Daliborka Dušanić

Rebeka Lucijana Berčič

Ivanka Cizelj

Simona Salmič

Mojca Narat

Dušan

Dušan Benčina
email: dusan.bencina@bfro.uni-lj.si

Rebeka Lucijana Berčič

Benčina Dušan

invades non-phagocytic chicken cells

Keywords: Mycoplasma synoviae invades non-phagocytic chicken cells in vitro M. synoviae, M. gallisepticum, invasion, erythrocytes, fibroblasts, chondrocytes

Introduction

Of the twelve recognized avian Mycoplasma species that infect chickens and/or turkeys, M. gallisepticum and M. synoviae are the most pathogenic [START_REF] Bradbury | Recovery of mycoplasmas from birds[END_REF][START_REF] Kleven | Mycoplasma synoviae infection[END_REF]. The factors responsible for considerable differences in tissue tropism, invasiveness and pathogenicity in strains of both species are largely unidentified.

Generally, in vitro passage of pathogenic microorganisms results in loss of pathogenicity. This has been shown also for M. gallisepticum and M. synoviae et al., 1986;[START_REF] Kleven | Mycoplasma synoviae infection[END_REF]. The low passage M. gallisepticum strain R low is invasive and pathogenic for poultry, whereas invasiveness and pathogenicity decreases in high passage strain R high (Levisohn et al., 1986;[START_REF] Winner | In vitro cell invasion of Mycoplasma gallisepticum[END_REF]Much et al., 2003;[START_REF] Vogl | Mycoplasma gallisepticum invades chicken erythrocytes during infection[END_REF]. The factors contributing to the decreased invasiveness of R high strain are most likely the mutation(s) in the gene(s) encoding cytadhesins and, possibly, decreased neuraminidase activity (NEAC) [START_REF] Vogl | Mycoplasma gallisepticum invades chicken erythrocytes during infection[END_REF]Berčič et al., 2008b). Sialic (neuraminic) acid is important in the process of infection of host cells, because M. gallisepticum and M. synoviae adhere via haemagglutinins to host-cell receptors containing sialic acid residues [START_REF] Aldridge | Growth and cytopathology of Mycoplasma synoviae in chicken embryo cell culture[END_REF][START_REF] Razin | Mycoplasma adherence[END_REF]Benčina, 2002). In M. synoviae, a haemagglutination-positive (HA + ) phenotype has also been associated with a higher frequency of synovitis in chickens inoculated into the hock joint (Narat et al., 1998).

The capacity to invade host cells has been demonstrated for four Mycoplasma species known to be human pathogens (Lo et al., 1989;[START_REF] Baseman | Interplay between mycoplasmas and host target cells[END_REF][START_REF] Giron | Adherence, fibronectin binding and introduction of cytoskeleton reorganization in cultured human cells by Mycoplasma penetrans[END_REF], as well as for poultry pathogen M. gallisepticum [START_REF] Winner | In vitro cell invasion of Mycoplasma gallisepticum[END_REF]Much et al., 2003;[START_REF] Vogl | Mycoplasma gallisepticum invades chicken erythrocytes during infection[END_REF]. Studies have shown close association of M. synoviae with chicken cells [START_REF] Aldridge | Immunofluorescence and electron microscopy of the attachment of Mycoplasma synoviae to chicken embryo fibroblasts[END_REF][START_REF] Walker | An ultrastructural study of avian synovium infected with an arthrotropic Mycoplasma, Mycoplasma synoviae[END_REF], but intracellular presence of M. synoviae has not been reported.

The main aim of this study was to determine whether M. synoviae is able to invade non-phagocytic chicken cells. Using a well established method, the gentamicin protection (invasion) assay, and three types of chicken cells, we assessed the invasion frequency of different M. synoviae strains and compared this with the invasiveness of M. gallisepticum R low .

Materials and methods

A c c e p t e d M a n u s c r i p t 4

Chicken cells

Blood was taken from adult specific pathogen-free chickens and mixed with citrate dextrose buffer (Sigma-Aldrich, Germany) in a 9:1 ratio. Erythrocytes (CER) were harvested from the collected blood by adding sterile phosphate buffer saline (PBS) pH 7.4 and centrifugation at 400×g for 10 minutes. CER were washed in PBS, centrifuged and resuspended in PBS to a final concentration of 5×10 5 to 10 6 cells/ml.

A stable transfected chicken embryonic fibroblast cell line (CEC-32) was donated by Prof. Bernd Kaspers (University of Munich, Munich, Germany). CEC-32 cells [START_REF] Kaaden | Establishment and characterization of chicken embryo fibroblast clone LSCC-H32[END_REF] were cultivated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 8% fetal bovine serum (FBS) and 2% chicken serum (all from Sigma-Aldrich, Germany).

For the isolation of chondrocytes from hyaline cartilage tissue, the protocol described by [START_REF] Barlič | Quantitative analysis of gene expression in human articular chondrocytes assigned for autologous implantation[END_REF] was modified. Briefly, cartilage tissue was obtained from adult chicken tarsometatarsal joints and digested in DMEM containing 1mg collagenase II/ml (Sigma-Aldrich, Germany) and 1µg gentamicin/ml (Krka, Slovenia) for 21 hours. The digested tissue was sieved through a 40 µm-pore nylon filter and the chondrocytes (CCH) seeded into culture flasks containing DMEM with 8% FBS and 2% chicken serum. The viability of CCH after isolation, as shown by Trypan Blue staining (Sigma-Aldrich, Germany), was 98 %. CCH were tested for mycoplasmas on Frey's agar and broth before the beginning of the study and in every experiment, and were found to be mycoplasma-free. CCH and CEC-32 cells were cultured in a CO 2 incubator at 38C in a 5% CO 2 atmosphere.

M. synoviae and M. gallisepticum cultures

A c c e p t e d M a n u s c r i p t 5 M. synoviae reference strains WVU 1853 and ULB 02/T6, also used in previous studies (Benčina et al., 2001;Lavrič et al., 2007;Berčič et al., 2008a), were used.

Recent field isolates M. synoviae ULB 08/T3 and ULB 08/T4 isolated from tracheas of chickens were also used. The culture of invasive M. gallisepticum strain R low , used in previous studies by [START_REF] Winner | In vitro cell invasion of Mycoplasma gallisepticum[END_REF] and [START_REF] Vogl | Mycoplasma gallisepticum invades chicken erythrocytes during infection[END_REF] was donated by Dr.

Michael Szostak (University of Veterinary Medicine of Vienna, Vienna, Austria).

Cultures were grown in modified Frey's medium with Bacto PPLO broth base (BD, US) containing 12% porcine serum (Invitrogen, US), 800000 IU of benzylpenicillin (Pliva, Croatia), glucose, vitamins and 0.1g of NAD/l (the latter three from Sigma-Aldrich, Germany) at 38C [START_REF] Kleven | Mycoplasma synoviae infection[END_REF]. The number of colony forming units (CFU) was calculated as described previously [START_REF] Rodwell | Methods for direct and indirect measurement of mycoplasma growth[END_REF].

Haemadsorption, haemagglutination and adherence test

Haemadsorption (HAD) of CER to Mycoplasma colonies was determined as described previously [START_REF] Gardella | Hemagglutination, Hemadsorption, and Hemolysis[END_REF]. A similar method was used to test M. synoviae colonies for adsorption of CEC-32 cells and CCH. Briefly, agar blocks (10 × 5 mm) bearing well-separated M. synoviae colonies were overlaid with ~ 50 μl samples of PBS pH 7.4 containing 10 3 -10 4 CEC-32 cells or CCH. After 30 min incubation at room temperature, blocks were gently washed with PBS. Binding of CEC-32 cells and CCH was evaluated at 40× and/or 100× magnification.

Haemagglutination (HA) tests were performed as described previously (Narat et al., 1998;Benčina et al., 1999).

Sialic acid linkages of CEC-32 and CCH glycoproteins

To demonstrate the presence of sialic acid receptors potentially used by Mycoplasma species to adhere to CEC-32 cells and CCH, proteins of both cell types were separated A c c e p t e d M a n u s c r i p t 6 by electrophoresis. The PhastGel (Gradient 8-25) and PhastSystem (Pharmacia, Sweden), followed by transfer to Immobilon P membrane were used for CEC-32 cells, whereas the proteins of CCH were separated in a larger, 12 % polyacrylamide gel (acrylamide mix, sodium dodecyl sulphate, ammonium persulphate and tetramethylethylenediamine, all from Sigma-Aldrich, Germany) and then transferred to Immobilon P membrane. The DIG Glycan Differentiation Kit (Roche, Slovenia) was used according to the instructions of the manufacturer to identify CEC-32 cells and/or CCH glycoprotein(s) containing sia(α-2-3) or sia(α-2-6) galactose linkages.

Determination of the minimal inhibitory concentration of gentamicin

The susceptibility of Mycoplasma strains for gentamicin was determined as the minimal inhibitory concentration (MIC) of gentamicin in broth cultures of M. synoviae strains. MIC was determined as described previously [START_REF] Hannan | Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary Mycoplasma species[END_REF]Bebear and Kempf, 2005). Briefly, 25 μl samples of rapidly growing M. synoviae cultures (10 5 -10 6 CFU/sample) were transferred into 1 ml of Frey´s broth without gentamicin or into 1 ml of Frey's broth supplemented with different concentrations of gentamicin.

For the gentamicin invasion assays, gentamicin was used in 500 μg/ml concentrations.

Experimental infection of chicken cells

CEC-32 cells and CCH were grown in 24-well culture plates until there were approximately 5×10 5 cells per well. The number of CER in samples used for infection ranged from 5×10 5 to 10 6 . CCH and CEC-32 cells had 98-100 % viability, as determined by Trypan blue staining. Broth cultures of Mycoplasma strains used for the infections were incubated till they reached the late logarithmic phase of growth. 100 µl of broth containing 10 7 -10 8 CFU was then used to inoculate 1 ml (~5×10 5 cells) samples of chicken cells suspended in/overlayed with Frey's broth. Thus, the A c c e p t e d M a n u s c r i p t multiplicity of infection (MOI) ranged from 10 to 100 mycoplasmas per chicken cell.

The infection of cells was performed at 37-38C. Uninfected samples of appropriate chicken cells were used as negative controls. In all experiments, the frequency of invasion (RIF) was assayed 24 hours after infection. In some experiments, the RIF after 2-4 hours and/or after 48 hours was also determined.

Gentamicin invasion assay

The number of intracellular mycoplasmas in CER, CEC and CCH was determined using a modified gentamicin invasion assay described by [START_REF] Winner | In vitro cell invasion of Mycoplasma gallisepticum[END_REF] and [START_REF] Vogl | Mycoplasma gallisepticum invades chicken erythrocytes during infection[END_REF]. Briefly, 2-4, 24 or 48 hours after infection, the whole contents of each well were centrifuged for 10 min at 300×g. The pellets were then resuspended either in 1 ml of Frey's broth without gentamicin, or in 1 ml of Frey's broth supplemented with 500µg of gentamicin/ml. Cells were resuspended 8 times during the 4 hours of incubation at 38C in 5% CO 2 . After treatment, the cell suspensions were washed three times with PBS. The infected pelleted cells treated with gentamicin were resuspended in approximately 150 μl of Frey's broth and 50 μl samples were plated onto Frey´s agar and incubated at 38C. The infected cells, which were not treated with gentamicin were resuspended in 1.5 ml of Frey's broth and plated as described above. Uninfected cell monolayers incubated in Frey's broth in the same way as the infected cells were used as negative controls. After 5-7 days, mycoplasma colonies on the plates were identified and counted as described in section 2.8. The relative invasion frequency (RIF) was calculated as the percentage of the number of mycoplasma colonies recovered after gentamicin treatment relative to the number of colonies recovered from samples that were not exposed to gentamicin [START_REF] Vogl | Mycoplasma gallisepticum invades chicken erythrocytes during infection[END_REF]. Data from independent assays, that were performed in triplicates, A c c e p t e d M a n u s c r i p t 8 were expressed as means ± S.E. The significance of the differences between RIF of the strains tested in one cell line and between cell lines infected with the same strain of mycoplasmas were assessed using Student's unpaired t-test. Differences were considered significant for p values under 0.05.

Identification of colonies

M. gallisepticum and M. synoviae colonies were identified using the indirect immunoperoxidase assay (IIPA) and/or direct immunofluorescence as described previously (Benčina et al., 1994;Benčina and Bradbury, 1992). The FITC-conjugated antibodies to M. synoviae used for the direct immunofluorescence (Supplementary Fig. 1), were also used in the differential immunofluorescent-antibody test to identify intracellular M. synoviae (Supplementary Figs. 2 and3).

Differential immunofluorescence microscopy

Double immunofluorescent-antibody test (Heesemann and Laufs, 1985) was modified as follows. Differentially labeled rabbit antibodies specific to M. synoviae (Benčina and Bradbury, 1992) were used for detecting M. synoviae on the surface and/or inside chicken cells in a direct immunofluorescent assay. Cells were fixed in 4 % PBSbuffered paraformaldehyde for 40 min. Antibodies conjugated with tetramethyl rhodamine isothiocyanate (TRITC) (diluted 1:100, 1 h at room temperature) were used for detecting M. synoviae on the surface of chicken cells. Following washing in PBS, cells were permeabilised with 1 % Triton X-100 (Sigma-Aldrich, Germany) in PBS for 10 min and incubated in 1 % bovine serum albumin in PBS for 30 min.

Results

Adherence of M. synoviae to chicken cells used in the invasion assays

Colonies of all the M. synoviae and M. gallisepticum R low cultures used were predominately HAD + , but all cultures also contained some colonies that did not adsorb chicken erythrocytes (HAD -). The pattern of adsorption of CCH and CEC-32 cells to colonies of M. synoviae was similar to that observed with erythrocytes. Strain ULB 02/T6 had a higher HA titre than other M. synoviae strains (Table 1).

Examination of CEC-32 cells and CCH using the DIG Glycan Differentiation Kit showed presence of glycoproteins containing sia(α2-3)gal and sia(α2-6)gal linkages.

CCH showed a major sialylated glycoprotein of about 70 kDa (Fig. 1), while CEC-32 cells contained a major sialylated glycoprotein of about 60 kDa (data not shown).

Invasion of chicken erythrocytes

The RIF for chicken erythrocytes was determined for two M. synoviae strains (WVU 1853 and ULB 02/T6) and compared to the RIF for M. gallisepticum strain R low (Tables 1,2), which invades sheep and chicken erythrocytes [START_REF] Vogl | Mycoplasma gallisepticum invades chicken erythrocytes during infection[END_REF]. M. synoviae strains were approximately 20-fold more susceptible to gentamicin than M. gallisepticum R low (Table 1).

Three hours after infection, M. gallisepticum and both M. synoviae strains were viable in erythrocytes treated with gentamicin, most probably because they had established intracellular residence. However, their estimated RIF values after 3 hours were much A c c e p t e d M a n u s c r i p t 10 lower (5-to 10-fold) than after 24 hours. After 24 hours, the mean RIF for M. gallisepticum R low (1.2 % ± 0.7) was similar to that reported by [START_REF] Vogl | Mycoplasma gallisepticum invades chicken erythrocytes during infection[END_REF].

The mean RIF for the M. synoviae strain ULB 02/T6 was also similar (Table 2). The type strain of M. synoviae, WVU 1853, had the greatest invasiveness for CER with a mean RIF (7.5 % ± 1.5) more than 6-fold higher than that of M. gallisepticum R low (Table 2). All three mycoplasma strains were capable of surviving within CER for 48 hours, but with no increase in the RIF after 24 hours for any strain. Invasion of CER was confirmed by immunofluorescent staining of internalized M. synoviae, which reacted with specific antibodies labeled with FITC after permeabilization of CER (data not shown).

Invasion of CEC-32 cells

Invasion of CEC-32 cells by M. gallisepticum R low was detected 4 hours after infection. After 24 hours, the mean RIF was 1.0 % ± 0.2 and did not increase over the following 24 hours (Table 2, data for 4 and 48-hour infection not shown). M. synoviae strains WVU 1853 and ULB 02/T6 also invaded CEC-32 cells after 4 hours of infection and survived inside them for 48 hours. However, after 24 hours of infection, the mean RIF of WVU 1853 (7.0 % ± 0.3) was almost 6-fold higher in comparison to ULB 02/T6. Lethality for CEC-32 cells was not observed over the first 24 hours (data not shown). Differential immunofluorescence microscopy confirmed the invasion of CEC-32 cells with WVU 1853 24 hours after infection. Besides numerous extracellular M.synoviae located at CEC-32 cells' surface, there were also distinct green foci inside these cells indicating the intracellular M. synoviae cells, which were immunostained only with antibodies conjugated with FITC. Superimposed images of micrographs showing red and green fluorescence showed extracellular M. synoviae as yellow foci or aggregates, whereas the presence of intracellular M. synoviae was

Invasion of chicken chondrocytes

After 24 hours of infection, RIF for the M. gallisepticum strain R low in CCH was quite similar (1.1 % ± 0.1) to that seen in CER and CEC-32 cells, as well as to that of M. synoviae strain WVU 1853 (1.2 % ± 0.3) in CCH (Table 2). On the other hand, the invasiveness of WVU 1853 was 6-fold lower in CCH than in CER and CEC-32 cells.

The capability to invade CCH within 2 hours after infection was determined for WVU 1853 (RIF ~ 0.25 %) and field strains ULB 08/T3 and ULB 08/T4. The latter two strains were used at a low passage, but after 24 hours of infection their RIF was not higher than that of WVU1853 (Table 2). M. synoviae strains remained viable within CCH for 48 hours after infection, but there was no evidence of further increase of their RIF (data not shown). The ability of WVU 1853 to invade CCH in 24 hours after infection was confirmed by differential immunofluorescence microscopy. In agreement with the gentamicin invasion assay results, the extracellular population of M. synoviae was predominant and appeared to form small aggregates on the surface of CCH. However, distinct foci of green fluorescence inside CCH, which were present only in CCH infected with M. synoviae, confirmed that it invades CCH (data not shown).

Discussion

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

Although M. gallisepticum and M. synoviae belong to different phylogenetic groups, they share hosts, tissue tropism and a number of horizontally transferred genes (Noormohammadi et al., 1998;Berčič et al., 2008a). Recent studies have provided evidence that some M. gallisepticum strains can invade cells, including chicken embryonic fibroblasts and erythrocytes [START_REF] Winner | In vitro cell invasion of Mycoplasma gallisepticum[END_REF][START_REF] Vogl | Mycoplasma gallisepticum invades chicken erythrocytes during infection[END_REF]. This is the first study to show that M. synoviae can invade chicken erythrocytes, chondrocytes and embryonic cells in vitro.

The well established gentamicin invasion assay, previously used to demonstrate invasion by M. gallisepticum [START_REF] Winner | In vitro cell invasion of Mycoplasma gallisepticum[END_REF][START_REF] Vogl | Mycoplasma gallisepticum invades chicken erythrocytes during infection[END_REF] was used with infected chicken cells exposed to 500 μg gentamicin/ml, a concentration almost 200fold higher than the MIC for the M. synoviae strains tested. The M. synoviae strains used were at least 20-fold more susceptible to gentamicin than M. gallisepticum R low (Table 1). This is consistent with the data published for MIC of these Mycoplasma species (Bebear and Kempf, 2005). Thus, it is very likely that all M. synoviae recovered from the infected chicken cells originated from M. synoviae cells that had invaded the chicken cells and thus escaped the mycoplasmacidal effect of gentamicin.

All M. synoviae cultures recovered from CER, CCH and CEC-32 cells after treatment with gentamicin had the same MIC as the original cultures, between 2 and 3 μg/ml.

It has been suggested that the HAD + phenotype of M. gallisepticum strain R low could be responsible for its higher invasiveness in erythrocytes [START_REF] Vogl | Mycoplasma gallisepticum invades chicken erythrocytes during infection[END_REF]. In this study, the type strain WVU 1853 of M. synoviae had the highest RIF in erythrocytes, but another M. synoviae strain (ULB 02/T6) had a higher HA titre (Tables 1 and2).

Thus, there must be additional factors that contribute to invasiveness in erythrocytes, as well as CEC-32 cells and CCH. These factors may differ for different host cells as WVU 1853 had about 6-fold higher RIF in CER and CEC-32 cells than in CCH.

A c c e p t e d M a n u s c r i p t 13

The adherence to chicken cells via receptors containing sialic acid is important in infections with M. gallisepticum and M. synoviae [START_REF] Aldridge | Growth and cytopathology of Mycoplasma synoviae in chicken embryo cell culture[END_REF][START_REF] Razin | Mycoplasma adherence[END_REF]Benčina, 2002). It is likely that major sialylated glycoprotein(s) of CCH (~ 70 kDa) (Figure 1) and CEC-32 cells (~ 60 kDa) could be involved in binding of M. synoviae and M. gallisepticum to these chicken cells. However, further investigations are required to confirm this and to identify genes encoding sialylated glycoproteins of CEC-32 and CCH cells.

It is probable that the gentamicin invasion assay indicates the number of infected host cells better than the number of invasive mycoplasmas [START_REF] Vogl | Mycoplasma gallisepticum invades chicken erythrocytes during infection[END_REF]. Differential immunofluorescent staining showed that CER and CEC-32 cells were mostly infected with M. synoviae attached to the surface these cells, but that a considerable number of cells contained intracellular M. synoviae (Supplementary Figs. 2 and3). Although the invasion frequency was not estimated with immunofluorescence microscopy, this finding confirms the capacity of M. synoviae to invade chicken cells in vitro.

It remains to be proved that M. synoviae also invades chicken cells in vivo. Infections of the upper respiratory tract with M. synoviae may be lifelong and are usually detectable by culturing swabs of the upper part of trachea and/or choanal cleft [START_REF] Kleven | Mycoplasma synoviae infection[END_REF]. Intracellular localization could help explain, at least partially, persistence of M. synoviae despite of specific local antibodies and phagocytosis and antibiotic treatment.

Conclusion

This study provides the first evidence for invasion and intracellular survival of M. synoviae in three different non-phagocytic chicken cell types in vitro. These findings Microbiol. 15,121,[278][279][280][281][282][283][284][285][286][287] Levisohn, S., Dykstra, M.J., Lin, M.Y., Kleven, S.H., 1986 b Numbers of in vitro passages before an appropriate mycoplasma culture was used for gentamicin invasion assay in this study.

c Minimal inhibitory concentration of gentamicin (μg/ml).

d Haemagglutination titer, the reciprocal of the highest dilution of mycoplasmal cell pellet which gave complete HA of 1 % suspension of chicken erythrocytes (Narat et al., 1998).

e CER: chicken erythrocytes, CEC-32: chicken embryonic cells, CCH: chicken chondrocytes. The numbers indicate the number of independent assays performed.

f NA: not assayed.

  Antibodies conjugated with fluorescein isothiocyanate (FITC) (diluted 1:100, 1 h at room temperature) were used for immunostaining of intracellular M. synoviae. Dako Fluorecence Mounting medium (Dako, USA) was used to prevent fluorescence A c c e p t e d M a n u s c r i p t fading. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.

  fluorescence (Supplementary Fig.2). Confocal scan of CEC-32 cells infected with WVU 1853, shown in Supplementary Fig.3, confirmed the intracellular localization of M. synoviae. M. synoviae labeled with FITC-conjugated antibodies were located in the cytoplasm of CEC-32 cells and became visible when the image was scanned from the top to the bottom.

  of how M. synoviae evades immune surveillance and persists in immunocompetent chickens. A c c e p t e d M a n u s c r i p t 15 Bebear, C.M., Kempf, I., 2005. Antimicrobial therapy and antimicrobial resistance. In: Blanchard, A., Browning, G. (Eds.), Mycoplasmas Molecular biology, pathogenicity and strategies for control, Horizon bioscience, pp. 535-569. Benčina D., 2002. Haemagglutinins of pathogenic avian mycoplasmas. Avian Pathol. 31, 6, 535-547. Benčina, D., Bradbury, J.M., 1992. Combination of immunofluorescence and immunoperoxidase techniques for serotyping mixtures of Mycoplasma species. J. Clin. Microbiol. 30, 407-410. Benčina, D., Kleven, S.H., Elfaki, M.G., Snoj, A., Dovč, P., Dorrer, D., Russ, I., 1994. Variable expression of epitopes on the surface of Mycoplasma gallisepticum demonstrated with monoclonal antibodies. Avian Pathol. 23, 19-36.Benčina, D., Narat, M., Dovc, P., Drobnič-Valič, M., Habe, F.,Kleven, S. H., 1999. The characterization of Mycoplasma synoviae EF-Tu protein and proteins involved in hemadherence and their N-terminal amino acid sequences. FEMS Microbiol.Lett. 173, 85-94. Benčina, D., Drobnič-Valič, M., Horvat, S., Narat, M., Kleven, S.H., Dovč, P., 2001. Molecular basis of the length variation in the N-terminal part of Mycoplasma synoviae hemagglutinin. FEMS Microbiol.Lett. 203, 115-123. Berčič, R.L., Slavec, B., Lavrič, M., Narat, M., Bidovec, A., Dovč, P., Benčina, D., 2008a. Identification of major immunogenic proteins of Mycoplasma synoviae isolates.Vet. Microbiol. 127,[147][148][149][150][151][152][153][154]

  .,Benčina, D., Kothlow, S., Kaspers, B., Narat, M., 2007. Mycoplasma synoviae lipoprotein MSPB, the N-terminal part of VlhA haemagglutinin, induces secretion of nitric oxide, IL-6 and IL-1beta in chicken macrophages. Vet.

Figure 1 :

 1 Figure captions

Figure 1 :

 1 Figure 1: Identification of the Mycoplasma synoviae WVU 1853 colonies by direct

Figure 2 :

 2 Figure 2: Differential immunofluorescence images of a CEC-32 cell infected with M.

Figure 3 :

 3 Figure 3: Confocal scan of a CEC-32 cell infected with M. synoviae WVU 1853 after

Fig. 1 :

 1 Fig. 1: Major sialylated glycoprotein of chicken chondrocytes. Proteins were separated by

  . Comparison of in vivo and in vitro methods for pathogenicity evaluation for Mycoplasma gallisepticum in respiratory infection. Avian Pathol. 15, 233-246. Lo, S.C., Dawson, M.S., Wong, D.M., Newton III, P.B., Sonoda, M.A., Engler, W.F., Wang, R.Y., Shih, J.W., Alter, H.J., Wear, D.J., 1989. Identification of

	Mycoplasma	incognitus	infection	in	patients	with	AIDS:	an
	immunohistochemical, in situ hybridization and ultrastructural study. Am. J. Trop.
	Med. Hyg. 41, 601-616.
	Much, P., Winner, F., Stipkovits, L., Rosengarten, R., Citti, C., 2003. Mycoplasma
	gallisepticum: influence of cell invasiveness on the outcome of experimental
	infection in chickens. FEMS Immunol. Med. Microbiol. 34, 181-186.
	Narat M., Benčina, D., Kleven, S.H., Habe, F., 1998. The hemagglutination-positive
	phenotype of Mycoplasma synoviae induces experimental infectious synovitis in
	chickens more frequently than does the hemagglutination-negative phenotype.
	Infect. Immun. 66, 12, 6004-6009.
	Noormohammadi, A.H., Markham, P.F., Duffy, M.F., Whithear, K.G., Browning,
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	phylogenetically distinct mycoplasmas. Infect Immun. 66, 7, 3470-3475.

Table 1 :

 1 Mycoplasma species, their strains and chicken cells used in the gentamicin invasion assay a

	Chicken cells e

Table 2 :

 2 Results of only three experiments were shown in Table 2. Relative invasion frequency of M. gallisepticum and M. synoviae strains for chicken cells a

			% of invasion for chicken cells b
	Species	Strain	CER	CEC-32	CCH
	M. gallisepticum	R low	1.2 ± 0.7	1.0 ± 0.2	1.1 ± 0.1
	M. synoviae	WVU 1853 c	7.5 ± 1.5	7.0 ± 0.3	1.2 ± 0.3
	M. synoviae M. synoviae M. synoviae	ULB 02/T6 ULB 08/T3 ULB 08/T4	M 1.4 ± 1.1 NA d a n u s c r i p t 1.2 ± 0.5 NA NA 0.6 ± 0.2 e NA NA 0.7 ± 0.2
	A c c e p t e d
					20

g a Evaluated and calculated 24 h after infection of appropriate cells with mycoplasmas (MOI ranged from 10 to 100 mycoplasmas per chicken cell).

b Evaluated and calculated as described in Materials and Methods (see also

[START_REF] Vogl | Mycoplasma gallisepticum invades chicken erythrocytes during infection[END_REF]

).

c

The difference in invasiveness of M. synoviae strain WVU 1853 was significant for CER versus CCH (p < 0.003) and for CEC-32 versus CCH (p ≤ 0.0001). The difference between RIF of M. synoviae strain WVU 1853 and both the M. synoviae ULB02/T6 and M. gallisepticum R low was also significant for both CER (p < 0.005 and <0.003, respectively) and CEC-32 (p ≤ 0.0001 for both Mycoplasma species). There was no significance between the differences in RIF of Mycoplasma species for CCH. d NA: not assayed e Extremely high RIF in one experiment (9.25 %) is not included in the mean RIF for ULB 08/T3. It was about one order of magnitude higher than all other RIF values in CCH in this study and was considered erroneous.

Acknowledgements

This work was supported by grant 1000-07-310121 from the Ministry of Education and Science of the Republic of Slovenia. For erythrocytes from SPF-chickens, we thank Prof. Dr. Olga Zorman Rojs. For the donation of Mycoplasma gallisepticum strain R low , we thank Dr. Michael Szostak and for the donation of CEC-32 cells Prof.

Bernd Kaspers. We would also like to thank Drs. Irena Oven and Nataša Obermajer for the help with the preparation of fluorescence images.

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Keywords: Equine Influenza virus, H3N8, surveillance, vaccine strain selection

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

Introduction

Equine influenza virus (EIV) is an Orthomyxovirus containing eight segments of single stranded negative sense RNA encoding 10 genes. Characteristically EIV can spread very rapidly in a susceptible population and continues to be an economically important pathogen of horses worldwide. Typical clinical signs of infection in fully susceptible animals include pyrexia, coughing, nasal discharge, dyspnoea, anorexia and very rarely ataxia [START_REF] Daly | Investigation of equine influenza cases exhibiting neurological disease: coincidence or association?[END_REF]. The H7N7 subtype of EIV was first isolated in 1956 in Eastern Europe [START_REF] Sovinova | Isolation of a virus causing respiratory disease in horses[END_REF]. However, no H7N7 subtype virus has been isolated from horses since 1977 (Webster, 1993). In 1963 the first EIV from the H3N8 subtype was the cause of a major epidemic in the USA that was thought to have entered the country with imported horses from Argentina (Waddell et al., 1963).

The virus, A/eq/Miami/63, was designated as the prototype virus and viruses of this subtype have circulated in horse populations ever since.

An early study suggested that H3N8 subtype viruses evolve as a single lineage [START_REF] Kawaoka | Evolution of the hemagglutinin of equine H3 influenza viruses[END_REF]. Later work has shown that two lineages of H3N8 subtype virus emerged in the late 1980s and have continued to circulate since [START_REF] Daly | Antigenic and genetic evolution of equine H3N8 influenza A viruses[END_REF]. The two lineages, American and Eurasian, were initially named based on their geographical location. However, it was apparent that American lineage viruses were also circulating in Europe. Work by Lai and colleagues (2001) studying North American EIV isolates identified a divergence of these viruses into three sublineages, namely a South American lineage, a Kentucky lineage, and a Florida lineage. Further evolution of the Florida sublineage has resulted in the emergence of two groups of viruses with divergent HA sequences which are provisionally referred to as Florida sublineage clade 1 and clade 2 viruses. Clade 1 includes the A/eq/Wisconsin/03-like A c c e p t e d M a n u s c r i p t 4 viruses while clade 2 is represented by the A/eq/Newmarket/5/03-like viruses [START_REF] Oie | Conclusions and recommendations from the Expert Surveillance Panel on Equine Influenza Vaccines[END_REF]. The Kentucky lineage has since been referred to as the classical American lineage.

In addition to the linear evolution of HA, the segmented nature of the influenza virus genome allows reassortment to take place resulting in rapid virus evolution (Webster et al., 1992). Reassortment between similar viruses may occur all the time but is only noticeable and significant if it occurs between distinct cocirculating viral strains. RNA hybridisation experiments and nucleotide analysis of H3N8 viruses have shown reassortment of RNA segments encoding NP [START_REF] Bean | Correlation of influenza A virus nucleoprotein genes with host species[END_REF], Gorman et al., 1990a), PB2 (Gorman et al., 1990b) and PA [START_REF] Okazaki | Evolutionary pathways of the PA genes of influenza A viruses[END_REF] between the equine H7N7 and H3N8 subtypes.

The non-structural protein (NS1) has been shown to be a virulence factor in influenza A virus infection in vivo [START_REF] Hale | The multifunctional NS1 protein of influenza A viruses[END_REF] so is of particular interest when studying the relative pathogenicity of isolated viruses. It functions as an antagonist of the innate immune response by blocking the activation of interferon [START_REF] Garcia-Sastre | Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems[END_REF] via regulatory factor-3 (Talon et al., 2000) and inhibiting the posttranscriptional processing of cellular mRNAs [START_REF] Fortes | Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport[END_REF]. NS1 proteins from some influenza viruses are also able to inhibit cellular gene expression, inhibiting the action of IFN [START_REF] Hayman | Variation in the ability of human influenza A viruses to induce and inhibit the IFN-beta pathway[END_REF]. NS1 sequences have been grouped into two different alleles (A or B) [START_REF] Ludwig | Phylogenetic relationship of the nonstructural (NS) genes of influenza A viruses[END_REF]. Allele A genes are found in human, swine, many avian and equine viruses, with the exception of a strain from an outbreak in China in 1989 (A/eq/Jilin/89), which is thought to be a relatively recent crossover from an avian source and contains an allele B gene. Other allele B genes are found in some American and Eurasian avian influenza viruses and a single equine isolate, A/eq/Prague/56 [START_REF] Suarez | Multiple alignment comparison of the nonstructural genes of influenza A viruses[END_REF].

A c c e p t e d M a n u s c r i p t 5 New virus variants can give rise to serious EIV epidemics, such as those in 1979 in Europe and North America (van Oirschot et al., 1981). As a result of this outbreak, mandatory vaccinations were introduced for race and competition horses in the UK. However, in 1989 an outbreak of EIV occurred in the UK and continental Europe caused by a virus, typified by the isolate A/eq/Suffolk/89, which infected both vaccinated and non-vaccinated horses. Antigenic and sequence analysis showed this virus was significantly different from the vaccine strains in use at the time (Livesay et al., 1993, Binns et al., 1993). Subsequent work using a Welsh Mountain pony challenge model showed that protection from virus challenge correlated with the antigenic relatedness of the vaccine to the challenge virus strain [START_REF] Mumford | Control of influenza from an international perspective[END_REF], Yates & Mumford, 2000[START_REF] Daly | Comparison of hamster and pony challenge models for evaluation of effect of antigenic drift on cross protection afforded by equine influenza vaccines[END_REF]. OIE recommendations from 1993 state that a modern virus variant should be incorporated into vaccines and that surveillance efforts should be intensified to keep track of all future developments (Mumford & Wood, 1993).

The last major outbreak of EIV in the UK was caused by a Florida sublineage clade 2 virus, designated A/eq/Newmarket/5/03, that caused disease in vaccinated and non-vaccinated horses in the field [START_REF] Lai | Diverged evolution of recent equine-2 influenza (H3N8) viruses in the Western Hemisphere[END_REF][START_REF] Newton | Description of the equine influenza (H3N8) outbreak in the United Kingdom during 2003, during which recent vaccination failed to prevent disease in racehorses in Newmarket[END_REF] et al., 2008[START_REF] Callinan | Equine Influenza: The August 2007 outbreak in Australia[END_REF].

In 2006 the OIE officially recommended that vaccines should contain a representative of the Florida lineage (clade 1) (A/eq/SouthAfrica/4/03 or A/eq/Ohio/03) and a representative of the Eurasian lineage (A/eq/Newmarket/2/93like) [START_REF] Oie | Conclusions and recommendations from the Expert Surveillance Panel on Equine Influenza Vaccines[END_REF]. Recommendations are reviewed on an annual basis and take into account up to date surveillance data from a number of countries. The previous recommendation was confirmed by the OIE in 2008 following its annual review of events in 2007 [START_REF] Oie | Conclusions and recommendations from the Expert Surveillance Panel on Equine Influenza Vaccines[END_REF]. This report is a summary of the EIV surveillance data collected at the Animal Health Trust in 2006 and 2007, including isolates submitted from the UK, Switzerland and the USA consisting of information on sequence analysis, antigenic and phylogenetic relationships.

Materials and methods

NP-ELISA and Directigen Flu A

A nucleoprotein enzyme-linked immunosorbent assay (NP-ELISA) was used to detect viral nucleoprotein (NP) in the nasal swab extract [START_REF] Cook | Detection of influenza nucleoprotein antigen in nasal secretions from horses infected with A/equine influenza (H3N8) viruses[END_REF].

Briefly, 100 l Tween-20 treated nasal swab extract (2l 10% Tween-20 in 200 l nasal wash for 5 min) was added to a 96-well plate coated with anti-A/eq/Sussex/89 rabbit polyclonal antibody and allowed to bind for 90 min. After washing 3 times in phosphate buffered saline-0.2%-Tween-20 (PBS-T), bound virus was incubated with the mouse monoclonal antibody 3G9, specific for NP, in a 60 min incubation step, then washed three times in PBS-T. Samples were then incubated with anti-mouse A c c e p t e d M a n u s c r i p t 7 peroxidase conjugated IgG antibody (Dako) for 30 min incubation step at 37 C followed by three washes, before being developed with the chromogen (3'3'5'5'-Tetramethylbenzidine) TMB (Sigma) for up to 5 minutes at room temperature. The ELISA was stopped using 2M sulphuric acid and plates were analysed spectrophotometrically at 450 nm.

North American samples were tested by the Directigen Flu A test kit (BD, NJ, USA) as instructed by the manufacturer.

Viruses

Isolation of viruses from the UK

Nasopharyngeal swabs (made in house) were taken from horses showing signs of acute respiratory disease. Swabs were placed in sterile tubes containing virus transport medium (VTM) consisting of PBS, 200 U/ml streptomycin, 150 U/ml penicillin, 5g/ml fungizone (Gibco) and 600 g/ml tryptone phosphate broth and chilled on ice immediately after collection. VTM samples that tested positive in the NP-ELISA were inoculated into the allantoic cavities of 10 day old embryonated hens' eggs. Eggs were incubated at 34  C and harvested 3 days post infection. Virus was detected by HA assay using 1% chicken erythrocytes in PBS. Viruses were serially passaged until stable titres were obtained, up to a maximum of five passages (Table 1).

Viruses from North American and Switzerland

A c c e p t e d M a n u s c r i p t 8 Viruses were isolated as described in eggs. Infected allantoic fluid was received frozen on dry ice. In order to obtain enough material for haemagglutination inhibition assays and sequencing, viruses were further amplified in eggs.

Haemagglutination inhibition assay

Serological analysis was conducted using haemagglutination inhibition assays (HI) as previously described using either native virus for virus differentiation or ether/Tween-80 treated virus for diagnostic purposes [START_REF] Daly | Antigenic and genetic evolution of equine H3N8 influenza A viruses[END_REF]. For virus differentiation the untreated viruses were assayed using ferret sera pre-treated with heat and periodate, using 1% chicken erythrocytes as previously described. Geometric mean titres were calculated for three HI tests for each combination. Viruses were tested against antisera specific for the strains A/eq/Newmarket/1/93, A/eq/Newmarket/2/93, A/eq/Kentucky/97, A/eq/Kentucky/98, A/eq/Lincolnshire/1/02, A/eq/Benelux/03, A/eq/Newmarket/5/03 and A/eq/South Africa/4/03.

Viral RNA isolation, RT-PCR and sequencing

Viral RNA was isolated from nasopharyngeal swabs or allantoic fluid using the QIAamp Viral RNA mini kit (Qiagen) according to the manufacturer's instructions. Genes of interest were amplified using the RobusT I RT-PCR kit (Finnzymes) with 2l of template RNA and 0.1 U/ml RNAsin (Promega) using the gene specific primers H3HA1/1 (5'-AGCAAAAGCAGGGGATATTTC) and H3HA1/2 (5'-GCTATTGCTCCAAAGATTC) for HA1 and primers NS1/EcoI (5'-

Phylogenetic trees, multiple sequence alignments and amino acid mapping

To determine the relationship between the EIV isolates a phylogenetic tree was constructed. MODELTEST was used to determine the best evolutionary model for the data (http://hcv.lanl.gov/content/sequence/findmodel/findmodel.html) [START_REF] Posada | Modeltest: testing the model of DNA substitution[END_REF]. Phylogenetic trees were constructed using PAUP version 4.0 [START_REF] Swofford | Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein[END_REF] under the General Time Reversible substitution model, as determined by MODELTEST, with branch swapping by tree-bisection-reconnection.

One hundred bootstrap replicates were conducted to assess the statistical support for the tree topology. Accession numbers of the virus strains in the trees are as follows (HA1, NS1): A/eq/Miami/63 (M29257, CY028840), A/eq/Fontainebleau/79 (CY032405, CY032409), A/eq/Newmarket/79 (D30677, AF001662), A/eq/Kentucky/2/81 (CY028820, CY028824), A/eq/Sussex/89 (X85090, FJ375212), A/eq/Rome/5/91 (D30684, AF001669), A/eq/Hong-Kong/92 (L27597, AF001670), and 2007 from the UK, and from 20 of these, virus was recovered following growth in eggs (Table 1). The following outlines the clinical and vaccination histories of the affected animals.

In 2006, A/eq/Cheshire/1/06 was isolated from a large mixed yard of horses of different ages. At least 10 animals were infected and showed typical equine influenza (EI) clinical signs, two of which were recently vaccinated. A/eq/Southampton/1/06 was isolated from one of four infected horses that had uncertain vaccination histories.

The NP-ELISA positive horse from Stowmarket in the UK was unvaccinated but had travelled from Ireland 2 days prior to showing typical clinical signs of EI.

A/eq/Lanark/1/06 was isolated from a horse with an uncertain vaccination history that was imported from Poland three days before showing clinical signs of infection.

A/eq/Lincolnshire/1/06 was isolated from an unvaccinated British spotted pony that had been in contact with a group of recently imported Friesian horses. Clinical signs were typical and included cough, nasal discharge and reduced appetite.

In 2007, A/eq/Horsham/1/07 was isolated from an infected horse on a large A/eq/Lincolnshire/107 was isolated from a horse with clinical signs imported into the UK from Spain. The lorry had been elsewhere in Europe prior to this and there were

Genetic characterisation

HA1

A c c e p t e d M a n u s c r i p t 14

The HA1 (1009 bp) from each virus isolate was sequenced and accession numbers are listed in table 1. EIV isolates were grouped into five well supported clades consisting of the Pre-divergent, Eurasian and American lineages with the Florida sublineage clades 1 and 2 (Fig. 1a). Phylogenetic analysis grouped 18 of the 20 isolates from the UK within the Florida clade 2 sublineage. The isolate designated A/eq/Lincolnshire/1/07 was unique among the UK isolates as it clustered with the Florida clade 1 sublineage viruses. The isolate designated A/eq/Cheshire/1/06 grouped with the American (Kentucky) lineage viruses. The isolate from Switzerland was different from those isolated in the UK over the same time period as it belonged to the Eurasian lineage and clustered alongside the older strain A/eq/Sussex/89 (Fig 1a). The HA1 of the North American virus isolates characterised here all clustered in the Florida clade 1 sublineage, typical of the viruses isolated in or originating from North America [START_REF] Lai | Diverged evolution of recent equine-2 influenza (H3N8) viruses in the Western Hemisphere[END_REF]) (Fig. 1a).

NS1

The NS1 (692 bp) coding sequence of each virus isolate was determined and accession numbers are listed in Table 1. The topology of the maximum-likelihood tree showed four clades corresponding to the Pre-divergent, American, Eurasian and Florida lineage viruses similar to those seen with the HA1 coding sequences. There was evidence of further differentiation into two different clades in the Florida sublineage cluster, as seen with HA1, the bootstrap value for this division in NS1 was 81% compared to 100% for HA1 (Fig. 1b). However, A/eq/Lincolnshire/1/06, which was within the Florida sublineage when analysing the HA1 coding sequence, grouped with the Eurasian lineage when analysing the NS1 coding sequence, and

A c c e p t e d M a n u s c r i p t 15 A/eq/Kentucky/98, which was American lineage by HA1 phylogeny was more similar to the Florida clade 1 viruses by NS1 phylogeny (Fig. 1b). The two isolates that were unusual according to their HA1 sequences, A/eq/Cheshire/1/06 and A/eq/Switzerland/P112/07, grouped as their HA1 sequences did, with the American lineage and Eurasian lineage NS1 sequences respectively. Both phylogenetic trees in Figure 1 contain only unique sequences and any isolates with identical sequences were omitted as described in the materials and methods.

Antigenic analysis of EIV isolates

The antigenic properties of the EIV isolates were evaluated using ferret antisera raised against a panel of 8 AHT reference strains (Table 2). The isolate A/eq/Berkshire/1/07 was not characterised by HI assay as it only grew to a low titre in eggs after repeated passage. The American lineage and Florida sublineage clade 1 and 2 viruses isolated in the UK and North America exhibited low reactivity to the antisera raised against Eurasian lineage isolates with the exception of A/eq/Pennsylvania/1/07, for which the highest titre obtained was against the A/eq/Benelux/03 antisera, although titres were generally low across the entire reference panel. The 17 Florida sublineage clade 2 viruses isolated from the UK and tested by HI all showed similar patterns of reactivity with the antisera, with 16 of the 17 strains showing an increase of between 2 and four fold in reactivity against A/eq/Newmarket/5/03 when compared to A/eq/South-Africa/4/03, and one strain showing a greater than 4 fold increase. Conversely 5 of the 7 Florida clade 1 viruses, one of which was isolated in the UK (A/eq/Lincolnshire/1/07) and the rest from North America, reacted 4-fold or more higher against A/eq/South-Africa/4/03 when A/eq/California/2/07 and A/eq/Pennsylvania/1/07 both reacted higher to the A/eq/South-Africa/4/03 but only to a level of 2-fold or less respectively. Another trend between the clade 1 and clade 2 Florida sublineage viruses was their reactivity with the antiserum raised against A/eq/Kentucky/97, an early member of the Florida sublineage. The majority of Clade 1 viruses raised relatively low titres (32) against A/eq/Kentucky/97, with the exception of A/eq/Kentucky/4/07 and A/eq/Kentucky/7/07, whereas the Clade 2 viruses raised higher titres of between 91 and 512. In contrast to the Florida sublineage clade 2 viruses isolated in the UK, A/eq/Cheshire/1/06, classified as an American lineage virus, had relatively low reactivity with the A/eq/Kentucky/97 antiserum and is more similar to those titres seen with the Florida clade 1 viruses from North America. It also reacted weakly across the panel when compared to A/eq/Newmarket/1/93, even though the predicted HA1 amino acid sequence is identical.

A/eq/Switzerland/P112/07, classified as a member of the Eurasian lineage on the basis of HA1 sequence, had low reactivity across the panel. It raised a weak titre of 16 against A/eq/Benelux/03 and A/eq/Kentucky/97 and was a low reactor to all sera.

Amino acid alignments

In order to identify which amino acids may play a role in the separation into different phylogenetic clades, amino acid sequence alignments of the HA1 and NS1 coding sequences were constructed with representative isolates aligned against A/eq/Newmarket/5/03. Any HA1 changes are summarised in 4. The numbering of the amino acid positions of the HA1 sequences starts with the serine residue immediately downstream of the predicted signal peptide cleavage site as 1. This was consistent with the numbering used for the H3 HA structure [START_REF] Ha | X-ray structure of the hemagglutinin of a potential H3 avian progenitor of the 1968 Hong Kong pandemic influenza virus[END_REF]. Negative numbers represent the predicted signal sequence. All the recent isolates had the substitutions at amino acids 190 and 193 when compared to A/eq/Newmarket/1/93, predicted to be within antigenic site B and first observed in A/eq/Newmarket/5/03 with the exception of A/eq/Cheshire/1/06 that was more similar to A/eq/Newmarket/1/93. The viruses belonging to the Florida sublineage clade 1 (North American isolates A/eq/Florida/2/06, A/eq/California/2/07, A/eq/Kentucky/4/07, A/eq/Kentucky/7/07, A/eq/Pennsylvania/1/07 and the UK isolate A/eq/Lincolnshire/1/07) all had very similar HA1 sequences in which the majority differ from American and Eurasian lineage viruses at residues -16, 62, 78, 104, 138 and 159. Between A/eq/South-Africa/4/03 and the latest Florida sublineage clade 1 isolates there were 10 different amino acid substitutions, one of which was in the signal sequence. The isolates A/eq/California/2/07 and A/eq/Kentucky/7/07 both contained 6 of these amino acid substitutions. A/eq/Pennsylvania/1/07 and A/eq/Ibaraki/07 shared the substitutions at residues V78A and N159S but not the changes at residues R62K, D104N or A138S with the other clade 1 isolates. They also had the additional unique changes at residues P162S and Q189K and when compared to A/eq/Newmarket/5/03. The Florida sublineage clade 2 viruses isolated in the UK including, A/eq/Linconshire/1/06, A/eq/Richmond/1/07 and A/eq/Newmarket/1/07, had a unique two amino acid insertion in the predicted signal peptide at residues -11 and -10, including one aromatic residue, combined with a leucine to phenylalanine substitution at -9, to generate a 'FIF' or 'FNF' motif . The predicted HA1 sequences of A/eq/Newmarket/1/93 and A/eq/Cheshire/06 were identical, with substitutions at 5,30,48,58,190,193,272 and 289 when compared to A/eq/Newmarket/5/03. The Eurasian lineage viruses A/eq/Sussex/89 and A/eq/Switzerland/P112/07 shared many of the amino acid changes listed above for the American lineage viruses, but also have some specific changes at T163I, K261R and I276T.

NS1 changes are summarised in Table 4. The previously observed truncation with a stop codon at position 220 was found in all 2006 to 2007 isolates apart from A/eq/Cheshire/1/06 and A/eq/Switzerland/P112/07. There were at least 8 amino acid substitutions seen between the NS1 from American (A/eq/Newmarket/1/93 and A/eq/Cheshire/1/06) and Eurasian lineage viruses (A/eq/Sussex/89 and A/eq/Switzerland/P112/07). There was also no stop codon at position 220 and the protein terminated at position 231. A/eq/Lincolnshire/1/06 NS1 also had these substitutions, making it most similar to the Eurasian lineage viruses, but with a further 7 substitutions unique to this isolate. Further substitutions between Florida sublineage clade 2, American and Eurasian lineage viruses occur consistently at I48S and I84V, and more recently H206Y. The differences between the Florida clade 2 sublineage viruses and the other lineages highlight A/eq/Lincolnshire/1/07 as a Florida clade 1 virus.

Discussion

EIV surveillance was conducted in order to identify and characterise currently circulating and new emerging virus strains to provide data for vaccine strain selection.

The surveillance data for the UK and Europe was consistent with the previous observations that there was a predominance of Florida sublineage clade 2 viruses in A c c e p t e d M a n u s c r i p t 19 circulation [START_REF] Newton | Description of the equine influenza (H3N8) outbreak in the United Kingdom during 2003, during which recent vaccination failed to prevent disease in racehorses in Newmarket[END_REF][START_REF] Damiani | Genetic characterization of equine influenza viruses isolated in Italy between 1999 and 2005[END_REF]). The index cases of the outbreaks that occurred in June 2007 in the UK were animals that had been recently imported into England, having been bought at a horse sale in County Kilkenny in the Republic of Ireland. It was therefore likely to be at least partially representative of the viruses circulating in the Republic of Ireland at that time [START_REF] Newton | Equine influenza in England[END_REF].

Florida sublineage clade 1 viruses were isolated in North America. Interestingly outbreaks of disease often follow horse movement and introduction into new environment. This may be due to stress associated with travelling making the animal more susceptible to infection.

The topology of the maximum-likelihood tree of the HA1 data was similar to that previously described (Fig. 1a) [START_REF] Daly | Antigenic and genetic evolution of equine H3N8 influenza A viruses[END_REF][START_REF] Lai | Diverged evolution of recent equine-2 influenza (H3N8) viruses in the Western Hemisphere[END_REF] and showed that viruses from the two Florida sublineage clades 1 and 2 continued to co-circulate in 2006 and 2007. Amino acid alignment of 2003 Florida clade 1 and clade 2 virus isolates suggested that the changes V78A and N159S were consistent between the clades (Table 3.). They also identified an insertion within the signal peptide that created a 'FIF motif at position -11 to -9 in the Florida sublineage clade 2 viruses isolated in the UK. This insertion was first seen in isolates from 2004 and has been seen in all UK isolates belonging to clade 2 since then (eg. A/eq/Wales/1/05, accession number EF541438). The authors note that the HA sequence of a virus isolated during the Mongolian outbreak in 2008 has been released to GenBank that indicates that this belongs to the Florida clade 2 sublineage (Accession number AB436910). The amino acid alignment in table 3 shows an isoleucine to an asparagine residue substitution at position -10 in A/eq/Newmarket/1/07, the last 2007 isolate described in this paper that changes the 'FIF' sequence to 'FNF'. This change A/eq/Cheshire/1/06 was characterised as a member of the American lineage, viruses of which have not been detected in the UK since isolation of A/eq/Moulton/98, A/eq/Snailwell/98 and A/eq/Edinburgh/98 [START_REF] Newton | Equine influenza in the United Kingdom in 1998[END_REF].

A/eq/Switzerland/P112/07 was the first virus to be characterised as a Eurasian lineage virus, since the isolate from Scotland in 2005 designated A/eq/Aboyne/05 (Accession number: EF541442). The lack of isolates belonging to these other lineages may be due to the relatively small numbers of samples received or the Eurasian lineage may be dying out. It also raises the possibility that circulating Florida sublineage virus strains may be able to acquire gene segments from a simultaneously circulating Eurasian strain or vice versa, potentially resulting in changes in pathogenicity or antigenicity. Reassortment such as this may be responsible for the Eurasian lineagelike NS1 found in A/eq/Lincolnshire/1/06, classified as a Florida sublineage clade 2 virus based on HA1 sequence. A/eq/Cheshire/1/06 and A/eq/Switzerland/P112/07 appeared to be more closely related to viruses isolated between 1989 and 1993 rather than the recent Florida sublineage. There are reports of viruses being isolated that appear to date from earlier in time, and it has been suggested it is due to a phenomenon called frozen evolution [START_REF] Endo | Evolutionary pattern of the H3 haemagglutinin of equine influenza viruses: multiple evolutionary lineages and frozen replication[END_REF][START_REF] Borchers | Characterisation of three equine influenza A H3N8 viruses from Germany (2000 and 2002): evidence for frozen evolution[END_REF]. This may result from a reduced amount of antigenic drift compared to the majority of circulating EIVs. However, these viruses are also very similar to virus reference strains A/eq/Newmarket/1/93 and A/eq/Sussex/89, suggesting a laboratory escape or vaccine origin as suggested previously [START_REF] Gupta | Characterisation of equine influenza isolates from the 1987 epizootic in India by nucleotide sequencing of the HA1 gene[END_REF]. The majority of vaccines currently available, especially in the UK, contain these older strains so they should provide good protection against infection. However, the presence of these viruses in As the UK and North America have two of the few active surveillance systems, it is not known which are the most widely circulating viruses globally and certain phylogenetic lineages may be over represented. Horse movement is a major contributing factor in transmitting EIV around the world, despite mandatory vaccination and quarantine procedures. Epidemiological and phylogenetic studies of EIV outbreaks have often found direct links with international horse movement such as in South Africa in 1986 [START_REF] Guthrie | The circumstances surrounding the outbreak and spread of equine influenza in South Africa[END_REF], Hong Kong in 1992 [START_REF] Lai | Genetic and antigenic analysis of the influenza virus responsible for the 1992 Hong Kong equine influenza epizootic[END_REF] and Newmarket in 2003 [START_REF] Newton | Description of the equine influenza (H3N8) outbreak in the United Kingdom during 2003, during which recent vaccination failed to prevent disease in racehorses in Newmarket[END_REF]. EIV was responsible for several large outbreaks of respiratory disease in 2007. In mid August 2007 an outbreak of EIV was detected in a partially vaccinated horse population in Japan (Yamanaka et al., 2008). Table 3 includes the partial sequence of A/eq/Ibaraki/07, a virus representative of the outbreak strain (Yamanaka et al., 2008). Horses were vaccinated with an inactivated vaccine containing A/eq/LaPlata/93 (American lineage),

A/eq/Avesta/93 (Eurasian lineage) and A/eq/Newmarket/77 (H7N7). These strains, although dated, are not unlike the strains used in the vaccines available in the UK (Fig 1). This raised the concern that currently available vaccines may also be vulnerable to breakdown when used to protect against this or a related virus, as seen in 2003 with the emergence of A/eq/Newmarket/5/03 which infected both vaccinated and nonvaccinated equids in Europe. A/eq/Ibaraki/07 had highest sequence identity with A/eq/Pennsylvania/1/07, a Florida sublineage clade 1 like virus with an HA1 sequence typical of those circulating in North America. However, the HI data presented in table 2 shows that sera raised against currently used vaccine strains and A/eq/South-Africa/4/03 virus that reacts highly to antisera raised against A/eq/Kentucky/98 (Table 2) and to antisera raised against itself only differs at these two amino acids within HA1, suggesting these two changes may be responsible for the differences in antigenicity observed. Residue 189 maps adjacent to the receptorbinding pocket on the structure of H3 and was exposed on the surface [START_REF] Ha | X-ray structure of the hemagglutinin of a potential H3 avian progenitor of the 1968 Hong Kong pandemic influenza virus[END_REF]. The substitution P162S has been previously observed in some pre-divergent strains such as A/eq/Tokyo/71 (Accession number: M24720) but this is on a substantially different backbone sequence that only has 85.8% sequence identity within HA1 when compared to A/eq/Newmarket/5/03. The isolate A/eq/California/2/07 also had low reactivity to the antisera in table 2. This strain contained 6 amino acid substitutions when compared to A/eq/South Africa/4/03, of which R62K, T192K and K207E mapped at or near the surface of the HA1 molecule when aligned with the three dimensional structure of H3 HA [START_REF] Ha | X-ray structure of the hemagglutinin of a potential H3 avian progenitor of the 1968 Hong Kong pandemic influenza virus[END_REF] (data not shown). Later in the same month in 2007 there was an outbreak of EIV in Australia, this time mainly in an unvaccinated population after the virus escaped from a quarantine station into the general horse population. Subsequently the two outbreaks have been linked and a representative virus from the Australian outbreak (A/eq/Sydney2888-8/07) was almost identical to A/eq/Ibaraki/07 [START_REF] Callinan | Equine Influenza: The August 2007 outbreak in Australia[END_REF], Yamanaka et al., 2008). The introduction of horse movement restrictions and horse free buffer zones in conjunction with vaccination using ProteqFlu (Merial) which

A c c e p t e d M a n u s c r i p t expressed both A/eq/Kentucky/94 and A/eq/Newmarket/2/93 HA, finally eradicated EIV from Australia.

Currently there are two recommended components to the vaccine, a Eurasian isolate and a Florida sublineage clade 1 isolate (OIE bulletin, 2008). Many vaccine manufacturers have yet to update their vaccines as recommended by the OIE, although recently 2 manufacturers have done so with the inclusion of A/eq/Ohio/03 (ProteqFlu, Merial and Calvenza, Boeringher Ingelheim). As the antigenic distance increases between vaccine strains and those circulating, so does the risk of EIV infection in the field associated with vaccine breakdown. If both Florida sublineage virus clades persist and diverge in the field, vaccines will have to be updated to allow both clades to be represented in order to confer clade specific immunity.

Vaccine studies have shown that currently available vaccines reduce virus shedding and clinical signs when compared to control ponies when challenged with recent strains of EIV [START_REF] Paillot | Protection, systemic IFNgamma, and antibody responses induced by an ISCOM-based vaccine against a recent equine influenza virus in its natural host[END_REF][START_REF] Bryant | Australian equine influenza: vaccine protection in the UK[END_REF]. However, in these studies, ponies were challenged two weeks post vaccination under optimal experimental conditions more accurately modelling vaccination in the face of infection. Vaccine breakdown in the field did occur in Japan among racehorses previously vaccinated 3 months before the outbreak (Yamanaka et al., 2008). Other experiments in ponies have shown protection induced by non-adjuvanted vaccines containing virus from one lineage against challenge infection with heterologous virus is reduced when compared to challenge with the homologous virus, as in the case of vaccination with A/eq/Newmarket/2/93 (Eurasian) and subsequent challenge with A/Newmarket/1/93 (American). Interestingly, this lack of protection occurs in one direction only in that vaccination with A/eq/Newmarket/1/93 does protect against A c c e p t e d M a n u s c r i p t 24 challenge with A/eq/Newmarket/2/93 [START_REF] Daly | Evidence supporting the inclusion of strains from each of the two co-circulating lineages of H3N8 equine influenza virus in vaccines[END_REF], suggesting the American lineage isolates may confer some protection against Eurasian isolates in the future.

Sequencing of the NS segment showed the truncation that was previously observed in A/eq/Kentucky/5/02 (Accession number AY855345) [START_REF] Quinlivan | Attenuation of equine influenza viruses through truncations of the NS1 protein[END_REF] and then in A/eq/Newmarket/5/03 in the UK (Table 4). The NS1 protein has been shown to play a role in virus pathogenicity. Differences in pathogenicity have been observed between Eurasian and American lineage viruses in experimental infection of ponies (Yates & Mumford, 2000[START_REF] Paillot | Antibody and IFN-gamma responses induced by a recombinant canarypox vaccine and challenge infection with equine influenza virus[END_REF] and it is possible NS1 plays a role similar to that seen for other influenza A viruses. However, the role of specific amino acid substitutions between the NS1 proteins of Eurasian and American lineage EIV isolates have yet to be elucidated.

In conclusion, 28 EIV strains from North America and Europe isolated in Lineage of new isolates is indicated on the left. Homologous titres are shown in bold. N/1/93 -A/eq/Newmarket/1/93, N/2/93 -A/eq/Newmarket/2/93, Ken/97 -A/eq/Kentucky/97, Ken/98 -A/eq/Kentucky/98, Lin/02 -A/eq/Lincolnshire/1/02, Ben/03 -A/eq/Benelux/03, N/5/03 -Newmarket/5/03, SA/4/03 -A/eq/South Africa/4/03, Am -American lineage, Eu -Eurasian lineage, FC1 -Florida sublineage clade 1, FC2-Florida sublineage Clade 2. Residues are numbered from 1 to 328 starting with the serine residue downstream of the predicted signal peptide (1-17aa) cleavage site. Amino acid identity to A/eq/Newmarket/5/03 (Accession Number: FJ375213) is shown with a dot. Other reference strains are A/eq/South-Africa/4/03 (Prof. Alan Guthrie, Personal communication), A/eq/Ibaraki/07 (Accession number: AB360549), A/eq/Newmarket/1/93 (Accession Number: X85088) and A/eq/Sussex/89 (Accession Number: X85090). A/eq/Richmond/1/07 is representative of A/eq/Cheshire/1/07, A/eq/Cheshire/2/07, A/eq/Maidstone/1/07, A/eq/Solihull/1/07, A/eq/Solihull/2/07, A/eq/Strathaven/1/07, A/eq/Horsham/1/07, A/eq/Lanark/1/06, A/eq/Southampton/1/07 and A/eq/Southampton/2/07; A/eq/Lincolnshire/1/07 is representative of A/eq/California/1/07; A/eq/Newmarket/1/07 is representative of A/eq/Berkshire/1/07, and A/eq/Cheshire/3/07. N/A -not available; ^ -not present. Amino acid residue identity to A/eq/Newmarket/5/03 is shown with a dot. Stop codons are represented with an asterisk (*). Residues are numbered from the N-terminal methionine. A/eq/California/1/07 is representative of A/eq/California/2/07 and A/eq/Kentucky/7/07; A/eq/Southampton/1/07 is representative of A/eq/Southampton/2/07; A/eq/Newmarket/1/07 is representative of A/eq/Cheshire/3/07, A/eq/Maidstone/2/07, A/eq/Lanark/1/06 and A/eq/Horsham/1/07; A/eq/Richmond/1/07 is representative of A/eq/Cheshire/2/07, A/eq/Strathaven/1/07, A/eq/Richmond/2/07, A/eq/Solihull/1/07 and A/eq/Solihull/2/07.

  of EIV from the UK A total of 27 NP-ELISA positive nasopharyngeal swabs were received in 2006

  dressage yard. The horse had an uncertain vaccination history but had recently been imported from Holland. Clinically it had pyrexia of 41.4C, nasal discharge and dyspnoea. The positive HI test from Stoke-on-Trent was from a non-vaccinated 6 year old gelding which presented with a marked pyrexia of 40.6C, anorexia, profuse serous nasal discharge, frequent harsh dry cough, lymphadenopathy and mild ataxia.The onset of clinical signs was reported 4-5 days earlier. Four foci of EIV infection in the Midlands, Kent and Hampshire were identified from the end of May to the end A c c e p t e d M a n u s c r i p t of June 2007 (Solihull, Maidstone, Southampton). Nasopharyngeal swabs were taken from horses typically showing clinical signs of pyrexia with rectal temperatures up to 41°C/, profuse and frequently mucopurulent nasal discharge and frequent, harsh, dry cough with substantially increased respiratory rates. In all four outbreaks the index cases were animals that were recently imported into the country, having been bought at a horse sale in County Kilkenny in the Republic of Ireland on the 26 th May. All these horses were reported to either be non-vaccinated or of unknown vaccination history. There was one case of vaccine breakdown where a 6 year old Irish sports horse resident at the Solihull yard showed clinical signs having previously been vaccinated with Equilis Prequenza TE (Intervet) on 13 th April 2007 and 8 th May 2007. A/eq/Strathaven/1/07 was isolated from a 13 year-old, non-vaccinated Shetland pony with typical EI clincial signs, inappetance and inco-ordination. Nine further ponies were reported to be affected. This pony was on premises where horses had returned back sick from the Royal Highland Show near Edinburgh. There were no mandatory vaccination requirements for horses attending the show and many of them were consequently not vaccinated. The horse from Hawick which tested posivitve by NP-ELISA was shipped over from Irish horse sales around 14 th August 2007. It was vaccinated with Equip FT on 10 th April 2006 and 8 th May 2006 and then with Prevac Pro (Intervet) on 11 th November 2006. Clinical signs of inappetance, pyrexia, cough, mucopurulent nasal discharge, swollen lymph nodes and lethargy were observed. The positive NP-ELISA from Stowmarket was an 8 year-old pony mare, last vaccinated with Prevac T Pro (Intervet) 12 th July 2005 showed clinical signs consisted of pyrexia of 39°C with unilateral nasal discharge and lethargy.

  of influenza in horses on the yard in Spain from where it came. Two in contact animals had clinical signs of influenza subsequently and one was identified as NP ELISA positive. Three separate viruses were isolated from Cheshire. The first, a 2 ½ year old gelding presented with a very mild pyrexia of 38.4°C with slight mucopurulent nasal discharge. Several in contact horses were coughing and had mucopurulent nasal discharges with mild pyrexia. At the second site, 9 horses were affected and there were 47 in contact horses. Vaccinated animals appeared healthy throughout. At the third site a disease outbreak occurred on a large livery yard that was restricted to unvaccinated young horses. A/eq/Berkshire/1/07 was recovered from a vaccinated horse newly imported from Holland although the exact vaccine was unknown. The two positive NP-ELISAs from Lambourn were from vaccinated national hunt horses. The first horse received ProteqFlu (Merial) for the last 3 vaccinations and was last boosted August 1 st 2007 (prior to this was 6 th January 2007).The second horse received Equilis Prequenza TE (Intervet) as the last vaccination on 28 th June 2007, ProteqFlu (Merial) before that on January 6 th 2007 and Duvaxyn IE-plus (Fort Dodge) before that on 13 th June 2006. A/eq/Newmarket/1/07 was isolated from an infected horse from one of three affected training yards. The animal had shown only mild signs and had been most recently vaccinated with ProteqFlu (Merial).

  /eq/Newmarket/5/03. The 2 remaining viruses, namely

  in the Mongolian isolate suggesting this virus had a greater similarity to the older UK clade 2 isolates (data not shown).

  addition to the Eurasian and Florida lineage viruses complicates matters when trying to determine the best possible combination of EIV strains for future vaccine recommendations.

  the OIE[START_REF] Oie | Conclusions and recommendations from the Expert Surveillance Panel on Equine Influenza Vaccines[END_REF] raise relatively low titres to A/eq/Pennsylvania/1/07. It remains to be determined whether this virus represents a new clade within the evolution of EIV or whether representative strains are still circulating in the field. The HA1 amino acid alignments show substitutions P162S and Q189K which are not found in the other clade 1 viruses described in this report.

  2006-2007 were characterised in detail, in order to aid the vaccine strain selection process. Viruses belonging to the American (Kentucky), Eurasian and Florida sublineages have been isolated with varying degrees of antigenic drift occurring from vaccine strains. The majority of isolates belonged to the Florida sublineage, sequence divergence and antigenic differences support the further division of this group into 2 clades. To date, Florida clade 1 viruses have been isolated in North America with outbreaks in Japan and Australia likely to be due to horse movements. Clade 2 viruses continue to predominate in Europe. This has reinforced the importance of continued surveillance in the field in order to identify any new emerging threats to the equine industry. J.T., Masurel, N., Huffels, A.D., Anker, W.J., 1981. Equine influenza in the Netherlands during the winter of 1978-1979; antigenic drift of the A-equi 2 virus. Vet Q., 3, 80-84 Waddell, G.H., Teigland, M.B., Sigel, M.M., 1963. A new influenza virus associated with equine respiratory disease. J Am Vet Med Assoc., 143, 587-590 Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., Kawaoka, Y., 1992. Evolution and ecology of influenza A viruses. Microbiol Rev. 56, 152-179 Webster, R.G., 1993. Are equine 1 influenza viruses still present in horses? Equine Vet J., 25, 537-538 Yamanaka, T., Niwa, H., Tsujimura, K., Kondo, T., Matsumura, T., 2008. Epidemic of equine influenza among vaccinated racehorses in Japan in 2007. J Vet Med Sci., 70, 623-625 Yates, P., Mumford, J.A., 2000. Equine influenza vaccine efficacy: the significance of antigenic variation. Vet Microbiol., 74, 173-177 A c c e p t e d M a n u s c r i p t

Figure legends Figure 1 :

 legends1 Figure legends

  Figure 1

  . Sporadic outbreaks of EIV have continued in the UK, Ireland and continental Europe and much larger outbreaks have occurred in Sweden (International Thoroughbred Breeders

	International	Collating	Centre	(ICC)	administered	by	AHT,

http://www.aht.org.uk/icc/linksicc.html), Japan, Northern China, Mongolia and Australia (Office International des Epizooties (OIE), World Animal Health Information Database, http://www.oie.int/). The EIV outbreaks in Japan and Australia in 2007 were attributable to closely related Florida sublineage clade 1 viruses, with

A c c e p t e d M a n u s c r i p t 6 similarity to A/eq/Wisconsin/03 and were probably of North American origin originally (Yamanaka

Table 3 ,

 3 NS1 changes

Table 2 .

 2 Characterisation of EIV isolates by HI assay using ferret antisera.

Table 3 .

 3 Amino acid alignment of predicted HA1 sequences compared to A/eq/Newmarket/5/03.

	STRAIN	-16	-11	-10	-9	-8	-2	5	7	26	30	48	58	62	78	104	105	138	158	159	162	163	189	190	192	193	207	213	244	261	265	272	273	275	276	289	291
	NEW/5/03	K	^^L	I	A	I	G	V	S	M	I	R	V	D	Y	A	G	N	P	T	Q	E	T	K	K	I	M	K	S	V	P	D	I	S	D
	LIN/06	.	F	I	F	.	.	.	D	I	.	.	.	.	.	.	.	.	.	.	.	.	.	.	K	.	.	M	.	.	G	.	.	.	.	.	.
	SOU/1/06	.	F	I	F	.	.	.	D	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	M	.	.	.	.	.	.	.	.	.
	RIC/1/07	.	F	I	F	.	.	.	N	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	E
	RIC/2/07	.	F	I	F	M	.	.	N	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	E
	MAI/2/07	.	F	I	F	.	.	.	N	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	NEW/07	.	F	N	F	.	.	.	N	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	LIN/07	T	.	.	.	.	.	.	.	.	.	.	.	K	A	N	.	S	.	S	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	SA/4/03	.	.	.	.	.	.	.	.	.	.	.	.	.	A	.	.	.	.	S	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	FLO/2/06	T	.	.	.	.	.	.	.	.	.	.	.	.	A	.	.	S	.	S	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	CAL/2/07	T	.	.	.	.	.	.	.	.	.	.	.	K	A	N	.	S	.	S	.	.	.	.	K	.	E	.	.	.	.	.	.	.	.	.	.
	KEN/4/07	T	.	.	.	.	.	.	.	.	.	.	.	K	A	N	H	S	.	S	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	KEN/7/07	T	.	.	.	.	.	.	.	.	.	.	.	K	A	N	.	S	.	S	.	.	.	.	.	.	.	.	.	.	.	L	.	N	.	.	.
	PEN/07	T	.	.	.	.	.	.	.	.	.	.	.	.	A	.	.	.	.	S	S	.	K	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	IBA/07	N/A	.	.	.	.	.	.	.	.	.	.	.	.	A	.	.	.	.	S	S	.	K	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	NEW/1/93	.	.	.	.	.	V	T	.	.	T	I	V	.	.	.	.	.	.	.	.	.	.	Q	.	E	.	.	.	.	.	A	.	.	.	P	.
	CHE/06	.	.	.	.	.	V	T	.	.	T	I	V	.	.	.	.	.	.	.	.	.	.	Q	.	E	.	.	.	.	.	A	.	.	.	P	.
	SUS/89	.	.	.	.	.	V	T	.	.	T	I	V	.	.	.	.	.	.	.	.	I	K	.	.	.	E	V	.	R	.	A	L	G	T	P	.
	SWI/07	.	.	.	.	.	V	T	.	.	T	I	V	.	.	.	.	.	E	.	.	I	K	.	.	.	E	V	T	R	.	A	.	.	T	P	.

Table 4 .

 4 Amino acid alignments of the predicted NS1 sequences compared to A/eq/Newmarket/5/03.

	STRAIN	5	22	44	48	53	56	59	66	71	76	77	84	86	95	96	111	124	140	156	179	180	185	194	206	210	212	213	216	220	227	228	230	231
	NEW/5/03	T	F	R	I	D	T	H	E	K	A	L	I	T	L	D	V	I	I	V	G	V	L	I	H	G	P	S	S	*	K	P	I	*
	SOU/1/06	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	I	.	.	.	.	.	.	.	.	.	*	.	.	.	*
	NEW/07	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	*	.	.	.	*
	LIN/07	.	.	.	S	.	.	.	.	.	.	.	V	.	.	.	.	.	.	.	.	.	.	.	.	R	.	.	.	*	.	.	.	*
	RIC/1/07	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	Y	.	.	.	.	*	.	.	.	*
	SOU/1/07	.	.	.	.	.	.	.	.	.	.	.	.	.	F	.	.	.	.	.	.	.	.	.	Y	.	.	.	.	*	.	.	.	*
	CHE/1/07	P	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	Y	.	.	.	.	*	.	.	.	*
	SA/4/03	.	V	.	S	.	.	.	.	.	.	.	V	.	.	.	.	.	.	I	.	.	.	.	.	.	.	.	.	*	.	.	.	*
	FLO/2/06	.	.	.	S	.	.	.	.	.	.	.	V	.	.	.	I	.	.	.	.	.	.	.	.	.	.	.	.	*	.	.	.	*
	CAL/1/07	.	.	.	S	.	.	.	.	.	.	.	V	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	*	.	.	.	*
	KEN/4/07	.	.	.	S	.	.	.	K	.	.	F	V	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	*	.	.	.	*
	PEN/07	.	.	.	S	.	.	.	.	.	.	.	V	.	.	G	.	.	.	.	.	.	.	.	.	.	.	.	.	*	.	.	.	*
	NEW/1/93	.	.	K	S	N	.	R	.	E	.	.	V	A	.	.	.	.	.	.	.	.	F	.	.	.	S	.	P	R	E	.	V	*
	CHE/06	.	.	K	S	N	.	R	.	D	.	.	V	A	.	.	.	.	.	.	.	.	F	.	.	.	S	.	P	R	E	.	V	*
	SUS/89	.	.	K	S	.	.	R	.	E	.	.	V	A	.	.	.	.	.	.	.	.	.	V	.	.	.	.	P	R	E	.	V	*
	LIN/06	.	.	K	S	N	I	R	.	E	E	.	.	V	.	.	.	V	K	.	E	I	F	V	Y	.	.	T	P	R	E	.	V	*
	SWI/07	.	.	K	S	.	.	R	.	E	.	F	V	A	.	.	.	.	.	.	.	.	.	V	.	.	.	.	P	R	E	S	V	*

Acknowledgements

We would like to thank Toni-Ann Hammond for excellent technical support for EIV