Detection of Clade 2.3.4.4b Avian Influenza A(H5N8) Virus in Cambodia, 2021.

In late 2021, highly pathogenic avian influenza A(H5N8) clade 2.3.4.4b viruses were detected in domestic ducks in poultry markets in Cambodia. Surveillance, biosafety, and biosecurity efforts should be bolstered along the poultry value chain to limit spread and infection risk at the animal-human interface.

Analysis of acidified feed components containing African swine fever virus.

Mitigation of African swine fever (ASF) virus in contaminated feed materials would assist control activities. Various finely-ground pig feed ingredients (5 cereals, 4 plant proteins, 2 animal proteins, 1 oil, 1 compound) were sprayed and mixed thoroughly with a buffered formic acid formulation (0, 1 or 2% vol/vol) to produce a consistent and durable level of formate (1% or 2%) with consistent acidification of cereal ingredients to less than pH 4. No such acidification was noted in other ingredients. Selected representative feed ingredients were further mixed with infectious ASF virus (106 TCID50) or media alone and incubated for 0, 6, 12, 24, 48, 72 or 168 h. The residual ASF virus at each timepoint was quantified using qPCR and a cell culture based TCID50 assay to determine survivability. Maize, rice bran and compound feed (with or without formate) all reduced infectious ASF virus to levels below the detection threshold of the cell culture assay (101.3 TCID50/mL). A consistent reduction in ASF virus DNA levels was observed by qPCR assay when maize containing ASF virus was mixed with 1% or 2% buffered formic acid. This reduction in viral DNA corresponded to the acidifying pH effect measured. No such reduction in ASF virus DNA levels was noted in non-cereal ingredients containing ASF virus, in which the pH had not been lowered below pH 4 following treatment. Interestingly, residual ASF virus levels in spiked meat/bone meal were greater than control levels, suggesting a buffering effect of that feed ingredient.

In vitro characterisation of SARS-CoV-2 and susceptibility of domestic ferrets (Mustela putorius furo).

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an emerging virus that has caused significant human morbidity and mortality since its detection in late 2019. With the rapid emergence has come an unprecedented programme of vaccine development with at least 300 candidates under development. Ferrets have proven to be an appropriate animal model for testing safety and efficacy of SARS-CoV-2 vaccines due to quantifiable virus shedding in nasal washes and oral swabs. Here, we outline our efforts early in the SARS-CoV-2 outbreak to propagate and characterize an Australian isolate of the virus in vitro and in an ex vivo model of human airway epithelium, as well as to demonstrate the susceptibility of domestic ferrets (Mustela putorius furo) to SARS-CoV-2 infection following intranasal challenge.

Correction: ADAMTS5 Is a Critical Regulator of Virus-Specific T Cell Immunity.

[This corrects the article DOI: 10.1371/journal.pbio.1002580.].

A Review of DNA Vaccines Against Influenza.

The challenges of effective vaccination against influenza are gaining more mainstream attention, as recent influenza seasons have reported low efficacy in annual vaccination programs worldwide. Combined with the potential emergence of novel influenza viruses resulting in a pandemic, the need for effective alternatives to egg-produced conventional vaccines has been made increasingly clear. DNA vaccines against influenza have been in development since the 1990s, but the initial excitement over success in murine model trials has been tempered by comparatively poor performance in larger animal models. In the intervening years, much progress has been made to refine the DNA vaccine platform-the rational design of antigens and expression vectors, the development of novel vaccine adjuvants, and the employment of innovative gene delivery methods. This review discusses how these advances have been applied in recent efforts to develop an effective influenza DNA vaccine.

Isolation of a divergent strain of Rickettsia japonica from Dew's Australian bat Argasid ticks (Argas (Carios) dewae) in Victoria, Australia.

A divergent strain of Rickettsia japonica was isolated from a Dew's Australian bat argasid tick, Argas (Carios) dewae, collected in southern Victoria, Australia and a full-genome analysis along with sequencing of 5 core gene fragments was undertaken. This isolate was designated Rickettsia japonica str. argasii (ATCC VR-1665, CSUR R179).

Enhanced immunogenicity following miR-155 incorporation into the influenza A virus genome.

Influenza A vaccine efficacy in the elderly is generally poor and so identification of novel molecular adjuvants to improve immunogenicity is important to reduce the overall burden of disease. Short non-coding RNAs, known as microRNAs (miRNAs) are known to regulate gene expression and have the potential to influence immune responses. One such miRNA, miR-155, has been shown to modulate T and B cell development and function. We incorporated miR-155 into the influenza A virus (IAV) genome creating a self-adjuvanting 'live vaccine' with the ability to modify immunogenicity. Infection of mice with a recombinant influenza virus encoding miR-155 in the NS gene segment altered epitope-specific expansion of influenza-specific CD8+ T cells and induced significantly higher levels of neutralising antibody.

ADAMTS5 Is a Critical Regulator of Virus-Specific T Cell Immunity.

The extracellular matrix (ECM) provides physical scaffolding for cellular constituents and initiates biochemical and biomechanical cues that are required for physiological activity of living tissues. The ECM enzyme ADAMTS5, a member of the ADAMTS (A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs) protein family, cleaves large proteoglycans such as aggrecan, leading to the destruction of cartilage and osteoarthritis. However, its contribution to viral pathogenesis and immunity is currently undefined. Here, we use a combination of in vitro and in vivo models to show that ADAMTS5 enzymatic activity plays a key role in the development of influenza-specific immunity. Influenza virus infection of Adamts5-/- mice resulted in delayed virus clearance, compromised T cell migration and immunity and accumulation of versican, an ADAMTS5 proteoglycan substrate. Our research emphasises the importance of ADAMTS5 expression in the control of influenza virus infection and highlights the potential for development of ADAMTS5-based therapeutic strategies to reduce morbidity and mortality.

miRNA modulation of SOCS1 using an influenza A virus delivery system.

Difficulties associated with efficient delivery and targeting of miRNAs to cells is hampering the real world application of miRNA technology. This study utilized an influenza A-based delivery system to express miR-155 in order to knockdown SOCS1 mRNA. Using qPCR and dual luciferase technology we show that miR-155 delivery resulted in a significant increase in cellular miR-155 which facilitated a downregulation of SOCS1 gene expression and a functional increase in IL-6 and IFN-ß cytokines.

Diversity of the 47-kD HtrA nucleic acid and translated amino acid sequences from 17 recent human isolates of Orientia.

Orientia tsutsugamushi, the etiologic agent of potentially fatal scrub typhus, is characterized by a high antigenic diversity, which complicates the development of a broadly protective vaccine. Efficacy studies in murine and nonhuman primate models demonstrated the DNA vaccine candidate pKarp47, based upon the O. tsutsugamushi Karp 47-kD HtrA protein gene, to be a successful immunoprophylactic against scrub typhus. To characterize 47-kD HtrA protein diversity among human isolates of Orientia, we sequenced the full open reading frame (ORF) of the 47-kD HtrA gene and analyzed the translated amino acid sequences of 17 patient isolates from Thailand (n=13), Laos (n=2), Australia (n=1), and the United Arab Emirates (UAE) (n=1) and 9 reference strains: Karp (New Guinea), Kato (Japan), Ikeda (Japan), Gilliam (Burma), Boryong (Korea), TA763, TH1811 and TH1817 (Thailand), and MAK243 (China). The percentage identity (similarity) of translated amino acid sequences between 16 new isolates and 9 reference strains of O. tsutsugamushi ranged from 96.4% to 100% (97.4% to 100%). However, inclusion of the recently identified Orientia chuto sp. nov. reduced identity (similarity) values to 82.2% to 83.3% (90.4% to 91.4%). These results demonstrate the diversity of Orientia 47-kD HtrA among isolates encountered by humans and therefore provide support for the necessity of developing a broadly protective scrub typhus vaccine that takes this diversity into account.

Promotion of Hendra virus replication by microRNA 146a.

Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease.

First probable Australian cases of human infection with Rickettsia felis (cat-flea typhus).

Human infection with Rickettsia felis has been reported in most parts of the world, and R. felis has recently been confirmed in cat fleas in Western Australia. The clinical presentations of R. typhi and R. felis are similar, and in the past, the incidence of R. felis infection may have been underestimated. We describe the first reported cases of probable human R. felis infection in Australia. Two adults and three children in Victoria contracted a rickettsial disease after exposure to fleas from kittens. Molecular testing of fleas demonstrated the presence of R. felis but not R. typhi.

Microbial ecology of autothermal thermophilic aerobic digester (ATAD) systems for treating waste activated sludge.

Despite their widespread use, our understanding of the microbial ecology of the autothermal thermophilic aerobic digesters (ATAD) used to dispose of sludge from wastewater treatment plants is poor. Applying both culture-dependent and molecular methods to two ATAD systems in Victoria, Australia treating different wastewaters revealed that their communities were highly specialized. Denaturing gradient gel electrophoresis (DGGE) profiling suggested differences in their population compositions and both changed over time. However, both showed low level biodiversity, and contained several novel bacterial populations. 16S rRNA clone library data and FISH analyses showed that Thermus thermophilus dominated both communities and that of a third ATAD plant in NSW (more than 90% of the total bacterial biovolume in repeated samples taken from each of the three ATAD plants). Culture-dependent methods also showed Geobacillus spp. were present in both Victorian communities. Nevertheless, the ecophysiology of these populations and their putative roles in sludge digestion remain unclear. FISH/microautoradiographic studies did not provide conclusive data elucidating which substrate/s T. thermophilus might utilize in the ATAD reactors.

Isolation of a novel Orientia species (O. chuto sp. nov.) from a patient infected in Dubai.

In July 2006, an Australian tourist returning from Dubai, in the United Arab Emirates (UAE), developed acute scrub typhus. Her signs and symptoms included fever, myalgia, headache, rash, and eschar. Orientia tsutsugamushi serology demonstrated a 4-fold rise in antibody titers in paired serum collections (1:512 to 1:8,192), with the sera reacting strongest against the Gilliam strain antigen. An Orientia species was isolated by the in vitro culture of the patient's acute blood taken prior to antibiotic treatment. The gene sequencing of the 16S rRNA gene (rrs), partial 56-kDa gene, and the full open reading frame 47-kDa gene was performed, and comparisons of this new Orientia sp. isolate to previously characterized strains demonstrated significant sequence diversity. The closest homology to the rrs sequence of the new Orientia sp. isolate was with three strains of O. tsutsugamushi (Ikeda, Kato, and Karp), with a nucleotide sequence similarity of 98.5%. The closest homology to the 47-kDa gene sequence was with O. tsutsugamushi strain Gilliam, with a nucleotide similarity of 82.3%, while the closest homology to the 56-kDa gene sequence was with O. tsutsugamushi strain TA686, with a nucleotide similarity of 53.1%. The molecular divergence and geographically unique origin lead us to believe that this organism should be considered a novel species. Therefore, we have proposed the name "Orientia chuto," and the prototype strain of this species is strain Dubai, named after the location in which the patient was infected.

Novel rickettsia in ticks, Tasmania, Australia.

A novel rickettsia was detected in Ixodes tasmani ticks collected from Tasmanian devils. A total of 55% were positive for the citrate synthase gene by quantitative PCR. According to current criteria for rickettsia speciation, this new rickettsia qualifies as Candidatus Rickettsia tasmanensis, named after the location of its detection.