Mosquito cells infected with Japanese encephalitis virus release slowly-sedimenting hemagglutinin particles in association with intracellular formation of smooth membrane structures

Arthropod-borne flaviviruses can grow in both arthropod and mammalian cells. Virion morphogenesis, though well studied in mammalian cells, is still unclear in arthropod cells. Here, we compared a mosquito cell line C6/36 and a mammalian cell line Vero in extracellular virus particles and intracellular ultrastructures triggered by infection with Japanese encephalitis virus (JEV). Sedimentation analyses of virion and slowly-sedimenting hemagglutinin (SHA) particles released by infection with the Nakayama strain revealed that C6/36 cells produced higher envelope (E) antigen levels in the SHA than the virion fraction in contrast to Vero cells that showed the opposite pattern. Specific infectivities per ng of E were similar in both cells, whereas specific hemagglutinating activities in the SHA fraction were lower in C6/36 than Vero cells. The precursor membrane protein was less efficiently cleaved to the membrane protein in SHA particles released from C6/36 than Vero cells. Ultrastructural studies showed more remarkable production of smooth membrane structures (SMSs) in C6/36 than in Vero cells. The differences in sedimentation patterns of extracellular virus particles between Nakayama-infected C6/36 and Vero cells were consistently observed in 5 other strains (Beijing P1, Beijing P3, JaTH-160, KE-093 and JaGAr-O1), except for KE-093-infected C6/36 cells which exhibited the Vero-type sedimentation profile under conditions of open cultivation. By electron microscopy, the production of SMSs from KE-093-infected C6/36 cells under open conditions was markedly less than that under closed conditions where the cells exhibited the C6/36-type sedimentation profile. Thus, intracellular SMS formations were associated with extracellular SHA production in JEV-infected mosquito cells.     

Rift Valley fever virus 78kDa envelope protein attenuates virus replication in macrophage-derived cell lines and viral virulence in mice

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus with a wide host range including ruminants and humans. RVFV outbreaks have had devastating effects on public health and the livestock industry in African countries. However, there is no approved RVFV vaccine for human use in non-endemic countries and no FDA-approved antiviral drug for RVFV treatment. The RVFV 78kDa protein (P78), which is a membrane glycoprotein, plays a role in virus dissemination in the mosquito host, but its biological role in mammalian hosts remains unknown. We generated an attenuated RVFV MP-12 strain-derived P78-High virus and a virulent ZH501 strain-derived ZH501-P78-High virus, both of which expressed a higher level of P78 and carried higher levels of P78 in the virion compared to their parental viruses. We also generated another MP-12-derived mutant virus (P78-KO virus) that does not express P78. MP-12 and P78-KO virus replicated to similar levels in fibroblast cell lines and Huh7 cells, while P78-High virus replicated better than MP-12 in Vero E6 cells, fibroblast cell lines, and Huh7 cells. Notably, P78-High virus and P78-KO virus replicated less efficiently and more efficiently, respectively, than MP-12 in macrophage cell lines. ZH501-P78-High virus also replicated poorly in macrophage cell lines. Our data further suggest that inefficient binding of P78-High virus to the cells led to inefficient virus internalization, low virus infectivity and reduced virus replication in a macrophage cell line. P78-High virus and P78-KO virus showed lower and higher virulence than MP-12, respectively, in young mice. ZH501-P78-High virus also exhibited lower virulence than ZH501 in mice. These data suggest that high levels of P78 expression attenuate RVFV virulence by preventing efficient virus replication in macrophages. Genetic alteration leading to increased P78 expression may serve as a novel strategy for the attenuation of RVFV virulence and generation of safe RVFV vaccines.     

Loss of Glycosaminoglycan Receptor Binding after Mosquito Cell Passage Reduces Chikungunya Virus Infectivity

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that can cause fever and chronic arthritis in humans. CHIKV that is generated in mosquito or mammalian cells differs in glycosylation patterns of viral proteins, which may affect its replication and virulence. Herein, we compare replication, pathogenicity, and receptor binding of CHIKV generated in Vero cells (mammal) or C6/36 cells (mosquito) through a single passage. We demonstrate that mosquito cell-derived CHIKV (CHIKV_mos) has slower replication than mammalian cell-derived CHIKV (CHIKV_vero), when tested in both human and murine cell lines. Consistent with this, CHIKV_mos infection in both cell lines produce less cytopathic effects and reduced antiviral responses. In addition, infection in mice show that CHIKV_mos produces a lower level of viremia and less severe footpad swelling when compared with CHIKV_vero. Interestingly, CHIKV_mos has impaired ability to bind to glycosaminoglycan (GAG) receptors on mammalian cells. However, sequencing analysis shows that this impairment is not due to a mutation in the CHIKV E2 gene, which encodes for the viral receptor binding protein. Moreover, CHIKV_mos progenies can regain GAG receptor binding capability and can replicate similarly to CHIKV_vero after a single passage in mammalian cells. Furthermore, CHIKV_vero and CHIKV_mos no longer differ in replication when N-glycosylation of viral proteins was inhibited by growing these viruses in the presence of tunicamycin. Collectively, these results suggest that N-glycosylation of viral proteins within mosquito cells can result in loss of GAG receptor binding capability of CHIKV and reduction of its infectivity in mammalian cells.     

Proteomics Profiling of Chikungunya-Infected Aedes albopictus C6/36 Cells Reveal Important Mosquito Cell Factors in Virus Replication

Chikungunya virus (CHIKV) is the only causative agent of CHIKV fever with persistent arthralgia, and in some cases may lead to neurological complications which can be highly fatal, therefore it poses severe health issues in many parts of the world. CHIKV transmission can be mediated via the Aedes albopictus mosquito; however, very little is currently known about the involvement of mosquito cellular factors during CHIKV-infection within the mosquito cells. Unravelling the neglected aspects of mosquito proteome changes in CHIKV-infected mosquito cells may increase our understanding on the differences in the host factors between arthropod and mammalian cells for successful replication of CHIKV. In this study, the CHIKV-infected C6/36 cells with differential cellular proteins expression were profiled using two-dimensional gel electrophoresis (2DE) coupled with the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 2DE analysis on CHIKV-infected C6/36 cells has shown 23 mosquito cellular proteins that are differentially regulated, and which are involved diverse biological pathways, such as protein folding and metabolic processes. Among those identified mosquito proteins, spermatogenesis-associated factor, enolase phosphatase e-1 and chaperonin-60kD have been found to regulate CHIKV infection. Furthermore, siRNA-mediated gene knockdown of these proteins has demonstrated the biological importance of these host proteins that mediate CHIKV infection. These findings have provided an insight to the importance of mosquito host factors in the replication of CHIKV, thus providing a potential channel for developing novel antiviral strategies against CHIKV transmission.     

Mosquito and mammalian cells grown on microcarriers for four-serotype dengue virus production: variations in virus titer, plaque morphology, and replication rate

Dengue (DEN) viruses consisting of four distinct serotypes cause diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome in humans. Most of the dengue viruses can be effectively propagated in some mosquito and mammalian cell lines. In this study, we applied microcarrier cell culture technology to study two relevant aspects involving dengue virus, one on biotechnology of cell growth and virus production, and the other on virus biology concerning genetic variation of a virus population. We investigated the growth of C6/36 mosquito cells and Vero cells grown on Cytodex 1 microcarriers. High-titer DEN virus production can be achieved in C6/36 and Vero cells infected at low cell inoculation density, in the lag-phase cell stage, and at low multiplicity of infection (MOI). The maximum titers produced for DEN-1, DEN-3, and DEN-4 viruses were approximately 10- to 10,000-fold lower than for DEN-2 virus produced in C6/36 and Vero cells grown on microcarriers. The DEN-2 virus produced in C6/36 cells displayed far more extensive plaque heterogeneity than in Vero cells. Microcarrier C6/36 mosquito cell culture appeared to be the most effective system for four-serotype DEN virus production. Interestingly, some selected variants of DEN virus may outgrow in Vero cells when using a T-flask culture. These results may provide useful information for DEN vaccine development.     

N-linked glycosylation of west nile virus envelope proteins influences particle assembly and infectivity

West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E). While the prM protein of all WNV strains contains a single N-linked glycosylation site, not all strains contain an N-linked site in the E protein. The presence of N-linked glycosylation on flavivirus E proteins has been linked to virus production, pH sensitivity, and neuroinvasiveness. Therefore, we examined the impact of prM and E glycosylation on WNV assembly and infectivity. Similar to other flaviviruses, expression of WNV prM and E resulted in the release of subviral particles (SVPs). Removing the prM glycosylation site in a lineage I or II strain decreased SVP release, as did removal of the glycosylation site in a lineage I E protein. Addition of the E protein glycosylation site in a lineage II strain that lacked this site increased SVP production. Similar results were obtained in the context of either reporter virus particles (RVPs) or infectious lineage II WNV. RVPs or virions bearing combinations of glycosylated and nonglycosylated forms of prM and E could infect mammalian, avian, and mosquito cells (BHK-21, QT6, and C6/36, respectively). Those particles lacking glycosylation on the E protein were modestly more infectious per genome copy on BHK-21 and QT6 cells, while this absence greatly enhanced the infection of C6/36 cells. Thus, glycosylation of WNV prM and E proteins can affect the efficiency of virus release and infection in a manner that is cell type and perhaps species dependent. This suggests a multifaceted role for envelope N-linked glycosylation in WNV biology and tropism.     

Source and Purity of Dengue-Viral Preparations Impact Requirement for Enhancing Antibody to Induce Elevated IL-1β Secretion: A Primary Human Monocyte Model

Dengue virus is a major global health threat and can lead to life-threatening hemorrhagic complications due to immune activation and cytokine production. Cross-reactive antibodies to an earlier dengue virus infection are a recognized risk factor for severe disease. These antibodies bind heterologous dengue serotypes and enhance infection into Fc-receptor-bearing cells, a process known as antibody-dependent enhancement of infection. One crucial cytokine seen elevated in severe dengue patients is IL-1β, a potent inflammatory cytokine matured by the inflammasome. We used a highly-physiologic system by studying antibody-dependent enhancement of IL-1β in primary human monocytes with anti-dengue human monoclonal antibodies isolated from patients. Antibody-enhancement increased viral replication in primary human monocytes inoculated with supernatant harvested from Vero cells infected with dengue virus serotype 2 (DENV-2) 16681. Surprisingly, IL-1β secretion induced by infectious supernatant harvested from two independent Vero cell lines was not enhanced by antibody. Secretion of multiple other inflammatory cytokines was also independent of antibody signaling. However, IL-1β secretion did require NLRP3 and caspase-1 activity. Immunodepletion of dengue virions from the infectious supernatant confirmed that virus was not the main IL-1β-inducing agent, suggesting that a supernatant component(s) not associated with the virion induced IL-1β production. We excluded RNA, DNA, contaminating LPS, viral NS1 protein, complement, and cytokines. In contrast, purified Vero-derived DENV-2 16681 exhibited antibody-enhancement of both infection and IL-1β induction. Furthermore, C6/36 mosquito cells did not produce such an inflammatory component, as crude supernatant harvested from insect cells infected with DENV-2 16681 induced antibody-dependent IL-1β secretion. This study indicates that Vero cells infected with DENV-2 16681 may produce inflammatory components during dengue virus propagation that mask the virus-specific immune response. Thus, the choice of host cell and viral purity should be carefully considered, while insect-derived virus represents a system that elicits antibody-dependent cytokine responses to dengue virus with fewer confounding issues.     

Identification of two surface proteins from C6/36 cells that bind dengue type 4 virus.

Dengue viruses infect cells by attaching to a surface receptor, probably through the envelope (E) glycoprotein, located on the surface of the viral membrane. However, the identity of the dengue virus receptor in the mosquito and in mammalian host cells remains unknown. To identify and characterize the molecules responsible for binding dengue virus, overlay protein blot and binding assays were performed with labeled virus. Two glycoproteins of 40 and 45 kDa located on the surface of C6/36 cells bound dengue type 4 virus. Virus binding by total and membrane proteins obtained from trypsin-treated cells was inhibited, while neuraminidase treatment did not inhibit binding. Periodate treatment of cell proteins did not reduce virus binding, but it modified the molecular weight of the polypeptide detected by overlay assays. Preincubation of C6/36 cells with electroeluted 40- and 45-kDa proteins or with specific antibodies raised against these proteins inhibited virus binding. These results strongly suggest that the 40- and 45-kDa surface proteins are putative receptors or part of a receptor complex for dengue virus.     

Dicer-2- and Piwi-Mediated RNA Interference in Rift Valley Fever Virus-Infected Mosquito Cells

Rift Valley fever virus (RVFV) is a Phlebovirus (Bunyaviridae family) transmitted by mosquitoes. It infects humans and ruminants, causing dramatic epidemics and epizootics in Africa, Yemen, and Saudi Arabia. While recent studies demonstrated the importance of the nonstructural protein NSs as a major component of virulence in vertebrates, little is known about infection of mosquito vectors. Here we studied RVFV infection in three different mosquito cell lines, Aag2 cells from Aedes aegypti and U4.4 and C6/36 cells from Aedes albopictus. In contrast with mammalian cells, where NSs forms nuclear filaments, U4.4 and Aag2 cells downregulated NSs expression such that NSs filaments were never formed in nuclei of U4.4 cells and disappeared at an early time postinfection in the case of Aag2 cells. On the contrary, in C6/36 cells, NSs nuclear filaments were visible during the entire time course of infection. Analysis of virus-derived small interfering RNAs (viRNAs) by deep sequencing indicated that production of viRNAs was very low in C6/36 cells, which are known to be Dicer-2 deficient but expressed some viRNAs presenting a Piwi signature. In contrast, Aag2 and U4.4 cells produced large amounts of viRNAs predominantly matching the S segment and displaying Dicer-2 and Piwi signatures. Whereas 21-nucleotide (nt) Dicer-2 viRNAs were prominent during early infection, the population of 24- to 27-nt Piwi RNAs (piRNAs) increased progressively and became predominant later during the acute infection and during persistence. In Aag2 and U4.4 cells, the combined actions of the Dicer-2 and Piwi pathways triggered an efficient antiviral response permitting, among other actions, suppression of NSs filament formation and allowing establishment of persistence. In C6/36 cells, Piwi-mediated RNA interference (RNAi) appeared to be sufficient to mount an antiviral response against a secondary infection with a superinfecting virus. This study provides new insights into the role of Dicer and Piwi in mosquito antiviral defense and the development of the antiviral response in mosquitoes.     

Identification of a putative coreceptor on Vero cells that participates in dengue 4 virus infection

Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (alpha-mannose or N-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells.     

An external loop region of domain III of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells

Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.     

Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome, causes a mosquito-borne disease that is endemic in sub-Saharan African countries and that also causes large epidemics among humans and livestock. Furthermore, it is a bioterrorist threat and poses a risk for introduction to other areas. In spite of its danger, neither veterinary nor human vaccines are available. We established a T7 RNA polymerase-driven reverse genetics system to rescue infectious clones of RVFV MP-12 strain entirely from cDNA, the first for any phlebovirus. Expression of viral structural proteins from the protein expression plasmids was not required for virus rescue, whereas NSs protein expression abolished virus rescue. Mutants of MP-12 partially or completely lacking the NSs open reading frame were viable. These NSs deletion mutants replicated efficiently in Vero and 293 cells, but not in MRC-5 cells. In the latter cell line, accumulation of beta interferon mRNA occurred after infection by these NSs deletion mutants, but not after infection by MP-12. The NSs deletion mutants formed larger plaques than MP-12 did in Vero E6 cells and failed to shut off host protein synthesis in Vero cells. An MP-12 mutant carrying a luciferase gene in place of the NSs gene replicated as efficiently as MP-12 did, produced enzymatically active luciferase during replication, and stably retained the luciferase gene after 10 virus passages, representing the first demonstration of foreign gene expression in any bunyavirus. This reverse genetics system can be used to study the molecular virology of RVFV, assess current vaccine candidates, produce new vaccines, and incorporate marker genes into animal vaccines.     

Nuclear localization of Japanese encephalitis virus core protein enhances viral replication

Japanese encephalitis virus (JEV) core protein was detected in both the nucleoli and cytoplasm of mammalian and insect cell lines infected with JEV or transfected with the expression plasmid of the core protein. Mutation analysis revealed that Gly(42) and Pro(43) in the core protein are essential for the nuclear and nucleolar localization. A mutant M4243 virus in which both Gly(42) and Pro(43) were replaced by Ala was recovered by plasmid-based reverse genetics. In C6/36 mosquito cells, the M4243 virus exhibited RNA replication and protein synthesis comparable to wild-type JEV, whereas propagation in Vero cells was impaired. The mutant core protein was detected in the cytoplasm but not in the nucleus of either C6/36 or Vero cell lines infected with the M4243 virus. The impaired propagation of M4243 in mammalian cells was recovered by the expression of wild-type core protein in trans but not by that of the mutant core protein. Although M4243 mutant virus exhibited a high level of neurovirulence comparable to wild-type JEV in spite of the approximately 100-fold-lower viral propagation after intracerebral inoculation to 3-week-old mice of strain Jcl:ICR, no virus was recovered from the brain after intraperitoneal inoculation of the mutant. These results indicate that nuclear localization of JEV core protein plays crucial roles not only in the replication in mammalian cells in vitro but also in the pathogenesis of encephalitis induced by JEV in vivo.     

The translational requirement for complete La Crosse virus mRNA synthesis is cell-type dependent.

The translational requirement to prevent premature termination during La Crosse virus S mRNA synthesis was found to be cell-type dependent. This requirement was present in the BHK, HEL, and Vero cell lines we examined, but not in C6/36 mosquito cells. The cell-dependent translational requirement could be reproduced in vitro by using either cell extracts or purified virions of BHK and C6/36 cells. In the BHK reactions, the polymerase terminated predominantly at nucleotide 175 in the absence of concurrent translation and required translation to read through this position. In the C6/36 reactions, however, the polymerase reads through nucleotide 175 efficiently independent of translation. Reconstitution studies suggested that the translational requirement was due to a factor(s) present in BHK, but not in C6/36, cells.     

Role of Bunyamwera Orthobunyavirus NSs Protein in Infection of Mosquito Cells

Background

Bunyamwera orthobunyavirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera virus in cultured mosquito cells other than the Aedes albopictus C6/36 line.

Methodology and Principal Findings

To determine potential functions of the NSs protein in mosquito cells, replication of wild-type virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and virus yields at various times post infection. Both viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced virus replication in U4.4 cells transfected with virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type virus, infection rates by the mutant virus were lower and more variable. If the NSs deletion virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type virus.

Conclusions/Significance

Bunyamwera virus NSs is required for efficient replication in certain mosquito cell lines and in Ae. aegypti mosquitoes.     

The envelope glycoprotein domain III of dengue virus serotypes 1 and 2 inhibit virus entry

Dengue virus (DV) is a flavivirus and its urban transmission is maintained largely by its mosquito vectors and vertebrate host, often human. In this study, investigation was carried out on the involvement of domain III of the envelope (E) glycosylated protein of dengue virus serotypes 1 and 2 (DV-1 and DV-2 DIII) in binding to host cell surfaces, thus mediating virus entry. Domain III protein of flavivirus can also serve as an attractive target in inhibiting virus entry. The respective DV DIII proteins were expressed as soluble recombinant fusion proteins before purification through enzymatic cleavage and affinity purification. The purified recombinant DV-1 and DV-2 DIII proteins both demonstrated the ability to inhibit the entry of DV-1 and DV-2 into HepG2 cells and C6/36 mosquito cells. As such, the DV DIII protein is indeed important for the interaction with cellular receptors in both human and mosquito cells. In addition, this protein induced antibodies that completely neutralized homologous dengue serotypes although not with the same efficiency among the heterologous serotypes. This observation may be of importance when formulating a generic vaccine that is effective against all dengue virus serotypes.     

Predicting the Mosquito Species and Vertebrate Species Involved in the Theoretical Transmission of Rift Valley Fever Virus in the United States

Rift Valley fever virus (RVFV) is a mosquito-borne virus in the family Bunyaviridiae that has spread throughout continental Africa to Madagascar and the Arabian Peninsula. The establishment of RVFV in North America would have serious consequences for human and animal health in addition to a significant economic impact on the livestock industry. Published and unpublished data on RVFV vector competence, vertebrate host competence, and mosquito feeding patterns from the United States were combined to quantitatively implicate mosquito vectors and vertebrate hosts that may be important to RVFV transmission in the United States. A viremia-vector competence relationship based on published mosquito transmission studies was used to calculate a vertebrate host competence index which was then combined with mosquito blood feeding patterns to approximate the vector and vertebrate amplification fraction, defined as the relative contribution of the mosquito or vertebrate host to pathogen transmission. Results implicate several Aedes spp. mosquitoes and vertebrates in the order Artiodactyla as important hosts for RVFV transmission in the U.S. Moreover, this study identifies critical gaps in knowledge which would be necessary to complete a comprehensive analysis identifying the different contributions of mosquitoes and vertebrates to potential RVFV transmission in the U.S. Future research should focus on (1) the dose-dependent relationship between viremic exposure and the subsequent infectiousness of key mosquito species, (2) evaluation of vertebrate host competence for RVFV among North American mammal species, with particular emphasis on the order Artiodactyla, and (3) identification of areas with a high risk for RVFV introduction so data on local vector and host populations can help generate geographically appropriate amplification fraction estimates.