Cationic Peptides and Peptidomimetics Bind Glycosaminoglycans as Potential Sema3A Pathway Inhibitors

Semaphorin3A (Sema3A) is a vertebrate-secreted protein that was initially characterized as a repulsive-guidance cue. Semaphorins have crucial roles in several diseases; therefore, the development of Sema3A inhibitors is of therapeutic interest. Sema3A interacts with glycosaminoglycans (GAGs), presumably through its C-terminal basic region. We used different biophysical techniques (i.e., NMR, surface plasmon resonance, isothermal titration calorimetry, fluorescence, and UV-visible spectroscopy) to characterize the binding of two Sema3A C-terminus-derived basic peptides (FS2 and NFS3) to heparin and chondroitin sulfate A. We found that these peptides bind to both GAGs with affinities in the low-micromolar range. On the other hand, a peptoid named SICHI (semaphorin-induced chemorepulsion inhibitor), which is positively charged at physiological pH, was first identified by our group as being able to block Sema3A chemorepulsion and growth-cone collapse in axons at the extracellular level. To elucidate the direct target for the reported SICHI inhibitory effect in the Sema3A signaling pathway, we looked first to the protein-protein interaction between secreted Sema3A and the Nrp1 receptor. However, our results show that SICHI does not bind directly to the Sema3A sema domain or to Nrp1 extracellular domains. We evaluated a new, to our knowledge, hypothesis, according to which SICHI binds to GAGs, thereby perturbing the Sema3A-GAG interaction. By using the above-mentioned techniques, we observed that SICHI binds to GAGs and competes with Sema3A C-terminus-derived basic peptides for binding to GAGs. These data support the ability of SICHI to block the biologically relevant interaction between Sema3A and GAGs, thus revealing SICHI as a new, to our knowledge, class of inhibitors that target the GAG-protein interaction.     

Genetic and Phylogenetic Characterization of a Chikungunya Virus Imported into Shenzhen,China

正Dear Editor,Chikungunya virus (CHIKV) is a mosquito-borne virus belonging to the family Togaviridae, genus Alphavirus, and was first isolated in Tanzania in the 1950s (Silva and Dermody 2017; Weaver and Lecuit 2015). Human infec-     

Glycosaminoglycans from marine sources as therapeutic agents

Glycosaminoglycans (GAGs) in marine animals are different to those of terrestrial organisms, mainly in terms of molecular weight and sulfation. The therapeutic properties of GAGs are related to their ability to interact with proteins, which is very much influenced by sulfation position and patterns. Since currently GAGs cannot be chemically synthesized, they are sourced from natural products, with high intra- but also inter-species variability, in terms of chain length, disaccharide composition and sulfation pattern. Consequently, sulfated GAGs are the most interesting molecules in the marine environment and constitute the focus of the present review. In particular, chondroitin sulfate (CS) appears as the most promising compound. CS-E chains [GlcA-GalNAc(4S,6S)] extracted from squid possess antiviral and anti-metastatic activities and seem to impart signalling properties and improve the mechanical performance of cartilage engineering constructs; Squid CS-E and octopus CS-K [GlcA(3S)-GalNAc(4S)], dermatan sulfate (DS) from sea squirts [-iK units, IdoA(3S)-GalNAc(4S)] and sea urchins [-iE units, IdoA-GalNAc(4S,6S)] and hybrids CS/DS from sharks (-B/iB [GlcA/IdoA(2S)-GalNAc(4S)], -D/iD [GlcA/IdoA(2S)-GalNAc(6S)] and –E/iE units [GlcA/IdoA-GalNAc(4S,6S)]) promote neurite outgrowth and could be valuable materials for nerve regeneration. Also displaying antiviral and anti-metastatic properties, a rare CS with fucosylated branches isolated from sea cucumbers is an anticoagulant and anti-inflammatory agent. In this same line, marine heparin extracted from shrimp and sea squirt has proven anti-inflammatory properties, with the added advantage of decreased risk of bleeding because of its low anticoagulant activity.     

Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 from Cattle in Argentina have Hypervirulent-Like Phenotypes

The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina.     

Proteoglycans Act as Cellular Hepatitis Delta Virus Attachment Receptors

The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved.     

Interferon Response Factors 3 and 7 Protect against Chikungunya Virus Hemorrhagic Fever and Shock

Chikungunya virus (CHIKV) infections can produce severe disease and mortality. Here we show that CHIKV infection of adult mice deficient in interferon response factors 3 and 7 (IRF3/7(-/-)) is lethal. Mortality was associated with undetectable levels of alpha/beta interferon (IFN-α/β) in serum, ～50- and ～10-fold increases in levels of IFN-γ and tumor necrosis factor (TNF), respectively, increased virus replication, edema, vasculitis, hemorrhage, fever followed by hypothermia, oliguria, thrombocytopenia, and raised hematocrits. These features are consistent with hemorrhagic shock and were also evident in infected IFN-α/β receptor-deficient mice. In situ hybridization suggested CHIKV infection of endothelium, fibroblasts, skeletal muscle, mononuclear cells, chondrocytes, and keratinocytes in IRF3/7(-/-) mice; all but the latter two stained positive in wild-type mice. Vaccination protected IRF3/7(-/-) mice, suggesting that defective antibody responses were not responsible for mortality. IPS-1- and TRIF-dependent pathways were primarily responsible for IFN-α/β induction, with IRF7 being upregulated >100-fold in infected wild-type mice. These studies suggest that inadequate IFN-α/β responses following virus infection can be sufficient to induce hemorrhagic fever and shock, a finding with implications for understanding severe CHIKV disease and dengue hemorrhagic fever/dengue shock syndrome.     

Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation

Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms.     

Host Immune Response to Mosquito-Transmitted Chikungunya Virus Differs from That Elicited by Needle Inoculated Virus

Background

Mosquito-borne diseases are a worldwide public health threat. Mosquitoes transmit viruses or parasites during feeding, along with salivary proteins that modulate host responses to facilitate both blood feeding and pathogen transmission. Understanding these earliest events in mosquito transmission of arboviruses by mosquitoes is essential for development and assessment of rational vaccine and treatment strategies. In this report, we compared host immune responses to chikungunya virus (CHIKV) transmission by (1) mosquito bite, or (2) by needle inoculation.

Methods and Findings

Differential cytokine expression was measured using quantitative real-time RT-PCR, at sites of uninfected mosquito bites, CHIKV-infected mosquito bites, and needle-inoculated CHIKV. Both uninfected and CHIKV infected mosquitoes polarized host cytokine response to a T_H2 profile. Compared to uninfected mosquito bites, expression of IL-4 induced by CHIKV-infected mosquitoes were 150 fold and 527.1 fold higher at 3 hours post feeding (hpf) and 6 hpf, respectively. A significant suppression of T_H1 cytokines and TLR-3 was also observed. These significant differences may result from variation in the composition of uninfected and CHIKV-infected mosquito saliva. Needle injected CHIKV induced a robust interferon-γ, no detectable IL-4, and a significant up-regulation of TLR-3.

Conclusions

This report describes the first analysis of cutaneous cytokines in mice bitten by CHIKV–infected mosquitoes. Our data demonstrate contrasting immune activation in the response to CHIKV infection by mosquito bite or needle inoculation. The significant role of mosquito saliva in these earliest events of CHIKV transmission and infection are highlighted.     

Respiratory Syncytial Virus Grown in Vero Cells Contains a Truncated Attachment Protein That Alters Its Infectivity and Dependence on Glycosaminoglycans

Human respiratory syncytial virus (RSV) contains a heavily glycosylated 90-kDa attachment glycoprotein (G). Infection of HEp-2 and Vero cells in culture depends largely on virion G protein binding to cell surface glycosaminoglycans (GAGs). This GAG-dependent phenotype has been described for RSV grown in HEp-2 cells, but we have found that it is greatly reduced by a single passage in Vero cells. Virions produced from Vero cells primarily display a 55-kDa G glycoprotein. This smaller G protein represents a post-Golgi compartment form that is lacking its C terminus, indicating that the C terminus is required for GAG dependency. Vero cell-grown virus infected primary well-differentiated human airway epithelial (HAE) cell cultures 600-fold less efficiently than did HEp-2 cell-grown virus, indicating that the C terminus of the G protein is also required for virus attachment to this model of the in vivo target cells. This reduced infectivity for HAE cell cultures is not likely to be due to the loss of GAG attachment since heparan sulfate, the primary GAG used by RSV for attachment to HEp-2 cells, is not detectable at the apical surface of HAE cell cultures where RSV enters. Growing RSV stocks in Vero cells could dramatically reduce the initial infection of the respiratory tract in animal models or in volunteers receiving attenuated virus vaccines, thereby reducing the efficiency of infection or the efficacy of the vaccine.Human respiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus in the family Paramyxoviridae, subfamily Pneumovirinae. RSV causes mild respiratory disease in all age groups, but the disease can be severe or fatal in infants and the elderly (4, 9, 11). Initial attempts to produce a killed vaccine were not successful, resulting instead in enhanced disease upon infection (26, 41). Efforts to produce a live attenuated vaccine are ongoing (6, 7, 51).RSV produces three glycoproteins which are important for infection. The largest glycoprotein (G) is involved in attachment to the host cell (35), the fusion (F) glycoprotein mediates virion membrane fusion with the target cell membrane (2), and the small hydrophobic (SH) glycoprotein may attenuate apoptosis (15). The F protein is the only glycoprotein that is absolutely required for infection of cultured immortalized cells (27, 45) and syncytium formation, the most obvious cytopathic effect of RSV in immortalized cell culture. Although the G protein is not absolutely required for infection, it enhances infection and syncytium formation (45). The G protein attaches to cultured, immortalized cell lines (35) primarily via glycosaminoglycans (GAGs) on the cell surface (13, 22, 23, 30). GAGs are repeating disaccharide units of hexuronic acid and hexosamine that form unbranched polysaccharide chains and are found on the surface of most mammalian cells. The GAG type that appears most important for RSV infection of HEp-2 cells is heparan sulfate (HS) (23, 30).The G protein is a type II integral membrane protein with its N terminus on the cytoplasmic side of the membrane and its C terminus as the extracellular ectodomain (49). An unglycosylated region in the center of the protein contains four cysteines held together by disulfide bonds in a cysteine noose (19, 24, 33), followed, to the C-terminal side, by a predicted heparin-binding domain (HBD) (12, 13). The 32-kDa G protein, while in the endoplasmic reticulum (ER), is modified by the addition of multiple N-linked carbohydrate chains, depending on the strain. These N-linked additions would increase the molecular mass of G to 45 to 60 kDa. Previous reports have found G protein forms of this size in cells and in virions at low levels (5, 20, 21, 50). All of these reports suggest that these smaller forms of the G protein are partially glycosylated processing intermediates.Maturation of the N-linked carbohydrates of the G protein occurs in the Golgi compartment, where a large number of O-linked carbohydrate chains are added, resulting in an 84- to 92-kDa mature protein (14, 32, 35, 49). This size variation of the G protein is probably due, in part, to the difficulty in sizing heavily glycosylated molecules and variations in molecular mass markers.The G protein shares no homology with the glycoproteins of paramyxoviruses outside the Pneumovirinae subfamily. The high serine and threonine content and the high O-linked glycosylation levels are similar to those found in mucins. The amount of O-linked glycosylation is partially dependent on the cell type used to produce the virus (18).In the present study, we examined virus produced in HEp-2 and Vero cells, which are both commonly used to grow RSV in the laboratory, for dependence on GAGs by the ability to infect cells expressing GAG or deficient in GAG expression. We also examined the ability of the viruses to infect primary, well-differentiated human airway epithelial (HAE) cell cultures. In both systems, infectivity was greatly dependent upon the cell line used to grow the virus. Biochemical characterization of purified virus grown in these two cell lines revealed a smaller form of the RSV G protein in virions from Vero cells. Using C terminus-specific antibodies and a six-His tag at the C terminus of the G protein, we determined that the smaller G protein form was lacking its C terminus. These results highlight the importance of the C-terminal portion of the G protein and suggest that the cell line used to produce a virus can alter its infectivity.     

Genetic Relatedness among Hepatitis A Virus Strains Associated with Food-Borne Outbreaks

The genetic characterization of hepatitis A virus (HAV) strains is commonly accomplished by sequencing subgenomic regions, such as the VP1/P2B junction. HAV genome is not extensively variable, thus presenting opportunity for sharing sequences of subgenomic regions among genetically unrelated isolates. The degree of misrepresentation of phylogenetic relationships by subgenomic regions is especially important for tracking transmissions. Here, we analyzed whole-genome (WG) sequences of 101 HAV strains identified from 4 major multi-state, food-borne outbreaks of hepatitis A in the Unites States and from 14 non-outbreak-related HAV strains that shared identical VP1/P2B sequences with the outbreak strains. Although HAV strains with an identical VP1/P2B sequence were specific to each outbreak, WG were different, with genetic diversity reaching 0.31% (mean 0.09%). Evaluation of different subgenomic regions did not identify any other section of the HAV genome that could accurately represent phylogenetic relationships observed using WG sequences. The identification of 2–3 dominant HAV strains in 3 out of 4 outbreaks indicates contamination of the implicated food items with a heterogeneous HAV population. However, analysis of intra-host HAV variants from eight patients involved in one outbreak showed that only a single sequence variant established infection in each patient. Four non-outbreak strains were found closely related to strains from 2 outbreaks, whereas ten were genetically different from the outbreak strains. Thus, accurate tracking of HAV strains can be accomplished using HAV WG sequences, while short subgenomic regions are useful for identification of transmissions only among cases with known epidemiological association.     

Sulfated Fibroin,a Novel Sulfated Peptide Derived from Silk,Inhibits Human Immunodeficiency Virus Replication in Vitro

We prepared two kinds of sulfated silk fibroins, SclFib30 and SclFib31, which contain different amounts of sulfate. These sulfated silk fibroins have anti-HIV-1 activity in vitro, apparently due to interference with the adsorption of virus particles to CD4⁺ cells, and completely blocked virus binding to the cells at a concentration of 100 μg/ml. Sulfated fibroins also abolished cell-to-cell infection-induced syncytium formation upon cocultivation of MOLT-4 and MOLT-4/HIV-1_IIIB cells, suggesting that they would interfere with gp120 and prevent the formation of gp120/CD4 complex. Silk is used in biomaterials such as surgical sutures and is believed to be a safe material for humans. In accordance with low anticoagulant activity and high anti-HIV-1 activity against both X4 HIV-1 and R5 HIV-1 strains, sulfated silk fibroins have potential as antiviral material such for a vaginal anti-HIV formulation.     

渤海湾两株H2亚型禽流感病毒的 遗传进化分析

野鸟是禽流感病毒的自然储存库,病毒可以随着宿主的迁徙传播给其他野鸟与家禽。渤海湾是鸟类南北迁徙的重要停歇地,也是东亚-澳大利西亚鸟类迁徙通道的重要组成部分,每年有大量水鸟在渤海湾停歇,促进了禽流感病毒的传播。为了解渤海湾地区禽流感病毒的传播及进化与水鸟迁徙的相关性,2018年春季鸟类迁徙期间的4和5月份,在渤海湾采集鸻鹬类粪便样品2 120份,对样品进行检测,分离出2株H2亚型禽流感病毒。对这2株H2亚型禽流感病毒进行了分子特征及遗传进化分析,并结合渤海湾水鸟的环志回收数据,对H2亚型病毒的重组及遗传进化与水鸟迁徙的联系进行了分析。结果表明,2株分离株的HA蛋白裂解位点符合低致病性禽流感病毒的分子特征,它们的8个基因片段同源性均不高,其中879-H2N7的8个基因片段分别与我国福建和澳大利亚的毒株同源性最高,遗传关系最近;854-H2N8的8个基因片段分别与我国湖南以及日本、韩国、孟加拉国和越南的毒株同源性最高,遗传关系最近。渤海湾水鸟的环志回收数据分析表明,879-H2N7随着野鸟的迁徙在渤海湾、福建沿海和澳大利亚之间进行传播与扩散;854-H2N8可能跨越东亚-澳大利西亚和中亚-印度两条通道之间进行基因重组和进化,并会随着鸟类迁徙进行传播和扩散。     

Genotypic and Phenotypic Characterization of Chikungunya Virus of Different Genotypes from Malaysia

Background

Mosquito-borne Chikungunya virus (CHIKV) has recently re-emerged globally. The epidemic East/Central/South African (ECSA) strains have spread for the first time to Asia, which previously only had endemic Asian strains. In Malaysia, the ECSA strain caused an extensive nationwide outbreak in 2008, while the Asian strains only caused limited outbreaks prior to this. To gain insight into these observed epidemiological differences, we compared genotypic and phenotypic characteristics of CHIKV of Asian and ECSA genotypes isolated in Malaysia.

Methods and Findings

CHIKV of Asian and ECSA genotypes were isolated from patients during outbreaks in Bagan Panchor in 2006, and Johor in 2008. Sequencing of the CHIKV strains revealed 96.8% amino acid similarity, including an unusual 7 residue deletion in the nsP3 protein of the Asian strain. CHIKV replication in cells and Aedes mosquitoes was measured by virus titration. There were no differences in mammalian cell lines. The ECSA strain reached significantly higher titres in Ae. albopictus cells (C6/36). Both CHIKV strains infected Ae. albopictus mosquitoes at a higher rate than Ae. aegypti, but when compared to each other, the ECSA strain had much higher midgut infection and replication, and salivary gland dissemination, while the Asian strain infected Ae. aegypti at higher rates.

Conclusions

The greater ability of the ECSA strain to replicate in Ae. albopictus may explain why it spread far more quickly and extensively in humans in Malaysia than the Asian strain ever did, particularly in rural areas where Ae. albopictus predominates. Intergenotypic genetic differences were found at E1, E2, and nsP3 sites previously reported to be determinants of host adaptability in alphaviruses. Transmission of CHIKV in humans is influenced by virus strain and vector species, which has implications for regions with more than one circulating CHIKV genotype and Aedes species.     

In Vitro Anti-influenza Virus Activities of Sulfated Polysaccharide Fractions from Gracilaria lemaneiformis

In this paper, in vitro anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were investigated. Cytotoxicities and antiviral activities of Gracilaria lemaneiformis polysaccharides (PGL), Gracilaria lemaneiformis polysaccharide fraction-1 (GL-1), Gracilaria lemaneiformis polysaccharide fraction-2 (GL-2) and Gracilaria lemaneiformis polysaccharide fraction-3 (GL-3) were studied by the Methyl thiazolyl tetrazolium (MTT) method, and the inhibitory effect against Human influenza virus H1-364 induced cytopathic effect (CPE) on MDCK cells were observed by the CPE method. In addition, the antiviral mechanism of PGL was explored by Plaque forming unit (PFU), MTT and CPE methods. The results showed: i) Cytotoxicities were not significantly revealed, and H1-364 induced CPE was also reduced treated with sulfated polysaccharide fractions from Gracilaria lemaneiformis; ii) Antiviral activities were associated with the mass percentage content of sulfate groups in polysaccharide fractions, which was about 13%, in polysaccharides (PGL and GL-2) both of which exhibited higher antiviral activity; iii) A potential antiviral mechanism to explain these observations is that viral adsorption and replication on host cells were inhibited by sulfated polysaccharides from Gracilaria lemaneiformis. In conclusion, Anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were revealed, and the antiviral activities were associated with content of sulfate groups in polysaccharide fractions.     

Genetic Subpopulations of Rift Valley Fever Virus Strains ZH548 and MP-12 and Recombinant MP-12 Strains

Rift Valley fever virus strain MP-12 was generated by serial plaque passages of parental strain ZH548 12 times in MRC-5 cells in the presence of a chemical mutagen, 5-fluorouracil. As a result, MP-12 encoded 4, 9, and 10 mutations in the S, M, and L segments, respectively. Among them, mutations in the M and L segments were responsible for attenuation, while the MP-12 S segment still encoded a virulent phenotype. We performed high-throughput sequencing of MP-12 vaccine, ZH548, and recombinant MP-12 (rMP-12) viruses. We found that rMP-12 contains very low numbers of viral subpopulations, while MP-12 and ZH548 contain 2 to 4 times more viral genetic subpopulations than rMP-12. MP-12 genetic subpopulations did not encode the ZH548 sequence at the 23 MP-12 consensus mutations. On the other hand, 4 and 2 mutations in M and L segments of MP-12 were found in ZH548 subpopulations. Thus, those 6 mutations were no longer MP-12-specific mutations. ZH548 encoded several unique mutations compared to other Egyptian strains, i.e., strains ZH501, ZH1776, and ZS6365. ZH548 subpopulations shared nucleotides at the mutation site common with those in the Egyptian strains, while MP-12 subpopulations did not share those nucleotides. Thus, MP-12 retains unique genetic subpopulations and has no evidence of reversion to the ZH548 sequence in the subpopulations. This study provides the first information regarding the genetic subpopulations of RVFV and shows the genetic stability of the MP-12 vaccine manufactured in MRC-5 cells.