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Background: Influenza is a contagious respiratory infectious disease out breaking in cold seasons of the year. The outbreak of the new influenza A (H1N1) virus in 2009 involved large populations of the world with considerable mortality. Hemagglutinin (HA) molecule, the main surface glycoprotein of the influenza virus, is one of the key factors for serological diagnostic kits and vaccine development. Thus establishment of HA gene bank of the circulating influenza viruses is essential in gaining quick access to large amounts of protein. Materials and Methods: The first step in providing such a bank is detection and isolation of HA full genome and its subunits by using specific primers and cloning them in proper vectors. For this purpose, using standard virus genome (A/New Caledonia/20/99(H1N1)) cultured on MDCK cell, HA coding gene was proliferated by RT-PCR using specific primers. Results: Isolation and cloning of the HA gene was verified by RT-PCR, enzyme digestion and determining nucleotide synonymy. Through the use of specific cloning primers, different HA gene constructs were propagated for expression of the gene in insect cells and E.coli bacteria. Conclusion: The results indicated the complete compatibility of the extracted HA gene with the influenza (A/New Caledonia/20/99(H1N1)) hemagglutinin. It makes it possible to use the gene as a source of cloning in a variety of eukaryotic and prokaryotic expression systems

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Background: Influenza is an acute respiratory disease caused by influenza virus. Influenza epidemics are reported every year and worldwide pandemics occur with varying frequencies. The majority of mortalities are due to underlying diseases and complications associated with influenza. In this study, we evaluated ten fatal cases caused by the established type A influenza (H1N1) infection in the 2009-2010 pandemic. Materials and Methods: This mortality survey was compiled by a review of the deceased patients’ files. The assessed variables were demographic data, underlying diseases, secondary infections, delayed commencement of therapy, and non-medication. Collected data were analyzed by measures of central tendency and dispersion using SPSS software. Results: In the ten deaths due to the established H1N1 virus, the median of age was 30 years and 90% of the cases had underlying diseases. Ninety percent of the deaths occurred during October and November and the rest took place in December and February. Conclusion: The main cause of death was the delayed commencement of antiviral treatment. This emphasizes the importance of timely treatment in high risk patients. In flu pandemics, physicians should swiftly start specific therapy in at-risk groups to reduce the mortality rates.

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Background: In recent years Influenza viruses have caused widely spread moderate to severe infection in all around the world and there is no Influenza vaccine which can protect people only with one dose injection till now. Therefore , producing a universal vaccine based on virus like particle (VLP) could be ideal. In this study one of the molecular structures was considered for VLP based Influenza vaccine. Materials and Methods: In this experimental study, the human influenza virus (A /New Caledonia 20/1999/ (H1N1)) was propagated in MDCK cell culture. Viral RNA was extracted using RNX-plus solution. Complementary DNA synthesis was carried out using uni-12 primer and random hexamer as specific and general primers, respectively. Neuraminidase open reading frame (1413-bp) was amplified by PCR and cloned into pBlue-script SK. Neuraminidase coding frame sub cloned into pFastBac11 plasmid through SalI/XhoI sites. After verification of cloned Neuraminidase by restriction analysis, it was subjected to automated sequencing bi-directionally. The recombinant pFastBac Neuraminidase vector was transformed to E.coli DH10Bac cells which harbor bacmid DNA and helper plasmid to create Neuraminidase recombinant bacmid. Results: Neuraminidase recombinant bacmid was created by homologous recombination between pFastBacNA and bacmid and was verified by PCR using Neuraminidase specific and M13 universal primers. Conclusion: Recombinant baculovirus expressing Neuraminidase gene can be also used with other individual recombinant baculoviruses expressing HA and M1 genes in production of influenza VLPs or proteins resulting from this structure could be purified in specific insects for vaccine research studies.

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Background: Genetic variability of influenza viruses causes new epidemics worldwide annually. Development of a new vaccine for prophylaxis of influenza virus has been amajor objective in recent years. The aim of this study was to construct a recombinant baculoviruscapable of expressing the two surficial antigenic glycoproteins, hemagglutininand neuraminidase, as well as matrix proteinsof swine influenza (H1N1) simultaneously and independently. Materials and Methods: In this experimental study, first, a triplet cassette providing simultaneous and independent expression of target proteins was designed and subjected to synthesis. It was then cloned into pFastBac1 donor plasmid. Competent E.ColiDH10Bac cells were transformed by donor clone and the recombinant bacmids were produced following homologous transposition. This construction was verified by PCR and then transfected into Sf9 insect cells to package new recombinant baculoviruses. Results: Restriction map of pFastBacI HNM1 donor plasmid confirmed the fidelity of the clone. The results of PCR done on the recombinant bacmidas template indicated that a proper homologous recombination has occurred between pFastBacI HNM1 donor plasmid and the bacmid in E.ColiDH10Bac host cells. Protein analysis of the infected Sf9 cells showed that all target proteins were efficiently expressed at the same time. Conclusion: The recombinant baculovirus constructed in this studypossesses proper characteristics to produce swine influenza virus-like particles in Sf9 cells.

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Background: Influenza type A virus is one of the most important viral agents in human respiratory diseases. The genetic variability of the influenza viruses leads to the incidence of new epidemics worldwide. Hence, there is a growing need for rapid and effective new methods capable of detection and differentiation of influenza virus circulating strains. This study was done to develop a method for rapid differentiation of the subtypes of influenza type A virus. Materials and Methods: In this experimental study, reverse-transcription and polymerase chain reaction (RT-PCR) were performed using a primer set based on M gene of H1N1, H3N2, H5N1, and H9N2 influenza subtypes. Then the amplified fragments were subjected to digestion using subtype specific restriction endonuclease enzymes. Results: The results of PCR reaction showed that the primer pair of the M gene was specific and capable of amplifying all influenza subtypes understudy. Also, different restriction fragment length polymorphism patterns (RFLP) were generated using enzyme digestion reaction on the amplified segment of M gene. Conclusion: RT-PCR and RFLP analysis of the M gene can be employed as a useful method for differentiating influenza virus subtypes

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Background: Today, the use of nano-materials is one of the most common methods of making modern medications these materials are very useful in increasing the accessibility of drugs to target. The aim of this study is to obtain immunogenic nano-vaccine against meningitis caused by Haemophilus influenza.

Materials and Methods: In this experimental study, the PRP (polyribosylribitol phosphate) antigen of Haemophilus influenza was conjugated to keyhole limpet hemocyanin (KLH), a powerful immunogen molecule, and a nanoparticle with high adsorption called poly lactic co-glycolic acid (PLGA). The two-part and three-part conjugated antigens were injected into male SW1 race mice. The animals were randomly divided into five groups, which received PRP, PRP+KLH, PRP-KLH, PRP-KLH-PLGA, and PRP-TT intramuscularly together with complete Freund’s adjuvant, respectively. Twenty eight days after injection, blood samples were obtained and increases in serum antibody titer were determined with ELISA technique. For evaluation of the amount of the produced antigen cell entrance into immune cells, immune cells uptake assay and flow cytometry technique were used.

Results: The results showed increases in serum IgG antibody titers of animals immunized with conjugate vaccines. The findings also suggest the higher phagocytosis of conjugated triplex-containing nanoparticle by host immune cells.

Conclusion: The findings of this study indicate that antigen-containing PLGA has considerably higher absorption and immunogenicity and can be more powerful vaccines against Haemophilus meningitis.

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Background: The outbreaks of new antigenic variants of influenza viruses in human populations have increased necessity the improvement of controlling programs. Influenza vaccines are formulated with adjuvant to enhance and direct the host immune responses. Currently, much effort is devoted to designing molecular adjutants. Hemokinin-1 (HK-1) activates T and B cells for proliferation, survival, differentiation into plasma cells, and antibody production. In this study, the effect of HK-1 as a molecular adjuvant for inducing humoral immune response against influenza virus was investigated.

Materials and Methods: The HK-1 coding sequence was cloned into pcDNA3.1 vector and used as adjuvant. Groups of mice were immunized with an inactivated influenza vaccine formulated with HK-1. The sera of vaccinated mice were collected prior to priming and boosting injections and at defined weeks, and analyzed with serological assays.

Results: The results showed that HK-1 was able to increase antibody titer against virus vaccine. The mice immunized with the adjuvanted vaccine produced higher antibody titers against influenza comparing to vaccine alone immunized group. Number of boosting had no effect on the enhancing of antibody titer.

Conclusion: These data revealed that HK-1 as a molecular adjuvant induces stronger humoral and memory responses against influenza immunization.

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Background: Direct transmission of avian influenza viruses with human receptor binding specificity to humans is a serious risk of newly emerging virus responsible for pandemy. The analysis of recent avian influenza hemagglutinin sequences and their glycans show their affinities to the human sialic acid receptors. The upregulation of proinflammatory cytokines and type I IFN genes, and host cell death responses contribute to the pathogenesis of influenza infection. Understanding the host cell-virus interactions and replication dynamic of the viruses in different cells is an essential step in surveillance and controlling programs against influenza.

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Background: Influenza A viruses are globally important respiratory pathogens which cause a high degree of morbidity and mortality during annual epidemics. M2 protein which expressed on the viral surface facilitates virus entry to the host cells. The extracellular domain of M2 protein (M2e) consists of N-terminal 24 residue which shows remarkable conservation among all subtypes of influenza A viruses. In this study, we evaluated the immunogenicity of three tandem repeats of M2e along with different adjuvants in BALB/C mice model.

Materials and Methods: Recombinant protein (3M2e) was expressed in Escherichia coli and purified. Six weeks old BALB/c mice were immunized interdermally with three doses of 3M2e alone or supplemented with Alum/CpG motif as adjuvant. Control group was injected with PBS. Two weeks after the last immunization, specific anti-M2 was measured using ELISA method and finally mice were challenged with one lethal dose (LD90) of PR8 virus.

Results: The results showed that 3M2e can induce specific antibody alone. However, 3M2e protein supplemented with Alum-CpG induced higher level of specific antibodies, so that, there was a significant difference with 3M2e group (p<0.05). Anti-M2 antibodies mostly consisted of IgG2a subclass which considered as activity index of TH1 Cells. Moreover, this group showed enhanced protection against wild-type virus (survival rate=60%).

Conclusion: Applying Alum-CpG as a complex adjuvant may play a crucial role in integrating innate and acquisitive immunity. We increased density of M2e in combination with complex adjuvant and showed that this vaccine induced power immune responses and semi-protected mice against lethal challenge.

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