Virological and Clinical Outcomes in Adults Treated with Ribavirin Aerosol for COVID-19 Pneumonia (preprint)

The emergence of new SARSCoV-2 variants can affect vaccine efficacy, laboratory diagnosis and therapies already available, triggering interest in the search for antiviral agents for SARSCoV-2 infections. Ribavirin (RBV) is a broad-spectrum antiviral with demonstrated in vitro activity against multiple viruses, including SARSCoV-2. Methods. This retrospective study evaluated the dynamic and viral clearance of SARSCoV-2 in hospitalized adult participants (PTs) with COVID-19 pneumonia who received RBV aerosol within a compassionate use study. Was also assessed the impact of RBV on the clinical outcome and the mutational profile of SARSCoV-2. Results. The median RNA values measured in nine PTs included in the study de-creased from baseline to discharge (at BL, threshold cycle (Ct)=22.4, IQR 19.84-5.07; at discharge, Ct=27.92, IQR 26.43-36.11). Seven/9 PTs experienced a clinical improvement, while two PTs deceased during hos-pitalization. In PTs with a favorable outcome the virus clearance rate at discharge was 28.6%. The cumulative clearance rate was 71.4% within day 14th from discharge. A mutational pattern after RBV was detected in 3/5 PTs in whom was available whole genome sequencing. Conclusions. RBV may limit SARSCOV-2 replication resulting in a favorable clinical outcome. Ribavirin may also contribute to the mutational spectrum of SARSCoV-2.

Epidemiological and Clinical Features of SARS-CoV-2 Variants Circulating between April-December 2021 in Italy (preprint)

SARS-CoV-2 is constantly evolving leading to new variants. We analysed data from 4,400 SARS-CoV-2-positive samples in order to continue variant surveillance in Italy to evaluate their epidemiological and relative impact on public health in the period April-December 2021. The main circulating strain (76.2%) was Delta followed by Alpha (13.3%), Omicron (5.3%) and Gamma variants (2.9%). B.1.1 lineages, Eta, Beta, Iota, Mu and Kappa variants represented around 1% of cases. Overall, 48.2% of subjects were not vaccinated with a lower median age compared to vaccinated subjects (47 vs. 61 years). An increasing number of infections in vaccinated subjects was observed overtime, with the highest proportion in November (85.2%). Variants correlated with clinical status; the largest proportion of symptomatic patients (59.6%) was observed among Delta variant, while subjects harboring Gamma variant showed the highest proportion of asymptomatics (21.6%), albeit also of deaths (5.4%). The Omicron variant was only found in vac-cinated subjects, of which 47% were hospitalized. Diffusivity and pathogenicity associated with the different SARS-CoV-2 variants are likely to have relevant public health implications, both at national and international level. Our study pro-vides data on the rapid changes in the epidemiological landscape of SARS-CoV-2 variants in Italy.

Discovering host protein interactions specific for SARS-CoV-2 RNA genome (preprint)

SARS-CoV-2 is a positive single-stranded RNA virus that interacts with proteins of infected cells at different stages of its life cycle. These interactions are necessary for the host to recognize and block the replication of the virus. Yet, if cells fail to block SARS-CoV-2, host proteins are recruited to translate, transcribe and replicate the genetic material of the virus. To identify the host proteins that bind to SARS-CoV-2 RNA, we adopted the RNA-Protein Interaction Detection coupled to Mass Spectrometry (RaPID-MS) technology, which allows the purification and identification by MS-based proteomics of the proteins associated with a specific RNA of interest expressed in mammalian cells. We specifically investigated proteins associated with the 5' and 3' end regions of SARS-CoV-2 RNA. As associations might involve non-physical protein-RNA interactions, we defined a set of reliable protein-RNA interactions by exploiting the predictive power of the catRAPID algorithm that assesses the direct binding potential of proteins to a given RNA region. Among these specific SARS-CoV-2 RNA end interactors, we identified the pseudouridine synthase PUS7 that binds to both 5' and 3' ends of viral RNA, which harbor the canonical consensus sequence modified by PUS7. We corroborated our results through SARS-CoV-2 RNA analysis by nanopore direct RNA sequencing. Indeed, these PUS7 consensus regions were found highly modified on viral RNAs, as demonstrated by ionic current features that are significantly different compared to the unmodified in vitro transcribed RNA. Overall, our data map the specific host protein interactions of SARS-CoV-2 RNA and point to a role for cellular pseudouridine synthases and the post-transcriptional pseudouridine modifications in the viral life cycle.

Nanopore ReCappable Sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs (preprint)

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

SARS-CoV-2 Spike Affinity and Dynamics Exclude the Strict Requirement of an Intermediate Host (preprint)

SARS-CoV-2 proximal origin is still unclear, limiting the possibility of foreseeing other spillover events with pandemic potential. Here we propose an evolutionary model based on the thorough dissection of SARS-CoV-2 and RaTG13 - the closest bat relative - spike dynamics, kinetics and binding to ACE2. Our results indicate that both spikes share nearly identical, high affinities for Rhinolophus affinis bat and human ACE2, pointing out to negligible species barriers directly related to receptor binding. Also, SARS-CoV-2 spike shows a higher degree of dynamics and kinetics optimization that favors ACE2 engagement. Therefore, we devise an affinity-independent evolutionary process that likely took place in R. affinis bats and limits the eventual involvement of other animal species in initiating the pandemic to the role of vector.

Recognition and inhibition of SARS-CoV-2 by humoral innate immunity pattern recognition molecules (preprint)

Summary The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in COVID-19. The present study was designed to conduct a systematic investigation of the interaction of humoral fluid phase pattern recognition molecules (PRM) with SARS-CoV-2. Out of 10 PRM tested, the long pentraxin PTX3 and Mannose Binding Lectin (MBL) bound the viral Nucleoprotein and Spike, respectively. MBL bound trimeric Spike, including that of variants of concern, in a glycan- dependent way and inhibited SARS-CoV-2 in three in vitro models. Moreover, upon binding to Spike, MBL activated the lectin pathway of complement activation. Genetic polymorphisms at the MBL locus were associated with disease severity. These results suggest that selected humoral fluid phase PRM can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.

Circulating SARS-CoV-2 Variants in Italy, October 2020-March 2021 (preprint)

A growing number of emerging SARS-CoV-2 variants is being identified worldwide, potentially impacting the effectiveness of current vaccines. We report the data obtained in several Italian regions involved in the SARS-CoV-2 variant monitoring from the beginning of the epidemic and spanning the period from October 2020 to March 2021.

Circulating SARS-CoV-2 Variants in Italy, October 2020-March 2021 (preprint)

A growing number of emerging SARS-CoV-2 variants is being identified worldwide, potentially impacting the effectiveness of current vaccines. We report the data obtained in several Italian regions involved in the SARS-CoV-2 variant monitoring from the beginning of the epidemic and spanning the period from October 2020 to March 2021.

Differential plasmacytoid dendritic cell phenotype and type I Interferon response in asymptomatic and severe COVID-19 infection (preprint)

SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure. Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro, asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum. This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo. These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients.

A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in VeroE6 cells (preprint)

We developed a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy (Airyscan, STORM, and STED) and easily matches the spatial scale of single virus-cell checkpoints. We deployed this toolbox to characterize subtle issues related to the entry phase of SARS-CoV-2 variants in VeroE6 cells. Our results suggest that the variant of concern B.1.1.7, currently on the rise in several countries by a clear transmission advantage, in these cells outcompetes its ancestor B.1 in terms of a much faster kinetics of entry. Given the molecular scenario (entry by the late pathway and similar fraction of pre-cleaved S protein for B.1.1.7 and B.1), the faster entry of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike RBD with ACE2. Remarkably, we also observed directly the significant role of clathrin as mediator of late entry endocytosis, which had been previously suggested in analogy with other CoVs and from experiments on pseudotyped virus models. On overall, we believe that our fluroescence microscopy-based approach is valuable for future studies addressing of how SARS-CoV-2 and its variants interact with cells.

Severity of SARS-CoV-2 infection as a function of the interferon landscape across the respiratory tract of COVID-19 patients. (preprint)

The COVID-19 outbreak driven by SARS-CoV-2 has caused more than 2.5 million deaths globally, with the most severe cases characterized by over-exuberant production of immune-mediators, the nature of which is not fully understood. Interferons of the type I (IFN-I) or type III (IFN-III) families are potent antivirals, but their role in COVID-19 remains debated. Our analysis of gene and protein expression along the respiratory tract shows that IFNs, especially IFN-III, are over-represented in the lower airways of patients with severe COVID-19, while high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity; also, IFN expression varies with abundance of the cell types that produce them. Our data point to a dynamic process of inter- and intra-family production of IFNs in COVID-19, and suggest that IFNs play opposing roles at distinct anatomical sites.

Maturation signatures of conventional dendritic cells in COVID-19 reflect direct viral sensing (preprint)

Growing evidence suggests that conventional dendritic cells (cDCs) undergo aberrant maturation in COVID-19, and this adversely affects T cell activation. Here, we find that cDC2 subtypes show similar infection-induced gene signatures with an increasing gradient of expression of interferon-stimulated genes from mild to severe patients and a down-regulation of major histocompatibility complex class II (MHC class II) molecules and some inflammatory cytokines compared to the baseline level of healthy donors. In vitro, the direct exposure of cDC2s to the virus recapitulates the type of activation observed in vivo. Our findings provide evidence that SARS-CoV-2 can directly interact with cDC2s and, by down-regulating crucial molecules required for T cell activation, implements an efficient immune escape mechanism.

Poor correlation between antibody titers and neutralizing activity in sera from SARS-CoV-2 infected subjects (preprint)

Plenty of serologic tests for SARS-CoV-2 have been developed so far, thus documenting the importance of evaluating the relevant features of the immune response to this viral agent. The performance of these assays is currently under investigation. Amongst them, LIAISON(R) SARS-CoV-2 S1/S2 IgG by DiaSorin and Elecsys Anti-SARS-CoV-2 cobas(R) by Roche are currently used by laboratory medicine hospital departments in Italy and many other countries. In the present study, we have firstly compared two serologic tests on serum samples collected at two different time points from forty-six laboratory-confirmed COVID-19 subjects. Secondly, eighty-five negative serum samples collected before the SARS-CoV-2 pandemic were analyzed. Thirdly, possible correlations between antibody levels and the resulting neutralizing activity against a clinical isolate of SARS-CoV-2 were evaluated. Results revealed that both tests are endowed with low sensitivity on the day of hospital admission, which increased to 97.8 and 100% for samples collected after 15 days for DiaSorin and Roche tests, respectively. The specificity of the two tests ranges from 96.5 to 100%, respectively. Importantly, a poor direct correlation between antibody titers and neutralizing activity levels was evidenced in the present study.

Molecular Tracing of SARS-CoV-2 in Italy in the First Three Months of the Epidemic (preprint)

The aim of this study is the characterization and genomic tracing by phylogenetic analyses of 59 new SARS-CoV-2 Italian isolates obtained from patients attending clinical centres in North and Central Italy until the end of April 2020. All but one of the newly characterized genomes belonged to the lineage B.1, the most frequently identified in European countries, including Italy. Only a single sequence was found to belong to lineage B. A mean of 6 nucleotide substitutions per viral genome was observed, without significant differences between synonymous and non-synonymous mutations, indicating genetic drift as a major source for virus evolution. tMRCA estimation confirmed the probable origin of the epidemic between the end of January and the beginning of February with a rapid increase in the number of infections between the end of February and mid-March. Since early February, an effective reproduction number (Re) greater than 1 was estimated, which then increased reaching the peak of 2.3 in early March, confirming the circulation of the virus before the first COVID-19 cases were documented. Continuous use of state-of-the-art methods for molecular surveillance is warranted to trace virus circulation and evolution and inform effective prevention and containment of future SARS-CoV-2 outbreaks.

Molecular tracing of SARS-CoV-2 in Italy in the first three months of the epidemic (preprint)

The aim of this study is the characterization and genomic tracing by phylogenetic analyses of 59 new SARS-CoV-2 Italian isolates obtained from patients attending clinical centres in North and Central Italy until the end of April 2020. All but one of the newly characterized genomes belonged to the lineage B.1, the most frequently identified in European countries, including Italy. Only a single sequence was found to belong to lineage B. A mean of 6 nucleotide substitutions per viral genome was observed, without significant differences between synonymous and non-synonymous mutations, indicating genetic drift as a major source for virus evolution. tMRCA estimation confirmed the probable origin of the epidemic between the end of January and the beginning of February with a rapid increase in the number of infections between the end of February and mid-March. Since early February, an effective reproduction number (Re) greater than 1 was estimated, which then increased reaching the peak of 2.3 in early March, confirming the circulation of the virus before the first COVID-19 cases were documented. Continuous use of state-of-the-art methods for molecular surveillance is warranted to trace virus circulation and evolution and inform effective prevention and containment of future SARS-CoV-2 outbreaks.

Type III interferons disrupt the lung epithelial barrier upon viral recognition (preprint)

Lower respiratory tract infections are a leading cause of mortality driven by infectious agents. RNA viruses such as influenza virus, respiratory syncytial virus and the new pandemic coronavirus SARS-CoV-2 can be highly pathogenic. Clinical and experimental evidence indicate that most severe and lethal cases do not depend on the viral burden and are, instead, characterized by an aberrant immune response. In this work we assessed how the innate immune response contributes to the pathogenesis of RNA virus infections. We demonstrate that type III interferons produced by dendritic cells in the lung in response to viral recognition cause barrier damage and compromise the host tissue tolerance. In particular, type III interferons inhibit tissue repair and lung epithelial cell proliferation, causing susceptibility to lethal bacterial superinfections. Overall, our data give a strong mandate to rethink the pathophysiological roles of this group of interferons and their possible use in the clinical practice against endemic as well as emerging viral infections.

Combined prophylactic and therapeutic use maximizes hydroxychloroquine anti-SARS-CoV-2 effects in vitro (preprint)

While the SARS-CoV-2 pandemic is hardly hitting the world, it is of extreme importance that significant in vitro observations guide the quick set up of clinical trials. In this study, we evidence that the anti-SARS-CoV2 activity of a clinically achievable hydroxychloroquine concentration is maximized only when administered before and after the infection of Vero E6 cells. This strongly suggests that only a combined prophylactic and therapeutic use of hydroxychloroquine may be effective in limiting viral replication in patients.