Adaptation and Molecular Characterization of Two Malaysian Very Virulent Infectious Bursal Disease Virus Isolates Adapted in BGM-70 Cell Line (original) (raw)
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Journal of pathogens, 2018
Two Malaysian very virulent infectious bursal disease virus (vvIBDV) strains UPM0081 (also known as B00/81) and UPM190 (also known as UPM04/190) isolated from local IBD outbreaks in 2000 and 2004, respectively, were separately passaged for 12 consecutive times in 11-day-old specific pathogen free (SPF) chicken embryonated eggs (CEE) via the chorioallantoic membrane (CAM) route. The CEE passage 8 (EP8) isolates were passaged once in BGM-70 cell line yielding UPM0081EP8BGMP1 and UPM190EP8BGMP1, while the EP12 isolates were passaged 15 times in BGM-70 cell line yielding UPM0081EP12BGMP15 and UPM190EP12BGMP15 using T25 tissue culture flask. These isolates were all propagated once in bioreactor using cytodex 1 as microcarrier at 3 g per liter (3 g/L) yielding UPM0081EP8BGMP1BP1, UPM190EP8BGMP1BP1, UPM0081EP12BGMP15BP1, and UPM190EP12BGMP15BP1 isolates. The viruses were harvested at 3 days after inoculation, following the appearance of cytopathic effects (CPE) characterized by detachment ...
Replication of classical infectious bursal disease virus in the chicken embryo related cell line
Avian Pathology, 2000
Infectious bursal disease (IBD) is an acute, highly contagious viral disease. The diagnosis of IBD depends on time-consuming and costly procedures, like virus isolation on chick embryos and histopathological examination. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunoperoxidase and reverse transcription polymerase chain reaction (RT-PCR) were applied in this study to detect classical IBD virus (IBDV) after three blind passages of the Lukert strain on chicken embryo related (CER) cell monolayer after different periods of infection: 6, 12, 24 and 48 h. Cytophatic effects were most evident 12 h post-infection (p.i.) but were observed at 6 h p.i. The maximum discrimination between IBDV-infected and uninfected cell suspensions obtained by the use of DAS-ELISA for virus detection corresponded to 0.597±0.02 and 0.010±0.01 after 12 h p.i., respectively. The RT-PCR was performed using the set of primers A3.1 and A3.2 to amplify the VP2 region of the IBDV genome. This molecular technique demonstrated that from 6 h p.i., it was possible to detect the viral RNA. The results show that the CER cell line can be used for classical IBDV propagation, confirmed by the DAS-ELISA, immunoperoxidase and RT-PCR assay.
IBDV outbreak had been reported to infect broiler chicken in Malaysia and cause high mortality. It had been reported that the virulence and pathogenicity of the virus is related to its coated protein, specifically the VP2 hypervariable regions. To study this, the nucleotides and protein of the local IBDV isolate was sequenced and analyzed by comparing it with other IBDV that had been reported previously. To obtain the virus sequence, local IBDV isolate strain 3529/92 was propagated in SPF eggs and RNA were extracted, amplify by rt(reverse transcriptase)-PCR before being inserted into pCR 2.1 TOPO for cloning and sequencing. The full length of the gene segment for the coat protein was constructed by concomitantly joining the fragments using the native restrictions sites of IBDV in pTrcHis2a expression vector. The sequence analysis revealed that the local IBDV strain 3529/92 consists of 3039 nucleotides which encodes for 1012 amino acids. BLAST analysis of the nucleotides sequence sho...
DNA Sequence, 2006
The present study was undertaken to characterize recent field isolates of infectious bursal disease virus (IBDV) by reverse transcription-polymerase chain reaction (RT-PCR) and partial sequencing of VP2 gene. The virus could be detected in 17 of 20 field samples from broiler chickens in Haryana state, India as well as in all the four vaccine strains. Nucleotide sequences of four field isolates and one vaccine strain were compared with 10 reported IBDV strains from different parts of the world. Nucleotide substitutions at 795G, 827T, 833C, 857C, 897A, 905T, 908T, 1011A and 1094G specific for very virulent (vv) strains, were maintained in all the four field isolates. However, unique nucleotide substitutions at 806A-G, 851C-T, 1010 T-C, 1019T-C and 1082T-C showed further divergence of these isolates from already reported vvIBDVs. Deduced amino acid substitutions at 222P-A, 256V-I, 279N-D, 294L-I and 299N-S specific for vvIBDV strains were also present in all the four isolates. The vaccine strain showed amino acid change 279D-N, a characteristic of attenuated vaccine strains. Phylogenetic analysis showed that all the field isolates in the present study were closely related to reported UK (UK661) and Japan (OKYM) field isolates. All the four field IBDV strains of the present study were closely related to each other but distinct from already reported vvIBDVs of India. On the basis of nucleotide sequencing and phylogenetic analysis, it is very likely that IBD causing strains in this part of India are of very virulent character and are still undergoing changes at genetic level.
2010
The objective of the present study was to isolate and identify infectious bursal disease viruses (IBDVs) from broiler and layer chickens of outbreaks of infectious bursal disease (IBD) in three districts of Bangladesh. A total of 70 bursal samples were collected from dead broiler (n=40) and layer (n=30) chickens showing specific lesions of IBD from seven commercial poultry farms of three different districts (Mymensingh, Chittagong and Tangail) of Bangladesh during the year 2007. Five representative bursal samples from each farm were used for the isolation of IBDVs using 9-day-old embryonated eggs of seronegative flock of layer birds and for identification the samples were subjected to agar gel immunodiffusion test (AGIDT), immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR). Out of 35 bursal samples, IBDVs were successfully isolated from 28 (80%) samples. By AGIDT, 32 (91.4%) samples were found positive for IBDV antigen. Results of AGIDT clearly indicated that IBDVs detected in 29 bursal samples of six affected farms were identical to each other but not to IBDVs present in the remaining three samples of another farm. Indirect immunoperoxidase staining of the bursal sections revealed the presence of IBDV antigen in 32 (91.4%) samples and the IBDV antigen was detected mainly in the cortex of the lymphoid follicles of the bursal tissues. In histopathology, cell depletion, atrophy and necrosis were observed in many bursal follicles with severe edema of interfollicular septa. Of the 35 bursal samples, 34 (97.1%) samples generated 254 bp product by RT-PCR. In conclusion, the results of virus isolation and identification by AGIDT, IHC and the analysis of viral genome by RT-PCR confirmed the outbreaks of acute IBD in commercial poultry of Bangladesh. Moreover, histopathological findings and results of AGIDT gave a clear indication that the isolates from six outbreaks were different from classical strain and it seems to be of very virulent strain. On the other hand, the isolates from the other outbreak were similar to the classical strain.
Journal of Applied Animal Research, 2007
An Indian classical infectious bursal disease virus (IBDV), adapted to grow in primary chicken embryo fibroblast culture, was characterized by RT-PCR IRFLP and sequence analysis of VP2 gene hyper variable region. The Indian classical virus displayed restriction profile similar to that already reported for attenuated IBDV strains. Sixteen nucleotide changes in Indian classical virus were revealed as compared to majority consensus with four unique changes 797A-T, 887C-A, 953A-C and 101 7A-G. Deduced amino acid sequence analysis showed two unique changes 270A-K and 2961-V. Presence of amino acid changes 253&-H, 2790-N a n d 284A-T, as well as 330s-R indicated this virus to have undergone attenuation after serial passaging in cell-culture. Phylogenetic anaiysis further confirmed this virus to be most closely related tQ, ,attenuated strain, PBG-98. Antigenic index analysis and hydrophilicity plots clearly show differences in the peaks of cell culture adapted, classical and very virulent viruses. The present study confirmed that Indian very virulent viruses having not originated from Indian classical virus.
This study was carried out to investigate the prevalence, molecular characterization and pathogenicity of field infectious bursal disease virus (IBDV) isolates. Nine isolates of IBDV were isolated from 13 naturally infected broiler flocks. Detection of IBDV antigen was carried out by agar gel precipitation test (AGPT), followed by virus isolation in specific pathogen free (SPF) embryonated chicken eggs (ECE) and finally molecularly characterized and identified using reverse transcriptase polymerase chain reaction (RT-PCR). The obtained nine strains of IBDV by RT-PCR were further classified by using restriction fragment length polymorphism (RFLP) technique into (4) classical, (3) variant and (2) very virulent (vv) IBDV serotype (I). The pathogenicity of the isolated IBDV strains was detected by three passages in SPF ECEs and by experimental infection of one hundred 14 days old maternally immune layer chicks. The results showed that the mortality rate of the embryos was increased by increase the number of passages till the third passage where it reached 100% for all IBDV strains and the embryos showed typical lesions of IBDV. Chicks inoculated with variant IBDV strains showed morbidity rates of 60-80 %, without mortalities. Sacrificed birds showed atrophied bursae and thymus glands and enlarged thickened proventriculus. Groups infected with classical IBDV strains showed morbidity rates 40-60,% with mortality 0-20%. The detectable lesions were muscular hemorrhages with variable bursal lesions. Inoculated chicks with vvIBDV strains showed 50-70% morbidity and mortality of rate was 30% with lesions of muscular hemorrhages, severe nephrosis with ureates in the ureters, hemorrhagic bursitis and pin point hemorrhages on the proventricular glands. Control negative non-infected group showed neither clinical signs nor mortalities along the observation period. The histopathological effect (lesion score) of IBDV strains on the bursa, spleen and thymus glands confirmed the previously mentioned results and revealed that the highest severity (score) for these organs were induced by vv IBDV strains.
2010
Infectious bursal disease (IBD) is an acute highly contagious viral infection of young chickens often resulting in immunosuppression. Inactivated vaccines play significant role in protection against IBD. Mammalian cell lines could be used for producing such vaccines. In present study twenty-five, local strains of IBD virus were inoculated into chicken embryo bursa cell culture, liver cell culture, kidney cell culture, fibroblast cell culture and Vero cell lines for cytopathic effect. Moreover comparative susceptibility of chicken embryo bursa organ, embryo liver organ and embryo kidney organ cultures, to infectious bursal disease virus were studied. Chicken embryo bursa cell line was found to be most susceptible (90%) followed by Vero cell lines (70%), fibroblast cell lines (65%), kidney cell lines (50%) and liver cell lines (45%). While chicken embryo bursa organ culture gave maximum cytopathic effect (80%) followed by chicken embryo liver (60%) and kidney organ (45%). From these studies it is concluded that after bursa cell lines, Vero cell lines gave maximum cytopathic effect yielding high number of virus particles and are easy to maintain. Thus Vero cell lines can be used to produce infectious bursal disease vaccines using local isolates.