Australia as a global sink for the genetic diversity of avian influenza A virus.

Most of our understanding of the ecology and evolution of avian influenza A virus (AIV) in wild birds is derived from studies conducted in the northern hemisphere on waterfowl, with a substantial bias towards dabbling ducks. However, relevant environmental conditions and patterns of avian migration and reproduction are substantially different in the southern hemisphere. Through the sequencing and analysis of 333 unique AIV genomes collected from wild birds collected over 15 years we show that Australia is a global sink for AIV diversity and not integrally linked with the Eurasian gene pool. Rather, AIV are infrequently introduced to Australia, followed by decades of isolated circulation and eventual extinction. The number of co-circulating viral lineages varies per subtype. AIV haemagglutinin (HA) subtypes that are rarely identified at duck-centric study sites (H8-12) had more detected introductions and contemporary co-circulating lineages in Australia. Combined with a lack of duck migration beyond the Australian-Papuan region, these findings suggest introductions by long-distance migratory shorebirds. In addition, on the available data we found no evidence of directional or consistent patterns in virus movement across the Australian continent. This feature corresponds to patterns of bird movement, whereby waterfowl have nomadic and erratic rainfall-dependant distributions rather than consistent intra-continental migratory routes. Finally, we detected high levels of virus gene segment reassortment, with a high diversity of AIV genome constellations across years and locations. These data, in addition to those from other studies in Africa and South America, clearly show that patterns of AIV dynamics in the Southern Hemisphere are distinct from those in the temperate north.

Fatal Human Neurologic Infection Caused by Pigeon Avian Paramyxovirus-1, Australia.

Avian paramyxovirus type 1 (APMV-1) is a virus of birds that results in a range of outcomes, from asymptomatic infections to outbreaks of systemic respiratory and neurologic disease, depending on the virus strain and the avian species affected. Humans are rarely affected; those who are predominantly experience mild conjunctivitis. We report a fatal case of neurologic disease in a 2-year-old immunocompromised child in Australia. Metagenomic sequencing and histopathology identified the causative agent as the pigeon variant of APMV-1. This diagnosis should be considered in neurologic conditions of undefined etiologies. Agnostic metagenomic sequencing methods are useful in such settings to direct diagnostic and therapeutic efforts.

A new Hendra virus genotype found in Australian flying foxes.

BACKGROUND: Hendra virus (HeV) has caused lethal disease outbreaks in humans and horses in Australia. Flying foxes are the wildlife reservoir from which the virus was first isolated in 1996. Following a heat stress mortality event in Australian flying foxes in 2013, a novel HeV variant was discovered. This study describes the subsequent surveillance of Australian flying foxes for this novel virus over a nine year period using qRT-PCR testing of tissues from flying foxes submitted primarily for Australian bat lyssavirus diagnosis. Genome sequencing and characterisation of the novel HeV variant was also undertaken. METHODS: Spleen and kidney samples harvested from flying fox carcasses were initially screened with two real-time qRT-PCR assays specific for the prototype HeV. Two additional qRT-PCR assays were developed specific for the HeV variant first detected in samples from a flying fox in 2013. Next-generation sequencing and virus isolation was attempted from selected samples to further characterise the new virus. RESULTS: Since 2013, 98 flying foxes were tested and 11 were positive for the new HeV variant. No samples were positive for the original HeV. Ten of the positive samples were from grey-headed flying foxes (GHFF, Pteropus poliocephalus), however this species was over-represented in the opportunistic sampling (83% of bats tested were GHFF). The positive GHFF samples were collected from Victoria and South Australia and one positive Little red flying fox (LRFF, Pteropus scapulatus) was collected from Western Australia. Immunohistochemistry confirmed the presence of henipavirus antigen, associated with an inflammatory lesion in cardiac blood vessels of one GHFF. Positive samples were sequenced and the complete genome was obtained from three samples. When compared to published HeV genomes, there was 84% sequence identity at the nucleotide level. Based on phylogenetic analyses, the newly detected HeV belongs to the HeV species but occupies a distinct lineage. We have therefore designated this virus HeV genotype 2 (HeV-g2). Attempts to isolate virus from PCR positive samples have not been successful. CONCLUSIONS: A novel HeV genotype (HeV-g2) has been identified in two flying fox species submitted from three states in Australia, indicating that the level of genetic diversity for HeV is broader than first recognised. Given its high genetic relatedness to HeV, HeV-g2 is a zoonotic pathogen.

Isolation and characterisation of an ISKNV-genotype megalocytivirus from imported angelfish Pterophyllum scalare.

Using cultures of the SKF-9 cell line, megalocytivirus AFIV-16 was isolated from imported angelfish Pterophyllum scalare held in quarantine at the Australian border. The cytopathic effect caused by isolate AFIV-16 presented as cell rounding and enlargement, but complete destruction of the infected cell cultures did not occur. The infected cells demonstrated immunocytochemical reactivity with monoclonal antibody M10, which is used for diagnosis of OIE-listed red sea bream iridoviral disease. Using electron microscopy, the virus particles, consisting of hexagonal nucleocapsids, were observed in the cytoplasm of SKF-9 cells. The replication of AFIV-16 in cultured SKF-9 cells was significantly greater at 28°C incubation than at 22 and 25°C incubation, whereas no difference in growth characteristics was observed for red sea bream iridovirus (RSIV) isolate KagYT-96 across this temperature range. Whole genome sequencing demonstrated that AFIV-16 has a 99.96% similarity to infectious spleen and kidney necrosis virus (ISKNV), the type species in the genus Megalocytivirus. AFIV-16 was classified into ISKNV genotype Clade 1 by phylogenetic analysis of the major capsid protein gene nucleotide sequence. This is the first report of whole genome sequencing of an ISKNV genotype megalocytivirus isolated from ornamental fish.

Pilchard orthomyxovirus (POMV). I. Characterisation of an emerging virus isolated from pilchards Sardinops sagax and Atlantic salmon Salmo salar.

An orthomyxo-like virus was first isolated in 1998 as an incidental discovery from pilchards Sardinops sagax collected from waters off the South Australian coast. In the following 2 decades, orthomyxo-like viruses have been isolated from healthy pilchards in South Australia and Tasmania. In 2006, an orthomyxo-like virus was also isolated from farmed Atlantic salmon Salmo salar in Tasmania during routine surveillance and, again, from 2012 onwards from diseased Atlantic salmon. Using transmission electron microscopy, these viruses were identified as belonging to the family Orthomyxoviridae. To further characterise the viruses, the genomes of 11 viral isolates were sequenced. The open reading frames (ORFs) that encode 10 putative proteins from 8 viral genome segments were assembled from Illumina MiSeq next generation sequencing (NGS) data. The complete genome of a 2014 isolate was also assembled from NGS, RNA-sequencing (RNA-seq) data, that included conserved motifs that shared commonalities with infectious salmon anaemia virus, rainbow trout orthomyxovirus and Influenzavirus A. The presence of 8 viral proteins translated from genome segments was confirmed by mass spectrometric analysis including 2 novel proteins with no known orthologs. Sequence analysis of the ORFs, non-coding regions and proteins indicated that the viruses had minimal diversity and hence were named pilchard orthomyxovirus (POMV), based on the fish host species of its first isolation. The low homology of POMV proteins with previously characterised orthomyxoviruses suggests that POMV is the first virus to be characterised from a new genus within the Orthomyxoviridae. To facilitate more rapid detection and subsequent diagnostic confirmation of POMV infections, TaqMan and conventional nested PCRs were designed.

Investigating the natural resistance of blackfoot p-a%%KERN_ERR%%ua Haliotis iris to abalone viral ganglioneuritis using whole transcriptome analysis.

The natural resistance of New Zealand blackfoot p-a%%%%%%%%%%%%%%KERN_ERR%%KERN_ERR%%KERN_ERR%%KERN_ERR%%KERN_ERR%%KERN_ERR%%KERN_ERR%%ua Haliotis iris to infection by haliotid herpesvirus-1 (HaHV-1) and to the disease abalone viral ganglioneuritis was investigated in experimentally challenged p-aua using high throughput RNA-sequencing. HaHV-1-challenged p-aua up-regulated broad classes of genes that contained chitin-binding peritrophin-A domains, which seem to play diverse roles in the p-aua immune response. The p-aua also up-regulated vascular adhesion protein-1 (VAP-1), an important adhesion molecule for lymphocytes, and chitotriosidase-1 (CHIT-1), an immunologically important gene in mammalian immune systems. Moreover, several blood coagulation pathways were dysregulated in the p-aua, possibly indicating viral modulation. We also saw several indications that neurological tissues were specifically affected by HaHV-1, including the dysregulation of beta-1,4-N-acetylgalactosaminyltransferase (B4GALNT), GM2 ganglioside, neuroligin-4 and the Notch signalling pathway. This research may support the development of molecular therapeutics useful to control and/or manage viral outbreaks in abalone culture.

Diversity and function of prevalent symbiotic marine bacteria in the genus Endozoicomonas.

Endozoicomonas bacteria are emerging as extremely diverse and flexible symbionts of numerous marine hosts inhabiting oceans worldwide. Their hosts range from simple invertebrate species, such as sponges and corals, to complex vertebrates, such as fish. Although widely distributed, the functional role of Endozoicomonas within their host microenvironment is not well understood. In this review, we provide a summary of the currently recognized hosts of Endozoicomonas and their global distribution. Next, the potential functional roles of Endozoicomonas, particularly in light of recent microscopic, genomic, and genetic analyses, are discussed. These analyses suggest that Endozoicomonas typically reside in aggregates within host tissues, have a free-living stage due to their large genome sizes, show signs of host and local adaptation, participate in host-associated protein and carbohydrate transport and cycling, and harbour a high degree of genomic plasticity due to the large proportion of transposable elements residing in their genomes. This review will finish with a discussion on the methodological tools currently employed to study Endozoicomonas and host interactions and review future avenues for studying complex host-microbial symbioses.

Contrasting dynamics of two incursions of low-pathogenicity avian influenza virus into Australia.

The current panzootic of high pathogenicity avian influenza virus H5N1 demonstrates how viral incursions can have major ramifications for wildlife and domestic animals. Herein, we describe the recent incursion into Australia of two low pathogenicity avian influenza virus subtypes, H4 and H10, that exhibited contrasting evolutionary dynamics. Viruses detected from national surveillance and disease investigations between 2020 and 2022 revealed 27 genomes, 24 of which have at least one segment more closely related to Eurasian or North American avian influenza lineages than those already circulating in Australia. Phylogenetic analysis revealed that H4 viruses circulating in shorebirds represent a recent incursion from Asia that is distinct from those circulating concurrently in Australian waterfowl. Analysis of the internal segments further demonstrates exclusive, persistent circulation in shorebirds. This contrasts with H10, where a novel lineage has emerged in wild waterfowl, poultry, and captive birds across Australia and has likely replaced previously circulating H10 lineages through competitive exclusion. Elucidating different dynamics for avian influenza incursions supports effective disease risk identification and communication that better informs disease preparedness and response.

The microbiome of the Red Sea coral Stylophora pistillata is dominated by tissue-associated Endozoicomonas bacteria.

Endozoicomonas bacteria were found highly associated with the coral Stylophora pistillata, and these bacteria are also ubiquitously associated with diverse corals worldwide. Novel Endozoicomonas-specific probes revealed that Endozoicomonas bacteria were abundant in the endodermal tissues of S. pistillata and appear to have an intimate relationship with the coral.

The bacterial community associated with the marine polychaete Ophelina sp.1 (Annelida: Opheliidae) is altered by copper and zinc contamination in sediments.

Tolerant species of polychaete worms can survive in polluted environments using various resistance mechanisms. One aspect of resistance not often studied in polychaetes is their association with symbiotic bacteria, some of which have resistance to metals and may help the organism to survive. We used "next generation" 454 sequencing of bacterial 16S rRNA sequences associated with polychaetes from a copper- and zinc-polluted harbor and from a reference site to determine bacterial community structure. We found changes in the bacteria at the polluted site, including increases in the abundance of bacteria from the order Alteromonadales. These changes in the bacteria associated with polychaetes may be relatively easy to detect and could be a useful indicator of metal pollution.

Comparative genomic analysis of the first Ehrlichia canis detections in Australia.

Ehrlichia canis (Rickettsiales; Anaplasmataceae) is one of the most prevalent tick-borne pathogens of dogs globally. The bacterium infects monocytes and is the aetiological agent of canine monocytic ehrlichiosis. For many decades Australia was thought to be free of the pathogen, but this abruptly changed in May 2020 when E. canis was detected in several dogs from Kununurra, Western Australia. Subsequent surveillance activities found unexpectedly large scale spread of E. canis throughout much of northern Australia. To gain insight into the genetic relationships of the Australian strain and its potential origin, we undertook a genomic analysis of E. canis positive domestic dog and tick (Rhipicephalus linnaei) samples from the north of Western Australia, the far north of South Australia and the Northern Territory, covering thousands of square kilometres. We obtained complete E. canis genomes from each of the three states, plus an additional 16 partial genomes, substantially increasing publicly available E. canis genetic resources. The Australian E. canis genomes were highly conserved across large geographic distances. Outside of Australia, the genomes were most similar to E. canis YZ-1 from China, although few reference sequences were available. We analysed the variable trp36 gene to obtain greater phylogenetic signal, which demonstrated that the Australian E. canis belonged to the Taiwan genotype, comprised of samples from Taiwan, China, Thailand and Turkey. Taken together, our findings suggest that E. canis in Australia may have originated from Asia or the Middle East and spread throughout northern and central Australia following its introduction.

Lagovirus Non-structural Protein p23: A Putative Viroporin That Interacts With Heat Shock Proteins and Uses a Disulfide Bond for Dimerization.

The exact function(s) of the lagovirus non-structural protein p23 is unknown as robust cell culture systems for the Rabbit haemorrhagic disease virus (RHDV) and other lagoviruses have not been established. Instead, a range of in vitro and in silico models have been used to study p23, revealing that p23 oligomerizes, accumulates in the cytoplasm, and possesses a conserved C-terminal region with two amphipathic helices. Furthermore, the positional homologs of p23 in other caliciviruses have been shown to possess viroporin activity. Here, we report on the mechanistic details of p23 oligomerization. Site-directed mutagenesis revealed the importance of an N-terminal cysteine for dimerization. Furthermore, we identified cellular interactors of p23 using stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics; heat shock proteins Hsp70 and 110 interact with p23 in transfected cells, suggesting that they 'chaperone' p23 proteins before their integration into cellular membranes. We investigated changes to the global transcriptome and proteome that occurred in infected rabbit liver tissue and observed changes to the misfolded protein response, calcium signaling, and the regulation of the endoplasmic reticulum (ER) network. Finally, flow cytometry studies indicate slightly elevated calcium concentrations in the cytoplasm of p23-transfected cells. Taken together, accumulating evidence suggests that p23 is a viroporin that might form calcium-conducting channels in the ER membranes.

Genomic Sequencing of Dengue Virus Strains Associated with Papua New Guinean Outbreaks in 2016 Reveals Endemic Circulation of DENV-1 and DENV-2.

Over the past decade, the Pacific region has experienced many arboviral outbreaks, including dengue, chikungunya, and Zika viruses. Papua New Guinea (PNG) has a high burden of arboviral diseases, but there is a paucity of knowledge about the epidemiology and circulation of these viruses in the country. In this study, we report investigations into suspected arboviral outbreaks of febrile disease in PNG from December 2015 to June 2017. DENV-1 and DENV-2 were the mostly commonly detected viruses, and low circulation of DENV-3 and ZIKV was also detected. DENV-4 and CHIKV were not detected during this period. Full genome sequencing of selected positive samples revealed that circulation was dominated by endemic indigenous strains belonging to DENV-1 (genotype IV) and DENV-2 (genotype C) that have been present in the country for up to a decade. A DENV-2 sublineage was also identified that has been associated with outbreaks of severe dengue in both PNG and the Solomon Islands.

Molecular detection and characterisation of the first Japanese encephalitis virus belonging to genotype IV acquired in Australia.

BACKGROUND: A fatal case of Japanese encephalitis (JE) occurred in a resident of the Tiwi Islands, in the Northern Territory of Australia in February 2021, preceding the large JE outbreak in south-eastern Australia in 2022. This study reports the detection, whole genome sequencing and analysis of the virus responsible (designated JEV/Australia/NT_Tiwi Islands/2021). METHODS: Reverse transcription quantitative PCR (RT-qPCR) testing was performed on post-mortem brain specimens using a range of JE virus (JEV)-specific assays. Virus isolation from brain specimens was attempted by inoculation of mosquito and mammalian cells or embryonated chicken eggs. Whole genome sequencing was undertaken using a combination of Illumina next generation sequencing methodologies, including a tiling amplicon approach. Phylogenetic and selection analyses were performed using alignments of the Tiwi Islands JEV genome and envelope (E) protein gene sequences and publicly available JEV sequences. RESULTS: Virus isolation was unsuccessful and JEV RNA was detected only by RT-qPCR assays capable of detecting all JEV genotypes. Phylogenetic analysis revealed that the Tiwi Islands strain is a divergent member of genotype IV (GIV) and is closely related to the 2022 Australian outbreak virus (99.8% nucleotide identity). The Australian strains share highest levels of nucleotide identity with Indonesian viruses from 2017 and 2019 (96.7-96.8%). The most recent common ancestor of this Australian-Indonesian clade was estimated to have emerged in 2007 (95% HPD range: 1998-2014). Positive selection was detected using two methods (MEME and FEL) at several sites in the E and non-structural protein genes, including a single site in the E protein (S194N) unique to the Australian GIV strains. CONCLUSION: This case represents the first detection of GIV JEV acquired in Australia, and only the second confirmed fatal human infection with a GIV JEV strain. The close phylogenetic relationship between the Tiwi Islands strain and recent Indonesian viruses is indicative of the origin of this novel GIV lineage, which we estimate has circulated in the region for several years prior to the Tiwi Islands case.

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.

Complete Genome Sequence of African Swine Fever Virus Isolated from a Domestic Pig in Timor-Leste, 2019.

Here, we report the complete genome sequence of the African swine fever virus (ASFV) isolate ASFV/Timor-Leste/2019/1, isolated from a domestic pig during the first outbreak of ASF in Timor-Leste in 2019. Using target enrichment short-read Illumina data combined with long-read Oxford Nanopore data, we assembled a full-length genome sequence of 192,237 bp.

ChAdOx1 nCoV-19 (AZD1222) vaccine candidate significantly reduces SARS-CoV-2 shedding in ferrets.

Vaccines against SARS-CoV-2 are likely to be critical in the management of the ongoing pandemic. A number of candidates are in Phase III human clinical trials, including ChAdOx1 nCoV-19 (AZD1222), a replication-deficient chimpanzee adenovirus-vectored vaccine candidate. In preclinical trials, the efficacy of ChAdOx1 nCoV-19 against SARS-CoV-2 challenge was evaluated in a ferret model of infection. Groups of ferrets received either prime-only or prime-boost administration of ChAdOx1 nCoV-19 via the intramuscular or intranasal route. All ChAdOx1 nCoV-19 administration combinations resulted in significant reductions in viral loads in nasal-wash and oral swab samples. No vaccine-associated adverse events were observed associated with the ChAdOx1 nCoV-19 candidate, with the data from this study suggesting it could be an effective and safe vaccine against COVID-19. Our study also indicates the potential for intranasal administration as a way to further improve the efficacy of this leading vaccine candidate.

Supporting pandemic response using genomics and bioinformatics: A case study on the emergent SARS-CoV-2 outbreak.

Pre-clinical responses to fast-moving infectious disease outbreaks heavily depend on choosing the best isolates for animal models that inform diagnostics, vaccines and treatments. Current approaches are driven by practical considerations (e.g. first available virus isolate) rather than a detailed analysis of the characteristics of the virus strain chosen, which can lead to animal models that are not representative of the circulating or emerging clusters. Here, we suggest a combination of epidemiological, experimental and bioinformatic considerations when choosing virus strains for animal model generation. We discuss the currently chosen SARS-CoV-2 strains for international coronavirus disease (COVID-19) models in the context of their phylogeny as well as in a novel alignment-free bioinformatic approach. Unlike phylogenetic trees, which focus on individual shared mutations, this new approach assesses genome-wide co-developing functionalities and hence offers a more fluid view of the 'cloud of variances' that RNA viruses are prone to accumulate. This joint approach concludes that while the current animal models cover the existing viral strains adequately, there is substantial evolutionary activity that is likely not considered by the current models. Based on insights from the non-discrete alignment-free approach and experimental observations, we suggest isolates for future animal models.

RNASeq Analysis of Aedes albopictus Mosquito Midguts after Chikungunya Virus Infection.

Chikungunya virus (CHIKV) is an emerging pathogen around the world and causes significant morbidity in patients. A single amino acid mutation in the envelope protein of CHIKV has led to a shift in vector preference towards Aedesalbopictus. While mosquitoes are known to mount an antiviral immune response post-infection, molecular interactions during the course of infection at the tissue level remain largely uncharacterised. We performed whole transcriptome analysis on dissected midguts of Aedes albopictus infected with CHIKV to identify differentially expressed genes. For this, RNA was extracted at two days post-infection (2-dpi) from pooled midguts. We initially identified 25 differentially expressed genes (p-value < 0.05) when mapped to a reference transcriptome. Further, multiple differentially expressed genes were identified from a custom de novo transcriptome, which was assembled using the reads that did not align with the reference genome. Thirteen of the identified transcripts, possibly involved in immunity, were validated by qRT-PCR. Homologues of seven of these genes were also found to be significantly upregulated in Aedes aegypti midguts 2 dpi, indicating a conserved mechanism at play. These results will help us to characterise the molecular interaction between Aedes albopictus and CHIKV and can be utilised to reduce the impact of this viral infection.

Attenuation of Bluetongue Virus (BTV) in an in ovo Model Is Related to the Changes of Viral Genetic Diversity of Cell-Culture Passaged BTV.

The embryonated chicken egg (ECE) is routinely used for the laboratory isolation and adaptation of Bluetongue virus (BTV) in vitro. However, its utility as an alternate animal model has not been fully explored. In this paper, we evaluated the pathogenesis of BTV in ovo using a pathogenic isolate of South African BTV serotype 3 (BTV-3) derived from the blood of an infected sheep. Endothelio- and neurotropism of BTV-3 were observed by immunohistochemistry of non-structural protein 1 (NS1), NS3, NS3/3a, and viral protein 7 (VP7) antigens. In comparing the pathogenicity of BTV from infectious sheep blood with cell-culture-passaged BTV, including virus propagated through a Culicoides-derived cell line (KC) or ECE, we found virus attenuation in ECE following cell-culture passage. Genomic analysis of the consensus sequences of segments (Seg)-2, -5, -6, -7, -8, -9, and -10 identified several nucleotide and amino-acid mutations among the cell-culture-propagated BTV-3. Deep sequencing analysis revealed changes in BTV-3 genetic diversity in various genome segments, notably a reduction of Seg-7 diversity following passage in cell culture. Using this novel approach to investigate BTV pathogenicity in ovo, our findings support the notion that pathogenic BTV becomes attenuated in cell culture and that this change is associated with virus quasispecies evolution.