key: cord-0429307-pruyy200 authors: Annand, Edward J.; Horsburgh, Bethany A.; Xu, Kai; Reid, Peter A.; Poole, Ben; de Kantzow, Maximillian C.; Brown, Nicole; Tweedie, Alison; Michie, Michelle; Grewar, John D.; Jackson, Anne E.; Singanallur, Nagendrakumar B.; Plain, Karren M.; Kim, Karan; Tachedjian, Mary; van der Heide, Brenda; Crameri, Sandra; Williams, David T.; Secombe, Cristy; Laing, Eric D.; Sterling, Spencer; Yan, Lianying; Jackson, Louise; Jones, Cheryl; Plowright, Raina K.; Peel, Alison J.; Breed, Andrew C.; Diallo, Ibrahim; Dhand, Navneet K.; Britton, Philip N.; Broder, Christopher C.; Smith, Ina; Eden, John-Sebastian title: Novel Hendra virus variant detected by sentinel surveillance of Australian horses date: 2021-09-22 journal: bioRxiv DOI: 10.1101/2021.07.16.452724 sha: c6c3463dc2db07f5747ba0818cb52991d1230014 doc_id: 429307 cord_uid: pruyy200 A novel Hendra virus (HeV) variant, not detected by routine testing, was identified and isolated from a Queensland horse that suffered acute, fatal disease consistent with HeV infection. Whole genome sequencing and phylogenetic analysis demonstrated the variant to have ~83% nucleotide identity to the prototype HeV strain. An updated RT-qPCR assay was designed for routine HeV surveillance. In silico and in vitro comparison of the receptor-binding protein with prototypic HeV showed that the human monoclonal antibody m102.4 used for post-exposure prophylaxis, as well as the current equine vaccine, should be effective against this variant. Genetic similarity of this virus to sequences detected from grey-headed flying-foxes suggests the variant circulates at-least in this species. Studies determining infection kinetics, pathogenicity, reservoir-species associations, viral–host co-evolution and spillover dynamics for this virus are urgently needed. Surveillance and biosecurity practices should be updated to appreciate HeV spillover risk across all regions frequented by flying foxes. qPCR) (28). Clinical, epidemiological and sample-related data were recorded including vaccination status and 109 perceived exposure to flying-foxes as variably reported by submitting veterinarians. All samples were archived 110 at -80°C. We applied a decision algorithm based on pathogenic-basis and syndromic analysis of clinical disease 111 to categorize each case by likelihood of having an infectious viral cause (Appendix Table) . Samples (EDTA 112 blood, serum, nasal swab, rectal swab) from cases assigned the highest likelihood of having infectious cause 113 (priority categories 1 & 2) were plated for serological screening and high-throughput nucleic acid extraction 114 using the MagMAX™ mirVANA and CORE pathogen kits (ThermoFisher, Australia). Pan-paramyxovirus RT-PCR screening 116 cDNA was prepared from extracted RNA using Invitrogen SuperScript IV VILO mastermix with 117 ezDNase (ThermoFisher, Australia). A nested reverse transcriptase-PCR (RT-PCR) assay targeting the 118 paramyxovirus L protein gene was adapted using primers developed by Tong et al (36) and the Qiagen AllTaq 119 PCR Core kit (QIAGEN, Australia) . Amplicons corresponding to the expected size (584 bp) were identified by 120 gel electrophoresis before purification with AMPure XP (Beckman Coulter, Australia). To capture any weak 121 detections, pools were also prepared by equal volume mixing of all PCR products across plated rows. Next-122 generation sequencing was performed using an Illumina iSeq with the Nextera XT DNA library preparation kit 123 (Illumina, Australia). For analysis, reads were assembled with MEGAHIT (37) before identification by 124 comparison to NCBI GenBank with BLAST (38) . 125 HeV-var whole-genome sequencing 126 Samples positive by paramyxovirus RT-PCR for the novel HeV-var were subjected to meta-127 transcriptomic sequencing to determine the complete genome sequence and identify any co-infecting agents. 128 RNA was reverse-transcribed with Invitrogen SSIV VILO mastermix (ThermoFisher, Australia) and FastSelect Assembly and comparative genomic and phylogenetic analyses 133 For genome assembly, RNA sequencing reads were trimmed and mapped to a horse reference genome 134 (GenBank GCA_002863925.1) using STAR aligner to remove host sequences. The non-host reads were de novo assembled with MEGAHIT (37) and compared with the NCBI GenBank nucleotide and protein databases 136 using blastn and blastx (38). The putative virus contig was extracted and reads were remapped to this draft 137 genome using bbmap v37.98 (https://sourceforge.net/projects/bbmap) to examine sequence coverage and 138 identify misaligned reads. The majority consensus sequence was extracted, aligned and annotated by reference 139 to the prototype HeV strain using Geneious Prime v2021.1.1, and submitted to GenBank (accession number 140 MZ318101). For classification, the paramyxovirus polymerase (L) protein sequence was aligned according to 142 International Committee on Taxonomy of Viruses (ICTV) guidelines (39). We prepared alignments of partial 143 nucleocapsid (N) and phosphoprotein (P) nucleotide sequences with known HeV strains from the GenBank 144 database. Phylogenies were prepared using a maximum likelihood approach in MEGA X 145 (https://www.megasoftware.net/) according to the best-fit substitution model and 500 bootstrap replicates. An existing RT-qPCR assay targeting the HeV M gene (28) was adapted to target the novel HeV-var. The duplex assay used the Applied Biosystems AgPath-ID One-Step RT-PCR kit (ThermoFisher, Australia) and 149 distinguishes prototype and variant HeV strains. Briefly, 4 µL RNA was combined with 10 µL 2´ RT-PCR 150 Buffer, 0.8 µL 25´ RT-PCR Enzyme Mix, 2 µL nuclease-free water and 3.2 µL primer/probe mix (0.6 µL each 151 primer, 0.3 µL each probe from 10 µM stock; Table 1 ). The reaction was performed as follows: 10 min at 50°C 152 for cDNA synthesis, 10 min at 95°C for RT inactivation, and 50 cycles of 95°C for 15s and 60°C for 30s with 153 FAM and HEX channels captured at the end of each cycle. As positive control, gene fragments were Serological analysis was performed using multiplex microsphere immunoassays with a Luminex 163 MAGPIX TM system (ThermoFisher, Australia). Initial screening for IgG antibodies was undertaken using an 164 extensive panel of bacterial (Leptospira, Brucella) and viral antigens (paramyxovirus, filovirus, coronavirus, 165 flavivirus, alphavirus) coupled to MagPlex beads (Bio-Rad, Australia) for multiplex screening. Briefly, blood or serum, diluted 1:100, was added to the beads, with binding detected following the addition of a combination of 167 biotinylated-Protein-G and -A and streptavidin-R-phycoerythrin. The median fluorescence intensity (MFI) was 168 read on the MagPix (Luminex) targeting 100 beads per antigen with a Bayesian latent class model used to 169 assess test performance and determine appropriate cut-offs for positive test classification (32). An 170 immunoglobulin (Ig)M assay was also applied in which biotinylated anti-equine IgM antibodies were used in 171 place of biotinylated Protein A and G. In silico analysis of the RBP homology and mAb binding 173 The translated protein sequence of the HeV-var RBP sequence was compared with the X-ray crystal 174 structures of the HeV RBP protein structure bound to mAb m102.4 (41) and to ephrin-B2 using SWISS- Given the case's assigned high-likelihood of zoonotic infectious cause (Supp table 1), both the EDTA 197 blood and pooled swab samples were screened by pan-paramyxovirus . This identified the partial 198 polymerase sequence of a novel paramyxovirus, most closely related to HeV (»11% nucleotide difference). Deep sequencing of blood RNA generated the near-full length genome of a novel HeV (mean coverage of 200 46.9x) ( Figure 1A) . The virus was less abundant in the pooled swab sample, with a mean coverage depth of 201 0.6X reads spanning only 9.9% of the genome ( Figure 1B) . Importantly, no other viruses were present in either 202 sample, and other microbial reads assembled were from common microflora including Staphylococcus aureus, 203 Aeromonas, Veillonella, Pseudarthrobacter, Streptococcus, Acinetobacter and Psychrobacter species. Figure 2 ). An RT-qPCR assay was designed to detect both prototype and variant HeV strains in duplex (Table 208 1, Supplementary Figures 1, 2) , which amplified the templates of each virus with similarly high efficiency Phylogenetic analyses of the novel HeV-var was performed with other known paramyxoviruses ( Figure 224 4A-C). Comparison of the nucleotide similarity of the novel HeV-var to the HeV prototype strain (GenBank phylogeny revealed that the branch lengths of prototype and variant HeV to their common node did not exceed 227 0.03 substitutions/site ( Figure 4A , B), thus according to ICTV criteria the viruses are considered of the same 228 species (39). However, the HeV-var is clearly well outside known HeV diversity ( Figure 4C ). Following this finding, comparison with a partial novel Henipavirus M gene sequence derived from a 230 GHFF from South Australia in 2013 (46) revealed 99% similarity to this HeV-var. This, along with subsequent 231 further flying-fox detections (47), suggests that this HeV-var represents a previous undescribed lineage, with 232 reservoir-host infection across at-least the range of this flying-fox species. Genomic sequence showed greatest variability in the non-coding regions with mean pairwise genome 235 identity higher (86.9%) across coding regions ( Figure 4D ). At the protein level, this HeV-var shared between 236 82.3% and 95.7% amino acid identity (mean 92.5%) to the HeV prototype (Table 2) . Notably, the HeV-var RBP Comparison of the translated amino-acid sequence of this HeV-var and prototypic HeV RBP in silico 255 revealed the ephrin-B2 entry receptor binding site and that of mAb m102.4 to be unchanged. Similarly, m102.4 256 neutralization was confirmed in vitro with HeV-var as for HeV. As such, it is expected that current PEP 257 utilizing mAb m102.4 will remain effective against this HeV-var. It should be emphasized, that the HeV RBP shares only 79% amino acid identity to NiV RBP, yet the HeV-sG subunit vaccine provides 100% protection 259 against lethal challenge with both HeV and NiV in animal models (11). Both the higher similarity between the 260 HeV-var and HeV RBP (92.5% amino acid identity) and structural consistency of critical epitopes mentioned 261 above, suggest that current vaccination utilizing the HEV RBP will elicit similarly protective antibodies against 262 this HeV-var. Current serological assays based on the HeV RBP are not expected to distinguish between 263 exposure to the variants. The 99% similarity between this HeV-var and a partial M-gene sequence detected in a GHFF from 265 Adelaide in 2013, highlights that: a greater diversity of HeV circulates among Australian flying-fox species than 266 has been previously recognized; and that this novel variant likely circulates as a relatively consistent sub- A novel henipavirus in 475 bats, Australia Genotype Found in Australian Flying Foxes Latitudinal range shifts in Australian flying-foxes: 481 a re-evaluation Unexpected result of Hendra virus outbreaks for veterinarians Emerging Infectious Diseases Hendra Virus Interagency Technical Working Group. Hendra Virus Infection Prevention Advice 485 -Hendra Virus Interagency Technical Working Group incl Animal Health Australia. Australia Veterinary Emergency Plan AUSVETPLAN, Response 489 policy brief Hendra virus infection Version 4.0 Low infectious disease suspect Non-infectious etiologies more common or most likely on differential diagnosis list, but infectious cause still possible Ataxia following known traumatic event. Traumatic wounds following unusual behavioral event. Acute lethargy following chronic noninfectious disease condition Category 5 No clinical signs of illness or no infectious cause considered likely Traumatic wounds in the absence of underlying disease. Screening in unvaccinated horses to manage biosecurity risk prior to invasive procedures addressing non-infectious disease such as is a common requirement for dentistry or admission to equine hospitals in Australia Category 6 Other infectious disease confirmed via diagnostic testing A case submitted for HeV testing, found negative and then testing positive for alternative known infectious disease such as ABLV, WNV, EHV or RRV* *ABLV, Australian bat lyssavirus; EHV, Equine herpes virus; HeV, Hendra virus; RRV, Ross River Virus; WNV, West Nile virus