The Intestinal Microbiome Restricts Alphavirus Infection and Dissemination through a Bile Acid-Type I IFN Signaling Axis.

Chikungunya virus (CHIKV), an emerging alphavirus, has infected millions of people. However, the factors modulating disease outcome remain poorly understood. Here, we show in germ-free mice or in oral antibiotic-treated conventionally housed mice with depleted intestinal microbiomes that greater CHIKV infection and spread occurs within 1 day of virus inoculation. Alteration of the microbiome alters TLR7-MyD88 signaling in plasmacytoid dendritic cells (pDCs) and blunts systemic production of type I interferon (IFN). Consequently, circulating monocytes express fewer IFN-stimulated genes and become permissive for CHIKV infection. Reconstitution with a single bacterial species, Clostridium scindens, or its derived metabolite, the secondary bile acid deoxycholic acid, can restore pDC- and MyD88-dependent type I IFN responses to restrict systemic CHIKV infection and transmission back to vector mosquitoes. Thus, symbiotic intestinal bacteria modulate antiviral immunity and levels of circulating alphaviruses within hours of infection through a bile acid-pDC-IFN signaling axis, which affects viremia, dissemination, and potentially transmission.

Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination.

Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 µg of nucleoside-modified ZIKV mRNA-LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 µg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA-LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.

Inactivation of chikungunya virus in blood components treated with amotosalen/ultraviolet A light or amustaline/glutathione.

BACKGROUND: Chikungunya virus, a mosquito-borne arbovirus, often co-circulates with the Zika, dengue, and yellow fever viruses in Aedes mosquito-infested areas where cases of arbovirus transfusion-transmitted infections have been reported. Building on past experience to help maintain the availability of safe components during major outbreaks of chikungunya virus in La Reunion, Italy, and Thailand and of Zika virus in the Pacific, the Caribbean, and the Americas, pathogen inactivation is a mitigation strategy to reduce the risk of transfusion-transmitted infection. Inactivation of chikungunya virus was investigated for platelets in 100% plasma using amotosalen/ultraviolet A light, and in red blood cells using amustaline/glutathione. STUDY DESIGN AND METHODS: Platelets in 100% plasma and red blood cells (RBCs) were spiked with chikungunya virus. Infectious chikungunya virus titers were measured in contaminated blood products before and after treatment with amotosalen/ultraviolet A light for platelets in 100% plasma and after treatment with amustaline/glutathione for RBCs. Viral infectivity was quantified by plaque assay. RESULTS: The mean chikungunya virus infectivity titers before inactivation were 6.50 log10 plaque-forming units/mL for platelets in 100% plasma and 7.60 log10 plaque-forming units/mL for RBCs. No infectivity was detected after amotosalen/ultraviolet A light or amustaline/glutathione treatment, corresponding to greater than 6.5 log10 plaque-forming units/mL and greater than 7.1 log10 plaque-forming units/mL of inactivation, respectively. CONCLUSION: Robust levels of chikungunya virus inactivation were achieved for platelets in 100% plasma and for RBC components. The licensed amotosalen/ultraviolet A light technology and the amustaline/glutathione pathogen-reduction system under development may provide an opportunity for comprehensive mitigation of the risk of chikungunya virus transfusion-transmitted infection by plasma, platelets, and RBCs.

A novel mosquito ubiquitin targets viral envelope protein for degradation and reduces virion production during dengue virus infection.

BACKGROUND: Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant human disease and mortality in the tropics and subtropics. By examining the effects of virus infection on gene expression, and interactions between virus and vector, new targets for prevention of infection and novel treatments may be identified in mosquitoes. We previously performed a microarray analysis of the Aedes aegypti transcriptome during infection with DENV and found that mosquito ubiquitin protein Ub3881 (AAEL003881) was specifically and highly down-regulated. Ubiquitin proteins have multiple functions in insects, including marking proteins for proteasomal degradation, regulating apoptosis and mediating innate immune signaling. METHODS: We used qRT-PCR to quantify gene expression and infection, and RNAi to reduce Ub3881 expression. Mosquitoes were infected with DENV through blood feeding. We transfected DENV protein expression constructs to examine the effect of Ub3881 on protein degradation. We used site-directed mutagenesis and transfection to determine what amino acids are involved in Ub3881-mediated protein degradation. Immunofluorescence, Co-immunoprecipitation and Western blotting were used to examine protein interactions and co-localization. RESULTS: The overexpression of Ub3881, but not related ubiquitin proteins, decreased DENV infection in mosquito cells and live Ae. aegypti. The Ub3881 protein was demonstrated to be involved in DENV envelope protein degradation and reduce the number of infectious virions released. CONCLUSIONS: We conclude that Ub3881 has several antiviral functions in the mosquito, including specific viral protein degradation. GENERAL SIGNIFICANCE: Our data highlights Ub3881 as a target for future DENV prevention strategies in the mosquito transmission vector.

Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.

Infection of myofibers contributes to increased pathogenicity during infection with an epidemic strain of chikungunya virus.

UNLABELLED: Chikungunya virus (CHIKV) is an alphavirus transmitted by mosquitoes that is known to cause severe arthritis and myositis in affected patients. The ongoing epidemic began in eastern Africa in 2004 and then spread to islands of the Indian Ocean, India, and Southeast Asia, ultimately afflicting millions. During this outbreak, more severe disease manifestations, including fatalities, have been documented. The reasons for this change in pathogenesis are multifactorial but likely include mutations that have arisen in the viral genome which could alter disease pathogenesis. To test this hypothesis, we used a murine model of CHIKV to compare the disease pathogeneses of two recombinant strains of CHIKV, the first derived from the La Reunion outbreak in 2006 (LR2006 OPY1) and the second isolated from Senegal in 1983 (37997). While the two strains exhibited similar growth in mammalian cells in vitro, we observed more severe clinical disease and pathology in mice infected with the LR2006 OPY1 strain of CHIKV, which included prolonged viremia and elevated viral titers and persistence in the muscle, resulting in devastating myonecrosis. Both CHIKV strains infected connective tissue fibroblasts of the muscle, but only the LR2006 OPY1 strain replicated within myofibers in vivo, despite similar growth of the two strains in these cell types in vitro. However, when the 37997 strain was administered directly into muscle, myofiber infection was comparable to that in LR2006 OPY1-infected mice. These results indicate that differences in the ability of the strain of CHIKV to establish infection in myofibers may contribute to the increased disease severity. IMPORTANCE: CHIKV is an emerging pathogen that causes significant morbidity. Little is known about the pathogenesis of the disease, and this study suggests that the ability of a recent epidemic strain to infect myofibers results in increased disease severity. Better understanding of how CHIKV causes disease contributes to the ultimate goal of creating therapeutics to alleviate the impact of this debilitating virus.

Chikungunya viruses that escape monoclonal antibody therapy are clinically attenuated, stable, and not purified in mosquitoes.

UNLABELLED: Chikungunya virus (CHIKV) is a reemerging mosquito-transmitted alphavirus that causes epidemics of debilitating polyarthritis in humans. A prior study identified two anti-CHIKV monoclonal antibodies ([MAbs] CHK-152 and CHK-166) against the E2 and E1 structural proteins, which had therapeutic efficacy in immunocompetent and immunocompromised mice. Combination MAb therapy was required as administration of a single MAb resulted in the rapid selection of neutralization escape variants and treatment failure in mice. Here, we initially evaluated the efficacy of combination MAb therapy in a nonhuman primate model of CHIKV infection. Treatment of rhesus macaques with CHK-152 and CHK-166 reduced viral spread and infection in distant tissue sites and also neutralized reservoirs of infectious virus. Escape viruses were not detected in the residual viral RNA present in tissues and organs of rhesus macaques. To evaluate the possible significance of MAb resistance, we engineered neutralization escape variant viruses (E1-K61T, E2-D59N, and the double mutant E1-K61T E2-D59N) that conferred resistance to CHK-152 and CHK-166 and tested them for fitness in mosquito cells, mammalian cells, mice, and Aedes albopictus mosquitoes. In both cell culture and mosquitoes, the mutant viruses grew equivalently and did not revert to wild-type (WT) sequence. All escape variants showed evidence of mild clinical attenuation, with decreased musculoskeletal disease at early times after infection in WT mice and a prolonged survival time in immunocompromised Ifnar1(-/-) mice. Unexpectedly, this was not associated with decreased infectivity, and consensus sequencing from tissues revealed no evidence of reversion or compensatory mutations. Competition studies with CHIKV WT also revealed no fitness compromise of the double mutant (E1-K61T E2-D59N) neutralization escape variant in WT mice. Collectively, our study suggests that neutralization escape viruses selected during combination MAb therapy with CHK-152 plus CHK-166 retain fitness, cause less severe clinical disease, and likely would not be purified during the enzootic cycle. IMPORTANCE: Chikungunya virus (CHIKV) causes explosive epidemics of acute and chronic arthritis in humans in Africa, the Indian subcontinent, and Southeast Asia and recently has spread to the New World. As there are no approved vaccines or therapies for human use, the possibility of CHIKV-induced debilitating disease is high in many parts of the world. To this end, our laboratory recently generated a combination monoclonal antibody therapy that aborted lethal and arthritogenic disease in wild-type and immunocompromised mice when administered as a single dose several days after infection. In this study, we show the efficacy of the antibody combination in nonhuman primates and also evaluate the significance of possible neutralization escape mutations in mosquito and mammalian cells, mice, and Aedes albopictus vector mosquitoes. Our experiments show that escape viruses from combination antibody therapy cause less severe CHIKV clinical disease, retain fitness, and likely would not be purified by mosquito vectors.

Mutagenesis analysis of T380R mutation in the envelope protein of yellow fever virus.

BACKGROUND: The RGD motif in the mosquito-borne flaviviruses envelope protein domain III (EDIII) FG loop was shown to bind negatively charged cellular molecules and mediate virus entry in mammals. However, its importance in virus entry in the mosquito has not yet been defined. The sequences of RGD motifs are conserved in JEV-serocomplex members primarily transmitted by Culex mosquitoes but absent from members of the DENV serocomplex, which utilize Aedes mosquitoes as vectors. Interestingly, the RGD sequence is present in the attenuated 17D strain of yellow fever virus as a result of the T380R mutation in the EDIII of Asibi strain following extensive in vitro passage in mice and chicken embryos and was found to contribute to the more rapid clearance in mice challenged with 17D. However, viral infectivity and dissemination in mosquitoes had not been evaluated for this mutant. FINDINGS: The study utilized the reverse genetics system of YFV and Ae. aegypti RexD WE mosquitoes to assess the impact of a T380R mutation in YFV Asibi and 17D/Asibi M-E chimera. The T380R mutation led to higher infection rates but similar dissemination rates when introduced into the YFV Asibi strain and 17D/Asibi M-E chimera. CONCLUSIONS: While the increase of the positive charge in EDIII may reduce the virulence of YFV in mice, this mutation favored the establishment of the viral infection in Ae. aegypti. However, such gain in viral infectivity did not increase dissemination in infected mosquitoes.

pH-Dependent entry of chikungunya virus fusion into mosquito cells.

BACKGROUND: Millions of human infections caused by arthropod-borne pathogens are initiated by the feeding of an infected mosquito on a vertebrate. However, interactions between the viruses and the mosquito vector, which facilitates successful infection and transmission of virus to a subsequent vertebrate host, are still not fully understood. FINDING: Here we describe early chikungunya virus (CHIKV) infectious events in cells derived from one of the most important CHIKV vectors, Aedes albopictus. We demonstrated that CHIKV infection of mosquito cells depended on acidification of the endosome as indicated by significant inhibition following prophylactic treatment with the lysosomotropic drugs chloroquine, ammonium chloride, and monensin, which is consistent with observations in mammalian cells. While all three agents inhibited CHIKV infection in C6/36 cells, ammonium chloride was less toxic to cells than the other agents. CONCLUSION: The observation of similar mechanisms for inhibition of CHIKV infection in mosquito and mammalian cell lines suggests that conserved entry pathways are utilized by CHIKV for vertebrate and invertebrate cell types.

Detection of anti-premembrane antibody as a specific marker of four flavivirus serocomplexes and its application to serosurveillance in endemic regions.

In the past few decades, several emerging/re-emerging mosquito-borne flaviviruses have resulted in disease outbreaks of public health concern in the tropics and subtropics. Due to cross-reactivities of antibodies recognizing the envelope protein of different flaviviruses, serosurveillance remains a challenge. Previously we reported that anti-premembrane (prM) antibody can discriminate between three flavivirus infections by Western blot analysis. In this study, we aimed to develop a serological assay that can discriminate infection or exposure with flaviviruses from four serocomplexes, including dengue (DENV), Zika (ZIKV), West Nile (WNV) and yellow fever (YFV) viruses, and explore its application for serosurveillance in flavivirus-endemic countries. We employed Western blot analysis including antigens of six flaviviruses (DENV1, 2 and 4, WNV, ZIKV and YFV) from four serocomplexes. We tested serum samples from YF-17D vaccinees, and from DENV, ZIKV and WNV panels that had been confirmed by RT-PCR or by neutralization assays. The overall sensitivity/specificity of anti-prM antibodies for DENV, ZIKV, WNV, and YFV infections/exposure were 91.7%/96.4%, 91.7%/99.2%, 88.9%/98.3%, and 91.3%/92.5%, respectively. When testing 48 samples from Brazil, we identified multiple flavivirus infections/exposure including DENV and ZIKV, DENV and YFV, and DENV, ZIKV and YFV. When testing 50 samples from the Philippines, we detected DENV, ZIKV, and DENV and ZIKV infections with a ZIKV seroprevalence rate of 10%, which was consistent with reports of low-level circulation of ZIKV in Asia. Together, these findings suggest that anti-prM antibody is a flavivirus serocomplex-specific marker and can be employed to delineate four flavivirus infections/exposure in regions where multiple flaviviruses co-circulate.

Characterization of Two Highly Specific Monoclonal Antibodies Targeting the Glycan Loop of the Zika Virus Envelope Protein.

Zika virus (ZIKV) is an emerging flavivirus associated with several neurological diseases such as Guillain-Barré syndrome in adults and microcephaly in newborn children. Its distribution and mode of transmission (via Aedes aegypti and Aedes albopictus mosquitoes) collectively cause ZIKV to be a serious concern for global health. High genetic homology of flaviviruses and shared ecology is a hurdle for accurate detection. Distinguishing infections caused by different viruses based on serological recognition can be misleading as many anti-flavivirus monoclonal antibodies (mAbs) discovered to date are highly cross-reactive, especially those against the envelope (E) protein. To provide more specific research tools, we produced ZIKV E directed hybridoma cell lines and characterized two highly ZIKV-specific mAb clones (mAbs A11 and A42) against several members of the Flavivirus genus. Epitope mapping of mAb A11 revealed glycan loop specificity in Domain I of the ZIKV E protein. The development of two highly specific mAbs targeting the surface fusion protein of ZIKV presents a significant advancement in research capabilities as these can be employed as essential tools to enhance our understanding of ZIKV identification on infected cells ex vivo or in culture.

Alterations in the Aedes aegypti transcriptome during infection with West Nile, dengue and yellow fever viruses.

West Nile (WNV), dengue (DENV) and yellow fever (YFV) viruses are (re)emerging, mosquito-borne flaviviruses that cause human disease and mortality worldwide. Alterations in mosquito gene expression common and unique to individual flaviviral infections are poorly understood. Here, we present a microarray analysis of the Aedes aegypti transcriptome over time during infection with DENV, WNV or YFV. We identified 203 mosquito genes that were ≥ 5-fold differentially up-regulated (DUR) and 202 genes that were ≥ 10-fold differentially down-regulated (DDR) during infection with one of the three flaviviruses. Comparative analysis revealed that the expression profile of 20 DUR genes and 15 DDR genes was quite similar between the three flaviviruses on D1 of infection, indicating a potentially conserved transcriptomic signature of flaviviral infection. Bioinformatics analysis revealed changes in expression of genes from diverse cellular processes, including ion binding, transport, metabolic processes and peptidase activity. We also demonstrate that virally-regulated gene expression is tissue-specific. The overexpression of several virally down-regulated genes decreased WNV infection in mosquito cells and Aedes aegypti mosquitoes. Among these, a pupal cuticle protein was shown to bind WNV envelope protein, leading to inhibition of infection in vitro and the prevention of lethal WNV encephalitis in mice. This work provides an extensive list of targets for controlling flaviviral infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.

ISG15 is critical in the control of Chikungunya virus infection independent of UbE1L mediated conjugation.

Chikungunya virus (CHIKV) is a re-emerging alphavirus that has caused significant disease in the Indian Ocean region since 2005. During this outbreak, in addition to fever, rash and arthritis, severe cases of CHIKV infection have been observed in infants. Challenging the notion that the innate immune response in infants is immature or defective, we demonstrate that both human infants and neonatal mice generate a robust type I interferon (IFN) response during CHIKV infection that contributes to, but is insufficient for, the complete control of infection. To characterize the mechanism by which type I IFNs control CHIKV infection, we evaluated the role of ISG15 and defined it as a central player in the host response, as neonatal mice lacking ISG15 were profoundly susceptible to CHIKV infection. Surprisingly, UbE1L⁻/⁻ mice, which lack the ISG15 E1 enzyme and therefore are unable to form ISG15 conjugates, displayed no increase in lethality following CHIKV infection, thus pointing to a non-classical role for ISG15. No differences in viral loads were observed between wild-type (WT) and ISG15⁻/⁻ mice, however, a dramatic increase in proinflammatory cytokines and chemokines was observed in ISG15⁻/⁻ mice, suggesting that the innate immune response to CHIKV contributes to their lethality. This study provides new insight into the control of CHIKV infection, and establishes a new model for how ISG15 functions as an immunomodulatory molecule in the blunting of potentially pathologic levels of innate effector molecules during the host response to viral infection.

Photochemical inactivation of chikungunya virus in plasma and platelets using the Mirasol pathogen reduction technology system.

BACKGROUND: Chikungunya virus (CHIKV) is a reemerging mosquito-borne virus that has been responsible for a number of large-scale epidemics as well as imported cases covering a wide geographical range. As a blood-borne virus capable of mounting a high-titer viremia in infected humans, CHIKV was included on a list of risk agents for transfusion and organ transplant by the AABB Transfusion-Transmitted Diseases Committee. Therefore, we evaluated the ability of the Mirasol pathogen reduction technology (PRT) system (CaridianBCT Biotechnologies) to inactivate live virus in contaminated plasma and platelet (PLT) samples. STUDY DESIGN AND METHODS: Plasma, PLTs, and phosphate-buffered saline controls were spiked with CHIKV and treated with riboflavin and varying doses of ultraviolet (UV) light using the Mirasol PRT system. Samples were tested before and after treatment for cytotoxicity, interference, and virus titer by titration and quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: A significant reduction in CHIKV titer of greater than 99% was recorded after treatment of plasma or PLTs with the Mirasol PRT system, and the titer reduction was directly proportional to the UV dose delivered to the samples. No cytotoxicity of interference was observed in any sample at any treatment dose. CONCLUSION: These data indicate that the Mirasol PRT system efficiently inactivated live CHIKV in plasma and PLTs and could therefore potentially be used to prevent CHIKV transmission through the blood supply.

Quantitative proteomic analysis of the Anopheles gambiae (Diptera: Culicidae) midgut infected with o'nyong-nyong virus.

Alphaviruses are arthropod-borne pathogens that infect a range of hosts. In humans and other mammals, alphavirus infection can cause severe disease. In mosquito hosts, however, there are generally few symptoms. Little is known about the cellular responses of mosquitoes that allow them to cope with infection. In this investigation, a six-plex tandem mass tagging proteomic approach was used to study protein accumulation changes in the midgut of Anopheles gambiae (Giles) (Diptera: Culicidae) mosquitoes infected with o'nyong-nyong virus (Togaviridae, Alphavirus). Five hundred thirty-six nonredundant proteins were identified. Twenty-two were found in significantly different quantities in infected midguts compared with controls. Of interest, analysis revealed molecular pathways possibly targeted by virus proteins, such as those involving TAF4 and DNA polymerase phi proteins. Also identified was an FK506-binding protein. FK506-binding protein orthologs have been described as conserved host resistance factors, which suppress dengue and West Nile virus infection in human HeLa cells. This investigation constitutes the first study of the midgut-specific proteome of An. gambiae in relation to alphavirus infection. Our findings offer insight into mosquito immunity, including factors that possibly contribute to the different pathological outcomes observed in vertebrate and insect hosts.

Comparison of Immunogenicity Between a Candidate Live Attenuated Vaccine and an Inactivated Vaccine for Cache Valley Virus.

Cache Valley virus (CVV) is a mosquito-borne bunyavirus that is enzootic throughout the new world. Although CVV is known as an important agricultural pathogen, primarily associated with embryonic lethality and abortions in ruminants, it has recently been recognized for its expansion as a zoonotic pathogen. With the increased emergence of bunyaviruses with human and veterinary importance, there have been significant efforts dedicated to the development of bunyavirus vaccines. In this study, the immunogenicity of a candidate live-attenuated vaccine (LAV) for CVV, which contains the deletion of the nonstructural small (NSs) and nonstructural medium (NSm) genes (2delCVV), was evaluated and compared with an autogenous candidate vaccine created through the inactivation of CVV using binary ethylenimine (BEI) with an aluminum hydroxide adjuvant (BEI-CVV) in sheep. Both 2delCVV and BEI-CVV produced a neutralizing antibody response that exceeds the correlate of protection, that is, plaque reduction neutralization test titer >10. However, on day 63 postinitial immunization, 2delCVV was more immunogenic than BEI-CVV. These results warrant further development of 2delCVV as a candidate LAV and demonstrate that the double deletion of the NSs and NSm genes can be applied to the development of vaccines and as a common attenuation strategy for orthobunyaviruses.

Immunogenicity of a Candidate Live Attenuated Vaccine for Rift Valley Fever Virus with a Two-Segmented Genome.

Rift Valley fever virus (RVFV) is an emerging arbovirus that affects both ruminants and humans. RVFV causes severe and recurrent outbreaks in Africa and the Arabian Peninsula with a significant risk for emergence into new locations. Although there are a variety of RVFV veterinary vaccines for use in endemic areas, there is currently no licensed vaccine for human use; therefore, there is a need to develop and assess new vaccines. Herein, we report a live-attenuated recombinant vaccine candidate for RVFV, based on the previously described genomic reconfiguration of the conditionally licensed MP12 vaccine. There are two general strategies used to develop live-attenuated RVFV vaccines, one being serial passage of wild-type RVFV strains to select attenuated mutants such as Smithburn, Clone 13, and MP12 vaccine strains. The second strategy has utilized reverse genetics to attenuate RVFV strains by introducing deletions or insertions within the viral genome. The novel candidate vaccine characterized in this report contains a two-segmented genome that lacks the medium viral segment (M) and two virulence genes (nonstructural small and nonstructural medium). The vaccine candidate, named r2segMP12, was evaluated for the production of neutralizing antibodies to RVFV in outbred CD-1 mice. The immune response induced by the r2segMP12 vaccine candidate was directly compared to the immune response induced by the rMP12 parental strain vaccine. Our study demonstrated that a single immunization with the r2segMP12 vaccine candidate at 105 plaque-forming units elicited a higher neutralizing antibody response than the rMP12 vaccine at the same vaccination titer without the need for a booster.

Infection of Feral Phenotype Swine with Japanese Encephalitis Virus.

Background: Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus and the leading cause of pediatric encephalitis in the Asian Pacific region. The transmission cycle primarily involves Culex spp. mosquitoes and Ardeid birds, with domestic pigs (Sus scrofa domestica) being the source of infectious viruses for the spillover of JEV from the natural endemic transmission cycle into the human population. Although many studies have concluded that domestic pigs play an important role in the transmission cycle of JEV, and infection of humans, the role of feral pigs in the transmission of JEV remains unclear. Since domestic and feral pigs are the same species, and because feral pig populations in the United States are increasing and expanding geographically, the current study aimed to test the hypothesis that if JEV were introduced into the United States, feral pigs might play a role in the transmission cycle. Materials and Methods: Sinclair miniature pigs, that exhibit the feral phenotype, were intradermally inoculated with JEV genotype Ib. These pigs were derived from crossing miniature domestic pig with four strains of feral pigs and were used since obtaining feral swine was not possible. Results: The Sinclair miniature pigs became viremic and displayed pathological outcomes similar to those observed in domestic swine. Conclusion: Based on these findings, we conclude that in the event of JEV being introduced into the United States, feral pig populations could contribute to establishment and maintenance of a transmission cycle of JEV and could lead to the virus becoming endemic in the United States.

Identification of the flavivirus conserved residues in the envelope protein hinge region for the rational design of a candidate West Nile live-attenuated vaccine.

The flavivirus envelope protein is a class II fusion protein that drives flavivirus-cell membrane fusion. The membrane fusion process is triggered by the conformational change of the E protein from dimer in the virion to trimer, which involves the rearrangement of three domains, EDI, EDII, and EDIII. The movement between EDI and EDII initiates the formation of the E protein trimer. The EDI-EDII hinge region utilizes four motifs to exert the hinge effect at the interdomain region and is crucial for the membrane fusion activity of the E protein. Using West Nile virus (WNV) NY99 strain derived from an infectious clone, we investigated the role of eight flavivirus-conserved hydrophobic residues in the EDI-EDII hinge region in the conformational change of E protein from dimer to trimer and viral entry. Single mutations of the E-A54, E-I130, E-I135, E-I196, and E-Y201 residues affected infectivity. Importantly, the E-A54I and E-Y201P mutations fully attenuated the mouse neuroinvasive phenotype of WNV. The results suggest that multiple flavivirus-conserved hydrophobic residues in the EDI-EDII hinge region play a critical role in the structure-function of the E protein and some contribute to the virulence phenotype of flaviviruses as demonstrated by the attenuation of the mouse neuroinvasive phenotype of WNV. Thus, as a proof of concept, residues in the EDI-EDII hinge region are proposed targets to engineer attenuating mutations for inclusion in the rational design of candidate live-attenuated flavivirus vaccines.

Detection of anti-premembrane antibody as a specific marker of four flavivirus serocomplexes and its application to serosurveillance in endemic regions.

In the past few decades, several emerging/re-emerging mosquito-borne flaviviruses have resulted in disease outbreaks of public health concern in the tropics and subtropics. Due to cross-reactivities of antibodies recognizing the envelope protein of different flaviviruses, serosurveillance remains a challenge. Previously we reported that anti-premembrane (prM) antibody can discriminate between three flavivirus infections by Western blot analysis. In this study, we aimed to develop a serological assay that can discriminate infection or exposure with flaviviruses from four serocomplexes, including dengue (DENV), Zika (ZIKV), West Nile (WNV) and yellow fever (YFV) viruses, and explore its application for serosurveillance in flavivirus-endemic countries. We employed Western blot analysis including antigens of six flaviviruses (DENV1, 2 and 4, WNV, ZIKV and YFV) from four serocomplexes. We tested serum samples from YF-17D vaccinees, and from DENV, ZIKV and WNV panels that had been confirmed by RT-PCR or by neutralization assays. The overall sensitivity/specificity of anti-prM antibodies for DENV, ZIKV, WNV, and YFV infections/exposure were 91.7%/96.4%, 91.7%/99.2%, 88.9%/98.3%, and 91.3%/92.5%, respectively. When testing 48 samples from Brazil, we identified multiple flavivirus infections/exposure including DENV and ZIKV, DENV and YFV, and DENV, ZIKV and YFV. When testing 50 samples from the Philippines, we detected DENV, ZIKV, and DENV and ZIKV infections with a ZIKV seroprevalence rate of 10%, which was consistent with reports of low-level circulation of ZIKV in Asia. Together, these findings suggest that anti-prM antibody is a flavivirus serocomplex-specific marker and can be employed to delineate four flavivirus infections/exposure in regions where multiple flaviviruses co-circulate.