Download The effect of histo-blood group antigen (HBGA)

Faculty of Bioscience Engineering
Academic year 2015 – 2016
Viral-bacterial interactions:
The effect of histo-blood group antigen (HBGA)-expressing
bacteria and probiotics on Noroviruses
Dries Loncke
Promotor: Prof. Dr. Ir. Mieke Uyttendaele
Tutor: Dr. Dan Li
Master’s dissertation submitted in partial fulfillment of the requirements for
the degree of
Master of Science in Bioscience Engineering:
Cell and Gene Biotechnology
Faculty of Bioscience Engineering
Academic year 2015 – 2016
Viral-bacterial interactions:
The effect of histo-blood group antigen (HBGA)-expressing
bacteria and probiotics on Noroviruses
Dries Loncke
Promotor: Prof. Dr. Ir. Mieke Uyttendaele
Tutor: Dr. Dan Li
Master’s dissertation submitted in partial fulfillment of the requirements for
the degree of
Master of Science in Bioscience Engineering:
Cell and Gene Biotechnology
Acknowledgements
While writing this thesis, it struck me how fast these past five years of studying Bioengineering went
by. Looking back at this period really convinces me that I made the right decision choosing this path. I
specialized in Cell and Gene Biotechnology, making the choice for a thesis concerning bacteria and
viruses a rather logic decision. However, this experience would not have been possible without the
help and support of certain people.
In the first place, I would like to thank Prof. Dr. Ir. Mieke Uyttendaele for offering me the chance to
work on the subject of viral-bacterial interactions as a master’s thesis as well as giving me advice on
how to improve this thesis. Special thanks go out to my tutor Dan. She guided me along this journey,
answered all my questions, read and corrected this thesis report, taught me experimental techniques
and procedures, helped me explain the obtained results, and made me a better bioengineer. I really
appreciate everything you have done for me!
I enjoyed working at the Department of Food Safety and Food Quality of Ghent University. Thank you
to Ann, Danny, and all the others in and around the lab for creating a nice working atmosphere. To my
fellow thesis students, it was fun having you guys around!
Last but not least, I want to express my gratitude towards my parents, brother and friends for always
supporting me. They have been there for me during the fun and a bit less fun moments, not only this
last year, but throughout my whole academic adventure.
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Permission for use of content
The author and the promotor give the permission to use this thesis for consultation and to copy parts
of it for personal use. Every other use is subjected to the copyright laws, more specifically the source
must be extensively specified when using results from this thesis.
Ghent, June 2016
Toelating tot bruikleen
De auteur en promotor geven de toelating deze scriptie voor consultatie beschikbaar te stellen en
delen ervan te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder de beperkingen van het
auteursrecht, in het bijzonder met betrekking tot de verplichting de bron te vermelden bij het aanhalen
van resultaten uit deze scriptie.
Gent, juni 2016
The author,
The promotor,
De auteur,
De promotor,
Dries Loncke
Prof. dr. ir. Mieke Uyttendaele
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Table of Contents
Acknowledgements .................................................................................................................................. i
Permission for use of content .................................................................................................................iii
Toelating tot bruikleen ............................................................................................................................iii
Table of Contents .....................................................................................................................................v
List of Abbreviations ............................................................................................................................... vii
Abstract ................................................................................................................................................... ix
Samenvatting........................................................................................................................................... xi
Chapter 1: Introduction ........................................................................................................................... 1
Chapter 2: Literature study ..................................................................................................................... 3
2.1 Introduction to Noroviruses ........................................................................................................ 3
2.1.1 Classification and taxonomy ................................................................................................. 3
2.1.2 Structure ............................................................................................................................... 4
2.1.3 Clinical symptoms and treatment ........................................................................................ 6
2.1.4 Transmission routes and prevalence .................................................................................... 7
2.1.5 Pathogenesis......................................................................................................................... 9
2.1.6 Non-cultivability and surrogates ........................................................................................ 10
2.2 Interactions between bacteria and viruses ............................................................................... 12
2.2.1 Types of interactions .......................................................................................................... 12
2.2.2 Histo-blood group antigen expressing bacteria ................................................................. 14
2.2.3 Probiotics ............................................................................................................................ 15
Chapter 3: Material and Methods ......................................................................................................... 21
3.1 Interaction between Norovirus and HBGA expressing bacteria ................................................ 21
3.1.1 Bacteria ............................................................................................................................... 22
3.1.2 Virus-like particles (VLPs) and antibodies........................................................................... 22
3.1.3 Monitoring of the bacterial growth .................................................................................... 22
3.1.4 HBGA expression test ......................................................................................................... 23
3.1.5 Direct ELISA ......................................................................................................................... 24
3.1.6 Mucin-binding ELISA ........................................................................................................... 24
3.2 Interaction between Norovirus and probiotic bacteria............................................................. 25
3.2.1 Bacteria and cell lines ......................................................................................................... 25
3.2.2 Viruses ................................................................................................................................ 26
3.2.3 Monitoring of the bacterial growth .................................................................................... 26
3.2.4 Culturing of the macrophage cell line RAW 264.7.............................................................. 26
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3.2.5 Plaque assay ....................................................................................................................... 26
3.3 Statistical analysis ...................................................................................................................... 27
Chapter 4: Results ................................................................................................................................. 29
4.1 Interaction between Norovirus and HBGA expressing bacteria ................................................ 29
4.1.1 Optimisation of the HBGA expression test ......................................................................... 29
4.1.1.1 Concentration of antibodies ........................................................................................ 29
4.1.1.2 Concentration of blocking reagent .............................................................................. 30
4.1.1.3 Cell numbers of the tested bacteria ............................................................................ 30
4.1.2 Influence of gas atmosphere of bacterial growth on their HBGA expression and viral
protective effects .......................................................................................................................... 31
4.1.2.1 Growth of E. coli LMG 8223 at different atmospheres ............................................... 31
4.1.2.2 HBGA expression of E. coli LMG 8223 and LFMFP 861 grown at different atmospheres
................................................................................................................................................... 33
4.1.2.3 Viral protective effects of E. coli LMG 8223 and LFMFP 861 grown at different
atmospheres .............................................................................................................................. 33
A) Direct ELISA ..................................................................................................................... 33
B) Mucin-binding ELISA ........................................................................................................ 34
4.2 Interaction between Norovirus and probiotic bacteria............................................................. 35
4.2.1 B. longum growth ............................................................................................................... 35
4.2.1.1 Growth in bacteria culture medium TSB ..................................................................... 35
4.2.1.2 Growth in a food matrix .............................................................................................. 36
4.2.2 Effect of B. longum on NoVs ............................................................................................... 36
Chapter 5: Discussion ............................................................................................................................ 41
5.1 Interaction between Norovirus and HBGA expressing bacteria................................................ 41
5.2 Interaction between Norovirus and probiotic bacteria............................................................. 43
Chapter 6: General Conclusions ............................................................................................................ 47
References ............................................................................................................................................. 49
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List of Abbreviations
ABO
Blood group system
ATCC
American Type Culture Collection
BCCM
Belgian Coordinated Collection of Microorganisms
BCS
Bacterial cell suspension
BGMT
Bacterial growth medium filtrate
BSA
Bovine serum albumin
CFU
Colony forming units
DMEM
Dulbecco’s modified Eagle’s medium
DMSO
Dimethyl sulfoxide
ELISA
Enzyme-linked immunosorbent assay
EMEM
Eagle’s minimum essential medium
EPS
Extracellular polymeric substances
FAO
Food and Agricultural Organization
FBS
Fetal bovine serum
FCV
Feline calicivirus
GI/II
Genogroup I / II
HBGA
Histo-blood group antigen
HRP
Horseradish peroxidase
HSV
Herpes simplex virus
IFN
Interferon
IgG
Immunoglobulin G
IL
Interleukin
INSERM
Institut National de la Santé et de la Recherche Médicale
LAB
Lactic acid bacteria
LFMFP
Laboratory of Food Microbiology and Food Preservation
LMG
Laboratory of Microbiology at the University of Ghent
LPS
Lipopolysaccharide
MMTV
Mouse mammary tumour virus
MNV
Murine norovirus
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NoV
Norovirus
NS
Non-structural protein
NTPase
Nucleoside triphosphate hydrolase
NV
Norwalk virus
OD
Optical density
ORF
Open reading frame
PBS
Phosphate buffered saline
PCR
Polymerase chain reaction
PFU
Plaque forming units
PPS
Peptone physiological salt
PVR
Poliovirus receptor
RdRp
RNA-dependent RNA polymerase
RGD
Arginyl-glycyl-aspartic acid
RNA
Ribonucleic acid
RT-PCR
Reverse transcriptase – polymerase chain reaction
SRSV
Small round structured viruses
TMB
Tetramethylbenzidine
TSA
Tryptic soy agar
TSB
Tryptic soy broth
TV
Tulane virus
VLP
Virus-like particle
VP
Viral protein
WHO
World Health Organization
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Abstract
It is known that noroviruses (NoVs) show affinity for histo-blood group antigen (HBGA) receptors. The
first part of this study aimed to investigate whether the gaseous atmosphere wherein HBGA-expressing
E. coli grow influences the receptor expression as well as the protective role these bacteria have on
NoV GII.4 virus-like particles (VLPs) from acute heat stress. E. coli LMG 8223 and E. coli LFMFP 861 were
included in this experiment. HBGA expression was identified by a method based on the principle of
ELISA. The VLP antigen integrity after heat treatment (2 min at 90°C) was detected by a direct ELISA
test. The receptor binding ability of the VLPs, after suffering from the same heat stress, was evaluated
by a porcine gastric mucin-binding assay. For E. coli LMG 8223, anaerobic growth led to more HBGA
expression and aerobic growth resulted in the highest receptor binding ability. No difference between
aerobic and anaerobic growth was seen for the VLP antigen integrity. For E. coli LFMFP 861, aerobic
growth favoured HBGA expression. Anaerobic growth maintained the VLP antigen integrity the most
and no difference was detected for the receptor binding ability.
The impact of probiotic bacteria on viral infections has recently gained a lot of interest. In the second
part of this study, the effect of Bifidobacterium longum against NoVs was investigated. Murine
norovirus type 1 (MNV-1), used as a human NoV surrogate, was detected by means of cell plaque
assays with murine macrophage RAW 264.7 cells. The direct effect of the probiotics was found to be
limited. Within all the countable results, the number of viruses decreased the most when MNV-1 was
co-incubated with cell-free bacteria culture filtrate for 48 h at 37°C: a 2.4 log reduction in plaque
forming units per mL (PFU/mL). A reduction greater than 5 log PFU/mL was observed when the virus
was co-incubated with a high number of bacterial cells in a skim milk-sucrose matrix. However, it is
possible that this reduction is not only caused by the antiviral effect of B. longum, but the viral
inhibiting effect of certain milk components might have played a role as well. Results were also
obtained that support the idea that the presence of B. longum could inhibit the multiplication of MNV1 on RAW 264.7 cells.
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Samenvatting
Het is reeds geweten dat norovirussen (NoVs) affiniteit vertonen voor een binding met histo
bloedgroep antigen (HBGA) receptoren. Daarnaast zijn er ook al bacteriën geïdentificeerd die deze
HBGA’s produceren en op hun oppervlak tot expressie brengen. Het eerste deel van deze studie had
als doel te onderzoeken indien de HBGA-expressie van dergelijke bacteriën, alsook de beschermende
rol die de bacteriën uitoefenen tegenover NoV GII.4 virus-like particles (VLPs) tijdens acute hittestress,
beïnvloed wordt door de bacteriën ofwel aeroob ofwel anaeroob op te groeien. E. coli LMG 8223 en E.
coli LFMFP 861 werden gebruikt in deze test. De meting van de HBGA-expressie was gebaseerd op de
ELISA-techniek. De integriteit van VLP-antigenen na hittebehandeling (2 min bij 90°C) werd gedetecteerd met behulp van een directe ELISA test. De mate waarin VLPs nog in staat zijn om aan hun
receptor te binden na hittebehandeling werd geëvalueerd aan de hand van een zogenaamde ‘porcin
gastric mucin-binding’ assay. Bij E. coli LMG 8223 werd HBGA-expressie bevoordeeld bij anaerobe
groei, maar aerobe groei van de bacteriën leverde dan weer de hoogste receptorbinding op. Er werd
geen verschil waargenomen tussen aerobe of anaerobe groei inzake de integriteit van VLP-antigenen.
Bij E. coli LFMFP 861 resulteerde aerobe groei in een hogere HBGA-expressie. Anaeroob opgegroeide
bacteriën konden de integriteit van VLP-antigenen het best bewaren, maar qua binding aan de
receptoren werd geen verschil waargenomen.
Het potentiële nut van probiotica in de strijd tegen virale infecties geniet steeds meer interesse van
wetenschappers. In het tweede deel van deze studie werd het effect van Bifidobacterium longum
tegen NoVs onderzocht. Murine norovirus type 1 (MNV-1) werd gebruikt als surrogaat voor het
menselijke NoV. De detectie van het virus gebeurde via cell plaque assays met RAW 264.7
macrofaagcellen van de muis. Het directe effect van B. longum op het virus bleek eerder beperkt te
zijn. Uit alle telbare resultaten bleek dat het virusaantal het meest daalde wanneer MNV-1 48 uur bij
37 °C geïncubeerd werd samen met celvrij filtraat van een B. longum cultuur: een reductie van 2,4 log
plaque vormende eenheden per mL (PVE/mL). Een daling van meer dan 5 log PVE/mL werd bekomen
door het virus te incuberen samen met een hoog aantal bacteriecellen in een medium van magere
melk met sucrose. Het is echter mogelijk dat deze daling niet enkel veroorzaakt werd door de
probiotische bacteriën, maar ook door welbepaalde melkproteïnen die de virale infectie van cellen
kunnen voorkomen. Daarnaast werden ook resultaten bekomen die de idee steunen dat de
aanwezigheid van B. longum de vermeerdering van MNV-1 in RAW 264.7 cellen inhibeert.
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Chapter 1: Introduction
Norovirus (NoV) is the most important cause of foodborne acute gastroenteritis worldwide. Annually,
the estimated number of people that get infected with NoVs lies around 20 million in the USA and 15
million in Europe (CDC a, n.d.; WHO, 2015). The most common clinical symptoms include vomiting,
non-bloody diarrhoea, abdominal cramps, nausea, fever and dehydration. NoV infections are mildly
virulent, transient, and self-limiting within 2-3 days. However, sometimes hospital care is required or
death occurs in infants, elderly, chronically ill or immunocompromised patients. Up to now, no vaccine
or therapeutic treatment is available for this viral disease. The genetic heterogeneity among the
different NoV strains and the inability to cultivate human NoVs make it very difficult to perform direct
studies on this virus.
Within the complicated transmission routes, evidences have been found that interactions occur
between NoVs and bacteria in the environment. For instance, it was shown that bacteria which express
histo-blood group antigen (HBGA) like structures have affinity to NoVs (Hutson et al., 2002; Li et al.,
2015; Lindesmith et al., 2003). Other studies indicated that probiotic bacterial strains can have antiviral
activities against NoVs (Aboubakr et al., 2014; Rubio-del-Campo et al., 2014). The main goal of this
thesis was to look deeper into the above mentioned viral-bacterial interactions and discover the
importance of specific bacterial strains when it comes to either protecting or fighting against the NoV.
The first part of this thesis focused on the protective effect that might occur when human NoVs bind
to a selection of HBGA expressing bacterial strains. Different growth conditions of the bacteria were
tested for their HBGA expression. The protective effect of aerobically and anaerobically grown E. coli
against heat treatment was investigated by looking into the integrity and functionality of NoVs in
presence or absence of bacterial suspensions. Respectively a direct enzyme-linked immunosorbent
assay (ELISA) and a porcine gastric mucin-binding ELISA were used for this.
The second part of this thesis was devoted to the influence of probiotics on NoVs. Viruses and probiotic
bacteria were combined at different ratios and incubated at the agreeable conditions for bacterial
growth (culture medium, temperature, atmosphere, etc.) for different time intervals. A food matrix
was attempted to be introduced in the treatment system in order to investigate the possibility of
applying this treatment in food industries. First of all, the virucidal effect of probiotics on virus particles
was investigated. This refers to the direct effect of the probiotics. Secondly, the inhibiting effect of
probiotic bacteria on the viral infection and replication on a tissue culture of macrophage cells was
examined. In both cases, cell plaque assays were carried out.
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Chapter 2: Literature study
2.1 Introduction to Noroviruses
2.1.1 Classification and taxonomy
In 1968, an elementary school in Norwalk, Ohio (USA) suffered from an outbreak of ‘winter vomiting
disease’. At that time there was no consensus on what the exact agent was that caused the acute
gastroenteritis. It took four years before Kapikian et al. (1972) discovered, with the use of immune
electron microscopy (Figure 1) on infectious stool filtrates, that a virus was the wrongdoer of the
outbreak in Norwalk. The virus got named after the place where it was first discovered: ‘Norwalk Virus’
(NV). Later on also other viruses with a similar morphology were identified and grouped together as
‘small round structured viruses’ (SRSVs). In the 1990’s, further typing and classification of the SRSVs
was possible with the help of molecular techniques. Because of this breakthrough, SRSVs were shown
to be members of the Caliciviridae family.
Figure 1: Immune electron microscopy image of Norwalk Virus from an infected stool filtrate. (Kapikian et al.,
1972)
The Caliciviridae family is subdivided into four genera: Vesivirus, Lagovirus, Norovirus (formerly known
as Norwalk-like viruses) and Sapovirus (the former Sapporo-like viruses) (Baert et al., 2000). Molecular
technology, more specifically reverse transcriptase polymerase chain reaction (RT-PCR), has allowed
the cloning and sequencing of many NoV strains. Within the genus of NoVs, at least five genetic groups
are distinguishable based on the sequence similarities across highly conserved regions in the genome
(e.g. the RNA-dependent RNA polymerase [RdRp] and the shell domain of the VP1 capsid protein).
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Such genetic groups are also termed ‘genogroups’. Genogroups I and II (GI and GII) are the ones
containing the majority of NoVs that can cause disease in humans. Human NoVs within one genogroup
share at least 60% amino acid sequence identity of the major capsid protein VP1. A genogroup can still
be subdivided into so-called ‘genetic clusters’. For example, GII.4 resembles NoV strains from genetic
cluster 4 of genogroup II. NoV strains within a genetic cluster share at least 80% VP1 amino acid
sequence identity with the cluster’s reference strain (Hutson et al., 2004). So NoVs are genetically and
antigenically diverse. An overview of all the genogroups and genetic clusters is presented in Figure 2.
Although GII.4 is currently considered the most predominant NoV strain causing viral gastroenteritis
worldwide, recent data from China suggest that NoV GII.17 could be the next big upcoming genotype
(Lu et al., 2015).
Figure 2: Phylogenetic relationship of the five known genogroups (GI to GV) within the NoV genus based on
the complete capsid amino acid sequences of NoV strains. (Tan & Jiang, 2011)
2.1.2 Structure
NoVs are small icosahedral viruses, with a diameter between 27-30 nm. A positive-stranded RNA
genome of 7.7 kilobases is linked with a VPg protein at the 5’-end and polyadenylated at the 3’-end
(Baert et al., 2000). The genome is protected from the environment by a protein capsid without
envelope. The viral genome is organized into three open reading frames (ORF 1-3), but murine NoVs
have a fourth ORF. This extra ORF overlaps ORF 2 but has an alternate reading frame (Karst et al.,
2014). The first ORF, at the 5’ proximal end, encodes for a large polyprotein of seven non-structural
protein products. Cleaving of this polyprotein is done by a viral protease called NS6 or Pro, which is
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also one of the seven mature protein products. The other six non-structural products are: NS1, NS2
(p48), NS3 (NTPase), NS4 (p22), NS5 (VPg) and NS7 (RdRp). The functions of all these and other viral
proteins are given in Table 1. A visual representation of the NoV genome and the proteins it encodes
is shown in Figure 3.
Figure 3: The NoV genome. The 5’ proximal ORF 1 (shown in orange) encodes a non-structural polyprotein,
which is cleaved into mature products by the virally encoded protease (NS6 or Pro). Other non-structural
proteins include NS1/2 (also referred to as p48), NS3 (NTPase), NS4 (p22), NS5 (VPg), and NS7 (the RNAdependent RNA polymerase; RdRp). ORF 2 encodes the capsid protein referred to as VP1; this protein can be
divided into shell (S; shown in yellow) and protruding (P) domains. The P domain is further subdivided into the
P1 stalk domain (shown in blue) and the hypervariable P2 domain comprising the tips of the arches (shown in
red). ORF 3 encodes the minor structural protein VP2, and ORF 4 of murine NoV genomes encodes virulence
factor 1, or VF1. NoV genomes are covalently linked to VPg at their 5’ ends and polyadenylated at their 3’ ends.
(Karst et al., 2014)
Table 1: Names and functions of the proteins that are encoded by a NoV genome. (Karst, 2010)
Protein name
Function
NS1
Unknown
1
NS2 / p48
Inhibits trafficking of host proteins to the cell surface
Involved in replication complex formation?
Has NTPase activity
1
Inhibits secretion of host proteins
Involved in replication complex formation?
Primes viral RNA replication following its uridylylation
Recruits host translation initiation factors
Mediates cleavage of ORF1 polyprotein
1
Replicates the viral genome
Generates uridylylated VPg
Major structural role
1
Increases expression of VP1 and stabilizes particles
Recruits genomes into progeny virions?
3
NS3 / NTPase
NS4 / p22
NS5 / VPg
NS6 / Pro
NS7 / Pol
VP1
VP2
Encoded by ORF
1
1
1
2
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The capsid of NoVs is mainly composed of one major structural protein: viral protein 1 (VP1), which is
encoded by ORF 2. This VP1-protein consists of two parts: a well-conserved shell domain (S) and a
protruding domain (P). The P domain can be further subdivided into subdomains P1 and P2. Next to
VP1, the capsid also contains a few copies of a minor structural protein called VP2. ORF 3 is responsible
for encoding this small protein. VP1 and VP2 are translated from subgenomic RNA. With the help of
cryo-electron microscopy and x-ray crystallography, Prasad et al. (1999) were able to determine the
capsid structure of NoV virus-like particles (VLPs) as shown in Figure 4.
Figure 4: The NoV VLP structure. NoV VLPs have 90 dimers of capsid protein (left, ribbon diagram) assembled
in T = 3 icosahedral symmetries. Each monomeric capsid protein (right, ribbon diagram) is divided into an Nterminal arm region (green) facing the interior of the VLP, a shell domain (S-domain, yellow) that forms the
continuous surface of the VLP, and a protruding domain (P-domain) that emanates from the S-domain surface.
The P-domain is further divided into subdomains, P1 and P2 (red and blue, respectively) with the P2subdomain at the most distal surface of the VLPs. (Hutson et al., 2004)
2.1.3 Clinical symptoms and treatment
People from all ages are prone to NoV infections. Infections can occur in settings like day care centres,
schools, hospitals, hotels, recreational camps, and so on. NoV outbreaks are more likely to occur in
cooler winter months, although people can get infected at any time during the year. In addition, there
can be an increase of 50% in the number of NoV illnesses in years when there is a new strain of the
virus circulating (CDC a, n.d.). Moreover, the infectious dose of NoV is very low: it is estimated that 10100 viral particles may already be sufficient to infect an individual (ECDC, n.d.).
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The incubation period of a NoV infection can take 12 to 48 hours (CDC b, n.d.). The most common
clinical symptoms include vomiting, non-bloody diarrhoea, abdominal cramps, nausea, fever and
dehydration. NoV infections are generally mildly virulent, transient, and self-limiting within 2-3 days.
Although most of those infected fully recover, sometimes hospital care may be required or the
infection might lead to death in infants, elderly, chronically ill or immunocompromised patients (Baert
et al., 2000). There is no specific medicine or cure available to treat or prevent a human NoV infection.
An oral rehydration providing electrolyte replacement and sugar (glucose or sucrose) is commonly
used as the clinical treatment (Thistlethwaith, 2015). Sometimes patients can suffer from severe
dehydration and are unable to tolerate oral fluids. In these cases, intravenous replacement of
electrolytes is the method of choice. Since NoV infection has a viral origin, an antibiotic therapy will
not be effective. In 2011, Siddiq et al. suggested nitazoxanide, a broad-spectrum antimicrobial agent,
as a potential therapeutic treatment for NoV infections because it was successfully used to resolve
NoV gastroenteritis of an immunocompromised patient.
2.1.4 Transmission routes and prevalence
Worldwide, NoV is the leading cause of acute gastroenteritis (inflammation of the stomach or
intestines or both). The virus has been identified as the cause of between 73% to more than 95% of
global epidemic nonbacterial gastroenteritis outbreaks and approximately half of all gastroenteritis
outbreaks (Atmar & Estes, 2006). In the USA e.g. more than 20 million people per year get infected by
this virus, leading to 570-8000 deaths (CDC a, n.d.). WHO estimates that NoV is the most common
cause of foodborne illness in the European region with close to 15 million cases each year, causing
more than 400 deaths (WHO, 2015). An estimated 200,000 deaths occur annually among young
children in developing countries (Patel et al., 2008).
Apart from being highly contagious, constantly evolving, and evoking only limited immunity, another
attribute that makes NoVs an ideal infectious agent is the fact that multiple transmission routes exist.
Factors such as the high stability of the viruses in the environment and their high prevalence facilitate
the transmission (Hutson et al., 2004). Furthermore, it is also possible that an infected person can still
shed infectious virus particles after recovery for up to three weeks after exposure to NoVs (Rockx et
al., 2002).
NoVs are foodborne viruses and their primary transmission route is a person-to-person spread through
the faecal-oral route. They can also be transmitted through food, water, the environment,
contaminated surfaces or aerosols. A schematic overview of the most important NoV transmission
routes is presented in Figure 5.
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Figure 5: Main transmission routes of NoVs. (Leutenez, 2016)
The most efficient way of transmitting NoVs is via direct person-to-person contact or via aerosols that
are produced while vomiting. Food handlers that are infected with NoV play an important role in
spreading the virus, because they can easily transmit viruses to mainly ready-to-eat food (Bidawid et
al., 2000). Two other categories concerning the transmission of NoVs through food are fresh produce
and shellfish. Fresh produce poses a threat because it is mostly consumed without first undergoing a
treatment that could effectively destroy the virus (Ethelberg et al., 2010). Bivalve molluscan shellfish,
such as mussels or oysters, are filter-feeders. They take up surrounding water and filter out nutritive
components. However, when human pathogens are present in the water, they can also be filtered out
and accumulate in the shellfish. Contaminated water is not only an infection source for shellfish
(Westrell et al., 2010), but it can also directly infect humans if it is used as drinking water or recreation
water (Hoebe et al., 2004).
As was noted by FAO and WHO (2008), large-scale outbreaks are often a result of a combination of
several transmission routes. For example, the virus can be introduced in a sensitive population by
contaminated food, water or an asymptomatic shedder. Direct person-to-person contact or a
contaminated environment may lead to an efficient spread of the virus through the population.
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2.1.5 Pathogenesis
The possibility of a NoV infection is not only dependent on the viral dose and strain one is exposed to.
Research has shown that the specific human susceptibility plays an important role as well. In 2002,
Hutson et al. were the first to report that the risk of a Norwalk virus infection and disease was
associated with someone’s ABO blood type. In following research, volunteer studies (Lindesmith et al.,
2003) and outbreak investigations (Rockx et al., 2005) delivered increasing evidence of HBGAs being
receptors of NoV infection. HBGAs are complex carbohydrates present on the surfaces of red blood
cells and mucosal epithelium of the respiratory, digestive and urogenital tracts. They can also be found
as free oligosaccharides in biologic fluids such as saliva, intestinal contents, milk and blood (Tan &
Jiang, 2005). As reviewed by Hutson et al. (2004), these carbohydrates are synthetically related triand tetrasaccharide moieties that are located at the distal ends of carbohydrate chains on cellular
glycolipids and glycoproteins found on the exterior cell surface. The variety of expressed carbohydrates
is determined by the presence or absence of specific glycotransferase enzymes as a result of a person’s
genetics. The human HBGA system is controlled by multiple gene families, that contain silent alleles
(Marionneau et al., 2001).
The recognition of human HBGAs by NoVs occurs through a typical protein-carbohydrate interaction,
in which the protruding domain of the viral capsid protein forms an interface with the oligosaccharide
side-chains of the antigens. The P2 subdomain forms the most exterior surface of the capsid (Figure
4). A plausible binding pocket in the P2 subdomain was identified by sequence homology modelling
based on the receptor binding patterns of different NoV strains (Tan et al., 2003). The pocket consists
of a RGD-like motif and three scattered, binding pattern-specific amino acids. Nearby this pocket,
another binding site that is composed of three amino acid residues on the NoV capsid was identified
(Chakravarty et al., 2005). Thus, the two binding sites could belong to one binding interface and act
cooperatively with respective sugar side-chains of the HBGAs. Figure 6 displays a close-up starting from
a NoV particle up to the HBGA binding site.
Although a lot of efforts have already been made to investigate the human NoV-HBGA interaction and
since the human NoVs are still non-cultivable, the complete picture of virus-host interaction could be
more complex.
9
Figure 6: The four panels show structures of NoVs at different levels: (from left to right) an electron microscopy
image of NoVs, a single VLP, a P dimer with indication of the carbohydrate-binding interface (coloured region),
and the crystal structure of the HBGA-binding interface. (Tan & Jiang, 2010)
2.1.6 Non-cultivability and surrogates
Numerous efforts have been made to establish an in vitro cultivation technique of human NoVs. In
2004, Duizer and his colleagues tried to grow NoV on multiple routinely cultured cell lines (Caco-2,
HeLa, and many more). Even with the addition of bioactive digestive enzymes, substances to induce
cell differentiation (insulin, DMSO or butyric acid) or using different inoculation methods, successful
virus propagation could not be achieved. Three years later, Straub et al. (2007) were the first to report
that NoVs can infect and replicate in a 3-dimensional (3-D), organoid model of human small intestinal
epithelium. The cells were grown on porous collagen-I coated microcarrier beads under conditions of
physiological fluid shear in rotating wall vessel bioreactors. The NoV infection was proven using
microscopy, PCR and fluorescent in situ hybridisation. However, no follow-up experiments were found
until the same research group studied human NoV infectivity using a 3-D model of large intestinal
epithelium (Straub et al., 2011). Culturing human NoVs was made possible to a certain extent using
this system, although other laboratories have not yet been able to verify and repeat the findings of
Straub.
In an attempt to develop an in vitro infection model of human NoVs, Jones et al. (2014) identified B
cells as a cellular target of the virus. A BJAB human B cell line was inoculated with the Sydney human
NoV GII.4-positive stool sample. The viral genome copy numbers increased 10 to 25 times at
respectively three and five days after infection. Notably, the replication in B cells required the presence
of HBGA-expressing enteric bacteria. Nowadays, a lot of attention is paid on this topic and further
research is being done to confirm the model of Jones.
The study of human NoVs faces some difficulties due to the inability to distinguish infectious human
NoVs from the non-infectious ones, and the unavailability of large amounts of infectious viruses. To
counteract these problems, surrogates are used to study the stability and inactivation of human NoVs.
10
The surrogates should share pathological and/or biological features with the human NoVs and they
should be able to propagate in cell culture. Much of the current knowledge on NoV biology was
acquired by studies with surrogates. As an example, studies with murine NoVs have led to the discovery
of the important role of the STAT-1 molecule in the resistance of a NoV disease in vivo and the control
of virus growth in vitro (Mumphrey et al., 2007).
Feline calicivirus (FCV) shows similarities with human NoVs in primary sequence and genomic
organisation. In addition, the virus grows well in vitro. These characteristics made FCV a frequently
used surrogate virus in previous studies (e.g. Aboubakr et al., 2014; Lee et al., 2012; Sosnovtsev et al.,
2005). The main disadvantages of this cat virus are: it does not cause gastro-intestinal diseases but
respiratory ones, and it does not bind to HBGAs but to sialic acid (reviewed by Vashist et al., 2011).
Murine norovirus type 1 (MNV-1) was firstly identified in 2003 by Karst et al. Reasons why MNV-1
nowadays is often the model of choice for human NoV research include its close relatedness to human
NoVs, the natural host is relatively cost-effective and genetically well-characterised, and it is the only
NoV that can be efficiently propagated in vitro because it has a tropism for dendritic cells and
macrophages (Vashist et al., 2011). This last feature allows the propagation of MNV-1 in RAW 264.7
murine macrophage cells (Wobus et al., 2004). MNV-1 and human NoV not only share a similar size
and shape, but also the three big ORFs are characteristic (Wobus et al., 2006). However, the use of
MNV-1 as a surrogate for human NoVs has some limitations. First of all, MNV-1 uses sialic acid as a
receptor for attachment to specific cell lines instead of HBGAs. Secondly, MNV-1 does not cause
gastroenteritis in its host, but some of the occurring symptoms include encephalitis, meningitis or
vasculitis.
Another attractive surrogate model could be a virus that infects a non-human primate. An example in
this category is the Tulane virus, a calicivirus discovered in rhesus monkeys (Farkas et al., 2008).
Although this virus can recognize HBGAs as receptors, it does not belong to the NoV genus and it does
not cause gastroenteritis.
The use of bacteriophages as surrogates can be considered because of their non-pathogenic properties
and easy detection and cultivation (Li, 2012). Coliphage φX174 is an examples of a bacteriophage
surrogate for human NoV that has already been used for experimental means (Li et al., 2011).
The use of recombinant VLPs has been a valuable method to investigate the virus-host interaction. The
discovery of NoVs being able to bind to HBGAs, as described earlier, was achieved by performing
experiments with human NoV VLPs (e.g. Harrington et al., 2002 & 2004). VLPs are generated by the
expression of the viral major capsid protein (VP1) with or without the minor capsid protein (VP2). The
produced proteins then self-assemble to form VLPs, having the same morphological characteristics of
11
the original human NoV virion (Vashist et al., 2011). Expressing the viral capsid proteins in insect cells
using baculovirus expression systems is a frequently used strategy (Belliot et al., 2001; Caddy et al.,
2014; Li et al., 2015). In some cases, only the P domain of the NoV capsid is considered for experimental
use. The domain forms a 24-mer subviral particle, called P particle. P particles can easily be produced
in E. coli, are very stable and also highly immunogenic, making them an interesting platform for vaccine
development (Tan & Jiang, 2012).
2.2 Interactions between bacteria and viruses
2.2.1 Types of interactions
There is an estimation of 1031 viruses on earth (Breitbart & Rohwer, 2005). Scientists have estimated
the total number of bacteria on earth to be 5 x 1030 (University of Georgia, 1988). The human body is
colonized by an immense population of microorganisms. A widely cited statement says that bacteria
outnumber the amount of human cells by a ratio 10:1 in humans. However, a recent study rejects this
theory, saying that a “reference man” has approximately the same number of bacteria and human cells
in his body (Sender et al., 2016). Considering that the mean weight of a cell equals one nanogram, a
person of 70 kg would consequently harbour about 70 trillion bacteria. As humans also often get
infected by viruses, it would not be a surprise if an interaction would occur between viruses and
bacteria inside the body. Since this thesis mainly focuses on NoVs, this section will focus on interactions
between enteric bacteria and NoVs.
A first category of mechanisms involves the direct facilitation of viral infection, including bacterial
stabilization of viral particles and the facilitation of viral attachment to host target cells (Karst, 2016).
Binding of enteric viruses to bacterial surface polysaccharides enhances virion stability towards a heat
treatment or inactivation by chlorine bleach (Robinson et al., 2014). The same research group also
discovered that bacterial polysaccharides enhance poliovirus cell attachment by increasing binding to
the viral receptor PVR. An example of increased cell invasion is the stimulation of B cell infection by
NoVs in presence of HBGA expressing bacteria (Jones et al., 2014). A graphical presentation of these
examples is given in Figure 7.
12
Figure 7: (A) The binding of poliovirus to lipopolysaccharide (LPS) leads to an increase in viral thermostability
and resistance to inactivation by dilute chlorine bleach. (B) Poliovirus associated with LPS binds to the
poliovirus receptor (PVR) more efficiently on the surface of target cells. Several lines of evidence show that
LPS enhancement is conferred by facilitating viral binding to the known PVR: pre-treatment of permissive cells
with PVR-specific antibody inhibits viral binding in both the presence and absence of LPS; virus bound to LPS
does not gain competency to infect non-PVR-expressing cells; and virus incubated with LPS has increased
binding to soluble PVR compared with virus alone. (C) Human NoV infection of B cells is facilitated by
commensal bacteria that express the appropriate HBGA. The first indication that commensal bacteria
stimulate human NoV infection of B cells was provided by the observation that the filtration of virus-positive
stool to remove commensal bacteria reduced viral infectivity. Providing direct evidence, the supplementation
of filtered stool with HBGA-expressing bacteria fully restored infectivity. (Karst, 2016)
13
A second category concerns mechanisms that indirectly influence the antiviral immune response in a
way that promotes viral infection (Karst, 2016). The strategy mouse mammary tumour virus (MMTV)
uses to evade the host’s immune responses has been described by Kane et al. (2011). The binding of
MMTV to bacterial lipopolysaccharide triggered Toll-like receptor 4 on macrophages or dendritic cells.
These cells subsequently started producing interleukin-6 (IL-6), leading to the induction of the
inhibitory cytokine IL-10 by B cells. An immunosuppressive microenvironment was created by this
means. As a consequence, MMTV could persist and a tolerance for their antigens was established. In
2014, Kernbauer, Ding, & Cadwell found that commensal bacteria possess the ability to suppress the
type III interferon (IFN) response by diminishing the production of antiviral IFNλ. If IFNλ is produced,
which is the case in microorganism-depleted mice, it will activate the type III interferon receptor
(IFNλR) in enterocytes or in other bystander cells to indirectly inhibit norovirus persistence. So
suppressing IFNλ production facilitates MNV persistence. In mice lacking functional type III IFN
signaling pathways, interactions with commensal bacteria are not necessary to gain persistence
because the bystander cells are impaired in their ability to respond to IFNλ (Karst, 2016).
2.2.2 Histo-blood group antigen expressing bacteria
HBGAs play an important role in multiple viral infections. It has been mentioned before that human
NoVs contain binding pockets in their P2 domain, through which they are able to bind carbohydrate
structures on the surfaces of red blood cells and mucosal epithelium of the respiratory, digestive and
urogenital tracts (Tan et al., 2003). These receptors were identified as HBGAs. NoVs are not the only
viruses that can bind HBGAs. In 2012, Huang et al. discovered that human rotaviruses, the most
important cause of severe gastroenteritis in children, also recognize HBGAs. The spike protein VP8* of
the virus is responsible for this interaction.
A number of gram-negative bacteria show blood group expression as well (Springer et al., 1961). The
discovery that human NoVs are capable of binding to these HBGA expressing bacteria has only recently
been confirmed. Transmission electron microscopy demonstrated that VLPs of human NoVs mainly
bound to extracellular polymeric substances (EPS) of an enteric bacterium strain (SENG-6), closely
related to Enterobacter cloacae (Miura et al., 2013). This is also the location bearing HBGA-like
molecules. NoV VLPs of a GI.1 wild-type strain and a GII.6 strain that can recognize HBGA type A were
shown by ELISA to bind to the EPS extracted from Enterobacter sp. SENG-6.
The study of Jones et al. (2014) identified B cells as a cellular target of both murine and human NoVs.
The presence of enteric HBGA expressing Enterobacter cloacae bacteria was essential to facilitate viral
attachment to, and infection of, B cells. M12 and WEHI-231 mouse B cell lines were infected with MNV14
1 or MNV-3 to check whether MNVs infect B cells in culture. Both virus strains replicated efficiently in
B cells. In vivo experiments confirmed that B cells are indeed targets of the NoVs: significant reductions
in the number of viruses were found in B cell-deficient mice compared to normal mice. Antibiotic
depletion of normal intestinal flora in mice led to a significant drop in MNV titers, demonstrating that
enteric bacteria play an important role during the process of NoV infection.
For human NoVs, a BJAB human B cell line was inoculated with the Sydney human NoV GII.4-positive
stool sample. The viral genome copy number increased 10 to 25 times at respectively three and five
days after infection. Filtering the infected stool sample over a 0.2 µm membrane reduced genome
replication. Adding Enterobacter cloacae to the filtered stool before inoculation of BJAB B cells
restored infectivity of the virus in a dose-dependent way. Enterobacter cloacae is an HBGA type H
expressing bacterial strain to which the GII.4 Sydney human NoV strain can bind. The presence of
synthetic H antigen together with the filtered stool was sufficient to restore the attachment of the
virus to B cells.
In 2015, a study was done by the Laboratory of Food Microbiology and Food Preservation (LFMFP) at
Ghent University on the effect of HBGA expressing bacteria on human NoV (Li et al., 2015). Both E. coli
strains LMG 8223 and LFMFP 861 were found to express HBGAs and bind to human NoV VLP GI.1 and
GII.4. E. coli ATCC 8739 served as a negative control, since it shows no HBGA expression and no VLP
binding. VLPs pre-incubated with HBGA expressing or non-HBGA expressing bacteria were heated at
90°C for 2 min. Afterwards, the antigenicity of the VLPs was measured by a direct ELISA test. A porcine
gastric mucin-binding ELISA test examined the receptor binding ability of VLPs after the heat
treatment. As a result, the presence of HBGA expressing E. coli always secured higher antigen integrity
as well as mucin-binding ability of the VLPs after exposure to heat stress, indicating a protective effect
of HBGA expressing E. coli on human NoVs from acute heat treatment.
2.2.3 Probiotics
The consumption of antibiotics to kill harmful bacterial infections is huge: from 2000 to 2010, the
global antibiotic consumption grew from approximately 30 billion to 50 billion standard units, based
on data gathered from 71 countries (Van Boeckel et al., 2014). Having the wrong bacteria in the wrong
place can cause health problems. However, the right bacteria in the right place can lead to benefits.
This is the part where probiotics have their importance.
Probiotics are defined as “living micro-organisms, which upon ingestion in certain numbers, exert
health benefits beyond inherent basic nutrition” (Gionchetti et al., 2012). One of the largest groups of
probiotic organisms are the lactic acid bacteria (LAB), containing e.g. Lactobacillus sp, Bifidobacterium
15
sp and Enterococcus sp (Ljungh & Wadstrom, 2006). The presence of living bacteria inside the human
body is not abnormal. There are up to 1013 - 1014 bacteria found in the gastrointestinal tract, divided
over more than 400 bacteria species (FAO & WHO, 2001). In order for those bacteria to be probiotic,
they should be able to survive both the stomach and bile acids, sufficient quantities must arrive in the
intestines and they must show some beneficial effects on human health.
A few examples of the beneficial health effects probiotics promote, include alleviation of lactose
intolerance, prevention and treatment of diarrhoea, maintenance of normal intestinal flora,
antagonism against pathogens, stimulation of the immune system, anticarcinogenic activity, and
reduction of serum cholesterol levels (BC Dairy, 2016). Effects on the human or animal host may be
caused directly by preventing the infection and fighting against the causative agent of an intestinal
disorder, or indirectly by balancing the disrupted equilibrium of the enteric flora and raising the host’s
immune response (Maragkoudakis et al., 2010). Probiotics are not only found in the human body.
Other sources are certain food products (e.g. fermented dairy products or soybean foods) or dietary
supplements. Figure 8 gives an overview of the main mechanisms of action of probiotics.
Figure 8: Mechanisms of action of probiotics. The probiotics are coloured in yellow. IEC: epithelial cells, DC:
dendritic cells, T: T cells, Tn: naïve T cells, Th: helper T cells, Treg: regulatory T cells, B: B cells, IL: interleukin,
TGF: transforming growth factor. (Custom Probiotics Inc., n.d.)
16
LAB strains generally produce antimicrobial substances with a narrow or broad spectrum against
homologous gram-positive bacteria. They additionally often produce microbicidal substances with dual
functions: showing an effect against gastrointestinal pathogens and other microbes, or competing for
cell surface and mucin binding sites (Ljungh & Wadstrom, 2006). The LAB metabolites causing
antimicrobial activity include organic acids (lactic and acetic acid), hydrogen peroxide, ethanol,
diacetyl, acetaldehyde, carbon dioxide, reuterin, and bacteriocins (Suskovic et al., 2010).
A few studies demonstrated the possibility of probiotics or the substances they naturally produce to
not only possess antimicrobial characteristics, but also enhance the antiviral effect on viruses. For
instance, by blocking binding sites on epithelial cells, some Bifidobacterium species can inhibit the
growth of viral pathogens (Colbère-Garapin et al., 2007). Bacteriocins could help in reducing the viral
coat protein synthesis (Wachsman et al., 1999), decreasing the infectious virus yield (Serkedjieva et al.,
2000), or blocking the host cell receptor required for viral attachment (Berkhout et al., 1997).
The use of probiotics as a possible method to control and treat viral infections is an interesting topic
for investigation. In the case of human NoVs for example, several key challenges remain in assessing
the efficacy of vaccines and antiviral drugs. There is for instance no robust cell culture system, limiting
the direct study of these viruses. Also the genetic heterogeneity among NoV strains makes it difficult
to find a suitable vaccine. If probiotics have antiviral activity against NoV, this will offer a promising
alternative to finding a cure for NoV infections.
A lot of researches have already been done to examine the influence of probiotics on viruses. The
studies listed in Table 2 aimed at examining possible antiviral activities of probiotic LAB on viruses.
Frequently used techniques to detect the viruses included cell plaque assays, RT-qPCR and ELISA. In
most experiments, the presence of LAB probiotics reduced the infectivity of viruses. However, this
reduction was very variable. For instance, a 6 to 7 log infectivity reduction was measured when FCV
was co-incubated with either bacterial growth medium filtrate or bacterial cells of the Lactococcus
lactis strain (Aboubakr et al., 2014). On the other hand, no direct effect was found on MNV-1 and
Tulane virus (TV) when they were co-incubated with cell-free supernatant of a commercial probiotic
mixture (Shearer et al., 2014). It is difficult to draw general conclusions. A lot of different LAB strains
and viruses were used in the listed experiments, and there is still a lack of enough thorough studies on
this topic.
17
Table 2: Antiviral studies of probiotics.
Bacteria
Virus
Cell line
Effect
Reference
Lactobacillus delbrueckii
subsp. bulgaricus 1043
Lactobacillus paracasei/
rhamnosus Q85, Lb. paracasei A14 and F19, Bifidobacterium longum Q46
Lb. paracasei, Lactobacillus
reuteri SD2112, Lb. rhamnosus GG, Lb. paracasei NCC
2461, Lactobacillus johnsonii
NCC 533, Streptococcus
thermophilus NCC 2496
Lb. rhamnosus GG, Lb. casei
Shirota
Influenza virus A
chick embryo fibroblast cells
Serkedjieva et al. (2000)
Vesicular stomatitis
virus
pig alveolar macrophage cells
Rhesus rotavirus
MA104 cells (mouse)
No inactivation effect on extracellular virus
Viral reproduction considerably reduced
Lactobacilli and bifidobacteria decrease viral
infection by establishing the antiviral state in
macrophages, by production of NO and
inflammatory cytokines
Lactobacillus rhamnosus strain GG had the
strongest influence in reducing prevalence,
duration and severity of diarrhoea
Rotavirus,
Transmissible
gastroenteritis virus
Human
immunodeficiency
virus type 1
CLAB porcine epithelial cells
Maragkoudakis et al. (2010)
Herpes simplex
virus (HSV) type 1
Vero African green monkey
kidney cells and murine
monocyte/macrophage cell
line J774 cells
RAW 264.7 and CrandellReese feline kidney cells
Co-incubation of the CLAB cell line with the
bacteria resulted in increased survival percentages,
up to 80%
Greatest virus inhibition with Lactobacillus
curvatus VM25 (55.5%), Lactobacillus fermentum
VMA (52.5%), and Pediococcus pentosaceous
VM95 (49.0%)
Lb. rhamnosus significantly increased cell viability
(47.6 – 49 %) of the HSV-1 infected Vero cells and
it reduced plaque forming (15 – 18 %). Similar
effect for E. coli.
At the end of the fermentation (20 days), the
population of FCV and MNV decreased about 4.12
and 1.47 log units, respectively
Pre-treatment of FCV with BGMF or BCS:
1 to 2 log reduction
Co-treatment of FCV with BGMF or BCS: 6 to 7 log
reduction
The intracellular level of Coxsackievirus B3 RNA
was inhibited by 50%
Kim et al. (2014)
38 bacterial strains (15
different species), isolated
from breastmilk of healthy
women
Lb. rhamnosus PTCC 1637,
Escherichia coli PTCC 25923
TZM-bI cells
LAB species in general after
Dongchimi fermentation
Murine norovirus,
Feline calicivirus
Lactococcus lactis subsp.
lactis LM0230 (bacterial
growth medium filtrate
(BGMT) or bacterial cell
suspension (BCS))
Bifidobacterium
adolescentis SPM 1605
Feline calicivirus
Crandell-Reese feline kidney
cells
Coxsackievirus B3
HeLa cells
Ivec et al. (2007)
Pant et al. (2007)
Martín et al. (2010)
Khani et al. (2012)
Lee et al. (2012)
Aboubakr et al. (2014)
18
E. coli Nissle 1917,
Lactobacillus casei BL23
Norovirus P
particles
HT-29 cells
Commercial mixture of
Lactobacillus acidophilus,
Lb. rhamnosus, Bifidobacterium bifidum, Lactobacillus
salivarius, and S. thermophilus
LAB species in general after
oyster fermentation
Murine norovirus 1, Tulane virus
RAW 264.7 and LLCMK2 cells
Murine norovirus,
Feline calicivirus
RAW 264.7 and CrandellReese feline kidney cells
Lactobacillus bulgaricus
761N
Hepatitis C virus
Human liver hepatocellular
carcinoma (HepG2) cells,
Lactobacillus ruminis SPM
0211, B. longum SPM 1205
and SPM 1206
Human rotavirus
Caco-2 and Vero cells,
In vivo in mice
E. coli Nissle, Lb. rhamnosus
GG
Human rotavirus
In vivo in neonatal gnotobiotic
piglets
B. adolescentis
Murine norovirus 1, Human norovirus
GI.1 VLP
RAW 264.7, Caco-2 and HT-29
cells
Total inhibition when co-incubating E. coli with the
virus particles and the cells. The effect was limited
with Lb. casei
A lack of direct effect of cell free supernatant on
the virions
Rubio-del-Campo et al.
(2014)
Fermentation with 5 % NaCl for 15 days: MNV and
FCV decreased with 1.60 and 3.01 log units, the
number of LAB increased from 3.62 to 8.77 log
CFU/g
Pre-treatment (adding bacteria to the cells before
incubation with HCV): 70.9 % reduction in viral
load
Post-treatment (adding bacteria after incubation):
88.7 % reduction
Similar results for Caco-2 and Vero cells: Lb.
ruminis SPM 0211 and B. longum SPM 1206
decreased plaque formation up to 45 %
(dependent on the bacterial dose).
In vivo: Lb. ruminis SPM0211 caused a decrease of
56 % in viral gene expression, B. longum SPM 1205
and SPM 1206 respectively 39 % and 47 %.
Mean peak virus shedding titers were significantly
lower in the E. coli- colonized compared with Lb.
rhamnosus-colonized or uncolonized piglets.
Presence of B. adolescentis inhibits multiplication
of MNV-1 on RAW 264.7 cells within 48 h of coincubation and it also decreased the binding of
human NoV GI.1 VLPs to both Caco-2 and HT-29
cells.
Seo et al. (2014)
Shearer et al. (2014)
El-Adawi et al. (2015)
Kang et al. (2015)
Kandasamy et al. (2016)
Li et al. (unpublished)
19
20
Chapter 3: Material and Methods
3.1 Interaction between Norovirus and HBGA expressing
bacteria
The objective of this study is to investigate whether the gaseous atmosphere, in which HBGA
expressing bacteria grow, could affect the protective effect of these bacteria on NoVs from heat stress.
Due to a limitation of the facility availability, flow cytometry was not used like previously (Li et al.,
2015). Alternatively, an ELISA based method was firstly established to measure HBGA expression of
the bacteria. Optimisation was performed to determine the most favourable antibody concentration,
blocking reagent concentration and starting bacterial cell numbers.
The investigation of the influence of the gaseous atmosphere started by following the growth of HBGA
expressing E. coli aerobically and anaerobically. Subsequently, the effect of bacterial growth
atmosphere on the viral protection capability was explored by three different tests. The pre-optimised
HBGA expression test was employed to compare the HBGA expressing levels produced by the bacteria.
The direct ELISA method was employed to measure the antigenicity of NoVs after they suffered a heat
treatment in the presence of (an)aerobically grown HBGA expressing bacteria. The mucin-binding
ELISA test was employed to study the receptor binding ability of the viruses after heat stress in the
presence of (an)aerobically grown HBGA expressing bacteria. These three tests were performed with
the use of two different HBGA expressing E. coli strains, in order to observe the consistency between
strains.
Figure 9 displays a flowchart of the procedures described above.
Optimisation
HBGA measuring test
• Concentration
antibodies
• Concentration
blocking reagent
• Bacterial cell
numbers
E. coli growth:
aerobic vs
anaerobic
atmosphere
E. coli LMG
8223: aerobic
vs anaerobic
growth
E. coli LFMFP
861: aerobic
vs anaerobic
growth
• HBGA expression
• Direct ELISA
• Mucin-binding
ELISA
• HBGA expression
• Direct ELISA
• Mucin-binding
ELISA
Figure 9: Flowchart showing the different steps that were performed to study the protective effect of HBGA
expressing bacteria on NoVs when the bacteria were grown in different gaseous conditions.
21
3.1.1 Bacteria
An overview of the bacterial strains that were used to study the interactions of NoVs and HBGA
expressing bacteria, is presented in Table 3. Escherichia coli LMG 8223 was selected because it was
found by Li et al. (2015) to express type A HBGAs and it is also capable to bind to human NoV VLP GII.4.
This strain was obtained from the Belgian Coordinated Collection of Microorganisms (BCCM/LMG). E.
coli LFMFP 861 expresses type B HBGAs and can also bind human NoV VLP GII.4. This strain was isolated
at the Laboratory of Food Microbiology and Food Preservation (LFMFP), Ghent University. E. coli ATCC
8793 was obtained from the American Type Culture Collection (ATCC). This strain was used as a
negative control because it does not express HBGAs. All bacterial strains were cultured in tryptic soy
broth (TSB, Oxoid, Thermo; Hampshire, UK) at 37°C.
Table 3: Characteristics of the E. coli strains that were used for the experiments with HBGAs. The percentage
of positively stained cells determined by flow cytometry analysis were grouped as in the following. < 1%: - ;
1-5%: + ; 5-10%: ++ ; > 10%: +++. (Dan Li et al., 2015)
Bacteria
Strain
Biological origin
Escherichia
coli
E. coli
E. coli
LMG 8223
Clinical isolate
ATCC 8739
LFMFP 861
Feces
Thick whey
products
HBGA expression
Type A
Type B
+++
++
-
+++
VLP GII.4 binding
+++
+++
3.1.2 Virus-like particles (VLPs) and antibodies
VLPs were used as an alternative for human NoV, due to the non-cultivability of the latter. VLPs of
human NoV GII.4, based on the NoV isolated in Dijon in 1996, were obtained from the Institut National
de la Santé et de la Recherche Médicale (INSERM, Paris, France). Anti-HBGA antibodies (anti-A #21 and
anti-B #49) and anti-VLP antibodies (lp132) were also supplied by INSERM. Horseradish peroxidase
(HRP) congjugated anti-mouse IgG and HRP-conjugated anti-rabbit IgG antibodies were purchased
from Promega (Madison, WI, USA).
3.1.3 Monitoring of the bacterial growth
Bacteria culture was made by dilution in peptone physiological salt solution (PPS; 1 g of neutralised
bacteriological peptone [Oxoid, Basingstoke, UK] and 8.5 g of NaCl [Sigma Aldrich, St. Louis, MO, USA]
per liter) and inoculation into fresh TSB (30 g of tryptic soy broth powder [Oxoid] per liter) from an
estimated concentration of 105 to 106 CFU/ml. Aerobic culture was made by incubation at 37°C
22
overnight while shaking at a speed of 200 rpm (IKA®-Werke GmbH & Co. KG, Staufen, Germany).
Anaerobic culture was made by incubation at 37°C overnight with the use of ANAEROGENTM COMPACT
(Oxoid, Thermo).
Before each experiment, a first passage was made aerobically as described above from the defrozen
culture to activate the bacteria. The defrozen culture was stored at 4°C for maximally one month.
Experimentally applied bacteria were always prepared from the activated first passage either
aerobically or anaerobically as described above (the second passage).
In order to follow the bacterial growth, five tubes were prepared from the first passage in parallel and
incubated under the conditions defined above for 0, 3, 6, 12 and 24h respectively. For the bacteria
from each tube, the optical density at 600 nm (OD600) and viable bacteria numbers were measured.
The optical density was measured by using a VersaMax ELISA Microplate Reader (Molecular Devices,
Sunnyvale, CA, USA). In order to obtain viable bacteria numbers, a tenfold dilution series in PPS was
made for each tube, and 100 µL of each dilution was plated onto tryptic soy agar (TSA; 40 g of tryptic
soy agar powder [Oxoid] per liter). After overnight incubation at 37°C, the colonies that were formed
on the spread plates were counted. The following formula was used to calculate the number of
bacteria in the original second passage tubes:
X=
A∗V
I
X equals the number of CFUs per mL in the second passage tube, A represents the number of counted
colonies, V is the dilution factor and I the inoculation volume in mL.
3.1.4 HBGA expression test
Bacterial cells (concentration to be determined by preliminary test) were collected by centrifuging for
5 min at 6,000 rpm (Eppendorf Micro Centrifuge 5415C). The pellet of each sample was washed with
100 µL phosphate buffered saline (PBS, pH 7.4 [Lonza, Verviers, Belgium]) by centrifuging, resuspended with 100 µL of bovine serum albumin (BSA; Sigma Aldrich; concentration to be determined
by preliminary test) in PBS and incubated at 37°C for 1 h to block the nonspecific binding.
After centrifugation, 100 µL of mouse anti-HBGA monoclonal antibodies (anti-A #21 for E. coli LMG
8223, anti-B #49 for E. coli LFMFP 861) that were diluted 250 times in 0.1% BSA-PBS were added to the
pellet. The mixtures of bacteria and antibodies got vortexed and then incubated at 37°C for 1 h.
Subsequently, two washing steps by centrifugation with PBS were performed. One hundred µL
secondary antibodies, anti-mouse antibodies conjugated with the enzyme HRP at a 1:10,000 dilution
23
in 0.1% BSA-PBS, were added and incubated at 37°C for 1 h. Two washing steps by centrifugation were
performed with PBS. Per sample, 100 µL 3,3′,5,5′-Tetramethylbenzidine (TMB) One Solution (Promega,
Madison, WI, USA) was added as the substrate for HRP. After adding 50 µL 0.10 N phosphoric acid
(H3PO4), the optical density was measured at 450 nm by a VersaMax ELISA Microplate Reader.
3.1.5 Direct ELISA
E. coli cells were washed with PBS and mixed with NoV GII.4 VLPs (100 µL per sample, 1:100 diluted in
PBS to get 5 µg/mL). These mixtures of VLPs and bacteria in 0.5 mL thin-walled PCR tubes were heated
at 90°C for 2 min in a PCR cycler (Arktik™ Thermal Cycler, Thermo Fisher Scientific; Waltham, MA, USA)
and cooled down immediately on ice. The treated VLP-bacteria mixtures were diluted at 1:10 in a
carbonate buffer (CBS, 1.50 g Na2CO3 [Sigma Aldrich] and 1.92 g NaHCO3 [Merck KGaA, Darmstadt,
Germany] per liter, pH 9.6) and then coated on a Nunc Maxisorp immunoplate (100 µL per well; Sigma
Aldrich) overnight at 4°C. Three non-coated wells were included as blank controls.
The plate was washed three times with PBS-T (0.05% Tween 20 [polyoxyethylenesorbitan
monolaurate, C58H114O26, Sigma Aldrich] in PBS) and blocked with 5% skim milk (Difco TM; Becton,
Dickinson and Company; Sparks, MD, USA) in PBS (‘5% milk-PBS’) for 1 h at 37°C. One hundred µL
primary antibodies, anti-VLP rabbit polyclonal antibodies lp132 diluted at 1:1,000 in 5% milk-PBS, were
added per well and incubated for 1 h at 37°C. After washing, 100 µL secondary antibodies, HRPconjugated anti-rabbit IgG antibodies, diluted 1:2,500 in 5% milk-PBS were added per well and
incubated 1 h at 37°C. After washing, 100 µL TMB One Solution was added as the substrate of the
enzyme, followed by adding of 50 µL phosphoric acid to stop the reaction. The optical density was
measured at 450 nm (OD450).
3.1.6 Mucin-binding ELISA
A Nunc Maxisorp immunoplate was pre-coated with porcine gastric mucin (Sigma-Aldrich, 2µg/well).
After overnight incubation at 4°C, the wells were washed three times with PBS-T and then blocked
with 5% milk-PBS for 1 h at 37°C. E. coli cells were washed with PBS and mixed with NoV GII.4 VLPs
(100 µL per sample, 1:100 diluted in PBS to get 5 µg/mL). These mixtures, either untreated or treated
at 90°C for 2 min in a PCR cycler and then cooled down on ice, were added to each well. Five per cent
milk-PBS without VLPs was added to mucin-coated wells to include blank controls. The plates were
incubated for 1 h at 37°C. The rest of the procedure – adding antibodies, TMB, H3PO4 and measuring
the absorbance at 450 nm – was performed as was described for the direct ELISA.
24
3.2 Interaction between Norovirus and probiotic bacteria
The second part of this thesis was dedicated to the influence of probiotic bacteria on NoVs.
Bifidobacterium longum was chosen for this study based on the previous results generated from
LFMFP, Ghent University (data not published).
The growth of B. longum was followed both in bacteria culture medium and in skim milk, a
representative model for food matrices being natural sources of probiotics.
Murine Norovirus 1 (MNV-1) was used as a surrogate of human NoVs. Plaque assays were used to test
the antiviral effect of B. longum on MNV-1. First of all, the survival of MNV-1 in different solutions
wherein the treatment with B. longum would occur were tested. In the following, the effect of B.
longum on MNV-1 was investigated with different bacterial cell numbers, treatment time intervals and
food matrices. Figure 10 shows an overview of the experimental set-ups.
Growth of B.
longum in TSB
Choice of a food
matrix model
Cell plaque assay
without bacteria
Cell plaque assay
with bacteria
• TSB
• Milk + sucrose
• Cell-free bacteria
culture filtrate
• 1010 bacterial cells
for 1 h
• Bacteria growing in
TSB for 0 to 48 h
• 1011 bacterial cells
in TSB or milk +
sucrose for 48 h
Figure 10: Flowchart showing the different steps that were performed to study the effect of B. longum as a
probiotic bacterium on a NoV infection.
3.2.1 Bacteria and cell lines
Bifidobacterium longum (LMG 10502, biological origin: infant intestines) was obtained from the
Belgian Coordinated Collection of Microorganisms (BCCM/LMG). B. longum was cultured in TSB at 37°C
in an anaerobic atmosphere, generated with the use of ANAEROGENTM COMPACT.
Cells of the murine macrophage cell line RAW 264.7 (ATCC TIB-71) were kindly provided by Prof. H. W.
Virgin, Washington University School of Medicine, St. Louis, MO, USA. The RAW 264.7 cells were
cultivated in complete DMEM medium and grown at 37°C under a 5 % CO2 atmosphere. The complete
DMEM medium contained Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Walkersville, MD, USA)
containing 10 % low-endotoxin fetal bovine serum (FBS; HyClone, Logan, UT, USA), 100 U/mL penicillin
and 100 µg/mL streptomycin (Lonza), 10 mM Hepes (Lonza), and 2 mM L-glutamine (Lonza).
25
3.2.2 Viruses
MNV-1, which was used as a surrogate for human NoV, was kindly provided by Prof. H. W. Virgin,
Washington University School of Medicine, St. Louis, MO, USA. The virus lysate was prepared by Dr.
Dan Li from LFMFP as described previously (Li et al., 2011).
3.2.3 Monitoring of the bacterial growth
The growth of B. longum was monitored as described in part 3.1.3 except that the first passage was
grown for 48 h before making a second passage. Samples of the second passage were taken at 0, 12,
24 and 48 h. For the viable bacteria numeration, the inoculated plates were incubated at 37°C for 48
h before counting the colonies.
3.2.4 Culturing of the macrophage cell line RAW 264.7
RAW 264.7 cells were maintained in low-endotoxin medium (DMEM with 10 % low-endo FBS, 1 %
Hepes, 1 % penicillin/streptomycin, 1 % L-glutamine). The cells were put in 20 mL of this medium and
grown at 37°C under a 5 % CO2 atmosphere in 75 cm2 tissue culture flasks. The cells were splitted when
a confluence of 70-75 % was reached. The old medium was removed and 10 mL fresh DMEM medium
was added to the flask before scraping off the cells. A single-cell suspension was made by sucking up
the medium with cells and then forcefully squeezing it through a pipette tip pressed onto the flask
surface. This was repeated 5 times. Finally, 1 mL (1:10 dilution) or 2 mL (1:5 dilution) of the cell
suspension was transferred into a new flask, already containing 19 mL respectively 18 mL new fresh
complete DMEM before pursuing cell growth. Cell growth always occurred in a CO2 incubator C150
(Binder GmbH, Tuttlingen, Germany). All other steps were performed in a MSC-Advantage™ Class II
Biological Safety Cabinet (Thermo Fisher Scientific).
3.2.5 Plaque assay
The plaque assay for MNV-1 was always executed in a MSC-Advantage™ Class II Biological Safety
Cabinet. On the first day, RAW 264.7 cells were seeded into 6-well plates (3.5 cm diameter) at a density
of 2 x 106 viable cells per well. The plates were rocked and put overnight in a CO2 incubator C150 at
37°C and 5% CO2.
On the next day, tenfold serial dilutions in antibiotics-free complete DMEM (low endo DMEM, 10% low
endo FBS, 1% Glutamine and 1% Hepes) of a viral-bacterial mixture were prepared in 24-well plates.
26
The next step comprised rapidly pouring out the medium of the 6-well plates and adding 500 µL of the
desired diluted viral-bacterial sample in duplicate wells. The 6-well plates were incubated at room
temperature for 1 h, manually rocking the plates every 15 min. After aspirating the inoculum, the cells
in each well were overlaid with 2 mL 1.5% SeaPlaque agarose (42°C; Cambrex, Rockland, ME, USA) in
antibiotics-free complete Eagle’s minimum essential medium (37°C; complete EMEM; EMEM [Lonza],
10% low endo FBS, 2% L-glutamine and 1% Hepes) at a 1:1 ratio. The plates were placed in a tissue
culture incubator at 37°C and 5 % CO2 for 2 days. Overlaying the cells in each well with 2 mL of a 1:1
mixture of 1.5% SeaKem agarose (50°C; Cambrex, Rockland, ME, USA) and antibiotics-free complete
EMEM containing 1% neutral red (Sigma Aldrich), allowed for visualizing the plaques after incubation
for 4 h at 37°C and 5% CO2.
The following formula was used to calculate the number of viruses in the original virus-bacteriamedium mixture:
X = log(A ∗ V)
X equals the log number of PFU per mL in the original mixture, A represents the total number of
counted plaques in the duplicate wells and V is the dilution factor.
3.3 Statistical analysis
Statistical analyses of the results were performed by independent samples t-tests with SPSS Statistics
23 for Windows (SPSS Inc., Chicago, IL, USA). Significant differences were considered when p was
smaller than 0.05.
27
28
Chapter 4: Results
4.1 Interaction between Norovirus and HBGA expressing
bacteria
4.1.1 Optimisation of the HBGA expression test
In the previous study of our research group (Li et al., 2015), the HBGA expression of bacteria was
identified by flow cytometry analysis with the use of BDTM LSRII flow cytometer at INSERM, Nantes,
France. In this study, due to the inaccessibility of this equipment, a substitutive method based on the
principle of ELISA was attempted. Therefore, primary tests had to be performed in order to optimise
the experimental conditions.
4.1.1.1 Concentration of antibodies
Different concentrations of the primary and secondary antibodies were examined. For the primary
antibodies (mouse anti-A antibodies #21), dilutions 1:250 and 1:1,000 were made in 0.1% BSA-PBS.
Secondary anti-mouse antibodies were diluted to concentrations 1:2,500 and 1:10,000 in 0.1% BSAPBS. The effect of the four possible combinations of these primary and secondary antibody dilutions
were compared in HBGA expression tests.
Besides the detection of HBGA on E. coli LMG 8223 (previously confirmed as an HBGA expression
positive strain), two types of negative controls were also included in parallel. Control 1: Detection of
E. coli LMG 8223 without the addition of primary antibody, in order to control the non-specific binding
of E. coli LMG 8223 with the secondary antibody; Control 2: Detection of E. coli ATCC 8739 (previously
confirmed as an HBGA expression negative strain), in order to control the binding of non-HBGA
materials with primary and/or secondary antibody.
The results of the corresponding HBGA tests are given in Table 4. Combinations 1 and 3 gave the
highest OD450 values for E. coli LMG 8223, but the negative controls also had a high OD value (higher
than 1). This indicates that nonspecific binding took place. Combination 4, having the highest dilutions
of both antibodies, gave a rather small difference between the positive value and the negative
controls. Combination 2 had low values for the controls, the positive value was high and the difference
was big enough to make a good differentiation. This makes combination 2, with primary antibodies
diluted 250 times and secondary antibodies 10,000 times, the desired combination for further HBGA
tests.
29
Table 4: OD450 values indicating HBGA expression with different antibody concentrations (primary
antibody:secondary antibody). 1 = combination 1:250 and 1:2,500; 2 = combination 1:250 and 1:10,000; 3 =
combination 1:1,000 and 1:2,500; 4 = combination 1:1,000 and 1:10,000. Control 1: Detection of E. coli LMG
8223 without the addition of primary antibody. Control 2: Detection of E. coli ATCC 8739.
Combination 1
Combination 2
Combination 3
Combination 4
3.122
1.602
1.001
1.859
0.860
0.490
1.964
1.388
0.984
1.113
0.984
0.538
E. coli LMG 8223
Control 1
Control 2
4.1.1.2 Concentration of blocking reagent
BSA was used in the HBGA expression test to prevent non-specific bindings. This test was performed
to determine the appropriate concentration of BSA as a blocking reagent. Similar with section 4.1.1.1,
two types of negative controls were included in parallel. Control 1: Detection of E. coli LMG 8223
without the addition of primary antibody; Control 2: Detection of E. coli ATCC 8739.
The results are shown in Table 5. The group with the use of 0.1% BSA as blocking reagent induced a
better differentiation between positive detection and negative controls than the use of 1% BSA. It
could be possible that the higher amount of BSA also covered some of the HBGA receptors so that
these carbohydrates were not recognisable for the primary antibodies. Therefore 0.1% BSA was chosen
as blocking reagent concentration for further HBGA tests.
Table 5: OD450 values indicating HBGA expression with different blocking reagent concentrations. Control 1:
Detection of E. coli LMG 8223 without the addition of primary antibody. Control 2: Detection of E. coli ATCC
8739.
E. coli LMG 8223
Control 1
Control 2
1% BSA
0.1% BSA
0.959
0.506
0.160
1.556
0.775
0.377
4.1.1.3 Cell numbers of the tested bacteria
The amount of cells in a bacterial solution will influence the outcome of the HBGA expression test. To
investigate this effect, cultures of E. coli LMG 8223 and E. coli LFMFP 861 (both previously confirmed
as HBGA expression positive strains) were grown for 6 h and 24 h in aerobic atmosphere. For this HBGA
test, only one type of control was carried out: Detection of E. coli LMG 8223 without the addition of
primary antibody.
The test results are presented in Table 6. Although the OD450 values were higher for the 24 h cultures,
the difference between positive detection and the negative control was rather low. A bigger
30
differentiation was acquired with the 6 h samples. The optical density at 600 nm which indicates the
bacterial cell mass is also given in Table 6. The 6 h cultures had an OD600 of around 0.3. So in the
following tests, the bacteria used in the HBGA expression test were always normalised to an OD600 of
0.3, since this concentration is expected to give a good distinction between positive and negative
results for the HBGA expression.
Table 6: OD values at 450 nm (HBGA expression) and 600 nm (cell mass) of E. coli LMG 8223 and E. coli LFMFP
861 cultures. Bacteria were grown for 6 h and 24 h in aerobic conditions. Control: Detection of E. coli without
the addition of primary antibody. Each time the mean and standard deviation of three independent tests are
given.
OD 450 nm (HBGA expression)
E. coli LMG 8223
E. coli LFMFP 861
6h
6 h, control
24 h
24 h, control
1.45 ± 0.08
0.61 ± 0.08
1.71 ± 0.29
1.48 ± 0.10
0.81 ± 0.05
0.49 ± 0.04
0.96 ± 0.05
0.88 ± 0.04
0.32 ± 0.02
0.67 ± 0.09
0.27 ± 0.01
0.56 ± 0.01
OD 600 nm (cell mass)
6h
24 h
4.1.2 Influence of gas atmosphere of bacterial growth on their
HBGA expression and viral protective effects
E. coli are facultative anaerobe bacteria. This means that they are capable of growing in both aerobic
or anaerobic conditions. In this section, the goal is to audit if the amount of HBGA expression and the
protective effect on viruses can be linked to the gas environment wherein the bacteria grow.
4.1.2.1 Growth of E. coli LMG 8223 at different atmospheres
First of all, growth of E. coli LMG 8223 was followed in aerobic (shaking at 200 rpm, 37°C) and anaerobic
(ANAEROGENTM COMPACT, 37°C) gaseous atmospheres. The number of viable bacteria (colony forming
units per mL, CFU/mL) and the cell mass (OD600) were measured at different points in time: after 0, 3,
6, 12 and 24 h. The data are shown in Figure 11 for aerobic growth and Figure 12 for anaerobic growth.
In both cases, considering the viable bacteria numbers, a plateau phase was reached after 6 h of
growth. The total number of living bacteria stayed approximately constant from this point onward and
was similar for aerobic and anaerobic growth. Even though both atmospheres showed a similar
number of alive cells, the OD600 is much higher in the aerobic case. This difference can be explained by
assuming that E. coli cells die faster in aerobic conditions and that these dead cells help increase the
turbidity of the solution.
31
For further experiments on the influence of the gas atmosphere, a similar starting number of bacteria
was pursued so that it would be easier to compare results. Based on the two figures, the choice was
made to let the bacteria grow aerobically for 6 h or anaerobically for 24 h. In these conditions, a similar
OD600 (0.3) and number of CFU/mL (9 log) was reached.
10
0,8
9
0,6
8
0,4
7
0,2
6
5
OD 600 nm
Concentration (log[CFU/mL])
Aerobic growth E. coli LMG8223
0
0
5
10
15
20
25
Time (h)
log(CFU/mL)
OD 600 nm
Figure 11: Growth of E. coli LMG 8223 in TSB over 24 h in aerobic atmosphere (shaking at 200 rpm) at 37°C,
visualizing the log(CFU/mL) with circles and OD600 with triangles.
10
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
9
8
7
6
5
0
5
10
15
20
25
OD 600 nm
Concentration (log [CFU/mL])
Anaerobic growth E. coli LMG8223
30
Time (h)
log(CFU/mL)
OD 600 nm
Figure 12: Growth of E. coli LMG 8223 in TSB over 24 h in anaerobic atmosphere (ANAEROGENTM bags) at 37°C,
visualizing the log(CFU/mL) with circles and OD600 with triangles.
32
4.1.2.2 HBGA expression of E. coli LMG 8223 and LFMFP 861 grown
at different atmospheres
In a next step in investigating the influence of gas, HBGA expression tests were performed with E. coli
LMG 8223 and LFMFP 861 that were cultured for 6 h in aerobic conditions and 24 h in anaerobic
conditions. The control comprises the detection of E. coli without addition of primary antibodies.
For both growth conditions, the OD600 values were around 0.3 (Table 7). Assuming this means a
comparable number of cells was present, HBGA tests were performed. In the same table, also the
results of the HBGA expression test are given. Comparing the two different gas atmospheres, E. coli
LMG 8223 gave a significantly higher HBGA expression for the 24 h anaerobe samples (p value 0.009),
however E. coli LFMFP 861 showed a significantly higher HBGA expression for the 6 h aerobe samples
(p value 0.034). No consistent conclusions on the effect of the different atmosphere could be drawn
from these results for both strains.
Table 7: OD values at 450 nm (HBGA expression) and 600 nm (cell mass) of E. coli LMG 8223 and E. coli LFMFP
861 cultures. Bacteria were grown for 6 h in aerobic atmosphere or 24 h in anaerobic atmosphere. Control:
Detection of E. coli without the addition of primary antibody. (*) indicates a significantly higher result between
the aerobic and anaerobic samples. Each time the mean and standard deviation of three independent tests
are given.
OD 450 nm (HBGA expression)
E. coli LMG 8223
E. coli LFMFP 861
6 h aerobic
6 h aerobic, control
24 h anaerobic
24 h anaerobic, control
1.45 ± 0.08
0.61 ± 0.08
1.87 ± 0.13 (*)
0.57 ± 0.07
0.81 ± 0.05 (*)
0.49 ± 0.04
0.64 ± 0.07
0.43 ± 0.02
0.32 ± 0.02
0.30 ± 0.04
0.27 ± 0.01
0.26 ± 0.01
OD 600 nm (cell mass)
6 h aerobic
24 h anaerobic
4.1.2.3 Viral protective effects of E. coli LMG 8223 and LFMFP 861
grown at different atmospheres
A) Direct ELISA
Previous experiments have demonstrated that the binding of NoV VLPs to HBGA expressing bacteria
protects the VLPs from a heat treatment (Hirneisen & Kniel, 2013; Li et al., 2015). This study aims to
find out whether there is a difference in this protective role if the bacteria are grown in different
atmospheres. The direct ELISA test is performed in order to measure the antigenicity of the VLP
antigens after heat treatment of 2 minutes at 90°C. The results of the test for E. coli LMG 8223 and
LFMFP 861 are listed in Figure 13.
33
For both E. coli strains, detection of the VLP epitopes increased after heat treatment in the presence
of the bacteria. However, no significant difference was present for E. coli LMG 8223 grown in different
atmospheres (p value 0.134). E. coli LFMFP 861 gave a significantly higher result after heat treatment
when the bacteria were grown in anaerobic conditions compared to aerobic conditions (p value 0.006).
Similarly, no consistent conclusions on the effect of the different atmosphere could be drawn from
these results for both strains.
Direct ELISA
2,50
OD 450 nm
2,00
1,50
1,00
0,50
0,00
6h aer + VLP
6h aer + VLP, after 24h anaer + VLP 24h anaer + VLP,
heat
after heat
E. coli LMG 8223
Only VLP
Only VLP, after
heat
E. coli LFMFP 861
Figure 13: Protection of HBGA-expressing bacteria (E. coli LMG 8223 [dots] and E. coli LFMFP 861 [striped]) on
the antigenicity of human NoV GII.4 VLPs towards heat treatment (90°C for 2 min). Antigen detection of NoV
GII.4 in the presence of 6 h cultured bacteria in aerobic conditions (TSB, 37°C), 24 h cultured in anaerobic gas
atmosphere (TSB, 37°C) or in absence of bacteria; each time before and after heat treatment. Every bar is an
average of three independent tests, and every error bar represents the data range.
B) Mucin-binding ELISA
The second test to investigate the protective role of bacteria grown in different atmospheres is a
mucin-binding ELISA. Porcine gastric mucin contains a mixture of HBGAs. This test determines if the
VLPs lose their ability to bind receptors (HBGAs) after the same heat treatment as before: 2 min at
90°C. The same two E. coli strains were tested as for the direct ELISA test. The results are presented in
Figure 14.
Heating decreased the mucin-binding ability drastically. However, the measured optical density was
always slightly higher in the tests with bacteria. When E. coli LMG 8223 was used, the aerobic cultures
could preserve the binding ability of the VLPs better than anaerobic cultures (p value 0.001). For E. coli
LFMFP 861, no significant difference could be made between incubation of the VLPs with aerobic or
34
anaerobic cultures (p value 0.374). Still, no consistent conclusions on the effect of the different
atmosphere could be drawn from these results for both strains.
Mucin-binding ELISA
2,00
OD 450 nm
1,60
1,20
0,80
0,40
0,00
6h aer + VLP
6h aer + VLP, after 24h anaer + VLP 24h anaer + VLP,
heat
after heat
E. coli LMG 8223
Only VLP
Only VLP, after
heat
E. coli LFMFP 861
Figure 14: Protection of HBGA-expressing bacteria (E. coli LMG 8223 [dots] and E. coli LFMFP 861 [striped]) on
the receptor binding ability of human NoV GII.4 VLPs towards heat treatment (90°C for 2 min). Mucin-binding
ability detection of NoV GII.4 in the presence of 6 h cultured bacteria in aerobic conditions, 24 h cultured in
anaerobic gas atmosphere or in absence of bacteria; each time before and after heat treatment. Each bar is an
average of three independent tests, and each error bar represents the data range.
4.2 Interaction between Norovirus and probiotic bacteria
4.2.1 B. longum growth
4.2.1.1 Growth in bacteria culture medium TSB
The anaerobic growth of B. longum in bacteria culture medium TSB at 37°C was followed (Figure 15).
ANAEROGENTM bags created the anaerobic atmosphere. The number of alive B. longum cells showed
a noticeably increase after 12 h of incubation. The bacteria culture could be considered as fully grown
after 24 h, when approximately 9 to 10 log CFU/mL were present and a plateau phase was reached. In
addition, the cells mass remained constant from 24 h on. The OD600 measurements reached a value of
0.2 in this case.
35
12
0,4
10
0,3
8
6
0,2
4
OD 600 nm
Concentration (Log CFU/mL)
Growth of B. longum
0,1
2
0
0
0
5
10
15
20
25
30
35
40
45
50
Time (h)
log(CFU/mL)
OD 600 nm
Figure 15: Growth of B. longum over 48 h, visualizing the log(CFU/mL) with circles and OD600 with triangles.
The bacteria were anaerobically grown (ANAEROGENTM bags) at 37°C.
4.2.1.2 Growth in a food matrix
Since dairy products are the main vehicles for probiotic supplementation, the choice was made to test
skim milk as a food matrix model. Around 106 B. longum cells were inoculated in skim milk and
incubated anaerobically at 37°C. After 48 h, 8 log CFU/mL were present in the milk culture (Table 8).
This was still approximately 2 log less than growth in TSB. Consequently, an extra carbon source (5 %
sucrose or 5 % honey) was added to the milk to stimulate bacterial growth. Adding sucrose resulted in
the biggest increase of growth, almost 1 log CFU/mL. Therefore, the skim milk supplemented with 5%
sucrose was chosen as the food model in the following tests.
Table 8: The growth of B. longum cultured in different media. The bacteria were grown anaerobically for 48 h
at 37°C.
Log CFU/mL
TSB
Skim milk
Skim milk +
5 % sucrose
Skim milk +
5 % honey
9.86
8.00
8.95
8.60
4.2.2 Effect of B. longum on NoVs
The survival of MNV-1 in TSB, milk with 5 % sucrose, and cell-free bacteria culture filtrate was firstly
investigated. Bacteria cultures were centrifuged at 6000 rpm for 5 min in an Eppendorf centrifuge 5804
R (Novolab, Geraardsbergen, Belgium) and the supernatants were recovered and filtered over a 0.22
36
µm Millex-GV syringe filter (Merckx Millipore, Carrigtwohill, Co. Cork, Ireland) to remove residual
bacteria and end up with the filtrate. In each case, 1 mL of the virus was added to 10 mL of the medium
and anaerobically incubated for 48 h at 37°C before performing the plaque assays. The results are
presented in Table 9. Incubating the virus with cell-free filtrate, containing secondary metabolites of a
fully grown B. longum culture, delivered the highest viral reduction: 2.4 log PFU/mL. This is almost 1
log more than in TSB or milk with sucrose. Figure 16 displays an example of the plaque assay, with the
transparent spots as plaques caused by MNV-1.
Table 9: Results of the survival of MNV-1 in different media detected by cell plaque assays. Each time the mean
log PFU/mL and standard deviation of three independent tests are given.
0h
12 h
24 h
48 h
Log reduction after
48 h
10 mL TSB +
1 mL MNV-1
10 mL skim milk with
5 % sucrose
+ 1 mL MNV-1
10 mL cell-free
bacteria culture filtrate
(in TSB) + 1 mL MNV-1
4.15 ± 0.06
3.40 ± 0.09
3.06 ± 0.06
2.50 ± 0.17
6.60 ± 0.12
6.73 ± 0.12
5.17 ± 0.15
4.33 ± 0.35
1.65 ± 0.18
1.43 ± 0.19
2.40 ± 0.37
Figure 16: An example of the plaque assay, with the transparent spots as plaques caused by MNV-1.
37
The direct effect of B. longum cells on MNV-1 was investigated in the following. As a first test, 1010 B.
longum cells were added to 1 mL of the virus. After 1 h of incubation at 37°C, no reduction in the
number of PFU was found (Test 1, Table 10). This test was repeated with a lowered virus concentration,
assuming MNV-1 might become more susceptible for the treatment. However similarly, only a very
small reduction was observed (Test 2, Table 10).
Table 10: Effect of B. longum on MNV-1 with 1010 bacterial cells, incubated anaerobically for 1 h at 37°C. MNV1 lysate was used for Test 1, 1,000 times diluted virus lysate for Test 2. Each time the mean log PFU/mL and
standard deviation of three independent tests are given.
1 mL MNV-1 + 1010 B. longum cells
0h
1h
Log reduction after 1 h
Test 1
Test 2
8.15 ± 0.06
8.58 ± 0.06
5.15 ± 0.06
4.89 ± 0.04
No reduction
0.26 ± 0.07
Since the effect of the bacteria after 1 h was very limited, the treatment time was prolonged up to 48
h in a next phase of the experiments. For Test 3, 1 mL of MNV-1 was added to 10 mL of a growing
bacteria culture in TSB. Based on the bacterial growth indicated in Figure 14, bacteria numbers starting
from 106 going up to 1011 after 48 h were expected. Test 3 showed only a 1.28 ± 0.27 log reduction in
PFU/mL after 48 h. For similar reasons as with Test 2, the virus concentration was lowered to repeat
the test. In this case (Test 4), a 1.74 ± 0.21 log reduction in PFU/mL occurred after 12 h, but no plaques
were observed after 24 and 48 h. The uncountable results remained questionable, as it is known that
higher B. longum cell numbers were present in the specific wells for plaque assay compared to other
wells (with higher virus concentrations and therefore further dilutions or less bacteria culture time),
and it is known based on our previous experience that the presence of B. longum in the plaque assay
wells could inhibit virus multiplication, therefore inducing smaller and less plaques (data not
published). The results of Tests 3 and 4 are shown in Table 11.
From the countable results of the four tests described above, not a single one gave a reduction that
was similar or higher to the one obtained with cell-free bacterial filtrate. The starting number of B.
longum bacteria was increased to 1011 bacterial cells for the next tests. The bacteria were put together
with 1 mL of MNV-1 and 10 mL of TSB, incubated anaerobically for 48 h at 37°C, and the plaque assay
was performed subsequently in Test 5. All the same was done in Test 6, but this time milk with 5 %
sucrose was the matrix of choice. Table 12 gives a summary of the results obtained by these two tests.
Test 5 resulted in a 2.14 ± 0.29 log PFU/mL reduction after 48 h, whereas a procedure as in Test 6 led
to a reduction that might be bigger than 5.73 log.
38
Table 11: Results of the cell plaque assays for MNV-1 in a growing bacteria culture in TSB, incubated
anaerobically up to 48 h at 37°C. MNV-1 lysate was used for Test 3, 1,000 times diluted virus lysate for Test 4.
Each time the mean log PFU/mL and standard deviation of three independent tests are given.
10 mL growing bacteria culture in TSB + 1 mL MNV-1
0h
3h
6h
12 h
24 h
48 h
Log reduction
Test 3
Test 4
5.86 ± 0.04
5.66 ± 0.02
5.58 ± 0.03
5.44 ± 0.13
5.13 ± 0.10
4.58 ± 0.27
4.76 ± 0.13
3.79 ± 0.10
3.40 ± 0.17
3.02 ± 0.16
<1?
<1?
1.28 ± 0.27 log reduction after
48 h
1.74 ± 0.21 log reduction after
12 h; > 3.76 log after 24 h?
Table 12: Results of the cell plaque assays for MNV-1 with 1011 bacterial cells, incubated anaerobically in
different media for 48 h at 37°C. TSB was used for Test 5, milk with 5 % sucrose for Test 6. Each time the mean
log PFU/mL and standard deviation of three independent tests are given.
10 mL matrix + 1 mL MNV-1 + 1011 B. longum cells
0h
48 h
Log reduction after 48 h
Test 5, in TSB
Test 6, in milk + 5 % sucrose
6.73 ± 0.12
4.59 ± 0.26
6.73 ± 0.13
<1?
2.14 ± 0.29
> 5.73 ?
39
40
Chapter 5: Discussion
5.1 Interaction between Norovirus and HBGA expressing
bacteria
Previous research at LFMFP investigated the HBGA expression of multiple bacterial strains (Li et al.,
2015). The study also indicated that if NoV VLPs were heated for 2 min at 90°C in the presence of HBGA
expressing E. coli, a higher antigen integrity as well as mucin-binding ability of the VLPs was detected
compared to the same test with the same cell numbers of non-HBGA expressing bacteria. This led to
the conclusion that HBGA expressing bacteria can have a protective role on human NoV from acute
heat stress. However, it was also noticed that large variations can occur in HBGA expression between
different bacterial strains, even if they are closely related. Different cultural batches from one
particular bacterial strain were susceptible to varying HBGA levels as well. HBGA-like structures can be
secondary metabolites of bacteria and their production could be regulated by some currently unknown
factors. In this thesis, further explorations were done to tackle the rising question of which factors are
influencing HBGA expression levels.
E. coli are facultative anaerobe bacteria, meaning that they are capable of growing in both aerobic or
anaerobic environments. It has already been demonstrated that the production of secondary
metabolites in E. coli can be influenced by the gaseous atmosphere in which the bacteria grow (Gray
et al., 1966). In this study, the goal is to investigate the role or influence of the gaseous atmosphere
on both the expression of HBGAs and the viral protective effect of the bacteria.
Due to a limitation in the available facilities, flow cytometry could not be used to measure HBGA
expression like was done in the previously mentioned article from Li et al. (2015). The first challenge
was thus to create a new HBGA detection method. An ELISA test was chosen as a substitute. Therefore,
the amounts of antibodies, blocking reagent and bacterial cell numbers had to be optimised in the first
place. Based on the results of Li et al. (2015), the choice was made to use E. coli LMG 8223 and E. coli
LFMFP 861 since they showed the highest HBGA expression and could both bind to NoV VLPs. Besides
the ELISA-based HBGA expression test, direct ELISA assays were used to measure the NoV VLP antigen
integrity after heat treatment and porcine gastric mucin-binding assays were performed with the
prospect of measuring the receptor binding ability of the NoV VLPs after the same treatment.
Table 13 gives an overview of the main findings from all the tests. Looking at the two strains separately,
no consistent link could be made between the HBGA expression and the protection of the virus by the
bacteria that were cultured in either an aerobic or anaerobic gaseous atmosphere. In addition, the
41
conclusions that were observed for E. coli LMG 8223 were not consistent with those for E. coli LFMFP
861.
Table 13: Summary of the differences of aerobically or anaerobically grown E. coli LMG 8223 and E. coli LFMFP
861 on their HBGA expression on the one hand, and the NoV VLP antigen integrity and receptor binding ability
after heat treatment on the other hand. The p values were calculated by an independent samples t-test that
compared the means of the 24 h anaerobic and 6 h aerobic groups.
HBGA expression
NoV VLP antigen integrity after
heat treatment
Receptor binding ability after
heat treatment
E. coli LMG 8223
E. coli LFMFP 861
24 h anaerobic > 6 h aerobic
p = 0.009
No significant difference
p = 0.134
24 h anaerobic < 6 h aerobic
p = 0.001
24 h anaerobic < 6 h aerobic
p = 0.034
24 h anaerobic > 6 h aerobic
p = 0.006
No significant difference
p = 0.374
This inconsistency in the results might be described partially to the normalisation of bacteria. In this
study, it was determined to normalise the optical density of a bacteria culture at 600 nm to 0.3 before
each experiment. The purpose was to make sure that the cell mass was similar in each sample to be
compared with. However as shown in the results, the growth dynamics of E. coli in aerobic and
anaerobic atmospheres, determined by measuring the viable cell numbers and optical density
respectively, were rather different. Therefore, although the cell mass was normalised, it is possible
that other factors exist to influence the HBGA expression and NoV protective effects that we
investigated. In this study, the HBGA expression was measured by an ELISA based method. Compared
with the flow cytometry used previously (Li et al., 2015) which measures the fluorescence of the cells
one by one, ELISA measures the total fluorescence signal generated from all the bacterial cells from
one sample. Therefore, it is possible that bigger variation can be caused in the ELISA based method
due to aggregation of bacterial cells or background noise.
A clear link between the gaseous atmosphere and the level of HBGA expression that accounts for both
E. coli strains was not found (see Table 13). However, it was noticed that after 24 h of aerobic growth,
for both of the two bacterial strains, it was difficult to differentiate positive HBGA expression signals
from the negative controls (too high background signals, Table 6). Greater differences were present
for 24 h of growth in the anaerobic case (Table 7). It has been demonstrated in this study that the E.
coli bacteria grow faster aerobically than anaerobically. It was also shown that the gaseous atmosphere
can influence the production of secondary metabolites (Gray et al., 1966). Taken together, we can
assume that certain secondary metabolites are earlier formed when the bacteria are grown
aerobically, and some of these metabolites may as well have affinity towards the antibodies that would
interfere with the detection of HBGAs. It is also possible that other factors apart from the gaseous
42
environment, such as the prevalent pH in the intestines of the human body, have a greater influence
on HBGA expression. This can be the subject for further examination on the HBGA topic.
The direct ELISA assay revealed that NoV VLPs gained a higher immunoreactivity after exposure to heat
stress. This can be due to partial degradation of the VLPs when they are heated, causing otherwise
masked epitopes to now reach the surface. The results from the direct ELISA and mucin-binding ELISA
suggest that HBGA expressing bacteria can protect NoVs during heat treatment, even though these
assays do not completely reflect an authentic NoV infection. This outcome is consistent with the
findings of Li et al. (2015).
5.2 Interaction between Norovirus and probiotic bacteria
Several key challenges remain in assessing the efficacy of vaccines and antiviral drugs for human NoV
infections. There is for example no robust cell culture system for human NoVs, limiting the direct study
of these viruses. Additionally, the genetic heterogeneity among NoV strains makes it difficult to find a
good vaccine. Consequently, the use of probiotics as a possible method to control and treat a NoV
infection is an interesting topic for investigation.
Nowadays, research on the effect of probiotic bacteria on NoVs is still in a preliminary phase. This is
mainly due to two reasons. First of all, as a result of the non-cultivability of human NoVs, surrogates
are used to conduct in vitro experiments (e.g. FCV, Aboubakr et al., 2014; MNV, Lee et al., 2012; TV,
Shearer et al., 2014; NoV P particles, Rubio-del-Campo, 2014). Secondly, because of a lack of enough
in-depth studies on this topic, it is difficult to draw general conclusions. As previously shown in part
2.2.3, the decrease in viral infectivity caused by probiotic bacteria can be very variable. The
inconsistency in the results could be caused by differences between the used LAB strains and viruses
(or its surrogates), as well as the experimental set-ups.
The purpose of this study was to investigate the potential antiviral effect of B. longum against NoVs.
MNV-1 was employed as a surrogate. B. longum is a recognized probiotic (Tamaki et al., 2016; Vieira
et al., 2016) and it was also reported to show antiviral effect against rotavirus (Kang et al., 2015). The
survival of MNV-1 in different media without bacteria was firstly tested. In a next phase, the potential
antiviral effect of B. longum was examined by looking into the outcome of multiple co-incubation setups with the bacteria and virus.
Skim milk was chosen as a food matrix, because it is relatively easy to define its compounds and dairy
products are known to be the main vehicles of probiotic supplementation. Bifidobacteria are normal
inhabitants of the human gastrointestinal tract and when they are added to foods, exterior
43
environmental factors (e.g. the compatibility with the starter lactic acid culture, oxygen stress, acid
stress, etc.) can greatly influence the stability and functionality of these bacteria (reviewed by Granato
et al., 2010). Indeed, in this study, B. longum grew slower in skim milk compared with the growth in
TSB. Previous research reported enhanced growth by bifidobacteria grown in skim milk in the presence
of carbohydrate components such as honey, fructooligosaccharides, and galactooligosaccharides (Shin
et al., 2000; Ustunol & Gandhi, 2001; Ustunol, 2000). Based on a preliminary test, 5% of sucrose was
added into the slim milk in this study to support the growth of B. longum.
Based on the results in this research, it was indicated that the direct effect of B. longum is limited.
Within all the countable results of the plaque assays, the highest reduction was obtained with cell-free
bacteria culture filtrate. Even with high bacterial cell numbers, not much difference was noticed
compared to the filtrate. This filtrate contained secondary metabolites that were secreted by the
bacteria. It is known that probiotic bacteria can produce metabolites such as organic acids (lactic and
acetic acid), hydrogen peroxide, ethanol, diacetyl, acetaldehyde, carbon dioxide, reuterin, and
bacteriocins (Suskovic et al., 2010). There is not much information available concerning bacteriocins
produced by B. longum. Kang et al. (1989) described the bacteriocin bifilong, Saleh and El-Sayed (2004)
informed about bifilong Bb-46, and Lee et al. (2011) identified the lantibiotic bisin. Some characteristics that were determined for these B. longum bacteriocins are given in Table 14. Recently,
metabolites of B. adolescentis were shown to have an antiviral effect on rotavirus by decreasing the
production of the viral protein NSP4, being a viral enterotoxin protein (Olaya Galán et al., 2016). What
specific metabolites caused this reduction and how they did it, was not mentioned. No studies have
been performed yet that investigated a possible antiviral effect of B. longum metabolites.
Table 14: Bacteriocins from B. longum and their main characteristics. (Martinez et al., 2013)
Bacteriocin
Species
Mol. wt.
and strain
(kDa)
Heat
range
stability
pH
range
stability
Production
phase
Inhibitory
spectrum
Bifilong
B. longum
120
(100°C –
30 min)
2.5 – 5.0
(-)
Bifilong Bb46
B. longum
Bb-46
25-127
(121°C –
15 min)
4.0 – 7.0
(-)
Lantibiotic
(Bisin)
B. longum
DJO10A
(-)
(-)
(-)
1–8h
gram-positive &
gram-negative
bacteria
Staphylococcus
aureus, Salmonella
typhimurium,
Bacillus cereus,
Escherichia coli
Streptococcus
thermophilus
ST403, Clostridium
perfringens,
Staphylococcus
epidermidis,
Bacillus subtilis, E.
coli DH5a.
Reference
Kang et
al. (1898)
Saleh &
El-Sayed
(2004)
Lee et al.
(2011)
44
Incubation of the bacteria with the virus for 1 h resulted in no or only very small viral reductions.
Because of this, the decision was made to prolong the treatment time. It was not known beforehand
if and when a significant antiviral effect could happen, so multiple incubation durations were tested
ranging from 3 h up to 48 h. A NoV infection is self-limiting within two to three days. Testing treatment
times longer than 48 h would thus not be very useful, because a NoV infection might already be over
again by that time. From a clinical point of view, it would be most interesting if a meaningful viral
reduction could be found at low treatment periods.
It was demonstrated in this study that the presence of B. longum could inhibit the multiplication of
MNV-1 on RAW 264.7 cells. The experiments support the idea that B. longum does not show direct
virucidal effect or prevent the viruses from binding to the cells, but that it is more likely that the viral
replication phase would be influenced. Several reasons can be brought up to confirm this statement.
First of all, previous research from our laboratory has revealed that the presence of probiotic bacteria
could induce smaller plaques in the plaque assay of MNV-1 (Li et al., unpublished). When uncountable
results occurred in the test, always a high number of bacterial cells were present in the cell culture
wells for plaque assay. In addition, in Test 4, for the groups incubated with 12 h cultured bacteria, the
plaques were found to be too small to count in some replicates. In Test 5, where MNV-1 was coincubated with 1011 bacterial cells in TSB, plaques could only be counted when a ten-thousand-time
dilution of the original viral-bacterial sample was made. Lower dilutions did not allow for counting
plaques, since they were too small to be seen.
Co-incubation of MNV-1 with the highest number of bacterial cells in milk supplemented with sucrose
led to viral numbers below the detection limit. However, in this case it was not sure whether the
reduction was caused by the antiviral effect of B. longum, by the viral inhibiting effect of certain milk
components, or by a combination of these two. Some milk proteins were reported to interfere with
viral infections, which might lead to greater virus inhibition effect in milk. Lactoferrin could for instance
prevent viral entry of host cells by blocking cellular receptors or by binding to the virus particles (Pan
et al., 2006). In addition, it was found that human milk glycans inhibits binding between NoV and its
host glycan receptor (Shang et al., 2013). This might be an interesting lead to combat with NoV
infection in young children.
Rotaviruses and NoVs cause gastroenteritis and the symptoms of both infections are similar. Although
some of the articles mentioned in Table 2 studied the effect of probiotics on rotaviruses, these viruses
cannot be considered as real surrogates for NoVs. The viruses differ too much in their virion structure
and genome structure (Estes & Cohen, 1989). As a result, mechanisms that are effective against
rotaviruses might not always work against NoVs.
45
46
Chapter 6: General Conclusions
Overall we observed that growing the bacteria in different gas atmospheres had indeed influenced the
HBGA expression as well as the protection of the integrity and functionality of NoV VLPs during heat
stress. However, performing the tests for two different E. coli strains always led to different
conclusions that could be drawn. So in the end, the inconsistency in these results makes it impossible
to make general conclusions that account for both strains. The gas atmosphere might not be the
biggest factor influencing HBGA expression and virus protection. Perhaps for further research on this
topic, attention could be paid to the influence of pH. As the bacteria should be capable of passing
through the stomach and intestines, it might be useful to mimic this route and expose HBGA expressing
bacteria to fluids similar to those present in the stomach (pH 1.5 - 3.5) and small intestines (pH 6.8),
before checking their receptor expression and virus protection ability.
Currently, research on the effect of probiotics on NoVs is still preliminary. This is mainly caused by the
non-cultivability of human NoVs and the lack of sufficient in-depth studies on this topic. This study
intended to demonstrate the antiviral effect of B. longum against NoVs. The direct effect was found to
be limited. The actual effect should mainly be found in the replication phase of the virus. Co-incubation
of the virus with a high concentration of bacteria in a mixture of skim milk and sucrose could drastically
decrease the infectivity of the virus after 48 h, although it was not sure whether this effect was fully
attributed to the probiotic bacteria or if the milk proteins also played a significant role. Further
research in the topic of antiviral effects of probiotics on NoVs could be for example based on the effect
of probiotic bacteria metabolites on NoVs, the effect of probiotic bacterial cells on NoV replication or
the possible synergistic effect of dairy products with probiotics.
47
48
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