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Molecular epidemiology used to track viral outbreaks–norovirus and beyond
Viruses in May 2018 - The Carrington Hotel, Katoomba NSW18th May 2018
Professor Peter WhiteMolecular Microbiology Lab
School of Biotechnology and Biomolecular SciencesUniversity of New South Wales
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Norovirus
▪ Leading cause of viral gastroenteritis
▪ Highly infectious and environmentally stable
▪ Nursing home, hospital wards, cruise ships, childcare centre
▪ Massive social-economic cost each year
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Norovirus transmission
▪ Person to person, most common via faecal – oral route
▪ Consumption of contaminated food or drink
▪ Poor food-handling
▪ Secondary spread - aerosols, fomites
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Classification
Caliciviridae (family)• Lagovirus [rabbits]
• Nebovirus [bovine]
• Sapovirus [swine/humans]
• Vesivirus [feline/sea lion/swine]
• Norovirus (genus)
[swine/humans/mice/feline/dogs]
− Norwalk virus (prototype species)
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Norovirus classification
▪
▪ Family: Caliciviridae▪ Genus: Norovirus▪ Classified into 7 genogroups▪ > 40 genotypes
▪ Genogroup II, genotype 4 (GII.4) is the most prevalent (65-80% of
all norovirus infections)
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GII.4 global pandemic variants
2017
First reported
pandemic strain-
US1995/96
Farmington Hills
2002
Hunter 2004 New Orleans 2009
Sydney 2012
Osaka 2007
Apeldoorn 2008Yerseke 2006a
Den Haag 2006b
1995 2002 2004 2009 20122007
Pandemic
Epidemic
Key
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Viral evolution mechanisms
▪ ANTIGENIC DRIFT : point mutations within the capsid region
▪ ANTIGENIC SHIFT : exchange of genetic material between two different strains
New pandemic variants
New recombinants
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Recombination
+Replication
Recombinant
Requires dual infection
Bull et al., EID, 2005
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Norovirus genotyping
ORF1ORF2
ORF3
GII.Pe/GII.4 Sydney 2012
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Emergence of GII.4 recombinants in 2016
74%
37.5%
10.8%
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Emergence of GII.4 recombinants in 2016
74%
37.5%
10.8%
10%
14.6%
15.9%
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Emergence of GII.4 recombinants in 2016
74%
37.5%
10.8%
20.5%
8.3%
Lun et al. Emerging Microbes & Infections (2018) 7:50
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Emergence of GII.4 recombinants in 2016
23.6%
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Emergence of GII.4 recombinants in 2016
▪ GII.P4 NO 2009/GII.4 Sydney 2012▪ GII.P16/GII.4 Sydney 2012▪ GII.P12/GII.3▪ GII.P16/GII.2▪ GII.P17/GII17
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Aim
Examine the molecular epidemiology of circulating clinical norovirus in NSW from January to October 2017
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Cruise Ships in Sydney
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M o n th -Y e a r
Nu
mb
er
of
gast
roe
nte
riti
s o
utb
rea
ks
Jan -1
0
Jul-1
0
Jan -1
1
Jul-1
1
Jan -1
2
Jul-1
2
Jan -1
3
Jul-1
3
Jan -1
4
Jul-1
4
Jan -1
5
Jul-1
5
Jan -1
6
Jul-1
6
Jan -1
7
Jul-1
7
0
5 0
1 0 0
1 5 0
2 0 0
A g e d c a r e fa c il ity
C h ild c a r e c e n tr e
H o s p ita l
O th e rs
An increase in outbreaks of gastroenteritis, 2017
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The number of gastroenteritis and norovirus outbreaks reported in NSW, Australia and New Zealand, 2017.
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GII.4 global pandemic variants
2017
First reported
pandemic strain-
US1995/96
Farmington Hills
2002
Hunter 2004 New Orleans 2009
Sydney 2012Den Haag 2006b
1995 2002 2004 2009 20122007
New strain?
2017
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Monthly distribution of norovirus genotypes identified in Australia and New Zealand, 2017.
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Norovirus GII distribution in clinical samples from Australian and New Zealand, 2013-2017
NSW
New Zealand
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Problems with current screening methods
▪ We only use clinical samples
▪ Symptomatic cases only
▪ Norovirus-related gastroenteritis is generally self-limiting
▪ Over-representation of childhood infection
▪ A system to screen for norovirus at a population level
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Where can we find norovirus at population level?
Wastewater treatment plants
▪ Collection of waste from within a population
▪ Prolong and high viral load shedding
▪ Abundance of enteric viruses
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Study aims
▪ To detect and quantify norovirus RNA levels in wastewater samples
▪ Examine the norovirus genetic diversity in wastewater
▪ Compare norovirus diversity in clinical and wastewater samples
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Wastewater sample collection
▪ Monthly collections throughout 2016 and 2017, from 3 sites;▪ Sydney – Bondi and Malabar▪ Melbourne – Western Treatment Plant
Bondi(population 296,350)
Malabar(population 1,667,460)
Melbourne WTP(population 2,400,000)
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Sample preparation for NGS
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Sample preparation for NGS
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NGS data analysis
▪ Average of 580,000 reads per sample
▪ Geneious program for data analysis
▪ Paired end reads merged, primers trimmed
▪ Reads mapped to NoV reference sequences
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Norovirus genotype distribution in wastewater samples 2017
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Norovirus GII distribution in clinical samples from Australian and New Zealand, 2013-2017
NSW
New Zealand
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▪ No ethics
▪ Less sample to be processed= cheaper
▪ Represents a whole population
▪ Non-biased
▪ Greater diversity
▪ Possible predictive tool
Benefits Improvements?
▪ Recombinant noroviruses
▪ GI noroviruses
▪ Other enteric viruses
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Summary▪ The molecular surveillance of norovirus is important to
predict and identify new noroviruses and potential pandemic variants
▪ Recombinant GII.P16/GII.4 Sydney 2012 is responsible for majority of acute viral gastroenteritis in 2017 – pandemic?
▪ Clinical samples are biased towards symptomatic cases and over-represent childhood infections
▪ Wastewater samples provide a better understanding of noroviruses within a population
▪ Genotype dominance correlates, although not exactly.
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Jennifer Lun - PhD student
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Acknowledgements
▪ Douglass Hanly MoirRichard Jones
▪ White Lab Prof. Peter WhiteDr Tulio FumianNatalie NetzlerDaniel Enosi TuipulotuAlice RussoLeigh Morrell Grace Yan
▪ University of SydneyProf. Edward HolmesDr John Sebastian Eden
▪ Prince of Wales HospitalProf. William RawlinsonJuan Merif
▪ NSW Public HealthProf. Mark FersonDr Kelly-Anne Ressler
▪ Ramaciotti Centre for Genomics
Alper YasarJason Koval
▪ Westmead HospitalDr Ian CarterProf. Dominic DwyerDr Jen Kok
▪ Melbourne Water Corporation
Nick Crosbie
▪ Sydney WaterPeter Beatson
▪ Water FuturesDaniel Deere
▪ Canberra HospitalEmma Malinsky
▪ NZ Public HealthDr Joanne Hewitt
▪ QLD Public Health David WarrilowDr. Ben Huang
▪ Hunter New England Population Health Prof. David DurrheimDr. Belinda JonesJames Flint Julie Collins