Ph.D Arthur R Frampton
email: framptona@uncw.edu

Hiroaki Uchida

Jens Von Einem

William F Goins

Paola Grandi

Justus B Cohen

Nikolaus Osterrieder

Joseph C Glorioso

Equine herpesvirus type 1 (EHV-1) utilizes microtubules, dynein, and ROCK1 to productively infect cells

Keywords: EHV-1, trafficking, microtubules, dynein, ROCK1

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

Introduction

Equine herpesvirus 1 (EHV-1) is a major pathogen of horses. Most horses are exposed to the virus within 6 months to a year after birth and are frequently re-infected throughout their lifetime [START_REF] Allen | Molecular epizootiology, pathogenesis, and prophylaxis of equine herpesvirus-1 infections[END_REF]. Clinical signs that appear early after infection include fever, inappattence, malaise, coughing, and mucopurulent discharge (O' Callaghan et al., 1983). As the infection progresses, horses may exhibit signs of serious neurological illness including ataxia, disorientation, and partial to full paralysis. In addition to the neurological sequelae, EHV-1 also causes abortigenic disease in pregnant mares.

At the cellular level, EHV-1 must engage host cellular proteins and induce specific signaling pathways very early in infection in order to successfully infect cells. EHV-1 initially binds to target cells via an interaction between viral glycoproteins B and C and glycosaminoglycans on the plasma membrane [START_REF] Sugahara | Adaptation of equine herpesvirus 1 to unnatural host led to mutation of the gC resulting in increased susceptibility of the virus to heparin[END_REF][START_REF] Osterrieder | Construction and characterization of an equine herpesvirus 1 glycoprotein C negative mutant[END_REF]. After attachment, a putative entry receptor is recognized [START_REF] Frampton | Equine herpesvirus 1 utilizes a novel herpesvirus entry receptor[END_REF] by A c c e p t e d M a n u s c r i p t 3 glycoprotein D [START_REF] Csellner | EHV-1 glycoprotein D (EHV-1 gD) is required for virus entry and cell-cell fusion, and an EHV-1 gD deletion mutant induces a protective immune response in mice[END_REF]. The next step in the EHV-1 entry pathway varies depending upon the cell type [START_REF] Frampton | Equine Herpesvirus 1 Enters Cells by Two Different Pathways, and Infection Requires the Activation of the Cellular Kinase ROCK1[END_REF]. EHV-1 fuses with the plasma membrane and deposits de-enveloped particles into the cytoplasm of equine dermal (ED) and rabbit kidney (RK-13) cells. On the contrary, fully enveloped particles are endocytosed in CHO-K1 cells and naked capsids are then released into the cytosol after the virus fuses with the endosomal membrane. Regardless of the initial mode of entry, we recently showed that the cellular kinase Rho associated coiled-coil kinase 1 (ROCK1) must be induced for the virus to complete the infection process [START_REF] Frampton | Equine Herpesvirus 1 Enters Cells by Two Different Pathways, and Infection Requires the Activation of the Cellular Kinase ROCK1[END_REF].

Once inside the cells, EHV-1 must traverse the intricate meshwork of the intracellular cytoskeleton in order to reach the nucleus and deposit the viral genome. The mechanisms employed by EHV-1 to traffic to the nucleus have yet to be defined, but certain clues are provided by studies with other viruses that utilize microtubules and their associated machinery for intracellular trafficking. Microtubules are composed of  and  dimers of tubulin and contain a structural polarity with a positive end that is oriented toward the periphery of the cell and a negative end that is anchored at the microtubule organizing center (MTOC) adjacent to the nucleus [START_REF] Nogales | Structural insights into microtubule function[END_REF]. To reach the nucleus, many viruses attach themselves to, and are directionally propelled by dynein, a minus-end directed microtubule motor, towards the MTOC [START_REF] Kelkar | Cytoplasmic dynein mediates adenovirus binding to microtubules[END_REF][START_REF] Alonso | African swine fever virus protein p54 interacts with the microtubular motor complex through direct binding to light-chain dynein[END_REF][START_REF] Suikkanen | Exploitation of microtubule cytoskeleton and dynein during parvoviral traffic toward the nucleus[END_REF][START_REF] Petit | Targeting of incoming retroviral Gag to the centrosome involves a direct interaction with the dynein light chain 8[END_REF][START_REF] Ye | The herpes simplex virus 1 U(L)34 protein interacts with a cytoplasmic dynein intermediate chain and targets nuclear membrane[END_REF][START_REF] Douglas | Herpes simplex virus type 1 capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and plays a role in retrograde cellular transport[END_REF][START_REF] Jacob | Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein[END_REF][START_REF] Raux | Interaction of the rabies virus P protein with the LC8 dynein light chain[END_REF][START_REF] Florin | Identification of a dynein interacting domain in the papillomavirus minor capsid protein l2[END_REF]. Sodeik et al. initially showed that the alphaherpesvirus herpes simplex virus type 1 (HSV-1) uses microtubules to traffic to the nucleus. Their data revealed that nuclear transport of HSV-1 was significantly inhibited in the presence of the microtubule-depolymerizing drugs, nocodazole, vinblastine, or colchicine [START_REF] Sodeik | Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus[END_REF]. The movement of HSV-1 capsids along microtubules was also shown to require dynein as expression of the dynein inhibitor, dynamitin [START_REF] Dohner | Function of dynein and dynactin in herpes simplex virus capsid transport[END_REF], and specific inhibitors of dynein ATPase [START_REF] Lee | Reconstitution of herpes simplex virus microtubule-dependent trafficking in vitro[END_REF][START_REF] Kobayashi | Inhibition of dynein ATPase by vanadate, and its possible use as a probe for the role of dynein in cytoplasmic motility[END_REF][START_REF] Kristensson | Neuritic transport of herpes simplex virus in rat sensory neurons in vitro. Effects of substances interacting with microtubular function and axonal flow [nocodazole, taxol and erythro-9-3-(2-hydroxynonyl)adenine][END_REF][START_REF] Penningroth | Dynein ATPase is inhibited selectively in vitro by erythro-9-[3-2-(hydroxynonyl)]adenine[END_REF] decreased HSV-1 transport to the nucleus. The movement of virus particles was also shown to be independent of microtubule turnover or treadmilling (microtubule polymerization/depolymerization) as the microtubule-stabilizing drug paclitaxel had no effect on nucleocapsid transport [START_REF] Sodeik | Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus[END_REF].

A c c e p t e d M a n u s c r i p t 4 In addition to utilizing microtubules for transport, viruses also generate an intracellular environment that is conducive for nuclear transport by triggering various signaling pathways. One common theme is the induction of signaling pathways that lead to the reorganization of cytoskeletal components. Many viruses, including HSV-1, stimulate members of the Ras GTPase superfamily and this induction can lead to a wide array of morphological changes of infected cells, including, but not limited to, stabilization of microtubules [START_REF] Naranatt | Kaposi's sarcomaassociated herpesvirus modulates microtubule dynamics via RhoA-GTP-diaphanous 2 signaling and utilizes the dynein motors to deliver its DNA to the nucleus[END_REF], formation of focal adhesions [START_REF] Cheshenko | Focal adhesion kinase plays a pivotal role in herpes simplex virus entry[END_REF] and endocytosis or phagocytosis [START_REF] Clement | A novel role for phagocytosis-like uptake in herpes simplex virus entry[END_REF].

In this study, we show that EHV-1 causes the stabilization of microtubules via the acetylation of tubulin and utilizes the minus-end directed microtubule motor dynein to traffic to the nucleus along stabilized microtubules.

In addition, we show that nuclear accumulation of capsids is decreased in the presence of the ROCK1 inhibitor, Y-27632.

Materials and methods

Cells, viruses, and plasmids

RK-13 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA). Chinese hamster ovary (CHO-K1) cells were kindly provided by Dr. Patricia Spear (Northwestern University, Chicago, IL) and grown in F12-K media (Invitrogen) supplemented with 10% FBS. Equine dermal cells (ED), a gift from Dr. Ron Montelaro (University of Pittsburgh, Pittsburgh, PA), were maintained in minimal essential medium (MEM) supplemented with 10% FBS. All cells were maintained at 37C in 5% CO 2 .

The EHV-1 recombinant virus L11gIgE contains a lacZ reporter cassette in place of gI and gE [START_REF] Frampton | Contribution of gene products encoded within the unique short segment of equine herpesvirus 1 to virulence in a murine model[END_REF]. The EHV-1 recombinant virus L11VP26mRed was described previously (J. von Einem et al., 24 th annual ASV meeting 2005). This recombinant virus contains an mRFP1 fluorescent protein fused to the EHV-1 VP26 small capsid protein.

Drug inhibition assays

A c c e p t e d M a n u s c r i p t 5 For entry assays, 2.5 x 10 4 ED or CHO-K1 cells were seeded in a 96-well plate. The next day, cells were incubated with increasing amounts of the microtubule depolymerizing drugs, nocodazole, vinblastine, colchicine, the microtubule stabilizing drug, paclitaxel (PTX), the dynein inhibitor, EHNA (erythro-9-(2hydroxy-3-nonyl) adenine, or the kinesin inhibitor AMP-PNP (adenosine 5-(, -imido) triphosphate tetralithium salt) (Sigma, St. Louis, MO) at 37C for 30 min. Cells were infected with EHV-1 (L11gIgE) at an MOI of 5 and incubated for 5.5 h in the presence of the drugs at 37C. Cells were washed once with PBS and then ONPG was added to the cells and the absorbance read at 405nm. Triplicate samples were measured for each concentration of drug. A 1mM stock of paclitaxel was prepared in distilled H 2 O. One-hundred mM stock solutions of EHNA and nocodazole were prepared in DMSO. A 100 mM stock solution of AMP-PNP was prepared in H 2 O. 10 mM stocks of the ROCK1 inhibitor, Y-27632 (EMD Biosciences, La Jolla, CA) were made in H 2 O. 10mM stocks of vinblastine, and colchicine were also made in H 2 O. Cell viability was measured in triplicate using the CellTiter 96 aqueous non-radioactive cell proliferation assay (Promega, Madison, WI) following the manufacturer's protocol.

Western Blot Analyses

Confluent ED cells in a 12-well plate were serum starved for 1 h and cells were then mock-treated or treated with paclitaxel (10 M) or Y-27632 (100 M). Cells treated with paclitaxel were washed with MEM at 15, 30, and 60 min and cell lysates were harvested with PARP lysis buffer (6 M urea, 2% SDS, 10% glycerol, 62.5 mM Tris-HCl, pH 6.8, and 5% -mercaptoethanol). Mock-treated cells or cells treated with Y-27632 were infected with EHV-1 (L11VP26mRed) at an MOI of 10 in the presence or absence of Y-27632. At 15, 30, and 60 min post-infection, cells were washed once with MEM and lysates were harvested with PARP lysis buffer. Samples were run on a 10% SDS-PAGE gel and transferred to an immobilon-P membrane (Millipore, Billerica, MA).

The membranes were blocked for 1 h in 10% non-fat dry milk (NFDM) at room temperature (RT) before monoclonal anti-acetylated tubulin antibody 6-11B-1 (Sigma) was added at a concentration of 1:2000 in TBST with 5% NFDM for 16 h at 4C. The membranes were washed 3x for 10 min/wash and then anti-mouse-HRP A c c e p t e d M a n u s c r i p t 6 antibody SC-2031 (Santa Cruz Biotechnology, Santa Cruz, CA) was added at a concentration of 1:3,000 in TBST with 5% NFDM for 1 h at RT. Membranes were washed 3x for 10 min/wash then ECL detection reagent (Amersham Biosciences, Buckinghamshire, England) was added and the membranes exposed to film. After stripping the membrane with stripping buffer (10% SDS, 0.5M Tris pH 6.8, 0.5% -mercaptoethanol) for 30 min at 50C, the membranes were washed 2x for 10 min/wash with TBST and then blocked for 1 h with 10% NFDM. Total tubulin was detected by adding monoclonal anti--tubulin antibody B-5-1-2 (Sigma) at 1:2,000

for 16 h at 4C with the secondary antibody and detection steps as described earlier.

Infectious virus recovery assay

4 x 10 5 cells in 24-well plates were incubated with 100 M of Y-27632 in DMEM or DMEM alone for 30 min at 37C. Plates were placed on ice for 5 min and then EHV-1 (MOI = 10) in DMEM or DMEM containing 100 M Y-27632 was incubated on the cells at 4C for 3 h. Cells were washed once with cold DMEM and then warm media (37C) was added to the cells. At each time-point, cells were washed with glycine (pH 3.0) for 30 s, washed once with DMEM, and harvested. Virus samples were freeze-thawed once then sonicated 3x for 15 s.

Virus harvested from each time-point was titered on RK13 cells. Triplicate samples were measured for each time-point.

Confocal microscopy of EHV-1 infected cells

CHO-K1 cells were seeded to confluency in collagen-coated 35 mm dishes (MatTek Corporation, Ashland, MA). Cells were mock-treated or treated with 100 M Y-27632 or 10 M paclitaxel for 30 min at 37C. Cells were chilled on ice for 10 min and then L11VP26mRed was added to the cells at an MOI of 10. Virus was incubated on the cells at 4C for 2 h in the presence or absence of drug. After 2 h, cells for the 0 time-point were fixed with 4% paraformaldehyde (PFA) at 4C. Media pre-warmed to 37C, with or without drug, was added to the remaining cells and then the cells were fixed with 4% PFA at 15, 30, 60, and 120 min post temperature shift.

After 30 min of fixation with 4% PFA, cells were rehydrated with PBS for 15 min at 4C. Cells were washed A c c e p t e d M a n u s c r i p t 7 once more with PBS for 15 min and then cells were examined using an inverted Olympus Fluo-View 1000 confocal microscope (Olympus America Inc., Center Valley, PA). Prior to imaging, cells were stained with Hoechst dye (1 g/mL) and wheat germ agglutinin (5 g/mL) to label the nucleus and plasma membrane, respectively. All images were captured at a 60x magnification.

Results

EHV-1 utilizes microtubules for infection

To determine if EHV-1 requires microtubules for infection, a virus entry assay was performed in the presence or absence of microtubule depolymerizing agents. Equine dermal (ED) cells were untreated or treated with increasing concentrations of nocodazole, vinblastine, or colchicine for 30 min prior to infection and the drugs were kept on the cells throughout the experiment. Cells were infected with L11gIgE, an EHV-1 recombinant virus containing a lacZ reporter gene, at an MOI of 5 for 5.5 hours. EHV-1 infection, as measured by -galactosidase expression, was significantly inhibited in a dose-dependent manner by all of the drugs.

Nocodazole inhibited infection by 80% at the highest concentration tested (Figure 1A) and vinblastine (Figure 1B) and colchicine (Figure 1C) inhibited infection by 82% and 56%, respectively. This inhibition of infection was not the result of drug-associated cell toxicity as cell viability was similar for all of the concentrations used.

In addition to inhibiting infection on ED cells, these drugs also inhibited infection on CHO-K1 cells (data not shown). These results indicate that an intact microtubule network is required regardless of the mode of EHV-1 entry.

To shown to be a reliable indicator of stable or fixed microtubules [START_REF] Piperno | Microtubules containing acetylated alpha-tubulin in mammalian cells in culture[END_REF]. Western blotting was performed to detect acetylated tubulin at 15, 30, and 60 minutes post-treatment with 10 M paclitaxel. As shown in Figure 3, paclitaxel treatment resulted in the hyperacetylation of tubulin as early as 15 minutes posttreatment and the level of acetylation steadily increased the longer the cells were treated with the drug.

The ability of EHV-1 to travel to the nucleus after microtubule stabilization was assessed by infecting CHO-K1 cells with the fluorescently tagged-EHV-1 recombinant L11VP26mRed in the presence or absence of paclitaxel. This recombinant virus contains a red fluorescent protein fused to the VP26 capsid protein and thus the subcellular localization of this virus can be detected throughout a time course of infection by confocal microscopy. CHO-K1 cells were untreated or treated with 10 M of paclitaxel for 30 minutes and then virus was added and allowed to attach to the cells at 4C for 2 hours to synchronize the entry process. After attachment, medium warmed to 37C, with or without addition of the drug, was added to the cells to allow infection to ensue. At 0 and 60 minutes post-temperature shift, cells were stained with Hoechst dye and wheat germ agglutinin to label the nucleus (blue) and plasma membrane (green), respectively. As shown in Figure 4, attached virus (red) was detected on the surface of mock-treated and paclitaxel-treated cells at the 0 time-point.

At the 60-minute time-point, a similar number of capsids were observed at the nucleus in both mock-treated and paclitaxel-treated cells. The ability of virus to reach the nucleus after paclitaxel stabilization of microtubules in a manner similar to that observed in mock-treated cells suggests that EHV-1 is efficiently transported along stable microtubules and that there is not a requirement for a continuous process of microtubule polymerization/depolymerization (treadmilling) for successful delivery of EHV-1 to the nucleus.

EHV-1 infection induces the acetylation of tubulin

The ability of EHV-1 to traffic along stable microtubules led us to ask whether EHV-1 actively induces the stabilization of microtubules. To test this possibility, we compared the amount of acetylated tubulin between mock and EHV-1 infected cells by western blot assay. Other viruses, including HSV-1 [START_REF] Elliott | Herpes simplex virus type 1 tegument protein VP22 induces the stabilization and hyperacetylation of microtubules[END_REF], KSHV [START_REF] Naranatt | Kaposi's sarcomaassociated herpesvirus modulates microtubule dynamics via RhoA-GTP-diaphanous 2 signaling and utilizes the dynein motors to deliver its DNA to the nucleus[END_REF][START_REF] Raghu | Lipid rafts of primary endothelial cells are essential for Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8-induced phosphatidylinositol 3-kinase and RhoA-GTPases critical for microtubule dynamics and nuclear delivery of viral DNA but dispensable for binding and entry[END_REF] and African Swine Fever Virus [START_REF] Jouvenet | Transport of African swine fever virus from assembly sites to the plasma membrane is dependent on microtubules and conventional kinesin[END_REF] A c c e p t e d M a n u s c r i p t 9 have been shown to induce the acetylation of tubulin upon infection and in some cases this event has been linked to the activity of Rho-signaling proteins [START_REF] Naranatt | Kaposi's sarcomaassociated herpesvirus modulates microtubule dynamics via RhoA-GTP-diaphanous 2 signaling and utilizes the dynein motors to deliver its DNA to the nucleus[END_REF][START_REF] Raghu | Lipid rafts of primary endothelial cells are essential for Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8-induced phosphatidylinositol 3-kinase and RhoA-GTPases critical for microtubule dynamics and nuclear delivery of viral DNA but dispensable for binding and entry[END_REF]. Since ROCK1 is a proximal effector of Rho signaling, we also tested whether the inhibition of ROCK1 would have any effect on the ability of EHV-1 to induce the acetylation of tubulin. ED cells were untreated or treated with the ROCK1 inhibitor Y-27632 for 30 min and then either mock-infected or infected with EHV-1 at an MOI of 10 for 15, 30, and 60 minutes. As shown in Figure 5, EHV-1 infection resulted in a significant induction of acetylated tubulin as early as 15 minutes post-infection and the amount of acetylated tubulin increased over time. The ROCK1 inhibitor had no effect on the ability of EHV-1 to cause the acetylation of tubulin. These results indicate that EHV-1 actively induces the acetylation of microtubules and that this process is not dependent upon the activation of ROCK1.

The microtubule motor, dynein, is essential for EHV-1 infection

Dynein is a minus-end directed motor that transports intracellular cargo along microtubules and many intracellular pathogens, including HSV [START_REF] Sodeik | Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus[END_REF], have been shown to utilize dynein for transport to the nucleus. To determine if EHV-1 needs dynein for efficient transport to the nucleus and thus infection, we utilized the dynein inhibitor, EHNA, which blocks the ability of dynein to hydrolyze ATP [START_REF] Ravikumar | Dynein mutations impair autophagic clearance of aggregate-prone proteins[END_REF][START_REF] Penningroth | Dynein ATPase is inhibited selectively in vitro by erythro-9-[3-2-(hydroxynonyl)]adenine[END_REF][START_REF] Bouchard | erythro-9-[3-(2-Hydroxynonyl)]adenine is an inhibitor of sperm motility that blocks dynein ATPase and protein carboxylmethylase activities[END_REF][START_REF] Dhani | Evidence for a functional interaction between the ClC-2 chloride channel and the retrograde motor dynein complex[END_REF][START_REF] Cheung | p150(Glued), Dynein, and microtubules are specifically required for activation of MKK3/6 and p38 MAPKs[END_REF]. ED cells were infected with L11gIgE in the presence or absence of EHNA and -galactosidase expression was measured as an indicator of virus infection (Fig. 6A). As a control, cells were treated with AMP-PNP, which inhibits the plus-end directed motor, kinesin. Infection was significantly reduced in cells treated with EHNA, but not in cells treated with AMP-PNP. At the highest concentration of EHNA tested (1 mM), infection was inhibited by 68%. Neither drug was toxic to the cells at the concentrations used as measured by an MTS cell viability assay.

The inhibition of EHV-1 infection by EHNA indicates that EHV-1 utilizes dynein for movement to the nucleus post-entry.

Since the initial entry process of EHV-1 in CHO-K1 cells is different from entry into ED cells, we asked whether dynein was also employed for intracellular movement of EHV-1 in CHO-K1 cells. Entry of EHV-1

A c c e p t e d M a n u s c r i p t 10 into CHO-K1 cells was also inhibited by EHNA in a dose-dependent manner (Fig 6B). These data suggest that, while the initial entry of EHV-1 into different cell types is dissimilar, the virus uses the same molecular machinery for transport post-entry.

Inhibition of ROCK1 deregulates the intracellular processing of EHV-1

In a previous study, we showed that the activation of ROCK1 is critical for productive EHV-1 infection [START_REF] Frampton | Equine Herpesvirus 1 Enters Cells by Two Different Pathways, and Infection Requires the Activation of the Cellular Kinase ROCK1[END_REF]. One possible step that ROCK1 might be required for in the EHV-1 life cycle is at the initial stage of virus uptake into cells. To test this possibility, we performed an infectious virus recovery assay on CHO-K1 cells in the presence or absence of the ROCK1 inhibitor, Y-27632. This assay allows for the recovery of fully enveloped virus particles from endosomal vesicles at different time points post-infection.

CHO-K1 cells were untreated or treated with Y-27632 for 30 min at 37C (Fig. 7). Cells were then chilled to 4C and EHV-1 was added with or without drug and allowed to attach to cells for 3 hours at 4C. After virus attachment, the temperature was raised to 37C and virus was incubated on cells for up to 3 hours in the presence or absence of Y-27632. At the indicated times post-temperature shift, extracellular virus was inactivated by washing the cells with an acidic buffer, and intracellular virus was harvested and titered as described in materials and methods. No difference was observed in the amount of infectious virus recovered at 15 minutes, indicating that ROCK1 did not exert an effect on initial EHV-1 uptake into cells. However, there was a significant spike in the amount of virus recovered at the 30-minute time-point in the presence of Y-27632.

After the 30-minute time point the amount of virus recovered from untreated and Y-27632 cells was virtually the same. The increased recovery of virus at 30 minutes in the presence of the ROCK1 inhibitor suggests that ROCK1 mediates its effect at a time post virus entry.

Decreased nuclear localization of EHV-1 in the presence of the ROCK1 inhibitor, Y-27632

To examine if ROCK1 inhibition disrupts EHV-1 trafficking, CHO-K1 cells were infected with the fluorescent EHV-1 recombinant virus L11VP26mRed in the presence or absence of the ROCK1 inhibitor, Y-A c c e p t e d M a n u s c r i p t 11 27632 (Fig. 8). Cells were untreated (Fig. 8A) or treated with Y-27632 (Fig. 8B) for 30 minutes at 37C and then virus was added and allowed to attach to cells for 2 hours at 4C. After the attachment period, cells were warmed to 37C to allow for virus entry. Infected cells were fixed and imaged at 0 to 120 minutes posttemperature shift using a confocal microscope. At the 0 time-point, virus particles were attached to the plasma membrane (yellow circles) and no particles were observed inside either the untreated or Y-27632-treated cells.

At 15 minutes post-infection, internal and external virus particles were observed in both groups. Interestingly, as shown in Figure 8A about one-third of the virus particles were localized to the nucleus in the mock-treated cells (white circles) at 15 minutes post-infection, but only two virus particles were observed at the nucleus in the Y-27632-treated cells (Fig. 8B). Over the course of infection, more virus particles accumulated at the nucleus in the untreated cells (Fig. 8A) compared to the cells treated with Y-27632 (Fig. 8B) and the majority of particles in the Y-27632-treated cells remained associated with the plasma membrane. These data show that treatment of cells with Y-27632 significantly inhibits the movement of EHV-1 to the nucleus and indicate that ROCK1 is critical for the intracellular trafficking of EHV-1.

Discussion

Many viruses exploit the cellular machinery to travel intracellularly. One common theme that has emerged as an integral part of the lifecycle of viruses is the co-opting of cellular motors, which are normally used to traffic various cargoes throughout the cell. Upon entry into cells, many viruses bind to molecular motors that are tethered to microtubules [for review see [START_REF] Dohner | Viral stop-and-go along microtubules: taking a ride with dynein and kinesins[END_REF]]. Microtubules are dynamic structures that act as cellular tracks, which viruses utilize to move directionally toward the nucleus of the cell where replication ensues. After replication and assembly, viruses utilize another set of cellular motors for egress. Without the use of these cellular motors for transport, viruses would never be able to find their way through the dense and intricate cytoskeletal structures and would thus fail to replicate and release progeny virus.

In this study, we show that EHV-1 utilizes the microtubule network and the minus-end directed motor protein dynein to productively infect cells. Disruption of the microtubular network with the microtubule A c c e p t e d M a n u s c r i p t depolymerizing agents nocodazole, vinblastine, and colchicine inhibited the ability of EHV-1 to productively infect cells and deliver its payload to the nucleus. In addition, the introduction of dynein, but not kinesin inhibitors, also reduced infection. Interestingly, microtubules and dynein were required for virus trafficking in cells that were infected via an endocytic route or by direct fusion of virus at the plasma membrane. These data suggest at least two possible models for intracellular trafficking of EHV-1 particles that we have designated as the direct and the indirect trafficking model. In the direct model, nucleocapsids that are still surrounded by some tegument proteins would bind to dynein via a viral capsid or tegument protein and this event would therefore occur only after completion of the fusion event between the viral envelope and either the plasma or endosomal membrane. In the indirect model, EHV-1 would be transported within an endosome and an endosomal protein or complex would interact with dynein. Virus would then fuse and be released from the endosome once it is in close proximity to the nucleus. The ability of EHV-1 to enter some cells by direct fusion at the cell membrane implies that a direct interaction between a viral protein and a dynein component most likely has to occur for the virus to traffic to the nucleus. However, since EHV-1 enters other cell types via an endocytic mechanism, it is possible that the indirect model of trafficking applies in these cells and thus the mechanism of intracellular movement of virus toward the nucleus may be cell-type dependent. Studies are currently underway using L11VP26mRed in conjunction with dynein and endosomal marker antibodies to further explore these models.

An intriguing finding from this study is the importance of the Rho kinase, ROCK1, for intracellular trafficking of EHV-1 particles. Previously, we showed that inhibition of this kinase significantly reduced EHV-1 infection [START_REF] Frampton | Equine Herpesvirus 1 Enters Cells by Two Different Pathways, and Infection Requires the Activation of the Cellular Kinase ROCK1[END_REF], but we did not address the mechanism. In the current study, we showed that treatment with a ROCK1 inhibitor significantly decreased the number of capsids that accumulated at the nucleus suggesting a role for ROCK1 in the intracellular trafficking of EHV-1.

While this is the first report to connect ROCK1 activation with virus trafficking, previous studies have shown that other molecules involved in the ROCK1 signaling pathway also contribute to virus movement within cells. Naranatt and colleagues reported that inactivation of the RhoA GTPase, which acts directly upstream of ROCK1, blocks the delivery of KSHV to the nucleus [START_REF] Naranatt | Kaposi's sarcomaassociated herpesvirus modulates microtubule dynamics via RhoA-GTP-diaphanous 2 signaling and utilizes the dynein motors to deliver its DNA to the nucleus[END_REF]. In addition, their data support a model in which virus binding to cells activates RhoA, which in turn leads to microtubule stabilization A c c e p t e d M a n u s c r i p t 13 via acetylation and an increased ability of the virus to traffic towards the nucleus [START_REF] Raghu | Lipid rafts of primary endothelial cells are essential for Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8-induced phosphatidylinositol 3-kinase and RhoA-GTPases critical for microtubule dynamics and nuclear delivery of viral DNA but dispensable for binding and entry[END_REF]. This microtubule stabilization was further shown to occur after induction of Dia-2 [START_REF] Naranatt | Kaposi's sarcomaassociated herpesvirus modulates microtubule dynamics via RhoA-GTP-diaphanous 2 signaling and utilizes the dynein motors to deliver its DNA to the nucleus[END_REF] a member of the diaphanous-related formin family involved in cytoskeletal rearrangements and a downstream effector of RhoA. Consistent with these reports, other groups have shown that many viruses including adenoviruses and the alphaherpesvirus, HSV-1, travel along stabilized microtubules. Sodeik et al. showed that stabilization of microtubules with paclitaxel prior to HSV-1 infection had no effect on the nuclear accumulation of capsids [START_REF] Sodeik | Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus[END_REF] and Mabit et al. showed that paclitaxel treatment slightly increased nuclear delivery of capsids [START_REF] Mabit | Intact microtubules support adenovirus and herpes simplex virus infections[END_REF]. These studies revealed that microtubule dynamics, or treadmilling, are not required for HSV-1 capsid trafficking. The finding in our current study that EHV-1 infection actively induces the stabilization of microtubules and that infection is not negatively affected by the prior stabilization of microtubules suggests a common mechanism of microtubule transport of nucleocapsids that is shared amongst alphaherpesviruses.

The connection between EHV-1-induced microtubule stabilization, virus trafficking along stabilized microtubules, and the dependence of virus trafficking on ROCK1 function would suggest a direct role for the RhoA-ROCK1 signaling pathway in microtubule stabilization. However, one group showed that while RhoA is required for this effect, it is not mediated through ROCK1 [START_REF] Palazzo | mDia mediates Rhoregulated formation and orientation of stable microtubules[END_REF]. Consistent with these data, our results revealed that the inhibition of EHV-1 infection by Y-27632 did not inhibit EHV-1-induced stabilization of microtubules. We therefore concluded that the contribution of ROCK1 to virus trafficking is most likely separate from virus-induced microtubule stabilization.

The observation that many virus particles remain associated with the plasma membrane in the presence of the ROCK1 inhibitor Y-27632 suggests other possible roles for ROCK1 in virus trafficking. Two intriguing possibilities are that 1) ROCK1 activity is required in order to mediate an interaction between virus and microtubules and/or other cytoskeletal components or 2) ROCK1 is required for efficient fusion of the viral envelope with a cellular membrane. Inhibition of either of these processes could account for the inability of EHV-1 to traffic to the nucleus. Future studies will examine which virus components are involved in the

Conclusion

In the current study we examined some of the early interactions between EHV-1 and host cell factors and, in particular, investigated how EHV-1 utilizes the cell's microtubular network and associated proteins to efficiently infect susceptible cells. Our results showed that disruption of microtubules by the addition of microtubule-depolymerizing agents and the inhibition of the microtubule motor protein, dynein, significantly abrogated EHV-1 infection in ED and CHO-K1 cells. Our data also revealed that stabilization of microtubules by paclitaxel prior to infection had no impact on the efficiency of EHV-1 infection in either cell type.

Interestingly, our data showed that microtubules are acetylated very early after infection with EHV-1. Whether this acetylation is an absolute requirement for EHV-1 infection is an intriguing question and efforts are underway to address this possibility. Finally, we showed that inhibition of the cellular kinase ROCK1 with the drug Y-27632, inhibited the movement of EHV-1 capsids to the nucleus. These ROCK1 data follow-up on our earlier study, showing that ROCK1 activation is critical for EHV-1 infection [START_REF] Frampton | Equine Herpesvirus 1 Enters Cells by Two Different Pathways, and Infection Requires the Activation of the Cellular Kinase ROCK1[END_REF]. Future studies will aim to define how ROCK1 is activated by EHV-1, what specific cellular events are reliant upon this activation, and how these cellular events contribute to a productive infection. Knowledge obtained from these studies may be translated into the development of antiviral compounds that can be used to inhibit the identified critical processes that EHV-1 needs to complete its pathogenic program within the horse.

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

% cell viability

  address the importance of microtubule dynamics or treadmilling (polymerization/depolymerization of microtubules) in virus transport, microtubules were first stabilized with paclitaxel or left untreated before and during infection with EHV-1. Virus entry was assessed by measuring -galactosidase activity 5.5 h postinfection. In both ED (Fig 2A) and CHO-K1 (Fig 2B) cells, stabilization of microtubules prior to the addition of EHV-1 had no effect on infection as measured by viral reporter gene expression. To assure that paclitaxel treatment of ED cells induced microtubule stabilization, we examined the acetylation of tubulin, which has beenA c c e p t e d M a n u s c r i p t 8

  pathways that lead to both ROCK1 activation and separately the acetylation of microtubules.

Fig. 1 .

 1 Fig. 1. Depolymerization of microtubules inhibits EHV-1 infection. Triplicate cultures of ED cells were mock-

Fig. 2 .

 2 Fig. 2. Stabilization of microtubules does not inhibit EHV-1 infection. Triplicate cultures of ED (A) or CHO-

Fig. 3 .

 3 Fig. 3. Paclitaxel (PTX) stabilization of microtubules. ED cells were serum-starved for 1 h and then mock-

Fig. 5 .

 5 Fig. 5. EHV-1 induces the acetylation of tubulin. ED cells were serum starved for 1 h and then mock-treated or

Fig. 6 .

 6 Fig. 6. Inhibition of dynein with EHNA blocks EHV-1 infection. Triplicate cultures of (A) ED or (B) CHO-K1

Fig. 7 .

 7 Fig. 7. Increased infectious EHV-1 recovery in the presence of the ROCK inhibitor, Y-27632. Triplicate

Fig. 8 .A

 8 Fig. 8. (A) CHO-K1 cells were mock treated (top panels) or (B) treated with 100 M Y-27632 for 30 min at

Figure

  Figure

Figure

  Figure

Acknowledgments

We thank Patricia Spear for providing the CHO cell lines and Ron Montelaro for the ED cells. We thank Simon Watkins for the use of the confocal microscope. This work was supported by NIH grants CA119298, NS44323, NS040923, and HL066949.

% infection % cell viability

120

Figure


Giovanni Manarolla
email: giovanni.manarolla@unimi.it

Usutu virus in wild birds in northern Italy

Keywords: Usutu virus, wild birds, immunohistochemistry, RT-PCR, sequencing

Usutu virus (USUV) infection was diagnosed in two free living blackbirds and in three captive owls belonging to two different species in northern Italy in the summers of 2006-2008. Diagnosis was established by immunohistochemistry and RT-PCR. RT-PCR was performed on frozen and on paraffin-embedded tissues (PET), respectively. From the frozen samples a partial sequence of the putative USUV E and NS1 proteins (1229 bp) was determined, whereas partial sequences of the putative NS3 (278 bp) and NS5 (159 bp) proteins were obtained from PET. Additionally, one partial sequence (163 bp) of the putative 3'UTR region was determined from all samples. Sequencing of the amplification products revealed 99.8 to 100% nucleotide identity of the Italian USUV strains to those from other central European countries.

 [START_REF] Kuno | Phylogeny of the genus Flavivirus[END_REF][START_REF] Bakonyi | Complete genome analysis and molecular characterization of Usutu virus that emerged in Austria in 2001: comparison with the South African strain SAAR-1776 and other flaviviruses[END_REF]. USUV infection emerged in Austria in 2001, and has been associated with avian mortality for five consecutive summers since, affecting predominantly blackbirds (Turdus merula) [START_REF] Weissenböck | Emergence of Usutu virus, an African mosquito-borne flavivirus of the Japanese encephalitis virus group, central Europe[END_REF][START_REF] Bakonyi | Emergence of Usutu virus in Hungary[END_REF]. USUV outbreaks have also been recently reported from Hungary [START_REF] Bakonyi | Emergence of Usutu virus in Hungary[END_REF] and Switzerland [START_REF] Steinmetz | Emergence of Usutu virus in Switzerland[END_REF]. The disease in birds is characterized by encephalitis, myocardial degeneration, and necrosis in liver and spleen [START_REF] Chvala | Pathology and viral distribution in fatal Usutu virus infections of birds from the 2001 and 2002 outbreaks in Austria[END_REF]. This paper reports the first detection of USUV antigens and nucleic acid in wild birds in northern Italy.

MATERIALS AND METHODS

Animals and samples

2006:

The first case suspicious of USUV infection in Italy was identified nearby Milan in 2006, in a collection of 70 owls from all over the world belonging to 32 species. In August 2006, one great grey owl (Strix nebulosa) showed suddenly signs of apathy associated with ruffled plumage and increased water intake. The bird died within 2-3 days after onset of clinical symptoms on August 5 th , 2006. Further 5 birds of the same species died within a time span of 6 weeks after showing similar signs. Other owls of different species belonging to the same owl collection remained healthy. The first-mentioned dead owl was submitted for diagnostic investigations, and was necropsied. Samples of liver, spleen, lung, kidney, ovary, adrenals, thyroids, bone marrow, proventriculus, gizzard, intestines and pancreas were collected, fixed in 10% buffered formalin, and routinely processed for histopathology. After 24 hours they stood on the aviary floor looking for drinking water, and died within three days after onset of the first symptoms. Both birds were necropsied. Samples of liver, spleen, lung, kidneys ovary and adrenals were processed for histopathology as described below. Six offspring of the same species and birds of two other species -Eurasian pygmy owl (Glaucidium passerinum) and northern hawk-owl (Surnia ulula) -died with similar symptoms in the same time period but were not examined due to post mortal autolysis. On August 28 th , 2007, one free-living adult female Eurasian blackbird (Turdus merula) was found in downtown Milan unable to fly, dull, and had been easily caught by hand. It died spontaneously a few minutes after submission to the Department and was immediately necropsied. Samples of liver, spleen, heart, lung, kidney, brain, eyes, proventriculus, gizzard, intestines and pancreas were fixed in 10% buffered formalin and routinely processed for histopathology. Additional samples of the same organs were stored at -20°C.

2008:

One year later, in July 2008, a male wild Eurasian blackbird was found dead in the garden of the owl breeder in the Milan area. The bird was necropsied and samples of liver, spleen, lung, kidneys and heart were collected for histopathology.

Histopathology and immunohistochemistry

Four micrometer thick paraffin-wax sections of the investigated birds were stained with haematoxylin-eosin and examined by light microscopy. Additional sections from the great grey owl and the boreal owls, and from the first-mentioned Eurasian blackbird were stained for demonstration of viral antigen using a rabbit polyclonal antiserum to USUV (produced at the Department of Microbiology, Faculty of Veterinary Science, Budapest) and the avidin-A c c e p t e d M a n u s c r i p t 5 biotin complex technique, as described previously (Weissenböck et al., 2004). Brain sections of experimentally infected suckling mice were included as positive controls, and sections of the samples incubated with normal rabbit serum served as negative controls.

RT-PCR, sequencing, and sequence analysis

Paraffin-embedded tissue (PET) samples (from the owls and from the second ( 2008) blackbird) and frozen, unfixed organ samples (from the first blackbird) were submitted for molecular investigations. The PET samples were deparaffinated by xylol and ethanol treatment, and digested with proteinase K prior to nucleic acid extraction [START_REF] Weissenböck | Screening for West Nile virus infections of susceptible animal species in Austria[END_REF]. Unfixed organ samples were homogenized, suspended in RNase-free water, centrifuged at 6,000 ×g for 5 min, and the supernatants were used for RNA extraction. Viral nucleic acids were extracted by the QiaAmp Viral RNA Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. The viral nucleic acid was reverse-transcribed and amplified in continuous RT-PCR assays, using USUV-specific oligonucleotide primers and the Qiagen One-Step RT-PCR kit [START_REF] Bakonyi | Complete genome analysis and molecular characterization of Usutu virus that emerged in Austria in 2001: comparison with the South African strain SAAR-1776 and other flaviviruses[END_REF]. From the frozen samples (2007 blackbird), one partial sequence of the putative E and NS1 proteins (1229 bp) was obtained.

In the case of PET-derived samples, shorter genome regions were amplified, i.e. partial sequences of the putative NS3 protein (278 bp) from the great grey owl and of the putative NS5 protein (159 bp) for the boreal owls. In addition, one partial sequence (163 bp) of the putative 3'UTR region was determined for all samples. Detailed information on the primers is summarized in Table 1. The thermal profile of the amplifications contained a reverse transcription step at 50°C for 30 min, followed by HotStarTaq activation at 95°C for 15 min, and 40 cycles of amplification at 94°C -40 sec, 57°C -50 sec, 72°C -1 min, respectively.

After a final elongation step at 72°C for 7 min, the reaction mixtures were stored cooled until PCR products with expected size were excised from the gel, purified, and the nucleotide sequences were determined in both directions using fluorescence-based direct sequencing reactions [START_REF] Bakonyi | Complete genome analysis and molecular characterization of Usutu virus that emerged in Austria in 2001: comparison with the South African strain SAAR-1776 and other flaviviruses[END_REF]. The nucleotide sequences were identified by BLAST search against the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). After manual editing and excluding primer regions, the sequences were compiled and aligned with the USUV complete genome sequences deposited in GenBank database (accession numbers AY453411, AY453412 and EF206350) with the help of the Align Plus 4 (Scientific and Educational Software) and ClustalX (Thomson et al., 1997) programs. The putative amino acid sequences were also deduced.

RESULTS

Gross and microscopic findings 2006:

The great grey owl showed at necropsy an enlarged and congested liver. No other gross changes were noticed. Lympho-plasmacytic infiltrates were detected in liver, spleen and kidney. USUV positive immunolabelling was observed in liver, lung, spleen and ovary.

2007:

The boreal owls were in good body condition. At necropsy the female showed only nephromegaly. Hepatomegaly associated with enlargement and discoloration of the kidneys were observed in the male. Histologically, liver and kidney sections of the female boreal owl were characterized by locally extensive lympho-plasmacytic infiltrates. In male boreal owl, liver sections revealed periportal lympho-plasmacytic infiltrates. The spleen was characterized by zonal necrosis centred on the sheathed arteries associated with histiocytic hyperplasia. Minimal lympho-plasmacytic perivascular cuffings were detectable in the lungs. The blackbird was moulting and lean. Liver and spleen were severely enlarged and hyperaemic (Figures 12). The liver was characterized by myriads of small (up to 1 mm) yellowish foci. The gallbladder was enlarged and distended. Meninges and brain were hyperaemic. The intestine was enlarged and contained numerous trematodes.

Histopathological investigations revealed multifocal to coalescing areas of non zonal, coagulative necrosis in the liver associated with small, multifocal haemorrhages. Small, multifocal lympho-plasmacytic aggregates were also observed. The spleen was also characterized by small areas of coagulative necrosis. The cardiac sections revealed multifocal infiltrations of lymphocytes and plasma cells with lesser numbers of macrophages centred on fragmented cardiomyocytes. The brain was characterized by increased numbers of microglia with satellitosis and neuronophagia. Sections of the other organs were without pathological findings. Immunohistological examination revealed scattered foci of USUV-immunoreactivity in the encephalon (Figure 3), cerebellum and in some endothelial cells of lung and heart.

2008

The second blackbird showed no remarkable gross changes except for numerous trematodes in the coelom and respiratory tract. Histologically, small, scattered periportal lympho-plasmacytic infiltrates were observed in the liver sections. USUV immunolabelling was detected in liver, spleen, lung, kidneys and heart.

Positive and negative controls gave the expected results.

RT-PCR, sequencing, and sequence analysis. RT-PCR assays employing USUV-specific primer pairs generated amplification products of the expected molecular sizes. The nucleotide sequences of the amplification products were determined, and BLAST searches verified that they are USUV-specific sequences. The partial E protein coding region and the partial 3' UTR A c c e p t e d M a n u s c r i p t 8 were 100% identical with the corresponding regions of the "USUV-Vienna-2001-blackbird" strain (AY453411), and differed in one nucleotide (C 2185 to U substitution) from the "USUV-Budapest-2005-blackbird" strain (EF206350, [START_REF] Bakonyi | Complete genome analysis and molecular characterization of Usutu virus that emerged in Austria in 2001: comparison with the South African strain SAAR-1776 and other flaviviruses[END_REF]2007). Within the partial NS3 coding region the great grey owl-derived sequence showed one nucleotide difference (A 4900 to G), and in the partial NS5 coding region three nucleotide substitutions (U 9087 to C, C 9138 to U, and U 9153 to C) were observed, compared to the Viennese and Budapest USUV strains. The nucleotide substitution in the NS3 region caused an Ile 1602 to Val substitution in the deduced amino acid sequence, while the NS5 nucleotide substitutions remained silent.

DISCUSSION

This report is the first virological proof of USUV in Italy. USUV was detected in free-living blackbirds and in captive owls of two different species during summertime. Gross lesions observed in the dead birds consisted of general congestion, hepatomegaly and splenomegaly of varying severity. Microscopically, encephalitis, myocardial degeneration and necrosis of liver and spleen were found, and point towards multi-organ failure as the cause of death. The species of the affected birds, the season, the clinical and pathological similarities to the USUV cases reported earlier in Austria [START_REF] Weissenböck | Emergence of Usutu virus, an African mosquito-borne flavivirus of the Japanese encephalitis virus group, central Europe[END_REF][START_REF] Chvala | Pathology and viral distribution in fatal Usutu virus infections of birds from the 2001 and 2002 outbreaks in Austria[END_REF] [START_REF] Weissenböck | Screening for West Nile virus infections of susceptible animal species in Austria[END_REF]. Also, these sequence similarities could be considered in a possible future development of an USUV vaccine, which could be potentially valuable to protect endangered and worthy birds such as owls.

In two previous reports [START_REF] Rizzoli | West Nile virus circulation detected in northern Italy in sentinel chickens[END_REF]Lelli et al., 2008) antibodies to USUV had been demonstrated in sentinel chickens in northern Italy; USUV-associated bird mortality, however, was not reported in these publications. Italy has implemented a flaviviral surveillance system for more than a decade including sentinel horses, chicken and cattle. This plan also includes investigations of wild birds found dead. Unfortunately, it is well known that only a fraction of birds dying in the wild are noticed and still only a small proportion of those are collected and made available for laboratory diagnosis [START_REF] Chvala | Monitoring of Usutu virus activity and spread by using dead bird surveillance in Austria, 2003-2005[END_REF].

Moreover, USUV diagnosis requires adequately conserved tissues. This is infrequent for birds, especially for wild birds, which are usually submitted for laboratory diagnosis with severe post mortal changes. Consequently, it can be argued that USUV infections remain underestimated and unnoticed unless associated with considerable avian mortality. Therefore, dead bird surveillance should be particularly encouraged as suggested by previous experiences [START_REF] Chvala | Monitoring of Usutu virus activity and spread by using dead bird surveillance in Austria, 2003-2005[END_REF]. Besides, this report points out that zoos and rescue centres can play an important role in monitoring USUV infections. Increased dead bird surveillance would uncover the geographic distribution of USUV-infected birds in Italy, the most frequently affected species, the seasonality of USUV infections, changes in the severity of pathological lesions, and similarities among strains through RT-PCR and subsequent sequencing of the amplification products. Our laboratory diagnosed relatively few cases of USUV infection in wild birds since summer 2007, and no particular mortality has been recorded. On the contrary, checking our database, unexplained outbreaks of blackbird mortality had been observed in Italy since the late 1990's. Blackbirds are considered to be one of the most susceptible host species for USUV infections. Unfortunately, no frozen material A c c e p t e d M a n u s c r i p t 10 from those cases was available. Preliminary results of RT-PCR applied to paraffin embedded tissues of selected cases revealed no positivity for USUV (data not shown). Positivity for West Nile virus is still to be ascertained. More extensive retrospective studies are necessary to establish whether those outbreaks of blackbird mortality in the late nineties were related to flavivirus infections. In that case, herd immunity could justify the few USUV cases recorded in the last few years in Italy, as hypothesized for Austria. Both blackbirds of this report were parasitized by trematodes. As a rule, free-ranging wild birds are commonly afflicted with numerous parasites [START_REF] Cole | Miscellaneous parasitic diseases[END_REF] which weaken their hosts, and thus might contribute also to facilitate USUV infections. All these aspects need to be ascertained.

Therefore, further retrospective studies on archived material as well as prospective surveillance systems should be encouraged worldwide to unveil the origin and other epidemiological characteristics of USUV infection.

  USUV) is a mosquito-borne flavivirus closely related to important human pathogens such as Japanese encephalitis virus, Murray Valley encephalitis virus, Saint Louis encephalitis virus and West Nile virus

  In summer 2007, 2 adult boreal owls (Aegolius funerius), one male and one female, of the same collection where the great grey owls had died in 2006, got sick. First, they looked apathetic and anorectic.

  A c c e p t e d M a n u s c r i p t 6 electrophoresis. Amplification products were electrophoresed in 1.5% agarose gel, and following ethidium bromide staining they were visualized under UV light.

  was detected in liver, lung, kidney, spleen and ovary from the female boreal owl, and in lung, kidney, spleen and gizzard from the male.

  supported a presumptive diagnosis of USUV infection, which was confirmed by IHC and RT-PCR. The partial nucleotide sequences of the Italian USUV strains revealed 99.8 to 100% identity to other central European strains. This result indicates that the USUV strains, which circulate in Italy, are closely related to other European strains, suggesting the possibility that these USUV strains spread to Italy from neighbouring central European countries, where the infection emerged earlier. On the other hand it should be mentioned that the affected bird species in those countries are quite sedentary with little tendency to fly during the season of virus

  .,Bakonyi, T., Chvala, S., Nowotny, N., 2004. Experimental Usutu virus infection of suckling mice causes neuronal and glial cell apoptosis and demyelination. ActaNeuropathol. 108,[453][454][455][456][457][458][459][460]

  Figures 1 and 2: Pathological findings in an USUV-infected blackbird: enlarged and

Figure 3 :

 3 Figure 3: Brain section of an USUV-infected blackbird. Positive immunolabelling is

  Figure 1

  Figure 2

  Figure 2

Figure 3

 3 Figure 3

Table 1 .

 1 USUV-specific oligonucleotide primers used in RT-PCR assays, amplified genomic 1 Primers used for virus RNA detection in PET-derived samples 3 b Positions refer to the genome of USUV strain SAAR-1776 (GenBank accession number AY453412)

	14

a in Italics:

Acknowledgements

This study was partially supported by the grants OTKA K67900, OMFB00738/07 and by funds from Regione Lombardia (Italy). T. Bakonyi is grantee of the Bolyai Research Fellowship Grant of the Hungarian Academy of Sciences.


A Loeffler
email: aloeffler@rvc.ac.uk

D U Pfeiffer

J A Lindsay

R Soares-Magalhaes

D H Lloyd

Lack of transmission of methicillinresistant Staphylococcus aureus (MRSA) between apparently healthy dogs in a rescue kennel

Keywords: MRSA, dogs, carriage, transmission

Although it is widely accepted that methicillin-resistant Staphylococcus aureus (MRSA) can be transmitted between humans and animals in both directions, little is known about the dynamics of animal-to-animal transfer. This study aimed to investigate aspects of dog-to-dog MRSA transfer in a rescue facility in the South-East of England during an MRSA outbreak.

One hundred and twenty nine apparently healthy dogs, mostly housed in pairs, were swabbed at nasal, oral, axillary and perianal sites. Swabs were enriched in selective broth and staphylococci identified using standard biological methods. MRSA isolates were confirmed by demonstration of the thermonuclease gene (nuc) and mecA. After initial swabbing, a dog excluded from the study design but housed at the same facility was discovered to have a wound infection due to MRSA.

MRSA carriage was identified in 10/129 dogs (7.8%) and all isolates were of the same lineage as the one isolated from the infected dog. All carrier dogs lived in shared kennels and their 16 kennel partners sampled negative on two occasions. Concurrently with successful antimicrobial treatment of the infected patient, MRSA carriage resolved spontaneously in all dogs within two weeks.

In conclusion, MRSA did not transmit readily between apparently healthy dogs, MRSA carriage was not supported for long periods in a regularly cleaned environment and exposure alone may not lead to MRSA acquisition by dogs without the presence of additional risk factors.

A c c e p t e d M a n u s c r i p t Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is now widely accepted as a human and animal pathogen. For dogs in particular, indirect evidence from case reports and epidemiological strain typing suggests that MRSA transmission between human and animal hosts can occur in both directions [START_REF] Baptiste | Methicillin-resistant staphylococci in companion animals[END_REF][START_REF] Hanselman | Methicillin-resistant staphylococcal colonization in dogs entering a veterinary teaching hospital[END_REF][START_REF] Loeffler | Prevalence of methicillin-resistant Staphylococcus aureus among staff and pets in a small animal referral hospital in the UK[END_REF][START_REF] Van Duijkeren | Human-todog transmission of methicillin-resistant Staphylococcus aureus[END_REF][START_REF] Weese | Suspected transmission of methicillin-resistant Staphylococcus aureus between domestic pets and humans in veterinary clinics and in the household[END_REF][START_REF] Cefai | Human carriage of methicillin-resistant Staphylococcus aureus linked with pet dog[END_REF][START_REF] Manian | Asymptomatic nasal carriage of mupirocin-resistant, methicillin-resistant Staphylococcus aureus (MRSA) in a pet dog associated with MRSA infection in household contacts[END_REF]. Thus, dogs are considered as potential vectors and reservoirs for MRSA with implications for public health [START_REF] Tisinger | Empowering your patients in the fight against methicillin-resistant Staphylococcus aureus[END_REF]. In contrast, little is known about the epidemiology of MRSA within the dog population and whether dogs can support MRSA carriage or contamination over time. While the term "reservoir" may imply the spread of an organism within the reservoir host to maintain the pathogen indefinitely [START_REF] Ashford | When is a reservoir not a reservoir?[END_REF], this has not been established for MRSA in animals. Unfortunately, temporal studies of the duration of MRSA carriage and studies on transmission of MRSA between animals in privately owned dogs is associated with practical and ethical difficulties as leaving any detected carrier animals or infected pets untreated may allow the spread of a human pathogen. In one case, MRSA carriage was maintained in a healthy dog over several weeks but the dog lived with immunocompromised owners previously infected with MRSA and continuing to suffer from open wounds. Therefore, contamination of the environment was highly likely and could have accounted for positive samples recovered from the dog [START_REF] Manian | Asymptomatic nasal carriage of mupirocin-resistant, methicillin-resistant Staphylococcus aureus (MRSA) in a pet dog associated with MRSA infection in household contacts[END_REF]. In horses, it has been proposed that MRSA carriage is short lived, but again specific longitudinal studies on animal carriage have not been published [START_REF] Weese | Attempted eradication of methicillin-resistant Staphylococcus aureus colonisation in horses on two farms[END_REF]. Such information is urgently needed though to evaluate whether decolonisation of carrier animals is possible and required. Decolonisation is used in humans as part of comprehensive infection control strategies to prevent MRSA infection. However, its value remains controversial even in people due to inconsistent long-term efficacy and increasing drug resistance amongst MRSA.

The aim of this study was to examine the potential for animal-to-animal transmission of MRSA in apparently healthy carrier dogs housed in shared kennels during an MRSA outbreak.

Materials and methods

Study design

Apparently healthy dogs at a large rescue centre in the Greater London area were sampled for MRSA carriage as part of a cross-sectional prevalence study in companion animals. Dogs were considered healthy if they were (1) free of non-neoplastic skin disease, fresh wounds and abnormal discharge from body openings on inspection,(2) had not received within the previous three weeks either antimicrobial or immunosuppressive therapy, (3) had not required within the previous three weeks veterinary attention other than for routine health checks, vaccination, neutering and antiparasitic treatment administered by the facility's own veterinary surgeon on site and (4) were considered fit for rehoming. Dogs were sampled irrespective of their length of stay at the facility and dogs in contact with diseased animals were excluded from the screening. The precision of the prevalence estimate was expressed using the 95% confidence interval (WinEpiscope 2.0) and groups were compared using the chi-squared test with a P-value of less than 0.05 considered as significant.

A dog (index case) with a surgical wound infection had been housed at the premises for five weeks before study begin. Four days after the first prevalence screening, MRSA isolation from the wound was reported. An epidemiological link between the MRSA carriage rate in apparently healthy dogs and the infected dog was proposed based on the temporal and geographical overlap and both were investigated as an outbreak. The infected dog and its kennel partner were sampled for carriage as part of this outbreak investigation. Both dogs had been kennelled in a wing separate from the healthy screened dogs but communal areas and staff movement along shared corridors provided communication between the wings.

Most dogs were housed in pairs without the opportunity for contact with neighbouring kennels.

Equipment such as food bowls and toys were either dog or dog pair specific or they were washed between use. Kennels were cleaned and disinfected (TriGene, Medichem International, Sevenoaks, UK) twice daily. Infection control policies at the facility remained unchanged during the study period.

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

To minimise contamination during sampling, hands were washed when macroscopically dirty or after handling a group of animals; alcohol hand gel was used before and after handling of each dog. All investigators handling swabs or broth vials confirmed fortnightly that their own nasal swabs were MRSA negative following the methods described for animal samples.

The study was approved by the Royal Veterinary College Ethics and Welfare Committee. Animals positive for MRSA were re-sampled seven to ten days after the first swab and if follow-up swabs were also positive, topical antimicrobial therapy with products licensed for use in dogs would be initiated to pursue elimination of MRSA from carrier sites before rehoming.

Swab collection and identification of MRSA

Saline-moistened swabs from both nostrils, the buccal mucosa, the axillary skin and the anal mucosa were pooled immediately tryptone soya broth (Oxoid, Basingstoke, UK) supplemented with sodium chloride (Sigma-Aldrich, Gillingham, UK) to a total salt concentration of 10% and incubated at 37° C for 48 h. Samples were streaked out and incubated at 37° C on blood agar (Oxoid) containing 5% ovine blood (TCS Biosciences, Buckingham, UK) for isolation of total staphylococci and on mannitol salt agar (Oxoid) with 6 mg/l oxacillin (MSA)(Sigma-Aldrich) for selective isolation of MRSA and examined after 24 and 48 h respectively. Colonies from MSA with oxacillin were subcultured on blood agar for further identification. Presumptive staphylococci were identified based on colony morphology, haemolysis, Gram staining and salt resistance. S. aureus was identified phenotypically by positive results for DNase, clumping factor with dog plasma or, if negative, tube coagulase using rabbit plasma, Voges-Proskauer reaction (VP-A and VP-B, bioMérieux, France) and fermentation tests with trehalose and lactose [START_REF] Barrow | Cowan and Steel's Manual for the Identification of Medical Bacteria[END_REF]. Resistance to methicillin was verified by disc diffusion tests according to CLSI guidelines on Mueller-Hinton agar using 5μg methicillin discs (Oxoid) and overnight incubation at 30°C (CLSI, 2004).

A c c e p t e d M a n u s c r i p t 6 MRSA were confirmed genotypically by the demonstration of the S. aureus-specific thermonuclease gene (nuc) and mecA after gene replication by polymerase-chain reaction (PCR) [START_REF] Becker | Thermonuclease gene as a target for specific identification of Staphylococcus intermedius isolates: Use of a PCR-DNA enzyme immunoassay[END_REF]Brakstad et al., 1993) and typed by determination of the S. aureus lineage-specific restriction modification enzyme system [START_REF] Cockfield | Rapid determination of hospitalacquired meticillin-resistant Staphylococcus aureus lineages[END_REF]. Reference strains for bacteriological and genetic assays were ATCC29663, PM62 [START_REF] Moore | Molecular characterisation of the dominant UK methicillin-resistant Staphylococcus aureus strains, EMRSA-15 and EMRSA-16[END_REF], ATCC49545, LMG22219.

Results

Carrier animals

A total of 129 dogs was sampled on two days during December 2006 and January 2007. These accounted for 71.7% of the 180 dogs present at the facility during the study period while 51 dogs, including the index case and its kennel partner, were excluded from the screening due to concurrent or preceding disease, behavioural problems or imminent rehoming. The first sampling involved 84 dogs of which six (7.1%) were positive for MRSA; on the second study day, three weeks later, 4/45 dogs (8.9%) yielded MRSA. Proportions of positive dogs were similar on the two days (P=0.72) and a prevalence estimate of 7.8% MRSA carriage was calculated for the total population of 129 dogs (standard error 2.5%, confidence interval 95%).

The 129 dogs sampled included 82 crossbred dogs and 47 presumed pure breeds (27 terriers, 5 spaniels, 18 large breeds such as Rottweiler, German shepherd dog, Doberman, Setter, Border collie and 3 toy dog breeds). Sixty-seven dogs were male (34 neutered) and 62 female (33 neutered). Their approximate ages ranged from three months to 16 years (mean 2.1 years) and at the time of sampling their mean length of stay at the premises was 3.6 weeks (range between six hours to three months).

Of the ten carrier dogs, none was housed alone, five shared a kennel with one other dog and three shared with two other dogs. The remaining two MRSA carriers were two of seven three-month-old puppies from the same litter, all kept in the same kennel. They were swabbed on the day of arrival at A c c e p t e d M a n u s c r i p t 7 the centre after they had been rescued and transported to the facility in the same container over a period of 48 hours. Thus, sixteen dogs in total had close direct contact with at least one of the ten MRSA carrier dogs; all sixteen in-contacts sampled negative for MRSA on the same occasion.

Follow-up swabs taken from the ten original carrier dogs and from the sixteen in-contact kennel mates which had remained in the same kennels were all negative for MRSA; samples from these 26 dogs all yielded non-MRSA staphylococci.

Index case

The infected dog and its kennel partner were sampled for carriage nine days after the first screening of healthy dogs. Systemic antimicrobial treatment of the infected wound with trimethoprimsulfamethoxazole had been initiated five days earlier. While the infected dog also carried MRSA in the nostrils and on the perineum, swabs from its axilla and the mouth as well as all four swabs from the in-contact dog were negative. Due to the need for prompt rehoming and the zoonotic risk of MRSA, the dog was isolated and decolonisation using fusidic acid and chlorhexidine topically was started. MRSA was no longer isolated from samples taken one week later, nor from a swab of the closed wound on the dorsum. Topical antimicrobial therapy was discontinued five days and systemic treatment fifteen days after the last sampling. A final follow-up sample from carrier sites was submitted prior to rehoming one month later and was again negative.

Genotyping

The infection isolate was obtained from the veterinary laboratory, confirmed as MRSA and typed as described above. All ten carriage isolates from apparently healthy dogs, the MRSA from the wound and the two MRSA isolates from the infected dog's nostrils and perineum were of clonal cluster 22, represented in the UK by EMRSA-15 [START_REF] Johnson | Dominance of EMRSA-15 and -16 among MRSA causing nosocomial bacteraemia in the UK: analysis of isolates from the European Antimicrobial Resistance Surveillance System (EARSS)[END_REF].

Discussion

The study findings indicate that MRSA is not always acquired by healthy dogs despite exposure and that it is not transmitted readily between in-contact dogs. In particular, the dog sharing a kennel with the infected animal for four weeks prior to isolation and therefore the animal thought to be most at risk from exposure sampled negative at all four carrier sites. Although the sensitivity of surface swabbing may be similar to the 77% quoted for human nasal sampling [START_REF] Sewell | Sensitivity of surveillance cultures for the detection of methicillin-resistant Staphylococcus aureus in a nursing-home-care unit[END_REF], false negative results cannot be ruled out especially since the use of a high salt concentration in enrichment broth (10%) and oxacillin rather than cefoxitin discs may have underestimated the occurrence of MRSA [START_REF] Pottumarthy | Evaluation of alternative disk diffusion mehtods for detecting mecA-mediated oxacillin resistance in an international collection of staphylococci: Validation report from the SENTRY Antimicrobial Surveillance Program[END_REF]. However, the lack of MRSA isolation from this incontact dog strongly suggests that exposure alone does not necessarily result in carriage in healthy dogs and that other factors may be required to facilitate MRSA acquisition.

Similarly, another sixteen in-contact kennel partner dogs, each sharing a kennel with at least one MRSA carrier, did not yield MRSA when sampled on two occasions. While the differences between exposure, contamination, carriage and colonisation may be ill defined, the lack of MRSA isolation from those 16 in-contact dogs suggests that dog-to-dog transmission of MRSA is infrequent. Dog-todog transmission cannot be excluded in the two cohabitating puppies though, but as they were sampled within hours of arrival at the facility, separate MRSA acquisition prior to or during transport is also possible. In people, contact with other carriers or infected patients, antimicrobial treatment, advanced age and association with hospitals or healthcare facilities are well documented as the main risk factors for MRSA carriage [START_REF] Harbarth | A predictive model for identifying surgical patients at risk of methicillinresistant Staphylococcus aureus carriage on admission[END_REF]. None of these was identified in the in-contact animals but unfortunately only limited histories were available for most rescue dogs.

MRSA exposure may have occurred from a contaminated environment, from human vectors, from direct contact with the infected dog or with carrier dogs or via inanimate objects, and all routes would be compatible with the studied outbreak scenario. In people, MRSA is most frequently transferred by hand contact but acquisition from contaminated environments and inanimate objects A c c e p t e d M a n u s c r i p t 9 has also been documented [START_REF] Sexton | Environmental reservoirs of methicillin-resistant Staphylococcus aureus in isolation rooms: correlation with patient isolates and implications for hospital hygiene[END_REF]. Although environmental swabs were not taken, the regular use of detergents and disinfectants is likely to have reduced environmental contamination.

As dog-to-dog transmission could not be demonstrated, human vectors or inanimate objects may have been the main routes of transfer that led to the high MRSA prevalence detected.

Although MRSA exposure may have been reduced in the dogs sampled on the second study day as antimicrobial treatment had by then been initiated for the index case, carriage rates were similar for both groups (7.1% and 8.9% respectively; P=0.72). The combined MRSA carriage prevalence of 7.8% amongst apparently healthy dogs in a rescue facility in the South-East of England was alarmingly high. A previous study from the North-West of England did not detect any MRSA carriers in 22 dogs sampled in the community [START_REF] Baptiste | Methicillin-resistant staphylococci in companion animals[END_REF]. Similarly, MRSA carriage was rare in healthy dogs in Hong Kong (6/815, 0.72%) [START_REF] Boost | Characterisation of methicillin-resistant Staphylococcus aureus isolates from dogs and their owners[END_REF] and not detected in Slovenia (n=200) [START_REF] Vengust | Methicillin-resistant staphylococcal colonization in clinically normal dogs and horses in the community[END_REF]. In contrast, MRSA was isolated from 14% of dogs at a university referral hospital in the South-East of England but all were diseased and had received veterinary treatment prior to referral [START_REF] Loeffler | Prevalence of methicillin-resistant Staphylococcus aureus among staff and pets in a small animal referral hospital in the UK[END_REF]. These data suggest that the prevalence of MRSA carriage amongst healthy dogs is low unless risk factors predispose or if particular routes of transmission, as in the presented study, exist.

In line with other MRSA reports from UK dogs, all MRSA isolated in this study were typed as EMRSA-15 [START_REF] Baptiste | Methicillin-resistant staphylococci in companion animals[END_REF][START_REF] Loeffler | Prevalence of methicillin-resistant Staphylococcus aureus among staff and pets in a small animal referral hospital in the UK[END_REF]. This lineage is considered highly clonal in the UK so that further genotypic distinction of isolates by additional typing methods would not have been helpful. More discriminatory genetic information on isolates within this lineage could, for example, be gained from comparative genome microarray analysis and this might have helped to support a common origin of the two isolates from puppies. However, it would not have contributed to the study objective which was to investigate dog-to-dog transmission of MRSA.

  all healthy carrier dogs was transient and resolved without antimicrobial decolonisation therapy in the regularly cleaned and disinfected environment. In addition, MRSA was not transferred readily between healthy dogs despite close contact. This is the first study to describe the dynamics of MRSA transmission in a dog kennel environment; similar research is now required within a home environment.

Acknowledgements

This work was funded by PetPlan Charitable Trust, UK, as part of a larger study into the prevalence of MRSA carriage in companion animals. The authors are very grateful to Chris Laurence, Dogs Trust, for his supportive collaboration and to Sian Buckingham for her skilled help with the sampling.