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. 2023 Aug 9;15(8):1711.
doi: 10.3390/v15081711.

Frequency of Atypical Mutations in the Spike Glycoprotein in SARS-CoV-2 Circulating from July 2020 to July 2022 in Central Italy: A Refined Analysis by Next Generation Sequencing

Maria Concetta Bellocchi  1 , Rossana Scutari  2 , Luca Carioti  1 , Marco Iannetta  3 , Greta Marchegiani  1 , Lorenzo Piermatteo  4 , Luigi Coppola  3 , Simona Tedde  3 , Leonardo Duca  1 , Vincenzo Malagnino  3 , Lorenzo Ansaldo  3 , Neva Braccialarghe  3 , Stefano D Anna  1 , Maria Mercedes Santoro  1 , Andrea Di Lorenzo  3 , Romina Salpini  1 , Elisabetta Teti  3 , Valentina Svicher  4 , Massimo Andreoni  3 , Loredana Sarmati  3 , Francesca Ceccherini-Silberstein  1 , On Behalf Of The Ptv-Utv-Id-Covid Group
Affiliations

Frequency of Atypical Mutations in the Spike Glycoprotein in SARS-CoV-2 Circulating from July 2020 to July 2022 in Central Italy: A Refined Analysis by Next Generation Sequencing

Maria Concetta Bellocchi et al. Viruses. .

Abstract

In this study, we provided a retrospective overview in order to better define SARS-CoV-2 variants circulating in Italy during the first two years of the pandemic, by characterizing the spike mutational profiles and their association with viral load (expressed as ct values), N-glycosylation pattern, hospitalization and vaccination. Next-generation sequencing (NGS) data were obtained from 607 individuals (among them, 298 vaccinated and/or 199 hospitalized). Different rates of hospitalization were observed over time and among variants of concern (VOCs), both in the overall population and in vaccinated individuals (Alpha: 40.7% and 31.3%, Beta: 0%, Gamma: 36.5% and 44.4%, Delta: 37.8% and 40.2% and Omicron: 11.2% and 7.1%, respectively, both p-values < 0.001). Approximately 32% of VOC-infected individuals showed at least one atypical major spike mutation (intra-prevalence > 90%), with a distribution differing among the strains (22.9% in Alpha, 14.3% in Beta, 41.8% in Gamma, 46.5% in Delta and 15.4% in Omicron, p-value < 0.001). Overall, significantly less atypical variability was observed in vaccinated individuals than unvaccinated individuals; nevertheless, vaccinated people who needed hospitalization showed an increase in atypical variability compared to vaccinated people that did not need hospitalization. Only 5/607 samples showed a different putative N-glycosylation pattern, four within the Delta VOC and one within the Omicron BA.2.52 sublineage. Interestingly, atypical minor mutations (intra-prevalence < 20%) were associated with higher Ct values and a longer duration of infection. Our study reports updated information on the temporal circulation of SARS-CoV-2 variants circulating in Central Italy and their association with hospitalization and vaccination. The results underline how SARS-CoV-2 has changed over time and how the vaccination strategy has contributed to reducing severity and hospitalization for this infection in Italy.

Keywords: COVID-19; N-glycosylation; SARS-CoV-2; epidemiology; spike; variants of concern.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Temporal distribution of variants from July 2020 to July 2022. Monthly distribution of variants according to Nextstrain Clades and Pangolin for 607 SARS-CoV-2 Spike sequences collected from individuals attending the University Hospital of Rome Tor Vergata in Central Italy. The main waves of COVID-19 are clearly visible.
Figure 2
Figure 2
Prevalence of different sublineages in Delta and Omicron VOCs. The percentage of individuals infected with a specific sublineages was calculated in the subset of 185 and 188 individuals infected with the Delta and Omicron VOC, respectively. * Indicates sublineages with a percentage of individuals greater than 10%.
Figure 3
Figure 3
Distribution of atypical major (prevalence > 20%) and minor (prevalence > 1% and <20%) mutations observed in each VOC. The histogram reports for each VOC the percentage of individuals with at least one atypical major mutation and with at least one atypical minor mutation. * p-values were calculated by chi-squared test. Those remaining statistically significant after Bonferroni’s correction are reported in bold.
Figure 4
Figure 4
Distribution of atypical mutational profiles in spike glycoprotein observed in each VOC. Abbreviations: Wa: without atypical mutations, Ma: only major atypical mutations; ma: only minor atypical mutations; Mma: major + minor atypical mutations. * p-values were calculated by chi-squared test: Wa of Omicron sublineages vs. all others; Ma of Delta sublineages vs. all others, and of Gamma VOC vs. all others without Delta; ma of Delta sublineages vs. all others, and of Alpha VOC vs. all others without Beta; Mma of Gamma VOC vs. all others. p-values remaining statistically significant after Bonferroni’s correction are reported in black.
Figure 5
Figure 5
Distribution of atypical mutational variabilities according to: (a) vaccination status, (b) hospitalization status in vaccinated individuals, (c) vaccination status in hospitalized individuals within each VOC. Abbreviations: Mma: major + minor atypical mutations; Ma: only major atypical mutations; ma: only minor atypical mutations; Wa: without atypical mutations. * The p-value was calculated by chi-squared test. Those remaining statistically significant after Bonferroni’s correction are reported.
Figure 6
Figure 6
Distribution of individuals in Alpha, Gamma, Delta and Omicron VOCs according to (a) hospitalization status, (b) vaccination status and (c) hospitalization status in vaccinated individuals. None of the few individuals infected with the Beta VOC was hospitalized. * The p-value was calculated by chi-squared test, comparing Omicron sublineages vs. all others. p-values remaining statistically significant after Bonferroni’s correction are reported.
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