Annick C Gryspeerdt

A P Vandekerckhove

B Garré

F Barbé

G R Van De Walle

H J Nauwynck

Van De

Walle Gerlinde
email: gerlinde.vandewalle@ugent.be

Differences in replication kinetics and cell tropism between neurovirulent and non-neurovirulent EHV1 strains during the acute phase of infection in

Keywords: EHV1, pathogenesis, strain differences, carrier cells

come

Differences in replication kinetics and cell tropism between neurovirulent and non-neurovirulent EHV1 strains during the acute phase of infection in horses

A c c e p t e d M a n u s c r i p t Introduction Infectious respiratory tract disease has been recognized as the cause of major health problems in horses. The most important respiratory pathogen is equine herpesvirus 1 (EHV1) and infections with this virus cause serious economic losses in the horse industry worldwide [START_REF] Allen | Molecular epizootiology, pathogenesis, and prophylaxis of equine herpesvirus-1 infections[END_REF][START_REF] Ostlund | The equine herpesviruses[END_REF]van Maanen, 2002). The upper respiratory tract is the first line of defence against respiratory pathogens and is also the primary replication site of EHV1, where it causes upper respiratory disorders (Kydd et al., 1994a, b). In addition, EHV1 can spread via infected blood leukocytes to internal organs, for example the pregnant uterus, causing symptoms such as abortion and neonatal foal death [START_REF] Allen | Molecular epizootiology, pathogenesis, and prophylaxis of equine herpesvirus-1 infections[END_REF].

EHV1 can also reach the central nervous system, where replication in endothelial cells results in severe nervous system disorders with frequent fatal outcome. Neurological disease in horses caused by infection with certain isolates of EHV1 is a severe condition which is poorly understood [START_REF] Mccartan | Clinical, serological and virological characteristics of an outbreak of paresis and neonatal foal disease due to equine herpesvirus-1 on a stud farm[END_REF][START_REF] Stierstorfer | Equine herpesvirus type 1 (EHV-1) myeloencephalopathy: a case report[END_REF]van der Meulen et al., 2003a). The pathogenesis for developing this devastating condition is largely unknown and full protection against these secondary and severe symptoms can not be obtained by vaccination with the currently available vaccines [START_REF] Heldens | Clinical and virological evaluation of the efficacy of an inactivated EHV1 and EHV4 whole virus vaccine (Duvaxyn EHV1,4). Vaccination/challenge experiments in foals and pregnant mares[END_REF]Goodman et al., 2006;van der Meulen et al., 2007). Therefore, an improvement of the existing vaccines and/or the development of alternative strategies for the prevention and treatment of EHV1-induced infections are necessary. Importantly, this implies a firm notice about the pathogenesis of EHV1 during the acute phase of infection.

It has been suggested that distinct isolates of EHV1, differing in pathogenic capacity, circulate in the field. [START_REF] Nugent | Analysis of equid herpesvirus 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus nonneuropathogenic disease outbreaks[END_REF] indicated that a single nucleotide polymorphism (SNP) in the DNA polymerase is strongly associated with neurological versus nonneurological disease outbreaks. Studies in naturally infected horses with nervous system disorders have revealed a more robust cell-associated viremia in horses infected with A c c e p t e d M a n u s c r i p t 4 neurovirulent isolates of EHV1, in contrast to horses infected with non-neurovirulent isolates [START_REF] Allen | Quantification by real-time PCR of the magnitude and duration of leucocyte-associated viraemia in horses infected with neuropathogenic vs. non-neuropathogenic strains of EHV-1[END_REF]. This was further confirmed by reverse genetic experiments of EHV1 strains with a sole mutation of the DNA polymerase SNP, where experimental inoculation with such strains altered neurologic disease in horses [START_REF] Goodman | A point mutation in a herpesvirus polymerase determines neuropathogenicity[END_REF][START_REF] Van De Walle | A Single-Nucleotide Polymorphism in a Herpesvirus DNA Polymerase Is Sufficient to Cause Lethal Neurological disease[END_REF]. Moreover, in vitro experiments with these strains revealed differences in leukocyte tropism which could explain the difference in clinical outcome [START_REF] Goodman | A point mutation in a herpesvirus polymerase determines neuropathogenicity[END_REF]. Still, the exact identity of carrier cells susceptible to EHV1 infection and hereby responsible for dissemination of infectious virus to sites of secondary replication, is a matter of debate. Blood samples from experimentally infected ponies were collected and examined for the presence of neurovirulent EHV1 [START_REF] Scott | In vivo harboring of equine herpesvirus-1 in leukocyte populations and subpopulations and their quantitation from experimentally infected ponies[END_REF]. Hereby, T-lymphocytes were found to be the most important cell fraction to harbor virus in vivo. However, no full characterization of these cells was performed. Different studies with in vitro EHV1-infected leukocytes have also been performed to determine the identity of these cells. Hereby, mainly monocytes were susceptible to infection with non-neurovirulent EHV1 (van der Meulen et al., 2000). After stimulation with mitogens, T-lymphocytes became more susceptible, followed by B-lymphocytes (van der Meulen et al., 2001). Despite these studies, information on possible differences between neurovirulent and non-neurovirulent EHV1 strains at the port of entry, and identity of carrier cells during viremia remains limited.

To investigate this, six Shetland ponies were experimentally inoculated with a neurovirulent EHV1 strain and six with a non-neurovirulent EHV1 strain. These animals were subsequently euthanized to collect different tissues of the upper respiratory tract. Tissues that were positive for EHV1 on titration were stained with markers for different cell types to determine and quantify the carrier cells of EHV1 in the upper respiratory tract. In addition, peripheral blood mononuclear cells (PBMC) were also collected to quantify and identify the infected cell type during cell-associated viremia.

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

Materials and methods

Horses

Twelve male Shetland ponies, between the age of 6 months and 2 years, were used in this study. They were housed inside isolated stables. They were fed daily with a commercial, complete feed. Drinking water and hay were supplied ad libitum.

Prior to the experiment, ponies were monitored for 4 weeks. Rectal temperatures were measured daily and complement-dependent seroneutralization (SN)-tests and immunoperoxidase monolayer assays (IPMA) were performed weekly to determine EHVspecific antibody titers.

Virus and inoculation

Ponies were divided into two groups of 6. For the first group the Belgian EHV1 strain 03P37, isolated from the blood of a paralytic horse in 2003, was used for inoculation [START_REF] Garre | Evaluation of orally administered valacyclovir in experimentally EHV1-infected ponies[END_REF]van der Meulen et al., 2003a). The second group was inoculated with the Belgian strain 97P70, which was isolated from an aborted fetus (van der [START_REF] Van Der Meulen | Absence of viral envelope proteins in equine herpesvirus 1-infected blood mononuclear cells during cellassociated viremia[END_REF]. The strains were typed in the DNA polymerase as D 752 and N 752 respectively in cooperation with 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 neuropathogenic versus nonneuropathogenic disease outbreaks[END_REF]. Based on their origin and genotyping, these strains will be referred as neurovirulent and non-neurovirulent in this paper. Virus stocks used for inoculation were at the 6 th passage; 2 passages in rabbit kidney cells (RK13) and 4 subsequent passages in equine embryonic lung cells (EEL). Both viruses had virtually identical single-step growth properties in RK13 cells for intra-and extracellular virus yields at all tested time points post infection (data not shown).

A c c e p t e d M a n u s c r i p t 6 Ponies were inoculated oronasally with 20 ml of a virus suspension containing 10 6.5 tissue culture infectious dose 50 (TCID 50 ). Ten ml of the virus suspension was administered intranasally (5 ml per nostril) using a thin probe and 10 ml was inoculated orally with a syringe. The virus titer was confirmed by titration of the inoculum.

Serological examination

For the seroneutralization (SN)-test, two-fold dilution series of the sera were prepared in MEM. Fifty micro liter of these serial dilutions were incubated for 23 hours (h) at 37°C with a fixed number of infectious virus (300 TCID 50 of EHV1 strain Arabica in 50 μl).

Hereafter, 25 µl of guinea pig complement was added. After 1 h of incubation, the mixture of serum, virus and complement was added to RK13 cells. Inoculated cultures were further incubated at 37°C in an atmosphere containing 5% CO 2 . After 7 days of incubation, the cultures were analysed for the presence of cytopathic effect. The neutralization titer was calculated as the reciprocal of the highest dilution of the serum that was able to completely block EHV1 infection in RK13 cells.

For the immunoperoxidase monolayer assay (IPMA), RK13 cells were inoculated with 10 3 TCID 50 of the EHV1 strain 97P70. After 28 h, cells were washed, dried at 37°C for 1 h and stored at -20°C until use. Plates were thawed and subsequently fixed with 4% paraformaldehyde and a solution containing 1% hydrogen peroxide in methanol. Following extensive washing, serial 2-fold dilutions of the sera were added and cells were incubated for 1 h at 37°C. Cells were incubated with peroxidase-labeled goat anti-horse antibodies (Jackson ImmunoResearch Laboratories Inc., PA, USA) and after 1 h, a substrate solution of 3-amino-9-ethylcarbazole in 0.05 M acetate buffer with 0.05% hydrogen was added to each well.

Following 20 minutes of incubation at 37°C, substrate solution was replaced with acetate buffer to block the enzymatic staining reaction. The IPMA titer was calculated as the

Clinical examination

Horses were monitored daily for clinical signs by physical examination and measurement of rectal temperatures. Fever was defined as a rectal temperature ≥ 38.5°C.

During clinical examination, nasal discharge (serous, mucous or purulent), lymph node swelling, tachypnea and coughing were evaluated. In addition, neurological exams were performed.

Virological examination

Nasopharyngeal mucus samples were taken daily until euthanasia. Immediately after collection, swabs were immersed in transport medium containing phosphate-buffered saline (PBS) supplemented with 10% fetal calf serum (FCS), 1000 U/ml penicillin, 1 mg/ml streptomycin and 0.5 mg/ml kanamycin. EHV1 in nasopharyngeal secretions was titrated on monolayers of RK13 cells, exactly as described previously (van der Meulen et al., 2003b).

On 1, 2, 3, 4, 5 and 7 days post inoculation, one pony of each group was euthanized with an overdose of Natriumpentobarbital ® (Kela, Hoogstraten, Belgium). After euthanasia, different tissue samples were collected from the upper respiratory tract. Tissues from slaughterhouse horses were collected and served as uninfected control samples. Per tissue, two equal squares of 1 cm 2 were collected from both left and right side of the head. From each side, one square was frozen immediately with (immunofluorescence) or without (virus titration) methylcellulose medium (Methocel ® MC, Sigma-Aldrich, St. Louis, MO, USA) at -70°C. To determine viral replication, 20% suspensions of collected tissues were made and A c c e p t e d M a n u s c r i p t 8 titrated on RK13 cells. Titers of infected tissues were determined after 7 days by means of Reed and Muench's formula [START_REF] Reed | A simple method of estimating fifty per cent endpoints[END_REF].

The experimental design was approved by the local ethical committee of the Faculty of Veterinary Medicine.

Quantification and characterization of individual infected cells in tissues of the upper respiratory tract

Immunofluorescent double stainings were used to quantify and characterize individual infected cells in tissues that were EHV1-positive after titration on RK13 cells. First, cryosections (16 µm) were incubated with either monoclonal antibody (mAb) HT23A, DH59B or 1.9/3.2 (VMRD Inc, Pullman, WA, USA) against CD5 (T-lymphocytes), CD172a (cells from the monocyte lineage, CML) or IgM (B-lymphocytes) respectively, and subsequently incubated with Texas Red ® -labeled goat anti-mouse Abs (Molecular Probes, Eugene, OR, USA). Epithelial cells were identified based on the pancytokeratin marker MNF116 (Abcam, Cambridge, UK) and/or morphological features. Second, viral EHV1 proteins were stained by incubation with biotinylated polyclonal horse anti-EHV1 IgG (van der Meulen et al., 2000), followed by streptavidin-FITC ® (Molecular Probes, Eugene, OR, USA). As controls, (i) stainings were performed on uninfected tissue and (ii) isotype control antibodies were included. All cryosections were analyzed using confocal microscopy (Leica TCS SP2 Laser scanning spectral confocal system, Leica Microsystems GmbH, Wetzlar, Germany). Number and latitude of EHV1-induced plaques were calculated using the software program ImageJ. Latitude was used to evaluate plaque size and therefore the degree of cell-tocell spread. In order to quantify infected cells in nasal septum, two main regions were taken into account, on the one hand regions with and on the other hand regions without plaques in the epithelium. These regions were then subdivided into three equal regions underneath the respectively). All regions were evaluated for the presence of plaques and/or individual infected cells. In addition, the total amount of infected cells (positive for staining with anti-EHV1 Abs), equine immune cells (positive for staining with the mAbs against immune cell markers) and infected equine immune cells (double positive) were determined.

Quantification and characterization of viremia

Heparinized blood samples were taken daily until euthanasia and peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation on Ficoll-Paque, according to manufacturer's instructions (Pharmacia Biotech AB, Uppsala, Sweden). To determine the magnitude of viremia, co-cultivation of PBMC on RK13 cells was performed as previously described (van der Meulen et al., 2003b). Remaining PBMC were resuspended in RPMI supplemented with 30% FCS and 20% Dimethyl Sulfoxide (DMSO) and frozen in liquid nitrogen for later examination to identify the nature of infected cells. Hereby, a double immunofluorescence staining was performed on acetone-fixed cell smears of PBMC. EHV1 expression was detected using a biotinylated polyclonal horse anti-EHV1 IgG (van der Meulen et al., 2003b), followed by streptavidin-FITC ® . The identity of infected cells was determined using the mAbs HT23A, DH59B or 1.9/3.2, as described above, followed by the incubation with Texas Red ® -labeled goat anti-mouse Abs. Samples were analyzed using confocal laser scanning microscopy.

Results

A c c e p t e d M a n u s c r i p t 10 EHV-status of the ponies before inoculation

The inclusion criterium for ponies used in this study was a complete absence of EHVspecific antibodies by either SN-test (<2) or IPMA (<10). In addition, during the 4-week observation period prior to the experiment, (i) no raise in temperature was noted, (ii) no EHVspecific antibodies were detected, and (iii) no virus was isolated from nasopharyngeal swabs.

The use of naïve horses is an important tool to minimize variation in clinical and virological outcome due to differences in immunological status [START_REF] Cornick | Safety and efficacy of a thymidine kinase negative equine herpesvirus-1 vaccine in young horses[END_REF][START_REF] Heldens | Clinical and virological evaluation of the efficacy of an inactivated EHV1 and EHV4 whole virus vaccine (Duvaxyn EHV1,4). Vaccination/challenge experiments in foals and pregnant mares[END_REF][START_REF] Matsumura | Lack of virulence of the murine fibroblast adapted strain, Kentucky A (KyA), of equine herpesvirus type 1 (EHV-1) in young horses[END_REF].

Experimental inoculation with either strain induces fever and upper respiratory tract disease in ponies

All ponies developed fever upon EHV1 inoculation with exception of the two ponies that were euthanized 24 hours post inoculation (hpi). Fever started at 48 hpi for the neurovirulent strain (5/5 animals) and at 36 (3/5 animals) or 48 hpi (2/5 animals) for the nonneurovirulent strain. High temperatures lasted for the complete observation period, with peak temperatures of 40.7°C for the neurovirulent strain and 40.1°C for the non-neurovirulent strain. Serous nasal discharge was noted after inoculation with both strains, starting at 1 day post inoculation (dpi). A transition to purulent nasal discharge was seen in both groups from 3 dpi. Breathing problems, consisting of tachypnea or coughing, were observed in two ponies of each group. Swelling of mandibular lymph nodes was observed in the majority of the ponies and occurred from 1 dpi in 2/6 animals inoculated with a neurovirulent strain and in 4/6 animals inoculated with a non-neurovirulent strain. Painful retropharyngeal lymph nodes were noted in 5/6 ponies inoculated with a non-neurovirulent strain, in contrast to only 2 ponies inoculated with a neurovirulent strain. None of the ponies showed neurological signs, irrespective of the strain used for inoculation. The latter is not too unexpected, as the A c c e p t e d M a n u s c r i p t 11 incubation period of EHV1-induced neurological disorders is normally 6 to 10 days [START_REF] Edington | Endothelial cell infection and thrombosis in paralysis caused by equid herpesvirus-1: equine stroke[END_REF][START_REF] Jackson | Equine herpesvirus 1 infection of horses: studies on the experimentally induced neurologic disease[END_REF][START_REF] Mumford | EHV1 and equine paresis[END_REF]. Moreover, nervous system disorders have been described to occur more frequently in older horses and certain breeds [START_REF] Garre | Evaluation of orally administered valacyclovir in experimentally EHV1-infected ponies[END_REF][START_REF] Goehring | Equine herpesvirus type 1-associated myeloencephalopathy in The Netherlands: a four-year retrospective study (1999-2003)[END_REF][START_REF] Greenwood | Clinical report of a paralytic syndrome affecting stallions, mares and foals on a thoroughbred studfarm[END_REF][START_REF] Jackson | Equine herpesvirus 1 infection of horses: studies on the experimentally induced neurologic disease[END_REF][START_REF] Mccartan | Clinical, serological and virological characteristics of an outbreak of paresis and neonatal foal disease due to equine herpesvirus-1 on a stud farm[END_REF].

Experimental inoculation with either strain results in nasal shedding and viral replication in the upper respiratory tract, but differences were observed in the preferential epithelial replication site between strains When evaluating nasal shedding, by titrations of nasal swab samples, five out of six ponies inoculated with the neurovirulent strain shed virus from 1 dpi, with a peak excretion at 2 dpi (Fig 1). Nasal shedding then slowly decreased until 7 dpi. For the non-neurovirulent strain, 6 out of 6 ponies shed virus at 1 dpi, reaching a peak at 2 dpi with remaining high titers induced plaques in the intermedial nasal mucosa at different time points pi for both strains. In the epithelium of the nasopharynx, plaques could be found for the non-neurovirulent strain from 2 till 7 dpi, with the exception of 4 dpi (Fig 3B). In contrast, no plaques were found for the neurovirulent strain in the nasopharyngeal epithelium, with the exception of one small plaque at 3 dpi (Fig 3B). Interestingly, plaques were not able to cross the BM at any time point pi, but single EHV1-infected cells could be noticed in the connective tissue from 1 dpi.

Taken together, these results show that EHV1 infection results in nasal shedding and replication in several tissues of the upper respiratory tract, irrespective of its neuropathogenic potential. However, whereas both types of EHV1 replicate equally in the epithelium of the nasal mucosa, replication in the nasopharynx was limited to non-neurovirulent EHV1.

Neurovirulent EHV1 uses mainly CD172a + cells as carrier cells in tissues of the upper respiratory tract, whereas non-neurovirulent EHV1 shows a tropism for both CD172a + cells and CD5 + cells

As mentioned before, EHV1-induced plaques never crossed the BM, irrespective of the strain used. However, individual infected cells were observed in the underlying connective tissue and the total number was 3-7 times lower with non-neurovirulent EHV1 compared to neurovirulent EHV1. To identify the nature of these cells, cryosections of tissues were made and double immunofluorescence stainings were performed.

Nasal septum. In the connective tissue of the nasal septum, no differences were observed in the total amount of infected cells present beneath epithelium with plaques or without plaques, with the exception of 5 and 7 dpi for the neurovirulent strain, where a Immunofluorescence stainings, using mAbs against immune cell surface markers showed that in all tissues of the upper respiratory tract, infected cells were largely CD172a + CML, followed by a slightly lower amount of infected CD5 + lymphocytes when the A c c e p t e d M a n u s c r i p t neurovirulent EHV1 strain was used for inoculation (Table I). These results were very similar to what was observed with the non-neurovirulent strain, although a more substantial amount of infected CD5 + lymphocytes was observed in these different tissues of the upper respiratory tract (Table I). IgM + lymphocytes were always present, but rarely infected with the exception of the mandibular (Table I) and retropharyngeal (data not shown) lymph nodes, were these cells were an important target cell type for both strains.

Ponies became viremic upon experimental inoculation with either strain, and CD172a + cells were the most important carrier cells in the blood

Transmission of EHV1 from PBMC to susceptible RK13 cells, as determined by cocultivation, was observed in 5 out of 6 ponies upon experimental inoculation with neurovirulent EHV1 (Fig 8). No differences in the amount of infected PBMC was noted, except for one pony, which showed a higher level of viremia at 4 and 5 dpi compared to the other infected ponies (Fig 8). For the non-neurovirulent strain, viremia was observed in all six infected ponies, with no differences in magnitude for the first 4 dpi (Fig A ). However, at day 5 and 7 pi, the non-neurovirulent strain seemed to retain a higher viremia level then the neurovirulent strain, but due to the low amount of animals left at those days, no conclusions could be drawn (Fig 8).

In addition, around 2 x 10 7 mononuclear cells for each strain were analyzed by confocal microscopy to detect infected cells and further characterize their nature. These cells were a mixture of PBMC from three ponies collected at 2, 3 and 4 dpi. For the neurovirulent strain, fourteen EHV1-infected cells were found and eleven were characterized as CD172a + cells (78.5%), whereas three were CD5 + (21.5%). No infected B-lymphocytes were found. For the non-neurovirulent strain, nine EHV1-infected cells were identified, consisting of seven Also here, no infected B-lymphocytes were found.

These data indicate that CD172a + cells appear to be the main target cell during EHV1induced viremia, irrespective of the neuropathogenicity of EHV1.

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

Discussion

Despite several studies conducted on the pathogenesis of equine herpesvirus type 1 (EHV1), it still remains unclear how this virus invades the tissues of the upper respiratory tract and invades the deeper tissues to initiate a cell-associated viremia. Furthermore, two strains of EHV1 circulate in the field, namely non-neurovirulent and neurovirulent strains, the latter causing neurological disease, a severe condition which is poorly understood. In the present study, we used the two strains of EHV1 to gain more insight into the pathogenesis of these strains during the acute phase of infection in the upper respiratory tract in vivo. To this end, Shetland ponies were experimentally inoculated with either a non-neurovirulent or a neurovirulent EHV1 strain and upon euthanasia, several tissues from the upper respiratory tract were collected and analyzed.

It was found that EHV1 has different approaches to successfully establish an infection in the upper respiratory tract. Firstly, we observed that EHV1, irrespective of the strain, can replicate in the epithelium of the nasal septum and/or nasopharynx by means of virus-induced plaques. This replication appears to occur in a restricted way, as no increase in number or size of plaques was observed over time. In addition, a repulsion of EHV1-induced plaques was observed from 5 dpi onwards for the neurovirulent strain, and a decrease in plaque latitude was seen for the non-neurovirulent strain starting at day 3 pi. This reduction in replication might be attributed to the production of interferon (IFN), as (i) the presence of interferon in nasal secretions after experimental inoculation of horses with EHV1 has previously been described and (ii) peak concentrations of IFN-α were noted at day 4 and day 7 pi [START_REF] Bridges | Innate immunity during Equid herpesvirus 1 (EHV-1) infection[END_REF][START_REF] Edington | Equine interferons following exposure to equid herpesvirus-1 or -4[END_REF]. In the present study, IFN-α was also found in nasal swabs of experimentally infected ponies (data not shown), which further points towards the importance of IFN to limit EHV1 replication in the epithelium of the upper respiratory tract.

Interestingly, even though the non-neurovirulent strain was able to replicate in a plaquewise manner in the epithelium of the nasopharynx, no plaques could be observed at any time point pi in the nasopharynx after inoculation with the neurovirulent strain. As the epithelial structure of both tissues is identical, with the exception of the presence of lymphoid aggregates in the nasopharynx, the reason for this observation remains elusive. Another interesting observation for both EHV1 strains was that we were unable to observe EHV1infected plaques under the basement membrane (BM) at any time point pi, indicating that EHV1 does not reach the underlying tissue by breaking down the BM. This is in striking contrast to previous findings with another and closely related alphaherpesvirus, pseudorabies virus (PRV), which has been shown to cross the BM in a plaque wise manner between 12 and 24 hpi in a porcine respiratory explant model [START_REF] Glorieux | Different replication characteristics of historical pseudorabies virus strains in porcine respiratory nasal mucosa explants[END_REF]. This indicates that different alphaherpesviruses may use different mechanisms to invade the underlying connective tissue. Indeed, whereas PRV was shown to break down the BM and replicates further in the underlying tissue in a plaquewise manner, our study with EHV1 demonstrated only single infected cells below the BM. This is in agreement with previous studies on EHV1, where the presence of single infected cells in tissues of the upper respiratory tract has been reported [START_REF] Edington | Endothelial cell infection and thrombosis in paralysis caused by equid herpesvirus-1: equine stroke[END_REF][START_REF] Kydd | Distribution of equid herpesvirus-1 (EHV-1) in the respiratory tract of ponies: implications for vaccination strategies[END_REF]. We could further demonstrate that these infected cells in the underlying tissue were immune cells (as discussed in more detail below), which makes it tempting to speculate that immune cells in the epithelium become infected with EHV1 and are responsible for transporting the virus to deeper connective tissue. Indeed, we did found EHV1-infected cells in the epithelium of nasal septum and/or nasopharynx, which were further characterized to belong to the monocytic lineage (CML) by using the cell surface maker CD172a. Not only differences concerning the identity of the EHV1-infected immune cells present in the underlying tissues were noticed between both strains, moreover, differences concerning there localization were also observed. When analyzing the identity of the single EHV1-infected cells in the connective tissue of nasal septum and nasopharynx, A c c e p t e d M a n u s c r i p t 19 several differences could be observed between the two EHV1 strains. Firstly, the total number of infected cells in the nasal septum was 10 to 20 times higher for the neurovirulent strain compared to the non-neurovirulent strain. Secondly, while individual infected cells in the nasopharynx were mainly present in the connective tissue upon neurovirulent EHV1 inoculation, individual cells infected with the non-neurovirulent EHV1 seemed to migrate deeper into the lymphoid follicles of the nasopharynx in a time wise manner. Thirdly, CD172a + CML were the most important carrier cells for both neurovirulent and nonneurovirulent EHV1, although these cells seemed to be more important for the neurovirulent strain. In addition, both strains were able to infect CD5 + T-lymphocytes, with a higher amount of infected cells for the non-neurovirulent strain (Table I). When analyzing the data on the identity of infected cells in lymphoid tissues of the upper respiratory tract, we again observed that the most prominent target cells for EHV1 were CD172a + CML, irrespective of the strain used. This is in line with previous studies, where EHV1-infected macrophages and lymphoblasts were detected in lymph nodes after inoculation with EHV1 isolates from both an aborted fetus and a paralyzed mare [START_REF] Patel | Variation in cellular tropism between isolates of equine herpesvirus-1 in foals[END_REF]. Moreover, another EHV1 experimental inoculation study showed that lymphocytes, monocytes, macrophages and plasma cells in interstitium and lymph nodes all expressed EHV1 antigens, as determined by immunohistochemical staining using anti-EHV1 polyclonal Abs [START_REF] Kydd | Distribution of equid herpesvirus-1 (EHV-1) in the respiratory tract of ponies: implications for vaccination strategies[END_REF]. In the present study, the majority of EHV1-positive cells was found in the marginal sinus of lymph nodes. At those sites, transport is possible to extra follicular interdigitating cells in the lymph node where both mature and virgin lymphocytes have access to antigen, hereby initiating humoral immune responses [START_REF] Tew | Follicular dendritic cells as accessory cells[END_REF]. Surprisingly, and in contrast to what was observed in nasal septum and nasopharynx, IgM + B-lymphocytes were also an important target cell for EHV1. In vitro it has been previously shown that a 6-to 14-fold increase of EHV1-infected peripheral blood mononuclear cells (PBMC) could be obtained after in vitro A c c e p t e d M a n u s c r i p t 20 stimulation with proliferating drugs such as pokeweed mitogen (PWM), concanavalin A (ConA), phytohaemagglutinin (PHA), or ionomycin and phorbol dibutyrate (IONO/PDB) (van der Meulen et al., 2001). Together with the fact that B-lymphocytes are known to undergo extensive proliferation in lymphoid follicles [START_REF] Fu | Development and maturation of secondary lymphoid tissues[END_REF], we would like to reason that the elevated percentage of EHV1-infected B-lymphocytes observed in lymphoid tissues, is due to the proliferation stage of these immune cells.

The onset of a cell-associated viremia could be detected as early as one day after experimental inoculation with EHV1, indicating that EHV1-infected cells can enter the blood stream very rapidly. Moreover, we identified these EHV1-positive cells as mainly CD172a + CML. Despite the fact that only 14 infected cells were found, we strongly feel that this low number is of relevance, as it is known that EHV1-induced viremia is generally very low with numbers ranging from 1 to 10 positive cells/0.8 x 10 7 PBMC (van der [START_REF] Van Der Meulen | Absence of viral envelope proteins in equine herpesvirus 1-infected blood mononuclear cells during cellassociated viremia[END_REF].

Combining the results on the identity of EHV1-infected cells in the connective tissues of the primary port of entry, the upper respiratory tract, with the results on the identity of the EHV1infected cells in the blood, we would like to hypothesize that the CD172a + cells we observed could be dendritic cells (DCs). The reasoning behind such hypothesis is as follows. The marker DH59B that was used in the present study to identify CD172a + cells is a pan granulocyte/monocyte marker which has been used in several studies as a DC marker [START_REF] Ahn | Scavenger receptor cysteine-rich domains 9 and 11 of WC1 are receptors for the WC1 counter receptor[END_REF][START_REF] Tumas | Monoclonal antibodies to the equine CD2 T-lymphocyte marker, to a pangranulocyte monocyte marker and to a unique pan-B lymphocyte marker[END_REF]. In addition, there is evidence indicating that local tissue DCs are able to re-enter the bloodstream, which might facilitate the spread of pathogens from tissue to tissue, carried by DCs serving as Trojan horses [START_REF] Randolph | Migration of dendritic cell subsets and their precursors[END_REF]. Finally, DCs have been shown to play an important role during the pathogenesis of alphaherpesviruses in general [START_REF] Bosnjak | Dendritic cell biology in herpesvirus infections[END_REF][START_REF] Novak | Dancing with the enemy: the interplay of herpes simplex virus with dendritic cells[END_REF][START_REF] Pollara | Dendritic cells in viral pathogenesis: protective or defective?[END_REF], and for EHV1 in specific, both murine and equine blood-derived DCs are susceptible to EHV1 in vitro [START_REF] Siedek | Equine herpesvirus type 1 infects dendritic cells in vitro: stimulation of T lymphocyte proliferation and cytotoxicity by infected dendritic cells[END_REF][START_REF] Steinbach | Dendritic cells presenting equine herpesvirus-1 antigens induce protective antiviral immunity[END_REF]. Unfortunately, little is known about equine DCs in results presented here are in striking contrast to another in vivo study, where mainly blood Tlymphocytes were found to harbor EHV1 [START_REF] Scott | In vivo harboring of equine herpesvirus-1 in leukocyte populations and subpopulations and their quantitation from experimentally infected ponies[END_REF]. However, in the latter study, leukocyte populations were solely separated by glass-bead columns and no direct characterization was performed. A possible explanation for the discrepancy between cell types infected during cell-associated viremia could be the strain used. In a previous study an American strain, isolated from an aborted fetus, was used [START_REF] Scott | In vivo harboring of equine herpesvirus-1 in leukocyte populations and subpopulations and their quantitation from experimentally infected ponies[END_REF] whereas in the present study, we used two Belgian EHV1 strains. Surprisingly, we observed CD172a + cells as the main carrier cell type for both the neurovirulent and the non-neurovirulent strain, indicating that the EHV1 strain does not seem to correlate with a difference in cell tropism during viremia. This is again in contrast to another study, where a difference in leukocyte tropism between neuro-and non-neurovirulent EHV1 strains has been demonstrated [START_REF] Goodman | A point mutation in a herpesvirus polymerase determines neuropathogenicity[END_REF]. Hereby it was observed that the EHV1 strain Ab4, which has the neuropathogenic genotype, mainly infected CD4 + lymphocytes, whereas mutating this strain into the non-neuropathogenic genotype resulted in a preferential infection of monocytes and B-cells. However, these experiments were performed with in vitro infected PBMC, which most likely explains the discrepancy with our in vivo data.

Conclusion

Based on all the observations presented in this study, we would like to propose following model on the pathogenesis of different EHV1 strains in the upper respiratory tract.

Upon infection, EHV1 replication in epithelial cells of the nasal septum, and nasopharynx for the non-neurovirulent strain, results in a productive infection with plaque formation and nasal virus shedding. This nasal shedding allows for transmission of virus to susceptible contact A c c e p t e d M a n u s c r i p t 22 animals and ensures that EHV1 can persist in the equine population. This productive infection in epithelial cells, however, is self-limiting, most likely because of IFN-α production. In addition, EHV1 seems to cross the BM through the help of CD172a + carrier cells, probably mucosal dendritic cells. We postulate that these carrier cells which become infected with EHV1 can transport the virus across the basement membranes and through connective tissues in the direction of vascular endothelial cells on the one hand and lymphatic epithelium on the other hand. As a result, a cell-associated viremia is originated, together with a migration of infected immune cells to local lymph nodes, hereby initiating the start of a specific immune response. Although not observed in this study, the higher potential of the neurovirulent strain to infect large amounts of carrier cells, could lead to a prolonged viremia, with a higher risk of developing nervous system disorders.

Despite the fact that only a limited number of animals could be included in this experiment, we still feel that this paper revealed important new insights about the pathogenesis of neurovirulent and non-neurovirulent strains in the acute phase of infection in horses. Ponies were inoculated with a neurovirulent or a non-neurovirulent strain of EHV1. At 0, 1, 2, 3, 4, 5 and 7 days post inoculation (dpi), blood samples were collected and cocultivation of PBMC was performed as mentioned in materials and methods.

  the highest serum dilution that induced visual staining of infected RK13 cells as determined by light microscopy.

  , B and C for regions with epithelial plaques and D, E and F for regions without epithelial plaques (Fig 2A). For nasopharynx, tubal/nasopharyngeal tonsils and lymph nodes, subdivisions into different zones of functional importance were made (Fig 3A and 4A

  until 7 dpi (Fig 1). Next, different tissue samples were collected from the upper respiratory tract of one pony per group per day for viral titrations. Virus could not be isolated from any tissue at 1 dpi for the neurovirulent strain, but was found in the nasopharynx of ponies inoculated with the non-neurovirulent strain (Fig 2). From 2 dpi, virus was mainly recovered from different parts of the nasal septum and the nasopharynx for both strains (Fig 2). Septum and nasopharynx remained positive for EHV1 until 7 dpi, with the exception of day 5 for the neurovirulent strain where no virus was detected in the nasopharynx (Fig 2) and day 4 for the nonneurovirulent strain where the distal nasal septum was negative (Fig 2). No difference in virus titers could be noticed in the nasal septum, but titers found in the nasopharynx seemed to be higher after inoculation with the non-neurovirulent strain of EHV1 compared to the neurovirulent strain (Fig 2). However, because of the limited number of animals used, we A c c e p t e d M a n u s c r i p t 12 should be careful to draw any conclusions based on these results. Virus was also found in lymphoid tissues such as the tubal and nasopharyngeal tonsils and mandibular lymph nodes (Fig 2). In general, titers found in tubal and nasopharyngeal tonsils (Fig 2) were higher for the non-neurovirulent strain. Titers detected in mandibular lymph nodes started rising from 3 dpi (neurovirulent strain) or 4 dpi (non-neurovirulent strain), and at 7dpi, mandibular lymph nodes were positive for the non-neurovirulent strain only (Fig 2). EHV1 could also be isolated for both strains at different time points pi from the ethmoid, the cranial part of the trachea and 3 different tonsils (data not shown).Finally, replication of EHV1 was evaluated in the epithelium of different tissues of the upper respiratory tract by immunofluorescence. Hereby, EHV1-induced plaques were analyzed and plaque latitudes measured. In the epithelium of the entire nasal septum, EHV1induced plaques were seen at different time points pi for both strains(Fig 3A). These plaques were defined as groups of infected epithelial cells. Since results for proximal, intermedial and distal nasal septum were virtually identical, only results for the intermedial nasal septum are shown. Plaques were not present in the epithelium at 0 and 1 dpi (Fig3A). However, individual infected cells were observed at 1 dpi, which were identified as CD172a + cells, as determined by the marker DH59B, or epithelial cells, as determined by the pancytokeratin marker (data not shown). For the neurovirulent strain, EHV1-induced plaques were observed in the epithelium of the nasal mucosa starting from 2 till 7 dpi(Fig 3A). At 2, 3 and 4 dpi, these plaques consisted of epithelial cells and rarely one or two CD172a + cells. From 5 dpi, EHV1-positive cells became separated from the basement membrane (BM) and these cells were exclusively positive for the DH59B marker which identifies cells of the monocytic lineage (CML)(Fig 3A). This observation was not seen in ponies inoculated with a nonneurovirulent strain. There, plaques were present in the epithelium of the nasal mucosa and nasopharynx from 2 till 7 dpi(Fig 3A & B). These plaques consisted of epithelial cells and A c c e p t e d M a n u s c r i p t 13 rarely some CD172a + cells, but no separation of EHV1-positive cells was observed (Fig 3A). No differences were observed in both number (data not shown) and size (Fig 3A) of EHV1-

  infected cells was observed in region A (Fig 4). Such increase was not seen in the regions located underneath epithelium without plaques (Fig 4). Nasopharynx. EHV1-infected cells were present in the connective tissue of the nasopharynx from 1 till 7 dpi, irrespective of the strain used, however, the localization of these cells appeared to be strain-dependent (Fig 5). Whereas for the neurovirulent strain, single infected cells were mainly found in epithelium (region A) and connective tissue (region B), single infected cells were located in connective tissue (region B) and lymphocyte aggregates (region C) for the non-neurovirulent strain (Fig 5). Tubal and nasopharyngeal tonsils. For both EHV1 strains, single infected cells were observed starting from 2 dpi (Fig 6). They were mostly present in the connective tissue underneath the epithelium (region B) and lymphocyte aggregates (region C, Fig 6), but single infected cells could also be observed in epithelium/fundus (region A), deeper connective tissue (region D), blood vessels (region E) and glands (region F) (Fig 6). Mandibular lymph nodes. For the neurovirulent strain, most of the infected cells were found in the marginal sinus of the mandibular lymph nodes (region C, Fig 7). Their number increased until 4 dpi and rapidly decreased afterwards, with rarely any infected cells left at 7 dpi. Furthermore, infected cells were also observed in the capsule and trabecula (region A), the medulla (region F) and in fewer amounts in vessels of the capsule and the cortical nodules (regions B and D, Fig 7). In these regions a peak number of infected cells was observed at 4 and 5 dpi (Fig 7). For the non-neurovirulent strain, single cells were only found from 4 till 7 dpi and were mostly found in the marginal sinus (region C) and medulla (region F) (Fig 7).

  (77.8%), one CD5 + T-lymphocyte (11.0%) and one cell remained unidentified.

  DCs in specific in this species, and more research is needed to elucidate the role of DCs in EHV1 infection and/or other viral infections in equines. Regardless, the

  , K.M., Nauwynck, H.J., Buddaert, W., Pensaert, M.B., 2000, Replication of equine herpesvirus type 1 in freshly isolated equine peripheral blood mononuclear cells and changes in susceptibility following mitogen stimulation. J Gen Virol 81, 21-25. van der Meulen, K.M., Nauwynck, H.J., Pensaert, M.B., 2001, Mitogen stimulation favours replication of equine herpesvirus-1 in equine blood mononuclear cells by inducing cell proliferation and formation of close intercellular contacts. J Gen Virol 82, 1951-1957. van der Meulen, K.M., Nauwynck, H.J., Pensaert, M.B., 2003b, Absence of viral antigens on the surface of equine herpesvirus-1-infected peripheral blood mononuclear cells: a strategy to avoid complement-mediated lysis. J Gen Virol 84, 93-97. van Maanen, C., 2002, Equine herpesvirus 1 and 4 infections: an update. Vet Q 24, 58-78.

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Viral replication in different tissues of the upper respiratory tract. Ponies were

Figure 3 .

 3 Figure 3. Plaque formation in epithelium of intermedial nasal septum and nasopharynx.

Figure 4 .

 4 Figure 4. Quantification and identification of single EHV1-infected cells in different

Figure 5 .

 5 Figure 5. Quantification and identification of single EHV1-infected cells in different

  The

  identification of single infected cells are shown in the lower graphs and are represented as the mean of ten sections ± SEM. ROI: region of interest.

Figure 6 .

 6 Figure 6. Quantification and identification of single EHV1-infected cells in different

Figure 7 .

 7 Figure 7. Quantification and identification of single EHV1-infected cells in different

Figure 8 .

 8 Figure 8. Quantification of viremia for neurovirulent and non-neurovirulent strains.

  Figure

Figure

  Figure

Figure

  Figure

Table I .

 I Percentage of marker positive EHV1-infected individual cells in different tissues of the upper respiratory tract

	Region of interest		Percentage of marker positive EHV1-infected individual cells
		Neurovirulent strain		Non-neurovirulent strain
		CD172a +	CD5 +	IgM +	CD172a +	CD5 +	IgM +
	Nasal septum	53.4 ± 22,8	16.2 ± 11.7	0.3 ± 0.8	43.9 ± 12.8	23.6 ± 8.6	0
	Nasopharynx	69.5 ± 20.7	0	8.3 ± 20.4	37.9 ± 35.5	13.6 ± 5.7	1.1 ± 2.7
	Tubal and nasopharyngeal tonsils	66.4 ± 20.7	11.2 ± 12.8	4.8 ± 5.6	28.7 ± 3.6	21.2 ± 5.9	0.5 ± 1.0
	Mandibular lymph nodes	71.3 ± 17.9	7.3 ± 9.5	8.3 ± 8.8	42.6 ± 21	26.7 ± 5.7	7.7 ± 10.8
							30

Acknowledgements

This research was supported by the Institute for the promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). The authors like to thank C. Boone and N. Monteiro for titrations, and C. Bracke for isolation of PBMC and performing SN-tests.

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

Conflict of interest

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.


Zoltán Demeter
email: demeter.zoltan@aotk.szie.hu

Elena Alina Palade

Ákos Hornyák

Miklós Rusvai

Controversial results of the genetic analysis of a canine distemper vaccine strain

Keywords: canine distemper, vaccine strain, phylogenetic analysis Running title: Canine distemper vaccine strain ambiguity

Canine distemper (CD) is a highly contagious, often fatal, multisystemic viral disease of receptive carnivores. The presence of a PsiI cleavage site on a specific location of the hemagglutinin (H) gene was found to be a hallmark of vaccine strains, thus, a previously published restriction fragment length polymorphism (RFLP) test using PsiI theoretically allows the distinction between all currently used vaccine strains and virulent field strains. The RFLP test was carried out on all brands of CD vaccines available in Hungary. The present work describes the extensive sequencing and phylogenetic study of the strain present in Vanguard (Pfizer Animal Health) vaccines, which following the PsiI based RFLP test reacted as a wild-type strain. Based on the product description provided by the manufacturer, all batches should have contained a virus strain (Snyder Hill) belonging to the group of vaccine strains (America-1). Extensive genetic analysis involving the full nucleic acid sequence of four other genes (N, M, P and F) of the CDV genome revealed that the incriminated virus *Manuscript strain showed a higher level of genetic identity to wild-type strains from the America-2 group than to any of the strains belonging to America-1 group, therefore the vaccine does not contain the virus strain stated by the manufacturer in its product description and has not been containing it since at least 1992.

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

Introduction

Canine distemper (CD) is a highly contagious viral disease that has a wide range of receptive carnivore animal families, such as the Canidae, Mustelidae, Procyonidae, Felidae and many others. It is caused by the canine distemper virus (CDV) which belongs to the Morbillivirus genus of the Paramyxoviridae virus family [START_REF] Appel | Canine distemper virus[END_REF][START_REF] Kabakci | Immunohistochemical investigation of cerebellum in dogs infected with canine distemper virus[END_REF][START_REF] Lednicky | Genetically distant American canine distemper virus lineages have recently caused epizootics with somewhat different characteristics in raccoons living around a large suburban zoo in the USA[END_REF]. Although live attenuated vaccines have been used for many years to control distemper [START_REF] Chappuis | Control of canine distemper[END_REF]Barrett, 1996;[START_REF] Patel | Review of companion animal viral diseases and prophylaxis[END_REF], CDV continues to cause outbreaks in receptive domestic carnivores all around the world, in particular young dogs in breeding units and shelter facilities which most often suffer fatal disease [START_REF] Appel | Canine distemper virus[END_REF][START_REF] Blixenkrone-Møller | Studies on manifestations of canine distemper virus infection in an urban dog population[END_REF]Barrett, 1996;[START_REF] Gemma | Epidemiological observations on recent outbreaks of canine distemper in Tokyo area[END_REF][START_REF] Demeter | Experiences on the control of endemic distemper at the flaying-house of Budapest[END_REF][START_REF] Martella | Canine distemper virus[END_REF]. Previous genetic and phylogenetic studies have revealed that the virus strains used in the currently applied vaccines form a distinct group (America-1) from the large group of wild-type strains that were reported worldwide in the previous decades [START_REF] Martella | Genotyping canine distemper virus (CDV) by a hemi-nested multiplex PCR provides a rapid approach for investigation of CDV outbreaks[END_REF][START_REF] Demeter | Polymerase chain reaction based investigations of canine distemper and parvovirus strains from Hungary[END_REF]. The most frequently used strains in the manufacturing process of CDV vaccines are the Onderstepoort, Snyder Hill, Rockborn, Lederle, and Convac. The Onderstepoort strain, used worldwide as an attenuated live vaccine, dates back to a disease A c c e p t e d M a n u s c r i p t 3 outbreak among North American ranched foxes in the 1930's [START_REF] Haig | Canine distemper: immunization with avianized virus[END_REF][START_REF] Martella | Genotyping canine distemper virus (CDV) by a hemi-nested multiplex PCR provides a rapid approach for investigation of CDV outbreaks[END_REF]. The Snyder Hill strain was isolated in Ithaca, USA in the 1950's from the brain of a dog. The virus was then passaged in vivo in dogs before being adapted to cell growth in NL-DKC cells [START_REF] Brown | Immune response of pups to modified live-virus canine distemper-measles vaccine[END_REF][START_REF] Martella | Genotyping canine distemper virus (CDV) by a hemi-nested multiplex PCR provides a rapid approach for investigation of CDV outbreaks[END_REF]. The Rockborn strain is undistinguishable from the Snyder Hill strain [START_REF] Greene | Canine distemper[END_REF], while all the other vaccine strains (Lederle, Convac etc.) also belong to the America-1 group. Nowadays it is not clear whether these strains are still circulating in the field, since they have not been detected over the last five decades [START_REF] Martella | Genotyping canine distemper virus (CDV) by a hemi-nested multiplex PCR provides a rapid approach for investigation of CDV outbreaks[END_REF].

In recent years the number of vaccinated dogs that got infected with CDV and developed characteristic clinical signs of CD has increased [START_REF] Appel | Canine distemper virus[END_REF][START_REF] Blixenkrone-Møller | Studies on manifestations of canine distemper virus infection in an urban dog population[END_REF][START_REF] Kai | Use of B95a cells for isolation of canine distemper virus from clinical cases[END_REF][START_REF] Shin | Detection of canine distemper virus nucleocapsid protein gene in canine peripheral blood mononuclear cells by RT-PCR[END_REF][START_REF] Gemma | Epidemiological observations on recent outbreaks of canine distemper in Tokyo area[END_REF][START_REF] Lan | Comparative analyses of canine distemper viral isolates from clinical cases of canine distemper in vaccinated dogs[END_REF]. The molecular analysis of the isolates from the vaccinated yet infected canines demonstrated that the causative agent in these cases was not closely related to any of the currently used vaccine strains [START_REF] Pardo | Phylogenetic characterization of canine distemper viruses detected in naturally infected dogs in North America[END_REF][START_REF] Lan | Comparative analyses of canine distemper viral isolates from clinical cases of canine distemper in vaccinated dogs[END_REF][START_REF] Calderon | Detection by RT-PCR and genetic characterization of canine distemper virus from vaccinated and non-vaccinated dogs in Argentina[END_REF][START_REF] Martella | Genotyping canine distemper virus (CDV) by a hemi-nested multiplex PCR provides a rapid approach for investigation of CDV outbreaks[END_REF]. On the other hand, cases when vaccinated animals shed the vaccine strain for a limited period of time were also described [START_REF] Greene | Canine distemper[END_REF]. Considering the fact that in case of recently vaccinated animals the virus strain present in the vaccine can interfere with polymerase chain reaction (PCR) based diagnostic tests, leading to diagnostically false positive results, in order to rule out the mentioned misleading factors, a PCR coupled with restriction fragment length polymorphism (RFLP) assay using a PsiI cleavage site of the hemagglutinin (H) gene of the viral genome was designed [START_REF] Demeter | Genetic diversity of Hungarian canine distemper virus strains[END_REF]. According to the nucleotide sequences deposited in the publicly accessible GenBank, the presence of PsiI cleavage site is characteristic only for vaccine strains, thus, PsiI RFLP was expected to allow a reliable differentiation of all CDV vaccine strains from the wild-type ones. During the examination of CDV vaccines marketed in Hungary by PsiI RFLP, the vaccine strain present

Materials and methods

Vaccine batches

Products of four different manufacturers were purchased and tested during this study. A total number of ten vaccines were investigated, of which six were of the same brand (vaccines number 1 to 6), but purchased in different countries (Hungary, Israel, Malta and United States of America) or were from batches dated back to 1992, 1994 and 2006 (Table 1). All modified live CD vaccines were stored at the recommended temperature of 4 °C.

RNA purification

Vaccines were prepared according to the protocol provided by each manufacturer. Following dilution of the lyophilized component with the solution recommended and supplied together with the vaccine, viral RNA was isolated from each vaccine using QIAamp viral RNA Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. After purification, the RNA samples were stored at -80 °C until the following steps of the investigations.

Primers

The primers used in the present study were designed using the Primer Designer 4 software (Scientific & Educational Software, USA). Sequences of different vaccine and wild-type CDV strains were retrieved from GenBank and were aligned to ensure the attaching of the

RT-PCR assays, RFLP and gel electrophoresis

Reverse transcription and amplifications were performed in one-step RT-PCR method using the QIAGEN OneStep RT-PCR Kit (Qiagen, Germany), according to the manufacturer's instructions. Reverse transcription was carried out at 50 °C for 30 min. Following an initial denaturation at 95 °C for 15 min, the reaction mixture was subjected to 40 cycles of heat denaturation at 94 °C for 45 sec, the corresponding annealing temperature for each primer pair for 1 min, and DNA extension at 72 °C for 2 min, followed by a final extension of 10 min at 72 °C. The reactions were performed in a SPRT001 PCR Sprint Thermal Cycler (Hybaid Ltd, UK).

Following RT-PCR 7.5 l of amplicons was electrophoresed at 80 V for 80 min in a 1.2% Tris acetate-EDTA-agarose gel (Merck, Germany), stained with ethidium bromide. The bands were visualized by UV transillumination at 312 nm. Product sizes were determined with reference to 100 bp and 1 kb molecular weight ladders (Fermentas, Lithuania). The PsiI (SibEnzyme, Russia) based RFLP was carried out according to the previously described protocol [START_REF] Demeter | Genetic diversity of Hungarian canine distemper virus strains[END_REF].

Sequence analysis and phylogeny

DNA was extracted from the gel with the QiaQuick Gel Extraction Kit (Qiagen, Germany).

Fluorescence-based direct sequencings were performed in both directions on the amplicons by BIOMI Kft. (Gödöllő, Hungary) using an ABI 3100 genetic analyzer (Applied Biosystems, USA). Nucleotide sequences were identified by BLAST search against gene bank databases (http://www.ncbi.nlm.nih.gov/BLAST/). The complete sequences of each gene were compiled http://darwin.uvigo.es/rdp/rdp.html) was used [START_REF] Martin | RDP: detection of recombination amongst aligned sequences[END_REF].

Results

RT-PCRs and RFLP analysis

The RT-PCRs resulted amplicons of the expected size (Table 1) in case of every pair of primers used. The RFLP resulted two clearly differentiable bands in case of the currently used vaccine strains at the predicted heights of 294 bp and 816 bp, while the amplicons obtained from field viruses and vaccines 1 to 6 (Table 1) remained undigested (1110 bp).

Different batches of Vanguard vaccines (Pfizer Animal Health, USA) dating back to 1992, 1994 and 2006 were also involved in the study. Furthermore, batches of the same brand were purchased in different countries as well, such as Hungary, Israel, Malta and the United States of America (Table 1). The RFLP test unequivocally resulted in undigested amplicons in case of all batches from different time and geographical origin.

Genetic analyses

To identify the strain present in the Vanguard vaccines, primers were designed to cover more than half of the viral genome. The nucleotide sequences of all CDV genes, except the highly conserved L gene were determined. The genetic analyses of all investigated genes of the Vanguard vaccines (Table 1) have revealed that the virus strain present in all of them is more closely related to different wild-type strains from the America-2 group, than to the one stated by the manufacturer (Snyder Hill strain) or any of the viruses from the group of vaccine strains (Table 2). As a next step the full nucleotide sequences of the highly variable H gene of all vaccine brands currently used in Hungary were included in the study (Table 1). Since the obtained nucleotide sequences of vaccines 1 to 6 turned out to be 100% similar in case of all batches, the subsequent investigations performed on other domains of the virus genome were carried out using only vaccine number 6. According to the investigation of the complete sequence of the H gene, the investigated virus strain was not positioned anywhere close to the Snyder Hill strain, but it showed a significantly higher similarity to a lesser panda isolate (99.45%) and a wild-type strain (98.90%) isolated from a naturally infected dog (strain 25259, accession number: AY964114) in North America (Figure 1). As expected following the initial RFLP, the virus strains present in vaccines 7-10 were positioned in the group of vaccine strains (America-1). The findings of the neighbour-joining method-based phylogenetic analysis were confirmed by the results of the Bayesian and ML methods as well: the Vanguard vaccine strain showed a considerably higher level of identity with wild-type viruses from the America-2 group (data not shown). RDP could not identify any recombination in case of any of the analyzed genes of the Vanguard vaccine strain (data not shown).

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

The genetic analyses of the full nucleic acid sequences of the F, N, M and P genes only confirmed the initial observations of the H gene analysis: in each case the virus strain present in the Vanguard vaccine was not positioned in the group of vaccine strains (America-1). Unfortunately the nucleic acid sequence of the Snyder Hill strain is not publicly available for these genomic segments, except for a partial, 335 bps long F gene sequence: AY288312 and a 390 bps long partial P gene segment: AY286481. Even tough it cannot be considered as reliable as complete gene sequence analyses, the genetic analysis of such small segments yielded results identical with the initial investigations performed on the H gene.

Discussion

In the present study, we have applied a previously described PsiI-based RFLP test [START_REF] Demeter | Genetic diversity of Hungarian canine distemper virus strains[END_REF] that theoretically allows the differentiation of all wild-type strains with known nucleotide sequence from all vaccine CDV strains. The differentiating RFLP analysis carried out on CD vaccine brands currently used in Hungary revealed that the CDV strain present in the Vanguard (Pfizer Animal Health, USA) vaccine reacted as a wild-type strain. According to the statement of the manufacturer, the European Vanguard products used to immunize dogs against CD contain the Snyder Hill strain. Based on the nucleotide sequence available in the GenBank (complete sequence of the H gene of the Snyder Hill strain: accession number: AF259552), the amplicons of the virus strain present in the Vanguard vaccine should have been digested by the PsiI enzyme, but they remained undigested, hence reacted as a wild-type virus. In order to elucidate the dilemma, an extensive genetic and phylogenetic analysis of the incriminated virus strain was conducted. Primer pairs that enabled the amplification of complete genes of the CDV genome were designed and the nucleotide sequences of all CDV genes (H, N, M, P and F), except the highly conserved L gene were determined. For phylogenetic purposes the H gene was chosen, because of its vital role in the infectious A c c e p t e d M a n u s c r i p t 9 process [START_REF] Murphy | Veterinary Virology[END_REF] this gene is considered as one of the most variable one in the CDV genome [START_REF] Haas | Analysis of the haemagglutinin gene of current wild-type canine distemper virus isolates from Germany[END_REF]Harder & Osterhaus, 1997;Iwastsuki et al., 1997;[START_REF] Mochizuki | Genotypes of canine distemper virus determined by analysis of the hemagglutinin genes of recent isolates from dogs in Japan[END_REF]Uema et al., 2005;[START_REF] Martella | Heterogeneity within the hemagglutinin genes of canine distemper virus (CDV) strains detected in Italy[END_REF][START_REF] Mccarthy | Pathogen evolution and disease emergence in carnivores[END_REF], therefore it is profoundly suitable for phylogenetic analysis. H gene analysis further emphasized the previous findings: the investigated virus strain was not positioned anywhere close to the Snyder Hill strain, but turned out to be most closely related to a wild-type virus strain isolated from a lesser panda (AF178039). Similar observations were reported by another research group [START_REF] Pardo | Phylogenetic characterization of canine distemper viruses detected in naturally infected dogs in North America[END_REF], but the investigated segment was too small to obtain relevant results (979 bases). Based on the findings of this research group the virus strain present in the Vanguard vaccine was most closely related to the A75/17 wild-type strain isolated from a dog, but the investigation of the complete sequence of the H gene has revealed that it is more closely related to the lesser panda isolate (99.45 %) and a wild-type strain (98.90 %) isolated in North America from a naturally infected dog (strain 25259, accession number: AY964114) by the same research group [START_REF] Pardo | Phylogenetic characterization of canine distemper viruses detected in naturally infected dogs in North America[END_REF]. The analysis of other genes of CDV has only confirmed the findings of the H gene analysis.

In order to extend our investigations in time and geographical respect, Vanguard vaccine vials produced in different time (1992 and 1994) and different geographical origin (Malta, Israel, and USA) were purchased and the same RFLP test was applied to determine whether these vaccines contain the same virus strain as the vaccine purchased in Hungary in 2006. The result of the RFLP test and the complete sequencing of the H gene of the virus strains present in all these Vanguard vaccine batches revealed that all of them contain exactly the same virus strain, showing not even one nucleotide difference on this relatively large and highly variable gene. Due to these findings, the vaccine batches dated back to 1992 and 1994

and the ones purchased in other countries were not investigated on other segments of the CDV genome, since those results most probably would have only confirmed the previous A c c e p t e d M a n u s c r i p t 10 observations. On the other hand, these findings led to the conclusion that the Vanguard vaccines have been containing the analyzed virus strain since at least 1992.

There could be several possible explanations for the findings of the present study, such as (1) the incorrect labelling of the Snyder Hill strain deposited in the GenBank (AF259552)contradicted by its positioning among the group of vaccine strains in case of the phylogenetic trees constructed based on the nucleic acid sequences of the full length of the H gene (Figure 1) and partial segment of the F gene (data not shown);

(2) a possible recombination of the seed virus with a wild-type strainthis theory is eliminated by the simultaneous RDP-based analysis of several different genes;

(3) due to a taxonomical and technical error (i.e. erroneous laboratory registries) the strain was incorrectly labelled when the vaccine was registered and finally (4) contamination of the seed virus stock used in the vaccine production, as also suggested by [START_REF] Pardo | Phylogenetic characterization of canine distemper viruses detected in naturally infected dogs in North America[END_REF]. At this point the last two explanations cannot be demonstrated neither eliminated. The significance of these findings is inconclusive without further studies, as phylogeny can not reveal whether or not a virus is virulent, and there may be little genetic distance between virus passages with dramatic changes in their phenotypes, including virulence. At this point it can only be declared that the vaccine does not contain the virus strain erroneously stated by the manufacturer in its product description and has not been containing it since at least as 1992. Surely the vaccine strain underwent all the required tests before being licensed and proved to be safe to be used in animals, and no claims of inefficacy of the Vanguard vaccines have been reported. There are only a few reports of animals vaccinated with Vanguard vaccines that still developed clinical disease [START_REF] Mcinnes | Possible vaccine-induced canine distemper in a South American bush dog (Speothos venaticus)[END_REF][START_REF] Pardo | Phylogenetic characterization of canine distemper viruses detected in naturally infected dogs in North America[END_REF], but the exact cause of the lack of protection and the exact role of the vaccine strain in the infection was not elucidated, therefore no relevant conclusions can be drawn regarding the efficacy of the Vanguard vaccines, as there are numerous aspects that can interfere with the successful immunization of an animal [START_REF] Povey | Distemper vaccination of dogs: factors which could cause vaccine failure[END_REF][START_REF] Greene | Canine distemper[END_REF] A c c e p t e d M a n u s c r i p t 11 2006). As no documented concerns regarding the safety and efficacy of the Vanguard strains have been raised since at least 1992, it is possible that this recently and apparently successfully attenuated wild-type strain could provide, at least theoretically, a better protection against the currently circulating wild-type CDV strains.

On the other hand, these findings suggest that the previously described PsiI-based identification of America-1 (vaccine) strains could lead to diagnostically false results in case of animals recently vaccinated with any Vanguard vaccine, or in case of animals with uncertain vaccination history that could have been vaccinated with a Vanguard vaccine.

Acknowledgements

The authors would like to thank Pfizer Animal Health division of Pfizer Inc. for the support and assistance during the investigations described in the present study.

Follow-up

Following the notification of the research group, the manufacturer has performed the sequencing of the vaccine master-seed strain used globally for the production of the vaccine.

The work has resulted in a regulatory review of, and a change in, the strain designation in the product literature globally in early 2009. This work demonstrated that the distemper strain used in vaccine production was misidentified back in 1956 when it was first acquired. Documentary evidence of these findings was supplied to the European regulatory agencies, and they have accepted the evidence of the misclassification, and the integrity of the registration study data and subsequent production batches. The strain is now identified in the product descriptions as N-CDV. * result of a recombination between an "Asia-1" and an "European wildlife" strain [START_REF] Han | Cross-species recombination in the haemagglutinin gene of canine distemper virus[END_REF] A c c e p t e d M a n u s c r i p t

  vaccine (Pfizer Animal Health, USA) exhibited wild-type genetic feature remaining undigested by PsiI. This study presents the results of the extensive sequencing and phylogenetic investigation of batches of this vaccine brand purchased in different countries (Hungary, Malta, Israel, and USA), as well as batches dating back to 1992 and 1994.

  to the vaccine strains and wild-type strains as well. Primers were synthesized by BIOMI Kft. (Gödöllő, Hungary).

  the complete genome sequence of CDV using the Align Plus 4 software (Scientific & Educational Software, USA). The nucleotide sequences of the Vanguard vaccine strain were deposited in the GenBank under the following accession numbers: EF095750 (H gene), EU072198 (F gene), EU072199 (M gene), EU072200 (N gene) and EU072201 (P gene). Phylogenetic trees of the nucleic acid and putative amino acid (aa) sequences were established by using sequence data of the analyzed vaccine strains and the ones retrieved from the GenBank (most of the available complete sequences of the analyzed genes of vaccine and wild-type strains as well). In case of the Vanguard vaccine, each gene of the CDV genome was analyzed, except the highly conserved large polymerase (L) gene. Phylogenetic analyses were performed with the help of the ClustalX program and the robustness of the groupings in the neighbor-joining analysis was assessed with 1000 bootstrap resampling. Results of the phylogenetic analysis were verified using Bayesian and maximum likelihood (ML) approaches[START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under mixed models[END_REF] with the MrBayes 3.1.2 program. In order to

  ., Ohashi, K., Wakasa, C.,Kai, C., 2005. Phylogenetic and restriction fragment length polymorphism analyses of hemagglutinin (H) protein of canine distemper virus isolates from domestic dogs in Japan. VirusRes. 109,[59][60][61][62][63]

Figure 1 :

 1 Figure 1: Phylogenetic tree constructed based on a total number of 165 complete nucleotide

Table 2 :

 2 The level of identity between the nucleotide and amino acid sequence of the H gene and H protein of the strain present in the Vanguard vaccines and those of the viruses from the group of vaccine strains (shaded) and of the wild-type strains, which showed the highest level of identity and the Snyder Hill strain (bolded). The values in italic represent the level of identity at the amino acid level.

		Vanguard	Panda	Dog	Snyder Hill	Convac	Canigen	Onder.	Eurican	Japan
	Vanguard	-	98.84	98.35	91.77	90.95	90.95	91.44	90.13	91.44
	Panda	99.45	-	97.86	91.61	91.44	90.78	91.28	89.96	91.28
	Dog	98.90	98.57	-	90.78	89.96	89.96	90.46	89.14	90.46
	Snyder Hill	92.92	92.70	92.32	-	95.39	95.23	95.72	93.91	95.72
	Convac	93.09	93.31	92.48	97.09	-	96.87	97.53	95.55	97.36
	Canigen	93.03	92.81	92.43	96.87	98.46	-	99.01	98.19	99.17
	Onder.	93.25	93.03	92.65	97.09	98.68	99.39	-	97.69	99.50
	Eurican	92.59	92.37	91.99	96.32	97.91	99.23	98.84	-	97.69
	Japan	93.25	93.03	92.65	97.09	98.68	99.45	99.61	98.90	-
	Vanguard: EF095750; Snyder Hill: AF259552; Convac: Z35493; Canigen: DQ903854; Onder: AF378705, Eurican: AF014953; Japan:
	AB212966; Panda (Lesser Panda): AF178039; Dog (25259): AY964114

Table 2

 2