Evaluation of Exploratory Fluid Biomarker Results from a Phase 1 Senolytic Trial in Mild Alzheimer's Disease.

Senescent cell accumulation contributes to the progression of age-related disorders including Alzheimer's disease (AD). Clinical trials evaluating senolytics, drugs that clear senescent cells, are underway, but lack standardized outcome measures. Our team recently published data from the first open-label trial to evaluate senolytics (dasatinib plus quercetin) in AD. After 12-weeks of intermittent treatment, we reported brain exposure to dasatinib, favorable safety and tolerability, and modest post-treatment changes in cerebrospinal fluid (CSF) inflammatory and AD biomarkers using commercially available assays. Herein, we present more comprehensive exploratory analyses of senolytic associated changes in AD relevant proteins, metabolites, lipids, and transcripts measured across blood, CSF, and urine. These analyses included mass spectrometry for precise quantification of amyloid beta (Aß) and tau in CSF; immunoassays to assess senescence associated secretory factors in plasma, CSF, and urine; mass spectrometry analysis of urinary metabolites and lipids in blood and CSF; and transcriptomic analyses relevant to chronic stress measured in peripheral blood cells. Levels of Aß and tau species remained stable. Targeted cytokine and chemokine analyses revealed treatment-associated increases in inflammatory plasma fractalkine and MMP-7 and CSF IL-6. Urinary metabolites remained unchanged. Modest treatment-associated lipid profile changes suggestive of decreased inflammation were observed both peripherally and centrally. Blood transcriptomic analysis indicated downregulation of inflammatory genes including FOS, FOSB, IL1ß, IL8, JUN, JUNB, PTGS2. These data provide a foundation for developing standardized outcome measures across senolytic studies and indicate distinct biofluid-specific signatures that will require validation in future studies. ClinicalTrials.gov: NCT04063124.

Spatial proteomics of hippocampal subfield-specific pathology in Alzheimer's disease and primary age-related tauopathy.

INTRODUCTION: Alzheimer's disease (AD) and primary age-related tauopathy (PART) both harbor 3R/4R hyperphosphorylated-tau (p-tau)-positive neurofibrillary tangles (NFTs) but differ in the spatial p-tau development in the hippocampus. METHODS: Using Nanostring GeoMx Digital Spatial Profiling, we compared protein expression within hippocampal subregions in NFT-bearing and non-NFT-bearing neurons in AD (n = 7) and PART (n = 7) subjects. RESULTS: Proteomic measures of synaptic health were inversely correlated with the subregional p-tau burden in AD and PART, and there were numerous differences in proteins involved in proteostasis, amyloid beta (Aß) processing, inflammation, microglia, oxidative stress, and neuronal/synaptic health between AD and PART and between definite PART and possible PART. DISCUSSION: These results suggest subfield-specific proteome differences that may explain some of the differences in Aß and p-tau distribution and apparent pathogenicity. In addition, hippocampal neurons in possible PART may have more in common with AD than with definite PART, highlighting the importance of Aß in the pathologic process. HIGHLIGHTS: Synaptic health is inversely correlated with local p-tau burden. The proteome of NFT- and non-NFT-bearing neurons is influenced by the presence of Aß in the hippocampus. Neurons in possible PART cases share more proteomic similarities with neurons in ADNC than they do with neurons in definite PART cases.

Demographic and Clinical Characteristics Associated With Serum GFAP Levels in an Ethnically Diverse Cohort.

BACKGROUND AND OBJECTIVES: Elevations in circulating glial fibrillary acidic protein (GFAP), a putative marker of reactive astrocytosis, have been found to associate with cognitive decline and dementia status. Further validation in diverse cohorts and evaluation of potential health disparities are necessary for broader generalization. The goal of this study was to examine the associations between demographics, cardiovascular risk factors, and APOE ε4 status with serum GFAP levels among Mexican American and non-Hispanic White older adults across the continuum from cognitively unimpaired to Alzheimer disease dementia. METHODS: Serum GFAP levels were assayed using a Simoa HD-1 analyzer in older adults enrolled in the observational Texas Alzheimer Research and Care Consortium. Associations between demographic and clinical characteristics with serum GFAP levels were evaluated using linear regression. The diagnostic accuracy of serum GFAP was further examined using area under the receiver operating characteristic curves (AUROC) in univariate and adjusted models, and optimal cut points were derived using the maximum Kolmogorov-Smirnov metric. All models were also stratified by ethnicity and disease stage. RESULTS: A total of 1,156 Mexican American and 587 non-Hispanic White participants were included (mean age = 68 years, standard deviation = 10; 65% female). Older age (ß = 0.562 (95% CI 0.515-0.609), p < 0.001), apolipoprotein ε4 status (ß = 0.139 (95% CI 0.092-0.186), p < 0.001), and cognitive impairment (ß = 0.150 (95% CI 0.103-0.197), p < 0.001) were positively associated with serum GFAP. By contrast, higher body mass index (ß = -0.181 (95% CI -0.228 to -0.134), p < 0.001), diabetes (ß = -0.065 (95% CI -0.112 to -0.018), p < 0.001), and tobacco use (ß = -0.059 (95% CI -0.106 to -0.012), p < 0.001) were inversely associated with serum GFAP. AUROC values were generally comparable across ethnicities and model fit improved with inclusion of additional covariates. However, optimal cut-off values were consistently lower in Mexican Americans relative to non-Hispanic White participants. DISCUSSION: The study results highlight the importance of understanding the role of broader demographic and clinical factors on circulating GFAP levels within diverse cohorts to enhance precision across clinical, research, and community settings.

Senolytic therapy in mild Alzheimer's disease: a phase 1 feasibility trial.

Cellular senescence contributes to Alzheimer's disease (AD) pathogenesis. An open-label, proof-of-concept, phase I clinical trial of orally delivered senolytic therapy, dasatinib (D) and quercetin (Q), was conducted in early-stage symptomatic patients with AD to assess central nervous system (CNS) penetrance, safety, feasibility and efficacy. Five participants (mean age = 76 + 5 years; 40% female) completed the 12-week pilot study. D and Q levels in blood increased in all participants (12.7-73.5 ng ml-1 for D and 3.29-26.3 ng ml-1 for Q). In cerebrospinal fluid (CSF), D levels were detected in four participants (80%) ranging from 0.281 to 0.536 ml-1 with a CSF to plasma ratio of 0.422-0.919%; Q was not detected. The treatment was well-tolerated, with no early discontinuation. Secondary cognitive and neuroimaging endpoints did not significantly differ from baseline to post-treatment further supporting a favorable safety profile. CSF levels of interleukin-6 (IL-6) and glial fibrillary acidic protein (GFAP) increased (t(4) = 3.913, P = 0.008 and t(4) = 3.354, P = 0.028, respectively) with trending decreases in senescence-related cytokines and chemokines, and a trend toward higher Aß42 levels (t(4) = -2.338, P = 0.079). In summary, CNS penetrance of D was observed with outcomes supporting safety, tolerability and feasibility in patients with AD. Biomarker data provided mechanistic insights of senolytic effects that need to be confirmed in fully powered, placebo-controlled studies. ClinicalTrials.gov identifier: NCT04063124 .

Association of MRI Visible Perivascular Spaces and Neurofilament Light Chain: The Framingham Heart Study.

BACKGROUND: Neurofilament light chain (NfL) is a marker of neuronal injury. Perivascular spaces (PVS) visible on magnetic resonance imaging (MRI) represent cerebral small vessel disease (CSVD) but their role as markers of neuronal injury needs further clarification. OBJECTIVE: To relate PVS burden according to brain topography and plasma NfL. METHODS: Framingham Heart Study (FHS) participants with brain MRI and NfL measurements were included. PVS were rated in the basal ganglia (BG) and centrum semiovale (CSO) using validated methods and categorized based on counts. A mixed region variable representing high burden PVS in either BG or CSO was assessed. Multivariable linear regression analyses were used to relate PVS burden to log-transformed NfL levels in models adjusted for age, sex, FHS cohort, time between MRI and clinic exam, and image view (model 1), vascular risk factors (model 2), and white matter hyperintensity volume, covert brain infarcts, and cerebral microbleeds (model 3). RESULTS: Among 1,457 participants (68.1±8.5 years, 45% males), NfL levels increased with higher PVS burden. Multivariable analysis showed an association of high PVS burden strictly in BG with NfL (ß= 0.117, 95% CI 0.014-0.221; p = 0.027), but attenuated in model 3. The associations were mainly in participants≥65 years (ß= 0.122, 95% CI 0.015-0.229, p = 0.026), women (ß= 0.156, 95% CI 0.024-0.288, p = 0.021), and APOE ɛ4 non-carriers (ß= 0.140, 95% CI 0.017-0.263, p = 0.026). CONCLUSIONS: The association of strictly BG high PVS burden with NfL suggests a role for PVS as markers of neuroaxonal injury, but our results are hypothesis generating and require further replication.

Cerebral small vessel disease burden is associated with decreased abundance of gut Barnesiella intestinihominis bacterium in the Framingham Heart Study.

A bidirectional communication exists between the brain and the gut, in which the gut microbiota influences cognitive function and vice-versa. Gut dysbiosis has been linked to several diseases, including Alzheimer's disease and related dementias (ADRD). However, the relationship between gut dysbiosis and markers of cerebral small vessel disease (cSVD), a major contributor to ADRD, is unknown. In this cross-sectional study, we examined the connection between the gut microbiome, cognitive, and neuroimaging markers of cSVD in the Framingham Heart Study (FHS). Markers of cSVD included white matter hyperintensities (WMH), peak width of skeletonized mean diffusivity (PSMD), and executive function (EF), estimated as the difference between the trail-making tests B and A. We included 972 FHS participants with MRI scans, neurocognitive measures, and stool samples and quantified the gut microbiota composition using 16S rRNA sequencing. We used multivariable association and differential abundance analyses adjusting for age, sex, BMI, and education level to estimate the association between gut microbiota and WMH, PSMD, and EF measures. Our results suggest an increased abundance of Pseudobutyrivibrio and Ruminococcus genera was associated with lower WMH and PSMD (p values < 0.001), as well as better executive function (p values < 0.01). In addition, in both differential and multivariable analyses, we found that the gram-negative bacterium Barnesiella intestinihominis was strongly associated with markers indicating a higher cSVD burden. Finally, functional analyses using PICRUSt implicated various KEGG pathways, including microbial quorum sensing, AMP/GMP-activated protein kinase, phenylpyruvate, and ß-hydroxybutyrate production previously associated with cognitive performance and dementia. Our study provides important insights into the association between the gut microbiome and cSVD, but further studies are needed to replicate the findings.

Senolytic therapy to modulate the progression of Alzheimer's Disease (SToMP-AD) - Outcomes from the first clinical trial of senolytic therapy for Alzheimer's disease.

Cellular senescence has been identified as a pathological mechanism linked to tau and amyloid beta (Aß) accumulation in mouse models of Alzheimer's disease (AD). Clearance of senescent cells using the senolytic compounds dasatinib (D) and quercetin (Q) reduced neuropathological burden and improved clinically relevant outcomes in the mice. Herein, we conducted a vanguard open-label clinical trial of senolytic therapy for AD with the primary aim of evaluating central nervous system (CNS) penetrance, as well as exploratory data collection relevant to safety, feasibility, and efficacy. Participants with early-stage symptomatic AD were enrolled in an open-label, 12-week pilot study of intermittent orally-delivered D+Q. CNS penetrance was assessed by evaluating drug levels in cerebrospinal fluid (CSF) using high performance liquid chromatography with tandem mass spectrometry. Safety was continuously monitored with adverse event reporting, vitals, and laboratory work. Cognition, neuroimaging, and plasma and CSF biomarkers were assessed at baseline and post-treatment. Five participants (mean age: 76±5 years; 40% female) completed the trial. The treatment increased D and Q levels in the blood of all participants ranging from 12.7 to 73.5 ng/ml for D and 3.29-26.30 ng/ml for Q. D levels were detected in the CSF of four participants ranging from 0.281 to 0.536 ng/ml (t(4)=3.123, p=0.035); Q was not detected. Treatment was well-tolerated with no early discontinuation and six mild to moderate adverse events occurring across the study. Cognitive and neuroimaging endpoints did not significantly differ from baseline to post-treatment. CNS levels of IL-6 and GFAP increased from baseline to post-treatment (t(4)=3.913, p=008 and t(4)=3.354, p=0.028, respectively) concomitant with decreased levels of several cytokines and chemokines associated with senescence, and a trend toward higher levels of Aß42 (t(4)=-2.338, p=0.079). Collectively the data indicate the CNS penetrance of D and provide preliminary support for the safety, tolerability, and feasibility of the intervention and suggest that astrocytes and Aß may be particularly responsive to the treatment. While early results are promising, fully powered, placebo-controlled studies are needed to evaluate the potential of AD modification with the novel approach of targeting cellular senescence.

A population-based meta-analysis of circulating GFAP for cognition and dementia risk.

OBJECTIVE: Expression of glial fibrillary acidic protein (GFAP), a marker of reactive astrocytosis, colocalizes with neuropathology in the brain. Blood levels of GFAP have been associated with cognitive decline and dementia status. However, further examinations at a population-based level are necessary to broaden generalizability to community settings. METHODS: Circulating GFAP levels were assayed using a Simoa HD-1 analyzer in 4338 adults without prevalent dementia from four longitudinal community-based cohort studies. The associations between GFAP levels with general cognition, total brain volume, and hippocampal volume were evaluated with separate linear regression models in each cohort with adjustment for age, sex, education, race, diabetes, systolic blood pressure, antihypertensive medication, body mass index, apolipoprotein E ε4 status, site, and time between GFAP blood draw and the outcome. Associations with incident all-cause and Alzheimer's disease dementia were evaluated with adjusted Cox proportional hazard models. Meta-analysis was performed on the estimates derived from each cohort using random-effects models. RESULTS: Meta-analyses indicated that higher circulating GFAP associated with lower general cognition (ß = -0.09, [95% confidence interval [CI]: -0.15 to -0.03], p = 0.005), but not with total brain or hippocampal volume (p > 0.05). However, each standard deviation unit increase in log-transformed GFAP levels was significantly associated with a 2.5-fold higher risk of incident all-cause dementia (Hazard Ratio [HR]: 2.47 (95% CI: 1.52-4.01)) and Alzheimer's disease dementia (HR: 2.54 [95% CI: 1.42-4.53]) over up to 15-years of follow-up. INTERPRETATION: Results support the potential role of circulating GFAP levels for aiding dementia risk prediction and improving clinical trial stratification in community settings.

Plasma biomarkers predict cognitive trajectories in an ethnoracially and clinically diverse cohort: Mediation with hippocampal volume.

Introduction: We examine whether the association between key plasma biomarkers (amyloid ß [aß] 42/40, total tau (t-tau), neurofilament light [NfL]) and cognitive trajectories (executive function [EF] and episodic memory [EM]) is mediated through neurodegeneration. Methods: All participants were recruited from the University of California, Davis-Alzheimer's Disease Research Center (n = 473; baseline age range = 49-95 years, 60% women). We applied an accelerated longitudinal design to test latent growth models for EF and EM, and path and mediation analyses. Age was centered at 75 years, and all models were adjusted for sex, education, and ethnicity. Results: HV differentially mediated the association aß 42/40 and NfL on EF and EM level and change. Hippocampal volume (HV) did not mediate the association between t-tau and cognitive performance. Discussion: Neurodegeneration as represented with HV selectively mediates the association between key non-invasive plasma biomarkers and cognitive trajectories in an ethnoracially and clinically diverse community-based sample.

Meta-analysis of genome-wide association studies identifies ancestry-specific associations underlying circulating total tau levels.

Circulating total-tau levels can be used as an endophenotype to identify genetic risk factors for tauopathies and related neurological disorders. Here, we confirmed and better characterized the association of the 17q21 MAPT locus with circulating total-tau in 14,721 European participants and identified three novel loci in 953 African American participants (4q31, 5p13, and 6q25) at P < 5 × 10-8. We additionally detected 14 novel loci at P < 5 × 10-7, specific to either Europeans or African Americans. Using whole-exome sequence data in 2,279 European participants, we identified ten genes associated with circulating total-tau when aggregating rare variants. Our genetic study sheds light on genes reported to be associated with neurological diseases including stroke, Alzheimer's, and Parkinson's (F5, MAP1B, and BCAS3), with Alzheimer's pathological hallmarks (ADAMTS12, IL15, and FHIT), or with an important function in the brain (PARD3, ELFN2, UBASH3B, SLIT3, and NSD3), and suggests that the genetic architecture of circulating total-tau may differ according to ancestry.

Blood biomarkers for cognitive decline and clinical progression in a Mexican American cohort.

Introduction: The clinical translation of biofluid markers for dementia requires validation in diverse cohorts. The study goal was to evaluate if blood biomarkers reflecting diverse pathophysiological processes predict disease progression in Mexican American adults. Methods: Mexican American adults (n = 745), 50 years of age and older, completed annual assessments over a mean of 4 years. Serum collected at baseline was assayed for total tau, neurofilament light (NFL), ubiquitin carboxyl-terminal hydrolase LI, glial fibrillary acidic protein (GFAP), soluble cluster of differentiation 14 (sCD14), and chitinase-3-like protein 1 (YKL-40). Results: Higher GFAP and NFL were associated with global cognitive decline. Only GFAP was associated with increased incident dementia risk (hazard ratio: 1.611 (95% confidence interval: 1.204-2.155)) and inclusion of additional biomarkers did not improve model fit. Discussion: Among a panel of six blood biomarkers previously associated with neurodegenerative disease, only GFAP predicted incident dementia in our cohort. The findings suggest that blood GFAP levels may aid dementia-risk prediction among Mexican American adults.

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

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.

Negeviruses Reduce Replication of Alphaviruses during Coinfection.

Negeviruses are a group of insect-specific viruses (ISVs) that have been found in many arthropods. Their presence in important vector species led us to examine their interactions with arboviruses during coinfections. Wild-type negeviruses reduced the replication of several alphaviruses during coinfections in mosquito cells. Negev virus (NEGV) isolates were also used to express green fluorescent protein (GFP) and anti-chikungunya virus (CHIKV) antibody fragments during coinfections with CHIKV. NEGV expressing anti-CHIKV antibody fragments was able to further reduce replication of CHIKV during coinfections, while reductions of CHIKV with NEGV expressing GFP were similar to titers with wild-type NEGV alone. These results are the first to show that negeviruses induce superinfection exclusion of arboviruses and to demonstrate a novel approach to deliver antiviral antibody fragments with paratransgenic ISVs. The ability to inhibit arbovirus replication and express exogenous proteins in mosquito cells makes negeviruses a promising platform for control of arthropod-borne pathogens. IMPORTANCE Negeviruses are a group of insect-specific viruses (ISVs), viruses known to infect only insects. They have been discovered over a wide geographical and species range. Their ability to infect mosquito species that transmit dangerous arboviruses makes negeviruses a candidate for a pathogen control platform. Coinfections of mosquito cells with a negevirus and an alphavirus demonstrated that negeviruses can inhibit the replication of alphaviruses. Additionally, modifying Negev virus (NEGV) to express a fragment of an anti-CHIKV antibody further reduced the replication of CHIKV in coinfected cells. This is the first evidence to demonstrate that negeviruses can inhibit the replication of important arboviruses in mosquito cells. The ability of a modified NEGV to drive the expression of antiviral proteins also highlights a method for negeviruses to target specific pathogens and limit the incidence of vector-borne diseases.

A Low Fidelity Virus Shows Increased Recombination during the Removal of an Alphavirus Reporter Gene.

Reporter genes for RNA viruses are well-known to be unstable due to putative RNA recombination events that excise inserted nucleic acids. RNA recombination has been demonstrated to be co-regulated with replication fidelity in alphaviruses, but it is unknown how recombination events at the minority variant level act, which is important for vaccine and trans-gene delivery design. Therefore, we sought to characterize the removal of a reporter gene by a low-fidelity alphavirus mutant over multiple replication cycles. To examine this, GFP was inserted into TC-83, a live-attenuated vaccine for the alphavirus Venezuelan equine encephalitis virus, as well as a low-fidelity variant of TC-83, and passaged until fluorescence was no longer observed. Short-read RNA sequencing using ClickSeq was performed to determine which regions of the viral genome underwent recombination and how this changed over multiple replication cycles. A rapid removal of the GFP gene was observed, where minority variants in the virus population accumulated small deletions that increased in size over the course of passaging. Eventually, these small deletions merged to fully remove the GFP gene. The removal was significantly enhanced during the passaging of low-fidelity TC-83, suggesting that increased levels of recombination are a defining characteristic of this mutant.

Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles.

Mutations are incorporated into the genomes of RNA viruses at an optimal frequency and altering this precise frequency has been proposed as a strategy to create live-attenuated vaccines. However, determining the effect of specific mutations that alter fidelity has been difficult because of the rapid selection of the virus population during replication. By deleting residues of the structural polyprotein PE2 cleavage site, E3D56-59, in Venezuelan equine encephalitis virus (VEEV) TC-83 vaccine strain, non-infectious virus particles were used to assess the effect of single mutations on mutation frequency without the interference of selection that results from multiple replication cycles. Next-generation sequencing analysis revealed a significantly lower frequency of transversion mutations and overall mutation frequency for the fidelity mutants compared to VEEV TC-83 E3D56-59. We demonstrate that deletion of the PE2 cleavage site halts virus infection while making the virus particles available for downstream sequencing. The conservation of the site will allow the evaluation of suspected fidelity mutants across alphaviruses of medical importance.

Viral RNA-dependent RNA polymerase mutants display an altered mutation spectrum resulting in attenuation in both mosquito and vertebrate hosts.

The presence of bottlenecks in the transmission cycle of many RNA viruses leads to a severe reduction of number of virus particles and this occurs multiple times throughout the viral transmission cycle. Viral replication is then necessary for regeneration of a diverse mutant swarm. It is now understood that any perturbation of the mutation frequency either by increasing or decreasing the accumulation of mutations in an RNA virus results in attenuation of the virus. To determine if altering the rate at which a virus accumulates mutations decreases the probability of a successful virus infection due to issues traversing host bottlenecks, a series of mutations in the RNA-dependent RNA polymerase of Venezuelan equine encephalitis virus (VEEV), strain 68U201, were tested for mutation rate changes. All RdRp mutants were attenuated in both the mosquito and vertebrate hosts, while showing no attenuation during in vitro infections. The rescued viruses containing these mutations showed some evidence of change in fidelity, but the phenotype was not sustained following passaging. However, these mutants did exhibit changes in the frequency of specific types of mutations. Using a model of mutation production, these changes were shown to decrease the number of stop codons generated during virus replication. This suggests that the observed mutant attenuation in vivo may be due to an increase in the number of unfit genomes, which may be normally selected against by the accumulation of stop codons. Lastly, the ability of these attenuated viruses to transition through a bottleneck in vivo was measured using marked virus clones. The attenuated viruses showed an overall reduction in the number of marked clones for both the mosquito and vertebrate hosts, as well as a reduced ability to overcome the known bottlenecks in the mosquito. This study demonstrates that any perturbation of the optimal mutation frequency whether through changes in fidelity or by alterations in the mutation frequency of specific nucleotides, has significant deleterious effects on the virus, especially in the presence of host bottlenecks.

Clinical Importance of Placental Testing among Suspected Cases of Congenital Zika Syndrome.

Contemporaneous Zika virus (ZIKV) strains can cause congenital Zika syndrome (CZS). Current ZIKV clinical laboratory testing strategies are limited and include IgM serology (which may wane 12 weeks after initial exposure) and nucleic acid testing (NAT) of maternal serum, urine, and placenta for (+) strand ZIKV RNA (which is often transient). The objectives of this study were to determine if use of additional molecular tools, such as quantitative PCR and microscopy, would add to the diagnostic value of current standard placental ZIKV testing in cases with maternal endemic exposure and indeterminate testing. ZIKV RNA was quantified from dissected sections of placental villi, chorioamnion sections, and full cross-sections of umbilical cord in all cases examined. Quantitation with high-resolution automated electrophoresis determined relative amounts of precisely verified ZIKV (74-nt amplicons). In order to localize and visualize stable and actively replicating placental ZIKV in situ, labeling of flaviviridae glycoprotein, RNA ISH against both (+) and (⁻) ZIKV-specific ssRNA strands, and independent histologic examination for significant pathologic changes were employed. We demonstrate that the use of these molecular tools added to the diagnostic value of placental ZIKV testing among suspected cases of congenital Zika syndrome with poorly ascribed maternal endemic exposure.

RNA Virus Fidelity Mutants: A Useful Tool for Evolutionary Biology or a Complex Challenge?

RNA viruses replicate with low fidelity due to the error-prone nature of the RNA-dependent RNA polymerase, which generates approximately one mutation per round of genome replication. Due to the large population sizes produced by RNA viruses during replication, this results in a cloud of closely related virus variants during host infection, of which small increases or decreases in replication fidelity have been shown to result in virus attenuation in vivo, but not typically in vitro. Since the discovery of the first RNA virus fidelity mutants during the mid-aughts, the field has exploded with the identification of over 50 virus fidelity mutants distributed amongst 7 RNA virus families. This review summarizes the current RNA virus fidelity mutant literature, with a focus upon the definition of a fidelity mutant as well as methods to confirm any mutational changes associated with the fidelity mutant. Due to the complexity of such a definition, in addition to reports of unstable virus fidelity phenotypes, the future translational utility of these mutants and applications for basic science are examined.

Low-fidelity Venezuelan equine encephalitis virus polymerase mutants to improve live-attenuated vaccine safety and efficacy.

During RNA virus replication, there is the potential to incorporate mutations that affect virulence or pathogenesis. For live-attenuated vaccines, this has implications for stability, as replication may result in mutations that either restore the wild-type phenotype via reversion or compensate for the attenuating mutations by increasing virulence (pseudoreversion). Recent studies have demonstrated that altering the mutation rate of an RNA virus is an effective attenuation tool. To validate the safety of low-fidelity mutations to increase vaccine attenuation, several mutations in the RNA-dependent RNA-polymerase (RdRp) were tested in the live-attenuated Venezuelan equine encephalitis virus vaccine strain, TC-83. Next generation sequencing after passage in the presence of mutagens revealed a mutant containing three mutations in the RdRp, TC-83 3x, to have decreased replication fidelity, while a second mutant, TC-83 4x displayed no change in fidelity, but shared many phenotypic characteristics with TC-83 3x. Both mutants exhibited increased, albeit inconsistent attenuation in an infant mouse model, as well as increased immunogenicity and complete protection against lethal challenge of an adult murine model compared with the parent TC-83. During serial passaging in a highly permissive model, the mutants increased in virulence but remained less virulent than the parent TC-83. These results suggest that the incorporation of low-fidelity mutations into the RdRp of live-attenuated vaccines for RNA viruses can confer increased immunogenicity whilst showing some evidence of increased attenuation. However, while in theory such constructs may result in more effective vaccines, the instability of the vaccine phenotype decreases the likelihood of this being an effective vaccine strategy.

Mosquito bottlenecks alter viral mutant swarm in a tissue and time-dependent manner with contraction and expansion of variant positions and diversity.

Viral diversity is theorized to play a significant role during virus infections, particularly for arthropod-borne viruses (arboviruses) that must infect both vertebrate and invertebrate hosts. To determine how viral diversity influences mosquito infection and dissemination Culex taeniopus mosquitoes were infected with the Venezuelan equine encephalitis virus endemic strain 68U201. Bodies and legs/wings of the mosquitoes were collected individually and subjected to multi-parallel sequencing. Virus sequence diversity was calculated for each tissue. Greater diversity was seen in mosquitoes with successful dissemination versus those with no dissemination. Diversity across time revealed that bottlenecks influence diversity following dissemination to the legs/wings, but levels of diversity are restored by Day 12 post-dissemination. Specific minority variants were repeatedly identified across the mosquito cohort, some in nearly every tissue and time point, suggesting that certain variants are important in mosquito infection and dissemination. This study demonstrates that the interaction between the mosquito and the virus results in changes in diversity and the mutational spectrum and may be essential for successful transition of the bottlenecks associated with arbovirus infection.