A new genotype of HeV has been identified in flying foxes in Australia. This follows the first detection of HeV-G1 in Australian flying foxes in 1996 (8), and Cedar virus in 2012 (2). Histopathology, including IHC, indicated that this virus replicates within blood vessels and is able to cause significant vascular disease in flying foxes. Molecular characterisation of the HeV-G2 demonstrated highest genetic similarity to HeV-G1, when compared to other members of the genus. Sequence variation was observed within HeV-G2 despite high sequence similarity (> 99% at genome level) between each other. This is similar to the findings from HeV (G1) in flying foxes and horses (18; 19). Due to the high genetic similarity between HeV-G1 and HeV-G2 it is highly possible that HeV-G2 is a zoonotic pathogen posing potential risk to other species, including horses and humans. To date, there has been no report of spill-over events of HeV-G2 in the areas where the virus has been detected in flying foxes. However, the recent retrospective identification of a virus belonging to this sub-lineage from a 2015 case of equine neurological disease in Queensland shows the HeV-G2 is able to infect horses and potentially cause disease (Annand et al. submitted 2021). Further studies on epidemiology and pathogenicity of HeV-G2 are warranted.
In this study, 98 bats were tested by PCR and HeV-G2 was detected in 11, with a detection rate of 11.2%. In a previous study (20), 2840 individual black flying-foxes were tested by qRT-PCR, and HeV was detected in 43 animals with 1.48% prevalence of detection. This is significantly lower than that for HeV-G2 in the current study, however in Edson’s study (20) the sample type was not tissues; they sampled urine and swabs suggesting that those samples have a lower sensitivity for the detection of virus. Additionally, the sensitivity and specificity of the assay used in Edson’s study may not have been optimal for the detection of different henipaviruses, and they were sampling healthy bats in the wild.
Ten of the eleven bats that tested positive for HeV-G2 were GHFF (Table 3). In another study by Edson et al. (21), HeV was not detected in 1168 GHFF. Once again, the sample types were different; they were not tissues, but urine, packed blood cells, serum and swabs. This, along with other studies where HeV was either not found or difficult to find in excreted samples from GHFF (22–26) strongly suggests that excretion from GHFF is significantly lower than in BFF. However, it is still unclear whether infection of HeV-G2 occurs in BFF, and what level of prevalence exists, as only very limited numbers (n = 3) of BFF were tested in this study. It is interesting that one of three LRFF from Western Australia tested positive for HeV-G2. However, this was at the limit of detection and could not be confirmed by sequencing. Additional surveillance in these populations is needed.
Hendra and Nipah virus are serologically cross-protective (10). Although serologic evidence of HeV infection has been found in all four flying foxes species in Australia, including the spectacled flying-fox, BFF, LRFF and GHFF (27), the discovery of HeV-G2 raises the question of which variant the flying foxes have been exposed to – HeV-G1, HeV-G2 and/or other variants. Current serological tests would not be able to differentiate between the different variants.
Recent epidemiological studies have suggested that only the BFF and spectacled flying-fox, are the primary reservoir hosts for HeV (20; 21; 28–30). Neither GHFF nor LRFF have been identified as the principal source of HeV in spill-over events to horses, despite reports of high levels of seroprevalence to HeV in these species, and the isolation of virus from GHFF (8; 23; 31). It is notable that in this study HeV-G2 appears to have a broad geographic distribution, in areas of Australia that were previously deemed as low risk for HeV spill-over events.
Co-infection with HeV-G2 and ABLV was detected in one GHFF. This is the first time that this has been reported. Virus co-infection in flying foxes has not been studied extensively but is known to occur. While mean viral prevalence was low, multi-viral shedding from flying fox populations was common in one report, with up to eight paramyxoviruses, which included HeV-G1, detected in one mixed colony (containing BFF and GHFF) at a single point in time, and referred to as a ‘synchronous shedding pulse’ (32).
A qRT-PCR assay targeting the HeV N gene was used during initial detection of the HeV-G2 from GHFF from South Australia in 2013, in addition to the HeV-G1-specific M gene-specific assay that failed to detect HeV-G2. This demonstrates the benefit of using diagnostic qRT-CR tests that target multiple gene for disease investigations to address circulating genetic variants. Subsequently, a new qRT-PCR assay based on the HeV-G2 M gene was developed and applied to the screening of samples from flying foxes. This assay is more sensitive than the HeV N gene assay for detection of HeV-G2, and it is also able to detect HeV belonging to the prototype lineage. The broad reactivity of this assay could potentially be beneficial to the detection of mutant/variant forms of HeV in the future. Through obtaining more sequences of HeV-G2 from GHFFs during this study, we developed an additional HeV-G2 specific qRT-PCR assay targeting the N gene. This assay is specific for HeV-G2 and is used as the confirmatory test in our laboratory. Preliminary validation of both assays using bat samples submitted for disease exclusion, equine samples from previous outbreaks and NiV isolates indicated that these tests perform with high levels of sensitivity and specificity. Further assessment of these tests will be required for full validation according to World Organisation for Animal Health principles for veterinary diagnostic tests (33).
As it is likely that HeV-G2 can spill over from flying foxes to other species, this novel virus is a zoonotic pathogen that threatens animal and human health. These new assays enhance diagnostic capability through rapid and specific detection of HeV-G2. The addition of capability for detection of HeV-G2 to current laboratory diagnostic testing algorithms for the diagnosis of HeV infections in animals of different species, particularly horses, is of critical importance. An updated testing protocol to incorporate these tests has been applied at ACDP.
In the present study, virus isolation was attempted using the continuous Vero cell line and primary Pteropus alecto (kidney) cell culture. HeV-G2 was unable to be isolated from selected PCR positive GHFF samples. Previously, HeV has been isolated from flying fox urine using these cell cultures, with equal success in either Vero cells or primary bat cell lines (19). The unsuccessful attempts for isolation of HeV-G2 may be due to non-viability of the virus in the samples. These samples were collected either from carcasses exposed to ambient temperature for unknown periods, or euthanised and stored under suboptimal conditions for the preservation of live virus. The apparent low virus load in some of the samples, as indicated by qRT-PCR results, may have also been a contributing factor.
The finding of viral antigen within an artery of the heart in association with a prominent mononuclear cell inflammation indicates that HeV is able to cause vasculitis. This is the first time, to our knowledge, that a henipavirus has been demonstrated to cause significant pathology in a flying fox. The lesion was localised and limited in extent and therefore is unlikely to have caused serious illness in that individual. Nevertheless, this finding challenges the notion that henipavirus infections in flying boxes do not cause disease. Further investigation on the ability of these viruses to cause disease and illness in flying foxes is warranted.