Novel coronavirus-like particles targeting cells lining the respiratory tract

<div><p>Virus like particles (VLPs) produced by the expression of viral structural proteins can serve as versatile nanovectors or potential vaccine candidates. In this study we describe for the first time the generation of HCoV-NL63 VLPs using baculovirus system. Major structural proteins of HCoV-NL63 have been expressed in tagged or native form, and their assembly to form VLPs was evaluated. Additionally, a novel procedure for chromatography purification of HCoV-NL63 VLPs was developed. Interestingly, we show that these nanoparticles may deliver cargo and selectively transduce cells expressing the ACE2 protein such as ciliated cells of the respiratory tract. Production of a specific delivery vector is a major challenge for research concerning targeting molecules. The obtained results show that HCoV-NL63 VLPs may be efficiently produced, purified, modified and serve as a delivery platform. This study constitutes an important basis for further development of a promising viral vector displaying narrow tissue tropism.</p></div

APOBEC3-mediated restriction of RNA virus replication

Abstract APOBEC3 family members are cytidine deaminases with roles in intrinsic responses to infection by retroviruses and retrotransposons, and in the control of other DNA viruses, such as herpesviruses, parvoviruses and hepatitis B virus. Although effects of APOBEC3 members on viral DNA have been demonstrated, it is not known whether they edit RNA genomes through cytidine deamination. Here, we investigated APOBEC3-mediated restriction of Coronaviridae. In experiments in vitro, three human APOBEC3 proteins (A3C, A3F and A3H) inhibited HCoV-NL63 infection and limited production of progeny virus, but did not cause hypermutation of the coronaviral genome. APOBEC3-mediated restriction was partially dependent on enzyme activity, and was reduced by the use of enzymatically inactive APOBEC3. Moreover, APOBEC3 proteins bound to the coronaviral nucleoprotein, and this interaction also affected viral replication. Although the precise molecular mechanism of deaminase-dependent inhibition of coronavirus replication remains elusive, our results further our understanding of APOBEC-mediated restriction of RNA virus infections

Staphylococcus aureus proteases degrade lung surfactant protein a potentially impairing innate immunity of the lung

The pulmonary surfactant is a complex mixture of lipids and proteins that is important for respiratory lung functions, which also provides first line of innate immune defense. Pulmonary surfactant protein-A (SP-A) is a major surfactant component with immune functions that importance during S. aureus infections was demonstrated in numerous studies. The current study showed that S. aureus can efficiently cleave the SP-A protein using its arsenal of proteolytic enzymes. This degradation appears to be mediated by cysteine proteases, in particular staphopain A (ScpA). The staphopain-mediated proteolysis of SP-A resulted in a decrease or complete abolishment of SP-A biological activity, including the promotion of S. aureus phagocytosis by neutrophils, aggregation of Gram-negative bacteria, and bacterial cell adherence to epithelium. Significantly, ScpA has also efficiently degraded SP-A in complete bronchi-alveolar lavage fluid (BALF) from human lungs. This indicates that staphopain activity in the lungs is resistant to protease inhibitors thus suggesting that SP-A can be cleaved in vivo. Collectively, this study showed that the S. aureus protease ScpA is an important virulence factor that may impair innate immunity of the lungs

HTCC: Broad Range Inhibitor of Coronavirus Entry.

To date, six human coronaviruses have been known, all of which are associated with respiratory infections in humans. With the exception of the highly pathogenic SARS and MERS coronaviruses, human coronaviruses (HCoV-NL63, HCoV-OC43, HCoV-229E, and HCoV-HKU1) circulate worldwide and typically cause the common cold. In most cases, infection with these viruses does not lead to severe disease, although acute infections in infants, the elderly, and immunocompromised patients may progress to severe disease requiring hospitalization. Importantly, no drugs against human coronaviruses exist, and only supportive therapy is available. Previously, we proposed the cationically modified chitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and its hydrophobically-modified derivative (HM-HTCC) as potent inhibitors of the coronavirus HCoV-NL63. Here, we show that HTCC inhibits interaction of a virus with its receptor and thus blocks the entry. Further, we demonstrate that HTCC polymers with different degrees of substitution act as effective inhibitors of all low-pathogenic human coronaviruses

Infection with human coronavirus NL63 enhances streptococcal adherence to epithelial cells

Understanding the mechanisms of augmented bacterial pathogenicity in post-viral infections is the first step in the development of an effective therapy. This study assessed the effect of human coronavirus NL63 (HCoV-NL63) on the adherence of bacterial pathogens associated with respiratory tract illnesses. It was shown that HCoV-NL63 infection resulted in an increased adherence of Streptococcus pneumoniae to virus-infected cell lines and fully differentiated primary human airway epithelium cultures. The enhanced binding of bacteria correlated with an increased expression level of the platelet-activating factor receptor (PAF-R), but detailed evaluation of the bacterium–PAF-R interaction revealed a limited relevance of this process

Novel polymeric inhibitors of HCoV-NL63

The human coronavirus NL63 is generally classified as a common cold pathogen, though the infection may also result in severe lower respiratory tract diseases, especially in children, patients with underlying disease, and elderly. It has been previously shown that HCoV-NL63 is also one of the most important causes of croup in children. In the current manuscript we developed a set of polymer-based compounds showing prominent anticoronaviral activity. Polymers have been recently considered as promising alternatives to small molecule inhibitors, due to their intrinsic antimicrobial properties and ability to serve as matrices for antimicrobial compounds. Most of the antimicrobial polymers show antibacterial properties, while those with antiviral activity are much less frequent. A cationically modified chitosan derivative, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and hydrophobically-modified HTCC were shown to be potent inhibitors of HCoV-NL63 replication. Furthermore, both compounds showed prominent activity against murine hepatitis virus, suggesting broader anticoronaviral activity

Novel polymeric inhibitors of HCoV-NL63

The human coronavirus NL63 is generally classified as a common cold pathogen, though the infection may also result in severe lower respiratory tract diseases, especially in children, patients with underlying disease, and elderly. It has been previously shown that HCoV-NL63 is also one of the most important causes of croup in children. In the current manuscript we developed a set of polymer-based compounds showing prominent anticoronaviral activity. Polymers have been recently considered as promising alter- natives to small molecule inhibitors, due to their intrinsic antimicrobial properties and ability to serve as matrices for antimicrobial compounds. Most of the antimicrobial polymers show antibacterial properties, while those with antiviral activity are much less frequent. A cationically modified chitosan derivative, N -(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and hydrophobically-modified HTCC were shown to be potent inhibitors of HCoV-NL63 replication. Furthermore, both compounds showed prominent activity against murine hepatitis virus, suggesting broader anticoronaviral activity

Primers and probes used for a quantitative RT-PCR.

<p>Primers and probes used for a quantitative RT-PCR.</p

HTCCs with different DS effectively inhibit HCoV-NL63, HCoV-229E HCoV-OC43, and HCoV-HKU1 infection.

<p>LLC-Mk2 cells were infected with HCoV-NL63 in the presence of HTCCs with DS ranging from 57% to 77% at a concentration of 200 μg/ml or control medium. Likewise, MRC-5 cells were infected with HCoV-229E and HCT-8 cells were infected with HCoV-OC43 in the presence of HTCCs with DS ranging from 57% to 77% at 200 μg/ml or control medium. HAE cultures were infected with HCoV-HKU1 in the presence of HTCCs with DS ranging from 57% to 77% (200 μg/ml) or PBS (control). <b>(A)</b> Virus replication in cell culture supernatants (cell lines) or apical lavages (HAE) was evaluated using quantitative RT-PCR. Data are presented as Log Removal Value (LRV) compared to untreated samples. The assays were performed in triplicate, and average values with standard errors are presented. * <i>P</i> < 0.05. <b>(B)</b> Cytotoxicity of HTCCs with DS ranging from 57% to 77% in cell lines and HAE cultures. Cell viability was assessed via XTT assay. Data on the y-axis represent the percentage values obtained for the untreated reference samples. All assays were performed in triplicate in at least two independent experiments, and average values with standard errors are presented. The differences in cytotoxicity of HTCCs were not statistically significant.</p