Jim Gray Professor of Clinical Virology, UEA & Consultant Virologist, NNUH

Gastroenteric viruses

Virus evolution and diversity: Keys to their success.

Viruses infecting the gut Viruses associated with gastroenteritis

• rotaviruses • caliciviruses

• noroviruses • sapoviruses

• astroviruses • adenoviruses 40, 41

Noroviruses

Sapoviruses

Rotaviruses

Astroviruses

Adenoviruses

• Family Reoviridae

• Unenveloped, icosahedral,

triple layered capsid (75nm diameter)

• Genome: 11 segments of dsRNA ~ 18,550bp (660bp – 3300bp) • Responsible for more than 500,000 deaths/ year in young children (1 in 285 children)

100nm

Rotavirus

Rotavirus structure

VP4

VP2

VP6

VP7

Aqueous channel

dsRNA

Rotavirus evolution

Three mechanisms are important for the evolution and diversity of rotaviruses • Genome rearrangement

• Antigenic drift

• Antigenic shift

Rotavirus evolution

Antigenic drift: • Rate of mutation within rotavirus genes is relatively high because RNA replication is error-prone. • A rotavirus genome differs from its parental genome by at least one mutation. • Point mutations can accumulate to give rise to intratypic variation.

DC14 DC2 DC4 S2 DC17 DC15 DC12 DC24 DC3 399/99 625/96 544/96 543/96 617/96 563/96 538/96 621/96 583/96 351/96 366/96 721/99 KU

G2 G2 G2 G2 G2 G2 G2 G2 G2 G2 G2 G2 G2 G2 G2 G2 G2 G2 G2 G2 G2

G1

P[4] P[4] P[4] P[4] P[4] P[4] P[4] P[4] P[4] P[4] P[4] P[4]

P[8]

London London London

London London London London London

Cambridge Cambridge Cambridge Cambridge Cambridge Cambridge Cambridge Cambridge Newcastle Newcastle

Aberdeen

Aberdeen

M11164 Cluster A (1991)

Cluster B (1996-99)

Phylogenetic tree constructed with cDNA sequences of 882bp fragments of the VP7 genes of G2 rotavirus strains.

G2 rotavirus strains – VP7

Antigenic Site A

87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 DSI A E A K N E I S D D E W N T ES2 . . . . . . . . . . . . . . . HN126 T . . . . . . . . . . . . . . DC2/91 . . . . . . . . . . . . . . . DC3/91 . . . . . . . . . . . . . . . DC4/91 . . . . . . . . . . . . . . . DC12/91 . . . . . . . . . . . . . . . DC14/91 . . . . . . . . . . . . . . . DC15/91 . . . . . . . . . . . . . . . DC17/91 . . . . . . . . . . . . . . . DC24/91 . . . . . . . . . . . . . . . DC24/91 . . . . . . . . . . . . . . . DC22/91 . . . . . . . . . . . . . . . 543/96 T . . . . . . . . N . . . . . 544/96 T . . . . . . . . N . . . . . 536/96 T . . . . . . . . N . . . . . 583/96 T . . . . . . . . N . . . . . 538/96 T . . . . . . . . N . . . . . 621/96 T . . . . . . . . N . . . . . 625/96 T . . . . . . . . N . . . . . 366/91 T . . . . . . . . N . . . . . 617/96 T . . . . . . . . N . . . . . 351/96 T . . . . . . . . N . . . . . 399/99 T . . . . . . . . N . . . . . 721/99 T . . . . . . . . N . . . . .

E

Serotyped

Did not serotype

aa position

Alanine: threonine Aspartic acid: asparagine

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 combinations • However, reassortment is not a random phenomenon and is influenced by the selection of progeny with viable gene combinations, the host cell and the immune status of the host.

Dual Infection of an enterocyte by 2 parent viruses (A, B)

Progeny viruses

Rotavirus gene reassortment

G9P[6]

G1P[8]

G9P[6]

G1P[8]

G9P[8]

G1P[6]

1995/96 G1P[8] 407 G1P[6] 2 G9P[6] 18 G9P[8] 1 G1+G9/P[8] 5 G1+G9/P[6] 2

Parental and daughter stains and multiple type combinations of rotavirus strains 1995/96

1995/96 3 locations

1996/97 6 locations

1997/98 8 Locations

Geographical Distribution of Rotavirus G9 Strains in the UK during Three Consecutive Seasons

Several findings suggest that rotavirus G9P[6] strains were introduced into the human population recently

• Relative lack of diversity between the VP7 genes and the

chronological clustering of all G9 strains.

• The associations between infection with G9P[8] strains and; Symptomatic infections in older individuals (older children

and adults) More severe disease requiring hospitalisation and iv-

rehydration Infection in a neonatal unit (lack of maternal protection) Infections predominantly in the urban population

• G12 found in association with many P-types • G12 found throughout the world • Does this represent multiple introductions? • Can we identify an animal reservoir?

G12 rotavirus strains

100

9896949290

466-96/97892-96/97632-96/97873-96/97884-96/97480-96/97221-96/97

431-96/97414-97/98436-97/98107481-97/98114-97/98

GOS51117-95/96GOS51426-95/96GOS51996-95/96GOS52006-95/96GOS49314-95/96US1205 95/96651-95/96107071-97/98104907-97/98132-97/98422-97/98435-97/98426-97/98MY9919878-97/98MY919877-97/980.10-97/980.7-97/980.9-97/98103053-97/98101647-98/990.8-97/98105041-97/98

116E-L14072

G9G9G9G9G9G9G9G9G9G9G9G9

G9G9G9G9G9G9G9

G9G9G9G9G9G9G9G9G9G9G9G9G9G9G9

G9

P[8]P[8]P[8]P[8]P[8]P[8]P[8]P[8]P[8]P[8]P[8]P[8]

P[6]P[6]P[6]P[6]P[6]P[6]P[6]

P[8]P[6]P[8]P[8]P[8]P[8]

P[6]P[6]P[6]P[8]P[8]P[6]P[6]

P[11]

BirminghamPlymouthReadingBirminghamRhylBirminghamNewcastle

GOS51203-95/96 G9 P[6] London

LeedsLeedsLeedsBirminghamPeterborough

LondonLondonLondonLondonLondonUSALeedsBirminghamBirminghamPeterboroughLeedsLeedsLeedsMysore IndiaMysore IndiaLondon (OB)London (OB)London (OB)BirminghamBirminghamLondon (OB)Birmingham

India

II

III

IA

IB

100

9896949290

466-96/97892-96/97632-96/97873-96/97884-96/97480-96/97221-96/97

431-96/97414-97/98436-97/98107481-97/98114-97/98

GOS51117-95/96GOS51426-95/96GOS51996-95/96GOS52006-95/96GOS49314-95/96US1205 95/96651-95/96107071-97/98104907-97/98132-97/98422-97/98435-97/98426-97/98MY9919878-97/98MY919877-97/980.10-97/980.7-97/980.9-97/98103053-97/98101647-98/990.8-97/98105041-97/98

116E-L14072

G9G9G9G9G9G9G9G9G9G9G9G9

G9G9G9G9G9G9G9

G9G9G9G9G9G9G9G9G9G9G9G9G9G9G9

G9

P[8]P[8]P[8]P[8]P[8]P[8]P[8]P[8]P[8]P[8]P[8]P[8]

P[6]P[6]P[6]P[6]P[6]P[6]P[6]

P[8]P[6]P[8]P[8]P[8]P[8]

P[6]P[6]P[6]P[8]P[8]P[6]P[6]

P[11]

BirminghamPlymouthReadingBirminghamRhylBirminghamNewcastle

GOS51203-95/96 G9 P[6] London

LeedsLeedsLeedsBirminghamPeterborough

LondonLondonLondonLondonLondonUSALeedsBirminghamBirminghamPeterboroughLeedsLeedsLeedsMysore IndiaMysore IndiaLondon (OB)London (OB)London (OB)BirminghamBirminghamLondon (OB)Birmingham

India

II

III

IA

IB

G9 rotavirus strains

• Relative lack of diversity between the VP7 genes and the chronological clustering of all G9 strains. • May suggest single introduction

G and P Genotype Combinations (%) 1995/96 1996/97 1997/98 1998/99 2005/06 2006/07

G1P[8] 56.9 86.5 73.1 92.1 51.8 59.7

G2P[4] 17.9 1.9 15.9 3.9 5.1 13.0

G3P[8] 6.3 4.2 0.4 0.3 9.8 5.2

G4P[8] 11.6 4.2 4.7 2.0 1.5 1.4

G1P[4] 1.5 1.2 0.8 0.1 0.0 0.3

G2P[8] 0.8 0.0 0.3 0.0 0.5 1.0

G4P[4] 0.1 0.2 0.2 0.0 0.2 0.0

G1P[6] 0.3 0.0 0.0 0.0 0.0 0.0

G1P[9] 0.0 0.1 0.2 0.3 0.0 0.0

G3P[6] 1.5 0.0 0.0 0.0 0.0 0.0

G3P[9] 0.1 0.0 0.0 0.0 0.0 0.0

G4P[6] 0.1 0.0 0.0 0.0 0.2 0.0

G8P[8] 0.0 0.0 0.2 0.0 0.0 0.3

G9P[8] 0.1 1.7 3.7 1.2 22.7 8.8

G9P[4] 0.0 0.0 0.0 0.0 0.0 0.4

G10P[4] 0.0 0.0 0.0 0.0 0.0 0.1

G12P[8] 0.0 0.0 0.0 0.0 2.4 1.0

G9P[6] 2.5 0.0 0.7 0.0 0.0 0.1

G12P[6] 0.0 0.0 0.0 0.0 0.0 0.1

Common genotypes

Reassortment among common genotypes

Zoonotic introduction

Reassortment between animal and human genotypes

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

P domain

S domain

RNA genome

Inner shell

Calicivirus structure

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 • faecal-oral route of transmission • limited heterotypic protection • absence of long term immunity

Mechanisms generating diversity among noroviruses

NTPase Vpg Pro RdRp Capsid AAA….A

5 5374 6950 7585

6950 5385

NTPase Vpg Pro RdRp Capsid AAA….A

5 5374 6950 7585

6950 5385

NTPase Vpg Pro RdRp Capsid AAA….A

5 5374 6950 7585

6950 5385

+

Hawaii virus

Mexico virus

Hawaii/Mexico recombinant virus

Norovirus recombination

GII-1 and GII-3 Recombinant norovirus

Hawaii GII-1

Mexico GII-3

Mechanisms generating diversity among noroviruses

• Accumulation of point mutations • emergence of variants • antibody escape mutants

Fort Lauderdale Saint Cloud

Alphatron

Jena

Newbury CH126

Blakemore

Winchester

DSV

Stavanger

KY89 Norwalk Hesse

Sindlesham

Southampton WhiteRose

Musgrove 318/S05/95

Thistle

Chiba

Malta

Koblenz

Bristol Lordsdale

Toronto Mexico

Seacroft

Sw43 Limburg

Leeds 273/Gwyned

M7

Amsterdam

VA97207 Idaho Fall

Fayetteville Kashiwa47

Erfurt 546

Snow Mountain Melksham Hillingdon

290/White River Girlington

Hawaii

Wortley/90 314/S19/94

GGII

GGIII

GGI

GGIV

MNV-1

GGV

Phylogenetic grouping among noroviruses

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 1 (5%)

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

genotypes

3 GI

6 GII

4 GII 2 GII 3 GII 3 GII 2 GII 4 GII 3 GI

6 GII

3 GII

Early = September/October, Middle = December, Late = March, GII = Genogroup II, GI = Genogroup I, * = February and March

Emergence of GII-4 variants

0%

20%

40%

60%

80%

100%

Sep

-03

Nov

-03

Jan-

04

Mar

-04

May

-04

Jul-0

4

Sep

-04

Nov

-04

Jan-

05

Mar

-05

May

-05

Jul-0

5

Sep

-05

Nov

-05

Jan-

06

Mar

-06

May

-06

Jul-0

6

Sep

-06

Nov

-06

Jan-

07

Mar

-07

May

-07

Jul-0

7

Sep

-07

v11v10v8v6v5v4v3v2

GII-4 variants: September 2003 to September 2007

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

A

B

A

B

A

B

A

B

A

B

Pre-2002 2002 epidemic

Structural changes on the P2 domain between GII-4 variants

Molecular surface

Electrostatic surface

Monoclonal antibodies raised against the pre-2002 GII-4 strains do not react with the 2002 GII-4 strain and vice versa.

Pre-2002 2002

Autum Winter Spring Summer Autum Winter Spring Summer

GII4 variant is selected, out of season outbreaks occur, becomes epidemic

Normal winter season

Narrowing diversity: GII4 predominates

GII4 variants emerge

Return to normal season, wide diversity at the beginning, narrowing as season progresses.

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.

Autum Winter Spring Summer

Normal winter season Normal summer activity

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.

Epidemic winter season Unusual summer activity

2002/03 epidemic

• Low infectious dose ~ 10-100 virus particles • Noroviruses 107 particles per gram/ml • Rotaviruses 1011 particles per gram/ml

• Stability in the environment • Norovirus survives up to 80oC • Rotavirus survives in the environment for months

• Protected by the matrix – faeces and vomit, which also inactivate chlorine-based disinfectants • Non-enveloped viruses – resistant to many disinfectants and alcohol • Short term immunity ~ 6 months • 16% of the population excrete NoV in the absence of symptoms • 14% of the population excrete rotavirus in the absence of symptoms

Keys to the success of gastroenteric viruses

• Multiple routes of transmission - person to person contact, through ingestion of contaminated water or food or by contact with contaminated environmental surfaces • RNA genome replication results in the accumulation of point mutations • Segmented rotavirus genome replication results in reassortment • Dual infections can result in recombination or reassortment • Rotaviruses are associated with zoonotic infection • Hospital-acquired infections are predominantly associated with GII-4 • There are multiple introductions into hospitals, of variants of GII-4, throughout the NoV season and many rotavirus strains co-circulate in the human population • Antibody-escape mutants and rotavirus reassortants are selected and driven by herd immunity resulting in epidemics in an immunologically naive population

Acknowledgements

Centre for Infections, HPA David Brown Miren Iturriza-Gomara Chris Gallimore David Allen Jacqueline Xerry Juliet Adigwe Farah Aladin Sameena Nawaz

Cambridge University Ulrich Desselberger

Norfolk and Norwich University Hospital Sarah Morter Stephen Brolley Emma Meader