Diverse Trichomonas lineages in Australasian pigeons and doves support a columbid origin for the genus Trichomonas.

Trichomonas is a significant protist genus, and includes T. vaginalis, the most prevalent sexually transmitted non-viral infection of humans, and T. gallinae of rock doves (Columba livia), one of the earliest known avian pathogens. New Trichomonas genotypes, including T. vaginalis-like isolates, have been discovered in American columbid hosts, suggesting geographically widespread cryptic diversity of Trichomonas in pigeons and doves. We sampled 319 birds from 22 columbid species in Australia, Papua New Guinea, New Zealand and southern Africa and uncovered 15 novel lineages of Trichomonas, more than doubling the known diversity of this parasite genus and providing evidence for frequent host-switching that eventually gave rise to T. vaginalis in humans. We show that Trichomonas has a columbid origin and likely underwent Miocene expansion out of Australasia. Our chronological topology for Trichomonas is calibrated on the evolution of a host phenotypic trait associated with ecological entrapment of the most basal extant lineage of Trichomonas in Ptilinopus fruit-doves.

Structural characterization of a short-chain dehydrogenase/reductase from multi-drug resistant Acinetobacter baumannii.

Acinetobacter baumannii (A. baumannii) is a clinically relevant, highly drug-resistant pathogen of global concern. An attractive approach to drug design is to specifically target the type II fatty acid synthesis (FASII) pathway which is critical in Gram negative bacteria and is significantly different to the type I fatty acid synthesis (FASI) pathway found in mammals. Enzymes involved in FASII include members of the short-chain dehydrogenase/reductase (SDR) superfamily. SDRs are capable of performing a diverse range of biochemical reactions against a broad spectrum of substrates whilst maintaining conserved structural features and sequence motifs. Here, we use X-ray crystallography to describe the structure of an SDR from the multi-drug resistant bacteria A. baumannii, previously annotated as a putative FASII FabG enzyme. The protein was recombinantly expressed, purified, and crystallized. The protein crystals diffracted to 2.0 Šand the structure revealed a FabG-like fold. Functional assays revealed, however, that the protein was not active against the FabG substrate, acetoacetyl-CoA. This study highlights that database annotations may show the necessary structural hallmarks of such proteins, however, they may not be able to cleave substrates that are typical of FabG enzymes. These results are important for the selection of target enzymes in future drug development.

Assessing circovirus gene flow in multiple spill-over events.

The establishment of viral pathogens in new host environments following spillover events probably requires adaptive changes within both the new host and pathogen. After many generations, signals for ancient cross-species transmission may become lost and a strictly host-adapted phylogeny may mimic true co-divergence while the virus may retain an inherent ability to jump host species. The mechanistic basis for such processes remains poorly understood. To study the dynamics of virus-host co-divergence and the arbitrary chances of spillover in various reservoir hosts with equal ecological opportunity, we examined structural constraints of capsid protein in extant populations of Beak and feather disease virus (BFDV) during known spillover events. By assessing reservoir-based genotype stratification, we identified co-divergence defying signatures in the evolution BFDV which highlighted primordial processes of cryptic host adaptation and competing forces of host co-divergence and cross-species transmission. We demonstrate that, despite extensive surface plasticity gathered over a longer span of evolution, structural constraints of the capsid protein allow opportunistic host switching in host-adapted populations. This study provides new insights into how small populations of endangered psittacine species may face multidirectional forces of infection from reservoirs with apparently co-diverging genotypes.

Pigeon circoviruses from feral pigeons in Australia demonstrate extensive recombination and genetic admixture with other circoviruses.

Like other avian circovirus species, Pigeon circovirus (PiCV) is known to be genetically diverse with a relatively small circular single-stranded DNA genome of 2 kb that encodes for a capsid protein (Cap) and a replication initiator protein (Rep). Recent paleoviral evidence hints towards a probable Gondwanan origin of avian circoviruses, paralleling the evolution and dispersal of their hosts. Limited availability of PiCV genome sequence data in Australia has hindered phylogeographic studies in this species, so we screened clinically normal rock doves (Columba livia) in regional New South Wales, and demonstrated a high prevalence (76%) of PiCV infection by PCR. We also recovered 12 complete novel PiCV genomes and phylogenetic analyses revealed that PiCV circulating in Australian feral pigeons formed two strongly supported monophyletic clades. One clade resided with PiCV genomes from Poland, Australia, United Kingdom, Belgium, China, and Japan, and another basal clade was more closely related to PiCV genomes from Poland. A novel more distantly-related PiCV rep gene formed a solitary clade with weak posterior support. So we further analysed all selected partial rep gene sequences to demonstrate a likely naturally occurring spillover infection from a passerine circovirus candidate. The findings suggest that there is a high degree of genetic variation within PiCV in Columbiformes with potential greater admixture between avian circoviruses within Australia than previously known. RESEARCH HIGHLIGHTS Confirmed high prevalence rate of PiCV circulating in Australian wild pigeons. Highlighted extensive recombination events within Australian PiCV. Demonstrated a likely naturally occurring spillover infection from a passerine circovirus candidate.

Structural insights into GDP-mediated regulation of a bacterial acyl-CoA thioesterase.

Thioesterases catalyze the cleavage of thioester bonds within many activated fatty acids and acyl-CoA substrates. They are expressed ubiquitously in both prokaryotes and eukaryotes and are subdivided into 25 thioesterase families according to their catalytic active site, protein oligomerization, and substrate specificity. Although many of these enzyme families are well-characterized in terms of function and substrate specificity, regulation across most thioesterase families is poorly understood. Here, we characterized a TE6 thioesterase from the bacterium Neisseria meningitidis Structural analysis with X-ray crystallographic diffraction data to 2.0-Å revealed that each protein subunit harbors a hot dog-fold and that the TE6 enzyme forms a hexamer with D3 symmetry. An assessment of thioesterase activity against a range of acyl-CoA substrates revealed the greatest activity against acetyl-CoA, and structure-guided mutagenesis of putative active site residues identified Asn24 and Asp39 as being essential for activity. Our structural analysis revealed that six GDP nucleotides bound the enzyme in close proximity to an intersubunit disulfide bond interactions that covalently link thioesterase domains in a double hot dog dimer. Structure-guided mutagenesis of residues within the GDP-binding pocket identified Arg93 as playing a key role in the nucleotide interaction and revealed that GDP is required for activity. All mutations were confirmed to be specific and not to have resulted from structural perturbations by X-ray crystallography. This is the first report of a bacterial GDP-regulated thioesterase and of covalent linkage of thioesterase domains through a disulfide bond, revealing structural similarities with ADP regulation in the human ACOT12 thioesterase.

Genomic characterization of two novel pathogenic avipoxviruses isolated from pacific shearwaters (Ardenna spp.).

BACKGROUND: Over the past 20 years, many marine seabird populations have been gradually declining and the factors driving this ongoing deterioration are not always well understood. Avipoxvirus infections have been found in a wide range of bird species worldwide, however, very little is known about the disease ecology of avian poxviruses in seabirds. Here we present two novel avipoxviruses from pacific shearwaters (Ardenna spp), one from a Flesh-footed Shearwater (A. carneipes) (SWPV-1) and the other from a Wedge-tailed Shearwater (A. pacificus) (SWPV-2). RESULTS: Epidermal pox lesions, liver, and blood samples were examined from A. carneipes and A. pacificus of breeding colonies in eastern Australia. After histopathological confirmation of the disease, PCR screening was conducted for avipoxvirus, circovirus, reticuloendotheliosis virus, and fungal agents. Two samples that were PCR positive for poxvirus were further assessed by next generation sequencing, which yielded complete Shearwaterpox virus (SWPV) genomes from A. pacificus and A. carneipes, both showing the highest degree of similarity with Canarypox virus (98% and 67%, respectively). The novel SWPV-1 complete genome from A. carneipes is missing 43 genes compared to CNPV and contains 4 predicted genes which are not found in any other poxvirus, whilst, SWPV-2 complete genome was deemed to be missing 18 genes compared to CNPV and a further 15 genes significantly fragmented as to probably cause them to be non-functional. CONCLUSION: These are the first avipoxvirus complete genome sequences that infect marine seabirds. In the comparison of SWPV-1 and -2 to existing avipoxvirus sequences, our results indicate that the SWPV complete genome from A. carneipes (SWPV-1) described here is not closely related to any other avipoxvirus genome isolated from avian or other natural host species, and that it likely should be considered a separate species.

Phylogenetic analysis of beak and feather disease virus across a host ring-species complex.

Pathogens have been hypothesized to play a major role in host diversity and speciation. Susceptibility of hybrid hosts to pathogens is thought to be a common phenomenon that could promote host population divergence and subsequently speciation. However, few studies have tested for pathogen infection across animal hybrid zones while testing for codivergence of the pathogens in the hybridizing host complex. Over 8 y, we studied natural infection by a rapidly evolving single-strand DNA virus, beak and feather diseases virus (BFDV), which infects parrots, exploiting a host-ring species complex (Platycercus elegans) in Australia. We found that host subspecies and their hybrids varied strikingly in both BFDV prevalence and load: both hybrid and phenotypically intermediate subspecies had lower prevalence and load compared with parental subspecies, while controlling for host age, sex, longitude and latitude, as well as temporal effects. We sequenced viral isolates throughout the range, which revealed patterns of genomic variation analogous to Mayr's ring-species hypothesis, to our knowledge for the first time in any host-pathogen system. Viral phylogeny, geographic location, intraspecific host density, and parrot community diversity and composition did not explain the differences in BFDV prevalence or load between subpopulations. Overall, our analyses suggest that functional host responses to infection, or force of infection, differ between subspecies and hybrids. Our findings highlight the role of host hybridization and clines in altering host-pathogen interactions, dynamics that can have important implications for models of speciation with gene flow, and offer insights into how pathogens may adapt to diverging host populations.

Evolution of circoviruses in lorikeets lags behind its hosts.

The presence of endogenous viral elements in host genomes hints towards much older host-virus relationships than predicted by exogenous phylogenies, with highly mutable single-stranded DNA (ssDNA) viruses and RNA viruses often occupying entangled multispecies ecological niches. The difficulty lies in unravelling the long-term evolutionary history of vertebrate virus-host relationships and determining the age of a potentially ancient tree based only fresh shoots at the tips. Resolving such lineages, and the sometimes great discrepancy amongst evolutionary timescales, is problematic, especially when purifying selection or recombination can significantly alter the accuracy of phylogenetic reconstruction methods. Pathogens which occupy entangled multispecies ecological niches add a further layer of complexity but we show that multi-host scenarios may also provide opportunities to identify allopatric or sympatric paleobiological signals that can unlock longer term phylogenies. We identified host-based, cryptic, sympatric differentiation in beak and feather disease virus in the Psittaciformes tribe Loriini along with endogenous circovirus motifs in Kea (Nestor notabilis) and Gondwanan vicariance estimates to infer the evolutionary timescale of the circoviruses. This demonstrated a chronology of psittacine circovirus speciation aligned to conservative Zealandic divergences for relic circovirus motifs in Kea and a 10million year divergence coinciding with the Papuan central range orogeny that triggered the radiation of Loriini and segregation of an antecedent viral clade in Australian lorikeets. Estimates of circovirus speciation in birds highlighted a Gondwanan dominant group in Neoaves with passerine, columbid and larid circoviruses deeply separated from those in waterfowl, consistent with a Triassic divergence of Galloanserae. The circovirus tree had a deep ancestry in invertebrates with a Palaeozoic expansion in fish and mammals. We show that longer term evolutionary relationships in viruses which have a high rate of mutation and admixture can be disentangled, highlighting that contemporary virus host-switching can be explained by deep intra-lineage host phylogeny.

Recombinantly expressed virus-like particles (VLPs) of canine circovirus for development of an indirect ELISA.

Canine circovirus (CanineCV) is an emerging pathogen in domestic dogs, detected in multiple countries in association with varying clinical and pathological presentations including diarrhoea, vasculitis, granulomatous inflammation, and respiratory signs. Understanding the pathology of CanineCV is confounded by the fact that it has been detected in asymptomatic dogs as well as in diseased dogs concurrently infected with known pathogens. Recombinantly expressed self-assembling Virus-like particles (VLPs) lack viral genomic material but imitate the capsid surface conformations of wild type virion, allowing arrays of biological applications including subunit vaccine development and immunodiagnostics. In this study, full length CanineCV capsid gene was expressed in Escherichia coli followed by two-step purification process to yield soluble capsid protein in high concentration. Transmission electron microscopy (TEM) confirmed the capsid antigen self-assembled into 17-20 nm VLPs in glutathione S-transferase (GST) buffer, later utilised to develop an indirect enzyme-linked immunosorbent assay (iELISA). The respective sensitivity and specificity of the proposed iELISA were 94.10% and 88.40% compared with those obtained from Western blot. The mean OD450 value for western blot positive samples was 1.22 (range 0.12-3.39) and negative samples was 0.21 (range 0.07-0.41). An optimal OD450 cut-off of 0.35 was determined by ROC curve analysis. Median inter-assay and intra-assay validation revealed that the iELISA test results were reproducible with coefficients of variation 7.70 (range 5.6-11.9) and 4.21 (range 1.2-7.4). Our results demonstrated that VLP-based iELISA is a highly sensitive method for serological diagnosis of CanineCV infections in dogs, suitable for large-scale epidemiological studies.

Beak and feather disease virus detected in the endangered Red Goshawk (Erythrotriorchis radiatus).

We report the first detection and prevalence of Beak and feather disease virus (BFDV) in Australia's Red Goshawk (Erythrotriorchis radiatus). This is a new host for this pervasive pathogen amongst a growing list of non-psittacine species including birds of prey from the orders Accipitriformes (hawks, eagles, kites), Falconiformes (falcons and caracas), and Strigiformes (owls). The Red Goshawk is the first non-psittacine species listed as Endangered to be diagnosed with BFDV. We report an initial case of infection discovered post-mortem in a dead nestling and subsequent surveillance of birds from across northern Australia. We reveal BFDV prevalence rates in a wild raptor population for the first time, with detections in 25% (n = 7/28) of Red Goshawks sampled. Prevalence appears higher in juveniles compared to adults, although not statistically significant, but is consistent with studies of wild psittacines. BFDV genotypes were associated with the Loriinae (lorikeets, budgerigar, and fig parrots), Cacatuini (Cockatoos), and Polytelini (long-tailed parrots) tribes; species which are preyed upon by Red Goshawks. A positive BFDV status may be associated with lower body mass but small sample sizes precluded robust statistical analysis. We postulate the possible impacts of the virus on Red Goshawks and discuss future research priorities given these preliminary observations.

Structural determination of importin alpha in complex with beak and feather disease virus capsid nuclear localization signal.

Circoviruses represent a rapidly increasing genus of viruses that infect a variety of vertebrates. Replication requires shuttling viral molecules into the host cell nucleus, a process facilitated by capsid-associated protein (Cap). Whilst a nuclear localization signal (NLS) has been shown to mediate nuclear translocation, the mode of nuclear transport remains to be elucidated. To better understand this process, beak and feather disease virus (BFDV) Cap NLS was crystallized with nuclear import receptor importin-α (Impα). Diffraction yielded structural data to 2.9Å resolution, and the binding site on both Impα and BFDV Cap NLS were well resolved. The binding mechanism for the major site is likely conserved across circoviruses as supported by the similarity of NLSs in circovirus Caps. This finding illuminates a crucial step for infection of host cells by this viral family, and provides a platform for rational drug design against the binding interface.

A Novel Pathogenic Avipoxvirus Infecting Vulnerable Cook's Petrel (Pterodroma cookii) in Australia Demonstrates a High Genomic and Evolutionary Proximity with South African Avipoxviruses.

Avipoxviruses are assumed to be restricted to avian hosts and are considered to be important viral pathogens that may impact the conservation of many vulnerable or endangered birds. Recent reports of avipoxvirus-like viruses from reptiles suggest that cross-species transmission may be possible within birds and other species. Most of the avipoxviruses in wild and sea birds remain uncharacterized, and their genetic variability is unclear. Here, cutaneous pox lesions were used to recover a novel, full-length Cook's petrelpox virus (CPPV) genome from a vulnerable Cook's petrel (Pterodroma cookii), and this was followed by the detection of immature virions using transmission electron microscopy (TEM). The CPPV genome was 314,065 bp in length and contained 357 predicted open-reading frames (ORFs). While 323 of the ORFs of the CPPV genome had the greatest similarity with the gene products of other avipoxviruses, a further 34 ORFs were novel. Subsequent phylogenetic analyses showed that the CPPV was most closely related to other avipoxviruses that were isolated mostly from South African bird species and demonstrated the highest sequence similarity with a recently isolated flamingopox virus (88.9%) in South Africa. Considering the sequence similarity observed between CPPV and other avipoxviruses, TEM evidence of poxvirus particles, and phylogenetic position, this study concluded that CPPV is a distinct candidate of avipoxviruses. IMPORTANCE Emerging viral disease is a significant concern with potential consequences for human, animal, and environmental health. Over the past several decades, multiple novel viruses have been found in wildlife species, including birds, and they can pose a threat to vulnerable and endangered species. Cook's petrel is currently listed as vulnerable. The threats to the species vary, but are, to a large degree, due to anthropogenic impacts, such as climate change, habitat loss, pollution, and other disturbances by humans. Knowledge of viral pathogens, including poxvirus of Cook's petrel is currently virtually nonexistent.

Lesions and viral loads in racing pigeons naturally coinfected with pigeon circovirus and columbid alphaherpesvirus 1 in Australia.

Columbid alphaherpesvirus 1 (CoHV1) is associated with oral or upper respiratory tract lesions, encephalitis, and occasional fatal systemic disease in naive or immunosuppressed pigeons. Clinical disease is often reported with CoHV1 and coinfecting viruses, including pigeon circovirus (PiCV), which may cause host immunosuppression and augment lesion development. A natural outbreak of CoHV1 and PiCV coinfection occurred in a flock of 60 racing rock pigeons (Columba livia), in which 4 pigeons succumbed within 7 d of clinical onset. Lesions included suppurative stomatitis, pharyngitis, cloacitis, meningitis, and tympanitis, with eosinophilic intranuclear inclusion bodies consistent with herpesviral infection. In addition, large numbers of botryoid intracytoplasmic inclusion bodies were present in the skin, oral mucosa, and bursa of Fabricius, suggestive of circoviral infection, which was confirmed by immunohistochemistry. The concurrent viral load of CoHV1 and PiCV was high in liver, oropharynx, and bursa of Fabricius. We found PiCV in oro-cloacal swabs from 44 of 46 additional birds of variable clinical status, PiCV alone in 23 birds, and coinfection with CoHV1 in 21 birds. Viral copy numbers were significantly higher (p < 0.0001) for both viruses in clinically affected pigeons than in subclinical qPCR-positive birds. The CoHV1-induced lesions might have been exacerbated by concomitant PiCV infection.

Visualizing neutrophil extracellular traps in septic equine synovial and peritoneal fluid samples using immunofluorescence microscopy.

Septic synovitis and peritonitis are routinely diagnosed in horses based on clinical examination findings and laboratory assessment of synoviocentesis and abdominocentesis samples, respectively. Diagnosis is difficult in some cases because of an overlap in laboratory results for septic and non-septic inflammation. Neutrophil extracellular trap (NET) formation is part of the innate immune response against pathogens. Identifying and quantifying NETs, which have not been explored in clinical samples from horses with septic synovitis and peritonitis, to our knowledge, may be helpful in detecting infectious processes. Our main objective was to determine whether NETs could be visualized in septic equine synovial and peritoneal fluid cytology samples using immunofluorescence with antibodies against citrullinated histone H3 (Cit-H3) and myeloperoxidase (MPO). We analyzed 9 synovial and 4 peritoneal fluid samples. NET percentages were quantified using a simple counting technique, which is suitable for high-quality, well-preserved, and stained cytospin smears. NETs were evident in all septic samples and were absent in a non-septic sample; NETs were better visualized with Cit-H3 than with MPO immunolabeling. Overall, we believe that there is the potential for NETs and associated markers to be used to investigate and understand septic inflammation in horses.

Australasian Pigeon Circoviruses Demonstrate Natural Spillover Infection.

Pigeon circovirus (PiCV) is considered to be genetically diverse, with a relatively small circular single-stranded DNA genome of 2 kb that encodes for a capsid protein (Cap) and a replication initiator protein (Rep). Australasia is known to be the origin of diverse species of the Order Columbiformes, but limited data on the PiCV genome sequence has hindered phylogeographic studies in this species. To fill this gap, this study was conducted to investigate PiCV in 118 characteristic samples from different birds across Australia using PCR and sequencing. Eighteen partial PiCV Rep sequences and one complete PiCV genome sequence were recovered from reservoir and aberrant hosts. Phylogenetic analyses revealed that PiCV circulating in Australia was scattered across three different subclades. Importantly, one subclade dominated within the PiCV sequenced from Australia and Poland, whereas other PiCV sequenced in this study were more closely related to the PiCV sequenced from China, USA and Japan. In addition, PiCV Rep sequences obtained from clinically affected plumed whistling duck, blue billed duck and Australian magpie demonstrated natural spillover of PiCV unveiled host generalist characteristics of the pigeon circovirus. These findings indicate that PiCV genomes circulating in Australia lack host adapted population structure but demonstrate natural spillover infection.

Development and applications of a TaqMan based quantitative real-time PCR for the rapid detection of Pigeon circovirus (PiCV).

TaqMan probe based quantitative polymerase reaction (TaqMan qPCR) is a robust and reliable technique for detecting and quantifying target DNA copies. Quantitative molecular diagnosis of genetically diverse single stranded DNA (ssDNA) virus such as Pigeon circovirus (PiCV) can be challenging owing to difficulties in primer binding or low abundance of template DNA copies in clinical specimens. Several methods have been described for the detection of PiCV, being qPCR the most simple and reliable. As far as is known, two qPCR systems described until now are based on SYBR green. This study reports development and validation of a highly sensitive TaqMan qPCR targeted to Rep for the detection of highly diverse PiCV in pigeon samples with excellent reproducibility, specificity, and sensitivity. The limit of detection was determined as low as 2 (two) plasmid copies. Estimations of 100 % specificity and 100 % sensitivity were obtained based on the qPCR results with panel of 60 samples (known PiCV positive, n = 30; known PiCV negative, n = 20; samples positive to Beak and feather disease virus (BFDV), n = 5 and samples positive to canine circovirus, n = 5). Co-efficient of variation (CV) for Ct values ranged between 0.27 % and 0.78 % in the same assay and 1.84-2.87 % in different assays.

Complete Mitochondrial Genome Sequence of a Seabird, Wedge-Tailed Shearwater (Ardenna pacifica).

Here, we report the complete mitochondrial genome sequence of a seabird, wedge-tailed shearwater (Ardenna pacifica). The circular genome has a size of 16,434 bp and contains 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. The study provides a reference mitochondrial genome of wedge-tailed shearwater for further molecular studies.

Molecular characterisation of a novel pathogenic avipoxvirus from an Australian passerine bird, mudlark (Grallina cyanoleuca).

Avipoxviruses have been recognised as significant pathogens in the conservation of numerous bird species. However, the vast majority of the avipoxviruses that infect wild birds remain uncharacterised. Here, we characterise a novel avipoxvirus, mudlarkpox virus (MLPV) isolated from an Australian passerine bird, mudlark (Grallina cyanoleuca). In this study, tissues with histopathologically confirmed lesions consistent with avian pox were used for transmission electron microscopy, and showed characteristic ovoid to brick-shaped virions, indicative of infectious particles. The MLPV genome was >342.7 Kbp in length and contained six predicted novel genes and a further six genes were missing compared to shearwaterpox virus-2 (SWPV-2). Subsequent phylogenetic analyses of the MLPV genome positioned the virus within a distinct subclade also containing recently characterised avipoxvirus genomes from shearwater, canary and magpie bird species, and demonstrated a high degree of sequence similarity with SWPV-2 (94.92%).

Complete Mitochondrial Genome Sequence of the Magpie-Lark (Grallina cyanoleuca).

Here, we report the complete mitochondrial genome sequence of an Australian passerine bird, magpie-lark (Grallina cyanoleuca). The circular genome has a size of 16,933 bp and contains 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. This study provides a reference mitochondrial genome of magpie-lark for further molecular studies.

The Structural Features of Henipavirus Matrix Protein Driving Intracellular Trafficking.

Henipaviruses are single-stranded RNA viruses that have recently emerged as zoonotic pathogens, capable of causing severe acute respiratory disease and encephalitis in humans. The prototypical henipaviruses, Hendra henipavirus and Nipah henipavirus, are a major health concern as they have high mortality rates and no currently approved human vaccine or drug therapy. Understanding the mechanisms of viral replication and pathogenicity is of critical importance for therapeutic developments. A novel target for such therapies is the Henipavirus Matrix (M) protein, a multifunctional protein that drives viral assembly and inhibits the innate immune response. These multifunctional attributes promote a complicated lifecycle: while viral replication occurs in the cytoplasm, M traffics to the nucleus, where it is ubiquitinated, for correct cellular targeting and virion packaging. In this study, we review the relationship between the structure and functions of M. In specific cases, the compatibility between structural accessibility and protein functionality is not always evident, and we highlight areas that require further investigation.