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.

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.

Expression of the major inner capsid protein of the epizootic haemorrhagic disease virus in baculovirus and potential diagnostic use.

The RNA 7 encoding the major capsid protein (VP7) of epizootic haemorrhagic disease virus (EHDV), Australian serotype 2 (strain CS439), was cloned and the complete nucleotide sequence was determined. The coding region contained 1047 nucleotides (nt) capable of encoding a predicted 349 amino acid (aa) polypeptide with a calculated molecular size of 38.087 kDa. When the VP7 gene was expressed in bacterial or yeast expression systems, the expression product showed weak or no reactivity with polyclonal antibodies to EHDV. Therefore, the expression of the VP7 gene in baculovirus was pursued. The expressed EHDV VP7 was similar in antigenicity to that of the native virus as revealed by its reactivity in ELISA with monoclonal antibody (MAb) specific to EHDV. Preliminary ELISA results indicated that the recombinant protein binds to EHDV antibodies in serum and that these antibodies block the binding of EHDV-specific MAb. The availability of a reliable EHDV recombinant VP7 could enhance our existing assay for detection of EHDV-specific antibodies.

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.

Serotype identification of Australian bluetongue viruses using a rapid fluorescence inhibition test.

Rapid serotyping of bluetongue virus (BTV) isolates is required to facilitate the choice of an appropriate serotype-specific vaccine in a disease situation or to improve surveillance of BTV serotype prevalence. This communication describes the development and validation of a bluetongue virus fluorescent inhibition test (BTV FIT) as a rapid method to serotype Australian BTV isolates. The BTV FIT uses virus neutralisation principles similar to those used in the rabies rapid fluorescent focus inhibition test. The BTV FIT has the ability to provide an accurate serotype identification within 24 h thereby abbreviating the serotyping process by 3-4 days relative to conventional virus neutralisation assays and making the BTV FIT comparable time-wise with the polymerase chain reaction technique. The development of the BTV FIT is described using BTV reference viruses which have been isolated in Australia, and validation of the assay by assessment of five Australian BTV isolates of unknown serotype by comparison with the plaque inhibition method. The use of the BTV FIT readily facilitated rapid and accurate serotype identification of Australian BTV reference viruses and five unknown BTV isolates with results indicating full agreement with the plaque inhibition method.

A rapid indirect ELISA for the serogrouping of Australian orbiviruses.

This communication describes the development and evaluation of a simple and rapid method for the classification of Australian orbiviruses into one of seven established serogroups (i.e. bluetongue, epizootic haemorrhagic disease of deer, Palyam, Eubenangee, Corriparta, Wallal, Warrego) or an 'ungrouped' category. The Australian orbivirus serogrouping ELISA (SG-ELISA) utilised a sodium deoxycholate-treated cell lysate preparation from infected BHK cells which was subsequently probed in an indirect ELISA format with polyclonal antibodies representative of each serogroup. Bound immunoglobulin was detected by the use of a recombinant streptococcal protein G-HRPO conjugate and subsequent reaction with the chromogenic substrate. All reference orbiviruses tested in the SG-ELISA were identified and were in agreement with the serogroups originally designated. Minimal inter-serogroup cross-reactions were observed. One-way cross-reactions were observed between Warrego and Mitchell River viruses.

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.

The use of an indirect ELISA with protein G-peroxidase conjugate and a blocking ELISA to demonstrate recent bluetongue infection in sheep.

The humoral immune response of sheep infected with bluetongue virus serotypes 3, 9 and 16 was monitored by plaque inhibition (PI), blocking ELISA (BELISA) and indirect ELISA over a period of 63 days post-infection. Results indicated that testing of a single plasma or serum sample by both a BELISA and an indirect ELISA using a recombinant streptococcal protein G (PrG) peroxidase conjugate enabled an assessment of the proximity of a recent infection based on the failure of PrG to bind ovine IgM class antibodies. When BELISA and indirect ELISA results were expressed as a ratio, values indicative of recent infection (> or = 5) were observed for an average duration of 16.5 days (range 8 to 23 days) following the initial detection of antibody by BELISA. This approach has potential to improve diagnosis of a wide range of virus infections by providing an indicator for the relationship of serological status with a recent infection. However, where reinfection may occur, as with bluetongue virus, alternative methods may be required for definitive diagnosis.

Identification of epizootic haemorrhagic disease of deer virus serotypes using a fluorescence inhibition test.

A fluorescence inhibition test (FIT) is described for serotyping rapidly isolates of epizootic haemorrhagic disease of deer virus (EHDV). The test used a serogroup-reactive monoclonal antibody in a immunofluorescence procedure to detect virus which resisted neutralisation by antisera to any of the eight known EHDV serotypes. The EHDV FIT provided an accurate serotype identification procedure for all eight reference serotypes and, in comparison with the plaque inhibition assay, abbreviated the serotyping process by three to four days.

A modified liquid phase (LP) blocking ELISA used to assess type O foot-and-mouth disease virus antigenic variation in Thailand.

A selection of type O foot-and-mouth disease (FMD) viruses isolated in Thailand between 1986 and 1989 were compared to the reference viruses O1 Thailand 1960 (O BKK/60) and O Nakorn Pathom 1965 (O NPT/65) using a liquid-phase blocking ELISA (LP ELISA) to derive serum titres and associated r values. Interpolation techniques were used to increase the precision for estimation of r values through a more accurate estimation of serum titres at predicted equivalent levels of antigen input. Mean r values were 0.45 (for 56 field viruses) relative to O BKK/60 reference virus and 0.56 (for 51 field viruses) relative to O NPT/65. While only two viruses showed considerable difference (r < 0.20) to a reference virus (O BKK/60), 41% and 31% gave r values less than 0.4 for O BKK/60 and O NPT/65 respectively. This indicated antigenic differences between reference and field viruses which may result in a reduction in vaccine efficacy.

Studies with enzyme-linked immunosorbent assays for the serodiagnosis of bluetongue and epizootic haemorrhagic disease of deer.

An ELISA for the detection of serum antibody in sheep, cattle and goats to the viruses of bluetongue (BTV) and epizootic haemorrhagic disease of deer (EHDV) has been developed. Two methods of antigen preparation were analysed for efficacy in the ELISA and inter-group seroreactivity. A freeze-thaw (F/T) antigen appeared to have a narrower specificity than a cytoskeletal preparation from infected cells (P200) which contained all viral proteins. A higher background reactivity was seen when using the P200 antigen, suggesting that a F/T antigen, perhaps as a composite of serotypes, would be of greater value in an ELISA to replace current methods for antibody screening. The effect of multiple infections with unrelated orbiviruses was found to have no effect on the detection of antibody to BTV and EHDV by ELISA. The ELISA was able to demonstrate development and persistence of antibody to BTV in cattle over the course of 120 days.

A monoclonal antibody blocking ELISA detects antibodies specific for epizootic haemorrhagic disease virus.

The isolation of a monoclonal antibody (1G9/C9) with specificity for the epizootic haemorrhagic disease (EHD) serogroup has enabled the development of a highly sensitive and specific blocking ELISA (B-ELISA) for the detection of serum antibodies to EHD viruses. The assay was sensitive to blocking antibodies present in hyperimmune reference antisera to all six EHD serotypes tested but was unaffected by reference antisera to 19 South African and eight Australian serotypes of the related orbivirus bluetongue virus (BTV). The sensitivity of the EHD B-ELISA exceeded that of an indirect ELISA (I-ELISA) for EHD-specific antibody detection. Serum antibody titres to BTV and EHD in experimental and field sera, including a sentinel herd from which virus isolations were made, were examined in both the BTV and EHD B-ELISA tests. These results showed the B-ELISA was only sensitive to antibodies specific for the homologous serogroup in each case, even where sequential and mixed infections with each virus type occurred.

Detection by ELISA of bluetongue antigen directly in the blood of experimentally infected sheep.

An antigen-capture ELISA (Ag-ELISA) was developed to detect bluetongue virus (BTV) antigen directly from blood samples. Four blood preparations [whole blood, buffy coat, washed red blood cells (RBC) and plasma] taken pre-inoculation and on days 6 to 9 post-inoculation (PI) were used in the ELISA to study antigenaemia in forty sheep, each experimentally infected with one of 20 South African BTV serotypes. Seventeen of the 20 serotypes were detected and 27 of the 40 sheep were at some stage Ag-ELISA positive. Over the period of sampling, Ag-ELISA positive results were most frequently returned from whole blood taken on days 6 and 7 PI. However in some instances the quantity and/or duration of BTV antigenaemia was greater in buffy coat and washed RBC preparations. In a selection of samples examined, positive Ag-ELISA results were generally obtained when samples had an infectious virus titre in eggs of > 10(3.2) egg lethal doses (ELD50/ml). The appearance and duration of detectable antigenaemia was compared with the development of clinical signs and antibody responses of infected sheep. On days 6 and 7 PI the presence of fever (> 40 degrees C) was indicative to the likelihood of detectable antigenaemia. After day 5 PI antigenaemia declined and clinical signs of swollen face and inflamed feet appeared together with the first detectable antibody response. The Ag-ELISA, when used in conjunction with clinical observations and serologic data, should be useful as a rapid diagnostic procedure for bluetongue disease.

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.

EHDV-1, a new Australian serotype of epizootic haemorrhagic disease virus isolated from sentinel cattle in the Northern Territory.

In 1992, a virus (DPP2209) isolated from sentinel cattle located at Coastal Plains Research Station, latitude 12 degrees 39'S, longitude 131 degrees 20'E, approximately 60 km east of Darwin, Northern Territory. This virus was identified as a serotype of epizootic haemorrhagic disease (EHD) of deer virus previously undescribed in Australia. An additional 17 isolation of this virus were made from eight animals during the period February to May. Electron microscopic studies showed the presence of orbivirus-like structures. Serogrouping ELISA, indirect immunofluorescence assay and the serogrouping plaque reduction neutralisation test indicated the virus was a member of the epizootic haemorrhagic disease serogroup. Serotype specific plaque reduction neutralisation tests, indicated the virus was a member of the epizootic haemorrhagic disease serogroup not previously isolated in Australia. Analysis of the VP3 gene confirmed this observation. Cross neutralisation testing of the isolate with known epizootic haemorrhagic disease serotype viruses including endemic Australian and exotic strains identified isolate DPP2209 as epizootic haemorrhagic disease virus serotype 1.

Use of combined Shewhart-CUSUM control charts in internal quality control of enzyme-linked immunosorbent assays for the typing of foot and mouth disease virus antigen.

An enzyme-linked immunosorbent assay (ELISA) for the typing of foot and mouth disease virus (FMDV) antigen was employed for the routine laboratory diagnosis of FMD at a regional veterinary laboratory in northern Thailand. An objective procedure was developed to monitor the test performance of the ELISA, using absolute test control limits in a Shewhart-CUSUM (cumulative sum) control chart method. The procedure detected significant data trends and 'beyond control limit' situations for each antigen typing system (types O, A and Asia 1), using an assay variable (gamma i). Retrospective analysis using Shewhart-CUSUM control charts of data from 42 ELISAs demonstrated that control limits were exceeded in two assays for FMDV type A. The Shewhart-CUSUM control chart is a simple and reliable internal quality control method for the detection of significant random and systematic variation in assays.

Serological comparison of type A foot and mouth disease virus isolates from Thailand.

Antigenic variation of type A foot and mouth disease (FMD) virus in Thailand was examined using a total of 82 field viruses isolated between 1986 and 1989. A two-dimensional serum microneutralisation test was used to compare these isolates to a reference strain, A15 Bangkok 1960 (A BKK/60). Viruses regarded as unrelated to A BKK/60 were compared to another reference strain, A22 Nakhon Pathom 1986 (A NPT/86). This approach divided the viruses into two groups. Most of the viruses shared a close antigenic relationship with A BKK/60. Only twelve viruses were regarded as unrelated to A BKK/60, and these were related to A NPT/86. All but one of these twelve isolates were from two provinces in one administrative region of the country. Future type A vaccines in Thailand will need to confer protection against both groups of viruses.

Establishment of a typing enzyme-linked immunosorbent assay for foot and mouth disease antigen, using reagents against viruses endemic in Thailand.

Antisera were produced at a central laboratory in Thailand against the endemic serotypes (O, A and Asia 1) of foot and mouth disease (FMD) virus. At a regional veterinary laboratory, these antisera were used in an indirect sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and serotyping of FMD virus (FMDV) antigen. ELISA readings of < 0.10 optical density (OD) units were considered negative. This was verified using fifty tissue samples which were known to be negative for FMDV. The highest mean sample value for three different dilutions was 0.02 OD units. Of a total of 93 samples submitted for antigen typing, 80 (86%) tested positive by ELISA and 13 (14%) were negative. No FMDV was detected in ELISA-negative samples following attempted tissue-culture virus isolation.

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.