A chimeric vaccine protects farmed saltwater crocodiles from West Nile virus-induced skin lesions.

West Nile virus (WNV) causes skin lesions in farmed crocodiles leading to the depreciation of the value of their hides and significant economic losses. However, there is no commercially available vaccine designed for use in crocodilians against WNV. We tested chimeric virus vaccines composed of the non-structural genes of the insect-specific flavivirus Binjari virus (BinJV) and genes encoding the structural proteins of WNV. The BinJV/WNV chimera, is antigenically similar to wild-type WNV but replication-defective in vertebrates. Intramuscular injection of two doses of BinJV/WNV in hatchling saltwater crocodiles (Crocodylus porosus) elicited a robust neutralising antibody response and conferred protection against viremia and skin lesions after challenge with WNV. In contrast, mock-vaccinated crocodiles became viraemic and 22.2% exhibited WNV-induced lesions. This suggests that the BinJV/WNV chimera is a safe and efficacious vaccine for preventing WNV-induced skin lesions in farmed crocodilians.

Nucleic Acid Preservation Card Surveillance Is Effective for Monitoring Arbovirus Transmission on Crocodile Farms and Provides a One Health Benefit to Northern Australia.

The Kunjin strain of West Nile virus (WNVKUN) is a mosquito-transmitted flavivirus that can infect farmed saltwater crocodiles in Australia and cause skin lesions that devalue the hides of harvested animals. We implemented a surveillance system using honey-baited nucleic acid preservation cards to monitor WNVKUN and another endemic flavivirus pathogen, Murray Valley encephalitis virus (MVEV), on crocodile farms in northern Australia. The traps were set between February 2018 and July 2020 on three crocodile farms in Darwin (Northern Territory) and one in Cairns (North Queensland) at fortnightly intervals with reduced trapping during the winter months. WNVKUN RNA was detected on all three crocodile farms near Darwin, predominantly between March and May of each year. Two of the NT crocodile farms also yielded the detection of MVE viral RNA sporadically spread between April and November in 2018 and 2020. In contrast, no viral RNA was detected on crocodile farms in Cairns during the entire trapping period. The detection of WNVKUN and MVEV transmission by FTATM cards on farms in the Northern Territory generally correlated with the detection of their transmission to sentinel chicken flocks in nearby localities around Darwin as part of a separate public health surveillance program. While no isolates of WNVKUN or MVEV were obtained from mosquitoes collected on Darwin crocodile farms immediately following the FTATM card detections, we did isolate another flavivirus, Kokobera virus (KOKV), from Culex annulirostris mosquitoes. Our studies support the use of the FTATM card system as a sensitive and accurate method to monitor the transmission of WNVKUN and other arboviruses on crocodile farms to enable the timely implementation of mosquito control measures. Our detection of MVEV transmission and isolation of KOKV from mosquitoes also warrants further investigation of their potential role in causing diseases in crocodiles and highlights a "One Health" issue concerning arbovirus transmission to crocodile farm workers. In this context, the introduction of FTATM cards onto crocodile farms appears to provide an additional surveillance tool to detect arbovirus transmission in the Darwin region, allowing for a more timely intervention of vector control by relevant authorities.

Evidence of Infection with Zoonotic Mosquito-Borne Flaviviruses in Saltwater Crocodiles (Crocodylus porosus) in Northern Australia.

The risk of flavivirus infections among the crocodilian species was not recognised until West Nile virus (WNV) was introduced into the Americas. The first outbreaks caused death and substantial economic losses in the alligator farming industry. Several other WNV disease episodes have been reported in crocodilians in other parts of the world, including Australia and Africa. Considering that WNV shares vectors with other flaviviruses, crocodilians are highly likely to also be exposed to flaviviruses other than WNV. A serological survey for flaviviral infections was conducted on saltwater crocodiles (Crocodylus porosus) at farms in the Northern Territory, Australia. Five hundred serum samples, collected from three crocodile farms, were screened using a pan-flavivirus-specific blocking ELISA. The screening revealed that 26% (n = 130/500) of the animals had antibodies to flaviviruses. Of these, 31.5% had neutralising antibodies to WNVKUN (Kunjin strain), while 1.5% had neutralising antibodies to another important flavivirus pathogen, Murray Valley encephalitis virus (MVEV). Of the other flaviviruses tested for, Fitzroy River virus (FRV) was the most frequent (58.5%) in which virus neutralising antibodies were detected. Our data indicate that farmed crocodiles in the Northern Territory are exposed to a range of potentially zoonotic flaviviruses, in addition to WNVKUN. While these flaviviruses do not cause any known diseases in crocodiles, there is a need to investigate whether infected saltwater crocodiles can develop a viremia to sustain the transmission cycle or farmed crocodilians can be used as sentinels to monitor the dynamics of arboviral infections in tropical areas.

Mosquito-Independent Transmission of West Nile virus in Farmed Saltwater Crocodiles (Crocodylus porosus).

West Nile virus, Kunjin strain (WNVKUN) is endemic in Northern Australia, but rarely causes clinical disease in humans and horses. Recently, WNVKUN genomic material was detected in cutaneous lesions of farmed saltwater crocodiles (Crocodylus porosus), but live virus could not be isolated, begging the question of the pathogenesis of these lesions. Crocodile hatchlings were experimentally infected with either 105 (n = 10) or 104 (n = 11) TCID50-doses of WNVKUN and each group co-housed with six uninfected hatchlings in a mosquito-free facility. Seven hatchlings were mock-infected and housed separately. Each crocodile was rotationally examined and blood-sampled every third day over a 3-week period. Eleven animals, including three crocodiles developing typical skin lesions, were culled and sampled 21 days post-infection (dpi). The remaining hatchlings were blood-sampled fortnightly until experimental endpoint 87 dpi. All hatchlings remained free of overt clinical disease, apart from skin lesions, throughout the experiment. Viremia was detected by qRT-PCR in infected animals during 2-17 dpi and in-contact animals 11-21 dpi, indicating horizontal mosquito-independent transmission. Detection of viral genome in tank-water as well as oral and cloacal swabs, collected on multiple days, suggests that shedding into pen-water and subsequent mucosal infection is the most likely route. All inoculated animals and some in-contact animals developed virus-neutralizing antibodies detectable from 17 dpi. Virus-neutralizing antibody titers continued to increase in exposed animals until the experimental endpoint, suggestive of persisting viral antigen. However, no viral antigen was detected by immunohistochemistry in any tissue sample, including from skin and intestine. While this study confirmed that infection of saltwater crocodiles with WNVKUN was associated with the formation of skin lesions, we were unable to elucidate the pathogenesis of these lesions or the nidus of viral persistence. Our results nevertheless suggest that prevention of WNVKUN infection and induction of skin lesions in farmed crocodiles may require management of both mosquito-borne and water-borne viral transmission in addition to vaccination strategies.

Crocodilepox Virus Evolutionary Genomics Supports Observed Poxvirus Infection Dynamics on Saltwater Crocodile (Crocodylus porosus).

Saltwater crocodilepox virus (SwCRV), belonging to the genus Crocodylidpoxvirus, are large DNA viruses posing an economic risk to Australian saltwater crocodile (Crocodylus porosus) farms by extending production times. Although poxvirus-like particles and sequences have been confirmed, their infection dynamics, inter-farm genetic variability and evolutionary relationships remain largely unknown. In this study, a poxvirus infection dynamics study was conducted on two C. porosus farms. One farm (Farm 2) showed twice the infection rate, and more concerningly, an increase in the number of early- to late-stage poxvirus lesions as crocodiles approached harvest size, reflecting the extended production periods observed on this farm. To determine if there was a genetic basis for this difference, 14 complete SwCRV genomes were isolated from lesions sourced from five Australian farms. They encompassed all the conserved genes when compared to the two previously reported SwCRV genomes and fell within three major clades. Farm 2's SwCRV sequences were distributed across all three clades, highlighting the likely mode of inter-farm transmission. Twenty-four recombination events were detected, with one recombination event resulting in consistent fragmentation of the P4c gene in the majority of the Farm 2 SwCRV isolates. Further investigation into the evolution of poxvirus infection in farmed crocodiles may offer valuable insights in evolution of this viral family and afford the opportunity to obtain crucial information into natural viral selection processes in an in vivo setting.