Transcript Noroviruses

Enteric viruses
Mechanisms for
generating diversity
Jim Gray
Enteric Virus Unit
CfI, HPA
Viruses infecting the gut
Viruses associated with gastroenteritis
• rotaviruses
• caliciviruses
• noroviruses
• sapoviruses
• astroviruses
Rotaviruses
• adenoviruses 40, 41
Sapoviruses
Adenoviruses
Astroviruses
Noroviruses
Viruses infecting the gut
Viruses associated with systemic infections
• enteroviruses
• parechoviruses
enteroviruses
Viruses associated with infection in the
immunocompromised
• adenovirus types 42-48
• cytomegalovirus
• human immunodeficiency virus
Viruses infecting the gut
Presumptive enteric viruses
• Torovirus
• Coronavirus
• Parvovirus
• Picobirnavirus
• Aichi virus
Torovirus
Coronavirus
Parvovirus
Viral gastroenteritis – testing strategies
• Outbreak investigation
• Sporadic cases
• Environmental contamination
• Contamination of food
• Recombinants, reassortants and variants
• Zoonosis
Rotavirus
• Family Reoviridae
• Unenveloped, icosahedral,
triple layered capsid (75nm diameter)
• Genome: 11 segments of dsRNA
~ 18,550bp (660bp – 3300bp)
100nm
Genotype
Common
Human
Strains
G1:P[8]
G2:P[4]
G3:P[8]
G4:P[8]
G9:P[8]
Reassortant
of Human
Strains
G2:P[8]
G3:P[4]
G4:P[4]
G9:P[4]
Potential
Zoonotic
Strains
G1:P[6]
G1:P[4]
G2:P[6]
G3:P[6]
G3:P[9]
G4:P[6]
G4:P[14]
G6:P[8]
G6:P[9]
G6:P[11]
G8:P[4]
G8:P[6]
G8:P[8]
G8:P[14]
G9:P[6]
G9:P[9]
G10:P[6]
G10:P[8]
G10:P[14]
G10:P[4]
G12:P[6]
G12:P[8]
Partially
typed strains
Mixed types
TOTAL
2005/06
No
%
2006/07
No
%
2007/08
No
%
TOTAL
No
%
1939
922
51
81
90
795
91.1
43.3
2.4
3.8
4.2
37.3
3552
1686
598
125
377
766
88.1
41.8
14.8
3.1
9.4
19.0
2514
1690
175
128
190
331
92.4
62.1
6.4
4.7
7.0
12.2
8005
4298
824
334
657
1892
88.1
47.3
9.1
3.7
7.2
20.8
23
17
1
3
2
1.1
0.8
0.1
0.1
0.1
28
22
1
1
4
0.7
0.6
0.0
0.0
0.1
8
6
0
1
1
0.3
0.2
0.0
0.0
0.0
59
45
2
5
7
0.7
0.5
0.0
0.1
0.1
42
3
2
1
1
0
6
1
0
0
0
2
0
1
2
4
0
1
2
0
0
0
16
2.0
0.1
0.1
0.1
0.1
0.0
0.3
0.1
0.0
0.0
0.0
0.1
0.0
0.1
0.1
0.2
0.0
0.1
0.1
0.0
0.0
0.0
0.8
69
0
13
4
0
1
5
0
1.7
0.0
0.3
0.1
0.0
0.0
0.1
0.0
0.0
0.1
0.0
0.0
0.1
0.1
0.0
0.1
0.0
0.0
0.0
0.1
0.0
0.1
0.5
34
0
8
1
0
1
0
0
2
0
0
1
1
1
0
0
0
0
2
0
1
3
13
1.3
0.0
0.3
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.0
0.1
0.5
145
3
23
6
1
2
11
1
2
4
1
3
5
4
2
9
1
2
4
5
2
7
47
1.6
0.0
0.3
0.1
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.0
0.1
0.0
0.0
0.0
0.1
0.0
0.1
0.5
100
25
2194
4.7
1.2
150
231
4127
3.7
5.7
104
60
2762
3.8
2.2
354
316
9083
3.9
3.5
4
1
0
4
2
0
5
1
1
0
5
1
4
18
Virus diversity
Rotavirus Genotype distribution
This extrapolates to 57,265 cases out of
the 3,579,070 expected in the population
under surveillance by EuroRotaNet
Potential reservoir for reassortment
Rotavirus evolution
Three mechanisms are important for the evolution and
diversity of rotaviruses
• Genome rearrangement
• Antigenic drift
• the possible vaccine-induced emergence of antibody
escape mutants
• Antigenic shift
• the possible emergence in the general population of
reassortants between two co-circulating rotavirus
strains
• Zoonosis
• the possible emergence of animal/human
rotavirus reassortants
Rotavirus evolution
Antigenic shift
• Shuffling of gene segments through reassortment
can occur during dual infection of one cell
• If reassortment occurs at random, the 11 segments
of the 2 parental strains can reassort into 211 possible
gene combination
Rotavirus gene reassortment
G9P[6]
G1P[8]
Dual Infection of an enterocyte
by 2 parent viruses (A, B)
Progeny
viruses
G9P[6] G9P[8]
G1P[6] G1P[8]
Parental and daughter stains and multiple type
combinations of rotavirus strains 1995/96
1995/96
G1P[8]
G1P[6]
G9P[6]
G9P[8]
G1+G9/P[8]
G1+G9/P[6]
407
2
18
1
5
2
Geographical Distribution of Rotavirus G9 Strains
in the UK during Three Consecutive Seasons
1995/96
1996/97
1997/98
3 locations
6 locations
8 Locations
Identification of zoonotic infections
VP7 sequences of the Vellore strains were more closely
related to sequences of Indian Bovine strains than to
human G10 strains from the UK or Japan.
KK3 - Bovine Thailand
B75 – Bovine India
A64-Human UK
B69 – Bovine India
I321 – Human India
Mc35-Human Japan
99-D/78
00-2KD/472
00-2KD/851
MF53-Bovine India
8
99-D/93
99-D/265
99-D/214
99-D/258
99-D/264
00-2KD/399
00-2KD/495
00-2KD/665
99-D/472
00-2KD/348
99-D/228
99-D/220
98-V10-20
G10P[11] rotavirus
strains from
Vellore
• This segregation or interspecies
clustering is observed with the
genes encoding NSP4 and VP6
Animal
Lp14- Lamb China
Rotavirus Surveillance in the UK
2005-2006
No.
%
Common Human Strains
G1P[8]
211
54.0
G2P[4]
21
5.4
G3P[8]
40
10.2
G4P[8]
5
1.3
G9P[8]
91
23.3
Reassortment of Human Strains
G1P[4]
0
0.0
G2P[8]
2
0.5
G3P[4]
0
0.0
G4P[4]
1
0.3
G9P[4]
0
0.0
Potential Zoonotic Strains
G2P[9]
0
0.0
G2P[10]
0
0.0
G4P[6]
1
0.3
G8P[4]
0
0.0
G8P[8]
0
0.0
G9P[6]
0
0.0
G10[P4]
0
0.0
G10P[10]
0
0.0
G12P[4]
0
0.0
G12P[6]
0
0.0
G12P[8]
10
2.6
Partially Typed/Mixed Strains
Partially Typed
2
0.5
Mixed Types
7
1.8
Sub-Total
391
Negative
14
1.2
TOTAL
405
2006-2007
No.
%
2007-2008
No.
2008-2009
No.
498
114
44
11
71
61.9
14.2
5.5
1.4
8.8
638
65
73
51
46
68.2
7.0
7.8
5.5
4.9
395
83
122
72
49
48.1
10.1
14.9
8.8
6.0
4
8
1
1
3
0.5
1.0
0.1
0.1
0.4
0
1
0
1
0
0.0
0.1
0.0
0.1
0.0
3
7
2
3
1
0.4
0.9
0.2
0.4
0.1
1
1
0
0
2
1
1
0
0
1
8
0.1
0.1
0.0
0.0
0.2
0.1
0.1
0.0
0.0
0.1
1.0
0
0
0
0
0
0
1
1
0
2
2
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.1
0.0
0.2
0.2
0
0
0
29
0
0
0
0
8
0
2
0.0
0.0
0.0
3.5
0.0
0.0
0.0
0.0
1.0
0.0
0.2
13
22
805
28
833
1.6
2.7
18
36
935
54
989
1.9
3.9
10
35
821
13
834
1.2
4.3
3.4
5.5
1.6
Total
No.
2700
1742
283
279
139
257
38
7
18
3
6
4
77
1
1
1
29
2
1
2
1
8
3
22
%
91.5
59.0
9.6
9.5
4.7
8.7
1.3
0.2
0.6
0.1
0.2
0.1
2.6
0.0
0.0
0.0
1.0
0.1
0.0
0.1
0.0
0.3
0.1
0.7
43
100
2952
109
3061
1.5
3.4
3.6
Geographical distribution of G12 and G8 strains
G12
G8P[4]
G8P[8]
26 strains clustered within Yorkshire in 2009
G8 VP7 encoding gene: Three genetic clusters
Bovine and human strains
associated with P[6] or P[14]
Australia, Argentina, Itly, Japan, US, Japan
and UK
1980-2007
Bovine, porcine, simian and human strains
associated with P[6], P[10] or P[14]
Malawi, Kenya, Congo, Cameroon, South Africa,
Egypt, India, Thailand and Tajikistan
1990-2007
Multiple zoonotic
introductions
Human strains
Associated with P[6], P[4] or P[8]
Cameroon, Tunisia, Ivory Coast, Ethiopia, Slovenia and UK
2000-2009 with less diversity seen in the last 3 years
Reassortment leading to
adaptation in the human host
Noroviruses
• Family : Caliciviridae
• Non-enveloped small round
structured viruses (27-32 nm
diameter)
• Genome: pos sense ssRNA ~ 7.5kb
• Predominantly epidemic
• The most common cause of
outbreaks of gastroenteritis
Noroviruses
Phylogenetic grouping among noroviruses
Alphatron
Fort Lauderdale
Saint Cloud
Fayetteville
Snow Mountain
Melksham Hillingdon
Kashiwa47
Erfurt 546
290/White River
Girlington
Hawaii
Idaho Fall
VA97207
314/S19/94
Wortley/90
Amsterdam
GGIII
Jena
M7
273/Gwyned
Leeds
GGIV
Limburg
Sw43
Seacroft
Newbury
CH126
Mexico
Toronto
GGII
Bristol
Lordsdale
Blakemore
MNV-1
Virus diversity
GGV
Chiba
Koblenz
Thistle
Winchester
Malta
318/S05/95
Musgrove
DSV
Stavanger
WhiteRose
Southampton
Sindlesham
Norwalk KY89
Hesse
GGI
Mechanisms generating
diversity among noroviruses
Genetic Recombination
Requirements
• co-infection of a single cell
• relatedness of parental strains
Noroviruses
• endemic co-circulation of genotypes
• multiple infections associated with food and water borne spread
• environmental contamination and virus survival
• faecal-oral route of transmission
• limited heterotypic protection
• absence of long term immunity
ARGUS outbreak
• Thirty-seven (10%) of the ships
company presented with abrupt
onset of gastrointestinal illness
• The presentation was watery
diarrhoea, colicky abdominal pain,
and nausea with and without vomiting
RFA Argus,
Falmouth 2004
• Clinically the illness was consistent with a viral aetiology
• Outbreak was controlled by standard infection controls measures
• Routine bacteriology on board was negative for the common enteric bacteria
• Specimens were sent to CfI(Colindale) and six enteric viruses were detected in six
patients; including noroviruses, sapoviruses and rotavirus
Patient
Virus
Genotype
1
Rotavirus
Group A rotavirus
2
Nvd
3
Norovirus
4-7
Nvd
8
Strain
designation
GII-6 (Seacroft/1990/UK)
Argus-3/2003/IQ
Norovirus
GI-6
(Sindlesham/1995/UK)
Argus-4/2003/IQ
9
Sapovirus
Argus-2/2003/IQ
Argus-2/2003/IQ
10
Nvd
11
Norovirus
GI-3 (Desert
Shield/1990/SA)
Argus-1/2003/IQ
12
Nvd
13
Norovirus
GI-6
(Sindlesham/1995/UK)
Argus-5/2003/IQ
A common food source was
implicated in the outbreak and
epidemiological analysis
showed a statistically
significant association with
eating salad on a date 24-48
hours preceding the outbreak
Environmental monitoring for
noroviruses in food outlets
Environmental swabs
• Dipped in 0.1M PBS pH 7.2
Applied to sites within toilet facilities
• Toilet flush handle
• Toilet door handle
• Wash basin taps
Applied to sites within kitchens
• Fridge door handles
• Preparation sites
Norovirus detected and characterised using
• Real-time RT-PCR
• DNA sequencing
Premises
• A total of 39 premises in Hertfordshire were sampled
• Noroviruses were detected in 16/39 (41%)
• Noroviruses were detected on more than one visit in
4/16 (25%) contaminated premises
• Two norovirus genotypes were predominant, GII-3 and GII-4
• Virus genotypes and variants were those seen in the
community at the same time
Premises
Cafés
Restaurants
Take away
Coffee shop
Staff canteen
No. visited
12
17
7
2
1
No. NoV detected (%)
7 (58.3)
7 (41.2)
2 (28.6)
0 (0)
0 (0)
Contaminated sites within 16 premises
• Toilet flush
• Toilet door handle
• Wash hand basin taps
• Fridge handle
• Fridge door
• Preparation surfaces
10
7
7
6
3
5
24 sites associated with the toilets and 14 with kitchen areas
Virus detected on one site on 11 occassions and on multiple
sites on 8 occasions
Mechanisms generating
diversity among noroviruses
• Accumulation of point mutations
• emergence of variants
• antibody escape mutants
Inter- and Intra-seasonal diversity of NoV genotypes during 2003 to
2006. Early, mid and late season outbreaks characterised.
Genotype
2003/04
2004/05
2005/06
Early
Mid
Late
Early
Mid
Late
Early
Mid
Late
GI-1
0
0
0
0
0
0
0
1 (5%)
0
GI-2
1 (5%)
0
0
0
0
0
0
0
0
GI-3
1 (5%)
0
0
0
0
0
0
1 (5%)
0
GI-4
0
0
0
0
0
0
0
3 (15%) 0
GI-6
2 (10%)
0
0
0
0
0
0
0
0
GII-1
0
0
0
0
0
1 (5%)
0
1 (5%)
0
GII-2
3 (15%)
1 (5%)
0
4 (20%)
2 (10%)
0
0
1 (5%)
0
GII-3
7 (35%)
3 (15%)
2 (10%)
0
0
0
0
2 (10%) 0
GII-4
1 (5%)
14 (70%)
18 (90%) 14 (70%) 18 (90%)
18* (90%)
9 (45%)
7 (35%) 18 (90%)
GII-6
1 (5%)
0
0
2 (10%)
1 (5%)
0
2 (10%)
0
GII-7
3 (15%)
2 (10%)
0
0
0
1(5%)
6 (30%)
3 (15%) 1 (5%)
GII-8
1 (5%)
0
0
0
0
0
0
1 (5%)
0
Total
20
20
20
20
20
20
20
20
20
Total of
3 GI
4 GII
2 GII
3 GII
3 GII
2 GII
4 GII
3 GI
3 GII
genotypes
6 GII
1 (5%)
6 GII
Early = September/October, Middle = December, Late = March, GII = Genogroup II, GI = Genogroup I, * = February and March
Highlights the fitness of GII-4 to infection the human
population against a background of herd immunity
Sep-07
Jul-07
May-07
Mar-07
Jan-07
Nov-06
Sep-06
Jul-06
May-06
Mar-06
Jan-06
Nov-05
Sep-05
Jul-05
May-05
Mar-05
Jan-05
Nov-04
Sep-04
Jul-04
May-04
Mar-04
Jan-04
Nov-03
Sep-03
GII-4 variants: September 2003 to September 2007
Emergence of GII-4 variants
100%
80%
v11
v10
60%
v8
v6
40%
v5
v4
v3
20%
v2
0%
pre-2002
A
2002 epidemic
2006 epidemic
E Surface
Molecular
A
J
A
Homology modelling of
amino acid changes at sites
A and B on VA387 model
structure
A
B
B
B
A
E
A
B
J
A
B
Electrostatic Surface
A
B
Monoclonal antibodies against
sites A and B of the 2002
epidemic strain did not bind to
sites A and B of the pre-2002
strains suggesting that the 2002
epidemic strain was an
antibody-escape mutant
Neutral Networks: A model for NoV evolution
Method of representing random neutral drift between related proteins
Genotype populations that are linked by point mutation but are selectively neutral
Groups are defined by epitope structure, not sequence diversity
Motif
Site A Site B
SHD
N-N
SHD
S-N
THD
NSN
THN
NGT
TRT
STA
TRT
SST
SRN
STT
TQN
STT
TQN
NTT
TQE
STT
TQE
NTT
TQE
SAT
TQH
STT
Number of
Strains
14
pre-2002
3
2
6
2002
1
epidemic
2
through to
1
2006
16
9
9
2006
1
epidemic
1
1
n=
66
* as in Gallimore et al (2007)
Year
S Domain P2 Domain
1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 Variant*
Cluster
1
10
3
v2
A
1
2
v2
B
1
1
v1
C
2
4
v2
D
1
v1
E
1
1
v2
F
1
v6
G
10
4
2
v3
H
9
v3
I
9
v2, v4
J
1
v8
K
1
v4
L
1
v3
M
Epidemiological
Variant
3 neutral networks
blue
pre-2002 epidemic
2 clusters
orange
2002 epidemic – 2006
7 clusters
yellow
2006 epidemic
4 clusters
Allen DJ et al. PLoS One, 2008
2002/03 epidemic
Strain diversity
Autum
Winter
Spring
Normal winter season
Summer
Unusual
summer
activity
Autum
Winter
Spring
Summer
Autum
Normal
summer
activity
Epidemic winter season
Winter
Spring
Summer
Normal winter season
Narrowing diversity:
GII4 predominates
GII4 variants emerge
GII4 variant is selected, out of season outbreaks occur, becomes
epidemic
Return to normal season, wide diversity at the
beginning, narrowing as season progresses.
Lack of short-term herd immunity to a new variant
•Population protected in the short term
against variant GII4
•Population susceptible to other
genotypes due to short-term immune
protection.
GII4 dominate and have an advantage over other co-circulating genotypes.
• replicative advantage
• greater transmissibility associated with a lower infectious dose
• larger proportion of the population susceptible through inherited genetic factors,
• better survival of the virus in the environment,
• a mechanism that allows the virus to evade immune surveillance to some degree.
Factors associated with enteric virus
diversity and disease transmission
• Associated with endemic and epidemic
patterns of disease
• The ability of the viruses to survive in the
environment
• Multiple transmission routes – food, water,
person-to-person, environmental – infection with multiple
strains
• Low infectious dose (Norovirus: 10 virus particles)
• Short term immunity may allow infection by multiple strains
• Diversity through genetic recombination or reassortment
• Lack of proof reading during RNA replication – leads to
genetic variants
• Re-infections and asymptomatic infections are common –
large reservoir of infection
• Enteric virus infections are common in children and their carers