Transfected Vero Cells Research Articles

Multiple Porcine Innate Immune Signaling Pathways Are Involved in the Anti-PEDV Response.

Porcine epidemic diarrhea virus (PEDV) has caused great damage to the global pig industry. Innate immunity plays a significant role in resisting viral infection; however, the exact role of innate immunity in the anti-PEDV response has not been fully elucidated. In this study, we observed that various porcine innate immune signaling adaptors are involved in anti-PEDV (AJ1102-like strain) activity in transfected Vero cells. Among these, TRIF and MAVS showed the strongest anti-PEDV activity. The endogenous TRIF, MAVS, and STING were selected for further examination of anti-PEDV activity. Agonist stimulation experiments showed that TRIF, MAVS, and STING signaling all have obvious anti-PEDV activity. The siRNA knockdown assay showed that TRIF, MAVS, and STING are also all involved in anti-PEDV response, and their remarkable effects on PEDV replication were confirmed in TRIF-/-, MAVS-/- and STING-/- Vero cells via the CRISPR approach. For further verification, the anti-PEDV activity of TRIF, MAVS, and STING could be reproduced in porcine IPEC-DQ cells treated with siRNAs. In summary, this study reveals that multiple pattern-recognition receptor (PRR) signaling pathways of porcine innate immunity play an important role in the anti-PEDV infection, providing new and useful antiviral knowledge for prevention and control of PEDV spreading.

Development of a cell-based reporter assay for detection of Human alphaherpesviruses

Immunosuppressed patients can suffer from Human alphaherpesvirus (HSV) infection with fast evolution, severe atypical symptomatology, and often-fatal outcome. Thus, the development and validation of new methods in vitro and in vivo to promote an early diagnosis and effective treatment of these patients are crucial. Therefore, this work aimed to develop a cell-based reporter assay for the detection of HSV through the transfection of Vero cells with the ICP10 promoter from HSV-2 linked to the pZsGreen1-1 plasmid. The assay was evaluated on Vero cells infected with HSV-1 or HSV-2 and followed by treating them with anti-HSV agents (acyclovir, gallic acid, convallatoxin, and Uncaria sp. extract) or with no anti-HSV activity agents (Passiflora edulis extract and cardenolide derivatives). The GFP expression was increased by both HSV cellular infection, which was detected by flow cytometry and fluorescence microscopy. F2R Zsgreen1-1 cells infection with 200 and 600 PFU/mL of HSV-2 increased the fluorescence intensity, when compared to the controls, by approximately 30% and 60%, respectively. Infection with 100 and 600 PFU/mL of HSV-1 also increased the fluorescence intensity by approximately 20% and 35%, when compared to the controls, respectively. The F2R ZsGreen1-1 system revealed to be an efficient assay, which can be used for clinical diagnosis, antiviral resistance evaluation, HSV cycle studies, and new antiviral drug research.

Construction of the vaccine strain of the influenza B virus with chimeric hemagglutinin to induce a cross-protective immune response

BACKGROUND: Influenza viruses cause worldwide epidemics, and the most effective method to prevent influenza disease is regular vaccinations. The development of new generation vaccines is aimed primarily at the formation of an immune response against a wide range of influenza viruses. One of the promising approaches is sequential vaccination with chimeric influenza viruses with identical stem domains of the hemagglutinin surface protein.
 AIM: The development of an experimental vaccine strain of influenza B virus with chimeric hemagglutinin consisting of head and stem domains of influenza B viruses belonging to different genetic lineages.
 MATERIALS AND METHODS: A chimeric influenza hemagglutinin gene was obtained by genetic engineering from the genetic material of B/Victoria and B/Yamagata influenza strains. The gene was inserted into the vector for the reverse genetics of the influenza virus. The influenza B virus strain with chimeric hemagglutinin was obtained by transfection of Vero cells using an 8-plasmid system. The rest of the genes were obtained from the attenuated influenza B virus with cold-adapted and temperature-sensitive phenotypes. The biological properties of the obtained recombinant strain, its infectious titer in developing chicken embryos and MDCK cell culture were evaluated.
 RESULTS: A recombinant vaccine strain has been successfully rescued. The head domain of the hemagglutinin of the virus is inherited from the B/Victoria influenza virus, and the stem domain from the B/Yamagata virus. The virus actively replicated in eggs and MDCK cells, with temperature-sensitive and cold-adapted phenotypes identical to classical live attenuated influenza vaccine viruses. The thermal stability of the chimeric hemagglutinin did not differ significantly from the thermal stability of the hemagglutinins of the donor viruses.
 CONCLUSIONS: The results obtained indicate the possibility of creating a strain with chimeric hemagglutinin, fragments of which are inherited from different genetic lineages. The growth characteristics and biological properties of the strain make it a promising candidate for the experimental evaluation of the possibility of inducing a cross-protective immune response by sequential vaccination with vaccine strains with identical stem hemagglutinin domains.

Generation and in vitro characterization of engineered cold-adapted influenza A strains with modified NS gene

BACKGROUND: The high variability of influenza strains and the emergence of new variants of viruses lead to the need for constant updating of the composition of influenza vaccines. One of the options for solving this problem is the development of vaccines with enhanced cross-protection against a wide range of influenza strains. Genetically engineered preparations based on live influenza vaccine can be used for targeted stimulation of the cellular immune response. It has been experimentally established that CTL epitopes inserted into the NS gene of the live influenza vaccine strain cause the activation of lymphocytes and the formation of a pool of resident memory T-cells in the lungs of model animals. It is optimal to use experimentally confirmed immunogenic regions for insertion.
 AIM: The aim of this study was to rescue a panel of experimental cold-adapted live attenuated influenza vaccine strains with a modified NS gene using A/Leningrad/134/17/57 backbone and recent influenza strains of H1N1, H3N2 and H7N9 subtypes, and evaluate their properties in vitro.
 MATERIALS AND METHODS: A cassette encoding immunogenic, conserved among a wide range of influenza strains T-cell epitopes of the influenza virus PB1 protein restricted by common HLA-allotypes was inserted into the gene encoding the NS1 protein. The modified NS gene was cloned into the pCIPolISapIT influenza virus reverse genetics vector. Chimeric influenza viruses were rescued by transfection of Vero cells by electroporation using a standard 8-plasmid system. The growth characteristics of viruses were assessed in developing chicken embryos.
 Results: Three strains were successfully obtained based on the live influenza vaccine master donor virus A/Leningrad/ 134/17/57 with a modified NS gene and influenza viruses of the H1N1, H3N2, H7N9 subtypes. Thus, modification of NS gene by insertion of immunogenic PB1 epitopes did not affect the viability and replicative activity of the rescued chimeric live influenza vaccine strains, regardless of the composition of the surface proteins. The strains replicated well at an optimal temperature, had temperature-sensitive phenotype and were able to grow at low temperature.
 CONCLUSIONS: The strains will be further studied as candidates for influenza prophylaxis as an experimental universal influenza vaccine.

Growth characteristics of experimental live influenza vaccine strains with modified NP and NS genes

BACKGROUND: Vaccination is the most effective means of fighting influenza epidemics, but the immunogenicity of licensed influenza vaccines is not always satisfactory. One of the ways to increase the immunogenicity of an attenuated live influenza vaccine is to shorten the open reading frame of the NS1 protein, a modulator of innate antiviral immunity. In addition, the T-cell response to vaccination can be optimized by including the NP gene from the epidemic parental virus into the genome of vaccine strains.
 MATERIALS AND METHODS: The open reading frame of the NS1 protein of the master donor virus A/Leningrad/134/17/57 was truncated to 126 amino acids by site-directed mutagenesis. The HA, NA, and NP genes of the model virus A/Anhui/1/2013 (H7N9) were cloned into the pCIPolISapIT vector. The rescue of recombinant influenza viruses was performed by transfection of Vero cells with a desired set of plasmids. The growth properties of the recombinant viruses were determined in embryonated chicken eggs incubated at different temperatures, as well as in the tissues of the respiratory tract of mice (nasal turbinates, lungs).
 RESULTS: Experimental live influenza vaccine strains of subtype H7N9 with genome compositions 6:2 and 5:3 and carrying a full-length or truncated NS1 gene were actively replicated in eggs under optimal conditions, while maintaining the temperature-sensitive and cold-adapted phenotypes characteristic of classical live influenza vaccine strains. All viruses lacked the ability to grow in the lungs of C57BL/6J mice, which confirms the attenuated phenotype of the viruses. In the nasal passages of mice, only viruses with the full-length NS1 gene replicated, while viruses expressing the truncated NS1 protein were not detected in the respiratory tract of animals.
 CONCLUSIONS: The results indicate that modification of the NS1 gene of the vaccine virus and the inclusion of wild-type NP gene in its genome does not affect its growth characteristics in eggs. A decrease in the activity of viral replication in the upper respiratory tract of mice with a shortening of the NS1 open reading frame indicates an increase in the attenuating properties of modified vaccines, which opens up prospects for the use of new vaccines in children under three years of age.

The anti-HIV drug nelfinavir mesylate (Viracept) is a potent inhibitor of cell fusion caused by the SARSCoV-2 spike (S) glycoprotein warranting further evaluation as an antiviral against COVID-19 infections.

Severe acute respiratory syndrome coronavirus‐2 (SARS CoV‐2) is the causative agent of the coronavirus disease‐2019 (COVID‐19) pandemic. Coronaviruses enter cells via fusion of the viral envelope with the plasma membrane and/or via fusion of the viral envelope with endosomal membranes after virion endocytosis. The spike (S) glycoprotein is a major determinant of virus infectivity. Herein, we show that the transient expression of the SARS CoV‐2 S glycoprotein in Vero cells caused extensive cell fusion (formation of syncytia) in comparison to limited cell fusion caused by the SARS S glycoprotein. Both S glycoproteins were detected intracellularly and on transfected Vero cell surfaces. These results are in agreement with published pathology observations of extensive syncytia formation in lung tissues of patients with COVID‐19. These results suggest that SARS CoV‐2 is able to spread from cell‐to‐cell much more efficiently than SARS effectively avoiding extracellular neutralizing antibodies. A systematic screening of several drugs including cardiac glycosides and kinase inhibitors and inhibitors of human immunodeficiency virus (HIV) entry revealed that only the FDA‐approved HIV protease inhibitor, nelfinavir mesylate (Viracept) drastically inhibited S‐n‐ and S‐o‐mediated cell fusion with complete inhibition at a 10‐μM concentration. In‐silico docking experiments suggested the possibility that nelfinavir may bind inside the S trimer structure, proximal to the S2 amino terminus directly inhibiting S‐n‐ and S‐o‐mediated membrane fusion. Also, it is possible that nelfinavir may act to inhibit S proteolytic processing within cells. These results warrant further investigations of the potential of nelfinavir mesylate to inhibit virus spread at early times after SARS CoV‐2 symptoms appear.

Study of the activation of the PI3K/Akt pathway by the motif of σA and σNS proteins of avian reovirus.

The present study was conducted to determine whether avian reovirus (ARV) activates the phosphatidylinositol 3-kinase-dependent Akt (PI3K/Akt) pathway according to the PXXP or YXXXM motifs of σA and σNS proteins. Gene splicing by overlap extension PCR was used to change the PXXP or YXXXM motifs of the σA and σNS genes. Plasmid constructs that contain mutant σA and σNS genes were generated and transfected into Vero cells, and the expression levels of the corresponding genes were quantified according to immunofluorescence and Western blot analyses. The Akt phosphorylation (P-Akt) profile of the transfected Vero cells was examined by flow cytometry and Western blot. The results showed that the σA and σNS genes were expressed in the Vero cells, and P-Akt expression in the σA mutant groups (amino acids 110–114 and 114–117) was markedly decreased. The results indicated that the σA protein of ARV activates the PI3K/Akt pathway via the PXXP motif. The results of this study reveal the mechanisms by which ARV manipulates the cellular signal transduction pathways, which may provide new ideas for novel drug targets.

A GFP-tagged version of the pseudorabies virus protein UL56 localizes to the Golgi and trans-Golgi network through a predicted C-terminal leucine-rich helix in transfected cells

BackgroundPseudorabies virus (PRV) protein UL56 (pUL56) has been implicated in viral dissemination and virulence in vivo. However, the properties of PRV pUL56 remain largely unknown. In the present study, we aim to investigate the subcellular localization of pUL56 and the underlying molecular basis in transfected cells.MethodsConstructs of N-terminal green fluorescent protein (GFP) fused pUL56 and its truncations were employed for investigating subcellular localization and further identifying amino acids crucial for pUL56 localization in transfected Vero cells. Finally, the identified amino acids were replaced with alanine for confirming if these mutations could impair the specific localization of pUL56.ResultsThe pUL56 predominantly localized at the Golgi and trans-Golgi network (TGN) through its predicted C-terminal transmembrane helix in transfected Vero cells. A Golgi-associated protein Rab6a, independent of interaction with pUL56, was significantly downregulated by pUL56. Further, we found three truncated pUL56 C-terminal fragments (174–184, 175–185 and 191–195) could restrict GFP in the perinuclear region, respectively. Within these truncations, the 174proline (P), 181leucine (L), 185L and 191L were essential for maintaining perinuclear accumulation, thus suggesting an important role of leucine. Alanine (A) mutagenesis assays were employed to generate a series of pUL56 C-terminal mutants on the basis of leucine. Finally, a pUL56 mutant M10 (174P/A-177L/A-181L/A-185L/A-191L/A-194L/A-195I/A-196-197L/A-200L/A) lost Golgi-TGN localization. Thus, our data revealed that the leucine-rich transmembrane helix was responsible for pUL56 Golgi-TGN localization and retention, probably through specific intracellular membrane insertion.ConclusionOur data indicated that the C-terminal transmembrane helix was responsible for the Golgi-TGN localization of pUL56. In addition, the leucines within C-terminal transmembrane helix were essential for maintaining pUL56 Golgi-TGN retention in cells. Further, the pUL56 can induce downregulation of Golgi-associated protein Rab6a.

Plasmid DNA launches live-attenuated Japanese encephalitis virus and elicits virus-neutralizing antibodies in BALB/c mice

We describe novel plasmid DNA that encodes the full-length Japanese encephalitis virus (JEV) genomic cDNA and launches live-attenuated JEV vaccine in vitro and in vivo. The synthetic cDNA based on the sequence of JEV SA14-14-2 live-attenuated virus was placed under transcriptional control of the cytomegalovirus major immediate-early promoter. The stability and yields of the plasmid in E. coli were optimized by inserting three synthetic introns that disrupted JEV cDNA in the structural and nonstructural genes. Transfection of Vero cells with the resulting plasmid resulted in the replication of JEV vaccine virus with intron sequences removed from viral RNA. Furthermore, a single-dose vaccination of BALB/c mice with 0.5 − 5μg of plasmid resulted in successful seroconversion and elicitation of JEV virus-neutralizing serum antibodies. The results demonstrate the possibility of using DNA vaccination to launch live-attenuated JEV vaccine and support further development of DNA-launched live-attenuated vaccine for prevention of JEV infections.

Avian reovirus σA and σNS proteins activate the phosphatidylinositol 3-kinase-dependent Akt signalling pathway.

The present study was conducted to identify avian reovirus (ARV) proteins that can activate the phosphatidylinositol 3-kinase (PI3K)-dependent Akt pathway. Based on ARV protein amino acid sequence analysis, σA, σNS, μA, μB and μNS were identified as putative proteins capable of mediating PI3K/Akt pathway activation. The recombinant plasmids σA-pcAGEN, σNS-pcAGEN, μA-pcAGEN, μB-pcAGEN and μNS-pcAGEN were constructed and used to transfect Vero cells, and the expression levels of the corresponding genes were quantified by immunofluorescence and Western blot analysis. Phosphorylated Akt (P-Akt) levels in the transfected cells were measured by flow cytometry and Western blot analysis. The results showed that the σA, σNS, μA, μB and μNS genes were expressed in Vero cells. σA-expressing and σNS-expressing cells had higher P-Akt levels than negative control cells, pcAGEN-expressing cells and cells designed to express other proteins (i.e., μA, μB and μNS). Pre-treatment with the PI3K inhibitor LY294002 inhibited Akt phosphorylation in σA- and σNS-expressing cells. These results indicate that the σA and σNS proteins can activate the PI3K/Akt pathway.

Strategies to improve the immunogenicity of prM+E dengue virus type-2 DNA vaccine.

An important goal for dengue vaccines is to induce a high and durable level of neutralizing antibody. Three strategies were investigated for improving the immunogenicity of a prM+E dengue serotype 2 (DENV-2) DNA vaccine: 1) expression in two different plasmids; 2) adjustment of dose; and, 3) introduction of the E sequence of Japanese encephalitis virus (JEV) at the carboxy-terminal portion of DENV-2 E. Expression cassettes were designed to encode a full-length prM+E sequence of DENV-2 virus employing human-preferred codons (D2prMEopt), or a chimeric prM+E sequence in which the 100-residue carboxy-terminal region of E was derived from JEV (D2prMEJE20opt). pHIS and pCMVkan in the presence and absence of CpG motif, respectively, were used for cassette expression. The immunogenicity was compared in mice. Three injections of full-length-D2prMEopt in pHIS and pCMVkan induced a comparable neutralizing antibody titer at post-week-2-injection and post-week-4-injection. The 100-μg DNA dose induced a numerically but not statistically higher neutralizing antibody titer than the 10-μg dose. The chimeric-D2prMEJE20opt produced higher extracellular prM and E protein levels in transfected Vero cells, but had a tendency to induce a lower neutralizing antibody titer in mice when compared with the full-length-D2prMEopt. To optimize the immunogenicity of D2prMEopt-DNA candidate, both expression plasmids can be used to generate reproducible high neutralizing titer. A higher dose of DNA immunogen may induce a higher neutralizing antibody response. The strategy of the C-terminal region chimeric counterpart with JE20 did not improve but may have reduced the induction of neutralizing antibodies.

Accumulation of asoluble form of human nectin-2 is required for exerting the resistance against herpes simplex virus type 2 infection in transfected cells.

Cell entry of herpes simplex virus type 2 (HSV-2) requires the interaction of viral glycoprotein D (gD) with the receptor nectin-1 and herpesvirus entry mediator (HVEM). In addition, it is known that nectin-2 is also functional as areceptor for HSV-2, although the binding to the gD is weak. To examine an antiviral potential of asoluble form of human nectin-2 (hNectin-2Ig), transfected Vero cells expressing the entire ectodomain of nectin-2 fused to the Fc portion of human IgG were established. Specific binding of hNectin-2Ig to HSV-2 gD was confirmed by ELISA. Competitive ELISA demonstrated that accumulation of hNectin-2Ig in transfected cells increased significantly in acell culture time dependent manner. Viral growth of several HSV-2 strains was significantly inhibited in the transfected cells that were cultured for 72 hr compared with control Vero cells, but not in cells that were cultured for 24 hr. These results indicate that accumulation of asoluble form of nectin-2 is required for exerting the resistance against HSV-2 infection.

Development of a stable Gaussia luciferase enterovirus 71 reporter virus

We report a stable Gaussia luciferase enterovirus 71 (Gluc-EV71) reporter virus to facilitate drug discovery. The Gluc-EV71 reporter virus was generated by engineering the Gaussia luciferase (Gluc) gene between the 5′ untranslated region and VP4 gene of the EV71 genome. We could recover Gluc-EV71 after transfection of Vero cells with the cDNA clone-derived RNA. The reporter virus efficiently infects and replicates in various cell types (Vero, human rhabdomyosarcoma, and HeLa cells), producing robust luciferase activity. The Gluc-EV71 virus replicates slower than the wild-type virus in cell culture. The reporter virus is stable in maintaining the Gluc gene after five rounds of continuous passaging in Vero cells. Using known EV71 inhibitors, we demonstrate that the reporter virus can be used for antiviral testing. However, the Gluc-EV71 infection assay cannot be adapted to a homogenous format for high throughput screen, mainly due to the secreted nature of the Gluc protein and the short half-life of the Gluc luminescence signal. The Gluc-EV71 and its infection assay could be useful for antiviral drug discovery as well as for studying EV71 replication and pathogenesis.

Nordihydroguaiaretic acid (NDGA) inhibits replication and viral morphogenesis of dengue virus

Dengue is the most common mosquito borne viral disease in humans. The infection with any of the 4 dengue virus serotypes (DENV) can either be asymptomatic or manifest in two clinical forms, the mild dengue fever or the more severe dengue hemorrhagic fever that may progress into dengue shock syndrome. A DENV replicative cycle relies on host lipid metabolism; specifically, DENV infection modulates cholesterol and fatty acid synthesis, generating a lipid-enriched cellular environment necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with antioxidant and anti-inflammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for 24 and 48h with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated with different concentrations of NDGA. NDGA treatment significantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment also led to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number during DENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection by targeting genome replication and viral assembly.

Structure of the herpes simplex virus 1 genome: manipulation of nicks and gaps can abrogate infectivity and alter the cellular DNA damage response.

The herpes simplex virus 1 (HSV-1) virion DNA contains nicks and gaps, and in this study a novel assay for estimating the size and number of gaps in virion DNA was developed. Consistent with previous reports, we estimate that there are approximately 15 gaps per genome, and we calculate the average gap length to be approximately 30 bases. Virion DNA was isolated and treated with DNA-modifying enzymes in order to fill in the gaps and modify the ends. Interestingly, filling in gaps, blunting the ends, or adding random sequences to the 3' ends of DNA, producing 3' flaps, did not impair the infectivity of treated DNA following transfection of Vero cells. On the other hand, the formation of 5' flaps in the DNA following treatment resulted in a dramatic reduction (95 to 100%) in infectivity. Virion DNA stimulated DNA-PKcs activity in transfected cells, and DNA with 5' flaps stimulated a higher level of DNA-PKcs activity than that observed in cells transfected with untreated virion DNA. The infectivity of 5'-flapped DNA was restored in cells that do not express DNA-PKcs and in cells cotransfected with the immediate early protein ICP0, which degrades DNA-PKcs. These results are consistent with previous reports that DNA-dependent protein kinase (DNA-PK) and the nonhomologous end joining (NHEJ) repair pathway are intrinsically antiviral and that ICP0 can counteract this effect. We suggest that HSV-1 DNA with 5' flaps may induce an antiviral state due to the induction of a DNA damage response, primarily mediated by NHEJ, that renders the HSV-1 genome less efficient for lytic infection. For productive lytic infection to occur, HSV-1 must counteract a variety of cellular intrinsic antiviral mechanisms, including the DNA damage response (DDR). DDR pathways have been associated with silencing of gene expression, cell cycle arrest, and induction of apoptosis. In addition, the fate of viral genomes is likely to play a role in whether viral genomes adopt a configuration suitable for lytic DNA replication. This study demonstrates that virion DNA activates the cellular DDR kinase, DNA-PK, and that this response is inhibitory to viral infection. Furthermore, we show that HSV-1 ubiquitin ligase, ICP0, plays an important role in counteracting the negative effects of DNA-PK activation. These findings support the notion that DNA-PK is antiviral and suggest that the fate of incoming viral DNA has important consequences for the progression of lytic infection. This study underscores the complex evolutionary relationships between HSV and its host.

Expression of full length fusion (F) protein gene of Newcastle disease virus in mammalian expression system

The present study was carried out to clone and express fusion protein gene of Newcastle disease virus in mammalian expression system. The Newcastle disease virus (2K3) isolate from pigeon was propagated in 9–11 day-old embryonated chicken eggs and infected allantoic fluid was collected for RNA isolation. The fusion (F) gene of 1.68 Kb was amplified with cDNA as template and was cloned in TOPO cloning vector. The recombinant TOPO colonies were digested with NcoI and XhoI enzymes, the insert released was further ligated into pTriEx Neo 1.1 expression vector digested with the same enzymes and was transformed in Escherichia coli. The recombinant pTriEx colonies confirmed by colony PCR and restriction digestion were induced with 1mM IPTG which showed expressed fusion protein of 55 kDa at 4 h post induction and increased in overnight induced cultures. The recombinant pTriEx plasmid was transfected into vero cells. The cell lysate collected at 48 h and 72 h post transfection showed expressed fusion protein with the molecular weight of 55 kDa in 12% SDS-PAGE. The protein was further confirmed to be NDV specific by its immunoreactivity with NDV antiserum raised in rabbits showing fusion protein of 55 kDa. The immunofluorescence assay of transfected vero cells exhibited a bright cytoplasmic fluorescence confirming the fusion protein expression.

Human Enterovirus 71 DNA Vaccine Constructs Containing 5’UTR with Complete Internal Ribosome Entry Site Sequence Stimulated Improved Anti-Human Enterovirus 71 Neutralizing Immune Responses

Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for hand, foot and mouth disease (HFMD) have been developed. Here we examined the potential of improving the vaccines by inserting the EV71 5’ untranslated region (5’ UTR) containing the full length internal ribosome entry site (IRES) sequence to the EV71 VP1-based DNA vaccine constructs. Four vaccine constructs designated as 5’ UTR-VP1/EGFP, VP1/EGFP, 5’ UTR-VP1/pVAX and VP1/pVAX, were designed using the pEGFP-N1 and pVAX-1 expression vectors, respectively. Transfection of Vero cells with the vaccine constructs with the 5’-UTR (5’-UTR-VP1/EGFP and 5’ UTR-VP1/pVAX) resulted in higher percentages of cells expressing the recombinant protein in comparison to cells transfected with vectors without the 5’-UTR (67% and 57%, respectively). Higher IgG responses (29%) were obtained from mice immunized with the DNA vaccine construct with the full length 5’ UTR. The same group of mice when challenged with life EV71 produced significantly higher neutralizing antibody (NAb) titers (>5-fold). These results suggest that insertion of the EV71 5’ UTR sequence consisting of the full length IRES to the EV71 DNA vaccine constructs improved the efficacy of the constructs with enhanced elicitation of the neutralizing antibody responses.

Development of a novel DNA-launched dengue virus type 2 infectious clone assembled in a bacterial artificial chromosome

Major progress in Dengue virus (DENV) biology has resulted from the use of infectious clones obtained through reverse genetics. The construction of these clones is commonly based on high- or low-copy number plasmids, yeast artificial chromosomes, yeast-Escherichia coli shuttle vectors, and bacterial artificial chromosomes (BACs). Prokaryotic promoters have consistently been used for the transcription of these clones. The goal of this study was to develop a novel DENV infectious clone in a BAC under the control of the cytomegalovirus immediate-early promoter and to generate a virus with the fusion envelope-green fluorescent protein in an attempt to track virus infection. The transfection of Vero cells with a plasmid encoding the DENV infectious clone facilitated the recovery of infectious particles that increased in titer after serial passages in C6/36 cells. The plaque size and syncytia phenotypes of the recombinant virus were similar to those of the parental virus. Despite the observation of autonomous replication and the detection of low levels of viral genome after two passages, the insertion of green fluorescent protein and Renilla luciferase reporter genes negatively impacted virus rescue. To the best of our knowledge, this is the first study using a DENV infectious clone under the control of the cytomegalovirus promoter to facilitate the recovery of recombinant viruses without the need for in vitro transcription. This novel molecular clone will be useful for establishing the molecular basis of replication, assembly, and pathogenesis, evaluating potential antiviral drugs, and the development of vaccine candidates for attenuated recombinant viruses.

Enhancement of Dengue-2 E Protein Expression by the Expression of the Precursor Membrane Protein (Prm) of the Dengue-3 Virus

Several attempts to develop dengue recombinant subunit vaccines have failed due to insufficient levels of expression and incorrect folding of the E protein. In order to verify the importance of the precursor membrane protein to the level of E protein expression, we constructed two recombinant plasmids by cloning the full-length sequence of the prM gene from the dengue-2 and dengue-3 virus strains into a plasmid that expresses a truncated version of the E protein of the dengue-2 virus. Next, we evaluated these two constructs for their effect on the levels of E protein expression in vitro. Our results showed that both plasmids provided a correct expression of the E protein as E protein expression was detected in transfected Vero cells as demonstrated by indirect immunofluorescence and immunoblotting. Densitometry analysis of western blots of the cell extracts showed a 67.02% higher expression of E protein in the cells transfected with pCID2EtD3prM, indicating that the prM sequence of the dengue-3 virus may be more effective in assisting with the correct processing of the E protein. The results of this study may be used to increase the in vitro production of the E protein antigen for use in vaccines and for diagnostic purposes.

The simian varicella virus ORF A is expressed in infected cells but is non-essential for replication in cell culture.

The simian varicella virus (SVV) genome encodes ORF A, a truncated homolog of SVV ORF 4. The SVV ORF A was expressed as a 1.0-kb transcript in SVV-infected Vero cells. The ORF A promoter was active in infected Vero cells and was stimulated by the SVV immediate-early gene ORF 62 product (IE62), a viral transactivator of SVV genes. The SVV ORF A did not transactivate SVV IE, early, or late gene promoters in transfected Vero cells and was unable to augment IE62-mediated transactivation of SVV promoters. A SVV mutant lacking ORF A replicated as efficiently as wild-type SVV in infected Vero cells, indicating that ORF A expression is not essential for in vitro replication.