Clinico-demographic characteristics of the JN.1 cohortThe ongoing SARS-CoV-2 genomic surveillance activities at SPHL have identified the dramatic emergence of the JN.1 variant of omicron by replacing the XBB variant between November 2023 and December 2023 (Fig. 1A). The age of the JN.1 positive patients ranged from 1 to 89 years, with the median age during the study period being 51 years (Fig. 1B) and with an equal proportion of male-to-female ratio (Fig. 1C). More than 50% of the JN.1 patients had underlying diabetes mellitus, followed by 21% of the cases with hypertension, and 10% of the cases had both diabetes mellitus and hypertension (Fig. 1D). About 87% of the patients were presenting with fever, cold, cough and sore throat (pharyngitis) (Fig. 1E,I). On analysing the vaccination status of the JN.1 positive patients, 93.5% had received COVID-19 vaccinations, indicating a high proportion of vaccine breakthrough infections (Fig. 1F). In total, 63 (73.2%) patients had received two doses while 21 (24.4%) patients had received at least 3 doses of vaccines against COVID-19. Among the vaccinated, 98% had received two or more doses of the COVID-19 vaccines (Fig. 1G). About 45% of the JN.1 patients were hospitalized, of which 20% had severe illnesses requiring oxygen support and intensive care unit (ICU) or high dependency unit (HDU) care with ventilator support although no deaths were recorded (Fig. 1H). Among the different symptoms/signs presented, fever (pyrexia) was the most common presentation (69%) and was predominant among the 10–20 years (86%) and 80–90 years (80%) of age. The other common symptoms found were cough (55%) which was predominant in older age groups, rhinorrhea (20%) in younger age groups and sore throat predominant in 20–40 years (36%) and 60–80 years (32%) age groups. Breathlessness (6%) and myalgia (1.8%) were less prevalent, and were mostly noticed in the older age groups only (Fig. 1I). Among the vaccinated, 42% of patients required hospitalization, 12% required oxygen support and ICU/HDU support and 10% required ventilation support.Figure 1Distribution dynamics and clinico-demographic characteristics of the study population. (A) Analysis of the proportions of different SARS-CoV-2 variants circulating in Tamil Nadu over time. The x-axis represents the timeline, the y-axis represents the proportion, and different colors represent distinct variants. (B)–(H) Clinico-demographic characteristics, where (B) median age, (C) gender, (D) symptoms, (E) comorbidity, (F) vaccination status, (G) number of doses received, (H) medical support, and (I) Common symptoms among different age groups. Note: All patients recovered from COVID-19.Mutational analysis of spike protein of JN.1 omicron SARS-CoV-2 variantThe S gene sequences of the 66 JN.1 variants were analysed with different reference genomes, and revealed a slew of unique mutations in the different domains of the S protein including the receptor binding domain (RBD), signal peptide (SP) and N-terminal domain (NTD) (Fig. 2 and Table 1).Figure 2Amino acid changes in the different domains of JN.1 in comparison with Wuhan-Hu-1, BA.2, and XBB omicron variants. (Red color indicates the unique mutations, the blue color indicates the dynamic mutations, the black color indicates random mutations and pale red indicates universal mutations). Footnotes: FP Fusion peptide, HR1/2 heptad repeat, NTD N-terminal domain, RBD receptor binding domain, SP signal peptide, SD spike subdomain, TM transmembrane domain. The aforementioned are the different motifs of the SARS-CoV-2 spike glycoprotein.Table 1 Unique and dynamic mutational pattern in spike protein among different lineages.Comparison of spike protein sequences of JN.1 with Wuhan-Hu-1
While comparing the JN.1 isolates with the reference sequence of the Wuhan-Hu-1 strain, we identified 22 unique mutations and three dynamic mutations. Of the unique mutations, the majority were found in the NTD (n = 16), followed by heptad repeat (HR1/2) (n = 3) and spike subdomain (SD) (n = 2), whereas RBD showed only one mutation (V503A). Among the dynamic mutations, two were seen in NTD (P85K and V90Y) and one in the signal domain (P681H). Further, JN.1 showed six universal mutations (three in NTD, two in RBD-N440K and N501Y and one in SD-D614G). This also encompassed certain universal mutations, such as G142D, which have been reported in several countries10,11. Upon analysis of random mutations, we found that JN.1 variants showed 40 mutations across the spike protein as compared to that from Wuhan-Hu-1 with a maximum number of mutations located in the RBD (23) followed by the NTD (12) regions (Table 2 and Fig. 2A).Table 2 Unique, dynamic, universal, and random mutations in the spike protein of JN.1 compared to the Wuhan-Hu-1 sequence.Comparison of spike protein sequences of JN.1 with BA.2 omicron variantOur results from the comparison of JN.1 with the BA.2 variant showed unique mutations, including V83F, L84K, P85L, F86L, N87I, D88V, V90L, Y170E, Q218L, and T307R and dynamic mutations such as P85L, V90Y in the NTD domain, and the following unique mutations T333R, F456L, Q506P, and P507T within the RBD domain. There was no evidence of universal mutations within any of the aforesaid domains. This shows that the amino acid changes as compared to BA.2 were identified individually (Table 3 and Fig. 2B). We also found a total of 34 random mutations when comparing JN.1 and BA.2 with 10 mutations found in the NTD and 18 in the RBD regions. The fusion peptide (FP) domain showed one mutation viz., H681R.Table 3 Unique, dynamic, universal, and random mutations in the spike protein of JN.1 as compared to the BA.2 sequence.Comparison of spike protein sequences of JN.1 with the omicron variant XBBComparison of JN.1 with the XBB variant showed no trace of universal mutations, and hosted certain unique, dynamic, and random mutations. The unique mutations in the NTD domain were A83V, L84K, P85K, F86L, N87I, D88V, V90L, F92V, Y170E, and Q218L, besides two dynamic mutations viz., A83F and, V90Y. The unique mutations observed in the RBD domain included N331K, T333R, N334S, T346R, F456L, Q507P, and P507T, besides a dynamic mutation, T346I. The other unique mutations, T315V, S316C and F318I, were spotted in the intermediary region between the NTD and RBD domains (Table 4 and Fig. 2C). Our study showed 33 random mutations (13 in NTD, 14 in RBD, three in SD1/2, two in HR1-2 and one in the FP).Table 4 Unique, dynamic, universal, and random mutations in the spike protein of JN.1 as compared to the XBB sequence.Evolutionary mutation patterns in spike protein sequencesThe evolutionary relatedness of JN.1 is presented in a phylogenetic tree that depicts the hierarchy of the different lineages of the selected strain, and the position of the JN.1 lineage in the tree. The JN.1 lineage showed an extended branch length in both rooted and unrooted trees (Fig. 3).Figure 3The phylogenetic analysis of the JN.1 variant as compared with the wild-type Wuhan-Hu-1 genome. Phylogenetic analysis using a rooted tree (top panel) and an unrooted tree (bottom panel) illustrates the evolutionary relatedness among the sequences of JN.1 (n = 66) compared with the reference genome of SARS-CoV-2 (Wuhan-Hu-1).Mutational and conformational changes in the spike protein of JN.1 omicron variantWhen our study sequences were compared with the reference genomes of different variants, 19 mutations were observed in our strains and the previously reported JN.1 variants (but not seen in other lineages). This indicated that the mutations were unique to the JN.1 variant. The list of mutations and the specific motifs is given in Table 5, and the frequency of these mutations is given in Supplementary Table S1. The other 24 mutations that were observed in a few lineages/sub-lineages but not found in our 66 sequences were identified and listed in Table 6. Intrigued by these mutations, we next elucidated the resulting protein structural changes with the reference protein sequences. The 19 JN.1-specific mutations were created in the model and superimposed with the reference protein. The superimposed structure showed a root mean square deviation (RMSD) value of 0.071 Å (Fig. 4A). The 17 study-specific mutations were created and superimposed with the same model, which showed an RMSD value of 0.081 Å (Fig. 4B).Table 5 List of mutations observed in our strains and previously reported JN.1 variants.Table 6 List of mutations observed in a few lineages/sub-lineages but not in our current investigation.Figure 4Superimposition of protein models of SARS-CoV-2. (A) Superimposed protein model of Wuhan Hu-1 reference model (red) with JN.1 specific mutations (blue). (B) Superimposed protein model of JN.1 variant (green) with the study-specific unique mutation model identified in our study participants (red).Molecular docking revealed high binding affinity of the mutated structures of JN.1 compared with Wuhan Hu-1 reference SARS-CoV-2The results of docking different receptors (ACE2, CD147, CD209L and AXL) with wild-type and mutated structures of RBD and NTD are summarized in Table 7. The study indicated a high negative energy value of mutated RBD structures with ACE2, CD147 and CD209L compared to the wild-type. Of these, N481K and R403K showed a significantly low energy score (− 1011.9 kcal/mol, − 945 kcal/mol and − 1183.9 kcal/mol, respectively) demonstrating a strong binding affinity. In contrast, the energy value of mutated NTD structures with AXL was higher compared to the wild-type implying a weak binding affinity. The individual molecular interactions with different receptors are shown in Fig. 5. The key interacting residues of the selected mutated structures with strong binding affinity were analysed and listed in the Supplementary Table S1. The results signify high binding affinity with the mutated structures of JN.1 as compared with the Wuhan Hu-1 wild-type strain.Table 7 The binding energies of the interaction of wild-type and mutated structures with different receptors in the molecular docking investigation.Figure 5Molecular interactions of wild-type and mutated structures with host cell receptors. (A) Interaction of wild-type RBD with ACE2. (B) Interaction of N481K mutated RBD with ACE2. (C) Interaction of wild-type RBD with CD147. (D) Interaction of R403K mutated RBD with CD147. (E) Interaction of wild-type RBD with CD209L. (F) Interaction of R403K mutated RBD with CD209L. (G) Interaction of wild-type NTD with AXL. (H) Interaction of R158G mutated NTD with AXL. (I) Interaction of L216F mutated NTD with AXL. Footnotes: ACE2 angiotensin-converting enzyme-2, NTD N-terminal domain, RBD receptor binding domain. AXL is a tyrosine-protein kinase receptor, with potential oncogenic properties; CD147 is an alternate receptor for SARS-CoV-2 entry into host cells with low ACE2 expression; CD209L can also act as a receptor for SARS-CoV-2 entry into susceptible host cells.
Emergence of SARS-CoV-2 omicron variant JN.1 in Tamil Nadu, India – Clinical characteristics and novel mutations
