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.

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.

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.

Peanut stripe potyvirus resistance in peanut (Arachis hypogaea L.) plants carrying viral coat protein gene sequences

Peanut (Arachis hypogaea L.) lines exhibiting high levels of resistance to peanut stripe virus (PStV) were obtained following microprojectile bombardment of embryogenic callus derived from mature seeds. Fertile plants of the commercial cultivars Gajah and NC7 were regenerated following co-bombardmentwith the hygromycin resistance gene and one of two forms of the PStV coat protein (CP) gene, an untranslatable, full length sequence (CP2) or a translatable gene encoding a CP with an N-terminal truncation (CP4). High level resistance to PStV was observed for both transgenes when plants were challenged with the homologous virus isolate. The mechanism of resistance appears to be RNA-mediated, since plants carrying either the untranslatable CP2 or CP4 had no detectable protein expression, but were resistant or immune (no virus replication). Furthermore, highly resistant, but not susceptible CP2 T0 plants contained transgene-specific small RNAs. These plants now provide important germplasm for peanut breeding, particularly in countries where PStV is endemic and poses a major constraint to peanut production.

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.

The development of an improved PCR-based marker system for Sw-5, an important TSWV resistance gene of tomato

Sw-5 is an important disease resistance gene of tomato, providing broad resistance to Tomato spotted wilt virus (TSWV). A cleaved amplified polymorphic sequence (CAPS) marker, closely linked to the gene, has been reported. Although the Sw-5 locus has been characterised, a gene-specific marker has not been developed. This paper presents a PCR-based marker-system that consists of the co-amplification of a dominant marker representing the Sw-5 gene sequence, and the modified CAPS marker as a positive control and indicator of genotype.

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.

Developing and communicating strategies for controlling virus diseases in vegetable cucurbit crops

Virus diseases cause serious yield and quality losses in field grown cucurbit crops worldwide. In Australia, the main viruses of cucurbits are Papaya ringspot virus (PRSV), Squash mosaic virus (SqMV), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV). Plants infected early have severely distorted fruit. High infection incidences, of ZYMV and PRSV in crops cause losses of marketable fruit of up to 100% and infected crops are often abandoned. Two new alternative hosts of ZYMV were identified, the native cucurbit Cucumis maderaspatanus and wild legume Rhyncosia minima. No new alternative hosts of PRSV, SqMV or WMV were found in Western Australia or Queensland. Seed transmission of ZYMV (0.7%) was found in seedlings grown from ZYMV-infected fruit of zucchini but not of pumpkin. None was detected with PRSV or SqMV in zucchini or pumpkin seedlings, respectively. ZYMV spread to pumpkins by aphids was greater downwind than upwind of a virus source. Delaying sowing by 2 weeks decreased ZYMV spread. Millet non-host barriers between pumpkin plantings slowed ZYMV infection. Host resistance gene (zym) in cucumber cultivars was effective against ZYMV. Pumpkin cultivars with resistance gene (Zym) became infected under high virus pressure but leaf symptoms were milder and infected plants higher yielding with more market-acceptable fruit than those without Zym. Most zucchini cultivars with Zym developed severe leaf and fruit symptoms. ZYMV, PRSV, WMV and SqMV spread readily from infected to healthy cucurbit plants by direct leaf contact. ZYMV survives and remains infective on diverse surfaces for up to 6 hours but can be inactivated by some disinfectants. Phylogenetic analysis indicates at least three separate introductions of ZYMV into Australia, with new introductions rarely occurring. ZYMV isolates clustered into three groups according to collection location i) Kununurra, ii) Northern Territory and iii) Carnarvon, Qld and Vic. A multiplex Real-Time PCR was developed which distinguished between the three groups of Australian isolates. Integrated disease management (IDM) strategies for virus diseases of vegetable cucurbit crops grown in the field were improved incorporating the new information gathered. These strategies are aimed at causing using minimal extra expense, labour demands and disruption to normal practices.

Confirmed case of encephalitis caused by Murray Valley encephalitis virus infection in a horse

A 5-year-old Australian stock horse in Monto, Queensland, Australia, developed neurological signs and was euthanized after a 6-day course of illness. Histological examination of the brain and spinal cord revealed moderate to severe subacute, nonsuppurative encephalomyelitis. Sections of spinal cord stained positively in immunohistochemistry with a flavivirus-specific monoclonal antibody. Reverse transcription polymerase chain reaction assay targeting the envelope gene of flavivirus yielded positive results from brain, spinal cord, cerebrospinal fluid, and facial nerve. A flavivirus was isolated from the cerebrum and spinal cord. Nucleotide sequences obtained from amplicons from both tissues and virus isolated in cell culture were compared with those in GenBank and had 96-98% identity with Murray Valley encephalitis virus. The partial envelope gene sequence of the viral isolate clustered into genotype 1 and was most closely related to a previous Queensland isolate.

Encephalomyocarditis virus infection in a splenectomised calf

Background Encephalomyocarditis (EMC) caused by EMC virus (EMCV) was diagnosed in a 5-month-old splenectomised calf, which died suddenly on an experimental farm that had a high infestation of rodents. Results At postmortem examination, the lungs were dark purple and diffusely congested. On histological examination, the calf had severe necrotising myocarditis. EMCV was isolated from the heart. The polyprotein gene of the EMCV isolate was amplified by PCR and had 85–91% identity with published EMCV sequences, including 89% identity with isolates from Queensland. On phylogenetic analysis, the polyprotein gene had highest sequence identity with South Korean EMCV strain, CBNU. Conclusion This is the first report of naturally occurring EMC in cattle in Australia and the first report of naturally occurring bovine EMC from which EMCV has been isolated.

Routes of Hendra Virus Excretion in Naturally-Infected Flying-Foxes: Implications for Viral Transmission and Spillover Risk

Pteropid bats or flying-foxes (Chiroptera: Pteropodidae) are the natural host of Hendra virus (HeV) which sporadically causes fatal disease in horses and humans in eastern Australia. While there is strong evidence that urine is an important infectious medium that likely drives bat to bat transmission and bat to horse transmission, there is uncertainty about the relative importance of alternative routes of excretion such as nasal and oral secretions, and faeces. Identifying the potential routes of HeV excretion in flying-foxes is important to effectively mitigate equine exposure risk at the bat-horse interface, and in determining transmission rates in host-pathogen models. The aim of this study was to identify the major routes of HeV excretion in naturally infected flying-foxes, and secondarily, to identify between-species variation in excretion prevalence. A total of 2840 flying-foxes from three of the four Australian mainland species (Pteropus alecto, P. poliocephalus and P. scapulatus) were captured and sampled at multiple roost locations in the eastern states of Queensland and New South Wales between 2012 and 2014. A range of biological samples (urine and serum, and urogenital, nasal, oral and rectal swabs) were collected from anaesthetized bats, and tested for HeV RNA using a qRT-PCR assay targeting the M gene. Forty-two P. alecto (n = 1410) had HeV RNA detected in at least one sample, and yielded a total of 78 positive samples, at an overall detection rate of 1.76% across all samples tested in this species (78/4436). The rate of detection, and the amount of viral RNA, was highest in urine samples (>serum, packed haemocytes >faecal >nasal >oral), identifying urine as the most plausible source of infection for flying-foxes and for horses. Detection in a urine sample was more efficient than detection in urogenital swabs, identifying the former as the preferred diagnostic sample. The detection of HeV RNA in serum is consistent with haematogenous spread, and with hypothesised latency and recrudesence in flying-foxes. There were no detections in P. poliocephalus (n = 1168 animals; n = 2958 samples) or P. scapulatus (n = 262 animals; n = 985 samples), suggesting (consistent with other recent studies) that these species are epidemiologically less important than P. alecto in HeV infection dynamics. The study is unprecedented in terms of the individual animal approach, the large sample size, and the use of a molecular assay to directly determine infection status. These features provide a high level of confidence in the veracity of our findings, and a sound basis from which to more precisely target equine risk mitigation strategies.

Transcriptome Analysis of Capsicum Chlorosis Virus-Induced Hypersensitive Resistance Response in Bell Capsicum

Background Capsicum chlorosis virus (CaCV) is an emerging pathogen of capsicum, tomato and peanut crops in Australia and South-East Asia. Commercial capsicum cultivars with CaCV resistance are not yet available, but CaCV resistance identified in Capsicum chinense is being introgressed into commercial Bell capsicum. However, our knowledge of the molecular mechanisms leading to the resistance response to CaCV infection is limited. Therefore, transcriptome and expression profiling data provide an important resource to better understand CaCV resistance mechanisms. Methodology/Principal Findings We assembled capsicum transcriptomes and analysed gene expression using Illumina HiSeq platform combined with a tag-based digital gene expression system. Total RNA extracted from CaCV/mock inoculated CaCV resistant (R) and susceptible (S) capsicum at the time point when R line showed a strong hypersensitive response to CaCV infection was used in transcriptome assembly. Gene expression profiles of R and S capsicum in CaCV- and buffer-inoculated conditions were compared. None of the genes were differentially expressed (DE) between R and S cultivars when mock-inoculated, while 2484 genes were DE when inoculated with CaCV. Functional classification revealed that the most highly up-regulated DE genes in R capsicum included pathogenesis-related genes, cell death-associated genes, genes associated with hormone-mediated signalling pathways and genes encoding enzymes involved in synthesis of defense-related secondary metabolites. We selected 15 genes to confirm DE expression levels by real-time quantitative PCR. Conclusion/Significance DE transcript profiling data provided comprehensive gene expression information to gain an understanding of the underlying CaCV resistance mechanisms. Further, we identified candidate CaCV resistance genes in the CaCV-resistant C. annuum x C. chinense breeding line. This knowledge will be useful in future for fine mapping of the CaCV resistance locus and potential genetic engineering of resistance into CaCV-susceptible crops.