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 10^9.5 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.

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.

Mosquito-borne viruses encompass a range of virus families, comprising a number of significant human pathogens (e.g., dengue viruses, West Nile virus, Chikungunya virus). Virulent strains of these viruses are continually evolving and expanding their geographic range, thus rapid and sensitive screening assays are required to detect emerging viruses and monitor their prevalence and spread in mosquito populations. Double-stranded RNA (dsRNA) is produced during the replication of many of these viruses as either an intermediate in RNA replication (e.g., flaviviruses, togaviruses) or the double-stranded RNA genome (e.g., reoviruses). Detection and discovery of novel viruses from field and clinical samples usually relies on recognition of antigens or nucleotide sequences conserved within a virus genus or family. However, due to the wide antigenic and genetic variation within and between viral families, many novel or divergent species can be overlooked by these approaches. We have developed two monoclonal antibodies (mAbs) which show co-localised staining with proteins involved in viral RNA replication in immunofluorescence assay (IFA), suggesting specific reactivity to viral dsRNA. By assessing binding against a panel of synthetic dsRNA molecules, we have shown that these mAbs recognise dsRNA greater than 30 base pairs in length in a sequence-independent manner. IFA and enzyme-linked immunosorbent assay (ELISA) were employed to demonstrate detection of a panel of RNA viruses from several families, in a range of cell types. These mAbs, termed monoclonal antibodies to viral RNA intermediates in cells (MAVRIC), have now been incorporated into a high-throughput, economical ELISA-based screening system for the detection and discovery of viruses from mosquito populations. Our results have demonstrated that this simple system enables the efficient detection and isolation of a range of known and novel viruses in cells inoculated with field-caught mosquito samples, and represents a rapid, sequence-independent, and cost-effective approach to virus discovery. This paper describes a simple and cost-effective system for screening biological samples for virus-infection. The authors demonstrate the application of two antibodies to detect double-stranded RNA (dsRNA) which is a common molecule produced in infection by a number of different viruses. The use of antibodies which react with double-stranded RNA independently of sequence allows for detection of a diverse range of viruses and has been instrumental in the detection of known arboviruses from three different families and the discovery of a number of previously unknown viruses from Australian mosquito populations. This system provides a rapid and economical approach to virus surveillance and discovery. This is the first report of anti-dsRNA antibodies used in a streamlined system for virus detection and discovery in field-caught samples.

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.

Jingmenviruses are a group of viruses identified recently, in 2014, and currently classified by the International Committee on Taxonomy of Viruses as unclassified Flaviviridae. These viruses closely related to flaviviruses are unique due to the segmented nature of their genome. The prototype jingmenvirus, Jingmen tick virus (JMTV), was discovered in Rhipicephalus microplus ticks collected from China in 2010. Jingmenviruses genomes are composed of four to five segments, encoding for up to seven structural proteins and two non-structural proteins, both of which display strong similarities with flaviviral non-structural proteins (NS2B/NS3 and NS5). Jingmenviruses are currently separated into two phylogenetic clades. One clade includes tick- and vertebrate-associated jingmenviruses, which have been detected in ticks and mosquitoes, as well as in humans, cattle, monkeys, bats, rodents, sheep, and tortoises. In addition to these molecular and serological detections, over a hundred human patients tested positive for jingmenviruses after developing febrile illness and flu-like symptoms in China and Serbia. The second phylogenetic clade includes insect-associated jingmenvirus sequences, which have been detected in a wide range of insect species, as well as in crustaceans, plants, and fungi. In addition to being found in various types of hosts, jingmenviruses are endemic, as they have been detected in a wide range of environments, all over the world. Taken together, all of these elements show that jingmenviruses correspond exactly to the definition of emerging viruses at risk of causing a pandemic, since they are already endemic, have a close association with arthropods, are found in animals in close contact with humans, and have caused sporadic cases of febrile illness in multiple patients. Despite these arguments, the vast majority of published data is from metagenomics studies and many aspects of jingmenvirus replication remain to be elucidated, such as their tropism, cycle of transmission, structure, and mechanisms of replication and restriction or epidemiology. It is therefore crucial to prioritize jingmenvirus research in the years to come, to be prepared for their emergence as human or veterinary pathogens.