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Biomedical subjects

F Tsuda

Publications and source records attributed to F Tsuda.

At least 91 records · Page 5Linked to original sources
PubMed1994

Naturally occurring escape mutants of hepatitis B virus with various mutations in the S gene in carriers seropositive for antibody to hepatitis B surface antigen.

Hepatitis B virus (HBV) DNA was extracted from sera of six carriers with hepatitis B e antigen as well as antibody to hepatitis B surface antigen and sequenced within the pre-S regions and the S gene. HBV DNA clones from five of these carriers had point mutations in the S gene, resulting in conversion from Ile-126 or Thr-126 of the wild-type virus to Ser-126 or Asn-126 in three carriers and conversion from Gly-145 to Arg-145 in three of them; clones with Asn-126 or Arg-145 were found in one carrier. All 12 clones from the other carrier had an insertion of 24 bp encoding an additional eight amino acids between Thr-123 and Cys-124. In addition, all or at least some of the HBV DNA clones from these carriers had in-phase deletions in the 5' terminus of the pre-S2 region. These results indicate that HBV escape mutants with mutations in the S gene affecting the expression of group-specific determinants would survive in some carriers after they seroconvert to antibody against surface antigen. Carriers with HBV escape mutants may transmit HBV either by donation of blood units without detectable surface antigen or through community-acquired infection, which would hardly be prevented by current hepatitis B immuneglobulin or vaccines.

Adult↗
PubMed1994

Hypocomplementemia associated with hepatitis C viremia in sera from voluntary blood donors.

OBJECTIVES: Hepatitis C virus (HCV) infection induces extra-hepatic manifestations, most of which are considered to be mediated by circulating immune complexes. For evaluating this association in a wider perspective, complement activity was determined in sera from apparently healthy individuals, and hypocomplementemia was tested for correlation with HCV viremia. METHODS: Sera from 10,532 voluntary blood donors were stored at 4 degrees C overnight, serially diluted 2-fold, and tested for hemolytic activity by a microtitration method and antibody to HCV (anti-HCV) by passive hemagglutination with recombinant HCV antigens of the second generation. HCV RNA was determined in sera with anti-HCV or hypocomplementemia, or both, by polymerase chain reaction with nested primers deduced from the 5'-noncoding region of the HCV genome. RESULTS: Hypocomplementemia was detected in 53 (0.5%) of 10,532 donations and anti-HCV in 94 (0.9%). Anti-HCV was detected in 48 (91%) of the 53 sera with hypocomplementemia, more frequently than in 46 (0.44%) of 10,479 sera without (p < 0.001). Among 94 sera positive for anti-HCV, HCV RNA was detected in 45 (94%) of 48 sera with hypocomplementemia, more often than in 10 (22%) of 46 sera without (p < 0.001). CONCLUSIONS: A close association of hypocomplementemia with HCV viremia among apparently healthy blood donors would reflect circulating immune complexes which may cause extrahepatic diseases, such as cryoglobulinemia and membranoproliferative glomerulonephritis, in some HCV carriers. The storage of sera from HCV carriers at 4 degrees C before the test would have contributed to a decreased hemolytic activity due to the cold activation of complement by cryoglobulins involving HCV.

Adult↗
PubMed1994

Superinfection of chimpanzees carrying hepatitis C virus of genotype II/1b with that of genotype III/2a or I/1a.

Three chimpanzees persistently infected with hepatitis C virus of genotype II/1b were challenged with hepatitis C virus of genotype III/2a and 6 wk later with hepatitis C virus of genotype I/1a. They were tested for titers of total and genotype-specific hepatitis C virus RNA, as well as for serum transaminase levels, until 52 wk after the first challenge. One chimpanzee (CH489) with intermittent low hepatitis C virus RNA titers of genotype II/1b in serum was superinfected with hepatitis C virus of genotype III/2a between wk 1 and 7 after the challenge; superinfection was accompanied by fluctuating high transaminase levels. Later, the animal was superinfected with hepatitis C virus of genotype I/1a. Superinfection was accompanied by persistently high transaminase levels immediately after the challenge. Hepatitis C virus of genotype I/1a persisted, whereas hepatitis C virus of genotype II/1b was undetectable 22 wk after the challenge and thereafter. In another chimpanzee (CH353) with intermittent low hepatitis C virus RNA titers of genotype II/1b, hepatitis C virus of genotype III/2a induced fluctuating high levels of serum transaminases without revealing itself in serum. Then, HCV of genotype I/1a superinfected her, induced persistently high transaminase levels and took over HCV of genotype II/1b at 22 wk after the challenge and thereafter. The third chimpanzee (CH451) with persistently high HCV RNA titers of genotype II/1b did not reveal HCV RNA of genotype III/2a in serum after the challenge, although transaminases sharply increased. Low-titered HCV RNA of genotype I/1a was detected at 18 wk after the challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Hepatitis B virus subtypes and hepatitis C virus genotypes in patients with chronic liver disease in Nepal.

A total of 145 patients with chronic liver disease, including 20 with chronic hepatitis, 63 with cirrhosis and 62 with primary hepatocellular carcinoma from Nepal were tested for markers of hepatitis B virus or hepatitis C virus infection. HBsAg was detected in 57 (39%) and hepatitis C virus RNA in 12 (8%); the cause of liver disease was not known in the remaining 76 (52%). HBsAg was found in 5 (1.3%) of 379 normal controls, whereas hepatitis C virus-associated antibodies were detected in 13 (3.4%), none of whom was positive for serum hepatitis C virus RNA. Subtypes of 102 HBsAg samples, from patients and asymptomatic carriers, were adw in 35 (34%), adr in 4 (4%) and ayw in 48 (47%); the remaining 15 (15%) were of atypical subtypes such as ad, ay and a. Of 12 hepatitis C virus RNA samples, genotype I was detected in 1, genotype II in 5 and genotype V in 1; the remaining five samples were not to be classified by polymerase chain reaction with primers specific for genotypes I to V deduced from hepatitis C virus core sequences, despite high hepatitis C virus RNA titers in all of them. Sequences of 192 amino acids in the entire E1 region of unclassifiable hepatitis C virus isolates from five patients differed from each other in 17% to 23%, and varied from reported isolates of defined genotypes in 13% to 44%. These results indicate that atypical subtypes of hepatitis B virus and novel genotypes of hepatitis C virus would prevail in Nepal.

Adult↗
PubMed1993

High carrier rate after hepatitis B virus infection in the elderly.

An outbreak of hepatitis B virus infection occurred in a nursing facility; it involved 31 patients with sequelae of cerebral vascular accidents (15 men and 16 women; mean age, 77.4 +/- 9.3 yr). HBsAg disappeared within 6 mo in 9 patients and persisted during an observation period of more than 6 mo in 13; the remaining 9 patients were lost to follow-up while they carried HBsAg. Thus 13 of 22 patients followed (59%) became HBsAg carriers. We amplified a part of the S gene (436 nucleotides) with polymerase chain reaction on hepatitis B virus DNA from 12 randomly selected patients. The sequences of nine patients were the same as that of a nursing assistant who was an HBsAg carrier and suspected as the source of infection; it differed by only 1 or 2 (< 0.5%) nucleotides from those of the remaining three patients. Between the group of nine patients with transient HBV infection and the 13 patients with persistent HBV infection, we found no differences in age or sex or in parameters of nutrition or immunocompetence. These results indicate a high incidence of HBV carrier state in the elderly.

Adolescent↗
PubMed1993

Prevalence, genotypes, and an isolate (HC-C2) of hepatitis C virus in Chinese patients with liver disease.

China has not been extensively investigated for the prevalence of hepatitis C virus (HCV) infection among people with or without liver disease. We analyzed serum from 2,177 liver disease patients from 7 cities in different areas of China. Of 435 acute hepatitis patients, only 11% were positive for HCV RNA, while hepatitis B surface antigen (HBsAg) was detected in 33%. Of 1,668 patients with chronic liver disease, 14% and 74% were positive for HCV RNA and HBsAg, respectively. Nearly 80% of non-B chronic liver disease were negative for HCV RNA. The frequency of HCV RNA in chronic liver disease was significantly higher in Hami (32%) and Shenyang (30%) than in other cities (6-12%). The HCV genotype distribution varied by region. Genotype III was detected in 46-70% of HCV infections in Hami, Shenyang, and Lanzhou, while more than 90% of patients from southern cities (Nanjing, Nanning, and Chengdu) had genotype II. No evidence for genotype I or IV infections was found. A full-length HCV genome sequence (HC-C2) derived from a Beijing patient with genotype II was closely related to previous isolates from Japanese and Taiwanese patients. These results suggest that HCV prevalence and genotype distribution vary from region to region in China, and that the HCV now predominant in China may have evolved epidemiologically with infections in Japan and Taiwan. The study identified a high frequency of non-B, non-C chronic liver disease in China, suggesting possibly a new agent or infections with extreme variants of HCV.

Base Sequence↗
PubMed1993

A structurally flexible and antigenically variable N-terminal domain of the hepatitis C virus E2/NS1 protein: implication for an escape from antibody.

Hepatitis C virus persists in most infected hosts, and causes chronic hepatitis, liver cirrhosis, and/or hepatocellular carcinoma in humans. During the infection the RNA genome of hepatitis C virus undergoes frequent missense mutations at one or two "hypervariable" regions within a presumptive envelope gene (Okamoto et al., 1992, Virology 190, 894-899; Ogata et al., 1991, Proc. Natl. Acad. Sci. USA 88, 3392-3396). In the present study, we analyzed three cases of hepatitis C virus infection, two in chimpanzees and one in humans, for the antigenicity of peptides predicted from the hypervariable region of viral RNA obtained during the follow-up. Our results showed a successive appearance of hepatitis C virus mutants with antigenically distinct amino acid sequence within the domain; and the amino acid replacement was associated with an alteration of predicted local secondary structure of the epitope region. Hence, the hypervariable domain of the hepatitis C virus envelope appeared to be structurally flexible and antigenically variable, providing the virus a way to escape from host immunity.

Amino Acid Sequence↗
PubMed1993

Allelic subtypic determinants of hepatitis B surface antigen (i and t) that are distinct from d/y or w/r.

A monoclonal antibody (I-18) was raised against an enneapeptide representing amino acids 125 to 133 of the product of the S gene of hepatitis B virus DNA [S(125-133) segment] with a sequence of Thr-Ile-126-Pro-Ala-Gln-Gly-Thr-Ser-Met. Another monoclonal antibody (T-7) was raised against an S(125-133) segment in which Ile-126 was replaced by Thr-126. In a panel of 16 samples of hepatitis B surface antigen (HBsAg) with known S gene sequences, I-18 reacted with 5 with Ile-126. T-7 reacted with 10 HBsAg samples with Thr-126; it did not, however, react with the remaining one of subtype ayw with Thr-126 flanked by Met-125 and Thr-127. The two allelic subtypic determinants, specified by Ile-126 and Thr-126 and distinct from d/y or w/r, were named i and t after isoleucine and threonine, which regulate them. They were expressed in a mutually exclusive fashion in 216 (83%) of 260 HBsAg samples from asymptomatic carriers. They were not detected in 36 (14%) samples; the failure to detect an i or t determinant was particularly common in HBsAg samples of subtype ayw (26 [79%] of 33). A part of the S gene sequence was determined for eight HBsAg samples without a detectable i or t determinant. They had an Ile-126 or Thr-126 residue that was flanked by Thr-127, not the Pro-127 commonly possessed by HBsAg samples displaying an i or t determinant. Expression of the i/t allele, therefore, would require Pro-127. In eight (3%) of the samples, both i and t determinants were detected; the presence of i and t on the selfsame HBsAg particles was verified by sandwiching the particles between I-18 and T-7. A point mutation from thymine to cytosine at nucleotide 377 in the S gene, contributing different second letters to codon 126 (ATT for Ile and ACT for Thr), would have been responsible for the assembly of HBsAg particles with both i and t determinants by means of phenotypic mixing.

Alleles↗
PubMed1993

Genotype dependence of hepatitis C virus antibodies detectable by the first-generation enzyme-linked immunosorbent assay with C100-3 protein.

Hepatitis C virus (HCV) samples in 155 sera, from patients with chronic non-A, non-B liver disease and blood donors, were grouped into four genotypes (I, II, III, and IV) by amplification of core-gene sequences by polymerase chain reaction with type-specific primers. HCV genotypes were compared with various HCV-associated antibodies detectable by the first-generation ELISA (ELISA-1) with C100-3 protein and a second-generation immunoblot assay with four recombinant HCV proteins. Antibodies to C100-3 protein and those to its subsequence (5-1-1) were detected in 13 (93%) and 12 (86%), respectively, of 14 sera with genotype I HCV; 56 (79%) and 58 (82%) of 71 sera with genotype II; 13 (34%) and 6 (16%) of 38 sera with genotype III; and 11 (34%) and 4 (13%) of 32 sera with genotype IV. Amino acid sequences of C100-3 of genotype I HCV are conserved by approximately 90% in genotype II, but only by approximately 75% in genotypes III and IV. The sensitivity of ELISA-1, therefore, would be influenced by heterogeneity in C100-3 sequences of different genotypes.

Antigens, Viral↗
PubMed1992

Serodiagnosis of hepatitis C virus infection by ELISA for antibodies against the putative core protein (p20c) expressed in Escherichia coli.

The putative core gene of hepatitis C virus (HCV) was incorporated into a plasmid vector (pCC5-J4), and expressed in Escherichia coli. The product of 180 amino acids (p20c) was purified by gel electrophoresis in the presence of sodium dodecyl sulfate, and used in enzyme-linked immunosorbent assay for antibodies against the putative core protein of HCV (anti-p20c). Anti-p20c was detected in 13 (1.5%) of 873 apparently healthy blood donors. It was detected in 205 (86.5%) of 237 patients with acute or chronic non-A, non-B (NANB) hepatic disease, significantly more frequently (p less than 0.01) than antibodies against the C100-3 protein encoded by nonstructural regions of HCV (anti-C100-3) that was found in 178 (75.1%). Anti-p20c developed in the circulation of a patient with acute NANB hepatitis much earlier than anti-C100-3. HCV RNA was detected by polymerase chain reaction in serum samples from blood donors positive for anti-p20c in high titers, one of which was negative for anti-C100-3. These results indicated that anti-p20c would be useful in complementing anti-C100-3 for the diagnosis of NANB hepatitis and further decreasing the incidence of posttransfusion NANB hepatitis.

Adolescent↗
PubMed1992

Antibodies against synthetic oligopeptides deduced from the putative core gene for the diagnosis of hepatitis virus infection.

Immunoassays were developed to detect antibodies against oligopeptides deduced from the putative core gene of hepatitis C virus, and their performances were compared with that of the commercial immunoassay for antibodies against the product of nonstructural regions of hepatitis C virus (anti-C100-3). A 19-mer oligopeptide (CP10) and a 36-mer oligopeptide (CP9) were chemically synthesized, which represented hydrophilic regions of the product of the hepatitis C virus core gene. They were used to capture corresponding antibodies, anti-CP10 and anti-CP9, by enzyme-linked immunosorbent assay in sera from patients with acute or chronic non-A, non-B liver disease and in blood donations. At the onset of acute non-A, non-B hepatitis, anti-CP10 was detected in 15 of 20 patients (75%), and anti-CP9 was detected in 14 patients (70%). This was more frequent than anti-C100-3, which was found in only 9 patients (45%). In 186 patients with chronic non-A, non-B liver disease, anti-CP9, anti-CP10 or both were detected in 170 patients (91%). This was more frequent than anti-C100-3, which was found in 138 patients (74%). Blood with anti-CP10 as the single serological marker for hepatitis C virus infection transmitted non-A, non-B hepatitis by needlestick exposure. In sera from 558 apparently healthy blood donors, anti-CP10 was detected in 55 donors (9.9%), anti-CP9 was detected in 26 donors (4.7%) and anti-C100-3 was detected in 7 donors (1.3%).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Two distinct subtypes of hepatitis C virus defined by antibodies directed to the putative core protein.

Four distinct genotypes of hepatitis C virus types I, II, III and IV have been identified by comparison of nucleotide sequences of isolates from different areas of the world. We examined the possibility that hepatitis C virus may have serologically definable subtypes. Enzyme-linked immunosorbent assay systems were prepared by use of two synthetic peptides deduced from the putative core protein of hepatitis C virus. The following are the two peptides that were used: (a) IPKARRPEGRTWAQPGY (subtype-1) conserved in hepatitis C virus isolates with type I and type II genotypes; and (b) IPKDRRSTGKSWGKPGY (subtype-2) conserved in type III and type IV genotypes. With the enzyme-linked immunosorbent assays, the subtype-1 antibodies were detected in 26 (68%) of 38 subjects whose hepatitis C virus RNA had been genotyped as type I or type II, whereas subtype-2 antibodies were not detected. Inversely, the subtype-2 antibodies were detected in 10 (56%) of 18 subjects with hepatitis C virus RNA genotypes III or IV, whereas subtype-1 antibodies were detected in none of them. These results suggest that hepatitis C virus has two serologically distinguishable core antigen subtypes, corresponding to either genotype I/II or genotype III/IV. Subtyping of HCV by serological methods would contribute to tracking transmission routes of the virus, especially in cases where serum samples were not stored under conditions to preserve RNA or in infected hosts who have cleared the virus and therefore have only antibodies remaining to identify the infection.

Amino Acid Sequence↗
PubMed1992

Hepatitis B surface antigen particles of subtypes adw and adr, and compound subtype (adwr) in symptom-free carriers in Japan.

Of sera from 1,878 Japanese blood donors who carried hepatitis B surface antigen (HBsAg), 420 were subtyped as adw (22.4%) and 1,443 as adr (76.8%); only 15 (0.8%) contained HBsAg of subtype ayw or ayr. Sera with HBsAg/adr had higher HBsAg titres than those with HBsAg/adw (geometric mean of haemagglutination titre: 10.1 +/- 2.4 vs. 9.7 +/- 2.4, p less than 0.01), and a higher prevalence of hepatitis B e antigen (24% vs. 13%, p less than 0.001). Carriers of HBsAg/adr progressively predominated over those of HBsAg/adw with increasing age. Of sera from 1,863 carriers of HBsAg/adw or HBsAg/adr, 182 (9.8%) contained HBsAg particles with both subtypic determinants in the w/r allele. The presence of w and r determinants on the same particles was ascertained by sandwiching them between monoclonal antibody with the specificity for w and that with the specificity for r. HBsAg particles of compound subtype (adwr) were found more often in sera with hepatitis B e antigen than those without it (145/403 [36.0%] vs. 37/1,460 [2.5%], p less than 0.001). Sera with HBsAg/adwr particles had HBsAg titres higher than those without them (12.4 +/- 1.9 vs. 9.7 +/- 2.3, p less than 0.001). HBsAg/adwr particles arise from phenotypic mixing of the S-gene product of wild-type virus and that of mutants with point mutations for subtypic changes. The results obtained indicated that HBV strains of subtype adr have a higher replicative activity than those of adw, and suggested that mutations in the S gene for subtypic changes would be associated with an active replication of hepatitis B virus.

Adult↗
PubMed1992

Full-length sequence of a hepatitis C virus genome having poor homology to reported isolates: comparative study of four distinct genotypes.

Variable genomic sequences have been reported for RNA cloned from hepatitis C virus (HCV)-infected humans and chimpanzees. We found that four distinct genotypes of HCV could be differentially identified by PCR using type-specific primers. Full-length sequences have so far been reported for three of the four HCV genotypes, and we report herewith the sequence of the fourth type obtained from a Japanese blood donor. The entire nucleotide sequence of the HCV isolate (HC-J8) comprised 9481 bases plus a 3'-terminal poly(U) stretch of variable length. Like all previous isolates, the RNA contained a single, long open reading frame for a polyprotein of 3033 amino acids. HC-J8 differed from previously reported HCV isolates by 23.1-33.1% in nucleotide sequence and 15.9-28.8% in amino acid sequence. Based on genomic sequence homologies, a proposed phylogenetic tree of HCV, with a fourth branch represented by HC-J8, allowed a classification of all HCV isolates whose complete or partial sequences are now known. This classification suggests that all or most HCV genome sequences will fall into one of the proposed four types. The classification may be helpful in designing vaccine studies and for serological investigations of possible group- and type-specific antibodies.

Amino Acid Sequence↗
PubMed1992

Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources.

Based on variation in nucleotide sequence within restricted regions in the putative C (core) gene of hepatitis C virus (HCV), four groups of HCV have been postulated in a panel of 44 HCV isolates. They were provisionally designated types I, II, III and IV. A method for typing HCV was developed, depending on the amplification of a C gene sequence by polymerase chain reaction using a universal primer (sense) and a mixture of four type-specific primers (antisense). HCV types were determined by the size of the products specific to each of them. Type II was found in HCV samples from 131 (82%) of 159 blood donors, more often than in those from 48 (60%) of 80 patients with non-A, non-B (NANB) liver disease in Japan (P less than 0.01). In 11 haemophiliacs who had received imported coagulation factor concentrates, type I was found in five, as against type II in four. Double infection with two different HCV types was found in two patients with chronic NANB liver disease (types I and II; II and III) and two haemophiliacs (types I and II; I and III). HCV types were identical in mother and baby in each of two examples of perinatal transmission, and were also identical in donor and recipient in a case of accidental needle exposure.

Base Sequence↗
PubMed1992

Correlation between anti-HBc titers and HBV DNA in blood units without detectable HBsAg.

Hepatitis B virus (HBV) DNA was tested for in 294 blood units which had antibody against hepatitis B core antigen (anti-HBc) as the isolated serological marker of HBV infection. After amplification by polymerase chain reaction, HBV DNA was detected in 12 (6.9%) of 175 units that were positive for anti-HBc with hemagglutination inhibition titers greater than or equal to 2(6), significantly more often than in none of 119 units with titers less than or equal to 2(5) (p less than 0.01). These results indicate that the exclusion of blood units with isolated high-titer anti-HBc would be effective for further decreasing the risk of posttransfusion hepatitis B.

Base Sequence↗
PubMed1992

Mutations within the S gene of hepatitis B virus transmitted from mothers to babies immunized with hepatitis B immune globulin and vaccine.

A variant of hepatitis B virus (HBV) having a specific mutation within the S gene has been found to infect vaccinees. To know whether similar variants were involved in Japan, we analyzed two cases of maternal transmission of HBV in infants immunized with hepatitis B immune globulin and hepatitis B vaccine. DNA clones of HBV S genes were propagated from patients and family members and sequenced. In one family, the DNA clones from the baby patient had a Gly-to-Arg mutation at the 145th codon of the S gene, whereas those from her mother had no such mutations. In the other family, all the DNA clones obtained from the two infected children had the 145th codon intact, but they had a missense mutation at the 126th codon of the S gene, causing an amino acid substitution of Asn for Thr or Ile. This same mutation was observed in 12 of 17 clones of DNA obtained from their mother. In comparison with the wild type HBV-derived hepatitis B surface antigen, the two types of S gene mutations, either at the 145th or the 126th codon, were associated with a significant decrease in the antigenicity of some determinants on the hepatitis B surface antigen, measured by MAb. Amino acid substitution at these sites, therefore, would have induced the escape from conventional vaccines that were S gene products of wild type HBV and also from hepatitis B immune globulin, whose main components were probably also antibodies against the S gene products expressed by wild type HBV.

Adult↗