Complications arising from dengue virus infection include potentially fatal vascular leak, with severe disease being linked with excessive immune cell activation. Understanding the underlying triggers of this activation is critical for developing appropriate disease control strategies. We show here that the secreted form of the dengue virus non-structural protein NS1 is a pathogen-associated molecular pattern (PAMP). Highly purified NS1, devoid of bacterial endotoxin activity, directly activated mouse macrophages and human peripheral blood mononuclear cells (PBMCs) via toll-like receptor 4 (TLR4) leading to the induction and release of proinflammatory cytokines and chemokines. In an in vitro model of vascular leak, treatment with NS1 alone resulted in the disruption of endothelial cell monolayer integrity. Both NS1-mediated activation of PBMCs and NS1-induced vascular leak in vitro were inhibited by a TLR4 antagonist and by anti-TLR4 antibody treatment. The importance of TLR4 activation in vivo was confirmed by the reduction of capillary leak by a TLR4 antagonist in a mouse model of dengue virus infection. The results point to NS1 being a viral toxin counterpart of bacterial endotoxin. Similar to the role of LPS in septic shock, NS1 may contribute to vascular leak in dengue patients with TLR4 antagonists a therapeutic option in dengue disease.3

Flaviviruses such as dengue, yellow fever, Zika, West Nile, and Japanese encephalitis virus present substantial global health burdens. New vaccines are being sought to address safety and manufacturing issues associated with current live attenuated vaccines. Here, we describe a new insect-specific flavivirus, Binjari virus, which was found to be remarkably tolerant for exchange of its structural protein genes (prME) with those of the aforementioned pathogenic vertebrate-infecting flaviviruses (VIFs). Chimeric BinJ/VIF-prME viruses remained replication defective in vertebrate cells but replicated with high efficiency in mosquito cells. Cryo–electron microscopy and monoclonal antibody binding studies illustrated that the chimeric BinJ/VIF-prME virus particles were structurally and immunologically similar to their parental VIFs. Pilot manufacturing in C6/36 cells suggests that high yields can be reached up to 109.5 cell culture infectious dose/ml or ≈7 mg/liter. BinJ/VIF-prME viruses showed utility in diagnostic (microsphere immunoassays and ELISAs using panels of human and equine sera) and vaccine applications (illustrating protection against Zika virus challenge in murine IFNAR−/− mouse models). BinJ/VIF-prME viruses thus represent a versatile, noninfectious (for vertebrate cells), high-yield technology for generating chimeric flavivirus particles with low biocontainment requirements.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.

A protein kinase phosphorylation site in chicken gizzard myosin light chain kinase (MLCK) has been identified, and a synthetic peptide analogue of this site has been shown to be a high-affinity calmodulin binding peptide as well as a substrate for cyclic AMP dependent protein kinase. Phosphorylation of the site in MLCK is diminished when reactions are done in the presence of calmodulin. A fragment of MLCK containing the phosphorylation site was shown to have the amino acid sequence Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg-Ala-Ile-Gly-Arg-Leu- Ser-Ser. The interaction of calmodulin with a synthetic peptide based on this sequence was characterized by using circular dichroism and fluorescence spectroscopies and inhibition of calmodulin activation of MLCK. The peptide-calmodulin complex had an estimated dissociation constant in the range of 1 nM, underwent spectroscopic changes in the presence of calmodulin consistent with the induction of an alpha-helical structure, and interacted with calmodulin with an apparent 1:1 stoichiometry. Studies with other synthetic peptide analogues indicated that the phosphorylation of the serine residues diminished the ability of the peptide to interact with calmodulin even though the serines are not required for calmodulin binding. On the basis of the primary and secondary structural characteristics of these peptide analogues, a potential calmodulin binding region in another calmodulin binding protein, the gamma subunit of rabbit skeletal muscle phosphorylase kinase, was identified.(ABSTRACT TRUNCATED AT 250 WORDS)

The hallmarks of COVID-19 are higher pathogenicity and mortality in the elderly compared to children. Examining baseline SARS-CoV-2 cross-reactive immunological responses, induced by circulating human coronaviruses (hCoVs), is needed to understand such divergent clinical outcomes. Here we show analysis of coronavirus antibody responses of pre-pandemic healthy children (n = 89), adults (n = 98), elderly (n = 57), and COVID-19 patients (n = 50) by systems serology. Moderate levels of cross-reactive, but non-neutralizing, SARS-CoV-2 antibodies are detected in pre-pandemic healthy individuals. SARS-CoV-2 antigen-specific Fcγ receptor binding accurately distinguishes COVID-19 patients from healthy individuals, suggesting that SARS-CoV-2 infection induces qualitative changes to antibody Fc, enhancing Fcγ receptor engagement. Higher cross-reactive SARS-CoV-2 IgA and IgG are observed in healthy elderly, while healthy children display elevated SARS-CoV-2 IgM, suggesting that children have fewer hCoV exposures, resulting in less-experienced but more polyreactive humoral immunity. Age-dependent analysis of COVID-19 patients, confirms elevated class-switched antibodies in elderly, while children have stronger Fc responses which we demonstrate are functionally different. These insights will inform COVID-19 vaccination strategies, improved serological diagnostics and therapeutics.