Oxidation of 2-methoxynaphthalene by toluene, naphthalene and biphenyl dioxygenases:structure and absolute stereochemistry of metabolites.

2-Methoxynaphthalene was subjected to biooxidation by whole cells of six organisms: Pseudomonas putida F39/D containing toluene dioxygenase, Escherichia coli JM109(pDTG601), containing recombinant toluene dioxygenase from Pp F39/D, Pseudomonas sp. NCIB 9816/11, containing naphthalene dioxygenase. E. coli JM109(pDTG141), containing recombinant naphthalene dioxygenase from NCIB 98161/11, E. coli C534(ProR/Sac) containing recombinant naphthalene dioxygenase from Pp G7, and Beijerinckia sp. B8/36, containing biphenyl dioxygenase. The major product of oxidation by the naphthalene and biphenyl dioxygenases has been isolated and identified as (1R,2S)-dihydroxy-7-methoxy-1,2-dihydronaphthalene, 2c. A minor product, (1R,2S)-dihydroxy-6-methoxy-1,2-dihydronaphthalene, 3c, has also been detected. Oxidation by the toluene dioxygenase-containing organisms led to the isolation of 3c as the major product. Minor products detected in these reactions were 2c, and a third compound, (1S,2S)-dihydroxy-3-methoxy-1,2-dihydronaphthalene, 4c. Structural studies and dehydration of the diols to a mixture of naphthols are described. The absolute stereochemistry of these new diols has been established by correlation with known compounds. The organisms' potential in the production of new metabolites as useful chiral synthons by biooxidation of 2-substituted naphthalenes is indicated.

Unlinked anonymous screening of antenatal patients for antibody to human immunodeficiency virus type 1 (HIV-1).

OBJECTIVES: To determine the prevalence of HIV-1 infection in the general community by using a target population (antenatal patients) as an indicator of infection, to monitor any change in the prevalence of HIV infection in this population and provide baseline information on heterosexual spread of HIV infection into this low prevalence population. PATIENTS AND DESIGN: Between January 1989 and January 1992, 4537 unlinked anonymous antenatal sera were tested in two study groups at Westmead Hospital. Repeatedly reactive sera were confirmed by western blot and other supplementary assays as appropriate. RESULTS: No significant change in the seroprevalence of HIV-1 infection was detected between the two study periods. Of the 2208 sera tested in 1989-1990, one (0.05%) was confirmed as positive and one (0.05%) gave a non-specific reaction by enzyme immunoassay (EIA). Of the 2329 sera tested in 1991-1992, there was one (0.04%) HIV-1 antibody positive serum, and two gave non-specific EIA reactions. These results were compared with three other sample populations tested at Westmead Hospital during the same period: linked antenatal patients, antenatal methadone clinic attendees and all women tested. CONCLUSIONS: Periodic anonymous testing of a sample antenatal population (combined with screening of high risk patients) is useful for monitoring the prevalence of HIV infection in these populations and estimating any future need for generalised screening.

Patients infected with human immunodeficiency virus type 1 have low levels of virus in saliva even in the presence of periodontal disease.

In two consecutive studies, 80 subjects human immunodeficiency virus (HIV)-1-seropositive (21 asymptomatic, 6 persistent generalized lymphadenopathy, 13 AIDS-related complex, and 40 AIDS) were examined for oral lesions. Paired serum and saliva specimens were tested for HIV isolation, DNA, and antigen. HIV antigen was detected in sera from 31 patients, but not in saliva. HIV was isolated from blood mononuclear cells of 83% and saliva supernatants of 21%. In the second study of 25 patients, HIV was detected in plasma of 56% (titers, 1/10 to > 1/1000) but not in diluted saliva supernatants, even in those with severe periodontal disease. HIV DNA was detected using polymerase chain reaction in 2 of 7 saliva cell pellets and 4 of 5 blood samples. Hence, infectious HIV and DNA was found at very low concentrations in 21% and 28% of HIV-seropositive patients, respectively, at all stages of HIV infection.

Identification of infection of an Australian resident with the human immunodeficiency virus type 2 (HIV-2).

OBJECTIVE: To present the first confirmed case of human immunodeficiency virus infection type 2 (HIV-2) in an Australian resident. CLINICAL FEATURES: HIV-2 infection in a west African man resident in Sydney was diagnosed in 1992 at Westmead Hospital, Sydney, by serological testing. He was asymptomatic and the blood CD4 T-lymphocyte concentration was not significantly reduced. Infection was probably acquired before migration to Australia. The patient was initially tested for HIV-1 antibody as part of an application for permanent residency. He was in no obvious risk group or transmission category. His serum was repeatedly positive by Genetic Systems enzyme immunoassay (EIA) and borderline by Abbott EIA, was reactive to the HIV-2 peptide on a synthetic envelope peptide assay, and was strongly reactive to all HIV-2 specific viral protein bands on an HIV-2 western blot test. HIV-2 was isolated by co-cultivation of the patient's peripheral blood mononuclear cells and identified by hybridisation using HIV-2 specific oligonucleotide probes, with further confirmation by polymerase chain reaction. INTERVENTION AND OUTCOME: The patient was counselled regarding the clinical course and prognosis of HIV-2 infection, the possible indications for zidovudine therapy, modes of transmission of the virus and safer sex precautions. CONCLUSIONS: This is the first documented case of HIV-2 infection diagnosed in Australia and raises the possibility of other undetected cases. The cost effectiveness of general testing for HIV-2 needs to be assessed and formal epidemiological sentinel programs should be established to monitor specific Australian populations.

HIV-1 western blot: development and assessment of testing to resolve indeterminate reactivity.

OBJECTIVE: To reduce the number of HIV-1 Western blot (WB)-indeterminates requiring follow-up and the time taken to provide a clear positive or negative result. DESIGN: In the first of two stages, a testing and follow-up strategy was developed to resolve anti-HIV-1 status of WB-indeterminates. In the second stage, implementation of this strategy was assessed. METHODS: After dividing indeterminates into four groups according to WB profile, samples were tested for anti-HIV-1, anti-HIV-2, anti-HTLV-I antibodies, and HIV-1 antigen using the most sensitive assays available. When testing failed to clarify anti-HIV-1 status, follow-up samples were taken to monitor changes in antibody status. RESULTS: Samples in two out of the four indeterminate groups were negative for anti-HIV-1. The other two groups required additional testing and/or follow-up to distinguish reactivity caused by anti-HIV-1 from cross-reactivity. CONCLUSION: Grouping HIV-1 WB-indeterminates according to profile allows a significant percentage to be reported as anti-HIV-1-negative, while additional testing may allow others to be reported as anti-HIV-1-positive. The remainder require a maximum of 3 months' follow-up to resolve anti-HIV-1 status.

HIV-1 antibody testing strategy: evaluation of ELISA screening and western blot profiles in a mixed low risk/high risk patient population.

The Westmead HIV-1 antibody testing strategy showed that, regardless of ELISA screening kit manufacturer, sera which were repeatedly positive by two ELISA screening assays (one indirect and the other competitive format) had a 97-98% chance of being confirmed positive by Western blot for HIV-1 antibody or a less than 3% chance of either being identified as a seroconverter (1%) or a late stage AIDS patient (1.2%). Sera which were discordant by two ELISA screening assays had a less than 4% chance of either being confirmed positive by Western blot (2.5%) or identified as a seroconverter (1.3%). The incidence of non-specific indeterminate Western blot profiles were shown to be inversely proportional to the specificity of the ELISA screening kits used. The use of a recombinant envelope ELISA was able to confirm the viral specificity of HIV-1 envelope bands (gp160, 120 or 41) on Western blot. Guidelines suggested by the Australian National HIV Reference Laboratory, Fairfield Hospital, Melbourne, which categorized indeterminate or typical Western blot profiles into four reaction groups were found to be useful for the interpretation of Western blot patterns. A Western blot profile which is reactive for HIV-1 viral glycoproteins (gp160, 120 and 41) alone or in combination with not more than two other viral proteins (Indeterminate Group 4) and which is confirmed viral envelope specific by a recombinant envelope ELISA can be used as a predictor of seroconversion.

Fusion mutants of the influenza virus hemagglutinin glycoprotein.

The influenza virus hemagglutinin (HA) mediates viral entry into cells by a low pH induced membrane-fusion event in endosomal vesicles. Mutant viruses with altered pH dependence for both hemolysis and the HA conformational change required for fusion were selected for their ability to grow in cells treated with amantadine hydrochloride, which raises the endosomal pH. The amino acid sequence and three-dimensional location of 19 substitutions on the HA are reported. The mutations fall into two groups, one that results in the destabilization of the pH 7.0 location of the hydrophobic N-terminal HA2 peptide, and a second that results in the alteration of intersubunit contacts, suggesting a large distortion or disruption of these contacts in the "fusion-active" conformation.

A genetic and monoclonal analysis of high-yielding reassortants of influenza A virus used for human vaccines.

This paper describes two methods of analysis using monoclonal antibodies and RNA hybridization to characterize variation in the haemagglutinins of seven high-yielding influenza virus reassortants used for inactivated vaccine production. The results show that variants' were selected in producing these genetic reassortants. The haemagglutinins of two reassortants showed both antigenic and structural differences from their wild-type (wt) parents as detected by the two methods of analysis. These variants were more closely related to other subtype strains which had previously been differentiated from the wt parent by post-infection ferret sera and which also had amino acid sequence differences in antigenically significant sites on the HA 1 polypeptide chain of the haemagglutinin molecule. The haemagglutinins of four of the seven reassortants showed antigenic differences but no apparent structural differences from their wt parents. The haemagglutinin of only on reassortant was antigenically and structurally identical to its wt parent. The variants could not be reliably distinguished with hyperimmune rabbit serum or immune ferret serum to the wt parent virus. It is therefore important to use more discriminatory tests to identify influenza strains correctly. It is also essential for vaccine strains to be as completely characterized as possible. It is considered desirable that both methods of analysis be used to characterize influenza virus reassortant strains.

Host antigen as the sulphated moiety of influenza virus haemagglutinin.

Inorganic sulphate (35S) was incorporated into the haemagglutinin molecule of A/Memphis/1/71 (H3N2) influenza virus when a keratosulphate-like host antigen was also incorporated into the glycoproteins of virus grown in the chorioallantoic membrane of the embryonated hen's egg. Little or no 35S-sulphate was incorporated when this hose antigen was not present in the glycoproteins of virus grown in chick embryo kidney cells or in the chorioallantoic membrane of embryonated duck eggs. The presence of the keratosulphate-like host antigen was required for the stability of the haemagglutinin molecule in sodium dodecyl sulphate (SDS). The haemagglutinin molecules from virus grown in hens' eggs were stable in SDS, whereas those from virus grown in duck eggs or in chick embryo kidney cells were not and could not be isolated on cellulose acetate. Chemical analysis showed that there were 87 glucosamine residues and three molecules of sulphate per haemagglutinin subunit as calculated for a trimer molecule having a mol. wt. of 200 000. There was one sulphate molecule per HA1 polypeptide chain and this was associated with the slowest migrating carbohydrate-protein complex of an HA1 tryptic digest separated by polyacrylamide gel electrophoresis.

The ecology of influenza. Isolation of type 'A' influenza viruses from Australian pelagic birds.

Three different type A influenza viruses have been isolated from pelagic birds nesting on islands of the Great Barrier Reef. One of these, isolated in 1972, was of subtype Hav6Nav5. The other two, which are described in this paper, were isolated in 1975 and belonged to subtypes Hav5Nav2 and Hav3Nav6. Of eight isolates of the latter virus, seven were recovered from cloacal swabs and only one from the trachea.

Antigenic relationships among influenza virua A neuraminidase (N2) antigens by immunodiffusion and postinfection neutralization tests.

The antigenic relationships among the neuraminidases of influenza A strains from 1957 to 1973 were examined by postinfection application of neuraminidase antisera. This procedure causes inhibition of virus spread and apparent neutralization. Neuraminidase (apparent) neutralization and neuraminidase inhibition tests with chicken antisera gave similar results. Neuraminidase inhibition tests were more discriminating than neuraminidase neutralization tests when rabbit and goat antisera were used. Antibody absorption studies revealed that the neuraminidase, like the hemagglutinin, may possess two kinds of antigenic determinants, which can give rise to "common," or "cross-reacting," and "specific" antibodies. "Specific" antibody appears to be more effective in the inhibition of enzyme activity than in the inhibition of virus spread.

Diversity of the antibody response to the different antigenic determinants on the hemagglutinin subunits of influenza viruses.

Sera from rabbits hyperimmunized with hemagglutinin (HA) subunits isolated from the A/Port Chalmers/73 (H3N2)strain of influenza virus showed great differences in their cross-reactions with different strains of influenza virus. In hemagglutination-inhibition tests, some sera reacted to about the same titer with A/Port Chalmers/73 and A/Hong Kong/68 viruses, suggesting that these two strains were very closely related. Other sera, which reacted to high titer with A/Port Chalmers/73 virus, had only a low titer with the Hong Kong/68 strain, suggesting that the two viruses were distantly related. Evidence suggested that these diverse cross-reactions were due to widely different ratios, in the different sera, of antibodies to the "common" and the "specific" antigenic determinants on the HA subunits. Thus, some rabbits gave a stronger response to the "common" determinants than to the "specific", whereas in others, the reverse seemed to be the case. Sera from human volunteers injected with A/Port Chalmers/73 inactivated or subunit influenza virus vaccines, or from people infected with Port Chalmers/73 virus, contained, in most cases, antibodies predominantly to the "common" antigenic determinants on the HA subunits. These sera reacted to higher titer with Hong Kong/68 virus than with the Port Chalmers/73 strain. Absorption of these sera with Hong Kong/68 virus totally removed all detectable antibody, suggesting that they contained no antibody to the "specific" determinants of Port Chalmers/73 HA. Paradoxically, absorption of the sera with Port Chalmers virus did not remove all antibodies, suggesting that the sera contained antibodies to the "specific" determinants on Hong Kong/68 HA.

Inhibition of virus release by antibodies to surface antigens of influenza viruses.

When influenza virus was mixed with antisera to its surface subunits before inoculation of cell cultures, anti-hemagglutinin antibodies neutralized infectivity but anti-neuraminidase did not. When the antisera were added after infection of cell cultures, anti-hemagglutinin and anti-neuraminidase antibodies were equally effective in reducing virus titers in culture fluids. Decreased virus titers were not due to interference of antibody with assay and were not accompanied by a reduction in the synthesis of hemagglutinin and neuraminidase subunits. Both antisera also effectively prevented in vitro virus spread. Inhibition of virus release by neuraminidase antibody appeared unrelated to its antienzyme property. Hydrolysis of N-acetyl neuraminic acid residues of infected host cells proceeded unimpaired in the presence of subunit antisera. Anti-hemagglutinin and anti-neuraminidase antibodies may act to prevent virus release by binding newly formed virus subunits to each other and to anti-genically altered cell membranes.

The sequential appearance of antibody and immunoglobins in nasal secretion after immunization of volunteers with live and inactivated influenza B virus vaccines.

The sequential development of the immune response in nasal washings was studied in 54 volunteers immunized with either attenuated or inactivated influenza B/Eng/13/65 virus vaccines.Eleven of the 15 volunteers given the inactivated vaccine by deep subcutaneous inoculation showed no rise in nasal wash protein or immunoglobins due to the immunization procedure nor was specific neutralizing antibody detected in their nasal washings after immunization. Neutralizing antibody was detected in nasal washings of three volunteers in this group who also showed a 20-fold or greater increase in serum haemagglutinin-inhibiting antibody after immunization and in one volunteer who had antibody present in pre-trial nasal washings.Eleven of 15 volunteers who were successfully infected by the live attenuated vaccine showed a characteristic rise in protein and IgA and IgG immunoglobin concentrations in nasal washings 5-14 days after the administration of the live virus vaccine. Neutralizing antibody was detected in the nasal washings of these 11 volunteers and appeared at the same time as or 1-2 days after the initial rise of protein and immunoglobin. Neutralizing antibody was also detected in the nasal washings of one other volunteer who did not show a rise in protein or immunoglobin concentration in nasal washings after immunization.IgA was detected (>/= 3 mg./100 ml.) in the majority (84%) of nasal wash specimens which had a protein concentration of 0.2 mg./ml. or greater while IgG was not detected (>/= 4.5 mg./100 ml.) until the protein concentration rose to 0.4 mg./ml. or greater. The geometric mean concentration for normal nasal wash protein in this study was 0.3 +/- 0.1 mg./ml.Regression analysis indicated that the concentrations of both IgA and IgG immunoglobins were directly proportional to the protein concentration in nasal washings but that this relationship varied considerably between individuals.Absorption studies indicated that neutralizing and haemagglutinin-inhibiting antibodies in nasal secretion to influenza B/Eng/13/65 virus were predominantly associated with the IgA class of immunoglobin.