Zika virus (ZIKV) is a mosquito-transmitted virus that has caused significant public health concerns around the world, partly because of an association with microcephaly in babies born to mothers who were infected with ZIKV during pregnancy. As a recently emerging virus, little is known as to how the virus interacts with the host cell machinery. A yeast-2-hybrid screen for proteins capable of interacting with the ZIKV E protein domain III, the domain responsible for receptor binding, identified 21 proteins, one of which was the predominantly ER resident chaperone protein GRP78. The interaction of GRP78 and ZIKV E was confirmed by co-immunoprecipitation and reciprocal co-immunoprecipitation, and indirect immunofluorescence staining showed intracellular and extracellular co-localization between GRP78 and ZIKV E. Antibodies directed against the N-terminus of GRP78 were able to inhibit ZIKV entry to host cells, resulting in significant reductions in the levels of ZIKV infection and viral production. Consistently, these reductions were also observed after down-regulation of GRP78 by siRNA. These results indicate that GRP78 can play a role mediating ZIKV binding, internalization and replication in cells. GRP78 is a main regulator of the unfolded protein response (UPR), and the study showed that expression of GRP78 was up-regulated, and the UPR was activated. Increases in CHOP expression, and activation of caspases 7 and 9 were also shown in response to ZIKV infection. Overall these results indicate that the interaction between GRP78 and ZIKV E protein plays an important role in ZIKV infection and replication, and may be a potential therapeutic target.

Many mosquito transmitted viruses of the genera Alphavirus and Flavivirus are human pathogens of significant concern, and there is currently no specific antiviral for any member of these two genera. This study sought to investigate the broad utility of orlistat (tetrahydrolipstatin) in reducing virus infection for several mosquito borne viruses including flaviviruses (dengue virus (DENV; nine isolates analyzed), Japanese encephalitis virus (JEV; one isolate analyzed) and Zika virus (ZIKV; 2 isolates analyzed)) as well as an alphavirus (chikungunya virus; CHIKV; 2 isolates analyzed). Three different treatment regimens were evaluated, namely pre-treatment (only), post-treatment (only) and pre- and post-treatment, and three factors were evaluated, namely level of infection, virus titer and genome copy number. Results showed that all three treatment modalities were able to significantly reduce virus titer for all viruses investigated, with the exception of three isolates of DENV in the pre-treatment only regimen. Pre- and post-treatment was more effective in reducing the level of infection and genome copy number of all viruses investigated than either pre-treatment or post-treatment alone. Collectively, these results suggest orlistat has potential as a broad-spectrum agent against multiple mosquito transmitted viruses.

Infections with the mosquito transmitted dengue virus (DENV) are a significant public health burden in many parts of the world. Despite the introduction of a commercial vaccine in some parts of the world, the majority of the populations at risk of infection remain unprotected against this disease, and there is currently no treatment for DENV infection. Natural compounds offer the prospect of cheap and sustainable therapeutics to reduce the disease burden during infection, and thus potentially alleviate the risk of more severe disease. This study evaluated the potential anti-DENV 2 activity of five natural compounds namely melatonin, α-tocopherol, folic acid, acetyl-l-carnitine and resveratrol in two different cell lines. Screening of the compounds showed that one compound (acetyl-l-carnitine) showed no effect on DENV infection, three compounds (melatonin, α-tocopherol and folic acid) slightly increased levels of infection, while the 5th compound, resveratrol, showed some limited anti-DENV activity, with resveratrol reducing virus output with an EC50 of less than 25 μM. These results suggest that some commonly taken natural compounds may have beneficial effects on DENV infection, but that others may potentially add to the disease burden.

Kaempferol, a plant-derived flavonoid, has been reported to have activity against Japanese encephalitis virus (JEV) in BHK-21 cells. To determine the broader utility of this compound, we initially evaluated the activity of kaempferol against JEV and dengue virus (DENV) in HEK293T/17 cells. Results showed no significant antiviral activity against either virus. We subsequently investigated the activity of kaempferol against both JEV and DENV in BHK-21 cells. Results showed a significant inhibition of JEV infection but, surprisingly, a significant enhancement of DENV infection. The effect of kaempferol on both host protein expression and transcription was investigated and both transcriptional and translational inhibitory effects were observed, although a more marked effect was observed on host cell protein expression. Markedly, while GRP78 was increased in DENV infected cells treated with kaempferol, it was not increased in JEV infected cells treated with kaempferol. These results show that cellular alteration induced by one compound can have opposite effects on viruses from the same family, suggesting the presence of distinct replication strategies for these two viruses.

A lipid bilayer produced from the host membrane makes up around 20% of the weight of the dengue virus (DENV) virion and is crucial for virus entry. Despite its significance, the virion’s lipid composition is still poorly understood. In tandem with lipid profiles of the cells utilised to generate the virions, this work determined a partial lipid profile of DENV virions derived from two cell lines (C6/36 and LLC-MK₂). The results showed distinctive profiles between the two cell types. In the mammalian LLC-MK₂ cells, 30.8% (73/237 identified lipid species; 31 upregulated, 42 downregulated) of lipid species were altered in response to infection, whilst in insect C6/36 cells only 12.0% (25/208; 19 upregulated, 6 downregulated) of lipid species showed alterations in response to infection. For virions from LLC-MK₂ cells, 14 lipids were detected specifically in virions with a further seven lipids being enriched (over mock controls). For virions from C6/36 cells, 43 lipids were detected that were not seen in mock preparations, with a further 16 being specifically enriched (over mock control). These results provide the first lipid description of DENV virions produced in mammalian and mosquito cells, as well as the lipid changes in the corresponding infected cells.

Background: Zika virus (ZIKV) is a mosquito transmitted virus spread primarily byAedesspecies mosquitoes that can cause disease in humans, particularly when infection occurs in pregnancy where the virus can have a significant impact on the developing fetus. Despite this, there remains no prophylactic agent or therapeutic treatment for infection. Baicalein is a trihydroxyflavone, that is found in some traditional medicines commonly used in Asia, and has been shown to have several activities including antiviral properties. Importantly, studies have shown baicalein to be safe and well tolerated in humans, increasing its potential utilization.Methods: This study sought to determine the anti-ZIKV activity of baicalein using a human cell line (A549). Cytotoxicity of baicalein was determined by the MTT assay, and the effect on ZIKV infection determined by treating A549 cells with baicalien at different time points in the infection process. Parameters including level of infection, virus production, viral protein expression and genome copy number were assessed by flow cytometry, plaque assay, western blot and quantitative RT-PCR, respectively.Results: The results showed that baicalein had a half-maximal cytotoxic concentration (CC₅₀) of > 800 µM, and a half-maximal effective concentration (EC₅₀) of 124.88 µM. Time-of-addition analysis showed that baicalein had an inhibitory effect on ZIKV infection at the adsorption and post-adsorption stages. Moreover, baicalein also exerted a significant viral inactivation activity on ZIKV (as well as on dengue virus and Japanese encephalitis virus) virions.Conclusion: Baicalein has now been shown to possess anti-ZIKV activity in a human cell line.