Detection of natural infection of infectious spleen and kidney necrosis virus in farmed tilapia by hydroxynapthol blue-loop-mediated isothermal amplification assay.

AIMS: Infectious spleen and kidney necrosis virus (ISKNV) has recently been recognized as a causative agent of serious systemic disease in tilapia. Our objective was to establish a new colorimetric loop-mediated isothermal amplification (LAMP) assay with pre-addition of hydroxynapthol blue (blue-LAMP) to investigate ISKNV transmission in tilapia. METHODS AND RESULTS: The blue-LAMP, targeting a major capsid protein gene of ISKNV, was conducted at 65°C for 45 min, allowing unaided visual detection of the pathogen based on colour change without cross-amplification of other known fish pathogens tested. Comparison of blue-LAMP and PCR assays revealed a higher detection level for blue-LAMP assay (41·33%) in a population of farmed tilapia infected with ISKNV. The investigation of ISKNV transmission pattern in farmed red tilapia using the blue-LAMP revealed a possible matroclinical form. The presence of ISKNV in the gonad samples was confirmed by in situ LAMP assay. Positive signals only appeared in ovarian follicles, and not in oocytes. Moreover, tissue tropism assay revealed that the brain was the main target organ in both farmed red tilapia (40%) and Nile tilapia (20%). CONCLUSIONS: The developed blue-LAMP assay has the potential to be used as a viable tool for screening covert and natural infections of ISKNV in tilapia. The evidence of vertical transmission of ISKNV infection in tilapia indicates the seriousness of this disease and will require a close attention and collaboration between tilapia hatcheries and disease experts in order to find a solution. SIGNIFICANCE AND IMPACT OF THE STUDY: The new blue-LAMP assay is a time-saving and economically viable detection tool, which allows unaided visual detection for ISKNV in tilapia, and it could be applicable for field applications. Evidence on the vertical transmission of ISKNV in farmed tilapia suggests a need for developing farm management practices to control the spread of virus in aquaculture industries.

Generation of retroviral particles for the spleen necrosis virus (SNV)-based vector system and their use in transduction of various cell types.

Genetically engineered retroviruses are widely used for gene delivery into human cells. A number of investigators have studied spleen necrosis virus (SNV) as a vehicle for gene delivery. Vectors developed from SNV and its closely associated avian reticuloendotheliosis virus strain A (REV-A) can be used for gene transfer into a variety of cells, including primary hematopoietic cells and human brain and post-mitotic neuronal cells that are difficult to transduce with other vector systems. SNV-based vector systems have the advantage of being quite safe, because wild-type SNV is unable to infect human cells and has less preference for integration into transcriptionally active sites or genes. However, the generation of retroviral vectors requires cotransfection of more than one plasmid into a packaging cell line, which is a tedious process. The development of stable packaging cell lines expressing envelope (Env) proteins and the structural proteins Gag-Pol will enhance mass production of retroviral vectors for future gene therapy experiments both in vitro and in vivo. This protocol describes the generation of retroviral particles for the SNV-based vector system. These particles can then be used for transduction of various cell types; as an example, a technique for transduction of post-mitotic neurons is also presented.

Murine retroviruses re-engineered for lineage tracing and expression of toxic genes in the developing chick embryo.

We describe two replication incompetent retroviral vectors that co-express green fluorescent protein (GFP) and beta-galactosidase. These vectors incorporate either the avian reticuloendotheliosis (spleen necrosis virus; SNV) promoter or the chick beta-actin promoter, into the backbone of the murine leukemia (MLV) viral vector. The additional promoters drive transgene expression in avian tissue. The remainder of the vector is MLV-like, allowing high titer viral particle production by means of transient transfection. The SNV promoter produces high and early expression of introduced genes, enabling detection of the single copy integrated GFP gene in infected cells and their progeny in vivo. Substitution of the LacZ coding DNA with a relevant gene of interest will enable its co-expression with GFP, thus allowing visualization of the effect of specific and stable changes in gene expression throughout development. As the VSV-G pseudotyped viral vector is replication incompetent, changes in gene expression can be controlled temporally, by altering the timing of introduction.

Enhanced gene expression conferred by stepwise modification of a nonprimate lentiviral vector.

The practical application of gene transfer as a treatment for genetic diseases such as cystic fibrosis or hemophilia has been hindered, in part, by low efficiencies of vector delivery and transgene expression. We demonstrated that a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the envelope glycoprotein from the baculovirus Autographa californica (GP64) efficiently transduces and persistently expresses a reporter gene in respiratory epithelium in the absence of agents that disrupt cellular tight junction integrity. GP64-pseudotyped FIV also efficiently transduced murine hepatocytes after tail vein delivery. To improve the FIV-based vector, we tested the contribution of a series of modifications to luciferase expression in vitro and in vivo. These modifications included the addition of spleen necrosis virus U5 (SNV U5) and mutation of the major splice donor and gag start codon located in the packaging region of the FIV transgene plasmid. After vector modification, we observed significantly enhanced expression of luciferase in respiratory epithelia after nasal application and in the liver after tail vein delivery. In addition, we observed significantly enhanced human factor VIII production after tail vein delivery. These sequential modifications provide an improved FIV lentivirus platform for gene therapy applications and may be applied to other retroviral vectors.

RNA helicase A interacts with divergent lymphotropic retroviruses and promotes translation of human T-cell leukemia virus type 1.

The 5' untranslated region (UTR) of retroviruses contain structured replication motifs that impose barriers to efficient ribosome scanning. Two RNA structural motifs that facilitate efficient translation initiation despite a complex 5' UTR are internal ribosome entry site (IRES) and 5' proximal post-transcriptional control element (PCE). Here, stringent RNA and protein analyses determined the 5' UTR of spleen necrosis virus (SNV), reticuloendotheliosis virus A (REV-A) and human T-cell leukemia virus type 1 (HTLV-1) exhibit PCE activity, but not IRES activity. Assessment of SNV translation initiation in the natural context of the provirus determined that SNV is reliant on a cap-dependent initiation mechanism. Experiments with siRNAs identified that REV-A and HTLV-1 PCE modulate post-transcriptional gene expression through interaction with host RNA helicase A (RHA). Analysis of hybrid SNV/HTLV-1 proviruses determined SNV PCE facilitates Rex/Rex responsive element-independent Gag production and interaction with RHA is necessary. Ribosomal profile analyses determined that RHA is necessary for polysome association of HTLV-1 gag and provide direct evidence that RHA is necessary for efficient HTLV-1 replication. We conclude that PCE/RHA is an important translation regulatory axis of multiple lymphotropic retroviruses. We speculate divergent retroviruses have evolved a convergent RNA-protein interaction to modulate translation of their highly structured mRNA.

HIV-1 Vpr potently induces programmed cell death in the CNS in vivo.

The human immunodeficiency virus type I (HIV-1) accessory protein Vpr has been associated with the induction of programmed cell death (apoptosis) and cell-cycle arrest. Studies have shown the apoptotic effect of Vpr on primary and established cell lines and on diverse tissues including the central nervous system (CNS) in vitro. However, the relevance of the effect of Vpr observed in vitro to HIV-1 neuropathogenesis in vivo, remains unknown. Due to the narrow host range of HIV-1 infection, no animal model is currently available. This has prompted us to consider a small animal model to evaluate the effects of Vpr on CNS in vivo through surrogate viruses expressing HIV-1Vpr. A single round of replication competent viral vectors, expressing Vpr, were used to investigate the apoptosis-inducing capabilities of HIV-1Vpr in vivo. Viral particles pseudotyped with VSV-G or N2c envelopes were generated from spleen necrosis virus (SNV) and HIV-1-based vectors to transduce CNS cells. The in vitro studies have demonstrated that Vpr generated by SNV vectors had less apoptotic effects on CNS cells compared with Vpr expressed by HIV-1 vectors. The in vivo study has suggested that viral particles, expressing Vpr generated by HIV-1-based vectors, when delivered through the ventricle, caused loss of neurons and dendritic processes in the cortical region. The apoptotic effect was extended beyond the cortical region and affected the hippocampus neurons, the lining of the choroids plexus, and the cerebellum. However, the effect of Vpr, when delivered through the cortex, showed neuronal damage only around the site of injection. Interestingly, the number of apoptotic neurons were significantly higher with HIV-1 vectors expressing Vpr than by the SNV vectors. This may be due to the differences in the proteins expressed by these viral vectors. These results suggest that Vpr induces apoptosis in CNS cells in vitro and in vivo. To our knowledge, this is the first study to investigate the apoptosis-inducing capabilities of HIV-1Vpr in vivo in neonatal mice. We propose that this, in expensive animal model, may be of value to design-targeted neuroprotective therapeutics.

Full genome sequence and some biological properties of reticuloendotheliosis virus strain APC-566 isolated from endangered Attwater's prairie chickens.

Reticuloendotheliosis virus (REV) causes runting, high mortality, immunosuppression, and chronic neoplasia associated with T and/or B cell lymphomas in a variety of domestic and wild birds, including Attwater's prairie chickens (APC) (Tympanuchus cupido attwateri). The complete proviral sequence of a recent REV isolate from APC (REV APC-566) was determined. This virus was isolated from an APC maintained in captivity in a reproduction program intended to avoid its extinction. REV APC-566 was determined to be oncogenic in Japanese quail (Coturnix coturnix japonica), chickens (Gallus gallus) and turkeys (Meleagris gallopavo). Immune responses against bacteria and viruses were significantly reduced in turkeys infected with REV APC-566. The proviral genome is 8286 nucleotides in length and exhibits a genetic organization characteristic of replication-competent gammaretroviruses. The REV APC-566 provirus contains two identical long terminal repeats (LTR) and a complete set of genes including gag, gag-pol and env. As previously reported, alignments with other REV sequences showed high similarity with sequences found in the gag and pol genes from other REVs. The REV APC-566 env gene showed high nucleotide sequence homology with REV sequences inserted in fowl poxvirus (99.8%), and with spleen necrosis virus (SNV) (95.1%). Sequences coding for a previously reported immunosuppressive peptide contained in the transmembrane region of the env gene are well conserved among all REV sequences analyzed. The LTR was the most divergent region, exhibiting various deletions and insertions. REV APC-566 has a unique insertion of 23 bp in U3 and shares deletions of 19 and 5 bp with chicken syncytial virus and REV inserts in fowlpox virus.