In this study, we screened CEC-adapted RABV strain CTNCEC25 from the CNT-1 V strain through serial passages in CECs. The virus titer change indicated that the CTN-1 V strain gradually adapted to propagation in CECs following the passages. During this adaptation process, some of the viral characteristics changed. The most prominent was that the passage of the virus in CECs reduces its pathogenicity in adult mice. In adult mice, the CTNCEC25 strain was totally nonpathogenic at the highest tested dose (as high as 107.9 FFU/ml) after passage level 20. Conversely, the immunogenicity of the CTNCEC25 strain increased during the mouse immunization test along with the passage series in CECs, and the protection index reached up to 120,000 at passage 45, which was much higher than that of the parental CTN-1 V strain and the standard for the Pharmacopoeia of the People’s Republic of China (2010), Volume III. The NIH potency of inactivated vaccines without further concentration and based on the CTNCEC25 strain reached as high as 6.65 IU/ml, indicating that the immunogenicity of the CTNCEC25 strain is sufficient to permit inactivated vaccine production.
The G protein is the most relevant component of the RABV because of its multiple functions in the RABV replication cycle, such as its attachment to host cells, low pH-dependent membrane fusion, viral virulence [18–20] and for eliciting the production of neutralizing antibodies, etc. . Previous studies have demonstrated that the presence of Lys/Arg-333 in mature G proteins in RABV is essential for the lethality of the RABV strain in adult mice [18, 22, 23]. Our results indicated the Arg-333 in the CTN-1 V strain was mutated to Gln-333 during serial passages in CECs (Table 1). Accordingly, the pathogenicity of the CTNCEC25 strain also decreased greatly and totally lost viral virulence in adult mice totally after passage level 20 (Figure 1). However, it was noted that the amino acid mutation at position 333 and the pathogenicity reduction were not synchronous during CTN-1 V adaptation to CECs. The amino acid substitution of Arg-333 to Gln-333 was first detected at passage 6 in CECs, and the virulence of the CTNCEC25 strain started to decrease at passage 11; the strain became totally apathogenic to adult mice after passage 20. The delayed effect of mutating Arg-333 on the virulence of CTNCEC25 may be explained from two aspects. First, the Gln-333 substitution strain required natural screening through several passages to be purified because the gene mutations of all viral particles may not occur at the same time. Second, viral reproduction was improving more and more in CECs following the serial passages, which could be verified by the increasing virus titers from passage 6 to 10 (Figure 1). Therefore, the impact of increasing the virus titer may offset some of the pathogenicity reduction effects of the virus solution.
The mutation of Lys-147 to Glu-147, which is another amino acid substitutions observed in the CTNCEC25 strain, may affect the function of the virus. Previous studies have implicated the mutations of Lys-147 to Gln-147 mutation in mature G proteins in RABV for reducing its pathogenicity after i.m. inoculation of the virus into adult mice. Christophe Prehaud et al. 1988 also found that mutations in position aa 147 conferred partial or total resistance to most MAbs that recognized antigenic site II, suggesting this amino acid may be associated with the viral immunogenicity [24–28]. In our study, the CTNCEC25 strain had amino acid substitutions at position aa 147 in passage 30. However, the direct pathogenicity reduction of CTNCEC25 at passage 30 for adult mice could not be detected because the virus already had lost its pathogenicity for adult mice at passage 20, which could have occurred possibly because of a Gln-333 substitution. However, the immunogenicity effect of the mutation was obvious. The protection index increased to 4,266 at passage 30 from 600 at passage 25, and it drastically increased up to more than 10,000 after passage 33.
Likewise, amino acids at positions 34, 164, 182, 198, 200, 205, 210, 242, 255, 268, and 303 of mature G protein have been found to be associated with the pathogenicity of RABV strains in adult mice [22, 24, 29, 30]. However, none of these virulence-associated aa residues were changed during the cell culture adaptation of the CTN-1 V strain into the CTNCEC25 strain.
In addition to the two RABV pathogenicity-associated amino acids mutations mentioned above, there were five other amino acid substitutions in proteins G, L and M in the CTNCEC25 strain. Following passage in the CECs, these five amino acids were gradually changed. Among these changes, the amino acid substitutions at positions 389 and 485 of the G protein reverted to the same ones found at the corresponding genome positions in some other cell culture-adapted strains, such as strain PV and SAG2, suggesting that these changes might be involved in adapting to cell culture. Amino acid 1602 of the L protein may not be conserved because the mutation was found at this position in some other cell culture-adapted strain and street isolates. Two other aa site substitutions, namely aa 421 of the G protein and aa 99 of the M protein, were not found at the same genome positions in street rabies virus isolates and other vaccine strains, and their function requires further study.
In conclusion, we have successfully obtained a CEC-adapted RABV CTNCEC25 strain from the CTN-1 V strain by serial passage in CECs. The new adapted strain CTNCEC25 lost virulence in adult mice but retains its high immunogenicity and high propagation rate in cultured cells, which make it an ideal candidate for inactivated human vaccine production.