Development of an attenuated vaccine against Koi Herpesvirus Disease (KHVD) suitable for oral administration and immersion.

Since the end of the1990ies, Cyprinid herpesvirus 3 (also known as koi herpesvirus, KHV) has caused mass mortality events of koi and common carp all over the globe. This induced a high economic impact, since the KHV disease cannot be cured up to now, but only prevented by vaccination. Unfortunately, there is only one commercial vaccine available which is not approved in most countries. Therefore, there is an urgent need for new, safe and available vaccines. In this study, a live attenuated vaccine virus was generated by cell culture passages of virulent KHV, and shown to protect carp or koi after immersion or oral application against wild type challenge. An advantage of boost immunization was demonstrated, especially after oral application. Vaccination induced no or mild clinical signs and protecting antibodies have been measured. Additionally, the vaccine virus allowed differentiation of infected from vaccinated animals (DIVA) by PCR. The attenuation of the newly generated vaccine was tracked down to a partial deletion of open reading frame 150. This was confirmed by the generation of engineered ORF150 deletion mutants of wild-type KHV which exhibited a similar attenuation in vivo.

Genetic diversity and connectivity of the Ostreid herpesvirus 1 populations in France: A first attempt to phylogeographic inference for a marine mollusc disease.

The genetic diversity of viral populations is a key driver of the spatial and temporal diffusion of viruses; yet, studying the diversity of whole genomes from natural populations still remains a challenge. Phylodynamic approaches are commonly used for RNA viruses harboring small genomes but have only rarely been applied to DNA viruses with larger genomes. Here, we used the Pacific oyster mortality syndrome (a disease that affects oyster farms around the world) as a model to study the genetic diversity of its causative agent, the Ostreid herpesvirus 1 (OsHV-1) in the three main French oyster-farming areas. Using ultra-deep sequencing on individual moribund oysters and an innovative combination of bioinformatics tools, we de novo assembled twenty-one OsHV-1 new genomes. Combining quantification of major and minor genetic variations, phylogenetic analysis, and ancestral state reconstruction of discrete traits approaches, we assessed the connectivity of OsHV-1 viral populations between the three oyster-farming areas. Our results suggest that the Marennes-Oléron Bay represents the main source of OsHV-1 diversity, from where the virus has dispersed to other farming areas, a scenario consistent with current practices of oyster transfers in France. We demonstrate that phylodynamic approaches can be applied to aquatic DNA viruses to determine how epidemiological, immunological, and evolutionary processes act and potentially interact to shape their diversity patterns.

An ancient truncated duplication of the anti-Müllerian hormone receptor type 2 gene is a potential conserved master sex determinant in the Pangasiidae catfish family.

The evolution of sex determination (SD) in teleosts is amazingly dynamic, as reflected by the variety of different master sex-determining genes identified. Pangasiids are economically important catfishes in South Asian countries, but little is known about their SD system. Here, we generated novel genomic resources for 12 Pangasiids and characterized their SD system. Based on a Pangasianodon hypophthalmus chromosome-scale genome assembly, we identified an anti-Müllerian hormone receptor type Ⅱ gene (amhr2) duplication, which was further characterized as being sex-linked in males and expressed only in testes. These results point to a Y chromosome male-specific duplication (amhr2by) of the autosomal amhr2a. Sequence annotation revealed that the P. hypophthalmus Amhr2by is truncated in its N-terminal domain, lacking the cysteine-rich extracellular part of the receptor that is crucial for ligand binding, suggesting a potential route for its neofunctionalization. Reference-guided assembly of 11 additional Pangasiids, along with sex-linkage studies, revealed that this truncated amhr2by duplication is a male-specific conserved gene in Pangasiids. Reconstructions of the amhr2 phylogeny suggested that amhr2by arose from an ancient duplication/insertion event at the root of the Siluroidei radiation that is dated to ~100 million years ago. Together these results bring multiple lines of evidence supporting that amhr2by is an ancient and conserved master sex-determining gene in Pangasiids, a finding that highlights the recurrent use of the transforming growth factor ß pathway, which is often used for the recruitment of teleost master SD genes, and provides another empirical case towards firther understanding of dynamics of SD systems.

Application of the FTA elute card coupled with visual colorimetric loop-mediated isothermal amplification for the rapid diagnosis of Streptococcus agalactiae in farmed tilapia (Oreochromis niloticus).

A method combining the FTA Elute card and visual colorimetric loop-mediated isothermal amplification (FTA-e/LAMP) was tested to diagnose Streptococcus agalactiae infections in vitro and in vivo. FTA-e/LAMP consists of two main steps: first, the FTA card is used to extract DNA and then a colorimetric loop-mediated isothermal amplification (LAMP) reaction is carried out on the extracted DNA. In vitro sensitivity was 1.9 x 102  CFU/mL, and regarding specificity, all nine S. agalactiae strains tested positive. All Streptococcus spp. tested negative, except for S. dysgalactiae, thereby indicating the need for another set of primers to distinguish this species from S. agalactiae. To diagnose S. agalactiae infections using FTA-e/LAMP in vivo, two experimental trials on juvenile Oreochromis niloticus infected with bovine or piscine strains were carried out. Sensitivity in symptomatic fish was 100%, and 50.7% of fish without signs were positive. All negative control fish tested negative (n = 28). No bacteria were detected after 16 days post-infection (dpi). Accuracy during the first week (1-7 dpi) was 89% and decreased to 44% thereafter (10-22 dpi). FTA-e/LAMP results suggest that this method is a promising tool for early and fast diagnosis of S. agalactiae on tilapia farms.

Intra-Host Diversity of SARS-Cov-2 Should Not Be Neglected: Case of the State of Victoria, Australia.

Since the identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the etiological agent of the current COVID-19 pandemic, a rapid and massive effort has been made to obtain the genomic sequences of this virus to monitor (in near real time) the phylodynamic and diversity of this new pathogen. However, less attention has been given to the assessment of intra-host diversity. RNA viruses such as SARS-CoV-2 inhabit the host as a population of variants called quasispecies. We studied the quasispecies diversity in four of the main SARS-CoV-2 genes (ORF1a, ORF1b, S and N genes), using a dataset consisting of 210 next-generation sequencing (NGS) samples collected between January and early April of 2020 in the State of Victoria, Australia. We found evidence of quasispecies diversity in 68% of the samples, 76% of which was nonsynonymous variants with a higher density in the spike (S) glycoprotein and ORF1a genes. About one-third of the nonsynonymous intra-host variants were shared among the samples, suggesting host-to-host transmission. Quasispecies diversity changed over time. Phylogenetic analysis showed that some of the intra-host single-nucleotide variants (iSNVs) were restricted to specific lineages, highlighting their potential importance in the epidemiology of this virus. A greater effort must be made to determine the magnitude of the genetic bottleneck during transmission and the epidemiological and/or evolutionary factors that may play a role in the changes in the diversity of quasispecies over time.

Aquaculture at the crossroads of global warming and antimicrobial resistance.

In many developing countries, aquaculture is key to ensuring food security for millions of people. It is thus important to measure the full implications of environmental changes on the sustainability of aquaculture. We conduct a double meta-analysis (460 articles) to explore how global warming and antimicrobial resistance (AMR) impact aquaculture. We calculate a Multi-Antibiotic Resistance index (MAR) of aquaculture-related bacteria (11,274 isolates) for 40 countries, of which mostly low- and middle-income countries present high AMR levels. Here we show that aquaculture MAR indices correlate with MAR indices from human clinical bacteria, temperature and countries' climate vulnerability. We also find that infected aquatic animals present higher mortalities at warmer temperatures. Countries most vulnerable to climate change will probably face the highest AMR risks, impacting human health beyond the aquaculture sector, highlighting the need for urgent action. Sustainable solutions to minimise antibiotic use and increase system resilience are therefore needed.

Cyprinid herpesvirus 3 Evolves In Vitro through an Assemblage of Haplotypes that Alternatively Become Dominant or Under-Represented.

Viruses are able to evolve in vitro by mutations after serial passages in cell cultures, which can lead to either a loss, or an increase, of virulence. Cyprinid herpesvirus 3 (CyHV-3), a 295-kb double-stranded DNA virus, is the etiological agent of the koi herpesvirus disease (KHVD). To assess the influence of serial passages, an isolate of CyHV-3 (KHV-T) was passaged 99 times onto common carp brain (CCB) cells, and virus virulence was evaluated during passages through the experimental infections of common carp. After 78 CCB passages, the isolate was much less virulent than the original form. A comparative genomic analysis of these three forms of KHV-T (P0, P78 and P99) revealed a limited number of variations. The largest one was a deletion of 1363 bp in the predicted ORF150, which was detected in P78, but not in P99. This unexpected finding was confirmed by conventional PCR and digital PCR. The results presented here primarily suggest that, CyHV-3 evolves, at least in vitro, through an assemblage of haplotypes that alternatively become dominant or under-represented.

Resting Cyst Distribution and Molecular Identification of the Harmful Dinoflagellate Margalefidinium polykrikoides (Gymnodiniales, Dinophyceae) in Lampung Bay, Sumatra, Indonesia.

Margalefidinium polykrikoides, an unarmored dinoflagellate, was suspected to be the causative agent of the harmful algal blooms - associated with massive fish mortalities - that have occurred continually in Lampung Bay, Indonesia, since the first bloom event in October 2012. In this study, after examination of the morphology of putative M. polykrikoides-like cysts sampled in bottom sediments, cyst bed distribution of this harmful species was explored in the inner bay. Sediment samples showed that resting cysts, including several morphotypes previously reported as M. polykrikoides, were most abundant on the northern coast of Lampung Bay, ranging from 20.6 to 645.6 cysts g-1 dry sediment. Molecular phylogeny inferred from LSU rDNA revealed that the so-called Mediterranean ribotype was detected in the sediment while M. polykrikoides motile cells, four-cell chain forming in bloom conditions, belonged to the American-Malaysian ribotype. Moreover, hyaline cysts, exclusively in the form of four-cell chains, were also recorded. Overall, these results unequivocally show that the species M. polykrikoides is abundantly present, in the form of vegetative cells, hyaline and resting cysts in an Indonesian area.

Targeted genomic enrichment and sequencing of CyHV-3 from carp tissues confirms low nucleotide diversity and mixed genotype infections.

Koi herpesvirus disease (KHVD) is an emerging disease that causes mass mortality in koi and common carp, Cyprinus carpio L. Its causative agent is Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV). Although data on the pathogenesis of this deadly virus is relatively abundant in the literature, still little is known about its genomic diversity and about the molecular mechanisms that lead to such a high virulence. In this context, we developed a new strategy for sequencing full-length CyHV-3 genomes directly from infected fish tissues. Total genomic DNA extracted from carp gill tissue was specifically enriched with CyHV-3 sequences through hybridization to a set of nearly 2 million overlapping probes designed to cover the entire genome length, using KHV-J sequence (GenBank accession number AP008984) as reference. Applied to 7 CyHV-3 specimens from Poland and Indonesia, this targeted genomic enrichment enabled recovery of the full genomes with >99.9% reference coverage. The enrichment rate was directly correlated to the estimated number of viral copies contained in the DNA extracts used for library preparation, which varied between ∼5000 and ∼2×107. The average sequencing depth was >200 for all samples, thus allowing the search for variants with high confidence. Sequence analyses highlighted a significant proportion of intra-specimen sequence heterogeneity, suggesting the presence of mixed infections in all investigated fish. They also showed that inter-specimen genetic diversity at the genome scale was very low (>99.95% of sequence identity). By enabling full genome comparisons directly from infected fish tissues, this new method will be valuable to trace outbreaks rapidly and at a reasonable cost, and in turn to understand the transmission routes of CyHV-3.

Contribution of Next-Generation Sequencing to Aquatic and Fish Virology.

The recent technological advances in nucleic acid sequencing, called next-generation sequencing (NGS), have revolutionized the field of genomics and have also influenced viral research. Aquatic viruses, and especially those infecting fish, have also greatly benefited from NGS technologies, which provide a huge amount of molecular information at a low cost in a relatively short period of time. Here, we review the use of the current high-throughput sequencing platforms with a special focus on the associated challenges (regarding sample preparation and bioinformatics) in their applications to the field of aquatic virology, especially for: (i) discovering novel viruses that may be associated with fish mortalities, (ii) elucidating the mechanisms of pathogenesis, and finally (iii) studying the molecular epidemiology of these pathogens.

Plasticity of gene expression according to salinity in the testis of broodstock and F1 black-chinned tilapia, Sarotherodon melanotheron heudelotii.

The black-chinned tilapia Sarotherodon melanotheron heudelotii Rüppell 1852 (Teleostei, Cichlidae) displays remarkable acclimation capacities. When exposed to drastic changes of salinity, which can be the case in its natural habitat, it develops quick physiological responses and keeps reproducing. The present study focused on the physiological impact of salinity on male reproductive capacities, using gene expression as a proxy of acclimation process. Two series of experimental fish were investigated: the first one was composed of fish maintained in freshwater for several generations and newly acclimated to salinities of 35 and 70, whereas the second one consisted of the descendants of the latter born and were raised under their native salinity. Expression patterns of 43 candidate genes previously identified from the testes of wild males was investigated in the three salinities and two generations. Twenty of them showed significant expression differences between salinities, and their predicted function revealed that most of them are involved in the osmotic tolerance of sperm cells and/or in the maintenance of sperm motility. A high level of expression variation was evidenced, especially for fish maintained in freshwater. In spite of this, gene expression patterns allowed the differentiation between fish raised in freshwater and those maintained in hypersaline water in both generations. Altogether, the results presented here suggest that this high variability of expression is likely to ensure the reproductive success of this species under varying salinities.

Rapid detection and identification of nontuberculous mycobacterial pathogens in fish by using high-resolution melting analysis.

Mycobacterial infections in fish are commonly referred to as piscine mycobacteriosis, irrespectively of the specific identity of the causal organism. They usually cause a chronic disease and sometimes may result in high mortalities and severe economic losses. Nearly 20 species of Mycobacterium have been reported to infect fish. Among them, Mycobacterium marinum, M. fortuitum, and M. chelonae are generally considered the major agents responsible for fish mycobacteriosis. As no quick and inexpensive diagnostic test exists, we tested the potential of high-resolution melting analysis (HRMA) to rapidly identify and differentiate several Mycobacterium species involved in fish infections. By analyzing both the melting temperature and melting profile of the 16S-23S rRNA internal transcribed spacer (ITS), we were able to discriminate 12 different species simultaneously. Sensitivity tests conducted on purified M. marinum and M. fortuitum DNA revealed a limit of detection of 10 genome equivalents per reaction. The primers used in this procedure did not lead to any amplification signal with 16 control non-Mycobacterium species, thereby demonstrating their specificity for the genus Mycobacterium.

Permanent genetic resources added to molecular ecology resources database 1 December 2012-31 January 2013.

This article documents the addition of 268 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Alburnoides bipunctatus, Chamaerops humilis, Chlidonias hybrida, Cyperus papyrus, Fusarium graminearum, Loxigilla barbadensis, Macrobrachium rosenbergii, Odontesthes bonariensis, Pelteobagrus vachelli, Posidonia oceanica, Potamotrygon motoro, Rhamdia quelen, Sarotherodon melanotheron heudelotii, Sibiraea angustata, Takifugu rubripes, Tarentola mauritanica, Trimmatostroma sp. and Wallago attu. These loci were cross-tested on the following species: Alburnoides fasciatus, Alburnoides kubanicus, Alburnoides maculatus, Alburnoides ohridanus, Alburnoides prespensis, Alburnoides rossicus, Alburnoides strymonicus, Alburnoides thessalicus, Alburnoides tzanevi, Carassius carassius, Fusarium asiaticum, Leucaspius delineatus, Loxigilla noctis dominica, Pelecus cultratus, Phoenix canariensis, Potamotrygon falkneri, Trachycarpus fortune and Vimba vimba.

Osmoregulatory strategies in natural populations of the black-chinned tilapia Sarotherodon melanotheron exposed to extreme salinities in West African estuaries.

The effect of salinity was studied in natural populations of the black-chinned tilapia (Sarotherodon melanotheron) from West Africa. This euryhaline species colonizes nearly all coastal environments from bays to lagoons characterized by salinities ranging from fresh water to hypersaline water over 100 ‰. Individuals were sampled during the dry season at several locations characterized by different levels of salinity (3-102 ‰). Their osmotic status and their gills were analyzed. The branchial mitochondria-rich cells (MRC), localized at the basis of the filaments and along the lamellae in fish taken from the saline stations, showed a wide plasticity with significant differences in their number and size. The most striking results were a significant larger area (≈3x) and a higher number (≈55x) of MRC at high salinity (102 ‰) compared to low salinity (3 ‰). The major ion transporters and channels were localized by immunocytochemistry and different expression patterns have been recorded between stations. Despite an increased Na⁺/K⁺-ATPase (NKA) α-subunit expression and NKA activity, pointing to an increased monovalent ion excretion, a severe osmotic imbalance was recorded in animals living in hypersaline environments.

Investigation of Cyprinid herpesvirus-3 genetic diversity by a multi-locus variable number of tandem repeats analysis.

Cyprinid herpesvirus-3 (CyHV-3), or koi herpesvirus (KHV), is responsible for high mortalities in aquaculture of both common carp (Cyprinus carpio carpio) and koi carp (Cyprinus carpio koi) worldwide. The complete genomes of three CyHV-3 isolates showed more than 99% of DNA sequence identity, with the majority of differences located in short tandem repeats, also called VNTR (variable number of tandem repeats). By targeting these variations, eight loci were selected for genotyping CyHV-3 by multiple locus VNTR analysis (MLVA). CyHV-3 strains obtained after sequential in vivo infections exhibited identical MLVA profiles, whereas samples originating from a single isolate passaged 6 and 82 times in vitro exhibited mutations in two of the eight loci, suggesting a relatively slow genetic evolution rate of the VNTRs. The method was subsequently applied on 38 samples collected in Indonesia, France and the Netherlands. Globally, the isolates grouped in two main genetic clusters, each one divided in two subgroups including either CyHV-3-U/I or CyHV3-J. Interestingly, Indonesian strains were rather distant from CyHV-3-J isolate. The results of the present study indicate that these VNTR molecular markers are efficient in estimating the genetic diversity among CyHV-3 isolates and are therefore suitable for further molecular epidemiological studies.

A simple and innovative method for species identification of phytoplankton cells on minute quantities of DNA.

Dinoflagellates belonging to the genus Alexandrium are often involved in harmful algal blooms. Their ecological exploration is thus essential to increase our knowledge on these toxic events. Yet, population genetic studies, taxonomic identification and environmental monitoring are hampered by major constraints: the necessity to establish monoclonal cultures from environmental samples and the sensitivity of available molecular tools. The present work describes a very simple and sensitive method for extraction and amplification of DNA at the infra-single-cell level. Its on-slide format allows for easy visual control of both quality and quantity of the templates. Combined with a semi-multiplex PCR protocol designed on the 18S-28S rDNA-ITS region of Alexandrium catenella and Alexandrium tamarense, this procedure allowed the identification and discrimination of these species from both monoclonal cultures and natural samples.

Innovative integrated system for real-time measurement of hybridization and melting on standard format microarrays.

Despite the great popularity and potential of microarrays, their use for research and clinical applications is still hampered by lengthy and costly design and optimization processes, mainly because the technology relies on the end point measurement of hybridization. Thus, the ability to monitor many hybridization events on a standard microarray slide in real time would greatly expand the use and benefit of this technology, as it would give access to better prediction of probe performance and improved optimization of hybridization parameters. Although real-time hybridization and thermal denaturation measurements have been reported, a complete walk-away system compatible with the standard format of microarrays is still unavailable. To address this issue, we have designed a biochip tool that combines a hybridization station with active mixing capability and temperature control together with a fluorescence reader in a single compact benchtop instrument. This integrated live hybridization machine (LHM) allows measuring in real time the hybridization of target DNA to thousands of probes simultaneously and provides excellent levels of detection and superior sequence discrimination. Here we show on an environmental single nucleotide polymorphism (SNP) model system that the LHM enables a variety of experiments unachievable with conventional biochip tools.

Isolation and functional characterization of a new shrimp ovarian peritrophin with antimicrobial activity from Fenneropenaeus merguiensis.

Shrimp ovarian peritrophin (SOP), a major protein in jelly layer (JL) and cortical rods (CRs), is proposed to play a role in the protection of spawned eggs. The full sequence of SOP cDNA from Fenneropenaeus merguiensis (Fm-SOP) shares approximately 50% identity with other SOP sequences and contains several putative chitin-binding or peritrophin-A domains. Interestingly, Fm-SOP contains a putative 61-amino acid propeptide located at the N-terminal end, downstream of a 19-amino acid signal peptide, which is unique among penaeid SOP sequences described so far. This 61-amino-acid sequence constitutes a putative chitin-binding domain with six conserved cysteines, and is cleaved at a dibasic recognition site for a furin (subtilisin-like endoprotease). Expression analyses indicated that Fm-SOP mRNA is abundant in early vitellogenic ovaries and scarce in late-vitellogenic ovaries. Conversely, Fm-SOP protein is the most abundant at the end of vitellogenesis. To investigate its biological function, a recombinant Fm-SOP was expressed to generate a glycosylated protein in Spodoptera frugiperda Sf9 cells (rSOP-Sf9) and a nonglycosylated protein (rSOP-Ec) in Escherichia coli. rSOP-Sf9 and rSOP-Ec were found to bind to chitin, similarly to the native protein extracted from F. merguiensis ovaries. Most interestingly, rSOP-Ec displayed a chitinase activity and efficiently inhibited the growth of Vibrio harveyi and Staphylococcus aureus, with minimum inhibitory concentrations of 2.4 and 15.7 microM, respectively. This first report shows that a major component of CR and JL is biologically active against known pathogens and predicts a significant role of JL in the protection of the spawned eggs against pathogens.

Legumes symbioses: absence of Nod genes in photosynthetic bradyrhizobia.

Leguminous plants (such as peas and soybeans) and rhizobial soil bacteria are symbiotic partners that communicate through molecular signaling pathways, resulting in the formation of nodules on legume roots and occasionally stems that house nitrogen-fixing bacteria. Nodule formation has been assumed to be exclusively initiated by the binding of bacterial, host-specific lipochito-oligosaccharidic Nod factors, encoded by the nodABC genes, to kinase-like receptors of the plant. Here we show by complete genome sequencing of two symbiotic, photosynthetic, Bradyrhizobium strains, BTAi1 and ORS278, that canonical nodABC genes and typical lipochito-oligosaccharidic Nod factors are not required for symbiosis in some legumes. Mutational analyses indicated that these unique rhizobia use an alternative pathway to initiate symbioses, where a purine derivative may play a key role in triggering nodule formation.