Figure 1

Figure 1. Timeline of events from inclusion of flying foxes in Queensland through to housing at ACDP from study of natural HeV infections in captive Australian black flying foxes, Queensland, Australia. Bats...

We included in this study flying foxes submitted to bat rehabilitators because of minor injuries that were otherwise healthy but nonreleasable. The cohort was selected from 39 bats housed in outdoor rehabilitation enclosures in Queensland, separate from other bats and screened by an in-house Luminex indirect antibody assay as described previously (5) (Appendix Table). We selected 20 bats with the lowest HeV serostatus and best overall condition for transport (12 seronegative, 8 low-positive). We charted the age and sex of the cohort (Table), which included 15 adults (6 female, 9 male), 4 subadults (2 female, 2 male), and 1 juvenile male bat. Tests revealed no viral RNA for known zoonotic viruses (HeVg1, HeV genotype 2 variant, Menangle virus, and Australian bat lyssavirus) in swab or urine specimens before transport, and HeV serostatus remained stable over 2 sampling events for up to 6 months in captivity in Queensland. Three weeks after the final Queensland testing (April 2023), we transported the animals to ACDP in Victoria (Figure 1).

Figure 2

Figure 2. HeV serology of 20 flying foxes following arrival at Australian Centre for Disease Preparedness (ACDP) for a study of natural Hendra virus infections in captive Australian black flying foxes, Queensland,...

Samples collected 1 day after arrival at ACDP revealed that 11 bats had seroconverted positive to HeV during the 3 weeks between testing in Queensland and transport, including 6 previously seronegative and 5 low-seropositive bats (Figure 2). Nine of the 11 bats that had seroconverted to HeV according to the in-house Luminex assay also had neutralizing antibodies to HeV, with virus neutralization test (VNT) titers ranging from 10 to 160 (Figure 2). Two bats tested PCR-positive for HeVg1 RNA: 1 (bat R1) on day 1 postarrival in oral and rectal swab specimens (cycle threshold [Ct] = 33.9) and urine (Ct = 29) and 1 (bat 4) on day 8 in urine (Ct = 25). Both bats had already seroconverted in the 3 weeks between Queensland testing and arrival at ACDP, with bat R1 increasing from low Luminex seropositive to a VNT titer of 160, and bat 4 from seronegative to VNT titer of 10 (Figure 2). Urine collected from underneath individual cages tested negative for known zoonotic viruses, except for those from bat 4, which were HeVg1 positive from days 9–14 at ACDP.

Figure 3

Figure 3. Histopathology of lung from a naturally infected flying fox from a study of natural Hendra virus infections in captive Australian black flying foxes, Queensland, Australia. Immunohistochemistry of bat 4, left...

Because of biosafety constraints, we collected no tissues from bat R1, which was housed in a Biosafety Level (BSL) 3 facility (BSL3Z). Bat 4, housed in a BSL-4 facility (BSL4), had HeVg1 RNA detected at necropsy on day 14 in 2 of 14 tissues, the submandibular lymph node (Ct = 39.2) and spleen (Ct = 38.2), and in urine (Ct = 33). Virus isolation was unsuccessful from urine collected on arrival (bats R1 and 4) and from tissues collected from bat 4 at necropsy. Histopathologic analysis of tissues from bat 4 revealed apparent congestion of blood vessels throughout the lungs, as well as the expansion of perivascular tissues by edema. We also noted mild to moderate lymphoid follicular hyperplasia in bat 4, throughout lymphoid organs, but most pronounced in the spleen and mesenteric lymph nodes. No microscopic changes were evident in any other tissues. Viral antigen was only evident in isolated mononuclear cells scattered throughout the pulmonary interstitium (Figure 3).

This study provides evidence of natural HeV infections and seroconversion in a captive cohort of P. alecto bats, including detection of active HeV infections in apparently naive bats and in those with evidence of previous infections. Viral replication in seropositive animals implies that flying foxes undergo cycles of infection and reinfection, and it is thus unlikely that prior infection leads to lifelong immunity in P. alecto bats. This finding contrasts with those for some other bat species, which appear to undergo higher rates of seroconversion with immunity to reinfection even after antibodies have waned (6,7).

The timing of infection—within 3 weeks before transport—is consistent with infection having occurred in Queensland, either via exposure to HeV excretions from a wild bat outside the enclosure or because of recrudescence followed by horizontal transmission within the colony. Although evidence is insufficient to determine whether viral recrudescence occurred in the bats we studied, increasing evidence suggests that henipaviruses may establish latency and persist in humans and bats (8–11). Experimentally infected P. alecto bats typically begin shedding virus in throat and rectal swab specimens 2–7 days postinfection and in urine 7–19 days postinfection. Experimentally, development of HeV-neutralizing antibody has been inconsistent, but reports have noted low levels detected by 10 days postinfection (12–14). Thus, we speculate that the 2 flying foxes shedding HeV after arrival were likely infected with HeV ≥1 week before transport. Bat R1, which tested PCR-positive in both oral/rectal swab and urine specimen, likely experienced an active HeV infection within 10 days before transport to ACDP and continued shedding HeV upon arrival. In contrast, bat 4 may have undergone an active infection in Queensland that resolved before transport. Because those 2 bats were housed in separate rooms at ACDP, it is possible that bat 4 was reinfected immediately before transport or underwent viral recrudescence at ACDP.

We detected HeV neutralizing antibodies in 9 of the 11 seroconverted flying foxes upon arrival at ACDP; 3 had titers of 80 and 2 titers of 160. Prior reports have revealed inconsistent antibody responses in flying foxes experimentally infected with HeV, with around 50% seroconverting with neutralizing titers of 10–80 by 10 days after oronasal inoculation (14,15). Given the unknown infection histories of bats in our study, higher titers may reflect anamnestic responses to HeV infection or could allude to differences between natural and experimental infections. Further studies will be required to determine the nature of antibody responses to infection and reinfections in flying foxes.

Our findings underscore the complexity of HeV maintenance in bat populations and highlight the need for further studies on immune dynamics, latency, and environmental drivers of recrudescence. Those insights are critical for understanding spillover risk and informing public health strategies.