Yoichi Minamishima

Cytomegalovirus (CMV) antigenaemia for rapid diagnosis and monitoring of CMV‐associated disease after bone marrow transplantation

A technique for the rapid detection of cytomegalovirus (CMV) antigen‐positive blood leucocytes (CMV antigenaemia) was evaluated in 15 marrow transplant patients as a means of diagnosis and for monitoring CMV‐associated disease. CMV antigenaemia was determined by direct immunoperoxidase staining of leucocytes with a peroxidase‐labelled monoclonal antibody, HRP‐C7, which binds an immediate‐early antigen of human CMV.

Role of breast milk in acquisition of cytomegalovirus infection.

The prevalence of IgG antibody against cytomegalovirus (CMV) was compared between the age‐matched (0 month to 2 years of age) groups of 212 breast‐fed children and 223 bottle‐fed children to examine the role of breast milk for acquisition of CMV. Mothers of both groups of children were also examined for CMV IgG antibodies. Both the breast‐fed and bottle‐fed children groups showed high seropositivity for CMV at 0 to 2 months of age, which gradually decreased and bottomed at 6 to 8 months of age. Thereafter, in the breast‐fed children group, the seropositivity rate increased up to 70% by 1 year of age. In contrast, in the bottle‐fed children group, the seropositivity rate remained at the bottom level of lower than 30%, without showing any apparent increases. The serological data of the children whose mothers were confirmed to be seropositive, revealed that mother‐to‐child transmission of CMV occurred in 11 of 17 (64.7%) of the breast‐fed children and in 24 of 87 (27.6%) of the bottle‐fed children. All the bottle‐fed children born to seronegative mothers remained seronegative for CMV up to 1 year of age. The bottle‐fed children showed significantly lower seropositivity than the breast‐fed children, although most of both groups of children were born to seropositive mothers. The results strongly suggested that about 40% of the breast‐fed children acquire CMV via breast milk and breast‐feeding has certain protective effects on congenital CMV disease in the offspring.

Early viral complications following CD34-selected autologous peripheral blood stem cell transplantation for non-Hodgkin's lymphoma

A patient with non‐Hodgkins lymphoma who received a CD34‐selected autologous peripheral blood stem cell transplant (PBSCT) developed cytomegalovirus retinitis, adenovirus‐associated haemorrhagic cystitis (HC) and fatal herpes simplex virus pneumonia. Depletion of mature T cells from the graft and a persistent decrease in CD4+ lymphocytes following PBSCT may have predisposed this patient to such viral infections. Infusion of cryopreserved autologous PBSC (containing mature T cells) was effective for adenovirus‐associated HC. Immunosuppression and resultant viral infections may affect patients receiving CD34‐selected autologous transplantation.

Genetic analysis of a ganciclovir-resistant human cytomegalovirus mutant

We isolated a ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) from a laboratory strain, AD169, and analysed the mutant. Attempts were also made to identify directly the mutated gene. The 50% inhibitory concentration (IC50) of GCV for the mutant strain was five times higher than that of the wild-type strain. The mutant strain showed similar sensitivity to phosphonoacetic acid and cidofovir as the wild-type strain. These data suggest mutation in the UL97 gene encoding for the phosphotransferase that phosphorylates GCV. Molecular analysis of the mutant strain revealed that a single base substitution of adenine by cytosine occurred at the 1796 nucleotide position of the UL97 gene region, resulting in the substitution of lysine by threonine at codon 599 in the UL97 gene product. Marker transfer experiment confirmed that this mutation conferred HCMV resistance to GCV. The mutation at codon 599 was easily identified by means of RsaI digestion of the selected PCR product.

Detection of human T-cell lymphotropic virus type 1 infection by the polymerase chain reaction using dried blood specimens on filter papers.

A simple method for detection of proviral DNA sequences of human T-cell lymphotropic virus type 1 (HTLV-1) was developed using dried blood specimens on filter papers. The whole blood was blotted onto the Guthrie paper. After the blood has dried, the blotted paper was punched out into small discs. The discs were then boiled to prepare the template for PCR (filter paper-PCR method). The filter paper-PCR method detected even a single HTLV-1-infected cell in three discs. The sensitivity of the filter paper-PCR method was equivalent to that of the method in which DNA was extracted with phenol and used as the template for PCR (DNA extraction-PCR method). In addition, DNA in the blotted filter paper was still utilizable as the template after the storage at 25 degrees C for at least 7 wk. A total of 53 clinical specimens from 30 seropositive and 23 seronegative individuals who were screened by particle agglutination (PA) test were analysed for HTLV-1 DNA by both PCR methods. Of 30 PA-positive specimens, 28 were also positive for HTLV-1 antibody by Western blot (WB) analysis, but two were indeterminate. The twenty eight WB-positive and one of the two indeterminate specimens were positive for HTLV-1 proviral DNA by both PCR methods. Of 23 PA-negative specimens, 22 were negative for HTLV-1 proviral DNA by both PCR methods. However, one PA-negative specimen was positive by both PCR methods. This patient was a 16-mth-old infant who was born to an HTLV-1 carrier mother and fed thereafter without her breast milk. In comparison to DNA extraction-PCR method, the sensitivity and specificity of the filter paper-PCR method was 100%, respectively.

“Transformation” of Human Endothelial Cells by SV40 Virions

Human endothelial cells derived from the umbilical vein were transformed with SV40 virions. A cell line subcultured for over 60 serial passages was characterized in comparison with its untransformed counterpart which was culturable for less than five passages.

Murine cytomegalovirus DNA polymerase: Purification, characterization and role in the antiviral activity of acyclovir

Murine cytomegalovirus (MCMV) neither induces a viral thymidine kinase (TK) nor enhances the activity of a cellular TK. Nevertheless, MCMV is highly susceptible to 9-(2-hydroxyethoxymethyl)guanine (acyclovir, ACV). The cellular TK is neither responsible for phosphorylation of ACV nor its anti-MCMV activity. This is clear from the findings that little ACV triphosphate is formed in MCMV-infected mouse embryo fibroblasts (MEF) and that the replication of MCMV is inhibited equally well by ACV in TK+ and TK- cells. Even if trace amounts of ACV triphosphate would be formed by enzymes other than TK, and ACV triphosphate would be responsible for the anti-MCMV activity of ACV, then the MCMV DNA polymerase ought to be highly sensitive to ACV triphosphate. To examine this possibility, the MCMV DNA polymerase was partially purified and characterized. The apparent Ki value of the MCMV DNA polymerase for ACV triphosphate indicates that the sensitivity of the MCMV DNA polymerase to ACV triphosphate is equivalent to that of the HSV DNA polymerase. Therefore, the trace amounts of ACV triphosphate that are formed in MCMV-infected MEF seem to be insufficient to inhibit MCMV DNA polymerase and may not play a key role in the anti-MCMV activity of ACV.

A case of human herpesvirus-6 lymphadenitis with infectious mononucleosis-like syndrome.

The findings of a 20 year old woman with lymphadenopathy that was probably caused by an acute human herpesvirus‐6 (HHV‐6) infection are reported. She clinically demonstrated various signs of acute infection such as a high fever, skin rash, liver dysfunction, leukocytosis, an elevation of the erythrocyte sedimentation rate, and a positive change of C reactive protein, which mimicked the symptoms of infectious mononucleosis, but no positive titers for an Epstein‐Barr virus infection were observed. HHV‐6 DNA was detected using Southern blot analysis, polymerase chain reaction, and in situ hybridization in the affected node. Histologically, the lymph node showed an enlarged paracortex and infiltration of transformed lymphocytes and immunoblast‐like cells with some histiocytes and eosino‐phils. Almost all the transformed lymphocytes and immunoblasts were positive for UCHL‐1 (CD45RO), MT‐1 (CD43), and OPD‐4 (CD4), and some of these positive cells demonstrated HHV‐6 DNA.

Isolation of Multiple Cytomegalovirus Strains from a Patient with Adult T Cell Leukemia

A 66-year-old male with adult T cell leukemia had an ulcer on the left medial thigh. The biopsy of the skin lesion revealed enlarged endothelial cells with acidophilic intranuclear inclusion bodies, suggesting cytomegalovirus (CMV) infection. At autopsy, CMV was isolated from a nodular skin lesion of the scrotum. The urine constantly tested positive for CMV. Restriction endonuclease cleavage analysis of DNA of the isolates from the skin and urine indicated that this patient was infected with two different strains of CMV.

Induction of Fc (IgG) Receptor(s) by Simian Cytomegaloviruses in Human Embryonic Lung Fibroblasts

Human embryonic lung (HEL) fibroblasts infected with simian cytomegalovirus (SCMV) were found to bind nonspecifically to the Fc portion of human immunoglobulin (Ig) G (IgG). Binding of IgG to SCMV-infected HEL fibroblasts, but not to uninfected HEL cells, was visualized as cytoplasmic fluorescence by the indirect immunofluorescence test, regardless of the presence of anti-CMV antibodies. The receptor(s) reacted with the IgG class of different species, but not with IgM and IgA. The purified Fc fragment reacted with the receptor(s), but the Fab fragment reacted poorly. The reaction was blocked by pretreatment of infected cells with the Fc fragment, but was not blocked with the Fab fragment. The appearance of the Fc receptor(s) required RNA and protein synthesis, whereas a requirement for DNA synthesis remains to be answered by a more sensitive assay. The development of the Fc receptor(s) was inhibited by 2-deoxy-D-glucose, thus indicating that the Fc receptor(s) may be a glycoprotein(s). The Fc receptor(s) was induced by all strains of SCMV tested so far. These included one laboratory strain (GR2757), four fresh isolates from primary kidney cell cultures of African green monkeys, and four fresh isolates from the salivary glands of the Macaca monkeys captured from the wild in Japan.