Shin Koyano

Etiology of Severe Sensorineural Hearing Loss in Children: Independent Impact of Congenital Cytomegalovirus Infection and GJB2 Mutations

BACKGROUND Sensorineural hearing loss (SNHL) is the most common congenital disease. Longitudinal studies of infants with congenital cytomegalovirus (CMV) infection have demonstrated an association between CMV and SNHL. However, because of the lack of suitable neonatally collected specimens, the proportion of CMV-associated SNHL has not been defined, nor has the relationship between CMV and the major genetic causes of SNHL, such as mutations in the GJB2 gene. METHODS Sixty-seven children with severe SNHL were analyzed for CMV and human herpesvirus 6 (HHV-6) infections and for GJB2 mutations. DNA specimens were prepared from dried umbilical cords, which are available for everyone born in Japan. Four children with typical symptomatic infection at birth served as positive control subjects. RESULTS Congenital CMV infection and GJB2 mutations were identified in 15% and 24% of the patients, respectively. HHV-6 was not detected. All children with CMV-associated cases developed SNHL before they were 2 years old. Most children with CMV-associated SNHL had no obvious clinical abnormality at birth, and their viral loads were lower than those of symptomatic children. CONCLUSIONS Congenital CMV infection is an important cause of severe SNHL, and it has an incidence comparable to that of GJB2-associated SNHL.

Characterization of Entry Mechanisms of Human Herpesvirus 8 by Using an Rta-Dependent Reporter Cell Line

ABSTRACT To analyze the mechanisms of entry of human herpesvirus 8 (HHV-8), we established a reporter cell line T1H6 that contains the lacZ gene under the control of the polyadenylated nuclear RNA promoter, known to be strongly activated by a viral transactivator, Rta. We found that infection with cell-free virus, as well as cocultivation with HHV-8-positive primary effusion lymphoma cell lines, activated the lacZ gene of T1H6 in a sensitive and dose-dependent manner. Addition of Polybrene and centrifugation enhanced, but polysulfonate compounds inhibited, the HHV-8 infectivity. RGD-motif-containing polypeptides and integrins did not decrease the infectivity, suggesting the presence of an additional cellular receptor other than the reported one. The entry was dependent on pH acidification but not on the clathrin pathway. Although conditioned media obtained from human immunodeficiency virus (HIV)-infected cells did not have any effect on the early steps of HHV-8 infection, intracellular expression of a proviral HIV type 1, but not of Tat alone, increased the HHV-8-dependent reporter activation slightly, suggesting a potential of HIV-mediated enhancement of an early step of HHV-8 infection.

Encephalopathy associated with haemophagocytic lymphohistiocytosis following rotavirus infection

Abstract A 2-year-old Japanese boy with a haemophagocytic lymphohistiocytosis (HLH) associated encephalopathy which developed after rotavirus infection is described. The neurological symptoms consisted of coma, seizures and spastic quadriplegia. On therapy with steroids, etoposide and cyclosporin A, the patient recovered without any neurological deficits. The interferon-gamma levels in serum and CSF were elevated at onset of the disease but had returned to normal at the time of clinical remission. Brain MRI revealed diffuse white matter abnormalities and parenchymal volume loss. Proton magnetic resonance spectroscopy revealed elevated lactate in the abnormal lesions observed on MRI, indicating that macrophages not exhibiting aerobic metabolism had infiltrated the CNS. At the time of clinical remission, the white matter abnormalities and brain lactate had disappeared. These findings suggested that the neurological symptoms resulted from the overproduction of cytokines by activated T-cells and macrophages. The pathophysiology of a HLH associated encephalopathy was considered to be a local immune response within the CNS, because interferon-gamma can induce the expression of major histocompatibility complex class I and II antigens on glial cells in the CNS. Conclusion Haemophagocytic lymphohistiocytosis associated encephalopathy should be considered early in the differential diagnosis of cases with acute onset neuropathy.

Evaluation of screening tests for congenital cytomegalovirus infection.

Although the use of dried blood spots has been proposed for screening of newborns with congenital cytomegalovirus infection, viral loads in blood were significantly smaller than those in urine (P < 0.001), and DNA recovery from dried blood spots using the thermal shock procedure was inefficient. In contrast, our urine-based screening program identified asymptomatic cases with low viral loads in blood.

Real-Time PCR Assay Using Specimens on Filter Disks as a Template for Detection of Cytomegalovirus in Urine

ABSTRACT Since congenital cytomegalovirus (CMV) infection causes late-onset sequelae, the identification of CMV-infected newborns is important. For this purpose, we established a simple real-time PCR assay using a filter disk. Combined with the collection of urine using filter papers placed in the diaper, this assay can make CMV screening more feasible and cost-effective.

Dried Umbilical Cords in the Retrospective Diagnosis of Congenital Cytomegalovirus Infection as a Cause of Developmental Delays

To clarify the impact of congenital cytomegalovirus (CMV) infection on developmental disabilities, 20 children with disabilities of unknown cause were analyzed. Five children were CMV positive and had no clinical manifestations at birth. Intracranial calcification was observed in 4 cases. Thus, congenital CMV infection is a significant cause of developmental disabilities.

Analysis of the Relationship between Cellular Thymidine Kinase Activity and Virulence of Thymidine Kinase-Negative Herpes Simplex Virus Types 1 and 2

The virulence of thymidine kinase‐negative herpes simplex virus type 1 (HSV‐1; VRTK− strain) and type 2 (HSV‐2; UWTK− strain) was studied in comparison with that of their parental strains (VR‐3 and UW‐268, respectively) in an encephalitis model of adult (4‐week‐old) and newborn (3‐day‐old) mice. Viral thymidine kinase (TK) activity was essential for the maximum expression of virulence of HSV‐1, because the 50% lethal dose (LD50) of VRTK− was 60 times higher than that of VR‐3 in the brains of newborn mice expressing high levels of cellular TK activity. However, the UWTK− strain showed the same virulence as the parental strain in newborn mice, despite the lack virulence in adults, suggesting that replication of the UWTK− strain was completely supported by cellular TK activity. This difference in the role of viral and cellular TKs for virus growth between HSV‐1 and HSV‐2 was confirmed with the one‐step growth of virus strains in L‐M and L‐M(TK−) cells.

Polymorphisms in TLR-2 are associated with congenital cytomegalovirus (CMV) infection but not with congenital CMV disease

BACKGROUND Cytomegalovirus (CMV) is the most common cause of congenital virus infection. However, the risk factors for infection in utero and for progression to a severe clinical outcome remain uncertain. Recent studies have identified associations of specific single nucleotide polymorphisms (SNPs) in Toll-like receptor (TLR) genes with susceptibility to infections of some viruses and with their clinical outcome. METHODS Genetic polymorphisms in the TLR-2, TLR-4, and TLR-9 genes were analyzed in 87 children with congenital CMV infections by the TaqMan allelic discrimination assay. The frequencies of genotypes in the general Japanese population were obtained from the National Center for Biotechnology Information (NCBI) databases. Associations between the analyzed SNPs and congenital CMV infection or disease were evaluated. RESULTS The CC genotype at SNP rs3804100 in the TLR-2 gene was significantly associated with congenital CMV infection but not with congenital CMV disease. Furthermore, the AG genotype at SNP rs1898830 in the TLR-2 gene tended to be identified less frequently in children with congenital CMV infection. There were no statistically significant associations between SNPs in the TLR-4 and TLR-9 genes and congenital CMV infection or disease. CONCLUSION TLR-2 polymorphisms may have some association with congenital CMV infection, although the mechanism underlying this effect remains to be clarified.

Establishment of a Cell-Based Assay for Screening of Compounds Inhibiting Very Early Events in the Cytomegalovirus Replication Cycle and Characterization of a Compound Identified Using the Assay

ABSTRACT To simplify the detection of infectious human cytomegalovirus (HCMV), we generated a cell line that produced luciferase in a dose-dependent manner upon HCMV infection. Using this cell line, we identified anti-HCMV compounds from a diverse library of 9,600 compounds. One of them, 1-(3,5-dichloro-4-pyridyl)piperidine-4-carboxamide (DPPC), was effective against HCMV (Towne strain) infection of human lung fibroblast cells at a 50% effective concentration of 2.5 μM. DPPC also inhibited the growth of clinical HCMV isolates and guinea pig and mouse cytomegaloviruses. Experiments using various time frames for treatment of the cells with DPPC demonstrated that DPPC was effective during the first 24 h after HCMV infection. DPPC treatment decreased not only viral DNA replication but also IE1 and IE2 expression at mRNA and protein levels in the HCMV-infected cells. However, DPPC did not inhibit the attachment of HCMV particles to the cell surface. DPPC is a unique compound that targets the very early phase of cytomegalovirus infection, probably by disrupting a pathway that is important after viral entry but before immediate-early gene expression.

Newborn screening of congenital cytomegalovirus infection using saliva can be influenced by breast feeding

Congenital cytomegalovirus (cCMV) infection occurs in 0.2–2% of all births in developed countries and causes developmental abnormalities.1 In addition to patients symptomatic at birth, asymptomatic newborns can develop late-onset sequelae, including sensorineural hearing loss and developmental delay. As the early identification of congenitally infected newborns may allow early intervention and antiviral treatment options, it is important to establish newborn cCMV screening programmes. Since newborn screening assays using dried blood spots for cCMV infection were …