Guidelines for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples (preprint)

BackgroundSARS-CoV-2 genotyping has been instrumental to monitor virus evolution and transmission during the pandemic. The reliability of the information extracted from the genotyping efforts depends on a number of aspects, including the quality of the input material, applied technology and potential laboratory-specific biases. These variables must be monitored to ensure genotype reliability. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in studies of viral spread and evolution. ResultsWe used clinical samples and synthetic viral genomes to evaluate the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination using an amplicon-based approach. We found that at least 1000 viral genomes are necessary to confidently detect variants in the genome at frequencies of 10% or higher. The broad applicability of our recommendations was validated in >200 clinical samples from six independent laboratories. The genotypes of clinical isolates with viral load above the recommended threshold cluster by sampling location and period. Our analysis also supports the rise in frequency of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was favoured by travelling during the summer 2020. ConclusionsWe present much-needed recommendations for reliable determination of SARS-CoV-2 genome sequence and demonstrate their broad applicability in a large cohort of clinical samples.

Role of Long-range Allosteric Communication in Determining the Stability and Disassembly of SARS-COV-2 in Complex with ACE2 (preprint)

Severe acute respiratory syndrome (SARS) and novel coronavirus disease (COVID-19) are caused by two closely related beta-coronaviruses, SARS-CoV and SARS-CoV-2, respectively. The envelopes surrounding these viruses are decorated with spike proteins, whose receptor binding domains (RBDs) initiate invasion by binding to the human angiotensin-converting enzyme 2 (ACE2). Subtle changes at the interface with ACE2 seem to be responsible for the enhanced affinity for the receptor of the SARS-CoV-2 RBD compared to SARS-CoV RBD. Here, we use Elastic Network Models (ENMs) to study the response of the viral RBDs and ACE2 upon dissassembly of the complexes. We identify a dominant detachment mode, in which the RBD rotates away from the surface of ACE2, while the receptor undergoes a conformational transition which stretches the active-site cleft. Using the Structural Perturbation Method, we determine the network of residues, referred to as the Allostery Wiring Diagram (AWD), which drives the large-scale motion activated by the detachment of the complex. The AWD for SARS-CoV and SARS-CoV-2 are remarkably similar, showing a network that spans the interface of the complex and reaches the active site of ACE2, thus establishing an allosteric connection between RBD binding and receptor catalytic function. Informed in part by the AWD, we used Molecular Dynamics simulations to probe the effect of interfacial mutations in which SARS-CoV-2 residues are replaced by their SARS-CoV counterparts. We focused on a conserved glycine (G502 in SARS-CoV-2, G488 in SARS-CoV) because it belongs to a region that initiates the dissociation of the complex along the dominant detachment mode, and is prominent in the AWD. Molecular Dynamics simulations of SARS-CoV-2 wild-type and G502P mutant show that the affinity for the human receptor of the mutant is drastically diminished. Our results suggest that in addition to residues that are in direct contact with the interface those involved in long range allosteric communication are also a determinant of the stability of the RBD-ACE2 complex.

Graphene nanoplatelet and Graphene oxide functionalization of face mask materials inhibits infectivity of trapped SARS-CoV-2 (preprint)

Recent advancements in bidimensional nanoparticles such as Graphene nanoplatelets (G) and the derivative Graphene oxide (GO) have the potential to meet the need for highly functional personal protective equipment (PPE) that confers increased protection against SARS-CoV-2 infection and the spread COVID-19. The ability of G and GO to interact with and bind microorganisms as well as RNA and DNA provides an opportunity to develop engineered textiles for use in PPE. The face masks widely used in health care and other high-risk settings for COVID transmission provide only a physical barrier that decreases likelihood of infection and do not inactivate the virus. Here, we show pre-incubation of viral particles with free GO inhibits SARS-CoV-2 infection of VERO cells. Highly relevant to PPE materials, when either polyurethane or cotton material was loaded with G or GO and culture medium containing SARS-CoV-2 viral particles either filtered through or incubated with the functionalized materials, the infectivity of the medium was nearly completely inhibited. The findings presented here constitute an important nanomaterials-based strategy to significantly increase face mask and other PPE efficacy in protection against the SARS-CoV-2 virus and COVID-19 that may be applicable to additional anti-SARS-CoV-2 measures including water filtration, air purification, and diagnostics. One Sentence SummaryCotton and polyurethane materials functionalized with bidimensional Graphene nanoplatelets trap SARS-CoV-2 and have the potential to reduce spread of COVID-19.

Monitoring COVID-19 transmission risks by RT-PCR tracing of droplets in hospital and living environments (preprint)

SARS-CoV-2 environmental contamination occurs through droplets and biological fluids released in the surroundings from patients or asymptomatic carriers. Surfaces and objects contaminated by saliva or nose secretions represent a risk for indirect transmission of COVID-19. We assayed surfaces from hospital and living spaces to identify the presence of viral RNA and the spread of fomites in the environment. Anthropic contamination by droplets and biological fluids was monitored by detecting the microbiota signature using multiplex RT-PCR on selected species and massive sequencing on 16S-amplicons. A total of 92 samples (flocked swab) were collected from critical areas during the pandemic, including indoor (3 hospitals and 3 public buildings) and outdoor surfaces exposed to anthropic contamination (handles and handrails, playgrounds). Traces of biological fluids were frequently detected in spaces open to the public and on objects that are touched with the hands (>80%). However, viral RNA was not detected in hospital wards or other indoor and outdoor surfaces either in the air system of a COVID-hospital, but only in the surroundings of an infected patient, in consistent association with droplets traces and fomites. Handled objects accumulated the highest level of multiple contaminations by saliva, nose secretions and faecal traces, further supporting the priority role of handwashing in prevention. In conclusion, anthropic contamination by droplets and biological fluids is widespread in spaces open to the public and can be traced by RT-PCR. Monitoring fomites can support evaluation of indirect transmission risks for Coronavirus or other flu-like viruses in the environment. ImportanceSeveral studies searched for SARS-CoV-2 in the environment because saliva and nasopharyngeal droplets can land on objects and surfaces creating fomites. However, the ideal indicator would be the detection of the biofluid. This approach was not yet considered, but follows a traditional principle in hygiene, using indicators rather than pathogens. We searched for viral RNA but also for droplets on surfaces at risk. For the first time, we propose to monitor droplets thorugh their microbiota, by RT-PCR or NGS. Even if performed during the pandemic, SARS-CoV-2 wasnt largely spread on surfaces, unless in proximity of an infectious patient. However, anthropic contamination was frequently at high level, suggesting a putative marker for indirect transmission and risk assessment. Moreover, all SARS-CoV-2-contaminated surfaces showed the droplets microbiota. Fomites detection may have an impact on public health, supporting prevention of indirect transmission also for other communicable diseases such as Flu and Flu-like infections. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/20179754v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@b15c11org.highwire.dtl.DTLVardef@1398ddorg.highwire.dtl.DTLVardef@98f501org.highwire.dtl.DTLVardef@1fd5282_HPS_FORMAT_FIGEXP M_FIG C_FIG

Nasopharyngeal Microbiota Profiling of SARS-CoV-2 Infected Patients (preprint)

We analyzed the bacterial communities of the nasopharynx in 40 SARS-CoV-2 infected and uninfected patients. All patients had a mild COVID-19 disease. We did not find statistically significant differences in either bacterial richness and diversity or composition. These findings suggest a nasopharyngeal microbiota at least early resilient to SARS-CoV-2 infection.

Enhanced binding of SARS-CoV-2 Envelope protein to tight junction-associated PALS1 could play a key role in COVID-19 pathogenesis (preprint)

Background: The Envelope (E) protein of SARS-CoV-2 is the most enigmatic protein among the four structural ones on the viral genome. Most of the current knowledge on the E protein is based on the direct comparison to the SARS E protein, initially mistakenly undervalued and subsequently proved to be a key factor in the ER-Golgi localization and in tight junction disruption. Methods: We compared the genomic sequences of E protein of SARS-CoV-2, SARS-CoV and the closely related genomes of bats and pangolins obtained from the GISAID and GenBank databases. Multiple sequence alignments were done with the Geneious software using the MAFFT algorithm. In silico modelling analyses of E proteins conformation and docking with PALS1 were performed with the Schrodinger Suite.Results: When compared to the known SARS E protein, we observed a different amino acidic sequence in the C-terminal of SARS-CoV-2 E protein which might have a key role in the current COVID-19 pathogenesis. In silico docking results provide evidence of a strengthened binding of SARS-CoV-2 E protein with the tight junction-associated PALS1 protein.Conclusions: We suggest that SARS-CoV-2 E protein may interfere with the tight junction stability and formation leading to an enhanced epithelial barrier disruption, amplifying the inflammatory processes, and promoting tissue remodelling. These findings raise a warning on the underestimated role of the E protein in the pathogenic mechanism and could open the route to detailed experimental investigations.