[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.

Single dose recombinant VSV based vaccine elicits robust and durable neutralizing antibody against Hantaan virus.

Hantaan virus (HTNV) is a pathogenic orthohantavirus prevalent in East Asia that is known to cause hemorrhagic fever with severe renal syndrome (HFRS), which has a high fatality rate. However, a Food and Drug Administration (FDA)-approved vaccine is not currently available against this virus. Although inactivated vaccines have been certified and used in endemic regions for decades, the neutralizing antibody (NAb) titer induced by inactivated vaccines is low and the immunization schedule is complicated, requiring at least three injections spanning approximately 6 months to 1 year. Replication-competent vesicular stomatitis virus (VSV)-based vaccines provide prolonged protection after a single injection. In this study, we successfully engineered the HTNV glycoprotein (GP) in the VSV genome by replacing the VSV-G open reading frame. The resulting recombinant (r) rVSV-HTNV-GP was rescued, and the immunogenicity of GP was similar to that of HTNV. BALB/c mice immunized with rVSV-HTNV-GP showed a high titer of NAb against HTNV after a single injection. Notably, the cross-reactive NAb response induced by rVSV-HTNV-GP against Seoul virus (an orthohantavirus) was higher than that induced by three sequential injections of inactivated vaccines. Upon challenge with HTNV, rVSV-HTNV-GP-immunized mice showed a profoundly reduced viral burden in multiple tissues, and inflammation in the lungs and liver was nearly undetectable. Moreover, a single injection of rVSV-HTNV-GP established a prolonged immunological memory status as the NAbs were sustained for over 1 year and provided long-term protection against HTNV infection. The findings of our study can support further development of an rVSV-HTNV-GP-based HTNV vaccine with a simplified immunization schedule.

[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.

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.

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.

Low-noise fluorescent detection of cardiac troponin I in human serum based on surface acoustic wave separation.

Acute myocardial infarction (AMI) is a life-threatening disease when sudden blockage of coronary artery occurs. As the most specific biomarker, cardiac troponin I (cTnI) is usually checked separately to diagnose or eliminate AMI, and achieving the accurate detection of cTnI is of great significance to patients' life and health. Compared with other methods, fluorescent detection has the advantages of simple operation, high sensitivity and wide applicability. However, due to the strong fluorescence interference of biological molecules in body fluids, it is often difficult to obtain high sensitivity. In order to solve this problem, in this study, surface acoustic wave separation is designed to purify the target to achieve more sensitive detection performance of fluorescent detection. Specifically, the interference of background noise is almost completely removed on a microfluidic chip by isolating microbeads through acoustic radiation force, on which the biomarkers are captured by the immobilized detection probe. And then, the concentration of cTnI in human serum is detected by the fluorescence intensity change of the isolated functionalized beads. By this way, the detection limit of our biosensor calculated by 3σ/K method is 44 pg/mL and 0.34 ng/mL in PBS buffer and human serum respectively. Finally, the reliability of this method has been validated by comparison with clinical tests from the nephelometric analyzer in hospital.

[Preparation of mouse monoclonal antibodies against human adenovirus 55 Hexon (HAdV55 Hexon) protein].

Objective To prepare specific mouse monoclonal antibody (mAb) against human adenovirus type 55 Hexon protein (HAdV55 Hexon). Methods The Hexon genes of HAdV55, 3, 4, 7, 16 and 21 were chemically synthesized as templates for PCR amplification. The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon were constructed respectively. The pET28a-HAdV55 Hexon plasmid was transformed into E. coli competent cell BL21 (DE3) and was induced by IPTG. After the purified inclusion body was denatured and renatured, Hexon55 protein was purified by tangential flow filtration system. pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping, and HAdV55 Hexon protein was used to booster immunization. The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined. The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay (IFA) using BHK cells transfected with pCAGGS-HAdV55 Hexon. Both clones with high titer were selected, and the cross-reactivity of pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA. Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon, 3, 4, 7, 16 and 21 expression plasmids were successfully constructed. BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG. The HAdV55 Hexon protein was mainly expressed in the form of inclusion body. After denaturation and renaturation, the purified HAdV55 Hexon protein was obtained by ultrafiltration. Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained. The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b. Two specific HAdV55 Hexon antibodies with high titer were obtained, and there was no cross-reactivity with HAdV3, 4, 7, 16, 21 Hexon. Conclusion The specific mice mAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.

Multiple virus sorting based on aptamer-modified microspheres in a TSAW device.

Due to the overlapping epidemiology and clinical manifestations of flaviviruses, differential diagnosis of these viral diseases is complicated, and the results are unreliable. There is perpetual demand for a simplified, sensitive, rapid and inexpensive assay with less cross-reactivity. The ability to sort distinct virus particles from a mixture of biological samples is crucial for improving the sensitivity of diagnoses. Therefore, we developed a sorting system for the subsequent differential diagnosis of dengue and tick-borne encephalitis in the early stage. We employed aptamer-modified polystyrene (PS) microspheres with different diameters to specifically capture dengue virus (DENV) and tick-borne encephalitis virus (TBEV), and utilized a traveling surface acoustic wave (TSAW) device to accomplish microsphere sorting according to particle size. The captured viruses were then characterized by laser scanning confocal microscopy (LSCM), field emission scanning electron microscopy (FE-SEM) and reverse transcription-polymerase chain reaction (RT‒PCR). The characterization results indicated that the acoustic sorting process was effective and damage-free for subsequent analysis. Furthermore, the strategy can be utilized for sample pretreatment in the differential diagnosis of viral diseases.

An algal lectin griffithsin inhibits Hantaan virus infection in vitro and in vivo.

Hantaan virus (HTNV) is the etiological pathogen of hemorrhagic fever with renal syndrome in East Asia. There are currently no effective therapeutics approved for HTNV and other hantavirus infections. We found that griffithsin (GRFT), an algae-derived lectin with broad-spectrum antiviral activity against various enveloped viruses, can inhibit the growth and spread of HTNV. In vitro experiments using recombinant vesicular stomatitis virus (rVSV) with HTNV glycoproteins as a model revealed that the GRFT inhibited the entry of rVSV-HTNV-G into host cells. In addition, we demonstrated that GRFT prevented authentic HTNV infection in vitro by binding to the viral N-glycans. In vivo experiments showed that GRFT partially protected the suckling mice from death induced by intracranial exposure to HTNV. These results demonstrated that GRFT can be a promising agent for inhibiting HTNV infection.

Dihydropyridine-derived calcium channel blocker as a promising anti-hantavirus entry inhibitor.

Hantaviruses, the causative agent for two types of hemorrhagic fevers, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), are distributed from Eurasia to America. HFRS and HPS have mortality rates of up to 15% or 45%, respectively. Currently, no certified therapeutic has been licensed to treat hantavirus infection. In this study, we discovered that benidipine hydrochloride, a calcium channel blocker, inhibits the entry of hantaviruses in vitro. Moreover, an array of calcium channel inhibitors, such as cilnidipine, felodipine, amlodipine, manidipine, nicardipine, and nisoldipine, exhibit similar antiviral properties. Using pseudotyped vesicular stomatitis viruses harboring the different hantavirus glycoproteins, we demonstrate that benidipine hydrochloride inhibits the infection by both HFRS- and HPS-causing hantaviruses. The results of our study indicate the possibility of repurposing FDA-approved calcium channel blockers for the treatment of hantavirus infection, and they also indicate the need for further research in vivo.

Axl-/- neurons promote JEV infection by dampening the innate immunity.

Japanese encephalitis virus (JEV) causes the most commonly diagnosed viral encephalitis in Asia. JEV is a highly neurotropic flavivirus that can replicate efficiently in the brain. Axl belongs to the TAM (Tyro3, Axl, Mer) family, a group of tyrosine kinase receptors involved in the viral entry, micked as apoptotic bodies and regulation of innate immunity. However, the underlying mechanisms on its regulation in the neurons for JEV are unclear. Here, we found that Axl was upregulated in neurons after JEV infection. Unexpectedly, Axl deficient (Axl-/-) mice were more susceptible to JEV infection with increased viral loads in neurons. The RNA-sequencing analysis between the wild type neurons and Axl-/- neurons infected with JEV showed that many interferon-stimulated genes were downregulated in the Axl-/- neurons which innate immunity was attenuated largely. The rescue experiment in Axl-/- neurons indicated that Axl may be positively involved in the regulation of antiviral immunity. Taken together, our data demonstrated that Axl may play an antiviral role in JEV replication within neurons by modulating neuronal innate immunity.

Corrigendum: Remdesivir (GS-5734) Impedes Enterovirus Replication Through Viral RNA Synthesis Inhibition.

[This corrects the article DOI: 10.3389/fmicb.2020.01105.].

Remdesivir (GS-5734) Impedes Enterovirus Replication Through Viral RNA Synthesis Inhibition.

Human enteroviruses are responsible for diverse diseases, from mild respiratory symptoms to fatal neurological complications. Currently, no registered antivirals have been approved for clinical therapy. Thus, a therapeutic agent for the enterovirus-related disease is urgently needed. Remdesivir (GS-5734) is a novel monophosphoramidate adenosine analog prodrug that exhibits potent antiviral activity against diverse RNA virus families, including positive-sense Coronaviridae and Flaviviridae and negative-sense Filoviridae, Paramyxoviridae, and Pneumoviridae. Currently, remdesivir is under phase 3 clinical development for disease COVID-19 treatment. Here, we found that remdesivir impeded both EV71 viral RNA (vRNA) and complementary (cRNA) synthesis, indicating that EV71 replication is inhibited by the triphosphate (TP) form of remdesivir. Moreover, remdesivir showed potent antiviral activity against diverse enteroviruses. These data extend the remdesivir antiviral activity to enteroviruses and indicate that remdesivir is a promising antiviral treatment for EV71 and other enterovirus infections.

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.

Corrigendum: An Improved Enzyme-Linked Focus Formation Assay Revealed Baloxavir Acid as a Potential Antiviral Therapeutic Against Hantavirus Infection.

[This corrects the article DOI: 10.3389/fphar.2019.01203.].

An Improved Enzyme-Linked Focus Formation Assay Revealed Baloxavir Acid as a Potential Antiviral Therapeutic Against Hantavirus Infection.

Hantaviruses, etiologic pathogens responsible for two severe human diseases, exist in areas ranging from Eurasia to America and remain global public health concerns. Conventionally, plaque formation assays have been used for hantavirus titering. However, hantaviruses replicate slowly within cells and produce minimal cytopathic effects, making this technique difficult to master. The improved enzyme-linked immunosorbent assay-based antigen detection method is easier to perform but is still time consuming. Here, we established an enzyme-linked focus formation assay (FFA) for Hantaan virus titering that is twice as fast as traditional assays. Moreover, using this method, we evaluated the effects of favipiravir (T-705) and another influenza virus drug, baloxavir acid (BXA), on hantavirus replication. We found that the endonuclease inhibitor BXA exerted similar anti-hantavirus effects as T-705. Overall, we developed a time-saving method for hantavirus titering and suggest BXA as a potential treatment choice for hantavirus-exposed individuals.

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.

[Isolation and purification of host factors interacting with Hantaan virus nucleocapsid protein].

Objective To construct the plasmid expressing the fusion protein of Hantaan virus nucleocapsid protein (HTNV NP) with affinity tag, and isolate the host factors interacting with NP using the affinity purification. Methods The synthetic streptavidin-FLAG (SF) gene and HTNV NP gene were cloned into the mammalian eukaryotic expression vector to obtain the recombinant expression plasmid (pCAGGS-SF-NP). The plasmid pCAGGS-SF-NP was transfected into HEK293T cells, and the expression of SF-NP was detected by Western blotting. Next, cell lysates were mixed with StrepTrapTM HP agar beads. After incubating overnight at 4DegreesCelsius, the agar beads were transferred into affinity chromatography column and washed with elution buffer. Finally, the binding proteins that interacted with SF-NP were collected by competitive elution buffer with desthiobiotin, and then were subjected to SDS-PAGE. Results The recombinant SF-NP proteins were highly expressed in eukaryotic cells. The host factors interacting with SF-NP were successfully enriched by affinity purification, and confirmed by SDS-PAGE. Conclusion The host factors interacting with HTNV NP can be isolated by affinity purification.

Antibody-induced uncoating of human rhinovirus B14.

Rhinoviruses (RVs) are the major causes of common colds in humans. They have a nonenveloped, icosahedral capsid surrounding a positive-strand RNA genome. Here we report that the antigen-binding (Fab) fragment of a neutralizing antibody (C5) can trigger genome release from RV-B14 to form emptied particles and neutralize virus infection. Using cryo-electron microscopy, structures of the C5 Fab in complex with the full and emptied particles have been determined at 2.3 Å and 3.0 Å resolution, respectively. Each of the 60 Fab molecules binds primarily to a region on viral protein 3 (VP3). Binding of the C5 Fabs to RV-B14 results in significant conformational changes around holes in the capsid through which the viral RNA might exit. These results are so far the highest resolution view of an antibody-virus complex and elucidate a mechanism whereby antibodies neutralize RVs and related viruses by inducing virus uncoating.