Mitochondrial DNA supports the identification of two endangered river sharks (Glyphis glyphis and Glyphis garricki) across northern Australia

The river sharks (genus Glyphis) are a small group of poorly known sharks occurring in tropical rivers and estuarine waters across northern Australia, south-east Asia and the subcontinent. The taxonomy of the genus has long been unclear due to very few individuals having been caught and examined, resulting in a paucity of data regarding their distribution, biology and ecology. Only recently has attention focussed on the two Australian species, G. glyphis and G. garricki. This study is a result of a rare opportunity to collate the few samples that have been collected from these species and the bull shark Carcharhinus leucas, which shares an overlapping range. These samples were analysed using the DNA barcoding approach (cox1 mitochondrial gene), compared with six other species of carcharhinids and evaluated in light of the current taxonomic classification. Nine species-specific nucleotide differences were found between G. glyphis and G. garricki and no intra-specific variation provides strong support for the separation into distinct species. Significant differences were also observed at the inter-generic level, with Glyphis forming a distinct clade from Carcharhinus. This study provides the basis for future molecular studies required to better address conservation issues confronting G. glyphis and G. garricki in Australia.

Should the 40-year-old practice of releasing virulent myxoma virus to control rabbits (Oryctolagus cuniculus) be continued?

Release of virulent myxoma virus has been a key component of rabbit-control operations in Queensland, Australia, since the 1960s but its use rests on anecdotal reports. During a routine operation to release virulent myxoma virus we found no evidence to support the continued regular use of the technique in south-west Queensland. Radio-tagged rabbits inoculated with virulent myxoma virus contracted the disease but failed to pass enough virus to other rabbits to spread the disease. Rabbits with clinical signs of myxomatosis that were shot were infected with field strain derived from the original laboratory strain released in 1950 rather than the virulent strain that has been released annually. There was no change in rabbit survival or abundance caused by the release. Nevertheless, the release of virulent virus may be useful against isolated pockets of rabbits mainly because field strains are less likely to be present. Such pockets are more common now that rabbit haemorrhagic disease virus is established in Queensland.

Susceptibility of source plants to sugarcane Fiji disease virus influences the acquisition and transmission of the virus by the planthopper vector Perkinsiella saccharicida

Fiji leaf gall (FLG) caused by Sugarcane Fiji disease virus (SCFDV) is transmitted by the planthopper Perkinsiella saccharicida. FLG is managed through the identification and exploitation of plant resistance. The glasshouse-based resistance screening produced inconsistent transmission results and the factors responsible for that are not known. A series of glasshouse trials conducted over a 2-year period was compared to identify the factors responsible for the erratic transmission results. SCFDV transmission was greater when the virus was acquired by the vector from a cultivar that was susceptible to the virus than when the virus was acquired from a resistant cultivar. Virus acquisition by the vector was also greater when the vector was exposed to the susceptible cultivars than when exposed to the resistant cultivar. Results suggest that the variation in transmission levels is due to variation in susceptibility of sugarcane cultivars to SCFDV used for virus acquisition by the vector.

Transmission of Japanese Encephalitis Virus from the Black Flying Fox, Pteropus alecto, to Culex annulirostris Mosquitoes, Despite the Absence of Detectable Viremia.

To determine the potential role of flying foxes in transmission cycles of Japanese encephalitis virus (JEV) in Australia, we exposed Pteropus alecto (Megachiroptera: Pteropididae) to JEV via infected Culex annulirostris mosquitoes or inoculation. No flying foxes developed symptoms consistent with JEV infection. Anti-JEV IgG antibodies developed in 6/10 flying foxes exposed to infected Cx. annulirostris and in 5/5 inoculated flying foxes. Low-level viremia was detected by real-time reverse transcriptase polymerase chain reaction in 1/5 inoculated flying foxes and this animal was able to infect recipient mosquitoes. Although viremia was not detected in any of the 10 flying foxes that were exposed to JEV by mosquito bite, two animals infected recipient mosquitoes. Likewise, an inoculated flying fox without detectable viremia infected recipient mosquitoes. Although infection rates in recipient mosquitoes were low, the high population densities in roosting camps, coupled with migratory behavior indicate that flying foxes could play a role in the dispersal of JEV.

Development of a New Zealand database of plant virus and virus-like organisms.

The recent 8th Australasian plant virology workshop in Rotorua, New Zealand, discussed the development of a New Zealand database of plant virus and virus-like organisms. Key points of discussion included: (i) the purpose of such a database; (ii) who would benefit from the information in a database; (iii) the scope of a database and its associated collections; (iv) database information and format; and (v) potential funding of such a database. From the workshop and further research, we conclude that the preservation and verification of specimens within the collections and the development of a New Zealand database of plant virus and virus-like organisms is essential. Such a collection will help to fulfil statutory requirements in New Zealand and assist in fulfilling international obligations under the International Plant Protection Convention. Sustaining such a database will assist New Zealand virologists and statutory bodies to undertake scientifically sound research. Establishing reliable records and an interactive database will help to ensure accurate and timely diagnoses of diseases caused by plant viruses and virus-like organisms. Detection of new incursions and their diagnosis will be further enhanced by the use of such reference collections and their associated database. Connecting and associating this information to similar overseas databases would assist international collaborations and allow access to the latest taxonomic and diagnostic resources. Associated scientists working in the areas of plant breeding, export phytosanitary assurance and in the area of the conservation estate would also benefit from access to verified specimens of plant viruses and virus-like organisms. We conclude that funding of a New Zealand database of virus and virus-like organisms and its associated collections should be based partly on Crown funds, as it is a nationally significant biological resource.

First report of Colombian datura virus from Australia.

Colombian datura virus was identified from the ornamental plant Brugmansia sp., showing leaf mosaic symptoms. The nucleotide sequence of the 3 untranslated region and the amino acid sequence of the 3 portion of the coat protein were 100% identical to those from a Hungarian isolate of the virus. This represents the first record of this virus in Australia.

High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira.

A new test for pathogenic Leptospira isolates, based on RAPD-PCR and high-resolution melt (HRM) analysis (which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used to define serovars or detect genotypes. Ten serovars, of leptospires from the species Leptospira interrogans (serovars Australis, Robinsoni, Hardjo, Pomona, Zanoni, Copenhageni and Szwajizak), L. borgpetersenii (serovar Arborea), L. kirschneri (serovar Cynopteri) and L. weilii (serovar Celledoni), were typed against 13 previously published RAPD primers, using a real-time cycler (the Corbett Life Science RotorGene 6000) and the optimised reagents from a commercial kit (Quantace SensiMix). RAPD-HRM at specific temperatures generated defining amplicon melt profiles for each of the tested serovars. These profiles were evaluated as difference-curve graphs generated using the RotorGene software package, with a cut-off of at least 8 'U' (plus or minus). The results demonstrated that RAPD-HRM can be used to measure serovar diversity and establish identity, with a high degree of stability. The characterisation of Leptospira serotypes using a DNA-based methodology is now possible. As an objective and relatively inexpensive and rapid method of serovar identification, at least for cultured isolates, RAPD-HRM assays show convincing potentia.

Diagnosis of a naturally occurring dual infection of layer chickens with fowlpox virus and gallid herpesvirus 1 (infectious laryngotracheitis virus).

An outbreak of acute respiratory disease in layers was diagnosed as being of dual nature due to fowlpox and infectious laryngotracheitis using a multidisciplinary approach including virus isolation, histopathology, electron microscopy and polymerase chain reaction (PCR). The diagnosis was based on virus isolation of gallid herpesvirus 1 (GaHV-1) in chicken kidney cells and fowlpox virus (FWPV) in 9-day-old chicken embryonated eggs inoculated via the chorioallantoic membrane. The histopathology of tracheas from dead birds revealed intra-cytoplasmic and intra-nuclear inclusions suggestive of poxvirus and herpesvirus involvement. The presence of FWPV was further confirmed by electron microscopy, PCR and histology. All FWPV isolates contained the long terminal repeats of reticuloendotheliosis virus as demonstrated by PCR. GaHV-1 isolates were detected by PCR and were shown to have a different restriction fragment length polymorphism pattern when compared with the chicken embryo origin SA2 vaccine strain; however, they shared the same pattern with the Intervet chicken embryo origin vaccine strain. This is a first report of dual infection of chickens with GaHV-1 and naturally occurring FWPV with reticuloendotheliosis virus insertions. Further characterization of the viruses was carried out and the results are reported here.

DNA bar-coding reveals source and patterns of Thaumastocoris peregrinus invasions in South Africa and South America.

Thaumastocoris peregrinus is a recently introduced invertebrate pest of non-native Eucalyptus plantations in the Southern Hemisphere. It was first reported from South Africa in 2003 and in Argentina in 2005. Since then, populations have grown explosively and it has attained an almost ubiquitous distribution over several regions in South Africa on 26 Eucalyptus species. Here we address three key questions regarding this invasion, namely whether only one species has been introduced, whether there were single or multiple introductions into South Africa and South America and what the source of the introduction might have been. To answer these questions, bar-coding using mitochondrial DNA (COI) sequence diversity was used to characterise the populations of this insect from Australia, Argentina, Brazil, South Africa and Uruguay. Analyses revealed three cryptic species in Australia, of which only T. peregrinus is represented in South Africa and South America. Thaumastocoris peregrinus populations contained eight haplotypes, with a pairwise nucleotide distance of 0.2-0.9% from seventeen locations in Australia. Three of these haplotypes are shared with populations in South America and South Africa, but the latter regions do not share haplotypes. These data, together with the current distribution of the haplotypes and the known direction of original spread in these regions, suggest that at least three distinct introductions of the insect occurred in South Africa and South America before 2005. The two most common haplotypes in Sydney, one of which was also found in Brisbane, are shared with the non-native regions. Sydney populations of T. peregrinus, which have regularly reached outbreak levels in recent years, might thus have served as source of these three distinct introductions into other regions of the Southern Hemisphere.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, interserovar convergence, and evidence of attenuation in Leptospira reference collections.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD-HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD-HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD-HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD-HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.

DNA-based identification of Quambalaria pitereka causing severe leaf blight of Corymbia citriodora in China

Quambalaria spp. include serious plant pathogens, causing leaf and shoot blight of Corymbia and Eucalyptus spp. In this study, a disease resembling Quambalaria leaf blight was observed on young Corymbia citriodora trees in a plantation in the Guangdong Province of China. Comparisons of rDNA sequence data showed that the causal agent of the disease is Q. pitereka. This study provides the first report of Quambalaria leaf blight from China, and it is also the first time that this pathogen has been found on trees outside the native range of Eucalypts.

Raw data for project "Development of a DNA based aging technique for use in fisheries assessments" Fisheries and Development Corporation project 2007/033

The primary aim of this study was to determine the relationship between telomere length and age in a range of marine invertebrates including abalone (Haliotis spp) oysters (Saccostrea glomerata), spiny lobsters (Sagmariasus verreauxi formerly Jasus verreauxi and Jasus edwardsii) and school prawns (Metapenaeus macleayi). Additionally, this relationship was studied in a vertebrate organism using the freshwater fish Silver perch (Bidyanus bidyanus). Telomere length differences between tissues were also examined in some species such as Saccostrea glomerata, Sagmariasus verreauxi and Bidyanus bidyanus. In some cases cultured specimens of known age were used and this is quoted in the spreadsheets. For other wild-caught specimens where age was not known, size was used as a proxy for age. This may be a broad size class, or be determined by shell size or carapace length depending on the organism. Each spreadsheet contains raw data of telomere length estimates from Terminal Restriction Fragment Assays (TRF) for various individuals of each species including appropriate details such as age or size and tissue. Telomere length estimates are given in base pairs (bp). In most cases replicate experiments were conducted on groups of samples three times but on a small number of occasions only two replicate experiments were conducted. Further description of the samples can be found in final report of FRDC 2007/033. The arithmetic average for each individual (sample ID) across the two or three replicate experiments is also given. Bidyanus bidyanus (SilverPerch) Two sheets are contained within. a) Comparison of telomere length between different tissues (heart, liver and muscle) within the three year old age class - two replicate experiments were conducted. b) Comparison of telomere length between fish of different but known ages (0.25, 1, 2, and 3 years old) in each of three tissues, heart, liver and muscle – three replicate experiments were conducted per tissue. Haliotis spp (Abalone species) Three species were tested. H. asinina Telomere length was compared in two age classes-11 month and 18 month old abalone using muscle tissue from the foot. Within gel-variation was also estimated using a single sample run three times on one gel (replicate experiment). H. laevigata x H. rubra hybrids Telomere length was compared in three known age classes – two, three and four years old using muscle tissue from the foot. H. rubra Telomere length was compared in a range of different sized abalone using muscle tissue from the foot. Shell size is also given for each abalone Saccostrea glomerata Three sheets are contained within the file. a) Samples came from Moreton Bay Queensland in 2007. Telomere length was compared in two tissues (gill and mantle) of oysters in three age groups (1, 3 and 4 years) b) Samples came from Moreton Bay Queensland in 2009. Telomere length was compared in three age classes using DNA from gill tissue only c) Samples came from Wallis Lake, New South Wales. Telomere length was estimated from whole body minus the shell from 1 year old oysters, gill tissue of 3 age classes (1.5 years, 3 and 4 years), mantle tissue of two age classes (3 and 4 years). Sagmariasus verreauxi (formerly Jasus verreauxi) Telomere length was estimated from abdomen tissue of puerulus, gill and muscle tissue of 3 year old, large and very large size classes of lobsters. Jasus edwardsii Telomere length was measured in two size classes of lobsters- adults of varying sizes using muscle tissue and puerulus using tissues from the abdomen minus the exoskeleton. Metapenaeus macleayi Telomere length was measured in three size classes of school prawns adults. Muscle tissue was used, minus the exoskeleton.

A proposal to change existing virus species names to non-Latinized binomials.

A proposal has been posted on the ICTV website (2011.001aG.N.v1.binomial_sp_names) to replace virus species names by non-Latinized binomial names consisting of the current italicized species name with the terminal word "virus" replaced by the italicized and non-capitalized genus name to which the species belongs. If implemented, the current italicized species name Measles virus, for instance, would become Measles morbillivirus while the current virus name measles virus and its abbreviation MeV would remain unchanged. The rationale for the proposed change is presented.

The Interplay Between KSHV-encoded Viral Cyclin and CDK-inhibitors p27Kip1 and p21Cip1 -implications for Viral Lymphomagenesis

Cell division, which leads to the birth of two daughter cells, is essential for the growth and development of all organisms. The reproduction occurs in a series of events separated in time, designated as the cell cycle. The cell cycle progression is controlled by the activity of cyclin-dependent kinases (CDK). CDKs pair with cyclins to become catalytically active and phosphorylate a broad range of substrates required for cell cycle progression. In addition to cyclins, CDKs are regulated by inhibitory and activating phosphorylation events, binding to CDK-inhibitory proteins (CKI), and also by subcellular localization. The control of the CDK activity is crucial in preventing unscheduled progression of the cell cycle with mistakes having potentially hazardous consequences, such as uncontrolled proliferation of the cells, a hallmark of cancer. The mammalian cell cycle is a target of several DNA tumor viruses that can deregulate the host s cell cycle with their viral oncoproteins. A human herpesvirus called Kaposi s sarcoma herpesvirus (KSHV) is implicated in the cause of Kaposi s sarcoma (KS) and lymphoproliferative diseases such as primary effusion lymphomas (PEL). KSHV has pirated several cell cycle regulatory genes that it uses to manipulate its host cell and to induce proliferation. Among these gene products is a cellular cyclin D homologue, called viral cyclin (v-cyclin) that can activate cellular CDKs leading to the phosphorylation of multiple target proteins. Intriguingly, PELs that are naturally infected with KSHV consistently express high levels of CDK inhibitor protein p27Kip1 and still proliferate actively. The aim of this study was to investigate v-cyclin complexes and their activity in PELs, and search for an explanation why CKIs, such as p27Kip1 and p21Cip1 are unable to inhibit cell proliferation in this type of lymphoma. In this study, we found that v-cyclin binds to p27Kip1 in PELs, and confirmed this novel interaction also in the overexpression models. We observed that p27Kip1 associated with v-cyclin was also phosphorylated by a v-cyclin-associated kinase and identified cellular CDK6 as the major kinase partner of v-cyclin responsible for this phosphorylation. Analysis of the p27Kip1 residues targeted by v-cyclin-CDK6 revealed that serine 10 (S10) is the major phosphorylation site during the latent phase of the KSHV replication cycle. This phosphorylation led to the relocalization of p27Kip1 to the cytoplasm, where it is unable to inhibit nuclear cyclin-CDK complexes. In the lytic phase of the viral replication cycle, the preferred phosphorylation site on p27Kip1 by v-cyclin-CDK6 changed to threonine 187 (T187). T187 phosphorylation has been shown to lead to ubiquitin-mediated degradation of p27Kip1 and downregulation of p27Kip1 was also observed here. v-cyclin was detected also in complex with p21Cip1, both in overexpression models and in PELs. Phosphorylation of p21Cip1 on serine 130 (S130) site by v-cyclin-CDK6 functionally inactivated p21Cip1 and led to the circumvention of G1 arrest induced by p21Cip1. Moreover, p21Cip1 phosphorylated by v-cyclin-associated kinase showed reduced binding to CDK2, which provides a plausible explanation why p21Cip1 is unable to inhibit cell cycle progression upon v-cyclin expression. Our findings clarify the mechanisms on how v-cyclin evades the inhibition of cell cycle inhibitors and suggests an explanation to the uncontrolled proliferation of KSHV-infected cells.