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Abstract
Since 1994, several different inactivated rabies vaccines have been used to immunize domestic animals such as dogs, cats, and cattle in South Korea. The Korean Veterinary Authority has conducted safety and efficacy testes of inactivated vaccines using laboratory animals. In this study, we applied a molecular method to investigate the genetic characterization of the rabies virus (RABV) genes in six commercial inactivated rabies vaccines, and determined the efficiency of two extraction reagents (i.e., sodium citrate or isopropyl myristate) to separate the vaccine antigens from the antigen/adjuvant complexes. Six partial nucleocapsid (N: 181 bp) and five partial glycoprotein (G: 306 bp) genes were successfully amplified with specific primer sets, which demonstrated that sodium citrate is more efficient than isopropyl myristate in extracting viral RNA from inactivated gel vaccines. In addition, we identified the viral strain of the vaccine by analyzing the nucleotide sequences of the N and the G genes. The nucleotide similarity of the partial N and G genes ranged from 97.1 to 99.4% and from 91.8 to 100% among rabies vaccine strains, respectively, indicating that each manufacturer used different rabies virus strains to produce their vaccines. The molecular method used in this study could also be used to identify viral strains in other inactivated vaccines.
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Figure 1.
Amplification of the N gene from the rabies virus using RNA extracted from sodium citrate-treated antigen (A) and isopropyl myristate-treated antigen (B) with specific primer sets for the N gene. The expected size was 181 bp. M: 100-bp DNA ladder; lane 1-6: company A, B, C, D, E, F, respectively.
Figure 2.
Amplification of the G gene from the rabies virus using RNA extracted from sodium citrate-treated antigen (A) and isopropyl myristate-treated antigen (B) with specific primer sets for the G gene. The expected size was 181 bp. M: 100-bp DNA ladder; lane 1: company A; lane 2: company C; lane 3: company B; lane 4~6: D, E and F, respectively.
Figure 3.
Phylogenetic analysis based on the partial N-gene nucleotide sequences of the vaccine strains and other sequences obtained from the GenBank database. Numbers at each key node indicate the degree of bootstrap support and only those with >70% support are shown.
Figure 4.
Phylogenetic analysis based on the partial G gene nucleotide sequences of the vaccine strains and other sequences obtained from the GenBank database. Numbers at each key node indicate the degree of bootstrap support and only those with >70% support are shown.
Figure 5.
Comparison of the nucleotide sequences of nucleocapsid (A) and glycoprotein (B) genes of the vaccine strains and representative Korean RABV isolates. Dots indicate nucleotides agreeing with the first line sequence.
Table 1.
List of the oligonucleotide primers used for RT-PCR of the rabies vaccine
| Primer | Nucleotide sequences (5′-3′) | Nucleotide positiona | Sense | RABV gene | Size of amplicon (bp) |
|---|---|---|---|---|---|
| RVNDF | GRA ATT GGG CTT TGA CTG GA | 353~372 | + | N | 181 |
| RFNDR | AAA GGG GCT GTC TCG AAA AT | 514~533 | – | ||
| RVGDF | CCC GAG AAT CCG AGA CTA | 595~612 | + | G | 306 |
| RFGDR | CTC TCT CTT CCT GAC CAA CTC | 880~900 | – |
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