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 current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.

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.

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.

Arboviruses cycle between, and replicate in, both invertebrate and vertebrate hosts, which for Zika virus (ZIKV) involves Aedes mosquitoes and primates1. The viral determinants required for replication in such obligate hosts are under strong purifying selection during natural virus evolution, making it challenging to resolve which determinants are optimal for viral fitness in each host. Herein we describe a deep mutational scanning (DMS) strategy2–5 whereby a viral cDNA library was constructed containing all codon substitutions in the C-terminal 204 amino acids of ZIKV envelope (E) protein. The cDNA library was transfected into C6/36 (Aedes) and Vero (primate) cells, with subsequent deep sequencing and computational analyses of recovered viruses showing that K316Q and S461G, or Q350L and T397S substitutions conferred substantial replicative advantages in mosquito and primate cells, respectively. A 316Q/461G virus was constructed and shown to be replication-defective in mammalian cells due to severely compromised virus particle formation and secretion. The 316Q/461G virus was also highly attenuated in human brain organoids, and illustrated utility as a vaccine in mice. This approach can thus imitate evolutionary selection in a matter of days and identify amino acids key to regulating virus replication in specific host environments.