Virulence genes, resistome and mobilome of Streptococcus suis strains isolated in France.

Streptococcus suis is a leading cause of infection in pigs, causing extensive economic losses. In addition, it can also infect wild fauna, and can be responsible for severe infections in humans. Increasing antimicrobial resistance (AMR) has been described in S. suis worldwide and most of the AMR genes are carried by mobile genetic elements (MGEs). This contributes to their dissemination by horizontal gene transfer. A collection of 102 strains isolated from humans, pigs and wild boars in France was subjected to whole genome sequencing in order to: (i) study their genetic diversity, (ii) evaluate their content in virulence-associated genes, (iii) decipher the mechanisms responsible for their AMR and their association with MGEs, and (iv) study their ability to acquire extracellular DNA by natural transformation. Analysis by hierarchical clustering on principal components identified a few virulence-associated factors that distinguish invasive CC1 strains from the other strains. A plethora of AMR genes (n=217) was found in the genomes. Apart from the frequently reported erm(B) and tet(O) genes, more recently described AMR genes were identified [vga(F)/sprA, vat(D)]. Modifications in PBPs/MraY and GyrA/ParC were detected in the penicillin- and fluoroquinolone-resistant isolates respectively. New AMR gene-MGE associations were detected. The majority of the strains have the full set of genes required for competence, i.e for the acquisition of extracellular DNA (that could carry AMR genes) by natural transformation. Hence the risk of dissemination of these AMR genes should not be neglected.

Development and evaluation of a core genome multilocus sequence typing scheme for Paenibacillus larvae, the deadly American foulbrood pathogen of honeybees.

Paenibacillus larvae is the causative agent of the fatal American foulbrood disease in honeybees (Apis mellifera). Strain identification is vital for preventing the spread of the disease. To date, the most accessible and robust scheme to identify strains is the multilocus sequence typing (MLST) method. However, this approach has limited resolution, especially for epidemiological studies. As the cost of whole-genome sequencing has decreased and as it becomes increasingly available to most laboratories, an extended MLST based on the core genome (cgMLST) presents a valuable tool for high-resolution investigations. In this study, we present a standardized, robust cgMLST scheme for P. larvae typing using whole-genome sequencing. A total of 333 genomes were used to identify, validate and evaluate 2419 core genes. The cgMLST allowed fine-scale differentiation between samples that had the same profile using traditional MLST and allowed for the characterization of strains impossible by MLST. The scheme was successfully used to trace a localized Swedish outbreak, where a cluster of 38 isolates was linked to a country-wide beekeeping operation. cgMLST greatly enhances the power of a traditional typing scheme, while preserving the same stability and standardization for sharing results and methods across different laboratories.

Evaluation of un-methylated DNA enrichment in sequencing of African swine fever virus complete genome.

African swine fever is a febrile hemorrhagic fever disease that is caused by the African swine fever virus (ASFV) and is lethal for domestic pigs and wild boar. ASFV also infects soft ticks of the genus Ornithodoros, some species of which can act as a vector for ASFV. Whole genome sequencing of ASFV is a challenge because, due to the size difference of the host genome versus the viral genome, the higher proportion of host versus virus DNA fragments renders the virus sequencing poorly efficient. A novel approach of DNA enrichment, based on the separation of methylated and un-methylated DNA, has been reported but without an evaluation of its efficacy. In this study, the efficiency of the un-methylated DNA enrichment protocol was evaluated for pig and tick samples infected by ASFV. As expected, fewer reads corresponding to ASFV were found in the methylated fraction compared to the un-methylated fraction. However, the sequencing coverage of the un-methylated fraction was not improved compared to the untreated DNA. In our hands, the ASFV DNA enrichment was inefficient for tick samples and very limited for pig samples. This enrichment process represents extra work and cost without a significant improvement of ASFV genome coverage. The efficiency of this enrichment approach and the cost/benefit ratio are discussed.

In vitro, in cellulo and structural characterizations of the interaction between the integrase of Porcine Endogenous Retrovirus A/C and proteins of the BET family.

Retroviral integrase (IN) proteins catalyze the permanent integration of the viral genome into host DNA. They can productively recruit cellular proteins, and the human Bromodomain and Extra-Terminal domain (hBET) proteins have been shown to be co-factors for integration of gamma-retroviruses such as Murine Leukemia Virus (MLV) into human cells. By using two-hybrid, co-immunoprecipitation and in vitro interaction assays, we showed that IN of the gamma- Porcine Endogenous Retrovirus-A/C (PERV IN) interacts through its C-terminal domain (CTD) with hBET proteins. We observed that PERV IN interacts with the BRD2, BRD3 and BRD4 proteins in vitro and that the BRD2 protein specifically binds and co-localizes with PERV IN protein in the nucleus of cells. We further mapped the interaction sites to the conserved Extra-Terminal (ET) domain of the hBET proteins and to several amino acids of the of the C-terminal tail of the PERV IN CTD. Finally, we determined the first experimental structure of an IN CTD - BET ET complex from small-angle X-ray scattering data (SAXS). We showed that the two factors assemble as two distinct modules linked by a short loop which confers partial flexibility. The SAXS-restrained model is structurally compatible with the binding of the PERV intasome to BRD2. Altogether, these data confirm the important role of host BET proteins in the gamma-retroviruses' targeting site and efficiency of integration.

Targeted Next Generation Sequencing to study insert stability in genetically modified plants.

The EU directive 2001/18/EC requires any genetically modified (GM) event to be stable. In the present work, a targeted Next-Generation Sequencing (NGS) approach using barcodes to specifically tag each individual DNA molecules during library preparation was implemented to detect mutations taking into account the background noise due to amplification and sequencing errors. The method was first showed to be efficient in detecting the mutations in synthetic samples prepared with custom-synthesized mutated or non-mutated P35S sequences mixed in different proportions. The genetic stability of a portion of the P35S promoter targeted for GM detection was then analyzed in GM flour samples. Several low frequency mutations were detected in the P35S sequences. Some mutated nucleotides were located within the primers and probes used in the P35S diagnostic test. If present not as somatic mutations but as the consensus sequence of some individuals, these mutations could influence the efficiency of the P35S real time PCR diagnostic test. This methodology could be implemented in genetic stability studies of GM inserts but also to detect single nucleotide mutant GM plants produced using "new breeding techniques".

Genetic identification of two Acipenser iridovirus-European variants using high-resolution melting analysis.

Acipenser iridovirus-European (AcIV-E) is an important pathogen of sturgeons. Two variants differing by single-nucleotide polymorphisms (SNP) in the Major Capsid Protein gene have been described, but without any indication as to their prevalence in farms. To facilitate epidemiological studies, we developed a high-resolution melting (HRM) assay to distinguish between two alleles (var1 and var2) differing by five point substitutions. The HRM assay detected as little as 100 copies of plasmids harboring cloned sequences of var1 and var2, which have melting temperatures (Tm) differing by only 1 °C. The assay was specific of AcIV-E as demonstrated by the absence of signal when testing a related, yet distinct, virus as well as DNA from an AcIV-E-negative sturgeon sample. Experiments with mixtures of two distinct plasmids revealed abnormal melting curve patterns, which showed dips just before the main melting peaks. These dips in the curves were interpreted as the dissociation of heteroduplexes fortuitously created during the PCR step. Screening AciV-E-positive field samples of Russian sturgeons from three farms revealed the presence of var2, based on the Tm. However, for a few samples, the melting curves showed patterns typical of var2 as the dominant viral genome, mixed with another minor variant which proved to be var1. In conclusion, HRM is a simple method to screen for AcIV-E var1 and var2 and can be used on a large scale in Europe to trace these two variants which likely represent two genetic lineages.

Development of a pig infection model with colistin-resistant Escherichia coli.

Colistin-resistant Escherichia coli are isolated from pigs suffering from post-weaning diarrhea (PWD). This study was designed to develop an experimental model of PWD using mcr-1-carrying shiga toxin-producing E. coli (STEC) or enterotoxigenic E. coli (ETEC), for the future evaluation of control measures. Three groups of eight piglets, kept in high biosecurity units, were orally inoculated with mcr-1-positive STEC or ETEC, and one unchallenged group was used as a control. Clinical signs were recorded. Regularly-collected fecal samples and samples obtained from the digestive tract of animals sacrificed one month after inoculation were cultured in selective media and isolates were characterized. Blood samples were used to genotype the polymorphisms of the pigs' intestinal receptors for F4 and F18 E. coli adhesins. Diarrhea was more frequent and more fecal samples contained the inoculated strain in the group inoculated with the O149-F4 ETEC strain than with the O141-F18 or O139-F18 STEC strains. However, fewer positive samples were obtained from the two pigs with the F4 resistant genotype. The three inoculated strains could be re-isolated up to the end of the experiment. Excretion peaked on the first week after inoculation with the O149-F4 ETEC strain, and later for the other two. An mcr-1 gene transfer to other commensal isolates was observed only for O139-F18 STEC, while the loss of mcr-1 from the inoculated strain occurred in all groups. The O149-F4 ETEC challenge may be used to evaluate alternative solutions to combat PWD caused by colistin-resistant E. coli in pigs.

Characterization of plasmids harboring blaCTX-M genes in Escherichia coli from French pigs.

Resistance to extended-spectrum cephalosporins is prevalent in French pig E. coli isolates. The aim of this study was to characterize the plasmids and genes present in pathogenic and commensal extended-spectrum cephalosporins -resistant isolates. The resistance plasmids of 26 strains were sequenced and then analyzed to identify resistance and virulence genes. Results showed that resistance to extended-spectrum cephalosporins in French pig E. coli isolates is-as in other food animals in France-mainly carried by highly similar blaCTX-M-1 IncI1/ST3 plasmids. These plasmids very often bear other resistance genes such as resistance to sulphonamides (sul2), trimethoprim (dfrA17) and aminoglycosides (aadA5), and occasionally to tetracycline (tet(A)), macrolides (mph(A) and erm genes), phenicols (floR) or streptomycin (strA, strB). Few virulence genes were detected, including colicins, heat-stable enterotoxins, adhesins or temperature-sensitive hemagglutinins. The other cefotaximases detected were blaCTX-M-27 and blaCTX-M-14, the latter being on an IncF plasmid which showed very close identity to a human epidemic plasmid. Importantly, resistance genes for quinolones or polymyxins were never detected on the extended-spectrum cephalosporins resistance plasmids. These results are helpful to evidence the risk of co-selecting cephalosporins -resistance using antibiotics outside this group. They also highlight the occasional presence in pigs of human epidemic plasmids.

Acipenser iridovirus-European encodes a replication factor C (RFC) sub-unit.

New genomic sequence data were acquired for the Acipenser iridovirus-European (AcIV-E), a virus whose complete genome and classification still remain to be elucidated. Here, we obtained the first full-length Major capsid protein (MCP) gene sequence for AcIV-E, as well as two additional open reading frames (ORFs) adjacent to the MCP gene. BLAST searches of the first ORF (α) resulted in no match to any gene or protein in the public databases. The other ORF (ß) was identified as a subunit of a replication factor C (RFC), known to function as a clamp loader in eukaryotes, archae and some viruses. The presence of similar RFC genes was confirmed in two distinct, yet related, viruses, the white sturgeon iridovirus and a European variant of Namao virus. The existence of an RFC gene in AcIV-E suggests a genome size larger than that of other classifiable members of the family Iridoviridae along with a mode of replication involving an interaction between a clamp loader and a proliferating nuclear cell antigen. Sequencing and comparison of the full-length RFC gene from various sturgeon samples infected with AcIV-E revealed two distinct clusters of sequences within one particular sample in which the coexistence of two lineages had previously been predicted based on analysis of the partial MCP gene sequence. These genetic data provide further evidence of the circulation of at least two concurrent AcIV-E lineages, sometimes co-infecting cultured European sturgeon.

Longitudinal study of Escherichia coli plasmid resistance to extended-spectrum cephalosporins in free-range broilers.

Resistance to extended-spectrum cephalosporins (ESCs) is mostly borne by conjugative plasmids. The aim of the present study was to evaluate the characteristics and diversity of ESC resistance plasmids in Escherichia coli from different free-range broiler flocks in France, and their persistence in flocks during rearing. Two hatcheries were selected. Faecal samples from 11 flocks were collected from before their arrival on the broiler production farm up to their slaughter at the end of the rearing period. A selection of 25 E. coli isolates obtained at different times from different flocks but all harbouring an ESC resistance gene was characterised. The plasmids coding for ESC resistance were sequenced using Mi-seq Illumina technology or the ion proton system (Ion Torrent). Ten IncI1 ST12 plasmids carried the blaCMY-2 gene, and most of them had no other resistance genes. All blaCMY-2 plasmids were obtained from day-old to 7-day-old chicks from four flocks hatched at the same hatchery and sent to three different farms. Sequence comparisons showed identity percentages higher than 99%. Fifteen IncI1 ST3 plasmids were obtained from day-old to 77-day-old broilers from seven flocks on six farms. These plasmids harboured the blaCTX-M-1 gene, and most also had the tet(A) and sul2 genes, with sequence identity higher than 99%. For both types of plasmid, very high identity percentages were also obtained with published sequences of plasmids isolated from broilers in other countries or from other animal species. Thus, unlike the IncI1 ST12 blaCMY-2 plasmids, the epidemic nature of the IncI1 ST3 blaCTX-M-1 plasmids in the French poultry production makes it difficult to determine the origin of a contamination which may persist for weeks in a flock.

Infectious bursal disease virus in Algeria: Detection of highly pathogenic reassortant viruses.

Infectious bursal disease (IBD) is an immunosuppressive viral disease, present worldwide, which causes mortality and immunosuppression in young chickens. The causative agent, the Avibirnavirus IBDV, is a non-enveloped virus whose genome consists of two segments (A and B) of double-stranded RNA. Different pathotypes of IBDV exist, ranging from attenuated vaccine strains to very virulent viruses (vvIBDV). In Algeria, despite the prophylactic measures implemented, cases of IBD are still often diagnosed clinically and the current molecular epidemiology of IBDV remains unknown. The presence of the virus and especially of strains genetically close to vvIBDV was confirmed in 2000 by an unpublished OIE report. In this study, nineteen IBDV isolates were collected in Algeria between September 2014 and September 2015 during clinical outbreaks. These isolates were analyzed at the genetic, antigenic and pathogenic levels. Our results reveal a broad genetic and phenotypic diversity of pathogenic IBDV strains in Algeria, with, i) the circulation of viruses with both genome segments related to European vvIBDV, which proved as pathogenic for specific pathogen-free chickens as vvIBDV reference strain, ii) the circulation of viruses closely related - yet with a specific segment B - to European vvIBDV, their pathogenicity being lower than reference vvIBDV, iii) the detection of reassortant viruses whose segment A was related to vvIBDV whereas their segment B did not appear closely related to any reference sequence. Interestingly, the pathogenicity of these potentially reassortant strains was comparable to that of reference vvIBDV. All strains characterized in this study exhibited an antigenicity similar to the cognate reference IBDV strains. These data reveal the continuous genetic evolution of IBDV strains in Algerian poultry through reassortment and acquisition of genetic material of unidentified origin. Continuous surveillance of the situation as well as good vaccination practice associated with appropriate biosecurity measures are necessary for disease control.

Characterization of plasmids harboring blaCTX-M and blaCMY genes in E. coli from French broilers.

Resistance to extended-spectrum cephalosporins (ESC) is a global health issue. The aim of this study was to analyze and compare plasmids coding for resistance to ESC isolated from 16 avian commensal and 17 avian pathogenic Escherichia coli (APEC) strains obtained respectively at slaughterhouse or from diseased broilers in 2010-2012. Plasmid DNA was used to transform E. coli DH5alpha, and the resistances of the transformants were determined. The sequences of the ESC-resistance plasmids prepared from transformants were obtained by Illumina (33 plasmids) or PacBio (1 plasmid). Results showed that 29 of these plasmids contained the blaCTX-M-1 gene and belonged to the IncI1/ST3 type, with 27 and 20 of them carrying the sul2 or tet(A) genes respectively. Despite their diverse origins, several plasmids showed very high percentages of identity. None of the blaCTX-M-1-containing plasmid contained APEC virulence genes, although some of them were detected in the parental strains. Three plasmids had the blaCMY-2 gene, but no other resistance gene. They belonged to IncB/O/K/Z-like or IncFIA/FIB replicon types. The blaCMY-2 IncFIA/FIB plasmid was obtained from a strain isolated from a diseased broiler and also containing a blaCTX-M-1 IncI1/ST3 plasmid. Importantly APEC virulence genes (sitA-D, iucA-D, iutA, hlyF, ompT, etsA-C, iss, iroB-E, iroN, cvaA-C and cvi) were detected on the blaCMY-2 plasmid. In conclusion, our results show the dominance and high similarity of blaCTX-M-1 IncI1/ST3 plasmids, and the worrying presence of APEC virulence genes on a blaCMY-2 plasmid.

Molecular investigations of outbreaks of Perch perhabdovirus infections in pike-perch.

In 2016, a total of 5 massive mortality episodes each affecting hundreds of thousands of pike-perch Sander lucioperca larvae occurred at 2 sites in 2 Western European countries. For each episode, perhabdoviruses related to the perch rhabdovirus (PRV) were detected in samples, using either PCR or cell culture combined with PCR. The sequences of the glycoprotein (g), phosphoprotein (p) and nucleoprotein (n) genes of these samples demonstrated that 2 different genotypes were present at 1 site, each associated with 1 of the 3 episodes. At the other site, a single genotype was associated with the 2 outbreaks. Furthermore, this genotype was strictly identical to 1 genotype involved in the outbreaks of the first site, strongly suggesting a common origin for these 2 viruses. The common origin was confirmed a posteriori because some larvae introduced to both sites had exactly the same geographic origin in Eastern Europe. Taken together, the molecular and epidemiological data suggest that both horizontal and vertical transmission of 2 distinct strains of perhabdoviruses were involved in the various outbreaks affecting pike-perch.

The expression level of gC1qR is down regulated at the early time of infection with porcine circovirus of type 2 (PCV-2) and gC1qR interacts differently with the Cap proteins of porcine circoviruses.

Porcine circoviruses (PCV) are small, non-enveloped single-stranded DNA-viruses. Porcine circovirus type 2 (PCV-2) is the causal agent of post-weaning multisystemic wasting syndrome (PMWS) whereas porcine circovirus of type 1 (PCV-1) is non- pathogenic. gC1qR is a membrane-located receptor of the complement protein subunit C1q and interacts with PCV capsid proteins. The mechanisms associated with the triggering of PMWS are not well known and gC1qR may have a role in the life cycle and eventually in the pathogenicity of PCV. The objectives of this study were to determine the level of expression of gC1qR during early PCV-2 infection, to determine the region of PCV-2 capsid protein (Cap) required for the interaction with gC1qR and to evaluate the interaction of gC1qR with Cap proteins of different PCV strains. The results indicate that gC1qR transcripts are downregulated in the tonsils and the tracheo-bronchial lymph nodes of piglets infected by PCV-2 at the early time of the infection. The N-terminal amino acids (a.a. 1-59) of PCV-2b Cap, an arginine rich region, are involved in the interaction with gC1qR. Porcine gC1qR interacts with Cap proteins of two pathogenic viral strains, PCV-2a and PCV-2b, while interaction has been observed with only one Cap protein of two investigated strains of PCV-1. The amino acids 30 and 49 of PCV-1Cap, solely, were not responsible of the difference of interaction observed. We have also shown that gC1qR interacts strongly with PCV-2Caps and PCV-1 GER Cap. This result suggests that the different interaction of gC1qR with PCV Cap proteins may have an impact on the pathogenicity of the PCV.

Impact of the administration of a third-generation cephalosporin (3GC) to one-day-old chicks on the persistence of 3GC-resistant Escherichia coli in intestinal flora: An in vivo experiment.

The aim of the experiment was to evaluate under controlled conditions the impact on the excretion of 3GC-resistant Escherichia coli of the injection of one-day-old chicks with ceftiofur, a third-generation cephalosporin (3GC). Three isolators containing specific-pathogen-free chicks were used. In the first one, 20 birds were injected with ceftiofur then ten of them were orally inoculated with a weak inoculum of a 3GC-resistant E. coli field isolate containing an IncI1/ST3 plasmid encoding a blaCTX-M-1 beta-lactamase. The other chicks were kept as contact birds. None of the 20 birds in the second isolator were injected with ceftiofur, but ten of them were similarly inoculated with the 3GC-resistant strain and the others kept as contact birds. A third isolator contained ten non-injected, non-inoculated chicks. Fecal samples were collected regularly over one month and the E. coli isolated on non-supplemented media were characterized by antimicrobial agar dilution, detection of selected resistance genes and determination of phylogenetic group by PCR. The titers of 3GC-resistant E. coli in individual fecal samples were evaluated by culturing on 3GC-supplemented media. Results showed that the inoculated strain rapidly and abundantly colonized the inoculated and contact birds. The ceftiofur injection resulted in significantly higher percentages of 3GC-resistant E. coli isolates among the analyzed E. coli. No transfer of the 3GC-encoding plasmid to other isolates could be evidenced. In conclusion, these results highlight the dramatic capacity of 3GC-resistant E. coli to colonize and persist in chicks, and the selecting pressure imposed by the off-label use of ceftiofur.

Draft Genome Sequence of Taylorella equigenitalis Strain MCE529, Isolated from a Belgian Warmblood Horse.

Taylorella equigenitalis is the causative agent of contagious equine metritis (CEM), a sexually transmitted infection of horses. We herein report the genome sequence of T. equigenitalis strain MCE529, isolated in 2009 from the urethral fossa of a 15-year-old Belgian Warmblood horse in France.

Evolutionary dynamics and genetic diversity from three genes of Anguillid rhabdovirus.

Wild freshwater eel populations have dramatically declined in recent past decades in Europe and America, partially through the impact of several factors including the wide spread of infectious diseases. The anguillid rhabdoviruses eel virus European X (EVEX) and eel virus American (EVA) potentially play a role in this decline, even if their real contribution is still unclear. In this study, we investigate the evolutionary dynamics and genetic diversity of anguiillid rhabdoviruses by analysing sequences from the glycoprotein, nucleoprotein and phosphoprotein (P) genes of 57 viral strains collected from seven countries over 40 years using maximum-likelihood and Bayesian approaches. Phylogenetic trees from the three genes are congruent and allow two monophyletic groups, European and American, to be clearly distinguished. Results of nucleotide substitution rates per site per year indicate that the P gene is expected to evolve most rapidly. The nucleotide diversity observed is low (2-3 %) for the three genes, with a significantly higher variability within the P gene, which encodes multiple proteins from a single genomic RNA sequence, particularly a small C protein. This putative C protein is a potential molecular marker suitable for characterization of distinct genotypes within anguillid rhabdoviruses. This study provides, to our knowledge, the first molecular characterization of EVA, brings new insights to the evolutionary dynamics of two genotypes of Anguillid rhabdovirus, and is a baseline for further investigations on the tracking of its spread.

Genetic diversity of perch rhabdoviruses isolates based on the nucleoprotein and glycoprotein genes.

Despite the increasing impact of rhabdoviruses in European percid farming, the diversity of the viral populations is still poorly investigated. To address this issue, we sequenced the partial nucleoprotein (N) and complete glycoprotein (G) genes of nine rhabdoviruses isolated from perch (Perca fluviatilis) between 1999 and 2010, mostly from France, and analyzed six of them by immunofluorescence antibody test (IFAT). Using two rabbit antisera raised against either the reference perch rhabdovirus (PRhV) isolated in 1980 or the perch isolate R6146, two serogroups were distinguished. Meanwhile, based on partial N and complete G gene analysis, perch rhabdoviruses were divided into four genogroups, A-B-D and E, with a maximum of 32.9% divergence (G gene) between isolates. A comparison of the G amino acid sequences of isolates from the two identified serogroups revealed several variable regions that might account for antigenic differences. Comparative analysis of perch isolates with other rhabdoviruses isolated from black bass, pike-perch and pike showed some strong phylogenetic relationships, suggesting cross-host transmission. Similarly, striking genetic similarities were shown between perch rhabdoviruses and isolates from other European countries and various ecological niches, most likely reflecting the circulation of viruses through fish trade as well as putative transfers from marine to freshwater fish. Phylogenetic relationships of the newly characterized viruses were also determined within the family Rhabdoviridae. The analysis revealed a genetic cluster containing only fish viruses, including all rhabdoviruses from perch, as well as siniperca chuatsi rhabdovirus (SCRV) and eel virus X (EVEX). This cluster was distinct from the one represented by spring viraemia of carp vesiculovirus (SVCV), pike fry rhabdovirus (PFRV) and mammalian vesiculoviruses. The new genetic data provided in the present study shed light on the diversity of rhabdoviruses infecting perch in France and support the hypothesis of circulation of these viruses between other hosts and regions within Europe.

Very virulent infectious bursal disease virus: reduced pathogenicity in a rare natural segment-B-reassorted isolate.

The purpose of this study was to compare the molecular epidemiology of infectious bursal disease virus (IBDV) segments A and B of 50 natural or vaccine IBDV strains that were isolated or produced between 1972 and 2002 in 17 countries from four continents, with phenotypes ranging from attenuated to very virulent (vv). These strains were subjected to sequence and phylogenetic analysis based on partial sequences of genome segments A and B. Although there is co-evolution of the two genome segments (70 % of strains kept the same genetic relatives in the segment A- and B-defined consensus trees), several strains (26 %) were identified with the incongruence length difference test as exhibiting a significantly different phylogenetic relationship depending on which segment was analysed. This suggested that natural reassortment could have occurred. One of the possible naturally occurring reassortant strains, which exhibited a segment A related to the vvIBDV cluster whereas its segment B was not, was thoroughly sequenced (coding sequence of both segments) and submitted to a standardized experimental characterization of its acute pathogenicity. This strain induced significantly less mortality than typical vvIBDVs; however, the mechanisms for this reduced pathogenicity remain unknown, as no significant difference in the bursal lesions, post-infectious antibody response or virus production in the bursa was observed in challenged chickens.