Acute infectious bursal disease in poultry: Isolation and characterisation of a highly virulent strain

A highly virulent strain of infectious bursal disease virus (IBDV) was isolated from the field and propagated in SPF chickens, causing up to 100% mortality. Although it still belongs to the standard serotype 1 IBD viruses, serological typing with monoclonal antibodies showed an antigenic drift in this pathogenic strain. Conventional 'intermediate' IBD vaccines are probably more antigenically related to the pathogenic strain than the mild ones and were effective in protecting SPF chickens against challenge.     

Acute infectious bursal disease in poultry: A review

This review is focused on the acute form of infectious bursal disease (IBD) caused by very virulent IBD virus (vvIBDV). First described in Europe about 10 years ago, this new form of the disease has rapidly spread all over the world, causing dramatic losses; after a decade, it still represents a considerable threat to the poultry industry. Emergence of the acute forms of the disease has drastically changed the epidemiology of IBD. Although their origin is still under investigation, vvIBDVs have spread all over the world in a very explosive but conserved manner. This raises the question of the origin of vvIBDVs, of the possible existence of reservoirs and of the possible emergence of new, distinct lineages in the future. While it has become clear that amino acids within the variable region of virus protein VP2 account for the molecular basis of antigenic variation, no definite hot spot that determines pathogenicity has been identified. Fingerprints of VP2 on vvIBDVs have to be considered more as common evolutionary markers than as virulence markers. The search for such markers is in progress. Pathogenesis of the disease is still poorly understood, and cytokines might play a crucial role in the onset of the disease and in the development of immunosuppression. Mechanisms such as apoptosis and necrosis have been described in lymphoid organs and are involved in the severity of the disease. Macrophages, especially, could play a specific role in the acute phase. Classical serotype 1 vaccines still induce good protection, but the actual problem for control of the disease has became the interference of maternally derived antibody in the establishment of the vaccination schedule. The development of safe vaccines that could either transmit a high passive immunity which could protect broilers during the whole growing period or prime an immune response before or at hatching in the presence of passive immunity might be established in the near future. In this context, recombinant vaccines and virus-neutralizing factor technology might have an advantage over other approaches.     

Nucleotide sequence and phylogenetic analysis of a segment of a highly virulent strain of infectious bursal disease virus

The complete nucleotide sequences encoding precursor polyprotein (VP2-VP3-VP4) and VP5 of a highly virulent (hv) infectious bursal disease virus (IBDV), UPM97/61 was determined. Comparison of the deduced amino acid sequences with the published ones revealed 8 common amino acid substitutions, which were found only in the hv IBDV including the UPM97/61 strain. Three of the amino acid substitutions (222 Ala, 256 Ile and 294 Ile) were used as a marker for determining hv IBDV strains. The other five substitutions (685 Asn, 715 Ser, 751 Asp, 990 Val and 1005 Ala) were also conserved in hv IBDV strains isolated in various countries. UPM97/61 strain demonstrated also 8 unique amino acid substitutions of which 3 were in VP2, 4 in VP3 and 1 in VP4. There was 1 unique amino acid substitution in VP5 at position 19 (Asp-->Gly) not found in other strains. However, all the strains have a conserved 49 Arg. The amino acid sequence of UPM97/61 strain differed by 1.09% from the Japanese (OKYM) and Hong Kong (HK46) strains, and by 1.48% from the Israeli (IBDVKS) and European (UK661) strains. Hence, UPM97/61 is more closely related to the hv strains from Asia. However, phylogenetic analysis indicated that the origin of UPM97/61 might be the same as that of other hv strains isolated from other parts of the world.     

Sequence and phylogenetic analyses of highly virulent infectious bursal disease virus

Summary.  The nucleotide sequences of the genome segment A and B encoding the precursor polyprotein (NH₂-VP2-VP4-VP3-COOH) and VP1 were determined for a highly virulent strain of infectious bursal disease virus (IBDV). The precursor polyprotein and VP1 coding regions of highly virulent OKYM strain consisted of 3 039 nucleotides (1 012 deduced amino acids) and 2 640 nucleotides (879 deduced amino acids), respectively. Comparison of the deduced amino acid sequences of the highly virulent IBDV (HV-IBDV) with other serotype 1 and 2 sequences revealed 17 amino acid residues which were conserved only in the HV-IBDV. Among the 17 unique amino acid differences, 8 were in VP1, 4 were in VP2, 3 were in VP3 and 2 were in VP4. Although it is impossible to predict the effect of the unique amino acid residues without detailed knowledge of the three-dimensional structure and function of the proteins, they could affect the virulence of HV-IBDV. Alignment of the nucleic acid sequences of precursor polyprotein, VP1, VP2, VP3 and VP4 coding regions followed by distance analysis allowed the generation of phylogenetic trees. The same tree topology was obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. On the other hand, the tree topology of VP1 was quite different from that obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. These findings indicate that not a genetic recombination but a genetic reassortment may play an important role in the emergence of HV-IBDV. Accepted January 16, 1997; Received October 25, 1996     

Characterisation of an Indonesian very virulent strain of infectious bursal disease virus

Summary.  An Indonesian very virulent (vv) strain of infectious bursal disease virus (IBDV), designated Tasik94, was characterised both in vivo and at the molecular level. Inoculation of Tasik94 into 5-week-old specific-pathogen-free (SPF) chickens resulted in 100% morbidity and 45% mortality. The complete nucleotide and predicted amino acid sequences of genomic segments A and B were determined. Across each of the three deduced open reading frames (ORFs), Tasik94 shared the greatest nucleotide homology to Dutch vv strain D6948. Phylogenetic analyses were performed using 15 full-length polyprotein sequences and a total of 105 VP2 hypervariable region sequences from geographically and pathogenically diverse strains. In each case, Tasik94 grouped closely with vv strains, particularly those from Europe. The deduced VP1, VP2, VP3, VP4 and VP5 protein sequences of Tasik94 were aligned with those from published strains and putative virulence determinants were identified in VP2, VP3 and VP4. Alignment of additional protein sequences across the VP2 hypervariable region confirmed that residues Ile[242], Ile[256] and Ile[294] were highly-conserved amongst vv strains, and may account for their enhanced virulence. Received January 11, 2002; accepted February 26, 2002 Published online May 24, 2002     

Sequence and phylogenetic analysis of a Chinese very virulent infectious bursal disease virus

The complete genome sequence of a Chinese very virulent infectious bursal disease virus (vvIBDV) strain, Harbin-1, was determined. Based on the sequence analysis, the molecular characteristics and potential virulence determinants and origin of vvIBDV strains were identified. Phylogenetic analysis indicated that a reassortment and/or recombination event may have occurred in the emergence of Chinese vvIBDV strains.     

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.     

Acute infectious bursal disease in poultry: protection afforded by maternally derived antibodies and interference with live vaccination

Maternally derived antibodies (MDA) were found insufficient to protect broiler chicks against a highly pathogenic strain of IBDV during the growing period even if the parent flocks had been boostered at point of lay by using oil emulsion vaccines. Whatever the vaccination scheme of the parent flocks, maximum mortality was observed after a challenge performed at 38 days of age in broiler and layer chicks, suggesting that the offspring need to be vaccinated with live vaccines before that age. MDA were demonstrated to interfere with live vaccination but strain D78 was shown to be more efficient as it could establish an infection even at higher antibody levels than the other strains. Given this pathogenicity and the lack of uniformity in the transmitted immunity, there is always a critical period where immune and susceptible birds coexist in the same flock. This period seems to be broader when parental flocks were vaccinated with an inactivated oil emulsion vaccine before lay.     

High sequence diversity in infectious bursal disease virus serotype 1 in poultry and turkey suggests West-African origin of very virulent strains

Fifty-eight outbreaks of Infectious bursal disease virus (IBDV) were observed in vaccinated chicken flocks in four Southwestern states of Nigeria between 1995 and 2000. Bursa samples from 40 flocks were found virus-positive in VP2-specific nested RT-PCR. Sequences of the hypervariable region of VP2 were compared to reference strains of the different IBDV variants including also 1988 isolates from Nigeria. Sequence analysis revealed that all 40 Nigerian isolates belonged to the very virulent (vv) variant. The maximum sequence diversity of 5.7% was higher than in all other vvIBDV sequences listed in Genbank (3.6%). Two clusters within Nigerian isolates are unique to this region. Serotype 1 IBDV was also detected in four symptomatic turkey flocks. The turkey isolates were found within 2 of the 3 VV-clusters of chicken isolates. Full length sequence of a turkey isolate (NIE009t) confirmed its close relation to vvIBDV strain D6948NET for both segment A (1.4% sequence diversity) and segment B (2.1%). Thus, turkeys should be considered susceptible to vvIBDV infection. The unusually high sequence diversity of vvIBDV may be an indication of a West-African origin of this virus, from where it spread to other continents.     

Critical amino acid changes in VP2 variable domain are associated with typical and atypical antigenicity in very virulent infectious bursal disease viruses

Summary.  Classical serotype 1 infectious bursal disease viruses (IBDV), but not very virulent (vv) isolates, react with neutralizing monoclonal antibody (NMab) 3 in virus neutralization tests or antigen-capture ELISA. Two other NMabs, 6 and 8, bind to both classical and most vv strains, but not to the atypical 94 432 and 91 168 vv strains, respectively. The basis for such reactivities was investigated by sequencing the genome region encoding the VP2 major immunogenic domain. In classical, variant, vaccine or vv IBDV strains, negative reactions with NMab3 were associated with changes in the Proline-Glycine pair at amino-acid (aa) positions 222–223 (hydrophilic peak A), and negative reactions with NMabs 6 and 8 with aa changes from positions 318 to 324 (hydrophilic peak B). The 91 168 and 94 432 viruses are the first vvIBDVs to present aa changes in peak B. Received January 9, 1998 Accepted March 20, 1998     

Attenuation of very virulent infectious bursal disease virus and comparison of full sequences of virulent and attenuated strains

A very virulent strain of infectious bursal disease virus (IBDVks) was isolated from the bursae of Fabricius of IBDV-affected broiler chickens. Following 43 serial passages in specific pathogen-free embryonated eggs, an attenuated strain was established (IBDVmb). Dosages of IBDVmb in the range 10² to 10⁴ embryo infective dose of 50% were found to be safe and protective for commercial chicks. Chickens vaccinated with live vaccine containing IBDVmb responded with precipitating and type-specific neutralizing antibodies, and were immune to subsequent challenge with a very virulent IBDV. IBDVmb has been used as an attenuated vaccine throughout the world since 1993. A comparison of the full sequences of the virulent and attenuated strains (IBDVks and IBDVmb, respectively) revealed seven nucleotides that were different, four of them leading to changes in the amino-acid sequence. Comparison of the protein sequencse of these strains and published sequences of very virulent and attenuated phenotypes lead us to sugget that the novel difference responsible for virulence of the Israeli strains are: residue 272 (VP2, very conserved site) and residue 527 (VP4), both in segment A, and in segment B (VP1) residues 96 and 161 (both conserved). Our study strengthens the possibility that more than one protein is involved in IBDV attenuation. In all reports, including ours, virulence was reduced without affecting antigenicity of the neutralizing epitopes in VP2. This could have practical implications for attenuated-vaccine development.     

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.     

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.     

Demonstration of receptor binding properties of VP2 of very virulent strain infectious bursal disease virus on Vero cells

Tissue culture adaptation of infectious bursal disease virus (IBDV) results in alternation of three residues on its major capsid protein VP2 and these residues may engage in receptor binding. Although the key of successful infection of tissue culture adapted IBDV in tissue cultures was defined as the virus entering steps, mechanism of the adaptation is poorly understood. In this study, recombinant VP2s of an attenuated strain (D78) and a very virulent strain (HK46) of IBDV tagged with rabbit immunoglobulin G heavy chain were expressed in mammalian cells, generating RAVP2 and RVVP2, respectively, in high purity. Using flow cytometry, both RAVP2 and RVVP2 were demonstrated to bind with Vero cells while these bindings were blocked by D78 viral particles, implying both very virulent IBDVs (vvIBDVs) and attenuated IBDVs bind to Vero cells through the same receptor(s). Since vvIBDVs cannot be propagated directly in tissue cultures, the specific binding between RVVP2 and Vero cells suggests the barrier for tissue culture adaptation may be beyond the virus attachment process.     

Purification and preliminary characterisation of a pathogenic strain of infectious bursal disease virus

A pathogenic strain of infectious bursal disease virus has been purified by density gradient centrifugation, principally on sucrose and tartrate gradients. Examination of gradient peak fractions by electron microscopy has revealed two populations of particles of average size 62 nm and 20 nm, which band together on sucrose and tartrate gradients. Purified virus has been shown to reproduce the typical symptoms and lesions of infectious bursal disease. The possible classification of the larger virus particle in the diplornavirus group and the origin of the smaller particle are discussed.     

Histopathology and immunohistochemistry of renal lesions due to avian infectious bronchitis virus in chicks uninoculated and previously inoculated with highly virulent infectious bursal disease virus.

A nephropathogenic strain of infectious bronchitis virus (IBV) was inoculated intra-tracheally into 14-day-old specific-pathogen-free chicks or ones previously inoculated with highly virulent infectious bursal disease virus (IBDV) at 7 days of age. The renal lesions were examined histopathologically and immunohistochemi-cally at intervals up to 30 days post-inoculation. The mortality was 20% in the IBDV + IBV-inoculated group, but not in the IBV-inoculated one. Swollen and pale kidneys due to IBV infection were more severe and of longer duration in dually infected chicks. At the early stage of infection, the histopathological changes in the kidneys were similar in both groups, but the ducto-tubular damage was more severe in the dually infected chicks. At the late stage of infection, the renal lesions were characterized by chronic interstitial nephritis with formation of lymphoplasmacytic nodules in IBV-inoculated chicks and by chronic active nephritis which consisted of tubular degeneration, lymphoid cell reaction and interstitial fibrosis in IBDV + IBV-inoculated ones. More IBV antigen-positive cells persisted longer in the kidneys of dually infected chicks than in those of IBV-inoculated ones.     

Isolates of infectious bursal disease virus from India are of very virulent phenotype

Four Indian field isolates, a classical virulent and an attenuated vaccine strains of Infectious bursal disease virus (IBDV) have been characterized by sequence analysis of part of the VP1 gene (from nucleotide 1538-1979) comprising one of viral RNA dependent RNA polymerase motifs. Sequence alignment of these viruses with reported viruses of other countries revealed Indian IBDV field isolates to be 100% similar to very virulent Japanese (OKYM), European (UK661) and Bangladesh (BD3/99) IBD viruses at amino acid level, whereas they had 0.2-0.9% divergence at nucleotide level. Out of the total 24 nucleotide changes found in the Indian field isolates, as well as reported very virulent viruses, only one resulted in amino acid change S-P at 562 position. The Indian field isolates displayed nucleotide divergence of 10.6-11.6% and amino acid divergence of 2.8-3.5% from the classical virulent and attenuated vaccine strains. The RNA dependent RNA polymerase motif from amino acid 528-541, present in the sequence analyzed, was conserved among all the viruses, irrespective of pathotype and serotype. In the phylogenetic tree, based on nucleotide sequence, Indian field viruses were grouped with reported very virulent viruses in one lineage whereas, classical virulent, attenuated vaccine and serotype 2 strains formed part of the second lineage. But in the phylogenetic tree based on amino acid sequence alignment, the serotype 2 strain OH grouped with Indian field isolates and reported very virulent viruses in one lineage and classical virulent and attenuated vaccine strains formed the second lineage.     

Susceptibility of vaccinated and unvaccinated Egyptian chickens to very virulent infectious bursal disease virus

The responses of vaccinated and unvaccinated chickens of different breeds to infection with very virulent infectious bursal disease virus (vvIBDV) were investigated. Five-week-old chickens of five Egyptian breeds (Fayoumi, Balady, Golden, Mandarah, and Gimmizah), and foreign White Leghorn pullets were tested. In unvaccinated birds, mortality, relative bursa and spleen weight, bursal lesion score, antibody titres and the response of blood lymphocytes to mitogens were examined. The Gimmizah and Fayoumi birds showed the greatest susceptibility to infection with mortalities of 85 and 47%, respectively. Mandarah birds were resistant (11% mortality), while the Leghorn, Golden and Balady birds were of intermediate susceptibility (20, 21 and 37% mortality rates, respectively). Vaccinated birds were administered a live intermediate classic vaccine and challenged 10 days later with vvIBDV. Mortality following challenge was about 3% in the Fayoumi, Gimmizah and Balady birds, whereas no mortality was seen in Mandarah, Golden, and Leghorn birds. The classic vaccine induced significant protection. However, it did not prevent histological bursal lesions, especially in the Fayoumi and Leghorn birds. Neither pathological nor immunological measures correlated closely with susceptibility or resistance of the different breeds. The findings suggest that innate non-immunogenic factor(s) may play a critical role in resistance.     

Phylogeographic distribution of very virulent infectious bursal disease virus isolates in the Iberian Peninsula

Viral population dynamics of very virulent infectious bursal disease virus (vvIBDV) field strains isolated in the Iberian Peninsula since the first outbreak in the 1990s have been analysed. Low levels of genetic variability and a global purification selection pattern were reported in 480 base pairs of the hypervariable region of the VP2 gene, indicating a lack of a selection-driven immune escape in the evolutive pathway of the virus. The viral population structure of vvIBDV strains in the Iberian Peninsula showed a strong relationship between geography and phylogeny, with two main groups observed. A global comparison among vvIBDV strains also showed an association with sequences from the same country. The low variability, the strong purifying selection and the geographical pattern observed point to a picture where the virus evolves slowly, occupying the same geographical niche for a long time. The scenario depicted fits well with the biological features of the virus: being able to remain viable for long periods of time due to a strong environmental resistance, and as an immunosuppressive agent, capable per se of annihilating temporally the immune system of the host.