La Crosse virus reassortants highlight genomic determinants of infection and pathogenesis.

The genomic determinants that contribute to orthobunyavirus infection and pathogenesis are not well-defined. In this study, we harnessed the process of reassortment to understand which viral factors drive change in the replication and pathogenesis of La Crosse virus (LACV). We systematically reassorted the genomic segments of two genetically similar Lineage I LACV isolates into six unique reassortants. Despite the parental isolates having high levels of RNA and protein consensus, the reassortants demonstrate how minimal changes in RNA and protein structure can have significant changes in viral growth and reproduction in vitro in mammalian and insect models. We observed that swapping the S segment between isolates led to differences in replication and assembly resulting in one non-rescuable reassortant and one viable reassortant that exhibited an increase in viral growth dynamics. Switching the M segment led to changes in viral plaque phenotype and growth kinetics. L segment reassortants similarly differed in changes in viral growth dynamics. We further explored the M segment reassortants in a neonate mouse model and observed a role for the M segment in neuroinflammation and virulence. Through reassortment of the La Crosse virus genomic segments, we are able to further understand how genomic determinants of infection and pathogenesis operate in orthobunyaviruses. Future investigations will focus on identifying the specific molecular elements that govern the observed phenotypes in vitro and in vivo . Importance: La Crosse virus is the leading cause of pediatric arboviral encephalitis in the United States, yet it is largely unknown how each of the three genomic segments contribute to pathogenesis and disease. Our study utilizes genomic reassortment between two similar Lineage I LACV isolates to understand genomic determinants for differences in infection and pathogenesis phenotypes in vitro and in vivo. By identifying roles for each segment in observed outcomes, we are able to plan further studies for molecular characterization of these phenotypes. Additionally, it is imperative to continue to characterize orthobunyavirus function since climate change will expand the range and prevalence of arthropod-borne diseases such as LACV in the United States.

The chikungunya virus E1 glycoprotein fusion loop and hinge alter glycoprotein dynamics leading to cell and host specific changes in infectivity.

Alphaviruses infect both mammals and insects, yet the distinct mechanisms that alphaviruses use to infect different hosts are not well defined. In this study, we characterize CHIKV E1 variants in the fusion loop (E1-M88L) and hinge region (E1-N20Y) in vitro and in vivo to understand how these regions of the E1 glycoprotein contribute to host-specific infection. Through cell culture assays, we found that CHIKV E1-N20Y enhanced infectivity in mosquito cells while the CHIKV E1-M88L variant enhanced virus binding and infectivity in both BHK-21 and C6/36 cells, and led to changes in the virus cholesterol-dependence in BHK-21 cells. Given these in vitro results and that residue E1-M88L is in a defined Mxra8 interacting domain, we hypothesized that this residue may be important for receptor usage. However, while the CHIKV E1-M88L variant increased replication in Mxra8-deficient mice compared to WT CHIKV, it was attenuated in vitro in mouse fibroblasts, suggesting that residue E1-M88 may function in a cell-type dependent manner to alter entry. Finally, using molecular dynamics to understand how potential changes in the E1 glycoprotein may impact the CHIKV glycoprotein E1-E2 complex, we found that E1-M88L and other E1 domain II variants lead to changes in both E1 and E2 dynamics. Taken together, these studies show that key residues in the CHIKV E1 fusion loop and hinge region function through changes in E1-E2 dynamics to facilitate cell- and host-dependent entry. Importance: Arthropod-borne viruses (arboviruses) are significant global public health threats, and their continued emergence around the world highlights the need to understand how these viruses replicate at the molecular level. The alphavirus class II glycoproteins are critical for virus entry in mosquitoes and mammals, yet how these proteins function is not completely understood. Therefore, to address these gaps in our knowledge, it is critical to dissect how distinct glycoprotein domains function in vitro and in vivo . Here, we show that changes in the CHIKV E1 fusion loop and hinge contribute to host-specific entry and E1-E2 dynamics, furthering our knowledge of how alphaviruses infect mammals and insects.

The La Crosse virus class II fusion glycoprotein ij loop contributes to infectivity and replication in vitro and in vivo.

Arthropod-borne viruses (arboviruses) are an emerging and evolving global public health threat, with limited antiviral treatments or vaccines available. La Crosse virus (LACV) from the Bunyavirales order is responsible for pediatric encephalitis cases in the United States, yet little is known about the infectivity of LACV. Given the structural similarities between class II fusion glycoproteins of LACV and chikungunya virus (CHIKV), an alphavirus from the Togaviridae family, we hypothesized that LACV would share similar entry mechanisms with CHIKV. To test this hypothesis, we performed cholesterol-depletion and repletion assays and used cholesterol-modulating compounds to study LACV entry and replication. We found that LACV entry was cholesterol dependent, while replication was less affected by cholesterol manipulation. In addition, we generated single-point mutants in the LACV Gc ij loop that corresponded to known CHIKV residues important for virus entry. We found that a conserved histidine and alanine residue in the Gc ij loop impaired virus infectivity and attenuated LACV replication in vitro and in vivo. Finally, we took an evolution-based approach to explore how the LACV glycoprotein evolves in mosquitoes and mice. We found multiple variants that cluster in the Gc glycoprotein head domain, providing evidence for the Gc glycoprotein as a contributor to LACV adaptation. Together, these results begin to characterize the mechanisms of LACV infectivity and how the LACV glycoprotein contributes to replication and pathogenesis. IMPORTANCE Vector-borne viruses are significant health threats that lead to devastating disease worldwide. The emergence of arboviruses, in addition to the lack of effective antivirals or vaccines, highlights the need to study how arboviruses replicate at the molecular level. One potential antiviral target is the class II fusion glycoprotein. Alphaviruses, flaviviruses, and bunyaviruses encode a class II fusion glycoprotein that contains strong structural similarities at the tip of domain II. Here, we show that the bunyavirus La Crosse virus uses a cholesterol-dependent entry pathway similar to the alphavirus chikungunya virus, and residues in the ij loop are important for virus infectivity in vitro and replication in mice. These studies show that genetically diverse viruses may use similar pathways through conserved structure domains, suggesting that these viruses may be targets for broad-spectrum antivirals in multiple arboviral families.

The La Crosse virus class II fusion glycoprotein ij loop contributes to infectivity and cholesterol-dependent entry.

Arthropod-borne viruses (arboviruses) are an emerging and evolving global public health threat with little to no antiviral treatments. La Crosse virus (LACV) from the Bunyavirales order is responsible for pediatric encephalitis cases in the United States, yet little is known about the infectivity of LACV. Given the structural similarities between class II fusion glycoproteins of LACV and chikungunya virus (CHIKV), an alphavirus from the Togaviridae family, we hypothesized that LACV would share similar entry mechanisms to CHIKV. To test this hypothesis, we performed cholesterol-depletion and repletion assays and used cholesterol modulating compounds to study LACV entry and replication. We found that LACV entry was cholesterol-dependent while replication was less affected by cholesterol manipulation. In addition, we generated single point mutants in the LACV ij loop that corresponded to known CHIKV residues important for virus entry. We found that a conserved histidine and alanine residue in the Gc ij loop impaired virus infectivity and attenuate LACV in vitro and in vivo . Finally, we took an evolution-based approach to explore how the LACV glycoprotein evolution in mosquitoes and mice. We found multiple variants that cluster in the Gc glycoprotein head domain, supporting the Gc glycoprotein as a target for LACV adaptation. Together, these results begin to characterize the mechanisms of LACV infectivity and how the LACV glycoprotein contributes to infectivity and pathogenesis. Importance: Vector-borne arboviruses are significant health threats leading to devastating disease worldwide. This emergence and the fact that there are little to no vaccines or antivirals targeting these viruses highlights the need to study how arboviruses replicate at the molecular level. One potential antiviral target is the class II fusion glycoprotein. Alphaviruses, flaviviruses, and bunyaviruses encode a class II fusion glycoprotein that contain strong structural similarities in the tip of domain II. Here we show that the bunyavirus La Crosse virus uses similar mechanisms to entry as the alphavirus chikungunya virus and residues in the ij loop are important for virus infectivity. These studies show that genetically diverse viruses use similar mechanisms through concerned structure domains, suggesting these may be a target for broad-spectrum antivirals to multiple arbovirus families.

Structurally Conserved Domains between Flavivirus and Alphavirus Fusion Glycoproteins Contribute to Replication and Infectious-Virion Production.

Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants, including an envelope glycoprotein variant in ß-strand c (V114M) of domain II. We have previously shown that the analogous ß-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E ß-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that shared domains of the alphavirus and flavivirus class II fusion glycoproteins harbor structurally analogous residues that are functionally important and contribute to virus infection in vivo.IMPORTANCE Arboviruses are a significant global public health threat, yet there are no antivirals targeting these viruses. This problem is in part due to our lack of knowledge of the molecular mechanisms involved in the arbovirus life cycle. In particular, virus entry and assembly are essential processes in the virus life cycle and steps that can be targeted for the development of antiviral therapies. Therefore, understanding common, fundamental mechanisms used by different arboviruses for entry and assembly is essential. In this study, we show that flavivirus and alphavirus residues located in structurally conserved and analogous regions of the class II fusion proteins contribute to common mechanisms of entry, dissemination, and infectious-virion production. These studies highlight how class II fusion proteins function and provide novel targets for development of antivirals.

Atovaquone and Berberine Chloride Reduce SARS-CoV-2 Replication In Vitro.

Epidemic RNA viruses seem to arise year after year leading to countless infections and devastating disease. SARS-CoV-2 is the most recent of these viruses, but there will undoubtedly be more to come. While effective SARS-CoV-2 vaccines are being deployed, one approach that is still missing is effective antivirals that can be used at the onset of infections and therefore prevent pandemics. Here, we screened FDA-approved compounds against SARS-CoV-2. We found that atovaquone, a pyrimidine biosynthesis inhibitor, is able to reduce SARS-CoV-2 infection in human lung cells. In addition, we found that berberine chloride, a plant-based compound used in holistic medicine, was able to inhibit SARS-CoV-2 infection in cells through direct interaction with the virion. Taken together, these studies highlight potential avenues of antiviral development to block emerging viruses. Such proactive approaches, conducted well before the next pandemic, will be essential to have drugs ready for when the next emerging virus hits.

Microbial signatures in the lower airways of mechanically ventilated COVID-19 patients associated with poor clinical outcome.

Respiratory failure is associated with increased mortality in COVID-19 patients. There are no validated lower airway biomarkers to predict clinical outcome. We investigated whether bacterial respiratory infections were associated with poor clinical outcome of COVID-19 in a prospective, observational cohort of 589 critically ill adults, all of whom required mechanical ventilation. For a subset of 142 patients who underwent bronchoscopy, we quantified SARS-CoV-2 viral load, analysed the lower respiratory tract microbiome using metagenomics and metatranscriptomics and profiled the host immune response. Acquisition of a hospital-acquired respiratory pathogen was not associated with fatal outcome. Poor clinical outcome was associated with lower airway enrichment with an oral commensal (Mycoplasma salivarium). Increased SARS-CoV-2 abundance, low anti-SARS-CoV-2 antibody response and a distinct host transcriptome profile of the lower airways were most predictive of mortality. Our data provide evidence that secondary respiratory infections do not drive mortality in COVID-19 and clinical management strategies should prioritize reducing viral replication and maximizing host responses to SARS-CoV-2.

Combination of a Sindbis-SARS-CoV-2 Spike Vaccine and αOX40 Antibody Elicits Protective Immunity Against SARS-CoV-2 Induced Disease and Potentiates Long-Term SARS-CoV-2-Specific Humoral and T-Cell Immunity.

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is a major global public threat. Currently, a worldwide effort has been mounted to generate billions of effective SARS-CoV-2 vaccine doses to immunize the world's population at record speeds. However, there is still a demand for alternative effective vaccines that rapidly confer long-term protection and rely upon cost-effective, easily scaled-up manufacturing. Here, we present a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (αOX40) as a novel, highly effective vaccine approach. We show that SV.Spike plus αOX40 elicits long-lasting neutralizing antibodies and a vigorous T-cell response in mice. Protein binding, immunohistochemical, and cellular infection assays all show that vaccinated mice sera inhibits spike functions. Immunophenotyping, RNA Seq transcriptome profiles, and metabolic analysis indicate a reprogramming of T cells in vaccinated mice. Activated T cells were found to mobilize to lung tissue. Most importantly, SV.Spike plus αOX40 provided robust immune protection against infection with authentic coronavirus in transgenic mice expressing the human ACE2 receptor (hACE2-Tg). Finally, our immunization strategy induced strong effector memory response, potentiating protective immunity against re-exposure to SARS-CoV-2 spike protein. Our results show the potential of a new Sindbis virus-based vaccine platform to counteract waning immune response, which can be used as a new candidate to combat SARS-CoV-2. Given the T-cell responses elicited, our vaccine is likely to be effective against variants that are proving challenging, as well as serve as a platform to develop a broader spectrum pancoronavirus vaccine. Similarly, the vaccine approach is likely to be applicable to other pathogens.

Two-dimensional multiplexed assay for rapid and deep SARS-CoV-2 serology profiling and for machine learning prediction of neutralization capacity.

Antibody responses serve as the primary protection against SARS-CoV-2 infection through neutralization of viral entry into cells. We have developed a two-dimensional multiplex bead binding assay (2D-MBBA) that quantifies multiple antibody isotypes against multiple antigens from a single measurement. Here, we applied our assay to profile IgG, IgM and IgA levels against the spike antigen, its receptor-binding domain and natural and designed mutants. Machine learning algorithms trained on the 2D-MBBA data substantially improve the prediction of neutralization capacity against the authentic SARS-CoV-2 virus of serum samples of convalescent patients. The algorithms also helped identify a set of antibody isotype-antigen datasets that contributed to the prediction, which included those targeting regions outside the receptor-binding interface of the spike protein. We applied the assay to profile samples from vaccinated, immune-compromised patients, which revealed differences in the antibody profiles between convalescent and vaccinated samples. Our approach can rapidly provide deep antibody profiles and neutralization prediction from essentially a drop of blood without the need of BSL-3 access and provides insights into the nature of neutralizing antibodies. It may be further developed for evaluating neutralizing capacity for new variants and future pathogens.

Combination of a Sindbis-SARS-CoV-2 spike vaccine and αOX40 antibody elicits protective immunity against SARS-CoV-2 induced disease and potentiates long-term SARS-CoV-2-specific humoral and T-cell immunity.

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is a major global public threat. Currently, a worldwide effort has been mounted to generate billions of effective SARS-CoV-2 vaccine doses to immunize the world's population at record speeds. However, there is still demand for alternative effective vaccines that rapidly confer long-term protection and rely upon cost-effective, easily scaled-up manufacturing. Here, we present a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (αOX40) as a novel, highly effective vaccine approach. We show that SV.Spike plus αOX40 elicits long-lasting neutralizing antibodies and a vigorous T-cell response in mice. Protein binding, immunohistochemical and cellular infection assays all show that vaccinated mice sera inhibits spike functions. Immunophenotyping, RNA Seq transcriptome profiles and metabolic analysis indicate a reprogramming of T-cells in vaccinated mice. Activated T-cells were found to mobilize to lung tissue. Most importantly, SV.Spike plus αOX40 provided robust immune protection against infection with authentic coronavirus in transgenic mice expressing the human ACE2 receptor (hACE2-Tg). Finally, our immunization strategy induced strong effector memory response, potentiating protective immunity against re-exposure to SARS-CoV-2 spike protein. Our results show the potential of a new Sindbis virus-based vaccine platform to counteract waning immune response that can be used as a new candidate to combat SARS-CoV-2. Given the strong T-cell responses elicited, our vaccine is likely to be effective against variants that are proving challenging, as well as, serve as a platform to develop a broader spectrum pancoronavirus vaccine. Similarly, the vaccine approach is likely to be applicable to other pathogens.

Impaired Humoral Immunity to SARS-CoV-2 Vaccination in Non-Hodgkin Lymphoma and CLL Patients.

Patients with hematologic malignancies are a high priority for SARS-CoV-2 vaccination, yet the benefit they will derive is uncertain. We investigated the humoral response to vaccination in 53 non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), or CLL patients. Peripheral blood was obtained 2 weeks after first vaccination and 6 weeks after second vaccination for antibody profiling using the multiplex bead-binding assay. Serum IgG, IgA, and IgM antibody levels to the spike specific receptor binding domain (RBD) were evaluated as a measure of response. Subsequently, antibody-positive serum were assayed for neutralization capacity against authentic SARS-CoV-2. Histology was 68% lymphoma and 32% CLL; groups were: patients receiving anti-CD20-based therapy (45%), monitored with disease (28%), receiving BTK inhibitors (19%), or chemotherapy (all HL) (8%). SARS-CoV-2 specific RBD IgG antibody response was decreased across all NHL and CLL groups: 25%, 73%, and 40%, respectively. Antibody IgG titers were significantly reduced (p < 0.001) for CD20 treated and targeted therapy patients, and (p = 0.003) for monitored patients. In 94% of patients evaluated after first and second vaccination, antibody titers did not significantly boost after second vaccination. Only 13% of CD20 treated and 13% of monitored patients generated neutralizing antibodies to SARS-CoV-2 with ICD50s 135 to 1767, and 445 and > 10240. This data has profound implications given the current guidance relaxing masking restrictions and for timing of vaccinations. Unless immunity is confirmed with laboratory testing, these patients should continue to mask, socially distance, and to avoid close contact with non-vaccinated individuals. STATEMENT OF TRANSLATIONAL RELEVANCE: Non Hodgkin lymphoma (NHL) and Chronic Lymphocytic leukemia (CLL) patients who are treated with anti-CD20 antibody therapy, BTK inhibitor therapy, or who are monitored with active disease, have decreased antibody response to SARS-CoV-2 vaccination and decreased antibody titers compared to healthy controls. Antibody titers do not boost following second vaccination, and very few patients generate neutralizing antibodies against SARS-CoV-2. This data is of particular importance, given the recent guidance from the CDC that vaccinated patients no longer need to be masked indoors as well as outdoors. Patients with NHL or CLL who fall into these categories should not consider their immunity from vaccination to be assured. If infected with SARS-CoV-2, they should be a high priority for monoclonal antibody directed therapy. Unless immune response to vaccination is confirmed with laboratory testing, they should continue to mask, socially distance, and to avoid close contact with non-vaccinated individuals.

Microbial signatures in the lower airways of mechanically ventilated COVID19 patients associated with poor clinical outcome.

Mortality among patients with COVID-19 and respiratory failure is high and there are no known lower airway biomarkers that predict clinical outcome. We investigated whether bacterial respiratory infections and viral load were associated with poor clinical outcome and host immune tone. We obtained bacterial and fungal culture data from 589 critically ill subjects with COVID-19 requiring mechanical ventilation. On a subset of the subjects that underwent bronchoscopy, we also quantified SARS-CoV-2 viral load, analyzed the microbiome of the lower airways by metagenome and metatranscriptome analyses and profiled the host immune response. We found that isolation of a hospital-acquired respiratory pathogen was not associated with fatal outcome. However, poor clinical outcome was associated with enrichment of the lower airway microbiota with an oral commensal ( Mycoplasma salivarium ), while high SARS-CoV-2 viral burden, poor anti-SARS-CoV-2 antibody response, together with a unique host transcriptome profile of the lower airways were most predictive of mortality. Collectively, these data support the hypothesis that 1) the extent of viral infectivity drives mortality in severe COVID-19, and therefore 2) clinical management strategies targeting viral replication and host responses to SARS-CoV-2 should be prioritized.