1 Graduate Institute of Veterinary Microbiology, National Chung Hsing University, Taichung 40227, Taiwan, Republic of China
2 Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan, Republic of China
3 Department of Veterinary Medicine, National Chung Hsing University, Taichung 40227, Taiwan, Republic of China
Correspondence
Shiow-Her Chiou
shchiou{at}dragon.nchu.edu.tw
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY179166, AY613984, AY613985, AY772190 and AY772191.
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INTRODUCTION |
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In humans, EBV infects B lymphocytes via CD21, a C3d receptor (Fingeroth et al., 1984; Yoshiyama et al., 1997
). Interestingly, introduction of the human CD21 gene into canine or murine cells was found to render cells susceptible to EBV infection (Volsky et al., 1980
; Cantaloube et al., 1990
; Chodosh et al., 2000
; Yang et al., 2000
). Moreover, whilst canine cells are permissive for the latent EBV origin (oriP) to function, rodent cells require an additional human genomic DNA of about 20 kb to support oriP replication (Yates et al., 1985
; Heinzel et al., 1991
; Krysan & Calos, 1993
).
Infection of mice with murine gammaherpesvirus 68 (MHV-68) has been used as an experimental model for EBV pathogenesis (Flaño et al., 2002). However, as a member of the gammaherpesvirus genus Rhadinovirus, MHV-68 is genetically related to herpesvirus saimiri and Human herpesvirus 8 (known as Kaposi's sarcoma-associated herpesvirus) (Doherty et al., 1997
; Virgin et al., 1997
), the viral genomes of which differ from that of EBV; thus, the results of such studies may not be applicable to interpretation of EBV-associated clinical observations. Infection of dogs with canine herpesvirus 1 also cannot be used as a comparative model for EBV because, as an alphaherpesvirus, it is related more closely to varicella-zoster virus (Remond et al., 1996
) and thus is related only distantly to EBV (Davison & Taylor, 1987
). Nonetheless, the intimate contact between human beings and dogs over the last few millennia has provoked anticipation that an unspecified gammaherpesvirus or LCV that shares a common origin with the widespread EBV might exist in the dog.
Traditionally, identification of viruses has relied mainly on virus culture, which has its own limitations, particularly when the virus infection is latent or when the incubation time is longer than expected. In recent years, antibody reactivity and nucleic acid-based techniques have been used successfully to detect viruses before the results of virus culture are available. For example, the utilization of antibodies that cross-reacted with previously known human hantaviruses, together with RT-PCR, led to the discovery of Sin Nombre virus, the causative agent of hantavirus-associated pulmonary syndrome (Nichol et al., 1993).
In this study, we used recombinant EBV-encoded antigens, including EBV-specific DNA polymerase (EDP), EBV-encoded DNA-binding protein (DBP) and EBV-specific thymidine kinase (TK) (Chow et al., 1997), to measure antibody reactivity in the peripheral blood of pet dogs. The EBV-related BamHI W fragment DNA sequence (Baer et al., 1984
) and EBV-encoded RNA (EBER) signal (Howe & Steitz, 1986
) were also examined by PCR and in situ hybridization (ISH), respectively.
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METHODS |
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Western blot analysis of antibodies to EBV antigens.
Purified recombinant EBV-encoded proteins (Chow et al., 1997) EDP, DBP and TK were mixed (250 ng per lane) and subjected to 10 % SDS-PAGE. After electrophoresis, the proteins were electrotransferred to a nitrocellulose membrane. The membrane was incubated with buffer A (PBS with 5 % non-fat dried milk, 1 % rabbit pre-immune serum) for 1 h at room temperature, before being layered onto a Miniblotter apparatus (Immunetics) containing 25 lanes of incubation chambers. Dog serum (diluted 1 : 100 in buffer A) was added to each incubation chamber and incubation was continued for 1 h. The membrane was then removed from the apparatus, washed three times with buffer B (PBS with 0·05 % Tween 20) and incubated for 1 h at room temperature with 1 : 1000-diluted alkaline phosphatase-conjugated anti-canine IgG rabbit antibodies (Jackson ImmunoResearch Laboratories), before chromogenic development with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Sigma).
PCR detection of EBV-related DNA sequences.
Canine leukocyte DNA was subjected to 35 cycles of PCR in a total volume of 50 µl containing 0·5 U Taq DNA polymerase, reaction buffer, 0·2 mM dNTPs, 20 pmol primers and 200 ng DNA, using the standard procedure of denaturation at 94 °C for 1 min, hybridization at 60 °C for 1 min and elongation at 72 °C for 30 s. DNA extracted from the EBV-infected lymphoma cell line BC-2 (ATCC) was used as a positive control (Callahan et al., 1999). The primer sequences specific for the BamHI W region of EBV (GenBank accession no. NC_001345; Baer et al., 1984
) were 5'-GCCAGAGGTAAGTGGACTTT-3' and 5'-TGGAGAGGTCAGGTTACTTA-3'. Two sets of control PCR were carried out by using primer pairs specific for the dog cardiac actin gene (5'-AGCACTGTTAGAGACACCTG-3' and 5'-CGGATAGCACGTTGTTGGCA-3'; Brouillette et al., 2000
) and the human cardiac actin gene (5'-CTGCAGTGTGTCTTATAGGG-3' and 5'-GAATACCAAGACCTGCCTCG-3'; Hamada et al., 1982
). PCR products were resolved by electrophoresis in a 2 % agarose gel.
Cloning of PCR products and DNA sequencing.
PCR-amplified DNA fragments were cloned into the pCRII-TOPO vector (Invitrogen). DNA sequencing was performed by using an automated ABI sequencing system (Applied Biosystems).
Alignment and phylogenetic analysis of DNA sequences.
Alignment of DNA sequences was performed by using GeneWorks software (IntelliGenetics). Based on the percentage difference of homologous nucleotide sequences between viruses, phylogenetic trees were generated by the unweighted pair group mean average (UPGMA) method (Sokal & Michener, 1958; Jobes et al., 1998
), using Kodon sequence-analysis software (Applied Maths BVBA).
ISH.
Paraffin-embedded bone-marrow sections of dog 27 were deparaffinized, dehydrated and predigested with proteinase K. For peripheral leukocytes, cells from dog 28 were isolated, cultured for 2 weeks in RPMI 1640 medium with 10 % fetal bovine serum, centrifuged and fixed in formalin/ethanol solution (4 % formaldehyde in 70 % ethanol) at 4 °C for 10 min.
ISH was performed as described previously (Chow et al., 1992). Briefly, hybridization was carried out in hybridization buffer containing 50 % formamide, 6x SSC, 0·1 % Brij 35 (Sigma), 0·25 % non-fat dried milk and a fluorescein-labelled oligonucleotide probe (250 ng ml1; 5'-fluoresceinTACAGCCACACACGTCTCCTCCCTAGCAAAACCT-3') complementary to EBER-1. Hybridization products were detected with alkaline phosphatase-conjugated rabbit antibodies to fluorescein (Amersham Biosciences). Chromogenic development was carried out by using NBT/BCIP and the slides were counterstained. Cells with purple/blue precipitate in the nucleus were identified as positive for the presence of EBER-1.
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RESULTS |
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DISCUSSION |
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Nonetheless, unlike seropositive carriers and patients with infectious mononucleosis, in whom cell-free EBV can be detected in peripheral blood (Gan et al., 1994), no viral signal was detected in the sera of these dogs. The presence of EBER in bone-marrow cells and peripheral lymphocytes (Fig. 5
) further indicated that an EBV-like virus infection could be latent in these dogs. The persistent presence of antibodies to the viral gene products, which reflects continuous stimulation of the host immune system, on the other hand, suggested that the virus in these dogs might occasionally set off aberrant viral gene expression, reactivation of virus or a mixture of both (Chow et al., 1997
). The prevalence and type of virus infection, as well as the pathophysiological regulation of viral gene expression in the dog that occurs following virus infection, require further studies.
As noted previously, the BamHI W-related sequence detected in dog leukocyte DNA had 99·2100 % identity to that of EBV, and the degree of sequence identity between human EBV and dog EBV-like DNA was higher than that between human EBV and other primate LCVs, e.g. CeHV-12 and CeHV-15, which showed only 80 % identity to EBV. Furthermore, exon W2 of EBNA-LP, which is part of the 241 bp BamHI W fragment, was highly conserved among EBV strains (Fig. 3). The sequence similarity, however, decreased markedly in CeHV-12 and CeHV-15 (Fig. 4
). By determining the sequence identity of dog BamHI W-like DNA to that of human EBV, our results indicated that EBV-like sequences in the dog leukocytes were related far more closely to EBV than to other known LCVs, suggesting that both sequences may originate from the same source.
In terms of other viral gene homologues, it is worth noting that only LCVs encode the EBNA-LP gene and that LCVs have only been detected in primates (Peng et al., 2000; McCann et al., 2001
; Rivailler et al., 2002
; Jenson et al., 2002
). Detection of the EBNA-LP sequence in the dog would indicate the position of canine EBV-like DNA in the evolutionary pedigree of the virus. In an ongoing study, lytic induction of virus and the search for viral particles by electron microscopy are being undertaken.
At present, the results of our immunological and molecular genetic analyses show clearly that a widespread virus could be detected in pet dogs in Taiwan. These results may indicate that this putative canine virus is related closely to EBV. Our data suggest that hosts of LCV may not be restricted to primates. Although much remains to be studied to consolidate the biological significance of this canine EBV-like virus, our results serve as a foundation for further investigations, which may shed light on the correlation with human disorders.
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ACKNOWLEDGEMENTS |
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Received 28 November 2004;
accepted 10 January 2005.
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