Application of the Serodia-HCV particle agglutination for the detection of antibodies to hepatitis C virus.

The Serodia-HCV Particle Agglutination (HCV-PA) for the detection of HCV antibodies was compared with the Enzyme Immunoassay Test (UBI HCV EIA) for possible in-house use. A total of 150 specimens were analysed using UBI HCV EIA and Serodia-HCV PA. Of these, 80 (53.3%) were both PA and EIA positive and 59 (39.3%) were negative by both techniques. Eleven sera (7.4%) were found to be EIA-positive but PA-negative. These 11 discordant sera were further tested by the LiaTek-HCV III Immunoassay (Organon Teknika). Ten were found to be line immunoassay negative and one was line immunoassay positive. Failure of the PA to detect the HCV positive serum meant that a small proportion of HCV antibody positives may be missed by the PA test. We conclude that (i) EIA should continue to be the first line screening test in our laboratory, (ii) PA with its 100% specificity could be a useful supplementary screen for all EIA-positive sera and finally (iii) line immunoassay could be used on sera to resolve discordant results in the EIA and PA assays.

Hepatitis C--the Malaysian story.

We studied the presence of Hepatitis C Virus (HCV) antibodies in a defined Malaysian population and examined the association, if any, between HCV and the Hepatitis B Virus (HBV), using sensitive recombinant DNA second generation Enzyme Immunoassay (EIA) test kits. This sero-prevalence study comprised 1,434 sera from eleven distinct groups comprising intravenous drug users (IVDU), haemophiliacs, male homosexuals, female prostitutes, healthy blood donors, staff of dialysis unit and laboratory personnel, chronic renal failure patients undergoing dialysis (CRFD), patients with liver cirrhosis, chronic active hepatitis, chronic persistent hepatitis and primary liver cancer. Except in laboratory personnel and dialysis staff, HCV antibodies were detected in each group of patients ranging from 3% in blood donors to 85% in IVDU. The main modes of HCV transmission identified were parenteral drug use, transfusion and/or dialysis related. The HBV was found to be the major viral etiological agent in 75% of chronic liver disease (CLD); while in 10% of cases both HCV and HBV were detected. HCV was implicated as the sole viral agent in only a small proportion (1.5%) of patients with chronic liver disease.

Baculovirus vectors for multiple gene expression and for occluded virus production.

The nature and properties of a variety of plasmids are described that facilitate the construction of baculovirus vectors for expression of one or more heterologous genes. The plasmids are designed for use with Autographa californica nuclear polyhedrosis virus, AcMNPV, as a vector for protein production in insect cells and/or insect larvae. Several plasmids described here facilitate the simultaneous insertion and expression of two different genes. Some vector systems allow high and equal levels of transcription of both genes while others employ two different promoters for differential transcription. Four of the plasmids described here are designed for expression of both the viral polyhedrin-encoding gene and a heterologous gene. Such recombinants form polyhedral occlusion bodies which serve as visible markers of recombination and facilitate oral infection of insect larvae for mass-scale protein production. A synthetic promoter with a unique sequence can be used at a variety of sites in the viral genome and avoids sequence duplication. A series of plasmids are also described that supply an N terminus with an efficient translational initiation signal and convenient multiple cloning sites in the three different translational reading frames. The modular nature of all the constructs allows the use of other promoters with different temporal regulation to be utilized in the construction of additional plasmids for customized expression work.

The influence of antisense RNA on transcriptional mapping of the 5' terminus of a baculovirus RNA.

S1 nuclease protection and primer extension analyses were used to determine the 5' end of the Autographa californica nuclear polyhedrosis virus 603 open reading frame (ORF) transcript upstream of and on the opposite strand to the polyhedrin gene. These analyses suggested that the 5' end of the 603 ORF was located near the initiation site for polyhedrin gene transcription. Primer extension products of reverse transcription of 603 RNA were dependent on the presence of the TAAG sequence, an essential polyhedrin promoter element. The results could be interpreted as indicating bidirectional transcription from the TAAG element. However, the data could also be due to an artefact of antisense transcripts and RNA duplex formation in this region. Since the bidirectional transcriptional model is not consistent with other mapping data and Northern blot analysis, we conclude that the presence of antisense RNA can result in mapping artefacts and that caution must be taken in interpreting data where overlapping sense and antisense RNAs are present.

Mutational analysis of the Escherichia coli glpFK region with Tn5 mutagenesis and the polymerase chain reaction.

Transposon Tn5 mutagenesis of the Escherichia coli chromosome was used to isolate 21 independent insertion mutations conferring an altered colony color phenotype on MacConkey-glycerol plates. The polymerase chain reaction was used to map 16 of these Tn5 insertions within the glpFK region at 88 min. The most polar Tn5 insertion was shown by nucleotide sequencing to be in the proposed glpF open reading frame. The data suggest that the glpF and glpK genes are in an operon with a bent DNA segment (BENT-6) involved in transcriptional regulation of this operon.

Transcription of the baculovirus polyhedrin gene reduces the levels of an antisense transcript initiated downstream.

A late 3.2-kilobase (kb) RNA initiated approximately 2 kb downstream of the 3' end of the Autographa californica nuclear polyhedrosis virus polyhedrin-coding sequence and traversed the polyhedrin gene in an antisense direction. This RNA was sense RNA for the two open reading frames flanking the polyhedrin gene. A mutant virus, vXpoly, which differs from wild-type virus only at the essential RNA initiation site in the polyhedrin promoter, exhibited higher levels of the 3.2-kb RNA than did wild-type virus during the polyhedrin transcriptional phase. Thus, transcription of the polyhedrin gene down regulates the levels of this 3.2-kb RNA.

Downstream sequences augment transcription from the essential initiation site of a baculovirus polyhedrin gene.

A series of recombinant baculoviruses containing linker-substituted polyhedrin promoters attached to a reporter gene encoding chloramphenicol acetyl transferase (CAT) were constructed and tested for expression of the gene. The major determinant for promoter activity was narrowed to within eight nucleotides, TAAGTATT, at the start point of polyhedrin mRNA transcription. Mutations within TAAGTATT blocked initiation of transcription from this site and resulted in a 2000-fold decrease in CAT activity. Linker mutations from 12 to 22 bases upstream from the TAAGTATT sequence increased the steady-state levels of RNAs initiated within TAAGTATT and increased CAT expression by up to 50%. Mutations downstream from TAAGTATT and within the region specifying the untranslated RNA leader diminished transcriptional initiation at TAAGTATT and decreased CAT activity two- to 20-fold. The half-lives of CAT RNAs were not noticeably affected by mutations in the untranslated RNA leader region and thus RNA turn-over was not responsible for the reduced levels of these CAT RNAs. Nuclear run-on analysis of two mutant viruses showed that these mutations decrease the rate of transcriptional initiation. Transcriptional initiation thus appears to be the major means of polyhedrin gene regulation. The data define promoter-related roles for TAAGTATT and the sequences specifying the untranslated mRNA leader in transcriptional initiation. A model by which the viral-induced RNA polymerase distinguishes late and very late initiation sites is proposed.

Prevalence of hepatitis delta virus infection in Malaysia.

The prevalence of coinfection, superinfection and chronic infection with the hepatitis delta virus (HDV) was studied in 324 hepatitis B surface antigen (HBsAg)-positive Malaysians. Of these, 10.0% (5/50) had coinfection, 5.7% (11/194) had superinfection, but none of the 80 patients with chronic liver disease (CLD) or primary hepatocellular carcinoma (PHC) had chronic infection with HDV. The overall HDV infection was 4.9% (16/324). One of the coinfection cases acquired the HDV infection as early as 1982. HDV superinfection was detected mainly among IV drug abusers (20% or 7/35) and promiscuous males and females (13.6% or 3/22). They were all asymptomatic. Only 0.8% (1/125) apparently healthy blood donors was infected with HDV. None of the 12 multi-transfused patients examined were positive. Malaysia is the only Southeast Asian country examined so far in which HDV infection was detected. The reason could be that the IV drug abusers and the sexually promiscuous groups missed being examined in the other countries. Comparing the HDV infection rates in 4 categories of infected Malaysians (viz. acute hepatitis B patients, IV drug abusers, blood donors and CLD patients) with those of other countries, it was noted that the Malaysian rates were similar to the lowest in the range of prevalence rates of each category in the latter group. The rate of coinfection in a preliminary study in 1982-84 (9.0% or 1/11) was not very different from that obtained to date (10.0% or 5/50).(ABSTRACT TRUNCATED AT 250 WORDS)

Eight base pairs encompassing the transcriptional start point are the major determinant for baculovirus polyhedrin gene expression.

The effects of mutations within the 92-bp region immediately upstream from the translational initiation ATG of the polyhedrin gene of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) were determined by plasmid transient expression assays in the presence of wild-type (wt) AcMNPV DNA. Clustered point mutations were generated by substitution of 10-bp stretches of the polyhedrin promoter/leader region with a 10-bp HindIII linker. Three of these linker scan (LS) mutations in the region from nucleotides (nt) -62 to -84 (relative to the original polyhedrin ATG at +1, +2, +3) had no effect or a mild stimulatory effect on reporter gene expression. One mutation immediately upstream (nt -52 to -60) from the transcription start point (at nt -50) reduced expression four-fold. Three overlapping mutations affecting 8 bp from nt -44 to -51 (encompassing the transcriptional start point) reduced expression by 1000-fold. A 1000-fold reduction was also observed for a total deletion of nt -1 and -92. Five LS mutations between nt -1 and -43 each reduced expression two- to five-fold, whereas combining an LS mutation and a 9-bp deletion mutation in the leader reduced expression approx. nine-fold. Reversing the orientation of the reporter gene along with the wt 92-bp upstream polyhedrin promoter/leader sequences resulted in slightly higher expression levels than those observed for the normal orientation indicating that all the essential cis-acting promoter elements, with the possible exception of long-range enhancer sequences, are located downstream from nt -92. Sequences of the AcMNPVhr5 enhancer (homologous region No. 5 of AcMNPV) had only a minor effect on expression from the polyhedrin promoter in transient assays. The results show that 8 bp encompassing the transcriptional start point, a sequence which is conserved in all late AcMNPV promoters, is essential for polyhedrin gene expression. Additional nucleotides within the leader region are necessary for optimal expression.

Regulation of host RNA levels during baculovirus infection.

During infection of the permissive host insect cell line Spodoptera frugiperda IPLB-SF-21 by the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV), the levels of host actin, histone, and heat shock 70 (hsp70) RNAs are reduced substantially. Reduction of the host RNA levels occurs primarily during a narrow window of the replication process, from approximately 12 to 18 hr postinfection (p.i.), corresponding to the phase in which the extracellular form of the virus buds into the media. A late viral protein appears to be required for this reduction since cycloheximide, an inhibitor of cytosolic protein synthesis, and aphidicolin, an inhibitor of host and viral DNA polymerases, inhibit the reduction of actin and histone RNA levels. A cDNA corresponding to the carboxyl half of the S. frugiperda mitochondrial cytochrome c oxidase subunit III (COIII) gene was isolated, sequenced, and characterized. Two differentially regulated mitochondrial transcripts of this gene are observed. The level of the larger of these transcripts, which is dependent on active cytosolic protein synthesis, is reduced during virus infection in a fashion similar to that of the nuclear host genes. The smaller COIII transcript is stable until at least 24 hr p.i. but the level of this RNA eventually declines by 48 hr p.i.

Biogenesis of mitochondria: a mutation in the 5'-untranslated region of yeast mitochondrial oli1 mRNA leading to impairment in translation of subunit 9 of the mitochondrial ATPase complex.

A temperature-conditional mit- mutant of Saccharomyces cerevisiae has been characterized; the mutant strain h45 cannot grow at 36 degrees C on nonfermentable substrates yet appears to be normal at 28 degrees C. The mutation in strain h45 maps genetically to the oli1 region of the mitochondrial DNA (mtDNA) genome, and prevents the synthesis at 36 degrees C of the oli1 gene product, subunit 9 of the mitochondrial ATPase complex. Since the level of oli1 mRNA in mutant h45 is close to normal at 36 degrees C, it is concluded that there is a specific block in translation of this mRNA at the non-permissive temperature. DNA sequence analysis of mtDNA from strain h45 reveals an additional T residue inserted 88 bp upstream of the oli1 coding region, in the A,T-rich sequence that is transcribed into the 5'-untranslated region of the oli1 mRNA. Sequence data on two revertants show that one returns to wild-type parental (J69-1B) mtDNA sequence, whilst the other contains an inserted A residue adjacent to the T inserted in the original h45 mutant. The results are discussed in terms of the stability of folds in RNA upstream of putative ribosome-binding sites in mitochondrial mRNA, and the potential action of nuclear-coded proteins that might be activators of the translation of specific mitochondrial mRNAs in yeast mitochondria.

Hepatitis B markers in non-icteric medical patients in Malaysia.

Sera were obtained from 494 non-icteric patients admitted with illnesses other than overt hepatitis into the medical wards of the rural and urban hospitals in Malaysia. They were tested for HBsAg, HBeAg, and anti-HBs by enzyme immunoassay. The overall HBsAg carrier rate was 18.0% ranging from 9.6% in children, (10 years and under), to a maximum of 23.5% in the adolescents (11 to 20 years), the rates decreasing subsequently to 16.5% and 20.8% in the adult and middle-age groups, respectively. The Chinese (18.6%) and Malays (19.9%) had similar HBsAg carrier rates but the rate in the Indians (9.0%) was distinctly lower. Similar rates were observed in the males (16.5%) and the females (19.8%). The carrier rate was 17.1% in rural patients compared with 21.4% in the urban ones. The 'e' antigen was found in 14 of the 89 HBsAg carriers (15.7%). The overall prevalence was 14/494 (2.8%) rising sharply from childhood (2.9%) to adolescence (5.3%), subsequently declining with advancing age. The Chinese had the highest rate (6.2%) followed by the Indians (1.5%) and the Malays (1.1%). Males had a rate of 3.3% compared to the females with 2.3%. Anti-HBs was found in 33.8% of the patients, increasing steadily from childhood (18.3%) to middle-age (46.4%). The Chinese had a higher prevalence rate (41.6%) than the Indians (32.8%) and the Malays (29.3%). The rates were similar for the males (35.6%) and the females (31.5%). Rural patients (46.1%) had a higher rate than urban patients (35.7%). Both areas showed rising prevalence with increasing age.(ABSTRACT TRUNCATED AT 250 WORDS)

A seroepidemiologic study of hepatitis A in Malaysia.

Sera from 494 non-icteric patients admitted with illnesses other than overt hepatitis into the various hospitals in rural and urban Malaysia were tested for IgG antibody to hepatitis A virus. The overall antibody prevalence rate was 67.0% with rates increasing steadily from childhood 10 years old and under (39.4%) to middle-age and above (96.0%). No significant differences were noted between males (68.4%) and females (65.3%). The highest rate was in the Indians (80.6%), the lowest in the Chinese (55.9%) with Malays occupying intermediate position (70.3%). The rate in the rural patients (74.7%) was higher than that in the urban patients (65.5%) especially in the 21 to 40 year age-group where the rural patients had a rate of 96.7% compared with that in urban patients (61.1%). A comparison of antibody prevalence rates in different countries was made.

Aetiology of acute hepatitis in Malaysia.

Icteric patients with clinical and biochemical evidence of liver disease, admitted into various hospitals in Malaysia, were investigated to determine the cause of their infection. Of these patients, 11.0% (16/145) were found positive for IgM anti-HAV (EIA), 4.1% (6/145) for IgM anti-HBc (EIA), 1.0% (1/102) for IgM anti-CMV (ELISA), 17.2% (16/64) for rising titres of leptospiral agglutinin, and none for heterophile antibody of EBV. Hepatitis NANB accounted for 67.9% of cases. The mean serum transaminases (ALT and AST) values in patients with hepatitis A and B were higher (more than 500IU) than in patients with leptospirosis or non-A, non-B hepatitis, whereas serum bilirubin levels were higher in patients with hepatitis A and leptospirosis than in patients with hepatitis B.

Amino acid substitutions in mitochondrial ATPase subunit 9 of Saccharomyces cerevisiae leading to oligomycin or venturicidin resistance.

A series of isonuclear oligomycin-resistant mutants of Saccharomyces cerevisiae carrying mutations in the mitochondrial oli1 gene has been studied. DNA sequence analysis of this gene has been used to define the amino acid substitutions in subunit 9 of the mitochondrial ATPase complex. A domain of amino acids involved in oligomycin resistance can be recognized which encompasses residues in each of the two hydrophobic portions of the subunit 9 polypeptide that are thought to span the inner mitochondrial membrane. Certain amino acid substitutions also confer cross-resistance to venturicidin: these residues define an inner domain for venturicidin resistance. The expression of venturicidin resistance resulting from one particular substitution is modulated by nuclear genetic factors.

The oli1 gene and flanking sequences in mitochondrial DNA of Saccharomyces cerevisiae: the complete nucleotide sequence of a 1.35 kilobase petite mitochondrial DNA genome covering the oli1 gene.

As part of our genetic and molecular analysis of mutants of Saccharomyces cerevisiae affected in the oli1 gene (coding for mitochondrial ATPase subunit 9) we have determined the complete nucleotide sequence of the mtDNA genome of a petite (23-3) carrying this gene. Petite 23-3 (1,355 base pairs) retains a continuous segment of the relevant wild-type (J69-1B) mtDNA genome extending 983 nucleotides upstream, and 126 nucleotides downstream, of the 231 nucleotide oli1 coding region. There is a 15-nucleotide excision sequence in petite 23-3 mtDNA which occurs as a direct repeat in the wild-type mtDNA sequence flanking the unique petite mtDNA segment (interestingly, this excision sequence in petite 23-3 carries a single base substitution relative to the parental wild-type sequence). The putative replication origin of petite 23-3 is considered to be in its single G,C rich cluster, which differs in just one nucleotide from the standard oriS sequence. The DNA sequences in the intergenic regions flanking the oli1 gene of strain J69-1B (and its derivatives) have been systematically compared to those of the corresponding regions of mtDNA in strains derived from the D273-10B parent (sequences from the laboratory of A. Tzagoloff). The nature and distribution of the sequence divergences (base substitutions, base deletions or insertions, and more extensive rearrangements) are considered in the context of functions associated with mitochondrial gene expression which are ascribed to specialized sequences in the intergenic regions of the yeast mitochondrial genome.

DNA sequence analysis of the oli1 gene reveals amino acid changes in mitochondrial ATPase subunit 9 from oligomycin-resistant mutants of Saccharomyces cerevisiae.

The nucleotide sequence of the oli1 gene encoding mitochondrial ATPase subunit 9 (76 amino acids) has been determined for five oligomycin-resistant mutants of Saccharomyces cerevisiae. Three of the mutations affect amino acids in the vicinity of the glutamic acid residue 59 at which dicylohexyl carbodiimide binds. Two other mutations lead to substitution of amino acid 23, which would lie very close to residue 59 in the folded hairpin conformation that this protein is thought to adopt in the inner mitochondrial membrane. The apposition of residues 23 and those adjacent to residue 59, lying respectively in the two hydrophobic membrane-spanning arms of subunit 9, is considered to constitute an oligomycin-binding domain. By consideration of the amino acid substitutions in those mutants cross-resistant to venturicidin, a domain of resistance for venturicidin is defined to lie within the oligomycin-binding domain, also centered on residues 23 and 59. These data also clarify the genetic recombination behaviour of alleles previously defined to form part of the oli3 locus (mutants characterized by resistance to both oligomycin and venturicidin) together with alleles defined to form part of the oli1 locus (mutants not cross-resistant to venturicidin). The oli1 and oli3 loci can now be seen to form two overlapping extended groups within the oli1 gene, with sequenced oli3 mutations being as far apart as 125 nucleotides within the subunit 9 coding region of 231 nucleotides.

Biogenesis of mitochondria: DNA sequence analysis of mit- mutations in the mitochondrial oli1 gene coding for mitochondrial ATPase subunit 9 in Saccharomyces cerevisiae.

The nucleotide sequence of the yeast mitochondrial olil gene has been obtained in a series of mit- mutants with mutations in this gene, which codes for subunit 9 of of the mitochondrial ATPase complex. Subunit 9 is the proteolipid, 76 amino acids in length, necessary for the proton translocation function of the membrane Fo-sector. These mutants were classified on the basis of their rescue by a petite strain shown here to retain the entire wild-type olil gene. The mutation in one mit- strain removes a positively charged residue (Arg39----Met) which is likely to be located in a segment of subunit 9 that protrudes from the inner mitochondrial membrane. In a second mit- mutant, a negatively charged residue replaces a conserved glycine residue (Gly18----Asp) in a glycine-rich segment of the protein that is most likely embedded within the membrane. Other mit- mutations result in frameshifts with predicted products 7, 65 and 68 amino acid residues long. In each mit- mutant, there is the loss of one or more of the amino acid residues that are highly conserved among diverse species. The location and nature of specific changes pinpoint amino acid residues in subunit 9 essential to the activity of the mitochondrial ATPase complex.

Biogenesis of mitochondria: the mitochondrial gene (aap1) coding for mitochondrial ATPase subunit 8 in Saccharomyces cerevisiae.

A mitochondrial gene (denoted aap1) in Saccharomyces cerevisiae has been characterized by nucleotide sequence analysis of a region of mtDNA between the oxi3 and oli2 genes. The reading frame of the aap1 gene specifies a hydrophobic polypeptide containing 48 amino acids. The functional nature of this reading frame was established by sequence analysis of a series of mit- mutants and revertants. Evidence is presented that the aap1 gene codes for a mitochondrially synthesized polypeptide associated with the mitochondrial ATPase complex. This polypeptide (denoted subunit 8) is a proteolipid whose size has been previously assumed to be 10 kilodaltons based on its mobility on SDS-polyacrylamide gels, but the sequence of the aap1 gene predicts a molecular weight of 5,815 for this protein.