Validation of a serum neutralization test for detection of antibodies specific to cyprinid herpesvirus 3 in infected common and koi carp (Cyprinus carpio).

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a serious infective, notifiable disease affecting common carp and varieties. In survivors, infection is generally characterized by a subclinical latency phase with restricted viral replication. The CyHV-3 genome is difficult to detect in such carrier fish that represent a potential source of dissemination if viral reactivation occurs. In this study, the analytical and diagnostic performance of an alternative serum neutralization (SN) method based on the detection of CyHV-3-specific antibodies was assessed using 151 serum or plasma samples from healthy and naturally or experimentally CyHV-3-infected carp. French CyHV-3 isolate 07/108b was neutralized efficiently by sera from carp infected with European, American and Taiwanese CyHV-3 isolates, but no neutralization was observed using sera specific to other aquatic herpesviruses. Diagnostic sensitivity, diagnostic specificity and repeatability of 95.9%, 99.0% and 99.3%, respectively, were obtained, as well as a compliance rate of 89.9% in reproducibility testing. Neutralizing antibodies were steadily detected in infected carp subjected to restrictive or permissive temperature variations over more than 25 months post-infection. The results suggest that this non-lethal diagnostic test could be used in the future to improve the epidemiological surveillance and control of CyHV-3 disease.

Outbreak of betanodavirus infection in tilapia, Oreochromis niloticus (L.), in fresh water.

A betanodavirus associated with a massive mortality was isolated from larvae of tilapia, Oreochromis niloticus, maintained in fresh water at 30 degrees C. Histopathology revealed vacuolation of the nervous system, suggesting an infection by a betanodavirus. The virus was identified by indirect fluorescent antibody test in the SSN1 cell line and further characterized by sequencing of a PCR product. Sequencing of the T4 region of the coat protein gene indicated a phylogenetic clustering of this isolate within the red-spotted grouper nervous necrosis virus type. However, the tilapia isolate formed a unique branch distinct from other betanodavirus isolates. The disease was experimentally reproduced by bath infection of young tilapia at 30 degrees C. The reservoir of virus at the origin of the outbreak remains unidentified. To our knowledge, this is the first report of natural nodavirus infection in tilapia reared in fresh water.

Differentiation between Cyprinid herpesvirus type-3 lineages using duplex PCR.

To date, all the isolates of Cyprinid herpesvirus type-3 (CyHV3) responsible for serious outbreaks in carps Cyprinus carpio have been found to be very similar or identical on the basis of DNA sequences of a few reference genes. However, two genetic lineages (U/I and J) are distinguished by full-length genome sequencing. Two molecular markers presenting genetic variations were targeted for developing a duplex PCR assay able to distinguish CyHV3-U/I from CyHV3-J while avoiding DNA sequencing. The method was validated on a series of 42 samples of infected carps from France, The Netherlands and Poland collected from 2001 to 2008. Among these samples, both the U/I and J genotypes were identified, but also a third genotype representing a genetic intermediate between U/I and J for one of the two molecular markers. A classification of CyHV3 genotypes, based on the alleles of the two molecular markers, is proposed. The assay is easy to perform and provides a genotype information with samples moderately or highly concentrated. This tool should improve our knowledge regarding the present distribution and future diversification of this emerging virus.

Study of the viral interference between infectious pancreatic necrosis virus (IPNV) and infectious haematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss).

The resistance of rainbow trout (Oncorhynchus mykiss) to an infectious haematopoietic necrosis virus (IHNV) challenge following a preceding non-lethal infection with infectious pancreatic necrosis virus (IPNV) was investigated through experimental dual infections. Trout initially infected with IPNV were inoculated 14 days later with IHNV. Single infections of trout with 1 of the 2 viruses or with cell culture supernatant were also carried out and constituted control groups. No mortality was noted in fish after a single infection with IPNV. This virus had no influence on the head kidney leucocyte phagocytic activity and plasma haemolytic complement activity. IHNV induced a high mortality (72%) and reduced the macrophage phagocytic activity and complement haemolytic activity. It also induced a late production of anti-IHNV antibodies which occurred after clearance of the virus in the fish. In trout co-infected with both viruses, a mortality rate of 2% occurred and the immune parameters were similar to those observed in the fish infected with IPNV only, demonstrating that in co-infected trout IPNV inhibits the effects of IHNV. The studied parameters did not allow us to define the mechanism of interference occurring between these 2 viruses, but some hypothesis are put forward to explain the interference between the 2 viruses.

Experimental transmission of sleeping disease in one-year-old rainbow trout, Oncorhynchus mykiss (Walbaum), induced by sleeping disease virus.

Sleeping disease (SD) is a serious disease of rainbow trout, Oncorhynchus mykiss, reared in fresh water caused by sleeping disease virus (SDV). In this study a detailed clinical, histological, virological and serological description of the experimental reproduction of SD in 1-year-old rainbow trout exposed to SDV was carried out. Two hundred disease-free fish were intraperitoneally inoculated with a SDV isolate and 100 fish were inoculated with an uninfected cell culture lysate as a negative control. Infected and control fish were randomly removed at days 4, 7, 14, 21, 42 and 70 post-infection. Blood and tissues were collected for virus isolation, histopathological examination and serum neutralization. SDV was detected in serum, kidney and brain of infected fish from 4 to 21 days post-infection (dpi). Characteristic pathological lesions were observed in infected fish as early as 7 dpi. Lesions were first detected in exocrine pancreas and subsequently observed in heart and skeletal muscle. Neutralizing antibodies to SDV were detected in infected fish from 14 to 70 dpi. Infected fish displayed typical signs of SD 1-month pi and the mortality reached 18.7% within 44 days. This study experimentally reproduced all the pathognomonic features of natural outbreaks of SD in 1-year-old rainbow trout.

Studies on pathogenesis following single and double infection with viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) were bath challenged with viral hemorrhagic septicemia (VHS) virus or infectious hematopoietic necrosis (IHN) virus or with both viruses simultaneously. The viral distribution and development of histologic lesions were examined using immunohistochemistry, while virus titer in kidney was determined by viral titration in cell culture. Single infections with VHS virus and IHN virus showed similar distributions of virus in internal organs. The early identification of virus in gill epithelium, 1 and 2 days postinfection (PI) for VHS virus and IHN virus, respectively, indicates that this organ is the point of entry for both viruses. The detection of VHS virus at 1 day PI and 3 days PI for IHN virus is indicative of kidney and spleen being the target organs for these viruses. A simultaneous infection of VHS virus and IHN virus resulted in both viruses establishing an infection. Further double infection did not result in a statistically significant lower titer of both viruses in kidney but a more restricted distribution of IHN virus in internal organs compared with the single infected group. The most striking finding is that, for IHN virus, virus was not detected in the brain in situ in the double-infected group. This study provides support for the conclusion that simultaneous infection with two piscine rhabdoviruses in a susceptible host results in some degree of interaction at the cell level, leading to a reduced systemic distribution of IHN virus.

Phylogenetic analysis of viral haemorrhagic septicaemia virus (VHSV) isolates from France (1971-1999).

The nucleotide sequences of a specific region of the glycoprotein gene were compared among 63 strains of viral haemorrhagic septicaemia virus (VHSV) isolated from fish in France between 1971 and 1999. The analysis was performed on a region corresponding to amino acids 238 to 331 of the glycoprotein gene, also designated the V2 region and previously shown to accumulate most of the mutations. The sequences of many VHSV isolates were found to be identical or very conserved. An isolate, designated L59X, obtained from elver in the Loire estuary, depicted a higher degree of divergence compared to the other French isolates. The deduced amino-acid sequences were analysed together with the results of neutralisation tests performed using monoclonal antibody 168m4 specific to serotype 1. Non-neutralised VHSV strains had mutations in the region corresponding to the previously described 168m4 epitope. Phylogenetic analysis showed that all the VHSV isolates studied, except L59X, belong to genotype I, previously described as containing VHSV strains isolated from continental Europe. Most of the VHSV isolates studied were found to be genetically related to one of the previously described VHSV strains representative of the major serotypes. Isolate L59X, which was the only French marine strain studied, was found to belong to genotype II, previously shown to encompass the VHSV strains isolated from the British Isles coastal waters. Overall there was a good correlation between the geographical origin of the studied isolates and their genetic characteristics.

Isolation of salmon pancreas disease virus (SPDV) in cell culture and its ability to protect against infection by the 'wild-type' agent.

A Scottish salmon pancreas disease virus (SPDV) has been isolated and its optimum growth conditions determined. Although several fish cell lines have been tested, successful culture was achieved only with CHSE-214 cells. Cytopathic effects were observed after 5 days. The highest virus titres, calculated by microtitration assay, were reached at 15 degrees C. After 7-9 days post-inoculation, CHSE-214 cell supernatants contained between 10(7)-10(5) TCID50 ml(-1) The cultured isolate is chloroform- and pH 3.0-sensitive, and virions are 50-60 nm in diameter. These characteristics are similar to the Irish SPDV isolates. The culture isolate induced typical pancreas disease (PD) lesions in experimentally infected Atlantic salmon and convalescent fish were resistant to experimental infection with PD-infective kidney homogenates obtained by serial in vivo passages from a PD-infected farmed salmon (termed wild-type SPDV). Furthermore, fish immunised with the inactivated cultured virus were protected against a cohabitation challenge with the wild-type virus. Immunised fish sera showed virus-neutralising activity before challenge (7 weeks post-immunisation) and from 3-6 weeks post-challenge, when sera from non-immunised fish did not neutralise the virus. At 6 weeks post-cohabitation challenge, previously immunised fish had neutralising titres of up to 1:65. Following intraperitoneal (i.p.) challenge, immunised fish showed neutralising titres as high as 1:226 at 8 weeks post-challenge. Non-immunised fish injected i.p. with the wild-type virus developed serum-neutralising activity against the cultured isolate when sampled 8 weeks after infection, confirming an antigenic relationship between the wild-type and cultured virus. The results demonstrate that the tissue culture-adapted isolate of SPDV could be successfully used to protect against challenge by the wild-type virus and could therefore have potential use as an inactivated vaccine against PD.

An RT-PCR-based method for the diagnosis of the sleeping disease virus in experimentally and naturally infected salmonids.

The sleeping disease (SD) of rainbow trout (Oncorhynchus mykiss) is a worldwide disease for which the causative agent, the sleeping disease virus (SDV), has been recently characterized as an atypical alphavirus (Villoing et al. 2000). Up to now, no diagnostic tools were available and thus no epidemiological studies have been undertaken to evaluate the occurrence of this disease on the field. We present in this paper a sensitive and highly specific 1 working day method, which allows the detection of SDV from experimentally and naturally infected fishes. This method, based on a reverse transcriptase/polymerase chain reaction (RT-PCR) assay on total RNA extracted from SDV-infected fish organs, enables the specific DNA amplification of part of the gene encoding the SDV glycoprotein E2, as early as 2 d post-infection (d.p.i.) and as late as 70 d.p.i., at which time clinical signs of infection are no longer apparent. Moreover, we show that this RT-PCR method can be successfully used for the diagnosis of fish infected by a closely related virus, namely salmon pancreas disease virus (SPDV). This report is the first description of a very powerful diagnostic assay which could provide a more accurate replacement for the classical virological, histological and immunochemistry methods.

Rainbow trout sleeping disease virus is an atypical alphavirus.

Sleeping disease (SD) is currently a matter of concern for salmonid fish farmers in most parts of the world. A viral etiology of SD has recently been suspected, since virus-like particles have been observed in infected rainbow trout cells. In salmonid-derived cell lines, the maximal rate of virus production was observed at 10 degrees C, while little virus was produced at 14 degrees C. Through biochemical, physicochemical, and morphological studies, SD virus (SDV) was shown to be an enveloped virus of roughly 60 nm in diameter. The genome consists of 12 kb of RNA, with the appearance of a 26S subgenomic RNA during the time course of SDV replication. The screening of a random-primed cDNA library constructed from the genomic RNA of semipurified virions facilitated the identification of a specific SDV cDNA clone having an open reading frame related to the alphavirus E2 glycoproteins. To extend the comparison between SDV structural proteins and the alphavirus protein counterparts, the nucleotide sequence of the total 4.1-kb subgenomic RNA has been determined. The 26S RNA encodes a 1,324-amino-acid polyprotein exhibiting typical alphavirus structural protein organization. SDV structural proteins showed several remarkable features compared to other alphaviruses: (i) unusually large individual proteins, (ii) very low homology (ranging from 30 to 34%) (iii) an unglycosylated E3 protein, and (iv) and E1 fusion domain sharing mutations implicated in the pH threshold. Although phylogenetically related to the Semliki Forest virus group of alphaviruses, SDV should be considered an atypical member, able to naturally replicate in lower vertebrates.

Natural outbreak of viral encephalopathy and retinopathy in juvenile sea bass, Dicentrarchus labrax: study by nested reverse transcriptase-polymerase chain reaction.

In order to improve the sensitivity of the diagnosis of viral encephalopathy and retinopathy (VER) in sea bass, a nested reverse transcriptase-polymerase chain reaction (RT-PCR) detection method was developed. The reverse transcription step and the first stage PCR were performed using outer primers specific for the coat protein gene, whereas a new primer set was used as inner primers for the second stage PCR. Fish were collected just before, during and after a VER outbreak occurring in a mediterranean fish farm. For each time point, ten different fish were analysed individually by nested RT-PCR, single step PCR and virus cultivation. The results showed that the frequency of positive samples was always higher using the nested RT-PCR assay. In particular, it was possible to detect nodavirus specific signals 1 month before the appearance of the first mortalities, but only by nested RT-PCR. Altogether these results showed that the sensitivity of nodavirus detection is greatly improved using a nested RT-PCR method. In particular, it was possible to monitor the presence of viral genome in asymptomatic carrier fish using this method.

Comparative study of viral encephalopathy and retinopathy in juvenile sea bass Dicentrarchus labrax infected in different ways.

The transmission of viral encephalopathy and retinopathy (VER) was investigated in juvenile sea bass (3 g) Dicentrarchus labrax by using cell culture supernatant (SSN-1 cell line) containing nodavirus. Five methods of infection were tested: intramuscular injection (IM), intraperitoneal injection (IP), oral infection, bath exposure and cohabitation of healthy fish with infected fish. Some differences were observed in time of disease onset and severity of symptoms depending on the mode of infection used. Clinical symptoms such as whirling swimming and lethargic or hyperactive behaviour were generally reproduced, except for fish infected via oral and IP infection. First mortalities occurred 3 d after IM and IP infection and 6 d after for the other modes of infection. Cumulative mortalities were also variable: 100% after IM infection, 10% after IP infection, 32% for bath exposure, 43% after cohabitation and 24% via oral infection. Histopathologically, vacuolation was observed in the central nervous tissues and in the retina. The observed lesions were more or less severe depending on the mode of infection, the sampling time and the organs: lesions on the surviving fish (42 days post infection, d p.i.) seemed to be generally more conspicuous in the retina than in the brain of the same fish. In most cases, the presence of nodavirus was confirmed in the same samples of brain and retina by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). The virus was not detected in other organs examined. The present results suggest that 2 forms of VER can be induced: IM injection leads to an acute form (severe nervous disorders with high and fast mortality) whereas oral infection, bath exposure and cohabitation induce a subacute form (less severe disorders and weak daily mortality). This experiment demonstrates experimentally induced horizontal transmission of VER in sea bass for the first time.

Eighteen years of vaccination against viral haemorrhagic septicaemia in France.

Viral haemorrhagic septicaemia (VHS) has been considered for many years to be a major cause of loss in the French trout industry. The high prevalence of VHS in certain geographic areas made a control strategy based on control policy unfeasible. This provided the impetus for immunoprophylaxis development that resulted in 3 successive types of vaccines: inactivated, live attenuated and recombinant vaccines. When delivered by intraperitoneal injection, the 2 propiolactone-inactivated VHS virus was immunogenic and/or protective for trout all of sizes, but it was not suitable for the practical immunization of alevin, the trout life stage that is the most sensitive to VHS. A carp cell-passed, attenuated variant of the VHS virus was effective after both immersion or injection delivery and met the practical requirements of juvenile vaccination. However, this vaccine was discarded because it retained some virulence that discouraged the launching of its commercialization. Then came the era of genetically engineered vaccines. The recombinant glycoprotein of VHSV produced in Escherichia coli or in Saccharomyces cerevisiae failed to protect fish whatever the route of delivery. A recombinant baculovirus vaccine was found to be immunogenic and protective against VHS, but only when delivered by injection. Due to its cost and route of delivery, the latter vaccine was not licensed. Simultaneously, the sudden occurrence of another rhabdovirosis, infectious haematopoietic necrosis (IHN), in France, rendered vaccination against VHS questionable. Indeed, no cross-protection between these 2 rhabdoviroses exists. If vaccination is still believed to be an effective control method for VHS, it should be based in the future upon an autoreplicative vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)