Host Subtraction, Filtering and Assembly Validations for Novel Viral Discovery Using Next Generation Sequencing Data.

The use of next generation sequencing (NGS) to identify novel viral sequences from eukaryotic tissue samples is challenging. Issues can include the low proportion and copy number of viral reads and the high number of contigs (post-assembly), making subsequent viral analysis difficult. Comparison of assembly algorithms with pre-assembly host-mapping subtraction using a short-read mapping tool, a k-mer frequency based filter and a low complexity filter, has been validated for viral discovery with Illumina data derived from naturally infected liver tissue and simulated data. Assembled contig numbers were significantly reduced (up to 99.97%) by the application of these pre-assembly filtering methods. This approach provides a validated method for maximizing viral contig size as well as reducing the total number of assembled contigs that require down-stream analysis as putative viral nucleic acids.

A viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing.

Here we describe a virus discovery protocol for a range of different virus genera, that can be applied to biopsy-sized tissue samples. Our viral enrichment procedure, validated using canine and human liver samples, significantly improves viral read copy number and increases the length of viral contigs that can be generated by de novo assembly. This in turn enables the Illumina next generation sequencing (NGS) platform to be used as an effective tool for viral discovery from tissue samples.

Clustering of immunoreceptor tyrosine-based activation motif-containing signalling subunits in CD4(+) T cells is an optimal signal for IFN-gamma production, but not for the production of IL-4.

CD4(+) T cells with pre-defined MHC-unrestricted specificity to type II collagen (CII) were engineered for cell-based anti-inflammatory gene therapy of autoimmune arthritis. To this end, recombinant chimeric immunoreceptors, C2gamma or C2zeta, were expressed in primary mouse keyhole limpet hemocyanin (KLH)-specific T(h)1 and T(h)2 cells using retrovirus vector-based somatic cell gene transfer. The ectodomain of these tyrosine-based activation motif (ITAM)-containing immunoreceptors is a single-chain IgG variable domain of an anti-CII mAb. The engineered cells might arrest migration when they encounter CII in articular cartilage. Up to 19 and 55% of transduced CD4(+) T cells expressed respectively C2gamma and C2zeta. The expression of C2gamma or C2zeta on the surface of CD4(+) T cells was down-regulated upon binding CII, and cells activated in such a way proliferated, up-regulated CD25 expression and produced cytokines. Comparison of cytokine levels normalized by the number of producer cells revealed that C2gamma and C2zeta were as potent as TCR in the induction of IFN-gamma, but induced lower levels of IL-4. It appears that the reason why CD4(+) T cells stimulated through C2gamma and C2zeta produce low levels of IL-4 is a lack of integration between co-stimulatory signals required for the optimal production of this cytokine and the ITAM-dependent signals generated by the immunoreceptors. The significance of these data for the development of anti-inflammatory gene therapy based on CD4(+) T cells targeted to a tissue-specific protein is discussed.

Highly efficient gene transfer into antigen-specific primary mouse lymphocytes with replication-deficient retrovirus expressing the 10A1 envelope protein.

BACKGROUND: Introduction of recombinant genes in the genome of primary lymphocytes by virtue of a replication-deficient retrovirus can be used in immunological studies and for cell-based gene therapy. METHODS: Packaging cells GP+E86 producing replication-deficient retrovirus incorporating the genes of enhanced green fluorescent protein (eGFP), C2gamma or C2xi, were generated by calcium phosphate-mediated transfection. Clones with the highest titres of retrovirus vector were isolated from them and their supernatants were used for transduction of PT67 cells. Primary mouse lymphocytes and T-cell hybridoma MD.45 were transduced by centrifugation with retroviral stock. The retroviral content of packaging cell supernatants was determined by dot blotting and hybridization with a DNA probe. RESULTS: PT67 cells produced approximately 50 times more retrovirus vector than the original GP+E86 clones. When retroviral stocks of PT67 and GP+E86 cells were used at 1/50 dilution and undiluted, respectively (to normalize them for retroviral RNA content), the transduction efficiency of mouse T-cell hybridoma was 40% and 5%, respectively. Centrifugation of target cells with retroviral stock at 2000 g for 60 min increased the percentage of transduced cells two- to three-fold. Within a population of cells isolated from the draining lymph nodes of an immunized mouse and reactivated with an antigen, up to 60% of CD4(+) T cells and up to 80% of B cells could be transduced with a transgene in replication-deficient retrovirus packaged by PT67 cells using the optimized gene transfer protocol. CONCLUSIONS: This protocol allows for the generation of packaging cells producing high titres of retrovirus vector. The 10A1 envelope protein is superior to the ecotropic one for the transduction of mouse lymphocytes.