University of Veterinary Medicine Hannover

Institute of Virology

Infection of differentiated epithelial cells by

viruses of the bovine respiratory disease complex

THESIS

Submitted in partial fulfillment of the requirements for the degree

-Doctor rerum naturalium-

(Dr. rer. nat.)

awarded by the University of Veterinary Medicine Hannover

by

Jana Kirchhoff

(Herzberg am Harz)

Hannover, Germany 2014

Supervisor: Prof. Dr. Georg Herrler

Advisory Committee: Prof. Dr. Georg Herrler

Prof. Dr. Christiane Pfarrer

Prof. Dr. Evgeni Ponimaskin

1st Evaluation: Prof. Dr. Georg Herrler

Institute of Virology

University of Veterinary Medicine Hannover, Foundation

Prof. Dr. Christiane Pfarrer

Department of Anatomy

University of Veterinary Medicine Hannover, Foundation

Prof. Dr. Evgeni Ponimaskin

Institute for Cellular Neurophysiology

Hannover Medical School

2nd Evaluation: Prof. Dr. Michael Veit

Institute of Virology

Free University Berlin

Date of oral exam: 22st April 2014

This work was financed by a grant of Deutsche Forschungsgemeinschaft (SFB 587,

TPA1).

To my family

Contents

List of Abbreviations ................................................................................................. I

List of Figures .......................................................................................................... III

Abstract ..................................................................................................................... V

Zusammenfassung ................................................................................................. VII

1 Introduction ........................................................................................................ 1

1.1 Bovine respiratory disease complex .............................................................. 1

1.1.1 Infection of cattle ..................................................................................... 1

1.1.2 Infection of other ruminant species ......................................................... 3

1.2 Respiratory syncytial virus (RSV) .................................................................. 4

1.2.1 Epidemiology .......................................................................................... 4

1.2.2 Clinical signs and Pathology ................................................................... 4

1.2.3 Vaccination ............................................................................................. 5

1.2.4 Taxonomy ............................................................................................... 6

1.2.5 Virus structure ......................................................................................... 6

1.2.6 Viral life cycle .......................................................................................... 8

1.3 Bovine parainfluenza virus type 3 (BPIV3) .................................................... 9

1.4 Bovine herpesvirus type 1 (BHV-1) ............................................................. 11

1.4.1 Epidemiology ........................................................................................ 11

1.4.2 Clinical signs and Pathology ................................................................. 11

1.4.3 Vaccination ........................................................................................... 12

1.4.4 Taxonomy ............................................................................................. 13

1.4.5 Virus structure ....................................................................................... 13

1.4.6 Viral life cycle ........................................................................................ 14

1.5 The respiratory epithelial barrier .................................................................. 16

1.5.1 Basic functions of the respiratory epithelium ......................................... 16

1.5.2 Polarized virus entry ............................................................................. 17

1.5.3 Cultures for differentiated respiratory epithelial cells ............................. 18

1.6 Aim of the study ........................................................................................... 20

2 Publication I ...................................................................................................... 23

Three viruses of the bovine respiratory disease complex apply different strategies

to initiate infection

3 Publication II ..................................................................................................... 25

Infection of differentiated airway epithelial cells from caprine lungs by viruses of the

bovine respiratory disease complex

4 Discussion ........................................................................................................ 27

4.1 In vitro cultures for differentiated respiratory epithelial cells ........................ 27

4.2 Infection strategies of bovine parainfluenza virus type 3 ............................. 29

4.2.1 Infection of well-differentiated bovine airway epithelial cells ................. 29

4.2.2 Infection of well-differentiated caprine airway epithelial cells ................ 31

4.3 Infection strategies of bovine herpesvirus type 1 ......................................... 32

4.3.1 Infection of well-differentiated bovine airway epithelial cells ................. 32

4.3.2 Infection of well-differentiated caprine airway epithelial cells ................ 34

4.4 Infection strategies of bovine respiratory syncytial virus .............................. 35

4.4.1 Infection of well-differentiated bovine airway epithelial cells ................. 35

4.4.2 Infection of well-differentiated caprine airway epithelial cells ................ 38

4.5 Summary and Outlook ................................................................................. 40

4.5.1 Strategies of viruses of the BRDC to infect the bovine host .................. 40

4.5.2 Goats as a virus reservoir ..................................................................... 41

5 References ........................................................................................................ 43

6 Appendix ........................................................................................................... 62

7 Acknowledgements ......................................................................................... 63

I List of Abbreviations

List of Abbreviations

ALI Air-liquid interface

BAEC Bovine airway epithelial cells

BHV-1 Bovine herpesvirus type 1

BPIV3 Bovine parainfluenza virus type 3

BRD Bovine respiratory disease

BRDC Bovine respiratory disease complex

BRSV Bovine respiratory syncytial virus

BSA Bovine serum albumin

CapHV-1 Caprine herpesvirus type 1

CO2 Carbon dioxide

CPE Cytopathic effect

CRSV Caprine respiratory syncytial virus

Cy3 Indocarbocyanine

DAPI 4’,6’-Diamidino-2-phenylindole

DCs Dendritic cells

DMEM Dulbecco’s modified Eagle medium

dpi days post infection

et al. et alii (and others)

FFU Focus-forming units

Fig. Figure

FITC Fluorescine isothiocyanate

h hour

hpi hours post infection

HRSV Human respiratory syncytial virus

IFN Interferon

L liter

M Molarity, - molar

MDBK Madin-Darby bovine kidney

MDCK Madin-Darby canine kidney

II List of Abbreviations

min minute

MOI Multiplicity of infection

mRNA messenger RNA

NA Neuraminidase

PBS Phosphate buffered saline

PCLS Precision-cut lung slices

PFA Paraformaldehyde

pH potentia Hydrogenii

TEER Transepithelial electrical resistance

TTF-1 Thyroid transcription factor-1

ZO-1 Zonulae occludentes-1 protein

°C degree Celsius

α anti (antibodies) or alpha (sialic acids)

µ micro (gram or liter for example)

Ω ohm

III List of Figures

List of Figures

Figure 1-1 Factors contributing to the bovine respiratory disease complex ................ 2

Figure 1-2 Structure of respiratory syncytial virus ....................................................... 7

Figure 1-3 Schematic drawing of an Alphaherpesvirus particle ................................ 14

Figure 1-4 composition of the respiratory epithelium ................................................ 16

Figure 1-5 ALI culture system. .................................................................................. 19

Figure 1-6 Light microscopic (A) and confocal (B) picture of a typical PCLS ............ 20

IV -

-

V Abstract

Abstract

Infection of differentiated epithelial cells by viruses of the bovine

respiratory disease complex

Jana Kirchhoff

Respiratory tract infections of cattle are often associated with the bovine respiratory

disease complex (BRDC). BRDC is a multifactorial disease and several factors like

stress or reduced immunity contribute to the severity of disease. Bovine respiratory

syncytial virus (BRSV), bovine parainfluenza virus type 3 (BPIV3) and bovine

herpesvirus type 1 (BHV-1) are important viral pathogens associated with this

disease complex.

The aim of the thesis was to analyze the susceptibility of bovine airway epithelial

cells (BAEC) to the three viruses of the BRDC and the different viral strategies

concerning entry and release. For this purpose, two culture systems for well-

differentiated BAEC were used, which were previously established in our institute:

the air-liquid interface (ALI) system, where filter-grown respiratory BAEC undergo

mucociliary differentiation and precision-cut lung slices (PCLS), where cells are

maintained in their original structural conformation and stay vital for more than one

week. When the viruses were applied to these culture systems, different infection

strategies were observed: BPIV3 efficiently infected ciliated epithelial cells and

replicated to high titers. Entry and release were a) sialic acid-dependent and b)

localized to the apical side of the ALI cultures. In contrast, BAEC were largely

resistant to infection by BHV-1, which could be overcome by injury of the cell

monolayer or destruction of tight junctions. Moreover, BHV-1 infection was primarily

directed to basal cells. BRSV infected the respiratory epithelial cells inefficiently; in

contrast to BHV-1, this infection was not enhanced when the integrity of the epithelial

barrier was destroyed. Subepithelial cells and pneumocytes type I and II appeared to

be the target of infection by this virus.

VI Abstract

Furthermore, non-bovine ruminants such as goats can be considered as a target for

viruses of BRDC and therefore may serve as a virus reservoir as well. To address

this question, a culture system for differentiated caprine epithelial cells, caprine

PCLS, was established for the first time. When the three viruses of the BRDC were

applied to the cultures, it was observed that the infection pattern was similar to that

reported for bovine cultures. While infection of caprine cells by BPIV3 and BRSV was

as efficient as that observed for bovine cells, infection of BHV-1 required a tenfold

higher dose of infectious virus in comparison to the bovine culture system.

Nevertheless, BHV-1 displayed the same tropism for basal cell. These results

indicate that non-bovine ruminants are also important hosts for viruses of the BRDC

and should be considered in the development of control strategies.

VII Zusammenfassung

Zusammenfassung

Infektion differenzierter Epithelzellen durch Viren des Rindergrippe-

Komplexes

Jana Kirchhoff

Die Enzootische Bronchopneumonie (oder Rindergrippe-Komplex) ist eine

Infektionskrankheit des Respirationstraktes bei Rindern, die multifaktoriellen

Ursprungs ist. Neben zahlreichen viralen und bakteriellen Erregern tragen auch der

Immunstatus des Tieres sowie Umweltbedingungen zum Schweregrad der

Erkrankung bei. Wichtige Viren sind das bovine respiratorische Synzytialvirus

(BRSV), das bovine Parainfluenzavirus 3 (BPIV3) sowie das bovine Herpesvirus 1

(BHV-1).

Das Ziel dieser These war es, die Empfänglichkeit von bovinen primären

Lungenepithelzellen gegenüber den obig genannten Viren zu untersuchen sowie

deren Strategien zum Ein- und Austritt aus dem Epithelgewebe zu analysieren. Um

dieses Ziel zu erreichen, wurden zwei Kultursysteme für primäre respiratorische

Epithelzellen genutzt, die zuvor in unserer Arbeitsgruppe etabliert worden waren:

Zum einen handelt es sich hierbei um das Air-liquid interface (ALI)-System, in dem

Zellen aus der Rinderlunge isoliert und anschließend auf Filterkultureinsätzen zur

Ausdifferenzierung gebracht werden. Als Vergleichskultur wurden

Lungenpräzisionsschnitte (PCLS; precision-cut lung slices) verwendet. Diese

Schnitte haben den Vorteil, dass die respiratorischen Epithelzellen im ursprünglichen

Gewebeverband verbleiben und zudem für mindestens eine Woche vital sind.

Infektionsversuche zeigten, dass die drei Viren unterschiedliche Infektionsstrategien

verfolgen: BPIV3 infizierte die zilientragenden Epithelzellen sehr effizient und

replizierte zu hohen Titern. Des Weiteren ist die Infektion lokal ausgeprägt und war

abhängig von der Gegenwart von Sialinsäuren auf der Zelloberfläche. Im Gegensatz

dazu waren die Epithelzellen resistent gegenüber einer Infektion mit BHV-1. Diese

Resistenz konnte jedoch überwunden werden, wenn den Viren Zugang zur basalen

VIII Zusammenfassung

Seite des Epithels verschafft wurde, z.B. durch chemische oder manuelle Eingriffe.

Zudem konnte gezeigt werden, dass nicht die differenzierten Epithelzellen selbst

infiziert wurden, sondern die Basalzellen, die als Vorläuferzellen der differenzierten

Epithelzellen fungieren. Wie schon in vorherigen Studien beschrieben wurde, hatte

das Flimmerepithel nur eine geringe Empfänglichkeit gegenüber einer BRSV-

Infektion; diese Unempfänglichkeit konnte nicht durch die oben bei BHV-1

beschriebenen Eingriffe am Epithel überwunden werden. Durch Infektion der

Lungenpräzisionsschnitte wurde deutlich, dass subepitheliale Zellen sehr effizient

durch BRSV infiziert wurden; bei diesen Zellen könnte es sich um dendritische Zellen

handeln. Zudem stellte sich heraus, dass Typ-I- und Typ-II-Pneumozyten sehr

empfänglich für eine BRSV-Infektion sind.

Es wird angenommen, dass auch kleine Wiederkäuer wie Ziegen durch Viren des

Rindergrippe-Komplexes infiziert werden und somit als Virusquelle dienen können.

Um diese Thematik zu untersuchen, wurde ein Kultursystem für enddifferenzierte

Epithelzellen aus der Ziegenlunge etabliert: caprine Lungenpräzisionsschnitte.

Infektionsversuche mit den obig genannten Viren zeigten, dass die

Infektionsstrategien ähnlich waren wie im bovinen System sowie gleiche Zielzellen

infiziert wurden. Zudem wurde deutlich, dass die Infektion mit BPIV3 und BRSV

ähnlich effizient war wie in den bovinen Kultursystemen; die Infektion mit BHV-1

benötigte jedoch einen zehnfach höheren Titer. Auch hier waren die Basalzellen Ziel

der Infektion. Diese Ergebnisse zeigen auf, dass auch nicht-bovine Wiederkäuer als

Reservoir für Viren des Rindergrippe-Komplexes dienen können und somit bei

Interventionsmaßnahmen berücksichtigt werden sollten.

Introduction 1

1 Introduction

1.1 Bovine respiratory disease complex

1.1.1 Infection of cattle

Virus infections of the respiratory tract are the most frequent cause of viral diseases

worldwide. They range from mild infections of the upper respiratory tract such as

common colds to severe lower respiratory tract infections like life-threatening

pneumonia (Garibaldi et al., 1985). Bovine respiratory disease complex (BRDC) is a

common term for respiratory disease in cattle. Other names are “shipping fever” and

“enzootic bronchopneumonia”. BRDC is considered to be the major cause of

morbidity and mortality within feedlot and dairy cattle (Yates, 1982; Taylor et al.,

2010; Iglseder et al., 2011).

Bovine respiratory disease (BRD) has a multifactorial origin; therefore several factors

contribute to the severity of disease (Figure 1-1). Among viruses, bovine respiratory

syncytial virus (BRSV), bovine parainfluenza virus type 3 (BPIV 3) and bovine

herpesvirus type 1 (BHV-1) are causative agents (Cusack et al., 2003; Ellis, 2009).

These were identified by immunohistochemistry (Fulton et al., 2000; Juarez Barranco

et al., 2003) or serological surveys (Durham et al., 1991; Fulton et al., 2000;

Hagglund et al., 2007). According to these reports, viral infection can result in

disease without bacterial co-infection, but the most common scenario is that viruses

predispose to subsequent bacterial invasion. This is facilitated due to the viral ability

to decrease the clearance system of the lung (Lopez et al., 1976; Jones, 1983;

Cusack et al., 2003). The bacterial pathogens which are associated with disease are

Pasteurella multocida, Mannheimia haemolytica, Mycoplasma bovis and Histophilus

somni (Allen et al., 1991; Gagea et al., 2006; Rice et al., 2007; Srikumaran et al.,

2007; Confer, 2009). These were identified by isolation from animals suffering from

BRD (Harris and Janzen, 1989; Welsh et al., 2004; Gagea et al., 2006; Fulton et al.,

2009) or by serological examinations (Martin et al., 1989; Booker et al., 1999).

Further factors, which predispose to disease are the special anatomy of the bovine

Introduction 2

lung and the immune status of the host which for example might be impaired in

young animals due to deficient colostral transfer (Müller, 2012). Also, environmental

stressors are considered to be an important issue. One main stressor is

transportation to different places e.g. auction markets (Pinchak et al., 2004;

Sanderson et al., 2008). Others are weaning prior to sale, crowding and introduction

of new animals into a herd. Also, poor housing conditions have an important effect on

BRD (Sanderson et al., 2008; Ohlson et al., 2010; Taylor et al., 2010). How and to

what extent these factors contribute to the disease is widely debated (Taylor et al.,

2010).

Figure 1-1 Factors contributing to the bovine respiratory disease complex (see text for references).

BRD causes high economic losses worldwide; it was estimated for the US that BRD-

associated costs for the cattle industry amount to about 1 billion dollars per year

(Griffin, 1997; McVey, 2009). Costs are not only due to death losses but also include

treatment costs, reduction in feed efficiency, reproductive performance and milk

production. Expenses easily accumulate because reinfection is common (Van der

Poel et al., 1993; Griffin, 1997; Jim, 2009; Fulton and Confer, 2012). Diagnosis of

disease is difficult due to its multifactorial nature. Characteristic symptoms are

Introduction 3

elevated body temperature, respiratory signs, low appetite and depression (Allen et

al., 1991; Barbour et al., 1997; Booker et al., 1999).

1.1.2 Infection of other ruminant species

Pneumonia is also a serious disease in other ruminant species like sheep and goats

(Ackermann and Brogden, 2000). Many aspects concerning epidemiology,

pathogenesis and clinical signs are comparable to the outcomes in cattle populations

(Dungworth, 1993). Accordingly, infection of goats and sheep is caused by similar

pathogens as observed in the bovine counterpart. Alphaherpesviruses for instance

can be isolated from a wide variety of ruminant species and many of them share

antigenic properties (Yates, 1982). Especially caprine herpesvirus 1 (CapHV-1) and

BHV-1 exhibit a close relationship (Nixon et al., 1988; Whetstone and Evermann,

1988), also bovine and caprine RSV (CRSV), which have been suggested to belong

to one subgroup (Duncan and Potgieter, 1993; Alansari et al., 1999; Eleraky et al.,

2003), share antigenic relationship. As far as bacterial pathogens are concerned, M.

haemolytica is considered to be an important agent contributing to the severity of

disease (Brogden et al., 1998). Therefore, concerns about BRD eradication schemes

came up because it seems likely that viruses of BRDC can cross the species barrier.

Since respiratory viruses are transmitted directly through aerosols or by close contact

among infected animals, interspecies transmission is common as ruminants are often

kept in close contact to each other. Accordingly, antibodies to BRSV, BHV-1 and

BPIV3 were found in goats and sheep (Fulton et al., 1982; Elazhary et al., 1984; Van

der Poel et al., 1995; Yener et al., 2005). Moreover, sheep and goats developed

clinical signs after experimental infection with BHV-1 and BRSV (Lehmkuhl and

Cutlip, 1979; Engels et al., 1992; Meehan et al., 1994; Six et al., 2001). Also BPIV3

was found to infect sheep and to induce clinical signs (Woods et al., 1965; Hore,

1966; Stevenson and Hore, 1970; Rudolph et al., 2007). For goats, data on BPIV3

infection are rare (Yener et al., 2005). Based on these studies, sheep and goats have

been designated as a reservoir for viruses of BRD (Van der Poel et al., 1995; Thiry et

al., 2006). Further research addressing this question is important to analyze whether

virus isolates cocirculate in ruminant populations and whether cross-transmission of

these viruses is common (Geraghty et al., 1998).

Introduction 4

1.2 Respiratory syncytial virus (RSV)

1.2.1 Epidemiology

Bovine respiratory syncytial virus (BRSV) is the most common cause of respiratory

disease in cattle, especially in calves (Stott and Taylor, 1985). It was first isolated in

Europe in the early 1970s (Paccaud and Jacquier, 1970). BRSV is distributed all over

the world, and virus was isolated in different countries from both, beef and dairy

cattle (Smith et al., 1975; Lehmkuhl and Gough, 1977; Van Vuuren, 1990; Gagea et

al., 2006; Hagglund et al., 2006; Bidokhti et al., 2009; Saa et al., 2012). Infections

normally occur in autumn and wintertime (Stott et al., 1980; Van der Poel et al.,

1993), with a seroprevalence of 28-95% (Lehmkuhl and Gough, 1977; Collins et al.,

1988; Van Vuuren, 1990; Van der Poel et al., 1994; Elvander, 1996; Hagglund et al.,

2006; Saa et al., 2012), but can also take place in summer (Gershwin, 2007; Sacco

et al., 2012). Morbidity is high (60-80%) and mortality can increase up to 20% (Baker

et al., 1986; Elvander, 1996; Valarcher and Taylor, 2007). Especially young animals

are under high risk of getting infected (Kimman et al., 1988; Hagglund et al., 2006)

and infection even occurs under the protection of maternal antibodies (Van der Poel

et al., 1994). Though calves are under higher risk of getting severe clinical signs

(Stott et al., 1980; Kimman et al., 1988), adult animals are also at risk mainly in naïve

herds (Van der Poel et al., 1994). Reinfection is common but severity is reduced in

these cases (Kimman et al., 1987). It is not completely understood how the virus can

survive in a cattle population (Stott et al., 1980), but there is some evidence that

BRSV can persist in its host (Van der Poel et al., 1993; De Jong et al., 1996;

Valarcher et al., 2001).

1.2.2 Clinical signs and Pathology

BRSV is transmitted by aerosols or upon direct contact (Mars et al., 1999). The

incubation time takes between 2-5 days. The course of disease can be either

asymptomatic, restricted to the upper respiratory tract or spread to the lower airways

as well (Verhoeff et al., 1984). Clinical symptoms associated with mild cases are

cough and ocular and nasal discharge. Severe cases result in depression,

hyperthermia, anorexia and polypnea (Verhoeff et al., 1984). Microscopic lesions

Introduction 5

include necrotic and apoptotic epithelial cells, also occurrence of syncytia in the

bronchiolar or alveolar epithelium and infiltration of mononuclear cells is

characteristic (Viuff et al., 1996; Viuff et al., 2002; Brodersen, 2010).

BRSV predominantly infects respiratory epithelial cells but was also found to replicate

in type II pneumocytes (Viuff et al., 1996; Viuff et al., 2002). In vitro studies

demonstrated that RSV in general causes little or no cytopathic effect in epithelial cell

cultures; therefore, it is assumed that the pathology results from the host immune

response which is triggered by a skewed Th2 response (Hussell et al., 1997;

Kristjansson et al., 2005; Bueno et al., 2008; Antonis et al., 2010). In this context,

eosinophils, neutrophils and lymphocytes are activated which produce pro-

inflammatory cytokines and chemokines leading to destruction of the epithelium

(Valarcher and Taylor, 2007).

1.2.3 Vaccination

Vaccine design for prevention of BRSV and human RSV (HRSV) infection is still a

challenging task. For HRSV, formalin-inactivated vaccines were produced in the

1960s, but they were not only unable to prevent RSV infection but rather intensified

the clinical response upon natural infection. Until now, there is no approved vaccine

available (Kim et al., 1969; Prince et al., 1986; Gershwin et al., 1998; Schreiber et al.,

2000). As far as BRSV is concerned, there are several vaccines on the market, killed

as well as modified live virus vaccines. But also for the bovine counterpart, infections

combined with severe disease symptoms were observed in vaccinated animals

(Schreiber et al., 2000; Antonis et al., 2003). Successful vaccination in calves at the

age of 2 to 6 months is hampered by the presence of maternal antibodies (Valarcher

and Taylor, 2007). Furthermore, protection is not long-lasting (Sacco et al., 2012).

Efforts to develop improved vaccines are ongoing. Modified live vaccines lacking e.g.

the NS2 or NS1 genes appeared to be promising candidates (Valarcher et al., 2003)

but never got to the stage approved vaccines. Problems concerning vaccine design

include the heterogeneity within the RSV genomes since there is no proofreading

mechanism during virus replication (Valarcher et al., 2000; Brodersen, 2010). So far,

there is only one medication available for high risk patients; Palivizumab is a

Introduction 6

humanized monoclonal antibody, which prevents infection by binding to the F protein

of HRSV or reduces the severity of disease in already infected infants (Malley et al.,

1998; Singleton et al., 2003).

1.2.4 Taxonomy

Bovine respiratory syncytial virus (BRSV) belongs to the family Paramyxoviridae

within the order Mononegavirales. Among paramyxoviruses, it is classified in the

subfamily Pneumovirinae and the genus Pneumovirus (ICTV, 2012b). Other

pneumoviruses are ovine RSV (ORSV), caprine RSV (CRSV) (Lehmkuhl et al., 1980)

and pneumonia virus of mice (PVM). CRSV is most closely related to BRSV. A

characteristic feature of all pneumoviruses is the presence of two non-structural

proteins (Smith et al., 1979; Trudel et al., 1989).

BRSV strains are assigned to either of four antigenic subgroups: A, AB, B and

untyped (Schrijver et al., 1996). Within an antigenic subtype, the genetic variability of

the G protein between strains is less than 15 % (Prozzi et al., 1997). In contrast,

HRSV has been divided into two subgroups (Anderson et al., 1985; Cristina et al.,

1990) which exhibit a greater variability (Collins et al., 1990).

BRSV and HRSV are closely related and similar in their pathogenesis and

epidemiology (Sacco et al., 2012). Therefore, research about the bovine counterpart

also provides information about the human virus and vice versa (Van der Poel et al.,

1994; Valarcher and Taylor, 2007). Nevertheless, the tropism of BRSV and HRSV in

vivo is restricted to the natural host. The F and the NS proteins are major

determinants of the host range (Bossert and Conzelmann, 2002; Schlender et al.,

2003).

1.2.5 Virus structure

BRSV particles are surrounded by an envelope which is derived from the plasma

membrane of the host cell. The genome consists of single-stranded, non-segmented

RNA in a negative orientation. It comprises ~15,000 nucleotides and sequentially

encodes for 11 proteins which are translated from a gradient of transcription which

Introduction 7

decreases from the mRNA for the NS proteins to the mRNA for the L protein

(Valarcher and Taylor, 2007).

Figure 1-2 Structure of respiratory syncytial virus; adapted from Valarcher and Taylor (2007).

RSV encodes for three different surface glycoproteins which are incorporated as

separate spikes into the envelope (see Figure 1-2): the glycoprotein (G), the fusion

protein (F) and the small hydrophobic protein (SH). The glycoprotein G is a type II

membrane protein which is heavily glycosylated. It serves as an attachment protein;

blocking of G abolishes the virus binding (Levine et al., 1987). G is unique to RSV,

and its structure differs from those of other paramyxovirus attachment proteins

(Wertz et al., 1985; Langedijk et al., 1996). Apart from its attachment function, it is

the target of neutralizing antibodies (Valarcher and Taylor, 2007). The F protein

contributes to virus attachment. In addition, it has fusion activity inducing the fusion of

the viral membrane with the host cell membrane and the formation of syncytia

between adjacent cells. It is a type I membrane protein and highly conserved among

BRSV isolates (Valarcher et al., 2000). It is synthesized as a precursor protein F0

and proteolytic cleavage at two multibasic furin cleavage sites generates a fusion-

Introduction 8

active heterodimer made up from an F1 and an F2 subunit which are connected by a

disulphide bridge (Gonzalez-Reyes et al., 2001; Zimmer et al., 2001). The process

goes along with the removal of a small intervening peptide with the length of 27

amino acids (pep 27) (Zimmer et al., 2001; Zimmer et al., 2003), which is released as

a virokinin that can induce bronchoconstriction (Zimmer et al., 2003) and eosinophil

recruitment (Valarcher and Taylor, 2007). The role of the SH protein in virus

replication is not known (Karger et al., 2001). The envelope of the virus surrounds a

helical nucleocapsid which comprises the genomic RNA. It is associated with the

nucleoprotein (N), which protects against degradation by RNases (Samal et al.,

1991; Mallipeddi et al., 1996), the phosphoprotein (P), which regulates transcription

and replication of the viral genome (Alansari and Potgieter, 1994; Valarcher and

Taylor, 2007), and a viral RNA-dependent polymerase protein (L) (Yunus et al.,

1998). In addition to the structural proteins, there are two non-structural proteins NS1

and NS2, which are important for counteracting the cellular interferon activity

(Bossert and Conzelmann, 2002; Bossert et al., 2003). Additionally, RSV has three

different matrix proteins; the non-glycosylated M protein is associated with the inner

surface of the viral envelope and is important for virion assembly (Collins et al., 2001;

Valarcher and Taylor, 2007).The M2 mRNA encodes for 2 proteins, M2-1 and M2-2

(Collins et al., 1990); M2-1 serves as an elongation factor and M2-2 has regulatory

functions in the transcription of the viral RNA (Hardy and Wertz, 1998; Bermingham

and Collins, 1999).

Virions are pleomorphic and can exhibit a spherical or filamentous shape. The

spherical type ranges in its size between 100 and 350 nm in diameter and the

filamentous type has a length up to 5 µm (Trudel et al., 1989).

1.2.6 Viral life cycle

Attachment of RSV is mediated by the surface glycoprotein G which binds to

glycosaminoglycans (Krusat and Streckert, 1997) as well as by F, which binds to

both glycosaminoglycans and a specific protein receptor (Feldman et al., 2000;

Techaarpornkul et al., 2002). The glycoprotein F plays a crucial role in cell entry by

inducing fusion of the viral envelope with the cellular membrane; recently nucleolin

Introduction 9

was identified as a putative receptor for HRSV (Tayyari et al., 2011). So far, no

receptors for BRSV have been identified. After fusion, the genome and the

nucleocapsids are released into the cytoplasm. Subsequently, the polymerase

transcribes the viral RNA which results in the generation of subgenomic RNAs. After

a sufficient amount of protein has been translated from the mRNAs, full length RNAs

are transcribed which are associated with the N, P and L proteins to form

ribonucleoproteins (RNPs). RNPs are enveloped by a budding process. The M

protein plays a crucial role in the assembly of the virions (Valarcher and Taylor,

2007). Budding occurs either directly at the host plasma membrane or at the

membrane of cytoplasmic vesicles (Arslanagic et al., 1996). In vitro studies showed

that HRSV buds at the apical surface of differentiated human respiratory epithelia

cells (Zhang et al., 2002), which was also observed for BRSV (Valarcher and Taylor,

2007).

1.3 Bovine parainfluenza virus type 3 (BPIV3)

Bovine parainfluenza virus type 3 was first isolated in 1959 in the United States

(Reisinger et al., 1959). It belongs to the genus Respirovirus within the subfamily

Paramyxovirinae and the family Paramyxoviridae (ICTV, 2012b). BPIV3 can be

divided into 2 genotypes (Chanock et al., 2001; Horwood et al., 2008). It is closely

related to human parainfluenza virus type 3 (HPIV3) (Bailly et al., 2000); additional

members of the genus are human parainfluenza virus type 1 (HPIV1) and Sendai

virus (SeV). Like RSV, PIV3 is an enveloped negative-stranded and non-segmented

RNA virus. The pleomorphic virion is 150 to 200 nm in size (Chanock et al., 2001).

The nucleocapsid is surrounded by a lipid envelope and consists of the viral genome,

the nucleoprotein (N), the phosphoprotein (P) and a large RNA polymerase (L). In

addition to the structural proteins, the genome encodes for the non-structural proteins

C, D and V, the functions of which are not well known but are assumed to inhibit IFN-

α and β activity (Durbin et al., 1999; Chanock et al., 2001). The matrix protein (M)

mediates the interaction of the nucleocapsid with the envelope and is responsible for

maturation of the virion and for budding (Chanock et al., 2001; Lamb and Kolakofsky,

Introduction 10

2001). PIV3 has two surface glycoproteins; the fusion protein F mediates cell entry

by inducing fusion of the viral envelope with the cellular membrane. This process is

pH-independent and requires the cooperation with the hemagglutinin-neuraminidase

(HN) (Heminway et al., 1994; Lamb and Kolakofsky, 2001; Takimoto et al., 2002).

Primary attachment occurs via HN which binds to (and cleaves) sialic acid-containing

cellular glycoconjugates on host cells (Scheid et al., 1972; Markwell and Paulson,

1980; Chanock et al., 2001). Replication occurs in the cytoplasm and budding at the

cytoplasmic membrane. It has been shown for HPIV3 that virus entry and release

occurs via the apical surface of polarized A549 cells (Bose et al., 2001) and of

human differentiated airway epithelia cells (Zhang et al., 2005). Although most

infections are mild or subclinical, it is considered to be the second most common

cause of severe respiratory tract infections in children and calves (Glezen and

Denny, 1973; Haanes et al., 1997). Transmission occurs via droplet infection. PIV3

predominantly infects the epithelial cells of the upper respiratory tract and only in

severe cases enters the lower airways (Chanock et al., 2001). Infection of the lower

respiratory tract may result in bronchitis, bronchiolitis or alveolitis. Microscopic lesions

include loss of ciliated cells, inclusion bodies and syncytia formation (Craighead and

Brennan, 1968; Bryson et al., 1983). As in the case of RSV, disease mainly occurs in

the winter months (Stott et al., 1980). Vaccines which are available on the market are

most often combination vaccines which also include other respiratory viruses or

bacteria (Salt et al., 2007; Ellis, 2010). There exist inactivated vaccines as well as

modified live vaccines which are still under improvement (Matsuoka et al., 1966; Salt

et al., 2007; Vangeel et al., 2009). Obstacles in vaccine design are similar but maybe

not as pronounced as those for RSV, e.g. inhibition due to maternal antibodies in

young animals (Vangeel et al., 2009) or boosting of the host immune response (Ellis,

2010).

Introduction 11

1.4 Bovine herpesvirus type 1 (BHV-1)

1.4.1 Epidemiology

Bovine herpesvirus was first isolated in 1955 (Madin et al., 1956). It is the etiological

agent of two major clinical cattle diseases: infectious bovine rhinotracheitis (IBR)

(Schroeder and Moys, 1954) as well as infectious pustular vulvovaginitis (IPV)/

balanoposthitis (IPB) (Kendrick et al., 1958; McKercher, 1964; van Oirschot, 1995).

Outbreaks can occur in the absence of acute infection and are caused by

maintenance of latent virus within a cattle herd and reactivation after specific stimuli

(Kaashoek et al., 1996; Ellis, 2009). Morbidity is high and can approach 100% in

unvaccinated herds; mortality rates range between 1% and 10% (Yates, 1982). In

recent years, eradication programs were initiated to eliminate BHV-1 from several

countries like Denmark, Switzerland and Austria (Ackermann and Engels, 2006).

Intervention strategies applied are mainly based on the detection and elimination of

latently infected animals within a herd.

1.4.2 Clinical signs and Pathology

Transmission occurs via aerosols at short distance or direct nose-to-nose contact in

case of respiratory infections (Mars et al., 1999; Mars et al., 2000); genital forms are

transmitted through mating and virus contaminated semen (Kupferschmied et al.,

1986). BHV-1 has an incubation period of 2-7 days (Yates, 1982). Clinical symptoms

are fever, anorexia, coughing, nasal discharge, conjunctivitis and dyspnea (Jericho

and Darcel, 1978; Jones and Chowdhury, 2007), but most infections remain

subclinical (Curtis et al., 1966; Hage et al., 1998). In fatal cases abortion and

systemic disease can occur, especially in newborn animals (Curtis et al. 1966, Bryan

1994, Muylkens 2007). The primary targets of BHV-1 are mucosal surfaces in the

upper airways (or genital tract) which results in massive cell destruction due to

programmed cell death and necrosis. Erosions and ulcers within the mouth, nose and

trachea are common (Curtis et al., 1966; Jericho and Darcel, 1978; Yates, 1982;

Muylkens et al., 2007). The cytopathic effect by BHV-1 causes epithelial damage

comprising cell ballooning and intranuclear inclusion bodies (Curtis et al., 1966;

Jericho and Darcel, 1978; Bryan et al., 1994). Respiratory disease lasts up to 10

Introduction 12

days (Curtis et al., 1966; Yates, 1982). Production of antibodies can be observed

between 8 and 12 days post-infection (Rouse and Babiuk 1978). After infection, virus

is released at high titers being responsible for the rapid transmission within a cattle

population (Yates, 1982). Once infected, BHV-1 causes lifelong latency (Stevens

1978). Latency is mainly established in ganglionic neurons, particularly the trigeminal

ganglion, but is assumed to take place also in tonsils and lymph nodes (Mweene et

al., 1996; Winkler et al., 1999; Winkler et al., 2000). Viremia is possible, therefore,

BHV-1 can also enter other organs and tissues and thus lead to atypical clinical

symptoms (Bryan et al., 1994; Kaashoek et al., 1998). BHV-1 plays a role in the

initiation of BRD as it can suppress the host immune system after infection. This

implies downregulation of IFN-α (Henderson et al., 2005), induction of apoptosis in

leukocytes, especially CD4+ T cells (Devireddy and Jones, 1999; Winkler et al., 1999)

as well as downregulation of MHC I molecules. Therefore, antigen presentation is

suppressed and the removal of virus-infected cells is hampered (Nataraj et al., 1997;

Koppers-Lalic et al., 2005). The impairment of the immune system in turn clears the

way for secondary bacterial infections which contribute to severe lower respiratory

tract infections (Jericho and Langford, 1978; Yates, 1982). Important bacterial

pathogens which are associated with BHV-1 are M. haemolytica or P. multocida

(Allan and Msolla, 1980; Ellis, 2009).

1.4.3 Vaccination

There exist several commercially available vaccines, either killed whole virus or

modified live attenuated vaccines (van Drunen Littel-van den Hurk, 2006), but they

can lead to disease in young calves due to immunosuppression and subsequent

induction of BRD (Jones and Chowdhury, 2007). Thus, so called vaccine outbreaks

have occurred in herds of vaccinated feedlot cattle (Bryan et al., 1994; van Drunen

Littel-van den Hurk et al., 2001). In recent years, many research efforts have been

directed to modified live virus vaccines and gE- and Us9- deletion mutants have been

suggested to be the most promising candidates (Kaashoek et al., 1998; Chowdhury

et al., 1999; Muylkens et al., 2006; Butchi et al., 2007).

Introduction 13

1.4.4 Taxonomy

BHV-1 belongs to the order Herpesvirales within the family Herpesviridae and the

subfamily Alphaherpesvirinae (ICTV, 2012a). Viruses belonging to the subfamily

Alphaherpesvirinae cause similar respiratory tract infections in a wide variety of

species. All members share a large host range, short replication cycles and the ability

to establish latent infections. Within the Alphaherpesvirinae, BHV-1 is classified in the

genus Varicellovirus. This genus consists of several viruses which are closely

related. Apart from BHV-1, the most prominent ones within this genus are Varicella

zoster virus (human herpesvirus 3), which causes chickenpox in humans and

pseudorabies virus (suid herpesvirus 1), which is responsible for Aujesky’s disease

(Roizman and Knipe, 2001; Roizman and Pellett, 2001).

On the basis of genomic and antigenetic surveys, three different subtypes of BHV-1

have been identified, BHV-1.1, BHV-1.2a and BHV-1.2b (Metzler et al., 1985).

Subtype 1.1 isolates were associated with IBR and found in the respiratory tract and

aborted fetuses. Subtype 1.2a can be associated with the respiratory and genital

tract and is considered to be the etiologic agent for IPV/IPB, IBR and abortions.

Subtype 1.2b is the causative agent of IPV/IPB, mild respiratory disease but has no

abortigenic potential (Miller et al., 1991; van Oirschot, 1995; D'Arce et al., 2002). Up

to now, the subtype tropism of BHV-1 for either the respiratory or the genital tract is

not supported by molecular (Muylkens et al., 2007) or experimental data (Steukers et

al., 2011). All subtypes exhibit more than 95% homology when compared by DNA

sequence analysis (Seal et al., 1985; Engels et al., 1986).

1.4.5 Virus structure

Herpesviruses are large (135 kb), linear double-stranded DNA viruses. The core is

surrounded by an icosahedral nucleocapsid which has a diameter of 100 to 110 nm

and is composed of 150 hexamers and 12 pentamers. The envelope consists of a

lipid bilayer with more than a dozen of glycoproteins (see Figure 1-3); among them

the glycoproteins gB, gC and gD are present in large amounts. Nucleocapsid and

envelope are connected by a protein-rich tegument. The size of a virus particle varies

between 120 and 300 nm in diameter (Roizman and Pellett, 2001). The genome of all

Introduction 14

members belonging to the genus Varicellovirus is divided into one long unique unit

(UL) and one short unique unit (US), which are flanked by repeat sequences. The

genome consists of a set of genes which are under temporal regulation; therefore,

one distinguishes between immediate early (IE), early (E) and late (L) genes, which

encode more than 70 proteins (Wirth et al., 1989). Herpesviruses are equipped with a

DNA polymerase that has proofreading activity (Ellis, 2009).

Figure 1-3 Schematic drawing of an Alphaherpesvirus particle; adapted from Flint et al. (2004).

1.4.6 Viral life cycle

Alphaherpesviruses like BHV-1, herpes simplex virus (HSV-1) or pseudorabies virus

utilize the glycoproteins gB and/or gC for primary attachment to heparan sulfate

structures (Liang et al., 1991; Okazaki et al., 1991). To mediate entry, the virions bind

via the surface glycoprotein gD to one or more of three known receptors (nectin-1,

herpesvirus entry mediator HVEM, 3-O sulfated heparan sulfate) (Johnson and

Spear, 1989; Montgomery et al., 1996; Geraghty et al., 1998; Shukla et al., 1999;

Campadelli-Fiume et al., 2000). Nectin-1 (also known as herpesvirus entry mediator

Introduction 15

C or poliovirus receptor-related-1) is a member of a family of cellular adhesion

molecules which are characterized by three extracellular immunoglobulin-like

domains (Ogita and Takai, 2006). It is ubiquitously expressed and can be found as a

protein of the adherens junctions (Rikitake et al., 2012). HVEM belongs to the tumor

necrosis factor family (Montgomery et al., 1996). Up to now, only nectin-1 was

identified as an entry receptor for BHV-1 (Geraghty et al., 1998). Due to the broad

tissue distribution of this receptor, BHV-1 infects many different cell types in the

respiratory tract as well as in other organs (Yates, 1982; Ellis, 2009). After receptor

binding, fusion is accomplished by interaction of the surface glycoproteins

gB/gD/gH/gL (Liang et al., 1995; Meyer et al., 1998; Gerdts et al., 2000; Pertel et al.,

2001). Once the fusion process has taken place, the capsids are transported to the

nucleus via the microtubuli. This process is up to know not well understood (Antinone

et al., 2006; Muylkens et al., 2007). After entry into the nucleus, the genome is

assumed to circularize (Garber et al., 1993). Within the nucleus, the transcription of

viral genes and the replication of the viral DNA takes place. Gene expression is

divided into three phases: immediate early (IE), early (E) and late (L) (Wirth et al.

1989). Immediate early gene expression starts after release of the genome from the

capsid and mRNAs for regulatory proteins are transcribed which initiate expression of

early genes by the host cell RNA polymerase. The expression of late genes is

delayed and starts after DNA replication to build structural proteins (Roizman and

Knipe, 2001; Schynts et al., 2001). Viral capsids are formed which contain the viral

DNA. They acquire a first envelope from the inner nuclear membrane and are

released from the nucleus into the cytoplasm by fusion with the outer nuclear

membrane. From the cytoplasm, they bud into a trans-Golgi compartment to be

finally released from the cell (Granzow et al., 2001; Muylkens et al., 2007).

To establish latency, herpesviruses enter sensory neurons via direct cell-to-cell

spread. Spread to neighboring cells is accomplished by the interaction of gD; deletion

of gD abolishes cell-to-cell spread (Fehler et al., 1992). Viral gene expression and

generation of infectious virus are arrested, so that only viral genomes are detectable

during this phase (Schang and Jones, 1997; Jones, 1998). Reactivation from latency

is induced by different stimuli like high levels of corticosteroids or suppression of the

Introduction 16

immune system; subsequently, gene expression in the neurons is reactivated and

secretion of infectious virus occurs from nasal or ocular regions (Sheffy and Davies,

1972; Rock et al., 1992; Jones, 2003; Jones et al., 2006b).

1.5 The respiratory epithelial barrier

1.5.1 Basic functions of the respiratory epithelium

The respiratory airways are common sites of infection (Greenberg, 2002; Sloots et

al., 2008), because they are in close contact with the environment and therefore a

target for airborne transmission of microorganisms (Vareille et al., 2011). The airway

epithelium which lines the respiratory tract represents the first barrier to infection. It is

composed of different cell types e.g. ciliated cells and goblet cells as well as basal

cells which serve as progenitor cells for the differentiated epithelial cells (Figure 1-4).

The goblet cells continuously secrete mucins which form the mucus barrier on the

surface of the respiratory epithelium. The ciliary activity of specialized cells transports

the mucus – and entrapped foreign material – out of the lung; therefore, ciliated cells

are an important part of the clearance system of an organism.

Figure 1-4 composition of the respiratory epithelium; adapted from Flint et al. (2004).

Introduction 17

The epithelial cells are connected to adjacent cells via cell-cell junctions. These

include tight junctions, adherens junctions, gap junctions and desmosomes (Roche et

al., 1993; Davies and Garrod, 1997). Cell-cell contacts form a mechanical barrier

which divides the epithelium into an apical, surface-orientated domain and a

basolateral domain which faces the internal environment. Due to the expression of

different proteins and enzymes on each domain, the epithelium obtains its polarity.

An ionic gradient is built across the epithelium which allows the directional transport

of molecules (Davies and Garrod, 1997). Especially the tight junctions are essential

to preserve the epithelial integrity (Roche et al., 1993).

1.5.2 Polarized virus entry

Cell-cell junctions serve as a barrier to viral entry into and egress from the respiratory

tissue. Viruses usually enter the epithelium through either the apical or basolateral

side. Apical entry may be hampered by the mucin-rich layer which covers the ciliated

epithelium throughout the respiratory tract. The mucus consists not only of mucins,

but also of antiviral substances like IFNs and nitric oxide being part of the innate

immune response (Nicholas et al., 2006; Rose and Voynow, 2006). It is estimated

that about 90% of particles which enter the respiratory tract are removed by the

mucociliary clearance system (Voynow and Rubin, 2009; Vareille et al., 2011). There

are several functions which help airway epithelial cells in their fight against invading

pathogens; for instance they express pattern recognition receptors like toll-like

receptors (TLRs) which recognize viruses and subsequently induce the onset of the

immune response (Diamond et al., 2000; Akira, 2009). Upon activation, they release

cytokines and chemokines in the submucosal environment to recruit immune cells to

the site of infection (McNamara et al., 2004; McNamara et al., 2005). Also the

basolateral membrane consists of several proteins which serve as interaction

partners for respiratory viruses (Bergelson, 2003; Bergelson, 2009). Therefore, the

question arises how these viruses can cross the epithelia barrier to successful infect

their host.

Many research efforts have been made in recent years to understand how viruses

can cross the epithelial barrier. Some viruses directly bind to the luminal surface. An

Introduction 18

example of this strategy are human and avian influenza viruses (Matrosovich et al.,

2004; Thompson et al., 2006) as well as HPIV3 (Zhang et al., 2005) or SeV

(Villenave et al., 2010). Also for HRSV, it was shown that it infects differentiated

human air-liquid interface cultures from the apical side (Zhang et al., 2002; Wright et

al., 2005; Villenave et al., 2012). Other viruses enter epithelial cells from the

basolateral surface. One example is measles virus (MV), which was found to

preferentially enter well-differentiated human airway epithelial cells from the

basolateral side (Sinn et al., 2002). Also human adenovirus predominantly infects

from the basolateral side (Lam et al., 2011; Lutschg et al., 2011) as well as vaccinia

virus (Vermeer et al., 2007).

1.5.3 Cultures for differentiated respiratory epithelial cells

As mentioned above, the primary target cells for many respiratory viruses are cells of

the respiratory epithelium. Differentiated respiratory epithelial cells are an interesting

tool to analyze virus infections since they resemble the in vivo situation more closely

than immortalized cell lines do. Two successful culture systems for differentiated

respiratory epithelial cells are the air-liquid interface system and precision-cut lung

slices. Respiratory epithelial cells can be cultured at the air-liquid interface to

generate a pseudostratified and differentiated epithelium, which closely resembles

the morphology of a respiratory epithelium in vivo. For ALI cultures, cells are seeded

on a filter membrane (Figure 1-5 A) and the medium initially covers the apical and

basolateral compartment to establish a confluent monolayer. Afterwards, the medium

in the upper chamber is removed and cells require about 3-4 weeks to grow in the

presence of retinoic acid to a multilayered and differentiated epithelium. Figure 1-5

shows characteristic features of such an epithelium like cilia development (B, upper

panel), multiple layers of nuclei (B, lower panel) and the development of tight

junctions (C). Integrity of epithelia can be evaluated by a stable transepithelial

electrical resistance (TEER). Fig. 1-5 D shows a typical time course of the TEER of

an ALI culture with three phases: 1) increase to a peak indicating polarization, 2)

phase of decrease to a lower value and (3) subsequent increase to a stable level.

Introduction 19

Figure 1-5 ALI culture system. A) shows schematic drawing of an ALI culture system and (B) confocal scanning microscopy of a differentiated bovine epithelium indicated by the expression of cilia (upper panel, top view) and multilayered structure of nuclei (lower panel, z-scan). (C) Epithelial cells are connected by tight junctions which are visualized by staining against ZO-1 (green). (D) shows a typical TEER time course. The arrow indicates the time point, where ALI conditions were established. Mean values and standard deviation of 3 independent cultures are shown. Scale bars = 50 µm (B, upper panel), 25 µm (B, lower panel) and 25 µm (C). Cilia were visualized by staining against β-tubulin (red), Nuclei were stained by DAPI (blue).

The second culture system are PCLS, they exhibit several relevant features, e.g.

ciliated and mucus-producing cells are maintained in their original setting. Also

subepithelial cells are present in this system, which can provide further interesting

insights as they may be involved in virus entry and spread. Furthermore, slices can

be generated in large amounts and cells are viable for at least one week. Fig. 1-6

shows a light-microscopic picture (A) as well as a confocal picture (B) of a

characteristic slice consisting of the respiratory epithelium which lines the lumen of a

bronchiole. This is surrounded by the peribronchiolar connective tissue. Further,

alveolar regions extend through the border areas of the slice. A detailed description

and further confocal pictures of the PCLS can be found in Publication II.

Introduction 20

Figure 1-6 Light microscopic (A) and confocal (B) picture of a typical PCLS, scale bar = 100 µm.

1.6 Aim of the study

The aim of the thesis was to analyze strategies of respiratory viruses of the BRDC to

infect the bovine host. This problem was addressed with the help of ex vivo/ in vitro

respiratory epithelial cell cultures which exhibit in vivo characteristics of respiratory

epithelial cells more closely than immortalized cell lines do. These culture systems

have been established in our laboratory: The air-liquid interface system where

respiratory epithelial cells are grown on filter supports to establish a pseudostratified,

differentiated epithelium and precision-cut lung slices where slices are obtained from

the lobus accessorius after having stabilized the airways by low-melting point

agarose. In PCLS, the differentiated epithelial cells are maintained in their original

setting. Both culture systems showed that differentiated respiratory epithelial cells

were susceptible to infection by bovine parainfluenza virus type 3 (BPIV3) but not to

bovine respiratory syncytial virus (BRSV). These interesting observations were

subject to further detailed investigations within this thesis. In this context, another

important virus of the BRDC, bovine herpesvirus type 1 (BHV-1) was included into

the study. For BHV-1, it is not known which cells are the primary target cells within

the respiratory epithelium. Also for the human counterpart, herpes simplex virus type

1, it is not completely understood how the virus enters the respiratory airways and

which cells are the main target of infection. Relating to this question, it should be

determined whether infection by BHV-1 and BRSV is triggered by mechanical or

Introduction 21

chemical intervention of the epithelium. Further investigations should compare the

growth characteristics of the viruses. In the case of BRSV, further efforts addressed

the identification of primary target cells (Publication I).

Since it was observed that viruses of the BRDC can cross the species barrier, it is of

interest to analyze whether viruses can also infect in vitro cultures of non-bovine

origin. Since goats are considered as an important source of transmission, it was a

further aim of the thesis to establish PCLS of caprine lungs and to analyze them for

susceptibility to the three bovine viruses. Investigations like these are important to

understand interspecies transmission of viruses among ruminants (Publication II).

Introduction 22

Publication I 23

2 Publication I:

Three viruses of the bovine respiratory disease complex

apply different strategies to initiate infection

Jana Kirchhoff1, Sabine Uhlenbruck1, Katherina Goris1, Günther M. Keil2, Georg

Herrler1

1Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany

2Institute of Molecular Biology, Friedrich-Loeffler-Institut, Greifswald - Insel Riems, Germany

Veterinary Research 2014, 45(1):20

DOI: 10.1186/1297-9716-45-20.

Authors’ contributions

JK and GH conceived and designed the experiments; JK, SU (technical assistance)

and KG performed the experiments; JK, SU, GK, KG and GH analyzed the data; GK

contributed reagents/materials/analysis tools; JK and GH wrote the paper. All authors

read and approved the final manuscript.

The extent of contribution from Jana Kirchhoff to this article:

A) Performance of experiments: 80%

B) Analysis of experiments: 90%

C) Writing of the paper: 80%

Publication I 24

Abstract

Bovine respiratory disease complex (BRDC) is the major cause of serious respiratory

tract infections in calves. The disease is multifactorial, with either stress or reduced

immunity allowing several pathogens to emerge. We investigated the susceptibility of

bovine airway epithelial cells (BAEC) to infection by the three major viruses

associated with the BRDC: bovine respiratory syncytial virus (BRSV), bovine

herpesvirus type 1 (BHV-1) and bovine parainfluenza virus type 3 (BPIV3). For this

purpose, two culture systems for well-differentiated BAEC were used: the air-liquid

interface (ALI) system, where filter-grown BAEC differentiate into a pseudostratified

respiratory epithelium and precision-cut lung slices (PCLS) where BAEC are

maintained in the original tissue organisation. Comparative infection studies

demonstrated that entry and release of BPIV3 occurred specifically via the apical

membrane with ciliated cells being the major target cells. By contrast, airway

epithelial cells were largely resistant to infection by BHV-1. When the epithelial

barrier was abolished by opening tight junctions or by injuring the cell monolayer,

BHV-1 infected mainly basal cells. Respiratory epithelial cells were also refractory to

infection by BRSV. However, this virus infected neither differentiated epithelial cells

nor basal cells when the integrity of the epithelial barrier was destroyed. In contrast to

cells of the airway epithelium, subepithelial cells were susceptible to infection by

BRSV. Altogether, these results indicate that the three viruses of the same disease

complex follow different strategies to interact with the airway epithelium. Possible

entry mechanisms are discussed.

Keywords

Bovine respiratory disease complex, bovine respiratory syncytial virus, bovine

herpesvirus type 1, bovine parainfluenza virus type 3, air-liquid interface, precision-

cut lung slices.

Publication II 25

3 Publication II:

Infection of differentiated airway epithelial cells from

caprine lungs by viruses of the bovine respiratory disease

complex

Jana Kirchhoff1, Sabine Uhlenbruck1, Günther M Keil2, Christel Schwegmann-

Wessels1, Martin Ganter3, Georg Herrler1

1Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany

2Institute of Molecular Biology, Friedrich-Loeffler-Institut, Greifswald – Insel Riems, Germany

3Clinic for Swine and Small Ruminants, Forensic Medicine and Ambulatory Service, University of Veterinary Medicine, Hannover, Germany

Veterinary Microbiology 2014, 170:58-64

DOI: 10.1016/j.vetmic.2014.01.038.

Authors’ contributions

JK, MG and GH conceived and designed the experiments; JK and SU (technical

assistance) performed the experiments; JK, SU, GMK, CSW and GH analyzed the

data; GMK, CSW and MG contributed reagents/materials/analysis tools; JK and GH

wrote the paper. All authors read and approved the final manuscript.

The extent of contribution from Jana Kirchhoff to this article:

A) Performance of experiments: 80%

B) Analysis of experiments: 95%

C) Writing of the paper: 90%

Publication II 26

Abstract

Bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus type 3 (BPIV3)

and bovine herpesvirus type 1 (BHV-1) are important pathogens associated with the

bovine respiratory disease complex (BRDC). Non-bovine ruminants such as goats

may also be infected and serve as a virus reservoir to be considered in the

development of control strategies. To evaluate the susceptibility of caprine airway

epithelial cells to infection by viruses of BRDC, we established a culture system for

differentiated caprine epithelial cells. For this purpose, we generated precision-cut

lung slices (PCLS), in which cells are retained in their original structural configuration

and remain viable for more than a week. The three bovine viruses were found to

preferentially infect different cell types. Ciliated epithelial cells were the major target

cells of BPIV3, whereas BHV-1 preferred basal cells. Cells infected by BRSV were

detected in submucosal cell layers. This spectrum of susceptible cells is the same as

that reported recently for infected bovine PCLS. While infection of caprine cells by

BRSV and BPIV3 was as efficient as that reported for bovine cells, infection of

caprine cells by BHV-1 required a tenfold higher dose of infectious virus as compared

to infection of bovine airway cells. These results support the notion that non-bovine

ruminants may serve as a reservoir for viruses of BRDC and introduce a culture

system to analyze virus infection of differentiated airway epithelial cells from the

caprine lung.

Keywords

Bovine respiratory disease complex, Goat respiratory disease, Bovine respiratory

syncytial virus, Bovine herpesvirus type 1, Bovine parainfluenza virus type 3,

Precision-cut lung slices

Discussion 27

4 Discussion

4.1 In vitro cultures for differentiated respiratory epithelial cells

Culture systems for primary bronchial epithelial cells were widely used within the last

decade to study replication strategies of respiratory viruses. When cultured under air-

liquid interface (ALI) conditions, cells undergo mucociliary differentiation to generate

a pseudostratified epithelium including ciliated cells, secretory cells and progenitor

cells; they closely resemble the protective epithelial barrier pathogens have to face

when they invade a host. Therefore, they mimic the in vivo situation better than

immortalized cell lines do; even cell lines like MDCK cells which grow as polarized

cells when seeded on filter membranes are not directly applicable to the natural

situation since they do not resemble the phenotype of differentiated epithelia.

Nevertheless, so far, protocols for culturing differentiated epithelial cells are often a

challenge and it is not trivial to transfer them from one species to another. Until now,

ALI cultures have been widely used to study entry strategies of human respiratory

viruses, e.g. it was found that human influenza viruses cause localized infection with

an initial preference for non-ciliated cells (Matrosovich et al., 2004; Ibricevic et al.,

2006). Human adenoviruses have been reported to preferentially enter the epithelium

from the basolateral side (Pickles et al., 1998; Lam et al., 2011) as it was also

observed for measles virus (Sinn et al., 2002). Moreover, several studies have been

performed on the entry and infectivity of HRSV (Zhang et al., 2002; Villenave et al.,

2011; Villenave et al., 2012) with different outcomes regarding the susceptibility of

cells (see chapter 4.4). In summary, studies on well-differentiated ALI cultures helped

to elucidate infection strategies of respiratory viruses in terms of entry pathways,

targeted cell types, tropism, receptor-expression, cytopathogenesis and immune

response of the epithelium. Furthermore, the application of this culture system to

cells from species other than human has been successful in the case of cattle (Goris

et al., 2009), swine (Lam et al., 2011; Bateman et al., 2013) and mouse (Ibricevic et

al., 2006); but in general the establishment of ALI cultures for other species is still a

challenging task. A big advantage of cultures grown under ALI conditions is the

formation of two compartments, so that viruses can be applied to the apical and/or

Discussion 28

basolateral side. Disadvantages are the long differentiation time and the cost-

intensive maintenance of the cultures. Morphological analyses showed that

differentiated human airway epithelial cultures closely resemble the airway epithelium

in vivo (Karp et al., 2002). Another study compared cells grown under ALI conditions

with cells derived from tracheal and bronchial brushings via microarray analysis and

concluded that the transcriptional profile was similar (Pezzulo et al., 2011).

Interestingly, it was also found in that study that cells isolated from tracheal and

bronchial regions were similar in their transcriptional profile. A more recent

development are ALI cultures which are co-cultured with e.g. dendritic cells or

macrophages. Such culture systems allow to address questions concerning the

influence of immune cells on viral entry or cytopathogenesis (Lutschg et al., 2011).

The involvement of immune cells on virus infection can also be studied with PCLS.

Slices of 200-300 µm thickness can be prepared from the lower respiratory tract. The

advantage of this culture system is that the differentiated epithelial cells are

maintained in their original setting and the above mentioned inclusion of other cell

types like immune cells and other cells of the peribronchiolar and alveolar regions.

Further interesting features are the high yield of slices which can be produced from

one lung, the inexpensive maintenance for more than a week, easy monitoring of the

ciliary activity and the possibility to induce bronchoconstriction. However, this culture

system is less convenient to study polarized entry of viruses since there is no direct

access to either side of the polarized cells. PCLS have been widely used to address

physiological and pharmacological questions (Martin et al., 2001; Vietmeier et al.,

2007; Henjakovic et al., 2008), whereas infection studies are only emerging (Ebsen

et al., 2002; Goris et al., 2009; Abd El Rahman et al., 2010; Punyadarsaniya et al.,

2011; Meng et al., 2013). Previously two different in vitro airway systems for well-

differentiated bovine epithelial cells have been established at our institute, ALI

cultures and PCLS (Goris et al., 2009), and infection studies with these culture

systems were extended during this thesis (Kirchhoff et al., 2014a). Furthermore, a

culture system for caprine bronchial epithelial cells was established to study the

influence of other ruminant species on virus transmission (Kirchhoff et al., 2014b).

The application of two culture systems, ALI cultures and PCLS, has the advantage

Discussion 29

that (i) results can be confirmed by different approaches to exclude artifacts and (ii)

results can complement each other by making use of the strength of each system.

4.2 Infection strategies of bovine parainfluenza virus type 3

4.2.1 Infection of well-differentiated bovine airway epithelial cells

When BPIV3 was applied to the apical surface of airway epithelial cells the virus was

able to efficiently infect the epithelium. Ciliated cells were observed to be the primary

target of infection as indicated by co-staining of β-tubulin. Within the ALI system, only

ciliated cells were infected, whereas other cell types, e.g. mucus-producing cells

were not targeted. The same result was obtained when virus was applied to PCLS.

Here, the viral antigen also colocalized with β-tubulin expression (Goris et al., 2009;

Kirchhoff et al., 2014a). Titration of the supernatants of BPIV3 infected ALI cultures

on MDBK cells revealed that BPIV3 replicated to high titers which peaked at 96 h pi.

and that infected BAEC remain highly productive during the course of the

experiment. Moreover, progeny virus was released exclusively from the apical

surface; no virus release into the basolateral compartment was observed. It is

therefore proposed that BPIV3 causes a localized infection with entry and release

being restricted to the apical side of the respiratory epithelium. These conclusions

are supported by the findings that no infection occurred when the inverted,

basolateral side of the filter membrane was exposed to the virus (data not shown).

Virus entry and release from the apical side of well-differentiated human cultures

have also been reported for the human counterpart of BPIV3, HPIV3 (Zhang et al.,

2005). These results are consistent with previous studies performed with HPIV1,

Sendai virus (SeV), HRSV as well as human and avian influenza viruses (Zhang et

al., 2002; Thompson et al., 2006; Bartlett et al., 2008; Villenave et al., 2010).

Cytopathic effects were not detected after one week; these observations go in line

with the finding that there was not decline in TEER. Therefore, infection by BPIV3

does not affect the integrity of the epithelium. The preference for the apical side may

be explained by the expression of receptors. It has been shown for influenza viruses

that sialic acids present on the apical surface of respiratory epithelia are crucial for

Discussion 30

entry into target cells (Matrosovich et al., 2004; Ibricevic et al., 2006; Thompson et

al., 2006). Glycoconjugates of ciliated cells have been reported to contain both, α2-6-

linked and α2-3-linked sialic acid residues whereas α2-6 linked sialic acids are

predominantly expressed on non-ciliated cells. Both types are well-known receptor

determinants for avian and human strains, respectively (Matrosovich et al., 2004;

Ibricevic et al., 2006; Thompson et al., 2006). Also for some coronaviruses, ciliated

cells are supposed to be the main entry port (Dijkman et al., 2013). Prior

neuraminidase treatment of PCLS abolished infection of BAEC by BPIV3; thus, sialic

acids present on the cell surface are also crucial for BPIV3 entry into bovine epithelial

cells. It has been reported that infection of human ALI cultures by HPIV3 is sensitive

to pretreatment with a neuraminidase that is specific for α2-6-linked sialic acid

residues (Zhang et al., 2005). By contrast, two studies that employed glycan array

analyses showed that HPIV3 preferred α2-3-linked sialic acids (Suzuki et al., 2001;

Amonsen et al., 2007). Therefore, further studies on well-differentiated airway

epithelial cells are necessary to specify the linkage type of the sialic acids required.

Up to now, studies were mostly performed with plant lectins, which bind to specific

linkage types of sialic acids. However, those plant lectins only recognize a small

subset of sialylated N-glycans. Comparative studies with influenza viruses have

shown that the spectrum of sialoglycans preferentially recognized by the viral

hemagglutinin may be quite different from that bound by plant lectins. Therefore, it

has been questioned whether plant lectins are a reliable marker for detection of

potential receptors for influenza viruses in general (Sauer et al., 2014). Because of

these considerations, plant lectins may also not provide the correct information about

the presence of receptors for BPIV3. Therefore, there is a need for more research

directed to the entry mechanism of BPIV3. For instance, the role of the

neuraminidase as well as the expression of sialic acids on bovine cultures has to be

analyzed in more detail in the future.

Apart from the susceptibility of ciliated cells of well-differentiated BAEC to BPIV3

infection, immortalized cell lines as well as non-polar BAEC were also found to be

readily infected by this virus. Interestingly, polarized BAEC, which were in a pre-

differentiating status, were refractory to infection (personal observations; J. Klein,

Discussion 31

Master thesis, University of Veterinary Medicine Hannover). Also Zhang et al. (2005)

observed a reduced susceptibility to infection during the differentiation phase and

concluded that there might be other, sialic acid-independent, pathways being

involved in the infection of non-polarized or immortalized cell lines. Studies on an

immortalized alveolar cell line (A549) showed that HPIV3 was able to bind to both,

sialic acid and heparan sulfate (HS) residues (Bose and Banerjee, 2002).

Concordantly, HS-moieties were found on the surface of non-polarized human airway

epithelial cells, but were restricted to basal cells when cultures were fully

differentiated (Zhang et al., 2005).

In PCLS, when BPIV3 infection was monitored over a period of one week, a strong

cytopathic effect (CPE) was observed as indicated by ciliated cells shedding into the

lumen of the bronchioli. As mentioned above, there was no visible BPIV3-mediated

CPE observed in infected ALI cultures after one week. Since it is suggested for

BPIV3 that viral clearance is mediated via an interaction of innate and adaptive

immunity (Ellis, 2010) which targets both, the virus and the infected cell, the lack of

cytopathology within the ALI cultures might be due to the lack of immune cells which

respond to the infection and therefore contribute to destruction of the epithelium. In

summary, an entry strategy is proposed where BPIV3 enters the epithelium via the

apical side by the use of sialic acid as receptor determinants and then replicates

within the ciliated cells. Spread of virus to adjacent cells is achieved by virus released

from the apical membranes and subsequent movement due to the ciliary activity of

BAEC.

4.2.2 Infection of well-differentiated caprine airway epithelial cells

As observed for the bovine cultures, also caprine PCLS were highly susceptible to

BPIV3. Co-staining of viral antigen and β-tubulin revealed that exclusively ciliated

cells were infected, which were shed into the lumen of the bronchioles 5 days after

infection. Occasionally, also infection of alveolar epithelial cells was observed,

consistent with the spread of virus to the lower airways in severe cases of BPIV3

infection. Therefore, it is assumed, that the course of infection by this virus is

comparable in the bovine and caprine lung. The pathogenic role of BPIV3 has been

Discussion 32

reported to be similar in cattle and sheep (Dungworth, 1993), but so far, there exist

only few studies about experimental infections of goats (Afshar and Terlecki, 1979).

In that study, BPIV3 was recovered from nasal, tracheal and bronchial regions up to

6 days after infection and the authors detected BPIV3-specific antibodies as well as

characteristic histological lesions. However, goats did not develop clinical signs.

Moreover, serological surveys based on indirect immunofluorescence assays

revealed antibodies against BPIV3 in goats (Fulton et al., 1982; Elazhary et al., 1984;

Trahman and Singh, 1990; Yesilbag and Gungor, 2009). Furthermore, Yener et al.

(2005) analyzed paraffin sections of pneumonic caprine lungs and detected BPIV3-

specific antigen in the majority of cases. Virus-positive staining was predominantly

found in the cytoplasm of bronchiolar luminal epithelial cells and type II pneumocytes.

Thus, it appears likely that BPIV3 plays a role in caprine pneumonia; however, since

data on BPIV3 infection of goats is rare, further in vivo and in vitro studies are

needed to clarify the role of goats in BPIV3 transmission.

4.3 Infection strategies of bovine herpesvirus type 1

4.3.1 Infection of well-differentiated bovine airway epithelial cells

When BHV-1 was applied to the apical side of ALI cultures, infection was detectable

only occasionally even when the viral titer was increased 100-fold compared to the

titer applied in the case of BPIV3. In contrast, when cell-cell contacts were disrupted

by mechanical injury or depletion of calcium, viral entry was strongly enhanced. Also,

when BHV-1 was applied to the basolateral surface of the filter-grown cells, the

epithelial cells were susceptible to infection. Thus, BHV-1 needs to get access to the

basal side to successfully invade the epithelium. Furthermore, infection studies with

the ALI cultures revealed that nonlumenal airway epithelial cells were infected by

BHV-1. Immunostaining showed that these cells were basal cells, which serve as

progenitor cells for the differentiated epithelial cells. Spread of infection occurred in a

plaquewise manner. These findings were confirmed with the PCLS, where also basal

cells were found to be the primary site of replication. Recently, Steukers et al. (2011)

established bovine organ cultures derived from the upper respiratory tract of cattle

Discussion 33

and found that BHV-1 can invade the mucosa across the basement membrane when

an infectious dose of 107 TCID50/mL was used. As stated above, occasional infection

from the apical side was also observed within this study when ALI cultures were

infected with a comparable titer. However, this titer is 100-fold higher compared to

that which was used to establish successful infection from the basal side, as well as

100-fold higher than the titer which is required for successful infection by BPIV3 from

the apical side. Numerous studies were performed to analyze the entry mechanism

of HSV-1. Studies with MDCK cells grown under air-liquid interface conditions

revealed similar entry strategies with HSV-1 being able to infect the polarized

epithelium after disrupting the tight junctions or injuring the cell monolayer

(Schelhaas et al., 2003; Marozin et al., 2004). However, other studies proposed

HSV-1 entry from both sides (Sears et al., 1991; Tran et al., 2000) or exclusively via

the apical membrane (Galen et al., 2006). Indeed, monolayer cultures do not

resemble the morphology of a differentiated epithelium and therefore are not suitable

to identify the cell type which is targeted by a virus. This can only be achieved with

cultures of well-differentiated cells. In this context two studies which were conducted

on primary keratinocyte cultures are of special interest: Here it was found that

successful infection by HSV-1 requires access to the basal side (Schelhaas et al.,

2003) and it was therefore concluded that viral replication is initiated in the basal cells

by passing through microabrasions within the uppermost layers (Visalli et al., 1997).

Thus, also for BHV-1 it appears likely that the virus enters through small lesions

within the epithelium (Tran et al., 2000; Yoon and Spear, 2002; Schelhaas et al.,

2003; Marozin et al., 2004), replicates within the basal cell layer and then either

reaches blood vessels via cell-to-cell spread to cause viremia or enters sensory

neurons to establish lifelong latency. This model is consistent with the cell-to-cell

spread observed within the PCLS when the incubation time was increased up to five

days. In general, it cannot be ruled out that alphaherpesvirus entry comprises

different routes and involves multiple receptors. The most prominent one is nectin-1,

a member of the immunoglobulin superfamily (Geraghty et al., 1998; Spear et al.,

2000), which colocalizes to adherent junctions of epithelial cells (Takai and

Nakanishi, 2003) but was also reported to be expressed on the apical side of the

Discussion 34

epithelium (Schelhaas et al., 2003; Marozin et al., 2004). However, since the

distribution of nectin-1 differs depending on the study and the cell system used,

further entry receptors like HVEM may play a role (Montgomery et al., 1996). So far,

only nectin-1 was shown to be important in the entry process of BHV-1 (Geraghty et

al., 1998). Due to the complex infection cycle of alphaherpesviruses in general, one

cannot exclude that studies on polarized and differentiated epithelia mimic different

steps in herpesvirus entry and spread, e.g. invasion through the mucosa upon

primary infection and/or infection from the basolateral side and subsequent shedding

after reactivation (Geraghty et al., 1998). Which receptors and entry pathways are

explicitly used by BHV-1 remains to be elucidated.

4.3.2 Infection of well-differentiated caprine airway epithelial cells

In comparison to BPIV3 and BRSV, BHV-1 infected caprine PCLS very inefficiently.

When the virus was applied to the caprine slices with the same infectious dose as in

the bovine system, there was no infection visible. Only when the viral titer was

increased tenfold, infection occurred; basal cells were the primary site of infection as

it was also observed in the bovine system. Moreover, the disruption of tight junctions

facilitates viral entry. The group of alphaherpesviruses consists of different ruminant

viruses which are closely related. Therefore, interspecies transmission is considered

to be likely and was observed for almost all of them (Thiry et al., 2006). Accordingly,

it has been concluded that also non-bovine ruminant species have to be considered

as a reservoir for BHV-1 (Engels and Ackermann, 1996). Natural BHV-1 infections

have been recognized in goats, sheep and swine as indicated by virus isolation and

serological surveys (Fulton et al., 1982; Elazhary et al., 1984; Engels et al., 1992;

Clark et al., 1993). Especially BHV-1 and caprine HV-1 (CapHV-1) exhibit a close

relationship (Nixon et al., 1988; Whetstone and Evermann, 1988) and experimental

infections revealed that cross-infection is possible with higher susceptibility of goats

for BHV-1 than calves for CapHV-1 (Engels and Ackermann, 1996; Six et al., 2001).

In comparison, one study (Hage et al., 1997) analysed BHV-1 transmission from

experimentally infected sheep and concluded that sheep do not play a major role in

viral spread. For goats, so far such studies have not been conducted, but will be an

important step in the analysis of virus transmission. The fact that BHV-1 is less

Discussion 35

infectious for goats when compared to BPIV3 and BRSV might be due to the fact that

there exists also a species-specific component. Alphaherpesviruses contain several

different surface glycoproteins; some of them are closely related but others are very

distinct within members of the same genus. One example is glycoprotein C, which is

involved in the primary attachment to the host cell and considered to be the most

host-specific surface glycoprotein (Ackermann et al., 1986; Engels et al., 1992).

4.4 Infection strategies of bovine respiratory syncytial virus

4.4.1 Infection of well-differentiated bovine airway epithelial cells

When BRSV was applied to the apical surface of ALI cultures, infection was

predominantly found in ciliated cells and only occasionally in non-ciliated cells. In

comparison to BPIV3, the infection level was low since GFP-expression was

detected in only a few, non-contiguous cells when equal amounts of virus were

applied. Concordantly, titers of infectious progeny virus were up to 600 fold lower

than those observed for BPIV3. Moreover, progeny virus was released exclusively

via the apical membrane; therefore, results from ALI cultures indicate that also BRSV

causes localized infections in the respiratory tract. No syncytia formation was

observed and no cytopathogenic effect. The infection pattern resembles that reported

for HRSV by Zhang et al. (2002) where HRSV was applied to well-differentiated

human airway epithelial cells and basically the same mechanism of infection was

observed; but infection levels were high in comparison to the titers which were

observed in this thesis. However, these outcomes result from an unusual high viral

titer which was applied to the human ALI cultures. Contrary to that study and in

accordance with results obtained in this thesis, Wright et al. (2005) merely observed

low replication of HRSV in the airway epithelium of human nasopharyngeal mucosa,

and in studies by Villenave et al. (2012), RSV infection of pediatric bronchial

epithelial cells was only present in a subset of ciliated cells. Therefore, the authors

concluded that the entry receptor is not expressed on all ciliated cells and needs to

be identified in the future. Since disruption of tight junctions as well as wounding of

the epithelium did not enhance infection, it appears likely that BRSV applies other

Discussion 36

entry strategies than herpesviruses do. One explanation might be the upregulation of

a potential receptor molecule. In this context, environmental stressors may have an

influence which renders the epithelium susceptible to infection. Since severe BRSV

infections predominantly occur in young cattle (Kimman et al., 1988; Hagglund et al.,

2006), stressful events within the first month of live like weaning, transport, or

introduction into new herds can make the animals susceptible. Also co-infection with

other viruses or bacteria of the BRDC may predispose them to disease. Bacteria like

P. multocida, M. haemolytica, M. bovis and H. somni may contribute to severe

respiratory tract infections (Virtala et al., 1996; Dabo et al., 2007; Booker et al.,

2008). Gershwin et al. (2005) showed that experimental co-infection of calves with H.

somni and BRSV results in a more severe disease as compared to infection with

either of the pathogens alone. In this study it has been suggested that viral infection

causes immunosuppression of the host which leads to increased colonization of

commensal bacteria; however an inverse order of events cannot be excluded:

primary attachment of bacteria may also facilitate subsequent viral entry into the

respiratory tract. This model was also proposed by Nguyen et al. (2010) who showed

that HRSV infection was enhanced in primary human epithelial cells after treatment

with a synthetic bacterial lipopeptide. So far, such experimental co-infection studies

are rare and should be performed in the future both in vivo and in vitro. Especially

well-differentiated respiratory epithelial cell cultures are a useful tool to address such

questions. Work on BRSV infection is still hampered by the fact that the entry

receptor is not known to date. Recently, the core protein nucleolin was proposed as a

receptor for HRSV (Tayyari et al., 2011). Nucleolin has the capacity to shuttle

between the nucleus and the surface especially in cells which undergo mitosis

(Ginisty et al., 1999). A two-step process of entry has been proposed with initial

attachment to cellular proteoglycans via the G protein and subsequent fusion of the

viral envelope with the cellular membrane mediated by a specific receptor, e.g.

nucleolin (Mastrangelo and Hegele, 2013). Since nucleolin is ubiquitously expressed,

it cannot explain the tropism of RSV for the respiratory tract; therefore, it appears

likely that other co-receptors are involved in the entry of RSV. Identification of these

Discussion 37

co-receptors will be helpful to understand the limited susceptibility of bovine airway

epithelial cells to BRSV.

When BRSV was applied to PCLS, infection was observed in subepithelial cells and

only occasionally within the epithelium. Unfortunately, antibodies for the bovine

system are limited and those who are available displayed a high background. Thus,

so far it was not possible to identify the type of infected cells within this region.

Nevertheless, the morphological appearance suggests that at least some of them

might be dendritic cells (DCs). Staining with an antibody against CD86, which was

shown to be expressed on bovine dendritic cells (Yamakawa et al., 2008) indicated

some colocalization with BRSV-infected cells (data not shown). Since there exist

more than one subtype of dendritic cells in the bovine lung, more immune markers

have to be included in the analysis to truly identify the targeted cell type. DCs are

antigen-presenting cells which take up antigen and migrate to lymph nodes to induce

a specific immune response (Banchereau and Steinman, 1998); however viruses

may also use them as a “Trojan horse” to migrate to other regions of the body

(Yewdell and Brooke, 2012), either to establish persistence or to cause viremia. For

bovine DCs, it has indeed been shown that they can be infected by BRSV (Werling et

al., 2002) as well as it has been reported for human DCs and HRSV (Jones et al.,

2006a; Hobson and Everard, 2008; Everard et al., 2009). Involvement of dendritic

cells in the uptake of viruses was proposed for other viruses as well, e.g. measles

virus (Sinn et al., 2002). As this virus was found to preferentially infect well-

differentiated respiratory epithelial cells from the basolateral side, it has been

suggested that immune cells like macrophages or DCs may take up the virus from

the lumen and transport it to lymph nodes or the bloodstream (Sinn et al., 2002;

Mesman et al., 2012). Also for HIV-1, it has been reported that DCs are involved in

viral entry and spread (Geijtenbeek et al., 2000). Therefore, the possibility has to be

considered that also RSV exploits immune cells like DCs to overcome the epithelial

barrier.

When the incubation time of BRSV was increased to one week, spread of infected

cells into the alveolar epithelium was observed. Staining with a marker against TTF-1

Discussion 38

showed that type II pneumocytes were targeted. These cells are cuboidal in shape

and are typically located at the intersections of two or more alveoli. Thyroid-

transcription factor (TTF)-1 has regulatory functions in the transcription of genes in

the thyroid and lung, e.g. expression of surfactant gene and Clara cell secretory

protein. Originally, it was used as a marker for lung adenocarcinoma (Lau et al.,

2002). A positive staining for TTF-1 has been reported for type II pneumocytes and

Clara cells; e.g. in the lungs of sheep infected with sheep pox virus (Beytut, 2010) or

in a study where pigs were experimentally infected with porcine reproductive and

respiratory syndrome virus (PRRSV) (Balka et al., 2013). Moreover, also TTF-1-

negative cells within the alveolar epithelium were infected. These cells had a

flattened morphology being characteristic for type I pneumocytes. Thus, it is

concluded that BRSV can infect both, type I and type II pneumocytes. Replication of

BRSV in type I and type II pneumocytes was found in histological sections of calves

experimentally infected with BRSV (Viuff et al., 2002). Moreover, this group showed

that severe clinical outcomes were correlated to spread of BRSV to deeper regions of

the lung, e.g. the alveoli, and concluded that both, the host immune response and

deficient clearance of the virus with a subsequent spread to alveoli can lead to

severe BRSV infection. Infection of alveolar epithelial cells within PCLS is a striking

feature and may help to understand the strategies of BRSV to establish infection in

the upper and lower respiratory tract as well as to identify possible entry pathways in

the future.

4.4.2 Infection of well-differentiated caprine airway epithelial cells

When BRSV was applied to the caprine PCLS, subepithelial cells were found to be

the primary target of infection as described above for the bovine system. When the

titer was increased tenfold, only occasionally epithelial cells became infected and

were identified as ciliated cells by staining with a marker against β-tubulin. This

refractory behaviour against BRSV infection could not be overcome by increasing the

incubation time to 5 days; under these conditions, cells rather spread to the alveolar

regions of the slice. Within the alveolar regions both, cells with a cuboidal and those

with a flattened morphology were identified as being infected; thus it is concluded

that, like in the bovine system, type I and type II pneumocytes were infected. To

Discussion 39

confirm these observations, antibody staining with the marker of TTF-1 would be the

next step. Unfortunately, this marker was obtained at the very end of the thesis,

where infection studies on caprine slices had already been finished. Thus, these

staining procedures could not be included in the thesis but will be done afterwards.

However, it appears likely that BRSV infects the same cell types in the bovine and in

the caprine system, therefore, cross- infection of goats may be a characteristic

feature of BRSV.

Antibodies to BRSV were found in goats and to a lesser extent in sheep (Elazhary et

al., 1984; Van der Poel et al., 1995; Langedijk et al., 1996; Yesilbag and Gungor,

2009). Van der Poel et al. (1995) performed an RSV-specific ELISA with sera from

different animal species and humans which all had prior contact to cattle. In this

study, it has been observed that more than 50% of the sera were BRSV-positive with

the strongest impact being directed to goats. In contrast, samples of human and

sheep were not positive. Antibody prevalence against BRSV was also found in

serological examinations of goats in North-Western Turkey (Yesilbag and Gungor,

2009) with values similar to those observed for cattle; thus, BRSV is considered to be

the most common cause of respiratory infections within this region. Prevalence of

BRSV antibodies in goats was also reported in studies from Canada (Elazhary et al.,

1984) and USA (Fulton et al., 1982).

Based on immunological and nucleotide sequence data on the G-protein, the most

variable protein among RSV isolates, it has been suggested that there exist at least

two ruminant RSV subgroups, one is represented by strains isolated from cattle and

goat and the other one by RSV isolated from sheep (Duncan and Potgieter, 1993;

Alansari et al., 1999; Eleraky et al., 2003). Experimental infection of sheep resulted in

mild clinical signs and BRSV could be isolated from the lungs of all infected animals.

Accordingly, antigen was mainly detected in the bronchiolar and alveolar epithelium,

but the overall amount of BRSV-positive cells was low (Meehan et al., 1994).

Interestingly, this group infected ovine lung slice organ cultures with BRSV at a titer

of 106 CCID50/mL and could not find antigen in any of the lung slices even after 72 h.

In comparison, slices were positive for BPIV3 when the virus was applied at similar

Discussion 40

titers, and infection was observed within the bronchiolar epithelium and occasionally

within the alveolar regions. Another group (Lehmkuhl and Cutlip, 1979) infected

lambs and also detected mild clinical symptoms, necrotic lesions and

seroconversion. Moreover, virus was recovered from 2 of 5 animals. Unfortunately,

information about experimental infection of goats is rare (Fulton et al., 1982).

It has been suggested that BRSV can survive in cattle populations since outbreaks

occur in the absence of initial infection or detection of clinical signs. So far, it is not

known, if BRSV can somehow persist in its host, although there is some evidence

that the virus can persist in e.g. lymph nodes (Van der Poel et al., 1993; De Jong et

al., 1996; Valarcher et al., 2001). Therefore, it is also possible that BRSV is

transmitted from other sources, e.g. goats and reintroduced into cattle populations.

4.5 Summary and Outlook

4.5.1 Strategies of viruses of the BRDC to infect the bovine host

Viruses of the BRDC are causative agents of respiratory disease in cattle and

especially young calves. In this thesis, the entry strategies of three bovine viruses

associated with the BRDC - BPIV3, BRSV and BHV-1 - were analyzed. In summary,

it can be concluded that the viruses display different strategies to infect their bovine

host: BPIV3 enters the cell via sialic acid containing glycoconjugates located on the

apical side of the respiratory epithelium, replicates to high titers in the ciliated

epithelial cells and progeny virus is shed into the lumen of the airways being

characteristic for a localized infection. Spread of BPIV3 to adjacent cells is

accomplished via the ciliary activity. BHV-1 is supposed to enter through small

lesions into the epithelium and primarily replicates within the basal cells, the

progenitor cells of the respiratory epithelium. Then the virus can either be released

from the apical side to be shed into the environment during acute infection or

establish a persistent infection in neurons. BRSV is likely to enter the epithelium from

the apical surface via a putative receptor molecule which is enhanced after specific

stimuli or may be taken up from immune cells like dendritic cells to cross the

Discussion 41

epithelial barrier. The potential of BRSV to somehow persist in its host remains

speculative. Since BRDC is a multifactorial disease, future research should include

co-infection studies with other viruses or bacteria to analyze, whether the

susceptibility of cells to BRSV can be enhanced under these conditions. Also, the

finding that pneumocytes are readily infected by BRSV may help to understand the

infection strategy of BRSV in the future.

Since respiratory viruses are primarily transmitted through the upper airways

including mouth, pharynx and trachea, it would be useful to establish well-

differentiated epithelial cell cultures from the uppermost airways like the nose to

clarify whether the replication of the viruses, especially BRSV, is enhanced in those

regions of the respiratory tract.

Taken together, analyses of the entry strategies of viruses of the BRDC are important

to understand the pathogenicity of these viruses and will help to create new

intervention strategies. Moreover, such studies will also help to design new or

improve existing vaccines in the future.

4.5.2 Goats as a virus reservoir

Pneumonia is considered to be an important disease in small ruminant species like

sheep and goats (Ackermann and Brogden, 2000; Yener et al., 2005) and it has been

suggested that viruses of the BRDC are causative agents in the naturally occurring

caprine pneumonia (Van der Poel et al., 1995; Thiry et al., 2006). Therefore, studies

as they were conducted in this thesis are important to analyze the role of goats as a

reservoir of viruses of the BRDC. Thus, one aim of the thesis was to establish

caprine PCLS and to infect these with viruses of the BRDC. In summary, it was found

that BPIV3 efficiently infected the epithelial cells while BRSV was mostly found in

submucosal cell layers. The infection efficiency of these viruses was similar to that

seen in bovine PCLS. In contrast, BHV-1 was much less efficient in infecting caprine

cells, but displayed the same tropism to basal cells as seen in bovine samples. The

thesis describes for the first time the preparation of caprine PCLS and demonstrates

that these slices can represent a first step in investigating pathogen dynamics among

co-housed closely related species. It can be concluded that in vitro studies are a

Discussion 42

useful ex vivo tool to investigate such interactions. A next consequent step is to carry

out infection studies of goats in vivo to close gaps in terms of infectivity, antibody-

production, clinical outcomes and shedding of viruses. Furthermore, transmission

studies have to be performed to analyze whether virus can be transmitted to

seronegative cattle. Also for small ruminant species like goats, it has been suggested

that viruses of the BRDC can increase the susceptibility to secondary bacterial

infections with e.g. M. haemolytica. Therefore, also in this context it would be

interesting to perform co-infections in order to see if they enhance the susceptibility

of caprine PCLS to BRSV. Studies like these are important to understand the role of

goats in the BRDC and are an essential step for vaccine design.

References 43

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Appendix 62

6 Appendix

Parts of the thesis have already been published or communicated.

Publications:

Kirchhoff, J., Uhlenbruck, S., Goris, K., Keil, G.M., Herrler, G. Three viruses of the

bovine respiratory disease complex apply different strategies to initiate infection. Vet.

Res. 45(1):20.

Kirchhoff, J., Uhlenbruck, S, Keil, G.M., Schwegmann-Wessels, C., Ganter, M and

Herrler, G. Infection of differentiated airway epithelial cells from caprine lungs by

viruses of the bovine respiratory disease complex. Vet. Microbiol. 170:58-64

Oral presentation:

Kirchhoff, J., Uhlenbruck, S., Keil, G.M. and Herrler, G. Different strategies of host

infection by bovine respiratory viruses. 5th International Congress of Virology, Lyon,

France, 11-14 September 2013.

Poster presentation (selected):

Kirchhoff, J., Uhlenbruck, S., Goris, K. and Herrler, G. Different strategies of host

infection by viruses of the bovine respiratory disease complex. 8th International RSV

Symposium, New Mexico, USA, 27-30 September 2012.

Best Poster Award, 5th Graduate School Day 23/24, November 2012, HGNI

Acknowledgements 63

7 Acknowledgements

I am particularly grateful to Prof. Dr. Georg Herrler for providing this very interesting

topic. Thank you for your continuous advice, encouragement and support throughout

my research and Ph.D. study.

Prof. Dr. Christiane Pfarrer and Prof. Dr. Evgeni Ponimaskin I would like to thank for

interesting discussions and helpful remarks during our meetings and for evaluation of

this thesis. Also, I thank Prof. Dr. Michael Veit for external review of this work.

Furthermore, I thank Prof. Martin Ganter for the collaboration during the goat project,

Prof. Günther Keil for providing recombinant BHV-1, Prof. Klaus Conzelmann for

providing recombinant BRSV and Dr. Wiebke Köhl for generating BRSV-GFP.

I am very grateful to Sabine Uhlenbruck for the assistance and all the good time we

had especially during our excursions. In this context, I also like to thank Günter

Thiem and Holger Mosch for the “shuttle-service”.

I would also like to thank Markus Hoffmann for his help, for sharing his incredible

knowledge and to be patience itself.

Also I like to thank all recent and former members of our institute, in particular Anna,

Ann-Kathrin, Anne, Christel, Christine, Fandan, Yang-Wei, Katarina, Karim, Mareike,

Martina, Moni, Nadine, Nai-Huei, Sarah, Sandra B., Sandra S. (especially), Trust and

Tim. Working with all of you was inspiring and family-like.

At the end I like to thank Tjardo and my family ….for everything; I can´t put into

words.