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The unprecedented emergence of Japanese encephalitis (JE) in mainland Australia represents an outbreak of high clinical and public health significance. JE is a zoonosis spread by mosquitoes and is one of the most important causes of endemic viral encephalitis in South-East Asia and the Indian subcontinent. While occasional cases of human Japanese encephalitis virus (JEV) infection have occurred in far north Australia, its detection in pigs and the substantial number of locally acquired human cases across multiple jurisdictions in early 2022 prompted the declaration of this outbreak as a Communicable Disease Incident of National Significance. Laboratory testing for JEV is complex, and most cases are diagnosed by serology, for which interpretation is difficult. This review provides a comprehensive outline of currently available methods for JEV diagnosis including serology, nucleic acid amplification testing, virus isolation, sequencing and metagenomics. The relative advantages and disadvantages of the diagnostic tests are presented, as well as their value in clinical and public health contexts. This review also explores the role of mosquito, veterinary and human surveillance as part of the laboratory response to JEV. As JEV may become endemic in Australia, a collaborative and coordinated One Health approach involving animal, human and environmental health is required for optimal disease response and control.

Introduction: In May 2020, The Communicable Diseases Network of Australia (CDNA) case definition introduced serological criteria to support the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). We present findings that support the utility of SARS‐CoV‐2‐specific serology for public health investigations. Methods: From 24 January to 31 July 2020, the following information was collected from individuals with positive SARS‐CoV‐2‐specific immunofluorescence antibody tests: history of contact with COVID‐19 cases; recent travel; symptoms consistent with COVID‐19; and SARS‐CoV‐2 nucleic acid testing (NAT) results. Individuals were classified as confirmed or probable by CDNA criteria or additionally as possible (SARS‐CoV‐2‐specific IgG positive with compatible symptoms or epidemiologic risk) or indeterminate (SARS‐CoV‐2‐specific IgA/IgM positive only) cases. Results: A total of 10,595 individuals were tested in the six‐month period. Of these, 9.8% (1,037) individuals had positive SARS‐CoV‐2‐specific serology of which 566 (53.6%) were NAT‐confirmed COVID‐19 cases and 286 (27.6%) were part of a cruise ship outbreak sero‐survey. The remaining 185 individuals (NAT negative) were individually classified as serologically confirmed (4, 0.4%), probable (72, 6.9%) possible (66, 6.4%) and indeterminate (38, 3.7%) cases. Maternal antibody transfer was inferred in one infant and four were unclassified. Conclusion: SARS‐CoV‐2‐specific serology is a key diagnostic tool for retrospective identification of COVID‐19 infection. Implications for public health: SARS‐CoV‐2 specific serology can enhance the ability to find cases, link missing cases in clusters of infection and identify the epidemiological extent of SARS‐CoV‐2 outbreaks. A combination of epidemiological criteria, clinical criteria and a quantitative serological test can be used as an adjunct to classify SARS‐CoV‐2 cases. Our study confirms the low level of community transmission in NSW during the first year of the COVID‐19 pandemic

Objectives To estimate SARS‐CoV‐2‐specific antibody seroprevalence after the first epidemic wave of coronavirus disease 2019 (COVID‐19) in Sydney. Setting, participants People of any age who had provided blood for testing at selected diagnostic pathology services (general pathology); pregnant women aged 20–39 years who had received routine antenatal screening; and Australian Red Cross Lifeblood plasmapheresis donors aged 20–69 years. Design Cross‐sectional study; testing of de‐identified residual blood specimens collected during 20 April – 2 June 2020. Main outcome measure Estimated proportions of people seropositive for anti‐SARS‐CoV‐2‐specific IgG, adjusted for test sensitivity and specificity. Results Thirty‐eight of 5339 specimens were IgG‐positive (general pathology, 19 of 3231; antenatal screening, 7 of 560; plasmapheresis donors, 12 of 1548); there were no clear patterns by age group, sex, or location of residence. Adjusted estimated seroprevalence among people who had had general pathology blood tests (all ages) was 0.15% (95% credible interval [CrI], 0.04–0.41%), and 0.29% (95% CrI, 0.04–0.75%) for plasmapheresis donors (20–69 years). Among 20–39‐year‐old people, the age group common to all three collection groups, adjusted estimated seroprevalence was 0.24% (95% CrI, 0.04–0.80%) for the general pathology group, 0.79% (95% CrI, 0.04–1.88%) for the antenatal screening group, and 0.69% (95% CrI, 0.04–1.59%) for plasmapheresis donors. Conclusions Estimated SARS‐CoV‐2 seroprevalence was below 1%, indicating that community transmission was low during the first COVID‐19 epidemic wave in Sydney. These findings suggest that early control of the spread of COVID‐19 was successful, but efforts to reduce further transmission remain important.

Immunofluorescence (IF) is an important technique used in the diagnosis of many infectious diseases. In virology, it has proven to be particularly suited to detecting antibody directed against newly emerging viruses able to be cultivated in cell culture. It permits visualization of antibody and allows for antibody class to be determined which is critical to understanding the timing of infection. The procedure used to determine IgG, IgA, and IgM antibody directed against SARS-CoV-2 in humans is described in this chapter. These methods were developed for routine diagnosis of SARS-CoV-2 infection in Australia at the start of the global pandemic in 2020.

Background: This study evaluates trends in tetanus immunity and epidemiology over the last two decades in Australia, drawing on two national serological surveys and national tetanus morbidity data, to justify current Australian adult tetanus booster recommendations. Methods: We compare tetanus immunity level between two national serosurveys, and examine incidence trends using the most accurate estimation of the true number of cases by correcting for under-ascertainment. Results: Tetanus immunity in people aged Conclusion: Despite a positive decrease in tetanus incidence, there remains a significant burden in the elderly population of an entirely preventable disease. Supplying a funded booster dose of dTpa at 65 years would be, potentially, an effective strategy to prevent tetanus cases in Australia.

Zika virus (ZIKV) has recently emerged as an important human pathogen due to the strong evidence that it causes disease of the central nervous system, particularly microcephaly and Guillain–Barré syndrome. The pathogenesis of disease, including mechanisms of neuroinvasion, may include both invasion via the blood–brain barrier and via peripheral (including cranial) nerves. Cellular responses to infection are also poorly understood. This study characterizes the in vitro infection of laboratory-adapted ZIKV African MR766 and two Asian strains of (1) brain endothelial cells (hCMEC/D3 cell line) and (2) olfactory ensheathing cells (OECs) (the neuroglia populating cranial nerve I and the olfactory bulb; both human and mouse OEC lines) in comparison to kidney epithelial cells (Vero cells, in which ZIKV infection is well characterized). Readouts included infection kinetics, intracellular virus localization, viral persistence and cytokine responses. Although not as high as in Vero cells, viral titres exceeded 104 plaque-forming units (p.f.u.) ml−1 in the endothelial/neuroglial cell types, except hOECs. Despite these substantial titres, a relatively small proportion of neuroglial cells were primarily infected. Immunolabelling of infected cells revealed localization of the ZIKV envelope and NS3 proteins in the cytoplasm; NS3 staining overlapped with that of dsRNA replication intermediate and the endoplasmic reticulum (ER). Infected OECs and endothelial cells produced high levels of pro-inflammatory chemokines. Nevertheless, ZIKV was also able to establish persistent infection in hOEC and hCMEC/D3 cells. Taken together, these results provide basic insights into ZIKV infection of endothelial and neuroglial cells and will form the basis for further study of ZIKV disease mechanisms.

Background As of mid-2021, Australia’s only nationwide coronavirus disease 2019 (COVID-19) epidemic occurred in the first 6 months of the pandemic. Subsequently, there has been limited transmission in most states and territories. Understanding community spread during the first wave was hampered by initial limitations on testing and surveillance. To characterize the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody seroprevalence generated during this time, we undertook Australia’s largest national SARS-CoV-2 serosurvey. Methods Between June 19 and August 6, 2020, residual specimens were sampled from people undergoing general pathology testing (all ages), women attending antenatal screening (20–39 years), and blood donors (20–69 years) based on the Australian population’s age and geographic distributions. Specimens were tested by Wantai total SARS-CoV-2-antibody assay. Seroprevalence estimates adjusted for test performance were produced. The SARS-CoV-2 antibody-positive specimens were characterized with microneutralization assays. Results Of 11 317 specimens (5132 general pathology; 2972 antenatal; 3213 blood-donors), 71 were positive for SARS-CoV-2-specific antibodies. Seroprevalence estimates were 0.47% (95% credible interval [CrI], 0.04%–0.89%), 0.25% (CrI, 0.03%–0.54%), and 0.23% (CrI, 0.04%–0.54%), respectively. No seropositive specimens had neutralizing antibodies. Conclusions Australia’s seroprevalence was extremely low (

In late November 2021, the World Health Organization declared the SARS-CoV-2 lineage B.1.1.529 the fifth variant of concern, Omicron. This variant has acquired 15 mutations in the receptor binding domain of the spike protein, raising concerns that Omicron could evade naturally acquired and vaccine-derived immunity. We utilized an authentic virus, multicycle neutralisation assay to demonstrate that sera collected one, three and six months post-two doses of Pfizer-BioNTech BNT162b2 has a limited ability to neutralise SARS-CoV-2. However, four weeks after a third dose, neutralising antibody titres are boosted. Despite this increase, neutralising antibody titres are reduced four-fold for Omicron compared to lineage A.2.2 SARS-CoV-2.

A total of 1080 individual patient samples (158 positive serology samples from confirmed, predominantly mildly symptomatic COVID-19 patients and 922 serology negative including 496 collected pre-COVID) from four states in Australia were analysed on four commercial SARS-CoV-2 serological assays targeting antibodies to different antigens (Roche Elecsys and Abbott Architect: nucleocapsid; Diasorin Liaison and Euroimmun: spike). A subset was compared to immunofluorescent antibody (IFA) and micro-neutralisation. Sensitivity and specificity of the Roche (n = 1033), Abbott (n = 806), Diasorin (n = 1034) and Euroimmun (n = 175) were 93.7 %/99.5 %, 90.2 %/99.4 %, 88.6 %/98.6 % and 91.3 %/98.8 %, respectively. ROC analysis with specificity held at 99 % increased the sensitivity for the Roche and Abbott assays from 93.7% to 98.7% (cut-off 0.21) and 90.2 % to 94.0 % (cut-off 0.91), respectively. Overall seropositivity of samples increased from a maximum of 23 % for samples 0-7 days-post-onset of symptoms (dpos), to 61 % from samples 8-14dpos and 93 % from those >14dpos. IFA and microneutralisation values correlated best with assays targeting antibodies to spike protein with values >80 AU/mL on the Diasorin assay associated with neutralising antibody. Detectable antibody was present in 22/23 (96 %), 20/23 (87 %), 15/23 (65 %) and 9/22 (41 %) patients with samples >180dpos on the Roche, Diasorin, Abbott and microneutralisation assays respectively. Given the low prevalence in this community, two-step algorithms on initial positive results saw an increase in the positive predictive value (PPV) of positive samples (39 %-65 % to ≥98 %) for all combinations. Similarly accuracy increased from a range of 98.5 %-99.4 % to ≥99.8 % assuming a 1 % seroprevalence. Negative predictive value (NPV) was high (≥99.8 %) regardless of which assay was used initially.

Objectives To determine population-level immunity to mumps in Australia. Methods We tested randomly selected specimens from people aged 1-49 years using the Enzygnost anti-parotitis IgG enzyme immunoassay from an opportunistically collected serum bank in 2012-2013. Weighted estimates of the proportion seropositive and equivocal for mumps-specific IgG antibody were determined by age group and compared with two previous national serosurveys conducted in 2007-2008 and 1997-1998. Results Overall, 82.1% (95% CI 80.6-83.5%) of 2,729 specimens were positive or equivocal for mumps-specific IgG antibodies (71.1% positive [95% CI 69.4-72.9%]; 10.9% equivocal [95% CI 9.8-12.2%]). The proportion positive or equivocal was higher in 2012-2013 (82.1%) than in 2007-2008 (75.5%) and 1997-1998 (72.5%), but varied by age. The proportion positive or equivocal in 2012-2013 was above 80% for all age groups older than 1 year except for 30-34 year olds, corresponding to the 1978-1982 birth cohort previously identified as most likely to have missed out on a second MMR vaccine dose. Conclusion Seropositivity to mumps in 2012-2013 was well-maintained compared with previous serosurveys. Low mumps notifications over this period in Australia suggest an absence of community-based transmission of mumps infection in the general population, but recent outbreaks among Aboriginal adolescents and young adults in close-contact settings, despite high 2-dose MMR coverage, suggest that seroprotection may be insufficient in other similar settings in Australia.Seropositivity to mumps in 2012-2013 was well-maintained compared with previous serosurveys. Low mumps notifications over this period in Australia suggest an absence of community-based transmission of mumps infection in the general population, but recent outbreaks among Aboriginal adolescents and young adults in close-contact settings, despite high 2-dose MMR coverage, suggest that seroprotection may be insufficient in other similar settings in Australia.

BackgroundTesting for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific antibodies has become an important tool, complementing nucleic acid tests (NATs) for diagnosis and for determining the prevalence of coronavirus disease 2019 (COVID-19) in population serosurveys. The magnitude and persistence of antibody responses are critical for assessing the duration of immunity.MethodsA SARS-CoV-2-specific immunofluorescent antibody (IFA) assay for immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) was developed and prospectively evaluated by comparison to the reference standard of NAT on respiratory tract samples from individuals with suspected COVID-19. Neutralizing antibody responses were measured in a subset of samples using a standard microneutralization assay.ResultsA total of 2753 individuals were eligible for the study (126 NAT-positive; prevalence, 4.6%). The median “window period” from illness onset to appearance of antibodies (range) was 10.2 (5.8–14.4) days. The sensitivity and specificity of either SARS-CoV-2 IgG, IgA, or IgM when collected ≥14 days after symptom onset were 91.3% (95% CI, 84.9%–95.6%) and 98.9% (95% CI, 98.4%–99.3%), respectively. The negative predictive value was 99.6% (95% CI, 99.3%–99.8%). The positive predictive value of detecting any antibody class was 79.9% (95% CI, 73.3%–85.1%); this increased to 96.8% (95% CI, 90.7%–99.0%) for the combination of IgG and IgA.ConclusionsMeasurement of SARS-CoV-2-specific antibody by IFA is an accurate method to diagnose COVID-19. Serological testing should be incorporated into diagnostic algorithms for SARS-CoV-2 infection to identify additional cases where NAT was not performed and resolve cases where false-negative and false-positive NATs are suspected. The majority of individuals develop robust antibody responses following infection, but the duration of these responses and implications for immunity remain to be established.

Background Australia is one of only a few countries with a long-standing and consistent serosurveillance program. We conducted a national serosurvey in 2012–2013 to estimate population seroprevalence of measles-specific IgG and the effective reproduction number, R, and compare the results with the three previous serosurveys (1996–1999, 2002 and 2007) to examine trends following a decade of sustained measles control. Methods 2729 residual sera from 1 to 49 year olds were tested using the Enzygnost anti-measles IgG enzyme immunoassay (EIA). All sera in the equivocal range by EIA on re-testing and a random sample of low positive and negative sera were later tested by a microneutralisation assay. R was calculated from weighted estimates of the proportion seronegative by age using a previously developed contact matrix. Results In the 2012–13 serosurvey, anti-measles IgG seropositivity for 1–49 year olds was 80.8% (95% CI: 79.4–82.3%) and 8.9% (95% CI: 7.8–10.0%) had equivocal antibody levels. The increasing proportion of seronegative and equivocal individuals in age groups 10–39 years continued a trend seen in previous serosurveys. There was also an increase in equivocal results among 2–4 and 5–9 year old children, >90% of whom were recently vaccinated. R increased from 0.57 in 1999 to above the epidemic threshold of 1 in 2012–13 (R = 1.7). All 20 EIA negative sera, 238/241 (98.8%) equivocal sera, and 89/92 (96.7%) low positive sera had a titre Conclusions A number of countries with sustained measles control have now demonstrated that measles-specific IgG antibodies decline with time since vaccination. As there is good epidemiologic evidence of population-level protection, the implications of declining measles-specific IgG antibody levels for maintaining measles elimination are unclear. Novel studies to determine correlates of protection against measles transmission and disease in the post-elimination era are needed to help answer this question.

There are limited long-term data on seroprevalence of neutralising antibody (nAb) to the three poliovirus serotypes following the switch from oral polio vaccine (OPV) to inactivated polio vaccine (IPV). In Australia, combination vaccines containing IPV replaced OPV in late 2005. Using serum and plasma specimens collected during 2012 and 2013, we compared prevalence of nAb to poliovirus type 1 (PV1), type 2 (PV2) and type 3 (PV3) in birth cohorts with differing IPV and OPV eligibility from an Australian population-based sample. In the total sample of 1673 persons aged 12 months to 99 years, 85% had nAb against PV1, 83% PV2 and 67% PV3. In the cohort 12 to18 years (eligible for 4 OPV doses, last dose 8-14 years prior), a significantly lower proportion had nAb than in the 7 to12 year cohort (eligible for 3 OPV doses and an IPV booster, last dose 3-8 years prior) for all poliovirus types: [PV1: 87.1% vs. 95.9% (P = 0.01), PV2: 80.4% vs. 92.9% (P = 0.003) and PV3: 38.1% vs. 84.0% (P 0.0001)]. These data suggest individual-level immunity may be better maintained when an OPV primary schedule is boosted by IPV, and support inclusion of an IPV booster in travel recommendations for young adults who previously received only OPV.

TO THE EDITOR—A recent study by He et al found that interleukin 1β (IL-1β) production during Zika virus (ZIKV) infection was driven by NLRP3 inflammasome activation [1]. NLRP3 activation was mediated by ZIKV nonstructural protein 5 (NS5), leading to IL-1β activation in a process also involving reactive oxygen species. This important study provides new insights into the role of IL-1β signaling in the pathogenesis of ZIKV disease and highlights a new avenue for therapeutic options.

Background: This study assessed the impact of the staged introduction of universal infant and adolescent catch-up hepatitis B vaccination programs on the prevalence of immunity and past hepatitis B virus (HBV) infection in targeted cohorts over almost a decade in Australia. Methods: We compared the prevalence of immunity in relevant cohorts of children and adolescents in repeated national serological surveys conducted in 1998-99, 2002 and 2007. Residual sera (n =2210) collected opportunistically from Australian laboratories in 2007 were tested for antibody to hepatitis B surface antigen (anti-HBs) indicating vaccine-induced immunity; sera from individuals aged 12-29 years with anti-HBs detected (n =386) were then tested for hepatitis B core antibody (anti-HBc) to identify past hepatitis B infection. Results: In 2007, compared with the baseline period of 1998-99, anti-HBs prevalence had increased significantly in all age groups below 24 years, by more than double in target children. Prevalence of anti-HBc was zero in the 12-14 years and reduced by 71% in those aged 15-19 years. The hepatitis B vaccination protected a significant number of targeted adolescents with a modest vaccine uptake (57% to 60% nationally). Conclusion: In a setting without incentives or school entry requirements, adolescent vaccination coverage was significantly higher when delivered by school-based rather than GP-based mechanisms. A cohort of children was growing up in Australia with a high prevalence of vaccineinduced immunity against hepatitis B, providing the best opportunity for controlling HBV infection in Australia.

Background The World Health Organization has targeted rubella virus for elimination regionally. Australia was one of the first countries to implement a nationally funded rubella immunisation program, in 1971, and conducts regular national rubella serosurveillance studies. We aimed to estimate the seroprevalence of rubella-specific IgG antibody in the Australian population by age and sex in 2012–2013, to compare the results with three previous serosurveys conducted in 1996–1999, 2002 and 2007 and to estimate the effective reproduction numbers (Rn). Methods This study used 2729 serum and plasma specimens, randomly selected from a specimen bank collected in 2012–2013 across Australia. Age groups included in the sample ranged from 1 to 49 years. Sera were tested for rubella-specific IgG-antibody using the Enzygnost anti-rubella IgG enzyme immunoassay and classified as positive, negative or equivocal according to rubella-specific IgG concentrations of >7 IU/ml, Results The overall proportions seropositive, seronegative and equivocal for rubella-specific IgG were 92.1% (95% CI, 91.0–93.2), 6.7% (95% CI, 5.7–7.7) and 1.2% (95% CI, 0.8–1.6), respectively. The proportion of males seropositive was significantly lower than females in the 30–34 (83.1% vs. 96.8%, p = 0.003), 35–39 (86.1% vs. 96.3%, p = 0.02) and 40–44 (86.1% vs. 95.7%, p = 0.03) year age groups. Rn for rubella in 2012–2013 was estimated to be 0.33 (95% CI 0.28–0.39). Discussion The 2012–2013 national serosurvey showed levels of rubella-specific IgG seropositivity in the Australian population are relatively high with no evidence of decrease compared to previous serosurveys conducted in 1996–1999, 2002 and 2007. The lower proportion of seropositive males aged 30–44 years likely reflects the initial immunisation program targeting females only. To our knowledge this study represents the longest period of serosurveillance following introduction of a nationally funded rubella immunisation program. The lack of evidence of decreasing rubella-specific IgG seropositivity is therefore reassuring for Australia and other countries with longstanding high vaccine coverage.

The recent global resurgence of arthritogenic alphaviruses, in particular chikungunya virus (CHIKV), highlights an urgent need for the development of therapeutic intervention strategies. While there has been significant progress in defining the pathophysiology of alphaviral disease, relatively little is known about the mechanisms involved in CHIKV-induced arthritis or potential therapeutic options to treat the severe arthritic symptoms associated with infection. Here, we used microcomputed tomographic (μCT) and histomorphometric analyses to provide previously undescribed evidence of reduced bone volume in the proximal tibial epiphysis of CHIKV-infected mice compared to the results for mock controls. This was associated with a significant increase in the receptor activator of nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) ratio in infected murine joints and in the serum of CHIKV patients. The expression levels of the monocyte chemoattractant proteins (MCPs), including MCP-1/CCL2, MCP-2/CCL8, and MCP-3/CCL7, were also highly elevated in joints of CHIKV-infected mice, accompanied by increased cellularity within the bone marrow in tibial epiphysis and ankle joints. Both this effect and CHIKV-induced bone loss were significantly reduced by treatment with the MCP inhibitor bindarit. Collectively, these findings demonstrate a unique role for MCPs in promoting CHIKV-induced osteoclastogenesis and bone loss during disease and suggest that inhibition of MCPs with bindarit may be an effective therapy for patients affected with alphavirus-induced bone loss. IMPORTANCE Arthritogenic alphaviruses, including chikungunya virus (CHIKV) and Ross River virus (RRV), cause worldwide outbreaks of polyarthritis, which can persist in patients for months following infection. Previous studies have shown that host proinflammatory soluble factors are associated with CHIKV disease severity. Furthermore, it is established that chemokine (C-C motif) ligand 2 (CCL2/MCP-1) is important in cellular recruitment and inducing bone-resorbing osteoclast (OC) formation. Here, we show that CHIKV replicates in bone and triggers bone loss by increasing the RANKL/OPG ratio. CHIKV infection results in MCP-induced cellular infiltration in the inflamed joints, and bone loss can be ameliorated by treatment with an MCP-inhibiting drug, bindarit. Taken together, our data reveal a previously undescribed role for MCPs in CHIKV-induced bone loss: one of recruiting monocytes/OC precursors to joint sites and thereby favoring a pro-osteoclastic microenvironment. This suggests that bindarit may be an effective treatment for alphavirus-induced bone loss and arthritis in humans.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus causing epidemics of acute and chronic arthritic disease. Herein we describe a comprehensive RNA-Seq analysis of feet and lymph nodes at peak viraemia (day 2 post infection), acute arthritis (day 7) and chronic disease (day 30) in the CHIKV adult wild-type mouse model. Genes previously shown to be up-regulated in CHIKV patients were also up-regulated in the mouse model. CHIKV sequence information was also obtained with up to ≈8% of the reads mapping to the viral genome; however, no adaptive viral genome changes were apparent. Although day 2, 7 and 30 represent distinct stages of infection and disease, there was a pronounced overlap in up-regulated host genes and pathways. Type I interferon response genes (IRGs) represented up to ≈50% of up-regulated genes, even after loss of type I interferon induction on days 7 and 30. Bioinformatic analyses suggested a number of interferon response factors were primarily responsible for maintaining type I IRG induction. A group of genes prominent in the RNA-Seq analysis and hitherto unexplored in viral arthropathies were granzymes A, B and K. Granzyme A-/-and to a lesser extent granzyme K-/-, but not granzyme B-/-, mice showed a pronounced reduction in foot swelling and arthritis, with analysis of granzyme A-/-mice showing no reductions in viral loads but reduced NK and T cell infiltrates post CHIKV infection. Treatment with Serpinb6b, a granzyme A inhibitor, also reduced arthritic inflammation in wild-type mice. In non-human primates circulating granzyme A levels were elevated after CHIKV infection, with the increase correlating with viral load. Elevated granzyme A levels were also seen in a small cohort of human CHIKV patients. Taken together these results suggest granzyme A is an important driver of arthritic inflammation and a potential target for therapy. Trial Registration: ClinicalTrials.gov NCT00281294 © 2017 Wilson et al.

Pertussis seroepidemiology and associated factors in older adults aged ≥40 years with and without acute myocardial infarction (AMI) were studied to investigate whether unrecognised pertussis may precipitate AMI. Sera were obtained from a previous case-control study investigating the role of influenza in precipitating AMIs. Baseline sera were considered pertussis toxin (PT) IgG seropositive at levels ≥5 IU/mL. Levels ≥v62.5 IU/mL were considered indicative of infection in the previous year, and recent infection was indicative at levels ≥125 IU/mL. Of the serum samples tested, 55% (122/222) were seropositive for PT IgG, 5% (11/222) had evidence of infection in the past year and 1.4% (3/222) had evidence of recent infection. Evidence of infection in the past year was found in 3.2% of those aged 65-74 years. Overall, 47.8% of 40-64 year olds and 43.2% of those aged ≥65 years were seronegative for pertussis. Serological evidence of pertussis was not associated with AMI (46/92, 50.0% cases vs. 76/130, 58.5% controls, p=0.2). After adjusting for age, AMI and self-reported pertussis and GP verified influenza vaccination, females (OR = 2.2, 95% CI = 1.1-4.1, p=0.02) were more likely to be seronegative. Just under half of participants had no detectable pertussis immunity and are therefore susceptible to infection. Our study supports the need for an adult pertussis booster to supplement current recommendations.

This study evaluates the evidence for elimination of rubella and congenital rubella syndrome (CRS) in Australia, drawing on three national serosurveys conducted between 1996 and 2007 and supported by statutory notification and vaccine coverage data. Anti-rubella IgG seropositivity was defined as ≥ 10 IU/ml by EIA. Between 1998 and 2007, rubella notifications fell >100-fold, to an average of 2 cases per million and there were five confirmed cases of CRS, two of which were locally acquired in 2003. Weighted overall seropositivity remained constant among 1-49 year-olds (89.6% in 1999; 88.1% in 2007). Between 2002 and 2009, 95% of children received at least one dose of the measles-mumps-rubella (MMR) vaccine. All three serosurveys provided estimates for R less than 0.5, well below the epidemic threshold of 1. All available data are supportive of Australia being considered for elimination status. Further reductions in incidence of CRS will require continued attention to vaccine coverage in overseas-born women, as well as the maintenance of current high coverage level of two-dose MMR vaccination.

A marine-derived compound, abalone hemocyanin, from Haliotis rubra was shown to have a unique mechanism of antiviral activity against herpes simplex virus 1 (HSV-1) infections. In vitro assays demonstrated the dose-dependent and inhibitory effect of purified hemocyanin against HSV-1 infection in Vero cells with a 50% effective dose (ED 50 ) of 40 to 50 nM and no significant toxicity. In addition, hemocyanin specifically inhibited viral attachment and entry by binding selectively to the viral surface glycoproteins gD, gB, and gC, probably by mimicking their receptors. However, hemocyanin had no effect on postentry events and did not block infection by binding to cellular receptors for HSV. By the use of different mutants of gD and gB and a competitive heparin binding assay, both protein charge and conformation were shown to be the driving forces of the interaction between hemocyanin and viral glycoproteins. These findings also suggested that hemocyanin may have different motifs for binding to each of the viral glycoproteins B and D. The dimer subunit of hemocyanin with a 10-fold-smaller molecular mass exhibited similar binding to viral surface glycoproteins, showing that the observed inhibition did not require the entire multimer. Therefore, a small hemocyanin analogue could serve as a new antiviral candidate for HSV infections.

Acute flaccid paralysis (AFP) has a changing epidemiology with ongoing polio outbreaks and emerging causes such as nonpolio enteroviruses and West Nile virus (WNV). We report a case of AFP from the Horn of Africa that was initially classified as probable polio but subsequently found to be due to WNV.

Background. Aboriginal children in central Australia have attach rates for acute lower respiratory tract infection (ALRI) that are similar to those in developing countries. Although mortality rates are much lower than in developing countries, morbidity is high and ALRI is still the leading cause of hospitalization. However, there are no data on the etiology of ALRI in this population. Methods. We prospectively studied 322 cases of ALRI in 280 Aboriginal children admitted to the hospital. Blood, urine and nasopharyngeal aspirate samples were examined for evidence of bacterial, viral and chlamydial infection. Results. The combination of blood culture, viral studies and chlamydial serology provided at least 1 etiologic agent in 170 of 322 (52.5%) cases. Assays for pneumolysin immune complex and pneumolysin antibody increased etiologic diagnosis to 219 (68.0%). Blood cultures were positive in 6% but pneumolysin immune complex and pneumolysin antibody studies were positive in one-third of cases. Evidence of viral infection was present in 155 (48%) of cases compared with 12% in controls (P < 001). There were only 7 possible cases and 2 definite cases of Chlamydia trachomatis and 3 cases of Chlamydia pneumoniae. Coinfection was common in these children. Conclusion. These findings have implications for both standard treatment protocols and vaccine strategies, The high rate of coinfection may make it difficult to develop simple clinical predictors of bacterial infection. In the setting of a developed country with efficient patient evacuation services, management algorithms that focus on disease severity and need for hospital referral will be most useful to health staff in remote. communities. Pneumococcal conjugate vaccines will be required to reduce the high attach rate of pneumococcal disease.

Barmah Forest virus (BFV) and Ross River virus (RRV) are mosquito-borne viruses with similar vectors and environmental requirements. They cause diseases characterised by arthralgia, arthritis and myalgia, often accompanied by fever and rash. Arthritis is more common and more prominent in RRV disease and rash is more common and florid with BFV infection, although the diseases cannot be reliably distinguished by their clinical symptoms. Diagnosis is based on serological tests and a definite diagnosis of recent infection requires the demonstration of rising titres of IgG. Arthralgia, myalgia and lethargy may continue for at least six months in up to half of patients with RRV, but in only about 10% of patients with BFV. Both diseases are managed symptomatically.

Murray Valley encephalitis (MVE) is an important mosquitoborne flavivirus infection endemic to Australia and Papua New Guinea. We report the first imported case of MVE in Europe. A 23-year-old tourist developed severe encephalitis after having returned to Germany from a long-term trip across the Australian continent. The diagnosis was suspected on the basis of clinical findings and the patient's travel history and was confirmed by serological findings. The patient made a prolonged but complete recovery. Our case coincides with a recently reported spread of MVE virus in Australia. This emphasizes the need for continuous surveillance in areas of endemicity and appropriate protection when traveling through regions in which the MVE virus is endemic.

The pertussis epidemic experienced in NSW in 2008–2009 was likely to be in part due to changes in diagnostic practice since 2007, which amplified disease notifications. We used population-based seroepidemiology as a less biased means of interpreting age-specific pertussis infection patterns in NSW from three serosurveys undertaken in 1997–98 (during an epidemic), 2002 (post-epidemic) and 2007 (inter-epidemic), using a standardised pertussis toxin IgG enzyme-linked immunosorbent assay (ELISA). There was a decrease in the proportion of high anti-pertussis toxin IgG titres (>62.5 ELISA Units/mL) across all age groups in the 2007 serosurvey compared to the previous two serosurveys. In the 2007 serosurvey, the proportion of undetectable (

BackgroundCross-sectional serosurveys using IgG antibody to pertussis toxin (IgG-PT) are increasingly being used to estimate trends in recent infection independent of reporting biases.Methods/principal findingsWe compared the age-specific seroprevalence of various levels of IgG-PT in cross-sectional surveys using systematic collections of residual sera from Australian diagnostic laboratories in 1997/8, 2002 and 2007 with reference to both changes in the pertussis vaccine schedule and the epidemic cycle, as measured by disease notifications. A progressive decline in high-level (≥62.5 EU/ml) IgG-PT prevalence from 19% (95% CI 16-22%) in 1997/98 to 12% (95% CI 11-14%) in 2002 and 5% (95% CI 4-6%) in 2007 was consistent with patterns of pertussis notifications in the year prior to each collection. Concomitantly, the overall prevalence of undetectable (Conclusions/significancePopulation seroprevalence of high levels of IgG-PT is accepted as a reliable indicator of pertussis disease activity over time within and between countries with varying diagnostic practices, especially in unimmunised age groups. Our novel findings suggest that increased prevalence of undetectable IgG-PT is an indicator of waning immunity useful for population level monitoring following introduction of acellular vaccines and/or schedule changes.

The impact of pandemic (H1N1) 2009 influenza in HIV-infected individuals is unknown. Determining the prevalence of pandemic influenza in this at-risk group will guide vaccination programs. After the first pandemic wave, the seroprevalence rate of pandemic influenza in HIV-infected individuals in western Sydney, New South Wales, Australia, was 34.2%, similar to the rate observed in the general population. However, true seroprevalence is more accurately determined by seroconversion, defined as a 4-fold or greater rise between preexposure and postexposure antibody levels, which was 14.6% in the present study. Seroconversion rates were independent of CD4+ T-lymphocyte count and HIV plasma load. Neither HIV infection, nor severe immunosuppression, was a significant risk factor for pandemic influenza during the first southern hemisphere pandemic wave.

Background The first wave of pandemic influenza A(H1N1)2009 (pH1N1) reached New South Wales (NSW), Australia in May 2009, and led to high rates of influenza-related hospital admission of infants and young to middle-aged adults, but no increase in influenza-related or all-cause mortality. Methodology/Principal Findings To assess the population rate of pH1N1 infection in NSW residents, pH1N1-specific haemagglutination inhibition (HI) antibody prevalence was measured in specimens collected opportunistically before (2007–2008; 474 specimens) and after (August–September 2009; 1247 specimens) the 2009 winter, and before the introduction of the pH1N1 monovalent vaccine. Age- and geographically-weighted population changes in seroprevalence were calculated. HI antibodies against four recent seasonal influenza A viruses were measured to assess cross-reactions. Pre- and post-pandemic pH1N1 seroprevalences were 12.8%, and 28.4%, respectively, with an estimated overall infection rate of 15.6%. pH1N1 antibody prevalence increased significantly - 20.6% overall - in people born since 1944 (26.9% in those born between 1975 and 1997) but not in those born in or before 1944. People born before 1925 had a significantly higher pH1N1 seroprevalence than any other age-group, and against any seasonal influenza A virus. Sydney residents had a significantly greater change in prevalence of antibodies against pH1N1 than other NSW residents (19.3% vs 9.6%). Conclusions/Significance Based on increases in the pH1N1 antibody prevalence before and after the first pandemic wave, 16% of NSW residents were infected by pH1N1 in 2009; the highest infection rates (27%) were among adolescents and young adults. Past exposure to the antigenically similar influenza A/H1N1(1918) is the likely basis for a very high prevalence (49%) of prepandemic cross-reacting pH1N1 antibody and sparing from pH1N1 infection among people over 85 years. Unless pre-season vaccine uptake is high, there are likely to be at least moderate rates including some life-threatening cases of pH1N1 infection among young people during subsequent winters.

Barmah Forest virus (BFV) is a commonly occurring arbovirus in Australia. Notifications of Barmah Forest infections diagnosed by a single positive IgM serology test have been increasing in coastal New South Wales north of Newcastle. We report on a 6 month prospective review of all routine notifications of BFV from the Lower Mid North Coast of New South Wales. Sera from 37 consecutive cases were sent for confirmatory testing by ELISA and neutralisation assays and 32 cases were interviewed. On confirmatory testing, 7 patients' sera (19%) was found to contain no BFV antibodies and 6 (16%) had BFV IgG only. Only 4 cases had antibody levels compatible with recent infection. A clinical presentation of fever with either rash or joint pain was associated with confirmation of recent BFV infection. On the basis of these findings, caution is advised in the interpretation of a single positive IgM for Barmah Forest disease and the clinical picture is an important factor in the diagnosis. Serological notifications of BFV alone should not prompt public health action such as public warning and targeted vector control in endemic areas.

The recent global resurgence of arthritogenic alphaviruses, in particular chikungunya virus (CHIKV), highlights an urgent need for the development of therapeutic intervention strategies. While there has been significant progress in defining the pathophysiology of alphaviral disease, relatively little is known about the mechanisms involved in CHIKV-induced arthritis or potential therapeutic options to treat the severe arthritic symptoms associated with infection. Here, we used microcomputed tomographic (μCT) and histomorphometric analyses to provide previously undescribed evidence of reduced bone volume in the proximal tibial epiphysis of CHIKV-infected mice compared to the results for mock controls. This was associated with a significant increase in the receptor activator of nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) ratio in infected murine joints and in the serum of CHIKV patients. The expression levels of the monocyte chemoattractant proteins (MCPs), including MCP-1/CCL2, MCP-2/CCL8, and MCP-3/CCL7, were also highly elevated in joints of CHIKV-infected mice, accompanied by increased cellularity within the bone marrow in tibial epiphysis and ankle joints. Both this effect and CHIKV-induced bone loss were significantly reduced by treatment with the MCP inhibitor bindarit. Collectively, these findings demonstrate a unique role for MCPs in promoting CHIKV-induced osteoclastogenesis and bone loss during disease and suggest that inhibition of MCPs with bindarit may be an effective therapy for patients affected with alphavirus-induced bone loss.Arthritogenic alphaviruses, including chikungunya virus (CHIKV) and Ross River virus (RRV), cause worldwide outbreaks of polyarthritis, which can persist in patients for months following infection. Previous studies have shown that host proinflammatory soluble factors are associated with CHIKV disease severity. Furthermore, it is established that chemokine (C-C motif) ligand 2 (CCL2/MCP-1) is important in cellular recruitment and inducing bone-resorbing osteoclast (OC) formation. Here, we show that CHIKV replicates in bone and triggers bone loss by increasing the RANKL/OPG ratio. CHIKV infection results in MCP-induced cellular infiltration in the inflamed joints, and bone loss can be ameliorated by treatment with an MCP-inhibiting drug, bindarit. Taken together, our data reveal a previously undescribed role for MCPs in CHIKV-induced bone loss: one of recruiting monocytes/OC precursors to joint sites and thereby favoring a pro-osteoclastic microenvironment. This suggests that bindarit may be an effective treatment for alphavirus-induced bone loss and arthritis in humans.

Dendritic cells (DCs) are an important early target cell for many mosquito-borne viruses, and in many cases mosquito-cell-derived arboviruses more efficiently infect DCs than viruses derived from mammalian cells. However, whether mosquito-cell-derived viruses differ from mammalian-cell-derived viruses in their ability to induce antiviral responses in the infected dendritic cell has not been evaluated. In this report, alphaviruses, which are mosquito-borne viruses that cause diseases ranging from encephalitis to arthritis, were used to determine whether viruses grown in mosquito cells differed from mammalian-cell-derived viruses in their ability to induce type I interferon (IFN) responses in infected primary dendritic cells. Consistent with previous results, mosquito-cell-derived Ross River virus (mos-RRV) and Venezuelan equine encephalitis virus (mos-VEE) exhibited enhanced infection of primary myeloid dendritic cells (mDCs) compared to mammalian-cell-derived virus preparations. However, unlike the mammalian-cell-derived viruses, which induced high levels of type I IFN in the infected mDC cultures, mos-RRV and mos-VEE were poor IFN inducers. Furthermore, the poor IFN induction by mos-RRV contributed to the enhanced infection of mDCs by mos-RRV. These results suggest that the viruses initially delivered by the mosquito vector differ from those generated in subsequent rounds of replication in the host, not just with respect to their ability to infect dendritic cells but also in their ability to induce or inhibit antiviral type I IFN responses. This difference may have an important impact on the mosquito-borne virus's ability to successfully make the transition from the arthropod vector to the vertebrate host.