Toward better monitoring of human noroviruses and F-specific RNA bacteriophages in aquatic environments using bivalve mollusks and passive samplers: A case study.

Monitoring pathogenic enteric viruses in continental and marine water bodies is essential to control the viral contamination of human populations. Human Noroviruses (NoV) are the main enteric viruses present in surface waters and foodstuff. In a context of global change, it is currently a challenge to improve the management of viral pollutions in aquatic environments and thereby limit the contamination of vulnerable water bodies or foodstuffs. The aim of this study is to evaluate the potential of specific accumulation systems for improving the detection of NoV in water bodies, compared to direct water analyses. Passive samplers (Zetapor filters) and three species of bivalve molluscan shellfish (BMS) (Dreissena polymorpha, Mytilus edulis and Crassostreas gigas) were used as accumulation systems to determine their performance in monitoring continental and marine waters for viruses. F-specific RNA bacteriophages (FRNAPH) were also analyzed since they are described as indicators of NoV hazard in many studies. During a one-year study in a specific area frequently affected by fecal pollution, twelve campaigns of exposure of passive samplers and BMS in continental and coastal waters were conducted. Using suitable methods, NoV (genome) and FRNAPH (infectious and genome) were detected in these accumulation systems and in water at the same time points to determine the frequency of detection but also to gain a better understanding of viral pollution in this area. The reliability of FRNAPH as a NoV indicator was also investigated. Our results clearly showed that BMS were significantly better than passive samplers and direct water analyses for monitoring NoV and FRNAPH contamination in water bodies. A dilution of viral pollution between the continental and the coastal area was observed and can be explained by the distance from the source of the pollution. Viral pollution is clearly greater during the winter period, and stakeholders should take this into consideration in their attempts to limit the contamination of food and water. A significant correlation was once again shown between NoV and FRNAPH genomes in BMS, confirming the reliability of FRNAPH as a NoV indicator. Moreover, a strong correlation was observed between NoV genomes and infectious FRNAPH, suggesting recent viral pollution since infectious particles had not been inactivated at sufficient levels in the environment. More generally, this study shows the value of using BMS as an active method for improving knowledge on the behavior of viral contamination in water bodies, the ranking of the contamination sources, and the vulnerability of downstream water bodies.

First isolation of Francisella halioticida strains from blue mussel (Mytilus edulis) in Normandy, France.

Mass mortality events affecting the blue mussels Mytilus edulis have been observed in France since 2014. The DNA of the bacterium Francisella halioticida, reported as pathogen of giant abalone (Haliotis gigantea) and Yesso scallop (Mizuhopecten yessoensis) has been detected recently in mussels from areas suffering mortalities. Isolation of this bacterium was attempted from individuals collected during mortality events. Identification was performed by 16S rRNA gene sequencing, real-time specific PCR and MALDI-ToF using spectra produced from the strain 8472-13A isolated from diseased Yesso scallop in Canada. Five isolates were identified as F. halioticida by real-time specific PCR and 16S rRNA sequencing. MALDI-ToF allowed the direct identification of four isolates (FR22a,b,c,d) which had 100% identity on the 16S rRNA gene with the known strains. On the other hand, one isolate (FR21) was not recognized by MALDI-ToF and had 99.9% identity on the 16S rRNA gene. The FR22 isolates showed difficult growth and required media optimization, which was not the case with the FR21 isolate. For these reasons, it was hypothesized that two type strains are present on French coasts, named FR21 and FR22. The FR21 isolate was selected for phenotypic analysis (growth curve, biochemical characteristics, electron microscopy), phylogenetic analysis and an experimental challenge. This isolate showed distinct differences compared to published F. halioticida strains, both at phenotypic and genotypic levels. Experimental infections of adult mussels led to 36% mortalities in 23 days following intramuscular injection with 3 × 107 CFU while a lower dose (3 × 103 CFU) did not lead to significant mortalities. In the context of this study, the strain FR21 was not virulent towards adult mussels.

How tibial tubercle torsion impacts patellar stability: A biomechanics study.

INTRODUCTION: A new factor for patellofemoral instability-external torsion of the tibial tubercle-has recently been described. The primary aim of this biomechanics study was to analyze the consequences of internal torsion tibial tubercle osteotomy (TTO) on an experimentally unstable patella. We hypothesized that internal TTO can stabilize an experimental patellar instability. MATERIALS AND METHODS: This in vitro study was conducted on six fresh anatomical specimens. The knees were flexed to 25°. The patella was destabilized by transecting the patellar retinaculae and the vastus medialis tendon and by applying continuous oblique traction on the quadriceps tendon. A 3D stereovision system was used to record patellar displacement and tilt and to determine whether patellar dislocation occurred. The measurements were done before the osteotomy then repeated on the same knee after a triangular internal torsion 30° TTO was completed, without medialization. RESULTS: There was a significant difference in the patellar displacement and tilt before and after the osteotomy (p<0.05). Patellar dislocation, which was induced by traction on all the knees before osteotomy, did not occur after the osteotomy was performed. DISCUSSION: Internal torsion of the tibial tubercle improves patellar stability, confirming our hypothesis. These findings confirm the stabilizing effect of placing the tibial tuberosity in internal torsion. Although a knee without instability factors is not the perfect model for patellar instability, our findings suggest that tibial tubercle torsion influences patellar stability. Internal TTO may be justified as a surgical treatment of patellofemoral instability.

Rearing practices identified as risk factors for ostreid herpesvirus 1 (OsHV-1) infection in Pacific oyster Crassostrea gigas spat.

Early detection of Pacific oyster spat infected with ostreid herpesvirus 1 (OsHV-1) could prevent introduction of OsHV-1-infected individuals into farming areas or onshore rearing facilities, thus reducing the risk of infection of naïve oysters in such production systems. Experiments were conducted on several hundred oyster spat provided by producers in order to examine whether early rearing practices could be considered as potential risk factors for (1) OsHV-1 infection as detected by molecular methods and (2) spat mortality experimentally induced through thermal challenge. Spat groups collected on oyster beds and hatchery spat reared in growout areas during summer exhibited higher viral DNA contamination and mortalities during the trial than spat kept in onshore rearing facilities. Quantification of viral DNA before and during the trial showed that infection prevalence and intensity changed over time and revealed latent infection initially unsuspected in 3 of 10 groups. Thermal challenge induced a clear increase in the probability of detecting infected individuals, particularly for groups exhibiting significant prevalence of OsHV-1-contaminated spat prior to the challenge. The use of detection methods are discussed in relation to early rearing practices and disease control strategies.

Physiological change under OsHV-1 contamination in Pacific oyster Crassostrea gigas through massive mortality events on fields.

BACKGROUND: Massive mortalities have been observed in France since 2008 on spat and juvenile Pacific oysters, Crassostrea gigas. A herpes virus called OsHV-1, easily detectable by PCR, has been implicated in the mortalities as demonstrated by the results of numerous field studies linking mortality with OsHV-1 prevalence. Moreover, experimental infections using viral particles have documented the pathogenicity of OsHV-1 but the physiological responses of host to pathogen are not well known. RESULTS: The aim of this study was to understand mechanisms brought into play against the virus during infection in the field. A microarray assay has been developed for a major part of the oyster genome and used for studying the host transcriptome across mortality on field. Spat with and without detectable OsHV-1 infection presenting or not mortality respectively were compared by microarray during mortality episodes. In this study, a number of genes are regulated in the response to pathogen infection on field and seems to argue to an implication of the virus in the observed mortality. The result allowed establishment of a hypothetic scheme of the host cell's infection by, and response to, the pathogen. CONCLUSIONS: This response shows a "sensu stricto" innate immunity through genic regulation of the virus OsHV-1 life cycle, but also others biological processes resulting to complex interactions between host and pathogens in general.