First records of the papaya strain of Papaya ringspot virus (PRSV-P) in French Polynesia and the Cook Islands

The papaya strain of Papaya ringspot virus (PRSV-P), the cause of papaya ringspot disease, was confirmed in French Polynesia and the Cook Islands by double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). In French Polynesia, the virus has probably been on the islands of Tahiti and Moorea for several years, but appears not to have spread to eight other islands. In contrast, PRSV-P has only recently appeared in the Cook Islands and is now the subject of an eradication campaign.

Nucleocapsid gene variability reveals two subgroups of Lettuce necrotic yellows virus

The complete nucleocapsid (N) genes of eight Australian isolates of Lettuce necrotic yellows virus (LNYV) were amplified by reverse transcription PCR, cloned and sequenced. Phylogenetic analyses of these sequences revealed two distinct subgroups of LNYV isolates. Nucleotide sequences within each subgroup were more than 96% identical but heterogeneity between groups was about 20% at the nucleotide sequence level. However, less than 4% heterogeneity was noted at the amino acid level, indicating mostly third nucleotide position changes and a strong conservation for N protein function. There was no obvious geographical or temporal separation of the subgroups in Australia.

Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy® or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to 1 infected in 800 samples with pepper but never detecting more than 1 infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

Inconsistent Transmission of Banana Bunchy Top Virus in Micropropagated Bananas and its Implication for Germplasm Screening

Banana bunchy top virus (BBTV) was readily transmitted through tissue culture in banana (Mum sp.) cv. Lady finger (AAB) and Cavendish cv. Williams (AAA). Lines derived from infected and healthy field plants had similar in vitro multiplication rates. BBTV infected in vitro cultures displayed symptoms of stunting, leaf curling, chlorotic and green flecks, and poor root growth. Symptoms became milder with time, and were often difficult to discern in older, rapidly multiplying cultures. A triple antibody sandwich ELISA using polyclonal and monoclonal antibodies was very efficient for detecting BBTV in vitro. Symptomless, ELISA-negative plants arose in 10 out of 11 lines derived from BBTV-infected field plants and first appeared after 9 months continuous in vitro culture at a constant 28OC. Meristem tip culture or heat therapy was not used. These plants remained symptomless and ELISA-negative after planting out in the glasshouse (individual plants checked for up to 16 months). The implications of this inconsistent transmission of BBTV for germplasm indexing and exchange are discussed.

Completion of the genome sequence of Lettuce necrotic yellows virus, type species of the genus Cytorhabdovirus

We completed the genome sequence of Lettuce necrotic yellows virus (LNYV) by determining the nucleotide sequences of the 4a (putative phosphoprotein), 4b, M (matrix protein), G (glycoprotein) and L (polymerase) genes. The genome consists of 12,807 nucleotides and encodes six genes in the order 3′ leader-N-4a(P)-4b-M-G-L-5′ trailer. Sequences were derived from clones of a cDNA library from LNYV genomic RNA and from fragments amplified using reverse transcription-polymerase chain reaction. The 4a protein has a low isoelectric point characteristic for rhabdovirus phosphoproteins. The 4b protein has significant sequence similarities with the movement proteins of capillo- and trichoviruses and may be involved in cell-to-cell movement. The putative G protein sequence contains a predicted 25 amino acids signal peptide and endopeptidase cleavage site, three predicted glycosylation sites and a putative transmembrane domain. The deduced L protein sequence shows similarities with the L proteins of other plant rhabdoviruses and contains polymerase module motifs characteristic for RNA-dependent RNA polymerases of negative-strand RNA viruses. Phylogenetic analysis of this motif among rhabdoviruses placed LNYV in a group with other sequenced cytorhabdoviruses, most closely related to Strawberry crinkle virus.

Transgenic gene silencing strategies for virus control.

Co-suppression of transgenes and their homologous viral sequences by RNA silencing is a powerful strategy for achieving high-level virus resistance in plants. This review provides a brief overview of RNA silencing mechanisms in plants and discusses important transgene construct design features underpinning successful RNA silencing-mediated transgenic virus control. Application of those strategies to protect horticultural and field crops from virus infection and results of field tests are also provided. The effectiveness and stability of RNA-mediated transgenic resistance are assessed taking into account effects of viral, plant and environmental factors.

Hendra virus infection in a veterinarian

A veterinarian became infected with Hendra virus (HeV) after managing a terminally ill horse and performing a limited autopsy with inadequate precautions. Although she was initially only mildly ill, serological tests suggested latent HeV infection. Nevertheless, she remains well 2 years after her initial illness. Recently emerged zoonotic viruses, such as HeV, necessitate appropriate working procedures and personal protective equipment in veterinary practice.

Abaca bunchy top virus, a new member of the genus Babuvirus (family Nanoviridae)

Two isolates of a novel babuvirus causing "bunchy top" symptoms were characterised, one from abaca (Musa textilis) from the Philippines and one from banana (Musa sp.) from Sarawak (Malaysia). The name abacá bunchy top virus (ABTV) is proposed. Both isolates have a genome of six circular DNA components, each ca. 1.0-1.1 kb, analogous to those of isolates of Banana bunchy top virus (BBTV). However, unlike BBTV, both ABTV isolates lack an internal ORF in DNA-R, and the ORF in DNA-U3 found in some BBTV isolates is also absent. In all phylogenetic analyses of nanovirid isolates, ABTV and BBTV fall in the same clade, but on separate branches. However, ABTV and BBTV isolates shared only 79-81% amino acid sequence identity for the putative coat protein and 54-76% overall nucleotide sequence identity across all components. Stem-loop and major common regions were present in ABTV, but there was less than 60% identity with the major common region of BBTV. ABTV and BBTV were also shown to be serologically distinct, with only two out of ten BBTV-specific monoclonal antibodies reacting with ABTV. The two ABTV isolates may represent distinct strains of the species as they are less closely related to each other than are isolates of the two geographic subgroups (Asian and South Pacific) of BBTV.

The environmental fate of diuron under a conventional production regime in a sugarcane farm during the plant cane phase

BACKGROUND: Field studies of diuron and its metabolites 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU), 3,4-dichlorophenylurea (DCPU) and 3,4-dichloroaniline (DCA) were conducted in a farm soil and in stream sediments in coastal Queensland, Australia. RESULTS: During a 38 week period after a 1.6 kg ha^-1 diuron application, 70-100% of detected compounds were within 0-15 cm of the farm soil, and 3-10% reached the 30-45 cm depth. First-order t1/2 degradation averaged 49 ± 0.9 days for the 0-15, 0-30 and 0-45 cm soil depths. Farm runoff was collected in the first 13-50 min of episodes lasting 55-90 min. Average concentrations of diuron, DCPU and DCPMU in runoff were 93, 30 and 83-825 µg L^-1 respectively. Their total loading in all runoff was >0.6% of applied diuron. Diuron and DCPMU concentrations in stream sediments were between 3-22 and 4-31 µg kg^-1 soil respectively. The DCPMU/diuron sediment ratio was >1. CONCLUSION: Retention of diuron and its metabolites in farm topsoil indicated their negligible potential for groundwater contamination. Minimal amounts of diuron and DCMPU escaped in farm runoff. This may entail a significant loading into the wider environment at annual amounts of application. The concentrations and ratio of diuron and DCPMU in stream sediments indicated that they had prolonged residence times and potential for accumulation in sediments. The higher ecotoxicity of DCPMU compared with diuron and the combined presence of both compounds in stream sediments suggest that together they would have a greater impact on sensitive aquatic species than as currently apportioned by assessments that are based upon diuron alone.

Drosophila melanogaster mounts a unique immune response to the rhabdovirus Sigma virus

Rhabdoviruses are important pathogens of humans, livestock, and plants that are often vectored by insects. Rhabdovirus particles have a characteristic bullet shape with a lipid envelope and surface-exposed transmembrane glycoproteins. Sigma virus (SIGMAV) is a member of the Rhabdoviridae and is a naturally occurring disease agent of Drosophila melanogaster. The infection is maintained in Drosophila populations through vertical transmission via germ cells. We report here the nature of the Drosophila innate immune response to SIGMAV infection as revealed by quantitative reverse transcription-PCR analysis of differentially expressed genes identified by microarray analysis. We have also compared and contrasted the immune response of the host with respect to two nonenveloped viruses, Drosophila C virus (DCV) and Drosophila X virus (DXV). We determined that SIGMAV infection upregulates expression of the peptidoglycan receptor protein genes PGRP-SB1 and PGRP-SD and the antimicrobial peptide (AMP) genes Diptericin-A, Attacin-A, Attacin-B, Cecropin-A1, and Drosocin. SIGMAV infection did not induce PGRP-SA and the AMP genes Drosomycin-B, Metchnikowin, and Defensin that are upregulated in DCV and/or DXV infections. Expression levels of the Toll and Imd signaling cascade genes are not significantly altered by SIGMAV infection. These results highlight shared and unique aspects of the Drosophila immune response to the three viruses and may shed light on the nature of the interaction with the host and the evolution of these associations.

Infection with Menangle virus in flying foxes (Pteropus spp.) in Australia

Objective: To examine flying foxes (Pteropus spp.) for evidence of infection with Menangle virus. Design: Clustered non-random sampling for serology, virus isolation and electron microscopy (EM). Procedure: Serum samples were collected from 306 Pteropus spp. in northern and eastern Australia and tested for antibodies against Menangle virus (MenV) using a virus neutralisation test (VNT). Virus isolation was attempted from tissues and faeces collected from 215 Pteropus spp. in New South Wales. Faecal samples from 68 individual Pteropus spp. and four pools of faeces were examined by transmission EM following routine negative staining and immunogold labelling. Results: Neutralising antibodies (VNT titres ≥ 8) against MenV were detected in 46% of black flying foxes (P. alecto), 41% of grey-headed flying foxes (P. poliocephalus), 25% of spectacled flying foxes (P. conspicillatus) and 1% of little red flying foxes (P. scapulatus) in Australia. Positive sera included samples collected from P. poliocephalus in a colony adjacent to a piggery that had experienced reproductive disease caused by MenV. Virus-like particles were observed by EM in faeces from Pteropus spp. and reactivity was detected in pooled faeces and urine by immunogold EM using sera from sows that had been exposed to MenV. Attempts to isolate the virus from the faeces and tissues from Pteropus spp. were unsuccessful. Conclusion: Serological evidence of infection with MenV was detected in Pteropus spp. in Australia. Although virus-like particles were detected in faeces, no viruses were isolated from faeces, urine or tissues of Pteropus spp.

Field isolates of Tomato spotted wilt virus overcoming resistance in capsicum in Australia

In 2002 at Virginia, South Australia, capsicum cultivars having the Tsw resistance gene against Tomato spotted wilt virus (TSWV) developed symptoms typical of TSWV infection and several glasshouse-grown crops were almost 100% infected. Samples reacted with TSWV antibodies in ELISA. Virus isolates from infected plants induced severe systemic symptoms, rather than a hypersensitive reaction, when inoculated onto capsicum cultivars and Capsicum chinense genotypes ( PI 152225 and PI 159236) that carry the Tsw resistance gene. Isolates virulent towards the Tsw gene had molecular and biological properties very similar to standard TSWV isolates, including a hypersensitive reaction in Sw-5 (TSWV-resistant) tomato genotypes. Tsw-virulent isolates were found during surveys at Virginia in 2002 and 2004 in both TSWV-resistant and susceptible cultivars of capsicum and tomato.

Identification of two distinct bovine parainfluenza virus type 3 genotypes

The partial gene sequencing of the matrix (M) protein from seven clinical isolates of bovine parainfluenza virus type 3 (BPIV-3), and the complete sequencing of a representative isolate (Q5592) was completed in this study. Nucleotide sequence analysis was initiated because of the failure of in-house BPIV-3 RT-PCR methods to yield expected products for four of the isolates. Phylogenetic reconstructions based on the nucleotide sequences for the M-protein and the entire genome, using all of the available BPIV-3 nucleotide sequences, demonstrated that there were two distinct BPIV-3 genotypes (BPIV-3a and BPIV-3b). These newly identified genotypes have implications for the development of BPIV-3 molecular detection methods and may also impact on BPIV-3 vaccine formulations.

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.

Capsicum chlorosis virus infecting Capsicum annuum in the East Kimberley region of Western Australia

Capsicum chlorosis virus (CaCV) was detected in field grown Capsicum annuum from Kununurra in northeast Western Australia. Identification of the Kununurra isolate (WA-99) was confirmed using sap transmission to indicator hosts, positive reactions with tospovirus serogroup IV-specific antibodies and CaCV-specific primers, and amino acid sequence comparisons that showed >97% identity with published CaCV nucleocapsid gene sequences. The reactions of indicator hosts to infection with WA-99 often differed from those of the type isolate from Queensland. The virus multiplied best when test plants were grown at warm temperatures. CaCV was not detected in samples collected in a survey of C. annuum crops planted in the Perth Metropolitan area.