Priya Mohan Das

Reversion of molecularly engineered, partially attenuated, very virulent infectious bursal disease virus during infection of commercial chickens

A molecularly cloned, tissue culture-adapted infectious bursal disease virus (BD-3tc) was generated from a very virulent strain by the reverse genetics approach following site-directed mutagenesis (Q253H and A284T in VP2). The pathogenicity of BD-3tc was tested in commercial chickens. The wild-type strain (BD-3wt) and the molecularly cloned parental strain (BD-3mc) were included for comparison. The subclinical course of the disease, with delayed and milder pathological lesions followed by quick follicular regeneration in the bursa of Fabricius in BD-3tc-inoculated birds, suggested that these amino acid substitutions made BD-3tc partially attenuated. However, severe bursa atrophy was observed at 14 days after inoculation. Reverse transcription-polymerase chain reaction coupled with restriction enzyme analysis revealed that both point mutations in BD-3tc had reverted 14 days after inoculation. Further investigations demonstrated that the codon for amino acid at position 284 had already reverted to the wild-type phenotype (T284A) 3 days after inoculation. Résumé Réversion dun virus de la bursite infectieuse, très virulent mais atténué partiellement par génie moléculaire, chez des poulets de chair durant linfection Un virus de la bursite infectieuse aviaire (BD-3tc) adapté à la culture cellulaire et cloné moléculairement a été généré à partir dune souche très virulente par une approche de génétique reverse suivant une mutagenèse dirigée (Q253H et A284T de VP2). La pathogénicité du virus BD-3tc a été étudiée chez des poulets de chair. La souche sauvage BD-3wt et la souche parentale clonée moléculairement (BD-3mc) ont été étudiées en comparaison. Chez les animaux inoculés avec la souche BD-3tc, l’évolution subclinique de la maladie, avec des lésions pathologiques moins sévères, plus tardives et qui ont été suivies par une régénération folliculaire rapide dans la bourse de Fabricius laisse supposer que les substitutions de ces acides aminés rendent la souche BD-3tc partiellement atténuée. Cependant, une atrophie sévère de la bourse a été observée 14 jours après inoculation. Une RT-PCR couplée à lanalyse dune restriction enzymatique a révélé que les deux points de mutation de la souche BD-3tc ont reversé 14 jours après inoculation. Des investigations complémentaires ont démontré que le codon pour lacide aminé en position 284 avait déjà reversé au phénotype sauvage (T284A) trois jours après inoculation. Zusammenfassung Reversion eines gentechnisch hergestellten, teilweise attenuierten hochvirulenten Stammes des Virus der infektiösen Bursitis nach der Infektion nicht-spezifiziert pathogen freier Hühner Unter Verwendung reverser Genetik wurde aus einem hochvirulenten Stamm des Virus der infektiösen Bursitis ein Zellkultur-adaptierter Stamm (BD-3tc) hergestellt, in dem mittels gerichteter Mutagenese die Aminosäuren Q253H und A284T in VP2 ausgetauscht worden sind. Die Pathogenität von BD-3tc wurde in nicht-spezifiziert pathogen freien Hühnern getestet. Der Wildtyp-Stamm (BD-3wt) und der gentechnisch hergestellte Elternstamm (BD-3mc) wurden in die Untersuchungen einbezogen. Der subklinische Verlauf der Krankheit mit verzögert einsetzenden und milderen pathologischen Veränderungen der Bursa Fabricii, gefolgt von einer schnellen Follikel-Regeneration in den BD-3tc-infizierten Hühnern, ließen vermuten, dass der Austausch der genannten Aminosäuren zu einer teilweisen Attenuierung des Stammes BD-3tc geführt hatte. Jedoch wurde 14 Tage nach der Inokulation eine schwere Atrophie der Bursa beobachtet. Untersuchungen mittels RT-PCR und nachfolgenden Restriktionsenzymanalysen ergaben, dass beide Punktmutationen in BD-3tc revertiert waren. Weitere Untersuchungen zeigten, dass die Reversion für das Codon der Aminosäure an Position 284 zum Phenotyp des Wildtyps (T284A) bereits drei Tage nach der Inokulation eingetreten war. Resumen Reversión de un virus de bursitis infecciosa aviar muy virulento, parcialmente atenuado mediante ingeniería genética, durante una infección en pollos comeciales Un virus de bursitis infecciosa aviar adaptado a cultivo celular y molecularmente clonado (BD-3tc) fue generado a partir de una cepa muy virulenta una aproximación con genética reversa seguido de site-directed mutagénesis (Q253H y A284T en VP2). La patogenicidad de BD-3tc fue probada en pollos comerciales. Se incluyeron la cepa original (BD-3wt) y la clonada molecularmente (BD-3mc) para poder ser comparadas. El curso subclínico de la enfermedad, con lesiones tardías y leves seguidas de una rápida regeneración folicular de la bolsa de Fabricio en las aves inoculadas con BD-3tc, sugieren que estas sustituciones aminoacídicas atenuaron parcialmente a BD-3tc. Aún así, se observo una atrofia grave de la bolsa a los 14 días tras la inoculación. La RT-PCR juntamente con el análisis con enzimas de restricción reveló que ambas mutaciones puntuales en BD-3tc revirtieron 14 días tras la inoculación. En investigaciones posteriores se demostró que el codon para la posición aminoacídica 284 había ya revertido al fenotipo original (T284A) tres días después de la inoculación.

Pathotypic and genotypic characterization of two Bangladeshi isolates of Newcastle disease virus of chicken and pigeon origin.

Two Bangladeshi isolates of Newcastle disease virus (NDV), one from a chicken and one from a pigeon, were characterized in this study. Pathogenicity of the isolates was evaluated on the basis of intracerebral pathogenicity index (ICPI). Both the isolates were found to be of velogenic pathotype having ICPI of 1.83 and 1.51 for the chicken and pigeon isolate, respectively. Genotype of the isolates was determined by phylogenetic analysis based on partial F gene sequences. A 766-bp genome fragment spanning partial M and F gene was amplified by RT-PCR and sequenced. The first 354xa0bp of the coding region of F gene and corresponding deduced amino acid sequences (residues 1-118) of these two NDV isolates were aligned with that of other NDV strains retrieved from GenBank. A phylogenetic tree constructed from the alignment showed that the chicken isolate (BD-C162) belonged to the newly described genotype XIII and the pigeon isolate (BD-P01) to genotype VI. Both the chicken and pigeon isolates possessed a virulent-like fusion protein cleavage site (112) RRQKRF(117) .

Further evidence for the association of distinct amino acid residues with in vitro and in vivo growth of infectious bursal disease virus

A cell-culture-adapted reverse genetics strain of very virulent infectious bursal disease virus (IBDV) of chickens, designated as BD-3tcC, having four amino acid substitutions (Gln253His, Asp279Asn, Ala284Thr and Ser330Arg) in the capsid protein VP2 was tested for its genetic stability during serial passage in chickens and chicken embryo fibroblast (CEF) cell culture. Results of in vitro and in vivo experiments demonstrated that all four introduced mutations in BD-3tcC remained stable during serial passage in CEF cell culture, but during passage in chickens, amino acid residues at position 253 and 284 reverted from histidine to glutamine and threonine to alanine, respectively. In a parallel experiment, the same substitutions also occurred in a conventionally attenuated vaccine strain D-78 on serial passage in chickens. However, no reversion or substitution took place at positions 279 and 330 during in vivo passage of the mutant virus BD-3tcC or vaccine virus D-78. The findings provide conclusive evidence that while IBDV requires histidine and threonine at positions 253 and 284 for cell culture adaptation, glutamine and alanine at these positions are selected preferentially during in vivo replication.

Phylogenetic analysis of Newcastle disease viruses from Bangladesh suggests continuing evolution of genotype XIII

A total of 23 Newcastle disease virus (NDV) isolates from Bangladesh taken between 2010 and 2012 were characterized on the basis of partial F gene sequences. All the isolates belonged to genotype XIII of class II NDV but segregated into three sub-clusters. One sub-cluster with 17 isolates aligned with sub-genotype XIIIc. The other two sub-clusters were phylogenetically distinct from the previously described sub-genotypes XIIIa, XIIIb and XIIIc and could be candidates of new sub-genotypes; however, that needs to be validated through full-length F gene sequence data. The results of the present study suggest that genotype XIII NDVs are under continuing evolution in Bangladesh.

Clinicopathological Investigation of Foot and Mouth Disease and Serotype Identification of the Viruses in Cattle of Bangladesh

Foot and Mouth Disease (FMD) is a highly contagious viral disease of farm animals. This study was aimed to identify symptoms, characteristics pathology and viral serotypes involved in infected cattle of Bangladesh. Dominant signs observed were salivation, vesicular eruption, lameness and maggot infestations in 66%, 80%, 36% and 14% cattle respectively. Out of 202 infected cattle investigated, 35 were below six months of age. A total of 18 young calves and 8 adult cattle were died due to FMD. Seven young calves and two adult cattle were examined at necropsy, Tiger heart disease was commonly seen both in the young and adult cattle. The currant jelly clot was common in the atrium, ventricles, aorta and veins of the heart of adult cattle. Congested and consolidated lungs, atrophied spleen, and fibrosed udder were grossly seen in the infected and dead cattle. Microscopically, hyalinization of cardiac muscle in young calf and degeneration, necrosis and multifocal lymphocytic infiltration in heart muscle were suggestive for infectivity due to FMD viruses. There were bronchiolitis, pulmonary emphysema, interstitial pneumonia and thickening of interlobular septa of lungs. Cattle survived from the acute infection developed respiratory distress in 15% cattle. Out of 202 samples tested in reverse transcriptase polymerase chain reaction (RT-PCR), 187 samples found to generate positive amplicons. Single infectivity due to FMD viral Serotypes O, A and Asia 1 were seen in 115 (62%), 26 (14%) and 34 (18%) cases, respectively. Seven cattle were co-infected with FMD viral Serotypes O and Asia 1 and four with FMD viral Serotypes O and A. FMD viral serotype O was dominating in cattle all over the country and was related to death of infected young and adult cattle. Cardiac myonecrosis and respiratory failure were the dominant lesions may be related to death of infected animals.