Jasmin Moran’s scientific contributions

Laboratory-based diagnostics such as plaque reduction neutralisation tests (PRNT) and ELISA are commonly used to detect seroconversion to flavivirus infections. However, faster, qualitative screening methods are essential for quicker diagnosis and improved patient outcomes. Lateral flow assays (LFAs) offer rapid results (5-15 mins) at the point-of-care, but few commercial flavivirus antibody detection LFAs are available. We developed an LFA using novel chimeric viral antigens produced by genetically modifying the mosquito restricted Binjari virus (BinJV) to display the outer virion proteins of pathogenic viruses like West Nile virus (WNV). The BinJV chimeric platform offers several advantages for diagnostic assay development, including rapid construction of new chimeras in response to emerging viral variants, safe, scalable antigen manufacturing, and structural indistinguishability to the wild-type pathogenic virion. To demonstrate feasibility, we applied the chimeric WNV (BinJV/WNV) antigen to LFA as the capture/test line reagent for detecting seroconversion in crocodilians to WNV-a virus affecting crocodilians across multiple continents. We confirmed the antigenic conservation of the chimera on the LFA detection surface using mono-clonal antibodies. Utilising well-characterised sera (n=60) from WNV-seropositive or flavivirus-naive Australian saltwater crocodiles (Crocodylus porosus), the assay exhibited 98.8 % sensitivity and 100 % specificity, with results obtained in under 15 minutes. The LFA also accurately detected seroconversion in animals experimentally infected with WNV. This qualitative screening method can be performed both inside and outside of a laboratory, and the assay design will guide the optimisation of similar tests for detecting vector-borne viral infections in humans and other animals.

Laboratory-based diagnostics like plaque reduction neutralization tests (PRNT) and ELISA are commonly used to detect seroconversion to flavivirus infections. However, faster, qualitative screening methods are needed for quicker diagnosis and better patient outcomes. Lateral flow assays (LFAs) can provide rapid results (5-15 mins) at the point-of-care, yet few commercial flavivirus antibody detection LFAs are available. We developed an LFA using novel chimeric viral antigens produced by genetically modifying the mosquito restricted Binjari virus (BinJV) to display the outer virion proteins of pathogenic viruses such as West Nile virus (WNV). The BinJV chimeric platform offers various advantages for diagnostic assay development, including rapid construction of new chimeras in response to emerging viral variants, safe, scalable antigen manufacturing, and structural indistinguishability to the wild-type pathogenic virion. As a demonstration of feasibility, we applied chimeric WNV (BinJV/WNV) antigen to LFA as the capture/test line reagent for detection of seroconversion of crocodilians to WNV – a virus affecting crocodilians on multiple continents. We verified the antigenic conservation of the chimera when applied to the LFA detection surface using monoclonal antibodies. Using well-characterised sera (n=60) from WNV seropositive or flavivirus naive Australian saltwater crocodiles ( Crocodylus porosus ), we illustrated 100% sensitivity and specificity, with results achieved in less than 15 minutes. The LFA further accurately detected seroconversion in animals experimentally infected with WNV. This qualitative screening method can be performed both inside and outside of a laboratory, and the assay design will guide the optimization of similar tests for vector borne virus infection detection in both humans and other animals.

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.

West Nile virus (WNV) causes skin lesions in farmed crocodiles leading to 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. Subcutaneous application 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 preventing WNV-induced skin lesions in farmed crocodilians. This is the first report of a vaccine that protects reptiles against viral infection.

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.

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.

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.

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.vir

Background The global crocodilian skin market is currently in oversupply. As a result, the tanneries can now be very selective about the quality of skins they purchase. The challenge to producers is to meet these quality standards. A part of this challenge is to understand the risk of pathogens to crocodile skin quality. Aims/objectives Collectively, the aims of this project were to understand crocodile skin microflora and to better understand the threats to skin quality. Methods used To characterise skin microflora, 16SrRNA tag sequencing was used to compare wild and captive skins, whilst the identification of blemish causation was achieved using specific genetic sequences to screen for particular organisms. A survey of crocodile skins allowed the infection dynamics of poxvirus to be understood. Results/key findings Dermatophilus sp infection is not isolated to focal lesions as first thought. Dermatophilus sp. is also implicated, at high reads, in linear lesions. It continues to be problematic in focal lesions but can be more subtle than when first described as “brown spot”. Dermatophilus sp can be easily confused with early active poxvirus lesions. Wild saltwater crocodile have a substantially lower presence of Dermatophilus sp. suggesting that intensification has allowed it to develop an ecological niche in farm production settings. While Dermatophilus sp. and poxvirus were identified from skin lesions with high prevalence, the Crocodyline herpesvirus and the Kunjin strain of West Nile virus were also identified. The prevalence of poxvirus is particularly high in grower pens but the infection dynamics on the two farms studied are contrary to each other. On one farm, poxvirus prevalence decreases as the animals approach finishing size and therefore has relatively little impact on production time. However, on the other farm, the number of lesions, particularly early stage lesions, increases and delays harvest times thus increasing production costs. Implications and Recommendations Developing quantitative genetic methods to detect these pathogens and assess different hygiene regimes is essential. This will allow producers to quantitatively assess the efficacy of their current regimes and make appropriate decisions. Reducing the impact of these pathogens will be crucial to delivering the skin quality demanded.