Development and validation of a real-time TaqMan PCR assay for the detection of betanodavirus in clinical specimens.

Betanodaviruses are the causal agents of viral encephalo-retinopathy, an infectious disease affecting more than 40 marine fish species, characterized by high morbidity and mortality. Because of its severe impact, robust diagnostic tools are required. The aim of this work was to develop and validate a real-time TaqMan PCR assay to detect betanodaviruses in clinical specimens by amplifying a conserved region of the RNA2 strand. The method proved to be specific and sensitive, being capable of detecting as low as 10 TCID(50)/ml. For clinical validation, samples from 100 marine fish were collected during a natural outbreak of disease and tested by three distinct laboratory methods, namely real-time TaqMan PCR, RT-seminested PCR and virus isolation. The results indicated optimal agreement between tests. The assay that was developed is capable of detecting members of all of the betanodavirus genetic groups currently described and can be considered a valid alternative to the time-consuming and contamination-prone nested PCR.

Direct identification of Photobacterium damselae subspecies piscicida by PCR-RFLP analysis.

Fish pasteurellosis is an infectious disease that affects several teleost species living in temperate marine waters. The pathogen responsible, Photobacterium damselae subspecies piscicida, shows high genetic similarity with P. damselae subsp. damselae, making subspecies discrimination extremely laborious. Here we report for the first time a PCR-RFLP method for the identification of P. damselae subsp. piscicida without prior isolation in pure culture. Genomic sequence information was obtained through cloning and sequencing of RAPD products. Two P. damselae-specific primer pairs were developed and tested on 17 strains of P. damselae subsp. piscicida, 10 strains of P. damselae subsp. damselae, and 6 closely related control species. High sensitivity was achieved in PCR amplification on serially diluted samples (<180 fg of pure bacterial DNA or <10 fg, depending on the amplified fragment). Restriction analysis of PCR products showed a unique digestion profile for all P. damselae subsp. piscicida strains. The same PCR-RFLP method was implemented on total DNA samples extracted from experimentally infected sea bream and sea bass. Positive results were obtained on fish with clear signs of the disease as well as on challenged, but asymptomatic, fish. The method presented here might provide a useful tool for both prevention and rapid diagnosis of fish pasteurellosis.

Sequence comparison and phylogenetic analysis of fish nodaviruses based on the coat protein gene.

We have amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced a 605-bp fragment covering the variable region of the coat protein gene of fish nodaviruses infecting European sea bass, Dicentrarchus labrax (n = 24), and shi drum, Umbrina cirrosa (n = 2), in the Mediterranean basin. Nine new isolates were identified and their sequences were combined with sequences in the literature to produce three different data sets. The first set, based on amino acid sequences, was used to verify the monophyly of fish nodaviruses. The second and third data sets, based on nucleic acids, were used to resolve the phylogenetic relationships between closely related fish nodaviruses. Phylogenetic analyses were performed according to the maximum parsimony and neighbor-joining methods. Our results support the monophyly of fish nodaviruses. Moreover, they confirm the subdivision of fish nodaviruses into four main clusters, in agreement with the previously suggested phylogeny of the genus Piscinodavirus, that was based on a smaller number of sequences and an alternative phylogenetic approach [14]. All the Mediterranean isolates were clustered in the group of the red-spotted grouper nervous necrosis virus and appear to have a restricted geographic distribution, except for one sequence-type (10 samples) that is widespread throughout the basin.

Optimization of single-strand conformation polymorphism and sequence analysis of the mitochondrial control region in Pagellus bogaraveo (Sparidae, Teleostei): rationalized tools in fish population biology.

We report the isolation and sequencing of 400-550 base pairs (bp) of the mitochondrial DNA (mtDNA) control region of eight species of Sparidae (Perciformes, Teleostei). This sequence information allowed us to design specific primers to one of these species (Pagellus bogaraveo). The new set of primers was used to test a rationalized approach to study the mtDNA nucleotide variability at the intraspecific level. The single-strand conformation polymorphism (SSCP) technique was applied to detect sequence variation in two non-overlapping fragments of the control region of 32 individuals of P. bogaraveo. To assess the sensitivity of the method, the nucleotide sequence of the analysed region was determined for all the specimens. The results showed that, for one of the two fragments, SSCP analysis was able to detect 100% of the underlying genetic variability. In sharp contrast, nucleotide variation of the second DNA fragment was completely unresolved by SSCP under different experimental conditions. This suggests that the resolution power of SSCP is crucially dependent on the nature of the fragment subjected to the analysis; therefore, a preliminary test of the sensitivity of the method should be performed on each specific DNA fragment before starting a large-scale survey. A rationalized approach, combining the SSCP technique and a simplified sequencing procedure, is proposed for studying intraspecific polymorphism at the mtDNA control region in fish.