Xinyang flavivirus, from Haemaphysalis flava ticks in Henan Province, China, defines a basal, likely tick-only Orthoflavivirus clade.

Tick-borne orthoflaviviruses (TBFs) are classified into three conventional groups based on genetics and ecology: mammalian, seabird and probable-TBF group. Recently, a fourth basal group has been identified in Rhipicephalus ticks from Africa: Mpulungu flavivirus (MPFV) in Zambia and Ngoye virus (NGOV) in Senegal. Despite attempts, isolating these viruses in vertebrate and invertebrate cell lines or intracerebral injection of newborn mice with virus-containing homogenates has remained unsuccessful. In this study, we report the discovery of Xinyang flavivirus (XiFV) in Haemaphysalis flava ticks from Xìnyáng, Henan Province, China. Phylogenetic analysis shows that XiFV was most closely related to MPFV and NGOV, marking the first identification of this tick orthoflavivirus group in Asia. We developed a reverse transcriptase quantitative PCR assay to screen wild-collected ticks and egg clutches, with absolute infection rates of 20.75 % in adult females and 15.19 % in egg clutches, suggesting that XiFV could be potentially spread through transovarial transmission. To examine potential host range, dinucleotide composition analyses revealed that XiFV, MPFV and NGOV share a closer composition to classical insect-specific orthoflaviviruses than to vertebrate-infecting TBFs, suggesting that XiFV could be a tick-only orthoflavivirus. Additionally, both XiFV and MPFV lack a furin cleavage site in the prM protein, unlike other TBFs, suggesting these viruses might exist towards a biased immature particle state. To examine this, chimeric Binjari virus with XIFV-prME (bXiFV) was generated, purified and analysed by SDS-PAGE and negative-stain transmission electron microscopy, suggesting prototypical orthoflavivirus size (~50 nm) and bias towards uncleaved prM. In silico structural analyses of the 3'-untranslated regions show that XiFV forms up to five pseudo-knot-containing stem-loops and a prototypical orthoflavivirus dumbbell element, suggesting the potential for multiple exoribonuclease-resistant RNA structures.

A novel tamanavirus (Flaviviridae) of the European common frog (Rana temporaria) from the UK.

Flavivirids are small, enveloped, positive-sense RNA viruses from the family Flaviviridae with genomes of ~9-13 kb. Metatranscriptomic analyses of metazoan organisms have revealed a diversity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. However, no flavivirus-like virus has been identified in amphibians. To remedy this, we investigated the virome of the European common frog (Rana temporaria) in the UK, utilizing high-throughput sequencing at six catch locations. De novo assembly revealed a coding-complete virus contig of a novel flavivirid ~11.2 kb in length. The virus encodes a single ORF of 3456 aa and 5' and 3' untranslated regions (UTRs) of 227 and 666 nt, respectively. We named this virus Rana tamanavirus (RaTV), as BLASTp analysis of the polyprotein showed the closest relationships to Tamana bat virus (TABV) and Cyclopterus lumpus virus from Pteronotus parnellii and Cyclopterus lumpus, respectively. Phylogenetic analysis of the RaTV polyprotein compared to Flavivirus and Flavivirus-like members indicated that RaTV was sufficiently divergent and basal to the vertebrate Tamanavirus clade. In addition to the Mitcham strain, partial but divergent RaTV, sharing 95.64-97.39 % pairwise nucleotide identity, were also obtained from the Poole and Deal samples, indicating that RaTV is widespread in UK frog samples. Bioinformatic analyses of predicted secondary structures in the 3'UTR of RaTV showed the presence of an exoribonuclease-resistant RNA (xrRNA) structure standard in flaviviruses and TABV. To examine this biochemically, we conducted an in vitro Xrn1 digestion assay showing that RaTV probably forms a functional Xrn1-resistant xrRNA.

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.

Chimeric viruses of the insect-specific flavivirus Palm Creek with structural proteins of vertebrate-infecting flaviviruses identify barriers to replication of insect-specific flaviviruses in vertebrate cells.

Here we report the generation of novel chimeric flaviviruses, which express the prM and E proteins of either dengue or Zika viruses on the genomic backbone of Palm Creek virus (PCV), an insect-specific flavivirus. The chimeric virus particles were antigenically indistinguishable from their parental prM-E donors, but were unable to infect vertebrate cells. An additional chimera (PCV structural genes in the backbone of West Nile virus - WNV/PCV-prME) was also unable to infect vertebrate cells, but transfection with RNA from this virus resulted in detectable RNA replication and translation but no infectious virion production. These data suggest multiple blocks at the entry, RNA replication and assembly/release stages of insect-specific flavivirus (ISF) infection in vertebrate cells. Serial passaging of these chimeric viruses in mosquito cells identified amino acid substitutions that may lead to increased replication efficiency. These chimeric viruses provide unique tools to further dissect the mechanisms of the host restriction of ISFs.

Reporter Flaviviruses as Tools to Demonstrate Homologous and Heterologous Superinfection Exclusion.

Binjari virus (BinJV) is a lineage II or dual-host affiliated insect-specific flavivirus previously demonstrated as replication-deficient in vertebrate cells. Previous studies have shown that BinJV is tolerant to exchanging its structural proteins (prM-E) with pathogenic flaviviruses, making it a safe backbone for flavivirus vaccines. Here, we report generation by circular polymerase extension reaction of BinJV expressing zsGreen or mCherry fluorescent protein. Recovered BinJV reporter viruses grew to high titres (107-8 FFU/mL) in Aedes albopictus C6/36 cells assayed using immunoplaque assays (iPA). We also demonstrate that BinJV reporters could be semi-quantified live in vitro using a fluorescence microplate reader with an observed linear correlation between quantified fluorescence of BinJV reporter virus-infected C6/36 cells and iPA-quantitated virus titres. The utility of the BinJV reporter viruses was then examined in homologous and heterologous superinfection exclusion assays. We demonstrate that primary infection of C6/36 cells with BinJVzsGreen completely inhibits a secondary infection with homologous BinJVmCherry or heterologous ZIKVmCherry using fluorescence microscopy and virus quantitation by iPA. Finally, BinJVzsGreen infections were examined in vivo by microinjection of Aedes aegypti with BinJVzsGreen. At seven days post-infection, a strong fluorescence in the vicinity of salivary glands was detected in frozen sections. This is the first report on the construction of reporter viruses for lineage II insect-specific flaviviruses and establishes a tractable system for exploring flavivirus superinfection exclusion in vitro and in vivo.

Extended characterisation of five archival tick-borne viruses provides insights for virus discovery in Australian ticks.

BACKGROUND: A subset of Australians who have been bitten by ticks experience a complex of chronic and debilitating symptoms which cannot be attributed to the known pathogenic species of bacteria present in Australia. As a result, there has been a renewed effort to identify and characterise viruses in Australian terrestrial ticks. Recent transcriptome sequencing of Ixodes and Amblyomma ticks has revealed the presence of multiple virus sequences. However, without virus isolates our ability to understand the host range and pathogenesis of newly identified viruses is limited. We have established a successful method for high-throughput virus discovery and isolation in mosquitoes using antibodies to double-stranded RNA. In this study we sought to characterise five archival tick-borne viruses to adapt our virus discovery protocol for Australian ticks. METHODS: We performed virus characterisation using a combination of bioinformatic sequence analysis and in vitro techniques including replication kinetics, antigenic profiling, virus purification and mass spectrometry. RESULTS: Our sequence analysis of Nugget virus, Catch-me-Cave virus and Finch Creek virus revealed marked genetic stability in isolates collected from the same location approximately 30 years apart. We demonstrate that the Ixodes scapularis-derived ISE6 cell line supports replication of Australian members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families, including Saumarez Reef virus (SREV), a flavivirus isolated from the soft tick Ornithodoros capensis. While antibodies against double-stranded RNA could be used to detect replication of a tick-borne reovirus and mosquito-borne flavivirus, the tick-borne flaviviruses Gadgets Gully virus and SREV could not be detected using this method. Finally, four novel virus-like sequences were identified in transcriptome sequencing of the Australian native tick Ixodes holocyclus. CONCLUSIONS: Genetic and antigenic characterisations of archival viruses in this study confirm that three viruses described in 2002 represent contemporary isolates of virus species first identified 30 years prior. Our findings with antibodies to double-stranded RNA highlight an unusual characteristic shared by two Australian tick-borne flaviviruses. Finally, comparative growth kinetics analyses of Australian tick-borne members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families in ISE6 and BSR cells will provide a useful resource for isolation of Australian tick-borne viruses using existing cell lines.

A unified route for flavivirus structures uncovers essential pocket factors conserved across pathogenic viruses.

The epidemic emergence of relatively rare and geographically isolated flaviviruses adds to the ongoing disease burden of viruses such as dengue. Structural analysis is key to understand and combat these pathogens. Here, we present a chimeric platform based on an insect-specific flavivirus for the safe and rapid structural analysis of pathogenic viruses. We use this approach to resolve the architecture of two neurotropic viruses and a structure of dengue virus at 2.5 Å, the highest resolution for an enveloped virion. These reconstructions allow improved modelling of the stem region of the envelope protein, revealing two lipid-like ligands within highly conserved pockets. We show that these sites are essential for viral growth and important for viral maturation. These findings define a hallmark of flavivirus virions and a potential target for broad-spectrum antivirals and vaccine design. We anticipate the chimeric platform to be widely applicable for investigating flavivirus biology.

Implications of Dengue Virus Maturation on Vaccine Induced Humoral Immunity in Mice.

The use of dengue virus (DENV) vaccines has been hindered by the complexities of antibody dependent enhancement (ADE). Current late-stage vaccine candidates utilize attenuated and chimeric DENVs that produce particles of varying maturities. Antibodies that are elicited by preferentially exposed epitopes on immature virions have been linked to increased ADE. We aimed to further understand the humoral immunity promoted by DENV particles of varying maturities in an AG129 mouse model using a chimeric insect specific vaccine candidate, bDENV-2. We immunized mice with mature, partially mature, and immature bDENV-2 and found that immunization with partially mature bDENV-2 produced more robust and cross-neutralizing immune responses than immunization with immature or mature bDENV-2. Upon challenge with mouse adapted DENV-2 (D220), we observed 80% protection for mature bDENV-2 vaccinated mice and 100% for immature and partially mature vaccinated mice, suggesting that protection to homotypic challenge is not dependent on maturation. Finally, we found reduced in vitro ADE at subneutralising serum concentrations for mice immunized with mature bDENV-2. These results suggest that both immature and mature DENV particles play a role in homotypic protection; however, the increased risk of in vitro ADE from immature particles indicates potential safety benefits from mature DENV-based vaccines.

The structure of an infectious immature flavivirus redefines viral architecture and maturation.

Flaviviruses are the cause of severe human diseases transmitted by mosquitoes and ticks. These viruses use a potent fusion machinery to enter target cells that needs to be restrained during viral assembly and egress. A molecular chaperone, premembrane (prM) maintains the virus particles in an immature, fusion-incompetent state until they exit the cell. Taking advantage of an insect virus that produces particles that are both immature and infectious, we determined the structure of the first immature flavivirus with a complete spike by cryo-electron microscopy. Unexpectedly, the prM chaperone forms a supporting pillar that maintains the immature spike in an asymmetric and upright state, primed for large rearrangements upon acidification. The collapse of the spike along a path defined by the prM chaperone is required, and its inhibition by a multivalent immunoglobulin M blocks infection. The revised architecture and collapse model are likely to be conserved across flaviviruses.

A broadly protective antibody that targets the flavivirus NS1 protein.

There are no approved flaviviral therapies and the development of vaccines against flaviruses has the potential of being undermined by antibody-dependent enhancement (ADE). The flavivirus nonstructural protein 1 (NS1) is a promising vaccine antigen with low ADE risk but has yet to be explored as a broad-spectrum therapeutic antibody target. Here, we provide the structural basis of NS1 antibody cross-reactivity through cocrystallization of the antibody 1G5.3 with NS1 proteins from dengue and Zika viruses. The 1G5.3 antibody blocks multi-flavivirus NS1-mediated cell permeability in disease-relevant cell lines, and therapeutic application of 1G5.3 reduces viremia and improves survival in dengue, Zika, and West Nile virus murine models. Finally, we demonstrate that 1G5.3 protection is independent of effector function, identifying the 1G5.3 epitope as a key site for broad-spectrum antiviral development.

Protective Efficacy of a Chimeric Insect-Specific Flavivirus Vaccine against West Nile Virus.

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 (WNVKUN) and the genome backbone of the insect-specific flavivirus Binjari virus (BinJV). This chimeric virus (BinJ/WNVKUN-prME) exhibits an insect-specific phenotype and does not replicate in vertebrate cells. Importantly, it authentically presents the prM-E proteins of WNVKUN, which is antigenically very similar to other WNV strains and lineages. Therefore BinJ/WNVKUN-prME represents an excellent candidate to assess as a vaccine against virulent WNV strains, including the highly pathogenic WNVNY99. When CD1 mice were immunized with purified BinJ/WNVKUN-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 WNVNY99, with no clinical or subclinical pathology observed in vaccinated animals. These data suggest that BinJ/WNVKUN-prME represents a safe and effective WNV vaccine candidate that warrants further investigation for use in humans or in veterinary applications.

A Unique Relative of Rotifer Birnavirus Isolated from Australian Mosquitoes.

The family Birnaviridae are a group of non-enveloped double-stranded RNA viruses which infect poultry, aquatic animals and insects. This family includes agriculturally important pathogens of poultry and fish. Recently, next-generation sequencing technologies have identified closely related birnaviruses in Culex, Aedes and Anopheles mosquitoes. Using a broad-spectrum system based on detection of long double-stranded RNA, we have discovered and isolated a birnavirus from Aedes notoscriptus mosquitoes collected in northern New South Wales, Australia. Phylogenetic analysis of Aedes birnavirus (ABV) showed that it is related to Rotifer birnavirus, a pathogen of microscopic aquatic animals. In vitro cell infection assays revealed that while ABV can replicate in Aedes-derived cell lines, the virus does not replicate in vertebrate cells and displays only limited replication in Culex- and Anopheles-derived cells. A combination of SDS-PAGE and mass spectrometry analysis suggested that the ABV capsid precursor protein (pVP2) is larger than that of other birnaviruses and is partially resistant to trypsin digestion. Reactivity patterns of ABV-specific polyclonal and monoclonal antibodies indicate that the neutralizing epitopes of ABV are SDS sensitive. Our characterization shows that ABV displays a number of properties making it a unique member of the Birnaviridae and represents the first birnavirus to be isolated from Australian mosquitoes.

Antigenic Characterization of New Lineage II Insect-Specific Flaviviruses in Australian Mosquitoes and Identification of Host Restriction Factors.

We describe two new insect-specific flaviviruses (ISFs) isolated from mosquitoes in Australia, Binjari virus (BinJV) and Hidden Valley virus (HVV), that grow efficiently in mosquito cells but fail to replicate in a range of vertebrate cell lines. Phylogenetic analysis revealed that BinJV and HVV were closely related (90% amino acid sequence identity) and clustered with lineage II (dual-host affiliated) ISFs, including the Lammi and Nounané viruses. Using a panel of monoclonal antibodies prepared to BinJV viral proteins, we confirmed a close relationship between HVV and BinJV and revealed that they were antigenically quite divergent from other lineage II ISFs. We also constructed chimeric viruses between BinJV and the vertebrate-infecting West Nile virus (WNV) by swapping the structural genes (prM and E) to produce BinJ/WNVKUN-prME and WNVKUN/BinJV-prME. This allowed us to assess the role of different regions of the BinJV genome in vertebrate host restriction and revealed that while BinJV structural proteins facilitated entry to vertebrate cells, the process was inefficient. In contrast, the BinJV replicative components in wild-type BinJV and BinJ/WNVKUN-prME failed to initiate replication in a wide range of vertebrate cell lines at 37°C, including cells lacking components of the innate immune response. However, trace levels of replication of BinJ/WNVKUN-prME could be detected in some cultures of mouse embryo fibroblasts (MEFs) deficient in antiviral responses (IFNAR-/- MEFs or RNase L-/- MEFs) incubated at 34°C after inoculation. This suggests that BinJV replication in vertebrate cells is temperature sensitive and restricted at multiple stages of cellular infection, including inefficient cell entry and susceptibility to antiviral responses.IMPORTANCE The globally important flavivirus pathogens West Nile virus, Zika virus, dengue viruses, and yellow fever virus can infect mosquito vectors and be transmitted to humans and other vertebrate species in which they cause significant levels of disease and mortality. However, the subgroup of closely related flaviviruses, known as lineage II insect-specific flaviviruses (Lin II ISFs), only infect mosquitoes and cannot replicate in cells of vertebrate origin. Our data are the first to uncover the mechanisms that restrict the growth of Lin II ISFs in vertebrate cells and provides new insights into the evolution of these viruses and the mechanisms associated with host switching that may allow new mosquito-borne viral diseases to emerge. The new reagents generated in this study, including the first Lin II ISF-reactive monoclonal antibodies and Lin II ISF mutants and chimeric viruses, also provide new tools and approaches to enable further research advances in this field.

Genetic, Morphological and Antigenic Relationships between Mesonivirus Isolates from Australian Mosquitoes and Evidence for Their Horizontal Transmission.

The Mesoniviridae are a newly assigned family of viruses in the order Nidovirales. Unlike other nidoviruses, which include the Coronaviridae, mesoniviruses are restricted to mosquito hosts and do not infect vertebrate cells. To date there is little information on the morphological and antigenic characteristics of this new group of viruses and a dearth of mesonivirus-specific research tools. In this study we determined the genetic relationships of recent Australian isolates of Alphamesonivirus 4 (Casuarina virus-CASV) and Alphamesonivirus 1 (Nam Dinh virus-NDiV), obtained from multiple mosquito species. Australian isolates of NDiV showed high-level similarity to the prototype NDiV isolate from Vietnam (99% nucleotide (nt) and amino acid (aa) identity). Isolates of CASV from Central Queensland were genetically very similar to the prototype virus from Darwin (95-96% nt and 91-92% aa identity). Electron microscopy studies demonstrated that virion diameter (≈80 nm) and spike length (≈10 nm) were similar for both viruses. Monoclonal antibodies specific to CASV and NDiV revealed a close antigenic relationship between the two viruses with 13/34 mAbs recognising both viruses. We also detected NDiV RNA on honey-soaked nucleic acid preservation cards fed on by wild mosquitoes supporting a possible mechanism of horizontal transmission between insects in nature.

Author Correction: Infectious DNAs derived from insect-specific flavivirus genomes enable identification of pre- and post-entry host restrictions in vertebrate cells.

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A recombinant platform for flavivirus vaccines and diagnostics using chimeras of a new insect-specific virus.

Flaviviruses such as dengue, yellow fever, Zika, West Nile, and Japanese encephalitis virus present substantial global health burdens. New vaccines are being sought to address safety and manufacturing issues associated with current live attenuated vaccines. Here, we describe a new insect-specific flavivirus, Binjari virus, which was found to be remarkably tolerant for exchange of its structural protein genes (prME) with those of the aforementioned pathogenic vertebrate-infecting flaviviruses (VIFs). Chimeric BinJ/VIF-prME viruses remained replication defective in vertebrate cells but replicated with high efficiency in mosquito cells. Cryo-electron microscopy and monoclonal antibody binding studies illustrated that the chimeric BinJ/VIF-prME virus particles were structurally and immunologically similar to their parental VIFs. Pilot manufacturing in C6/36 cells suggests that high yields can be reached up to 109.5 cell culture infectious dose/ml or ≈7 mg/liter. BinJ/VIF-prME viruses showed utility in diagnostic (microsphere immunoassays and ELISAs using panels of human and equine sera) and vaccine applications (illustrating protection against Zika virus challenge in murine IFNAR-/- mouse models). BinJ/VIF-prME viruses thus represent a versatile, noninfectious (for vertebrate cells), high-yield technology for generating chimeric flavivirus particles with low biocontainment requirements.

The taxonomy of an Australian nodavirus isolated from mosquitoes.

We describe a virus isolated from Culex annulirostris mosquitoes in Australia. Phylogenetic analysis of its RNA-dependent RNA polymerase sequence and that of other related viruses revealed 6 clades, two of which corresponded wholly or partly with existing genera in the family Nodaviridae. There was greater genetic diversity within the family than previously recognized prompting us to suggest that additional genera should be considered within the family.

The recently identified flavivirus Bamaga virus is transmitted horizontally by Culex mosquitoes and interferes with West Nile virus replication in vitro and transmission in vivo.

Arthropod-borne flaviviruses such as yellow fever (YFV), Zika and dengue viruses continue to cause significant human disease globally. These viruses are transmitted by mosquitoes when a female imbibes an infected blood-meal from a viremic vertebrate host and expectorates the virus into a subsequent host. Bamaga virus (BgV) is a flavivirus recently discovered in Culex sitiens subgroup mosquitoes collected from Cape York Peninsula, Australia. This virus phylogenetically clusters with the YFV group, but is potentially restricted in most vertebrates. However, high levels of replication in an opossum cell line (OK) indicate a potential association with marsupials. To ascertain whether BgV could be horizontally transmitted by mosquitoes, the vector competence of two members of the Cx. sitiens subgroup, Cx. annulirostris and Cx. sitiens, for BgV was investigated. Eleven to thirteen days after imbibing an infectious blood-meal, infection rates were 11.3% and 18.8% for Cx. annulirostris and Cx. sitiens, respectively. Cx. annulirostris transmitted the virus at low levels (5.6% had BgV-positive saliva overall); Cx. sitiens did not transmit the virus. When mosquitoes were injected intrathoracially with BgV, the infection and transmission rates were 100% and 82%, respectively, for both species. These results provided evidence for the first time that BgV can be transmitted horizontally by Cx. annulirostris, the primary vector of pathogenic zoonotic flaviviruses in Australia. We also assessed whether BgV could interfere with replication in vitro, and infection and transmission in vivo of super-infecting pathogenic Culex-associated flaviviruses. BgV significantly reduced growth of Murray Valley encephalitis and West Nile (WNV) viruses in vitro. While prior infection with BgV by injection did not inhibit WNV super-infection of Cx. annulirostris, significantly fewer BgV-infected mosquitoes could transmit WNV than mock-injected mosquitoes. Overall, these data contribute to our understanding of flavivirus ecology, modes of transmission by Australian mosquitoes and mechanisms for super-infection interference.

Infectious DNAs derived from insect-specific flavivirus genomes enable identification of pre- and post-entry host restrictions in vertebrate cells.

Flaviviruses such as West Nile virus (WNV), dengue virus and Zika virus are mosquito-borne pathogens that cause significant human diseases. A novel group of insect-specific flaviviruses (ISFs), which only replicate in mosquitoes, have also been identified. However, little is known about the mechanisms of ISF host restriction. We report the generation of infectious cDNA from two Australian ISFs, Parramatta River virus (PaRV) and Palm Creek virus (PCV). Using circular polymerase extension cloning (CPEC) with a modified OpIE2 insect promoter, infectious cDNA was generated and transfected directly into mosquito cells to produce infectious virus indistinguishable from wild-type virus. When infectious PaRV cDNA under transcriptional control of a mammalian promoter was used to transfect mouse embryo fibroblasts, the virus failed to initiate replication even when cell entry steps were by-passed and the type I interferon response was lacking. We also used CPEC to generate viable chimeric viruses between PCV and WNV. Analysis of these hybrid viruses revealed that ISFs are also restricted from replication in vertebrate cells at the point of entry. The approaches described here to generate infectious ISF DNAs and chimeric viruses provide unique tools to further dissect the mechanisms of their host restriction.