The interplay between the airway epithelium and tissue macrophages during the SARS-CoV-2 infection.

The first line of antiviral immune response in the lungs is secured by the innate immunity. Several cell types take part in this process, but airway macrophages (AMs) are among the most relevant ones. The AMs can phagocyte infected cells and activate the immune response through antigen presentation and cytokine release. However, the precise role of macrophages in the course of SARS-CoV-2 infection is still largely unknown. In this study, we aimed to evaluate the role of AMs during the SARS-CoV-2 infection using a co-culture of fully differentiated primary human airway epithelium (HAE) and human monocyte-derived macrophages (hMDMs). Our results confirmed abortive SARS-CoV-2 infection in hMDMs, and their inability to transfer the virus to epithelial cells. However, we demonstrated a striking delay in viral replication in the HAEs when hMDMs were added apically after the epithelial infection, but not when added before the inoculation or on the basolateral side of the culture. Moreover, SARS-CoV-2 inhibition by hMDMs seems to be driven by cell-to-cell contact and not by cytokine production. Together, our results show, for the first time, that the recruitment of macrophages may play an important role during the SARS-CoV-2 infection, limiting the virus replication and its spread.

SARS-CoV-2 infects an in vitro model of the human developing pancreas through endocytosis.

Recent studies showed that SARS-CoV-2 can infect adult human pancreas and trigger pancreatic damage. Here, using human fetal pancreas samples and 3D differentiation of human pluripotent cells into pancreatic endocrine cells, we determined that SARS-CoV-2 receptors ACE2, TMPRSS2, and NRP1 are expressed in precursors of insulin-producing pancreatic ß-cells, rendering them permissive to SARS-CoV-2 infection. We also show that SARS-CoV-2 enters and undergoes efficient replication in human multipotent pancreatic and endocrine progenitors in vitro. Moreover, we investigated mechanisms by which SARS-CoV-2 enters pancreatic cells, and found that ACE2 mediates the entry, while NRP1 and TMPRSS2 do not. Surprisingly, we found that in pancreatic progenitors, SARS-CoV-2 enters cells via cathepsin-dependent endocytosis, which is a different route than in respiratory tract. Therefore, pancreatic spheroids might serve as a model to study candidate drugs for endocytosis-mediated viral entry inhibition and to investigate whether SARS-CoV-2 infection may affect pancreas development, possibly causing lifelong health consequences.

Pseudanabaena galeata CCNP1313-Biological Activity and Peptides Production.

Even cyanobacteria from ecosystems of low biodiversity, such as the Baltic Sea, can constitute a rich source of bioactive metabolites. Potent toxins, enzyme inhibitors, and anticancer and antifungal agents were detected in both bloom-forming species and less commonly occurring cyanobacteria. In previous work on the Baltic Pseudanabaena galeata CCNP1313, the induction of apoptosis in the breast cancer cell line MCF-7 was documented. Here, the activity of the strain was further explored using human dermal fibroblasts, African green monkey kidney, cancer cell lines (T47D, HCT-8, and A549ACE2/TMPRSS2) and viruses (SARS-CoV-2, HCoV-OC43, and WNV). In the tests, extracts, chromatographic fractions, and the main components of the P. galeata CCNP1313 fractions were used. The LC-MS/MS analyses of the tested samples led to the detection of forty-five peptides. For fourteen of the new peptides, putative structures were proposed based on MS/MS spectra. Although the complex samples (i.e., extracts and chromatographic fractions) showed potent cytotoxic and antiviral activities, the effects of the isolated compounds were minor. The study confirmed the significance of P. galeata CCNP1313 as a source of metabolites with potent activity. It also illustrated the difficulties in assigning the observed biological effects to specific metabolites, especially when they are produced in minute amounts.

SARS-CoV-2 inhibition using a mucoadhesive, amphiphilic chitosan that may serve as an anti-viral nasal spray.

There are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of - 3 to - 4 at a concentration of 10-100 µg/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 µg/ mL (p < 0.05 compared to untreated controls). In vivo studies using transgenic mice expressing the ACE-2 receptor, dosed nasally with SARS-COV-2 (426,000 TCID50/mL) showed a trend for nasal GCPQ (20 mg/kg) to inhibit viral load in the respiratory tract and brain, although the study was not powered to detect statistical significance. GCPQ's electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 h after nasal dosing. With a no observed adverse effect level of 18 mg/kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.

The SARS-CoV-2 ORF10 is not essential in vitro or in vivo in humans.

SARS-CoV-2 genome annotation revealed the presence of 10 open reading frames (ORFs), of which the last one (ORF10) is positioned downstream of the N gene. It is a hypothetical gene, which was speculated to encode a 38 aa protein. This hypothetical protein does not share sequence similarity with any other known protein and cannot be associated with a function. While the role of this ORF10 was proposed, there is growing evidence showing that the ORF10 is not a coding region. Here, we identified SARS-CoV-2 variants in which the ORF10 gene was prematurely terminated. The disease was not attenuated, and the transmissibility between humans was maintained. Also, in vitro, the strains replicated similarly to the related viruses with the intact ORF10. Altogether, based on clinical observation and laboratory analyses, it appears that the ORF10 protein is not essential in humans. This observation further proves that the ORF10 should not be treated as the protein-coding gene, and the genome annotations should be amended.

Visualizing Coronavirus Entry into Cells.

Coronavirus entry encompasses the initial steps of infection, from virion attachment to genome release. Advances in fluorescent labeling of viral and cellular components and confocal imaging enable broad spectrum studies on this process. Here, we describe methods for visualization of coronavirus entry into immortalized cell lines and 3D tissue culture models.

HTCC as a Polymeric Inhibitor of SARS-CoV-2 and MERS-CoV.

Among seven coronaviruses that infect humans, three (severe acute respiratory syndrome coronavirus [SARS-CoV], Middle East respiratory syndrome coronavirus [MERS-CoV], and the newly identified severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) are associated with a severe, life-threatening respiratory infection and multiorgan failure. We previously proposed that the cationically modified chitosan N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC) is a potent inhibitor of human coronavirus NL63 (HCoV-NL63). Next, we demonstrated the broad-spectrum antiviral activity of the compound, as it inhibited all low-pathogenicity human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1). Here, using in vitro and ex vivo models of human airway epithelia, we show that HTCC effectively blocks MERS-CoV and SARS-CoV-2 infection. We also confirmed the mechanism of action for these two viruses, showing that the polymer blocks the virus entry into the host cell by interaction with the S protein.IMPORTANCE The beginning of 2020 brought us information about the novel coronavirus emerging in China. Rapid research resulted in the characterization of the pathogen, which appeared to be a member of the SARS-like cluster, commonly seen in bats. Despite the global and local efforts, the virus escaped the health care measures and rapidly spread in China and later globally, officially causing a pandemic and global crisis in March 2020. At present, different scenarios are being written to contain the virus, but the development of novel anticoronavirals for all highly pathogenic coronaviruses remains the major challenge. Here, we describe the antiviral activity of an HTCC compound, previously developed by us, which may be used as a potential inhibitor of currently circulating highly pathogenic coronaviruses-SARS-CoV-2 and MERS-CoV.

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Replication of SARS-CoV-2 in human respiratory epithelium

Currently, there are four seasonal coronaviruses associated with relatively mild respiratory tract disease in humans. However, there are also a plethora of animal coronaviruses, which have the potential to cross the species border. This regularly results in the emergence of new viruses in humans. In 2002 SARS-CoV emerged, to rapidly disappear in May 2003. In 2012 MERS-CoV was identified as a possible threat to humans, but its pandemic potential so far is minimal, as the human-to-human transmission is ineffective. The end of 2019 brought us information about the SARS-CoV-2 emergence, and the virus rapidly spread in 2020 causing an unprecedented pandemic. At present, the studies on the virus are carried out using a surrogate system based on the immortalized simian Vero E6 cell line. This model is convenient for diagnostics, but it has serious limitations and does not allow for the understanding of virus biology and evolution. Here we show that fully differentiated human airway epithelium cultures constitute an excellent model to study the infection with the novel human coronavirus SARS-CoV-2. We observed an efficient replication of the virus in the tissue, with the maximal replication at 2 days post-infection. The virus replicated in ciliated cells and was released apically. IMPORTANCE SARS-CoV-2 emerged by the end of 2019 to rapidly spread in 2020. At present, it is of utmost importance to understand the virus biology and to rapidly assess the potential of existing drugs and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in the Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on the SARS-CoV-2.