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. Author manuscript; available in PMC: 2022 Mar 7.
Published in final edited form as: Zoonoses. 2022 Jan 13;2:2. doi: 10.15212/zoonoses-2021-0016

Venezuelan Equine Encephalitis Virus V3526 Vaccine RNA-Dependent RNA Polymerase Mutants Increase Vaccine Safety Through Restricted Tissue Tropism in a Murine Model

Clint A Haines 1, Rafael K Campos 1, Sasha R Azar 2, K Lane Warmbrod 2,†, Tiffany F Kautz 1,‡, Naomi L Forrester 2,§,*, Shannan L Rossi 1,2,3,*
PMCID: PMC8900488  NIHMSID: NIHMS1772941  PMID: 35262074

Abstract

Background:

Venezuelan equine encephalitis virus (VEEV) is an arbovirus endemic to the Americas. There are no approved vaccines or antivirals. TC-83 and V3526 are the best-characterized vaccine candidates for VEEV. Both are live-attenuated vaccines and have been associated with safety concerns, albeit less so for V3526. A previous attempt to improve the TC-83 vaccine focused on further attenuating the vaccine by adding mutations that altered the error incorporation rate of the RNA-dependent RNA polymerase (RdRp).

Methods:

The research presented here examines the impact of these RdRp mutations in V3526 by cloning the 3X and 4X strains, assessing vaccine efficacy against challenge in adult female CD-1 mice, examining neutralizing antibody titers, investigating vaccine tissue tropism, and testing the stability of the mutant strains.

Results:

Our results show that the V3526 RdRp mutants exhibited reduced tissue tropism in the spleen and kidney compared to wild-type V3526, while maintaining vaccine efficacy. Illumina sequencing showed that the RdRp mutations could revert to wild-type V3526.

Conclusions:

The observed genotypic reversion is likely of limited concern because wild-type V3526 is still an effective vaccine capable of providing protection. Our results indicate that the V3526 RdRp mutants may be a safer vaccine design than the original V3526.

Keywords: Venezuelan equine encephalitis virus, TC-83, V3526, vaccine, fidelity

Introduction:

Venezuelan equine encephalitis virus (VEEV) is a member of the Alphavirus genus in the family Togaviridae with an 11.5 kb positive-sense single-stranded RNA genome [1, 2]. VEE commonly presents with mild flu-like symptoms, but severe symptoms can occur in approximately 4–14% of human patients [3–6]. Severe symptoms include encephalitis, confusion, coma, photophobia, and seizures [3]. Death as a result of VEE is rare but occurs in approximately 1% of cases depending on patient age and the outbreak [7, 8].

Multiple VEEV outbreaks have occurred across the Central and South America region with the most recent outbreak occurring in Peru in 2006 [9–15]. A Central American outbreak that occurred between 1969–1972 resulted in the deaths of 50,000 equines and 93 humans, and hundreds of additional cases of human disease were reported [13]. The outbreak reached southern Texas in 1971 resulting in 1,500 equine deaths and 110 cases of human disease [12]. Another major outbreak that occurred in Venezuela between 1992–1995 resulted in 4,000 equine deaths and between 75,000–100,000 cases of human disease [14]. The virus tends to recede and emerge in epizootic (outbreak) cycles every 14–20 years to cause disease in humans and equids [9, 14]. Despite this, VEEV is believed to still cause tens of thousands of human infections every year. VEEV is considered a biological risk agent due to high aerosol infectivity, numerous documented infections through laboratory exposure, and its potential for weaponization [16, 17]. The potential for re-emergence coupled with the biological risk factors means that a publicly available vaccine for VEE is needed to protect at-risk populations, healthcare providers, biomedical researchers, and emergency responders in the event of an outbreak.

There are numerous vaccine candidates for VEE, but TC-83 and V3526 are currently the most well-characterized [18, 19]. TC-83 was previously approved for limited use to protect military personnel and researchers, but V3526 has demonstrated improved protection, safety, and reduced chance of reversion or pseudo-reversion to wild type [19–21]. TC-83 was made by serial passaging the Trinidad donkey (TRD) strain of VEEV in fetal guinea pig heart cells 83 times. A single nucleotide polymorphism (SNP) in the 3rd position of the 5’UTR and a substitution in the 120th position of the E2 protein confer the majority of attenuation [18, 22]. Development of V3526 involved site-directed mutagenesis of a TRD cDNA clone to remove the PE2 furin cleavage site and create an E1 protein F253S point mutation [23]. However, there are safety concerns for both vaccine constructs. Vaccination with TC-83 can lead to clinical symptoms in 23% of human recipients, lack of neutralizing antibodies in 18% of recipients, and can potentially be transmitted by a mosquito vector [20, 24]. The safety concerns for V3526 include fever and flu-like symptoms in human recipients, the presence of viral antigen in the brains of vaccinated animals, indicating possible neurovirulence, and the potential for transmission of the virus by a mosquito vector [24–26].

A previous attempt to improve the TC-83 vaccine candidate focused on the use of mutations that altered the error incorporation rate of the RdRp [27–31]. The mutants included TC-83 3X, containing the G8R, E31G, and A90T mutations in the RdRp gene nsP4, and TC-83 4X containing these nsP4 mutations in addition to C482Y which was originally discovered in the related alphavirus, chikungunya virus (CHIKV) [27, 29]. The C482Y substitution has been shown to decrease the RdRp error frequency in CHIKV and is thus hypothesized to be a high-fidelity variant [29]. The research on TC-83 suggested that the 3X and 4X mutants possess reduced virulence in comparison to wild type (WT). Our research examined the impact of these 3X and 4X mutations in the V3526 platform, because V3526 has shown improved protection and safety over TC-83 in animal studies. Additionally, the furin cleavage site deletion should make reversion to WT TRD more difficult [19, 23, 32, 33]. Our study showed that V3526 3X and 4X have restricted tissue tropism in comparison to WT V3526, indicating improved vaccine safety without sacrificing immunogenicity.

Methods:

Animals and Viruses

The V3526, V3526 3X, and V3526 4X virus strains were cloned, in vitro transcribed, and electroporated to make the virus stocks as previously described for the TC-83 RdRp mutants [27]. Viral stocks were quantified using Vero cell plaque assays prior to each experiment to ensure consistent inoculation doses. All work with VEEV 3908 was performed in either BSL3 or ABSL3 laboratories, while all work with V3526 was performed in BSL2 facilities in accordance with UTMB regulations and all relevant state and federal laws.

All mice were ordered at 6-weeks old or as timed pregnant dams from Charles River, and they were housed in the Galveston National Laboratory ABSL2 facilities. CD-1 mice were chosen due to their use in testing for vaccine safety and efficacy and to allow for comparisons between this study and previous research on vaccines for VEE [27, 28, 34, 35]. Work was performed after IACUC approval and following all UTMB regulations and state and federal laws.

Plaque Assay

Plaque assays were performed by seeding Vero (ATCC® CCL-81™) cells at a density of 2×105 cells/well in a 12-well plate using Dulbecco’s modified Eagle medium (DMEM) supplemented with 1% penicillin and streptomycin and 10% fetal bovine serum (FBS). The cells were incubated overnight at 37°C with 5% CO2. Virus samples were serially diluted 10-fold in DMEM supplemented with 1% penicillin and streptomycin and 2% FBS. The plated cells were infected with the diluted viruses and allowed to incubate at 37°C with 5% CO2 for 1 hour with gentle rocking every 15 minutes. The cells were then overlayed with 0.4% agarose in DMEM supplemented with 1% penicillin and streptomycin and 2% FBS and incubated at 37°C with 5% CO2 for 36–48 hours. After this incubation, the cells were fixed with formalin and stained using 0.25% crystal violet. Plaques were counted and multiplied by the dilution factor to determine the final concentration.

Vaccine Efficacy

Adult female CD-1 mice were vaccinated subcutaneously with 104 or 105 PFU of either V3526 parent, V3526 3X, or V3526 4X. Phosphate buffered saline was used as a mock control. Weight and clinical signs of infection were monitored daily for the first 14-days post infection. Serum was collected on day 42 post-vaccination to assess neutralizing antibodies. Mice were challenged subcutaneously with 105 PFU of VEEV 3908 60-days post vaccination. Weight and clinical signs of infection were observed for 11-days post challenge. Survival statistics were performed using a log-rank (Mantel-Cox) test. Weight statistics were performed using Tukey’s multiple comparison test. All statistics were performed using the PRISM 8 software.

Plaque Reduction Neutralization Test (PRNT)

The PRNT50 and PRNT80 assays in this study were performed using standard methodology against VEEV V3526, V3526 3X, and V3526 4X [36]. Murine serum samples were incubated for 1 hour at 56°C for inactivation prior to a 10-fold dilution in 2% FBS and 1% penicillin and streptomycin supplemented Dulbecco’s modified Eagle medium (DMEM) media. The samples were then diluted 2-fold in the supplemented DMEM media from a range of 1:20 to 1:640. The diluted samples were mixed with a known concentration of virus and allowed to incubate for 1 hour. The virus-mixed serum samples were then added to a monolayer of Vero (ATCC® CCL-81™) cells that had been seeded onto a 12-well plate the previous day at a density of 2×105 cells/well. All plates were treated as a standard Vero cell plaque assay after infection. The limit of detection (LOD) was 1:20. Any sample below the limit of detection was set at 1:10 or half the LOD. The highest dilution was 1:640. Any sample above the highest dilution was set at 1:1280 or twice the highest dilution. This was done to differentiate between samples that were at the limits and samples that were past the limits.

Tissue Tropism

Adult female CD-1 mice were infected at 6 weeks of age with 104 PFU of either V3526 parent, V3526 3X, or V3526 4X. Blood and tissue samples were collected at 1-, 2-, and 7-days post vaccination. Full body perfusions were performed on the first two collection days to mitigate the impact of high viremia on tissue titers. Mice were checked daily for weight and clinical signs of infection. Statistical analysis of weight change was performed in the PRISM 8 software using Tukey’s multiple comparison test.

Eppendorf tubes containing 0.5mL of DMEM supplemented with 1% penicillin and streptomycin and 2% FBS and a ball bearing were used to collect the spleen, liver, brain, and kidney. A QIAGEN TissueLyser was utilized to homogenize samples prior to clarification via centrifugation. Vero cell plaque assays were used to titrate samples. Statistical analysis was performed in the PRISM 8 software using Sidak’s multiple comparison test.

Illumina Sequencing

V3526 parent, V3526 3X, and V3526 4X were used to intracranially infect 6-day-old murine pups with 104 PFU of virus in biological duplicates. The number of pups in each group was slightly variable based on the size of the litter, and all pups were randomized. Pup brains were harvested 36–48 hours post-vaccination, and a QIAGEN TissueLyser was used to homogenize the samples. Clarification via centrifugation was performed to remove excess tissue and collect virus. Titrations were performed using a Vero cell plaque assay prior to intracranial injection of the next round of pups out to a total of 5 passages.

RNA isolation was performed on the passage 5 brain homogenate supernatants for V3526, V3526 3X, and V3526 4X using TRIzol™ LS Reagent according to the instructions provided by the manufacturer. The UTMB Next Generation Sequencing Core performed the Illumina sequencing using the NextSeq 550 system. RT-PCR conversion of RNA into double stranded cDNA was used to prepare the sequencing library. Fragmentation of the cDNA was performed prior to ligation of each fragment to an adapter. A glass flow cell was then populated by the adapter ligated fragments, and template clusters were amplified. Reversible adapter-ligated terminator nucleotides were used to sequence each cluster. A previously described pipeline was utilized to assemble reads [37]. Starting plasmid sequences for each vaccine were compared against the sequencing results for identification of major nucleotide variants.

The Illumina sequencing data was analyzed by examining common SNPs that occurred in at least 3 of the 4 RdRp mutants. Only mutations present at or above 0.5% of the sequenced population were considered in the analyses. A list of 22 SNPs was created using these criteria. Each mutation was assessed for percent frequency in the population for each biological replicate tested, gene where the mutation occurred, and amino acid change encoded.

Results:

Vaccine Efficacy

Vaccine efficacy was examined for the V3526 3X and 4X mutants using adult CD-1 outbred mice. CD-1 mice were vaccinated with 105 plaque forming units (PFU) of the V3526 vaccines parent, 3X, or 4X. The post-vaccination survival for each group (Fig. 1A) showed that no animals succumbed after vaccination, as expected. The post-vaccination weights (Fig. 1B) indicated that all animals gained weight over the first 14 days and that there were no significant differences between mutants and the parent strain.

Figure 1: Percent Survival and Percent Weight Change After Vaccination and Challenge of CD-1 Mice.

Figure 1:

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Adult female CD-1 mice received 105 PFU of V3526 Parent, V3526 3x or V3526 4x for vaccination and 105 PFU of VEEV 3908 for challenge. Mice were monitored for A) post vaccination survival and C) post-challenge survival. Weight charts are represented as B) post-vaccination and D) post-challenge. In the post-challenge survival graph **** represents a P-value: <0.0001 when comparing each test group to mock. V3526 3X and 4X are offset from 100% for improved data visualization. In the post-challenge weight graph * represents a P-value: < 0.05 and **** represents a P-value: < 0.0001 when compared to mock at each indicated timepoint (2–5 DPC). A log-rank Mantel-Cox test was used for survival analysis, and Tukey’s multiple comparison test was used for weight analysis. Weight chart error bars represent SEM.

The CD-1 mice were challenged 60 days post-vaccination with 105 PFU of the epizootic VEEV 3908. All vaccinated mice survived challenge whereas unvaccinated (mock) mice all succumbed by day 5 post challenge (Fig. 1C). Further, all vaccinated animals maintained their weight after challenge, while mock-vaccinated animals steadily lost weight until they met the criteria for euthanasia (Fig. 1D). The average weight of mock vaccinated animals was significantly lower than each of the vaccine test groups for days 2–5 post-challenge as measured by Tukey’s multiple comparison test. There were no significant differences between mutants and their respective parent strain post-challenge for animal survival or weight.

Neutralizing Antibody Titers

Neutralizing antibody levels were measured by plaque reduction neutralization tests (PRNTs) using serum from mice vaccinated with either 105 PFU (Fig. 2A, B) or 104 PFU (Fig. 2C, D). PRNT levels in mice vaccinated with 105 PFU of V3526 3X or 4X mutants produced neutralizing antibody titers similar to parental V3526, and no significant differences were detected as measured by Sidak’s multiple comparison test. For the 104 PFU dose, there were no significant differences between the RdRp mutants and the V3526 parent strain. Interestingly, more mice vaccinated with 104 PFU produced titers outside of the assay’s detectable range (>1:640), indicating there may be a dose effect. Given that the lower dose may elicit higher neutralizing antibody titers, this dose was continued for subsequent studies.

Figure 2: Neutralizing Antibody Titers in CD-1 Mice After Vaccination With 105 or 104 PFU.

Figure 2:

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Neutralizing antibody titers were observed in adult female CD-1 mice after vaccination with 105 PFU of V3526 parent, V3526 3X, or V3526 4X for A) 80% plaque reduction and B) 50% plaque reduction. Neutralizing antibody titers were observed after vaccination with 104 PFU of V3526 parent, V3526 3X, or V3526 4X for C) 80% plaque reduction and D) 50% plaque reduction. Highest dilution is set at Log10 = 2.8 (1:640), and LOD is set at Log10 = 1.3 (1:20). No statistical differences were observed via Sidak’s multiple comparison test.

Vaccine Tissue Tropism

Vaccine tissue tropism was examined because previous TC-83 RdRp mutant studies found that the 3X and 4X mutants may have a reduced ability to overcome the host barriers to infection present during tissue dissemination and the establishment of neurovirulence [27]. CD-1 mice were vaccinated with 104 PFU of V3526 parent, 3X, or 4X. Serum, brain, spleen, kidneys, and liver were sampled on days 1, 2 and 7 post-vaccination to measure viral load. Mice were PBS-perfused on the first two days to eliminate potential viremia contamination. The weights for all infected animals (Fig. S1A) averaged between 95–105% of the initial weight over the course of the experiment, similar to the post-vaccination weights seen for the 105 PFU dose, without signs of disease.

Viral loads were undetectable in the serum (Fig. S1B), brain (Fig. 3A) or liver (Fig. 3D) at all timepoints for each of the V3526 vaccines tested. Viral load in the spleen was detectable for the V3526 parent strain on days 1 and 2 post-infection, but the V3526 mutants did not show detectable viral load at any timepoint (Fig. 3B). The V3526 parent strain had a significantly higher viral load when compared to the mutants on the first 2 days post-infection as measured by Sidak’s multiple comparison test. Viral load in the kidney was detectable in one mouse for the V3526 parent strain on the first day post-infection, but there was an undetectable viral load in the spleen at each timepoint for the V3526 mutants (Fig. 3C).

Figure 3: Viral Load in Tissues of Adult Female CD-1 Mice Vaccinated with 104 PFU V3526 parent, 3X, or 4X.

Figure 3:

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Vaccinated mice were monitored for viral load in A) brain, B) spleen, C) kidney, and D) liver at 1-, 2-, and 7-days post-vaccination. LOD was set at 50 PFU or PFU/sample (Log10) of 1.7. **** depicts a P-value = <0.0001 as measured by Sidak’s multiple comparison test when compared to V3526 parent. Error lines represent standard deviation.

Sequencing Vaccine Reversion During Neurovirulence Testing

V3526 and the 3X and 4X RdRp mutants were passaged 5 times in duplicate in neonatal murine pup brains to assess the safety and stability of the mutant constructs. Murine pup brains were chosen for this experiment because they are a highly permissive model that allows the virus to replicate to high titers and mutate rapidly in the process [38, 39]. Illumina sequencing of passage 5 virus was used to examine changes in the mutant repertoire of V3526 and the RdRp mutants over the course of this passaging.

The mutational frequencies for each major nucleotide variant (i.e. >0.5% prevalence) were compiled (Tables S1, S2, S3) and then narrowed (Table 1) for SNPs that occurred in at least three of the four RdRp mutant samples. Common mutations were found in all genetic regions except for the 5’UTR, capsid, and 6K genes. The 3 mutations C5724G, G5794A, and A5970G correspond to the R8G, G31E, and T90A genetic reversions to the V3526 parent strain genotype in the nsP4 region, respectively. The T7208C mutation corresponds to a silent substitution in the nsP4 protein coding region. The mutation A7147G was present in both replicates of the V3526 4X strain encoding a Y482C genetic reversion in the nsP4 coding region. Each of these substitutions, including the silent mutation, represent a genotypic reversion to the V3526 parent strain from the fidelity variant strains after serial passage in murine pup brains. All other major nucleotide variants found in Table 1 were present in at least one of the V3526 parent strain replicates.

Table 1:

Common mutations in the V3526 3X and 4X RdRp mutants compared to mutations in parental V3526. The criterion for including a major mutation in this table is presence in at least 3 of the 4 sequenced fidelity variants. Nucleotide position of SNPs is represented as “Position”. Nucleotide present in the starting plasmid is represented as “Reference”. Nucleotide present in Illumina sequencing data represented as “Mutation”. Percentage of mutation present in each Illumina sequenced population depicted as “Freq.” followed by name of sample. Gene where mutation occurred depicted as “Gene”. Substitution that occurred as a result of the mutation depicted as “Amino Acid Change”.

Commonly Observed Mutations
Position Reference Mutation Freq. Parent (A) Freq. Parent (B) Freq. 3X (A) Freq. 3X (B) Freq. 4X (A) Freq. 4X (B) Gene Amino Acid Change
1545 C T 98.02% 0.00% 99.93% 99.61% 97.82% 0.00% nsP1 R501C
1696 C A 99.72% 0.00% 99.91% 99.80% 98.10% 0.00% nsP2 A16D
5275 A G 99.35% 0.00% 99.94% 99.78% 98.00% 0.00% nsP3 H415R
5724* C G 0.00% 0.00% 100.00% 99.81% 98.30% 0.00% nsP4 R8G*
5794* G A 0.00% 0.00% 99.98% 99.85% 98.25% 0.00% nsP4 G31E*
5970* A G 0.00% 0.00% 99.97% 99.78% 97.97% 1.85% nsP4 T90A*
7208* T C 0.00% 0.00% 99.90% 99.70% 97.95% 0.00% nsP4 Silent
8549 G A 18.68% 0.00% 19.27% 19.76% 16.41% 0.00% E3 G55R
8552 T A 16.15% 0.00% 15.56% 15.11% 14.43% 0.00% E2 S1T
8572 G T 99.51% 1.36% 99.95% 99.62% 97.58% 77.76% E2 K7N
8804 C T 99.70% 0.00% 99.88% 99.70% 97.39% 0.00% E2 H85Y
8910 C G 99.70% 0.00% 99.95% 99.77% 97.77% 0.00% E2 T120R
9061 A G 99.73% 0.00% 99.95% 99.70% 97.54% 0.00% E2 Silent
9126 T A 99.77% 0.00% 99.94% 99.71% 97.45% 0.00% E2 V192D
9267 T A 99.69% 0.00% 99.94% 99.72% 97.80% 0.00% E2 I239N
9438 C T 99.76% 0.00% 99.92% 99.76% 98.03% 0.00% E2 T296I
9475 T C 99.80% 0.00% 99.95% 99.75% 97.86% 0.00% E2 Silent
9519 G A 99.56% 0.00% 99.78% 99.58% 97.30% 0.00% E2 G323E
10469 T A 99.71% 0.00% 99.93% 99.71% 97.98% 0.00% E1 L161I
10621 A T 99.73% 0.00% 99.94% 99.66% 97.73% 0.00% E1 Silent
10746 C T 99.70% 0.00% 99.90% 99.74% 98.01% 0.00% E1 S253F
11373 C T 0.95% 0.00% 0.79% 0.78% 1.09% 0.00% 3’UTR N/A
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*

in the Position column denotes a mutation that was present in the RdRp mutants but absent in the V3526 parent strain.

*

in the Amino Acid Change column represents a genotypic reversion to resemble V3526 parent strain.

Discussion:

V3526 and TC-83 are two of the most well-characterized vaccine candidates for VEE [18, 19]. TC-83 is currently the only vaccine candidate ever approved for limited military use, but V3526 has demonstrated similar or improved protection over TC-83 in previous animal studies [19, 32, 33]. The presence of a furin cleavage site deletion in V3526 means that reversion to wild-type TRD should be harder than it would be for the two SNPs that are associated with the attenuation observed in TC-83 [23]. Neither vaccine candidate has been approved for public use and there is limited accessibility to TC-83 vaccination for researchers and military personnel since the only distributor is the Special Immunizations Program [40]. Therefore, it is critical to improve the V3526 vaccine candidate safety. In this paper, we accomplished this by adding the 3X and 4X RdRp mutations previously described in TC-83 and CHIKV to develop a vaccine candidate that showed evidence of increased safety by a reduction in tissue tropism without sacrificing immunogenicity as measured by PRNTs [27].

Neutralizing antibody titers are a widely recognized correlate of protection for evaluating alphavirus vaccines [41–43]. As a result, PRNT80 titers are often discussed when evaluating the VEE vaccine candidates [17, 27, 44]. V3526 RdRp mutants produced titers equivalent to the parent strain after inoculation with 104 or 105 PFU of each construct for both PRNT80 and PRNT50. However, the PRNT80 results after inoculation with 104 PFU had more mice producing titers above the highest dilution of 1:640 than after inoculation with 105 PFU. Samples above 1:640 were not titrated beyond this cutoff, so it is possible that the titers could be even higher than shown in this study. Three mice had titers below the limit of detection for the PRNT80 after inoculation with 104 PFU, but the PRNT50 data demonstrate that all animals seroconverted. The neutralizing antibody data indicate that vaccination with the V3526 mutants produce neutralizing antibody titers equivalent to V3526 parent strain.

Antigens for V3526 have previously been detected in the brain and olfactory mucosa via immunohistochemistry, and undetectable viremia was previously described in multiple animal models [19, 25, 44, 45]. Accordingly, our study also failed to detect viremia in mice vaccinated with 104 PFU of any of the V3526 vaccines, which suggests that the vaccines cannot be transmitted to a mosquito vector [24, 46]. Viremia following TC-83 vaccination may have resulted in unintentional vaccine transmission among mosquito populations in the past, so this gives more support to the use of the V3526 vaccine over the TC-83 vaccine in the future [47]. Low viral titers were previously reported in the liver of C3H/HeN mice, so the undetectable liver viral load in our study was an expected result [48]. Undetectable viral loads in the brain indicates that the incorporated RdRp mutations do not result in neuroinvasion after subcutaneous injection. Reduced tissue tropism for the 3X and 4X mutants in the spleen and kidney on the first two days post vaccination may indicate further organ tropism restriction while still maintaining efficacy, resulting in a safer vaccine design than the V3526 parent strain. Some limitations of this study are the lack of data on viral load in additional lymphoid tissues and no description of the histopathological changes in the organs tested. These studies, as well as determining the capacity for mosquitoes to vector these vaccines, represent future studies.

Serially passaging virus in murine pup brains is a technique that allows viruses to replicate to high titers quickly while acquiring mutations that may occur during an infection [38, 39]. This technique is used to assess vaccine stability and screen for mutations with the potential for reversion or pseudo-reversion to wild type VEEV [39]. The Illumina sequencing data from this study shows a genotypic reversion of the 3X and 4X mutants encoding G8R, E31G, A90T, and C482Y back to the V3526 parent strain genotype after 5 passages in murine pup brains. However, the genotypic reversion is likely of limited concern since parental V3526 is still an effective vaccine capable of preventing disease [19]. The genotypic reversions to the parent strain may be explained by the fact that murine pup brains are a highly permissive model that allow the virus to grow with little selective pressure from a host immune system, which was enhanced by each passage performed prior to sequencing [38, 39]. The reduction in tissue tropism observed following vaccination with the mutants in the adult CD-1 mouse model suggests the risk of vaccines genotypically reverting in a healthy adult is low. This would be an interesting question to pursue in the future.

The Illumina sequencing data from this study also revealed a large difference in the mutations present between the biological replicates of V3526 and V3526 4X. Variations in the mutation profile of the two virus strains could be explained by a founder effect resulting from the high mutation rate associated with alphaviruses [49]. All three viral strains were rescued from cDNA clones in an effort to minimize founder effect and start the murine pup brain passaging from a well-defined stock. However, V3526 and the 3X and 4X RdRp mutants were rescued in cell culture before experimentation began which may have led to genetic diversity between replicates.

VEEV’s status as an agent of biological concern coupled with the increased risk of re-emergence and the impact of global warming on vector borne disease means that a publicly available vaccine for VEE is important for improving biosafety and biosecurity [50]. The research presented here represents an improvement on the V3526 vaccine candidate through the inclusion of RdRp mutations, which lead to a reduction in virus tissue tropism while maintaining the efficacy of the parent strain. Future work will focus upon increasing the stability of these mutations. These data provide further evidence that RdRp mutants can be used to create safer vaccines against alphavirus-caused diseases.

Supplementary Material

Figure S1
1

Acknowledgements

The data reported here were part of CAH Thesis entitled “Determining the Impact of Fidelity Mutations in the RNA-Dependent RNA Polymerase of V3526 Vaccine on Tissue Dissemination and Intrahost Variation.” This project was supported by an NIH R01 AI125902.

Footnotes

Conflicts of Interest

The authors have no Conflicts of Interest to declare.

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