Phylogenetic relationships in the family Alloherpesviridae.

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.

Revised phylogenetic relationships among herpesviruses isolated from sturgeons.

Initial phylogenetic comparisons based on a region of the DNA polymerase of seven herpes-like viruses found in sturgeons in North America and Europe indicated the presence of three distinct clades. A revised phylogenetic analysis of the same viruses, based on corrected DNA polymerase sequences and newly obtained sequence data from the putative ATP subunit of the terminase gene, indicate only two clades. These two clades correspond to the historical designations given to these herpes-like viruses from white sturgeon Acipenser transmontanus: white sturgeon herpesvirus type 1 (WSHV-1) and type 2 (WSHV-2). The identification of putative terminase gene sequences for all seven herpes-like viruses from sturgeons confirms their affinity with the family Herpesviridae (because this gene is unique to herpesviruses) and more distantly with T4-like bacteriophages. The two clades of sturgeon herpesviruses are therefore appropriately designated as Acipenserid herpesviruses 1 and 2, which correspond to the previous common names of white sturgeon herpesvirus types 1 and 2.

Genotypes and phylogeographical relationships of infectious hematopoietic necrosis virus in California, USA.

Infectious hematopoietic necrosis virus (IHNV) contains 3 major genogroups in North America with discreet geographic ranges designated as upper (U), middle (M), and lower (L). A comprehensive genotyping of 237 IHNV isolates from hatchery and wild salmonids in California revealed 25 different sequence types (a to y) all in the L genogroup; specifically, the genogroup contained 14 sequence types that were unique to individual isolates as well as 11 sequence types representing 2 or more identical isolates. The most evident trend was the phylogenetic and geographical division of the L genogroup into 2 distinct subgroups designated as LI and LII. Isolates within Subgroup LI were primarily found within waterways linked to southern Oregon and northern California coastal rivers. Isolates in Subgroup LII were concentrated within inland valley watersheds that included the Sacramento River, San Joaquin River, and their tributaries. The temporal and spatial patterns of virus occurrence suggested that infections among adult Chinook salmon in the hatchery or that spawn in the river are a major source of virus potentially infecting other migrating or resident salmonids in California. Serum neutralization results of the California isolates of IHNV corroborated a temporal trend of sequence divergence; specifically, 2 progressive shifts in which more recent virus isolates represent new serotypes. A comparison of the estimates of divergence rates for Subgroup LI (1 x 10(-5) mutations per nucleotide site per year) indicated stasis similar to that observed in the U genogroup, while the Subgroup LII rate (1 x 10(-3) mutations per nucleotide site per year) suggested a more active evolution similar to that of the M genogroup.

Genetic diversity of infectious hematopoietic necrosis virus from Feather River and Lake Oroville, California, and virulence of selected isolates for Chinook salmon and rainbow trout.

Infectious hematopoietic necrosis virus (IHNV) is a significant pathogen of young salmonid fishes worldwide but particularly within the historical range of the Pacific Northwest and California. In the Sacramento and San Joaquin River drainages of California, IHNV outbreaks in juvenile Chinook salmon Oncorhynchus tshawytscha have been observed regularly at large production hatcheries, including Coleman National Fish Hatchery (established in 1941) and Feather River State Fish Hatchery (FRH; established in 1967), since facility operations began. Recent severe epidemics at the FRH in 1998 and 2000-2002 prompted investigations into the characteristics and potential sources of virus at this facility. Both phylogenetic analyses of a variable portion of the glycoprotein gene and serologic comparisons based on neutralization with three polyclonal rabbit sera were used to characterize 82 IHNV isolates from the Feather River watershed between 1969 and 2004. All isolates examined were in the L genogroup and belonged to one of three serologic groups typical of IHNV from California. The IHNV isolates from the Feather River area demonstrated a maximum nucleotide sequence divergence of 4.0%, and new isolates appeared to emerge from previous isolates rather than by the introduction of more diverse subgroups from exogenous sources. The earliest isolates examined from the watershed formed the subgroup LI, which disappeared coincidently with a temporal shift to new genetic and serologic types of the larger subgroup LII. Experimental challenges demonstrated no significant differences in the virulence for juvenile Chinook salmon and rainbow trout O. mykiss from selected isolates representing the principal types of IHNV found historically and from recent epidemics at FRH. While most isolates were equally virulent for both host species, one isolate was found to be more virulent for Chinook salmon than for rainbow trout.

Prediction model for sequence variation in the glycoprotein gene of infectious hematopoietic necrosis virus in California, U.S.A.

The influence of spatio-temporal factors on genetic variation of infectious hematopoietic necrosis virus (IHNV) is an active area of research. Using host-isolate pairs collected from 1966 to 2004 for 237 IHNV isolates from California and southern Oregon, we examined genetic variation of the mid-G gene of IHNV that could be quantified across times and geographic locations. Information hypothesized to influence genetic variation was environmental and/or fish host demographic factors, viz. location (inland or coastal), year of isolation, habitat (river, lake, or hatchery), the agent factors of subgroup (LI or LII) and serotype (1, 2, or 3), and the host factors of fish age (juvenile or adult), sex (male or female), and season of spawning run (spring, fall, late fall, winter). Inverse distance weighting (IDW) was performed to create isopleth maps of the genetic distances of each subgroup. IDW maps showed that more genetic divergence was predicted for isolates found inland (for both subgroups: LI and LII) than for coastal watershed isolates. A mixed-effect beta regression with a logit link function was used to seek associations between genetic distances and hypothesized explanatory factors. The model that best described genetic distance contained the factors of location, year of isolation, and the interaction between location and year. Our model suggests that genetic distance was greater for isolates collected from 1966 to 2004 at inland locations than for isolates found in coastal watersheds during the same years. The agreement between the IDW and beta regression analyses quantitatively supports our conclusion that, during this time period, more genetic variation existed within subgroup LII in inland watersheds than within coastal LI isolates.

Evaluation of quantitative real-time PCR for rapid assessments of the exposure of sentinel fish to Myxobolus cerebralis.

Pathogen-free rainbow trout (Oncorhynchus mykiss) aged 735 degree days were experimentally exposed to a low dose of infectious Myxobolus cerebralis (20 triactinomyxons fish(-1)). Three time periods were chosen for sampling that included 10 days (d), 67 d, and 5 months (mo) post exposure. Five diagnostic assays were used: (1) conventional single-round polymerase chain reaction (PCR), (2) nested PCR, (3) real-time TaqMan PCR, (4) pepsin-trypsin digest, and (5) histopathology. M. cerebralis was detected among individual rainbow trout by all of the PCR diagnostic tests employed at each of the three sampling time points. This result demonstrates that any of these three diagnostic approaches are capable of detecting the parasite from infected fish tissues under the conditions tested. Real-time PCR provided good biological evidence that parasite replication increases temporally as shown by quantification values that were significantly different (P<0.0001) at 10 d as compared to 67 d and 5 mo postexposure. Although sampling at 10 d by real-time PCR may be too early to accurately predict quantities of the parasite that will be present at 5 mo, it does forecast the proportions of fish that are likely to be infected at 67 d and 5 mo postparasite exposure. Real-time PCR could potentially be used as a quantitative diagnostic PCR tool to predict parasite load and outcome of M. cerebralis infection.

Koi herpesvirus represents a third cyprinid herpesvirus (CyHV-3) in the family Herpesviridae.

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).

The severity of whirling disease among wild trout corresponds to the differences in genetic composition of Tubifex tubifex populations in central Colorado.

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.

Identification of a serine protease gene expressed by Myxobolus cerebralis during development in rainbow trout Oncorhynchus mykiss.

Serine proteases have been recognized as key factors in parasite physiology and disease development. We have identified a serine protease gene from Myxobolus cerebralis, MyxSP-1, the myxozoan parasite causing whirling disease in salmonid fishes. The amino acid sequence, as deduced from the cDNA sequence, included a catalytic residue arrangement similar to that of the chymotrypsin family of serine proteases. A real-time TaqMan polymerase chain reaction (PCR) analysis revealed differences in the transcription levels for the chymotrypsin-like protease as found in early, intermediate, and late developmental stages of the parasite in experimentally-infected rainbow trout Oncorhynchus mykiss. MyxSP-1 transcription differed between individual tissues at each sampling point and in the same tissues over time (p < 0.0001). A nonradioactive mRNA in situ hybridization (ISH) protocol was developed to detect MyxSP-1 transcripts. Using a mixture of 3 digoxigenin-labeled antisense mRNA probes, MyxSP-1 transcription was observed in developmental stages of the parasite during the acute and chronic phases of the disease over a 240 d time period in infected rainbow trout tissues. MyxSP-1 transcription observed by ISH in cartilage and as associated with cartilage destruction was consistent with our real-time TaqMan PCR findings that demonstrated high levels of MyxSP-1 transcription during lesion development. Identifying genes encoding these enzymes and characterization of their functions can lead to the development of new chemotherapeutic protocols and vaccine approaches to control parasitic diseases.

Evaluation of five diagnostic methods for the detection and quantification of Myxobolus cerebralis.

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.

Phylogenetic comparison of the myxosporea based on an actin cDNA isolated from Myxobolus cerebralis.

The full-length actin gene from Myxobolus cerebralis (McerAct-1), the first characterized from representatives in the phylum Myxozoa, encodes a 378-amino acid polypeptide with an estimated molecular weight of 41,580-Da. A phylogenetic comparison found M. cerebralis to branch outside the metazoans. This finding contrasts with previous reports that suggest an evolutionary affinity of the Myxozoa with either the Bilateria or Cnidaria.

Myxobolus cerebralis: identification of a cathepsin Z-like protease gene (MyxCP-1) expressed during parasite development in rainbow trout, Oncorhynchus mykiss.

Cysteine proteases are essential to the biological function of parasitic organisms and are gaining interest as the target of chemotherapeutics aimed at their control. We report a cysteine protease gene from the myxozoan Myxobolus cerebralis, the cause of whirling disease in salmonid fish. The mature gene (MyxCP-1) encodes a 248-amino acid polypeptide that includes catalytic residues similar to the papain family of cysteine proteases. MyxCP-1 features a propeptide region and sequence insertions that are characteristics of cathepsin Z proteases. Phylogenetic comparisons of M. cerebralis to other eukaryotes based on full-length cathepsin-like genes show that MyxCP-1 is the earliest lineage in the cathepsin Z group and separated from cathepsin L, B, and C-like proteases. Using TaqMan PCR differential levels of transcription of the cathepsin Z-like protease were found in earlier and later developmental stages of the parasite in experimentally infected rainbow trout (Oncorhynchus mykiss).

Prevalence and susceptibility of infection to Myxobolus cerebralis, and genetic differences among populations of Tubifex tubifex.

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.