Analysis of the JSRV env gene revealed a conserved tyrosine (597) and methionine (600) residue in the cytoplasmic tail within the transmembrane domain of the envelope, which creates a known binding site of SH2 domains in the p85 subunit of phosphatidylinositol 3-kinase. However, when this tyrosine residue was mutated to serine or alanine, transformation was not affected. Furthermore, mutation of the methionine residue to valine or leucine also failed to eliminate JSRV env-mediated transformation. These results are in contrast to mutational analysis performed in JSRV env-transformed murine NIH-3T3 cells in which both the tyrosine and methionine residues are necessary for transformation. These findings suggest that more than one mechanism may be involved in JSRV env-mediated transformation.

Chronic wasting disease (CWD) of cervids is associated with conversion of the normal cervid prion protein, PrP C , to a protease-resistant conformer, PrP CWD . Here we report the use of both nondenaturing amplification and protein-misfolding cyclic amplification (PMCA) to amplify PrP CWD in vitro. Normal brains from deer, transgenic mice expressing cervid PrP C [Tg(cerPrP)1536 mice], and ferrets supported amplification. PMCA using normal Tg(cerPrP)1536 brains as the PrP C substrate produced >6.5 ؋ 10 9 -fold amplification after six rounds. Highly efficient in vitro amplification of PrP CWD is a significant step toward detection of PrP CWD in the body fluids or excreta of CWD-susceptible species.Chronic wasting disease (CWD) of cervids is a transmissible spongiform encephalopathy (TSE), akin to sheep scrapie and bovine spongiform encephalopathy. TSE pathogenesis is associated with refolding of the normal prion protein, PrP C , into a partially protease resistant isomer termed PrP RES (2,15,17). A remarkable feature of CWD among prion diseases is its horizontal transmission in nature (12), suggesting that PrP C conversion is highly efficient in this TSE and perhaps is associated with the presence of infectious prions in the body fluids of deer (10). CWD is also transmissible to and pathogenic in ferrets (1) and transgenic mice expressing normal cervid PrP C (3, 8, 11). Raymond and colleagues (18) first demonstrated the conversion of cervid PrP C to PrP RES in vitro. Nondenaturing amplification without the use of radiolabeling (7, 9) further contributed to understanding of the mechanisms of PrP C -toPrP RES conversion due to its directness and technical simplicity. Soto, Castilla, and colleagues (5, 6, 21) greatly extended the process and power of in vitro PrP RES amplification by developing protein-misfolding cyclic amplification (PMCA). In PMCA, normal brain homogenates (NBH) supply PrP C , which, upon addition of an infected brain homogenate, is refolded into the protease-resistant isoform, PrP RES . Breakage of aggregates by use of sonic bursts releases the newly formed PrP RES and extends the enciphering process (19,20,22). To begin to address the mechanisms of PrP C -to-PrP RES conversion in CWD and to enhance the sensitivity of CWD prion (PrP CWD ) detection in deer, we developed two in vitro amplification assays: nondenaturing amplification patterned after the technique of Lucassen et al. (9) and serial PMCA modeled after the method of Soto and colleagues (19, 21). Here we report amplification using CWD-negative brain homogenates from white-tailed deer (Odocoileus virginianus), cervid PrP transgenic mice [Tg(cerPrP)1536 mice] (3), and ferrets (Mustela putorius furo), a species shown to be susceptible to CWD infection in vivo (1).Preparation of tissue homogenates. Whole brains were removed rapidly after sacrifice from CWD-free animals and were immediately frozen in liquid nitrogen. For PMCA experiments, animals were perfused at death with phosphate-buffered saline (PBS) containing 5 mM EDTA. NBH were prepare...

Teleost vertebral centra are often similar in size and shape, but vertebral-associated elements, i.e. neural arches, haemal arches and ribs, show regional differences. Here we examine how the presence, absence and specific anatomical and histological characters of vertebral centra-associated elements can be used to define vertebral column regions in juvenile Chinook salmon (Oncorhynchus tshawytscha). To investigate if the presence of regions within the vertebral column is independent of temperature, animals raised at 8 and 12 °C were studied at 1400 and 1530 degreedays, in the freshwater phase of the life cycle. Anatomy and composition of the skeletal tissues of the vertebral column were analysed using Alizarin red S whole-mount staining and histological sections. Six regions, termed I-VI, are recognised in the vertebral column of specimens of both temperature groups. Postcranial vertebrae (region I) carry neural arches and parapophyses but lack ribs. Abdominal vertebrae (region II) carry neural arches and ribs that articulate with parapophyses. Elastic- and fibrohyaline cartilage and Sharpey's fibres connect the bone of the parapophyses to the bone of the ribs. In the transitional region (III) vertebrae carry neural arches and parapophyses change stepwise into haemal arches. Ribs decrease in size, anterior to posterior. Vestigial ribs remain attached to the haemal arches with Sharpey's fibres. Caudal vertebrae (region IV) carry neural and haemal arches and spines. Basidorsals and basiventrals are small and surrounded by cancellous bone. Preural vertebrae (region V) carry neural and haemal arches with modified neural and haemal spines to support the caudal fin. Ural vertebrae (region VI) carry hypurals and epurals that represent modified haemal and neural arches and spines, respectively. The postcranial and transitional vertebrae and their respective characters are usually recognised, but should be considered as regions within the vertebral column of teleosts because of their distinctive morphological characters. While the number of vertebrae within each region can vary, each of the six regions is recognised in specimens of both temperature groups. This refined identification of regionalisation in the vertebral column of Chinook salmon can help to address evolutionary developmental and functional questions, and to support applied research into this farmed species.

A novel, fatal neurological disease of the Australian brushtail possum (Trichosurus vulpecula) was first identified in 1995 in a research facility and subsequently in free-living possums in New Zealand and termed wobbly possum disease (WPD). The results of previous transmission studies suggested that the aetiological agent of WPD is most likely a virus. However, the identity of the presumed viral agent had not been elucidated. In the current report, we describe identification of a novel virus from tissues of WPD-affected possums using a combination of next generation sequencing and traditional molecular methods. The proportion of possums positive for the novel virus by PCR was significantly higher (p<0.0001) among animals with WPD than clinically healthy possums, strongly suggesting an aetiological involvement of the virus in WPD. Analysis of the partial genomic sequence of the putative WPD virus indicated that it is a novel nidovirus, most closely related to the current members of the family Arteriviridae.

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