S Sánchez

R Martínez

J Rey

A García

J E Blanco

M Blanco

A Mora

S Herrera-León

A Echeita

J M Alonso

Pheno-genotypic characterization of Escherichia coli O157:H7 isolates from domestic and wild ruminants

Keywords: E. coli O157:H7, STEC, Phenotypic variant, Virulence genes, Phage typing, PFGE, Domestic and wild ruminants

Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major public health concern worldwide, with ruminants recognized as their main natural reservoir. The aim of this work was to determine the phenotypic features and genetic relationships of 46 E. coli O157:H7 isolates obtained from sheep, cattle, and deer faeces and from unpasteurized goat milk in Spain over a period of 11 years. Characterization was performed by PCR, phage typing, and pulsed-field gel electrophoresis (PFGE). An atypical E. coli O157:H7 strain (sorbitol-fermenting and β-glucuronidase positive) originating from deer faeces was detected. Genes encoding Shiga toxins were detected in 69.6% of isolates, all of them carrying only the stx 2 gene. The isolates were from nine different phage types, although 67.4% were restricted to only three: PT14, PT34, and PT54. PT54 was the most prevalent phage type and contained isolates from cattle, sheep, and deer. The majority of isolates were from phage types previously found in strains associated with human infection. XbaI-PFGE identified 33 different types and 11 groups of closely related types (more than 85% similarity), one of which included 21 (45.7%) isolates originating from different animal species, including deer. These results indicate common origin or inter-species spread of genetically similar E. coli O157:H7 isolates and contribute to earlier investigations identifying deer as a natural source of E. coli O157:H7. The study also highlights the emergence of phenotypic variants of E. coli O157:H7 which may not be identified by routine culture methods or by biochemical tests used to characterize serotype O157:H7.

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

Introduction

Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major public health concern worldwide. Human diseases ranging from mild diarrhoea to haemorrhagic colitis and the life-threatening haemolytic uraemic syndrome can be caused by STEC O157:H7, typically affecting children, the elderly, and immunocompromised patients (Centers for Disease [START_REF] Centers For | Outbreaks of Escherichia coli O157:H7 infections among children associated with farm visits -Pennsylvania and Washington, 2000[END_REF]. The pathogenic capacity of STEC resides in a number of virulence factors, including Shiga toxins (Stx1 and Stx2), intimin, and the enterohaemolysin [START_REF] Gyles | Shiga toxin-producing Escherichia coli: an overview[END_REF]. Unlike other E. coli strains, O157:H7 strains neither ferment sorbitol nor exhibit β-glucuronidase (GUD) activity after overnight incubation, and these differences facilitate the detection of this organism [START_REF] Kehl | Role of the laboratory in the diagnosis of enterohemorrhagic Escherichia coli infections[END_REF].

Healthy domestic ruminants can harbour STEC O157:H7 in their faeces and are thus natural reservoirs of this pathogen [START_REF] Rey | Serotypes, phage types and virulence genes of Shiga-producing Escherichia coli isolated from sheep in Spain[END_REF][START_REF] Blanco | Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from cattle in Spain and identification of a new intimin variant gene (eae-ξ)[END_REF][START_REF] Orden | A longitudinal study of verotoxin-producing Escherichia coli in two dairy goat herds[END_REF], although STEC O157:H7 strains have also been isolated from wild ruminants, especially from deer [START_REF] Renter | Escherichia coli O157:H7 in free-ranging deer in Nebraska[END_REF][START_REF] García-Sánchez | Presence of Shiga toxin-producing E. coli O157:H7 in a survey of wild artiodactyls[END_REF]. Sources of human infection include undercooked meat, unpasteurized milk and dairy products, vegetables or water, and contact with animal carriers or the environment [START_REF] Gyles | Shiga toxin-producing Escherichia coli: an overview[END_REF].

The aim of the current study was to characterize a collection of E. coli O157:H7 isolates obtained from domestic and free-ranging wild ruminants in Spain over a period of 11 years, with the objective of determining their phenotypic features and genetic relationships and therefore contributing to the knowledge of the epidemiology of this pathogen.

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

Materials and methods

E. coli O157:H7 isolates

A total of 46 E. coli O157:H7 isolates obtained from faeces of different healthy ruminants [sheep, extensive and beef cattle, and red deer (Cervus elaphus)] and from unpasteurized goat milk over a period of 11 years (1997 to 2008) were included in the present study. All of them originated from the Extremadura region in the South-West of Spain and comprised (i) 24 isolates epidemiologically related in different groups (15 isolates from sheep, 7 isolates from extensive cattle, and 2 isolates from deer) and (ii) 22 isolates not known to be epidemiologically related (14 isolates from sheep, 4 isolates from beef cattle, 3 isolates from deer, and 1 isolate from unpasteurized goat milk). Epidemiologically related isolates were E. coli O157:H7 isolates obtained from different animals at a single sheep flock, cattle herd, or game estate during the same periods. Some of the isolates included in the present study were obtained from previously published studies [START_REF] Rey | Serotypes, phage types and virulence genes of Shiga-producing Escherichia coli isolated from sheep in Spain[END_REF][START_REF] Rey | Prevalence, serotypes and virulence genes of Shiga toxin-producing Escherichia coli isolated from ovine and caprine milk and other dairy products in Spain[END_REF][START_REF] García-Sánchez | Presence of Shiga toxin-producing E. coli O157:H7 in a survey of wild artiodactyls[END_REF][START_REF] Sánchez | Shiga toxin-producing Escherichia coli O157:H7 from extensive cattle of the fighting bulls breed[END_REF], and the procedures for their isolation are described in detail in the reports of those studies.

Biotyping, serotyping, and phage typing

All E. coli O157:H7 isolates were confirmed biochemically as E. coli by the API 20E system (bioMérieux, Marcy L'Etoile, France). Fermentation of sorbitol and GUD activity were investigated on sorbitol MacConkey agar (Oxoid, Basingstoke, England) and Chromocult Coliform agar (Merck, Darmstadt, Germany), respectively, after overnight incubation at 37 ºC. The phage typing was performed as described by [START_REF] Khakhria | Extended phage-typing scheme for Escherichia coli O157:H7[END_REF] in the Centro Nacional de Microbiología (Instituto de Salud Carlos III, Madrid, Spain) using phages provided by the National Microbiology Laboratory (Public Health Agency of Canada, Winnipeg, Canada). The 16 different phages used were capable of identifying 90 phage types.

PCR of stx 1 , stx 2 , ehxA, eae, O157 rfbE, and fliCh7 genes

All isolates were tested as previously described [START_REF] Mora | Phage types and genotypes of Shiga toxin-producing Escherichia coli O157:H7 isolates from humans and animals in Spain: identification and characterization of two predominating phage types (PT2 and PT8)[END_REF] with primers specific for the genes encoding Stx1 and Stx2 (stx 1 and stx 2 ), enterohaemolysin (ehxA), intimin (eae and eae-γ1 variant), O157 antigen (O157 rfbE), and H7 antigen (fliCh7). Reference E. coli strains used as controls were EDL 933 (human, O157:H7, stx 1 , stx 2 , eae, ehxA) (ATCC No. 43895) and K12-185 (negative for stx 1 , stx 2 , eae, and ehxA genes) [START_REF] Blanco | Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from cattle in Spain and identification of a new intimin variant gene (eae-ξ)[END_REF].

Pulsed-field gel electrophoresis (PFGE)

PFGE was performed in accordance with the PulseNet-Europe protocol (http://www.pulsenet-europe.org/docs.htm). Genomic DNA was digested with XbaI (Roche Diagnostics, Mannheim, Germany) and analyzed in 1% agarose gels (Bio-Rad, Hemel Hempstead, United Kingdom) in 0.5 × TBE buffer at 14 ºC A c c e p t e d M a n u s c r i p t 6 using the CHEF MAPPER system (Bio-Rad). The runtime was 21.3 h at 6 V/cm, with initial and final switch times of 2.16 and 54.17 s, respectively. The XbaIdigested DNA from Salmonella enterica Braenderup H9812 was used as a molecular size marker. Resultant images were analyzed with the InfoQuestFP software (Bio-Rad). Isolates were allocated a different PFGE type when a genetic difference could be detected. Cluster analysis was performed using the Dice coefficient and the unweighted pair group method with arithmetic averages (UPGMA).

Results

Phenotypic properties and phage types

All but one of the isolates evaluated in the current study were biochemically typical of E. coli O157:H7 (non-sorbitol-fermenting and GUD negative) (Fig. 1).

A single isolate, originating from deer faeces, fermented sorbitol and exhibited GUD activity after overnight incubation.

The 46 E. coli O157:H7 isolates were from a total of nine different phage types (Table 1). However, among those nine, three phage types accounted for 67.4% of isolates analysed: PT14 (4 isolates), PT34 (3 isolates), and PT54 (24 isolates). PT54 was the most prevalent phage type among both sheep (62.1%) and deer (80.0%) isolates and one of the most frequently found among cattle isolates (18.2%). Seven isolates reacted with typing phages but did not conform to a recognized pattern (RDNC/NT = reacts but does not conform/non-typeable).

Forty-three (93.5%) of 46 E. coli O157:H7 isolates expressed the H7 antigen and three (6.5%) were nonmotile (NM). These NM isolates belonged to PT14 (1 isolate) and PT34 (2 isolates). The PCR procedure indicated that all E. coli O157:H7 isolates carried the eae-γ1, O157 rfbE, and fliCh7 genes. In addition, genes encoding Shiga toxins were detected in 32 (69.6%) isolates, all of them carrying only the stx 2 gene, and 44 (95.7%) isolates contained the ehxA gene (Fig. 1).

PFGE types and cluster analysis

PFGE of XbaI-digested genomic DNA of the 46 E. coli O157:H7 isolates produced a dendrogram indicating 33 different PFGE types with 15-21 discernible fragments, ranging from approximately 33 to 1100 kb in molecular size (Fig. 1).

Twenty-one types were identified among 29 sheep isolates, six types in 11 cattle isolates, and five types in five deer isolates, and no PFGE type contained isolates from more than one origin. However, these 46 isolates could be divided into 11 groups (I to XI, containing between 1 and 21 isolates per group) of closely related PFGE types (more than 85% similarity), according to the Dice coefficient of similarity (Fig. 1). Forty isolates (87.0%) were grouped in only five clusters (IV, V, VI, VII, and VIII), and one of them (group IV) included 21 (45.7%) isolates originating from sheep, cattle, and deer faeces, with PT54 being the predominant phage type in this cluster.

The major genetic relatedness was observed in four groups containing isolates with indistinguishable PFGE types (100% similarity): IV (6 isolates epidemiologically related from sheep and 2 isolates not known to be epidemiologically related from cattle), VI (2 isolates not known to be epidemiologically related from sheep), VII (5 isolates epidemiologically related A c c e p t e d M a n u s c r i p t 8 from cattle), and VIII (3 isolates epidemiologically related from sheep). The most divergent PFGE type was that of the atypical E. coli O157:H7 isolate obtained from deer faeces, which was distantly related to the other typical isolates (less than 64% similarity) (Fig. 1).

Discussion

The recognition of STEC O157:H7 has been largely facilitated by the availability of classical microbiological diagnostic procedures that are based on the characteristic phenotypic features of this pathogen, in particular, its inability to ferment sorbitol and lack of GUD activity after overnight incubation. However, other phenotypic variants of STEC O157 have been isolated during the last decade in Germany [START_REF] Ammon | A large outbreak of hemolytic uremic syndrome caused by an unusual sorbitol-fermenting strain of Escherichia coli O157:H[END_REF], the Czech Republic [START_REF] Bielaszewska | Isolation and characterization of sorbitol-fermenting Shiga toxin (Verocytotoxin)-producing Escherichia coli O157:H-strains in the Czech Republic[END_REF], Finland [START_REF] Saari | Phage types and genotypes of Shiga toxin-producing Escherichia coli O157 in Finland[END_REF], Italy [START_REF] Bonardi | Isolation of verocytotoxin-producing Escherichia coli O157:H7 from cattle at slaughter in Italy[END_REF], the United States [START_REF] Hayes | Isolation and characterization of a β-D-glucuronidase-producing strain of Escherichia coli serotype O157:H7 in the United States[END_REF], Australia [START_REF] Bettelheim | First isolation outside Europe of sorbitol-fermenting verocytotoxigenic Escherichia coli (VTEC) belonging to O group O157[END_REF], and Japan [START_REF] Nagano | Clonal structure of Shiga toxin (Stx)-producing and β-D-glucuronidase-positive Escherichia coli O157:H7 strains isolated from outbreaks and sporadic cases in Hokkaido, Japan[END_REF]. To our knowledge, the atypical STEC O157:H7 strain (sorbitol-fermenting and GUD positive) originating from deer faeces detected in the present study is the first phenotypic variant isolated in Spain and the first one originating from ruminants other than cattle in Europe, since such variants have been isolated before from deer in Japan and the United States [START_REF] Dunn | Prevalence of Escherichia coli O157:H7 in white-tailed deer from Louisiana[END_REF][START_REF] Nagano | Phenotypic and genotypic characterization of β-D-glucuronidase-positive Shiga toxin-producing Escherichia coli O157:H7 isolates from deer[END_REF]. This atypical STEC O157:H7 strain was distantly related to the other typical strains (less than 64% similarity) (Fig. 1). Similarly, GUD positive STEC O157:H7 strains isolated in Japan between 1996 and 2001, including human and deer strains, belonged to a single cluster only distantly related to the other typical STEC O157:H7 strains (less than 60% similarity by PFGE) [START_REF] Nagano | Clonal structure of Shiga toxin (Stx)-producing and β-D-glucuronidase-positive Escherichia coli O157:H7 strains isolated from outbreaks and sporadic cases in Hokkaido, Japan[END_REF] A c c e p t e d M a n u s c r i p t 9 [START_REF] Nagano | Phenotypic and genotypic characterization of β-D-glucuronidase-positive Shiga toxin-producing Escherichia coli O157:H7 isolates from deer[END_REF]. Based on these findings the authors suggested that such phenotypic variants may represent a distinct clone within STEC serogroup O157.

At least 90 phage types have been currently reported for STEC O157:H7 [START_REF] Ahmed | Emergence of new molecular and phage typing variants of E. coli O157:H7 in Canada[END_REF], but only seven of these (PT2, PT4, PT8, PT14, PT21/28, PT32, and PT54) account for the majority (more than 75%) of human strains isolated in Europe and Canada. Three of these phage types (PT4, PT14, and PT54) were identified in the present study and they accounted for 63.0% of the isolates.

PT54 was the most prevalent phage type and contained isolates from cattle, sheep, and deer. Actually, PT54 was the most prevalent phage type among isolates from deer. To our knowledge, this is the first report of the phage typing of STEC O157:H7 strains isolated from deer in the literature. Fourteen (30.4%) E. coli O157:H7 isolates were stx-negative, although all of them carried genes encoding other virulence-associated factors (enterohaemolysin and/or intimin). Whether this finding resulted from loss of stx gene(s) from initially stx-positive strains remains unclear. However, whilst the human health risks associated with stx-negative E. coli O157:H7 strains may be less significant, the acquisition of stx-carrying phages from the environment cannot be excluded [START_REF] Muniesa | Comparative survival of free Shiga toxin 2-encoding phages and Escherichia coli strains outside the gut[END_REF].

Among the 46 E. coli O157:H7 isolates characterized in the current study no PFGE type contained isolates from more than one origin, findings in contrast to those of [START_REF] Avery | Pulsed field gel electrophoresis of related Escherichia coli O157 isolates associated with beef cattle and comparison with unrelated isolates from animals, meats and humans[END_REF] and [START_REF] Mora | Phage types and genotypes of Shiga toxin-producing Escherichia coli O157:H7 isolates from humans and animals in Spain: identification and characterization of two predominating phage types (PT2 and PT8)[END_REF], with some of the PFGE types containing E. coli O157:H7 isolates from more than one origin, even though none of the samples were known to be epidemiologically related. Nevertheless, the PFGE group IV in the present study included closely related isolates (more than 85% similarity) obtained from different animal species, including deer, over a A c c e p t e d M a n u s c r i p t 10 period of 11 years (isolated in 1997, 2003, 2004, 2007, and 2008) with and without known epidemiological links (Fig. 1). These results indicate common origin or inter-species spread of genetically similar E. coli O157:H7 isolates. On the other hand, indistinguishable or closely related PFGE types were found in isolates recovered from samples from different animals at a single sheep flock, cattle herd, or game estate during the same periods (Fig. 1). This high similarity suggests the existence of horizontal transmission among animals, which has been demonstrated to be important in maintaining E. coli O157:H7 infections on farms [START_REF] Faith | Prevalence and clonal nature of Escherichia coli O157:H7 on dairy farms in Wisconsin[END_REF].

In conclusion, the current study contributes to earlier investigations identifying deer as a natural source of E. coli O157:H7 and reports the isolation of a sorbitol-fermenting and GUD positive strain from deer. This data highlights the emergence of phenotypic variants of E. coli O157:H7 which may not be identified by routine culture methods or by biochemical tests used to characterize serotype O157:H7. The study reports that the most common phage type among strains isolated from deer is also common among human strains, supporting the idea that ruminants are a principal reservoir. The current study also shows the natural occurrence of many genetic variants among E. coli O157:H7 isolated from domestic and wild ruminants in Spain but indicate common origin or inter-species spread of genetically similar isolates.

Conflict of interest statement

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

  O and H antigens was carried out as described by[START_REF] Guinée | Escherichia coli associated with neonatal diarrhoea in piglets and calves[END_REF] using O157 antiserum and the full range of H antisera from H1 to H56. All antisera were absorbed with corresponding cross-reacting antigens to remove non-specific agglutinins. O157 antiserum was produced in the Laboratorio de Referencia de E. coli (Lugo, Spain) and H antisera were obtained from the Statens Serum Institut (Copenhagen, Denmark).

  Gene detection: stx 1 , stx 2 , ehxA, eae, O157 rfbE, and fliCh7

Figure legends Fig. 1 .

 legends1 Figure legends

Acknowledgements

The authors thank R. Rubio and A. Aladueña for their skilful technical assistance. S. Sánchez acknowledges the Ministerio de Educación y Ciencia for his research fellowship (AP2002-3286). R. Martínez acknowledges the Junta de Extremadura for his research fellowship (PRE06053). A. Mora acknowledges the Ramón y Cajal programme from the Ministerio de Educación y Ciencia. This study was supported by grants from the Junta de Extremadura and FEDER (grant 3PR05A009-III Plan Regional de Investigación), from the Fondo de Investigación Sanitaria (Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, grants G03-025-COLIRED-O157 and RD06/0008-1018-REIPI), from the Xunta de Galicia (grants PGIDIT05BTF26101P, PGIDIT065TAL26101P, 07MRU036261PR), and from the Ministerio de Educación y Ciencia (AGL-2008-02129).

a Reacts but does not conform/non-typeable.


Wolfgang Gaede

Karl-Friedrich Reckling

Anette Schliephake

Dirk Missal

Helmut Hotzel

Konrad Sachse
email: konrad.sachse@fli.bund.de

Detection of Chlamydophila caviae and Streptococcus equi subsp. zooepidemicus in horses with signs of rhinitis and conjunctivitis

Keywords: Chlamydophila caviae, Streptococcus equi subsp. zooepidemicus, conjunctivitis, rhinitis, horse, real-time PCR, DNA microarray testing

At a stud farm of Trakehner horses, all 33 foals of a birth cohort developed conjunctivitis and serous to muco-purulent rhinitis, and 7 older horses showed recurrent signs of conjunctivitis.

Examination of nasal and conjunctival swabs by bacterial and cell culture, as well as real-time PCR, ArrayTube microarray analysis and DNA sequencing led to the identification of Chlamydophila (C.) caviae (first description in horses) and Streptococcus (S.) equi subsp. zooepidemicus. We presume a synergistic effect associated with these two agents by hypothesising that primary lesions were set by C. caviae and subsequently aggravated by Streptococcus equi subsp. zooepidemicus. Indications supporting this assumption include i) the conjunctivitis caused by mono-infection with C. caviae, ii) recurrent clinical symptoms in the affected animals, and iii) the absence of a sustained clinical effect of antibiotic therapy with trimethoprim-sulfonamide, enrofloxacin and amoxicillin. The detection of C. caviae in horses raises questions about the significance and natural host range of this agent.

Introduction

Infections of horses with chlamydiae have been known for decades, although it is not quite certain whether the horse represents a natural or aberrant host for these obligate intracellular bacteria. Up until the end of the 1990s, a number of papers reported clinical cases ranging from respiratory disease [START_REF] Mcchesney | Chlamydia psittaci induced pneumonia in a horse[END_REF][START_REF] Moorthy | Chlamydia psittaci infection of horses with respiratory disease[END_REF][START_REF] Popovici | Izolarea microorganismelor din grupul Bedsonia de la cabaline[END_REF], to conjunctivitis [START_REF] Moorthy | Chlamydia psittaci infection of horses with respiratory disease[END_REF][START_REF] Pienaar | The occurrence and pathology of chlamydioses in domestic and laboratory animals: A review[END_REF], polyarthritis [START_REF] Blanco Loizeiler | Clamidiosis equine (aborto y poliartritis clamidial en equinos)[END_REF][START_REF] Mcchesney | Chlamydial polyarthritis in a foal[END_REF][START_REF] Pienaar | The occurrence and pathology of chlamydioses in domestic and laboratory animals: A review[END_REF], encephalo-hepatitis [START_REF] Blanco Loizeiler | Las neo-rickettsiosis animales en España[END_REF]) and abortion [START_REF] Blanco Loizeiler | Clamidiosis equine (aborto y poliartritis clamidial en equinos)[END_REF][START_REF] Bocklisch | Chlamydien als Abortursache beim Pferd[END_REF][START_REF] Dilbeck | Optimized DNA microarray assay allows detection and genotyping of single PCR-amplifiable target copies[END_REF]Glávits et al., 1988, Lehmann and[START_REF] Lehmann | Keimspektrum infektiös bedingter Aborte bei Pferd, Rind, Schwein und Schaf von 1983-1993 in Nordwest-und Mittelthüringen[END_REF][START_REF] Pienaar | The occurrence and pathology of chlamydioses in domestic and laboratory animals: A review[END_REF][START_REF] Popovici | Izolarea microorganismelor din grupul Bedsonia de la cabaline[END_REF]). These cases were ascribed to Chlamydia psittaci (old classification), albeit diagnostic testing was often confined to serology. An isolate from a horse with serous nasal discharge (Wills et al., 1990) was later reclassified as Chlamydia pneumoniae (Storey et al., 1993). More recently, investigations of equine abortion cases revealed involvement of Chlamydophila (C.) psittaci [START_REF] Henning | Nachweis von Chlamydien bei Stutenabort[END_REF]Szeredi et al. 2005), which had been redefined as a species in a revised taxonomic scheme [START_REF] Everett | Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms[END_REF]. In a study on horses with recurrent airway obstruction, Theegarten et al.

(2008) identified both C. psittaci and C. abortus in healthy and diseased horses, but signs of acute chlamydial infection combined with inflammation of lung tissue were only observed in clinical cases. In contrast, [START_REF] Mair | Chlamydia psittaci infection in horses: results of a prevalence survey and experimental challenge[END_REF] isolated Chlamydia psittaci from 15 out of 300 horses, but found no association between the presence of these bacteria and signs of clinical ocular or respiratory disease.

A c c e p t e d M a n u s c r i p t 3 While S. equi subsp. zooepidemicus is considered to be part of the normal upper respiratory tract flora [START_REF] Clark | Bacterial isolates from equine infections in western Canada (1998-2003)[END_REF], Timoney, 2004), the agent was the most frequently isolated pathogen in clinical respiratory disease of young horses, typically associated with pharyngitis and purulent pneumonia (Clark et al., 2008, Rolle and[START_REF] Rolle | Medizinische Mikrobiologie[END_REF]. [START_REF] Chapman | Retrospective study of the relationships between age, inflammation and the isolation of bacteria from the lower respiratory tract of thoroughbred horses[END_REF] and [START_REF] Clark | Bacterial isolates from equine infections in western Canada (1998-2003)[END_REF] detected this bacterium at high rates in trachea samples. It seems that imbalances and disturbances in the interaction between pathogen and host defence, which can be triggered by viral co-infections, high summer temperatures, tissue injury or transport stress, are a prerequisite for the occurrence of clinical manifestations [START_REF] Oikawa | Pathology of equine pneumonia associated with transport and isolation of Streptococcus equi subsp. zooepidemicus[END_REF]Timoney, 2004).

In the present report, we describe an outbreak of conjunctivitis and rhinitis at a horse farm where mixed infection with chlamydiae and S. equi subsp. zooepidemicus was diagnosed.

Rather unexpectedly, the chlamydial agent involved was identified as C. caviae.

Materials and methods

Case history

In the period from April 27 to the end of June 2006, each newborn foal of the 7 Trakehner mares of a stud farm of 40 horses in the state of Saxony-Anhalt developed conjunctivitis, rhinitis and cough. The symptoms started at the age of 2-3 weeks. After about 4 days, the signs of rhinitis changed from serous to mucous-purulent in most cases. The body temperature was 38.6 -38.8 °C during the acute period. One foal died from pneumonia at the age of 8 weeks in July. In addition, diarrhoea was also observed in several foals beginning 1-2 days after the first respiratory symptoms and before antibiotic treatment. All horses of the farm periodically showed signs of conjunctivitis.

The initial antibiotic therapy (1st to 8th week) included two intramuscular applications of penicillin/streptomycin (given two times on two consecutive days) before the foals were sent to pasture. Subsequently, a trimethoprim/sulfonamide combination was given orally on five A c c e p t e d M a n u s c r i p t consecutive days, which stopped purulent inflammations, but recurrent symptoms were observed a few days later (repeated every other week by the owner). Amoxicillin was also given once in between (week 6). In the second treatment cycle (week 9), Baytril ® (enrofloxacin) was orally administered for five days. Although, in the third cycle (week 10), the administration period of trimethoprim/sulfonamide was extended to 10 days, clinical symptoms were recurring. Finally, another Baytril treatment for five days was conducted in week 14.

As the described manifestations of the disease recurred at the end of August of the same year, i.e. 14 days after the end of antibiotic therapy, the owner agreed to sampling of all diseased foals and laboratory testing of the specimens.

Samples and initial processing

Conjunctival and nasal swabs were taken for standard bacteriological, virological and DNA testing, as well as isolation of chlamydiae from 6 foals that survived the first infection. The

Real-time PCR

DNA extracts of swab samples were examined using a Chlamydiaceae family-specific assay targeting the 23S rRNA gene (23S-rtPCR) based on the protocol published previously [START_REF] Dilbeck | Optimized DNA microarray assay allows detection and genotyping of single PCR-amplifiable target copies[END_REF], but extended by inclusion of an internal amplification control [START_REF] Pantchev | New real-time PCR tests for species-specific detection of Chlamydophila psittaci and Chlamydophila abortus from tissue samples[END_REF].

ArrayTube (AT) DNA microarray testing

Species identification of chlamydial agents involved was performed using the ArrayTube ® DNA microarray assay as described previously [START_REF] Sachse | DNA microarray-based detection and identification of Chlamydia and Chlamydophila spp[END_REF][START_REF] Borel | Direct identification of chlamydiae from clinical samples using a DNA microarray assay -a validation study[END_REF].

DNA sequencing

To verify the 23S-rtPCR and AT test data, sequencing of a discriminatory region of the 16S rRNA gene [START_REF] Hotzel | Evidence of infection in tortoises by chlamydia-like organisms that are genetically distinct from known Chlamydiaceae species[END_REF] and the whole chlamydial ompA gene [START_REF] Borel | Direct identification of chlamydiae from clinical samples using a DNA microarray assay -a validation study[END_REF] was conducted as described previously. The sequences have been deposited at GenBank accession numbers GQ332574 and GQ332575, respectively.

Culture of chlamydial agents

To isolate chlamydial agents, Buffalo Green Monkey (BGM) cells and yolk sacs of embryonated chicken eggs were inoculated with sample material and cultured for several passages according to standard procedures [START_REF] Sachse | Detection of Chlamydia suis from clinical specimens: Comparison of PCR, antigen ELISA and culture[END_REF]Tang et al., 1957). Replication of chlamydiae was monitored by real-time PCR of culture aliquots.

Results

A c c e p t e d M a n u s c r i p t 6

The results of PCR testing and cultural bacteriological examination are shown in Table 1. In 5 of 6 tested foals, the real-time PCR assay specific for the family Chlamydiaceae was positive.

The highest titres of chlamydial agents were detected at the beginning of sampling, i.e. 14 days after the end of antibiotic treatment. Furthermore, the concentrations of Chlamydiaceae DNA in nasal swabs were higher than in conjunctival swabs. For foal no. 4, the real-time PCR assay was positive only in the nasal swab. From six nasal swabs of four foals, S. equi subsp.

zooepidemicus was successfully cultured and isolated as virtually pure or mixed culture. Antibiograms showed sensitivity of the isolated streptococci to β-lactam antibiotics, tetracyclines, fluoroquinolones and aminoglycosides (data not shown). Viral and mycoplasma testing of samples proved negative.

Identification of the chlamydial agent at species level was conducted using the AT DNA microarray assay, because this test allows parallel detection of all 9 currently defined species of Chlamydiaceae. AT testing revealed the presence of C. caviae as the only chlamydial species (Table 1). Typical hybridisation patterns obtained by AT testing of a nasal swab of foal no.1 at 14 days after the end of antibiotic treatment and the first passage of cell culture of the same sample are shown in Fig. 1.

The identity of the chlamydial agent was confirmed by sequencing of the signature sequence of the 16S ribosomal RNA gene (GenBank acc. no. GQ332574) and of the complete ompA gene (GenBank acc. no. GQ332575). PCR products from swab samples of foals 1-4 (cf. Table 1) were used as templates. BLAST analysis of the sequences revealed homologies of 99 and 92%, respectively, to C. caviae type strain GPIC. No sequence differences among individual samples were observed, thus suggesting that only one strain of C. caviae was involved.

Despite initial signs of chlamydial growth in BGM cell culture and embryonated chicken eggs we failed to obtain a continuously replicating cultural isolate.

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

Discussion

The present study revealed a mixed bacterial infection involving C. caviae and S. equi subsp.

zooepidemicus as the presumptive causative agents of the outbreak of respiratory and ocular disease in a stud farm.

So far, the list of chlamydial agents identified in horses has been limited to C. pneumoniae, C. psittaci and C. abortus. This report provides the first evidence on the occurrence of C. caviae in horses and its likely involvement in respiratory disease. Interestingly, after completion of the present study, the laboratory of one of the authors (KS) again detected this agent in respiratory samples from an unrelated horse breeding farm (data not shown), which might indicate a wider dissemination. In contrast to the present findings, C. caviae has been considered to possess high host specificity for guinea pigs [START_REF] Everett | Chlamydia and Chlamydiales: more than meets the eye[END_REF]. In any case, the detection of this agent raises new questions since the route of infection remains unclear. As there were no guinea pigs at the farm, nor do these animals roam freely in Central Europe, their possible role as source of infection can be ruled out.

As highly sensitive and species-specific diagnostic tools for chlamydiae became available only in the 1990s, it is not quite clear whether the present infection of horses with C. caviae represents a unique event. Moreover, the traditional taxonomy did feature C. caviae as a separate species, but rather subsumed it under the taxon of Chlamydia psittaci. Thus, studies of chlamydial infections in horses mentioning involvement of Chlamydia psittaci which were conducted before the molecular diagnostic age could unwittingly also have referred to C. caviae. In the present study, we used different DNA-based diagnostic tests targeting three different genomic sites, i.e. 23S rRNA gene (real-time PCR and AT test), ompA gene (sequencing) and 16S rRNA gene (sequencing), all of which identified C. caviae. In the case of the ompA gene sequence, a similarity of 92 % to the C. caviae type strain is still within the intra-species variability range of this highly variable membrane protein gene.

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

In view of the presence of C. caviae in nearly all swabs from the group of foals, it appears reasonable to assume a contribution of this agent to the described clinical manifestations. In addition, the fact that only C. caviae was found in the conjunctival swabs indicates an association with the conjunctivitis observed in the same animals, even more so since C. caviae is a known agent of conjunctivitis in guinea pigs. Nevertheless, its role as a primary causative agent cannot be defined unambiguously based on the present data.

In the present outbreak, C. caviae probably developed synergistic effects with S. equi subsp.

zooepidemicus. As a mucosal commensal in horses [START_REF] Hirsh | Veterinary Microbiology[END_REF], the latter requires precursors (Timoney, 2004). We hypothesise that primary lesions were set by C. caviae and subsequently aggravated by Streptococcus equi subsp. zooepidemicus. Indications supporting this assumption include i) the conjunctivitis caused by mono-infection with C. caviae, ii) recurrent clinical symptoms in the affected animals, and iii) the absence of a sustained clinical effect of antibiotic therapy with trimethoprim-sulfonamide, enrofloxacin and amoxicillin. The latter is usually effective against S. equi subsp. zooepidemicus.

The long-lasting antibiotic therapy of the diseased foals is the most likely explanation for our inability to grow the C. caviae strain(s) involved, as chlamydiae are known to pass into a state of persistence upon antibiotic treatment. Microbiol. 40, 3256-3260 Storey, C., Lusher, M., Yates, P., Richmond, S., 1993. Evidence for Chlamydia pneumoniae of non-human orign. J. Gen. Microbiol. 139, 2621-2626. Szeredi, L., Hotzel, H., Sachse, K., 2005. High prevalence of chlamydial (Chlamydophila psittaci) infection in fetal membranes of aborted equine fetuses. Vet. Res. Commun.

29 Suppl 1, 37-49.

Tang, F.F., Chang, H.L., Huang, Y.T., Wang, K.C., 1957. Studies on the etiology of trachoma with special reference to isolation of the virus in chick embryo. Chin. Med. J. 75, 429-447.

Theegarten, D., Sachse, K., Mentrup, B., Fey, K., Hotzel H., Anhenn, O., 2008. Chlamydophila spp. infection in horses with recurrent airway obstruction: similarities to human chronic obstructive disease. Respir. Res. 29, 9-14. Timoney, J.F., 2004. The pathogenic equine streptococci. Vet. Res. 35,[397][398][399][400][401][402][403][404][405][406][407][408][409] Van Kuppeveld, F.J., van der Logt, J.T., Angulo, A.F., van Zoest, M.J., Quint, W.G., Niesters, H.G., Galama, J.M., Melchers, W.J., 1992. Genus-and species-specific identification of mycoplasmas by 16S rRNA amplification. Appl Environ Microbiol. 58, 2606-2615. Erratum in: Appl Environ Microbiol. 59(1993)

  procedure for detection of bacterial agents comprised growth on blood agar (Oxoid, Wesel, Germany, Cat.-Nb. PB 5177 A) under aerobic and micro-aerobic conditions with 12% CO 2 at 37°C, as well as selective Gassner agar (Oxoid, Cat.-Nr. PO 5021 A), selective Pasteurella agar (Oxoid, Cat.-Nr. PB 5175 A) and selective Mycoplasma agar (Oxoid, Cat.-Nr. CM 0401, including supplement Cat.-Nr. SR 0059 C). For isolation of viruses, several cell cultures were inoculated and cultivated for three passages. PCR testing was conducted for Influenza A Virus (according to Spackman et al. 2002), Equine Herpesvirus Types 1 and 4 (Benetka et al., 2002), Equine Arteritis Virus (de Vries et al., 1990) and Mycoplasma spp. (van Kuppeveld et al., 1992). For simultaneous isolation of bacterial DNA, viral DNA and RNA from swabs, the High Pure Viral Nucleic Acid Isolation Kit (Roche Diagnostics, Mannheim, Germany) was used according to the instructions of the manufacturer. From culture fluids, DNA was extracted using the High Pure PCR Template Preparation Kit (Roche).

  As a general conclusion from the present findings, practitioners and laboratory diagnosticians should consider the possibility of C. caviae being involved in equine infections. Speciesspecific tests should be used to enable assessment of the clinical importance and zoonotic potential of the infectious agent involved. Finally, the rather unexpected identification of C. caviae in the present case also indicates that our current understanding of host ranges of Chlamydiaceae spp. is not yet complete.

  ., Laroucau, K.,[START_REF] Borel | Direct identification of chlamydiae from clinical samples using a DNA microarray assay -a validation study[END_REF]. Genotyping of Chlamydophila psittaci using a new DNA microarray assay based on sequence analysis of ompA genes. BMC Microbiol. 8, 63 Spackman E., Senne, D.A., Myers, T.J., Bulaga, L.L., Garber, L.P., Perdue, M.L., Lohman, K., Daum, L.T., Suarez, D.L., 2002. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin.

  :655. Wills, J.M., Watson, G. Lusher, M., Mair, T.S., Wood, D., Richmond, S.J., 1990. Characterisation of Chlamydia psittaci isolated from a horse. Vet. Microbiol. 24, 11-19. Legend to Figure 1 DNA microarray testing of equine samples using the ArrayTube ® assay. Results are shown for a nasal swab of foal no.1 at 14 days after the end of antibiotic treatment (A, B) and the first passage of cell culture of the same sample (C, D). A, C: Images of the stained microarrays. B, D: Bargraph showing specific hybridisation signals for Chlamydiaceae consensus probes (1), Chlamydophila genus-specific probes (2) and C. caviae species-specific probes (3), whereas the signals for the rest of the chlamydia species-specific probes (4) are absent or represent non-specific cross-hybridisation. (Signal 5 in Fig. D represents a cross-hybridisation with one of the three probes for Waddlia chondrophila.) A c c e p t e d M a n u s c r i p

  Fig. 1

Table

Acknowledgement

We thank Christine Grajetzki, Simone Bettermann and Karola Zmuda for excellent technical assistance.

Conflict of interest statement

None of the authors (Wolfgang Gaede, Karl-Friedrich Reckling, Anette Schliephake, Dirk Missal, Helmut Hotzel, and Konrad Sachse) has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the paper entitled "Detection of Chlamydophila caviae and Streptococcus equi subsp. zooepidemicus in horses with signs of rhinitis and conjunctivitis".


uVIPAC Massin Pascale

Kuntz-Simon Gaëlle

Barbezange Cyril

Deblanc Céline

Oger Aurélie

Marquet-Blouin Estelle

Bougeard Stéphanie

Werf Van Der

Sylvie

Jestin Véronique

Temperature sensitivity on growth and/or replication of H1N1, H1N2 and H3N2 influenza A viruses isolated from pigs and birds in mammalian cells

Keywords: Avian influenza virus, Swine influenza virus, Temperature-sensitivity, NPTr cells, Real-time quantitative PCR, PB2 627

Influenza A viruses have been isolated from a wide range of animal species, aquatic birds being the reservoir for their genetic diversity. Avian influenza viruses can be transmitted to humans, directly or indirectly through an intermediate host like pig. This study aimed to define in vitro conditions that could prove useful to evaluate the potential of influenza viruses to adapt to a different host. Growth of H1N1, H1N2 and H3N2 influenza viruses belonging to different lineages isolated from birds or pigs prior to 2005 was tested on MDCK or NPTr cell lines in the presence or absence of exogenous trypsin. Virus multiplication was compared at 33, 37 and 40°C, the infection site temperatures in human, swine and avian hosts, respectively. Temperature-sensitivity of PB2-, NPand M-RNA replication was also tested by quantitative real-time PCR.

Multiplication of avian viruses was cold-sensitive, whatever cell type. By contrast, temperature-sensitivity of swine viruses was found to depend on the virus and the host cell: for an H1N1 swine isolate from 1982, multiplication was cold-sensitive on NPTr cells and undetectable at 40°C. From genetic analyses, it appears that temperature-sensitivity could involve other residues than PB2 residue 627 and could affect other steps of the replication cycle than replication.

Introduction

Influenza A viruses infect a wide range of animal species. All 16 hemagglutinins (HA) and 9 neuraminidases (NA) that determine influenza virus subtypes have been isolated from wild aquatic birds. Thus, aquatic birds seem to be the reservoir for the genetic diversity of influenza A viruses, from which they can be transmitted to other species, like poultry, pig or human [START_REF] Webster | Evolution and ecology of influenza A viruses[END_REF]. This phenomenon of crossing the species barrier was responsible for the 1918, 1957 and 1968 influenza pandemics and as seen more recently with the emergence of the pandemic H1N1 2009 virus of swine origin is a major issue with respect to human and animal public health.

In humans and pigs, influenza A viruses first replicate in the upper respiratory tract at a temperature close to 33°C and 37°C, respectively, inducing a respiratory syndrome [START_REF] Alford | Human influenza resulting from aerosol inhalation[END_REF]. In aquatic birds, low pathogenic influenza A viruses preferentially replicate in the intestinal tract at a temperature close to 40°C, and the infection is usually asymptomatic [START_REF] Webster | Intestinal Influenza : Replication and Characterization of Influenza Viruses in Ducks[END_REF]. In domestic poultry, some subtypes of influenza A viruses can cause devastating epizootics leading to economic losses and sanitarian risks for humans and other domestic animals. The HA is involved in host range and adaptation of influenza A viruses through its binding specificity to the receptor i.e. sialic acids (SA). Indeed, the HA of human and avian influenza A viruses preferentially binds to SA α2,6and SA α2,3-galactose, respectively [START_REF] Connor | Receptor specificity in human, avian, and equine h2 and h3 influenza virus isolates[END_REF][START_REF] Ito | Recognition of Nglycolylneuraminic acid linked to galactose by the alpha2,3 linkage is associated with intestinal replication of influenza A virus in ducks[END_REF], which correlates well with their distribution at the site of virus multiplication: SA α2,6 on epithelial cells of the human upper respiratory tract and SA α2,3 on A c c e p t e d M a n u s c r i p t 4 epithelial cells of the avian intestinal tract [START_REF] Couceiro | Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory[END_REF][START_REF] Ito | Recognition of Nglycolylneuraminic acid linked to galactose by the alpha2,3 linkage is associated with intestinal replication of influenza A virus in ducks[END_REF].

In the tracheal epithelium of pigs, both types of SA are present, which could explain the susceptibility of pigs to both human and avian influenza A viruses (Scholtissek et al., 1998). Consequently, pigs are considered as a "mixing vessel" host, where reassortment among human, avian and swine influenza A viruses could lead to new influenza virus lineages with zoonotic potential [START_REF] Kuntz-Simon | Genetic and antigenic evolution of swine influenza viruses in Europe and evaluation of their zoonotic potential[END_REF][START_REF] Webster | Evolution and ecology of influenza A viruses[END_REF]. In addition to their possible transmission to humans, swine influenza A viruses can also be transmitted to poultry (Suarez et al., 2002) and wild ducks (Olsen et al., 2003).

Following receptor binding, entry involves the endocytosis of the virus particle and the low pH-dependent fusion of the virus envelope with the endosomal membrane. This process requires prior activation of the HA by protease cleavage. The enzymes responsible are trypsin-like proteases secreted by respiratory or intestinal tract cells that restrict virus multiplication to these tissues.

Therefore, in cell culture, the growth of influenza A viruses usually requires exogenously added trypsin, since culture cells generally lack appropriate endogenous proteases for HA cleavage [START_REF] Klenk | The molecular biology of influenza virus pathogenicity[END_REF]. However, some H5 and H7 influenza A viruses possess a HA cleavable by ubiquitous and intracellular proteases [START_REF] Stieneke-Grober | Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease[END_REF], allowing the virus to replicate in other tissues and to spread systemically within the infected host.

Although the HA is a major determinant for host restriction it is not necessarily sufficient. Indeed, some influenza A viruses of entire swine or avian origin, thus not reassortants, have been directly transmitted to humans (Banks et , 1998;[START_REF] Claas | Infection of children with avian-human reassortant influenza virus from pigs in Europe[END_REF][START_REF] Kendal | Identification and preliminary antigenic analysis of swine influenza-like viruses isolated during an influenza outbreak at Fort Dix, New Jersey[END_REF]. These viruses had specific characteristics that allowed them to replicate in humans, but were not able to transmit from human to human and then spread in the human population. Other determinants of host range or adaptation to a novel host were identified in the virus internal proteins, especially in the polymerase complex subunits PB1, PB2 and PA. For the 1957 and 1968 pandemics, it was demonstrated that the emerging viruses were reassortants between human and avian influenza viruses [START_REF] Yasuda | Molecular evidence for a role of domestic ducks in the introduction of avian H3 influenza viruses to pigs in southern China, where the A/Hong Kong/68 (H3N2) strain emerged[END_REF]. In both cases, reassortant viruses acquired not only NA and/or HA genes but also the viral polymerase subunit PB1 gene from an avian influenza virus, whereas other genes were acquired from a human influenza A virus [START_REF] Kawaoka | Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics[END_REF]Scholtissek and Murphy, 1978). Experiments on the compatibility of polymerase subunits showed that an avian origin of the PB1 gene may have conferred a selective advantage to the 1957 and 1968 reassortant viruses (Naffakh et al., 2000). In swine, reassortant viruses indicated that a PB1 gene of human origin allows the virus to be infectious in pigs, whatever the swine or avian origin of the two other subunits of the viral polymerase complex [START_REF] Karasin | Identification of human H1N2 and human-swine reassortant H1N2 and H1N1 influenza A viruses among pigs in Ontario, Canada (2003 to 2005)[END_REF].

Reassortment experiments further showed that genomic segments NP, M, NS and PB2 also seem to be involved in host restriction [START_REF] Clements | Use of single-gene reassortant viruses to study the role of avian influenza A virus genes in attenuation of wild-type human influenza A virus for squirrel monkeys and adult human volunteers[END_REF]Geiss et al., 2002;[START_REF] Snyder | The avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus polymerase genes each specify attenuation of avian-human influenza A/Pintail/79 reassortant viruses for monkeys[END_REF]Tian et al., 1985). A well-characterised host-range determinant is the residue 627 of PB2, a glutamic acid found in almost all avian influenza A viruses versus a lysine in human influenza A viruses [START_REF] Smith | Evolution and adaptation of H5N1 influenza virus in avian and human hosts in Indonesia and Vietnam[END_REF]Subbarao et al., 1993). The nature of residue 627 of PB2 mostly A c c e p t e d M a n u s c r i p t 6 determines the cold-sensitivity at 33°C of the replication of avian viruses in mammalian cells [START_REF] Labadie | Host-range determinants on the PB2 protein of influenza A viruses control the interaction between the viral polymerase and nucleoprotein in human cells[END_REF][START_REF] Massin | Residue 627 of PB2 is a determinant of cold sensitivity in RNA replication of avian influenza viruses[END_REF] and has been involved in the level of pathogenicity of the virus (Hatta et al., 2001;[START_REF] Munster | The molecular basis of the pathogenicity of the Dutch highly pathogenic human influenza A H7N7 viruses[END_REF] and in its ability to replicate in the upper respiratory tract (Hatta et al., 2007).

Altogether, published data suggest that host restriction is a complex mechanism, driven by specific gene constellations depending on the origin of the virus.

In an attempt to define in vitro conditions that could prove useful to evaluate the potential of viruses to adapt to a different host, a comparative analysis of multiplication capabilities of influenza A viruses isolated from pigs or birds was performed at three temperatures (33, 37 and 40°C), in three cell types (Madin-Darby Canine Kidney cell line or MDCK, Newborn Pig Tracheal cell line or NPTr, chicken fibroblast DF1 cell line), and in the presence or absence of exogenous trypsin. Three subtypes of influenza A viruses (H1N1, H1N2 and H3N2) were selected based on the fact that these subtypes have also been circulating in humans and are currently co-circulating in pigs and birds. The replication capabilities of two H1N1 viruses belonging to the same avian-like swine lineage and respectively isolated from turkey and swine were also investigated by real-time PCRs targeting the PB2-, NP-and M-genomic vRNAs.

Materials and Methods

Cells and viruses

Newborn Pig Trachea (NPTr, (Ferrari et al., 2003)

Phylogenetic analyses

For avian influenza A viruses, viral RNA was extracted from infectious allantoïc fluid according to a standardised procedure [START_REF] Alexander | Chapter 2.07.12 Avian Influenza[END_REF] Thompson et al., 1994). Phylogenetic analyses were conducted using the MEGA 3.1 software [START_REF] Kumar | MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment[END_REF]) by the Neighbor-joining method implemented with the Kimura-2 parameters model.

Topology of the trees was evaluated by 1000 bootstrap analyses. For a review of swine influenza virus lineages see [START_REF] Kuntz-Simon | Genetic and antigenic evolution of swine influenza viruses in Europe and evaluation of their zoonotic potential[END_REF]. Data are presented in Table 1.

Virus stock titration by TCID50 determination

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

and expressed as a tissue culture infectious dose 50% per millilitre (TCID 50 /ml).

MDCK or NPTr cells were seeded 24 h before titration in 96-well plates at a density of 3x10 4 cells/well in medium supplemented with FCS (10% or 5%, respectively). Serial 10-fold dilutions (10 -1 to 10 -10 ) of the virus stock or the viral sample were made in MEM without FCS and supplemented with 2 or 0.2 µg/ml of trypsin-TPCK (Whorthington Biochemical Corporation). Cells were washed twice with MEM without serum and then infected with virus dilutions (8 to 10 replicas per dilution). At 72 h post-infection, the cytopathic effect was observed using an inverted microscope and virus titres were calculated using the Reed and Muench method. Virus titration was performed twice at least and did not differ by more than one log 10 for a given virus suspension.

Virus growth kinetics in cell culture

NPTr and MDCK cells were seeded in 6-well plates at a density of 1x10 6 cells/well in medium supplemented with FCS (10% and 5%, respectively). 24 h later, cells were washed twice with MEM without serum and then infected at a multiplicity of infection (MOI) of 10 -3 TCID 50 /cell with either avian or swine influenza viruses, or mock infected. After adsorption for one hour at room temperature, the inoculum was removed. The cells were washed once with MEM without serum and then incubated at 33, 37 or 40°C with MEM without serum but supplemented, or not, with 2 µg/ml of trypsin-TPCK. At 0, 12, 24 and 48 h post-A c c e p t e d M a n u s c r i p t 10 infection, virus titres were determined in the supernatants, on MDCK cells only, as described in 2.3.

Virus replication kinetics in cell culture

NPTr cells were infected with either avian-or swine-H1N1 influenza viruses, or mock-infected as described in section 2.4. At 0, 12, 24 and 48 h postinfection, cells were washed twice in PBS and total RNA was extracted using the MagAttract RNA Tissue Mini M48 kit and the M48 automated platform (Qiagen).

Total RNA was eluted in 100 µl RNase-free water and quantified with the Quant-iT RNA kit and Qubit fluorometer (Invitrogen). Each sample was then subjected to real-time PCRs as described below, to determine copy numbers of NP-, M-, or PB2-vRNAs.

SYBR Green real-time PCR

Two-steps real-time PCRs specific for NP-, M-, or PB2-vRNA were developed. First, total RNA (60 ng) was denatured and annealed at 65°C for 5 min with 10 pmol of Uni12 primer [START_REF] Hoffmann | Universal primer set for the full-length amplification of all influenza A viruses[END_REF] and 1 µl of dNTP (10 mM each). RNA was reverse-transcribed at 42°C for 70 min in a total volume of 30 µl, using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's protocol. Real-time PCR reactions were next performed in a final volume of 25 µl with 12.5 µl of SYBR Green PCR Master Mix (Applied Biosystems), 10 µl of cDNA (diluted 1:10) and 200 nM of each forward and reverse primer (except for M PCR: 400 nM) (sequences available upon request).

A c c e p t e d M a n u s c r i p t 11 Amplifications were performed using the following conditions: 2 min at 50°C, 10 min at 95°C, followed by 45 amplification cycles (15 sec at 95°C and 1 min at 60°C for NP and M PCRs or 15 sec at 55°C and 1 min at 60°C for PB2 PCR). The program ended with a dissociation stage. Amplifications were carried out in 96well plates with 3 replicates per sample, using Applied Biosystems 7000 and 7500

Real-Time PCR Systems and analysed with the Sequence Detection Software. For optimisation and standard curves, full-length PCR products of each viral segment were used. RNA was extracted from allantoïc fluids infected with either avian or swine influenza viruses, using the RNeasy kit (Qiagen). cDNA corresponding to each virus was synthesized using the Uni12 primer and the Superscript II (Invitrogen) as described above. For each cDNA, specific amplification of NP-, M-, and PB2-genes were performed using primers derived from Hoffman et al. [START_REF] Hoffmann | Universal primer set for the full-length amplification of all influenza A viruses[END_REF] and the Taq Platinum High Fidelity polymerase For virus replication kinetics, statistical analyses were performed to test the effect of the virus (avian or swine H1N1 influenza virus), of the temperature (33°C, 37°C or 40°C) and of their potential interaction on the kinetics of replication of three different viral genes (PB2, NP and M), assessed by the log 10 of the copy number (three replicates). Linear mixed models [START_REF] Littell | Statistical analysis of repeated measures data using SAS procedures[END_REF] including repeated effects were used. The virus, the temperature and the virus by temperature interaction were introduced in the model as fixed effects in accordance with the design of the study. The time post-infection (0, 12, 24 and 48 h) was incorporated in the model as a repeated measurement factor in order to take into account the within-time covariability, specifying a first-order autoregressive structure for the covariance matrix. In the case of a significant virus by temperature interaction, an alternative to multiple comparison procedures, that are not available with linear mixed models, contrasts procedures were used to compare the different levels of the temperature, for each virus.

A c c e p t e d M a n u s c r i p t 13 Linear mixed models were computed with the MIXED procedure of the SAS software (SAS, 2004).

Results

Phylogenetic analyses

Phylogenetic analyses of genomic segments of each virus were performed as described in Materials and methods and are summarised in Table 1. Swine influenza viruses used in this study belonged to well-known swine lineages (for a review see [START_REF] Kuntz-Simon | Genetic and antigenic evolution of swine influenza viruses in Europe and evaluation of their zoonotic potential[END_REF]). Briefly, the sw-H1N1/82 belonged to the avian-like swine lineage. Its 8 genomic segments were of avian origin. The sw-H1N2/94 and sw-H3N2/84 belonged to human-like reassortant swine lineages: they possessed internal genes from the avian-like swine H1N1 lineage and HA and NA from human lineages. The av-H1N1/05 and av-H1N2/05 were found closely related to sw-H1N1/82 (avian-like swine lineage) and to sw-H1N2/94

(human-like reassortant swine lineage), respectively. The av-H1N1/02 possessed 8 genomic segments of avian origin. All genes of the av-H3N2/00 were closely related to human influenza viruses isolated in the 1970's.

Comparison of virus stock titres obtained on MDCK or NPTr cells in the presence of 2 or 0.2 µg/ml of trypsin

In order to perform virus titrations on MDCK, NPTr and DF1 cells, we first tested the ability of monolayers of each cell type to resist in a medium containing trypsin (data not shown). Monolayers of MDCK and NPTr cells The titres of H1N1, H1N2 and H3N2 influenza viruses isolated from pigs or birds (further referred to as avian (or av-) and swine (or sw-), respectively)

were determined on MDCK and NPTr cells in the presence of 2 or 0.2 µg/ml trypsin, as described in Materials and Methods. Calculated titres are presented in Fig. 1.

On MDCK cells (left panel), titres were about 2 log 10 higher with 2 µg/ml of trypsin than with 0.2 µg/ml of trypsin, whereas on NPTr cells in the same conditions, differences in titres were only about 0.4 log 10 . In the presence of 2 µg/ml of trypsin, titres for all viruses were slightly but significantly (p<0.0001) For viruses isolated from birds, in the presence of trypsin-TPCK (Fig. 2A, bold lines), the kinetics of multiplication gave very similar profiles whatever the host cell type or the virus used, with a delay in the titre raise at 33°C as compared to 37 and 40°C. For each temperature considered, av-H1N1/05, av-H1N2/05 and av-H3N2/00 viruses reached similar titres on MDCK and NPTr cells at 48 h postinfection (p.i.). Similar titres were also reached at 48 h p.i. on MDCK and NPTr cells for av-H1N1/02 virus, but only at 37 and 40°C. At 33°C, for av-H1N1/02

higher
virus the titre at 48 h p.i. was nearly three log 10 lower on NPTr cells than on MDCK cells.

In the absence of trypsin-TPCK (Fig. 2A, thin lines), the main profile observed for avian viruses at all three temperatures was a delay in growth, with titres starting to raise later than in the presence of trypsin-TPCK. Noticeably, for av-H1N1/05 virus, no virus growth could be detected on MDCK cells at either three temperatures. Similarly, av-H3N2/00 did not grow on MDCK cells when incubated at 40°C. For the viruses isolated from pigs, in the presence of trypsin-TPCK (Fig. 2B, bold lines), there was no general delay at 33°C as compared to 37°C on either MDCK or NPTr cells. However, for sw-H1N1/82 on NPTr cells, the titre reached at 48 h p.i. was more than one log 10 lower at 33°C than at 37°C. This difference at 33°C was not as clear on MDCK cells. Similarly, for sw-H1N2/94, the maximum titre was reduced by one to two log 10 at 33°C as compared to 37°C on both cell types. Moreover, at 40°C, virus growth was undetectable for sw-H1N1/82 on NPTr cells and significantly reduced on NPTr as compared to MDCK cells for sw-H1N2/94. As for sw-H3N2/84, whatever the temperature, it grew with similar capabilities on both MDCK and NPTr cells.

In the absence of trypsin-TPCK (Fig. 2B, thin, dotted lines) the swine viruses behaved differently from the avian viruses, since essentially no delay but only a slight reduction (one to two log 10 ) in the final titre reached at 48 h p.i. was observed. The only noticeable difference was observed for sw-H1N1/82 on MDCK cells at 40°C, for which the titre obtained was nearly three log 10 lower in the absence of exogenous trypsin.

Comparison of virus replication kinetics at different temperatures on NPTr cells in the presence of 2 µg/ml of trypsin

In order to determine if the temperature sensitivity observed in virus growth was due to a defect in replication, we analysed the viral RNA levels over time in NPTr cells infected with sw-H1N1/82 and av-H1N1/05 viruses using realtime quantitative PCRs. NPTr cells were infected with either av-H1N1/05 or sw-H1N1/82 at a low multiplicity of infection (MOI = 10 -3 TCID50/cell) and were incubated at 33, 37 or 40°C. As described in Materials and Methods, total RNAs were extracted and analysed by the three newly developed SYBR green real-time PCRs. Results are presented in Fig. 3. For av-H1N1/05, the kinetics of replication of each vRNA were very similar. At 12 h p.i., a delay was observed at 33°C as compared to 37°C and 40°C. The difference between 33°C and 37°C was statistically confirmed for the PB2 gene (p<0.05) and with a level of significance of 10% (p=0.068) for the M gene, but not for the NP gene (p>0.05). At 33°C the maximum level of vRNA seemed to be reached at 48 h p.i. whereas it was already reached at 24 h p.i. at 37 and 40°C. For sw-H1N1/82, the vRNA kinetics were also very similar for the three segments. A statistically significant delay in replication was observed at 33°C as compared to 37°C (p<0.05 for the three genes), and at 48 h p.i. the copy A c c e p t e d M a n u s c r i p t 18 number was nearly two log 10 lower than at 37°C. At 40°C, we failed to detect replication of any of the three vRNAs (p<0.05 for the three genes). Altogether, the real-time PCR results were very consistent with the virus growth results.

Discussion

The three subtypes of influenza A viruses that were used in this study (H1N1, H1N2 and H3N2) were selected because they have been circulating in the avian, swine and human populations. Furthermore, virus isolates were chosen in order to fall within major lineages based on the phylogenetic analyses which provided some insight on the genetic relationships between the viruses and their human counterparts.

Three cell lines were originally chosen to compare the avian and swine viruses. The MDCK cell line was derived from an adult female cocker spaniel in 1958 and was described to support replication of several influenza A viruses from human, swine, equine and avian origin, forming plaques with high efficiency by the addition of trypsin in the culture medium (Tobita et al., 1975). They are widely used for the isolation of influenza A viruses from respiratory specimens from human patients (Tobita et al., 1975) and for the multiplication of human and swine influenza A viruses. DF1 cells consist of an established chicken fibroblast cell line that has been used in recently published influenza studies [START_REF] Labadie | Host-range determinants on the PB2 protein of influenza A viruses control the interaction between the viral polymerase and nucleoprotein in human cells[END_REF][START_REF] Lee | Evaluation of chickenorigin (DF-1) and quail-origin (QT-6) fibroblast cell lines for replication of avian influenza viruses[END_REF]. The NPTr cell line was selected because of the tissue (pig trachea) from which it was established by serial culture, and the fact that it supports the multiplication of several influenza A viruses from human, swine and A c c e p t e d M a n u s c r i p t (Ferrari et al., 2003).

Unfortunately, the DF1 cells proved highly sensitive to the trypsin concentration in the culture medium and the cell monolayers inconsistently resisted a 0.2 µg/ml concentration of trypsin. In those conditions, titres obtained for avian virus stocks on DF1 cells were low (data not shown) and in the same range as those obtained on MDCK cells, i.e. two to three log 10 lower than titres obtained on MDCK cells with 2 µg/ml of trypsin. This seems to contrast with the results of [START_REF] Lee | Evaluation of chickenorigin (DF-1) and quail-origin (QT-6) fibroblast cell lines for replication of avian influenza viruses[END_REF], who evaluated the use of DF1 or QT6 cells, another avian cell line, for the study of avian influenza A viruses, instead of primary avian cells (Chicken Embryo Fibroblast, CEF) and MDCK cells, and concluded that DF1 cells supported the growth of avian influenza A viruses as well as MDCK cells. However, the trypsin concentrations used were different for each cell line (0.75 versus 0 µg/ml for MDCK cells and 0.05 versus 0 µg/ml for DF1 cells), making comparison difficult. As expected, on MDCK cells, the titres were higher in the presence of 2 µg/ml of trypsin than with only 0.2 µg/ml. In accordance with the results of Ferrari et al. (2003), NPTr cells supported the multiplication of the avian and swine viruses used and appeared less dependent on the addition of exogenous trypsin in the culture medium, as similar virus titres were obtained for both trypsin concentrations. Because of their tracheal origin, it is likely that NPTr cells produce trypsin-like proteases that are excreted in the culture medium.

However, in order to facilitate comparisons with other studies, we decided to use A c c e p t e d M a n u s c r i p t 20 MDCK cells in the presence of 2 µg/ml of trypsin-TPCK for all the subsequent titrations, as this is considered as a reference method for human viruses.

Multiplication kinetics were thus performed on MDCK and NPTr cells, in the absence or in the presence of 2 µg/ml of trypsin-TPCK. DF1 cells were judged too sensitive to exogenous trypsin and were not included. Three incubation temperatures were chosen with respect to the temperature of the primary infection site in human, swine and avian hosts, respectively. Indeed, human and swine influenza A viruses first replicate in the upper respiratory tract, at a temperature close to 33 and 37°C, respectively. In contrast, low pathogenic avian influenza A viruses preferentially replicate in the bird intestinal tract at a temperature close to 40°C. Exogenous trypsin is believed to cleave the HA proteins at the surface of virus particles. This step is required for the virus to gain infectiousness, as cleavage of the HA is necessary to allow its conformational change for the fusion process [START_REF] Skehel | Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin[END_REF]. The dependence on exogenous trypsin was expected on MDCK cells only and for all viruses. It was therefore surprising that the swine viruses did not appear to be highly dependent on exogenous trypsin to grow efficiently on both cell types. Indeed, considering that a one log 10 difference in titres simply reflects the accuracy error of the titration method, most differences observed in the absence or presence of trypsin-TPCK could be regarded as not significant. At 37 and 40°C, the av-H1N1/05 virus appeared extremely sensitive to the absence of trypsin-TPCK when infecting MDCK but not NPTr cells.

Interestingly, all avian viruses clearly showed a delay in titre raise in the absence of trypsin-TPCK on NPTr cells. It is possible that the proteases secreted by NPTr A clear dependence on trypsin was also noted for sw-H1N1/82 on MDCK cells, also only at 40°C. However, it is not clear at present why or how exogenous trypsin would be required only at 40°C for these viruses. Globally, for swine viruses, in the presence of trypsin-TPCK, there was little difference between the virus growth kinetics on MDCK or NPTr cells. The only two definite differences were for sw-H1N1/82 (that will be discussed below) and for sw-H1N2/94. The latter was the only swine virus to show a clear growth reduction on NPTr cells at all temperatures. This would suggest that this virus might be less adapted to the swine host. Noticeably, av-H1N2/02, with H1 and N2 sequences closely related to the H1 and N2 of swine and human viruses, did not display a similar profile suggesting that other genes could be involved.

Regarding the influence of temperature in the presence of trypsin-TPCK, the results showed a clear sensitivity of viruses isolated from birds to a low temperature of incubation, with a delay in growth observed at 33°C when compared to 37 and 40°C. This was consistent with the fact that low-pathogenic avian viruses replicate in the intestinal tract of infected birds, at a temperature around 40°C. No significant growth reduction at 33°C was observed for swine viruses. For swine influenza A viruses, to our knowledge, this is the first time that temperature sensitivity has been studied and reported. Previous studies have

shown that viral replication and polymerase activity of avian influenza A viruses are reduced at low temperature in several mammalian cells including MDCK cells A c c e p t e d M a n u s c r i p t 22 [START_REF] Labadie | Host-range determinants on the PB2 protein of influenza A viruses control the interaction between the viral polymerase and nucleoprotein in human cells[END_REF][START_REF] Massin | Residue 627 of PB2 is a determinant of cold sensitivity in RNA replication of avian influenza viruses[END_REF]. More specifically, the PB2 residue 627

(lysine for human (627K) and glutamic acid for most avian influenza A viruses(627E)) was described to be involved in this cold-sensitivity of the avian virus polymerase complex at 33°C in mammalian cells [START_REF] Labadie | Host-range determinants on the PB2 protein of influenza A viruses control the interaction between the viral polymerase and nucleoprotein in human cells[END_REF][START_REF] Massin | Residue 627 of PB2 is a determinant of cold sensitivity in RNA replication of avian influenza viruses[END_REF]. Another study also showed that a lysine at position 627 of PB2 confers an advantage for efficient growth of avian highly-pathogenic H5N1

viruses in the upper and lower respiratory tracts of mammals (Hatta et al., 2007).

In the present study, all avian, except av-H3N2/00, and swine viruses were characterised by a 627E at this position. Interestingly, a 627K was found for av-H3N2/00. Although the nature of this residue could account for the coldsensitivity observed in the growth kinetics of the avian H1N1 and H1N2 viruses, it cannot explain the apparent lack of cold-sensitivity of the swine viruses.

Moreover, it cannot account for the definite lower growth capabilities at 33°C noticed for av-H3N2/00 on MDCK cells. In fact, av-H3N2/00 was found to be more sensitive to a low temperature than human H3N2 viruses (data not shown), despite their close genetic relationship. This suggests that other determinants besides PB2 residue 627 are involved.

A striking observation was the total lack of growth of sw-H1N1/82 on NPTr cells at 40°C, which was not observed on MDCK cells nor for the closely related av-H1N1/05 virus. This prompted us to analyse the replication of these two viruses on NPTr cells using real-time PCRs targeting the PB2, NP and genomic vRNA segments. Very similar replication kinetics were obtained for the three genes tested, but differences might exist at earlier times of infection. We involving residue 627E may prevail to explain their sensitivity at 33°C. However, this sensitivity of replication at 33°C seemed more pronounced for sw-H1N1/82, suggesting that additional determinants might be involved. In addition, the absence of replication at 40°C was observed only for the swine virus and it seems unlikely that residue 627E of PB2 could account for the sensitivity observed for sw-H1N1/82 but not for other swine viruses. Thus, as clearly shown for H1N1

viruses, it seems that the temperature sensitivity of avian and swine viruses can be an intrinsic property of the virus and that, even if two viruses belong to the same genetic lineage, they might exhibit some features specific for the host they were isolated from.

Several studies have recently been published focusing on influenza A virus sensitivity to the incubation temperature and on host range determinants, all of them trying to better understand the mechanisms behind cross-species infection and host adaptation. Thus, using only human viruses as model, [START_REF] Steel | Transmission of influenza virus in a mammalian host is increased by PB2 amino acids 627K or 627E/701N[END_REF] showed that residue 627E of PB2 could be compensated by mutating residue 701 from aspartic acid (D) to asparagine (N), resulting in a wild-type temperature A c c e p t e d M a n u s c r i p t 24 sensitivity phenotype and restoring transmission to mammals [START_REF] Steel | Transmission of influenza virus in a mammalian host is increased by PB2 amino acids 627K or 627E/701N[END_REF].

This could not explain the slightly better growth capabilities observed at 33°C for av-H1N1/02 and av-H1N2/05 when compared to av-H1N1/05, as av-H1N2/05 and av-H1N1/05 were both characterised by the presence of a 701N residue in PB2, and it would not explain why av-H3N2/00 showed temperature sensitivity at 33°C on MDCK cells. Interestingly, Scull et al (2009) showed that PB2 residue 627 is not always sufficient to confer sensitivity to low temperature (Scull et al., 2009). Using human viruses as genetic background and HA and NA from an Italian H7N1 influenza virus isolated from chicken, they demonstrated that the glycoproteins could also be involved in the cold-sensitivity at 32°C. Although av-H3N2/00 contains HA and NA that were found closely related to human virus counterparts, it would be interesting to test this hypothesis to find an explanation to the sensitivity at 33°C observed for this virus on MDCK cells.

Other hypotheses could be tested to explain some of the temperature sensitivities we described. For example, the instability of the polymerase complex, that would prevent replication from occurring, was proposed to be responsible for a temperature sensitivity observed at 41°C for three mammalian influenza viruses (Dalton et al., 2006). Two recent studies also highlighted a putative role for NP in temperature sensitivity and as a determinant of host range.

Thus, residue 239 of NP was shown to be involved in the sensitivity at 39°C of the vRNA packaging, possibly by affecting the interaction between NP and M1 (Noton et al., 2009). Finally, Gabriel et al (2008) In conclusion, data reported in this study suggested that a virus closely related to a given genetic lineage defined by the host origin can possess specific phenotypic growth characteristics when isolated from another host. It seems that minor changes in nucleotide or amino acid sequences could be sufficient to allow the adaptation of a strain from one host lineage to another host. Temperature sensitivity might represent some specific signature of a virus, depending on the host from which it was isolated, and could be due to residues other than residue 627 of PB2 and could affect other steps of the virus life cycle besides RNA replication. Our results confirmed that temperature sensitivity of influenza viruses is variable for each virus and might depend on the cell type supporting the replication. More studies on various strains, including human influenza viruses, would be necessary to better understand the variability in growth and replication capabilities, and to possibly define in vitro conditions that could be used to predict the risk for a virus to cross the species barrier and efficiently infect a new host. a For a review, see [START_REF] Kuntz-Simon | Genetic and antigenic evolution of swine influenza viruses in Europe and evaluation of their zoonotic potential[END_REF]. 2 b Phylogenetic results were abbreviated as follows: Sw av , genes closely related to 3 avian-like swine influenza viruses; Sw hu , genes closely related to human-like 4 reassortant swine influenza viruses; Av, genes closely related to avian influenza 5 viruses; Hu, genes closely related to human influenza viruses. 6 c E = glutamic acid; K = lysine 7

Table 1

(

  Invitrogen), according to the manufacturer's protocol. PCR products of the expected size were purified from an agarose gel using the Qiaquick gel extraction kit (Qiagen). Purified products were then quantified with the Quant-iT DNAds kit and Qubit fluorometer (Invitrogen) to determine the copy number. Serial 10-fold dilutions of purified PCR products were used to establish standard curves. For all subsequent analyses, one full-length PCR product was chosen as a standard for each PB2-, NP-and M-specific real-time PCRs: av-H1N1-PB2 and sw-H3N2-NP and -M. Ten independent serial dilutions of each standard were analysed to determine limits of detection and quantification as described in a French reference norm(XPT 90-471 from April 2006).

  For virus titration experiments, analyses of variance were performed to test the effect of trypsin concentration and cell type on the virus titres, assessed by the log 10 of titres (TCID 50 /ml). Analyses of variances were computed with the GLM procedure of the SAS software(SAS, 2004). In addition, the Newman and Keuls multiple comparison procedure was used to compare the different trypsin concentrations, with respect to the different cell types.

  a medium containing up to 2 µg/ml of trypsin. In contrast, monolayers of DF1 cells were disrupted in such a medium. At 0.2 µg/ml of trypsin, DF1 cell monolayers did not reproducibly resist. Thus, virus stock titrations could only be performed on MDCK and NPTr cells.

  on MDCK cells (black bars, left panel) than on NPTr cells (black bars, right panel). In contrast, with 0.2 µg/ml of trypsin, titres were globally slightly but significantly (p<0.0001) higher on NPTr cells (grey bars, right panel) than on MDCK cells (grey bars, left panel). Taken as a whole, titres were significantly (p<0.0001) higher with 2 µg/ml of trypsin (black bars) than with 0.2 µg/ml (grey bars). These results indicated that multiplication of av-or sw-H1N1, H1N2 and H3N2 influenza viruses was less dependent on the trypsin-TPCK concentration onNPTr cells than on MDCK cells. of virus growth kinetics at different temperatures on MDCK and NPTr cells in the absence or presence (2 µg/ml) of trypsin-TPCKAs described in Materials and Methods, the growth capabilities of av-and sw-H1N1, H1N2 and H3N2 viruses were compared on MDCK and NPTr cells infected at a low multiplicity of infection (MOI = 10 -3 TCID50/cell) and then incubated at 33, 37 or 40°C. At different times post-infection, the production of infectious viral particles in the supernatants was evaluated by titration. The kinetics of virus multiplication are presented in Fig.2.

  SYBR green real-time PCRs were designed to specifically detect and quantify the PB2-, NP-, and M-viral RNA segments. In order to obtain a good efficiency of the real-time PCRs whatever the avian or swine origin of the viral genome, thermocycling conditions and primer concentrations were optimised on PB2-, NP-and M-full-length PCR products derived from avian and swine influenza A viruses used in this study (data not shown). No detection limit could be precisely determined for the three real-time PCRs, as they were below the lowest amounts tested. Quantification limits could be estimated to be approximately 220, 60 and 600 copies for our PB2-, NP-and M-specific real-time PCRs, respectively.

  fully efficient on the HA of avian viruses. More interestingly, we observed that av-H3N2/00 required trypsin-TPCK only at 40°C on MDCK cells.

  correlation with the growth kinetics determined by virus titres in the supernatant even if a light shift in time was observed, consistent with the fact that replication must take place before new viral particles can be produced. The delay in replication at 33°C confirmed the results of growth kinetics and was perfectly consistent with the previously published data regarding the role of residue 627E for avian viruses. Since av-H1N1/05 and sw-H1N1/82 are both related to the avian-like swine lineage, it seems likely that similar mechanisms

  described two point mutations (701 in PB2 and 319 in NP) involved in the adaptation of an avian influenza strain A c c e p t e d M a n u s c r i p t 25 to mouse, demonstrating that these two residues provides more efficient binding of the cellular importin 1 protein in mammalian cells, resulting in an increased hypotheses would be interesting to test using reverse genetics systems based on each virus background.

  role of the host cell in selection of hemagglutinin receptor specificity. Virus Research 29, 155-165. Dalton, R.M., Mullin, A.E., Amorim, M.J., Medcalf, E., Tiley, L.S., Digard, P., 2006, Temperature sensitive influenza A virus genome replication results from low thermal stability of polymerase-cRNA complexes. Virol J 3, 58. Ferrari, M., Scalvini, A., Losio, M.N., Corradi, A., Soncini, M., Bignotti, E., Milanesi, E., Ajmone-Marsan, P., Barlati, S., Bellotti, D., Tonelli, M., 2003, Establishment and characterization of two new pig cell lines for use in virological diagnostic laboratories. J Virol Methods 107, 205-212. Gabriel, G., Herwig, A., Klenk, H.D., 2008, Interaction of polymerase subunit PB2 and NP with importin alpha1 is a determinant of host range of influenza A virus. PLoS Pathog 4, e11. Geiss, G.K., Salvatore, M., Tumpey, T.M., Carter, V.S., Wang, X., Basler, C.F., Taubenberger, J.K., Bumgarner, R.E., Palese, P., Katze, M.G., Garcia-Sastre, A., 2002, Cellular transcriptional profiling in influenza A virusinfected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza. Proc Natl Acad Sci U S A 99, 10736-10741. Hatta, M., Gao, P., Halfmann, P., Kawaoka, Y., 2001, Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science 293., Massin, P., Escriou, N., Crescenzo-Chaigne, B., van der Werf, S., 2000, Genetic analysis of the compatibility between polymerase proteins from human and avian strains of influenza A viruses. J Gen Virol 81, 1283-1291. Noton, S.L., Simpson-Holley, M., Medcalf, E., Wise, H.M., Hutchinson, E.C., McCauley, J.W., Digard, P., 2009, Studies of an influenza A virus temperature-sensitive mutant identify a late role for NP in the formation of infectious virions. J Virol 83, 562-571. Olsen, C.W., Karasin, A., Erickson, G., 2003, Characterization of a swine-like reassortant H1N2 influenza virus isolated from a wild duck in the United States. Virus Res 93, 115-121. SAS, 2004, SAS OnLineDoc version 9.1. SAS Institute Inc., Cary, NC. Scholtissek, C., Hinshaw, V.S., Olsen, C.W., 1998, Influenza in pigs and their role as the intermediate host, In: Nicholson, K.G., Webster, R.G., Hay, A.J. (Eds.) Textbook of influenza. Blackwell Science, Ltd., Oxford, England, pp. 137-145. Scholtissek, C., Murphy, B.R., 1978, Host range mutants of an influenza A virus. Archives of Virology 58, 323-333. Scull, M.A., Gillim-Ross, L., Santos, C., Roberts, K.L., Bordonali, E., Subbarao, K., Barclay, W.S., Pickles, R.J., 2009, Avian Influenza virus glycoproteins restrict virus replication and spread through human airway epithelium at temperatures of the proximal airways. PLoS Pathog 5, e1000424.

FiguresFIG 01 :FIG 02 :FIG 03 :

 010203 Figures

  Sw hu Sw hu Sw av Sw av E Sw av Sw av Sw av Sw av

	/2899/82 (H1N1)	avian-like swine	Sw av Sw av	Sw av Sw av	E	Sw av Sw av Sw av Sw av
		human-like
	A/swine/Scotland/410440/94 (H1N2)	reassortant	Sw hu Sw hu Sw av Sw av	E	Sw av Sw av Sw av Sw av
		swine
		human-like
	A/swine/Gent/1/84 (H3N2)	reassortant
		swine
	Isolated from birds
	A/turkey/France/05016/05 (H1N1)	-	Sw av Sw av Sw av Sw av	E	Sw av Sw av Sw av Sw av
	A/mallard/France/710/02 (H1N1)	-	Av	Av	Av	Av	E	Av	Av	Av	Av
	A/turkey/France/05045c/05 (H1N2)	-	Sw hu Sw hu Sw av Sw av	E	Sw av Sw av Sw av Sw av
	A/duck/Ontario/05/00 (H3N2)	-	Hu	Hu	Hu	Hu	K	Hu	Hu	Hu	Hu

Acknowledgements

transport of the virus genome into the nucleus (Gabriel et al., 2008). All these The authors would like to thank Carole Guillemoto, Stéphane Queguiner, Stéphane Gorin for technical assistance, and François-Xavier Briand for help in avian influenza virus phylogenetic analyses. This work was supported by a "Programme transversal de recherche (PTR)" joined grant co-funded by the "Agence Française de Sécurité Sanitaire des Aliments" (AFSSA) and the Pasteur Institute.