Young-Chan Kwon

Long COVID frequently presents with persistent neurological symptoms, including cognitive impairment, fatigue, and sleep disturbances; however, its underlying mechanisms remain unclear. Here, we show that SARS-CoV-2 infection induces lasting cortical neuronal injury and hypothalamic orexin (hypocretin) dysfunction in vivo. In K18-hACE2 and wild-type BALB/c mice, viral RNA persisted in the brain and coincided with focal loss of Neuronal Nuclei (NeuN)-positive cortical neurons beyond acute infection. SARS-CoV-2, but not the influenza A virus, triggered rapid and sustained suppression of hypothalamic orexin expression, defining a virus-specific neuropathological signature. Considering the downregulation of orexin and focal cortical NeuN attenuation, we found that exogenous orexin-A/B supplementation increased NeuN abundance in vitro and in vivo under the tested conditions. Overall, these findings identify the orexin system as a candidate neural vulnerability to SARS-CoV-2 and suggest that orexinergic dysfunction may contribute to the neurological manifestations of Long COVID.

SUMMARY: Respiratory syncytial virus (RSV) remains the leading cause of severe respiratory infections in infants, the elderly, and the immunocompromised. Although stabilized full-length pre-fusion (pre-F) protein vaccines are promising, enhanced respiratory disease (ERD) remains a critical safety concern. Here, we used artificial intelligence to design a de novo immuno-focused antigen that structurally preserves the RSV F head region containing critical neutralising epitopes— site Ø, II and V–while replacing the non-neutralising stem with a computationally designed scaffold to minimise immunopathological risk. The lead candidate, aRF6, elicited robust protective immunity against RSV in mice and similar immunogenicity in non-human primates without detectable toxicity. Importantly, in stringent ERD-promoting models, aRF6 induced minimal pulmonary pathology and markedly attenuated Th2-biased cytokine responses, outperforming formalin-inactivated RSV and full-length-stabilized pre-F. The results of cryoelectron microscopy confirmed that the aRF6 structure precisely matched the computational predictions. These results demonstrated that computationally designed de novo immuno-focused antigens can yield safe and effective RSV vaccines, thereby providing a rational framework for next-generation vaccine development.

Long COVID frequently presents with persistent neurological symptoms, including cognitive impairment, fatigue, and sleep disturbances; however, its underlying mechanisms remain unclear. Here, we show that SARS-CoV-2 infection induces lasting cortical neuronal injury and hypothalamic orexin (hypocretin) dysfunction in vivo . In K18-hACE2 and wild-type BALB/c mice, viral RNA persisted in the brain and coincided with focal loss of Neuronal Nuclei (NeuN)-positive cortical neurones beyond acute infection. SARS-CoV-2, but not the influenza A virus, triggered rapid and sustained suppression of hypothalamic orexin expression, defining a virus-specific neuropathological signature. Considering the downregulation of orexin and focal cortical NeuN expression, we showed that augmenting orexin signalling using recombinant orexin-A/B restored NeuN expression in vitro and in vivo . Overall, these findings identify the orexin system as a selective neural vulnerability to SARS-CoV-2 and define orexinergic circuit disruption as a mechanistic axis underlying the neurological manifestations of Long COVID.

Many enveloped viruses engage phosphatidylserine (PS) receptors to enter cells, a phenomenon known as “apoptotic mimicry.” We previously reported that Zika virus (ZIKV), but not closely related West Nile virus (WNV) or dengue virus (DENV), utilized AXL to infect cells because only ZIKV could bind the AXL ligand GAS6, a PS-binding protein. In this study, we investigated the mechanisms underlying the differential ability of these viruses to bind GAS6. Although immature virions expose larger patches of the viral membrane than do mature ones, our data show that virion maturity levels did not contribute to GAS6 binding. Surprisingly, while ZIKV contains PS comparable to cellular membranes, PS on WNV and DENV is markedly reduced. These findings explain why only ZIKV can bind GAS6 and provide insights into a novel mechanism by which closely related flaviviruses differentially utilize cellular entry factors. IMPORTANCE: Among flaviviruses, Zika virus uniquely causes microcephaly and congenital defects. While no flavivirus-specific entry receptors have been identified, they commonly take advantage of phosphatidylserine (PS) receptors to enter cells. Our previous studies revealed that Zika virus uniquely utilizes AXL, found in immune-privileged sites, such as the brain and placenta, via binding to its ligand, GAS6. Our current study shows that despite being produced from the same cells, the Zika virus has substantially higher PS content than closely related dengue and West Nile viruses, which likely explains the Zika virus's unique ability to bind GAS6. These findings provide insight into how closely related flaviviruses can vary substantially in their use of cellular entry factors, potentially contributing to the distinct diseases they cause.

Respiratory syncytial virus (RSV) infection is the major cause of severe respiratory illnesses in infants and older adults. RSV forms phase-separated biomolecular condensates called inclusion bodies (IBs), which serve as hubs for viral replication. However, the contribution of IBs to host immune response evasion remains elusive. We report that RSV IBs protect viral RNA from the 2′-5′ oligoadenylate synthetase (OAS)-RNase L pathway, a critical antiviral defense mechanism that cleaves viral and cellular RNAs. RSV infection did not activate the OAS-RNase L pathway, and ectopically activated RNase L did not suppress viral replication. In RSV-infected cells, double-stranded RNA (dsRNA) was efficiently sequestered within liquid–liquid phase separation (LLPS)-mediated IBs, rendering its detection challenging. LLPS perturbation caused dsRNA release from IBs into the cytosol. dsRNA extracted from infected cells, which lacked LLPS shielding, triggered OAS-RNase L pathway activation. Thus, LLPS-driven IBs structurally sequester viral RNA, facilitating RSV to evade RNase-dependent genomic RNA degradation mediated by the OAS-RNase L antiviral pathway.

Mouse models expressing human ACE2 for coronavirus disease 2019 have been frequently used to understand its pathogenesis and develop therapeutic strategies against SARS-CoV-2. Given that human TMPRSS2 supports viral entry, replication, and pathogenesis, we established a double-transgenic mouse model expressing both human ACE2 and TMPRSS2 for SARS-CoV-2 infection. Co-overexpression of both genes increased viral infectivity in vitro and in vivo. Double-transgenic mice showed significant body weight loss, clinical disease symptoms, acute lung injury, lung inflammation, and lethality in response to viral infection, indicating that they were highly susceptible to SARS-CoV-2. Pretreatment with the TMPRSS2 inhibitor, nafamostat, effectively reduced virus-induced weight loss, viral replication, and mortality in the double-transgenic mice. Moreover, the susceptibility and differential pathogenesis of SARS-CoV-2 variants were demonstrated in this animal model. Together, our results demonstrate that double-transgenic mice could provide a highly susceptible mouse model for viral infection to understand SARS-CoV-2 pathogenesis and evaluate antiviral therapeutics against coronavirus disease 2019.

Zika virus (ZIKV) associated neurological impairments are an important global health problem. Our results suggest that ZIKV infection in the mouse brain can induce IL-1β-mediated inflammation and complement activation, thereby contributing to the development of neurological disorders.

During the 2015/16 Zika virus (ZIKV) epidemic, ZIKV associated neurological diseases were reported in adults, including microcephaly, Guillain-Barre syndrome, myelitis, meningoencephalitis, and fatal encephalitis. However, the mechanisms underlying the neuropathogenesis of ZIKV infection are not yet fully understood. In this study, we used an adult ZIKV-infection mouse model ( Ifnar1 ^−/− ) to investigate the mechanisms underlying neuroinflammation and neuropathogenesis. ZIKV infection induced the expression of proinflammatory cytokines, including IL-1β, IL-6, IFN-γ, and TNF-α, in the brains of Ifnar1 ^−/− mice. RNA-seq analysis of the infected mouse brain also revealed that genes involved in innate immune responses and cytokine-mediated signaling pathways were significantly upregulated at 6 days post infection. Furthermore, ZIKV infection induced macrophage infiltration and activation, and augmented IL-1β expression, whereas microgliosis was not observed in the brain. Using human monocyte THP-1 cells, we confirmed that ZIKV infection promotes inflammatory cell death and increases IL-1β secretion. In addition, the expression of complement component C3, which is associated with neurodegenerative diseases and known to be upregulated by proinflammatory cytokines, was induced by ZIKV infection through the IL-1β-mediated pathway. An increase in C5a produced by complement activation in the brains of ZIKV-infected mice was also confirmed. Taken together, our results suggest that ZIKV infection of the brain in this animal model augments IL-1β expression in infiltrating macrophages and elicits IL-1β-mediated inflammation, which can lead to the destructive consequences of neuroinflammation. Importance: Zika virus (ZIKV) associated neurological impairments are an important global health problem. Our results suggest that ZIKV infection of the mouse brain can induce IL-1β-mediated inflammation and complement activation, contributing to the development of neurological disorders. Thus, our findings reveal a mechanism by which ZIKV induces neuroinflammation in the mouse brain. Although we used adult type I IFN receptor IFNAR knockout ( Ifnar1 ^−/− ) mice owing to the limited mouse model of ZIKV pathogenesis, our conclusion could contribute to understanding ZIKV associated neurological diseases to develop treatment strategies based on these findings for the patients with ZIKV infection.

Although ocular manifestations are reported in patients with COVID-19, consensus on ocular tropism of SARS-CoV-2 is lacking. Here, we infect K18-hACE2 transgenic mice with SARS-CoV-2 using various routes. We observe ocular manifestation and retinal inflammation with production of pro-inflammatory cytokines in the eyes of intranasally (IN)-infected mice. Intratracheal (IT) infection results in dissemination of the virus from the lungs to the brain and eyes via trigeminal and optic nerves. Ocular and neuronal invasions are confirmed using intracerebral (IC) infection. Notably, the eye-dropped (ED) virus does not cause lung infection and becomes undetectable with time. Ocular and neurotropic distribution of the virus in vivo is evident in fluorescence imaging with an infectious clone of SARS-CoV-2-mCherry. The ocular tropic and neuroinvasive characteristics of SARS-CoV-2 are confirmed in wild-type Syrian hamsters. Our data can improve the understanding regarding viral transmission and clinical characteristics of SARS-CoV-2 and help in improving COVID-19 control procedures.