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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.

We used the 2002 national serosurvey to evaluate a primary care-based young adult vaccination campaign, measure the reproductive number (R) and, together with vaccination coverage estimates, predict R until 2012. The campaign had no impact on immunity in young adults. R was estimated to be 0.69 and predicted to stay well below the epidemic threshold of 1 until at least 2012, indicating that Australia should remain free of endemic measles in the medium term. To maintain elimination in the longer term, the timeliness and coverage of childhood vaccinations must improve and innovative strategies will be required to improve measles immunity among young adults. This experience is likely to apply to developed countries that have achieved or are approaching measles elimination.

To determine the prevalence of hepatitis A virus (HAV) infection in the general U.S. population, sera from participants in the Third National Health and Nutrition Examination Survey (NHANES III) conducted in 1988-1994 were tested for antibody to HAV (anti-HAV). Among 21,260 participants aged > or = 6 years tested, the overall prevalence of infection was 31.3%, and increased markedly with age. The age-adjusted prevalence was significantly higher among foreign- compared to U.S.-born participants, and was highest among Mexican-Americans and lowest among non-Hispanic whites. Among U.S.-born children, only Mexican-American ethnicity and income below the poverty level were associated with HAV infection in a multivariate model. During this period before hepatitis A vaccination, age, ethnicity and birthplace were the most important determinants of HAV infection in the United States.

Objectives: To weigh the evidence from outbreak data that Victoria has achieved, and is maintaining, elimination of measles. To identify age groups that measles vaccination has not protected adequately., Methods: Data on observed measles outbreaks in Victoria since the start of 1998 are used to estimate the reproduction number of cases, and the probability that it is maintained below unity, its threshold value for elimination. The relative susceptibility to measles is estimated as a function of age, with confidence intervals., Results: Seventeen measles introductions led to secondary cases, while 22 were single-case introductions. From these, the probability that the reproduction number for cases exceeds unity is estimated to be 0.044, or less, depending on assumptions made. There is no evidence that the reproduction number increased over time. Those aged between 19 and 32 years were most susceptible, followed by those in the first and second year of life., Conclusions: The data provide strong evidence that Victoria has maintained elimination of measles over the period 1998 to mid-2003. There is scope to improve the immunisation coverage. It is not clear how much outbreak intervention is contributing to the success in achieving apparent elimination., Implications: To prevent importations from causing a major epidemic of measles, Victoria must maintain its immunisation coverage and outbreak control at current levels, or better. It is important to monitor the control of measles even when elimination is achieved.

Objective: To determine the effect of maternal antibody on hepatitis A vaccine immunogenicity in infants. Study design Infants of mothers negative for antibody to hepatitis A virus (anti-HAV; group 1) were administered hepatitis A vaccine at 2, 4, and 6 months of age, and infants of anti-HAV-positive mothers were randomized to receive either hepatitis A vaccine (group 2) or hepatitis B vaccine (group 3) on the same schedule. Group 3 infants subsequently received hepatitis A vaccine at 8 and 10 months of age., Results: At 15 months of age, 100% of infants in group 1, 93% in group 2, and 92% in group 3 had protective levels of antibody. However, there were significant differences in the geometric mean concentration (GMC) of anti-HAV between groups. Group 1 GMC was 231 mIU/mL, compared with 85 mIU/mL for group 2 and 84 mIU/mL for group 3 (P<.001, group 1 vs group 3)., Conclusions: Passively acquired maternal anti-HAV resulted in a significantly lower final antibody response when infants were administered hepatitis A vaccine at 2, 4, and 6 months of age or at 8 and 10 months of age.

Two separate cases of acute hepatitis C virus (HCV) infection following medical procedures, arthroscopy and colonoscopy, are reported. In both episodes, patient risk factors were reviewed, and staff and other patients' sera were tested for HCV antibodies and RNA. HCV RNA positive samples were genotyped, sequenced, and subjected to phylogenetic analysis. No risk factors for HCV infection were identified for either case except for medical procedures. HCV RNA positive patients were identified preceding both cases on the respective theatre lists. HCV infection in a second low risk patient was also identified. Nucleic acid sequencing and phylogenetic analysis of HCV from the two putative source patients and the three recipient patients demonstrated a high degree of relatedness respectively. The results suggest that patient-to-patient transmission occurred in both episodes via contamination of intravenous anaesthetic ampoules with HCV used on multiple patients. Injectable medication ampoules should not be used for more than one patient.

Measles outbreaks in Victoria in 1999 and 2001 have suggested that a substantial proportion of young Victorian adults may be susceptible to measles infection. We performed a serosurvey of 300 18-30-year-old healthy blood donors and 312 sera retrieved after diagnostic testing for a non-rash illness in patients of the same age group, with the aim of estimating the proportion of young adults in Victoria immune to measles. We also aimed to define more precisely the birth cohorts at risk of measles infection, with cohorts reflecting the measles immunisation policies of previous years. There was no significant difference in measles immunity between the 300 blood donors (79.0%, 95% confidence interval 73.9-83.5) and the 312 patients whose sera had been stored (84.0%, 95% CI 79.4-87.9, p=0.11). There was, however, a significant difference in immunity by birth cohort. In the combined results from both samples, the proportion of people born between 1968 and 1974 who were immune to measles was 88.4 per cent (95% CI 84.1-91.6) while the proportion of those born between 1975 and 1981 was 74.1 per cent (95% CI 68.7-79.1). This study confirms that a substantial proportion of young Victorian adults are susceptible to measles, but also demonstrates that those born between 1975 and 1981 are more likely to be non-immune than those born before 1975. A review of published Australian data supports this conclusion and confirms the need for a measles control program aimed at young adults.

The 1998 Australian Measles Control Campaign had as its aim improved immunization coverage among children aged 1-12 years and, in the longer term, prevention of measles epidemics. The campaign included mass school-based measles-mumps-rubella vaccination of children aged 5-12 years and a catch-up programme for preschool children. More than 1.33 million children aged 5-12 years were vaccinated at school: serological monitoring showed that 94% of such children were protected after the campaign, whereas only 84% had been protected previously. Among preschool children aged 1-3.5 years the corresponding levels of protection were 89% and 82%. During the six months following the campaign there was a marked reduction in the number of measles cases among children in targeted age groups.

Objectives: To describe an outbreak of measles in Victoria., Design: Case series with cases identified through enhanced passive surveillance and outbreak-related active surveillance., Setting: State of Victoria, 1999., Main Outcome Measures: Number of cases; epidemiological links and patterns of transmission; patient demographic features and vaccination status; complications., Results: 75 cases were identified (74 laboratory-confirmed; and one epidemiologically linked to a laboratory-confirmed case), with onset between 11 February and 2 May 1999. The first case was in a 21-year-old woman who had recently holidayed in Bali and worked at a large cinema complex in Melbourne. Sixteen cases occurred in people who had contact with the index case at the cinema on one evening. The outbreak spread to regional Victoria and South Australia. Median age of patients was 22 years; 64 (85%) were born between 1968 and 1981, with only one patient in the age group targeted by the primary school component of the 1998 Australian Measles Control Campaign; this child had not been vaccinated. More than a third of patients (28) were hospitalised (total, 97 inpatient days), and five were healthcare workers., Conclusions: This outbreak was caused by international importation of measles virus. It highlights the change in epidemiology of measles in Australia, from a disease of childhood to one predominantly affecting young adults. A strong two-dose childhood vaccination program, vigilant surveillance, and rapid response to outbreaks will continue to be the basis of measles control, but better protection for young adults should be considered.

Objective: To describe results of the first two years of enhanced measles surveillance in Victoria., Design: Case series identified through enhanced measles surveillance., Participants and Setting: All measles cases notified to the Disease Control Section, Department of Human Services, Victoria, in 1997 and 1998., Main Outcome Measures: Proportion of notified cases laboratory confirmed as measles, rubella, or human parvovirus infection; identification of clusters (two or more linked cases of measles); and utility of the National Health and Medical Research Council clinical case definition for suspected measles., Results: Rates of laboratory testing of notified cases improved after introduction of a paediatric phlebotomy service in July 1997, from 21 of 90 notified patients (23%) in the preceding six months, to 258 of 317 notified patients (81%) between July 1997 and December 1998. Of the 317, only 19 (6%) were laboratory confirmed with measles, while a further 26 (8%) were laboratory confirmed with human parvovirus infection (18) or rubella (8). Three clusters of measles, involving 11 cases, were identified during 1998. Use of the NHMRC case definition did not greatly improve the positive predictive value for diagnosis of measles above that of notification alone (14% versus 8%)., Conclusions: Circulation of measles virus in Victoria in 1997 and 1998 appeared minimal. In this interepidemic period most notified cases of measles were not measles; to identify true cases, surveillance during an interepidemic period must include laboratory testing of notified cases.

Objective: Data from 2 National Health and Nutrition Examination Surveys (NHANES), NHANES II (1976-1980) and NHANES III (1988-1994), were analyzed to examine trends in the prevalence of hepatitis B infection in the United States., Methods: Serum specimens were tested for markers of hepatitis B virus infection, and risk factors were determined from questionnaires., Results: The overall age-adjusted prevalence of hepatitis B virus infection was 5.5% (95% confidence interval [CI] = 4.8, 6.2) in NHANES II, as compared with 4.9% (95% CI = 4.3, 5.6) in NHANES III. In both surveys, Black participants had the highest prevalence of infection (NHANES II, 15.8%; NHANES III, 11.9%). No differences in infection were found in the major racial groups between surveys, except for a decrease among those older than 50 years. Black race, increasing number of lifetime sexual partners, and foreign birth had the strongest independent associations with hepatitis B virus infection., Conclusions: Testing of participants in 2 national surveys demonstrates no significant decrease in hepatitis B virus infection, despite the availability of hepatitis B vaccine.

Precise estimates of the incidence of hepatitis B virus (HBV) infection are required to assess the impact of immunization and other prevention strategies in the United States. Race- and age-specific prevalence data obtained from the second and third National Health and Nutrition Examination Surveys (NHANES II, 1976-1980, and NHANES III, 1988-1994) were used to estimate the annual incidence of HBV infection by catalytic modeling. During the period covered by NHANES II, an estimated 323,462 persons were infected annually, and 334,863 were infected annually during the period covered by NHANES III. No statistically significant declines in prevalence of HBV infection occurred between the two surveys, a period during which hepatitis B vaccination targeted only limited numbers of high-risk adults.

Background: Naturally occurring viral proteins derived from cell culture and recombinant proteins expressed in procaryotic systems have been used extensively as target proteins in the development of immunoassay methods for the detection of antibodies. However, immunoassays utilizing these proteins often yield false-positive reactions suggesting that it may be possible to identify and remove regions responsible for these non-specific reactions., Objective: In this paper we describe a new strategy for the construction of immunoreactive recombinant proteins designed to improve immunoassay specificity., Study Design: A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligonucleotides by the polymerase chain reaction (PCR). The polypeptide comprises a mosaic of three antigenically dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione-S-transferase or beta-galactosidase., Results: Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by immunoblot analysis and by enzyme immunoassay (EIA) the presence and accessibility of all HEV-specific antigenic epitopes designed into the mosaic protein. Both hybrid proteins were shown by immunoblot analysis using a panel of human anti-HEV-positive and -negative sera to be HEV-specific. A sensitive and specific EIA was developed to detect IgG anti-HEV activity in human sera. A neutralization test using individual synthetic peptides corresponding to the epitopes designed into the mosaic protein was also developed to confirm IgG anti-HEV activity by absorbing the specimen before retesting by EIA., Conclusion: An artificial mosaic protein composed of short linear HEV-specific antigenic epitopes was constructed from synthetic oligonucleotides by PCR and used to develop a sensitive and specific EIA for the detection of anti-HEV activity in human sera.

Infection by hepatitis C virus (HCV)*, the aetiologic agent responsible for the majority of non-A-non-B posttransfusion hepatitis, is detected by assaying for antibodies against structural and nonstructural recombinant proteins or synthetic peptides. The aim of this study was to characterize the antibody reactivity of selected sera against antigenic peptides spanning immunodominant regions of the core, NS4 and NS5 HCV proteins. Reactivity to synthetic peptides was determined by enzyme immunoassay (EIA) for 11 selected sera from blood donors (good responders), for 27 selected sera from hemodialysis patients (poor responders), all positive for HCV antibodies (tested by different second and third-generation assays), and for 7 negative sera. Some peptides from the core and the NS4 region were widely recognized by the tested sera. Sera not reactive with core, NS4, or NS5 region by some immunoblot assays exhibited reactivity against peptides from these proteins. Autoimmune reactivity associated with HCV infection was evaluated by using a synthetic peptide derived from the GOR peptide; 8/11 HCV-positive sera were found reactive against this peptide. No correlation was found between reactivity to any of the peptides tested and the presence of HCV RNA in the serum or with HCV genotype. The EIA reactivity of peptides from the core region suggested a multideterminant antigenic structure, where reactivity of each epitope may be differentially affected by neighboring amino acids depending on individual sera. This situation was particularly evidenced in selected sera from poor responder specimens where a more restricted antibody response to core peptides was observed. Reactivity of sera from HCV-infected patients with synthetic peptides from the core, NS4, and NS5 regions indicated the presence of multiple linear epitopes (particularly in the core region) that may be used in a mixture for immunodiagnosis; however, the length and exact position of the synthetic peptides must be chosen carefully.

Endotoxin has been implicated as an aetiological factor in liver transplant rejection and acute graft-versus-host disease. To investigate the role of endotoxin in human single lung transplant rejection we measured the level of endotoxin in bronchoalveolar lavage (BAL) fluid from six subjects at baseline and during rejection, which was defined histologically from transbronchial biopsy. Differential cell counts of BAL fluid cells, the levels of protein and albumin in BAL fluid, and serum albumin levels were also examined. BAL fluid albumin to serum ratio was calculated to evaluate alveolar-capillary leakage. A significant elevation of BAL fluid endotoxin with rejection compared with baseline was observed. Standardizing endotoxin levels to BAL fluid volume, protein, or albumin were all of similar significance. Examination of BAL fluid cell population revealed a significant elevation in the percentage of lymphocytes with rejection. No significant difference between BAL fluid protein levels, BAL fluid albumin levels. BAL fluid albumin to protein ratio, serum albumin levels, or BAL fluid albumin to serum albumin ratio was seen with rejection. We conclude that BAL fluid endotoxin levels increase during lung rejection, endotoxin levels can be accurately standardized to millilitres of BAL fluid, and abnormal alveolar-capillary leakage does not appear to account for the increased BAL fluid endotoxin.

We studied the immune response to (re)vaccination with three 1-mL doses of recombinant hepatitis B vaccine administered intramuscularly on days 0, 30, and 180 to 75 public safety workers (PSWs) who had not developed antibody to hepatitis B surface antigen (anti-HBs) after three intradermal doses of hepatitis B vaccine; to 45 PSWs who had initially developed antibody but did not have detectable levels 11 months after intradermal vaccination; and to 16 hepatitis B-susceptible PSWs. Levels of anti-HBs were measured on days 14 and 210 after the first intramuscular dose. Overall, 46 (61%) of 75 PSWs in the initial-nonresponse group, 43 (96%) of 45 PSWs in the lost-response group, and 5 (31%) of 16 PSWs in the new-vaccinee group had anti-HBs titers of > or = 10 mIU/mL on day 14. On day 210 (after three doses), the figures were 62 (89%) of 70 PSWs in the initial-nonresponse group, 43 (98%) of 44 PSWs in the lost-response group, and 15 (94%) of 16 PSWs in the new-vaccine group. We conclude that persons who do not seroconvert after intradermal vaccination should receive three doses of hepatitis B vaccine by the intramuscular route.

A set of 150 synthetic peptides spanning the proteins NS3-NS4-NS5 of the hepatitis C virus (HCV) was synthesized and tested with a panel of 20 sera obtained from HCV-infected patients. Of 62 peptides prepared from the NS3 region, none exhibited strong antigenic reactivity. Rather, five peptides from this region demonstrated specific reactivity with only 5-10% of anti-HVC-positive sera. Nonetheless, it is well known that the NS3 region contains strong antigenic epitopes. These epitopes appear to be modeled in a functionally active manner with recombinant proteins and cannot be mimicked properly with short synthetic peptides. This finding suggests that the major NS3 antigenic epitopes are conformationally dependent. Seven of 20 peptides prepared from the NS4 region were immunoreactive. Five peptides from this region demonstrated very strong HCV-specific antigenic reactivity. Four of the five peptides belong to the recognized immunoreactive 5-1-1 region located inside the C100-3 antigen. One peptide demonstrating immunoreactivity with approximately 90% of anti-HCV-positive sera was found outside the C100-3 region at the C-terminal part of the NS4 protein. Of 68 peptides synthesized from the NS5 protein, 30 were immunoreactive. Six of the 30 demonstrated immunoreactivity with 35-50% of anti-HCV-positive sera. Thus, the NS4 and NS5 regions of the HCV polyprotein contain a large number of specific, broadly reactive, linear antigenic epitopes. The highly antigenic reactivity of the NS5 region suggests that this protein may have significant diagnostic potential.

A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and -negative sera. The data obtained strongly indicate a diagnostic potential for the mosaic protein.

Reactive oxygen species, including superoxide anion, have attracted increasing attention for their possible role in promoting inflammation in a variety of pulmonary diseases including asthma. However, reactive oxygen species metabolism of phagocytic cells may be substantially modified by therapeutic agents used for asthma. Peripheral blood mononuclear cells and alveolar macrophages from 15 normal subjects, and blood mononuclear cells from an additional 17 normal subjects, were studied to assess the effects of beta-receptor agonists and theophylline on phagocytic cell superoxide release. Isoproterenol produced a biphasic effect on spontaneous and phorbol ester stimulated alveolar macrophage and mononuclear cell superoxide production, augmenting release at 10(-5)M, and inhibiting release at 10(-4)M. These effects on spontaneous function in mononuclear cells were inhibited by 10(-5)M propranolol. Under conditions of phorbol ester-stimulation the enhancing effect of 10(-5)M isoproterenol on blood mononuclear cells was blocked by propranolol, but the inhibitory effect of 10(-4)M isoproterenol was not. Albuterol at equimolar concentrations with isoproterenol was not associated with altered spontaneous or stimulated superoxide release by alveolar macrophages or mononuclear cells. Further, spontaneous superoxide release by alveolar macrophages and mononuclear cells was significantly reduced by therapeutically achievable concentrations of theophylline (greater than 5 micrograms/ml). We conclude that the medication history must be controlled in studies of cell function in asthma, and that albuterol may be preferable to isoproterenol as a premedication for bronchoscopy when superoxide production of airspace cells is studied.