Susceptibility of juvenile european sea bass (dicentrarchus labrax) to different viral nervousnecrosis virus (VNNV) isolates

In this study, the susceptibility of 5-g juvenile European sea bass was evaluated by intramuscular injection (105 TCID50/g) using isolates belonging to the RGNNV and SJNNV genotypes as well as a reassortant isolate (RGNNV RNA1/SJNNV RNA2) obtained from Senegalese sole (Solea senegalensis). In these experimental infections, the cumulative mortality was determined. Furthermore, quantification of viral genome, by absolute real time PCR, and infective viral particles, by virus titration, was performed from brains of dead and survivor fish (30 days post-inoculation). In addition, anti-VNNV antibodies in sera from survivor animals were determined by indirect ELISA. Typical symptoms of VNN and mortality were only recorded in fish inoculated with the RGNNV (47% cumulative mortality) and the reassortant (33%) isolates. However, high levels of viral genome and infective viral particles were recorded in brain of survivor fish inoculated with the SJNNV isolate, although did not cause mortality or clinical signs. Specific antibody response, measured by indirect ELISA, was only observed in the VNNV-inoculated groups, with titres of 1/1024, 1/4096 and 1/8192 for RGNNV, SJNNV and reassortant inoculated animals, respectively.UNIVERSIDAD DE MÁLAGA. CAMPUS DE EXCELENCIA INTERNACIONAL ANDALUCÍA TEC

Detection of Chikunguny a virus by RT-LAMP

Background A single-tube one-step real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of chikungunya virus (CHIKV) targeting the conserved 6K-E1 target region was developed. The assay was validated with sera collected from a CHIKV outbreak in Senegal in 2015. Methodology/Principal findings A novel design approach by combining Principal Component Analysis and phylogenetic analysis of 110 available CHIKV sequences and the LAMP oligonucleotide design software LAVA was used. The assay was evaluated with an External Quality Assessment panel from the European Network for Diagnostics of "Imported" Viral Diseases and was shown to be sensitive and specific and did not cross-detect other arboviruses. The limit of detection as determined by probit analysis, was 163 molecules, and 100% reproducibility in the assays was obtained for 103 molecules (7/8 repetitions were positive for 102 molecules). The assay was validated using 35 RNA samples extracted from sera, and results were compared with those obtained by quantitative RT-PCR carried out at the Institut Pasteur Dakar, demonstrating that the RT-LAMP is 100% sensitive and 80% specific, with a positive predictive value of 97% and negative predictive value of 100%. Conclusions/Significance The RT-LAMP appeared to show superior performance with material stored for months compared to qRT-PCR and can be therefore recommended for use in infrastructures with poor settings

Distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens in nervous and non-nervous organs of European seabass (Dicentrarchus labrax) during the course of an experimental challenge

The distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens was examined by immunohistochemistry in the nervous and non-nervous organs of juvenile European seabass (Dicentrarchus labrax) during the course of an intramuscular infection. Histological changes resulting from the infection were evaluated from 3 days to 2 months post-infection. The specific antibody response was also studied 2 months post-challenge. Viral proteins were present throughout the experimental period in the retina (inner nuclear layer, ganglion layer, outer limiting membrane, and outer plexiform layer), brain (cerebellum and tectum opticum), and liver (hepatocytes and endothelial cells). These proteins were also observed in the renal tubular cells, white pulp of spleen, and in fibroblasts and cartilage of caudal fin. This is the first report of RGNNV proteins appearing in these organs, where the immunostaining was only detected at certain sampling times after the onset of mortality. Brain and retina of virus-exposed fish showed high levels of vacuolation, while accumulation of fat vacuoles was observed in the liver. RGNNV infection also induced a specific antibody response as measured by an ELISA. In summary, this is the first study demonstrating the presence of viral proteins in cells of caudal fin, kidney and spleen of European seabass

Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of Francisella noatunensis subsp. orientalis

Francisella noatunensis subsp. orientalis (Fno) is the causative agent of piscine francisellosis in warm water fish including tilapia. The disease induces chronic granulomatous inflammation with high morbidity and can result in high mortality. Early and accurate detection of Fno is crucial to set appropriate outbreak control measures in tilapia farms. Laboratory detection of Fno mainly depends on bacterial culture and molecular techniques. Recombinase polymerase amplification (RPA) is a novel isothermal technology that has been widely used for the molecular diagnosis of various infectious diseases. In this study, a recombinase polymerase amplification (RPA) assay for rapid detection of Fno was developed and validated. The RPA reaction was performed at a constant temperature of 42 degreesC for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in tilapia farms, but also by screening 78 Nile tilapia and 5 water samples. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other tested bacteria. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The RPA gave quicker results in approximately 6 min in contrast to the qPCR that needed about 90 min to reach the same detection limit, taking only 2.7- 3 min to determine Fno in clinical samples. Moreover, RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic 41 tool that will contribute to controlling the infection through prompt on-site detection of Fno

Detection of each DENV serotype by RT-LAMP

Background 4 one-step, real-time, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed for the detection of dengue virus (DENV) serotypes by considering 2,056 full genome DENV sequences. DENV1 and DENV2 RT-LAMP assays were validated with 31 blood and 11 serum samples from Tanzania, Senegal, Sudan and Mauritania. DENV3 and DENV4 RT-LAMP assays were validated with 25 serum samples from Cambodia Methodology/Principal findings 4 final reaction primer mixes were obtained by using a combination of Principal Component Analysis of the full DENV genome sequences, and LAMP primer design based on sequence alignments using the LAVA software. These mixes contained 14 (DENV1), 12 (DENV2), 8 (DENV3) and 3 (DENV4) LAMP primer sets. The assays were evaluated with an External Quality Assessment panel from Quality Control for Molecular Diagnostics. The assays were serotype-specific and did not cross-detect with other flaviviruses. The limits of detection, with 95% probability, were 22 (DENV1), 542 (DENV2), 197 (DENV3) and 641 (DENV4) RNA molecules, and 100% reproducibility in the assays was obtained with up to 102 (DENV1) and 103 RNA molecules (DENV2, DENV3 and DENV4). Validation of the DENV2 assay with blood samples from Tanzania resulted in 23 samples detected by RT-LAMP, demonstrating that the assay is 100% specific and 95.8% sensitive (positive predictive value of 100% and a negative predictive value of 85.7%). All serum samples from Senegal, Sudan and Mauritania were detected and 3 untyped as DENV1. The sensitivity of RT-LAMP for DENV4 samples from Cambodia did not quite match qRT-PCR. Conclusions/Significance We have shown a novel approach to design LAMP primers that makes use of fast growing sequence databases. The DENV1 and DENV2 assays were validated with viral RNA extracted clinical samples, showing very good performance parameters

Whole cell inactivated autogenous vaccine effectively protects red Nile tilapia (Oreochromis niloticus) against francisellosis via intraperitoneal injection

Francisella noatunensis subsp. orientalis is a pathogen of tilapia and other warm‐water fish for which no vaccines are commercially available. In this study, a whole cell formalin‐inactivated vaccine was developed for the first time using the highly virulent isolate STIR‐GUS‐F2f7 and the oil‐based adjuvant Montanide™ ISA 763A VG. The efficacy of the vaccine was assessed in red Nile tilapia via intraperitoneal (i.p.) injection using homologous experimental infection and correlates of protection such as seral antibody production and bacterial loads in the spleen. For immunization, fish were i.p. injected with 0.1 ml of the vaccine, the adjuvant alone or PBS. At 840 degree days post‐vaccination, all fish were i.p. injected with 4.0 × 103 CFU/fish of pathogenic bacteria. The RPS at the end of the trial was 100% in the vaccinated group with significantly higher survival than in the adjuvant and control groups. The RPS in the adjuvant group was 42%, and no significant difference was seen in survival between this and the PBS group. Moreover, significantly higher antibody titres in the serum and significantly lower bacterial loads in the spleen were detected in the vaccinated fish by ELISA and qPCR, respectively. These findings highlight the potential of autogenous vaccines for controlling francisellosis in tilapia

Fully automated point-of-care differential diagnosis of acute febrile illness

Background In this work, a platform was developed and tested to allow to detect a variety of candidate viral, bacterial and parasitic pathogens, for acute fever of unknown origin. The platform is based on a centrifugal microfluidic cartridge, the LabDisk (“FeverDisk” for the specific application), which integrates all necessary reagents for sample-to-answer analysis and is processed by a compact, point-of-care compatible device. Methodology/Principal findings A sample volume of 200 μL per FeverDisk was used. In situ extraction with pre-stored reagents was achieved by bind-wash-elute chemistry and magnetic particles. Enzymes for the loop-mediated isothermal amplification (LAMP) were pre-stored in lyopellet form providing stability and independence from the cold chain. The total time to result from sample inlet to read out was 2 h. The proof-of-principle was demonstrated in three small-scale feasibility studies: in Dakar, Senegal and Khartoum, Sudan we tested biobanked samples using 29 and 9 disks, respectively; in Reinfeld, Germany we tested spiked samples and analyzed the limit of detection using three bacteria simultaneously spiked in whole blood using 15 disks. Overall during the three studies, the FeverDisk detected dengue virus (different serotypes), chikungunya virus, Plasmodium falciparum, Salmonella enterica Typhi, Salmonella enterica Paratyphi A and Streptococcus pneumoniae. Conclusions/Significance The FeverDisk proved to be universally applicable as it successfully detected all different types of pathogens as single or co-infections, while it also managed to define the serotype of un-serotyped dengue samples. Thirty-eight FeverDisks at the two African sites provided 59 assay results, out of which 51 (86.4%) were confirmed with reference assay results. The results provide a promising outlook for future implementation of the platform in larger prospective clinical studies for defining its clinical sensitivity and specificity. The technology aims to provide multi-target diagnosis of the origins of fever, which will help fight lethal diseases and the incessant rise of antimicrobial resistance.Additional co-authors: Sieghard Frischmann, Konstantinos Mitsakaki

Experimental susceptibility of European sea bass and Senegalese sole to different betanodavirus isolates

The susceptibility of juvenile European sea bass and Senegalese sole to three VNNV isolates (a reassortant RGNNV/SJNNV, as well as the parental RGNNV and SJNNV genotypes) has been evaluated by challenges using two inoculation ways (bath and intramuscular injection). The results demonstrate that these two fish species are susceptible to all the VNNV isolates tested. In European sea bass, RGNNV caused the highest cumulative mortality, reaching maximum values of viral RNA and titres. Although the SJNNV isolate did not provoke mortality or clinical signs of disease in this fish species, viral production in survivor fish was determined; on the other hand the reassortant isolate did cause mortality and clinical signs of disease, although less evident than those recorded after RGNNV infection. These results suggest that the changes suffered by the SJNNV RNA2 segment of the reassortant isolate, compared to the parental SJNNV, may have involved host-specificity and/or virulence determinants for European sea bass. Regarding Senegalese sole, although the three isolates caused 100% mortality, the reassortant strain provoked the most acute symptoms, and more quickly, especially in the bath challenge. This was also the isolate showing less difference between the number of RNA copies and viral titre, reaching the highest titres of infective viral particles in nervous tissue of infected animals. The RGNNV isolate produced the lowest values of infective viral particles. All these results suggest that the RGNNV and the reassortant isolates are the most suited for infecting European sea bass and Senegalese sole, respectively

Effect of the coexistence on the replication of striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV) using an in vitro approach

Viral nervous necrosis virus (VNNV) is the aetiological agent of viral nervous necrosis (VNN), a widespread disease affecting different marine and freshwater fish species. Striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV) are the only genotypes of the Betanodavirus genus recorded in the Iberian Peninsula to date, but a high percentage of wild specimens simultaneously carrying both genotypes has been recently reported. The coexistence of the two viruses may affect the course of both viral infections. In the present study, viral genome quantification by two absolute real-time PCR protocols has been performed to characterise the effect of the RGNNV-SJNNV coexistence (coinfection and superinfection) on the replication of each genotype in E-11 cells. This is the first study showing the effect of the coexistence on the viral replication of two genotypes within the Betanodavirus genus. The results obtained in vitro showed the partial inhibition of SJNNV replication by the coexistence with RGNNV, whereas RGNNV replication was favoured in coinfection or superinfection with SJNNV

The effect of dietary n-3/n-6 polyunsaturated fatty acid ratio on salmonid alphavirus subtype 1 (SAV-1) replication in tissues of experimentally infected rainbow trout (Oncorhynchus mykiss)

Salmon pancreas disease (SPD) is one of the most commercially significant viral diseases of farmed Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) in Europe. In this study, the potential for dietary mitigation of the disease using different polyunsaturated fatty acid (PUFA) profiles was assessed in rainbow trout. We experimentally infected fish with salmonid alphavirus subtype 1 (SAV-1), the causative agent of SPD. These fish were fed two diets with different n-3/n-6 PUFA ratio (high omega 3, 3.08, and high omega 6, 0.87). We assessed the influence of the diets on the fatty acid composition of the heart at 0 days post infection (d.p.i.) (after 4 weeks of feeding the experimental diets prior to SAV-1 infection), and sampled infected and control fish at 5, 15 and 30d.p.i. Viral E1 and E2 glycoprotein genes were quantified by two absolute real-time PCRs in all the organs sampled, and significantly lower levels of the virus were evident in the organs of fish fed with high omega 6. Characteristic pathological lesions were identified in infected fish as early as 5d.p.i., with no significant differences in the pathology lesion scores between the two dietary regimes. This study shows that decreasing the n-3/n-6 PUFA ratio in experimental diets of rainbow trout changes the fatty acid content of the fish, and is associated with reduced SAV-1 replication in rainbow trout