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CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome engineering applications. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck in human cells. By encoding Cas9, sgRNA and Donor DNAs on a single, rapidly assembled baculoviral vector, we achieve with up to 30% efficacy whole-exon replacement in the intronic β-actin (ACTB) locus, including site-specific docking of very large DNA payloads. We use our approach to rescue wild-type podocin expression in steroid-resistant nephrotic syndrome (SRNS) patient derived podocytes. We demonstrate single baculovirus vectored delivery of single and multiplexed prime-editing toolkits, achieving up to 100% cleavage-free DNA search-and-replace interventions without detectable indels. Taken together, we provide a versatile delivery platform for single base to multi-gene level genome interventions, addressing the currently unmet need for a powerful delivery system accommodating current and future CRISPR technologies without the burden of limited cargo capacity.

Precise gene editing and genome engineering by CRISPR technology requires simultaneous delivery of multiple DNA-encoded components into living cells rapidly exceeding the cargo capacity of currently utilized viral vector systems. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck. We implement hybrid DNA techniques (MultiMate) for rapid and error-free assembly of currently up to 25 functional DNA modules in a single baculoviral vector enabling CRISPR-based genome engineering. Utilizing homology-independent targeted integration (HITI), we achieve up to 30% correct genome interventions in human cells, including precision docking of large DNA payloads in the ACTB locus. We demonstrate baculovirus-vectored delivery of prime-editing toolkits for seamless DNA search-and-replace interventions achieving, with a single vector, highly efficient cleavage-free trinucleotide insertion in the HEK3 locus without any detectable indels. Our approach thus unlocks a wide range of editing and engineering applications in human cell genomes.

Aim: Since HER2 targeted therapies have shown clinical benefit in breast cancer, in the present study recombinant baculovirus (BV) displaying anti-HER2 single-chain variable domain fragment (scFv) expressing Apoptin was generated. Methods: The binding specificity and surface display of anti-HER2 scFv were evaluated using enzyme-linked immunosorbent assay (ELISA) and electron microscopy, respectively. The targeting properties and cytotoxic effect on breast cancer cells determined by fluorescence microscopy and MTT assays. Results: The results demonstrated that recombinant BV could specifically bind to HER2-overexpressing SK-BR-3 cells but not to the HER2 negative MCF-7 cells and reduced the viability of SK-BR-3 cells by expressing Apoptin. Conclusion: These results suggest that the antitumor effect of Apoptin in combination with HER2 targeting of this recombinant BV makes it a promising vector in targeted cancer therapy.

Aim: To determine the molecular epidemiology and characterization of hepatitis D virus (HDV) genotypes circulating in different provinces of Iran. Patients & methods: In this study, the presence of HDV RNA was tested in sera that were positive for hepatitis B surface antigen and HDV antibody by nested-PCR. HDV genotypes were subsequently analyzed using restriction fragment length polymorphism (RFLP) assay and then confirmed by sequencing. Results: 86.5% of positive PCR patients had genotype I and 8.1% had genotype II while the genotype of 5.4% of the patients remained undetermined by RFLP. Sequencing followed by phylogenetic analysis demonstrated that all the Iranian isolates were from genotype I. Conclusion: Although analyzing the RFLP of RT-PCR is a simpler method, the gold standard of genotyping of HDV is the phylogenetic analysis based on sequencing.

Malignant mesothelioma (MMe) is a cancer with poor prognosis and resistance to standard treatments. Recent reports have highlighted the role of the BRCA1 associated protein 1 gene (BAP1) in the development of MMe. In this study, the chemosensitivity of human mesothelioma cell lines carrying BAP1 wild-type (WT), mutant and silenced was analysed. The BAP1 mutant cells were significantly less sensitive than BAP1 WT cell lines to the clinically relevant drug gemcitabine. Silencing of BAP1 significantly increased resistance of MMe cells to gemcitabine. Cell cycle analysis suggested that gemcitabine induced Sub-G1 phase accumulation of the BAP1 WT cells and increased in the S-phase in both BAP1 WT and mutant cells. Analysis of the role of BAP1 in apoptosis suggested that gemcitabine induced early apoptosis in both BAP1 WT and BAP1 mutant cells but with a much higher degree in the WT cells. Effects on the population of cells in late apoptosis, which can mark necrosis and necroptosis, could not be seen in the mutant cells, highlighting the possibility that BAP1 plays a role in several types of cell death. Significantly decreased DNA damage in the form of double-strand breaks was observed in gemcitabine-treated BAP1 mutant cells, compared to BAP1 WT cells under the same conditions. After BAP1 silencing, a significant decrease in DNA damage in the form of double-strand breaks was observed compared to cells transfected with scramble siRNA. Taken together, the results presented in this manuscript shed light on the role of BAP1 in the response of MMe cells to gemcitabine treatment and in particular in the control of the DNA damage response, therefore providing a potential route for more efficient MMe therapy.

Malignant pleural mesothelioma (MPM) is a rare neoplasm with a poor prognosis, as current therapies are ineffective. Despite the increased understanding of the molecular biology of mesothelioma, there is still a lack of drugs that dramatically enhance patient survival. The present review discusses recent and complete clinical trials supported by the NIH, other U.S. Federal agencies, universities and organizations found on clinicaltrials.gov. Firstly, chemotherapy-based trials are described, followed by immunotherapy and multitargeted therapy. Then drug repositioning and the use of drug docking as tools to find new interesting molecules are introduced. Finally, potential molecular pathways that may play a role in mesothelioma biology and therapy are highlighted. Numerous biases are present in the clinical trials due to a restricted number of cases, inappropriate endpoints and inaccurate stratification of patients which delay the finding of novel, successful treatments for MPM. The most crucial issue of independent research for MPM is the lack of more substantive funding to translate these findings to the clinical setting. Furthermore, many trials for mesothelioma adopt therapies utilised successfully in other cancers. However, this approach is not necessarily scientific due to the low mutational load of mesothelioma relative to other cancers. It is therefore clear that patients need a more solid rationale and bedrock of preclinical research to support these trials, to ultimately have a better chance of successful treatment.

Human Parvovirus B19 (B19V) is a prototype of the Erythroparvovirus genus in Parvoviridae family. B19V infections are often associated with fever and rash, and can be mistakenly reported as measles or rubella. Differential diagnosis of B19V illness is necessary for case management and also for public health control activities, particularly in outbreak situations in which measles or rubella is suspected. To investigate the causative role of B19V infection in children with measles- and rubella-like illness, a total of 583 sera from children with exanthema were tested for presence of B19V by determining anti-B19V IgG and IgM antibodies by ELISA as well as B19V DNA detection by nested PCR. DNA positive samples were assessed further for determination of viral load and sequence analysis by Real-Time PCR and Sanger sequencing method, respectively. Out of 583 patients, 112 (19.21%) patients were positive for B19V-IgM antibody, 110 (18.87%) were positive for B19V-IgG antibody, and 63 (10.81%) were positive for B19V viral DNA. The frequency of B19V-IgG antibodies were increased with age; that is children under 6 year old showed 7.11% seroprevalence for B19V-IgG as compared to 18.39% and 28.91% for age groups 6 to >11 and 11-14 years old, respectively. Phylogenetic analysis of the NS1-VPu1 overlapping region revealed that all sequenced B19V-DNA belonged to genotype 1. The results of this study may aid the surveillance programs aiming at eradicating measles/rubella virus in Iran, as infections with B19V can be mistakenly reported as measles or rubella if laboratory testing is not conducted.

Human Parvovirus B19 (B19V) is a prototype of the Erythroparvovirus genus in Parvoviridae family. B19V infections are often associated with fever and rash, and can be mistakenly reported as measles or rubella. Differential diagnosis of B19V illness is necessary for case management and also for public health control activities, particularly in outbreak situations in which measles or rubella is suspected. To investigate the causative role of B19V infection in children with measles- and rubella-like illness, a total of 583 sera from children with exanthema were tested for presence of B19V by determining anti-B19V IgG and IgM antibodies by ELISA as well as B19V DNA detection by nested PCR. DNA positive samples were assessed further for determination of viral load and sequence analysis by Real-Time PCR and Sanger sequencing method, respectively. Out of 583 patients, 112 (19.21%) patients were positive for B19V-IgM antibody, 110 (18.87%) were positive for B19V-IgG antibody, and 63 (10.81%) were positive for B19V viral DNA. The frequency of B19V-IgG antibodies were increased with age; that is children under 6 year old showed 7.11% seroprevalence for B19V-IgG as compared to 18.39% and 28.91% for age groups 6 to11 and 11-14 years old, respectively. Phylogenetic analysis of the NS1-VPu1 overlapping region revealed that all sequenced B19V-DNA belonged to genotype 1. The results of this study may aid the surveillance programs aiming at eradicating measles/rubella virus in Iran, as infections with B19V can be mistakenly reported as measles or rubella if laboratory testing is not conducted.

Occult HCV infection is a form of chronic HCV infection characterized by absence of detectable anti-HCV antibodies or plasma HCV-RNA but presence of HCV-RNA in liver biopsy and/or peripheral blood mononuclear cells (PBMCs). The aim of this study was to determine the presence of HCV-RNA in PBMCs of patients with lymphoproliferative disorders. One hundred and four consecutive patients with lymphoproliferative disorders admitted to Firouzgar Hospital from January 2010 to March 2011 were recruited in this cross-sectional study. A 6-ml sample of whole blood was taken from the patients, the total RNA was extracted from the samples after the separation of plasma and PBMCs. The HCV-RNA of the samples was amplified by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR). The HCV genotypes of the positive samples were tested using the INNO-LiPA™ HCV II kit, and the HCV genotypes were then confirmed by sequencing of the 5′-UTR fragments after the PCR products were cloned into a pJET1.2/blunt cloning vector. The mean age of the patients was 48.3 ± 1.76 years (range: 16–83). HCV-RNA was found in PBMCs from 2 (1.9%) of the 104 patients. Genotyping showed that the patients were infected with HCV subtype 1a. One patient suffered non-Hodgkin's lymphoma and the other suffered chronic lymphocytic leukemia. Patients with lymphoproliferative disorders with negative anti-HCV antibodies and negative plasma HCV-RNA may have occult HCV infection. Therefore, in the absence of a liver biopsy, the testing of PBMCs for the detection of genomic HCV-RNA may be beneficial. J. Med. Virol. 85:235–240, 2013. © 2012 Wiley Periodicals, Inc.