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.

Herpesviral haematopoietic necrosis virus (CyHV-2) infection: case studies from commercial goldfish farms

Herpesviral haematopoietic necrosis is a disease of goldfish, Carassius auratus, caused by Cyprinid herpesvirus-2 (CyHV-2) infection. Quantitative PCR was carried out on tissue homogenates from healthy goldfish fingerlings, broodfish, eggs and fry directly sampled from commercial farms, from moribund fish submitted to our laboratory for disease diagnosis, and on naturally-infected CyHV-2 carriers subjected to experimental stress treatments. Healthy fish from 14 of 18 farms were positive with copy numbers ranging from tens to 10(7) copies mu g(-1) DNA extracted from infected fish. Of 118 pools of broodfish tested, 42 were positive. The CyHV-2 was detected in one lot of fry produced from disinfected eggs. Testing of moribund goldfish, in which we could not detect any other pathogens, produced 12 of 30 cases with 10(6)-10(8) copies of CyHV-2 mu g(-1) DNA extracted. Subjecting healthy CyHV-2 carriers to cold shock (22-10 degrees C) but not heat, ammonia or high pH, increased viral copy numbers from mean copy number (+/- SE) of 7.3 +/- 11 to 394 +/- 55 mu g(-1) DNA extracted after 24 h. CyHV-2 is widespread on commercial goldfish farms and outbreaks apparently occur when healthy carriers are subjected to a sharp temperature drop followed by holding at the permissive temperature for the disease.

Australian Bat Lyssavirus

In Chapter 1, the literature relating to rabies virus and the rabies like lyssaviruses is reviewed. In Chapter 2, data are presented from 1170 diagnostic submissions for ABLV testing by fluorescent antibody test (Centocor FAT). All 27 non-bat submissions were ABLV-negative. Of 1143 bat accessions 74 (16%) were ABLV-positive, including 69 of 974 (7.1%) flying foxes (Pteropus spp.), 5 of 7 (71.4%) Saccolaimus flaviventris (Yellow-bellied sheathtail bats), none of 151 other microchiropteran bats, and none of 11 unidentified bats. Statistical analysis of data from 868 wild Black, Grey-headed, Little Red and Spectacled flying foxes (Pteropus alecto, P. poliocephalus, P. scapulatus, and P. conspicillatus) indicated that three factors; species, health status and age were associated with significant (p< 0.001) differences in the proportion of ABLV-positive bats. Other factors including sex, whether the bat bit a person or animal, region, year, and season submitted, were not associated with ABLV. Case data for 74 ABLV-positive bats, including the circumstances in which they were found and clinical signs, is presented. In Chapter 3, the aetiological diagnosis was investigated for 100 consecutive flying fox submissions with neurological signs. ABLV (32%), spinal and head injuries (29%), and neuro-angiostrongylosis (18%) accounted for most neurological syndromes in flying foxes. No evidence of lead poisoning was found in unwell (n=16) or healthy flying foxes (n=50). No diagnosis was reached for 16 cases, all of which were negative for ABLV by TaqMan PCR. The molecular diversity of ABLV was examined in Chapter 4 by sequencing 36 bases of the leader sequence, the entire N gene, and start of the P gene of 28 isolates from pteropid bats and 3 isolates from Yellow-bellied sheathtail (YBST) bats. Phylogenetic analysis indicated all ABLV isolates clustered together as a discrete group within the Lyssavirus genera closely related to rabies virus and European bat lyssavirus-2 isolates. The ABLV lineage consisted of two variants; one (ybst-ABLV) consisted of isolates only from YBST bats, the other (pteropid-ABLV) was common to Black, Grey-headed and Little Red flying foxes. No associations were found between the sequences and either the geographical location or year found, or individual flying fox species. In Chapter 5, 15 inocula prepared from the brains or salivary glands of naturally-infected bats were evaluated by intracerebral (IC) and footpad (FP) inoculation of Quackenbush mice in order to select and characterize a highly virulent inoculum for further use in bats (Inoculum 5). In Chapter 6, nine Grey-headed flying foxes were inoculated with 105.2 to 105.5 MICED50 of Inoculum 5 divided into four sites, left footpad, pectoral muscle, temporal muscle and muzzle. Another bat was inoculated with half this dose divided into the footpad and pectoral muscle only. Seven of 10 bats developed clinical disease of 1 to 4 days duration between PI-days 10 and 19 and were shown to be ABL-positive by FAT, HAM immunoperoxidase staining, virus isolation in mice, and TaqMan PCR. Five of the seven bats displayed overt aggression, one died during a seizure, and one showed intractable agitation, pacing, tremors, and ataxia. Viral antigen was demonstrated throughout the central and peripheral nervous systems and in the epithelial cells of the submandibular salivary glands (n=4). All affected bats had mild to moderate non-suppurative meningoencephalitis and severe ganglioneuritis. No ABLV was detected in three bats that remained well until the end of the experiment on day 82. One survivor developed a strong but transient antibody response. In Chapter 7, the relative virulence of inocula prepared from the brains and salivary glands of experimentally infected flying foxes was evaluated in mice by IC and FP inoculation and TaqMan assay. The effects in mice were correlated to the TaqMan CT value and indicated a crude association between virulence and CT value that has potential application in the selection of inocula. In Chapter 8, 36 Black and Grey-headed flying foxes were vaccinated with one (day 0) or two (+ day 28) doses of Nobivac rabies vaccine and co-vaccinated with keyhole limpet haemocyanin (KLH). All bats responded to the Nobivac vaccine with a rabies-RFFIT titer > 0.5 IU/mL that is nominally indicative of protective immunity. Plasma from bats with rabies titres >2 IU/mL had cross-neutralising ABLV titres >1:154. A specifically developed ELISA detected a strong but transient response to KLH.

The use of temperate species in the Australian subtropics.

Temperate species and tropical crop silage are the basis for forage production for the dairy industry in the Australian subtropics. Irrigation is the key resource needed for production, with little survival of temperate species under rain-grown conditions except for lucerne. Annual ryegrass (Lolium multiflorum), fertilised with either inorganic nitrogen or grown with clovers, is the main cool season forage for the dairy industry. It is sown into fully prepared seedbeds, oversown into tropical grasses, especially kikuyu (Pennisetum clandestinum) or sown after mulching. There has been a continual improvement in the performance of annual and hybrid ryegrass cultivars over the last 25 years. In small plot, cutting experiments, yields of annual ryegrass typically range from 15 to 21 t DM/ha, with equivalent on-farm yields of 7 to 14 t DM/ha of utilised material. Rust (Puccinia coronata) remains the major concern although resistance is more stable than in oats. There have also been major improvements in the performance of perennial ryegrass (L. perenne) cultivars although their persistence under grazing is insufficient to make them a reliable forage source for the subtropics. On the other hand, tall fescue (Festuca arundinacea) and prairie grass (Bromus willdenowii) cultivars perform well under cutting and grazing, although farmer resistance to the use of tall fescue is strong. White clover (Trifolium repens) is a reliable and persistent performer although disease usually reduces its performance in the third year after sowing. Persian (Shaftal) annual clover (T. resupinatum) gives good winter production but the performance of berseem clover (T. alexandrinum) is less reliable and the sub clovers (T. subterraneum) are generally not suited to clay soils of neutral to alkaline pH. Lucerne (Medicago sativa), either as a pure stand or in mixtures, is a high producing legume under both irrigation and natural rainfall. Understanding the importance of leaf and crown diseases, and the development of resistant cultivars, have been the reasons for its reliability. Insects on temperate species are not as serious a problem in the subtropics as in New Zealand (NZ). Fungal and viral diseases, on the other hand, cause many problems and forage performance would benefit from more research into resistance.

Cultivar-specific effects of pathogen testing on storage root yield of sweetpotato, Ipomoea batatas.

The accumulation and perpetuation of viral pathogens over generations of clonal propagation in crop species such as sweetpotato, Ipomoea batatas, inevitably result in a reduction in crop yield and quality. This study was conducted at Bundaberg, Australia to compare the productivity of field-derived and pathogen-tested (PT) clones of 14 sweetpotato cultivars and the yield benefits of using healthy planting materials. The field-derived clonal materials were exposed to the endemic viruses, while the PT clones were subjected to thermotherapy and meristem-tip culture to eliminate viral pathogens. The plants were indexed for viruses using nitrocellulose membrane-enzyme-linked immunosorbent assay and graft-inoculations onto Ipomoea setosa. A net benefit of 38% in storage root yield was realised from using PT materials in this study. Conversely, in a similar study previously conducted at Kerevat, Papua New Guinea (PNG), a net deficit of 36% was realised. This reinforced our finding that the response to pathogen testing was cultivar dependent and that the PNG cultivars in these studies generally exhibited increased tolerance to the endemic viruses present at the respective trial sites as manifested in their lack of response from the use of PT clones. They may be useful sources for future resistance breeding efforts. Nonetheless, the potential economic gain from using PT stocks necessitates the use of pathogen testing on virus-susceptible commercial cultivars. .

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.

Development of diagnotic capacity for Cotton Leaf Roll Virus from Thailand

Viral diseases of cotton are of economic significance in many parts of the world and several of these remain biosecurity threats to the Australian cotton industry, including Cotton Leaf Roll Virus (CLRV) from South East Asia. The proposed project will result in a greater understanding of the field symptoms of CLRV in Thailand and diagnostic assays used for its detection. I will also determine if the diagnostic assay being developed for Brazilian CLRDV as part of the CRDC project (11-12FRP00062) may also detect Thailand CLRV. It will provide educational opportunities to increase the knowledge base of staff currently working on cotton virus research and in doing so help to protect the Australian cotton industry from incursions of exotic viruses.

Detection and differentiation of bovine herpesvirus 1 and 5 using a multiplex real-time polymerase chain reaction.

A multiplex real-time PCR was developed for the detection and differentiation of two closely related bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5). The multiplex real-time PCR combines a duplex real-time PCR that targets the DNA polymerase gene of BoHV-1 and BoHV-5 and a real-time PCR targeting mitochondrial DNA, as a house-keeping gene, described previously by Cawthraw et al. (2009). The assay correctly identified 22 BoHV-1 and six BoHV-5 isolates from the Biosecurity Sciences Laboratory virus collection. BoHV-1 and BoHV-5 were also correctly identified when incorporated in spiked semen and brain tissue samples. The detection limits of the duplex assay were 10 copies of BoHV-1 and 45 copies of BoHV-5. The multiplex real-time PCR had reaction efficiencies of 1.04 for BoHV-1 and 1.08 for BoHV-5. Standard curves relating Ct value to template copy number had correlation coefficients of 0.989 for BoHV-1 and 0.978 for BoHV-5. The assay specificity was demonstrated by testing bacterial and viral DNA from pathogens commonly isolated from bovine respiratory and reproductive tracts. The validated multiplex real-time PCR was used to detect and differentiate BoHV-1 and BoHV-5 in bovine clinical samples with known histories.

Risk-based surveillance for avian influenza control along poultry market chains in South China: The value of social network analysis.

Over the past two decades, the poultry sector in China went through a phase of tremendous growth as well as rapid intensification and concentration. Highly pathogenic avian influenza virus (HPAIV) subtype H5N1 was first detected in 1996 in Guangdong province, South China and started spreading throughout Asia in early 2004. Since then, control of the disease in China has relied heavily on wide-scale preventive vaccination combined with movement control, quarantine and stamping out. This strategy has been successful in drastically reducing the number of outbreaks during the past 5 years. However, HPAIV H5N1 is still circulating and is regularly isolated in traditional live bird markets (LBMs) where viral infection can persist, which represent a public health hazard for people visiting them. The use of social network analysis in combination with epidemiological surveillance in South China has identified areas where the success of current strategies for HPAI control in the poultry production sector may benefit from better knowledge of poultry trading patterns and the LBM network configuration as well as their capacity for maintaining HPAIV H5N1 infection. We produced a set of LBM network maps and estimated the associated risk of HPAIV H5N1 within LBMs and along poultry market chains, providing new insights into how live poultry trade and infection are intertwined. More specifically, our study provides evidence that several biosecurity factors such as daily cage cleaning, daily cage disinfection or manure processing contribute to a reduction in HPAIV H5N1 presence in LBMs. Of significant importance is that the results of our study also show the association between social network indicators and the presence of HPAIV H5N1 in specific network configurations such as the one represented by the counties of origin of the birds traded in LBMs. This new information could be used to develop more targeted and effective control interventions.

Henipaviruses - unanswered questions of lethal zoonoses.

The highly lethal Hendra and Nipah viruses have been described for little more than a decade, yet within that time have been aetiologically associated with major livestock and human health impacts, albeit on a limited scale. Do these emerging pathogens pose a broader threat, or are they inconsequential 'viral chatter'. Given their lethality, and the evident multi-generational human-to-human transmission associated with Nipah virus in Bangladesh, it seems prudent to apply the precautionary principle. While much is known of their clinical, pathogenic and epidemiologic features in livestock species and humans, a number of fundamental questions regarding the relationship between the viruses, their natural fruit-bat host and the environment remain unanswered. In this paper, we pose and probe these questions in context, and offer perspectives based primarily on our experience with Hendra virus in Australia, augmented with Nipah virus parallels.

The meleagrid herpesvirus 1 genome is partially resistant to transposition

The propagation of herpesvirus genomes as infectious bacterial artificial chromosomes (iBAC) has enabled the application of highly efficient strategies to investigate gene function across the genome. One of these strategies, transposition, has been used successfully on a number of herpesvirus iBACs to generate libraries of gene disruption mutants. Gene deletion studies aimed at determining the dispensable gene repertoire of the Meleagrid herpesvirus 1 (MeHV-1) genome to enhance the utility of this virus as a vaccine vector have been conducted in this report. A MeHV-1 iBAC was used in combination with the Tn5 and MuA transposition systems in an attempt to generate MeHV-1 gene interruption libraries. However, these studies demonstrated that Tn5 transposition events into the MeHV-1 genome occurred at unexpectedly low frequencies. Furthermore, characterization of genomic locations of the rare Tn5 transposon insertion events indicated a nonrandom distribution within the viral genome, with seven of the 24 insertions occurring within the gene encoding infected cell protein 4. Although insertion events with the MuA system occurred at higher frequency compared with the Tn5 system, fewer insertion events were generated than has previously been reported with this system. The characterization and distribution of these MeHV-1 iBAC transposed mutants is discussed at both the nucleotide and genomic level, and the properties of the MeHV-1 genome that could influence transposition frequency are discussed. © American Association of Avian Pathologists.

Identification of diverse full-length endogenous betaretroviruses in megabats and microbats

Background: Betaretroviruses infect a wide range of species including primates, rodents, ruminants, and marsupials. They exist in both endogenous and exogenous forms and are implicated in animal diseases such as lung cancer in sheep, and in human disease, with members of the human endogenous retrovirus-K (HERV-K) group of endogenous betaretroviruses (βERVs) associated with human cancers and autoimmune diseases. To improve our understanding of betaretroviruses in an evolutionarily distinct host species, we characterized βERVs present in the genomes and transcriptomes of mega- and microbats, which are an important reservoir of emerging viruses.Results: A diverse range of full-length βERVs were discovered in mega- and microbat genomes and transcriptomes including the first identified intact endogenous retrovirus in a bat. Our analysis revealed that the genus Betaretrovirus can be divided into eight distinct sub-groups with evidence of cross-species transmission. Betaretroviruses are revealed to be a complex retrovirus group, within which one sub-group has evolved from complex to simple genomic organization through the acquisition of an env gene from the genus Gammaretrovirus. Molecular dating suggests that bats have contended with betaretroviral infections for over 30 million years.Conclusions: Our study reveals that a diverse range of betaretroviruses have circulated in bats for most of their evolutionary history, and cluster with extant betaretroviruses of divergent mammalian lineages suggesting that their distribution may be largely unrestricted by host species barriers. The presence of βERVs with the ability to transcribe active viral elements in a major animal reservoir for viral pathogens has potential implications for public health. © 2013 Hayward et al.; licensee BioMed Central Ltd.

Identification of a new Polerovirus (family Luteoviridae) associated with cotton bunchy top disease in Australia

Cotton bunchy top (CBT) disease has caused significant yield losses in Australia and is now managed by control of its vector, the cotton aphid (Aphis gossypii). Its mode of transmission and similarities in symptoms to cotton Blue Disease suggested it may also be caused by a luteovirus or related virus. Degenerate primers to conserved regions of the genomes of the family Luteoviridae were used to amplify viral cDNAs from CBT-affected cotton leaf tissue that were not present in healthy plants. Partial genome sequence of a new virus (Cotton bunchy top virus, CBTV) was obtained spanning part of the RNA-dependent-RNA-polymerase (RdRP), all of the coat protein and part of the aphid-transmission protein. CBTV sequences could be detected in viruliferous aphids able to transmit CBT, but not aphids from non-symptomatic plants, indicating that it is associated with the disease and may be the causal agent. All CBTV open-reading frames had their closest similarity to viruses of the genus Polerovirus. The partial RdRP had 90 % amino acid identity to the RdRP of Cotton leafroll dwarf virus (CLRDV) that causes cotton blue disease, while other parts of the genome were more similar to other poleroviruses. The sequence similarity and genome organization of CBTV suggest that it should be considered a new member of the genus Polerovirus. This partial genome sequence of CBTV opens up the possibility for developing diagnostic tests for detection of the virus in cotton plants, aphids and weeds as well as alternative strategies for engineering CBT resistance in cotton plants through biotechnology. © 2012 Australasian Plant Pathology Society Inc.

Seasonal abundance of thrips (Thysanoptera) in capsicum and chilli crops in south-east Queensland, Australia

Thrips can be important pests of capsicum and chilli crops, causing damage through their feeding and by vectoring viral diseases. As different species vary in their ability to transmit viruses and in their susceptibility to insecticides, it is important to know which species are present in a crop. The seasonal occurrence of thrips in capsicum and chilli crops in the Bundaberg district of south-east Queensland was investigated from July 2002 to June 2003. Fifty flowers were collected weekly from crops on seven farms and the adult thrips extracted and identified. Thrips palmi Karny and Frankliniella occidentalis (Pergande) were collected in the greatest numbers, with T. palmi predominant in autumn crops (March to July) and F. occidentalis predominant in spring crops (August to November). Pseudanaphothrips achaetus (Bagnall) was common, while Thrips tabaci Lindeman, Thrips imaginis Bagnall and Frankliniella schultzei (Trybom) were collected in low numbers.

In planta localization and interactions of impatiens necrotic spot tospovirus proteins

Impatiens necrotic spot tospovirus (INSV) is a significant pathogen of ornamentals. The tripartite negative- and ambi-sense RNA genome encodes six proteins that are involved in cytoplasmic replication, movement, assembly, insect transmission and defence. To gain insight into the associations of these viral proteins, we determined their intracellular localization and interactions in living plant cells. Nucleotide sequences encoding the nucleoprotein N, non-structural proteins NSs and NSm, and glycoproteins Gn and Gc of a Kentucky isolate of INSV were amplified by RTPCR, cloned, sequenced and transiently expressed as fusions with autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana. All proteins accumulated at the cell periphery and co-localized with an endoplasmic reticulum marker. The Gc protein fusion also localized to the nucleus. N and NSm protein self-interactions and an NSm-N interaction were observed by using bimolecular fluorescence complementation. A tospovirus NSm homotypic interaction had not been reported previously.