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Molecular detection, phylogenetic analysis, and identification of transcription motifs in feline leukemia virus from naturally infected cats in Malaysia.

1. Introduction

Feline leukaemia virus (FeLV) is a gammaretrovirus associated with anaemia, immunodeficiency, leukaemia, and lymphoma in cats [1]. FeLV has been studied extensively as a model for human immunodeficiency virus (HIV) and human T-cell lymphoma virus (HTLV) infections [2]. FeLV is distributed worldwide; however, prevalence varies greatly with geography and with risk factors that include age, health status, and population density [3,4]. A recent study reported FeLV seroprevalence of 5.1% and 18.9% in healthy and sick Malaysian cats, respectively [4]. On the other hand, studies carried out in other Asian regions reported 0% FeLV prevalence in Vietnam [5]; 14.7% among cats in Singapore [6]; 2.9% in Japan [7]; and 6% each from Taiwan and Thailand [8, 9]. In non-Asian countries, FeLV prevalence has been reported to be 4.8% on Prince Edward Island, Canada [10]; 5.3% and 3.7% in Raleigh and Gainesville, USA, respectively [11]; 3.4% in all Canada [12]; and 4.6% in Egypt [13]. These discrepancies in prevalence of FeLV may arise due to differences in cat's lifestyle and FeLV vaccination practices in different countries [4].

Diagnosis of FeLV is usually performed by detection of p27 antigen [14]. However, demonstrating p27 antigen is difficult during early viraemia and with latent infections. Studies have shown that FeLV viral RNA and provirus DNA are better predictors of progressive and latent infections, respectively [15,16].

Apart from the envelope gene of FeLV, the long terminal repeats (LTRs) play important role in determining disease outcome and in differentiating exogenous from endogenous FeLV [15, 17]. Vaccination against FeLV is not carried out in Malaysia and, to date, FeLV clinical status of Malaysian cats has not been investigated using molecular assays. Additionally, unlike the ubiquitous feline infectious peritonitis (FIP) [18] sequence and phylogenetic characteristics of the Malaysian FeLV isolates have not been elucidated. The objectives of this study are to evaluate the presence of FeLV viral RNA and provirus DNA in selected antigenaemic and nonantigenaemic cats, respectively. Sequence characteristics, enhancer motifs as well as phylogenetic relationships of the Malaysian FeLV also were determined.

2. Materials and Methods

2.1. Animals and Sampling. Heparinized blood samples were collected from cats presented at University Veterinary Teaching Hospital, Universiti Putra Malaysia (UVH-UPM). The samples were tested for the presence of FeLV p27 viral antigen using a commercially available test kit [4]. These cats were divided into p27 antigen positive and p27 antigen-negative groups. From each group, 39 cats were selected by convenience sampling method and the samples were subjected to PCR analysis. All cats had no history of vaccination against FeLV as vaccination against FeLV is not practiced in Malaysia. All samples were collected by the attending veterinary clinicians, as part of routine practices. In addition, consent for evaluation was obtained from the cat owners, prior to sampling.

2.2. Nucleic Acid and PCR Amplification. Viral RNA was extracted from the plasma of p27-positive cats, using high pure viral RNA purification kit (Roche, Germany). On the other hand, genomic DNA was isolated from whole blood of p27-negative cats, using QIAGEN DNA extraction kits (QIAGEN, Germany). All nucleic acid extraction procedures were carried out according to manufacturers' instructions. RNA was reverse transcribed and subjected to nested PCR, using a one-step access RT PCR (Promega, USA). Genomic DNA was amplified by nested PCR assay.

Two sets of primers (outer and inner primers) were synthesised (1st BASE, Malaysia) and used to amplify a 601 bp segment of FeLV-U3LTR and partial gag regions. This segment recognises exogenous but not endogenous FeLV segments presence in cat genome; thus the primers used in this study are specific for exogenous FeLV detection. Outer PCR reaction was carried out using U3-F(1) (57-ACA GCA GAA GTT TCA AGG CC -3') and G-R(1) (5'-GAC CAG TGA TCA AGG GTG AG-37) primers. The inner PCR reaction was carried out with U3-F(2) (57-GCT CCC CAG TTGACCAGAGT-3') and G-R(2) (5'-GCTTCGGTACCA AAC CGA AA-37) primers 15].

The PCR mixture was prepared in 25 [micro]L reaction volume containing 10 mM each of dNTPs mix, 0.2 mM Tfl DNA polymerase (5U/[micro]L), 0.1 U AMV (5U/[micro]L), 0.1 U recombinant RNasin ribonuclease inhibitor (400 U/[micro]L), 0.8 U MgS[O.sub.4] (25 mM), 20 pmol of each of the forward and reverse primer, 5.0 [micro]L of 1 times buffer, and 1 [micro]L RNA or DNA template. Nuclease-free water was used to bring the mixture to its final volume of 25 [micro]L. AMV reverse transcriptase enzymes and RNasin ribonuclease inhibitor were included only when RNA was a starting template for the PCR assay. In the nested PCR step, 1 [micro]L of outer PCR product was used as template.

In both inner and outer PCR steps, the target gene regions were amplified using the following conditions: reverse transcription: 45[degrees]C (45 min) (only in the case of RNA), initial denaturation: 94[degrees]C (2 min), denaturation: 94[degrees]C (45 sec), annealing: 58[degrees]C (30 sec), extension: 72[degrees]C (1min), 35 cycles of repeats, and final extension: 72[degrees]C (7 min). PCR product was electrophoresed using 1.5% agarose (SeaKem LE USA), stained with 0.5 [micro]g/mL ethidium bromide (Bio-Rad USA), and visualised under UV light (Geldoc system, Bio-Rad, USA). Extraction and amplification procedures were carried out in separate hood to reduce chances of contamination.

2.3. Sequence and Phylogenetic Analyses. In order to gain insight on the characteristics of Malaysian FeLV sequences, 29 nested PCR-positive samples (RNA n = 14; DNA provirus n = 15) were selected and purified using an Accuprep purification kit (Bioneer, Daejeon, Korea). Sequencing was carried out based on the amplified U3LTR-gag segment using a standard ABI Big Dye terminator version 3.1 sequence kit (Applied Biosystem). The obtained sequences were analysed for homology using the NCBI Basic Local Alignment Search Tool (BLAST: http://www.ncbi.nlm.nih.gov). In addition, multiple sequence alignment was carried out using ClustalW and the percentage nucleotide identity was determined using DNA identity matrix [19, 20]. On the other hand, single nucleotide polymorphism (SNP), DNA distance matrix, and transcription binding proteins prediction analyses were carried out using geneious software version R7 [20]. A neighbour-joining (NJ) phylogenetic tree was constructed based on the U3LTR-gag sequences using MEGA5 software. The tree reliability was assessed using 100 bootstrap replicates [21]. All nucleotide sequences were deposited with the NCBI GenBank (Table 1).

3. Results and Discussion

FeLV infection is of concern to cat owners due to its ability to induce tumours and immunodeficiency, thus predisposing cats to other secondary diseases. In this study, a U3-LTR and gag regions of exogenous without endogenous FeLV sequences were amplified by nested PCR methods. Post-PCR analysis using electrophoresis revealed an expected amplicon size of 770 bp in the outer PCR and 601 bp in the nested inner PCR assay (Figure 1). Overall, it was found that 97.4% (38/39) of p27 antigen-negative cats were positive for FeLV provirus DNA suggesting that this category of cats likely goes undetected when only p27 detection is used to judge their FeLV clinical status. Similar studies reported high prevalence of FeLV provirus DNA in Brazilian cats [22]. However, Hofmann-Lehmann et al. [23] reported a lower provirus DNA rate in cats in Switzerland. The observed differences in prevalence among different countries could be associated with cat lifestyle, as well as variations in factors known to favour FeLV transmission [3,4]. Provirus DNA detection rate observed in this study could be associated with regressive or latent FeLV infection, which is characterized by integration of DNA provirus into the host cell genome and absence of viral antigenincirculation 1,15].

The consequence of latent FeLV infection is that provirus DNA could reactivate to an infectious state, especially following stress and/or immunosuppression. Thus, cats that are p27 antigen-negative, but provirus DNA positive, could serve as sources of infection of FeLV-naive cats [24]. A previous study has established an association between feline lymphoma and provirus DNA positivity in p27 antigen-negative cats, though this has not been evaluated in the present study [25]. Moreover, transmission of FeLV has been shown to occur in cats following blood transfusion from cats with provirus DNA, thus highlighting the importance of screening blood donor cats for provirus DNA [26].

Viral RNA was detected in 87.2% (34/39) of p27 antigen-positive cats whereas 13% (5/39) tested negative using RTPCR assay. Since plasma viral RNA is an indicator of FeLV viraemia, cats that are positive for FeLV p27 antigen and viral RNA are likely to harbour replicating virus [27]. Cats in this category may progress to a persistent viraemic stage, succumbing to FeLV-associated illness [28].

Failure to detect FeLV viral RNA in about 13% p27 antigen-positive cats (p27-positive/viral RNA-negative) could result from atypical infection, wherein the virus is sequestered and replicates locally in tissues such as salivary gland, mammary gland, and urinary epithelium, causing intermittent or low-grade antigenaemia, although there is no detectable viraemia [28,29]. Our findings are consistent with the results of an earlier study that failed to isolate FeLV from about 10% of p27 antigen-positive cats, irrespective of the antigen detection methods used. Such cats were considered as "discordant," suggesting that p27 antigen-positive status may not always correlate with viraemia [30]. Another potential explanation for p27-positive-RNA-negative status might be false positive antigen or false negative RNA tests that arise occasionally because of low positive predictive value of p27 antigen tests in regions with low FeLV prevalence [31]. Clinical relevance of atypical FeLV infection is not well-understood, and it has been recommended to monitor the status of discordant cats over time [27, 28]. No additional follow-up was carried out in the present study, because most owners were not willing to subject cats to repeated venepunctures [4].

Based on the U3LTR and partial gag regions, nucleotide sequence analyses revealed homology of 91-100% among Malaysian FeLV isolates. However, homology decreased to 84.6% when local isolates were compared with reference isolates. Previous studies reported strong sequence conservation (>97%) among FeLV isolates of different geographic and temporal clusters [32, 33]. In agreement with Jackson et al. [17], we do observe point mutations and nucleotide deletion in Malaysian FeLV isolates (see Supplementary Material available online at http://dx.doi.org/10.1155/2014/760961). While U3LTR is conserved in FeLV, field isolates have been reported to exhibit sequence variation within the terminally repeated LTRs regions [17, 33]. Mutational changes in the LTR regions have been implicated with enhanced transcriptional and/or insertion activities of FeLV, thus supporting T-cell lymphomagenesis [34, 35].

In this study, several transcription binding motifs were predicted within the amplified U3LTR-gag region (Table 2). Of these, NF-GMa, Krox-20/WT1I-del2, BAF1, AP-2, TBP, TFIIF-beta, TRF, and TFIID motifs were found to be conserved between local FeLV isolates and the two characterized FeLV-Rickard subgroup A and FeLV-FAIDS reference isolates. On the other hand, E1A-F, ELP, Sp1, C\EBPbeta, BAF1, GCF, HNF-3, and PEA3 motifs are found in some local isolates but were absent in reference sequences. These motifs may have implication for viral oncogenicity or probably favours viral replication. For example, an Sp1 enhancer, a member of Sp/Kruppel-like factor, was reported to activate gene transcription and contribute to abnormal metabolism of cancer cells [36, 37] whereas C\EBPbeta regulates the growth and differentiation of myeloid as well as lymphoid cells [38]. Studies have shown that, the U3-LTR sequence contains multiple transcription binding sites that aid viral replication and pathogenesis. Interactions of different transcription binding factors, via the U3-LTRs, may contribute to cellular gene transactivation and viral leukemogenesis [39, 40]. Enhancer motifs observed in this study appeared in multiple locations such as in the case of E1A-F, BAF1, and TFIID, each occurring in duplicate; GCF appeared in triplicate while AP-2 is repeated 5 times at different positions. An enhancer duplication and triplication has been reported in naturally occurring cases of FeLV-induced T-cell lymphomas [41, 42]. The clinical relevance of multiple enhancers in cats used in the present study is not determined, although some FeLV positive cats had evidence of different tumour forms at post-mortem (result not shown). Previous studies reported that E1A-F, a member ets-oncogene family transcription factor, upregulates the multiple matrix metalloproteinase (MMP) genes thus contributing to the malignant phenotypic activity by increasing the invasion and metastatic activities of cancerous cells [43]. TFIID, a potential protooncogene with TATA-box protein and a TBP-associated factor also plays role in transcription initiation and genome expression [44]. On the other hand, AP2 and SP1 are known to activate epidermal growth factor receptor (EGFR) gene. In addition, overexpression of these gene has been reported to cause cellular transformation [45, 46]. Surprisingly we also identified a triplicate of GCF binding factor that has suppressor effect on EGR gene; these discrepancies, however, need further elucidation with quantitative real-time PCR [47].

Absence of length mutation (nucleotide position 473-481) in Malaysian FeLV isolates, as observed in FeLV isolates from Taiwan (FeLV-TW-25 and FeLV-TW-30) and a European isolate (FeLV-GM1), might suggest limited influence of geography in evolutionary patterns of FeLV, unlike its lentiviral counterpart, feline immunodeficiency virus [33, 48].

Phylogenetic analysis based on the U3LTR-gag sequence revealed that Malaysian FeLV isolates are closely related (Tables 3(a), 3(b), and 3(c)) but when compared with reference isolates, separated into two distinct clusters, with the majority (86.2%) being closely related to FeLV-K01803 isolate from UK. The remaining local FeLV isolates (13.8%) clustered with FeLV-GM1 (Figure 2). The reason for the observed similarity between local FeLV isolates and European isolates, but not with Taiwanese isolates, may suggest the lack of geographical influence, this should be explored further. It is possible also that FeLV might have been introduced into Malaysia as a result of translocation of domestic pets from Europe. Due to a somewhat conserved nature of the U3LTR region, conclusion about the FeLV subgroup requires further investigations of FeLV envelope protein gene.

4. Conclusion

This study revealed the occurrence of FeLV viral RNA and provirus DNA among naturally infected Malaysian cats.

Based on the U3LTR-gag sequence, Malaysian FeLV isolates are highly conserved and more closely related to K01803 isolate from UK compared to Taiwanese and other reference isolates. Presence of multiple enhancers some of which have been linked with FeLV induced tumours may contribute to the development of poor prognostic outcome in naturally infected Malaysian cats although this needs further investigation.

Overall, this is the first molecular study for evidence of FeLV in Malaysia. We also identified several motifs that have potential implications in FeLV-induced leukemogenesis.

Future studies need to explore association between FeLV positive status and occurrence of feline tumour in Malaysian cats. The present findings is useful in designing molecular diagnostics for clinical applications and for improved understanding of FeLV infection outcome and epidemiology.

http://dx.doi.org/10.1155/2014/760961

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

The authors thank cat owners and clinicians for their support, Dr. Dennis F. Lawler for proof reading the paper, and Saeid Kadkhodaei for assistance in sequence analysis. This project was funded by Science Fund, Ministry of Science and Technology and Innovation, Project no. 02-01-04-SF1070.

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Faruku Bande, (1,2) Siti Suri Arshad, (1) Latiffah Hassan, (3) and Zunita Zakaria (1)

(1) Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

(2) Department of Veterinary Services, Ministry of Animal Health and Fisheries Development, PMB 2109, Usman Faruk Secretariat, 840221 Sokoto, Sokoto State, Nigeria

(3) Department of Veterinary Laboratory Diagnostics, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Correspondence should be addressed to Siti Suri Arshad; suri@upm.edu.my

Received 26 June 2014; Revised 11 September 2014; Accepted 11 September 2014; Published 17 November 2014

Academic Editor: Philip H. Kass

Table 1: List of local and reference sequences used in phylogenetic
analysis of Malaysian FeLV isolates.

Isolate       Accession number  Country     Source
FeLV-UPM01        HQ197367      Malaysia  This study
FeLV-UPM02        HQ197368      Malaysia  This study
FeLV-UPM03        HQ197369      Malaysia  This study
FeLV-UPM04        HQ197370      Malaysia  This study
FeLV-UPM05        HQ197371      Malaysia  This study
FeLV-UPM06        HQ197372      Malaysia  This study
FeLV-UPM07        HQ197373      Malaysia  This study
FeLV-UPM08        HQ197374      Malaysia  This study
FeLV-UPM09        HQ197375      Malaysia  This study
FeLV-UPM10        HQ197376      Malaysia  This study
FeLV-UPM11        HQ197377      Malaysia  This study
FeLV-UPM12        HQ727890      Malaysia  This study
FeLV-UPM13        HQ727891      Malaysia  This study
FeLV-UPM14        HQ727892      Malaysia  This study
FeLV-UPM15        JF815538      Malaysia  This study
FeLV-UPM16        JF815539      Malaysia  This study
FeLV-UPM17        JF815540      Malaysia  This study
FeLV-UPM18        JF815541      Malaysia  This study
FeLV-UPM19        JF815542      Malaysia  This study
FeLV-UPM20        JF815543      Malaysia  This study
FeLV-UPM21        JF815544      Malaysia  This study
FeLV-UPM22        JF815545      Malaysia  This study
FeLV-UPM23        JF815546      Malaysia  This study
FeLV-UPM24        JF815547      Malaysia  This study
FeLV-UPM25        JF815548      Malaysia  This study
FeLV-UPM26        JF815549      Malaysia  This study
FeLV-UPM27        JF815550      Malaysia  This study
FeLV-UPM28        JF815551      Malaysia  This study
FeLV-UPM29        JF815552      Malaysia  This study
FeLV-914MG        EU048352       Brazil    GenBank
FeLV-1235MG       EU43664        Brazil    GenBank
FeLV-843MG        DQ821500       Brazil    GenBank
FeLV-887MG        DQ821501       Brazil    GenBank
FeLV-922MG        EU48356        Brazil    GenBank
FeLV-1286MG       EU090948       Brazil    GenBank
FeLV-328MG        AY745878       Brazil    GenBank
FeLV-Rickard      AF052723        USA      GenBank
FeLV-FAIDS         M18247         USA      GenBank
FeLV-GM1           D13922          UK      GenBank
FeLV-K01803        K01803          UK      GenBank
FeLV-TWK25        GQ465833       Taiwan    GenBank
FeLV-TW-K30       GQ327961       Taiwan    GenBank

Note: FeLV01-FeLV14 sequences were amplified from plasma viral RNA
while the remaining local sequence (FeLVUPM13-FeLVUPM29) were
amplified from proviral DNA.

Table 2: Predicted transcription binding motifs found within the
LTR-gag sequence of Malaysian FeLV isolates.

Motifs       Sequence      Seq   Coverage  Occurrence in  Occurrence in
                           len             local          reference
                           -gth            sequence       FeLV

NF-GMa       GAGGTTTCAT     10   523-532   All local seq  FeLV-Rickard;
                                           except UPM08   FeLV-FAIDs

E1A-F        CGGATGT        7    521-527   UPM08          NA
E1A-F        CGGATGT        7    70-76     UPM18          NA
ELP          CAAGGTC        7    523-527   UPM03,14,18,   NA
                                           20

Sp1          GGGGCTAGG      7    521-527   UPM03,18, 20   NA
C\EBPbeta    CTGGAAA        7    387-393   UPM18, 20      NA
Krox-20/     CGCCCCCGC      9    374-382   All local seq  FeLV-Rickard;
 WT1I- del2                                               FeLV-FAIDs
E2F          TTTTGGCGG      9    334-342   UPM03, 14,     FeLV-Rickard;
                                           18, 20         FeLV-FAIDs

BAF1         TCCTTGTATACG   12   301-312   All except     NA
                                           UPM03,14,18,
                                           20

BAF1         TCCTTGTATACG   12   158-169   All local seq  NA
AP-2         CCCAACCG       8    243-250   All local seq  FeLV-Rickard;
                                                          FeLV-FAIDs

AP-2         CCCAACCG       8    59-66     All local seq  FeLV-Rickard;
                                           except UPM17   FeLV-FAIDs

AP-2         CCCAACCG       8    58-65     UPM03, 14,     NA
                                           18, 20, 25

AP-2         CCCAACCG       8    4 to 11   All except     NA
                                           UPM03, 14

AP-2         CCCAACCG       8    3 to 11   All local seq  FeLV-Rickard
GCF          CCGCCCC        7    93-99     All local seq  FeLV-Rickard,
                                                          FeLV-FAIDs

GCF          CCGGCGC        7    64-70     All local seq  NA
                                           except UPM03,
                                           UPM 14

GCF          GCGCGCC        7    26-32     UPM27          NA
HNF-3        TGTTTGC        7    129-135   All local seq  NA
                                           except UPM06

PEA3         GCGGAAGT       8    69-76     UPM18          NA
TBP          TATAAAA        7    39-45     All local seq  FeLV-Rickard,
                                                          FeLV-FAIDs

TFIIF-beta   TATAAAA        7    39-45     All local seq  FeLV-Rickard,
                                                          FeLV-FAIDs

TRF          TATAAAA        7    39-45     All local seq  FeLV-Rickard,
                                                          FeLV-FAIDs

TFIID        TATAAAA        7    39-45     All local seq  FeLV-Rickard,
                                                          FeLV-FAIDs

TFIID        CTTCTCGC       8    10 to 17  UPM03, 14, 20  NA
MEP-1        TATAAAA        7    23-29     All local seq  FeLV-Rickard
                                           except
                                           UPM03,14, 18,
                                           20

MBF-I        TATAAAA        7    23-29     All local seq  FeLV-Rickard
                                           except
                                           UPM03,14, 18,
                                           21

MTF-1        TATAAAA        7    23-29     All except     FeLV-Rickard
                                           UPM03,14,18,
                                           22

NA: not applicable or not present; seq: sequences.

Table 3: Distance matrix showing sequence relationship among local
FeLV isolates.

    (a)
                FeLV-       FeLV-      FeLV-       FeLV-       FeLV-
               Rickard      FAIDS      UPM03       UPM18       UPM20
              [AF052723]  [M18247]   [HQ197369]  [JF815541]  [JF815543]

FeLV-Rickard                98.12      94.925      93.609      93.797
[AF052723]
FeLV-FAIDS      98.12                  95.301      93.985      94.173
[M18247]
FeLV-UPM03      94.925     95.301                  96.429      98.496
[HQ197369]
FeLV-UPM18      93.609     93.985      96.429                  96.053
[JF815541]
FeLV-UPM20      93.797     94.173      98.496      96.053
[JF815543]
FeLV-UPM29      96.805     96.429      94.737      93.797      93.609
[JF815552]
FeLV-UPM28      96.805     96.429      94.737      93.797      93.609
[JF815551]
FeLV-UPM12      96.992     96.617      94.925      93.985      93.797
[HQ727890]
FeLV-UPM06      96.617     96.617      94.549      93.609      93.421
[HQ197372]
FeLV-UPM23      96.805     96.429      94.737      93.797      93.609
[JF815546]
FeLV-UPM21      96.805     96.429      94.737      93.797      93.609
[JF815544]
FeLV-UPM24      96.805     96.429      94.737      93.797      93.609
[JF815547]
FeLV-UPM26      96.805     96.429      94.737      93.797      93.609
[JF815549]
FeLV-UPM09      96.805     96.429      94.737      93.797      93.609
[HQ197375]
FeLV-UPM17      96.617     96.241      94.549      93.609      93.421
[JF815540]
FeLV-UPM16      96.617     96.241      94.549      93.609      93.421
[JF815539]
FeLV-UPM25      96.429     96.053      94.549      93.797      93.421
[JF815548]
FeLV-UPM19      95.865     95.489      93.797      92.857      92.669
[JF815542]
FeLV-UPM27      96.617     95.865      94.173      93.233      93.045
[JF815550]
FeLV-UPM22      96.617     96.053      94.361      93.421      93.233
[JF815545]
FeLV-UPMIO      96.429     96.053      94.361      93.421      93.233
[HQ197376]
FeLV-UPM15      96.992     96.617      94.925      93.985      93.797
[JF815538]
FeLV-UPMll      96.992     96.617      94.925      93.985      93.797
[HQ197377]
FeLV-UPM13      96.805     96.429      94.737      93.797      93.609
[HQ727891]
FeLV-UPM05      96.429     96.053      94.361      93.421      93.233
[HQ197371]
FeLV-UPM07      96.805     96.429      94.737      93.797      93.609
[HQ197373]
FeLV-UPM02      96.805     96.429      94.737      93.797      93.609
[HQ197368]
FeLV-UPMOl      96.805     96.429      94.737      93.797      93.609
[HQ197367]
FeLV-UPM04      96.617     96.241      94.549      93.609      93.421
[HQ197370]
FeLV-UPM08      94.737     93.985      92.669      91.729      91.541
[HQ197374]
FeLV-UPMM       95.865     96.241      99.06       96.992      97.932
[HQ727892]

                 (a)
                FeLV-       FeLV-      FeLV-       FeLV-
                UPM29       UPM28      UPM12       UPM06
              [JF815552]  [JF815551] [HQ727890]  [HQ197372]

FeLV-Rickard    96.805     96.805      96.992      96.617
[AF052723]
FeLV-FAIDS      96.429     96.429      96.617      96.617
[M18247]
FeLV-UPM03      94.737     94.737      94.925      94.549
[HQ197369]
FeLV-UPM18      93.797     93.797      93.985      93.609
[JF815541]
FeLV-UPM20      93.609     93.609      93.797      93.421
[JF815543]
FeLV-UPM29                   100       99.812      99.436
[JF815552]
FeLV-UPM28       100                   99.812      99.436
[JF815551]
FeLV-UPM12      99.812     99.812                  99.624
[HQ727890]
FeLV-UPM06      99.436     99.436      99.624
[HQ197372]
FeLV-UPM23      99.248     99.248      99.436      99.06
[JF815546]
FeLV-UPM21      99.248     99.248      99.436      99.06
[JF815544]
FeLV-UPM24      99.248     99.248      99.436      99.06
[JF815547]
FeLV-UPM26      99.248     99.248      99.436      99.06
[JF815549]
FeLV-UPM09      99.248     99.248      99.436      99.06
[HQ197375]
FeLV-UPM17      99.06       99.06      99.248      98.872
[JF815540]
FeLV-UPM16      99.06       99.06      99.248      98.872
[JF815539]
FeLV-UPM25      98.872     98.872      99.06       98.684
[JF815548]
FeLV-UPM19      98.308     98.308      98.496      98.12
[JF815542]
FeLV-UPM27      98.684     98.684      98.872      98.496
[JF815550]
FeLV-UPM22      98.872     98.872      99.06       98.684
[JF815545]
FeLV-UPMIO      99.248     99.248      99.436      99.06
[HQ197376]
FeLV-UPM15      99.436     99.436      99.624      99.248
[JF815538]
FeLV-UPMll      99.436     99.436      99.624      99.248
[HQ197377]
FeLV-UPM13      99.624     99.624      99.812      99.436
[HQ727891]
FeLV-UPM05      99.248     99.248      99.436      99.06
[HQ197371]
FeLV-UPM07      99.624     99.624      99.812      99.436
[HQ197373]
FeLV-UPM02      99.624     99.624      99.812      99.436
[HQ197368]
FeLV-UPMOl      99.624     99.624      99.812      99.436
[HQ197367]
FeLV-UPM04      99.06       99.06      99.248      98.872
[HQ197370]
FeLV-UPM08      97.18       97.18      97.368      96.992
[HQ197374]
FeLV-UPMM       95.677     95.677      95.865      95.489
[HQ727892]

    (b)
                FeLV-      FeLV-      FeLV-      FeLV-      FeLV-
                UPM23      UPM21      UPM24      UPM26      UPM09
              [JF815546] [JF815544] [JF815547] [JF815549] [HQ197375]
FeLV-Rickard   96.805     96.805     96.805     96.805     96.805
[AF052723]
FeLV-FAIDS     96.429     96.429     96.429     96.429     96.429
[M18247]
FeLV-UPM03     94.737     94.737     94.737     94.737     94.737
[HQ197369]
FeLV-UPM18     93.797     93.797     93.797     93.797     93.797
[JF815541]
FeLV-UPM20     93.609     93.609     93.609     93.609     93.609
[JF815543]
FeLV-UPM29     99.248     99.248     99.248     99.248     99.248
[JF815552]
FeLV-UPM28     99.248     99.248     99.248     99.248     99.248
[JF815551]
FeLV-UPM12     99.436     99.436     99.436     99.436     99.436
[HQ727890]
FeLV-UPM06      99.06      99.06      99.06      99.06      99.06
[HQ197372]
FeLV-UPM23                  100        100        100        100
[JF815546]
FeLV-UPM21       100                   100        100        100
[JF815544]
FeLV-UPM24       100        100                   100        100
[JF815547]
FeLV-UPM26       100        100        100                   100
[JF815549]
FeLV-UPM09       100        100        100        100
[HQ197375]
FeLV-UPM17     99.812     99.812     99.812     99.812     99.812
[JF815540]
FeLV-UPM16     99.812     99.812     99.812     99.812     99.812
[JF815539]
FeLV-UPM25     99.624     99.624     99.624     99.624     99.624
[JF815548]
FeLV-UPM19      99.06      99.06      99.06      99.06      99.06
[JF815542]
FeLV-UPM27     99.436     99.436     99.436     99.436     99.436
[JF815550]
FeLV-UPM22     99.624     99.624     99.624     99.624     99.624
[JF815545]
FeLV-UPMIO     99.624     99.624     99.624     99.624     99.624
[HQ197376]
FeLV-UPM15     99.812     99.812     99.812     99.812     99.812
[JF815538]
FeLV-UPMll     99.812     99.812     99.812     99.812     99.812
[HQ197377]
FeLV-UPM13     99.624     99.624     99.624     99.624     99.624
[HQ727891]
FeLV-UPM05     99.248     99.248     99.248     99.248     99.248
[HQ197371]
FeLV-UPM07     99.624     99.624     99.624     99.624     99.624
[HQ197373]
FeLV-UPM02     99.624     99.624     99.624     99.624     99.624
[HQ197368]
FeLV-UPMOl     99.624     99.624     99.624     99.624     99.624
[HQ197367]
FeLV-UPM04      99.06      99.06      99.06      99.06      99.06
[HQ197370]
FeLV-UPM08     96.805     96.805     96.805     96.805     96.805
[HQ197374]
FeLV-UPMM      95.677     95.677     95.677     95.677     95.677
[HQ727892]

    (b)
                FeLV-      FeLV-      FeLV-      FeLV-      FeLV-
                UPM17      UPM16      UPM25      UPM19      UPM27
              [JF815540] [JF815539] [JF815548] [JF815542] [JF815550]
FeLV-Rickard   96.617     96.617     96.429     95.865     96.617
[AF052723]
FeLV-FAIDS     96.241     96.241     96.053     95.489     95.865
[M18247]
FeLV-UPM03     94.549     94.549     94.549     93.797     94.173
[HQ197369]
FeLV-UPM18     93.609     93.609     93.797     92.857     93.233
[JF815541]
FeLV-UPM20     93.421     93.421     93.421     92.669     93.045
[JF815543]
FeLV-UPM29      99.06      99.06     98.872     98.308     98.684
[JF815552]
FeLV-UPM28      99.06      99.06     98.872     98.308     98.684
[JF815551]
FeLV-UPM12     99.248     99.248      99.06     98.496     98.872
[HQ727890]
FeLV-UPM06     98.872     98.872     98.684      98.12     98.496
[HQ197372]
FeLV-UPM23     99.812     99.812     99.624      99.06     99.436
[JF815546]
FeLV-UPM21     99.812     99.812     99.624      99.06     99.436
[JF815544]
FeLV-UPM24     99.812     99.812     99.624      99.06     99.436
[JF815547]
FeLV-UPM26     99.812     99.812     99.624      99.06     99.436
[JF815549]
FeLV-UPM09     99.812     99.812     99.624      99.06     99.436
[HQ197375]
FeLV-UPM17                99.624     99.436     98.872     99.248
[JF815540]
FeLV-UPM16     99.624                99.436     98.872     99.248
[JF815539]
FeLV-UPM25     99.436     99.436                98.684      99.06
[JF815548]
FeLV-UPM19     98.872     98.872     98.684                98.496
[JF815542]
FeLV-UPM27     99.248     99.248      99.06     98.496
[JF815550]
FeLV-UPM22     99.436     99.436     99.248     98.684     99.248
[JF815545]
FeLV-UPMIO     99.436     99.436     99.248     98.684      99.06
[HQ197376]
FeLV-UPM15     99.624     99.624     99.436     98.872     99.248
[JF815538]
FeLV-UPMll     99.624     99.624     99.436     98.872     99.248
[HQ197377]
FeLV-UPM13     99.436     99.436     99.248     98.684      99.06
[HQ727891]
FeLV-UPM05      99.06      99.06     98.872     98.308     98.684
[HQ197371]
FeLV-UPM07     99.436     99.436     99.248     98.684      99.06
[HQ197373]
FeLV-UPM02     99.436     99.436     99.248     98.684      99.06
[HQ197368]
FeLV-UPMOl     99.436     99.436     99.248     98.684      99.06
[HQ197367]
FeLV-UPM04     98.872     98.872     98.684      98.12     98.496
[HQ197370]
FeLV-UPM08     96.617     96.617     96.429     96.053     96.617
[HQ197374]
FeLV-UPMM      95.489     95.489     95.489     94.737     95.113
[HQ727892]

   (c)
            FeLV-   FeLV-   FeLV-   FeLV-   FeLV-   FeLV-
            UPM22   UPM10   UPM15   UPM11   UPM13   UPM05
            [JF81-  [HQ19-  [JF81-  [HQ19-  [HQ72-  [HQ19-
            5545]   7376]   5538]   7377]   7891]   7371]

FeLV-       96.617  96.429  96.992  96.992  96.805  96.429
Rickard
[AF052723]
FeLV-FAIDS  96.053  96.053  96.617  96.617  96.429  96.053
[M18247]
FeLV-UPM03  94.361  94.361  94.925  94.925  94.737  94.361
[HQ197369]
FeLV-UPM18  93.421  93.421  93.985  93.985  93.797  93.421
[JF815541]
FeLV-UPM20  93.233  93.233  93.797  93.797  93.609  93.233
[JF815543]
FeLV-UPM29  98.872  99.248  99.436  99.436  99.624  99.248
[JF815552]
FeLV-UPM28  98.872  99.248  99.436  99.436  99.624  99.248
[JF815551]
FeLV-UPM12  99.06   99.436  99.624  99.624  99.812  99.436
[HQ727890]
FeLV-UPM06  98.684  99.06   99.248  99.248  99.436  99.06
[HQ197372]
FeLV-UPM23  99.624  99.624  99.812  99.812  99.624  99.248
[JF815546]
FeLV-UPM21  99.624  99.624  99.812  99.812  99.624  99.248
[JF815544]
FeLV-UPM24  99.624  99.624  99.812  99.812  99.624  99.248
[JF815547]
FeLV-UPM26  99.624  99.624  99.812  99.812  99.624  99.248
[JF815549]
FeLV-UPM09  99.624  99.624  99.812  99.812  99.624  99.248
[HQ197375]
FeLV-UPM17  99.436  99.436  99.624  99.624  99.436  99.06
[JF815540]
FeLV-UPM16  99.436  99.436  99.624  99.624  99.436  99.06
[JF815539]
FeLV-UPM25  99.248  99.248  99.436  99.436  99.248  98.872
[JF815548]
FeLV-UPM19  98.684  98.684  98.872  98.872  98.684  98.308
[JF815542]
FeLV-UPM27  99.248  99.06   99.248  99.248  99.06   98.684
[JF815550]
FeLV-UPM22          99.248  99.436  99.436  99.248  98.872
[JF815545]
FeLV-UPMIO  99.248          99.436  99.436  99.624  99.248
[HQ197376]
FeLV-UPM15  99.436  99.436          100     99.812  99.436
[JF815538]
FeLV-UPMll  99.436  99.436  100             99.812  99.436
[HQ197377]
FeLV-UPM13  99.248  99.624  99.812  99.812          99.624
[HQ727891]
FeLV-UPM05  98.872  99.248  99.436  99.436  99.624
[HQ197371]
FeLV-UPM07  99.248  99.624  99.812  99.812  100     99.624
[HQ197373]
FeLV-UPM02  99.248  99.624  99.812  99.812  100     99.624
[HQ197368]
FeLV-UPMOl  99.248  99.624  99.812  99.812  100     99.624
[HQ197367]
FeLV-UPM04  98.684  99.06   99.248  99.248  99.436  99.06
[HQ197370]
FeLV-UPM08  96.617  97.18   96.992  96.992  97.18   96.805
[HQ197374]
FeLV-UPMM   95.301  95.301  95.865  95.865  95.677  95.301
[HQ727892]

   (c)
            FeLV-   FeLV-   FeLV-   FeLV-   FeLV-   FeLV-
            UPM07   UPM02   UPM01   UPM04   UPM08   UPM14
            [HQ19-  [HQ19-  [HQ19-  [HQ19-  [HQ19-  [HQ72-
            7373]   7368]   7367]   7370]   7374]   7892]

FeLV-       96.805  96.805  96.805  96.617  94.737  95.865
Rickard
[AF052723]
FeLV-FAIDS  96.429  96.429  96.429  96.241  93.985  96.241
[M18247]
FeLV-UPM03  94.737  94.737  94.737  94.549  92.669  99.06
[HQ197369]
FeLV-UPM18  93.797  93.797  93.797  93.609  91.729  96.992
[JF815541]
FeLV-UPM20  93.609  93.609  93.609  93.421  91.541  97.932
[JF815543]
FeLV-UPM29  99.624  99.624  99.624  99.06   97.18   95.677
[JF815552]
FeLV-UPM28  99.624  99.624  99.624  99.06   97.18   95.677
[JF815551]
FeLV-UPM12  99.812  99.812  99.812  99.248  97.368  95.865
[HQ727890]
FeLV-UPM06  99.436  99.436  99.436  98.872  96.992  95.489
[HQ197372]
FeLV-UPM23  99.624  99.624  99.624  99.06   96.805  95.677
[JF815546]
FeLV-UPM21  99.624  99.624  99.624  99.06   96.805  95.677
[JF815544]
FeLV-UPM24  99.624  99.624  99.624  99.06   96.805  95.677
[JF815547]
FeLV-UPM26  99.624  99.624  99.624  99.06   96.805  95.677
[JF815549]
FeLV-UPM09  99.624  99.624  99.624  99.06   96.805  95.677
[HQ197375]
FeLV-UPM17  99.436  99.436  99.436  98.872  96.617  95.489
[JF815540]
FeLV-UPM16  99.436  99.436  99.436  98.872  96.617  95.489
[JF815539]
FeLV-UPM25  99.248  99.248  99.248  98.684  96.429  95.489
[JF815548]
FeLV-UPM19  98.684  98.684  98.684  98.12   96.053  94.737
[JF815542]
FeLV-UPM27  99.06   99.06   99.06   98.496  96.617  95.113
[JF815550]
FeLV-UPM22  99.248  99.248  99.248  98.684  96.617  95.301
[JF815545]
FeLV-UPMIO  99.624  99.624  99.624  99.06   97.18   95.301
[HQ197376]
FeLV-UPM15  99.812  99.812  99.812  99.248  96.992  95.865
[JF815538]
FeLV-UPMll  99.812  99.812  99.812  99.248  96.992  95.865
[HQ197377]
FeLV-UPM13  100     100     100     99.436  97.18   95.677
[HQ727891]
FeLV-UPM05  99.624  99.624  99.624  99.06   96.805  95.301
[HQ197371]
FeLV-UPM07          100     100     99.436  97.18   95.677
[HQ197373]
FeLV-UPM02  100             100     99.436  97.18   95.677
[HQ197368]
FeLV-UPMOl  100     100             99.436  97.18   95.677
[HQ197367]
FeLV-UPM04  99.436  99.436  99.436          96.617  95.489
[HQ197370]
FeLV-UPM08  97.18   97.18   97.18   96.617          93.609
[HQ197374]
FeLV-UPMM   95.677  95.677  95.677  95.489  93.609
[HQ727892]
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Title Annotation:Research Article
Author:Bande, Faruku; Arshad, Siti Suri; Hassan, Latiffah; Zakaria, Zunita
Publication:Veterinary Medicine International
Article Type:Report
Date:Jan 1, 2014
Words:7114
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