Abstract
Peste des Petits Ruminants (PPR) is an acute and highly contagious viral disease of small ruminants. The disease is endemic in Bangladesh and causes considerable economic losses due to its high morbidity and mortality in Black Bengal goats. A live attenuated conventional PPR vaccine was developed in the nineties and is currently being used to control PPR in Bangladesh. However, the farmers and the field veterinarians often raise the question about the efficacy of the vaccine. The poor efficacy of PPR vaccine in rural situation was thought to be due to break in cool chain during transportation. To solve this problem, a thermostable preparation of the former PPR vaccine was developed, nevertheless the efficacy still remained questionable. Studies are quite limited with regard to the molecular characteristics of the circulating viruses and the vaccine of Bangladesh. The present study was undertaken (i) to determine the efficacy of both the vaccines in goats, (ii) to study the gene sequences of the vaccine virus and its homology with the PPR virus of Bangladesh, (iii) to isolate and molecularly characterize the PPR virus circulating in Bangladesh, and (iv) to adapt local isolates of PPR virus of Bangladesh in cell culture for development of vaccine candidates. The efficacy of both the vaccines was determined by measuring the level of antibody in the vaccinated goats by a competitive Enzyme-Linked Immunosorbent Assay (cELISA). The partial F and N gene sequences of the vaccine virus were determined and compared with that of PPR virus available in the GenBank by Basic Local Alignment Search Tool (BLAST). Twelve (12) local isolates of Bangladeshi PPR virus (PPRV) were successfully isolated from post mortem samples of lymph node, spleen and trachea of goats during the year 2008 -2012. Out of 12 isolates, amplified PCR products of partial N gene of 10 isolates and F gene of 8 isolates were sequenced and submitted to the GenBank. Additionally full-length F gene of one of the isolates was sequenced. The phylogenetic analysis, homology study and substitution analysis at nucleotide and amino acid level of the sequences were carried out by MEGA (Version 5.10) and Lasergene DNASTAR software with the nucleotide sequences of PPRV obtained from GenBank. Vero cell was used for adaptation of local isolates of PPRV. The level of adaptation of the isolates was assessed by cytopathological study, gene sequencing and titration. Both thermostable and conventional vaccines available in Bangladesh provided partial sero- conversion (60-70%) that declined with time. The immunogenicity of the thermostable vaccine was not significantly affected (P>0.01) by storage temperature up to 14 days between 25o C and 40°C. Molecular analysis showed that Bangladeshi vaccine virus shared comparatively lesser homology with the locally circulated PPRV (94-95 % in case F gene and 87% in case of N gene). Whereas, Bangladeshi vaccine virus was genetically very close (99%) to early African isolates belonging to Lineage I. However, based on the 351 bp of N gene and 448 bp of F gene sequences, it was revealed that PPRVS circulating in Bangladesh for the last five years belonged to Lineage IV and they formed a separate cluster with the isolates from South and Southeast Asia (China, India, Pakistan, Nepal and Bhutan). It appeared that the N gene of PPRV is less conserved as compared to the F gene. Two unique amino acid substitutions were observed in Bangladeshi and Chinese isolates in the partial sequence of N protein. On full length sequence analysis of F gene, two unique amino acid substitutions were also found in Bangladeshi isolates, which were not marked in other Asian isolates. Field viruses successfully adapted to Vero cells showed vaculation, rounding and aggregation of cells. Acidophilic intracytoplasmic and intranuclear inclusion bodies were observed on stained infected Vero cell monolayers on coverslip when examined after 9th passage. A few substitutions in N gene at nucleotide and amino acid level were observed after 9th passage. Since the current vaccine virus of Bangladesh showed lesser degree of homology with the circulating strains of Bangladesh, a thermostable vaccine from the recent local isolate of PPRV needs to be developed. Further passages of the adapted virus in Vero cells may lead to the development of an attenuated vaccine candidate.