Linköping Medical Dissertations No. 1111

Norovirus Epidemiology

Prevalence, transmission,

and determinants of disease susceptibility

Johan Nordgren

Division of Medical Microbiology Department of Clinical and Experimental Medicine

Faculty of Health Sciences Linköping University

SE-581 85 Linköping, Sweden

Linköping 2009

II

The works in this thesis were supported by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (grant 245-2004-1821), and the Swedish Research Council (grant 10392). Cover: Norovirus particles as viewed in an electron microscope, with part of the cDNA code for norovirus in the background. © Johan Nordgren, 2009 ISBN: 978-91-7393-670-5 ISSN: 0345-0082 Printed in Sweden by LiU-Tryck, Linköping 2009

III

To Sarah

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Abstract

Norovirus (NoV) is today recognized as the most important agent of acute human

gastroenteritis, causing a high number of diarrheal episodes in both adults and children.

Outbreaks in hospitals, nursing homes, day-care centers, and from consumption of

contaminated food and drinking water are common. Wastewater can be a source of NoV

dissemination, e.g. when used for irrigation of crops, or due to shellfish cultivation near the

outlet of wastewater treatment plants. Today, at least 25 different genotypes of NoV

belonging to two major genogroups (GG) have been observed in humans. These genotypes

are associated with different transmission patterns and disease severity in humans. Also host

genetic factors, such as presence of ABO antigens and mutations in the FUT2 gene affect

susceptibility, and can even render complete resistance to symptomatic infections, but only

the most common NoV genotypes have been studied regarding this. In this thesis, we wanted

to find prevention strategies for NoV disease through four studies of NoV epidemiology:

Development of a sensitive real-time PCR assay for detection and quantification of human

NoVs, characterization of NoV in children with diarrhea in Nicaragua, investigation of the

prevalence and parameters influencing NoV concentration in a wastewater treatment plant in

Gothenburg, Sweden, and studying host susceptibility factors in a foodborne NoV outbreak in

Jönköping, Sweden.

First we developed a real-time PCR assay which can detect and quantify NoV in various

settings, both in stool samples of patients, and in wastewater samples from which virus was

first concentrated using ultracentrifugation. This assay was found to be more sensitive than

commercial immunological assays and conventional PCR methods. The assay is furthermore

able to differentiate between the two major human genogroups of NoV using melting curve

analysis, which provides valuable information about the circulating NoV strains.

The survey of NoV in pediatric diarrhea in Nicaragua revealed a large impact of NoV, both in

community and hospital based settings, with 15% of the severe diarrhea cases attributed to

NoV. Peaks of clinically diagnosed NoV gastroenteritis were associated with emerging

variants of genotype GGII.4, largely replacing the many different NoV genotypes circulating

before the peak of diarrheal cases. Children infected with the GGII.4 genotype were found to

shed more virus compared to children infected with other genotypes, which could partly

explain the high transmission of GGII.4.

VI

At the wastewater treatment plant in Gothenburg, both NoV GGI and GGII were detected

during a whole year, not only during the winter season when clinical cases are common. This

indicates that NoV infections are frequently occurring at clinical and/or sub-clinical levels in

the community. During winter, GGII was present in high concentrations, whereas GGI

concentration increased to higher levels than GGII in summer, possibly due to the emergence

of new genotypes following the winter outbreaks. The levels of NoV GGI were stable during

the year, and hence incoming concentrations were affected by dilution factors such as rain.

Primary treatment and treatment in a conventional, non-nitrifying activated sludge system

reduced the NoV concentration by a factor of about 30. The detection of NoV in outgoing

water, together with the low reduction and lack of correlation to indicator bacteria, suggest

that better monitoring tools for virus in wastewater are warranted to reduce environmental

contamination.

A foodborne NoV outbreak in Jönköping in October 2007, by a NoV GGI.3 strain, revealed a

surprising pattern of host susceptibility. In contrast to previous findings, this strain infected

individuals irrespective of secretor status and Lewis (Le) phenotype, with non-secretors and

Lea+b- individuals having a higher risk of disease. Individuals with blood group B had a partial

protection to symptomatic infection, but none of the host factors investigated mediated

complete resistance. Furthermore, we observed differences in susceptibility regarding

homozygosity and heterozygosity in the FUT2 gene, with heterozygous secretor-positive

individuals being more susceptible to symptomatic NoV infection than homozygous secretors.

In summary, the developed LUX real-time PCR assay was successfully used in all studies in

this thesis, which yielded important information about the prevalence and transmission of

NoV. We observed the emergence of GGII.4 variants, causing the majority of diarrheal cases

in children, largely replacing the other circulating genotypes, possibly due to better replication

leading to a higher viral shedding. After the peak of NoV-induced diarrheal episodes, the

incidence of GGII.4 decrease and other strains emerge which can infect people not previously

exposed. This was observed in the foodborne outbreak in Jönköping, where individuals

expected to be resistant to NoV were infected, and indeed had a higher risk of developing

disease. A similar seasonal pattern was also indirectly observed in wastewater, with high

levels of GGII in winter, which subsequently declined, followed by an increase of GGI in

summer. Taken together, these results provide a better insight into the epidemiology of the

virus, which hopefully can lead to better preventive measures for NoV gastroenteritis.

VII

Populärvetenskaplig sammanfattning

Norovirus (NoV) orsakar den s.k. vinterkräksjukan vilken är den vanligaste formen av akut

mag-tarm inflammation i världen. Symptomen kan variera men består oftast av våldsamma

kräkningar och diarré. Årligen infekteras över 250 miljoner människor med NoV vilket leder

till att ungefär 200 000 barn dör, de flesta i utvecklingsländer. NoV orsakar ofta stora

sjukdomsutbrott i slutna miljöer som sjukhus, vårdhem, dagis, kryssningsfartyg o.s.v. där

viruset lätt kan spridas från person till person. Viruset är mycket motståndskraftigt mot

desinfektionsmedel och temperaturskillnader och därför kan man lätt få vinterkräksjukan

genom kontaminerad mat och dryck. Avloppsvatten kan vara en källa till infektion,

exempelvis då skaldjursodlingar eller badvatten befinner sig i närheten av

avloppsvattenutsläpp och därigenom kontamineras av viruset. NoV är ett mycket genetiskt

instabilt virus med ett flertal underarter som har visat sig orsaka olika svåra symptom.

Ungefär en femtedel av Europas befolkning är vanligen resistent mot vinterkräksjukan men

denna resistens är bara kartlagd för några få av alla underarter av NoV. Den höga

smittsamheten, stabiliteten och det stora antalet underarter försvårar möjligheterna att

begränsa eller hindra NoV infektioner. I min avhandling har jag därför studerat NoV utifrån

olika perspektiv för att få klarhet i virusets förekomst och spridning i samhället:

• Utveckla en metod för att kunna påvisa och mäta mängden NoV i patientprover och i

avloppsvatten

• Bestämma förekomst av NoV hos barn med diarré i Nicaragua samt hur olika faktorer

påverkar svårighetsgrad av symptom

• Undersöka årlig förekomst och faktorer som påverkar mängden NoV i avloppsvatten

vid reningsverket Ryaverket i Göteborg

• Kartlägga genetiska faktorer hos människan som påverkar mottaglighet för NoV

infektion vid ett utbrott orsakat av kontaminerad mat i Jönköping

Vi utvecklade först en känslig molekylärbiologisk metod (realtids-PCR) som kan påvisa och

mäta NoV koncentration i patientprover samt i avloppsvatten. Utöver detta kan metoden

särskilja de två stora huvudgrupperna av NoV, vilket ger värdefull information om vilka

underarter av NoV som cirkulerar och orsakar sjukdom. Denna metod användes sedan i de tre

följande projekten.

VIII

I Nicaragua observerade vi att NoV orsakade en stor del av diarréfallen hos barn. Hela 15%

av de allvarliga diarréer som resulterade i inläggning på sjukhus var orsakad av NoV. De stora

ökningarna av diarré under året berodde på en utveckling av nya varianter av en specifik

underart av NoV (GGII.4) som verkar mer smittsam samt ger svårare symptom. Barn som var

infekterade med denna underart utsöndrade även mest virus, vilket delvis kan förklara dess

höga smittsamhet.

I avloppsvattnet från Ryaverket i Göteborg påvisades NoV under ett helt år, inte bara under

vinterhalvåret då kliniska fall är vanliga. Under sommarmånaderna dominerade mindre

vanliga underarter i avloppsvattnet, medan de vanligare underarterna främst observerades

under vintern. Mängden virus reducerades i genomsnitt 30 gånger under reningsprocessen i

Ryaverket, vilket var likvärdigt med den genomsnittliga reduktionen av bakterier. Inget klart

samband fanns dock om man jämförde bakterie och virus reduktion mellan enskilda månader.

Vid en konferens i Jönköping i oktober 2007 insjuknade många av deltagarna i kräkningar

och diarré. Vi upptäckte att det berodde på att maten var kontaminerad med NoV av en

ovanlig underart som sällan observeras. Överraskande nog fann vi att de personer som i regel

är resistenta mot vinterkräksjukan insjuknade i stor utsträckning. Ingen av de undersökta

genetiska faktorerna gav ett totalt skydd mot infektion, men personer med blodgrupp B

insjuknade i mindre utsträckning jämfört med personer som hade blodgrupp A eller 0.

Sammanfattningsvis så utvecklade vi en metod för att mäta koncentration av NoV som sedan

framgångsrikt användes i alla våra studier. Resultaten tyder på att NoV orsakar många

allvarliga diarréer hos barn och att det mestadels är varianter av en specifik underart, GGII.4,

som orsakar sjukdom under vinterkräksjukeperioden, dvs. vinterhalvåret i Europa och

regnsäsongen i tropiska länder. Under andra halvan av året minskar förekomsten av GGII4

och andra underarter av NoV börjar cirkulera. Ett liknande mönster såg vi i avloppsvattnet där

de mindre vanliga underarterna fanns i höga halter under sommaren. Dessutom fann vi att

vissa underarter av NoV kan infektera personer som ofta är resistenta mot vinterkräksjukan.

Våra resultat ger en större förståelse av virusets förekomst och spridningsvägar och kan

förhoppningsvis leda till bättre förebyggande åtgärder mot vinterkräksjukan i framtiden.

IX

List of papers The papers included in this thesis are listed below. They will be referred to in the text by their

roman numerals.

I Nordgren, J., Bucardo, F., Dienus, O., Svensson, L., and Lindgren, P-E. Novel

light-upon-extension real-time PCR assays for detection and quantification of

genogroup I and II noroviruses in clinical specimens

Journal of Clinical Microbiology 2008 Jan, 46(1): 164-170. Author´s correction

in: 2009 Apr, 47(4).

II Bucardo, F., Nordgren, J., Carlsson, B., Paniagua, M., Lindgren, P.E., Espinoza,

F., and Svensson, L. Pediatric norovirus diarrhea in Nicaragua

Journal of Clinical Microbiology 2008 Aug, 46(8): 2573-2580.

III Nordgren, J., Matussek, A., Mattsson, A., Svensson, L., and Lindgren, P-E.

Prevalence of norovirus and factors influencing virus concentrations in a full

scale wastewater treatment plant

Water Research 2009 43: 1117-1125.

IV Nordgren, J., Kindberg, E., Lindgren, P-E., Matussek, A., and Svensson L.

A FUT2 nonsense mutation (G428A) and Lewis-independent norovirus GI.3

outbreak

Submitted manuscript.

Papers I, II and III are reprinted with kind permission from the respective publisher.

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XI

Table of contents

ABSTRACT ........................................................................................................................................................V

POPULÄRVETENSKAPLIG SAMMANFATTNING................................................................................................VII

LIST OF PAPERS ............................................................................................................................................... IX

ABBREVIATIONS............................................................................................................................................. XII

INTRODUCTION................................................................................................................................................ 1

1. VIRAL GASTROENTERITIS.............................................................................................................................. 3

1.1 GENERAL INTRODUCTION TO DIARRHEAL DISEASE ..................................................................................................... 3 1.2 THE VIRAL PATHOGENS ....................................................................................................................................... 4 1.3 THE EMERGING IMPORTANCE OF NOROVIRUS .......................................................................................................... 5

2. DIVERSITY AND CLASSIFICATION OF NOROVIRUS......................................................................................... 7

2.1 A FAMILY WITH MANY MEMBERS .......................................................................................................................... 7 2.2 STRUCTURE ...................................................................................................................................................... 8 2.3 DETECTION AND MOLECULAR CHARACTERIZATION .................................................................................................... 9

3. TRANSMISSION OF NOROVIRUS IN THE HUMAN ENVIRONMENT............................................................... 11

3.1 DIFFERENT PROPERTIES OF NOROVIRUS STRAINS .................................................................................................... 11 3.2 CLINICAL SYMPTOMS AND FACTORS FACILITATING THE SPREAD OF DISEASE .................................................................. 13 3.3 NOROVIRUS IN WASTEWATER............................................................................................................................. 14

4. WHY ARE SOME PEOPLE RESISTANT TO WINTER VOMITING DISEASE?....................................................... 16

4.1 A BRIEF HISTORY OF THE GENETIC FACTORS DETERMINING SUSCEPTIBILITY ................................................................... 16 4.2 HISTO-BLOOD GROUP ANTIGENS AS THE PROBABLE RECEPTORS FOR NOROVIRUS .......................................................... 16

5. AIMS .......................................................................................................................................................... 19

6. DEVELOPMENT OF A NEW METHOD FOR DETECTING HUMAN NOROVIRUSES (PAPER I)............................ 21

7. CHARACTERIZATION OF NOROVIRUS IN CHILDREN´S DIARRHEA (PAPER II)................................................ 24

8. HIGH PREVALENCE OF NOROVIRUS IN A WASTEWATER TREATMENT PLANT (PAPER III) ............................ 27

9. NEW SUSCEPTIBILITY PATTERNS REVEALED IN A FOODBORNE NOROVIRUS OUTBREAK (PAPER IV)........... 30

10. CONCLUDING REMARKS ........................................................................................................................... 33

ACKNOWLEDGEMENTS .................................................................................................................................. 35

REFERENCES ................................................................................................................................................... 37

APPENDIX A: PCR PRIMERS ............................................................................................................................ 45

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Abbreviations

AGE Acute gastroenteritis

bp Base pair

cDNA Complementary DNA

ELISA Enzyme-linked immunosorbent assay

EM Electron microscopy

FUT2

GGI, GGII

Fucosyltransferase 2

Genogroup I and II respectively

HBGA Histo-blood group antigen

kDa kilo Dalton

Le a, b

NoV

Lewis antigen a, b

Norovirus

NSP Non-structural protein

OR

PBS

Odds ratio

Phosphate buffered saline

PCR Polymerase chain reaction

RdRp RNA-dependent RNA polymerase

RT Reverse-transcription

VLP Virus-like particle

VP Viral protein

WWTP Wastewater treatment plant

1

Introduction

Norovirus (NoV), causing the winter vomiting disease, is today recognized as the most

important agent of acute human gastroenteritis, causing ~270 million infections and an

estimate of 200,000 deaths in children annually. Second only to rotavirus in endemic pediatric

diarrhea, NoV is by far the most frequent pathogen in epidemic diarrhea in both developing

and industrialized countries, with outbreaks frequently occurring in closed settings such as

hospitals and day-care centers. The high stability and infectivity of the virus moreover make it

a common cause of food and waterborne gastroenteritis. Despite these facts, there are no

vaccines or therapeutic treatments available for this disease. Moreover, little is known about

the transmission pathways of the virus, and which measures that could be taken in order to

prevent contamination of the environment. This is partly due to the complex NoV

epidemiology, with several strains circulating, each having their own intrinsic properties

which are related to differences in severity, transmission pathways, and receptor specificities

in the human host.

Thus, before being able to find treatments or prevention strategies for this disease, many

questions about the epidemiology need to be answered. Are there certain strains of NoV that

are more clinically important, i.e. causing the majority of the severe gastroenteritis? What are

the mechanisms behind the seasonality, with most clinical cases appearing in the winter

months? How does the virus maintain its circulation in humans after the winter season? How

can we reduce the environmental contamination, e.g in water treatment plants to prevent

outbreaks? Which are the human receptors, and how do they differ between the various NoV

strains? This thesis aims at giving answers to some of these questions.

2

3

1. Viral gastroenteritis

1.1 General introduction to diarrheal disease

Despite improved water and food handling (1), diarrhea is still a common illness worldwide,

accounting for ~2 million deaths in children <5 years of age (2), mainly in developing

countries. Diarrhea is thus the largest killer after pneumonia and neonatal deaths (2), causing

approximately 17% of the fatalities (Figure 1).

Figure 1. Estimated causes of death in children <5 years of age. Modified from (2).

Diarrheal diseases also cause significant morbidity and economic loss (3); in children <5

years, estimates´ indicate that more than 700 million cases of acute diarrhea occur each year

(4). There are many bacterial and parasital pathogens causing diarrhea, some of the most

common include enterotoxigenic and enteropathogenic E.coli, Shigella spp, Campylobacter

jejuni, Salmonella spp, Entamoeba histolytica and Giardia (5-7). Historically, the bacterial

pathogens have received most attention, both due to lack of knowledge of, and sensitive

detection methods for, viral pathogens (8-10). Indeed, it was not until 1972 that the first virus

causing gastroenteritis was isolated (see section 1.2). However, as better tools developed for

the detection of viruses (8, 11-13), this view has changed dramatically. It is now widely

accepted that viruses are the cause of the majority of both sporadic and epidemic

gastroenteritis episodes world-wide (14-17). Also, many cases of gastroenteritis remain of

unknown etiology (9, 17), and it is likely that the majority of them are caused by viruses. The

rates of bacterial food borne illnesses in the industrialized world have declined, partly as a

result of improved refrigeration (1), a measure ineffective against many viruses (18), which

4

will likely increase the percentage of virus-induced gastroenteritis in the future. Bacteria are

estimated responsible for only <5% of the diarrheal cases in industrialized countries, whereas

in developing countries, bacteria are responsible for approximately 25% of the cases

occurring in children <2 years of age requiring hospitalization (19).

1.2 The viral pathogens

There are four major viral groups causing diarrhea: rotavirus, calicivirus (NoV and

sapovirus), astrovirus and adenovirus (serotypes 40, 41) (Table 1). Other, less common

viruses include toroviruses, picobirnaviruses, picornavirus (the Aichi virus), coronavirus,

pestivirus and enterovirus 22 (10, 20, 21). These four major families all cause endemic

childhood disease; this illness affects all children worldwide within the first few years of life

regardless of their level of hygiene, quality of water, food or sanitation (20). Rotavirus is the

most common endemic childhood pathogen, resulting in 2 million hospitalizations each year

(15), ~39% of all childhood diarrhea hospitalizations (22), and approximately 611,000 deaths

(22). Thus, rotavirus has received enormous attention during the last decades, which has lead

to the development and licensing of two new rotavirus vaccines, RotaTeq from Merck, and

Rotarix from GlaxoSmithKline (23). NoV is the second most important pathogen in endemic

childhood infections, causing approximately 12% of severe gastroenteritis in children <5

years of age, leading to an estimated mortality number of 200,000 deaths annually (24).

Sapovirus, related to NoV, has a lower incidence, and generally causes milder infections (25,

26), although the recent development of better detection tools (27, 28) is likely to increase the

incidence rate. Enteric adenoviruses have a reported incidence of 1-8% in industrialized

countries, whereas 2-31% has been reported in developing countries (10). Symptomatic

infection of astrovirus occurs mainly in young children and the elderly, with incidences

between 2.5 and 10% in children with sporadic diarrhea (29).

In contrast, looking at epidemic diarrheal disease affecting all ages, we observe a different

pattern. The absolute majority of all outbreaks of gastroenteritis are caused by NoV (14),

accounting for >90% of all outbreaks of viral origin, and ~50% of all-cause outbreaks

worldwide (30-32).

5

Table 1. The most important viruses causing gastroenteritis.

1.3 The emerging importance of norovirus

As early as 1929, Zahorsky first described “’Hyperemesis hemis” or “winter vomiting

disease”, an illness characterized by the sudden onset of self-limited vomiting and diarrhea

that typically peaked during the winter months (33). However, the winter vomiting virus was

not identified until 1972. Using immuno-electron microscopy, Kapikan and coworkers

identified the NoV particles in stool of volunteers challenged with stool filtrates from an

outbreak of gastroenteritis in a school in the town of Norwalk, Ohio in 1968 (34). NoV was

thus originally named Norwalk virus, and is still often referred to its original name. This was

also the first time a virus causing gastroenteritis was isolated. During the last two decades,

there has been a dramatic increase of reported cases of NoV-induced gastroenteritis (35). This

increase is partly due to the development of effective detection methods since the early years

of NoV research. However, the development of more sensitive detection methods cannot

account for all of this huge increase. Studies have indicated a recent emergence of highly

virulent NoV strains (described more in section 2), and that variants of these strains have

remained circulating, causing the high number of diarrheal cases that are now reported (36,

37). Our modern lifestyles also contribute to the increased impact of NoV. The world

population is increasing with more people living in densely populated areas. NoV outbreaks

Virus Family Size (nm)

Nucleic Acid Clinical features

Rotavirus

Reoviridae

70

dsRNA

The main etiological agent of severe gastroenteritis in children less than 5 years

Norovirus

Caliciviridae

28-38

+ssRNA

Causing >90% of all virus-induced outbreaks, and is second to rotavirus in endemic pediatric diarrhea

Sapovirus

Caliciviridae

28-35

+ssRNA

Generally causing milder and more infrequent infections as compared to NoV

Astrovirus

Astroviridae

28-30

+ssRNA

Primarily infects young children, many under 6 months

Adenovirus 40, 41

Adenoviridae

70-80

dsDNA

Primarily infects young children, can also give rise to respiratory symptoms

6

in day-care centers (38, 39), communal settings (40), nursing homes (41), and hospitals (42),

are frequently reported. In addition, we eat more foods that have been handled by a number of

potentially infectious people. There is a rise in the consumption of fresh vegetables and fruit,

food which is often grown in countries where crops are irrigated with potentially

contaminated wastewater (43). Also travelling has increased, augmenting the exposure risk at

hotels, cruise ships and airplanes.

Intensive research in the last years has thus led to that NoV today is recognized as the most

common cause of acute gastroenteritis in humans (30-32). The Center for Disease Control and

Prevention in the United States reported that NoVs account for over 96% of all foodborne

viral gastroenteritis, and over 23 million annual infections in the United States alone (44). It

has furthermore been estimated that each year, NoV causes 64,000 episodes of diarrhea

requiring hospitalizations, and 900,000 clinic visits among children in industrialized countries

(24). Moreover, NoV is estimated to cause up to 200,000 deaths of children less than 5 years

of age in developing countries (24), and other studies have indicated that NoV cause up to

half of all cases of gastroenteritis world-wide, resulting in the striking number of over

267 million annual infections (45). All these infections also cause considerably economic

loss, outbreaks of gastroenteritis in hospitals have been estimated to annual expenses

approaching or exceeding $184 million in supplies, staff time off, and closed beds in England

alone (3). These facts highlight the impact of NoV in the society, both in industrialized and

developing countries, with no vaccine, effective treatments, or prevention strategies currently

available.

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2. Diversity and classification of norovirus

2.1 A family with many members

NoV belongs to the family Caliciviridae, a virus family in which we also find sapovirus,

another virus causing gastroenteritis in humans (Table 1, Figure 2). The two other genera of

the Caliciviridae family are vesivirus, including feline calicivirus causing respiratory disease

in cats (46), and lagovirus, including the fatal rabbit hemorrhagic disease virus (47). NoV is

one of the most diverse human viruses described, and can be divided into five different

genogroups, where GGI, GGII, and GGIV cause disease in humans (48). The GGIII contains

bovine strains, whereas GV contains murine strains (Figure 2) (48). The NoV genogroups

exhibit a high diversity, with the amino acid sequence encoded by the capsid gene differing

up to 60% (48). This high diversity makes it plausible to consider the genogroups as

individual species. The genogroups can be further divided into genotypes based on sequence

diversity, with at least 8 genotypes for GGI and 17 for GGII have been described (48), and a

18th genotype for GGII pending (49) (Figure 2). The most frequently occurring, and clinically

severe genotype, GGII.4 (see section 3), is often further divided into specific variants to

account for the recurrent seasonal pandemics (36, 50, 51).

Figure 2. The Caliciviridae family, with human pathogenic groups marked in darker grey. Norovirus

is divided into five genogroups (GG), where GGI, GGII and GGIV cause disease in humans. The

vast majority of human illness is caused by strains belonging to GGI and GGII, which can be

divided into at least 25 different genotypes.

8

2.2 Structure

NoV is a non-enveloped, single-stranded positive sense RNA virus of ~7.5kbp in length,

having icosahedral capsid symmetry with a diameter of about 30 nm (21, 46, 48). The genome

of NoV is organized into three open reading frames (ORF), where ORF1 encompasses ~5 kb

(two-thirds of the NoV genome), and encodes a 200 kDa polyprotein, which is auto processed

by a virally encoded protease to yield the non-structural viral replicase proteins essential for

viral replication (Figure 3). ORF2 is ~1.8 kb in length, and encodes the 57 kDa major capsid

protein VP1, whereas ORF3 is ~0.6 kb and encodes a 33 kDa small protein, VP2 whose

function is unknown, but possibly plays a role in the expression and stability of VP1 (52), or

with the packaging of genomes into virions. The 3´end of the genome contains a poly(A) tail

(Figure 3) (53).

Figure 3. The norovirus genome consists of three open readings frames. ORF1 encodes the non-

structural proteins, with ORF2 and ORF3 encoding the structural proteins VP1, the major capsid

protein, and VP2, a minor structural protein. The non-structural polyprotein of ORF1 is processed

by the viral 3C-like protease (Pro), into six mature proteins: p48, an N-terminal protein of

unknown function; NTPase, a nucleoside triphosphatase; p20, a protein of unknown function; VPg,

which is found covalently attached to the 5´end of the viral genome; Pro, which is the 3C-like

proteinase, and Pol, which is the viral RNA dependent RNA polymerase. The major capsid protein is

further divided into the shell and protruding domains (P), with the P domain subsequently divided

into two different subdomains, P1 and P2.

The major capsid protein can be further divided into three domains, the N-terminal domain

within the capsid, the intermediate shell domain, and the protruding P domain (54). The P

domain is in turn divided into two major sub-domains, P1 and P2. Most of the cellular

receptor interactions and immune recognition epitopes are thought to be located in the P2 sub-

domain, extending above the viral surface, which is also the most variable part of the NoV

genome (55, 56). The P2 domain is moreover the key domain determining host ranges of

humans, with different NoV strains using different receptors (see more in section 4) (57, 58).

9

2.3 Detection and molecular characterization

After its discovery in the early 1970s, the only means to detect NoV was by electron

microscopy (EM). This technique can visualize norovirus particles from stool, but has a high

detection limit of about 106 -107 viral particles per gram of stool, and requires advanced

technical training in order to use (9). The inability to grow human NoVs in cell culture

hindered the development of reactive antibodies, and the first immunological assays (59) had

similarly low sensitivities as compared to EM. It was not until the molecular revolution with

the introduction of PCR based techniques in the beginning of the 1990s (13), that the

sensitivity of detection increased. With these new molecular techniques it was possible to

clone and develop virus-like particles (VLPs) of NoV, which lead to more reactive antibodies

for detection. With the successive accumulation of NoV sequence data over the following

years, it became possible to find relatively conserved regions even for this highly diverse

virus (12, 60, 61), enabling the use of broadly reactive RT-PCR assays. The next innovation

came with the introduction of real-time PCR techniques for detection of NoV (11, 12, 62, 63),

enabling additional sensitivity, and the ability to quantify the viruses. Real-time PCR has

now emerged as standard for diagnosis and detection of noroviruses, with the immunological

techniques still lagging behind in sensitivity (11). For example, two commercial

immunological kits, IDEIA Norovirus from OXOID and Ridascreen from R-Biopharm, were

estimated to have between 30-86% sensitivity as compared to RT-PCR methods (64-66). The

antigenic diversity, and the lack of cell culture systems for NoV, will likely maintain real-time

PCR as the best method for detection of NoVs in the foreseeable future.

It is possible to distinguish genogroups directly, without sequencing, using PCR or EIA based

detection methodology (11, 66, 67). To determine the genotype, however, sequencing of parts

of the NoV genome is required. No clear rule of thumb exists for a taxonomic assignment of

norovirus genotypes. Cut-off limits for differentiating between genotypes have been

suggested to 15-45% amino acid difference on the entire capsid protein, based on uncorrected

pairwise distance measurements (48). If there is less than 15% amino acid difference, the

strains belong to the same genotype, and if there is more than 45% amino acid difference, the

strains belong to different genogroups. However, obtaining the sequence for the whole capsid

is often difficult and not feasible when a large number of samples are to be investigated.

Therefore, various genotype-determining approaches, based on sequencing of smaller parts of

the genome, have been developed. They target different areas of the NoV genome, the RNA

polymerase (68), N/S region of the capsid (49, 60), and the P domain of the capsid (69). The

10

first attempts to classify NoV were based on sequencing of parts of the RNA polymerase

gene, but it has lately been shown not suitable for genotyping (60), although useful for

identification of recombinant strains (70). Today, sequencing of the N/S region is emerging as

standard methodology for quick assessment of genotypes, due to its broad detection range,

and relatively high resolution (49, 60), where cut-off limits have been tentatively assigned to

22.1 ± 6.3% (GGI), and 26.1 ± 10.5% (GGII) of mean pairwise nucleotide distances in order

to distinguish between genotypes (60). Other parts in the P2 domain (region D) of the capsid

have been suggested due to its high variability (69), but these assays have a less broad

detection range than the assays targeting the N/S region (49), and are thus not suitable for

large-scale epidemiological studies. The clinical importance of the emerging GGII.4 strains

has lead to a need to further differentiate them into variants. The assignment of variants can

be based on small sequence differences, or on amino acid substitutions in specific positions of

the N/S region of the capsid (71).

11

3. Transmission of norovirus in the human environment

3.1 Different properties of norovirus strains

It has been observed that different NoV genotypes and genogroups have different properties

regarding prevalence, transmission pathways, and host genetic factors. The most dominant

NoV genotype found in most outbreaks is GGII.4, which accounts for approximately 60-90%

of reported cases of NoV gastroenteritis (49, 71, 72). The pandemic outbreaks observed since

the mid-90s have been due to the emergence and spread of variants of this particular genotype

(36). These variants include the Camberwell cluster, which ranges from 1987 to 1995, the

Grimsby cluster from 1995 to 2002, the Farmington Hills cluster from 2002 to 2004, the

Hunter cluster from 2004 to 2006, and the Sakai cluster, which includes viruses isolated

2004–2006 (36, 51, 73). The reason why it is variants of GGII.4 that cause the pandemics is

unclear, but studies suggest that it could be due to one or a combination of the following

reasons: a higher biological fitness, larger viral shedding in infected patients (49), a more

diverse receptor specificity (74, 75) (see section 4), and a fast mutation frequency. Switching

human receptors, and altering antigenicity due to genetic drift would be a good way to avoid

the short-term herd immunity, which is probably in place after the large pandemics (36, 76).

Except for the virulence factors and larger impact of the GGII.4 strains, NoV genotypes and

genogroups exhibit differences regarding transmission pathways and seasonality (77, 78). In a

large collected system review of norovirus illnesses in Europe (78), the authors found

differences in transmission when stratifying disease spreading according to person-to-person,

waterborne or foodborne transmissions (Figure 4).

Figure 4. Distribution of transmission pathways between GGII.4, non-GGII and GGII non-4 NoV

genotypes. Modified from (78).

12

GGII.4 strains account for the absolute majority of the person-to-person spreading. This

genotype is found less in foodborne and waterborne outbreaks, as compared to other GGII

strains, in particular GGI strains. GGI strains account for the largest proportion of food and

waterborne outbreaks. It is possible that GGI is more resistant to environmental factors than

GGII (77), enhancing its long-term survival, thus causing the large proportion of food-and

waterborne NoV outbreaks.

Moreover, looking at the seasonality of NoV, the large recurrent outbreaks during the winter

months in Europe are mainly due to variants of the GGII.4 strains (78) (Figure 5). Then, after

the winter season, GGII.4 prevalence decreases until the next winter. In the tropics, the same

phenomenon has been observed, with the rain season associated with most GGII.4 induced

gastroenteritis (49). GGI strains however, have a more stable and lower occurrence in clinical

cases during the whole year (Figure 5). This concurs with the findings of paper III in this

thesis, where we observed a stable transmission of GGI in wastewater, as compared to GGII,

which exhibits a larger seasonality (77). Why the majority of clinical cases due to GGII.4

occur during the winter months is not clear, increased indoor dwelling and humidity factors

are possible explanations.

Figure 5. Number of outbreaks with genotypes and variants per month in Spain, Finland, France,

England and Wales, Hungary, The Netherlands, and Sweden (the subset of countries which have

outbreak reports with sequence data throughout the complete period). Reprinted with permission

(78).

13

3.2 Clinical symptoms and factors facilitating the spread of disease

The symptoms described for NoV-associated gastroenteritis include the following: nausea

(79%), vomiting (69%), diarrhea (66%), abdominal cramps (30%), headache (22%), low-

grade fever (37%), chills (32%) and myalgias (26%) (79). The diarrheal stool is non-bloody,

lacks mucus, and may be loose or watery. In children, it appears that vomiting occurs more

frequently than diarrhea, whereas in adults, diarrhea is more common (79). The average

incubation period is 24–48 hours, and the symptoms typically resolve in 12–72 hours (79),

although presentation of symptoms may be prolonged in some cases, particularly in the

elderly, young children or the immune compromised (55, 80, 81). A study from England

observed that symptoms in hospitalized persons lasted longer than for the NoV infected staff

(81). Another study found that the duration of illness was longer in children < 2 years as

compared to children between 2 and 5 years (7 days vs 3.5 days respectively) (80).

Furthermore, asymptomatic virus shedding occurs, and viruses have been detected up to 3

weeks after the resolution of symptoms (25, 80, 82), with even longer periods observed in

children <1 year (80). This provides a large opportunity for transmission of the virus to other

humans. There are also several cases of chronic NoV shedding in immunocompromised

individuals (55, 83), which has been suggested to have a potential role for the evolution of

new NoV strains (84).

All age groups of the human population are susceptible to NoV infection, particularly the

elderly, with many outbreaks occurring in nursing homes (32, 41). In addition to increased

susceptibility, the elderly often suffer from more severe disease and even death, as are the

very young and the immunocompromised (80, 85, 86). NoV infections have most often been

associated with consuming contaminated food or water, although spread during an outbreak is

predominantly by person-to-person transmission via direct contact, exposure to aerosols, or

the fecal–oral route (78). NoV outbreaks occur frequently where close human contact is

difficult to avoid, such as schools, nursing homes, retirement communities, hospitals, or day

care centers (38-42). There are many factors contributing to the transmission of NoV, and its

ability to cause outbreaks. Firstly, it is an extremely stable particle in the environment, highly

resistant to disinfectants such as chlorine, and to freezing and high temperatures (18).

Furthermore, a low dose, 10 viral particles, is required for symptomatic infection (87), and

prolonged asymptomatic shedding increasing the risk of secondary spread (82). Finally, the

14

high antigenic diversity (48), and the lack of long-term immunity (76), lead to many re-

infections from the same or different NoV strains (Table 2).

Table 2: Norovirus characteristics facilitating transmission. Modified from (14).

Characteristics Observations Consequence Stability

Highly resistant to freezing, heating (up to 60°C) and disinfections such as chlorine

Difficult to eliminate in water, leading to infections from: oysters, bathing water, and food irrigated with sewage. Increased risk of infections in closed settings such as hospitals

Asymptomatic shedding

Patients can shed NoV up to three weeks after resolution of symptoms

Increased risk of secondary spread, is especially a problem concerning food handlers

Diversity

Multiple genetic, antigenic and receptor specific strains exist

Developed detection methods may not be sensitive for all strains. Re-infections can occur more easily

Low infectious dose

Less than 10 virus particles are needed for symptomatic infection

Increases risk of infection from person-to-person spread, droplets, secondary spread, food contamination

Lack of long-term immunity

Symptomatic re-infection with the same strain can occur

Adults are not protected although infected as children. Hinders development of effective vaccines

3.3 Norovirus in wastewater

The norovirus particle is highly resistant to environmental degradation, and can withstand

different treatment processes (18, 88). Norovirus outbreaks resulting directly from swimming

and drinking water are frequently reported (89, 90). Moreover, many countries still use

sewage water to irrigate their crops, and since NoV is resistant to chemical treatment and

freezing, it can contaminate all types of food, and subsequently cause disease (91). Shellfish

are a common source of NoV disease. Often cultivated in water downstream a treatment plant,

pathogens such as NoV bio-accumulate in the shellfish, which can lead to outbreaks if the

shellfish are inadequately cleaned (92, 93). These findings highlight the importance of

treatment processes that are able to reduce virus contamination, for which most wastewater

treatment systems were not originally constructed. Numerous studies have shown that enteric

viruses are present in high levels in water, even after the treatment process (11, 94-98).

Bacterial indicators are often used to indirectly measure contamination, but they have proven

15

unreliable in terms of viral contamination (96, 99). There is an ongoing debate about finding a

reliable viral indicator, and many enteric viruses or bacteriophages have been suggested, such

as adenovirus and somatic coliphages (98, 100, 101), but no conclusions have yet been

reached. Many studies on enteric viruses in wastewater usually describe virus concentrations

from the influent and effluent water, but physiochemical parameters are often not considered

in the investigations (97, 102, 103). This approach fails in understanding which processes of

the wastewater treatment plant (WWTP) that are important for reduction of norovirus.

Therefore, little is still known about factors influencing viral reduction, and how to best

monitor virus contamination.

The sewage water, more than a potential health risk, can also be regarded as a mirror

reflecting what goes on in the community connected to the wastewater system. By measuring

the transmission of norovirus in wastewater, important epidemiological information can be

obtained. For example, does the quantity of NoV in wastewater reflect the clinical picture in

the community? Are there “silent” NoV shedding going on, perhaps from NoV types that

cause less severe or asymptomatic symptom? Are there unknown reservoirs of NoV, and do

the NoV types that circulate in wastewater relate to those found in persons with disease?

Some studies from Japan and the Netherlands have observed higher concentration of both

genogroups during the winter months, and that GGII is present in higher concentrations

during the whole year (94, 96, 98). Another recent study found similar genotypes in

wastewater as in the clinical material, suggesting that the genotypes in wastewater reflect well

to the viruses circulating in the community (104). However, the understanding of noroviral

transmission in the environment remains to a large extent unclear, and more studies are

clearly needed to elucidate this pattern.

16

4. Why are some people resistant to winter vomiting disease?

4.1 A brief history of the genetic factors determining susceptibility

In the 1970s, the first isolated NoV strain detected in the Norwalk outbreak (see section 1.3),

was used in a challenge study with 12 volunteers (76). Interestingly, 50% of the individuals

did not develop symptoms of NoV gastroenteritis, and when challenged again 27 and 42

months later, they remained asymptomatic while the other 50% of the individuals developed

symptoms at all times. A subsequent re-challenge was performed on the symptomatic

individuals, this time only 4-8 weeks later, in which only one individual developed symptoms.

Most symptomatic individuals had increased serum antibody titers after each challenge.

Taken together, these findings indicated that there was no long-lasting immunity to NoV. A

short-time immunity was noted, and other factors than serum antibodies appeared important

for immunity since increased antibody titers did not offer protection. The factors mediating

the resistance remained unknown for over two decades. In the beginning of the nineties, it

was shown that rabbit hemorrhagic fever virus (vesivirus), also belonging to the Caliciviridae

family, could agglutinate erythrocytes (105). Then, in 2000, it was observed that the rabbit

hemorrhagic virus also had the ability to agglutinate human erythrocytes in presence of ABO

antigens (blood groups) (106). Finally, it was demonstrated that the Norwalk prototype VLP

bound to surface epithelial cells of the gastroduodenal junction as well as to saliva, but only to

the so-called secretor positive individuals, which express histo-blood group antigens (HBGA)

in saliva and mucosa (107). Thus, the investigation of NoV receptors and genetic

determinants of susceptibility could begin in earnest.

4.2 Histo-blood group antigens as the probable receptors for norovirus

It is today recognized that human HBGAs are receptors for NoV. Several studies (108-111)

have associated NoV susceptibility to the presence of α1,2-linked fucose on HBGAs, which is

determined by the FUT2 gene (112, 113). Individuals carrying at least one functional FUT2

allele, and thus expressing the α1,2 fucosyl transferase (FucT-II) enzyme, are termed

secretor-positive (secretors), and can express the A and B blood group antigens, as well as H-

type 1 and Lewis b (Leb) antigens on mucosa and in secretions (Figure 6) (108-110).

Individuals lacking FucT-II are termed secretor-negative (non-secretors), and have been

shown to be highly protected from infections with the most common NoV genotype (GGII.4),

as well as the Norwalk virus prototype strain (GGI.1) (108, 109). Saliva binding studies have

17

demonstrated that different NoV strains exhibit different binding patterns (74, 75, 114), with

the prototype Norwalk virus (GGI.1) mainly recognizing saliva from secretors with blood

group A and O, while exhibiting low or no binding to saliva from non-secretors and carriers

of blood group B, all suggesting protection against infection among the latter two groups.

Similarly to GGI infection, the common GGII.4 strains have been found to bind saliva from

all secretors irrespective of blood group, but not to non-secretors (115). Some NoV strains

such as VA207 (GGII.9), OIF (GGII.13), Boxer (GGI.8) and Kashiwa645 (GGI.3) have been

shown to bind to saliva of non-secretors (74, 75), which indicates that these NoV strains also

can infect the normally resistant non-secretors. However, if the binding pattern to saliva is a

thorough indicator of susceptibility remains to be further investigated with authentic studies.

Figure 6. Schematic overview of the biosynthesis of ABH and Lewis histo-blood group antigens

(HBGAs) by stepwise addition of monosaccharides to precursor structures. The FUT2 gene encodes

an α1,2fucosyltransferase which adds a fucose residue in α1,2 linkage to the terminal galactose of

the H type 1 precursor. Non-secretors, having an inactivated FUT2 enzyme, lacks α1,2-linked

fucose containing HBGAs on many epithelial cells and secretions, and will not be able to synthesize

the H type 1 antigen from its precursor. Synthesis of the A and B antigens requires the presence of

the H type 1 antigen, adding an N-acetylgalactosamine (A) or a galactose (B) in a α1,3 linkage on

the galactose residue of the H type 1 antigen. The Lewis antigens are synthesized with the FUT3

enzyme which attaches a fucose residue on the N-acetylglucosamine of the precursor. Abbrv: Gal:

galactose; GlcNAc: N-acetylglucosamine; Fuc: fucose.

18

19

5. Aims

The general aim of this thesis project is to find ways of preventing NoV infection by studying

and understanding the epidemiology of the virus. NoV is a highly diverse virus, making it

difficult to develop effective detection methods. Furthermore, different NoV strains exhibit

differences regarding severity of symptoms, seasonal transmission pathways, and receptor

specificities in the human host. It is therefore important to detect and characterize the virus at

different time-points and in different settings, enabling a fuller understanding of NoV disease.

Our approach to this can be summarized as follows:

• Develop real-time PCR assays for detecting and quantifying human NoV in clinical

and environmental samples

• Characterize the most predominantly circulating NoV strains and their prevalence in

children’s diarrhea in Nicaragua during one year

• Elucidate the presence, seasonal variation and parameters influencing the reduction of

NoV in a wastewater treatment plant during one year

• Investigate host susceptibility factors to NoV infection in a foodborne outbreak

20

21

6. Development of a new method for detecting human noroviruses (paper I)

An important aspect of all papers included in this thesis is the detection and characterization

of NoV. This can be difficult since NoVs are highly variable, which makes it problematic to

develop a broad detection assay able to target all the various strains. It is also important to

consider the detection limit. For stool samples this is generally not a problem, since they

contain high amounts of virus particles. Finding NoV in wastewater with molecular methods

is much more difficult, mainly due to two reasons: First, virus in wastewater is highly diluted

as compared to stool samples. Second, wastewater has a high density of other particles that

can work inhibitory in RT and/or PCR reactions. This means that a very sensitive method for

detection and/or a method for enriching the virus fraction in the water are warranted. We

therefore established a real-time PCR assay, a method previously shown to be highly sensitive

for NoV detection. For analysis of NoV in wastewater samples, we established an

ultracentrifugation assay to concentrate the virus fraction before proceeding with the real-time

PCR detection (Figure 7).

The real-time PCR assay was developed using fluorescently labeled primers based on the

Light Upon Extension (LUX) technique. The LUX technique uses a fluorophore attached near

the 3´end of one of the primers, constructed to form a hairpin loop, and thus rendering a

sterical fluorescence quenching capability (Appendix A). When the primer becomes

incorporated into the double-stranded PCR product, the fluorophore is de-quenched, resulting

in an increase of the fluorescence signal (116). The advantage with LUX is that it offers high

sensitivity and specificity, without the use of probe or quencher molecules. This structure also

hinders primer-dimer formation, which can decrease efficiency (117). The incorporation of a

fluorophore moreover enables the use of melting curve analysis, which can differentiate

amplicons based on sequence differences, in our case two human NoV genogroups.

Our developed real-time PCR assay was validated against stool specimens collected from

both Sweden (n= 61) and Nicaragua (n=42), and against a reference panel from the Swedish

Center for Infectious Disease control (n=15). The same samples were also tested with other

methods for comparison of sensitivity and specificity. A commercial ELISA kit (DAKO

6044), a published conventional PCR method (118), and a TaqMan real-time PCR method

22

modified from Kageyama and coworkers (12) were used for this comparison. Cloning of gene

fragments, obtained after PCR amplification of GGI.4 and GGII.4 strains, into plasmids was

performed in order to have a reference for determination of detection limits and PCR

efficiency of the real-time PCR assay, and also to be able to quantify virus in stool and

wastewater.

Figure 7. Flow scheme of NoV detection and quantification from stool and wastewater samples.

Analysis using bioinformatics revealed a highly conserved region in the ORF1-ORF2 junction

for both GGI and GGII, which was chosen as target for the LUX primers. LUX primers were

manually designed and evaluated with conventional PCR, where the best primer pairs were

chosen to be used in the real-time PCR assay (Appendix A).

The real-time PCR assay was able to detect ≤10 gene copies in reference samples investigated

for GGII and GGI, respectively. Ten gene copies per PCR reaction is equivalent to ~20 000

virus particles per gram of stool, or ~10 000 virus particles per liter of wastewater after being

processed as described in Figure 7. These two assays were further evaluated with clinical

23

specimens positive for NoV GGI, NoV GGII, rotavirus, sapovirus, adenovirus, astrovirus and

feline calicivirus, respectively, and no cross-reactivity was observed.

The LUX real-time PCR assay was able to detect all NoV positive specimens and assign the

correct genogroup in the reference panel. We furthermore found a 99% correlation between

our LUX based assay and the TaqMan real-time PCR assay with all specimens tested, with

one specimen negative in the LUX assay being positive in the TaqMan assay. The LUX assay

was more sensitive compared to the conventional PCR and the ELISA based methods (Table

3, paper I).

For each PCR-product a specific melting temperature interval was determined, and the

melting temperature range between the genogroups was clearly distinguishable. The LUX

real-time PCR assay was able to simultaneously detect and distinguish between NoV GGI and

GGII positive specimens and mixed infections of these, using a duplex assay containing

primers for both GGI and GGII (Figure 3, paper I). This is the first assay of its type able to

distinguish between GGI and GGII based on melting curve analysis. Distinguishing between

the NoV genogroups yields valuable epidemiological information, and a multiplex assay

saves time and considerably lowers the screening costs.

To summarize, we developed and established a novel real-time PCR assay for detection and

quantification of NoV GGI and GGII. Using specimens both from Sweden and Nicaragua, we

have shown that the assays can be applied in different geographic regions, and the use of

melting curve analysis can successfully distinguish between the two main human NoV

genogroups. This assay can also be used to detect, quantify and assign genogroups of NoV in

wastewater samples. The LUX system is simple and cost-effective, since it does not use

probes or various fluorophores. The system can be used on most real-time PCR platforms, and

there is no need for post PCR processing which reduces the time and possibility of

contamination.

24

7. Characterization of norovirus in children’s diarrhea (paper II)

Earlier believed to mainly be a pathogen important in adult gastroenteritis, the big impact of

NoV in children’s diarrhea is now beginning to be realized. Second only to rotavirus, NoV

has an estimated prevalence of between 10-15% in all severe diarrhea episodes in children

(24). Considering this, we wanted to elucidate the impact of NoV, and to characterize

circulating strains in pediatric diarrhea in Nicaragua during a whole year. This is the first

study of its kind in the Central American region.

The clinical specimens investigated for NoV were collected from children living in the city of

León, Nicaragua. Nicaragua is located in Central America with an estimated population of

5,500,000 inhabitants; approximately 12.3% are children 1-4 years of age (119). The

mortality rate in Nicaragua was 26.4 per 1000 live births between 2000 and 2005 (119), with

respiratory and diarrhea illness as the leading causes of death among children 1- 4 years of

age (120). The climate is tropical; the rainy season starts in June, and lasts until November,

when the dry season starts. Sanitary conditions are insufficient in large sections of the city of

León, especially in peripheral areas.

From March 2005 to February 2006, a total of 542 children ≤ 5 years of age suffering from

sporadic acute diarrhea were enrolled at five different health facilities in León, in a

longitudinal prospective manner. The clinical information was obtained by reviewing the

clinical records of the cases. The information was registered in a paper file containing answers

to questions about symptoms such as; fever, nausea, vomiting, loss of appetite, abdominal

cramps, abdominal distension (gas), number of loose stools during the past 24 hours,

dehydration status and treatment plan. The disease was then classified according to

dehydration status in three levels: “severe dehydration”, “some dehydration”, and “no

dehydration” (Integrated Management of Childhood Illness, WHO ref

WHO/FCH/CAH/01.01). Detection of norovirus was performed with commercial ELISA kits,

IDEIA k6043 and 6044, and NoV positive specimens were subsequently quantified with the

LUX real-time PCR (paper I), followed by genotyping by sequencing of the N/S region of the

capsid gene (Figure 8, Appendix A).

25

Figure 8: Schematic overview over the detection and molecular characterization of norovirus in

pediatric diarrhea in Nicaragua.

Norovirus was detected in 65 (12%) of the 542 stool samples analyzed, 11% in children from

the community, and 15% in a total of 133 hospitalized children. The high prevalence of NoV

in hospitalized children is noteworthy, since it is more than earlier described in France,

Australia and the United States (118, 121). Considering that the sensitivity of the ELISA

screening assay used is less as compared to RT-PCR methods, the true impact in Nicaragua is

probably 5-10% higher, indicating a very high prevalence of NoV. Surprisingly, girls (15%)

were significantly more infected than boys (10%) (p=0.04), with children less than 2 years

more frequently infected than children 2-5 years (Table 1, paper II). No gender specific NoV-

susceptibility mutation is known, thus socio-economic factors are the likely explanation to the

difference of NoV prevalence between girls and boys.

GGII was the most common genogroup observed, found in 88% (57/65) of the children,

followed by GGI in 11% (7/65). The highest diversity of NoV genotypes was observed in

April when at least four genotypes circulated, GGII.2, GGII.4, GGII.7 and a novel cluster,

tentatively termed GGII.18. This novel cluster was confirmed by sequencing of the region D

of the capsid gene (Appendix A). During June, GGI.4 and GGII.4 were observed, and in July

the genotype GGII.4 variant 3 become predominant (Figures 1 and 2, paper II). During the

26

following months the NoV positive specimens decreased but in October, once again, GGII.4

re-appeared. In November, the uncommon genotype GGII.17 appeared, and during January

2006 the number of NoV-positive isolates increased up to 36%, which was associated with

the re-emergence of GGII.4 variant 2 (Figures 1 and 2, paper II). Our observations extend

previous knowledge about the emergence and selection of GGII.4 variants, and suggest that

particular variants with increased fitness are selected from a pool of co-circulating strains.

Using the LUX real-time PCR (paper I), we quantified NoV shedding in children and

compared the measured viral quantity to severity of symptoms, and to which genotype the

child was infected with. The geometric mean viral loads of NoV GGI and GGII was 5.7 x 106

and 3.8 x 107 genome equivalents per gram of fecal specimen, respectively. Virus

concentrations in specimens from children infected with NoV GGII.4 were approximately 15

fold higher as compared to those infected with other GGII genotypes (7.2 x 107

vs. 4.8 x 106) ,

and 13 fold higher than other GGI genotypes (7.2 x 107

vs. 5.7 x 106). The highest viral load

was observed in the group of children infected with GGII.4 and requiring intravenous re-

hydration (mean 3.2 x 108) (Figure III, paper II).

To summarize, we found a high impact of NoV in children’s diarrhea in Nicaragua, both in

community and hospital based settings. The peak of NoV-induced diarrheal episodes were

associated with variants of the GGII.4 genotype, emerging and replacing the many different

genotypes circulating before the increase of diarrheal cases. Children infected with the GGII.4

genotype shed more virus as compared to children infected with other genotypes, which could

provide an explanation to the high prevalence of GGII.4 in person-to-person transmissions.

27

8. High prevalence of norovirus in a wastewater treatment plant (paper III)

Viruses are usually not monitored in treatment plants, thus little is known about their

prevalence, and which parameters that influence reduction. Furthermore, wastewater can

reflect NoV shedding in the community connected to the treatment plant, where sub-clinical

infections will not be observed in the clinical data. We therefore performed a NoV study at

the wastewater treatment plant (WWTP) in Gothenburg, Sweden, where we sampled

wastewater during a whole year (Oct 2005–Sep 2006) (Figure 9). The Gothenburg WWTP is

one of the largest WWTP in the Nordic countries, receiving wastewater from nearly 830,000

person equivalents, with an average daily incoming water volume of ~350,000 m3 (~4 m3/s).

The WWTP is designed for biological nitrogen removal, utilizing pre-denitrification in a non-

nitrifying activated sludge system, and post-nitrification in a trickling filter. During primary

settling, heavy particles are removed. After the primary settling, iron sulfate is added which

aggregates phosphor (Figure 9).The activated sludge contains high levels of biomass and is

divided into two phases: an anaerobic phase, where the denitrification occurs, and an aerobic

phase for decomposition of organic material. During secondary settling, sludge and

phosphorous aggregates are removed, and the sludge is collected and pumped to the primary

settling. After the secondary settling, ~50% of the water goes out into the recipient water, and

the rest goes back into circulation via the nitrifying trickling filter. Sludge is extracted from

the primary settlers and digested in completely mixed mesophilic anaerobic digesters with a

retention time of 20-30 days. The digested sludge is centrifuged and the reject water is

returned to the WWTP (Figure 9).

Our wastewater samples were taken monthly at eight different key sites in the wastewater

treatment process (Figure 9), and stored at 4°C until processed with ultracentrifugation as

described in Figure 7, and quantified with the developed real-time PCR assay (paper I).

Physicochemical parameters were also measured in incoming and outgoing water for all

sampling months, to determine their effect on viral concentrations. These measurements were

performed as part of the routine at Gryaab laboratory, Ryaverket, Gothenburg, Sweden.

28

Figure 9. Schematic overview of the municipal WWTP Ryaverket in Gothenburg, Sweden. Sampling

sites are indicated with numerals and arrows.

We found that NoV GGII exhibited higher concentration levels at all sites during the winter

months, while NoV GGI exhibited higher concentration levels during the summer months.

Moreover, NoV GGI exhibited smaller variation regarding virus concentrations than NoV

GGII (Figures 2 and 3, paper III). The reason for the increase of GGI during summer, which

was associated with a decrease of GGII, is probably due to the emergence of new GGI strains

after the GGII-induced winter outbreaks.

The reduction between incoming and outgoing water was on average 1.5 log10 units (Table 2,

paper III), which was largely the same between the two genogroups, although GGI at many

times was not detected in the outgoing water, making reduction estimations uncertain. Virus

concentration was reduced in the primary settling (average 0.7 log10 units) and in the activated

sludge in combination with the secondary settling (average 0.9 log10 units). The trickling filter

exhibited a limited reduction for the few occasions that the remaining virus was detected in

the influent to the trickling filters (Table 2, paper III). The reduction averages were similar to

the reduction of indicator bacteria, coliform bacteria and Escherichia coli, which were on

average 1.2 and 1.0 log10 units, although no correlation was observed with the viral reduction

at individual sampling months. This emphasizes the importance of finding better indicators

for monitoring of virus contamination.

29

We observed that the reduction of NoV in the WWTP varied between months, and thus

related this to incoming virus concentrations, and to different physicochemical parameters.

We found that higher incoming concentrations correlated to higher reductions of both

genogroups, particularly NoV GGI, and that a higher inflow was associated with less

reduction (Table 3, paper III). This negative correlation could be related to the fact that low

flow gives less dilution and thus higher NoV concentrations, creating a higher potential for

reduction. We furthermore observed that the incoming concentration of NoV GGI is

significantly correlated to inflow, the less inflow the higher concentration of NoV GGI,

probably due to dilution effects (Table 4, paper III). However, no such correlation exists for

NoV GGII. This could be due to the fact that the presence of NoV GGII is more seasonal

dependent than NoV GGI, thus disguising the effect of dilution. Levels of NoV GGII peaked

during the winter months when clinical cases are more common, making it difficult to detect a

decrease of concentration due to a higher inflow of wastewater. However, this is observed for

NoV GGI, since it exhibited more stable concentration levels in wastewater, which could

indicate that infections of NoV strains belonging to this genogroup occur at a stable rate

throughout the whole year.

To summarize, we found that NoV was present in wastewater throughout the year, not only

during the winter months. GGI levels increased in summer, possibly due to emerging

circulations of new genotypes after the winter outbreaks. The transmission of NoV GGI was

stable during the year, hence incoming concentrations was affected by dilution factors. This

stable transmission in wastewater indicates that infections of this genogroup occur at a stable

rate in the community, perhaps giving rise to sub-clinical or mild disease, since GGI is not

frequently observed in clinical data. Primary treatment and treatment in a conventional, non-

nitrifying activated sludge system reduced the NoV content by about a factor 30, and water

flow and incoming virus concentration were associated with reduction.

30

9. New susceptibility patterns revealed in a foodborne norovirus outbreak (paper IV)

In October 2007, a NoV outbreak occurred in Jönköping, Sweden, at a seminar for health care

improvement. NoV GGI was identified in the stool from some of the ill, and we understood

that this would be an excellent opportunity to study host susceptibility factors to infection,

since we had access to the clinical data and patient material. Moreover, GGI strains have not

been studied much with regards to host susceptibility. Epidemiological investigations

indicated that the lunch meal on the first day was contaminated with NoV, and subsequently

the cause of the outbreak. The cook was ill four days before the outbreak started, and three

days later other employees of the restaurant became ill, suggesting the restaurant employees

as the probable source of NoV contamination in the food.

A total of 112 health care workers from different parts of Sweden joined the seminar. The

health care workers were asked to take part of this case control study, and 83 individuals,

including 4 employees from the restaurant, decided to participate in the study. In total 33

(40%) of these 83 individuals acquired acute gastroenteritis during or after the seminar. NoV

disease was determined by at least one of the following symptoms: vomiting, diarrhea, or

nausea combined with stomach-ache, from ~12 to 60 hours after the ingested meal.

Descriptions of symptoms were obtained through a questionnaire sent out to all participants of

the study. Saliva samples, for geno- and phenotyping of host susceptibility factors, were

collected from all participants of the study (n=83) and stored at -20°C until further use (Figure

10). Furthermore, stool samples (n=4) were obtained from the cook, two employees, and one

participant of the seminar with symptoms of NoV gastroenteritis, which enabled us to perform

molecular characterization of the virus. Informed consent was received from all participants.

31

Figure 10. Schematic overview over the determination of host susceptibility factors for

symptomatic norovirus infection.

In contrast to earlier findings with GGII.4, GGII.3 and GGI.1 strains, we observed that 7 out

of the 15 non-secretors were symptomatically infected (Table 1, paper IV), and the risk of

developing symptomatic infection was approximately twice as high among non-secretors

compared to secretors (Table 2, paper IV). Consistent with the secretor association, Lea+b-

individuals had the highest susceptibility (OR [Odds Ratio] 2.42), compared to Lea+b+or Lea-b-

individuals (OR 0.73 and 0.61 respectively) (Table 2, paper IV). Moreover, none of the non-

secretors who were also Lewis-negative (n=3), hence lacking the Lea and ABO antigens in

saliva, were symptomatically infected. These findings indicate but do not prove that the Lea

antigen is one putative receptor for this NoV strain. The clinical symptoms were not affected

by secretor status or HBGA profile (Table 3, paper IV).

We furthermore found that blood type B individuals had reduced risk of symptomatic

infection of the outbreak strain (OR 0.27, p=0.11) (Table 2, paper IV). Nevertheless, 2 out of

12 individuals with blood group B developed symptomatic illness. It is possible that the α-gal

in the blood type B structure partly covers an epitope needed for binding, and hence decreased

the ability of the outbreak strain to infect carriers of blood type B.

32

Sequencing of the entire capsid gene revealed that the outbreak strain was a genotype GGI.3

virus (Figure 3, paper IV). Interestingly, Shirato and coworkers (75), found that the

Kashiwa645 strain, another GGI.3 strain, sharing high aa homology with the outbreak strain

in the P2 region (Figure 4, paper IV), bound to both secretor and non-secretor saliva to the

same extent. Shirato et al. (75) also found that the Kashiwa645 strain bound to synthetic Lea

carbohydrates, but not to synthetic Leb ,which is in concordance with the disease pattern in

our study, with Lea+b- individuals having the highest OR for symptomatic infection of all

HBGA investigated. Also, the Kashiwa645 strain bound weaker to B type saliva as compared

to A or O type saliva. This indicates that saliva binding studies may be used as a reliable

indicator of host susceptibility factors for individual NoV strains.

FUT2 G428A genotyping revealed, for the first time to our knowledge, that heterozygous

secretors were more susceptibility as compared to homozygous secretors, with twice the risk

of symptomatic infection for heterozygous individuals (Table 2, paper IV). Being a

heterozygous secretor may lead to lower FucT-II expression as compared to a homozygous

secretor, which could increase the possibility for FucT-III (building the Lewis antigens on H-

type 1 or its’ precursor) to compete with FucT-II for the H-type 1 precursor, increasing the

concentration of the HBGA Lea. Possibly, there is a correlation between Lea concentration

and susceptibility to this NoV strain, since Lea+b- individuals had the highest susceptibility of

all the HBGAs investigated in this study

To summarize, this study describes for the first time a foodborne NoV outbreak infecting

individuals irrespective of secretor status, with non-secretors and Lea+b- individuals having a

higher risk of disease. Furthermore, we observed differences in susceptibility regarding

homozygosity and heterozygosity in the FUT2 gene, with heterozygous secretors more

susceptible to symptomatic NoV infection than homozygous secretors.

.

33

10. Concluding remarks

The transmission of noroviral populations in the community and the environment is complex,

and more studies need to be performed in order to understand the underlying mechanisms.

However, on the first page of this concluding section, I have brought together some of the

main findings from the different papers in a speculative attempt to describe the seasonal

transmission of NoV strains (Figure 11). The year is divided into “high” and “low” NoV

season, depending on the number of clinical cases associated with NoV.

Figure 11: Overview of NoV transmission in humans during one year. During high season, many

NoV-induced diarrheal cases are reported, which are mainly due to infections of GGII.4 strains

(paper II). During this season we also observed high levels of GGII in wastewater (paper III), as

determined by the LUX real-time PCR (paper I). During low season, there is less number of clinical

cases attributed to NoV, and many different genotypes are found in patients (paper II). During the

low season we observed high levels of GGI in wastewater (paper III). Some of these strains cause

gastroenteritis in people not previously exposed in the high season, partly due to a different

receptor usage than GGII.4 (paper IV).

Many different

genotypes emerge

(paper II)

Less common strains cause disease in people

resistant to GGII.4 (paper IV)

GGII.4 variants re-emerge replacing other

strains, partly due to a better replication (paper II)

Many clinical cases due to

GGII.4 (paper II)

Short term herd

immunity to GGII.4?

NoV “high season”

NoV “low season”

NoV “high season” NoV “low season”

34

The results from the papers included in this thesis provide valuable information, which could

be used to develop preventive approaches to NoV disease. We observed that a specific

genotype of NoV, GGII.4, gives rise to severe symptoms, and most of the clinical cases

during the high season (paper II). This may provide a focus for development of prophylactic

treatment, such as vaccines. However, as also observed in our investigations, there is a

constant emergence of new GGII.4 variants (paper II), which indicates a mechanism of

GGII.4 to avoid host responses, which could hinder the effectiveness of prophylactic

treatments. We also found a possible explanation for the high transmission of the GGII.4

strains, since children infected with this genotype shed a higher number of virus (paper II).

The fact that ~20% of the Caucasian population is highly resistant to the GGII.4 has also been

suggested to provide means for developing new treatments. By identification of functional

receptors, it would be possible to develop medicines that block NoV infection. However, as

we discovered in the foodborne outbreak (paper IV), the expectedly resistant individuals were

the most susceptible to the more uncommon genotype, GGI.3, which warrants the need for

additional investigations of host genetic factors before developing such treatments. This

foodborne outbreak occurred before peak of NoV-induced diarrhea, a time when according to

our studies, many NoV genotypes circulate (papers II and III) before the re-emergence of

GGII.4 strains, highlighting the importance of molecular characterization of the virus.

The continual occurrence of NoV in wastewater, particularly the GGI, during the whole year

(paper III) indicates that infections are frequently occurring even in the low season. These

variants probably give rise to more mild or asymptomatic infections, since the clinical cases

are less frequent during this season. The NoV was often detected in high concentration in

outgoing water (paper III), which could lead to environmental contamination in the river

downstream the WWTP, and no correlation between reduction of bacteria and NoV was

observed. These facts clearly demonstrate the need for improved monitoring tools for viruses

to account for environmental contamination, and that a viral reduction strategy at the WWTPs

needs to be implemented. The assays we have developed, with ultracentrifugation and LUX

real-time PCR (papers I & III), are easy to perform and could be used for monitoring of NoV

in environmental samples. In conclusion, this thesis has demonstrated the ubiquitous presence

of NoV in the human environment and its high impact in diarrheal disease. The obtained

insights into the epidemiology of the virus can hopefully be used towards finding preventive

measures, which will reduce the number of NoV-induced gastroenteritis in the future.

35

Acknowledgements

There are many people who have been a support, directly or indirectly, during the years

working with this thesis. I would primary like to thank all my colleagues at the Divisions of

Medical Microbiology and Molecular Virology at Linköping University for making it a

pleasant and interesting experience. I furthermore seize the moment to specifically mention

the following persons:

Per-Eric Lindgren, my main supervisor, who has been of invaluable support in most aspects

during my stay in his group, and from whom I have learned a lot, from science to the history

of Visingsö. Lennart Svensson, my co-supervisor, who has been an encouraging mentor and

taught me much about the world of viruses. Andreas Matussek, my co-supervisor, who has

cycled across the Alps but still hasn’t managed Vasaloppet. For all your valuable help with

the projects, and for being at the right place at the right time.

The group at Medical Microbiology with Stefan Börjesson, friend and schlager-aficionado

who has been a support since the beginning. Carina Sundberg, ex-colleague, it was nice to

get to know you and the family, good luck with your post-doc! Peter Wilhelmsson, office-

partner and friend from “little Munkfors”, enjoy the apartment and the new life! Pontus

Lindblom, for friendship, all the interesting discussions, and valuable technical expertise.

Björn Berglund, for standing up to the FRA and other injustices in life, Magalí Martí, for

keeping the dream of a norovirus DGGE alive (and a free Catalunya), and all the others

Elisabet Hollén, Charlotte Sandgren, and Fredrik Nyström for creating a superb group to

be working in. Thanks again to Fredrik for help with the cover image.

The group at Molecular Virology with Elin Kindberg, I am really grateful for our friendship,

interesting and fun conversations as well as scientific collaboration. Thanks for putting up

with my sometimes rudimentary organization skills. Dr. Filemón Bucardo-Riviera, mi

mejor amigo Nicaragüense, for showing me another perspective of life, and for the

encouraging “flor de caña” evenings. I hope to see you and your family again soon. Beatrice

Carlsson, for friendship, scientific collaboration and for valuable input about wedding

planning, Malin Vildevall, Caroline Jönsson and Marie Hagbom, for your nice company

and valuable help in our virus group.

36

Che Karl-Hans Fru. Xintin bono! It´s been great to get to know you, next time we meet I

will be playing in team Africa. Vegard and Veronica Tjomsland, for your nice company at

work or at various running activities, good luck with the marathon. Ana Vujic, for your

friendship, enjoy yourself in Oxford! Lena Svensson, Caroline Jönsson, Christina

Samuelsson for creating a pleasant and social atmosphere, and organization of fun events.

Katarina, Christina, and Mary, for kindly reminding me when I have forgotten to make

coffee (which always never happens). Karl-Eric Magnusson, for interesting discussions and

for helping me solve various ultracentrifugation problems. Alexander Persson, for nice

company and entertaining discussions, especially during the long student labs. Thanks also to

all the people I have met through our collaboration with UNAN-León in Nicaragua.

The Jönköping crew with Andreas Matussek, Sture Löfgren, Olaf Dienus, Sara Melin,

Lisa Stark for making me feel at home at Ryhov with surroundings. Thanks also to Ing-

Marie Einemo for help with sample collection during the Jönköping outbreak. Britt

Åkerlind, Tina Lundqvist, and all the others at level 10 for always helping me when I

wanted to find viruses. Ann Mattsson, for help with all things related to the wastewater

treatment plant Gryaab in Gothenburg. Thanks also to Lucica Enache and Åsa Nilsson for

help with the collection of wastewater samples.

Ida and Amanda for interesting conversation about life and the advantages of doing science,

Sofie L, for nice discussions and for helping me stay in shape. G, Sofia, and Blixten for

relaxing evenings after hard days at work, and for providing ticks to our lab. Gerzon and

Tobias, for a generous friendship. Östlin, Mårten, Peer, Olof, Henrik, because you are

friends, and it is tradition. Especially thanks to Mårten for finding the Eyedropper tool,

making it possible to finish this thesis.

My Belgian family with Johan, Renilt, Han, Sven, Rita and Alfons. Hopelijk krijgen we de

kans elkaar wat vaker te zien in de toekomst!

To my parents, Anki and Lars, for support and for making an excellent job raising me, as

well as the organization of the highly successful “Fästing” lecture series in the Munkfors area.

My little brother Marcus for help with the correction of the thesis, and other things.

, and to Sarah, my greatest source of strength and inspiration.

37

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45

Appendix A: PCR primers

Pri

mer

pai

rs

Seq

uen

ce (5´

-3´)

Pos

itio

na

Funct

ion

Ref

eren

ce

NV

G1f

1b

NV

G1r

lux

CG

Y T

GG

AT

G C

GN

TT

C C

AT

GA

ga

tga

GT

C C

TT

AG

A C

GC

CA

T C

A T

C

5291

-531

0 53

78-5

360

LU

X r

eal-

tim

e P

CR

for

NoV

GG

I;

OR

F1-

OR

F2

junc

tion

Nor

dgre

n et

al

(pap

er I

) (1

1)

NV

G2f

lux1

C

OG

2R

gara

a A

TG

TT

Y A

GR

TG

G A

TG

AG

R T

TY

TC

T

CG

AC

G C

CA

TC

T T

CA

TT

C A

CA

50

12-5

034

5100

-508

0 L

UX

rea

l-ti

me

PC

R f

or N

oV G

GII

; O

RF

1-O

RF

2 ju

ncti

on

Nor

dren

et

al.,

(pap

er I

) (1

1)

Kag

eyam

a et

al.

, (12

)

NV

G1f

1b

G1S

KR

C

GY

TG

G A

TG

CG

N T

TC

CA

T G

A

CC

A A

CC

CA

R C

CA

TT

R T

AC

A

5291

-531

0 56

71-5

653

Gen

otyp

ing

by N

/S r

egio

n of

NoV

GG

I

Nor

dgre

n et

al.

, (pa

per

I) (

11)

Koj

ima

et a

l., (

67)

NV

G2f

lux1

G

2SK

R

gara

a A

TG

TT

Y A

GR

TG

G A

TG

AG

R T

TY

TC

C

CR

CC

N G

CA

TR

H C

CR

TT

R T

AC

AT

50

12-5

034

5389

-536

7b G

enot

ypin

g by

N/S

reg

ion

of N

oV G

GII

N

ordg

ren

et a

l., (

pape

r I)

(11

) K

ojim

a et

al.

, (67

) C

ap A

C

ap B

2 C

ap B

1

GG

C W

GT

TC

C C

AC

AG

G C

TT

T

AT

GT

I G

AY

CC

W G

AC

AC

T

AT

GT

T G

AC

CC

T G

AT

AC

6897

–691

4 67

38–6

754

6738

–675

4

Gen

otyp

ing

by r

egio

n D

in

the

P d

omai

n of

N

oV G

GI

V

inje

et

al.,

(69)

Cap

C

Cap

D3

Cap

D1

CC

T T

YC

CA

K W

TC

CC

A Y

GG

T

GY

CT

Y I

TI

CC

H C

AR

GA

A T

GG

T

GT

CT

R S

TC

CC

C C

AG

GA

A T

G

6667

–668

4 64

32–6

452

6432

–645

1

Gen

otyp

ing

by r

egio

n D

in

the

P d

omai

n of

N

oV G

GII

V

inje

et

al.,

(69)

298h

28

9i

290h

29

0i

290j

29

0k

TG

A C

GA

TT

T C

AT

CA

T C

AC

CA

T A

T

GA

CG

A T

TT

CA

T C

AT

CC

C C

GT

A

GA

T T

AC

TC

C A

GG

TG

G G

AC

TC

C A

C

GA

T T

AC

TC

C A

GG

TG

G G

AC

TC

A A

C

GA

T T

AC

TC

C A

CC

TG

G G

AT

TC

A A

C

GA

T T

AC

TC

C A

CC

TG

G G

AT

TC

C A

C

4865

-488

6 48

65-4

886

4568

-459

0 45

68-4

590

4568

-459

0 45

68-4

590

Pri

mer

s ta

rget

ing

the

RdR

p re

gion

of

NoV

G

GI,

GG

II a

nd s

apov

irus

Zin

tz e

t al

., (1

18)

Cap

GI3

fw

Cap

GI3

rv

GA

T C

TC

CT

G C

CC

GA

T T

AT

GT

A A

AT

GA

T G

AT

G

CA

T T

AT

GA

T C

TC

CT

A A

TT

CC

A A

GC

CT

A C

GA

GC

53

36-5

366

6952

-692

1 A

mpl

ific

atio

n of

the

com

plet

e ca

psid

gen

e fo

r N

oV G

I.3

Nor

dgre

n et

al.

, (pa

per

IV)

428-

F(b

ioti

n)

428-

R

Seq

uenc

ing

GA

G G

AA

TA

C C

GC

CA

C A

TC

CC

G G

GG

GA

G T

AC

A

TG

GA

C C

CC

TA

C A

AA

GG

T G

CC

CG

G C

CG

GC

T

GG

T G

GT

GG

T A

GA

AG

G T

C

403-

432

597-

568

478-

462

PC

R a

mpl

ific

atio

n an

d py

ro-s

eque

ncin

g of

pa

rts

of

the FUT2

ge

ne

for

dete

rmin

ing

secr

etor

sta

tus

Buc

ardo

et

al.,

(122

) K

elly

et

al.,

(112

) K

indb

erg

et a

l., (

123)

a) P

osit

ions

for

NoV

GG

I co

rres

pond

to

refe

renc

e st

rain

Nor

wal

k68

(M87

661)

; po

siti

ons

for

NoV

GG

II c

orre

spon

d to

ref

eren

ce s

trai

n L

ords

dale

(X

8655

7);

posi

tion

s fo

r FUT2

cor

resp

onds

to

Gen

Ban

k ac

c.no

DQ

3212

7.

b) P

osit

ions

mod

ifie

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