tissue structure and cell composition of the developing human brain in health and disease. Here, we developed a human fetal organotypic brain slice culture (hfOBSC) model and determined its value in mimicking the HSE neuropathology in vitro. METHODS: Cell viability and tissues integrity were determined by lactate dehydrogenase release in supernatant and immunohistological (IHC) analyses. Brain slices were infected with green fluorescent protein (GFP-) expressing HSV-1 and HSV-2. Virus replication and spread were determined by confocal microscopy, PCR and virus culture. Expression of pro-inflammatory cytokines and chemokines were detected by PCR. Cell tropism and HSV-induced neuropathology were determined by IHC analysis. Finally, the in situ data of HSV-infected hfOBSC were compared to the neuropathology detected in human HSE brain sections. RESULTS: Slicing and serum-free culture conditions were optimized to maintain the viability and tissue architecture of ex vivo human fetal brain slices for at least 14 days at 37 °C in a CO2 incubator. The hfOBSC supported productive HSV-1 and HSV-2 infection, involving predominantly infection of neurons and astrocytes, leading to expression of pro-inflammatory cytokines and chemokines. Both viruses induced programmed cell death-especially necroptosis-in infected brain slices at later time points after infection. The virus spread, cell tropism and role of programmed cell death in HSV-induced cell death resembled the neuropathology of HSE. CONCLUSIONS: We developed a novel human brain culture model in which the viability of the major brain-resident cells-including neurons, microglia, astrocytes and oligodendrocytes-and the tissue architecture is maintained for at least 2 weeks in vitro under serum-free culture conditions. The close resemblance of cell tropism, spread and neurovirulence of HSV-1 and HSV-2 in the hfOBSC model with the neuropathological features of human HSE cases underscores its potential to detail the pathophysiology of other neurotropic viruses and as preclinical model to test novel therapeutic interventions.

Monkeypox virus infection of human astrocytes causes gasdermin B cleavage and pyroptosis.

Monkeypox virus (MPXV) infections in humans cause neurological disorders while studies of MPXV-infected animals indicate that the virus penetrates the brain. Pyroptosis is an inflammatory type of regulated cell death, resulting from plasma membrane rupture (PMR) due to oligomerization of cleaved gasdermins to cause membrane pore formation. Herein, we investigated the human neural cell tropism of MPXV compared to another orthopoxvirus, vaccinia virus (VACV), as well as its effects on immune responses and cell death. Astrocytes were most permissive to MPXV (and VACV) infections, followed by microglia and oligodendrocytes, with minimal infection of neurons based on plaque assays. Aberrant morphological changes were evident in MPXV-infected astrocytes that were accompanied with viral protein (I3) immunolabelling and detection of over 125 MPXV-encoded proteins in cell lysates by mass spectrometry. MPXV- and VACV-infected astrocytes showed increased expression of immune gene transcripts (IL12, IRF3, IL1B, TNFA, CASP1, and GSDMB). However, MPXV infection of astrocytes specifically induced proteolytic cleavage of gasdermin B (GSDMB) (50 kDa), evident by the appearance of cleaved N-terminal-GSDMB (30 kDa) and C-terminal- GSDMB (18 kDa) fragments. GSDMB cleavage was associated with release of lactate dehydrogenase and increased cellular nucleic acid staining, indicative of PMR. Pre-treatment with dimethyl fumarate reduced cleavage of GSDMB and associated PMR in MPXV-infected astrocytes. Human astrocytes support productive MPXV infection, resulting in inflammatory gene induction with accompanying GSDMB-mediated pyroptosis. These findings clarify the recently recognized neuropathogenic effects of MPXV in humans while also offering potential therapeutic options.

Attenuation of neurovirulence of chikungunya virus by a single amino acid mutation in viral E2 envelope protein.

BACKGROUND: Chikungunya virus (CHIKV) has reemerged as a major public health concern, causing chikungunya fever with increasing cases and neurological complications. METHODS: In the present study, we investigated a low-passage human isolate of the East/ Central/South African (ECSA) lineage of CHIKV strain LK(EH)CH6708, which exhibited a mix of small and large viral plaques. The small and large plaque variants were isolated and designated as CHIKV-SP and CHIKV-BP, respectively. CHIKV-SP and CHIKV-BP were characterized in vitro and in vivo to compare their virus production and virulence. Additionally, whole viral genome analysis and reverse genetics were employed to identify genomic virulence factors. RESULTS: CHIKV-SP demonstrated lower virus production in mammalian cells and attenuated virulence in a murine model. On the other hand, CHIKV-BP induced higher pro-inflammatory cytokine levels, compromised the integrity of the blood-brain barrier, and led to astrocyte infection in mouse brains. Furthermore, the CHIKV-SP variant had limited transmission potential in Aedes albopictus mosquitoes, likely due to restricted dissemination. Whole viral genome analysis revealed multiple genetic mutations in the CHIKV-SP variant, including a Glycine (G) to Arginine (R) mutation at position 55 in the viral E2 glycoprotein. Reverse genetics experiments confirmed that the E2-G55R mutation alone was sufficient to reduce virus production in vitro and virulence in mice. CONCLUSIONS: These findings highlight the attenuating effects of the E2-G55R mutation on CHIKV pathogenicity and neurovirulence and emphasize the importance of monitoring this mutation in natural infections.

Capsid protein mediated evasion of IRAK1-dependent signalling is essential to Sindbis virus neuroinvasion and virulence in mice.

ABSTRACTAlphaviruses are arthropod-borne, single-stranded positive-sense RNA viruses that are recognized as rapidly emerging pathogens. Despite being exquisitely sensitive to the effects of the innate immune response alphaviruses can readily replicate, disseminate, and induce pathogenesis in immunologically competent hosts. Nonetheless, how alphaviruses evade the induction of an innate immune response prior to viral gene expression, or in non-permissive infections, is unknown. Previously we reported the identification of a novel host/pathogen interaction between the viral Capsid (CP) protein and the host IRAK1 protein. The CP/IRAK1 interaction was determined to negatively impact IRAK1-dependent PAMP detection in vitro, however, the precise importance of the CP/IRAK1 interaction to alphaviral infection remained unknown. Here we detail the identification of the CP/IRAK1 interaction determinants of the Sindbis virus (SINV) CP protein and examine the importance of the interaction to alphaviral infection and pathogenesis in vivo using an interaction deficient mutant of the model neurotropic strain of SINV. Importantly, these interaction determinants are highly conserved across multiple Old-World alphaviruses, including Ross River virus (RRV), Mayaro virus (MAYV), Chikungunya virus (CHIKV), and Semliki Forest virus (SFV). In the absence of a functional CP/IRAK1 interaction, SINV replication is significantly restricted and fails to disseminate from the primary site of inoculation due to the induction of a robust type-I Interferon response. Altogether these data indicate that the evasion of IRAK1-dependent signalling is critical to overcoming the host innate immune response and the in vivo data presented here demonstrate the importance of the CP/IRAK1 interaction to neurovirulence and pathogenesis.

Tick-Borne Encephalitis (TBE): From Tick to Pathology.

Tick-borne encephalitis (TBE) is a viral arthropod infection, endemic to large parts of Europe and Asia, and is characterised by neurological involvement, which can range from mild to severe, and in 33-60% of cases, it leads to a post-encephalitis syndrome and long-term morbidity. While TBE virus, now identified as Orthoflavivirus encephalitidis, was originally isolated in 1937, the pathogenesis of TBE is not fully appreciated with the mode of transmission (blood, tick, alimentary), viral strain, host immune response, and age, likely helping to shape the disease phenotype that we explore in this review. Importantly, the incidence of TBE is increasing, and due to global warming, its epidemiology is evolving, with new foci of transmission reported across Europe and in the UK. As such, a better understanding of the symptomatology, diagnostics, treatment, and prevention of TBE is required to inform healthcare professionals going forward, which this review addresses in detail. To this end, the need for robust national surveillance data and randomised control trial data regarding the use of various antivirals (e.g., Galidesivir and 7-deaza-2'-CMA), monoclonal antibodies, and glucocorticoids is required to improve the management and outcomes of TBE.

In vitro and in vivo models for the study of EV-D68 infection.

Enterovirus D68 (EV-D68) is one of hundreds of non-polio enteroviruses that typically cause cold-like respiratory illness. The first EV-D68 outbreak in the United States in 2014 aroused widespread concern among the public and health authorities. The infection was found to be associated with increased surveillance of acute flaccid myelitis, a neurological condition that causes limb paralysis in conjunction with spinal cord inflammation. In vitro studies utilising two-dimensional (2D) and three-dimensional (3D) culture systems have been employed to elucidate the pathogenic mechanism of EV-D68. Various animal models have also been developed to investigate viral tropism and distribution, pathogenesis, and immune responses during EV-D68 infection. EV-D68 infections have primarily been investigated in respiratory, intestinal and neural cell lines/tissues, as well as in small-size immunocompetent rodent models that were limited to a young age. Some studies have implemented strategies to overcome the barriers by using immunodeficient mice or virus adaptation. Although the existing models may not fully recapitulate both respiratory and neurological disease observed in human EV-D68 infection, they have been valuable for studying pathogenesis and evaluating potential vaccine or therapeutic candidates. In this review, we summarise the methodologies and findings from each experimental model and discuss their applications and limitations.

Severe pneumonia and pathogenic damage in human airway epithelium caused by Coxsackievirus B4.

Coxsackievirus B4 (CVB4) has one of the highest proportions of fatal outcomes of other enterovirus serotypes. However, the pathogenesis of severe respiratory disease caused by CVB4 infection remains unclear. In this study, 3 of 42 (7.2%, GZ-R6, GZ-R7 and GZ-R8) patients with severe pneumonia tested positive for CVB4 infection in southern China. Three full-length genomes of pneumonia-derived CVB4 were sequenced and annotated for the first time, showing their high nucleotide similarity and clustering within genotype V. To analyze the pathogenic damage caused by CVB4 in the lungs, a well-differentiated human airway epithelium (HAE) was established and infected with the pneumonia-derived CVB4 isolate GZ-R6. The outcome was compared with that of a severe hand-foot-mouth disease (HFMD)-derived CVB4 strain GZ-HFM01. Compared with HFMD-derived CVB4, pneumonia-derived CVB4 caused more intense and rapid disruption of HAE polarity, leading to tight-junction barrier disruption, loss of cilia, and airway epithelial cell hypertrophy. More pneumonia-derived CVB4 were released from the basolateral side of the HAE than HFMD-derived CVB4. Of the 18 cytokines tested, only IL-6 and IL-1b secretion significantly increased on bilateral sides of HAE during the early stage of pneumonia-derived CVB4 infection, while multiple cytokine secretions significantly increased in HFMD-derived CVB4-infected HAE. HFMD-derived CVB4 exhibited stronger neurovirulence in the human neuroblastoma cells SH-SY5Y than pneumonia-derived CVB4, which is consistent with the clinical manifestations of patients infected with these two viruses. This study has increased the depth of our knowledge of severe pneumonia infection caused by CVB4 and will benefit its prevention and treatment.

Rift Valley fever virus modulates apoptosis and immune response during infection of human astrocytes.

Rift Valley fever (RVF) is an arboviral disease of zoonotic origin that causes recurrent epidemics in Africa, the Arabic Peninsula, and islands of the South West of the Indian Ocean. RVF occurs mainly in livestock but also affects humans with severe clinical manifestations, including neurological disorders. However, human neuropathogenesis of Rift Valley fever virus (RVFV) is still poorly characterized. To study the interactions between RVFV and the central nervous system (CNS), we focused on RVFV infection of astrocytes, the major glial cells of the CNS that have several supporting roles including immune response regulation. We confirmed the permissiveness of astrocytes to RVFV infection and highlighted a strain-dependent infectivity. We showed that RVFV infection of astrocytes induced cell apoptosis and observed that the RVFV Non-Structural protein NSs, a known virulence factor, potentially delayed apoptosis by sequestrating activated-caspase 3 in the nucleus. Our study also showed that RVFV-infected astrocytes upregulated expression of genes associated with inflammatory and type I interferon responses at the mRNA level, but not at the protein level. This inhibition of immune response is potentially due to a NSs-dependent mechanism of mRNA nuclear export inhibition. Together, these results highlighted the direct impact of RVFV infection on the human CNS through the induction of apoptosis and a possible inhibition of early-onset immune responses that are crucial for the host survival.

The roles of XJ13 and XJ44-specific mutations within the Candid #1 GPC in Junin virus attenuation.

Junin virus (JUNV) is a member of the Arenaviridae family of viruses and is the pathogen responsible for causing Argentine hemorrhagic fever, a potentially lethal disease endemic to Argentina. A live attenuated vaccine for human use, called Candid#1, is approved only in Argentina. Candid#1 vaccine strain of Junin virus was obtained through serial passage in mouse brain tissues followed by passage in Fetal Rhesus macaque lung fibroblast (FRhL) cells. Previously, the mutations responsible for attenuation of this virus in Guinea pigs were mapped in the gene encoding for glycoprotein precursor (GPC) protein. The resulting Candid#1 glycoprotein complex has been shown to cause endoplasmic reticulum (ER) stress in vitro resulting in the degradation of the GPC. To evaluate the attenuating properties of specific mutations within GPC, we created recombinant viruses expressing GPC mutations specific to key Candid#1 passages and evaluated their pathogenicity in our outbred Hartley guinea pig model of Argentine hemorrhagic fever. Here, we provide evidence that early mutations in GPC obtained through serial passaging attenuate the visceral disease and increase immunogenicity in guinea pigs. Specific mutations acquired prior to the 13th mouse brain passage (XJ13) are responsible for attenuation of the visceral disease while having no impact on the neurovirulence of Junin virus. Additionally, our findings demonstrate that the mutation within an N-linked glycosylation motif, acquired prior to the 44th mouse brain passage (XJ44), is unstable but necessary for complete attenuation and enhanced immunogenicity of Candid#1 vaccine strain. The highly conserved N-linked glycosylation profiles of arenavirus glycoproteins could therefore be viable targets for designing attenuating viruses for vaccine development against other arenavirus-associated illnesses.

Interaction of the Hemagglutinin Stalk Region with Cell Adhesion Molecule (CADM) 1 and CADM2 Mediates the Spread between Neurons and Neuropathogenicity of Measles Virus with a Hyperfusogenic Fusion Protein.

Measles virus (MeV), the causative agent of measles, is an enveloped RNA virus of the family Paramyxoviridae, which remains an important cause of childhood morbidity and mortality. MeV has two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins. During viral entry or virus-mediated fusion between infected cells and neighboring susceptible cells, the head domain of the H protein initially binds to its receptors, signaling lymphocytic activation molecule family member 1 (SLAM) and nectin-4, and then the stalk region of the H protein transmits the fusion-triggering signal to the F protein. MeV may persist in the human brain and cause a fatal neurodegenerative disease, subacute sclerosing panencephalitis (SSPE). Recently, we showed, using in vitro cell culture, that cell adhesion molecule (CADM) 1 and CADM2 are host factors that trigger hyperfusogenic mutant F proteins, causing cell-to-cell fusion and the transfer of the MeV genome between neurons. Unlike conventional receptors, CADM1 and CADM2 interact in cis (on the same membrane) with the H protein and then trigger membrane fusion. Here, we show that alanine substitutions in part of the stalk region (positions 171-175) abolish the ability of the H protein to mediate membrane fusion triggered by CADM1 and CADM2, but not by SLAM. The recombinant hyperfusogenic MeV carrying this mutant H protein loses its ability to spread in primary mouse neurons as well as its neurovirulence in experimentally infected suckling hamsters. These results indicate that CADM1 and CADM2 are key molecules for MeV propagation in the brain and its neurovirulence in vivo. IMPORTANCE Measles is an acute febrile illness with skin rash. Despite the availability of highly effective vaccines, measles is still an important cause of childhood morbidity and mortality in many countries. The World Health Organization estimates that more than 120,000 people died from measles worldwide in 2021. Measles virus (MeV), the causative agent of measles, can also cause a fatal progressive neurological disorder, subacute sclerosing panencephalitis (SSPE), several years after acute infection. There is currently no effective treatment for this disease. In this study, using recombinant MeVs with altered receptor usage patterns, we show that cell adhesion molecule (CADM) 1 and CADM2 are host factors critical for MeV spread in neurons and its neurovirulence. These findings further our understanding of the molecular mechanism of MeV neuropathogenicity.

NS1 Protein N-Linked Glycosylation Site Affects the Virulence and Pathogenesis of Dengue Virus.

Live attenuated vaccine is one of the most effective vaccines against flavivirus. Recently, site-directed mutation of the flavivirus genome using reverse genetics techniques has been used for the rapid development of attenuated vaccines. However, this technique relies on basic research of critical virulence loci of the virus. To screen the attenuated sites in dengue virus, a total of eleven dengue virus type four mutant strains with deletion of N-glycosylation sites in the NS1 protein were designed and constructed. Ten of them (except for the N207-del mutant strain) were successfully rescued. Out of the ten strains, one mutant strain (N130del+207-209QQA) was found to have significantly reduced virulence through neurovirulence assay in suckling mice, but was genetically unstable. Further purification using the plaque purification assay yielded a genetically stable attenuated strain #11-puri9 with mutations of K129T, N130K, N207Q, and T209A in the NS1 protein and E99D in the NS2A protein. Identifying the virulence loci by constructing revertant mutant and chimeric viruses revealed that five amino acid adaptive mutations in the dengue virus type four non-structural proteins NS1 and NS2A dramatically affected its neurovirulence and could be used in constructing attenuated dengue chimeric viruses. Our study is the first to obtain an attenuated dengue virus strain through the deletion of amino acid residues at the N-glycosylation site, providing a theoretical basis for understanding the pathogenesis of the dengue virus and developing its live attenuated vaccines.

RNA Structure in the 5' Untranslated Region of Enterovirus D68 Strains with Differing Neurovirulence Phenotypes.

Enterovirus-D68 (EV-D68) is a positive-sense single-stranded RNA virus within the family Picornaviridae. EV-D68 was initially considered a respiratory virus that primarily affected children. However, in 2014, EV-D68 outbreaks occurred causing the expected increase in respiratory illness cases, but also an increase in acute flaccid myelitis cases (AFM). Sequencing of 2014 outbreak isolates revealed variations in the 5' UTR of the genome compared to the historical Fermon strain. The structure of the 5' UTR RNA contributes to enterovirus virulence, including neurovirulence in poliovirus, and could contribute to neurovirulence in contemporary EV-D68 strains. In this study, the secondary and tertiary structures of 5' UTR RNA from the Fermon strain and 2014 isolate KT347251.1 are analyzed and compared. Secondary structures were determined using SHAPE-MaP and TurboFold II and tertiary structures were predicted using 3dRNAv2.0. Comparison of RNA structures between the EV-D68 strains shows significant remodeling at the secondary and tertiary levels. Notable secondary structure changes occurred in domains II, IV and V. Shifts in the secondary structure changed the tertiary structure of the individual domains and the orientation of the domains. Our comparative structural models for EV-D68 5' UTR RNA highlight regions of the molecule that could be targeted for treatment of neurotropic enteroviruses.