D Chachra

J G Coote #

R Parton

# S K Jand

Haemolytic and cytotoxic activities of the Tween 80-extracted putative haemolysin of Pasteurella multocida B:2

The objective of this study was to investigate the haemolytic and cytotoxic activity of P. multocida B:2 strains, originally from cases of haemorrhagic septicaemia in cattle. All six P. multocida B:2 strains were non-haemolytic on sheep blood agar (SBA) overlay revealed clear haemolytic zones but suggested that Tween was bound to some component of the P. multocida B:2 fractions and was responsible, to some extent, for the haemolytic activity observed. However, the effect of heat and other reagents on the Tween-extracted fractions and the lack of haemolytic activity in different Tweenextracted cell fractions of organisms other than P. multocida suggested that some proteinaceous component of the organism could indeed act as a haemolysin. This putative haemolysin may be one of the virulence attributes of P. multocida, but its characterisation and role in pathogenesis require further study.

and horse blood agar (HBA) when grown aerobically and on SBA anaerobically but they were haemolytic on HBA when grown anaerobically. No haemolytic activity against horse red blood cells was detected in culture supernates from aerobically or anaerobically grown cultures and only very weak haemolytic activity was obtained in supernates or pellet fractions from sonicated cells. However, after repeated extraction of sonicated cells with Tween 80, haemolytic activity was found in various cell fractions, both Tweensoluble and -insoluble. The Tween-extracted putative haemolysin and other bacterial fractions were also cytotoxic for mouse macrophage-like J774.2 cells. Further characterisation of the putative haemolysin revealed it to be a heat-labile, non-poreforming protein of molecular weight >10 KDa whose activity was completely destroyed by trypsin and greatly reduced with protease and proteinase K treatment. Congo red also reduced the haemolytic activity. Non-denaturing gel-electrophoresis and RBC agar A c c e p t e d M a n u s c r i p t

Introduction

Haemolysis is associated with pathogenicity of a number of bacteria but P.

multocida is not classified as a haemolytic species (Krieg and Holt, 1984). However, browning on blood agar plates as a sign of erythrocyte damage was reported by [START_REF] Carter | Pasteurella In: Bergy's Manual of Systematic Bacteriology[END_REF], and [START_REF] Diallo | Characteristics of a haemolytic extract from avian Pasteurella multocida[END_REF] observed a diffuse but obvious zone of haemolysis around avian P. multocida (strain VP21) colonies on horse blood agar under anaerobic conditions. Treatment of P. multocida strain VP 21 with Tween 80 yielded an extract that lysed red blood cells (RBCs) from the chicken, rabbit, horse, bovine and human, with the highest titres observed on chicken RBCs incubated aerobically [START_REF] Diallo | Characteristics of a haemolytic extract from avian Pasteurella multocida[END_REF] The effect that some bacterial haemolysins produce is not always apparent on diagnostic blood agar plates and special methods may be needed to demonstrate their activity. For example, non-ionic detergents and various macromolecules were used to extract streptolysin S from Group A Streptococcus [START_REF] Ginsburg | Oxygen stable hemolysins of Group A Streptococci II. Chromatographic and electrophoretic studies[END_REF][START_REF] Loridan | Purification of RNA-core induced streptolysin S, and isolation and haemolytic characteristics of carrier-free toxin[END_REF][START_REF] Kent | Production, purification and molecular weight determination of the haemolysin of Treponema hyodysenteriae[END_REF][START_REF] Lysons | A cytotoxic haemolysin from Treponema hyodysenetriae-a probable virulence determinant in swine dysentery[END_REF]. The haemolysin obtained by such methods was assumed to be stabilised by being bound to the carrier-molecule (Tsaihong , 1983). The aim of this study was to further investigate the haemolytic activity of P. multocida and to determine its cytotoxic properties.

Materials and Methods

Strains, media and growth conditions

A total of six Pasteurella multocida B:2 strains were used in this study. These included strains 90016, 85020, 90025, 8402, 86006 and 85009 originally from cases of haemorrhagic septicaemia in cattle in Sri Lanka. A porcine isolate, P. multocida CW5519/93 (serotype D), a bovine strain of P. multocida (serotype A3), a bovine strain of Mannheimia haemolytica and Escherichia coli K12 were included as controls. All strains were stored as lyophilized cultures in the Institute of Infection, Immunity and Inflammation, University of Glasgow.

Bacteria were grown aerobically at 37 o C for 18 h in 500-ml volumes of brain heart infusion (BHI) broth (Difco) in 2-L dimpled flasks in an orbital incubator at 150 rpm. For anaerobic growth, bacteria were inoculated on BHI agar plates and incubated

for 48 h at 37 o C in an anaerobic jar with Gas Pak. The resulting growth was scraped off in a minimum volume (2-3 ml) of phosphate-buffered saline (PBS). BHI blood agar was prepared by adding 8% v/v horse or sheep blood to BHI agar (Difco). Haemolysin production was evaluated on BHI blood agar after growth aerobically for 24 h or anaerobically, in an anaerobic jar with Gas Pak (Oxoid), for 48 h at 37 o C.

Extraction of haemolysin

a Sonication

Bacteria from BHI broth cultures grown aerobically, or grown anaerobically on After sonication, the suspensions were centrifuged at 1800 g for 15 min. The supernates and the pellets, resuspended to the same volume in PBS, were stored at -20 o C until used.

P. multocida B:2 strain Pm 90016 was also grown aerobically for 18 h as described in section 2.1 but in BHI broth containing bovine serum albumin (30 mg/ml) (Sigma). The culture was then processed as described above.

2.2.b Tween 80 extraction

Tween 80 (0.5% v/v) (Sigma) in sterile PBS (Sigma) (pH 7.3) was used to extract the haemolysin from cultures grown both aerobically and anaerobically. Bacterial extracts were prepared as shown in Fig. 1. Briefly, cultures grown for 18h in BHI broth were pelleted by centrifugation and the bacteria suspended in PBS and sonicated as described in section 2.2a. After sonication, the cell fragments were suspended in an equal volume of Tween 80 (0.5 % v/v) and incubated for 2 h at 37 o C. After low-speed centrifugation to remove the first Tween-insoluble fraction, the first Tween extract (Fig. were tested for haemolytic activity as described.

Cytotoxicity test

Preparation of cells

Mouse macrophage-like cells (J774.2 cells) were grown in RPMI-1640 medium (Cambrex Bioscience) containing L-glutamine (2 mM, Gibco), penicillin-streptomycin (100 IU/ml penicillin, 100 µg/ml streptomycin (Gibco)) and fungizone (amphotericin B 2.5 µg/ml) (Gibco) and heat-inactivated foetal bovine serum (Biowest) 10% v/v. After 2-3 days, the spent medium was decanted and cells were washed twice with fresh, nonphenol red RPMI-1640 medium (Cambrex Bioscience). The cells were scraped off into 5 ml of fresh, non-phenol red medium and centrifuged for 5 min at 1100 g. The cells were then resuspended in fresh non-phenol red medium, counted by Trypan Blue exclusion and the cell concentration was adjusted to 5 x 10 5 cells/ml. A 50 µl aliquot of this suspension was added to each well of 96-well flat-bottomed cell culture microtitre plates and incubated for 1 h at 37 o C, in CO 2 (5% v/v).

MTT assay

Samples of bacterial extracts were diluted in non-phenol red RPMI-1640 medium and 50 µl of each dilution were added to wells containing 50 µl of cells, mixed gently and plates were incubated for 2 h at 37 o C in CO 2 (5% v/v). After this time, 15 µl of MTT dye

Polyacrylamide gel electrophoresis and RBC agar overlay

Tween 80 extracts (fraction H) were separated by non-denaturing gel electrophoresis with 12% acrylamide in the separating gel, according to the method of [START_REF] Bollag | Protein Methods[END_REF]. A c c e p t e d M a n u s c r i p t

Results

Effect of media and growth conditions on haemolysin production

All six P. multocida B:2 strains were non-haemolytic on sheep blood agar (SBA)

and horse blood agar (HBA) when grown aerobically and on SBA anaerobically but they were haemolytic on HBA when grown anaerobically. The approximate size of zones was 1-2 mm, with no discolouration. The P. multocida A:3 and D strains, M. haemolytica and E. coli K12 were non-haemolytic on HBA and SBA grown either aerobically or anaerobically.

3.2.a Effect of cell sonication on recovery of haemolysin

No haemolytic activity was detected against horse RBCs in supernates from cultures grown aerobically or anaerobically and only very weak haemolytic activity was obtained from supernates of sonicated cells of P. multocida B: 2 strains grown in BHI broth under either aerobic or anaerobic conditions. The sonicated supernates were concentrated 25x and 4x by ultrafiltration through 30 kDa and 10 kDa molecular weight cut-off Amicon filters (Microcon), respectively, but no increase in haemolysis was detected. Pellets from cells grown aerobically or anaerobically, then sonicated, did not show any haemolytic activity (data not shown). None of the above cell fractions were cytotoxic for the J774.2 cells in the MTT assay (data not shown). No haemolytic activity was observed in culture supernates or supernates from sonicated cells of P. multocida B:2 grown in BHI broth containing BSA.

3.2.b Effect of Tween 80 on extraction of haemolysin

In preliminary experiments, both aerobically-and anaerobically-grown P. multocida B: 2 strains 90016 and 85020 whole bacterial cells, and supernates and pellets from sonicated (data not shown). For convenience, the subsequent extractions were done on cells grown aerobically. After Tween extraction of cells grown aerobically (as in Fig. 1), haemolytic activity was found in various cell fractions, both Tween soluble and insoluble. Fig. 2 shows the percent haemolysis of different cell fractions from P. multocida B:2 strain 90016. The highest activity was found in fractions D, H and A, with 50% haemolytic titres of >16, 8-16 and 8-16, respectively. There was lower activity in fractions B and G, with titres of 2-4 and 4-8, respectively. Fraction F had activity similar to fraction D whereas fraction E had little haemolytic activity (data not shown). Similar haemolytic activity was present in fractions A, B, D, G, H (Fig. 1) derived from P. multocida serotypes A3 (bovine strain) and D (pig strain). P. multocida A3 showed highest haemolytic activity in fractions A, D and G, all with 50% haemolytic titres of 8-16. There was lower activity in fractions H and B, with titres of 4-8 and 2-4, respectively. In P.

multocida serotype D, maximum haemolytic activity with 50% haemolytic titres of >16

were observed in fraction G. Titres of 8-16, 4-8 and 2-4 were observed in fractions A, H and D, and fraction B, respectively. However, no haemolytic activity was seen in any fraction of E. coli K12 or M. haemolytica, using the same method of extraction, or with Tween alone at concentrations corresponding to those in the bacterial extract samples. No haemolytic activity could be detected when BSA was used in place of Tween 80 for the extraction. The Tween extractions described above (as in Fig. 1) were repeated twice, with similar results.

A c c e p t e d M a n u s c r i p t 10

Cytotoxic activity of the Tween-extracted putative haemolysin

In order to test whether the factor responsible for the haemolytic activity was also cytotoxic to cells other than RBCs, the same fractions were tested for cytotoxicity on a J774.2 mouse macrophage cell line (Fig. 3). Fractions A, D, G and H showed similar cytotoxicity with 50% cytotoxicity titres of around 16. The cytotoxicity data, therefore, correlated to some extent with the haemolytic activity (Fig. 2). Fractions D, H and A were highly active in both assays but fraction G was more active against J774.2 cells than against RBCs. Fraction B had low activity in both assays.

Effect of heat, enzymes and Congo red on the haemolytic activity of P. multocida

After heating a 1 in 2 dilution of the third Tween extract (fraction H, in Fig. 1) of P. multocida B:2 strain 90016 to different temperatures for 30 min, there was a decline in haemolytic activity from 100% haemolysis with the unheated sample to 60% haemolysis at 60 o C, 40% at 80 o C and no haemolysis after heating to 100 o C.

Haemolytic activity in fraction H was completely destroyed by trypsin treatment and greatly reduced with the other two proteases. The haemolytic activity of a 1 in 2 dilution of fraction H was reduced from 100% to 32% and 67%, when treated with protease (0.5mg/ml w/v) and proteinase K (0.1 mg/ml w/v), respectively.

Treatment of fraction H (1 in 4 dilution) with Congo red (0.25% w/v) also reduced the haemolytic activity, from 85% haemolysis to 20%.

Detection of haemolysin by non-denaturing electrophoresis and RBC agar overlay

A c c e p t e d M a n u s c r i p t 11 A number of samples were subjected to non-denaturing polyacrylamide gel electrophoresis. These included fraction H from P. multocida strain 90016 that was either unconcentrated or concentrated 30-fold by ultrafiltration (Centiplus YM-30, Amicon).

The RBC agar overlay showed clear haemolytic zones after 18 h (Fig. 4) particularly with the P. multocida B:2 concentrated samples but also with the unconcentrated samples. The M. haemolytica Tween-extract and the solution of Tween 80 also showed zones of haemolysis but they had not migrated as far as the P. multocida B: 2 fractions. No distinct bands coinciding with the zones of clearing were seen in a Coomassie blue-stained duplicate gel before overlay.

Discussion

The aim of this study was to determine and characterise the haemolytic and cytotoxic activity of P. multocida B:2 strains. The presence of haemolysis on HBA plates incubated under anaerobic but not aerobic conditions indicated either that the active component is inactivated in the presence of oxygen or that it is released from the bacteria only under anaerobic conditions. However, the latter explanation is most likely as haemolytic activity could be extracted from both aerobically-and anaerobically-grown bacteria, as described below. No haemolytic activity or cytotoxic activity for mouse macrophage J774.2 cells was detected in culture supernates of P. multocida B:2 strains even after concentration with 10 KDa and 30 KDa cut-off membranes. [START_REF] Shah | Vacuolating cytotoxic activity of Pasteurella multocida causing haemorrhagic septicaemia in buffalo and cattle[END_REF] and [START_REF] Diallo | Characteristics of a haemolytic extract from avian Pasteurella multocida[END_REF] had also reported little vacuolating and cytotoxic activity in the culture supernates of P. multocida B:2 strains. Moreover, in the present study, little haemolytic activity was detected in supernate or pellet fractions from sonicated cells of P. multocida B:2.

A c c e p t e d M a n u s c r i p t 12 A non-ionic detergent Tween-80 was used to extract the putative haemolysin, as reported previously by [START_REF] Diallo | Characteristics of a haemolytic extract from avian Pasteurella multocida[END_REF] with an avian strain of P. multocida.

Repeated extractions with Tween 80 to extract the haemolysin into the soluble fraction led to the idea that the component is either in an inactive form before extraction or is cellbound. However, other possibilities, as assumed for the haemolysin of Group B streptococci, were that the haemolysin was continually synthesized and released by the bacteria but was rapidly inactivated unless it was associated with other molecules, like

Tween 80, that act as carriers for this haemolysin (Marchlewicz and [START_REF] Marchelwicz | Properties of a hemolysin produced by group B Streptococci[END_REF].

The Tween-extracted haemolytic activity from both aerobically and anaerobically-grown bacteria was found to be genus-specific since extracts from P.

multocida A:3 and D strains showed haemolytic activity whereas those from E. coli and M. haemolytica treated in the same way did not. The haemolytic extract from P. multocida B:2 was heat-labile and was inactivated by various proteases which suggested it was a protein. These contrasts with the findings of [START_REF] Diallo | Characteristics of a haemolytic extract from avian Pasteurella multocida[END_REF], working with a Tween extract from an avian P. multocida, who found it to be heat-stable. The extraction procedure adopted by [START_REF] Diallo | Characteristics of a haemolytic extract from avian Pasteurella multocida[END_REF] was different in that whole cells were treated with Tween 80, rather than the sonicated cells as used in the present study.

The apparent molecular weight of the putative haemolysin was >10 KDa as the haemolytic activity was seen in the concentrate, rather than the filtrate of haemolytic extract, when passed through a 10 KDa cut-off filter (data not shown).

Oxygen-stable haemolysins of streptococci have been shown to be induced by certain agents like serum and serum albumin [START_REF] Ginsburg | Oxygen stable hemolysins of Group A Streptococci II. Chromatographic and electrophoretic studies[END_REF]. The 13 haemolysins induced by these agents were all non-antigenic and their activity was inhibited by trypan blue and Congo red [START_REF] Ginsburg | Oxygen stable hemolysins of Group A Streptococci II. Chromatographic and electrophoretic studies[END_REF]. In our study, haemolysin was extracted by Tween 80 but not by bovine serum albumin. [START_REF] Duncan | Characteristics of streptolysin S hemolysis[END_REF] suggested that, in the case of streptolysin S, certain agents removed the active peptide from the surface of the bacteria and served as carrier molecules for the haemolysin. The carrier molecule Tween 80 is thought to bind to streptolysin S by substituted oxyethylene polymers via hydrogen bonding [START_REF] Ginsburg | Oxygen stable hemolysins of Group A Streptococci II. Chromatographic and electrophoretic studies[END_REF].

There is also a report of a requirement for a carrier of the extracellular haemolysin released by S. agalactiae during growth [START_REF] Tsaihong | Effect of carrier molecules on production and properties of extracellular hemolysin produced by Streptococcus agalactiae[END_REF]. In this report, there was enhanced production of extracellular haemolysin in the presence of Tween or starch added to the culture medium and there was evidence of an association of the haemolysin with the molecules eliciting its release and determining some of its properties. With P. multocida, Tween might be sequestering some haemolytic component of the organism and may be acting as a carrier molecule, as with streptolysin S, but the mechanisms by which this is achieved are unknown.

The Tween-extracted putative haemolysin and other bacterial fractions as prepared in Fig. 1 were cytotoxic for J774.2 cells. The cytotoxicity results correlated to some extent with the haemolytic activity, suggesting the cytotoxic effect of the putative haemolysin against J774.2 cells. Haemolysins and cytolysins are membrane-damaging agents. They play a multifunctional role in disease due to their ability to lyse erythrocytes, mast cells, polymorphs and other cells (Braun and Focarte, 1991).

Membrane-damaging toxins form a large, heterogenous group comprising substances which are characterized functionally by their ability to act either as surfactants,

A c c e p t e d M a n u s c r i p t 14 solubilizing the membrane through a detergent-like action, or through enzymatic or poreforming activity [START_REF] Braun | Activation and secretion of Serratia hemolysin[END_REF][START_REF] Titball | Bacterial phospholipases C[END_REF][START_REF] Rowe | Assays of haemolytic toxins[END_REF]. The poreforming cytotoxins from Gram-negative bacteria are considered to be virulence determinants [START_REF] Welch | Pore-forming cytolysins of Gram-negative bacteria[END_REF], although their precise role in the pathogenesis of any disease has been difficult to define. In the present study, the putative haemolysin of P. multocida B:2 did not seem to be a pore-forming toxin as no osmotic protection was afforded by suspending the RBCs in solutions of polyethylene glycols of different sizes [START_REF] Kuga | Pore-size distribution analysis of gel substances by size exclusion chromatography[END_REF] (data not shown).

The results of non-denaturing gel electrophoresis and RBC agar overlay showed clear haemolytic zones in lanes containing samples of P. multocida B:2 haemolysin, especially when concentrated by ultrafiltration. However, there were haemolytic zones in lanes containing M. haemolytica extract and Tween alone although the mobility of the putative haemolysin was greater in the P. multocida B:2 samples. These results suggest that PAGE concentrates the Tween and it stays within the gel, despite the washing step.

They could also suggest that, in the Tween-extracted samples, the Tween remains bound to some component of the P. multocida B:2 fractions and it is this concentrated detergent that is responsible, to some extent, for the haemolytic activity observed. However, the production of haemolysis on HBA under anaerobic conditions, the effect of heat and other reagents on the Tween-extracted fractions and the lack of activity in different Tween-extracted cell fractions of organisms other than P. multocida suggests that some, presumably proteinaceous, component of the organism can indeed act as a haemolysin.

This putative haemolysin may be one of the virulence attributes of P. multocida, but its characterization and role in pathogenesis require further study.

  and suspended in PBS, were pelleted by centrifugation at 1100g for 15 min. The supernate was removed and the cell pellet resuspended in a minimum volume of PBS, and then sonicated on ice for 10 min, with 10 s on and 10 s off, using a standard ½" diameter probe in a Vibra cell sonicator (Sonics and Materials, Danburg, CT, USA).

1

  , fraction A) was centrifuged at 70,000 g for 30 min at 4 o C to obtain the cytoplasmic fraction (B) and the second Tween-insoluble fraction (C). This fraction, C, was further treated with Tween 80 for 2 h at 37 o C to obtain fraction D then centrifuged at 1100 g for 15 min to obtain the third Tween-insoluble fraction E, and the second Tween extract, F.Fraction F, was centrifuged at 70,000 g for 30 min at 4 o C to obtain the fourth Tweeninsoluble fraction (G) and the third Tween extract (fraction H). Tween insoluble fractions were resuspended in Tween to the same volume as the corresponding soluble fractions for haemolysis assay2.3 Haemolysis assayDefibrinated horse blood was used except where stated. RBCs were washed thrice with PBS and resuspended at 2% v/v in PBS. Two-fold serial dilutions of the bacterial extracts were made in 500 µl-volumes of PBS in 1.5 ml tubes. To these, equal volumes of the 2% v/v horse RBC suspension were added and, after incubation aerobically for 4 h at 37 o C on a shaker, unlysed RBCs were removed by centrifugation at 1100 g for 15 min and the OD 540nm of each supernate was determined. The controls for the test included RBCs in the appropriate concentrations of Tween 80 in PBS as negative controls and RBCs in TritonX-100 (0.1% v/v) as a positive control. All assays were performed in duplicate. Percent haemolysis was calculated as: OD of ((test sample)-(negative control) / (positive control)-(negative control)Putative haemolysin (fraction H, Fig.1) was subjected to various degrees of heat (60, 80, 100 o C) treatment for 30 min and then tested for haemolytic activity. Unheated sample was included as control. Various enzymes were tested for their effect on haemolytic activity. Trypsin (0.1 mg/ml), protease (0.5 mg/ml) from Streptomyces griseus and proteinase K (0.1 mg/ml) (all from Sigma) dissolved in PBS were mixed 1:1 with test sample, incubated for 30 min at 37 o C, and the suspension was then measured for haemolytic activity, as described. Controls included Tween 80 and enzymes at appropriate concentrations, RBCs in PBS and RBCs in Triton X-100 (0.1% v/v). Congo A c c e p t e d M a n u s c r i p t 6 red dissolved in PBS was added to the Tween 80 extracted-soluble fraction (fraction H) at a final concentration of 0.25% w/v, incubated at 37 o C for 30 min and then the samples

  (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (CellTiter 96 Non-Radioactive Cell Proliferation Assay, Promega) were added to each well and plates were incubated for 4 h at 37 o C in CO 2 (5% v/v) . A 100 µl volume of stop solution was then A c c e p t e d M a n u s c r i p t 7 added and plates were incubated overnight at 37 o C in CO 2 (5% v/v). Controls included cells in RPMI medium alone and cells in the appropriate concentration of Tween 80 (negative control). Cells treated with Triton X-100 (0.55% v/v concentration) was used as a positive control. The OD 540nm of the samples was measured and the percentage cell death was obtained from the following equation: 1-((OD of test sample-OD of negative control)/ (OD of positive control-OD of negative control)) x 100.

  The gel was run at 70 volts overnight at 4 o C. After electrophoresis, the gel was removed from the cast and washed three times with Tween 80 (0.5% v/v) in PBS at 37 o C on a shaker, changing the Tween every 15-20 min. The gel was then washed three times with PBS at 37 o C on a shaker changing the PBS every 15-20 min. The washed gel was placed in a square Petri dish and overlaid with 30 ml of molten 0.7% w/v microagar (Oxoid) in PBS which had been allowed to cool to 45 o C before adding 10 ml of washed horse RBCs (2% v/v). The agar was allowed to set at room temperature for half an hour and then the Petri dish was incubated at 37 o C. The RBC overlay was observed every hour for development of haemolytic zones. If no haemolytic zone appeared after 6 h, incubation was continued overnight. A sample containing Tween 80 (0.5% v/v) alone was run on the gel alongside the test samples, as a control.

  with Tween 80 (0.5% v/v) for 4h at 37 o C and tested for haemolytic activity. Most of the haemolytic activity was found in the pellet fraction of the sonicated cells irrespective of whether the bacteria had been grown aerobically or anaerobically

Fig. 1 .Fig. 2 .

 12 Fig. 1. Flow diagram of the method used for the extraction of putative haemolysin of P.

Fig. 3 .

 3 Fig. 3. Cytotoxicity for J774.2 cells in different bacterial fractions, prepared as described

Acknowledgement

The author is thankful to the Commonwealth Scholarship Commission, UK for the Commonwealth split-site doctoral scholarship.

6.


Mcmonagle El

Willett Bj

Hosie Mj

Margaret J Hosie
email: margaret.hosie@glasgow.ac.uk

Phylogenetic characterisation of naturally occurring feline immunodeficiency virus in the United Kingdom

Keywords: Feline immunodeficiency virus, FIV, vaccine, phylogeny

Feline Immunodeficiency Virus (FIV) is a significant pathogen of domestic and non-domestic felids worldwide. In domestic cats, FIV is classified into 5 distinct subtypes (A-E) with subtypes A and B distributed most widely. However, little is known about the degree of intrasubtype viral diversity and this may prove critical in determining whether monovalent vaccines are likely to protect against FIV strains within a single subtype. Here, we characterise novel env sequences from 47 FIV strains recovered from infected cats in the United Kingdom and its environs. Phylogenetic analyses revealed that all bar one sequence belonged to subtype A, the predominant subtype in Western Europe. A single sequence was identified as a likely subtype A/C

recombinant, intriguing given that subtype C does not appear to exist in either the UK or North Western Europe and suggestive of a recombination event predating its introduction into the UK. Subtype A strains from the UK were not significantly differentiated from representative subtype A isolates found elsewhere suggesting multiple introductions of FIV into the country.

Divergence among isolates was comparable to that observed for subtype A isolates worldwide, indicating that FIV in the UK covers the full spectrum of subtype A diversity seen globally. This study demonstrates that while subtype A is predominant in the UK, novel introductions may result in the emergence of novel subtypes or intersubtype recombinants, potentially circumventing vaccine strategies. However, the dominance of subtype A suggests that the development of a regional or subtype-specific protective vaccine for the UK could be achievable.

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

Introduction

Feline immunodeficiency virus (FIV) is a widespread pathogen of the domestic cat and infection results in a progressive immune dysfunction similar to AIDS in human immunodeficiency virus (HIV) infection. Since its discovery in 1986 from a cat with an immunodeficiency like syndrome [START_REF] Pedersen | Isolation of a T-lymphotropic virus from domestic cats with an immunodeficiency syndrome[END_REF], FIV has been recognized widely as the feline equivalent of human immunodeficiency virus (HIV) and both viruses share significant physical, biochemical and pathogenic features [START_REF] Johnson | FIV as a model for AIDS vaccination[END_REF]. Such are the similarities between FIV and HIV that FIV serves as a valuable animal model for both prophylactic and therapeutic studies of HIV [START_REF] Elder | Feline immunodeficiency virus as a model for development of molecular approaches to intervention strategies against lentivirus infections[END_REF]as well as being the only non-primate lentivirus that induces an AIDSlike syndrome in its natural host (reviewed in [START_REF] Willett | FIV infection of the domestic cat: an animal model for AIDS[END_REF].).

Like other retroviruses, FIV has high mutation rate, mainly due to the errorprone reverse transcriptase [START_REF] Shankarappa | Consistent viral evolutionary changes associated with the progression of human immunodeficiency virus type 1 infection[END_REF]; accordingly, diverse viral variants emerge continually in the infected host. Since the env gene is the key determinant of viral diversity amongst FIV [START_REF] Olmsted | Nucleotidesequence analysis of feline immunodeficiency virus -genome organization and relationship to other lentiviruses[END_REF], FIV phylogeny studies have focussed mainly on env sequences. According to the nucleotide sequence diversity of the V3-V5 region of env, FIV is classified currently into five distinct phylogenetic subtypes, designated A to E [START_REF] Sodora | Identification of 3 feline immunodeficiency virus (FIV) env gene subtypes and comparison of the FIV and human immunodeficiency virus type-1 evolutionary patterns[END_REF][START_REF] Pecoraro | Genetic diversity of Argentine isolates of feline immunodeficiency virus[END_REF]; subtypes A and B are the most commonly occurring worldwide [START_REF] Martins | Phylogenetic and genetic analysis of feline immunodeficiency virus gag, pol, and env genes from domestic cats undergoing nucleoside reverse transcriptase inhibitor treatment or treatmentnaive cats in Rio de Janeiro, Brazil[END_REF]. Furthermore, similar to HIV-1, several inter-subtype FIV recombinants have been recognized in natural populations following co-infection; inter-subtype recombinants A/B, A/C and A c c e p t e d M a n u s c r i p t B/D have been identified [START_REF] Bachmann | Genetic diversity of feline immunodeficiency virus: dual infection, recombination, and distinct evolutionary rates among envelope sequence clades[END_REF][START_REF] Hayward | Recombination in feline immunodeficiency virus from feral and companion domestic cats[END_REF].

Phylogenetic studies on FIV sequences have revealed significant heterogeneity (up to 30%) in the sequence of the env gene of FIV isolates worldwide [START_REF] Burkhard | Transmission and immunopathogenesis of FIV in cats as a model for HIV[END_REF] similar to that estimated for HIV-1 [START_REF] Rong | Unique mutational patterns in the envelope alpha 2 amphipathic helix and acquisition of length in gp120 hypervariable domains are associated with resistance to autologous neutralization of subtype C human immunodeficiency virus type 1[END_REF]. Subtype A isolates are common in Australia, New Zealand, western United States, South Africa and northwestern Europe [START_REF] Bachmann | Genetic diversity of feline immunodeficiency virus: dual infection, recombination, and distinct evolutionary rates among envelope sequence clades[END_REF][START_REF] Steinrigl | Phylogenetic analysis of feline immunodeficiency virus in Central Europe: a prerequisite for vaccination and molecular diagnostics[END_REF]Kann et al., 2006a;Kann et al., 2006b;Kann et al., 2007a;Kann et al., 2007b). Subtype B isolates have been identified in the central and eastern United States, central Europe, Brazil and eastern Japan [START_REF] Sodora | Identification of 3 feline immunodeficiency virus (FIV) env gene subtypes and comparison of the FIV and human immunodeficiency virus type-1 evolutionary patterns[END_REF][START_REF] Kakinuma | Nucleotide sequence of feline immunodeficiency virus: classification of Japanese isolates into two subtypes which are distinct from non-Japanese subtypes[END_REF][START_REF] Pistello | Analysis of the genetic diversity and phylogenetic relationship of Italian isolates of feline immunodeficiency virus A c c e p t e d M a n u s c r i p t indicates a high prevalence and heterogeneity of subtype B[END_REF][START_REF] Nishimura | Genetic heterogeneity of env gene of feline immunodeficiency virus obtained from multiple districts in Japan[END_REF][START_REF] Steinrigl | Phylogenetic analysis of feline immunodeficiency virus in Central Europe: a prerequisite for vaccination and molecular diagnostics[END_REF][START_REF] Martins | Phylogenetic and genetic analysis of feline immunodeficiency virus gag, pol, and env genes from domestic cats undergoing nucleoside reverse transcriptase inhibitor treatment or treatmentnaive cats in Rio de Janeiro, Brazil[END_REF]. Subtype C has been recognized in Canada, New Zealand and southeast Asia [START_REF] Uema | Feline immunodeficiency virus subtype C is prevalent in northern part of Taiwan[END_REF][START_REF] Nakamura | Phylogenetic analysis of Vietnamese isolates of feline immunodeficiency virus: genetic diversity of subtype C[END_REF][START_REF] Reggeti | Feline immunodeficiency virus subtypes A, B and C and intersubtype recombinants in Ontario, Canada[END_REF][START_REF] Hayward | Phylogenetic analysis of feline immunodeficiency virus in feral and companion domestic cats of New Zealand[END_REF]. Finally, subtypes D and E are infrequent but were identified originally in southwestern Canada and Japan, and Argentina, respectively [START_REF] Pecoraro | Genetic diversity of Argentine isolates of feline immunodeficiency virus[END_REF][START_REF] Nishimura | Genetic heterogeneity of env gene of feline immunodeficiency virus obtained from multiple districts in Japan[END_REF].

Such heterogeneity in env gene sequence poses problems for the design of a broadly protective vaccine. It has been reported previously that effective protection was obtained following a homologous FIV challenge using an inactivated whole virus vaccine, although, despite the relative success of Fel-O-Vax against heterologous subtype B isolates [START_REF] Pu | Dual-subtype FIV vaccine (Fel-O-Vax FIV) protection against a heterologous subtype B FIV isolate[END_REF], protection did not extend to a heterologous challenge [START_REF] Dunham | Limited efficacy of an inactivated feline immunodeficiency virus vaccine[END_REF]. Such A c c e p t e d M a n u s c r i p t different outcomes highlight the impact of genetic diversity on vaccine strategies against FIV and the importance of assessing the genetic diversity of local subtypes for vaccine development, or before introducing a commercial vaccine to a particular geographical area. Furthermore, identification of the predominant strains in a given region is necessary in order to develop appropriate reagents for the molecular diagnosis of FIV infection [START_REF] Leutenegger | Rapid feline immunodeficiency virus provirus quantitation by polymerase chain reaction using the TaqMan fluorogenic real-time detection system[END_REF][START_REF] Hosie | Evolution of replication efficiency following infection with a molecularly cloned feline immunodeficiency virus of low virulence[END_REF].

In order to understand better the degree of intrasubtype viral diversity and the likelihood of a monovalent subtype A FIV vaccine offering broad intrasubtype immunity, we investigated the diversity of FIV within the United Kingdom, a country in which FIV infection is prevalent and where evidence to date suggests that a single viral subtype may dominate. Glasgow. Cats were either companion pets or had been placed into re-homing shelters. Blood samples were centrifuged at 2000 rpm for 10 minutes and the plasma removed. Cell pellets were diluted in 1 ml phosphate buffered saline (PBS) and transferred to 20ml pre-warmed red blood cell lysis buffer (0.88% NH 4 Cl, 10 mM; pH 7.4) and incubated at room temperature for 5 minutes (red blood cell lysis). The intact white blood cell were then pelleted by centrifugation at 1000 rpm for 5 minutes. The supernatant was then discarded and the pelleted cells washed twice by centrifugation through icecold PBSA buffer (1% bovine serum albumin in PBS). 10 7 cells were used for DNA extraction using QIAamp DNA Blood Mini Kit (Qiagen, Crawley, UK), as per the manufacturer's protocol.

Cloning of FIV env genes and nucleic acid sequence determination.

Env genes were amplified by using the polymerase chain reaction (PCR) using a high fidelity enzyme mix (Expand High Fidelity PCR system, Roche Diagnostics Ltd., Burgess Hill, UK) and the degenerate primers 1F4 Sequencing was performed by using Applied Biosystems 3730xl genetic analyser. Raw chromatographic data were analysed by using 'Contig Express' sequence analysis software within the Vector NTI suite of programs (Invitrogen Ltd., Paisley, UK).

Multiple sequence alignments and phylogenetic analyses.

Nucleotide sequence analysis was performed on a 651 nucleotide fragment spanning the V3-V5 region of env gene. The generated consensus included sequences of the 47 isolates included in the study, as well as selected representative sequences from subtypes A-E. Multiple alignment was performed using Clustal X (version 2.0) [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF], followed by manual adjustment.

Alignments were translated and the resulting amino acid-based alignments used as an exact guide for re-positioning of improper gapping, particularly, where sequences were different in length. Final alignments are available from the authors upon request. DNA distance matrices were calculated with Paup [START_REF] Swofford | Paup -A Computer-Program for Phylogenetic Inference Using Maximum Parsimony[END_REF][START_REF] Wilgenbusch | Inferring evolutionary trees with PAUP*[END_REF] under a GTR + I + G model, selected in Modeltest [START_REF] Posada | MODELTEST: testing the model of DNA substitution[END_REF] and parameterized using Maximum Likelihood (ML). A Bayesian phylogeny was estimated in

MrBayes under a SDR06 model of evolution [START_REF] Huelsenbeck | MRBAYES: Bayesian inference of phylogenetic trees[END_REF] based on two independent runs of 10 Million generations, with samples taken every 2000 generations. Because initial analyses indicated a problem with inflated branch lengths [START_REF] Brown | When trees grow too long: investigating the causes of highly inaccurate bayesian branchlength estimates[END_REF], a ML tree (generated in A c c e p t e d M a n u s c r i p t

Paup) was added as a starting tree and the branch length prior was adjusted according to the formula provided in [START_REF] Brown | When trees grow too long: investigating the causes of highly inaccurate bayesian branchlength estimates[END_REF].

Spatial coordinates of the sampled isolates were used to determine their Euclidian distances. In order to test for any spatial patterns consistent with isolation by distance, the correlation between spatial and genetic ML distances was assessed based on a Mantel test using the geodist package in program R (http://cran.r-project.org).

Recombination analysis.

A putative recombinant UK sequence (101070) was assessed for evidence of recombination using three different approaches: Bootscan analysis as implemented in the program Simplot, as well as and RDP and GeneConv in program RDP3 [START_REF] Martin | RDP3: a flexible and fast computer program for analyzing recombination[END_REF]. Along with the 101070 sequence, consensus sequences for each of the five subtypes (A-E) were included in the analysis.

Nucleotide sequence accession numbers. GenBank accession

numbers for control reference sequences in the phylogenetic analysis are: A c c e p t e d M a n u s c r i p t

Results

The sample population

Clinical blood samples were examined from 47 animals that had been infected naturally with FIV; the cats were either pet cats or stray cats waiting to be rehomed from animal protection shelters. Samples were collected from across the UK. While the majority of samples originated from England, Scotland or

Wales, a single sample came from Jersey (171069) and another from the Republic of Ireland (179200) (Figure 1). The majority of the cats (35 of 47)

were male (74%), a finding that is consistent with previous studies demonstrating the predominant infection of male cats by FIV [START_REF] Hosie | Prevalence of feline leukaemia virus and antibodies to feline immunodeficiency virus in cats in the United Kingdom[END_REF][START_REF] Reggeti | Feline immunodeficiency virus subtypes A, B and C and intersubtype recombinants in Ontario, Canada[END_REF][START_REF] Hayward | Phylogenetic analysis of feline immunodeficiency virus in feral and companion domestic cats of New Zealand[END_REF], and likely to be associated with aggressive social behaviour in males increasing the risk of transmission via biting [START_REF] Natoli | Bold attitude makes male urban feral domestic cats more vulnerable to Feline Immunodeficiency Virus[END_REF].

The ages were known for 34 of the 47 cats and these ranged from 6 months to 16 years, with only 3 cats less than 1 year of age. Infected cats were at different stages of infection, with clinical signs ranging from asymptomatic to symptomatic; clinical signs recorded on the submission forms included lethargy, pyrexia, anaemia, stomatitis, gingivitis, upper respiratory tract infections, urinary tract infections and central nervous system disorders (Table 1).

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

Phylogenetic Analysis of naturally occurring UK isolates of FIV

A Bayesisan phylogenetic tree was generated based on a 651 nucleotide alignment of the V3-V5 region of the env gene, comprising 47 novel sequences from naturally occurring isolates of FIV together with reference sequences from subtypes A, B, C, D and E (Figure 1). The tree demonstrated that all the novel sequences fell within subtype A with 100% posterior support, except for 171070, which did not cluster with any of the known subtypes.

The genetic distance between any two sequences within subtype A ranged between 0.0 and 0.365, with distances among UK isolates (median: 0.110) being virtually identical to those seen among non-UK subtype A isolates worldwide (median: 0.110). These values were lower than the divergence for other subtypes described in previous studies [START_REF] Sodora | Identification of 3 feline immunodeficiency virus (FIV) env gene subtypes and comparison of the FIV and human immunodeficiency virus type-1 evolutionary patterns[END_REF][START_REF] Duarte | Phylogenetic analysis of Portuguese Feline Immunodeficiency Virus sequences reveals high genetic diversity[END_REF], indicating that subtype A may be less genetically heterogeneous in comparison with other FIV subtypes.

Although the phylogenetic structure of subtype A sequences was poorly resolved, a number of UK sequences clustered with strong support. This included several pairs (180140/180115; 182455/179288; 171536/171069; 171025/179466) with highly similar V3-V5 sequences, despite having being isolated from geographically distinct locations and regions (Figure 1). In each case, these sample pairs had been collected and processed on different days;

hence sample mix-up or cross-contamination was highly unlikely. A high

Recombination analysis

Isolate 171070 was the only British isolate that did not cluster within subtype A and comparisons with sequences representative of subtypes B to E also failed to yield a definitive match. 171070 was isolated from an adopted feral cat and circumstantial evidence leaned towards an introduction to the UK from overseas, possibly from Eastern Europe. Because the virus could not be assigned to any given subtype, we postulated that 171070 may represent a recombinant virus. While further analysis of the sequence using RDP (recombination detection program, [START_REF] Martin | RDP3: a flexible and fast computer program for analyzing recombination[END_REF] and GeneConv [START_REF] Sawyer | Statistical tests for detecting gene conversion[END_REF]found no evidence for a recombination event, bootscan analysis suggested that 171070 may represent a recombinant of subtypes A and C, with a putative breakpoint approximately 260 nucleotides from the start of the env sequence (Figure 2). Phylogenetic trees were constructed based on the criteria of maximum likelihood for the sequences before and after the Within the UK itself, the virus population appears to be highly admixed, as no relationship between spatial and genetic distances was apparent from the data. The absence of spatial clustering, likely a consequence of high human mobility, suggests that any new virus subtype being introduced into the UK has the potential to quickly become spatially disseminated. This also includes neighbouring areas, as two of our sequences, originating from Ireland and Jersey, exhibited very limited genetic divergence from strains circulating within the British mainland.

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

As the only exception to the dominance of subtype A, isolate 171070 was identified as a possible A/C recombinant. This virus was isolated from a feral cat that had been adopted in a suburb near a sea port, raising the possibility that the cat had originated from outside the UK. According to statistics published by the Department for Environment Food and Rural Affairs (DEFRA), 74786 cats entered the UK from 2000 to 2010 under the Pet Travel Scheme (PETS), the system that allows pet dogs, cats and ferrets from a range of European Union (EU) and non-EU countries to enter the UK providing they have been microchipped, vaccinated against rabies and blood tested, and treated for ticks and tapeworms. Cats are not tested for FIV prior to entry into the UK, therefore, animals from areas of the world known to harbour subtype C FIVs e.g. the USA, may enter the UK provided they have met the requirements for entry under the PETS scheme. The subtype A-like part of the 171070 sequence grouped with, but did not fall within, the known UK viruses (Fig. 3). Therefore, while the specific origin of the putative recombinant is not clear, our results suggest that the recombination event did not involve UK viruses and thus likely took place prior to the virus' introduction into the country. Since a potential subtype A parent sequence was not found in our data set, it is also unlikely that recombination arose via PCR-mediated recombination. Furthermore, the high fidelity DNA polymerase used to amplify the env gene has a low error rate and overcomes many of the limitations of Taq polymerase [START_REF] Gilje | Highfidelity DNA polymerase enhances the sensitivity of a peptide nucleic acid clamp PCR assay for K-ras mutations[END_REF]. Interestingly, the position of the putative breakpoint for 171070 (approximately at position 265) was similar to that reported for isolate 214 from New Zealand [START_REF] Hayward | Phylogenetic analysis of feline immunodeficiency virus in feral and companion domestic cats of New Zealand[END_REF] [START_REF] Pistello | Analysis of the genetic diversity and phylogenetic relationship of Italian isolates of feline immunodeficiency virus A c c e p t e d M a n u s c r i p t indicates a high prevalence and heterogeneity of subtype B[END_REF][START_REF] Steinrigl | Phylogenetic analysis of feline immunodeficiency virus in Central Europe: a prerequisite for vaccination and molecular diagnostics[END_REF][START_REF] Duarte | Phylogenetic analysis of Portuguese Feline Immunodeficiency Virus sequences reveals high genetic diversity[END_REF][START_REF] Steinrigl | Phylogenetic analysis suggests independent introduction of feline immunodeficiency virus clades A and B to Central Europe and identifies diverse variants of clade B[END_REF].

Accordingly, the presence of subtype B viruses has been confirmed recently in Turkey [START_REF] Oguzoglu | First molecular characterization of feline immunodeficiency virus in Turkey[END_REF]. Given the physical separation of the UK from mainland Europe, and its nearest neighbors also having subtype A viruses in circulation, the FIV subtypes in the UK cat population may have remained relatively free from the introduction of novel subtypes. The presence of a subtype C recombinant in the UK would suggest a non-European country of origin. The advent of more frequent transport of cats in and out of the UK under the PETS scheme will increase the likelihood of introducing novel subtypes into the UK. Given the prevalence of FIV in the UK is estimated at approximately 5% [START_REF] Hosie | Prevalence of feline leukaemia virus and antibodies to feline immunodeficiency virus in cats in the United Kingdom[END_REF]) and the total cat population at approximately 11 million, this would equate to approximately 550,000 FIVpositive cats in the UK. In comparison, in the USA, the prevalence of FIV has A c c e p t e d M a n u s c r i p t been recorded at 2.5% across North America [START_REF] Levy | Seroprevalence of feline leukemia virus and feline immunodeficiency virus infection among cats in North America and risk factors for seropositivity[END_REF], while approaching 3.6% in the southern states of Louisiana, Mississippi and Texas [START_REF] Levy | Seroprevalence of Dirofilaria immitis, feline leukemia virus, and feline immunodeficiency virus infection among dogs and cats exported from the 2005 Gulf Coast hurricane disaster area[END_REF]. If we were to consider an extreme scenario whereby all the cats entering the UK were to come from a country such as the USA where subtype C was present, assuming no other biases (for example differences in transmission rates), over a ten year period with 74,786 introductions only 2.5% or approximately 1870 FIV positive cats would enter the UK; 1870 FIV positive cats added to the existing 550,000 subtype A positive cats (0.34%).

Thus, in extremis, we might expect to detect one novel introduced subtype in approximately every ~300 cats sampled. Our study examined 47 FIV-positive cats, it is therefore somewhat fortuitous that we detected the novel A/C recombinant. This theoretical consideration does emphasise that it may take a considerable time for the composition of subtypes in the UK to alter.

Since Env, the major target for virus neutralization, displays immense diversity, it has been proposed that it may be necessary to develop regional vaccines that are each based on the sequence of the predominant circulating viruses in particular regions or countries, at least as a preliminary controlling measure [START_REF] Pistello | Analysis of the genetic diversity and phylogenetic relationship of Italian isolates of feline immunodeficiency virus A c c e p t e d M a n u s c r i p t indicates a high prevalence and heterogeneity of subtype B[END_REF][START_REF] Steinrigl | Phylogenetic analysis of feline immunodeficiency virus in Central Europe: a prerequisite for vaccination and molecular diagnostics[END_REF]. There is some evidence that protection can be achieved against an intra-subtype B heterologous FIV challenge using an attenuated FIV vaccine [START_REF] Pistello | AIDS vaccination studies using an ex vivo feline immunodeficiency virus model: protection from an intraclade challenge administered systemically or mucosally by an attenuated vaccine[END_REF]. However this trial was conducted solely with FIV isolates from subtype B and this finding has not been reproduced in trials involving other subtypes or in a wider geographical area. It has been suggested that subtype B isolates may be more ancient and host adapted and as a consequence may be less A c c e p t e d M a n u s c r i p t virulent [START_REF] Bachmann | Genetic diversity of feline immunodeficiency virus: dual infection, recombination, and distinct evolutionary rates among envelope sequence clades[END_REF]. In countries where a more heterogeneous FIV population exists, an effective vaccine would be required to protect against diverse strains which may belong to more than one subtype.

Moreover, in such circumstances, recombination between FIV isolates of different subtypes may occur as a result of co-infection and result in chimaeric strains which may be resistant to vaccine-induced protection. Our results

suggest that such a scenario is less likely for the UK, which appears to be strongly dominated by subtype A virus and thus may a suitable area for a subtype A vaccine trial, should such a vaccine become available.

In conclusion, this study has demonstrated that FIV subtype A, if not exclusively the circulating subtype, predominates in the UK. Further characterisation of the genetic diversity of FIV investigating a greater number of isolates may provide more information about whether subtype A is the only subtype circulating within the UK, since an A/C recombinant isolate has been recognised. Because of the immense diversity of the virus, developing regional or sub-type specific vaccine could be of significant use.

  samples, PBMC collection and DNA extraction. Blood samples were collected from 47 FIV sero-positive samples from naturally infected cats submitted to the Companion Animals Diagnostic Services at the University of

  )TCCCAGTCCACCCTT-3', primers we have found previously to amplify laboratory strains of viral subtypes A, B and C. The FIV env genes were then cloned into the VR1012 eukaryotic expression vector (Vical Inc., San Diego, CA, USA) and their nucleic acid sequences determined using a BigDye Terminator v1.1 kit (Applied Biosystems, Warrington, UK).

A

  ); AB010401 (PTH-BM3); EF154053 (WST01); EF154076 (WST05); D37813 (Sendai 1); U02404 (USCAlemy_01A); U02411 ; X69501 (M2); X69502 (M3); U02418 (brny03); U02419 (boy03); U02420 (glwd03) U02422 (mtex03); D84497 (Lp9); AB010397 (AICO2).Subtype C: AB016025 (TI1); AB016026 (TI2); AB016027 (TI3); AB016028 (TI4); U02392 (pady02); U02393 (pbar01); U02394 (pbar02); U02395 (pbar03); U02397 (pbar07). Subtype D: AB010400 (OKA01D). Subtype E: D84496 (Lp3); D84498 (Lp20); D84500 (Lp24). GenBank accession numbers for sequences generated in this study are HQ456781 to HQ456827 inclusive.

  admixture among UK sequences was also evident from the Mantel test, which failed to reveal a positive correlation between genetic and spatial distances (r = -0.010, CI: -0.090 -0.044; p = 0.539) Notably, British isolates almost never grouped with isolates of other geographic origin; the sole exception being a moderately supported clade that included three UK sequences (171265/171175/172325) and two sequences from continental Europe, DEBAD59 (Germany) and ATVID02 (Austria).

  s c r i p t putative breakpoint. Although the subtype A-like sequence before the putative breakpoint that clustered with subtype A as expected, it failed to fall within the known subtype A. For the subtype C-like portion of the 171070 sequence, assignment was not clear as it did not group with any of the known subtypes (Figure3). It is possible that the latter sequence was derived from a subtype C isolate that is distant from reference sequences used to calculate the tree, perhaps derived from Eastern Europe. Unfortunately, reference sequences from this region were not available.

  the V3-V5 region of the env gene have been used extensively in the genetic subtyping of FIV strains as well as in molecular epidemiological studies. Although it had been suspected that the FIV isolates circulating within the UK largely belong to subtype A, this is the first phylogenetic study to have been performed in the UK. The results of the current study, based on the sequence of 45 naturally occurring isolates of FIV from across the UK, confirm that subtype A predominates. The diversity of subtype A viruses is thereby remarkably high in the UK, rivaling levels of diversity seen for this subtype at a global scale. This suggests a large number of independent introductions of the virus into the UK. At the same time, phylogenetic resolution of the V3-V5 sequences was relatively poor, precluding insights regarding the possible geographic sources of these introductions as well as introductions into other countries from the UK.

Figure 1 .

 1 Figure Legends

Figure 2 .

 2 Figure 2. Bootscan analysis of env V3-V5 from 171070. Bootscan analysis

Figure 3 .

 3 Figure 3. Maximum Likelihood phylogenetic trees based on the nucleotide

  appears likely that this is the first isolation of a subtype C-like isolate of FIV from either the UK or Western Europe. Reviewing the NCBI database confirmed that no FIV sequences from Eastern Europe were available for comparison. Is the predominance of a subtype A in the UK unexpected? It is likely that the dominant subtypes of FIV around the world were established many years ago among geographically isolated populations of cats and may represent multiple introductions of FIV into the cat population. Previous studies have hinted that subtype A viruses may predominate not only in the UK, but in the west of Europe per se, in the UK, France, The Netherlands and Switzerland, while subtype B viruses are more common towards the east and south of Europe in Portugal, Italy and Germany

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

, which could indicate that this site is a recombination hotspot within env V3-V5. Hence it

Acknowledgements

We thank Michael McDonald, Leah, Andrea, Maria and Janet from the Companion Animals Diagnostic Services at the University of Glasgow for kindly providing blood samples from FIV sero-positive cats, and for the helpful assistance which enabled us to perform this study. This study was supported by the Wellcome Trust.