FATAL HENDRA VIRUS DISEASE IN A HORSE IN 2015 DUE TO A PREVIOUSLY UNRECOGNISED HENDRA VIRUS VARIANT ASSOCIATED WITH MULTIPLE FLYING-FOX SPECIES.

Background Hendra virus (HeV) causes severe acute respiratory and encephalitic disease mediated by endothelial vasculitis in horses and humans. Sixty-three natural spillovers have been detected resulting in 105 horse deaths, seven human cases, and four human fatalities. Around 1000 horses are tested annually for HeV, mostly from regions within the ranges of the black flying-fox and the spectacled flying-fox, and less than 0.5% of these cases test positive. Causative diagnosis is rarely identified in the remaining cases, including those with high indications of an infectious cause. We hypothesised that some severe equine diseases consistent with HeV, but negative when tested, were due to infection with other paramyxoviruses spilling over from flying-foxes to horses, and plausibly posing zoonotic risks. Methods A biobank of samples from horses tested for HeV by the Queensland Biosecurity Sciences Laboratory was constructed and found to be negative. Subject, geographic, clinical and sample details and results were captured using a purpose-built, de-identified SQL database. Cases were classified via a syndromic disease decision algorithm according to likelihood of infectious cause to guide selection and processing for subsequent batched molecular and serological testing pathways that included meta-transcriptomic next-generation-sequencing (RNAseq-NGS) coupled with nested-conventionalPAN-Paramyxovirus RT-PCR screening and a 33-plex microsphere immuno-serological assay screening for both IgM and IgG against emerging infectious diseases. Results A previously unrecognised variant of HeV was detected in a case of acute severe disease in a 12-yearold Arabian gelding clinically indistinguishable to that caused by the prototypic HeV. The case, which occurred in September 2015 near Gympie in South-East Queensland, presented clinically with severely ‘injected’ mucous membranes, tachycardia (75 bpm), tachypnoea (60 bpm), rectal temperature of 38.0C, muscle fasciculations, head pressing and recumbency, with rapid deterioration over 24 hours resulting in euthanasia on humane grounds. A complete viral genome sequence was obtained by RNAseq-NGS from the blood sample. While the variant is sufficiently consistent in amino-acid sequence and protein structure to be of equivalent pathogenicity (mean 92.5% phenotypic consistency), it was sufficiently divergent in nucleotide sequence (82.9% genotypic similarity) to fail detection by standard molecular surveillance testing for HeV. Updated PCR approaches for routine use to detect both this variant and the formally-known HeV were developed, optimised and made available to state and national human and animal health laboratories. In-silico analysis of the variant’s receptor-binding protein (RBP or G glycoprotein), using established x-ray structures of the HeV RBP, revealed unaltered epitope structures relevant to the binding of both the Ephrin-B2 & -B3 virus entry receptors and the monoclonal antibody mAb 102.4 used as post-exposure prophylaxis in humans. These findings support the observed consistent pathogenicity and indicate that equivalent immune protection should be afforded by the Equivac® HeV vaccine against this variant. Comparison of a partial viral sequence identified from a grey-headed flying-fox from Adelaide in January 2013 and genomic sequence of this HeV variant (2015 horse) showed them to be >99% similar. Recommendations Equine veterinarians should consider HeV a differential diagnosis in unvaccinated horses anywhere in Australia where flying-foxes are present. Suspect diseased horses should be screened for this variant via routine state biosecurity HeV testing. Biosecurity recommendations should be updated to reflect the link between HeV variants and flying-fox populations while emphasising the protected status and importance of flying-foxes. [ABSTRACT FROM AUTHOR]