Transcriptome analysis reveals a complex response to the RGNNV/SJNNV reassortant Nervous Necrosis Virus strain in sea bream larvae.

The gilthead sea bream (Sparus aurata) is a marine fish of great importance for Mediterranean aquaculture. This species has long been considered resistant to Nervous Necrosis Virus (NNV), an RNA virus that causes massive mortalities in several farmed fish animals. However, the recent appearance of RGNNV/SJNNV reassortant strains started to pose a serious threat to sea bream hatcheries, as it is able to infect larvae and juveniles of this species. While host response to NNV has been extensively studied in adult fish, little attention has been devoted to early life history stages, which are generally the most sensitive ones. Here we report for the first time a time-course RNA-seq analysis on 21-day old fish gilthead sea bream larvae experimentally infected with a RGNNV/SJNNV strain. NNV-infected and mock-infected samples were collected at four time points (6 h, 12 h, 24 h, and 48 h post infection). Four biological replicates, each consisting of five pooled larvae, were analysed for each time point and group. A large set of genes were found to be significantly regulated, especially at early time points (6 h and 12 h), with several heat shock protein encoding transcripts being up-regulated (e.g. hspa5, dnaj4, hspa9, hsc70), while many immune genes were down-regulated (e.g. myd88 and irf5 at T06, pik3r1, stat3, jak1, il12b and il6st at T12). A gene set enrichment analysis (GSEA) identified several altered pathways/processes. For instance, the formation of peroxisomes, which are important anti-viral components as well as essential for nervous system homeostasis, and the autophagy pathway were down-regulated at 6 h and 24 h post infection (hpi). Finally, two custom "reactomes" (i.e. significant gene sets observed in other studies) were defined and used. The first reactome integrated the transcriptomic response to NNV in different fish species, while the second one included all genes found to be stimulated either by interferon (IFN) or by IFN and Chikungunya virus in zebrafish. Genes in both reactomes showed predominant up-regulation at 6hpi and 12hpi and a general down-regulation at 24hpi. Such evidence suggest a certain degree of similarity between the response of sea bream and that of other fish species to NNV, while the observed down-regulation of IFN- and viral-stimulated pathways argues for a possible interference of NNV against the host response.

Development of a real-time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues.

The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, is essential for managing fish pasteurellosis outbreaks in farmed fish. A single-step, high-sensitivity real-time PCR assay for simultaneous detection and quantification of P. damselae was designed targeting partial of the sequence of the bamB gene and tested for specificity and sensitivity on laboratory-generated samples as well as on experimentally infected seabream tissue samples. With a limit of detection (LOD) of one copy in pure bacterial DNA, the sensitivity was higher than all methods previously reported. Validation in target and non-target bacterial species proved the assay was able to discriminate Phdd-Phdp subspecies from diverse hosts/geographical origins and between non-target species. In addition, two SNPs in the target amplicon region determine two distinctive qPCR dissociation curves distinguishing between Phdp-Phdd. This is the first time that a molecular method for P. damselae diagnosis combines detection, quantification and subspecies identification in one step. The assay holds the potential to improve the knowledge of infection dynamics and the development of better strategies to control an important fish disease.

Emergence of carp edema virus (CEV) and its significance to European common carp and koi Cyprinus carpio.

Carp edema virus disease (CEVD), also known as koi sleepy disease, is caused by a poxvirus associated with outbreaks of clinical disease in koi and common carp Cyprinus carpio. Originally characterised in Japan in the 1970s, international trade in koi has led to the spread of CEV, although the first recognised outbreak of the disease outside of Japan was not reported until 1996 in the USA. In Europe, the disease was first recognised in 2009 and, as detection and diagnosis have improved, more EU member states have reported CEV associated with disease outbreaks. Although the structure of the CEV genome is not yet elucidated, molecular epidemiology studies have suggested distinct geographical populations of CEV infecting both koi and common carp. Detection and identification of cases of CEVD in common carp were unreliable using the original PCR primers. New primers for conventional and quantitative PCR (qPCR) have been designed that improve detection, and their sequences are provided in this paper. The qPCR primers have successfully detected CEV DNA in archive material from investigations of unexplained carp mortalities conducted >15 yr ago. Improvement in disease management and control is possible, and the principles of biosecurity, good health management and disease surveillance, applied to koi herpesvirus disease, can be equally applied to CEVD. However, further research studies are needed to fill the knowledge gaps in the disease pathogenesis and epidemiology that, currently, prevent an accurate assessment of the likely impact of CEVD on European koi and common carp aquaculture and on wild carp stocks.

Molecular identification of iridoviruses infecting various sturgeon species in Europe.

Iridoviridae are known to cause disease in sturgeons in North America. Here, histological and molecular methods were used to screen for this family of virus in sturgeons from various European farms with low-to-high morbidity. Some histological samples revealed basophilic cells in the gill and labial epithelia, strongly suggesting the accumulation of iridovirus particles. Newly developed generic PCR tests targeting the major capsid protein (MCP) gene of sturgeon iridoviruses identified in North America, namely the white sturgeon iridovirus and the Namao virus (NV), produced positive signals in most samples from four sturgeon species: Russian (Acipenser gueldenstaedtii), Siberian (A. baerii), Adriatic (A. naccarii) and beluga (Huso huso). The sequences of the PCR products were generally highly similar one another, with nucleotide identities greater than 98%. They were also related to (74-88%), although distinct from, American sturgeon iridoviruses. These European viruses were thus considered variants of a single new virus, provisionally named Acipenser iridovirus-European (AcIV-E). Moreover, three samples infected with AcIV-E showed genetic heterogeneity, with the co-existence of two sequences differing by five nucleotides. One of our European samples carried a virus distinct from AcIV-E, but closely related to NV identified in Canada (95%). This study demonstrates the presence of two distinct sturgeon iridoviruses in Europe: a new genotype AcIV-E and an NV-related virus.

Efficacy of domestic cooking inactivation of human hepatitis A virus in experimentally infected manila clams (Ruditapes philippinarum).

AIM: The aim of this work was to evaluate the efficacy of domestic cooking in inactivating Manila clams experimentally infected with human hepatitis A virus (HAV). METHODS AND RESULTS: Electronic temperature probes were positioned to measure the internal temperature of Manila clams during domestic cooking. Two batches were infected with 10(7) and 10(5) TCID50  ml(-1) of HAV. The infected whole-in-shell clams were divided into three replicates and cooked on a conventional stove both singularly and in group and removed from the pan at fixed intervals. Pools of three digestive glands were examined by virus isolation for three blind passages and cell culture supernatant tested with real-time PCR. CONCLUSION: Results showed that 2-min cooking by a traditional domestic method at a temperature close to 100°C, after the opening up of the valves of all the clams, can completely devitalize the HAV in high viral load-infected clams. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on inactivation of HAV in experimentally infected Manila clams subjected to domestic cooking. At present, labelling all lagoon products as 'requiring cooking before consumption' is highly recommended, but no specifications are given on how long and at what temperature they should be cooked. Considering the high commercial value of Manila clams, our results can provide both the producers and the consumer with useful indications on how to cook clams to prevent the risk of HAV foodborne illness.

Betanodavirus ability to infect juvenile European sea bass, Dicentrarchus labrax, at different water salinity.

Viral encephalopathy and retinopathy (VER) is one of the most devastating and economically relevant diseases for marine aquaculture. The presence of betanodavirus in freshwater fish is recorded, but very little is known about VER outbreaks in marine species reared in freshwater. Our study investigated the ability of betanodavirus to cause disease in European sea bass, Dicentrarchus labrax, reared at different salinity levels. Fish were challenged with RGNNV or mock infected by bath at different salinity levels (freshwater, 25‰ and 33‰). Fish were checked twice a day and the dead ones were examined by standard virological techniques, by rRT-PCR and by histochemical and immunohistochemical analyses. All the infected groups showed a significant higher mortality rate than the one of the mock-infected group. VERv presence was confirmed by rRT-PCR. Histochemical and immunohistochemical analyses highlighted the typical lesions associated with VER. Our results highlight that salinity does not affect the ability of betanodavirus to induce clinical signs and mortality in European sea bass infected under experimental conditions. These results underline the great adaptation potential of VERv, which in combination with its already known high environmental resistance and broad host range, may explain the diffusion of this disease and the threat posed to aquaculture worldwide.

Quantitative immunoenzymatic detection of viral encephalopathy and retinopathy virus (betanodavirus) in sea bass Dicentrarchus labrax.

Viral encephalopathy and retinopathy disease caused by betanodavirus, genus of the family Nodaviridae, affects marine, wild and farmed species including sea bass, one of the most important farmed species in Europe. This work describes a reliable and sensitive indirect ELISA assay to detect betanodavirus in biological samples using a polyclonal antiserum (pAb 283) against the 283/I09 virus strain, the most common red-spotted grouper nervous necrosis virus (RGNNV) genotype in the Mediterranean area, and a capture-based ELISA using a monoclonal antibody (mAb 4C3) specific to a common epitope present on the capsid protein. Using adsorbed, purified VERv preparation, the detection limit of indirect ELISA was 2 µg mL(-1) (3 × 10(5) TCID50 per mL), whereas for capture-based ELISA, the sensitivity for the antigen in solution was 17 µg mL(-1) (35 × 10(5) TCID50 per mL). The capture-based ELISA was employed to detect VERv in brain homogenates of in vivo infected sea bass and resulted positive in 22 of 32 samples, some of these with a high viral load estimates (about 1.1 × 10(8)  TCID50 per mL). The ELISA system we propose may be helpful in investigations where coupling of viral content in fish tissues with the presence of circulating VERv-specific IgM is required, or for use in samples where PCR is difficult to perform.

First generic one step real-time Taqman RT-PCR targeting the RNA1 of betanodaviruses.

The detection of betanodavirus genomic components is a major issue for diagnostics and control of viral nervous necrosis (VNN), a devastating disease affecting fish worldwide. Despite a number of published molecular-based tests, most of them targeting the RNA2 molecule of the virus, diagnostics is still a challenge due to the high genetic diversity within this genus. In the present study, a new one-step real-time RT-PCR (rRT-PCR), targeting RNA1 of most genotypes of betanodaviruses, was proposed and validated. The test detected successfully various isolates of betanodavirus representatives of the four species RGNNV, SJNNV, TPNNV and BFNNV, either produced on cell culture or from clinical samples. It was specific as shown by the absence of signal on samples from healthy sea bass or from field samples of six other fish species without clinical signs of VNN. The assay detected reliably 50-100 copies of plasmids containing the targeted cloned RNA1 region, as well as an infectious dose of virus of 10(2.5)-10(2.85) TCID50/ml. A set of samples was tested by two different laboratories, with similar results, demonstrating the robustness of the test. This is the first one step generic rRT-PCR method for betanodaviruses. It is simple to perform and may be used for first intention diagnostics as well as for confirmation in case of doubtful results obtained with other published tests targeting RNA2.

Comparative pathogenicity study of ten different betanodavirus strains in experimentally infected European sea bass, Dicentrarchus labrax (L.).

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a severe pathological condition caused by RNA viruses belonging to the Nodaviridae family, genus Betanodavirus. The disease, described in more than 50 fish species worldwide, is considered as the most serious viral threat affecting marine farmed species in the Mediterranean region, thus representing one of the bottlenecks for further development of the aquaculture industry. To date, four different genotypes have been identified, namely red-spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus and barfin flounder nervous necrosis virus, with the RGNNV genotype appearing as the most widespread in the Mediterranean region, although SJNNV-type strains and reassortant viruses have also been reported. The existence of these genetically different strains could be the reason for the differences in mortality observed in the field. However, very little experimental data are available on the pathogenicity of these viruses in farmed fish. Therefore, in this study, the pathogenicity of 10 isolates has been assessed with an in vivo trial. The investigation was conducted using the European sea bass, the first target fish species for the disease in the Mediterranean basin. Naive fish were challenged by immersion and clinical signs and mortality were recorded for 68 days; furthermore, samples collected at selected time points were analysed to evaluate the development of the infection. Finally, survivors were weighed to estimate the growth reduction. The statistically supported results obtained in this study demonstrated different pathogenicity patterns, underlined the potential risk represented by different strains in the transmission of the infection to highly susceptible species and highlighted the indirect damage caused by a clinical outbreak of VER/VNN.

The effectiveness of domestic cook on inactivation of murine norovirus in experimentally infected Manila clams (Ruditapes philippinarum).

AIM: The aim of this work was to evaluate the efficacy of domestic cooking in inactivating Manila clams experimentally infected with murine norovirus (MNV). METHODS AND RESULTS: A cooking pan was modified to enable electronic temperature probes to be positioned to record both flesh and environment temperature. Manila clams were infected with 10(4) TCID 50% ml(-1) of MNV. The infected whole-in-shell clams, divided into three replicates, were cooked on an electric stove, and groups of nine clams were removed from the pan at fixed intervals. Pools of three digestive glands were examined by virus isolation to ascertain residual viral load. CONCLUSION: Results showed that 10 min of cooking by a traditional domestic method at a temperature close to 100°C, for at least 2 min, can completely devitalize the MNV in infected clams. This is generally the time needed for the majority of valves to open up. SIGNIFICANCE AND IMPACT OF THE STUDY: At present, it is highly recommended to label all lagoon products as 'requiring cooking before consumption', but no specifications are given on how long and at what temperature they should be cooked. Our results can provide the consumer with useful indications on how to cook clams to prevent any risk of foodborne illness.

Detection of Cyprinid herpesvirus 2 in association with an Aeromonas sobria infection of Carassius carassius (L.), in Italy.

Sixteen specimens of female crucian carp, Carassius carassius (L.), during the breeding season, were investigated for post-mortem and full diagnostic examination during a mortality outbreak in a tributary stream of the Arno River in Tuscany in 2011. Necropsy highlighted the presence of a swollen anus and widespread haemorrhages in the body, fins, gills and eyes. Haemorrhages in internal organs and spleen granulomas were also observed. Bacteria isolated from the brain, kidney and spleen of affected fish were identified as A. sobria. Microscopic lesions observed in gills were characterized by necrosis of the secondary lamellae, congestion and multifocal lamellar fusion. The kidney showed necrosis, oedema, fibrin exudation and areas of haemorrhages, while in the spleen the main lesions were by multifocal necrosis of the lymphoid tissue. In the gills, transmission electron microscopy revealed herpesvirus-like particles, subsequently identified as Cyprinid herpesvirus-2 (CyHV-2) with a nested PCR protocol. Although it was not possible to attribute a pathogenic role to CyHV-2 in this mortality event, the identification of this herpesvirus in crucian carp increases the concern about its potential role in this species.

Pathologic findings of highly pathogenic avian influenza virus A/Duck/Vietnam/12/05 (H5N1) in experimentally infected pekin ducks, based on immunohistochemistry and in situ hybridization.

The ongoing H5N1 Asian epidemic is currently affecting a number of avian species including ducks. These birds are an important part of the poultry industry in the affected countries, and it is likely that they are acting as a reservoir of infection. Ten Pekin ducks were challenged with 100 microl containing 10(7) 50% egg infective dose of the highly pathogenic avian influenza virus (HPAIV) A/Duck/Vietnam/12/05 (H5N1), administered by an intra-nasal and oral route. Clinical symptoms were recorded twice a day up to 14 days postinfection (dpi). Clinical signs were first noted at 2 dpi, with conjunctivitis and slight depression, and progressed over a period of 1-3 days to severe neurologic signs consisting of torticollis, incoordination, tremors, and seizures. Survival times varied from 3 to 7 dpi. On postmortem examination, hemorrhages were observed in the duodenum, ceca, proventriculus, ventriculus, trachea, pancreas, and brain. Histologic lesions, as well as immunohistochemistry positivity, were recorded in the pancreas and brain. In situ hybridization revealed viral antigen associated with acinar pancreatic cells, bronchial epithelial cells, and with cells of the central nervous system as well as neurons of the submucosal plexus of the duodenum. Our experimental findings agree with those previously observed in ducks naturally infected with HPAIV H5N1 viruses, confirming the acquired viral neurotropism and pancreatotropism, as previously noted in other avian species, as well as in humans.

Conventional H5N9 vaccine suppresses shedding in specific-pathogen-free birds challenged with HPAI H5N1 A/chicken/Yamaguchi/7/2004.

Avian influenza represents one of the greatest concerns for public health that has emerged in recent times. Highly pathogenic avian influenza viruses belonging to the H5N1 subtype are endemic in Asia and are spreading in Europe and Africa. Vaccination is now considered a tool to support eradication efforts, provided it is appropriately managed. This study was carried out to establish the degree of clinical protection and reduction of viral shedding induced by a high-specification, commercially available avian influenza vaccine of a different lineage and containing a strain with a heterologous neuraminidase (H5N9 subtype) to the challenge virus isolate A/chicken/Yamaguchi/7/2004 (H5N1 subtype).

Co-circulation of distinct genetic lineages of astroviruses in turkeys and guinea fowl.

Viruses belonging to the genus Astrovirus have been increasingly associated with enteritis in mammalian and avian species, including turkeys. More recently, astroviruses have also been detected in diseased guinea fowl. In turkeys, two genetically distinct types of astrovirus have been reported, namely turkey astrovirus 1 and 2 (TAstV1 and TAstV2). The prevalence and the pathogenesis of astrovirus infections in this species is currently unknown, with the exception of data generated in the USA. In the present report, we have demonstrated the co-circulation of distinct genetic lineages of astroviruses infecting turkeys and guinea fowl. The predominant lineage infecting turkeys is genetically related to the American TAstV2, but genetic variability within this lineage was demonstrated. Other isolates appeared to be either TastV1-related or unrelated to any other known isolate. Astroviruses infecting guinea fowl were more closely related to TAstV2, and interspecies transmissions between turkeys and guinea fowl is suggested as a possible mechanism by which these viruses may have evolved, based on the genetic data available. This investigation provides genetic and epidemiological information contributing to a better understanding of enteric viral infections in turkeys and guinea fowl.

Comparison of three rapid detection systems for type A influenza virus on tracheal swabs of experimentally and naturally infected birds.

The present paper reports of the comparison between three rapid virus detection systems and virus isolation (VI) from pooled tracheal swabs collected from naturally and experimentally infected birds with a low pathogenicity avian influenza virus of the H7N3 subtype. The relative sensitivity, specificity and agreement (K value) were calculated for a commercial antigen capture enzyme immunoassay (AC-EIA) and for two nucleic acid detection tests, a one-step reverse transcriptase-polymerase chain reaction (RT-PCR) and a real-time RT-PCR (RRT-PCR), both targeting the M gene. The results indicate that in experimentally infected turkeys VI was positive from the pooled tracheal swabs collected from day 3 to day 10. One-step RT-PCR was able to detect influenza RNA from samples collected from day 3 to day 12, while RRT-PCR amplified influenza RNA in swabs collected from day 3 to day 15. The AC-EIA test yielded positive results between day 5 and day 10 post-infection. On field samples, the K value between the AC-EIA and VI tests was 0.82. Compared with VI, the relative sensitivity of this test was 88.9% (CI95 = 85.2-92.6) and the relative specificity was 95.7% (CI95 = 93.7-97.7). The K value between the RT-PCR and VI tests was 0.88. Compared with virus isolation, the relative sensitivity of the one-step RT-PCR was 95.6% (CI95 = 93.1-98.0) and the relative specificity was 96.3% (CI95 = 94.4-98.1). The K value between the RRT-PCR and VI tests was 0.92. Compared with virus isolation, the relative sensitivity and specificity of RRT-PCR was 93.3% (CI95 = 90.4-96.3) and 98.4% (CI95 = 97.2-99.6), respectively. Generally speaking, comparison between virus isolation, the AC-EIA test and the two nucleic acid detection methods indicated excellent agreement. Data obtained from both experimental and field study suggest a higher sensitivity of the PCR-based methods compared with the AC-EIA. The economical and practical implications of using one of the rapid tests as an alternative to VI during an avian influenza epidemic are discussed.