SARS-CoV-2 variant N.9 identified in Rio de Janeiro, Brazil

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease 2019 (COVID-19) pandemic has reached every continent since December 2019.⁽¹⁾ A novel variant of the virus emerged at the end of January 2020 in lineage B.1 presented the spike protein substitution D614G. This variant replaced the initial strain identified in Wuhan (Wuhan-Hu1) and became the globally dominant strain of SARS-CoV-2.^{(2, 3)} Since then, several circulating variants of SARS-CoV-2 have raised significant concerns about the impact on viral fitness, transmissibility, pathogenicity, and immunological escape. The first wave of the COVID-19 epidemic in Brazil in 2020 was dominated by the presence of B.1.1.28 and B.1.1.33 lineages.^{(4, 5)} In Rio de Janeiro, the B.1.1.33 reached the dominance of 80% in the early pandemic phase.⁽⁵⁾ A second and most significant wave of COVID-19 epidemic in Brazil started in December 2020 in the Amazonas State, and it was associated with the emergence of the variant of concern (VOC) P.1 and described as the prevalent SARS-CoV-2 lineage detected in this state.^{(6, 7)} By March 2021, the lineage P.1 was detected as the most prevalent lineage all across the country (http://www.genomahcov.fiocruz.br, accessed on May 2nd, 2021). On the other hand, the lineage P.2 VOI Zeta, probably emerged in Brazil in July 2020,^{(8, 9)} and has been detected as the most prevalent variant in several states across the country until late 2020 and early 2021 (http://www.genomahcov.fiocruz.br, accessed on May 2nd, 2021). Both lineages have emerged from lineage B.1.1.28. In the same way, at least two B.1.1.33-derived lineages have recently emerged in Brazil: N.9⁽¹⁰⁾ and N.10.⁽¹¹⁾ The lineage N.9 probably emerged in August 2020 in the State of São Paulo, has spread across different Brazilian states (Southeast, South, North, and Northeast regions), and comprise a high fraction (35%) of the B.1.1.33 sequences detected between November 2020 and February 2021. The lineage N.9 is also considered a potential VOI due to the presence of E484K amino acid substitution at the receptor-binding domain (RBD) of the Spike (S) protein.⁽¹⁰⁾ This mutation has been described as one of the most important immune evasion mechanisms, as it confers resistance to several monoclonal antibodies and reduces the neutralisation effect of sera obtained from convalescent and vaccinated individuals.^{(12, 13, 14)}

MATERIALS AND METHODS

In the present study, we described the first detection of N.9 in the State of Rio de Janeiro. The genome of this N.9 strain was detected in two clinical specimens, nasopharyngeal swab and saliva, collected on December 9th, 2020, from a 32 years old diabetic patient, male, resident in the city of Rio de Janeiro. The patient had no history of traveling in the last two months. He reported self-limited fatigue, myalgia, cough, headache, and mild fever during five days, with no loss of taste or smell and no need for hospitalisation. SARS-CoV-2 infection was confirmed by real time RT-PCR by using the 2019-nCoV RUO Kit (Integrated DNA Technologies, Coralville, USA), two days after the symptoms onset (December 9th) in the nasopharyngeal swab and saliva clinical specimens. The subject provided written, informed consent before participation in the study, and a medical assistant filled a standardised medical questionnaire form during an interview with the participant. The institutional review boards at Fundação Oswaldo Cruz (FIOCRUZ) and National Research Ethics Commission approved the study protocol (CAAE: 30639420.0.0000.5262/032458/2020). The SARS-CoV-2 whole-genome nucleotide sequencing was generated with a RT-PCR scheme containing 25 overlapping amplicons of 1000 to 1500 bp in length covering the entire genome (Supplementary data I - Tables I-II). The DNA sequencing was performed at the sequencing facility of Fiocruz-RJ (RPT01A - Sequenciamento de DNA - RJ). Sequences were edited and assembled to obtain the whole-genome consensus sequence using the SeqMan Pro version 8.1.5 software (DNASTAR, Madison, USA) and the complete genome sequence of Wuhan Hu-1 (MN908947.3) as reference. The whole-genome SARS-CoV-2 consensus sequence was initially classified as N.9 according to the PANGO Lineage web application (https://pangolin.cog-uk.io),⁽³⁾ and it was later confirmed through a phylogenetic analysis (Fig. 1). Three other N.9 recognised by the Pango Lineage were later detected in Rio de Janeiro, one of them by our research group. This N.9 genome detected in the present study was generated at the Laboratory of Respiratory Viruses and Measles, FIOCRUZ, using an in-house methodology as previously described.⁽⁹⁾ All available N.9 (n = 156) and all Brazilian B.1.1.33 genomes (n = 1458) by August 13th, 2021 in EpiCoV database in the GISAID (https://www.gisaid.org/) were downloaded (Supplementary data II and III). To preserve B.1.1.33 diversity but also limit its dimension, a downsampling was done with CD-HIT v.4.8.1.⁽¹⁵⁾ The final dataset (n = 353) was aligned by using MAFFT online service v.7.⁽¹⁶⁾ The alignment was manually edited by using AliView software v.1.26⁽¹⁷⁾ and subjected to a maximum likelihood (ML) phylogenetic analysis with IQ-TREE v2.1.3,⁽¹⁸⁾ using a GTR + I + F + ?4 nucleotide substitution model as selected by ModelFinder.⁽¹⁹⁾ The reliability of the obtained tree topology was estimated with the approximate likelihood-ratio test.⁽²⁰⁾ The ML tree generated was visualised by using FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). The mutational profile was investigated using the Nextclade v.0.14.2 (https://clades.nextstrain.org, Nextclade 2020-2021 Nextstrain developers), and the CoVSurver mutations App (https://www.gisaid.org/epiflu-applications/covsurver-mutations-app/, A*STAR Bioinformatics Institute, Singapore).

RESULTS AND DISCUSSION

The six synapomorphic mutations of SARS-CoV-2 lineage N.9⁽¹⁰⁾ were confirmed in the first one of the N.9 genomes obtained in this study (EPI_ISL_2086268) and it grouped in the N.9 cluster in our phylogenetic analysis (Fig. 1, upper part). The genome of SARS-CoV-2 from saliva could not be recovered, although several attempts were made to amplify it through RT-PCR SARS-CoV-2 whole-genome protocol. Perhaps, it was due to the low viral load of the sample as determined by the high Ct value obtained at the real time RT-PCR (Ct = 31,02). The other one (EPI_ISL_2557401), assigned by PANGO Lineage as N.9 lineage, only has one of the six synapomorphic mutations: the G23012A, S: E484K (Fig. 2). Other three strains defined as N.9 in the EpiCoV database at GISAID have the same genomic profile. These four strains are grouped within the B.1.1.33 lineage and basal to the N.9 lineage in our phylogenetic analysis (Fig. 1, middle part). They seem to be another example of convergent evolution of the E484K mutation in different lineages, and we call them “N.9-like / B.1.1.33 + E484K”. This convergent evolution of E484K can be evidenced by its appearance multiple times independently around the world in four variants: VOC Beta, VOC Gamma, VOC Kappa and VOI Zeta.^{(8, 21)} By phylogenetic reconstruction the four N.9 genomes described in Rio de Janeiro were four independent introductions in the State of Rio de Janeiro in October and December 2020 and January and March 2021.

There is no evidence of the clinical impact of N.9 in the literature.⁽¹⁰⁾ And, in the same way, by analysing the metadata associated with each of the 138 genomes available in the EpiCoV database at GISAID (by June 28th), only 24% (n = 33) of them have epidemiological information without clinical symptoms description. From these data we observed the next epidemiological information: (i) the patients age range were from 16 to 90 years old, with a media age of 50.63 years old, (ii) 60% of the cases were from patients older than 60 years old, (iii) four patient died (all males), and three of them were older than 75 years old, the other were a patient of 51 years old, and (iv) 18/33 were male, and 15/33 were female. There is no information in the EpiCoV database at GISAID related to treatment, hospitalisation requirement or clinical symptoms.

The plot of the frequency of P.2 and N.9 lineages that co-circulated in São Paulo State, and P.2 in Rio de Janeiro, between August/2020 and April/2021 (Fig. 3), allows us to hypothesise that probably there was a competition between N.9 and P.2 lineages in these states, and it seems that P.2 was able to quickly spread in these populations, and, this way, limited the N.9 dissemination in the same populations. Both lineages have the same unique beneficial mutation (E484K) and are very similar genotypically. So, we think that other factors external to viral fitness allowed P.2 to be prevalent over N.9, for example the population behavior regarding non pharmaceutical interventions at the moment of emergence and introduction of this lineage. In this sense, an important fact to be considered is that P.2 emerged earlier than N.9. The first genome of P.2 strain identified in Brazil, as accessed in the EpiCoV database at GISAID, was collected in April/2020, and, on the other hand, the first genome of N.9 was collected in October/2020.

In conclusion, we report the first genome description of the N.9 lineage in the state of Rio de Janeiro, where the SARS-CoV-2 VOC Alpha, VOC Gamma and the VOI Zeta also circulated. We also described an apparently B.1.1.33-derived sublineage that we called as “N.9-like / B.1.1.33 + E484K”. In this sense, the detection of N.9 and “N.9-like / B.1.1.33 + E484K” reinforces the importance of continuous real-time genomic surveillance to monitor the emergence and behavior of the SARS-CoV-2 variants in the population.

The SARS-CoV-2 genomes generated and analysed in this study are available at GISAID (https://www.gisaid.org/), under the ID: EPI_ISL_2086268 and EPI_ISL_2557401.

ACKNOWLEDGEMENTS

To Raiane do Carmo Milagre from Instituto Nacional de Infectologia - Ambulatório de Doenças Febris Agudas - INI/FIOCRUZ-RJ for helping with samples and data collection; the Plataforma Genômica-Sequenciamento de DNA/RPT01A/FIOCRUZ; Ighor Arantes for his valuable collaboration with the phylogenetic analysis; the GISAID team and all the EpiCoV database?s submitters (GISAID acknowledgment table containing sequences used in this study is available in Supplementary data II and III), and Nathália Dias Furtado, Iasmim Silva de Mello and Déberli Ruiz Fernandes for their assistance in the lab work.

AUTHORS’ CONTRIBUTION

LFLT, IPR, LSRM, PB, LD and MCB - Conceived the study design and data collection; IPR and MPS - carried out viral RNA extraction from the biological specimens and the diagnosis by RT-qPCR; AACS - designed the primers sets; LFLT, IPR and AACS - carried out genome amplification and sequencing; LFLT - performed phylogenetic and mutational profile analysis; LFLT, IPR, PCRS, MAMTS and MCB - prepared the manuscript. All authors critically read and approved the final version of the manuscript. The authors declare any conflict of interest related to their research work.