Carlos P. Dopazo

Comparative analysis of both genomic segments of betanodaviruses isolated from epizootic outbreaks in farmed fish species provides evidence for genetic reassortment.

Sequencing of the full coding region of both genomic segments of seven betanodavirus strains isolated from different farmed species in Spain and Portugal revealed that six were reassortants, exhibiting a red-spotted grouper nervous necrosis virus (RGNNV)-type RNA1 and a striped jack nervous necrosis virus (SJNNV)-type RNA2. Analysis of sequences of reassortant strains at both the genomic and protein levels revealed the existence of differences compared with type strains of both genotypes. These differences were greater in the polymerase sequence, which is remarkable because viral structural proteins generally diverge more rapidly than non-structural proteins. Changes in two amino acids observed in the SJNNV capsid protein might be involved in the colonization of new host species by these reassortant strains. In addition, a more extensive phylogenetic analysis, including partial sequences of both RNA segments of 16 other Iberian nodaviruses, confirmed the existence of reassortment between RGNNV and SJNNV.

Bacillary necrosis in hatcheries of Ostrea edulis in Spain

Abstract Two hatcheries of flat oyster (Ostrea edulis) located in the North-West of Spain, which were affected by massive larval mortalities during summer months, were monitored in order to look for the causative organism(s) and the source(s) of the pathogens. Qualitative and quantitative analyses of the bacterial flora, conducted in different parts of the hatcheries, revealed that the total bacterial count (TVC) and the presumptive vibrios (PVC) increased during the spring, following the rise of temperature, up to the start of the outbreaks. Very high TVC levels were found in the phytoplanktonic food of broodstock and larvae; these bacterial populations were composed mainly of Flavobacterium and Pseudomonas but no vibrios were detected. Whereas the number of vibrios in the incoming water was effectively reduced by filtration and U.V. light, Vibrio strains were detected as part of the normal flora in the broodstock and larval tanks favoured by the conditions of nutrients and temperature provided in the hatcheries. The fact that vibrios were present in the gonads of hatchery-conditioned broodstock but not in wild oysters seems to indicate that these opportunistic pathogens can invade and proliferate in the oyster tissues, and subsequently infect the larvae. The agents responsible for the vibriosis outbreaks in both hatcheries resembled Vibrio tubiashii which produces lethal exotoxins for larvae and the typical symptoms of bacillary necrosis. Chloramphenicol was the most effective drug for controlling the disease.

Host range, host specificity and hypothesized host shift events among viruses of lower vertebrates

The successful replication of a viral agent in a host is a complex process that often leads to a species specificity of the virus and can make interspecies transmission difficult. Despite this difficulty, natural host switch seems to have been frequent among viruses of lower vertebrates, especially fish viruses, since there are several viruses known to be able to infect a wide range of species. In the present review we will focus on well documented reports of broad host range, variations in host specificity, and host shift events hypothesized for viruses within the genera Ranavirus, Novirhabdovirus, Betanodavirus, Isavirus, and some herpesvirus.

Restriction Fragment Length Polymorphisms and Sequence Analysis: an Approach for Genotyping Infectious Pancreatic Necrosis Virus Reference Strains and Other Aquabirnaviruses Isolated from Northwestern Spain

ABSTRACT Reference strains of infectious pancreatic necrosis virus resembling the 10 recognized serotypes and local isolates of aquabirnaviruses isolated in northwestern Spain from reservoirs (mollusks) and from asymptomatic and carrier cultured fish were genotyped by restriction fragment length polymorphism (RFLP) and nucleic acid sequence analyses. The RFLP analysis yielded seven genogroups, each of which was clearly correlated with a serotype. Sequence analysis of the three open reading frames provided quite similar results in terms of genogrouping. Based on the results of this study and in order to unify the two types of assays, we propose placing aquabirnaviruses into six genogroups, four of which can be subdivided into two genotypes based on a two-step restriction analysis. The genotyping corresponds with serotyping as follows: genogroup I includes two genotypes corresponding to serotypes A9 (genotype I.1) and A1 (genotype I.2); genogroup II corresponds to serotype A3; genogroup III includes genotypes III.1 (serotype A2) and III.2 (serotype B1); genogroups IV and V include two genotypes, each corresponding to serotypes A5, A6, A7, and A8 (genotypes IV.1, IV.2, V.1, and V.2, respectively);and genogroup VI corresponds to serotype A4. As expected, most local isolates belonged to genotype III.1 and genogroup II. However, a few local isolates corresponded to the American types of genogroup I. Finally, based on the results of this study and due to its simplicity, the two-step restriction analysis assay is proposed as a method for typing new isolates of aquabirnaviruses, and the results correspond to the results of conventional serotyping.

Antemortem versus postmortem methods for detection of betanodavirus in Senegalese sole (Solea senegalensis)

The suitability of nested reverse transcription polymerase chain reaction (nRT-PCR) to detect betanodavirus in blood samples from naturally infected Senegalese sole (Solea senegalensis) was evaluated in comparison with other diagnostic methods. Results indicated that histologic examination of brain lesions could be regarded as the most consistent indicator of nodavirus infection in this species. The nRT-PCR showed low to moderate levels of detection; the best values were obtained in brain samples followed by blood samples. Inoculation of SSN-1 and SAF-1 cells with fish samples did not cause cytopathic effect, although virus was detected by reverse transcription polymerase chain reaction in approximately 25% of the SSN-1 inoculated wells. The efficiency of detection of the viral genome was dramatically increased by the use of nRT-PCR, reaching 90.6% of positives in brain samples and 84.4% in blood samples. The sensitivity and the negative predictive value of nRT-PCR in blood samples were slightly lower than those obtained using brain samples. Nevertheless, it is suggested that the advantage of being able to perform diagnosis on live fish adequately counterbalances the slightly lower sensitivity of nRT-PCR on blood samples. This technique is proposed as a useful tool, not only for the selection of nodavirus-free breeders but also to check the fish status during ongrowing.

Diversity of infectious pancreatic necrosis virus strains isolated from fish, shellfish, and other reservoirs in Northwestern Spain

ABSTRACT A comparison was done of 231 strains of birnavirus isolated from fish, shellfish, and other reservoirs in a survey study that began in 1986 in Galicia (northwestern Spain). Reference strains from all of the infectious pancreatic necrosis virus serotypes were included in the comparison, which was done by neutralization tests and agarose and polyacrylamide gel electrophoresis of the viral genome. The neutralization tests with antisera against the West Buxton, Spajarup (Sp), and Abild (Ab) strains showed that most of the Galician isolates were European types Sp and Ab; however, many isolates (30%) could not be typed. Results from agarose gels did not provided information for grouping of the strains, since all were found to have genomic segments of similar sizes. Analysis of polyacrylamide gels, however, allowed six electropherogroups (EGs) to be differentiated on the basis of genome mobility and separation among segments, and a certain relationship between EGs and serotypes was observed. A wide diversity of electropherotypes was observed among the Galician isolates, and as neutralization tests showed, most of the isolates were included in EGs corresponding to European types Ab and Sp. Only 6.5% of the isolates had the electropherotype characteristic of American strains.

Marine environment as reservoir of birnaviruses from poikilothermic animals

Abstract In this study 51 aquatic birnaviruses were isolated from environmental samples (molluscs, wild fish and sediments) during routine surveys around fish farms located in Galicia (NW of Spain). These viruses were identified as IPN-like viruses based on their physical and chemical characteristics. Furthermore, these isolates were molecular and serologically compared to the reference strains VR-299, Sp and Ab. Although they caused no mortalities in oysters and showed varying degrees of virulence in trout, all isolates were recovered from the inoculated animals, indicating that oysters can become carriers. The results obtained in this study indicate the role of the molluscs, sediments or fish feed as important agents to consider in the epidemiology of birnaviruses.

Use of reverse transcription-real time polymerase chain reaction (real time RT-PCR) assays with Universal Probe Library (UPL) probes for the detection and genotyping of infectious pancreatic necrosis virus strains isolated in Chile

Reverse transcription-real time polymerase chain reaction (real time RT-PCR) assay with Universal Probe Library (UPL) probes has been developed for the detection and genotyping of Chilean infectious pancreatic necrosis virus (IPNV) isolates from infected cell culture. Partial nucleotide sequences (1175 bp) of the VP2 coding region from a selection of 7 Chilean IPNV isolates showed that they clustered into two main groups strongly correlated with Genogroups 1 and 5 proposed by Blake et al. (2001), corresponding to types West Buxton (WB) and Spajarup (Sp), respectively. Based on the VP2 gene sequences of those 7 Chilean isolates and different reference IPNV strains, 2 sets of candidate primer/UPL probes (# 8 and # 117) were designed and evaluated with a total of 32 field isolates isolated from Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and Pacific salmon (Oncorhynchus kisutch) farms from 2006 to 2010 in Chile. The UPL probes clearly differentiated the same two major Genogroups that those recognized by sequencing analysis. Among the Chilean isolates examined, 18 yielded amplification with UPL probe # 8, and 14 with probe # 117, respectively corresponding to types Sp and WB, as demonstrated by typing by sequencing. Based on the findings reported below, it has been demonstrated that the combined real time RT-PCR protocol with UPLs approach was efficient in discriminating distinct Genogroups of IPNV cultured in fish cell lines and, therefore, recommended its use for detection and typing of IPN viruses. The study also confirmed the existence of two IPNV type strains in Chilean salmonid aquaculture.

Relationships among virulence for fish, enterotoxigenicity, and phenotypic characteristics of motile Aeromonas

Abstract In this study we have analysed the biochemical, enzymatic, and cell surface properties of 59 Aeromonas strains isolated from fish culture systems, with the aim of establishing the possible relationships among some of these phenotypic characters and pathogenicity. The cytotoxic activity of extracellular products was also evaluated. Virulence assays showed that 64.3% of the strains were pathogenic for fish. However, both pathogenic and non-pathogenic Aeromonas isolates were able to produce enterotoxins. The majority of the strains were proteolytic, amylolytic and produced DNAase. Nevertheless, elastase and staphylolytic activities were present only in A. hydrophila. Although 96% of the isolates produced haemolysins, a clear specificity toward trout or human erythrocytes was not found in the pathogenic strains. Similarly there was no specificity in the haemagglutinating activity. Statistical analysis of the association between virulence and phenotypic traits revealed a positive relationship among virulence for fish and arabinose and sucrose fermentation, elastase and haemolysis of human erythrocytes. However, only the LDC test showed a significant relation with enterotoxin production. These findings suggest that different mechanisms are involved in the invasion by Aeromonas of their poikilotherm and homeotherm hosts. Extracellular products of the Aeromonas strains displayed cytotoxicity on fish cell-lines regardless of the virulence capacities of the strains, which indicates that cytotoxic activity is not an adequate criterion of pathogenicity.

Presence of viruses in wild eels Anguilla anguilla L, from the Albufera Lake (Spain)

A virological analysis was conducted on wild eels from the Albufera Lake (Spain). A total of 179 individuals at different growth stages were collected in two different surveys (2004 and 2008). Presence of anguillid herpesvirus (AngHV-1), aquabirnavirus and betanodavirus was confirmed by PCR procedures in both surveys, although the number of detections was clearly higher in 2008 (83% of the eels analysed resulted positive for virus presence). AngHV-1 was the viral agent most frequently detected, followed by aquabirnaviruses. Betanodaviruses were detected by the first time in wild eels, and although the detections were only made by nested PCR, high percentage of positives were achieved. In addition, in 2008, seven aquabirnaviruses were isolated. Phylogenetic analysis performed using partial sequences of both genomic segments of aquabirnaviruses indicated that the seven isolates could be typed as WB (genogroup I) on the basis of segment A sequences, but when segment B was used six of them clustered with C1 strain (genogroup V) and one was typed as Ab (genogroup II). These results indicate natural reassortment between different strains of aquabirnaviruses in the eels. Although betanodaviruses were not isolated in cell culture, the analysis of the sequence of the nested PCR product indicated that they clustered with SJNNV genotype. The diversity of viral agents and the high level of viral detections suggest that viral infections may play a more prominent role in the decline of the European eel than initially thought.