Dual-role FilmArray® diagnostics for high-impact viral diseases.

In this study, we explored the potential utility of the human-focused FilmArray® Respiratory Panel for the diagnosis of a broad range of influenza viruses of veterinary concern as compared with the standard portfolio of recommended TaqMan®-based diagnostic tests. In addition, we discuss some potential operational advantages associated with the use of such integrated sample extraction, amplification and analysis devices in the context of a future long-term, dual-role strategy for the detection of emergency diseases of both human and veterinary concern.

Design and evaluation of consensus PCR assays for henipaviruses.

Henipaviruses were first discovered in the 1990s, and their potential threat to public health is of increasing concern with increasing knowledge. Old-world fruit bats are the reservoir hosts for these viruses, and spill-over events cause lethal infections in a wide range of mammalian species, including humans. In anticipation of these spill-over events, and to investigate further the geographical range of these genetically diverse viruses, assays for detection of known and potentially novel strains of henipaviruses are required. The development of multiple consensus PCR assays for the detection of henipaviruses, including both SYBR Green and TaqMan real-time PCRs and a conventional heminested PCR is described. The assays are highly sensitive and have defined specificity. In addition to being useful tools for detection of known and novel henipaviruses, evaluation of assay efficiency and sensitivity across both biological and synthetic templates has provided valuable insight into consensus PCR design and use.

Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples.

OBJECTIVE: To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. DESIGN: A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. RESULTS: The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per microl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. CONCLUSION: The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis.

Generation of chicken single chain antibody variable fragments (scFv) that differentiate and neutralize infectious bursal disease virus (IBDV).

Phage-displayed recombinant antibody libraries derived from splenic mRNA of chickens immunized with an Australian strain of infectious bursal disease virus (IBDV) were constructed as single chain variable fragments (scFv) by either overlap extension polymerase chain reaction (PCR) or sequential ligation of the individual heavy (V(H)) and light (V(L)) chain variable gene segments. Sequential cloning of the individual V(H) and V(L) genes into a newly constructed pCANTAB-link vector containing the synthetic linker sequence (Gly(4)Ser)(3) was more efficient than cloning by overlap extension PCR, increasing the library size 500 fold. Eighteen IBDV specific antibodies with unique scFv sequences were identified after panning the library against the immunizing antigen. Eight of the clones contained an identical V(H) gene but unique V(L) genes. In ELISA analysis using a panel of Australian and overseas IBDV strains, one scFv antibody was able to detect all strains, whilst 3 others could discriminate between Australian and overseas strains, classical and variant strains and Australian field strains and vaccine strains. In addition, some scFvs showed significant neutralization titres in vitro. This report shows that generation of chicken antibodies in vitro by recombinant means has considerable potential for producing antibodies of diverse specificity and neutralizing capacity.

A capripoxvirus detection PCR and antibody ELISA based on the major antigen P32, the homolog of the vaccinia virus H3L gene.

Sheeppoxvirus (SPV), goatpoxvirus (GPV) and lumpy skin disease virus (LSDV) of cattle belong to the Capripoxvirus genus of the Poxviridae family and can cause significant economic losses in countries where they are endemic. Capripox diagnosis by classical virological methods dependent on live capripox virus is not suitable in countries such as Australia where the virus is exotic and live virus is not available. To develop diagnostic tests based on recombinant material, we cloned and sequenced a 3.7 kb viral DNA fragment of SPV that contained open reading frames homologous to the vaccinia virus J6R, H1L, H2R, H3L and H4L genes. A capripoxvirus specific PCR assay was developed that differentiated between SPV and LSDV on the basis of unique restriction sites in the corresponding PCR fragments. The vaccinia virus H3L homolog was identified as the capripoxvirus P32 antigen. The P32 proteins of SPV and LSDV were expressed in Escherichia coli as a fusion protein with a poly-histidine tag and affinity purified on metal binding resin. The full-length P32 protein contained a transmembrane region close to the carboxy terminus and was membrane associated but could be solubilised in detergent and used as trapping antigen in an antibody detection ELISA. The ELISA was specific for capripoxvirus as only sera from sheep infected with capripoxvirus but not orf or vaccinia virus reacted with the capripoxvirus P32 antigen.

Recombinant fowlpox virus vaccines against Australian virulent Marek's disease virus: gene sequence analysis and comparison of vaccine efficacy in specific pathogen free and production chickens.

We have cloned and sequenced the glycoprotein genes gB, gC and gD of the Australian virulent Marek's disease virus (MDV) isolate Woodlands No. 1. The glycoprotein gB and gC sequences were identical to the homologs of other virulent MDV type 1 strains, and the glycoprotein gD sequence contained only one unique amino acid substitution. Recombinant fowlpox viruses (rFPVs) expressing the MDV glycoprotein genes were constructed and their efficacy as vaccines was evaluated in specific pathogen free (SPF) and production chickens. Vaccination with the FPV-gB recombinant protected SPF chickens from Marek's disease mortality and tumour formation following challenge with virulent MDV Woodlands No. 1. The degree of protection from Marek's disease was dependent on the vaccine dose and route of inoculation. The rFPVs expressing gC or gD did not provide protection from Marek's disease. A rFPV expressing both gB and gC did not provide enhanced protection in comparison with the rFPV-gB alone. The rFPV-gB vaccine failed to protect commercial chickens from MD mortality and provided little protection from tumour formation in comparison with the commercial herpesvirus of turkey (HVT) vaccine. The failure to provide protection against MD may be related to the impact of maternally derived immunity to MDV and FPV and possibly the genotype of the chickens.

All-fiber optical frequency shifter.

A new type of all-fiber frequency shifter is demonstrated. It is capable of operation at lower frequencies than traditional approaches based on surface acoustic waves and is believed to be unique in not requiring the removal of the fiber buffer coat. For 2.3-W electrical drive the prototype achieved a 2.5% conversion efficiency and greater than 20-dB unwanted sideband suppression.