Novel Hendra Virus Variant Circulating in Black Flying Foxes and Grey-Headed Flying Foxes, Australia

A novel Hendra virus variant, genotype 2, was recently discovered in a horse that died after acute illness and in Pteropus flying fox tissues in Australia. We detected the variant in flying fox urine, the pathway relevant for spillover, supporting an expanded geographic range of Hendra virus risk to horses and humans.


The Study
We collected pooled urine samples from plastic sheets placed underneath flying fox roosts in southeastern Queensland and mid-to north-coast NSW during December 2016-September 2020 (Figure). We placed sheets in areas of the roost where P. alecto flying foxes were roosting, although other species were often also present. We recorded the number and species of bats immediately above the sheets. We also captured individual bats in mist nests; recorded species, sex, and age class; then collected urine samples directly from each anaesthetised bat or from a urine collection bag attached to its holding bag. Shortly after collection, we placed samples into viral lysis buffer, virus transport media, or an empty cryovial and stored them at −80°C (Appendix, https://wwwnc.cdc.gov/EID/ article/28/5/21-2338-App1.pdf).
We used the QIAamp Viral RNA Kit using a QIAcube HT automated system (QIAGEN, https:// www.qiagen.com) to extract RNA, then eluted it in 150 µL of TE buffer and first screened it for HeV-g1 using a qRT-PCR assay targeting the P gene (Table 1). We stored extracted RNA at −80°C and then screened it for HeV-g2 using the new multiplexed qRT-PCR assay, targeting the M gene with primers specific for HeV-g1 and HeV-g2 (2,3) (Table 1; Appendix). We used 10-fold dilutions with a known number of genome copies to construct a standard curve, calculate copy numbers/mL, and estimate limit of detection. We amplified the partial cytochrome b gene from all positive samples (10,11) (Table 1) and confirmed host species identity based on sequence identity across 402-bp sequences (Appendix).
We screened 4,539 pooled urine samples collected from 129 underroost sampling sessions and 1,674 urine samples collected from individual bats over 39 catching sessions during July 2017-September 2020 (Appendix Tables 1, 2). Eight pooled urine samples and 2 samples from individual flying foxes tested positive for HeV-g2 (Table 2). Positive samples were from Sunnybank in Queensland and Clunes, Lismore, Dorroughby, Maclean, and Nambucca Heads in NSW.
Individual flying foxes that tested positive included a P. poliocephalus juvenile female captured in 1044 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 28, No. 5, May 2022 Maclean, NSW, and a P. alecto adult male captured in Clunes, NSW (Appendix Table 3). We detected HeV-g2 in pooled samples from mixed-species roosts containing P. alecto and P. poliocephalus flying foxes. Cytochrome b sequencing identified DNA from P. alecto flying foxes in 6/8 positive underroost samples and from P. poliocephalus flying foxes in 2/8 ( Table 2).

Conclusions
Urine is the route of HeV excretion from flying foxes and the source of virus transmission to horses. Detecting the novel Hendra variant HeV-g2 in the urine of flying foxes helped identify its distribution range, associated host species, transmission dynamics, and spillover risk. We show evidence that P. alecto and P. poliocephalus flying foxes excrete HeV-g2 in urine and both are likely competent reservoir hosts. We did not screen urine samples from P. conspicillatus or P. scapulatus flying foxes, so the potential of these species to excrete HeV-g2 in urine remains unconfirmed. Although HeV-g1 has been detected in flying fox urine samples collected across all seasons, prevalence peaks in winter in subtropical regions (4,12), which is consistent with our preliminary HeV-g2 seasonality findings (5/8 detections in late May-late August) in the study area. The significantly lower prevalence of HeV-g2 than HeV-g1 could indicate actual lower prevalence in the sampled population. Alternatively, repeated freeze-thaw cycles in our samples or the bias toward collecting P. alecto urine in our sampling design might have led to lower detection. Tissue samples from flying foxes submitted for lyssavirus  †HeV-g2 viral copies/mL: the minimum copy number which would be expected to reliably give a positive PCR result in all replicates in the quantitative reverse transcription PCR assay (the limit of detection) was 5-10 copies per reaction (1,070-2,140 copies/mL).
testing after contact with humans or pets showed higher HeV-g2 prevalence than our samples from wild populations (7), which might reflect higher prevalence in sick or stressed bats or geographical differences. HeV-g2 was previously detected in tissue samples from South Australia (3 positives from 4 samples), Victoria (7/64), and Western Australia (1/2) (7). Our findings extend the known distributional range of HeV-g2 to southeastern Queensland and mid-to north-coast NSW, areas proximate to the 2 known cases of HeV-g2 spillover to horses (3,5). Our findings support expanding the expected geographic risk area for HeV spillover to include the distribution of P. poliocephalus flying foxes. Screening flying fox urine samples from a broader geographic range, including regions where P. alecto flying foxes are absent, should better inform epidemiologic relationships and relative prevalence of HeV variants. Given that data on the true diversity of HeV and related viruses in flying fox populations are incomplete, unbiased or Paramyxoviridae family-level viral surveillance in reservoir and spillover hosts might identify further variants. Developing a panel of diagnostic tools to detect a more comprehensive range of the viruses capable of spillover would substantially advance our ability to forecast spillover risk, manage biosecurity, and provide guidance to horse owners, veterinarians, and other stakeholders.  Table 1).
To address host species associations, we selected samples attributed to either P. alecto or P.
poliocephalus. Initially, we screened all available samples collected from individual bats where the species was identified in the field (674 urine samples collected from individual bats over 39 catching sessions during August 2017-September 2020, Appendix Table 2). Because the number of samples from captured bats was biased towards P. alecto, we included an additional 217 under-roost samples from 2 sessions with high proportions of P. poliocephalus (Maclean and Stewarts Brook, Appendix Table 1).

Cytochrome b Sequencing for Species Identification
Partial cytochrome b gene was amplified by PCR from all positive samples using previously described primers validated for species identification ( https://www.mn-net.com). PCR products were Sanger sequenced (ACGT) and species confirmed based on >98% sequence identity across 402 bp length sequences. Cytochrome b sequences are listed in Appendix Table 3 below.