Quantification and molecular characterization of the feline leukemia virusA receptor

A. Katrin Helfer-Hungerbuehler a, Valentino Cattori a, Barbara Bachler a,1, Sonja Hartnack b,Barbara Riond a, Pete Ossent c,2, Hans Lutz a, Regina Hofmann-Lehmann a,⇑aClinical Laboratory, Vetsuisse Faculty, University of Zurich, Zurich, Switzerlandb Section of Epidemiology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerlandc Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland

a r t i c l e i n f o

Article history:Received 25 May 2011Received in revised form 30 July 2011Accepted 17 August 2011Available online 25 August 2011

Keywords:Thiamine transporterReceptorFeLVRetrovirusSusceptibilityReference genes

a b s t r a c t

Virus receptors and their expression patterns on the cell surface determine the cell tropism of the virus,host susceptibility and the pathogenesis of the infection. Feline thiamine transport protein 1 (fTHTR1) hasbeen identified as the receptor for feline leukemia virus (FeLV) A. The goal of the present study was todevelop a quantitative, TaqMan real-time PCR assay to investigate fTHTR1 mRNA expression in tissuesof uninfected and FeLV-infected cats, cats of different ages, in tumor tissues and leukocyte subsets. More-over, the receptor was molecularly characterized in different feline species. fTHTR1 mRNA expressionwas detected in all 30 feline tissues investigated, oral mucosa scrapings and blood. Importantly, identi-fication of significant differences in fTHTR1 expression relied on normalization with an appropriate ref-erence gene. The lowest levels were found in the blood, whereas high levels were measured in the oralmucosa, salivary glands and the musculature. In the blood, T lymphocytes showed significantly higherfTHTR1 mRNA expression levels than neutrophil granulocytes. In vitro activation of peripheral bloodmononuclear cells with concanavalin A alone or followed by interleukin-2 led to a transient increaseof fTHTR1 mRNA expression. In the blood, but not in the examined tissues, FeLV-infected cats tendedto have lower fTHTR1 mRNA levels than uninfected cats. The fTHTR1 mRNA levels were not significantlydifferent between tissues with lymphomas and the corresponding non-neoplastic tissues. fTHTR1 washighly conserved among different feline species (Iberian lynx, Asiatic and Indian lion, European wildcat,jaguarundi, domestic cat). In conclusion, while ubiquitous fTHTR1 mRNA expression corresponded to thebroad target tissue range of FeLV, particularly high fTHTR1 levels were found at sites of virus entry andshedding. The differential susceptibility of different species to FeLV could not be attributed to variationsin the fTHTR1 sequence.

� 2011 Elsevier B.V. All rights reserved.

1. Introduction

Feline leukemia virus (FeLV) infection is initiated by the interac-tion of the viral envelope (Env) glycoprotein surface unit with a spe-cific cellular receptor. Therefore, the expression pattern of specificcell surface molecules determines the target cell tropism of FeLV.

After virus entry into the host cell and expression of the viral surfaceunit, the specific cellular receptor is typically down-modulated, so itis unavailable for use by another virion (Hunter, 1997; Overbaughet al., 2001; Weiss, 1993). This process is known as superinfectioninterference and can be mediated by mechanisms including inter-nalization of the receptor, disruption of the transport of the receptorto the cell surface or competition with the receptor binding domain(Hunter, 1997). Exogenous FeLV isolates are grouped into threemajor groups according to their superinfection interference andneutralization patterns, FeLV-A, -B, -C (Jarrett et al., 1973a; Sarmaand Log, 1971, 1973; Sarma et al., 1975). These subgroups are linkedto specific feline diseases (Hardy, 1993; Neil et al., 1991). Each FeLVsubgroup has defined receptor requirements. The variable region Awithin the N-terminal half of the Env is predominantly involved inmediating receptor binding (Rigby et al., 1992). Small changes inthe variable region Awere sufficient to yield an env thatwas capableof using a different receptor (Mazari et al., 2009; Sarangi et al.,

1567-1348/$ - see front matter � 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.meegid.2011.08.015

⇑ Corresponding author. Address: Clinical Laboratory, Vetsuisse Faculty, Univer-sity of Zurich, Winterthurerstr. 260, 8057 Zurich, Switzerland. Tel.: +41 44 635 8322; fax: +41 44 635 89 23.

E-mail addresses: khungerbuehler@vetclinics.uzh.ch (A. Katrin Helfer-Hungerbuehler), vcattori@vetclinics.unizh.ch (V. Cattori), barbara_bachler@dfci.harvard.edu (B. Bachler), sonja.hartnack@access.uzh.ch (S. Hartnack), briond@vetclinics.uzh.ch (B. Riond), hlutz@vetclinics.uzh.ch (H. Lutz), rhofmann@vetclinics.uzh.ch(R. Hofmann-Lehmann).

1 Present address: Cancer Immunology & AIDS, Dana-Farber Cancer Institute,Harvard Medical School, Boston, MA, USA.

2 Deceased.

Infection, Genetics and Evolution 11 (2011) 1940–1950

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2007).The receptor of the most transmissible form of FeLV, FeLV-A,has been identified using a gene transfer approach (Mendoza et al.,2006). Because of its high sequence identity with human thiaminetransport protein 1, the receptor was designated feline thiaminetransport protein 1 (fTHTR1), and it is the protein product of theSLC19A2 gene. Thiamine, vitamin B1, is a water soluble essentialmicronutrient, and the active form of thiamine, thiamine diphos-phate, serves as a cofactor for several enzymes involved primarilyin carbohydratemetabolism (Said et al., 2001; Singleton andMartin,2001). Physiologically, thiamine transporters 1 and 2 are used inmammals for intestinal absorptionof thiamine and for the reabsorp-tion of thiamine in the kidney to prevent loss via urine.

Mutations in the THTR1 gene have been linked to thiamine-responsive megaloblastic anemia (Diaz et al., 1999; Labay et al.,1999; Raz et al., 2000). Because this disease has been described insome cats suffering from FeLV-infection, it has been speculated thatFeLV-A infection may disrupt the thiamine transport function(Mendoza et al., 2006). We hypothesized that the FeLV-A receptorexpression patterns in different host tissues may influence thepathogenesis of the infection and that the virusmay lead to changesin the receptor transcription in different tissues. In addition,wehypothesized that receptor expressionmayvary at different timepoints during a cat’s life, thereby altering the susceptibility of the catto exogenous FeLV. Furthermore, sequence variations in the FeLV-Areceptor in different feline speciesmay contribute to the differentialsusceptibility to FeLV infection. Therefore, the purpose of the pres-ent study was to (i) develop and optimize a TaqMan real-time PCRassay for the quantification of fTHTR1 mRNA expression levels, (ii)quantify fTHTR1 mRNA expression in different tissues from unin-fected, clinically healthy cats and cats experimentally infected withFeLV-A and (iii) compare the fTHTR1 sequences in various wildfelids.

2. Materials and methods

2.1. Sample collection from domestic cats

All domestic cats included in this study were in studies officiallyapproved by the veterinary office of the Swiss Canton of Zurich(197/89, 43/90, 66/91, 131/91, 329/91, 56/95, 30/2003, 99/2007and 160/2010). The cats were kept in groups under optimal etho-logical conditions as previously described (Geret et al., 2011a;Museux et al., 2009). In order to assess fTHTR1 mRNA expressionlevels, tissue samples from uninfected, clinically healthy cats(Kessler et al., 2009) and FeLV-A/Glasgow-1 infected cats wereexamined (Geret et al., 2011b; Hofmann-Lehmann et al., 2006;Lehmann et al., 1991; Tandon et al., 2008). All cats were eutha-nized for reasons unrelated to the present study. The uninfectedand the FeLV-infected cats were age-matched. At the time ofeuthanasia, the cats ranged in age from 1.2 to 9.6 years (med-ian = 3.1 years; median of uninfected cats: 2.9 years; median ofFeLV-infected cats = 3.8 years). The FeLV-positive cats had been in-fected with FeLV between 0.9 and 8.5 years (median = 3.1 years)before euthanasia.

The following 30 tissues were collected: adrenal gland, pan-creas, parathyroid, thyroid, bone marrow, mesenteric lymph node(ln.), popliteal ln., sternal ln., submandibular ln., spleen, tonsil, thy-mus, parotid gland, mandibular gland, duodenum, ileum, jejunum,colon, rectum, brain, ischiadic nerve, spinal cord, kidney, urinarybladder, aorta, diaphragm, femoral muscle, myocardium, lungand liver. The tissues were collected from four uninfected, clini-cally healthy cats. From the FeLV-infected cats, six samples weretaken from each tissue, with the exception of the parathyroid(n = 3); adrenal gland and pancreas (n = 4); submandibular ln., thy-mus, jejunum, ischiadic nerve, spinal cord and femoral muscle

(n = 5); thyroid, mesenteric ln., mandibular gland, duodenum,ileum, brain, kidney and urinary bladder (n = 7); and liver (n = 8).All tissues were snap-frozen following collection and stored at�80 �C until nucleic acids were extracted. In addition, EDTA-anti-coagulated whole blood samples and oral mucosa scrapings werecollected from seven healthy, uninfected, 14-week-old, SPF blooddonor and seven approximately 3-year-old, FeLV-infected cats(Major et al., 2010; Tandon et al., 2008). Complete hemogramswere performed using a Sysmex XT-2000iV (Sysmex Corporation,Kobe, Japan) (Weissenbacher et al., 2011). For the white blood celldifferential, microscopic blood smear evaluation was performed, asdescribed (Novacco et al., 2011).

To study fTHTR1 mRNA expression in leukocyte subsets, sam-ples were analyzed that had been collected from five long-termpersistently FeLV-infected (#10, #19, #32, #38 and #43) and fiveFeLV exposed but nonantigenemic, nonviremic cats (#11, #12,#20, #49 and #69) and sorted by fluorescence activated cell sortingas described previously (Pepin et al., 2007). Briefly, cell subsetswere sorted from Percoll purified PBMC on a BD FACS Aria (BD Bio-science, Allschwil, Switzerland) using the following antibodies: amouse anti-feline CD4 antibody (Fel7, Southern Biotech, Birming-ham, Alabama, USA); a mouse anti-feline CD8 antibody (FT2,Southern Biotech); a fluoresceinisothiocyanate (FITC)-conjugatedmouse anti-feline CD5 antibody (f43, Southern Biotech; recogniz-ing T lymphocytes) and a Peridinin chlorophyll-a Protein (PerCP)-conjugated rat anti-mouse CD45R/B220 antibody (BD, Bioscience;recognizing B lymphocytes). Because the number of monocytesin the peripheral blood is low, they were enriched from Percollpurified PBMC using magnetic beads and a CD14 antibody (TüK4,Dako Cytomation, Zug, Switzerland) prior to cell sorting. Finally,granulocytes were stained from defibrinated blood after blockingthe Fc receptors using mouse anti-feline granulocyte antibodies(CL35A, VMRD Inc., Pullman, WA, USA).

Additionally, fTHTR1mRNA expression was quantified in tissuesfrom three FeLV-infected cats with terminal lymphoma (Hofmann-Lehmann et al., 2007; Tandon et al., 2008). At the time of euthana-sia, the cats ranged in age from2.3 to 3.8 years (median = 3.1 years),and they had been infectedwith FeLV-A/Glasgow-1 for between 1.9and 3.4 years (median = 2.8 years). The following neoplastic tissueswere collected: from one cat, mesenteric ln., sternal ln., bladder,diaphragm and ovary; from a second cat, mesenteric ln., ileumand kidney; and from a third cat, kidney. All lymphomas were con-firmed histologically. For each lymphoma tissue sample a controlsample was collected from the same cat and tissue but withoutapparent lymphoma as determined macroscopically and verifiedhistologically.

In order to assess the potential influence of the age of the cat onthe fTHTR1 expression level, EDTA-anticoagulated whole bloodand oral mucosa scrapings were collected from five uninfected,healthy SPF cats (Major et al., 2010). Samples were collected atthe age of 10, 12, 14, 17, 25, 31 and 37 weeks.

2.2. Samples from wild felids

The following samples were available from wild felids fromunrelated studies: lung and kidney samples from two Iberianlynxes (Lynx pardinus) from southern Spain, a spleen sample froma jaguarundi (Herpailurus yagouaroundi) from Brazil and a brainsample from an African lion (Panthera leo bleyenberghi) from Bots-wana. In addition, a spleen sample from an Asiatic lion (Pantheraleo persica) from the zoo of Zurich and a lung and kidney samplefrom two European wildcats (Felis silvestris silvestris) from theWildpark Langenberg in Switzerland were available. All sampleswere retrieved during necropsy performed for unrelated reasonsand immediately stored at �80 �C until RNA extraction.

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2.3. Cell culture and stimulation

Heparin anticoagulated blood samples (24 ml) were collectedfrom four SPF cats. Peripheral blood mononuclear cells (PBMC)were Ficoll-Hypaque purified (Histopaque�-1077, Sigma–Aldrich,Buchs, Switzerland) as described (Robert-Tissot et al., 2011).They were grown in RPMI-1640 medium (Sigma) containing10% fetal calf serum (BioConcept, Allschwil, Switzerland), 1%penicillin–streptomycin (Gibco, Invitrogen, San Diego, CA, USA),1% L-glutamine (Gibco). They were kept either unstimulated(medium alone) or stimulated with concanavalin A (Con A;10 lg/ml; Sigma–Aldrich), 50 U/ml recombinant human interleu-kin-2 (IL-2; Sandoz Pharmaceuticals AG, Cham, Switzerland) or acombination thereof. For the latter, Con A was added at the ini-tiation of the cultures and IL-2 was added after 24 h as described(Robert-Tissot et al., 2011). Cells were harvested after 0, 4, 8, 24,48 and 72 h for all cultures and after 28 and 32 h for the Con Aand IL-2 combination and analyzed for fTHTR1 mRNAexpression.

2.4. RNA extraction from blood, oral mucosa scrapings and tissuesamples

RNA was extracted from 1 ml EDTA-anticoagulated whole bloodsamples within 60 min of collection or from PBMC directly afterharvesting using the QIAamp Blood Mini Kit (Qiagen, Hombrechti-kon, Switzerland) and stored at �80 �C until further use. RNA fromleukocyte subsets had been extracted in a previous study (Pepinet al., 2007). Oral mucosa scrapings were collected, and RNA wasextracted similar to saliva swabs, as previously described (Cattoriet al., 2009; Gomes-Keller et al., 2006) with the following modifi-cations: oral mucosa scrapings were obtained using swabs afterroughening the oral mucosa with a sterile scalpel. In the labora-tory, 600 ll of RLT buffer with b-mercaptoethanol was added.The samples were incubated at 37 �C for 10 min and sporadicallyvortexed and centrifuged as described (Gomes-Keller et al.,2006). RNA extraction was performed using the QIAamp BloodMini Kit (Qiagen). Tissues samples collected upon necropsy werehomogenized as previously described (Kessler et al., 2009) andprocessed using the RNeasy Mini Kit (Qiagen), according to themanufacturer’s instructions. For all RNA extractions, negative con-trols consisting of 100 ll of phosphate buffered saline wereprepared with each batch to monitor for cross-contamination.

2.5. First-strand cDNA synthesis

All RNA samples, with the exception of those for the age-dependency study, the cell subsets and the PBMC stimulation, werereverse transcribed into cDNA. First-strandcDNAwas synthesized induplicate using the High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Rotkreuz, Switzerland) and random primersaccording to the manufacturer’s instructions.

2.6. Probe and primer design for the fTHTR1 TaqMan assay

The TaqMan probe and primers were designed using PrimerExpress software (version 3, Applied Biosystems; Table 1). Infor-mation regarding the sequence and the gene organization wereretrieved from GenBank (http://www.ncbi.nlm.nih.gov). In orderto reduce possible amplification of gDNA, the assay was designedto span an exon–exon junction of the SLC19A2 gene. Optimal posi-tioning of the primers and the probe was based on sequence align-ments of the feline fTHTR1 [GenBank: DQ391281] with the human[GenBank: NM_006996], murine [GenBank: NM_054087] and ratTHTR1 [GenBank: BC099811]. All DNA and RNA samples were

quantified on a Rotor-Gene 6000 real-time rotary analyzer(Corbett, Mortlake, Australia).

2.7. TaqMan fluorogenic real-time PCR assay

In order to determine fTHTR1 expression in cDNA samples aTaqMan fluorogenic real-time PCR assay was designed. Primerand probe concentrations for the newly designed TaqMan real-time assay were optimized and tested for potential amplificationof pseudogenes as described (Kessler et al., 2009). The optimizedPCR reactions contained 12.5 ll TaqMan Fast Universal PCR MasterMix (Applied Biosystems), a final concentration of 300 nM forwardand 900 nM reverse primers, 200 nM of fluorogenic probe (Micros-ynth, Balgach, Switzerland; Table 1) and 5 ll of template in a totalvolume of 25 ll. The thermocycling conditions consisted of an ini-tial denaturation of 20 s at 95 �C, followed by 45 cycles of 95 �C for3 s and 60 �C for 45 s.

2.8. Construction and production of an fTHTR1 cDNA standard forabsolute quantification

To generate a standard template for absolute quantification,RNA was isolated from feline embryonic fibroblast (FEA) cells thathad been cultured until confluence as described previously (Tan-don et al., 2005). RNA was isolated from 6 � 106 cells using theRNeasy Mini Kit (Qiagen). First strand cDNA was produced usingthe Euroscript Reverse Transkriptase (Eurogentec). A 200-bpsequence in the 50 portion of the SLC19A2 gene containing the87-bp long TaqMan template was amplified using fTHTR1-specificprimers (Table 1) and cloned into the TOPO TA Cloning vector(Invitrogen, Basel Switzerland). The insert in selected clones wasverified by sequencing (Microsynth). The fTHTR1 reference plas-mid was linearized by restriction digestion using the enzyme KpnI(Roche, Rotkreuz, Switzerland), gel purified (Gen Elute PCR Clean-Up Kit, Sigma–Aldrich, Buchs, Switzerland) and the copy numberwas determined spectrophotometrically (NanoDrop ND-1000;Nanodrop Technologies, Witec, Littau, Switzerland). Tenfold serialdilutions of the standard templates in 30 lg/ml carrier salmonsperm DNA (Invitrogen) were aliquoted and frozen at �20 �C.The fTHTR1 expression in the tissues was normalized to two refer-ence genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Table 1Oligonucleotides used in this study.

Assay Oligo Sequence Amplicon size(bp)

Production of standarda

fTHTR1 Forward GAG CCC TTC CTG ACC CCT TA 200Reverse AGC AGC ATG AAC CAT GTG AC

Real-time PCR and RT-PCR assayfTHTR1 Forward GAG CCC TTC CTG ACC CCT TA 87

Probe CGG ACA AGA ACC TGA CGG AGAGGG AGb

Reverse AGT CCA TAC TGG GTA AAT TTC ATTGAA

fTHTR1 sequencinga

176F Forward CTC CGC ACC GCG AAT GGA TG192F Forward GAT GTG CCC GGC CCG GTG TC579F Forward ATC GCC ACA GCC ACT GAA ATC741F Forward CTG TTC AGC CTG AAT GTC741R Reverse GAC ATT CAG GCT GAA CAG837R Reverse GGT GAA AGA ACA GGC TCT TC1037R Reverse GTA GCA CAT CAG GAA GTC1067R Reverse CAC AGA CCA GCA GAG CAG AG1789R Reverse TAG GAG CAC AAG GTA GAG

a All primers were used at a final concentration of 500 nM.b 50 FAM (6-carboxyfluorescein)/30 TAMRA (5-(6) carboxytetramethylrhodamine).

1942 A. Katrin Helfer-Hungerbuehler et al. / Infection, Genetics and Evolution 11 (2011) 1940–1950

(Leutenegger et al., 1999), the most commonly used referencegene, and ribosomal protein S7 (RPS7), the best reference genefor various feline tissues (Kessler et al., 2009). In addition, bloodsamples were also normalized to the expression of tryptophan5-monooxygenase activation protein, zeta polypeptide (YWHAZ),the optimal reference gene for feline blood samples (Kessleret al., 2009).

2.9. TaqMan fluorogenic real-time RT-PCR assay

In order to assess the fTHTR1mRNA levels in the age-dependencystudy, the cell subsets and the PBMC stimulation assays, 5 ll of RNAwasamplified ina total reactionvolumeof25 ll using theEuroscriptReverse Transcriptase (Eurogentec, Seraing, Belgium), with primersand probe at the concentrations mentioned above. Reverse tran-scription for 30 min at 48 �C was followed by a denaturation stepof 95 �C for 10 min and 45 cycles of 95 �C for 15 s and 60 �C for 60 s.

2.10. Construction and production of an fTHTR1 RNA standard forabsolute quantification

The linearized fTHTR1 reference plasmid was subjected toin vitro transcription using the Large Scale T7 Transcription Kit(Novagen, Juro Supply, Lucerne, Switzerland), and the generatedRNA was purified using the RNeasy Mini Kit (Qiagen). In vitro tran-scribed RNA was quantified using a fluorescent nucleic acid stain,RiboGreen, as previously described (Tandon et al., 2005). RNA copynumbers were calculated, and 10-fold serial dilutions of the stan-dard template in 30 lg/ml of carrier yeast tRNA (Sigma) werealiquoted and frozen at �80 �C.

2.11. Efficiency, analytical sensitivity, linear range and precision of thefTHTR1 real-time PCR and RT-PCR assays

The efficiency of the newly designed assays was calculated aspreviously described (Klein et al., 1999) using the equationE = (10(�1/slope)) � 1. The analytical sensitivity of the newlydesigned fTHTR1 real-time PCR and RT-PCR assays was determinedin an endpoint dilution experiment using 10 replicates of dilutionscontaining 103, 102, 101, and 100 standard template copies perreaction. The sensitivity of the assay is given by the highest dilu-tion in which at least 7 of 10 replicates are positive (Lockeyet al., 1998). The linear range of amplification and the precisionwere determined using 10-fold serial dilutions of the linearizedreference plasmid or a 10-fold serial dilution of standard RNA tem-plate in carrier RNA. Intra-run (n = 10) and between-run (n = 10)analytical performances of the real-time PCR and RT-PCR measure-ments were determined using a standard template copy numberthat was characteristic of the fTHTR1 expression levels (104 cop-ies/reaction for cDNA and 3 � 104 copies/reaction for RNA).

2.12. FeLV viral loads

FeLV viral loads were quantified from cDNA using TaqMan real-time PCR (U3 region), as previously described (Tandon et al., 2005).

2.13. Sequencing of the fTHTR1 gene in different feline species

For the analysis of the almost full-length fTHTR1 cDNA of thedomestic cat and the Iberian lynx, fTHTR1 cDNA obtained from tis-sue samples was amplified using PCR with primers 192F and1789R (Table 1), resulting in a 1597-bp fragment. It was necessaryto amplify the fTHTR1 cDNA of the lion, wildcat and jaguarundi intwo parts: the 50 region was amplified with the primers 176F and1067R, and the 30 region was amplified with the primers 741F and1789R (Table 1), resulting in an 891-bp and a 1048-bp fragment,

respectively. To ensure high fidelity of amplification, the PCR wasperformed using Phusion polymerase and HF buffer (Finnzyme,Ipswich, UK).

PCR products were either sequenced directly after purificationusing the GenElute PCR Clean-Up Kit (Sigma) or separated byagarose gel electrophoresis, excised from the gel, purified usingthe GenElute Gel Extraction Kit (Sigma) and cloned into thepCRII-TOPO vector (Invitrogen) according to the manufacturer’sinstructions. The obtained clones were then sequenced eitherin-house or by Microsynth (Balgach, Switzerland) using variousprimers (Table 1).

2.14. Data evaluation and statistics

Statistical analyses were firstly performed using GraphPadPrism for Windows, Version 4.03 (GraphPad software, San Diego,CA). Differences among three and more groups were analyzed bythe non-parametric Kruskal-Wallis one-way ANOVA by Ranks(pKW) for unpaired samples or the non-parametric Friedman testfor paired samples (pF) followed by the Dunn’s Multiple Compari-son test (pD). Differences between two groups were tested for sig-nificance using the non-parametric Mann-Whitney U-test forunpaired samples (pMWU) or the Wilcoxon signed rank test forpaired samples (pW). A p-value less than 0.05 was considered sig-nificant. Since observations within a cat were not independent,an adjustment became necessary to avoid inflated p-values. Statis-tical analysis using a repeated measures ANOVA was performed inNCSS, and mixed effects models were analyzed in R (R Develop-ment Core Team, 2010) with the package nlme (Pinheiro et al.,2009). The values for the expression levels were log transformed.The explanatory variables ‘‘FeLV status’’ and ‘‘tissue’’ were ana-lyzed as fixed or random effects and ‘‘cat’’ as random effect. Fol-lowing the repeated measures ANOVA, the Tukey-KramerMultiple-Comparison Test (TKMC-Test) was used as a multiplecomparison test to examine all pairwise differences betweenmeans.

2.15. Nucleotide sequence accession numbers

The following sequences were submitted to GenBank: fTHTR1from Iberian lynx [HQ018798], from Brazilian jaguarundi[HQ018797], from Indian lion [HQ018800] and from Europeanwildcat [HQ018799].

3. Results

3.1. fTHTR1 real-time PCR assay design

A real-time PCR assay was developed to detect and quantifymRNA encoding the FeLV-A receptor, fTHTR1. The fTHTR1 real-time PCR assay did not amplify gDNA (data not shown).

3.2. Linear range of amplification and analytical sensitivity of thefTHTR1 PCR assay

The fTHTR1 real-time PCR and RT-PCR assays showed linearityover eight orders of magnitude. The highest dilution that stillresulted in a positive signal in the PCR assay contained an averageof one copy of the standard per reaction; in an endpoint dilutionexperiment, 7 out of 10 replicates of this dilution were positive.The lower limit of detection of the real-time RT-PCR was 300 cop-ies of the RNA standard per reaction; in an endpoint dilution exper-iment, 10 out of 10 replicates of this dilution were positive.

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3.3. Amplification efficiency and precision of the real-time PCR andRT-PCR assay

The calculated amplification efficiency of the real-time PCR andRT-PCR assays was 99% and 91%, respectively. The real-time PCRand RT-PCR assays displayed a coefficient of variation of 0.92%and 1.10% for the intra-run precision analysis and of 0.95% and5.31% for the between-run analysis, respectively.

3.4. fTHTR1 mRNA expression levels in various tissues of healthy cats

Using the real-time PCR assay, fTHTR1 cDNA was detectable inall samples investigated (Fig. 1A and B). However, the levels werelow when compared with those of the reference genes RPS7 andGAPDH (10- to 10,000-fold lower).

When either RPS7 or GAPDH were used for normalization, thefTHTR1 expression level varied significantly among the differenttissues (Fig. 1A and B, pKW < 0.0001). After normalization to RPS7,fTHTR1 expression was significantly higher in the oral mucosa thanin the blood (pD < 0.001), the spleen (pD < 0.01) and the bone mar-row (pD < 0.05). Furthermore, fTHTR1 expression in the blood wassignificantly lower than the expression in the diaphragm, femoralmuscle, myocardium (pD < 0.01) and the mandibular gland(pD < 0.05) (Fig. 1A). Normalization to GAPDH resulted in partiallycongruent results. fTHTR1 expression was significantly lower inthe blood when compared with oral mucosa (pD < 0.001), pancreas,parotid gland, mandibular gland, kidney, lung (pD < 0.01) and ischi-adic nerve (pD < 0.05). In addition, fTHTR1 expression was signifi-cantly higher in the oral mucosa when compared with thefemoral muscle (pD < 0.01), diaphragm and liver (pD < 0.05). Finally,expression was higher in the pancreas than in the femoral muscle(pD < 0.05) (Fig. 1B).

In order to account for the different origin of some tissues (notall tissues were from the same four healthy cats), a subset of thedata was reanalyzed using a linear mixed effects model (likeli-hood ratio test). Balanced data from 27 tissues with the explana-tory variable ‘‘cat’’ as a random effect was used to determine theeffects of different tissues. Following normalization to RPS7, theTKMC-Test revealed significantly higher fTHTR1 expression inthe femoral muscle (24 out of 27 tissues) and the diaphragm(14 out of 27 tissues) when compared with the other collectedtissues. Normalization to GAPDH resulted in a significantly higherfTHTR1 expression level in the parotid gland (25 out of 27 tis-sues) and the mandibular gland (14 out of 27 tissues). Becausethe oral mucosa scrapings and blood samples could not be in-cluded in these statistical analyses, they were analyzed using anon-paired nonparametric test. Significantly higher fTHTR1expression was confirmed in the oral mucosa compared withother tissues (normalized to RPS7: pMWU < 0.05 for 25 out of 29tissues; normalized to GAPDH: pMWU < 0.05 for 24 out of 29 tis-sues). In addition, significantly lower fTHTR1 expression was con-firmed in the blood compared with the other collected tissues(normalized to RPS7: pMWU < 0.05 for 26 out of 29 tissues; nor-malized to GAPDH: pMWU = 0.0061 for all 29 tissues). Significantdifferences were also detected between the oral mucosa and theblood when normalized to RPS7, GAPDH or YWHAZ using a non-parametric, paired test (pW = 0.0156).

Finally, when fTHTR1 expression was inspected according tofunctional groups of tissues (classified as depicted in Fig. 1), highlevels were seen in the musculature, gastrointestinal and urinarytissues, whereas the lowest expression levels were found in thelymphoid tissues and the blood (Fig. 2A, normalized to RPS7). Afternormalization to GAPDH, the urinary, endocrine and gastrointesti-nal tissues were highest, whereas the blood levels were lowest(Fig. 2B).

3.5. Comparison of fTHTR1 expression in uninfected and FeLV-infectedcats

fTHTR1 mRNA expression was compared between tissues fromuninfected (Fig. 1A and B) and FeLV-infected cats (Fig. 1C and D). Ina mixed effects model and using the explanatory variables ‘‘tissue’’and ‘‘cat’’ as random effects, FeLV status did not have a significanteffect independent of the reference gene used for normalization.Moreover, no significant differences were observed between thetwo groups for any of the tissues when values were normalizedto RPS7 (pMWU > 0.05). In contrast, normalization to GAPDH re-sulted in significantly higher fTHTR1 expression in FeLV-infectedcats in the diaphragm (pMWU = 0.01) and the femoral muscle(pMWU = 0.04) and significantly lower expression in the kidney(pMWU = 0.02) when compared with uninfected healthy cats. Whentissues from FeLV-infected cats were examined, no significantassociation between FeLV viral load and receptor expression wasfound.

The fTHTR1 expression was significantly lower in the bloodsamples (normalized to YWHAZ, the optimal reference gene forblood samples) from FeLV-infected than from FeLV-uninfected cats(pMWU = 0.0070, Fig. 2C). Haematological analysis revealed thatFeLV-infected cats had significant lower total white blood cellcounts (pMWU = 0.0026) and lymphocyte counts (pMWU = 0.0012)compared with FeLV-uninfected cats.

A significant difference was found among fTHTR1 mRNAexpression levels of different leukocyte subsets (pF = 0.0050): Tlymphocytes (CD5+) had significantly higher expression levelsthan granulocytes (pD < 0.01, Fig. 2D). The FeLV status of the cats(p27 positive and FeLV-exposed but nonantigenemic, nonviremiccats) had no significant effect on the fTHTR1 mRNA expression lev-els of the leukocyte subsets (pMWU > 0.05).

3.6. fTHTR1 expression in PBMC after in vitro stimulation

In order to investigate the fTHTR1 expression in leukocytes afteractivation, PBMC were purified and cultured in the presence of ConA, IL-2 or a sequential combination thereof (Fig. 3). IL-2 alone didnot significantly increase fTHTR1 expression compared with med-ium alone. In contrast, a significant transient increase of fTHTR1expression was observed 8 h after stimulation with Con A com-pared with IL-2 and medium alone (pMWU = 0.0381). Moreover,fTHTR1 expression tended to increase in the PBMC stimulated withthe combination of Con A and IL-2 72 h after the initiation of theculture, when compared with the supplements alone and medium(pMWU = 0.0503).

3.7. Tumor samples

The mRNA expression of fTHTR1 was compared in nine neoplas-tic tissues (all lymphomas) and their corresponding non-tumor tis-sues. No significant differences in fTHTR1 expression weredetected after normalization to either RPS7 or GAPDH (data notshown).

3.8. Association between age and fTHTR1 expression

To determine whether the high susceptibility of young cats toFeLV is associated with an increased expression of fTHTR1, fTHTR1expression was quantified in whole blood and oral mucosa scrap-ings, a primary site of FeLV entry, collected from five young catsover a period of 25–27 weeks (6–7 collections). In the blood sam-ples, fTHTR1 expression tended to decline (linear mixed effectsmodel with ‘‘cat’’ as random effect using balanced data, weeks10–37; p = 0.02, Fig. 4), while in the oral mucosa fTHTR1 expres-sion tended to increase (weeks 12–37, p = 0.002, Fig. 4). The

1944 A. Katrin Helfer-Hungerbuehler et al. / Infection, Genetics and Evolution 11 (2011) 1940–1950

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Endocrine Lymphoid Gastrointestinal Nerve Urinary Muscle Others Blood

Endocrine Lymphoid Gastrointestinal Nerve Urinary Muscle Others Blood

Endocrine Lymphoid Gastrointestinal Nerve Urinary Muscle Others Blood

Fig. 1. fTHTR1 mRNA expression in tissues from uninfected and FeLV-infected cats. fTHTR1 expression levels in 30 different tissues collected during necropsy and in ex vivoblood and oral mucosa scrapings from uninfected and FeLV-infected cats were quantified using real-time PCR. (A) fTHTR1 cDNA copy numbers from uninfected cats aredepicted normalized to 1000 RPS7 cDNA copies (kRPS7). (B) fTHTR1 cDNA copy numbers from uninfected cats were normalized to 1000 GAPDH cDNA copies (kGAPDH). (C)fTHTR1 cDNA copy numbers from FeLV-infected cats, normalized to kRPS7. (D) fTHTR1 cDNA copy numbers from FeLV-infected cats, normalized to kGAPDH. The data areshown as box plots, and the boxes extend from the 25th to the 75th percentile. The horizontal line represents the median, and the whiskers extend from the smallest to thelargest value. The grey and white shading emphasize the division into functional groups of tissues.

A. Katrin Helfer-Hungerbuehler et al. / Infection, Genetics and Evolution 11 (2011) 1940–1950 1945

approximately 100- to 1000-fold greater fTHTR1 expression levelin the oral mucosa compared with blood was confirmed through-out the entire observation period.

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Fig. 2. fTHTR1 expression according to tissue groups and FeLV status in blood. A comparison of the fTHTR1 mRNA expression levels among functional groups of tissues fromuninfected cats, normalized to (A) 1000 RPS7 cDNA copies (kRPS7) or (B) 1000 GAPDH cDNA copies (kGAPDH). (C) A comparison of fTHTR1 mRNA expression levels betweenblood samples from uninfected and FeLV-infected cats, normalized to 1000 YWHAZ cDNA copies (kYWHAZ). The fTHTR1 expression levels were tested for statisticallysignificant differences using the Mann-Whitney U-test (pMWU as indicated). (D) fTHTR1 mRNA expression levels normalized to kGAPDH of leukocyte subsets sorted byfluorescence activated cell sorting. GAPDH was detectable in all leukocyte subsets, while fTHTR1 was undetectable in many of the granulocytes, monocytes and CD8+samples. To calculate the load in these samples, fTHTR1 was set to 0.1 copies. Loads were tested for statistical differences by the Friedman test (pF as indicated) and the Dunn’spost test (⁄⁄p < 0.01 as indicated). CD4+, CD4+ lymphocytes; CD8+, CD8+ lymphocytes; CD5+, T lymphocytes; B, B lymphocytes; Mc, monocytes; Gc granulocytes. The data (A,B and D) are shown as box plots, and the boxes extend from the 25th to the 75th percentile. The horizontal line represents the median, and the whiskers extend from thesmallest to the largest value.

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Fig. 3. fTHTR1 expression in PBMC after in vitro stimulation. fTHTR1 expression wascompared in leukocytes after stimulation with Con A, IL-2, a sequential combinationthereof or incubated with medium alone for up to 72 h (x axes). The fTHTR1 RNAcopy numbers were normalized to 1000 copies of GAPDH RNA (kGAPDH).Experiments were conducted with PBMC from four cats and the mean (m) isdepicted. fTHTR1 expression was tested for significant differences betweenparticular stimulation conditions by the Mann-Whitney U-test. Significantlyincreased expression was found at 8 h after stimulation with Con A (marked byan asterisk; pMWU = 0.0381) and a tendency of higher levels was found at 72 h afterstimulation with Con A followed by IL-2 (marked by a circle; pMWU = 0.0503).

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Fig. 4. Correlation between age and fTHTR1 expression in blood and oral mucosascrapings. Expression levels of fTHTR1 in blood and oral mucosa scrapings from catsobserved from the age of 10–37 weeks. The fTHTR1 RNA copy numbers arenormalized to 1000 GAPDH RNA copies (kGAPDH). For the oral mucosa, only foursamples were available at week 10; therefore, this time point was not included inthe statistical analysis using paired samples.

1946 A. Katrin Helfer-Hungerbuehler et al. / Infection, Genetics and Evolution 11 (2011) 1940–1950

3.9. fTHTR1 sequence in different felids

The fTHTR1 cDNA of different feline species, including thedomestic cat, the European wildcat, the Asiatic and African lion,the Brazilian jaguarundi and the Iberian lynx, was sequenced.The fTHTR1 sequences were highly conserved, sharing at least99% nucleotide and amino acid identity with the fTHTR1 sequenceof the domestic cat (Table 2).

4. Discussion

The present study is the first to develop a real-time TaqManassay for the quantification of the feline FeLV-A receptor, fTHTR1,and to apply this method to measure fTHTR1 expression levels invarious feline tissues. mRNA expression of the FeLV-A receptorwas identified in all 30 tested tissues, the blood samples and theoral mucosa scrapings. The detection of fTHTR1 expression in abroad variety of tissues is in accordance with the observation thatFeLV-A can infect many cat organs (Hoover et al., 1977; Rojko et al.,1979) and - because fTHTR1 is a thiamine transporter – it is consis-tent with the fact that thiamine is an essential vitamin. The fTHTR1expression was highest at the presumed sites of FeLV-A entry andshedding (the oral mucosa and the salivary glands), and lowest inthe peripheral blood. A trend towards lower fTHTR1 expression inblood but not in the tissues of infected cats was detected whencompared with uninfected cats.

Gene expression analysis by real-time PCR is highly dependenton co-analysis of suitable reference genes for accurate normaliza-tion of the results. Previous experiments in healthy cats haverevealed that no single gene was optimal for accurate normaliza-tion in all tissues (Kessler et al., 2009). It could be argued that, ide-ally, the best reference gene for each tissue should be applied;however, that would not allow for comparison of expression levelsamong the different tissues. Overall, for feline tissues RPS7 wasrated the best among 10 common mammalian reference genes(Kessler et al., 2009). In the present study, we therefore usedRPS7 as a reference gene. In addition, for comparison we measuredGAPDH, which is less well suited but has been frequently appliedin the field. Finally in blood samples, we also used YWHAZ, whichwas found optimal for normalization in feline blood samples (Kess-ler et al., 2009). While it has been suggested to calculate a geneexpression normalization factor from multiple reference genesfor each sample (Andersen et al., 2004; Vandesompele et al.,2002), this method was not used in the present study becausewe knew that GAPDH and YWHAZ were not well-suited for manytissues (Kessler et al., 2009). Therefore, we expected that the qual-ity of the normalization with a good reference gene, such as RPS7,would be reduced by adding the effect of less well-suited referencegenes. Instead, the limitations and effects of the reference geneswere considered when interpreting the results and are discussedbelow.

To date, only limited data regarding fTHTR1 expression wereavailable from a study that quantified fTHTR1 in one uninfectedcat using northern blot (Mendoza et al., 2006). In this semi-quantitative analysis of seven tissues and purified monocytes andT cells, the fTHTR1 expression was highest in kidney, small intes-tine, liver and the two cell subsets investigated (Mendoza et al.,2006). However, because of the very low basal expression ofTHTR1, northern blot analysis may be inaccurate and result in dis-parities found between different studies and species (Dutta et al.,1999; Oishi et al., 2001). In addition, depending on the probe andstringency conditions used, northern blot may detect differentsplice variants or cross-reactive sequences. In the present study,using quantitative real-time PCR and tissues from several unin-fected and FeLV-infected cats, fTHTR1 expression was particularlyhigh in the oral mucosa scrapings independent of the referencegene chosen for normalization and the FeLV status of the cats(Fig. 1), suggesting that carrier-mediated thiamine uptake maybe an important feature of the feline oral mucosa, as reported forhumans (Evered and Mallett, 1983). The oral mucosa representsa primary site for FeLV entry (Hardy et al., 1973; Jarrett et al.,1973b); therefore, high expression of its receptor may be advanta-geous for the virus. As remarked by Mendoza and co-workers(2006), it will be interesting to identify the cell populations inthe oral mucosa that actually express the receptor at the high lev-els detected in the present study.

High fTHTR1 expression was also found in the muscular tissuescompared with most other tissues investigated, but only when nor-malized to RPS7. When normalized to GAPDH, fTHTR1 expressionin the muscular tissues of healthy cats was rather low, whichmay be related to the reported increased GAPDH expression infeline and human tissues with high energy demands, such as themyocardium, skeletal muscle and regions of the brain (Barberet al., 2005; Kessler et al., 2009). Because of this altered expressionof GAPDH relative to other tissues, the results from normalizationto RPS7 rather than GAPDH should be considered for the muscula-ture. As a result, we suppose that the expression level of fTHTR1 infeline muscle is high. This finding is in agreement with results fromhuman tissues, where the highest levels of hTHTR1 expressionwere found in skeletal muscle, followed by placenta, heart, liverand kidney when detected using northern blot (Diaz et al., 1999;Dutta et al., 1999). There is no indication that musculature playsa particular role in FeLV pathogenesis.

A similar phenomenon may be responsible for the high fTHTR1expression observed in the pancreas when the results were nor-malized to GAPDH but not to RPS7 (Fig. 1A and B). The apparentlyhigh levels may be related to low levels of the GAPDH in the pan-creas, as has been described for human tissues (Barber et al., 2005).Therefore, we think that the fTHTR1 expression level followingnormalization to RPS7, which was not higher in the pancreas thanin other tissues, reflects the situation more accurately. In summary,for some tissues the calculated expression level of fTHTR1 wasmarkedly influenced by the choice of the reference gene, and sta-tistically significant differences among tissues were either presentor absent depending on the normalization chosen. In particular,normalization with the widely used reference gene GAPDH mayproduce misleading results for certain tissues. These results con-firm the importance of using well-chosen reference gene(s) andunderstanding the limitations and effects of normalization to dif-ferent reference genes. However, in contrast to these examples,consistent results independent of the reference gene used for nor-malization were found for some other tissues, including the oralmucosa, the parotid and mandibular glands, and the blood sam-ples.fTHTR1 expression on the cell surface is a prerequisite forthe susceptibility of the cell to FeLV-A (Mendoza et al., 2006). Forother retroviruses, the specific receptor is typically downregulatedafter virus entry into the host cell so that it is unavailable for use by

Table 2Sequence identity of fTHTR1 from different felids compared with the domestic catsequence.

Gene sequenceidentity (%)

Proteinsequenceidentity (%)

Mutations Accession no.[Pubmed]

Iberianlynx

99.4 99.6 [A368T],[E481T]

HQ018798

Europeanwildcat

99.9 99.8 [E260Q] HQ018799

Jaguarundi 99.3 99.8 [E481A] HQ018797Asiatic/

Africanlion

99.0 99.8 [E481A] HQ018800

A. Katrin Helfer-Hungerbuehler et al. / Infection, Genetics and Evolution 11 (2011) 1940–1950 1947

another virion (Hunter, 1997). This mechanisms is mediated, e.g.by internalization of the receptor, disruption of the transport ofthe receptor to the cell surface or competition with the receptorbinding domain (Hunter, 1997). So far no changes have beendescribed on the mRNA level related to the downregulation ofthe receptor. In the present study, significantly higher fTHTR1expression was found in the musculature, and lower levels werefound in the kidney of FeLV-infected cats when compared withuninfected cats and when GAPDH was used for normalization. Thisfinding may be related to altered expression of either the receptoror GAPDH. Since we did not see these differences following nor-malization to RPS7, we suppose that they are most likely relatedto altered expression of the reference gene.

Significantly lower fTHTR1 expression levels were detected inblood samples from FeLV-infected cats compared with uninfectedcats. In search for an explanation for this observation, we specu-lated that the blood composition and in particular the distributionof the leukocyte subsets could have played a role. The FeLV-infected cats of the present study had significantly lower lympho-cyte counts than the uninfected cats. In an attempt to evaluate theinfluence of varying cell subsets on the fTHTR1 expression, we per-formed RT-PCR from RNA extracted from previously sorted cellsubsets (Pepin et al., 2007). Indeed the T lymphocytes showed sig-nificantly higher fTHTR1 mRNA expression levels than the neutro-phil granulocytes. Thus, the lower fTHTR1 expression levels in theblood from FeLV-infected cats could have been caused by the sig-nificantly lower number of cells with high fTHTR1 mRNA expres-sion (lymphocytes).

We further evaluated whether propagation of leukocytes in cul-ture in the presence of IL-2 and/or Con A increases fTHTR1 mRNAexpression. Indeed, activation of PBMC in vitro led to a transientupregulation of fTHTR1 expression. This was primarily observedafter the stimulation with Con A but not IL-2 alone. Only whenIL-2 was used for stimulation after the cells had been primed withCon A, fTHTR1 mRNA expression tended also to increase. The lattermay be due to upregulation of IL-2 receptors in PBMC upon Con Astimulation (Rey Nores and McCullough, 1996). Further experi-ments would be necessary to determine whether activation ofthe PBMC and upregulation of the viral receptor would also leadto an increased capacity of the cells to support the viral infectionas had been demonstrated, e.g. for the rinderpest virus (Rey Noresand McCullough, 1996). Increased replication after Con A stimula-tion has been demonstrated for some other viruses in vitro or invivo, such as the porcine circovirus (Lefebvre et al., 2008; Yuet al., 2009), but not for all viruses (Mulder et al., 1995). An earlystudy on FeLV demonstrated that in vitro stimulation using Con Acompletely obviated virus replication in peripheral blood lympho-cytes; however, it was suggested that the failure of productiveinfection was effected at the post virus adsorption step (Rojkoet al., 1981).

Thiamine plays an important role during neonatal growth. Inmice, the intestinal and renal thiamine uptake is regulated duringdevelopmental maturation (Reidling et al., 2006). Expression ofthiamine transporters at both the mRNA and protein level in theintestine and kidney was higher in suckling mice than in weanlingmice, and adults had the lowest expression of THTR1 (Reidlinget al., 2006). Therefore, we hypothesized that the well-documentedage dependency of FeLV susceptibility in cats may be related toage-dependent changes in the expression of the FeLV-A receptor.Newborn kittens, which show a FeLV-susceptibility of 70–100%,may have higher fTHTR1 levels than adults, which only display aFeLV-susceptibility of 10–20% (Hoover et al., 1976; Jarrett, 1980).To identify any correlations between the age of the cat and theFeLV susceptibility, fTHTR1 expression levels were quantified inwhole blood and oral mucosa scrapings from healthy cats of differ-ent ages. When we included the samples originating from five cats

followed over time (repeated observations) in the analyses, fTHTR1levels tended to decline in blood samples with age, while theyincreased in the oral mucosa. A decrease of the virus receptor inthe blood could result in a lower number of persistently viremiccats upon infection, as has been observed in older cats. We thenattempted to confirm our observations by increasing the numberof samples and investigated time points. Therefore, additionalsamples from four unrelated non-SPF kittens (at the age of1.1–8.4 weeks), five adult cats (at the age of 3–3.3 years) and twoolder cats (at the age of 13.7 years) were included. However, thetrends could not be confirmed (data not shown), although expres-sion of fTHTR1 in the oral mucosa and the ileum in two stillbirthswas lower when compared with adult cats. Taken together, thesedata suggest that the FeLV susceptibility of cats at different agesis not primarily or solely related to the FeLV-receptor mRNAexpression levels at the site of virus entry or in the blood.

Differences in susceptibility to FeLV have also been observedamong different feline species. The fTHTR1 cDNA from several wildfelids (including the Iberian lynx, the European wildcat, the Asiaticand African lion and the Brazilian jaguarundi) was sequenced andfound to be highly conserved among different species. Interest-ingly, despite the large distance in the geographic origin of the Asi-atic and the African lions, the fTHTR1 sequence of these twosubspecies was identical. Our findings indicate that the differencesin FeLV susceptibility among distinct species may not primarily berelated to sequence variations in the FeLV-A receptor. This assump-tion is supported by the observation that the Iberian lynx, which ishighly susceptible to FeLV, shares one of the two amino acidchanges with the Asiatic lion, which is believed to be immuneagainst FeLV (Harrison et al., 2010; Hofmann-Lehmann et al.,1996; Meli et al., 2009). On the basis of the topology of humanor mouse THTR1, all non-synonymous polymorphisms in the felinesequences were located in the predicted cytoplasmic or transmem-brane regions of the protein (Dutta et al., 1999; Oishi et al., 2001).Therefore, interaction of these sites with the virus surface proteinsseem unlikely.

The development of feline lymphoma is a common manifesta-tion of FeLV infection (Jarrett et al., 1964). The risk of FeLV-infectedcats to develop lymphomas is greatly increased compared withuninfected cats (Hardy et al., 1973). Moreover, the association ofcertain vitamin deficiencies and cancer is well known; a connec-tion between the thiamine status and breast carcinoma was showndecades ago (Basu and Dickerson, 1976). Patients with advancedcancer frequently present with thiamine deficiency; therefore, thi-amine supplementation is given as nutritional support (Comin-Anduix et al., 2001). In its coenzyme form, thiamine pyrophosphateplays an important role in carbohydrate metabolism (Said et al.,2001; Singleton and Martin, 2001). Thus, because of its involve-ment during the synthesis of pentoses, which are used as nucleicacid precursors, thiamine utilization may be increased in tumorcells (Singleton and Martin, 2001) and excessive thiamine supple-mentation may be responsible for therapeutic failure by enhancingtumor cell proliferation (Boros et al., 1998; Comin-Anduix et al.,2001). It has been hypothesized that the increased thiamine usagemay result in an upregulation of genes associated with thiaminetransport in tumors compared with the analogous normal tissue.While THTR1 levels were unchanged in 50 human breast tumorsexamined (Liu et al., 2003), a second thiamine transporter gene,SLC19A3 (THTR2), was downregulated in human breast cancer,which could have contributed to an increased resistance to apopto-sis (Liu et al., 2003). Because FeLV uses fTHTR1 as specific receptor,it was of particular interest to investigate whether FeLV-associatedtumors had altered expression of fTHTR1. The fTHTR1 expressionlevel in tumor tissue was therefore analyzed by comparing non-tumor tissues with the corresponding tumor tissues from the samecat. All tumors investigated were lymphomas and developed

1948 A. Katrin Helfer-Hungerbuehler et al. / Infection, Genetics and Evolution 11 (2011) 1940–1950

during FeLV-infection. No significant effect on the expression offTHTR1 was detected.

5. Conclusions

This is the first study to quantitate the fTHTR1 expression levelsin various tissues from FeLV-infected and uninfected cats. Wedemonstrated detectable expression in all the tissues tested andfound significant differences among some of them, which may beprimarily related to functional differences in the tissues. Remark-ably, fTHTR1 expression was particularly high at the site of FeLVentry and shedding. We were not able to demonstrate that youngcats have higher FeLV-A receptor expression levels, a potentialexplanation for their higher susceptibility to FeLV. Moreover,sequence variations in the FeLV-A receptor do not seem to be theprimary mechanism underlying differences in the susceptibilityof distinct feline species to FeLV infection. Our study emphasizesthe importance of selecting an appropriate reference gene for accu-rate normalization of gene expression. The real-time PCR describedherein will be an important prerequisite for future studies in whichthe mRNA levels of fTHTR1 are quantified.

Acknowledgments

The authors would like to thank the Environmental Council ofthe government of Andalusia, southern Spain, for providing theIberian lynx samples. We also would like to thank Dr. C. Filoniand Dr. K. Baipoledifor for providing the Brazilian Jaguarundi andthe African lion samples, respectively, and the Veterinary Pathol-ogy of the Vetsuisse Faculty for the Asiatic lion and European wild-cat samples. We would especially like to thank Dr. M. Meli forexcellent assistance and helpful discussions. We are grateful tothe animal caretakers for expert technical aid with the cats andthe technicians and doctoral students of the Clinical Laboratory,particularly T. Meili, B. Weibel and B. Zellweger for excellent labo-ratory assistance. The molecular biology work was performedusing the logistics of the Center for Clinical Studies, Vetsuisse Fac-ulty, University of Zurich. R.H.L. was the recipient of a professor-ship by the Swiss National Science Foundation (PP00P3-119136).

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