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.

Blood B Cell Depletion Reflects Immunosuppression Induced by Live-Attenuated Infectious Bursal Disease Vaccines.

Immunosuppression in poultry production is a recurrent problem worldwide, and one of the major viral immunosuppressive agents is Infectious Bursal Disease Virus (IBDV). IBDV infections are mostly controlled by using live-attenuated vaccines. Live-attenuated Infectious Bursal Disease (IBD) vaccine candidates are classified as "mild," "intermediate," "intermediate-plus" or "hot" based on their residual immunosuppressive properties. The immunosuppression protocol described by the European Pharmacopoeia (Ph. Eur.) uses a lethal Newcastle Disease Virus (NDV) infectious challenge to measure the interference of a given IBDV vaccine candidate on NDV vaccine immune response. A Ph. Eur.-derived protocol was thus implemented to quantify immunosuppression induced by one mild, two intermediate, and four intermediate-plus live-attenuated IBD vaccines as well as a pathogenic viral strain. This protocol confirmed the respective immunosuppressive properties of those vaccines and virus. In the search for a more ethical alternative to Ph. Eur.-based protocols, two strategies were explored. First, ex vivo viral replication of those vaccines and the pathogenic strain in stimulated chicken primary bursal cells was assessed. Replication levels were not strictly correlated to immunosuppression observed in vivo. Second, changes in blood leukocyte counts in chicks were monitored using a Ph. Eur. - type protocol prior to lethal NDV challenge. In case of intermediate-plus vaccines, the drop in B cells counts was more severe. Counting blood B cells may thus represent a highly quantitative, faster, and ethical strategy than NDV challenge to assess the immunosuppression induced in chickens by live-attenuated IBD vaccines.

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.

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.

Antigenicity, pathogenicity and immunosuppressive effect caused by a South American isolate of infectious bursal disease virus belonging to the "distinct" genetic lineage.

Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious immunosuppressive disease affecting young chickens. The recently described "distinct IBDV" (dIBDV) genetic lineage encompasses a group of worldwide distributed strains that share conserved genetic characteristics in both genome segments making them unique within IBDV strains. Phenotypic characterization of these strains is scarce and limited to Asiatic and European strains collected more than 15 years ago. The present study aimed to assess the complete and comprehensive phenotypic characterization of a recently collected South American dIBDV strain (1/chicken/URY/1302/16). Genetic analyses of both partial genome segments confirmed that this strain belongs to the dIBDV genetic lineage and that it is not a reassortant. Antigenic analysis with monoclonal antibodies indicated that this strain has a particular antigenic profile, similar to that obtained in a dIBDV strain from Europe (80/GA), which differs from those previously found in the traditional classic, variant and very virulent strains. Chickens infected with the South American dIBDV strain showed subclinical infections but had a marked bursal atrophy. Further analysis using Newcastle disease virus-immunized chickens, previously infected with the South American and European dIBDV strains, demonstrated their severe immunosuppressive effect. These results indicate that dIBDV strains currently circulating in South America can severely impair the immune system of chickens, consequently affecting the local poultry industry. Our study provides new insights into the characteristics and variability of this global genetic lineage and is valuable to determine whether specific control measures are required for the dIBDV lineage. Research Highlights A South American strain of the dIBDV lineage was phenotypically characterized. The strain produced subclinical infections with a marked bursal atrophy. Infected chickens were severely immunosuppressed. The dIBDV strains are antigenically divergent from other IBDV lineages.

Propagation and titration of infectious bursal disease virus, including non-cell-culture-adapted strains, using ex vivo-stimulated chicken bursal cells.

Infectious bursal disease virus (IBDV) is a Birnaviridae family member of economic importance for poultry. This virus infects and destroys developing B lymphocytes in the cloacal bursa, resulting in a potentially fatal or immunosuppressive disease in chickens. Naturally occurring viruses and many vaccine strains are not able to grow in in vitro systems without prior adaptation, which often affects viral properties such as virulence. Primary bursal cells, which are the main target cells of lymphotropic IBDV in vivo, may represent an attractive system for the study of IBDV. Unfortunately, bursal cells isolated from bursal follicles undergo apoptosis within hours following their isolation. Here, we demonstrate that ex vivo stimulation of bursal cells with phorbol 12-myristate 13-acetate maintains their viability long enough to allow IBDV replication to high titres. A wide range of field-derived or vaccine serotype 1 IBDV strains could be titrated in these phorbol 12-myristate 13-acetate -stimulated bursal cells and furthermore were permissive for replication of non-cell-culture-adapted viruses. These cells also supported multistep replication experiments and flow cytometry analysis of infection. Ex vivo-stimulated bursal cells therefore offer a promising tool in the study of IBDV.

Protection patterns in duck and chicken after homo- or hetero-subtypic reinfections with H5 and H7 low pathogenicity avian influenza viruses: a comparative study.

Avian influenza viruses are circulating continuously in ducks, inducing a mostly asymptomatic infection, while chickens are accidental hosts highly susceptible to respiratory disease. This discrepancy might be due to a different host response to the virus between these two bird species and in particular to a different susceptibility to reinfection. In an attempt to address this question, we analyzed, in ducks and in chickens, the viral load in infected tissues and the humoral immune response after experimental primary and secondary challenge infections with either homologous or heterologous low pathogenicity avian influenza viruses (LPAIV). Following homologous reinfection, ducks were only partially protected against viral shedding in the lower intestine in conjunction with a moderate antibody response, whereas chickens were totally protected against viral shedding in the upper respiratory airways and developed a stronger antibody response. On the contrary, heterologous reinfection was not followed by a reduced viral excretion in the upper airways of chickens, while ducks were still partially protected from intestinal excretion of the virus, with no correlation to the antibody response. Our comparative study provides a comprehensive demonstration of the variation of viral tropism and control of the host humoral response to LPAIV between two different bird species with different degrees of susceptibility to avian influenza.

Field monitoring of avian influenza viruses: whole-genome sequencing and tracking of neuraminidase evolution using 454 pyrosequencing.

Adaptation of avian influenza viruses (AIVs) from waterfowl to domestic poultry with a deletion in the neuraminidase (NA) stalk has already been reported. The way the virus undergoes this evolution, however, is thus far unclear. We address this question using pyrosequencing of duck and turkey low-pathogenicity AIVs. Ducks and turkeys were sampled at the very beginning of an H6N1 outbreak, and turkeys were swabbed again 8 days later. NA stalk deletions were evidenced in turkeys by Sanger sequencing. To further investigate viral evolution, 454 pyrosequencing was performed: for each set of samples, up to 41,500 reads of ca. 400 bp were generated and aligned. Genetic polymorphisms between duck and turkey viruses were tracked on the whole genome. NA deletion was detected in less than 2% of reads in duck feces but in 100% of reads in turkey tracheal specimens collected at the same time. Further variations in length were observed in NA from turkeys 8 days later. Similarly, minority mutants emerged on the hemagglutinin (HA) gene, with substitutions mostly in the receptor binding site on the globular head. These critical changes suggest a strong evolutionary pressure in turkeys. The increasing performances of next-generation sequencing technologies should enable us to monitor the genomic diversity of avian influenza viruses and early emergence of potentially pathogenic variants within bird flocks. The present study, based on 454 pyrosequencing, suggests that NA deletion, an example of AIV adaptation from waterfowl to domestic poultry, occurs by selection rather than de novo emergence of viral mutants.

Truncation of the NS1 protein converts a low pathogenic avian influenza virus into a strong interferon inducer in duck cells.

The NS1 protein of influenza A viruses is known as a nonessential virulence factor inhibiting type I interferon (IFN) production in mammals and in chicken cells. Whether NS1 inhibits the induction of type I IFNs in duck cells is currently unknown. In order to investigate this issue, we used reverse genetics to generate a virus expressing a truncated NS1 protein. Using the low pathogenic avian influenza virus A/turkey/Italy/977/1999 (H7N1) as a backbone, we were able to rescue a virus expressing a truncated NS1 protein of 99 amino acids in length. The truncated virus replicated poorly in duck embryonic fibroblasts, but reached high titers in the mammalian IFN-deficient Vero cell line. Using a gene reporter system to measure duck type I IFN production, we showed that the truncated virus is a potent inducer of type I IFN in cell culture. These results show that the NS1 protein functions to prevent the induction of IFN in duck cells and underline the need for a functional NS1 protein in order for the virus to express its full virulence.