lessons learned/examples from the molecular …lessons learned/examples from the molecular...

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.



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

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




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




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




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



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

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143081

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143081

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/28.14

MVs/Ohio.USA/27.14











MVs/Ohio.USA/21.14



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

lessons learned/examples from the molecular …lessons learned/examples from the molecular...

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