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Omicron has been shown to predominantly use the endosomal entry pathway, resulting in reduced lung tropism and reduced disease severity; however, the underlying mechanism is not fully understood. In addition, whether the most recent Omicron subvariants, including BA.5 and BA.2.75, use the same pathway as their ancestor for entry is currently not known.

Given that neutralizing antibodies play a key role in protection of SARS-CoV-2 infection, it is important to define the neutralization levels in vaccinated individuals when they contracted breakthrough infections. In this study, we analyzed the neutralization levels from 64 vaccinated patients on days 0 to 5 before they tested positive for Delta breakthrough infections.

Pathogenic coronaviruses represent a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified several small-molecule inhibitors that potently block the replication of the newly emerged severe acute respiratory syndrome virus 2 (SARS-CoV-2). Among them, JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with an EC50 of 695 nM, with a specificity index of greater than 1,000. JIB-04 showed in vitro antiviral activity in multiple cell types against several DNA and RNA viruses, including porcine coronavirus transmissible gastroenteritis virus. In an in vivo porcine model of coronavirus infection, administration of JIB-04 reduced virus infection and associated tissue pathology, which resulted in improved weight gain and survival. These results highlight the potential utility of JIB-04 as an antiviral agent against SARS-CoV-2 and other viral pathogens.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a historic pandemic of respiratory disease (coronavirus disease 2019 [COVID-19]), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2 signaling restricts the viral burden in the lung. We find that a recently developed mouse-adapted SARS-CoV-2 (MA-SARS-CoV-2) strain as well as the emerging B.1.351 variant trigger an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Single-cell RNA sequencing (scRNA-Seq) analysis of lung homogenates identified a hyperinflammatory monocyte profile. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a potential CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts the viral burden in the lung. We find that SARS-CoV-2 triggers an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using RNA sequencing and flow cytometry approaches, we demonstrate that SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Mechanistically, CCR2 signaling promoted the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified that the CCR2 pathway is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.

ZIKV is a recently emerged mosquito-borne flavivirus that can cause devastating congenital Zika syndrome in pregnant women and Guillain-Barré syndrome in adults. The molecular mechanism of ZIKV virion assembly is largely unknown. Here, we report that ZIKV NS2A plays a central role in recruiting viral RNA, structural protein prM/E, and viral NS2B/NS3 protease to the virion assembly site and orchestrating virion morphogenesis. One mutation that impairs these interactions does not significantly affect viral RNA replication but selectively abolishes virion assembly, demonstrating the specific role of these interactions in virus morphogenesis. We also show that the 3ʹ UTR of ZIKV RNA may serve as a “recruitment signal” through binding to NS2A to enter the virion assembly site. Following a coordinated cleavage of C-prM-E at the virion assembly site, NS2A may present the viral RNA to C protein for nucleocapsid formation followed by envelopment with prM/E proteins. The results have provided new insights into flavivirus virion assembly., The flavivirus virion consists of an envelope outer layer, formed by envelope (E) and membrane (M) proteins on a lipid bilayer, and an internal core, formed by capsid (C) protein and genomic RNA. The molecular mechanism of flavivirus assembly is not well understood. Here, we show that Zika virus (ZIKV) NS2A protein recruits genomic RNA, the structural protein prM/E complex, and the NS2B/NS3 protease complex to the virion assembly site and orchestrates virus morphogenesis. Coimmunoprecipitation analysis showed that ZIKV NS2A binds to prM, E, NS2B, and NS3 (but not C, NS4B, or NS5) in a viral RNA-independent manner, whereas prM/E complex does not interact with NS2B/NS3 complex. Remarkably, a single-amino-acid mutation (E103A) of NS2A impairs its binding to prM/E and NS2B/NS3 and abolishes virus production, demonstrating the indispensable role of NS2A/prM/E and NS2A/NS2B/NS3 interactions in virion assembly. In addition, RNA-protein pulldown analysis identified a stem-loop RNA from the 3ʹ untranslated region (UTR) of the viral genome as an “RNA recruitment signal” for ZIKV assembly. The 3ʹ UTR RNA binds to a cytoplasmic loop of NS2A protein. Mutations of two positively charged residues (R96A and R102A) from the cytoplasmic loop reduce NS2A binding to viral RNA, leading to a complete loss of virion assembly. Collectively, our results support a virion assembly model in which NS2A recruits viral NS2B/NS3 protease and structural C-prM-E polyprotein to the virion assembly site; once the C-prM-E polyprotein has been processed, NS2A presents viral RNA to the structural proteins for virion assembly.

Recent Zika virus (ZIKV) outbreaks have identified sexual transmission as a new route of disease spread not reported for other flaviviruses. ZIKV crosses the blood-testis barrier and establishes infection in seminiferous tubules, the site for spermatozoa development. Currently, there are no therapies to treat ZIKV infection, and the immune mechanisms underlying testicular persistence are unclear. We found that multiple human testicular cell types, except Leydig cells, support ZIKV infection. Axl receptor, which plays a pivotal role in maintaining the immunosuppressive milieu of the testis, is highly expressed in Sertoli cells and augments ZIKV infection by promoting virus entry and negatively regulating the antiviral state. By using testicular organoids, we further describe the antiviral role of Axl inhibition. The significance of our research lies in defining cross talk between Axl and type I interferon signaling as an essential mechanism of immune control that can inform therapeutic efforts to clear ZIKV from the testis., Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in its ability to be sexually transmitted. Persistent ZIKV infection in the testes, which are immune privileged organs, long after peripheral clearance suggests involvement of immunosuppressive pathways; however, the underlying mechanisms remain undetermined. We recently demonstrated that ZIKV infects human Sertoli cells (SC), the major cell type of the seminiferous epithelium responsible for maintaining the immune privileged compartment of seminiferous tubules. Recent reports have identified the TAM (Tyro3, Axl, Mer) receptor tyrosine kinase Axl as an entry receptor and/or immune modulator for ZIKV in a cell type-specific manner. Interestingly, the seminiferous epithelium exhibits high basal expression of the Axl receptor where it is involved in clearance of apoptotic germ cells and immunosuppression. Here, we show that Axl was highly expressed in SC compared to Leydig cells (LC) that correlated with robust ZIKV infection of SC, but not LC. Further, neutralization of Axl receptor and its ligand Gas6 strongly attenuated virus entry in SC. However, inhibition of Axl kinase did not affect ZIKV entry but instead led to decreased protein levels of suppressor of cytokine signaling 1 (SOCS1) and SOCS3, increased expression of interferon-stimulated genes (ISGs), and reduced ZIKV replication. Similarly, treatment of multicellular human testicular organoids with an Axl kinase inhibitor attenuated ZIKV replication and increased ISG expression. Together, our data demonstrate that Axl promotes ZIKV entry and negatively regulates the antiviral state of SC to augment ZIKV infection of the testes and provides new insights into testis antiviral immunity and ZIKV persistence.

Glioblastoma (GBM) is the deadliest type of brain tumor, and glioma stem cells (GSCs) contribute to tumor recurrence and therapeutic resistance. Thus, an oncolytic virus targeting GSCs may be useful for improving GBM treatment. Because Zika virus (ZIKV) has an oncolytic tropism for infecting GSCs, we investigated the safety and efficacy of a live attenuated ZIKV vaccine candidate (ZIKV-LAV) for the treatment of human GBM in a GSC-derived orthotopic model. Intracerebral injection of ZIKV-LAV into mice caused no neurological symptoms or behavioral abnormalities. The neurovirulence of ZIKV-LAV was more attenuated than that of the licensed Japanese encephalitis virus LAV 14-14-2, underlining the superior safety of ZIKV-LAV for potential GBM treatment. Importantly, ZIKV-LAV significantly reduced intracerebral tumor growth and prolonged animal survival by selectively killing GSCs within the tumor. Mechanistically, ZIKV infection elicited antiviral immunity, inflammation, and GSC apoptosis. Together, these results further support the clinical development of ZIKV-LAV for GBM therapy.

Glioblastoma (GBM) is the deadliest type of brain tumor, and glioma stem cells (GSCs) contribute to tumor recurrence and therapeutic resistance. Thus, an oncolytic virus targeting GSCs may be useful for improving GBM treatment. Because Zika virus (ZIKV) has an oncolytic tropism for infecting GSCs, we investigated the safety and efficacy of a live attenuated ZIKV vaccine candidate (ZIKV-LAV) for the treatment of human GBM in a GSC-derived orthotopic model. Intracerebral injection of ZIKV-LAV into mice caused no neurological symptoms or behavioral abnormalities. The neurovirulence of ZIKV-LAV was more attenuated than that of the licensed Japanese encephalitis virus LAV 14-14-2, underlining the superior safety of ZIKV-LAV for potential GBM treatment. Importantly, ZIKV-LAV significantly reduced intracerebral tumor growth and prolonged animal survival by selectively killing GSCs within the tumor. Mechanistically, ZIKV infection elicited antiviral immunity, inflammation, and GSC apoptosis. Together, these results further support the clinical development of ZIKV-LAV for GBM therapy. IMPORTANCE Glioblastoma (GBM), the deadliest type of brain tumor, is currently incurable because of its high recurrence rate after traditional treatments, including surgery to remove the main part of the tumor and radiation and chemotherapy to target residual tumor cells. These treatments fail mainly due to the presence of a cell subpopulation called glioma stem cells (GSCs), which are resistant to radiation and chemotherapy and capable of self-renewal and tumorigenicity. Because Zika virus (ZIKV) has an oncolytic tropism for infecting GSCs, we tested a live attenuated ZIKV vaccine candidate (ZIKV-LAV) for the treatment of human GBM in a human GSC-derived orthotopic model. Our results showed that ZIKV-LAV retained good efficacy against glioblastoma by selectively killing GSCs within the tumor. In addition, ZIKV-LAV exhibited an excellent safety profile upon intracerebral injection into the treated animals. The good balance between the safety of ZIKV-LAV and its efficacy against human GSCs suggests that it is a potential candidate for combination with the current treatment regimen for GBM therapy.