Arnon Dishon

Description of an as Yet Unclassified DNA Virus from Diseased Cyprinus carpio Species

ABSTRACT Numerous deaths of koi and common carp (Cyprinus carpio) were observed on many farms throughout Israel, resulting in severe financial losses. The lethal viral disease observed is highly contagious and extremely virulent, but morbidity and mortality are restricted to koi and common carp populations. Diseased fish exhibit fatigue and gasping movements in shallow water. Infected fish had interstitial nephritis and gill necrosis as well as petechial hemorrhages in the liver and other symptoms that were not consistent with viral disease, suggesting a secondary infection. Here we report the isolation of carp nephritis and gill necrosis virus (CNGV), which is the etiologic agent of this disease. The virus propagates and induces severe cytopathic effects by 5 days postinfection in fresh koi or carp fin cell cultures (KFC and CFC, respectively), but not in epithelioma papillosum cyprini cells. The virus harvested from KFC cultures induced the same clinical signs, with a mortality of 75 to 95%, upon inoculation into naive koi and common carp. Using PCR, we provide final proof that the isolated virus is indeed the etiologic agent of food and ornamental carp mortalities in fish husbandry. Electron microscopy revealed viral cores with icosahedral morphology of 100 to 110 nm that resembled herpesviruses. Electron micrographs of purified pelleted CNGV sections, together with viral sensitivities to ether and Triton X-100, suggested that it is an enveloped virus. However, the genome of the isolated virus is a double-stranded DNA (dsDNA) molecule of 270 to 290 kbp, which is larger than known herpesviruses. The viral DNA seems highly divergent and bears only small fragments (16 to 45 bp) that are similar to the genomes of several DNA viruses. Nevertheless, amino acid sequences encoded by CNGV DNA fragments bear similarities primarily to members of the Poxviridae and Herpesviridae and to other large dsDNA viruses. We suggest, therefore, that the etiologic agent of this disease may represent an as yet unclassified virus species that is endemic in C. carpio (carp).

Detection of Carp Interstitial Nephritis and Gill Necrosis Virus in Fish Droppings

ABSTRACT Carp interstitial nephritis and gill necrosis virus (CNGV) is an unclassified large DNA virus that morphologically resembles members of the Herpesviridae but contains a large (ca. ∼280-kbp) linear double-stranded DNA. This virus has also been named koi herpesvirus, koi herpes-like virus, and cyprinid herpesvirus 3. CNGV is the cause of a lethal disease that afflicts common carp and koi. By using immunohistochemistry, molecular analysis, and electron microscopy we previously demonstrated that this virus is present mainly in the intestine and kidney of infected fish. Based on these observations, we postulated that viruses and/or viral components may appear in droppings of infected carp. Here we report that (i) by using PCR we demonstrated that fish droppings contain viral DNA, (ii) fish droppings contain viral antigens which are useful for CNGV diagnosis, and (iii) fish droppings contain active virus which can infect cultured common carp brain cells and induce the disease in naïve fish following inoculation. Thus, our findings show that CNGV can be identified by using droppings without taking biopsies or killing fish and that infectious CNGV is present in the stools of sick fish. The possibility that fish droppings preserve viable CNGV during the nonpermissive seasons is discussed.

Herpesviruses that Infect Fish

Herpesviruses are host specific pathogens that are widespread among vertebrates. Genome sequence data demonstrate that most herpesviruses of fish and amphibians are grouped together (family Alloherpesviridae) and are distantly related to herpesviruses of reptiles, birds and mammals (family Herpesviridae). Yet, many of the biological processes of members of the order Herpesvirales are similar. Among the conserved characteristics are the virion structure, replication process, the ability to establish long term latency and the manipulation of the host immune response. Many of the similar processes may be due to convergent evolution. This overview of identified herpesviruses of fish discusses the diseases that alloherpesviruses cause, the biology of these viruses and the host-pathogen interactions. Much of our knowledge on the biology of Alloherpesvirdae is derived from research with two species: Ictalurid herpesvirus 1 (channel catfish virus) and Cyprinid herpesvirus 3 (koi herpesvirus).

Persistence of cyprinid herpesvirus 3 in infected cultured carp cells.

ABSTRACT Cyprinid herpesvirus 3 (CyHV-3), previously designated carp interstitial nephritis and gill necrosis virus or koi herpesvirus, is the cause of a worldwide mortal disease of koi and carp. Morphologically, the virus resembles herpesviruses, yet it bears a genome of 277 to 295 kbp, which is divergent from most of the genomic sequences available in GenBank. The disease afflicts fish in the transient seasons, when the water temperature is 18 to 28°C, conditions which permit virus propagation in cultured cells. Here we report that infectious virus is preserved in cultured cells maintained for 30 days at 30°C. CyHV-3-infected vacuolated cells with deformed morphology converted to normal, and plaques disappeared following shifting up of the temperature and reappeared after transfer to the permissive temperature. Viral propagation and viral gene transcription were turned off by shifting cells to the nonpermissive temperature. Upon return of the cells to the permissive temperature, transcription of viral genes was reactivated in a sequence distinguished from that occurring in naïve cells following infection. Our results show that CyHV-3 persists in cultured cells maintained at the nonpermissive temperature and suggest that viruses could persist for long periods in the fish body, enabling a new burst of infection upon a shift to a permissive temperature.

Characterization of a Novel Virus Causing a Lethal Disease in Carp and Koi

SUMMARY Since 1998 a lethal disease of carp and ornamental koi (Cyprinus carpio) has afflicted fisheries in North America, Europe, and Asia, causing severe economic losses to the fish farming industry. This review summarizes the isolation and identification of the disease-causing agent and describes the currently known molecular characteristics of this newly isolated virus, distinguishing it from other known large DNA viruses. In addition, we summarize the clinical and histopathological manifestations of the disease. Providing information on the immune response to this virus and evaluating the available means of diagnosis and protection should help to reduce the damage induced by this disease. This review does not discuss the economic aspects of the disease or the debate on whether the disease should be registered; both of these issues were recently reviewed in detail (O. L. M. Haenen, K. Way, S. M. Bergmann, and E. Ariel, Bull. Eur. Assoc. Fish Pathol. 24:293-307, 2004; D. Pokorova, T. Vesely, V. Piackova, S. Reschova, and J. Hulova, Vet. Med. Czech. 50:139-147, 2005).

The outbreak of carp disease caused by CyHV-3 as a model for new emerging viral diseases in aquaculture: a review

Aquacultured koi and common carp (Cyprinus carpio) are intensively bred for ornamental purposes and for human consumption worldwide. The carp and koi farming industries have suffered enormous economic losses over the past decade due to an epizootic disease caused by Cyprinus herpesvirus-3 (CyHV-3) also known as koi herpesvirus and carp interstitial nephritis gill necrosis virus. CyHV-3 is a large dsDNA virus, morphologically similar to herpesviruses, yet contains genetic elements similar to those of pox, irido- and herpesviruses. Considering the phylogenic distance between CyHV-3 and higher vertebrate herpesviruses, CyHV-3 represents the prototype of viruses assigned to the novel family Alloherpesviridae. Although emergence of a new virus rarely initiates a pandemic so severe that it reduces the life expectancy of a population, CyHV-3 is exceptional because of its enormous impact on the world carp population. High population density is the major contributing factor to the epizootic disease caused by CyHV-3.

Coordinated and sequential transcription of the cyprinid herpesvirus-3 annotated genes

Cyprinid herpesvirus-3 (CyHV-3) is the cause of a fatal disease in carp and koi fish. The disease is seasonal and appears when water temperatures range from 18 to 28°C. CyHV-3 is a member of the Alloherpesviridae, a family in the Herpesvirales order that encompasses mammalian, avian and reptilian viruses. CyHV-3 is a large double-stranded DNA (dsDNA) herpesvirus with a genome of approximately 295kbp, divergent from other mammalian, avian and reptilian herpesviruses, but bearing several genes similar to cyprinid herpesvirus-1 (CyHV-1), CyHV-2, anguillid herpesvirus-1 (AngHV-1), ictalurid herpesvirus-1 (IcHV-1) and ranid herpes virus-1 (RaHV-1). Here we show that viral DNA synthesis commences 4-8h post-infection (p.i.), and is completely inhibited by pre-treatment with cytosine β-d-arabinofuranoside (Ara-C). Transcription of CyHV-3 genes initiates after infection as early as 1-2h p.i., and precedes viral DNA synthesis. All 156 annotated open reading frames (ORFs) of the CyHV-3 genome are transcribed into RNAs, most of which can be classified into immediate early (IE or α), early (E or β) and late (L or γ) classes, similar to all other herpesviruses. Several ORFs belonging to these groups are clustered along the viral genome.

Susceptibility of cyprinid cultured cells to cyprinid herpesvirus 3

SummaryCyprinid herpesvirus 3 is a highly contagious and lethal virus that affects ornamental koi and common carp worldwide. However, it is not yet known whether other cyprinids are infected and/or harbor the virus. Here, we report that cultured cells derived from common carp, koi, silver carp and goldfish allow CyHV-3 propagation, while cyprinid cells derived from fathead minnow and non-cyprinid cells derived from the channel catfish ovary are resistant to CyHV-3 infection. Interestingly, the epithelioma papulosum Cyprini cells derived from Cyprinus carpio are restrictive to the virus. These results indicate that CyHV-3 is not restricted to common carp and koi, but other cyprinids are also vulnerable to the virus.

Cyprinid herpes virus-3 (CyHV-3) bears genes of genetically distant large DNA viruses

A large DNA virus, designated koi herpes virus (KHV), carp interstitial nephritis gill necrosis virus (CNGV) and Cyprinid herpes virus‐3 (CyHV‐3), causes massive mortality of carp. Morphologically, the virus resembles herpes viruses, but it contains a genome of ca 295 kbp, larger than that of any Herpesviridae member. Interestingly, three CyHV‐3 genes, thymidylate monophosphate kinase (TmpK), ribonucleotide reductase and thymidine kinase, which are involved in deoxynucleotide tri‐phosphate synthesis, resemble those of pox viruses. In addition to the TmpK gene, which is nonexistent in the genome of herpes viruses, CyHV‐3 contains a B22R‐like gene, exclusively expressed by pox viruses. These results raise questions on the phylogenic origin of CyHV‐3.

Down-regulation of the cyprinid herpesvirus-3 annotated genes in cultured cells maintained at restrictive high temperature

Cyprinid herpesvirus-3 (CyHV-3) is a member of the Alloherpesviridae, in the order Herpesvirales. It causes a fatal disease in carp and koi fish. The disease is seasonal and is active when water temperatures ranges from 18 to 28 °C. Little is known about how and where the virus is preserved between the permissive seasons. The hallmark of the herpesviruses is their ability to become latent, persisting in the host in an apparently inactive state for varying periods of time. Hence, it could be expected that CyHV-3 enter a latent period. CyHV-3 has so far been shown to persist in fish maintained under restrictive temperatures, while shifting the fish to permissive conditions reactivates the virus. Previously, we demonstrated that cultured cells infected with CyHV-3 at 22 °C and subsequently transferred to a restrictive temperature of 30 °C preserve the virus for 30 days. The present report shows that cultured carp cells maintained and exposed to CyHV-3 at 30 °C are abortively infected; that is, autonomous viral DNA synthesis is hampered and the viral genome is not multiplied. Under these conditions, 91 of the 156 viral annotated ORFs were initially transcribed. These transcripts were down-regulated and gradually shut off over 18 days post-infection, while two viral transcripts encoded by ORFs 114 and 115 were preserved in the infected cells for 18 days p.i. These experiments, carried out in cultured cells, suggest that fish could be infected at a high non-permissive temperature and harbor the viral genome without producing viral particles.