Development and preliminary validation of a MERS-CoV ELISA for serological testing of camels and alpacas.

This study describes the development and preliminary validation of a new serological assay using MERS-CoV S1 protein in an indirect enzyme-linked immunosorbent assay (ELISA) format. This assay has the advantage of being able to test MERS-CoV serum samples in a PC2 laboratory without the need for a high-level biocontainment laboratory (PC3 or PC4), which requires highly trained and skilled staff and a high level of resources and equipment. Furthermore, this MERS-CoV S1 ELISA enables a larger number of samples to be tested quickly, with results obtained in approximately five hours. The MERS-CoV S1 ELISA demonstrated high analytical specificity, with no cross-reactivity observed in serum of animals infected with other viruses, including different coronaviruses. We tested 166 positive and 40 negative camel serum samples and have estimated the diagnostic sensitivity (DSe) to be 99.4% (95% CI: 96.7 - 100.0%) and diagnostic specificity (DSp) to be 100% (95% CI: 97.2%-100.0%) relative to the assigned serology results (ppNT and VNT) using a S/P ratio cut-off value of >0.58. The findings of this study showed that our MERS-CoV S1 ELISA was more sensitive than the commercial EUROIMMUN ELISA (Se 99.4% vs 84.9%) and comparable to the ppNT assay, and therefore could be used as a diagnostic aid in countries in the Middle East where MERS-CoV is endemic in dromedary camels. The assay reagents and protocol were easily adapted and transferred from an Australian laboratory to a laboratory in the University of Hong Kong. Thus, the results described here show that the MERS-CoV S1 ELISA represents a cheap, rapid, robust, and reliable assay to support surveillance of MERS-CoV in camels in endemic regions.

Evidence of a Possible Viral Host Switch Event in an Avipoxvirus Isolated from an Endangered Northern Royal Albatross (Diomedea sanfordi).

Avipoxviruses have been characterized from many avian species. Two recent studies have reported avipoxvirus-like viruses with varying pathogenicity in reptiles. Avipoxviruses are considered to be restricted to avian hosts. However, reports of avipoxvirus-like viruses from reptiles such as the green sea turtle (Chelonia mydas) and crocodile tegu (Crocodilurus amazonicus) suggest that cross-species transmission, within avian species and beyond, may be possible. Here we report evidence for a possible host switching event with a fowlpox-like virus recovered from an endangered northern royal albatross (Diomodea sanfordi)-a species of Procellariiformes, unrelated to Galliformes, not previously known to have been infected with fowlpox-like viruses. Complete genome sequencing of this virus, tentatively designated albatrosspox virus 2 (ALPV2), contained many fowlpox virus-like genes, but also 63 unique genes that are not reported in any other poxvirus. The ALPV2 genome contained 296 predicted genes homologous to different avipoxviruses, 260 of which were homologous to an American strain of fowlpox virus (FWPV). Subsequent phylogenetic analyses indicate that ALPV2 likely originated from a fowlpox virus-like progenitor. These findings highlight the importance of host-switching events where viruses cross species barriers with the risk of disease in close and distantly related host populations.

Genomic characterisation of a novel avipoxvirus, magpiepox virus 2, from an Australian magpie (Gymnorhina tibicen terraereginae).

Avipoxviruses are large, double-stranded DNA viruses and are considered significant pathogens that may impact on the conservation of numerous bird species. The vast majority of avipoxviruses in wild birds remain uncharacterised and their genetic variability is unclear. Here, we fully sequenced a novel avipoxvirus, magpiepox virus 2 (MPPV2), which was isolated 62 years ago (in 1956) from an Australian black-backed magpie. The MPPV2 genome was 298,392 bp in length and contained 419 predicted open-reading frames (ORFs). While 43 ORFs were novel, a further 24 ORFs were absent compared with another magpiepox virus (MPPV) characterised in 2018. The MPPV2 genome contained an additional ten genes that were homologs to shearwaterpox virus 2 (SWPV2). Subsequent phylogenetic analyses showed that the novel MPPV2 was most closely related to other avipoxviruses isolated from passerine and shearwater bird species, and demonstrated a high degree of sequence similarity (95.0%) with MPPV.

Genomic Characterisation of a Novel Avipoxvirus Isolated from an Endangered Northern Royal Albatross (Diomedea sanfordi).

Marine bird populations have been declining globally with the factors driving this decline not fully understood. Viral diseases, including those caused by poxviruses, are a concern for endangered seabird species. In this study we have characterised a novel avipoxvirus, tentatively designated albatrosspox virus (ALPV), isolated from a skin lesion of an endangered New Zealand northern royal albatross (Diomedea sanfordi). The ALPV genome was 351.9 kbp in length and contained 336 predicted genes, seven of which were determined to be unique. The highest number of genes (313) in the ALPV genome were homologs of those in shearwaterpox virus 2 (SWPV2), while a further 10 were homologs to canarypox virus (CNPV) and an additional six to shearwaterpox virus 1 (SWPV1). Phylogenetic analyses positioned the ALPV genome within a distinct subclade comprising recently isolated avipoxvirus genome sequences from shearwater, penguin and passerine bird species. This is the first reported genome sequence of ALPV from a northern royal albatross and will help to track the evolution of avipoxvirus infections in this endangered species.

Characterisation of an Australian fowlpox virus carrying a near-full-length provirus of reticuloendotheliosis virus.

Fowlpox virus (FWPV), which is the type member of the genus Avipoxvirus, subfamily Chordopoxvirinae, family Poxviridae, can lead to significant losses to the poultry industry. Although a large number of fowlpox virus genomes have been sequenced and characterised globally, there are no sequences available at the genomic level from Australian isolates. Here, we present the first complete genome sequence of a fowlpox virus vaccine strain (FWPV-S) containing an integrated near-full-length reticuloendotheliosis virus (REV) provirus. The genome of FWPV-S showed the highest sequence similarity to a fowlpox virus from the USA (97.74% identity). The FWPV-S genome contained 16 predicted unique genes, while a further two genes were fragmented compared to previously reported FWPV genome sequences. Subsequent phylogenetic analysis showed that FWPV-S was most closely related to other fowlpox viruses. This is the first reported genome sequence of FWPV from Australia.

Genomic Characterisation of a Novel Avipoxvirus Isolated from an Endangered Yellow-Eyed Penguin (Megadyptes antipodes).

Emerging viral diseases have become a significant concern due to their potential consequences for animal and environmental health. Over the past few decades, it has become clear that viruses emerging in wildlife may pose a major threat to vulnerable or endangered species. Diphtheritic stomatitis, likely to be caused by an avipoxvirus, has been recognised as a significant cause of mortality for the endangered yellow-eyed penguin (Megadyptes antipodes) in New Zealand. However, the avipoxvirus that infects yellow-eyed penguins has remained uncharacterised. Here, we report the complete genome of a novel avipoxvirus, penguinpox virus 2 (PEPV2), which was derived from a virus isolate obtained from a skin lesion of a yellow-eyed penguin. The PEPV2 genome is 349.8 kbp in length and contains 327 predicted genes; five of these genes were found to be unique, while a further two genes were absent compared to shearwaterpox virus 2 (SWPV2). In comparison with penguinpox virus (PEPV) isolated from an African penguin, there was a lack of conservation within the central region of the genome. Subsequent phylogenetic analyses of the PEPV2 genome positioned it within a distinct subclade comprising the recently isolated avipoxvirus genome sequences from shearwater, canary, and magpie bird species, and demonstrated a high degree of sequence similarity with SWPV2 (96.27%). This is the first reported genome sequence of PEPV2 from a yellow-eyed penguin and will help to track the evolution of avipoxvirus infections in this rare and endangered species.

Extended sequencing of vaccine and wild-type capripoxvirus isolates provides insights into genes modulating virulence and host range.

The genus Capripoxvirus in the subfamily Chordopoxvirinae, family Poxviridae, comprises sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which cause the eponymous diseases across parts of Africa, the Middle East and Asia. These diseases cause significant economic losses and can have a devastating impact on the livelihoods and food security of small farm holders. So far, only live classically attenuated SPPV, GTPV and LSDV vaccines are commercially available and the history, safety and efficacy of many have not been well established. Here, we report 13 new capripoxvirus genome sequences, including the hairpin telomeres, from both pathogenic field isolates and vaccine strains. We have also updated the genome annotations to incorporate recent advances in our understanding of poxvirus biology. These new genomes and genes grouped phenetically with other previously sequenced capripoxvirus strains, and these new alignments collectively identified several recurring alterations in genes thought to modulate virulence and host range. In particular, some of the many large capripoxvirus ankyrin and kelch-like proteins are commonly mutated in vaccine strains, while the variola virus B22R-like gene homolog has also been disrupted in many vaccine isolates. Among these vaccine isolates, frameshift mutations are especially common and clearly present a risk of reversion to wild type in vaccines bearing these mutations. A consistent pattern of gene inactivation from LSDV to GTPV and then SPPV is also observed, much like the pattern of gene loss in orthopoxviruses, but, rather surprisingly, the overall genome size of ~150 kbp remains relatively constant. These data provide new insights into the evolution of capripoxviruses and the determinants of pathogenicity and host range. They will find application in the development of new vaccines with better safety, efficacy and trade profiles.

Probe capture enrichment next-generation sequencing of complete foot-and-mouth disease virus genomes in clinical samples.

Next-generation sequencing (NGS) techniques offer an unprecedented "step-change" increase in the quantity and quality of sequence data rapidly generated from a sample and can be applied to obtain ultra-deep coverage of viral genomes. This is not possible with the routinely used Sanger sequencing method that gives the consensus reads, or by cloning approaches. In this study, a targeted-enrichment methodology for the simultaneous acquisition of complete foot-and-mouth disease virus (FMDV) genomes directly from clinical samples is presented. Biotinylated oligonucleotide probes (120 nt) were used to capture and enrich viral RNA following library preparation. To create a virus capture panel targeting serotype O and A simultaneously, 18 baits targeting the highly conserved regions of the 8.3 kb FMDV genome were synthesised, with 14 common to both serotypes, 2 specific to serotype O and 2 specific to serotype A. These baits were used to capture and enrich FMDV RNA (as cDNA) from samples collected during one pathogenesis and two vaccine efficacy trials, where pigs were infected with serotype O or A viruses. After enrichment, FMDV-specific sequencing reads increased by almost 3000-fold. The sequence data were used in variant call analysis to identify single nucleotide polymorphisms (SNPs). This methodology was robust in its ability to capture diverse sequences, was shown to be highly sensitive, and can be easily scaled for large-scale epidemiological studies.

A lumpy skin disease virus deficient of an IL-10 gene homologue provides protective immunity against virulent capripoxvirus challenge in sheep and goats.

Sheep and goat pox continue to be important livestock diseases that pose a major threat to the livestock industry in many regions in Africa and Asia. Currently, several live attenuated vaccines are available and used in endemic countries to control these diseases. One of these is a partially attenuated strain of lumpy skin disease virus (LSDV), KS-1, which provides cross-protection against both sheep pox and goat pox. However, when used in highly stressed dairy cattle to protect against lumpy skin disease (LSD) the vaccine can cause clinical disease. In order to develop safer vaccines effective against all three diseases, a pathogenic strain of LSDV (Warmbaths [WB], South Africa) was attenuated by removing a putative virulence factor gene (IL-10-like) using gene knockout (KO) technology. This construct (LSDV WB005KO) was then evaluated as a vaccine for sheep and goats against virulent capripoxvirus challenge. Sheep and goats were vaccinated with the construct and the animals were observed for 21days. The vaccine appeared to be safe, and did not cause disease, although it induced minor inflammation at the injection site similar to that caused by other attenuated sheep and goat pox vaccines. In addition, no virus replication was detected in blood, oral or nasal swabs using real-time PCR following vaccination and low levels of neutralising antibodies were detected in both sheep and goats. Leukocytes isolated from vaccinated animals following vaccination elicited capripoxvirus-specific IFN-γ secretion, suggesting that immunity was also T-cell mediated. Following challenge with virulent capripoxvirus, vaccinated sheep and goats were found to be completely protected and exhibited no clinical disease. Furthermore, real-time PCR of blood samples at various time points suggested that viremia was absent in both groups of vaccinated animals, as opposed to capripoxvirus-related clinical disease and viremia observed in the unvaccinated animals. These findings suggest that this novel knockout strain of LSDV has potential as a vaccine to protect livestock against sheep pox and goat pox.

Fit-for-purpose curated database application in mass spectrometry-based targeted protein identification and validation.

BACKGROUND: Mass spectrometry (MS) is a very sensitive and specific method for protein identification, biomarker discovery, and biomarker validation. Protein identification is commonly carried out by comparing MS data with public databases. However, with the development of high throughput and accurate genomic sequencing technology, public databases are being overwhelmed with new entries from different species every day. The application of these databases can also be problematic due to factors such as size, specificity, and unharmonized annotation of the molecules of interest. Current databases representing liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based searches focus on enzyme digestion patterns and sequence information and consequently, important functional information can be missed within the search output. Protein variants displaying similar sequence homology can interfere with database identification when only certain homologues are examined. In addition, recombinant DNA technology can result in products that may not be accurately annotated in public databases. Curated databases, which focus on the molecule of interest with clearer functional annotation and sequence information, are necessary for accurate protein identification and validation. Here, four cases of curated database application have been explored and summarized. FINDINGS: The four presented curated databases were constructed with clear goals regarding application and have proven very useful for targeted protein identification and biomarker application in different fields. They include a sheeppox virus database created for accurate identification of proteins with strong antigenicity, a custom database containing clearly annotated protein variants such as tau transcript variant 2 for accurate biomarker identification, a sheep-hamster chimeric prion protein (PrP) database constructed for assay development of prion diseases, and a custom Escherichia coli (E. coli) flagella (H antigen) database produced for MS-H, a new H-typing technique. Clearly annotating the proteins of interest was essential for highly accurate, specific, and sensitive sequence identification, and searching against public databases resulted in inaccurate identification of the sequence of interest, while combining the curated database with a public database reduced both the confidence and sequence coverage of the protein search. CONCLUSION: Curated protein sequence databases incorporating clear annotations are very useful for accurate protein identification and fit-for-purpose application through MS-based biomarker validation.

Increased bacterial cell density and recombinant protein yield using a commercial microbial cultivation system.

EnBase (BioSilta, Finland) is a microbial cultivation system that replicates fed-batch systems through sustained release of glucose by enzymatic degradation of a polymeric substrate. Achievable bacterial cell densities and recombinant capripoxvirus protein expression levels, solubility, and antigenicity using the EnBase system were assessed. BL21-AI Escherichia coli expressing capripoxvirus proteins achieved up to eightfold higher cell densities when grown in EnBase media compared with standard media. Greater yields of capripoxvirus proteins were attained using EnBase media, either through increases in the amount of expressed protein per cell in conjunction with higher cell density or through the increase in cell density alone. Addition of EnBase booster enhanced protein yield for one of the proteins tested but reduced yield for the other. However, the amount of soluble forms of the capripoxvirus proteins tested was not different from that observed from cultures grown under standard conditions. Purified capripoxvirus proteins expressed using EnBase or standard media were assessed for their performance by enzyme-linked immunosorbent assay (ELISA) and were shown to be equally capable of specifically binding capripoxvirus antibodies.

Menangle virus, a pteropid bat paramyxovirus infectious for pigs and humans, exhibits tropism for secondary lymphoid organs and intestinal epithelium in weaned pigs.

This study is the first report of experimental infection and transmission of Menangle virus (MenPV) in pigs. Isolated in 1997 from piglets that were stillborn at a large commercial piggery in New South Wales, Australia, MenPV is a recently identified paramyxovirus of bat origin that causes severe reproductive disease in pigs and an influenza-like illness, with a rash, in humans. Although successfully eradicated from the infected piggery, the virus was only isolated from affected fetuses and stillborn piglets during the period of reproductive disease, and thus the mode of transmission between pigs was not established. To investigate the pathogenesis of MenPV, we undertook time-course studies in 6-week-old pigs following intranasal administration of a low-passage, non-plaque-purified isolate from the lung of an infected stillborn piglet. Viraemia was of short duration and low titre, as determined by real-time RT-PCR and virus isolation. Following an incubation period of 2-3 days, virus was shed in nasal and oral secretions, faeces and urine, typically for less than 1 week. Cessation of shedding correlated with the development of neutralizing antibodies in sera. Secondary lymphoid organs and intestine were identified, using quantitative real-time RT-PCR, as major sites of viral replication and dissemination, and this was confirmed by positive immunolabelling of viral antigen within various lymphoid tissues and intestinal epithelium. These data provide new insights into the pathogenesis of MenPV in weaned pigs, and will facilitate future control and eradication programmes should it ever re-emerge in the pig population.

Validation of a high-throughput real-time polymerase chain reaction assay for the detection of capripoxviral DNA.

Capripoxviruses, which are endemic in much of Africa and Asia, are the aetiological agents of economically devastating poxviral diseases in cattle, sheep and goats. The aim of this study was to validate a high-throughput real-time PCR assay for routine diagnostic use in a capripoxvirus reference laboratory. The performance of two previously published real-time PCR methods were compared using commercially available reagents including the amplification kits recommended in the original publication. Furthermore, both manual and robotic extraction methods used to prepare template nucleic acid were evaluated using samples collected from experimentally infected animals. The optimised assay had an analytical sensitivity of at least 63 target DNA copies per reaction, displayed a greater diagnostic sensitivity compared to conventional gel-based PCR, detected capripoxviruses isolated from outbreaks around the world and did not amplify DNA from related viruses in the genera Orthopoxvirus or Parapoxvirus. The high-throughput robotic DNA extraction procedure did not adversely affect the sensitivity of the assay compared to manual preparation of PCR templates. This laboratory-based assay provides a rapid and robust method to detect capripoxviruses following suspicion of disease in endemic or disease-free countries.

Preclinical efficacy studies of influenza A haemagglutinin precursor cleavage loop peptides as a potential vaccine.

A universal influenza vaccine that does not require annual reformulation would have clear advantages over the currently approved seasonal vaccine. In this study, we combined the mucosal adjuvant alpha-galactosylceramide (αGalCer) and peptides designed across the highly conserved influenza precursor haemagglutinin (HA(0)) cleavage loop as a vaccine. Peptides designed across the HA(0) of influenza A/H3N2 viruses, delivered to mice via the intranasal route with αGalCer as an adjuvant, provided 100 % protection following H3N2 virus challenge. Similarly, intranasal inoculation of peptides across the HA(0) of influenza A/H5N1 with αGalCer completely protected mice against heterotypic challenge with H3N2 virus. Our data suggest that these peptide vaccines effectively inhibited subsequent influenza A/H3N2 virus replication. In contrast, only 20 % of mice vaccinated with αGalCer-adjuvanted peptides spanning the HA(0) of H5N1 survived homologous viral challenge, possibly because the HA(0) of this virus subtype is cleaved by intracellular furin-like enzymes. Results of these studies demonstrated that HA(0) peptides adjuvanted with αGalCer have the potential to form the basis of a synthetic, intranasal influenza vaccine.

Detection of antibodies specific for sheeppox and goatpox viruses using recombinant capripoxvirus antigens in an indirect enzyme-linked immunosorbent assay.

Viruses in the genus Capripoxvirus, family Poxviridae, cause sheeppox, goatpox and lumpy skin disease, which are the most serious poxvirus diseases of production animals. Despite the considerable threat that these viruses pose to livestock production and global trade in sheep, goats, cattle and their products, convenient and effective serodiagnostic tools are not readily available. To develop a more effective antibody detection capability, selected open reading frames from capripoxvirus DNA were amplified and expressed in Escherichia coli as His-tagged fusion proteins. By screening 42 candidate antigens, two sheeppox virus virion core proteins that were expressed efficiently, purified readily using affinity chromatography and reactive against capripoxvirus immune sera in an indirect enzyme-linked immunosorbent assay (ELISA) were identified. The ELISA performed favourably when sera from sheep and goats infected experimentally with virulent capripoxvirus isolates were tested, with sensitivity and diagnostic specificity ranging between 95 and 97%, but it was unable to detect antibodies reliably in vaccinated sheep or goats. Furthermore, no cross-reactivity with antibodies against orf virus was detected. This assay offers the prospect of a convenient and standardised ELISA-based serodiagnostic test, with no requirement for infectious reagents, that is well suited to high-throughput capripoxvirus surveillance on a flock or herd basis.

Intranasal flu vaccine protective against seasonal and H5N1 avian influenza infections.

BACKGROUND: Influenza A (flu) virus causes significant morbidity and mortality worldwide, and current vaccines require annual updating to protect against the rapidly arising antigenic variations due to antigenic shift and drift. In fact, current subunit or split flu vaccines rely exclusively on antibody responses for protection and do not induce cytotoxic T (Tc) cell responses, which are broadly cross-reactive between virus strains. We have previously reported that gamma-ray inactivated flu virus can induce cross-reactive Tc cell responses. METHODOLOGY/PRINCIPAL FINDING: Here, we report that intranasal administration of purified gamma-ray inactivated human influenza A virus preparations (gamma-Flu) effectively induces heterotypic and cross-protective immunity. A single intranasal administration of gamma-A/PR8[H1N1] protects mice against lethal H5N1 and other heterotypic infections. CONCLUSIONS/SIGNIFICANCE: Intranasal gamma-Flu represents a unique approach for a cross-protective vaccine against both seasonal as well as possible future pandemic influenza A virus infections.

Yemen and Vietnam capripoxviruses demonstrate a distinct host preference for goats compared with sheep.

Sheeppox and goatpox are caused by viruses that are members of the genus Capripoxvirus, and globally result in significant production losses. To improve the understanding of disease pathogenesis and evaluate host species preferences, sheep and goats were inoculated either with a capripoxvirus isolate from Yemen or from a recent outbreak in Vietnam. Blood, swabs and tissues were collected at various time points following experimental challenge and assessed for viral DNA content using real-time PCR and infectivity using virus isolation. The Yemen isolate was considerably more pathogenic in goats with 100 % mortality and morbidity compared with sheep with 0 % mortality and 100 % morbidity. The Vietnam isolate was also more pathogenic in goats with 100 % morbidity and an estimated 33 % mortality rate compared with mild morbidity and a 0 % mortality rate in sheep. Higher viral titres were observed in nasal, oral and conjunctival swabs from goats inoculated with either the Yemen or Vietnam isolate compared with those collected from sheep. Although the highest viral titres were detected in primary and secondary skin lesions in sheep and goats, the severity of clinical disease observed in each species varied according to the inoculum used. Whereas both the Yemen and Vietnam isolates clearly caused more severe disease in goats, the Yemen isolate was also moderately pathogenic in sheep. The Vietnam isolate, in contrast, caused only very mild disease in sheep. Limited DNA sequencing revealed ORF 074 of the Vietnam isolate to be identical to that of several goatpox virus isolates from China, suggesting a possible Chinese origin.

Capripoxvirus tissue tropism and shedding: A quantitative study in experimentally infected sheep and goats.

Sheeppox virus and goatpox virus cause systemic disease in sheep and goats that is often associated with high morbidity and high mortality. To increase understanding of the pathogenesis of these diseases, we undertook quantitative time-course studies in sheep and goats following intradermal inoculation of Nigerian sheeppox virus or Indian goatpox virus in their respective homologous hosts. Viremia, determined by virus isolation and real-time PCR, cleared within 2 to 3 weeks post inoculation. Peak shedding of viral DNA and infectious virus in nasal, conjunctival and oral secretions occurred between 10 and 14 days post inoculation, and persisted at low levels for up to an additional 3 to 6 weeks. Although gross lesions developed in multiple organ systems, highest viral titers were detected in skin and in discrete sites within oronasal tissues and gastrointestinal tract. The temporal distribution of infectious virus and viral DNA in tissues suggests an underlying pathogenesis that is similar to smallpox and monkeypox where greatest viral replication occurs in the skin. Our data demonstrate that capripoxvirus infections in sheep and goats provide additional and convenient models which are suitable not only for evaluation of poxvirus-specific vaccine concepts and therapeutics, but also study of poxvirus-host interactions.

Evaluation of an ovine testis cell line (OA3.Ts) for propagation of capripoxvirus isolates and development of an immunostaining technique for viral plaque visualization.

An ovine testis cell line (OA3.Ts) was evaluated and compared with primary lamb kidney (LK) cells for its utility in capripoxvirus propagation and titration. A comparison of OA3.Ts cell growth kinetics and morphology at low (<33) and high (34-36) passage levels indicated a difference in both characteristics. However, viral titers determined in low and high passage OA3.Ts cells were comparable with those obtained using LK cells. Capripoxvirus infection of OA3.Ts and LK cells resulted in a similar cytopathic effect, which allowed for the detection of discrete viral plaques following immunostaining with capripoxvirus-specific antiserum.

Generation of menangle virus nucleocapsid-like particles in yeast Saccharomyces cerevisiae.

Menangle virus (MenV), which was isolated in Australia in 1997 during an outbreak of severe reproductive disease in pigs, is a novel member of the genus Rubulavirus in the family Paramyxoviridae. Although successfully eradicated from the affected piggery, fruit bats are considered to be the natural reservoir of the virus and therefore an ongoing risk of re-introduction to the pig population exists. Accordingly, reagents to facilitate serological surveillance are required to enhance the diagnostic capability for MenV, which is a newly recognized cause of disease in pigs with the potential to severely affect production in naive breeding herds. To address this need, recombinant MenV nucleocapsid (N) protein was expressed in the yeast Saccharomyces cerevisiae. Using the expression vector pFGG3 under control of the GAL7 promoter, high yields of recombinant MenV N protein were obtained. Electron microscopy demonstrated that purified recombinant N protein self-assembled into nucleocapsid-like particles which were identical in density and morphology, although not in length, to authentic nucleocapsids from virus-infected cells. Electron microscopy analysis also showed that yeast-expressed N protein which lacked the C-terminal tail (amino acid residues 400-519) formed significantly longer and denser nucleocapsid-like particles. Nucleocapsid-like particles derived from the full-length recombinant protein were stable and readily purified by CsCl gradient ultracentrifugation. When used as coating antigen in an indirect ELISA, the recombinant N protein reacted with sera derived from pigs experimentally infected with MenV and a simple serological assay to detect MenV-specific antibodies in pigs, fruit bats and humans could be designed on this basis.