Determination of the cleavage site of enterovirus 71 VP0 and the effect of this cleavage on viral infectivity and assembly.

Hand, foot, and mouth disease (HFMD) is a major public health concern, especially among infants and young children. The primary pathogen of HFMD is enterovirus 71 (EV71), whose capsid assembly mechanism including capsid protein processing has been widely studied. However, some of its mechanisms remain unclear, such as the VP0 cleavage. This study aimed to identify the cleavage site of the EV71 VP0 capsid protein and to elucidate the effects of EV71 VP0 cleavage on viral infectivity and assembly. A mass spectrometry analysis indicated that the cleavage site of EV71 VP0 is located between residues Lys69 and Ser70. To analyze the importance of either residue to cleavage, we designed single mutations of Lys69, Ser70 and double mutations respectively and implemented these genomes to encapsulation. The results indicated that Ser70 is more important for VP0 cleavage and EV71 infectivity. In addition, exogenous expression of EV71 protease 2A and 3C was used to verify whether they play roles in VP0 cleavage. Analyses also showed that none of them participate in this process. This study provides novel insights into the mechanisms of EV71 capsid maturation, which may be a potential target to improve the productivity and immunogenicity of EV71 vaccines.

Exosome-Mediated Delivery of Inducible miR-423-5p Enhances Resistance of MRC-5 Cells to Rabies Virus Infection.

The human diploid cell line Medical Research Council -5 (MRC-5) is commonly utilized for vaccine development. Although a rabies vaccine developed in cultured MRC-5 cells exists, the poor susceptibility of MRC-5 cells to the rabies virus (RABV) infection limits the potential yield of this vaccine. The underlying mechanism of MRC-5 cell resistance to RABV infection remains unknown. In this study, we demonstrate that viral infection increased exosomal release from MRC-5 cells; conversely, blocking exosome release promoted RABV infection in MRC-5 cells. Additionally, RABV infection up-regulated microRNA (miR)-423-5p expression in exosomes, resulting in feedback inhibition of RABV replication by abrogating the inhibitory effect of suppressor of cytokine signaling 3 (SOCS3) on type I interferon (IFN) signaling. Furthermore, intercellular delivery of miR-423-5p by exosomes inhibited RABV replication in MRC-5 cells. We also show that RABV infection increased IFN-ß production in MRC-5 cells and that blocking the type I IFN receptor promoted RABV infection. In conclusion, MRC-5 cells were protected from RABV infection by the intercellular delivery of exosomal miR-423-5p and the up-regulation of IFN-ß. These findings reveal novel antiviral mechanisms in MRC-5 cells against RABV infection. miR-423-5p, exosomes, and IFN signaling pathways may therefore be potential targets for improving MRC-5 cell-based rabies vaccine production.

An Adenovirus-Based Recombinant Herpes Simplex Virus 2 (HSV-2) Therapeutic Vaccine Is Highly Protective against Acute and Recurrent HSV-2 Disease in a Guinea Pig Model.

Genital herpes (GH) has become one of the most common sexually transmitted diseases worldwide, and it is spreading rapidly in developing countries. Approximately 90% of GH cases are caused by HSV-2. Therapeutic HSV-2 vaccines are intended for people already infected with HSV-2 with the goal of reducing clinical recurrences and recurrent virus shedding. In our previous work, we evaluated recombinant adenovirus-based vaccines, including rAd-gD2ΔUL25, rAd-ΔUL25, and rAd-gD2, for their potency as prophylactic vaccines. In this study, we evaluated these three vaccines as therapeutic vaccines against acute and recurrent diseases in intravaginal challenged guinea pigs. Compared with the control groups, the recombinant vaccine rAd-gD2ΔUL25 induced a higher titer of the binding antibody, and rAd-gD2 + rAd-ΔUL25 induced a higher titer of the neutralizing antibody. Both rAd-gD2ΔUL25 and rAd-gD2 + rAd-ΔUL25 vaccines significantly enhanced the survival rate by 50% compared to rAd-gD2 and reduced viral replication in the genital tract and recurrent genital skin disease. Our findings provide a new perspective for HSV-2 therapeutic vaccine research and provide a new technique to curtail the increasing spread of HSV-2.

Comparison of effects of multiple adjuvants and immunization routes on the immunogenicity and protection of HSV-2 gD subunit vaccine.

Genital herpes caused by herpes simplex virus type 2 (HSV-2) poses a global health issue. HSV-2 infection increases the risk of acquiring HIV infection. Studies have demonstrated that HSV-2 subunit vaccines have potential benefits, but require adjuvants to induce a balanced Th1/Th2 response. To develop a novel, effective vaccine, in this study, a truncated glycoprotein D (aa 1-285) of HSV-2 was formulated with an Al(OH)3 adjuvant, three squalene adjuvants, zMF59, zAS03, and zAS02, or a mucosal adjuvant, bacterium-like particles (BLPs). The immunogenicity of these subunit vaccines was evaluated in mice. After three immunizations, vaccines formulated with Al(OH)3, zMF59, zAS03, and zAS02 (intramuscularly) induced higher titers of neutralizing antibody than that formulated without adjuvant, and in particular, mice immunized with the vaccine plus zAS02 had the highest neutralizing antibody titers and tended to produce a more balanced immune reaction than others. Intranasal gD2-PA-BLPs also induced excellent IgA levels and a more balanced Th1 and Th2 responses than intranasal gD2. After challenge with a lethal dose of HSV-2, all five adjuvants exhibited a positive effect in improving the survival rate. zAS02 and gD2-PA-BLPs enhanced survival by 50% and 25%, respectively, when compared with the vaccine without adjuvant. zAS02 was the only adjuvant that resulted in complete vaginal virus clearance and genital lesion healing within eight days. These results demonstrate the potential of using zAS02 as a subunit vaccine adjuvant, and BLPs as a mucosal vaccine adjuvant.

Interferon Inhibition Enhances the Pilot-Scale Production of Rabies Virus in Human Diploid MRC-5 Cells.

Inactivated vaccines based on cell culture are very useful in the prevention and control of many diseases. The most popular strategy for the production of inactivated vaccines is based on monkey-derived Vero cells, which results in high productivity of the virus but has a certain carcinogenic risk due to non-human DNA contamination. Since human diploid cells, such as MRC-5 cells, can produce a safer vaccine, efforts to develop a strategy for inactivated vaccine production using these cells have been investigated using MRC-5 cells. However, most viruses do not replicate efficiently in MRC-5 cells. In this study, we found that rabies virus (RABV) infection activated a robust interferon (IFN)-ß response in MRC-5 cells but almost none in Vero cells, suggesting that the IFN response could be a key limiting factor for virus production. Treatment of the MRC-5 cells with IFN inhibitors increased RABV titers by 10-fold. Additionally, the RABV titer yield was improved five-fold when using IFN receptor 1 (IFNAR1) antibodies. As such, we established a stable IFNAR1-deficient MRC-5 cell line (MRC-5IFNAR1-), which increased RABV production by 6.5-fold compared to normal MRC-5 cells. Furthermore, in a pilot-scale production in 1500 square centimeter spinner flasks, utilization of the MRC-5IFNAR1- cell line or the addition of IFN inhibitors to MRC cells increased RABV production by 10-fold or four-fold, respectively. Thus, we successfully established a human diploid cell-based pilot scale virus production platform via inhibition of IFN response for rabies vaccines, which could also be used for other inactivated virus vaccine production.

Myristoylation of EV71 VP4 is Essential for Infectivity and Interaction with Membrane Structure.

The Enterovirus 71 (EV71) VP4 is co-translationally linked to myristic acid at its amino-terminal glycine residue. However, the role of this myristoylation in the EV71 life cycle remains largely unknown. To investigate this issue, we developed a myristoylation-deficient virus and reporter (luciferase) pseudovirus with a Gly-to-Ala mutation (G2A) on EV71 VP4. When transfecting the EV71-G2A genome encoding plasmid in cells, the loss of myristoylation on VP4 did not affect the expression of viral proteins and the virus morphology, however, it did significantly influence viral infectivity. Further, in myristoylation-deficient reporter pseudovirus-infected cells, the luciferase activity and viral genome RNA decreased significantly as compared to that of wild type virus; however, cytopathic effect and viral capsid proteins were not detected in myristoylation-deficient virus-infected cells. Also, although myristoylation-deficient viral RNA and proteins were detected in the second blind passage of infection, they were much fewer in number compared to that of the wild type virus. The replication of genomic RNA and negative-strand viral RNA were both blocked in myristoylation-deficient viruses, suggesting that myristoylation affects viral genome RNA release from capsid to cytoplasm. Besides, loss of myristoylation on VP4 altered the distribution of VP4-green fluorescent protein protein, which disappeared from the membrane structure fraction. Finally, a liposome leakage assay showed that EV71 myristoylation mediates the permeability of the model membrane. Hence, the amino-terminal myristoylation of VP4 is pivotal to EV71 infection and capsid-membrane structure interaction. This study provides novel molecular mechanisms regarding EV71 infection and potential molecular targets for antiviral drug design.

Exosomes Released from Rabies Virus-Infected Cells May be Involved in the Infection Process.

Exosomes are cell-derived vesicles that are secreted by many eukaryotic cells. It has recently attracted attention as vehicles of intercellular communication. Virus-infected cells release exosomes, which contain viral proteins, RNA, and pathogenic molecules. However, the role of exosomes in virus infection process remains unclear and needs to be further investigated. In this study, we aimed to evaluate the effects of exosomes on rabies virus infection. OptiPrep™ density gradient centrifugation was used to isolate exosomes from rabies virus-infected cell culture supernatants. A rabies virus G protein enzyme-linked immunosorbent assay and acetylcholinesterase activity assays were performed to verify the centrifugation fractions. Exosomes were then characterized using transmission electron microscopy and Western blotting. Our results showed that rabies virus infection increased the release of exosomes. Treatment with GW4869 and si-Rab27a, two exosomal secretion inhibitors, inhibited exosome release. Furthermore, the inhibitors reduced the levels of extracellular and intracellular viral RNA. These data indicated that exosomes may participate in the viral infection process. Moreover, our results establish a basis for future research into the roles of exosomes in rabies virus infection and as potential targets for developing new antiviral strategies.

[Study on the extraction of polysaccharides from Marasmius androsaceus Mycelium].

OBJECTIVE: To optimize the extraction condition for the polysaccharides from Marasmius androsaceus Mycelium. METHODS: The single factor tests and response surface methodology (RSM) were applied. Overall research was proceeded by rational desingn. Technological condition parameters were determined according to regression analysis by SAS. RESULTS: The optimum condition was as follows: extraction temperature was 78 degrees C , extraction-duration was 1. 5h, water to material ratio was 36: 1. The optimum extracting rate for the polysaccharide was 11. 59%. CONCLUSION: The RSM is for optimum extraction process and it will improve the extraction conditions of polysaccharides.

Characterization of human enterovirus71 virus-like particles used for vaccine antigens.

Human enterovirus 71 (EV71) is a major causative pathogen of hand, foot and mouth disease (HFMD) and has caused outbreaks with significant mortality among young children in the Asia-Pacific region in recent years. Towards developing a vaccine for this disease, we have expressed and purified EV71 virus-like particles (VLPs), which resemble the authentic virus in appearance, capsid structure and protein sequence, from insect cells (Sf9) using a multistep chromatography process. We demonstrated intracellular localization of the VLPs in host cells by in situ immunogold detection, electron microscopy and immunofluorescence. Characteristics of these EV71 VLPs were studied using a variety of immunological and physicochemical techniques, which aimed to reveal that the purified EV71 VLPs have good morphology and structure consistent with natural EV71 empty capsids. Results of the amino acid analysis, SDS-PAGE, Western blotting and high-performance liquid chromatography confirmed the high purity of the EV71 VLPs. However the sedimentation coefficient of the VLPs showed that they were smaller than that of secreted EV71 VLPs purified by discontinuous cesium chloride density gradients, they were similar to the empty capsids of natural EV71 virions reported previously. Combined with the previous study that EV71 VLPs purified by a multistep chromatography process were able to elicit strong humoral immune responses in mice, our results further supported the conclusion that our EV71 VLPs had well-preserved molecular and structural characteristics. The EV71 VLPs produced from the baculovirus expression system and purified by a multistep chromatography process displayed key structural and immunological features, which would contribute to their efficacy as a HFMD vaccine.

Antiviral effects of Retro-2cycl and Retro-2.1 against Enterovirus 71 in vitro and in vivo.

Enterovirus 71 (EV71) is one of the causative pathogens of hand, foot and mouth disease (HFMD), especially the form associated with fatal neurological disorders. Sustained outbreaks of EV71 infections remain a serious health threat worldwide. However, no antiviral agent against EV71 for clinical therapy has been approved. Retro-2cycl and Retro-2.1 are inhibitors of several pathogens specifically targeting the intracellular vesicle transport, which also participates in the EV71 lifecycle processes including progeny virus release. Here, we reported that Retro-2cycl and Retro-2.1, respectively, could inhibit EV71 infection with 50% effective concentrations of 12.56 µM and 0.05 µM in a cytopathic effect inhibition assay and showed relatively low cytotoxicity with 50% cytotoxicity concentrations of more than 500 µM and 267.80 µM. Preliminary mechanism studies revealed that Retro-2cycl and Retro-2.1 did not inhibit EV71 protein synthesis or RNA replication but could block progeny EV71 release specifically. Furthermore, administration of Retro-2cycl at the dose of 10 mg/kg significantly protected 90% of newborn mice from lethal EV71 challenge. Consequently, our results for the first time identified Retro-2cycl and Retro-2.1 as effective inhibitors of EV71 as well as lead compounds, which would contribute to anti-EV71 drug development. We also identified progeny virus release and the intracellular vesicle transport as antiviral targets for EV71.

Synonymous codon usage analysis of hand, foot and mouth disease viruses: A comparative study on coxsackievirus A6, A10, A16, and enterovirus 71 from 2008 to 2015.

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) have been considered major pathogens of hand, foot and mouth disease (HFMD) throughout the world for decades. In recent years, coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) have raised attention as two other serious pathogens of HFMD. The present study focused on the synonymous codon usage of four viruses isolated from 2008 to 2015, with particular attention on P1 (encoding capsid proteins) and P2-P3 regions (both encoding non-structural proteins) in the genomic RNA. Relative synonymous codon usage, effective number of codons, neutrality and correspondence were analyzed. The results indicated that these viruses prefer A/T at the third position in codons rather than G/C. The most frequent codons of 4 essential and 2 semi-essential amino acids, as well as a key amino acid of metabolic junctions (Glu) used in the four viruses are also the most frequently used in humans. Effective number of codons (ENC) values indicated weak codon usage bias in all the viruses. Relatively, the force of mutation pressure in the P1 region was found to be stronger than that in the P2-P3 region, and this force in the P1 region of CVA6 and EV71 was stronger than that of CVA10 and A16. The neutrality analysis results implied that mutation pressure plays a minor role in shaping codon bias of these viruses. Correspondence analysis indicated that the codon usage of EV71 strains varied much more than that of other viruses. In conclusion, the present study provides novel and comparative insight into the evolution of HFMD pathogens at the codon level.