Comparison of COVID-19 cases in Pune and those diagnosed at Bharati Hospital (March 2020-June 2021)
Figure 1A shows the number of COVID-19 cases each month in the city of Pune as reported by the Pune Municipal Corporation . The first case was detected on March 19, 2020, and the number of cases increased steadily until June, with a sharp rise in July 2020. At the peak in September 2020, 49,918 cases were reported, and this peak was followed by a sharp decline until February 2021. In March 2021, there was a rapid increase in the number of COVID-19 cases, which reached a peak of 150,175 cases in April 2021. Starting in the third week of April, the number declined sharply again, continuing through May and June 2021. During June 2021, 8,590 cases were reported. Figure 1B presents the number of patients advised and the number with confirmed COVID-19 diagnosis at BVDUMCH after permission was given by the government to provide diagnostic services. At Bharati Hospital, during the first wave of disease, the number of COVID-19 cases increased steadily from June to September 2020. The highest number (n = 800) was recorded in September 2020, followed by a sharp decline (n = 285) in October 2020. An increasing trend in the number of cases was observed during March 2021 that reached a peak (n = 3203) in April 2021 during the second wave of the disease. Clearly, the patterns of the number of COVID-19 cases detected at different times were identical when the city of Pune and Bharati Hospital were compared.
RBD sequence analysis
In view of the role of RBD in the attachment of the virus to the ACE2 receptor, in neutralization of the virus by antibodies, and as a major target for vaccine development, RBD sequences were monitored over time. Table 1 shows the frequency of selected mutations in the RBD region over a period of one year (May 2020 to June 2021). Until December 2020, none of the samples exhibited the characteristic mutations (K417N/T, E484K, N501Y, T478K) found in the variants of concern that have been identified so far. Two of the 15 samples from December 2020 contained the N440K mutation. At the beginning of March 2021, when the number of cases started showing an upward trend (Fig. 1B), we did not find the N501Y mutation, which is a characteristic of the UK variant (and shared by the Brazil and SA variants), or the K417N mutation that is found in the Brazil/SA variants.
Strikingly, 70% of the NPS specimens sequenced during the beginning of March 2021 and 83% of the NPS samples sequenced at the end of March contained the L452R mutation observed in the California variant. In addition, instead of the E484K mutation seen in strains from Brazil and South Africa, Indian strains exhibited an E484Q mutation, defined later as the Kappa variant. Overall, in the month of March, 33 out of 45 samples (73%) harboured the India-specific L452R/E484Q mutations. Week-wise analysis revealed the same trend, with 70% and 76% of Kappa-related cases in the first and last week of March 2021. A simultaneous exponential rise in the number of COVID-19 cases (Fig. 1B) revealed a clear association of the emergence of this mutant with the second wave of the disease. Unfortunately, we did not collect samples during January or February, and hence, the possibility of earlier detection was missed. As shown in Table 1, we identified the V382L mutation in 4.8% of NPS specimens in 2021.
Analysis of 91 RBD sequences from the samples collected during the month of April led to striking observations. At that time, 34 variants contained L452R/E484Q (37%), while 54 sequences harboured L452R/T478K mutations (59%), characteristic of the Delta variant. One sequence each exhibited the N501Y mutation characteristic of the UK strain, an N440K mutation, and no mutation. Overall, the L452R mutation increased from 0/30 in December to 123/136 (90%) in April (p < 0.001). Importantly, as compared to March (35/45), a significant rise in L452R mutants was seen in April (88/91, p = 0.001). Interestingly, at the same time, the frequency of the T478K mutation increased dramatically from 4% in March to 59% in April (p < 0.001). The increasing frequency of the dominant mutation L452R/T478K continued in May and June 2021, but we experienced a significant decline in the number of patients seeking COVID-19 diagnosis at the hospital (Table 1). Our results indicated that the dominant clade G virus strains in Pune seem to have been replaced by the variant of concern Delta (L452R/T478K) during the second wave of the disease.
Analysis of SARS-CoV-2 full genome sequences from Pune, India
During 2020-21, 20 full SARS-CoV-2 genomes from Pune were sequenced (two in May and two in September of 2020; 10 in March, two in April, and four in May of 2021). The four mutations, C241T, C3037T, C14408T, and A23403G, were observed in all 20 genome sequences from the clade “G” isolates (named after the D614G mutation). Of the 10 genomes from March 2021 selected for sequencing, four were wild type, while six were the Kappa variant (B.1.617.1) according to RBD analysis.
As shown in Table 2, two sequences each from May and September 2020 belonged to lineage B.1.1.306 and clade 20B. Of the 10 sequences from March 2021, two (CD211295 [MW969753] and CD210761 [MZ021503] belonged to the original prevalent lineage B.1.1.306, clade 20B, while the other two sequences, CD210922 (MW969752) and CD210896 (EPI_ISL_1710598) belonged to separate lineages, B.1.1 (clade 20B) and B.1.36.29 (clade 20A), respectively, suggesting simultaneous low-level circulation of the wild-type virus. The remaining six sequences from March 2021 (CD210871 [MZ021506], CD210927 [MZ021505], CD210929 [MZ021504], CD211290 [MW969754], CD211294 [MW969755], and CD211406 [MW969756] formed a distinct lineage, B.1.617.1, Kappa variant (clade 21B). Two sequences from April 2021 (CD212095, CD212098) and four sequences from May 2021 (CD213366, CD213522, CD213523, and CD213570) formed a distinct clade, 21A, and belonged to the B.1.617.2 lineage (Delta variant, VoC).
Complete genome sequence analysis of sample CD210896 (EPI_ISL_1710598) revealed an in-frame stop codon in Orf3a at amino acid position 261 (nucleotide position 26,173). The first strain with this amino acid change was reported in Germany (hCoV-19/Germany/NW-KRO-2355/2020) in March 2020 and more recently in England in June 2021 (hCoV-19/England/HSLL-17FBAD3/2021). It has been reported in 35 countries so far (https://www.gisaid.org/epiflu-applications/covsurver-mutations-app/).
Next, spike protein sequences from the Indian variants were compared with those of other known variants (Table 2). The Indian variants formed two distinct clusters, B.1.617.1 (Kappa) and B.1.617.2 (Delta). The presence of the D614G mutation in all of the Indian sequences revealed that clade G continued to be the only clade circulating so far in Pune. Mutations specific to the recently emergent variants (UK, South Africa, Brazil, and California) were not shared by the Indian variants; however, signature mutations of the Delta variant were observed in all six sequences obtained in April and May of 2021.
Two sequences from 2021 (CD211295 [MW969753] and CD210761 [MZ021503]) belonging to the original prevalent lineage B.1.1.306 had four characteristic mutations, L18F, A27S, E484K, and Q675H, in the spike protein. None of these mutations were present in the four sequences from 2020, suggesting that SARS-CoV-2 is continuously evolving. Two isolates, CD210922 (MW969752) and CD210896 (EPI_ISL_1710598), had the unique mutations V143F, Q677H, and N440K (Table 2). All six Indian variants of the B.1.617.1 lineage exhibited five unique mutations: G142D, L452R, E484Q, P681R, and Q1071H. Additionally, the E154K mutation was found in five and the V382L and D1153Y mutations were found in four of the six Kappa variants. For the Delta variant, all six sequences belonged to B.1.617.2 lineage and had six unique mutations (T19R, G142D, L452R, T478K, P681R, and D950N). Together with the substitution E156G, two deletions at F157 and R158 were identified. Notably, the position of the P681R mutation in the Kappa and Delta variants is immediately adjacent to the furin cleavage site (682-685). In vitro site-directed mutagenesis experiments have shown that this mutation leads to an increase in the fusion activity of the SARS-CoV-2 spike protein . Importantly, P681H was present in the UK variant B.1.1.7 (Table 2). The acquisition of A222V and K417N by the Delta variant (AY.2 lineage) was associated with an upsurge in COVID-19 cases in Europe and the USA, and importantly, these amino acid changes were not found in the Delta variants from this study.
The Indian variants were distinct in this protein as well (Table 3) and did not share the D3L and S236F mutations and the P80R mutation seen in the UK and Brazilian variants, respectively. The two Indian sequences (CD211295, CD210761) had tyrosine (Y) instead of leucine (L) at the third amino acid position, as was observed in the UK strain. The R203M and D377Y mutations were observed in all twelve Kappa and Delta variants. At position 203, the change is from a hydrophilic arginine to a hydrophobic methionine, while at position 377, aspartic acid is changed to tyrosine. The biological significance of these mutations needs to be evaluated. The remaining Indian sequences continued to harbour the R203K and G204R mutations, which were present since May 2020. One Indian sequence (CD210896_EPI_ISL_1710598) was unique, without amino acid substitutions at positions 203 and 204. Like the Delta variant, the six Indian variants contained the unique mutation D63G.
Other genomic regions
The other genome regions were also compared with the Wuhan strain as well. The Kappa-variant-specific mutations were Nsp3-T749I, Nsp6-T77A (Orf1a), Nsp12-P323L, Nsp13-M429I, Nsp15-K259R (Orf1b), S26L (Orf3a), I33T (Orf6), and V82A (Orf7a). The other proteins (E, M, Orf7b, Orf8, and Orf10) remained unchanged in the Kappa variant. The Indian Delta variant-specific mutations were G210T (5’UTR) Nsp2-P129L, Nsp3-P822L, Nsp4-A446V, Nsp6-V149A (Orf1a), Nsp12-P323L, Nsp12-G671S, Nsp13-P77L (Orf1b), S26L (Orf3a), I82T (M), V82A, T120I (Orf7a), and deletions of D119 and F120 in Orf8. The other proteins (E, Orf6, Orf7b, and Orf10) remained unchanged in the Delta variant.