Serological characterization of lineage II insect-specific flaviviruses compared with pathogenic mosquito-borne flaviviruses.

The genus Flavivirus includes pathogenic tick- and mosquito-borne flaviviruses as well as non-pathogenic insect-specific flaviviruses (ISFVs). Phylogenetic analysis based on whole amino acid sequences has indicated that lineage II ISFVs have similarities to pathogenic flaviviruses. In this study, we used reactive analysis with immune serum against Psorophora flavivirus (PSFV) as a lineage IIa ISFV, and Barkeji virus (BJV) as a lineage IIb ISFV, to evaluate the antigenic similarity among lineage IIa and IIb ISFVs, and pathogenic mosquito-borne flaviviruses (MBFVs). Binding and antibody-dependent enhancement assays showed that anti-PSFV sera had broad cross-reactivity with MBFV antigens, while anti-BJV sera had low cross-reactivity. Both of the lineage II ISFV antisera were rarely observed to neutralize MBFVs. These results suggest that lineage IIa ISFV PSFV has more antigenic similarity to MBFVs than lineage IIb ISFV BJV.

Improved detection of flaviviruses in Australian mosquito populations via replicative intermediates.

Mosquito-borne flaviviruses are significant contributors to the arboviral disease burdens both in Australia and globally. While routine arbovirus surveillance remains a vital exercise to identify known flaviviruses in mosquito populations, novel or divergent and emerging species can be missed by these traditional methods. The MAVRIC (monoclonal antibodies to viral RNA intermediates in cells) system is an ELISA-based method for broad-spectrum isolation of positive-sense and double-stranded RNA (dsRNA) viruses based on detection of dsRNA in infected cells. While the MAVRIC ELISA has successfully been used to detect known and novel flaviviruses in Australian mosquitoes, we previously reported that dsRNA could not be detected in dengue virus-infected cells using this method. In this study we identified additional flaviviruses which evade detection of dsRNA by the MAVRIC ELISA. Utilising chimeric flaviviruses we demonstrated that this outcome may be dictated by the non-structural proteins and/or untranslated regions of the flaviviral genome. In addition, we report a modified fixation method that enables improved detection of flavivirus dsRNA and inactivation of non-enveloped viruses from mosquito populations using the MAVRIC system. This study demonstrates the utility of anti-dsRNA monoclonal antibodies for identifying viral replication in insect and vertebrate cell systems and highlights a unique characteristic of flavivirus replication.

Novel monoclonal antibodies against Australian strains of negeviruses and insights into virus structure, replication and host -restriction.

We report the isolation of Australian strains of Bustos virus and Ngewotan virus, two insect-specific viruses in the newly identified taxon Negevirus, originally isolated from Southeast Asian mosquitoes. Consistent with the expected insect-specific tropism of negeviruses, these isolates of Ngewotan and Bustos viruses, alongside the Australian negevirus Castlerea virus, replicated exclusively in mosquito cells but not in vertebrate cells, even when their temperature was reduced to 34 °C. Our data confirmed the existence of two structural proteins, putatively one membrane protein forming the majority of the virus particle, and one glycoprotein forming a projection on the apex of the virions. We generated and characterized 71 monoclonal antibodies to both structural proteins of the two viruses, most of which were neutralizing. Overall, these data increase our knowledge of negevirus mechanisms of infection and replication in vitro.

New genotypes of Liao ning virus (LNV) in Australia exhibit an insect-specific phenotype.

Liao ning virus (LNV) was first isolated in 1996 from mosquitoes in China, and has been shown to replicate in selected mammalian cell lines and to cause lethal haemorrhagic disease in experimentally infected mice. The first detection of LNV in Australia was by deep sequencing of mosquito homogenates. We subsequently isolated LNV from mosquitoes of four genera (Culex, Anopheles, Mansonia and Aedes) in New South Wales, Northern Territory, Queensland and Western Australia; the earliest of these Australian isolates were obtained from mosquitoes collected in 1988, predating the first Chinese isolates. Genetic analysis revealed that the Australian LNV isolates formed two new genotypes: one including isolates from eastern and northern Australia, and the second comprising isolates from the south-western corner of the continent. In contrast to findings reported for the Chinese LNV isolates, the Australian LNV isolates did not replicate in vertebrate cells in vitro or in vivo, or produce signs of disease in wild-type or immunodeficient mice. A panel of human and animal sera collected from regions where the virus was found in high prevalence also showed no evidence of LNV-specific antibodies. Furthermore, high rates of virus detection in progeny reared from infected adult female mosquitoes, coupled with visualization of the virus within the ovarian follicles by immunohistochemistry, suggest that LNV is transmitted transovarially. Thus, despite relatively minor genomic differences between Chinese and Australian LNV strains, the latter display a characteristic insect-specific phenotype.

Unleashing Nature's Allies: Comparing the Vertical Transmission Dynamics of Insect-Specific and Vertebrate-Infecting Flaviviruses in Mosquitoes.

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.

A chimeric vaccine derived from Australian genotype IV Japanese encephalitis virus protects mice from lethal challenge.

In 2022, a genotype IV (GIV) strain of Japanese encephalitis virus (JEV) caused an unprecedented and widespread outbreak of disease in pigs and humans in Australia. As no veterinary vaccines against JEV are approved in Australia and all current approved human and veterinary vaccines are derived from genotype (G) III JEV strains, we used the recently described insect-specific Binjari virus (BinJV) chimeric flavivirus vaccine technology to produce a JEV GIV vaccine candidate. Herein we describe the production of a chimeric virus displaying the structural prM and E proteins of a JEV GIV isolate obtained from a stillborn piglet (JEVNSW/22) in the genomic backbone of BinJV (BinJ/JEVNSW/22-prME). BinJ/JEVNSW/22-prME was shown to be antigenically indistinguishable from the JEVNSW/22 parental virus by KD analysis and a panel of JEV-reactive monoclonal antibodies in ELISA. BinJ/JEVNSW/22-prME replicated efficiently in C6/36 cells, reaching titres of >107 infectious units/mL - an important attribute for vaccine manufacture. As expected, BinJ/JEVNSW/22-prME failed to replicate in a variety of vertebrate cells lines. When used to immunise mice, the vaccine induced a potent virus neutralising response against JEVNSW/22 and to GII and GIII JEV strains. The BinJ/JEVNSW/22-prME vaccine provided complete protection against lethal challenge with JEVNSW/22, whilst also providing partial protection against viraemia and disease for the related Murray Valley encephalitis virus. Our results demonstrate that BinJ/JEVNSW/22-prME is a promising vaccine candidate against JEV.

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.

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.

The Chimeric Binjari-Zika Vaccine Provides Long-Term Protection against ZIKA Virus Challenge.

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 ZIKVPRVABC59. 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.

Structural analysis of 3'UTRs in insect flaviviruses reveals novel determinants of sfRNA biogenesis and provides new insights into flavivirus evolution.

Subgenomic flaviviral RNAs (sfRNAs) are virus-derived noncoding RNAs produced by pathogenic mosquito-borne flaviviruses (MBF) to counteract the host antiviral response. To date, the ability of non-pathogenic flaviviruses to produce and utilise sfRNAs remains largely unexplored, and it is unclear what role XRN1 resistance plays in flavivirus evolution and host adaptation. Herein the production of sfRNAs by several insect-specific flaviviruses (ISFs) that replicate exclusively in mosquitoes is shown, and the secondary structures of their complete 3'UTRs are determined. The xrRNAs responsible for the biogenesis of ISF sfRNAs are also identified, and the role of these sfRNAs in virus replication is demonstrated. We demonstrate that 3'UTRs of all classical ISFs, except Anopheles spp-asscoaited viruses, and of the dual-host associated ISF Binjari virus contain duplicated xrRNAs. We also reveal novel structural elements in the 3'UTRs of dual host-associated and Anopheles-associated classical ISFs. Structure-based phylogenetic analysis demonstrates that xrRNAs identified in Anopheles spp-associated ISF are likely ancestral to xrRNAs of ISFs and MBFs. In addition, our data provide evidence that duplicated xrRNAs are selected in the evolution of flaviviruses to provide functional redundancy, which preserves the production of sfRNAs if one of the structures is disabled by mutations or misfolding.