Formulation and Biomedical Activity of Oil-in-Water Nanoemulsion Combining Tinospora smilacina Water Extract and Calophyllum inophyllum Seeds Oil.

Introduction: Tinospora smilacina is a native plant used in traditional medicine by First Nations peoples in Australia to treat inflammation. In our previous study, an optimised Calophyllum inophyllum seed oil (CSO) nanoemulsion (NE) showed improved biomedical activities such as antimicrobial, antioxidant activity, cell viability and in vitro wound healing efficacy compared to CSO. Methods: In this study, a stable NE formulation combining T. smilacina water extract (TSWE) and CSO in a nanoemulsion (CTNE) was prepared to integrate the bioactive compounds in both native plants and improve wound healing efficacy. D-optimal mixture design was used to optimise the physicochemical characteristics of the CTNE, including droplet size and polydispersity index (PDI). Cell viability and in vitro wound healing studies were done in the presence of CTNE, TSWE and CSO against a clone of baby hamster kidney fibroblasts (BHK-21 cell clone BSR-T7/5). Results: The optimised CTNE had a 24 ± 5 nm particle size and 0.21± 0.02 PDI value and was stable after four weeks each at 4 °C and room temperature. According to the results, incorporating TSWE into CTNE improved its antioxidant activity, cell viability, and ability to promote wound healing. The study also revealed that TSWE has >6% higher antioxidant activity than CSO. While CTNE did not significantly impact mammalian cell viability, it exhibited wound-healing properties in the BSR cell line during in vitro testing. These findings suggest that adding TSWE may enhance CTNE's potential as a wound-healing treatment. Conclusion: This is the first study demonstrating NE formulation in which two different plant extracts were used in the aqueous and oil phases with improved biomedical activities.

Toward higher-performance bionic limbs for wider clinical use.

Most prosthetic limbs can autonomously move with dexterity, yet they are not perceived by the user as belonging to their own body. Robotic limbs can convey information about the environment with higher precision than biological limbs, but their actual performance is substantially limited by current technologies for the interfacing of the robotic devices with the body and for transferring motor and sensory information bidirectionally between the prosthesis and the user. In this Perspective, we argue that direct skeletal attachment of bionic devices via osseointegration, the amplification of neural signals by targeted muscle innervation, improved prosthesis control via implanted muscle sensors and advanced algorithms, and the provision of sensory feedback by means of electrodes implanted in peripheral nerves, should all be leveraged towards the creation of a new generation of high-performance bionic limbs. These technologies have been clinically tested in humans, and alongside mechanical redesigns and adequate rehabilitation training should facilitate the wider clinical use of bionic limbs.

Optimisation of Calophyllum inophyllum seed oil nanoemulsion as a potential wound healing agent.

BACKGROUND: Efficient delivery systems of Calophyllum inophyllum seed oil (CSO) in the form of nanoemulsion were optimised to enhance its stability and ensure its therapeutic efficiency as a potential agent for various biomedical applications. METHOD: Response Surface Methodology (RSM) was used to determine the effects of independent variables (oil, surfactant, water percentage and homogenisation time) on physicochemical characteristics, including droplet size, polydispersity index and turbidity. RESULTS: The optimised CSO nanoemulsion (CSONE) has a 46.68 nm particle size, 0.15 Polydispersity index value and 1.16 turbidity. After 4 weeks of storage at 5 ± 1 °C and 25 ± 1 °C, the CSONE was physically stable. The optimised CSO nanoemulsion showed enhancement in cell viability and wound healing in baby hamster kidney a clone BHK-21 (BSR) cells as compared to the CSO. The wound healing property of CSONE was higher than CSO. CONCLUSION: Thus, our in vitro wound healing results demonstrated that CSO in the nanoemulsion form can promote wound healing by enhancing the proliferation and migration of epidermal cells. The coarse emulsion of Calophyllum inophyllum seed oil nano emulsion was prepared using high shear homogeniser techniques. The optimised CSONE with the droplet size of 46.68 nm was prepared from a mixture of CSO, Tween 80, and high pure water (HPW), then used for the biological investigation. The in vitro cell monolayer scratch assay revealed that CSONE in the lowest concentration of CSO resulted in 100% wound closure after 48 hrs. The optimised CSO nanoemulsion was found to be a promising and effective approach in the treatment of wounds by boosting the proliferation and migration of epidermal cells.

Cellulose-acetate coating of carbon cloth diffusion layer for liquid-fed fuel cell applications.

Direct glucose fuel cells (DGFCs) and direct methanol fuel cells (DMFCs) commonly supply the reducing agent in liquid (aq.) form. In this work, we present key characteristics of implementing cellulose acetate (CA) coatings, which can subsequently be deacetylated toward cellulose, on carbon cloth used as a fuel diffusion layer in aqueous fuel-fed cells. Specifically, we illustrate functionality with an abiotic glucose fuel cell. Carbon cloth with and without a CA coating (with varying deacetylation) was characterized in terms of liquid permeation rate, electronic conductivity, and roll-off angle wetting characteristics. Additionally, fuel cell power production was measured over a variety of fuel concentrations and alkalinities by generating polarization curve data. These coatings facilitated a significant increase in aqueous solution permeation and adhesion properties, as well as providing up to two-fold increases in maximum power generation in an alkaline DGFC, despite experiencing some decreased conductivity of the carbon cloth diffusion layer.

Comparison of Myoelectric Control Schemes for Simultaneous Hand and Wrist Movement using Chronically Implanted Electromyography: A Case Series.

OBJECTIVE: A current biomedical engineering challenge is the development of a system that allows fluid control of multi-functional prosthetic devices through a human-machine interface. Here we probe this challenge by studying two subjects with trans-radial limb loss as they control a virtual hand and wrist system using 6 or 8 chronically implanted intramuscular electromyographic (iEMG) signals. The subjects successfully controlled a 4, 5, and 6 Degrees of Freedom (DoF's) virtual hand and wrist systems to perform a target matching task. APPROACH: Two control systems were evaluated where one tied EMG features directly to movement directions (Direct Control) and the other method determines user intent in the context of prior training data (Linear Interpolation). MAIN RESULTS: Subjects successfully matched most targets with both controllers but differences were seen as the complexity of the virtual limb system increased. The Direct Control method encountered difficulty due to crosstalk at higher DoF's. The Linear Interpolation method reduced crosstalk effects and outperformed Direct Control at higher DoF's. This work also studied the use of the Postural Control Algorithm to control the hand postures simultaneously with wrist degrees of freedom. SIGNIFICANCE: This work presents preliminary evidence that the PC algorithm can be used in conjunction with wrist control, that Direct Control with iEMG signals allows stable 4-DoF control, and that EMG pre-processing using the Linear Interpolation method can improve performance at 5 and 6-DoF's.

Genome-scale molecular and phylogenetic characterization of Middle Point orbiviruses from Australia.

Middle Point orbivirus (MPOV) is an Australian arbovirus, belongs to the Yunnan orbivirus species found in China. First detected and reported from Beatrice Hill, Northern Territory (NT), MPOV has to date, only been exclusively reported from the NT, Australia. Whilst genetic characterization of MPOV has been previously described, only restricted to sequence information for segments 2 and 3 coding core protein VP2 and outer capsid protein VP3, respectively. This study presents for the first time nearly full-length genome sequences of MPOV, which represent 24 isolates collected over a span of more than 20 years from 1997 to 2018. Whilst the majority of isolates were sampled at Beatrice Hill, NT where MPOV is most frequently isolated, this report also describes the first two isolations of MPOV from Queensland (QLD), Australia. One of which is the first non-bovine isolate obtained from the mosquito vector Aedes vittiger. We further compared these MPOV sequences with known sequences of the Yunnan orbivirus and other known orbivirus sequences of mosquito origin found in Australia. The phylogenetic analyses indicate the Australian MPOV sequences are more closely related to each other than other known sequences of Yunnan orbivirus. Furthermore, MPOV sequences are closely related to sequences from the Indonesian isolate JKT-8650. The clustering of Australian sequences in the phylogenetic tree suggests the monophyletic lineage of MPOV circulating in Australia. Further, ongoing surveillance is required to assess the existence and prevalence of this or other yet undetected lineages of MPOV and other orbiviruses in Australia.

Optical vagus nerve modulation of heart and respiration via heart-injected retrograde AAV.

Vagus nerve stimulation has shown many benefits for disease therapies but current approaches involve imprecise electrical stimulation that gives rise to off-target effects, while the functionally relevant pathways remain poorly understood. One method to overcome these limitations is the use of optogenetic techniques, which facilitate targeted neural communication with light-sensitive actuators (opsins) and can be targeted to organs of interest based on the location of viral delivery. Here, we tested whether retrograde adeno-associated virus (rAAV2-retro) injected in the heart can be used to selectively express opsins in vagus nerve fibers controlling cardiac function. Furthermore, we investigated whether perturbations in cardiac function could be achieved with photostimulation at the cervical vagus nerve. Viral injection in the heart resulted in robust, primarily afferent, opsin reporter expression in the vagus nerve, nodose ganglion, and brainstem. Photostimulation using both one-photon stimulation and two-photon holography with a GRIN-lens incorporated nerve cuff, was tested on the pilot-cohort of injected mice. Changes in heart rate, surface electrocardiogram, and respiratory responses were observed in response to both one- and two-photon photostimulation. The results demonstrate feasibility of retrograde labeling for organ targeted optical neuromodulation.

Optogenetic stimulation of cholinergic fibers for the modulation of insulin and glycemia.

Previous studies have demonstrated stimulation of endocrine pancreas function by vagal nerve electrical stimulation. While this increases insulin secretion, expected concomitant reductions in circulating glucose do not occur. A complicating factor is the non-specific nature of electrical nerve stimulation. Optogenetic tools, however, provide the potential for cell-type specific neural stimulation using genetic targeting and/or spatially shaped excitation light. Here, we demonstrate light-activated stimulation of the endocrine pancreas by targeting parasympathetic (cholinergic) axons. In a mouse model expressing ChannelRhodopsin2 (ChR2) in cholinergic cells, serum insulin and glucose were measured in response to (1) ultrasound image-guided optical stimulation of axon terminals in the pancreas or (2) optical stimulation of axons of the cervical vagus nerve. Measurements were made in basal-glucose and glucose-stimulated conditions. Significant increases in plasma insulin occurred relative to controls under both pancreas and cervical vagal stimulation, while a rapid reduction in glycemic levels were observed under pancreatic stimulation. Additionally, ultrasound-based measurements of blood flow in the pancreas were increased under pancreatic stimulation. Together, these results demonstrate the utility of in-vivo optogenetics for studying the neural regulation of endocrine pancreas function and suggest its therapeutic potential for the control of insulin secretion and glucose homeostasis.

The Point Digit II: Mechanical Design and Testing of a Ratcheting Prosthetic Finger.

INTRODUCTION: People with partial hand loss represent the largest population of upper limb amputees by a factor of 10. The available prosthetic componentry for people with digit loss provide various methods of control, kinematic designs, and functional abilities. Here, the Point Digit II is empirically tested and a discussion is provided comparing the Point Digit II with the existing commercially available prosthetic fingers. MATERIALS AND METHODS: Benchtop mechanical tests were performed using prototype Point Digit II prosthetic fingers. The battery of tests included a static load test, a static mounting tear-out test, a dynamic load test, and a dynamic cycle test. These tests were implemented to study the mechanisms within the digit and the ability of the device to withstand heavy-duty use once out in the field. RESULTS: The Point Digit II met or exceeded all geometric and mechanical specifications. The device can withstand over 300 lbs of force applied to the distal phalange and was cycled over 250,000 times without an adverse event representing 3 years of use. Multiple prototypes were utilized across all tests to confirm the ability to reproduce the device in a reliable manner. CONCLUSIONS: The Point Digit II presents novel and exciting features to help those with partial hand amputation return to work and regain ability. The use of additive manufacturing, unique mechanism design, and clinically relevant design features provides both the patient and clinician with a prosthetic digit, which improves upon the existing devices available.

Bluetongue virus serotype 12 enters Australia - a further incursion of novel western lineage genome segments.

Bluetongue virus (BTV) is an arbovirus (genus: Orbivirus) that occurs worldwide. It infects domestic and wild ruminant species and can cause disease in livestock, producing high economic impact. Recently, it gained extra prominence throughout Europe, with disease occurring in regions traditionally free of BTV. BTV enters Australia from Southeast Asia via wind-borne infected Culicoides spp. The first Australian isolation was 1975 (BTV-20) and further serotypes were isolated between 1979-86 (BTV-1, -3, -9, -15, -16, -21, -23). Despite increased, more sensitive, monitoring, no more were detected in over two decades, implying a stable BTV episystem of eastern ancestry. Isolations of BTV-2, -7 and -5 then occurred between 2007-15, with the latter two possessing genome segments with high sequence identity to western isolates. We report on the first isolation and genomic characterization of BTV-12, which revealed that three more novel western topotype gene segments have entered northern Australia.

Spatiotemporal analyses suggest the role of glacial history and the ice-free corridor in shaping American badger population genetic variation.

Recurring glacial cycles through the Quaternary period drastically altered the size and distribution of natural populations of North American flora and fauna. The "southerly refugia model" has been the longstanding framework for testing the effects of glaciation on contemporary genetic patterns; however, insights from ancient DNA have contributed to the reconstruction of more complex histories for some species. The American badger, Taxidea taxus, provides an interesting species for exploring the genetic legacy of glacial history, having been hypothesized to have postglacially emerged from a single, southerly refugium to recolonize northern latitudes. However, previous studies have lacked genetic sampling from areas where distinct glacial refugia have been hypothesized, including the Pacific Northwest and American Far North (Yukon, Alaska). In order to further investigate the phylogeographic history of American badgers, we collected mitochondrial DNA sequence data from ancient subfossil material collected within the historical range (Alaska, Yukon) and combined them with new and previously published data from across the species' contemporary distribution (n = 1,207). We reconstructed a mostly unresolved phylogenetic tree and star-like haplotype network indicative of emergence from a largely panmictic glacial refugium and recent population expansion, the latter further punctuated by significantly negative Tajima's D and Fu's Fs values. Although directionality of migration cannot be unequivocally inferred, the moderate to high levels of genetic variation exhibited by American badgers, alongside the low frequency of haplotypes with indels in the Midwest, suggest a potential recolonization into central North America after the hypothesized ice-free corridor reopened ~13,000 years ago. Overall, the expanded reconstruction of phylogeographic history of American badgers offers a broader understanding of contemporary range-wide patterns and identifies unique genetic units that can likely be used to inform conservation of at-risk populations at the northern periphery.

A Myoelectric Postural Control Algorithm for Persons With Transradial Amputations: A Consideration of Clinical Readiness.

BACKGROUND: The bottleneck in upper limb prosthetic design is the myoelectric control algorithm. Here we studied the clinical readiness of the myoelectric postural control algorithm in a laboratory setting with two trans-radial amputees using a commercially available prosthetic limb system. TECHNIQUE: The postural control algorithm was integrated into prosthetic limb systems using standard of care components. A comparison between a commercial state of the art system (the i-limb revolution state-based myoelectric controller) and the postural controller was performed with two people with trans-radial amputation using a self-contained prosthesis system. DISCUSSION: The performance using the i-limb revolution state-based controller versus the postural controller was mixed based on the Southampton Hand Assessment Procedure. The SHAP scores indicate that the postural controller with i-limb revolution provided an average of 66% of hand function compared to an intact limb. Future work will study the advantages of the postural control algorithm in everyday use.

Hayes Yard virus: a novel ephemerovirus isolated from a bull with severe clinical signs of bovine ephemeral fever is most closely related to Puchong virus.

Bovine ephemeral fever is a vector-borne disease of ruminants that occurs in tropical and sub-tropical regions of Africa, Asia and Australia. The disease is caused by a rhabdovirus, bovine ephemeral fever virus (BEFV), which occurs as a single serotype globally. Although several other closely related ephemeroviruses have been isolated from cattle and/or arthropods, only kotonkan virus from Nigeria and (tentatively) Mavingoni virus from Mayotte Island in the Indian Ocean have been previously associated with febrile disease. Here, we report the isolation of a novel virus (Hayes Yard virus; HYV) from blood collected in February 2000 from a bull (Bos indicus) in the Northern Territory of Australia. The animal was suffering from a severe ephemeral fever-like illness with neurological involvement, including recumbency and paralysis, and was euthanised. Histological examination of spinal cord and lung tissue identified extensive haemorrhage in the dura mata with moderate perineuronal oedema and extensive emphysema. HYV displayed cone-shaped morphology, typical of rhabdoviruses, and was found to be most closely related antigenically to Puchong virus (PUCV), isolated in 1965 from mosquitoes in Malaysia. Analysis of complete genome sequences of HYV (15 025 nt) and PUCV (14 932 nt) indicated that each has a complex organisation (3' N-P-M-G-GNS-α1-α2-ß-γ-L 5') and expression strategy, similar to that of BEFV. Based on an alignment of complete L protein sequences, HYV and PUCV cluster with other rhabdoviruses in the genus Ephemerovirus and appear to represent two new species. Neutralising antibody to HYV was also detected in a retrospective survey of cattle sera collected in the Northern Territory.

Combination of Simultaneous Artificial Sensory Percepts to Identify Prosthetic Hand Postures: A Case Study.

Multiple sources of sensory information are combined to develop hand posture percepts in the intact system, but the combination of multiple artificial somatosensory percepts by human prosthesis users has not been studied. Here, we report on a case study in which a person with transradial amputation identified prosthetic hand postures using artificial somatosensory feedback. He successfully combined five artificial somatosensory percepts to achieve above-chance performance of 95.0% and 75.7% in identifying four and seven postures, respectively. We studied how artificial somatosensation and the extant hand representation are combined in the decision-making process by providing two mappings between the prosthetic sensor and the location of the sensory percept: (1) congruent, and (2) incongruent. The participant's ability to combine and engage with the sensory feedback significantly differed between the two conditions. The participant was only able to successfully generalize prior knowledge to novel postures in the congruent mapping. Further, he learned postures more accurately and quickly in the congruent mapping. Finally, he developed an understanding of the relationships between postures in the congruent mapping instead of simply memorizing each individual posture. These experimental results are corroborated by a Bayesian decision-making model which tracked the participant's learning.

Ecology and Infection Dynamics of Multi-Host Amdoparvoviral and Protoparvoviral Carnivore Pathogens.

Amdoparvovirus and Protoparvovirus are monophyletic viral genera that infect carnivores. We performed surveillance for and sequence analyses of parvoviruses in mustelids in insular British Columbia to investigate parvoviral maintenance and cross-species transmission among wildlife. Overall, 19.1% (49/256) of the tested animals were parvovirus-positive. Aleutian mink disease virus (AMDV) was more prevalent in mink (41.6%, 32/77) than martens (3.1%, 4/130), feline panleukopenia virus (FPV) was more prevalent in otters (27.3%, 6/22) than mink (5.2%, 4/77) or martens (2.3%, 3/130), and canine parvovirus 2 (CPV-2) was found in one mink, one otter, and zero ermines (N = 27). Viruses were endemic and bottleneck events, founder effects, and genetic drift generated regional lineages. We identified two local closely related AMDV lineages, one CPV-2 lineage, and five FPV lineages. Highly similar viruses were identified in different hosts, demonstrating cross-species transmission. The likelihood for cross-species transmission differed among viruses and some species likely represented dead-end spillover hosts. We suggest that there are principal maintenance hosts (otters for FPV, raccoons for CPV-2/FPV, mink for AMDV) that enable viral persistence and serve as sources for other susceptible species. In this multi-host system, viral and host factors affect viral persistence and distribution, shaping parvoviral ecology and evolution, with implications for insular carnivore conservation.

Design of a High-Speed Prosthetic Finger Driven by Peano-HASEL Actuators.

Current designs of powered prosthetic limbs are limited by the nearly exclusive use of DC motor technology. Soft actuators promise new design freedom to create prosthetic limbs which more closely mimic intact neuromuscular systems and improve the capabilities of prosthetic users. This work evaluates the performance of a hydraulically amplified self-healing electrostatic (HASEL) soft actuator for use in a prosthetic hand. We compare a linearly-contracting HASEL actuator, termed a Peano-HASEL, to an existing actuator (DC motor) when driving a prosthetic finger like those utilized in multi-functional prosthetic hands. A kinematic model of the prosthetic finger is developed and validated, and is used to customize a prosthetic finger that is tuned to complement the force-strain characteristics of the Peano-HASEL actuators. An analytical model is used to inform the design of an improved Peano-HASEL actuator with the goal of increasing the fingertip pinch force of the prosthetic finger. When compared to a weight-matched DC motor actuator, the Peano-HASEL and custom finger is 10.6 times faster, has 11.1 times higher bandwidth, and consumes 8.7 times less electrical energy to grasp. It reaches 91% of the maximum range of motion of the original finger. However, the DC motor actuator produces 10 times the fingertip force at a relevant grip position. In this body of work, we present ways to further increase the force output of the Peano-HASEL driven prosthetic finger system, and discuss the significance of the unique properties of Peano-HASELs when applied to the field of upper-limb prosthetic design. This approach toward clinically-relevant actuator performance paired with a substantially different form-factor compared to DC motors presents new opportunities to advance the field of prosthetic limb design.

Estimating local need for mental healthcare to inform fair resource allocation in the NHS in England: cross-sectional analysis of national administrative data linked at person level.

BACKGROUND: Equitable access to mental healthcare is a priority for many countries. The National Health Service in England uses a weighted capitation formula to ensure that the geographical distribution of resources reflects need. AIMS: To produce a revised formula for estimating local need for secondary mental health, learning disability (intellectual disability) and psychological therapies services for adults in England. METHOD: We used demographic records for 43 751 535 adults registered with a primary care practitioner in England linked with service use, ethnicity, physical health diagnoses and type of household, from multiple data-sets. Using linear regression, we estimated the individual cost of care in 2015 as a function of individual- and area-level need and supply variables in 2013 and 2014. We sterilised the effects of the supply variables to obtain individual-need estimates. We aggregated these by general practitioner practice, age and gender to derive weights for the national capitation formula. RESULTS: Higher costs were associated with: being 30-50 years old, compared with 20-24; being Irish, Black African, Black Caribbean or of mixed ethnicity, compared with White British; having been admitted for specific physical health conditions, including drug poisoning; living alone, in a care home or in a communal environment; and living in areas with a higher percentage of out-of-work benefit recipients and higher prevalence of severe mental illness. Longer distance from a provider was associated with lower cost. CONCLUSIONS: The resulting needs weights were higher in more deprived areas and informed the distribution of some 12% (£9 bn in 2019/20) of the health budget allocated to local organisations for 2019/20 to 2023/24.

Intravascular injections of adenoassociated viral vector serotypes rh10 and PHP.B transduce murine sciatic nerve axons.

Adenoassociated viral vectors provide a safe and robust method for expression of transgenes in nondividing cells such as neurons. Intravenous injections of these vectors provide a means of transducing motoneurons of peripheral nerves. Previous research has demonstrated that serotypes 1, rh10 and PHP.B can transduce motor neuron cell bodies in the spinal cord, but has not quantified expression in the peripheral nerve axon. Axonal labeling is crucial for optogenetic stimulation and detection of action potentials in peripheral nerve. Therefore, in this study, serotypes 1, PHP.B, and rh10 were tested for their ability to label axons of the murine sciatic and tibial nerve following intravenous injection. Serotype rh10 elicits expression in 10% of acetylcholine transferase positive axons of the sciatic nerve in immunohistochemically-stained sections. Serotype rh10 transduces a variety of axon diameters from <1-12 µm, while PHP.B transduces larger axons of diameter (4-16 µm). Expression was not seen with serotype 1. These results show the potential of serotypes PHP.B and rh10 delivery of transgenic products to axons of the peripheral nerve.

Identification and genomic characterization of the first isolate of bluetongue virus serotype 5 detected in Australia.

Bluetongue virus (BTV), transmitted by midges (Culicoides sp), is distributed worldwide and causes disease in ruminants. In particular, BT can be a debilitating disease in sheep causing serious trade and socio-economic consequences at both local and global levels. Across Australia, a sentinel cattle herd surveillance program monitors the BTV activity. Prior to 2014, BTV-1, -2, -3, -7, -9, -15, -16, -20, -21 and -23 had been isolated in Australia, but no bluetongue disease has occurred in a commercial Australian flock. We routinely use a combination of serology, virus isolation, RT-PCR and next generation and conventional nucleotide sequencing technologies to detect and phylogenetically characterize incursions of novel BTV strains into Australia. Screening of Northern Territory virus isolates in 2015 revealed BTV-5, a serotype new to Australia. We derived the complete genome of this isolate and determined its phylogenetic relationship with exotic BTV-5 isolates. Gene segments 2, 6, 7 and 10 exhibited a close relationship with the South African prototype isolate RSArrrr/5. This was the first Australian isolation of a Western topotype of segment 10. Serological surveillance data highlighted the antigenic cross-reactivity between BTV-5 and BTV-9. Phylogenetic investigation of segments 2 and 6 of these serotypes confirmed their unconventional relationships within the BTV serogroup. Our results further highlighted a need for a revision of the current serologically based system for BTV strain differentiation and importantly, implied a potential for genome segments of pathogenic Western BTV strains to rapidly enter Southeast Asia. This emphasized a need for continued high-level surveillance of vectors and viruses at strategic locations in the north of Australia The expansion of routine characterization and classification of BTV to a whole genome approach is recommended, to better monitor the presence and level of establishment of novel Western topotype segments within the Australian episystem.