Isolation of infectious bursal disease virus from turkeys

A virus, which is morphologically identical and antigenically related to two previously known isolates of infectious bursal disease (IBD) virus, was isolated from pooled faeces of 6-week-old turkeys with diarrhoea. It is concluded that this virus, designated TY89, is an isolate of IBD virus. The isolation of TY89 was heavily dependent upon the use of electron microscopy and the immunofluorescence technique. Antibody to TY89 virus was detected by both indirect immunofluorescence and serum neutralisation tests in 28 of 95 (29%) sera collected from 20-week-old turkeys. The available evidence suggests that this virus has only recently been introduced into turkeys in Northern Ireland. It is believed that this is the first report of natural infection of turkeys with IBD virus.     

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.     

Comparison of four infectious bursal disease viruses isolated from different bird species

Summary Four isolates of infectious bursal disease virus (IBDV), isolated from chicken, duck, goose and sparrow in Jiangsu province of China in 2002, were compared. The viruses were stable to the treatments of 60 °C for 1 h, pH 2.0 and lipid solvents. Their antigenic relatedness values (R) were from 0.76 to 0.78. Chickens infected with the chicken isolate showed severe clinical symptoms of IBD and the mortality rate was 33.3% (2/6). Chickens infected with the other three viruses survived but their bursas were damaged and the bursa/body-weight ratios were lower than those of the uninfected control (p< 0.01). The titers of anti-IBDV antibody in infected chicken sera reached up to 1600 by virus neutralization and 6400 by ELISA at 10 days post infection. The sequences of the variable region of VP2 were aligned and compared, showing nucleotide variations ranging from 1.5 to 6.7% and deduced aminoacid variations from 0.8 to 2.2%. All had the same heptapeptide, S-W-S-A-S-G-S, Asp279, and Ala284. The four viruses clustered on a phylogenetic tree and were distant from the STC strain. These findings suggested that different bird species naturally infected with IBDV could serve as carriers or reservoirs in IBDV transmission and might play a role in the emergence of variant IBDV.     

Generation of serotype 1/serotype 2 reassortant viruses of the infectious bursal disease virus and their investigation in vitro and in vivo

Infectious bursal disease virus (IBDV) is the causative agent of acute or immunosuppressive disease in chickens. Serotype 1 strains are pathogenic whereas serotype 2 strains neither cause disease nor protect against infection with the serotype 1 strains. The target organ of serotype 1 strains is the bursa Fabricii (BF). The molecular determinants of this tropism, and therefore pathogenicity, are poorly understood. IBDV is a non-enveloped icosahedral virus particle of 60 nm in diameter, which contains two genome segments of double-stranded RNA. Here, the generation of interserotypic reassortants using the reverse genetics approach is reported. The results of in vitro and in vivo investigations show that genome segment A determines the bursa tropism of IBDV, whereas segment B is involved in the efficiency of viral replication; they further indicate the significance of the interaction of the polymerase (segment B) with the structural protein VP3 (segment A) or the viral genome for efficient virus formation and replication.     

Serological studies with reoviruses in chickens, turkeys and ducks

Fluorescent antibody (FA) studies with avian reoviruses in chickens, turkeys and ducks are described. Detection of the group-specific antigen by FA test was investigated by titrating a reovirus antiserum on chick embryo liver cell cultures infected with 18 reovirus strains fixed on multitest slides. With 16 of the viruses, test titres were similar, indicating presence of a common antigen. The titre observed with a duck reovirus isolate was considerably lower, suggesting partial cross-reactivity. One virus (Kosters) was not stained. A comparison of the agar gel immuno-diffusion (AGID) test with the FA test on serial dilutions of antiserum demonstrated the greater sensitivity of the FA test. In a survey of chicken and turkey sera for reovirus antibodies by both tests, a higher percentage positive was recorded by immunofluorescence with CS108 virus as antigen. No antibodies to this virus were detected in 46 duck sera but 4 sera were positive for the duck reovirus isolate. Of 100 chicken sera, 19 were positive for duck reovirus and 6 for Kosters virus. No antibodies to either of these viruses were found in turkey sera. The results demonstrate the usefulness of the indirect FA test with multitest slides for avian reovirus serology and indicate the existence of atypical strains with no or partial group antigen reactivity.     

Isolation of avian pneumovirus from mallard ducks that is genetically similar to viruses isolated from neighboring commercial turkeys

Our earlier studies demonstrating avian pneumovirus (APV) RNA in wild geese, sparrows, swallows, starlings and mallard ducks suggested that wild birds might be involved in the circulation of APV in the United States. To determine whether turkey virus can be transmitted to the free flying birds, we placed APV-negative mallard ducks next to a turkey farm experiencing a severe APV outbreak and in an area with a large population of waterfowls. The sentinel ducks did not develop clinical APV disease but infectious APV (APV/MN-12) was recovered from choanal swabs after 2 weeks, and anti-APV antibodies detected after 4 weeks. Four APV isolates recovered from the neighboring turkeys that were experiencing an APV outbreak at the same time shared 95-99% nucleotide identity and 97-99% predicted amino acid identity with the duck isolate. In addition experimental infection of turkey poults with APV/MN-12 resulted in detection of viral RNA in nasal turbinates and APV-specific IgG in serum. These results indicate that the APV isolates from turkeys and ducks shared a common source, and the viruses from different avian species can cross-infect.     

Acute infectious bursal disease in poultry: Isolation and characterisation of a highly virulent strain

A highly virulent strain of infectious bursal disease virus (IBDV) was isolated from the field and propagated in SPF chickens, causing up to 100% mortality. Although it still belongs to the standard serotype 1 IBD viruses, serological typing with monoclonal antibodies showed an antigenic drift in this pathogenic strain. Conventional 'intermediate' IBD vaccines are probably more antigenically related to the pathogenic strain than the mild ones and were effective in protecting SPF chickens against challenge.     

Rescue of infectious bursal disease virus from mosaic full-length clones composed of serotype I and II cDNA

Summary.  Infectious Bursal Disease Virus (IBDV) is the causative agent of one of the most important and wide-spread infectious diseases among commercial chicken flocks. IBDV causes a depletion of B-lymphoid cells in the bursa of Fabricius, inducing immunosuppression, morbidity, or even acute mortality. Because currently used live IBDV vaccines are derivatives from field isolates no serologic discrimination between field isolates and live vaccines can be made. The recently developed reverse genetics techniques for IBDV allows one to generate genetically modified IBDVs which might have altered biological and antigenic properties. Here, we describe the rescue of mosaic serotype I IBDVs, of which the polyprotein encoding region was partly replaced by the corresponding region of a serotype II strain. A mosaic virus, containing the C-terminal part of serotype II VP3 showed only a slightly delayed release of progeny virus compared to unmodified serotype I virus, while maximum viral titers at 25 h post infection were equal. Since serotype specific epitope(s) are present in the C-terminal part of VP3, we were able to discriminate this rescued virus from serotype I and II IBDV strains. These findings make the use of a chimeric VP3 a promising approach to develop an IBDV marker vaccine. Accepted February 22, 2001 Received July 24, 2000     

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.     

Biochemical studies with infectious bursal disease virus: Comparison of some of its properties with infectious pancreatic necrosis virus

Summary Infectious bursal disease (IBD) virus was purified from the bursae of infected chickens. Two morphologically indistinguishable populations of virus particles were separated in sucrose gradients and possessed sedimentation coefficients of 395S and 460S. Both populations contained RNA and had identical polypeptide compositions. IBD virus banded at a density of 1.31 g/ml in CsCl and at 1.24 g/ml in sodium potassium tartrate.IBD virus contained two RNA segments with mol. wts. of 2.4×10⁶ and 2.2×10⁶ as estimated by polyacrylamide-agarose gel electrophoresis, but sedimented in sucrose gradients at 15S. Virus RNA was resistant to 0.1 µg/ml ribonuclease treatment under conditions in which ribosomal RNA was completely hydrolysed, but was sensitive to 1.0 and 10 µg/ml treatments. These results suggest that the RNA consists of either double-stranded or highly ordered single-stranded molecules. IBD virus contained seven polypeptides with mol. wts. in the range 97,000 to 24,000. Two polypeptides were absent in empty particles of IBD virus.IBD and infectious pancreatic necrosis (IPN) viruses were morphologically indistinguishable. IPN virus possessed a sedimentation coefficient of 440S and banded at a density of 1.32 g/ml in CsCl. In addition the electrophoretic mobilities of IBD and IPN virus RNAs were almost identical. Polyacrylamide slab gel electrophoresis showed that while the number and size of the polypeptides were different for each virus there were similarities in the overall pattern.With 5 Figures     

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.     

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.     

Isolation, propagation, and characterization of a second equine rotavirus serotype.

A rotavirus designated strain H-2 was isolated in primary African green monkey kidney cells from a foal with diarrhea. This cell culture-adapted strain was found to be similar, if not identical, to simian rotavirus (strains MMU18006 and SA-11) and canine rotavirus (strain CU-1) and, in addition, demonstrated a one-way antigenic relationship with five human rotavirus strains (P, B, no. 14, no. 15, and YO) of the third human rotavirus serotype by the plaque reduction neutralization test. This is the fifth example of an animal rotavirus which shares serotypic specificity with a human rotavirus. The H-2 strain is distinct from the H-1 strain (Y. Hoshino et al., J. Clin. Microbiol., in press) of equine rotavirus not only in serotypic specificity by neutralization but also in subgroup specificity, hemagglutinating activity, and RNA electrophoretic migration pattern, thus establishing the existence of a second equine rotavirus serotype. This H-2 isolate is also distinct by neutralization from three other human rotavirus serotypes, 1 (Wa), 2 (DS-1), and 4 (St. Thomas no. 4), as well as bovine (NCDV), and porcine (OSU) rotaviruses.     

Isolation of rotaviruses from turkeys and chickens: demonstration of distinct serotypes and RNA electropherotypes

Six isolates of rotavirus were made from turkeys and two from chickens. Three of these required trypsin treatment for isolation and serial passage in cell cultures. The remainder were isolated without trypsin treatment. Most of these viruses were isolated in chick embryo liver cell cultures from the faeces of birds aged under 1 week. In six of the eight instances, rotavirus isolation was associated with enteric disturbance, characterised by signs such as diarrhoea, poor or abnormal appetite, abnormally fluid or gaseous intestinal contents or increased mortality. Cross immunofluorescence tests showed that while avian and mammalian rotaviruses shared a common group antigen, the avian viruses were more closely related to each other than to the Nebraska calf rotavirus isolate. On the basis of serum neutralisation tests seven of the eight avian rotaviruses were grouped into three serotypes, with two turkey isolates (Ty1 and Ty3) and a chicken (Ch1) virus being the prototype strains. The remaining virus, Ty2, was intermediate in type between Ty1 and Ch1. Analysis of the RNA of these viruses by polyacrylamide gel electrophoresis showed that they could also be grouped into a number of electropherotypes. The isolates which were serologically distinct were also electrophoretically distinct. Similarly the five isolates which belonged to the Ty3 sero-type were electrophoretically identical. Analysis of the serological and electrophoretic differences suggested that RNA segment 5 may code for a type-specific antigen.     

Precipitating antigens associated with Marek's disease viruses and a herpesvirus of turkeys

Precipitating antigens associated with a number of Marek's disease virus strains and with a turkey herpesvirus have been analyzed. The 'A' antigen has been defined as the major soluble antigen in feather follicles of infected chickens, which is identical with the major antigen usually present in supernatants of chicken kidney cell cultures infected with strains of Marek's disease virus. 'BC' antigens are 2 or more antigens which are usually not noted in skin extracts but present in cultured cells infected with Marek's disease virus or turkey herpesvirus, in addition to the 'A' antigen. Some of the virus strains examined were positive and others negative for 'A' antigen, but all contained the 'BC' antigens. Results of agar-gel precipitin tests suggested a serological classification of the group of avian herpesviruses formed by Marek's disease viruses and turkey herpesvirus into 3 types. Pathogenic strains of Marek's disease virus and their attenuated A- variants, represented by the HPRS-16 strain (HPRS-16, JM, GA, VC, Oldenburg). Apathogenic Marek's disease virus, represented by the HPRS-24 strain. Turkey herpesvirus and its A- variants, represented by the FC126 strain. A serological subdivision corresponding to the different grades of pathogenicity of virus strains of the first type was not possible. Differences between antigens associated with the 3 types of virus were apparent from the antigen and antibody titres against homologous and heterologous reagents. Precipitin bands produced by homologous antigen and antibody were stronger than those produced by heterologous reagents. Differences between 'A' antigens of the 3 virus types were characterized by spur patterns of precipitin bands indicating a partial identity. At least 3 'BC' precipitin bands were noted; at least one was group-specific and one appeared to be type-specific.     

Amino acid changes in the variable region of VP2 in three infectious bursal disease viruses with different virulence,originating from a common ancestor

An infectious bursal disease virus (IBDV), Hyd(C), from an outbreak was isolated and plaque-purified in BGM-70 cells. From the moderately virulent plaque-purified virus, two IBDVs with relatively high and low virulence were obtained by passaging the virus in specific pathogen free chickens and BGM-70 cells 10 and seven times, respectively. Comparison of amino acid sequences of the VP2 variable region of these viruses revealed that three amino acids at positions 279, 284 and 300 (Asp, Thr and Glu, respectively, in the plaque-purified virus) were changed. In in vitro- passaged virus, amino acid residues 279, 284 and 300 were Asn, Thr and Glu, whereas these were Asp, Ala and Gln in the in vivo -passaged virus. Change of residue 284 (Thr M Ala) had a critical role in cell culture infectivity, whereas the change in residue 279 (Asp M Asn) was associated with attenuation of the virus. No correlation could be observed between amino acid changes at position 300 and virulence or cell culture infectivity. Moreover, residue 330 (Arg) in heptapeptide motif SWSAR 330 GS was not found to be associated with the cell culture infectivity or virulence.