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.

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.

NS4/5 mutations enhance flavivirus Bamaga virus infectivity and pathogenicity in vitro and in vivo.

Flaviviruses such as yellow fever, dengue or Zika viruses are responsible for significant human and veterinary diseases worldwide. These viruses contain an RNA genome, prone to mutations, which enhances their potential to emerge as pathogens. Bamaga virus (BgV) is a mosquito-borne flavivirus in the yellow fever virus group that we have previously shown to be host-restricted in vertebrates and horizontally transmissible by Culex mosquitoes. Here, we aimed to characterise BgV host-restriction and to investigate the mechanisms involved. We showed that BgV could not replicate in a wide range of vertebrate cell lines and animal species. We determined that the mechanisms involved in BgV host-restriction were independent of the type-1 interferon response and RNAse L activity. Using a BgV infectious clone and two chimeric viruses generated as hybrids between BgV and West Nile virus, we demonstrated that BgV host-restriction occurred post-cell entry. Notably, BgV host-restriction was shown to be temperature-dependent, as BgV replicated in all vertebrate cell lines at 34°C but only in a subset at 37°C. Serial passaging of BgV in Vero cells resulted in adaptive mutants capable of efficient replication at 37°C. The identified mutations resulted in amino acid substitutions in NS4A-S124F, NS4B-N244K and NS5-G2C, all occurring close to a viral protease cleavage site (NS4A/2K and NS4B/NS5). These mutations were reverse engineered into infectious clones of BgV, which revealed that NS4B-N244K and NS5-G2C were sufficient to restore BgV replication in vertebrate cells at 37°C, while NS4A-S124F further increased replication efficiency. When these mutant viruses were injected into immunocompetent mice, alongside BgV and West Nile virus chimeras, infection and neurovirulence were enhanced as determined by clinical scores, seroconversion, micro-neutralisation, viremia, histopathology and immunohistochemistry, confirming the involvement of these residues in the attenuation of BgV. Our studies identify a new mechanism of host-restriction and attenuation of a mosquito-borne flavivirus.

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.

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.

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

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

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.

Differential Diagnosis of Flavivirus Infections in Horses Using Viral Envelope Protein Domain III Antigens in Enzyme-Linked Immunosorbent Assay.

In Australia, infection of horses with the West Nile virus (WNV) or Murray Valley encephalitis virus (MVEV) occasionally results in severe neurological disease that cannot be clinically differentiated. Confirmatory serological tests to detect antibody specific for MVEV or WNV in horses are often hampered by cross-reactive antibodies induced to conserved epitopes on the envelope (E) protein. This study utilized bacterially expressed recombinant antigens derived from domain III of the E protein (rE-DIII) of MVEV and WNV, respectively, to determine whether these subunit antigens provided specific diagnostic markers of infection with these two viruses. When a panel of 130 serum samples, from horses with known flavivirus infection status, was tested in enzyme-linked immunosorbent assay (ELISA) using rE-DIII antigens, a differential diagnosis of MVEV or WNV was achieved for most samples. Time-point samples from horses exposed to flavivirus infection during the 2011 outbreak of equine encephalitis in south-eastern Australia also indicated that the rE-DIII antigens were capable of detecting and differentiating MVEV and WNV infection in convalescent sera with similar sensitivity and specificity to virus neutralization tests and blocking ELISAs. Overall, these results indicate that the rE-DIII is a suitable antigen for use in rapid immunoassays for confirming MVEV and WNV infections in horses in the Australian context and warrant further assessment on sensitive, high-throughput serological platforms such as multiplex immune assays.

A New Clade of Insect-Specific Flaviviruses from Australian Anopheles Mosquitoes Displays Species-Specific Host Restriction.

Flaviviruses are arthropod-borne viruses found worldwide and are responsible for significant human and veterinary diseases, including dengue, Zika, and West Nile fever. Some flaviviruses are insect specific and replicate only in mosquitoes. We report a genetically divergent group of insect-specific flaviviruses from Anopheles mosquitoes that do not replicate in arthropod cell lines or heterologous Anopheles species, exhibiting unprecedented specialization for their host species. Determination of the complete sequences of the RNA genomes of three of these viruses, Karumba virus (KRBV), Haslams Creek virus, and Mac Peak virus (McPV), that are found in high prevalence in some Anopheles mosquito populations and detection of virus-specific proteins, replicative double-stranded RNA, and small interfering RNA responses in the host mosquito species provided strong evidence of a functional replicating virus in the mosquito midgut. Analysis of nucleotide composition in the KRBV and McPV sequences also revealed a pattern consistent with the virus evolving to replicate only in insects. These findings represent a significant advance in our knowledge of mosquito-borne flavivirus ecology, host restriction, and evolution. IMPORTANCE Flaviviruses like dengue, Zika, or West Nile virus infect millions of people each year and are transmitted to humans via infected-mosquito bites. A subset of flaviviruses can only replicate in the mosquito host, and recent studies have shown that some can interfere with pathogenic flaviviruses in mosquitoes and limit the replication and transmission of the latter. The insect-specific flaviviruses (ISFs) reported here form a new Anopheles mosquito-associated clade separate from the Aedes- and Culex-associated ISF clades. The identification of distinct clades for each mosquito genus provides new insights into the evolution and ecology of flaviviruses. One of these viruses was shown to replicate in the midgut of the mosquito host and exhibit the most specialized host restriction reported to date for ISFs. Understanding this unprecedented host restriction in ISFs could help identify the mechanisms involved in the evolution of flaviviruses and their emergence as mosquito-borne pathogens.

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.

Commensal Viruses of Mosquitoes: Host Restriction, Transmission, and Interaction with Arboviral Pathogens.

Recent advances in virus detection strategies and deep sequencing technologies have enabled the identification of a multitude of new viruses that persistently infect mosquitoes but do not infect vertebrates. These are usually referred to as insect-specific viruses (ISVs). These novel viruses have generated considerable interest in their modes of transmission, persistence in mosquito populations, the mechanisms that restrict their host range to mosquitoes, and their interactions with pathogens transmissible by the same mosquito. In this article, we discuss studies in our laboratory and others that demonstrate that many ISVs are efficiently transmitted directly from the female mosquito to their progeny via infected eggs, and, moreover, that persistent infection of mosquito cell cultures or whole mosquitoes with ISVs can restrict subsequent infection, replication, and transmission of some mosquito-borne viral pathogens. This suggests that some ISVs may act as natural regulators of arboviral transmission. We also discuss viral and host factors that may be responsible for their host restriction.

The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition.

Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1-35 and 140-210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.