Pan India 1000 SARS-CoV-2 RNA Genome Sequencing Consortium - PowerPoint Presentation

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Pan India 1000 SARS-CoV-2 RNA Genome Sequencing Consortium An Initiative of DBT-AIsCoordinated by National Institute of Biomedical Genomics (NIBMG), Kalyani Participating Institutes:

Teams:

NIBMG:

Arindam

Maitra

, Nidhan Biswas, Saumitra Das ILS: Sunil Raghav, Arup Ghosh, Ajay Parida CDFD: Ashwin Dalal, Murali Bashyam, Debasis Mitra NCCS: Yogesh Souche, Manoj Kumar Bhat InStem-NCBS: Aswin Sai Narain, Dasaradhi Palakodepi, Satyajit Mayor, Apurva Sarin

Objectives:Short term:i) Sequencing of 1000 viral genomes from samples collected from different zones within India.ii) Molecular phylogeny of the virus, which will indicate the track of how the virus is evolving in India

iii) Correlate

sequence variation in viral RNA to disease severity and transmission efficiency, ultimately leading to identification of strains which are associated with enhanced pathogenicity.

 Long term:

Identify host genetic polymorphisms that either confers susceptibility or protection from the viral infections.

Viral and host genetic determinants of disease severity

The PAN–INDIA consortium launched the programme on April 27, 2020, to

get the countrywide

landscape of the variations in SARS-CoV-2 RNA genome sequences.

Coordinated by National Institute of Biomedical Genomics (NIBMG-Kalyani), West Bengal. Four other National clusters, ILS-Bhubaneswar, CDFD-Hyderabad, InStem-NCBS, Bangalore and NCCS-Pune have actively participated in sequencing and analysis. Other collaborating National Institutes and clinical organizations involved are ICMR-NICED, IPGMER-Kolkata, IISc-Bangalore, AIIMS-Uttarakhand, MAMC-Delhi, THSTI-Faridabad, GMC-Aurangabad, MGIMS-Wardha, RMRC-Bhubaneswar

,

AFMC

and

BJMC-Pune

and other hospitals

.

Pan India 1000 SARS-Cov-2 Genome Sequencing Consortium

Phylodynamic Time Tree:A2a (20A/B/C) is predominant Along with fewparental haplotype 19A/BMultiple lineages introduced which are evolving over time

Haplotype diversities peaked between March-May, early part of the outbreak.By June A2a (20A/B/C) emerged as predominant haplotypeThe temporal haplotype diversities landscape appears to be similar PAN India

North (

Uttarakhand

, Haryana & Delhi)

West (Maharashtra)

South (Karnataka & Telangana)

East (West Bengal & Odisha)

Pan India

Temporal Clade Diversities: Pan India

Haplotype distribution: Region wiseWithin haplotypes different states appear to have different proportionsEast and North are similarWest and South are similar

Predicted Origin &Introduction of Viral Lineages19A and 19B mostly came from China and 20A, 20B and 20C from the United Kingdom, Italy and Saudi Arabia

20A came from Italy and Saudi Arabia in all Indian regions, but in the case of Eastern Indian, 20A came additionally from the United Kingdom and Switzerland.

20B was introduced mostly from the United Kingdom in all regions, additionally in Western India from Brazil and in

Southern-India

introduced from Italy and Greece

Haplotype nodes with majority of the genomes from West Bengal, Odisha and a small percentage of the samples belonging to Uttarakhand. Geographically Odisha and West Bengal share borders and the shared SARS-CoV-2 haplotypes might be because of the high interstate travelling.Maharashtra, Delhi, Haryana and Uttarakhand

grouped together with 2-4 single nucleotide variants (SNVs),

suggesting the infection might have spread in a short duration of time.

There

is a portion of the samples from Haryana and Karnataka sharing same parent haplotype, representing possible transmission by migration.

The negative estimate Tajima’s D (D = -2.26817, AMOVA p(D<=-2.26817) = 0.00281

) [PMID

: 2513255] is consistent with the rapid expansion of SARS-CoV-2 population in India

Haplotype Networks

ILS team

Differences Between SARS-CoV (2003) and SARS-CoV-2 (2019)Both are Genus: Betacoronavirus (lineage B) Overall base similarity 82.3%SL4’SL1

SL2

SL3

SL4

SL5

SARS-

CoV

(2003)

SARS-CoV-2 (2019)

SL1SL2SL3SL4SL5In 2 sequences contain 134 (U to C) mutation which disrupts the SL4’ Stem-LoopIn ~90% of SARS-CoV-2 sequences contain 241 (C to U) mutation in SL5 which comes under A2a clade.Emergence of New Stem-loop in 5’UTR in SARS-CoV-2SL4’The Fatality rate of SARS-CoV (2003): 9.6% (WHO) The Fatality Rate of SARS-CoV (2019): ~3.9% (till now) (WHO)SARS-CoV (2003)

Variant D614G in Spike, most common across the country, is declining in frequency in Delhi wherein a different set of variants (nsp3 T1198K, RdRp A97V, N P13L) is dominating. Increase of one is correlated with a decrease in the other across states.Variant nsp3_A994D is highly prevalent, and increasing in frequency, in Maharashtra; less so in other statesEffect of these variants on virulence or infectivity are being tested.

A sub-type seems to have attained high frequencies in multiple states; this one is characterized by two consecutive point mutations

in

Nucleocapsid

gene (

N RG203KR

) and appears to have originated on the background of type

ST4/A2a

Pan

India 42%North 37%East 9%West 85%South 70%

Within Spike (S) two mutations (D614G and G1124V) specific for A2a clade are found in most of the samples. D614G is supposed to confer flexibility in Sd domain and mutation in G1124V might impart partial rigidity in the conformation of S2 domain. Both the mutations are away from receptor binding domain (RBD), but can affect positioning of the residues involved in receptor binding

.

In the

Nucleocapsid (N)

two mutations (

R203K, G204R

) are observed in samples from West Bengal, which might have consequence in virus assembly and packaging of the virus particle inside host.

Mutation (

P323L

) in the RNA dependent RNA polymerse (RdRp) is also observed in India which might have implications in viral RNA replication.(Maitra and Chawla et al, 2020, J. Biosciences, Vol 45, 0076)

N RG203KR affects miRNA binding sites & N protein structure

Maitra, A., Sarkar, M.C.,

Raheja

, H. et al. Mutations in SARS-CoV-2 viral RNA identified in Eastern India: Possible implications for the ongoing outbreak in India and impact on viral structure and host susceptibility.

J

Biosci

45, 76 (2020)

Mutations in N gene reported, might result in altered binding ability of host miRNAs to viral RNA, that might affect host susceptibility.

225 COVID-19 Genomes from OdishaVirus clade distributionClade evolution with timeSalient findingsWith migration from 9 Indian StatesAll five reported clades

19A, 19B, 20A, 20B and 20C found.Total of 247 single nucleotide variants

were identified.Europe and Southeast Asia as

two major routes of disease transmission in India.Recently evolved clades 20A and 20B showed prevalence of

four common mutations

.

ILS

team

Clade90 COVID-19 Genomes from MaharashtraVirus clade distributionSalient findingsThree clades 19A, 20A and 20B found.Total of 125 single nucleotide variants were identified.Europe and Southeast Asia as two major routes of disease transmission in India.

Recently evolved clades 20A and 20B showed prevalence of four common mutations.

Clade evolution with time

NCCS

Team

SARS-CoV-2 genome analysis: Hyderabad & Telangana>200 COVID-19 genomes sequenced (includes symptomatic and asymptomatic patients)Patient samples representing Hyderabad and additional districts from TelanganaGenomic position

No of samples with alteration

Preliminary indications -

Presence of A3i clade defining mutations C6312A, C13730T and C28311T

Relatively higher number of mutations in ORF1a (

RdRp gene), higher frequency of mutations in N gene

, relative to S gene

Technical inputs:

Sabarinathan, NCBS

CDFD team

As on date, the Consortium has achieved its initial goal of completing the sequencing of 1000 SARS-CoV-2 genomes from nasopharyngeal and oropharyngeal swabs collected from individuals testing positive for COVID19 by Real Time PCR. The samples were collected across 10 states covering different zones within India. Given the importance of this information for public health response initiatives investigating transmission of COVID-19, the sequence data will soon be released in public domain (GISAID database). Information will improve our understanding on how the virus is spreading, ultimately helping to interrupt the transmission chains, prevent new cases of infection, and provide impetus to research on intervention measures. Initial results indicate that multiple lineages of SARS-CoV-2 are circulating in India, probably introduced by travel from Europe, USA and East Asia. In particular, there is a predominance of the A2a haplotype (20A/B/C) with D614G mutation, which is found to be emerging in almost all regions of the country. This particular haplotype is globally reported to be associated with enhanced transmission efficiency. 

Additionally

, mutations in important regions of the viral genome with significant geographical clustering have also been observed.

Detailed

mutational analysis to understand the gradual emergence of mutants at different regions of the country and its possible impact on the disease management is in progress.

Highlights:

CDFD, HyderabadDr Ashwin Dalal, Dr Murali Bashyam, Dr Pratyusha Bala, Mr Vinay

Donipadi,

Dr Divya

Vashisht, Dr

Debashis

Mitra

,

InStem

-NCBS, Bengaluru

Dr. Apurva Sarin, Dasaradhi Palakodeti, Aswin Sai Narain Seshasayee, Uma Ramakrishnan, Shah-e-Jahan Gulzar, Varadharajan Sundaramurthy, Srikar Krishna, Vanessa Molin Paynter, Awadhesh Pandit, Farhan Ali, Mohak Sharda, Dr. Satyajit Mayor, Dr. Apurva SarinILS, BhuabneswarDr. Sunil Raghav. Mr. Arup Ghosh and Dr. Ajay ParidaNIBMG, Kalyani

Dr

.

Arindam

Maitra,

Dr.

Nidhan

K. Biswas,

Dr.

Sreedhar

Chinnaswamy

, Mr

.

Shekhar

Ghosh, Mr.

Sumanta

Sarkar,

Dr.

Subrata

Patra,

Dr.

Rajib

Mondal

,

Dr.

Trinath

Ghosh, Mr. Arnab Ghosh, Mr.

Shouvik

Chakraborty,

Dr.

Saumitra

Das,

NCCS, Pune

Dhiraj

Paul,

Kunal

Jani,

Janesh

Kumar,

Radha

Chauhan,

Vasudevan

Seshadri

,

Girdhari

Lal,

Dr.

Arvind

Sahu

,

Dr.

Yogesh

S

Shouche

,

Dr.

Manoj

Kumar Bhat,

Experimental & Computational Team

Coordinated by NIBMG,

Kalyani

IPGMER, KolkataDr. Monimoy Banerjee, Dr. Raja Ray, Dr. Jayeeta Halder, Dr. Aritra

Biswas

ICMR-NICED

Dr. Shanta

D

utta, Dr.

Mamta

Chawla Sarkar, Ms.

Ananya

Chatterjee, Ms. Hasina Banu, Mr. Agniva Majumdar Clinical CollaboratorsGMC, AurangabadDr. Jyoti Iravan, Mr Dhaval Khatri, Mr Maitrik Dave AIIMS, Rishikesh Prof Ravi Kant, Dr Deepjyoti Kalita, Dr Amit ManglaMAMC. Delhi Dr Sonal Saxena, Dr Vikas Manchanda, Dr Oves Siddiqui MGIMS, WardhaDr Vijayshri Deotale, Dr Rahul Narang, Dr Deepashri Maraskolhe

IISc

, Bengaluru

Dr.

Bharath

K

Sundararaj

,

Harsha

Raheja

,

Prof.

N

. Srinivasan,

Prof

.

Deepak K

Saini

,

Prof.

Amit

Singh,

Prof

.

K N

Balaji

,

Prof.

Umesh

Varshney

THSTI, Faridabad

Dr.

Guruprasad

Medigeshi

,

Dr.

Gagandeep

Kang,

Sharanabasava

Patil

,

Anbalagan

Ananthraj

,

Madhu

Pareek

, Imran

Khan,

ESIC Hospital and Medical College, Faridabad,

Gurugram

Civil Hospital,

Gurugram

and

Palwal

Civil Hospital,

Palwal

NIMS, Hyderabad

Dr

Madhumohan

Rao, Dr

Vijay Dharma

Teja

AFMC, Pune

Sourav

Sen,

Santosh

Karade

,

KavitaBala

Anand

,

Shelinder

Pal Singh

Shergill

,

Rajiv Mohan Gupta

BJMC, Pune

Rajesh

Karyakarte

,

Suvarna

Joshi,

Murlidhar

Tambe

RMRC, 

Bhubaneswar

Dr.

Sanghamitra

Pari

,

Dr.

Jyotirmayee

Turuk