An integrated QTL mapping and transcriptome sequencing provides further molecular insights and candidate genes for stem strength in rapeseed (Brassica napus L.).

KEY MESSAGE: We detected the major QTL- qSR.A07, which regulated stem strength and was fine-mapped to 490 kb. BnaA07G0302800ZS and BnaA07G0305700ZS as the candidate functional genes were identified at qSR.A07 locus. The stem's mechanical properties reflect its ability to resist lodging. In rapeseed (Brassica napus L.), although stem lodging negatively affects yield and generates harvesting difficulties, the molecular regulation of stem strength remains elusive. Hence, this study aimed to unravel the main loci and molecular mechanisms governing rapeseed stem strength. A mapping population consisting of 267 RILs (recombinant inbred lines) was developed from the crossed between ZS11 (high stem strength) and 4D122 (low stem strength), and two mechanical properties of stems including stem breaking strength and stem rind penetrometer resistance were phenotyped in four different environments. Four pleiotropic QTLs that were stable in at least two environments were detected. qSR.A07, the major one, was fine-mapped to a 490 kb interval between markers SA7-2711 and SA7-2760 on chromosome 7. It displayed epistatic interaction with qRPR.A09-2. Comparative transcriptome sequencing and analysis unveiled methionine/S-adenosylmethionine cycle (Met/SAM cycle), cytoskeleton organization, sulfur metabolism and phenylpropanoid biosynthesis as the main pathways associated with high stem strength. Further, we identified two candidate genes, BnaA07G0302800ZS and BnaA07G0305700ZS, at qSR.A07 locus. Gene sequence alignment identified a number of InDels, SNPs and amino acid variants in sequences of these genes between ZS11 and 4D122. Finally, based on these genetic variants, we developed three SNP markers of these genes to facilitate future genetic selection and functional studies. These findings offer important genetic resources for the molecular-assisted breeding of novel rapeseed stem lodging-resistant varieties.

Research Progress of Selenium-Enriched Foods.

Selenium is an essential micronutrient that plays a crucial role in maintaining human health. Selenium deficiency is seriously associated with various diseases such as Keshan disease, Kashin-Beck disease, cataracts, and others. Conversely, selenium supplementation has been found to have multiple effects, including antioxidant, anti-inflammatory, and anticancer functions. Compared with inorganic selenium, organic selenium exhibits higher bioactivities and a wider range of safe concentrations. Consequently, there has been a significant development of selenium-enriched foods which contain large amounts of organic selenium in order to improve human health. This review summarizes the physiological role and metabolism of selenium, the development of selenium-enriched foods, the physiological functions of selenium-enriched foods, and provides an analysis of total selenium and its species in selenium-enriched foods, with a view to laying the foundation for selenium-enriched food development.

Integrating biochemical and anatomical characterizations with transcriptome analysis to dissect superior stem strength of ZS11 (Brassica napus).

Stem lodging resistance is a serious problem impairing crop yield and quality. ZS11 is an adaptable and stable yielding rapeseed variety with excellent resistance to lodging. However, the mechanism regulating lodging resistance in ZS11 remains unclear. Here, we observed that high stem mechanical strength is the main factor determining the superior lodging resistance of ZS11 through a comparative biology study. Compared with 4D122, ZS11 has higher rind penetrometer resistance (RPR) and stem breaking strength (SBS) at flowering and silique stages. Anatomical analysis shows that ZS11 exhibits thicker xylem layers and denser interfascicular fibrocytes. Analysis of cell wall components suggests that ZS11 possessed more lignin and cellulose during stem secondary development. By comparative transcriptome analysis, we reveal a relatively higher expression of genes required for S-adenosylmethionine (SAM) synthesis, and several key genes (4-COUMATATE-CoA LIGASE, CINNAMOYL-CoA REDUCTASE, CAFFEATE O-METHYLTRANSFERASE, PEROXIDASE) involved in lignin synthesis pathway in ZS11, which support an enhanced lignin biosynthesis ability in the ZS11 stem. Moreover, the difference in cellulose may relate to the significant enrichment of DEGs associated with microtubule-related process and cytoskeleton organization at the flowering stage. Protein interaction network analysis indicate that the preferential expression of several genes, such as LONESOME HIGHWAY (LHW), DNA BINDING WITH ONE FINGERS (DOFs), WUSCHEL HOMEOBOX RELATED 4 (WOX4), are related to vascular development and contribute to denser and thicker lignified cell layers in ZS11. Taken together, our results provide insights into the physiological and molecular regulatory basis for the formation of stem lodging resistance in ZS11, which will greatly promote the application of this superior trait in rapeseed breeding.

VEGFR-3 signaling restrains the neuron-macrophage crosstalk during neurotropic viral infection.

Upon recognizing danger signals produced by virally infected neurons, macrophages in the central nervous system (CNS) secrete multiple inflammatory cytokines to accelerate neuron apoptosis. The understanding is limited about which key effectors regulate macrophage-neuron crosstalk upon infection. We have used neurotropic-virus-infected murine models to identify that vascular endothelial growth factor receptor 3 (VEGFR-3) is upregulated in the CNS macrophages and that virally infected neurons secrete the ligand VEGF-C. When cultured with VEGF-C-containing supernatants from virally infected neurons, VEGFR-3+ macrophages suppress tumor necrosis factor α (TNF-α) secretion to reduce neuron apoptosis. Vegfr-3ΔLBD/ΔLBD (deletion of ligand-binding domain in myeloid cells) mice or mice treated with the VEGFR-3 kinase inhibitor exacerbate the severity of encephalitis, TNF-α production, and neuron apoptosis post Japanese encephalitis virus (JEV) infection. Activating VEGFR-3 or blocking TNF-α can reduce encephalitis and neuronal damage upon JEV infection. Altogether, we show that the inducible VEGF-C/VEGFR-3 module generates protective crosstalk between neurons and macrophages to alleviate CNS viral infection.

Multi-Functional Development and Utilization of Rapeseed: Comprehensive Analysis of the Nutritional Value of Rapeseed Sprouts.

Rapeseed is the third largest oil crop in the world and the largest oil crop in China. The multi-functional development and utilization of rapeseed is an effective measure for the high-quality development of rapeseed industry in China. In this study, several basic nutrients of eight rapeseed sprouts and five bean sprouts (3-5 varieties each) were determined, including sugar, crude protein, crude fiber, vitamin E, minerals, fatty acids, amino acids, and glucosinolates. Data analysis revealed that compared with bean sprouts, rapeseed sprouts were nutritionally balanced and were richer in active nutrients such as glucose, magnesium, selenium, vitamin E, and glucosinolate. Moreover, rapeseed sprouts exhibited reasonable amino acid composition and abundant unsaturated fatty acids (accounting for 90.32% of the total fatty acids). All these results indicated the potential of rapeseed sprout as a functional vegetable. Subsequently, three dominant nutrients including vitamin E, glucosinolate, and selenium were investigated in seeds and sprouts of 44 B. napus L. varieties. The results showed that germination raised the ratio of α-tocopherol/γ-tocopherol from 0.53 in seeds to 9.65 in sprouts, greatly increasing the content of α-tocopherol with the strongest antioxidant activity among the eight isomers of vitamin E. Furthermore, germination promoted the conversion and accumulation of glucosinolate components, especially, glucoraphanin with strong anti-cancer activity with its proportion increased from 1.06% in seeds to 1.62% in sprouts. In addition, the contents of selenium, vitamin E, and glucosinolate in rapeseed sprouts were highly correlated with those in seeds. Furthermore, these three dominant nutrients varied greatly within B. napus varieties, indicating the great potential of rapeseed sprouts to be further bio-enhanced. Our findings provide reference for the multi-purpose development and utilization of rapeseed, lay a theoretical foundation for the development of rapeseed sprout into a functional vegetable, and provide a novel breeding direction.

Recovery Infectious Enterovirus 71 by Bac-to-Bac Expression System in vitro and in vivo.

Enterovirus 71 (EV71) is one of the most important etiological agents for hand-foot-mouth disease. Compared with coxsackievirus A16 infection, EV71 infection is often associated with severe central nervous system complications, such as encephalitis, encephalomyelitis, and acute flaccid paralysis in infants and young children. In this study, we constructed a recombinant baculovirus with T7 ribonucleic acid polymerase under the control of a cytomegalovirus promoter and simultaneously engineered the T7 promoter upstream of a full-length EV71 complementary deoxyribonucleic acid. After transduction into mammalian cells, typical cytopathic effects (CPEs) and VP1 signals were detected in cells transfected with recombinant baculovirus. Additionally, viral particles located in the cytoplasm of human rhabdomyosarcoma cells (Rd) and Vero cells were observed by electron microscope, indicating that EV71 was recovered using a Bac-to-Bac expression system in vitro. After four passages, the rescued virus had a growth curve and plaque morphology similar to those of the parental virus. Furthermore, the Vp1 gene and the protein from the mouse brain were detected by reverse transcription polymerase chain reaction and immunohistochemistry after intracerebral injection of purified recombinant baculovirus. Typical CPEs were observed after inoculation of the supernatant from mouse brain to Rd cells, revealing a reconstruction of EV71 in vivo. Thus, we established a new approach to rescue EV71 based on a baculovirus expression system in vitro and in vivo, which may provide a safe and convenient platform for fundamental research and a strategy to rescue viruses that currently lack suitable cell culture and animal models.

Nitric oxide affects seed oil accumulation and fatty acid composition through protein S-nitrosation.

Nitric oxide (NO) is a key signaling molecule regulating several plant developmental and stress responses. Here, we report that NO plays an important role in seed oil content and fatty acid composition. RNAi silencing of Arabidopsis S-nitrosoglutathione reductase 1 (GSNOR1) led to reduced seed oil content. In contrast, nitrate reductase double mutant nia1nia2 had increased seed oil content, compared with wild-type plants. Moreover, the concentrations of palmitic acid (C16:0), linoleic acid (C18:2), and linolenic acid (C18:3) were higher, whereas those of stearic acid (C18:0), oleic acid (C18:1), and arachidonic acid (C20:1) were lower, in seeds of GSNOR1 RNAi lines. Similar results were obtained with rapeseed embryos cultured in vitro with the NO donor sodium nitroprusside (SNP), and the NO inhibitor NG-Nitro-L-arginine Methyl Ester (L-NAME). Compared with non-treated embryos, the oil content decreased in SNP-treated embryos, and increased in L-NAME-treated embryos. Relative concentrations of C16:0, C18:2 and C18:3 were higher, whereas C18:1 concentration decreased in rapeseed embryos treated with SNP. Proteomics and transcriptome analysis revealed that three S-nitrosated proteins and some key genes involved in oil synthesis, were differentially regulated in SNP-treated embryos. Therefore, regulating NO content could be a novel approach to increasing seed oil content in cultivated oil crops.

Baculovirus Surface Display of Zika Virus Envelope Protein Protects against Virus Challenge in Mouse Model.

Zika virus (ZIKV) is emerging as a significant pathogen worldwide and may cause severe neurological disorders such as fetal microcephaly and Guillain-Barre syndrome. No drug or listed vaccines are currently available for preventing ZIKV infection. As a major target of neutralizing, ZIKV envelop (E) protein usually used for vaccine development. Nevertheless, the immunogenicity of ZIKV envelop (E) protein expressed by baculovirus display system has never been assessed. In this study, we reported a new strategy for surface display of ZIKV E protein by a recombinant baculovirus vector derived from Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and assessed its immunogenicity in mice. We produced recombinant fusion ZIKV E protein linked with signal peptide (SP) and transmembrane domain (TM) of AcMNPV GP64. The results showed that the recombinant protein was easy to produce by baculovirus display system. BALB/c mice immunized with this recombinant E protein developed ZIKV specific serum antibodies. The anti-E protein sera from the mice were able to effectively neutralize ZIKV in vitro. More importantly, AG6 (IFN-α/ß and IFN-γ receptor deficient) mice immunized with recombinant E protein were protected against lethal ZIKV challenge. Together, these findings demonstrated that the recombinant E protein displayed by baculovirus can be conveniently prepared and displayed good immunogenicity in immunized mice. It is a promising practical approach for prompting the development of vaccine and related immunology research.

Enterovirus A71 Oncolysis of Malignant Gliomas.

Malignant gliomas, the most lethal type of primary brain tumor, continue to be a major therapeutic challenge. Here, we found that enterovirus A71 (EV-A71) can be developed as a novel oncolytic agent against malignant gliomas. EV-A71 preferentially infected and killed malignant glioma cells relative to normal glial cells. The virus receptor human scavenger receptor class B, member 2 (SCARB2), and phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1)-mediated cell death were involved in EV-A71-induced oncolysis. In mice with implanted subcutaneous gliomas, intraneoplastic inoculation of EV-A71 caused significant tumor growth inhibition. Furthermore, in mice bearing intracranial orthotopic gliomas, intraneoplastic inoculation of EV-A71 substantially prolonged survival. By insertion of brain-specific microRNA-124 (miR124) response elements into the viral genome, we improved the tumor specificity of EV-A71 oncolytic therapy by reducing its neurotoxicity while maintaining its replication potential and oncolytic capacity in gliomas. Our study reveals that EV-A71 is a potent oncolytic agent against malignant gliomas and may have a role in treating this tumor in the clinical setting.

Characterization of Zika Virus Endocytic Pathways in Human Glioblastoma Cells.

Zika virus (ZIKV) infections can cause microcephaly and neurological disorders. However, the early infection events of ZIKV in neural cells remain to be characterized. Here, by using a combination of pharmacological and molecular approaches and the human glioblastoma cell T98G as a model, we first observed that ZIKV infection was inhibited by chloroquine and NH4Cl, indicating a requirement of low intracellular pH. We further showed that dynamin is required as the ZIKV entry was affected by the specific inhibitor dynasore, small interfering RNA (siRNA) knockdown of dynamin, or by expressing the dominant-negative K44A mutant. Moreover, the ZIKV entry was significantly inhibited by chlorpromazine, pitstop2, or siRNA knockdown of clathrin heavy chain, indicating an involvement of clathrin-mediated endocytosis. In addition, genistein treatment, siRNA knockdown of caveolin-1, or overexpression of a dominant-negative caveolin mutant impacted the ZIKV entry, with ZIKV particles being observed to colocalize with caveolin-1, implying that caveola endocytosis can also be involved. Furthermore, we found that the endocytosis of ZIKV is dependent on membrane cholesterol, microtubules, and actin cytoskeleton. Importantly, ZIKV infection was inhibited by silencing of Rab5 and Rab7, while confocal microscopy showed that ZIKV particles localized in Rab5- and Rab7-postive endosomes. These results indicated that, after internalization, ZIKV likely moves to Rab5-positive early endosome and Rab7-positive late endosomes before delivering its RNA into the cytoplasm. Taken together, our study, for the first time, described the early infection events of ZIKV in human glioblastoma cell T98G.

Zika virus promotes CCN1 expression via the CaMKIIα-CREB pathway in astrocytes.

Zika virus (ZIKV) infection in the human central nervous system (CNS) causes Guillain-Barre syndrome, cerebellum deformity, and other diseases. Astrocytes are immune response cells in the CNS and an important component of the blood-brain barrier. Consequently, any damage to astrocytes facilitates the spread of ZIKV in the CNS. Connective tissue growth factor/Nephroblastoma overexpressed gene family 1 (CCN1), an important inflammatory factor secreted by astrocytes, is reported to regulate innate immunity and viral infection. However, the mechanism by which astrocyte viral infection affects CCN1 expression remains undefined. In this study, we demonstrate that ZIKV infection up-regulates CCN1 expression in astrocytes, thus promoting intracellular viral replication. Other studies revealed that the cAMP response element (CRE) in the CCN1 promoter is activated by the ZIKV NS3 protein. The cAMP-responsive element-binding protein (CREB), a transacting factor of the CRE, is also activated by NS3 or ZIKV. Furthermore,a specific inhibitor of CREB, i.e. SGC-CBP30, reduced ZIKV-induced CCN1 up-regulation and ZIKV replication. Moreover, co-immunoprecipitation, overexpression, and knockdown studies confirmed that the interaction between NS3 and the regulatory domain of CaMKIIα could activate the CREB pathway, thus resulting in the up-regulation of CCN1 expression and enhancement of virus replication. In conclusion, the findings of our investigations on the NS3-CaMKIIα-CREB-CCN1 pathway provide a foundation for understanding the infection mechanism of ZIKV in the CNS.

Genomic and transcriptional analyses of novel parvoviruses identified from dead peafowl.

To identify potential pathogens responsible for a disease outbreak of cultured peafowls in China in 2013, metagenomic sequencing was conducted. The genomes of two closely related parvoviruses, namely peafowl parvovirus 1 (PePV1) and PePV2, were identified with size of 4428 bp and 4348 bp, respectively. Phylogenetic analysis revealed that both viruses are novel parvoviruses, belonging to the proposed genus Chapparvovirus of Parvoviridae. The transcriptional profile of PePV1 was analyzed by transfecting a nearly complete PePV1 genome into HEK-293T cells. Results revealed that PePV1 employs one promoter and two polyadenylation sites to start and terminate its transcriptions, with one donor site and two acceptor sites for pre-mRNA splicing. PePV1 DNA and structural protein were detected in several tissues of a dead peafowl, which appeared to have suffered enteritis, pneumonia and viremia. These results provide novel information of chapparvoviruses, and call for attention to the potential pathogens.

A Novel Chimeric Mitochondrial Gene Confers Cytoplasmic Effects on Seed Oil Content in Polyploid Rapeseed (Brassica napus).

Cytoplasmic effects (CEs) have been discovered to influence a diverse array of agronomic traits in crops, and understanding the underlying mechanisms can help accelerate breeding programs. Seed oil content (SOC) is of great agricultural, nutritional, and economic importance. However, the genetic basis of CEs on SOC (CE-SOC) remains enigmatic. In this study, we use an optimized approach to sequence the cytoplasmic (plastid and mitochondrial) genomes of allotetraploid oilseed rape (Brassica napus) cultivars, 51218 and 56366, that bear contrasting CE-SOC. By combining comparative genomics and genome-wide transcriptome analysis, we identify mitochondria-encoded orf188 as a potential CE-SOC determinant gene. Functional analyses in the model system Arabidopsis thaliana and rapeseed demonstrated that orf188 governs CE-SOC and could significantly increase SOC, strikingly, through promoting the yield of ATP. Consistent with this finding, transcriptional profiling with microarray and RNA sequencing revealed that orf188 affects transcriptional reprogramming of mitochondrial energy metabolism to facilitate ATP production. Intriguingly, orf188 is a previously uncharacterized chimeric gene, and the presence of this genetic novelty endows rapeseed with positive CE-SOC. Our results shed light on the molecular basis of CEs on a key quantitative trait in polyploid crops and enrich the theory of maternal control of oil content, providing new scientific guidance for breeding high-oil rapeseed germplasms.

N6-methyladenosine modification and METTL3 modulate enterovirus 71 replication.

N6-methyladenosine (m6A) constitutes one of the most abundant internal RNA modifications and is critical for RNA metabolism and function. It has been previously reported that viral RNA contains internal m6A modifications; however, only recently the function of m6A modification in viral RNAs has been elucidated during infections of HIV, hepatitis C virus and Zika virus. In the present study, we found that enterovirus 71 (EV71) RNA undergoes m6A modification during viral infection, which alters the expression and localization of the methyltransferase and demethylase of m6A, and its binding proteins. Moreover, knockdown of m6A methyltransferase resulted in decreased EV71 replication, whereas knockdown of the demethylase had the opposite effect. Further study showed that the m6A binding proteins also participate in the regulation of viral replication. In particular, two m6A modification sites were identified in the viral genome, of which mutations resulted in decreased virus replication, suggesting that m6A modification plays an important role in EV71 replication. Notably, we found that METTL3 interacted with viral RNA-dependent RNA polymerase 3D and induced enhanced sumoylation and ubiquitination of the 3D polymerase that boosted viral replication. Taken together, our findings demonstrated that the host m6A modification complex interacts with viral proteins to modulate EV71 replication.

Viperin Inhibits Enterovirus A71 Replication by Interacting with Viral 2C Protein.

Enterovirus A71 (EVA71) is a human enterovirus belonging to the Picornaviridae family and mostly causes hand-foot-and-mouth disease in infants. Viperin is an important interferon-stimulated gene with a broad antiviral activity against various viruses. However, the effect of viperin on human enteroviruses and the interaction mechanism between EVA71 and viperin remains elusive. Here, we confirmed the EVA71-induced expression of viperin in a mouse model and cell lines and showed that viperin upregulation by EVA71 infection occurred on both the mRNA and protein level. Viperin knockdown and overexpression in EVA71-infected cells indicated that this protein can markedly inhibit EVA71 infection. Interestingly, immunofluorescent confocal microscopy and co-immunoprecipitation assays indicated that viperin interacts and colocalizes with the EVA71 protein 2C in the endoplasmic reticulum. Furthermore, amino acids 50⁻60 in the N-terminal domain of viperin were the key residues responsible for viperin interaction with 2C. More importantly, the N-terminal domain of viperin was found responsible for inhibiting EVA71 replication. Our findings can potentially aid future research on the prevention and treatment of nervous system damage caused by EVA71 and may provide a potential target for antiviral therapy.

ZIKV infection activates the IRE1-XBP1 and ATF6 pathways of unfolded protein response in neural cells.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Zika Virus Attenuation by Codon Pair Deoptimization Induces Sterilizing Immunity in Mouse Models.

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.

Tick-Borne Encephalitis Virus Nonstructural Protein NS5 Induces RANTES Expression Dependent on the RNA-Dependent RNA Polymerase Activity.

Tick-borne encephalitis virus (TBEV) is one of the flaviviruses that targets the CNS and causes encephalitis in humans. The mechanism of TBEV that causes CNS destruction remains unclear. It has been reported that RANTES-mediated migration of human blood monocytes and T lymphocytes is specifically induced in the brain of mice infected with TBEV, which causes ensuing neuroinflammation and may contribute to brain destruction. However, the viral components responsible for RANTES induction and the underlying mechanisms remain to be fully addressed. In this study, we demonstrate that the NS5, but not other viral proteins of TBEV, induces RANTES production in human glioblastoma cell lines and primary astrocytes. TBEV NS5 appears to activate the IFN regulatory factor 3 (IRF-3) signaling pathway in a manner dependent on RIG-I/MDA5, which leads to the nuclear translocation of IRF-3 to bind with RANTES promoter. Further studies reveal that the activity of RNA-dependent RNA polymerase (RdRP) but not the RNA cap methyltransferase is critical for TBEV NS5-induced RANTES expression, and this is likely due to RdRP-mediated synthesis of dsRNA. Additional data indicate that the residues at K359, D361, and D664 of TBEV NS5 are critical for RdRP activity and RANTES induction. Of note, NS5s from other flaviviruses, including Japanese encephalitis virus, West Nile virus, Zika virus, and dengue virus, can also induce RANTES expression, suggesting the significance of NS5-induced RANTES expression in flavivirus pathogenesis. Our findings provide a foundation for further understanding how flaviviruses cause neuroinflammation and a potential viral target for intervention.

SpyCatcher-SpyTag mediated in situ labelling of progeny baculovirus with quantum dots for tracking viral infection in living cells.

A non-invasive labelling strategy is proposed to label baculovirus via genetic insertion of a SpyTag into the viral glycoprotein, followed by specific conjugation with the SpyCatcher protein on modified quantum dots (QDs) through an isopeptide bond. The labelling method is convenient and efficient and shows little attenuation of viral infectivity. Therefore, it is a biologically compatible technique for tracking viral infection.

The 3C protease of enterovirus A71 counteracts the activity of host zinc-finger antiviral protein (ZAP).

Enterovirus A71 (EV-A71) is a positive-strand RNA virus that causes hand-foot-mouth disease and neurological complications in children and infants. Although the underlying mechanisms remain to be further defined, impaired immunity is thought to play an important role. The host zinc-finger antiviral protein (ZAP), an IFN-stimulated gene product, has been reported to specifically inhibit the replication of certain viruses. However, whether ZAP restricts the infection of enteroviruses remains unknown. Here, we report that EV-A71 infection upregulates ZAP mRNA in RD and HeLa cells. Moreover, ZAP overexpression rendered 293 T cells resistant to EV-A71 infection, whereas siRNA-mediated depletion of endogenous ZAP enhanced EV-A71 infection. The EV-A71 infection stimulated site-specific proteolysis of two ZAP isoforms, leading to the accumulation of a 40 kDa N-terminal ZAP fragment in virus-infected cells. We further revealed that the 3C protease (3Cpro) of EV-A71 mediates ZAP cleavage, which requires protease activity. Furthermore, ZAP variants with single amino acid substitutions at Gln-369 were resistant to 3Cpro cleavage, implying that Gln-369 is the sole cleavage site in ZAP. Moreover, although ZAP overexpression inhibited EV-A71 replication, the cleaved fragments did not show this effect. Our results indicate that an equilibrium between ZAP and enterovirus 3Cpro controls viral infection. The findings in this study suggest that viral 3Cpro mediated ZAP cleavage may represent a mechanism to escape host antiviral responses.