Alejandro Banda

Genetic Characterization of Very Virulent Infectious Bursal Disease Viruses from Latin America

Abstract Very virulent infectious bursal disease viruses (vvIBDVs) were detected in phenol inactivated bursal samples obtained from Brazil, the Dominican Republic, and Venezuela.After nucleotide sequence analysis of the hypervariable region of VP2 gene, the vvIBDVs from Brazil and Venezuela exhibited all of the 14 nucleotide changes that are conserved in the European UK-661 and most other vvIBDV strains. However, the vvIBDV from the Dominican Republic presented 11 nucleotide changes that are conserved in vvIBDV strains. After phylogenetic analysis, the Latin American strains were found to be related to other vvIBDV strains from Europe, Asia, and Africa. However, Brazilian and Dominican vvIBDVs clustered in two separate subgroups, while the vvIBDVs from Venezuela were closely related to other strains from other parts of the world. By deduced amino acid sequence, the three conserved amino acid residues in vvIBDV strains (222 Ala, 256 Ile, and 294 Ile) were confirmed in the Latin American viruses, and one amino acid change (300 Ala) was unique to all vvIBDVs from the Dominican Republic. The occurrence of this change in the Dominican vvIBDVs may have an impact in their antigenic makeup. Results of this study indicate that the vvIBDVs detected in Latin America are genetically similar to IBDV strains from other parts of the world. However, vvIBDVs from Venezuela were more similar to the vvIBDV strains from Europe and Asia. Of all the samples analyzed, vvIBDVs from Brazil and the Dominican Republic exhibited more genetic changes. These changes may have emerged as a result of the different management practices and environmental conditions present in each particular geographic area.

Molecular Characterization of Infectious Bursal Disease Virus from Commercial Poultry in the United States and Latin America

SUMMARY. 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.

Molecular Characterization of Seven Field Isolates of Infectious Bursal Disease Virus Obtained from Commercial Broiler Chickens

Specific-pathogen-free sentinel birds were used as an initial biological system to isolate infectious bursal disease virus (IBDV) field isolates from commercial broiler farms exhibiting recurrent respiratory problems and poor performance. Reverse transcription (RT)-polymerase chain reaction (PCR) was used to amplify a 248-bp product encompassing the hypervariable region of the IBDV VP2 gene. Restriction fragment length polymorphism (RFLP) analysis of the RT-PCR products was performed with the restriction endonucleases DraI, SadI, TaqI, StyI, BstNI, and SspI. Two isolates (619 and 850) exhibited a RFLP pattern characteristic of Delaware variant E IBDV. Restriction enzyme digestion for four isolates (625, 849, 853, and 11,153) revealed unmatched RFLP patterns when compared with reference IBDV strains. Nucleotide and deduced amino acid sequence analyses of the VP2 hypervariable region for these six isolates revealed identity (96.3% up to 98%) with Delaware E variant IBDV strain. However, serine at position 254, which is characteristic of Delaware variant strains, was substituted by asparagine in these six isolates. The seventh IBDV isolate (9109) also exhibited a unique RFLP pattern, which included the SspI restriction site, which is characteristic of very virulent (vv) IBDV strains. Nucleotide and amino acid sequence analyses of the hypervariable region for this isolate revealed identity (90%) with the standard challenge strain. However, the leucine residue at position 294 was substituted by isoleucine. This substitution corresponds to one of the amino acids that are conserved in the vvIBDV strains. Antigenic index studies of the predicted amino acid sequence of the hypervariable region of VP2 from isolates 619, 625, 849, 850, 853, and 11,153 exhibited a profile almost identical to variant E, whereas the isolate 9109 exhibited a profile characteristic of standard IBDV strains.

Genetic analysis of a duck circovirus detected in commercial pekin ducks in New York

Abstract The genetic organization of the duck circovirus (DuCV) 33753-52 detected in commercial Pekin duck flocks from Long Island, NY, is described. The nucleotide sequence of virus 33753-52 exhibited high similarity with DuCVs previously detected in Germany and Hungary. It is possible that this DuCV from New York shares the same ancestor with the European counterparts. The virus 33753-52 exhibited genetic features characteristic of other circoviruses, such as the presence of two major open reading frames (rep and cap), two intergenic regions, one stem-loop structure, four intergenic direct repeats, and the conserved motifs for the rolling circle replication and for the dNTP binding domain in the Rep protein. This report is the first report of the presence of DuCV in commercial Pekin duck farms in the United States. The clinical and pathologic significance of DuCV in the duck farms located on Long Island needs to be clarified. DuCv was detected in culled birds, due to low body development, leg deformities, or arthritis. Staphylococcus aureus and Riemerella anatipestifer serotype 4 were isolated from some of the DuCV-positive birds. The apparent low prevalence of the virus suggests that at this time, this infection is not a significant problem for the duck industry in New York. However, the immunosuppressive properties of this virus need to be clarified as well as its role as a predisposing agent for other diseases.

Detection of Very Virulent Strains of Infectious Bursal Disease Virus (VVIBDV) in Commercial Broilers from Uruguay

Abstract Bursal samples were collected from commercial broiler flocks exhibiting clinical signs suggestive of infectious bursal disease (IBD). The presence of IBD virus (IBDV) was confirmed by partial amplification of the VP2 and VP1 genes by reverse transcription and polymerase chain reaction. The Uruguayan viruses were identified as very virulent strains of IBDV (vvIBDV) by nucleotide and amino acid sequence analysis. The comparison of the VP2 nucleotide sequences among the Uruguayan samples revealed the presence of single-nucleotide polymorphisms suggestive of different viral subpopulations or quasispecies in the same flock. The comparative analysis indicated that these Uruguayan viruses were genetically close to the European strain UK661 and to the vvIBDVs previously detected in Venezuela. Our analyses provided new information about the distribution, variability, and evolutionary trends of vvIBDV strains in the Americas.

Phylodynamic analysis of avian infectious bronchitis virus in South America.

Infectious bronchitis virus (IBV) is a coronavirus of chickens that causes great economic losses to the global poultry industry. The present study focuses on South American IBVs and their genetic relationships with global strains. We obtained full-length sequences of the S1 coding region and N gene of IBV field isolates from Uruguay and Argentina, and performed Phylodynamic analysis to characterize the strains and estimate the time of the most recent common ancestor. We identified two major South American genotypes, which were here denoted South America I (SAI) and Asia/South America II (A/SAII). The SAI genotype is an exclusive South American lineage that emerged in the 1960s. The A/SAII genotype may have emerged in Asia in approximately 1995 before being introduced into South America. Both SAI and A/SAII genotype strains clearly differ from the Massachusetts strains that are included in the vaccine formulations being used in most South American countries.

Development and validation of a TaqMan-MGB real-time RT-PCR assay for simultaneous detection and characterization of infectious bursal disease virus

Rapid and reliable detection and classification of infectious bursal disease viruses (IBDVs) is of crucial importance for disease surveillance and control. This study presents the development and validation of a real-time RT-PCR assay to detect and discriminate very virulent (vv) from non-vv (classic and variant) IBDV strains. The assay uses two fluorogenic, minor groove-binding (MGB) TaqMan probes targeted to a single nucleotide polymorphism (SNP) embedded in a highly conserved genomic region. The analytical sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA. The assay demonstrated a wide dynamic range between 10(2) and 10(8) standard RNA copies per reaction. Good reproducibility was also detected, with intra- and inter-assay coefficients of variation ranging from 0.13% to 2.23% and 0.26% to 1.92%, respectively. The assay detected successfully all the assessed vv, classical, and variant field and vaccine strains and correctly discriminated all vvIBDV strains from non-vvIBDV strains. Other common avian RNA viruses tested negative, indicating high specificity of the assay. The high sensitivity, rapidity, reproducibility, and specificity of the real-time RT-PCR assay make this method suitable for general and genotype-specific detection and quantitation.

Sequence variability and evolution of the terminal overlapping VP5 gene of the infectious bursal disease virus

The infectious bursal disease virus (IBDV; Birnaviridae family) constitutes one of the main threats to the poultry industry worldwide. Most of the progress in the molecular epidemiology of this virus has been achieved through the study of the coding region of the capsid protein VP2. Little research has been done regarding the molecular evolution and the epidemiological implications of genetic variability of other IBDV genome regions. In this article, the gene that codes the non-structural protein VP5 was analyzed. Although this protein is not essential for the virus replication, recent evidence indicates that it could be related to the virulent phenotype and the adaptive capacity of the virus. The VP5 gene is also of evolutionary interest because it has an open reading frame that terminally overlaps with the pVP2–VP4–VP3 polyprotein coding region. In the first part of this study, the full VP5 gene of a South American strain was characterized. The results revealed that the VP5 gene of Uruguayan hypervirulent IBDV strains (vvIBDV) lacks the alternative AUG start codon characteristic of the vvIBDV strains that have been described to date. Instead, as occurs in classic and variant strains, this VP5 gene has an AUG start site located four codons downstream and, consequently, it codes for a 145 amino acid long protein rather than the putative 149 amino acid long protein of other vvIBDV. In spite of this, these viruses conserved the VP5 and VP2 amino acid signature of the hypervirulent strains and clustered with reference vvIBDV sequences. This finding may represent evidence that the VP5 gene could be evolving by changing the translation initiation site. In the second part of this study, an evolutionary analysis including the sequences reported in this study together with most of VP5 sequences available in the GenBank, showed the existence of a complex system of selective pressures controlling the evolution of the VP5 gene. Using the dN/dS index, we found a strong purifying selection exerted on the 5′ terminal overlapping region of VP2 that would be constraining the evolution of VP5. These results reinforce the hypothesis that the VP5 gene was originated late in the IBDV evolution by a mechanism of genetic overprinting. The results described in this study provided new information about the dynamics of the IBDV genome and revealed some of the mechanisms at play in the evolution of this virus. Since VP5 seems to be related to viral pathogenicity, this evolutionary information might be useful to highlight the impact of the genetic variation of this protein on the epidemiology of IBDV.

Novel multiplex RT-PCR/RFLP diagnostic test to differentiate low- from high-pathogenic strains and to detect reassortant infectious bursal disease virus.

SUMMARY. Three types of infectious bursal disease virus (IBDV) strains are currently circulating worldwide: the low-pathogenic classic and variant strains and the high-pathogenic very virulent strains. There are also natural reassortant viruses that combine genomic segments A and B from different strains and exhibit particular pathogenic characteristics. Detection and characterization of the different IBDVs is extremely critical for improving disease control and performing epidemiologic studies. Here, we present a novel detection and genotyping method based on the simultaneous characterization of both IBDV genomic segments followed by a simple restriction fragment length polymorphism (RFLP) assay. This single restriction enzyme, multiplex reverse transcriptase-PCR/RFLP diagnostic test not only distinguished typical high-pathogenic from low-pathogenic strains but also detected natural reassortant IBDV. The test was based on the detection of single nucleotide polymorphisms (SNP), in both segments, which were strongly linked to the pathogenic phenotype. These SNPs are embedded in highly conserved genomic regions and can be identified with TfiI endonuclease. The application of this methodology in field samples confirmed that the assay is fast, specific, and may be easily adopted by any molecular diagnostic laboratory as an economical and routine method.

Protection Conferred by Coarse Spray Vaccination Against Challenge with Infectious Bursal Disease Virus in Commercial Broilers

Abstract The efficacy of coarse spray vaccination against pathogenic infectious bursal disease virus (IBDV) in commercial broilers was evaluated. Different coarse spray vaccination schedules using a commercial 2512 strain vaccine were compared with single or double drinking water application at 1 and/or 10 days of age. At 29 days of age, the chickens were challenged with the virulent Edgar strain of IBDV. Seven days postchallenge, severe gross bursal atrophy was observed in the unvaccinated-challenged birds. After challenge and regardless of the method of vaccination used, moderate-to-severe lymphoid depletion was observed, indicating challenge virus replication, later confirmed by reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism analysis. Coarse spray and drinking water vaccination induced protection against body weight loss. Significant differences (P < 0.05) were observed between the unvaccinated-challenged group (1483 g) and the birds vaccinated at 10 days of age by coarse spray (1812 g). The coarse spray vaccination also induced protection against challenge-induced gross bursal atrophy, as determined by bursal index values. After challenge, significant bursal atrophy was observed in the birds orally vaccinated at 1 day (0.61), 10 days (0.66), and 1 and 10 days (0.63) as well as the unvaccinated-challenged birds (0.62), but not in the coarse-spray-vaccinated groups that exhibited bursal indexes above 0.70 and did not differ from the unvaccinated-unchallenged control group. These results suggest that coarse spray vaccination can be considered as another tool to control IBDV in the field.