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.

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.

Bromus catharticus striate mosaic virus: a new mastrevirus infecting Bromus catharticus from Australia.

Although monocotyledonous-plant-infecting mastreviruses (in the family Geminiviridae) are known to cause economically significant crop losses in certain areas of the world, in Australia, they pose no obvious threat to agriculture. Consequently, only a few Australian monocot-infecting mastreviruses have been described, and only two have had their genomes fully sequenced. Here, we present the third full-genome sequence of an Australian monocot-infecting mastrevirus from Bromus catharticus belonging to a distinct species, which we have tentatively named Bromus catharticus striate mosaic virus (BCSMV). Although the genome of this new virus shares only 57.7% sequence similarity with that of its nearest known relative, Digitaria didactyla striate mosaic virus (DDSMV; also from Australia), it has features typical of all other known mastrevirus genomes. Phylogenetic analysis showed that both the full genome and each of its probable expressed proteins group with the two other characterised Australian monocot-infecting mastreviruses. Besides the BCSMV genome sequence revealing that Australian monocot-infecting mastrevirus diversity rivals that seen in Africa, it has enabled us, for the first, to time detect evidence of recombination amongst the Australian viruses. Specifically, it appears that DDSMV possesses a short intergenic region sequence that has been recombinationally derived from either BCSMV or a close relative that has not yet been identified.

Baker's yeast expressing the Japanese encephalitis virus envelope protein on its cell surface: induction of an antigen-specific but non-neutralizing antibody response

Live recombinant Saccharomyces cerevisiae yeast expressing the envelope antigen of Japanese encephalitis virus (JEV) on the outer mannoprotein layer of the cell wall were examined for their ability to induce antigen-specific antibody responses in mice. When used as a modelantigen, parenteral immunization of mice with surface-expressing GFP yeast induced a strong anti-GFP antibody response in the absence of adjuvants. This antigen delivery approach was then used for a more stringent system, such as the envelope protein of JEV, which is a neurotropic virus requiring neutralizing antibodies for protection.Although 70% of cells were detected to express the total envelope protein on the surface by antibodies raised to the bacterially expressed protein, polyclonal anti-JEV antibodies failed to react with them. In marked contrast, yeast expressing the envelope fragments 238-398, 373-399 and 373-500 in front of a Gly-Ser linker were detected by anti-JEV antibodies as well as a monoclonal antibody but not by antibodies raised to the bacterially expressed protein. Immunization of mice with these surface-expressing recombinants resulted in a strong antibody response. However, the antibodies failed to neutralize the virus, although the fragments were selected based on neutralizing determinants.

A native Wolbachia Endosymbiont does not limit Dengue virus infection in the mosquito Aedes notoscriptus (Diptera: Culicidae)

The endosymbiotic bacterium Wolbachia pipientis infects many species of insects and has been transinfected into the mosquito Aedes aegypti (L.), the primary vector of dengue virus (DENV). Recently, it has been shown that Wolbachia blocks the replication and transmission of RNA viruses, such as DENV, in a number of mosquito species including Ae. aegypti and Aedes albopictus (Skuse), which is naturally infected with Wolbachia and considered a secondary vector for DENV. The mosquito species Aedes notoscriptus (Skuse) is highly prevalent in Australia, including in areas where DENV outbreaks have been recorded. The mosquito has been implicated in the transmission of Ross River and Barmah Forest viruses, but not DENV. We investigated whether Wolbachia naturally infects this mosquito species and whether it has an impact on the ability of Ae. notoscriptus to transmit DENV. We show, for the first time, that Ae. notoscriptus is naturally infected with a strain of Wolbachia that belongs to supergroup B and is localized only in the ovaries. However, Wolbachia infection in Ae. notoscriptus did not induce resistance to DENV and had no effect on overall DENV infection rate or titer. The presence of a native Wolbachia in Ae. notoscriptus cannot explain why this mosquito is an ineffective vector of DENV.

Virus identification and development of long-term management strategies for the rhubarb industry

The purpose of this report is to present the final results of all activities conducted under HAL Project VG05053 ‘Virus identification and development of long-term management strategies for the rhubarb industry’. The report provides a summary of project findings, a description of technology transfer activities, and recommendations arising from the outcomes of the project. The overall objective of this project was to devise a strategy for the control of rhubarb decline disease through 1) knowledge of the viruses present and their epidemiology, 2) production of virus-free planting material via tissue culture, and 3) formation of a national grower group to represent industry.

Resistance to tomato yellow leafcurl virus in tomato

Resistance to tomato yellow leafcurl virus in tomato.

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.

ACIAR Producing virus-free sweetpotato planting material in Papua New Guinea

The aim of this small research activity (SRA) is to provide a foundation for establishing a national 'clean seed system' for sweetpotato in Papua New Guinea.

Management of virus diseases in vegetables

• To undertake an audit of management systems used for tomato spotted wilt virus (TSWV) in greenhouse and field production with the aim of improving disease management determining knowledge gaps in virus-vector relationships. • To investigate the basis for the development of resistance breaking strains of TSWV in capsicums and apply this to virus management in capsicums. • To further develop effective virus management systems in vegetable cucurbit crops. Aspects to be investigated include value of barrier crops, non-insecticide products and cultivar tolerance to virus. • To further develop and assess the adoption and impact of integrated viral disease management systems in field grown and protected cropping systems as part of the vegetable industry development plan.

Surveillance and monitoring for endemic and exotic virus diseases of cotton

The aims of this project will provide capacity in virology expertise to help protect Australian cotton from virus diseases including both existing and those that pose significant biosecurity threats. This project will also provide continued capacity in virology to support the cotton industry.

Modern diagnosis of Epstein-Barr virus infections and post-transplant lymphoproliferative disease

Infection by Epstein-Barr virus (EBV) occurs in approximately 95% of the world s population. EBV was the first human virus implicated in oncogenesis. Characteristic for EBV primary infection are detectable IgM and IgG antibodies against viral capsid antigen (VCA). During convalescence the VCA IgM disappears while the VCA IgG persists for life. Reactivations of EBV occur both among immunocompromised and immunocompetent individuals. In serological diagnosis, measurement of avidity of VCA IgG separates primary from secondary infections. However, in serodiagnosis of mononucleosis it is quite common to encounter, paradoxically, VCA IgM together with high-avidity VCA IgG, indicating past immunity. We determined the etiology of this phenomenon and found that, among patients with cytomegalovirus (CMV) primary infection a large proportion (23%) showed antibody profiles of EBV reactivation. In contrast, EBV primary infection did not appear to induce immunoreactivation of CMV. EBV-associated post-transplant lymphoproliferative disease (PTLD) is a life threatening complication of allogeneic stem cell or solid organ transplantation. PTLD may present with a diverse spectrum of clinical symptoms and signs. Due to rapidity of PTLD progression especially after stem cell transplantation, the diagnosis must be obtained quickly. Pending timely detection, the evolution of the fatal disease may be halted by reduction of immunosuppression. A promising new PTLD treatment (also in Finland) is based on anti-CD-20 monoclonal antibodies. Diagnosis of PTLD has been demanding because of immunosuppression, blood transfusions and the latent nature of the virus. We set up in 1999 to our knowledge first in Finland for any microbial pathogen a real-time quantitative PCR (qPCR) for detection of EBV DNA in blood serum/plasma. In addition, we set up an in situ hybridisation assay for EBV RNA in tissue sections. In collaboration with a group of haematologists at Helsinki University Central Hospital we retrospectively determined the incidence of PTLD among 257 allogenic stem cell transplantations (SCT) performed during 1994-1999. Post-mortem analysis revealed 18 cases of PTLD. From a subset of PTLD cases (12/18) and a series of corresponding controls (36), consecutive samples of serum were studied by the new EBV-qPCR. All the PTLD patients were positive for EBV-DNA with progressively rising copy numbers. In most PTLD patients EBV DNA became detectable within 70 days of SCT. Of note, the appearance of EBV DNA preceded the PTLD symptoms (fever, lymphadenopathy, atypical lymphocytes). Among the SCT controls, EBV DNA occurred only sporadically, and the EBV-DNA levels remained relatively low. We concluded that EBV qPCR is a highly sensitive (100%) and specific (96%) new diagnostic approach. We also looked for and found risk factors for the development of PTLD. Together with a liver transplantation group at the Transplantation and Liver Surgery Clinic we wanted to clarify how often and how severely do EBV infections occur after liver transplantation. We studied by the EBV qPCR 1284 plasma samples obtained from 105 adult liver transplant recipients. EBV DNA was detected in 14 patients (13%) during the first 12 months. The peak viral loads of 13 asymptomatic patients were relatively low (<6600/ml), and EBV DNA subsided quickly from circulation. Fatal PTLD was diagnosed in one patient. Finally, we wanted to determine the number and clinical significance of EBV infections of various types occurring among a large, retrospective, nonselected cohort of allogenic SCT recipients. We analysed by EBV qPCR 5479 serum samples of 406 SCT recipients obtained during 1988-1999. EBV DNA was seen in 57 (14%) patients, of whom 22 (5%) showed progressively rising and ultimately high levels of EBV DNA (median 54 million /ml). Among the SCT survivors, EBV DNA was transiently detectable in 19 (5%) asymptomatic patients. Thereby, low-level EBV-DNA positivity in serum occurs relatively often after SCT and may subside without specific treatment. However, high molecular copy numbers (>50 000) are diagnostic for life-threatening EBV infection. We furthermore developed a mathematical algorithm for the prediction of development of life-threatening EBV infection.

Tobacco Streak Virus (TSV) in Cotton - scoping study

This project aims to examine the possible impact of Tobacco Streak Virus (TSV) on the Australian cotton industry. TSV is transmitted by thrips, causes a disease which has had a significant impact on grain crops in Central Queensland and a preliminary study in 2007 has shown that cotton is also susceptible to field infection in this region, but many questions remain unanswered. This project aims to: • Determine the impact of TSV in “normal” seasons. • Survey New South Wales and Queensland crops and determine alternative weed and crop hosts. • Assess yield-loss in cotton due to TSV, and factors that lead to systemic infection. • Assess thrips vector species present in cotton • Provide extension material on the impact and management of TSV in cotton

Management of Tobacco streak virus in sunflower and pulse crops

Management of Tobacco streak virus in sunflower and pulse crops.