key: cord-0895748-4kii9ir9
authors: Ranasinghe, D.; Jayathilaka, D.; Jeewandara, C.; Gunasinghe, D.; Ariyaratne, D.; Jayadasa, T.; Kuruppu, H.; Wijesinghe, A.; Bary, F.; Madushanka, D.; Pushpakumara, P.; Guruge, D.; Wijayamuni, R.; Ogg, G.; Malavige, G. N.
title: Molecular epidemiology of AY.28 and AY.104 delta sub-lineages in Sri Lanka
date: 2022-02-07
journal: nan
DOI: 10.1101/2022.02.05.22270436
sha: 44d4c8d749ff768122d8c498d9009c1646a82f0c
doc_id: 895748
cord_uid: 4kii9ir9

Background: The worst SARS-CoV-2 outbreak in Sri Lanka was due to the two Sri Lankan delta sub-lineages AY.28 and AY.104. We proceeded to further characterize the mutations and clinical disease severity of these two sub-lineages. Methods: 705 delta SARS-CoV-2 genomes sequenced by our laboratory from mid-May to November 2021 using Illumina and Oxford Nanopore were included in the analysis. The clinical disease severity of 440/705 individuals were further analyzed to determine if infection with either AY.28 or AY.104 was associated with more severe disease. Sub-genomic RNA (sg-RNA) expression was analyzed using periscope. Results: AY.28 was the dominant variant throughout the outbreak, accounting for 67.7% of infections during the peak of the outbreak. AY.28 had three lineage defining mutations in the spike protein: A222V (92.80%), A701S (88.06%), and A1078S (92.04%) and seven in the ORF1a: R24C, K634N, P1640L, A2994V, A3209V, V3718A, and T3750I. AY.104 was characterized by the high prevalence of T95I (90.81%) and T572L (65.01%) mutations in the spike protein and by the absence of P1640L (94.28%) in ORF1a with the presence of A1918V (98.58%) mutation. The mean sgRNA expression levels of ORF6 in AY.28 were significantly higher compared to AY.104 (p < 0.0001) and B.1.617.2 (p < 0.01). Also, ORF3a showed significantly higher sgRNA expression in AY.28 compared to AY.104 (p < 0.0001). There was no difference in the clinical disease severity or duration of hospitalization in individuals infected with these sub lineages. Conclusions: Therefore, AY.28 appears to have a fitness advantage over the parental delta variant (B.1.617.2) and AY.104 possibly due to the A222V mutation. AY.28 also had a higher expression of sg-RNA compared to other sub-lineages. The clinical implications of these should be further investigated.

The SARS-CoV-2 virus continues to result in outbreaks in many geographical regions, with the number of cases exponentially increasing in many countries due to the rapid transmission of Omicron 1 . Of the five variants of concern (VOCs) that have been identified so far, the delta variant is associated with more severe disease compared to other variants 2,3 . Until Omicron emerged, the delta variant was the most transmissible variant and rapidly displaced all other VOCs and variants of interest 4 . Due to the higher transmissibility and increased virulence of the delta variant, outbreaks due to the delta wave were associated with the highest mortality, intensive care admissions and hospitalizations so far in all countries 1 .

As the delta variant was the dominant variant globally before emergence of Omicron for the longest time period during the COVID-19 pandemic, it gave rise to over 100 sub lineages.

Phylogenetic Assignment of Named Global Outbreak (PANGO) nomenclature has currently assigned 122 sub lineages of delta (AY.1 -AY.122) that are distributed in different geographical regions 4 . The sub-lineages of the delta variants have not shown to be functionally different to the parental delta variant (B.1.617.2) and have shown to have a similar susceptibility to neutralizing antibodies 5 . However, some of these sub lineages such AY.4.2 have been assigned as a variant of interest due to its possible higher transmissibility compared to B.1.617.2 the parental delta variant 6 . While certain mutations such as the presence of A222V has shown to cause slightly higher viral titres, which is thought to result in a higher transmissibility 7 , mutations such as the E484K have a possibility of enhanced immune evasion 8 . Therefore, it is important to study the . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 7, 2022. ; https://doi.org/10.1101/2022.02.05.22270436 doi: medRxiv preprint evolution and spread of different delta sub lineages to understand their transmission and to detect possible changes associated with virulence and immune evasion.

The largest Sri Lankan SARS-CoV-2 outbreak was due to the delta variant and its sub lineages from July to end of October 2021 9 . During the peak of this 'delta wave' the PCR positivity rates rose above 30% with case fatality rates reaching 6.35% 9 . Apart from the delta parental lineage B.1.617.2, two other delta sub lineages AY.28 and AY.104 were the predominant variants observed during this time period 4 . The AY.28 and AY.104 were assigned as Sri Lanka delta sub-lineages as they were found to originate in Sri Lanka and to be transmitted to all continents in the world 10,11 . In this study, we discuss lineage defining mutations, sub genomic RNA expression, relative frequency over time and clinical disease severity of individuals infected with either AY.28, AY.104 or B.1.617.2 in Sri Lanka.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The clinical disease severity and vaccination status of 440/705 individuals who were found to be infected with the delta variant during this time period were further analyzed in order to determine if infection with either AY.28 or AY.104 was associated with more severe disease or with the type of vaccination. Those who were not hospitalized or who were hospitalized and were not given oxygen were considered as having mild illness, whereas those who were given oxygen or required intensive care admission were classified as moderate/severe based on the WHO guidelines in COVID-19 clinical disease classification 12 . Pearson's Chi-squared test was used to determine the . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 7, 2022. ; https://doi.org/10.1101/2022.02.05.22270436 doi: medRxiv preprint associations between categorical variables (gender, vaccination, vaccine dose, and disease severity) and sub-lineages while pairwise T-tests with Bonferroni correction to compare means of age and hospitalization period. All statistical tests were done using R version 4.1.2.

In addition to the delta variants sequenced by us, all the AY.28 (n=519) and AY.104 (n=493) sequences available at GISAID were downloaded from the GISAID database and aligned to the reference sequence using Nextalign (https://github.com/neherlab/nextalign). Amino acid mutations and their frequencies for Spike, ORF1a, ORF1b, and N proteins were calculated, and frequencies of ambiguous amino acids derived from ambiguous nucleotides were removed using in-house python scripts. The phylogenetic tree was inferred by Maximum Likelihood in IQ-Tree (version 1.6.12) using the GTR+G model of nucleotide substitution and 1000 replicates of ultrafast bootstrapping (-B 1000) and SH-aLRT branch test (-alrt 1000). The ML tree was then time stamped with TreeTime 13 (version 0.7.5) using least-squares criteria and the evolutionary rate of 1.1*10 -3 subs/site/year as described by Duchene et a 14 . Six sequenced with inconsistent temporal signal were removed from the analysis. The tree was rendered using ggtree in R version 4.1.2.

Sub-genomic RNA expression of 705 delta genomes (335 AY.28, 217 AY.104 and 68 B.1.617.2) were analyzed using periscope (https://github.com/sheffield-bioinformatics-core/periscope). This algorithm aligns raw reads against the SARS-CoV-2 reference genome (MN908947.3) and identifies reads that contain the leader sequence at their start position. Depending on the amplicon . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 7, 2022. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 7, 2022. ; https://doi.org/10.1101/2022.02.05.22270436 doi: medRxiv preprint

We have previously described the changes in the SARS-CoV-2 variants from the onset of the pandemic to May 2021 in Sri Lanka 15 . The first delta variant was identified in Sri Lanka on 22 nd May (AY.28), and the relative frequency of delta and sub lineages in Sri Lanka is shown in Figure   1A . 

Of the 440 individuals who were infected with the delta variant, the mean age of those infected with AY.28 was 38.39 (SD± 15.32) and AY.104 was 38.27 (SD± 17.36) and therefore was not significantly different (p=0.86). There was no difference in the gender of those who were infected . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 7, 2022. ; https://doi.org/10.1101/2022.02.05.22270436 doi: medRxiv preprint association between the number of vaccine doses and infection with different sub-lineages (p= 0.69). Association between vaccine and sub-lineage was insignificant (p= 0.83).

In this study we have described the molecular epidemiology of the delta variant and its sub lineages during a massive outbreak in Sri Lanka, which occurred from July to November 2021. We 19 . Although our data showed that there was no difference in the total sgRNA levels between the two sub-lineages and the parental delta, sg RNA expression was significantly higher in AY.28 for ORF3a, ORF6 and ORF7a compared to the other two lineages.

All these ORFs play an important role in evading the host interferon responses by suppressing STAT1 and STAT2 phosphorylation, inhibiting STAT1 complex nuclear translocation and interacting with STING and preventing nuclear translocation of NFκβ [19] [20] [21] . Therefore, increased . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 7, 2022. ; https://doi.org/10.1101/2022.02.05.22270436 doi: medRxiv preprint expression of sgRNA of ORF3a, ORF6 and ORF7a by AY.28 compared to other lineages, may associate with increased virulence due to suppression of host IFN responses. AY.104 had a higher sgRNA expression of ORF8 and the N genes compared to the other lineages. ORF8 has shown to inhibit IRF3 nuclear translocation and N protein has shown to inhibit RIG-1 signaling 19 . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 7, 2022. ; https://doi.org/10.1101/2022.02.05.22270436 doi: medRxiv preprint

In conclusion, the massive outbreak due to the delta variant in 2021 was predominantly due to two delta sub lineages AY.28 and AY.104. These two Sri Lankan sub lineages accounted for over 80% of the sequenced delta variants in Sri Lanka from July to December, while AY.28 was the predominant sub lineage. AY.28 is possibly more transmissible than the other two lineages potentially due to the A222V mutation and significantly higher sgRNA expression was detected in ORF3a, ORF6 and ORF7a suggesting possible enhanced suppression of interferon genes compared to the parental delta variant. It would be important to further investigate the relevance of the findings by isolating these viruses, in order to understand the evolution and virulence of SARS-CoV-2 variants during this pandemic.

World Health Organization; World bank, Sri Lanka Covid 19 Emergency Response and Health Systems Preparedness Project (ERHSP) of Ministry of Health Sri Lanka funded by World Bank.

All data are available in the manuscript and the supporting files. The source code and data used to produce the results and analyses presented in this manuscript are available on GitHub repository https://github.com/aicbu.

None of the authors have any conflicts of interest.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 7, 2022. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 7, 2022. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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