The L, M, and S Segments of Rift Valley Fever Virus MP-12 Vaccine Independently Contribute to a Temperature-Sensitive Phenotype

Abstract

Rift Valley fever (RVF) is endemic to Africa, and the mosquito-borne disease is characterized by "abortion storms" in ruminants and by hemorrhagic fever, encephalitis, and blindness in humans. Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) has a tripartite negative-stranded RNA genome (L, M, and S segments). A live-attenuated vaccine for RVF, the MP-12 vaccine, is conditionally licensed for veterinary use in the United States. MP-12 is fully attenuated by the combination of the partially attenuated L, M, and S segments. Temperature sensitivity (ts) limits viral replication at a restrictive temperature and may be involved with viral attenuation. In this study, we aimed to characterize the ts mutations for MP-12. The MP-12 vaccine showed restricted replication at 38°C and replication shutoff (100-fold or greater reduction in virus titer compared to that at 37°C) at 39°C in Vero and MRC-5 cells. Using rZH501 reassortants with either the MP-12 L, M, or S segment, we found that all three segments encode a temperature-sensitive phenotype. However, the ts phenotype of the S segment was weaker than that of the M or L segment. We identified Gn-Y259H, Gc-R1182G, L-V172A, and L-M1244I as major ts mutations for MP-12. The ts mutations in the L segment decreased viral RNA synthesis, while those in the M segment delayed progeny production from infected cells. We also found that a lack of NSs and/or 78kD/NSm protein expression minimally affected the ts phenotype. Our study revealed that MP-12 is a unique vaccine carrying ts mutations in the L, M, and S segments.

Importance: Rift Valley fever (RVF) is a mosquito-borne viral disease endemic to Africa, characterized by high rates of abortion in ruminants and severe diseases in humans. Vaccination is important to prevent the spread of disease, and a live-attenuated MP-12 vaccine is currently the only vaccine with a conditional license in the United States. This study determined the temperature sensitivity (ts) of MP-12 vaccine to understand virologic characteristics. Our study revealed that MP-12 vaccine contains ts mutations independently in the L, M, and S segments and that MP-12 displays a restrictive replication at 38°C.

FIG 1

Schematics of RVFV MP-12 mutations. Mutations of MP-12 vaccine compared to the parental ZH548 strain are shown. The S segment encodes 3 silent mutations and 1 amino acid substitution. The M segment encodes 4 silent mutations and 5 amino acid substitutions, and the L segment encodes 7 silent mutations and 3 amino acid substitutions. Amino acid substitutions are in red. Reported attenuation mutations in the M segment (11) are also indicated.

FIG 2

Restrictive temperatures for MP-12 vaccine and the mutants. Viral growth curve kinetics for MP-12 (A and B), rMP12-ΔNSm21/384 (C and D), or rMP12-C13type (E) in Vero (A, C, and E) or MRC-5 (B and D) cells are shown (MOI, 0.01). Cells were infected with virus at 37°C for 1 h and subsequently incubated at the indicated temperatures. Means and standard deviations from 3 independent experiments are shown.

FIG 3

Temperature sensitivity phenotype of rZH501 and reassortants. Vero cells were infected with rZH501 or virus carrying the MP-12 S segment (RST-MP12-S), the M segment (RST-MP12-M), the L segment (RST-MP12-L) or both the MP-12 S and M segments (RST-MP12-S/M) at 37°C for 1 h (MOI, 0.01). Subsequently, cells were incubated at either 37°C (white) or 41°C (black). Virus titers at 72 hpi (A) and viral S-RNA copy numbers measured by droplet digital PCR at 1 and 72 hpi (B) are shown. The detection limit of the plaque assay is shown by the dotted line (A). Means and standard deviations from 3 independent experiments are shown. Changes (n-fold) in virus titers or S-RNA copy numbers at 41°C compared to 37°C are also shown as log₁₀ values above the bars. Statistically significant differences (versus the 37°C value), determined by Student's unpaired t test, are indicated (*, P < 0.05; **, P < 0.01).

FIG 4

Temperature sensitivity phenotype of rZH501 encoding an MP-12 mutation in the M or L segment. Vero cells were infected with rZH501 encoding an MP-12 mutation in the M (A and B) or L (C and D) segment at 37°C for 1 h (MOI, 0.01). Subsequently, cells were incubated at either 37°C (white) or 41°C (black). Virus titers at 72 hpi (A and C) and viral S-RNA copy numbers measured by droplet digital PCR at 1 and 72 hpi (B and D) are shown. Additionally, rZH501 viruses carrying both U795C and A3564G or lacking nt 21 to 384 (Δ78kD/NSm) in the M segment were also analyzed (A). Means and standard deviations from 3 independent experiments are shown. Changes (n-fold) in virus titers or S-RNA copy numbers at 41°C compared to 37°C are also shown as log₁₀ values above the bars. Statistically significant differences (versus the 37°C value), determined by Student's unpaired t test, are shown (*, P < 0.05; **, P < 0.01).

FIG 5

Functional analysis of temperature sensitivity mutations, L-V172A and L-M1244I. (A) Plaque phenotypes of rZH501 and the same virus encoding V172A or M1244I in VeroE6 cells at 37°C. (B and C) BHK/T7-9 cells were transfected with plasmid carrying an RVFV M segment minigenome including the Renilla luciferase (rLuc) ORF (20, 21) or plasmids expressing RVFV ZH501 N or L, as indicated. For reporter assays, a plasmid encoding firefly luciferase (fLuc) was also transfected to normalize the rLuc activities among samples. After incubation at 37°C or 41°C, total RNA or cell lysates were harvested at 48 h posttransfection. (B) Northern blot images obtained with rLuc RNA probes which detect sense (top) and antisense (bottom) rLuc ORFs. Mv, viral-sense M segment minigenome; Mvc, antiviral-sense M segment minigenome; Primary transcript, RNA transcribed by T7 RNA polymerase, which is not cleaved by HDV ribozyme; rLuc mRNA, rLuc mRNA transcribed by viral L proteins. (C) Minigenome rLuc reporter activities normalized by fLuc activities. Values are averages and standard deviations from three independent experiments. Statistically significant differences, determined by Student's unpaired t test (compared to the value obtained with the minigenome with N and parental L), are indicated (*, P < 0.05; **, P < 0.01). wt, parental ZH501 L protein.

FIG 6

Temperature sensitivity phenotype of rZH501 lacking both NSs and 78kD/NSm. (A and B) Vero cells were infected with rZH501 lacking NSs and 78kD/NSm or rZH501 lacking NSs and 78kD/NSm, both of which also contain MP-12 M and/or L segment mutations, at 37°C for 1 h (MOI, 0.01). Subsequently, cells were incubated at either 37°C or 41°C. Virus titers at 72 hpi (A) and viral S-RNA copy number measured by droplet digital PCR at 1 and 72 hpi (B) are shown. Statistically significant differences, determined by Student's unpaired t test (versus the 37°C value), are indicated (*, P < 0.05; **, P < 0.01). (C) Viral replication kinetics of rZH501 lacking NSs and 78kD/NSm (blue) and rZH501 lacking NSs and 78kD/NSm, which also encodes MP-12 M segment mutations (red), in Vero cells either at 37°C or 41°C (MOI, 5). Changes (n-fold) in virus titers or S-RNA copy numbers at 41°C compared to 37°C are shown as log₁₀ values above the bars. Data are means and standard deviations from 3 independent experiments.

FIG 7

Reversion mutations at V172A and/or M1244I do not induce a temperature-resistant phenotype. Vero cells were infected with rZH501 lacking NSs and 78kD/NSm, encoding L-V172A and/or L-M1244I, rMP-12 lacking NSs and 78kD/NSm, or rMP-12 lacking NSs and 78kD/NSm encoding reversion mutations (L-A172V and/or L-I1244M) at 37°C for 1 h (MOI, 0.01). Cells were further incubated either at 37°C or 41°C. Virus titers at 72 hpi are shown. Changes (n-fold) in virus titers at 41°C compared to 37°C are shown as log₁₀ values above the bars. Data are means and standard deviations from 3 independent experiments.