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S A Locarnini

Publications and source records attributed to S A Locarnini.

At least 19 recordsLinked to original sources
PubMed2001

Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region.

There is currently no universally accepted numbering convention for the antiviral drug-related resistance mutations in the reverse transcriptase (rt) domain of the human hepatitis B virus (HBV) polymerase. The published inconsistencies have resulted from different HBV genotypes. A standardized numbering system for HBV polymerase is proposed. The new system is based on functional observations of HBV surface gene proteins (preS1, preS2, and HBsAg) and on the current convention used for human immunodeficiency virus type 1 (HIV-1) polymerase proteins (protease, rt, and integrase), in which the amino acid numbering restarts at the first codon position of each domain. The HBV polymerase protein can be divided into 4 domains (terminal protein, spacer, rt, ribonuclease H) and each of these can be numbered separately. In this proposal, the HBV rt domain starts with the highly conserved EDWGPCDEHG motif, contains 344 amino acids, and the lamivudine-related resistance mutations are found at amino acid rtL180M (previously amino acid 528, 526, 515, or 525) and rtM204V/I (previously 552, 550, 539, or 549). The new consensus rt domain numbering system is genotype independent and allows investigators to number any previously and newly discovered antiviral-related amino acid change in a standardized manner.

Amino Acid Sequence↗
PubMed2000

Molecular analysis of human immunodeficiency virus strains associated with a case of criminal transmission of the virus.

An investigation was done of the evidence for transmission of human immunodeficiency virus (HIV) from an HIV-positive man to several male and female sex contacts. Phylogenetic analysis of sequences from the gag and env genes showed a close relationship between the predominant virus strains from the source and 2 contacts. However, the likelihood that a female contact was infected by the source could not be determined, despite contact tracing indicating that this may have occurred. One male, shown by contact tracing and molecular evidence to have been infected by the source, subsequently transmitted HIV to his female sex partner. HIV sequence from a plasma sample used as a control in the phylogenetic analysis contained env and gag sequences that were closely related to those from the source. An epidemiologic link between these 2 individuals was subsequently confirmed by contact tracing.

Adult↗
PubMed1999

Lamivudine therapy in patients undergoing liver transplantation for hepatitis B virus precore mutant-associated infection: high resistance rates in treatment of recurrence but universal prevention if used as prophylaxis with very low dose hepatitis B immune globulin.

Recurrent hepatitis B virus (HBV) infection remains a major cause of morbidity and mortality after liver transplantation. Recently, antiviral therapy, such as lamivudine, has become available for prophylaxis against HBV reactivation posttransplantation and for the treatment of HBV recurrent disease. We report our initial experience with lamivudine therapy in patients with precore mutant-associated HBV infection undergoing liver transplantation (n = 29). Outcomes were compared in three patient groups: group 1, precore mutant HBV infection not receiving lamivudine (n = 10); group 2, recurrent precore mutant HBV infection posttransplantation subsequently treated with lamivudine (n = 10); and group 3, HBV precore mutant patients undergoing liver transplantation and receiving lamivudine and low-dose hepatitis B immune globulin (HBIG) from the time of transplantation (n = 9). In group 1, HBV recurred in 9 of 10 patients, with subsequent graft loss in all 9 patients. In group 2, all patients developed HBV recurrence at a mean of 7.3 months posttransplantation and started lamivudine therapy at a median of 16 months posttransplantation. Follow-up on lamivudine therapy was for a median of 11 months. Six of these 10 patients developed mutations in the HBV polymerase gene associated with lamivudine resistance. There were two liver failure-related deaths in this group. In group 3 patients, there was one death from graft-versus-host disease. The remaining 8 patients have been followed up for a mean of 15.6 months posttransplantation, and all remain hepatitis B surface antigen negative and HBV DNA negative. In conclusion, lamivudine therapy in association with low-dose HBIG is effective in preventing HBV reactivation posttransplantation. Rescue therapy with lamivudine in patients with HBV recurrence is only moderately effective, with a 60% lamivudine resistance rate in patients treated for longer than 6 months.

Adult↗
PubMed1999

Preclinical aspects of lamivudine and famciclovir against hepatitis B virus.

Chemotherapy for chronic hepatitis B virus (HBV) infection is inherently difficult for a variety of reasons that are related to unusual features of both HBV replication strategy and host cell metabolism. Previous attempts to treat chronic HBV infection using nucleoside analogues have been almost universally disappointing, but several recently developed nucleoside analogues have been identified as potent, non-toxic inhibitors of HBV replication. These fall into two broad categories: nucleosides having the 'unnatural' L-configuration, and deoxyguanosine analogues with modified-sugar configurations, represented by lamivudine and penciclovir respectively. Both lamivudine and penciclovir (in its orally available form, famciclovir) have progressed to phase III clinical trials against chronic HBV infection, with promising preliminary results. However, chemotherapy for chronic HBV is necessarily long term, which increases the risks for development of viral resistance and cumulative toxicity. Such risks might be minimized by the use of appropriate drug combinations, rational selection of which requires knowledge of the pharmacokinetics and mechanisms of action of the individual agents. An appreciation of cellular deoxynucleoside metabolism and its regulation, the complexities of which are still emerging, is an indispensable aid to understanding the biological activities of deoxynucleoside analogues. The modes of action of lamividine and penciclovir, and how these two deoxynucleoside analogues may interact in vitro and in vivo as inhibitors of HBV replication, are examined here in the context of cellular deoxynucleoside metabolism.

2-Aminopurine↗
PubMed1999

Hepatitis G: viroprevalence and seroconversion in a high-risk group of children.

Hepatitis G virus (HGV), a recently discovered flavivirus, is parenterally transmitted and significantly associated with hepatitis C viraemia. Data on the viroprevalence of this agent in children is scant and its seroprevalence is unknown. The aim of this study was to determine the viroprevalence and seroprevalence of HGV in paediatric patients at risk of parenterally transmitted virus infection. Sera from 35 patients, previously tested for hepatitis C virus (HCV) infection, were analysed for the presence of HGV RNA by reverse transcription-polymerase chain reaction (RT-PCR) and for antibody to the E2 envelope protein (anti-E2) of HGV using the HGV-env kit. The mean age of the patients was 9.4 years (range 1-17 years), and risk factors included multiple transfusions and maternal HCV infection. Co-infection with HCV and HGV was a relatively common occurrence (31%). The prevalence of anti-E2, a marker of recovery from infection, was low (5%) when compared with overall viroprevalence (20%). This study highlights the significant association of HGV with HCV in children. The novel finding of a low ratio of anti-E2:HGV RNA contrasts with the pattern seen in adults and may reflect a higher risk of long-term carriage with acquisition of HGV infection at an early age.

Adolescent↗
PubMed1999

Treatment of chronic hepatitis B virus infection: an Asia-Pacific perspective.

Chronic hepatitis B infection is a serious health threat in the Asia-Pacific area. A consensus meeting on the treatment of chronic hepatitis B infection was conducted in Hong Kong, in August 1997. It was generally agreed that treatment of chronic hepatitis B infection should be based on the understanding of the natural history of chronic hepatitis B infection. To date, interferon alpha is the only Food and Drug Administration (FDA)-approved form of therapy for chronic hepatitis B infection. The overall response in Asian patients is unsatisfactory: approximately 15-20% will clear hepatitis B e antigen, but less than 5% will clear hepatitis B surface antigen. Newer immunomodulatory therapies are under trial. In contrast, nucleoside analogues, such as lamivudine (pending FDA approval) and famciclovir, have been shown to be potent suppressors of hepatitis B viral replication; however, their role as monotherapy in the treatment of chronic hepatitis B infection remains to be defined. Also, the issues of resistance to nucleoside analogues and withdrawal rebound need to be carefully studied. The future direction of therapy in chronic hepatitis B infection is probably a combination of nucleoside analogues or nucleoside analogues with immunomodulatory therapy.

2-Aminopurine↗
PubMed1999

Only the non-glycosylated fraction of hepatitis E virus capsid (open reading frame 2) protein is stable in mammalian cells.

Hepatitis E virus (HEV) is a non-enveloped, positive-strand RNA virus, with the genome encoding three open reading frames (ORFs) of which ORF 2 directs translation of the capsid protein, PORF2. Following pulse-labelling and cell fractionation of PORF2 expressed in mammalian cells using the Semliki Forest virus replicon, the capsid protein was detected as three major species of 78 (PORF2), 82 and 86 kDa, with P82 and P86 being N-glycosylated (gPORF2 and ggPORF2, respectively). Although gPORF2 and ggPORF2 species represented 79% of total PORF2 after 20 min metabolic labelling and were largely membrane-associated, the glycosylated PORF2 species were much less stable than non-glycosylated PORF2, which was present in the cytosol and represented the major product accumulated in the cell. In the absence of detectable surface expression or export of PORF2, this suggests that glycosylated ORF 2 proteins may not be intermediates in HEV capsid assembly.

Animals↗
PubMed1998

Role of alcohol in the progression of liver disease caused by hepatitis C virus infection.

In patients with chronic hepatitis C, alcohol consumption has been proposed as a risk factor for the progression of liver disease; however, evidence for this remains conflicting. Two hundred thirty-four anti-hepatitis C virus (HCV)-positive patients who had a liver biopsy performed within the past 24 months were studied. Demographic data and information on risk factors were recorded. A detailed lifetime alcohol consumption history was obtained. Viral studies included HCV viral titer and HCV genotype. Mean age (+/- SEM) of the group was 40.8 +/- 0.7 years. One hundred sixty-six (71%) were male. A risk factor for HCV infection was found in 195 patients (86%). Genotype distribution was: 1b: 22%; 1a: 15%; 1(nonsubtypable): 15%; 3a: 34%; and 2: 7%. Fifty (21%) patients had cirrhosis. Patients with cirrhosis were older (51.6 +/- 1.8 years) than those with chronic hepatitis (37.6 +/- 0.6 years; P = .0001), were infected at an older age (25.9 +/- 2.0 vs. 20.9 +/- 0.6 years; P = .001), and had a longer duration of infection (20.5 +/- 1.3 vs. 16.2 +/- 0.5 years; P = .0008). Patients with cirrhosis had a greater total lifetime alcohol consumption (288,765 +/- 58,115 g) than those with chronic hepatitis (189,941 +/- 15,453 g; P = .018). Cirrhotic patients also had greater total alcohol consumption during the period of infection with HCV (240,962 +/- 63,756 g vs. 146,510 +/- 12,862 g; P = .02). On multivariate analysis, subject age and total alcohol consumption were independently associated with the presence of cirrhosis. Total lifetime alcohol consumption is a risk factor for the progression of liver disease caused by HCV.

Adult↗
PubMed1998

The sequential occurrence of viral mutations in a liver transplant recipient re-infected with hepatitis B: hepatitis B immune globulin escape, famciclovir non-response, followed by lamivudine resistance resulting in graft loss.

BACKGROUND/AIMS: The purpose of this study was to characterize the clinical, histological and virological events in an orthotopic liver transplant (OLT) recipient with recurrent hepatitis B infection who was initially managed with hepatitis B immune globulin (HBIg) and when viral recurrence occurred, with nucleoside analogue salvage therapy. The aims were to document the mutations occurring in the hepatitis B virus (HBV) polymerase gene as a consequence of HBIg escape, famciclovir non-response and subsequent lamivudine resistance. METHODS: Throughout the follow-up of 796 days, the patient was seen at least at 4-week intervals. Clinical, biochemical and virological data were registered according to protocol. HBV DNA was quantified throughout the treatment period. The viral polymerase gene was sequenced from serum samples collected at representative time intervals. Consecutive liver biopsies were scored according to the modified Knodell classification. RESULTS: Clinically, the patient was in excellent condition until the development of acute hepatitis during the lamivudine therapy period, 765 days post-OLT. Until this terminal event, serum transaminase activity was only 1-2 times the upper limit of normal with serum bilirubin and prothrombin time within the normal range. Subsequent liver biopsies showed chronic active hepatitis with no signs of fibrosis. The post-mortem biopsy showed severe acute hepatitis B with massive necrosis. The HBV polymerase gene was sequenced during HBIg, famciclovir and lamivudine treatment. One mutation I533L was detected during HBIg treatment. No amino acid changes were selected during famciclovir treatment. Three amino acid changes were selected while the patient was on lamivudine treatment, which include L533I, S559T and M550I. CONCLUSIONS: We have documented HBV recurrence in a liver transplant recipient with the emergence of a multidrug resistant HBV which caused graft loss. The primary resistance to famciclovir in spite of therapeutic penciclovir levels may be as a result of a combination of the mutations found in the polymerase region. After 300 days of lamivudine treatment, a drug-resistant population emerged which was associated with a greater than three log increase in HBV DNA and contributed to loss of graft function. This is the first report of such an adverse clinical outcome due to the emergence of a mutant virus as a consequence of immunoprophylactic and antiviral therapy in a liver transplant recipient.

2-Aminopurine↗
PubMed1998

Inhibition of duck hepatitis B virus replication by 9-(2-phosphonylmethoxyethyl)adenine, an acyclic phosphonate nucleoside analogue.

The use of regimens that use nucleoside analogues for the treatment of chronic hepatitis B virus infection is often limited because of their high relapse rates. This is thought to be due to the persistence of virus in nonhepatocyte reservoirs and/or the viral covalently closed circular (CCC) DNA species in the nucleus of infected hepatocytes. We have evaluated the novel nucleoside analogue 9-(2-phosphonylmethoxyethyl)adenine (PMEA) in the duck model of hepatitis B. Eight Pekin-Aylesbury ducks congenitally infected with the duck hepatitis B virus (DHBV) were treated with PMEA at a dosage of 15 mg/kg of body weight/day via the intraperitoneal route for 4 weeks. At the end of the treatment period, four animals were killed and the remainder were monitored for a further 4-week drug-free period before analysis. The results were compared with those for eight age-matched, untreated controls. The levels of viremia, the total intrahepatic DHBV load, and CCC DNA, viral RNA, and protein levels were measured by Southern hybridization, Northern hybridization, and immunoblotting of the appropriate specimen, respectively. Viral proteins and DNA were also measured by immunohistochemistry (IHC) and in situ hybridization (ISH) of sections of liver and pancreatic tissue. PMEA treatment reduced the viremia to undetectable levels, while the total viral DNA load in the liver was reduced by 95% compared to the control level. Viral RNA and protein levels decreased by approximately 30%. ISH and IHC confirmed the PMEA-related intrahepatic changes and established that the amount of virus in bile duct epithelial cells (BDEC) was reduced by 70% during therapy. During the follow-up period all parameters of active virological replication returned to those for the age-matched controls. PMEA had no significant effect upon the number of virus-infected islet or acinar cells in the pancreas. PMEA at a dosage of 15 mg/kg/day has potent activity against DHBV found within hepatocytes and BDEC and inhibits DHBV replication in BDEC.

Adenine↗
PubMed1997

Amino-terminal epitopes are exposed when full-length open reading frame 2 of hepatitis E virus is expressed in Escherichia coli, but carboxy-terminal epitopes are masked.

We constructed a panel of overlapping and non-overlapping fragments of cDNA derived from open reading frame 2 (ORF2) of hepatitis E virus (HEV) and fused to the gene encoding glutathione S-transferase (GST), from which proteins were expressed in Escherichia coli. IgG-specific immunoreactivity against each protein was measured by Western immunoblotting using sera from experimentally infected Rhesus macaques (Macaca mulatta) or from HEV-infected patients. Under these conditions, full-length ORF2 protein (GST-ORF2) was strongly reactive with acute-phase sera from either macaques or patients, but was poorly reactive with convalescent sera. Recombinant protein GST-ORF2.3, representing amino acids 1-110 of the 660 encoded by ORF2, demonstrated a pattern of reactivity largely indistinguishable from the full-length protein. Conversely, GST-ORF2.1, representing amino acids 394-660 of the ORF2 protein was strongly reactive with both acute- and convalescent-phase sera. Extension of GST-ORF2.1 towards the N-terminus led to a progressive loss of convalescent-phase reactivity, apparent with as few as 20 additional HEV-specific amino acids. Deletion of 40 or more amino acids from the N-terminus of ORF2.1 also led to reduced convalescent-phase reactivity, however a protein representing this "reactive" region, containing amino acids 394-473, was poorly reactive, suggesting that the convalescent-reactive epitopes are conformational. Expression of full-length ORF2 protein in E. coli therefore masks the convalescent-reactive epitopes within the C-terminal part of the protein, without affecting N-terminal, acute-reactive epitopes.

Animals↗
PubMed1997

Transmission of human and feline immunodeficiency viruses via reused suture material.

Several documented cases of human immunodeficiency virus (HIV) infection have involved unconventional or unknown modes of transmission of the virus. Some such cases have occurred within a surgical setting. We investigated the potential for transmission of HIV on suture material that had been reused following passage through an HIV-infected patient. Initial experiments were conducted in vitro using HIV. To provide stronger evidence that HIV could be transmitted via this route, further experiments were undertaken in vivo using a feline immunodeficiency virus (FIV)/cat model. Both methods indicated the possibility of transmission of virus if suture materials were reused.

Animals↗
PubMed1997

Demonstration of duck hepatitis B virus in bile duct epithelial cells: implications for pathogenesis and persistent infection.

Hepatitis B virus (HBV) has been demonstrated in bile duct epithelial cells (BDEC) during chronic infection. The persistence of virus in BDEC may play an important role in disease pathogenesis, and may be at least partly responsible for the relapse phenomenon observed in antiviral treatments using nucleoside analogues. The aims of this study were to examine the morphological changes within the liver in the duck hepatitis B model following bile duct ligation (BDL), and to assess the effect of biliary hyperplasia upon viral DNA and proteins. Seven-day-old ducklings, congenitally infected with the duck hepatitis B virus (DHBV), were subject to BDL. The pathological and virological changes were then followed at 5, 10, 15, and 20 days after ligation. All results were compared with age-matched unligated control birds congenitally infected with DHBV. To assess the early morphological changes, additional animals were sacrificed at 1, 2, 3, and 4 days post-BDL. The proportion of DHBV-infected BDEC, was examined by immunohistochemistry and in situ hybridization. BDL induced rapid biliary hyperplasia, with a doubling time for BDEC of 1.3 days. The proliferated BDEC displayed immunohistochemical features identical to resting BDEC. More than 50% of BDEC in unligated controls, and more than 46% of proliferated BDEC in ligated animals were positive for DHBV DNA and structural proteins. The intensity of immunohistochemical staining and in situ hybridization signal in the BDEC was consistently greater than that of the hepatocytes, both before and after BDL. BDL induces biliary hyperplasia in the duck model, and BDEC division does not reduce the viral burden in infected cells.

Animals↗
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↗