Aphanomyces invadans (Saprolegniaceae) is a peronosporomycete fungus associated with the serious fish disease, epizootic ulcerative syndrome (EUS), also known as mycotic granulomatosis. In this study, interspecific relationships were examined between A. invadans isolates and other aquatic animal pathogenic Saprolegniaceae, and saprophytic Saprolegniaceae from EUS-affected areas. Restriction fragment length polymorphisms and sequences of ribosomal DNA confirmed that A. invadans is distinct from all other species studied. A sequence from the internal transcribed spacer region ITS1, unique to A. invadans, was used to design primers for a PCR-based diagnostic test. Intraspecific relationships were also examined by random amplification of polymorphic DNA using 20 isolates of A. invadans from six countries. The isolates showed a high degree of genetic homogeneity using 14 random ten-mer primers. This provides evidence that the fungus has spread across Asia in one relatively rapid episode, which is consistent with reports of outbreaks of EUS. Physiological distinctions between A. invadans and other Aphanomyces species based on a data set of 16 growth parameters showed remarkable taxonomic congruence with the molecular phylogeny.
The complete genome of the snakehead fish retrovirus has been cloned and sequenced, and its transcriptional profile in cell culture has been determined. The 11.2-kb provirus displays a complex expression pattern capable of encoding accessory proteins and is unique in the predicted location of the env initiation codon and signal peptide upstream of gag and the common splice donor site. The virus is distinguishable from all known retrovirus groups by the presence of an arginine tRNA primer binding site. The coding regions are highly divergent and show a number of unusual characteristics, including a large Gag coiled-coil region, a Pol domain of unknown function, and a long, lentiviral-like, Env cytoplasmic domain. Phylogenetic analysis of the Pol sequence emphasizes the divergent nature of the virus from the avian and mammalian retroviruses. The snakehead virus is also distinct from a previously characterized complex fish retrovirus, suggesting that discrete groups of these viruses have yet to be identified in the lower vertebrates.
MATERIALS AND METHODS
Cell culture and virus purification.Cultures of retrovirus-infected SSN-1 cells and noninfected SSN-2 cells, both derived from striped snakehead fish, were grown and maintained in serum-supplemented Leibovitz L-15 medium. Retrovirus particles were purified by sedimentation velocity and equilibrium density gradient centrifugation. Pelleted virus from clarified SSN-1 medium was resuspended in TNE buffer (10 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM EDTA), layered onto a 5 to 20% step sucrose gradient, and centrifuged at 100,000 ϫ g for 20 min. The visible viral band was collected, layered onto a 20 to 50% continuous sucrose gradient, and centrifuged at 150,000 ϫ g for 3 h. Gradient fractions were collected and assayed for RT activity as described previously (18). Fractions with peak activity were pooled and stored at Ϫ80ЊC.
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