Lack of antibodies against the antigen domain 2 epitope of cytomegalovirus (CMV) glycoprotein B is associated with CMV disease after renal transplantation in recipients having the same glycoprotein H serotypes as their donors.

Cytomegalovirus (CMV) reinfection of seropositive individuals has been associated with adverse outcomes in organ transplantation and is a frequent cause of congenital infection. Previously we demonstrated that mismatching of CMV glycoprotein H (gH) serotypes was associated with CMV disease after renal transplantation. Because the antigen domain 2 (AD2) epitope of glycoprotein B (gB) is conserved among CMV isolates and is one of the known targets of neutralizing antibodies, in this study we investigated whether antibodies against the epitope contribute to protection from CMV reinfection in renal transplantation, irrespective of gH serological matching. For this purpose, the gB and gH serology and clinical outcomes were analyzed retrospectively for 77 transplant recipients in the donor positive/recipient positive setting, who were managed by preemptive strategy. We found that there was a good negative correlation between the numbers of antigenemia-positive cells and the levels of antibodies against gB AD2 in the CMV-gH antibody matched group, but not in the CMV-gH antibody mismatched group. None of the recipients with antibodies against both gB AD2 and strain-specific epitopes of gH have experienced CMV disease during 6 month after transplantation, while 28% of those who lacked either/both antibody response needed preemptive therapy. Because the outcome was statistically significant, antibodies against gB AD2 can be a useful indicator to predict emergence of CMV disease for preemptive therapy, in addition to antibodies against the mismatched gH types.

Participation of type I interferon in the decreased virulence of the UL13 gene-deleted mutant of herpes simplex virus type 1.

We isolated a UL13 gene-deleted mutant of the herpes simplex virus type 1 (HSV-1) strain VR3 (VRDelta13) and its revertant virus (VRDelta13R). This deletion mutant still had virus host shutoff (vhs) activity, although a previous report had suggested the possibility of a functional relation between the UL13 product, that is protein kinase (PK), and vhs activity. We compared the in vivo growth of these viruses in BALB/c mice. VRDelta13 was cleared in the early period of intraperitoneal infection. VRDelta13 had a higher sensitivity to the mouse type I interferon (IFN) and showed a higher level of IFN induction in the study period of infection than did VR3 and VRDelta13R. These results suggest that a nonspecific antiviral response (i.e., the IFN system) may contribute to this rapid inhibition of viral replication in vivo.

Susceptibility of protein kinase (ORF47)-deficient varicella-zoster virus strains to anti-herpesvirus nucleosides.

To clarify whether varicella-zoster virus (VZV) protein kinase (PK; ORF47) takes part in phosphorylation of anti-herpesvirus nucleosides, thymidine kinase (TK) deficient, and PK/TK double deficient recombinant VZV strains were isolated and their susceptibility, and that of wild type and PK-deficient strains to various nucleoside analogs was evaluated. The PK-deficient VZV strains showed a sensitivity equal to that of the wild type strain against all compounds tested, including ganciclovir. This indicates that PK is not involved in phosphorylation of the tested nucleosides in VZV-infected cells.

Structure-activity relationship of the affinity of 5-substituted uracil nucleoside analogues for varicella-zoster virus thymidine kinase and their activity against varicella-zoster virus.

We investigated structure-activity relationships of 5-substituted uracil nucleoside analogues for their selective antiviral activity against varicella-zoster virus (VZV) and affinity for VZV thymidine kinase (TK). Anti-proliferative activity of the compounds was measured using human lymphoblastoid cells. Most 2'-deoxyribofuranosyluracil, arabinofuranosyluracil (araU) and 2'-deoxy-2'-fluoro-arabinofuranosyluracil derivatives showed selective anti-VZV activity as well as activity against herpes simplex virus types 1 and 2. 2'-Deoxyuridine derivatives showed higher affinity than the corresponding araU analogues. A correlation was seen between the 50% effective doses for VZV and the Ki values for VZV TK, except for 5-ethyl-2'-deoxyuridine and 5-ethyl araU that showed relatively high affinity for VZV TK without showing any activity against VZV. 5-Halogenovinyluracil nucleosides showed the highest affinity and the most potent and selective anti-VZV activity. 2'-Deoxy-2'-fluoro-arabinofuranosyluracil derivatives exhibited high anti-VZV potency though they showed relatively low affinity for VZV TK. Some 3'-deoxythymidine analogues having anti-human immunodeficiency virus activity were inactive against herpesviruses.

Synthesis and anti-influenza virus activity of novel pyrimidine derivatives.

Efficient synthetic routes of 2-amino-4-(omega-hydroxyalkylamino)pyrimidine derivatives were investigated in relation to the anti-influenza virus activity of these compounds. The derivatives in which cyclobutyl and cyclopentyl groups were introduced to the beta-position of the aminoalkyl group (especially the cyclobutyl group substituted by a phenylalkyl group at the 3'-position) resulted in improved antiviral potency: i.e. an average 50% effective concentration for inhibition of plaque formation (EC50, microM) of 0.1-0.01 microM for both types A and B influenza virus. The antiviral efficacies were in the order of amino group > hydroxyiminomethyl group > halogen substitution at the 5-position, and chlorine or methoxy group > hydrogen at the 6-position of the pyrimidine ring. The antiviral indices of these compounds were 2-6 with respect to the 50% inhibitory concentration for cell proliferation (IC50, microM) for growing cells, but > 500 to > 10(4) with respect to the IC50 for stationary cells, indicating that these compounds may be efficacious for the topical treatment of influenza virus infection.

Analysis of toxic and mutagenic activities of antiherpesvirus nucleosides against HeLa cells and herpes simplex virus type 1.

The toxic and mutagenic activities of five antiherpesvirus agents to HeLa cells and herpes simplex virus type 1 (HSV-1) were investigated. 5-Iodo-2'-deoxyuridine (IDU) and 9-beta-D-arabinofuranosyl-adenine (araA) showed very potent inhibitory effects on cell growth and the cloning efficiency of HeLa cells, whereas 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU), E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 9-(2-hydroxyethoxymethyl)guanine (ACV) showed less inhibitory effect. 50% inhibitory doses of BV-araU and BVDU for cell growth were 657 and 253 micrograms/ml, respectively. Although the growth inhibitory activity of BVDU was very weak, as above, the mutagenic activity of this drug to the cells, estimated by induction of colchicine-resistant mutants, was observed to be 4 micrograms/ml, which was a markedly smaller dose than the inhibitory dose for cell growth, and the highest frequency of mutation of the cells was shown at 100 micrograms/ml of BVDU. This activity was more potent than that of IDU. No mutagenic activity of BV-araU, araA and ACV to cells was observed within the concentration range of 1-800 micrograms/ml. IDU showed high mutagenic activity to HSV-1 growing in human embryo lung fibroblasts, and IDU-resistant mutants were induced at a high frequency. BVDU also induced a small amount of BVDU-resistant mutant virus, although this drug induced many mutant cells. No mutagenic activity of BV-araU, araA and ACV to HSV-1 was observed.

Analysis of nucleotide sequence variations in herpes simplex virus types 1 and 2, and varicella-zoster virus.

To analyze the difference in the degree of divergence between genes from identical herpesvirus species, we examined the nucleotide sequence of genes from the herpes simplex virus type 1 (HSV-1) strains VR-3 and 17 encoding thymidine kinase (TK), deoxyribonuclease (DNase), protein kinase (PK; UL13) and virion-associated host shutoff (vhs) protein (UL41). The frequency of nucleotide substitutions per 1 kb in TK gene was 2.5 to 4.3 times higher than those in the other three genes. To prove that the polymorphism of HSV-1 TK gene is common characteristic of herpesvirus TK genes, we compared the diversity of TK genes among eight HSV-1, six herpes simplex virus type 2 (HSV-2) and seven varicella-zoster virus (VZV) strains. The average frequency of nucleotide substitutions per 1 kb in the TK gene of HSV-1 strains was 4-fold higher than that in the TK gene of HSV-2 strains. The VZV TK gene was highly conserved and only two nucleotide changes were evident in VZV strains. However, the rate of nonsynonymous substitutions in total nucleotide substitutions was similar among the TK genes of the three viruses. This result indicated that the mutational rates differed, but there were no significant differences in selective pressure. We conclude that HSV-1 TK gene is highly diverged and analysis of variations in the gene is a useful approach for understanding the molecular evolution of HSV-1 in a short period.

Identification of a cell membrane receptor for interferon induction by poly rI:rC.

PR-RK, a cell line derived from rabbit kidney cells (RK-13), was insensitive to the cytotoxic effect and interferon (IFN) inducing activity of the copolymer of riboinosinic and ribocytidylic acid (poly rI:rC). However, PR-RK was sensitive to the cytotoxic effect of the copolymer of riboadenylic and ribouridylic acid (poly rA:rU). Comparison of PR-RK cells and RK-13 cells by cytofluorometric analysis revealed that the binding of poly rI:rC was considerably reduced on PR-RK cells. These results suggested that the receptor for poly rI:rC might be different from the receptor for poly rA:rU, and this difference could provide a basis for the identification of the dsRNA receptor on cell surface. Western blot analysis of the components of cell membrane fraction prepared from RK-13 cells was performed by using a monoclonal antibody, which binds to cell membrane of RK-13 cells but not to PR-RK cells, and which blocks IFN induction by poly rI:rC in RK-13 cells. The 60K protein was identified as one of the poly rI:rC receptor protein.

Activity of anthocyanins from fruit extract of Ribes nigrum L. against influenza A and B viruses.

Earlier, we have detected antiviral activity in an extract from Ribes nigrum L. fruits ("Kurokarin", name of the one species of black currant in Japanese) against influenza A and B viruses, and herpes simplex virus 1 (Knox et al., Food Processing 33, 21-23, 1998). In the present study, the antiviral activity of constituents of a Kurokarin extract and the mechanism of its antiviral action were examined. Kurokarin extracts were separated to fractions A to D by column chromatography. The major constituents of the fraction D were estimated as anthocyanins. The fraction D was further fractionated by thin-layer chromatography (TLC) to fractions A' to G'. The fraction E' consisted of 3-O-alpha-L-rhamnopyranosyl-beta-D-glucopyranosyl-cyanidin and 3-O-beta-D-glucopyranosyl-cyanidin, and the fraction F' consisted of 3-O-alpha-L-rhamnopyranosyl-beta-D-glucopyranosyl-delphinidin and 3-O-beta-D-glucopyranosyl-delphinidin, identified by high performance liquid chromatography (HPLC) with standards and by high resolution mass spectrometry. The fractions D' to G' showed potent antiviral activity against influenza viruses A and B. The additive antiviral effect of a combination of the fractions E' and F' was assessed. Anthocyanins in the fraction F' did not directly inactivate influenza viruses A and B, but they inhibited virus adsorption to cells and also virus release from infected cells.

[The relationship between gene function and virulence in alpha-herpesviruses].

Virulent alpha-herpesvirus genes, though not essential for virus replication in cell culture, play important roles in virus replication in vivo. In this paper, I classify the virulent genes and discuss the relationship between gene function and virulence. The products of the virulent genes of herpes simplex virus, described in this paper, are enzymes (thymidine kinase, ribonucleotide reductase, deoxyuridine triphosphatase, DNA polymerase, and two protein kinases), glycoproteins (gC, gE), immediate early gene product (ICP47) and gamma 34.5. To identify the virulent genes of varicellazoster virus, mutation in the Oka vaccine strain was studied. The low levels of gV expression and mutation found in the immediate early gene were predicted as the cause of the attenuation of the Oka vaccine strain.

[Antiviral compounds: current approaches for development].

Recent dynamic advances of molecular biology have been discovering the specific virus functions and replication strategies that differ greatly from their host cells. These studies made it possible to design the antiviral agents which block virus replication specifically. In this paper, we review the recent approach for development of anti-HIV agents and the problems of anti-herpes compounds applied in clinical treatment.

[Investigations on ashing of animal tissues for the determinations of trace metals by means of polarography or atomic-absorption spectrometry (author's transl)].

A comparison has been made of three ashing methods for animal tissues with subsequent determination of minute quantities of metals by polarography or atomic-absorption spectrometry. Wet oxidation method with many disadvantages and few advantages has been shown not to be useful for the purpose. Both dry ashing in a muffle-furnace at 530 degrees C and low-temperature ashing are equally applicable to atomic-absorption spectrometry, but dry ashing in a furnace is more excellent with polarography. For obtaining ashes with constant compositions it is far more difficult with low-temperature ashing than with dry ashing in a furnace. Hence, dry ashing in a furnace is most widely applicable.

[Interaction between electrode mercury of polarography and dissolved oxygen in sample solution (author's transl)].

The dissolved oxygen in sample solution produces the reduction wave with remarkable maximum by D.C. polarograph. The height of the maximum wave decreases with the increase of surface and duration of contact with electrode mercury. It has been demonstrated that the decrease of height of maximum wave depends on the decrease of dissolved oxygen in sample solution and the authors have assumed that the decrease of dissolved oxygen is due to the adsorption of oxygen on electrode mercury.

[Studies on medico-legal diagnosis in cold district. 1. On the production of ethylalcohol in the outdoor cadavers in winter (author's transl)].

Some diagnostic problems in legal medicine were studied in dog cadavers left outdoors in the snow during winter in Sapporo. It was found that the cadaveric blood did not deteriorate till the end of 7 weeks and ethylalcohol or other Widmark positive substances were not produced in the cadavers during this period.

[Studies on the estimation of the postmortem interval. 5. The turbidity of the cornea (author's transl)].

The authors have investigated the relationship between the postmortem interval and the turbidity of the cornea of 410 persons who had died at known times and had been subjected to medico-legal autopsies in our laboratory. The conclusions are as follows: 1) Differences due to the states of the eyelids of cadavers, either open or closed, gave rise to slight variations of the turbidity of the cornea only within 12 hours after death. Slight variations due to age and sex were seen only from 12 to 24 hours after death and only in the age group of 0 to 19 years. Seasonal variation was not seen on the cadavers which had remained indoors at the Hokkaido districts. 2) In death from exsanguination none became a state of the strong turbidity. In death from intracranial injuries and asphyxia the strong turbidity developed earlier than in the other types of death. 3) When the cornea is transparent, the time which has elapsed since death is less than 36 hours, with few exceptions, with eyelids either open or closed. On the cadavers with closed eyelids, the week turbidity of the cornea is seen between 6 hours and 3 days, the moderate turbidity between 12 hours and 2 weeks and the strong turbidity over 18 hours.

[Studies on the estimation of the postmortem interval. 8. Burned bodies (author's transl)].

The authors have investigated 15 kinds of findings of 30 bodies burned at known times and autopsied in our laboratory. The conclusions are as follows: As the criteria for determining the time of death, external findings of cadavers burned lose value considerably, but internal findings, especially undigested food in the stomach remain as valuable as in ordinary circumstances. The usual criteria can be applied with the same degree of success to burned bodies, because appearances of findings caused by cadaveric phenomena show neither acceleration nor retardation in bodies burned so far as they are not severely destroyed.

[Studies on the estimation of the postmortem interval. 6. The putrefaction (author's transl)].

The authors have investigated ten kinds of putrefactive findings on 368 cadavers which were subjected to medico-legal autopsies in our laboratory and have come to the following conclusions. 1) Concerning the occurrence of the putrefactive findings, seasonal variation was not seen on the cadavers which had remained indoors in the Hokkaido districts. No variation due to age and sex was seen. The occurrence of the findings was less frequent in death from exsanguination, traumatic shock and cardiac tamponade. 2) The putrefactive findings can be utilized as a criterion for estimating the postmortem interval; the interval is estimated to be over 6 hours when the pericardial fluid is turbid, over 12 hours when exudation of putrefactive fluid, gas formation, blood-stained peritoneal fluid or blood-stained pericardial fluid is present, over 18 hours when greenish discoloration of the skin, network of the skin or blood staining of the endocardium or the endothelium is seen, over 2 days when bullae is present on the skin, over 5 days when the pericardial cavity is dry, within 6 days when none of them are present.

[Studies on medico-legal diagnosis in cold district. 2. Cadaveric phenomena in low temperature surroundings (author's transl)].

The authors have investigated 14 kinds of findings of 15 cadavers in low temperature surroundings which had remained at known postmortem interval and had been subjected to medico-legal autopsies in our laboratory, comparing with those at normal temperature. In low temperature surroundings, the onset and progress of the findings due to autolysis or putrefaction are retarded in a marked degree but the turbidity of the cornea which is considered to be due to physical or chemico-physical phenomenon is retarded merely in a slight degree. Hence, an erroneous determination of the postmortem interval can be avoided by taking care of the peculiarity. Even the cadavers at low temperature display in 3 to 5 months after death the findings whose degrees correspond to those of the cadavers at normal temperature in 4 to 7 days after death. Some fly-larvae pass the winter on cadavers demonstrating that the time of death was towards the end of autumn of the preceding year.

[Studies on medico-legal diagnosis in cold district. 3. Autopsy findings of the victims of cold exposure (author's transl)].

The authors have investigated the autopsy findings of 8 bodies died from cold. The conclusions are as follows: Pink colour of the blood in the left ventricle or the pulmonary veins, pink colour of the lungs and pink patches of the skin are fairly pathognomic findings of death from cold. Seven out of eight bodies have presented at least one of these findings. Subserous or submucous hemorrhage is less pathognomic and has been presented less frequently.

[Studies on medico-legal diagnosis in cold district. 4. An experimental study on the rise of temperature of dead body caused by the radiation of sunlight (author's transl)].

A sausage of 4 cm diameter and 15 cm length was chosen as a model of a part of human extremities. On the snowfield the model was stood on end and its temperature was recorded continuously by means of thermistor thermorecorder. The conclusions are as follows: In fine weather the temperature directly under the sunny surface of a model rose higher than the air temperature by 18 degrees C in the most remarkable case. Even in cloudy weather it rose fairly though lower than in fine weather. The rise of a model temperature became higher in February than in January. Even at a depth of 1.0 cm the temperature rose fairly though lower than directly under the surface. The temperature of the shady side of a model was equal to air temperature or snow temperature. It is presumed from these data that the temperature of a human dead body on snowfield becomes higher and reaches a optimum temperature for putrefaction at least partly by the radiation of sunlight. The presumption is supported by our experiences at the autopsies in cold district.