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.

Characterization and susceptibility to antiviral agents of herpes simplex virus type 1 containing a unique thymidine kinase gene with an amber codon between the first and the second initiation codons.

A herpes simplex virus type 1 (HSV-1) containing a thymidine (TK) gene with an amber mutation at the 8th position counted from the first AUG codon was isolated from a child with acute gingivostomatitis. The virus was predicted to express a mutant viral translated from the 2nd AUG codon at the 46th amino acid position and consisting of 331 amino acids. The virus was as sensitive to acyclovir (ACV), 5-bromovinyl-2'-deoxyuridine (BVdU), 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), and 1-beta-D-arabinofuranosylthymine (araT) as a wild-type HSV-1. The mutant TK showed the same level of TK activity as the wild-type TK at reaction temperatures of 34 degrees C, 37 degrees C and 39 degrees C. ACV, BVdU, BVaraU, and araT inhibited the replication of the TK-deficient and drug-resistant HSV-1 and HSV-2 in 293T cells in which the mutant TK was expressed to the same extent as in cells in which intact HSV-1-TK was expressed, whereas BVdU and BVaraU inhibited the replication of these viruses less strongly in cells in which HSV-2-TK was expressed. It can be concluded that the mutant HSV-1 exists in nature as a variant and possesses the necessary phosphorylation activities to form ACV-monophosphate from ACV, to form BVdU-diphosphate through BVdU-monophosphate from BVdU, and to form BVaraU-diphosphate through BVaraU-monophosphate from BVaraU. These results indicate that the mutant HSV-1-TK with a deletion of the first 45 amino acid residues is phenotypically the same as that of wild-type HSV-1-TK in terms of the phosphorylation activity of TK-associated anti-herpes virus drugs.

The membrane-type collectin CL-P1 is a scavenger receptor on vascular endothelial cells.

Collectins are a family of C-type lectins that have collagen-like sequences and carbohydrate recognition domains (CRD). They are involved in host defense through their ability to bind to carbohydrate antigens of microorganisms. The scavenger receptors type A and MARCO are classical type scavenger receptors that have internal collagen-like domains. Here we describe a new scavenger receptor that is a membrane-type collectin from placenta (collectin placenta 1 (CL-P1)), which has a typical collectin collagen-like domain and a CRD. The cDNA has an insert of about 2.2 kilobases coding for a protein containing 742 amino acid residues. The deduced amino acid sequence shows that CL-P1 is a type II membrane protein, has a coiled-coil region, a collagen-like domain, and a CRD. It resembles type A scavenger receptors because the scavenger receptor cysteine-rich domain is replaced by a CRD. Northern analyses, reverse transcription-polymerase chain reaction, and immunohistochemistry show that CL-P1 is expressed in vascular endothelial cells but not in macrophages. By immunoblotting and flow cytometry CL-P1 appears to be a membrane glycoprotein of about 140 kDa in human umbilical vein or arterial endothelial cells, placental membrane extracts, and CL-P1 transfected Chinese hamster ovary cells. We found that CL-P1 can bind and phagocytose not only bacteria (Escherichia coli and Staphylococcus aureus) but also yeast (Saccharomyces cerevisiae). Furthermore, it reacts with oxidized low density lipoprotein (OxLDL) but not with acetylated LDL (AcLDL). These binding activities are inhibited by polyanionic ligands (polyinosinic acid, polyguanylic acid, dextran sulfate) and OxLDL but not by polycationic ligands (polyadenylic acid or polycytidylic acid), LDL, or AcLDL. These results indicate that CL-P1 might play important roles in host defenses that are different from those of soluble collectins in innate immunity.

Herpes simplex virus type 1 suppresses the interferon signaling pathway by inhibiting phosphorylation of STATs and janus kinases during an early infection stage.

We examined the influence on the interferon (IFN) signaling pathway of infection with herpes simplex virus type 1 (HSV-1) strain VR3. Data from reporter gene assays showed that expression of both type I and type II IFN-inducible genes was dramatically suppressed during the early stage of HSV-1 infection (2 to 3 h postinfection). During these periods, phosphorylation levels of janus kinases (JAKs) and STATs did not increase after treatment of HSV-1-infected FL cells with IFN-alpha or IFN-gamma, although cellular protein levels of the JAKs and the STATs were not significantly changed. In contrast, the inhibitory effect of HSV-1 on phosphorylation of STAT1 was not observed in U937 cells, which show resistance to steady-state accumulation of RNA for HSV-1 immediate-early genes. The phosphorylation of STAT1 in FL cells was not inhibited by infection with a UV-inactivated virus. These results indicate that viral gene expression or viral protein production is necessary for the inhibition of phosphorylation by HSV-1.

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.

Direct and mononuclear cell mediated effects on interleukin 6 production by glioma cells in infection with herpes simplex virus type 1.

To clarify the mechanism of interleukin (IL)-6 elevation in the cerebrospinal fluid of viral meningitis and/or encephalitis patients, we investigated how herpes simplex virus type 1 (HSV1)-infection enhances IL-6 production in human glioma cells (the U373MG and T98G cells). Although human glioma cells did not show enhanced IL-6 production by direct HSV1-infection, the cell-free supernatant from HSV1-stimulated mononuclear cells (MNC) culture and lipopolysaccharide, as a positive control, markedly elevated IL-6 production at both mRNA and polypeptide levels. Ultra violet-irradiated HSV1 induced the secretion of the IL-6 inducing factor(s) from MNC, whereas heat-inactivated HSV1 did not show this activity. This finding indicated that the adsorption of virus on the surface of MNC may be sufficient for induction of secretion. The supernatant from the culture of HSV1-stimulated MNC contained detectable amounts of IL-1beta, tumor necrosis factor (TNF) alpha, interferon (IFN) gamma and IL-6, and its IL-6-inducing activity was inhibited only by anti-IL-1beta antibodies. Moreover, recombinant IL-1beta markedly enhanced IL-6 production in glioma cells with a concomitant elevation of its mRNA level. Taken together, the results suggest that in HSV1-infection of the CNS, enhancement of IL-6 production in glial cells is mediated not by direct infection to glial cells but rather by IL-1beta released from HSV1-stimulated MNC. These findings may help elucidate the mechanisms underlying cerebro-parenchymal inflammatory progression and repair in herpes simplex encephalitis.

Role of the UL25 gene product in packaging DNA into the herpes simplex virus capsid: location of UL25 product in the capsid and demonstration that it binds DNA.

Recent studies have suggested that the herpes simplex type 1 (HSV-1) UL25 gene product, a minor capsid protein, is required for encapsidation but not cleavage of replicated viral DNA. This study set out to investigate the potential interactions of UL25 protein with other virus proteins and determine what properties it has for playing a role in DNA encapsidation. The UL25 protein is found in 42 +/- 17 copies per B capsid and is present in both pentons and hexons. We introduced green fluorescent protein (GFP) as a fluorescent tag into the N terminus of UL25 protein to identify its location in HSV-1-infected cells and demonstrated the relocation of UL25 protein from the cytoplasm into the nucleus at the late stage of HSV-1 infection. To clarify the cause of this relocation, we analyzed the interactions of UL25 protein with other virus proteins. The UL25 protein associates with VP5 and VP19C of virus capsids, especially of the penton structures, and the association with VP19C causes its relocation into the nucleus. Gel mobility shift analysis shows that UL25 protein has the potential to bind DNA. Moreover, the amino-terminal one-third of the UL25 protein is particularly important in DNA binding and forms a homo-oligomer. In conclusion, the UL25 gene product forms a tight connection with the capsid being linked with VP5 and VP19C, and it may play a role in anchoring the genomic DNA.

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.

Comparison of polymorphism of thymidine kinase gene and restriction fragment length polymorphism of genomic DNA in herpes simplex virus type 1.

The polymorphism of the thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1) was analyzed and was compared with the restriction fragment length polymorphism (RFLP) of the whole genome to evaluate the relative efficiency of the TK gene as a potential probe for identification and discrimination of HSV-1. The effectiveness of using the polymorphism of the TK gene in classifying HSV-1 strains was comparable to that of RFLP analysis of 66 sites, suggesting that TK gene sequencing may have important applications in epidemiological studies of HSV-1.

The role of the UL41 gene of herpes simplex virus type 1 in evasion of non-specific host defence mechanisms during primary infection.

The UL41 gene product (vhs) of herpes simplex virus (HSV) is packaged in the virion, and mediates host protein synthesis shutoff at the early stage of the virus replication cycle. In order to clarify the role of vhs in virus replication and virulence, we isolated a completely UL41-deficient mutant (the VRDelta41 strain) and its revertant (the VRDelta41R strain). In the mouse encephalitis model, the replication of strain VRDelta41 was inhibited after 2 days post-infection, resulting in low virulence, by gamma-ray-sensitive cells such as lymphocytes and/or neutrophils. The result suggested that some cytokines, produced in VRDelta41-inoculated brains, activate and induce the migration of gamma-ray-sensitive cells to the infection site. Therefore, cytokines produced by HSV-1-infected human cells were screened, and potent inductions of interleukin (IL)-1beta, IL-8 and macrophage inflammatory protein-1alpha by VRDelta41 infection were observed. Moreover, the VRDelta41 strain showed 20- and 5-fold higher sensitivity to interferon-alpha and -beta compared to the wild-type strain, respectively. These results indicate that one important role of vhs in vivo is evasion from non-specific host defence mechanisms during primary infection through suppression of cytokine production in HSV-infected cells and reduction of the anti-HSV activity of interferon-alpha and -beta.

Rapid phenotypic characterization method for herpes simplex virus and Varicella-Zoster virus thymidine kinases to screen for acyclovir-resistant viral infection.

A rapid phenotypic screening method for herpes simplex virus (HSV) and varicella-zoster virus (VZV) thymidine kinase (TK) genes was developed for monitoring acyclovir-resistant viruses. This method determines the biochemical phenotype of the TK polypeptide, which is synthesized in vitro from viral DNA using a procedure as follows. The TK gene of each sample virus strain is amplified and isolated under the control of a T7 promoter by PCR. The PCR products are transcribed with T7 RNA polymerase and translated in a rabbit reticulocyte lysate. Using this method, enzymatic characteristics and the size of the TK polypeptides encoding HSV and VZV DNA were defined in less than 2 days without virus isolation. The assay should be a powerful tool in monitoring drug-resistant viruses, especially in cases in which virus isolation is difficult.

[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.

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.

Herpesvirus alkaline deoxyribonuclease; a possible candidate as a novel target for anti-herpesvirus therapy.

Herpesvirus alkaline deoxyribonucrease (DNase) is coded in the genome of all herpesvirus species determined total sequence and is conserved in structure. In order to determine whether the enzyme could be a target for a novel antiherpesvirus therapy, the anti-herpes simplex virus type 1 (HSV-1) activity of antisense oligonucleotide for HSV-1 alkaline DNase was studied. Six antisense phosphorothioate oligonucleotides, targeted to an internal AUG start codon, were designed and evaluated. One of the oligonucleotides, UL12-4, inhibited wild type and thymidine kinase-deficient HSV-1 replication to 21.5 and 19.5% at 40 microM, respectively. The quantity of alkaline DNase mRNA and DNase activity in HSV-1-infected Vero cells was reduced to one eighth and 66.9% of control, respectively, by treatment with 40 microM of UL12-4, but no effect was observed on the quantity of HSV-1 glycoprotein H mRNA (gamma2 gene) or on the replication of Vero cells. These results indicate that UL12-4 inhibits HSV-1 replication by decreasing the amount of alkaline DNase mRNA. The herpesvirus alkaline DNase could be a novel target for anti-herpesvirus drug.

Insufficient resistance of trehalose-6,6-dimycolate-treated T-cell receptor delta gene mutant (TCR delta-/-) mice against influenza virus infection.

Normal mice inoculated intravenously with 50 microg trehalose-6,6'-dimycolate, a glycolipid component of the cell wall of Mycobacterium, in an oil-in-water emulsion (TDM emulsion) acquired a high resistance to intranasal lethal infection of an influenza virus. In contrast, TDM emulsion-treated T-cell receptor delta gene mutant (TCR delta-/-) mice acquired insufficient resistance against the lethal influenza virus infection. The patterns of insufficient resistance were identical to the results obtained previously with mice which were depleted of T-lymphocytes bearing gammadelta T-cell receptors (gammadelta T-cells) by in vivo administration of anti-gammadelta T-cell receptor monoclonal antibody (Hoq et al, J. Gen. Virol. 78: 1597-1603, 1997). These results strongly suggest that the gammadelta T-cells play an important non-specific role in resistance against influenza virus infection.

Nucleotide sequence of thymidine kinase gene of sequential acyclovir-resistant herpes simplex virus type 1 isolates recovered from a child with Wiskott-Aldrich syndrome: evidence for reactivation of acyclovir-resistant herpes simplex virus.

Recurrent acyclovir (ACV)-resistant (ACV-r) herpes simplex virus type 1 (HSV-1) infections occurred in a patient with Wiskott-Aldrich syndrome, an X-linked recessive immunodeficiency syndrome composed of three clinical characteristics of immunodeficiency, thrombocytopenia, and an eczematous dermatitis. The patient had severe and recurrent ACV-r herpes simplex and was treated with vidarabine in a satisfactory manner from 1993 to 1997. During the 4-year observation period, two ACV-sensitive (ACV-s) HSV-1 isolates and five ACV-r HSV-1 isolates were recovered. The nucleotide sequence of the thymidine kinase (TK) gene from these sequential ACV-r isolates was compared with the ACV-s isolates. A single nucleotide deletion of cytosine (C) from homopolymer stretch of four C residues between nucleotide 1061 and 1064 of the open reading frame was found in all ACV-r isolates. No other differences were observed in the TK nucleotide sequence between ACV-s and ACV-r isolates. The TK nucleotide sequences of the two ACV-s isolates were identical to each other and those of the five ACV-r isolates were identical to one another. These results suggest that the ACV-r HSV-1 might have derived from the ACV-s strain in the patient body and that TK-associated ACV-r HSV-1 can reactivate from latency.

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.

Recurrent aciclovir-resistant herpes simplex in a child with Wiskott-Aldrich syndrome.

A boy with Wiskott-Aldrich syndrome suffered from thymidine kinase (TK)-altered and aciclovir-resistant herpes simplex virus type 1 (HSV-1) skin infections. He presented with severe herpes simplex around the left eye in March 1993 at the age of 8 years. HSV-1 strain TAS was isolated and was shown to be susceptible to aciclovir (50% inhibitory concentration (IC50) 0.23 microg/mL). He was treated with intravenous (i.v.) high dose aciclovir, 2 mg/kg per h, which produced an improvement. About 1 year later (May 1994), a severe herpes simplex infection appeared on his face, arm, genitalia, back and foot. Treatment with i.v. aciclovir, 2 mg/kg per h, was initiated, but the skin lesions did not improve. HSV-1 strain TAR was isolated and was shown to be resistant to aciclovir (IC50 36 microg/mL). HSV-1 TAR and TAS were susceptible to vidarabine (IC50 4. 4 and 2.9 microg/mL, respectively). The skin lesions were treated with i.v. vidarabine, 15-20 mg/kg per day, and healed satisfactorily. However, in March 1995, the patient again experienced a severe herpes simplex infection around the left eye. HSV-1 strain R95 was isolated and was shown to be resistant to aciclovir (IC50 36 microg/mL). Diminished sensitivity of HSV-1 TAR and R95 to aciclovir was associated with reduced viral TK activity and loss of aciclovir phosphorylation activity.

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.

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.