Rapid Establishment of Omicron Variant in Rio Grande do Sul State, Brazil

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Mariana Soares da Silva¹, Juliana Schons Gularte¹, Ana Cristina Sbaraini Mosena¹, Meriane Demoliner¹, Micheli Filippi¹, Alana Witt Hansen¹, Vyctoria Malayhka de Abreu Góes Pereira¹, Fágner Henrique Heldt¹, Matheus Nunes Weber¹, Paula Rodrigues de Almeida¹, Juliane Deise Fleck¹, Fernando Rosado Spilki¹

¹Universidade Feevale, Laboratório de Microbiologia Molecular, Rodovia ERS-239, Nº

2755, Prédio Vermelho, Piso 1, sala 103, Vila Nova, CEP 93525-075, Novo Hamburgo,

RS, Brazil.

In November 2021, researchers in Botswana and South Africa identified a new SARS-CoV-2 variant through whole-genome sequencing (WGS), posteriorly named as Omicron (B.1.1.529) [1]. This variant, announced by WHO as a Variant of Concern (on November 26, 2021), presents more than 50 mutations in its genome; of which, 32 were in the receptor binding domain (RBD) of the spike protein [2]. Omicron has spread rapidly, increasing COVID-19 cases through the world [3]. In Brazil, the first description was reported a few weeks after the first case in South Africa, from an airplane passenger that arrived in São Paulo State from South Africa, in late November.

A total of 87 SARS-CoV-2 complete genomes were sequenced through Ilumina MiSeq platform at Laboratório de Microbiologia Molecular - Feevale University, one of the institutions linked to the Corona-ômica.BR-MCTI Network. The positive samples were collected between late November and mid-January, from patients belonging to eleven cities from Rio Grande do Sul State (Arroio do Meio (1), Campo Bom (15), Canoas (9), Estância Velha (1), Garibaldi (5), Ivoti (1), Novo Hamburgo (41), Santa Cruz do Sul (11), São Sebastião do Caí (1), Sobradinho (1) and Triunfo (1).

Regarding genetic characterization, the sequences were aligned with complete SARS-CoV-2 genomes of different lineages through the NextClade online tool (https://clades.nextstrain.org/) and the phylogenetic tree was inferred (Figure 1). Of the total samples, 26 (30%) were classified as Delta variant (21J) and 61 (70%) as Omicron (21K). Subsequently, the sequences were analyzed through the Pangolin online tool (https://github.com/hCoV-2019/pangolin) and characterized as belonging to the following Delta: AY.99.2 (15/26 – 57.7%), AY.101 (3/26 – 11.5%), AY.122 (2/26 – 7.6%), AY.43 (2/26 – 7.6%), AY.112 (1/26 – 3.9%), AY.25 (1/26 – 3.9%), AY.43.2 (1/26 – 3.9%), AY.46 (1/26 – 3.9%) and Omicron sub lineages: BA.1 (38/61 – 62%) and BA.1.1 (23/61 – 38%).

Figure 1. Phylogenetic tree performed through NextClade online tool. In green, 26 sequences characterized as Delta variant and in red 61 Omicron variant sequences.

Performing a retrospective analysis, it is interesting to note that, among the samples sequenced herein, the Delta variant represented 100% of frequency in November 2021. In December of the same year, the first Omicron variant detections were performed, however not very expressive (3/30 – 10%), and the Delta variant still predominated (27/30 – 90%). Less than a month later, in January 2022, the scenario reversed, and the Delta comprised only 4 samples (4/62 – 6%) while Omicron established with 58 detections (58/62 – 94%). The complete data can be seen in Figure 2.

Figure 2. Lineages distribution of the SARS-CoV-2 samples sequenced by Laboratório de Microbiologia Molecular at Feevale University between January 2021 and January 2022.

According to our results, Omicron variant established in a short period of time, which was also observed in other countries [3]. It spreads faster and can evade natural and vaccine-induced immunity better than its predecessor variants [4]. Even the milder severity, especially in fully vaccinated individuals, the public health impact should be strongly considerable since the cases increased exponentially. It is likely that others SARS-CoV-2 variants will emerge and the continuous case control as well as epidemiological genomic surveillance must continue.

References

1. World Health Organization. Classification of Omicron (B.1.1.529): SARS-CoV-2 variant of concern. Geneva, Switzerland: World Health Organization; 2021. Accessed December 3, 2021. Classification of Omicron (B.1.1.529): SARS-CoV-2 Variant of Concern
2. Callaway E. Heavily mutated Omicron variant puts scientists on alert. Nature. 2021;600:21.
3. Mohapatra, R.K., Sarangi, A.K., Kandi, V., Azam, M., Tiwari, R. and Dhama, K. (2022), Omicron (B.1.1.529 variant of SARS-CoV-2); an emerging threat: Current global scenario. J Med Virol. https://doi.org/10.1002/jmv.27561.
4. Rössler, A., Riepler, L., Bante, D., Laer, D. von & Kimpel, J. SARS-CoV-2 B.1.1.529 variant (Omicron) evades neutralization by sera from vaccinated and convalescent individuals. medRxiv (2021) https://doi.org/10.1101/2021.12.08.21267491.

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Update information – samples analysis from late July and early September

Since the last update, we have additionally sequenced a total of 92 SARS-CoV-2 samples collected between late January and early March 2022. The patients were from 13 Rio Grande do Sul state cities: Brochier (1), Campo Bom (15), Canoas (9), Caxias do Sul (14), Estância Velha (19), Garibaldi (10), Lindolfo Collor (1), Montenegro (4), Novo Hamburgo (13), Portão (1), Porto Alegre (1), Presidente Lucena (1), São Leopoldo (2) and one sample from an Australian resident. The 14 samples from Caxias do Sul were obtained in a partnership with Immuneshare. Complete SARS-CoV-2 genomes were first analyzed through online Pangolin tool ( https://github.com/hCoV-2019/pangolin ) , and characterized as VOC Ômicron, sublineages: BA.1 (24), BA.1.1 (35), BA.1.14 (5), BA.1.15 (12), BA.1.17 (10), BA.1.9 (3), e BA.2 (3). BA.1, BA.1.1 and BA.2 are the predominants sublineages. Despite the notorious increase in BA.2 sublineage cases worldwide, in our samples its detection is still low, and we can observe a greater genomic diversification of BA.1. In order to confirm the data obtained, the sequences were aligned with complete SARS-CoV-2 genomes from different lineages through the NextClade online platform and the phylogenetic tree was inferred corroborating previous analysis (Figure 1).

Figure 1. Phylogenetic tree performed through NextClade online tool.

Performing a retrospective analysis, among the samples sequenced herein, since its emergence, Ômicron variant established in a short period of time, representing 95% of the samples in January, 2022 and 100% in the subsequently months (February and March). Complete temporal analysis results, performed since the beginning of 2021 at Laboratório de Microbiologia Molecular, Universidade Feevale, could be observed in Figure 2.

Figure 2. Lineages distribution of the SARS-CoV-2 samples sequenced by Laboratório de Microbiologia Molecular at Feevale University between January 2021 and March 2022.

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Update Information - The finding of a new Omicron-L452R mutant in Southern Brazil

SARS-CoV-2 Omicron variant (B.1.1.529) established in a short period of time in Brazil, which was also observed in other countries, contaminating an expressive number of individuals in a short period of time. Mutational SARS-CoV-2 analysis have been an important information since the beginning of the pandemic, especially in the S glycoprotein, which mediates virus attachment to ACE2 receptor, membrane fusion, and entry into the host cell, and also acts as a primary target for neutralizing antibodies elicited by the host immune response.This current research, describe the identification of a new Omicron variant of concern: Omicron-L452R in two patients from Southern, Brazil.

Post

Two sequences, classified as BA.1 Omicron sub lineage and named as hCoV-19/Brazil/LMM69880 (Gisaid accession number EPI_ISL_11514450) and hCoV-19/Brazil/LMM71052 (Gisaid accession number EPI_ISL_11514436), draw attention to the presence of a Delta signature S:L452R mutation. Both sequences presented Omicron signatures mutations sharing ORF1a:L2084I, ORF1a:I3758V, ORF1b:P314L, ORF1b:I1566V, ORF9b:P10S, N:P13L, N:R203K, N:G204R, S:G339D, S:L452R, S:T478K, S:D614G, S:H655Y, S:N856K, S:Q954H and S:N764K. Additional uncommon mutations were also found in hCoV-19/Brazil/LMM69880/2022 (ORF1b:P1570S and ORF7a:S83Y) and hCoV-19/Brazil/LMM71052/2022 (ORF1a:F1501S, ORF1a:V1535A, ORF1a:F3624, ORF1a:M3626T, ORF1a:F3628P and ORF1b:F2283L). No evidence of recombination was found for the analyzed sequences through RDP v.4.101 software analysis (Martin et al., 2015).

These patients presented different clinical presentations. The patient LMM69880, 60 years old, BMI 34 and a unique Oxford/AstraZeneca Covid-19 vaccine (2021/06/18) dose presented mild symptoms with no complications. The patient LMM71052, 43 years old, BMI 37 and two Pfizer vaccine doses (last dose: 2021/10/18) had severe symptoms and was hospitalized requiring mechanical ventilation. Our virus genome surveillance effort found a new variant of concern (VOC), the Omicron-L452R variant, which should be closely monitored.

Until now, the L452R, an exclusive Delta mutation, is related to increased SARS-CoV-2 fusogenicity and infectivity (Motozono et al., 2021). In our study, we report a natural Omicron-L452R in two patients. Through genetic engineering, a recent research, produced a mutated Omicron-L452R with the enhanced ability of viral fusogenicity and capacity of infect lung tissues of humanized mice (Zhang et al., 2022) and SARS-CoV-2 pathogenicity in patients is strongly related to fusogenicity capability (Motozono et al., 2021). This mechanism could be the reason for, despite the big COVID-19 wave associated with this variant, less hospitalization rates and clinical severity, observed in Omicron infections (Maslo et al., 2022). The clinical signs of the infected Omicron-L52R reported in this study were divergent, one of them presented serious COVID-19 complications, and the other, only mild clinical signs. Despite this, it is important to note that the severity of the disease is multifactorial, and, to link L452R with specific clinical signs it’s not possible without further studies.

In summary, we reported a natural infection by Omicron-L452R, an important S protein mutation present in Delta variant. Our study has provided a comprehensive investigation concerning the epidemiological and genetic characteristics of the major wave caused by Omicron in Rio Grande do Sul state, Southern Brazil, and contributed to important scientific findings enriching the knowledge about SARS-CoV-2.

References
Martin, D.P., Murrell, B., Golden, M., Khoosal, A., Muhire, B., 2015. RDP4 : Detection and analysis of recombination patterns in virus genomes 1, 1–5. https://doi.org/10.1093/ve/vev003

Motozono, C., Toyoda, M., Zahradnik, J., Saito, A., Nasser, H., Tan, T.S., Ngare, I., Kimura, I., Uriu, K., Kosugi, Y., Yue, Y., Shimizu, R., Ito, J., Torii, S., Yonekawa, A., Shimono, N., Nagasaki, Y., Minami, R., Toya, T., Sekiya, N., Fukuhara, T., Matsuura, Y., Schreiber, G., Ikeda, T., Nakagawa, S., Ueno, T., Sato, K., 2021. SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity. Cell Host Microbe 29, 1124-1136.e11. https://doi.org/https://doi.org/10.1016/j.chom.2021.06.006

Zhang, Y., Zhang, T., Fang, Y., Liu, J., Ye, Q., Ding, L., 2022. SARS-CoV-2 spike L452R mutation increases Omicron variant fusogenicity and infectivity as well as host glycolysis. Signal Transduct. Target. Ther. 7, 76. SARS-CoV-2 spike L452R mutation increases Omicron variant fusogenicity and infectivity as well as host glycolysis | Signal Transduction and Targeted Therapy

Maslo, C., Friedland, R., Toubkin, M., Laubscher, A., Akaloo, T., Kama, B., 2022. Characteristics and Outcomes of Hospitalized Patients in South Africa During the COVID-19 Omicron Wave Compared With Previous Waves. JAMA 327, 583–584. Characteristics and Outcomes of Hospitalized Patients in South Africa During the COVID-19 Omicron Wave Compared With Previous Waves | Global Health | JAMA | JAMA Network

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Update Information – Predominance of Omicron BA.2 sublineage in the latest SARS-CoV-2 positive samples

Since the last update, we have additionally sequenced a total of 40 SARS-CoV-2 samples collected between late January and early May 2022. The patients were from 9 Rio Grande do Sul state cities: Campo Bom (13), Canoas (2), Caxias do Sul (4), Estância Velha (2), Esteio (1), Ivoti (1), Montenegro (2), Novo Hamburgo (13), Porto Alegre (2), in a joint initiative between Corona-ômica BR – MCTI Network and the Immuneshare Project.

The sequenced genomes were analyzed through the Nextclade online platform (https://clades.nextstrain.org/) and characterized as belonging to the Ômicron lineage (100% of the samples) of the following sublineages: BA.1 (1), BA.1.1 (23), BA.1.14 (1), BA.1.15 (1), BA.1.1.1 (1), recombinant XQ (3) and BA.2 (10) (Table 1). Currently, the VOC Omicron is worldwide predominant. Corroborating with the previous analysis, the sequences were aligned with complete SARS-CoV-2 genomes of different variants through the NextClade online platform (Nextclade) and a phylogenetic tree was generated (Figure 1). All the data is being made available in national (Corona-Ômica. BR – MCTI) and international (GISAID) public databases with subsequent labor submission to the scientific journal.

Figure 1 . Phylogenetic tree performed through NextClade online tool.

Through the epidemiological monitoring that we see being carried out by our laboratory since the emergence of SARS-Cov-2 in December 2019, it was possible to demonstrate that VOC Omicron has been predominant since January 2022 (Figure 2), accompanying the world scenario. The most common sublineages are BA.1, BA.1.1 (clade 21K), and BA.2 (clade 21L). The proportion of globally reported sequences designated as BA.2 has increased when compared to BA.1 (Statement on Omicron sublineage BA.2), which can be observed in this sequencing, once all the samples from May 2022 belong to BA.2 sublineage, being, therefore, the most recent samples (Figure 3). Recent studies have been presenting that the BA.2 would be more transmissible than BA.1 and more efficient in infecting vaccinated people, even with a third dose, when compared to the previous variants.

Figure 2. SARS-CoV-2 lineages distribution of the SARS-CoV-2 samples sequenced by Laboratório de Microbiologia Molecular at Feevale University between January 2021 and May 2022.

Figure 3. Sublineages distribution of the Omicron SARS-CoV-2 samples sequenced by Laboratório de Microbiologia Molecular at Feevale University between January to May 2022.

Furthermore, recombinant sequences have been reported worldwide, such as the XD sublineage, a recombinant between Delta and Omicron (“Deltacron”); the XG and XE, recombinants from BA.1 and BA.2, with different mutations; and the XQ, recombinant between the lines BA.1.1 and BA.2, and detected in our study (3 samples). This recombinant was reported for the first time in Brazil (São Paulo) in April of this year, in a couple that did not have a third dose SARS-CoV-2 vaccine. The temporal analysis of the sublineages carried out by our laboratory in 2022 can be observed in more detail in Figure 3. The expansion of the BA.2 and the recombinant XQ have also been observed from the data inserted in the GISAID international platform by others Rio Grande do Sul State research groups. These events occur concomitantly with a considerable increase in the number of unexplained cases and reinforce the need for caution in relation to this moment of the pandemic.