Biophysical Properties of Australia Antigen

Biophysical studies with Australia complement-fixing (CF) antigen showed it to be a particle with a buoyant density of 1.20 g/cm(3) in CsCl, a sedimentation coefficient of 110, and an average diameter of 25 nm. The CF antigen was not inactivated by ether, 1% deoxycholate, 1% Tween 80 or overnight heating at 56 C. The antigen was unstable when treated with 1% sodium dodecyl sulfate. A procedure is described for the isolation and partial purification of Australia antigen from serum by using isopycnic banding and rate separation techniques. Treatment of the 1.20 g/cm(3) Australia antigen with 1% Tween 80 yielded a minor peak of CF activity with a buoyant density of 1.39 g/cm(3) in CsCl.     

Compound d+y+ particles of Australia antigen.

Australia antigen (HB8 Ag) particles vary in their antigenic structure. They are generally found to carry either determinant d or determinant y, but not both. This report describes seven sera which contain Australia antigen carrying both d and y on the same particle.     

New Specificity of Australia Antigen

THE immunodiffusion laboratory at the Institute for Cancer Research frequently acts as a reference laboratory to test anti-Australia antigen sera for our colleagues in many parts of the world. Because Australia antigen is known to possess different antigenic specificities^1–4, a panel was established which consisted of Australia antigen specimens selected from hepatitis and Down's syndrome patients and from clinically normal residents of the Lau area in Malaita, British Solomon Islands. Sera from normal blood donors without Australia antigen were included as negative controls. All antisera received after August 1971 were tested against this panel to detect heterogeneity among both the antibodies tested and the antigens included in the panel. Immunodiffusion was performed in a seven-hole Ouchterlony pattern with the antiserum in the centre well and a positive Australia antigen control serum from a Pennsylvania Down's syndrome patient in the top and bottom wells. The patterns were cut in a layer of 1.1% agarose in veronal buffer, pH 8.2, on glass lantern slides^6,7.     

Conformational Studies of Australia Antigen by Optical Rotatory Dispersion and Circular Dichroism

The optical rotatory dispersion and circular dichroism of intact, 8 m urea- or sodium dodecyl sulfate-treated, and carbamidomethylated Australia antigen indicated that the antigen possesses a high alpha-helical content similar to human high-density lipoproteins.     

α-Antitrypsin Deficiency and Neonatal Hepatitis

Five out of 28 infants investigated in a regional survey of neonatal hepatitis were found to have genetically-determined deficiency of α₁-antitrypsin (ZZ phenotype). The clinical course and pathological changes varied considerably. All five infants had an acute hepatitis-like illness, and although this subsided cirrhosis later developed in three cases. The remaining two infants had minimal abnormalities of the liver function tests at 12 and 18 months of age, and one had increased hepatic fibrosis. Australia antigen was found in the serum of three infants, and Australia antigen or antibody in one or both parents of these and of one further case whose serum was negative. It is suggested that the association of neonatal hepatitis with α₁-antitrypsin deficiency may be commoner than previously realized and that Australia antigen acts as a trigger factor in these cases.     

Adaptation to infectious disease. Australia antigen and hepatitis

Infectious diseases have probably acted as selective forces to generate polymorphisms in human populations. An example is given of a study related to this concept. There is evidence that “Australia antigen” is a hepatitis virus or closely associated with it. In some tropical populations there appears to be an inherited susceptibility to chronic infection with “Au(1) virus” controlled by a simple genetic mechanism. There are strikingly different frequencies of Australia antigen in different populations and these may have been determined by differences in the selective pressures in different environments.     

Changes induced by Hepatitis Serum in Cultured Liver Cells

THE discovery of the relationship between the Australia (hepatitis-associated) antigen and serum hepatitis has provided a specific serological marker of infection for type B viral hepatitis. The precise biological nature of the Australia antigen remains uncertain and so far there has been no convincing demonstration of the successful infection of tissue culture preparations in spite of extensive efforts¹. We now report the nuclear involvement of cultured primary human embryo liver cells inoculated with the serum of a well-documented former blood donor (P. J. G.) whose blood had been implicated in the transmission of serum hepatitis in transfused recipients and had induced serum hepatitis in human volunteers. Australia antigen has been repeatedly demonstrated in the serum of this blood donor^1,2.     

Total Screening of Blood Donations for Australia (Hepatitis Associated) Antigen and its Antibody

During a period of one year all of 105,724 blood donations were tested for Australia (Au) antigen and its antibody by rapid immunoelectro-osmophoresis—86 (1 in 1,229) were positive for antigen and 67 (1 in 1,578) positive for antibody. Second donations by previously negative donors reduce the overall incidence of positives. Men prisoners have a significantly higher incidence of Au antigen (1 in 153) than non-institutionalized men (1 in 803). The latter have a significantly higher incidence of antigen than women (1 in 2,019). Only one antigen-positive donor was incubating acute viral hepatitis. Failure to detect one strong and one weak antigen was responsible for two cases of posttransfusion Au-antigen-positive hepatitis.     

Expanding distribution of human serotype G6 rotaviruses in Australia

Serotype G6 rotaviruses are common pathogens of cattle but are rarely found in humans. In Australia, human G6 isolates have previously been detected in two major southern population centres. A new isolate, ASG6.02, was detected in central Australia (Alice Springs) in 1997. Comparison of the deduced amino acid sequence of the major neutralizing antigen, VP7, indicated that ASG6.02 was related to human G6 viruses isolated from children in Italy and Australia. Phylogenetic analysis supported the close relationship between ASG6.02 and other Australian isolates and indicated that G6 VP7 sequences generally clustered according to the species of origin (human, bovine or porcine). The VP4 type of ASG6.02 was determined as P-type [14], in common with other isolates from Australia and Italy. The detection of ASG6.02 indicates that the distribution of this serotype is increasing in this country and may have implications for successful vaccine development.     

RNA of Australia Antigen

ALTHOUGH the exact nature of Australia (Au) antigen is not resolved, increasing evidence suggests that it is the causal agent of viral hepatitis. This supposition is based chiefly on the frequent occurrence of Au antigen in the sera of patients with viral hepatitis^1–4 and on its virus-like appearance under the electron microscope^5–7. Biochemical studies have shown that Au antigen consists largely of protein, with a minor lipid moiety^{8, 9}. So far, however, no genetic material has been detected in the Au antigen and it has been suggested that the Au antigen might be “a unique infectious particle with little or no nucleic acid”¹⁰. We wish to present evidence, however, that RNA is an essential component of Au antigen.     

Preparation of Australia Antigen Free Human Albumin with Polyethylene Glycol

Albumin was prepared from human plasma diluted In 0.05M phosphate buffer pH 8.6, by the precipitation of approximately 95% of the associated plasma proteins with 357. polyethylene glycol. More than 70% of the albumin In the original plasma was recoverable as a viscous liquid on lowering the pH to 5.5. The albumin prepared by this technique is associated with 5 to 10% or and B globulin. Plasma, positive for Australia antigen (Au(SH)ag) yields an albumin preparation negative for the antigen.     

Chronic Liver Disease and Mitochondrial Antibodies: A Family Study

Two sisters had primary biliary disease and associated autoimmune thyroiditis with high titres of mitochondrial and other autoantibodies. Their deceased mother possibly suffered from similar disorders. In the same family two brothers had multiple autoimmune reactions, including mitochondrial antibodies, but liver function tests gave normal results. Ten other close relatives were investigated. Australia antigen was not found in the proband or her relatives.     

Identification of antigenic differences that discriminate between cattle vaccinated with Anaplasma centrale and cattle naturally infected with Anaplasma marginale

Monoclonal antibodies were raised against the vaccine strain of Anaplasma centrale used in Australia. A monoclonal antibody that reacted with an 80 kDa antigen was used to develop an A. centrale-specific fluorescent antibody test that will be useful for confirming species identity in patent infections. Another monoclonal antibody that reacted with a 116 kDa antigen was used to develop an A. centrale-specific competitive inhibition enzyme-linked immunosorbent assay (ELISA) for the serological identification of vaccinated cattle. The sensitivity of the ELISA was 100% in cattle experimentally infected with A. centrale, 97.1% in a vaccinated beef herd and 98.3% in a vaccinated dairy herd. The specificity of the ELISA was 98.6% in non-vaccinated cattle outside the Anaplasma marginale-endemic area, 97.9% in non-vaccinated cattle within the A. marginale-endemic area and 100% in cattle experimentally infected with A. marginale. The ELISA detected antibodies to A. centrale in cattle up to 9 years after vaccination with no apparent decrease in sensitivity. The assay has proved extremely valuable in Australia for investigating reported failures of multivalent live vaccines used to protect cattle against anaplasmosis and babesiosis, and should be similarly useful elsewhere in the world where these types of vaccines are used, e.g. Israel and South America.     

Sensitive Matrix Gel Diffusion Test for the Detection of Australia Antigen

A matrix gel diffusion (MGD) procedure with a sensitivity comparable to the complement fixation test (CF) has been developed for detecting Australia antigen in serum. The test utilizes a thin layer of agar (0.1 mm) with an applied plastic matrix. Reactants are introduced directly onto the surface of the agar through wells in the plastic matrix. End points obtained by CF with a panel of 11 sera varied from 1:8 to 1:512. When these sera were tested by MGD, end points for detection of antigen were within one dilution of that obtained by CF.     

Interactions of Standard Antibody with Australia Antigens in Au Ag-Ab Radioimmunoassay

Human antisera against Australia (Au) antigen have been characterized by liquid-phase radioimmunoassay (RIA) for their precipitation of (125)I-labeled Au antigen. The end-point dilutions of sera (anti-Au) which precipitated 50% of (125)I-Au antigen by RIA correlated well with complement fixation titers but had a much wider range, indicating a greater precision and perhaps a better sensitivity of assay. Anti-Au serum diluted to precipitate 50% of (125)I-labeled Au antigen was used as standard antibody in RIA tests to detect either inhibition or enhancement of the reaction by preincubated mixtures of Au antigen and antibody specimens. Without free Au antigen or antibody in the resultant mixtures there was no inhibition or enhancement; the mixtures presumably contained immunoreactively equivalent proportions of Au antigen and antibody. RIA data for diagnostic specimens indicated an end-point sensitivity which was proportional to the dilution of the standard anti-Au sera used in the test. High concentrations of the standard antibody permitted detectable inhibition of (125)I-Au antigen precipitation at lower antigen specimen concentrations. Similarly, low concentrations of the standard antibody permitted detectable enhancement of (125)I-Au antigen precipitation at lower antibody specimen concentrations. Omitting the standard antibody altogether resulted in a more sensitive RIA for Au antibody in test sera.     

Australia Antigen: Large-Scale Purification from Human Serum and Biochemical Studies of Its Proteins

Biophysical techniques are described for the large-scale isolation of Australia antigen (Au) from unit quantities of human serum by using the batch-type zonal centrifuge rotors. A three-step procedure involving isopycnic banding of the particle in CsCl density gradients and rate-zonal centrifugation on sucrose gradients resulted in a highly purified Au preparation which was used for biochemical studies of Au proteins and as immunizing antigen for the production of reagent antiserum in animals. The spherical form of Au, which was devoid of detectable nucleic acid, was composed of two major proteins (AuP1 and AuP2) and a minor protein (AuP3) of 26,000, 32,000, and 40,000 molecular weight, respectively, as determined by acrylamide gel electrophoresis. The significance of these findings to the possibility of Au subtypes is discussed.     

Australian Antigen and Autoantibodies in Chronic Hepatitis

The sera of 110 patients with chronic hepatitis and adequate controls were examined for antibodies to smooth muscle (S.M.), mitochondria (M.), and for antinuclear factors by the immunofluorescence method, and for Australia (Au(1)) antigen by a modified micro-Ouchterlony immunodiffusion technique. Twelve out of 13 patients with primary biliary cirrhosis had M. antibodies, two had antinuclear factor, and none had Au(1) in their sera. In chronic aggressive hepatitis 23·5% of the sera contained antinuclear factor, 13% S.M. antibodies, 10·5% M. antibodies, and 22% Au(1) antigen. Of the 12 patients with chronic persistent hepatitis, one had antinuclear factor, one S.M. antibodies, and three Au(1) antigen.The most striking finding was a mutual exclusion between Au(1) antigen and M. and S.M. antibodies. None of the 33 patients with one or the other form of chronic hepatitis and M. or S.M. antibodies had Au(1) antigen; 22 out of 77 (28%) patients without such antibodies were positive.     

Identification of a novel polyomavirus from patients with acute respiratory tract infections

We report the identification of a novel polyomavirus present in respiratory secretions from human patients with symptoms of acute respiratory tract infection. The virus was initially detected in a nasopharyngeal aspirate from a 3-year-old child from Australia diagnosed with pneumonia. A random library was generated from nucleic acids extracted from the nasopharyngeal aspirate and analyzed by high throughput DNA sequencing. Multiple DNA fragments were cloned that possessed limited homology to known polyomaviruses. We subsequently sequenced the entire virus genome of 5,229 bp, henceforth referred to as WU virus, and found it to have genomic features characteristic of the family Polyomaviridae. The genome was predicted to encode small T antigen, large T antigen, and three capsid proteins: VP1, VP2, and VP3. Phylogenetic analysis clearly revealed that the WU virus was divergent from all known polyomaviruses. Screening of 2,135 patients with acute respiratory tract infections in Brisbane, Queensland, Australia, and St. Louis, Missouri, United States, using WU virus-specific PCR primers resulted in the detection of 43 additional specimens that contained WU virus. The presence of multiple instances of the virus in two continents suggests that this virus is geographically widespread in the human population and raises the possibility that the WU virus may be a human pathogen.     

Preparation and Standardization of an Australia Antigen Antibody of Equine Origin

A horse has been immunized with Australia antigen (Au/SH) purified 20-fold by a procedure employing gel filtration of Cohn fraction IV derived from an Au/SH-positive human plasma pool. Hyperimmunization was initiated by the intramuscular injection of 20 ml of a mixture of equal parts of purified Au/SH and complete Freund's adjuvant. The 20-ml volume was divided into four 5-ml doses, two of which were administered on each side of the horse's neck. Booster doses of antigen alone were given as follows: 10 ml intravenously 30 days later and 5 ml intramuscularly on each of days 77 and 205. Au/SH antibody formed readily, beginning on day 17, and was demonstrated by the agar gel double-diffusion technique and the complement fixation test during the subsequent 6 months. Antihuman plasma protein antibodies were effectively removed from the horse serum by one absorption with 1 to 3 volumes of normal human plasma. Abrupt rises in anticomplementary activity observed shortly after the third and fourth antigen injections, when the horse had developed elevated and steady levels of Au/SH antibody, could possibly be due to formation of antigen-antibody complexes. After optimal conditions were determined, an Au/SH antibody reagent pool which met official requirements was prepared. It was found equally suitable for the agar gel double-diffusion, complement fixation, and counterimmunoelectrophoresis test procedures.