Differences in the entire nucleotide sequence between hepatitis B virus genomes from carriers positive for antibody to hepatitis B e antigen with and without active disease.

The entire nucleotide sequence was determined for eight hepatitis B virus (HBV) genomes from three symptom-free carriers, two patients with chronic persistent hepatitis and one patient with chronic active hepatitis, who were positive for antibody to hepatitis B e antigen (HBeAg). The two patients with chronic persistent hepatitis were tested again after they developed chronic active or fulminant hepatitis, making a total of eight samples. Six had a point mutation in the preC region prohibiting the encoding of HBeAg precursor, while the remaining two had a deletion of 8 or 21 nucleotides within the X gene upstream of the preC transcription initiation sites which would affect the X gene and the putative preC/C promoter. Most genomes from the three symptom-free carriers and the two patients with chronic persistent hepatitis, with HBV DNA levels of 10(2)-10(3)/ml, had deletion, frameshift mutation, initiation failure or a premature stop codon, rendering them replication-incompetent. In contrast, such mutations were rarely seen in HBV genomes from the two patients with chronic persistent hepatitis after they had developed active or fulminant hepatitis and from the patient with chronic active hepatitis, all of whom had vigorous HBV replication with serum HBV DNA from 10(6) to 10(9)/ml. Unique mutations for amino acid changes were more frequent in HBV genomes with a higher replicative activity. These results indicate two kinds of HBV genomes with an HBeAg-minus phenotype, one with defects seriously affecting viral replication and the other without such defects, which would account for different clinical profiles in carriers with antibody to HBeAg.

Prevalence of HCV-antibodies and HCV-RNA in donor blood in Tokushima Prefecture.

The incidence of C100-3 among the blood specimens qualified for transfusion according to the conventional criteria was 1.1%. The incidence of C100-3 in donor blood in Tokushima Prefecture is not significantly different from that reported for all Japan. Of the donors positive for the conventional screening test and C100-3, 73.6% showed high ALT levels. For all antibodies, the incidence of HCV-RNA was very low in the donors positive for a single antibody, but was high in those positive for multiple antibodies. All of the donors showing the 3 antibodies were positive for HCV-RNA. While a test for multiple antibodies is thought to be effective for the screening of HCV, more blood needs to be discarded, having a serious cost-performance problem. The O.D. value for C100-3 and the 2nd antibody seem to be useful reference value for antibody titers.

Hepatitis C virus variants from Nepal with novel genotypes and their classification into the third major group.

Five isolates of hepatitis C virus (HCV) RNA from patients with chronic liver disease in Nepal were not classifiable into the known genotypes I/1a, II/1b, III/2a, IV/2b or V/3a using PCR with type-specific primers deduced from the HCV core gene. Their nucleotide sequences were determined for the 5'-terminal 1.5 kilobases and 3'-terminal 1.2 kilobases, covering 30% of the entire genome, and compared with each other and with reported sequences of HCV isolates of various genotypes. They were more similar to a reported HCV isolate (NZL1) of genotype V/3a (in 81.6 to 84.1% of their nucleotides and 85.7 to 88.7% of the deduced amino acid sequence) compared with the genotypes I/1a to IV/2b (in 69.3 to 74.7% and 72.3 to 77.4%, respectively). Hence they were considered to be variants of the third major group (group 3). The five HCV isolates shared 81.3 to 85.2% of nucleotide sequence and 85.4 to 89.3% of deduced amino acid sequence. Thus they were substantially different from each other. One of them was classified as genotype VI/3b due to an 88.2% similarity in nucleotide sequence to that of the reported HCV isolates of this genotype, whereas the remaining four were classified into provisional genotypes 3c, 3d, 3e and 3f. These HCV variants have evolved and remained in Nepal, and have not been observed in the other areas of the world.

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.

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.

Characterization of the genomic sequence of type V (or 3a) hepatitis C virus isolates and PCR primers for specific detection.

We have identified four new hepatitis C virus (HCV) isolates whose genomic RNA could be amplified by PCR using primers from the 5' untranslated region (UTR), but the RNA could not be detected with genotype I to IV (or types 1a, 1b, 2a and 2b respectively)-specific core region-derived primers. We compared the nucleotide sequences of the new isolates from positions 65 to 1850 (3' end of 5' UTR, C, E1 and 5' end of E2/NS1) and 8276 to 9394 (3' end of NS5 and 3' UTR) with those for genotypes I to IV. The four isolates had the following characteristics: (i) the overall nucleotide sequence similarity between the four isolates was 95 to 96%, compared to 73 to 74%, 73%, 70% or 69 to 70% against genotypes I, II, III or IV, respectively; (ii) the sequence similarity to other reported 'type V (3a)' isolates was 88 to 100%; (iii) the hypervariable region 1 [(HVR)-1] was present but HVR-2 was absent within the E2/NS1 region; (iv) only one in-frame termination codon was present for the presumed polyprotein; (v) the 3'UTR preceding a terminal poly(U) stretch was significantly shorter than in genotype I to IV isolates. We classified the four isolates as genotype V (3a), and searched for uniquely conserved nucleotide sequences that could be used for type-specific PCR. A core region-derived primer pair (no. 104V: 5' CGTAAAACTTCT GAACGGTC, sense and no. 339: 5' GCTGAGCCCA GGACCGGTCT, antisense) was identified and successfully used to diagnose genotype V (3a) HCV infection.

Prevention of posttransfusional non-A, non-B hepatitis by a screening test for hepatitis C virus antibody of donor bloods.

The preventive effect on posttransfusional non-A, non-B hepatitis (PTH) of screening out HCV-Ab positive blood and the prevalence of HCV-Ab-positive donor blood were examined. The incidence of HCV-Ab-positivity in donor blood A was 0.9% and that in donor blood B was 1.35%. The mean ALT and guanase levels were 11.5 +/- 5.8 and 0.58 +/- 0.24 IU/l in HCV-Ab negative blood and 17.3 +/- 7.9 and 0.84 +/- 0.23 IU/l in HCV-Ab-positive blood. Both levels were significantly higher in HCV-Ab-positive blood. These differences were considered to be nonspecific, but there may be some relationship between the levels of ALT and guanase in donor blood and the HCV carrier status. After adoption the screening test for HCV-Ab positive blood, there was no case of a definite diagnosis of PTH, although 4 patients (6.6%) suspected of developing PTH. So, the incidence of PTH was clearly lower than the lowest incidence before adoption of this test. Therefore, we conclude that screening for HCV-Ab in donor blood should be routinely used for prevention of PTH.