ABSTRACT
On February 2020, the municipality of Vo, a small town near Padua (Italy), was quarantined due to the first coronavirus disease 19 (COVID-19)-related death detected in Italy. The entire population was swab tested in two sequential surveys. Here we report the analysis of the viral genomes, which revealed that the unique ancestor haplotype introduced in Vo belongs to lineage B and, more specifically, to the subtype found at the end of January 2020 in two Chinese tourists visiting Rome and other Italian cities, carrying mutations G11083T and G26144T. The sequences, obtained for 87 samples, allowed us to investigate viral evolution while being transmitted within and across households and the effectiveness of the non-pharmaceutical interventions implemented in Vo. We report, for the first time, evidence that novel viral haplotypes can naturally arise intra-host within an interval as short as two weeks, in approximately 30% of the infected individuals, regardless of symptoms severity or immune system deficiencies. Moreover, both phylogenetic and minimum spanning network analyses converge on the hypothesis that the viral sequences evolved from a unique common ancestor haplotype, carried by an index case. The lockdown extinguished both viral spread and the emergence of new variants, confirming the efficiency of this containment strategy. The information gathered from household was used to reconstructs possible transmission events. AUTHOR SUMMARYIt is of great interest and importance to understand SARS-CoV-2 ability to mutate generating new viral strains, and to assess the impact of containment strategies on viral transmission. In this study we highlight the rapid intra-host haplotype evolution regardless of symptom severity and immune deficiencies that we observed during the first wave of the pandemic in the municipality of Vo in Italy. The confirmation that all the haplotypes found in this small community derive from a common ancestor haplotype, has allowed us to track the rapid emergence of new variants but lockdown and mass testing efficiently prevented their spread elsewhere.
ABSTRACT
SARS-CoV-2 genetic variants are emerging as a major threat to vaccination efforts worldwide as they may increase virus transmission rate and/or confer the ability to escape vaccine induced immunity with knock on effects on the level of herd immunity and vaccine efficacy respectively. These variants concern the Spike protein, which is encoded by the S gene, involved in virus entry into host cells and the major target of vaccine development. We report here that genetic variants of the N gene can impair our ability to utilize antigenic tests for both diagnosis and mass testing efforts aimed at controlling virus transmission. While conducting a large validation study on the Abbott Panbio COVID-19 Ag test, we noticed that some swab samples failed to generate a positive result in spite of a high viral load in Rt-PCR assays. Sequencing analysis of viruses showing discordant results in the RT-PCR and antigen assays revealed the presence of multiple disruptive amino-acid substitutions in the N antigen (the viral protein detected in the antigen test) clustered from position 229 to 374 a region known to contain an immunodominant epitope. A relevant fraction of the variants, undetected by the antigen test, contained the mutations A376T coupled to M241I. Intriguingly we found that virus sequences with this mutation were over-represented in the antigen-test-negative and PCR-positive samples and progressively increased in frequency over time in Veneto, a region of Italy that has aggressively scaled up the utilization of antigen tests, which reached nearly 68% of all the SARS-CoV-2 swab assays performed there. We speculate that mass utilization of antigen assays could create a selection pressure on the target that may favor the spread of undetectable virus variants.
ABSTRACT
Identification and development of effective drugs active against SARS-CoV-2 are urgently needed. Here, we report on the anti-SARS-CoV-2 activity of MEDS433, a novel inhibitor of human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of the de novo pyrimidines biosynthesis. MEDS433 inhibits in vitro virus replication in the low nanomolar range, and through a mechanism that stems from its ability to block hDHODH activity. MEDS433 thus represents an attractive candidate to develop novel anti-SARS-CoV-2 agents.
ABSTRACT
In viral diseases T cells exert a prominent role in orchestrating the adaptive immune response and yet a comprehensive assessment of the T-cell repertoire, compared and contrasted with antibody response, after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is currently lacking. A prior population-scale study of the municipality of Vo', Italy, conducted after the initial SARS-CoV-2 outbreak uncovered a high frequency of asymptomatic infected individuals and their role in transmission in this town. Two months later, we sampled the same population's T-cell receptor repertoire structure in terms of both diversity (breadth) and frequency (depth) to SARS-CoV-2 antigens to identify associations with both humoral response and protection. For this purpose, we analyzed T-cell receptor and antibody signatures from over 2,200 individuals, including 76 PCR-confirmed SARS-CoV-2 cases (25 asymptomatic, 42 symptomatic, 9 hospitalized). We found that 97.4% (74/76) of PCR confirmed cases had elevated levels of T-cell receptors specific for SARS-CoV-2 antigens. The depth and breadth of the T-cell receptor repertoire were both positively associated with neutralizing antibody titers; helper CD4+ T cells directed towards viral antigens from spike protein were a primary factor in this correlation. Higher clonal depth of the T-cell response to the virus was also significantly associated with more severe disease course. A total of 40 additional suspected infections were identified based on T-cell response from the subjects without confirmatory PCR tests, mostly among those reporting symptoms or having household exposure to a PCR-confirmed infection. Taken together, these results establish that T cells are a sensitive, reliable and persistent measure of past SARS-CoV-2 infection that are differentially activated depending on disease morbidity.
ABSTRACT
Lactoferrin, a multifunctional cationic glycoprotein, secreted by exocrine glands and neutrophils, possesses an antiviral activity extendable to SARS-CoV-2. We performed in vitro assays proving lactoferrin antiviral activity through direct attachment to both virus and cell surface components. This activity varied according to concentration (100/500g/ml), multiplicity of infection (0.1/0.01) and cell type (Vero E6/Caco-2 cells). Interestingly, the in silico results strongly supported the hypothesis of a direct recognition between the lactoferrin and the Spike S glycoprotein, thus hindering the viral entry into the cells. Hence, we conducted a clinical trial to investigate effect and tolerability of a liposomal lactoferrin formulation as a supplementary nutraceutical agent in mild-to-moderate and asymptomatic COVID-19 patients. A total of 92 mild-to-moderate (67/92) and asymptomatic (25/92) COVID-19 patients were recruited and divided in 3 groups according to the administered regimen. Thirty-two patients, 14 hospitalised and 18 in home-based insolation received oral and intranasal liposomal bovine lactoferrin (bLf), 32 hospitalised patients were treated with standard of care treatment (hydroxychloroquine, azitromicin and lopinavir/darunavir), and 28, in home-based isolation, did not take any medication. Furthermore, 32 COVID-19 negative, not-treated, healthy subjects were added as a control group for ancillary analysis. bLf-supplemented COVID-19 patients obtained an earlier and significant (p < 0,0001.) median rRT-PCR SARS-COV-2 RNA negative conversion than standard of care-treated and non-treated COVID-19 patients (14.25 vs 27.13 vs 32.61 days, respectively). In addition, bLf-supplemented COVID-19 patients showed significant fast clinical symptoms recovery than standard of care-treated and non-treated COVID-19 patients. Moreover, in bLf-supplemented patients, a significant decrease of either serum ferritin or IL-6 levels or host iron overload, all parameters characterizing inflammatory processes, were observed. Serum D-dimers was also found significantly decreased following bLf supplement. No adverse events were reported. These in vitro and in vivo observations led us to speculate a potential and safe supplementary role of Blf in the management of mild-to-moderate and asymptomatic COVID-19 patients.
ABSTRACT
On the 21st of February 2020 a resident of the municipality of Vo, a small town near Padua, died of pneumonia due to SARS-CoV-2 infection1. This was the first COVID-19 death detected in Italy since the emergence of SARS-CoV-2 in the Chinese city of Wuhan, Hubei province2. In response, the regional authorities imposed the lockdown of the whole municipality for 14 days3. We collected information on the demography, clinical presentation, hospitalization, contact network and presence of SARS-CoV-2 infection in nasopharyngeal swabs for 85.9% and 71.5% of the population of Vo at two consecutive time points. On the first survey, which was conducted around the time the town lockdown started, we found a prevalence of infection of 2.6% (95% confidence interval (CI) 2.1-3.3%). On the second survey, which was conducted at the end of the lockdown, we found a prevalence of 1.2% (95% CI 0.8-1.8%). Notably, 43.2% (95% CI 32.2-54.7%) of the confirmed SARS-CoV-2 infections detected across the two surveys were asymptomatic. The mean serial interval was 6.9 days (95% CI 2.6-13.4). We found no statistically significant difference in the viral load (as measured by genome equivalents inferred from cycle threshold data) of symptomatic versus asymptomatic infections (p-values 0.6 and 0.2 for E and RdRp genes, respectively, Exact Wilcoxon-Mann-Whitney test). Contact tracing of the newly infected cases and transmission chain reconstruction revealed that most new infections in the second survey were infected in the community before the lockdown or from asymptomatic infections living in the same household. This study sheds new light on the frequency of asymptomatic SARS-CoV-2 infection and their infectivity (as measured by the viral load) and provides new insights into its transmission dynamics, the duration of viral load detectability and the efficacy of the implemented control measures.
