Migraine inhibitor olcegepant reduces weight loss and IL-6 release in SARS-CoV-2-infected older mice with neurological signs.

COVID-19 can cause neurological symptoms such as fever, dizziness, and nausea. However, such neurological symptoms of SARS-CoV-2 infection have been hardly assessed in mouse models. In this study, we infected two commonly used wild-type mouse lines (C57BL/6J and 129/SvEv) and a 129S calcitonin gene-related peptide (αCGRP) null-line with mouse-adapted SARS-CoV-2 and demonstrated neurological signs including fever, dizziness, and nausea. We then evaluated whether a CGRP receptor antagonist, olcegepant, a "gepant" antagonist used in migraine treatment, could mitigate acute neuroinflammatory and neurological signs of SARS-COV-2 infection. First, we determined whether CGRP receptor antagonism provided protection from permanent weight loss in older (>18 m) C57BL/6J and 129/SvEv mice. We also observed acute fever, dizziness, and nausea in all older mice, regardless of treatment. In both wild-type mouse lines, CGRP antagonism reduced acute interleukin 6 (IL-6) levels with virtually no IL-6 release in mice lacking αCGRP. These findings suggest that migraine inhibitors such as those blocking CGRP receptor signaling protect against acute IL-6 release and subsequent inflammatory events after SARS-CoV-2 infection, which may have repercussions for related pandemic or endemic coronavirus outbreaks.IMPORTANCECoronavirus disease (COVID-19) can cause neurological symptoms such as fever, headache, dizziness, and nausea. However, such neurological symptoms of severe acute respiratory syndrome CoV-2 (SARS-CoV-2) infection have been hardly assessed in mouse models. In this study, we first infected two commonly used wild-type mouse lines (C57BL/6J and 129S) with mouse-adapted SARS-CoV-2 and demonstrated neurological symptoms including fever and nausea. Furthermore, we showed that the migraine treatment drug olcegepant could reduce long-term weight loss and IL-6 release associated with SARS-CoV-2 infection. These findings suggest that a migraine blocker can be protective for at least some acute SARS-CoV-2 infection signs and raise the possibility that it may also impact long-term outcomes.

Adaptive immune cells are necessary for SARS-CoV-2-induced pathology.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the ongoing global pandemic associated with morbidity and mortality in humans. Although disease severity correlates with immune dysregulation, the cellular mechanisms of inflammation and pathogenesis of COVID-19 remain relatively poorly understood. Here, we used mouse-adapted SARS-CoV-2 strain MA10 to investigate the role of adaptive immune cells in disease. We found that while infected wild-type mice lost ~10% weight by 3 to 4 days postinfection, rag-/- mice lacking B and T lymphocytes did not lose weight. Infected lungs at peak weight loss revealed lower pathology scores, fewer neutrophils, and lower interleukin-6 and tumor necrosis factor-α in rag-/- mice. Mice lacking αß T cells also had less severe weight loss, but adoptive transfer of T and B cells into rag-/- mice did not significantly change the response. Collectively, these findings suggest that while adaptive immune cells are important for clearing SARS-CoV-2 infection, this comes at the expense of increased inflammation and pathology.

Migraine inhibitor olcegepant reduces weight loss and IL-6 release in SARS-CoV-2 infected older mice with neurological signs.

COVID-19 can result in neurological symptoms such as fever, headache, dizziness, and nausea. However, neurological signs of SARS-CoV-2 infection have been hardly assessed in mouse models. Here, we infected two commonly used wildtype mice lines (C57BL/6 and 129S) with mouse-adapted SARS-CoV-2 and demonstrated neurological signs including motion-related dizziness. We then evaluated whether the Calcitonin Gene-Related Peptide (CGRP) receptor antagonist, olcegepant, used in migraine treatment could mitigate acute neuroinflammatory and neurological responses to SARS-COV-2 infection. We infected wildtype C57BL/6J and 129/SvEv mice, and a 129 αCGRP-null mouse line with a mouse-adapted SARS-CoV-2 virus, and evaluated the effect of CGRP receptor antagonism on the outcome of that infection. First, we determined that CGRP receptor antagonism provided protection from permanent weight loss in older (>12 m) C57BL/6J and 129 SvEv mice. We also observed acute fever and motion-induced dizziness in all older mice, regardless of treatment. However, in both wildtype mouse lines, CGRP antagonism reduced acute interleukin 6 (IL-6) levels by half, with virtually no IL-6 release in mice lacking αCGRP. These findings suggest that migraine inhibitors such as those blocking CGRP signaling protect against acute IL-6 release and subsequent inflammatory events after SARS-CoV-2 infection, which may have repercussions for related pandemic and/or endemic coronaviruses.

Endothelial SARS-CoV-2 infection is not the underlying cause of COVID-19-associated vascular pathology in mice.

Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. In the present study, we utilized a set of new mouse genetic tools developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID-19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells do not contribute significantly to the diverse vascular pathology associated with COVID-19.

Age-dependent acquisition of pathogenicity by SARS-CoV-2 Omicron BA.5.

Pathology studies of SARS-CoV-2 Omicron variants of concern (VOC) are challenged by the lack of pathogenic animal models. While Omicron BA.1 and BA.2 replicate in K18-hACE2 transgenic mice, they cause minimal to negligible morbidity and mortality, and less is known about more recent Omicron VOC. Here, we show that in contrast to Omicron BA.1, BA.5-infected mice exhibited high levels of morbidity and mortality, correlating with higher early viral loads. Neither Omicron BA.1 nor BA.5 replicated in brains, unlike most prior VOC. Only Omicron BA.5-infected mice exhibited substantial weight loss, high pathology scores in lungs, and high levels of inflammatory cells and cytokines in bronchoalveolar lavage fluid, and 5- to 8-month-old mice exhibited 100% fatality. These results identify a rodent model for pathogenesis or antiviral countermeasure studies for circulating SARS-CoV-2 Omicron BA.5. Further, differences in morbidity and mortality between Omicron BA.1 and BA.5 provide a model for understanding viral determinants of pathogenicity.

Endothelial SARS-CoV-2 infection is not the underlying cause of COVID19-associated vascular pathology in mice.

Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. Here, we utilized a set of new mouse genetic tools 1 developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells does not contribute significantly to the diverse vascular pathology associated with COVID-19.

Thermophobic Trehalose Glycopolymers as Smart C-Type Lectin Receptor Vaccine Adjuvants.

Herein, this work reports the first synthetic vaccine adjuvants that attenuate potency in response to small, 1-2 °C changes in temperature about their lower critical solution temperature (LCST). Adjuvant additives significantly increase vaccine efficacy. However, adjuvants also cause inflammatory side effects, such as pyrexia, which currently limits their use. To address this, a thermophobic vaccine adjuvant engineered to attenuate potency at temperatures correlating to pyrexia is created. Thermophobic adjuvants are synthesized by combining a rationally designed trehalose glycolipid vaccine adjuvant with thermoresponsive poly-N-isoporpylacrylamide (NIPAM) via reversible addition fragmentation chain transfer (RAFT) polymerization. The resulting thermophobic adjuvants exhibit LCSTs near 37 °C, and self-assembled into nanoparticles with temperature-dependent sizes (90-270 nm). Thermophobic adjuvants activate HEK-mMINCLE and other innate immune cell lines as well as primary mouse bone marrow derived dendritic cells (BMDCs) and bone marrow derived macrophages (BMDMs). Inflammatory cytokine production is attenuated under conditions mimicking pyrexia (above the LCST) relative to homeostasis (37 °C) or below the LCST. This thermophobic behavior correlated with decreased adjuvant Rg is observed by DLS, as well as glycolipid-NIPAM shielding interactions are observed by NOESY-NMR. In vivo, thermophobic adjuvants enhance efficacy of a whole inactivated influenza A/California/04/2009 virus vaccine, by increasing neutralizing antibody titers and CD4+ /44+ /62L+ lung and lymph node central memory T cells, as well as providing better protection from morbidity after viral challenge relative to unadjuvanted control vaccine. Together, these results demonstrate the first adjuvants with potency regulated by temperature. This work envisions that with further investigation, this approach can enhance vaccine efficacy while maintaining safety.

Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection.

Angiotensin-converting enzyme 2 (ACE2) is the cell-surface receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). While its central role in Coronavirus Disease 2019 (COVID-19) pathogenesis is indisputable, there remains significant debate regarding the role of this transmembrane carboxypeptidase in the disease course. These include the role of soluble versus membrane-bound ACE2, as well as ACE2-independent mechanisms that may contribute to viral spread. Testing these roles requires in vivo models. Here, we report humanized ACE2-floxed mice in which hACE2 is expressed from the mouse Ace2 locus in a manner that confers lethal disease and permits cell-specific, Cre-mediated loss of function, and LSL-hACE2 mice in which hACE2 is expressed from the Rosa26 locus enabling cell-specific, Cre-mediated gain of function. Following exposure to SARS-CoV-2, hACE2-floxed mice experienced lethal cachexia, pulmonary infiltrates, intravascular thrombosis and hypoxemia-hallmarks of severe COVID-19. Cre-mediated loss and gain of hACE2 demonstrate that neuronal infection confers lethal cachexia, hypoxemia, and respiratory failure in the absence of lung epithelial infection. In this series of genetic experiments, we demonstrate that ACE2 is absolutely and cell-autonomously required for SARS-CoV-2 infection in the olfactory epithelium, brain, and lung across diverse cell types. Therapies inhibiting or blocking ACE2 at these different sites are likely to be an effective strategy towards preventing severe COVID-19.

Multivalent viral particles elicit safe and efficient immunoprotection against Nipah Hendra and Ebola viruses.

Experimental vaccines for the deadly zoonotic Nipah (NiV), Hendra (HeV), and Ebola (EBOV) viruses have focused on targeting individual viruses, although their geographical and bat reservoir host overlaps warrant creation of multivalent vaccines. Here we explored whether replication-incompetent pseudotyped vesicular stomatitis virus (VSV) virions or NiV-based virus-like particles (VLPs) were suitable multivalent vaccine platforms by co-incorporating multiple surface glycoproteins from NiV, HeV, and EBOV onto these virions. We then enhanced the vaccines' thermotolerance using carbohydrates to enhance applicability in global regions that lack cold-chain infrastructure. Excitingly, in a Syrian hamster model of disease, the VSV multivalent vaccine elicited safe, strong, and protective neutralizing antibody responses against challenge with NiV, HeV, or EBOV. Our study provides proof-of-principle evidence that replication-incompetent multivalent viral particle vaccines are sufficient to provide protection against multiple zoonotic deadly viruses with high pandemic potential.

A TMPRSS2 inhibitor acts as a pan-SARS-CoV-2 prophylactic and therapeutic.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle5,6. Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 106 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids7. In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.

Ecology, evolution and spillover of coronaviruses from bats.

In the past two decades, three coronaviruses with ancestral origins in bats have emerged and caused widespread outbreaks in humans, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the first SARS epidemic in 2002-2003, the appreciation of bats as key hosts of zoonotic coronaviruses has advanced rapidly. More than 4,000 coronavirus sequences from 14 bat families have been identified, yet the true diversity of bat coronaviruses is probably much greater. Given that bats are the likely evolutionary source for several human coronaviruses, including strains that cause mild upper respiratory tract disease, their role in historic and future pandemics requires ongoing investigation. We review and integrate information on bat-coronavirus interactions at the molecular, tissue, host and population levels. We identify critical gaps in knowledge of bat coronaviruses, which relate to spillover and pandemic risk, including the pathways to zoonotic spillover, the infection dynamics within bat reservoir hosts, the role of prior adaptation in intermediate hosts for zoonotic transmission and the viral genotypes or traits that predict zoonotic capacity and pandemic potential. Filling these knowledge gaps may help prevent the next pandemic.

SARS-CoV-2 infection of olfactory epithelial cells and neurons drives acute lung injury and lethal COVID-19 in mice.

Lethal COVID-19 is associated with respiratory failure that is thought to be caused by acute respiratory distress syndrome (ARDS) secondary to pulmonary infection. To date, the cellular pathogenesis has been inferred from studies describing the expression of ACE2, a transmembrane protein required for SARS-CoV-2 infection, and detection of viral RNA or protein in infected humans, model animals, and cultured cells. To functionally test the cellular mechanisms of COVID-19, we generated hACE2 fl animals in which human ACE2 (hACE2) is expressed from the mouse Ace2 locus in a manner that permits cell-specific, Cre-mediated loss of function. hACE2 fl animals developed lethal weight loss and hypoxemia within 7 days of exposure to SARS-CoV-2 that was associated with pulmonary infiltrates, intravascular thrombosis and patchy viral infection of lung epithelial cells. Deletion of hACE2 in lung epithelial cells prevented viral infection of the lung, but not weight loss, hypoxemia or death. Inhalation of SARS-CoV-2 by hACE2 fl animals resulted in early infection of sustentacular cells with subsequent infection of neurons in the neighboring olfactory bulb and cerebral cortexâ€" events that did not require lung epithelial cell infection. Pharmacologic ablation of the olfactory epithelium or Foxg1 Cre mediated deletion of hACE2 in olfactory epithelial cells and neurons prevented lethality and neuronal infection following SARS-CoV-2 infection. Conversely, transgenic expression of hACE2 specifically in olfactory epithelial cells and neurons in Foxg1 Cre ; LSL- hACE2 mice was sufficient to confer neuronal infection associated with respiratory failure and death. These studies establish mouse loss and gain of function genetic models with which to genetically dissect viral-host interactions and demonstrate that lethal disease due to respiratory failure may arise from extrapulmonary infection of the olfactory epithelium and brain. Future therapeutic efforts focused on preventing olfactory epithelial infection may be an effective means of protecting against severe COVID-19.

Drivers and Distribution of Henipavirus-Induced Syncytia: What Do We Know?

Syncytium formation, i.e., cell-cell fusion resulting in the formation of multinucleated cells, is a hallmark of infection by paramyxoviruses and other pathogenic viruses. This natural mechanism has historically been a diagnostic marker for paramyxovirus infection in vivo and is now widely used for the study of virus-induced membrane fusion in vitro. However, the role of syncytium formation in within-host dissemination and pathogenicity of viruses remains poorly understood. The diversity of henipaviruses and their wide host range and tissue tropism make them particularly appropriate models with which to characterize the drivers of syncytium formation and the implications for virus fitness and pathogenicity. Based on the henipavirus literature, we summarized current knowledge on the mechanisms driving syncytium formation, mostly acquired from in vitro studies, and on the in vivo distribution of syncytia. While these data suggest that syncytium formation widely occurs across henipaviruses, hosts, and tissues, we identified important data gaps that undermined our understanding of the role of syncytium formation in virus pathogenesis. Based on these observations, we propose solutions of varying complexity to fill these data gaps, from better practices in data archiving and publication for in vivo studies, to experimental approaches in vitro.

Headless Henipaviral Receptor Binding Glycoproteins Reveal Fusion Modulation by the Head/Stalk Interface and Post-receptor Binding Contributions of the Head Domain.

Cedar virus (CedV) is a nonpathogenic member of the Henipavirus (HNV) genus of emerging viruses, which includes the deadly Nipah (NiV) and Hendra (HeV) viruses. CedV forms syncytia, a hallmark of henipaviral and paramyxoviral infections and pathogenicity. However, the intrinsic fusogenic capacity of CedV relative to NiV or HeV remains unquantified. HNV entry is mediated by concerted interactions between the attachment (G) and fusion (F) glycoproteins. Upon receptor binding by the HNV G head domain, a fusion-activating G stalk region is exposed and triggers F to undergo a conformational cascade that leads to viral entry or cell-cell fusion. Here, we demonstrate quantitatively that CedV is inherently significantly less fusogenic than NiV at equivalent G and F cell surface expression levels. We then generated and tested six headless CedV G mutants of distinct C-terminal stalk lengths, surprisingly revealing highly hyperfusogenic cell-cell fusion phenotypes 3- to 4-fold greater than wild-type CedV levels. Additionally, similarly to NiV, a headless HeV G mutant yielded a less pronounced hyperfusogenic phenotype compared to wild-type HeV. Further, coimmunoprecipitation and cell-cell fusion assays revealed heterotypic NiV/CedV functional G/F bidentate interactions, as well as evidence of HNV G head domain involvement beyond receptor binding or G stalk exposure. All evidence points to the G head/stalk junction being key to modulating HNV fusogenicity, supporting the notion that head domains play several distinct and central roles in modulating stalk domain fusion promotion. Further, this study exemplifies how CedV may help elucidate important mechanistic underpinnings of HNV entry and pathogenicity. IMPORTANCE The Henipavirus genus in the Paramyxoviridae family includes the zoonotic Nipah (NiV) and Hendra (HeV) viruses. NiV and HeV infections often cause fatal encephalitis and pneumonia, but no vaccines or therapeutics are currently approved for human use. Upon viral entry, Henipavirus infections yield the formation of multinucleated cells (syncytia). Viral entry and cell-cell fusion are mediated by the attachment (G) and fusion (F) glycoproteins. Cedar virus (CedV), a nonpathogenic henipavirus, may be a useful tool to gain knowledge on henipaviral pathogenicity. Here, using homotypic and heterotypic full-length and headless CedV, NiV, and HeV G/F combinations, we discovered that CedV G/F are significantly less fusogenic than NiV or HeV G/F, and that the G head/stalk junction is key to modulating cell-cell fusion, refining the mechanism of henipaviral membrane fusion events. Our study exemplifies how CedV may be a useful tool to elucidate broader mechanistic understanding for the important henipaviruses.

A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced against emerging variants of concern (VOCs) 1,2 . Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against VOCs 3,4 . Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs), such as TMPRSS2, whose essential role in the virus lifecycle is responsible for the cleavage and priming of the viral spike protein 5-7 . Here, we identify and characterize a small-molecule compound, N-0385, as the most potent inhibitor of TMPRSS2 reported to date. N-0385 exhibited low nanomolar potency and a selectivity index of >10 6 at inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids 8 . Importantly, N-0385 acted as a broad-spectrum coronavirus inhibitor of two SARS-CoV-2 VOCs, B.1.1.7 and B.1.351. Strikingly, single daily intranasal administration of N-0385 early in infection significantly improved weight loss and clinical outcomes, and yielded 100% survival in the severe K18-human ACE2 transgenic mouse model of SARS-CoV-2 disease. This demonstrates that TTSP-mediated proteolytic maturation of spike is critical for SARS-CoV-2 infection in vivo and suggests that N-0385 provides a novel effective early treatment option against COVID-19 and emerging SARS-CoV-2 VOCs.

Roles of Cholesterol in Early and Late Steps of the Nipah Virus Membrane Fusion Cascade.

Cholesterol has been implicated in various viral life cycle steps for different enveloped viruses, including viral entry into host cells, cell-cell fusion, and viral budding from infected cells. Enveloped viruses acquire their membranes from their host cells. Although cholesterol has been associated with the binding and entry of various enveloped viruses into cells, cholesterol's exact function in the viral-cell membrane fusion process remains largely elusive, particularly for the paramyxoviruses. Furthermore, paramyxoviral fusion occurs at the host cell membrane and is essential for both virus entry (virus-cell fusion) and syncytium formation (cell-cell fusion), central to viral pathogenicity. Nipah virus (NiV) is a deadly member of the Paramyxoviridae family, which also includes Hendra, measles, mumps, human parainfluenza, and various veterinary viruses. The zoonotic NiV causes severe encephalitis, vasculopathy, and respiratory symptoms, leading to a high mortality rate in humans. We used NiV as a model to study the role of membrane cholesterol in paramyxoviral membrane fusion. We used a combination of methyl-beta cyclodextrin (MßCD), lovastatin, and cholesterol to deplete or enrich cell membrane cholesterol outside cytotoxic concentrations. We found that the levels of cellular membrane cholesterol directly correlated with the levels of cell-cell fusion induced. These phenotypes were paralleled using NiV/vesicular stomatitis virus (VSV)-pseudotyped viral infection assays. Remarkably, our mechanistic studies revealed that cholesterol reduces an early F-triggering step but enhances a late fusion pore formation step in the NiV membrane fusion cascade. Thus, our results expand our mechanistic understanding of the paramyxoviral/henipaviral entry and cell-cell fusion processes.IMPORTANCE Cholesterol has been implicated in various steps of the viral life cycle for different enveloped viruses. Nipah virus (NiV) is a highly pathogenic enveloped virus in the Henipavirus genus within the Paramyxoviridae family, capable of causing a high mortality rate in humans and high morbidity in domestic and agriculturally important animals. The role of cholesterol for NiV or the henipaviruses is unknown. Here, we show that the levels of cholesterol influence the levels of NiV-induced cell-cell membrane fusion during syncytium formation and virus-cell membrane fusion during viral entry. Furthermore, the specific role of cholesterol in membrane fusion is not well defined for the paramyxoviruses. We show that the levels of cholesterol affect an early F-triggering step and a late fusion pore formation step during the membrane fusion cascade. Thus, our results expand our mechanistic understanding of the viral entry and cell-cell fusion processes, which may aid the development of antivirals.

Finding proteases that make cells go viral.

The activation of influenza virus hemagglutinin (HA) glycoprotein via cleavage by host cell proteases is essential for viral infectivity, and understanding the mechanisms for HA protein cleavage and how they may differ depending on the biological context is important for the development of flu treatments. However, the HA proteases involved in the activation of many viral strains remain unidentified. In this issue, Harbig et al. identify a repertoire of proteases that cleave HA and determine the proteases' functionality against specific HA glycoproteins.

Modified Sialic Acids on Mucus and Erythrocytes Inhibit Influenza A Virus Hemagglutinin and Neuraminidase Functions.

Sialic acids (Sia) are the primary receptors for influenza viruses and are widely displayed on cell surfaces and in secreted mucus. Sia may be present in variant forms that include O-acetyl modifications at C-4, C-7, C-8, and C-9 positions and N-acetyl or N-glycolyl at C-5. They can also vary in their linkages, including α2-3 or α2-6 linkages. Here, we analyze the distribution of modified Sia in cells and tissues of wild-type mice or in mice lacking CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme, which synthesizes N-glycolyl (Neu5Gc) modifications. We also examined the variation of Sia forms on erythrocytes and in saliva from different animals. To determine the effect of Sia modifications on influenza A virus (IAV) infection, we tested for effects on hemagglutinin (HA) binding and neuraminidase (NA) cleavage. We confirmed that 9-O-acetyl, 7,9-O-acetyl, 4-O-acetyl, and Neu5Gc modifications are widely but variably expressed in mouse tissues, with the highest levels detected in the respiratory and gastrointestinal (GI) tracts. Secreted mucins in saliva and surface proteins of erythrocytes showed a high degree of variability in display of modified Sia between different species. IAV HAs from different virus strains showed consistently reduced binding to both Neu5Gc- and O-acetyl-modified Sia; however, while IAV NAs were inhibited by Neu5Gc and O-acetyl modifications, there was significant variability between NA types. The modifications of Sia in mucus may therefore have potent effects on the functions of IAV and may affect both pathogens and the normal flora of different mucosal sites.IMPORTANCE Sialic acids (Sia) are involved in numerous different cellular functions and are receptors for many pathogens. Sia come in chemically modified forms, but we lack a clear understanding of how they alter interactions with microbes. Here, we examine the expression of modified Sia in mouse tissues, on secreted mucus in saliva, and on erythrocytes, including those from IAV host species and animals used in IAV research. These Sia forms varied considerably among different animals, and their inhibitory effects on IAV NA and HA activities and on bacterial sialidases (neuraminidases) suggest a host-variable protective role in secreted mucus.

Expression of 9-O- and 7,9-O-Acetyl Modified Sialic Acid in Cells and Their Effects on Influenza Viruses.

Sialic acids (Sia) are widely displayed on the surfaces of cells and tissues. Sia come in a variety of chemically modified forms, including those with acetyl modifications at the C-7, C-8, and C-9 positions. Here, we analyzed the distribution and amounts of these acetyl modifications in different human and canine cells. Since Sia or their variant forms are receptors for influenza A, B, C, and D viruses, we examined the effects of these modifications on virus infections. We confirmed that 9-O-acetyl and 7,9-O-acetyl modified Sia are widely but variably expressed across cell lines from both humans and canines. Although they were expressed on the cell surfaces of canine MDCK cell lines, they were located primarily within the Golgi compartment of human HEK-293 and A549 cells. The O-acetyl modified Sia were expressed at low levels of 1 to 2% of total Sia in these cell lines. We knocked out and overexpressed the sialate O-acetyltransferase gene (CasD1) and knocked out the sialate O-acetylesterase gene (SIAE) using CRISPR/Cas9 editing. Knocking out CasD1 removed 7,9-O- and 9-O-acetyl Sia expression, confirming previous reports. However, overexpression of CasD1 and knockout of SIAE gave only modest increases in 9-O-acetyl levels in cells and no change in 7,9-O-acetyl levels, indicating that there are complex regulations of these modifications. These modifications were essential for influenza C and D infection but had no obvious effect on influenza A and B infection.IMPORTANCE Sialic acids are key glycans that are involved in many different normal cellular functions, as well as being receptors for many pathogens. However, Sia come in diverse chemically modified forms. Here, we examined and manipulated the expression of 7,9-O- and 9-O-acetyl modified Sia on cells commonly used in influenza virus and other research by engineering the enzymes that produce or remove the acetyl groups.