Osamu Hashimoto

Molecular Diagnosis of Human Enteroviruses by Phylogeny-Based Classification by Use of the VP4 Sequence

Human enteroviruses (EVs) are the major cause of a variety of acute and chronic illnesses. Virus isolation and neutralization tests are usually done to identify the causative virus, but these tests are labor intensive, time consuming, and sometimes require suckling mice from which certain viruses have been isolated. This study investigated a rapid and reliable method based on reverse-transcription polymerase chain reaction and phylogenetic analysis. The phylogenetic tree constructed by neighbor-joining on the basis of the VP4 sequence from 66 prototypes grouped all human EVs into 5 distinct clusters. These clusters correspond closely to the 5 newly designated species-human EV A-D and poliovirus. The VP4 sequences of 89 isolates from 26 serotypes obtained over >30 years plus those of 66 prototype strains were analyzed. Each isolate formed a monophyletic cluster along with its respective prototype strain, allowing for serotype identification (with the exception of E-8). VP4-based classification appears to be an effective tool for the molecular epidemiology study of EVs.

Production and release of infectious hepatitis C virus from human liver cell cultures in the three-dimensional radial-flow bioreactor.

Lack of efficient culture systems for hepatitis C virus (HCV) has been a major obstacle in HCV research. Human liver cells grown in a three-dimensional radial-flow bioreactor were successfully infected following inoculation with plasma from an HCV carrier. Subsequent detection of increased HCV RNA suggested viral replication. Furthermore, transfection of HCV RNA transcribed from full-length cDNA also resulted in the production and release of HCV virions into supernatant. Infectivity was shown by successful secondary passage to a new culture. Introduction of mutations in RNA helicase and polymerase regions of HCV cDNA abolished virus replication, indicating that reverse genetics of this system is possible. The ability to replicate and detect the extracellular release of HCV might provide clues with regard to the persistent nature of HCV infection. It will also accelerate research into the pathogenicity of HCV, as well as the development of prophylactic agents and new therapy.

Japanese external quality assessment program to standardize HIV-1 drug-resistance testing (JEQS2010 program) using in vitro transcribed RNA as reference material.

To design appropriate antiretroviral therapy regimens and avoid the emergence of human immunodeficiency virus (HIV)-1 variants with reduced susceptibility to antiretroviral drugs, genotypic drug-resistance testing (HIV genotyping) is strongly recommended. To monitor the quality of HIV genotyping in Japan, we performed an external quality assessment (EQA), named the Japanese external quality assessment program, to standardize HIV genotyping (JEQS). To accurately evaluate the quality of HIV genotyping, we employed as reference material (RM) a well-characterized sample, in vitro transcribed RNA (trRNA) that includes the HIV gag-pol sequence, and created a JEQS2010 panel consisting of three single variant and three mixed trRNA samples. All 11 participating laboratories showed high concordance rates (>96%) for the single variant samples. Eight laboratories also showed good rates of detecting minor variants, but three laboratories failed to detect the variants comprising one-half of the sample. These three laboratories used a common primer that had four internal mismatches to the minor trRNA clone. This program showed the usefulness of trRNA as RM, the high quality of HIV genotyping, and extensive interlaboratory variation in the ability to detect minor variants. These results suggest that improving the quality of HIV genotyping in Japan requires regularly implementing the EQA program and improving the HIV genotyping protocol in each laboratory.

The incidence of viremia and the heterogeneity of hepatitis C Virus genotypes among blood donors, hemophiliacs and patients with chronic liver disease

&NA; Hepatitis C Virus (HCV) is the major cause of parentally transmitted non‐A, non‐B hepatitis. We studied the incidence of HCV Viremia in blood donors, hemophiliacs and patients with chronic liver disease who are positive for antibodies to HCV, and then correlated the HCV genotypes among the three groups. 23 blood donors, 10 hemophiliacs and 97 patients with chronic liver disease were found to be positive for anti‐HCV during this study period from June 1993 to December 1993. Only 3 (13%) blood donors, 6 (60%) hemophiliacs and 71 (73%) patients with chronic liver disease were found to be viremic when tested for HCV RNA by reverse transcriptase‐polymerase chain reaction (RT‐PCR). The low incidence of viremia among blood donors may be due to any one of the following three reasons. 1, the level of viremia was below the level of detection. 2, the viremia was intermittent with persistent infection. 3, the majority of cases represented resolved infection. The HCV genotypes were heterogeneous among the three groups. All the blood donors with viremia and 35 (50%) of patients with chronic liver disease, belonged to type II (1b). However only one (17%) of the hemophiliacs belonged to type II (1b). Studies have shown that the genotype I (1a) is the predominant type in the USA and Europe, whereas type II (1b) is more frequent in the Far East. It is also suggested that type II (1b) is associated with non‐responsiveness to interferon therapy. Our hemophiliacs were treated with imported coagulation factors, thus they were probably exposed to the genotypes in the west. There was significant difference in the incidence of HCV type II (1b) among local blood donors and hemophiliacs (P = 0.005). However the difference between the hemophiliacs and the patients with chronic liver disease was not statistically significant. The number of patients in this study was too small to draw any firm conclusions. However the findings highlight the importance of studying the genotypes of patients with Hepatitis C infection due to their relevance in the management of these cases with interferon therapy.

Effects of benzodiazepine on neurotransmitter receptors in the brain of rats with acute ischemic hepatic failure: With special relations to the kinetics of .GAMMA.-aminobutyric acid binding.

肝不全時の脳内神経伝達物質レセプターに対するベンゾジアゼビン(BDZ)投与の影響をGABA結合能を指標として検討した.対照ラットより調製した脳シナプス膜では,BDZ10-12MでBDZ非添加時に比し14%と最大のGABA結合能の増強を示し,より高濃度ではむしろ増強効果の減弱を認めた.一方,肝不全ラットでは,10-12Mで7%, 10-11Mで32%とGABA結合能は濃度依存性に増強効果を示し,10-10～10-8Mで34%と平衡に達した.この結合能の増強はkinetic studyの結果,対照ラットでは低親和性結合部位のaffinityの増強,肝不全ラットでは同部位のaffinityのより強い増強とdensityの増加の両者によることが判明した.以上より肝不全時には,BDZ投与によりGABA結合能が対照群に比し,より増強すること,すなわち脳内神経伝達物質レセプターレベルでの異常が存在する可能性が強く示唆された.

Enhanced transport of neutral amino acids by brain microvessels isolated from rats with hepatic filure, with special relation to hyperammonemia.

肝不全時の脳内芳香族アミノ酸(AAA)増加の機序に関し,門脈-下大静脈吻合(PCA),急性虚血性肝不全(AIHF)およびアンモニア負荷ラット(NH3-R)ではトリプトファン(Trp)のbrain uptake indexが有意に増加していることをすでに報告した.今回は,単離脳微小血管(BMV)を用い,Trp,フェニルアラニン,イソロイシン(Ile)の取り込みを検討した.その結果,PCA, AIHFでは中性アミノ酸(NAA)取り込みの亢進が認められ,Trpにおけるkineticsの検討から,saturable influxのVmaxの変化が重要であることが判明した.また,NH3-RでもTrp, Ileの取り込みが増加し,さらに正常ラットBMVでも高濃度グルタミン(Gln)を予め添加した場合,Trp取り込みが増加した.以上より,肝不全時には高NH3血症によって増加した脳内GlnとNAAとのexchange transportが亢進し,すなわちNAAの脳内進入が亢進することによって脳内NAAの増加がもたらされる可能性が強く示唆された.