Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Recent studies showed that serum from convalescent patients can display variable neutralization capacities. Still, it remains unclear whether there are specific signatures that can be used to predict neutralization. Here, we performed a detailed analysis of sera from a cohort of 101 recovered healthcare workers and we addressed their SARS-CoV-2 antibody response by ELISA against SARS-CoV-2 Spike receptor binding domain and nucleoprotein. Both ELISA methods detected sustained levels of serum IgG against both antigens. Yet, the majority of individuals from our cohort generated antibodies with low neutralization capacity and only 6% showed high neutralizing titers against both authentic SARS-CoV-2 virus and the Spike pseudotyped virus. Interestingly, higher neutralizing sera correlate with detection of -IgG, IgM and IgA antibodies against both antigens, while individuals with positive IgG alone showed poor neutralization response. These results suggest that having a broader repertoire of antibodies may contribute to more potent SARS-CoV-2 neutralization. Altogether, our work provides a cross sectional snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides preliminary evidence that possessing multiple antibody isotypes can play an important role in predicting SARS-CoV-2 neutralization.

Understanding the fundamental mechanisms of arbovirus transmission and pathogenesis is essential to develop strategies for treatment and prevention. We previously took an in vivo evolution-based approach and identified the chikungunya virus E1 glycoprotein residue 80 to play a critical role in viral transmission and pathogenesis. In this study, we address the genetic conservation and function of position 80 and demonstrate that this residue is a key determinant in alphavirus infectivity and dissemination through modulation of viral fusion and cholesterol dependence. In addition, in studying the evolution of position 80, we identified a network of glycoprotein residues, including epidemic determinants, that regulate virus dissemination and infectivity. These studies underscore the importance of taking evolution-based approaches to not only identify key viral determinants driving arbovirus transmission and pathogenesis but also to uncover fundamental aspects of arbovirus biology.

Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Here, we determine the ability of sera from 101 recovered healthcare workers to neutralize both authentic SARS-CoV-2 and SARS-CoV-2 pseudotyped virus and address their antibody titers against SARS-CoV-2 nucleoprotein and spike receptor-binding domain. Interestingly, the majority of individuals have low neutralization capacity and only 6% of the healthcare workers showed high neutralizing titers against both authentic SARS-CoV-2 virus and the pseudotyped virus. We found the antibody response to SARS-CoV-2 infection generates antigen-specific isotypes as well as a diverse combination of antibody isotypes, with high titers of IgG, IgM and IgA against both antigens correlating with neutralization capacity. Importantly, we found that neutralization correlated with antibody titers as quantified by ELISA. This suggests that an ELISA assay can be used to determine seroneutralization potential. Altogether, our work provides a snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides evidence that possessing multiple antibody isotypes may play an important role in SARS-CoV-2 neutralization.

Understanding the fundamental mechanisms of arbovirus transmission and pathogenesis is 30 essential to develop new strategies for treatment and prevention. We previously took an in vivo 31 evolution-based approach and identified the chikungunya virus E1 glycoprotein residue 80 to 32 play a critical role in viral transmission and pathogenesis. In this study, we address the genetic 33 robustness and function of position 80 and demonstrate that this highly conserved residue is 34 a key determinant in alphavirus infectivity and dissemination through modulation of viral fusion 35 and cholesterol dependence. In addition, in studying the evolution of position 80, we identified 36 a network of glycoprotein residues, including epidemic determinants, that regulate virus 37 dissemination and infectivity. These studies underscore the importance of taking evolution-38 based approaches to not only identify key viral determinants driving arbovirus transmission 39 and pathogenesis but also to uncover fundamental aspects of arbovirus biology. 40 41 42 43 fusion, viral dissemination. 44 45 46 47 48 49 50 51 52 53 54 55 56the mature virion, E1 and the attachment protein E2 are arranged to form 80 trimeric spikes 87 constituted of trimers of E1-E2 heterodimers (Kielian and Rey, 2006; Sun et al., 2013). These 88 protein complexes then mediate CHIKV internalization by receptor mediated endocytosis and 89 fusion within the early endosome (Hoornweg et al., 2016), where low endosomal pH triggers 90 E1-E2 dissociation, E1 fusion loop insertion into the target membrane, and concomitant 91 membrane fusion (Kielian and Rey, 2006; van Duijl-Richter et al., 2015). 92The CHIKV glycoproteins play a significant role in CHIKV transmission, emergence, 93 and spread. In particular, an adaptive mutation in the CHIKV E1 glycoprotein, A226V, gave 94 rise to the Indian Ocean Linage (IOL) of CHIKV and represents one of the most emblematic 95 examples of the role of viral glycoproteins in transmission and adaptation (Schuffenecker et 96 al. , 2006). The emergence of the E1-A226V mutation was found to increase transmission by 97 Ae. albopictus mosquitoes conferring a selective advantage over the vector Ae. aegypti 98 (Tsetsarkin et al., 2007; Vazeille et al., 2007). As a consequence, this mutation allowed for 99 rapid spread from the Indian Ocean, to India, Italy and eventually France, due to its ability to 100 infect and to be transmitted by the widespread Ae. albopictus mosquito (Vignuzzi and Higgs, 101 2017). Since then, CHIKV has continued to evolve, accumulating mutations in the viral 102 attachment glycoprotein E2 (e.g., L210Q, K252Q), which further increased CHIKV fitness in 103 Ae. albopictus (Tsetsarkin et al., 2014; Tsetsarkin and Weaver, 2011). This continuous step-104 wise evolution of CHIKV again highlights the viral glycoproteins as key determinants of 105 arbovirus transmission and infectivity. 106In a previous study, we took an in vivo evolution-based approach to study novel and 107 emerging viral determinants of CHIKV transmission and path...

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.

Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets. Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and a versatile reagent and genetic toolbox. However, adult mice do not transmit SARS-CoV-2. Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2) and omicron (B.1.1.529). We found that an index shedding threshold is a key determinant for viral transmissibility. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in delayed and reduced transmission. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing for the first time a role for an accessory protein this context. We now have a tractable small animal model to help us decipher and counteract some of the most decisive aspects of SARS-CoV-2 continued spread in the human population.

Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets. Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not robustly transmit SARS-CoV-2. Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron BA.1 and Omicron BQ.1.1. We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission in our model. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing a role for an accessory protein in this context.

Chikungunya virus (CHIKV) infection has been associated with severe cardiac manifestations, yet, how CHIKV infection leads to heart disease remains unknown. Here, we leveraged both mouse models and human primary cardiac cells to define the mechanisms of CHIKV heart infection. Using an immunocompetent mouse model of CHIKV infection as well as human primary cardiac cells, we demonstrate that CHIKV directly infects and actively replicates in cardiac fibroblasts. In immunocompetent mice, CHIKV is cleared from cardiac tissue without significant damage through the induction of a local type I interferon response from both infected and non-infected cardiac cells. Using mice deficient in major innate immunity signaling components, we found that signaling through the mitochondrial antiviral-signaling protein (MAVS) is required for viral clearance from the heart. In the absence of MAVS signaling, persistent infection leads to focal myocarditis and vasculitis of the large vessels attached to the base of the heart. Large vessel vasculitis was observed for up to 60 days post infection, suggesting CHIKV can lead to vascular inflammation and potential long-lasting cardiovascular complications. This study provides a model of CHIKV cardiac infection and mechanistic insight into CHIKV-induced heart disease, underscoring the importance of monitoring cardiac function in patients with CHIKV infections.