Infectious Bursal Disease Virus Serotype Research Articles

Identification of Extremely Virulent Infectious Bursal Disease Virus Via Molecular and Histological Methods in Broiler Chickens

Infectious bursal disease (IBD) is caused by an RNA virus belonging to the Avibirnavirus genus within the Birnaviridae family. The global prevalence of infectious bursal disease virus (IBDV) is a significant concern, affecting birds of all ages. Birds infected with IBDV exhibit symptoms, such as depression, bleeding in the thighs and pectoral muscles, and enlargement of the bursa. This study aimed to identify predominant IBDV serotypes using molecular methods and to gain insights into the resulting pathological conditions in infected chickens. Additionally, the study investigated the viral sequence and the relationship between a local Diyala isolate and reference strains from the Genebank. In the current study, the IBDV was isolated from broiler chickens aged 2-3 weeks from 15 farms in the Diyala Governorate of Iraq. A total of 15 samples, each from a different farm, were collected. Necropsy samples were obtained from various organs of broiler chickens, including the bursa of Fabricius, lungs, liver, and kidneys. Specific primers targeting the VP2 gene were used for reverse transcription-polymerase chain reaction (RT-PCR) analysis. The RT-PCR analysis yielded a 727 bp fragment, confirming the presence of IBDV in 10 out of the 15 samples. One strain was assigned the accession number LC498531 in the NCBI database. Phylogenetic analysis using the neighbor-joining tree program revealed three distinct groups. All examined regional samples (S1) were situated within the constructed tree. Five samples formed a specific group, indicating a close relationship. Histological examination of the tissues showed visible alterations such as degeneration, necrosis, and infiltration of inflammatory cells, particularly heterophils, providing clear evidence of the disease. In conclusion, this study confirmed the presence of IBDV in broiler chickens from multiple farms in Iraq’s Diyala Governorate, highlighting distinct clustering patterns in viral sequences. Moreover, the study confirmed the virus's presence using conventional RT-PCR, with histological examination supporting the findings. Keywords: Broiler chicken, Bursal enlargement, Infectious bursal disease, RT-PCR

An Orally Administered Nonpathogenic Attenuated Vaccine Virus Can Be Used to Control SARS-CoV-2 Infection: A Complementary Plan B to COVID-19 Vaccination.

BackgroundCoronavirus disease 2019 (COVID-19) vaccination has substantially altered the course of the pandemic, saving tens of millions of lives globally. The problem is that despite such spectacular results, vaccination alone will not be able to control the COVID-19 pandemic because of the rapid evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) even in vaccinated human populations. Therefore, the development of a post-infection, broad-based, orally administered antiviral therapy that would complement vaccination efforts is urgently needed.MethodologyThe so-called viral superinfection therapy (SIT) administers a nonpathogenic attenuated double-stranded RNA (dsRNA) vaccine virus drug candidate, the infectious bursal disease virus serotype R903/78 (IBDV-R903/78) that activates the interferon (IFN) genes, which are the natural, antiviral defense system of host cells.ResultsHere we present two cases of properly vaccinated (with BNT162b2-Pfizer) and booster-dosed COVID-19 patients with vaccine breakthrough infections whose disease duration was shortened to a few days by oral SIT.ConclusionsSIT has already been demonstrated to be safe and effective against five different families of viruses, hepatitis A virus, hepatitis B virus, hepatitis C virus, SARS-CoV-2, and herpes zoster virus. The R903/78 drug candidate is simple to manufacture and easy to administer in an outpatient setting. The expected cost of SIT will be affordable even in resource-limited countries.

The nucleotide sequence of the vp2 gene of the epizootic strain of infectious bursal disease virus and comparing it with classical and highly virulent strains

Infectious bursal disease (IBD) virus is the causative agent of a severe immunosuppressive disease in young birds. Although the disease was first discovered more than 60 years ago, it continues to pose a significant threat to the poultry industry worldwide [4]. The causative agent is RNA-containing virus, which belongs to the genus Avibirnavirus of the family Birnaviridae [17].The virion includes five viral proteins, designated VP1, VP2, VP3, VP4, and VP5 [15] with an approximate molecular weight of 97 kDa, 41 kDa, 32 kDa, 28 kDa, and 21 kDa, respectively. Additional proteins such as VPX or pVP2 are also noted [12]. The capsid protein VP2 has long been a focus of recombinant subunit vaccine development because it is responsible for eliciting a protective immune response against IBD. However, reports of many authors [2,3,6] point to the antigenic heterogeneity of IBD virus strains isolated in Russia and other countries, which is associated with the failure of existing vaccines in preventing the disease; therefore, to create and successfully use recombinant vaccines, it is necessary to study epizootic strains isolated in the Russian Federation as well.Therefore, the purpose of this work was to perform genetic analysis of the VP2 gene of the epizootic strain "Sinyavinsky" and compare it with the classical strains of IBD virus serotypes 1 and 2 isolated in Europe and highly virulent strains isolated in the Russian Federation.

Six decades of infectious bursal disease in poultry: The journey so far and challenges ahead


 
 
 Despite six decades of concerted efforts, Infectious bursal disease (IBD) still remains a major threat to the poultry industry worldwide. Most importantly, the emergence of variant and very virulent strains of infectious bursal disease virus (IBDV) has dramatically changed the epidemiology of the disease, thus resulting in the renewed efforts in the search for effective control measures. Currently, live attenuated, inactivated, and immune-complex vaccines are among the immune-therapeutic approaches employed for the control of IBD in the field alongside adequate biosecurity, albeit with various degrees of success and limitations. Progress in genetic engineering has allowed the generation of reverse genetic IBDV mutants, recombinant live viral vectors expressing the IBDV VP2 immunodominant protein, intra-serotypic recombinant IBDV viral-like particle co-expressing the outer capsid protein structures derived from 2 or more serotype 1 strains or the incorporation of either VP2 or VP2-4-3 polyprotein sequences alongside molecular adjuvants that can be used as IBD vaccine candidates to elicit an immune response. However, despite these advances, outbreaks are still reported even in flocks that have up to date vaccination records and somewhat excellent management practices. This paper reviews aspect of genetic characteristics of IBDV and reflects on the progress and future challenges in providing effective IBD vaccine to achieve effective control of both classical and very-virulent IBDV serotypes that constitute a major devastation to poultry production and health.
 
 

Persistence and Tissue Distribution of Infectious Bursal Disease Virus Serotypes 1 and 2 in Turkeys.

Two experiments were conducted to determine the persistence and tissue distribution of serotypes 1 and 2 of infectious bursal disease virus (IBDV) in specific-pathogen-free and vaccinated turkeys. In Experiment 1, three groups of 2-wk-old turkey poults, including a negative control group, were used. In groups 1 and 2, 13 poults in each group were challenged with either serotype 1 (STC) or serotype 2 (OH) strains using an inoculum of 10(4) 50% embryo infectious dose (EID50)/0.2 ml/bird. Thymus, bursa, spleen, kidney, lungs, liver, pancreas, caecum, and breast and thigh muscles were sampled at predetermined intervals. The bursal tissues from birds inoculated with either serotype were reverse transcriptase-PCR (RT-PCR) positive up to 21 days postinoculation (DPI). In both groups virus isolation from bursas was possible up to 14 DPI. Except for the bursas and spleens in birds inoculated with serotype 1 and bursas in birds inoculated with serotype 2, all other tissues were RT-PCR negative. In Experiment 2, five groups of turkey poults were used. At 4 wk of age, group 1 was challenged with a serotype 1 STC strain and group 2 with serotype 2 OH strain using an inoculum size of 10(2) EID50/0.2 ml for both serotypes. Groups 3 and 4 were vaccinated at 2 wk of age using an inactivated serotype 1 IBDV vaccine. At 2 wk postvaccination, groups 3 and 4 were challenged with STC and OH strains respectively. From group 1, bursal, spleen, and liver tissues were RT-PCR positive up to 14 DPI; breast muscle and kidney tissues were positive up to 7 DPI; and lungs and pancreatic tissues were positive up to 3 DPI. From group 2, bursal tissues were RT-PCR positive up to 14 DPI and lung tissues up to 3 DPI. All of the tissue samples collected from groups 3, 4, and 5 were RT-PCR negative. Virus could not be isolated from RT-PCR positive bursal homogenate. In this work, it was confirmed that the virus persisted in the bursa longer than in any other tissues. The difference in the results between Experiments 1 and 2 could be due to the age of poults at vaccination and the higher inoculum size used in Experiment 1. This study indicates that turkeys are more resistant to IBDV as compared to chickens. Viruses of serotypes 1 and 2 infect turkeys and persist in bursal tissue for 14 days and RNA was detected up to 21 days.

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.

SURVEY FOR ANTIBODIES TO INFECTIOUS BURSAL DISEASE VIRUS SEROTYPE 2 IN WILD TURKEYS AND SANDHILL CRANES OF FLORIDA, USA

Captive-reared Whooping Cranes (Grus americana) released into Florida for the resident reintroduction project experienced unusually high mortality and morbidity during the 1997-98 and 2001-02 release seasons. Exposure to infectious bursal disease virus (IBDV) serotype 2 as evidenced by seroconversion was suspected to be the factor that precipitated these mortality events. Very little is known about the incidence of IBD in wild bird populations. Before this study, natural exposure had not been documented in wild birds of North America having no contact with captive-reared cranes, and the prevalence and transmission mechanisms of the virus in wild birds were unknown. Sentinel chickens (Gallus gallus) monitored on two Whooping Crane release sites in central Florida, USA, during the 2003-04 and 2004-05 release seasons seroconverted, demonstrating natural exposure to IBDV serotype 2. Blood samples collected from Wild Turkeys (Meleagris gallopavo) and Sandhill Cranes (Grus canadensis) in eight of 21 counties in Florida, USA, and one of two counties in southern Georgia, USA, were antibody-positive for IBDV serotype 2, indicating that exposure from wild birds sharing habitat with Whooping Cranes is possible. The presence of this virus in wild birds in these areas is a concern for the resident flock of Whooping Cranes because they nest and raise their chicks in Florida, USA. However, passively transferred antibodies may protect them at this otherwise vulnerable period in their lives.

Exchange of the VP5 of infectious bursal disease virus in a serotype I strain with that of a serotype II strain reduced the viral replication and cytotoxicity

Infectious bursal disease virus (IBDV), belonging to Avibirnavirus genus in the Birnaviridae family, consists of two segments of double-strand RNA. There are two distinct serotypes of IBDV, the pathogenic serotype I and the non-pathogenic serotype II. Comparison of the deduced amino acid sequences of a panel of VP5 genes retrieved from GenBank revealed a high identity among strains within the serotype I or serotype II group but a low identity between strains across two serotypes. In this study, we rescued two mosaic viruses, rGtGxVP5 and rGt2382VP5 by exchanging the VP5 gene of a cell culture-adapted serotype I Gt strain with its counterpart of the very virulent IBDV Gx strain, or a non-pathogenic 23/82 strain of the serotype II. In comparison to the parental strain rGt virus, the rGtGxVP5 showed the similar viral replication, cytotoxicity and the ability of inducing apoptosis; however, the other mosaic virus rGt2382VP5 had a lower titer and a reduced cytotoxicity. Although exchange of VP5 within serotype I group did not alter the viral replication and cytotoxicity of Gt strain, exchange of VP5 in the serotype I with that of a serotype II reduced the viral replication and cytotoxicity on chicken embryo fibroblast (CEF) cells. Therefore, the VP5 of serotype II may be one of the factors responsible for the distinct pathogenic features of two serotypes.

Detection of Infectious Bursal Disease Virus Isolates with Unknown Antigenic Properties by Reverse Genetics

Infectious bursal disease virus (IBDV) serotype 1 is the causative agent of a highly contagious immunosuppressive disease of young chickens. In the past, a number of antigenic, as well as pathogenic, subtypes have been described. The determination of the antigenic makeup of circulating strains is of vital interest to the poultry industry because changes in the antigenicity of circulating field strains have an impact on the use of vaccines. To obtain a more comprehensive overview of the relationship between the nucleotide and amino acid sequence and the antigenic makeup of field isolates, a system based on reverse genetics of IBDV was established. Using this approach, a database for field isolates from three different states in the United States (Georgia, Alabama, and Louisiana), consisting of nucleotide sequence, amino acid sequence, and a reaction pattern based on a panel of monoclonal antibodies, was established. The obtained results showed that phylogenic analysis, which is based on the similarity of sequences, would lead to false conclusions regarding a possible antigenic makeup of the particular isolate. Sequences of field samples were divided into three groups: 1) those that grouped with variant strain E/Del sequences but were antigenically different, 2) those that did not group with sequences of E/Del but were similar in their antigenic makeup, and 3) those that did not group with E/Del sequences and were antigenically different. In addition, using the reverse-genetics approach, a number of field isolates showed no reactivity with any of the used monoclonal antibodies, indicating that an unknown, antigenic subtype of IBDV serotype 1 is circulating in the field.

Molecular Detection and Differentiation of Infectious Bursal Disease Virus

Vaccination of hens, with the subsequent maternal immunity imparted to chicks, is the primary means of controlling infectious bursal disease virus (IBDV). Effective vaccination depends on rapid and accurate diagnosis of the subtype present in a flock because vaccines based on the classic subtype of IBDV can fail to protect against challenge with a variant subtype. This review describes the various methods available to detect and differentiate between IBDV subtypes. Serotype 1 IBDV causes economically significant immunosuppressive disease in young chickens. Within serotype 1, two subtypes, classic and variant, can be differentiated by the virus neutralization assay. Antigen capture enzyme-linked immunosorbent assay (AC-ELISA) with MAbs has been successful at differentiating the very virulent IBDV phenotype (vvIBDV) from less pathogenic types. More rapid and sensitive molecular diagnostic methods based on reverse transcription-polymerase chain reaction (RT-PCR) for amplification of the IBDV VP2 gene have been a major focus of investigation in recent years. Conventional RT-PCR has been useful in detecting IBDV serotypes and, to a lesser extent, differentiating IBDV subtypes. One of the approaches has been the use of SspI and NgoM IV restriction enzymes, for restriction endonuclease (RE) analysis of RT-PCR products (RT-PCR-RE) and BstNI and MboI for restriction fragment length polymorphism (RFLP) analysis (RT-PCR-RFLP) to find unique banding patterns associated with antigenic variation within the variable region of the IBDV VP2 protein. However, these approaches were ultimately found to be unreliable because subtypes could not be consistently distinguished with restriction enzymes. These limitations led to studies in differentiating subtypes by detection of single nucleotide differences in sequence through real-time RT-PCR or DNA sequencing of RT-PCR products. Conventional RT-PCR, amplifying the VP2 hypervariable region, in combination with DNA sequencing of the PCR product, can differentiate classic, variant, and vvIBDV strains because variant and vvIBDV have characteristic nucleotide and amino acid substitutions. Real-time RT-PCR, targeting different regions of the IBDV genome, including VP1, VP2, and VP4 genes, in conjunction with melting-curve analysis is being investigated as a promising tool for molecular diagnosis of IBDV infection. These methods potentially allow for more rapid, sensitive, and specific detection and differentiation of IBDV classic, very virulent, and variant subtypes.

Detection of Antibodies Against Serotypes 1 and 2 Infectious Bursal Disease Virus by Commercial ELISA Kits

Two distinct serotypes of infectious bursal disease virus (IBDV) are recognized in chicken and turkey flocks in the United States. Serologic testing of chicken flocks for serotype 1 viruses is routinely performed to monitor disease status and vaccination. Earlier studies indicated that enzyme-linked immunosorbent assay (ELISA) test detects antibodies to both serotypes of the virus, while the virus neutralization (VN) test is serotype specific. It is useful to evaluate currently available commercial ELISA kits for their ability to differentiate between antibodies elicited by the two serotypes. Three trials were performed in which chickens were orally inoculated with either a high or a low dose of serotype 1 STC or serotype 2 OH strains of IBDV. Sera collected at 0, 7, 14, and 21 days from these chickens and antisera procured from naturally infected broiler (n=20) and layer (n=30) flocks were tested with five different commercial ELISA kits and by VN. All ELISA kits detected different levels of antibodies elicited against serotype 1 of the virus and moderate and high levels of antibodies against serotype 2 virus. A correlation existed between the ELISA and the VN titers of experimentally infected chickens. All serum samples tested from the commercial layer flocks and 65% of the broiler flocks had antibodies against the OH strain. However, no correlation between the VN titers and ELISA titers was observed for the commercial broilers and layers sera by the majority of the kits. The results indicated that currently available commercial ELISA kits detect antibodies elicited by the two serotypes of IBDV. Hence, the prevalence of serotype 2 antibodies in the flocks should be considered while determining antibody profiles of the flocks against serotype 1 viruses.

Infectious bursal disease virus (IBDV) induces apoptosis in chicken B cells

The ability of infectious bursal disease virus (IBDV) serotypes 1 and 2, and the role of VP4 of both serotypes as well as the capacity of three IBDV intermediate serotype 1-specific vaccine strains to induce apoptosis in a chicken B-lymphocyte cell line, DT40, were investigated using the TUNEL technique. It was observed that IBDV serotype 1 infected the DT40 cell line and directly induced apoptosis. In contrast, the non-pathogenic serotype 2 neither infected nor induced apoptosis, but was able to reduce the serotype 1-induced apoptosis when the two viruses were present in combination. VP4 of both serotypes did not induce apoptosis. IBDV VP2 of serotype 2 induced apoptosis in the same proportion and intensity as VP2 of serotype 1. IBDV intermediate vaccines varied in their ability to induce apoptosis in the DT40 cell line, which was also decreased-delayed in presence of serotype 2 IBDV. We hypothesize that both serotypes compete for the same receptor in DT-40 cells, and suggest that IBDV-induced apoptosis is a multistep process involving virus replication, protein expression, and release of virions.

Validation of five commercially available ELISAs for the detection of antibodies against infectious bursal disease virus (serotype 1)

In this study, the results are reported from a validation study of five commercially available enzymelinked immunosorbent assays (ELISAs) for the detection of antibodies against infectious bursal disease virus (IBDV), serotype 1. The specificity of the ELISAs varied from 63.8 to 100%. All ELISAs reached a sensitivity of 100% on sera between 14 and 21 days post-vaccination (d.p.v.) with two classical vaccines and a Delaware variant-E virus. Overall, most birds became positive between 8 and 11 d.p.v. As expected, the ELISA with the lowest specificity showed the highest sensitivity at 5 d.p.v. When the decrease in maternally derived antibodies against IBDV was measured, a highly significant correlation (P < 0.001) was found for all ELISAs and the virus neutralization test (VNT). R 2 varied from 0.44 to 0.76, whereas the slope varied from 0.33 to 0.57. This indicates that there is a certain correlation between VNT and ELISA titres, but that the correlation differs from ELISA to ELISA. For all ELISAs, no significant (P < 0.05) differences in level of antibodies were detected between antibody titres in breeder serum, egg yolk and progeny at 1 and 4 days of age. This validation of five commercially available ELISAs for the detection of antibodies against IBDV (serotype 1) shows that there are significant differences in their performance. Therefore, choosing an ELISA or interpreting ELISA results without knowing the technical performance should be avoided.

Chimeras in noncoding regions between serotypes I and II of segment A of infectious bursal disease virus are viable and show pathogenic phenotype in chickens.

Two serotypes, I and II, have been identified for infectious bursal disease virus (IBDV), a member of the family BIRNAVIRIDAE: Here, the generation by reverse genetics of IBDV chimeras in segment A of the bisegmented genome is reported. The 5- and 3'-noncoding regions (NCRs) of a serotype II strain were exchanged with the NCRs of a full-length cDNA clone of segment A of a serotype I strain. Isolated chimeric viruses were characterized in cell culture and susceptible chickens. The results show that IBDV chimeras in segment A were able to replicate in cell culture and that VP1 encoded by a serotype I segment B is functionally active with serotype I NCRs as well as with serotype II NCRs. Chimeric viruses infected susceptible chickens and caused mild depletion of bursal cells. Thus, the noncoding regions of segment A are not responsible for the different pathotypes of IBDV serotypes I and II.

Heat inactivation of serotype 1 infectious bursal disease virus

Heat inactivation curves for infectious bursal disease virus serotype 1 (IBDV) strain 52/70 in bursal homogenate supernatant were constructed for 70°C, 75°C and 80°C. Biphasic, multiple kinetics curves were produced with an initial rapid drop in infectivity followed by a more gradual decline. In this second phase the approximate times taken to reduce the infectivity by 1 log10 at each temperature were: 70°C (18.8 min), 75°C (11.4 min) and 80°C (3.0 min), confirming the heat resistance of this virus.

Antigenic Characterization of an Arkansas Isolate of Infectious Bursal Disease Virus

The s977 strain of infectious bursal disease virus (IBDV) was isolated in northwest Arkansas in 1977 from the bursae of young broilers with high maternal antibody titers to the Moulthrop strain of IBDV (BursaVac). The comparison of a plaque-purified isolate of s977 with other IBDV serotype 1 and serotype 2 strains using virus neutralization indicates that s977 is a subtype of serotype 1 vaccine viruses and the MD variant strain of IBDV and has no relatedness to the Delaware Variant A (VarA) virus. In vivo cross-protection studies in specific-pathogen-free white leghorn chickens showed that an inactivated vaccine using s977 antigen was 2.5 times more protective against challenge with s977 than was an inactivated IBDV Variant E (VarE) vaccine. The vaccination of maternally immune broiler chicks with live s977 did not provide protection against subsequent challenge, indicating that s977 does not have enough antigenic difference to break through maternal immunity. Analysis of denatured viral polypeptides using polyacrylamide gel electrophoresis showed that s977 and two reported variant strains, 51 and VarE, share three protein bands, 90 kD (VP1), 40 kD (VP2), and 31 kD (VP3), that were not observed in BursaVac. BursaVac and s977 shared a 74 kD precursor band that was absent or very faint in the VarE and 51 strains. The most unique characteristic of s977 was the relative abundance of a wide, 56-63 kD band that contained two distinct immunoreactive bands when blotted with antiserum to s977. BursaVac contained a 56 kD band that failed to react with s977 antiserum. Analysis of polypeptide bands using laser densitometry indicated the presence of a number of bands between 20 kD and 25 kD in the s977, 51, and VarE preparations but only a 25 kD band in BursaVac. The number of bands decreased with the degree of relatedness to standard vaccine strains. It appears that, antigenically, S977 may hold an intermediate position between the classic virus strains and the more recently reported serotype 1 variants.

Susceptibility of chicken lymphoid cells to infectious bursal disease virus does not correlate with the presence of specific binding sites

Pathogenic serotype 1 strains of infectious bursal disease virus (IBDV) replicate efficiently in lymphoid cells of the bursa of Fabricius of chicken. Lymphoid cells in other organs are not susceptible. Apathogenic serotype 2 strains do not replicate in lymphoid bursa cells or in other lymphoid cells. Chicken embryo fibroblasts (CEF), however, efficiently replicate strains of either serotype. Binding studies showed that strains of both IBDV serotypes bind to lymphoid cells isolated from the bursa, thymus or spleen, indicating that restriction of IBDV replication to lymphoid B cells is not determined by the presence of specific receptor sites. The specificity of binding was demonstrated by saturation and competition experiments. These revealed the presence of different receptors: CEF had receptors common to both serotypes and specific ones for each serotype. Receptor sites common to both serotypes were also present on lymphoid cells; however, additional serotype-specific sites were only demonstrated for the apathogenic serotype 2 strain. Strains of both serotypes specifically bound to proteins with molecular masses of 40 kDa and 46 kDa, exposed on the surface of CEF and lymphoid cells. Competition experiments indicated that these proteins might represent the common receptor sites of IBDV.

Restriction fragment profiles of genome segment A of infectious bursal disease virus correlate with serotype and geographical origin of avibirnaviruses

Previous analysis of the two serotypes of infectious bursal disease virus (IBDV) have demonstrated the correlation between antigenicity and similarities of nucleotide and amino acid sequences of the VP2 coding region in genome segment A. Restriction fragment profiles of genomic segment A cDNA of five IBDV isolates (QC-2 and QT-1 of serotype 1, SK9, and Nos. 39 and 52 of serotype 2) were determined in order to establish the genetic relationship of these viruses to other avibirnaviruses. The restriction fragment profiles using three of seven restriction enzymes (SacI which cuts in the VP2 region, DraIII which cuts in the VP3 region, and EcoRI which cuts in the VP4 region) were used to place QC-2, QT-1, SK9, No. 39, and No. 52 within the phylogenetic tree among seven other avibirnaviruses of known sequence. The two IBDV serotypes corresponded to two genotypes on the basis of the presence or absence of the SacI restriction site. The serotype 1 cluster of strains was further differentiated into five minor clusters on the basis of the PstI, EcoRI, BamHI, HindIII, DraIII, and Bsu361 restriction sites, which emphasized the geographical origins of the strains. It is concluded that restriction analysis of cDNA of the whole viral genomic segment A allows differentiation of IBDV isolates on the basis of their antigenicity and geographical origin.

Rapid Differentiation of Infectious Bursal Disease Virus Serotypes by Polymerase Chain Reaction

Infectious bursal disease (IBD) remains one of the most important contributors to economic loss in the poultry industry. The disease primarily affects the bursa of Fabricius of young chickens, resulting in reduced feed efficiency, reduced weight gain, increased mortality, and immunosuppression. Immunosuppression may increase susceptibility to other infectious disease agents. Infectious bursal disease virus (IBDV) is highly contagious and very resistant to disinfectants. Once the virus becomes established on the premises, it tends to persist for a long time. Infectious bursal disease virus, a member of the Bimaviridae, is a double-stranded RNA virus and contains 2 segments designated A and B. 3,5 Genome segment A encodes the structural proteins, VP-2, VP-3, and VP-4. Genome segment B encodes VP-l, a viral polymerase. There are at least two distinct serotypes of IBDV, serotypes 1 and 2. Only serotype 1 viruses cause disease, whereas all known serotype 2 viruses are not pathogenic to chickens. However, both serotypes are widespread in chickens. Polyclonal and monoclonal antibodies have been developed and used in differentiating IBDV serotypes by the virus neutralization (VN) test. Most IBD field samples cannot be directly used in the VN test. The viruses for the VN test are usually obtained by inoculating field samples into chicken embryos or are adapted to grow in primary cell cultures or mammalian cell lines. The essential element of the VN test requires that the amounts of IBDV or its replication products should be high enough for antigen-antibody reaction and serotype differentiation. Furthermore, the VN test is time consuming and inconvenient. Early diagnosis of IBD is necessary for the control of the disease, and rapid detection of IBDV and differentiation of IBDV serotypes is warranted. Recently, the polymerase chain reaction (PCR) was applied to the detection of IBDV and resulted in a very specific and sensitive assay. Although both IBDV serotypes were recognized by the PCR technique in that study, the PCR does offer several advantages. First, there is no need to grow or isolate virus from clinical specimens before PCR amplification, which significantly reduces the time and labor. Second, the PCR results in exponential amplification of a specific IBDV cDNA sequence flanked by a pair of primers selected from a known cDNA sequence of IBDV genome. By carefully analyzing the published genomic

Chemiluminescent detection of infectious bursal disease virus with a PCR-generated nonradiolabeled probe.

A polymerase chain reaction (PCR)-generated digoxigenin-labeled nonradioactive oligonucleotide probe was developed and utilized in slot-blot hybridization coupled with chemiluminescence for the detection of infectious bursal disease virus (IBDV). The probe was prepared from the RNA of the standard challenge strain (STC) of IBDV serotype 1 by reverse transcription followed by 2 PCR amplifications with 2 separate sets of primers. RNA of STC viruses prepared from bursae infected with STC viruses was subjected to the first PCR with the outer primers V8 and V9 that amplified a 607-base pair (bp) segment. The PCR product from the first PCR was eluted following agarose gel electrophoresis and subjected to the second PCR with the nested primers V6 and V7 that flanked a 351-bp segment. In the second PCR, dTTP was substituted by digoxigenin-11-dUTP in the PCR reaction mixture so that the amplified 351-bp DNA products were labeled with digoxigenin. The specificity of the PCR-generated digoxigenin-labeled probe was tested on different strains of IBDV, several unrelated avian viruses, and bacteria by slot-blot hybridization assay. Hybridization was detected by chemiluminescence. The sensitivity of the probe was assayed using 10-fold serial dilutions of purified RNA from the STC strain of IBDV. The PCR-generated digoxigenin-labeled probe hybridized with genomic RNA of STC and variant strains A, D, E, G, and GLS-5 of IBDV serotype 1 but not OH strain of IBDV serotype 2. The probe did not react with avian reovirus, infectious bronchitis virus, Salmonella enteritidis, Escherichia coli, or Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS)