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In spite of the promising properties of small interfering RNAs (siRNAs) in the treatment of infectious diseases, safe and efficient siRNA delivery to target cells is still a challenge. In this research, an effective siRNA delivery approach (against HIV-1) has been reported using targeted modified superparamagnetic iron oxide nanoparticles (SPIONs). Trimethyl chitosan-coated SPION (TMC-SPION) containing siRNA was synthesized and chemically conjugated to a CD4-specific monoclonal antibody (as a targeting moiety). The prepared nanoparticles exhibited a high siRNA loading efficiency with a diameter of about 85 nm and a zeta potential of +28 mV. The results of the cell viability assay revealed the low cytotoxicity of the optimized nanoparticles. The cellular delivery of the targeted nanoparticles (into T cells) and the gene silencing efficiency of the nanoparticles (containing anti-nef siRNA) were dramatically improved compared to those of nontargeted nanoparticles. In conclusion, this study offers a promising targeted delivery platform to induce gene silencing in target cells. Our approach may find potential use in the design of effective vehicles for many therapeutic applications, particularly for HIV treatment.

The innate immune response in humans involves a wide variety of factors, including the tripartite motif-containing 5α (TRIM5α) and 22 (TRIM22) as a cluster of genes on chromosome 11 that have exhibited antiviral activity in several viral infections. We analyzed the correlation of the expression of TRIM5α and TRIM22 with the severity of Coronavirus Disease 2019 (COVID-19) in blood samples of 330 patients, divided into two groups of severe and mild disease, versus the healthy individuals who never had contact with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).The transcription level of TRIM5α and TRIM22 was determined by quantitative real-time polymerase chain reaction (qPCR). The laboratory values were collected from the patients' records.The expression of both genes was significantly lower in the severe group containing the hospitalized patients than in both the mild group and the control group. However, in the mild group, TRIM22 expression was significantly higher (p0.0001) than in the control group while TRIM5α expression was not significantly different between these two groups. We found a relationship between the cycle threshold (Ct) value of patients and the expression of the aforementioned genes.The results of our study indicated that lower Ct values or higher RNA viral load might be associated with the downregulation of TRIM5α and TRIM22 and the severity of COVID-19. Additional studies are needed to confirm the results of this study.

Infection with human immunodeficiency virus (HIV)‐1 causes immunological disorders and death worldwide which needs to be further assisted by novel anti‐retroviral drug delivery systems. Consequently, finding newer anti‐retroviral pharmaceuticals by using biocompatible, biodegradable nanomaterials comprising a nanoparticle as core and a therapeutic agent is of high global interest. In this experiment, a second generation of a negatively charged nano‐biopolymer linear globular G2 dendrimer was carefully conjugated and loaded with well‐known anti‐HIV drugs lamivudine and efavirenz, respectively. They were characterised by a variety of analytical methods such as Zetasizer, Fourier‐transform infrared spectroscopy, elemental analysis and liquid chromatography‐mass spectroscopy. Additionally, conjugated lamivudine and loaded efazirenz with globular PEGylated G2 dendrimer were tested on an HEK293 T cell infected by single‐cycle replicable HIV‐1 virion and evaluated using XTT test and HIV‐1 P24 protein load. The results showed that lamivudine‐conjugated G2 significantly decreased retroviral activity without any cell toxicity. This effect was more or less observed by efavirenz‐loaded G2. These nano‐constructs are strongly suggested for further in vivo anti‐HIV assays.

Introduction: Hepatitis E virus (HEV) causes emerging diseases in poor regions of the world. The ORF2 is the only protein encoded by the virus to make the viral capsid. The aggregation of proteins into inclusion bodies (IBs) while expressing ORF2 is a major challenge in bioengineering. Methods: The ORF2 conserved sequence was expressed in Escherichia coli BL21 and assessed by a modified SDS-PAGE containing a weak denaturing environment to solubilize the recombinant ORF2 protein and Western blotting. The protein of interest was evaluated by secondary structure prediction using SOPMA, homology modeling by I-TASSER and Circular Dichroism analyses. The function of the recombinant protein was investigated by an in-house ELISA using serum specimens of HEV infected patients. Results: The solubilized form of ORF2 protein was successfully expressed in E. coli BL21 and was confirmed by SDS-PAGE and Western blotting. Secondary structure prediction, homology modeling and CD analysis of the protein of interest demonstrated that the native structure of ORF2 was almost intact. The specific anti-HEV antibody was detected using this recombinant protein and an in-house ELISA test. Conclusion: We achieved new combinations of chemical agents, consisted of low concentrations of urea and detergents to overcome the aggregation of ORF2 protein in IBs inside E. coli BL21.

Background The recent pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has elevated several clinical and scientific questions. These include how host genetic factors influence the pathogenesis and disease susceptibility. Therefore, the aim of this study was to evaluate the impact of interferon lambda 3 and 4 (IFNL3/4) gene polymorphisms and clinical parameters on the resistance and susceptibility to coronavirus disease 2019 (COVID-19) infection. Methods A total of 750 SARS-CoV-2 positive patients (375 survivors and 375 nonsurvivors) were included in this study. All single-nucleotide polymorphisms (SNPs) on IFNL3 (rs12979860, rs8099917, and rs12980275) and IFNL4 rs368234815 were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) method. Results In this study, a higher viral load (low PCR Ct value) was shown in nonsurvivor patients. In survivor patients, the frequency of the favorable genotypes of IFNL3/4 SNPs (rs12979860 CC, rs12980275 AA, rs8099917 TT, and rs368234815 TT/TT) was significantly higher than in nonsurvivor patients. Multivariate logistic regression analysis has shown that a higher low-density lipoprotein (LDL), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and PCR Ct value, and lower 25-hydroxyvitamin D, and also IFNL3 rs12979860 TT, IFNL3 rs8099917 GG, IFNL3 rs12980275 GG, and IFNL4 rs368234815 ∆G/∆G genotypes were associated with the severity of COVID-19 infection. Conclusions The results of this study proved that the severity of COVID-19 infection was associated with clinical parameters and unfavorable genotypes of IFNL3/IFNL4 SNPs. Further studies in different parts of the world are needed to show the relationship between severity of COVID-19 infection and host genetic factors.

Introduction: To date, while many studies have investigated antiviral agents or vaccines against HIV, success has been limited. In this field, mutagenesis of the viral genome often contributes to viral escape from the antiretroviral therapies and the emergence of HIV-1 strains resistant to the drugs. Therefore, developing alternative methods, including more effective vaccines, antiviral therapies (such as RNAi therapy) and delivery systems, seems to be necessary to compensate for these issues. The aim of this study was to establish an efficient system for siRNA delivery as a safe anti-HIV therapeutic approach. Methods: In this research, the use of chitosan-coated SPION was investigated as a method for RNA delivery. So, after generating a stable cell line of HEK293 (expressing HIV-1 tat), we designed a potent siRNA against HIV-1 tat, and then the effectiveness of the modified SPION in siRNA delivery to HEK293 cells was evaluated. Results: The results of this study showed that the optimal concentration (50 µg/mL) of the modified SPION containing anti-tat siRNA (with a range size of 50-70 nm and average zeta potential of +25 mV) were significantly internalized into the cells and decreased the expression of HIV-1 tat over than 80%. Moreover, the nanoparticles showed no considerable toxicity on the cells. Conclusion: It would be concluded that SPION could be optimized as a probable RNA/vaccine delivery system into target cells. Therefore, this study offers a therapeutic strategy against HIV or other infectious diseases.

Chitosan has emerged as a promising polysaccharide for gene/siRNA delivery. However, additional works will be required to modify chitosan nanoparticles. In the present study, chitosan nanoparticles were well modified to introduce anti-HIV siRNA into two mammalian cell lines, macrophage RAW 264.7 and HEK293. We first generated two stable cell lines expressing HIV-1 Tat, and then designed and generated an efficient anti-tat siRNA. The nanoparticles were prepared by using different concentrations of chitosan, polyethylenimine (PEI) and carboxymethyl dextran (CMD) in various formulations and then their physicochemical and biological properties were investigated. The results demonstrated that the combination of chitosan with both CMD and PEI significantly improved both cell viability and siRNA delivery. The modified chitosan nanoparticles (ChNPs) at the N:P ratio of 50 were approximately uniform spheres with sizes ranging from 100 to 150 nm and a positive zeta potential of about +22 mV. In both cell types, the nanoparticles noticeably increased siRNA delivery efficiency with no significant cytotoxicity or apoptosis-inducing effects compared to the control cells. In addition, the nanoparticles significantly reduced the RNA and protein expression of HIV-1 tat in both stable cells. These data show that the nanoparticle formulation could potentially be used in gene therapy, especially against HIV infection.

BackgroundHepatitis C virus (HCV), non-structural 5A (NS5A), and non-structural 5B (NS5B) resistance-associated substitutions (RASs) are the main causes of failure to direct-acting antiviral agents (DAAs). NS5A and NS5B RASs can occur in patients with HCV infection naturally and before exposure to DAAs.ObjectivesThis study aimed to evaluate naturally-occurring NS5A and NS5B RASs in Iranian patients with HCV genotype 1a (HCV-1a) and -3a infections.MethodsIn this cross-sectional study, viral RNA was extracted from serum specimens. NS5A and NS5B regions were amplified using RT-PCR followed by DNA sequencing. The results of nucleotide sequences were aligned against reference sequences of HCV-1a and -3a and the amino acid substitutions were analyzed using geno2pheno [hcv] web application.ResultsAmong 135 patients with hepatitis C, NS5A amino acid substitutions/RASs were identified in 26.4% and 15.9% of patients with HCV-1a and -3a infections, respectively. The identified amino acid substitutions/RASs in the NS5A region of patients with HCV-1a infection were M28T/V/I 11.1%, Q30R/H 4.2%, L31M 1.4%, and H58Y/P/C/D/Q/S/T 16.7%. Y93H substitution was not found in HCV-1a sequences. In patients with HCV-3a infection, NS5A amino acid substitutions/RASs were A30T/K 9.5%, L31F 1.6%, P58S/T/C 3.2%, Y93H 3.2%, and Y93N 3.2%. No resistance substitutions were identified in NS5B sequences from patients with HCV-1a and -3a infections.ConclusionIn this study, baseline amino acid substitutions/RASs were only identified in the NS5A region in Iranian patients with HCV-1a and -3a infections, and the prevalence of these amino acid substitutions/RASs were in accordance with similar studies. There were no RASs in the HCV-1a and -3a NS5B region.

Human diseases like viral organisms for example, hepatitis, HIV and etc., attack the health and caused large mortality in populations by many years. So finding novel delivery vehicles based antiviral drugs employing nano-materials is of high universal interest. In current approach a very biocompatible biodegradable nano-biopolymer anionic linear globular dendrimer second generation G2 was elaborately conjugated to a well-known anti-HIV drug Azidovudine and thereafter was characterized by different analytical techniques like AFM, Zeta sizer, 1HNMR, FTIR and LC-Mass spectroscopy. Then, Anionic Linear Globular DendrimerG2-Zidovudine Nano-Conjugate was assessed on human normal cells (toxicity assay by XTT test) and also HIV cell model and the results showed that Anionic Linear Globular DendrimerG2-Zidovudine Nano-Conjugate Significantly Decreased Retroviral Activity without any human cell toxicity respectively. Based on current experimental data such nano-compositions is proposed for further in vivo anti-HIV assays as well.

Introduction: Hepatitis E virus (HEV) is a fecal-oral transmitting virus which causes a chronic liver disease. ORF2 is an immunogen capsid protein of HEV that has been proposed to be used for Hepatitis E vaccine design. It is a 660-amino acid protein which includes an immunogenic region (residues 112-607). This protein has been expressed in complete and truncated forms, using different expression vectors such as pRSET-C, pMAL, pSG and baculovirus expression systems. Escherichia coli BL21 which is used as a host for protein expression was utilized as a host for pET26b vector in this study. We evaluated the expression of ORF2 as Hepatitis E vaccine candidate in presence of several IPTG concentrations. Methods: First, orf2 gene was sub-cloned into a pET26b vector which adds a C-terminal His-tag to the coding sequence. The procedure was confirmed by gel electrophoresis and double digestion. Subsequently, the recombinant pet26b-ORF2 was transformed into E. coli BL21 cells for protein expression and the resulted recombinant protein was analyzed by Bradford assay, SDS-PAGE and Western blotting. Results: SDS-PAGE and Western blotting confirmed the proper protein expression while there was no significant difference among the expressions of protein in presence of different IPTG concentrations. Conclusion: The expression of HEV ORF2 protein was successfully performed in E. coli BL21 and it showed that ORF2 can be expressed in presence of different concentration of IPTG with no significant difference in protein expression. The produced recombinant protein could be used in further vaccine-related studies and also its expression can be studied at several different temperatures.

Background Inhibition of viral genes through siRNA seems to be promising for treatment of complicated viral infections like human immunodeficiency virus (HIV-1). HIV-1 Tat (Trans Activator of Transcription) and Nef (Negative regulatory Factor) proteins are very interesting targets for designing siRNAs. Methods The effectiveness of suppressing Tat and Nef was investigated using three specific siTATs and three siNEFs. They were used to transfect the developed stable and infected Human Embryonic Kidney cells (HEK293) as an ex-vivo model. Both stable and virus infected HEK293 cells were transfected with each siTAT and siNEF. The inhibitory effect was evaluated using qRT-PCR, western blot analysis, and HIV P24 ELISA. Results siTAT-100, siTAT-162, and siNEF-136 and at a concentration of 100 nM/mL showed the most inhibitory effect on their target genes. Conclusions Utilization of more developed molecular inhibition strategies such as RNAi or even a combination of different molecular approaches could be promising to overcome emerging HIV escape mutants.

Background Rabies is a fatal zoonotic infectious disease, which can be prevented by prompt post-exposure prophylaxis that could be expensive in countries with a large population. The Essen protocol with injection of 5 single doses of human rabies vaccine on separate days is a well-known rabies prophylaxis schedule. Decreasing the number of vaccine doses and the number of clinical visits due to an effective alternative schedule is strongly needed. The 2-1-1 regimen, known as Zagreb, is one of the best candidates to succeed Essen. Methods To evaluate the effectiveness of Zagreb regimen in the Iranian population by using the Purified Vero cell Rabies Vaccine (PVRV), anti-rabies antibody titer was measured in volunteers with second and third exposure through Rapid Fluorescent Focus Inhibition Test (RFFIT) and Enzyme Linked Immunosorbent Assay (ELISA) test and compared with patients, who were treated according to the Essen protocol. Results In all participants, anti-rabies antibody titer reached the protective level with no suppressive effect of rabies immunoglobulin in patients with third exposure in Zagreb regimen. Conclusions Zagreb regimen could be considered a suitable alternative for the Essen protocol.

Objectives In this study, the optimal dose of Lipofectamine 3000 and Turbofect to transfect adherent cell lines such as CHO-K1 and HEK293 cells in comparison with non-adherent H9T-cells with pEGFP-N1 and pCDH was identified. Background Lipofectamine 3000 is a new transfection reagent which is claimed to be more efficient than other transfection reagents like Turbofect. Transfection efficiency could be affected by the nature of target cell line and vector. Methods Transfection efficiency and cytotoxicity of each reagent was identified by using flow cytometry and XTT assay, respectively. Results Lipofectamine 3000 was more efficient in transfecting pCDH, while Turbofect was more efficient in separate transfection of CHO-K1 and HEK293 with pEGFP-N1. Lipofectamine 3000 could be cytotoxic in transfecting H9T-cells with pCDH. Also, H9T-cells were not sufficiently transfected with each plasmid vector by using each Lipofectamine 3000 and Turbofect. Turbofect had less cytotoxicity effect on all three cell lines than Lipofectamine 3000.Transfection of suspended cells like H9T-cells by using Lipofectamine 3000 and Turbofect would not result in sufficient transfection. Conclusion Lipofectamine 3000 is the best choice for transfection of CHO-K1 and HEK293 with pCDH while Turbofect is preferably used in transfecting these cell lines with pEGFP-N1 (Tab. 1, Fig. 2, Ref. 26).

Gene therapy, including small interfering RNA (siRNA) technology, is one of the leading strategies that help to improve the outcomes of the current therapeutic systems against HIV-1 infection. The successful therapeutic application of siRNAs requires their safe and efficient delivery to specific cells. Here, we introduce a superparamagnetic iron oxide nanoparticle (SPION) for delivering siRNA against HIV-1 nef (anti-nef siRNA) into two cell lines, HEK293 and macrophage RAW 264.7. SPIONs were coated with trimethyl chitosan (TMC), and thereafter, different concentrations of SPION-TMC were coated with different ratios of a carboxymethyl dextran (CMD) to modify the physicochemical properties and improve the biological properties of the nanocarriers. The nanoparticles exhibited a spherical shape with an average size of 112 nm. The obtained results showed that the designed delivery route enhanced the uptake of siRNA into both HEK293 and RAW 264.7 cells compared with control groups. Moreover, CMD-TMC-SPIONs containing anti-nef siRNA significantly reduced the expression of HIV-1 nef in HEK293 stable cells. The modified siRNA-loaded SPIONs also displayed no toxicity or apoptosis-inducing effects on the cells. The CMD-TMC-SPIONs are suggested as potential nanocarriers for siRNA delivery in gene therapy of HIV-1 infection.

Background HCV E2 glycoprotein is one of the most attractive proteins for designing an effective vaccine. Deletion of hydrophobic carboxyl-terminal region of this protein is necessary for its secretion, especially when it is expressed in E-coli. In this study we expressed this protein in truncated form and evaluated its application in developing an ELISA test and induction of humoral response in immunized mice. Objectives The purpose of this study was expression of HCV truncated E2 protein from JFH1 strain in E-coli BL21(DE3) and evaluation of its antigenicity. Methods Truncated E2 region from HCV genotype 2a (JFH1) was amplified by PCR and cloned into a pET28a (+) vector and was used to transform the E-coli DH5α strain. The recombinant E2 protein was evaluated both in an ELISA test and induction of humoral immunity in mice. Results Truncated E2 protein was expressed in BL21(DE3). Its specific antibody was detected in serum samples from HCV infected patients. Also, it could elicit a significant humoral immunity in mice. Conclusion Truncated form of E2 protein which has been expressed in E-coli could be used as an effective antigen both in diagnostic tests such as ELISA and also, as a protein-based vaccine candidate.

One of the significant problems in vaccination projects is the lack of an effective vaccine against hepatitis C virus (HCV). The goal of the current study is to evaluate and compare two DNA constructs encoding HCV core and coreE1E2 genes alone or complexed with MPG peptide as a delivery system for stimulation of antibody responses and IFN-γ secretion in Balb/c mice model. Indeed, MPG cell penetrating peptide was used to improve DNA immunization in mice. Our results demonstrated that MPG forms stable non-covalent nanoparticles with pcDNA-core and pcDNA-coreE1E2 at an N/P ratio of 10:1. The in vitro transfection efficiency of core or coreE1E2 DNA using MPG and TurboFect delivery systems was confirmed by western blot analysis. The results indicated the expression of the full-length core (∼21 kDa), and coreE1E2 (∼83 kDa) proteins using an anti-His monoclonal antibody. In addition, the expression of HCV core and coreE1E2 proteins was performed in bacteria and the purified recombinant proteins were injected to mice with Montanide 720 adjuvant. Our data showed that the immunized mice with HCV core and coreE1E2 proteins generated the mixture of sera IgG1 and IgG2a isotypes considerably higher than other groups. Furthermore, DNA constructs encoding core and coreE1E2 complexed with MPG could significantly induce IFN-γ secretion in lower concentrations than the naked core and coreE1E2 DNAs. Taken together, the DNA formulations as well as protein regimens used in this study triggered high-level IFN-γ production in mice, an important feature for the development of Th1 immune responses.

Introduction: The development of an effective vaccine against Hepatitis C virus (HCV) is still a target of intensive vaccine research. The HCV envelope proteins E1 and E2 which can induce broadly neutralizing antibodies are the major candidate for this purpose. Different types of expression systems have been used to express these glycoproteins. In this study, an expression system using Pichia pastoris was used to express E1 and E2 in full length. Methods: E1 and E2 regions containing the restriction sites from HCV 1b were separately amplified and cloned into a pPICZAa vector. The km71h strain of P. pastoris was transfected with the confirmed vectors separately using electroporation. The recombinant E1 and E2 proteins were evaluated for their antigenicity in an ELISA test and the induction of humoral immunity in mice. Results: The expression of full length HCV glycoproteins E1 and E2 in P. pastoris strain km71h was successfully achieved and their specific antibody was detected in serum samples from HCV infected patients. Furthermore, the recombinant glycoproteins could elicit a significant humoral immunity in mice as a vaccine candidate. Conclusion: P. pastoris is one of the best eukaryotic expression systems for the production of HCV glycoproteins in full length and the expressed proteins could be used in diagnostic tests such as ELISA. The induction of humoral immune responses in mice should lead to further studies on these glycoproteins for designing an effective vaccine.

Background Hepatitis C virus infection is one of global health concern. No vaccine is available so far and nonstructural protein 3 (NS3) is one of the main target antigens for developing studies on therapeutic vaccine and diagnostic application. In the current study, we expressed a truncated recombinant HCV-NS3 protein under native condition and evaluated its potential applications in immunization and diagnosis. Methods The recombinant pET-NS3 containing a truncated form of HCV NS3 region was constructed, confirmed by sequencing reactions, and expressed into E.coli BL21-DE3 strain. The expressed protein was purified by affinity chromatography under native condition. Characterization of diagnostic value and immunogenicity of this recombinant protein were analyzed by using an indirect ELISA method. Results Our data showed that a truncated NS3 which contains the immunodominant region of this protein was successfully expressed and purified with a high yield of 3 mg/L. Our data showed that the immunogenicity of this truncated protein can induce a specific humoral response, as well as the usage for screening of HCV positive blood samples. Conclusions Altogether, the present study provides a simple and efficient system for protein expression and purification of an immunodominant region of NS3-HCV in native conformation and its potential application for diagnosis and vaccine development in future.

Human parechoviruses (HPeV) are classified into 14 genotypes. HPeV1 and HPeV2 are the most prevalent genotypes in young children, which have been associated with mild to severe diseases. This study was conducted to investigate the involvement of HPeVs in aseptic meningitis and sepsis-like illness in Iran. Viral RNA was extracted from 148 cerebrospinal fluid samples from children

HIV-1 Vpr is an accessory protein that induces proteasomal degradation of multiple proteins. We recently showed that Vpr targets class I HDACs on chromatin for proteasomal degradation. Here we show that Vpr induces degradation of HDAC1 and HDAC3 in HIV-1 latently infected J-Lat cells. Degradation of HDAC1 and HDAC3 was also observed on the HIV-1 LTR and as a result, markers of active transcription were recruited to the viral promoter and induced viral activation. Knockdown of HDAC1 and HDAC3 activated the latent HIV-1 provirus and complementation with HDAC3 inhibited Vpr-induced HIV-1 reactivation. Viral reactivation and degradation of HDAC1 and HDAC3 was conserved among Vpr proteins of HV-1 group M. Serum Vpr isolated from patients or the release of virion-incorporated Vpr from viral lysates also activated HIV-1 in latently infected cell lines and PBMCs from HIV-1 infected patients. Our results indicate that Vpr counteracts HIV-1 latency by inducing proteasomal degradation of HDAC1 and 3 leading to reactivation of the viral promoter.

BACKGROUND Hepatitis C virus (HCV) genome contains an overlapping reading frame which results in alternative core protein (ARFP). Baculovirus expression system was used as a powerful eukaryotic vector system to express core+1/F protein for the first time. This recombinant core+1/F protein was used to assess the anti-core+1 antibody in anti-HCV drug resistant and sustained virologic response (SVR) patients. METHODS The core+1 coding sequence from HCV genotype 1 was designed and synthesized in pUC57 vector. It was subcloned into baculovirus donor plasmid pFastBacTM HTA and transposed into baculovirus shuttle vector (bacmid) to transfect Sf9 cells. Recombinant core+1 protein was purified using Ni-NTA agarose under native condition and verified using SDS-PAGE electrophoresis and Western blotting. An enzyme-linked immunosorbent assay (ELISA) was developed using this purified protein to assess anti-core+1 antibody in 28 anti-HCV drug resistant patients and in 34 patients with sustained virologic response (SVR) in comparison with 31 healthy volunteers used as the negative control. RESULTS Expression of HCV core+1 protein in Sf9 cells was confirmed by using SDS-PAGE and Western blotting. Antibody titer against core+1 protein in anti-HCV drug resistant patients was significantly higher than that in both the healthy volunteers and SVR patients (p < 0.0001). CONCLUSIONS HCV core+1 protein was expressed successfully in a baculovirus expression system in high yield in order to develop an ELISA to assess the anti-core+1 antibody. Further studies are needed to reveal the potential application of core+1 protein in anti-HCV treatment prognosis.

Background: Since the importance of poliovirus has diminished, as a result of its elimination in the majority of countries, non-polioviruses are emerging as causative agents of severe central nervous system (CNS) involvement. Outbreaks of enterovirus 71 (EV71)-associated CNS infections have recently been reported in Asia, Australia, and Europe. Objectives: This is the first study on genotyping of EV71 in children with meningoencephalitis to be carried out in Iran, and it was conducted in order to obtain an improved understanding of the disease burden of this virus, particularly with regard to CNS involvement. Patients and Methods: Viral RNA was extracted from 170 cerebrospinal fluid samples obtained from children aged under 8 years with a primary diagnosis of aseptic meningitis. Specific EV71 PCR was conducted to identify the genotype of the detected EV71 viruses. Results: Human enteroviruses (HEVs) were detected in 89 patients (52.3%). EV71 infection was detected in 19 (21.3%) of the 89 EV71-positive patients, and the C genotype was identified in 15 isolates. Conclusions: The C genotype should be considered as the prevalent EV71 circulating genotype in Iran, particularly in cases of aseptic meningitis.

HIV is commonly caused to a very complicated disease which has not any recognized vaccine, so designing and development of novel antiretroviral agents with specific application of nanomedicine is a globally interested research subject worldwide. In the current study, a novel structure of silver complexes with anionic linear globular dendrimer was synthesized, characterized and then assessed against HIV replication pathway in vitro as well. The results showed a very good yield of synthesis (up to 70%) for the nano-complex as well as a very potent significant (P < 0.05) antiretroviral activity with non-severe toxic effects in comparison with the Nevirapine as standard drug in positive control group. According to the present data, silver anionic linear globular dendrimers complex may have a promising future to inhibit replication of HIV viruse in clinical practice.

Objective Post exposure prophylaxis using one of the WHO-approved vaccines is the method of choice for preventing rabies. Abnormal immune function in patients with some specific medical conditions, such as pregnancy, chronic hepatitis B virus infection, different types of cancers like lymphoma, diabetes I and II, corticosteroid consumption by patients with rheumatoid arthritis and lupus erythematosus, could impair the immunologic response to various vaccines. The immune response to rabies vaccination has never been examined in patients with any of these described medical conditions. This study purposed to evaluate the neutralyzing antibody response after vaccination with purified Vero cell rabies vaccine (PVRV) according to the WHO-recommended Post–Exposure Prophylaxis (PEP) "ESSEN" regimen. Methods Thirty healthy volunteers and 50 volunteers with different medical conditions who were exposed to a suspected rabid animal in the 2nd or 3rd category of exposure received 5 doses of PVRV under the ESSEN protocol. Three blood samples were collected on days 0 (before the first dose), 14, and 35. The anti-rabies antibody titer was measured using the Rapid Fluorescent Foci Inhibition Test (RFFIT) and an ELISA Bio-Rad, Platelia, Rabies II kit. Results All subjects reached NAb titers above 0.5 IU/ml by day 14 after vaccination. On day 35 (1 week after receiving the last rabies vaccine), anti-rabies antibodies were in the protective level (>0.5 IU/ml) in both groups. There was no statistically significant difference in anti-rabies antibody response due to the type of exposure (category 2 or 3), and successful seroconversion was confirmed in both groups. Conclusion In conclusion, the ESSEN protocol using the PVRV vaccine is sufficient for rabies prophylaxis in patients with specific medical conditions.

Background: Rabies is a major zoonotic viral disease and is detected using the World Health Organization standard diagnostic techniques. Rabies detection is preferably done using the fluorescent antibody technique (FAT) that provides reliable diagnosis with almost 100% accuracy for all variant strains, if a proper conjugate is used. Rabies virus nucleoprotein (NP) is the most important protein used in production of a specific diagnostic conjugate. Objectives: The aim of this study was to extract the cell-associated rabies virus NP from infected Baby Hamster Kidney cell clone (BSR) with rabies virus (Pasteur vaccine strain/PV) and purify for a future project to produce an anti-NP conjugate. Materials and Methods: Pasteur vaccine strain (PV) as the standard rabies vaccine strain with a focus-forming dose (FFD) of 105 was inoculated in to the BSR cell culture at a concentration of 10 6 cells per milliliter. Infected cells were harvested 72 hours after infection and the rabies NP was extracted from these cells by low-speed centrifugation and purification by ultracentrifugation in cesium chloride (CsCl) gradient. For analysis, the purified NP was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: The volume of the lysate was 15 mL and it became 2.5 mL after purification, with a concentration of 3.25 mg/mL. The corresponding band to the cell lysate protein on the SDS-PAGE had a molecular weight of 50 KDa, similar to the molecular weight of NP in rabies virus. Conclusions: The rabies virus NP could be extracted and purified in an appropriate amount from infected cell culture. The results of SDSPAGE analysis showed that the intact rabies virus NP had been purified properly and thus could be used for further steps to produce the specific diagnostic rabies conjugate.

In 2008, African swine fever was introduced into Georgia, after which it spread to neighboring Armenia, Azerbaijan, and the Russian Federation. That same year, PCR and sequence analysis identifi ed African swine fever virus in samples from 3 dead female wild boars in northwestern Iran. Wild boars may serve as a reservoir.

Background: Viral meningitis is an inflammation of the leptomeninges as a manifestation of central nervous system (CNS) infection. More than 85% of viral meningitis cases are caused by non-polio enteroviruses. This is the first study on the description of the enteroviruses and the related serotypes involved in viral meningitis in Iran. Objectives: This project was conducted to improve our knowledge about the role of enteroviruses and their circulating serotypes in viral meningitis in Iran. Patients and Methods: Cerebrospinal fluids from 118 children under 13 years old with primary clinical diagnosis of aseptic meningitis were collected in Children Medical Center in Tehran and sent to Department of Virology of Pasteur Institute of Iran. To investigate the enteroviruses, 5`-noncoding regions were amplified by Real-Time PCR method using Pan-EV primers and a specific probe. Serotype identification in enterovirus positive samples was conducted by RT-PCR. Results: Enterovirus detection rate in all 118 Cerebrospinal fluid specimens was 10.16%. Most patients were 0 - 2 years old (40.67%). Serotyping was achieved from 6 specimens with two polio viruses type 1 (vaccine type), two echoviruses 14, one echovirus 5 and one echovirus 30. Conclusions: Enteroviruses should be considered as the main cause of viral meningitis in Iran. Molecular detections of 5-'NCR and VP1-2A RT-PCR with sequence analysis were found to be better than the conventional methods, for direct diagnosis and EVs typing that may lead to decrease of the unnecessary hospitalizations.

BACKGROUND Human enterovirus 71 (HEV71) was isolated for the first time from an infant with encephalitis in California in 1969 and then spread through the world. It has emerged as a major cause of a vast variety of diseases such as epidemics of hand, foot and mouth disease (HFMD), aseptic meningitis (AM), acute flaccid paralysis, and encephalitis. The aim of this study was to determine the prevalence of enterovirus 71 in children < 8 years old who were hospitalized due to primary diagnosis of AM in Tehran. METHODS One hundred cerebrospinal fluid samples (CSF) were collected by physicians from children with a diagnosis of AM and transported on ice to the Pasteur Institute of Iran for further processing. Viral RNA was extracted and EV71 infection was detected by RT-PCR method using the specific primers. RESULTS EV71 infection was detected in 14 patients (14%). Eight (57.14%) patients were younger than 2 years old, 11 (78.57%) were male and 3 (21.43%) were female. The seasonal peaks of EV71 were observed during autumn and winter with 6 (42.86%) and 5 (35.71%) cases, respectively. CONCLUSIONS EV71 should be considered as a causative agent of AM in Iran with the epidemiological pattern similar to that of other enteroviruses as males are more susceptible to be affected by these viruses. Further studies on this virus are needed to improve our knowledge about them in our country.

Cancer is one of the most fatal diseases in the world and it has been years that finding new drugs and chemotherapeutic techniques with lowest side effects become one of the most important challenging matters needs really hard efforts. Chlorambucil (CBL), an ancient direct-acting alkylating anticancer agent, is commonly used for initial treatment of some kinds of cancers but the use of CBL is often limited because of the unpleasant side effects due to its lack of specificity for targeting cancer cells. In this research we tried to increase the specificity of CBL by producing a novel conjugate by using glutamine amino acid (Glut). Based on previous studies, poly amines and nitrogen compounds noticeably are used by cancer cells increasingly; therefore we decided to increase the efficiency and specificity of CBL by designing and producing a novel anti cancer conjugate using glutamine amino acid as an uptake enhancer, CBL, and Adipic acid Dihydrazide (ADH) as a spacer and linker. The biological tests were carried out on HT29 colorectal cancer cell line to evaluate its anticancer properties. Biological tests like MTT assay, finding IC50, evaluating the induced mechanism of the death of our novel CBL-Glutamine conjugate on HT29 cells, testing abnormal toxicity of this conjugate on mice in comparison with CBL drug were careid out. We found that not only CBL-Glutamine conjugate preserved its anti cancer property with regard to CBL drug, but also it represent lower abnormal toxicity in mice. Apoptosis was detected as its mechanism of the death. Our present study provides a promising strategy for targeting cancer cells using amino acids nano-conjugate drugs. The future perspectives have also been highlighted in continuing similar and relative researches.

Background Rabies is an endemic fatal zoonotic disease, commonly transmitted to humans through contact (bites and scratches) with infected animals. Objectives During the years 1990–2010, six patients with the clinical symptoms of rabies (fever, tinnitus, buzzing, delirium and hydrophobia), with no history of a bite, were diagnosed by physicians in Iran. To obtain laboratory confirmation of rabies infection, different clinical specimens from each patient were sent to the World Health Organisation (WHO) Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran. The first case was a 39-year-old male veterinary technician who entered his uncovered scratched hand into the mouth of a rabid bovine and became infected. Two years later, a herd of sheep being tended by a shepherd and his two sons were attacked by a rabid wolf. All three individuals were infected when they applied burnt thorny wool to the sheep's wounds as a bandage. Their hands were scratched and then infected through contact with the remaining saliva of the rabid wolf on the sheep's wounds. In 1994, two other human cases occurred through corneal transplantation from the same donor who had died with the clinical signs of food poisoning (according to his hospital record), which probably was a misdiagnosis of rabies infection. Study design This is a case series study that describes human rabies cases without biting incidents. According to the WHO recommendation, human rabies cases are notifiable, therefore, in Iran, a rabies surveillance system has been established to follow these cases. During the last decade, six patients with no ‘history of a bite’ were hospitalised with growing symptoms of rabies. The data were collected from each patient by the physicians and transferred to the Ministry of Health and Medical Education of Iran, and to the WHO Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran as the only testing laboratory. Thus, they came to the attention of the surveillance system. Ante-mortem diagnosis was performed on saliva, cerebrospinal fluid and blood samples that were collected from the first patient by the physicians. Fresh brain specimens from all patients were kept in a mixture of 50% glycerol in phosphate-buffered saline and transported on ice to the WHO Collaborating Center for Reference and Research on Rabies. Results For the first patient, rabies virus was investigated in saliva using the rapid tissue cell inoculation test (RTCIT) and the mouse inoculation test (MIT). Anti-rabies antibodies in this patient's serum and cerebrospinal fluid (CSF) were examined using the mouse neutralisation test (MNT). Fresh brain specimens from all patients were examined using the fluorescence antibody test (FAT) as recommended by the WHO laboratory manual in rabies as the post-mortem diagnostic test for rabies. Rabies infection was confirmed in all of the deceased patients. Anti-rabies antibodies were identified only in one patient's serum specimen. Testing also showed that the rabies virus isolated was the classic rabies virus (serotype 1), which is widespread in Iran. Conclusions Prevention and control of this fatal disease require a sensitive surveillance system to follow ‘suspected’ animal and human rabies cases thoroughly through the improved reporting system, which contains the history of exposure, clinical examinations, symptoms and laboratory results. This study describes some notable human rabies infections and their transmission modes to prevent occupational accidents.

International audience; Background: The 66 serotypes of human enteroviruses (EVs) are classified into four species A-D, based on phylogenetic relationships in multiple genome regions. Partial VP1 amplification and sequence analysis are reliable methods for identifying non-polio enterovirus serotypes, especially in negative cell culture specimens from patients with residual paralysis. Objectives: In Iran during the years 2000-2002, there were 29 residual paralysis cases with negative cell (RD, HEp2 and L20B) culture results. Study design: The genomic RNA was extracted from stool specimens from cases of residual paralysis and detected by amplification of the 5 -nontranslated region using RT-PCR with Pan-EV primers. Partial VP1 amplification by semi-nested RT-PCR (snRT-PCR) and sequence analysis were done. Results: Specimens from the 29 culture-negative cases contained echoviruses of six different serotypes. Conclusions: The global eradication of wild polioviruses is near and study of non-polio enteroviruses, which can cause poliomyelitis, is increasingly important to understand their pathogenesis. The VP1 sequences, derived from the snRT-PCR products, allowed rapid molecular analysis of these non-polio strains.