A network approach for provisional assay recognition of a Hendra virus antibody ELISA: test validation with low sample numbers from infected horses.

Obtaining statistically sound numbers of sera from Hendra virus (HeV)-infected horses is problematic because affected individuals usually die or are euthanized before developing a serum antibody response. As a consequence, test validation becomes a challenge. Our approach is an extension of OIE principles for provisional recognition and included 7 validation panels tested across multiple laboratories that provided estimates for test performance characteristics.

Development and validation of an immunoperoxidase antigen detection test for improved diagnosis of rabies in Indonesia.

Rabies continues to pose a significant threat to human and animal health in regions of Indonesia. Indonesia has an extensive network of veterinary diagnostic laboratories and the 8 National laboratories are equipped to undertake diagnostic testing for rabies using the commercially-procured direct fluorescent antibody test (FAT), which is considered the reference (gold standard) test. However, many of the Indonesian Provincial diagnostic laboratories do not have a fluorescence microscope required to undertake the FAT.

Detection of highly pathogenic zoonotic influenza virus H5N6 by reverse-transcriptase quantitative polymerase chain reaction.

Background: Variant high pathogenicity avian influenza (HPAI) H5 viruses have recently emerged as a result of reassortment of the H5 haemagglutinin (HA) gene with different neuraminidase (NA) genes, including NA1, NA2, NA5, NA6 and NA8. These viruses form a newly proposed HA clade 2.3.

Reassortant highly pathogenic influenza A(H5N6) virus in Laos.

In March 2014, avian influenza in poultry in Laos was caused by an emergent influenza A(H5N6) virus. Genetic analysis indicated that the virus had originated from reassortment of influenza A(H5N1) clade 2.3.

Development and validation of a 3ABC antibody ELISA in Australia for foot and mouth disease.

Objective: To measure the diagnostic performance of an Australian-developed ELISA for the detection of antibodies against the non-structural proteins (NSP) 3ABC of the foot and mouth disease (FMD) virus.

Design: Test development and validation study.

Methods: The diagnostic specificity was determined using 2535 sera from naïve animals and 1112 sera from vaccinated animals.

Development of veterinary laboratory networks for avian influenza and other emerging infectious disease control: the southeast asian experience.

The outbreak of highly pathogenic H5N1 avian influenza, with its international spread, confirmed that emerging infectious disease control must be underpinned by effective laboratory services. Laboratory results are the essential data underpinning effective surveillance, case diagnosis, or monitoring of responses. Importantly, laboratories are best managed within national and international networks of technological support rather than in isolation.

Genome sequence conservation of Hendra virus isolates during spillover to horses, Australia.

Bat-to-horse transmission of Hendra virus has occurred at least 14 times. Although clinical signs in horses have differed, genome sequencing has demonstrated little variation among the isolates. Our sequencing of 5 isolates from recent Hendra virus outbreaks in horses found no correlation between sequences and time or geographic location of outbreaks.

Screening for Nipah virus infection in West Kalimantan province, Indonesia.

Compared to other viruses, research on Nipah virus has been limited in Indonesia because attributable disease outbreaks have not been reported. However, Nipah virus is a zoonotic Biosafety Level 4 (BSL4) agent, so strategic monitoring is prudent. Farmer interviews and a serologic survey of 610 pig sera and 99 bat sera from West Kalimantan province were conducted.

Development and evaluation of a rapid immunomagnetic bead assay for the detection of classical swine fever virus antigen.

Classical swine fever (CSF) is a highly contagious and severe viral disease of swine resulting in substantial production losses in different farming systems in many regions of the world. The accurate and rapid detection of CSF outbreaks is reliant on sensitive and specific laboratory testing and is a key component of disease control. Specific detection of CSF virus can be achieved by virus isolation in tissue culture, antigen capture or the detection of viral RNA using molecular techniques.

Experimental Nipah virus infection in pteropid bats (Pteropus poliocephalus).

Seventeen grey-headed fruit bats (Pteropus poliocephalus) were inoculated subcutaneously with an isolate of Nipah virus derived from a fatally infected human. A control group of eight guinea-pigs was inoculated intraperitoneally with the same isolate in order to confirm virulence. Three of eight infected guinea-pigs developed clinical signs 7-9 days post-inoculation.

A comparative indirect ELISA for the detection of henipavirus antibodies based on a recombinant nucleocapsid protein expressed in Escherichia coli.

The indirect ELISA is a simple and useful method for detection of pathogen-specific antibodies in animal sera. However, non-specific or background binding is often a problem, especially when recombinant proteins from Escherichia coli are used. In this study, a comparative indirect ELISA in which the total reactivity and the background binding were determined simultaneously on the same ELISA plate was reported.

Porcine interleukin-3 enhances DNA vaccination against classical swine fever.

DNA vectors can be used to deliver vaccine antigens that stimulate effective protective immunity in mice, but in larger, outbred animal species, the protective efficacy is lower or large doses of DNA are required. These data demonstrate that porcine interleukin-3 (IL-3) when delivered to pigs by DNA vector or in low doses as recombinant protein, can enhance antibody responses to classical swine fever virus antigen expressed from co-delivered DNA, and improve the protective efficacy of the DNA vaccine. The effect was further enhanced when IL-3 was expressed as a fusion protein with the potyvirus coat protein.

Detection and quantitative pathogenesis study of classical swine fever virus using a real time RT-PCR assay.

A real time reverse transcription (RT) TaqMan PCR assay for the detection of classical swine fever virus (CSFV) previously described for use on a SmartCycler was validated on the Applied Biosystems AB 7700 Sequence Detection System using the Roche MagNA pure instrument for nucleic acid extraction and reaction set up. The primers and probe were specific for the CSFV strains (NSW, Baker and Weybridge) and did not react with other pestiviruses (BDV Tobias, BDV #327, BVDV non-CPE and BVDV C24V). Analysis of blood samples collected from pigs 1-6 and 8 days post-oronasal infection showed that over >10(6) range there was a linear relationship between log10TCID50ml-1 blood and the log10 normalised genetic load measured by quantitative TaqMan assay.

Characterization of epitopes for neutralizing monoclonal antibodies to classical swine fever virus E2 and Erns using phage-displayed random peptide library.

Infection of cells with classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoproteins E2 and Erns with receptor molecules on the cell surface. These proteins are also the major antigens for eliciting neutralizing antibodies and conferring protective immunity. Here we report the identification of multiple neutralizing epitopes on these proteins by screening a phage-displayed random peptide library with CSFV-specific neutralizing monoclonal antibodies.

Protection of pigs against 'in contact' challenge with classical swine fever following oral or subcutaneous vaccination with a recombinant porcine adenovirus.

A recombinant porcine adenovirus expressing the classical swine fever virus (CSFV) gp55 gene (rPAdV-gp55) was administered to commercially available outbred pigs via the subcutaneous or oral route and their susceptibility to 'in contact' challenge with classical swine fever determined. Animals vaccinated subcutaneously with a single dose of recombinant vaccine and challenged by 'in contact' exposure were protected from disease, whereas pigs given an equivalent single oral dose did not survive challenge. However, pigs given two oral doses of rPAdV-gp55, 22 days apart, were completely protected from disease.

Experimental Nipah virus infection in pigs and cats.

A human isolate of Nipah virus from an outbreak of febrile encephalitis in Malaysia that coincided with a field outbreak of disease in pigs was used to infect eight 6-week-old pigs orally or subcutaneously and two cats oronasally. In pigs, the virus induced a respiratory and neurological syndrome consistent with that observed in the Malaysian pigs. Not all the pigs showed clinical signs, but Nipah virus was recovered from the nose and oropharynx of both clinically and sub-clinically infected animals.

Oral and sub-cutaneous vaccination of commercial pigs with a recombinant porcine adenovirus expressing the classical swine fever virus gp55 gene.

A recombinant porcine adenovirus expressing the classical swine fever virus (CSFV) gp55/E2 gene was administered to commercially available pigs via oral or subcutaneous routes and their susceptibility to oral and subcutaneous challenge with CSFV was determined. 100% of animals vaccinated and challenged subcutaneously were protected. In the groups of pigs vaccinated either orally or subcutaneously and then challenged orally, 60% of animals were protected.

A rapid immune plaque assay for the detection of Hendra and Nipah viruses and anti-virus antibodies.

Rapid immune plaque assays have been developed to quantify biohazard level 4 agents Hendra and Nipah viruses and detect neutralising antibodies to both viruses. The methods rely on the fact that both viruses rapidly generate large syncytia in monolayers of Vero cells within 24 h and that monospecific antiserum to the Hendra virus phosphoprotein (P) detects that protein in both Hendra and Nipah virus-induced syncytia after methanol fixation of virus-infected cells. The P protein is a constituent of the ribonucleoprotein core of the viruses and a component of the viral RNA-dependent RNA polymerase and is made in significant amounts in infected cells.

Serological examination for evidence of infection with Hendra and Nipah viruses in Queensland piggeries.

Objective: To examine piggeries in Queensland for evidence of infection with Hendra virus and Nipah virus.

Design: A serological survey was designed to provide 99% confidence of detecting at least one infected pig herd in Queensland, assuming that for each virus, at least 5% of herds would have been exposed to virus and that at least 40% of the finisher pigs in these herds would have detectable antibodies to virus.

Procedure: A two stage sampling regimen was used.

Vaccination of pigs with a recombinant porcine adenovirus expressing the gD gene from pseudorabies virus.

Five week old, commercially available large white pigs were vaccinated with either a single dose or two doses of a recombinant porcine adenovirus expressing the glycoprotein D gene from pseudorabies virus (PRV). Pigs were monitored for the development of serum neutralizing antibodies to PRV and challenged 3 weeks after final vaccination. Prior to challenge, pigs given 2 doses of the vaccine demonstrated boosted levels of antibody compared with those given a single dose, and all surviving pigs had increased neutralization titres over pre-challenge levels.

Nipah virus infection in bats (order Chiroptera) in peninsular Malaysia.

Nipah virus, family Paramyxoviridae, caused disease in pigs and humans in peninsular Malaysia in 1998-99. Because Nipah virus appears closely related to Hendra virus, wildlife surveillance focused primarily on pteropid bats (suborder Megachiroptera), a natural host of Hendra virus in Australia. We collected 324 bats from 14 species on peninsular Malaysia.