April 29th, 2022

PhD Open Seminar on

Exploring the enigmatic role of

Epstein-Barr virus in neuroinflammation
and neurodegeneration associated with
Alzheimer’s disease
Presented by:
Deeksha Tiwari
PhD scholar, Infection Bioengineering Group, BSBE, IIT Indore
Roll no.: 1701171004

Principle Investigator:
Dr. Hem Chandra Jha
Contents :-
1. Introduction

2. Chapter 1: Does Epstein-Barr virus have potential to initiate the neurodegenerative pathology associated
with Alzheimer’s disease ?

3. Chapter 2: Investigating the biochemical response in microglial cells upon EBV infection through Raman
microspectroscopy

4. Chapter 3: Examining the probable interaction of apolipoprotein variant E (ApoE) with Epstein-Barr virus
proteins

5. Chapter 4: Identification of potential phytochemical inhibitors of EBV-dUTPase: a viral neuromodulatory

protein

6. Future Prospects

7. Publications

8. Acknowledgements 2
INTRODUCTION
 1.1 Viral infections in Central Nervous System
Relevant viruses in humans allocated to the brain region they may affect

Meninges:
HIV, JEV, MuV, HEV, MV, CP/ependyma:
VZV, EBV, HPeV and HEV, MuV, CMV, HIV, VZV,
CHIKV NPEV, HPeV and CHIKV

White matter:
HIV, CMV, CHIKV, Cerebral cortex:
and HHV-6 JEV, HSV-1, HIV, MV,
WNV, CMV, and TBEV
Thalamus:
HHV6, WNV,
RV, and EEEV
Cerebellum:
Brain stem/ spinal cord: NPEV and WNV
HIV, VZV, EBV, PV, WNV,
RV, and EEEV

CHIKV, chikungunya virus; CMV, cytomegalovirus; EBV, Epstein Barr virus; EEEV, eastern equine encephalitis virus; HHV-6, human herpes virus-6; HIV, human immunodeficiency virus; HPeV,
human parechovirus; HSV-1, herpes simplex virus-1; JCV, John Cunningham virus; JEV, Japanese encephalitis virus; MV, measles virus; MuV, mumps virus; NPEV, nonpolio enterovirus; PV,
poliovirus; RV, rabies virus; TBEV, tick-borne encephalitis virus; WNV, West Nile virus; VZV, varicella zoster virus
Source: Dahm, T., Rudolph, H., Schwerk, C., Schroten, H. & Tenenbaum, T. Neuroinvasion and Inflammation in Viral Central Nervous System Infections. Mediators of Inflammation 2016, 1–16
4
(2016)
 1.2 Effects of viral infections on CNS
• Viral infections in CNS (Brain and spinal cord)
could give rise to various pathologies such as:
 Meningitis
 Encephalitis
 Neuritis
 Myelitis

• Associated Disease Conditions:

 Alzheimer’s disease (AD)
 Parkinson's disease (PD)
 Multiple sclerosis (MS)

Viral  Amyotrophic lateral sclerosis (ALS)

infection
 Autism spectrum disorders (ASD)
5
Source: Lotz, S. K., Blackhurst, B. M., Reagin, K. L. & Funk, K. E. Microbial Infections Are a Risk Factor for Neurodegenerative Diseases. Front Cell Neurosci 15, 691136 (2021).
1.3 Alzheimer’s Disease

6
 1.4 EBV and Alzheimer’s Disease

Pathogen enters the brain.

Amyloid beta peptides bind to pathogen.

Immune responses initiate.
Amyloid beta plaque is formed surrounding Involvement of Pathogens in Alzheimer’s disease (AD).
pathogen. Neurotropic viral pathogens such as EBV, HSV, etc can become
Plaque activates microglia which releases entrapped by amyloid-β after entering the brain. Amyloid fibrils
inflammatory cytokines. form in response to these pathogens, and infection may play a role
in accelerating AD pathology by stimulating inflammation and
Neuronal death leading to neurodegeneration and
AD pathogenesis. neurodegeneration.
7
Source: Naughton, S. X., Raval, U. & Pasinetti, G. M. The Viral Hypothesis in Alzheimer’s Disease: Novel Insights and Pathogen-Based Biomarkers. JPM 10, 74 (2020)
 Chapter 1
Does Epstein-Barr virus have potential to
initiate the neurodegenerative pathology
associated with Alzheimer’s disease ?
 2.1 Introduction
 A study reported peptide derived from glycoprotein B of Hypothesis
Herpesviridae family member (HSV-1) form β-amyloid-

like aggregates [1].

 Glycoprotein B- conserved among members of

Herpesvirus family. (HSV-1 gB: 29% identical and 43%

similar to EBV gB) [2].

 Recent study by Singh V. et.al., with HSV-1 protein gK

revealed high aggregation potential in in-silico as well as

in-vitro experiments [2].

1. Cribbs, D. H.; Azizeh, B. Y.; Cotman, C. W.; LaFerla, F. M. “Fibril Formation and Neurotoxicity by a Herpes Simplex Virus Glycoprotein B Fragment with Homology to the Alzheimer’s Aβ
Peptide. Biochemistry, 2000, 39, 5988−5994.
9
2. Singh, Vikas Kumar, Sandeep Kumar et.al., "Aggregation Propensities of Herpes Simplex Virus-1 Proteins and Derived Peptides: An In Silico and In Vitro Analysis." ACS Omega (2020).
 Study Design

Peptide
Oligomers
Aggregation
evaluation Proteasomal
EBV cleavage
proteins
Prediction NetChop20S-3.1 EBV Peptides

EBV Peptide
aggregate

• Congo red assay

• Thioflavin-S assay In-vitro
• Secondary structure analysis: Raman aggregation
spectroscopy analysis
• AFM
• Cytotoxicity assay

10
Source: Tiwari, D. et al. Indication of Neurodegenerative Cascade Initiation by Amyloid-like Aggregate-Forming EBV Proteins and Peptide in Alzheimer’s Disease. ACS Chem. Neurosci.
12, 3957–3967 (2021)
 2.2 In-silico screening of aggregation-prone EBV proteins
Calculation of Aggregation Score (using TANGO and AGGRESCAN)

 On scanning ~180 protein sequences of EBV

using online softwares (TANGO and

AGGRESCAN) we found following EBV proteins

shows comparable aggregation score with Aβ1-42 :

• Glycoprotein-M
• BMRF-2,
• LMP-1,
• LMP-2A,
• LMP-2B,
Average aggregation scores of EBV proteins according to TANGO
• BILF-1

11
Source: Tiwari, D. et al. Indication of Neurodegenerative Cascade Initiation by Amyloid-like Aggregate-Forming EBV Proteins and Peptide in Alzheimer’s Disease. ACS Chem. Neurosci.
12, 3957–3967 (2021)
 Graphical representation of aggregation scores of each residue across the entire length of
the test subjects

(b) Aggregation score of EBV-gM plotted along the entire length of the protein compared to the positive and negative control as calculated by
TANGO.
(c) as calculated by AGGRESCAN. Both the plots show that aggregation score of EBV-gM along the entire protein length is higher than the
positive control.

12
Source: Tiwari, D. et al. Indication of Neurodegenerative Cascade Initiation by Amyloid-like Aggregate-Forming EBV Proteins and Peptide in Alzheimer’s Disease. ACS Chem. Neurosci.
12, 3957–3967 (2021)
 Representation of predictive cleavage site distribution

Source: Tiwari, D. et al. Indication of Neurodegenerative Cascade Initiation by Amyloid-like Aggregate-Forming EBV Proteins and Peptide in Alzheimer’s Disease. ACS Chem. Neurosci.
12, 3957–3967 (2021) 13
 Various properties of EBV-gM peptide depicting its aggregation capability

Amino acid residues

• On analysing the aggregation score of individual fragments, we found a 11 amino acid long (146-157) fragment
derived from EBV-gM showing comparable hydrophobicity and average aggregation score values with Aβ1-42.
• Also, aggregation score per residue of EBV-gM146-157 showed comparable values with that of Aβ1-42.

14
Source: Tiwari, D. et al. Indication of Neurodegenerative Cascade Initiation by Amyloid-like Aggregate-Forming EBV Proteins and Peptide in Alzheimer’s Disease. ACS Chem. Neurosci.
12, 3957–3967 (2021)
Congo-Red Absorption assay
 Thioflavin-S Fluorescence assay
• The selective binding of Thioflavin-S (ThS) with amyloid like aggregates is known to exhibit increased
fluorescence emission.

Concentration (µM)

Thioflavin-S fluorescence emission by EBV-gM146-157 peptide after The quantification of normalized fluorescence intensity
incubation. at maxima plotted with increasing dosage of aggregates
16
Source: Tiwari, D. et al. Indication of Neurodegenerative Cascade Initiation by Amyloid-like Aggregate-Forming EBV Proteins and Peptide in Alzheimer’s Disease. ACS Chem. Neurosci.
12, 3957–3967 (2021)
 Fluorescence intensity (a.u.)
Thioflavin-S fluorescence intensity of 125µM with time. (a) Fluorescence intensity spectrum of 125uM EBV-gM146-157 peptide recorded in the range of 400-
600 nm. The spectrum shows maximum fluorescence emission at 2 hours. (b) Quantification of fluorescence at maxima revealed gradual increase till 2 hours
followed by a decrease depicting temporal pattern in aggregate formation.

17
Source: Tiwari, D. et al. Indication of Neurodegenerative Cascade Initiation by Amyloid-like Aggregate-Forming EBV Proteins and Peptide in Alzheimer’s Disease. ACS Chem. Neurosci.
12, 3957–3967 (2021)
 Structural analysis of aggregates

Cytotoxicity analysis of EBV-gM146-157 peptide (125 µM ) Atomic Force Microscopic (AFM) image of EBV-gM146-157
using MTT assay at 72 hour post treatment. aggregates

Source: Tiwari, D. et al. Indication of Neurodegenerative Cascade Initiation by Amyloid-like Aggregate-Forming EBV Proteins and Peptide in Alzheimer’s Disease. ACS Chem.18
Neurosci.
12, 3957–3967 (2021)
 Cytotoxicity analysis of EBV-gM

80

70

60

Cytotoxicity (%age)
50

40

30

20

10 IC50 = 37.05 µM
0
Media Ctrl 2.5 5 10 20 50 100
Peptide Concentration (µM)

Cytotoxicity analysis of EBV-gM146-157 peptide (125 µM ) using MTT assay at 72 hour post treatment.

Source: Tiwari, D. et al. Indication of Neurodegenerative Cascade Initiation by Amyloid-like Aggregate-Forming EBV Proteins and Peptide in Alzheimer’s Disease. ACS Chem.19
Neurosci.
12, 3957–3967 (2021)
 2.4 Conclusion
 EBV proteins could give rise to amyloidogenic peptides upon proteasomal cleavage.

One such 11 aa long (146-157) peptide generated from EBV glycoprotein-M (gM) is
demonstrated to exhibit amyloid-like aggregate forming capability.

The 11-mer EBV-gM146-157 showed properties similar to amyloid- β42 aggregates.

MTT assay revelated neurotoxic behaviour of aggregates formed.

EBV may possibly initiate degenerative cascade in Alzheimer’s or other such

amyloidosis conditions by following the proposed aggregation pathway.

20
 Chapter 2
Investigating the biochemical response in
microglial cells upon EBV infection through
Raman microspectroscopy
 3.1 Study of the viral infection progression in glial cells
• Objective: To study and identify the EBV infection progression markers in glial
cells using Raman Spectroscopy.
• Study Design:
Signature
Raman spectra
of Protein

Signature
EBV Raman spectra
Infection of Lipids

Signature
Raman spectra
Microglia (HMC-3) of Nucleic acids
Raman Spectroscopy

Monitor infection
progression 22
 3.2 Signature spectrum identification
Signature Raman spectral range of biomolecules reported to be associated with viral infection.

Wavenumber Wavenumber Associated Probable function in

Reports on association with virus infection
Range (cm-1) (cm-1) Biomolecule cell metabolism

481 DNA Genetic Material

1. HCMV enhances glycolytic flux to fuel fatty acid
synthesis. [111]
2. HSV-1 gears glycolytic metabolism toward the
460-480 production of pyrimidine nucleotide components. [111]
484-490 Glycogen Energy storage molecule
3. EBV infected NPC cell lines show increased
glycolysis levels; LMP-1 of EBV induces Hexokinase-
2 to induce glycolysis and upregulation of GLUT-1.
[120]

Cell membrane 1. Cellular Cholesterol Facilitates the Post-entry

constituent (maintains Replication Cycle of Herpes Simplex Virus 1 [102]
Cholesterol/ membrane fluidity), 2. A nuclear receptor involved in cholesterol metabolism
548 Cholesterol Involved in cell regulates herpesvirus latency and reactivation [121]
esters signalling, transport 3. EBV: LMP-2A is secreted in exosomes in Cholesterol
processes and nerve dependent manner [122]
conduction 4. KSHV- decreased cholesterol synthesis. [123]
540-560 Glucose-
Energy currency of the [124]
540 saccharide 1. HBV- Increased Gluconeogenesis & glycolysis.
cell
band
1. Conserved Tryptophan Motifs in the Large Tegument
Essential amino acid, Protein pUL36 Are Required for Efficient Secondary
involved in synthesis of Envelopment of Herpes Simplex Virus Capsids [125]
573 Tryptophan
brain serotonin and 2. Indoleamine-2,3-Dioxygenase (IDO) plays role in IFN-
kynurenine gamma mediated Antiviral Effects against HSV
Infections [126]
23
Source: Tiwari, D., et. al. Temporal In Vitro Raman Spectroscopy for Monitoring Replication Kinetics of Epstein–Barr Virus Infection in Glial Cells. ACS Omega 5, 29547–29560 (2020).
Signature Raman spectral range of biomolecules reported to be associated with viral infection.
Glucose and
Energy currency of the
1117 Saccharide
cell
band
Polysaccharid Energy storage or
1122
es structural support
1112-1124 Conformational structure
of lipids; involved in
C-C long term energy storage,
1. HCMV- increased lipid biosynthesis. [128]
1124 stretching cell membrane
mode of lipids constitution, and 2. KSHV- Increased lipid synthesis [129]
intercellular
transmembrane transport
Structural constituent of
1258-60 Amide III 1. HCMV- increased anaplerotic use of glutamine. [130]
proteins in the body
1257-1263 Protein band Plays multiple roles in
the cells; involved in cell
1260
Second amide signalling, proliferation
etc; structural role.
1. HSV, EBV and CMV- Infection facilitates cytokine-
induced alterations in lipid and lipoprotein metabolism,
leading to decreased serum levels of total cholesterol
1264 Triglycerides Main component of
(TC), HDL-C, LDL-C, apoA1, apoB and Lp(a), as well
dietary fats, act as long- as increased triglyceride (TG) and apoE concentrations.
1270-1280
term energy storage [131]
molecule
Unsaturated
1270 1. DENV- increased fatty acid biosynthesis [132]
fatty acids
1270 Phospholipids
1. Vaccinia virus- Increased de-novo fatty acid
Structural constitution of
Lipids and biosynthesis and β-oxidation [133]
2860-2880 2853-2881 the cell; involved in
proteins 2. HBV- Disturbed lipid synthesis [134]
signalling and transport
3. HCV- Decreased lipid secretion [135]

The table enlists the specific Raman signature of the biomolecules that are known to be altered on infection with different viruses and how the virus utilizes them.
ɫHCMV - Human cytomegalovirus; HSV-1 - Herpes simplex virus-1; EBV - Epstein-Barr virus; KSHV - Kaposi’s sarcoma associated herpes virus; HBV - Hepatitis B
virus; DENV - Dengue virus.

24
Source: Tiwari, D., et. al. Temporal In Vitro Raman Spectroscopy for Monitoring Replication Kinetics of Epstein–Barr Virus Infection in Glial Cells. ACS Omega 5, 29547–29560 (2020).
 3.3 Raman spectral analysis
Expression of various biomolecules at different time points post EBV infection in HMC-3 cells

25
Source: Tiwari, D., et. al. Temporal In Vitro Raman Spectroscopy for Monitoring Replication Kinetics of Epstein–Barr Virus Infection in Glial Cells. ACS Omega 5, 29547–29560 (2020).
 Representative view of changes in biomolecular activity upon continuous infection
progression.

Periphery (HMC-3) Nucleus (HMC-3)

Source: Tiwari, D., et. al. Temporal In Vitro Raman Spectroscopy for Monitoring Replication Kinetics of Epstein–Barr Virus Infection in Glial Cells. ACS Omega 5, 29547–29560 (2020). 26
 Connectome: molecular crosstalk between various EBV influenced biochemical factors
associated with neurodegenerative disorders

27
Source: Tiwari, D., et. al. Temporal In Vitro Raman Spectroscopy for Monitoring Replication Kinetics of Epstein–Barr Virus Infection in Glial Cells. ACS Omega 5, 29547–29560 (2020).
 3.4 Conclusion
 Unique spectral changes associated with specific biomolecules were obtained in microglial cells post
EBV infection.

 The biomolecular changes observed in time dependent manner correspond to characteristic events
implicated in virus-host interaction.

 Early events from 2-4 hour post infection (pi) correlate with the attachment & entry of the virus in host
cell; the investigation directed us to believe that EBV enters the glial cells probably in the first 2 hpi
by utilizing PIP-dependent signaling pathways.

 Late events post 12 hours of infection correspond to replication of the virus and it’s subsequent
egress from the glial cells. During its nuclear hijack process from 6-12 hpi, the virus manipulates
glycogen and amino acid metabolism in the microglial cells.

 Interestingly our study reflected a possible role of ApoE and cholesterol metabolism in EBV infection.
28
Source: Tiwari, D., et. al. Temporal In Vitro Raman Spectroscopy for Monitoring Replication Kinetics of Epstein–Barr Virus Infection in Glial Cells. ACS Omega 5, 29547–29560 (2020).
 Chapter 3
Examining the probable interaction of
apolipoprotein variant E (ApoE) with
Epstein-Barr virus proteins
4.1 Introduction

30
 Study Design:
Structure of ApoE3
• Objective: To study probable
interaction between viral and host
apolipoprotein conspiring to create
a deleterious microenvironment in
neuronal milieu culminating in AD

• Approach: Employing an in-silico

approach to check the interaction
between ApoE3 and EBV antigens.

Source: Source: Iurescia, Sandra et.al., Journal of Alzheimer's Disease, vol. 21, 31
no. 1,
pp. 35-48, 2010.
 4.2 ApoE3 structure retrieval and modelling

ApoE3
(Alphafold structure)

(PDB-1LPE) (iTASSER Model)

ApoE3-NTD Region ApoE3-CTD Region

(41-184) (192-299)

32
(Manuscript under preparation)
4.3 ApoE3-CTD modelled structure validation

33
(Manuscript under preparation)
4.4 Molecular docking using HADDOCK and HDOCK
Sr.
No.
Protein Name Abbrev. PDB ID
• Site specific docking of EBV
1 Apolipoprotein E3 (NTD: 41-184) ApoE3-NTD 1LPE
proteins with ApoE3-NTD and CTD
2 Apolipoprotein E3 (CTD: 192-299) ApoE3-CTD iTASSER
structures (listed below) using
1 Secreted protein BARF1 BARF1 2CH8
2 Portal protein BBRF1 6LQN
3 Viral interleukin-10 homolog BCRF1 1VLK HADDOCK and HDOCK.
4 Apoptosis regulator BHRF1 BHRF1 2WH6
5 DNA polymerase processivity factor
BMRF1
BMRF1 2ZOL
• MD simulation of the complexes
6 Trans-activator protein BZLF1 BZLF1 2C9L
7 EBV- deoxyuridine 5'-triphosphate EBV- 2BSY showing similar structural
nucleotidohydrolase dUTPase
8 Epstein-Barr nuclear antigen 1 EBNA1 6VHZ orientation as predicted by both
9 Envelope glycoprotein H gH 7CZE
10 Envelope glycoprotein L gL 7CZE
11 Glycoprotein 42 gp42 6LYJ the software (HADDOCK and
12 Capsid scaffolding protein 40 p40 3FD4
13 Uracil-DNA glycosylase UDG 1O6E HDOCK). 34
(Manuscript under preparation)
4.5 Molecular dynamic simulation analysis
Stability of bound complexes

Root-mean-square deviations (RMSDs) of the respective

EBV protein backbone atoms in complex with the ApoE3
during MD simulation
35
(Manuscript under preparation)
 Compactness of bound complexes

Rg plot of respective EBV proteins bound with Solvent accessible surface area (SASA) analysis
ApoE3-NTD and CTD

36
(Manuscript under preparation)
 Hydrogen bond analysis
EBNA1- ApoE (NTD) EBNA1- ApoE (CTD)

BZLF1- ApoE (NTD) BZLF1- ApoE (CTD)

37
 4.6 Interaction analysis
a) ApoE3 (NTD) - EBNA-1 Complex
(Chain A) (Chain B)

Before MD simulation After MD simulation

EBNA1 ApoE3 (NTD)
EBNA1 ApoE3 (NTD)

38
(Manuscript under preparation)
 b) ApoE3 (CTD)- EBNA-1 Complex
(Chain A) (Chain B)
Before MD simulation After MD simulation

ApoE3 (CTD)
ApoE3 (CTD)

EBNA1
EBNA1

39
(Manuscript under preparation)
 c) ApoE3 (NTD)- BZLF-1 Complex
(Chain A) (Chain B)

Before MD simulation After MD simulation

ApoE3 (NTD)
ApoE3 (NTD)

BZLF1 BZLF1

40
 d) ApoE3 (CTD)- BZLF-1 Complex
(Chain A) (Chain B)
Before MD simulation After MD simulation

BZLF1 BZLF1

ApoE3 (CTD)

ApoE3 (CTD)

41
 4.7 Energy Decomposition

van der Waal Electrostattic Polar solvation energy SASA energy Binding energy
Protein-complexes energy (kJ/mol) energy (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol)

EBNA1 + ApoE3 (NTD) -258.961 -680.593 939.931 -35.227 -34.848

EBNA1 + ApoE3 (CTD) -632.575 -2438.544 1846.063 -82.89 -1307.947

BZLF1 + ApoE3 (NTD) -159.783 -118.708 256.527 -18.186 -40.149

BZLF1 + ApoE3 (CTD) -537.615 -3655.62 2524.269 -73.806 -1742.772

(Manuscript under preparation)

42
 4.8 Conclusion
Based on the preliminary results, EBV proteins EBNA-1 and BZLF-1 seems

to bind stably with lipid binding region at CTD of ApoE3.

The interaction might hinder the normal functioning of ApoE3 and

increase the chances of aggregate formation by accumulation of free

floating Aβ in the cytoplasm thus, initiation AD pathogenesis.

43
 Chapter 4
Identification of potential phytochemical
inhibitors of EBV-dUTPase: a viral
neuromodulatory protein
 5.1 Introduction to EBV-dUTPase

Figure 1 Structural details of the EBV-dUTPase. a) Location and orientation of the domains I (1-116), II (117-219), and III (220-278) forming the secondary structure
of EBV-dUTPase is shown in red, blue, and green color, respectively. b) The conserved motifs I (magenta), II (orange), III (red), and IV (blue) constitute the active site of
the EBV-dUTPase. Due to its flexibility and disordered structure, motif V is generally invisible.
45
(Manuscript communicated)
 5.2 EBV v/s human dUTPase

Figure 1 Structural details of the EBV-dUTPase. c) The sequence alignment of the human dUTPase with that of EBV. The location of each of the motifs I, II, III, IV,
and V is highlighted respectively in magenta, yellow, cyan, green, and grey.
46
(Manuscript communicated)
 5.3 Active binding site

Figure 1 Structural details of the EBV-dUTPase. The active site of EBV-dUTPase is depicted in blue residues; inset: The residues involved in the active site formation
are shown in red.

47
(Manuscript communicated)
 5.4 Screening of phytochemicals and
Molecular docking
Binding
Compound
Sr. Compound Name Molecular Energy
Class identifier
No. (mol. wt.) formula (kCal/
(PubChem)
mol)
dUTP (deoxyuridine
Natural substrate C9H15N2O14P3 CID-65070 -7.6
triphosphate)
1 Anti-inflammatory
K3R (902.8g/mol) C27H30O15 CID-122173234 -9.1
2 Neuroprotective,
Anti-inflammatory, MANG (422.3g/mol) C19H18O11 CID-5281647 -9
Anti-viral
3 Neuroprotective
SARA (416.6g/mol) C27H44O3 CID-92095 -8.8
4 Neuroprotective,
DHED (301.3g/mol) C19H15N3O CID-9817839 -8.6
Anti-viral
K3R: Kaempferol-3-rutinoside; MANG: Mangiferin; SARA: Sarsasapogenin; DHED: Dehydroevodiamine 48
(Manuscript communicated)
 5.5 MD Simulation
Root-mean-square deviations (RMSDs) of the EBV- Root-mean-square fluctuations (RMSFs) analysis of Cα
dUTPase backbone atoms during MD simulation during MD simulation

49
(Manuscript communicated)
Solvent accessible surface area (SASA) Rg plot of EBV-dUTPase bound and Hydrogen bond analysis of the
analysis unbound with phytochemicals docked complexes.

50
(Manuscript communicated)
 5.6 Energy decomposition analysis of bound
complexes
Binding Energy van der Waals Electrostatic Solvation
Complex
(kJ/mol) Energy (kJ/mol) energy (kJ/mol) Energy (kJ/mol)

dUTPase/dUTP -65956.70 -7515.65 -45758.80 -12682.25

dUTPase/DHED -69358.97 -7519.74 -48210.74 -13799.67
dUTPase/SARA -68777.60 -7492.05 -48945.86 -12339.68
dUTPase/K3R -68746.01 -7472.04 -48802.74 -12471.24

dUTPase/MANG -68194.67 -7472.45 -47749.60 -12972.63

51
(Manuscript communicated)
5.7 2D visualization of ligand-bound protein
before and after MD simulation
EBV-dUTPase +Dehydroevodiamine

(Manuscript communicated)
52
5.8 Analysis of ADMET and drug likeliness
properties of phytochemical ligands

Predicted Value
Property Model Name (Unit)
DHED K3R MANG SARA

Drug likeness Lipinski (Yes/No) Yes No No Yes

Ghose (Yes/No) Yes No No No

Veber (Yes/No) Yes No No Yes
Egan (Yes/No) Yes No No Yes
Muegge (Yes/No) Yes No No No
Bioavailability Score 0.55 0.17 0.17 0.55
Medicinal Chemistry Lead likeness (Yes/No) Yes No No No

53
(Manuscript communicated)
 5.9 Conclusion
The in-silico study shows DHED as a suitable drug development candidate for
treating the EBV mediated neurodegenerative pathologies by targeting EBV-
dUTPase.

Virus infection to the Immunomodulatory

host cell effect Phytochemicals
EBV

EBV gHg
L

dUTPase Dehydroevodiamine
Active viral Computational studies (DHE)
proteins dUTP dUDP • Neuroprotective
• Anti-inflammatory
• Anti-viral
Host cell
54
(Manuscript communicated)
 Future Prospects
Could EBV
peptide be In-vivo experiments to validate amyloid seeding and AD
amyloidogenic pathogenesis
in-vivo as
well?
How EBV
manipulates the Biochemical assays to explore the role of EBV in
biochemistry of modulating cholesterol and lipid metabolism
glial cells?

Does DHED
have potential In-vitro validation of DHED’s effect on EBV replication
to affect EBV
efficiency in neurons
replication in-
vitro?
55
 Publications:
 From Thesis:
1. Tiwari, D1. et al. “Indication of Neurodegenerative Cascade Initiation by Amyloid-like Aggregate-Forming EBV
Proteins and Peptide in Alzheimer’s Disease”. ACS Chem. Neurosci. 12, 3957–3967 (2021).
DOI:10.1021/acschemneuro.1c00584.
2. Tiwari D1, Jakhmola S1, et.al., “Temporal In-vitro Raman Spectroscopy for Monitoring Replication Kinetics of
Epstein-Barr virus Infection in Glial Cells”, ACS Omega (2020), DOI: 10.1021/acsomega.0c04525
3. Tiwari, D., & Jha, H.C., “Detection and Analysis of human brain disorders”. In M. Tanveer & R. B. Pachori
(Eds.), Machine Intelligence and Signal Analysis (Vol. 748, pp717-726). Springer Singapore. DOI:
https://doi.org/10.1007/978-981-13-0923-6_61
4. Tiwari D1, Murmu S1, et.al., “Targeting Epstein-Barr virus dUTPase, an immunomodulatory protein using anti-
viral, anti-inflammatory and neuroprotective phytochemicals” (Communicated)
5. Tiwari D1, Mittal N., Jha H.C*, “Epstein - Barr virus-mediated cell-cycle modulation: A possible mode of
neurodegenerative pathogenesis in Alzheimer’s and Parkinson’s disease” (Communicated)
6. Tiwari, D1. et al., “An in-silico approach to examine probable novel interactions of EBV proteins with
Apolipoprotein E” (Under preparation)
7. Jha H.C.; Tiwari, D.; Indian Patent filed under application number: 202121032715, “Viral peptides as a target
for the treatment of virus-mediated neuropathologies”. (Under process)
56
Outside Thesis:

1. Kashyap, D., Jakhmola, S., Tiwari, D., Kumar, R., Natércia F. Brás, NS Hari Narayana Moorthy,
Elangovan, M., & Jha, H.C*. “Plant derived active compounds as potential anti SARS-CoV-2 agents:
An in-silico study”. Journal of biomolecular structure and dynamics (2021),
DOI:10.1080/07391102.2021.1947384
2. Indari, O.1#, Singh, A.K. 2#, Tiwari, D.1, Jha, H.C.1* and Jha, A.N.2*, “Repurposing antimalarial
compounds to target viral pathogens associated with malaria” (Communicated)
3. Indari, O.a , Tiwari, D.a , Tanwar, M.b , Kumar, R.b* , Jha, H.C.a*, “Early biomolecular changes in
brain microvascular endothelial cells under Epstein Barr Virus influence: a Raman microspectroscopy
approach” (Communicated)
4. Tiwari, D.*, Rani, A.*, & Jha, H.C., “Homocysteine and Folic acid metabolism” In Dubey, G.P., Misra,
K., Kesharwai, R.K., & Ojha, R.P. (Eds.), Frontier’s in Homocysteine Metabolism: Implications in
Nutrition and Health. Springer (In Press)
5. Varshney N, Rani A, Kashyap D, Tiwari D, Jha H.C., “Aurora kinase: An emerging potential target in
therapeutics”. Protein Kinase Inhibitors: From Discovery to Therapeutics, Elsevier (In Press)

57
 Conferences and Workshops Attended
 Poster Presentation

1. Tiwari D., Jha H.C.* “Exploring the enigmatic role of Epstein-Barr virus in neuroinflammation” at Research and Industrial Conclave (RIC), organised by
IIT Guwahati from 20-23rd January 2022.

2. Jakhmola S., Indari O., Baral B., Kashyap D., Tiwari D., Varshney N., Rani A., Sonkar C., Verma T.P., Kandpal M., Jha H.C.* “COVID-19 pandemic from
coinfection and comorbidity aspect” at World Hindi Day Celebration, 2022 organised by IIT Indore on 5th January 2022.

3. Jakhmola S., Tiwari D., Rani A., Jha H.C.* “Kissing virus can play with your mind” at Madhya Pradesh Vigyan Sammelan (MPVS), 2021 orgnised by
MGM Medical College, Indore from 22-25th December 2021.

4. Tiwari D., Jakhmola S., Jha H.C.* “Raman spectroscopy analysis of biochemical changes in glial cells on Epstein-Barr virus infection” at Emerging
Areas in Biosciences and Biomedical Technology (eBBT) 2020 organised by Indian Institute of Technology, Indore from 7-9th February 2020.

5. Tiwari D., Jakhmola S., Jha H.C.* “Epstein Barr Virus infection as an inducer of neurodegeneration” at Emerging Areas in Biosciences and Biomedical
Technology (eBBT) 2018 organised by Indian Institute of Technology, Indore from 5-6th January 2018.

6. Shyam Singh, Jakhmola S. Charu Sonkar, Tiwari D., Indari O., Jha H.C.* “Potential Roles of Bacteria and Viruses in Disease Pathogenesis and Water
Quality Monitoring” at Industry-Academia Conclave (IAC) 2017 organised by Indian Institute of Technology, Indore from 5-6th September 2017.

58
 Seminar Oral Presentations:

• Tiwari D., Jha H.C.*, “Unraveling the mysterious affair of Epstein-Barr virus with neuroinflammation“ in
Research and Industrial Conclave (RIC), 2022 at IIT Indore on 10-12th February 2022.
• Tiwari D., Jha H.C.*, “Detection and Analysis of human brain disorders“ in International Conference on
Machine Learning and Signal Processing (MISP) 2017 at Indian Institute of Technology, Indore during 22-
24th December 2017.

 Awards and Accolades:

• 1st Prize for Poster presentation (external category) | Title: “ Exploring the enigmatic role of Epstein-Barr
virus in neuroinflammation“ in Research and Industrial Conclave (RIC), 2022 organized by Indian Institute
of Technology (IIT) Guwahati | Duration: 20-23rd January 2022.
• Indian Patent filed under Application number: 202121032715 | Name of the Inventors: Hem Chandra
Jha, Deeksha Tiwari | Title: Viral peptides as a target for the treatment of virus-mediated
neuropathologies.
• Best Poster Award for the poster titled “Epstein Barr Virus infection as an inducer of neurodegeneration“
presented at the international conference on “Emerging Areas in Biosciences and Biomedical Technology
(eBBT)- 2018“
59
 Acknowledgements
• Principal Investigator: Dr. Hem Chandra Jha
• Dr. Suman Tapryal and lab members (Dr. Vikas
Kumar; Collaborator, Central Univ. of Rajasthan)
• Dr. Sunil Kumar and lab members (Collaborator,
IARI New Delhi)
• Dr. Jayabalan, J. (RR-CAT Indore)
• Dr. Gaurava Shrivastava (Collaborator, CDRI,
Lucknow)
• Dr. Rajesh Kumar and lab members (Pathak D.,
Tanwar M.), IIT Indore
• PSPC Members (Dr. Abhijeet Joshi, Dr. Rajesh
Kumar, and Dr. M.Tanveer)
• Lab members & colleagues
• IIT Indore
• Ministry of Human Resource and Development
(MHRD)
• Sophisticated Instrumentation Centre (SIC), IIT-I
• Family and Friends 60
Thank You