Vaccines are the major control measure to protect animals from infection with RVFV and preventing transmission of infection to humans. In Egypt, inactivated RVFV vaccine is produced by Veterinary Serum and Vaccines Research Institute (VSVRI), from the wild-type ZH-501 strain (El-Nimr, 1980). The vaccine is licensed for national use to vaccinate livestock in Egypt. Periodical analysis of the vaccine seed virus, following OIE guidelines for conventional vaccines production (OIE, 2018), is required to check for virus identity genetic stability. Previous studies analyzed the master seed of VSVRI vaccine virus nucleotide/amino acids sequences at different genome loci (Atwa et al., 2011; Abo Hatab et al., 2019). In this study, we used the same fragment of the GN ectodomain (eGN) which has been used before by Atwa et al. (2011) to analyze the identity and stability of the ZH501-TC adapted strain which evolved from the parental ZH501-VSVRI vaccine virus after ten years of vaccine production and adaptation in tissue culture.
Although, RVFV neutralizing antibodies are elicited against the dimeric surface glycoproteins GN and GC which are involved in virus attachment and entry and encoded by the viral M segment. The GC is imbedded in the viral envelope bilayer forming the iscosahedral scaffold and the ridges between capsomers, whereas the GN ectodomain (eGN) forms the protrusion spikes exposed to IgG antibodies (Schmaljohn et al., 1989; Rusu et al., 2012 and Wu et al., 2017). This highlights the importance of this fragment for analyzing its stability in the tissue culture adapted vaccine strain, ZH501-TC, used for vaccine production in Egypt.
A truncated 714 bases M segment fragment (nt. 612 – nt. 1325) nucleotide sequence, partially covering the eGN of the TC adapted ZH-501 strain (MZ218760), was analyzed in this study. Although the TC adapted strain showed close genetic relation to the virulent ZH501 strain in phylogenetic analysis, analysis of nucleotide sequence revealed two sites of nucleotides substitutions; C1033T and A1206C, corresponding to two amino acids changes; T287I and S336R, at the M segment of the TC adapted ZH-501 strain (MZ218760) compared to the parental virulent RVFV isolate ZH501 Egy Sh 77 (DQ380200). These mutations evolved in the parental vaccine strain ZH-501-VSVRI sequence (GU953292), as reported before by Atwa et al. (2011), and are stable by passaging in tissue culture for vaccine production as obtained for the TC adapted ZH-501 strain (MZ218760).
In our current study, we report a missense nucleotide substitution A1312G corresponding to the amino acid mutation N371S, in the ZH501-TC (MZ218760) compared to the parental virulent RVFV isolate ZH501 Egy Sh 77 (DQ380200) and other sequences published in the Gen Bank (Fig. 2 and Fig. 4). Unfortunately, that stretch of eGn sequence is not covered by the sequence published for the parental vaccine strain ZH-501-VSVRI (GU953292) (Atwa et al., 2011) despite using the same primers set for M segment fragment amplification. We assume this missense mutation is evolved in the parental virulent RVFV isolate ZH501 Egy Sh 77 (DQ380200) due to passaging in tissue culture. This assumption comes in accordance with the results obtained by Lokugamage et al. (2017), where potential risk of reversion mutation at the L-G3750A temperature-sensitive mutation after excessive viral passages in Vero culture cells was proved. Moreover, Moutailler al. (2011) reported deletion of virulence factors encoded sequences at the NSs gene at the 15th serial passage onwards in BHK21 cells.
RVFV Gn 3D structure was predicted by Rusu et al. (2012) as a mixture of α- helical, ß-strands, and coil secondary structural elements where the Gn ectodomain (eGn) was rich in ß-strands than the transmembrane (TMD) and cytoplasmic tail domains (CTD). We have analyzed the protein folding structure of that stretch of eGn using Protein Homology/analogy Recognition (PHYR2) Engine V2.0 for protein modeling, prediction and analysis (Kelley et al., 2015). It is noteworthy that the missense mutation [ASN]N371S[SER] (Fig. 4) enhanced ß sheet formation in ZH501-TC 367AQYASAYCS375 motif (Fig. 4), which might increase antigenicity of the eGn protein and hence increasing antigenicity of ZH501-TC vaccine strain.
Ikegami et al., (2015) identified a combination of three amino acid changes; two in M segment (Y259H [Gn] and R1182G [Gc]) and one in L segment (R1029K) to induce full attenuation of the wild type ZH-501 strain driving the MP12 vaccine strain. In addition, in vivo attenuation of RVFV was demonstrated in swiss albino mice through a single, P82I, amino acid mutation in the NSs protein (Borrego et al., 2021). At the same context, RVFV mutants lacking the M segment non-structural protein, Nsm, was strongly attenuated in mice (Kreher, et al., 2014).
At this study, pathogenicity test in mice was performed to investigate whether the aforementioned missense mutations and changes in the Gn protein folding affected the virulence of the tissue culture adapted ZH501-TC strain (MZ218760) in comparison to the parental virulent RVFV isolate ZH501 Egy Sh 77 (DQ380200) or not. Here we denote that the aforementioned mutations at the M segment T287I, S336R, and N371R did not affect virus virulence in Swiss albino mice, as fast progression of the disease and death of all tested mice was observed, indicating keeping of virulence of the tissue culture adapted vaccine virus strain (ZH501-TC) (MZ218760). This result comes in accordance with the high homology between ZH501-TC and the parental virulent strains isolated in 1977–1979. ZH501-TC strain was clustered with the parental ancestral virulent strains of ZH-501 isolated from Human, Bovine, Ovine and Mosquito in Egypt during 1977–1979 time period. The allocation of ZH501-TC strain at the A designated lineage (Fig. 2) indicating the genomic stability of ZH-501-TC vaccine strain and compatibility for vaccine preparation.
In addition, Atwa et al. (2011) mentioned other one missense nucleotide mutation; A1252G corresponding to the amino acid mutation K351R, and two other silent nucleotide substitutions; T1257A and G1258C, in ZH-501-VSVRI (GU953292) M segment; compared to the virulent parental RVFV isolated in 1977, ZH501 Egy Sh 77 (DQ380200). The TC adapted ZH-501 strain (MZ218760), did not show any nucleotide substitutions at the aforementioned three positions.In parallel, the chromatogram obtained for the tissue culture adapted vaccine seed virus strain (ZH501-TC) (MZ218760) showed clear sharp peaks for that part of the sequence (Fig. 3A). This misleading sequence reading could be explained by sequence artifacts at the end of sequence read of the truncated M segment fragment obtained by Atwa et al. (2011) as shown in Fig. 3B. Noteworthy, Atwa et al. (2011) obtained a shorter truncated sequence of 586 bases (GU953292), but we have obtained 714 bases (MZ218760) nucleotide sequence fragment of the eGn, despite using the same primer set indicating a better quality of the sequencing reaction.
In conclusion, reanalysis and data recall for the eGn partial nucleotide sequence of the TC adapted ZH501 seed vaccine virus confirmed the stability of the nucleotide mutations C1033T and A1206C, corresponding to two amino acids changes; T287I and S336R, by passaging in tissue culture cells. Here, we report a missense nucleotide substitution A1312G corresponding to the amino acid mutation N371S, in the ZH501-TC vaccine strain (MZ218760). This mutation enhanced ß sheet formation at the eGn antigenic motif 367AQYASAYCS375 which might increase antigenicity of the TC adapted seed virus vaccine. Despite these mutations at the eGn, the TC adapted ZH501 kept its virulence when infected mice. In addition, we resolved the incorrectness of the previously published two nucleotide mutations at the same fragment of eGn. These results confirms the compatibility of ZH501-TC virus for inactivated RVFV vaccine production.