Inactivation of dengue, chikungunya, and Ross River viruses in platelet concentrates after treatment with ultraviolet C light.

BACKGROUND: Arboviruses, including dengue (DENV 1-4), chikungunya (CHIKV), and Ross River (RRV), are emerging viruses that are a risk for transfusion safety globally. An approach for managing this risk is pathogen inactivation, such as the THERAFLEX UV-Platelets system. We investigated the ability of this system to inactivate the above mentioned arboviruses. STUDY DESIGN AND METHODS: DENV 1-4, CHIKV, or RRV were spiked into buffy coat (BC)-derived platelet (PLT) concentrates in additive solution and treated with the THERAFLEX UV-Platelets system at the following doses: 0.05, 0.1, 0.15, and 0.2 J/cm(2) (standard dose). Pre- and posttreatment samples were taken for each dose, and the level of viral infectivity was determined. RESULTS: At the standard ultraviolet C (UVC) dose (0.2 J/cm(2) ), viral inactivation of at least 4.43, 6.34, and 5.13 log or more, was observed for DENV 1-4, CHIKV, and RRV, respectively. A dose dependency in viral inactivation was observed with increasing UVC doses. CONCLUSIONS: Our study has shown that DENV, CHIKV, and RRV, spiked into BC-derived PLT concentrates, were inactivated by the THERAFLEX UV-Platelets system to the limit of detection of our assay, suggesting that this system could contribute to the safety of PLT concentrates with respect to these emerging arboviruses.

Dengue and chikungunya viruses in plasma are effectively inactivated after treatment with methylene blue and visible light.

BACKGROUND: Arboviruses, such as dengue viruses (DENV) and chikungunya virus (CHIKV), pose a risk to the safe transfusion of blood components, including plasma. Pathogen inactivation is an approach to manage this transfusion transmission risk, with a number of techniques being used worldwide for the treatment of plasma. In this study, the efficacy of the THERAFLEX MB-Plasma system to inactivate all DENV serotypes (DENV-1, DENV-2, DENV-3, DENV-4) or CHIKV in plasma, using methylene blue and light illumination at 630 nm, was investigated. STUDY DESIGN AND METHODS: Pooled plasma units were spiked with DENV-1, DENV-2, DENV-3 DENV-4, or CHIKV and treated with the THERAFLEX MB-Plasma system at four light illumination doses: 20, 40, 60, and 120 (standard dose) J/cm(2) . Pre- and posttreatment samples were collected and viral infectivity was determined. The reduction in viral infectivity was calculated for each dose. RESULTS: Treatment of plasma with the THERAFLEX MB-Plasma system resulted in at least a 4.46-log reduction in all DENV serotypes and CHIKV infectious virus. The residual infectivity for each was at the detection limit of the assay used at 60 J/cm(2) , with dose dependency also observed. CONCLUSIONS: Our study demonstrated the THERAFLEX MB-Plasma system can reduce the infectivity of all DENV serotypes and CHIKV spiked into plasma to the detection limit of the assay used at half of the standard illumination dose. This suggests this system has the capacity to be an effective option for managing the risk of DENV or CHIKV transfusion transmission in plasma.

Antiviral Evaluation of UV-4B and Interferon-Alpha Combination Regimens against Dengue Virus.

Dengue virus (DENV) is a flavivirus associated with clinical manifestations ranging in severity from self-limiting dengue fever, to the potentially life threatening condition, severe dengue. There are currently no approved antiviral therapies for the treatment of DENV. Here, we evaluated the antiviral potential of four broad-spectrum antivirals, UV-4B, interferon-alpha (IFN), sofosbuvir (SOF), and favipiravir (FAV) against DENV serotype 2 as mono- and combination therapy in cell lines that are physiologically relevant to human infection. Cell lines derived from human liver (HUH-7), neurons (SK-N-MC), and skin (HFF-1) were infected with DENV and treated with UV-4B, IFN, SOF, or FAV. Viral supernatant was sampled daily and infectious viral burden was quantified by plaque assay on Vero cells. Drug effect on cell proliferation in uninfected and infected cells was also assessed. UV-4B inhibited DENV in HUH-7, SK-N-MC, and HFF-1 cells yielding EC50 values of 23.75, 49.44, and 37.38 µM, respectively. Clinically achievable IFN concentrations substantially reduced viral burden in HUH-7 (EC50 = 102.7 IU/mL), SK-N-MC (EC50 = 86.59 IU/mL), and HFF-1 (EC50 = 163.1 IU/mL) cells. SOF potently inhibited DENV in HUH-7 cells but failed to produce the same effect in SK-N-MC and HFF-1 cells. Finally, FAV provided minimal suppression in HUH-7 and SK-N-MC cells, but was ineffective in HFF-1 cells. The two most potent anti-DENV agents, UV-4B and IFN, were also assessed in combination. UV-4B + IFN treatment enhanced antiviral activity in HUH-7, SK-N-MC, and HFF-1 cells relative to monotherapy. Our results demonstrate the antiviral potential of UV-4B and IFN against DENV in multiple physiologically relevant cell types.

Understanding Zika Virus Stability and Developing a Chimeric Vaccine through Functional Analysis.

Compared with other flaviviruses, Zika virus (ZIKV) is uniquely associated with congenital diseases in pregnant women. One recent study reported that (i) ZIKV has higher thermostability than dengue virus (DENV [a flavivirus closely related to ZIKV]), which might contribute to the disease outcome; (ii) the higher thermostability of ZIKV could arise from an extended loop structure in domain III of the viral envelope (E) protein and an extra hydrogen-bond interaction between E molecules (V. A. Kostyuchenko, E. X. Y. Lim, S. Zhang, G. Fibriansah, T.-S. Ng, J. S. G. Ooi, J. Shi, and S.-M. Lok, Nature 533:425-428, 2016, https://doi.org/10.1038/nature17994). Here we report the functional analysis of the structural information in the context of complete ZIKV and DENV-2 virions. Swapping the prM-E genes between ZIKV and DENV-2 switched the thermostability of the chimeric viruses, identifying the prM-E proteins as the major determinants for virion thermostability. Shortening the extended loop of the E protein by 1 amino acid was lethal for ZIKV assembly/release. Mutations (Q350I and T351V) that abolished the extra hydrogen-bond interaction between the E proteins did not reduce ZIKV thermostability, indicating that the extra interaction does not increase the thermostability. Interestingly, mutant T351V was attenuated in A129 mice defective in type I interferon receptors, even though the virus retained the wild-type thermostability. Furthermore, we found that a chimeric ZIKV with the DENV-2 prM-E and a chimeric DENV-2 with the ZIKV prM-E were highly attenuated in A129 mice; these chimeric viruses were highly immunogenic and protective against DENV-2 and ZIKV challenge, respectively. These results indicate the potential of these chimeric viruses for vaccine development. IMPORTANCE: Analysis of a recently observed high-resolution structure of ZIKV led to a hypothesis that its unusual stability may contribute to the associated, unique disease outcomes. Here we performed a functional analysis to demonstrate that viral prM-E genes are the main determinants for the high stability of ZIKV. The extra hydrogen-bond interaction (observed in the high-resolution structure) between ZIKV E proteins did not enhance virion stability, whereas the extended loop of E protein (CD loop in domain III) was essential for ZIKV assembly. More importantly, we found that a chimeric ZIKV with DENV-2 prM-E genes and a chimeric DENV-2 with ZIKV prM-E genes were highly attenuated in A129 mice. Mice immunized with these chimeric viruses generated robust neutralizing antibody responses and were fully protected from DENV-2 and ZIKV challenge, respectively, indicating that these chimeric viruses could be further developed as vaccine candidates.

Inactivation of dengue virus by methylene blue/narrow bandwidth light system.

Peracetic acid was one of the most commonly used disinfectants on solid surfaces in hospitals or public places. However, peracetic acid is an environmental toxin. Therefore, safer, alternative disinfectants or disinfectant systems should be developed. Because photodynamic virus inactivation with methylene blue (MB)/light system has proven effective in blood banking, MB was selected as a photosensitizing agent, dengue virus as a model virus for enveloped RNA viruses, and an in-house fabricated narrow bandwidth light system overlapping the absorption spectrum of MB as the light source. Dengue virus was mixed with different concentrations of MB, and illuminated by the narrow bandwidth light system under different illumination distances and times. The amount of dengue virus remaining was evaluated by plaque forming assays. Results showed that the concentration of MB working solution, illumination intensity of light source, illumination distance and time were four key factors affecting efficiency of virus inactivation using the MB/narrow bandwidth light system. Dengue virus could be completely inactivated at 2.5 m in 5 min when MB >/= 1.0 microg/ml. However, when the distance reached 3.0 m, only greater concentrations of MB (2.0 microg/ml) could completely inactivate virus in a reasonably short time (20 min), and smaller concentrations of MB (1.0 microg/ml) could only completely inactivate virus using longer times (25 min). The results of this virus inactivation model indicate that our MB/narrow bandwidth light system provides a powerful, easy way to inactivate dengue viruses.

Dengue virus-specific cross-reactive CD8+ human cytotoxic T lymphocytes.

Stimulation with live dengue virus of peripheral blood mononuclear cells from a dengue virus type 4-immune donor generated virus-specific, serotype-cross-reactive, CD8+, class I-restricted cytotoxic T lymphocytes (CTL) capable of lysing dengue virus-infected cells and cells pulsed with dengue virus antigens of all four serotypes. These CTL lysed autologous fibroblasts infected with vaccinia virus-dengue virus recombinant viruses containing the E gene or several nonstructural dengue virus type 4 genes. These results demonstrate that both dengue virus structural and nonstructural proteins are targets for the cytotoxic T-cell-mediated immune response to dengue virus and suggest that serotype-cross-reactive CD8+ CTL may be important mediators of viral clearance and of virus-induced immunopathology during secondary dengue virus infections.

MIP-1 alpha and MIP-1 beta induction by dengue virus.

Dengue virus (DV) infection can result in either a mild febrile illness known as dengue fever (DF) or a life-threatening disease called dengue hemorrhagic fever (DHF). DHF is more prevalent in patients undergoing secondary DV infection. This observation has led to the hypothesis that DHF may be the result of immune reactions to the secondary DV infection; an event termed immunopathology. Two cellular factors, MIP-1 alpha and MIP-1 beta, have been found to be induced by infection with DV. MIP-1 induction by DV infection was observed in a myelomonocytic cell line, as well as in peripheral blood mononuclear cells isolated from a dengue naive donor. MIP-1 induction was not due to factors secreted by infected cells. In fact, replication-competent virus was required to induce MIP-1. Evidence is also provided that MIP-1 genes are expressed in patients with dengue disease. It is hypothesized that these chemokines may have roles in the immunopathology of dengue infections and may contribute to fever and bone marrow suppression observed in patients with DV infections.

Light-independent inactivation of dengue-2 virus by carboxyfullerene C3 isomer.

Carboxyfullerene (C60) is known as a photosensitizer for virus inactivation. Its regioisomer with C3 symmetry, named the C3 isomer, could also inactivate the dengue-2 virus without light when the dose of C3 isomer was increased to 40 microM, indicating the possible involvement of a light-independent mechanism. Further analysis showed that the C3 isomer blocked viral replication at the attachment and penetration stages, suggesting that a direct interaction between the C3 isomer and the virion is required for inactivation. The C3 isomer with a bipolar structure showed better lipid interaction and dengue-2 virus suppression than D3, another isomer that contains evenly distributed hydrophilic side chains. Moreover, the C3 isomer selectively inactivated enveloped viruses (viz., dengue-2 virus and Japanese encephalitis virus) instead of nonenveloped viruses (viz., enterovirus 71 and coxsackievirus B3). Collectively, these findings support the hypothesis that C3 isomer suppression of enveloped viruses is effected through its hydrophobic interaction with the viral lipid envelope. Our report, which demonstrates the light-dependent and -independent mechanisms of C60 on viral inactivation, will aid in the development of novel anti-viral agents for use against enveloped viruses.