Kazufumi Ikuta

Anti-viral and anti-bacterial activities of an extract of blackcurrants (Ribes nigrum L.).

The inhibitory effects of an extract of the blackcurrant (Ribes nigrum L.) against pathogens associated with oral, nasopharyngeal and upper respiratory infectious diseases; namely respiratory syncytial virus (RSV), influenza virus A and B (IFV‐A and IFV‐B), adenovirus (AdV), herpes simplex virus type 1, Haemophilus influenzae type B, Streptococcus pneumoniae and Streptococcus mutans, were investigated. Less than 1% concentration of extract of blackcurrant inhibited replication of RSV, IFV‐A and ‐B and HSV‐1 by over 50% and a 10% extract inhibited adsorption of these viruses onto the cell surface by over 95%. The effects on AdV were much less pronounced; the half minimal inhibitory concentration of AdV replication was 2.54 ± 0.26, and a 10% concentration of the extract inhibited AdV adsorption on the cell surface by 72.9 ± 3.4%. The antibacterial activities of the blackcurrant were evaluated based on its efficacy as a disinfectant. A 10% extract disinfected 99.8% of H. Influenzae type B and 78.9% of S. pneumoniae in 10 min, but had no demonstrable effect against S. mutans. The blackcurrant extract still showed antiviral and antibacterial activities after the pH had been made neutral with sodium hydroxide, suggesting that these activities are not the result of acidic reactions or of components precipitated at a neutral pH. These findings demonstrate the potential of blackcurrant extract as a functional food for oral care.

Oral valganciclovir treatment for congenital cytomegalovirus infection

Congenital cytomegalovirus (CMV) infection is the most common intrauterine infection. Approximately 10–15% of congenitally infected neonatal infants exhibit clinical evidence of congenital infection at birth. This group is more likely to experience sequelae, including microcephalus, sensor neural hearing loss, cognitive, motor and visual deficits and seizures. Previous studies have shown that approximately half of the children with symptomatic congenital CMV infection develop hearing loss, and the majority of these children experience continued postnatal deterioration of their hearing. Ganciclovir is an antiviral agent that acts against herpes viruses and has been used successfully to treat CMV infection. In addition, it has been reported that ganciclovir therapy, begun in the neonatal period in symptomatic infants with a CMV infection involving the central nervous system, prevents hearing deterioration. However, the efficacy of ganciclovir for hearing deterioration in patients beyond the neonatal period is unknown. In this report we present the case of a five-month-old girl who was treated with oral valganciclovir for progressive hearing loss resulting from congenital CMV infection.

Cytomegalovirus (CMV) glycoprotein H-based serological analysis in Japanese healthy pregnant women, and in neonates with congenital CMV infection and their mothers

BACKGROUND Congenital cytomegalovirus (CMV) infection is caused by maternal primary infection as well as CMV reinfection or reactivation during pregnancy, although differences in the clinical impact between these modes of infection remain to be clarified. OBJECTIVES To investigate the latest prevalence and risk of multiple CMV infection in healthy pregnant women, as well as the types of maternal CMV infection associated with congenital CMV infection. STUDY DESIGN Seroprevalence against CMV and IgG subclasses were determined in 344 serum samples from healthy pregnant women in Japan. CMV genotype and serotype were also determined in 18 pairs of mothers and neonates with congenital CMV infection identified in our CMV screening program. RESULTS Thirty-two percent of the pregnant women were seronegative, while 66% of CMV seropositive women had IgG3 antibodies against one epitope on glycoprotein H (gH) as the major subclass, and 52% had IgG1 antibodies against one epitope on glycoprotein B (gB). Only a single genotype determined by CMV gH neutralizing epitope was found in the urine from the 18 neonates with congenital CMV infection, even though one case possessed antibodies against multiple CMV strains. In that case, the antibodies against the strain not detected in the urine from the infant disappeared within one month after birth, whereas the antibodies against the infecting CMV strain continued to be detected at 12 months after birth. CONCLUSIONS Two (11%) of 18 cases of congenital CMV infection occurred via maternal CMV reinfection. Maternal humoral immunity did not prevent congenital CMV infection with another gH subtype.

A novel real-time PCR method for determination and quantification of each cytomegalovirus glycoprotein H subtype in clinical samples.

ABSTRACT To investigate reinfection in patients with congenital cytomegalovirus (CMV) infection, we established a CMV subtype-specific real-time quantitative PCR method targeting the CMV gH epitope region that can be used for evaluating pathogenic CMV strains in cases of mixed CMV infection.

Restricted infection of murine cytomegalovirus (MCMV) in neonatal mice with MCMV-induced sensorineural hearing loss

BACKGROUND Congenital infection with human Cytomegalovirus (HCMV) is known to be a causative agent of sensorineural hearing loss (SNHL). OBJECTIVES To clarify the nongenetic etiology of SNHL by identifying the Cytomegalovirus (CMV)-infected region in the cochleae. STUDY DESIGN We established an animal model of SNHL by injecting neonatal Balb/c mice with intracerebral murine Cytomegalovirus (MCMV) within 24h after delivery. RESULTS At 3 weeks of age, unilateral and bilateral SNHL were observed in 24% (5/21) and 29% (6/21) of the mice, respectively. SNHL thereafter progressed, with 79% of mice developing bilateral SNHL by 6 weeks of age. MCMV antigens and DNA were detected in the spiral ganglion, and cells surrounding the meninges and scala tympani at 1 week of age. However, both MCMV antigens and DNA had completely disappeared by 2 weeks of age. It is possible that the MCMV reached the spiral ganglion via cerebrospinal fluid as the result of meningitis, as the stria vascularis was found to be MCMV antigen negative. Myosin VI expression in the outer hair cells was lost at 3 weeks of age. MCMV and myosin VI expression disappeared before and during SNHL progression, respectively. CONCLUSIONS There was a definite lag time between the period in which MCMV antigens/DNA-positive cells were observed and that in which SNHL developed and myosin VI-negative hair cells were observed. Further study is needed to explore the role of MCMV in the loss of myosin VI expression in the outer hair cells.

Herpes simplex virus type 1 virion-derived US11 inhibits type 1 interferon-induced protein kinase R phosphorylation

The herpes simplex virus type 1 (HSV‐1) VRTK− strain that was previously isolated in our laboratory as an acyclovir‐resistant thymidine kinase (TK)‐deficient mutant, is more sensitive to type 1 interferon than is the parent strain VR3. The properties of this mutant were investigated to clarify the mechanism for its hyper‐sensitivity to interferon (IFN). It was found that: (i) IFN‐pretreated cells, but not those treated with IFN after adsorption, are hyper‐sensitive to IFN; (ii) the mutant cannot inhibit protein kinase R phosphorylation efficiently during the early stage of replication (2 hrs post‐infection); (iii) expression of US11 in infected cells and its incorporation into the virion is reduced in the mutant compared to the wild type, despite the fact that a similar degree of DNA synthesis occurs during replication of both strains and; (iv) over‐expression of wild‐type viral TK has no effect on the phenotype of the VRTK− strain, indicating that the phenotype is induced by a mutation(s) that does not involve the TK gene. These results suggested that the presence of US11 in the virion, but not that expressed after infection, plays an important role in the escape function of HSV‐1 from the antiviral activity of type 1 IFN.

Involvement of herpes simplex virus type 1 UL13 protein kinase in the induction of SOCS genes, the negative regulator of cytokine signaling

The suppressor of cytokine signaling (SOCS) family has eight members and suppresses various cytokine signaling pathways, including IFN signaling. Therefore, some viruses have evolved molecular mechanisms for inducing SOCS proteins and thus escaping host immunity. Herpes simplex virus type 1 (HSV‐1) has a mechanism for escaping from type I IFN by induction of both SOCS1 and SOCS3. In this study, expression of the eight members of the SOCS family stimulated by HSV‐1 infection was comparatively analyzed by qRT‐PCR. It was found that SOCS1 and SOCS3 are induced by HSV‐1‐infection at 4 hr post infection. However, such induction was not observed in UL13 deficient virus‐infected cells, suggesting that UL13 protein kinase participates in induction of both genes. The transcription factor Sp1‐binding sites of SOCS3 promoter/enhancer region were identified as the regulatory elements for induction of SOCS3 in HSV‐1 infected cells. Accumulation of activated Sp1 was detectable in the nuclei of HSV‐1‐infected cells before induction of SOCS3. Taken together, these results suggest that HSV‐1 has a potent mechanism for escaping from the IFN system.

Congenital CMV infection via a re-infected mother with original antigenic sin: a case report.

A 27-year-old pregnant woman who was positive for anti-cytomegalovirus (CMV) antibodies gave birth to a congenitally CMV-infected neonate at 40 weeks of gestation. According to strain-specific serological analysis, which is able to determine the two types of CMV glycoprotein H (gH), the mother possessed anti-gH(To) antibodies only, but the neonate possessed anti-gH(AD) and anti-gH(To) antibodies at 4 weeks after birth. As the anti-gH(To) IgG was decreased in the neonate at 8 months post-delivery, these antibodies are thought to have been transferred from the mother as maternal antibodies. The anti-gH(AD) IgG level was maintained in the child even after 8 months post-delivery. Congenital infection with a CMV gH(AD) type strain was confirmed by strain-specific real-time PCR using a urine specimen from the child. On the other hand, anti-gH(AD) IgG was not detected even after 8 months post-delivery in a maternal specimen. The mother only produced antibodies against the CMV strain identified as the primary infection, which is characteristic of original antigenic sin.

Modification of the HCMV-specific IFN-γ release test (QuantiFERON-CMV) and a novel proposal for its application

Human cytomegalovirus (HCMV) is universally distributed among humans without any adverse effects; however, it induces severe diseases in immunocompromised patients such as organ transplant recipients and AIDS patients. To manage these immunocompromised patients, an easy clinical examination for the monitoring of disease risk is required. In this study, we modified the interferon-γ (IFN-γ) release test (QuantiFERON®-CMV) using HCMV immediate early-1 (IE-1) or pp65 whole proteins, or UV-inactivated HCMV particles as an antigen. The response of heparinized peripheral blood from healthy volunteers to the pp65 protein showed an obvious dose-dependent sigmoid curve, although no correlation was observed between results of this assay and an ELISPOT assay. The addition of pp65 to the blood samples at a final concentration of 1×103 to 1×105 pg/ml was found to be optimum. Using this assay, we observed a significant enhancement in cellular immunity in volunteers after the daily ingestion of yogurt for 8 weeks, which suggested a novel application of the assay in addition to monitoring HCMV infection risk. IFN-γ secretion from peripheral blood cells on HCMV-antigen stimulation differed significantly between individuals; therefore, the assay could not be normalized. Nevertheless, it was found to be particularly useful for observing fluctuations in cellular immune activity on an individual level.

Protection from lethal herpes simplex virus type 1 infection by vaccination with a UL41-deficient recombinant strain

UNLABELLED The UL41 gene of herpes simplex virus type 1 (HSV-1) encodes a virion host shut off protein which is involved in immune evasion.　The growth and virulence of HSV-1 is markedly reduced by the deletion of UL41.　In this report, the UL41-deleted recombinant HSV-1 strain VR∆41 was evaluated as a prophylactic live attenuated vaccine against lethal HSV-1 infection in a mouse model.　Intraperitoneal (i.p.) inoculation with the VR∆41 strain clearly inhibited lethal wild-type HSV-1 (VR-3 strain) infection after both i.p. and intracerebral (i.c.) inoculations.　Vaccination with the VR∆41 strain was safer than VR-3 vaccination and was able to protect against a wild-type challenge to the same degree as VR-3 vaccination.　In contrast, i.p. inoculation with ultraviolet-irradiated VR-3 induced resistance against i.p. infection, but not against i.c. INFECTION Although replication of the VR∆41 strain in mice was greatly reduced compared to that of the VR-3 strain, VR∆41 strain maintained the ability to spread to the central nervous system (CNS) from a peripheral inoculation site. These results indicated that the VR∆41 strain evoked a potent immune reaction through viral protein expression within CNS without the induction of lethal encephalitis.　The entry of antigens into the CNS was essential for the establishment of protective immunity against the lethal HSV encephalitis.　We concluded that only a live attenuated vaccine is able to afford a prophylactic effect against CNS infection with HSV.　In order to fulfill this requirement, UL41-deleted viruses provide a strong candidate for use as a recombinant live vaccine.