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. IMPORTANCERift 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.

Rift Valley fever vaccines: an overview of the safety and efficacy of the live-attenuated MP-12 vaccine candidate

“…A total of 23 mutations of the MP-12 strain compared to the parental ZH548 strain are also specified. When individual MP-12 mutations were introduced into the pathogenic ZH501 strain, the resulting mutant viruses displayed attenuation and/or temperature-sensitivity (ts) phenotypes, as summarized in the box under the genome schematics [61,98]. …”

Attenuation of pathogenic Rift Valley fever virus strain through the chimeric S-segment encoding sandfly fever phlebovirus NSs or a dominant-negative PKR

“…Viral loads in livers and spleens were also analyzed by droplet digital PCR (ddPCR) using Taqman probe specific to RVFV S-segment and N mRNA. 20,21 The mean RNA copy numbers in liver samples were 3.0 £ 10 5 (rZH501), 7.3 £ 10 4 (rZH501-TOSNSs), 2.0 £ 10 4 (rZH501-PTANSs), 2.7 £ 10 4 (rZH501-PTBNSs), and 2.8 £ 10 4 (rZH501-DNSm21/ 384) copies per mg total RNA, whereas viral RNA was not detected in liver samples infected with either rZH501-DNSs16/198 or rZH501-SFSNSs (Fig. 3c).…”

“…19 The MP-12 vaccine is attenuated by a combination of mutations within the L-, M-and S-segments, and complete attenuation happens with the combination of all attenuated segments. 20 Moreover, each L-and M-segment encode 2 independent temperature-sensitive mutations, which limit MP-12 replication above 38 C. 21 A potential disadvantage of the MP-12 vaccine is a lack of a DIVA marker (Differentiation of Infected from Vaccinated Animals). 22 Without a DIVA marker, vaccinated animals elicit antibodies toward the MP-12 vaccine strain, which is a similar response to pathogenic RVFV.…”

Attenuation and protective efficacy of Rift Valley fever phlebovirus rMP12-GM50 strain

“…Two amino acid substitutions (Gn-Y259H and Gc-R1182G) in the M-segment are independently responsible for the attenuation [35]. Those two mutations in the M-segment and two other amino acid substitutions (V172A and M1244I) in the L-segments display a temperature-sensitive phenotype, which restricts viral replication at 38°C and above [37]. The attenuation strength of each L-, M-, and S-segment was previously determined as M > L > S, based on the virulence of reassortant ZH501 strains that encode one of either the MP-12 L-, M-, or S-segments.…”