Discovery of Epstein–Barr virus (EBV)-encoded RNA signal and EBV nuclear antigen leader protein DNA sequence in pet dogs

Shiow-Her Chiou1, Kuan-Chih Chow2, Chih-Huan Yang3, Shu-Fen Chiang1 and Chun-Hao Lin1

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


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The aim of this study was to investigate Epstein–Barr virus (EBV)-related virus infection in pet dogs. The presence of antibodies to EBV antigens and EBV-related DNA was determined by Western blot analysis and PCR, respectively. Among 36 pet dogs examined for serum antibodies, 32 (88·9 %) were positive for EBV-specific thymidine kinase, 15 (41·7 %) for EBV-encoded DNA-binding protein and 10 (27·8 %) for EBV-specific DNA polymerase. A BamHI W fragment sequence encoding part of the EBV nuclear antigen leader protein was detected by PCR in corresponding leukocyte DNA samples. Among 21 dogs tested, 15 (71·4 %) were positive for the BamHI W fragment sequence. The specificity of the amplified DNA fragments was confirmed by DNA sequencing. Within the amplified region of the BamHI W fragment (241 bp), DNA sequences detected in 10 dogs had 99·2 % (two nucleotide variations), 99·6 % (one nucleotide variation) or 100 % identity to that of EBV. Furthermore, an EBV-encoded RNA signal was detected by in situ hybridization in dog lymphocytes, as well as in bone-marrow sections, indicating a latent infection with EBV or an EBV-like virus. In conclusion, although the sample size was small, these results showed that a widespread EBV-related gammaherpesvirus could be detected in the peripheral blood and bone marrow of pet dogs. Although no evident zoonotic transmission was detected, further studies are imperative for disclosing the biological significance of this canine EBV-like virus, which may correlate with human disorders.

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY179166, AY613984, AY613985, AY772190 and AY772191.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Epstein–Barr virus (EBV) is a widespread gammaherpesvirus in the human population, with over 90 % of adults being seropositive for the virus (Henle et al., 1969). EBV can cause infectious mononucleosis and is associated with several malignancies, including Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease and T-cell lymphoma (Epstein et al., 1964; Henle et al., 1968, 1970; Pallesen et al., 1993). Although other species of the gammaherpesvirus genus Lymphocryptovirus have been identified in primates, EBV is the only lymphocryptovirus (LCV) that has been detected in humans (Chang et al., 1994; Virgin et al., 1997). In vivo, EBV has been used to infect New World monkeys, including marmosets and owl monkeys (Epstein et al., 1973; Shope et al., 1973; Wedderburn et al., 1984). In addition, LCVs isolated from primates, such as Cercopithecine herpesvirus 12 (CeHV-12) from baboons (also known as herpesvirus papio) (McCann et al., 2001), CeHV-15 from rhesus monkeys (rhesus EBV-like herpesvirus) (Peng et al., 2000), EBV-related herpesvirus from cynomolgus macaques (Ohara et al., 2000) and callitrichine herpesvirus 3 from the common marmoset (Cho et al., 2001; Rivailler et al., 2002), have been used as animal models for investigating EBV pathogenesis. However, the high cost of maintaining a large quantity of primates is the inevitable burden for these studies to obtain a significant outcome. Studies of EBV pathogenesis are, therefore, still reliant on clinical subjects.

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Dog sera and leukocyte DNA.
After obtaining permission from the owners, blood samples from 36 pet dogs were collected from general households. Total cellular DNA was extracted from leukocytes that were isolated by using Ficoll-Hypaque (Sigma). Leukocytes were then resuspended in STE buffer [100 mM NaCl, 50 mM Tris/HCl (pH 8·0), 15 mM EDTA], lysed with 0·4 % SDS and incubated with 200 µg proteinase K ml–1 at 65 °C for 1 h. After phenol/chloroform extraction, DNA was precipitated by using ethanol and dissolved in TE buffer [10 mM Tris/HCl (pH 8·0), 1 mM EDTA].

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 ml–1; 5'-fluorescein–TACAGCCACACACGTCTCCTCCCTAGCAAAACCT-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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Characteristics of the dogs
The mean age of the 36 pet dogs was 3·0±3 years. The ratio of females to males was 1 : 1 (Table 1). Dog breeds included Maltese (8·3 %; dogs 30, 31 and 36), Akita (2·8 %; dog 14), Shetland sheepdog (2·8 %; dog 32), Chihuahua (2·8 %; dog 33) and mongrel (83·3 %). Most of these dogs appeared healthy when their blood samples were collected, with the exception of dog 27, which had been diagnosed with anaemia, thrombocytopenia and a mammary-gland tumour. Unfortunately, dog 27 died within 1 week of blood collection; with the owner's permission, we collected a piece of bone for further examination.


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Table 1. Details of dogs and detection of antibodies to EBV proteins, EBV-related DNA sequence and EBER signal

+, Positive result; ++, strongly positive result; –, negative result; ND, not determined.

 
Detection of serum antibodies to EBV antigens
By using purified recombinant EBV proteins as antigens (Chow et al., 1997), the presence of serum antibodies to EBV was determined by Western blot analysis. The results showed that, among the 36 pet dogs examined, 10 (27·8 %) were seropositive for EBV-specific EDP, 15 (41·7 %) for EBV-encoded DBP and 32 (88·9 %) for EBV-specific TK. Among them, dogs 19 and 22 possessed strong antibody signals against EDP, DBP and TK. Serum from dog 16 reacted strongly with both DBP and TK proteins. Dogs 6, 7, 9, 17 and 35 were strongly seropositive for TK only (Fig. 1 and Table 1). The molecular masses of the recombinant EBV proteins were 110 (EDP), 76 (DBP) and 73 (TK) kDa.



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Fig. 1. Western blot analysis of antibodies to recombinant EBV proteins in dog serum. Each nitrocellulose-membrane strip contained 250 ng purified recombinant EDP, DBP or TK. P, The three EBV proteins stained with Coomassie blue; M, molecular mass marker. Serum from individual dogs (dogs 1–36 in lanes 1–36, respectively) was used in each hybridization strip. The positions of the three EBV proteins in the gel are indicated.

 
Detection of EBV-related DNA sequences
Specific PCR primers were synthesized to detect a DNA sequence (241 bp) within the BamHI W fragment of EBV (Hayward et al., 1982; Baer et al., 1984). Interestingly, 15 out of 21 (71·4 %) leukocyte DNA samples were positive for this sequence (Fig. 2, Table 1). Nonetheless, no EBV-related sequence was ever detected in serum (data not shown).



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Fig. 2. EBV-related DNA sequences detected in dog leukocyte DNA. The presence of DNA sequences related to the BamHI W fragment of EBV was detected by PCR using primers specific to the gene. –, Negative control without DNA template; BC-2, DNA from EBV-infected BC-2 lymphoma cells was used as template. PCR products were amplified from leukocyte DNA of the dogs indicated above the lanes.

 
Alignment and phylogenetic analysis of EBV-related DNA sequences
PCR-amplified BamHI W-related sequences from the leukocyte DNA of 10 dogs were cloned and sequenced. Nucleotide sequences of the DNA fragments from dogs 12, 14, 17, 30, 33 and 36 had 100 % identity, those from dogs 27, 32 and 34 had 99·6 % identity (varied in 1 nt) and that from dog 28 had 99·2 % identity (varied in 2 nt) to the 241 bp stretch of the EBV BamHI W region (Fig. 3). The sequences of the PCR-amplified products from dogs 27, 28, 32 and 34 were verified repeatedly and the variations were confirmed. Interestingly, in addition to the variant sequences (Fig. 3), these dogs concurrently carried sequences that had 100 % identity to the 241 bp stretch of the BamHI W region of EBV. Within this region, EBV sequences of BC-2 cells and of the B95-8 (Baer et al., 1984), P3HR-1 (Jenson et al., 1987) and Elijah (McCann et al., 2001) strains are identical, whereas EBV strain Akata (McCann et al., 2001) has a 1 nt deletion in the non-coding region (Fig. 3). In fact, the BamHI W region of EBV is differentially spliced and encodes the EBV nuclear antigen leader protein (EBNA-LP) (Dillner et al., 1986; Rowe et al., 1987). The 241 bp stretch contains partial exon W1' and complete exon W2 of the EBNA-LP gene (Peng et al., 2000). However, phylogenetic analysis of the EBNA-LP gene (or BamHI W region) showed that, within this 241 bp region, the percentage difference in nucleotide sequence between EBV and other primate LCVs, including CeHV-12, CeHV-15, Pongine herpesvirus 1 (PoHV-1) and PoHV-3 (Peng et al., 2000; McCann et al., 2001; Rivailler et al., 2002), varied from 17 to 42 % (Fig. 4, Table 2). Interestingly, the BamHI W sequences from the EBV strains and dog leukocyte DNA were related more closely to CeHV-12 and CeHV-15 than to PoHV-1 and PoHV-3 (Fig. 4, Table 2).



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Fig. 3. Sequence alignment of BamHI W fragment homologues. The BamHI W-related sequence (241 bp) amplified by PCR from leukocyte DNA of dogs 12, 14, 17, 30, 33 and 36 (nt 1–241, GenBank accession no. AY179166) completely matched the BamHI W region of EBV strains B95-8 (nt 14613–14853, NC_001345), P3HR-1 (nt 1400–1640, M15973) and Elijah (nt 90–330, AJ311192). However, EBV strain Akata (nt 90–329, AJ311190) had a 1 nt deletion, dogs 27 (nt 1–241, AY772190), 32 (nt 1–241, AY613985) and 34 (nt 1–241, AY613984) had one nucleotide variation and dog 28 (nt 1–241, AY772191) had two nucleotide variations within this region. Amino acid sequences encoded by the exon regions of EBNA-LP, which are interrupted by an intron spanning nt 8–88, are boxed. One of the nucleotide variations (A->G) detected in dog 28 would change the encoded amino acid from glutamate (E) to glycine (G) (denoted by *).

 


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Fig. 4. Phylogenetic relationship of BamHI W-related sequences. Phylogenetic analysis indicated that, after the BamHI W-related sequences detected in dogs, the LCV related most closely to EBV is CeHV-12 (17·52 % nucleotide difference).

 

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Table 2. Nucleotide difference (%) of BamHI W-related sequences

Nucleotide difference between the BamHI W fragment of EBV (nt 14613–14853, GenBank accession no. NC_001345) and the BamHI W-related sequences from leukocyte DNA of dogs 12, 14, 17, 30, 33 and 36 (nt 1–241, AY179166) was 0 %. The percentage nucleotide differences among BamHI W-related sequences from CeHV-12, CeHV-15, PoHV-1 and PoHV-3, which were retrieved from GenBank (nt 90–329, AJ311198; nt 791–1032, AF200821; nt 136–363, AJ311197; and nt 133–360; AJ311195), are listed.

 
Detection of EBER signal by ISH
EBERs are EBV-encoded, small, non-polyadenylated RNAs that are expressed abundantly in latently infected cells (Howe & Steitz, 1986). To detect EBER expression, ISH was performed on paraffin-embedded bone-marrow sections from dog 27 and peripheral leukocytes of dog 28. A positive EBER signal was detected in the nuclei of bone-marrow cells from dog 27 (Fig. 5a) and in atypical lymphocytes from dog 28 (Fig. 5b). Depending on the microscopic fields, the proportion of EBER-positive cells varied from 1 to 15 % among six slides examined.



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Fig. 5. Detection of EBER signal by ISH. EBER (indicated by arrows) was detected in the nuclei of bone-marrow cells from dog 27 (original magnification x1000) (a) and in atypical lymphocytes from dog 28 (original magnification x400) (b).

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The results of immunological and nucleic acid analyses presented above showed that an EBV-like virus infection was detected frequently in the peripheral blood of pet dogs in Taiwan. Moreover, as EBV-specific TK, DBP and EDP are expressed during the early stage of virus infection (de Turenne-Tessier et al., 1986; Fixman et al., 1992), the presence of strong antibody signals to these EBV antigens indicated that some of these dogs (6, 7, 9, 16, 17, 19, 22 and 35; Fig. 1 and Table 1) could be experiencing virus reactivation.

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 99·2–100 % 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.


   ACKNOWLEDGEMENTS
 
We would like to thank Ms Yun-Ping Chang for collecting blood samples from the pet dogs. We are grateful to Ms Chih-Yo Kuan and Ms Chao-Min Wang for their technical assistance.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 28 November 2004; accepted 10 January 2005.



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