Type I Interferon acts as a major barrier to the establishment of infectious bursal disease virus (IBDV) persistent infections.

Infectious bursal disease virus (IBDV), the best characterized member of the Birnaviridae family, is a highly relevant avian pathogen causing both acute and persistent infections in different avian hosts. Here, we describe the establishment of clonal, long-term, productive persistent IBDV infections in DF-1 chicken embryonic fibroblasts. Although virus yields in persistently-infected cells are exceedingly lower than those detected in acutely infected cells, the replication fitness of viruses isolated from persistently-infected cells is higher than that of the parental virus. Persistently-infected DF-1 and IBDV-cured cell lines derived from them do not respond to type I interferon (IFN). High-throughput genome sequencing revealed that this defect is due to mutations affecting the IFNα/ß receptor subunit 2 (IFNAR2) gene resulting in the expression of IFNAR2 polypeptides harbouring large C-terminal deletions that abolish the signalling capacity of IFNα/ß receptor complex. Ectopic expression of a recombinant chicken IFNAR2 gene efficiently rescues IFNα responsiveness. IBDV-cured cell lines derived from persistently infected cells exhibit a drastically enhanced susceptibility to establishing new persistent IBDV infections. Additionally, experiments carried out with human HeLa cells lacking the IFNAR2 gene fully recapitulate results obtained with DF-1 cells, exhibiting a highly enhanced capacity to both survive the acute IBDV infection phase and to support the establishment of persistent IBDV infections. Results presented here show that the inactivation of the JAK-STAT signalling pathway significantly reduces the apoptotic response induced by the infection, hence facilitating the establishment and maintenance of IBDV persistent infections.IMPORTANCE Members of the Birnaviridae family, including infectious bursal disease virus (IBDV), exhibit a dual behaviour, causing acute infections that are often followed by the establishment of life-long persistent asymptomatic infections. Indeed, persistently infected specimens might act as efficient virus reservoirs, hence potentially contributing to virus dissemination. Despite the key importance of this biological trait, information about mechanisms triggering IBDV persistency is negligible. Our report evidences the capacity of IBDV, a highly relevant avian pathogen, to establishing long-term, productive, persistent infections in both avian and human cell lines. Data presented here provide novel and direct evidence about the crucial role of type I IFNs on the fate of IBDV-infected cells and their contribution to controlling the establishment of IBDV persistent infections. The use of cell lines unable to respond to type I IFNs opens a promising venue to unveiling additional factors contributing to IBDV persistency.

Exacerbated Apoptosis of Cells Infected with Infectious Bursal Disease Virus upon Exposure to Interferon Alpha.

Infectious bursal disease virus (IBDV) belongs to the Birnaviridae family and is the etiological agent of a highly contagious and immunosuppressive disease (IBD) that affects domestic chickens (Gallus gallus). IBD or Gumboro disease leads to high rates of morbidity and mortality of infected animals and is responsible for major economic losses to the poultry industry worldwide. IBD is characterized by a massive loss of IgM-bearing B lymphocytes and the destruction of the bursa of Fabricius. The molecular bases of IBDV pathogenicity are still poorly understood; nonetheless, an exacerbated cytokine immune response and B cell depletion due to apoptosis are considered main factors that contribute to the severity of the disease. Here we have studied the role of type I interferon (IFN) in IBDV infection. While IFN pretreatment confers protection against subsequent IBDV infection, the addition of IFN to infected cell cultures early after infection drives massive apoptotic cell death. Downregulation of double-stranded RNA (dsRNA)-dependent protein kinase (PKR), tumor necrosis factor alpha (TNF-α), or nuclear factor κB (NF-κB) expression drastically reduces the extent of apoptosis, indicating that they are critical proteins in the apoptotic response induced by IBDV upon treatment with IFN-α. Our results indicate that IBDV genomic dsRNA is a major viral factor that contributes to the triggering of apoptosis. These findings provide novel insights into the potential mechanisms of IBDV-induced immunosuppression and pathogenesis in chickens.IMPORTANCE IBDV infection represents an important threat to the poultry industry worldwide. IBDV-infected chickens develop severe immunosuppression, which renders them highly susceptible to secondary infections and unresponsive to vaccination against other pathogens. The early dysregulation of the innate immune response led by IBDV infection and the exacerbated apoptosis of B cells have been proposed as the main factors that contribute to virus-induced immunopathogenesis. Our work contributes for the first time to elucidating a potential mechanism driving the apoptotic death of IBDV-infected cells upon exposure to type I IFN. We provide solid evidence about the critical importance of PKR, TNF-α, and NF-κB in this phenomenon. The described mechanism could facilitate the early clearance of infected cells, thereby aiding in the amelioration of IBDV-induced pathogenesis, but it could also contribute to B cell depletion and immunosuppression. The balance between these two opposing effects might be dramatically affected by the genetic backgrounds of both the host and the infecting virus strain.

High antigenic diversity of serotype 1 infectious bursal disease virus revealed by antigenic cartography.

The antigenic characterization of IBDV, a virus that causes an immunosuppressive disease in young chickens, has been historically addressed using cross virus neutralization (VN) assay and antigen-capture enzyme-linked immunosorbent (AC-ELISA). However, VN assay has been usually carried out either in specific antibody negative embryonated eggs, for non-cell culture adapted strains, which is tedious, or on chicken embryo fibroblasts (CEF), which requires virus adaptation to cell culture. AC-ELISA has provided crucial information about IBDV antigenicity, but this information is limited to the epitopes included in the tested panel with a lack of information of overall antigenic view. The present work aimed at overcoming those technical limitations and providing an extensive antigenic landscape based on original cross VN assays employing primary chicken B cells, where no previous IBDV adaptation is required. Sixteen serotype 1 IBDV viruses, comprising both reference strains and documented antigenic variants were tested against eleven chicken post-infectious sera. The VN data were analysed by antigenic cartography, a method which enables reliable high-resolution quantitative and visual interpretation of large binding assay datasets. The resulting antigenic cartography revealed i) the existence of several antigenic clusters of IBDV, ii) high antigenic relatedness between some genetically unrelated viruses, iii) a highly variable contribution to global antigenicity of previously identified individual epitopes and iv) broad reactivity of chicken sera raised against antigenic variants. This study provides an overall view of IBDV antigenic diversity. Implementing this approach will be instrumental to follow the evolution of IBDV antigenicity and control the disease.

Genome Evolution of Two Genetically Homogeneous Infectious Bursal Disease Virus Strains During Passages in vitro and ex vivo in the Presence of a Mutagenic Nucleoside Analog.

The avibirnavirus infectious bursal disease virus (IBDV) is responsible for a highly contagious and sometimes lethal disease of chickens (Gallus gallus). IBDV genetic variation is well-described for both field and live-attenuated vaccine strains, however, the dynamics and selection pressures behind this genetic evolution remain poorly documented. Here, genetically homogeneous virus stocks were generated using reverse genetics for a very virulent strain, rvv, and a vaccine-related strain, rCu-1. These viruses were serially passaged at controlled multiplicities of infection in several biological systems, including primary chickens B cells, the main cell type targeted by IBDV in vivo. Passages were also performed in the absence or presence of a strong selective pressure using the antiviral nucleoside analog 7-deaza-2'-C-methyladenosine (7DMA). Next Generation Sequencing (NGS) of viral genomes after the last passage in each biological system revealed that (i) a higher viral diversity was generated in segment A than in segment B, regardless 7DMA treatment and viral strain, (ii) diversity in segment B was increased by 7DMA treatment in both viruses, (iii) passaging of IBDV in primary chicken B cells, regardless of 7DMA treatment, did not select cell-culture adapted variants of rvv, preserving its capsid protein (VP2) properties, (iv) mutations in coding and non-coding regions of rCu-1 segment A could potentially associate to higher viral fitness, and (v) a specific selection, upon 7DMA addition, of a Thr329Ala substitution occurred in the viral polymerase VP1. The latter change, together with Ala270Thr change in VP2, proved to be associated with viral attenuation in vivo. These results identify genome sequences that are important for IBDV evolution in response to selection pressures. Such information will help tailor better strategies for controlling IBDV infection in chickens.

Tetrahymena Glutathione Peroxidase Family: A Comparative Analysis of These Antioxidant Enzymes and Differential Gene Expression to Metals and Oxidizing Agents.

In the present work, an extensive analysis of the putative glutathione peroxidases (GPx) of the eukaryotic microorganism model Tetrahymena thermophila is carried out. A comparative analysis with GPx present in other Tetrahymena species and other very taxonomically diverse ciliates is also performed. A majority of ciliate GPx have replaced the selenocysteine (Sec) by Cys in its catalytic center, so they can be considered as phospholipid hydroperoxide glutathione peroxidases (PHGPx). Selenocysteine insertion sequence (SECIS) elements have been detected in several ciliate GPx that do not incorporate Sec in their amino acid sequences, and conversely, in other ciliate GPx with Sec, no SECIS elements are detected. These anomalies are analyzed and discussed. From the phylogenetic analysis using the ciliate GPx amino acid sequences, the existence of extensive intra- and interspecific gene duplications that produced multiple GPx isoforms in each species is inferred. The ancestral character of the selenoproteins is also corroborated. The analysis by qRT-PCR of six selected T. thermophila GPx genes has shown a quantitative differential expression between them, depending on the stressor (oxidizing agents, apoptotic inducer or metals) and the time of exposure.

Ex vivo rescue of recombinant very virulent IBDV using a RNA polymerase II driven system and primary chicken bursal cells.

Infectious Bursal Disease Virus (IBDV), a member of the Birnaviridae family, causes an immunosuppressive disease in young chickens. Although several reverse genetics systems are available for IBDV, the isolation of most field-derived strains, such as very virulent IBDV (vvIBDV) and their subsequent rescue, has remained challenging due to the lack of replication of those viruses in vitro. Such rescue required either the inoculation of animals, embryonated eggs, or the introduction of mutations in the capsid protein (VP2) hypervariable region (HVR) to adapt the virus to cell culture, the latter option concomitantly altering its virulence in vivo. We describe an improved ex vivo IBDV rescue system based on the transfection of an avian cell line with RNA polymerase II-based expression vectors, combined with replication on primary chicken bursal cells, the main cell type targeted in vivo of IBDV. We validated this system by rescuing to high titers two recombinant IBDV strains: a cell-culture adapted attenuated strain and a vvIBDV. Sequencing of VP2 HVR confirmed the absence of unwanted mutations that may alter the biological properties of the recombinant viruses. Therefore, this approach is efficient, economical, time-saving, reduces animal suffering and can be used to rescue other non-cell culture adapted IBDV strains.

Activation of the autophagy pathway by Torovirus infection is irrelevant for virus replication.

Autophagy is a conserved eukaryotic process that mediates lysosomal degradation of cytoplasmic macromolecules and damaged organelles, also exerting an important role in the elimination of intracellular pathogens. Despite the antiviral role of autophagy, many studies suggest that some positive-stranded RNA viruses exploit this pathway to facilitate their own replication. In this study, we demonstrate that the equine torovirus Berne virus (BEV), the prototype member of the Torovirus genus (Coronaviridae Family, Nidovirales Order), induces autophagy at late times post-infection. Conversion of microtubule associated protein 1B light chain 3 (LC3) from cytosolic (LC3 I) to the membrane associated form (LC3 II), a canonical marker of autophagosome formation, is enhanced in BEV infected cells. However, neither autophagy induction, via starvation, nor pharmacological blockade significantly affect BEV replication. Similarly, BEV infection is not altered in autophagy deficient cells lacking either Beclin 1 or LC3B protein expression. Unexpectedly, the cargo receptor p62, a selective autophagy receptor, aggregates within the region where the BEV main protease (Mpro) localizes. This finding, coupled with observation that BEV replication also induces ER stress at the time when selective autophagy is taking place, suggests that the autophagy pathway is activated in response to the hefty accumulation of virus-encoded polypeptides during the late phase of BEV infection.