Ken Ishioka

Differential mutation patterns in thymidine kinase and DNA polymerase genes of herpes simplex virus type 1 clones passaged in the presence of acyclovir or penciclovir.

ABSTRACT A total of 21 clones of acyclovir (ACV)-resistant (ACVr) herpes simplex virus type 1 (HSV-1) and 23 clones of penciclovir (PCV)-resistant (PCVr) HSV-1, emerging during serial passages in the presence of ACV or PCV, were isolated under conditions excluding contamination of resistant mutants in the starting virus culture, and their mutations in the thymidine kinase (TK) and DNA polymerase (DNA Pol) genes were analyzed comparatively. Mutations in the TK genes from ACVr mutants consisted of 50% single nucleotide substitutions and 50% frameshift mutations, while the corresponding figures for the PCVr mutants were 4 and 96%, respectively (P < 0.001). Eight of the 21 ACVr clones, but none of the 23 PCVr clones, had mutations in DNA Pol. Only nucleotide substitution(s) could be detected in the DNA Pol gene, as the gene is essential for virus replication. Therefore, the results for the DNA Pol mutants are concordant with those for the TK mutants in that a single nucleotide substitution was commonly observed in the ACVr, but not in the PCVr, mutants. These results clearly point to differential mutation patterns between ACVr and PCVr HSV-1 clones.

RSV replication is attenuated by counteracting expression of the suppressor of cytokine signaling (SOCS) molecules.

Human RSV causes an annual epidemic of respiratory tract illness in infants and in elderly. Mechanisms by which RSV antagonizes IFN-mediated antiviral responses include inhibition of type I IFN mRNA transcription and blocking signal transduction of JAK/STAT family members. The suppressor of cytokines signaling (SOCS) gene family utilizes a feedback loop to inhibit cytokine responses and block the activation of the JAK/STAT signaling pathway. To evaluate the potential of SOCS molecules to subvert the innate immune response to RSV infection, eight SOCS family genes were examined. RSV infection up-regulated SOCS1, SOCS3, and CIS mRNA expression in HEp-2 cells. Suppression of SOCS1, SOCS3 and CIS by short interfering ribonucleic acid (siRNA) inhibited viral replication. Furthermore, inhibition of SOCS1, SOCS3, or CIS activated type I IFN signaling by inducing STAT1/2 phosphorylation. These results suggest that RSV infection escapes the innate antiviral response by inducing SOCS1, SOCS3 or CIS expression in epithelial cells.

Identification of a highly conserved region in the human cytomegalovirus glycoprotein H gene and design of molecular diagnostic methods targeting the region

Genomic polymorphism of human cytomegalovirus (HCMV) leads to difficulties in the design of molecular diagnostic systems; therefore, a suitable target region was determined in the glycoprotein H (gH) gene, which has been reported to be the most conserved gene. A highly conserved region was identified from codon 1,282 to 1,988 of the gH gene by alignment of 23 nucleotide sequences (14 registered in the DNA Data Bank of Japan and 9 sequenced in this laboratory). Diagnostic methods based on nested PCR, real-time PCR and loop-mediated isothermal temperature amplification (LAMP) were designed for this region. Primers and a probe for nested and real-time PCR were designed for the completely conserved sequences in all HCMV strains. However, a few mismatched nucleotides could not be excluded from the LAMP primers due to the need for eight primer-binding sites in a 200bp-region. The sensitivities of the nested PCR, real-time PCR and LAMP reactions were 5, 10 and 100 copies/tube, respectively. An analysis of clinical specimens showed that both nested and real-time PCR detected HCMV with greater sensitivity than did a pp65 antigenemia assay and were expected to minimize the incidence of false-negative results, whereas the sensitivity of the LAMP reaction was comparable with that of the antigenemia assay.

DSCG reduces RSV-induced illness in RSV-infected mice.

Respiratory syncytial virus (RSV) is one of the pathogens generally associated with the common cold, lower respiratory infection, and exacerbation of asthma. Disodium cromoglycate (DSCG) is a safe and widely used drug for the prevention of bronchial asthma and allergic rhinitis attacks. The effect of DSCG on acute upper respiratory tract viral infections remains controversial. The purpose of the study was to investigate the effects of DSCG on parameters of RSV induced‐illness. Using a well‐characterized murine model of RSV infection, the effect of DSCG on RSV‐induced illness was evaluated by body weight, respiratory function, viral replication, level of IFN‐γ in lungs, serology, and histopathology. Mice treated with DSCG were protected against RSV‐induced weight loss. The baseline Penh in RSV‐infected mice treated with DSCG was less than that in mice treated with saline. In methacholine challenge, the increase in Penh in RSV‐infected mice treated with DSCG was suppressed to the same level as that in the mock‐infected group. Further, there were no differences in viral replication between the mice treated with DSCG and those treated with saline, and the level of inflammation observed in the lungs in RSV‐infected mice treated with DSCG was not as severe as that in mice treated with saline. These findings indicate that DSCG may be an effective agent for the prevention of RSV induced disease and the relief of symptoms of RSV infection. J. Med. Virol. 81:354–361, 2009.

Discrimination of Herpes Simplex Virus Type 2 Strains by Nucleotide Sequence Variations

ABSTRACT We determined the polymorphous 400-bp regions in UL53, US1, and US4 for the discrimination of herpes simplex virus type 2 (HSV-2) strains. Thirty-six HSV-2 clinical strains could be differentiated into 35 groups using these three regions and into 36 groups by additional analysis of three noncoding regions previously reported as polymorphous.

Evaluation of mixed infection cases with both herpes simplex virus types 1 and 2

Herpes simplex virus type 1 (HSV‐1) is isolated principally from the upper half of the body innervated by the trigeminal ganglia whereas herpes simplex virus type 2 (HSV‐2) is generally isolated from the lower half of the body innervated by the sacral ganglia. However, recent reports suggest that HSV‐1 and HSV‐2 can each infect both the upper and lower half of the body causing a variety of symptoms and there is a possibility that HSV‐1 and HSV‐2 infections can occur simultaneously with both causing symptoms. HSV type in clinical isolates from 87 patients with genital herpes and 57 with ocular herpes was determined by the polymerase chain reaction (PCR), and six cases of mixed infection with both HSV‐1 and HSV‐2 were identified. Of the six cases, three were patients with genital herpes and three were ocular herpes patients. Analysis of the copy number of the HSV‐1 and HSV‐2 genome by a quantitative real time PCR demonstrated that HSV‐1 was dominant at a ratio of approximately 100:1 in the ocular infections. In contrast, the HSV‐2 genome was present at a 4–40 times higher frequency in isolates from genital herpes patients. There was no obvious difference between the clinical course of mixed infection and those of single HSV‐1 or HSV‐2 infections. This study indicated that the frequency of mixed infection with both HSV‐1 and HSV‐2 is comparatively higher than those of previous reports. The genome ratio of HSV‐1 and HSV‐2 reflects the preference of each HSV type for the target organ. J. Med. Virol. 80:883–887, 2008.

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.

The quantitative detection of herpes simplex virus, varicella zoster virus, and cytomegalovirus DNAs in recipient corneal buttons.

Purpose: We detected herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), varicella zoster virus (VZV), and cytomegalovirus (CMV) DNAs in recipient corneal buttons taken at the time of penetrating keratoplasty. Methods: Twenty-seven corneal buttons were obtained from 27 patients (10 men and 17 women), 7 of whom had a history of HSV keratitis. Excised corneal buttons were immediately frozen in liquid nitrogen in the operating theater and then stored at −80°C until DNA extraction. The detection of HSV-1, HSV-2, VZV, and CMV DNAs was carried out by a nested polymerase chain reaction (PCR) method. The genome copy numbers for the nested PCR-positive samples were subsequently quantified by real-time PCR. Results: HSV-1, HSV-2, VZV, and CMV DNAs were detected in 10, 1, 9, and 2 of the 27 recipient corneal buttons, respectively. HSV-1 or HSV-2 DNAs were also detected in 5 of 7 patients with a history of HSV keratitis. Both CMV-positive patients (patients 2 and 3) had ocular pemphigoid. Among the nested PCR-positive samples, 2 HSV-1, 1 HSV-2, 1 VZV, and 1 CMV sample could be quantified by real-time PCR. Copy numbers ranged from 19 to 928 copies. Conclusions: All 4 herpesviruses, including CMV, were detected in the corneal buttons. The relationship between CMV in the cornea and ocular diseases of the anterior segment should be further evaluated.

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.