Analysis of the interaction between bovine neutrophils and rotavirus
by Angela Jean Hanson
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Veterinary Molecular Biology
Montana State University
© Copyright by Angela Jean Hanson (2002)
Abstract:
Neutrophils play an essential role in defending the host against bacterial and fungal pathogens.
Recently, it has been discovered that neutrophils also play a role in viral clearance. In addition to
inhibiting viruses, neutrophils can also be modulated by viruses, which can either exacerbate illness or
increase susceptibility to other infections. Viruses mediate these effects either directly, by binding to
neutrophils, or indirectly, by the induction of neutrophil-acting molecules. Because neutrophils express
putative rotavirus receptors, neutrophils are found at sites of rotavirus infection in several animals, and
rotavirus induces secretion of neutrophil activators in vitro, it was hypothesized that neutrophils can
bind rotavirus and that this interaction would modulate host defense responses. Since rotavirus strains
are species-specific, studies were designed to characterize a homologous system consisting of bovine
neutrophils and the B641 calf strain of rotavirus. Studies were performed to first determine whether
neutrophils bound rotavirus using three complementary methods. These studies showed that rotavirus
specifically bound to neutrophils in a dose-dependent manner. In contrast, rotavirus was not
internalized by neutrophils after an extended period of incubation at a temperature permissive for
internalization. Together, these data suggest that like many cell lines, neutrophils possess a rotavirus
attachment site but not the co-receptors or mechanisms for internalization. In addition, exposure of
virus to neutrophils had no effect on virus replication, suggesting that neutrophils do not play a role in
clearance of this particular pathogen. We next evaluated the ability of rotavirus to elicit or modulate
neutrophil responses such as respiratory burst, chemotaxis, and phagocytosis. In addition, the ability of
rotavirus-infected MA1 04 cells to secrete neutrophil activators or inhibitors was evaluated. We found
that neither rotaviruses, nor supernatants from cells infected with rotavirus, elicited neutrophil
responses in any of the parameters analyzed. Thus, the binding of rotavirus by neutrophils basically
results in a nonproductive interaction.
Since these experiments were performed in vitro, it is possible that this interaction could be different in
the infected calf intestine. Nevertheless, the data obtained in these studies provides an initial framework
for developing our understanding of the role of neutrophils in rotavirus infection. ANALYSIS OF THE INTERACTION BETWEEN BOVINE
NEUTROPHILS AND ROTAVIRUS
by
Angela Jean Hanson
\
A thesis submitted in partial fulfillment
o f the requirements for the degree
of
M aster o f Science
in
Veterinary Molecular Biology
MONTANA STATE UNIVERSITY-BOZEMAN
Bozeman, M ontana
April 2002
APPROVAL
o f a thesis submitted by
Angela Jean Hanson
This thesis has been read by each member o f the thesis committee and has been
found to be satisfactory regarding content, EngUsh usage, format, citations, bibliographic
style, and consistency, and is ready for submission to the College o f Graduate Studies.
V//7-/6 Z-
Mark T. Quinn, Ph D.
Chair
Date
'
Approved for the Department o f Veterinary Molecular Biology
AUen G. Harmsen, Ph D.
Department Head
Date
Approved for the CoUege o f Graduate Studies
Bruce R. McLeod,
Graduate Dean
7
Date
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment o f the requirements for a master’s
degree at M ontana State University, I agree that the Library shall make it available to
borrowers under rules o f the Library.
I f I have indicated my intention to copyright this thesis by including a copyright
notice page, cppying is allowable only for scholarly purposes, consistent with “fair use”
as prescribed in the U.S. Copyright Law. Requests for permission for extended quotation
from or reproduction o f this thesis in whole or in parts may be granted only by the
copyright holder.
Signature
Date
10 /
ACKNOWLEDGMENTS
This publication was made possible by the joint efforts o f Dr. Mark Quinn and Dr.
Michele Hardy. Together they helped me design and carry out my project. I would also
like to thank Dr. Steve Swain, an assistant research professor, for his expertise and
assistance with designing experiments.
I would also like to thank my committee members, Dr. M ark Quinn, Dr. Michele
Hardy, and Dr. CliffBond for their assistance reviewing my thesis material.
This project was funded in part by the Bayard Taylor Fellowship.
TABLE OF CONTENTS
1. INTRODUCTION................................................................................................................... I
Neutrophils.....................................................................................
Neutrophil Functional Responses................................................................................ 2
Role o f Neutrophils in Viral Infections..................................................................
7
Rotavirus..................................................................................................................................... 12
Rotavirus Proteins.........................................................................................................13
Cellular Rotavirus Receptors............................................................
15
Receptors for Rotavirus In Vivo ................................................................................ 23
Disease Caused by R otavirus...........................
28
Immune Response to Rotavirus..................................................................................33
Evidence for a Role o f Neutrophils in Rotavirus Infection....................................39
Project G oals.............................................................................................................................. 43
2. ROTAVIRUS BINDING AND INTERNALIZATION
BY BOVINE N EUTROPHILS..............................................................,..................44
Introduction................................................................................................................................ 44
Materials and M ethods.........................
47
M aterials........................................................................................................................ 47
Maintenance o f Cell L in es......................................................................................... 47
Preparation o f Purified Viral Particles..........................
48
Production o f Polyclonal A ntibodies........................................................................ 49
Isolation o f Bovine N eutrophils................................................................................ 50
Flow Cytometry............................................................................................................50
Virus Overlay Protein Blot Assays (V O PB A )..................................
51
Radiolabeling o f Viral Particles................................................................................ 51
Radiolabeled Virus Binding A ssays.........................................................!.............. 52
Statistical Analysis....................................................................................................... 52
R esults..............................................................:........................................................................ 53
Analysis o f Purified Rotavirus and Polyclonal A ntiserum .................................... 53
Flow Cytometry............................................................................................................53
Virus Overlay Protein Blot Assays (V O PB A ).............................
55
Radioactive Virus-Binding A ssays......................................................................
57
Discussion...........................................................................................
3. ANALYSIS OF BOVINE ROTAVIRUS EFFECTS
ON NEUTROPHIL FUNCTIONAL RESPONSES............................................... 68
Introduction................................................................................................................................ 68
Materials and M ethods................................................................
TABLE OF CONTENTS (CONTINUED)
M aterials.........................................................................................................................71
Preparation o f Purified Viral Particles...................................................................... 71
Isolation o f Bovine N eutrophils................................................................................ 73
Superoxide Production.................................................................................................73
Chemotaxis........................................................................ ;..........................................74
Phagocytosis..................................................................................................................75
Exposure o f Neutrophils to R otavirus...................................................................... 75
Collection o f M A l 04 Cell Supernatants...................................................................75
Plaque A ssays............................................................................................................... 76
Statistical Analysis....................................................................................................... 76
R esults.........................................................................................................................................76
Analysis o f Neutrophil Effects on Rotavirus Replication...................................... 76
Analysis o f Neutrophil Responses Elicited by R otavirus..............................
77
Analysis o f Rotavirus Effects on Neutrophil R esponses....................................... 80
Analysis o f Neutrophil Responses to Virus-Infected M A l 04 Supernatants.......83
Discussion................................................................................................................................... 87
4. CONCLUSION................................................................................................................... 96
REFERENCES C IT E D ............................................................................................................ 99
APPENDIX A: PRODUCTION OF MONOCLONAL ANTIBODIES
111
vii
LIST OF TABLES
Table
Page
L I: Rotavirus P roteins........................................................................ ,...................................14
1.2: Putative Integrin Receptors............................................................................................21
vm
LIST OF FIGURES
Figure
Page
L I: Bovine Neutrophil Microbicidal M echanism s................................................................3
1.2: Structure o f the Rotavirus Virion Showing Viral Proteins......................................... 15
2.1: Analysis o f Purified Rotavirus by SDS-PAGE............................................................ 54
2.2: Western Blot Analysis o f Anti-Rotavirus Mouse A nti-Serum .................................. 54
2.3: Flow Cytometric Analysis o f Rotavirus B inding........................................................ 56
2.4: Rotavirus Binding to Neutrophil M em branes............................................................... 56
2.5: Radioactive Virus-Binding A ssay...................................................................................58
2.6: Scatchard Analysis o f Rotavirus Binding Curve for N eutrophils..............................58
2.7: Competition o f Radiolabeled Rotavirus Binding with Unlabeled V irus.................. 60
2.8: Analysis o f Rotavirus Internalization by Neutrophils..................................................60
3.1: Plaque Assay ofV iruses Exposed to N eutrophils........................................................78
3.2: Superoxide Production Induced by R otavirus............................................................. 78
3.3: Superoxide Production Induced by Opsonized R otavirus..........................................79
3.4: Chemotaxis to Triple Layered Particles........................................................................ 79
3.5: Chemotaxis to Double Layered Particles...................................................................... 81
3.6: Effect o f Rotavirus on Neutrophil Superoxide Production.........................................81
3.7: Effect o f Rotavirus on Neutrophil Migration, Calcein M ethod.................................82
3.8: Effect o f Rotavirus on Neutrophil Migration, LDH M ethod..................................... 82
3.9: Effect o f Rotavirus on Phagocytosis.............................................................................84
3.10: Neutrophil Chemotaxis to M A l04 Supernatants....................................................... 84
3.11: Chemotactic Activity o f 2.5 M OI Supernatants.........................................................86
3.12: Oxidative Burst Stimulated by 2.5 MOI Supernatants..............................................86
3.13: Chemotactic Activity o f 0.1 MOI Supernatants.................................................... ,...88
3.14: Neutrophil Chemotaxis After Exposure to Virus-Infected Cell Supernatants....... 88
3.15: Oxidative Burst o f Neutrophils After Exposure to Virus-Infected Supernatants.. 89
A .I: Testing Specificity o f M ab1.......................................................................................... 117
A.2: W estern Blot o f M abI ......... ......................................................................................... 117
A.3: Testing Specificity o fM ab 2 .......................................................................................... 119
A.4: Western Blot o f M ab2.................................................................................................... 119
IX
ABSTRACT
Neutrophils play an essential role in defending the host against bacterial and
fungal pathogens. Recently, it has been discovered that neutrophils also play a role in
viral clearance. In addition to inhibiting viruses, neutrophils can also be modulated by
viruses, which can either exacerbate illness or increase susceptibility to other infections.
Viruses mediate these effects either directly, by binding to neutrophils, or indirectly, by
the induction o f neutrophil-acting molecules. Because neutrophils express putative
rotavirus receptors, neutrophils are found at sites o f rotavirus infection in several animals,
and rotavirus induces secretion o f neutrophil activators in vitro, it was hypothesized that
neutrophils can bind rotavirus and that this interaction would modulate host defense
responses. Since rotavirus strains are species-specific, studies were designed to
characterize a homologous system consisting o f bovine neutrophils and the B641 calf
strain o f rotavirus. Studies were performed to first determine whether neutrophils bound
rotavirus using three complementary methods. These studies showed that rotavirus
specifically bound to neutrophils in a dose-dependent manner. In contrast, rotavirus was
not internalized by neutrophils after an extended period o f incubation at a temperature
permissive for internalization. Together, these data suggest that like many cell lines,
neutrophils possess a rotavirus attachment site but not the co-receptors or mechanisms for
internalization. In addition, exposure o f virus to neutrophils had no effect on virus
replication, suggesting that neutrophils do not play a role in clearance o f this particular
pathogen. We next evaluated the ability o f rotavirus to elicit or modulate neutrophil
responses such as respiratory burst, chemotaxis, and phagocytosis. In addition, the ability
o f rotavirus-infected M A l 04 cells to secrete neutrophil activators or inhibitors was
evaluated. We found that neither rotaviruses, nor supernatants from cells infected with
rotavirus, elicited neutrophil responses in any o f the parameters analyzed. Thus, the
binding o f rotavirus by neutrophils basically results in a nonproductive interaction.
Since these experiments were performed in vitro, it is possible that this interaction could
be different in the infected calf intestine. Nevertheless, the data obtained in these studies
provides an initial framework for developing our understanding o f the role o f neutrophils
in rotavirus infection.
I
INTRODUCTION
Neutrophils
Phagocytes, such as neutrophils, play a key role in the body’s innate immune
response to infection. These cells travel throughout the body in search o f pathogens and
are rapidly mobilized to sites o f infection or inflammation [1,2]. Indeed, neutrophils or
polymorphonuclear leukocytes (PMNs) are among the first responders o f the immune
system [3]. In addition to killing microbes themselves, neutrophils also contribute to the
acquired immune system through various signaling pathways [4]. Neutrophils are present
in all mammals, and cells with similar phagocytic functions are found throughout the
animal kingdom.
Neutrophils are produced from bone marrow stem cells in a process called
hematopoiesis. Activation o f neutrophilic stem cells results in formation o f myeloblasts,
which develop into myelocytes via active cell division. Subsequently, the myelocytes
withdraw from the cell cycle and develop into band cells and then mature neutrophils,
which have approximately an eight-hour lifespan in the bloodstream. Additionally,
certain stressors, such as bacterial infections, can accelerate neutrophil production [5].
Mature or segmented neutrophils account for 50 to 70 percent o f circulating leukocytes in
humans, but only 20 to 30 percent in cattle [6].
The essential role played by the neutrophils in protecting the host from infection
is unequivocally demonstrated by genetic disorders in neutrophils that impair either their
production or function. These disorders lead to severe, life-long bacterial and fungal
2
infections and must be treated with antimicrobial agents, neutrophil growth factors, and
possibly bone marrow transplants [5].
Neutrophil Functional Responses
The immediate host response to infection is a transient neutropenia resulting from
increased margination and accelerated delivery o f neutrophils to sites o f infection.
Within an hour, neutrophils are released from the bone marrow reserve into the
bloodstream. In the early phases o f infection, the circulating half-life o f neutrophils is
shortened, and cell turnover is accelerated. However, the circulating half-life normalizes
over time. In prolonged inflammation or stress, there are increased numbers o f band cells
(immature neutrophils) in the circulating blood representing a depletion o f the neutrophil
storage pools. In addition to changes in the neutrophil blood count, neutrophil
morphology and function can be altered during infection [7],
Neutrophils undergo a number o f functional responses in response to the presence
o f pathogens, outlined in Figure 1.1. In order for neutrophils to respond effectively to
infections, they must exit the bloodstream and move into the tissue. This process
involves a complex interplay o f rolling, tight adhesion, and transmigration [7,8].
Currently, the molecular aspects o f these systems are being defined. Neutrophils survey
the vascular endothelium for inflammatory signals by rolling along the margin o f blood
flow. This process is mediated, in part, by L-selectin, which binds to adhesion molecules
on the endothelial surface, causing the cell to slow down and “roll” along the endothelial
wall [9]. From this location, neutrophils can detect chemical signals derived either from
the invading organism, such as chemotactic peptides or endotoxin, or from the
O
Figure L I. Bovine Neutrophil Microbicidal Mechanisms
Reprinted with permission from Peggy Bunger
4
damaged tissue, such as interleukin -8 (TL-8) [10,11], DiBusion o f these signals from the
site o f infection creates a concentration gradient, which can be sensed by the rolling
neutrophil, even at a 0.1% difference [12]. These chemical signals modulate neutrophil
responses by altering actin polymerization, activating or up-regulating adhesion
receptors, such as L-selectin [9,13], and stimulating intracellular calcium fluxes [11].
The encounter o f rolling neutrophils with inflammatory signals induces the
activation or upregulation o f adhesion receptors such as the (32 integrins, which mediate
tight adhesion to the vascular wall and subsequent shedding o f L-selectin [14,15], The
adherent cells then squeeze through (i.e., diapedesis) the endothelial layer and move into
the tissues, where they subsequently migrate (i.e., chemotaxis) to the site o f injury [16].
The importance o f this extravasation process is noted in humans and cattle with a
mutation in the fb integrin molecule CD 18 [17], This defect is known as leukocyte
adhesion deficiency (LAD in humans or BLAD in bovines), and is characterized by
delayed wound healing, recurrent subcutaneous and mucosal infections, and chronic skin
ulcers without pus [5],
Once neutrophils have entered the tissue, their main functions are to search, find
and destroy pathogens and to interact with the other cells o f the immune system to clean
up and repair sites o f infection and inflammation. Foreign particles, such as a bacteria or
fungi, are ingested by these cells in a process called phagocytosis. Neutrophils can ingest
some particles without coating by serum components (opsonization); however,
opsonization with either antibodies or the complement component C3b dramatically
increases the efficiency o f phagocytosis [I I]. Foreign particles are ingested into a special
5
endocytotic vesicle called the phagosome. Adter this vesicle is internalized, cytoplasmic
granules fuse with the phagosomal membrane to form the phagolysosome. These
cytoplasmic granules contain stores o f antimicrobial enzymes and peptides that can be
quickly delivered to the phagosome upon neutrophil activation [18]. Neutrophil granules
possess oxygen-independent microbicidal mechanisms (cationic proteins, lactoferrin, acid
hydrolases, etc.) as well as oxygen-dependent microbicidal mechanisms (NADPH
oxidase, myeloperoxidase, etc.) [19]. These mechanisms will act in concert to kill the
intruders [20,21]. While human neutrophils contain three distinct granules (azurophil,
specific, and secretory), bovine neutrophils contain a fourth granule known as the large
granule. Large granules contain bactenecins, which are cationic antimicrobial proteins
[22]. Humans lack large granules as well as bactenecins [18], but instead store lysozyme
and catalase [23]. In both humans and cattle, it appears that the various neutrophil
granule species are differentially mobilized depending on the severity o f the stimulus or
type o f pathogen encountered [19,24].
The generation o f microbicidal oxidants by neutrophils (also known as the
respiratory burst) results from the activity o f a multi-protein enzymatic complex known
as the NADPH oxidase, which can be found either in the phagolysosomal membrane or
in the plasma membrane. Thus, neutrophils can utilize the NADPH oxidase to kill
organisms intra- and extracellularly. Altogether, there are currently seven proteins
reported to be associated with the NADPH oxidase assembly [25]. In resting neutrophils,
these protein components are segregated into cytoplasmic and plasma membrane
compartments. However, during assembly and activation o f the NADPH oxidase, the
6
cytosolic protein components translocate to the membrane where they assemble around a
central membrane-bound protein known as flavocytochrome b. During the respiratory
burst, electrons are transferred from NADPH to molecular oxygen, producing superoxide
anion (CV), the direct product o f the NADPH oxidase. CV is then rapidly converted to
secondary toxic oxygen species such as hydrogen peroxide (H 2O 2), hydroxyl radical
(OHe), and hypochlorous acid (H0C1), which can efficiently kill microorganisms and, in
combination with the primary and secondary granule contents, are the chief host defense
mechanism used by neutrophils [25,26], Mutations in any o f the oxidase components
cause chronic granulomatous disease (CGD), a genetic defect characterized by frequent
bacterial and fungal infections [ 11].
Because products o f degranulation and the oxidative burst are toxic, neutrophils
employ several mechanisms to minimize host tissue damage. First, the neutrophils must
be specifically activated to release these compounds. In addition, neutrophils often do
not respond to a stimulus unless they are first “primed.” Priming agents include
bacterially derived factors, such as endotoxin (lipopolysaccharide or LPS), and tissuederived factors, such as tumor necrosis factor-a (TNF- a), granulocyte-macrophage
colony-stimulating factor (GM-CSF), complement component C5a, and platelet­
activating factor (PAF) [27]. For example, prior exposure o f bovine neutrophils to TNFa or C5a increases their ability to phagocytose Staphylococcus aureus [28].
In addition to the regulatory mechanisms governing neutrophil activation, as
described above, neutrophils also possess a number o f defense mechanisms to scavenge
neutrophil-generated reactive oxygen species. These include superoxide dismutase
7
(SOD), which converts O2 ' into H 2O2 [29], and catalase, which detoxifies H 2O2. Given
the vast amount o f toxic agents neutrophils bring to bear against pathogens, it is clear that
the regulation o f neutrophil reactivity is essential to avoid inadvertent damage to host
tissues. Indeed, inappropriate neutrophil responses have been implicated in causing
tissue damage associated with inflammatory diseases such as ischenua-reperfusion injury,
rheumatoid arthritis, and acute respiratory distress syndrome [27].
Role o f Neutrophils in Viral Infections
The role that neutrophils play in viral infections is not well understood.
Rhinovirus, influenza A virus (IAV), and Coxsackie virus all induce neutrophilia in the
blood, especially early in infection. Neutrophils are found at sites o f viral infection with
varicella-zoster and respiratory syncytial virus (RSV), and neutrophils have also been
found in cerebral spinal fluid during viral meningitis [30]. Importantly, these results have
been confirmed in vitro, since many viruses induce the secretion o f neutrophil
chemokines by host cells. For example, endothelial cells infected with cytomegalovirus
(CMV) secrete CXC chemokines, such as GRO-a and IL- 8. In fact, these cells secreted
nearly IO"9 M, which is a sufficient concentration to induce chemotaxis o f both human
and bovine neutrophils [31].
Since neutrophils are found at sites o f viral infection, it is not surprising that they
can play a role in defense against viral pathogens. However, anti-viral activity does not
appear to be essential in many cases, as neither LAD nor neutropenia patients have
increased susceptibility to viral infections [5]. Nevertheless, the neutrophil’s role may be
subtle and may go unnoticed in diseases with abundant bacterial and fungal infections.
8
Several viruses can be phagocytosed by neutrophils, including vaccinia, herpes simplex
virus (HSV), IAV, poliovirus, adenovirus, coxsackie virus, and CMV [30]. Human
neutrophils are capable o f inactivating HSV in vitro, especially when it is opsonized with
either antibodies or complement [32]. The bovine bac7 bactenecin, an antimicrobial
protein stored in neutrophil large granules, can inactivate both H SV l and 2 in vitro
[22,33], Neutrophils can also inactivate unopsonized vaccinia virus, an effect most likely
due to O 2" production, since neutrophils from CGD patients were not able to inactivate
vaccinia [34]. Neutrophils can degrade Japanese encephalitis virus as well [35]. This
degradation is often assisted by toxic oxygen species, which can attack both lipid and
protein components o f the viral particle [34,35].
Some viruses take advantage o f neutrophil phagocytosis in order to spread. CMV
infects neutrophils in the bloodstream [31], and although its replication is abortive, there
is evidence that migrating neutrophils help spread CMV infection in the body (abortive
transfer). It has been suggested that HIV may also spread to T cells from neutrophils in a
similar fashion [36]. In immunocompromised patients, CMV is often associated with
neutrophils, whereas in patients with healthy immune systems, CMV is primarily found
in monocytes [31].
In addition to directly inactivating viruses, neutrophils also kill virus-infected
cells. Bovine neutrophils inhibited replication o f bovine rhinotracheitis virus (IBR) in
cell culture in the absence o f antibody or complement [30]. Similarly, human and mouse
neutrophils were able to kill cells infected with vesicular stomatitis virus or reovirus [30].
However, this process usually requires antibodies, and killing is mediated through
9
antibody-dependent cellular cytotoxicity (ADCC). ADCC occurs when virus-infected
cells have been coated with antibodies. Various leukocytes have receptors that recognize
the Fe portion o f the antibody, and after binding will induce cell lysis [37]. ADCC by
neutrophils occurred with !BR, HSV, VZV, and IAV, although neutrophils were not as
effective as lymphocytes at killing virus-infected cells. This killing was not superoxidedependent, since neutrophils from CGD patients were able to perform similarly [30].
Complement can also mediate cell killing, and bovine neutrophils are capable o f
destroying herpesvirus-infected cells in the presence o f complement [38].
Many viruses have evolved ways o f circumventing or inactivating various arms o f
the immune response. Viral infection often causes transient neutropenia, especially in
children, and this decrease o f neutrophils in the bloodstream can cause an increase in
bacterial or fungal colonization [5]. A German physician named Clemens von Pirquet
noted in 1908 that viral infections could induce susceptibility to bacteria. He observed
that a “filterable” substance could lead to measles as well as superinfection with other
organisms [39]. Today scientists recognize that many different viruses behave in this
manner, somehow altering the host and paving the way for future disease. This viralinduced susceptibility to bacterial infection is no doubt caused by several factors, one o f
which may be simply tissue damage. However, growing evidence suggests that this story
is more complex, and involves the inactivation o f neutrophils by viruses.
The best understood example o f viral-induced neutrophil dysfunction occurs with
influenza A, and patients with IAV become susceptible to certain bacterial and fungal
superinfections, especially otitis media, pneumonia and sepsis [39]. Lung damage cannot
10
account for this phenomenon, since other respiratory viruses capable o f causing similar or
greater lung pathology are not associated with these superinfections [39]. Neutrophils
incubated with unopsonized IAV particles have many altered functions in vitro, including
impaired chemotaxis, oxidative burst, and bacterial phagocytosis and killing [40].
Studies performed in vivo confirmed these results. For example, neutrophils from the
sputum o f patients with IAV were isolated and tested for their ability to secrete lysozyme
and kill bacteria. Both functions were downregulated in IAV patients [41]. IAV binds
directly to sialylated glycoproteins on neutrophils (e.g., CD43 or CD45) via its HA, or
hemagglutinin, antigen and thus interferes with receptor-mediated signaling pathways
involving G proteins and calcium. The net result is an interference with superoxide
production, phagosome-lysosome Iusionj, and normal response to activators [42].
Other viruses inhibit neutrophil responses in a similar fashion. The echo-9 virus
induces changes in the human neutrophil membrane, which alters its ability to respond to
the potent chemoattractant V-formyl-methionine-leucine-phenylalanine (fMLF) [43,44].
Neutrophils that were incubated with Newcastle disease virus (NDV) exhibited a 50%
reduction in their phagocytic ability, an 80% reduction in reactive oxidant generation
stimulated by zymosan and phorbol myristate acetate (PMA), and a decreased ability to
kill S. aureus. Part o f this inhibition was mediated by the interaction between the viral
neuraminidase and the cell membrane, and blocking this protein abrogated virusmediated inhibition o f neutrophil function [45].
In addition to direct inactivation by viruses, neutrophils can also be inactivated by
viral-induced cytokines. For example, neutrophils from both children and adults infected
11
with HIV showed a decreased ability to produce O2" and H 2O2 [46]. Neutrophils from
AIDS patients had impaired ADCC and complement systems [47,48], decreased
production o f the inflammatory mediator leukotriene B 4 [49], and decreased surface IL -8
receptor expression [50]. Since HIV does not integrate its genome into neutrophils, it is
thought that it alters tissue cytokine concentrations, resulting in neutrophil inactivation
[49]. M ost likely, HIV either alone or in combination with opportunistic infections
disrupts the normal cytokine profile. Indirect evidence indicates that the specific effects
include a decrease in granulocyte colony-stimulating factor (G-CSF) and an increase in
interleukin-4 (IL-4) and interleukin-10 (IL-10), although this has not been proven
conclusively [51].
Since AIDS is characterized by the inability to clear normally harmless bacterial
and fungal infections, reversing these neutrophil dysfunctions is an attractive idea for
treatment. For example, interleukin-15 (IL-15), a recently discovered potentiator o f
neutrophil responses, increased chemotaxis and fungicidal activity o f neutrophils from
HIV patients in vitro [51]. HIV exposed to defensins, which are potent antimicrobial
cationic proteins produced by human neutrophils, did not replicate as efficiently [52].
Patients who were treated with the neutrophil growth factor GM-CSF showed an increase
in their leukotriene B 4 production [49]. Since the HIV cocktail drug therapy is expensive
and often ineffective, these are promising avenues for inclusion in treatment o f AIDS
patients.
Rhino virus, a respiratory virus that causes the common cold, also alters the
neutrophil response via effects on cytokine profiles. In this case, however, the result is
12
the over-activation o f neutrophils. This non-enveloped virus induces the release o f
inflammatory cytokines such as IL -8 and G-CSF, which cause an influx o f leukocytes,
including neutrophils, and thus an exacerbation o f airway inflammation [53,54], Based
on this host response, it is not surprising that bacterial superinfections are not associated
with rhinovirus infection [53]. Japanese encephalitis virus will also induce the secretion
o f IL- 8, and higher levels o f this cytokine correlate with greater severity o f illness [55].
Respiratory syncytial virus (RSV) is the most frequent cause o f bronchiolitis, and is
linked to asthma [56]. Neutrophils are found in abundance in the airway upon RSV
infection and are recruited by IL -8 released from infected epithelial cells. Neutrophils
adhere to these cells and kill them, which contributes to the ensuing tissue damage. It is
intriguing that neutrophil apoptosis is upregulated in RSV patients and is perhaps the
body’s way o f trying to limit neutrophil-mediated damage. Unlike with HIV, treatment
o f these viral infections could involve inhibition o f neutrophil responses. For example,
treating RSV-infected cells with the corticosteroid fluticasone propionate inhibited their
IL -8 production [56] and could potentially limit the level o f inflammatory cell
recruitment in vivo.
Rotavirus
Rotaviruses are non-enveloped viruses within the family Reoviridae. They are a
major cause o f acute gastroenteritis in mammals and birds, infecting cells in the small
intestine [57,58]. Six different rotavirus groups have been characterized, and whereas
groups A - C cause disease in both humans and animals, groups D - F only infect
13
animals [59]. Bovine rotavirus was the first group A rotavirus to be isolated in cell
culture and was characterized by C A . Mebus in 1969 [60]. Ruth Bishop then discovered
human rotaviruses in 1973 [61]. Group A rotaviruses are the most common, causing
severe diarrheal disease in the young o f many mammalian species. Group B rotaviruses
can infect adults, although cases are localized primarily to China and India [62,63].
Group C rotaviruses were first reported in Australia, but were later found throughout
Europe and Japan [64]. Little is known about non-group A viruses, since they are
difficult to culture and have no routine diagnostic test [64].
Rotavirus Proteins
The rotavirus genome consists o f 11 segments o f double-stranded RNA, which
code for at least one protein (Table I). Fully infectious particles are triple layered, with
the three layers consisting o f the core (VP I, VP2, VP3, and RNA), the second layer made
up o f V P 6, and the third layer made up o f VP4 and VP7. A schematic structure o f the
rotavirus virion showing the locations o f the viral proteins and a 3D reconstruction based
on the virion crystal structure are shown in Figure 1.2. VP 6 makes up 51% o f the total
rotavirus protein. VP4 is the viral attachment protein, although VP7 most likely plays a
role in viral entry and uncoating [65]. Trypsin cleaves VP4 into two proteins, VP5* and
VP 8*. This cleavage is necessary for injectivity but not for binding and somehow
stabilizes the VP4 spikes [66]. VP7 is glycosylated, binds calcium, and resides in the
endoplasmic reticulum (ER) until virus is shed. The SAl I rotavirus strain VP7 contains
I glycosylation site, whereas the UK bovine strain contains 3; therefore its molecular
weight varies among strains [67]. After the double-layered particle is formed, the
14
particles bud through the E R and acquire a transient cellular envelope, which is lost as
mature particles exit the cell [65].
Protein
M W (kD a)
RNA
VPl
VP2
VP3
VP4
125
94
I
VP5*
VP 6
VP7
V P 8*
N SPl
NSP2
NSP3
NSP4
NSP5
N SP 6
60
41
38
28
53
35
34
28
26
?
88
88
2
3
4
4
6
9
4
5
8
7
10
11
11
Function
RNA polymerase
Binding to RNA
Guanylyltransferase
Viral attachment, hemagglutinin,
putative fusion protein, forms dimers
Trypsin cleavage product o f VP4
Forms trimers, major structural role
Budding and assembly o f virus in ER
Trypsin cleavage product o f VP4
Binding to RNA
Binding to RNA
Binding to RNA
Receptor in ER, enterotoxin
Binding to RNA
Unknown, truncated form o f NSP5
Location
Core
Core
Core
Outer layer
Outer layer
Inner layer
Outer layer
Outer layer
Nonstructural
Nonstructural
Nonstructural
Nonstructural
Nonstructural
Nonstructural
Table 1.1: Rotavirus Proteins. Modified from Fundamental Virology, Bernard N Fields
et al, 3rd Edition, 1996, Lippincott Wilhams and Wilkins, Chapter 25 by MK Estes [59].
Molecular weights are from the simian S A -II strain.
Because it has a segmented genome, rotavirus is capable o f reassortment, which
makes both serotyping and vaccine development problematic. There are two antigens
that define the genotypes, G and P. The G serotypes are defined by the sequence o f the
glycosylated VP7, and the P serotypes are defined by the proteolyticahy cleaved VP4.
As o f 1992, there were 101 different G genotypes and 78 P genotypes described for
Group A rotaviruses [68]. Human rotaviruses are generally G types 1-4, but recently G 6,
8, and 10 have been isolated from infected children [69]. Bovine rotaviruses tend to be
15
G 6, 8, or 10, and P5 or P l I, with 90% o f isolates containing one o f these serotypes [70].
The strain used in this study, B641, is serotype G 6 and P5 [70,71]. This strain has a 94%
similarity with the UK calf strain in the VP4 protein, but only a 73% similarity with the
NCDV strain, which drops to 53% when comparing the VP 8* cleavage product [70].
Figure 1.2: Structure o f the Rotavirus Virion Showing Viral Proteins. Figure modified
from dsRNA Virus, RNA/ Protein Tables: Edited by P.P.C. Mertens & D.H. Bamford
(Original by Mossel, Estes, and Ramig).
Cellular Rotavirus Receptors
After rotavirus enters the host digestive tract, the next step is binding and entry
into the host cell. Viruses utilize specific receptors to invade, and these receptors are an
absolute requirement. However, the presence o f the receptor does not automatically mark
a cell for viral infection; it must also have the correct machinery. For instance, a virus
that requires a nucleus for replication will soon be in trouble if it enters a mature
enucleated red blood cell. Since rotavirus exhibits specificity on several levels including
age, species, and tissue, determining what factors mediate its tropism are o f great interest
to researchers. One major component o f this tropism is most certainly the receptor. In an
16
exhaustive study, it was shown that if rotavirus was introduced into cells by Hpofection,
thus bypassing the binding and entry steps o f infection, it was able to replicate in all cells,
albeit not equally [72]. These results indicate that infection with rotavirus is primarily
limited by the presence or absence o f receptors; however, its replication efficiency can be
mediated by other factors as well.
In general, viruses can often bind non-specifically to host cells and can also have
alternate receptors [59]. Because o f these problems, the cellular receptor(s) for any
rotavirus strain are still uncharacterized. M uch o f what is known about these receptors
has been gleaned from studies in tissue culture with the primate strains RRV or SAl I, or
the human W a strain [73]. The most common cell line used to propagate rotavirus is
M A l 04, an African green monkey kidney cell line. From M Al 04 studies, it was
determined that the initial binding o f the virus to the cell requires sodium and calcium,
and can occur between pH 5.5 and 8, consistent with the small intestine tropism o f
rotavirus [73]. There are about 13,000 binding units per cell, all o f which appear to have
the capability to internalize [73]. The virus most likely enters the cell directly, since
endocytosis inhibitors such as sodium azide or dinitrophenol did not decrease viral entry
[74]. Direct penetration seems to require trypsin-cleaved VP4; however, a trypsin-treated
porcine strain (OSU) was found associated w ith coated pits, vesicles, and secondary
lysosomes, indicating that endocytosis can be a mode o f entry for some strains [75]. The
emerging paradigm for rotavirus is that two or three receptors are involved: one for initial
attachment and one or two for inactivity [72]. Therefore, several different interactions
between virus and cell surface receptors occur during infection [76]. However, it is still
17
unclear whether these receptors are glycoproteins or glycolipids. Cells extracted with a
low concentration o f detergent, followed by treatment with cyclohexamide could still
replicate the porcine strain CRW 8, but not NCDV or Wa, indicating that the former may
be involve a glycolipid-based receptor, and the latter two a glycoprotein [77].
One possible approach to elucidate viral receptors is to separate membranes on a
gel and probe with virus. This method, known as the virus overlay protein blot assay, or
VOPBA, has given mixed results [77-79]. NCDV bound to several bands between 80
and 116 kD a in HT29 membranes, and both Wa and NCDV bound to a 95 kDa protein on
M A l 04 membranes [77]. This same laboratory previously reported that NCDV
recognized three other bands on M A l 04 cells between 66 and 97 kDa, and that galactosespecific lectins were important [78]. Yet another group found five proteins in M Al 04
membranes that bound to KRV, at 30, 45, 57, 75, and HO kDa [79]. However, binding
studies such as this are complicated by several problems. These studies utilized reducing
gels and, therefore, the native protein conformations were lost or changed. Additionally,
contamination with particles that have lost their outer layer (i.e., double layer particles or
DLPs) could be contributing to non-specific binding. DLPs did not bind to whole cells or
to membrane fractions in some studies [80,81] but did bind in a W estern blot to
. asialogangliosides isolated from cells [82]. Perhaps the most problematic aspect o f
binding studies is that rotavirus binds promiscuously to many cell types. A recent study
o f 46 cell lines from a variety o f tissues and species showed that human and simian
rotaviruses bound to all cell lines tested, but did not necessarily infect and replicate in the
18
same cells [72]. Thus, the conclusions about rotavirus infection derived from binding
studies may be misleading.
Binding studies performed with various inhibitors have shown that ganglioside
residues are important in binding to rotavirus both in vivo and in vitro. A ganglioside is
an anionic glycolipid containing one or more units o f sialic acid [83]. A study with SAl I
showed that G M l gangliosides were important in binding to LLK cells [84]. Also,
NCDV and UK bovine viruses bound to gangliosides, but each to a slightly different
profile [80]. Infection studies overcome the problems with binding described earlier. It
was found that treating M A l 04 cells with tunicamycin, which blocks N-glycosylation,
decreased inactivity o f several primate rotavirus strains, but did not decrease binding
[79]. N-glycosylation links sugars to asparagine residues, and O-glycosylation links
sugars to a serine or threonine residue [83]. Therefore, these studies suggest one o f the
receptors may also be an N-glycosylated protein.
The role o f sialic acid as a component o f rotavirus receptors is still under
investigation, and seems to vary depending on not only the strain, but also the host
species [82,85-87]. Sialic acid is a generic name for a 40-member family o f acidic 9carbon monosaccharides. These residues are found on gangliosides and other
glycolipids, as well as glycoproteins. M ost researchers report that animal viruses such as
rhesus rotavirus (RRV) are sialic acid-dependent and utilize V P 8*, the smaller o f the VP4
cleavage products, for attachment, but human strains such as Wa are sialic acidindependent and utilize VP5* [85,88,89]. However, exceptions to this rule do exist. The
ability o f the milk lactadherin to bind to four human strains o f rotavirus was lost after
19
treatment with neuraminidase, inconsistent with the classification o f these viruses as
sialic acid-independent [90]. Additionally, two RRV variants were isolated which no
longer needed sialic acids to infect cells, and treatment o f cells with two different
nemaminidases did not affect infectivity [88]. A study comparing two strains o f bovine
rotavirus found that NCDV is neuraminidase-sensitive and hemagglutinates, and UK is
neuraminidase-insensitive and does not hemagglutinate [80]. Because B641, the bovine
strain used in this study, is the same P serotype as UK (P5), it is probably also
neuraminidase-insensitive and non-hemaggutinating; however, this has not been tested.
Usually sialic acid dependence and hemagglutination activity are related, but some RRV
variants have been described that were neuraminidase-insensitive but could still .
agglutinate red blood cells [88].
Conflicting reports in the literature may be due to the different activities o f
various neuraminidases [83]. In one study, neuraminidase from C. perfringens was
ineffective at abolishing rotavirus binding to brush border membranes in a Western blot,
but neuraminidase from A. ureaficans was able to block binding [91]. Some researchers,
have suggested that all strains are actually sialic acid-dependent, including all human
strains, and the “escape variants” are really binding to internal sialic acids, that are missed
by neuraminidase [80]. This conclusion is supported by the fact that a cell line lacking
sialic acid residues could not sustain human rotavirus infection [82]. However, there
could be side effects to these mutant cells, since tunicamycin, which blocks all N-Iinked
glycosylation, can also inhibit protein synthesis [83]. Other groups feel that all strains are
actually sialic acid independent, and that observed effects were due to the presence o f a
20
similar glycan [77]. The data suggest that both groups are partially correct - some
viruses are sialic acid-dependent under certain circumstances, but can escape that
dependence with mutations or by infecting a different cell line. It is clear that the viruses
classified as sialic acid-dependent are very selective about the specific sugar residues.
For instance, the sialic acid containing lactadherin binds to human RV, but other sialic
acid containing oligosaccharides in milk do not, which suggests the requirement for a.
highly specific carbohydrate structure for full anti-rotaviral activity [90]. This specificity
no doubt also applies to actual virus receptors.
For strains requiring a protein receptor, several lines o f evidence indicate that one
o f these proteins could be an integrin. P2 integrins are N-glycosylated with high-mannose
type oligosaccharides and also contain sialic acid residues [92]. These glycoproteins are
heterodimers composed o f one a and one p subunit, are expressed in many cells, and
implicated in several pathways from fetal development to immune function. Their
ligands vary depending on the specific function, but often contain an RGD protein
sequence [93]. Because combinations o f these subunits can confer diversity in ligand
recognition, integrins are attractive virus receptors, and may confer species- or tissuespecific tropisms. Table 2 highlights the integrins that may be receptors for rotavirus.
Several viruses, including adenovirus, Coxsackievirus, echoviruses, hantaviruses,
papillomavirus, and foot-and-mouth disease virus all use integrins as a receptor, ctvPs
being the most common [94]. Similarly, in vitro studies indicate that integrins could act
as rotavirus receptors. One group measured integrin expression via flow cytometry
between permissive and non-permissive cell lines and found a correlation between
21
Integrin
a2J31
a2
BI
a4Bl
a4
BI
aXB2
aX
B2
a5B3
a5
B3
AKA
VLA-2
CD49b
CD29
VLA-4
CD49d
CD29
kDa
300
170
130
280
150
130
p i 50/95 245
C D llc 150
95
CDl 8
CD51
CD61
265
150
115
Experimental Data
Inducible on rat, human neutrophils
N ot present on resting K562s
Upregulated by PMA in K562s
Found on RV-permissive cells
Transfection into K562 cells conferred
binding and infection o f SA l I virus
Transfection into CHO cells conferred
infection o f several strains
Antibodies do not block binding o f RV
VP4 contains ligand site DGE
Expressed on most cells o f adult and
infant small intestine
fibronectin Found on neutrophils
N ot on M A l04, Caco-2 or K562s
VCAM l
but antibodies blocked RV infection
Transfection into K562 cells conferred
SA l I binding, infection
VP7, NSP2 contains ligand site LDV
Ligands
collagen I
laminin
fibrinogen
fibrinogen Found on neutrophils
N ot present on K562s
LPS
VP7 contains ligand site GPR
C3bi
Expressed on adult human enterocytes
vitronectin Found on RV-permissive cells
N ot on CHO, BHK, K562s
Upregulated by PMA in K562s
Abs block infection, not binding, o f
S A -Il in M A l04s
Transfection into CHO cells conferred
S A ll infection
Expressed on adult, infant enterocytes
Refs
224
24
16
16
243
243
6
180
24
6,24
16
6
37
24
6
27
14
14
14
14
14
175
Table 1.2: Putative Integrin Receptors.
expression o f several integrins and ability to sustain replication o f monkey and human
rotaviruses [95]. Both permissive and non-permissive cell lines expressed Pi, whereas all
permissive adherent cell lines expressed c^pi and UxPz- Only the permissive KD cells, a
22
human rhabdomyosarcoma line, expressed the a 4Pi integrin. Rotavirus growth levels also
matched expression o f a? integrin in all permissive cell lines. The K562 cell line, which
is often used as a negative control, did not express any o f these integrins except Pi [95].
Transfection studies have provided direct evidence that integrins can function as
virus receptors. Transfection o f CHO cells with OtvPs increased viral infectivity o f three
primate strains, whether or not they were neuraminidase sensitive [94]. Antibodies to
this integrin or vitronectin (the receptor ligand) blocked replication but not binding.
Interestingly, this particular interaction was RGD-independent [94]. Insertion o f either
a 2pi or OLtpi into K562 cells allowed them to replicate SA l I rotavirus, whereas a control
<23transfection did not. Again, adding antibody to the appropriate integrin blocked
infection. PM A treatment o f the K562 cells also increased their ability to sustain
rotavirus infection, which is known to upregulate the a 2 subunit [96].
Viral sequence data offers more p ro o f that integrins are receptors, since rotaviral
proteins appear to contain three separate integrin ligand motifs [97]. VP5*, the larger b f
the VP4 cleavage products, contains a m otif similar to type I collagen, which binds a 2pi.
In the SA l I strain, this sequence is DGE, and in the bovine strains it is DGV. VP7, the
other outer layer capsid protein, contains the other two motifs. A fibrinogen-like
sequence, GPR, which binds to axpz, was found in all mammalian rotaviruses. A
fibronectin-like site, which binds to a 4pi, was also found in VP7. SA l I contains the
sequence LDV, but variations such as LDI or IDI are seen in other animals. In addition
to these motifs, about 87% o f rotaviruses have the classic RGDE sequence motif, a
hallmark o f integrin binding, and peptides containing these sequences blocked infection
23
o f viruses to cells by 34 to 90%. Anti-integrin antibodies also blocked infection, and
combining antibodies produced an additive effect, implying that more than one receptor
acts to internalize rotavirus, at least in M A l 04 cells [97].
Receptors for Rotavirus In Vivo
In vitro studies indicate that rotavirus can bind to nearly all cells tested and can
replicate to some extent in these same cells if entry was bypassed [72]. Thus, the virus
specificity could be primarily mediated by the presence o f the secondary internalization
receptor or receptors, but efficient replication involves other factors. Since rotavirus
infects such a narrow population o f cells in vivo, this seems to contradict the above
findings indicating that rotavirus can bind to and infect cells from many types o f tissues
including bone, breast, stomach, and lung, as well as intestine [72]. One resolution o f
this paradox could be that rotavirus does not infect these tissues in vivo because it never
gets the chance. Clinical cases have been described where rotavirus does occasionally
find its way into other tissues and induces damage. This phenomenon is seen in
immunocompromised patients such as transplant recipients [98]. In one study, twenty
percent o f adult kidney transplant patients (4 out o f 20) developed a nosocomiallyacquired rotavirus infection [98]. Other extra-digestive disorder associations have been
found with rotavirus, indicating that it does have the potential to bind and infect other
tissues. A recent report o f two fatal rotavirus infections, one in a I year old and one in a
4 year old, detected viral RNA by in situ PCR in the heart and the central nervous system.
These results correlated with the clinical observations o f a systemic virus infection in
those organs [99]. However, lack o f exposure to tissues cannot be the only explanation
24
for the usually narrow tropism. Rotaviruses have been found in aerosols and, although
they infect a lung-derived cell line in vitro, rotavirus replication in the lungs is not
observed. Aerosolized rotavirus is thought to contribute to nosocomial outbreaks, but
'
most likely through the transfer o f particles to the digestive tract by mucociliary action
[100].
Since rotavirus infects enterocytes in the small intestine, this is obviously a good
place to start looking for the receptor. The stem cells in the gut are called crypt cells, and
these give rise to four cell types: enterocytes, goblet cells, Paneth cells, and
enteroendocrine cells, which together make up the intestinal brush border membrane
(BBM) [101]. Crypt cells will divide and chum out undifferentiated cells, which at some
point undergo a switch, probably due to some signal, such as a protein called M athl in
the Notch pathway. These cells then stop dividing, express specialized proteins, and
migrate up the villi. Once the cells reach the top, they undergo apoptosis. This process
causes a complete replacement o f the intestinal epithelium every 2-3 days in rats and
every 4-6 days in humans [102]. The factors that determine each particular cell lineage
are still not clear; however, the position along the crypt-villus axis is an important
determinant [ 101].
Several laboratories have tried to find the receptor in actual intestinal epithelial
cells, such as with the VOPBA assay described earlier. In one report, murine brush
border membranes were probed with RRV. The virus did bind more to villus cells than
crypt cells, protein was required for binding, and both N- and O-Iinked sialic acids were
implicated in the binding. Adult BBM did not bind virus as well, correlating with the
25
invincibility o f adult animals to RRV disease. The JEull triple layered particles (TLP)
bound to two bands at 300 and at 330 kDa, but when DLPs were used, the authors noted
several contaminating bands between 120-200 kD a [91]. The bands appearing with TLPs
were significantly higher than those found with M A l04 cells (bands between 30 to HO
kDa), calling into question the relevance o f these studies [78,79]. I f the proteins are in
fact integrins, then perhaps they have different glycosylation patterns than those found in
cell culture. The possibility that the higher bands correspond to intact integral
heterodimers is remote, since all labs used the same reducing, denaturing gels.
Similar studies were carried out with porcine OSU strain and pig enterocytes.
Interestingly, DLP were unable to bind these blots [103]. These studies highlighted the
importance o f monoganglio sides with internal sialic acid residues. The specific
gangliosides which bound virus correlated with the ganglioside pattern o f susceptibleaged pigs (NeuGcGM3 and NeuAcGMB) [104]. These results invite speculation that
perhaps age-related infection is due to loss o f the appropriate ganglioside or other sugarbased receptors as the animal weans. In the mouse small intestine, a 2-week-old has
GM3, G M l, and G D la gangliosides, which all decrease after weaning [105]. In the rat
intestine, there was a decrease in the amount o f sialic acid residues, and a concomitant
increase in JEucosyl residues during the weaning period [106]. Since rats and mice both
lose susceptibility to rotavirus infection shortly after weaning, this hypothesis has some
credence. However, the human and bovine situations must be different, since children
are susceptible to infection at least a year or tw o after the cessation o f breast-feeding
[119]. In addition, the ganglioside pattern in bovine intestine does not correlate as well
26
with age and rotavirus susceptibility. For instance, GM l gangliosides bound to the calf
U K strain, but these glycolipids are not decreased in the intestine with age or cessation o f
weaning [80].
I f integrins do function as co-receptors, then they should be expressed in the
appropriate intestinal cells. Adult human enterocytes express ctxPi, cbpi, and U5Ps [107].
The 012P1 integrin is ubiquitously expressed in the adult and infant small intestine, but is
more associated with crypt cells, whereas 03Pi was found more on villus enterocytes
[108,109]. This finding is hard to reconcile with in vitro data that transfection o f the 013
integrin subunit did not upregulate the ability o f K562 cells to replicate virus, whereas 0.2
and (X4 did [96]. However, there have been conflicting studies with (X2P 1, with some
reports finding it only in crypts, and others finding it on apical surfaces o f cells [HO].
The
Ot5 P3 integrin
is also found in neonates, is a co-receptor for adenovirus, and localizes
to the basolateral surface [111]. These results indicate that while integrins could be
functioning as receptors, they cannot be the primary determinant o f virus tropism, since
they are on the non-permissive crypt cells as well as adult tissue.
N ot only do the receptors need to be present in enterocytes, they also need to be
accessible. Studies done in Caco-2 cells are helpful to elucidate in vivo information,
because they differentiate into a fetal enterocyte-like cell. Caco-2 cells show a diffuse Pi
expression pattern, but when grown in co-culture with mesenchymal cells, this integrin
became associated with the basolateral surface, implicating its involvement in
epithelial/mesenchymal interactions [112]. In fact, expression o f m ost integrins was
increased in immature Caco-2 enterocytes and was found on the apical surfaces. As the
27
cells matured, the integrins migrated to the basolateral side o f the cell [108]. Unlike the
ganglioside studies, the in vivo data do not support the theory that rotavirus utilizes
integrins since it infects mature, but not immature enterocytes. In addition, if the mature
cells do have these proteins, they express them on the basolateral but not apical surface.
However, since rotaviruses have been shown to alter tight junctions in Caco-2 cells [94],
they may gain access to even basolateral surfaces o f host cells [113].
Overall, these data suggest that in vivo, specific gangliosides or other glycolipids
or glycoproteins confer specificity for rotavirus to the correct intestinal tissue and that
integrins may be involved in internalization. Studies that directly link integrins to
intestinal virus infection need to be completed. In addition, these studies need to account
for the fact that integrins can change their binding avidity for their ligands upon
activation [93]. Perhaps rotaviruses recognize an epitope that requires both subunits, or
recognizes an activated integrin, and these interactions are missed on reducing gels.
Obviously these questions about rotavirus receptors do not have easy answers. It
is widely recognized that multiple receptors are involved in vitro, and thus probably in
vivo as well. Some authors caution against assuming normal receptor-ligand kinetics to
analyze rotavirus-binding data, because rotavirus may not saturate cells by conventional
methods. Binding o f IAV to host cells is the result o f multivalency, which means that
more than one viral protein binds to low-afGnity cell surface sialic acid epitopes,
resulting in a high affinity binding [103]. Perhaps rotavirus binding exhibits multivalent
properties as well. The working hypothesis is that the virus-cell interaction may require
several key molecules including gangliosides with or without sialic acids, N-Iinked
28
glycoproteins, and other proteins organized in a specific array [79,94]. Since extracting
cholesterol from M A l 04 cells decreased inactivity ofR R V , nar3, and Wa by 90%,
rotavirus-specific receptors may be a part o f lipid rafts [79]. These rafts consist o f
glycosphingolipid-enriched plasma membrane microdomains important in cell signaling
[76]. Elucidating the exact roles o f YP4 and VP7 will also help answer the receptor
questions. It is thought that the first interaction between rotavirus and its host cell occurs
with VP4 binding to a sialic acid or other sugar residue. This attachment then brings VP4
into close contact with other cell receptors [88]. At some point, VP7 initiates direct
penetration into the cell, and the uncoating process occurs, possibly by the removal o f
calcium [65,74], When designing experiments to study rotavirus receptors, it is
important to remember that binding and internalization are active, dynamic processes.
Disease Caused by Rotavirus
In a study done by Snodgrass in Scotland in 1986, it was determined that rotavirus
was the most common diarrheal-causing pathogen in calves, especially in the more severe
outbreaks [114]. In N orth America, it is estimated that five percent o f calves die from a
diarrheal disease before they reach one month o f age, which costs the cattle industry from
$1 to 7 billion a year [71]. In cattle, the normal age range o f rotavirus infection is one
week to tw o months [115], but the timing and severity o f disease varies from strain to
strain [116]. The peak prevalence o f rotavirus diarrhea is from I to 3 weeks, which is
also the nursing period o f calves [60]. There is an age-dependent association with
disease, but not viral excretion, as even older calves shed rotavirus in their stools despite
lack o f illness [116]. Symptoms in a one-day-old calf occur 26 hours post-infection. The
29
first symptoms to occur are change in demeanor and anorexia, which is defined as the
failure to eat offered food within one hour (normal cattle will eat within 5 to 10 minutes).
Then fecal output becomes light yellow and increases to over 500 g/day. A normal calf
has an average fecal output o f 320 g/day. Also, rectal temperature will increase above
39.2°C [117]. Histopathological studies comparing one-day and ten-day old calves
showed that viral antigen was found in the enterocytes, but not in crypt cells, and mostly
localized to the jejunum, ileum, and colon. Virus was detected in the lamina propria,
Peyer’s patches, and the mesenteric lymph nodes, but not in the liver, lung or spleen. The
ten-day-old calves did sustain rotavirus infection, but antigen staining was splotchier in
the intestine, whereas the younger calves had more uniform staining [118].
For humans, rotavirus is also the most common cause o f diarrhea in children
[119]. According to the Center for Disease Control, approximately 600,000 children die
annually worldwide due to rotavirus infection [120]. In the United States, the mortality
rate is much lower, but rotavirus is still responsible for the hospitalization o f 55,000
children [120] and 20 to 40 deaths annually [121], which incurs over $2.6 billion in direct
medical costs [122]. In humans, rotavirus is most common between 4 to 24 months o f
age, but is rarely seen before four months [119]. Symptoms in children are remarkably
consistent, and similar to afflicted cattle. After an incubation period o f 2 to 4 days,
children suffer from sudden vomiting, fever, dehydration, and diarrhea [123]. Disease
occurs mostly during the winter months in temperate climate zones [124].
Studies have been performed using several animal models to determine exactly
how rotavirus causes disease. In all cases, rotaviruses infect small intestinal enterocytes,
30
and this invasion results in a loss o f mature cells and a subsequent repopulation with
immature cells generated from crypt stem cells [119]. Obviously, removal o f enterocytes
is part o f the disease pathology, since these new cells have a limited capacity to absorb
nutrients as well as dysfunctions in the sodium-glucose and sodium-alanine cotransport
systems [124]. However, rotavirus disease is more complex than just stripping off cells
from the intestine. In addition to causing cell lysis, rotavirus also causes extensive
cytoplasmic vacuolization. Histopathology studies in rats indicate that rotavirus induces
relocation o f the nucleus and causes the disappearance o f tight junctions [57]. There is
also evidence that rotavirus activates the enteric nervous system, thereby, further
activating secretion o f Cl' ion [125]. Two strains o f rotavirus, bovine C486 and simian
RRV, were recently reported to induce fusion in M A l04 cells. This fusion is most likely
mediated by VP5*, which has a region with 72% identity to the fusion proteins o f
Semliki forest and Sindbis virus. Fusion only occurred if the media was supplemented
with cholesterol, so it is not normally observed in cell culture. This study is the first
report o f a non-enveloped virus externally inducing fusion o f cells [126]. The in vivo
relevance o f this finding is unknown but could perhaps help to explain the loss o f tight
junctions in Caco-2 cells [94].
Rotavirus protein NSP4 was characterized as the first viral enterotoxin [127].
This non-structural protein normally functions as the intracellular receptor for sub-viral
particles in the E R membrane, and is important for acquisition o f the outer capsid by
immature particles. Administration o f NSP4 induces calcium-dependent chloride ion
secretion in young mice [128]. At the cellular level, NSP4 inhibits the Na+/D-Glucose
31
symporter in rabbit brush border cells [129], alters the distribution o f filamentous actin
[130] , blocks cellular protein trafficking by accumulating in a post-ER compartment [57],
and reduces the electrical resistance o f polarized MDCK cells [130].
In all species susceptible to rotavirus infection, an age-dependency is observed for
disease, even though non-susceptible adults can shed virus [119]. The mechanisms that
make the young susceptible are unknown, but are most likely due to a number o f factors.
The receptors for rotavirus are obviously in adults, but perhaps they are downregulated so
that infection is more limited. This fits with the splotchy verses uniform epithelial cell
damage seen in old versus young calves [118]. Other factors may include an increased
fecal-oral exposure for infants, a nascent immune system, or other differences in the
intestinal biology [119]. Indeed, the number o f intestinal plasma cells is much lower at
1-2 years o f age in humans, which correlates with rotavirus susceptibility. However,
other immune functions do not correlate as well. Children secrete “adult” secretory IgA
levels by 4-6 weeks o f age, but do not achieve adult serum IgA levels until 10-14 years
[131] . Differences in gut biology may also account for the age-dependent disease in
rotavirus susceptibility. Infants have a larger extracellular fluid volume and a higher
daily fluid turnover and are, therefore, more sensitive to body fluid losses. Infants also
have increased sensitivity and response to enterotoxin, which rotavirus is now known to
produce [119]. Age-dependent differences are seen in animals as well. In rats, there is a
profound decrease in membrane fluidity in the brush border during maturation, which is
best explained by a steep increase in the cholesterohphospholipid ratio [132]. In one
cattle study, age-related disease resistance was not due to an inability o f rotavirus to
32
infect and replicate in enterocytes but rather was associated with a slowing o f the
pathogenic process in the older calf intestines [118]. AU o f these differences may
contribute to increased disease susceptibility in the young. Adding to the confusion, it is
StiU unclear why human infants less than four months old rarely suffer from disease,
although they can stiU shed virus [119].
CompUcating the age-dependence o f rotavirus infection even further is the rare
outbreak o f rotavirus strains among adults in both humans and cattle. For example, three
adult cattle in Japan (ages 3-7 years) became infected with an unusual rotavirus serotype .
(G 8/P 6) [133]. In humans, an outbreak o f G2/P4 rotavirus occurred in a coUege dining
hall in Washington, DC. This outbreak was not only unusual because it infected adults
but also because rotavirus was traced back to the food [134]. These outbreaks o f
rotavirus indicate that factors at the virus level can contribute to defining the agedependence barrier. A virulent form o f a bovine strain was found to replicate more
rapidly, destroying enterocytes at a greater rate [116]. Another group compared bovine
strains with different levels o f virulence and confirmed that virulence was associated with
a greater colonization o f the intestine and more enterocyte damage and was determined
by variants o f VP4 [135]. Thus, it appears that both virus and host factors act in concert
to determine disease susceptibility. Perhaps further study o f the groups B and C
rotaviruses, which frequently affect adult humans, will help characterize the
pathogenicity o f rotavirus. Whether they utilize similar receptors or induce the same
disease pathology is currently unknown [62,64].
33
Diagnosis o f Group A rotaviruses is usually performed by an ELISA or a latex
agglutination test and can be confirmed by electron microscopy [136]. Treatment o f
rotavirus in humans is limited to oral rehydration therapy, such as with a glucose +
electrolyte solution [137]. Beverages high in carbohydrates and low in sodium should be
avoided, such as soda, chicken broth, apple juice, or sports drinks [136]. One new
treatment is one oral administration o f 300 mg/kg human serum Ig, which speeds up
recovery from the disease [138].
Immune Response to Rotavirus
In order for a virus or bacterium to be pathogenic to the digestive tract, it must
overcome innate gut defenses and evade the immune system. Understanding hdw
rotavirus manages these tasks has been difficult, since animal models have limited
usefulness. Infaint mice and rats are not susceptible long enough to test active protection
o f vaccines for humans, but they still serve as useful models for the bovine system, and
an adult mouse model has been developed [139]. Pigs also make a good model since
their infection closely resembles humans and they are also susceptible to more than one
strain [140]. A new rat model was just described, in which several group A rotaviruses
cause symptoms similar to natural infection, and will provide other avenues for immune
studies and vaccine developments [57].
In all species, non-immune factors play a major role in viral clearance, since most
symptoms o f infection are resolved before the adaptive immune system has a chance to
respond. Some o f these factors include increased peristalsis o f the gut, gastric acidity,
mucous production, and motility o f intestinal villi In addition, the fact that regenerated
34
enterocytes are less susceptible to infection creates a self-limiting infection [141]. It is
clear, however, that these mechanisms are not enough to cause complete clearance o f
rotavirus. Mice without any functional T or B cells, such as rag-2 knockouts, develop
chronic rotavirus disease [142], as do their human counterparts [143].
After infection with rotavirus, antigen enters M cells in the mucosa. These
specialized epithelial cells transport viral antigen to immune inductive sites called
Peyer' s patches. In these organs, antigen-presenting cells (APCs) pick up, process, and
present antigens to naive T cells. Pre-CTLs and pre-B cells are then generated which
enter the systemic circulation and home to effector sites in the lamina propria. Here these
cells undergo maturation, causing the secretion o f antibodies from B cells and IL-4, IL-5,
and IL -6 from CTLs [141]. Expression o f 04(3? integrin is essential for homing o f both
memory B and T cells back to the gut [142]. It is unknown why some enteric viruses like
rotavirus limit themselves to epithelium, whereas other viruses, such as reovirus or
poliovirus, spread to the Peyer’s patches and subsequently invade other tissues.
Differential M cell involvement may be a crucial factor [59].
Defining the specific roles o f T and B cells in clearing rotavirus infection has
been somewhat difficult. In some studies, complete clearance has been seen in mice
lacking T cells [142]. However, [B2-Inicroglobulin knockout mice, which have no
functional CD 8+ T cells, showed delayed clearance o f rotavirus [144]. Mice lacking
CD4+ T cells could still generate IgA, but complete resolution o f disease required a(3 T
cells [145]. Children with HIV are infected with rotavirus at the same rate as tUV-firee
children, and exhibit the same symptoms. However, AIDS patients are more likely to
35
shed rotavirus [146]. The role o f B cells is also confusing. Surprisingly, people deficient
in IgA production do not have increased incidences o f gastrointestinal infections; In fact,
IgA-knock out mice did just as well as their wild-type counterparts at clearing a
secondary infection, perhaps with compensatory IgG [147]. In addition, IL -6 is thought
to be important in IgA secretion, and mice lacking the gene for IL -6 cleared rotavirus and
were protected from a secondary infection [144]. These reports may be misleading, since
many immune functions are redundant. Natural killer (NK) cells play a major role in
responding to viral infections, but their role in rotavirus has not yet been addressed.
Since N K cells are activated by interferons and IL -12, and rotavirus typically does not
induce this T hl type response o f cytokines, they may not play that crucial o f a role in
clearance [148].
The digestive system also possesses some specific immune defenses. For
example, enterocytes may act as antigen presenting cells, since they have many o f the
same CD expression molecules as APCs, such as CD25, C D l 8, and M HC class II. It is
possible, however, that these molecules have different functions in the gut [107]. T cells
resident in the gut, termed the intraepithelial lymphocytes or IELs, have some unusual
features. They are generated locally rather than within the thymus. Since IELs can
therefore be auto-reactive, there must be other mechanisms to keep them from attacking
host tissue. These cells are more similar to effector than to naive T cells because they do
not need to be primed by antigen. In addition, they tend to have an unusual CD 8 pattern
(yS versus a[3) and an uncommon MHC type [149]. Interestingly, human infants do have
fewer IELs than adults [150]. In a calf, these y8 IEL cells probably play an important
36
role in gut defense, since they are so abundant in the intestine. Because they are mainly
associated with the villi and not the crypts, it is thought that y5 T cells play a role in
renewal o f epithelial cells by inducing apoptosis [151]. Studies with rotavirus need to
further address these intestinal-specific defenses.
Protection from a second natural infection appears to be mediated by secretory
IgA in humans and mice, since their levels correlate with a decrease in disease severity
[141]. However, virus-specific IgA disappears after a year, making this protection
ineffective for children, who can be susceptible for 2-5 years. In contrast, many
vaccination studies indicate that IgA levels do not correlate with protection from a second
challenge. The amount o f protection does depend on the species, the dose, and the route
o f inoculation. It has been shown that while rotavirus-specific memory T and B cells do
not protect against disease, their presence does shorten both duration and illness severity
[141]. Since rotavirus causes disease in young mammalian species, passive, immunity
from breast milk is thought to be important. Breast milk contains IgA and other Igs,
complement, lactoferrin, interferons, anti-microbial proteins, and even T cells and
macrophages [131]. Human milk also contains lactadherin, which specifically binds to
rotavirus and inhibits its replication [90]. In cattle, colostrum contains proteins called
collectins, which are a family o f oligomeric proteins that can bind to VP7 o f rotavirus,
and also opsonize particles for phagocytic cells [152]. One study found that the rate o f
infection was similar in breast-fed and formula-fed children; however, the severity o f the
symptoms was less in breast-fed kids [90]. Still another study indicated that
breastfeeding only postponed, but did not prevent, disease from rotavirus [136].
37
Because o f the widespread morbidity and mortality due to rotavirus, attempts
have been made to develop effective vaccines for cattle and humans. Two vaccination
strategies in cattle have been utilized. The first was to stimulate the calves’ own active
immunity by orally administrating a five, attenuated strain on the day o f birth. This
approach worked well in gnotobiotic calves, but has several problems in field cattle.
First, calves in a normal environment are quickly exposed to more virulent strains before
the vaccine has had a chance to stimulate a protective response. Second, maternal
antibodies passively acquired through colostrum and milk can neutralize the vaccine,
making it unable to induce a response [60].
The second strategy was to immunize dams and generate passive immunity,
which is passed through colostrum and milk to calves. Natural antibodies to rotavirus do
exist in teat secretions regardless o f the vaccination status o f the mother. However, these
antibodies do not provide full protection for several reasons, including the presence o f
high doses o f virus in confined areas, dilution o f milk antibodies w ith feed supplements,
and early weaning. Therefore, vaccines that boost passive immunity are promising
options. Unlike humans, ungulates (pigs and calves) are bom agammaglobulinemic and
only acquire serum antibody from colostrum for up to 48 hours after birth. In ruminants
such as calves, serum-derived IgG is the most common antibody found in both colostrum
and milk, whereas secretory IgA, produced by the mammary glands, is the predominant
antibody in humans. One advantage o f this difference is that a bovine vaccine can be
administered intramuscularly or through other non-oral routes, since serum IgG and not
mucosal IgA is the goal. This means that vaccines such as virus-like particles (VLPs) or
38
other recombinant vaccines can be used because they will not be inactivated in the gut
[60]. VLPs and inactivated viruses are safer than attenuated viruses and increase and
prolong antibody secretion in both colostrum and milk [153,154]. Feeding calves
colostrum from a vaccinated cow at 12 hours after birth did not reduce shedding but did
reduce severity o f disease [154]. Several live attenuated vaccines are commercially
available for cattle, but their efficacy varies from place to place [60].
In humans, vaccination is a possible strategy because there is protection from
natural infection after the first exposure. However, rotavirus is not as easy to vaccinate
against as other viruses since it is a non-systemic virus with a short incubation time (1-4
days). In contrast, the M MR vaccine is effective because measles, mumps and rubella
are systemic viruses with a longer incubation time (8-14 days) [141]. The RotashieM©
vaccine, which has a 49 to 68% efficacy in preventing diarrhea and a 69 to 91% efficacy
in preventing severe disease, was withdrawn in October o f 1999 due to a temporal
association with intussusception, resulting in the death o f one child [155].
Intussusception is the invagination o f a proximal bowel segment into a distal one, which
can cause bleeding and abundant mucous secretion, responsible for the “red currant jelly
stools” seen in afflicted patients. Intussusception can be caused by a number o f factors
including hypertrophy o f infected lymph nodes or Peyer’s patches [140]. Because 82%
o f the cases o f intussusception occurred after the first dose o f the RRV-TV, the vaccine
was removed from the market. Since studies have found no link between natural
rotavirus infection and intussusception, the intestinal damage could have come from the
non-human components o f the vaccine [156]. Host factors, however, play a role in
39
determining risk o f intussusception. This study noted that males were at a 3x higher risk
than females, and well-nourished children also tended to be at a higher risk. Since the
lymphoid tissue o f the gastrointestinal tract is less prominent in very young and
malnourished infants, it is less likely that the P ey efs patches or the mesenteric lymph
node would enlarge enough to cause invagination o f the bowel [140].
One problem that any effective rotavirus vaccine must overcome is the serotype
diversity problem for group A viruses. Homologous strains are better inducers o f slgA
than heterologous strains and also require a smaller viral dose. However, heterotypic
vaccinations can w ork for several reasons. Antibody may be directed at cross-reactive
neutralizing epitopes, some antibodies like those against VP6 may ablate infectious virus
as they pass through epithelial cells on the way to the lumen. CTLs can also broadly
cross-react with other rotavirus serotypes [141]. One group has developed a DNA
vaccine that codes for bovine rotavirus VP4. The response was biased toward a T h l,
with IFNy being more common than with regular immunization, which produced more o f
a balance between IFNy and IL-4 . Interestingly, mice did not produce antibody from the
DNA vaccine but were primed to produce antibody upon challenge [148].
Evidence for a Role o f Neutrophils in Rotavirus Infection
Several lines o f evidence indicate that rotavirus and neutrophils could directly
interact. For example, a number o f the proposed receptors for rotavirus are found on
neutrophils. Neutrophils express ax $ 2 and have recently been found to upregulate the Pi
integrals upon extravasation (eta, 04, as, and as) [14,157]. It is also likely that neutrophils
are recruited to sites o f rotavirus infection. Studies in HT29 cells demonstrated that
40
rotavirus increases the transcription factors NF-k B, STATI, and !SG. These in turn
upregulate several CXC cytokines including IL-8, GROa, IP -10, and also the CC
chemokines RANTES and M CP-I [158]. IL-8 is a potent chemotactic agent and
neutrophil activator in human and bovine cells, and GRO-a is chemotactic for human
neutrophils [159,160]. The secretion o f RANTES, GRO-a, and IL-8 in HT29 cells is
dependent on the dose o f rotavirus as well as the time course o f infection [160].
M ore direct evidence for a role o f neutrophils in rotavirus infection comes from
studies showing that neutrophils are found in biopsies o f human rotavirus patients [160],
in the lamina propria and the cecum o f chickens and turkeys infected with rotavirus [58],
and also in the splenic sinuses o f infected lambs [161]. Band cell production was
increased in 21% o f children infected with rotavirus, indicating this virus could be
inducing neutrophil production [162]. However, these findings were not confirmed in a
recently described rat model o f rotavirus, where the leukocyte content o f the lamina
propria o f sham-infected was similar to that o f rotavirus-infected rats. Nevertheless, the
rat may not be a perfect model for all'rotavirus infections, since these rats did not develop
a severe disease, both clinically and pathologically [57].
Ifrotavirus infection results in neutrophil recruitment, this effect alone may
contribute to disease pathology. Parkos found that neutrophils might contribute to the
pathology o f secretory diarrhea, mainly as a response to influx and perturbation o f cell­
cell junctions. This transmigration not only perturbs the lining physically, but also
neutrophils secrete modulatory chemicals as well, such as 5’AMP, which can cause
epithelial cells to excrete ions and water [163]. Neutrophil migration through polarized
41
T84 epithelial cells in response to Yersinia pseudoiuberculosis infection caused damage
to the lining such that the pi integrins were now exposed to the luminal side [164].
Neutrophils have also been shown to exacerbate inflammation during other gut diseases,
such as helminth infection [165].
I f neutrophils are arriving at the site o f rotavirus replication, then they may be
playing a role in viral clearance. Neutrophils are not known to play a major role in viral
infections, because neither LAD nor neutropenia patients have increased susceptibility to
viral infections [5]. However, since innate defenses are so important to rotavirus, it is
possible that neutrophils play a role in clearing this infection before the adaptive immune
system even arrives.
Finally, there is indirect evidence that rotavirus does induce a transient bacterial
susceptibility. One group found an association between rotavirus and secondary bacterial
infections in mice. Mice infected with rotavirus were challenged with either Clostridium
perfringens or Clostridium difficile. Even though rotavirus infection did not seem to alter
the establishment o f normal intestinal microflora, the pathogenic bacteria did develop an
earlier and more severe disease than in the control mice [166]. Rotavirus infection also
increased mouse susceptibility to enterotoxigenic E. coli, and mice with both pathogens
had more severe symptoms than from either pathogen alone [167]. Another study found
this synergistic effect in cattle [168]. In contrast, a study o f horses in Ireland and Britain
demonstrated that there were no synergistic effects between rotavirus, Cryptosporidia, and
pathogenic E. coli [115]. Studies in humans are conflicting as well. One study found that
90% o f patients had a mixed infection with rotavirus and enterotoxic E. coli and, thus,
42
had a more protracted illness [123]. A study in Italy found 28 cases o f rotavirus with
other infections, and the mixed infections were more severe as evidenced by being
associated more with the inpatient than the outpatient group [169]. In contrast, 59% o f
children in a special-baby care unit had rotavirus, but no other enteric pathogens were
found [170]. Thus, although the role o f rotavirus infection in facilitating superinfection is
suggested, the data are mixed and further work is necessary to evaluate this relationship.
Nevertheless, the high frequency o f rotavirus-associated bacterial superinfections does
suggest that an active neutrophil response is likely present.
Project Goals
Based on the studies outlined above and the evidence showing that neutrophils
participate in many anti-viral activities, we hypothesized that neutrophils bind to
rotavirus and that this interaction could modulate the various neutrophil host defense
responses. In addition, we hypothesized that rotavirus could induce secretion o f
cytokines from host cells, which could also either up- or down-regulate neutrophil
functions. To evaluate this hypothesis, we utilized the B641 strain o f bovine rotavirus
and the M A l 04 host cell line. In Chapter 2, we investigated whether neutrophils are
capable o f binding rotavirus particles using three complementary methods: flow
cytometry, VOPBAs, and radioactive-binding assays. Radioactive particles were also
used to carry out internalization studies. In Chapter 3, we investigated the effect o f
exposure to neutrophils on rotavirus viability. We next evaluated what modulator effect
rotavirus might have on various neutrophil functional responses. To carry out these
43
analyses, purified viral particles were first tested as direct activators o f neutrophil
oxidative burst and chemotaxis, and then later as primers or inhibitors o f neutrophil
respiratory burst, chemotaxis, and phagocytosis. To investigate whether rotavirus was
capable o f inducing neutrophil cytokines, M A l 04 cells were infected with virus, and at
various time points supernatant was collected and tested in the same functional assays.
Based on these studies, we hope to further develop our understanding o f the interaction o f
bovine neutrophils and rotavirus and the potential role o f this interaction in rotavirus
disease. An overall discussion o f the findings is presented in Chapter 4.
44
ROTAVIRUS BINDING AND INTERNALIZATION
BY BOVINE NEUTROPHILS
Introduction
Neutrophils are known to directly interact with many different viruses, both in
vitro and in vivo [39]. This interaction can result in several outcomes, from neutrophils
aiding the clearance o f viral infection, as with herpes simplex virus [32]; neutrophils
aiding the spread o f viral infections, as with cytomegalovirus [36]; or neutrophils
becoming inactivated, as with influenza virus [39]. Since the interactions between
neutrophils and rotaviruses have not been evaluated, this project was designed to
characterize the neutrophil response to a bovine strain o f rotavirus. The first step o f any
virus-cell interaction is specific viral attachment. Therefore, we hypothesized that
neutrophils could specifically bind rotavirus, and performed several types o f binding
assays to evaluate this possibility.
Because the cellular receptors for rotavirus most likely contribute to its age,
species, and tissue-specific tropism, their identification is an area o f active research.
None o f the rotavirus receptors have yet been characterized, but clues have been
illuminated from several studies, mainly with the monkey kidney M A l 04 cell line.
Binding o f rotavirus to this cell requires sodium, and occurs between pH 5.5 and 8.
Binding is initiated by VP4, and although trypsin cleavage o f this outer protein is
required for viral entry, it is not required for initial attachment [73]. Studies performed
with various inhibitors have shown that specific ganglioside residues on glycolipids and
45
N-Iinked carbohydrate residues on glycoproteins can bind to several strains o f animal and
human rotaviruses [79,80,84]. The role o f sialic acid, which is a sugar residue found on
all gangliosides and many glycoproteins, is still under investigation and seems to vary
depending on not only the strain, but also the host species [82,85-87]. Transfection
studies have indicated that integrins including aaPi and axPz can act as functional
receptors in vitro [72,94,96]. The emerging paradigm for rotavirus is that two or three
receptors are involved - one for initial attachment and one or two for infectivity [72].
Therefore, several different interactions between virus and cell surface receptors can
occur during infection [76]. In addition, the virus most likely enters through direct cell
penetration, since endocytosis inhibitors did not decrease viral entry [74].
M ost o f the information on rotavirus binding to host cells has been gleaned from
studies on primate rotaviruses. However, several results indicate that the species-specific
differences among rotaviruses are not trivial. Porcine strains o f rotavirus, for example,
require a glycolipid for infection, whereas most other strains seem to require a
glycoprotein [77]. Also, one o f the porcine strains appears to enter through endocytosis,
even after trypsin cleavage [75]. In addition, most o f these studies were performed on
tissue-adapted cell lines, not from in vivo or even ex vivo cells. Since this project
involves characterizing binding o f a bovine rotavirus to bovine neutrophils, information
obtained from this study will hopefully be useful not just to the specific project at hand
but also to the virus research community in general. It is possible that neutrophils do
bind rotavirus, since neutrophils contain sialic acids [42] and express most o f the putative
integrin receptors [93,95,157]. I f it is found that rotavirus can bind to neutrophils and
46
induce functional responses, then characterization o f this interaction may give clues to
pathogenesis o f rotavirus.
Testing whether neutrophils bind rotavirus was performed using three methods.
The first method involved flow cytometry, which allowed for easy quantitation o f
binding, and comparisons between neutrophil binding and cell line binding. However, it
does not give information as to specificity. The second method consisted o f probing
blots o f neutrophil membranes with purified viral particles. The advantage o f this
method is that it is possible to determine the molecular weights o f any putative virus­
binding components, but a disadvantage is that the proteins are rarely in their native state.
The final method used was a radioactivity binding assay. This technique allows testing
o f specificity, saturation, and internalization. Neutrophils are known to phagocytose
several different viruses, including herpes simplex, influenza, and cytomegalovirus [30],
and if they can internalize rotavirus, this may implicate the neutrophil in viral clearance
mechanisms.
These studies indicated that neutrophils bound rotavirus as well as, or possibly
greater than M A l 04 cells. Binding was specific and was not affected by prior
stimulation o f the neutrophils with PMA. The kinetics o f binding resembled the
traditional linear relationship between a ligand and a receptor. However, the virus did not
internalize after one hour at 37°C. These results suggest that while neutrophils probably
do not play a role in direct viral inactivation through phagocytosis, it is possible that
rotavirus could be eliciting functions or otherwise altering these important leukocytes.
47
Materials and Methods
Materials
M l 99 media powder was purchased from Irvine Scientific. Flasks and roller
bottles for cell w ork was from Fisher. Fetal bovine serum-premium was bought from
Atlanta Biologicals. Buffers.used for neutrophil studies, HBSS and D PBS, were
purchased from Gibco. Trypsin used to activate virus was obtained from Worthington
Biochemicals. Radioactive S-35 RediVue Pro Mix was obtained from Amersham
Pharmacia. FITC-Iabeled goat-anti mouse antibody was purchased from Jackson
Scientific. Dr. Michele Hardy kindly provided original stocks o f the B641 bovine strain
o f rotavirus, as well as M A l 04 cells. Kathy Jutila prepared the anti-gp91 phox
monoclonal antibody. AU animal use was approved by the MSU Institutional Animal
Care and Use Committee.
Maintenance o f CeU Lines
K562 cells were derived from a human hematopoietic leukemia ceU line. They
were maintained in suspension at IO5 to IO6 per ml in RPMI supplemented with 5% FBS,
2 mM L-glutamine, 50 U/ml penicillin, and 50 U/ml streptomycin. The day before
experiments, ceUs were counted and spUt appropriately to ensure log phase growth. On
the day o f the experiments, K562 ceUs were removed, counted, and washed with warm
PBS. M A l 04 ceUs, an adherent epitheUal ceU line o f rhesus monkey kidney origin, were
maintained on M l 99 media supplemented with 5% FBS, 10 mM sodium bicarbonate, and
4 mM L-glutamine in T -162 flasks. CeUs were harvested for experiments when they
48
reached 90-95% confluency. On the day o f the experiments, cells were lifted with 5 mM
EDTA in PBS for 10 min at 37°C.
Preparation o f Purified Viral Particles
The bovine rotavirus strain B641 was propagated by infection o f M A l04 cells
with virus stocks. Cells were grown to 90-95% confluency in roller bottles with M199
complete medium supplemented with 10 mM sodium bicarbonate, 20% FBS, and 4 mM
T-glutamine On the day o f the infection, cells were incubated with media lacking FBS
for 2 to 3 hours, since protease inhibitors in fetal bovine serum inhibit viral replication.
Meanwhile, a virus stock was treated with 10 pg/ml trypsin (Worthington) to cleave VP4,
which enhances inactivity [59]. Media was removed from the rollers bottles, and virus
was added in a volume o f 5 ml at an MOI o f 1-10. The MOI, or multiplicity o f infection,
was calculated by the following formula,
ml o f virus = IMODtcells/rollerl
(Titer o f virus)
in which cells/roller is 7x107, and the virus titer is the amount o f plaque-forming units
(PFU) per ml, as determined by a plaque assay. Cells were inoculated for one hour at
37°C, then 10 ml o f post-infection media was added (M l 99 complete, no FBS, plus I
pg/ml trypsin, and 15 mM sodium bicarbonate). Roller bottles were checked daily to
determine the cytopathic effect (CPE), which is the amount o f detachment o f infected
cells. Once 90% CPE had been reached, roller bottles were placed in - 20°C and
subjected to three freeze/thaw cycles to break up the cells and release viral particles. The
supernatant was harvested, and virus was purified according to previously described
49
methods [80]. Briefly, supernatant was centrifuged at 90,000 x g with a 20% sucrose
cushion to concentrate the virus. The virus-containing supernatant was then centrifuged
on a cesium chloride gradient at 190,000 x g for 18 hours. This gradient separates the
fully infectious triple layered particles (TLP), which appear at the 1.36 g/ml density band,
from the VP4- and VP7-lacking double layered particles (DLP), which appear at the 1.38
g/ml band. Bands were then collected and pelleted at 190,000 x g for I hour. The
concentration o f particles was determined by a spectrometry reading at A26o, since they
contain RNA. The protein concentration in pg/ml was then determined by multiplying
the reading by the dilution factor and then by 185. Particles per ml were calculated by
multiplying the pg /ml by 3.33x10s. These conversion factors were previously obtained
by counting viral particles via electron microscopy, then correlating particle counts with
protein and RNA measurements [171]. N ote that these calculations assume all particles
are triple layered. On average, the particle to PFU ratio was 100, meaning that for every
100 particles, only one is capable o f replication. Particles were then stored in Tris buffer
(10 mM Tris, 100 mM NaCl, and 5 mM CaCl2) at -80°C until use.
Production o f Polyclonal Antibodies
Purified triple layered particles were obtained and diluted 1:1 in Titermax
adjuvant (Sigma) to a final concentration o f 0.9 pg/pl o f protein, i.e. 3x108 particles/ml,
and adjuvant and virus were emulsified for 10 minutes. Two female BALB/c mice at 2
months o f age were inoculated with 100 pi antigen via the intra-peritoneal route, then
boosted in two weeks with an additional 100 pi. After 2 months, mice were euthanized
and exsanguinated. Blood was allowed to clot at 37°C for over one hour, then
50
centrifuged. Serum was removed, supplemented with 0.02% sodium azide as a
preservative, and serum was stored at 4°C. Dilutions were prepared on the day o f
experiments and were not re-used.
Isolation o f Bovine Neutrophils
Blood was obtained via jugular venipuncture from healthy Holstein calves and
purified as described in [172]. Two water lyses were performed to remove red cells, and
remaining cells were centrifuged through a gradient o f 1077 and 1119 Histopaque. This
gradient separates the mononuclear cells, which remain in the upper layer, from the
neutrophils, which pellet to the bottom. The mononuclear cell layer was then removed,
and the neutrophils were washed in PBS and counted with a hemacytometer. Viability
was determined by trypan blue staining.
Flow Cytometry
AU cells to be tested were aliquotted into tubes in 250 pi aliquots. Buffer used
throughout was DPBS + 1% BSA. Where indicated, cells were treated with 20 nM PMA,
a phorbol ester that activates neutrophils, for 10 minutes at 37°C, then washed. Viruses
were added, and tubes were placed on a rocker at 4°C for one hour. The ceUs were
directly fixed with 1% paraformaldehye in DPBS, washed, then stained with polyclonal
mouse anti-rotavirus serum for one hour at room temperature or 4°C. CeUs were again
washed and stained with secondary antibody (goat anti-mouse FITC IgG) for one hour at
either room temperature or 4°C. CeUs were washed, transferred to FACS tubes, and
analyzed by flow cytometry in a FACS CaUbur using the FL l channel.
51
Virus Overlay Protein Blot Assays fVOPBAs)
Membranes were obtained from purified bovine neutrophils through nitrogen
cavitation followed by centrifugation through a sucrose gradient as described elsewhere
[173]. Membranes were sonicated, diluted to 300 pg/ml, and separated on a reducing
10% SDS-PAGE gel with 15 pg total protein per lane. Then gels were transferred to
nitrocellulose, dried, and stored at 4°C until use. To probe the blots with virus, the blots
were first blocked w ith blotto buffer (5% non-fat dry milk, 0.2% Tween-20, in PBS)
overnight at 4°C. Virus at 4.47 jig /ml was added in 5 ml, and blots were again incubated
overnight at 4°C. Blots were washed with TBS + 0.2% Tween 20, incubated with
primary antibody (mouse serum) for one hour at room temperature, washed again, arid
stained w ith secondary antibody (alkaline phosphatase-conjugated goat anti-mouse IgG)
for one hour at room temperature. Blots were washed and subsequently developed with
an alkaline phosphatase development kit (Biorad). Negative control blots for each
antibody were simultaneously processed, as well as a positive control using a monoclonal
antibody developed in our laboratory to gp91 phox o f the bovine NADPH oxidase.
Radiolabeling o f Viral Particles
M A l 04 cells were grown to confluency in roller bottles supplemented with 10%
fetal bovine serum. Virus was activated with 10 pg/ml trypsin, added to rollers with an
MOI o f 2.5, and inoculated for two hours. Inoculum was then removed, and depletion
media was added to each roller. This media consisted o f DMEM without cysteine or
methionine, and supplemented with 10 mM L-glutamine, 15 mM sodium bicarbonate and
I Iig/ml trypsin. After a 30 minute incubation, 50 pCi/ml o f S-35 labeled cysteine and
52
methionine (RediVue Pro-Mix, Amersham) was added to each roller. When CPE was
reached, rollers were frozen at -20°C. A non-radioactive infection was done
simultaneously to provide virus for competition experiments. Viruses were purified as
described above.
Radiolabeled Virus-Binding Assays
Cells were chilled in TBS (20 mM Tris, 140 mM NaCl, 5 mM KC1, 0.4 mM
MgCfi5 0.7 mM CaCl2, pH 7.2). M A l 04 cell experiments were carried out in a 96-well
plate, and K562 and neutrophils were performed in suspension in Eppendorf tubes, all in
100 pi volumes . Viruses at various concentrations were added to the cells and were
allowed to bind at 4°C for 60 to 90 minutes. Cells were then washed w ith TBS, lysed in
100 pi RIPA buffer (150 m M N aC l, 1% DOC, 1% Triton-X 100, 0.1% SDS, 5 pg/ml
pepstatin A, 10 pg/ml aprotinin, 10 mM Tris, pH 7.2). Samples were vigorously
vortexed and incubated for 15 minutes at 4°C to allow complete lysis, then transferred to
scintillation vials for counting. For the internalization studies, cells were washed with
TBS and placed at 37°C for one hour, then washed and lysed with RIPA buffer. To
remove viruses from the membrane, cells were treated with proteinase K buffer for 30
minutes at 4°C (10 mM EDTA + 0.5 mg/ml proteinase K in TBS), then washed and lysed
with RIPA buffer.
Statistical Analysis
AU error bars represent the SEM (standard error o f the mean). Where indicated,
the student’s two-taUed t test was performed to ascertain statistical significance.
53
Results
Analysis o f Purified Rotavirus and Polyclonal Antiserum
After collecting virus bands from the gradient, a sample o f each band was run on
a gel to confirm that the viral particles were intact, as evidenced by the presence o f the
outer VP4 and VP7 proteins. One representative gel is shown in Figure 2 .1. Lane A
shows a TLP preparation. It has a strong band at around 40 kDa representing the most
abundant protein, VP6, and another intense band at 94, which is VP2. A band at 38 kDa
shows VP7, and thin bands at 60 and 28 show the VP4 cleavage products VP5* and
VP8*. Lane B represents the DLP band from the gradient. In this preparation, the band
at 38 kD a is much fainter, indicating that most o f these particles do not have the outer
capsid. Lane C represents the top band from a cesium chloride separation, which
contains various proteins that have fallen off the particles such VP6, VP7, and VP8.
Although the particle :PFU ratio varied between experiments, this should not affect the
results since all particles should be equally capable o f binding even if they are not fully
infectious.
To show that the mouse polyclonal antiserum recognizes these rotavirus proteins,
a sample o f TLPs was run on a gel, transferred to nitrocellulose, and blotted with the
serum (Figure 2.2). It recognized all the major rotavirus proteins even at a 1:200 dilution.
Flow Cvtometrv
A flow cytometry binding study was designed to test whether cells in suspension
could bind rotavirus. K562 cells, M A l04 cells, and bovine neutrophils were incubated
54
A B C
<
MW
----
' e—■
id
^
-
H !
■
97
66
=3 I
Figure 2.1: Analysis o f Purified Rotavirus by SDS-P AGE. Bands from a cesium chloride
separation were separated on a 4-20% SDS-PAGE pre-cast mini gel, then stained with
Gel-Code. Lane A is from the triple layer band, lane B is from the double layer band,
and lane C is from the top layer o f the gradient. Prestained standards were used on all
gels, and the relative standard molecular masses are indicated.
Figure 2.2: Western Blot Analysis o f Anti-Rotavirus Mouse Anti-Serum. Triple layered
particles were separated by SDS-PAGE and transferred to nitrocellulose. Blots were
probed with polyclonal serum at a 1:2000 dilution, then with alkaline phosphataseconjugated goat-anti mouse IgG. Blots were developed with an alkaline phosphatase kit.
Lane A, on the left, is control mouse serum, lanes B and C are serum from two different
immunized mice. Prestained standards are shown flanking each o f the viral protein lanes,
and the relative standard molecular masses are indicated.
55
with rotavirus at 4°C. It has been previously shown that rotavirus will bind, but not
internalize, at this temperature [73]. Cells were then fixed, stained with the mouse
rotavirus antiserum, followed by FITC-Iabeled goat-anti mouse antibody (Jackson).
Samples were then analyzed by flow cytometry. Since each cell line has a different
forward and side scatter profile, these parameters were set differently for each cell line.
Thus, in order to make comparisons, the binding data was expressed as percent above
control cells treated without virus. Figure 2.3 shows a compilation o f three separate
experiments. It appears that all cells tested do bind rotavirus particles, since fluorescent
staining for each cell type was always above control. Neutrophils bound the most
rotavirus, followed by M A l 04 cells, then K562 cells. Stimulation with 20 nM PMA for
15 minutes did not change the binding profiles o f any o f the cell populations.
Virus Overlay Protein Blot Assays (VOPBAsI
Neutrophil membrane proteins were separated on a gel and transferred to
nitrocellulose. These blots were then probed w ith purified rotavirus and stained with the
appropriate antibodies. In the first blot, bands were observed in the test lanes, but not in
the control lanes, including a very faint band at around 95 kDa (data not shown). The
strong bands that only appeared in the test lane were all less than 30 kDa. However,
there were several bands, albeit not as many, that also appeared in the controls around 30
kDa. The second set o f overlay blots showed similar results (Figure 2.4). The main
difference was a lack o f a band at 95 kDa. Again, bands appeared in the test lane around
the 30 kDa marker, but there were several similar bands in the controls. Therefore,
determining which bands were specific to the virus was difficult. These non-specific
56
No PMA
+ PMA
Figure 2.3: Flow Cytometric Analysis o f Rotavirus Binding. Cells were either treated
with buffer or 20 nM PMA for 15 minutes at 37°C. Cells were washed and allowed to
bind purified triple layer particles, at a ratio o f 10,000 viruses to I cell for I hour at 4°C
Cells were then fixed with paraformaldehyde, stained with appropriate antibodies, and
analyzed by flow cytometry for FITC staining. The data are expressed as the average
percent o f binding, determined by dividing data by the no-virus controls. This is a
compilation o f three separate experiments (mean ± SEM).
220kDa-I
97 k D a -,
66 kDa--i
45 kDa30 kDa-i
20 kDa14 kDa-l
I
Virus
I 0 Ab
2° Ab
Virus
2° Ab
I 0 Ab
2° Ab
2° Ab
anti-gp91p/70x
2° Ab
Figure 2.4: Rotavirus Binding to Neutrophil Membranes. Neutrophil membranes were
separated on a gel and transferred to nitrocellulose. Blots were probed with purified
TLPs and stained with reagents as indicated. Each blot consists o f the membrane
proteins in the right lane and prestained molecular weight markers in the left lane.
57
bands appeared both in the primary and secondary antibody controls, but not in the
dilution buffer, implying that both antibodies may bind neutrophil membranes directly.
One blot was stained with an antibody that recognizes bovine gp91 phox (arrowhead), as
a positive control. A 91 kDa band appeared, but so did the non-specific bands at 30 kDa.
Radioactive Virus-Binding Assays
M A l 04 cells, K562 cells and bovine neutrophils were incubated with radiolabeled
virus at 4°C, washed, lysed, and read in a scintillation counter. Figure 2.5 shows the
results from all three cell types. Similar to the flow cytometry data, all cells bound
rotavirus in a dose-dependent manner. However, the pattern from the radioactive assay
differs from the flow analysis. In both experiments, neutrophils bound the most virus out
o f the three cells tested, but in the radioactive assay, the K562 cells bound more virus
than the M A l 04 cells. This difference was surprising but could be because adherent
M A l 04 cells were tested in the radioactive assay, whereas in the FACS assay, they were
in suspension. Using the previously measured CPM/pg for the radiolabeled virus
preparation, the counts per minute data was converted to bound virus. Then it was
possible to calculate the free virus and the bound/free ratio. The bound/free ratio was
then plotted against the amount o f bound virus for each cell type, known as a Scatchard
plot, which can be useful in determining the kinetics o f the virus/receptor interaction. I f
the plot is linear, then the amount o f receptors per cell is represented by the x-intercept.
The M A l 04 data did not show a linear Scatchard plot unless several data points were
excluded (data not shown), however the neutrophil plot did resemble a linear curve to
some extent (Figure 2.6) as did the K562 graph (data not shown).
58
15000
12000
-
9000-
6000-
3000-
300
1100
6000
30000
VirusiCell Ratio
Figure 2.5: Radioactive Virus-Binding Assay. Cells were washed and incubated with
viruses at the indicated ratios for 90 minutes at 4°C, either in suspension (neutrophils and
K562 cells) or in a plate (MAI 04 cells). Cells were washed thoroughly to remove
unbound virus, lysed with RIPA buffer, added to 2 ml scintillation fluid, and read in a
scintillation counter. The data are expressed as the mean counts per minute (CPM) ±
SEM, and each point represents 3 samples.
0) 0.4-
O 0.2-
1000
2000
3000
4000
Particles/Cell
Figure 2.6: Scatchard Analysis o f Rotavirus Binding Curve for Neutrophils. Bound virus
was determined by measuring the CPM, and free virus was calculated by subtracting the
bound virus from the total virus added. The Y axis represents the ratio o f bound:free
particles, and the X-axis represents the amount o f particles per cell bound.
59
To determine if this virus binding to neutrophils was specific, a competition assay
was performed. The addition o f 10 times the amount o f non-radiolabeled virus reduced
the radioactivity to basal levels, indicating the binding was specific (Figure 2.7). Next,
an internalization study was performed. In this experiment, cells were allowed to bind to
the virus for one hour at 4°C, washed to remove unbound virus, then either incubated for
I hour at 37°C, or immediately analyzed for the 0 hour time point. For each time point,
cells were divided into tw o groups. Cells in the first group were lysed with RIPA buffer
and read in the scintillation counter to determine total virus counts. Cells in the second
group were treated with proteinase K, which has been reported to remove 94 to 98 % o f
bound viruses [174]. Cells were then lysed with RIPA buffer and read to provide an
estimate o f internalized virus amounts (Figure 2.8). I f virus was being internalized, then
the I hour-incubated virus (the black bar on the right) would be increased over the 0 hour
time point (the black bar on the left). In fact, the bars stayed the same, indicating that the
virus was not being internalized.
Discussion
Using three different methods, it was shown that the bovine strain B641 o f
rotavirus could bind to bovine neutrophils. These approaches included flow cytometry,
blot overlays, and radiolabeled virus-binding assays. Since binding studies have several
difficulties to overcome, it is a good idea to use more than one method for confirmation.
The advantage o f flow cytometry, or FACS analysis, is that contaminating viral
particles can be gated out, which is important, since aggregation o f rotavirus can be a
60
40001
□ 5-35 Virus Alone
m s-3 5 + IOx CoIdV
3000-
5
O 2000-1
1000-
100:1
SJ
1000:1
Vims:Cell Ratio
Figure 2.7: Competition o f Radiolabeled Rotavirus Binding with Unlabeled Virus.
Neutrophils were isolated and incubated with either radiolabeled rotavirus particles alone
(dotted bars) or radiolabeled particles plus ten times non-radiolabeled particles (black
bars) for 90 minutes at 4°C. Then cells were washed thoroughly to remove unbound
virus, lysed with RIPA buffer, added to 2 ml scintillation fluid, and read in a scintillation
counter. The data are expressed as the mean counts per minute (CPM) ± SEM, and each
bar represents five samples.
□ T o tal
Internalized
4000
30002000 1000
I Hour
0 Hours
Incubation Time
Figure 2.8: Analysis o f Rotavirus Internalization by Neutrophils. Neutrophils were
isolated and incubated with radiolabeled viral particles at a virusxell ratio o f 1000:1 for
60 minutes at 4°C. Cells were washed to remove unbound virus, then either incubated 0
or I hour at 37°C. Cells were then either lysed and read to obtain the total virus count
(dotted bars), or treated with proteinase K then lysed and read to obtain the internal virus
count (black bars). The data are expressed as the mean counts per minute (CPM) ± SEM,
and each bar represents five samples.
61
problem for binding studies [82]. In addition, wash steps do not have to be as stringent.
FACS analysis showed that neutrophils bound more viral particles than M A l 04 and
K562 cells. The fact that K562s bound to rotavirus despite being non-permissive for
infection confirms the reports that there is currently no true negative control for virus
binding [72]. However, flow cytometric analysis did not address specificity. In addition
to non-specific binding o f TLPs to cells, double layered viral particles may also
contaminate results since the mouse serum is polyclonal. There is currently no
consensus as to whether double layered particles can bind to cells and thus skew binding
results. DLPs did not bind to whole cells [81] or to ganglioside membrane fractions [80],
but did bind to asialoganglioside fractions in a thin-layer chromatography assay [82] and
to intestinal membranes in a VOPBA assay [91]. One way to eliminate DLP
contamination is to use an antibody specific for the outer layer VP4 or VP7 proteins. An
attempt was made to isolate a monoclonal antibody that only recognized VP4.
Unfortunately, the antibody generated recognized DLPs as well (Appendix).
PMA, or phorbol myristate acetate, is a potent neutrophil activator and induces
degranulation and an oxidative burst [19,24], Stimulation o f neutrophils with 20 nM
PMA did not alter neutrophil’s ability to bind rotavirus. In fact, it appeared to decrease
their ability to bind, but this decrease was not statistically significant. PM A will mobilize
secretory granules in bovine neutrophils which upregulates a number o f proteins
including p2 integrins, complement and endotoxin receptors, and alkaline phosphatase.
However, this response is biphasic, with PMA causing an increase in secretory
degranulation, as measured by the expression o f alkaline phosphatase, at I and 100 ng/ml
62
but a decrease at 10 ng/ml [19]. Bovine specific granules are also mobilized by PMA,
with a peak at 10 ng/ml, but not in a biphasic manner. These granules are known to
contain (Ba integrals in addition to fibrinogen and laminin receptors [24]. Since the
neutrophils in these experiments were treated with 20 nM PMA, which corresponds to 12
ng/ml, it is unclear what receptors would have been upregulated. To conclusively show
whether PM A regulates the rotavirus receptor expression on bovine neutrophils, a more
complete dose-response curve would have to be performed.
VOPBAs, or virus overlay protein blot assays, are a useful way to study virus
binding. They are simply modified Western blots o f cell membrane o f interest, using
purified viral particles as a probe. An advantage o f this method includes identification o f
the molecular mass o f the protein that binds the virus. A major disadvantage is that the
receptors are rarely in their native state after membrane isolation and separation on a
reducing and denaturing gel. For example, integrals are proposed to be rotavirus
receptors, and they become separated into their respective subunits in a denaturing
solution. Other receptors may not be in their native conformation as well. Indeed, this
aspect o f VOPBAs may explain some o f the discrepancies in the literature. The bovine
strain NCDV bound to several bands between 80 and 116 kDa in HT29 cells and to a 95
kD a protein on M A l 04 cells [77]. The same lab had previously reported that the bovine
NCDV strain recognized three bands on M A l 04 cells between 66 and 97 kDa [78]. The
axP 2 integral is a proposed receptor for rotavirus, and the 02 subunit is 95 kDa. Still
another group found that a rhesus virus strain, RRV, bound to five proteins in M A l 04
cells at 3 0 ,4 5 ,5 7 ,7 5 , and 110 kDa, none o f which appear to be integrals [79]. When
63
these studies are done with intestinal fractions, still different results are obtained. For
example, when murine brush border membranes were probed with RRV, tw o bands
appeared at much higher molecular weights than the cell line VOPBAs, occurring at 300
and 330 kD a [91]. Appearance o f the bands correlated with both the cell type and the age
o f susceptible mice. When double-layered particles were used, the authors noted
contaminating bands from 120-200 kDa [91]. Since the intestinal blots should
theoretically contain the in vivo viral receptor, these results call into question the validity
o f the cell line VOPB As. One such resolution o f these different results is that the
receptors in the intestine are the same as the cell lines, but have different patterns o f
glycosylation. Another possibility is that rotavirus utilizes different receptors in cell lines
than it does in the intestine.
Unfortunately, the neutrophil VOPBAs did not provide consistent results and had
significant background, staining in the antibody control lanes. Perhaps the membranes
were binding antibody through Fe receptors. Neutrophils possess three types o f Fe
receptors, FcRI, FcRII, and FcRIII. FcRIII is the most common antibody receptor on
neutrophils. It is 50 to 80 kDa normally, but 21 to 24 kDa after removal o f carbohydrate
residues [175]. This receptor will bind IgG complexes, but not monomeric IgG [176].
FcRII is the second most common, and is 40 kDa. FcRI is 72 kDa, but after removal o f
N-Iinked glycoproteins is only 55 kDa [175]. It is typically only expressed in activated
neutrophils, such as after IFN-y treatment [176]. Whether or not the non-specific bands
correlate with these Fe receptors is unknown, but the strongest contaminating band
appeared at 20 kDa, and could perhaps be the result o f the FcRIII receptor. The fact that
64
one set o f VOPBAs showed a band at 95 kDa was exciting, since that is the weight o f the
p2 subunit o f the axpz integrin, which is expressed on bovine neutrophils. This band was
also seen in studies o f another bovine virus binding to M A l 04 and HT29 cells [77]. I f
this band had been darker, and more consistent, it would have been interesting to see if
antibodies specific to this integrin inhibited this band.
Assays with radiolabeled viral particles were performed to obtain even more
information about the neutrophil and rotavirus binding interactions. These assays can
provide data on saturation, specificity, and internalization. A main disadvantage is that
once again, viral aggregates, and contamination with DLPs could be skewing the results.
In addition, it was difficult to achieve the high virus:cell ratios necessary for saturation
studies, since cells at or lower than IO4Zml were lost in the wash steps. Despite these
difficulties, binding curves were generated for each cell type, and analyzed with a
Scatchard plot. This plot can help ascertain the mode o f viral attachment, which can
occur one o f three ways [177]. Mode I, or monovalent attachment, occurs when one
virus binds to only one receptor either because it is too small to bind more than one, or
the number o f receptors on the cell is low. In this case, the virus site number equals the
receptor number, and the Scatchard plot gives a straight line with the x axis indicating the
number o f receptors per cell. Mode 2, or receptor saturation with multivalent binding, is
used to evaluate viruses which first attach to a single receptor, but then undergo a series
o f reversible binding interactions with other receptors. The Scatchard plot shows a
reverse sigmoid line, instead o f a straight line. In this mode, the number o f receptors for
a virus is different than the number o f viral attachment sites. Thus, a way to test for a
65
mode 2 interaction is to do a binding assay with fully infectious particles, then with
purified viral attachment proteins, and see if the numbers agree. I f the first number is less
than the second, mode 2 kinetics may be involved. Mode 3 describes spatial saturation
with multivalent binding, which is often the case for cells that have a very high number
o f receptors, or for very large viruses. In this mode, the Scatchard plot is a straight line
similar to mode I, but the virus site number is less than the receptor number. There are
perhaps other modes o f binding, but these three seem to encompass the vast majority o f
viruses [177].
The data from Figure 2.5 was converted into Scatchard plots, and the neutrophil
graph is shown in Figure 2.6. The cells non-permissive for viral infection, K562 cells
and neutrophils, appear to follow mode one. This fits nicely with the theory that most
cells have the initial attachment receptor for rotavirus, but not the internalization receptor
[72]. M A l 04 cells, on the other hand, have all the necessary receptors and since most
data indicate that rotavirus utilizes more than one receptor to gain entry. However, some
caution must be placed here. The data that was used to generate the Scatchard plots did
not reach saturation for any o f the cell lines tested, and thus may be not indicative o f the
full interaction. In addition, the data would need to be repeated many more times.
The radioactive virus-binding studies do confirm the other tw o experiments that
rotavirus does indeed bind to neutrophils, and added the information that it is specific,
with the cold competition. Further studies could be done to characterize the neutrophilrotavirus interaction. First, treating neutrophils with neuraminidase would determine if
sialic acid residues were mediating attachment. Since B641 is the same P5 serotype as
66
the sialic-acid independent UK strain, no difference in binding would be expected after
neuraminidase treatment [71,80]. Blocking studies using antibodies to the integrins
present on neutrophils, which include c^fh, a 4[3i, and OtxP2, would also be useful.
The radioactive virus-binding assays also allowed internalization tests. In this
study, internalization was not seen after a one-hour incubation. This was surprising,
since neutrophils are known to internalize many different viruses [30]. One possibility is
that virus needs to be opsonized with antibody or complement for internalization.
Neutrophils will phagocytose HSV at an increased rate when the particles are coated with
viral-specific antibodies or complement [32]. Another interesting study would be to see
if adherent neutrophils are more susceptible to rotavirus infection. Adherent neutrophils
were tested in a radiolabeled virus-binding study, but this study did not work due to the
loss o f nearly all the adherent cells after the required wash steps.
I f internalization with one o f these methods did occur, more studies could be done
to determine how virus entry was taking place. Rotavirus is thought to enter enterocytes
through direct membrane penetration, not by endocytosis like many other viruses [74].
However, neutrophils are a professional phagocytic cell. To test which method was
being utilized, inhibitors o f endocytosis such as sodium azide or dinitrophenol could be
added. I f internalization occurred in the presence o f these inhibitors, then the virus was
most likely entering through penetration.
The results showing that neutrophils bind rotavirus but do not internalize it after I
hour support the conclusion that rotavirus binds to a wide variety o f cell lines, but
binding does not mean internalization and infectivity [72]. Nevertheless, since specific
67
binding does occur, it is possible that rotavirus could modulate cellular functions, similar
to influenza A virus. IAV binds directly to sialylated glycoproteins on neutrophils via its
hemagglutinin antigen and initiates a signal cascade without needing to internalize. The
net result o f this signal is an interference with superoxide production, phagosomelysosome fusion, and normal response to activators [42]. In these cases, the virus acts
like a chemokine, binding to the receptor and exerting the effects from outside the cell.
Whether or not rotavirus can mediate any effects by binding to neutrophils was evaluated
in the following chapter (Chapter 3).
68
ANALYSIS OF BOVINE ROTAVIRUS EFFECTS ON
NEUTROPHIL FUNCTIONAL RESPONSES
Introduction
Neutrophils, or polymorphonuclear leukocytes (PMN), are an essential part o f the
host defense system against bacterial and fungal pathogens [11,178], After these
leukocytes arrive at the infection site through a process called chemotaxis, they kill
invading microbes with a plethora o f functions such as phagocytosis and oxidative burst
[2], Recently, it has been noted that neutrophils play a role in viral clearance as well. In
addition to inhibiting virus replication, neutrophils can also be modulated by viruses
[30,39,56], creating profound effects on the host. Elucidating the interactions between
viruses and neutrophils will increase our understanding o f how viruses cause disease.
A neutrophil has the capacity to interact with viruses in several ways. First,
viruses can elicit direct responses from neutrophils, which may implicate these
leukocytes in viral defense. Neutrophils phagocytose viral particles including
adenovirus, poliovirus, vaccinia virus, Coxsackie virus, herpes simplex (HSV), influenza
A virus (IAV), and cytomegalovirus [30]. This ingestion obviously has the potential to
impair viral replication and spread. Neutrophils inhibit HSV, Japanese encephalitis virus
and vaccinia virus replication by phagocytosis and degradation. Often this inactivation is
augmented by the coating o f viruses with complement Or serum, which are both known to
increase neutrophil ingestion o f particles [32,34,35]. Viruses can also take advantage o f
neutrophil phagocytosis to spread themselves. CMV infects neutrophils in the
69
bloodstream, and there is evidence that migrating neutrophils facilitate virus
dissemination [31]. H IV may also spread to T cells from neutrophils in a similar fashion
[36]. In addition to phagocytosis, neutrophils inhibit viral replication in other ways.
Vaccinia viruses induce oxidative metabolism in human neutrophils in vitro, which leads
to their inactivation [34]. Since neutrophils have been found at the scene o f many viral
infections, it is possible that these actions do occur in vivo [30].
In addition to activation, viruses can also directly inhibit neutrophil functions. It
has been recognized for nearly a century that viral infections can lead to bacterial
susceptibility, due in part to the ability o f viruses to shut down this major player in
bacterial defense. The adverse effects o f influenza A virus on neutrophils have been
well-documented [39]. The H antigen o f IAV binds to sialic acid residues on certain
neutrophil proteins, which induces a calcium-dependent signal cascade resulting in
decreased chemotaxis, oxidative burst, and bacterial phagocytosis and killing [40,42],
These alterations result in bacterial superinfections o f IAV patients [179]. Other viruses
can inhibit neutrophils in a similar manner. Echo-9 virus impairs chemotaxis to £MLF
[43], and Newcastle disease virus (NDV) causes a reduction in ROS generation [45].
These described effects require that the neutrophils bind to the virus, either
directly, or to molecules coating the virus such as antibodies or complement. However,
viruses can also modulate neutrophils indirectly through the induction o f cytokines or
other mediators. These bioactive molecules, in turn, either enhance or suppress
neutrophil functions. Rhinovirus, one o f the causes o f the common cold, induces airway
epithelial cells to release inflammatory mediators such as IL-8. Consequently,
70
neutrophils are recruited and activated, resulting in exacerbation o f airway inflammation
[54]. HIV infection, on the other hand, results in neutrophil inhibition. Neutrophils from
AIDS patients have severely impaired functions including reduced superoxide
production, antibody-dependent cellular cytotoxicity (ADCC), and complement
responses [46-48]. Since HIV does not directly interact with neutrophils, most likely it
inhibits them by altering the cytokine profile, especially by decreasing the levels o f GCSF, an important neutrophil growth factor [49,51].
Based on these previous studies, it was hypothesized that rotavirus could mediate
neutrophil effects in one or more o f these ways. Neutrophils are capable o f binding
rotavirus (Chapter 2), and they have been found at the site o f rotavirus infection in
several species [58,160,161]. In addition, cells infected with rotavirus secrete neutrophil
chemokines and cytokines in vitro [160]. Since the interaction between rotavirus and
neutrophils has never been examined, these studies were designed to characterize the
bovine neutrophil response to a bovine-specific rotavirus. To look for direct interactions
between rotavirus and neutrophils, purified viral particles were first tested as direct
activators, and then as primers or inhibitors, in several functional assays. To test whether
rotavirus is capable o f inducing neutrophil cytokines, M A l 04 cells were infected with
virus and at their supernatants were collected at various time points and tested in the
same functional assays. From these studies, it was determined that rotavirus does not
elicit neutrophil responses in any o f the conducted tests. These experiments provide the
first lines o f evidence that rotavirus does not induce host defense responses in bovine
neutrophils in vitro.
71
Materials and Methods
Materials
M l 99 media powder was purchased from Irvine Scientific. Flasks and roller
bottles for virus propagation in M A l 04 cells were from Fisher. Fetal bovine serumpremium was bought from Atlanta Biologicals. Buffers used for neutrophil studies,
HBSS and DPBS, were purchased from Gibco. Trypsin used to activate virus was
obtained from Worthington Biochemicals. Fluorescent Staphylococcus aureus particles
and calcein-AM were purchased from Molecular Probes. AU animal use was approved
by the M SU Institutional Animal Care and Use Committee. Dr. Michele Hardy kindly
provided original stocks o f the B641 bovine strain o f rotavirus, as weU as M A l 04 ceUs.
Preparation o f Purified Viral Particles
The bovine rotavirus strain B641 was propagated by infection o f M A l 04 ceUs
with virus stocks. Cells were grown to 90-95% confiuency in roUer bottles with M l99
complete medium supplemented with 10 mM sodium bicarbonate, 20% FBS, and 4 mM
L-glutamine. On the day o f the infection, ceUs were incubated with media lacking FBS
for 2 to 3 hours, since protease inhibitors in fetal bovine serum inhibit viral repUcation.
MeanwhUe, a virus stock was treated with 10 pg/ml trypsin (Worthington) to cleave VP4,
which enhances inactivity [59]. Media was removed from the roUer bottles, and virus
was added in a volume o f 5 ml at an MOI o f 1-10. The MOI, or multiplicity o f infection,
was calculated by the foUowing formula.
72
ml o f virus = CMOIVcells/rollef)
(Titer o f virus)
in which cells/roller is 7x1075 and the virus titer is the amount o f plaque-forming units
(PFU) per ml, as determined by a plaque assay. Cells were inoculated for one hour at
37°C, then 10 ml o f post-infection media was added (M199 complete, no FBS, plus I
pg/ml trypsin, and 15 mM sodium bicarbonate). Roller bottles were checked daily to
determine the cytopathic effect (CPE), which is the amount o f detachment o f infected
cells. Once 90% CPE had been reached, roller bottles were placed in - 20°C and
subjected to three freeze/thaw cycles to break up the cells and release viral particles. The
supernatant was harvested, and viruses were purified according to previously described
methods [80]. Briefly, supernatant was centrifuged at 90,000 x g with a 20% sucrose
cushion to concentrate the virus. The virus-containing supernatant was then centifuged
on a cesium chloride gradient at 190,000 x g for 18 hours. This gradient separates the
fully infectious triple layered particles (TLP), which appear at the 1.36 g/ml density band,
from the VP4- and VP7-lacking double layered particles (DTP), which appear at the 1.38
g/ml band. Bands were then collected and pelleted at 190,000 x g for I hour. The
concentration o f particles was determined by a spectrometry reading at A 260, since they
contain RNA. The protein concentration in pg/ml was then determined by multiplying
the reading by the dilution factor and then by 185. Particles per ml were calculated by
multiplying the pg /ml by 3.33x108. These conversion factors were previously obtained
by counting viral particles via electron microscopy, then correlating particle counts with
protein and RNA measurements [171]. Note that these calculations assume all particles
are triple layered. On average, the particle to PFU ratio was 100, meaning that for every
73
100 particles, only one is capable o f replication. Particles were then stored in Tris buffer
(10 mM Tris, 100 mM NaCl, and 5 mM CaCl2) at -80°C until use.
Isolation o f Bovine Neutrophils
Blood was obtained via jugular venipuncture from healthy Holstein calves and
purified as described in [172]. Two water lyses were performed to remove red cells, and
remaining cells were centrifuged through a gradient o f 1077 and 1119 Histopaque. This
gradient separates the mononuclear cells, which remain in the upper layer, from the
neutrophils, which pellet to the bottom. The mononuclear cell layer was then removed,
and the neutrophils were washed in PBS and counted with a hemacytometer. Viability
was determined by trypan blue staining.
Superoxide Production
External superoxide production was measured by the reduction o f cytochrome c
as described in [180]. 20 pl o f I mM cytochrome c and 160 pi o f buffer (BBSS + 10 mM
Hepes + I mM CaCl2) were placed in wells o f a 96-well microtiter plate. To some wells,
50 U/ml superoxide dismutase (SOD) was added in 2 pi volumes as an inhibitor. This
enzyme will scavenge any O2' generated, preventing it from reducing cytochrome c.
Next, the activators were added to wells in 2 pi volumes. PMA, a phorbol ester that
induces superoxide in neutrophils, was used at 10 to 100 ng/ml final concentration as a
positive control. The cells, which were previously diluted to IO7Zml in HBSS without
calcium, were added last in 20 pi volumes. The plate was immediately read in a
Molecular Devices THERMOmax plate reader for 15 minutes, at 37°C, at 550 nm.
74
Chemotaxis
Chemotaxis was measured by two different methods. The LDH method used
Falcon 24-well plates and 3 pm pore size tissue culture inserts. Chemotactic reagents or
controls were placed in wells in 250 pi volumes, then inserts were placed in the wells.
IO6 cells in a volume o f 250 pi buffer (HESS + 10 mM Hepes + 0.1% BSA) were then
pipetted into the inserts. The plate was placed in a 37°C incubator with 5% CO 2. After I
hour, inserts were removed, and 0.9% Triton-X 100 was added to each well to lyse the
cells. Quantitation o f cell migration was performed using a Cytotox 96® kit (Promega).
This kit detects the presence o f lactate dehydrogenase (LDH), which is proportional to
the amount o f cells present. After cell lysis was complete, 50 pi from each well was
transferred into a clear 96-well plate, 50 pi o f assay buffer was added, and the plate was
incubated at room temperature for 30 minutes. Finally, 50 pi o f stop solution was added,
and the plate was read in a microplate reader at 490 nm. OD readings were converted to
% cell migration by comparing them against cell standard controls.
The second chemotaxis method involved using ftuorescently-labeled cells.
Neutrophils were first loaded with 2 pi o f stock calcein-AM (Molecular Probes, final
concentration 2 pg/ml) for 30 minutes at 37°C, then washed and placed in chemotaxis
buffer (HESS + 10 mM Hepes + 0.1% ESA). ChemoTx 96® plates (Neuroprobe) were
set up with chemotactic agents in 25 pi volumes in the bottom wells, allowing them to
form a positive meniscus. Then the filter with a hydrophobic coating was fitted on top o f
the plate. Cells at 4x106Zml were pipetted in 25 pi volumes on the top o f the filter above
the wells. The chamber was then placed in a 37°C incubator with 5% CO 2 for I hour.
75
Next the filter was removed, and fluorescence o f each well was read in a plate reader
with excitation 485, emission 538. Fluorescent readings were converted to % cell
migration by comparing them against cell standard controls.
Phagocytosis
To test phagocytosis, neutrophils were incubated with FITC-Iabeled S. aureus at a
ratio o f 15 bacteria to I cell for 30 minutes in a 37°C shaking water bath, in HBSS + I
mM Hepes + I mM CaCl2. Then cells were washed, and incubated for another 30
minutes in HBSS without CaCl2 to allow for complete internalization. Cells were then
washed, and analyzed by reading in the flow cytometer using the FL l channel.
Exposure o f Neutrophils to Rotavirus
The virus incubation step was optimized by trying various times and buffers, then
testing the cells for viability and for ability to produce O2". It was determined that
rocking the cells for 30 minutes at 37°C, in HBSS + 10 mM Hepes + 0.1% BSA, was the
optimal incubation method, and used in all experiments unless otherwise indicated.
Collection o f M A l 04 Cell Supernatants
M A l 04 cells were grown to confluency in Costar 6-well plates w ith M l 99
supplemented with 20 mM sodium bicarbonate, 20% FBS, and 4 mM L-glutamine. On
the day o f virus infection, media was replaced with media lacking FBS. Virus was
activated with 10 pg/ml trypsin for 30 minutes at 37°C, diluted to various MOIs (from 0.1
to 5) and added to wells in 500 pi volumes. After I hour, fresh M l99 complete media
was added to each well. Mock-infected control plates were prepared in the same manner.
76
Supernatant was collected at various time points after infection, centrifuged to remove
cell debris, transferred and aliquotted to fresh tubes, and frozen at -20°C until use.
Aliquots were not reused after thawing.
Plaque Assays
Virus stocks were exposed to neutrophils, and their titers were tested in a standard
plaque assay as follows. M A l 04 cells were grown in Costar 6-well plates and inoculated
with serial dilutions o f virus supernatant for one hour. Inoculum was removed, and a 1:1
mixture o f 1.2% Seakem agarose and M l 99 media supplemented with pancreatin and
DEAE dextran was overlaid in 3 ml volumes. Two days later, another mixture o f agarose
and media, supplemented with neutral red, was overlaid in 2 ml volumes. Over the next
three days, plaques were counted. Titers were determined by multiplying the average
plaque numbers by the dilution factor.
Statistical Analysis
AU error bars represent the SEM (standard error o f the mean). Where indicated,
the student’s two-tailed t test was performed to ascertain statistical significance.
Results
Analysis o f NeutrophU Effects on Rotavirus RepUcation
To test whether neutrophUs can inhibit rotavirus rephcation, virus supernatant at a
titer o f 2x10 6 PFU/ml was incubated with either unstimulated or PMA-stimulated
neutrophUs, than plated in a plaque assay. Control virus samples were incubated in
77
buffer without neutrophils, and plated in the same manner. No differences were seen in
the plaque numbers for two different viral titers (Figure 3.1). This indicates neutrophils
are unable to inactivate virus directly, even after maximal activation with PMA.
Analysis o f Neutrophil Responses Elicited bv Rotavirus
Purified triple layered particles were tested for their ability to induce several
neutrophil functions. Rotavirus at 10 to 1000 times the concentration o f the neutrophils
did not induce a superoxide burst after a 15-minute incubation, while the positive control
PMA induced a robust response (Figure 3.2). SOD was used as an inhibitor to
demonstrate that the reduction o f cytochrome c was solely due to O2". To test whether
opsonization with bovine serum is required for a rotavirus-induced oxidative burst,
viruses were pre-incubated with 5 pi pooled calf serum for 15 minutes at 37°C, then
tested for their ability to induce an oxidative burst (Figure 3.3). None o f the virus
concentrations caused a superoxide release above control, even after opsonization. It
appears that the highest dose o f opsonized virus may have slightly inhibited spontaneous
oxidative burst. However, this decrease was not significant.
To test whether rotavirus was chemotactic for neutrophils, viruses were diluted
and placed in the lower half o f a neuroprobe chamber, and calcein-loaded cells were
placed on top. Over forty percent o f the cells migrated to the positive control, IL -8 at
IO-8M (Figure 3.4). In contrast, rotavirus did not stimulate a chemotactic response above
the buffer control. The IO9Zml well does look slightly higher, but the difference from the
78
CU Virus A lon e
BgSNeut + Virus
■ A ct N eut + Virus
lO'S
10'
Dilution of Virus
Figure 3.1: Plaque Assay o f Viruses Exposed to Neutrophils. Viruses were divided into
three pools: virus alone (dotted bars), virus plus inactivated neutrophils (checkered bars)
and virus plus 10 ng/ml PMA-stimulated neutrophils (black bars). Incubation took place
at 3T C for 30 minutes. Neutrophils were removed by centrifugation, and titer was
determined by a plaque assay. Data is expressed as average number o f plaques on day 3,
in duplicate. The original virus titer was 2x106 PFU/ml, and neutrophils were at IO7Zml.
.2 30-
Buffer
PMA
PMA+I 2x107
2x108
2x10'
Activator
Figure 3.2: Superoxide Production Induced by Rotavirus. Viruses were added to wells, at
the concentrations indicated per ml, in a 96-well plate containing 2x10 neutrophils in
triplicate. Superoxide production was measured in a cytochrome c assay for 15 minutes.
PMA at 100 ng/ml was used as a positive control, and the wells with SOD are
represented by the PMA + I bar. The data are expressed as the mean Vmax ± SEM.
79
QPM A
■ Buffer
^ 107 v/ml
E llO 9 v/ml
1 ^ 1 0 10VZmI
Activator + Serum
Activator
Figure 3.3: Superoxide Production Induced by Opsonized Rotavirus. Virus particles
were incubated with pooled calf serum for 15 minutes at 37 0C. Then virus was added to
wells, at the concentrations indicated, in a 96-well plate containing 2x106 neutrophils in
triplicate. Superoxide production was measured in a cytochrome c assay for 15 minutes.
The data are expressed as the mean Vmax ± SEM.
.2> 30
V iruses per ml
Figure 3.4: Chemotaxis to Triple Layered Particles. Calcein-Ioaded neutrophils were
tested for chemotaxis to purified TLPs at the indicated concentrations. The
chemoattractant IL -8 was used as a positive control at IO 8 M. The data are expressed as
% PMN migration, determined by comparing fluorescent readings against cell standards.
Each bar represents 5 samples.
80
buffer control was not statistically significant. Additionally, double layered particles
failed to induce chemotaxis (Figure 3.5).
Analysis o f Rotavirus Effects on Neutrophil Responses
Rotavirus was tested for its ability to alter three neutrophil functions: oxidative
burst, chemotaxis, and phagocytosis. First, the virus incubation method was optimized to
insure that this step by itself was not substantially altering neutrophil function. Cells
were incubated for either 30 or 45 minutes with a variety o f buffers. The cells were then
washed, recounted, stained for viability with trypan blue, and tested for the ability to
produce an oxidative burst in response to PMA. It was determined that rocking the cells
for 30 minutes at 37°C in HBSS + IOmM Hepes + 0.1% BSA gave the highest cell
count, viability and superoxide production, and therefore these conditions were used in
all experiments unless otherwise indicated (data not shown).
Neutrophils incubated with rotavirus in this manner were tested for their ability to
produce O 2" to PMA. Even at a virusmeutrophil ratio o f 1000:1, viruses did not affect
superoxide burst (Figure 3.6). Next, viruses were tested to see if they interfered with
chemotaxis. Using the calcein method, it was shown that neutrophils exposed to virus
migrated at the same rate as cells exposed to control buffer (Figure 3.7). Because calcein
incubation is performed before the virus exposure step, there was some concern that
perhaps the calcein was preventing the viruses from binding or otherwise altering the
neutrophils. To confirm the calcein data, the LDH chemotaxis assay was performed,
which did not involve an additional incubation step. Even at a virus:cell ratio o f I G00:1,
no difference in migration was observed (Figure 3.8). This pattern was true for both
81
.g> 30-
Ib
4x10'
4
V iruses per ml
Figure 3.5: Chemotaxis to Double Layered Particles. Calcein-Ioaded neutrophils were
tested for chemotaxis to DLPs at the indicated concentrations. IL -8 was used as a
positive control at I O 8 M. The data are expressed as % PMN migration, determined by
comparing fluorescent readings against cell standards. Each bar represents 5 samples.
Control
EHj PMA S S PMA+ SOD
I
c
O
=3
T3
2
CL
a>
-o
X
2m
CL
3
CO
1000:1
Virus: Neutrophil Ratio
Figure 3.6: Effect o f Rotavirus on Neutrophil Superoxide Production. Purified
neutrophils were incubated with the indicated concentrations o f virus for 30 minutes at
37°C. Cells were then washed, placed in a 96 well plate in triplicate, and tested for
superoxide production in a cytochrome c assay with buffer alone (black bars), 100 ng/ml
PMA (dotted bars), and PMA plus SOD (checkered bars). The data are expressed as the
mean Vmax ± SEM.
82
™ 30-
100:1
1000:1
VirusiNeutrophiI ratio
Figure 3.7: Effect o f Rotavirus on Neutrophil Migration, Calcein Method. Calceinloaded neutrophils were incubated with the indicated concentrations o f rotavirus for 15
minutes at 37°C. Cells were washed and allowed to migrate to IO'8 M IL -8 in a
neuroprobe chemotaxis chamber. The data are expressed as % PMN migration,
determined by comparing fluorescent readings against cell standards. Each bar represents
5 data points.
V irusPM N
I
□ 0:1
E5320:1
■ 200:1
T
1 302
2 _
I
CL
Buffer
IL-8
Chemoattractant
LTB4
Figure 3.8: Effect o f Rotavirus on Neutrophil Migration, LDH Method. Purified
neutrophils were incubated with the indicated concentrations o f virus for 30 minutes at
37°C, washed, and tested for chemotaxis using the LDH method. Both IL -8 and LTB 4
were used at IO"8 M. This graph is the compilation o f three experiments (two in
triplicate, one in duplicate). The data are expressed as % PMN migration, determined by
comparing OD 490 readings against cell standards.
83
agents tested: IL- 8, a protein chemokine, and LTB4, a lipid chemoattractant. Together,
these two experiments indicate that rotavirus has no effect on neutrophil chemotaxis.
Another important bactericidal function o f neutrophils is phagocytosis. To test
whether rotavirus altered this function, phagocytosis o f Euorescently labeled
Staphylococcus aureus particles was analyzed. Pre-incubating neutrophils with viral
particles did not alter their ability to phagocytose the bacteria (Figure 3.9). In addition,
neutrophils exposed to the bacteria and viruses simultaneously had no problems
internalizing S. aureus. In control samples, background staining o f neutrophils alone was
negligible (data not shown).
Analysis o f Neutrophil Responses to Virus-Infected M A l 04 Supernatants
Rotavirus was tested for its ability to induce secretion o f neutrophil activating
factors, e.g. chemotactic agents, in M A l04 cells. Since the LDH method relies on a red
colorimetric agent, and the cell culture supernatants contain a red dye, all the chemotaxis
studies were done using the calcein method. For the first experiment, M A l 04 cells were
infected w ith either no virus for a control, or with an MOI o f I, 2.5, or 5. Virus inoculum
was left on to ensure preservation o f all chemokines produced, and then supernatants
were collected at 0, 4, and 12 hours post-infection. Supernatants were then tested in the
calcein assay. There was a statistically significant difference in chemotaxis at M O I I and
2.5 after 12 hours, compared to the no-virus control (Figure 3.10).
Since the M OI 2.5 supernatant at 12 hours induced the highest level of
chemotaxis, this infection was repeated in two time course studies. The first study
included 0, 15, 21, and 23 hours after infection; however, none o f these supernatants
84
10000
Staph
Staph + V V then S
Figure3.9: Effect o f Rotavirus on Phagocytosis. Neutrophils were purified and treated in
three ways: incubated with bacteria alone, incubated with bacteria and virus together, or
preincubated with rotavirus then incubated with bacteria. FITC-Iabeled S. aureus was
used at a bacteria:cell ratio o f 15:1. Fluorescence was measured with a flow cytometer.
The data is expressed as the average FLl reading, with 5 replicates per sample.
□ No Virus
□ MOI 1
BSMOI 2.5
■ MOI 5
z
CL
Hours Post Infection
Figure 3.10: Neutrophil Chemotaxis to M A l04 Supernatants. M A l04 cells were infected
with rotavirus with several MOIs and supernatant was collected at the times indicated.
Neutrophil chemotaxis to supernatants was measured using the calcein assay. The data
are expressed as % PMN migration, determined by comparing fluorescent readings
against cell standards. The data is represented by 13 replicates pooled from 3
experiments. The * specifies p<.01, and ** specifies p< 001, when compared to the no­
virus control at the same time point.
85
induced chemotaxis (data not shown). This experiment was repeated to include more
time points, and to pay careful attention to the health o f the M A 104 cells as time
progressed. Many cells started to reach CPE and lift off the plates about 13 hours post
infection. Sixty to 90% o f the cells had lifted from the plates at 24 hours post-infection,
but a few cells were resilient and did not lift until 87 hours later. Control cells remained
adherent even past 52 hours. These supernatants were then tested for their ability to
induce neutrophil migration. Once again, none o f the time points were chemotactic
(Figure 11). Migration to IL -8 was 12 times-higher than it was to buffer control (data not
shown). This second set o f supernatants was also tested for its ability to induce an
oxidative burst. Neither the mock- nor the virus-infected supernatants induced an
oxidative burst above the buffer control (Figure 12). As a control, PM A induced a
characteristic oxidative response.
It was unclear why the results from Figure 3.10 were not reproducible. One
possibility is that the M A l04 cells were being killed before they could secrete cytokines
or other putative factors. This theory is supported by the fact that MOT 5 supernatants did
not induce chemotaxis, unlike I and 2.5 MOI supernatants. Therefore M A l04s were
infected with a much lower MOI o f 0.1, and supernatants were collected approximately
every 4 hours for 49 hours. In this experiment, complete CPE was not reached at the
time points tested, due to the low MOL At around 25 hours post-infection, 35 to 40 % o f
the cells showed CPE. Compared to the 2.5 M OI study, more cells were alive at all time
points in the 0.1 MOI experiment. These supernatants were then tested for their ability to
cause neutrophil chemotaxis, compared to uninfected cell supernatants. Three slightly
86
-"-Virus
-o-Control
Hours Post Infection
Figure 3.11: Chemotactic Activity o f 2.5 MOI Supernatants. M A l04 cells were grown to
90% confluency, then infected with either no virus (dotted line) or virus at an MOI o f 2.5
(solid line). Supernatants were collected from 0 to 87 hours post infection and tested for
their ability to induce chemotaxis using the calcien method. The data are expressed as %
PMN migration, determined by comparing fluorescent readings against cell standards.
Each point represents duplicate samples, and this is a representative o f three experiments.
- a -is -"-Virus
I
-o-Control
6 t T PMA
5
• Buffer
2
EMA
Buffer
Hours Post Infection
Figure 3.12: Oxidative Burst Stimulated by 2.5 MOI Supernatants. M A l04 cells were
grown to 90% confluency, then infected with either no virus (dotted line) or virus at an
MOI o f 2.5 (solid line). Supernatants were collected from O to 87 hours post infection
and tested for their ability to induce superoxide production in a cytochrome c assay. The
triangle represents the PMA positive control, and the circle represents background 0%
production. The data are expressed as the mean Vmax ± SEM, done in triplicate.
87
different migration patterns were noted from three separate experiments, so the data were
pooled to look for any overall patterns (Figure 3.13). There was a statistically significant
difference at 40 and at 49 hours, but not at 46 hours, making it unlikely that a
chemotactic response occurred. It was hypothesized that perhaps the concentrations o f
the chemoattractants was too high in the pure supernatant. Therefore, supernatants at all
time points were diluted 1:10 and 1:100, and tested for chemotaxis. No differences were
seen between the mock- and the virus-infected cell supernatants (data not shown).
Virus-infected cell supernatants were also tested for their ability to modulate
neutrophil functions. Supernatant collected 23 hours post infection was tested for its
ability to alter neutrophil migration to a known chemotactic agent. Neutrophils were
incubated with buffer, control cell supernatant, or virus-infected cell supernatant for 30
minutes at 37°C, and then placed in a chemotactic chamber. Both the mock- and the
virus-infected supernatants increased neutrophil chemotaxis to IL-8 (Figure 3.14).
However, there was no significant difference between the virus and the mock
supernatants. Neutrophils exposed to these agents were also tested for superoxide release.
In contrast to the chemotaxis results, exposure to both supernatants slightly decreased the
PMA-induced superoxide burst but again, there was no difference between the mock and
the virus supernatants (Figure 3.15).
v
Discussion
Several lines o f evidence indicate that rotavirus and neutrophils could directly
interact. Our previous studies showed that neutrophils are capable o f binding to
88
-"-V irus
-o-Control
Hours Post Infection
Figure 3.13: Chemotactic Activity o f 0.1 MOI Supernatants. M A l04 cells were grown to
90% confluency, then infected with either no virus (dotted line) or virus at an MOI o f 0 .1
(solid line). Supernatants were taken at time points from 0 to 49 hours post infection as
indicated, and their chemotactic activity was tested on neutrophils using the calcein
method. The data are expressed as the mean fluorescence o f the wells. This data is a
compilation o f three experiments, each done in replicates o f 5. The * indicates p<0.05.
X 300-
Buffer
No Virus
Virus
Figure 3.14: Neutrophil Chemotaxis After Exposure to Virus-Infected Cell Supernatants.
M A l04 cells were infected with either no virus or rotavirus at an MOI o f 2.5, and the
supernatants were collected at 23 hours. Neutrophils were incubated with 100 ul o f either
buffer, mock-infected supernatant (labeled “No Virus”), or virus-infected supernatant
(labeled “Virus”), for 30 minutes at 37°C. Cells were then washed and tested for
chemotaxis toward IO 8M IL-8 with the calcein method. The data are expressed as the
mean fluorescence o f the wells, and each bar represents 5 samples.
89
ICells only CHl PMA
I
204
I
I 1W
^
PMA + SOD
3
CL
I
0)
TD
I
"
104
0)
CL
3
CO
Buffer
No Virus
Virus
Figure 3.15: Oxidative Burst o f Neutrophils After Exposure to Virus-Infected
Supernatants. M A 104 cells were infected with either no virus, or virus at an MOI o f 5,
and the supernatants were collected at 8 hours post infection. Cells were incubated with
100 ul o f either buffer, mock-infected supernatant (labeled “No Virus”) or virus-infected
supernatant (labeled “Virus”) for 30 minutes at 37°C. Cells were then washed and tested
for oxidative burst in response to 100 ng/ml PMA. The data are expressed as the mean
Vmax ± SEM, and each bar represents 3 samples.
rotavirus. They also express some o f the proposed viral receptors such as c^pi and axP 2
integrins [14,157]. In addition, neutrophils have been found at sites o f rotavirus
infections in humans [160], chickens and turkeys [58], and lambs [161]. Band cell
production was increased in 21% o f children infected with rotavirus alone, indicating this
virus could be inducing neutrophil production [162]. Indirect interactions may occur
with rotavirus as well, since cells infected in culture produce neutrophil chemokines such
as GROa and IL-8 [160]. Because rotavirus is usually cleared before the adaptive
immune system has time to respond, it is possible that neutrophils play a role in
clearance. Therefore, these studies were undertaken to see if bovine neutrophils could
affect, or be affected by, the B641 bovine strain o f rotavirus.
90
Several viruses can be inactivated by neutrophils. Neutrophils will phagocytose
HSV, especially when the particles are coated with viral-specific antibodies or
complement [32]. Human neutrophils degrade both Japanese encephalitis and vaccinia
virions via phagocytosis and subsequent reactive oxygen species production, even
without opsonization [34,35]. IAV, Sendai, adenovirus, and NDV are also known to
induce an oxidative burst in neutrophils [30]. In IAV, this burst can be enhanced by the
addition o f IFN-a [40].
Since our previous studies determined that neutrophils bind rotavirus, but do not
internalize unopsonized particles after one hour (Chapter 2), further studies were done to
see if this binding leads to any measurable effects on neutrophil functional responses.
Neither double nor triple layered particles were chemotactic, which was not surprising
since viral particles rarely induce cell migration by themselves [30]. Viruses exposed to
neutrophils were as capable o f repheating as the control viruses in a plaque assay, even
when the neutrophils were activated with PM A Since the dose o f PM A used (10 ng/ml)
is capable o f producing both an oxidative burst and specific granule release, it appears
that superoxide, as well as other defense mechanisms mobilized by PM A such as
lactoferrin, are ineffective at inhibiting rotavirus replication [24]. Also, rotavirus itself
does not induce an oxidative burst in neutrophils. To determine whether serum
components were required to elicit neutrophil functions, virus was first opsonized with
pooled calf serum. The addition o f serum, which should contain neutrophil-activating
complement components such as C3b [37], did not increase rotavirus’s ability to induce
an oxidative burst. However, the interaction may require viral-specific antibodies, which
91
were probably not present since the serum could not out-compete anti-rotavirus mouse
serum in a western blot to viral particles (data not shown). It is possible that opsonization
with serum from a calf that recently recovered from rotavirus infection might have
caused a different response; however, this was not evaluated.
Many viruses down-regulate neutrophil functions after they are incubated
together. Incubating unopsonized IAV with neutrophils alters their functions in vitro,
including chemotaxis, oxidative burst, and bacterial phagocytosis and killing [40]. This
aspect o f IAV has serious clinical consequences. In the United States, IAV is responsible
for about 10,000 deaths per year, and most o f these deaths are due to bacterial
superinfections, especially Staphylococcus aureus and Streptococcus pneumoniae [179].
Other viruses also downregulate neutrophil functions. Chemotaxis is inhibited by
measles, IAV, CMV, RSV, hepatitis B, arid HSV. IAV, NDV, and mumps inhibited
phagocytosis, and NDV, CMV, hepatitis B and IAV decrease intracellular bacterial
killing [30].
Rotaviral particles were tested for their ability to similarly inhibit neutrophil
functions to known activators. In these studies, rotavirus was unable to alter the
neutrophil superoxide burst to PMA, chemotaxis to IL-8 or LTB4, or phagocytosis o f S.
aureus particles. However, the role o f virus-specific antibodies, serum components, or
priming agents was not investigated. It is also possible that rotavirus induces other
neutrophil functions that were not tested, such as actin polymerization or calcium flux.
However, usually a change in actin or calcium will alter chemotaxis, phagocytosis and
oxidative burst.
92
In vivo, viruses may modulate neutrophils by increasing or decreasing the levels
o f certain cytokines, especially neutrophil chemokines. Neutrophils are found at the site
o f several viral infections, including varicella zoster virus and respiratory syncytial virus
[30]. Several viruses are known to induce the production o f neutrophil chemotactic
agents in vitro, including herpes simplex, mumps, and Newcastle disease virus [30].
Endothelial cells infected with CMV in vitro will secrete IL -8 at nearly I O"9 M, which is
sufficient to induce human and bovine neutrophil migration [31]. Chemokines are not the
only type o f cytokine induced by viral infections. Rhinovirus causes epithelial cells to
release TNF-a, a potent priming agent o f neutrophils [53], and it is thought that HIV
decreases the release o f G-CSF, a growth factor and activator o f neutrophils [51].
The net effects o f altering cytokine profiles vary depending on the virus. The
amount o f IL -8 induced by Japanese encephalitis virus has been correlated with greater
severity o f illness [55]. There is a transient increase in neutrophils seen in rhino virusinfected patients in the early phase o f infection, which matches IL-8 production. Since
IL-8 is chemotactic and activating for neutrophils, it is not surprising that bacterial
superinfections are uncommon with rhinovirus [53]. Instead, the neutrophils themselves
help create the pathology. RSV is the most frequent cause o f bronchiolitis and is linked
to asthma. Neutrophils are found in abundance in the airway upon RSV infection, due to
chemokine release (e.g. IL-8) from infected epithelial cells. Neutrophils adhere to the
cells and kill them, exacerbating airway damage. In these illnesses, inhibiting cytokine
production and therefore neutrophil damage is a possible treatment. For example,
93
treating RSV-infected cells with the corticosteroid fluticasone propionate inhibits their
IL-8 production [56].
Viruses do not always induce the secretion o f neutrophil-enhancing cytokines.
Patients with HIV have a myriad o f impaired neutrophil functions including superoxide
production [46], antibody-dependent cellular cytotoxicity (ADCC) [47], decreased IL-8
receptors [50], and decreased production o f LTfL [49]. Since HIV does not integrate its
genome into neutrophils, it is thought that it alters neutrophils at a distance by changing
the cytokine profile [49]. It is hypothesized that HIV, either alone or in conjunction with
opportunistic pathogens, induces a decrease in G-CSF and an increase in IL-4 and IL-10
[51]. These results suggest that boosting neutrophil function is a possible treatment for
HIV. AIDS patients who were treated with the neutrophil growth factor GM-CSF
demonstrated an increase in the production o f the potent neutrophil chemokine
leukotriene B4 [49]. Also, IL-15, a newly discovered neutrophil activator, increases
chemotaxis and fungicidal activity o f neutrophils from HIV patients in vitro, even those
who no longer respond to antiviral therapy [51].
To determine whether rotavirus induces any neutrophil-altering cytokines in host
cells, supernatants from virus-infected M A l 04 cells were tested for their ability to induce
chemotaxis and other functions. Virus-infected supernatants did not induce oxidative
burst in neutrophils. In addition, incubating neutrophils with virus-infected cell
supernatants did not alter their ability to produce an oxidative burst to PM A or to migrate
to IL-8, compared to neutrophils that were incubated with mock-infected supernatant.
This implies that M A l 04 cells infected with rotavirus do not release any neutrophil
Z
94
priming or neutrophil inhibiting agents. Occasionally, supernatant was chemotactic for
neutrophils; however, this result was neither reproducible nor consistent. This was
surprising since other labs have reported the production o f IL-8 by virally-infected cells
at similar MOIs and times tested [158,160], However, their IL-8 concentration measured
by ELISA was 2.5 x IO"10 M, which is a full two logs lower than used in these studies.
They were also using HT-29 cells, a human colon cell line, whereas M A l 04 cells are a
monkey kidney cell line. It is noteworthy that the m ock infected cell supernatants often
induced over a 20% cell migration, whereas media alone only induced 5% or less.
Perhaps M A l 04 cells constitutively produce these chemotactic factors, and a slight virusinduced increase is not detectable by standard neutrophil assays.
In conclusion, rotavirus does not appear to induce standard neutrophil hostdefense responses, such as respiratory burst, chemotaxis, or phagocytosis. Since all o f
these studies were done in vitro, the situation may be different in the infected calf or
human intestine. However, these results do correlate well with the observations that
rotavirus generally causes an acute infection and is not associated with inflammation
[117,141]. Also, neutrophils are probably not playing a role in viral clearance, since
neutrophils do not internalize viral particles (Chapter 2), viruses with or without
opsonization with serum do not elicit superoxide, and activated neutrophils do not inhibit
viral replication in a plaque assay. Yet more tests need to be done to conclusively answer
this question. It is possible that either rotavirus-specific antibody or a priming agent is
required for the virus to elicit or inhibit neutrophil functions. Also, perhaps neutrophils
mediate effects on rotavirus-infected cells. In a calf model o f RSV infection, neutrophils
95
adhered to and killed epithelial cells infected with RSV [56], and bovine neutrophils are
also capable o f inhibiting the replication o f infectious bovine rhinotracheitis virus (!BR)
in the absence o f antibody or complement [30]. It would be interesting to see if
neutrophils could similarly kill M A 104 cells infected with rotavirus.
Understanding how viruses and neutrophils interact can provide treatments for
viral infections. For example, to treat neutrophil-activating illnesses such as rhinovirus or
RSV, agents could be designed to inhibit neutrophil influx or action. In other illnesses
where neutrophil impairment augments disease, such as IAV or HIV, drugs could be
designed to increase neutrophil activity. Since rotavirus is the most common diarrhealcausing pathogen in calves [114], and vaccines have limited efficacy [60], other
treatments for this infection are desirable. To our knowledge, these are the first studies
specifically investigating rotavirus and neutrophil interactions, and one o f few studies
investigating bovine-specific virus effects on bovine cells. Rotavirus itself was unable to
induce either neutrophil responses, or secretion o f neutrophil-acting agents in M Al 04
cells. In addition, rotavirus did not alter these responses in bovine neutrophils stimulated
with known inflammatory agents. Overall, my studies suggest that neutrophils may not
be involved with either rotaviral clearance or pathology.
96
CONCLUSION
Neutrophils are an essential component o f the mammalian host defense system
against bacterial and fungal pathogens, but they are also implicated in most inflammatory
diseases. These leukocytes have recently been discovered to play a role in viral clearance
and pathology as well. Viruses may interact with neutrophils on several levels. First,
viruses elicit direct responses from neutrophils, such as phagocytosis or oxidative burst.
Second, viruses directly inhibit neutrophil responses to other activators. Third, viruses
modulate neutrophils indirectly through the induction o f cytokines. Based on these
observations, it was hypothesized that rotavirus could mediate neutrophil effects in one or
more o f these ways. Since the interaction between rotavirus and neutrophils has never
been examined, these studies were designed to characterize bovine neutrophil responses
to the B641 calf strain o f rotavirus.
In order for rotavirus to directly activate or inactivate neutrophils, binding must
take place. Rotavirus bound to purified bovine neutrophils via three different methods:
flow cytometry, VOPBA, and radiolabeled virus binding studies. Flow cytometry and
radioactivity assays showed that neutrophils bound more viral particles than M A l 04 and
K562 cells. Since putative rotavirus receptors such as integrins are contained in bovine
secretory and specific granules, binding o f rotavirus to degranulated neutrophils was also
evaluated. Stimulation o f neutrophils with 20 nM PMA did not alter their ability to bind
rotavirus. However, since this dose has varying effects on cattle neutrophil
degranulation, a dose-response curve would have to be performed to conclusively show
97
whether PMA regulates receptor expression. VOPBA analysis indicated that rotavirus
binds to either low molecular weight proteins around 30 kDa or to a protein at 95 kDa on
neutrophil membranes. The |32 integrin subunit is 95 kDa, providing circumstantial
evidence that neutrophils are utilizing this protein as a receptor. Unfortunately, this band
was not consistently demonstrated. Radioactivity assays demonstrated that binding was
specific, since cold virus competed out the radioactivity at non-saturating doses.
However, the virus did not internalize after I hour at 37°C. The Scatchard plot o f the
binding data was fairly linear, which differed from the permissive cell line M A l 04, but
was similar to the non-permissive K562 cells. This pattern may indicate a mode o f
binding in which the receptor number is equal to the virus number. Along with the
internalization studies, this data suggests that like most cell lines, neutrophils possess an
attachment site but not the co-receptors or mechanisms for internalization.
Functional effects o f rotavirus on neutrophils were also investigated. Neither
double nor triple layered particles were chemotactic for bovine neutrophils. Viruses
exposed to neutrophils did not have a decrease in replication in a plaque assay, even
when the neutrophils were activated with PMA. In addition, viruses did not induce an
oxidative burst, either alone, or when opsonized by bovine serum. These studies suggest
that neutrophils do not play a major role in clearance o f rotaviral infections, at least in
cattle. Rotaviral particles were then tested for their ability to inhibit neutrophil functions
to known activators. In these studies, rotavirus was unable to alter the neutrophil
superoxide burst to PMA, chemotaxis to IL-8 or LTB4, or phagocytosis o f S. aureus
98
particles. However, the role o f virus-specific antibodies, serum components, or priming
agents in virus-neutrophil interactions was not investigated.
To determine whether rotavirus induces the production o f neutrophil-altering
cytokines, supernatants from virus-infected M A l 04 cells were tested for their ability to
induce chemotaxis and other functions. Occasionally, supernatants were chemotactic for
neutrophils; however, this result was not consistent. Additionally, supernatants did not
induce an oxidative burst. These supernatants were also incapable o f modulating
neutrophil oxidative burst and chemotaxis to known inflammatory agents, indicating that
infected M A l 04 cells do not release agents that prime or inhibit neutrophil functions.
In conclusion, although rotavirus does bind to neutrophils in a specific manner, it
does not appear to internalize as it does with rotavirus-susceptible cells. This binding
does not induce standard neutrophil host defense responses, such as respiratory burst or
chemotaxis, and it does not modulate these responses to known activators. In addition,
rotavirus does not induce the release o f neutrophil-modulating cytokines or compounds in
M A l 04 cells. To our knowledge, these are the first studies specifically investigating
rotavirus and neutrophil interactions. Since these experiments were performed in vitro, it
is also possible that this interaction could be different in the infected calf intestine.
Nevertheless, the data obtained in these studies provides an initial framework for
developing an understanding o f the role o f neutrophils in rotavirus infection.
99
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I ll
APPENDIX A
PRODUCTION OF MONOCLONAL ANTIBODIES
112
Introduction
Monoclonal antibodies have been an invaluable tool in biological research. Their
production was developed by Georges Kohler and Cesar Milstein in 1975, who shared the
Nobel Prize in Medicine in 1984 for this discovery. The principle behind making
monoclonal antibodies relies on two observations. First, B cells secrete the same
antibody throughout their lifespan that recognizes a specific epitope on an antigen, and
second, tum or cells have the potential to grow and divide indefinitely. To obtain
monoclonal antibodies, mice are immunized with antigen, and after two months, their
spleens will contain antigen-specific B cells. These cells are harvested and then fused to
cancerous myeloma cells, which generates immortal antibody-secreting cells. The fusion
is carried out either with a syncytium-inducing virus or a chemical such as po lyetheylene
glycol (PEG).
Cells that fuse successfully are selected based on their ability to synthesize purine
nucleotides for DNA [37]. A cell can make nucleotides in two ways: the de novo
pathway from simpler carbon compounds, and the salvage pathway^ from other purine
bases. Myeloma cells are deficient in enzymes for the salvage pathway, so if the de novo
pathway is blocked with aminopterin, the cells are unable to grow. The B cells from the
spleen contain the genes for the salvage pathway, but are not normally capable o f
extended growth in vitro. However, if these two cell types are fused together, they are
now not only capable o f growing via the salvage pathway if hypoxanthine and thymidine
are provided but are also able to grow indefinitely. The supplement used for selection o f
113
fused cells is called “HAT” for hypoxanthine, aminopterin, and thymidine. These cells
are plated, and the ones secreting the antibodies o f interest are grown in mass quantities.
The purpose o f this particular monoclonal antibody endeavor was to obtain
specific antibodies that recognize various proteins o f the bovine B641 strain o f rotavirus.
Antibodies that recognized the outer capsid, i.e. VP4 or VP7, were especially desired, so
they could be used in binding studies.
Methods
Immunization o f Mice
Purified triple layered particles were obtained and diluted 1:1 in Titermax
adjuvant (Sigma) to a final concentration o f 0.9 pg/(il o f protein, i.e. 3x108 particles/ml,
and adjuvant and virus were emulsified for 10 minutes. Two female BALB/c mice at 2
months o f age were inoculated with 100 jxl o f the antigen/adjuvant mixture via the intraperitoneal route, then boosted in two weeks with an additional 100 pi. Serum was tested
at the time o f boosting to ensure the mice were producing an immune response to
rotavirus antigen. After either one week or two months, mice were euthanized, and their
spleens were collected. Spleens were homogenized in a tissue grinder to obtain white
cells which were washed in RPMI/HEPES and counted. Cells were then frozen in fetal
bovine serum (FBS) + 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen
until day o f fusion. AU animal use was approved by the MSU Institutional Animal Care
and Use Committee.
114
Fusion o f Spleen Cells with Hvbridoma Cells
A week prior to the fusion, SP2/0 m ouse.myeloma cell cultures were started and
maintained on complete RPMI (10 mM Hepes, 50 U/ml penicillin, 50 U/ml streptomycin,
I mM sodium pyruvate, 0.1 mM non-essential amino acids, and 2 mM L-glutamine)
supplemented with 20% FB S. On the day o f the fusion, spleen cells were thawed rapidly,
counted and resuspended in RPMI + 10 mM Hepes. SP2/0 cells were also washed and
counted. Cells were mixed at a ratio o f spleenrmyeloma cell at 2:1 in 40 ml RPMI/Hepes
and centrifuged at 1500 rpm for 10 minutes at room temperature. The pellet was then
carefully resuspended with 2 ml o f 50% PEG, and clumps were gently broken up for
three minutes. Cells were then centrifuged at 500 x g for 6 minutes. The PEG layer was
removed, and HAT media was added to the pellet very slowly. HAT media consisted o f
complete RPMI supplemented with 20% FBS, 2% HAT solution (10 pM hypoxanthine,
0.04 pM aminopterin, 1.6 pM thymidine), and 10% Origen (IGEN International). The
HAT reagent selects for cells that fused successfully, now termed “hybridoma cells,” and
Origen contains necessary growth factors. Hybridomas were plated in a 48-well plate
and incubated at 37°C with 10% CO 2. Four to five days later, cells were fed and
observed for growth o f large clones.
Screening and Subcloning
Either double (DTP) or triple layered (TLP) particles were separated on a 10%
SDS-PAGE curtain gel and transferred to nitrocellulose. Supernatants from wells with
large clones were collected and screened in a miniblot apparatus that creates 28 separate
lanes from the nitrocellulose blot. Clones positive for proteins o f interest were then
115
subjected to two rounds o f subcloning to guarantee that the final cloned population was
derived from only one cell, and thus the resulting antibody represents a true monoclonal.
HAT supplement is no longer needed at this point, so subcloning was carried out in
complete KPMI supplemented with 20% FBS. To subclone, cells from positive clones
were lifted off wells, counted, diluted, and plated in a 96-well plate such that each well
theoretically received only one cell. Clones were grown until they covered at least onefourth o f the well, subcloned again, and re-screened using a Western mini-blot.
Propagation and Purification o f Antibody
After positive clones had been subjected to two rounds o f subcloning, they were
expanded and grown in T -162 flasks to obtain enough antibodies to purify. At this time,
cells were weaned from RPMI into H BlO l medium (Irvine Scientific), which is
specifically designed for use in hybridoma cells. Then cells were slowly weaned from
FBS until they were growing in serum-free media. This procedure ensures that the only
protein in the supernatant is the secreted antibody. Both weaning procedures can be
problematic, causing cells to sometimes die off unexpectedly. Therefore, it was
important to maintain a flask o f cells in the original growth media. In addition, some o f
the cells were also frozen in FBS + 10% DMSO and stored in liquid nitrogen, so more
antibody could be generated in the future.
After growing up several flasks, supernatant was harvested and centrifuged to
remove cells. Antibodies were purified by running supernatant through a protein G
Sepharose column, which binds to Fe receptors o f antibodies, and then eluted with 0.1M
glycine, pH 2.2. Eluted fractions were collected and reconstituted to neutral pH with 2M
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Tris base. Each fraction was then tested in a Biorad assay to determine the protein
concentration, and separated on a gel to check for both the light and heavy antibody
chains. Fractions containing an adequate amount o f protein were then concentrated,
aliquotted, and frozen at -80°C.
Results
One o f the immunized mice developed an ascites tumor; therefore it was
euthanized only one week after boosting. The other mouse was healthy, and its spleen
was harvested two months after the boost. N ot surprisingly, the spleen from the ascites
mouse gave more rotavirus-positive B cells from the initial screen, ahd supplied both
antibodies developed here. From two separate fusion screens, bands corresponding to
VP4 and VP6 molecular weights appeared on the blots. Therefore these two clones,
named m abl and mab2, were subcloned twice, expanded, purified, and characterized.
Since VP6 makes up the second layer o f the virion, DLPs could be used for
screening m ab l. Normally, VP6 forms trimers, which are dissociated by boiling.
Therefore, boiled and non-boiled DLP were prepared, separated on an SDS-PAGE gel,
transferred to nitrocellulose, and probed with the VP6 antibody. The molecular weights
o f the bands were determined with imaging software. A band at 41 kD a appeared on the
boiled sample lane, but shifted to 120 kDa on the non-boiled sample lane, indicating that
the antibody specifically recognizes VP6 (Figure A l ) . After purification and
concentration, approximately 500 pi o f VP6 antibody at 3.9 mg/ml was obtained. This
monoclonal recognized VP6 in a western blot even at a dilution o f 1: 15,000 (Figure A.2).
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B NB
220
MM
97
66
Single
45
30
20
14
Figure A .l: Testing Specificity o f M abI. DLP samples were either boiled or not boiled,
separated on a gel, transferred to nitrocellulose, and blotted with m abl. The lane labeled
“B” shows a band at 41 kDa, the weight o f a single VP6, and the non-boiled sample lane
“NB” shows a band at 120 kDa, corresponding to a VP6 trimer. Prestained standards
were used for all blots, and the relative standard molecular masses are indicated.
1:500
1:1000
1:1500
1:5000
1:10000 1:15000
Control
Figure A.2: Western Blot o f M abI . Purified DLPs were run on a gel, and probed with the
VP6 antibody at the indicated dilutions. A band appears at 41 kDa, which is the
molecular weight o f VP6. Prestained standards were used for all blots which appear on
the right side o f each blot, and their relative standard molecular masses are indicated.
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At the lower dilutions, other bands appeared, but disappeared with increasing dilutions.
These bands were most likely background staining, which can occur if antibodies are too
concentrated.
To determine whether mab2 recognized VP4, equal concentrations o f DLP and
TLP were separated on an SDS-PAGE gel, transferred to nitrocellulose, and probed with
supernatant. I f the antibody is specific for VP4, than it should only stain the TLP lane at
86 kDa. Two bands appeared on the TLP band, one at 92 and one at 86 (Figure A.3).
However, even though the 86 kD a band disappeared in the DLP lane as expected, the 92
kDa band remained. To further determine what antigens mab2 recognized, viruses were
cleaved with trypsin and tested in a screen. I f mab2 is specific for VP4, then it should
recognize one o f the cleavage products, either VP5* at 60 kDa or VP8* at 28 kDa, in the
trypsin-cleaved virus preparation. A band appeared at around 65 kDa with the trypsincleaved product which could be VP5*, but a contaminating band remained at 92 kDa.
This protein is most likely YP2, which is 94 kDa. Bovine VP2 and VP4 do not have any
sequence similarities according to a BLAST alignment, so it is unclear why the antibody .
seems to recognize both proteins. One likely explanation is that the band at 65 kDa
represents a VP2 degradation product that is recognized by mab2. After purification and
concentration, approximately 500 pi o f antibody at 2 mg/ml was obtained. The antibody
still recognized protein in a western blot even at a dilution o f 1:15,000 (Figure A.4). In
the higher dilutions, only a band corresponding to VP2 was stained. In conclusion, two
rotavirus-specific antibodies were generated: one to the inner layer protein VP6 and
possibly one to the core protein VP2.
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TL DL
Figure A.3: Testing Specificity o f Mab2. Purified TL and DL particles were separated on
a gel, transferred to nitrocellulose, and probed with mab2. A band at 86 kDa appears
only in the TL lane at the size o f VP4, but a contaminating band is observed in both lanes
at 92 kDa. Prestained standards were used for all blots, and the relative standard
molecular masses are indicated.
1:500
1:1000
1:1500 1:5000 1:10000 1:15000
Control
Figure A.4: Western Blot o f Mab2. Purified TLPs were separated on a gel and probed
with mab2 at the indicated dilutions. A band appears at 92 kDa, which is the
approximate weight o f VP2, not VP4. Prestained standards were used for all blots which
appear on the right side o f each blot, and their relative standard molecular masses are
indicated.
MONTANA STATE UNIVERSITY
BOZEMAN