The combined application of next-generation sequencing platforms has provided an economical approach to unlocking the potential of the turkey genome.

RNA interference (RNAi) has recently emerged as a promising antiviral technique in vertebrates. Although most studies have used exogenous short interfering RNAs (siRNAs) to inhibit viral replication, vectors expressing short hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) are more efficient and are practical for in vivo delivery. In this study, replication competent retroviral vectors were designed to deliver shRNA-mirs targeting subgroup B avian leukosis virus (ALV), the most effective of which reduced expression of protein targets by as much as 90% in cultured avian cells. Cells expressing shRNA-mirs targeting the tvb receptor sequence or the viral env(B) sequence significantly inhibited ALV(B) replication. This study demonstrates efficient antiviral RNAi in avian cells using shRNA-mirs expressed from pol II promoters, including an inducible promoter, allowing for the regulation of the antiviral effect by doxycycline.

We have designed and characterized two new replication-competent avian sarcoma/leukosis virus-based retroviral vectors with amphotropic and ecotropic host ranges. The amphotropic vector RCASBP-M2C(797-8), was obtained by passaging the chimeric retroviral vector RCASBP-M2C(4070A) (6) in chicken embryos. The ecotropic vector, RCASBP(Eco), was created by replacing the env-coding region in the retroviral vector RCASBP(A) with the env region from an ecotropic murine leukemia virus. It replicates efficiently in avian DFJ8 cells that express murine ecotropic receptor. For both vectors, permanent cell lines that produce viral stocks with titers of about 5 ؋ 10 6 CFU/ml on mammalian cells can be easily established by passaging transfected avian cells. Some chimeric viruses, for example, RCASBP(Eco), replicate efficiently without modifications. For those chimeric viruses that do require modification, adaptation by passage in vitro or in vivo is a general strategy. This strategy has been used to prepare vectors with altered host range and could potentially be used to develop vectors that would be useful for targeted gene delivery.Retroviral vectors are widely used in studies of gene structure and function in cultured cells and in animal models. Retroviral vectors have also been used for clinical applications, including human somatic cell gene therapy. A number of retroviral vectors have been developed; most are based on avian and mammalian retroviruses. The majority of these vectors are replication-defective derivatives of the murine leukemia virus (MLV). In general, MLV vectors lack all genes for the viral structural proteins that are required for viral replication. The viral genes are usually expressed either by cotransfection or by a packaging cell line that supplies the viral proteins in trans. There are replication-competent MLV vectors; however, the insert size is limited (71, 72). Replication-competent vectors based on avian sarcoma/leukosis viruses (ASLV) can accept larger inserts. Naturally occurring ASLV can have several different envelopes (subgroups A to E). The various ASLV envelopes are distinguished based on host range; none of these envelopes allows the ASLV (or the vectors derived from them) to efficiently infect mammalian cells.We developed the replication-competent chimeric retroviral vector RCASBP-M2C(4070A) by replacing the subgroup A env gene of the ASLV-based retroviral vector RCASBP(A) with the env-coding sequence of an amphotropic MLV (6). The original amphotropic RCASBP replicated poorly; passage of the virus selected for a variant that has a single change, P242I, in gp70. The adapted vector, RCASBP-M2C(4070A), replicates efficiently in chicken embryo fibroblasts (CEF) or in DF-1 cells (25, 64) and can efficiently transfer genes into cultured mammalian cells; however, the virus is replication defective in mammalian cells. The RCASBP-M2C(4070A) vector has advantages compared with replication-defective MLVbased vectors. Since the RCASBP-M2C(4070A) vector is replication competent in avian cells, it s...

The interactions between the subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and soluble forms of the ALV(A) receptor Tva were analyzed both in vitro and in vivo by quantitating the ability of the soluble Tva proteins to inhibit ALV(A) entry into susceptible cells. Two soluble Tva proteins were tested: the 83-amino-acid Tva extracellular region fused to two epitope tags (sTva) or fused to the constant region of the mouse immunoglobulin G heavy chain (sTva-mIgG). Replication-competent ALV-based retroviral vectors with subgroup B or C env were used to deliver and express the two soluble tv-a (stva) genes in avian cells. In vitro, chicken embryo fibroblasts or DF-1 cells expressing sTva or sTva-mIgG proteins were much more resistant to infection by ALV(A) (∼200-fold) than were control cells infected by only the vector. The antiviral effect was specific for ALV(A), which is consistent with a receptor interference mechanism. The antiviral effect of sTva-mIgG was positively correlated with the amount of sTva-mIgG protein. In vivo, the stva genes were delivered and expressed in line 0 chicken embryos by the ALV(B)-based vector RCASBP(B). Viremic chickens expressed relatively high levels ofstva and stva-mIgG RNA in a broad range of tissues. High levels of sTva-mIgG protein were detected in the sera of chickens infected with RCASBP(B)stva-mIgG. Viremic chickens infected with RCASBP(B) alone, RCASBP(B)stva, or RCASBP(B)stva-mIgG were challenged separately with ALV(A) and ALV(C). Both sTva and sTva-mIgG significantly inhibited infection by ALV(A) (95 and 100% respectively) but had no measurable effect on ALV(C) infection. The results of this study indicate that a soluble receptor can effectively block infection of at least some retroviruses and demonstrates the utility of the ALV experimental system in characterizing the mechanism(s) of viral entry.