Optimization and diagnostic evaluation of monoclonal antibody-based blocking ELISA formats for detection of neutralizing antibodies to Hendra virus in mammalian sera.

Maintenance of Hendra virus (HeV) in pteropid bat populations has been associated with spillover events in horses, humans and dogs. Experimental studies have demonstrated infections for several other species including guinea pigs, cats and ferrets. The criteria of a sensitive and specific serological test that is effective for a range of species, but which does not require use of live virus, has not been satisfactorily addressed by currently available tests. We have evaluated the use of two HeV neutralizing monoclonal antibodies (mAbs) in a blocking format enzyme-linked immunosorbent assay (bELISA) to detect serum antibody against a recombinant expressed HeV G protein (sol G) in several animal species. The human mAb m102.4 neutralises both HeV and the closely related Nipah virus (NiV); the mouse mAb 1.2 neutralises only HeV. Given these functional differences, we have investigated both antibodies using a bELISA format. Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were optimized using individual thresholds for mAb 1.2 and m102.4. For mAb 1.2 the positive threshold of >33% inhibition yielded DSe and DSp values of 100% (95% CI 95.3-100.0) and 99.5 (95% CI 98.8-99.8) respectively; for mAb m102.4 a positive threshold of >49% inhibition gave DSe and DSp values of 100 (95% CI 95.3-100.0) and 99.8 (95% CI 99.2-100.0) respectively. At these thresholds the DSe was 100% for both tests relative to the virus neutralization test. Importantly, the occurrence of false positive reactions did not overlap across the assays. Therefore, by sequential and selective application of these assays, it is possible to identify false positive reactions and achieve a DSp that approximates 100% in the test population.

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. Diagnostic sensitivity was calculated from the data for 995 sera from experimentally and field-infected animals from FMD-endemic countries in South East Asia. A commercial ELISA detecting antibodies against FMD virus NSP was used as the reference test to establish relative sensitivity and specificity. Bayesian latent class analysis was performed to corroborate results. The diagnostic window and rate of detection were determined at different times using sera from cattle, sheep and pigs before and after infection, and after vaccination and subsequent infection. Repeatability and reproducibility data were established. RESULTS: At 35% test cut-off, the 3ABC ELISA had an overall diagnostic sensitivity of 91.5% and diagnostic specificity of 96.4%. The diagnostic sensitivity in vaccinated and subsequently infected cattle was 68.4% and diagnostic specificity in vaccinated cattle was 98.0%. CONCLUSIONS: The 3ABC ELISA identified field and experimentally infected animals, as well as vaccinated and subsequently infected animals. Diagnostic sensitivity and specificity estimates for other FMD NSP tests are comparable with the results obtained in this study. This NSP ELISA was found to be 'fit for purpose' as a screening assay at the herd level to detect viral infection and also to substantiate absence of infection.

Henipavirus microsphere immuno-assays for detection of antibodies against Hendra virus.

Hendra and Nipah viruses (HeV and NiV) are closely related zoonotic pathogens of the Paramyxoviridae family. Both viruses belong to the Henipavirus genus and cause fatal disease in animals and humans, though only HeV is endemic in Australia. In general and due to the acute nature of the disease, agent detection by PCR and virus isolation are the primary tools for diagnostic investigations. Assays for the detection of antibodies against HeV are fit more readily for the purpose of surveillance testing in disease epidemiology and to meet certification requirements in the international movement of horses. The first generation indirect ELISA has been affected by non-specific reactions which must be resolved using virus neutralisation serology conducted at laboratory bio-safety level 4 containment (PC4). Recent developments have enabled improvements in the available serology assays. The production of an expressed recombinant truncated HeV G protein has been utilised in ELISA and in Luminex-based multiplexed microsphere assays. In the latter format, two Luminex assays have been developed for use in henipavirus serology: a binding assay (designed for antibody detection and differentiation) and a blocking assay (designed as a surrogate for virus neutralisation). Equine and canine field sera were used to evaluate the two Luminex assays relative to ELISA and virus neutralisation serology. Results showed that Luminex assays can be effective as rapid, sensitive and specific tests for the detection of HeV antibody in horse and dog sera. The tests do not require PC4 containment and are appropriate for high throughput applications as might be required for disease investigations and other epidemiological surveillance. Also, the results show that the Luminex assays detect effectively HeV vaccine-induced antibodies.

Epizootics of sudden death in tammar wallabies (Macropus eugenii) associated with an orbivirus infection.

Epizootics of sudden death in tammar wallabies (Macropus eugenii) occurred at six research facilities and zoological gardens in New South Wales, Australia, in late 1998 and at one Queensland research facility in March 1999. There were 120 confirmed tammar wallaby deaths during this period; however, population censuses indicated that up to 230 tammar wallabies may have died. The majority of animals died without premonitory signs. A small proportion of wallabies exhibited increased respiratory rate, sat with a lowered head shortly before death or were discovered in lateral recumbency, moribund and with muscle fasciculations. Gross postmortem findings consistently included massive pulmonary congestion, mottled hepatic parenchyma and subcutaneous oedema throughout the hindlimbs and inguinal region. Approximately 30% of the animals examined also had extensive haemorrhage within the fascial planes and skeletal muscle of the hindlimb adductors, inguinal region, ventral thorax, dorsal cervical region and perirenal retroperitoneal area. The tissues of affected animals became autolytic within a short period after death. Bacteriological examination of tissues from 14 animals did not provide any significant findings. Toxicological examination of the gastric and colonic contents of four animals did not reveal evidence of brodifacoume or other rodenticides. Viruses from the Eubenangee serogroup of the Orbivirus genus were isolated from the cerebral cortex of nine, and the myocardium of two, tammar wallabies and the liver and intestine of another tammar wallaby. A similar orbivirus was also isolated from the cerebrospinal fluid of another tammar wallaby that died suddenly. The disease agent appears to be a previously unrecognised orbivirus in the Eubenangee serogroup. This is the first report of epizootics of sudden deaths in tammar wallabies apparently associated with an orbivirus infection.

The first identified case of pandemic H1N1 influenza in pigs in Australia.

A 300-sow farrow-to-finish herd in New South Wales was infected with influenza pandemic (H1N1) 2009 (H1N1/09) virus in July 2009 and became the first recorded case of influenza in pigs in Australia. The outbreak resulted from human-to-pig transmission. Clinical signs in affected pigs were mild compared with overseas reports of 'classical' swine influenza virus and included coughing and decreased appetite in a small proportion of non-lactating breeding stock, weaners, growers and finishers. A diagnosis of H1N1/09 influenza virus infection was confirmed using a combination of serology (haemagglutination inhibition, blocking enzyme-linked immunosorbent assay) and real-time reverse transcription polymerase chain reaction. Attempts at virus isolation were unsuccessful. Results of a longitudinal study of pigs on this farm suggested that the virus continued to circulate for 9 weeks after the onset of infection, but was not present 6 months later. This report highlights the difficulties in preventing transmission of H1N1/09 influenza virus from infected humans to pigs during a human pandemic.

Experimental studies of the role of the little raven (Corvus mellori) in surveillance for West Nile virus in Australia.

OBJECTIVE: To study the potential role of an Australian corvid, the little raven (Corvus mellori), in the surveillance for exotic West Nile virus (WNV) in Australia. METHOD: In a series of trials, little ravens were infected with WNV (strain 4132 New York 1999) and Kunjin virus (strain K42886) by the intramuscular route. They were observed for 20 days during which blood and swab samples were taken for virus isolation. Tissue samples were taken from ravens humanely killed during the acute infection period, and at the termination of the trials, for virus isolation, histopathology and immunohistochemistry. RESULTS: Ravens infected with WNV became mildly ill, but all recovered and seroconverted. Blood virus titres peaked around 3 to 4 days after inoculation at levels between 10(3.0) to 10(7.5) plaque forming units/mL. Virus or viral antigen was detected in spleen, liver, lung, kidney, intestine, testis and ovary by virus isolation and/or immunohistochemistry. WNV was detected in oral and cloacal swabs from 2 to 7 days post inoculation. The molecular and pathogenic characteristics of the inocula were consistent with them being of high virulence, as expected for this isolate. Ravens infected with Kunjin virus developed viraemia and seroconverted, although they did not develop disease. CONCLUSIONS: Little ravens do not develop severe disease in response to virulent WNV infection and for this reason may not be important sentinel hosts in the event of an outbreak of WNV, as in North America. However, as they have relatively high viraemias, they may be able to support virus cycles.

Cultured skin fibroblast cells derived from bluetongue virus-inoculated sheep and field-infected cattle are not a source of late and protracted recoverable virus.

A recent hypothesis to explain the recurrence of bluetongue disease after winter seasonal absences of the vector has suggested a role for persistent infection of sheep. This report presents combined independent work from two laboratories investigating the possible recovery of Bluetongue virus (BTV) over a protracted period after infection of both sheep and cattle. Prior to infection with either cell-culture-adapted or non-culture-adapted BTV, sheep were subjected to a preliminary exposure to Culicoides sp. insects, which reportedly facilitates recovery of virus from infected sheep several months post-infection (p.i.). A series of skin biopsies at different intervals p.i. was used to establish skin fibroblast (SF) cultures from which attempts were made to detect virus by isolation and by molecular and immunological methods. Also examined was the effect on virus recovery of additional exposure to Culicoides sp. prior to skin biopsy during the post-inoculation period. A herd of cattle sentinels for surveillance of natural BTV infection in northern Australia was monitored prospectively for seroconversion. Evidence of infection initiated attempted virus recovery by establishing SF cultures. It was found that in both cattle and sheep there was not a protracted period over which BTV could be recovered from SF cultures. The data do not support a general hypothesis that BTV persists in either sheep or cattle.

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.

Japanese encephalitis in a racing thoroughbred gelding in Hong Kong.

A horse in Hong Kong that had been vaccinated against Japanese encephalitis suffered a pyrexic episode that culminated in a hyperexcitable state and self-inflicted trauma. Japanese encephalitis was diagnosed on the basis of clinical, pathological and serological observations, and confirmed by the detection of genomic sequences of the virus in spinal cord tissue. Phylogenetic analyses of E gene and NS5-3'UTR sequences revealed divergent clustering of these segments with previously described genotypes, suggesting the possibility that the horse might have been infected with a recombinant between genotype I and genotype II viruses. Horses are considered to be dead-end hosts for the disease, but the occurrence of an infected horse in a population may have implications for the health status of the national herd. The effect that this case had on the horse industry in Hong Kong is discussed with specific reference to the movement of horses and the vaccination programme for Japanese encephalitis.

Genetic diversity of bluetongue viruses in south east Asia.

Bluetongue viruses (BTV) were isolated from sentinel cattle in Malaysia and at two sites in Indonesia. We identified eight serotypes some of which appeared to have a wide distribution throughout this region, while others were only isolated in Malaysia or Australia. Nearly half of the 24 known BTV serotypes have now been identified in Asia. Further, we investigated the genetic diversity of their RNA segments 3 and 10. Using partial nucleotide sequences of the RNA segment 3 (540 bp) which codes for the conserved core protein (VP3), the BTV isolates were found to be unique to the previously defined Australasian topotype and could be further subdivided into four distinct clades or genotypes. Certain of these genotypes appeared to be geographically restricted while others were distributed widely throughout the region. Similarly, the complete nucleotide sequences of the RNA segment 10 (822 bp), coding for the non-structural protein (NS3/3A), were also conserved and grouped into the five genotypes; the BTV isolates could be grouped into three Asian genotypes and two Nth American/Sth African genotypes.

Genetic diversity of bluetongue viruses in Australasia.

The authors have characterised the genetic diversity of the bluetongue virus (BTV) RNA segments 3 and 10 from Indonesia, Malaysia and Australia. Analysis of RNA segment 3, which codes for the core protein VP3, showed conserved sequences in the previously defined Australasian topotype, but which further divided into four distinct clades or genotypes. Certain genotypes appeared to be geographically restricted while others were distributed widely throughout South-East Asia. Ongoing surveillance programmes in Australia have identified the movement of Indonesian genotypes into northern Australia and possible reassortment among them. Similarly, analysis of RNA segment 10, which codes for the non-structural protein NS3/3A, showed they were also conserved and grouped into five clades or genotypes, three Asian and two North American/South African.

Pathogenesis studies with Australian bat lyssavirus in grey-headed flying foxes (Pteropus poliocephalus).

OBJECTIVE: To examine the susceptibility of the grey-headed flying fox (Pteropus poliocephalus) to Australian bat lyssavirus (ABL), and to provide preliminary observations on the pathogenesis of the disease in flying foxes. PROCEDURE: Ten flying foxes were inoculated intramuscularly with ABL, and four with a bat-associated rabies virus. Inoculated animals were observed daily, and clinical samples collected every 9 to 14 days. Animals with abnormal clinical signs were euthanased, and samples collected for histological, serological, virological and immunohistological examinations. At 3 months post inoculation (PI), all survivors were euthanased, and each submitted to a similar examination. RESULTS: Three ABL-inoculated flying foxes, and two rabies-inoculated animals developed abnormal clinical signs between 15 and 24 days PI. All three ABL-inoculated animals had histological lesions consistent with a lyssavirus infection, and lyssaviral antigen was identified in the central nervous system (CNS) of each. Virus was isolated from the brain of two affected animals. Of the rabies-inoculated flying foxes, both had histological lesions and viral antigen in the CNS. Virus was recovered from the brain of only one. None of the five affected flying foxes developed anti-lyssavirus antibodies, but, by 3 months PI, five of the seven ABL-inoculated survivors, and one of the two rabies virus-inoculated survivors, had seroconverted. The dynamics of the immune responses were quite variable. CONCLUSIONS: The response of flying foxes to ABL, administered by a peripheral route of inoculation, was similar to that of bats inoculated peripherally with bat-derived rabies viruses.

Experimental infections of pigs with Japanese encephalitis virus and closely related Australian flaviviruses.

The flavivirus Japanese encephalitis (JE) virus has recently emerged in the Australasian region. To investigate the involvement of infections with related enzootic flaviviruses, namely Murray Valley encephalitis (MVE) virus and Kunjin (KUN) virus, on immunity of pigs to JE virus and to provide a basis for interpretation of serologic data, experimental infections were conducted with combinations of these viruses. Antibody responses to primary and secondary infections were evaluated using panels of monoclonal antibody-based blocking enzyme-linked immunosorbent assays and microtiter serum neutralization tests (mSNTs). Identification of the primary infecting virus was possible only using the mSNTs. Following challenge, unequivocal diagnosis was impossible due to variation in immune responses between animals and broadened and/or anamnestic responses. Viremia for JE virus was readily detected in pigs following primary infection, but was not detected following prior exposure to MVE or KUN viruses. Boosted levels of existing cross-neutralizing antibodies to JE virus suggested a role for this response in suppressing JE viremia.

Potential exposure to Australian bat lyssavirus, Queensland, 1996-1999.

Two human deaths caused by Australian bat lyssavirus (ABL) infection have been reported since 1996. Information was obtained from 205 persons (mostly adults from south Brisbane and the South Coast of Queensland), who reported potential ABL exposure to the Brisbane Southside Public Health Unit from November 1,1996, to January 31, 1999. Volunteer animal handlers accounted for 39% of potential exposures, their family members for 12%, professional animal handlers for 14%, community members who intentionally handled bats for 31%, and community members with contacts initiated by bats for 4%. The prevalence of Lyssavirus detected by fluorescent antibody test in 366 sick, injured, or orphaned bats from the area was 6%. Sequelae of exposure, including the requirement for expensive postexposure prophylaxis, may be reduced by educating bat handlers and the public of the risks involved in handling Australian bats.

Epidemic of blindness in kangaroos--evidence of a viral aetiology.

OBJECTIVE: To determine the cause of an epidemic of blindness in kangaroos. DESIGN AND PROCEDURES: Laboratory examinations were made of eyes and brains of a large number of kangaroos using serological, virological, histopathological, electron microscopical, immunohistochemical methods, and PCR with cDNA sequencing. In addition, potential insect viral vectors identified during the disease outbreak were examined for specific viral genomic sequences. SAMPLE POPULATION: For histopathological analysis, 55 apparently blind and 18 apparently normal wild kangaroos and wallabies were obtained from New South Wales, Victoria, South Australia, and Western Australia. A total of 437 wild kangaroos and wallabies (including 23 animals with apparent blindness) were examined serologically. RESULTS: Orbiviruses of the Wallal and Warrego serogroups were isolated from kangaroos affected with blindness in a major epidemic in south-eastern Australia in 1994 and 1995 and extending to Western Australia in 1995/96. Histopathological examinations showed severe degeneration and inflammation in the eyes, and mild inflammation in the brains. In affected retinas, Wallal virus antigen was detected by immunohistochemical analysis and orbiviruses were seen in electron microscopy. There was serological variation in the newly isolated Wallal virus from archival Wallal virus that had been isolated in northern Australia. There were also variations of up to 20% in genotype sequence from the reference archival virus. Polymerase chain reactions showed that Wallal virus was present during the epidemic in three species of midges, Culicoides austropalpalis, C dycei and C marksi. Wallal virus nucleic acid was also detected by PCR in a paraffin-embedded retina taken from a blind kangaroo in 1975. CONCLUSION: Wallal virus and perhaps also Warrego virus are the cause of the outbreak of blindness in kangaroos. Other viruses may also be involved, but the evidence in this paper indicates a variant of Wallal virus, an orbivirus transmitted by midges, has the strongest aetiological association, and immunohistochemical analysis implicates it as the most damaging factor in the affected eyes.

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.

Characterisation of a novel lyssavirus isolated from Pteropid bats in Australia.

A novel lyssavirus isolated from Pteropid bats in Australia (Australian Bat Lyssavirus, ABLV) has been characterised using gene sequence analyses, electron microscopy and a panel of monoclonal antibodies. Electron microscopic examination of Pteropid bat and mouse brain material as well as virus isolated from tissue culture medium, showed the presence of bullet-shaped rhabdovirus particles and structures characteristic of lyssavirus. Analysis using nucleocapsid (N) specific monoclonal antibodies, showed a strong relationship between this new lyssavirus and serotype 1 rabies. The nucleotide sequence of the prototype strain of ABLV was determined from the initiator methionine codon for the nucleocapsid protein (N protein) to the amino terminus of the polymerase gene (L protein), a distance of 5344 nucleotides. Comparisons of the deduced N, phosphoprotein (P), matrix protein (M), and glycoprotein (G) proteins showed that ABLV was more closely related to serotype 1 classic rabies viruses than to other members of the Lyssavirus genus. The percent relatedness of the ABLV proteins when compared to the cognate proteins of PV (Pasteur vaccine strain) rabies was 92, 75, 87 and 75% for the N, P, M and G proteins, respectively. Phylogenetic studies of N protein sequences showed clearly that ABLV is an unrecognised member of the Lyssavirus genus and represents a new genotype, genotype 7.

Rapid identification of Australian bunyavirus isolates belonging to the Simbu serogroup using indirect ELISA formats.

The Bunyavirus genus, belonging to the Bunyaviridae family, is comprised of a large group of antigenically and geographically disparate arthropod-borne viruses of medical and veterinary significance. In Australia, viruses belonging to the Simbu serogroup of the Bunyavirus genus, Akabane, Tinaroo, Peaton, Aino, Douglas, Thimiri and Facey's Paddock have been isolated. In this communication we describe two indirect ELISAs, referred to as the Simbu serogroup ELISA (SG-ELISA), and the Simbu typing ELISA (ST-ELISA), for the identification of these Simbu serogroup viruses. Infected cell lysate antigens prepared from Simbu serogroup virus isolates were assessed in the SG-ELISA for reactivity with a mouse monoclonal antibody (4H9/B11/F1). The monoclonal antibody reacted strongly with all Australian members of Simbu serogroup reference viruses and is proposed for use as a serogrouping reagent for Simbu viruses. Furthermore, the ST-ELISA enabled specific identification of viruses from within this group by recognition of characteristic reaction patterns between infected cell lysate antigens and a panel of polyclonal antisera raised to Simbu serogroup viruses.

Australian bat lyssavirus infection in three fruit bats from north Queensland.

We report the case findings of Australian bat lyssavirus infection in two black flying foxes (Pteropus alecto) and one little red flying fox (Pteropus scapulatus) from north Queensland between January 1995 and August 1996. Although the P. alecto case in January 1995 is the first recognised case of Australian bat lyssavirus infection in Australia, this was a retrospective diagnosis made after identification of the index case at Ballina in May 1996. Eight persons had exposure to the three bats. Serum antibodies to classical rabies virus were measured in six of these persons; the only one seropositive was a veterinarian who had previously been vaccinated against rabies. Six persons received rabies vaccine following exposure. None of the in-contact humans developed signs of lyssavirus infection. For people exposed to Australian bat lyssavirus-positive bats who have not been scratched or bitten or had mucosal contamination by these bats, we suggest a post-exposure regime of five inoculations of the human diploid cell inactivated rabies vaccine.