Molecular characterization of infectious bursal disease virus isolates from Nepal based on hypervariable region of VP2 gene

Two Infectious bursal disease virus (IBDV) isolates, NP1SSH and NP2K were obtained from a severe infectious bursal disease (IBD) outbreak in Nepal in 2002. The hypervariable (HV) region of VP2 gene (1326 bp) of the isolates was generated by RT-PCR and sequenced. The obtained nucleotide sequences were compared with those of twenty other IBDV isolates/strains. Phylogenetic analysis based on this comparison revealed that NP1SSH and NP2K clustered with very virulent (vv) IBDV strains of serotype 1. In contrast, classical, Australian classical and attenuated strains of serotype 1 and avirulent IBDV strains of serotype 2 formed a different cluster. The deduced amino acid sequences of the two isolates showed a 98.3% identity with each other and 97.1% and 98.3% identities, respectively with very virulent IBDV (vvIBDV) isolates/strains. Three amino acids substitutions at positions 300 (E-->A), 308 (I-->F) and 334 (A-->P) within the HV region were common for both the isolates. The amino acids substitutions at positions 27 (S-->T), 28 (I-->T), 31 (D-->A), 36 (H-->Y), 135 (E-->G), 223 (G-->S), 225 (V-->I), 351 (L-->I), 352 (V-->E) and 399 (I-->S) for NP1SSH and at position 438 (I-->S) for NP2K were unique and differed from other IBDV isolates/strains. NP1SSH and NP2K showed highest similarity (97.8%) with the BD399 strain from Bangladesh as compared with other vvIBDV isolates/strains. We conclude that the NP1SSH and NP2K isolates of IBDV from Nepal represent vvIBDV of serotype 1.     

Characterization of three infectious bursal disease virus isolates obtained from layer chickens in Iran

Three infectious bursal disease viruses (IBDVs) were isolated from field outbreaks in IBDV-vaccinated and non-vaccinated layer chicken flocks. Agar gel precipitation test (AGPT), immunoperoxidase staining, transmission electron microscopy (TEM), inoculation into embryonated eggs, and chicken embryo fibroblasts (CEFs) confirmed that the isolates were IBDVs. RT-PCR, restriction fragment length polymorphism (RFLP), and phylogenetic analysis demonstrated that the isolates were very virulent IBDV (vvIBDV) and showed a nucleotide sequence similarity of 96.3 to 99.8% in comparison with other vvIBDV strains. It was concluded that the Iranian isolates represented vvIBDV of serotype 1 originating from Europe, Japan, and China.     

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.     

Molecular characterization of recent infectious bursal disease virus isolates from Malaysia

Three isolates of Infectious bursal disease virus (IBDV), designated UPM04178, UPM04190 and UPM04238, were obtained from severe outbreaks of infectious bursal disease (IBD) in Malaysia in 2004. The hypervariable region (HPVR) of VP2 gene of these isolates was sequenced. The obtained sequences were compared with those of other isolates. The highest similarity (98%) concerning both nucleotide and amino acid sequences was found to very virulent IBDV (vvIBDV) strains. Phylogenetic analysis revealed clustering of the three isolates with vvIBDV strains. Evolutionary relatedness of the three isolates to vvIBDV strains was demonstrated by three phylogenetic methods: bootstrap values of 100%, 95% and 90% for nucleotide sequences and those of 58%, 86% and 96% for amino acid sequences were obtained by the distance, maximum parsimony and maximum likehood methods, respectively. It is concluded that UPM04178, UPM04190 and UPM04238 are vvIBDV isolates of serotype 1, which originate from a common ancestor of IBDV strains present in Malaysia.     

Pathogenicity of SspI-positive infectious bursal disease virus and molecular characterization of the VP2 hypervariable region

The pathogenicity of four isolates of infectious bursal disease virus (IBDV) that have restriction fragment length polymorphism patterns of very virulent IBDV (vvIBDV), based on the presence of SspI and TaqI sites in the VP2 hypervariable region, was studied in specific pathogen free chickens. Chickens inoculated with isolates 92/04, 94/B551 and 97/61 developed severe clinical signs with a high mortality ranging from 70 to 80%, whereas the 94/273 isolate caused 10% mortality. Regardless of the isolates, significant differences were noted in the bursal lesion scores and bursa:body weight ratio index in the infected groups in comparison with the control groups. However, the presence of lesions in non-bursal tissues, muscles, thymus and at the junction of the proventriculus and gizzard were found only in the 92/04, 97/61 and 94/B551 isolates. Restriction fragment length polymorphism and sequence analysis of the VP2 hypervariable region indicated that all the isolates can be classified as vvIBDV based on the presence of SspI and TaqI sites at nucleotide positions 1011 and 833, respectively. In addition, all the isolates had amino acid substitutions at P222A, V256I and L294I, which are characteristic for vvIBDV isolated from different parts of the world. All the isolates except 94/273 also had a StyI site at nucleotide position 888. The absence of a StyI site in this isolate was associated with amino acid substitution at 254 from G to S. The 94/273 also had an amino acid substitution at position 270 from A to E, which is variable in the STC, Cu1 and OH strains. The presence of amino acid substitutions from G254S andA270E in SspI- and TaqI-positive vvIBDV strains is very uncommon and has not been reported previously. These amino acid variations might have caused the 94/273 to become less virulent in specific pathogen free chickens and resemble a classical virulent IBDV strain.     

Cloning and nucleotide analysis of the vp2 gene of a very virulent infectious bursal disease virus isolate from Iran

An Iranian field isolate (IR01) of Infectious bursal disease virus (IBDV) was characterized by sequence analysis of its VP2 gene and protein. Comparison of the obtained sequences with those of IBDV isolates from other countries revealed that IR01 was similar to very virulent IBDV (vvIBDV) strains with the identities at nucleotide and amino acid levels reaching 98.198.9% and 99.199.3%, respectively. On the other hand, it was less similar to non-vvIBDV strains; with nucleotide and amino acid identities of 95.295.7% and 96.097.3%, respectively. Out of nine unique nucleotide differences found between IR01 and some other serotype 1 strains only two resulted in amino acid substitutions (Ile296Val and Thr359Lys). In phylogenetic analysis, IR01 was closely related to Asian and European vvIBDV strains. Based on these results, IR01 obviously belongs to vvIBDV strains.     

Sequence analysis of both genome segments of two very virulent <Emphasis Type="Italic">Infectious bursal disease virus</Emphasis> field isolates with distinct pathogenicity

Summary. The deduced amino acid sequences of segment A and B of two very virulent Infectious bursal disease virus (vvIBDV) isolates, UPM94/273 and UPM97/61 were compared with 25 other IBDV strains. Twenty amino acid residues (8 in VP1, 5 in VP2, 2 in VP3, 4 in VP4, 1 in VP5) that were common to vvIBDV strains were detected. However, UPM94/273 is an exceptional vvIBDV with usual amino acid substitutions. The differences in the divergence of segment A and B indicated that the vvIBDV strains may have been derived from genetic reassortment of a single ancestral virus or both segments have different ability to undergo genetic variation due to their different functional constraints.     

Identification and pathogenicity of a natural reassortant between a very virulent serotype 1 infectious bursal disease virus (IBDV) and a serotype 2 IBDV

Infectious bursal disease virus (IBDV) causes an economically important, immunosuppressive disease in chickens. There are two serotypes of the virus that contain a bi-segmented double-stranded RNA genome. In December 2008, the first very virulent (vv)IBDV was identified in California, USA and in 2009 we isolated reassortant viruses in two different locations. Genome segment A of these reassortants was typical of vvIBDV serotype 1 but genome segment B was most similar to IBDV serotype 2. The CA-K785 reassortant caused 20% mortality in chickens but no morbidity or mortality in commercial turkey poults despite being infectious. There have been previous reports of natural reassortants between vvIBDV and other serotype 1 strains, but a natural reassortant between IBDV serotypes 1 and 2 has not been described. The apparent reassorting of California vvIBDV with an endemic serotype 2 virus indicates a common host and suggests vvIBDV may have entered California earlier than originally thought.     

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.     

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.     

The enhanced virulence of very virulent infectious bursal disease virus is partly determined by its B-segment

Summary. There is a remarkable difference in virulence of infectious bursal disease virus (IBDV) strains ranging from sub-clinical infections for serotype 2 and cell culture adapted serotype 1 strains, to 100% mortality for very virulent serotype 1 strains in young SPF chickens. It is known that cell culture adaptation related attenuation is determined by distinct mutations in the hypervariable region of the VP2 outer capsid protein, encoded on the A-segment. Amino acid mutations in the hypervariable VP2 region however, offer no explanation for the difference in virulence of classical and very virulent serotype 1 strains. Here we show by in vitro and in vivo analysis of rescued segment reassorted IBDVs that virulence factors are not only located on the A-segment, but on the RNA Dependent RNA Polymerase (VP1) encoding B-segment as well. Insight into the virulence factors of very virulent IBDV will contribute to the improvement of live IBDV vaccines.     

Chicken recombinant antibodies specific for very virulent infectious bursal disease virus

Summary. A phage-displayed single chain variable fragment (scFv) antibody library was constructed from the immune spleen cells of chickens immunized with very virulent infectious bursal disease virus (vvIBDV) strain CS89. A library consisting of around 9.2 × 10⁷ clones was subjected to 3 rounds of panning against captured CS89 virus. Analysis of individual clones by nucleotide sequencing revealed at least 22 unique scFv antibodies binding to vvIBDV in ELISA. Testing of the scFv antibody panel in ELISA against classical, variant or vaccine strains and a wide variety of vvIBDV isolates from the UK, China, France, Belgium, Africa, Brazil, Indonesia and the Netherlands identified one antibody, termed chicken recombinant antibody 88 (CRAb 88) that was specific for vvIBDV. CRAb 88 was capable of recognizing all vvIBDV strains tested regardless of their country of origin and showed no reactivity with classical, variant or vaccine strains, lending support to the use of this scFv as a powerful diagnostic tool for the differentiation of vvIBDV strains. Immunoprecipitation studies revealed that CRAb 88 was directed towards a highly conformational epitope located within the major neutralizing protein VP2. Sequence analysis of the hypervariable region of VP2 of the IBDV strains tested indicate that Ile(256) and Ile(294) may play roles in binding of CRAb 88. This is the first reagent of its type capable of positively distinguishing vvIBDV from other IBDV strains.     

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.     

Antigenic and genetic diversity of early European isolates of <Emphasis Type="Italic">Infectious bursal disease virus</Emphasis> prior to the emergence of the very virulent viruses: early European epidemiology of <Emphasis Type="Italic">Infectious bursal disease virus</Emphasis> revisited?

Summary. Eleven Polish and Hungarian isolates of Infectious bursal disease virus (IBDVs) obtained in the 70/80s (early IBDV) and in the 90s (recent IBDV) were characterized in an Antigen-Capture-ELISA with a panel of neutralizing monoclonal antibodies (Mabs), and by nucleotide sequencing of the VP2 variable domain (vVP2). The viruses were compared with reference IBDV strains, among others with Faragher 52/70 (F52/70, classical, isolated 1970), 89163 (typical very virulent-vvIBDV, isolated 1989) and 91168 (antigenically modified vvIBDV, isolated 1991). Only one of the early isolates (Hungarian strain P1) proved antigenically and genetically similar to F52/70. Other early isolates exhibited no reactivity versus Mabs 3, 4, 5 and/or 8 and had a common previously unrecognized combination of amino acid changes in vVP2. The recent isolates all proved antigenically and genetically related to typical vvIBDV strain 89163, except the Polish isolate 93/35 which proved related to the 91168 strain although no epidemiological relationship had been documented between these viruses in the field. Phylogenetic analysis confirmed that the non-P1 early IBDVs represent a previously unrecognized group among serotype 1 IBDVs. It is discussed whether these early isolates are derivatives of the F52/70-like viruses that might still be present in the field, or whether they represent early IBDV strains that might have been present prior to and progressively replaced by the F52/70-like viruses, as the latter have been replaced by vvIBDVs in the late eighties.