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.

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.

Rapid development of a DNA vaccine for Zika virus.

Zika virus (ZIKV) was identified as a cause of congenital disease during the explosive outbreak in the Americas and Caribbean that began in 2015. Because of the ongoing fetal risk from endemic disease and travel-related exposures, a vaccine to prevent viremia in women of childbearing age and their partners is imperative. We found that vaccination with DNA expressing the premembrane and envelope proteins of ZIKV was immunogenic in mice and nonhuman primates, and protection against viremia after ZIKV challenge correlated with serum neutralizing activity. These data not only indicate that DNA vaccination could be a successful approach to protect against ZIKV infection, but also suggest a protective threshold of vaccine-induced neutralizing activity that prevents viremia after acute infection.

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.

Deliberate attenuation of chikungunya virus by adaptation to heparan sulfate-dependent infectivity: a model for rational arboviral vaccine design.

Mosquito-borne chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus from the genus Alphavirus, family Togaviridae, which causes fever, rash and severe persistent polyarthralgia in humans. Since there are currently no FDA licensed vaccines or antiviral therapies for CHIKV, the development of vaccine candidates is of critical importance. Historically, live-attenuated vaccines (LAVs) for protection against arthropod-borne viruses have been created by blind cell culture passage leading to attenuation of disease, while maintaining immunogenicity. Attenuation may occur via multiple mechanisms. However, all examined arbovirus LAVs have in common the acquisition of positively charged amino acid substitutions in cell-surface attachment proteins that render virus infection partially dependent upon heparan sulfate (HS), a ubiquitously expressed sulfated polysaccharide, and appear to attenuate by retarding dissemination of virus particles in vivo. We previously reported that, like other wild-type Old World alphaviruses, CHIKV strain, La Réunion, (CHIKV-LR), does not depend upon HS for infectivity. To deliberately identify CHIKV attachment protein mutations that could be combined with other attenuating processes in a LAV candidate, we passaged CHIKV-LR on evolutionarily divergent cell-types. A panel of single amino acid substitutions was identified in the E2 glycoprotein of passaged virus populations that were predicted to increase electrostatic potential. Each of these substitutions was made in the CHIKV-LR cDNA clone and comparisons of the mutant viruses revealed surface exposure of the mutated residue on the spike and sensitivity to competition with the HS analog, heparin, to be primary correlates of attenuation in vivo. Furthermore, we have identified a mutation at E2 position 79 as a promising candidate for inclusion in a CHIKV LAV.

Photochemical inactivation of chikungunya virus in human apheresis platelet components by amotosalen and UVA light.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that recently re-emerged in Africa and rapidly spread into countries of the Indian Ocean basin and South-East Asia. The mean viremic blood donation risk for CHIKV on La Réunion reached 1.5% at the height of the 2005-2006 outbreaks, highlighting the need for development of safety measures to prevent transfusion-transmitted infections. We describe successful inactivation of CHIKV in human platelets and plasma using photochemical treatment with amotosalen and long wavelength UVA illumination. Platelet components in additive solution and plasma units were inoculated with two different strains of high titer CHIKV stock (6.0-8.0 logs/mL), and then treated with amotosalen and exposure to 1.0-3.0 J/cm² UVA. Based on in vitro assays of infectious virus pre- and post-treatment to identify endpoint dilutions where virus was not detectable, mean viral titers could effectively be reduced by > 6.4 ± 0.6 log10 TCID50/mL in platelets and ≥ 7.6 ± 1.4 logs in plasma, indicating this treatment has the capacity to prevent CHIKV transmission in human blood components collected from infected donors in or traveling from areas of CHIKV transmission.

Role of the yellow fever virus structural protein genes in viral dissemination from the Aedes aegypti mosquito midgut.

Live-attenuated virus vaccines are key components in controlling arboviral diseases, but they must not disseminate in or be transmitted by mosquito vectors. Although the cycles in which many mosquito-borne viruses are transmitted are well understood, the role of viral genetics in these processes has not been fully elucidated. Yellow fever virus (YFV) is an important arbovirus and the prototype member of the family Flaviviridae. Here, YFV was used in Aedes aegypti mosquitoes as a model to investigate the genetic basis of infection and dissemination in mosquitoes. Viruses derived from infectious clones and chimeric viruses with defined sequential manipulations were used to investigate the influence of specific sequences within the membrane and envelope structural protein genes on dissemination of virus from the mosquito midgut. Substitution of domain III of the envelope protein from a midgut-restricted YFV into a wild-type YFV resulted in a marked decrease in virus dissemination, suggesting an important role for domain III in this process. However, synergism between elements within the flavivirus structural and non-structural protein genes may be necessary for efficient virus escape from the mosquito midgut.