Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike glycoprotein that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera , including the nine human coronaviruses, through recognition of a conserved motif that includes the S2´ site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice expressing human ACE2 against infection when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae , including SARS-CoV-2 variants.

Arthropod-borne flaviviruses are human pathogens of global medical importance, against which no effective small molecule-based antiviral therapy has currently been reported. Arbidol (umifenovir) is a broad-spectrum antiviral compound approved in Russia and China for prophylaxis and treatment of influenza. This compound shows activities against numerous DNA and RNA viruses. The mode of action is based predominantly on impairment of critical steps in virus-cell interactions. Here we demonstrate that arbidol possesses micromolar-level anti-viral effects (EC50 values ranging from 10.57 ± 0.74 to 19.16 ± 0.29 µM) in Vero cells infected with Zika virus, West Nile virus, and tick-borne encephalitis virus, three medically important representatives of the arthropod-borne flaviviruses. Interestingly, no antiviral effects of arbidol are observed in virus infected porcine stable kidney cells (PS), human neuroblastoma cells (UKF-NB-4), and human hepatoma cells (Huh-7 cells) indicating that the antiviral effect of arbidol is strongly cell-type dependent. Arbidol shows increasing cytotoxicity when tested in various cell lines, in the order: Huh-7 < HBCA < PS < UKF-NB-4 < Vero with CC50 values ranging from 18.69 ± 0.1 to 89.72 ± 0.19 µM. Antiviral activities and acceptable cytotoxicity profiles suggest that arbidol could be a promising candidate for further investigation as a potential therapeutic agent in selective treatment of flaviviral infections.

BackgroundThe recent Zika virus (ZIKV) outbreak has linked ZIKV with microcephaly and other central nervous system pathologies in humans. Astrocytes are among the first cells to respond to ZIKV infection in the brain and are also targets for virus infection. In this study, we investigated the interaction between ZIKV and primary human brain cortical astrocytes (HBCA).ResultsHBCAs were highly sensitive to representatives of both Asian and African ZIKV lineages and produced high viral yields. The infection was associated with limited immune cytokine/chemokine response activation; the highest increase of expression, following infection, was seen in CXCL-10 (IP-10), interleukin-6, 8, 12, and CCL5 (RANTES). Ultrastructural changes in the ZIKV-infected HBCA were characterized by electron tomography (ET). ET reconstructions elucidated high-resolution 3D images of the proliferating and extensively rearranged endoplasmic reticulum (ER) containing viral particles and virus-induced vesicles, tightly juxtaposed to collapsed ER cisternae.ConclusionsThe results confirm that human astrocytes are sensitive to ZIKV infection and could be a source of proinflammatory cytokines in the ZIKV-infected brain tissue.

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BackgroundTick-borne encephalitis (TBE) is a severe neuropathological disorder caused by tick-borne encephalitis virus (TBEV). Brain TBEV infection is characterized by extensive pathological neuroinflammation. The mechanism by which TBEV causes CNS destruction remains unclear, but growing evidence suggests that it involves both direct neuronal damage by the virus infection and indirect damage caused by the immune response. Here, we aimed to examine the TBEV-infection-induced innate immune response in mice and in human neural cells. We also compared cytokine/chemokine communication between naïve and infected neuronal cells and astrocytes.MethodsWe used a multiplexed Luminex system to measure multiple cytokines/chemokines and growth factors in mouse serum samples and brain tissue, and in human neuroblastoma cells (SK-N-SH) and primary cortical astrocytes (HBCA), which were infected with the highly pathogenic TBEV strain Hypr. We also investigated changes in cytokine/chemokine production in naïve HBCA cells treated with virus-free supernatants from TBEV-infected SK-N-SH cells and in naïve SK-N-SH cells treated with virus-free supernatants from TBEV-infected HBCA cells. Additionally, a plaque assay was performed to assess how cytokine/chemokine treatment influenced viral growth following TBEV infection.ResultsTBEV-infected mice exhibited time-dependent increases in serum and brain tissue concentrations of multiple cytokines/chemokines (mainly CXCL10/IP-10, and also CXCL1, G-CSF, IL-6, and others). TBEV-infected SK-N-SH cells exhibited increased production of IL-8 and RANTES and downregulated MCP-1 and HGF. TBEV infection of HBCA cells activated production of a broad spectrum of pro-inflammatory cytokines, chemokines, and growth factors (mainly IL-6, IL-8, CXCL10, RANTES, and G-CSF) and downregulated the expression of VEGF. Treatment of SK-N-SH with supernatants from infected HBCA induced expression of a variety of chemokines and pro-inflammatory cytokines, reduced SK-N-SH mortality after TBEV infection, and decreased virus growth in these cells. Treatment of HBCA with supernatants from infected SK-N-SH had little effect on cytokine/chemokine/growth factor expression but reduced TBEV growth in these cells after infection.ConclusionsOur results indicated that both neurons and astrocytes are potential sources of pro-inflammatory cytokines in TBEV-infected brain tissue. Infected/activated astrocytes produce cytokines/chemokines that stimulate the innate neuronal immune response, limiting virus replication, and increasing survival of infected neurons.

Vector-borne flaviviruses (VBFs) affect human health worldwide, but no approved drugs are available specifically to treat VBF-associated infections. Here, we performed in silico screening of a library of U.S. Food and Drug Administration-approved antiviral drugs for their interaction with Zika virus proteins. Twelve hit drugs were identified by the docking experiments and tested in cell-based antiviral assay systems. Efavirenz, tipranavir, and dasabuvir at micromolar concentrations were identified to inhibit all VBFs tested; i.e., two representatives of mosquito-borne flaviviruses (Zika and West Nile viruses) and one representative of flaviviruses transmitted by ticks (tick-borne encephalitis virus). The results warrant further research into these drugs, either individually or in combination, as possible pan-flavivirus inhibitors.

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Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a causative agent of the pandemic coronavirus disease 2019 (COVID-19), which has resulted in over two million deaths worldwide to date. Diphyllin and diphyllinosides are known as natural blockers of cellular vacuolar ATPases, and so can act as inhibitors of the pH-dependent fusion of viral envelopes with host cell endosomal membranes. Such pH-dependent fusion is a critical early step during the SARS-CoV-2 replication cycle. Accordingly, the anti-SARS-CoV-2 profiles and cytotoxicities of diphyllin, diphyllinoside cleistanthin B, and two structurally related compounds, helioxanthin 8-1 and helioxanthin 5-4-2, are evaluated here using in vitro cell-based assay systems. Neither helioxanthin exhibits any obvious anti-SARS-CoV-2 effects in vitro. By contrast diphyllin and cleistanthin B do exhibit anti-SARS-CoV-2 effects in Vero cells, with respective 50% effective concentrations (EC50) values of 1.92 and 6.51 µM. Diphyllin displays anti-SARS-CoV-2 effect also in colorectal adenocarcinoma (CaCo-2) cells. Moreover, when diphyllin is added at various times post infection, a significant decrease in viral titer is observed in SARS-CoV-2-infected Vero cells, even at high viral multiplicities of infection. Importantly, neither diphyllin nor cleistanthin B are found cytotoxic to Vero cells in concentrations up to 100 µM. However, the cytotoxic effect of diphyllin is more pronounced in Vero E6 and CaCo-2 cells. Overall, our data demonstrate that diphyllin and diphyllin analogues might be perfected as anti-SARS-CoV-2 agents in future preclinical studies, most especially if nanomedicine approaches may be invoked to optimize functional drug delivery to virus infected cells.