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.     

Differentiation of infectious bursal disease virus (IBDV) genome segment B of very virulent and classical lineage by RT-PCR amplification and restriction enzyme analysis

Molecular characterization of IBDV usually relies on the analysis of segment A of the bi-segmented, double-stranded RNA genome. Although segments B of classical and very virulent IBDVs differ significantly, re-assortment of genome segments does occur, and molecular epidemiological studies demand the analysis of both segments. An RT-PCR and restriction enzyme analysis for molecular discrimination between genome segment B of classical and very virulent IBDVs is described. Tested on eight IBDV strains/isolates, the protocol successfully identified very virulent and classical IBDVs as well as a segment reassortant. This approach is a valuable tool for molecular epidemiological studies on IBDV.     

Sequence analysis of the VP2 gene hypervariable region of infectious bursal disease viruses from India

Attempts have been made to characterize infectious bursal disease virus (IBDV) isolates collected from different parts of India during 1993 to 1999. Phylogenetic analysis was performed on a sequence generated by cycle sequencing comprising the variable region of the VP2 gene of 14 isolates. Indian IBDV isolates had divergence of 0.2 to 4.3% at nucleotide and 0 to 2.2% at amino acid levels among themselves. Nine nucleotide changes were found in Indian IBDV field isolates, resulting in the four specific amino acid changes at 222P-A, 256V-I, 294L-I and 299N-S, reported regularly in very virulent isolates. One of the Indian IBDV isolates, UP1/99, had change D to N at position 212 in the first hydrophilic region. The serine-rich heptapeptide sequence 'SWSASGS' was conserved in all the isolates. Phylogenetically, all Indian field isolates were found to be closely related to very virulent IBDV isolates from Europe, Japan, China and Israel.     

Differentiation of infectious bursal disease virus strains using real-time RT-PCR and high resolution melt curve analysis

Differentiation of infectious bursal disease virus (IBDV) strains is crucial for effective vaccination programs and epidemiological investigations. In this study, a combination of real-time RT-PCR and high resolution melt (HRM) curve analysis was developed for simultaneous detection and differentiation of IBDV strains/isolates. The hypervariable region of VP2 gene was amplified from several IBDV strains and subjected to HRM curve analysis. The method could readily differentiate between classical vaccines/isolates and variants. Analysis of the nucleotide sequence of the amplicons from each strain revealed that each melt curve profile was related to a unique DNA sequence. The real-time RT-PCR HRM curve analysis was also able to differentiate IBDV strains/isolates directly in bursal tissues from field submissions and from vaccinated commercial flocks. The differences between melting peaks generated from IBDV strains were significantly different (P < 0.0001) demonstrating the high discriminatory power of this technique. The results presented in this study indicated that real-time RT-PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping IBDV isolates/strains and can contribute to effective control of IBDV outbreaks.     

Full-length sequence analysis of four IBDV strains with different pathogenicities

Characterization of field isolate 9109, Lukert, Edgar cell culture-adapted (CCA), and Edgar chicken embryo-adapted (CEA) serotype 1 IBDV strains using full-length genomic sequences is reported. IBDV genomic segments A and B were sequenced and the nucleotide and deduced amino acid (aa) sequences were compared with previously reported full-length sequenced IBDV strains. We found that the viral protein VPX and amino acid sequences between aa 202-451 and 210-473 of VP2 but not the entire VP2 protein are the best representatives of the entire IBDV genome. The greatest variability was found in the VP2 and 5' non-coding region of segment B among IBDV strains. The deduced amino acid sequences of the VP1 protein varies in length among the strains analyzed. The RNA-dependent, RNA-polymerase motifs within VP1 and the VP5 protein were highly conserved among isolates. Although within the VP2 processing site, amino acid sequence of Lukert was similar to the classical while the Edgar CCA, and CEA were more similar to the very virulent strains, it was determined that these strains have sequence characteristics of the classical strains. In addition, close relatedness between Lukert, Edgar CCA and CEA was observed. Although phylogenetic analysis of the VP1, VP3, and VP4 proteins indicated that 9109 is a classical type virus, this isolate shares unique amino acid changes with very virulent strains within the same proteins. Phylogenetic analysis of the 3' and 5' non-coding regions of segment A revealed that 9109 is more similar to the very virulent strains compared to the classical strains. In the VP2 protein, several amino acids were conserved between variant E and 9109 strains. Thus, it appears that 9109 isolate has characteristics of classical, very virulent, and variant strains. Our analysis indicates that although VPX amino acid comparison may be initially useful for molecular typing, full-length genomic sequence analysis is essential for thorough molecular characterization as partial sequences may not designate a particular strain as very virulent, classical, or variant.     

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.     

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.     

Characterization of field and vaccine infectious bursal disease viruses from Nigeria revealing possible virulence and regional markers in the VP2 minor hydrophilic peaks

Outbreaks of infectious bursal disease in vaccinated chicken flocks are frequent in Nigeria. For the control of infectious bursal disease, live vaccines based on foreign infectious bursal disease virus (IBDV) strains are used. The present study investigated the phylogenetic relationship between field and vaccine IBDV strains from northwestern Nigeria. Thirty field IBDV strains and three commercial vaccines strains were characterized through sequencing the VP2 hypervariable region. In addition, the complete genome segment A coding region for two vaccines and two field strains was sequenced. The deduced amino acid sequences (position 212 to 331) of IBDV strains from Nigeria and other regions of the world were aligned and possible regional and virulence markers were identified associated with VP2 minor hydrophilic peaks. Reversion to virulence of a vaccine strain with a Q to L mutation at position 253 was observed. Phylogenetic analyses revealed a unique cluster of northwest Nigerian field IBDV strains alone or related to imported characterized classical and very virulent IBDV vaccines. The results suggest that when IBDV strains spread from their region of origin to a different region they mutate alongside indigenous field strains but may retain their identity on the VP2 region.     

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.     

The genome segment B encoding the RNA-dependent RNA polymerase protein VP1 of very virulent infectious bursal disease virus (IBDV) is phylogenetically distinct from that of all other IBDV strains

A full-length cDNA clone of the segment B of the very virulent infectious bursal disease virus (IBDV) strain BD 3/99 was constructed and the full-length nucleotide sequence was established. The nucleotide sequence encoding VP1, an RNA-dependent RNA polymerase, of BD 3/99 was aligned with that of 17 other IBDV strains including six very virulent, three classical virulent, five classical attenuated, one antigenic variant and two serotype 2 strains. The VP1 genes of all very virulent strains were 97.5% to 99.8% identical. With the exception of an atypical Australian strain, 002-73, all of the classical virulent or attenuated and antigenic variant strains were also 97.5% to 100% identical. Serotype 2 strains showed only 4-6% divergence from serotype 1 classical virulent or attenuated strains; in contrast, however, the very virulent strains were 10.5% to 12.5% divergent from the classical virulent or attenuated strains as well as serotype 2 strains. Analysis of the deduced amino acid sequence of VP1 revealed 17 common, including 8 unique amino acid substitutions in the very virulent strains. In the phylogenetic tree the very virulent strains formed a distinct cluster and all other strains including classical virulent, attenuated and antigenic variant strains and even serotype 2 strains were grouped together. It is suggested that the VP1 of very virulent IBDV is phylogenetically distinct from that of all other IBDV strains and probably originated from a hitherto unidentified source.     

Phylogenetic and restriction fragment length polymorphism analyses of hemagglutinin (H) protein of canine distemper virus isolates from domestic dogs in Japan

We conducted phylogenetic and restriction fragment length polymorphism (RFLP) analyses of 995 nucleotides within the hemagglutinin (H) gene open reading frame (ORF) of field isolates of 23 canine distemper virus (CDV) strains isolated from domestic dogs in Japan between 1982 and 1998. The phylogenetic analysis showed that Japanese field isolates could be separated into three groups. Eighteen out of the twenty-three strains constituted one cluster consisting of Japanese CDVs, four strains formed a second Japanese CDV group, and only one strain belonged to a group containing foreign CDV strains. By RFLP analysis using SspI, we could distinguish all the Japanese field isolates from the vaccine strains. Thus, the RFLP method is useful for differentiating the infections with field CDV strains from the vaccine strains in clinical cases.