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GenovGenov Scientific project of SARS-CoV-2 genomic surveillance in Brazil

20/08/2021

Sumário

Introduction...................................................................................3 Case Study ...................................................................................4 Main variants of SARS-CoV-2 and nomenclature according to the PANGO classification ..................................................................5

Perfil mutacional das principais linhagens derivadas de P.1 (Gama) descritas neste relatório.............................................6 Ethical Concerns .........................................................................6 Sample Selection........................................................................7 Sample Processing ......................................................................7

Sequencing of viral genomes.................................................... 7 Variant classification...................................................................8 Results ...........................................................................................8

Predominance of P.1 (Gamma)..................................................9

P.1 Diversification (Gamma) .....................................................10

Evolution of P.1 samples with mutation in S:P681...............10

Delta variant (B.1.617.2)............................................................12

Intra-Hospital Outbreak in Vaccinated Patients.................12

References...................................................................................14

APPENDIX - Variants per State..............................................15

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In this second report, we offer data from the complete sequencing of 1,297 samples collected in the first half of June 2021 from Dasa patients with positive RT-PCR for SARS-CoV-2. The entire process has been improved in order to reach the level of 3,000 sequenced samples a month. Currently, the sequencing platform is transitioning from the NextSeq equipment to the higher-capacity NovaSeq equipment, which will speed up the project. The data for June/21 is similar to that of May/21 with the P.1 variant (Gamma) continuing to predominate, which corroborates/substantiates its enormous spread in 2021. A small increase in lineages derived from P.1 such as P.1.1, P.1.2 etc. can already be perceived, indicating the evolution of the variant. Among these, we have noticed in some areas of the country a significant increase in isolates with mutation in the human furin cleavage sites, position P681 of the Spike (S) protein. In June/21, we confirmed the first 4 cases of variant 1.617.2 (Delta) in our casuistry. This report also includes data from the investigation involving an outbreak among fully vaccinated health workers. We hope this report is as interesting for those who read it as it is for those of us who generate the data. Enjoy!

Dr. José Eduardo Levi - on behalf of the Genov teamCRBio 23.407/01-D

Introduction

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CASE STUDY1.Number of samples/region

2. Sex

* Except São Paulo city and Rio de Janeiro city

* Except São Paulo city and Rio de Janeiro city

01 to 15 May 2021 01 to 15 June 2021 Total

Northern Region 21 30 51

Northeastern Region 49 198 247

Midwest Region 140 216 356

Southeast Region* 117 212 329

São Paulo City 36 254 290

Rio de Janeiro City 190 124 314

Southern Region 76 263 339

Total 626 1297 1926

01 to 15 May 2021 01 to 15 June 2021

Male Female Male Female

Northern Region 47.6% 52.4% 60% 40.0%

Northeastern Region 53.1% 46.9% 47.0% 53.0%

Midwest Region 49.3% 50.7% 53.7% 46.3%

Southeast Region* 50.4% 49.6% 40.6% 59.4%

São Paulo City 59.55 40.5% 50% 50%

Rio de Janeiro City 44.7% 55.3% 446.8% 53.2%

Southern Region 47.4% 52.6% 55.5% 44.5%

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PANGO WHO Place of 1st identification

VARIANTS OF CONCERN (VOCs)

B.1.1.7 Alfa United Kingdom

B.1.351 Beta South Africa

P.1 Gama Brazil (Amazonas)

B.617.2 Delta India

VARIANTS OF INTEREST (VOIs)

B.1.525 Eta Several countries

B.1.526 Iota United States of America

B.1.617.1 Kappa india

C.37 Lambda Peru

Table 1 – Main variants of SARS-CoV-2 and nomenclature according to the PANGO classification (https://cov-lineages.org/) and new classification by the World Health Organization (WHO).

Available at: https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/ 12 JULY 21

3. Age

* Exceto cidade de São Paulo e cidade do Rio de Janeiro

01 to 15 May 2021 01 to 15 June 2021

Median (min-max) Average Median (min-max) Average

Northern Region 36 years of age (1 - 84) 36.10 34.50 anos (13-67) 37.40

Northeastern Region 35 years of age (3-78) 37.12 37 anos (0-88) 38.24

Midwest Region 39 years of age (0-83) 39.98 40.5 anos (0-80) 41.01

Southeast Region* 39 years of age(3-75) 39.70 36 anos (0-92) 36.17

São Paulo City 35 years of age(3-66) 32.76 35 anos (0-90) 36.31

Rio de Janeiro City 42 years of age (1-83) 41.62 40 anos (1-83) 40.13

Southern Region 36.5 years of age (6-78) 37.25 37 anos (0-85) 37.54

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Ethical Concerns: Only the viral genomes of the samples are sequenced and associated exclusively with basic patient data such as sex, age and place of collection, with anonymity maintained. Therefore, the Research Ethics Committee of the 9 de Julho Hospital, affiliated to CONEP, approved the study and the waiver of the Informed Consent Form (CAAE: 45540421.0.0000.5455).

Tabela 2 - Mutational profile of the main P.1 (Gamma) derived lineages described in this report.

Legend. Table with the characteristic mutations of the P.1.1; P.1.2; P.1.6; P.1.7 and P.1.8 lineages. The P681 position located at the human furin cleavage site in the Spike (S) protein of SARS-CoV-2 is shown in red. Courtesy Dr. Camila Romano IMT-FMUSP.

Methods

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Sample selection:

This report adds 127 samples from the 1st half of May in addition to the 502 contained in the 1st report, bringing the total to 629. In addition 1,297 complete genomes of SARS-CoV-2 sampled in June/2021 from different regions of the country is presented. According to the attachment, 24 Brazilian states had at least 1 sample from June/21 sequenced, with only the samples from Amapá, Roraima and Espírito Santo state absent. The choice of samples aimed to represent all regions of the country, while reflecting the incidence of SARS-CoV-2 in the period. For technical reasons, only samples with positive RT-PCR and Ct< 30 (Cycle Threshold) value were selected, corresponding to viral loads that allow the sequencing of the complete genome with high quality. The monthly number of sequenced samples will progressively increase until it reaches 3,000/month, which is the limit of GENOV's logistic-operational capacity.

Sample Processing

The samples are submitted to the RNA extraction process in Chemagic equip-ment, part of the automated sample pipetting and extraction system (Janus, Perkin-Elmer) using magnetic bead technology with the Viral DNA/RNA 960 kit (Perkin Elmer), in groups of 96 samples. The extracted RNA is eluted in 96 plates and subjected to cDNA synthesis and amplification of the viral genome by PCR in overlapping fragments of approximately 300 base pairs, with 2 pools of pri-mers already provided in the kit Illumina COVIDSeq Test kit (Illumina, CA, USA).

Sequencing of viral genomes

The viral genome sequences are obtained by Next Generation Sequencing (NGS) using reagents from the Illumina COVIDSeq Test and Mid Flowcell kits in NextSeq 550 equipment (Illumina, CA, USA).The processing of the raw data is conducted using the COVID Lineage pipeline on the Dragen analysis platform. Consensus sequence assembly, mapping and identification of variant sites with respect to the reference lineage (NC_045512.2) are carried out after quality control.

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Variant classification:

The classification of the lineages followed the dynamic classification system proposed by Rambaut and collaborators (2020), through the software Phylogenetic Assignment of Named Global Outbreak LINeages, available at https://github.com/cov-lineages/pangolin and also by NextClade. The definitive classification of the lineages was made after confirmation by phylogenetic analysis containing representative sequences of the main circulating lineages.

Results

TABLE 3 - LINEAGES DETECTED PER REGION

01 to 15 May 2021 01 to 15 June 2021

Northern Region P.1 P.1 P.1.2 P.1.2

95.2% (2021)95.2% (2021)4.8% (1/21)4.8% (1/21)

B.1.1P.1P.1.4P.1.7

3.3% (1/30)80.0 (24/30)3.3% (1/30)13.4% (4/30)

Northeastern Region P.1 P.1.7 P.1.2 B B.1.1 B.1 B.1.247 N.10

69.5% (34/49)4.1% (2/49)4.1% (2/49)2.0% (1/49)4.1% (2/49)10.2% (5/49)2.0% (1/49)2.0% (1/49)2.0% (1/49)

P.1 P.1.4 P.1.2. P.1.7 B B.1.566 N.10

88.4% (175/198)1.0 % (2/198)0.5 % (1/198)8.6 % (17/198) 0.5 % (1/198)0.5 % (1/198)0.5 % (1/198)

Midwest Region B.1 B.1.1.7 P.1 P.1.1 P.1.2

0.7% (1/140)2.2% (3/140)92.1% (129/140)4.3% (6/140) 0.7% (1/140)

B B.1.1.7 P.1 P.1.1 P.1.2 P.1.7

0.5% (1/216) 1.9% (4/216)87.5% (189/216)0.9 % (2/216)0.9% (2/216)8.3% (18/216)

Southeast Region* B.1.1.7 P.1 P.1.2 P.4

5.1% (6/117)91.4% (107/117)2.6% (3/117)0.9% (1/117)

P.1 P.1.1 P.1.2 P.1.7 P.1.8 P.4 P.1.4 B.1.1.28 B B.1.595

89.6% (190/212)0.5% (1/212)3.7 % (8/212)2.8 % (6/212)0.9% (2/212)0.5% (1/212)0.5% (1/212)0.5% (1/212)0.5% (1/212)0.5% (1/212)

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São Paulo City P.1 P.1.1 P.1.2

91.7% (33/36)5.5% (2/36)2.8% (1/36)

B.1.1.7 B.1.1.28 B.1.1 P.1 P.1.2 P.1.1 P.1.7

0.4% (1/254)0.4% (1/254)0.4 % (1/254)95.3% (242/254)1.6% (4/254)1.2% (3/254)0.7 % (2/254)

Rio de Janeiro City B.1.1.7 P.1 P.1.2 P.1.1 P1.8

1.1% (2/190)93.2% (177/190)2.6% (5/190)2.6% (5/190)0.5% (1/190)

P.1 P.1.1 P.1.2 P.1.3 P.1.7 P.1.8 B.1.617.2

92.0% (114/124)0.8% (1/124)2.4% (3/124)0.8% (1/124)1.6% (2/124)1.6% (2/124)0.8%% (1/124)

Southern Region B.1.1.7 B.1.1 P.1 P.1.2 P.1.1

1.3% (1/76)1.3% (1/76)89.5% (68/76)1.3% (1/76)6.6% (5/76)

P.1 P.1.2 P.1.1 P.1.7 B.1.617.2

86.4% (227/263)6.8% (18/263)0.4% (1/263)5.3% (14/263)1.1% (3/263)

*Except São Paulo city and Rio de Janeiro city

Predominance of P.1 (Gamma)

In May/2021, 605 of the 629 sequenced samples belonged to the P.1 variant and in June/2021 the number was higher, 1,277 of the 1,297 sequenced (Figure 1).

Figure 1. Frequency of the P.1 and non-P.1 variants in May and June/2021 among GENOV samples.

MAY/21 (N=629) JUNE/21 ( N=1.277)

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P.1 Diversification (Gamma)

Evolution of P.1 samples with mutation in S:P681 ("Gamma-Plus")(“Gama-Plus”)

Among the 605 samples of P.1 in May/21, 37 were classified as a derived lineage of P.1, here collectively referred to as P.1.n. In June/21, this number was slightly higher, totalling 116 out of the 1,277 P.1 samples (Figure 2).

A significant percentage (29%) of P.1 samples found in patients from Amazonas state in May/21 contained the P681H mutation. This variant (P.1+P681H) was called "P.1-plus" by the authors who initially described it, together with the variants P.1+NTDel (with deletions in the N-terminal domain of protein S) and P.1+N679K [Naveca F, et al, 2021]. Subsequently, variants containing P.1+P681H were given the denomination P.1.6 or P.1.7 according to the set of mutations present (see Table 2). In our previous report we described the identification of 12 "Gamma-plus" cases among 489 May/21 samples classified as Gamma (12/489= 2.4%). Eleven of these cases were P.1.7 and one was P.1.8 (P681R).

In order to monitor the evolution of these variants, we analysed the P681 site in another 210 Gamma samples from May/21 and in 1,232 Gamma sequences from June/2021:

1,372 have Proline at position 681 (ancestor) and 70 have a mutation at this position:

1 sample from SP has an L at position (P681L) - P.15 samples from RJ have R at position (P681R) - P.1.8 64 samples have H at position (P681H) - P.1.7. 1 AL; 2 CE; 4 DF; 6 GO; 12 MA; 1 MG; 8 MS; 3 PA; 3 PE; 11 PR; 1 RJ; 2 RS; 1 SC; 7 SP; 2 TO

It must be pointed out that particularly in the state of Maranhão (MA), 12 of the 18

Figura 2. Frequency of the P.1 and P.1 variants (P.1.n) in May and June/2021 among GENOV samples.

MAY/21 (N=629) JUNE/21 ( N=1.277)

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P.1 like-II

We also found 15 samples that have an intermediate phylogenetic relationship among P.1 strains and their direct ancestor B.1.1.28 (Figure 3). This strain, called P.1 like-II, was described a few months ago by the FIOCRUZ COVID-19 Surveillance Centre [Gräf, 2021].  The nomenclature of the strain, however, continues to be P.1 according to the adopted system (pangolin.cog).

These viruses have genetic characteristics that correspond to B.1.1.28 in addition to 15 of the mutations that define classical P.1. P.1 like-II also has 6 unique mutations (see Table 2) but, unlike what was described previously, some P.1 like-II do not have the synonymous mutation in ORF1ab C8905T.  This sublineage is believed to be an intermediate between B.1.1.28 and P.1, both of which originated in Brazil. These viruses were found by FIOCRUZ in different Brazilian states, with a predominance in the South and Southeast regions. This report shows the following distribution for the 15 GENOV samples that were classified as belonging to this sublineage: SC (1 sample), RS (2 samples), PR (11 samples) and for the first time, in the Federal District (1 sample) (see Figure 3). 

Figure 3. Maximum likelihood phylogenetic tree containing sequences of B.1.1.28, P.1 and P.1 like-II.

randomly sequenced samples contained the P.1.7 variant (see appendix). Therefore, the frequency of “Gamma-Plus” variants among P.1 samples went from 16/699 (2.3%) in May/21 to 66/1,232 (5.3%) in June/21.

Legend: The 15 P.1-like II samples detected by GENOV are highlighted in green in the large P.1-like II clade.

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Variant Delta (B.1.617.2)

Intra-Hospital Outbreak in Vaccinated Patients

Four cases of Delta variant were identified in June/21, three from Paraná state and one from Rio de Janeiro state, most probably due to two different introductions. We intend to further explore the analysis of this variant when a more significant number of samples are available, which should occur in the next 2 months (July and August) according to data from other Brazilian SARS-CoV-2 Genomic Surveillance programmes.

One of Genov's goals is to sequence samples from vaccinated people, who developed Covid-19 in more severe forms, or even outbreaks in vaccinated people indicating variants with potential vaccine escape ability.

We investigated an outbreak that occurred in a kidney transplant unit of a public hospital in the city of São Paulo. The outbreak occurred between 24 and 29/06/2021 and involved a total of 3 healthcare professionals and 7 kidney transplant patients. All healthcare workers had received full vaccination for COVID-19 with two doses of CoronaVac in January and February 2021, and all had asymptomatic or oligosymptomatic infections. The vaccination schedule for COVID-19 of kidney transplant patients was heterogeneous, with patients not having received any doses, partially vaccinated patients, and patients with a full vaccination schedule. All evolved with COVID-19 symptoms.

Ten RT-PCR+ samples were retrieved for sequencing; 9 were sequenced and one sample did not amplify SARS-CoV-2 RNA. All nine sequenced samples were identified as the Gamma variant, but they presented the same set of specific mutations that led us to conclude that all cases of the outbreak were infected by the same variant (Figure 3).

Figure 4. Phylogenetic tree clipping generated with 1,438 GENOV Gamma samples, shown in purple 8 samples from the outbreak of Covid-19 among healthcare professionals and patients in a renal transplant unit in São Paulo-SP, all vaccinated.

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Legend. The numbers correspond to the support of each cluster obtained by bootstrap analysis with 1,000 replicates.

Figure 4. Phylogenetic tree clipping generated with 1,438 GENOV Gamma samples, shown in purple 8 samples from the outbreak of Covid-19 among healthcare professionals and patients in a renal transplant unit in São Paulo-SP, all vaccinated.

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Gräf  T, et al. (2021) Identification of SARS-CoV-2 P.1-related lineages in Brazil provides new insights about the mechanisms of emergence of Variants of Concern. Available at: https://virological.org.

Naveca F, Nascimento V, Souza V, Corado A, Nascimento F. (2021) Emergen-ce and spread of SARS-CoV-2 P.1 (Gamma) lineage variants carrying Spike mutations 141-144, N679K or P681H during persistent viral circulation in Ama-zonas, Brazil. Available at: https://virological.org.

Rambaut, A., Holmes, E.C., O’Toole, Á. et al. A dynamic nomenclature pro-posal for SARS-CoV-2 lineages to assist genomic epidemiology. Nat Micro-biol 2020; 5:1403–1407. https://doi.org/10.1038/s41564-020-0770-5.

References

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APPENDIX - Variants per State

Northern Region

Northeastern Region

Date of collection between 01/Jun/2021 and 15/Jun/2021

AmazonasN=1P.1 100% (1/1)

BahiaN=49P.1 100% (49/49)

ParáN=18P.1 77.8% (14/18)P.1.7 16.7% (3/18)B1.1 5.5% (1/18)

CearáN=10P.1 80% (8/10)P.1.4 10% (1/10)P1.7 10% (1/10)

PernambucoN= 88B 1.1% (1/88)B.1.566 1.1% (1/88)P.1 92.1% (81/88)P1.2 1.1% (1/88)P1.4 1.1% (1/88)P1.7 3.5% (3/88)

AcreN=1P.1 100% (1/1)

MaranhãoN=18N.10 5.5% (1/18) P.1 27.8% (5/18)P.1.7 66.7% (12/18)

ParaíbaN= 6P.1 100% (6/6)

TocantinsN=7P.1 85.7% (6/7)P.1.7 14.3% (1/7)

AmapáN=0

RondôniaN=3P.1 66.7% (2/3)P.1.4 33.3% (1/3)

RoraimaN=0

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Midwest Region

Southeast Region

PiauíN=9P.1 100% (9/9)

Rio Grande do NorteN=6P.1 100% (6/6)

SergipeN=5P.1 100% (5/5)

Mato GrossoN=18P.1 100% (18/18)

São PauloN=408B.1.1 0.2% (1/408)B.1.1.28 0.5% (2/408)B.1.1.7 0.2% (1/408)B.1.595 0.2% (1/408)P.1 93.9% (383/408)P.1.1 1.0% (4/408) P.1.2 1.8% (7/408)P.1.4 0.2% (1/408)P.1.7 1.8% (7/408)P.4 0.2% (1/408)

Mato Grosso do Sul

N=52P.1 80.8% (42/52)P.1.1 1.9% (1/52)P.1.2 1.9% (1/52)P.1.7 15.4% (8/52)

Rio de Janeiro

N= 167B 0.6% (1/167)B.1.617.2 0.6% (1/167)P.1 90.4% (151/167)P.1.1 0.6% (1/167)P.1.2 3.6% (6/167)P.1.3 0.6% (1/167)P.1.7 1.2% (2/167)P1.8 2.4% (4/167)

GoiásN= 56B.1.1.7 1.8% (1/56)P.1 87.5% (49/56)P.1.7 10.7% (6/56)

Federal DistrictN=90B 1.1% (1/90)B.1.1.7 3.3% (3/90)P.1 84.6% (76/90)P.1.1 1.1% (1/90)P.1.2 5.5% (5/90)P.1.7 4.4% (4/90)

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Southern Region

Minas GeraisN=15P.1 80.0% (12/15)P.1.2 13.3% (2/15)P.1.7 6.7% (1/15)

ParanáN= 209B.1.617.2 1.4% (3/209)P.1 86.6% (181/209)P.1.1 0.5% (1/209)P.1.2 5.7% (13/209)P.1.7 5.3% (11/209)

Espirito Santo

N=0

Santa Catarina

Rio Grande do Sul

N= 28P.1 89.3% (25/28)P.1.2 7.1% (2/28)P.1.7 3.6% (1/28)

N= 26P.1 80.8% (21/26)P.1.2 11.5% (3/26)