Identification of an essential virulence gene of cyprinid herpesvirus 3

The genus Cyprinivirus consists of a growing list of phylogenetically related viruses, some of which cause severe economic losses to the aquaculture industry. The archetypal member, cyprinid herpesvirus 3 (CyHV-3) causes mass mortalities worldwide in koi and common carp. A CyHV-3 mutant was described previously that is attenuated in vivo by a deletion affecting two genes (ORF56 and ORF57). The relative contributions of ORF56 and ORF57 to the safety and efficacy profile of this vaccine candidate have now been assessed by analysing viruses individually deleted for ORF56 or ORF57. Inoculation of these viruses into carp demonstrated that the absence of ORF56 did not affect virulence, whereas the absence of ORF57 led to an attenuation comparable to, though slightly less than, that of the doubly deleted virus. To demonstrate further the role of ORF57 as a key virulence factor, a mutant retaining the ORF57 region but unable to express the ORF57 protein was produced by inserting multiple in-frame stop codons into the coding region. Analysis of this virus in vivo revealed a safety and efficacy profile comparable to that of the doubly deleted virus. These findings show that ORF57 encodes an essential CyHV-3 virulence factor. They also indicate that ORF57 orthologues in other cypriniviruses may offer promising targets for the rational design of attenuated recombinant vaccines

Cyprinid herpesvirus 3 : an intersting virus for applied and fundamental research

peer reviewe

Proteomic and functional analyses of the virion transmembrane proteome of cyprinid herpesvirus 3

Virion transmembrane proteins (VTPs) mediate key functions in the herpesvirus infectious cycle. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses. The present study was devoted to CyHV-3 VTPs. Using mass spectrometry approaches, we identified 16 VTPs of the CyHV-3 FL strain. Mutagenesis experiments demonstrated that eight of these proteins are essential for viral growth in vitro (ORF32, ORF59, ORF81, ORF83, ORF99, ORF106, ORF115, and ORF131), and eight are non-essential (ORF25, ORF64, ORF65, ORF108, ORF132, ORF136, ORF148, and ORF149). Among the non-essential proteins, deletion of ORF25, ORF132, ORF136, ORF148, or ORF149 affects viral replication in vitro, and deletion of ORF25, ORF64, ORF108, ORF132, or ORF149 impacts plaque size. Lack of ORF148 or ORF25 causes attenuation in vivo to a minor or major extent, respectively. The safety and efficacy of a virus lacking ORF25 were compared to those of a previously described vaccine candidate deleted for ORF56 and ORF57 (Δ56-57). Using quantitative PCR, we demonstrated that the ORF25 deleted virus infects fish through skin infection and then spreads to internal organs as reported previously for the wild-type parental virus and the Δ56-57 virus. However, compared to the parental wild-type virus, the replication of the ORF25 deleted virus was reduced in intensity and duration to levels similar to those observed for the Δ56-57 virus. Vaccination of fish with a virus lacking ORF25 was safe but had low efficacy at the doses tested. This characterization of the virion transmembrane proteome of CyHV-3 provides a firm basis for further research on alloherpesvirus VTPs. IMPORTANCE Virion transmembrane proteins play key roles in the biology of herpesviruses. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses and the causative agent of major economic losses in common and koi carp worldwide. In this study of the virion transmembrane proteome of CyHV-3, the major findings were: (i) the FL strain encodes 16 virion transmembrane proteins; (ii) eight of these proteins are essential for viral growth in vitro; (iii) seven of the non-essential proteins affect viral growth in vitro, and two affect virulence in vivo; and (iv) a mutant lacking ORF25 is highly attenuated but induces moderate immune protection. This study represents a major breakthrough in understanding the biology of CyHV-3 and will contribute to the development of prophylactic methods. It also provides a firm basis for the further research on alloherpesvirus virion transmembrane proteins

Virus-induced interference as a means for accelerating fitness-based selection of cyprinid herpesvirus 3 single nucleotide variants in vitro and in vivo

Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses and is advantageous to research because, unlike many herpesviruses, it can be studied in the laboratory by infection of the natural host (common and koi carp). Previous studies have reported a negative correlation among CyHV-3 strains between viral growth in vitro (in cell culture) and virulence in vivo (in fish). This suggests the existence of genovariants conferring enhanced fitness in vitro but reduced fitness in vivo, and vice versa. Here, we identified syncytial plaque formation in vitro as a common trait of CyHV-3 strains adapted to cell culture. Comparison of the sequences of virion transmembrane protein genes in CyHV-3 strains, and the use of various recombinant viruses, demonstrated that this trait is linked to a single nucleotide polymorphism (SNP) in the ORF131 coding sequence (C225791T mutation) that results in codon 183 encoding either an alanine (183A) or a threonine (183T) residue. In experiments involving infections with recombinant viruses differing only by this SNP, the 183A genovariant associated with syncytial plaque formation was the more fit in vitro but the less fit in vivo. In experiments involving co-infection with both viruses, it was observed that in addition to the more fit genovariant contributing to the purifying selection of the less fit genovariant by outcompeting the latter, we observed that this process may be accelerated by viral stimulation of interference at a cellular level, and stimulation of resistance to superinfection at a host level. Collectively, this study illustrates how the fundamental biological properties of some viruses and their hosts may have a profound impact on the degree of diversity that arises within viral populations

Pregnancy length and health in giant pandas: what can metabolic and urinary endocrine markers unveil?

Mature female giant pandas usually ovulate once a year. This is followed by an obligatory luteal phase, consisting of a long-lasting corpus luteum dormancy phase (CLD; primary increase in progestogens) and a much shorter active luteal phase (AL; secondary increase in progestogens). Varying duration of both the dormant (embryonic diapause) and AL (post-embryo reactivation) phases has hampered unambiguous pregnancy length determination in giant pandas until today. Additionally, progestogen profiles have been considered not to differ between pregnant and pseudopregnant cycles. Only ceruloplasmin, 13,14-dihydro-15-keto-PGF2α (PGFM) and – more recently – estrogens have been assigned diagnostic power so far. Our study investigated the competence of metabolic (fecal output) and Urinary Specific Gravity (USpG)-normalized urinary endocrine (progestogens, PGFM, glucocorticoids (GCM) and ceruloplasmin) markers for pregnancy monitoring including defining the duration of the AL phase length. Research on 24 (6 pregnant, 8 pseudopregnant and 10 non-birth) cycles of 6 giant pandas revealed a fixed AL phase length of 42 days in giant pandas, e.g. representing 6 weeks of post- diapause development in case of pregnancy. Progestogen concentrations were significantly higher in pregnant cycles throughout the majority of the AL phase, with significant higher values during the AL phase in healthy twin compared to singleton pregnancies. GCM concentrations were also markedly higher in giant pandas expecting offspring, with a clear increase towards birth in the final 2 weeks of pregnancy. This increase in GCM was running in parallel with elevating estrogen and PGFM concentrations, and decreasing progestogens. In addition, during the AL phase, a more pronounced decrease in fecal output was obvious for pregnant females. The combined profiles of non-invasive metabolic and endocrine markers, the latter normalized based on USpG, showed a true pregnancy signature during the AL phase. The findings of this study are applicable to retrospective evaluations of non-birth cycles facilitating categorizing those into pseudopregnant or lost pregnancies, with USpG-normalization of the urinary endocrine markers as a prerequisite

Communiquer les recherches de l'ULiege en BD

La bande dessinée, de par son visuel attrayant et sa lecture rapide, apparaît comme un format de choix pour vulgariser de façon attrayante une publication scientifique. Pour concrétiser ce projet, une collaboration a été établie avec avec un dessinateur liégeois, Emilio Licata (http://www.emiliolicata.be). Le première bande dessinée réalisée fut conçue comme une introduction présentant le projet «Essaimeurs de Savoir».Essaimeurs de Savoi

The virion transmembrane proteome: a glance to the evolution and to the biology of Cyprinid herpesvirus 3

Cyprinid herpesvirus 3 (CyHV-3) is the etiological agent of a highly contagious and lethal disease affecting koi and common carp worldwide. Beyond its economic importance, this virus turned out to be an interesting subject for fundamental research and is currently considered as the archetype of Alloherpesviridae. The divergence of this family with the Herpesviridae is ancestral, with almost no significant homology between CyHV 3 genes and those of Herpesviridae. Consequently, the extensive knowledge acquired for the latter cannot be used to predict CyHV 3 biological features. Virion transmembrane proteins (VTPs) are well documented in Herpesviridae for their involvement in crucial processes such as entry, immune evasion, morphogenesis and egress of progeny virions from the host cells. In contrast, very little is known about the functions of these proteins in CyHV 3 or in any of the alloherpesviruses. The main objective of this thesis was to provide a first functional characterization of CyHV-3 virion transmembrane proteome. The experimental work performed is summarized in two main chapters. The first study aimed to update the list of known CyHV-3 VTPs and to determine those that are essential to viral growth in vitro. Using mass spectrometry approaches and mutagenesis experiments, we identified 16 VTPs in the CyHV-3 FL strain, among which 8 turned out to be essential to viral growth in vitro. The non-essential VTPs were further assessed quantitatively for their relative importance in vitro and in vivo. ORF25, ORF64, ORF108, ORF132, ORF136, ORF148, and ORF149 were shown to affect viral growth in vitro; while the lack of ORF148 or ORF25 caused attenuation to a minor or major extent in vivo, respectively. Finally, we showed that a mutant lacking ORF25 was highly attenuated but induced a moderate immune protection under the conditions tested. Bioinformatic analyses suggested that CyHV-3 ORF27 encodes a VTP. However, no protein corresponding to this ORF was detected in our proteomic analyses. Analyses of genome sequence and protein expression demonstrate that the FL strain, like several other laboratory strains, do not express ORF27 due to presence of various mutations, while it encodes a VTP in field strains. These observation led us to hypothesize that the deletion of ORF27 could confer a selective advantage to viral growth in vitro, while the expression of a functional pORF27 could confer a selective advantage in vivo. These hypotheses were addressed by producing recombinant viruses. In vitro, the CyHV 3 strain lacking ORF27 expression was shown to have a replicative advantage, especially during co-infection with a viral strain expressing this protein. In contrast, in vivo, no difference between both genotypes could be detected in the experimental conditions tested. This observation suggests that the biological functions of ORF27 cannot be revealed in the laboratory conditions used. In conclusion, this thesis provides a first functional characterization of the virion transmembrane proteome of CyHV-3. It represents a firm basis for further research on alloherpesvirus VTPs

Communicating high impact research through innovative formats

High scored research papers are usually out of reach of the global society. The University of Liège specialised in decoding and disseminating the content of top articles to different publics and stakeholders by using unconventional innovative formats. This contribution will present ULiège last creation: a research outcome placemat for restaurants. Research papers are highly valued among the university community since they are representative of excellence of authors. However, because of their highly technical and specialised content, they are poorly accessible to the society. Moreover, science popularization by journalists and media suffers from several drawbacks: lack of significant information, excessive simplification, overshadow of research process and researchers’ joint working. This finding led us to develop new initiatives for disseminating the content of publications outside of the authors’ research units. The project is based on two main axes: 1) Deciphering of published articles (vocabulary, evolution of knowledge in the discipline, contribution of authors and research units, research network,…) 2) Disseminating of science Targets are: the university community (researchers from other groups, PhDs, students), senior high schools students (15-18 years) and the large public. Attention is paid to the creation of attractive formats for disseminating results as well as the research process. We developed comic strips, animations, quiz, mind maps,… This contribution will focus on one of these formats: a placemat distributed in restaurants. The different steps of the creation will be detailed: choice of a significant publication, collaboration with researchers, adaptation to the target, selection of images and drawings, examples and analogies, layout, review by a non-expert panel and distribution. The critical analysis of the impact will be presented.Essaimeurs de Savoi

La recherche en Médecine Vétérinaire - 4 enquêtes pour un virus

Dans le cadre de la journée de l’enseignement secondaire du 29 janvier 2019 à l’ULiège, quatre publications scientifiques du laboratoire d’Immunologie et Vaccinologie (Faculté de Médecine Vétérinaire) ont été vulgarisées sous forme de quizz interactifs. L’atelier offrait ainsi aux élèves l’opportunité de se glisser dans la peau d’un chercheur. Leur mission ? Mener l’enquête sur un virus qui fait des ravages chez les poissons. Une belle occasion de découvrir des exemples concrets de sujets de recherche, des outils utilisés par les chercheurs, leur environnement et leur méthode de travail.Essaimeurs de Savoi