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

ABSTRACT: Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Arabian Peninsula that affects sheep, cattle, goats, camels, and humans. Effective vaccination of susceptible ruminants is important for the prevention of RVF outbreaks. Live-attenuated RVF vaccines are in general highly immunogenic in ruminants, whereas residual virulence might be a concern for vulnerable populations. It is also important for live-attenuated strains to encode unique genetic markers for the differentiation from wild-type RVFV strains. In this study, we aimed to strengthen the attenuation profile of the MP-12 vaccine strain via the introduction of 584 silent mutations. To minimize the impact on protective efficacy, codon usage and codon pair bias were not de-optimized. The resulting rMP12-GM50 strain showed 100% protective efficacy with a single intramuscular dose, raising a 1:853 mean titer of plaque reduction neutralization test. Moreover, outbred mice infected with one of three pathogenic reassortant ZH501 strains, which encoded rMP12-GM50 L-, M-, or S-segments, showed 90%, 50%, or 30% survival, respectively. These results indicate that attenuation of the rMP12-GM50 strain is significantly attenuated via the L-, M-, and S-segments. Recombinant RVFV vaccine strains encoding similar silent mutations will be also useful for the surveillance of reassortant strains derived from vaccine strains in endemic countries.

SUBMITTER: Ly HJ

PROVIDER: S-EPMC5696010 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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

Ly Hoai J HJ Nishiyama Shoko S Lokugamage Nandadeva N Smith Jennifer K JK Zhang Lihong L Perez David D Juelich Terry L TL Freiberg Alexander N AN Ikegami Tetsuro T

Vaccine 20171020 48 Pt B

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Arabian Peninsula that affects sheep, cattle, goats, camels, and humans. Effective vaccination of susceptible ruminants is important for the prevention of RVF outbreaks. Live-attenuated RVF vaccines are in general highly immunogenic in ruminants, whereas residual virulence might be a concern for vulnerable populations. It is also important for live-attenuated strains to encode unique genetic markers for the di ...[more]

PMID: 29061350

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Project description:Rift Valley fever (RVF) in ungulates and humans is caused by a mosquito-borne RVF phlebovirus (RVFV). Live attenuated vaccines are used in livestock (sheep and cattle) to control RVF in endemic regions during outbreaks. The ability of two or more different RVFV strains to reassort when co-infecting a host cell is a significant veterinary and public health concern due to the potential emergence of newly reassorted viruses, since reassortment of RVFVs has been documented in nature and in experimental infection studies. Due to the very limited information regarding the frequency and dynamics of RVFV reassortment, we evaluated the efficiency of RVFV reassortment in sheep, a natural host for this zoonotic pathogen. Co-infection experiments were performed, first in vitro in sheep-derived cells, and subsequently in vivo in sheep. Two RVFV co-infection groups were evaluated: group I consisted of co-infection with two wild-type (WT) RVFV strains, Kenya 128B-15 (Ken06) and Saudi Arabia SA01-1322 (SA01), while group II consisted of co-infection with the live attenuated virus (LAV) vaccine strain MP-12 and a WT strain, Ken06. In the in vitro experiments, the virus supernatants were collected 24 h post-infection. In the in vivo experiments, clinical signs were monitored, and blood and tissues were collected at various time points up to nine days post-challenge for analyses. Cell culture supernatants and samples from sheep were processed, and plaque-isolated viruses were genotyped to determine reassortment frequency. Our results show that RVFV reassortment is more efficient in co-infected sheep-derived cells compared to co-infected sheep. In vitro, the reassortment frequencies reached 37.9% for the group I co-infected cells and 25.4% for the group II co-infected cells. In contrast, we detected just 1.7% reassortant viruses from group I sheep co-infected with the two WT strains, while no reassortants were detected from group II sheep co-infected with the WT and LAV strains. The results indicate that RVFV reassortment occurs at a lower frequency in vivo in sheep when compared to in vitro conditions in sheep-derived cells. Further studies are needed to better understand the implications of RVFV reassortment in relation to virulence and transmission dynamics in the host and the vector. The knowledge learned from these studies on reassortment is important for understanding the dynamics of RVFV evolution.

Project description:Rift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV infection, host factors affecting susceptibility are not well understood. To identify the host factors or genes essential for RVFV replication, we conducted CRISPR-Cas9 knockout screening in human A549 cells. We then validated the putative genes using siRNA-mediated knock-downs and CRISPR-Cas9-mediated knock-out studies. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers were analyzed using plaque assay or TCID50 assay. We identified approximately 900 genes with potential involvement in RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knock-downs revealed that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of the LRP1 gene in RVFV replication was previously described in detail. WDR7 knockout A549 cell lines were generated and used to dissect the effect of WRD7 on a bunyavirus, RVFV, and an orthobunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knockout cells on both intracellular RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24 h) when compared with the LACV replication, which was affected in an earlier replication phase (12 h). In summary, we identified WDR7 as an essential host factor for the replication of two different viruses, RVFV and LACV, both of which belong to the Bunyavirales order. Future studies will investigate the mechanistic role through which WDR7 facilitates phlebovirus replication.

Dataset Information

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

Publications

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

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