FULL PAPER Virology

Identification of the Porcine Major Capsid Protein Gene

Vasantha RUPASINGHE1), Kiyoko IWATSUKI-HORIMOTO2), Shunji SUGII1) and Taisuke HORIMOTO2)*

1)Department of Veterinary Microbiology, Graduate School of Osaka Prefecture University, 1–1 Gakuen, Sakai, Osaka 599–8531 and 2)Division of Virology, Institute of Medical Science, University of Tokyo, 4–6–1 Shirokanedai, Minato-ku, Tokyo 108–8639, Japan

(Received 13 November 2000/Accepted 14 February 2001)

ABSTRACT. A major capsid protein (MCP) gene homologue of porcine cytomegalovirus (PCMV) was identified. Sequence analysis indi- cated that the PCMV MCP gene is 4,026 nucleotides in length encoding a protein of 1,341 amino acid residues. The predicted molecular weight of the PCMV MCP is 151,456 Da, equivalent to those of other herpesvirus MCP counterparts. Phylogenetic analysis using her- pesviral MCP gene sequences confirmed that PCMV is a betaherpesvirus with higher homology with human herpesvirus-6 and -7 than human and mouse . The serum of pig experimentally infected with PCMV did not react with bacterially expressed MCP, suggesting that the PCMV MCP may not be related to the humoral immune response in the course of PCMV infection. Also, we established polymerase chain reaction (PCR) protocols using primers corresponding to MCP gene sequences for detection of PCMV infection. The PCR protocol would be effective for the diagnosis of slow-growing PCMV infection, for which traditional methods involving -isolation are not useful. KEY WORDS: betaherpesvirus, major capsid protein, polymerase chain reaction, porcine cytomegalovirus. J. Vet. Med. Sci. 63(6): 609–618, 2001

Porcine cyomegalovirus (PCMV), first described in 1955 established a polymerase chain reaction (PCR) protocol for [6], usually induces a silent infection in adult pigs but often specific detection of PCMV infection. a fatal, generalized infection in piglets. In utero infection in sows can cross the placenta and infect the fetuses leading to MATERIALS AND METHODS fetal death or birth of weak piglets [3, 17]. As PCMV exhib- its a relatively protracted replication cycle, a slowly devel- Cells and virus: Pig fallopian tube fibroblast cells (PFT- oping cytopathology characterized by cytomegaly, and a F) [11] were grown in Dulbecco’s modified Eagle’s medium restricted host range, it is grouped into the subfamily Beta- (DMEM) supplimented with 5% fetal calf serum at 37°C. herpesvirinae [18]. Recently, we and other research groups The OF-1 strain of PCMV [11] was propagated in the PFT- confirmed that PCMV is a betaherpesvirus based on F cell line and used in this study. The virus was harvested sequence analyses of DNA polymerase gene fragments [19, exclusively from the culture supernatant at 7–10 days after 29]. infection as guided by the appearance of cytopathic effects The major capsid protein (MCP) gene appears to be con- (CPE). Other Japanese PCMV strains, J1, Chiba-2, Chiba- served among the herpesviruses [5, 15], suggesting that 3, Chiba-C, Hiroshima and Kagawa, and a British isolate, MCP should play essential roles in vital processes in the B6, were also used. All Japanese strains except J1 [21] were viral replication cycle such as assembly and maturation. isolated in our laboratory [24]. In addition, -1 (HSV-1) has been shown to encode virus (strain Indiana) was propagated in the RK-13 cell line seven capsid proteins. Of these, MCP is the structural sub- and used. RK-13 cells were grown in DMEM with 10% unit of the hexons and pentons, and is the principal struc- fetal calf serum. tural element making up the matrix of the icosahedral capsid Preparation of PCMV DNA: Extrachromosomal DNA [16]. In addition, in some herpesviruses such as HSV-1 and containing viral DNA was extracted from virus-infected varicella-zoster virus (VZV), MCPs serve as an antigen for cells by a modification of Hirt’s method [9]. Briefly, cell the humoral immune response during the course of infec- pellets were suspended gently in 20 mM Tris-HCl buffer, tion, being highly reactive with human antisera as well as pH 7.5, containing 0.6% SDS and 0.01 M EDTA, and incu- being responsible for cell-mediated immunity [7, 20, 27]. bated at room temperature for 20 min. Then, 1/3 volume of The MCPs of many herpesviruses have been shown to 5N NaCl was added and mixed by inverting the tube. The have similar molecular masses, e.g., between 135 kDa and sample was incubated at 4°C for at least 8 hr, and the lysate 160 kDa for human herpesviruses [2, 4, 10, 14, 15]. In this recovered after centrifugation (15,000 rpm, 30 min) was fur- study, we identified the MCP gene of PCMV and expressed ther mixed with the same volume of 2 × lysis buffer (20 mM the protein in a bacterial expression system. In addition, we Tris-HCl, pH 7.5, 2 mM EDTA, 0.1% SDS, and 40 µg/ml also analyzed the evolutionary position of PCMV in the her- proteinase K) and incubated at 37°C for 1 hr. Following pesvirus group using MCP sequence information. We also phenol-chloroform extraction, DNA was ethanol-precipi- tated and suspended in deionized water. *CORRESPONDENCE TO: HORIMOTO, T., Division of Virology, Insti- Cloning of PCMV DNA fragments: The Hirt’s DNA from tute of Medical Science, University of Tokyo, 4–6–1 Shirokane- dai, Minato-ku, Tokyo 108–8639, Japan. virus-infected cells was digested with BamHI endonuclease 610 V. RUPASINGHE ET AL. and the digested fragments were directly ligated with reading frame (ORF) by PCR using a pair of primers, MCP/ BamHI-digested vector (pBluescript SK-; Stratagene, La I/E and MCP/T/X (Table 1). These primers included a Jolla, USA). These constructs were then used for transfor- unique restriction site for EcoRI and XhoI, respectively, to mation of Escherichia coli strain (DH5α). The transfor- facilitate cloning. PCR was performed using the B18 tem- mants were selected on 2YT agar plates with ampicillin. plate DNA with LA Taq DNA polymerase (Takara, Tokyo, DNA sequencing and genetic analyses: Plasmids were Japan); for 35 cycles of denaturation at 94°C for 1 min, isolated from each transformant by mini-preparation and annealing at 50°C for 1 min, and strand-extension at 72°C partially sequenced by the dye-terminator protocol using for 5 min. The amplified product was ligated into the M13 primers. Briefly, the plasmid DNA (0.5 µg) was mixed pCR2.1 vector (Invitrogen, Carlsbad, U.S.A.) and then the with 3.2 nmol of primer, -21M13 or M13 Rev, in a sequenc- construct was used for transformation of E. coli. The plas- ing mixture (BigDye-terminator reagent; Applied Biosys- mid extracted from the transformant was digested with tems Japan, Tokyo, Japan) and reacted in a thermal cycler EcoRI and XhoI, and the fragment containing MCP-ORF (MJ Research Inc., Waltham, U.S.A.) according to the man- was subcloned into EcoRI and XhoI sites of the expression ufacturer’s instructions. The reaction products were etha- vector pGEX 6P-3 (Amersham Pharmacia Biotech, Little nol-precipitated and suspended in template suppression Chalfont, England) in-frame with the glutathione s-trans- reagent (Applied Biosystems) for sequencing in an autose- ferase (GST) gene, generating pGEX/MCP. A series of quencer (ABI PRISM 310). deletion mutants of the carboxyl (C)-terminal portion of the Sequencing of the MCP gene, which was included in MCP-ORF were prepared by endonuclease digestion using clone B18 (with an insert consisting of a 9 kbp BamHI frag- combinations of XhoI with XbaI, BglII, NdeI or HindIII, ment), was performed by the “primer walking” method with unique recognition sites for which are present in the ORF. primers designed from the known sequence towards the The portion between two enzyme recognition sites was unknown region. The primers are listed in Table 1. The deleted and the resulting plasmid was self-ligated in a blunt- homology search was carried out using the BLAST program ended manner and used for transformation. These plasmids (DNA Data Bank of Japan [DDBJ]). Multiple alignment were used for expression of deletion forms of the fusion pro- with other herpesviral MCPs was performed using the tein, generating GST/MCP∆Xb/Xh, -∆Bg/Xh, -∆Nd/Xh and CLUSTAL W program [25] with bootstrap analysis -∆Hd/Xh. (DDBJ). A phylogenic tree was generated using Tree View Expression of GST/MCP fusion protein or GST/MCP software (ver. 1.5). deletion proteins was examined using anti-GST antibody by Expression of MCP in E. coli.: To generate a vector plas- immunoblotting assay. Briefly, the lysate of the isopropyl- mid for MCP expression, we amplified the MCP gene open β-D-thiogalactosidase (IPTG)-induced cells transformed

Table 1. Oligonucleotide primers used in this study Primer Sequence (5’ → 3’)a) Positionb) (Polarity) Purpose B18F(10) TCCAATGCAGTCCGTAGACG PCP:downstream (-) Seq.c) B18F(11) AACTCGCCTTCTCTTCTATG PCP:829–810 (-) Seq. B18F(12) GAGGCAGTACCAGCTCCCAG PCP:388–369 (-) Seq. MCP/T/X AACTCGAGTTAGGCGCTCAGTATGCTGG MCP:4026–4007 (-) PCR cloning B18F(13) AACCTGGTCTCTAATAGAGC MCP:3935–3916 (-) Seq. B18F(14) TCACCGGGGTTATGATCGCC MCP:3463–3444 (-) Seq., PCR B18F(15) AGTGCATGGCCGACAGAGTG MCP:2962–2943 (-) Seq., PCR B18F(16) GTTCTCCGTCATGAAACAG MCP:2588–2569 (-) Seq., Probe B18F(17) GGCATCGTGTGCATGAACGG MCP:2175–2146 (-) Seq. B18R(5) TCGCAGTAGCAAGGCACCG MCP:upstream (+) Seq. MCP/I/E ATGAATTCATGGAAGACTGGAGGGCCACMCP:1–20 (+) PCR cloning B18R(6) AATGACACTCGGGAAAATGC MCP:255–274 (+) Seq. B18R(7) ACCTATAAATCTAGCGATGAG MCP:778–798 (+) Seq. B18R(8) CTTTGCCACCCGGCGGTCAAC MCP:1345–1365 (+) Seq. B18R(9) ATCCTGCTGTTCTGCAATAG MCP:1927–1946 (+) Seq. B18R(C1) GCGTCATACCCGCACTGACG MCP:2351–2370 (+) Seq., PCR B18R(10) TCTCACTCTCATACAGGATG MCP:2493–2512 (+) Seq., PCR B18R(11) ACAGACAATATCCTCTATAC MCP:3055–3074 (+) Seq., PCR MCP(P1) AGCGCGTAAAGACAGACATG MCP:3224–3243 (+) Probe B18R(C2) GATCATAACCCCGGTGACCG MCP:3447–3466 (+) Seq. B18R(12) TGTACGATCACTCCAACACC MCP:3566–3585 (+) Seq. B18R(13) TCCGATGTCAGCAAACTGTG PCP:43–62 (+) Seq. B18R(14) GCATGCATAGCATTTTCTATG PCP:580–600 (+) Seq. a) Restriction enzyme recognition sites are underlined; XhoI site for MCP/T/X and EcoRI site for MCP/I/E were included to facilitate cloning of PCR products. b) Positions are shown as nucleotide positions in the open reading frame for major capsid protein (MCP) or putative capsid protein (PCP). c) Seq.=for sequencing. MCP OF PORCINE CYTOMEGALOVIRUS 611 with pGEX/MCP or its deletion derivatives was separated preparation with cellular DNA. We randomly selected by 10% SDS-polyacrylamide gel electrophoresis (SDS- some clones and partially sequenced them using M13 prim- PAGE) and transferred onto polyvinylidene difluoride ers. A homology search of these sequences with other herp- (PVDF) membranes (Nihon Millipore, Tokyo, Japan). esviral sequences in databases indicated that several clones After blocking with Blockace reagent (Dainippon Seiyaku, might include PCMV DNA fragments. From these, we Osaka, Japan), the membranes were reacted with anti-GST selected one clone, B18, with a large insert (approximately antibody (1:2000) followed by horseradish peroxidase- 9 kbp) for further analysis to find undefined PCMV genes. labeled second antibody (1:1000) and a substrate (0.25 mg/ Sequence analysis of B18 DNA and a homology search ml 3,3’-diaminobendizine tetrahydrochloride, 0.03% H2O2) indicated that this fragment included the entire MCP gene to detect fusion proteins. homologue of PCMV with its surrounding regions. PCR and Southern blotting: For PCR, we selected some Figure 1 shows the entire PCMV MCP gene sequence from the primers that were synthesized for sequencing of the and its adjacent sequence. The MCP-ORF is 4,026 nucle- OF-1 strain MCP gene. Here, we used three pairs of prim- otides in length encoding a protein of 1,341 amino acid res- ers, F(15)/R(C1), F(15)/R(10) and F(14)/R(11). Template idues. The predicted molecular size of MCP is 151,456 Da, DNAs were prepared from cells infected with the OF-1 equivalent with those of other herpesvirus MCP counter- strain as well as other PCMV isolates and pseudorabies parts (e.g., 149 kDa for HSV-1 MCP and 154 kDa for virus (PRV) as a control. Template DNA (1 µg) was reacted HCMV MCP). in a mixture containing 32 nmol of each primer, 20 µM of In addition, we found another ORF (888 bases) down- each deoxynucleotide triphosphate, 2 mM MgCl2 and 2U stream of the MCP-ORF, encoding a protein with 295 amino Taq DNA polymerase (Takara) in 50 µl of reaction buffer. acids (33,392 Da) (Fig. 1). Homology search predicted that The mixture was then amplified in a thermal cycler (MJ this gene was the PCMV homologue of the herpesviral Research Inc.) by 30 cycles of denaturation at 94°C for 1 “probable capsid protein (PCP)” gene (e.g., HSV-1 UL18, min, annealing at 50°C for 1 min, and strand-extension at HCMV UL85), whose biological functions have been unde- 72°C for 2 min. After cycling, the reaction mixture was fined. incubated at 72°C for 5 min. The amplified products were Expression of the MCP in E. coli: To confirm that the analyzed by electrophoresis in 2% agarose gels, stained with MCP-ORF encodes a protein, we inserted the ORF ethidium bromide, and visualized on a UV transilluminator. sequence in the bacterial expression vector pGEX. Anti- The gels were soaked in denaturing buffer (0.5 N NaOH, GST antibody was used for detection of GST/MCP fusion 0.6 M NaCl) for 1 hr, then neutralized in a solution of 1 M protein, because of the lack of an anti-MCP antibody. Tris-HCl, pH 7.2, 0.6 M NaCl, and transferred overnight Expression of GST/MCP fusion protein with a predicted onto nylon membranes (Hybond N+: Amersham Pharmacia size of 178 kDa (includes 27 kDa GST) was hardly detected Biotech) through 10 × SSC buffer (0.15 M sodium citrate, (Fig. 2). However, the deletion derivatives of the GST/MCP 1.5 M NaCl, pH 7.0). The membranes were dried, baked at with the appropriate molecular weights were detected (44 120°C for 2 hr, and used for hybridization. According to the kDa for GST/MCP∆Xb/Xh, 79 kDa for GST/MCP∆Bg/Xh, MCP sequence, we prepared two kinds of probe DNA; 119 kDa for GST/MCP∆Nd/Xh, and 152 kDa for GST/ F(16) for the PCR products generated with primer pairs of MCP∆Hd/Xh), indicating that low expression level of the F(15)/R(C1) and F(15)/R(10), and MCP-P(1) for the prod- GST/MCP was due to its large insert. Nevertheless, these ucts generated with F(14)/R(11). These probe DNAs were observations confirmed that MCP-ORF encodes a protein. labeled with digoxigenin (DIG) and used for hybridization. Immunogenicity of MCP in PCMV-infected pigs: We Following pretreatment of the membranes with hybridiza- examined immunogenicity of MCP using experimentally tion buffer (5 × SSC, 0.1% N-lauroylsarcosine, 0.02% SDS, PCMV-infected SPF pig serum. Immunoblotting assay did 2% blocking reagent [Roche Diagnostics, Basel, Switzer- not detect specific antibody to bacterially expressed MCP in land], and 50% formamide), hybridization was performed in the pig serum, although this serum reacted a considerable the same buffer with probes at 42°C overnight. Washing number of viral proteins in infected PFT-F cell lysate (data and detection were performed according to the instructions not shown). supplied with a commercial kit (DIG high prime DNA label- Genetic relationship of PCMV-MCP to other herpesvirus ing and detection kit; Roche Diagnostics). MCPs: To determine the phylogenetic position of PCMV, we examined the genetic relationships among this and other RESULTS herpesviruses based on their MCP genes (Fig. 3). Three clusters of MCP genes obviously represented each herpesvi- Cloning and sequencing of PCMV MCP gene: There has rus subfamily, i.e. alpha-, beta- and gammaherpesviruses, been no previous report of PCMV DNA physical maps or confirming that PCMV is a betaherpesvirus. The phyloge- genes except for DNA polymerase gene fragments [19, 29]. netic tree also indicated that PCMV is genetically closer to Here, we cloned unfractionated BamHI fragments of Hirt’s human herpesvirus-6 (HHV-6) and -7 (HHV-7) than human DNA from PCMV OF-1-infected cells in the vector plas- (HCMV) or mouse (MCMV) cytomegaloviruses. Align- mid. However, not all of the clones included PCMV DNA ment of the amino acid sequences among PCMV, HHV-6, fragments because of contamination of the Hirt’s DNA and HHV-7 is shown in Fig. 4. 612 V. RUPASINGHE ET AL.

Fig. 1. Sequences of major capsid protein and probable capsid protein genes of PCMV. Deduced amino acid sequences are also shown. Region of probable capsid protein gene was shaded. Only open reading frames are shown. The sequence has been assigned DDBJ/EMBL/GenBank accession number AB051069. MCP OF PORCINE CYTOMEGALOVIRUS 613

Fig. 2. Expression of the GST/MCP fusion protein and its deletion derivatives in E. coli. Fusion proteins were detected on immunoblotting assay using anti-GST antibody. Dots indicate positions of fusion proteins. Molecular size markers are shown on the left.

Table 2 shows percentages of amino acid identity of based on the MCP gene sequences and used them for PCR PCMV MCP to other herpesvirus MCPs. The highest amplification of gene fragments. As shown in Fig. 5A, all degree of homology was seen with HHV-6 MCP (53.2% of the three primer pairs amplified DNA fragments from identity), followed by other betaherpesvirus MCPs (around Hirt’s DNA preparations extracted from OF-1-infected cells 50%). The extent of amino acid identity shared with alpha- but not from mock-infected or PRV-infected cells. herpesviruses was between 23–25% and that with gamma- Although amplified DNAs had expected molecular sizes herpesviruses was between 28–30%. (611bp for F(15)/R(C1), 469bp for F(15)/R(10), and 408bp PCR detection of PCMV infection: PCR would be useful for F(14)/R(11) primers), we confirmed by Southern blot- for detection of PCMV infection in animals, since virus-iso- ting analysis that they contained virus-specific sequences lation is not a sensitive method due to the lack of highly sus- (Fig. 5B). ceptible cell lines and to its slow replication cycle with Specific PCR amplification was also observed from all of ambiguous CPE. Therefore, we designed three primer pairs the additional PCMV isolates examined including a British 614 V. RUPASINGHE ET AL.

Fig. 3. Phylogenetic tree of the herpesviral MCPs. Bootstrap values are indicated at each node of the branches. Virus abbreviations are the same as those in the footnote of Table 2. isolate (Fig. 5A), indicating that the sequences of the prim- than those of antibodies to envelope glycoproteins [7, 27]. ers are conserved on the MCPs among PCMV strains. Antibodies specific to MCPs are barely detected in HCMV- or EBV-infected human serum or PRV-infected pig serum DISCUSSION [10, 30]. These observations suggested that the humoral immune response of herpesvirus MCPs is poor. In this study, we determined the entire sequence of In this study, we found the PCP-ORF downstream of the PCMV MCP and successfully expressed it in E. coli. This MCP-ORF. It is intresting to note that these two ORFs are is the first large-scale analysis of sequences of the PCMV in-frame and have no intermediate sequence. Further stud- genome. The deduced amino acid sequence of the PCMV ies are required to determine the transcriptional and transla- MCP had a high predicted molecular weight similar to those tional regulation for these two genes. of other herpesvirus MCPs [12–15, 30], suggesting that her- PCMV infection causes an important known as pesvirus MCP plays an important molecule forming the “inclusion body rhinitis” in pigs. The traditional method for capsid structure. diagnosing PCMV infection involves virus-isolation and Amino acid identity and phylogenetic analysis of the detection of virus-antigen or specific antibodies by indirect PCMV MCP with other herpesvirus MCPs indicated that immunofluorescence test or enzyme-linked immunosorbent PCMV is a betaherpesvirus with higher degrees of homol- assay [1, 22, 23]. PCMV can be isolated from affected ani- ogy to HHV-6 and -7 than HCMV or MCMV. These results mals by sampling the nasal mucosa, lung and kidney, and by confirmed our earlier conclusions based on partial sequence making direct cultures of lung macrophages if possible. analysis of the DNA polymerase gene of PCMV [19, 29]. It However, good quality of samples including large amounts is interesting to note that the biological properties of PCMV of infectious is required for successful virus-growth appeared to be quite different from those of HHV-6 and in cell culture. Furthermore, since PCMV is a slow growing HHV-7, which are lymphotropic, although these viruses are virus, it takes about 7–10 days to detect the CPE of cytome- genetically close. galy in cell culture. Thus, PCR would be a more sensitive Bacterially expressed MCP or its deletion derivatives and more rapid method for detecting PCMV infection in could not be detected by immunoblotting with serum from a organs from affected animals as well as in cell culture. The pig experimentally infected with PCMV, suggesting that three primer pairs designed for use in our PCR protocols PCMV MCP may not be related to the humoral immne would be more useful for detecting heterogenous wild-type response in the course of natural PCMV infection, or that PCMV strains, compared to the previous PCR protocols antigenicity of expressed MCP may be different from that of based on DNA polymerase gene sequences whose conserva- intact MCP. It has been shown that sera from humans tiveness among herpesvirus family may result in false posi- infected with HSV-1 or VZV are reactive with each MCP. tive signals [8, 29]. Studies are currently in progress to However, the antibody titers are variable and much lower assess whether this PCR protocol can be applied for detec- MCP OF PORCINE CYTOMEGALOVIRUS 615

Fig. 4. Alignment of amino acid sequences of PCMV, HHV-6 and HHV-7 MCPs. Asterisks indicate conserved amino acid residues, and dots indicate conservative amino acid substitutions. 616 V. RUPASINGHE ET AL.

Table 2. PCMV MCP amino acid identity to other herpesvirus MCP Alphaherpesvirinaea) Betaherpesvirinaea) Gammaherpesvirinaea) Viruses A.A.b) % Viruses A.A. % Viruses A.A. % VZV 1397 23.9 HHV-6 1346 53.2 HHV-8 1377 28.4 HSV-1 1375 23.1 HHV-7 1346 51.9 EHV-2 1382 29.4 HVT 1413 23.5 HCMV 1371 44.3 EBV 1382 30.9 BHV-1 1386 25.1 MCMV 1353 46.8 AtHV 1372 29.3 EHV-1 1377 24.8 HVS 1372 29.0 PRV 1330 24.4 AIHV 1371 29.5 a) include varicella-zoster virus [VZV] (GenBank accession no. X04370.1), herpes simplex virus 1 [HSV-1] (X04467), herpesvirus of turkey [HVT] (Z54369), bovine herpesvirus 1 [BHV-1] (AJ004801), equine herpesvirus 1 [EHV-1] (M86664), and pseudorabies virus (PRV) [30]. include [HHV-6] (X83413), human herpesvirus 7 [HHV-7] (U43400), human cytomegalovirus [HCMV] (M25411), and mouse cytomegalovirus [MCMV] (U68299). include human herpesvirus 8 [HHV- 8] (U75698), equine herpesvirus 2 [EHV-2] (U20824), Epstein-Barr virus [EBV] (V01555.1), ateline herpesvirus [AtHV] (AF083424), saimirine herpesvirus [HVS] (X64346) and alcelaphine herpesvirus [AlVH] (AF005370). b) A. A.=amino acid. tion of PCMV infection in clinical samples or latent infec- ruses. J. Gen. Virol. 64: 927–1942. tion in organs. 6. Done, J. T. 1955. An “inclusion-body” rhinitis of pigs. Vet. Xenotransplantation of pig organs to humans is a promis- Rec. 67: 525–527. ing but exceedingly complex issue. There are a number of 7. Eberle R. and Mou, S.-W. 1983. Relative titers of antibodies to major barriers to successful xenotransplantation, including individual polypeptide antigens of herpes simplex virus type 1 in human sera. J. Infect. Dis. 148: 436–444. immunological, physiological, anatomical, infectious and 8. Hamel, A. L., Lin, L., Sachvie, C., Grudeski, E. and Nayar, G. ethical problems. A recent report demonstrating transmis- P. S. 1999. PCR assay for detecting porcine cytomegalovirus. sion of an infectious agent from pigs to mice via trans- J. Clin. Microbiol. 37: 3767–3768. planted organs has shown one example of such barriers [28]. 9. Hirt, B. 1967. Selective extraction of polyoma DNA from One report described evaluation of PCMV as a potential infected mouse cell cultures. J. Mol. Biol. 26: 365–369. zoonotic agent in xenotransplantation [26]. In this report, 10. Jahn, G., Scholl, B.-C., Traupe, B. and Fleckenstein, B. 1987. PCMV did not infect some cell cultures of human origin. The two major structural phosphoproteins (pp65 and pp150) of However, this result does not necessarily exclude the possi- human cytomegalovirus and their antigenic properties. J. Gen. bility of infection to human cells by PCMV in vivo. The Virol. 68: 1327–1337. PCR system described in the present report could be useful 11. Kawamura, H., Tajima, T., Hironao, T., Kajikawa, T. and Kotani, T. 1992. Replication of porcine cytomegalovirus in the for sensitive detection of PCMV infection in organs used in 19-PFT cell line. J. Vet. Med. Sci. 54: 1209–1211. xenotransplantation. 12. Killington, R. A., Yeo, J., Honess, R. W., Watson, D. H., Dun- can, B. E., Halliburton, I. W. and Mumford, J. 1977. Compara- ACKNOWLEDGMENT. This study was supported in part tive analysis of the proteins and antigens of five herpesviruses. by Grants-in-Aid from the Ministry of Education, Science, J. Gen. Virol. 37: 297–310. Culture and Sports of Japan. 13. Kopacek, J., Klucar, L., Koptidesova, D., Turna, J., Pastorek, J. and Zelnik, V. 2000. Nucleotide sequence of the gene encoding REFERENCES the major capsid protein of herpesvirus of turkeys. Virus Genes 20: 107–115. 1. Assaf, R., Bouillant, A. M. and Di Franco, E. 1982. Enzyme 14. Littler, E., Lawrence, G., Liu, M. Y., Barrell, B. G. and Arrand, linked immunosorbent assay (ELISA) for the detection of anti- J. R. 1990. Identification, cloning, and expression of the major bodies to porcine cytomegalovirus. Can. J. Comp. Med. 46: capsid protein gene of human herpesvirus 6. J. Virol. 64: 714– 183–185. 722. 2. Chee, M., Udolph, S. A., Plachter, B., Barrell, B. and Jahn, G. 15. Mukai, T., Isegawa, Y. and Yamanishi, K. 1995. Identification 1989. Identification of the major capsid protein gene of human of the major capsid protein gene of human herpesvirus 7. Virus cytomegalovirus. J. Virol. 63: 1345–1353. Res. 37: 55–62. 3. Corner, A. H., Mitchell, D., Julian, R. J. and Meads, E. B. 16. Newcomb, W. W., Trus, B. L., Booy, F. P., Steven, A. C., 1964. A generalized disease in piglets associated with the pres- Wall, J. S. and Brown, J. C. 1993. Structure of the herpes sim- ence of cytomegalic inclusion. J. Comp. Pathol. 74: 192–199. plex virus capsid. Molecular composition of the pentons and 4. Davison, A. J. and Scott, J. E. D. N. 1986. A sequence of the the triplexes. J. Mol. Biol. 232: 499–511. major capsid protein gene of herpes simplex virus type 1. J. 17. Ohlinger, V. 1989. pp. 326–333. In: Herpesvirus of Gen. Virol. 67: 2279–2286. Cattle, Horses, and Pigs (Wittmann, G. ed.), Kluwer, Boston. 5. Davison, A. J. and Wilkie, N. M. 1983. Location and orienta- 18. Roizman, B., Carmichael, L. E., Deinhardt, F., de-The, G., tion of homologous sequences in the genome of five herpesvi- Nahmias, A. J., Plowright, W., Rapp, F., Sheldrick, P., Taka- MCP OF PORCINE CYTOMEGALOVIRUS 617

Fig. 5. PCR amplification of MCP gene fragments. (A) Three pairs of primers, F(15)/R(C1), F(15)/R(10) and F(14)/R(11), were tested for PCR using each PCMV strain or PRV-infected cell DNA (V) or mock-infected cell DNA (C) as a template. (B) Southern blotting analysis was performed to confirm reaction specificity. Size marker is shown on the left .

hashi, M. and Wolf, K. 1981. . Definition, provi- 19. Rupasinghe, V., Tajima, T., Maeda, K., Iwatsuki-Horimoto, sional nomenclature, and taxonomy. The Herpesvirus Study K., Sugii, S. and Horimoto, T. 1999. Analysis of porcine Group, the International Committee on Taxonomy of Viruses. cytomegalovirus DNA polymerase by consensus primer PCR. Intervirology 16: 201–217. J. Vet. Med. Sci. 61: 1253–1255. 618 V. RUPASINGHE ET AL.

20. Scherba, G., Turek, J. J. and Gustafson, D. P. 1983. Pseudora- 1999. Evaluation of porcine cytomegalovirus as a potential bies nucleocapsid antigen for skin testing in swine. J. Clin. zoonotic agent in xenotransplantation. Transplant. Proc. 31: Microbiol. 17: 539–544. 915. 21. Shirai, J., Narita, M., Iijima, Y. and Kawamura, H. 1985. A 27. Vafai, A., Wroblewska, Z. and Graf, L. 1990. Antigenic cross- cytomegalovirus isolation from swine testicle culture. Jpn. J. reaction between a varicella-zoster virus nucleocapsid protein Vet. Sci. 45: 697–703. encoded by gene 40 and a herpes simplex virus nucleocapsid 22. Tajima, T., Hironao, T., Kajikawa, T. and Kawamura, H. 1993. protein. Virus Res. 15: 163–174. Application of enzyme-linked immunosorbent assay for the 28. Van den Laan, L. J. W., Lockey, C., Griffeth, B. C., Frasier, F. seroepizootiological survey of antibodies against porcine S., Wilson, C. A., Onions, D. E., Hering, B. J., Long, Z., Otto, cytomegalovirus. J. Vet. Med. Sci. 55: 421–424. E., Torbett, B. E. and Salomon, D. R. 2000. Infection by por- 23. Tajima, T., Hironao, T., Kajikawa, T., Suzuki, Y. and Kawa- cine endogenous retrovirus after islet xenotransplantation in mura, H. 1994. Detection of the antibodies against porcine SCID mice. Nature (Lond.) 407: 90–94. cytomegalovirus from whole blood collected on the blood sam- 29. Widen, B. F., Lowings, J. P., Belak, S. and Banks, M. 1999. pling paper. J. Vet. Med. Sci. 56: 189–190. Development of a PCR system for porcine cytomegalovirus 24. Tajima, T. and Kawamura, H. 1998. Serological relationship detection and determination of the putative partial sequence of among porcine cytomegalovirus Japanese isolates and a UK its DNA polymerase gene. Epidemiol. Infect. 123: 177–180. isolate. J. Vet. Med. Sci. 60: 107–109. 30. Yamada, S., Imada, T., Watanabe, W., Honda, Y., Nakajima- 25. Thomopson, J. D., Higgins, D. G. and Gibson, T. J. 1994. Iijima, S., Shimizu, Y. and Sekizaki, K. 1991. Nucleotide CLUSTALW. Nucleic Acids Res. 22: 4673–4680. sequence and transcriptional mapping of the major capsid pro- 26. Tucker, A. W., Galbraith, D., McEwan, P. and Onions, D. tein gene of pseudorabies virus. Virology 185: 56–66.