Annelies P Vandekerckhove
email: annelies.vandekerckhove@ugent.be

S Glorieux

A C Gryspeerdt

L Steukers

J Van Doorsselaere

N Osterrieder

G R Van De Walle

H J Nauwynck

Equine alphaherpesviruses (EHV-1 and EHV-4) differ in their efficiency to infect mononuclear cells during early steps of infection in nasal mucosal explants

Keywords: EHV-4, hampered spread, basement membrane barrier, mononuclear cells

Equine herpesvirus type 1 (EHV-1) replicates extensively in the epithelium of the upper respiratory tract, after which it can spread throughout the body via a cell-associated viremia in mononuclear leukocytes reaching the pregnant uterus and central nervous system. In a previous study, we were able to mimic the in vivo situation in an in vitro respiratory mucosal explant system. A plaquewise spread of EHV-1 was observed in the epithelial cells, whereas in the connective tissue below the basement membrane (BM), EHV-1-infected mononuclear leukocytes were noticed. Equine herpesvirus type 4 (EHV-4), a close relative of EHV-1, can also cause mild respiratory disease, but a cell-associated viremia in leukocytes is scarce and secondary symptoms are rarely observed. Based on this striking difference in pathogenicity, we aimed to evaluate how EHV-4 behaves in equine mucosal explants. Upon inoculation of equine mucosal explants with the EHV-4 strains VLS 829, EQ 1 012 and V01-3-13, replication of EHV-4 in epithelial cells was evidenced by the presence of viral plaques in the epithelium.

Interestingly, EHV-4-infected mononuclear leukocytes in the connective tissue below the BM were extremely rare and were only present for one of the three strains. The inefficient capacity of EHV-4 to infect mononuclear cells explains in part the rarity of EHV-4-induced viremia, and subsequently, the rarity of EHV-4-induced abortion or EHM.

Introduction

At first, the equine alphaherpesviruses equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4) were considered as 2 subtypes of the same virus, designated EHV-1 subtype 1 and EHV-1 subtype 2 respectively [START_REF] Sabine | Differentiation of sub-types of equine herpesvirus 1 by restriction endonuclease analysis[END_REF][START_REF] Studdert | Differentiation of respiratory and abortigenic isolates of equine herpesvirus 1 by restriction endonucleases[END_REF]. Additional sequence information confirmed that EHV-1 and EHV-4 are actually two closely related, but distinct viruses, with an amino acid (aa) sequence identity ranging between 54.9% and 96.4% [START_REF] Telford | The DNA sequence of equine herpesvirus-4[END_REF]. To date, EHV-1 and EHV-4 are both classified as members in the order Herpesvirales, subfamily Alphaherpesvirinae, genus Varicellovirus [START_REF] Davison | The order Herpesvirales[END_REF].

EHV-1 and EHV-4 are major causative agents of respiratory disease in the horse [START_REF] Allen | Molecular epizootiology, pathogenesis, and prophylaxis of equine herpesvirus-1 infections[END_REF][START_REF] Crabb | Equine herpesvirus 4 (equine rhinopneumonitis virus) and 1 (equine abortion virus)[END_REF][START_REF] Reed | Equine herpesvirus 1 and 4[END_REF]. Initial infection starts with replication of the virus in epithelial cells lining the upper respiratory tract (URT). EHV-1 does not only have a tropism for epithelial cells, but also targets mononuclear leukocytes, misusing these cells for viral transportation through the basement membrane (BM) barrier [START_REF] Kydd | Distribution of equid herpesvirus-1 (EHV-1) in the respiratory tract associated lymphoid tissue: Implications for cellular immunity[END_REF][START_REF] Gryspeerdt | Differences in replication kinetics and cell tropism between neurovirulent and non-neurovirulent EHV-1 strains during the acute phase of infection in horses[END_REF]. Indeed, we showed in a previous study that EHV-1-induced plaques cannot breach the BM, but that EHV-1 penetrates the deeper tissues of the respiratory tract in a more discrete manner, using migrating individual mononuclear cells as a Trojan horse [START_REF] Vandekerckhove | Replication kinetics of neurovirulent versus non-neurovirulent equine herpesvirus type 1 strains in equine nasal mucosal explants[END_REF]. Carried by these mononuclear leukocytes, EHV-1 can easily spread through the body via a cell-associated viremia, reaching its target organs, the pregnant uterus and the central nervous system. At these secondary replication sites, EHV-1 replicates in endothelial cells, causing vasculitis, thrombosis and disseminated ischemic necrosis, resulting in abortion and nervous system disorders [START_REF] Smith | A comparison of equid herpesvirus-1 (EHV-1) vascular lesions in the early versus late pregnant equine uterus[END_REF][START_REF] Wilson | Equine herpesvirus 1 myeloencephalopathy[END_REF][START_REF] Smith | A study of the pathogenesis of equid herpesvirus-1 (EHV-1) abortion by DNA in-situ hybridization[END_REF]2001;[START_REF] Reed | Equine herpesvirus 1 and 4[END_REF][START_REF] Brosnahan | Equine Herpesvirus-1: A review and update[END_REF].

In contrast, EHV-4 infection remains mostly restricted to the URT and a cell-associated viremia, following primary replication in the URT is very rare [START_REF] Patel | Variation in cellular tropism between isolates of equine herpesvirus-1 in foals[END_REF][START_REF] Van Maanen | Equine herpesvirus 1 and 4 infections: an update[END_REF] [START_REF] Van Maanen | Equine herpesvirus 1 and 4 infections: an update[END_REF][START_REF] Patel | Equine herpesviruses 1 (EHV-1) and 4 (EHV-4)epidemiology, disease and immunoprophylaxis: a brief review[END_REF]. What determines this difference in pathogenic potential between EHV-1 and EHV-4 is unknown up till now. Strikingly, pathogenicity seems to be correlated with host cell range. EHV-1 has a broad host range and can replicate in equine, human, mouse, monkey, hamster, rabbit, pig, bovine, canine, feline and even avian cells [START_REF] Studdert | Equine herpesviruses: on the differentiation of respiratory from foetal strains of type 1[END_REF][START_REF] Trapp | Potential of equine herpesvirus 1 as a vector for immunization[END_REF], whilst EHV-4 replication seems to be mainly restricted to equine cells. Furthermore, EHV-1 can infect mice and cause respiratory disease while this is not the case for EHV-4 [START_REF] Awan | The pathogenesis of equine herpesvirus type 1 in the mouse: a new model for studying host responses to the infection[END_REF][START_REF] Azmi | Interactions between equine herpesvirus 1 and equine herpesvirus 4: T cell responses in a murine infection model[END_REF].

In this study, we aimed to evaluate how EHV-4 interacts with the respiratory mucosa upon inoculation of equine respiratory mucosal explants and we wanted to compare these replication kinetics with those of its close relative, EHV-1.

Materials and Methods

Donor horses

Material from slaughter horses was used to obtain nasal explants. Horses negative for nasal/ocular discharge and lung pathology were selected. All horses were between 5 and 7 years old, as determined by inspection of dental incisive architecture [START_REF] Muylle | Ageing horses by an examination of their incisor teeth: An (im)possible task?[END_REF]. A complement-dependent seroneutralization (SN)-test was performed on the serum of all horses and EHV-specific antibody titres ranged between 24 and 96. Nasal explants of at least three individual horses were used to evaluate replication kinetics of several EHV-1 and EHV-4 isolates.

A c c e p t e d M a n u s c r i p t 5

Cultivation of the nasal mucosal explants

The cultivation of nasal mucosal explants was performed exactly as previously described [START_REF] Vandekerckhove | In vitro culture of equine respiratory mucosa explants[END_REF]. In brief, immediately after slaughter, the head was removed from the carcass and longitudinally sawn into 2 equal sections. et al., 2007). Strain EQ 1 012 was obtained from Intervet [START_REF] Heldens | Clinival and virological evaluation of the efficacy of an inactivated EHV-1 and EHV-4 whole virus vaccine (Duvaxyn EHV-1,4). Vaccination/challenge experiments in foals and pregnant mares[END_REF] and strain V01-3-13 was isolated from a horse with respiratory disease and obtained from the Institut für Virologie in Berlin (kind gift of Dr. K. Borchers).

The EHV-1 isolate 03P37 was isolated from peripheral blood mononuclear cells (PBMC) of a paralytic horse in 2003 and typed as neurovirulent by the Animal Health Trust in the United Kingdom [START_REF] Nugent | Analysis of equid herpesvirus 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neurovirulent versus nonneurovirulent disease outbreaks[END_REF]. EHV-1 strain RacL11 was isolated in the late 1950s from an aborted foal and exhibits high virulence in the natural host and laboratory animals [START_REF] Reczko | Uber die feinsruktur eines vom pferd isolierten virus den herpesgruppe. (kurze mitteilung)[END_REF][START_REF] Mayr | Untersuchungen zur Entwicklung eines Lebendimpfstoffes gegen die Rhinopneumonitis (Stutenabort) der Pferde[END_REF].

Inoculation of the nasal mucosal explants

All explants were inoculated after 24 h of culture. Inoculation took place by immersion of the explant in 1 ml of inoculum, containing 10 6.5 TCID 50 of EHV-1 or EHV-4 for 1 h at 37 °C and 5 % CO 2 . After incubation, explants were washed twice with warm medium and transferred back to their gauze. At several time points post inoculation (pi), explants were collected, embedded in methylcellulose medium (Methocel ® MC, Sigma-Aldrich) and frozen at -70 °C.

Nasal explants of three horses were collected. Of each horse, several explants were made and inoculated with different EHV-1 isolates (03P37, parental RacL11) and EHV-4 isolates (VLS 829, EQ 1 012, V01-3-13), and collected at different time points pi (0, 24, 48 and 72 hpi).

Plaque analysis and quantification of individual infected cells

At 0, 24, 48 and 72 hpi, one hundred consecutive cryosections of 20 µm were made of the frozen explants and the cryosections were fixed in methanol for 20 min (-20 °C, 100 %).

Subsequently, the basement membrane (BM) of the tissues was stained with monoclonal mouse anti-collagen VII antibodies (Sigma-Aldrich), followed by secondary Texas Red ® - This polyclonal antibody against EHV-1 shows cross-reactivity with EHV-4 and was therefore also used for staining of EHV-4. Antibodies were incubated for 1 h at 37 °C and 5 % CO 2 . Finally, cryosections were washed three times in PBS and mounted with glycerin-DABCO (Janssen Chimica, Beerse, Belgium). To reproducibly analyse the replication characteristics of several EHV-1 isolates, we used a system set up by [START_REF] Glorieux | Different replication characteristics of historical pseudorabies virus strains in porcine respiratory nasal mucosa explants[END_REF] and optimized by [START_REF] Vandekerckhove | Replication kinetics of neurovirulent versus non-neurovirulent equine herpesvirus type 1 strains in equine nasal mucosal explants[END_REF]. Briefly, penetration of the virus through the BM was inspected and virus plaque latitudes were measured in one hundred consecutive cryosections using the ImageJ 1.

Statistical analysis

The data were processed by the SPSS software (SPSS) for analysis of variance (ANOVA).

The data are presented as means + standard deviations. Results with P values of ≤ 0.05 were considered significant.

A c c e p t e d M a n u s c r i p t 8

Results

EHV-4 strains replicate plaquewise in the epithelial cells of respiratory mucosal explants but are severely impaired in infecting mononuclear cells

Replication kinetics of several EHV-4 strains were evaluated in our respiratory nasal mucosal explant system. To this end, nasal explants of three individual horses were inoculated with 10 6.5 TCID 50 /ml of the EHV-4 isolates VLS 829, EQ 1 012, V01-3-13 and of each animal and for every strain used, one explant was examined at each collected time point (0, 24, 48 and 72 hpi).

No significant differences were seen in the results obtained with different EHV-4 strains, and hence, results will not be discussed separately for each strain. In general, viral epithelial plaques were visible starting from 24 hpi (Figure 1A) and their latitudes increased over time (Figure 1B).

These results are similar to what is normally observed upon inoculation of nasal mucosal explants with EHV-1. Indeed, when comparing the replication kinetics of the EHV-4 strains with the replication kinetics of the EHV-1 strains 03P37 and RacL11 (Figure 2), following observations were made. Viral plaques in the epithelium were seen from 24 hpi onwards. In addition, plaque latitude of these epithelial plaques increased over time, with significant differences between the EHV-1 plaque latitudes and the EHV-4 plaque latitudes at all observed time points (Figure 2B). The number of plaques and plaque latitudes increased significantly between 24 and 48 hpi, but no longer between 48 and 72 hpi (Figure 2A).

When evaluating infection below the BM, no individual infected cells were noticed at any observed time point pi for the EHV-4 strains VLS 829 and V01-3-13, and for strain EQ 1 012, only three EHV-4-infected cells were found below the BM in the explant of 1 horse at 72 hpi (Table I). As these individual infected cells below the BM were only noticed very rarely, this implies that there is hardly any passing of virus through the BM via individual infected Indeed, upon inoculation with strains 03P37 and RacL11, the presence of single infected cells in the underlying connective tissue was observed from 24 hpi onwards and the number of these single EHV-1-infected cells increased over time with an average of 21 ± 4.4 cells at 24 hpi, 135 ± 58.3 cells at 48 hpi, and 546 ± 94.3 cells at 72 hpi for strain 03P37, and an average of 6 ± 1.2 cells at 24 hpi, 78.7 ± 20.3 cells at 48 hpi, and 156.3 ± 16.3 at 72 hpi for strain RacL11 (Table I). EHV-1 strain 03P37 infected significantly more cells below the BM than EHV-1 strain RacL11.

In conclusion, these experiments indicate that (i) EHV-4 spreads plaquewise in epithelial cells of nasal mucosal explants, similar to EHV-1 albeit to a lesser extent, and (ii) EHV-4, in contrast to EHV-1, has no marked tropism for mononuclear leukocytes as single infected cells were rarely observed below the basement membrane at all time points pi.

Discussion

The subfamily of the Alphaherpesvirinae is, within the family of the Herpesviridae, an extensive subfamily containing numerous mammal, bird and reptile viruses [START_REF] Davison | The order Herpesvirales[END_REF]. Pseudorabies virus (PRV or Suid herpesvirus 1, SHV-1), bovine herpesvirus 1 (BoHV-1), equine herpesvirus 1 (EHV-1) and -4 (EHV-4) are clinically and economically the most relevant veterinary mammalian viruses belonging to the alphaherpesvirus subfamily.

Although these viruses share many similarities, differences between the viruses become apparent when considering the clinical picture and pathogenesis at the level of the upper respiratory tract (URT). While EHV-1 and EHV-4 cause mild respiratory problems, PRV and

BoHV-1 have the possibility to cause severe respiratory disease accompanied by pustular, necrotic lesions that progress to large haemorrhagic and ulcerated areas in the respiratory A c c e p t e d M a n u s c r i p t 10 mucosa (Allen andBryans, 1986, Crabb and[START_REF] Crabb | Equine herpesvirus 4 (equine rhinopneumonitis virus) and 1 (equine abortion virus)[END_REF][START_REF] Reed | Equine herpesvirus 1 and 4[END_REF][START_REF] Nauwynck | Cell biological and molecular characteristics of pseudorabies virus infections in cell cultures and in pigs with emphasis on the respiratory tract[END_REF][START_REF] Nandi | Bovine herpes virus infections in cattle[END_REF]. Besides EHV-1, PRV and BoHV-1 can easily spread through the body via a cell-associated viremia, whereas EHV-4-induced viremia is an exceptional event [START_REF] Nyaga | Pathogenesis of bovine herpesvirus 1 (BHV-1) infections: interactions of the virus with peripheral bovine blood cellular components[END_REF][START_REF] Patel | Variation in cellular tropism between isolates of equine herpesvirus-1 in foals[END_REF][START_REF] Wang | Flow cytometric analysis of porcine peripheral blood leukocytes infected with Aujeszky's disease virus[END_REF][START_REF] Nauwynck | Cell-free and cell-associated viremia in pigs after oronasal infection with Aujeszky's disease virus[END_REF][START_REF] Van Maanen | Equine herpesvirus 1 and 4 infections: an update[END_REF][START_REF] Patel | Equine herpesviruses 1 (EHV-1) and 4 (EHV-4)epidemiology, disease and immunoprophylaxis: a brief review[END_REF]. These differences can partially be explained by the diverse replication and invasion strategies of these viruses in the respiratory mucosa. By means of respiratory mucosal explants, it was found that PRV and BoHV-1 spread horizontally as well as vertically in a plaquewise manner in the epithelium, and that virus-induced plaques penetrated the BM barrier [START_REF] Glorieux | In vitro culture of porcine respiratory nasal mucosa explants for studying the interaction of porcine viruses with the respiratory tract[END_REF][START_REF] Steukers | Kinetics of BoHV-1 dissemination in an in vitro culture of bovine upper respiratory tract mucosa explants[END_REF]. In contrast, the spread of EHV-1-induced plaques was solely lateral, as plaques never crossed the BM at any time point pi, implying that the BM functions as an absolute barrier [START_REF] Gryspeerdt | Differences in replication kinetics and cell tropism between neurovirulent and non-neurovirulent EHV-1 strains during the acute phase of infection in horses[END_REF][START_REF] Vandekerckhove | Replication kinetics of neurovirulent versus non-neurovirulent equine herpesvirus type 1 strains in equine nasal mucosal explants[END_REF]. However, a marked tropism of EHV-1 for mononuclear leukocytes was observed and EHV-1-infected mononuclear leukocytes were present both in the epithelium and below the BM in the vicinity of virus-induced plaques in vitro but also in vivo [START_REF] Gryspeerdt | Differences in replication kinetics and cell tropism between neurovirulent and non-neurovirulent EHV-1 strains during the acute phase of infection in horses[END_REF]Vandekerckhove et al., 2010). Hence, these data suggest that closely related alphaherpesviruses use distinct ways of crossing the BM barrier and invading the respiratory mucosa. In the present study, replication of several EHV-4 strains was assessed for the first time in nasal mucosal explants of the horse. Thereby, it was found that EHV-4-induced virus plaques were present in the epithelium, starting from 24 hours post inoculation (hpi), although the lateral spread of EHV-4 was less extensive when compared to EHV-1. This is in accordance with the in vivo situation as respiratory signs following an infection with EHV-4 are less severe when compared to those following an EHV-1 infection [START_REF] Heldens | Clinival and virological evaluation of the efficacy of an inactivated EHV-1 and EHV-4 whole virus vaccine (Duvaxyn EHV-1,4). Vaccination/challenge experiments in foals and pregnant mares[END_REF][START_REF] Patel | Equid herpesvirus 1 (EHV-1) live vaccine strain C147: efficacy against respiratory diseases following EHV types 1 and 4 challenges[END_REF][START_REF] Patel | Equine herpesviruses 1 (EHV-1) and 4 (EHV-4)epidemiology, disease and immunoprophylaxis: a brief review[END_REF]. Remarkably, individual EHV-4-infected cells below the BM were only noticed very rarely and were only present for one of the three strains, implying that infection is not at all consistent [START_REF] Patel | Variation in cellular tropism between isolates of equine herpesvirus-1 in foals[END_REF]. This was in agreement with a study on kinetics of EHV-4 viral DNA load in peripheral blood leukocytes (PBL) from foals during a field outbreak of respiratory disease, showing that EHV-4 DNA loads in PBLs were low, which indicates that EHV-4 viremia is rare (Pusterla et al., 2005). Hence, the induction of abortion or nervous system disorders upon EHV-4 infection is also scarce [START_REF] Patel | Variation in cellular tropism between isolates of equine herpesvirus-1 in foals[END_REF].

Indeed, EHV-4 accounts for only 1 to 16% of herpesviral-induced abortions [START_REF] Ostlund | The equine herpesviruses[END_REF]Whitwell et al., 1995a, b) and apart from a single case report in Europe [START_REF] Meyer | Characterization of two equine herpesvirus (EHV) isolates associated with neurological disorders in horses[END_REF],

EHV-4 has not been found in association with herpesviral-induced neurologic disease [START_REF] Ostlund | The equine herpesviruses[END_REF]. This limited capability of EHV-4, in contrast to EHV-1, to initiate a cellassociated viremia might be due to an inefficient infection of these circulating mononuclear cells. Indeed, it has been previously shown that EHV-4 is incapable of efficiently infecting in vitro cultured peripheral blood mononuclear cells (PBMC) and moreover, it was found by using mutational analysis that an RSD motif, present in EHV-1 gD but not in EHV-4 gD, is an important determinant for proper infection of mononuclear cells in vitro (Van de [START_REF] Van De Walle | Equine herpesvirus 1 entry via endocytosis is facilitated by alphaV integrins and an RSD motif in glycoprotein D[END_REF][START_REF] Osterrieder | Pathogenic potential of equine herpesviruses: The importance of the mononuclear cell compartment in disease outcome[END_REF]. As our study indicates that some strains are incapable of infecting nasal mucosal mononuclear cells, while some strains are capable of infecting very few mononuclear cells, this might imply that the latter strains are the ones able to cause abortion or nervous system disorders in the field.

Another interesting observation in our present study was that the EHV-1 strain RacL11

showed a mild infection pattern in mucosal explants when compared to infection patterns obtained upon inoculation with several other EHV-1 strains such as Ab4, NY03 and different Belgian field isolates [START_REF] Vandekerckhove | Replication kinetics of neurovirulent versus non-neurovirulent equine herpesvirus type 1 strains in equine nasal mucosal explants[END_REF]. Epithelial plaques were smaller in size A c c e p t e d M a n u s c r i p t

and, when compared to other previously tested EHV-1 strains, only a fraction of individual infected cells below the BM was observed at 72 hpi [START_REF] Vandekerckhove | Replication kinetics of neurovirulent versus non-neurovirulent equine herpesvirus type 1 strains in equine nasal mucosal explants[END_REF]. This is in agreement with results obtained in in vivo studies comparing the pathogenic potential of EHV-1 strains in the horse. Upon infection with Ab4, horses displayed a significantly longer period of fever, a higher nasal virus excretion and a longer duration of viremia, when compared to RacL11 [START_REF] Goodman | A point mutation in a herpesvirus polymerase determines neuropathogenicity[END_REF]G. Van de Walle, personal communication).

This seems to indicate that RacL11 is an attenuated EHV-1 strain with less virulent potential in the horse than other EHV-1 strains, such as Ab4. In mice however, inoculation with RacL11 does result in severe symptoms such as a dramatic body weight loss within days of infection, a strong inflammatory infiltration in the lungs and even death [START_REF] Von Einem | In vitro and in vivo characterization of equine herpesvirus type 1 (EHV-1) mutants devoid of the viral chemokine-binding glycoprotein G (gG)[END_REF]. The murine model is frequently used as an in vivo model for investigation of virological and histological aspects of EHV-induced disease in the horse [START_REF] Awan | The pathogenesis of equine herpesvirus type 1 in the mouse: a new model for studying host responses to the infection[END_REF][START_REF] Walker | Comparison of the pathogenesis of acute equine herpesvirus 1 (EHV-1) infection in the horse and the mouse model : a review[END_REF]. However, to which extent valid comparisons and extrapolations can be made from mouse to horse remains highly questionable [START_REF] Walker | Comparison of the pathogenesis of acute equine herpesvirus 1 (EHV-1) infection in the horse and the mouse model : a review[END_REF]. Concerning the pathogenesis of equine herpesviruses, we conclude that our in vitro model appears to relate more closely to the in vivo situation in the natural host than the murine model. Indeed, the pathogenesis of an infection with EHV-1 strain RacL11 in the explant model closely resembles the situation in the natural host, whereas the clinical picture in the mouse model is much more dramatic [START_REF] Von Einem | In vitro and in vivo characterization of equine herpesvirus type 1 (EHV-1) mutants devoid of the viral chemokine-binding glycoprotein G (gG)[END_REF]. Also, EHV-4 replication in equine explants is similar to the in vivo situation, with a solid replication in epithelial cells but an incapability to infect mononuclear cells, whereas EHV-4 is incapable of infecting murine respiratory mucosa [START_REF] Awan | The pathogenesis of equine herpesvirus type 1 in the mouse: a new model for studying host responses to the infection[END_REF][START_REF] Azmi | Interactions between equine herpesvirus 1 and equine herpesvirus 4: T cell responses in a murine infection model[END_REF]. This indicates that our in vitro respiratory mucosal explant system is a valuable alternative model to provide novel information on pathogenesis or differences in pathogenic potential of different EHV-4 strains, in addition to the currently existing EHV-1/EHV-4 models. remains to be developed [START_REF] Patel | Equine herpesviruses 1 (EHV-1) and 4 (EHV-4)epidemiology, disease and immunoprophylaxis: a brief review[END_REF][START_REF] Kydd | The equine immune respons to equine herpesvirus-1: the virus and its vaccines[END_REF][START_REF] Rosas | Equine herpesvirus type 1 modified live virus vaccines: quo vaditis?[END_REF][START_REF] Brosnahan | Equine Herpesvirus-1: A review and update[END_REF]Pusterla et al., 2009). The effect of the existing vaccines, both inactivated and attenuated, seems to be limited to the alleviation of viral shedding and clinical signs. An adequate protection against viremia, and thus severe EHV-1-induced symptoms such as abortion and EHM, might be reached by impeding the virus to cross the basement membrane (BM) barrier via single mononuclear leukocytes. To this end, it is important to induce a local immunity. The best way to raise local immunity is to administer an attenuated EHV strain intranasally. For safety reasons, this EHV vaccine may no longer infect mononuclear cells. An attenuated EHV-4 strain cannot be used as a vaccine virus since a study in ponies has shown that the immunity induced by an EHV-4 infection is not crossprotective against an EHV-1 challenge [START_REF] Edington | One way protection between equid herpesvirus 1 and 4 in vivo[END_REF]. The most promising route for the design of more efficacious vaccines remains recombinant technology. For instance, when the glycoprotein(s) of EHV-1 responsible for efficient infection of the nasal mucosal mononuclear cells is (are) identified, genes coding for this (these) glycoproteins in EHV-4 could be introduced in the genome of EHV-1. This could result in a recombinant virus that is able to induce a powerful local immune response upon local intranasal administration, but is safe due to its inefficiency to infect mononuclear cells, markedly reducing its capability to initiate a cell-associated viremia.

Conclusion

The following conclusions can be drawn from the observations presented in this study. EHV-4 spreads plaquewise in epithelial cells of nasal mucosal explants, similar to EHV-1 albeit to a

  -mouse antibodies (Molecular Probes (Invitrogen)). In a second step, viral proteins were stained by incubation with biotinylated equine polyclonal anti-EHV-1 IgG (van der[START_REF] Van Der Meulen | A local epidemic of equine herpesvirus 1-induced neurological disorders in Belgium[END_REF], followed by streptavidin-FITC ® (Molecular Probes (Invitrogen)).

  s c r i p t immune cells, which is in striking contrast to what is normally observed for EHV-1 strains.

  virus through the BM via individual infected mononuclear leukocytes is an exceptional event in comparison with EHV-1. Also this observation can be linked to the in vivo pathogenesis, since a cell-associated viremia following an EHV-4-induced respiratory

  from researchers worldwide, an adequate vaccine preventing the severe EHV-1-induced symptoms such as abortion and equine herpes myeloencephalopathy (EHM)

  Remarkably, and in contrast to EHV-1, the EHV-4 strains in the present study have no marked tropism for mononuclear leukocytes as single EHV-4-infected cells are only rarely observed below the BM at all time points pi. Both these observations can be linked to the in vivo situation since respiratory signs following an infection with EHV-4 are less severe when compared to those following an EHV-1 infection, and since a cell-associated viremia following an EHV-4-induced respiratory infection is not at all consistent, and hence neither is the induction of abortion or nervous system disorders upon EHV-4 infection. The excellent correlation between the in vitro model and the in vivo situation in the horse indicates that our in vitro respiratory mucosal explant system is a valuable tool to provide novel information on pathogenesis or differences in pathogenic potential of different EHV-4 strains.

  Figure legends

Figure 2 .

 2 Figure 2. Evolution of plaque formation in the epithelial cells of nasal explants for EHV-1 and EHV-4. Equine respiratory mucosal explants were inoculated (10 6.5 TCID 50 /ml of

  Quantification of EHV-infected cells below the BM 502Virus designationNo of EHV-infected cells below the BM at … hpi

  Figure 1 Click here to download high resolution image

Figure 2

 2 Figure 2 Click here to download high resolution image

Acknowledgements

This research was supported by the Institute for the promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). We thank Carine Boone and Chantal Vanmaercke for their excellent technical assistance.

Conflict of interest statement

None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.


Airborne transmission of a highly pathogenic avian influenza virus strain H5N1 between groups of chickens quantified in an experimental setting

Keywords: Avian Influenza, H5N1, HPAI, transmission, chicken, airborne

Highly Pathogenic Avian Influenza (HPAI) is a devastating viral disease of poultry and quick control of outbreaks is vital. Airborne transmission has often been suggested as a route of transmission between flocks, but knowledge of the rate of transmission via this route is sparse. In the current study, we quantified the rate of airborne transmission of an HPAI H5N1 virus strain between chickens under experimental conditions. In addition, we quantified viral load in air and dust samples. Sixteen trials were done, comprising a total of 160 chickens housed in cages, with three treatment groups. The first group was inoculated with strain A/turkey/Turkey/1/2005 H5N1, the second and third group were not inoculated, but housed at 0.2 and 1.1m distance of the first group, respectively. Tracheal and cloacal swabs were collected daily of each chicken to monitor virus transmission. Air and dust samples were taken daily to quantify virus load in the immediate surroundings of the birds. Samples were tested by quantitative RRT-PCR and virus isolation. In 4 out of 16 trials virus was transmitted from the experimentally inoculated chickens to the non-inoculated chickens. The transmission rate was 0.13 and 0.10 new infections per infectious bird at 0.2m and 1.1 m, respectively. The difference between these estimates was, however, not significant. Two air samples tested positive in virus isolation, but none of these samples originated from the trials with successful transmission. Five dust samples were confirmed positive in virus isolation. The results of this study demonstrate that the rate of airborne transmission between chickens over short distances is low, suggesting that airborne transmission over a long distance is an unlikely route of spread. Whether or not this also applies to the field situation needs to be examined.

A c c e p t e d M a n u s c r i p t

Introduction

Highly Pathogenic Avian Influenza (HPAI), caused by avian influenza viruses of subtype H5 or H7, is one of the most important poultry diseases worldwide [START_REF] Alexander | An overview of the epidemiology of avian influenza[END_REF]. The infection spreads rapidly among chickens and between flocks, causing high mortality rates and severe economic losses. Moreover, HPAI virus strains have caused infections in humans (Kallthof et al., 2010) and are considered a risk for a human influenza pandemic. As a consequence, outbreaks of HPAI virus in poultry flocks need to be controlled quickly.

Control measures aiming to eliminate HPAI virus often include stamping out infected flocks, pre-emptive culling of flocks at risk to become infected, movement restrictions and bio-safety measures. These control measures may, however, not be sufficient to control a major epidemic in poultry dense regions [START_REF] Capua | The use of vaccination as an option for the control of avian influenza[END_REF][START_REF] Stegeman | Avian influenza A virus (H7N7) epidemic in The Netherlands in 2003: course of the epidemic and effectiveness of control measures[END_REF][START_REF] Boender | Risk maps for the spread of highly pathogenic avian influenza in poultry[END_REF]. Moreover, the costs associated with pre-emptive culling are high and the killing of large numbers of uninfected birds evokes ethical discussion in society. Consequently, improvement of the culling strategy, making it both more efficient and acceptable is needed.

To increase the effectiveness of control strategies, quantitative information of the possible routes of virus transmission between farms is essential. It has been demonstrated that the probability of between-flock virus transmission decreases with increasing distance between an infected and an uninfected flock [START_REF] Boender | Risk maps for the spread of highly pathogenic avian influenza in poultry[END_REF], but the underlying mechanism of transmission still shows considerable gaps. Several routes are considered to be important during AI epidemics, such as movements of visitors, materials, and fomites, but, as shown for some other viral diseases [START_REF] Gloster | Airborne spread of foot-and-mouth disease-model intercomparison[END_REF][START_REF] Otake | Long-distance airborne transport of infectious PRRSV and Mycoplasma hyopneumoniae from a swine population infected with multiple viral variants[END_REF][START_REF] Li | Occurrence and transmission of Newcastle disease virus aerosol originating from infected chickens under experimental conditions[END_REF] also airborne transmission has been hypothesised [START_REF] Chen | Ambient influenza and avian influenza virus during dust storm days and background days[END_REF][START_REF] Tsukamoto | Impact of different husbandry conditions on contact and airborne transmission of H5N1 highly pathogenic avian influenza virus to chickens[END_REF]Yee et al., 2009). Although some of the routes could be controlled by stringent hygienic [START_REF] Tsukamoto | Impact of different husbandry conditions on contact and airborne transmission of H5N1 highly pathogenic avian influenza virus to chickens[END_REF] demonstrated the possibility of airborne transmission of HPAI H5N1 virus in an isolator. Moreover, they showed that the likelihood of infection was dependent on the number of infectious birds. From their results, however, we cannot quantify the transmission probability. Moreover, they did not examine the presence of virus in the air.

In this paper we describe two experiments which enabled us to quantify the rate of airborne transmission of H5N1 virus strain between chickens at various distances. In addition, we quantified virus concentrations in air and dust samples in the immediate surroundings of the birds.

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Materials en methods

Animals

Embryonated eggs from White Leghorn chickens were purchased from a commercial AI-free poultry breeder farm and hatched at the Central Veterinary Institute (CVI), Lelystad, The Netherlands. After hatching, the chickens were housed in one room. At 5 weeks of age, the chickens were tested for the presence of antibodies against AI using a modified indirect double antibody sandwich (IDAS) nucleoprotein (NP)-blocking ELISA. The chickens were subsequently randomly divided in 4 groups, each housed in a separate room. Two experiments were carried out consecutively. In the first experiment 4 groups of 8 chickens were formed. Based on the results of this experiment, we increased the number of chickens in the second experiment in which 4 groups of 32 chickens were used. Feed and water were provided ad libitum.

Experimental design

Two experiments were carried out, each in 4 isolation rooms under BSL3+ conditions at the CVI . The lay-out of the rooms of the first experiment is shown in Figure 1, of the second experiment in Figure 2. The volume of the rooms was 22m 3 , which were ventilated 0.8/h. The temperature was kept at 20ºC and the relative humidity at 55%. The rooms contained two rows of 3 cages each. A cage was constructed of gaze and hardboard and had a size of 1x0.5m 2 . In the first experiment, 2 chickens were placed in the first cage of every row and 1 chicken in the second and third cage each. In the second experiment, 7 chickens were placed in the first and second cage and 2 chickens were placed in the third cage of every row.

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The distance between the first and second cage was 0.2m and between the second and third cage 0.4m. Chickens from the first cage of every row were inoculated at day 0. Chickens in the second and third cage were not inoculated. The air circulation in the rooms was determined with a smoke test. The rows were placed in favour of the transmission route. The experiments complied with the Dutch law on animal experiments and were reviewed by an ethical committee.

Inoculum

The HPAI virus strain A/turkey/ Turkey/1/2005 H5N1 (clade 2.2) was used as challenge strain for inoculation [START_REF] Londt | Pathogenenis of highly pathogenic avian influenza A/turkey/Turkey/1/2005 H5N1 in Pekin Ducks (Anas platyrhynchos) infected experimentally[END_REF][START_REF] Spekreijse | The effect of inoculation dose of a highly pathogenic avian influenza virus strains H5N1 on the infectiousness of chickens[END_REF]. The virus was grown in embryonated SPF eggs and vials with a known egg infectious dose (EID 50 ) titer were stored at -70ºC until use. On the day of challenge, one vial was thawed and diluted in 10-fold dilution steps in tryptose phosphate buffer (TPB) to obtain the necessary inoculation dose of 10 4 EID 50. In both experiments, the chickens were inoculated with 0.1 ml inoculum applied intra-nasally and 0.1 ml inoculum applied intra-tracheally using a blunt needle, according to standard protocol [START_REF] Van Der Goot | Quantification of the effect of vaccination on transmission of avian influenza (H7N7) in chickens[END_REF][START_REF] Spekreijse | The effect of inoculation dose of a highly pathogenic avian influenza virus strains H5N1 on the infectiousness of chickens[END_REF].

Sampling procedures

At day 1 post-inoculation (p.i.), swabs from trachea and cloaca were collected from inoculated chickens, and at days 2 to 10, 14, 17 and 21 p.i., from all chickens. The noninoculated chickens were sampled first, and between the rooms clothes and gloves of the animal handlers were changed. The swabs were put in 2 ml of 2.95% TPB with 5 x 10 3 IU of penicillin-sodium and 5 mg streptomycin per ml and stored at -70 o C until analysed.

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Serum blood samples were taken from the ulnar vein 7 days before and at days 7, 14 and 21 after inoculation from all chickens. The samples were stored at -20 o C until analysed.

In two of four rooms air samples were taken and in the remaining 2 rooms dust was sampled. Dust and air samples were collected from day 1 to day 10 p.i.

Air samples were taken with an MD8 air-scan air sampling device (Sartorius, Nieuwegein, The Netherlands) using sterile gelatine filters of 80mm diameter and 3μm pore size. Samples were taken at an air speed of 8m 3 /h for 10 minutes, according to the manual of the manufacturer 1 . In both rooms 2 samples were taken; one above the first cage of one row, another above the second cage of the second row. After sampling, the gelatine filters were dissolved in 10 ml of 2.95% TPB with 5 x 10 3 IU of penicillin-sodium and 5 mg streptomycin per ml at a temperature of 37 0 C. Dissolved filter solutions were stored at -70 o C until analysed.

Dust samples were taken using electrostatic dust cloths (Swiffer, Procter and Gamble, U.S.) that were placed in a Petri disk. Dust was sampled for 24 hours. In both rooms 4 samples were taken; two per row. The Petri disks were placed on both sides of the second cage. The dust cloths were put in 10 ml of 2.95% TPB with 5 x 10 3 IU of penicillin-sodium and 5 mg streptomycin per ml and stored at -70 o C until analysed.

The experiments were terminated 21 days p.i. by euthanizing surviving birds with an intracardiac injection of T-61.

RNA isolation and quantitative real-time reverse transcriptase PCR (RT-qPCR)

RNA isolation was performed with the MagNA Pure LC 2.0 instrument (Roche Applied Science, Mannheim, Germany) with the MagNA Pure LC total Nucleic Acid Isolation Kit (Roche Applied Science, Mannheim, Germany). The viral RNA was isolated 1 Jaschhof, H. Sammlung von virusaerosolenvergleichende untersuchungen zur effektivität von gelatinemembranfiltern, schlitzsammler und impinger. Biotec. October 1992 against known virus titers. Titers of the samples were expressed as EID 50 equivalents.

Laboratory tests

Sera were incubated for 30min at 56 ºC. A modified indirect double antibody sandwich (IDAS) nucleoprotein (NP)-blocking ELISA that detects antibodies against the nucleoprotein of influenza A was performed as described by de Boer et al. (1990).

For virus isolation, per swab three embryonated SPF chicken eggs incubated for 9 days were inoculated with 0.2 ml swab fluid per egg. After 72 h the allantoic fluid was harvested and a standard hemagglutination assay (HA) with chicken red blood cells was performed [START_REF] Oie | Avian influenza. In: Manual of diagnostic tests and vaccines for terrestrial animals 2008[END_REF]. When at least one egg was positive in HA the swab was considered to be positive. The effect of the gelatine filters on the concentration of virus particles was determined.

The effect of the gelatine filter on the concentration of HPAI virus

Gelatine filters were placed in Petri dishes and inoculated either with 1 ml of 10 3 , 10 4 or 10 5 EID 50 /ml. Two gelatine filters per dose were used. The first filter per dose was dissolved in 9 ml of PBS (at 37 degrees) after 10 minutes of incubation. The second filter per dose was dissolved in 9 ml of PBS (at 37 degrees) after 30 minutes of incubation. This number of minutes was considered to be representative for the time between sampling and processing in the lab. As control, the virus stock was treated similarly and dissolved in the same volume of medium. Virus concentration was determined by RNA isolation and RT-qPCR.

Data analysis

Airborne transmission was based on the number of infected non-inoculated chickens.

A chicken was considered infected if it met one or more of the following criteria: the occurrence of HPAI-like symptoms, a positive RT-qPCR, or both. Positive RT-qPCR results from chickens that did not die were confirmed by virus isolation.

The day of infection of the non-inoculated chickens was defined as the first day of virus excretion minus a one day latent period [START_REF] Spekreijse | The effect of inoculation dose of a highly pathogenic avian influenza virus strains H5N1 on the infectiousness of chickens[END_REF]. Non-inoculated chickens got infected either through air by the inoculated chickens, through air by infected non-inoculated chickens from another cage, or non-inoculated chickens were contact exposed by shedding cage mates.

A generalized linear model (GLM) assuming a stochastic SIR (susceptible-infectiousremoved) transmission process was used to estimate a separate transmission rate parameter (the average number of infections caused by one infectious bird per day) for every distance (Velthuis et al, 2003).

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The mean latent and infectious period and the total amount of virus detected in the samples of the inoculated chickens in the two experiments were compared using ANOVA. All statistical tests were performed assuming a 2-sided alternative hypothesis; p values smaller than 0.05 were considered significantly different. Analysis was performed using commercially available statistical software (SPSS 16.0; SPSS Inc., Chicago, Illinois).

Results

Infection of inoculated chickens

None of the inoculated chickens had pre-existing antibodies against avian influenza virus. In the first experiment 3 out of 16 inoculated chickens escaped infection and remained serologically negative; the other thirteen died. The mean latent period of the inoculated chickens was 1.1 day (95% confidence interval (C.I.): 0.9 -1.2 days). The mean infectious period of the inoculated chickens (days of virus shedding) was 1.6 day (95% C.I.: 1.4 -1.7 days). In the first experiment, the mean amount of virus shedding on day 1 p.i. was 10 3.5 EID 50 (95% C.I.: 10 2.5 -10 4.5 EID 50 ), and on day 2 p.i., 10 5.3 EID 50 (95% C.I.: 10 5.2 -10 5.5 EID 50 ).

In the second experiment all 56 inoculated chickens died. The mean latent period of these chickens was 1.1 day (95% C.I.: 1.0 -1.2 days). The mean infectious period was 1.7 day (95% C.I.: 1.6 -1.8). In the second experiment, the mean shedding of virus on day 1 p.i. was 10 4.3 (95% C.I.: 10 3.8 -10 4.8 EID 50 ), and on day 2 p.i., 10 6.3 EID 50 (95% C.I.: 10 5.3 -10 6.9 EID 50 ). The mean latent period, the mean infectious period and the total amount of virus shedding did not differ significantly between the two experiments.

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Infection of non-inoculated chickens

None of the non-inoculated chickens had pre-existing antibodies against AI. Chickens that died during the experiments showed AI-like symptoms, and had one or more positive RT-qPCR results for the tracheal and/or cloacal swabs. The most common AI-like symptoms were loss of appetite, depression and conjunctivitis.

In the first experiment none of the non-inoculated chickens became infected, showed clinical signs of infection or developed detectable amounts of antibodies. In the second experiment, 20 out of 72 non-inoculated chickens got infected. In total 17 of these 20 infected chickens died from infection. The infected chickens originated from 7 cages, 16 birds were located in cage 2, and 4 birds in cage 3. Based on the time of infection, we concluded that 6 of these 20 birds became infected by the inoculated chickens and 14 by either their cage mates or by "air" with virus shed by infectious chickens from another cage. Three non-inoculated infected chickens, one from a second cage and two from a third cage, tested positive once, both in the RT-qPCR and virus isolation, but did not die from infection nor seroconverted. From the moment of infection we derived that 2 birds became infected by the inoculated chickens and 1 became infected from infected chickens from another cage.

In 5 rows, the virus did not transmit to the non-inoculated chickens. In 2 rows, the inoculated chickens succeeded in transmission to the non-inoculated chickens in both cages.

The distribution of the infected chickens over the different isolation rooms and rows are summarized in Figure 3.

Virus detection in air and dust samples

A c c e p t e d M a n u s c r i p t

No effect of the gelatine filters was seen on the concentration of HPAI virus (Table 1) and processing filters after 30 minutes did not reduce the quantities measured in RT-qPCR.

In the first experiment, 2 air samples tested positive in RT-qPCR and virus isolation. On day 2 p.i., one air sample tested 10 1.6 EID 50 , and on day 3 p.i., one air sample tested 10 1.3 EID 50 . Three dust samples tested positive in RT-qPCR and virus isolation. On day 1 p.i., one dust sample tested 10 1.6 EID 50 , and on day 2 p.i., one dust sample tested 10 1.7 EID 50 , and on day 4 p.i., one dust sample tested 10 2.4 EID 50 .

In the second experiment, on day 2 and day 3 dust samples tested positive in RT-qPCR and virus isolation (10 3.9 and 10 2.2 EID 50 , respectively), but none of the air samples tested positive.

Quantification of transmission parameters

In the first experiment, no transmission of virus from the inoculated chickens to noninoculated chickens occurred. In the second experiment transmission did occur and combined with the first experiment, the transmission rate parameters for the various distances were calculated. The transmission parameter for the inoculated chickens to the non-inoculated chickens at a distance of 0.2m was estimated at 0.13 new infections per infectious chicken per day (95% C.I.: 0.01 -2.73), for the distance of 0.4m between the inoculated and noninoculated chickens at 0.21/day (95% C.I.: 0 -9.31), and for the distance 1.1m at 0.10/day (95% C.I.: 0.02 -0.40). The estimates did not differ significantly for the various distances.

The transmission rate parameter for directly exposed chickens was estimated at 1.43/day (95% C.I.: 0.27 -7.56). This parameter differed significantly from the combined transmission rate parameter (0.12/day; 95% C.I.: 0.06 -0.26) of the non-inoculated chickens (p < 0.05).

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Discussion

The aim of this study was to quantify airborne transmission of an HPAI H5N1 virus strain between chickens housed at various distances. Virus was transmitted to chickens exposed to inoculated chickens over distances of 0.2, 0.4 and 1.1m, but most exposed chickens escaped infection. No statistical difference was found between the rates of transmission over the above mentioned distances. Nevertheless, the combined β of airborne infection was significant lower than the transmission rate between chickens in the same pen (β = 1.43/day). Our findings indicate that airborne transmission over a short distance can occur, but that the rate at which it takes place is low.

Throughout the experiments, strict hygienic measures were taken to exclude transmission via other routes than by air, and the flow in the rooms was in favour of airborne transmission. Inoculated chickens shed virus in large quantities, but the amount of virus detected in air samples was low and most times undetectable. Moreover, the test results of the air samples did not correlate with the occurrence of airborne transmission. An explanation for the low proportion of virus positive air samples could be the length of the sampling time.

According to the manual of the manufacturer, samples were taken daily for 10', a protocol that had also been used by Weesendorp et al. (2008) to detect classical swine fever virus in ambient air. Moreover, chickens are exposed to virus containing particles much longer, which may have increased the probability of contracting the infection. Stochastic processes may also have occurred. Sampling for a longer time might have increased the number of positive air samples. Optimization of the sampling procedure may be an option for further research.

In the first experiment the amount of virus produced by the inoculated chickens was apparently not sufficient for transmission of virus over a distance of 0.2m. We therefore increased the number of inoculated chickens in the second experiment in order to increase the A c c e p t e d M a n u s c r i p t probability of airborne transmission [START_REF] Tsukamoto | Impact of different husbandry conditions on contact and airborne transmission of H5N1 highly pathogenic avian influenza virus to chickens[END_REF]. In the second experiment, virus shedding of the inoculated chickens resulted in airborne infection of 6 chickens in 6 cages.

The other 14 non-inoculated chickens that got infected were most likely infected either by cage mates (13 chickens) or by airborne-infected chickens from another cage (one chicken).

In the second experiment, virus was isolated from three chickens exposed to airborne transmission that did not die from infection nor did they seroconvert. This is remarkable, but we made a similar observation in a previous experiment [START_REF] Spekreijse | The effect of inoculation dose of a highly pathogenic avian influenza virus strains H5N1 on the infectiousness of chickens[END_REF]. In that experiment a small number of chickens in direct contact with experimentally infected chickens tested positive in RT-qPCR and virus isolation, but did not die from infection, nor seroconverted. These findings suggest that airborne introduction of a HPAI virus may not always result in a major outbreak. In the field, however, the amount of dust produced by large poultry flocks may be high, and dust-borne infection may be facilitated via artificial ventilation. Moreover, in an infected flock the number of infectious birds may be much higher. Therefore, it is difficult to extrapolate the experimental results to a field situation.

In studies using influenza virus in other host species, airborne transmission was observed. Stark et al. (1999) for example showed that airborne transmission of influenza virus between pigs was feasible, and [START_REF] Tellier | Aerosol transmission of influenza A virus: a review of new studies[END_REF] demonstrated airborne transmission of influenza virus among mice and guinea pigs. Our results showed that the probability of airborne transmission over a distance of 1m is low. Although in our study the relation between distance and probability of infection was not straightforward, as we did not find a significant difference between transmission over the various distances, our results can be used as input in transmission kernels to better understand the indirect transmission of HPAI virus [START_REF] Keeling | Models of foot-and-mouth disease[END_REF].

The possibility of long-range transport of other livestock viruses has been reported for other diseases such as foot-and-mouth disease [START_REF] Amaral Doel | Airborne transmission of foot-andmouth disease in pigs: evaluation and optimisation of instrumentation and techniques[END_REF][START_REF] Gloster | Airborne spread of foot-and-mouth disease-model intercomparison[END_REF], A c c e p t e d M a n u s c r i p t porcine reproductive and respiratory syndrome virus (PRRSV) [START_REF] Dee | Evidence of long distance airborne transport of porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae[END_REF] and Newcastle disease [START_REF] Li | Occurrence and transmission of Newcastle disease virus aerosol originating from infected chickens under experimental conditions[END_REF]. This long-range transport was linked to aerosol transmission and meteorological data, but not with the dispersion of dust, suggesting at least air-born transmission. Our findings of transmission via air seems to be consistent with these field observations.

In our experiments, dust samples tested positive, although no clear link between transmission and positive dust samples was demonstrated. Infectious particles in dust were previously demonstrated by [START_REF] Sedlmaier | Generation of avian influenza virus (AIV) contaminated fecal fine particulate matter (PM 2.5): genome and infectivity detection and calculation of immission[END_REF]) and, moreover, Chen et al. (2010) suggested the possibility of long-range transport of influenza virus through air by dust storms as the attachment of viruses to dust particles could increase their chances of survival. [START_REF] Pitkin | Use of a production region model to assess the airborne spread of porcine reproductive and respiratory syndrome virus[END_REF] confirmed in a production region model that the use of air filtration under controlled field conditions could significantly reduce airborne transmission of PRRSV between two pig populations. Their results could be useful for the implications of filter systems in the field. Unfortunately, the amount of dust produced by flocks of poultry exceeds the dust produced by pigs and, therefore, it is questionable if the filter system could be implicated easily in poultry houses. To substantiate the hypothesis of long-range dust-borne infection it would be necessary to quantify the amount of dust-borne virus produced by an infected flock and combine that with the dispersion pattern of dust emitted by that flock. This field research can, however, only be done with other viruses than AI, and whether or not this applies to AI should then be evaluated. Nevertheless, our experimental work showed that transmission via air is possible and our estimates may be of value for models used to simulate between flock transmission.

  s c r i p t measures, prevention of virus introduction via airborne route seems hardly feasible in commercial poultry industry. It is therefore important to establish the contribution of airborne infection in the between-farm spread.During an AI epidemic it is difficult to quantify the rate of airborne virus transmission between farms. The rate at which such an epidemic evolves, the need for immediate implementation of control measures and the presence of other routes of transmission that can act as confounding factors hamper a thorough investigation during epidemic episodes. An alternative way to quantify airborne transmission is by carrying out animal experiments. In experiments the occurrence of airborne transmission can be established and the relation between distance to an infectious bird and probability of infection can be quantified in the absence of confounding factors.

  A c c e p t e d M a n u s c r i p t from 200μl of swab fluid or filter solution according to the manufacturer"s instructions. The nucleic acids were collected in elution buffer and stored at -70 o C or directly processed for the quantitative real-time reverse transcriptase PCR (RT-qPCR). The RT-qPCR and data analysis were performed using the MX4000 Quantitative PCR system (Stratagene) with version 4.20 software. We used 5μl of the elution buffer with extracted RNA for RT-qPCR as described in van der Goot (2008) to detect the matrix gene of the influenza virus. The viral RNA concentration of each sample could be calculated using a calibration curve of serial dilutions of a standard batch of the virus with a known EID 50 titer. Dilutions of the standard batch were run along with the unknown samples. Quantification of the viral concentration in each sample was based on the calibration curve generated by plotting the cycle threshold value (C t -value)

  A c c e p t e d M a n u s c r i p t

Figure 1 .

 1 Figure 1. Lay-out of the isolation room of the first experiment.represents the inoculated

Figure 2 .

 2 Figure 2. Lay-out of the isolation room of the second experiment.represents the

Figure 3 .

 3 Figure 3. Summary of the RT-qPCR results of the swabs and mortality data of the second

Table 1 :

 1 Effect of gelatine filters on the concentration of HPAI virus determined by RT-qPCR

	Dose groups	Time		C t value		Titer
	(EID 50 /ml)	(min)		(cycles)	(log 10 EID 50 /ml)
			filter	virus stock	filter	virus stock
	10 3	10	36.6	36.8	1.9	1.9
		30	36.6	37.6	1.9	1.7
	10 4	10	29.8	30.0	3.8	3.7
		30	29.8	30.1	3.8	3.7
	10 5	10	26.0	26.6	4.7	4.8
		30	26.2	26.6	4.7	4.8

Acknowledgements

This work was financed by the framework "Fonds Economische Structuurverstrekking/ Economic Challenges Fund" (FES) financed by the Dutch Ministry of Economical affairs.


Jose L Gonzales
email: jose.gonzales@wur.nl

J A Van Der Goot

J A Stegeman

A R W Elbers

G Koch

Transmission between chickens of an H7N1 Low Pathogenic Avian Influenza virus isolated during the epidemic of 1999 in Italy

Keywords: Avian Influenza, LPAI, H7N1, Transmission, Transmission parameters, Reproduction ratio

The transmissibility of an H7N1 Low Pathogenic Avian Influenza (LPAI) virus isolated from a turkey flock during the large epidemic in Italy in 1999, was experimentally studied in chickens. Four group transmission experiments were performed. Infection and transmission were monitored by means of virus isolation on swab samples and antibody detection in serum samples. From the results of these groups, we estimated the mean infectious period at 7.7 (6.7 -8.7) days, the transmission rate parameter at 0.49 (0.30 -0.75) infections per infectious chicken per day and the basic reproduction ratio at 3.8 (1.3 -6.3). These estimates can be used for the development of surveillance and control programmes of LPAI in poultry.

Introduction

Highly pathogenic Avian influenza (HPAI) is caused by AI viruses of H7 or H5 subtypes, that have evolved from preceding Low Pathogenic Avian Influenza viruses (LPAIv) [START_REF] Banks | Changes in the haemagglutinin and the neuraminidase genes prior to the emergence of highly pathogenic H7N1 avian influenza viruses in Italy[END_REF][START_REF] Capua | The avian influenza epidemic in Italy, 1999-2000[END_REF][START_REF] Defra | Highly pathogenic avian influenza -H7N7, Oxfordshire[END_REF][START_REF] Garcia | Heterogeneity in the haemagglutinin gene and emergence of the highly pathogenic phenotype among recent H5N2 avian influenza viruses from Mexico[END_REF]. The H7N1 LPAI epidemic in Italy in 1999, which preceded the HPAI (1999HPAI ( -2000) ) epidemic, is one of the largest LPAI epidemics reported in a western country. In the absence of control measures -no compulsory regulations for control of LPAI outbreaks were in place -a total of 199 LPAI infected poultry farms were detected before the HPAI virus (HPAIv) took over [START_REF] Busani | Risk factors for highly pathogenic H7N1 avian influenza virus infection in poultry during the 1999-2000 epidemic in Italy[END_REF][START_REF] Capua | The avian influenza epidemic in Italy, 1999-2000[END_REF]. Current knowledge does not allow to predict when an H5 or H7 LPAIv strain will mutate into a HPAIv strain. However, assuming that the molecular A c c e p t e d M a n u s c r i p t changes necessary for the virulence transformation occur at random, the probability that a LPAIv strain will mutate into a HPAIv strain will depend on the number of virus replicates, which in turn is associated with the number of birds acquiring infection. Hence, besides knowledge of the molecular biology of these viruses, quantitative knowledge of the transmission of LPAIv is important for the development of surveillance programmes that target early detection (see for example Graat et al., (2001)), hence reducing the probability of both undesired mutations and/or large epidemics.

To our knowledge, until now only one study quantifying LPAI virus transmission between chickens has been published in the peer reviewed literature -a chicken-derived H5N2 LPAI precursor virus of the 1983 HPAI epidemic in Pennsylvania ( Van der Goot et al., 2003) -and there is no information about the transmissibility between chickens of H7 LPAIv subtypes . The objective of this study was to characterize -by means of transmission experiments -the betweenchicken transmission of an H7N1 LPAIv isolated from turkey during the 1999 epidemic in Italy.

Material and methods

Virus and inoculation

The LPAIv used for this study was the H7N1 LPAI A/turkey/Italy/1067/99 virus isolate. Its intravenous pathogenicity index and amino acid sequence of the hemagglutinin gene (GenBank accession number AF364134) have been published elsewhere [START_REF] Banks | Changes in the haemagglutinin and the neuraminidase genes prior to the emergence of highly pathogenic H7N1 avian influenza viruses in Italy[END_REF][START_REF] Capua | The avian influenza epidemic in Italy, 1999-2000[END_REF]. Birds were inoculated both intranasally and intratracheally with 0.1 ml/route of inoculum containing 10 6 EID 50 (50% egg infectious dose)/ml.

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M a n u s c r i p t

Birds and experimental procedure

A total of two transmission experiments were performed and each experiment consisted of two replicates (here referred to as trials). A trial comprised 10 six-weekold specific pathogen free (SPF) white leghorn chickens (Charles Rivers, The Netherlands). Five of the 10 birds were inoculated and the remaining five kept as contacts. The experiments were carried out in the High Containment Unit at the Central Veterinary Institute of Wageningen UR in Lelystad using biosafety level 3 procedures. Birds were housed in different pens in the same experimental room. Next to each experimental pen, 2 pairs of birds, housed in separate smaller pens, were placed as sentinels to detect whether between-pen (indirect) transmission should be considered and monitor independence of the trials. Chickens were housed 1 week before challenge to allow adaptation and during this period they were tested for presence of antibodies against influenza A virus using an in-house competitive NP ELISA [START_REF] De Boer | An ELISA for detection of antibodies against influenza A nucleoprotein in humans and various animal species[END_REF] and for virus by virus isolation. The day of inoculation (day = 0), contacts were removed from their pen and placed back 24 hours later.

Trachea and cloaca swabs were collected daily from 1 to 10 days post inoculation (dpi) and then on 14, 17 and 21 dpi. Serum samples were taken at 7, 14 and 21 dpi.

All surviving birds were euthanized at 21dpi. All experiments complied with the Dutch Law on Animal Experiments and were reviewed by an ethical committee.

Trachea and cloaca swabs were used for virus isolation using SPF embryonated chicken eggs that were incubated for 9 days. Serum samples were tested for antibodies using the Hemagglutinin Inhibition test (HI) using 8 Hemagglutination units (HAU) of H7N1 as antigen. Diagnostic procedures were similar as those described by van der Goot et al. (2003). A bird was considered infected when it

Data analysis

The basic reproduction ratio (R 0 ), which is defined as the average number of new infections caused by one infectious individual in a susceptible population, was estimated by the final size (FS) and the generalized linear model (GLM) methods as described by van der Goot et al. (2003;2005). The latent period was estimated as the time (in days) from inoculation to the first day the inoculated chicken was positive to virus isolation. The infectious period was estimated by fitting a parametric survival regression model with a Weibull distribution. In some cases, infected birds were detected positive by virus isolation, late, for example at 10 dpi and were tested again at 14 dpi when they became negative and assumed to have recovered (Table 1). These cases were treated as interval censoring in the survival analysis. Survival analysis and the GLM method were carried out with the statistical software R (R Development Core Team, 2005).

Results

In the first experiment, all inoculated birds in both trials became infected and transmitted virus to 4 and 5 contact birds in the first and second trial respectively (Tables 1a and1b). In the second experiment (trials 3 and 4) all inoculated and contact birds became infected (Tables 1c and1d). No apparent clinical signs were observed in infected birds. The sentinels remained negative during the course of the experiment, which confirmed the independence of the trials.

A c c e p t e d M a n u s c r i p t

Inoculated birds were positive to virus isolation at 1 dpi. The estimated mean length of the infectious period (IP) was 7.7 (95% Confidence Interval (CI): 6.7 -8.7) days and the mean estimate of the transmission parameter β was equal to 0.49 (95% CI: 0.30 -0.75) infections/day per chicken. R 0 was estimated to be 3.8 (95% CI: 1.3 -6.3) by the GLM method and 4.0 (95% CI: 1.7 -11.0) by the FS method (Table 2).

Discussion

Herein the transmissibility in chickens of the H7N1 LPAIv, which caused a severe epidemic in poultry in Italy in 1999 and later evolved into a HPAI epidemic, was characterized. We have, in particular, estimated β and the IP. These parameters give an indication of how fast the virus would spread to another susceptible bird and how long an infected chicken would remain infectious. Due to, mainly, the subclinical presentation of LPAI infections, it is difficult to accurately follow the course of the infection in field conditions, and characterization of transmission in the field would be limited to estimates of the R 0 [START_REF] Comin | Estimate of basic reproduction number (R 0 ) of low pathogenicity avian influenza outbreaks using a bayesian approach[END_REF]. Our experimental approach allowed us to follow the course of the infection under controlled conditions and have objective estimates of the transmission parameters.

The mean estimates of the IP, β and R 0 in chickens for this H7N1 LPAIv are higher than those observed for an H5N2 LPAIv (van der Goot et al., 2003). This is remarkable because the latter strain was isolated from a chicken whereas our strain originated from turkey. Upon introduction, the virus here studied would reach its peak (highest prevalence of infectious animals) faster and at a higher prevalence than the H5N2 virus. Thus, it could be hypothesized that the infectivity of the flock would be higher, resulting in a higher probability of transmission (assuming similar contact rate) to other flocks. In this scenario, surveillance programmes would need to sample A c c e p t e d M a n u s c r i p t at a higher frequency if the objective is to detect in an early stage infection and to reduce the probability of between-flock transmissions.

Of the two methods used to estimate R 0 , the GLM method yielded a more precise estimate (Table 2). This is because the GLM method uses all the detailed (day to day) information about the time course of the infection chain, while the FS method uses only the total number of animals that got infected throughout the experiment. We also used the FS method, because with that method there is no dependency on whether or not there is a latency period, as intrinsically assumed in the GLM method.

In conclusion, this study revealed valuable information on transmission parameters for LPAI virus in chickens. Such information can be used to improve or develop surveillance programmes and control measures.

  s c r i p t scored positive in virus isolation or serology. A bird was considered infectious for the time it remained positive in virus isolation.

  Graat, E.A.M., de Jong, M.C.M., Frankena, K., Franken, P., 2001, Modelling the effect of surveillance programmes on spread of bovine herpesvirus 1 between certified cattle herds. Veterinary Microbiology 79, 193-208. R Development Core Team 2005. R: A Language and Evironment for Statistical Computing. ( Vienna, Austria: Foundation for Statistical Computing, 2005). van der Goot, J.A., De Jong, M.C.M., Koch, G., van Boven, M., 2003, Comparison of the transmission characteristics of low and high pathogenicity avian influenza A virus (H5N2). Epidemiology and Infection 131, 1003-1013. van der Goot, J.A., Koch, G., de Jong, M.C.M., van Boven, M., 2005, Quantification of the effect of vaccination on transmission of avian influenza (H7N7) in chickens PNAS 102 18141-18146.

Acknowledgement

The research was financially supported by EU contract QLK2-CT-2002-01454 AVIFLU and the Dutch Ministry of Economic Affairs, Agriculture and Innovation.

We thank Giovanni Cattoli (Instituto Zooprofilattico Sperimentale delle Venezie) for providing the virus for this study.

a +|+, +|-, -|+, -: virus isolation results of "trachea | cloaca" swab samples. The single "-" means that both type of samples were negative. b nd: test not done