Effects of ultra-filtration purification of infectious bursal disease virus on immune responses and cytokine activation in specific pathogen free chickens.

Purification is an important step in the production of viral vaccines that strongly affects product recovery and subsequent immune responses. The present study was carried out with the aim of improving the purification of infectious bursal disease virus (IBDV) by the tangential flow filtration (TFF) method. Then, the effect of the purified virus on the induction of immune responses against IBDV in specific pathogen free (SPF) chickens was investigated. The IBD07IR strain was propagated in embryonated SPF eggs. The virus was purified using a 100 kDa cassette. The quality of the recovered viruses was evaluated by titration. A total number of 60 SPF chickens were randomly divided into three groups (n = 20) and received the concentrated viral antigen, commercial live IBDV vaccine and phosphate-buffered saline at the age of 3 weeks by eye drop method. The bursa of Fabricius was examined histopathologically for possible changes. Sera were collected at 1-week intervals from day 0 until the end of 6 weeks after vaccination. The IBDV-specific antibody levels, induction of cell-mediated immunity and mRNA expression levels of cytokines were evaluated. The results showed that despite a relative raise in virus titer from 7.66 to 8.17 embryo infectious dose (EID)50 mL-1 following purification, both the purified IBDV and commercial vaccine are able to induce strong immune responses against the virus. Within a context of egg-based IBDV vaccine production, a single-step TFF can be applied for the relatively purification. This platform requires a further study in the selection of multiple membranes to optimize the operating conditions and final product.

Down-regulating CD19 surface markers expression correlates with infectious bursal disease virus replication.

The infectious bursal disease virus (IBDV) causes an acute and highly contagious immunosuppressive response in young chickens by targeting B lymphocytes in immune organs. Changes in regulatory T-cell ratio and apoptosis have been demonstrated during IBDV infection in these cells. The possible change in CD19 expression as the precursor of B cells after IBDV replication was detected in this study. Raji cells were infected with an IBDV isolate at MOIs of 1.0 and 3.0. The viral kinetics were determined using the characteristic virus-induced CPE, cell viability, and infectious titer. Induction of apoptosis and also changes in the CD19 expression within the virus infection were assessed by flow cytometry. The Raji cells were found to be susceptible to IBDV infection by producing marked CPEs dependent on MOI. The infectivity titers were determined in intra- and extracellular samples at the defined hours. The kinetics of early IBDV replication in Raji cells were nearly identical for both MOIs, but a significant difference in the infectivity titer was observed at 48 hpi. The quick apoptotic events were observed to be significantly higher in MOI 3.0, which was correlated with the lower virus titer. A significant CD19 expression change in the IBDV-infected Raji cells was revealed. The results suggested that Raji cells mimic the IBDV replication in lymphoid organs and the virus replication is related to CD19 expression frequencies in the lymphoid cells.

SARS-CoV-2 Variant-Specific mRNA Vaccine: Pros and Cons.

Emerging severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants have raised concerns about the efficacy of vaccines. The present study aimed to compare the potential of Delta and Omicron variant-specific mRNA vaccines in inducing immune responses. B cell and T cell epitopes and population coverage of spike (S) glycoprotein of the variants were predicted using the Immune Epitope Database. The molecular docking was carried out between the protein and different toll-like receptors, as well as the receptor-binding domain (RBD) protein and angiotensin-converting-enzyme 2 (ACE2) cellular receptor using ClusPro. The molecular simulation was done for each docked RBD-ACE2 using YASARA. The mRNA secondary structure was predicted through the RNAfold. The simulation of immune responses to the mRNA vaccine construct was performed using C-ImmSim. Apart from a few positions, no significant difference was observed in the prediction of S protein B cell and T cell epitopes of these two variants. The lower amounts of Median consensus percentile in the Delta variant in similar positions signify its stronger affinity to major histocompatibility complex (MHC) II binding alleles. Docking of Delta S protein with TLR3, TLR4, and TLR7 and also its RBD with ACE2 showed striking interactions with the lower binding energy than Omicron. In the immune simulation, elevated levels of cytotoxic T lymphocytes, helper T lymphocytes, and memory cells in both the active and resting states and the main regulators of the immune system suggested the capacity of mRNA constructs to elicit robust immune responses against SARS-CoV-2 variants. Considering slight differences in the binding affinity to MHC II binding alleles, activation of TLRs, mRNA secondary structure stability, and concentration of immunoglobulins and cytokines, the Delta variant is suggested for the mRNA vaccine construction. Further studies are being done to prove the efficiency of the design construct.

A Novel Multi-Epitope Edible Vaccine Candidate for Newcastle Disease Virus: In Silico Approach.

Background: Newcastle disease, is one of the most important illnesses in the aviculture industry which shows a constant threat. In this case, the vaccine could be considered an important solution to prevent and control this disease. So, the development of a new and more effective vaccine against Newcastle disease is an urgent need. Immune informatics is an important field that provides insight into the experimental procedure and could facilitate the analysis of large amounts of immunological data generated by experimental research and help to design a new vaccine candidate. Objectives: This study is aimed at bioinformatics to investigate and select the most immunogenic and conserved epitopes derived from F and HN glycoproteins, which play a key role in pathogenesis and immunity. This strategy could cover a wide range of Newcastle disease viruses. Materials and Method: For expression in both E. coli (as an injectable recombinant vaccine candidate) and maize plant (as an edible vaccine candidate) host, two constructs were designed and analyzed separately. Furthermore, the role of LTB as an effective bio-adjuvant for general eliciting of the immune system and simultaneous expressions with those two antigens was evaluated. Hence, here a multimeric recombinant protein with the abbreviation LHN2F from the highly immunogenic part of HN, F and LTB proteins were designed. The synthetic construct was analyzed based on different bioinformatics tools. Results: The proper immunogenicity and stability of this multimeric fusion protein have been shown by immunoinformatic methods from various servers. To confirm the function of the designed protein, the final molecule was docked to chicken MHC class I using the Pyrex-python 0.8 program. the results of Immune Epitope analysis were confirmed by the docking results between protein and receptor. Conclusions: The results of structural and immunological computational studies proposed that the protein deduced from this novel construct could act as a vaccine candidate for Newcastle disease virus control and prophylactic.

A Combination of Recombinant HA1-and Nucleoprotein-Based Chitosan Nanoparticles Induces Early and Potent Immune Responses Against the H9N2 Influenza Virus.

The effectiveness of inactivated H9N2 influenza vaccines is doubtful due to changes in antigenic regions of the virus hemagglutinin (HA) protein. One strategy for the development of the efficacious vaccine is the use of nanoparticles that display more immunogenic regions of the influenza virus. In this study, chitosan (CS)-based nanoparticles were developed as a delivery system for intranasal immunization using recombinant H9N2 virus HA1 and nucleoprotein (NP), for the induction of humoral and cellular responses. CS-HA1 and CS-NP nanoparticles were prepared by the ionic gelation method and characterized for their physicochemical properties and shape. The immunogenicity and the protective efficacy were evaluated by measuring antibody titers, T cell proliferation response, CD4+/CD8+ ratio, and quantitative real-time RT-PCR following intranasal administration of the prepared nanoparticles alone or in combination in chickens compared to an inactivated H9N2 vaccine. The average size, surface charge, and spherical structure of the synthesized nanoparticles showed high quality. Serologic analysis revealed that the immunization of inactivated vaccine groups resulted in strong influenza antibodies, which were significantly (p < 0.05) higher compared to the other groups. The vaccinated chickens with CS-HA1+CS-NP developed higher specific anti-influenza antibodies than in those vaccinated with each of rHA1 and rNP. Administration of a combination of the protein-based nanoparticles has stimulated the activation of both CD4+ and CD8+T cells and induced a significantly higher T cell proliferation. The viral shedding was significantly lower in CS-HA1+CS-NP and inactivated vaccine groups compared with other challenged groups. The data demonstrate the potential of CS-HA1+CS-NP nanoparticles for eliciting specific influenza antibodies and conferring protection in chickens.

Modulation of Immune Responses Against HA1 Influenza Vaccine Candidate by B-lymphocyte Stimulator Cytokine in Mice.

Utilizing subunit vaccines is one of the strategies to address influenza infection. Recent innovations have focused on high doses of vaccine antigens and immune enhancers or adjuvant to induce more robust and long-lasting immune responses. Here, an effect of the B cell-activating factor receptor (BAFF-R) to increase the magnitude and durability of immune responses of the recombinant HA1 (rHA1) protein against the H1N1 influenza virus was studied. The HA1 protein and the effector domain of BAFF-R were expressed in the pET-28a (+) vector. Eight-week-old BALB/c mice were equally grouped into five groups (n=20). The 15 and 25 µg/µL of rHA1 were mixed with 2 µg/µL of rBAFF-R and injected three times for vaccinated groups. Three control groups were received normal saline and two concentrations of rHA1. The ability of rBAFF-R in eliciting HA-specific antibody response and stimulating T lymphocyte proliferation to induce the cell-mediated immunity was assayed. Induction of protection was evaluated following the challenge with PR8 strain. Analysis of immune responses showed that the co-administration of rBAFF-R with rHA1 boosted HI responses to the antigen in mice, whilst it was not able to promote the T cell proliferation responses against influenza. Compared to rHA1alone, the rBAFF-R/rHA1 generated efficient protection for the animals. There were no significant differences in eliciting the immune responses in mice immunized with the lower dose of rHA1 than that with the higher dose. The data indicate the rBAFF-R can enhance the primary and memory immune responses to protect against influenza infection.

Evaluation of cellular and humoral immune response in chickens immunized with flagellin-adjuvanted inactivated newcastle disease virus.

The aim of the present study was to evaluate the potential effect of flagellin as adjuvant in Newcastle disease virus (NDV) vaccine on the cellular and humoral immunity in chickens. Fifty-six specific pathogen-free chickens were assigned to seven groups of eight chickens and immunized twice with a two-week interval, intramuscularly. Group 1, received phosphate buffered saline as control (C), groups 2, 3, 4, 5, 6 and 7 were immunized with inactivated NDV [Ag], Ag + full FliC protein [AgF], Ag + truncated Flic protein [AgT], Ag + native Flic protein [AgN], commercial NDV vaccine [Vac] and Vac + N [VacN], respectively. After 45 days, spleen and bursa of Fabricius samples were collected and analyzed by flow cytometry and responses in control/vaccinated chickens were studied by immunophenotyping. Humoral response was also, evaluated by ELISA during the experiment. Results showed that immunized chickens with Ag + flagellin proteins had significantly higher frequency of circulating CD3+, CD4+ and CD8+ T cells in bursa of Fabricius in AgF, AgT and AgN, respectively, compared with other groups. Similar results were observed for spleen; however, the highest frequency of circulating CD3+, CD4+ and CD8+ T cells belonged to AgT and AgF, respectively. ELISA results showed that all flagellin-adjuvanted groups had higher antibody titers than other groups with the highest antibody response in VacN. It can be concluded that flagellin may induce both humoral and cellular immune responses against ND and is suggested for use as an efficient adjuvant.

Tween 80 improves the infectivity of BCL1 cell-adapted infectious bursal disease virus.

Non-ionic surfactants have the ability to alter the cell membrane's permeability for enhancing virus replication. The impact of non-ionic surfactant Tween 80 (TW80) on the infectivity of infectious bursal disease virus (IBDV) was studied in BCL1 cells. The toxicity of different concentrations of TW80 for BCL1 cells was determined for five-time passages. The confluent monolayer of BCL1 was infected by IBDV and subsequently passaged. The adaptation was confirmed by virus titration and RT-PCR assay. Replication kinetics of the cell-adapted IBDV was evaluated in pre-treatment and simultaneous treatment with TW80 at 0.01% concentration. The IBDV infectivity patterns were determined by virus titration, FRAP assay, and transmission electron microscopy. Sequence analysis, RNA secondary structure, and potential N-glycosylation site were conducted for IBDV VP2. Despite the similar cytopathic effects found in both TW80-treated cells and similar ROS levels, the IBDV titer was higher in TW80 pre-treated cells compared to the simultaneous treatment one. Such an increase in IBDV titer did not associate with changes in the VP2 sequence and RNA secondary structure. The possible antioxidant capacity of TW80 can attenuate the ROS damage and improve the cell viability, thereby improving IBDV infectivity.

Comparison of flagellin and an oil-emulsion adjuvant in inactivated Newcastle disease vaccine in stimulation of immunogenic parameters.

The present study was designed to investigate the potential application of native (N) and recombinant (truncated modified [tmFliC] and full-length [flFliC]) flagellin proteins along with inactivated Newcastle disease virus (NDV). Fifty six SPF chickens were immunized twice with PBS (control), inactivated NDV (Ag), inactivated NDV/flFliC (AgF), inactivated NDV/tmFliC (AgT), inactivated NDV/N (AgN), commercial vaccine containing Montanide (Vac) and Vac/N (VacN), with a two-week interval. Blood was collected weekly and spleens were harvested after chickens were sacrificed. Interleukin-6 (IL-6) and tumor necrotic factor-α (TNF-α) gene expression in peripheral blood mononuclear cells were analyzed by Real-Time PCR. Antibody response was assessed by haemagglutination inhibition (HI). Cellular activity was quantified by MTT assay. Results showed that the most IL-6 and TNF-α gene expression was observed in AgF group (P < 0.01). The lowest gene expression among vaccinated groups was observed in Ag group for IL-6 and Ag and Vac group for TNF-α. The highest HI titer was observed in Vac, VacN, AgF and AgT groups. The AgF group showed the highest cellular activity (P < 0.01). In conclusion, flagellin-adjuvanted groups showed a pro-inflammatory effect and acted similarly to or better than the Vac group. Hence, flagellin can be proposed as a potential adjuvant for ND vaccine.

Isolation, identification and chemical inactivation of infectious laryngotracheitis virus for use as a vaccine candidate.

Infectious laryngotracheitis (ILT) is a poultry respiratory disease associated with considerable mortality in chicken and decreasing egg production. Vaccination along with biosecurity measures are considered as the main strategy for ILT control. This study was aimed to evaluate the potency of an inactive ILT vaccine candidate derived from a local ILTV isolate. The isolated virus was characterized and treated with various chemicals and their concentrations. The virus infectivity was entirely abolished by treatment of 3 mM binary ethylene imine following 16 h incubation. The immune response of inactivated ILTV suspension with adjuvans was evaluated in both SPF chickens (experiment-I) and Hyline pullets (experiment-II). Efficacy of the inactivated and live ILT vaccines combination was compared. The results of experiment-I showed that the inactivated antigen induced specific antibody titers against ILTV. In experiment-II, despite the increase in serum antibody level administration of the inactivated antigen alone did not offer sufficient protection. The full protection was found in chickens that received the combination regimen. We conclude that simultaneous administration of the inactivated and live ILT vaccines was efficient for induction of immunity against ILTV. Keywords: infectious laryngotracheitis virus; vaccine; inactivation; immune response.

The Immunogenicity of a Novel Chimeric Hemagglutinin-Neuraminidase-Fusion Antigen from Newcastle Disease Virus by Oral Delivery of Transgenic Canola Seeds to Chickens.

Newcastle disease (ND) is considered as one of the most devastating infectious diseases targeting domestic birds and has considerable threat to the commercial poultry production. Two surface glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F), act as antigens in the virus structure and also play important roles in infecting host cells. In the current study, the expression of the chimeric HN-F protein in canola seeds and its immunogenicity in chickens were investigated. The HN-F gene was cloned downstream of the fatty acid elongase 1 (FAE1) promoter in the binary expression vector, pBI1400-HN-F, and introduced into rapeseed (Brassica napus L.) using Agrobacterium-mediated transformation. The amount of the HN-F glycoprotein was estimated up to 0.18% and 0.11% of the total soluble protein (TSP) in transgenic seeds and leaves of canola, respectively. Confirmatory analyses of 36 transgenic lines revealed that the HN-F gene was integrated into the genome. Subsequently, HN-F protein could be expressed and accumulated in the seed tissue. Specific pathogen-free (SPF) chickens immunized orally with recombinant HN-F showed a significant rise in specific and hemagglutination inhibition (HI) antibodies 35 to 42 days post the first administration. The results implied the potential of transgenic canola seed-based expression for oral delivery of NDV immunogenic glycoproteins.

TIR-TLR7 as a Molecular Adjuvant: Simultaneous Enhancing Humoral and Cell-Mediated Immune Responses Against Inactivated Infectious Bursal Disease Virus.

Despite the robust induction of humoral immune responses, a limitation of many adjuvants is their weak stimulation of cellular immunity. The development of synthetic gene-encoding adjuvants for simultaneous induction of both humoral and cell-mediated immune responses is under study. In this study, we examined the impact of toll/interleukin-1 receptor (TIR) domain of toll-like receptor 7 (TLR7) as molecular adjuvants on potency of inactivated infectious bursal disease (IBD) vaccines. A total of 60 specific pathogen-free week-old chicks were randomized grouped to receive either TIR-TLR7-adjuvanted IBD-inactivated vaccine or inactivated IBD antigen along with an unvaccinated control. Serum antibody titers were measured to estimate the humoral immunity, as well as lymphocyte proliferation activity for cellular immune responses. The protection was estimated after challenge with a very virulent IBD virus (IBDV) strain at 4 weeks postvaccination. The results indicated that one dose of IBD/TIR-TLR7 vaccine induced specific antibody responses, whereas a lower response after administration of inactivated IBD antigen was observed. The stimulation of splenocytes results indicated that the TIR-TLR7 adjuvanted IBD vaccine is capable of modulating cell-mediated immune response in treated chickens. A full protection against IBDV infection was achieved by injection of one dose IBD/TIR-TLR7 vaccine in the challenge trial. This study demonstrated that codelivery of TIR-TLR7 with inactivated IBD antigen resulted in simultaneous enhancing immune responses against IBD.

Improvement Efficacy of Influenza Nanovaccine in Combination with Hemokinin-1 Molecular Adjuvant.

BACKGROUND: H9N2 avian influenza viruses have the potential to become the next human pandemic threat and next generation vaccine technologies are needed. Current studies introduce nanoparticles as a proper vaccine delivery vehicle for induction of protective immunity. In this study, the efficacy of chitosan nanoparticle-based H9N2 influenza vaccine with and without hemokinin-1 (HK-1) as a molecular adjuvant to induce protective immunity against the virus was examined. METHODS: The H9N2 antigen was prepared in MDCK cells and inactivated with formalin. The inactivated antigen alone and in combination with HK-1 was encapsulated into chitosan nanoparticles. Groups of BALB/c mice received chitosan nanoparticle-based H9N2 antigen alone or in combination with HK-1 in a prime/boost platform via eye drop method. To evaluate the efficacy of the adjuvanted-nanovaccine candidate, systemic antibody responses were compared among the groups of animals. RESULTS: Serological analysis indicated that mice receiving the HK-1/H9N2 nanoparticles formulation induced higher antibody titers that were sustained until the end of experiment. However, in the immunized mice, influenza specific antibody titers were comparable to that in the animals which were immunized either with inactivated antigen alone or the H9N2 nanoparticles without HK-1 adjuvant. CONCLUSION: The data demonstrate the synergy between HK-1 as an adjuvant and chitosan nanoparticles as a delivery antigen/adjuvant carrier in the improvement of influenza immune responses.

Immunogenicity of the Multi-Epitopic Recombinant Glycoproteins of Newcastle Disease Virus: Implications for the Serodiagnosis Applications.

BACKGROUND: Newcastle disease virus (NDV) is a dangerous viral disease, infecting a broad range of birds, and has a fatal effect on the poultry industries. The attachment and consequently fusion of the virus to the host cell membrane is directed by the two superficial glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) which is considered as the important targets for the poultry immune response. OBJECTIVES: The principal goal of this investigation was to realize the potential efficacy of the E. coli expression system for the production of the multi-epitopic HN, and F proteins with respect to the ability for the stimulation of the immune system and production of the cross-reactive antibodies in mice. MATERIALS AND METHODS: The recombinant HN and F (rHN, rF) have accumulated almost 40% of the total bacterial proteins. The presence of rHN and rF proteins recognized by the Western blotting with specific anti-HN, anti-F, anti-Newcastle B1, and anti-poly 6x His-tag antibodies. Furthermore, both rHN and rF have shown the specific reactivity against the Newcastle B1 antiserum as a standard strain. RESULTS: The ELISA analysis showed that the higher dilutions of the antibody against Newcastle B1 could react with the as least quantity as 100 ng of the purified rHN, and rF. Cross-reactivity analysis of the sera from the mice immunized with Newcastle B1 in two time points indicated that the raise of anti-Newcastle B1, anti-HN and anti-F antibodies peaked at 28 days post immunization (dpi). Moreover, temporal variation in IgG titration between both time points was significant at 5% probability level. CONCLUSION: The results provided valuable information about the cross-reactivity patterns and biological activity of the multi-epitopic proteins compared to the NDV standard strain which was determined by the Western blotting and ELISA.

Interfering With Lipid Raft Association: A Mechanism to Control Influenza Virus Infection By Sambucus Nigra.

Sambucus nigra (elder) are broadly used species to treat microbial infections. The potential antiviral activity and mechanism action of elder fruit (EF) in human epithelium cell (A549) cultures infected with H9N2 influenza virus were determined. The effect of various concentrations of EF on influenza virus replication was examined by using virus titration, quantitative real time RT-PCR, fusion and lipid raft assays following two treatment procedures: A) pre-treated H9N2 virus with each concentration of EF extract and transfection of A549 cell cultures, and B) each concentrations of EF was added to H9N2 virus infected-cell cultures following virus adsorption. In both treatments with lower doses of EF increased viral titer as well as synthesized viral nucleoprotein as indicating the herb had no inhibitory effects on virus replication. In (B) trial with higher doses, 40 and 80 µg/mL of EF, a significant decrease in virus titer and viral protein synthesis were shown in EF treated cells indicating the herb affect either entry of viruses or inhibition virus particle release. The results suggest that EF treatment of the influenza virus infected-human epithelial cells may involve in lipid raft association which function as platform for formation of viral membrane fusion and budding. Differencesin treatment time and dose of EF extract in infected cells with influenza virus have a marked effect on the efficacy of the herb.

Improvement influenza HA2 DNA vaccine cellular and humoral immune responses with Mx bio adjuvant.

Immunization with DNA vaccines as a novel alternative to conventional vaccination strategy requires adjuvant for improving vaccine efficacy. The conserved immunogenic HA2 subunit, which harbors neutralizing epitopes is a promising vaccine candidate against influenza viruses. In this study, for the first time we explore the idea of using host interferon inducible Mx protein to increase the immunogenicity of HA2 H9N2 influenza DNA vaccine. The potency and safety of the Mx adjuvanted-HA2 vaccine was evaluated in BALB/c mice by different prime-boost strategies. To assess the effect of the vaccination on the virus clearance rate, mice were challenged with homologous influenza virus. Administration of the adjuvanted vaccine and boosting with the same regimen could effectively enhance both humoral and cellular immune responses in treated mice. These data demonstrated that Mx as host defense peptide can be potentiated for improving influenza vaccine efficacy.

Design of a heterosubtypic epitope-based peptide vaccine fused with hemokinin-1 against influenza viruses.

Influenza viruses continue to emerge and re-emerge, posing new threats for public health. Control and treatment of influenza depends mainly on vaccination and chemoprophylaxis with approved antiviral drugs. Identification of specific epitopes derived from influenza viruses has significantly advanced the development of epitope-based vaccines. Here, we explore the idea of using HLA binding data to design an epitope-based vaccine that can elicit heterosubtypic T-cell responses against circulating H7N9, H5N1, and H9N2 subtypes. The hemokinin-1 (HK-1) peptide sequence was used to induce immune responses against the influenza viruses. Five conserved high score cytotoxic T lymphocyte (CTL) epitopes restricted to HLA-A*0201-binding peptides within the hemagglutinin (HA) protein of the viruses were chosen, and two HA CTL/HK-1 chimera protein models designed. Using in silico analysis, which involves interferon epitope scanning, protein structure prediction, antigenic epitope determination, and model quality evaluation, chimeric proteins were designed. The applicability of one of these proteins as a heterosubtypic epitopebased vaccine candidate was analyzed.

Necrotic Response to Low Pathogenic H9N2 Influenza Virus in Chicken Hepatoma Cells.

BACKGROUND: Limited knowledge about the molecular mechanism of avian influenza H9N2 virus pathogenicity in birds as well as human hosts has limited the development of effective control against the disease. To overcome this issue detailed understanding of the infectious characteristics of the virus in host cells should be obtained. OBJECTIVES: In this study we examined the replication kinetics of H9N2 virus in a chicken hepatoma cell line to obtain insight into the pathogenesis of H9N2 viruses. MATERIALS AND METHODS: The kinetic replication of H9N2 influenza virus in chicken hepatoma and fibroblastic cells was studied in the presence and absence of supplemental trypsin. High viral titers observed in liver cells in a short time correlated with the degree of cytopathic effects. To determine whether the ultimate outcome of infection results in programmed cell death, the infected cells were observed by the cell viability assay, DNA fragmentation, caspase cascade activation, and quantified lactate dehydrogenase release. RESULTS: The degree of viability was significantly reduced in infected hepatoma cells. Observations of caspase activation and cell DNA laddering in infected cells were not indicative of apoptosis. The infected hepatoma cells released lactate dehydrogenase, which is consistent with cell death by necrosis. CONCLUSIONS: Taken together, these data reveal that cellular protease of chicken liver cells allows the replication of high yields of H9N2 virus in the absence of trypsin and also cell death in the infected cells is due to necrosis.

Expression of Factor X in BHK-21 Cells Promotes Low Pathogenic Influenza Viruses Replication.

A cDNA clone for factor 10 (FX) isolated from chicken embryo inserted into the mammalian cell expression vector pCDNA3.1 was transfected into the baby hamster kidney (BHK-21) cell line. The generated BHK-21 cells with inducible expression of FX were used to investigate the efficacy of the serine transmembrane protease to proteolytic activation of influenza virus hemagglutinin (HA) with monobasic cleavage site. Data showed that the BHK-21/FX stably expressed FX after ten serial passages. The cells could proteolytically cleave the HA of low pathogenic avian influenza virus at multiplicity of infection 0.01. Growth kinetics of the virus on BHK-21/FX, BHK-21, and MDCK cells were evaluated by titrations of virus particles in each culture supernatant. Efficient multicycle viral replication was markedly detected in the cell at subsequent passages. Virus titration demonstrated that BHK-21/FX cell supported high-titer growth of the virus in which the viral titer is comparable to the virus grown in BHK-21 or MDCK cells with TPCK-trypsin. The results indicate potential application for the BHK-21/FX in influenza virus replication procedure and related studies.

In Silico Analysis of HA2/Mx Chimera Peptide for Developing an Adjuvanted Vaccine to Induce Immune Responses Against Influenza Viruses.

PURPOSE: The direct transmission of avian influenza viruses to human and increasing drug resisted strains posing new threats for public health. Therefore, development of efficient vaccines is needed to generate protective and persistent immunity to the viruses. METHODS: Three motifs of Mx protein sequence in human, mouse and poultry located in interferon induced (GTP ase) domain were candidate as biologic adjuvant for enhancing the immune responses against influenza virus. Chimera proteins composed with the conserved HA2 subunit of influenza virus and the Mx motifs named HA2/Mx were modeled and evaluated by in silico analysis includes bioinformatics algorithms in order to explore biological characteristics of these peptides. RESULTS: Amongst the predicted models, HA2/Mx1 peptide showed the better results following protein structures prediction, antigenic epitopes determination and model quality evaluation. Comparative homology modeling was performed with Swiss Model and the model was validated using ProSA. Epitope predictions revealed the construct could induce both B and T cell epitopes that expect a high immune response. CONCLUSION: Taken together, these data indicate that the HA2/Mx1 chimera peptide can be potentiated for developing an adjuvant-fused influenza vaccine capable of stimulating effective immune response.