Evolutionary relationships between bat coronaviruses and their hosts

Recent studies have suggested that bats are the natural reservoir of a range of coronaviruses (CoVs), and that rhinolophid bats harbor viruses closely related to the severe acute respiratory syndrome (SARS) CoV, which caused an outbreak of respiratory illness in humans during 2002-2003. We examined the evolutionary relationships between bat CoVs and their hosts by using sequence data of the virus RNA-dependent RNA polymerase gene and the bat cytochrome b gene. Phylogenetic analyses showed multiple incongruent associations between the phylogenies of rhinolophid bats and their CoVs, which suggested that host shifts have occurred in the recent evolutionary history of this group. These shifts may be due to either virus biologic traits or host behavioral traits. This finding has implications for the emergence of SARS and for the potential future emergence of SARS-CoVs or related viruses.

Henipavirus in Pteropus vampyrus Bats, Indonesia

The emergence of Nipah virus (NiV) in Malaysia in 1999 resulted in 265 known human infections (105 fatal), widespread infection in pigs (with >1 million culled to control the outbreak), and the collapse of the Malaysian pig export market. As with the closely related Hendra virus (HeV) that emerged in Australia in 1994 and caused fatal disease in horses and humans, bats of the genus Pteropus (commonly known as flying foxes) were identified as the major reservoir of Nipah virus in Malaysia. This report describes a serologic survey of Pteropus vampyrus in neighboring Indonesia.

Concordant molecular and phenotypic data delineate new taxonomy and conservation priorities for the endangered mountain yellow-legged frog

The mountain yellow-legged frog Rana muscosa sensu lato, once abundant in the Sierra Nevada of California and Nevada, and the disjunct Transverse Ranges of southern California, has declined precipitously throughout its range, even though most of its habitat is protected. The species is now extinct in Nevada and reduced to tiny remnants in southern California, where as a distinct population segment, it is classified as Endangered. Introduced predators (trout), air pollution and an infectious disease (chytridiomycosis) threaten remaining populations. A Bayesian analysis of 1901 base pairs of mitochondrial DNA confirms the presence of two deeply divergent clades that come into near contact in the Sierra Nevada. Morphological studies of museum specimens and analysis of acoustic data show that the two major mtDNA clades are readily differentiated phenotypically. Accordingly, we recognize two species, Rana sierrae, in the northern and central Sierra Nevada, and R. muscosa, in the southern Sierra Nevada and southern California. Existing data indicate no range overlap. These results have important implications for the conservation of these two species as they illuminate a profound mismatch between the current delineation of the distinct population segments (southern California vs. Sierra Nevada) and actual species boundaries. For example, our study finds that remnant populations of R. muscosa exist in both the southern Sierra Nevada and the mountains of southern California, which may broaden options for management. In addition, despite the fact that only the southern California populations are listed as Endangered, surveys conducted since 1995 at 225 historic (1899-1994) localities from museum collections show that 93.3% (n=146) of R. sierrae populations and 95.2% (n=79) of R. muscosa populations are extinct. Evidence presented here underscores the need for revision of protected population status to include both species throughout their ranges.

Review of bats and SARS

Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, including henipaviruses and variants of rabies viruses. Recently, we and another group independently identified several horse-shoe bat species (genus Rhinolophus) as the reservoir host for a large number of viruses that have a close genetic relationship with the coronavirus associated with severe acute respiratory syndrome (SARS). Our current research focused on the identification of the reservoir species for the progenitor virus of the SARS coronaviruses responsible for outbreaks during 2002-2003 and 2003-2004. In addition to SARS-like coronaviruses, many other novel bat coronaviruses, which belong to groups 1 and 2 of the 3 existing coronavirus groups, have been detected by PCR. The discovery of bat SARS-like coronaviruses and the great genetic diversity of coronaviruses in bats have shed new light on the origin and transmission of SARS coronaviruses.

Pimelea trichostachya poisoning (St George disease) in horses

A dense population of Pimelea trichostachya plants (Family Thymelaeaceae) in pasture poisoned a horse herd in southern inland Queensland in October-November 2005. Plant density was 2 to 45 g wet weight/m2 (mean 16 g/m2) from 5 to 69 plants/m2 (mean 38 plants/m2) representing 3 to 20% (mean 9%) of the volume of pasture on offer. Ten of 35 mares, fillies and geldings were affected. Clinical signs were loss of body weight, profound lethargy, serous nasal discharge, severe watery diarrhoea and subcutaneous oedema of the intermandibular space, chest and ventral midline. Pathological findings were anaemia, leucocytopenia, hypoproteinaemia, dilatation of the right ventricle of the heart, dilated hepatic portal veins and periportal hepatic sinusoids (peliosis hepatis), alimentary mucosal hyperaemia and oedema of mesenteric lymph nodes. Cattle grazing the same pasture were affected by Pimelea poisoning simultaneously. Removal of the horses to Pimelea-free pasture initiated recovery. The one other incident of this syndrome, previously only recognised in cattle in Australia, occurred in horses, in South Australia in 2002, with access to a dense Pimelea simplex population.

Rapid and sensitive detection of Avibacterium paragallinarum in the presence of other bacteria using a 5' Taq nuclease assay: A new tool for diagnosing infectious coryza

A 5' Taq nuclease assay specific for Avibacterium paragallinarum was designed and optimized for use in diagnosing infectious coryza. The region chosen for assay design was one of known specificity for Av. paragallinarum. The assay detected Av. paragallinarum reference strains representing the three Page and the eight Kume serovars, and field isolates from diverse geographical locations. No cross-reactions were observed with other Avibacterium species, with other bacteria taxonomically related to Av. paragallinarum nor with bacteria and viruses likely to be present in swabs collected from suspected infectious coryza cases. The detection limit for the assay was 6 to 60 colony-forming units per reaction. Twenty-two out of 53 swabs collected from sick birds reacted in the 5' Taq nuclease assay, whereas Av. paragallinarum was not isolated from any of the swabs. All of the 22 swabs yielded other bacteria in culture. The presence of Av. paragallinarum in the swabs was also demonstrated by sequencing, thereby confirming the ability of the assay to detect Av. paragallinarum in the presence of other bacteria. The ability to quantify bacterial load in the swabs using the 5' Taq nuclease assay was demonstrated.

Characterization of disease resistance gene candidates of the nucleotide binding site (NBS) type from banana and correlation of a transcriptional polymorphism with resistance to Fusarium oxysporum f.sp. cubense race 4

Most plant disease resistance (R) genes encode proteins with a nucleotide binding site and leucine-rich repeat structure (NBS-LRR). In this study, degenerate primers were used to amplify genomic NBS-type sequences from wild banana (Musa acuminata ssp. malaccensis) plants resistant to the fungal pathogen Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) race 4. Five different classes of NBS-type sequences were identified and designated as resistance gene candidates (RGCs). The deduced amino acid sequences of the RGCs revealed the presence of motifs characteristic of the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses grouped the banana RGCs within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS sequences. Southern hybridization showed that each banana RGC is present in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to FOC race 4. RGC1, 3 and 5 showed a constitutive expression profile in both resistant and susceptible plants whereas no expression was detected for RGC4. Interestingly, RGC2 expression was found to be associated only to FOC race 4 resistant lines. This finding could assist in the identification of a FOC race 4 resistance gene.

Tan spot: A new disease of pyrethrum caused by Microsphaeropsis tanaceti sp. nov

The isolation frequency of Microsphaeropsis sp. in spring in association with necrotic lesions on leaves in Tasmanian pyrethrum (Tanacetum cinerariifolium) fields has increased substantially since first identification in 2001. Examination of morphological features and sequencing of the internal transcribed spacer region (ITS) resulted in the identification of a new species, herein described as Microsphaeropsis tanaceti sp. nov. The pathogenicity of three M. tanaceti isolates to two pyrethrum cultivars was confirmed by inoculating glasshouse-grown plants in three experiments. No significant differences in the susceptibility of the two cultivars to infection by M. tanaceti were found. Symptoms were tan-coloured spots which coalesced around the margins of the leaves. Therefore, the name 'tan spot' is proposed for this new disease of pyrethrum. The sensitivity of seven M. tanaceti isolates to difenoconazole and azoxystrobin, commonly used fungicides for the management of foliar diseases in spring, was assessed under in vitro conditions. Sensitivity testing for difenoconazole was conducted using a mycelial growth assay on potato dextrose agar, whilst testing for sensitivity to azoxystrobin used a conidial germination assay on water agar. Microsphaeropsis tanaceti was found to be more sensitive to azoxystrobin than difenoconazole, with complete inhibition of conidial germination at concentrations above 0.625 µg a.i. mL-1. By comparison, concentrations of 50 µg a.i. difenoconazole mL-1 or greater were required for significant inhibition of mycelial growth. It therefore appears likely that there is currently some control of tan spot as a result of the use of azoxystrobin and to a lesser extent, difenoconazole, for the control of other diseases.

The essential and non-essential genes of Bovine herpesvirus 1

Bovine herpesvirus 1 (BoHV-1) is an economically important pathogen of cattle associated with respiratory and reproductive disease. To further develop BoHV-1 as a vaccine vector, a study was conducted to identify the essential and non-essential genes required for in vitro viability. Randominsertion mutagenesis utilizing a Tn5 transposition system and targeted gene deletion were employed to construct gene disruption and gene deletion libraries, respectively, of an infectious clone of BoHV-1. Transposon insertion position and confirmation of gene deletion were determined by direct sequencing. The essential or non-essential requirement of either transposed or deleted open reading frames (ORFs) was assessed by transfection of respective BoHV-1 DNA into host cells. Of the 73 recognized ORFs encoded by the BoHV-1 genome, 33 were determined to be essential and 36 to be non-essential for virus viability in cell culture; determining the requirement of the two dual copy ORFs was inconclusive. The majority of ORFs were shown to conform to the in vitro requirements of BoHV-1 homologues encoded by human herpesvirus 1 (HHV-1). However, ORFs encoding glycoprotein K (UL53), regulatory, membrane, tegument and capsid proteins (UL54, UL49.5, UL49, UL35, UL20, UL16 and UL7) were shown to differ in requirement when compared to HHV-1-encoded homologues.

Black Sigatoka disease: new technologies to strengthen eradication strategies in Australia

In 2001, an incursion of Mycosphaerella fijiensis, the causal agent of black Sigatoka, was detected in Australia's largest commercial banana growing region, the Tully Banana Production Area in North Queensland. An intensive surveillance and eradication campaign was undertaken which resulted in the reinstatement of the disease-free status for black Sigatoka in 2005. This was the first time black Sigatoka had ever been eradicated from commercial plantations. The success of the eradication campaign was testament to good working relationships between scientists, growers, crop monitors, quarantine regulatory bodies and industry. A key contributing factor to the success was the deployment of a PCR-based molecular diagnostic assay, developed by the Cooperative Research Centre for Tropical Plant Protection (CRCTPP). This assay complemented morphological identification and allowed high throughput diagnosis of samples facilitating rapid decision-making during the eradication campaign. This paper describes the development and successful deployment of molecular diagnostics for black Sigatoka. Shortcomings in the gel-based assay are discussed and the advantages of highly specific real-time PCR assays, capable of differentiating between Mycosphaerella fijiensis, Mycosphaerella musicola and Mycosphaerella eumusae are outlined. Real-time assays may provide a powerful diagnostic tool for applications in surveillance, disease forecasting and resistance testing for Sigatoka leaf spot diseases.

Absolute quantitation of Marek's disease virus and Herpesvirus of turkeys in chicken lymphocyte, feather tip and dust samples using real-time PCR

The further development of Taqman quantitative real-time PCR (qPCR) assays for the absolute quantitation of Marek's disease virus serotype 1 (MDV1) and Herpesvirus of turkeys (HVT) viruses is described and the sensitivity and reproducibility of each assay reported. Using plasmid DNA copies, the lower limit of detection was determined to be 5 copies for the MDV1 assay and 75 copies for the HVT assay. Both assays were found to be highly reproducible for Ct values and calculated copy numbers with mean intra- and inter-assay coefficients of variation being less than 5% for Ct and 20% for calculated copy number. The genome copy number of MDV1 and HVT viruses was quantified in PBL and feather tips from experimentally infected chickens, and field poultry dust samples. Parallelism was demonstrated between the plasmid-based standard curves, and standard curves derived from infected spleen material containing both viral and host DNA, allowing the latter to be used for absolute quantification. These methods should prove useful for the reliable differentiation and absolute quantitation of MDV1 and HVT viruses in a wide range of samples.

Cucumber mosaic virus infection of kava (Piper methysticum) and implications for cultural control of kava dieback disease

Cucumber mosaic virus (CMV) was found by reverse transcription polymerase chain reaction (RT-PCR) to be not fully systemic in naturally infected kava (Piper methysticum) plants in Fiji. Twenty-six of 48 samples (54%) from various tissues of three recently infected plants were CMV-positive compared with 7/51 samples (14%) from three long-term infections (plants affected by dieback for more than 1 year). The virus was also found to have a limited ability to move into newly formed stems. CMV was detected in only 2/23 samples taken from re-growth stems arising from known CMV infected/dieback affected plants. Mechanical inoculation experiments conducted in Fiji indicate that the known kava intercrop plants banana (Musa spp.), pineapple (Ananas comosus), peanut (Arachis hypogaea) and the common weed Mikania micrantha are potential hosts for a dieback-causing strain of CMV It was not possible to transmit the virus mechanically to the common kava intercrop plants taro (Colocasia esculenta), Xanthosoma sp., sweet potato (Ipomoea batatas), yam (Dioscorea alata), papaya (Carica papaya) or the weed Momordica charantia. Implications of the results of this research on a possible integrated disease management strategy are discussed.

Variation in acquisition of Fiji disease virus by Perkinsiella saccharicida (Hemiptera: Delphacidae).

Fiji leaf gall, caused the Fiji disease virus (genus Fijivirus, family Reoviridae, FDV), is a serious disease of sugarcane, Saccharum officinarum L., in Australia and several other Asia-Pacific countries. In Australia FDV is transmitted only by the planthopper Perkinsiella saccharicida Kirkaldy (Hemiptera: Delphacidae), in a propagative manner. Successful transmission of FDV by single planthoppers confined to individual virus free plants is highly variable, even under controlled conditions. The research reported here addresses two possible sources of this variation: 1) gender, wing form, and life stage of the planthopper; and 2) genotype of the source plant. The acquisition of FDV by macropterous males, macropterous females, brachypterous females, and nymphs of P. saccharicida from infected plants was investigated using reverse transcription-polymerase chain reaction to diagnose FDV infection in the vector. The proportion of individuals infected with FDV was not statistically related to life stage, gender, or adult wing form of the vector. The acquisition of FDV by P. saccharicida from four cultivars of sugarcane was compared to assess the influence of plant genotype on acquisition. Those planthopper populations reared on diseased 'NCo310' plants had twice as many infected planthoppers as those reared on 'Q110', 'WD1', and 'WD2'. Therefore, variation in FDV acquisition in this system is not the result of variation in the gender, wing form and life stage of the P. saccharicida vectors. The cultivar used as the source plant to rear vector populations does affect the proportion of infected planthoppers in a population.

Bats and Emerging Zoonoses: Henipaviruses and SARS.

Nearly 75% of all emerging infectious diseases (EIDs) that impact or threaten human health are zoonotic. The majority have spilled from wildlife reservoirs, either directly to humans or via domestic animals. The emergence of many can be attributed to predisposing factors such as global travel, trade, agricultural expansion, deforestation habitat fragmentation, and urbanization; such factors increase the interface and or the rate of contact between human, domestic animal, and wildlife populations, thereby creating increased opportunities for spillover events to occur. Infectious disease emergence can be regarded as primarily an ecological process. The epidemiological investigation of EIDs associated with wildlife requires a trans-disciplinary approach that includes an understanding of the ecology of the wildlife species, and an understanding of human behaviours that increase risk of exposure. Investigations of the emergence of Nipah virus in Malaysia in 1999 and severe acute respiratory syndrome (SARS) in China in 2003 provide useful case studies. The emergence of Nipah virus was associated with the increased size and density of commercial pig farms and their encroachment into forested areas. The movement of pigs for sale and slaughter in turn led to the rapid spread of infection to southern peninsular Malaysia, where the high-density, largely urban pig populations facilitated transmission to humans. Identifying the factors associated with the emergence of SARS in southern China requires an understanding of the ecology of infection both in the natural reservoir and in secondary market reservoir species. A necessary extension of understanding the ecology of the reservoir is an understanding of the trade, and of the social and cultural context of wildlife consumption. Emerging infectious diseases originating from wildlife populations will continue to threaten public health. Mitigating and managing the risk requires an appreciation of the connectedness between human, livestock and wildlife health, and of the factors and processes that disrupt the balance.

Rapid Global Expansion of the Fungal Disease Chytridiomycosis into Declining and Healthy Amphibian Populations.

The fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis, is enigmatic because it occurs globally in both declining and apparently healthy (non-declining) amphibian populations. This distribution has fueled debate concerning whether, in sites where it has recently been found, the pathogen was introduced or is endemic. In this study, we addressed the molecular population genetics of a global collection of fungal strains from both declining and healthy amphibian populations using DNA sequence variation from 17 nuclear loci and a large fragment from the mitochondrial genome. We found a low rate of DNA polymorphism, with only two sequence alleles detected at each locus, but a high diversity of diploid genotypes. Half of the loci displayed an excess of heterozygous genotypes, consistent with a primarily clonal mode of reproduction. Despite the absence of obvious sex, genotypic diversity was high (44 unique genotypes out of 59 strains). We provide evidence that the observed genotypic variation can be generated by loss of heterozygosity through mitotic recombination. One strain isolated from a bullfrog possessed as much allelic diversity as the entire global sample, suggesting the current epidemic can be traced back to the outbreak of a single clonal lineage. These data are consistent with the current chytridiomycosis epidemic resulting from a novel pathogen undergoing a rapid and recent range expansion. The widespread occurrence of the same lineage in both healthy and declining populations suggests that the outcome of the disease is contingent on environmental factors and host resistance.