Preliminary characterization of Tasmanian aquareovirus (TSRV) isolates.

In an attempt to determine whether or not genetic variants of the Tasmanian strain of Atlantic salmon aquareovirus (TSRV) exist, 14 isolates of TSRV, originating from various locations in Tasmania, covering a 20-year period (1990-2010), obtained from various host species and tissues, and isolated on different cell lines, were selected for this study. Two categories, termed "typical" and "atypical", of variants of TSRV were identified based on preliminary genotypic and phenotypic characterization carried out on these 14 different isolates. In addition, electron microscopic examination indicated the existence of at least three variants based on viral particle size. Finally, this study demonstrated the existence of at least one new variant of TSRV isolates, other than the more commonly isolated typical TSRV isolates, in farmed Tasmanian Atlantic salmon.

Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry.

Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the 'lynchpin' for size expansion is highly specific. We demonstrate that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection.

Effective Coordination and Management of Emerging Infectious Diseases in Wildlife Populations.

A transdisciplinary, One Health approach is proposed for the coordination of wildlife health diagnostics, research, and policy development. In some countries, considerable effort has been made to establish specific activities including surveillance and integration of wildlife health within diagnostic and research laboratories. We suggest that some of these activities can be improved and many countries still require national structures to deal with wildlife disease investigation and management. We also suggest that scientists in this field should actively engage with national and international organizations and conferences to influence the development of policy, diagnostics, research, and management of emerging wildlife diseases.

Isolation of a novel aquatic birnavirus from rainbow trout Oncorhynchus mykiss in Australia.

In November 2010, a rainbow trout (Oncorhynchus mykiss) hatchery in Victoria reported increased mortality rates in diploid and triploid female fingerlings. Live and moribund fish were submitted for laboratory investigation. All fish showed hyperpigmentation of the cranial half of the body. Histological lesions were seen in all areas of skin examined despite the localised nature of the gross lesions. There was irregular hyperplasia and spongiosis, alternating with areas of thinning and architectural disturbance. Occasionally, particularly in superficial layers of epithelium, cells showed large, eosinophilic inclusions that obscured other cellular detail. A small number of fish had necrosis in dermis, subcutis and superficial muscles. Bacteriological culture of skin and gills was negative for all bacterial pathogens, including Flavibacterium columnare, the agent of columnaris disease. Attempts at virus isolation from the skin of affected fish resulted in the development of a cytopathic effect in RTG-2 cell cultures suggestive of the presence of a virus. Negative contrast electron microscopy of cell culture supernatant demonstrated the presence of viral particles with the typical morphology of birnaviruses. Preliminary molecular characterisation identified an aquabirnavirus that differed from both the Tasmanian aquabirnavirus (TABV) and other aquabirnaviruses exotic to Australia. Previous isolates of aquabirnaviruses in Australia and New Zealand have been from healthy fish in a marine environment. This is the first report of an aquabirnavirus isolated from young salmonids at a freshwater hatchery in Australia. The role of the virus in the mortality event on the farm is uncertain as no further deaths attributable to this virus have occurred in the 4 yr since its initial discovery. The virus has been provisionally named Victorian trout aquabirnavirus (VTAB).

Novel phlebovirus with zoonotic potential isolated from ticks, Australia.

Recently discovered tick-borne phleboviruses have been associated with severe disease and death among persons in Asia and the United States. We report the discovery of a novel tick phlebovirus in Tasmania State, Australia, that is closely related to those zoonotic viruses found in Asia and North America.

Mesenchymal stromal cells integrate and form longitudinally-aligned layers when delivered to injured spinal cord via a novel fibrin scaffold.

Mesenchymal stromal cells (MSCs) have been shown to promote healing and regeneration in a number of CNS injury models and therefore there is much interest in the clinical use of these cells. For spinal cord injuries, a standard delivery method for MSCs is intraspinal injection, but this can result in additional injury and provides little control over how the cells integrate into the tissue. The present study examines the use of a novel fibrin scaffold as a new method of delivering MSCs to injured spinal cord. Use of the fibrin scaffold resulted in the formation of longitudinally-aligned layers of MSCs growing over the spinal cord lesion site. Host neurites were able to migrate into this MSC architecture and grow longitudinally. The length of the MSC bridge corresponded to the length of the fibrin scaffold. MSCs that were delivered via intraspinal injection were mainly oriented perpendicular to the plane of the spinal cord and remained largely restricted to the lesion site. Host neurites within the injected MSC graft were also oriented perpendicular to the plane of the spinal cord.

The novel 'Candidatus Amphibiichlamydia ranarum' is highly prevalent in invasive exotic bullfrogs (Lithobates catesbeianus).

Knowledge concerning microbial infectious diseases in the current amphibian crisis is rudimentary and largely limited to ranavirosis and chytridiomycosis. The family Chlamydiaceae is gaining attention as a common cause of disease in amphibians and may harbour new and emerging amphibian pathogens. We identified a novel species of Chlamydiales (Candidatus Amphibiichlamydia ranarum) with a prevalence of 71% in exotic invasive bullfrog tadpoles (Lithobates catesbeianus) from an introduced population in the Netherlands. The sequence of a 1474 bp 16S rRNA gene fragment showed that the novel taxon forms a well-defined clade with 'Candidatus Amphibiichlamydia salamandrae' within the Chlamydiaceae family. Although none of the tadpoles examined showed signs of clinical disease, urgent evaluation of its pathogenic potential for native amphibian species is required.

Visualising single molecules of HIV-1 and miRNA nucleic acids.

BACKGROUND: The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19-26 nucleotides in length. RESULTS: We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1-2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection. CONCLUSIONS: Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1-2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.

Cygnet River virus, a novel orthomyxovirus from ducks, Australia.

A novel virus, designated Cygnet River virus (CyRV), was isolated in embryonated eggs from Muscovy ducks in South Australia. CyRV morphologically resembles arenaviruses; however, sequencing identified CyRV as an orthomyxovirus. The high mortality rate among ducks co-infected with salmonellae suggests that CyRV may be pathogenic, either alone or in concert with other infections.

Batrachochytrium dendrobatidis zoospore secretions rapidly disturb intercellular junctions in frog skin.

Global amphibian declines are in part driven by the chytrid fungus Batrachochytrium dendrobatidis, causing superficial dermatomycosis with epidermal hyperplasia and hyperkeratosis in infected amphibians. The susceptibility to chytridiomycosis and the severity of epidermal lesions in amphibians with chytridiomycosis are not consistent across species or even among individuals. Severe infections cause death of the animal most likely through disturbance of ion homeostasis. The mechanism by which this superficial skin infection results in epidermal lesions has so far eluded precise definition. It was the aim of this study to unravel how B. dendrobatidis causes alterations that affect skin integrity. Exposure of Xenopus laevis skin to B. dendrobatidis zoospore supernatant using skin explants and Ussing chambers caused rapid disruption of intercellular junctions, demonstrated using histology and transmission electron microscopy. The loss of intercellular junctions led to detachment-induced cell apoptosis, or anoikis. The zoospore supernatant induced neither apoptosis nor necrosis in isolated primary keratinocytes of X. laevis. This supports the idea that the loss of cell contacts triggered apoptosis in the skin explants. Mass spectrometric analysis of the protein composition of the supernatant revealed a complex mixture, including several new virulence associated proteins, such as proteases, biofilm-associated proteins and a carotenoid ester lipase. Protease and lipase activity of the supernatant was confirmed with a protease and lipase assay. In conclusion, B. dendrobatidis zoospores produce a complex mixture of proteins that quickly disturbs epidermal intercellular junctions leading to anoikis in the anuran skin. The role of the identified proteins in this process remains to be determined.

Release of vancomycin and gentamicin from a contact lens versus a fibrin coating applied to a contact lens.

PURPOSE: To develop a contact lens capable of releasing antibiotics for a minimum of 8 hours for the treatment of bacterial keratitis. METHODS: Fibrin gel was loaded with vancomycin or gentamicin and then shaped into a curved disc. The disc was then used to coat the surface of a commercial contact lens or was sealed between two lenses. Separate contact lenses were soaked in solutions of vancomycin or gentamicin. The in vitro release kinetics for each system was determined using PBS at 37°C and a particle-enhanced turbidimetric inhibition immunoassay. The bioactivity of the antibiotics released from the fibrin was confirmed by using a microbiological assay. RESULTS: Vancomycin and gentamicin were released at similar rates from soaked contact lenses and a coating of fibrin gel; however, the amounts of antibiotic delivered by the two systems differed considerably. The fibrin coating released over three times more gentamicin but less than one-fifth that of the lenses soaked in vancomycin. When fibrin was encapsulated between two contact lenses, significantly more controlled release was observed. For all systems, bactericidal amounts of vancomycin and gentamicin were released throughout the three-day testing period. CONCLUSIONS: As a delivery system, fibrin gel loaded with gentamicin performs better than contact lenses soaked in gentamicin. The opposite is true for vancomycin, where soaked lenses outperform fibrin gel. These systems could potentially be used as a treatment for bacterial keratitis.

Agricultural intensification, priming for persistence and the emergence of Nipah virus: a lethal bat-borne zoonosis.

Emerging zoonoses threaten global health, yet the processes by which they emerge are complex and poorly understood. Nipah virus (NiV) is an important threat owing to its broad host and geographical range, high case fatality, potential for human-to-human transmission and lack of effective prevention or therapies. Here, we investigate the origin of the first identified outbreak of NiV encephalitis in Malaysia and Singapore. We analyse data on livestock production from the index site (a commercial pig farm in Malaysia) prior to and during the outbreak, on Malaysian agricultural production, and from surveys of NiV's wildlife reservoir (flying foxes). Our analyses suggest that repeated introduction of NiV from wildlife changed infection dynamics in pigs. Initial viral introduction produced an explosive epizootic that drove itself to extinction but primed the population for enzootic persistence upon reintroduction of the virus. The resultant within-farm persistence permitted regional spread and increased the number of human infections. This study refutes an earlier hypothesis that anomalous El Niño Southern Oscillation-related climatic conditions drove emergence and suggests that priming for persistence drove the emergence of a novel zoonotic pathogen. Thus, we provide empirical evidence for a causative mechanism previously proposed as a precursor to widespread infection with H5N1 avian influenza and other emerging pathogens.

Viruses of fish: an overview of significant pathogens.

The growing global demand for seafood together with the limited capacity of the wild-capture sector to meet this demand has seen the aquaculture industry continue to grow around the world. A vast array of aquatic animal species is farmed in high density in freshwater, brackish and marine systems where they are exposed to new environments and potentially new diseases. On-farm stresses may compromise their ability to combat infection, and farming practices facilitate rapid transmission of disease. Viral pathogens, whether they have been established for decades or whether they are newly emerging as disease threats, are particularly challenging since there are few, if any, efficacious treatments, and the development of effective viral vaccines for delivery in aquatic systems remains elusive. Here, we review a few of the more significant viral pathogens of finfish, including aquabirnaviruses and infectious hematopoietic necrosis virus which have been known since the first half of the 20th century, and more recent viral pathogens, for example betanodaviruses, that have emerged as aquaculture has undergone a dramatic expansion in the past few decades.

Pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission.

Bats of the genus Pteropus have been identified as the reservoir hosts for the henipaviruses Hendra virus (HeV) and Nipah virus (NiV). The aim of these studies was to assess likely mechanisms for henipaviruses transmission from bats. In a series of experiments, Pteropus bats from Malaysia and Australia were inoculated with NiV and HeV, respectively, by natural routes of infection. Despite an intensive sampling strategy, no NiV was recovered from the Malaysian bats and HeV was reisolated from only one Australian bat; no disease was seen. These experiments suggest that opportunities for henipavirus transmission may be limited; therefore, the probability of a spillover event is low. For spillover to occur, a range of conditions and events must coincide. An alternate assessment framework is required if we are to fully understand how this reservoir host maintains and transmits not only these but all viruses with which it has been associated.

Assessment of virally vectored autoimmunity as a biocontrol strategy for cane toads.

BACKGROUND: The cane toad, Bufo (Chaunus) marinus, is one of the most notorious vertebrate pests introduced into Australia over the last 200 years and, so far, efforts to identify a naturally occurring B. marinus-specific pathogen for use as a biological control agent have been unsuccessful. We explored an alternative approach that entailed genetically modifying a pathogen with broad host specificity so that it no longer caused disease, but carried a gene to disrupt the cane toad life cycle in a species specific manner. METHODOLOGY/PRINCIPAL FINDINGS: The adult beta globin gene was selected as the model gene for proof of concept of autoimmunity as a biocontrol method for cane toads. A previous report showed injection of bullfrog tadpoles with adult beta globin resulted in an alteration in the form of beta globin expressed in metamorphs as well as reduced survival. In B. marinus we established for the first time that the switch from tadpole to adult globin exists. The effect of injecting B. marinus tadpoles with purified recombinant adult globin protein was then assessed using behavioural (swim speed in tadpoles and jump length in metamorphs), developmental (time to metamorphosis, weight and length at various developmental stages, protein profile of adult globin) and genetic (adult globin mRNA levels) measures. However, we were unable to detect any differences between treated and control animals. Further, globin delivery using Bohle iridovirus, an Australian ranavirus isolate belonging to the Iridovirus family, did not reduce the survival of metamorphs or alter the form of beta globin expressed in metamorphs. CONCLUSIONS/SIGNIFICANCE: While we were able to show for the first time that the switch from tadpole to adult globin does occur in B. marinus, we were not able to induce autoimmunity and disrupt metamorphosis. The short development time of B. marinus tadpoles may preclude this approach.

Controlled release of chondroitinase ABC from fibrin gel reduces the level of inhibitory glycosaminoglycan chains in lesioned spinal cord.

Chondroitinase ABC (ChABC) is a bacterial enzyme that can enhance plasticity following injury to the central nervous system (CNS) by degrading the glycosaminoglycan (GAG) side chains of proteoglycans. CNS lesions treated with ChABC often show enhanced axonal sprouting and improved functional recovery and there is therefore much interest in the potential use of ChABC as a clinical treatment in humans. When highly concentrated fibrin gel containing ChABC was implanted adjacent to a spinal cord lesion, bioactive ChABC was detectable in the spinal cord for at least three weeks. Nearly six times more bioactive ChABC was detected in the spinal cord 3 weeks after injury when the fibrin delivery system was used vs. an intraspinal injection of ChABC (61+/-30 mU vs. 11+/-4 mU). Furthermore, 3 weeks after injury the level of inhibitory GAG found in injured spinal cord treated with the delivery system was 37% lower than the level of GAG in spinal cord treated with an injection of ChABC. When using the delivery system, 24.4% of the initial ChABC dose could still be detected in the lesion after 3 weeks, compared to just 4.4% when using an intraspinal injection of ChABC.

Broome virus, a new fusogenic Orthoreovirus species isolated from an Australian fruit bat.

This report describes the discovery and characterization of a new fusogenic orthoreovirus, Broome virus (BroV), isolated from a little red flying-fox (Pteropus scapulatus). The BroV genome consists of 10 dsRNA segments, each having a 3' terminal pentanucleotide sequence conserved amongst all members of the genus Orthoreovirus, and a unique 5' terminal pentanucleotide sequence. The smallest genome segment is bicistronic and encodes two small nonstructural proteins, one of which is a novel fusion associated small transmembrane (FAST) protein responsible for syncytium formation, but no cell attachment protein. The low amino acid sequence identity between BroV proteins and those of other orthoreoviruses (13-50%), combined with phylogenetic analyses of structural and nonstructural proteins provide evidence to support the classification of BroV in a new sixth species group within the genus Orthoreovirus.