Daral J. Jackwood

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.

The Diagnosis of Very Virulent Infectious Bursal Disease in California Pullets

Abstract This report documents the occurrence of a very virulent infectious bursal disease virus (vvIBDV) in Northern California commercial brown pullets. Diagnosis was made from multiple accessions from two neighboring and epidemiologically related ranches submitted to the California Animal Health and Food Safety (CAHFS) laboratory. Pullets, 11 and 14 wk of age from ranch A (rA) and ranch B (rB) respectively, were submitted from infectious bursal disease virus vaccinated flocks experiencing a drastic increase in mortality. The December 2008 outbreak resulted in 26% and 34% mortality on rA and rB respectively. Gross and histologic lesions characteristic of acute vvIBDV were observed. Gross lesions included edematous bursas, hemorrhages at the junction of the proventriculus and gizzard as well as hemorrhages on skeletal muscles. Microscopic lesions included severe lymphoid necrosis and inflammation in edematous bursas, lymphoid necrosis in thymus, spleen, Peyers patches and cecal tonsils. Diagnosis of vvIBDV was confirmed by molecular characterization of the IBDV from bursas as well as viral pathogenicity in specific-pathogen-free birds. RT- PCR and nucleotide sequencing of the hypervariable region of the VP2 (vVP2) gene segment of the IBDV genome was performed on rA, rB and embryo passaged rA virions.The amino acids compatible with vvIBDV isolates: 222(Ala), 242(Ile), 256(Ile), 294(Ile) and 299(Ser) were reported from both ranches. In addition, nucleotide sequencing of a fragment of the VP1 gene demonstrated the viruses have the segment B genotype associated with highly pathogenic vvIBDV. Inocula of 105.5 50% egg infective dose of vvIBDV virus from rA and rB were introduced orally into two groups (g1 and g2 respectively) of 4 wk 2-day-old SPF leghorns. At 4 days postinoculation, there was 100% (22/22) morbidity in g1 and g2; 91% (20/22) mortality in g1; 100% (22/22) mortality for g2; 0% (0/20) morbidity and 0% (0/20) mortality was reported in the control group. This is the first occurrence of vvIBDV reported from birds in the United States.

Molecular Characteristics of Infectious Bursal Disease Viruses from Asymptomatic Broiler Flocks in Europe

Abstract Infectious bursal disease virus (IBDV) exists in several different antigenic and pathogenic forms. The immune suppression caused by this virus in young chickens is not always associated with clinical signs of disease. The antigenic variant viruses originally described in the United States typically do not cause clinical signs of disease but can cause a marked immune suppression via the destruction of B lymphocytes. Using a reverse transcription–polymerase chain reaction (RT-PCR) assay we conducted a survey of asymptomatic broiler chicken flocks in Europe for IBDV. Restriction fragment length polymorphisms in the viral protein 2 (VP2) gene of four isolates from Spain and four isolates from France indicated they may be different from the classic and very virulent (vv) IBDV strains found throughout Europe. Nucleotide sequence and phylogenetic analysis of the hypervariable region of the VP2 gene indicated that all eight viruses were more similar to U.S. variant viruses than classic viruses. In two viruses, one from France and one from Spain, threonine was observed at amino acid position 222 and serine was found at position 254. These two substitution mutations are characteristic of Delaware variant viruses. In addition, all eight viruses had mutated amino acid position 318 from glycine to aspartic acid, another substitution mutation commonly found in U.S. variant viruses. Although importation restrictions prevented us from directly testing the antigenicity of these viruses, their nucleotide and predicted amino acid sequences suggest they could be antigenically distinctive compared to classic and vvIBDV commonly found in Europe. Confirmation of the presence of antigenic variant IBDV strains in Europe requires additional immunologic studies to elucidate the exact nature of the viral epitopes. Our data support the need for these immunologic studies.

Characteristics of a Very Virulent Infectious Bursal Disease Virus from California

Abstract An outbreak of infectious bursal disease (IBD) in two California layer flocks resulted in the isolation of two infectious bursal disease viruses designated rA and rB. Increased mortality plus gross and histopathology in the layer flocks suggested rA and rB could be very virulent infectious bursal disease virus (vvIBDV). Preliminary studies indicated that high mortality resulted when bursa homogenates from the layer farms were used to inoculate specific-pathogen-free (SPF) chicks. In addition, rA and rB contained VP2 amino acid sequences typically seen in vvIBDV. Molecular and in vivo studies were conducted to more thoroughly identify and characterize the rA and rB viruses. Nucleotide sequence analysis demonstrated that rA and rB had identical sequences across the hypervariable VP2 (hvVP2) and segment B regions examined, and their amino acid sequences in the hvVP2 region were identical to the vvIBDV type strains UK 661, OKYM, and Harbin. Furthermore, the genome segment B nucleotide sequences examined for rA and rB were a 98.1% match with vvIBDV and only an 88.0% match with classic IBDV strains. Phylogenetic analysis placed the rA and rB viruses with other vvIBDV and confirmed these viruses were close genetic descendants of vvIBDV seen around the world. Pathogenicity studies in 4-wk-old SPF chicks demonstrated that at a high dose (105.5 50% egg infective dose [EID50]) and a low dose (102.0 EID50) of rA and rB, mortality ranged from 91% to 100%. A pathogenic classic virus, standard challenge (STC), at similar doses did not cause mortality in the SPF chicks. In addition, mortality occurred in three out of four SPF birds exposed by direct contact to rA and rB inoculated chicks. Serum from convalescent birds inoculated with rA had high titers to IBDV but were negative for antibodies to infectious bronchitis virus, avian influenza virus, chicken anemia virus, Newcastle disease virus, Mycoplasma gallisepticum, and Mycoplasma synoviae. Virus isolation attempts on the rA and rB bursa homogenate inocula also indicated that no contaminating microorganisms contributed to the high mortality and pathology observed in the SPF chicks. In one experiment, broilers with maternal immunity to IBDV were protected from infection and disease when they were challenged with 102 EID50 and 105 EID50 of the STC virus. When challenged with 102 EID50 of the rA virus, the maternally immune broilers were protected from disease but not infection as evidenced by a positive reverse transcription–polymerase chain reaction (RT-PCR) assay for the virus. When the broilers were challenged with 105 EID50 of the rA virus, they had typical gross and histopathologic signs of IBD but no mortality by 7 days postinoculation. It was concluded that the rA and rB viruses meet the genotypic and phenotypic characteristics of a vvIBDV.

Molecular Epidemiologic Evidence of Homologous Recombination in Infectious Bursal Disease Viruses

SUMMARY. Nucleotide and predicted amino acid sequences of the infectious bursal disease virus (IBDV) surface protein VP2 have been used to identify strains of the virus and place them into phylogenetic groups. The amino acids across the hypervariable sequence region of VP2 (hvVP2) vary, but typically variant viruses have amino acids 222T, 249K, 286I, and 318D and classic viruses have 222P, 249Q, 286T, and 318G. A molecular epidemiologic study was conducted from 2001 to 2011 in commercial chickens (Gallus gallus) from Mexico, Colombia, and Venezuela. Although many IBDVs were identified, most had the typical variant or classic amino acid sequences across the hvVP2 region. Four viruses identified in 2004, one in 2006, and 10 in 2011 from Mexico had the amino acids 222T, 249Q, 286T, and 318D. Six samples from Venezuela in 2001, one sample from Colombia in 2001, two samples from Venezuela in 2004, and one sample from Venezuela in 2005 had the amino acids 222P, 249K, 286I, and 318G. These combinations of classic and variant amino acid sequence markers had not been identified previously in any IBDV strains. The VP2 amino acid sequences in the PBC and PHI loop structures of the Venezuela and Colombia viruses were similar to most classic viruses, whereas their minor PDE and PFG loop sequences were typical of Delaware variant strains. The Mexico viruses had VP2 PBC loop sequences that were typical of variant IBDV strains, but their minor PDE and PFG loop structures contained amino acids that were similar but not identical to classic strains. The PHI loop sequences of the Mexico viruses had 318D that is typical of a Delaware variant virus, but the other amino acids in this loop structure distinguished them from all other IBDV strains. The data suggest that one or more recombination events may have occurred to create this type of sequence diversity. Because of importation regulations, immunologic studies could not be conducted in the United States to determine the antigenicity of the viruses examined in this study. The amino acid sequence data, however, suggest they would contain antigenic epitopes of both variant and classic IBDVs.

Sequence of precursor polyprotein gene (segment A) of infectious bursal disease viruses isolated in Korea.

The coding regions of segment A of two recent Korean very virulent (vv) infectious bursal disease virus (IBDV) isolates (KK1 and KSH) and one atypical IBDV isolate (K310) were amplified by reverse transcriptase-polymerase chain reaction, sequenced, and compared with published sequences for IBDV. The overall amino acid sequence similarity of the KK1 and KSH strains compared with foreign vvIBDV strains was between 97.43% and 98.02%. The KK1 and KSH strains, like vvIBDV strains, share unique amino acid residues at positions 222(A), 256(I), 294(I), and 299(S). The sequence of K310 strain was markedly different from other IBDV strains. The K310 strain had 12, 2, and 1 unique amino acid substitutions in the VP2 hypervariable region, VP4, and VP3 gene, respectively, and 3 of 12 substitutions in a VP2 hypervariable region were found in two hydrophilic regions known to be involved in antigenic determination. Also, the K310 strain had 222(S) and 254(S), which were found in variant IBDV strains. The SWSASGS heptapeptide is conserved in all Korean IBDV isolates. By phylogenetic analysis, KK1 and KSH were categorized in one group with foreign vvIBDV isolates, but K310 isolate was categorized in a separate group that was differentiated from the other IBDV strains compared. The K310 strain seemed to be evolved from a separate lineage of IBDV strain.

Detection and characterization of infectious bursal disease viruses in broilers at processing

The presence of infectious bursal disease virus (IBDV) in broilers entering processing plants was examined. The dissemination of IBDV and the introduction of non-native strains for example very virulent (vv) IBDV have had a negative economic impact on poultry production in many countries. Restrictions have been placed on the import and export of poultry products by some countries. There is a perceived risk that IBDV can be spread through transportation and contamination of processing equipment, poultry protein products and processing plant personnel. This risk, however, is fundamentally unknown because scientific studies have not been conducted on the presence of IBDV in birds entering processing plants or the variables that may affect this risk during and post-harvest. The goal of this study was to determine if infectious IBDV was present in broilers entering processing plants. A total of 47 pooled bursa samples from 26 processing plants in the Eastern U.S. were examined. Molecular testing indicated that an IBDV specific RT-PCR was positive in 12 (25.5%) of the samples from 11 different processing plants. Nucleotide sequence analysis was conducted on the 12 RT-PCR positive samples and indicated the IBDV was not commercially available attenuated vaccine strains. Most of the sequences were unique and a phylogenic analysis indicated they were most closely related to variant strains of IBDV. Five RT-PCR positive samples were selected at random for testing in specific-pathogen-free chickens. All five samples contained infectious IBDV as evidenced by macroscopic lesions and bursa/body weight ratios that were significantly lower in infected birds than in the non-inoculated controls. The five viruses were re-identified in bursa tissue from chickens in their respective groups at necropsy using RT-PCR and nucleotide sequencing. The results indicate that infectious and pathogenic IBDV are entering processing plants in this geographic region of the U.S. Additional studies are needed on post-harvest variables that could increase or decrease the risk that these viruses are being disseminated during this process.

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.

Antigenic and Molecular Characterization of Three Infectious Bursal Disease Virus Field Isolates

Three infectious bursal disease field viruses, identified as U28, 3212, and MISS and isolated in the early 1980s from the southeastern United States, were characterized both antigenically and genotypically. A panel of monoclonal antibodies (MAbs) was utilized in an antigen-capture enzyme-linked immunosorbent assay (ELISA) for antigenic characterization. The ELISA data indicated that U28 and the Delaware variants are antigenically related. The 3212 and the GLS variants were more closely related antigenically to each other than to other viruses analyzed. However, the MISS isolate reacted with MAbs that were specific for both classic and variant strains of infectious bursal disease virus (IBDV). Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify nucleotide sequences from the genome coding for the variable region of VP2 from IBDV field isolates U28, 3212, and MISS. Phylogenetic analysis of the deduced amino acid sequences revealed that U28 was most similar (98.3%) to the IBDV Delaware variants and that 3212 was most similar (97.1%) to the GLS variant. The MISS isolate was most similar (97.4%) to the classic 52/70 strain. Positive correlation occurred with the use of different methods to characterize IBDV isolates.

Multivalent virus-like-particle vaccine protects against classic and variant infectious bursal disease viruses.

SUMMARY. Nucleotide sequences that encode the pVP2 proteins from a variant infectious bursal disease virus (IBDV) strain designated USA08MD34p and a classic IBDV strain designated Mo195 were produced with the use of reverse-transcriptase–polymerase chain reaction (RT-PCR) and cloned into a pGEM-T Easy vector. A nucleotide sequence that encodes the VP3 protein was also produced from the USA08MD34p viral genome with the use of RT-PCR and cloned into a pGEM-T Easy vector. The VP3 and pVP2 clones were inserted into the pVL1393 baculovirus transfer vector and sequenced to confirm their orientation to the promoter and to ensure they contained uninterrupted open reading frames. Recombinant baculoviruses were constructed by transfection in Sf9 cells. Three recombinant baculoviruses were produced and contained the USA08MD34p-VP3, USA08MD34p-pVP2, or Mo195-pVP2 genomic sequences. Virus-like particles (VLPs) were observed with the use of transmission electron microscopy when the USA08MD34p-VP3 baculovirus was co-inoculated into Sf9 cells with either of the pVP2 constructs. VLPs were also observed when the USA08MD34p-pVP2 and Mo195-pVP2 were coexpressed with USA08MD34p-VP3. These multivalent VLPs contained both classic and variant pVP2 molecules. Stability tests demonstrated the VLPs were stable at 4 and 24 C for 8 wk. The USA08MD34p, Mo195, and multivalent VLPs were used to vaccinate chickens. They induced an IBDV-specific antibody response that was detected by enzyme-linked immunosorbent assay (ELISA), and virus-neutralizing antibodies were detected in vitro. Chickens vaccinated with the multivalent VLPs were protected from a virulent variant IBDV strain (V1) and a virulent classic IBDV strain (STC). The results indicate the multivalent VLPs maintained the antigenic integrity of the variant and classic viruses and have the potential to serve as a multivalent vaccine for use in breeder-flock vaccination programs. RESUMEN. Protección conferida por una vacuna multivalente elaborada con partículas similares a virus contra cepas clásicas y variantes de la enfermedad infecciosa de la bolsa. Las secuencias de nucleótidos que codifican las proteínas pVP2 de una cepa variante de virus de la enfermedad infecciosa bursal (IBDV) designada USA08MD34p y una cepa clásica designada Mo195 fueron producidas mediante transcripción reversa y reacción en cadena de la polimerasa (RT-PCR) y se clonaron en el vector pGEM -T Easy vector. También se produjo una secuencia de nucleótidos que codificaba para la proteína VP3 a partir del genoma viral de la cepa USA08MD34p mediante RT-PCR y se clonó en el vector pGEM-T Easy. Los clones VP3 y pVP2 fueron insertados en el vector de transferencia baculovirus pVL1393 y fueron secuenciados para confirmar su orientación con el promotor y para asegurar que contenía marcos de lectura continua ininterrumpidos. Los baculovirus recombinantes fueron construidos por la transfección en células Sf9. Se produjeron tres baculovirus y contenían las secuencias genómicas USA08MD34p-VP3, USA08MD34p-pVP2, o Mo195-pVP2. Con el uso de microscopía electrónica de transmisión se observaron virus semejantes a partículas cuando el baculovirus USA08MD34p-VP3 fue co-inoculado en células Sf9 con cualquiera de los constructos de pVP2. También se observaron partículas semejantes a virus cuando los vectores USA08MD34p-pVP2 y Mo195-pVP2-se fueron co-expresados con el vector USA08MD34p-VP3. Estas partículas semejantes a virus polivalentes contenían moléculas de pVP2 tanto clásicas como variantes. Las pruebas de estabilidad demostraron que las partículas semejantes a virus eran estables entre 4 y 24 C, durante 8 semanas. Los vectores USA08MD34p, Mo195 y las moléculas semejantes a virus polivalentes se utilizaron para vacunar pollos. Se indujo una respuesta de anticuerpos específico contra el virus de Gumboro que fue detectada por el ensayo de inmunoabsorción con enzimas ligadas (ELISA), y se detectaron anticuerpos neutralizantes in vitro. Los pollos vacunados con las partículas semejantes a virus estuvieron protegidos contra una cepa variante virulenta (V1) y una cepa clásica virulenta (STC). Los resultados indican que las partículas semejantes a virus multivalentes mantuvieron la integridad antigénica de la variante y de los virus clásicos y tienen el potencial de servir como una vacuna multivalente para su uso en programas de vacunación en aves reproductoras.