Genotyping and its utility in verification for both measles and rubella elimination

Webinar: Measles and Rubella Genotyping

Mick N. Mulders PhD | FWC/IVB/EPI | Geneva, Switzerland

05 December 2017, 11:00 – 13:00 GMT

Moderators

• Mick N. Mulders PhD – Coordinator VPD Laboratory Networks, WHO/HQ

• Kevin E. Brown MD MRCP FRCPath – Deputy Director and Lead Clinical Virologist, Virus Reference Department, National Infection Service, Public Health England, London / WHO Global Specialised Laboratory for Measles and Rubella

• Paul A. Rota PhD – Acting Chief, Viral Vaccine Preventable Diseases Branch, Division of Viral Diseases, CDC, Atlanta / WHO Global Specialised Laboratory for Measles and Rubella

Introduction, goals and roles

• Strengthening and maintaining global laboratory capacity

Verification of elimination and genotyping

• Methods and nomenclature

Examples of genotyping and how it is used for verification

• Capacity of genotyping in network and expectations

• Lessons learned

• Enhancing resolution

Measles and rubella genotyping

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Webinar

Documentation of the interruption of endemic measles, or rubella virus transmission for a period of at least 36 months from the last known endemic case

The presence of a high-quality surveillance system that is sensitive and specific enough to detect imported and import-related cases

Genotyping evidence that supports the interruption of endemic transmission

Indicators of the quality of field and laboratory surveillance

• Timeliness of reporting

• Reporting rate of discarded non-measles non-rubella cases

• Representativeness of reporting

• Laboratory confirmation

• Viral detection

• Adequacy of investigation

• Timeliness of specimen transport

• Timeliness of reporting laboratory results

Criteria for verifying elimination

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http://www.who.int/wer/2013/wer8809.pdf

Framework for Verifying Elimination of Measles and Rubella

A detailed description of the epidemiology of measles and rubella since the introduction of measles and rubella vaccine in the national

immunization programme

Population immunity presented as a birth cohort analysis with the addition of evidence related to any marginalized and migrant groups

Quality of epidemiological and laboratory surveillance systems for measles and rubella

Sustainability of the national immunization programme including resources for mass campaigns, where appropriate, in order to sustain

elimination

Genotyping evidence that measles and rubella virus transmission is interrupted

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Lines of evidence

Surveillance

• Classifying cases

• Provide evidence for verification of elimination

• Monitor laboratory performance in meeting quality indicators

Accreditation

• All countries with an elimination goal must have access to an measles-rubella laboratory accredited by GMRLN, with ongoing quality control

Molecular surveillance

• Documenting pre-elimination genotypes

• Monitoring genetic characteristics of circulating strains

• Verifying absence of endemic transmission

Population immunity

• Supporting studies on population immunity/serosurveys

Global Measles and Rubella Laboratory Network (est. 2000)

Role of GMRLN in verification

Monitor virus strain distribution to characterize outbreaks and to identify transmission pathways

Provide evidence for interruption of endemic virus circulation

Molecular surveillance should be conducted during all phases of measles and rubella control

Indicator for quality of laboratory surveillance

Measles and rubella virus

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http://www.who.int/wer/2012/wer8709.pdf?ua=1 and

Molecular Epidemiology

Primers Amplify 104 Copies of RNA Template

• Primers MeV214 and MeV216 are designed to amplify a 634 nucleotide region coding for the 3’ terminus of the nucleoprotein (N)

gene in a conventional RT-PCR reaction.

• 11 different genotypes tested

• MV 214 and 216 are also used in sequencing reactions

Measles Genotyping

N P/C/V M F H L

seq window

1104 1131 1233 1747 1686

seq window MeV214 MeV216

Target sequence for measles genotyping

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MeV216 (nt 1105-1124)

1105 TGG AGC TAT GCC ATG GGA GTA GGA GTG GAA CTT GAA AAC TCC ATG GGA 1152

1153 GGT TTG AAC TTT GGC CGA TCT TAC TTT GAT CCA GCA TAT TTT AGA TTA 1200

start of N-450 window

1201 GGG CAA GAG ATG GTA AGG AGG TCA GCT GGA AAG GTC AGT TCC ACA TTA 1248

1249 GCA TCT GAA CTC GGT ATC ACT GCC GAG GAT GCA AGG CTT GTT TCA GAG 1296

1297 ATT GCA ATG CAT ACT ACT GAG GAC AAG ATC AGT AGA GCG GTT GGA CCC 1344

1344 AGA CAA GCC CAA GTA TCA TTT CTA CAC GGT GAT CAA AGT GAG AAT GAG 1392

1394 CTA CCG AGA TTG GGG GGC AAG GAA GAT AGG AGG GTC AAA CAG AGT CGA 1440

1441 GGA GAA GCC AGG GAG AGC TAC AGA GAA ACC GGG CCC AGC AGA GCA AGT 1488

1489 GAT GCG AGA GCT GCC CAT CTT CCA ACC GGC ACA CCC CTA GAC ATT GAC 1536

1537 ACT GCA TCG GAG TCC AGC CAA GAT CCG CAG GAC AGT CGA AGG TCA GCT 1584

1585 GAG CCC CTG CTT AGG CTG CAA GCC ATG GCA GGA ATC TCG GAA GAA CAA 1632

1633 GGC TCA GAC ACG GAC ACC CCT ATA GTG TAC AAT GAC AGA AAT CTT CTA 1680

end of N-450 window MeV214

1681 GAC TAG GTG CGA GAG GCC GAG GGC CAG AAC AAC ATC CGC CTA CCC TCC 1728

1729 ATC ATT GTT ATA AAA AA

3

Measles Virus Clades and Genotypes

3

Groups are defined by phylogeny of the last 450 nt of N protein coding gene

0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08

subst itut ions per 450 nucleot ides

MVi/Illinois.USA/50.99/[D7]

MVi/Victoria.AUS/16.85/[D7]

MVi/Yunnan.CHN/47.09/[D11]

MVi/Manchester.GBR/30.94/[D8]

MVi/Montreal.CAN/0.89/[D4]

MVi/Victoria.AUS/12.99/[D9]

MVi/Palau.PLW/0.93/[D5]

MVi/Bangkok.THA/0.93/[D5]

MVi/Illinois.USA/0.89/[D3]

MVi/Johannesburg.ZAF/0.88/[D2]

MVi/Kampala.UGA/51.00/[D10]

MVi/Bristol.GBR/0.74/[D1]

MVi/New Jersey.USA/0.94/[D6]

MVi/Ibadan.NGA/0.97/[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]

MVi/Erlangen.DEU/0.90/[C2]

MVi/Maryland.USA/0.77/[C2]

MVi/Tokyo.JPN/0.84/[C1]

MVi/Goett ingen.DEU/0.71/[E]

MVi/Gresik.IDN/18.02/[G3]

MVi/Amsterdam.NLD/49.97/[G2]

MVi/Berkeley.USA/0.83/[G1]

MVi/Beijing.CHN/0.94/[H2]

MVi/Hunan.CHN/0.93/7[H1]

D

B

FCEGH

A Clades

MeaNS update 2017

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http://www.who-rubella.org/ and http://www.who-measles.org

Sequences submitted to and RubeNS

0

50

100

150

200

250

300

350

400

450

0

1000

2000

3000

4000

5000

6000

7000

8000

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017

Nu

mb

er

of

seq

ue

nce

s

Measles

Rubella

MMWR 2016, 65/17; WER 2016, 91/18

Global Distribution of Measles Genotypes: 2010-2015

Data Source: MeaNS database (Genotypes) and IVB Database (Incidence) as of 2017-11-10 and covering the period 2016-10-01 to 2017-09-30 - Pie charts proportional to the number of sequenced viruses

Distribution of measles genotypes (last 12 months)

13

Rubella virus genotyping

8258 8731 9469 9700

Molecular window (739-nt)

Genomic RNA

E1 coding region

1 41 3943 6391 6512* 9700* 9762*

P150 5’ Cap polyA 3’ P90 C E2 E1

Non-structural proteins (NSP) Structural proteins (SP)

1 2 M

Over interpretation of sequence results

• Identical measles sequences don’t imply epidemiological link!

Genotype information provides only limited information

• Need to analyze sequences at nucleotide level

New methods, new technology and protocols being developed to enhance resolution of molecular epidemiology

• GSL-RRL level mostly

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Caveats genotyping Rubella Virus Clades and Genotypes

3 Measles sequence diversity

Groups are defined by phylogeny of the last 739 nt window of E1 gene

1

2

Clades

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14

subst itut ions per site over 739 nucleot ides

RVi/Minsk.BLR/29.04/[1G]

RVi/Ontario.CAN/0.05/[1G]

RVi/Kam pala.UGA/20.01/[1G]

RVi/Minsk.BLR/28.05/2[1H]

RVi/Ryazan.RUS/09.08/[1H]

RVi/Bene Berak.ISR/0.79/[1B]

RVi/Tiberius.ISR/0.88/[1B]

RVi/Jerusalem.ISR/0.75/[1B]

RVi/Miyazaki.JPN/10.01/[1J]

RVi/Kagoshim a.JPN/22.04/[1J]

RVi/Shandong.CHN/0.02/[1E]

RVi/Kuala Lum pur.MYS/0.01/[1E]

RVi/Pennsylvania.USA/0.64/[1a]

RVi/Toyama.JPN/0.67/[1a]

RVi/Brussels.BEL/0.63/[1a]

RVi/New Jersey.USA/0.61/[1a]

RVi/Anhui.CHN/0.00/2[2B]

RVi/Washington.USA/16.00/[2B]

RVi/Tel Aviv.ISR/0.68/[2B]

RVi/Beijing.CHN/0.79/[2A]

RVi/Beijing.CHN/0.80/[2A]

RubeNS update 2017 3

MMWR 2016, 65/17; WER 2016, 91/18

Global Distribution of Rubella Genotypes: 2010-2015

Data Source: RubNS database (Genotypes) and IVB Database (Incidence) as of 2017-11-10 and covering the period 2016-10-01 to 2017-09-30 - Pie charts proportional to the number of sequenced viruses

Distribution of rubella genotypes (last 12 months)

Capacity building - Training

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Timely and complete reporting

• Diagnostic and genotyping data

Accreditation

• Ensure WHO performance indicators are met

• Timely reporting of lab data, incl genotyping

Quality control

• Confirmatory testing by supervisory laboratory

Quality assurance

• Proficiency testing serologic and molecular

Monitoring Laboratory Performance

FTA cards; CDC and INSTAND e.V.

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Molecular EQA program

Network laboratories with existing molecular infrastructure (RT-qPCR, sequencing) trained for measles and rubella molecular testing

• Infrastructure mostly developed through GPLN, influenza programme, or GHSA

Workshops have been repeatedly conducted in all WHO Regions

Molecular external quality assurance programme established to closely monitor the performance of labs

• Including abiltiy to analyse and upload quality sequences to MeaNS and RubeNS

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Conclusions