key: cord-0942835-8fiswlb2
authors: Singh, Pushpendra; Sharma, Kuldeep; Singh, Priyanka; Bhargava, Anudita; Negi, Sanjay Singh; Sharma, Pratibha; Bhise, Mayuri; Tripathi, Manish Kumar; Jindal, Atul; Nagarkar, Nitin M.
title: Genomic characterization unravelling the causative role of SARS-CoV-2 Delta variant of lineage B.1.617.2 in 2nd wave of COVID-19 pandemic in Chhattisgarh, India
date: 2022-01-19
journal: Microb Pathog
DOI: 10.1016/j.micpath.2022.105404
sha: 4e28bd2f4beab07ccac3b0e9bea32c974d9c617c
doc_id: 942835
cord_uid: 8fiswlb2

COVID-19 pandemic 2nd wave catastrophic effect in the state of Chhattisgarh, India, from where no exclusive genomic data yet published, has prompted us to undertake this study to unearth the causative variant. Whole-genome sequencing of SARS-CoV-2 isolated from COVID-19 infected nine vaccinated healthcare workers (HCW), thirty mild/moderate, seventeen severe, and twenty-seven deceased patients, was performed. The significant predominance of the SARS-CoV-2 variant of concern (VOC), Delta (lineage B.1.617.2) identified in sixty-four (77.1%) cases in contrast to B.1 and its sublineage in eleven (13.2%), variant under monitoring (VUM), Kappa (lineage B.1.617.1) in five (6.0%) and another VOC Alpha (lineage B.1.1.7) in three (3.6%) cases respectively (p < 0.05, χ(2) = 162.49). 88.8% vaccine breakthrough, 60% mild/moderate, 94.4% severe and 81.5% dead patients were infected by Delta. Kappa presents exclusively in mild/moderate, Alpha in vaccine breakthrough, mild/moderate, and dead patient and B.1 and its sublineages in mild, severe, and dead patient categories. Delta variant spike mutation of T19R, G142D, E156G, L452R, and deletion (F157 and R158) helps in escaping antibody response, T478K and D614G enhance viral affinity with ACE2 receptor while P681R and D950N result in higher replication and transmissibility by cleaving S1/S2 at furin site. We conclude that Delta variant predominant role along with co-occurrence of Kappa, Alpha, and B.1 variant during COVID-19 2nd wave pandemic in Chhattisgarh may pose a potential threat of future outbreak through hybrid variant evolution. Thus, intensive genomic surveillance for monitoring variant evolution and a more efficacious vaccine against the Delta and Alpha variants are required.

COVID-19 disease pandemic caused by a novel coronavirus (nCoV) named SARS-CoV-2 has so far posed the biggest public health challenge of the 21st century across the globe since its emergence in December 2019 from Wuhan, China. India, the second most affected country after the United States of America (USA), although managed first wave reasonably well, was unfortunately hit hard by 2 nd wave of COVID-19 pandemic. 2 nd wave of the pandemic that started around 1 st March 2021 has accounted for eighteen million laboratory-confirmed cases and two hundred five thousand death at average daily death of over 2000 till 11 th June, 2021 [1] . The highest single-day number of cases rose to four hundred fourteen thousand on 6 th May, 2021. The worst affected states reported were Maharashtra, Karnataka, Kerala, Delhi, Andhra Pradesh, Telengana, Madhya Pradesh, and Chhattisgarh. This second wave was largely uncontrollable and unmanageable as it nearly crippled the nation's healthcare system and led to an unprecedented rise in COVID cases and deaths. The severe shortage of hospitals beds, oxygen supply, and medicine have haunted humanity as overwhelmed crematoria were seen working round the clock to keep up with the pace of dead bodies. Patients presented more with the clinical features of difficulty in breathing, fever, headache, myalgia, acute respiratory distress syndrome (ARDS), pneumonia, severe lung damage, gastrointestinal symptoms, and multiorgan failure. National guidelines have classified the patients into mild (normal saturation), moderate (pneumonia with no severe disease sign), and severe (severe pneumonia) [2] . Strict implementation of early diagnosis, treatment, contact tracing, lockdown, masking, and restricting social gathering has eventually brought down 2 nd wave distratous effect under control by mid of June, 2021.

The emergence of different variants of SARS-CoV-2 has been reported as the plausible explanation of 2 nd wave [3, 4] . As per WHO information latest on 25 th The causative nCoV variant of pandemic 2 nd wave from various states of India has been reported [3, 4] [3, 4] .

Chhattisgarh, one of the largest central states of India, was also severely affected by COVID-19 2 nd wave, with majorly affected cities were Raipur (capital city of Chhattisgarh), Bhilai and Durg. Around 662,325 people were found infected, including 9275 deaths [1] . So, the need for whole-genome sequencing of SARS-CoV-2 isolated from COVID-19 patients categories of vaccine breakthrough, mild/ moderate, severe and deceased from these districts of Chhattisgarh was utmost felt to unravel the variant/s responsible for this menace.

J o u r n a l P r e -p r o o f Accordingly, the present study was undertaken to genetically characterize SARS-CoV-2 strains obtained from COVID-19 patients in Chhattisgarh by sequencing the whole genome to identify accumulated viral mutation/s, strains, and lineages. The information, thus generated, would be utilized for formulating policies for effective management of the future course of the pandemic.

Samples collection 

The isolated RNA from all the eighty-three cases was used for their cDNA and library preparation 

Individual consensus sequences of eighty-three nCoV isolates obtained using QIAGEN CLC Table) . The subsequent mapping and alignment of these sequences against the SARS-CoV-2 Wuhan variant reference sequence (GenBank ID NC_045512) were done to analyze mutational changes. The phylogenetic tree was constructed by the Neighbour-joining (NJ) method using MEGA-X version 10.2.

The 3D modeling of spike glycoprotein of the Wuhan reference prototype and our predominant identified lineage of Alpha, Delta and Kappa was performed using the SWISS-MODEL server and PyMOL to study the effect of the specific amino acid (AA) mutational changes [7] .

The whole-genome sequence analysis of SARS-CoV-2 from all eighty-three cases showed major nonsynonymous mutational AA changes primarily in viral receptor spike gene in comparison with reference Wuhan prototype ( harbored the nonsynonymous mutations of T19R, E156G, L452R, T478K, D614G, P681R, D950N, and two deletions of F157del and R158del, respectively. Among them, six vaccinated cases showed one more G142D AA change, while the other two vaccinated cases did not exhibit this mutation (Fig. 4.A) . The one case of Alpha nCoV variant was found with mutations as described above (Fig. 5A and 6 ).

Among mild/moderate symptomatic cases, eighteen cases were found infected by Delta, five by Kappa, one by Alpha and six by nCoV B.1 variant having various sublineages respectively (Fig. 6) . The sublineages revealed two infected with B. Fig. 5B ). The important observation included exclusive finding of Kappa variant with its earlier described characteristic mutational pattern only in the mild/moderate symptomatic cases (Fig. 5B) . The one case of Alpha variant exhibited similar mutational changes as observed in Alpha variant infected vaccinated patients. In the rest six cases of different B.1 sublineages, the mutation of D614G was found in all cases, while N440K was found in three cases (Fig. 5B ).

Among seventeen severe symptomatic cases, sixteen cases were found infected with Delta variant while only one case by B.1.1.326 variant (Fig. 5C, 6 (Fig. 5C ).

In twenty-seven deceased patients, the majority of twenty-two cases were found succumbed to the Delta variant (Fig. 5D, 6) . In other five cases, one case was found infected with Alpha variant while the remaining four were found infected with B. in fifteen cases respectively (Fig. 5D) . The whole-genome analysis also showed mutations in specific genes/ regions other than spike either among all the lineages or restricted exclusively to Delta and/or Kappa variant (Table 2) .

Notably, all variants showed P323L mutation in nonstructural protein (NSP) 12 encoding RNA dependent RNA polymerase (RdRP) gene, and Delta variant exclusively showed I82T mutation in membrane protein-encoding gene. The 3D modeling of spike glycoprotein has depicted positional changes in AA residue of SARS-CoV-2 variant of VOC and VUM in comparison to the Wuhan prototype ( Fig. 7A-D) . 

The phylogenetic analysis identified our eighty-three sequences belonged to 10 different Epidemiological and genomic data of the eighty-three cases revealed that males were twice more infected than females. The majority of the infected cases were adults with a collective mean age of 47.49 yrs. Since Delta variants was detected in all different patient groups, no specific mutation inclination pattern or change was seen in severe or dead cases. So, it appeared have altered a recognized monoclonal antibody (mAb) recognition site N1 and N3 loop to helps the virus in escaping antibody response [4, 11] . The two significant deletions of F157 and R158

and one substitution E156G at NTD antigenic supersite in Delta variant have also been reported as 'surge-associated mutations' to increase infectivity by 10 fold due to mechanism of evading neutralizing antibodies [12, 13] . L452R, T478K occurred in the RBD region of the spike protein. L452R has already been earlier reported for escape from antibody immune response as these mutations reduces the antibody binding to mutation variants compared to wild type [3] . An earlier study also reports that L452R mutation had abolished the neutralizing effect of 14 mAb out of around 35 RBD mAb [14] . L452R mutation was further reported to help in virus escape from cellular immunity induced with human leukocyte antigen (HLA)-24 to eventually result in increased viral replication and infectivity [15] . It was reported earlier that in T478K, mutated Lysine reduced the gap to the extent of 8.3Å between spike and ACE2 receptor in comparison to wild prototype which with threonine measured as 10Å to eventually enhance the viral affinity with ACE2 resulting in increased viral infectivity [4] . Remarkably, L452R and T478K observed at the critical RBD in Delta variant have specifically been responsible for enhancing ACE2 binding [16, 17] . D614G mutation has been reported earlier as the first mutation adapted by the variants evolved from original Wuhan nCoV [4] . It has been identified distally to the furin cleavage site to enhance viral cell entry efficiency by increased affinity to ACE2 cell receptors [4] . This may lead to increased viral loads in the upper respiratory tract COVID-19. D614G mutation earlier reported producing an allosteric conformational change in the furin cleavage site to result in RBD opening [18] . The detection of D614G in all our reported lineages has strongly suggested that the emergence of all variants of SARS-CoV-2 occurred after the worldwide adaptation and rise of the D614G mutants. The two mutations of J o u r n a l P r e -p r o o f P681R, D950N were found close to the S1/S2 cleavage site to enhance viral replication and transmissibility [19] . P681R was seen in Delta, whereas P681H was observed in Alpha variant.

Although both mutations increase the basicity of the S1-S2 poly-basic stretch at the furin cleavage site, P681R has inflicted more advantage to the virus by cleaving more efficiently S1/S2 cleavage at the furin site leading to the enhanced entry of the virus into host cells. This may augment the rate of viral membrane fusion and internalization into the host cell to result in higher replication and transmissibility, facilitating Delta enhanced fitness over Alpha variant [20] . Both L452R and P681R were reported to also aid in escaping the virus from specific monoclonal antibody responses [14, [21] [22] [23] . In totality, these nonsynonymous mutations in reported in around six percent of the nCoV sequenced across India [28] .

Mutational analysis of genes/regions other than spike has revealed the mutational changes seen either in all lineages or restricted to the Delta and/or kappa variant. All lineages have shown P323L mutation in the interface domain of the RdRP region. P323L leads to stability of the tertiary protein structure of the virus to help in virus successful survival in different geographical conditions [29] . Although mutation in membrane gene relatively uncommon,

I82T exclusive presence in Delta variant helps in viral host cell attachment, heparin sulfate proteoglycan, protein assembly in conjunction with nucleocapsid(N) and envelope (E) genes, and enhanced glucose transport to provide more rigidity and stability resulting in increased transmissibility and pathogenicity [30] .

The limitation of the study included less number of studied cases, especially the vaccine breakthrough cases. There can be several reasons for this, including the acquisition of protective immune response in vaccinated people, continued adherence to COVID appropriate behavior, limited data on breakthrough cases, and poor prospective follow-up of completely vaccinated people for acquiring SARS-nCoV-2 infection. Nonetheless, we believe that even nine vaccinated infected patients have provided essential baseline information that Delta and 

Ministry of Health & Family Welfare. COVID-19 India Statewise Status: Chhattisgarh. Https // Www Mohfw Gov / 2021

Government of India Ministry of Health & Family Welfare. CLINICAL MANAGEMENT PROTOCOL FOR COVID-19

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Transmission, infectivity, and neutralization of a spike L452R SARS-CoV-2 variant

Preliminary report on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike mutation T478K

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Structures and dynamics of the novel S1/S2 protease cleavage site loop of the SARS-CoV-2 spike glycoprotein

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Infection and vaccineinduced neutralizing antibody responses to the SARS-CoV-2 B

Neutralization of variant under investigation B.1.617 with sera of BBV152 vaccinees

SARS-CoV-2 spike E484K mutation reduces antibody neutralisation. The Lancet Microbe

Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies

Recurrent emergence and transmission of a SARS-CoV-2 spike deletion H69/V70

Spike mutation D614G alters SARS-CoV-2 fitness

SARS-CoV-2 genomics: An Indian perspective on sequencing viral variants

Concurrent mutations in RNA-dependent RNA polymerase and spike protein emerged as the epidemiologically most successful SARS-CoV-2 variant

Emerging variants of concern in SARS-CoV-2 membrane protein: a highly conserved target with potential pathological and therapeutic implications