Patterns of within-host genetic diversity in SARS-CoV-2 -

Monitoring the spread of SARS-CoV-2 and reconstructing transmission chains has become a major public health focus for many governments around the world. The modest mutation rate and rapid transmission of SARS-CoV-2 prevents the reconstruction of transmission chains from consensus genome sequences, but within-host genetic diversity could theoretically help identify close contacts. Here we describe the patterns of within-host diversity in 1,181 SARS-CoV-2 samples sequenced to high depth in duplicate. 95% of samples show within-host mutations at detectable allele frequencies. Analyses of the mutational spectra revealed strong strand asymmetries suggestive of damage or RNA editing of the plus strand, rather than replication errors, dominating the accumulation of mutations during the SARS-CoV-2 pandemic. Within and between host diversity show strong purifying selection, particularly against nonsense mutations. Recurrent within-host mutations, many of which coincide with known phylogenetic homoplasies, display a spectrum and patterns of purifying selection more suggestive of mutational hotspots than recombination or convergent evolution. While allele frequencies suggest that most samples result from infection by a single lineage, we identify multiple putative examples of co-infection. Integrating these results into an epidemiological inference framework, we find that while sharing of within-host variants between samples could help the reconstruction of transmission chains, mutational hotspots and rare cases of superinfection can confound these analyses.

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Potent in vitro anti-SARS-CoV-2 activity by gallinamide A and analogues via inhibition of cathepsin L -

The emergence of SARS-CoV-2 in late 2019, and the subsequent COVID-19 pandemic, has led to substantial mortality, together with mass global disruption. There is an urgent need for novel antiviral drugs for therapeutic or prophylactic application. Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is recognized as a promising drug target. The marine natural product, gallinamide A and several synthetic analogues, were identified as potent inhibitors of cathepsin L activity with IC50 values in the picomolar range. Lead molecules possessed selectivity over cathepsin B and other related human cathepsin proteases and did not exhibit inhibitory activity against viral proteases Mpro and PLpro. We demonstrate that gallinamide A and two lead analogues potently inhibit SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range, thus further highlighting the potential of cathepsin L as a COVID-19 antiviral drug target.

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A SARS-CoV-2 spike binding DNA aptamer that inhibits pseudovirus infection in vitro by an RBD independent mechanism -

The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2. Notwithstanding, infection studies revealed potent and specific inhibition of pseudoviral infection by the aptamer. The present study opens up new vistas in developing SARS-CoV2 infection inhibitors, independent of blocking the ACE2 interaction of the virus and harnesses aptamers as potential drug candidates and tools to disentangle hitherto inaccessible infection modalities, which is of particular interest in light of the increasing number of escape mutants that are currently being reported.

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SARS-CoV-2 infecting the inner ear results in potential hearing damage at the early stage or prognosis of COVID-19 in rodents -

Objectives: In order to find out the association between the sensorineural hearing loss and COVID-19, we detected the expression ACE2 and TMPRSS2 in the mouse the hamster. Design: Using the public data from NCBI and GISAID, we assessed the expression of ACE2 and TMPRSS2 at the transcriptomic, DNA, and protein levels of ACE2 in the brain, inner ear, and muscle from the golden Syrian hamster (Mesocricetus auratus) and mouse (Mus musculus). Results: We identified ACE2 and TMPRSS2 expressed at different level in the inner ear and brain at DNA and transcriptomic levels of both mouse and the hamster. The protein expression shows a similar pattern of the brain and inner ear, while the expression of ACE2 from the inner ear was relatively higher than it from the muscle. Conclusion: SARS-CoV-2 could infect the hearing system potentially and SSNHL could be a characteristic to detect asymptomatic patients of COVID-19.

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REAL-TIME MECHANISTIC BAYESIAN FORECASTS OF COVID-19 MORTALITY -

The COVID-19 pandemic emerged in late December 2019. In the first six months of the global outbreak, the US reported more cases and deaths than any other country in the world. Effective modeling of the course of the pandemic can help assist with public health resource planning, intervention efforts, and vaccine clinical trials. However, building applied forecasting models presents unique challenges during a pandemic. First, case data available to models in real-time represent a non-stationary fraction of the true case incidence due to changes in available diagnostic tests and test-seeking behavior. Second, interventions varied across time and geography leading to large changes in transmissibility over the course of the pandemic. We propose a mechanistic Bayesian model (MechBayes) that builds upon the classic compartmental susceptible-exposed-infected-recovered (SEIR) model to operationalize COVID-19 forecasting in real time. This framework includes non-parametric modeling of varying transmission rates, non-parametric modeling of case and death discrepancies due to testing and reporting issues, and a joint observation likelihood on new case counts and new deaths; it is implemented in a probabilistic programming language to automate the use of Bayesian reasoning for quantifying uncertainty in probabilistic forecasts. The model has been used to submit forecasts to the US Centers for Disease Control, through the COVID-19 Forecast Hub. We examine the performance relative to a baseline model as well as alternate models submitted to the Forecast Hub. Additionally, we include an ablation test of our extensions to the classic SEIR models. We demonstrate a significant gain in both point and probabilistic forecast scoring measures using MechBayes when compared to a baseline model. We show that MechBayes ranks as one of the top models out of those submitted to the COVID-19 Forecast Hub. Finally, we demonstrate that MechBayes performs significantly better than the classical SEIR model.

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Prioritizing Risk Groups for SARS-CoV-2 Vaccination "By the Numbers" -

Background Given the limited supply of two COVID-19 vaccines, it will be important to choose which risk groups to prioritize for vaccination in order to get the most health benefits from that supply. Method In order to help decide how to get the maximum health yield from this limited supply, we implemented a logistic regression model to predict COVID-19 death risk by age, race, and sex and did the same to predict COVID-19 case risk. Results Our predictive model ranked all demographic groups by COVID-19 death risk. It was highly concentrated in some demographic groups, e.g. 85+ year old Black, Non-Hispanic patients suffered 1,953 deaths per 100,000. If we vaccinated the 17 demographic groups at highest COVID-19 death ranked by our logistic model, it would require only 3.7% of the vaccine supply needed to vaccinate all the United States, and yet prevent 47% of COVID-19 deaths. Nursing home residents had a higher COVID-19 death risk at 5,200 deaths/100,000, more than our highest demographic risk group. Risk of prison residents and health care workers (HCW) were lower than that of our demographic groups with the highest risks. We saw much less concentration of COVID-19 case risk in any demographic groups compared to the high concentration of COVID-19 death in some such groups. We should prioritize vaccinations with the goal of reducing deaths, not cases, while the vaccine supply is low. Conclusion SARS-CoV-2 vaccines protect against severe COVID-19 infection and thus against COVID-19 death per vaccine studies. Allocating at least some of the early vaccine supplies to high risk demographic groups could maximize lives saved. Our model, and the risk estimate it produced, could help states define their vaccine allocation rules.

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Investigating the origin of the Belgian second SARS-CoV-2 wave by using (pre)admission screening samples. -

Background: Belgium has been struggling with a second pandemic wave caused by SARS-CoV-2. Aim: The goal of this study was to estimate rates of carriership and viral loads in the general population in order to evaluate the dynamics leading to the second wave. Methods: Since the major Antwerp hospitals implemented extensive (pre-)admission SARS-CoV-2 screening of patients (eg. prior to elective surgery), they have gathered valuable information regarding the viral prevalence, incidence, and viral loads in the general population throughout the pandemic period. Prevalences and incidences were calculated and compared with available governmental data (numbers of positively tested and contacted cases). Major government coronavirus responses were taken into account. Results: The overall positivity rate of (pre)admission screenings was 1.3% (35.4% of positive cases carrying high viral loads). The highest prevalence of carriership was found in the elderly (2.6% for +80 y). 0-18 year-olds tested positive in 0.9% of cases. We estimated that, by extrapolation of cohort data, 20.3 % of the Antwerp population contracted the virus, whereas only 3.0 % was tested positive. In September, restriction measures were eased at a time when increased incidences were being observed. Conclusion: The estimation that only a small proportion of the positive cases (including cases with high viral loads) was detected and traced, in combination with a country-wide easing of restriction measures within a period of increasing incidences (and within an overall high base-line prevalence of the virus), were, in our opinion, the major drivers in the origin of the second pandemic wave.

🖺 Full Text HTML: Investigating the origin of the Belgian second SARS-CoV-2 wave by using (pre)admission screening samples.

Probability-based approaches for identifying low-titer antibody responses against SARS-CoV-2 -

The levels of the antibody response against SARS-CoV-2 varies widely between individuals, which together with the decline of antibody responses over time, complicates the correct classification of seropositivity using conventional assay cut-offs. All subjects in a cohort of SARS-CoV-2 PCR+ individuals representing different disease severity categories (n=105), and a group of PCR+ hospital staff (n=33), developed IgG against pre-fusion-stabilized spike (S) trimers and 97% did against the receptor-binding domain (RBD). The levels differed by several orders of magnitude and associated with disease phenotype. Concomitant analysis of a cohort of healthy blood donors and pregnant women (n=1,000), representing individuals who had undergone milder infections, demonstrated highly variable IgG titers, including several that scored between the classical 3SD and 6SD cut-offs. Since the correct classification of seropositivity is critical for epidemiological estimates, we trained probabilistic algorithms to assign likelihood of past infection using anti-S and -RBD IgG data from PCR+ individuals and a large cohort of historical negative controls (n=595). Applied to blood donors and pregnant women, this probabilistic approach provided a more accurate way to interpret antibody titers spread over a large continuum offering a probability-based diagnosis. The methods described here are directly applicable to serological measurements following natural infection and vaccination.

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ace2 expression is higher in intestines and liver while being tightly regulated in development and disease in zebrafish -

Human Angiotensin I Converting Enzyme 2 (ACE2) that acts as a receptor for SARS-CoV-2 entry is highly expressed in human type II pneumocytes and enterocytes and similarly in other mammals and zebrafish (Danio rerio). The zebrafish genome has a highly conserved, one-to-one ortholog of ACE2, i.e., ace2, whose expression profile however has not yet been studied during development or in pathologies relevant to COVID-19. Herein, we identified significant development-, tissue- and gender-specific modulations in ace2 expression based on meta-analysis of zebrafish Affymetrix transcriptomics datasets (ndatasets=107, GPL1319 in GEO database). Co-expression network analysis of ace2 revealed distinct positively correlated (carboxypeptidase activity and fibrin clot formation), and negatively correlated (cilia biogenesis/transport and chromatin modifications) STRING network modules. Using additional transcriptomics datasets, we showed zebrafish embryos before 3 days post fertilization (dpf) exhibited low levels of ace2 that increased significantly until 4 dpf implicating a role for ace2 in organogenesis. Re-analysis of RNA-seq datasets from zebrafish adult tissues demonstrated ace2 was expressed highly in intestines, variably in liver, and at lower levels in other organs. In addition, zebrafish females and males showed significant dimorphism in their age-dependent expression of ace2, and between ovary and testis where the latter had higher levels. Moreover, we demonstrated ace2 expression was significantly modulated under different physiological and pathological conditions associated with development, diet, infection, and inflammation. Our findings implicate a novel translational role for zebrafish ace2 in differentiation and pathologies predominantly found in intestines and liver, in which the effects of SARS-CoV-2 could be detrimental.

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HLA-C* 04:01 is a Genetic Risk Allele for Severe Course of COVID-19 -

Background Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, there has been increasing demand to identify predictors of severe clinical course in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human leukocyte antigen alleles (HLA) have been suggested as potential genetic host factors. We sought to evaluate this hypothesis by conducting an international multicenter study using HLA sequencing with subsequent independent validation. Methods We analyzed a total of 332 samples. First, we enrolled 233 patients in Germany, Spain, and Switzerland for HLA and whole exome sequencing. Furthermore, we validated our results in a public data set (United States, n=99). Patients older than 18 years presenting with COVID-19 were included, representing the full spectrum of the disease. HLA candidate alleles were identified in the derivation cohort (n=92) and tested in two independent validation cohorts (n=240). Results We identified HLA-C* 04:01 as a novel genetic predictor for severe clinical course in COVID-19. Carriers of HLA-C* 04:01 had twice the risk of intubation when infected with SARS-CoV-2 (hazard ratio 2.1, adjusted p-value=0.0036). Importantly, these findings were successfully replicated in an independent data set. Furthermore, our findings are biologically plausible, as HLA-C* 04:01 has fewer predicted bindings sites with relevant SARS-CoV-2 peptides as compared to other HLA alleles. Exome sequencing confirmed findings from HLA analysis. Conclusions HLA-C* 04:01 carriage is associated with a twofold increased risk of intubation in patients infected with SARS-CoV-2. Testing for HLA-C* 04:01 could have clinical implications to identify high-risk patients and individualize management.

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Novel air sterilization process for clean air production and microbial spread limitation using protection devices -

The aim of this work is to develop and simulate a novel process based on sterilizing natural air by heating at high temperatures followed by a rapid cooling. The new sustainable process is called “Air Rapid Heating Rapid Cooling Sterilization” (ARHRCS). Thus, it can be used in Heating, Ventilation, and Air Conditioning (HVAC) system in hospitals to produce safe air, free of pathogenic airborne microbes including bacteria and viruses such as tuberculosis (TB) and coronavirus (SARS-CoV-2). A crown fixed on the head of the medical staff may be connected to the HVAC system to produce a constant flow of clean laminar air. Hence, this crown is capable of keeping airborne microorganisms at a safe distance from Health Care Workers (HCWs), avoiding any potential microbial infection. Accordingly, HCWs will be able to work in a more suitable and safe conditions, especially in high infection risk areas. Depending on its need, the newly developed process may be implemented in different other locations such as laboratories, malls, buildings and other crowded spaces. Further applications of this method may arise including its usage in the sterilization of recirculated air in biosafety cabinets and the development of a portable air sterilizing unit.

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A Neanderthal OAS1 Isoform Protects Against COVID-19 Susceptibility and Severity: Results from Mendelian Randomization and Case-Control Studies -

Proteins detectable in peripheral blood may influence COVID-19 susceptibility or severity. However, understanding which circulating proteins are etiologically involved is difficult because their levels may be influenced by COVID-19 itself and are also subject to confounding factors. To identify circulating proteins influencing COVID-19 susceptibility and severity we undertook a large-scale two-sample Mendelian randomization (MR) study, since this study design can rapidly scan hundreds of circulating proteins and reduces bias due to reverse causation and confounding. We identified genetic determinants of 931 circulating proteins in 28,461 SARS-CoV-2 uninfected individuals, retaining only single nucleotide polymorphism near the gene encoding the circulating protein. We found that a standard deviation increase in OAS1 levels was associated with reduced COVID-19 death or ventilation (N = 4,336 cases / 623,902 controls; OR = 0.54, P = 7x10-8), COVID-19 hospitalization (N = 6,406 / 902,088; OR = 0.61, P = 8x10-8) and COVID-19 susceptibility (N = 14,134 / 1,284,876; OR = 0.78, P = 8x10-6). Results were consistent in multiple sensitivity analyses. We then measured OAS1 levels in 504 patients with repeated plasma samples (N=1039) with different COVID-19 outcomes and found that increased OAS1 levels in a non-infectious state were associated with protection against very severe COVID-19, hospitalization and susceptibility. Further analyses suggested that a Neanderthal isoform of OAS1 affords this protection. Thus, evidence from MR and a case-control study supported a protective role for OAS1 in COVID-19 outcomes. Available medicines, such as phosphodiesterase-12 inhibitors, increase OAS1 and could be explored for their effect on COVID-19 susceptibility and severity.

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Aerosolized Hydrogen Peroxide Decontamination of N95 Respirators, with Fit-Testing and Virologic Confirmation of Suitability for Re-Use During the COVID-19 Pandemic -

In response to the current demand for N95 respirators by healthcare workers responding to the COVID-19 pandemic, we evaluated decontamination of N95 respirators using a low concentration aerosolized hydrogen peroxide (aHP) system. This system dispenses a consistent atomized spray of micron-sized, low concentration, hydrogen peroxide (H2O2) particles over a treatment cycle. Multiple N95 respirator models were subjected to ten or more cycles of respirator decontamination, and masks were periodically assessed for qualitative and quantitative fit testing to verify respirator integrity. In parallel, we assessed the ability of aHP treatment to inactivate multiple viruses absorbed onto masks, including phi6 bacteriophage, HSV-1, CVB3, and SARS-CoV-2. Given that SARS-CoV-2 is transmitted via expired respiratory droplets and aerosols, it is critical to address respirator safety for reuse. This study provided experimental validation of a suitable aHP treatment process that decontaminates the respirators while maintaining N95 function. After ten rounds of aHP treatment, respirators passed N95 filtration efficiency testing. Virus inactivation by aHP was comparable to the sterilization of commercial spore-based biological indicators. These data demonstrate that the aHP process is effective, on the basis of zero failure rate on fit-testing of respirators, effective decontamination of multiple virus species including SARS-CoV-2, successful sterilization of bacterial spores, and filtration efficiency maintained at or greater than 95%. Collectively, these studies support the use of specific aHP decontamination protocol that permits safe reuse of N95 respirators by healthcare providers.

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Shortcomings of SARS-CoV-2 genomic metadata -

Metadata is integral to data-driven association studies relevant to epidemiology, viral population dynamics and public health response. However, SARS-CoV-2 metadata quality remains inadequate. Here I exemplify this through a brief analysis of two metadata categories in the GISAID SARS-CoV-2 genomic database: “originating lab” and “submitting lab”. My analysis reveals a startling prevalence of spelling errors and inconsistent naming conventions, which together occur in an estimated ~9.8% and ~11.6% of “originating labs” and “submitting labs” respectively. In addition, I find numerous ambiguous lab names, such as “Biology Dpt” and “Hospital” which provide very little information with regard to the actual source of a sample and could easily associate with multiple sources worldwide. Importantly, all of these issues can impair the ability and accuracy of association studies by deceptively causing a group of samples to identify with multiple sources when they truly all identify with one source, or vice versa. GISAID’s “originating lab” and “submitting lab” categories are specifically relevant to identifying problematic sites in SARS-CoV-2 genomic data through lab association. Thus, I advocate that both data submitters and maintainers strive for a higher metadata quality standard now and in the future.

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Substantial Impact of Post Vaccination Contacts on Cumulative Infections during Viral Epidemics -

Background: The start of 2021 will be marked by a global vaccination campaign against the novel coronavirus SARS-CoV-2. Formulating an optimal distribution strategy under social and economic constraints is challenging. Optimal distribution is additionally constrained by the potential emergence of vaccine resistance. Analogous to chronic low-dose antibiotic exposure, recently inoculated individuals who are not yet immune play an outsized role in the emergence of resistance. Classical epidemiological modelling is well suited to explore how the behavior of the inoculated population impacts the total number of infections over the entirety of an epidemic. Methods: A deterministic model of epidemic evolution is analyzed, with 7 compartments defined by their relationship to the emergence of vaccine-resistant mutants and representing three susceptible populations, three infected populations, and one recovered population. This minimally computationally intensive design enables simulation of epidemics across a broad parameter space. The results are used to identify conditions minimizing the cumulative number of infections. Results: When an escape variant is only modestly less infectious than the originating strain within a naive population, there exists an optimal rate of vaccine distribution. Exceeding this rate increases the cumulative number of infections due to vaccine escape. Analysis of the model also demonstrates that inoculated individuals play a major role in the mitigation or exacerbation of vaccine-resistant outbreaks. Modulating the rate of host-host contact for the inoculated population by less than an order of magnitude can alter the cumulative number of infections by more than 20%. Conclusions: Mathematical modeling shows that optimization of the vaccination rate and limiting post-vaccination contacts can affect the course of an epidemic. Given the relatively short window between inoculation and the acquisition of immunity, these results might merit consideration for an immediate, practical public health response.

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