Introduction of RNAvirus evolution

Several viruses, in particular RNA viruses, have high mutation rates and relatively short generation times. Particle stability during infection in nature or in laboratory triggers the evolutionary event toward different mechanisms such as genome segmentation, point mutation and recombination. The frequency of mutant genomes increase and modify the previous distribution, which, consequently, lead to emergence of a new infectious particle. Mutation and selection are the most fundamental processes in evolution. High mutation rate of RNA viruses has an important role in viral fitness. Therefore, it increase our understanding about molecular biology of viral infections and their evolution by selection, mutation could reliably determine our ability to challenge destructive viruses. This review focuses on existing impressions of genetic organization and mechanisms of RNA viruses evolution

Further stimulation of cellular immune responses through association of HPV-16 E6, E7 and L1 genes in order to produce more effective therapeutic DNA vaccines in cervical cancer model

Cervical cancer has been shown to be highly associated with human papillomavirus [HPV] infection. The viral oncogenes E6 and E7 are constantly expressed by the tumor cells and are therefore potent targets for therapeutic genetic vaccination. In the present study, it was investigated the potential effect of HPV-16 E6, E7 and L1 co-administration to activate specific cytotoxic T lymphocytes in tumor mice models. The HPV-16 E6, E7 and L1 genes from Iranian isolate were separately inserted into the mammalian expression vector, pcDNA3, to construct the DNA vaccine candidates. Tumor-bearing Animals [C57BL/6 mice] were immunized with the vaccine candidate; then, Lymphocyte Proliferation Assay [LPA] and relative tumor volume measurements were carried out in order to examine the immunological effects of the vaccine. Obtained results showed that co-administration of the HPV-16 E6, E7 and L1 DNA induced HPV-16 specific cellular immune responses and also protected against TC-1-induced tumor in vivo compared with negative controls. The results showed that mixed delivery systems might be valuable to improve the magnitude of the induced immune responses and confirmed therapeutic effects of HPV-16 E6, E7 through cytotoxic T lymphocyte induction and illustrate the new promising role for HPV-16 L1 CTL epitopes as a suitable CTL inducer

Efficient lentiviral transduction of adipose tissue-derived mouse mesenchymal stem cells and assessment of their penetration in female mice cervical tumor model

Although the incidence of cervical cancer has reduced during last years, but it causes mortality among women. Many efforts have performed to develop new drugs and strategy for treatment of cervical cancer. Adipose Tissue-Derived mouse Mesenchymal Stem Cells [MSCs] has many advantages which make them a suitable choice as a cell therapeutic agent in cancer treatment. In this study, we aimed to develop an improved protocol for Mouse MSCs transduction as well as assess the homing capacity and incorporation of MSCs in cervical cancer model. MScs were isolated from the mouse adipose tissue and characterized by differentiation and flow cytometry. In our study, lentiviral vector transductions of MSCs performed. Their penetrations were detected in tissue sections after injection of transduced MSCs to female C57BL/6 mice as a cervical cancer model. Results showed that MSCs were efficiently transduced with lentiviral vector resulting in efficient tumor penetration. The results provide evidence that MSCs were able to penetrate into the tumor mass of cervical tumor model and are good vehicles for gene transfer to cervical cancer

Determining influenza virus shedding at different time points in madin-darby canine kidney cell line

Monitoring of influenza virus shedding and optimization of multiplicities of infection [MOI] is important in the investigation of a virus one step growth cycle and for obtaining a high yield of virus in vaccine development and conventional basic diagnostic methods. However, eluted infectious viruses may still be present immediately after virus inoculation and when cells are washed following virus cultivation which may lead to a false positive virus infectivity assay. In this experimental study, we investigated influenza virus progeny production in Madin-Darby canine kidney [MDCK] cells with five different MOI at determined time points. The results were analyzed by end point titration tests and immunofluorescence assay. Higher titers of eluted virus were observed following a high MOI inoculation of virus in cell culture. Most probably, this was the result of sialic acid residues from viral hemagglutin in proteins that were cleaved by neuraminidase glycoproteins on the surface of the influenza virus, which promoted viral spread from the host cell to the culture supernatant or during endocytosis, where viruses recycle to the cell surface by recycling endosomes which culminated in virus shedding without replication. We demonstrated that the pattern of influenza virus progeny production was dose-dependent and not uniform. This production was influenced by several factors, particularly MOI. Understanding the exact features of viral particle propagation has a major impact in producing high virus yields in the development of vaccines. Use of lower MOI [0.01] could result in accurate, precise quantitative assays in virus diagnosis and titration methods

[Rearranged bovine rotavirus production through cultivation of virus by high multiplicity of infection [MOI] in cell culture and amplification of non-structural genes using RT-PCR]

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. 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. 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. 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

Comparison of rotavirus rf strain and hsv-1 titration by CCID50% and plaque assays

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. 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. 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. 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 CCID 50% method is not applicable for purification of homogenous viruses, nor is this technique reproducible

Comparison of qualitative PCR and pp65 antigenemia for the diagnosis of CMV infection in hematopoietic stem cell transplanted patients

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. 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. 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] 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

[Construction of pcDNA3.1+ vector containing FMDV type O/IRN/1/2007-VP1 gene, confirmation of protein expression in BHKT7 cells and evaluation of immune response in mice model]

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. 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. 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]. 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

Efficacy of HPV-16 E7 based vaccine in a TC-1 tumoric animal model of cervical cancer

The human papillomavirus as an etiological agent of cervical cancer does not grow adequately in tissue culture systems. The tumor cell line TC-1 continuously expresses the E6 and E7 oncogenic proteins of HPV, and is considered a suitable tool in laboratory investigations and vaccine researches against cervical cancer The TC-1 cell line was grown in RPMI 1650 supplemented with 10% FBS, glutamine and antibiotics, and was used for tumor development in mice. Six to seven week-old tumor bearing C57BL/6 mice were divided into 3 groups consisting of 7 mice per group. The first group received pcDNA-E7, the second group received pcDNAS, and the third group received phosphate buffered saline [PBS]. The treated animals were monitored for their tumor size progression and survival. At last, the tumoric tissues from autopsied animals were fixed and examined with Mayer's hematoxylin and eosin [H and E]. All experiments were done in accordance with guidelines of the Laboratory Animal Ethical Commission of Tarbiat Modares University. Data analysis was performed using the oneway ANOVA followed by Tukey's test in both experimental and control groups. A p-value <0.05 was considered significant. There were significant decreases in tumor growth; there were also improvements in survival among mice in the treated groups [p<0.041]. H and E stained sections from untreated mice were studied independently in a blinded fashion by two observers and showed malignant neoplasms composed of severely pleomorphic tumor cells with nuclear enlargement, high nuclear-cytoplasmic [N/C] ratios, and prominent nucleoli in solid and fascicular patterns of growth. High mitotic activity with extensive necrosis was also noted in both test and control groups. The TC-1 lung metastatic model can be used to test the efficacy of various E7-based therapeutic cancer vaccine strategies for cervical cancer and the prevention of HPV-related neoplasia

Strong Immune Responses Induced by a DNA vaccine containing HPV16 truncated E7 C-terminal linked to HSP70 gene

Vaccines capable of controlling tumor virus based infections are found difficult to develop due to the consistence latent infection in the host. DNA vaccines are attractive tools for the development of HPV vaccines and inducing antigen-specific immunity owing to the stability, simplicity of delivery, safety and cost effectiveness. However, there is a need to increase their potency by procedures such as using HSP70 gene as an adjuvant. To evaluate a DNA vaccine containing HPV16 truncated E7 C-terminal cytotoxic T-lymphocyte epitopes linked to HSP70 gene [HSP70-tE7] in an animal model. Mice were immunized with the plasmid DNA after pre-treatment with cardiotoxin. The splenocytes of immunized mice were then tested for CTL activity by detecting the apoptosis and necrosis in target cells, cytokine production by ELISA, CD4 and CD8 frequencies by flow cytometry, and lymphocyte stimulation by MTT assay. The recombinant expression vector was able to elicit immune responses close to that of full length E7 complete gene. Although the use of a small part of a target antigen can induce immune responses equivalent to the full length antigen, it fails to elicit statistically significant stronger immune responses when fused with HSP70 compared to the complete E7 gene alone. The potent immunogenicity of HPV16 E7 was preserved in the HSP70-tE7 vaccine and may represent a target of choice for the therapeutic vaccination strategies. However, to improve the immunogenicity polytope DNA vaccines which elicit multiple effector and memory CTL responses should be considered in future studies of DNA-based cancer vaccines

Effect of LIGHT adjuvant on kinetics of T-cell responses induced by HSV-1 DNA Immunization

Studies on efficacy of various vaccines that prevent or reduce the primary and recurrent HSV-1 infection have demonstrated the importance of cellular immunity for protection against the infection. We previously used DNA vaccination to induce cellular immunity against HSV-1 infection in mice. The aim of our study was to evaluate the effect of LIGHT; a member of TNF super family, on the kinetic of CTL response induced by HSV-1 glycoprotein B based DNA vaccine. Using a granzyme B ELISA for detection and analysis of CD8+ T cells, CTL activity was determined in the spleen of BALB/c mice at various time points after primary and booster dose of vaccination. The kinetics of CTL response to primary and secondary HSV-1 infection and DNA vaccination were compared to those induced by DNA vaccination in combination with LIGHT adjuvant in the present study. In primary and secondary immunization, the CTL activity in the HSV injected group peaked 7 days and 12 hours post immunization, respectively. After 5 days, LIGHT could neither accelerate the CTL response compared to DNA vaccination alone nor could enhance the CTL activity in the primary and the first peak of memory response, the amount of granzyme B induced by the LIGHT containing vaccine was significantly higher than that induced by the vaccine without the adjuvant. Although LIGHT enhances the cellular response in the booster dose of vaccination, it does not accelerate the CTL response

Cloning, expression, purification and characterization of recorabinant UreB[229-561] from Helicobacter pylori

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 a H. pylori vaccine is growing up rapidly. We selected a fragment of B subunit of H. pylori urease enzyme consist of four important epitopes, involving in elevating host immune responses. This 1070bp fragment was amplified by PCR from genomic DNA isolated from H. pylori 22596 and then cloned into the pET28a expression vector. UreB229-561 was expressed and then affinity-purified by Ni2+-Sepharose resin. The recombinant UreB229-561 was reacted with the serum of/. pylori-infected human and rabbit anti-H. pylori polyclonal antibody in western-blot analysis. Having transformed competent E.coli DH5 alpha with ligation product of digested ureB fragment and pET28a, plasmid extraction from single colonies appeared in LB-agar plate after 18-24 h incubation at 37°C, using plasmid extraction kit [Bioneer, Korea]. Applying both infected human serum and rabbit anti-H. pylori polyclonal antibody, brown strip corresponding to the location of the recombinant protein appeared on PVDF membrane after adding DAB solution, hence confirming the antigenicity of the protein. This recombinant fragment showed urease activity. Our findings confirmed that a prokaryotic expression system of rUreB[229-561] was successfully constructed. The results of SDS-PAGE showed that our constructed prokaryotic expression system pET28 alpha- ureB[229-561]-BL21DE3 efficiently produces target recombinant protein in the form of dissoluble inclusion body. Therefore we can suggest that these epitopes can effectively be a vaccine candidate

Localization of herpes simplex virus type 1 DNA in latently infected BALB/c mice neurons using in situ polymerase chain reaction

Herpes simplex virus type-1 [HSV-1] establishes a lifelong latent infection in neurons following primary infection. The existence of latent HSV-1 DNA in the trigeminal ganglia of infected BALB/c mice was examined using a direct in situ PCR technique, based on Digoxigenin-11-dUTP detection system with anti-digoxigenin-peroxidase and 3,3f-diaminobenzidine [DAB] substrate. Eight-week-old male BALB/c mice were inoculated via the eye by 10[4] plaque forming unit of wild type Iranian isolates of HSV-1. After establishment of latency, trigeminal ganglia were removed and examined using in situ PCR to detect HSV-1 genome. Finally, the results of in situ PCR were verified by a two-round PCR method, using amplification cocktail of in situ reaction, as a template for a conventional gel base PCR. The results suggest that a direct in situ PCR method using a peroxidase and DAB detection system is a useful means for detection of latent HSV-1 DNA in the latently infected ganglia

Development of a western blot assay for detection of antibodies against HSV using purified HSV virions prepared by sucrose density gradient

Herpes simplex viruses [HSVs] have widespread and ubiquitous prevalence in the human population and they have received a great deal of attention due to the range of diseases, they caused as a result of an infection. It seems that the fast and reliable diagnostic methods are needed for detecting the herpes simplex virus type 1 [HSV1] antibodies especially in patients with HSV encephalitis, immunocompromised people, and neonatal infections. The aim of this study was designing a Western blotting method for HSV1 antibody detection, using the purified virus by sucrose density gradient centrifugation procedure. The most reliable method for HSV detection is virus neutralization test but it needs cell culture preparation, high expertise, as well as the high amounts of serum samples. Considering the difficulties of this method, we tried to run a new one for HSV antibody detection by propagating the viruses and then purify them by sucrose density gradient centrifugation method. The purified viruses used as antigens in Western blotting assay. Diluted sera [1:100, and 1:200 dilutions] used in Western blotting and two-fold dilutions of the sera applied in virus neutralization test. Five of twenty seven samples were negative in Western blotting and the same results obtained in virus neutralization test. Comparing with our gold standard, the sensitivity and specificity of the developed assay were both 100%. Our results show that the designed method is a reliable method for replacing the virus neutralization test in diagnostic laboratories. It can also, be used for confirming the ELISA results

effect of herpes simplex virus virion host shutoff gene- a new suicide gene- on tumor cells

The herpes simplex virus [HSV] UL41 gene product, virion host shutoff [Vhs] protein, mediates the rapid degradation of both viral and cellular mRNA. This ability suggests that Vhs protein can be used as a suicide gene in cancer gene therapy applications. The recent reports have shown that the degradation of cellular mRNA during herpes simplex infection is selective. RNA containing AU-rich elements [ARE] in their 3' untranslated ends are the targets for the Vhs protein. RNA that are not subject to Vhs protein-dependent degradation are up-regulated during HSV infection. ARE are frequently found in mRNA that encode proto-oncogenes, nuclear transcription factors, and cytokines. In many human cancers, the AU-rich stretch of proto-oncogenes and regulatory genes has impaired. To investigate whether Vhs protein might be useful for inhibition of tumor cell proliferation, a eukaryotic expression vector containing Vhs protein gene was constructed. Cell degradation and RNA content of HeLa and MRC-5 tumor cells after transfection with the constructed vector were studied. The results showed a strong inhibitory activity in proliferation of transfected tumor cells and a sharp decrease in their RNA content. These data suggest that Vhs protein can be considered as a candidate for suicide cancer gene therapy

CTL responses to a DNA vaccine encoding E7 gene of human papillomavirus type 16 from an Iranian isolate

Cervical cancer is the most prevalent tumor in developing countries and the second most frequent cancer among female population worldwide. Specific human papillomaviruses and, most notably, HPV types 16 and 18 are recognized as being causally associated with cervical carcinomas. The early HPV type 16 genes, E6 and E7, directly participate in the in vitro transformation of primary human keratinocytes and represent an excellent target for immune therapy of HPV related disease. The aim of this study was the evaluation of the efficacy of a DNA vaccine containing human papillomaviruse type 16 E7 gene [Iranian isolate] in induction of CTL responses in an animal model. In this study, the expression vector containing HPV type 16 E7 gene was constructed and chosen as a model antigen in the development of a therapeutic DNA vaccine in an animal model. CTL responses, cytokine assay, lymphocyte stimulation test, CD4 and CD8 staining and flowcytometry were done for evaluating of the immune responses. Our findings indicate that the target DNA vaccine can induce an E7-specific CTL response, which is important in the lysis of infected tumor cells, compared to negative control [p < 0.005] after in vivo immunization in the mouse system. The developed vaccine may be promising as an anti-cancer vaccine

[Development of ELISA test for determining the antibody titer against herpes simplex virus-1 and comparison of the results by VNT]

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. 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]. 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]. 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

Isolation and cloning of human papillomavirus 16 L1 gene from Iranian isolate

To isolate and construct cloning and expression vectors containing human papillomavirus [HPV16-L1] gene as target for application as recombinant vaccine. The study was performed in 2007 in Tarbiat Modares University, Tehran, Iran. Four genital specimens DNAs were amplified with the use of HPV type-specific primers based on HPV-16 L1, E6, and E7 genes. The polymerase chain reaction products were cloned into suitable cloning and expression vectors and were confirmed by restriction enzyme analysis and sequencing. The desired plasmids were sequenced and indicated 99% homology with those submitted full length L1 sequences in the GenBank. The results showed that there was 99% homology between our product and those mentioned in the GenBank. Nowadays empty viral capsids, termed virus-like particles, are synthesized with the use of microbial or cellular expression systems. Therefore, it can be concluded that the Iranian HPV16 full length L1 sequence is very similar to the other sequences in the GenBank and it can be used as a candidate gene in prophylactic vaccine for cervical cancer