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.

Exposure to hepatitis B and C of tattooists in Victoria in 1984.

Although tattooing is recognized as a risk factor for transmission of hepatitis C, the efficiency with which transmission occurs is unknown. Sera stored from a serosurvey of tattooists undertaken in 1984 to test for human immunodeficiency virus (HIV) provided the opportunity to determine the prevalence of serological markers of hepatitis B virus (HBV) and hepatitis C virus (HCV) in tattooists at that time. The stored sera had been obtained from five unregistered and 36 of 37 (97%) of the registered tattooists operating in 1984. Serological status for hepatitis B (hepatitis B surface antigen (HBsAg), antibody to hepatitis B surface antigen (HBsAb) and antibody to hepatitis B core antigen (HBcAb) in standard assays) or hepatitis C (HCV antibody reactivity in second and third generation tests, confirmed by recombinant immunoblot assay) was determined. No sera was HIV positive or HBsAg positive. Of 35 specimens tested for HCV specific antibody, only two (5.6%) were positive despite markers of HBV in 48.6% of the same sera. As acute HBV infection was common amongst tattooists prior to 1984, it is clear that hepatitis B vaccination would be of benefit to this group. Despite frequent needlestick injuries reported by tattooists at the time, the low seroprevalence of HCV in this group suggests that HCV may not be transmitted efficiently by intradermal inoculation using solid-bore tattooing needles.

Blood-borne virus infections among Australian injecting drug users: implications for spread of HIV.

To describe the epidemiology of infection with hepatitis C virus (HCV), hepatitis B virus (HBV) and human immunodeficiency virus (HIV) among injecting drug users (IDUs) in Australia, in relation to the potential for further spread of HIV in IDUs, a cross-sectional analysis was performed on data from a sample of injecting drug users, correlating markers of exposure to blood-borne viruses with sex, age, sexual orientation, primary current drug injected and duration of injecting in rural and metropolitan Victoria, Australia. The subjects were currently active IDUs from a wide spectrum of age, sex, sexual orientation, geographical location and social background, contacted and recruited through their social networks and from community agencies and prisons by trained peer workers who interviewed and collected blood from them in the field. Sera were tested for antibody to HIV, HCV and hepatitis B core antigen (HBcAg), for hepatitis B surface antigen (HBsAg), and for HCV RNA using reverse transcription and polymerase chain reaction (RT-PCR). At entry to the study, 4.5% (14/311) had antibody to HIV, 47% (146/308) to HBcAg and 68% (206/303) to HCV. Prevalence of HBsAg was 1.8% overall (5/282), and 50% (84/168) were positive for HCV RNA. By multivariate analysis, HIV seropositivity was strongly associated with a history of homosexual contact in males and with exposure to HBV but not to HCV. Those who reported their current primary injected drug to be amphetamines were at greater and continuing risk of HIV infection than were current heroin injectors, while the reverse applied for HCV.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatitis C virus infection among a cohort of Victorian injecting drug users.

OBJECTIVE: To describe the epidemiology of infection with hepatitis C virus (HCV) among injecting drug users (IDUs) in Victoria. DESIGN AND SUBJECTS: Subjects were current IDUs from a wide spectrum of age, sex and social background, enrolled in a prospective study of injecting drug use. They were contacted by peer workers through their social networks and through community agencies and prisons, and were regularly followed for interview and blood collection in the field. Sera were tested for presence of antibody to HCV (anti-HCV), for the presence of HCV RNA directly in serum, and for measures of liver function. The results were correlated with demographic variables. SETTING: Rural and metropolitan Victoria. MAIN OUTCOME MEASURES: Presence of anti-HCV and demonstration of HCV RNA. RESULTS: Two-thirds (68%, 206/303) of the current cohort of IDUs were seropositive for HCV, risk being particularly associated with duration of injecting, and independently for men with opiate use and prison history, and for women with a history of methadone therapy. HCV RNA was detected in 48% (76/160) by polymerase chain reaction (PCR); 61% (74/122) of these subjects were HCV seropositive and 5% (2/38) seronegative. Of 32 HCV seronegative subjects followed for a mean period of 291 days, five seroconverted to HCV, an incidence of 20 infections per 100 person-years. Those who seroconverted were older, more likely to be male, had been injecting longer, more often reported opiate use, and were more likely to be based in the country. Serum liver enzyme levels were higher and more likely to be abnormal in HCV seropositive than seronegative subjects, and were highest in those seropositive subjects in whom HCV RNA was detected. CONCLUSIONS: This population of IDUs has a very high rate of exposure to HCV, related to duration of injecting and independently to opiate use and prison history, perhaps reflecting increased risk in particular social networks. There is evidence of high rates of carriage of HCV, of continuing transmission of HCV, and of ongoing liver disease among these IDUs. If these IDUs are at all representative of all IDUs in Australia, we estimate that 80,000 current and former IDUs may be at risk of chronic liver disease from HCV, and that 8000-10,000 new infections may be occurring each year. Two subjects who were seronegative had HCV RNA detectable in sera. These data have important implications for screening programs and document the need for further measures to prevent spread of blood-borne viruses including HIV among IDUs.

Presence of antibodies to human immunodeficiency virus-1 in reference reagents of human origin in diagnostic kits.

We tested 54 reagents of human origin that were included in a number of diagnostic kits to be used as positive or negative control material, and for quality assurance, for the presence of antibodies to human immunodeficiency virus-1 (HIV-1). Of these, 12 (22%) reagents were found to give positive results for antibodies to HIV-1 by enzyme-linked immunosorbent assay and/or supplementary tests. These results suggest that some diagnostic reagents of human origin may be contaminated with HIV-1 and potentially may be infectious.

Immunochemical analyses of Australian bluetongue virus serotypes using monoclonal antibodies.

In order to characterize the immunochemical role of bluetongue virus (BTV)-specified proteins and provide reagents capable of defining the serological relatedness of bluetongue (BT) serotypes and their relationship with other orbiviruses, a panel of 16 IgG monoclonal antibodies was raised to the Australian BTV serotypes, isolate CSIRO156 (BTV 1), CSIRO19 (BTV20) and CSIRO154 (BTV21). Analyses of virus-coded polypeptide specificities of these monoclonals using enzyme-linked immunosorbent assay (ELISA), a radioimmunoprecipitation assay (RIPA), and a virus neutralization assay, revealed the outer coat viral protein P2 to have a major role in the neutralization of both CSIRO156 and CSIRO19. Presumptive evidence for the involvement of the P3 protein in the neutralization of CSIRO19 was also obtained. The virus-specified non-structural protein P6A induced a group reactive immune response to all 3 serotypes. Antigenic relationships between P3 of CSIRO156 and P2 of CSIRO19 were found, and an analysis of the relationships between epitopic regions on P2 and P3 of both viruses revealed several distinct immunogenic sites exist on the P2 protein.

The major surface glycoprotein of simian rotavirus (SA11) contains distinct epitopes.

The polypeptide specificities on monoclonal antibodies previously derived against the SA11 simian, NIC bovine, and Wa human strains of rotavirus were determined by radioimmunoprecipitation of infected cell lysates. All the monoclonal antibodies derived using NIC and Wa were found to be directed against the major component of the inner capsid, while most of the SA11 monoclones were directed against the major outer capsid glycoprotein. When several SA11 glycoprotein-specific monoclonal antibodies were used in competitive binding studies, four distinct epitopes, which correlated with the functional activities of the antibodies, were defined. One epitope appeared most critical for virus neutralization, another was involved to a lesser extent, and the remaining two epitopes seemed to have no role. A possible topographical arrangement of these epitopes is suggested.

Anti-oxazolone hybridomas and strain distribution of the oxazolone idiotype.

Antibodies produced in the primary response to the hapten 2-phenyloxazolone (OX) express a cross-reactive idiotype in BALB/c and DBA/2 mice. We studied the response in hyperimmunized mice, using ascites produced after multiple immunizations with an OX conjugate and by generating monoclonal antibodies. A competitive radioimmunoassay was developed using a rabbit anti-idiotype antiserum raised against purified hyperimmune anti-OX antibodies. Mice from strains including CBA, C3H/He and B10.BR expressed all the determinants found in BALB/c serum, although at a lower titre. C57BL/6 mice, however, only expressed some of the BALB/c determinants. We isolated a monoclonal antibody, 1F9, which expressed some of the determinants found in BALB/c serum. All the cross-reactive idiotopes expressed on 1F9 were also expressed in the above strains including C57BL/6. Almost every BALB/c anti-OX antibody also expressed the 1F9 determinants. There are thus determinants of the BALB/c OX idiotype which are expressed in a number of different strains previously thought to be negative for the idiotype.

Derivation of neutralizing monoclonal antibodies against rotavirus.

Monoclonal antibodies were derived against the SA11 simian, NIC bovine, and Wa human rotavirus strains and characterized by enzyme-linked immunosorbent assay, plaque neutralization, and hemagglutination inhibition. Several strain SA11-specific antibodies were found to have neutralizing and hemagglutination-inhibiting capacity.

Characterization of the Hallé SSPE measles virus isolate.

The Hallé subacute sclerosing panencephalitis (SSPE) measles virus isolate and its plaque-purified progeny were investigated to determine whether any unusual properties could be associated with its ability to cause persistent infection. Three types of plaque-purified progeny were isolated. One population appeared to be similar in biological and biochemical properties to laboratory-adapted measles virus and had the ability to induce syncytia (syn+). A second population (syn-) plaqued more efficiently at 39 degrees C than at 33 degrees C, did not cause normal cell fusion at either temperature, and produced particles that interfered with the replication of other measles virus isolates in vivo and in vitro. This syn- virus was further plaque-purified to eliminate the interfering particles, producing the syn- P2 virus. This virus also plaqued more efficiently at 39 degrees C than at 33 degrees C, but caused cell fusion only at 39 degrees C. Both syn- viruses and the parental virus were significantly less virulent in vivo than the syn+ virus and caused a more prolonged infection. Biochemical analysis showed that the syn- P2 population produced particles that banded at two different densities in potassium tartrate gradients; both densities were less than those of the standard laboratory measles virus and the syn+ virus. Although the syn- P2 virus did not cause cell fusion at 33 degrees C, [35S]methionine labelling demonstrated that the haemolysin/cell fusion protein was present in syn- P2 virions. The production of interfering particles, the inability to cause cell fusion at 33 degrees C, and the cold-sensitive nature of the syn- population appear to play a role in the ability of the Hallé SSPE virus to establish persistent infection.

SSPE-BIKEN: a naturally arising hemagglutination-defective mutant of measles virus.

Biochemical and genetic methods have been used to investigate a defective variant of measles virus previously isolated from a patient with subacute sclerosing panencephalitis (SSPE). Since its isolation, this syncytiogenic strain (SSPE-BIKEN) has remained cell-associated; infected cells do not hemadsorb and do not release infectious virus. Immune precipitation and polyacrylamide gel electrophoresis were used to study the synthesis of measles virion proteins in SSPE-BIKEN-infected cells. All of the virion proteins were detected in immune precipitated whole cell extracts. However, the hemagglutinin (HA) protein was not detected on the cell surface by lactoperoxidase iodination. These results suggest that the failure of the HA protein to insert into the cell membrane accounts for the block in the release of infections virus. Radioactively labeled, noninfectious, virus-like particles have been purified from the media of SSPE-BIKEN-infected cells. These particles contain virus nucleocapsid, nucleocapsid-associated, and membrane proteins, but very little HA and hemolysin proteins. Genetic complementation between SSPE-BIKEN and a temperature-sensitive mutant of measles virus was observed and suggests that the SSPE isolate defect is due to a mutation. Additional evidence of a mutation is provided by the detection of low frequency revertant progeny in SSPE-BIKEN stocks. Our results support the hypothesis that genetic variants of measles virus are involved in the etiology of SSPE.

Measles virus meningoencephalitis. Immunofluorescence study of brains infected with virus mutants.

The localization of measles virus antigens in hamster brains inoculated with a temperature-sensitive mutant of measles virus r its revertant was studied by immunofluorescence. We found significantly less virus antigen in mutant-inoculated than in revertant-inoculated brains. In mutant-inoculated brains the virus antigens were very often found in the meninges. Possible mechanisms for the development of experimental hydrocephalus and meningoencephalitis are discussed.

Complementation analysis of measles virus temperature-sensitive mutants.

Two sets of independently isolated measles virus temperature-sensitive mutants were quantitatively tested for complementation. Analysis of the nine possible combinations of representative mutants indicated that only one pair of mutants is noncomplementing. Thus, the measles virus mutants studied to date define five complementation groups.

Neurovirulence and induction of hydrocephalus with parental, mutant, and revertant strains of measles virus.

The relationship between neurovirulence and induction of hydrocephalus was investigated for a measles virus temperature-sensitive mutant and its revertant. The revertant regained the neurovirulence of the parental strain. At appropriate doses the parental, mutant, and revertant strains induced hydrocephalus.

Genetic evidence for an additional function of phage T4 gene 32 protein: interaction with ligase.

Gene 32 of bacteriophage T4 is essential for DNA replication, recombination, and repair. In an attempt to clarify the role of the corresponding gene product, we have looked for mutations that specifically inactivate one but not all of its functions and for compensating suppressor mutations in other genes. Here we describe a gene 32 ts mutant that does not produce progeny, but in contrast to an am mutant investigated by others, is capable of some primary and secondary DNA replication and of forming "joint" recombinational intermediates after infection of Escherichia coli B at the restrictive temperature. However, parental and progeny DNA strands are not ligated to covalently linked "recombinant" molecules, and single strands of vegetative DNA do not exceed unit length. Progeny production as well as capacity for covalent linkage in this gene 32 ts mutant are partially restored by additional rII mutations. Suppression by rII depends on functioning host ligase [EC 6.5.1.2; poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming, NMN-forming)]. This gene 32 ts mutation (unlike some others) in turn suppresses the characteristic plaque morphology of rII mutants. We conclude that gene 32 protein, in addition to its role in DNA replication and in the formation of "joint" recombinational intermediates, interacts with T4 ligase [EC 6.5.1.1; poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming)] when recombining DNA strands are covalently linked. The protein of the mutant that we describe here is mainly defective in this interaction, thus inactivating T4 ligase in recombination. Suppressing rII mutations facilitate substitution of host ligase. There is suggestive evidence that these interactions occur at the membrane.