Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was declared pandemic by WHO in march 2020 soon after its emergence in Wuhan, China in December 2019 due to its rapid spread in the whole world. After its emergence, different waves were observed in many parts of the world due to the mutations in the genome of COVID-19. These waves were caused by different variants and their lineages and these lineages differ from each other in terms of their transmission potential and immune escape potential. The number of COVID-19 cases is on continuous rising trend all across the world.

India has observed three different waves of COVID-19 since its emergence. The different variant that was responsible for these waves were known by the names of alpha, beta, gamma, delta and so on. These variants were identified with the help of whole genome sequencing (WGS) which serves an important tool for various reasons. Firstly, it is helpful for the study of virus evolution over time and also predicting the trends of disease transmission. Also, it helps to determine the geographic prevalence of disease. Lastly, it can help understand the most effective design and platform for developing vaccines and therapeutics.

The second wave of coronavirus disease 2019 was due to delta variant that emerged in April 2021.⁹ This variant was responsible for high morbidity and mortality as well as increase in number of hospitalization, high requirement of oxygen and cases of mucormycosis too. These were the reasons why sequencing the genome of coronavirus became important to know the variant responsible for high morbidity and mortality.

Omicron variant of COVID-19 emerged in month of November 2021 in South Africa and spread to other parts of the world. This study was conducted to reveal the most predominant lineage of Omicron variant that emerged as third wave of COVID-19 in January 2022 in different districts of Uttarakhand.

In the present study, a total of 2149 samples were studied during a period of 09 months (January 2022 till September 2022). These samples were positive for COVID-19 and were received from different districts of Uttarakhand. Demographic data was collected for all the samples and mean age of presentation of the patient included in the study was 37.9±18.45 years and most common age group to be affected was 21-40 years (45.3%) followed by 41-60 years (24.8%) as shown in Table 1. This finding of our study was consistent with the study conducted by Raju et al. in Chennai, Garg et al. in Delhi and Zaman et al. in Gorakhpur with the mean age of 37 years, 3 years and 33 years respectively.^10,11,12  Male preponderance over females was also a finding in our study which also corroborated with the findings of the study mentioned above.

Table (1):
Demographic data of all the samples tested

In the present study, sequencing of the samples from January 2022 showed that majority of the samples belonged to Omicron variant and only two samples were positive for delta variant which suggested that the peak in the third wave of COVID-19 was due to Omicron variant and delta variant (predominant in second wave) was replaced by Omicron variant. Figure 1 shows all the lineages that were detected in the samples sequenced. Table 2 shows the predominant lineages of omicron variant in which BA.2 was detected in the majority of the samples followed by BA.2.75. Delta variant was predominant till December 2021 and this finding was also consistent with the findings of the study conducted by Sharma et al. in Rajasthan who performed WGS analysis of SARS-CoV-2 positive cases from Nov. 2021 to Jan. 2022.¹³ Potdar et al. from Indian Council of Medical Research, National Institute of Virology, Pune also studied the effect of Omicron on Indian population after its emergence in South Africa and found that B.1.1.529 was responsible for infection in the initial cases whereas BA.1 lineage was responsible for infection in later contacts which also led to third wave of COVID-19.¹⁴ In more than 50 countries including United Kingdom, the United States, France, Italy, Netherlands, and India, Omicron variant caused new wave and this was reported in the study done by Ranjan in the year 2022.¹⁵

Table (2):
Incidence of predominant lineages that were circulating in population in total sample sequenced

In the present study, BA.1 lineage of Omicron was first detected on 3^rd January 2022 along with some samples positive for BA.2 detected simultaneously. This sub-lineage (BA.1) of Omicron variant was considered as original form of Omicron variant as described by Rana et al.⁷ The findings of the present study depict that sub-lineage BA.2 started peaking from January 2022 soon after it was identified as shown in Table 3. This variant was also replaced by other subvariant of Omicron BA.2.38 which was first detected in May 2022 and peaked in June 2022 as shown in Figure 2. INSACOG (Indian SARS-CoV-2 Genomic Consortium), a national agency also reported that BA.2 Omicron variant was replaced with BA.2.38 as seen in this study.¹⁶

Table (3):
Trends in evolution of variant of concern from January 2022 till September 2022

However, this variant was also soon replaced by BA.2.75 that was first identified in the month of June 2022 but peaked in July 2022. BA.2.75, BA.2.12.1, BA.4 and BA.5 were also placed as Variant of Concern Lineage Under Monitoring (VOC-LUM) in July 2022 according to WHO.¹⁷ In India, at least 23 samples of BA.2.75 were recorded at the time of its emergence in the data uploaded on Nextstrain which is an open-source platform of genomic data and these were reported from areas such as Maharashtra, Karnataka, and Jammu & Kashmir. This variant was also detected outside India, such as in Australia, Canada, Germany and New Zealand.¹⁸ In the present study also, BA.2.75 was mainly reported near the month of July and August 2022 which makes it a consistent finding. Tamil Nadu and Telangana were those southern states from where BA.5 was mainly reported. In the present study, various other mutations were also identified from NGS such as BA.2.75.1, BA.2.75.2, BA.2.76 and BA.5.2, however, the most predominant lineage of them all was BA.2.75 which was present in maximum number of samples as shown in Figure 2.

In the present study, amongst the most predominant lineages that were circulating in Uttarakhand, we observed that in Dehradun district majority of the cases were of BA.2 lineage which was replaced by its sub-lineages BA.2.75 and BA.2.75.1 in the month of July, whereas, in Haridwar district the majority of the cases were of BA.2 lineage followed by BA.2.75 lineage which was present in few cases. Other districts majorly showed the presence of BA.2 lineage as shown in Figure 3 and Figure 4. There were some other lineages also that were circulating in the population of Uttarakhand such as BA.2.76, BA.2.75.1, BA.2.75.2 and BA.5. These all lineages were reported from all districts of Uttarakhand with majority being reported from Dehradun and Haridwar. This observation was between the period of 9 months (Jan. 2022 till Sept. 2022). Similarly, in a study done in Delhi, initial few cases were of BA.1 lineage (73.1%) while BA.2 lineage was present in 26.8% cases amongst all the cases in December 2021. Zaman et al. from Gorakhpur also found a shift of sub-lineages of Omicron variant in their study where amongst the samples of January and February 2022 the majority were of BA.2 sub-lineage (82.3%).¹²

The presence of this high number of Omicron lineage and sub-variant in the population of Uttarakhand and this shift of lineages from one to other at different point of time suggests that there was persistence of Omicron variant in the population and the virus kept on mutating to survive in the population. The presence of these mutant strain can also be due to the fact that Uttarakhand being high in tourism, so the people coming from other states can also bring the mutant strain from outside. It was also noticed that the peaks of these mutant strain corresponded to the month that were high in tourism since no major lockdown was imposed in the state so there was free interstate transmission of people.

In the present study, there were several mutations that were identified by Next Generation Sequencing in the structure of Omicron variants that confers various benefits to the virus for its maintenance and survival in the population as shown in Table 4. These mutations are responsible for immune escape, increasing binding capacity, improves cell surface entrance and effective cellular invasion. However, due to the fact that vaccination was given to mass population worldwide and also due to the active immunity acquired during infection, the severity of the disease was not much increased in spite of these high number of alterations in the Omicron variant.¹⁹ Figure 5 shows the phylogenetic distribution of SARS-CoV-2 genome in the population of Uttarakhand where all the genome was identified by next generation sequencing. However, phylogenetic tree of the predominant lineage that was identified (BA.2.75 and BA.2) was also constructed through INSACOG portal which is depicted in Figure 6a and 6b. The major mutations that were present in all the lineages were G339D, S373P, S375F, K417N, S477N, Q493R, N501Y, N440K, G446S, T478K, E484A, Q498R, Y505H. The location of these mutations was in the spike protein (receptor binding domain). These amino acid substitutions are also described by CDC.⁸ In other structural gene, E protein contains T9I mutation, M protein contains Q19E and A63T mutation and N protein contains P13L, R203K, G204R and S413R mutations. N gene in different lineages of Omicron also contains deletion of 31-33 amino acid position. Rest of the genome analysis showed that in ORF1a gene, in all the variants, common mutations were present such as S135R, L3027F, T3090I, P314L, R1315C, T2163I and L3201F. However, in lineages such as BA.2, BA.2.38 and BA.5.2, BA.2.75, one deletion in the gene del3675/3677 was also present and its sub-lineages also comprised of additional mutations such as S1221L, P1640S, N4060S and G662S. These mutations being exclusive for this lineage. In all detected lineages of Omicron, ORF6 showed D61L mutation except BA.5.2, while ORF8 showed mutation S84L.

Table (4):
Lineages that were identified in the study, including their mutations detected by NGS

Note: red and bold indicates the major mutations present in different lineages of Omicron variant in ORF1a, S and N gene.

In the lineage BA.2 which was the most predominant lineage as identified by NGS in our study, the mutations that were identified were responsible for the increased affinity of S-protein towards the ACE2 receptors and also lead to increased transmissibility of the Omicron variant.²⁰ Mutations such as N764K, D796Y, Q954H and N969K have been responsible for change in entry of virus in the host cell and also transmissibility.²¹

Mutations such as P681H, H655Y, and N679K present in the variants are associated with increased pathogenicity by facilitating S-protein cleavage by furin and thereby helping in transmission.¹⁸ Increase in transmissibility and infectiousness of the disease is also associated with several other mutations such as Q498R, S477N, and N501Y which are also responsible for increase in binding capacity of S-protein with ACE2 receptors. However, mutations such as T478K and E484A are those that are responsible for increase in neutralization of antibody resistance and higher immune escape.^22,23 Additionally, the infectivity of SARS-CoV-2 virus was increased due to the D614G substitution which is located in the S protein and is conserved in all the variant of Omicron.²⁴ The mutations present in nucleocapsid protein (R203K and G204R) are linked with enhanced sub genomic RNA expression and viral replication.²⁵

The sub-variant BA.2.75 soon displaced other sub-lineages due to its adaptation to new mutations. This variant also showed large number of mutations in the viral spike protein amongst which some were similar to the mutations that were present in the other lineages but some mutations were different such as G257S, D339H, N460K, and G446S. Higher infectivity of BA.2.75 (up to 44-fold) was due to the mutation such as Q493R, N460K and alteration of G339D to G339H which in all together impose fusogenicity.²⁶ The mutation G446Shave potential to affect immune evasion and ACE2 binding. It can lead to considerable immunological escape which suggest that reinfection and breakthrough infections may be leading to the spread of BA.2.75 and thus increasing the number of cases.^27,28 Immune evasion was reported more in BA.2.75 lineage than BA.5 lineage especially in cases which were infected earlier with delta variant.²⁹ This fast transmission of BA.2.75 could be due to the mutation such as S1221L, P1640S present in ORF1a gene which was absent in BA.2.

Limitation
The present study’s limitation include that no data was available about the clinical status of the patient due to which a potential relationship between the predominant lineage and the severity of the disease could not be made. Also, the travel status, reinfection could not be retrieved from the data which also led to failing of defining any relationship between the predominant lineage to be established.