ijvp

Indian Journal of Veterinary Pathology

0250-47580973-970X

Indian Association of Veterinary Pathologists

ijvp-34-1-abs024

ABSTRACTS OF DISSERTATIONS OR ARTICLES

Molecular epidemiology and pathogenesis of rabies

Reddy

G.B Manjunatha

¹Singh

Rajendra

Dr.¹

¹Degree/year: Ph.D./2010, Name of University: Deemed University IVRI, Izatnagar-243122 (UP)

162010

341102102

2010

IndianJournals.com

Despite immense research work in rabies pathogenesis, the basis of innate immune mechanisms involved and role of blood - brain barrier (BBB) in the disease are still enigmatic. After the development of the clinical signs, the survival of the host is almost impossible as peripheral host immune response against rabies virus is being prevented from reaching brain by BBB together with late generation of immune response in brain. Further, there is scanty report on molecular epidemiology of rabies virus circulating in the country. Therefore, the present study was conducted with an aim to investigate: i) Involvement of TLR-3 and its associated cytokines (IL1á, TNF- á, and IFN- á) in the pathogenesis of rabies with LPS as BBB modulator and ii) Sequencing and phylogenetic analysis of Indian rabies virus isolates. For pathogenesis study, a total of 200 young Swiss albino mice were divided into 8 groups of 25 mice each. The mice were inoculated with Poly I:C, a TLR-3 agonist, I/P @ 25 íg/mouse three days prior (GP), LPS I/P on day 0 @ 2ig/mouse (GL), Challenge Virus Standard (CVS-18) rabies virus I/C @ 100 LD50/mouse (GI), Poly I:C and CVS (GPI), Poly I:C and LPS (GPL), Poly I:C, LPS and CVS (GPLI) and PBS I/C @ 0.03ml/mouse (GC). Different parameters were studied at 2, 4, 6, 8, 10 and 12^th DPI. Clinical history score revealed mild and delayed onset of clinical signs and more survival of mice on different time points in GPI, GPLI and GLI than in GI. The non suppurative encephalitic changes were scored less in GPI, GPLI and GLI than in GI. The lesions were more severe in medulla oblongata followed by hippocampus, particularly CA1 and CA2 regions. The CD4⁺: CD8⁺ ratio decreased in GL, GLP, GLI and GPLI than in GI and GPI. The percentage of NK cells in blood and spleen were found to be more in GI followed by GPI, GPLI and GLI than in their respective controls. The â-catenin signals were mild in LPS treated groups (GLI, GPLI) than in GI and GC, which suggested opening of BBB. The TLR-3 and its associated cytokines showed higher expression in GPI, GPLI, and GLI than in GI. Further, the presence of virus in brain was detected using N gene specific primers (200 bp) in all the infected groups. The study clearly showed that TLR-3 induction by rabies virus had some role in reducing the development of disease and increased survival through pro-inflammatory cytokines and opening of BBB. Further studies are needed to explore the possibility of TLR-3 for therapeutic purposes in rabies and other diseases.

In second set of study, a total of 48 rabies positive brain samples were subjected to amplification of partial N and P genes for sequencing. These samples were belonged to different species of animals from different geographical regions of the country. Partial N gene (806bp) could be amplified from 30 samples (14 dogs, 4 cattle, 1 buffaloes, 2 goats, lbeer, 2 hyenas and 6 humans) and got sequenced commercially. The phylogenetic analyses revealed 3 distinct genetic groups (GrI, GrII and Grill). In GrL rabies samples were from southern part of India and in GrII the samples were from northern part of India. No species specific variation could be recorded. The Grin rabies samples were from different parts of India, but this group was more closely related to Artie lineage of rabies virus. The partial P gene (544bp) could be amplified from 41 samples (16 dogs, 1 cat, 5 cattle, 3 buffaloes, 2 goats, 3 horse, lbeer, 2 hyenas, 1 jackal and 7 humans) and got sequenced commercially. The phylogenetic analyses revealed almost all Indian isolates forming GrI and the samples form Punjab and Gujarat forming GrII. The N and P partial gene sequences from hyena and jackal samples were more closely related to dog isolates, which might be due to species spill over. The deduced amino- acid sequences in both phosphoprotein and nucleoprotein also found conserved, which differed from other isolates of world. These conserved amino- add sequences in N & P genes can serve as epidemiological marker in future for Indian rabies virus isolates. From the study, it was concluded that all the isolates drculating in India belong to dassical rabies virus geno- type-I and are genetically related to one another (>95% homology). The N gene of 30 samples showed 3 genetic variants which were regionalized as South India, North India and other parts of India. The partial P gene analysis showed dustering of genetic variants into two dusters: most parts of India and Punjab & Gujarat. From the study, it was evident that rabies is region specific, but not host specific in India. Dog rabies vims variants are the single major variants circulating in India. Continuing molecular epidemiology of rabies virus is required for better understanding the rabies dynamics to achieve reduction of rabies burden and its incidence. The data of gene sequences were submitted to GenBank for future studies.