Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation.

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10-15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1-2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

The chicken chorioallantoic membrane model for isolation of CRISPR/cas9-based HSV-1 mutant expressing tumor suppressor p53.

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, which selectively target and kill cancer cells while sparing normal ones. Among them, engineered Herpes simplex virus type 1 (HSV-1) has been proposed as a potential treatment for cancer and was moved to phase III clinical trials. Previous studies showed that design of OV therapy combined with p53 gene therapy increases the anti-cancer activities of OVs. Here, the UL39 gene of the ICP34.5 deleted HSV-1 was manipulated with the insertion of the EGFP-p53 expression cassette utilizing CRISPR/ Cas9 editing approach to enhance oncoselectivity and oncotoxicity capabilities. The ΔUL39/Δγ34.5/HSV1-p53 mutant was isolated using the chorioallantoic membrane (CAM) of fertilized chicken eggs as a complementing membrane to support the growth of the viruses with gene deficiencies. Comparing phenotypic features of ΔUL39/Δγ34.5/HSV1-p53-infected cells with the parent Δγ34.5/HSV-1 in vitro revealed that HSV-1-P53 had cytolytic ability in various cell lines from different origin with different p53 expression rates. Altogether, data presented here illustrate the feasibility of exploiting CAM model as a promising strategy for isolating recombinant viruses such as CRISPR/Cas9 mediated HSV-1-P53 mutant with less virus replication in cell lines due to increased cell mortality induced by exogenous p53.

human adenoviruses role in ophthalmic pterygium formation.

BACKGROUND: Ophthalmic pterygium is a common benign lesion of unknown origin and the pathogenesis might be vision-threatening. This problem is often associated with exposure to solar light. Recent evidence suggests that potentially oncogenic viruses such as human papillomavirus and Epstein-Barr virus may be involved in the pathogenesis of pterygia. Expression of specific adenovirus genes such as E1A and E1B, which potentially have many functions, may contribute to their oncogenic activity as well as relevance to cellular immortalization. OBJECTIVES: For the first time, we aimed to investigate involvement of adenoviruses in pterygium formation. PATIENTS AND METHODS: Fifty tissue specimens of pterygium from patients undergoing pterygium surgery (as cases), 50 conjunctival swab samples from the same patients and 10 conjunctival biopsy specimens from individuals without pterygium such as patients undergoing cataract surgery (as controls) were analyzed for evidence of adenovirus infection with polymerase chain reaction using specific primers chosen from the moderately conserved region of the hexon gene. Furthermore, ß-globin primers were used to access the quality of extracted DNA. Data was analyzed using SPSS (version 16) software. RESULTS: Of 50 patients, 20 were men and 30 women with mean age of 61.1 ± 16.9 years ranged between 22 and 85 years. All samples of pterygia had positive results for adenoviruses DNA with polymerase chain reaction, but none of the negative control groups displayed adenoviruses. The pterygium group and the control groups were ß-globin positive. Direct sequencing of PCR products confirmed Adenovirus infection. CONCLUSIONS: Adenoviruses might act as a possible cause of pterygium formation and other factors could play a synergistic role in the development. However, further larger studies are required to confirm this hypothesis.

Chitosan nanoparticles as a potential nonviral gene delivery for HPV-16 E7 into mammalian cells.

Chitosan nanoparticles (CS NPs) were prepared as a carrier for Human papillomavirus type 16 HPV-16) E7 gene and their gene transfection ability were evaluated in vitro. The plasmid expressing green fluorescent protein (pEGFP) was used as a reporter gene. Gel electrophoresis demonstrated full binding of CS NPs with the pDNA. The transfection of CS-pEGFP NPs was efficient in CHO cells and the expression of green fluorescent proteins was well observed. The expression of E7 proteins was confirmed under SDS-PAGE and western blot analysis. As a conclusion CS NPs may serve as an effective nonviral carrier for delivery of nucleotides into eukaryotic cells.

Antitumor effect of therapeutic HPV DNA vaccines with chitosan-based nanodelivery systems.

BACKGROUND: Cervical cancer is the second-most-common cause of malignancies in women worldwide, and the oncogenic activity of the human papilloma virus types (HPV) E7 protein has a crucial role in anogenital tumors. In this study, we have designed a therapeutic vaccine based on chitosan nanodelivery systems to deliver HPV-16 E7 DNA vaccine, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer. We have developed a Nano-chitosan (NCS) as a carrier system for intramuscular administration using a recombinant DNA vaccine expressing HPV-16 E7 (NCS-DNA E7 vaccine). NCS were characterized in vitro for their gene transfection ability. RESULTS: The transfection of CS-pEGFP NPs was efficient in CHO cells and the expression of green fluorescent proteins was well observed. In addition, NCS-DNA E7 vaccine induced the strongest E7-specific CD8+ T cell and interferon γ responses in C57BL/6 mice. Mice vaccinated with NCS-DNA E7 vaccine were able to generate potent protective and therapeutic antitumor effects against challenge with E7-expressing tumor cell line, TC-1. CONCLUSIONS: The strong therapeutic effect induced by the Chitosan-based nanodelivery suggest that nanoparticles may be an efficient carrier to improve the immunogenicity of DNA vaccination upon intramuscular administration and the platform could be further exploited as a potential cancer vaccine candidate in humans.

GB virus C Viremia and Anti-E2 Antibody Response Among Hemodialysis Patients in Gorgan, Iran.

BACKGROUND: GB Virus C is a blood-borne virus and a member of Flaviviridae, like hepatitis C that is distributed globally and puts hemodialysis patients at high risk of developing liver disease. The clinical significance of GBV-C in this population remains unclear. OBJECTIVES: The current study aimed to evaluate GBV-C infection among hemodialysis patients. PATIENTS AND METHODS: Totally, 149 patients receiving hemodialysis were included in the study. The detection of GBV-C sequences in plasma was done by the nested Reverse transcription polymerase chain reaction (RT-PCR) using specific primers selected from highly conserved regions of 5' UTR of GBV-C and antibodies to the envelope protein of GBV-C (anti-E2 GBV-C antibody) were analyzed by also serological methods. In addition, Hepatitis B surface antigen (HBsAg), Hepatitis B core antibody (HBcAb) IgM, anti- Hepatitis C virus (HCV) and anti- hepatitis E virus (HEV) Ab was determined in patients who were GBV-C RNA and anti-E2 GBV-C antibody positive. RESULTS: The total prevalence of GBV-C infection was 14.7% (95%CI: 0.09-0.21) among patients receiving hemodialysis. The rate of GBV-C viremia and anti-E2 antibody positivity were 6.04% and 10.73%, respectively. Among the subjects who were positive for GBV-C, 27.27% (95% CI: 0.02-0.09), 45.45% (95% CI: 0.03-0.11), 59.9% (95% CI: 0.06-0.16) and 0% (95% CI: 0.01-0.07) were positive for anti-HCV, anti-HBsAg, anti-HBc IgM and anti-(HEV) Ab, respectively. In addition, the rate of both anti-HBc IgM /anti-HCV/ HBsAg and anti-HBc IgM /anti-HCV positivity in GBV-C infected cases were 9.09%. The liver enzymes were normal in all of them. There was significant difference between GBV-C exposures with viral hepatitis co-infection, but there was no correlation between GBV-C exposure with gender, age, ethnicity, time on dialysis and history of blood transfusions. A relatively high frequency of positivity GBV-C-exposure among hemodialysis patients suggested that the transmission route for GBV-C may be nosocomial transmission, and via transfusions. CONCLUSIONS: The current study found a relatively high frequency of positivity GBV-C-exposure among the patients receiving hemodialysis in the area understudy. Nosocomial transmission seems to be the main route of GBV-C infection in the area.