Lessons Learned/Examples from the
Molecular Epidemiology of Measles
Paul Rota*
Centers for Disease Control and Prevention
Topic: Webinar on Molecular Genotyping for MR
Date: Tuesday, 5. December 2017
National Center for Immunization & Respiratory Diseases
Division of Viral Diseases
*The findings and conclusions in this report are those of the authors and do not necessarily represent the official position
of the Centers for Disease Control and Prevention.
National Center for Immunization & Respiratory Diseases
Division of Viral Diseases
Lessons Learned from > 20 years of Genetic Characterization of
Measles Viruses
• All vaccines strains are in genotype A
• Wild-type viruses in genotype A no longer circulate
• Rapid confirmation of vaccine reactions is an important part of laboratory
surveillance in elimination settings
• Sequence information allowed development of RT-PCR based
assays for rapid confirmation of vaccines reactions
3
Rapid Assay to Detect Measles Vaccine Strains
• MeVA allows confirmation of MR, MMR vaccine reactions in 3-4 hours
compared to 24-48 for sequencing
• Validations in progress
4
National Center for Immunization & Respiratory Diseases
Division of Viral Diseases
Lessons Learned from > 20 years of Genetic Characterization of
Measles Viruses
• Countries with endemic measles have multiple, co circulating lineages
of measles virus, endemic genotype or genotypes (e.g. genotype H1 in
China, B3 in sub Saharan Africa)
Endemic Measles/Endemic Genotypes
6
National Center for Immunization & Respiratory Diseases
Division of Viral Diseases
Lessons Learned from > 20 years of Genetic Characterization of
Measles Viruses
• In countries that have eliminated measles, the pattern of genotypes
reflects the sources of imported virus
• Lack of endemic genotype is an essential criterion for verification of
elimination
Measles Eliminated/Multiple Genotypes
8
National Center for Immunization & Respiratory Diseases
Division of Viral Diseases
Lessons Learned from > 20 years of Genetic Characterization of
Measles Viruses
• In countries where measles has been reintroduced, often only a single
lineage is detected; there may be a “switch” in genotypes indicating
single source of importation and single chain of transmission
• Tracking by using named strains can help to identify sources
• Sequence data can help documents switch in genotypes after
elimination and following reintroduction
0
10000
20000
30000
40000
50000
60000
70000
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014
Measles cases by year, Philippines, 2000-2014
National Center for Immunization & Respiratory Diseases
Division of Viral Diseases
2000-2004
Genotype D3 is the
endemic genotype in
the Philippines and
D3 imports to other
countries are traced
to the Philippines
2007-2009
Genotypes D9 and
G3 introduced and
detected in
outbreaks
2014-2015
Genotype B3
introduced,
genotype B3 and D9
continues to
circulate and cause
outbreaks
2004-2005
Measles
campaign in 2004,
no measles
detected in 2005
Genotype D3 not detected globally after 2004
Measles Genotypes In the Philippines
Nu
mb
er
of m
ea
sle
s c
ase
s
Mapping Transmission Pathways Following the Outbreak in the Philippines in 2014: Tracking
Genotype B3 “Harare”
11
Tracking Measles Importations by Named
Strain (Lineage)
12
1 2 3 4 5 6 7 8 9 10 11 12
A Genotype B3
B B3-name 1
B3-name 2
B3-name 3
Tracking Measles Lineages by using Named Strains
Time in months
• In both cases, genotype B3 is reported every month for 1 consecutive months
• Example A, reporting only genotype: continuous transmission of genotype B3
• Example B, tracking named strains: Multiple importations of three different named
strain/lineages
13
0
1
2
3
4
5
6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52
Nu
mb
er
of
case
s
Week number
Lineage #7
Lineage #6
Lineage #5
Lineage #4
Lineage #3
Lineage #2
Lineage #1
IDN
IDN
IDN
ID
N
IDN
IDN
IDN
IDN
IDN
VN
M
IND
IN
D
NZL
NP
L
NP
L
THA
THA
Country codes of importation associated with measles cases are indicated (IDN=Indonesia, VNM=Viet Nam; IND=India; NZL=New Zealand; NPL=Nepal; THA=Thailand
Weekly distribution of measles genotype D8
lineages identified at VIDRL in 2016.
14
MVs/Michigan.USA/49.14/1 (3000050723) MVs/Michigan.USA/48.14/1 (3000050657) MVs/Michigan.USA/48.14/2 (3000050658) MVs/Michigan.USA/49.14/3 (3000050725) MVs/Michigan.USA/49.14/2 (3000050724) MVs/Kosrae.FSM/21.14/4 (3000040041) MVs/California.USA/6.14/1 (CDC COE-2014-9) MVs/Alaska.USA/37.14/ (CDC COE-2014-173)
MVs/California.USA/5.14/ (CDC COE-2014-7) + 81 identical strains MVs/Pohnpei.FSM/28.14/2 (3000040292) MVs/California.USA/4.14/ (CDC COE-2014-3) MVs/Missouri.USA/22.14 (3000040083) MVs/Kansas.USA/24.14 (3000040147)
MVs/Kosrae.FSM/21.14/2 (3000040038) + 69 identical strains MVs/NewYork.USA/8.14/6 (CDC COE-2014-24) MVs/Chuuk.FSM/34.14 (3000040384)
MVs/California.USA/8.14/3 (CDC COE-2014-18) MVs/California.USA/10.14/4 (CDC COE-2014-35) MVs/California.USA/10.14/ (CDC COE-2014-32)
MVs/New York.USA/15.14/1 (2014729334) MVs/Washington.USA/14.14/ (CDC COE-2014-71) MVi/Washington.USA/13.14/ (CDC COE-2014-70) MVs/Washington.USA/14.14/4 (CDC COE-2014-74) MVs/Washington.USA/14.14/3 (CDC COE-2014-73)
MVi/Ibadan.NGE/0.97/1 (B3) MVi/New York.USA/0.94 (B3)
MVi/Yaounde.CMR/12.83 (B1) MVi/Libreville.GAB/0.84 (B2)
MVi/Maryland.USA/0.54 (A) MVs/Madrid.ESP/0.94 (F)(SSPE)
MVi/Goettingen.DEU/0.71 (E) MVi/Tokyo.JPN/0.84 (C1)
MVi/Erlangen.DEU0.90 (C2) MVi/Maryland.USA/0.77 (C2) MVi/Amsterdam.NLD/49.97 (G2)
MVi/Gresid.IDN/17.02 (G3) MVi/Berkeley.USA/0.83 (G1)
MVi/Beijing.CHN/0.94/1 (H2) MVi/Hunan.CHN/0.93/7 (H1)
MVs/Texas.USA/36.14/ (CDC COE-2014-171) MVs/California.USA/1.14/ (CDC COE-2014-23)
MVs/California.USA/18.14/ (CDC COE-2014-95) MVs/Indiana.USA/30.14/ (3000040342)
MVi/Bristol.GBR/0.74 (D1) MVi/New Jersey.USA/0.94/1 (D6)
MVi/Kampala.UGA/51.01/1 (D10) MVi/Johannesburg.ZAF/0.88/1 (D2)
MVs/Ohio.USA/28.14 (3000040284) + 37 identical strains MVs/Ohio.USA/16.14/1 (2014729385)
MVi/Victoria.AUS/12.99 (D9) MVi/Montreal.CAN0.89 (D4)
MVi/Illinois.USA/0.89/1 (D3) MVi/Bangkok.THA/0.93/1 (D5)
MVi/Palau.PLW/0.93 (D5) MVi/Victoria.AUS/16.85 (D7)
MVi/Illinois.USA/50.99 (D7) MVi/Menglian.Yunnan.CHN/47.09 (D11)
MVi/Manchester.GBR/30.94 (D8) MVs/Hawaii.USA/43.14/3 (3000050390) MVs/Hawaii.USA/42.14 (3000050659) MVs/New Mexico.USA/47.14 (3000050691)
MVs/California.USA/20.14/ (CDC COE-2014-118) MVi/Massachusetts.USA/14.14 (2014729283)
MVs/Virginia.USA/19.14 (3000039924) MVs/Washington.USA/12.14/ (CDC COE-2014-69) MVs/Washington.USA/14.14/2 (CDC COE-2014-72)
MVs/California.USA/4.14/2 (CDC COE-2014-11) MVs/California.USA/8.14/ (CDC COE-2014-14)
MVs/Minnesota.USA/17.14/ (CDC COE-2014-119) MVs/Massachusetts.USA/19.14 (3000039975)
MVs/California.USA/8.14/2 (CDC COE-2014-17) MVs/California.USA/15.14/2 (CDC COE-2014-86) MVs/California.USA/15.14/ (CDC COE-2014-85) MVs/California.USA/15.14/3 (CDC COE-2014-88)
MVs/California.USA/14.14/3 (CDC COE-2014-89) MVs/California.USA/14.14/2 (CDC COE-2014-87) MVs/California.USA/12.14/8 (CDC COE-2014-90)
5
Genotype B3
Genotype H1
Genotype D9
Genotype D8
Phylogenetic tree based on the N-
450 gene sequences of measles
viruses detected in the US in 2014.
Reference strains are underlined
and color code for the genotypes
is given at bottom left.
15
Measles Representative Strain Gen. Number of Strains MeaNS Exact Match
MVs/New York.USA/15.14/2 B3 + 4 identical strains NEM
MVs/Missouri.USA/22.14 B3 + 1 identical strains NEM
MVs/Michigan.USA/48.14/1 B3 + 4 identical strains NEM
MVs/Alaska.USA/37.14/ (CDC_COE-2014-173) B3 NEM
MVs/California.USA/4.14/ (CDC_COE-2014-3) B3 NEM
MVs/California.USA/6.14/1 (CDC_COE-2014-9) B3 NEM
MVs/California.USA/8.14/3 (CDC_COE-2014-18) B3 + 2 identical strains NEM
MVs/NewYork.USA/8.14/6 (CDC_COE-2014-24) B3 NEM
MVs/California.USA/5.14/ (CDC_COE-2014-7) B3 + 81 identical strains MVi/Harare.ZWE/38.09/
MVs/Kosrae.FSM/21.14/2 B3 + 69 identical strains MVi/Harare.ZWE/38.09/
MVs/Kosrae.FSM/21.14/4 B3 NEM
MVs/Pohnpei.FSM/28.14/2 B3 NEM
MVs/Chuuk.FSM/34.14 B3 NEM
MVs/Indiana.USA/30.14/ H1 + 1 identical strains MVs/Liaoning.CHN/23.14/2, MVi/Tianjin.CHN/22.14/4,
MVi/Shanghai.CHN/20.14/, MVs/Anhui.CHN/19.14/8
MVs/California.USA/1.14/ (CDC_COE-2014-23) H1 MVs/Hong Kong.CHN/49.12
MVs/Texas.USA/36.14/ (CDC_COE-2014-171) H1 MVs/Hong Kong.CHN/42.11/
MVs/Ohio.USA/28.14 D9 NEM
MVs/Ohio.USA/16.14/1 D9 + 37 identical strains MVs/Hong Kong.CHN/08.14/2
MVs/Washington.USA/12.14/ (CDC_COE-2014-69) D8 + 2 identical strains MVs/Taunton.GBR/27.12/
MVs/California.USA/4.14/2 (CDC_COE-2014-11) D8 NEM
MVs/California.USA/8.14/ (CDC_COE-2014-14) D8 NEM
MVs/Hawaii.USA/43.14/3 D8 + 2 identical strains
MVs/Queensland.AUS/45.14/, MVs/London.GBR/30.14/
MVs/NewSouth Wales.AUS/27.14/
MVs/WesternAustralia.AUS/23.14/
MVi/Massachusetts.USA/14.14 D8 MVs/London.GBR/22.12/3, MVs/Victoria.AUS/6.11/
MVs/Ludwigsburg.DEU/13.10/
MVs/Virginia.USA/19.14 D8 MVs/Maastricht.NLD/14.14, MVi/Pune.IND/38.13
MVs/WesternAustralia.AUS/51.13
MVs/Massachusetts.USA/19.14 D8 + 2 identical strains MVs/Heidelberg.DEU/45.13/
MVs/California.USA/20.14/ (CDC_COE-2014-118) D8 MVi/HuluLangat.MYS/26.11
MVs/California.USA/8.14/2 (CDC_COE-2014-17) D8 MVs/FrankfurtMair.DEU/17.11/
MVs/California.USA/12.14/8 (CDC_COE-2014-90) D8 + 4 identical strains MVs/Western Australia.AUS/12.14/, MVs/London.GBR/9.14/2
MVi/Singapore.SGP/13.14/
Lineages of Measles Viruses Detected in the US during 2014. Strains from the US are listed in the left column. Strains with exact matches
in N-450 are listed on the right column. Named lineages (from MeaNS) are shown in red. NEM refers to no exact match.
16
MeV – Genotype B3
imported into the USA
in 2015, multiple
importations from a
single source and
spread to multiple
states
MVs/California.USA/1.15.3 (CDC RC-2015-7) MVs/Washington.USA/3.15/ (CDC RC-2015-42) MVs/Utah.USA/3.15/ (CDC RC-2015-41) MVs/Utah.USA/1.15/ (CDC RC-2015-1) MVs/Illinois.USA/7.15 (3000373357) MVs/Illinois.USA/7.15/4 (3000375770) MVs/Illinois.USA/7.15/2 (3000375767) MVs/California.USA/3.15/20 (CDC RC-2015-146) MVs/California.USA/3.15/21 (CDC RC-2015-144) MVs/California.USA/3.15/17 (CDC RC-2015-141) MVs/Illinois.USA/6.15/4 (3000373355) MVs/Illinois.USA/6.15/2 (3000373329) MVs/Illinois.USA/5.15/2 (3000373273) MVs/California.USA/9.15/2 (CDC RC-2015-119) MVs/California.USA/8.15/4 (CDC RC-2015-104) MVs/California.USA/8.15/ (CDC RC-2015-96) MVs/California.USA/7.15/5 (CDC RC-2015-106) MVs/California.USA/6.15/3 (3000373614) MVs/California.USA/53.14 (3000373479) MVs/California.USA/5.15/8 (CDC RC-2015-78) MVs/California.USA/5.15/6 (CDC RC-2015-75) MVs/California.USA/5.15/4 (CDC RC-2015-57) MVs/California.USA/5.15/2 (CDC RC-2015-55) MVs/California.USA/5.15/ (CDC RC-2015-54) MVs/California.USA/4.15/5 (CDC RC-2015-63) MVs/California.USA/4.15/3 (CDC RC-2015-35) MVS/California.USA/4.15/ (CDC RC-2015-33) MVs/California.USA/3.15/8 (CDC RC-2015-129) MVs/California.USA/3.15/6 (CDC RC-2015-127) MVs/California.USA/3.15/6 (3000373474) MVs/California.USA/3.15/4 (CDC RC-2015-40) MVs/California.USA/3.15/2 (CDC RC-2015-38) MVs/California.USA/3.15/13 (CDC RC-2015-134) MVs/California.USA/3.15/11 (CDC RC-2015-132) MVs/California.USA/3.15/ (CDC RC-2015-37) MVs/California.USA/2.15/8 (CDC RC-2015-20) MVs/California.USA/2.15/6 (CDC RC-2015-18) MVs/California.USA/2.15/4 (CDC RC-2015-16) MVs/California.USA/2.15/2 (CDC RC-2015-5) MVs/California.USA/2.15/15 (CDC RC-2015-28) MVs/California.USA/2.15/13 (CDC RC-2015-26) MVs/California.USA/2.15/11 (CDC RC-2015-23) MVs/California.USA/2.15/ (CDC RC-2015-3) Mvs/California.USA/10.15/ (CDC RC-2015-117) MVs/California.USA/1.15/4 (CDC RC-2015-9) MVs/California.USA/1.15/ (CDC RC-2015-4)
MVs/Arizona.USA/5.15/ (CDC RC-2015-59) MVs/Washington.USA/9.15/ (CDC RC-2015-114) MVs/Washington.USA/2.15/ (CDC RC-2015-30) MVs/Utah.USA/1.15/2 (CDC RC-2015-2)
MVs/Nevada.USA/7.15/ (CDC RC-2015-67) MVs/Nevada.USA/9.15/2 (CDC RC-2015-115) MVs/Nevada.USA/5.15/ (CDC RC-2015-47) MVs/Nevada.USA/9.15/ (CDC RC-2015-113)
MVs/Illinois.USA/8.15/ (3000375773) MVs/Illinois.USA/7.15/5 (3000375772) MVs/Illinois.USA/7.15/3 (3000375768) MVs/California.USA/5.15/10 (CDC RC-2015-147) MVs/California.USA/3.15/19 (CDC RC-2015-145) MVs/California.USA/3.15/18 (CDC RC-2015-142) MVs/Illinois.USA/6.15/5 (3000373356) MVs/Illinois.USA/6.15/3 (3000373330) MVs/Illinois.USA/6.15/1 (3000373328) MVs/Illinois.USA/5.15/1 (3000373272) MVs/California.USA/9.15/3 (CDC RC-2015-120) MVs/California.USA/8.15/5 (CDC RC-2015-110) MVs/California.USA/8.15/2 (CDC RC-2015-100) MVs/California.USA/7.15/6 (CDC RC-2015-107) MVs/California.USA/7.15/4 (3000373612) MVs/California.USA/7.15/2 (CDC RC-2015-95) MVs/California.USA/6.15/2 (CDC RC-2015-73) MVs/California.USA/5.15/9 (CDC RC-2015-79) MVs/California.USA/5.15/7 (CDC RC-2015-77) MVs/California.USA/5.15/5 (CDC RC-2015-58) MVs/California.USA/5.15/3 (CDC RC-2015-56) MVs/California.USA/4.15/6 (CDC RC-2015-64) MVs/California.USA/4.15/4 (CDC RC-2015-36) MVs/California.USA/4.15/2 (CDC RC-2015-34) MVs/California.USA/3.15/9 (CDC RC-2015-130) MVs/California.USA/3.15/7 (CDC RC-2015-128) Mvs/California.USA/3.15/6 (CDC RC-2015-126) MVs/California.USA/3.15/5 (3000373473) MVs/California.USA/3.15/3 (CDC RC-2015-39) MVs/California.USA/3.15/14 (CDC RC-2015-135) MVs/California.USA/3.15/12 (CDC RC-2015-133) MVs/California.USA/3.15/10 (CDC RC-2015-131) MVs/California.USA/2.15/9 (CDC RC-2015-21) MVs/California.USA/2.15/7 (CDC RC-2015-19) MVs/California.USA/2.15/5 (CDC RC-2015-17) MVs/California.USA/2.15/3 (CDC RC-2015-8) MVs/California.USA/2.15/16 (CDC RC-2015-29) MVs/California.USA/2.15/14 (CDC RC-2015-27) MVs/California.USA/2.15/12 (CDC RC-2015-25) MVs/California.USA/2.15/10 (CDC RC-2015-22) MVs/California.USA/10.15/2 (CDC RC-2015-123) MVs/California.USA/1.15/5 (CDC RC-2015-24) MVs/California.USA/1.15/2 (CDC RC-2015-6)
MVs/California.USA/7.15/3 (3000373480) MVs/Hawaii.USA/1.15 (3000373195-96)
MVs/California.USA/5.15/10 (3000373477) MVs/Nevada.USA/6.15 (3000373363)
MVi/Ibadan.NGE/0.97/1 (B3)
5 17
Increasing the Resolution of Molecular Epidemiology
for Measles
18
Moss & Griffin, 2006
WHO standard
genotyping N target
450 nt
Alternate genotyping target:
H ORF, 1854 nt
MF-NCR new
target
1018 nt
WGS-t
15,875 nt
• WHO N.E.W. Working Group to report in January
• Sequencing addition regions or the entire genome can
increase resolution in some cases
Identical N450 (a) and H (b) gene
target sequences
Courtesy: A Severini, NML, Canada 19
The MF-NCR (B) offers a resolution comparable
to WGS (A)
Courtesy: A Severini, NML, Canada 20
Fig 3. Phylogenetic
analysis of the N-450, H, N
and M/F NCR sequences
of D8 outbreak strains.
Penedos AR, Myers R, Hadef B, Aladin F, Brown KE (2015) Assessment of the Utility of Whole Genome Sequencing of Measles Virus in the Characterisation of Outbreaks. PLOS ONE 10(11): e0143081. https://doi.org/10.1371/journal.pone.0143081 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143081
21
Genotype D9 from Single Importation into the USA 2014: Identical
N-450 and MF-NCR Sequences
N-450 MF-NCR
MVs/Ohio.USA/19.14/3
MVs/Ohio.USA/17.14/6
MVs/Ohio.USA/18.14/10
MVs/Ohio.USA/18.14/9
MVs/Ohio.USA/18.14/8
MVs/Ohio.USA/18.14/7
MVs/Ohio.USA/19.14/2
MVs/Ohio.USA/18.14/6
MVs/Ohio.USA/19.14/1
MVs/Ohio.USA/18.14/5
MVs/Ohio.USA/18.14/4
MVs/Ohio.USA/18.14/3
MVs/Ohio.USA/17.14/5
MVs/Ohio.USA/17.14/4
MVs/Ohio.USA/18.14/2
MVs/Ohio.USA/17.14/3
MVs/Ohio.USA/17.14/2
MVs/Ohio.USA/17.14/1
MVs/Ohio.USA/16.14/3
MVs/Ohio.USA/16.14/2
MVs/Ohio.USA/16.14/1
MVs/Ohio.USA/28.14
MVs/Ohio.USA/27.14
MVs/Ohio.USA/25.14/1
MVs/Ohio.USA/23.14/3
MVs/Ohio.USA/23.14/2
MVs/Ohio.USA/22.14/2
MVs/Ohio.USA/22.14/1
MVs/Ohio.USA/23.14/1
MVs/Ohio.USA/20.14/1
MVs/Ohio.USA/20.14/3
MVs/Ohio.USA/19.14/5
MVs/Ohio.USA/19.14/4
MVs/Ohio.USA/21.14
MVs/Ohio.USA/20.14/2
MVs/Ohio.USA/18.14/11
MVs/Ohio.USA/23/14/4
MVi/Victoria.AUS/12.99 D9
62
0.005
22
Isolated Cases 2 Case Chains
Outbreaks with 3-5
Cases
Outbreaks with >5
Cases
All Chains of
Transmission
Cases Total Genotyped Total Genotyped Total Genotyped Total Genotyped Total Genotyped
2016 509 233 (46%) 77 47 (63%) 61 47 (77%) 50 47 (96%) 748 523 (71%)
Completeness of Molecular Surveillance: USA
2001-2015
23
Summary
• Genetic characterization of measles virus by the GMRLN has
made substantial contributions to understanding both the
biology and evolution of measles viruses, and has become
and integral part of routine laboratory surveillance for measles
• The GMRLN needs to continue to build capacity for genetic
characterization of both measles and rubella and to integrate
new testing schemes and new technologies
24
What RVC members should expect from GMRLN regarding
molecular epidemiology of measles and rubella
• Strategically increasing capacity for molecular testing
– Network laboratories with existing molecular infrastructure (RT-
qPCR, sequencing) trained for measles and rubella molecular
testing
• Ability to analyze and upload quality sequences to MeaNS and RubeNS
– Workshops will be conducted in as needed in all WHO Regions
– Molecular external quality assurance program expanded to monitor
the performance of laboratories
25
• Laboratories must work with program staff to obtain adequate samples
for viral detection (>80% of chains of transmission, with genotype)
• Obtain virologic samples from all cases and report genotypes even
from sporadic cases
• Timely reporting of all sequence data to MeaNS and RubeNS
• Implementation molecular methods for case classification/confirmation • RT-PCR
RVC Members should look for
26
• Rapid confirmation of measles vaccine reactions is essential
• Description of the named strains/lineages in addition to the
genotypes
• Always include a phylogenetic tree
• Always include matches with named strain or identical
sequence on MeaNS/RubeNS
• Consider using expanded sequencing windows to increase
resolution when necessary
• Consider additional training in molecular methods including
sequence analysis and database submission (keep using
webinars)
Continued…..
27
Questions??
Discussion
28