Autophagy is a common strategy for cell protection; however, some viruses can in turn adopt cellular autophagy to promote viral replication. Zika virus (ZIKV) is the pathogen that causes Zika viral disease, and it is a mosquito-borne virus. However, its pathogenesis, especially the interaction between ZIKV and target cells during the early stages of infection, is still unclear. In this study, we demonstrate that infecting human umbilical vein endothelial cells (HUVEC) with ZIKV triggers cellular autophagy. We observed both an increase in the conversion of LC3-I to LC3-II and increased accumulation of fluorescent cells with LC3 dots, which are considered to be the two key indicators of autophagy. The ratio of LC3-II/GAPDH in each group was significantly increased at different times after ZIKV infection at different MOIs, indicating that the production of lipidated LC3-II increased. Moreover, both the ratio of LC3-II/GAPDH and the expression of viral NS3 protein increased with increasing time of viral infection. The expression level of p62 decreased gradually from 12 h post-infection. Expression profile of double fluorescent protein labelling LC3 indicated that the autophagy induced by ZIKV infection was a complete process. We further investigated the role of autophagy in ZIKV replication. We demonstrated that either the treatment with inhibitors of autophagosomes formation or short hairpin RNA targeting the Beclin-1 gene, which is critical for the formation of autophagosomes, significantly reduced viral production. Taken together, our results indicate that ZIKV infection induces autophagy of HUVEC, and inhibition of ZIKV-induced autophagy restrains viral replication.

T lymphopenia, occurring in the early phase of sepsis in response to systemic inflammation, is commonly associated with morbidity and mortality of septic infections. We have previously shown that a sufficient number of T cells is required to constrain Toll-like receptors (TLRs) mediated hyperinflammation. However, the underlying mechanisms remains unsolved. Herein, we unveil that CD4⁺ T cells engage with MHC II of macrophages to downregulate TLR pro-inflammatory signaling. We show further that the direct contact between CD4 molecule of CD4⁺ T cells or the ectodomain of CD4 (soluble CD4, sCD4), and MHC II of resident macrophages is necessary and sufficient to prevent TLR4 overactivation in LPS and cecal ligation puncture (CLP) sepsis. sCD4 serum concentrations increase after the onset of LPS sepsis, suggesting its compensatory inhibitive effects on hyperinflammation. sCD4 engagement enables the cytoplasmic domain of MHC II to recruit and activate STING and SHP2, which inhibits IRAK1/Erk and TRAF6/NF-κB activation required for TLR4 inflammation. Furthermore, sCD4 subverts pro-inflammatory plasma membrane anchorage of TLR4 by disruption of MHC II-TLR4 raft domains that promotes MHC II endocytosis. Finally, sCD4/MHCII reversal signaling specifically interferes with TLR4 but not TNFR hyperinflammation, and independent of the inhibitive signaling of CD40 ligand of CD4⁺ cells on macrophages. Therefore, a sufficient amount of soluble CD4 protein can prevent excessive inflammatory activation of macrophages via alternation of MHC II-TLR signaling complex, that might benefit for a new paradigm of preventive treatment of sepsis.

The emergence of significant arboviruses and their spillover transmission to humans represent a major threat to global public health. No approved drugs are available for the treatment of significant arboviruses in circulation today. The repurposing of clinically approved drugs is one of the most rapid and promising strategies in the identification of effective treatments for diseases caused by arboviruses. Here, we screened small-molecule compounds with anti-tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus activity from 2580 FDA-approved drugs. In total, 60 compounds showed antiviral efficacy against all four of the arboviruses in Huh7 cells. Among these compounds, ixazomib and ixazomib citrate (inhibitors of 20S proteasome β5) exerted antiviral effects at a low-micromolar concentration. The time-of-drug-addition assay suggested that ixazomib and ixazomib citrate disturbed multiple processes in viruses’ life cycles. Furthermore, ixazomib and ixazomib citrate potently inhibited chikungunya virus replication and relieved virus-induced footpad swelling in a mouse model. These results offer critical information which supports the role of ixazomib as a broad-spectrum agent against arboviruses.

Diseases transmitted by arthropod-borne viruses such as West Nile virus (WNV) and chikungunya virus (CHIKV) pose threat to global public health. Unfortunately, to date, there is no available approved drug for severe symptoms caused by both viruses. It has been reported that reverse transcriptase inhibitors can effectively inhibit RNA polymerase activity of RNA viruses. We screened the anti-WNV activity of the FDA-approved reverse transcriptase inhibitor library and found that 4 out of 27 compounds showed significant antiviral activity. Among the candidates, etravirine markedly inhibited WNV infection in both Huh 7 and SH-SY5Y cells. Further assays revealed that etravirine inhibited the infection of multiple arboviruses, including yellow fever virus (YFV), tick-borne encephalitis virus (TBEV), and CHIKV. A deeper study at the phase of action showed that the drug works primarily during the viral replication process. This was supported by the strong interaction potential between etravirine and the RNA-dependent RNA polymerase (RdRp) of WNV and alphaviruses, as evaluated using molecular docking. In vivo, etravirine significantly rescued mice from WNV infection-induced weight loss, severe neurological symptoms, and death, as well as reduced the viral load and inflammatory cytokines in target tissues. Etravirine showed antiviral effects in both arthrophlogosis and lethal mouse models of CHIKV infection. This study revealed that etravirine is an effective anti-WNV and CHIKV arbovirus agent both in vitro and in vivo due to the inhibition of viral replication, providing promising candidates for clinical application.

Chikungunya virus (CHIKV) is a reemerging arbovirus causing disease on a global scale, and the potential for its epidemics remains high. CHIKV has caused millions of cases and heavy economic burdens around the world, while there are no available approved antiviral therapies to date. In this study, nifuroxazide, an FDA-approved antibiotic for acute diarrhea or colitis, was found to significantly inhibit a variety of arboviruses, although its antiviral activity varied among different target cell types. Nifuroxazide exhibited relatively high inhibitory efficiency in yellow fever virus (YFV) infection of the hepatoma cell line Huh7, tick-borne encephalitis virus (TBEV) and west nile virus (WNV) infection of the vascular endothelial cell line HUVEC, and CHIKV infection of both Huh7 cells and HUVECs, while it barely affected the viral invasion of neurons. Further systematic studies on the action stage of nifuroxazide showed that nifuroxazide mainly inhibited in the viral replication stage. In vivo, nifuroxazide significantly reduced the viral load in muscles and protected mice from CHIKV-induced footpad swelling, an inflammation injury within the arthrosis of infected mice. These results suggest that nifuroxazide has a potential clinical application as an antiviral drug, such as in the treatment of CHIKV infection.

Introduction: Coronaviruses and influenza viruses are significant respiratory pathogens that cause severe disease burdens and economic losses for society. Due to their diversity and evolution, vaccines typically require periodic updating to remain effective. An additional challenge is imposed by the possible coinfection of SARS-CoV-2 and influenza, which could increase disease severity.Methods: We developed a vaccinia vaccine, named rTTV-RBD-HA2, broadly targeting coronaviruses and influenza viruses. This vaccine expresses three fusion proteins, each comprising the receptor-binding domain (RBD) from one of the three highly pathogenic coronaviruses (SARS-CoV-2, SARS-CoV, and MERS-CoV) and the conserved HA stalk region from two influenza viruses (pdmH1N1 and nH7N9) belonging to groups 1 and 2, respectively.Results: The multi-targeting nature of this vaccine was validated by its success in inducing antibody responses to the three RBDs and both group 1 and 2 HAs in mice. Importantly, it also generated robust T cell responses to all the immunogens, which could be mobilized to the lung through intranasal vaccination. Consistent with this broad immunogenicity profile, when administered via intramuscular priming and two intranasal boosts, rTTV-RBD-HA2 effectively protected vaccinated mice against challenges of the wild-type SARS-CoV-2 virus, the Omicron XBB variant, and the influenza A H1N1 and H3N2 viruses.Discussion: Our results collectively support the candidacy of recombinant rTTV-RBD-HA2 as a novel respiratory virus vaccine that provides cross-protection against coronaviruses and influenza viruses, surpassing the breadth of previous vaccines. Additionally, they underscore the importance of establishing a strong mucosal T cell response in the development of a universal respiratory virus vaccine.