Humoral cross-reactivity towards SARS-CoV-2 in young children with acute respiratory infection with low-pathogenicity coronaviruses.

Background: SARS-CoV-2 infection in children frequently leads to only asymptomatic and mild infections. It has been suggested that frequent infections due to low-pathogenicity coronaviruses in children, impart immunity against SARS-CoV-2 in this age group. Methods: From a prospective birth cohort study prior to the pandemic, we identified children with proven low-pathogenicity coronavirus infections. Convalescent sera from these children were tested for antibodies against respective seasonal coronaviruses (OC43, NL63, and 229E) and SARS-CoV-2 by immunofluorescence and virus microneutralization assay respectively. Results: Forty-two children with proven seasonal coronavirus infection were included. Convalescent sera from these samples demonstrated antibodies against the respective seasonal coronaviruses. Of these, 40 serum samples showed no significant neutralization of SARS-CoV-2, while 2 samples showed inconclusive results. Conclusion: These findings suggest that the antibodies generated in low-pathogenicity coronavirus infections offer no protection from SARS-CoV-2 infection in young children.

Resolution of viral load in mild COVID-19 patients is associated with both innate and adaptive immune responses.

Over 90% of the COVID-19 patients manifest mild/moderate symptoms or are asymptomatic. Although comorbidities and dysregulation of immune response have been implicated in severe COVID-19, the host factors that associate with asymptomatic or mild infections have not been characterized. We have collected serial samples from 23 hospitalized COVID-19 patients with mild symptoms and measured the kinetics of SARS-CoV-2 viral load in respiratory samples and markers of inflammation in serum samples. We monitored seroconversion during the acute phase of illness and quantitated the amount of total IgG against the receptor-binding domain of SARS-CoV-2 and estimated the virus neutralization potential of these antibodies. Viral load decreased by day 8 in all the patients but the detection of viral RNA in saliva samples did not correlate well with viral RNA detection in nasopharyngeal/oropharyngeal swab samples. 25% of the virus-positive patients had no detectable neutralizing antibodies in the serum and in other cases, the efficiency of antibodies to neutralize SARS-CoV-2 B1.1.7 strain was lower as compared to the circulating virus isolate. Decrease in viral load coincided with increase in neutralizing antibodies and interferon levels in serum. Most patients showed no increase in inflammatory cytokines such as IL-1ß or IL-6, however, elevated levels of IL-7 and other inflammatory mediators such as TNF-α and IL-8 was observed. These data suggest that most mild infections are associated with absence of inflammation coupled with an active innate immune response, T-cell activation and neutralizing antibodies.

Humoral cross-reactivity towards SARS-CoV-2 in young children with acute respiratory infection with low-pathogenicity coronaviruses

SARS-CoV-2 infection in children frequently leads to only asymptomatic and mild infections. It has been suggested that frequent infections due to low-pathogenicity coronaviruses in children, imparts immunity against SARS-CoV-2 in this age group. From a prospective birth cohort study prior to the pandemic, we identified children (n=42) with proven low-pathogenicity coronavirus infections. Convalescent sera from these samples had antibodies against the respective seasonal CoVs as demonstrated by immunofluorescence assay. We tested these samples for neutralization of SARS-CoV-2 using virus microneutralization assay. Forty serum samples showed no significant neutralization of SARS-CoV-2, while 2 samples showed inconclusive results. These findings suggest that the antibodies generated in low-pathogenicity coronavirus infections offer no protection from SARS-CoV-2 infection in young children.

Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein Based Novel Epitopes Induce Potent Immune Responses in vivo and Inhibit Viral Replication in vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) initiates infection by attachment of the surface-exposed spike glycoprotein to the host cell receptors. The spike glycoprotein (S) is a promising target for inducing immune responses and providing protection; thus the ongoing efforts for the SARS-CoV-2 vaccine and therapeutic developments are mostly spiraling around S glycoprotein. The matured functional spike glycoprotein is presented on the virion surface as trimers, which contain two subunits, such as S1 (virus attachment) and S2 (virus fusion). The S1 subunit harbors the N-terminal domain (NTD) and the receptor-binding domain (RBD). The RBD is responsible for binding to host-cellular receptor angiotensin-converting enzyme 2 (ACE2). The NTD and RBD of S1, and the S2 of S glycoprotein are the major structural moieties to design and develop spike-based vaccine candidates and therapeutics. Here, we have identified three novel epitopes (20-amino acid peptides) in the regions NTD, RBD, and S2 domains, respectively, by structural and immunoinformatic analysis. We have shown as a proof of principle in the murine model, the potential role of these novel epitopes in-inducing humoral and cellular immune responses. Further analysis has shown that RBD and S2 directed epitopes were able to efficiently inhibit the replication of SARS-CoV-2 wild-type virus in vitro suggesting their role as virus entry inhibitors. Structural analysis revealed that S2-epitope is a part of the heptad repeat 2 (HR2) domain which might have plausible inhibitory effects on virus fusion. Taken together, this study discovered novel epitopes that might have important implications in the development of potential SARS-CoV-2 spike-based vaccine and therapeutics.

Oxidative stress specifically inhibits replication of dengue virus.

Reactive oxygen species (ROS) are chemically active species which are involved in maintaining cellular and signalling processes at physiological concentrations. Therefore, cellular components that regulate redox balance are likely to play a crucial role in viral life-cycle either as promoters of viral replication or with antiviral functions. Zinc is an essential micronutrient associated with anti-oxidative systems and helps in maintaining a balanced cellular redox state. Here, we show that zinc chelation leads to induction of reactive oxygen species (ROS) in epithelial cells and addition of zinc restores ROS levels to basal state. Addition of ROS (H2O2) inhibited dengue virus (DENV) infection in a dose-dependent manner indicating that oxidative stress has adverse effects on DENV infection. ROS affects early stages of DENV replication as observed by quantitation of positive and negative strand viral RNA. We observed that addition of ROS specifically affected viral titres of positive strand RNA viruses. We further demonstrate that ROS specifically altered SEC31A expression at the ER suggesting a role for SEC31A-mediated pathways in the life-cycle of positive strand RNA viruses and provides an opportunity to identify drug targets regulating oxidative stress responses for antiviral development.

Kinetics of viral load, immunological mediators and characterization 1 of a SARS-CoV-2 isolate in mild COVID-19 patients during acute phase of infection

Over 95% of the COVID-19 cases are mild-to-asymptomatic who contribute to disease transmission whereas most of the severe manifestations of the disease are observed in elderly and in patients with comorbidities and dysregulation of immune response has been implicated in severe clinical outcomes. However, it is unclear whether asymptomatic or mild infections are due to low viral load or lack of inflammation. We have measured the kinetics of SARS-CoV-2 viral load in the respiratory samples and serum markers of inflammation in hospitalized COVID-19 patients with mild symptoms. We observed a bi-phasic pattern of virus load which was eventually cleared in most patients at the time of discharge. Viral load in saliva samples from a subset of patients showed good correlation with nasopharyngeal samples. Serum interferon levels were downregulated during early stages of infection but peaked at later stages correlating with elevated levels of T-cell cytokines and other inflammatory mediators such as IL-6 and TNF- which showed a bi-phasic pattern. The clinical recovery of patients correlated with decrease in viral load and increase in interferons and other cytokines which indicates an effective innate and adaptive immune function in mild infections. We further characterized one of the SARS-CoV-2 isolate by plaque purification and show that infection of lung epithelial cells (Calu-3) with this isolate led to cytopathic effect disrupting epithelial barrier function and tight junctions. Finally we showed that zinc was capable of inhibiting SARS-CoV-2 infection in this model suggesting a beneficial effect of zinc supplementation in COVID-19 infection. IMPORTANCEA majority of COVID-19 patients are asymptomatic or exhibit mild symptoms despite high viral loads suggesting a key role for the acute phase innate immune response in limiting the damage and clearing the virus. Therefore, it is important to understand the early phase response to SARS-CoV-2 infection in such patients to devise strategies for clinical management of the disease. Our study shows the kinetics of immune mediators in the serum samples collected from hospitalized COVID-19 patients with mild symptoms. We further characterize a virus isolate from one of these patients and demonstrate its effect on epithelial barrier functions and show that zinc was capable of inhibiting SARS-CoV-2 infection under these conditions. Our results suggest a key role for the innate immune responses in the early phase of infection in mitigating clinical symptoms, clearing the virus and recovery from illness and suggest an antiviral role for zinc in COVID-19 infection.

Respiratory Syncytial Virus-Induced Oxidative Stress Leads to an Increase in Labile Zinc Pools in Lung Epithelial Cells.

Zinc supplementation in cell culture has been shown to inhibit various viruses, like herpes simplex virus, rotavirus, severe acute respiratory syndrome (SARS) coronavirus, rhinovirus, and respiratory syncytial virus (RSV). However, whether zinc plays a direct antiviral role in viral infections and whether viruses have adopted strategies to modulate zinc homeostasis have not been investigated. Results from clinical trials of zinc supplementation in infections indicate that zinc supplementation may be beneficial in a pathogen- or disease-specific manner, further underscoring the importance of understanding the interaction between zinc homeostasis and virus infections at the molecular level. We investigated the effect of RSV infection on zinc homeostasis and show that RSV infection in lung epithelial cells leads to modulation of zinc homeostasis. The intracellular labile zinc pool increases upon RSV infection in a multiplicity of infection (MOI)-dependent fashion. Small interfering RNA (siRNA)-mediated knockdown of the ubiquitous zinc uptake transporter ZIP1 suggests that labile zinc levels are increased due to the increased uptake by RSV-infected cells as an antiviral response. Adding zinc to culture medium after RSV infection led to significant inhibition of RSV titers, whereas depletion of zinc by a zinc chelator, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) led to an increase in RSV titers. The inhibitory effect of zinc was specific, as other divalent cations had no effect on RSV titers. Both RSV infection and zinc chelation by TPEN led to reactive oxygen species (ROS) induction, whereas addition of zinc blocked ROS induction. These results suggest a molecular link between RSV infection, zinc homeostasis, and oxidative-stress pathways and provide new insights for developing strategies to counter RSV infection.IMPORTANCE Zinc deficiency rates in developing countries range from 20 to 30%, and zinc supplementation trials have been shown to correct clinical manifestations attributed to zinc deficiency, but the outcomes in the case of respiratory infections have been inconsistent. We aimed at understanding the role of zinc homeostasis in respiratory syncytial virus (RSV) infection. Infection of lung epithelial cell lines or primary small-airway epithelial cells led to an increase in labile zinc pools, which was due to increased uptake of zinc. Zinc supplementation inhibited RSV replication, whereas zinc chelation had an opposing effect, leading to increases in RSV titers. Increases in labile zinc in RSV-infected cells coincided with induction of reactive oxygen species (ROS). Both zinc depletion and addition of exogenous ROS led to enhanced RSV infection, whereas addition of the antioxidant inhibited RSV, suggesting that zinc is part of an interplay between RSV-induced oxidative stress and the host response to maintain redox balance.

Zinc Chelation Specifically Inhibits Early Stages of Dengue Virus Replication by Activation of NF-κB and Induction of Antiviral Response in Epithelial Cells.

Zinc is an essential micronutrient which regulates diverse physiological functions and has been shown to play a crucial role in viral infections. Zinc has a necessary role in the replication of many viruses, however, antiviral action of zinc has also been demonstrated in in vitro infection models most likely through induction of host antiviral responses. Therefore, depending on the host machinery that the virus employs at different stages of infection, zinc may either facilitate, or inhibit virus infection. In this study, we show that zinc plays divergent roles in rotavirus and dengue virus infections in epithelial cells. Dengue virus infection did not perturb the epithelial barrier functions despite the release of virus from the basolateral surface whereas rotavirus infection led to disruption of epithelial junctions. In rotavirus infection, zinc supplementation post-infection did not block barrier disruption suggesting that zinc does not affect rotavirus life-cycle or protects epithelial barriers post-infection suggesting the involvement of cellular pathways in the beneficial effect of zinc supplementation in enteric infections. Zinc depletion by N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) inhibited dengue virus and Japanese encephalitis virus (JEV) infection but had no effect on rotavirus. Time-of-addition experiments suggested that zinc chelation affected both early and late stages of dengue virus infectious cycle and zinc chelation abrogated dengue virus RNA replication. We show that transient zinc chelation induces ER stress and antiviral response by activating NF-kappaB leading to induction of interferon signaling. These results suggest that modulation of zinc homeostasis during virus infection could be a component of host antiviral response and altering zinc homeostasis may act as a potent antiviral strategy against flaviviruses.

Diffusion of Carbon Dioxide and Nitrogen in the Small-Pore Titanium Bis(phosphonate) Metal-Organic Framework MIL-91 (Ti): A Combination of Quasielastic Neutron Scattering Measurements and Molecular Dynamics Simulations.

The diffusivity of CO2 and N2 in the small-pore titanium-based bis(phosphonate) metal-organic framework MIL-91(Ti) was explored by using a combination of quasielastic neutron scattering measurements and molecular dynamics simulations. These two techniques were used to determine the loading dependence of the self-diffusivity, corrected and transport diffusivities of these two gases to complement our previously reported thermodynamics study, which revealed that this material was a promising candidate for CO2 /N2 separation. The calculated and measured diffusivities of both gases were shown to be of an order of magnitude sufficiently high, from 10-9 to 10-10  m2 s-1 , and N2 diffused faster than CO2 through the small channel of MIL-91(Ti). Consequently, the separation process does not involve any kinetic-driven limitations. This study further revealed that the global diffusion mechanism involves motions of gases along the channels by a jump sequence, and the residence times for CO2 in the region close to the specific PO⋅⋅⋅H⋅⋅⋅N zwitterionic sites are much higher than those for N2 , which explains the faster diffusivity observed for N2 .

Identification and characterization of the role of c-terminal Src kinase in dengue virus replication.

We screened a siRNA library targeting human tyrosine kinases in Huh-7 cells and identified c-terminal Src kinase (Csk) as one of the kinases involved in dengue virus replication. Knock-down of Csk expression by siRNAs or inhibition of Csk by an inhibitor reduced dengue virus RNA levels but did not affect viral entry. Csk partially colocalized with viral replication compartments. Dengue infection was drastically reduced in cells lacking the three ubiquitous src family kinases, Src, Fyn and Yes. Csk knock-down in these cells failed to block dengue virus replication suggesting that the effect of Csk is via regulation of Src family kinases. Csk was found to be hyper-phosphorylated during dengue infection and inhibition of protein kinase A led to a block in Csk phosphorylation and dengue virus replication. Overexpression studies suggest an important role for the kinase and SH3 domains in this process. Our results identified a novel role for Csk as a host tyrosine kinase involved in dengue virus replication and provide further insights into the role of host factors in dengue replication.