Kanji Hirai

Protection of chickens from Newcastle disease by vaccination with a linear plasmid DNA expressing the F protein of Newcastle disease virus.

To evaluate the usefulness of a DNA vaccine for chickens, we constructed a plasmid vector expressing the Newcastle disease virus F protein (NDV-F) under the control of the human cytomegalovirus immediate early enhancer and chicken beta-actin gene promoter. One-week-old chickens injected intramusculary with the circular plasmid DNA did not produce significant levels of antibody against NDV-F. However, two of five birds injected with the linearized plasmid DNA produced high levels of the antibody. Moreover, four of five birds injected with a mixture of the linearized-plasmid and Lipofectin produced the antibody efficiently. At 9 weeks post-injection, chickens were challenged with the velogenic NDV Sato strain. These chickens that had the antibody against NDV-F were protected from lethal NDV challenge. These results demonstrate that the DNA vaccine conferred efficient protection against the disease.

Interaction of Epstein-Barr Virus Nuclear Antigen Leader Protein (EBNA-LP) with HS1-Associated Protein X-1: Implication of Cytoplasmic Function of EBNA-LP

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) consists of W1W2 repeats and a unique C-terminal Y1Y2 domain and has been suggested to play an important role in EBV-induced transformation. To identify the cellular factors interacting with EBNA-LP, we performed a yeast two-hybrid screen, using EBNA-LP cDNA containing four W1W2 repeats as bait and an EBV-transformed human peripheral blood lymphocyte cDNA library as the source of cellular genes. Our results were as follows. (i) All three cDNAs in positive yeast colonies were found to encode the same cellular protein, HS1-associated protein X-1 (HAX-1), which is localized mainly in the cytoplasm and has been suggested to be involved in the regulation of B-cell signal transduction and apoptosis. (ii) Mutational analysis of EBNA-LP revealed that the association with HAX-1 is mediated by the W1W2 repeat domain. (iii) A purified chimeric protein consisting of glutathioneS-transferase fused to EBNA-LP specifically formed complexes with HAX-1 transiently expressed in COS-7 cells. (iv) When EBNA-LP and HAX-1 were coexpressed in COS-7 cells, EBNA-LP was specifically coimmunoprecipitated with HAX-1. (v) Careful cell fractionation experiments of an EBV-infected lymphoblastoid cell line revealed that EBNA-LP is localized in the cytoplasm as well as in the nucleus. (vi) When EBNA-LP containing four W1W2 repeats was expressed in COS-7 cells, EBNA-LP was detected mainly in the nucleus by immunofluorescence assay. Interestingly, when EBNA-LP containing a single W1W2 repeat was expressed in COS-7 cells, EBNA-LP was localized predominantly in the cytoplasm and was colocalized with HAX-1. These results indicate that EBNA-LP is in fact present and may have a significant function in the cytoplasm, possibly by interacting with and affecting the function of HAX-1.

Expression of human immunodeficiency virus type 1 (HIV-1) gag antigens on the surface of a cell line persistently infected with HIV-1 that highly expresses HIV-1 antigens

MT-4 cells persistently infected with human immunodeficiency virus type 1 (HIV-1) (MT-4/HIV-1) were recently isolated (K. Ikuta, C. Morita, M. Nakai, N. Yamamoto, and S. Kato, Japan. J. Cancer Res. (Gann), 79, 418-423, 1988). Mouse hybridoma cell clones producing monoclonal antibodies (MoAbs) to HIV-1 gag p24 and p18, and pol reverse transcriptase (RT) were isolated by using this MT-4/HIV-1 cell line for the screening of MoAb production by the immunofluorescence (IF) test. By indirect IF tests of acetone-fixed cells with these MoAbs, the IF intensities in MT-4/HIV-1 cells were found to be higher than those in the other HIV-1 infected cells, such as MOLT-4/HIV-1, HL-60/HIV-1, and U937/HIV-1 cells. Cell surface expression of the HIV-1 gag p24 and p18 antigens examined by IF and radioimmune techniques with these MoAbs revealed the p24 and p18 antigens to be expressed strongly on the cell surface of MT-4/HIV-1 cells and faintly on the cell surface of MOLT-4/HIV-1 cells, respectively. However, monoclonal antibody isolated in the present study failed to detect pol RT antigen on the surface of MT-4/HIV-1 cells. These results indicate that the gag p24 and p18 antigens are expressed, at least in part, on the surface of HIV-1-infected cells.

Clonal lymphoproliferation following chronic active Epstein-Barr virus infection and hypersensitivity to mosquito bites.

In order to elucidate the possibility of lymphoproliferation in cases of chronic active Epstein‐Barr virus infection (CAEBV), to clarify the clonality and genotype of proliferating lymphocytes, and to search for the factors that induce lymphoproliferation, we studied 11 cases of CAEBV, using genetical and immunological techniques.

Induction of α-type DNA polymerases in human cytomegalovirus-infected WI-38 cells

Abstract Productive infection of WI-38 cells with human cytomegalovirus (HCMV) induced the increase in the activity of DNA polymerases as well as the synthesis of viral and cellular DNA. Sedimentation analyses in sucrose gradients of high ionic strength showed that the HCMV infection caused marked increase in the activity of α-type polymerases (resolved into α 1 , 8 S, and α 2 , 6 S, in the present experiments), while the infection little affected the level of β-type polymerase (about 3.5 S) activity in both the nuclei and cytoplasm. Such increase in α-type polymerases was also observed when DNA synthesis in WI-38 cells was enhanced by SV40 infection or by an increased concentration of serum in medium. Phosphonoacetate, which selectively blocked the synthesis of HCMV DNA, did not significantly affect the HCMV-mediated induction of DNA polymerases. However, phosphonoacetate added in the reaction mixture for DNA polymerase assay inhibited the activity of the HCMV-induced polyperase α 1 but not of the polymerases α 2 and β. These results support the idea that α-type polymerases are involved in the replicative synthesis of cellular and viral DNA.

Epstein–Barr virus-encoded protein kinase BGLF4 mediates hyperphosphorylation of cellular elongation factor 1δ (EF-1δ): EF-1δ is universally modified by conserved protein kinases of herpesviruses in mammalian cells

Translation elongation factor 1δ (EF-1δ) is hyperphosphorylated in various mammalian cells infected with alpha-, beta- and gammaherpesviruses and EF-1δ modification is mediated by viral protein kinases, including UL13 of herpes simplex virus type 1 and UL97 of human cytomegalovirus. In this study, the following is reported. (i) BGLF4 encoded by the prototype gammaherpesvirus Epstein–Barr virus was purified as a fusion protein that was labelled with [γ-32P]ATP and labelling was eliminated by phosphatase. (ii) The ratio of the hyperphosphorylated form of human EF-1δ was increased both in Sf9 cells after infection with baculoviruses expressing GST–BGLF4 fusion proteins and in COS-7 cells after transfection with a BGLF4 expression plasmid. These results indicate that purified BGLF4 possesses protein kinase activity and mediates EF-1δ hyperphosphorylation. These data also support the hypothesis that the protein kinases that are conserved by herpesviruses universally mediate EF-1δ modification in mammalian cells.

Detection of human cytomegalovirus DNA in dried newborn blood filter paper

To detect human cytomegalovirus (HCMV) DNA, filter paper spotted with peripheral blood from newborns 4 to 7 days after birth was dried and subjected to the polymerase chain reaction (PCR) followed by Southern blot hybridization with a nonradioactive oligonucleotide probe. The detection rates were 25.1% in healthy individuals and 33.0% in low body weight neonates weighing not more than 2500 g at birth, most of whom appeared to have been infected transplacentally or by other means within the uterus. HCMV was detected after only heating the dried blood on the filter paper, and may be applied as a screening method for the early diagnosis of HCMV.

Restriction enzyme map of herpesvirus of Turkey DNA and its collinear relationship with Marek's disease virus DNA

The genome of herpesvirus of turkey (HVT) was shown to consist of long and short unique regions flanked by inverted repeats (J. Cebrian, Kaschka-Dietrich, C., Berthelot, N., and Sheldrick, P., 1982, Proc. Natl. Acad. Sci. USA 79, 555-558). In this paper we report the construction of the linkage map of HVT DNA for BamHI, HindIII, and PstI restriction endonucleases. The maps were constructed by hybridization of 19 cloned BamHI fragments of HVT DNA to electrophoretically separated digests of genomic DNA. Our results indicate that the terminal and internal inverted repeats (TRL and IRL) flanking the long unique sequences (UL) are spanned by BamHI-F fragment and a -F-related terminal fragment, respectively, whereas the terminal and internal inverted repeats (TRS and IRS) flanking the short unique sequences (US) are mostly contained in BamHI-A fragment. Both BamHI-A and -F showed a heterogeneity in size, suggesting the presence of amplification of certain sequences in the inverted repeats. We also report that the HVT genome is collinear with the genetically related Mareks disease virus (MDV) genome, as determined by hybridization of labeled cloned HVT DNA fragments with electrophoretically separated MDV DNA fragments.

Molecular detection and differentiation of enteroviruses in endomyocardial biopsies and pericardial effusions from dilated cardiomyopathy and myocarditis.

Enteroviruses (EVs), especially group B coxsackieviruses, have been implicated in the pathogenesis of myocarditis and dilated cardiomyopathy (DCM). To determine whether a specific type of EV is present in DCM hearts, we examined the genotypes of EVs detected in endomyocardial biopsies and pericardial effusions by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. Positive PCR results were obtained from biopsies in 6 (19 percent) of 31 patients with DCM, 5 (18 percent) of 28 with myocarditis, 5 (22 percent) of 23 with other cardiac diseases, and from pericardial effusions in 4 (57 percent) of 7 patients with pericarditis. SSCP profiles of most of the clinical samples were different and were not identical to any of the standard group B coxsackie viruses. Our findings suggest that EV genomes are involved in the myocardium of patients with various cardiac conditions and that a particular type of EV is not present in DCM hearts.

Similarities and dissimilarities in the structure and expression of viral genomes of various virus strains immunologically related to Marek's disease virus.

SummaryVarious strains immunologically related to Mareks disease virus (MDV) have been subdivided into three serotypes: serotype 1, pathogenic strains of MDV and attenuated or apathogenic variants derived from them; serotype 2, naturally occuring apathogenic strains of MDV; serotype 3, herpesvirus of turkey (HVT). The viral genome structures of these three serotypes were compared by a simple, practical method using total DNA extracted from virus-infected cells instead of viral DNA purified from virions. The restriction endonuclease-cleavage patterns of serotype 2 viral DNA were found to differ from those of either serotype 1 MDV or serotype 3. Under stringent conditions, no significant DNA homology was detected among the three serotype viruses, except in a restricted portion of these viral genomes. Northern blot hybridization experiments suggested that virus-specific polyadenylated RNA of about 2.4 kilobases was transcribed from a restricted portion showing close homology in these viruses. Southern blot hybridization under less stringent conditions revealed that regions with weak homology were distributed over most of the viral genomes of the three serotypes. Two types of virus-specific glycoproteins, gA and gB, were identified in the immunoprecipitates of the culture medium and cell lysates, respectively, of serotype 2-infected cultures with monoclonal antibodies or hyperimmune antisera cross reactive with serotype 1 MDV and serotype 3 HVT, and detected on the surface of serotype 2-infected cells by the membrane immunofluorescence test. These results indicate a close evolutionary relationship among these three viral serotypes.