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.

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.

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.

Re-Examining the Importance of Pigs in the Transmission of Japanese Encephalitis Virus.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of pediatric encephalitis in Southeast Asia. The enzootic transmission of JEV involves two types of amplifying hosts, swine and avian species. The involvement of pigs in the transmission cycle makes JEV a unique pathogen because human Japanese encephalitis cases are frequently linked to the epizootic spillover from pigs, which can not only develop viremia to sustain transmission but also signs of neurotropic and reproductive disease. The existing knowledge of the epidemiology of JEV largely suggests that viremic pigs are a source of infectious viruses for competent mosquito species, especially Culex tritaeniorhynchus in the endemic regions. However, several recently published studies that applied molecular detection techniques to the characterization of JEV pathogenesis in pigs described the shedding of JEV through multiple routes and persistent infection, both of which have not been reported in the past. These findings warrant a re-examination of the role that pigs are playing in the transmission and maintenance of JEV. In this review, we summarize discoveries on the shedding of JEV during the course of infection and analyze the available published evidence to discuss the possible role of the vector-free JEV transmission route among pigs in viral maintenance.

The Use of Arthropod-Borne Challenge Models in BSL-3Ag and BSL-4 Biocontainment.

The study of many arthropod-borne pathogens requires high biosafety considerations, including the use of specialized facilities and equipment for arthropod containment. Mosquito- and tick-borne viruses such as yellow fever, West Nile, and Crimean Congo hemorrhagic fever viruses require facilities that are suitable for housing vertebrates. Multidisciplinary studies that incorporate the vector, vertebrate, and pathogens are essential for a complete understanding of the interactions between these transmission cycle components, especially if they aim to evaluate and model relative susceptibilities of different arthropods and vertebrates to infection and transmission between these. Under laboratory conditions, these studies can be relatively simple, for example, involving colonized arthropods, small animals, and attenuated viruses. Other studies are complex with large animals, high-biocontainment pathogens, and field-collected arthropods. These require a higher level of containment and special design considerations. Both of these types of experiments have their relative merits. A thorough understanding of the issues related to these types of studies and the benefits and drawbacks to using various challenge models will enable the researcher to develop realistic goals for various experiments. This review examines the varied issues that should be considered prior to starting these experiments and covers the basics from the procurement of various arthropods, rearing, high-containment facilities and operational issues specific to work with arthropods, types of infection experiments, and specific issues with arthropod and animal experiments in biosafety levels 3 and 4.

Inactivation of SARS-CoV-2 in All Blood Components Using Amotosalen/Ultraviolet A Light and Amustaline/Glutathione Pathogen Reduction Technologies.

No cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transfusion-transmitted infections (TTI) have been reported. The detection of viral RNA in peripheral blood from infected patients and blood components from infected asymptomatic blood donors is, however, concerning. This study investigated the efficacy of the amotosalen/UVA light (A/UVA) and amustaline (S-303)/glutathione (GSH) pathogen reduction technologies (PRT) to inactivate SARS-CoV-2 in plasma and platelet concentrates (PC), or red blood cells (RBC), respectively. Plasma, PC prepared in platelet additive solution (PC-PAS) or 100% plasma (PC-100), and RBC prepared in AS-1 additive solution were spiked with SARS-CoV-2 and PR treated. Infectious viral titers were determined by plaque assay and log reduction factors (LRF) were determined by comparing titers before and after treatment. PR treatment of SARS-CoV-2-contaminated blood components resulted in inactivation of the infectious virus to the limit of detection with A/UVA LRF of >3.3 for plasma, >3.2 for PC-PAS-plasma, and >3.5 for PC-plasma and S-303/GSH LRF > 4.2 for RBC. These data confirm the susceptibility of coronaviruses, including SARS-CoV-2 to A/UVA treatment. This study demonstrates the effectiveness of the S-303/GSH treatment to inactivate SARS-CoV-2, and that PRT can reduce the risk of SARS-CoV-2 TTI in all blood components.

SARS-CoV-2 and Arthropods: A Review.

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that led to the unprecedented COVID-19 pandemic exemplifies how a lack of understanding and preparedness for emerging viruses can result in consequences on a global scale. Statements that SARS-CoV-2 could not be transmitted by arthropod vectors were made without experimental support. Here we review laboratory-based research, field studies, and environmental studies to evaluate the potential for the virus to be transmitted either biologically or mechanically by arthropods. Based on these data, we conclude that transmission by arthropods is highly unlikely to play a significant epidemiological role in the transmission of SARS-CoV-2.

Replication Kinetics of a Candidate Live-Attenuated Vaccine for Cache Valley Virus in Aedes albopictus.

Background: The emergence or re-emergence of several orthobunyaviruses (order: Bunyavirales; family: Peribunyaviridae), including Cache Valley virus (CVV) and Oropouche virus, warrants the development and evaluation of candidate live-attenuated vaccines (LAVs). Ideally, these vaccines would elicit long-lasting immunity with one single immunization. Materials and Methods: Since the deletion of two virulence factors, NSs and NSm, has been shown to attenuate the virulence phenotype of orthobunyaviruses, phleboviruses, and nairoviruses, genetic manipulation of the viral genome is considered an effective strategy for the rational design of candidate LAVs for bunyaviruses across multiple families. In addition, the deletion of Rift Valley fever virus NSs and NSm genes has been shown to reduce transmission by mosquitoes. Results: In this study, the ability of a CVV mutant lacking the NSs and NSm genes (2delCVV) to replicate in intrathoracically injected Aedes albopictus was compared with the parental wild-type CVV (wtCVV) 6V633 strain. In contrast to the robust replication of wtCVV in injected mosquitoes, the multiplication kinetics of the 2delCVV mutant was reduced by more than a 100-fold. Conclusion: These results suggest that the deletion of NSm and NSs genes is a feasible approach to rationally design candidate orthobunyavirus LAVs that are highly attenuated in mosquitoes and, therefore, pose little risk of reversion to virulence and transmission.

Impact of yellow fever virus envelope protein on wild-type and vaccine epitopes and tissue tropism.

The envelope (E) protein of flaviviruses is functionally associated with viral tissue tropism and pathogenicity. For yellow fever virus (YFV), viscerotropic disease primarily involving the liver is pathognomonic for wild-type (WT) infection. In contrast, the live-attenuated vaccine (LAV) strain 17D does not cause viscerotropic disease and reversion to virulence is associated with neurotropic disease. The relationship between structure-function of the E protein for WT strain Asibi and its LAV derivative 17D strain is poorly understood; however, changes to WT and vaccine epitopes have been associated with changes in virulence. Here, a panel of Asibi and 17D infectious clone mutants were generated with single-site mutations at the one membrane residue and each of the eight E protein amino acid substitutions that distinguish the two strains. The mutants were characterized with respect to WT-specific and vaccine-specific monoclonal antibodies (mAbs) binding to virus plus binding of virus to brain, liver, and lung membrane receptor preparations (MRPs) generated from AG129 mice. This approach shows that amino acids in the YFV E protein domains (ED) I and II contain the WT E protein epitope, which overlap with those that mediate YFV binding to mouse liver. Furthermore, amino acids in EDIII associated with the vaccine epitope overlap with those that facilitate YFV binding mouse brain MRPs. Taken together, these data suggest that the YFV E protein is a key determinant in the phenotype of WT and 17D vaccine strains of YFV.

Treatment with dry hydrogen peroxide accelerates the decay of severe acute syndrome coronavirus-2 on non-porous hard surfaces.

BACKGROUND: Disinfection of contaminated or potentially contaminated surfaces has become an integral part of the mitigation strategies for controlling coronavirus disease 2019. Whilst a broad range of disinfectants are effective in inactivating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), application of disinfectants has a low throughput in areas that receive treatments. Disinfection of large surface areas often involves the use of reactive microbiocidal materials, including ultraviolet germicidal irradiation, chlorine dioxide, and hydrogen peroxide vapor. Albeit these methods are highly effective in inactivating SARS-CoV-2, the deployment of these approaches creates unacceptable health hazards and precludes the treatment of occupied indoor spaces using existing disinfection technologies. In this study, the feasibility of using dry hydrogen peroxide (DHP) in inactivating SARS-CoV-2 on contaminated surfaces in large indoor spaces was evaluated. METHODS: Glass slides were inoculated with SARS-CoV-2 and treated with DHP between 5 and 25 ppb for up to 24 hours. Residual infectious virus samples were eluted from three replicates at each time point and titrated in African green monkey VeroE6 cells. RESULTS: In comparison with the observed relatively high stability of SARS-CoV-2 on contaminated glass slides (control group), residual infectious titers of glass slides inoculated with SARS-CoV-2 were significantly reduced after receiving 120 minutes of DHP treatment. CONCLUSIONS: The accelerated decay of SARS-CoV-2 on contaminated glass slides suggests that treatment with DHP can be an effective surface disinfection method for occupied indoor spaces.