Molecular epizootiology of infectious bursal disease (IBD) in Korea

We conducted a molecular epizootiological study of infectious bursal disease (IBD) in Korea by analyzing 85 IBD viruses (IBDVs) obtained from vaccinated or unvaccinated flocks between 1980 and 2007. Phylogenetic analysis of the partial nucleotide sequence of the hypervariable region of the VP2 gene (nucleotides 661–1020) and pathogenicity tests revealed more genetic and phenotypic diversity of IBDV in Korea than has been reported previously. We showed that very virulent IBDVs (vvIBDVs) were already present in Korea in 1986. Moreover, vvIBDVs were repeatedly detected in Korean poultry that had been vaccinated, which casts doubt on the IBD vaccine programs. We also identified novel putative antigenic variant (AV)-like IBDV isolates on the basis of their antigenic indices and the presence of amino acid changes (P222S or P222T-A321D) that are known to affect the antigenicity of VP2. These observations suggest that future studies examining the efficacy of conventional vaccines against atrophy of the bursa of Fabricius and vvIBDV shedding may be useful. Moreover, it will be of interest to determine the prevalence of putative Korean antigenic variants and whether these strains exert immunosuppressive effects in vaccinated birds.     

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.     

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.     

Genomic sequence of an infectious bursal disease virus isolate from Zambia: classical attenuated segment B reassortment in nature with existing very virulent segment A

We determined the complete nucleotide sequence of an infectious bursal disease (IBD) virus (IBDV) isolate (designated KZC-104) from a confirmed IBD outbreak in Lusaka in 2004. The genome consisted of 3,074 and 2,651 nucleotides in the coding regions of segments A and B, respectively. Alignment of both nucleotide and deduced amino acid sequences and phylogenetic analysis revealed that the genome segment A of KZC-104 was derived from a very virulent (VV) strain, whereas its segment B was derived from a classical attenuated strain. On BLAST search, the full-length segment A and B sequences showed 98 % nucleotide sequence identity to the VV strain D6948 and 99.8 % nucleotide sequence identity to the classical attenuated strain D78. This is a unique IBDV reassortant strain that has emerged in nature, involving segment B of a cell-culture-adapted attenuated vaccine.     

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.     

Changes in VP3 and VP5 genes during the attenuation of the very virulent infectious bursal disease virus strain Gx isolated in China

A very virulent infectious bursal disease virus (vvIBDV) Gx strain with high pathogenicity was attenuated through replication in specific-pathogen free (SPF) chicken embryos and in chicken embryo fibroblast (CEF) cell cultures. The changes in nucleotide sequences and the deduced amino acid sequences of VP3 and VP5 genes during attenuation were obtained. Sequence analysis of selected passages from numbers 0 to 20 in CEF’s (designated here Gx to CEF-20) showed that there were no amino acid changes detected in the VP3 and VP5 genes before CEF-9. There were some changes in the nucleotide sequence and amino acid substitutions in the VP3 and VP5 genes at CEF-9. CEF-9 was an intermediate with some amino acid changes which were possibly related to virulence. The amino acid sequences of VP2 and VP5 genes remained unchanged from CEF-10 to CEF-20. The results of pathogenicity test showed that the mortalities of vvIBDV-Gx, CEF-5, CEF-8, and CEF-9 were 64, 60, 60, and 28%, respectively, while there were no mortalities observed for CEF-10, CEF-15 and CEF-20. There was also no bursal atrophy when chickens were inoculated with CEF-10, CEF-15, and CEF-20. Virus neutralization tests with the Gx strain and sera from inoculated chickens showed that the antigenicity was similar from Gx to CEF-20. The implications of these findings for the study of IBDV virulence and a more effective control of vvIBDV are discussed.     

Molecular epidemiology studies on partial sequences of both genome segments reveal that reassortant infectious bursal disease viruses were dominantly prevalent in southern China during 2000-2012

A molecular epidemiology study of infectious bursal disease viruses (IBDVs) isolated from seven provinces in southern China during the years 2000-2012 was performed based on partial sequences of genome segments A and B, namely the hypervariable region of the A-VP2 gene (A-vVP2) and the b fragment of VP1 gene (B-VP1b) from a total of 91 field isolates. Sequence analysis based on vVP2 revealed that 72 out of 91 isolates had the same characteristic amino acid (aa) sequences as vvIBDV. The mutation of D212N in A-vVP2 has become prevalent in the recent isolates. The origin of the field isolates with vvIBDV characteristic amino acid residues was complex, evidenced by the findings that more than one subgroup of strains prevailed in each province. When B-VP1b was analyzed, there were three lineages among the field isolates, and none of the isolates had a relationship to vvIBDV-related segment B. Phylogenetic analysis of both segments revealed that only a few isolates (13/91) had the same genetic relatives in consensus trees based on segments A and B, whereas the majority of the isolates (85.71%, 78/91) were identified to be naturally reassorted strains. Based on the origin of each segment, at least six types of reassortant IBDVs prevailed in southern China, three of which were shown to be dominant: segment A from vvIBDV and B from attenuated IBDV, segment A of vvIBDV and B from 002-73-like IBDV, and segment A of vvIBDV and B from HLJ0504 or a similar strain. Our findings suggest that both genomic segments of field IBDVs has been evolving, and continuous monitoring of the evolution of field IBDV genome is therefore urgently needed in the control of IBDV.     

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     

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.     

Cloning and Sequence Analysis of the Nucleocapsid Gene of Porcine Epidemic Diarrhea Virus Chinju99

The nucleocapsid (N) gene of the porcine epidemic diarrhea virus (PEDV) Chinju99 which was previously isolated in Chinju, Korea was cloned and sequenced to establish the information for the development of genetically engineered diagnostic reagents. Also, sequences of the nucleotides and deduced amino acids of the Chinju99 N gene were analyzed by alignment with those of CV777 and Br1/87. The nucleotide sequence encoding the entire N gene open reading frame (ORF) of Chinju99 was 1326 bases long and encoded a protein of 441 amino acids with predicted M _r of 49 kDa. It consisted of 405 adenine (30.5%), 293 cytosine (22.1%), 334 guanines (25.2%) and 294 thymines (22.2%) residues. The Chinju99 N ORF nucleotide sequence was 96.5% and 96.4% homologous with that of the CV777 and Br1/87, respectively. The Chinju99 N protein revealed 96.8% amino acid identity with that of Br1/87 and CV777, respectively. The amino acid sequence contained seven potential sites for threonine (T)- or serine (S)-linked phosphorylation by each protein kinase C and casein kinase II.     

Cloning and Sequence Analysis of the Spike Gene of Porcine Epidemic Diarrhea Virus Chinju99

The spike (S) gene of the porcine epidemic diarrhea virus (PEDV) Chinju99 which was previously isolated in Chinju, Korea was cloned and sequenced to aid in the development of genetically engineered vaccines and diagnostic reagents against PEDV. The nucleotide sequence encoding the entire S gene open reading frame (ORF) of Chinju99 was 4152 bases long encoding 1383 amino acids. It consisted of 1001 adenine (24.1%), 849 cytosine (20.4%), 877 guanine (21.1%) and 1425 thymine (34.3%) residues. The Chinju99 S ORF nucleotide sequence was 94.5% homologous with that of the Br1/87 and CV777 strains, respectively. The Chinju99 S protein had 92.8% amino acid identity with that of Br1/87 and CV777, respectively. The amino acid sequence contained 27 potential sites for asparagine (N)-linked glycosylation and there was a stretch of highly hydrophobic residues at position 1325–1350.     

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.     

Genomic sequence analysis of a new reassortant infectious bursal disease virus from commercial broiler flocks in central China

We report the complete nucleotide sequence of a reassortant infectious bursal disease (IBD) virus (IBDV) HN isolate from commercial broiler flocks in central China. The genome consisted of 3,232 and 2,652 nucleotides in the coding regions of segments A and B, respectively. Alignment of both nucleotide and deduced amino acid sequences and phylogenetic analysis revealed that the genome segments A and B of HN were derived from the attenuated strain B87 and the VV strain OKYM. This is a new reassortant IBDV strain that has emerged in nature, involving segment A of a cell-culture-adapted attenuated vaccine strain B87.     

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.     

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.     

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.     

Base Usage and Dinucleotide Frequency of Infectious Bursal Disease Virus

Base usage and dinucleotide frequency have been extensively studied in many eukaryotic organisms and bacteria, but not for viruses. In this paper, a comprehensive analysis of these aspects for infectious bursal disease virus (IBDV) was presented. The analysis of base usage indicated that all of the IBDV genes possess equivalent overall nucleotide distributions. However when the base usage at each codon positions was analysed by using cluster analysis, the VP5 open reading frame (ORF) formed a different cluster isolated from the other genes. The unusual base usage of VP5 ORF may indicate that the gene was originated by the virus "overprinting strategy", a strategy in which virus may create novel gene by utilizing the unused reading frames of its existing genes. Meanwhile, the GC content of the IBDV genes and the chicken's coding sequences was comparable; suggesting the virus imitation of the host to increase its translational efficiency. The analysis of dinucleotide frequency indicated that IBDV genome had dinucleotide bias: the frequencies of CpG and TpA were lower and the TpG was higher than the expected. Classical methylation pathway, a process where CpG converted to TpG, may explain the significant correlation between the CpG deficiency and TpG abundance. "Principal component analysis of the dinucleotide frequencies" (DF-PCA) was used to analyse the overall dinucleotide frequencies of IBDV genome. DF-PCA on the hypervariable region and polyprotein (VPX-VP4-VP3) gene showed that the very virulent IBDV (vvIBDV) was segregated from other strains; which meant vvIBDV had a unique dinucleotide pattern. In summary, the study of base usage and dinucleotide frequency had unravelled many overlooked genomic properties of the virus.