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Biomedical subjects

S A Locarnini

Publications and source records attributed to S A Locarnini.

At least 37 records · Page 2Linked to original sources
PubMed1997

High level expression of the capsid protein of hepatitis E virus in diverse eukaryotic cells using the Semliki Forest virus replicon.

The capsid protein of hepatitis E virus (HEV) is encoded by open reading frame 2 (ORF 2) and exhibits variable processing when expressed in insect and COS cells, but nothing is known of its processing in cells relevant to its replication. The full-length ORF 2 protein was expressed at high levels in mammalian cells by insertion of ORF 2 in the Semliki Forest virus (SFV) replicon to generate rSFV/HEV ORF 2K. Expression of the capsid protein was detected readily by metabolic labelling and indirect immunofluorescence in BHK-21 cells transfected with RNA transcripts derived from rSFV/HEV ORF 2K. ORF 2 protein was also expressed at high levels in cells of diverse origin, including liver-derived cell lines Huh7 and HepG2, following infection with recombinant virus derived from cotransfection of BHK-21 cells with the rSFV/HEV ORF 2K and helper SFV replicon RNAs. The addition of hypertonic KCl during metabolic labelling reduced the level of host cell protein synthesis and enhanced the detection of intermediates in ORF 2 protein processing. The wide host range and high level expression directed by SFV replicon particles has particular utility in the analysis of cell-specific factors in the protein processing and assembly of non-cultivable viruses such as HEV.

Animals↗
PubMed1997

Hepatitis G virus and fulminant hepatic failure: evidence for transfusion-related infection.

BACKGROUND/AIMS: In the majority of cases of fulminant "viral" hepatitis in Australia, no known aetiological agent can be isolated. We have examined the possible role of the recently discovered hepatitis G virus (HGV) in such cases. METHODS: An HGV specific reverse transcription polymerase chain reaction (RT-PCR) was performed on pre- and post-liver transplant serum from 14 patients who were referred for transplantation at our unit between 1989 and 1995 for unexplained fulminant hepatic failure. Eleven patients successfully underwent transplantation and three died while waiting for a suitable donor organ. Hepatitis viruses A-E were excluded by standard serological and PCR based testing. HGV RT-PCR was also performed on 21 other, randomly selected, liver transplant recipients ("controls"). RESULTS: The 14 fulminant cases were HGV RT-PCR negative prior to transplantation while five of 21 controls were positive. Post-transplant, eight of the 11 fulminant patients were found to be HGV RT-PCR positive and the same five controls remained HGV RT-PCR positive. In three of the eight fulminant patients the HGV infection resolved. CONCLUSIONS: Our data indicate that HGV infection is unlikely to be responsible for fulminant hepatitis and that it is probably acquired from blood and/or blood products during the transplantation process. Furthermore, long-term carriage of HGV post-transplant is not associated with clinically apparent liver disease.

Amino Acid Sequence↗
PubMed1997

Hepatitis C virus genotypes in Australia.

The relative distribution of Australian hepatitis C virus (HCV) genotypes was determined for 500 isolates. Genotyping was performed using a commercial reverse phase hybridization assay after amplification of the 5' untranslated region of HCV by the polymerase chain reaction. Australian isolates comprised, predominantly, genotype 1 (55%) and genotype 3 (38%) with genotype 2 accounting for only 7%. Genotype 3a was the most common subtype. When the major risk groups of injecting drug users or transfusion-acquired hepatitis C were compared, there was a significantly higher incidence of genotype 1b in the transfusion-acquired group (P < 0.03). When the age of the patients was analysed, genotype 3a was more prevalent in the 21-40-year age group than the 41-60-year age group (P < 0.05). There was no significant difference in genotype distribution between males and females. HCV genotypes 1, 2 and 3 are most often found in developed countries but the relatively high prevalence of genotype 3a in Australia is unusual.

Adult↗
PubMed1997

Clinical experience with famciclovir against hepatitis B virus.

Famciclovir (FCV, the oral form of penciclovir, PCV) is a potent antiviral agent of hepatitis B virus (HBV) and is currently in phase III clinical trials. In this review, we examine the outcome of FCV treatment in preventing recurrent HBV in patients post transplantation. Resistance to FCV has now been documented in this setting, in which reduced sensitivity to FCV was associated with mutations upstream from the conserved 'YMDD' motif in the HBV polymerase gene. These mutations are in a region which has been designated as the B domain in RNA-dependent polymerases. To understand these mutations we have developed a model of the catalytic regions of the HBV polymerase and located mutations selected during antiviral treatment on this model.

2-Aminopurine↗
PubMed1996

Prevalence of hepatitis G virus in Queensland blood donors.

OBJECTIVE: To determine the prevalence of hepatitis G virus (HGV) carriage in Queensland blood donors. DESIGN: Cross-sectional survey with retrospective longitudinal study of HGV-positive donors. SETTING: Brisbane Red Cross Blood Bank, 1995. SUBJECTS: 100 consecutive blood donors attending the Blood Bank on two days in October 1995 and 20 blood donors with a raised plasma alanine aminotransferase (ALT) level on their last donation. OUTCOME MEASURES: Presence of HGV RNA by reverse transcription polymerase chain reaction (RT-PCR) in currently donated blood and in blood samples archived for up to 34 months. RT-PCR used two different reverse transcription methods and three different specific sets of primers and probes. RESULTS: Five of the 120 blood donors were positive for HGV RNA by all RT-PCR methods (four of the 100 with normal ALT levels [4%] and one of the 20 with raised ALT levels [5%]). Retrospective testing of archived samples showed that four of these five had been persistently HGV RNA-positive for at least two years, while the fifth had been HGV RNA-negative on two donations before becoming HGV RNA-positive. No risk factors were identified for this donor. CONCLUSIONS: A relatively large number of Queensland blood donors (4%) are persistently HGV RNA-positive.

Adult↗
PubMed1996

Mother-to-baby transmission of hepatitis G virus.

A mother tested positive for hepatitis G virus (HGV) by reverse transcription-polymerase chain reaction (RT-PCR) before and at delivery. Her baby tested HGV negative by RT-PCR at birth but serum samples at four and six weeks of age were HGV positive, suggesting transmission of HGV from mother to baby.

Adult↗
PubMed1996

New hepatitis viruses: are there enough letters in the alphabet?

Testing is now available for five recognised hepatitis viruses (A, B, C, D and E), and molecular technology is uncovering further hepatotropic viruses. An enteric agent isolated from human stool samples and transmitted experimentally to primates is a candidate hepatitis F virus. A provisionally designated blood-borne hepatitis G virus is associated with acute and chronic non-ABCDE hepatitis and has a worldwide distribution. A group of flavi-like viruses, the GB group, also blood borne, has also been reported. The role of two of these viruses, GBV-A and GBV-B, in human viral hepatitis has not been determined, but a third agent, GBV-C, is associated with acute and chronic hepatitis and appears to be a West African variant of hepatitis G. Our current knowledge suggests that the hepatitis alphabet may need to be extended even after inclusion of some of these new viruses.

Animals↗
PubMed1996

Inhibition of hepatitis B virus DNA polymerase by enantiomers of penciclovir triphosphate and metabolic basis for selective inhibition of HBV replication by penciclovir.

The deoxyguanosine analog penciclovir (PCV; 9-[4-hydroxy-3-hydroxymethyl-but-1-yl]guanine), has shown potent antiviral activity against herpes viruses and hepadnaviruses. Efficacy against chronic hepatitis B virus (HBV) infection has been demonstrated in an animal model and in recent clinical trials of famciclovir, the oral form of PCV. The antiviral activity of PCV is believed to be dependent on the intracellular formation of PCV-triphosphate (PCV-TP) which is presumed to inhibit HBV replication by interfering with viral DNA polymerase activity. The (S)-enantiomer is preferentially formed in herpes virus-infected cells, and is the more active against the herpes simplex virus; however, little is known about the biochemical mechanisms of PCV phosphorylation or of interference with viral replication in HBV-infected cells. Here, we report that in contrast with herpes simplex virus, the (R)-enantiomer of PCV-TP is a more potent inhibitor of HBV DNA polymerase-reverse transcriptase (pol-RT) in vitro than the (S)-enantiomer. In assays for HBV DNA pol-RT activity, in which purified viral core particles were the enzyme source, the IC50s for (R)- and (S)-enantiomers of PCV-TP were 2.5 micromol/L and 11 micromol/L, respectively. The estimated Kis for (R)- and (S)- PCV-TP were approximately 0.03 micromol/L and approximately .04 micromol/L, respectively, about 3-fold lower than the Km for deoxyguanosine triphosphate (dGTP) in the same system. In addition, we report that PCV metabolism is similar in both control (HepG2) and in HBV-transfected (2.2.15) hepatoblastoma cells in vitro, indicating that cellular enzyme(s) catalyze PCV phosphorylation. Peak PCV-TP concentrations of about .4 micromol/L were reached in both cell types in less than 12 hours, and intracellular PCV-TP was exceptionally stable with a half-life of about 18 hours. These observations provide a mechanistic basis for the potent activity of PCV against HBV.

Acyclovir↗
PubMed1996

Mutations in the hepatitis B virus precore/core gene and core promoter in patients with severe recurrent disease following liver transplantation.

Recurrent hepatitis B virus (HBV) infection is a major problem in patients undergoing liver transplantation. Previously, we reported that infection with HBV strains containing a mutation in the precore region (G-to-A at nucleotide 1896) was associated with severe recurrent disease posttransplantation. In this study we investigated other mutations in the precore/core gene and core promoter which may be associated with this severe recurrence. The precore/core gene and core promoter of HBV from pre and posttransplantation sera of 15 patients with HBV recurrence were amplified by polymerase chain reaction (PCR) and sequenced. Pre and posttransplant sequences were very similar for each patient. HBV from patients who developed severe recurrence had significantly more mutations in both the nucleotide (P < .05) and predicted amino acid (P < .05) sequences of the precore/core gene, but not in the core promoter, than virus from patients with mild recurrence. There was also an apparent link between severe disease and HBV strains of genotype D (P < .05). The number of nucleotide and amino acid mutations in the precore/core gene was strongly associated with the presence of the precore mutation (P < .01). Mutations were found throughout the entire gene, however, at the amino acid level clustering was observed in the B- and helper T-cell epitopes as well as nuclear localization signals. In the encapsidation signal, nucleotide mutations were found that were predicted to increase the stability of the stem-loop structure. Overall, our data shows that genotype D and accumulated mutations throughout the HBV precore/core gene, but not core promoter, were associated with severe recurrent disease posttransplantation. These mutations were strongly linked to the presence of the precore mutation at nucleotide position 1896 and may contribute to the poor outcome in these patients.

Adult↗
PubMed1995

Isolation of HIV-1 from experimentally contaminated multidose local anaesthetic vials.

OBJECTIVE: To investigate the hypothesis that HIV can be transmitted via contamination of multidose vials of local anaesthetic solution through reuse of needles and syringes. DESIGN AND SETTING: Laboratory study. (1) By experiments with multidose vials and disposable needles and syringes, we identified a sequence of events in which HIV could contaminate the anaesthetic solution. (2) Three anaesthetic solutions were contaminated with a laboratory strain of HIV and tested by viral culture and p24 enzyme immunoassay one, two and four hours later to see how long the virus remained active. RESULTS: (1) Needles and syringes retained small volumes of fluid after use (mean, 25 microL; in syringe alone, mean 16 microL) which could be transferred to multidose vials of local anaesthetic. (2) 10 mL of anaesthetic solution contaminated with 8 microL of HIV-infected solution (equivalent to 1% infected lymphocytes in vivo) contained active virus one hour later. In some settings, HIV could be isolated four hours after exposure. CONCLUSION: When inadvertently contaminated with HIV, multidose solutions represent a potential source of transmissible virus.

Anesthetics, Local↗
PubMed1995

Evaluation of blood donors with equivocal hepatitis C serological results.

OBJECTIVE: To characterise blood donors with equivocal hepatitis C serological results and to develop an algorithm for their diagnosis and follow-up. DESIGN: Prospective case survey. SUBJECTS AND SETTING: 100 consecutive blood donors referred to the St Vincent's Hospital Liver Clinic, Victoria, with equivocal hepatitis C serological results (positive result for second generation Abbott Enzyme Immunoassay 2.0, but at least one negative result on supplemental testing by first generation Abbott neutralisation assay and Abbott Supplemental Assay for antibody to specific viral antigens). OUTCOME MEASURES: Percutaneous risk factors for hepatitis C exposure, peak serum alanine aminotransferase (ALT) levels, results of alternative immunoassay (Monolisa) and polymerase chain reaction (PCR) to detect hepatitis C viraemia. RESULTS: Thirty subjects had positive results for alternative immunoassay. A risk factor was identified for 32 subjects and was significantly associated (P < 0.01) with positive results for alternative immunoassay (23/32) and PCR (11/32), abnormal ALT levels (7/32), and strong reactivity on initial immunoassay (23/32). Presence of antibodies to both structural and non-structural antigens was also associated with risk factors and positive alternative immunoassay results. CONCLUSIONS: A definitive diagnosis was possible in 87% of subjects. A diagnosis of hepatitis C infection was based on positive alternative immunoassay results together with positive PCR results or presence of a risk factor. Hepatitis C was excluded for 60% of patients. The diagnosis for the remaining 13% remained indeterminate, indicating the need for a definitive diagnostic test for hepatitis C.

Adult↗
PubMed1995

Spread of bloodborne viruses among Australian prison entrants.

OBJECTIVES: To assess spread of bloodborne viruses among prison entrants in Victoria, Australia. DESIGN: Voluntary confidential testing of all prison entrants for markers of exposure to bloodborne viruses with collection of minimal data on demography and risk factors over 12 months. SETTING: Her Majesty's Prisons, Pentridge and Fairlea, Victoria, Australia. SUBJECTS: 3429 male and 198 female prison entrants (> 99% of all prison entrants); 344 entered prison and were tested more than once. MAIN OUTCOME MEASURES: Prevalence and incidence of antibodies to HIV, hepatitis B, and hepatitis C viruses, and minimal data on risk factors. RESULTS: 1562 (46%) gave a history of use of injected drugs, 1171 (33%) had antibody to hepatitis B core antigen, 1418 (39%) were anti-hepatitis C positive including 914 (64%) of the men who injected drugs, 91 (2.5%) were positive for hepatitis B surface antigen, and 17 (0.47%) were positive for antibody to HIV. Incidence rates for infection with hepatitis B and C virus were 12.6 and 18.3 per 100 person years, respectively; in men who injected drugs and were aged less than 30 years (29% of all prison entrants) these were 21 and 41 per 100 person years. Seroconversion to hepatitis B or C was associated with young age and shorter stay in prison. Only 5% of those who were not immune to hepatitis B reported hepatitis B immunisation. CONCLUSIONS: Hepatitis B and C are spreading rapidly through some populations of injecting drug users in Victoria, particularly among men aged less than 30 years at risk of imprisonment in whom rates of spread are extreme; this group constitutes a sizeable at risk population for spread of HIV. This spread is occurring in a context of integrated harm reduction measures outside prisons for prevention of viral spread but few programmes within or on transition from prisons; it poses an urgent challenge to these programmes.

Adult↗
PubMed1995

Seroepidemiology of hepatitis E in selected Australian populations.

The presence of hepatitis E virus-specific antibodies (anti-HEV) was determined in selected Australian groups. Anti-HEV was detected initially using a recombinant antigen-based enzyme immunoassay (EIA). It was found that 1 of 279 (0.4%) blood donors, 14 of 182 (7.7%) Indochinese refugees, 2 of 89 (2.2%) sera submitted for amoebic serology (generally people who had visited developing countries), 1 of 13 (7.7%) patients with non-A, non-B hepatitis, none of 7 (0%) patients with fulminant non-A, non-B hepatitis, and none of 33 (0%) control sera were repeatedly reactive by the HEV EIA. The positive sera were subjected to further testing using a supplemental immunoblot. Preliminary data suggest that while potentially large numbers of people infected with HEV are entering Australia, no compulsive evidence was found in these particular groups for endemic HEV infection in Australia. This is the first seroepidemiological survey of HEV in Australia.

Adult↗
PubMed1995

Susceptibility of HIV to inactivation by disinfectants and ultraviolet light.

Assays were developed to assess a variety of conditions and presentations of infectious HIV to potential inactivating sources. A range of commercially available disinfectants with active constituents including glutaraldehyde, chlorine, phenolics, alcohol, iodine and quaternary ammonium compounds was tested. In addition, u.v. light was investigated as a potential inactivating source. All products were assessed against cell-free HIV in culture medium and cell-associated HIV suspended in medium or whole human blood. All products completely inactivated cell-free HIV following a 1 min exposure. However, cell-associated HIV was more resilient, requiring exposure of 5 min or more for some disinfectants. The effectiveness of the disinfectants was further compromised in the presence of blood.

Blood↗