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. Infected fruit bats developed a subclinical infection characterized by the transient presence of virus within selected viscera, episodic viral excretion and seroconversion. A range of histopathological changes was observed within the tissues of infected bats. Nipah virus was excreted in bat urine while neutralizing antibody was present in serum. This intermittent, low-level excretion of Nipah virus in the urine of bats may be sufficient to sustain the net reproductive value of the virus in a species where there is regular urine contamination of the fur, mutual grooming, and where urine droplets are a feature of the environment.

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. Natural infection of in-contact pigs, which was readily demonstrated, appeared to be acute and self-limiting. Subclinical infections occurred in both inoculated and in-contact pigs. Respiratory and neurological disease was also produced in the cats, with recovery of virus from urine as well as from the oropharynx. The clinical and pathological syndrome induced by Nipah virus in cats was comparable with that associated with Hendra virus infection in this species, except that in fatal infection with Nipah virus there was extensive inflammation of the respiratory epithelium, associated with the presence of viral antigen. Viral shedding via the nasopharynx, as observed in pigs and cats in the present study, was not a regular feature of earlier reports of experimental Hendra virus infection in cats and horses. The findings indicate the possibility of field transmission of Nipah virus between pigs via respiratory and oropharyngeal secretions.

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. Before challenge, neutralising antibodies to CSFV were detected in 60% of pigs vaccinated subcutaneously, but in none of those given the vaccine orally. CSFV antigen was found in the spleens of surviving pigs that had been vaccinated orally. In contrast, subcutaneous vaccination was shown to preclude the presence of CSFV in the spleen of animals that survived challenge.

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. All samples were tested with serum neutralisation tests developed and performed at the Australian Animal Health Laboratory. RESULTS: There was no evidence of antibody to either virus in the 500 samples collected from 100 herds. CONCLUSION: The results of the survey support a case that commercial pigs in Queensland are free of both Hendra virus and Nipah virus infections.

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. Following challenge, pigs were monitored for clinical signs of disease, with blood and nasal swabs collected for virus isolation. All control animals became sick with elevated temperatures for 6 days post challenge, whereas; vaccinated animals displayed an increase in body temperature for only 2-3 days. Control pigs and those given a single dose all lost condition, but the group given 2 doses remained healthy. At postmortem, gross lesions of pneumonia only occurred in control animals and those given a single dose of vaccine. Histology carried out on the brains of all animals demonstrated a difference in severity of infection and frequency of immunohistochemical antigen detection between test animals, with control and single dose groups being most severely affected and pigs given 2 doses the least. Virus isolation studies demonstrated that no viraemia could be detected, but virus was found in nasal swabs from some animals in both groups of vaccinates following challenge.

A prime-boost vaccination strategy using naked DNA followed by recombinant porcine adenovirus protects pigs from classical swine fever.

Weaned pigs (6-week-old) and 7-day-old pre-weaned piglets were vaccinated with naked plasmid DNA expressing the gp55/E2 gene from classical swine fever virus (CSFV). Both groups of pigs were then given a booster dose of recombinant porcine adenovirus expressing the gp55 gene (rPAV-gp55). Following challenge with CSFV, 100% of weaned pigs and 75% pre-weaned piglets were protected from disease. Weaned pigs given a single dose of rPAV-gp55 were also protected, but showed a slight increase in temperature immediately post-challenge. However, weaned animals given a DNA prime before rPAV-gp55 showed no fluctuation in body temperature following challenge and no pathology in spleen or lymph nodes upon post-mortem. In addition, no CSFV could be re-isolated from the rPAV vaccinated group and from only one pig in the prime-boost group following challenge, suggesting that both vaccination regimes have the potential to reduce or prevent virus shedding following experimental challenge.

The presence of cross-reactive antibodies to rabbit haemorrhagic disease virus in Australian wild rabbits prior to the escape of virus from quarantine.

Sera collected from Australian wild rabbits prior to the escape of rabbit haemorrhagic disease virus (RHDV) from Wardang Island were examined for RHDV antibodies using purified recombinant capsid protein VP60 expressed from baculovirus. A VP60-based indirect ELISA showed that 196 of 392 wild rabbit sera reacted (OD(450) >0.15) with VP60. Twenty sera (OD(450) ranging from 0.15-2.47), randomly chosen from the 196 positive sera, recognized the 64 kDa VP60 in Western blot analysis, indicating that the reactivity detected in ELISA is indeed specific to the VP60 antigen. In a separate study, sera of 23 rabbits from an RHD-free area after the escape of RHDV were tested by ELISA and 21 of the 23 rabbits were found to be positive. When these rabbits were challenged with a lethal dose of RHDV, 11 out of the 23 rabbits survived. The presence of RHDV-protective antibodies in some of these rabbits suggested that they had been exposed to a pre-existing non-virulent rabbit calicivirus closely related to RHDV. These results highlight the need to study the prevalence of, and to characterize, this viral agent in order to effectively control rabbit populations in Australia and New Zealand.

Protection of pigs against classical swine fever with DNA-delivered gp55.

Classical swine fever virus causes significant mortality and morbidity in commercial piggeries in many countries in Europe and Asia. The protective antigen, gp55, is highly conformation-dependent and thus killed virus or bacterially produced proteins are not protective. This report demonstrates that DNA vaccination with the gene encoding gp55 can provide protective immunity with inoculation of two doses of 25 microg DNA or a single shot of 200 microg. Furthermore, the DNA can be delivered intramuscularly or by a simple spring-loaded needleless inoculator. In addition it is shown that inoculation of the DNA at a single site conveys the same level of immunity as division of the dose between two sites.

Vaccination with a single dose of a recombinant porcine adenovirus expressing the classical swine fever virus gp55 (E2) gene protects pigs against classical swine fever.

A recombinant porcine adenovirus (rPAV) with the gp55 (E2) gene from the classical swine fever virus (CSFV) 'Weybridge' strain inserted into the right hand end of the PAV serotype 3 (PAV3) genome was constructed. Expression of gp55 was directed by the major late promoter and tri-partite leader sequences located and cloned from PAV3. No compensatory deletions of PAV DNA sequences were made. Vaccination of outbred pigs with a single dose of the recombinant virus (rPAV-gp55) resulted in complete protection from lethal challenge with CSFV. No adverse clinical signs were observed in vaccinated animals following administration of rPAV-gp55 and following challenge, no clinical signs of CSF were observed prior to, or at, post mortem. The insert made into the rPAV increased the genome length to 106.8% of wild type and therefore exceeded the expected maximum insert size for a stable recombinant by almost 2%. Thus rPAV-gp55 contains the largest stable insertion made into a non-deleted Mastadeno virus recombinant so far reported.

Immunohistochemistry in the identification of a number of new diseases in Australia.

Immunohistochemistry plays an important part in the diagnosis of some viral diseases. Demonstration of viral antigen in a lesion is an important contribution to diagnosis, either at the time of investigation or retrospectively. At the CSIRO Australian Animal Health Laboratory, the most frequent use of immunohistochemistry has been in the diagnosis of the important avian diseases, highly pathogenic avian influenza and Newcastle disease. The technology took key roles in the diagnoses of Hendra virus infections, and, later, an immunoperoxidase test gave the first indication of the existence of Australian bat lyssavirus. The test can often confirm that a virus isolated in an animal is the actual virus causing disease and not a coincidental isolation. Good examples of that in some more new diseases were the association of Wallal virus with blindness in kangaroos, and of the new porcine Menangle virus in natural and experimental cerebral disease in foetal piglets.

Fine mapping of a C-terminal linear epitope highly conserved among the major envelope glycoprotein E2 (gp51 to gp54) of different pestiviruses.

Envelope glycoprotein E2 (gp51 to gp54) is the major neutralizing antigen of pestiviruses, which include classical swine fever virus (CSFV), bovine viral diarrhoea virus (BVDV), and border disease virus (BVD). Previous studies carried out using a panel of monoclonal antibodies raised against CSFV strain Brescia have revealed the existence of four antigenic domains, A to D, of the E2 protein, all of which are located at the N-terminal half of the molecule. Here we report the detailed mapping, using three complementary techniques, of a novel linear epitope located at the C-terminal part of the molecule, which reacted with a monoclonal antibody (4-9D4) as well as polyclonal animal sera. This epitope is highly conserved in the three different members of pestiviruses and hence can be used as a genus-specific diagnosis tool. The observation that this epitope is not accessible on the native virus surface, together with its C-terminal location, supports a recently proposed structural model, indicating that the C-terminal part of E2 is membrane-bound while the N-terminal half of the molecule is exposed on the virus surface.

Presence of rabbit haemorrhagic disease virus antigen in rabbit tissues as revealed by a monoclonal antibody dependent capture ELISA.

The production of monoclonal antibodies (mAbs) to a common antigenic region on rabbit haemorrhagic disease virus (RHDV) has enabled the development of a capture ELISA for virus detection. The assay was shown to detect reliably the presence of viral antigen in crude homogenates of a range of rabbit tissues and has provided the first evidence for the presence of RHDV in blood, serum and heart tissue. A limited time course study of the progression of virus infection revealed viral antigen is first detected in the liver at between 18 and 24 h post-infection (p.i.) and in the spleen between 24 and 30 h p.i. The assay displayed a high level of specificity, clearly differentiating between groups of infected (lowest observed optical density (O.D.) 0.45) and uninfected (highest O.D. 0.06) rabbits. No viral antigen was detected in the urine or faeces collected from infected rabbits at post-mortem. Experiments employing the 'spiking' of pools of urine and faecal homogenates collected from uninfected rabbits, with a known amount of virus, indicated that RHDV may not be present in the faeces of infected animals and if present in urine, it appears to undergo substantial degradation.

Strong sequence conservation of African swine fever virus p72 protein provides the molecular basis for its antigenic stability.

The major capsid protein p72 of African swine fever virus (ASFV) has long been considered an important immunodominant antigen for serologic diagnosis. Here we describe the cloning and sequence analysis of two p72-coding genes from ASFV strains Uganda (UGA) and Dominican Republic-2 (DR2). Sequence comparison of these genes, together with those from two other ASFV strains (BA71V and E70), demonstrated that the p72 proteins are highly conserved (97.8% to 100% amino acid sequence identity) in strains isolated from different parts of the world. These results support previous observations indicating that p72 is antigenically stable, and provide a useful molecular basis for further development of ASFV serologic tests using this important antigenic molecule.

Self-assembly, antigenicity, and immunogenicity of the rabbit haemorrhagic disease virus (Czechoslovakian strain V-351) capsid protein expressed in baculovirus.

Rabbit haemorrhagic disease virus (RHDV) capsid protein was expressed in a baculovirus system. Analysis of the expressed product showed that the recombinant protein, which is 60 kDa in size, was antigenic as revealed by its reactions in ELISA and Western blot with the antibodies raised against RHDV. Direct electron microscopy of the cell culture supernatant and the purified protein demonstrated that the capsid protein expressed in insect cells self-assembled to form empty virus-like particles (VLP) which are similar in size and morphology to that of native virus. These particles were immunoreactive with polyclonal anti-RHDV antibodies and with four monoclonal antibodies which recognise conformational epitopes of the virus. The results indicated that the VLPs were morphologically and antigenically indistinguishable from native virus. The recombinant VLPs induced high levels of RHDV-specific antibodies in rabbits and mice following immunisation. The immune response to the VLPs protected the rabbits following challenge with the virulent RHDV. In haemagglutination assays, the VLPs bound to human red blood cells similar to the native virus particles. The recombinant protein and or VLPs is suitable for the development of a rapid, sensitive and reliable test for detection of antibodies to RHDV and for use as a vaccine for domestic rabbits.

High level expression of the envelope glycoprotein (gp53) of bovine viral diarrhoea virus (Singer) and its potential use as diagnostic reagent.

A 1.74-kb cDNA fragment containing the gp53 coding region has been cloned from bovine viral diarrhoea virus (BVDV) strain Singer by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis indicated that gp53 of BVDV strains Singer, NADL and SD-1 shared extensive sequence homology at both the RNA (85-94%) and protein (82-91%) levels. Nineteen cysteine residues and five potential N-linked glycosylation sites were identified within the sequenced region, all of which were conserved. These observations suggest that although the homology at the nucleotide sequence level may vary, there was strong structural conservation among bovine viral diarrhoea virus envelope proteins. Full-length gp53 was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST). The N-terminal half of gp53 was also synthesised in E. coli as a 28-KDa recombinant protein using the T7 RNA polymerase-directed expression system. Both recombinant proteins were expressed at high levels (approximately 30-50 mg/l). The recombinant proteins were recognised in ELISA and Western blot analyses by polyclonal serum raised against a mixture of BVDV and classical swine fever virus (CSFV). Rabbit antiserum raised against the 28-kDa recombinant protein reacted with different BVDV strains in ELISA and immunofluorescent antibody test, but not with CSFV in the same tests. These results demonstrated that the bacterial recombinant proteins have similar immunological properties to that of the native viral protein and, in conjunction with its homologous antisera, can be useful as diagnostic reagents.

A comparison of the pathogenicity of two strains of hog cholera virus. 1. Clinical and pathological studies.

The virulence of a strain of hog cholera virus isolated during an outbreak of mild disease in pigs in New South Wales in 1960/61 (the NSW strain) was compared over 11 days with that of a virulent strain by inoculating 8 pigs with each virus and comparing the ensuing clinical signs and pathology. Both viruses caused persistent pyrexia and leukopenia, the NSW strain 4 to 5 days and the virulent strain 3 days, after inoculation. Few other clinical signs were observed in the pigs inoculated with the NSW strain. In contrast, all pigs inoculated with the virulent strain became progressively depressed and incoordinated, and were killed between days 6 and 9. Bronchopneumonia and swollen, reddened lymph nodes were observed in pigs inoculated with both viruses. Few other gross lesions were observed with the NSW strain, but some pigs receiving the virulent strain had pustules in the tonsil and the anterior oesophagus, petechial haemorrhages in the kidney, and small infarcts at the margins of the spleen. There were marked differences in the histopathology, both in the variety of organs affected and the severity of lesions in individual organs. Suppurative bronchopneumonia occurred in both groups. Other changes in the pigs affected with the NSW strain were colitis, mild cerebral vasculitis, necrosis, haemorrhage and neutrophil infiltration in some lymph nodes and spleens. In pigs infected with virulent virus the cerebral vasculitis was so severe that there was necrosis of cells within the vessel walls.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of the pathogenicity of two strains of hog cholera virus. 2. Virological studies.

Quantitative and qualitative differences were demonstrated in the amount of virus in a range of tissues from pigs infected with either the Weybridge or New South Wales (NSW) strains of hog cholera (HC) virus. The titre of the Weybridge strain in samples, as assessed by either virus titration in cell culture or by the density of specific fluorescing cells in tissue sections, was higher than that for the NSW strain. This correlated with the greater severity of the clinico-pathological syndrome induced by the Weybridge strain. The implications of the differences in the virus content of tissues in the diagnosis of HC is discussed as is the use of monoclonal antibodies to differentiate HC and bovine virus diarrhoea viruses.