A Functional Minigenome of Parvovirus B19

Parvovirus B19 (B19V) is a human pathogenic virus of clinical relevance, characterized by a selective tropism for erythroid progenitor cells in bone marrow. Relevant information on viral characteristics and lifecycle can be obtained from experiments involving engineered genetic systems in appropriate in vitro cellular models. Previously, a B19V genome of defined consensus sequence was designed, synthesized and cloned in a complete and functional form, able to replicate and produce infectious viral particles in a producer/amplifier cell system. Based on such a system, we have now designed and produced a derived B19V minigenome, reduced to a replicon unit. The genome terminal regions were maintained in a form able to sustain viral replication, while the internal region was clipped to include only the left-side genetic set, containing the coding sequence for the functional NS1 protein. Following transfection in UT7/EpoS1 cells, this minigenome still proved competent for replication, transcription and production of NS1 protein. Further, the B19V minigenome was able to complement B19-derived, NS1-defective genomes, restoring their ability to express viral capsid proteins. The B19V genome was thus engineered to yield a two-component system, with complementing functions, providing a valuable tool for studying viral expression and genetics, suitable to further engineering for purposes of translational research.

Anti-Zika Virus Effects, Placenta Protection and Chemical Composition of Passiflora edulis Seeds Ethanolic Extract

Various beneficial biological activities have been reported for passion fruit (Passiflora edulis), grown in tropical and subtropical regions. However, no study has yet demonstrated its action against the Zika virus (ZIKV) infection. The present study aimed to investigate the anti-ZIKV of the ethanolic passion fruit seed extract (PFSE), from which, liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis allowed to putatively annotate the occurrence of piceatannol, astringin, scirpusin A, scirpusin B, isookanin-7-O-glucoside and naringenin-7-O-glucoside, two quadranguloside derivatives, tyrosine, and phenylalanine. PFSE is well tolerated by cell lines (Vero E6 and HTR-8/SVneo) and tissue explants from the human placenta. With 1- and 24-h treatments, PFSE consistently reduced the viral load and NS1 protein expression of the two strains of ZIKV (MR766 and PE243) in placental explants and cell lines. Thus, PFSE has a promising potential for developing anti-ZIKV treatments, protecting the placenta against infection caused by ZIKV, along with relevant antioxidant potential, represented by ferric reducing antioxidant power (FRAP) of 390.5 Trolox eq per g dry extract and half maximal inhibitory concentration (IC50) for 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) of 12.1 μg mL–1, and more than 90% protection from lipid peroxidation. Although further studies need to be conducted, PFSE treatment was safe and effective in trophoblast cells and placental explants, thus representing a promising class of all-accessible products based on the reuse of industrial by-products.

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.

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.

Dengue NS1 antigen kit shows high sensitivity for detection of recombinant dengue virus-2 NS1 antigen spiked with Aedes aegypti mosquitoes

Dengue, caused by the dengue virus (DENV) is a significant vector-borne disease. In absence of a specific treatment and vaccine, dengue is becoming a rising threat to public health. Currently, control of dengue mainly focuses on the surveillance of the mosquito vectors. Improved surveillance methods for DENV in mosquito populations would be highly beneficial to the public health. However, current methods of DENV detection in mosquitoes requires specialized equipment and expensive reagents and highly trained personnel. As an alternative, commercially available dengue NS1 antigen ELISA kits could be used for detection of DENV infection in Aedes aegypti mosquitoes. In this study, we explored the utility of commercially available Dengue NS1 antigen kit (J. Mitra & Co. Pvt. Ltd) for the detection of recombinant dengue virus-2 (rDENV-2) NS1 protein and serum of dengue infected patient spiked with Ae. aegypti mosquito pools. The kit was found to be highly sensitive and specific towards detection of all serotypes of DENV. Further, it could detect as low as 750 femto gram rDENV-2 NS1 protein. It was also observed that rDENV-2 NS1 antigen spiked with blood-fed and unfed mosquito pools could be detected. In addition, the kit also detected dengue infected patient serum spiked with Ae. aegypti mosquito pools. Overall, the Dengue NS1 antigen kit displayed high sensitivity towards detection of recombinant as well as serum NS1 protein spiked with Ae. aegypti mosquito pools and could be considered for the dengue virus surveillance after a field evaluation in Ae. aegypti mosquitoes.

Computational antigenic epitope prediction of clinical Indonesian Dengue virus NS1 protein

The identification of human Non-Structural-1 (NS1) protein epitopes will help us better understand Dengue virus (DENV) immunopathogenesis. In this study, several online and offline bioinformatic prediction tools were exploited to predict and analyze T-cell and B-cell epitopes of DENV NS1 consensus sequences originated from Indonesian clinical isolates. We identified a potential peptide at NS1155--163 (VEDYGFGIF) which interact with MHC-I allele HLA-B*40:01 and showed high binding affinity (IC50) scores ranging between 63.8 nM to 183.9 nM for all Indonesian DENV serotypes. Furthermore, we have succeeded identified a region at the C-terminal of Indonesian DENV NS1 protein between 325--344 as part of discontinuous antigenic epitope which conserved for all serotypes. Our analyses showed this region could induce strong and persistent antibody against all DENV serotypes by interacting with MHC-I molecule and also recognized by B-cell receptor. The identification of DENV NS1 T-cell and B-cell epitopes may help in the development of a new vaccine, drug discovery, and diagnostic system to help eradicate dengue infection.

In vitro and in silico Anti-dengue activity of Supercritical extract of medicinal plants against Dengue serotype-2

Dengue is transmitted by female Aedes mosquitoes. It has been reported that about 2.5 billion peoples are at the risk of dengue virus. Millions of cases of dengue virus occur worldwide each year. There is no antiviral drug available still. Hence, the researchers are in the search of new anti-dengue drugs from natural products. The present study is aimed to determine the anti-dengue activity of supercritical extracts of Andrographis paniculata, Berberis vulgaris, Carica papaya, Euphorbia hirta, Phyllanthus niruri and Tinospora cordifolia in vitro and in silico. The cell viability was evaluated in C6/36 cells line by using MTT assay using a microplate reader at 595 nm. The maximum non-toxic dose of C. papaya and B. vulgaris extracts were reported as 46.87µg/ml, 31.25µg/ml for A. paniculata, P. niruri and E. hirta and 23.43 µg/ml, for T. cordifolia. Further, the anti-dengue activity of plants extract was analyzed by real-time RT-PCR. 100 copies/ml of DENV-2 virus was used for the antiviral assay. A. paniculata supercritical extract showed complete inhibition against the dengue-2 virus at 40ºC temperature and 15Mpa pressure. The other plant extracts showed the inhibition in order of T. cordifolia (83.44%) > C. papaya (34.71%) > E. hirta (28.70%) whereas P. niruri and B. vulgaris failed to inhibit dengue virus. Andrographolide a major compound present in A. paniculata has been reported to have antiviral activity against hepatitis B, C virus, herpes simplex virus, influenza virus, chikungunya virus, dengue virus 2 and 4. Results of molecular docking showed that the interaction between andrographolide and NS1protein shows the maximum binding energy -7.30 Kcal/mol. The docking study was conducted to validate the result against the anti-dengue activity using dengue NS1 protein with andrographolide. It was concluded that A. paniculata could be a source for isolation of therapeutic compounds against the dengue-2 virus.

Zika Virus Seroprevalence in Two Districts of Chincha, Ica, Peru: A Cross-Sectional Study

In 2017, a major outbreak of Zika virus (ZIKV) infection took place in Chincha Province, Peru, where arboviral circulation had never been reported before. We conducted a cross-sectional survey (March–May 2019) in two districts of Chincha Province: Pueblo Nuevo and Chincha Baja. We included residents who were 20 to 40 years old and who had lived in these districts for at least 1 year. Serological testing combined screening with a commercial NS1 protein-based Zika IgG ELISA, and confirmation by a cytopathic effect-based virus neutralization test (VNT). Prevalence ratios (PRs) were calculated using Poisson regression with robust error variance. Four hundred participants, divided equally among districts, were enrolled. Anti-ZIKV IgG ELISA was positive for 42 participants (10.5%) and borderline for 12 (3%). Fifty-two of these 54 samples were confirmed positive by ZIKV VNT (13% of the total population). The Pueblo Nuevo district exhibited a greater ZIKV seroprevalence based on VNT results than the Chincha Baja district (23.5% versus 2.5%), with participants from the Pueblo Nuevo district being 9.4 times more likely to have a positive ZIKV VNT result. Average monthly income greater than the minimum wage and adequate water storage were found to be protective factors (PR, 0.29 and 0.24, respectively). In multivariate analysis, living in the Pueblo Nuevo district and a personal history of fever and rash were strong predictors of ZIKV positivity by VNT. The low ZIKV seroprevalence should prompt health authorities to stimulate interventions to prevent potential future outbreaks. In the Pueblo Nuevo district, the seroprevalence was greater but presumably not sufficient to ensure protective herd immunity.

Respiratory Syncytial virus NS1 protein targets the transactivator binding domain of MED25

Respiratory syncytial virus has evolved a unique strategy to evade host immune response by coding for two non-structural proteins NS1 and NS2. Recently it was shown that in infected cells, nuclear NS1 could be involved in transcription regulation of host genes linked to innate immune response, via an interaction with chromatin and the Mediator complex. Here we identified the MED25 Mediator subunit as an NS1 interactor in a yeast two-hybrid screen. We demonstrate that NS1 directly interacts with MED25 in vitro and in cellula, and that this interaction involves the C-terminal α3 helix of NS1 and the MED25 ACID domain. More specifically we showed by NMR that the NS1 α3 sequence primarily binds to the MED25 ACID H2 face, which is a transactivation domain (TAD) binding site for transcription regulators such as ATF6α, a master regulator of ER stress response activated upon viral infection. Moreover, we found out that the NS1 α3 helix could compete with ATF6α TAD binding to MED25. This finding points to a mechanism of NS1 interfering with innate immune response by impairing recruitment by cellular TADs of the Mediator via MED25 and hence transcription of specific genes by RNA polymerase II.

The Combined Expression of the Non-structural Protein NS1 and the N-Terminal Half of NS2 (NS2 1-180 ) by ChAdOx1 and MVA Confers Protection against Clinical Disease in Sheep upon Bluetongue Virus Challenge

Bluetongue, caused by bluetongue virus (BTV), is a widespread arthropod-borne disease of ruminants that entails a recurrent threat to the primary sector of developed and developing countries. In this work, we report MVA and ChAdOx1-vectored vaccines designed to simultaneously express the immunogenic NS1 protein and/or NS2-Nt, the N-terminal half of protein NS2 (NS2 1-180 ). A single dose of MVA or ChAdOx1 expressing NS1-NS2-Nt improved the protection conferred by NS1 alone in IFNAR(-/-) mice. Moreover, mice immunized with ChAdOx1/MVA-NS1, ChAdOx1/MVA-NS2-Nt or ChAdOx1/MVA-NS1-NS2-Nt developed strong cytotoxic CD8+ T-cell responses against NS1, NS2-Nt or both proteins and were fully protected against a lethal infection with BTV serotypes 1, 4 and 8. Furthermore, although a single immunization with ChAdOx1-NS1-NS2-Nt partially protected sheep against BTV-4, the administration of a booster dose of MVA-NS1-NS2-Nt promoted a faster viral clearance, reduction of the period and level of viremia and also protected from the pathology produced by BTV infection. Importance Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and infected animals (DIVA strategy) and are serotype specific. In this work we have develop a DIVA multiserotype vaccination strategy based on adenoviral (ChAdOx1) and MVA vaccine vectors, the most widely used in current phase I and II clinical trials, and the conserved non-structural BTV proteins NS1 and NS2. This immunization strategy solves the major drawbacks of the current marketed vaccines.