Molecular and immunogenic characterization of BHK-21 cell line adapted CVS-11 strain of rabies virus and future prospect in vaccination strategy

Abstract

Development of a cost effective quality vaccine is a key issue in rabies control programme in developing countries. With this perspective, in the present study, challenge virus standard (CVS)-11 strain of rabies virus was adapted to grow in BHK-21 cells, characterized, compared with other viruses including global vaccine strains and field isolates from Indian subcontinent and China at molecular level. This cell adapted virus was evaluated for the production of cost effective veterinary vaccine. The maximum virus titre achieved was 10(7) fluorescent focus unit (FFU)/mL at 10th passage level. There was no nucleotide difference in the nucleoprotein (N) and glycoprotein (G) genes after adaptation in cell line. Phylogenetic analysis showed that adapted virus was grouped with global vaccine strains, closest being with other CVS strains but distinct from the Indian field isolates. Global vaccine strains including cell adapted CVS-11 virus have 83-87 % identity at nucleotide level of G gene with Indian field viruses. Growth kinetics of cell culture adapted virus showed that the optimum virus titer (around 10(7) FFU/mL) could be obtained at around 48 h post infection by co-cultivation method using 0.1 multiplicity of infection inoculums at 37 °C. These findings can be used for up scaling of vaccine production. The protective efficacy of test vaccine produced using 10(6.95) FFU/mL cell culture harvest showed 1.17 IU/mL relative potency by NIH test. Further, adapted virus was found to be suitable for use in rapid fluorescent focus inhibition test.

Keywords: Adaptation; BHK-21; CVS; NIH test; Rabies virus; Vaccine.

Fig. 1

Phylogenetic tree based on full length G gene nucleotide sequence of reference vaccine strains and recent Indian isolates of rabies virus. Phylogenetic analysis of G gene nucleotide sequence of mice origin (Accession No. KR105373) and BHK-21 adapted CVS-11 strain (Accession No. KR105372) of rabies, rabies vaccine strains used worldwide and recent Indian field isolates were used. The phylogenetic tree was constructed using neighbour-joining program of MEGA version-6. The numbers below the branches are bootstrap values for 1000 replicates. Note that most of the global vaccine strains clustered together, whereas Indian field isolates form a separate group

Fig. 2

Phylogenetic tree based on full length N gene nucleotide sequence of reference vaccine strains and various Indian isolates of rabies virus. Phylogenetic analysis of N gene nucleotide sequence of mice origin (Accession No. KR105375) and BHK-21 adapted CVS-11 strain (Accession No. KR105374) of rabies virus along with other vaccine strains and Indian field isolates available in GenBank were used. The tree was constructed using neighbour-joining program of MEGA version-6. The numbers below the branches are bootstrap values for 1000 replicates. Note that both sequences together clustered close to the other sequences of same strain, and also close to other vaccine strains. Whereas these stand entirely different and away from the Indian isolates

Fig. 3

Growth kinetics of BHK-21 adapted virus using different MOI. Growth kinetics of BHK-21 cell adapted CVS-11 rabies virus using 1, 0.1 and 0.01 MOI of virus by co-cultivation technique. Note that 0.1 MOI of virus is most suitable for optimum virus titre (about 10⁷), at around 48 h post infection

Fig. 4

Evaluation of BHK-21 adapted CVS-11 strain of rabies for rapid fluorescent focus inhibition test (RFFIT). BHK-21 adapted CVS-11 and PV-11 strain of rabies virus (100 FFU/mL) were incubated with variable dilution (tenfold) of anti-rabies antibody. Virus neutralization was assessed by FAT, after propagation of un-neutralized virus in BHK-21 cells. Note that, wells with higher dilution of antibody show poor or no neutralization and therefore positive by FAT; whereas, lower dilution of antibody show complete neutralization and therefore negative by FAT (Photographs were taken at × 200)