Potential Serological Misdiagnosis of Barmah Forest Virus and Ross River Virus Diseases as Chikungunya Virus Infections in Australia: Comparison of ELISA with Neutralization Assay Results.

To evaluate the frequency of errors in the diagnosis of medical laboratory-diagnosed Chikungunya virus (CHIKV) infections in Australia, we studied 42 laboratory-diagnosed CHIKV serum samples from one Queensland medical laboratory by ELISA IgG/IgM and measured the specific neutralization antibodies (Nab) against Barmah Forest virus (BFV), CHIKV and Ross River virus (RRV). The sero-positivity rates for the sera were as follows: anti-BFV IgG+ 19% (8/42), IgM+ 2.4% (1/42) and Nab+ 16.7% (7/42); anti-CHIKV IgG+ 90.5% (38/42), IgM+ 21.4% (9/42) and Nab+ 90.5% (38/42); anti-RRV IgG+ 88.1% (37/42), IgM+ 28.6% (12/42) and Nab+ 83.2% (35/42), respectively. Among the samples with multiple antibody positivity, 2.4% (1/42) showed triple ELISA IgM+, and 14.3% (6/42) exhibited double IgM RRV+CHIKV+; 9.5% (4/42) showed triple IgG+, 76.2% (32/42) displayed double IgG RRV+CHIKV+, 4.8% (2/42) showed IgG BFV+RRV+ and 4.8% (2/42) showed IgG BFV++CHIKV+; and 9.5% (4/42) showed triple Nab+ and 69% (29/42) exhibited double Nab RRV+CHIKV+, respectively. Our analysis of the single-virus infection control Nab results suggested no cross-neutralization between RRV and BFV, and only mild cross-neutralization between CHIKV and RRV, BFV and CHIKV, all with a ≥4-fold Nab titre ratio difference between the true virus infection and cross-reactivity counterpart virus. Subsequently, we re-diagnosed these 42 patients as 1 BFV+, 8 CHIKV+ and 23 RRV+ single-virus infections, along with five RRV+/BFV+ and four RRV+/CHIKV+ double infections, and one possible RRV+/BFV+ or RRV+CHIKV+, respectively. These findings suggests that a substantial proportion of medically attended RRV and BFV infections were misdiagnosed as CHIKV infections, highlighting the imperative need for diagnostic laboratory tests capable of distinguishing between CHIKV infections and actively co-circulating RRV and BFV. For a correct diagnosis, it is crucial to consider reliable diagnostic methods such as the neutralization assay to exclude RRV and BFV.

Prevalence of Barmah Forest Virus, Chikungunya Virus and Ross River Virus Antibodies among Papua New Guinea Military Personnel before 2019.

Barmah Forest virus (BFV), Chikungunya virus (CHIKV) and Ross River virus (RRV) belong to the Alphavirus genus of the family Togaviridae. All three virus infections have been reported in Papua New Guinea (PNG) previously, but the exact prevalence and distribution of these three alphaviruses in PNG has not been established. Sera collected from 204 PNG Military Personnel (PNGMP) study participants in April 2019 was tested for the presence of anti-BFV, anti-CHIKV and anti-RRV immunoglobulin G (IgG) antibodies using commercially available enzyme-linked immunosorbent assay (ELISA) IgG detection kits, as well as for specific neutralizing antibodies (NAb) against individual viruses. Overall, sero-positivity of the sera was anti-BFV IgG 12.3% (25/204), anti-BFV NAb 8.3% (17/204); anti-CHIKV IgG 47.1% (96/204), anti-CHIKV NAb 34.8% (71/204); and anti-RRV IgG 93.1% (190/204), anti-RRV NAb 56.4% (115/204), respectively. Of the 137/204 participants that were Nab-positive for at least one virus, we identified 4 BFV, 40 CHIKV and 73 RRV single infections, and 9 RRV+CHIKV and 11 BFV+RRV double infections. The lower proportion of NAb sero-positive compared to the ELISA IgG sero-positive assay samples suggests that the currently available commercial ELISA detection kits for these three alphaviruses may not be suitable for diagnostic/surveillance purposes in endemic areas such as PNG, due to serological cross-reactivity among these three alphaviruses. Laboratory testing using known positive control sera indicated no cross-neutralization between BFV and RRV; however, some RRV or BFV single infection human sera demonstrated low-level cross-neutralization against CHIKV (the ratio of RRV/CHIKV NAb titers or BFV/CHIKV ≥ 4). Our preliminary results indicate that the majority of PNGMP have previously been exposed to RRV, with mild exposure to CHIKV and low-level exposure to BFV, suggesting that multiple alphaviruses have been circulating among PNGMP. The transmission landscapes of these three alphaviruses across PNG should be prioritized for further investigation, including identification of specific vectors and hosts that mediate human spillover in order to mitigate future outbreaks. Ongoing education regarding precautionary and protective measures are needed to better protect individuals who travel to PNG.

Serological evidence of possible high levels of undetected transmission of Zika virus among Papua New Guinea military personnel, 2019.

Objectives: The Papua New Guinea (PNG) Health Department retrospectively reported six cases of Zika virus (ZIKV) from a cohort of febrile patients during outbreaks of dengue and malaria in 2016. However, the transmission of ZIKV remains unclear due to lack of testing capability. This study aimed to determine the level of immunity to ZIKV among PNG military personnel (PNGMP) in 2019. Methods: Sera of 208 PNGMP recruited in April 2019 was tested for the presence of anti-ZIKV immunoglobulin G (IgG) and M (IgM) antibodies using Euroimmun IgG/IgM detection kits, and anti-ZIKV neutralizing antibody (Nab) against a ZIKV African strain on all anti-ZIKV-IgG/IgM+ samples. Results: Anti-ZIKV seropositivity of these sera was as follows: IgG, 67%; IgM, 9%; and Nab, 65%. Five of 19 anti-ZIKV-IgM+ samples had anti-ZIKV-Nab titres ≥20, as well as an anti-ZIKV-Nab titre ratio ≥4 compared with the Nab titres of four anti-dengue serotypes, so met the criteria of the World Health Organization (WHO) for confirmed ZIKV infection. Conclusions: The prevalence of anti-ZIKV-Nab of 65% suggests that there are high levels of ZIKV exposure among PNGMP. Five of the 19 anti-ZIKV-IgM+ samples met the WHO criteria for confirmed ZIKV infection, suggesting a recent undetected outbreak in PNGMP. These results provide better understanding of the current ZIKV epidemic status in PNGMP.

Seroprevalence of chikungunya virus among military personnel in Papua New Guinea, 2019.

Objectives: The first outbreak of chikungunya virus (CHIKV) was reported in West Sepik, Papua New Guinea (PNG) in June 2012, and spread rapidly throughout PNG. CHIKV imported from PNG to Queensland has been reported occasionally, but transmission of CHIKV in PNG remains unclear due to the lack of testing capability. This study investigated the degree of CHIKV exposure among PNG military personnel (PNGMP) in 2019, 7 years after its first emergence. Methods: Sera of 204 PNGMP recruited in April 2019 was tested for the presence of anti-CHIKV immunoglobulin G (IgG) antibodies using a commercially available IgG detection kit, and anti-CHIKV neutralizing antibodies against a CHIKV Reunion strain using a neutralizing assay. Results: Anti-CHIKV seropositivity of the sera was 47% and 35%, respectively, using the enzyme-linked immunosorbent assay (ELISA) and neutralizing assay. Five percent (n=11) of samples were found to be IgG negative or borderline, but neutralizing antibody positive. Conclusions: The prevalence of anti-CHIKV neutralizing antibody of 35% suggests that CHIKV infection has become endemic among PNGMP. Current commercially available CHIKV ELISA detection kits may not be suitable for diagnostic purposes in multiple alphavirus endemic areas such as PNG, due to serological cross-reactivity among alphaviruses. Re-emergence of CHIKV in PNGMP is possible.

Circulation of 2 Barmah Forest Virus Lineages in Military Training Areas, Australia.

During 2017-2018, Barmah Forest virus was recovered from mosquitoes trapped in military training areas in Australia and from a soldier infected at 1 of these areas. Phylogenies of the nucleotide sequences of the envelope glycoprotein gene E2 and the 3' untranslated region suggest that 2 lineages are circulating in eastern Australia.

Genetic, Morphological and Antigenic Relationships between Mesonivirus Isolates from Australian Mosquitoes and Evidence for Their Horizontal Transmission.

The Mesoniviridae are a newly assigned family of viruses in the order Nidovirales. Unlike other nidoviruses, which include the Coronaviridae, mesoniviruses are restricted to mosquito hosts and do not infect vertebrate cells. To date there is little information on the morphological and antigenic characteristics of this new group of viruses and a dearth of mesonivirus-specific research tools. In this study we determined the genetic relationships of recent Australian isolates of Alphamesonivirus 4 (Casuarina virus-CASV) and Alphamesonivirus 1 (Nam Dinh virus-NDiV), obtained from multiple mosquito species. Australian isolates of NDiV showed high-level similarity to the prototype NDiV isolate from Vietnam (99% nucleotide (nt) and amino acid (aa) identity). Isolates of CASV from Central Queensland were genetically very similar to the prototype virus from Darwin (95-96% nt and 91-92% aa identity). Electron microscopy studies demonstrated that virion diameter (≈80 nm) and spike length (≈10 nm) were similar for both viruses. Monoclonal antibodies specific to CASV and NDiV revealed a close antigenic relationship between the two viruses with 13/34 mAbs recognising both viruses. We also detected NDiV RNA on honey-soaked nucleic acid preservation cards fed on by wild mosquitoes supporting a possible mechanism of horizontal transmission between insects in nature.

Genome Sequences of Barmah Forest Virus Strains Isolated from Mosquitoes Trapped in Australian Defence Force Training Areas Reveal Multiple Nucleotide Insertions in the 3' Untranslated Region.

The complete genome sequences of two Barmah Forest virus (BFV) strains isolated from mosquitoes trapped in the Australian Defence Force (ADF) training areas during 2017 and 2018 reveal multiple nucleotide insertions in the 3' untranslated region (UTR) of ADF BFV strains compared with the BFV prototype strain whole-genome sequence in GenBank.

Localized Outbreaks of Epidemic Polyarthritis among Military Personnel Caused by Different Sublineages of Ross River Virus, Northeastern Australia, 2016-2017.

Two outbreaks of epidemic polyarthritis occurred among Australian Defence Force personnel during and following short military exercises in the Shoalwater Bay Training Area, northeastern Australia, in 2016 and 2017. Ross River virus (RRV) IgM was detected in acute-phase serum samples from most patients (28/28 in 2016 and 25/31 in 2017), and RRV was recovered from 4/38 serum samples assayed (1/21 in 2016 and 3/17 in 2017). Phylogenetic analyses of RRV envelope glycoprotein E2 and nonstructural protein nsP3 nucleotide sequences segregated the RRV isolates obtained in 2016 and 2017 outbreaks into 2 distinct sublineages, suggesting that each outbreak was caused by a different strain of RRV. The spatiotemporal characteristics of the 2016 outbreak suggested that some of the infections involved human-mosquito-human transmission without any intermediate host. These outbreaks highlight the importance of personal protective measures in preventing vectorborne diseases for which no vaccine or specific prophylaxis exists.

Genome Sequences of Three Ross River Virus Isolates Obtained from the Australian Defence Force.

The complete genome sequences of three Ross River virus (RRV) isolates from infected Australian Defence Force (ADF) personnel and from mosquitoes collected in ADF training areas were determined. Phylogenetic analysis in comparison with all available complete RRV nucleotide sequences from GenBank split these three RRV isolates into two distinct sublineages.