Kinetics of BoHV-1 Dissemination in an in Vitro Culture of Bovine Upper Respiratory Tract Mucosa Explants

Lennert Steukers, Annelies P. Vandekerckhove, Wim Van den Broeck, Sarah Glorieux, and Hans J. Nauwynck

All authors are connected to the Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820, Belgium.  Lennert Steukers, DVM, is a PhD-student in the Department of Virology, Parasitology and Immunology; Annelies P. Vandekerckhove, DVM, is a member of the Department of Virology, Parasitology and Immunology; Wim Van den Broeck, DVM, Msc, PhD, is a Professor of Cell Biology and Histology at the Department of Morphology; Sarah Glorieux,^* Ir, PhD is a post-doctoral associate of the Department of Virology, Parasitology and Immunology; Hans J. Nauwynck,^* DVM, PhD, is a Full Professor in Virology and Director of the Laboratory of Virology, Department Virology, Parasitology and Immunology. All authors are connected to the Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820, Belgium. ^*Shared senior authorship.

Address correspondence and reprint requests to Hans J. Nauwynck, Laboratory of Virology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium; Phone: 00 32 9 264 73 73; Fax: 00 32 9 264 74 95 or email: hans.nauwynck@ugent.be.hans.nauwynck@ugent.be.

Abstract

Bovine herpesvirus 1 (BoHV-1) is a well-known disease-causing agent in cattle. There is little known detailed information on viral behavior with emphasis on host invasion at primary replication sites such as the mucosa of the upper respiratory tract. Therefore, an in vitro system of bovine upper respiratory tract (bURT) mucosa explants was set up to study BoHV-1 molecular/cellular host-pathogen interactions. We performed a thorough morphometrical analysis (epithelial integrity, basement membrane continuity, and lamina propria integrity) using light microscopy and transmission electron microscopy. We applied a terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining as a viability test. Bovine upper respiratory tract mucosa explants were maintained in culture for up to 96 hours without any significant changes in morphometry and viability. Next, bURT mucosa explants were infected with BoHV-1 (Cooper) and collected at 0, 24, 48, and 72 hours postinoculation (p.i.). Using a quantitative analysis system to measure plaque latitude and invasion depth, we assessed dissemination characteristics in relation to elapsed time p.i. and found a plaquewise spread of BoHV-1 across the basement membrane as early as 24h p.i., similar to pseudorabies virus (PRV). Moreover, we observed that BoHV-1 exhibited an increased capacity to invade in proximal tracheal tissues compared to tissues of the deeper part of the nasal septum and ventral conchae. Revealing a more distinct invasion of BoHV-1 in proximal trachea, we can conclude that, in order to study an important aspect of BoHV-1 pathogenesis, the bovine upper respiratory tract mucosa explant model is the best suited model.

Key Words: basement membrane; bovine herpesvirus 1 (BoHV-1); bovine upper respiratory tract (bURT); in vitro model; morphometry; pathogenesis; terminal deoxynucleotidyl transferase mediated dUTP nick end labeling; upper respiratory mucosa; viability

Introduction

The revised family Herpesviridae incorporates pathogenic members that can cause disease in mammals, birds, and reptiles. This family is the most important out of three families in the new order of the Herpesvirales (Davison et al. 2009). During evolution, these viruses developed different mechanisms to (1) penetrate different mucosal barriers and to reach internal organs via leukocytes and nerve endings (invasion), (2) evade both specific and aspecific immunity (immune-evasion), and (3) hide in the infected host (latency) (Favoreel et al. 2000, 2005; Field et al. 2006). Many viruses from the Alphaherpesvirinae subfamily use the epithelium of the upper respiratory tract as an important mucosal portal of entry. In contrast to other respiratory viruses, these viruses can penetrate through the basement membrane (BM¹) after local dissemination. If during this mucosal invasion nerve endings or blood vessels are reached, these viruses can spread in the host, resulting in neuronal symptoms and viremia  (Muylkens et al. 2007; Nauwynck et al. 2007).

Bovine herpesvirus 1 (BoHV-1¹) is an important pathogen of cattle and can cause two major clinical entities: infectious bovine rhinotracheitis and infectious pustular vulvovaginitis/balanoposthitis (Engels and Ackermann 1996). BoHV subtype 1.1 (infectious bovine rhinotracheitis) spreads in the respiratory mucosa which leads to extensive tissue destruction with ulceration in the upper respiratory tract. However, subtype 1.2 (infectious pustular vulvovaginitis/balanoposthitis) replicates at the peripheral genital tract and is associated with pustular lesions. From the primary site of replication, the virus will gain access to local sensory neurons, reach corresponding ganglia, and establish lifelong latency. Following viremia, abortion in cows and fatal systemic infections in young calves may occur (Miller et al. 1991). It is still unknown how BoHV-1, as well as many other mammalian alphaherpesviruses, can penetrate so easily through the mucosal layer, despite the presence of barriers including the BM.

Little is known about the mechanisms of replication and invasion in the respiratory tract for BoHV-1. An in vitro model, resembling the in vivo situation, would be useful to elucidate the BoHV-1 invasion mechanism. Recently, such an in vitro bovine respiratory organ culture system was set up (Niesalla et al. 2009). However, after 24h culture at air-liquid interface, degeneration started, especially of the lamina propria. Since we wanted to study the invasion mechanism of BoHV-1 through the BM towards the connective tissue, it is essential to keep this layer vivid. The aim of the present study was therefore to modify, optimize, and extend the in vitro system of bovine respiratory mucosal explants in order to study the BoHV-1 dissemination kinetics in respiratory mucosa.

Materials and Methods

Experimental Design of a Respiratory Mucosa Explant In in Vitro Culture

Bovine respiratory tissue was obtained at a slaughterhouse. Samples were taken from seven calves aged 6 months. The head was cut longitudinal into two pieces, exposing the nasal septum. We cautiously removed nasal septum from the caudal two thirds of the nasal cavity (septum), ventral conchae (conchae), and proximal trachea (trachea). Tissues were transported to the laboratory on ice in phosphate buffered saline (PBS), supplemented with 1 µg/mL gentamycin (Gibco), 1 mg/mL streptomycin (Certa), 1 mg/mL kanamycin (Sigma), 1000 U/mL penicillin (Continental Pharma), and 5 µg/mL fungizone (Bristol-Myers Squibb). We collected blood to perform a complement-dependent seroneutralization (SN)-test to determine BoHV-1 specific antibody titers as an animal exclusion criterion. Using surgical blades (Swann-Morton), respiratory mucosa was carefully stripped from the underlying layers. Mucosal explants covering a total area of 0.7 cm² were produced and placed in six well culture plates (Nunc) epithelium upwards on fine meshed gauze for culture. We added serum-free culture medium (50% DMEM (Gibco)/50% Ham’s F-12 GlutaMAX (Gibco)) supplemented with 0.3 mg/mL glutamine (BDM Biochemical), 0.1 mg/mL streptomycin (Certa), 100 U/mL penicillin (Continental Pharma)), and 1 µg/mL gentamycin (Gibco) until achieving an air-liquid interface.The explants were cultivated for up to 96h at 37°C and 5% CO₂.

Morphometric Analysis

The explants were checked for sufficient ciliary beating on a daily basis using a light microscope as a first viability parameter. At 0, 24, 48, 72, and 96h of cultivation, we gathered explants from the different tissues of each calf.

Light Microscopy

At each time interval, we harvested one explant from every calf and each tissue and fixed these tissues in a phosphate buffered 3.5% formaldehyde solution for 24h. Fixation was followed by paraffin embedding using an automated system (STP 420D, Micron, Praran, Merelbeke, Belgium). Sections of 20 µm thick were successively cut, deparaffinized in xylene, rehydrated in descending grades of alcohol, stained, dehydrated in ascending grades of alcohol and xylene, and mounted with DPX (DPX mountant, BDH Laboratory Supplies, Poole, UK).

After in vitro cultivation, a hematoxylin and eosin staining was used to evaluate the architecture of the explants. Epithelial integrity was evaluated as a parameter for the effect of in vitro culture on epithelial morphometry.

Using a reticulin staining, we evaluated the thickness and continuity of the BM. As a result, collagen type III reticular fibers of the lamina reticularis of the BM were stained. Five randomly selected places in five randomly chosen fields were measured in each explant.

We performed a Van Gieson staining to analyze the overall structure of the lamina propria. By setting a threshold, the relative amounts of collagen and nuclei were calculated in a defined region of interest (ROI) from five randomly chosen fields.

All stained sections were analyzed using a BX61 light microscope (Olympus) and the Cell F Software (Olympus) (magnification 40x).

Transmission Electron Microscopy

We performed a transmission electron microscopical analysis to evaluate the structure of the explant down to the subcellular level. Explants were fixed overnight at 4°C in Karnovsky’s Fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.4)) to ensure that the structure of the specimens was preserved (Karnovsky 1965). Later, explants were rinsed in 0.1 M sodium cacodylate buffer (pH 7.4) for 8h and underwent an overnight postfixation in 2% osmium tetroxide at 4°C. The samples underwent a stepwise dehydratation in ascending grades of alcohol, an infiltration in a low viscosity embedding (LVR) medium (Agar Scientific) for 2 days, and an embedding in LVR. Lastly, ultrathin sections of embedded material were cut using a diamond knife on an Ultramicrotome Ultracut EM UC6 and stained afterwards with a Leica Microsystems EM staining before analysis on a JEM-1010 transmission electron microscope (Jeol) operating at 60 kV.

Analysis of Viability

Tissues derived from the same three calves used in the morphometrical analysis were monitored for occurrence of apoptosis during in vitro culture. DNA fragmentation was evaluated using an In Situ Cell Death Detection Kit (Roche), which is based on terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL). This method is designed as a precise, fast and simple, non radioactive technique to detect and quantify apoptotic cell death at single cell level in cells and tissues. We performed the TUNEL reaction according to the manufacturer’s guidelines. TUNEL-positive cells were counted from five randomly chosen fields of 100 cells in the epithelium as well as in the lamina propria with a fluorescence microscope (Leica DM RBE microscope, Leica Microsystems).

Virus Inoculation and Assessment of Dissemination Kinetics

After optimization of the in vitro model, tissues from four different calves were used for this assessment. Inoculation of the explants with BoHV-1 Cooper strain (Colorado) took place after 24h of cultivation (York et al. 1957). Explants were taken from their gauze and placed epithelium upwards in a 24-well plate (Nunc). After double washing with warm medium, 1 mL medium containing 10⁷ TCID₅₀/mL virus was added in each well thereby submerging the explant. After incubation for 1 hour at 37°C and 5% CO₂, the explants were washed three times with warm medium and placed back on the gauze.

To monitor kinetics of viral dissemination, explants were gathered at different time points postinoculation (p.i.¹). After collection, samples were carefully embedded in methocel^® (Fluka) and frozen at -70°C. Cryosections were made, fixed in methanol (-20°C, 100%), and kept at -20°C until staining. For the evaluation of penetration through the BM, the BM in BoHV-1 infected explants was stained. Mouse anti collagen VII antibodies (Sigma), directed against anchoring fibrils residing in the BM, and goat anti-mouse Texas Red^® antibodies (Molecular Probes) were used in a first step to mark the BM barrier. Secondly, a FITC^®-labeled goat anti-IBR polyclonal antiserum (VMRD) staining was performed to visualize viral proteins. Mounted samples were analyzed using a confocal microscope (Leica TCS SP2 confocal microscope). Thereafter, plaque latitude and invasion depth (distance underneath the BM) were evaluated using the line-tool function of the software program ImageJ. Finally, at 24 and 48h p.i., the average number of plaques was measured in the entire evaluated surface of explants derived from either septum, conchae, or trachea.

Statistical Analysis

Analysis of variance was performed on the obtained data using SPSS software (ANOVA). The results represent means + standard deviation of triplicate and quadruple independent experiments of respectively morphometric/viability analysis and analysis of viral dissemination. Data with P values of ≤ 0.05 were considered significant.

Results

Ciliary Beating

The cilia covering the respiratory epithelium kept on beating during the entire period of in vitro culture (up to 96h, the end of the experiment).

Epithelial Morphometry

Light Microscopy

We observed no significant changes in epithelial integrity during the entire cultivation period for septum, conchae, and trachea (Fig. 1a). All samples showed a respiratory epithelium. However, concerning septum and conchae we found some zones possessing a stratified squamous epithelium.

 

Transmission Electron Microscopy

Transmission electron microscopy was applied to evaluate epithelial integrity. For all tissues at all collected time points, small intercellular spaces between basal epithelial cells were seen. These observations were not made between apical epithelial cells at 0h, 24h, and 48h of in vitro cultivation since apical cells were adjacent. However, it should be noted that starting from 72h of in vitro cultivation, intercellular spaces between basal epithelial cells became more distinct, and moreover, few intercellular spaces started to appear between apical epithelial cells. Figure. 2. shows intercellular spaces.

 

Basement Membrane Morphometry

Light Microscopy

No major changes were observed in the thickness of the reticular lamina during the in vitro culture of mucosae of the septum, conchae, and trachea (Fig. 1c). At 0h, 24h, 48h, and 72h of cultivation, we noticed a significant difference in lamina reticularis thickness when comparing proximal trachea to the deeper part of the nasal septum and ventral conchae. A smaller thickness of the lamina reticularis was observed in proximal trachea (P values ≤ 0.05) (Fig. 3c).

 

Transmission Electron Microscopy

The continuity of the lamina densa of the BM was evaluated. For all explants and at all time points (up to 96h), no significant changes in lamina densa integrity were observed (Fig. 4).

 

Morphometry of the Lamina Propria

When analyzing Van Gieson stainings, no significant changes in relative amounts of collagen and nuclei were observed during the entire cultivation period for all tissues (Figs. 1b and 3a-3b).

Viability of Bovine Upper Respiratory Tract Mucosa Explants

During 96h of in vitro culture, there was no major increase regarding the amount of TUNEL-positive cells in the epithelium. However, for each tissue, at 96h of cultivation, we noticed a small but significant increase in the amount of TUNEL-positive cells in the epithelium. Evaluating the viability of the underlying connective tissue, the percentage of apoptotic cells in all tissues ranged from 2.9 ± 1.7 to 19.3 ± 4.3 at 0h and 96h, respectively (Table 1). The occurrence of apoptosis was more clear in glandular structures .

Evaluation of Primary Viral Dissemination

All four animals showed an SN-titer of < 2 for BoHV-1 specific antibodies and were therefore selected for this study. We observed clear distinct infected cells at 24h, 48h, and 72h p.i. BoHV-1 was found to spread in a plaquewise manner in respiratory mucosa (Fig. 5). Starting at 48h p.i. and more pronounced at 72h p.i., infected epithelial cells loosened and detached from the viral plaque. Both plaque latitude and invasion depth in septum, conchae, and trachea were evaluated at different time points p.i.

Plaque Latitude

No plaques were visible at 0h p.i. for all tissues. Individual plaques were measured at 24h and 48h p.i. Since almost all present epithelial cells in the bovine upper respiratory tract (bURT¹) mucosa explants were infected, we were not able to measure individual plaques at 72h p.i. At 24h p.i., average plaque latitudes of 142.5 ± 67.4 µm in septum, 170.6 ± 32.4 µm in conchae, and 168.3 ± 43.4 µm in trachea were observed. Plaque latitude increased over time and at 48h p.i., we measured average plaque latitudes of 275.5 ± 55.0 µm in septum, 312.3 ± 14.4 µm in conchae, and 317.9 ± 12.1 µm in trachea (Fig. 6a). Plaque latitude rose significantly between 0h, 24h, and 48h p.i. However, no major changes concerning the latitude were seen when comparing septum, conchae, and trachea.

Plaque Depth

Plaque depth underneath the BM was evaluated at 0h, 24h, 48h, and 72h p.i. In septum average plaque depths of 0.0 ± 0.0 µm at 0h p.i., 1.3 ± 2.2 µm at 24h p.i., 14.1 ± 9.0 µm at 48h p.i.., and 34.6 ± 13.1 µm at 72h p.i. were observed. Average plaque depths in conchae ranged from 0.0 ± 0.0 to 2.8 ± 2.1 µm, 25.9 ± 7.0 µm and 36.8 ± 11.6 µm at 0h, 24h, 48h, and 72h p.i., respectively. When analyzing average plaque depth in trachea, values starting from 0.0 ± 0.0 µm at 0h p.i. to 7.4 ± 5.4 µm at 24h p.i., 50.7 ± 7.3 µm at 48h p.i., and 64.7 ± 16.2 µm at 72h p.i. were observed (Fig. 6b). There was a significant increase in plaque depth between 24h, 48h, and 72h p.i. for all tissues. Except in tracheal tissues, between 48h and 72h p.i. there was no significant change in plaque depth underneath the BM. There was a clear significant difference when evaluating plaque invasion depth underneath the BM in trachea compared with that in septum (at 24h, 48h, and 72h p.i.) and conchae (at 48h and 72h p.i.) (Fig. 5).

Plaque Number

We did not see any significant changes in plaque number between the deeper part of the nasal septum, ventral conchae, and proximal trachea at 24h and 48h p.i. Results of the average number of plaques are given per 5 mm². At 24h, an average plaque number of 21.2 ± 14.8, 42.3 ± 27.2, and 25.0 ± 0.8 was counted for septum, conchae, and septum. There was an average amount of 31.0 ± 5.6 plaques in septum, 24.5 ± 10.5 plaques in conchae, and 25.0 ± 12.3 plaques in trachea at 48h p.i. Moreover, no major differences in average plaque number were noticed when comparing time points 24h and 48h p.i. for septum, conchae, and trachea.

Discussion

Little is known about the primary replication and dissemination of different alphaherpesviruses at host mucosal entry ports. Nevertheless, getting a fundamental image on how the virus behaves at its primary replication site can provide insights for prevention and treatment on a rational basis. In the present study, a bURT mucosa explant model was set up to study BoHV-1 dissemination characteristics in bovine respiratory mucosa. The use of respiratory organ cultures already proved to be of importance in different scientific areas. Insights in drug transportation, metabolic pathways, and pathogen-host interactions are attained achievements based on organ culture arrays. The explant model is the perfect compromise between in vitro cell cultures and in vivo laboratory animals. In cell cultures, no tissue structure is present and therefore essential cell-cell and cell-extracellular matrix contacts are lost. Current in vivo model systems often used in alphaherpesvirus studies originated from rodents. However, these models create a heterologous situation since species specific cellular components, which may play a role in viral invasion, are absent.

The bovine mucosa explant system represents a homologue model strongly resembling the in vivo situation and implements reduction, refinement, and replacement (3R principles). These unique systems may be used for screening of different potent antiviral molecules. Since intraspecies variation is excluded, a specific effect of a certain condition or virus strain related to the mock situation may be examined. Explant models of respiratory tissue have already been described in the literature for use in human (Ali et al. 1996; Jackson et al. 1996; Jang et al. 2005; Schierhorn et al. 1995), rat (Fanucchi et al. 1999), canine (Anderton et al. 2004), swine (Glorieux et al. 2007; Pol 1990) and equine models (Hamilton et al. 2006; Lin et al. 2001; Vandekerckhove et al. 2009). In bovine models, respiratory organ cultures have been described in the literature but these explants were only maintained for up to 72h without extensive morphometrical analysis (Bouffard et al. 1982; Chemuturi et al. 2005; Fulton and Root 1978; Richter and Keipert 2004; Schmidt et al. 1999; Shroyer and Easterday 1968; Svitacheva et al. 1998). Recently, Niesalla and colleagues (2009) established an in vitro bovine respiratory organ culture system to use as a BoHV-1 infection model. Bovine tracheal and nasal mucosae were cultured for up to 72 hours and monitored for viability, integrity, and TNF-α gene expression. However, using latex beads clearance as a viability test only gives an indication about epithelial cell function. There was no information on the viability of the underlying layers. Moreover, a histological assessment of the organ culture using light microscopy and scanning electron microscopy showed evidence of progressive degeneration starting from 24h of cultivation. Although overall integrity of the epithelium was maintained, degeneration of mucus gland structures in the lamina propria, starting from 24h, and separation of collagen fibers from 48h onwards are reported.

We believe it is essential to maintain the viability and integrity of the underlying layers when studying a virus-host interaction at mucosal entry ports. Therefore, we modified, optimized, and extended the bovine respiratory organ culture. During in vitro culture up to 96h, no major changes in epithelium integrity and viability occurred. During in vitro culture, intercellular spaces became more clear between basal epithelial cells, and starting from 72h of in vitro culture, few intercellular spaces appeared between the adjacent apical cells. These findings were also evident in respiratory mucosa explants of swine and horse. A possible explanation for the gapping is a decreasing strength of cell-cell contacts at different regions in the explants (Glorieux et al. 2007; Vandekerckhove et al. 2009). Regarding BM integrity and continuity, no significant changes were noticed during the entire culture. The trachea showed significant differences in lamina reticularis thickness compared to the other tissues at 0h, 24h, 48h, and 72h of cultivation. We can conclude that trachea seems to have a smaller thickness of the lamina reticularis compared to septum and conchae. When evaluating the connective tissue, we did not observe major significant changes in tissue morphometry. Concerning viability of the connective tissue, starting from 24h of in vitro culture, we noticed an increase in the occurrence of apoptosis, especially inside glandular structures. This could be caused by a lack of sufficient nutrients. However, during the entire cultivation period (96h) occurrence of apoptosis did not exceed 19.3 ± 4.3% and no significant differences were observed between septum, conchae, and trachea. Since we did not notice any severe degeneration of mucus gland structures and collagen fibers, we considered this an acceptable percentage.

When comparing dissemination kinetics of BoHV-1 in septum, conchae, and trachea, we made some interesting findings. There was a significant rise in BoHV-1 invasion depth in tracheal tissues when compared to that in tissues of septum and conchae at 48h p.i. as well as 72h p.i. The reason for this difference may be the significantly smaller thickness of the lamina reticularis in trachea compared to septum and conchae, suggesting that the virus has to cross a thinner BM barrier in the trachea. Although speculative, an alternative hypothesis can be formed to explain the rapid and efficient invasion in tracheal tissues. Evans and colleagues (1993) reported an attenuated fibroblast sheath located just underneath the BM zone. This sheath consists of large flat fibroblasts covering up to 70% of the BM zone and makes contact with the basal lamina. These contacts are mainly situated underneath basal cells, which form the contact between epithelium and BM (Evans et al. 1990, 2001). Attenuated fibroblasts are only described so far in trachea, intrapulmonary bronchi, and terminal bronchioles in different species (Evans et al. 1993, 1999; Zhang et al. 1996). Alphaherpesviruses are known for their efficient cell-to-cell spread (Muylkens et al. 2007; Nauwynck et al. 2007). Since epithelial basal cells and attenuated fibroblast are in close proximity to each other, taken together with the observed smaller reticular lamina thickness, this could explain why invasion in trachea is more efficient than in conchae or septum. These findings put emphasis on the involvement of the trachea in the clinical picture (IBR) caused by BoHV-1 and are correlated with the in vivo situation.

Generally, we set up mucosa explant models of bovine, porcine, and equine in our laboratory to study aspects of viral mucosal invasion. BoHV-1 and pseudorabies virus (PRV) exhibit both a plaquewise spread across the BM in the in vitro mucosa explant model suggesting they have minor effort in passing a host defense line (Glorieux et al. 2007, 2008). This is confirmed by the severe upper respiratory tract in vivo symptoms of both viruses. On the contrary, equid herpesvirus 1 (EHV-1¹) spreads only laterally and does not pass the BM. EHV-1 developed another system to invade. It misuses local immune cells as carriers to penetrate the host (Gryspeerdt et al. 2010; Vandekerckhove et al. 2009, 2010). The attenuated EHV-1 replication characteristics in respiratory mucosa are similar to the mild in vivo respiratory symptoms. Moreover, in striking contrast with what was observed for BoHV-1, EHV-1 dissemination characteristics in tracheal tissue did not differ at all from those in other respiratory tissues (Vandekerckhove et al. 2010; A. P. Vandekerckhove, Ghent University, personal communication). These findings confirm the in vivo relevance of the mucosa explant in vitro model.

During evolution, what is the reason different mechanisms of viral invasion occurred between different alphaherpesviruses? Is this inherent to the species and merely an adaptation of the virus to its host? Or did some strains become more virulent by acquiring advantageous genetic material over time? Respiratory mucosal explants are ideal tools to answer these questions.

Conclusion

We conclude that bURT mucosa explants can be maintained in culture for up to 96h without any major changes in structural integrity and viability. These in vitro cultured mucosal explants are susceptible to BoHV-1 infection and therefore a good alternative for experiments on living animals (the three Rs of Russell and Burch). BoHV-1 was found to spread in a plaquewise manner when investigating the evolution of plaque formation at different time points p.i. in bURT mucosa explants. The crossing of the BM started from 24h p.i. onwards and all plaques crossed the BM at 48h p.i. Furthermore, BoHV-1 invaded more efficiently in depth across the BM in proximal tracheal tissue. Therefore, proximal trachea is an interesting target tissue to unravel BoHV-1 invasion mechanisms in respiratory mucosa.

¹Abbreviations used in this article: BM, basement membrane; BoHV-1, bovine herpesvirus 1; bURT, bovine upper respiratory tract; EHV-1, equid herpesvirus 1; p.i., postinoculation.

Acknowledgments

This work was supported by a Concerted Research Action of the Research Council of Ghent University and through funding from the Agency for Innovation by Science and Technology in Flanders (IWT). The authors thank A. Rekecki, M. Claeys, L. De Bels, J. De Craene, L. Pieters, and L. Standaert for their excellent technical support in preparing all the morphological samples and M. Bauwens for performing SN-tests.

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Table 1. Occurrence of apoptosis in epithelium and lamina propria as a parameter for the effect of in vitro culture on the viability of bovine respiratory mucosa explants. Values are given as means ± SD.

Tissue	Layer	% of TUNEL-positive cells at … h of cultivation
		0	24	48	72	96
Deeper part of the nasal septum	Epithelium	0.4 ± 0.2	2.6 ± 2.0	2.4 ± 2.6	1.5 ± 1.1	5.5 ± 2.7
	Lamina propria	2.9 ± 1.7	10.5 ± 7.2	11.2 ± 4.3	13.3 ± 6.7	19.3 ± 4.3
Ventral conchae	Epithelium	0.5 ± 0.4	2.0 ± 0.9	1.7 ± 1.6	1.3 ± 0.9	2.8 ± 1.7
	Lamina propria	3.0 ± 2.6	9.9 ± 4.0	11.6 ± 1.8	13.4 ± 4.3	17.5 ± 2.4
Proximal trachea	Epithelium	0.5 ± 0.3	0.9 ± 1.0	1.6 ± 0.7	1.3 ± 0.5	2.7 ± 0.6
	Lamina propria	3.0 ± 2.2	7.2 ± 1.1	9.5 ± 2.5	15.1 ± 1.9	17.2 ± 2.0