A herpesvirus was isolated from adult koi, a strain of common carp Cyprinus carpio, suffering mass mortality in two outbreaks-one in the mid-Atlantic region of the United States and the second in Israel. The principal external signs of dying fish were pale and irregularly colored gills. There were few consistent internal signs in either outbreak. The most prominent microscopic lesions were in the gills, where hyperplasia and necrosis of the epithelium were severe. Other lesions included interstitial nephritis, splenitis, and enteritis. Affected cells often contained nuclei with marginated chromatin and faint intranuclear inclusions. Typical herpesvirus particles were present in branchial epithelial cells, hepatocytes, and among circulating leukocytes. Inoculations of the koi fin (KF-1) cell line with tissue extracts from the gill and kidney-spleen resulted in cytopathic effects characterized by severe vacuolation first detected after 7 d incubation at 20°C. Exposures of adult koi to the herpesvirus as propagated in KF-1 cells by bath or intraperitoneal injections resulted in 80-100% mortality during a 26-d period, and the virus was reisolated from the gill, kidney, liver, spleen, intestine, and brain of dead fish. The viral agents from koi in Israel and the United States appear to be similar if not identical; both could be distinguished from Herpesvirus cyprini by indirect fluorescent antibody tests with rabbit anti-H. cyprini serum. Other factors should be examined but we strongly suspect that this newly recognized koi herpesvirus (KHV) has the potential to be a significant cause of mortality among koi and presumably common carp.

The Koi herpesvirus (KHV) is a herpes-like virus now recognized as a worldwide cause of mortality among populations of koi Cyprinus carpio koi and common carp Cyprinus carpio carpio.Temperature is a key factor influencing virus replication both in cell culture and in the tissues of experimentally infected fish. Genomic DNA sequences were used to optimize a rapid real-time TaqMan PCR assay to detect and quantify KHV DNA as found in the tissues of virus-exposed fish. The assay allowed analytical enumeration of target KHV genome copies ranging from 10 1 to 10 7 molecules as present in infected cell lines or fish tissues. The new assay was specific for KHV and did not detect DNA from 3 related herpes-like viruses found in fish, the Cyprinid herpesvirus 1 (CyHV-1), Cyprinid herpesvirus 2 (CyHV-2), Ictalurid herpesvirus 1 (IcHV-1) or the KF-1 cell line used for virus growth. Concentrations of KHV DNA were evaluated in 7 different tissues of replicate groups of virus-exposed koi held at water temperatures of 13, 18, 23 and 28°C. Viral DNA was detected among virus-exposed koi at all 4 water temperatures but mortality was only observed among fish at 18, 23, and 28°C. Time and temperature and the interactions between them affected concentrations of viral DNA detected in tissues of koi exposed to KHV. Although there were no recognized patterns to viral DNA concentrations as found in different tissues over time, KHV genome copies for all tissues increased with time post virus exposure and with water temperature. The remarkably rapid and systemic spread of the virus was demonstrated by the presence of viral DNA in multiple tissues 1 d post virus exposure. The greatest DNA concentrations found were in the gill, kidney and spleen,

In the last few years two factors have helped to significantly advance our understanding of the Myxozoa. First, the phenomenal increase in fin fish aquaculture in the 1990s has lead to the increased importance of these parasites; in turn this has lead to intensified research efforts, which have increased knowledge of the development, diagnosis. and pathogenesis of myxozoans. The hallmark discovery in the 1980s that the life cycle of Myxobolus cerebralis requires development of an actinosporean stage in the oligochaete. Tubifex tubifex, led to the elucidation of the life cycles of several other myxozoans. Also, the life cycle and taxonomy of the enigmatic PKX myxozoan has been resolved: it is the alternate stage of the unusual myxozoan, Tetracapsula bryosalmonae, from bryozoans. The 18S rDNA gene of many species has been sequenced, and here we add 22 new sequences to the data set. Phylogenetic analyses using all these sequences indicate that: 1) the Myxozoa are closely related to Cnidaria (also supported by morphological data); 2) marine taxa at the genus level branch separately from genera that usually infect freshwater fishes; 3) taxa cluster more by development and tissue location than by spore morphology; 4) the tetracapsulids branched off early in myxozoan evolution, perhaps reflected by their having bryozoan, rather than annelid hosts; 5) the morphology of actinosporeans offers little information for determining their myxosporean counterparts (assuming that they exist); and 6) the marine actinosporeans from Australia appear to form a clade within the platysporinid myxosporeans. Ribosomal DNA sequences have also enabled development of diagnostic tests for myxozoans. PCR and in situ hybridisation tests based on rDNA sequences have been developed for Myxobolus cerebralis, Ceratomyxa shasta, Kudoa spp., and Tetracapsula bryosalmonae (PKX). Lectin-based and antibody tests have also been developed for certain myxozoans, such as PKX and C. shasta. We also review important diseases caused by myxozoans, which are emerging or re-emerging. Epizootics of whirling disease in wild rainbow trout (Oncorhynchus mykiss) have recently been reported throughout the Rocky Mountain states of the USA. With a dramatic increase in aquaculture of fishes using marine netpens, several marine myxozoans have been recognized or elevated in status as pathological agents. Kudoa thyrsites infections have caused severe post-harvest myoliquefaction in pen-reared Atlantic salmon (Salmo salar), and Ceratomyxa spp., Sphaerospora spp., and Myxidium leei cause disease in pen-reared sea bass (Dicentrarchus labrax) and sea bream species (family Sparidae) in Mediterranean countries.

Within the past decade, iridoviruses have been identified as the causative agents of systemic disease in a variety of commercially and recreationally important fish. Here we examine nine iridoviruses from fish, reptiles, and amphibians and demonstrate that all isolates were more similar to frog virus 3, the type species of the genus Ranavirus, than to lymphocystis disease virus, the type species of the genus Lymphocystivirus. Comparison of viral protein synthesis profiles, restriction endonuclease digestion patterns, and the amino acid sequence of the major capsid protein indicated that iridoviruses isolated from the same geographic region were similar, if not identical, whereas viruses from different areas were distinct. Moreover, using primers complementary to the conserved major capsid protein, we found that both PCR and RT-PCR successfully amplified virus-specific nucleic acid from all nine isolates. These studies demonstrate that the piscine iridoviruses examined here were members of the genus Ranavirus, and suggest that surveys of pathogenic "fish viruses" may need to include neighboring amphibian and reptilian populations. In addition, the results indicate that PCR readily identified vertebrate iridoviruses and suggest that PCR will be useful in the diagnosis of fish disease.

Since the mid-1990s, lethal infections of koi herpesvirus (KHV) have been spreading, threatening the worldwide production of common carp and koi (both Cyprinus carpio). The complete genome sequences of three KHV strains from Japan, the United States, and Israel revealed a 295-kbp genome containing a 22-kbp terminal direct repeat. The finding that 15 KHV genes have clear homologs in the distantly related channel catfish virus (ictalurid herpesvirus 1) confirms the proposed place of KHV in the family Herpesviridae, specifically in the branch with fish and amphibian hosts. KHV thus has the largest genome reported to date for this family. The three strains were interpreted as having arisen from a wild-type parent encoding 156 unique protein-coding genes, 8 of which are duplicated in the terminal repeat. In each strain, four to seven genes from among a set of nine are fragmented by frameshifts likely to render the encoded proteins nonfunctional. Six of the affected genes encode predicted membrane glycoproteins. Frameshifts or other mutations close to the 3 ends of coding sequences were identified in a further six genes. The conclusion that at least some of these mutations occurred in vivo prompts the hypothesis that loss of gene functions might be associated with emergence of the disease and provides a basis for further investigations into the molecular epidemiology of the virus.