Hisao Iizuka

Molecular cloning and characterization of a novel DNA virus (TTV) associated with posttransfusion hepatitis of unknown etiology

Abstract The genomic DNA of a novel virus named TT virus (TTV), associated with posttransfusion hepatitis of unknown etiology, was cloned from plasma of a blood donor with an elevated transaminase level but without serological markers of known hepatitis viruses, and its sequence of 3739 bases was determined. TTV had a density of 1.26 g/cm 3 in sucrose, which did not change after the treatment with Tween 80. The viral genome was sensitive to DNase I and Mung Bean Nuclease. Hence, TTV would be an unenveloped, single-stranded DNA virus. Two possible open reading frames in different frames were identified, capable of encoding 770 and 202 amino acids, respectively. When a partial sequence of 356 bases was compared among TTV isolates from 78 sera from blood donors and hepatitis patients, it showed considerable divergence with differences of up to 30%. Oligonucleotide primers were designed on two well-conserved regions for the detection of TTV DNA in serum and biopsied liver tissues by polymerase chain reaction. TTV DNA was detected in sera from 9 of 19 (47%) patients with fulminant hepatitis and 41 of 90 (46%) patients with chronic liver disease of unknown etiology. TTV DNA was detected in liver tissues of all the five patients tested, in titers equal or 10–100 times higher than those in the corresponding sera. These results indicate that TTV would be responsible for a part of acute and chronic liver disease of unknown etiology.

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 ≧26, significantly more often than in none of 119 units with titers ≦25 (p<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 post‐transfusion hepatitis B.

Hepatitis C virus variants from Jakarta, Indonesia classifiable into novel genotypes in the second (2e and 2f), tenth (10a) and eleventh (11a) genetic groups.

Hepatitis C virus (HCV) isolates from 126 hepatitis patients in Jakarta, Indonesia were genotyped by PCR with genotype-specific primers deduced from the HCV core gene. Fifty-five isolates (44%) were classified as genotype II/1b, 15 (12%) as 1c, 33 (26%) as III/2a, and 1 (1%) as V/3a, while the remaining 22 (17%) were not classifiable into any of the five common genotypes (I/1a, II/1b, III/2a, IV/2b and V/3a) or 1c. Sequences of a part of the NS5b region [1093 bp (nucleotides 8279-9371)] of the 22 isolates of unclassifiable genotype were subjected to pair-wise comparison and phylogenetic analysis along with those of 62 isolates of 25 genotypes in nine genetic groups. Seven of the isolates were classified into 2e and two into 2f, representing novel genotypes in genetic group 2, while ten and three were classified into two new genetic groups, 10 and 11, respectively, and their genotypes were provisionally designated 10a and 11a. The isolates of genotype 10a (JK049) and 11a (JK046) were sequenced in full. Comparison of 24 HCV genomes including those of JK049 and JK046, over the entire genome and subgenomic regions, supported the classification of HCV into 11 genetic groups.

Nucleotide sequence of a cloned hepatitis B virus genome, subtype ayr: comparison with genomes of the other three subtypes.

The entire nucleotide sequence of genomic DNA was determined for hepatitis B virus (HBV) of subtype ayr, which had been derived from the blood of a Japanese asymptomatic carrier. The genome was 3215 nucleotides long, and differed in DNA sequence by 10% from that of subtypes adw or ayw, but by only 2% from that of subtype adr. Amino acid sequences coded for by the S, C, P and X genes, as well as by the pre-S region, closely resembled those of subtype adr, indicating that the evolution of HBV/ayr from HBV/adr was more recent than the differentiation of the other three subtypes. In the product of the S gene, the mutually exclusive subtypic determinants of the surface antigen, d and y, were associated with variation of amino acid residues at only the 68th and 122nd positions from the N terminus, in contrast to the variation at as many as seven positions for the other set of subtypic determinants, w and r. Sequences representing high local hydrophilicity in the product of the S gene were involved in subtypic variation, although such sequences in the pre-S region were shared by HBV genomes of the various subtypes. In particular, a hydrophilic sequence of 19 amino acid residues, coded for by the pre-S(2) region and implicated in the presumed hepatotropism of HBV, was possessed in common by HBV/adr, HBV/ayr and HBV/ayw, and differed in HBV/adw by only one residue at the 9th position. This amino acid sequence appears to be a promising candidate for a synthetic peptide vaccine.

THE ENTIRE NUCLEOTIDE SEQUENCES OF THREE HEPATITIS C VIRUS ISOLATES IN GENETIC GROUPS 7-9 AND COMPARISON WITH THOSE IN THE OTHER EIGHT GENETIC GROUPS

We have proposed that hepatitis C virus should be classified into eleven genetic groups (types) which further divide into more than 80 genotypes (subtypes). However, only eight genetic groups (1-6, 10 and 11) have been defined on the basis of the full-length sequence. Hence, the entire nucleotide sequences of three HCV isolates in genetic groups 7-9 have now been determined. Phylogenetic analysis over the full-length sequences of these three isolates, along with 30 more in the other eight genetic groups, indicated that genetic groups 6-9 and 11 have bifurcated from a common branch and groups 3 and 10 from another. In the former branch groups 7 and 11, and groups 8 and 9, are closely related. Consequently, HCV can be classified into either eleven (1-11) or six groups (1; 2; 3 and 10; 4; 5; 6-9 and 11), allowing a clear separation of group and genotype similarity within the NS5b region or a subregion of 1093 nt. When pairwise comparison of 1093 nt in the NS5b sequence was performed on 106 HCV isolates of 36 genotypes in eleven genetic groups, they were classified into either eleven (1-11) or six (1; 2; 3 and 10; 4; 5; 6-9 and 11) genetic groups. However, group and genotype similarities were not clearly separable in either classification. The overlapping range was smaller using the classification into eleven genetic groups as compared to six genetic groups (2.7 vs 4-7%). These results indicate that HCV might not have evolved in the two-tiered fashion, at least in a strict sense.

Distinct genotypes of a nonenveloped DNA virus associated with posttransfusion non-A to G hepatitis (TT virus) in plasma and peripheral blood mononuclear cells.

TT virus (TTV) is a nonenveloped, single‐stranded DNA virus with little sequence homology to known viruses, and associated with elevated transaminase levels in the patients with posttransfusion hepatitis of unknown etiology. The DNA of TTV was detected, by semi‐nested polymerase chain reaction, in peripheral blood mononuclear cells (PBMC) from the 30 healthy individuals with circulating virus in plasma. A sequence of 222 bases was determined on 6–10 TTV DNA clones each from plasma and 6 clones each from PBMC from eight individuals selected at random from this group. TTV can be classified into genotypes separated by an evolutionary distance > 0.30, which can be divided further into subtypes separated by that of 0.15. Three individuals possessed two different TTV variants of distinct genotypes, with predominant genotypes different between plasma and PBMC. Another possessed TTV of the same genotype in both the plasma and PBMC, but clones with a subtype not seen in plasma were observed in PBMC. A third individual had TTV variants with or without a deletion mutation, and those with the deletion mutation abounded only in PBMC. The remaining three individuals were infected with TTV with the same sequence both in plasma and PBMC. These results indicate that TTV variants with phylogenetic differences could infect the same individual, and that some variants would have a predilection for PBMC. It remains to be seen, however, if TTV replicates in PBMC or whether it has been sequestered before its evolution in the host. J. Med. Virol. 57:252–258, 1999.

Posttransfusion Fulminant Hepatitis B Associated with Precore‐Defective HBV Mutants

Abstract. Fulminant hepatitis B developed in 8 recipients of blood units without detectable hepatitis B surface antigen on routine screening. All 124 hepatitis B virus (HBV) DNA clones propagated from their sera possessed defects in the precore region. A point mutation from guanine to adenine at nucleotide 83, converting codon 28 for tryptophan (TGG) to a stop codon (TAG), was the commonest, and it was found in all 113 clones from 7 cases. The remaining case displayed 1 clone with this point mutation and 10 clones with an insertion of 2 base pairs after nucleotide 26. Antibody to hepatitis B core antigen (anti‐HBc) was detected in a high titer in 1 of 10 pilot plasma samples of blood units transfused to this case. HBV DNA clones propagated from it exhibited the same precore‐region defects as those from the recipient. On the basis of these results HBV mutants, defective in the precore region, would appear to be responsible for posttransfusion fulminant hepatitis B, and the exclusion of blood units with high‐titered anti‐HBc would be efficacious in preventing it.

Predictive value of screening tests for persistent hepatitis C virus infection evidenced by viraemia : Japanese experience

In November 1989, Japanese Red Cross Blood Centres started screening for heaptitis C virus (HCV) with enzyme‐linked immunosorbent assay (Elisa) for the C100‐3 viral peptide as the first such nationwide programme in the world. Thereafter post‐transfusion non‐A non‐B hepatitis (PTNANBH) was reduced by 61–80%, but this was not as complete a success as our programme to prevent post‐transfusion hepatitis B by screening for high titer hepatitis B core antibody, which we began in the same period. In order to acquire more effective control of PTNANBH, the HCV core‐related antigen (GOR, N14) and second‐generation Elisa (Ortho2, Abbott2)and second‐generation antigen agglutination (PA, PHA) tests have been employed. Among 16,500 donors in 11 blood centers, 365 were serologically positive by at least one of these tests. Among these, HCV RNA was detected in 138 units and the remaining 227 were HCV RNA negatives. The effectiveness of these serological tests to detect HCV RNA‐positive status were analyzed. Passive haemagglutination and particle agglutination (PHA and PA) tests were highly effective to predict HCV viraemia among blood donors. Also, these tests can easily determine antibody titre. By either PHA or PA, all units with ≧212 agglutination titre (120 and 122 units) were HCV RNA positive and all agglutination‐positive units with serum alanine aminotransferase level higher than 35 Karmen units were HCV RNA positive. These results have formed the basis for implementing a more effective screening for HCV viraemia in blood donors, where effectiveness is defined as enhancing the protection of patients from post‐transfusion hepatitis C and providing higher quality information to achieve more effective donor counselling.

The entire nucleotide sequences of two distinct TT virus (TTV) isolates (TJN01 and TJN02) remotely related to the original TTV isolates

Summary. TT virus (TTV) has a wide range of sequence divergence by which it is classified into at least 16 genotypes. A TTV isolate of genotype 12 (TJN01) and another of genotype 13 (TJN02) were sequenced in the entire genome, and compared with the reported TTV isolates. TJN01 and TJN02 had genomic lengths of 3787 and 3794 nucleotides (nt), respectively, which were shorter by 66 and 59 nt than the prototype TTV isolate of genotype 1 (TA278). TJN01 and TJN02 shared the nucleotide sequence with TA278 merely in 53.9% and 55.2%, respectively. They possessed two major open reading frames (ORFs) and the noncoding region with a GC-rich region forming stem-loop structures, which are characteristic of TTV. However, their amino acid sequences in ORF1 were similar to that of TA278 in only 35.4 and 34.0%, respectively; TJN01 was 45.4% similar to TJN02. Comparison with TTV isolates of the same genotype identified hypervariable regions in ORF1 of TJN01 and TJN02, as in the prototype TTV of genotype 1. However, quasispecies were barely observed in them. Furthermore, sequences of hypervariable regions scarcely changed during 2–5.5 years in both TJN01 and TJN02. These results indicate that TTV of genotypes 12 and 13 are much different from the prototype TTV of genotype 1.

Full-length genomic sequence of a hepatitis C virus genotype 2c isolate (BEBE1) and the 2c-specific PCR primers

SummaryWe sequenced the entire genome of an Italian isolate of hepatitis C virus: the first full-length sequence for the genotype 2c. We report hereby its characteristics and differential detection of 2c isolates using PCR.