Enterovirus 71 (EV71) has emerged as a major cause of neurological disease following the near eradication of poliovirus. Accumulating evidence suggests that mammalian microRNAs (miRNAs), a class of noncoding RNAs of 18 to 23 nucleotides (nt) with important regulatory roles in many cellular processes, participate in host antiviral defenses. However, the roles of miRNAs in EV71 infection and pathogenesis are still unclear. Here, hsa-miR-296-5p expression was significantly increased in EV71-infected human cells. As determined by virus titration, quantitative real-time PCR (qRT-PCR), and Western blotting, overexpression of hsa-miR-296-5p inhibited, while inhibition of endogenous hsa-miR-296-5p facilitated, EV71 infection. Additionally, two potential hsa-miR-296-5p targets (nt 2115 to 2135 and nt 2896 to 2920) located in the EV71 genome (strain BrCr) were bioinformatically predicted and validated by luciferase reporter assays and Western blotting. Genomic alignment of various EV71 strains revealed synonymous mutations in hsa-miR-296-5p target sequences. Furthermore, the introduction of synonymous mutations into the EV71 BrCr genome by site-directed mutagenesis impaired the viral inhibitory effects of hsa-miR-296-5p and facilitated mutant virus infection. Meanwhile, compensatory mutations in corresponding hsa-miR-296-5p target sequences of the EV71 HeN strain (GenBank accession number JN256064 ) restored the inhibitory effects of the miRNA. These results indicate that hsa-miR-296-5p inhibits EV71 replication by targeting the viral genome. Our findings support the notion that cellular miRNAs can inhibit virus infection and that the virus mutates to escape suppression by cellular miRNAs.

Many viruses depend on the extensive membranous network of the endoplasmic reticulum (ER) for their translation, replication, and packaging. Certain membrane modifications of the ER can be a trigger for ER stress, as well as the accumulation of viral protein in the ER by viral infection. Then, unfolded protein response (UPR) is activated to alleviate the stress. Zika virus (ZIKV) is a mosquito-borne flavivirus and its infection causes microcephaly in newborns and serious neurological complications in adults. Here, we investigated ER stress and the regulating model of UPR in ZIKV-infected neural cells in vitro and in vivo. Mice deficient in type I and II IFN receptors were infected with ZIKV via intraperitoneal injection and the nervous tissues of the mice were assayed at 5 days post-infection. The expression of phospho-IRE1, XBP1, and ATF6 which were the key markers of ER stress were analyzed by immunohistochemistry assay in vivo. Additionally, the nuclear localization of XBP1s and ATF6n were analyzed by immunohistofluorescence. Furthermore, two representative neural cells, neuroblastoma cell line (SK-N-SH) and astrocytoma cell line (CCF-STTG1), were selected to verify the ER stress in vitro. The expression of BIP, phospho-elF2α, phospho-IRE1, and ATF6 were analyzed through western blot and the nuclear localization of XBP1s was performed by confocal immunofluorescence microscopy. RT-qPCR was also used to quantify the mRNA level of the UPR downstream genes in vitro and in vivo. ZIKV infection significantly upregulated the expression of ER stress markers in vitro and in vivo. Phospho-IRE1 and XBP1 expression significantly increased in the cerebellum and mesocephalon, while ATF6 expression significantly increased in the mesocephalon. ATF6n and XBP1s were translocated into the cell nucleus. The levels of BIP, ATF6, phospho-elf2α, and spliced xbp1 also significantly increased in vitro. Furthermore, the downstream genes of UPR were detected to investigate the regulating model of the UPR during ZIKV infection in vitro and in vivo. The transcriptional levels of atf4, gadd34, chop, and edem-1 in vivo and that of gadd34 and chop in vitro significantly increased. Findings in this study demonstrated that ZIKV infection activates ER stress in neural cells. The results offer clues to further study the mechanism of neuropathogenesis caused by ZIKV infection.

There is growing evidence that porcine reproductive and respiratory syndrome virus (PRRSV) has developed mechanisms to subvert the host innate immune response. PRRSV non-structural protein 2 (Nsp2) was suggested previously as a potential interferon (IFN) antagonist. This study focused on Nsp2 to investigate its inhibitory mechanism of IFN induction. It was demonstrated that Nsp2 strongly inhibited IFN-β production by antagonizing activation of the IFN regulatory factor 3 (IRF-3) pathway induced by the Sendai virus (SeV). Further studies revealed that the cysteine protease domain (PL2) of Nsp2 was necessary for IFN antagonism. Additionally, both full-length Nsp2 and the PL2 protease domain of Nsp2 were found to inhibit SeV-induced phosphorylation and nuclear translocation of IRF-3. These findings suggest that Nsp2 is likely to play an important role in subversion of IRF-3-dependent innate antiviral defences, providing a basis for elucidating the mechanisms underlying PRRSV pathogenesis.

Enterovirus 71 (EV71), a single, positive-stranded RNA virus, has been regarded as the most important neurotropic enterovirus after the eradication of the poliovirus. EV71 infection can cause hand, foot, and mouth disease or herpangina. Cytokine storm with elevated levels of proinflammatory and inflammatory cytokines, including TNF-α, has been proposed to explain the pathogenesis of EV71-induced disease. TNF-α–mediated NF-κB signaling pathway plays a key role in inflammatory response. We hypothesized that EV71 might also moderate host inflammation by interfering with this pathway. In this study, we tested this hypothesis and identified EV71 2C protein as an antagonist of TNF-α–mediated activation of NF-κB signaling pathway. Expression of 2C protein significantly reduced TNF-α–mediated NF-κB activation in 293T cells as measured by gene reporter and gel mobility shift assays. Furthermore, overexpression of TNFR-associated factor 2-, MEK kinase 1-, IκB kinase (IKK)α-, or IKKβ-induced NF-κB activation, but not constitutively active mutant of IKKβ (IKKβ SS/EE)-induced NF-κB activation, was inhibited by 2C protein. These data together suggested that the activation of IKKβ is most likely targeted by 2C; this notion was further strengthened by immunoblot detection of IKKβ phosphorylation and IκBα phosphorylation and degradation. Coimmunoprecipitation and colocalization of 2C and IKKβ expressed in mammalian cells provided compelling evidence that 2C interacts with IKKβ. Collectively, our data indicate that EV71 2C protein inhibits IKKβ activation and thus blocks NF-κB activation.

Zika virus (ZIKV) infection during the large epidemics in the Americas is related to congenital abnormities or fetal demise. To date, there is no vaccine, antiviral drug, or other modality available to prevent or treat Zika virus infection. Here we designed novel live attenuated ZIKV vaccine candidates using a codon pair deoptimization strategy. Three codon pair-deoptimized ZIKVs (Min E, Min NS1, and Min E+NS1) were de novo synthesized and recovered by reverse genetics and contained large amounts of underrepresented codon pairs in the E gene and/or NS1 gene. The amino acid sequence was 100% unchanged. The codon pair-deoptimized variants had decreased replication fitness in Vero cells (Min NS1 ≫ Min E > Min E+NS1), replicated more efficiently in insect cells than in mammalian cells, and demonstrated diminished virulence in a mouse model. In particular, Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and a single immunization achieved complete protection against lethal challenge and vertical ZIKV transmission during pregnancy. More importantly, due to the numerous synonymous substitutions in the codon pair-deoptimized strains, reversion to wild-type virulence through gradual nucleotide sequence mutations is unlikely. Our results collectively demonstrate that ZIKV can be effectively attenuated by codon pair deoptimization, highlighting the potential of Min E+NS1 as a safe vaccine candidate to prevent ZIKV infections. IMPORTANCE Due to unprecedented epidemics of Zika virus (ZIKV) across the Americas and the unexpected clinical symptoms, including Guillain-Barré syndrome, microcephaly, and other birth defects in humans, there is an urgent need for ZIKV vaccine development. Here we provided the first attenuated versions of ZIKV with two important genes (E and/or NS1) that were subjected to codon pair deoptimization. Compared to parental ZIKV, the codon pair-deoptimized ZIKVs were mammal attenuated and preferred insect to mammalian cells. Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer and achieved complete protection against lethal challenge and vertical virus transmission during pregnancy. More importantly, the massive synonymous mutational approach made it impossible for the variant to revert to wild-type virulence. Our results have proven the feasibility of codon pair deoptimization as a strategy to develop live attenuated vaccine candidates against flaviviruses such as ZIKV, Japanese encephalitis virus, and West Nile virus.