The sequences of four complete genes were analysed in order to determine the relatedness of koi herpesvirus (KHV) to three fish viruses in the family Herpesviridae: carp pox herpesvirus (Cyprinid herpesvirus 1, CyHV-1), haematopoietic necrosis herpesvirus of goldfish (Cyprinid herpesvirus 2, CyHV-2) and channel catfish virus (Ictalurid herpesvirus 1, IcHV-1). The genes were predicted to encode a helicase, an intercapsomeric triplex protein, the DNA polymerase and the major capsid protein. The results showed that KHV is related closely to CyHV-1 and CyHV-2, and that the three cyprinid viruses are related, albeit more distantly, to IcHV-1. Twelve KHV isolates from four diverse geographical areas yielded identical sequences for a region of the DNA polymerase gene. These findings, with previously published morphological and biological data, indicate that KHV should join the group of related lower-vertebrate viruses in the family Herpesviridae under the formal designation Cyprinid herpesvirus 3 (CyHV-3).

Phylogenetic relationships among herpesviruses (HVs) of mammals, birds, and reptiles have been studied extensively, whereas those among other HVs are relatively unexplored. We have reconstructed the phylogenetic relationships among 13 fish and amphibian HVs using maximum likelihood and Bayesian analyses of amino acid sequences predicted from parts of the DNA polymerase and terminase genes. The relationships among 6 of these viruses were confirmed using the partial DNA polymerase data plus the complete sequences of the terminase, helicase, and triplex protein genes; the position of these viruses among all other sequenced HVs was also investigated using the complete terminase gene. The results established the monophyly of the fish and amphibian HVs (Alloherpesviridae) separate from the HVs of mammals, birds, and reptiles (Herpesviridae) and the single recognized HV of bivalve mollusks (Malacoherpesviridae) in the order Herpesvirales. Two major clades in the family Alloherpesviridae were recognized: one consisting of viruses from cyprinid and anguillid hosts and the other of viruses from ictalurid, salmonid, acipenserid, and ranid hosts. A comparison of virus and host phylogenies suggested that closely related HVs in this family may have coevolved with their hosts, whereas significant codiversification was not apparent for the more distantly related viruses.KEY WORDS: Alloherpesviridae · Fish herpesviruses · Frog herpesviruses · Phylogeny · Coevolution· DNA polymerase · Terminase Resale or republication not permitted without written consent of the publisherDis Aquat Org 84: [179][180][181][182][183][184][185][186][187][188][189][190][191][192][193][194] 2009 pesviridae, divided into the subfamilies Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae) containing mammalian, avian, and reptilian viruses, one (Alloherpesviridae) containing fish and amphibian viruses, and one (Malacoherpesviridae) containing a single virus, OsHV1. The taxonomic structure of the family Herpesviridae has been well established phylogenetically, and the viruses are characterized by approximately 43 homologous genes that were presumably inherited from a common ancestor (McGeoch et al. 2006). The erection of the 2 new families was driven by the findings that these viruses are but tenuously related to the fish and amphibian HVs, which share at least 13 homologous genes with each other (Aoki et al. 2007), and that OsHV1 is related only marginally to all other HVs (Davison et al. 2005b). Indeed, the best, and perhaps only, sequence-based evidence supporting a common ancestry for all HVs resides in a single gene, that encoding the putative ATPase subunit of terminase (hereafter terminase; an enzyme complex involved in packaging DNA into capsids), in which 5 regions are conserved (Davison 1992(Davison , 2002. This protein is also conserved to a lesser extent in T4-like bacteriophages in the family Myoviridae (Davison 1992). Indeed, the perceived utilization of a similar enzyme complex for packaging DNA into preformed capsids...

The prevalence of infection and susceptibility of the aquatic oligochaete Tubifex tubifex to Myxobolus cerebralis, was examined in 2 studies on the upper Colorado River, Colorado, USA, where whirling disease occurs in wild trout populations. In the first study, the prevalence of infection ranged from 0.4 to 1.5%, as determined by counting the number of T. tubifex releasing triactinomyxons of M. cerebralis directly following their collection from the field. The susceptibility of those T. tubifex not releasing triactinomyxons was assessed by the number of these oligochaetes releasing triactinomyxons 3 mo following experimental exposures to spores of M. cerebralis. The prevalence of infection following experimental exposures of these T. tubifex ranged from 4.2 to 14.1%. In a second study, all T. tubifex collected at 2 different times directly from the 2 field sites in Colorado were exposed to spores of M. cerebralis. Individual oligochaetes representing those groups of T. tubifex releasing and those groups not releasing triactinomyxons at 3 mo were screened with molecular genetic markers. T. tubifex populations found at the 2 study sites consisted of 4 genetically distinct lineages that varied with respect to their susceptibility to experimental exposure to M. cerebralis. Lineages I and III contained the most oligochaetes susceptible to M. cerebralis and were the most prominent lineages at Windy Gap Reservoir, a site of high infectivity for wild rainbow trout on the upper Colorado River. In contrast, at the Breeze Bridge site which is below Windy Gap Reservoir and where M. cerebralis infections are less severe in wild trout, oligochaetes in lineages V and VI that are resistant to M. cerebralis were more prominent. These results suggest that certain habitats, such as Windy Gap Reservoir, are conducive to large and more homogenous populations of susceptible T. tubifex lineages that may serve as point sources of infection for M. cerebralis. Although not a direct objective of this study, there was no evidence of M. cerebralis infections among any oligochaetes other than those that would be classified as T. tubifex by standard morphological characteristics. KEY WORDS: Whirling disease · Myxobolus cerebralis · Tubifex tubifex Resale or republication not permitted without written consent of the publisherDis Aquat Org 51: [113][114][115][116][117][118][119][120][121] 2002 clines among wild rainbow trout populations in the states of Idaho, Montana, Colorado and Utah, USA (Nehring & Walker 1996, Vincent 1996, Hedrick et al. 1998. M. cerebralis is the best known of 1300 parasites grouped in the phylum Myxozoa and the first shown to possess a 2 host life cycle including salmonid fish and an aquatic oligochaete, Tubifex tubifex (Markiw & Wolf 1983, Wolf & Markiw 1984, Wolf et al. 1986). The parasite has been observed among wild fish in 23 states in the USA, but without the same disastrous effects observed in the intermountain west (Nickum 1999). The differences in the impacts on wild fish between geographic region...

Diagnostic methods were used to identify and quantify Myxobolus cerebralis, a myxozoan parasite of salmonid fish. In this study, 7-week-old, pathogen-free rainbow trout (Oncorhynchus mykiss) were experimentally infected with M. cerebralis and at 7 months postinfection were evaluated with 5 diagnostic assays: 1) pepsin-trypsin digest (PTD) to detect and enumerate spores found in cranial cartilage, 2) 2 different histopathology grading scales that provide a numerical score for severity of microscopic lesions in the head, 3) a conventional single-round polymerase chain reaction (PCR), 4) a nested PCR assay, and 5) a newly developed quantitative real-time TaqMan PCR. There were no significant differences (P > 0.05) among the 5 diagnostic assays in distinguishing between experimentally infected and uninfected control fish. The 2 histopathology grading scales were highly correlated (P < 0.001) for assessment of microscopic lesion severity. Quantification of parasite levels in cranial tissues using PTD and real-time TaqMan PCR was significantly correlated r = 0.540 (P < 0.001). Lastly, 104 copies of the 18S rDNA gene are present in the M. cerebralis genome, a feature that makes this gene an excellent target for PCR-based diagnostic assays. Also, 2 copies of the insulin growth factor-I gene are found in the rainbow trout genome, whose detection can serve both as an internal quality control for amplifiable DNA and as a basis to quantify pathogen genome equivalents present in quantitative PCR assays.

We analyzed the geographic distribution of Tubifex tubifex from various river drainages in central Colorado by genetic screening with specific mitochondrial 16S ribosomal DNA (mt 16S rDNA) markers. Four distinct mt 16S rDNA lineages are evident. The sites varied with respect to land- and water-use practices. All sites represented habitats presumed capable of supporting oligochaetes. At the locations where whirling disease has had the greatest impact on resident rainbow trout, T. tubifex, representing lineages I and III (genotypes known to be susceptible to Mxyobolus cerebralis), were most commonly found. In contrast, at sites less affected by whirling disease, T. tubifex of lineages V and VI that are more resistant to M. cerebralis infections were more abundant. The predominance of resistant T. tubifex worms (lineages V and VI) at low-impact sites supports the conclusion that when these genotypes are in greater abundance, the potential for more severe effects of whirling disease on wild rainbow trout populations may be diminished.