CMPH Journal

Keywords: SARS-CoV-2; NGS; Variants; Lineages

Introduction

Since emergence of SARS-CoV-2 in December 2019, a large number of variants were reported across the globe [1]. However, emergence of Delta variant inflicted the most serious threat to public health and economy. This has alarmed for continuous monitoring for newly emerged variants for better preparedness and minimizing such catastrophic losses [2--6]. Thereafter, the other variant Omicron posed a serious threat to people and vaccine efficacy. The viral genetic makeup revealed unique changes in spike protein, which is the most important interacting viral protein with human receptors. The unique genetic patterns of spike protein are attributed to the enhancement in viral transmission patterns and evasion of human immune system [7-10]. Another important Omicron subvariant XBB was studied in depth by researchers due to its higher transmission among large population across the globe, evading the host immune response and failure of reported therapeutics [11].

The future challenges of emerging variants are not easy to tackle until continuous and detailed deciphering patterns of unique behaviours of each strain would be studied in depth to facilitate challenging public health interventions at global pace [12]. The next generation sequencing based continuous monitoring and deciphering unique viral characteristics with evolutionary dynamics can only help us to understand the behaviour of virus and changes required in existing diagnostic and therapeutic interventions. Here, we have reported the laboratory investigation of clinical samples, during the year 2024 and observed emergence of newer SARS-CoV-2 variants in central India.

Materials and Methods

Clinical samples: The clinical samples (Oro-pharyngeal/ Nasopharyngeal) were referred to Defence Research & Development Establishment (DRDE), Gwalior from sentinel District Hospitals of Madhya Pradesh, through the Indian SARS-CoV-2 genomic Consortium (INSACOG). Four referred positive SARS-CoV-2 clinical sample from Gwalior region of Madhya Pradesh state during the period of January to May 2024 were analyzed in this study.

Extraction of viral RNA and Confirmation of by SARS-CoV-2 specific RT-qPCR

The clinical swab samples were processed for extraction of viral nucleic acid employing viral RNA extraction kit (Qiagen, Germany) following the manufacturer’s instructions. Subsequently, the eluted viral RNA was used to perform SARS-CoV-2 gene specific multiplex RT-qPCR (Thermo, USA).

Next GenerationWhole Genome Sequencing (NG-WGS)

Library Preparation and nanopore sequencing

Firstly, SARS-CoV-2 positive RNA were processed for conversion of complimentary DNA (cDNA) employing Luna One-Step RTqPCR Kit (Sigma Aldrich, USA) followed by DNA amplification PCR using SARS-CoV-2 specific two sets of primer pools. Here the nanopore SARS-CoV-2 protocol with ‘midnight’ primer sets was carried out in two primer pools A and B respectively for each of amplified SARS-CoV-2 complementary DNA amplified through multiplex PCR reactions. These two pools were processed for multiplex PCR in a single tube with a reaction volume 10 µl (consisting of 1.5µl (100 M) primer pools in respective tubes, 8 µl of positive SARS-CoV-2 template RNA) further generated a tiled and non-overlapping amplicons of 1200 bp. The reaction of multiplex PCR was carried out through a reverse transcription (55 ⁰C, 30 minutes), denaturation (95 ⁰C, 1 minute) and a continuous annealing- extension of 34 cycles for each of primer pools A and B (denaturation (95 ⁰C, 20 sec) and annealing combined extension (60⁰C, 210 sec) followed by a final extension (65 ⁰C for 10 minutes). The amplified amplicons were pooled after multiplexing PCR represents the SARS-CoV-2 whole genome, which was processed for nanopore library preparation following the ONT Manufacturer’s instructions. We used a Rapid Barcoding Sequencing Kit protocol (ONT, Oxford, UK). Finally, sequencing was carried out in ONT MinION MK1B nanopore sequencer following manufacturers instructions under the MinKNOW software (ONT, UK). The COMMANDER software (Genotypic Technology, India) was used for bioinformatic analysis to generate the output files and interpret the results.

Library Preparation and Ion AmpliSeq sequencing

The SARS-CoV-2 whole genome sequencing was also carried out employing Ion Ampliseq technology of Ion Torrent S5 sequencer (Thermo, USA) following the manufacturer’s instructions. The SARS-CoV-2 whole genome Ion Ampliseq research panel was used to generate the 125-275bp amplicons designed against whole genome of SARS-CoV-2 virus. The Ion Ampliseq research insight primer pools contains 247 primer pairs to cover the whole genome of novel SARS-CoV-2. Firstly, the eluted viral RNA were converted to cDNA employing the SuperScript IV cDNA Synthesis Kit (Thermo Fisher Scientific,USA). Whole genome of SARS-CoV-2 was further amplified through PCR followed by ligation of adaptors. The ligated products were then purified using magnetic beads (Beckman Coulter, US) following the manufacturer’s instruction of library purification. These libraries were then quantified with the help of Qubit 4.0 Fluorimeter (Thermo Fisher, USA) employing DNA quantitation kits following the manufacturer’s instructions. Finally next generation sequencing of these samples were carried out in Ion Torrent S5 FNA sequencer (Thermo Fisher Scientifc, USA) in an Ion 540 sequencing chip to generate up to 1 million reads of paired-end per samples. The analysis of the results obtained trough Ion Torrent S5 sequencer were processed and analysed in Ion Torrent suite software version 5.2 (Thermo Fisher, USA) following the manufacturers instructions.

Mutation and evolutionary analysis of spike protein

A separate mutation and evolutionary pattern analysis of next generation sequencing amplified whole genome consensus fasta files were carried out using online software tools like Nextclade version 3.31 and Pangolin version 4.3.1 (https://clades.nextstrain. org). Here a comparative variation obtained against the different genes of SARS-CoV-2 were comparatively evaluated. We also did the divergence analysis manually employing MEGA 5.0 software. Further, we conducted a comparative mutation and phylogenetic analyses using open source Pangolin, Nextclade, and the Ion Reporter (Thermo Fisher, USA) softwares. The unique mutations were compared with the prototype Wuhan Hu-1 and other important SARS-CoV-2 variants. The phylogenetic tree was constructed for circulating SARS-CoV-2 to see viral evolutionary patterns with these unique variants circulating in India in 2024 employing the NextClade online software v.3.2.0. (https://clades.nextstrain.org/).

Results

Clinical samples and SARS-CoV-2 RT- qPCR

The ongoing SARS-CoV-2 genomic surveillance activities at DRDE Gwalior under INSACOG network received four SARS-CoV-2 positive clinical samples for sequence-based confirmation and whole genome molecular characterization during the period January to May 2024. The age profile of patients ranged above 50 years and with almost equal proportion of male and female. These samples were confirmed by SARS-CoV-2 specific qPCR with a Ct value ranging from 14 to 35 (Table 1).

Table 1: Details of SARS-CoV-2 positive samples

NGS based genome characterization and Mutational profiling of spike protein

We deciphered whole genome of SARS-CoV-2 (n=3) newly circulating variants in this particular part of India. The detailed genome coverage, generated SARS-CoV-2 genome reads were analyzed by variant calling on two different NGS sequencing platforms. Further, we assessed quality mapping scores, to find out the unaligned reads across the reference prototype novel 2019 SARS-CoV-2 genome. The genomic sequences deciphered by both Next Generation Sequencer revealed a high accuracy of approximately 99.99%. The details of genome wide coverage of each samples was listed in Tables 2. Here the samples with Ct values below 31 were successfully sequenced. However, we were unable to sequence the fourth sample of very high Ct value of 35. The amino acid substitutions of each isolate compared to prototype strain was depicted in Table 3.

Table 2: Genome analysis of SARS-CoV-2 sequenced in this study

Table 3: Amino acid substitutions in new SARS-CoV-2 variants.

Figure 1: Next Clade analysis.