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

ABSTRACT: 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.

SUBMITTER: Harrison JJ

PROVIDER: S-EPMC7300350 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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

Harrison Jessica J JJ Hobson-Peters Jody J Colmant Agathe M G AMG Koh Joanna J Newton Natalee D ND Warrilow David D Bielefeldt-Ohmann Helle H Piyasena Thisun B H TBH O'Brien Caitlin A CA Vet Laura J LJ Paramitha Devina D Potter James R JR Davis Steven S SS Johansen Cheryl A CA Setoh Yin Xiang YX Khromykh Alexander A AA Hall Roy A RA

mSphere 20200617 3

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 ...[more]

PMID: 32554715

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Project description: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.

Project description:The genus Flavivirus contains emerging arthropod-borne viruses (arboviruses) infecting vertebrates, as well as insect-specific viruses (ISVs) (i.e., viruses whose host range is restricted to insects). ISVs are evolutionary precursors to arboviruses. Knowledge of the nature of the ISV infection block in vertebrates could identify functions necessary for the expansion of the host range toward vertebrates. Mapping of host restrictions by complementation of ISV and arbovirus genome functions could generate knowledge critical to predicting arbovirus emergence. Here we isolated a novel flavivirus, termed Niénokoué virus (NIEV), from mosquitoes sampled in Côte d'Ivoire. NIEV groups with insect-specific flaviviruses (ISFs) in phylogeny and grows in insect cells but not in vertebrate cells. We generated an infectious NIEV cDNA clone and a NIEV reporter replicon to study growth restrictions of NIEV in comparison to yellow fever virus (YFV), for which the same tools are available. Efficient RNA replication of the NIEV reporter replicon was observed in insect cells but not in vertebrate cells. Initial translation of the input replicon RNA in vertebrate cells was functional, but RNA replication did not occur. Chimeric YFV carrying the envelope proteins of NIEV was recovered via electroporation in C6/36 insect cells but did not infect vertebrate cells, indicating a block at the level of entry. Since the YF/NIEV chimera readily produced infectious particles in insect cells but not in vertebrate cells despite efficient RNA replication, restriction is also determined at the level of assembly/release. Taking the results together, the ability of ISF to infect vertebrates is blocked at several levels, including attachment/entry and RNA replication as well as assembly/release. IMPORTANCE Most viruses of the genus Flavivirus, e.g., YFV and dengue virus, are mosquito borne and transmitted to vertebrates during blood feeding of mosquitoes. Within the last decade, an increasing number of viruses with a host range exclusively restricted to insects in close relationship to the vertebrate-pathogenic flaviviruses were discovered in mosquitoes. To identify barriers that could block the arboviral vertebrate tropism, we set out to identify the steps at which the ISF replication cycle fails in vertebrates. Our studies revealed blocks at several levels, suggesting that flavivirus host range expansion from insects to vertebrates was a complex process that involved overcoming multiple barriers.

Project description:BackgroundMosquito-borne diseases are rapidly spreading due to increasing international travel and trade. Routine mosquito surveillance and screening for mosquito-borne pathogens can be early indicators for local disease transmission and outbreaks. However, arbovirus detection in mosquito vectors has rarely been reported in Saudi Arabia.MethodsA total of 769,541 Aedes and Culex mosquitoes were collected by Black Hole traps during routine mosquito surveillance in the first half of 2016. Culex. quinquefasciatus and Ae. aegypti were the most prevalent species observed. Twenty-five and 24 randomly selected pools of Ae. aegypti and Cx. quinquefasciatus, respectively, were screened for arboviruses by RT-PCR.ResultsDengue 2 (DENV-2) and four strains of insect-specific flaviviruses, including one of cell-fusing agent virus (CFAV) and three of Phlebotomus-associated flavivirus (PAFV) were detected in pools of Ae. aegypti. We also detected 10 strains of Culex flavivirus (CxFV) in pools of Cx. quinquefasciatus. Phylogenetic analysis using whole genome sequences placed the DENV strain into the cosmopolitan 1 sub-DENV-2 genotype, and the CxFVs into the African/Caribbean/Latin American genotype. These analyses also showed that the DENV-2 strain detected in the present study was closely related to strains detected in China in 2014 and in Japan in 2018, which suggests frequent movement of DENV-2 strains among these countries. Furthermore, the phylogenetic analysis suggested at least five introductions of DENV-2 into Saudi Arabia from 2014 through 2018, most probably from India.ConclusionsTo our knowledge, this study reports the first detection of four arboviruses DENV, CFAV, PAFV, and CxFV in mosquitoes in Saudi Arabia, which shows that they are co-circulating in Jeddah. Our findings show a need for widespread mosquito-based arbovirus surveillance programs in Saudi Arabia, which will improve our understanding of the transmission dynamics of the mosquito-borne arboviruses within the country and help early predict and mitigate the risk of human infections and outbreaks.

Dataset Information

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

Publications

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

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