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TRANSCRIPT
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
05/12/2017 | Title of the presentation 6
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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8
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