Prokaryotic Expression of H1N1 Influenza A Virus Haemagglutinin Protein Globular Domain (HA1)

Background: Influenza viruses are a significant cause of morbidity and mortality. The influenza virus pandemics, 1918, 1977, and especially the most recent one, A/H1N1/2009, made evident the need for generating recombinant Influenza H1N1 antigens which are essential to develop both basic and applied research programs. Among influenza virus proteins, haemagglutinin (HA) is a major surface antigen of influenza virus, thus it is highly topical in influenza research and vaccine engineering programs. Alternatively, expression of fragments of the HA (HA1 and HA2) proteins in prokaryotic systems can potentially be the most efficacious strategy for manufacture of large quantities of influenza vaccine in a short period of time.Materials and Methods: The gene encoding the HA1 protein of the influenza A/Puerto Rico/8/34 was amplified by PCR, then cloned into pTZ57R/T cloning vector. The fidelity of the HA1 open reading frame was confirmed by bidirectional sequencing, then sub-cloned into pET28a prokaryotic expression plasmid, and proteins containing HA1 N-terminally fused to His-Tag were produced in Escherichia coli BL21 through IPTG inducing. The accuracy of the expression was confirmed by running time coursed fraction samples taken before and after the IPTG induction in SDS-PAGE, Western blot analysis were also used for confirmation of the recombinant protein.Results and Conclusion: The HA1 protein produced here could be considered and evaluated as a protective antigen, which its immunogenicity potential needs to be assessed in animal models along with proper control groups. Moreover, it could be subjected for polyclonal antibody preparation, which, in turn, may be used as an essential material in western blot analyses, as well as in other immunological applications, such as ELISA, immunocytochemistry, immunohistochemistry, and other immunological and serological studies

Cloning and prokaryotic expression of the globular head domain of hemagglutinin antigen (HA1) of influenza A (H3N2) virus in Escherichia coli and Bacillus subtilis

Background: The influenza virus hemagglutinin is the major surface protein of the influenza A virus which is composed of HA1 and HA2 subunits. HA1 has an important role in binding of virus to cells and designing neutralizing antibodies. Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) both are known as the most useful prokaryotic hosts to express recombinant proteins. The aim of this study was to clone and express recombinant HA1protein in E. coli and B. subtilis bacteria.Materials and Methods: HA1 gene was cloned into pET-28a vector and pHT43 shuttle vector and then, both transformed to E. coli. The recombinant plasmids were extracted and then transformed into the BL21 and WB600 as expressing hosts. After induction with isopropyl-β-d-thiogalactoside (IPTG), expressed recombinant protein was analyzed by SDS-PAGE. Finally, the expressed protein was confirmed by the Western blot.Results: HA1 gene was cloned into pET-28a vector and pHT43 shuttle vector and then, both transformed to E. coli.The recombinant plasmids were extracted and then transformed into the BL21 and WB600 as expressing hosts. After induction with isopropyl-β-d-thiogalactoside (IPTG), expressed recombinant protein was analyzed by SDS-PAGE. Finally, the expressed protein was confirmed by the Western blot.Conclusion: This study demonstrated a strategy for production and purification of recombinant protein in large scale to test as vaccine candidate against influenza and it’s potentially immunogenicity be assessed in animal models

Evaluation of fatty acid content and nutritional properties of selected native and imported hazelnut (Corylus avellana L.) varieties grown in Iran

Hazelnut (Corylus avellana L.) is one of the most important nuts rich in valuable nutrients. In this study chemical composition of Two Iranian native varieties namely ‘Pashmineh’ and ‘Garche’ and four imported varieties, ‘Ghafghaze’, ‘Zakatala’, ‘Ronde dupimont’ and ‘Fertile decotard’ were taken under investigation. The main fatty acid in hazelnut varieties were oleic (71.02%) and linoleic acid (14.45%). The hazelnut varieties showed oil content in a range from 53.36% to 63.5%; protein, 16.03-23.26%; energy, 653.4-707.65%; ash, 2.46-3.5%; carbohydrate, 13.16-20.14%; total phenolic content, 6.4-16.42 mg GAE /g; antioxidant capacity, 57.17-72.38%; oleic acid, 64.17 - 81.34%; Linoleic acid, 10 –21.07%; Linolenic acid, 0 – 2%; myristic acid, 0 – 0.5 %; stearic acid, 0 – 7.8%; eicosenoic acid, 0 – 1.69%; palmitic acid, 0.49 – 9.61%; palmitoleic acid, 0 – 1.6 % and behenic acid, 0 – 0.25%

Generation of truncated recombinant form of tumor necrosis factor receptor-1 to produce cancer vaccine

Purpose: To produce truncated recombinant form of tumor necrosis factor receptor 1 (TNFR1), cysteine-rich domain 2 (CRD2) and CRD3 regions of the receptor were generated using pET28a and E. coli/BL21.Methods: DNA coding sequence of CRD2 and CRD3 was cloned into pET28a vector and the corresponding protein was expressed under induction of isopropyl β-D-1-thiogalactopyranoside (IPTG) as 6×His tagged using E.coli BL21 (DE3) expression system. The protein was then purified by Ni-NTA affinity chromatography. The fragment insertion, expression of recombinant protein and the yield of expression were evaluated.Results: Protein expression was achieved by identifying a band with molecular weight of 1488.3 Da. The recombinant protein of CRD2 and CRD3 was most efficiently expressed in 0.5 mM IPTG and 3 h of incubation at 37 °C with high yield equal to 0.3 μg/μl. Also, the highest concentration of imidazole for purification of the recombinant protein was 250 mM.Conclusion: A truncated form of TNFR-1 has been successfully expressed in a bacterial expression system and purified on affinity column. The purified protein can be used in in vivo experiments to prepare specified agonist antibodies for TNFR-1.Keywords: Tumor necrosis factor receptor 1 (TNFR-1), Cysteine rich domain 2 (CRD2), Cysteine rich domain (CRD3), Apoptosis, Cancer vaccine, Antibodies, Recombinant protein, pET28

Autophagy induction regulates influenza virus replication in a time-dependent manner

Autophagy plays a key role in host defence responses against microbial infections by promoting degradation of pathogens and participating in acquired immunity. The interaction between autophagy and viruses is complex, and this pathway is hijacked by several viruses. Influenza virus (IV) interferes with autophagy through its replication and increases the accumulation of autophagosomes by blocking lysosome fusion. Thus, autophagy could be an effective area for antiviral research.Methodology. In this study, we evaluated the effect of autophagy on IV replication. Two cell lines were transfected with Beclin-1 expression plasmid before (prophylactic approach) and after (therapeutic approach) IV inoculation.Results/Key findings. Beclin-1 overexpression in the cells infected by virus induced autophagy to 26 %. The log10haemagglutinin titre and TCID50 (tissue culture infective dose giving 50 % infection) of replicating virus were measured at 24 and 48 h post-infection. In the prophylactic approach, the virus titre was enhanced significantly at 24 h post-infection (P≤0.01), but it was not significantly different from the control at 48 h post-infection. In contrast, the therapeutic approach of autophagy induction inhibited the virus replication at 24 and 48 h post-infection. Additionally, we showed that inhibition of autophagy using 3-methyladenine reduced viral replication. Conclusion. This study revealed that the virus (H1N1) titre was controlled in a time-dependent manner following autophagy induction in host cells. Manipulation of autophagy during the IV life cycle can be targeted both for antiviral aims and for increasing viral yield for virus production

Experimental validation and computational modeling of anti-influenza effects of quercetin-3-O-α-L-rhamnopyranoside from indigenous south African medicinal plant Rapanea melanophloeos

BACKGROUND : Influenza A virus (IAV) is still a major health threat. The clinical manifestations of this infection are related to immune dysregulation, which causes morbidity and mortality. The usage of traditional medication with immunomodulatory properties against influenza infection has been increased recently. Our previous study showed antiviral activity of quercetin-3-O-α-L-rhamnopyranoside (Q3R) isolated from Rapanea melanophloeos (RM) (L.) Mez (family Myrsinaceae) against H1N1 (A/PR/8/34) infection. This study aimed to confirm the wider range of immunomodulatory effect of Q3R on selective pro- and anti-inflammatory cytokines against IAV in vitro, to evaluate the effect of Q3R on apoptosis pathway in combination with H1N1, also to assess the physical interaction of Q3R with virus glycoproteins and RhoA protein using computational docking. METHODS : MDCK cells were exposed to Q3R and 100CCID50/100 μl of H1N1 in combined treatments (co-, pre- and post-penetration treatments). The treatments were tested for the cytokines evaluation at RNA and protein levels by qPCR and ELISA, respectively. In another set of treatment, apoptosis was examined by detecting RhoA GTPase protein and caspase-3 activity. Molecular docking was used as a tool for evaluation of the potential anti-influenza activity of Q3R. RESULTS : The expressions of cytokines in both genome and protein levels were significantly affected by Q3R treatment. It was shown that Q3R was much more effective against influenza when it was applied in co-penetration treatment. Q3R in combination with H1N1 increased caspase-3 activity while decreasing RhoA activation. The molecular docking results showed strong binding ability of Q3R with M2 transmembrane, Neuraminidase of 2009 pandemic H1N1, N1 and H1 of PR/8/1934 and Human RhoA proteins, with docking energy of − 10.81, − 10.47, − 9.52, − 9.24 and − 8.78 Kcal/mol, respectively. CONCLUSIONS : Quercetin-3-O-α-L-rhamnopyranoside from RM was significantly effective against influenza infection by immunomodulatory properties, affecting the apoptosis pathway and binding ability to viral receptors M2 transmembrane and Neuraminidase of 2009 pandemic H1N1 and human RhoA cellular protein. Further research will focus on detecting the detailed specific mechanism of Q3R in virus-host interactions.The National Research Foundation, South Africahttps://bmccomplementalternmed.biomedcentral.comam2020Paraclinical SciencesVeterinary Tropical Disease

Phylogenetic analysis and docking study of neuraminidase gene of influenza A/H1N1 viruses circulating in Iran from 2010 to 2019

Influenza A viruses (H1N1) have been consistently one of the most evolving viruses that escape from vaccine-induced immunity. Although there has been a rapid rise in human influenza virus knowledge since the 2009 pandemic, the molecular information about Iranian strains is still inadequate. The aim of this study was to analyze the neuraminidase (NA) segment of the Iranian isolates in terms of phylogenetic, antiviral resistance, and vaccine efficiency. Ninety-three NA sequences collected among 1758 nasopharyngeal swab samples during the 2015–2016 influenza season were sequenced and submitted to NCBI. Moreover, all the submitted Iranian influenza H1N1 NA sequences since 2010 till 2019 were included in the study.Software including MEGA-X, MODELLER, UCSF ChimeraX, Auto-Dock 4.2, and other online tools were used to analyze the phylogenetic relationship, vaccine efficiency, and binding affinity to sialic acid of the selected NA proteins. Moreover, the information about antiviral drug resistance mutations of NA were gathered and compared to the Iranian NA segments to check the presence of antiviral drug-resistant strains.The phylogenetic study showed that most Iranian NA sequences (between 2015 and 2016) were located in a single clade and following years were located in its subclade by 3 major mutations (G77R/K, V81A, and J188T). Resistant mutations in drug targets of NA including I117M, D151E, I223V, and S247N were ascertained in 10 isolates during the 2015–2016 flu seasons.Investigation of vaccination effect revealed that Iranian isolates in 2017 and 2018 were best matched to A/Brisbane/02/2018 (H1N1), and in 2019 to A/Guangdong-Maonan/SWL1536/2019 (H1N1).Furthermore, we performed an in-silico analysis of NA enzymatic activity of all Iranian sequences by assessment of enzyme stability, ligand affinity, and active site availability. Overall, the enzyme activity of four Iranian strains (AUG84119, AUG84157, AUG84095, and AUG84100) was assumed as the maximum enzyme activity. This study highlighted the evolutionary trend of influenza A virus/H1N1 circulating in Iran, which provides a preliminary viewpoint for a better comprehension of new emerging strains’ virulence and thus, more appropriate monitoring of influenza virus A/H1N1 during each outbreak season

A Comprehensive Comparison of Rapid RNA Extraction Methods for Detection of SARS-CoV-2 as the Infectious Agent of the Upper Respiratory Tract using Direct RT-LAMP Assay

Background: The current COVID-19 pandemic has highlighted the need for faster and more cost-effective diagnostic methods. The RNA extraction step in current diagnostic methods, such as real-time qPCR, increases the cost and time required for testing. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is a promising technique for developing diagnostic tests with desired sensitivity and specificity without the need for RNA extraction. Materials and Methods: An RT-LAMP assay was developed to detect SARS-CoV-2 with a sensitivity of 0.5 copies of positive control plasmid per microliter in 40 min. Several rapid RNA extraction protocols were evaluated using different reagents, including bovine serum albumin, Triton X-100, Tween 20, proteinase K, guanidine hydrochloride, guanidinium isothiocyanate (GITC), and thermal treatment. Finally, the sensitivity and specificity of the developed direct RT-LAMP were determined using 150 upper respiratory tract samples. Results: Method 10 was selected as the most efficient protocol for the RNA extraction step. The sensitivity and specificity of the developed direct RT-LAMP assay with clinical samples were estimated at 98.4% and 88.8%, respectively. Conclusion: These results suggest that the combination of GITC and Triton X-100 detergent is a highly efficient method for RNA extraction and direct RT-LAMP detection of SARS-CoV-2 in clinical samples, providing a valuable tool for the rapid and cost-effective diagnosis of COVID-19

In vitro and in vivo effects of Peganum harmala L. seeds extract against influenza A virus

Objective: Influenza A virus infections are still a major health problem and the choices available for the control and treatment of the disease are limited. This research evaluated in vitro and in vivo antiviral effects of Peganum harmala L. seeds (PHS) extract against influenza A virus. Materials and Methods: In this research, in vitro anti-influenza A virus activity of the extract was assessed in Madin-Darby canine kidney (MDCK) cells. In order to evaluate anti-influenza activity of PHS extract in vivo, BALB/c mice were infected with 5LD50 of mouse-adapted influenza virus (H1N1; PR8) and received 200 mg/kg/day of PHS extract or 20 mg/kg/day oseltamivir. Lungs of seven mice per group were removed on day 3 post-infection and lung virus titers were determined by qRT-PCR. Mice survival, body weights and general conditions were observed for up to 14 days post-infection. Results: The results demonstrated that, the ethanolic extract of PHS possesses high activity against influenza virus with IC50 value of 15.7 (CI95%:11.7-21) μg/ml in MDCK cells. Our results also showed that, oral administration of PHS extract (200 mg/kg/day) or oseltamivir (20 mg/kg/day) to infected mice, increased the survival rate, reduced body weight loss, and decreased lung virus titer. Conclusion: Based on our findings, P. harmala seeds extract can inhibit influenza A virus replication in vitro and in vivo. Therefore, isolation and characterization of the plant’s active compounds and investigation of the underlying mechanisms of its antiviral action are highly suggested