Heyy everyone, this is my class presentation of my MSc Dissertation which I presented in July 2017 at National Institute of Virology, Pune, Maharashtra, India. Description - Current WHO recommended genotyping of measles virus is based on terminal region of the N gene which has been proved to insufficiently diverse for genotyping purposes. Hence, as an alternate criterion we checked the utility of the hypervariable matrix-fusion (M/F) gene for the genotyping of Measles viruses(MeVs) using 35 Indian isolates (VerohSLAM cell line) obtained from 7 Indian regions during 2013-16. After sequencing the isolates, consensus sequences were built using BLASTn web tool and MEGA7 software. We obtained 30 whole Measles virus genomes (across 10 genotypes) from Genbank and created 2 reference lists,one for each gene. N gene data of the same was previously published. Two combined phylogenetic trees of all strains were constructed using MEGA7. Comparison revealed improved resolution by our criterion. In June 2017, our study concluded that our criterion may be useful for genotyping along with the routine N gene approach.

1. Application of Matrix (M) to Fusion (F) non coding
region
for the genotyping of Indian measles virus isolates
Aditya S Kulkarni
Roll no. IBB-NIV-2015-12
M.Sc Virology, Sem-IV
Guide - Dr. Sunil R. Vaidya
Measles Group Leader
Scientist ‘E’

2. Measles
Measles is a highly contagious disease characterized by a
prodromal illness of fever, cough, coryza, and conjunctivitis,
followed by generalized maculopapular rash.
 Rhazes, 10th
Century AD
Francis Home, 1757
J. Enders & T. Peebles, 1954 – David Edmonston
Vaccine preventable –
1.In India, since 1985 – 1 dose at 9 months
2.Since 2010 – 2nd
dose at 16-24 months
3.WHO-SEARO, Sept. 2013 - To be eliminate in SEA by 2020.
4.Since Feb. 2017 – 1 dose of MR vaccine
www.diseasepictures.com
www.healthline.com
www.healthline.com

3. Measles virus (MeV)
Family – Paramyxoviridae
Genus – Morbilivirus
(+)ssRNA virus
• 120-250 nm diameter pleomorphic,
spherical and enveloped particle.
• Receptors - CD46, SLAM/CD150
and PVRL4/Nectin-4
www.slideteam.net
Cynthia S. Goldsmith, CDC

4. MeV genome
 Genome – 15,894 nt
Nucleoprotein (525 nt), Phosphoprotoein (507 nt) [C & V], Large polymerase protein (2138 nt),
Matrix protein (335 nt), Hemagglutinin (617 nt) Fusion protein (550 nt).
 The 450 nt carboxy-terminal region of the N gene.
 24 different genotypes - A, B1-B3, C1, C2, D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, E, F, G1-
G3, H1 and H2
www.frontiersin.org

5. Global measles epidemiology (Feb 2016 – Jan
2017)
• 6 genotypes –
A, B3, D4, D8, D9 and H1 were
reported in 6 WHO regions all over
the world.
Most reported from -
• Western Pacific
• Europe
Most reported genotype -
• H1, endemic to China
Most distributed –
• B3 and D8
India subcontinent –
• D4 and D8
MeaNS database - http://www.who-measles.org/

6. Measles epidemiology in India (2011-16)
B3 strains reported from -
•AP, UP, KL & AS
D4 strains reported from
9 states, mostly from –
•GJ, MH, KA
D8 strains reported from 20 States
& UTs except –
•AP, JH
NIV unpublished data

7. N gene limitations
Vaidya et al,2016
D8 group D4 group
D8 = 351, D4 = 49 & B3 = 3

8. H gene and the MF region
www.frontiersin.org

9. Advantages of the MF region
Penedos et al,2015
N-450 MF region WGS
32 D8 - GBR [11 groups] (2012-13)

10. Objective
1. To determine the gene diversity of MF region of D4 and D8 MeV strains circulating in India.
2. To compare the sequence diversity of the MF region with that of N gene of the same virus strains.
3. Evaluate the genotyping potential of the MF region to characterize MeV outbreaks and to detect
importation/exportation in events.
Moss & Griffin, 2006

11. MeV Sequences included in the study
• Total 80 whole genome sequences were downloaded from GenBank on
December 30, 2016
• 50 sequences were rejected due to > 99.8 % identity.
• 30 whole genome sequences were selected to represent 10 genotypes -
• A, B3, D3, D4, D5, D6, D7, D8, G3, H1
• From each the MF region (1,012 nt) and N gene (450 nt) region was taken.

12. Measles virus isolates (n = 35)
State Age ( years ) Sex ( M/F ) Vaccinated Genotype
0-5 years 6-20 years >20 years D4 D8
Maharashtra 7 8 1 7 M/9 F 6 6 10
Gujarat 8 3 0 7 M/4 F 3 0 11
Uttarakhand 2 0 0 2 M 1 0 2
Haryana 1 0 0 1 M 0 0 1
Chhattisgarh 0 2 0 2 F 0 0 2
Madhya Pradesh 0 2 0 2 F 0 0 2
Delhi 1 0 0 1 F 0 0 1
19 15 1 17 M/ 18 F 10 6 29
Total = 35 Total = 35
2013 = 4, 2014 = 11, 2015 = 10, 2016 = 10

13. MF region primers
Primer name Primer
position (nt)
Primer sequence Expected
amplicon
size (bp)
1. MeV-L6-2F 3993 - 4011 5’-CGGGAACTTCAGGAGAAAG-3’ -
2. MeV-L6-3R 4930 - 4949 3’-CCCCCCGTCTTGGAYTGTCG-5’ 1000
3. MeV-4409F 4409-4429 5’-CATAAATGATGACCAAGGAC-3’ -
4. MeV-5145R 5126-5145 3’-GGTTGCCGTGGTCGTGTGTG-5’ 736
5. MeV-4801F 4801-4826 5’-CACAAGCGACCGAGGTGAC-3’ -
6. MeV-5609R 5586-5609 3’-CGAGTCATAACTTTGTAGCTTGC-5’ 808
7.MeV-216F 1105-1125 5’-TGGAGCTATGCCATGGGAGT-3’ -
8. MeV-214R 1736–1716 3’-TAACAATGATGGAGGGTAGG-5’ 634
1st
pair*
2nd
pair**
3rd
pair**
N gene *Brown et al
**Severini et al

14. Methodology
Measles virus isolates of suspected measles cases from year 2013-16 (n = 35).
Virus propagation – Vero/hSLAM cell line (n = 5, rest 30 procured from -80°C)
Viral RNA extraction – QIAmp viral RNA minikit(Qiagen)
Reverse transcription & Amplification–QIAGEN OneStep RT-PCR kit(Qiagen),Invitrogen SSIII OneStep RT-PCR kit
PCR product purification – ExoSAP-IT reagent
Cycle sequencing – Big Dye Terminator V 3.1 cycle sequencing kit (Applied Biosystems)
Cycle sequencing product purification – DyeEx 2.0 Spin kit
Capillary electrophoresis - AB 3130XL DNA analyser (Applied Biosystems, Tokyo, Japan)
Sequence Analysis – Sequence Analysis v2.0, Nucleotide BLAST & MEGA v7.0 softwares

15. Virus Propagation
5 MeV isolates were propagated in
Vero/hSLAM cell line.
A – Uninfected cells
B - +2 CPE –
•Syncytia formation
•Cell to cell fusion
C - +4 CPE –
•Cell detachment
•Plaque formation

16. One Step RT-PCR mix for N gene
Reagents Volume(µl) No. of
Reactions
Total
Volume(µl)
2X Reaction Mix 25 1 25
MgSO4 (5mM) 8 1 8
Rnasin (40U/ul) 1 1 1
MeV-216-F (20 µM) 0.5 1 0.5
MeV-214-R (20 µM) 0.5 1 0.5
Superscript enzyme 1 1 1
Rnase Free Water 9 1 9
45 1 45
45 µl mix + 5 µl eluted RNA
Steps Cycling
conditions
RT 55°C for 30 min
Hot start 94°C for 2 min
40 cycles of
Denaturation 94°C for 15 s
Annealing 55°C for 30 s
Extension 72°C for 30 s
Final extension 72°C for 7 min
Hold 4°C

17. One Step RT-PCR result for N gene
Representative 2% agarose gel
Lane 1: Positive control
Lane 2: Molecular weight marker (100 bp)
Lane 3: 634 bp amplicon of N gene

18. One Step RT-PCR mix for MF region
Reagents Volume(µl) No. of
Reactions
Total
Volume(µl)
RNase free water 18 3 54
5X Buffer 10 3 30
Q solution 10 3 30
dNTP 2 3 6
Forward primer (20 µM)* 1 1 each 1 each
Reverse primer (20 µM)** 1 1 each 1 each
Enzyme mix 2.5 3 7.5
Rnasin (40U/ul) 0.5 3 1.5
45 135
45 µl mix + 5 µl eluted RNA *MeV-L6-2F, 4409F, 4801F
**MeV-L6-3R, 5145R, 5609R

19. One Step RT-PCR reaction conditions for MF
region
Steps Cycling conditions
1st
pair 2nd
and 3rd
pair
RT 50°C for 30 min
Hot start 95°C for 15 min
40 cycles of
Denaturation 94°C for 1 min 20 s 95°C for 1 min
Annealing 55°C for 1min 30 s 55°C for 30 s
Extension 72°C for 2 min 68°C for 1 min 30 s
Final extension 72°C for 10 min 74°C for 10 min
Hold 4°C

20. One Step RT-PCR result for MF region
Representative 2% gel picture of 3 amplicons
Lane 1: Molecular weight marker (100 bp)
Lane 2: 1000 bp amplicon- MeV-L6-2F+MeV-L6-3R
Lane 3: 736 bp amplicon- 4409F+5145R
Lane 4: 808 bp amplicon- 4801F+5609R

21. Purification of PCR product
Representative 2% gel
to confirm the purification of amplified MF region
Lane 1: 1000 bp purified amplicon
Lane 2: 736 bp purified amplicon
Lane 3: 808 bp purified amplicon
Lane 4: Molecular weight marker
Similarly, purification of N gene amplicon was also
checked [ Image not shown ]

22. Cycle sequencing mix for N gene
Reagents Volume(µl)
No. of Reactions Total
volume(µl)
RNase free water 11
2 22
5X Buffer 2
2 4
Big Dye v3.1 4
2 8
Primer (3.2 µM)* 1 each
1 each 1 each
Template cDNA 2 each
2 each 2 each
20
2 40
Steps Cycling
conditions
25 cycles of
Denaturation 94°C for 15 s
Annealing 50°C for 5 s
Extension 60°C for 4 min
Hold 4°C
* MeV-216-F, MeV-214-R

23. Cycle sequencing mix for MF region
Reagents
Volume
(µl)
No. of Reactions Total volume (µl)
RNase free water 8.5
6 51
5X Buffer 2
6 12
Big Dye v3.1 4
6 24
Primer (20 µM* & 1 µM**) 1.5 each 1.5 each 1.5 each
Template cDNA 4 each 4 each 4 each
20 µl
6 120 µl
Steps Cycling
conditions
Hot start 96°C for 10 min
40 cycles of
Denaturation 98°C for 10 s
Annealing 50°C for 5 s
Extension 68°C for 4 min
Hold 4°C* MeV-L6-2F, MeV-L6-3R
** 4409F, 5145R, 4801F, 5609R

24. Cycle sequencing result & N gene
consensus
Primer Sequence (nt)
1. MeV-L6-2F 800-900
2. MeV-L6-3R 700-800
3. MeV-4409F 200-300
4. MeV-5145R 150-250
5. MeV-4801F 700-800
6. MeV-5609R 600-700
7. MeV-216F 400-500
8. MeV-214R 350-450
Representative sequence in ABI file
Consensus sequences of 500 nt (N gene)

25. Consensus of the MF region
MeV-L6-2F+MeV-L6-3R – 700-800 nt
4409F+5145R – 200-250 nt
4801F+5609R – 600-700 nt
+
+
WHOLE OVERLAPPING SEQUENCE – 1200 - 1400 nt
MF REGION – 1,012 nt (4339-5350 nt)
1.
2.
3.
Similarly, 450 nt region was selected from the consensus of the N gene (N-450)
REFERENCE WHOLE GENOME (D8/D4)
0 nt 15,894 nt

26. 1418678 MVs/Sa gar.IND/50.14(N)D8
KT283661/154489 MVs/Bha vnagar.IND/11.15/1(N)D8
KY120857/1616393 MVs/Pa lgha r.IND/39.16/2(N)D8
165553 MVs/Bhavnagar.IND/176/(N)D8
KT732231 MVs/London.GBR/22.12/3/D8(N)
KT382313/1512319 MVs/Panna.IND/28.15/(N)D8
KU571706/16496 MVs/Sa gar.IND/16/(N)D8
KF850705/1320036 MVi/Pune.IND/38.13(N)D8
KM043778/146224 MVs/Faridabad.IND/22.14(N)D8
161893 MVs/Pune.IND/76/(N)D8
1616872 MVs/Pune.IND/41.16(N)D8
KX033376/151659 MVs/Jamnaga r.IND/10.16/(N)D8
KX033376/163520 MVs/Jamnaga r.IND/106/(N)D8
KM034758/14587 MVs/Pune.IND/04.14/1(N)D8
KU571704/16333 MVs/Mumba i.IND/505/(N)D8
KC862249/132806 MVi/Pune .IND/10.13(N)D8
KU888840/161609 MVs/Jamnagar.IND/56/(N)D8
KM034764/141233 MVs/Ha ridw ar.IND/06.14/2(N)D8
KJ742849/14981 MVs/Pune.IND/05.14(N)D8
KT283625/15991 MVs/Bhavnaga r.IND/03.15/(N)D8
KU888841/161781 MVs/Ja mna gar.IND/66/2(N)D8
KT732230 MVs/Llandudno.GBR/7.12/2/D8(N)
KM034768/141254 MVs/Ha ridw ar.IND/06.14/5(N)D8
KU382090/1520639 MVs/Ana nd.IND/50.15/(N)D8
KU382091/1520640 MVs/Bija pur.IND/505/2(N)D8
KT283652/155516 MVs/Junaga rh.IND/13.15(N)D8
KM034757/1445 MVs/Pune .IND/01.14/1(N)D8
KT732232 MVs/London.GBR/28.12/D8(N)
KY120859/1615000 MVs/Morbi.IND/376/2(N)D8
1615001 MVs/Morbi.IND/376/2(N)D8
KM364586/147804 MVi/New Delhi.IND/26.14(N)D8
JN635405 MVi/Washington.USA/18.08/1/D5(N)
JN635406 MVi/Arizona.USA/11.08/2/D5(N)
GQ376026 MVi/Tokyo.JPN/37.99(Y) (MV99Y)/D3(N)
AB254456 MVs/Kobe .JPN/SSPE-Kobe -1/D3(N)
GQ376027 MVi/Tokyo.JPN/37.99(Y)C7 (MV99YC7)/D3(N)
AB481087 MVs/Tsukuba.JPN/T11w ild/D7(N)
JN635402 MVi/New York.USA/26.09/3/D4(N)
JN635403 MVi/Florida.USA/19.09/D4(N)
KC164757 MVi/Treviso.ITA/03.10/1D4(N)
KJ742848/141257 MVs/Pune.IND/06.14(N)D4
1418877 MVs/Gondia .Ind/50.14/5(N)D4
1418869 MVs/Gondia .Ind/50.14/2(N)D4
KF850709/1321231 MVi/Pune.IND/41.13(N)D4
KF850708/1320737 MVi/Pune.IND/40.13(N)D4
KT283655/155285 MVs/Gr.Mumbai.IND/125(N)D4
DQ227318 MVs/Zagre b.CRO/47.02/D6 SSPE(N)
DQ227321 MVs/Zagre b.CRO/17.98/D6(N)
DQ227320 MVs/Za greb.CRO/08.03/D6 SSPE(N)
KJ018970 MVi/BritishColumbia.CAN/12.10/1H1(N)
JN635411 MVi/Pennsylvania .USA/20.09/H1(N)
KT588921 MVs/Guizhou.CHN/29.13H1(N)
KJ755982 MVi/Zhejiang.CHN/10.11/2H1(N)
KC164758 MVi/Ve nice.ITA/06.11/1G3(N)
KT732221 MVs/London.GBR/3.14/3/B3(N)
KT732222 MVs/Luton.GBR/4.14/B3(N)
KT732219 MVs/London.GBR/3.14/B3(N)
KT732218 MVs/Da rtford.GBR/2.14/B3(N)
KT732214 MVs/Manchester.GBR/7.12/2B3(N)
FJ416067 MVi/Beijing.CHINA/Shanghai-191/A(N)
AY730614 MVi/Changchun.CHINA/Leningra d-4/A(N)
AF266290 MVi/Ne w York.USA/Edmonston(Zagreb vaccine)/A(N)
AF266291 MVi/New York.USA/Edmonston(Schw arz vaccine)/A(N)
AF266289 MVi/New York.USA/Edmonston(Rube ovax va ccine )/A(N)
60
81
99
30
29
99
99
74
99
88
29
65
87
95
85
99
99
70
22
67
92
44
99
90
60
78
99
90
65
46
75
99
99
32
32
73
52
29
27
24
53
57
98
98
68
64
65
60
63
63
33
32
0.0100
Phylogenetic analysis based on N
gene
In the D8 group –
1.Pune (2013/KF860706),
2.Faridabad (2014/KM043778)
3.Pune (2016) X 2
4.New Delhi (2014/KM364586)
In the D4 group -
1.Pune (2014/KJ742848)
2.Mumbai (2015/KT283655)
3.Pune (2013/KT850709)
4.Pune (2013/ KT850708)
•Weak resolution as per year and
location.

27. 132806 MVi/Pune.IND/10 3(MF-NCR)D8
1616393 MVs/Pa lghar.IND/406/2((MF-NCR))D8
163520 MVs/Ja mnagar.IND/106((MF-NCR))D8
14587 MVs/Pune.IND/04 4/1(MF-NCR)D8
1418678 MVs/Sa gar.IND/50 4(MF-NCR)D8
1512319 MVs/Pa nna.IND/28 5/(MF-NCR)D8
16496 MVs/Sagar.IND/16((MF-NCR))D8
154489 MVs/Bha vna gar.IND/11 5/1(MF-NCR)D8
KT732231 MVs/London.GBR/22.12/3/D8(MF-NCR)
1320036 MVi/Pune .IND/38.13(MF-NCR)D8
161893 MVs/Pune.IND/76((MF-NCR))D8
1616872 MVs/Pune.IND/((MF-NCR))D8
146224 MVs/Faridaba d.IND/22.14(MF-NCR)D8
161609 MVs/Ja mna gar.IND/56((MF-NCR))D8
16333 MVs/Mumbai.IND/505((MF-NCR))D8
165553 MVs/Bhavnagar.IND/176((MF-NCR))D8
147804 MVi/New Delhi.IND/26 4(MF-NCR)D8
1520639 MVs/Anand.IND/50 5/(MF-NCR)D8
1520640 MVs/Bijapur.IND/505/2((MF-NCR))D8
KT732230 MVs/Llandudno.GBR/7.12/2/D8(MF-NCR)
15991 MVs/Bha vnagar.IND/03 5/(MF-NCR)D8
161781 MVs/Jamnagar.IND/66/2((MF-NCR))D8
141233 MVs/Ha ridwa r.IND/06.14/2(MF-NCR)D8
14981 MVs/Pune.IND/05 4(MF-NCR)D8
141254 MVs/Haridw ar.IND/06.14/5(MF-NCR)D8
KT732232 MVs/London.GBR/28.12/D8(MF-NCR)
1445 MVs/Pune.IND/01 4/1(MF-NCR)D8
155516 MVs/Juna garh.IND/13 5(MF-NCR)D8
151659 MVs/Jamnagar.IND/10 6/(MF-NCR)D8
1615000 MVs/Morbi.IND/376/2((MF-NCR))D8
1615001 MVs/Morbi.IND/376/2((MF-NCR))D8
JN635405 MVi/Washington.USA/18.08/1/D5(MF-NCR)
JN635406 MVi/Arizona .USA/11.08/2/D5(MF-NCR)
AB481087 MVs/Tsukuba .JPN/T11w ild/D7(MF-NCR)
GQ376027 MVi/Tokyo.JPN/37.99(Y)C7 (MV99YC7)/D3(MF-NCR)
GQ376026 MVi/Tokyo.JPN/37.99(Y) (MV99Y)/D3(MF-NCR)
AB254456 MVs/Kobe.JPN/SSPE-Kobe-1/D3(MF-NCR)
JN635402 MVi/New York.USA/26.09/3/D4(MF-NCR)
KC164757 MVi/Treviso.ITA/03.10/1D4(MF-NCR)
JN635403 MVi/Florida.USA/19.09/D4(MF-NCR)
1418877 MVs/Gondia .Ind/50 4/5(MF-NCR)D4
1418869 MVs/Gondia .Ind/50 4/2(MF-NCR)D4
141257 MVs/Pune .IND/06 4(MF-NCR)D4
155285 MVs/Gr.Mumbai.IND/125((MF-NCR))D4
1321231 MVi/Pune.IND/41.13(MF-NCR)D4
1320737 MVi/Pune.IND/40 3(MF-NCR)D4
DQ227320 MVs/Zagreb.CRO/08.03/D6 SSPE(MF-NCR)
DQ227321 MVs/Zagre b.CRO/17.98/D6(MF-NCR)
DQ227318 MVs/Zagreb.CRO/47.02/D6 SSPE(MF-NCR)
KT732218 MVs/Dartford.GBR/2.14/B3(MF-NCR)
KT732222 MVs/Luton.GBR/4.14/B3(MF-NCR)
KT732219 MVs/London.GBR/3.14/B3(MF-NCR)
KT732221 MVs/London.GBR/3.14/3/B3(MF-NCR)
KT732214 MVs/Manchester.GBR/7.12/2B3(MF-NCR)
FJ416067 MVi/Beijing.CHINA/Sha nghai-191/A(MF-NCR)
AY730614 MVi/Changchun.CHINA/Leningra d-4/A(MF-NCR)
AF266289 MVi/New York.USA/Edmonston(Rube ova x vaccine)/A(MF-NCR)
AF266290 MVi/New York.USA/Edmonston(Zagreb vaccine )/A(MF-NCR)
AF266291 MVi/New York.USA/Edmonston(Schw arz va ccine)/A(MF-NCR)
KJ018970 MVi/BritishColumbia.CAN/12.10/1H1(MF-NCR)
JN635411 MVi/Pennsylva nia.USA/20.09/H1(MF-NCR)
KT588921 MVs/Guizhou.CHN/29.13H1(MF-NCR)
KJ755982 MVi/Zhejiang.CHN/10.11/2H1(MF-NCR)
KC164758 MVi/Venice .ITA/06.11/1G3(MF-NCR)
80
81
100
94
90
100
100
100
68
100
80
86
91
87
100
79
43
100
100
100
98
60
74
54
100
40
98
77
72
69
54
96
64
99
99
99
95
79
59
26
23
11
19
99
100
100
83
94
55
56
36
68
67
12
15
46
30
16
16
8
19
0.020
Phylogenetic analysis based on MF
region
In the D8 group –
1.Pune (2013/KF860706),
2.Faridabad (2014/KM043778)
3.Pune (2016) X 2
4.New Delhi (2014/KM364586)
In the D4 group -
1.Mumbai (2015/KT283655)
2.Pune (2013/KT850709)
3.Pune (2013/ KT850708)
4.Pune (2014/KJ742848)
•Strong resolution as per year
and location.

28. Mean genetic distance
• Genetic distance (%) –
The genetic divergence between species or between populations within a species.
• A genomic region is suitable for genotyping if –
• [Mean genetic distance between genotypes]>[Mean genetic distance within individual genotypes]

29. Mean genetic distance within and between genotypes (D8)
Mean genetic distance between genotypes (%) –
29 Indian D8 strains with 8 genotype groups
Mean genetic distance within genotypes (%) –
63 MeV strains in 9 genotype groups
Both MF region and N gene are suitable to for genotyping of Indian D8 strains.
Indian D8 & reference D8 strains are grouped together in the tree.
Phylogeny tree
N-450
MF region

30. Mean genetic distance within and between genotypes
(D4)
Mean genetic distance within genotypes (%) –
63 MeV strains in 9 genotype groups
Mean genetic distance between genotypes (%) –
6 Indian D4 strains with 8 genotype groups
Both MF region and N gene are suitable to for genotyping of Indian D4 strains.
Indian D4 & reference D4 strains are grouped separately in the
Phylogeny tree
N-450
MF region

31. Percentage nucleotide diversity & Mean genetic
diversity
MF region is more diverse than N gene in Indian D4 & D8 as well as international strains.
• Nucleotide diversity (%) [D8 & D4]–
1. 29 Indian D8 strains with 3 D8 reference strains
2. 6 Indian D4 strains with 3 D4 reference stains
• Mean genetic diversity (%) –
1. 65 MeV strains
2. 10 genotype groups

32. Unique stretch of nucleotides in MF region
(D4)
4661 nt to 4665 nt Mumbai (2015) - Italy (2010) & USA (2009) X 2

33. Unique stretch of nucleotides in N gene
(D8)
1351 nt to 1355 nt Pune (2014), Junagarh (2015) and Morbi (2016) X 2 - London (2012)

34. Conclusions
The sequence diversity of the MF region was investigated and compared with
that of the N gene of 35 circulating strains of MeV in India.
Gene diversity of the MF region > N gene among Indian strains.
Phylogeny by Neighbor-joining method reveals better resolution of related
strains as per year and location using the MF region as compared to N gene.
The MF region is suitable for genotyping of Indian viral strains.

35. • MeV is serologically monotypic, so vaccination can protect susceptible
individuals from circulating wild types.
• In 2015, vaccination coverage of MCV1 was 87% and that of MCV2 was 70%
and a drop in reported cases of measles to 300,000 was reported indicating
good coverage.
• Vaccination programmes interrupt endemic circulation of viruses.
• Genotyping - A way to measure effectiveness of ongoing vaccination
campaigns and helps us to formulate better immunization policies.
• To document the absence of the circulation endemic genotype of MeV in a
region is to declare Measles as eliminated.
• Better characterization of MeV = Better immunization policies = Elimination of
MeV by 2020
http://www.vaccine-info.com
http://www.wpro.who.int

36. Future prospects
1. More virus strains need to be sequenced for the MF region and more
reference strains from varying locations and time periods need to be included
in this study.
2. Mutation rate of the MF region needs to be calculated using the Bayesian-
skyline method using the BEAST online software.

37. References
• Harvala H, Wiman Å, Wallensten A, Zakikhany K, Englund H, Brytting M. Role of Sequencing the Measles Virus Hemagglutinin
Gene and Hypervariable Region in the Measles Outbreak Investigations in Sweden During 2013-2014. J Infect Dis. 2016 Feb
15;213(4):592-9.
• Measles virus nomenclature update: 2015. Wkly Epidemiol Rec. 2015 Jul 24;90(30):373-380. English, French.
• Penedos AR, Myers R, Hadef B, Aladin F, Brown KE. Assessment of the Utility of Whole Genome Sequencing of Measles Virus in
the Characterisation of Outbreaks.PLoS One. 2015 Nov 16;10(11):e0143081.
• Gardy JL, Naus M, Amlani A, Chung W, Kim H, Tan M, Severini A, Krajden M, Puddicombe D, Sahni V, Hayden AS, Gustafson R,
Henry B, Tang P. Whole-Genome Sequencing of Measles Virus Genotypes H1 and D8 During Outbreaks of Infection Following the
2010 Olympic Winter Games Reveals Viral Transmission Routes. J Infect Dis. 2015 Nov 15;212(10):1574-8.
• Vaidya SR. Commitment of measles elimination by 2020: challenges in India. Indian Pediatr. 2015 Feb;52(2):103-6.
• Rota PA, Brown K, Mankertz A, Santibanez S, Shulga S, Muller CP, Hübschen JM, Siqueira M, Beirnes J, Ahmed H, Triki H, Al-
Busaidy S, Dosseh A, Byabamazima C, Smit S, Akoua-Koffi C, Bwogi J, Bukenya H, Wairagkar N, Ramamurty N, Incomserb P,
Pattamadilok S, Jee Y, Lim W, Xu W, Komase K, Takeda M, Tran T, Castillo-Solorzano C, Chenoweth P, Brown D, Mulders MN,
Bellini WJ, Featherstone D. Global distribution of measles genotypes and measles molecular epidemiology. J Infect Dis. 2011
Jul;204 Suppl 1:S514-23.
• Vaidya SR, Chowdhury DT. Measles virus genotypes circulating in India, 2011-2015. J Med Virol. 2017 May;89(5):753-758.

38. Acknowledgements
• Dr. Sunil R Vaidya
• Dr. D T Mourya
• Mr. A. M. Walimbe
• Ranawade sir
• Measles group, NIV camp
• Bedekar sir, Neethi Ma’m and Dr. Vikram Ghole
• My family and M.Sc colleagues

39. THANK YOU

40. Supplementary information
• MF region – START: 3’-ACCGCAGTG- 5’, STOP: 3’-GTGTCCATC-5’ (relatively conserved)
• N gene – START: 3’-GTCAGTTCCACATTGG-5’, STOP: 3’-GAGATCTTCTA-5’
• MF region GC content – 63 % > Rest of the genome GC content – 47 %
• KT732261/MVs/Manchester.GBR/30.13 – D8 reference whole genome.
• KT732229/MVs/London.GBR/20.12 – D4 reference whole genome.
• Phylogeny as per MF region had a bootstrap value >70 between groups.
• Primers diluted from 100 µM stock to required concentration by C1V1 = C2V2 formula.
• MF region is non-coding and identical in vaccine strains.
• N gene sequences of 5 isolates were not available, hence propagated, rest 30 downloaded from GenBank.