Infectious Bursal Disease Virus Field Research Articles

Molecular Analysis of Field Infectious Bursal Disease Virus Isolated in Sulaimani/ Iraq.

Molecular Analysis of Field Infectious Bursal Disease Virus Isolated in Sulaimani/ Iraq.

Molecular epidemiology of infectious bursal disease virus in the Near East and Persian Gulf regions

ABSTRACT Infectious bursal disease virus (IBDV) is a ubiquitous immunosuppressive pathogen which is a severe burden to the poultry industry. Due to frequent mutation and reassortment events, its double-stranded, bi-segmented RNA genome displays a considerable heterogeneity, which in turn produces profound differences in antigenicity and pathogenicity. In recent years, the investigation of IBDV epidemiology has greatly benefited from the proposal of multiple phylogenetic classification systems which prompted the execution of molecular surveys all over the world, producing a wealth of standardized and easily sharable data. The present epidemiological survey was conducted in six Near East and Persian Gulf countries to characterize the field IBDVs circulating in the region. Ninety-three broiler flocks, immunized with different vaccination protocols, were investigated over a one-year period by collecting eight individual bursal samples at a time. Using molecular assays targeting both genome segments, field IBDVs belonging to four genotypes were detected from 42 flocks across all investigated countries. While traditional very virulent IBDVs (genotype A3B2) represented a minority, multiple atypical IBDV genotypes (A3B1, A4B1 and A6B1) were found to circulate in most of the region. Many of the detected strains were related to sequences from other continents, supporting the involvement of the Near and Middle East in complex networks of viral spreading. The obtained results, combined with the available literature, allowed us to update the epidemiological picture, hopefully favouring the identification of epidemiological threats and the planning of effective monitoring and control strategies. RESEARCH HIGHLIGHTS Different field IBDVs were found to circulate in the Near and Middle East. Multiple atypical genotypes (A3B1, A4B1, A6B1) were found to circulate extensively. Traditional very virulent IBDVs (A3B2) were a minority of the detected strains. Viral exchanges can be hypothesized between the region and different continents.

Serological study reveal different antigenic IBDV strains prevalent in southern China during the years 2000–2017 and also the antigenic differences between the field strains and the commonly used vaccine strains

The aim of this study was to determine the antigenic relatedness of Infectious Bursal Disease Viruses (IBDVs) in the field in southern China during the period 2000–2017, as well as the antigenic relationship between the field strains and the most commonly used vaccine strains by using a virus neutralization (VN) test in vitro. The antigenic relatedness (R) value and the difference in VN titers were analyzed, and the antigenic index based on the sequences of the hypervariable region of VP2 (vVP2) of the strains was further evaluated. As a result, the R value of representative field strains showed that there were three subtypes present in the field strains examined, with 7 strains belonging to subtype 1, while strains BH11 and JS7 belonged to subtype 2 and subtype 3, respectively. The commonly used vaccine strains B87 and FW2512 belonged to subtype 1. The analysis of the VN titer differences revealed that all the 136 field strains were classified into subtype 1, except BH11 and JS7. All the field strains in subtype 1 have been divided into at least 5 subgroups, suggesting the antigenic diversity among these strains. The antigenic index based on IBDV-VP2 sequences further confirmed the antigenic differences between the three subtype strains and also the antigenic diversity among the subtype 1. The results demonstrated the antigenic diversity of field IBDVs in southern China during the years 2000–2017 and the antigenic differences between the field strains and the commonly used vaccine strains. This would indicate that the commonly used vaccines are only partially effective. These results enhance our understanding of IBDV genetic evolution and should help to develop more effective vaccines for the control of this disease in the future.

Characterization of field infectious bursal disease viruses in Zambia: evidence of co-circulation of multiple genotypes with predominance of very virulent strains

ABSTRACT Infectious bursal disease (IBD) is a highly contagious, immunosuppressive disease of chickens and causes substantial economic losses to the poultry industry globally. This study investigated the genetic characteristics and pathological lesions induced by IBD viruses (IBDVs) that were associated with 60 suspected outbreaks in chickens during 2015–2016 in Lusaka Province, Zambia. Nucleotide sequences of VP2 hypervariable region (VP2-HVR) (n = 38) and part of VP1 (n = 37) of Zambian IBDVs were phylogenetically analysed. Phylogenetic analysis of the VP2-HVR and VP1 revealed that most viruses (n = 31 of each genome segment) clustered with the very virulent (vv) strains. The rest of the viruses clustered with the classical strains, with two of the viruses being closely related to attenuated vaccine isolates. Two of the viruses that belonged to the vv genotype had a unique amino acid (aa) substitution Q324L whereas one virus had two unique changes, N280S and E300A in the VP2-HVR aa sequence. Although Zambian strains with a vv genotype possessed virulence marker aa within VP1 at 145T, 146D and 147N, two viruses showed unique substitutions, with one virus having 147T while the other had 147H. Pathologically, it was noted that only viruses with a vv genotype appeared to be associated with inducing pathological lesions in non-lymphoid organs (proventriculus and gizzard). Whilst documenting for the first time the presence of classical virulent IBDVs, this study demonstrates the involvement of multiple genotypes, with predominance of vvIBDVs in the epidemiology of IBD in Zambia.

Genetic Characterization of Egyptian Field Isolates of Infectious Bursal Disease Virus

Detection and characterization of field infectious bursal disease virus (IBDV) isolates circulating among different broiler breeds representing three Egyptian governorates with a history of vaccination against IBDV, was done during 2013- 2015. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify and produce clear band of 620 Base pair (bp) product within the hypervariable region of the IBDV viral protein (VP2) gene. The IBDV was prevalent in thirty out one hundred flocks (30%). Subsequently, chosen PCR products were subjected to further analysis by sequencing and phylogenetic studies. Amino acid substitution in VP2 region used to establish a clear framework on the epidemiology of isolated IBDV . Genetic analysis showed that all examined IBDV isolates characterized as vvIBDV had amino acids residues(222A, 242I, 256I, 294I, 299S) which showed to be unique for all vvIBDV strains and clustered phylogenetically with the previously Egyptian IBDVs ( 99.3-100% ) identity. While, vaccinal IBDV strains (BURSA-VAC and UNIVAX) were clustered in another group with (91.1–91.9%), (91.9–92.6%) identity respectively which might leads to vaccination failure and reemergence of disease. Consequently, we recorded wide range of genetic similarities (94.8-95.6) with the recombinant vaccines (VP2 of Faragher 52/70) seed virus, which showed superior protective efficacy against recent Egyptian vvIBDV isolates. IBDV is still a threat against poultry industry. It is important to keep track on appearance and evolution of antigenically different IBDV circulating strains followed by regular updating of the vaccination strategy.

Biochemical and Histopathological Changes in Immune and Non Immune Broilers after Inoculation of Field Infectious Bursal Disease Virus

Biochemical and Histopathological Changes in Immune and Non Immune Broilers after Inoculation of Field Infectious Bursal Disease Virus

Molecular characterization of field infectious bursal disease virus isolates from Nigeria.

Aim:To characterize field isolates of infectious bursal disease virus (IBDV) from outbreaks in nine states in Nigeria through reverse transcription polymerase chain reaction (RT-PCR) and sequence analysis of portions of the VP2 and VP1 genes and to determine the presence or absence of reassortant viruses.Materials and Methods:A total of 377 bursa samples were collected from 201 suspected IBD outbreaks during 2009 to 2014 from nine states in Nigeria. Samples were subjected to RT-PCR using VP2 and VP1 gene specific primers, and the resulting PCR products were sequenced.Results:A total of 143 samples were positive for IBDV by RT-PCR. These assays amplified a 743 bp fragment from nt 701 to 1444 in the IBDV VP2 hypervariable region (hvVP2) of segment A and a 722 bp fragment from nt 168 to 889 in the VP1 gene of segment B. RT-PCR products were sequenced, aligned and compared with reference IBDV sequences obtained from GenBank. All but one hvVP2 sequence showed similarity to very virulent IBDV (vvIBDV) reference strains, yet only 3 of the VP1 67 VP1 sequences showed similarity to the VP1 gene of vvIBDV. Phylogenetic analysis revealed a new lineage of Nigerian reassortant IBDV strains.Conclusion:Phylogenetic analysis of partial sequences of genome segment A and B of IBDV in Nigeria confirmed the existence of vvIBDV in Nigeria. In addition, we noted the existence of reassortant IBDV strains with novel triplet amino acid motifs at positions 145, 146 and 147 in the reassorted Nigerian IBDV.

Role of naturally occurring genome segment reassortment in the pathogenicity of IBDV field isolates in Three-Yellow chickens

ABSTRACTReassortment among genome segments of infectious bursal disease virus (IBDV) field isolates was reported frequently worldwide, however the pathogenicity of the reassortant field IBDV is poorly understood. In this paper, a pathogenicity study on four representative IBDV field strains isolated from Southern China between 2005 and 2011 was conducted. Twenty-eight-day-old Three-Yellow chickens were divided into four groups and were inoculated intraocularly with one of the four field IBDV strains, namely NN1172, NN1005, GD10111 and JS7, respectively. The mortality and relative weight of bursa and thymus were subsequently determined in the acute phase of infection. In addition, B cells, T cells (CD4+ and CD8+) and virus were quantified in the bursa of Fabricius and thymus, respectively, by flow cytometry and real-time reverse transcription-polymerase chain reaction. The results showed that isolate NN1172, of which parts of segment A and B encoding the hypervariable (v) region of viral protein (VP2) and VP1, respectively, derived from vvIBDV strains, showed the most severe pathogenicity, and caused the most severe bursal B cell depletion as well as CD4+ and CD8+ T cell infiltration in the bursa of Fabricius. However, the virus induced the strongest decrease in CD4+ and CD8+ T cells in the thymus and exhibited the most efficient viral replication in the target organs. Isolate NN1005, whose vVP2 derived from vvIBDV and VP1 from unidentified origin, exhibited relatively lower pathogenicity compared to NN1172. The other two isolates, JS7 and GD10111, of which the vVP2 derived from vvIBDV and intermediate IBDV, and VP1 from 002–73 and attenuated IBDV, respectively, showed the lowest level of virulence. Our results suggest that various IBDV field isolates with different natural segment reassortments exhibit differential pathogenicity after infection of commercial Three-Yellow chickens.

Genetic characterization of South American infectious bursal disease virus reveals the existence of a distinct worldwide-spread genetic lineage

Infectious bursal disease virus (IBDV) is one of the most concerning health problems for world poultry production. IBDVs comprise four well-defined evolutionary lineages known as classic (c), classic attenuated (ca), variant (va) and very virulent (vv) strains. Here, we characterized IBDVs from South America by the genetic analysis of both segments of the viral genome. Viruses belonging to c, ca and vv strains were unambiguously classified by the presence of molecular markers and phylogenetic analysis of the hypervariable region of the vp2 gene. Notably, the majority of the characterized viruses (9 out of 15) could not be accurately assigned to any of the previously described strains and were then denoted as distinct (d) IBDVs. These dIBDVs constitute an independent evolutionary lineage that also comprises field IBDVs from America, Europe and Asia. The hypervariable VP2 sequence of dIBDVs has a unique and conserved molecular signature (272T, 289P, 290I and 296F) that is a diagnostic character for classification. A discriminant analysis of principal components (DAPC) also identified the dIBDVs as a cluster of genetically related viruses separated from the typical strains. DAPC and genetic distance estimation indicated that the dIBDVs are one of the most genetically divergent IBDV lineages. The vp1 gene of the dIBDVs has non-vvIBDV markers and unique nucleotide and amino acid features that support their divergence in both genomic segments. The present study suggests that the dIBDVs comprise a neglected, highly divergent lineage that has been circulating in world poultry production since the early time of IBDV emergence.

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.

Identification and molecular analysis of infectious bursal disease in broiler farms in the Kurdistan Regional Government of Iraq.

The present study was undertaken to characterize field isolates of infectious bursal disease virus (IBDV). The identification was done using reverse transcription-polymerase chain reaction (RT-PCR) and partial sequencing of the VP2 gene. Pooled bursal samples were collected from commercial broiler farms located in the Kurdistan Regional Government (KRG) of Iraq. The genetic material of the IBDV was detected in 10 out of 29 field samples. Sequences of the hypervariable VP2 region were determined for 10 of these viruses. Molecular analysis of the VP2 gene of five IBDVs showed amino acid sequences consistent with the very virulent (vv) IBDV. Two samples were identified as classic vaccine viruses, and three samples were classic vaccine viruses that appear to have mutated during replication in the field. Phylogenetic analysis showed that all five field IBDV strains of the present study were closely related to each other. On the basis of nucleotide sequencing and phylogenetic analysis, it is very likely that IBD-causing viruses in this part of Iraq are of the very virulent type. These IBDVs appear to be evolving relative to their type strains.

Simultaneous detection of vaccinal and field infectious bursal disease viruses in layer chickens challenged with a very virulent strain after vaccination

Infectious bursal disease virus is an important poultry pathogen. It is distributed worldwide and causes significant economic losses. In this study, a system was adopted for the simultaneous monitoring of vaccine and virulent strains using reverse transcription polymerase chain reaction (RT-PCR). After the decay of maternal antibodies, chickens were vaccinated at the age of 37 days with a virus of intermediate virulence and challenged at 5, 10 and 14 days post vaccination (dpv). The challenge was done with IBDV strain CH/99. Sequencing of the hypervariable region of VP2 has shown that CH/99 belongs to the very virulent group of viruses. The vaccine virus could be found in the bursa of Fabricius, spleen, thymus and bone marrow until 24 dpv. The CH/99 challenge virus was found in the bursa and lymphoid organs when chickens were challenged at 5 and 10 dpv. When challenge was performed at 14 dpv, the pathogenic virus could not be found in the bursa and other lymphoid organs.

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.

Sequence Analysis of Both Genome Segments of Three Croatian Infectious Bursal Disease Field Viruses

In order to determine the mutations responsible for virulence, three Croatian field infectious bursal disease viruses (IBDV), designated Cro-Ig/02, Cro-Po/00, and Cro-Pa/98 were characterized. Coding regions of both genomic segments were sequenced, and the nucleotide and deduced amino acid sequences were compared with previously reported full-length sequenced IBDV strains. Phylogenetic analysis, based on the nucleotide and deduced amino acid sequences of polyprotein and VP1, was performed. Eight characteristic amino acid residues, that were common to very virulent (vv) IBDV, were detected on polyprotein: 222A, 256I, 294I, 451L, 685N, 715S, 751D, and 1005A. All eight were found in Cro-Ig/02 and Cro-Po/00. C-Pa/98 had all the characteristics of an attenuated strain, except for glutamine on residue 253, which is common for vv, classical virulent, and variant strains. Between less virulent and vvIBDV, three substitutions were found on VP5: 49 G --> R, 79 --> F, and 137 R --> W. In VP1, there were nine characteristic amino acid residues common to vvwIBDV: 146D, 147N, 242E, 390M, 393D, 511S, 562P, 687P, and 695R. All nine residues were found in A-Ig/02, and eight were found in B-Po/00, which had isoleucine on residue 390. Based on our analyses, isolates Cro-Ig/02 and Cro-Po/00 were classified with vv IBDV strains. C-Pa/98 shared all characteristic amino acid residues with attenuated and classical virulence strains, so it was classified with those.

Restriction Fragment Length Polymorphism Typing of Infectious Bursal Disease Virus Field Strains in Turkey

Infectious bursal disease (IBD), also known as Gumboro disease, is a highly contagious, immunosuppressive disease of immature chickens. It is caused by IBD virus (IBDV) and is responsible for major economic losses in the poultry industry worldwide. In this study, 280 bursa samples from 56 commercially reared chicken flocks in Turkey with clinical symptoms of IBD were examined for IBDVs using the reverse transcription (RT)-polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) assay. The assay was conducted on a 743-bp fragment of the VP2 gene with the restriction enzymes BstNI, MboI, and SspI. The results indicate the existence of field isolates with new molecular patterns different from those previously published that may well be unique and specific to geographical regions.

Molecular characterization of Indian isolates of infectious bursal disease virus from broiler chickens

The present study was undertaken to characterize recent field isolates of infectious bursal disease virus (IBDV) by reverse transcription-polymerase chain reaction (RT-PCR) and partial sequencing of VP2 gene. The virus could be detected in 17 of 20 field samples from broiler chickens in Haryana state, India as well as in all the four vaccine strains. Nucleotide sequences of four field isolates and one vaccine strain were compared with 10 reported IBDV strains from different parts of the world. Nucleotide substitutions at 795G, 827T, 833C, 857C, 897A, 905T, 908T, 1011A and 1094G specific for very virulent (vv) strains, were maintained in all the four field isolates. However, unique nucleotide substitutions at 806A-G, 851 C-T, 1010 T-C, 1019T-C and 1082T-C showed further divergence of these isolates from already reported vvIBDVs. Deduced amino acid substitutions at 222P-A, 256V-I, 279N-D, 294L-I and 299N-S specific for vvIBDV strains were also present in all the four isolates. The vaccine strain showed amino acid change 279D-N, a characteristic of attenuated vaccine strains. Phylogenetic analysis showed that all the field isolates in the present study were closely related to reported UK (UK661) and Japan (OKYM) field isolates. All the four field IBDV strains of the present study were closely related to each other but distinct from already reported vvIBDVs of India. On the basis of nucleotide sequencing and phylogenetic analysis, it is very likely that IBD causing strains in this part of India are of very virulent character and are still undergoing changes at genetic level.

Heteroduplex Mobility Assay for Genotyping Infectious Bursal Disease Virus

A heteroduplex mobility assay (HMA) was developed to genotype infectious bursal disease virus (IBDV). This method analyzed 390-base pair (bp) polymerase chain reaction (PCR) products, encompassing the hypervariable region of the VP2 gene. IBDV strains from the United States and other countries were analyzed. The HMA was able to differentiate standard, antigenic variants and very virulent strains of IBDV. Minor differences between different strains from the same subtype were also detected. Close relationships between field IBDV with vaccines prepared with Delaware E strain were determined by HMA. The results obtained by HMA were confirmed by restriction fragment length polymorphism (RFLP) and phylogenetic analysis of nucleotide sequences. The HMA proved to be a useful technique to rapidly genotype different field strains of IBDV and should prove to be a useful tool in epidemiologic studies.

Sequence analysis of the variable VP2 gene of infectious bursal disease viruses passaged in Vero cells.

Korean field infectious bursal disease viruses (IBDVs) were isolated from IBDV suspected commercial chickens. A previous study revealed that these IBDV field isolates were virulent or very virulent IBDVs. The isolates were passaged three times in the chorioallantoic membrane of specific-pathogen-free embryonated chicken eggs and four times in Vero cells. After passage, viral RNAs were isolated and purified to determine the genetic changes. The hypervariable regions of the VP2 gene were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). To confirm the genetic changes, PCR products were cloned, sequenced and compared to the sequences of the parental IBDVs and published IBDV strains. By sequencing analysis, the passaged IBDVs had amino acid changes at positions 253 (Q --> H), 279 (D or N --> N) and 284 (A --> T) which were commonly found in the attenuated IBDV strains. Two serines in the serine-rich heptapeptide (residue 326-332) were substituted into other amino acids which were similar to the IBDV vaccine strains.

Sequence analysis of both genome segments of two very virulent infectious bursal disease virus field isolates with distinct pathogenicity.

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 infectious bursal disease virus from commercial poultry in the United States and Latin America.

From June 1999 to September 2001, 216 bursal samples from broiler farms in the United States and from countries of Latin America were submitted to the Poultry Diagnostic and Research Center at the University of Georgia for the purpose of genotyping field infectious bursal disease viruses (IBDVs). The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify a 248-bp product, encompassing the hypervariable region of VP2 gene. The genotyping was conducted by restriction fragment length polymorphism (RFLP) analysis with six restriction endonucleases, DraI, SacI, TaqI, Sty, BstNI, and SspI. For the 150 samples received from the United States, 125 samples (83.3%) were RT-PCR positive for the presence of IBDV. One hundred positive samples (80%) had RFLP identical to the variant Delaware E strain, whereas 10 samples (8.0%) exhibited a RFLP pattern similar to this antigenic variant. Other IBDV strains such as Grayson Laboratory strain (GLS), Lukert, PBG-98, Delaware A, and the vaccine strains Sal-1 and D-78 were also detected. Two samples exhibited a pattern similar to the standard challenge (STC) strain, and seven strains (5.6%) were not classified by RFLP. Sixty-six bursal samples previously inactivated with phenol were received from Latin American countries. IBDV strains with analyzed genotypes similar to the Lukert strain were predominantly detected in Mexico. IBDV strains similar to variant E were detected in Colombia and Ecuador. Peru and Venezuela exhibited a higher heterogeneity of IBDV strains due to the detection of classic Delaware type as well as GLS variant strains. IBDV strains detected from Brazil and Dominican Republic exhibited RFLP patterns identical to very virulent IBDV strains prevalent in several countries in Europe, Asia, and Africa.