Evidence of successive SARS-CoV-2 lineage replacements in Amazonas. To have a more in-depth understanding of the genetic diversity of the SARS-CoV-2 variants circulating in the Amazonas state since the early epidemic phase, we generated 250 SARS-CoV-2 high-quality whole-genome sequences from individuals living in 25 municipalities of the Amazonas state between 16th March 2020 and 13th January 2021 (Figs. 1a and 1b). Viral sequences were generated at FIOCRUZ Amazônia, which is part of both the Amazonas state health genomics network and the official consortium “FIOCRUZ Genomics Network” of the Brazilian Ministry of Health, for diagnostics and genomic surveillance of the SARS-CoV-2 in Brazil (http://www.genomahcov.fiocruz.br/). Our genomic survey revealed that most sequences were classified into five lineages: B.1.1.28 (n = 93; 37%), P.1 (n = 60; 24%), B.1.195 (n = 49; 20%), B.1.1.33 (n = 29; 12%) and P.2 (n = 9; 4%) (Supplementary Table 1), with a changing temporal prevalence over time (Fig. 1c). The lineage B.1.195 was the most prevalent variant during the first exponential growth phase. However, its prevalence gradually decreased after the first epidemic peak in early May and was surpassed by lineage B.1.1.28. This lineage persisted as the most prevalent one from May to December 2020, when the second lineage replacement took place, coinciding with the second phase of exponential growth. The VOC P.1 was first detected on 4th December 2020 in Manaus and displayed an extremely rapid increase in prevalence up to January 2021.
To better estimate the temporal trajectory of the P.1 emergence in the Amazonas state in late 2020 and early 2021, we designed a real-time PCR assay to detect the deletion at orf1b (NSP6: S106del, G107del, F108del), which is a genetic signature of the VOCs (P.1, B.1.1.7 and B.1.351). Thus, we evaluate all SARS-CoV-2 positive samples available between 1st November 2020 and 31st January 2021 that were not sequenced. None of the SARS-CoV-2 positive samples genotyped by real-time PCR before 16th December was positive for the NSP6 deletion, supporting our sequencing results that indicate a very low prevalence of VOC P.1 before mid-December 2020 in Amazonas. However, positive samples for the NSP6 deletion were quite frequent in the second half of December 2020 and January 2021. The combined results of genomic sequencing and real-time PCR testing support the sharp increase of lineage P.1 prevalence in the Amazonas state, from 0% in November 2020 (n = 0/88), to 4% in 1st-16th December 2020 (n = 2/54), 45% in 17th-31st December 2020 (n = 104/232), and 73% in 1st-15th January 2021 (n = 119/162) (Fig. 1d).
Major SARS-CoV-2 local clades drove the COVID-19 epidemic in the Amazonas state. Time-scale maximum likelihood (ML) phylogeographic analyses support at least 36 importation events of the widespread Brazilian lineages B.1.1.28 (n = 20), P.2 (n = 8), and B.1.1.33 (n = 8) into the Amazonas state; mostly from the Southeastern (69%) and Southern (14%) Brazilian regions (Figs. 2a and 2b). Despite multiple seeding events, most B.1.1.28 and B.1.1.33 viruses from the Amazonas state branched in three highly supported (approximate likelihood-ratio test [aLRT] > 80%) local clades (Figs. 2a and 2b). The clades 28-AM-I and 28-AM-II comprised 49% (n = 46) and 26% (n = 24) of all B.1.1.28 sequences from Amazonas, respectively, and clade 28-AM-II further gave origin to lineage P.1. The clade 33-AM-I comprises 48% (n = 14) of all B.1.1.33 sequences from Amazonas here detected. Bayesian phylogeographic analysis of the B.1.195 sequences sampled worldwide, by contrast, support a single importation event of this lineage in the Amazonas state either from other Brazilian states (posterior probability [PSP] = 0.61) or from abroad (PSP = 0.39) (Fig. 2c). The clustering of B.1.195 Amazonian sequences in a single highly supported (PP = 0.92) monophyletic clade (195-AM) was resolved after incorporating a nine nucleotide deletion at Nsp1 (delta 640–648: K141, S142, F143) as an informative trait in phylogenetic reconstruction using a Bayesian framework. Such deletion was the only molecular signature of clade 195-AM and was also detected in three sequences sampled outside Brazil that were nested within clade 195-AM: two from the Colombian Amazonian region and one from a Japanese traveler returning from Amazonas.
Analysis of the identified Amazonian clades revealed single lineage-defining mutations in clades 28-AM-I (C29284T), 28-AM-II (A6613G), and 33-AM-I (A28108C, ORF8:Q72P), relative to other B.1.1.28 and B.1.1.33 Brazilian sequences. Furthermore, only one signature mutation at the Spike (V1176F) distinguishes Amazonian lineages B.1.1.195 and B.1.1.28, which contrasts with the high number of mutations accumulated by the lineage P.1. A closer inspection of the genetic diversity within the Amazonian clade 28-AM-II further revealed a P.1-like sequence sampled in Manaus on 23rd December 2020 that branched basal to the P.1 lineage and also accumulated an unusually high number of genetic changes concerning other B.1.1.28 Brazilian sequences (Fig. 3a). The P.1-like sequence harbors 6/10 P.1 lineage-defining mutations in the Spike protein, including those at the RBD domain (K417T, E484K, and N501Y) (Supplementary Table 1). To investigate the events underlying P1 and P1-like lineages' emergence, we analyzed the intrahost SARS-CoV-2 variability in our samples. We found a total of 484 well-supported minor variants (MVs) with a predominance of non-synonymous (n = 368) over synonymous (n = 105) substitutions (Fig. 3b). However, no MVs corresponding to the Spike lineage-defining mutations of clades P.1 and P.1-like were identified (Supplementary Table 2). Furthermore, the frequency of MVs observed in samples taken during the early (March-September) and late (October-January) epidemic phases were comparable (Extended Data Fig. 1).
Differences in the epidemic trajectory of major SARS-CoV-2 Amazonian clades. Reconstruction of the spatiotemporal dissemination dynamic using a Bayesian phylogeographic approach supports that the early prevalent local clade 195-AM probably emerged in mid-March 2020 in the city of Manaus (Supplementary Table 3). Consequently, this lineage quickly spread from Manaus to several municipalities of the Manaus metropolitan region and inner Amazonas state (Figs. 4a and 4b). The origin of clades 28-AM-I and 28-AM-II was estimated to be in late March 2020 (Supplementary Table 3) in the area comprising Manacapuru, Iranduba, and Manaquiri municipalities, located on the Solimoes river, and from this area these clades were disseminated to Manaus and other inner municipalities (Figs. 4c to 4f). The clade 33-AM-I probably arose in late April (Supplementary Table 3) and remained restricted to the city of Manaus. Finally, our analyses indicate the VOC P.1 probably arose in Manaus around late November 2020 (Supplementary Table 3) and rapidly spread to other municipalities of the metropolitan region and also to municipalities located up to 1,100 km distant from Manaus, at the border with Peru, Colombia and Venezuela (Figs. 4e and 4f). These analyses further traced the most recent common ancestor of lineages P.1 and P.1-like to the Manaus city in late August (Supplementary Table 3).
We next applied the birth-death skyline (BDSKY) model to estimate the effective reproductive number (Re) of the Amazonian clades with more than 40 genomes. The estimated Re trajectories matched the relative prevalence of lineages and social distancing metrics (Fig. 5) very closely. The Re of clade 195-AM was high (2.6, 95% HPD: 1.6–3.8) in March, but displayed a steep decrease to 1.0 (95% HPD: 0.8–1.2) in April, coinciding with an increase of social distancing above 50% in Manaus. Clade 28-AM-I, which was estimated to have emerged in Amazonas countryside municipalities, also presented a high Re (2.1, 95% HPD: 1.2–3.4) in its initial spread, reducing to 0.9 (95% HPD: 0.7–1.2) in May, when the social distancing index reached 50% in the interior of Amazonas state. The increasing relative prevalence of clade 28.AM-I over clade 195-AM from April to June agrees with the estimated Re differences during April. From June to August 2020, the Re of clades 195-AM and 28-AM-I remained roughly stable around 1.0, as well as their relative prevalence. When the social distancing index decreased to below 40% in September 2020, the clade 195-AM became apparently extinct while the Re of the clade 28-AM-I increased to 1.2 (95% HPD: 0.9–1.6) and then remained roughly stable above 1.0 up to the end of 2020, leading to an increasing prevalence of clade 28-AM-I between September and November 2020. The lineage P.1 arose in late November and displayed a high Re of 2.6 (95% HPD: 1.5–4.5) during December 2020, becoming the predominant lineage. With the consequently increased social distance after the health system collapse, this VOC's Re was estimated to decrease to 1.2 (95% HPD: 0.9–1.6) in late December and January.
SARS-CoV-2 P.1 infections are associated with higher viral loads. The estimated Re trajectories of SARS-CoV-2 Amazonian lineages support that VOC P.1 could be more transmissible than earlier prevalent viral lineages circulating in Amazonas. To test whether this estimated Re difference might reflect a distinct virological phenotype, we used the real-time RT-PCR cycle threshold (Ct) scores as a proxy of the viral load in the upper respiratory tract (URT) of P.1-positive and P.1-negative samples collected at similar time from the onset of symptoms (Fig. 6 and Supplementary Table 4). Our analysis revealed a significant (Mann-Whitney test, P < 0.0001) lower median score in URT samples from P.1-infected (Ct = 19.8) compared with non-P.1-infected (Ct = 23.0) subjects, indicating that viral load was ~ 10-fold higher in P.1 infections than in non-P.1 infections. The Ct scores of P.1 infections were significantly lower than of non-P.1 infections in adult (18–59 years old) men (P = 0.0005), adult women (P < 0.0001), and old (> 59 years old) women (P = 0.0149); but not significantly different in old men (P = 0.4624). As expected, non-P.1 infected old men displayed higher viral load (Ct = 20.8) than adults male (Ct = 22.7) and female of all ages (Ct = 23.8). Old P.1-infected male, by contrast, displayed viral load (Ct = 19.8) similar to adult male (Ct = 19.4) and female of all ages (Ct = 20.1), suggesting that P.1-infected individuals above 18 years old could be equally infectious, irrespective of sex and age.