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Cyprinid herpesvirus 3 (CyHV-3) is causing severe economic losses worldwide in common and koi carp industries, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open reading frame 134 (ORF134), we unexpectedly obtained a clone with additional deletion of ORF56 and ORF57. This triple deleted recombinant replicated efficiently in vitro and expressed an in vivo safety/efficacy profile compatible with use as an attenuated vaccine. To determine the role of the double ORF56-57 deletion in the phenotype and to improve further the quality of the vaccine candidate, a series of deleted recombinants was produced and tested in vivo. These experiments led to the selection of a double deleted recombinant lacking ORF56 and ORF57 as a vaccine candidate. The safety and efficacy of this strain were studied using an in vivo bioluminescent imaging system (IVIS), qPCR, and histopathological examination, which demonstrated that it enters fish via skin infection similar to the wild type strain. However, compared to the parental wild type strain, the vaccine candidate replicated at lower levels and spread less efficiently to secondary sites of infection. Transmission experiments allowing water contamination with or without additional physical contact between fish demonstrated that the vaccine candidate has a reduced ability to spread from vaccinated fish to naïve sentinel cohabitants. Finally, IVIS analyses demonstrated that the vaccine candidate induces a protective mucosal immune response at the portal of entry. Thus, the present study is the first to report the rational development of a recombinant attenuated vaccine against CyHV-3 for mass vaccination of carp. We also demonstrated the relevance of the CyHV-3 carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin., Author Summary Common carp, and its colorful ornamental variety koi, is one of the most economically valuable species in aquaculture. Since the late 1990s, the common and koi carp culture industries have suffered devastating worldwide losses due to cyprinid herpesvirus 3 (CyHV-3). In the present study, we report the development of an attenuated recombinant vaccine against CyHV-3. Two genes were deleted from the viral genome, leading to a recombinant virus that is no longer capable of causing the disease but can be propagated in cell culture (for vaccine production) and infect fish when added to the water, thereby immunizing the fish. This attenuated recombinant vaccine also had a drastic defect in spreading from vaccinated to non-vaccinated cohabitant fish. The vaccine induced a protective mucosal immune response capable of preventing the entry of virulent CyHV-3 and is compatible with the simultaneous vaccination of a large number of carp by simply immersing the fish in water containing the vaccine. This vaccine represents a promising tool for controlling the most dreadful disease ever encountered by the carp culture industries. In addition, the present study highlights the importance of the CyHV-3 - carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin.

Transmission is a matter of life or death for pathogen lineages and can therefore be considered as the main motor of their evolution. Gammaherpesviruses are archetypal pathogenic persistent viruses which have evolved to be transmitted in presence of specific immune response. Identifying their mode of transmission and their mechanisms of immune evasion is therefore essential to develop prophylactic and therapeutic strategies against these infections. As the known human gammaherpesviruses, Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus are host-specific and lack a convenient in vivo infection model; related animal gammaherpesviruses, such as murine gammaherpesvirus-68 (MHV-68), are commonly used as general models of gammaherpesvirus infections in vivo. To date, it has however never been possible to monitor viral excretion or virus transmission of MHV-68 in laboratory mice population. In this study, we have used MHV-68 associated with global luciferase imaging to investigate potential excretion sites of this virus in laboratory mice. This allowed us to identify a genital excretion site of MHV-68 following intranasal infection and latency establishment in female mice. This excretion occurred at the external border of the vagina and was dependent on the presence of estrogens. However, MHV-68 vaginal excretion was not associated with vertical transmission to the litter or with horizontal transmission to female mice. In contrast, we observed efficient virus transmission to naïve males after sexual contact. In vivo imaging allowed us to show that MHV-68 firstly replicated in penis epithelium and corpus cavernosum before spreading to draining lymph nodes and spleen. All together, those results revealed the first experimental transmission model for MHV-68 in laboratory mice. In the future, this model could help us to better understand the biology of gammaherpesviruses and could also allow the development of strategies that could prevent the spread of these viruses in natural populations., Author Summary Epstein-Barr virus and the Kaposi's Sarcoma-associated Herpesvirus are two human gammaherpesviruses which are linked to the development of several cancers. Efficient control of these infections is therefore of major interest, particularly in some epidemiological circumstances. These viruses are however host-specific and cannot be experimentally studied in vivo. The identification of a closely related viral species, called Murid herpesvirus 4 with the main strain called murine gammaherpesvirus-68 (MHV-68), in wild rodents opened new horizons to the study of gammaherpesvirus biology. Surprisingly, despite 30 years of research, MHV-68 transmission had never been observed in captivity. In this study, using in vivo imaging, we showed that MHV-68 is genitally excreted after latency establishment in intranasally infected female mice. This allowed us to observe, for the first time, sexual transmission of MHV-68 between laboratory mice. In the future, this model should be important to better understand the biology of gammaherpesviruses and should also allow the development of strategies that could prevent the spread of these viruses in natural populations.

Viral diseases of the respiratory tract, which include influenza pandemic, children acute bronchiolitis, and viral pneumonia of the elderly, represent major health problems. Plasmacytoid dendritic cells play an important role in anti-viral immunity, and these cells were recently shown to express ChemR23, the receptor for the chemoattractant protein chemerin, which is expressed by epithelial cells in the lung. Our aim was to determine the role played by the chemerin/ChemR23 system in the physiopathology of viral pneumonia, using the pneumonia virus of mice (PVM) as a model. Wild-type and ChemR23 knock-out mice were infected by PVM and followed for functional and inflammatory parameters. ChemR23(-/-) mice displayed higher mortality/morbidity, alteration of lung function, delayed viral clearance and increased neutrophilic infiltration. We demonstrated in these mice a lower recruitment of plasmacytoid dendritic cells and a reduction in type I interferon production. The role of plasmacytoid dendritic cells was further addressed by performing depletion and adoptive transfer experiments as well as by the generation of chimeric mice, demonstrating two opposite effects of the chemerin/ChemR23 system. First, the ChemR23-dependent recruitment of plasmacytoid dendritic cells contributes to adaptive immune responses and viral clearance, but also enhances the inflammatory response. Second, increased morbidity/mortality in ChemR23(-/-) mice is not due to defective plasmacytoid dendritic cells recruitment, but rather to the loss of an anti-inflammatory pathway involving ChemR23 expressed by non-leukocytic cells. The chemerin/ChemR23 system plays important roles in the physiopathology of viral pneumonia, and might therefore be considered as a therapeutic target for anti-viral and anti-inflammatory therapies., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published