Homologous recombination is apparent in infectious bursal disease virus

Infectious bursal disease virus (IBDV) is a non-enveloped double-stranded RNA virus belonging to the Birnaviridae family. It shows substantial variation in the major antigen region of the viral capsid protein VP2, where a hypervariable region plays a key role in the virulence of IBDV and its epitope. This study identifies several putative recombinants from previously published data to suggest that homologous recombination may naturally occur between different IBDV strains. In addition, a novel very virulence sublineage emerges in the VP2 phylogenic tree, comprising three putative recombination strains isolated in Korea and China, KSH, KK1 and SH-h. The major putative parents of the three mosaics are descended from the vaccine lineage while their hypervariable regions from vvIBDV. These findings also suggest that vaccine coverage may have influence on the evolution and genetic diversity of IBDV, resulting in a novel group with vvIBDV phenotype through recombination with wild IBDV.     

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.     

Competitive replication of different genotypes of infectious bursal disease virus on chicken embryo fibroblasts

Infectious bursal disease virus (IBDV) causes immunosuppression in chickens. We investigated the molecular changes in chicken embryo fibroblasts (CEF) adapted IBDV by genomic sequencing. IBDV were serially passaged in CEF and chickens were infected with the IBDV obtained after different numbers of passages in CEF. Chicken infections showed that 16th, 20th, and 21st passage viruses were pathogenic, while 26th and 36th passage viruses were non-pathogenic. Sequencing demonstrated that the initial changes during the serial passage comprised of a single-nucleotide deletion in the 3′ non-coding region of segment B of the virus after 19th passage, followed by changes in the VP1 gene after the 20th passage of the virus and changes in VP2, VP5 after the 21st passage of the virus. These data suggested that the attenuation of very virulent IBDV was due to multigenic mutations and there are in vitro and in vivo competitive replications in IBDV quasispecies.     

Genetic characteristics of infectious bursal disease viruses from four continents

Following the initial discovery of very virulent infectious bursal disease virus (vvIBDV) strains in Europe, these viruses spread to many parts of the world. In this study, we examined the phylogenetic relationship of never-before-published IBDV from 18 countries on four continents. All the samples were collected between 1997 and 2005 and were reported to be from broiler flocks experiencing higher than expected mortality which is often associated with acute very virulent infectious bursal disease. A total of 113 samples were imported into the U.S. and viral genetic material was used to determine the nucleotide sequence of the VP2 gene hypervariable region. Although all the samples were reported to be associated clinically with high mortality, genetic analysis suggests that some were not vvIBDV strains. Two viruses from South Africa were genetically similar to U.S. variant viruses. A majority (71/113) of the viruses examined had the amino acid Alanine at position 222 and sixty-seven of these suspect vvIBDV also had amino acids I242, I256, I294 and S299 which are highly conserved among vvIBDV strains. Phylogenetic analysis placed putative vvIBDV strains from many different countries and geographic regions in a single clade with some minor non-significant branching.     

Identification of a European interserotypic reassortant strain of infectious bursal disease virus

Infectious bursal disease virus (IBDV, family Birnaviridae) is a bi-segmented double-stranded RNA virus for which two serotypes are described. Serotype 1 replicates in the bursa of Fabricius and causes an immunosuppressive and potentially fatal disease in young chickens. Serotype 2 is apathogenic in poultry species. Up to now, only one natural event of interserotypic reassortment has been described after the introduction of very virulent IBDV (vvIBDV) in the USA in 2009, resulting in an IBDV strain with its segment A related to vvIBDV and its segment B related to US serotype 2 strain OH. Here, we present the first European isolate illustrative of interserotypic reassortment. The reassorting isolate, named 100056, exhibits a genomic segment A typical of current European vvIBDV but a segment B close to European serotype 2 viruses, supporting an origin distinct from US strains. When inoculated into SPF chickens, isolate 100056 induced mild clinical signs in the absence of mortality but caused a severe bursal atrophy, which strongly suggests an immunosuppressive potential. These results illustrate that interserotypic reassortment is another mechanism that can create IBDV strains with a modified acute pathogenicity. As a consequence, and for a more precise inference of the possible phenotype, care should be taken that the molecular identification of IBDV strains is targeted to both genome segments.     

Susceptibility of chicken mesenchymal stem cells to infectious bursal disease virus

Infectious bursal disease virus (IBDV) is the causative agent of one of the most important viral diseases affecting the poultry industry worldwide. The virus causes an acute, highly contagious and immunosuppressive disease in chickens. Previous studies have demonstrated that in addition to B cells, macrophages can support the replication of IBDV. Since mesenchymal stem cells in bone marrow regulate the differentiation and proliferation of hematopoietic precursors, the interaction between IBDV and mesenchymal stem cells was investigated. Mesenchymal stem cells were isolated from chicken bone marrow. The classical IM strain and the variant strain-E of IBDV, both adapted to grow in a chicken macrophage cell line, were used to infect mesenchymal stem cells. Primary chicken mesenchymal stem cells were highly susceptible to replication of IBDV. Both viruses induced cytopathic effects and replicated to high titers in mesenchymal stem cells. The finding that IBDV can replicate in mesenchymal stem cells provides new information on the susceptible target cell population within the host and contributes to the understanding of the pathogenic potential of the virus.     

A molecular epidemiology study based on VP2 gene sequences reveals that a new genotype of infectious bursal disease virus is dominantly prevalent in Italy

A distinctive infectious bursal disease (IBD) virus genotype (ITA) was detected in IBD-live vaccinated broilers in Italy without clinical signs of IBD. It was isolated in specific-pathogen-free eggs and molecularly characterized in the hypervariable region of the virus protein (VP) 2. Phylogenetic analysis showed that ITA strains clustered separately from other homologous reference sequences of IBDVs, either classical or very virulent, retrieved from GenBank or previously reported in Italy, and from vaccine strains. The new genotype shows peculiar molecular characteristics in key positions of the VP2 hypervariable region, which affect charged or potentially glycosylated amino acids virtually associated with important changes in virus properties. Characterization of 41 IBDV strains detected in Italy between 2013 and 2014 showed that ITA is emergent in densely populated poultry areas of Italy, being 68% of the IBDV detections made during routine diagnostic activity over a two-year period, in spite of the immunity induced by large-scale vaccination. Four very virulent strains (DV86) and one classical strain (HPR2), together with eight vaccine strains, were also detected. The currently available epidemiological and clinical data do not allow the degree of pathogenicity of the ITA genotype to be defined. Only in vivo experimental pathogenicity studies conducted in secure isolation conditions, through the evaluation of clinical signs and macro/microscopic lesions, will clarify conclusively the virulence of the new Italian genotype.     

Molecular characteristic of VP2 gene of infectious bursal disease viruses isolated from a farm in two decades

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus in the Birnaviridae family. Four pathotypes, attenuated, virulent, antigenic variant, and very virulent, have been identified. In this study, we examined the phylogenetic relationship of 25 field isolates that were collected from a single farm during 1989–2008. A sequence analysis of PCR amplified 714 bp VP2 region showed that all the samples were derived from very virulent infectious bursal disease virus (vvIBDV) and were more closely related to the vvIBDV isolate UK661. From 1999, the isolate XA1999 had amino acids I228 and T394. XA2000, XA2001, XA2002, and XA2003-09 had amino acids E279 and T394. From 2004 to 2008, the isolates had amino acids H320, I349, S375, and R381 while the UK661 virus had T228, D279, Q320, V349, P375, K381, and A394. Such mutations do not change key amino acid residues in the domains which are essential for its virulence. It suggests that a virulent IBDV strain could maintain its virulence for a long period in the same chicken farm and the strain is highly stable under normal environments.     

Circulation of very virulent avian infectious bursal disease virus in Finland

In the spring of 2014 infectious bursal disease (IBD) was confirmed in a Finnish layer flock exhibiting clinical signs and increased mortality. Organ and blood samples were sent for diagnosis to the Finnish Food Safety Authority Evira. IBD virus (IBDV) was detected in RT–PCR studies. Altogether hens from six layer farms associated with increased mortality (7–10%, worst case 30%) were diagnosed with IBD during 2014. Antibodies were also detected with IBD-ELISA tests in hens on two farms. Phylogenetic analysis showed that the causative agent of the 2014 IBD outbreak was a non-reassortant very virulent type IBDV. The representative virus strains from previous IBD outbreaks in 1978, 1987 and 1993 were also included in the analysis. The strains isolated in 2014 and 1993 were very similar indicating circulation of a very virulent IBDV for over 20 years in the country. In spite of the comprehensive phylogenetic analysis, the definitive origin of the viruses from 2014 and previous outbreaks remains unclear.     

Very virulent infectious bursal disease virus isolated from wild birds in Korea: epidemiological implications

To explore the epidemiological link between infectious bursal disease virus (IBDV) in wild birds and domestic chickens in Korea, we examined 107 free-living wild birds, representing 7 species, that were found dead of apparent natural causes in Korea over the past two years for the presence of IBDV. Five birds were tested positive for IBDV by RT-PCR assay: black-billed magpie (n=1), mallard duck (n=2), bean goose (n=1) and white-fronted goose (n=1). IBDV was isolated from RT-PCR-positive tissues following chicken embryo inoculation. Sequence analysis of the VP2 gene indicated that all of the isolates from the wild birds encode amino acids A222, I242, I256, I294 and S299 of VP2, which are conserved among strains of very virulent IBDV (vvIBDV). Phylogenetic analysis revealed that the wild bird IBDV isolates are closely related to strains of vvIBDV. An IBDV isolate from a magpie showed 60% mortality in SPF chickens and severe bursal atrophy. The epidemiological implications of IBDV in free-living wild birds are discussed. To our knowledge, this is the first report of vvIBDV in free-living wild birds.     

Diversified bursal medullary B cells survive and expand independently after depletion following neonatal infectious bursal disease virus infection

The primary immunoglobulin repertoire of chickens is generated not by gene rearrangement but by a subsequent process of gene conversion in proliferating immature B cells within the follicles of a specialized gut-associated lymphoid organ, the bursa of Fabricius. Neonatal infection with infectious bursal disease virus can eliminate almost the entire bursal B-cell compartment. Thereafter, two types of follicle reappear. Larger follicles, with rapidly proliferating B cells and normal structure, are correlated with partial recovery of antibody response. Smaller follicles, lacking distinct cortex and medulla, appear unable to produce antigen-responsive B cells. To understand the genesis of the two types of follicle, we analysed their VL sequences and activation-induced deaminase mRNA levels. The results provide a model of bursal repopulation in which surviving bursal stem cells generate new follicles with normal morphology and function, while surviving medullary B cells continue to proliferate slowly, under the influence of stromal cells, giving rise to the smaller follicles. The latter remain fixed in a stage of development incapable of further gene diversification.     

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.

     

Real-time RT-PCR differentiation and quantitation of infectious bursal disease virus strains using dual-labeled fluorescent probes

A real-time RT-PCR assay was developed utilizing dual-labeled fluorescent probes binding to VP4 sequence that are specific to the classical (Cl), variant (V) and very virulent (vv) strains of infectious bursal disease virus (IBDV). The assay was highly sensitive and could detect as little as 3 x 10(2) to 3 x 10(3) copies of viral template. Viral genomic copy number could be accurately assayed over a broad range of 7-8 logs of viral genome. The variant sequence-specific probe was found to be highly specific in detecting isolates classified as variant A, D, E, G and GLS-5, and did not react with classical strains. A total of 130 field and experimental variant strain isolates were tested using this assay. The classical sequence-specific probe also demonstrated high sensitivity and specificity, and positively detected a total of 87 STC isolates, both field and experimental isolates, while differentiating between isolates that were variant and classical strains. The very virulent sequence-specific probe detected positively the Holland vvIBDV isolate and did not react with classical or variant strains. Rapid identification of viral strain is a primary concern to poultry flock health programs to ensure administered vaccines will protect against current strains of virus circulating in the flock. The ability to quantify virus concurrently is also of assistance in identifying the progression of disease outbreaks within the flock.     

Differentiation of Newcastle disease virus strains by one-dimensional peptide mapping

One-dimensional peptide mapping was used for the differentiation of Newcastle disease virus (NDV) strains. Virions were purified in one step, and digested with Staphylococcus aureus V8 protease or chymotrypsin without prior separation of their proteins. Peptides were separated by polyacrylamide gel electrophoresis and stained with Coomassie blue. This method proved to be a simple, economic and reproducible means of differentiating NDV strains.     

建立高分辨率熔解曲线法筛选含VEB-3型基因菌株

目的 建立高分辨率熔解曲线法筛选含VEB-3型基因菌株.方法 不重复收集2003年1-12月革兰阴性杆菌产超广谱β-内酰胺酶(ESBL)菌株292株,用酚-氯仿法抽提基因组DNA;首先以基因组DNA为模板,PCR扩增VEB基因;挑选阳性菌株34株再PCR扩增包含168位点的一段约110 bp的基因片段,高分辨率熔解曲线(high resolution melt,HRM)分析后测序验证.结果 VEB-3亚型与其他亚型基因通过HRM分析具有显著的差别;测序结果与HRM分型结果一致.结论 应用HRM技术可以准确快速筛选含VEB-3型基因菌株.