Hiroshi Ikegaya

Age-related urinary excretion of BK polyomavirus by nonimmunocompromised individuals.

ABSTRACT Two polyomaviruses, BK virus (BKV) and JC virus (JCV), are ubiquitous in the human population, generally infecting children asymptomatically and then persisting in renal tissue. It is generally thought that reactivation leads to productive infection for both viruses, with progeny shed in the urine. Several studies have shown that the rate of JC viruria increases with the age of the host, but a systematic approach to examine the shedding of BKV has not been developed. To elucidate the relationship between BK viruria and host age, we obtained urine from donors (healthy volunteers or nonimmunocompromised patients) who were divided into nine age groups, each containing 50 members. A high-sensitivity PCR was used to detect BKV and JCV DNA from urinary samples, and the specificity of amplification was confirmed by sequencing or restriction analysis of the amplified fragments. The rate of BK viruria was relatively low in subjects aged <30 years but gradually increased with age in subjects aged ≥30 years. However, BK viruria was less frequent than JC viruria in adults. The detected BKV isolates were classified into subtypes, and detection rates for individual subtypes were compared among age groups; this analysis showed that viruria of subtypes I (the most prevalent subtype) and IV (the second most prevalent subtype) occurred more frequently in older subjects. Therefore, our results reveal new aspects of BK viruria in nonimmunocompromised individuals.

Distribution patterns of BK polyomavirus (BKV) subtypes and subgroups in American, European and Asian populations suggest co-migration of BKV and the human race.

BK polyomavirus (BKV) is ubiquitous in the human population, infecting children asymptomatically and then persisting in the kidney. Based on serological and genotyping methods, BKV isolates worldwide are classified into four subtypes (I-IV), with subtype I prevalent throughout the world, subtype IV prevalent in Asia and part of Europe, and subtypes II and III rare throughout the world. Phylogenetic analyses of complete genome sequences have identified several geographically distinct subgroups of subtypes I and IV. To explain how the geographical distribution patterns of BKV subtypes and subgroups were formed, this study hypothesized that BKV co-migrated with human populations (the co-migration hypothesis), and examined this hypothesis by comparing the BKV subtype and subgroup profiles among two American populations in North-east USA and southern California, two European populations in Finland and Ireland/England, and two Asian populations in Japan and China (both American populations were composed mainly of European Americans). The frequency of subtype I was always the highest throughout the populations, but that of subtype IV was variable among populations. A subgroup of subtype I (I/b-2) was detected primarily in all of the European and American populations, whereas subgroup I/c was predominant in the Asian populations (the observed difference was statistically significant). Additionally, all of the five fully sequenced subtype IV isolates from the American and European populations belonged to subgroup IV/c-2, whereas all subtype IV isolates from the Asian populations belonged to the other subgroups. Collectively, the current findings provide support for the co-migration hypothesis.

Evaluation of mRNA-based approach for identification of saliva and semen

Multiplex mRNA profiling by a reverse transcription-polymerase chain reaction (RT-PCR) has been reported in the last few years as a new approach for the identification of body fluids. We have also demonstrated the feasibility of identifying body fluids by using a real-time RT-PCR assay. Statherin (STATH) and histatin (HTN3), the selected genes for saliva, and protamin 2 (PRM2) and semenogelin 1 (SEMG1), those selected for semen, showed high specificity to these body fluids. Thus, the sensitivity and specificity of target genes were examined in body fluid stains. All target genes were detected in 0.1 microL 6-day-old stains, and showed high specificity in 7-day-old 30 microL stains. Furthermore, the stability of HTN3 in saliva stains was examined under various environmental conditions over time. The results showed that the degradation of mRNA in the stains was highly affected by wet conditions, and that light was also an important factor. However, mRNA was detectable in an older saliva stain (6 years old) and in an older semen stain (3.5 years old), both of which had been kept under dry and dark conditions. The stability of mRNA beyond our supposition may play an important role in developing new techniques for body fluid identification.

Identification of Human Cytochrome P450 Isozymes Involved in Diphenhydramine N-Demethylation

Diphenhydramine is widely used as an over-the-counter antihistamine. However, the specific human cytochrome P450 (P450) isozymes that mediate the metabolism of diphenhydramine in the range of clinically relevant concentrations (0.14–0.77 μM) remain unclear. Therefore, P450 isozymes involved in N-demethylation, a main metabolic pathway of diphenhydramine, were identified by a liquid chromatography-mass spectrometry method developed in our laboratory. Among 14 recombinant P450 isozymes, CYP2D6 showed the highest activity of diphenhydramine N-demethylation (0.69 pmol/min/pmol P450) at 0.5 μM. CYP2D6 catalyzed diphenhydramine N-demethylation as a high-affinity P450 isozyme, the Km value of which was 1.12 ± 0.21 μM. In addition, CYP1A2, CYP2C9, and CYP2C19 were identified as low-affinity components. In human liver microsomes, involvement of CYP2D6, CYP1A2, CYP2C9, and CYP2C19 in diphenhydramine N-demethylation was confirmed by using P450 isozyme-specific inhibitors. In addition, contributions of these P450 isozymes estimated by the relative activity factor were in good agreement with the results of inhibition studies. Although an inhibitory effect of diphenhydramine on the metabolic activity of CYP2D6 has been reported previously, the results of the present study suggest that it is not only a potent inhibitor but also a high-affinity substrate of CYP2D6. Therefore, it is worth mentioning that the sedative effect of diphenhydramine might be caused by coadministration of CYP2D6 substrate(s)/inhibitor(s). In addition, large differences in the metabolic activities of CYP2D6 and those of CYP1A2, CYP2C9, and CYP2C19 could cause the individual differences in anti-allergic efficacy and the sedative effect of diphenhydramine.

An Asian Origin for Subtype IV BK Virus Based on Phylogenetic Analysis

Similarly to other members of the Polyomaviridae family, BK virus (BKV) is thought to have co-evolved with its human host. BKV has four subtypes that are distinguishable by immunological reactivity, with two (subtypes I and IV) being most prevalent in human populations. Subtype I is the major subtype worldwide, whereas subtype IV is prevalent in East Asia and Europe but rare in Africa. The geographic distribution pattern of subtype IV BKV is in apparent disagreement with the hypothesis that BKV co-evolved with humans, since subtype IV rarely occurs in Africa. To elucidate the origin of subtype IV, 53 complete subtype IV sequences derived from East Asians and Europeans were subjected to a detailed phylogenetic analysis using the maximum-likelihood and neighbor-joining methods. We identified six subgroups (a1, a2, b1, b2, c1, and c2) that formed a tree represented by the formula: “(a1, a2), ((b1, b2), (c1, c2)).” Interestingly, we found a close correlation between subtype IV subgroups and population geography; thus, all subgroups except c2 were prevalent in particular East Asian populations, with c2 occurring in both Europe and Northeast Asia. From these findings, we conclude that subtype IV of BKV now prevalent in modern humans is derived from a virus that infected ancestral Asians. We introduce two hypotheses to explain how ancestral Asians became infected with subtype IV BKV.

JC virus strains indigenous to northeastern Siberians and Canadian Inuits are unique but evolutionally related to those distributed throughout Europe and Mediterranean areas

Human polyomavirus JC virus (JCV) isolates around the world are classified into more than 10 geographically distinct genotypes (designated as subtypes). Evolutionary relationships among JCV subtypes were recently examined, and the following pattern of JCV evolution was indicated. The ancestral JCV first divided into three superclusters, designated Types A, B, and C. A split in Type A generated two subtypes, EU-a and -b, containing mainly European and Mediterranean isolates. The split in Type B generated Af 2 (the major African subtype), Bl-c (a minor European subtype), and various Asian subtypes. Type C generated a single subtype (Afl), consisting of isolates derived from western Africa. In this study, JCV isolates prevalent among northeastern Siberians and Canadian Inuits were evaluated in the context of the above-described pattern of JCV evolution. The Siberian/Arctic JCV isolates were classified as belonging mainly to Type A, based on the result of a preliminary phylogenetic analysis. We then examined, using the whole-genome approach, the phylogenetic relationships among worldwide Type A isolates. In neighbor-joining and maximum-likelihood analyses, Type A JCVs worldwide consistently diverged into three subtypes, EU-a, -b, and -c, with high bootstrap probabilities. EU-c was constructed only by northeastern Siberian isolates, derived mainly from Nanais living in the lower Amur River region, and was shown to have been generated by the first split in Type A. Most Siberian/Arctic isolates derived from Chukchis, Koryaks, and Canadian Inuits formed a distinct cluster within the EU-a subtype, with a high bootstrap probability. Based on the present findings, we discuss ancient human migrations, accompanied by Type A JCVs, across Asia and to Arctic areas of North America.

Subtype IV of the BK polyomavirus is prevalent in East Asia

Summary.BK polyomavirus (BKV) is ubiquitous in human populations, infecting children asymptomatically and then persisting in the kidney. Using either serological or genotyping methods, BKV isolates have been classified into four subtypes (I–IV), with subtype I mainly detected in all countries studied so far. To elucidate the subtype of BKV prevalent in East Asia, we examined BKV-positive urine samples collected from immunocompetent elderly patients in Mongolia, Northeast China, Northwest China, Southeast China, Southwest China, Vietnam and Japan. The 287-bp typing region of the viral genome in each of these samples was PCR-amplified and sequenced, and a phylogenetic tree was constructed. According to the tree, BKV isolates in East Asia were unambiguously classified into subtype I or IV (subtypes II and III were not detected). In Japan, subtype I was mainly detected and subtype IV was rare, whereas in the other regions subtype IV was detected frequently, at rates ranging from 24 to 100%. Thus, East Asia (excluding Japan) is a region in which subtype-IV BKV is prevalent, a finding that requires the view of the geographic distribution of BKV subtypes to be revised. Furthermore, we present evidence that the immunological states of urine donors do not affect the pattern of BKV subtypes.

Diagnosis of cyanide intoxication by measurement of cytochrome c oxidase activity

Cytochrome c oxidase (CCO), a mitochondrial enzyme, is inactivated by cyanide or carbon monoxide (CO) intoxication. We measured CCO activity, in the major organs of the rat at various times after death caused by cyanide intoxication. Tissue samples were homogenized, and the CCO activity in the mitochondrial fraction was measured using ferrous cytochrome c as the substrate. The CCO activity inhibition was highest in the brain, although the cyanide concentration was lowest level. As a result of this and the clinical symptoms displayed, we consider the brain to be the primary organ of cyanide intoxication. As cyanide is highly toxic to humans, in small amounts and many patients and victims have already had some medical care, it is difficult to detect cyanide in criminal investigations. The CCO activities in various organs remained significantly low for 2 days after the cyanide intoxication, suggesting that the diagnosis may be possible by measuring not only the cyanide concentration but also the CCO activity.

Detection of varicella-zoster virus DNA in 414 human trigeminal ganglia from cadavers by the polymerase chain reaction: a comparison of the detection rate of varicella-zoster virus and herpes simplex virus type 1.

Investigation of varicella‐zoster virus (VZV) is important epidemiologically, and determination of its prevalence rate in human trigeminal ganglia is important to provide surveillance data. To date, studies on VZV detection in trigeminal ganglia have used specimens obtained from a relatively limited number of cadavers. This study attempted to detect VZV DNA as well as Herpes simplex virus type 1 (HSV‐1) DNA by the polymerase chain reaction (PCR) from 414 samples of trigeminal ganglia obtained from 207 cadavers selected at random. The detection rate was examined to determine whether there were significant differences in the positive rate between the left and right trigeminal ganglia, males and females, and among age groups. A relationship was found between the positive rates for VZV and HSV‐1. VZV DNA was detected in 391 of the trigeminal ganglia (94.4%) and 201 of the cadavers (97.1%) in 121/124 males and 80/83 females. HSV‐1 DNA was detected in 251 of the samples (60.6%) and 134 of the cadavers (64.7%) in 72/124 males and 62/83 females. There was no significant difference for either virus in the detection rates between the left and right trigeminal ganglia and males and females. Age and positivity for HSV‐1, but not VZV, showed a significant relationship. All 134 cadavers positive for HSV‐1 were also positive for VZV. VZV and HSV‐1 become latent in bilateral trigeminal ganglia, and are not affected by gender. The prevalence of HSV‐1 was greater in advanced age, and the HSV‐1‐positive rate was correlated with the VZV‐positive rate. J. Med. Virol. 82:345–349, 2010.

Evaluation of Tamm-Horsfall Protein and Uroplakin III for Forensic Identification of Urine

Abstract:  In this study, Tamm‐Horsfall protein (THP), a major component of urinary protein, and uroplakin III (UPIII), a transmembrane protein widely regarded as a urothelium‐specific marker, were evaluated for forensic identification of urine by ELISA and/or immunohistochemistry. THP was detected in urine, but not in plasma, saliva, semen, vaginal fluid, or sweat by the simple ELISA method developed in this study. In addition, most aged urine stains showed positive results. The urine specificity of THP was confirmed by gene expression analysis. Therefore, as reported previously, ELISA detection of THP can be used as a presumptive test for urine identification. UPIII was specific for immunohistochemical staining of cells in centrifuged precipitate of urine. However, ELISA and RT‐PCR for UPIII were not specific for urine. UPIII may be applicable for forensic urine identification by immunohistochemistry.