Transcript Kate Bowman - web.biosci.utexas.edu

Determination and Examination of
Norovirus Genotypes Circulating
in Texas, 2009-2010
Cate Bowman
Public Health Internship Program
School of Biological Sciences
The University of Texas at Austin
Mentor: Aaron Benfield, PhD
Molecular Biology Group Manager
Texas Department of State Health Services
Introduction
Noroviruses
• Family Caliciviridae
• Genus Norovirus
• Genogroups -GI,GII, GIV
• Genoclusters - GII.4
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Subclusters:
Camberwell (1987)
Grimsby (1995)
Farmington Hills (2002)
Hunter (2004)
Minerva (2006)
Apeldoorn (2008)
Characteristics
• Non-enveloped
• Icosahedral
capsid symmetry
• Positive-sense,
single stranded
RNA
Genome
• Three open reading frames
– ORF1
• Encodes non-structural proteins
– ORF2
• Encodes VP1, major capsid protein
– ORF3
• Encodes VP2, minor capsid protein
ORF1
ORF1
Non-structural proteins
ORF2
ORF2
VP1
ORF3
ORF3
VP2
Entry into population
• Infected food
handlers
• Most common food
sources
– Prepared foods
– Fresh fruits and
vegetables
– Raw or undercooked
seafood
Norovirus Prevalence
• Most common cause
of viral acute
gastroenteritis
– 21 million cases per
year
• 50% of foodborne
disease outbreaks
• 232 outbreaks from
1997-2002
CDC. 2010. MMWR. 59:975.
Epidemiology
• At risk
populations:
– Prisons
– Nursing homes
– Schools
– Cruise Ships
Infection
•
•
•
•
Transmission – fecal oral route
Infectious dose <10 particles
Incubation period - 24-48 hour incubation
Disease characteristics:
– Self limiting - symptoms last 12-72 hours, up to 6
days
• Symptoms: watery diarrhea, nausea, vomiting
– Risk of dehydration
– Elderly, young, and immunocompromised at
greatest risk of death
Pathogenesis
• Localized infection of the
epithelial cells of the small
intestines
• P2 domain of major capsid
protein, VP1, binds HBGA cells
on the epithelial cell surface
• Virus replication
– disrupts normal function of the
cells
– interfering with absorption of
nutrients and water
Immune response
• Innate immune
response
– Polymorphonuclear and
mononuclear cells
• Adaptive immune
response
– B and T cells critical
– IgG, IgA, IgM
– No long-term immunity
Epochal evolution of Noroviruses
• Periods of evolutionary stasis, followed by
periods of rapid evolution
• Changes in VP1 – new subclusters
• Results in new phenotypes (subclusters)
• Most changes occur in P domain of VP1
Camberwell
1987-1995
Grimsby
Farmington Hills
1995-2002
2002-2004
Treatment and prevention
• Treatment
– No drugs or vaccines
– Supportive therapy
• Prevention
– Hand-washing
– Avoiding contact with
infected individuals
– Disinfection
Laboratory diagnosis
• Virus difficult to
grow in culture
• Real-time PCR is
most common
method
• Sequencing –
genotype samples
Tracking the source: Genotyping
ORF2 region of the
VP1 gene
5’
C
D
3’
CaliciNet
• Founded by the CDC
• Network of state and local health
departments and government agencies
like the FDA
• Purpose: to create a nationwide
database consisting entirely of
Norovirus genome sequences
Purpose
• To sequence region C of the genome of
noroviruses present in human stool
samples submitted to the Texas
Department of State Health Services in
2009-2010
• Contribute to the nationwide CacliciNet
database
Methods
Study population
• 55 patients with diarrheal disease in
Texas, 2009-2010
– 2 patient populations
• 46 from 20 male-only prisons in Texas
• 9 from 5 nursing homes
– Stool specimen and submitter forms
submitted to TDSHS from each patient
Demographic data
Submitter forms with
demographic data
Entered data into
excel spreadsheet
Analyzed variables
Gender
Age
Symptoms
Location
Report results
Stool specimens
Stool specimens submitted to
TDSHS, 2009 and 2010
Screened using real-time PCR
55 Norovirus-positive samples
Sequenced region C
Sequencing protocol
Extract RNA
Perform
RT-PCR
Agarose gel
electrophoresis
Ethanol
precipitation
PCR – cycle
sequencing
Gel purification
Sequencing
Sequence
analysis
Extraction
PCR and gel electrophoresis
Sequencing
Sequence analysis
Results
DEMOGRAPHIC ANALYSES
Gender
9
40
N=49
Number of patients
Age of population by location
N=37
Number of patients
Symptoms
N=16
Location in Texas by county
2
1
2 2
2
2
2
8
1
5
2
1
2
2
4
2
1
2
2
2
LABORATORY RESULTS
Electrophoretic profile from
reverse PCR of 15 Norovirus
samples
1
2 3
4 5
6 7
8 9 10 11 12 13 14 15 16
Sequence analysis
Sequencing results
• 38/55 successfully sequenced
– 9 were unable to be reverse transcribed
• Low virus levels in stool specimen
• PCR failed
– 8 were unable to be sequenced
• Poor quality DNA template
• Too much/little DNA
• Contaminants
Genotyping results
• 38 sequences were compared to other
sequences in CaliciNet database
• 12 - 2009 samples:
– 12/12 GII.4 Minerva
• 26 - 2010 samples:
– 24/26 GII.4 New Orleans
– 2/26 GII.12 (unnamed)
Number of patients
Genotyping by patient type
N=38
Conclusions
Conclusions: Demographic analyses
• Samples not
representative of the
general population in
the State
– Two populations
• Prisons – 40 men;
average age 41
• Nursing homes – 9
women; average age
78
Noroviruses in prisons – a
closed population?
• How do outbreaks spread through the
prison system?
– Food service
– Laundry service
– Prison hospitals
• 2010 Texas prison outbreak
Conclusions: Laboratory results
• Successfully sequenced 38 Norovirus
region C genome sequences
• Add 38 sequences to the TDSHS
database
Genoclusters and subclusters
Year
Genocluster Subcluster
2009
GII.4
Minerva
2010
GII.4
GII.12
New Orleans
Unnamed
GII.4: Minerva subcluster
Emerged in 2006
– Named after
cruise ship
– Caused outbreak
– Still circulating
GII.4: New Orleans subcluster
• Emerged in late 2009
– CDC announced new
subcluster in April, 2010
– Named for New Orleans
• Outbreak associated with
eating raw oysters
• Oyster beds closed
– Widespread
• 15 states
• 50% of norovirus cases to
date in 2010
New genocluster: G II.12
(Unnamed)
• Data from ViroMed Laboratory about norovirus
genotyping results, 2009-2010
•20/50 states GII.12 +
B. Lembke and C.
Cartwright, Abstr. 26th Ann.
Clinical Virol. Symp. 2010.
CaliciNet statistics
• Submissions to
CaliciNet
– 538 outbreaks
• 54% GII.4 New
Orleans
• 13% GII.12
L. Barclan, N. Gregoricus, E. Vega, and
J. Vinje, Abstr. IV Calicivirus Int. Conf.,
abstr.I-6, 2010.
Evolving genoclusters and
subclusters in Texas, 20092010
2009
Nursing Homes
Prisons
100% Minerva
100% Minerva
2010
100% New
Orleans
92% New
Orleans
8% GII.12
Limitations
• Small sample size, not representative of
the State
• Incomplete submitter forms –
incomplete data
• Unable to successfully genotype all 55
samples – technical difficulties
Future Studies
• Sequence region D of the genome
• Sequence more samples
• Compare Texas outbreaks with other
states
Acknowledgements
• Aaron Benfield, PhD
• Members of the
Molecular Biology
Laboratory
• Leanne Field, PhD;
Dr. Diane Kneeland
and Ms. Nancy
Elder
Thank you!
Funding generously provided by an
Association of Schools of Public
Health/Association of Public Health
Laboratories “Pathways to Public Health
Careers and Internships Grant”
From
The Centers for Disease Control and
Prevention