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Backgrounds: SARS-COV-2 infection is not always correlated with protection. Antibody seroprevalence in unvaccinated individuals, which is usually measured by N-specific antibodies, is not necessarily correlated with protection, while antibodies against S protein show a better correlation with protection due to its neutralizing epitopes. In this study, we tried to improve our conception of the hidden perspective of SARS-COV-2 in epidemiological reports and investigate anti-S antibody prevalence among anti-N antibody-positive asymptomatic and mildly symptomatic patients.
Materials & Methods: Blood samples were collected from asymptomatic or mildly symptomatic volunteer participants and symptomatic hospitalized patients with negative PCR results from May 30 to June 17, 2020. Detection of SARS-COV-2 antibodies was done using an ELISA kit targeting N or S protein.
Findings: Totally, 716 samples from volunteer participants and 81 samples from symptomatic hospitalized patients with negative PCR results were evaluated. The test performance-adjusted seroprevalence (95% CI) of SARS-COV-2 antibody was 17.3% (8.8-25.8%) for anti-N IgG in volunteers and 25.5% (12.8-39.7%) for anti-N and anti-S IgM in hospitalized patients. Among anti-N IgG positive infected individuals, 49.2% (21.4 and 78.8%) were anti-S antibody positive.
Conclusion: The results showed that SARS-COV-2 infection sometimes occurs in individuals without symptoms or with mild symptoms, but in more than half of them, the produced antibody is not protective. The findings of hospitalized patients showed that the combination of IgM assay with real-time PCR improved the disease diagnosis by more than 25% in cases with negative molecular test results.

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Objective: DNA vaccines have been widely used to develop immunity against various pathogens including parasites and viruses. The potential of DNA vaccine to induce an effective immune response is related to the expression levels of the encoded protein in eukaryotic cells. Therefore, optimization of plasmid DNA delivery system is a major concern in protein expression in order to make an efficient DNA vaccination. Non-viral vectors such as polymers and cationic peptides have been recently known as efficient gene delivery systems into eukaryotic cells. In this study, transfection efficiency of HPV16E7 gene was evaluated by two non-viral delivery systems in vitro. Materials and Methods: DNA construct encoding HPV16E7 (pEGFP-E7) was prepared in large scale with high purity. Then, two delivery systems including polymer PEI 25 kDa and polymer-peptide hybrid as PEI600-Tat conjugate were used to compare their efficiency for HPV16E7 DNA transfection in vitro. Results: Our data demonstrated that both delivery systems including PEI 25 kDa and PEI600-Tat are efficient tools for E7 gene transfection. Although the level of transfected COS-7 cells is higher using PEI 25 kDa in comparison with PEI600-Tat. Conclusion: Our study indicated that PEI potency for E7 gene transfection was higher than PEI600-Tat in vitro, but its toxicity was obstacle in vivo. Therefore, with regard to low toxicity of PEI600-Tat delivery system and its potent plasmid DNA delivery, it is critical issue to study its potency as new delivery system in vivo.

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Objective: Herpes simplex virus (HSVs) is a widespread human infectious agent, responsible for persistent and latent infections. Herpes simplex virus infections are usually continually recurrent in the normal population and represent a significant cause of complications in immunocompromised patients. Materials and Methods: In this study HSVs were propagated in BK cells and more than 502 samples were taken and analyzed for HSV IgG antibodies using Virus Neutralization Test (VNT) as golden standard test for evaluating in house Enzyme Linked Immunosorbent Assay (ELISA). Results: Based on the results 80.48 % and 81.67% were positive (1.8) in VNT and ELISA respectively. There was a significant correlation between the VNT and ELISA tests in the tested samples (Pearson’s r = 0.96). Conclusions: Our data showed that the in house ELISA can be used for screening and determination of the prevalence of HSV IgG antibodies, which can facilitates patient management using suitable and cost effective laboratory diagnostic tests.

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Objective: Helicobacter pylori is a widely distributed Gram negative bacterium that infects the human stomach and duodenum. Some antibiotic regimens are subjected to cure the infection but the cost of drugs, poor patient compliance and emerging of antibiotic-resistant strains are limiting the usefulness of these antibiotic therapies. Therefore, interest in developing a H. pylori vaccine is growing up rapidly. The aim of this study was to construct a recombinant vector containing fusion genes encoding a fragment of B subunit from Helicobacter pylori (H. pylori) urease (UreB332) and Helicobacter pylori adhesion A (HpaA) and expressed it in E. coli BL21, as well as determining its antigenicity as a vaccine candidate of H. pylori. Materials and Methods: The target genes encoding UreB332 and HpaA amplified from standard H. pylori chromosome by PCR, digested by restricted endonuclease enzyme and inserted into the prokaryotic expression vector pET28a(+) which was digested by corresponding restricted endonuclease enzyme. The target fusion protein was expressed in the BL21 (DE3) E.coli. Furthermore, UreB332-HpaA antigenicity was studied by western blotting after Ni-NTA agarose resin purification. Results: Enzyme digestion analysis, PCR and sequencing showed that the target genes were inserted correctly into the recombinant vector. The fusion protein UreB332-HpaA was recognized by the rabbit anti H. pylori polyclonal antibody and the human sera infected with H. pylori. Conclusion: Our results in addition to favorable properties of HpaA and UreB antigens, support the application of rUreB332-HpaA fusion protein, as a good candidate for the development of H. pylori vaccine.

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Objective: The aim of this study was to construct a pcDNA3.1+ vector containing FMDV type O/IRN/1/2007-VP1 gene, protein expression in BHKT7 cells and evaluation of immune response in BALB/c mice. Materials and Methods: FMDV type O/IRN/1/2007 was isolated from a cattle in Ray in 2007 and serotyped. The purified VP1 gene was sub-cloned into the PTZ57R/T vector and pcDNA3.1+ expression vector. The PCR product of Vp1 gene without stop codon was sub-cloned upstream of EGFP gene into the pEGFP-N1 vector to evaluate VP1-GFP fusion protein expression. The pcDNA3.1-VP1 and pEGFP-VP1 vectors were transfected into BHKT7 cell line. The expression of VP1 protein was evaluated by SDS-PAGE, western blotting and florescent analysis of VP1-GFP fusion protein. The mice were injected subcutaneously by pcDNA3.1-VP1 vector as DNA vaccine and titration of neutralizing antiserum and T cell proliferation assay were done to evaluate the immune response. Results: Insertion of VP1 gene was confirmed by double digestion of sub-cloned PTZ57R/T, pcDNA3.1+ and pEGFP-N1 vectors. The specific band in western blotting was also confirmed the VP1 protein expression in BHKT7 cells. The expression of VP1-GFP fusion protein was observed under the immune-florescent inverted microscopy as more green florescent spots versus expression of GFP protein, alone. The neutralizing antiserum titer and T cell proliferation increased significantly in the group of mice vaccinated with pcDNA3.1+-VP1 vector verses control groups (P<0.05). Conclusion: The results showed that the target gene was amplified, cloned in the cloning and expression vectors and protein expression was confirmed successfully. According to the confirmed VP1 protein expression and increasing neutralizing antiserum titer and T cell proliferation by pcDNA3.1+-VP1 vector (P<0.05), it can be used as DNA vaccine against FMDV type O/IRN/2007.

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Objectives: Human cytomegalovirus (CMV) is a major life-threatening pathogen for hematopoietic stem cell transplant recipients. Specific tests are used for the diagnosis and monitoring of CMV infection in transplant patients. This study evaluates the performance of pp65 antigenemia and qualitative PCR assays for monitoring CMV in such patients. Methods: We analyzed 179 clinical samples from 41 patients by using a validated home-brewed qualitative PCR and a commercial antigenemia assay. The obtained results were evaluated using quantitative real-time PCR as the gold standard. Results: CMV was observed in 26.8% of samples analyzed by the antigenemia assay and in 42.6% of the samples by qualitative PCR. Among 179 clinical samples, 50.8% were negative and 21.2% were positive by both assays. On the other hand, 26.3% were only positive by qualitative PCR whereas 1.7% were positive by the antigenemia assay. A comparison of the results with real-time PCR showed that qualitative PCR has a higher sensitivity than the antigenemia assay (98.7% vs. 45.7%). The specificity of both assays was equal (96.8%). Quantitative results of the antigenemia assay showed good correlation with real-time PCR (r=0.715; p<0.001). Conclusion: Both the qualitative PCR and antigenemia assays have special deficiencies for efficient diagnosis of CMV infection. Therefore, effective management of CMV infection in transplant patients requires the use of other sensitive quantitative methods such as qPCR.

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Objective: Titration of viruses is important to determine the quantity of virus in vaccine development, master virus seed stock preparation, viral vector studies and virus replication. In this study, we compared the CCID50% and plaque assay as a standard titration method for rotavirus (RF) and HSV-1.   Methods: The MA104 and Vero cells were inoculated by RF and HSV-1 in 6- and 96-well plates. Following infection and adsorption, the optimal time for the cytopathic effect caused by the viruses was noted and the results compared. Results: The CPE (Cytopathic Effect) of RF was observed in less than 18 hours, which increased until 72 hours after inoculation. In HSV-1, the CPE was observed 24 and 72 hours after inoculation. The virus titration in the plaque assay was monitored at 96 hours post-infection for RF and at 72 hours post-infection for HSV-1. In both viruses the plaque titer method was lower than the CCID50 method, since the results indicated that 1 CCID50% was equal to 0.7 PFU. Conclusion: The plaque assay is one of the most accurate methods for viral titration. For the plaque assay, individual lesions may be isolated, which the plaques can be counted. The CCID50% method is not applicable for purification of homogenous viruses, nor is this technique reproducible.

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Objective: Group A rotaviruses (GARV) are responsible for the vast majority of severe diarrhea worldwide that kills an estimated 600,000-870,000 children annually. Since infantile gastroenteritis is a main health problem, therefore diagnosis and treatment of this disease is crucial. Gene rearrangements have been detected in vitro during serial passages of the virus at a high multiplicity of infection (MOI) in cell culture, as well as in chronically infected immunodeficient individuals. In this study, we developed an RT-PCR method to detect and diagnose the standard and gene rearranged bovine rotavirus. Methods: Rotavirus RNA was extracted from confluent monolayers of infected MA-104 cells, stained with silver nitrate, and then electrophoresed in a 10% polyacrylamide gel. The full-length gene products that encoded the NSP1, 2, and 3 genes of the standard and rearranged rotavirus were amplified by RT-PCR using specific primers. Results: We observed rearranged NSP1 and NSP3 genes that had different migration patterns seen with polyacrylamide gel electrophoresis. NSP1, 2, and 3 gene segments from standard and rearranged rotaviruses were amplified by RT-PCR, then the complete nucleotide sequence of each gene was subjected to sequencing. The results showed the generation of gene rearrangement through serial passages of the bovine rotavirus RF strain. Conclusion: Serial passage of rotavirus in cell culture at a high MOI and chronic infection in immunodeficient target groups might alter rotavirus evolution. The methods utilized for detection and characterization of rotaviruses are continually evolving and being refined. Data collection is necessary to understand the molecular and antigenic features of the rotavirus in order to have a successful implementation of rotavirus studies and the development of a rotavirus vaccine. This study shows the importance of genetic variation and can provide valuable information about the amplification, diversity, biology, and evolution of rotaviruses.

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Objective: More than 99% of cervical cancers contain human papillomavirus (HPV), particularly the high-risk HPV type 16 (HPV-16). Among therapeutic HPV vaccines, DNA vaccines have emerged as a potentially promising approach. The main problem with DNA vaccination is the efficient delivery of the genes. A different delivery system has been used to bypass this problem. Archaeosomes have shown high stability during oxidative stress. In this study, we prepared the archaeosome Halobacterium salinarum polar lipid and used it as a delivery system and adjuvants for formulation with the E6/E7/L1 chimeric plasmid as an HPV vaccine candidate. Methods: The recombinant pIRES2-plasmid that contained an E6/E7/L1 chimeric gene of HPV were purified after extraction. Halobacterium salinarum total polar lipids were prepared according to a method by Bligh and Dyer. The archaeosome-pDNA complex was prepared by the addition of plasmid DNA to an archaeal lipid solution and the mixture kept at room temperature to allow for complex formation. Particle sizes and zeta potential of the samples were measured using dynamic light scattering. We measured the relative tumor volume after administration of TC-1 cells to C57BL/6 mice. Results: Zeta potential of the anionic archaeosomes was -6.84mV while archaeosome-pDNA complexes were -29 mV. The highly negative zeta potential of archaeosome-pDNA complexes demonstrated excellent loading of the plasmid on the nanoparticle surface and electrostatic stability. The results showed that the archaeosome-containing E6/E7/L1 chimeric gene significantly inhibited the rate of tumor growth in comparison with the control groups. Conclusion: Archaeosomes are easy and cost-economic to prepare and highly stable. They may hold tremendous promise as vaccine delivery vehicles

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SARS-COV-2, the latest member of Coronaviridea family as the cause of the recent global epidemic, has inflicted sever damages in different fields on most governments. Appropriate strategies such as identifying infected individuals and isolating them, trying to produce an effective vaccine, finding efficient treatment and making correct decisions in the social, economic and health fields are the current priorities of the world. Serological tests provide useful information in all of the above areas. In this article we will discuss the importance of performing SARS-COV-2 serological tests in diagnosis, vaccination, treatment and proper planning in the society.

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SARS-COV-2, the latest member of Coronaviridea family as the cause of the recent global epidemic, has inflicted sever damages in different fields on most governments. Appropriate strategies such as identifying infected individuals and isolating them, trying to produce an effective vaccine, finding efficient treatment and making correct decisions in the social, economic and health fields are the current priorities of the world. Serological tests provide useful information in all of the above areas. In this article we will discuss the importance of performing SARS-COV-2 serological tests in diagnosis, vaccination, treatment and proper planning in the society.

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