N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA.

Epitranscriptomic RNA modifications can regulate the stability of mRNA and affect cellular and viral RNA functions. The N4-acetylcytidine (ac4C) modification in the RNA viral genome was recently found to promote viral replication; however, the mechanism by which RNA acetylation in the host mRNA regulates viral replication remains unclear. To help elucidate this mechanism, the roles of N-acetyltransferase 10 (NAT10) and ac4C during the infection and replication processes of the alphavirus, Sindbis virus (SINV), were investigated. Cellular NAT10 was upregulated, and ac4C modifications were promoted after alphavirus infection, while the loss of NAT10 or inhibition of its N-acetyltransferase activity reduced alphavirus replication. The NAT10 enhanced alphavirus replication as it helped to maintain the stability of lymphocyte antigen six family member E mRNA, which is a multifunctional interferon-stimulated gene that promotes alphavirus replication. The ac4C modification was thus found to have a non-conventional role in the virus life cycle through regulating host mRNA stability instead of viral mRNA, and its inhibition could be a potential target in the development of new alphavirus antivirals.IMPORTANCEThe role of N4-acetylcytidine (ac4C) modification in host mRNA and virus replication is not yet fully understood. In this study, the role of ac4C in the regulation of Sindbis virus (SINV), a prototype alphavirus infection, was investigated. SINV infection results in increased levels of N-acetyltransferase 10 (NAT10) and increases the ac4C modification level of cellular RNA. The NAT10 was found to positively regulate SINV infection in an N-acetyltransferase activity-dependent manner. Mechanistically, the NAT10 modifies lymphocyte antigen six family member E (LY6E) mRNA-the ac4C modification site within the 3'-untranslated region (UTR) of LY6E mRNA, which is essential for its translation and stability. The findings of this study demonstrate that NAT10 regulated mRNA stability and translation efficiency not only through the 5'-UTR or coding sequence but also via the 3'-UTR region. The ac4C modification of host mRNA stability instead of viral mRNA impacting the viral life cycle was thus identified, indicating that the inhibition of ac4C could be a potential target when developing alphavirus antivirals.

GAS6 as a potential target to alleviate neuroinflammation during Japanese encephalitis in mouse models.

Viral encephalitis is characterized by inflammation of the brain parenchyma caused by a variety of viruses, among which the Japanese encephalitis (JE) virus (JEV) is a typical representative arbovirus. Neuronal death, neuroinflammation, and breakdown of the blood brain barrier (BBB) constitute vicious circles of JE progression. Currently, there is no effective therapy to prevent this damage. Growth arrest specific gene 6 (GAS6) is a secreted growth factor that binds to the TYRO3, AXL, and MERTK (TAM) family of receptor tyrosine kinases and has been demonstrated to participate in neuroprotection and suppression of inflammation in many central nervous system (CNS) diseases which has great potential for JE intervention. In this study, we found that GAS6 expression in the brain was decreased and was reversely correlated with viral load and neuronal loss. Mice with GAS6/TAM signalling deficiency showed higher mortality and accelerated neuroinflammation during peripheral JEV infection, accompanied by BBB breakdown. GAS6 directly promoted the expression of tight junction proteins in bEnd.3 cells and strengthened BBB integrity, partly via AXL. Mice administered GAS6 were more resistant to JEV infection due to increased BBB integrity, as well as decreased viral load and neuroinflammation. Thus, targeted GAS6 delivery may represent a strategy for the prevention and treatment of JE especially in patients with impaired BBB.

The protective role of Mertk in JEV-induced encephalitis by maintaining the integrity of blood-brain barrier.

Japanese encephalitis is an acute infectious disease of the central nervous system caused by neurotropic Japanese encephalitis virus (JEV). As a member of TAM (Tyro3, Axl and Mertk) family, Mertk has involved in multiple biological processes by engaging with its bridging ligands Gas6 and Protein S, including invasion of pathogens, phagocytosis of apoptotic cells, inflammatory response regulation, and the maintenance of blood brain barrier (BBB) integrity. However, its role in encephalitis caused by JEV infection has not been studied in detail. Here, we found that Mertk-/- mice exhibited higher mortality and more rapid disease progression than wild-type mice after JEV challenge. There were no significant differences in viral load and cytokines expression level in peripheral tissues between Wild type and Mertk-/- mice. Furthermore, the absence of Mertk had little effect on the inflammatory response and immunopathological damage while it can cause an increased viral load in the brain. For the in vitro model of BBB, Mertk was shown to maintain the integrity of the BBB. In vivo, Mertk-/- mice exhibited higher BBB permeability and lower BBB integrity. Taken together, our findings demonstrate that Mertk acts as a protective factor in the development of encephalitis induced by JEV infection, which is mainly associated with its beneficial effect on BBB integrity, rather than its regulation of inflammatory response.

N6-methyladenosine modification positively regulate Japanese encephalitis virus replication.

N6-methyladenosine (m6A) is present in diverse viral RNA and plays important regulatory roles in virus replication and host antiviral innate immunity. However, the role of m6A in regulating JEV replication has not been investigated. Here, we show that the JEV genome contains m6A modification upon infection of mouse neuroblast cells (neuro2a). JEV infection results in a decrease in the expression of m6A writer METTL3 in mouse brain tissue. METTL3 knockdown by siRNA leads to a substantial decrease in JEV replication and the production of progeny viruses at 48 hpi. Mechanically, JEV triggered a considerable increase in the innate immune response of METTL3 knockdown neuro2a cells compared to the control cells. Our study has revealed the distinctive m6A signatures of both the virus and host in neuro2a cells infected with JEV, illustrating the positive role of m6A modification in JEV infection. Our study further enhances understanding of the role of m6A modification in Flaviviridae viruses.

Modified exfoliated graphene functionalized with carboxylic acid-group and thionine on a screen-printed carbon electrode as a platform for an electrochemical enzyme immunosensor.

An enzyme immunoassay was developed based on the coulometric measurement of immunoglobulin M (IgM) against Hantaan viruses (HTNV) by using virus-like particles (VLPs) as recognition molecules. The surface functionalization of screen-printed carbon electrodes (SPCEs) was achieved through paste-exfoliated graphene that was modified with a COOH group and a thionine mediator through supramolecular-covalent scaffolds, on SPCEs by using the binder contained in the ink. After the covalent immobilization of the antibody, the sensor was used for the sandwich enzyme immunoassay of IgM against HTNV. By using HTNV VLPs as the second recognization molecules, the resulting sensor efficiently monitored the reaction of IgM against HTNV and anti-IgM antibody with high specificity. By attaching HTNV nucleocapsid protein antibody conjugate with horseradish peroxidase (HRP) onto VLPs, the signal response of the assay was derived from the coulometric measurement of H2O2 reduction mediated by thionine on the electrode surface after the application of a potential (- 0.2 V vs. Ag/AgCl). The ratio of charges measured before or after H2O2 addition was used to quantify IgM because these charges could be used as background charges or total charges, respectively. The ratio exhibited good agreement with IgM concentration within a range 0.1 to 1000 pg mL-1, and a detection limit of 0.06 pg mL-1 was obtained. The assay demonstrated high sensitivity and specificity toward HTNV-specific IgM in serum.

N6-methyladenosine modifications enhance enterovirus 71 ORF translation through METTL3 cytoplasmic distribution.

During replication, numerous viral RNAs are modified by N6-methyladenosine (m6A), the most abundant internal RNA modification. m6A is believed to regulate elements of RNA metabolism, such as splicing, stability, translation, secondary structure formation, and viral replication. In this study, we assessed the occurrence of m6A modification of the EV71 genome in human cells and revealed a preferred, conserved modification site across diverse viral strains. A single m6A modification at the 5' UTR-VP4 junction was shown to perform a protranslational function. Depletion of the METTL3 methyltransferase or treatment with 3-deazaadenosine significantly reduced EV71 replication. Specifically, METTL3 colocalized with the viral dsRNA replication intermediate in the cytoplasm during EV71 infection. As a nuclear resident protein, METTL3 relies on the binding of the nuclear import protein karyopherin to its nuclear localization signal (NLS) for nuclear translocation. We observed that EV71 2A and METTL3 share nuclear import proteins. The results of this study revealed an inner mechanism by which EV71 2A regulates the subcellular location of METTL3 to amplify its own gene expression, providing an increased understanding of RNA epitranscriptomics during the EV71 replication cycle.

IoT PCR for pandemic disease detection and its spread monitoring.

During infectious disease outbreaks, the centers for disease control need to monitor particular areas. Considerable effort has been invested in the development of portable, user-friendly, and cost-effective systems for point-of-care (POC) diagnostics, which could also create an Internet of Things (IoT) for healthcare via a global network. However, at present IoT based on a functional POC instrument is not available. Here we show a fast, user-friendly, and affordable IoT system based on a miniaturized polymerase chain reaction device. We demonstrated the system's capability by amplification of complementary deoxyribonucleic acid (cDNA) of the dengue fever virus. The resulting data were then automatically uploaded via a Bluetooth interface to an Android-based smartphone and then wirelessly sent to a global network, instantly making the test results available anywhere in the world. The IoT system presented here could become an essential tool for healthcare centers to tackle infectious disease outbreaks identified either by DNA or ribonucleic acid.

Phosphatidylethanolamine-binding protein 4 promotes the epithelial-to-mesenchymal transition in non-small cell lung cancer cells by activating the sonic hedgehog signaling pathway.

Phosphatidylethanolamine-binding protein 4 (PEBP4), a member of the PEBP family, has been reported to play a pivotal role in tumor progression. However, its role in epithelial-to-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) cells remains unclear. Here, we investigated the effects and underlying mechanism of PEBP4 in NSCLC EMT. Three human NSCLC cell lines (A549, H1299, and H460) were transfected with pcDNA3.1-PEBP4 or PEBP4-targeting small interfering RNA. Then, cell proliferation was analyzed by the MTT assay, and cell migration and invasion were analyzed by the transwell chamber assay. Protein and messenger RNA expression of the related genes and proteins were assessed by Western blot analysis and quantitative real-time polymerase chain reaction, respectively. Results showed that PEBP4 was highly expressed in the human lung cancer tissues and three human NSCLC cell lines. Pretreatment with pcDNA3.1-PEBP4 promoted the proliferation, invasion, and migration of NSCLC cells and increased EMT in vitro and lung tumor metastasis in vivo. Whereas knockdown of PEBP4 suppressed NSCLC cell migration, PEBP4, and invasion with prevented EMT. Furthermore, PEBP4 overexpression significantly promoted the transcriptional activity of sonic hedgehog (Shh) signaling in NSCLC cells. Further analysis showed that using cyclopamine to inhibit Shh signaling significantly ameliorated the effect on cell proliferation, invasion, and migration, as well as EMT triggered by PEBP4 overexpression. Together, these results suggest that PEBP4 may promote tumorigenesis in NSCLC by regulating cell proliferation and EMT via activation of the Shh signaling pathway.

Structure-based discovery of antiviral inhibitors targeting the E dimer interface of Japanese encephalitis virus.

Flaviviruses are emerging arthropod-borne viruses posing a great threat to human beings worldwide. The E dimer configuration of the flavivirus was prominent during viral assembly, maturation and entry. Neutralization antibodies targeting E dimer played the important role in controlling the flavivirus infection. Previously, the ideal drug target of small molecular inhibitors of JEV was viral proteases and polymerases. The crystal structure of JEV E protein showed a conserved pocket in it is important at membrane fusion step. Recently, a set of anti-virus drugs has been found by virtual screening. Here, we show that the fusion-loop pocket of JEV E protein was a conservative region and an ideal drug target. ChemDiv-3 from virtual screening as the lead compound was found to show a relatively modest inhibition effect for JEV in vitro and in vivo test and could interfere with the formation of JEV sE dimer. ChemDiv-3 interacts with the amino acid residues ASN 313, PRO 314, ALA 315, and VAL 323 in E protein via hydrogen bonds for occupation of the fusion-loop pocket. The key binding sites LYS 312, ALA 513 and THR 317 forming the fusion-loop pocket are the same and other auxiliary sites are similar among the flavivirus. Taken together, the fusion-loop pocket of the flavivirus could be one promising target for drug discovery.

The Long Noncoding RNA NEAT1 Exerts Antihantaviral Effects by Acting as Positive Feedback for RIG-I Signaling.

Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs (lncRNAs) play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus (HTNV) infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon (IFN-ß) production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I-IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein (SFPQ) by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs (lncRNAs) as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon (IFN) responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I-IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.

DDX50 inhibits the replication of dengue virus 2 by upregulating IFN-ß production.

Dengue virus (DENV) infects approximately 390 million people per year, and each of the four DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) is capable of causing infection. At present, there is no antiviral drug available for the treatment of DENV. Several DExD/H-box helicases have been shown to be involved in the antiviral immune response or viral replication. In the present study, we investigated the role of DDX50 in DENV-2 RNA replication. Our data showed that the level of DENV-2 RNA increased in DDX50 knockdown cells during an early stage of viral infection and decreased in DDX50-overexpressing cells. DDX50, in conjunction with RIG-I and MDA5, upregulated the production of IFN-ß in infected cells through an additive effect on the IFN-ß promoter. Furthermore, transcription of several IFN-stimulated genes was increased in DDX50-overexpressing cells infected with DENV-2. These results provide evidence that DDX50 negatively regulates DENV-2 replication during the early stages of infection by inducing IFN-ß production.

DDX21 translocates from nucleus to cytoplasm and stimulates the innate immune response due to dengue virus infection.

Successful DENV infection relies on its ability to evade the host innate immune system. By using iTRAQ labeling followed by LC-MS/MS analysis, DDX21 was identified as a new host RNA helicase involved in the DENV life cycle. In DENV infected cells, DDX21 translocates from nucleus to cytoplasm to active the innate immune response and thus inhibits DENV replication in the early stages of infection. DDX21 is then degraded by the viral NS2B-NS3 protease complex and the innate immunity is thus subverted to facilitate DENV replication. The results reveal a new mechanism in which DENV subverts the host innate immune system to facilitate its replication in host cells.

Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1.

Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3' end of dengue virus (DENV) 5'-3' UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions.

[Changes and intervention of reactive astrocyte activation patterns in the progression of Japanese encephalitis].

Objective To clarify the relationship between astrocyte activation patterns and disease progression in epidemic encephalitis B (Japanese encephalitis). Methods First, a mouse model of epidemic encephalitis B was constructed by foot-pad injection of Japanese encephalitis virus (JEV), and the expression of viral protein NS3 in different brain regions was detected by immunofluorescence assay (IFA). Next, IFA, RNA sequencing (RNA-seq) and real-time quantitative PCR (qRT-PCR) were used to clarify the changes in the astrocyte activation patterns at different stages of epidemic encephalitis B. Finally, intracerebroventricular administration of irisin was conducted to regulate the proportion of activation in complement C3-positive A1 astrocytes and S100A10-positive A2 astrocytes, investigating whether it could improve the body mass, behavioral scores, and brain tissue damage in a mouse model. Results NS3 protein was detected by IFA predominantly in the M1/M2 region of the motor cortex and the hippocampus. The number and volume of GFAP-positive astrocytes significantly increased in JEV-infected brain regions, in which the expression of multiple genes associated with A1/A2 astrocyte activation was significantly enhanced. Although intracerebroventricular or intraperitoneal injection of irisin did not improve the prognosis of epidemic encephalitis B, it inhibited the activation of A1 astrocytes and ameliorate neuroinflammation. Conclusion Neurons in the M1/M2 motor cortex and hippocampus are susceptible to JEV infection, in which the abnormal astrocyte activation contributes to the neuroinflammatory injury. Irisin administration may restrain A1 astrocyte activation and alleviate neuroinflammation following JEV infection.

[Exploring a novel instructional paradigm for the Fundamentals of Immunity and Infection module in our university: A study based on the O-PIRTAS flipped classroom approach].

Medical Microbiology and Medical Immunology are important components of our university's the modular teaching on fundamentals of immunity and infection. Among these, Bacterial Infection and Immunity serves as a bridge between Medical Microbiology and Medical Immunology. This chapter mainly introduces how pathogenic bacteria invade the body to cause infection and how the body's immune system resists bacterial infection. Studying this chapter, students can build a framework knowledge on infection-immunity. However, due to the complexity of the content and the limited duration of the course, the traditional teaching method struggles to help students clarify the knowledge structure, resulting in poor learning outcomes. Therefore, there is an urgent need for reforms. Using the bacterial infection and immunity chapter as an example, this article explores the teaching reform of the Fundamentals of Immunology and Infection module based on the O-PIRTAS flipped classroom model, providing valuable insights for subsequent teaching reforms.

Genomic sequencing revealed recombination event between clade 1 and clade 2 occurs in circulating varicella-zoster virus in China.

Varicella-zoster virus (VZV), a member of the Alphaherpesvirinae subfamily, causes varicella in primary infections and establishing a latent stage in sensory ganglia. Upon reactivation, VZV causes herpes zoster with severe neuralgia, especially in elderly patients. The mutation rate for VZV is comparatively lower than the other members of other alpha herpesviruses. Due to geographic isolation, different genotypes of VZV are circulating on separate continents. Here, we successfully isolated a VZV from the vesicular fluid of a youth zoster patient. Based on the single-nucleotide polymorphism profiles of different open reading frames that define the genotype, this newly isolated VZV primarily represents genotype clade 2 but also has characteristics of genotype clade 1. The next-generation sequencing provided a nearly full-length sequence, and further phylogenetic analysis revealed that this VZV isolate is distinct from clades 1 and 2. The Recombination Detection Program indicates that a possible recombinant event may occur between the VZV isolate and clade 1. In summary, we found that there is a circulating VZV isolate in China that may represent a recombinant between clade 1 and clade 2, providing new concerns that need to be considered in the future VZV vaccination program.

Viral myocarditis: From molecular mechanisms to therapeutic prospects.

Myocarditis is characterized as local or diffuse inflammatory lesions in the myocardium, primarily caused by viruses and other infections. It is a common cause of sudden cardiac death and dilated cardiomyopathy. In recent years, the global prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the widespread vaccination have coincided with a notable increase in the number of reported cases of myocarditis. In light of the potential threat that myocarditis poses to global public health, numerous studies have sought to elucidate the pathogenesis of this condition. However, despite these efforts, effective treatment strategies remain elusive. To collate the current research advances in myocarditis, and thereby provide possible directions for further research, this review summarizes the mechanisms involved in viral invasion of the organism and primarily focuses on how viruses trigger excessive inflammatory responses and in result in different types of cell death. Furthermore, this article outlines existing therapeutic approaches and potential therapeutic targets for the acute phase of myocarditis. In particular, immunomodulatory treatments are emphasized and suggested as the most extensively studied and clinically promising therapeutic options.

[Preparation of monoclonal antibodies against the envelope protein extracellular domain of Zika virus in mice].

Objective To prepare monoclonal antibodies against the envelope protein extracellular domain (Eecto) of Zika virus (ZIKV) in mice. Methods A prokaryotic expression plasmid, pET28a-ZIKV-Eecto of ZIKV Eecto, was constructed, transformed into Escherichia coli BL21 and induced by isopropyl ß-D-thiogalactoside (IPTG). The recombinant Eecto protein was expressed in the form of inclusion bodies, and purified proteins were obtained through denaturation, renaturation and ultrafiltration. After three rounds of immunization with the Eecto protein, the serum of BALB/c mice was obtained and the titer of polyclonal antibodies in serum was determined. The reactivity of polyclonal antibodies was analyzed with Western blotting and immunofluorescence assay in HEK293T cells expressing the ZIKV prME. Spleen cells from mice with higher antibody titers were prepared and fused with SP2/0 myeloma cells. The hybridoma cells secreting antibodies were screened through the limited dilution method, and the ascites containing antibody were harvested for titer measurement and subclass analysis. The Eecto from the envelope proteins of Japanese encephalitis virus (JEV), Yellow fever virus (YFV), Dengue virus (DENV1-4), and Tick borne encephalitis virus (TBEV) were coated and used to analyze the cross-reactivity of ZIKV monoclonal antibodies by ELISA. Further specificity analysis was conducted on antibodies with high titers and strong specificity. Results The plasmid pET28a-ZIKV-Eecto was successfully constructed. The purified Eecto protein was obtained with good immunogenicity. Four monoclonal antibodies were prepared and screened, namely 1D6, 4F11, 4H7, and 4F8. Among them, 1D6, 4H7, and 4F8 are IgG (K) type antibodies, and 4F11 is an IgM (K) antibody. The ascitic fluid titer of 1D6 was higher than 1:108. Antibodies 1D6 and 4H7 are ZIKV-specific and showed no cross-reactivity with other Flaviviruses. Conclusion The mice monoclonal antibodies against ZIKV-Eecto are produced successfully, which will provide experimental materials for the establishment of ZIKV detection methods and the study of its pathogenesis.

[Preparation of mouse monoclonal antibodies against the ectodomain of Western equine encephalitis virus E2 (E2ecto) protein].

Objective To prepare mouse monoclonal antibodies against the ectodomain of E2 (E2ecto) glycoprotein of Western equine encephalitis virus (WEEV). Methods A prokaryotic expression plasmid pET-28a-WEEV E2ecto was constructed and transformed into BL21 (DE3) competent cells. E2ecto protein was expressed by IPTG induction and presented mainly as inclusion bodies. Then the purified E2ecto protein was prepared by denaturation, renaturation and ultrafiltration. BALB/c mice were immunized with the formulated E2ecto protein using QuickAntibody-Mouse5W as an adjuvant via intramuscular route, boosted once at an interval of 21 days. At 35 days post-immunization, mice with antibody titer above 1×104 were inoculated with E2ecto intraperitoneally, and spleen cells were fused with SP2/0 cells three days later. Hybridoma cells secreting specific monoclonal antibodies were screened by the limited dilution method, and ascites were prepared after intraperitoneal inoculation of hybridoma cells. The subtypes and titers of the antibodies in ascites were assayed by ELISA. The biological activity of the mAb was identified by immunofluorescence assay(IFA) on BHK-21 cells which were transfected with eukaryotic expression plasmid pCAGGS-WEEV-CE3E2E1. The specificity of the antibodies were evaluated with E2ecto proteins from EEEV and VEEV. Results Purified WEEV E2ecto protein was successfully expressed and obtained. Four monoclonal antibodies, 3G6G10, 3D7G2, 3B9E8 and 3D5B7, were prepared, and their subtypes were IgG2c(κ), IgM(κ), IgM(κ) and IgG1(κ), respectively. The titers of ascites antibodies 3G6G10, 3B9E8 and 3D7G2 were 105, and 3D5B7 reached 107. None of the four antibody strains cross-reacted with other encephalitis alphavirus such as VEEV and EEEV. Conclusion Four strains of mouse mAb specifically binding WEEV E2ecto are successfully prepared.

Flexible nano-liposomes-encapsulated recombinant UL8-siRNA (r/si-UL8) based on bioengineering strategy inhibits herpes simplex virus-1 infection.

Herpes simplex virus-1 (HSV-1) infection can cause various diseases and the current therapeutics have limited efficacy. Small interfering RNA (siRNA) therapeutics are a promising approach against infectious diseases by targeting the viral mRNAs directly. Recently, we employed a novel tRNA scaffold to produce recombinant siRNA agents with few natural posttranscriptional modifications. In this study, we aimed to develop a specific prodrug against HSV-1 infection based on siRNA therapeutics by bioengineering technology. We screened and found that UL8 of the HSV-1 genome was an ideal antiviral target based on RNAi. Next, we used a novel bio-engineering approach to manufacture recombinant UL8-siRNA (r/si-UL8) in Escherichia coli with high purity and activity. The r/si-UL8 was selectively processed to mature si-UL8 and significantly reduced the number of infectious virions in human cells. r/si-UL8 delivered by flexible nano-liposomes significantly decreased the viral load in the skin and improved the survival rate in the preventive mouse zosteriform model. Furthermore, r/si-UL8 also effectively inhibited HSV-1 infection in a 3D human epidermal skin model. Taken together, our results highlight that the novel siRNA bioengineering technology is a unique addition to the conventional approach for siRNA therapeutics and r/si-UL8 may be a promising prodrug for curing HSV-1 infection.