Discovery of an unrecognized nidovirus associated with granulomatous hepatitis in rainbow trout.

Rainbow trout (Oncorhynchus mykiss) is the principal species of inland-farmed fish in the Western hemisphere. Recently, we diagnosed in farmed rainbow trout a disease in which the hallmark is granulomatous-like hepatitis. No biotic agents could be isolated from lesions. Still, unbiased high-throughput sequencing and bioinformatics analyses revealed the presence of a novel piscine nidovirus that we named "Trout Granulomatous Virus" (TGV). TGV genome (28,767 nucleotides long) is predicted to encode non-structural (1a and 1 ab) and structural (S, M, and N) proteins that resemble proteins of other known piscine nidoviruses. High loads of TGV transcripts were detected by quantitative RT-PCR in diseased fish and visualized in hepatic granulomatous sites by fluorescence in situ hybridization. Transmission electron microscopy (TEM) revealed coronavirus-like particles in these lesions. Together, these analyses corroborated the association of TGV with the lesions. The identification and detection of TGV provide means to control TGV spread in trout populations.

Inferring Protein Function in an Emerging Virus: Detection of the Nucleoprotein in Tilapia Lake Virus.

Emerging viruses impose global threats to animal and human populations and may bear novel genes with limited homology to known sequences, necessitating the development of novel approaches to infer and test protein functions. This challenge is dramatically evident in tilapia lake virus (TiLV), an emerging "orthomyxo-like" virus that threatens the global tilapia aquaculture and food security of millions of people. The majority of TiLV proteins have no homology to known sequences, impeding functionality assessments. Using a novel bioinformatics approach, we predicted that TiLV's Protein 4 encodes the nucleoprotein, a factor essential for viral RNA replication. Multiple methodologies revealed the expected properties of orthomyxoviral nucleoproteins. A modified yeast three-hybrid assay detected Protein 4-RNA interactions, which were independent of the RNA sequence, and identified specific positively charged residues involved. Protein 4-RNA interactions were uncovered by R-DeeP and XRNAX methodologies. Immunoelectron microscopy found that multiple Protein 4 copies localized along enriched ribonucleoproteins. TiLV RNA from cells and virions coimmunoprecipitated with Protein 4. Immunofluorescence microscopy detected Protein 4 in the cytoplasm and nuclei, and nuclear Protein 4 increased upon CRM1 inhibition, suggesting CRM1-dependent nuclear export of TiLV RNA. Together, these data reveal TiLV's nucleoprotein and highlight the ability to infer protein functionality, including novel RNA-binding proteins, in emerging pathogens. These are important in light of the expected discovery of many unknown viruses and the zoonotic potential of such pathogens. IMPORTANCE Tilapia is an important source of dietary protein, especially in developing countries. Massive losses of tilapia were identified worldwide, risking the food security of millions of people. Tilapia lake virus (TiLV) is an emerging pathogen responsible for these disease outbreaks. TiLV's genome encodes 10 major proteins, 9 of which show no homology to other known viral or cellular proteins, hindering functionality assessment of these proteins. Here, we describe a novel bioinformatics approach to infer the functionality of TiLV proteins, which predicted Protein 4 as the nucleoprotein, a factor essential for viral RNA replication. We provided experimental support for this prediction by applying multiple molecular, biochemical, and imaging approaches. Overall, we illustrate a strategy for functional analyses in viral discovery. The strategy is important in light of the expected discovery of many unknown viruses and the zoonotic potential of such pathogens.

Mapping of Tilapia Lake Virus entry pathways with inhibitors reveals dependence on dynamin activity and cholesterol but not endosomal acidification.

Tilapia Lake Virus (TiLV) is an emerging virus lethal to tilapia, which threatens the global tilapia aquaculture with severe implications for food security. TiLV possesses similar features to orthomyxoviruses but is classified in the sole and the monotypic genus Tilapinevirus of the family Amnoonviridae. TiLV enveloped virions encapsidate a genome comprising ten segments of single-stranded, negative RNA. Remarkably, nine of TiLV's ten major proteins lack sequence homology to any known viral or cellular proteins. The mode of TiLV entry into tilapia cells is not known. Following the measurement of the entry window of TiLV (∼3 h), we applied a panel of inhibitors of known regulators of endocytic functions to map the molecular requirements for TiLV entry. We identified productive entry by quantification of TiLV nucleoprotein expression and the generation of infectious particles. Inhibition of dynamin activity with dynasore or dynole, or depletion of cholesterol with methyl-ß-cyclodextrin, strongly inhibited TiLV protein synthesis and infectious virion production. Moreover, inhibition of actin cytoskeleton polymerization with latrunculin A or microtubule polymerization with nocodazole within the entry window resulted in partial inhibition of TiLV infection. In contrast, inhibitors of endosomal acidification (NH4Cl, bafilomycin A1, or chloroquine), an inhibitor of clathrin-coated pit assembly (pitstop 2), and erlotinib-an inhibitor of the endocytic Cyclin G-associated kinase (GAK), did not affect TiLV entry. Altogether, these results suggest that TiLV enters via dynamin-mediated endocytosis in a cholesterol-, cytoskeleton-dependent manner, and clathrin-, pH-independent manner. Thus, despite being an orthomyxo-like virus, when compared to the prototypical orthomyxovirus (influenza A virus), TiLV shows a distinct set of requirements for entry into cells.

Identification and Genetic Characterization of Viral Pathogens in Ruminant Gestation Abnormalities, Israel, 2015-2019.

Infectious agents including viruses are important abortifacients and can cause fetal abnormalities in livestock animals. Here, samples that had been collected in Israel from aborted or malformed ruminant fetuses between 2015 and 2019 were investigated for the presence of the following viruses: the reoviruses bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), the flaviviruses bovine viral diarrhea virus (BVDV) and border disease virus (BDV), the peribunyaviruses Shuni virus (SHUV) and Akabane virus (AKAV), bovine herpesvirus type 1 (BoHV-1) and bovine ephemeral fever virus (BEFV). Domestic (cattle, sheep, goat) and wild/zoo ruminants were included in the study. The presence of viral nucleic acid or antigen could be confirmed in 21.8 % of abnormal pregnancies (213 out of 976 investigated cases), with peribunyaviruses, reoviruses and pestiviruses being the most prevalent. At least four different BTV serotypes were involved in abnormal courses of pregnancy in Israel. The subtyping of pestiviruses revealed the presence of two BDV and several distinct BVDV type 1 strains. The peribunyaviruses AKAV and SHUV were identified annually throughout the study period, however, variation in the extent of virus circulation could be observed between the years. In 2018, AKAV even represented the most detected pathogen in cases of small domestic ruminant gestation abnormalities. In conclusion, it was shown that various viruses are involved in abnormal courses of pregnancy in ruminants in Israel.

Genomic Analysis Illustrated a Single Introduction and Evolution of Israeli Bluetongue Serotype 8 Virus Population 2008-2019.

Outbreaks of the European Bluetongue virus (BTV) serotype 8 (BTV-8), which are characterized by activity cycles separated by years of inactivity, may be influenced by genetic changes of the virus or by herd immunity. BTV activity in Israel is characterized by similar dynamics, but differs from European countries in its vector population, environmental conditions, and lack of cattle vaccination against this serotype. Comparison of these two geographical systems and characterization of their epidemiological connection is therefore of high interest in-order to better understand the factors influencing BTV-8 evolution. BTV-8, closely related to the European strain, was introduced to Israel in 2008. It was at the center of BT outbreaks in 2010 and 2015-2016 and thereafter was lastly isolated in Israel in 2019. We performed genetic analyses of twelve BTV-8 Israeli strains isolated between 2008 and 2019 and compared them with published sequences of BTV-8 isolated in other countries. The analysis revealed a single introduction of BTV-8 into Israel and thereafter extensive occurrence of genomic drifts and multiple reassortments with local BTV strains. Comparison of the Israeli and Cypriot BTV-8 from 2015 to 2016 suggests transmission of the virus between the two countries and a separate and parallel development from European or other Israeli BTV-8 strains. The parallel development of other BTV-8 strains was demonstrated by the identification of the Israeli BTV-8 ISR-1194/1/19 strain, which exhibited common origin with reassorted Israeli BTV-8 strains from 2010 and additional reassortment of seven segments. In order to reveal the source of BTV-8 introduction into Israel we performed BEAST analysis which showed that a probable common ancestor for both European and Israeli BTV-8 presumably existed in 2003-2004. In 2019, a possible new introduction occurred in Israel, where a novel BTV-8 strain was detected, sharing ~95% identity by segments 2 and 6 with Nigerian BTV-8NIG1982/07 and European-Middle Eastern strains. The results of the study indicate that Israel and neighboring countries consist a separate environmental and evolutionary system, distinct from European ones.

Bluetongue Serotype 3 in Israel 2013-2018: Clinical Manifestations of the Disease and Molecular Characterization of Israeli Strains.

In this paper, the results of the diagnostic activities on Bluetongue virus serotype 3 (BTV-3) conducted at Kimron Veterinary Institute (Beit Dagan, Israel) between 2013 and 2018 are reported. Bluetongue virus is the causative agent of bluetongue (BT), a disease of ruminants, mostly transmitted by competent Culicoides species. In Israel, BTV-3 circulation was first detected in 2013 from a sheep showing classical BT clinical signs. It was also evidenced in 2016, and, since then, it has been regularly detected in Israeli livestock. Between 2013 and 2017, BTV-3 outbreaks were limited in sheep flocks located in the southern area only. In 2018, BTV-3 was instead found in the Israeli coastal area being one of the dominant BTV serotypes isolated from symptomatic sheep, cattle and goats. In Israeli sheep, BTV-3 was able to cause BT classical clinical manifestations and fatalities, while in cattle and goats infection ranged from asymptomatic forms to death cases, depending on either general welfare of the herds or on the occurrence of viral and bacterial co-infections. Three different BTV-3 strains were identified in Israel between 2013 and 2018: ISR-2019/13 isolated in 2013, ISR-2153/16 and ISR-2262/2/16 isolated in 2016. Sequencing and phylogenetic analysis of these strains showed more than 99% identity by segment (Seg) 2, 5, 6, 7, and 8 sequences. In contrast, a wide range of diversity among these strains was exhibited in other viral gene segments, implying the occurrence of genome reassortment between these local circulating strains and those originating from Africa. The genome sequences of the BTV-3 isolated in 2017 and 2018 were most closely related to those of the ISR-2153/16 strain suggesting their common ancestor. Comparison of BTV-3 Israeli strains with those recently detected in the Mediterranean region uncovered high percentage identity (98.19-98.28%) only between Seg-2 of all Israeli strains and the BTV-3 Zarzis/TUN2016 strain. A 98.93% identity was also observed between Seg-4 sequences of ISR-2019/13 and the BTV-3 Zarzis/TUN2016 strain. This study demonstrated that BTV-3 has been circulating in the Mediterranean region at least since 2013, but, unlike the other Mediterranean strains, Israeli BTV-3 were able to cause clinical signs also in cattle.

Emergence of a Novel Reassortant Strain of Bluetongue Serotype 6 in Israel, 2017: Clinical Manifestations of the Disease and Molecular Characterization.

Reassortment contributes to the evolution of RNA viruses with segmented genomes, including Bluetongue virus (BTV). Recently, co-circulation of natural and vaccine BTV variants in Europe, and their ensuing reassortment, were proposed to promote appearance of novel European BTV strains, with potential implications for pathogenicity, spread and vaccination policies. Similarly, the geographical features of the Mediterranean basin, which spans over portions of three continents, may facilitate the appearance of clinically relevant reassortants via co-circulation of BTV strains of African, Asian and European origins. In August-October 2017, BTV serotype 6 (BTV-6) was identified in young animals exhibiting classical clinical signs of Bluetongue (BT) at Israeli sheep and cattle farms. Sequencing and pairwise analysis of this Israeli BTV-6 isolate revealed the closest sequence homology of its serotype-defining Segment 2 was with that of South African reference BTV-6 strain 5011 (93.88% identity). In contrast, the other viral segments showed highest homology (97.0%-99.47% identity) with BTV-3, -4 and -9 of Mediterranean and African origins. Specifically, four viral segments were nearly identical (99.13%-99.47%), with Tunisian and Italian BTV-3 strains (TUN2016 and SAD2018, correspondingly). Together, our data suggest that Mediterranean co-circulation and reassortment of BTV-3 and BTV-6 drove the emergence of a novel and virulent BTV-6 strain.

Pathological and molecular characterisation of peste des petits ruminants in Nubian ibex (Capra nubiana) in Israel.

Peste des petits ruminants (PPR) is a devastating disease that generally affects sheep and goats, mostly in Asia, the Middle East and Africa. The disease has been declared a target for global eradication. Despite its high prevalence in domestic flocks and its high seroprevalence among wildlife, it is rarely reported as a fulminant disease in wild ruminant species (with the exception of Central Asia). In this report, we describe a severe PPR outbreak in a zoo herd of Nubian ibex (Capra nubiana), causing the deaths of 2/3 of the herd. The clinical onset was acute with morbid animals exhibiting lethargy and watery-to-bloody diarrhea and death usually within 48 h. The most consistent gross pathologic findings were hemorrhagic abomasitis and enteritis. Oral lesions and pulmonary lesions were rare. Histology revealed necrohemorrhagic enteritis and abomasitis with myriad nuclear and cytoplasmic viral inclusion bodies. Molecular examinations confirmed the diagnosis of PPR and determined that the causative agent belongs to lineage IV. Further molecular examination showed that the virus belongs to the Asian clade of lineage IV and is closely related to a virus described in Turkey.

Shuni virus in Israel: Neurological disease and fatalities in cattle.

The insect-transmitted Shuni virus (SHUV) belongs to the Simbu serogroup of orthobunyaviruses and it is known to induce abortions, stillbirths and severe congenital malformations in ruminants and may cause neurological signs in infected horses. Here, SHUV was detected in brain samples of two Israeli cattle, which suffered from severe neurological signs that led to the deaths of the animals. During histopathological examination of the first case, a 5-month-old calf, small perivascular cuffs, composed mainly of neutrophils with few lymphocytes were observed in the brain stem and cerebrum. Similar infiltrates were also found to a lesser extent in the cerebellar meninges leading to the diagnosis of acute-subacute meningoencephalitis. The histological examination of the brainstem from the second case, a 16-month-old heifer, revealed perivascular infiltration composed of equal numbers of macrophages and neutrophils associated with cerebral and meningeal haemorrhages. In this case encephalitis was diagnosed. Viral RNA was extracted from brain samples of both cattle that suffered from severe neurological signs and was subsequently tested by a polymerase chain reaction PCR assay specific for Simbu serogroup viruses and found positive. The presence of SHUV was subsequently confirmed by the isolation of the virus from one sample and sequence analysis of both brain samples. The comparison of the complete sequences of the coding regions of all three genome segments from both cases revealed a close relationship to Shuni viruses detected in tissue samples of aborted or malformed calves or lambs born during the last years in Israel.

Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR.

Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen.

Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia.

UNLABELLED: Tilapia are an important global food source due to their omnivorous diet, tolerance for high-density aquaculture, and relative disease resistance. Since 2009, tilapia aquaculture has been threatened by mass die-offs in farmed fish in Israel and Ecuador. Here we report evidence implicating a novel orthomyxo-like virus in these outbreaks. The tilapia lake virus (TiLV) has a 10-segment, negative-sense RNA genome. The largest segment, segment 1, contains an open reading frame with weak sequence homology to the influenza C virus PB1 subunit. The other nine segments showed no homology to other viruses but have conserved, complementary sequences at their 5' and 3' termini, consistent with the genome organization found in other orthomyxoviruses. In situ hybridization indicates TiLV replication and transcription at sites of pathology in the liver and central nervous system of tilapia with disease. IMPORTANCE: The economic impact of worldwide trade in tilapia is estimated at $7.5 billion U.S. dollars (USD) annually. The infectious agent implicated in mass tilapia die-offs in two continents poses a threat to the global tilapia industry, which not only provides inexpensive dietary protein but also is a major employer in the developing world. Here we report characterization of the causative agent as a novel orthomyxo-like virus, tilapia lake virus (TiLV). We also describe complete genomic and protein sequences that will facilitate TiLV detection and containment and enable vaccine development.

Identification of a novel RNA virus lethal to tilapia.

Tilapines are important for the sustainability of ecological systems and serve as the second most important group of farmed fish worldwide. Significant mortality of wild and cultured tilapia has been observed recently in Israel. The etiological agent of this disease, a novel RNA virus, is described here, and procedures allowing its isolation and detection are revealed. The virus, denominated tilapia lake virus (TiLV), was propagated in primary tilapia brain cells or in an E-11 cell line, and it induced a cytopathic effect at 5 to 10 days postinfection. Electron microscopy revealed enveloped icosahedral particles of 55 to 75 nm. Low-passage TiLV, injected intraperitoneally in tilapia, induced a disease resembling the natural disease, which typically presents with lethargy, ocular alterations, and skin erosions, with >80% mortality. Histological changes included congestion of the internal organs (kidneys and brain) with foci of gliosis and perivascular cuffing of lymphocytes in the brain cortex; ocular inflammation included endophthalmitis and cataractous changes of the lens. The cohabitation of healthy and diseased fish demonstrated that the disease is contagious and that mortalities (80 to 100%) occur within a few days. Fish surviving the initial mortality were immune to further TiLV infections, suggesting the mounting of a protective immune response. Screening cDNA libraries identified a TiLV-specific sequence, allowing the design of a PCR-based diagnostic test. This test enables the specific identification of TiLV in tilapines and should help control the spread of this virus worldwide.

A pivotal role for the Streptococcus iniae extracellular polysaccharide in triggering proinflammatory cytokines transcription and inducing death in rainbow trout.

Streptococcus iniae is a major pathogen of fish, causing considerable economic losses in Israel, the United States and the Far East. Containment of mortalities through vaccination was recently compromised due to the emergence of novel vaccine-escape strains that are distinguished from previous strains by their ability to produce large amounts of extracellular polysaccharide (EPS) that is released to the medium. In vitro and in vivo data now indicate that the EPS is a major virulence factor, capable of triggering the proinflammatory cytokine machinery and inducing mortality of fish. Streptococcus iniae EPS might therefore be considered to be responsible for sepsis and death just as lipopolysaccharide is for Gram-negative pathogens.

Emergence of novel Streptococcus iniae exopolysaccharide-producing strains following vaccination with nonproducing strains.

Streptococcus iniae is a major pathogen of fish, producing fatal disease among fish species living in very diverse environments. Recently, reoccurrences of disease outbreaks were recorded in rainbow trout (Oncorhynchus mykiss, Walbaum) farms where the entire fish population was routinely vaccinated. New strains are distinguished from previous strains by their ability to produce large amounts of extracellular polysaccharide that is released into the medium. Present findings indicate that the extracellular polysaccharide is a major antigenic factor, suggesting an evolutionary selection of strains capable of extracellular polysaccharide production.

Transcytosis of Streptococcus iniae through skin epithelial barriers: an in vitro study.

By constructing a biological model based on in vitro culture of polarized rainbow trout primary skin epithelial cell monolayers, the series of early events that precede Streptococcus iniae infection, particularly colonization and translocation through external barriers, were analyzed. Streptococcus iniae promptly invades skin epithelial cells, but the rapid decline of viable intracellular bacteria points out the limited capability of intracellular survival for this bacterium. Translocation assays, supported by electron microscopy microphotographs, demonstrate that following successful in vitro invasion of skin epithelial cell, the bacterium exists free in the cytoplasm after release from the endosome, and translocates through the skin barrier. Bacterial invasion and transcytosis is not accompanied by apparent cell-line damages or disruption of host cells' tight junctions. It is hypothesized that the phenomenon of epithelial invasion coupled to the rapid translocation through the barrier plays a crucial role in Streptococcus iniae infection.

Genome sequences of three koi herpesvirus isolates representing the expanding distribution of an emerging disease threatening koi and common carp worldwide.

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.

Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis.

BACKGROUND: Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary. RESULTS: A robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK) was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV) and the channel catfish virus (CCV). The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection. CONCLUSION: The TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions.

Clonality and diversity of the fish pathogen Lactococcus garvieae in Mediterranean countries.

Infection with Lactococcus garvieae is considered the most important risk factor for the European trout industry, and the losses are approximately 50% of the total production. To improve our understanding of the genetic links among strains originating from different countries, we examined the population structure of L. garvieae by comparing 81 strains isolated from different sources and ecosystems (41 farms in six countries) in which the bacterium is commonly found. Genetic similarities (as assessed with molecular tools, including restriction fragment length polymorphism ribotyping with two endonucleases) were compared with serological data. The combined results reveal that in endemic sites the bacterial population displays a clonal structure, whereas bacterial diversity characterizes sites where the infection is sporadic.

Trojan horse effect: phagocyte-mediated Streptococcus iniae infection of fish.

The salmonid macrophage-like cell line RTS-11 and purified trout pronephros phagocytes were used to analyze in vitro entry and survival of two Streptococcus iniae serotypes. Efficient invasion by S. iniae occurred in both cells, but only the type II strain persisted in pronephros phagocytes for at least 48 h. Ex vivo models of opsonin-dependent phagocytosis by pronephros phagocytes demonstrated increased phagocytosis efficacy. Analysis of phagocytes collected from diseased fish demonstrated that approximately 70% of the bacteria contained in the blood during the septic phase of the disease were located within phagocytes, suggesting an in vivo intracellular lifestyle. In addition to the augmented levels of bacteremia and enhanced survival within phagocytes, S. iniae type II induces considerable apoptosis of phagocytes. These variabilities in intramacrophage lifestyle might explain differences in the outcomes of infections caused by different serotypes. The generalized septic disease associated with serotype II strains is linked not only to the ability to enter and multiply within macrophages but also to the ability to cause considerable death of macrophages via apoptotic processes, leading to a highly virulent infection. We assume that the phenomenon of survival within phagocytes coupled to their apoptosis plays a crucial role in S. iniae infection. In addition, it may provide the pathogen an efficient mechanism of translocation into the central nervous system.

Initial characteristics of koi herpesvirus and development of a polymerase chain reaction assay to detect the virus in koi, Cyprinus carpio koi.

Since 1998, episodes of mass mortality have occurred in populations of common carp Cyprinus carpio carpio in Israel and in populations of koi Cyprinus carpio koi in Israel and the USA. A herpesvirus isolated from infected fish has been shown in experimental studies to induce disease and mortality similar to those observed in outbreaks at infected farms. Initial characteristics of the virus show that it is clearly different from Herpesvirus cyprini (CHV), the most commonly known herpesvirus from cyprinid fish. The koi herpesvirus (KHV) has 31 virion polypeptides. Twelve of the virion polypeptides of KHV have similar molecular weights to those of CHV and 10 are similar to those of channel catfish virus (CCV). Both virion polypeptide and restriction fragment length polymorphism analyses of genomic DNA showed that the first KHV isolates from Israel and the USA were identical. In contrast, the genomic DNA restriction fragments clearly distinguish KHV from CHV and CCV. A polymerase chain reaction (PCR) assay to detect the virus in koi tissues was developed with sequences obtained from 1 restriction fragment of KHV DNA. The PCR assay effectively detected a 484 base pair sequence from KHV but did not amplify genomic DNA from either CHV or CCV. The PCR assay detected as little as 1 pg of KHV DNA mixed with 100 ng of host DNA. Viral sequences were amplified from koi obtained from field collections and from koi that were experimentally exposed to 10(2) TCID50 ml(-1) of KHV via the waterborne route. All KHV exposed fish dying of infection between 8 and 10 d post exposure or surviving to 14 d post exposure were found to be positive by PCR, while unexposed control koi were all negative. The assay also showed the presence of KHV DNA in tissues of koi obtained from farms in Israel. The PCR assay should assist virus isolation procedures and histologic and electron microscopic analyses now commonly used to detect KHV infection. Current studies are examining the possibility of using the PCR to detect KHV DNA in live fish and the relative sensitivity and specificity of the KHV PCR assay compared with other diagnostic tests.