Virulent strains of West Nile virus (WNV) are highly neuro-invasive and human infection is potentially lethal. However, no vaccine is currently available for human use. Here, we report the immunogenicity and protective efficacy of a vaccine derived from a chimeric virus, which was constructed using the structural proteins (prM and E) of the Kunjin strain of WNV (WNV_KUN) and the genome backbone of the insect-specific flavivirus Binjari virus (BinJV). This chimeric virus (BinJ/WNV_KUN-prME) exhibits an insect-specific phenotype and does not replicate in vertebrate cells. Importantly, it authentically presents the prM-E proteins of WNV_KUN, which is antigenically very similar to other WNV strains and lineages. Therefore BinJ/WNV_KUN-prME represents an excellent candidate to assess as a vaccine against virulent WNV strains, including the highly pathogenic WNV_NY99. When CD1 mice were immunized with purified BinJ/WNV_KUN-prME, they developed robust neutralizing antibody responses after a single unadjuvanted dose of 1 to 5 μg. We further demonstrated complete protection against viremia and mortality after lethal challenge with WNV_NY99, with no clinical or subclinical pathology observed in vaccinated animals. These data suggest that BinJ/WNV_KUN-prME represents a safe and effective WNV vaccine candidate that warrants further investigation for use in humans or in veterinary applications.

We recently developed a chimeric flavivirus vaccine technology based on the novel insect-specific Binjari virus (BinJV) and used this to generate a chimeric ZIKV vaccine (BinJ/ZIKA-prME) that protected IFNAR^-/- dams and fetuses from infection. Herein, we show that a single vaccination of IFNAR^-/- mice with unadjuvanted BinJ/ZIKA-prME generated neutralizing antibody responses that were retained for 14 months. At 15 months post vaccination, mice were also completely protected against detectable viremia and substantial body weight loss after challenge with ZIKV_PRVABC59. BinJ/ZIKA-prME vaccination thus provided long-term protective immunity without the need for adjuvant or replication of the vaccine in the vaccine recipient, both attractive features for a ZIKV vaccine.

Vector-borne flaviviruses are responsible for nearly half a billion human infections worldwide each year, resulting in millions of cases of debilitating and severe diseases and approximately 115,000 deaths. While approved vaccines are available for some of these viruses, the ongoing efficacy, safety and supply of these vaccines are still a significant problem. New technologies that address these issues and ideally allow for the safe and economical manufacture of vaccines in resource-poor countries where flavivirus vaccines are in most demand are urgently required. Preferably a new vaccine platform would be broadly applicable to all flavivirus diseases and provide new candidate vaccines for those diseases not yet covered, as well as the flexibility to rapidly pivot to respond to newly emerged flavivirus diseases. Here, we review studies conducted on novel chimeric vaccines derived from insect-specific flaviviruses that provide a potentially safe and simple system to produce highly effective vaccines against a broad spectrum of flavivirus diseases.

Insect-specific viruses (ISVs) include viruses that are restricted to the infection of mosquitoes and are spread mostly through transovarial transmission. Despite using a distinct mode of transmission, ISVs are often phylogenetically related to arthropod-borne viruses (arboviruses) that are responsible for human diseases and able to infect both mosquitoes and vertebrates. ISVs can also induce a phenomenon called “superinfection exclusion”, whereby a primary ISV infection in an insect inhibits subsequent viral infections of the insect. This has sparked interest in the use of ISVs for the control of pathogenic arboviruses transmitted by mosquitoes. In particular, insect-specific flaviviruses (ISFs) have been shown to inhibit infection of vertebrate-infecting flaviviruses (VIFs) both in vitro and in vivo. This has shown potential as a new and ecologically friendly biological approach to the control of arboviral disease. For this intervention to have lasting impacts for biological control, it is imperative that ISFs are maintained in mosquito populations with high rates of vertical transmission. Therefore, these strategies will need to optimise vertical transmission of ISFs in order to establish persistently infected mosquito lines for sustainable arbovirus control. This review compares recent observations of vertical transmission of arboviral and insect-specific flaviviruses and potential determinants of transovarial transmission rates to understand how the vertical transmission of ISFs may be optimised for effective arboviral control.

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 WNV_KUN (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 WNV_KUN. 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.

The Kunjin strain of West Nile virus (WNV_KUN) 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 WNV_KUN 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. WNV_KUN 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 WNV_KUN and MVEV transmission by FTA^TM 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 WNV_KUN or MVEV were obtained from mosquitoes collected on Darwin crocodile farms immediately following the FTA^TM card detections, we did isolate another flavivirus, Kokobera virus (KOKV), from Culex annulirostris mosquitoes. Our studies support the use of the FTA^TM card system as a sensitive and accurate method to monitor the transmission of WNV_KUN 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 FTA^TM 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.