Spread of Gamma (P.1) Sub-Lineages Carrying Spike Mutations Close to the Furin Cleavage Site and Deletions in the N-Terminal Domain Drives Ongoing Transmission of SARS-CoV-2 in Amazonas, Brazil

Abstract

The Amazonas was one of the most heavily affected Brazilian states by the COVID-19 epidemic. Despite a large number of infected people, particularly during the second wave associated with the spread of the Variant of Concern (VOC) Gamma (lineage P.1), SARS-CoV-2 continues to circulate in the Amazonas. To understand how SARS-CoV-2 persisted in a human population with a high immunity barrier, we generated 1,188 SARS-CoV-2 whole-genome sequences from individuals diagnosed in the Amazonas state from 1st January to 6th July 2021, of which 38 were vaccine breakthrough infections. Our study reveals a sharp increase in the relative prevalence of Gamma plus (P.1+) variants, designated Pango Lineages P.1.3 to P.1.6, harboring two types of additional Spike changes: deletions in the N-terminal (NTD) domain (particularly Δ144 or Δ141-144) associated with resistance to anti-NTD neutralizing antibodies or mutations at the S1/S2 junction (N679K or P681H) that probably enhance the binding affinity to the furin cleavage site, as suggested by our molecular dynamics simulations. As lineages P.1.4 (S:N679K) and P.1.6 (S:P681H) expanded (Re > 1) from March to July 2021, the lineage P.1 declined (Re < 1) and the median Ct value of SARS-CoV-2 positive cases in Amazonas significantly decreases. Still, we did not find an increased incidence of P.1+ variants among breakthrough cases of fully vaccinated patients (71%) in comparison to unvaccinated individuals (93%). This evidence supports that the ongoing endemic transmission of SARS-CoV-2 in the Amazonas is driven by the spread of new local Gamma/P.1 sublineages that are more transmissible, although not more efficient to evade vaccine-elicited immunity than the parental VOC. Finally, as SARS-CoV-2 continues to spread in human populations with a declining density of susceptible hosts, the risk of selecting more infectious variants or antibody evasion mutations is expected to increase. IMPORTANCE The continuous evolution of SARS-CoV-2 is an expected phenomenon that will continue to happen due to the high number of cases worldwide. The present study analyzed how a Variant of Concern (VOC) could still circulate in a population hardly affected by two COVID-19 waves and with vaccination in progress. Our results showed that the answer behind that was a new generation of Gamma-like viruses, which emerged locally carrying mutations that made it more transmissible and more capable of spreading, partially evading prior immunity triggered by natural infections or vaccines. With thousands of new cases daily, the current pandemics scenario suggests that SARS-CoV-2 will continue to evolve and efforts to reduce the number of infected subjects, including global equitable access to COVID-19 vaccines, are mandatory. Thus, until the end of pandemics, the SARS-CoV-2 genomic surveillance will be an essential tool to better understand the drivers of the viral evolutionary process.

Keywords: Brazil; COVID-19; SARS-CoV-2; coronavirus; variant gamma; virus evolution.

FIG 1

Temporal distribution and genetic diversity of SARS-CoV-2-positive samples from the Amazonas state during 2021. (A) Graph depicting the temporal evolution of SARI cases and SARI deaths based on the date of symptom onset (source, http://info.gripe.fiocruz.br) as a proxy for the COVID-19 epidemic curve in Amazonas state, along with the number of SARS-CoV-2 whole-genome sequences generated between January and July 2021. (B) Relative frequency of different P.1 lineage variants among SARS-CoV-2 positive cases sequenced in the Amazonas. (C) Relative frequency of different P.1 lineage variants among SARS-CoV-2 P.1 Brazilian sequences detected outside the Amazonas states.

FIG 2

Maximum likelihood phylogenetic tree of P.1 Amazonian sequences and P.1+NTDdel, P.1+N679K, and P.1+P681H sequences detected outside Amazonas. Tips were colored according to the S mutations as indicated in the legend. Major P.1 sublineages carrying additional mutations/deletions in the S protein were highlighted with colored boxes. The aLRT support values are indicated in key branches, and branch lengths are drawn to scale with the bar indicating nucleotide substitutions per site.

FIG 3

Temporal structure and phylogeographic reconstruction of the P.1+NTDdel, P.1+N679K, and P.1+P681H clades. (A) Root-to-tip regression of genetic divergence against dates of sample collection. P.1 sequences were colored green, while each P.1 subclade carrying deletions or additional mutations in S protein was colored following the legend. Time-resolved maximum clade credibility phylogenies of each P.1 subclade defined in the ML analysis: (B) P.1.4; (C) P.1.6; (D) P.1.7; (E) minor clades P.1.3, P.1.5, P.1.8 and P.1+Δ141-144AM. Tips and branches colors indicate the sampling state and the most probable inferred state of the nodes, respectively, as indicated in the legend for each tree. Bayesian posterior probabilities are indicated in key branches. All horizontal branch lengths are time-scaled, and the tree was automatically rooted under the assumption of the molecular clock model.

FIG 4

Epidemic trajectories of major SARS-CoV-2 lineages circulating in Amazonas in 2021. Graphs depicting the temporal variation in Re (median and 95% HPD) of Amazonian lineages P.1, P.1.4 and P.1.6 were estimated using the BDSKY approach.

FIG 5

Estimation by RT–PCR of viral load in the upper respiratory tract of SARS-CoV-2 infected patients in Amazonas. (A) Graph depicting the relative prevalence of P.1+ lineages estimated from whole-genome sequencing (dashed gray line) and the Ct (mean and 95% Confidence Interval) among SARS-CoV-2 positive cases (solid line) in Amazonas between March and July 2021. (B) Comparison of Ct values from March to July 2021. Horizontal bars represent Ct medians and IQR. Two-sided P values for the nonparametric Mann–Whitney test are shown for each group. Two-sided P-values <0.05 were considered statistically significant.

FIG 6

Binding between furin enzyme and the structural motif 679NSPRRARS686 of the SARS-CoV-2 Spike protein. (A) Representation of furin as electrostatic potential surface, showing the negative charge distribution around the loop substrate (represented in licorice cyan). Arrows indicate the native position of variant mutations N679K and P681H/R. (B) Free energy surface landscape of dissociation of modeled loops from furin enzyme depicted through the potential of mean force (PMF) as a function of the chosen CV. Dashed lines represent the lowest energy basin for the dissociation of the peptides.

FIG 7

Relative prevalence of P.1 and P.1+ lineages among unvaccinated and fully vaccinated groups. All breakthrough cases were detected among individuals fully vaccinated with CoronaVac. The P-value for the Chi–Square test is shown. P-values <0.05 were considered statistically significant.