Publications by authors named "Steven G Widen"

67 Publications

A genetically stable Zika virus vaccine candidate protects mice against virus infection and vertical transmission.

NPJ Vaccines 2021 Feb 17;6(1):27. Epub 2021 Feb 17.

Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston, TX, USA.

Although live attenuated vaccines (LAVs) have been effective in the control of flavivirus infections, to date they have been excluded from Zika virus (ZIKV) vaccine trials due to safety concerns. We have previously reported two ZIKV mutants, each of which has a single substitution in either envelope (E) glycosylation or nonstructural (NS) 4B P36 and displays a modest reduction in mouse neurovirulence and neuroinvasiveness, respectively. Here, we generated a ZIKV mutant, ZE4B-36, which combines mutations in both E glycosylation and NS4B P36. The ZE4B-36 mutant is stable and attenuated in viral replication. Next-generation sequence analysis showed that the attenuating mutations in the E and NS4B proteins are retained during serial cell culture passages. The mutant exhibits a significant reduction in neuroinvasiveness and neurovirulence and low infectivity in mosquitoes. It induces robust ZIKV-specific memory B cell, antibody, and T cell-mediated immune responses in type I interferon receptor (IFNR) deficient mice. ZIKV-specific T cell immunity remains strong months post-vaccination in wild-type C57BL/6 (B6) mice. Vaccination with ZE4B-36 protects mice from ZIKV-induced diseases and vertical transmission. Our results suggest that combination mutations in E glycosylation and NS4B P36 contribute to a candidate LAV with significantly increased safety but retain strong immunogenicity for prevention and control of ZIKV infection.
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http://dx.doi.org/10.1038/s41541-021-00288-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889622PMC
February 2021

Changes in Northern Elephant Seal Skeletal Muscle Following Thirty Days of Fasting and Reduced Activity.

Front Physiol 2020 6;11:564555. Epub 2020 Oct 6.

Department of Health and Kinesiology, Texas A&M University, College Station, TX, United States.

Northern elephant seals (NES, ) undergo an annual molt during which they spend ∼40 days fasting on land with reduced activity and lose approximately one-quarter of their body mass. Reduced activity and muscle load in stereotypic terrestrial mammalian models results in decreased muscle mass and capacity for force production and aerobic metabolism. However, the majority of lost mass in fasting female NES is from fat while muscle mass is largely preserved. Although muscle mass is preserved, potential changes to the metabolic and contractile capacity are unknown. To assess potential changes in NES skeletal muscle during molt, we collected muscle biopsies from 6 adult female NES before the molt and after ∼30 days at the end of the molt. Skeletal muscle was assessed for respiratory capacity using high resolution respirometry, and RNA was extracted to assess changes in gene expression. Despite a month of reduced activity, fasting, and weight loss, skeletal muscle respiratory capacity was preserved with no change in OXPHOS respiratory capacity. Molt was associated with 162 upregulated genes including those favoring lipid metabolism. We identified 172 downregulated genes including those coding for ribosomal proteins and genes associated with skeletal muscle force transduction and glucose metabolism. Following ∼30 days of molt, NES skeletal muscle metabolic capacity is preserved although mechanotransduction may be compromised. In the absence of exercise stimulus, fasting-induced shifts in muscle metabolism may stimulate pathways associated with preserving the mass and metabolic capacity of slow oxidative muscle.
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http://dx.doi.org/10.3389/fphys.2020.564555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573231PMC
October 2020

Evolution of resistance to fluoroquinolones by dengue virus serotype 4 provides insight into mechanism of action and consequences for viral fitness.

Virology 2021 Jan 1;552:94-106. Epub 2020 Oct 1.

Department of Biology, New Mexico State University, Las Cruces, NM, USA.

Drugs against flaviviruses such as dengue (DENV) and Zika (ZIKV) virus are urgently needed. We previously demonstrated that three fluoroquinolones, ciprofloxacin, enoxacin, and difloxacin, suppress replication of six flaviviruses. To investigate the barrier to resistance and mechanism(s) of action of these drugs, DENV-4 was passaged in triplicate in HEK-293 cells in the presence or absence of each drug. Resistance to ciprofloxacin was detected by the seventh passage and to difloxacin by the tenth, whereas resistance to enoxacin did not occur within ten passages. Two putative resistance-conferring mutations were detected in the envelope gene of ciprofloxacin and difloxacin-resistant DENV-4. In the absence of ciprofloxacin, ciprofloxacin-resistant viruses sustained a significantly higher viral titer than control viruses in HEK-293 and HuH-7 cells and resistant viruses were more stable than control viruses at 37 °C. These results suggest that the mechanism of action of ciprofloxacin and difloxacin involves interference with virus binding or entry.
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http://dx.doi.org/10.1016/j.virol.2020.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7528753PMC
January 2021

Acupuncture Reduces Hypertrophy and Cardiac Fibrosis, and Improves Heart Function in Mice with Diabetic Cardiomyopathy.

Cardiovasc Drugs Ther 2020 12 7;34(6):835-848. Epub 2020 Aug 7.

The Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA.

Purpose: To assess the effects of electro-acupuncture (EA) on glycemic control, myocardial inflammation, and the progression of diabetic cardiomyopathy in mice with type 2 diabetes.

Methods: Db/Db mice received EA at PC6+ST36 (DM-Acu), non-acupoint simulation (DM-Sham), or no treatment (DM). EA was applied for 30 min per day, 5 days a week for 4 weeks. Heart function was assessed by echocardiography. Myocardium was assessed by RT-PCR, immunoblotting, and histology. Serum TNF-α, IL-1α, IL-1β, IL-6, and IL-8 were measured.

Results: DM-Acu, but not DM-Sham, reduced fasting blood glucose without affecting body weight. DM decreased systolic function. DM-Acu, but not DM-Sham, attenuated the decrease in systolic function. Heart weight was significantly smaller in the DM-Acu than in the DM and DM-Sham groups. Percent fibrosis and apoptosis were reduced in the DM-Acu, but not the DM-Sham, group. Serum levels of IL-1α, IL-1β, IL-6, IL-8, ICAM-1, MCP-1, and TNF-α were significantly lower in the DM-Acu than in the DM or DM-Sham groups. Protein levels of P-Akt and P-AMPK and mRNA levels of phosphoinositide-3-kinase regulatory subunit 6 (PIK3r6) were significantly higher in the DM-Acu group. Myocardial mRNA and protein levels of insulin-like growth factor 1 receptor (IGF1R) were significantly lower in the DM and DM-Sham groups compared with the DM-Acu group.

Conclusions: EA reduced serum glucose; prevented DM-induced hypertrophy and deterioration of systolic function, inflammation, and fibrosis; and restored IGF1R, P-Akt, and P-AMPK levels in mice with type 2 diabetes mellitus.
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http://dx.doi.org/10.1007/s10557-020-07043-4DOI Listing
December 2020

Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient.

bioRxiv 2020 Mar 7. Epub 2020 Mar 7.

Centers for Disease Control and Prevention, Atlanta, GA, USA.

The etiologic agent of the outbreak of pneumonia in Wuhan China was identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) in January, 2020. The first US patient was diagnosed by the State of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into two virus repositories, making it broadly available to the public health and research communities. We hope that open access to this important reagent will expedite development of medical countermeasures.
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http://dx.doi.org/10.1101/2020.03.02.972935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239045PMC
March 2020

Principal component analysis of blood microRNA datasets facilitates diagnosis of diverse diseases.

PLoS One 2020 5;15(6):e0234185. Epub 2020 Jun 5.

Department of Anesthesiology, The University of Texas Medical Branch at Galveston, Galveston, Texas, United States of America.

Early, ideally pre-symptomatic, recognition of common diseases (e.g., heart disease, cancer, diabetes, Alzheimer's disease) facilitates early treatment or lifestyle modifications, such as diet and exercise. Sensitive, specific identification of diseases using blood samples would facilitate early recognition. We explored the potential of disease identification in high dimensional blood microRNA (miRNA) datasets using a powerful data reduction method: principal component analysis (PCA). Using Qlucore Omics Explorer (QOE), a dynamic, interactive visualization-guided bioinformatics program with a built-in statistical platform, we analyzed publicly available blood miRNA datasets from the Gene Expression Omnibus (GEO) maintained at the National Center for Biotechnology Information at the National Institutes of Health (NIH). The miRNA expression profiles were generated from real time PCR arrays, microarrays or next generation sequencing of biologic materials (e.g., blood, serum or blood components such as platelets). PCA identified the top three principal components that distinguished cohorts of patients with specific diseases (e.g., heart disease, stroke, hypertension, sepsis, diabetes, specific types of cancer, HIV, hemophilia, subtypes of meningitis, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, mild cognitive impairment, aging, and autism), from healthy subjects. Literature searches verified the functional relevance of the discriminating miRNAs. Our goal is to assemble PCA and heatmap analyses of existing and future blood miRNA datasets into a clinical reference database to facilitate the diagnosis of diseases using routine blood draws.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234185PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274418PMC
August 2020

Barrita Virus, a Novel Virus of the Patois Serogroup (Genus ; Family ).

Am J Trop Med Hyg 2020 07 21;103(1):190-192. Epub 2020 May 21.

Center for Tropical Diseases, Galveston, Texas.

During ecological investigations for arboviruses conducted in coastal Chiapas, Mexico, in 2007, isolate MP1078 was obtained from a pool of mosquitoes. Based on antigenic characterization, this isolate was classified as a strain of Patois virus (PATV) ( genus, family). Recently, we conducted nearly complete genome sequencing of this isolate to gain further insight into its genetic relationship with other members of the Patois serogroup. Based on the genetic characterization, we determined that MP1078 contains S, M, and L genome segments that are genetically distinct from other viruses within the Patois serogroup. Serological analyses confirmed the taxonomic classification of MP1078 as a new virus and species within the Patois serogroup, and we propose the name Barrita virus (BITV).
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http://dx.doi.org/10.4269/ajtmh.19-0906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356424PMC
July 2020

Using Next Generation Sequencing to Study the Genetic Diversity of Candidate Live Attenuated Zika Vaccines.

Vaccines (Basel) 2020 Apr 3;8(2). Epub 2020 Apr 3.

Department of Pathology, Sealy Institute for Vaccine Sciences, Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555, USA.

(ZIKV) is a mosquito-transmitted positive-sense RNA virus in the family . Candidate live-attenuated vaccine (LAV) viruses with engineered deletions in the 3' untranslated region (UTR) provide immunity and protection in animal models of ZIKV infection, and phenotypic studies show that LAVs retain protective abilities following in vitro passage. The present study investigated the genetic diversity of wild-type (WT) parent ZIKV and its candidate LAVs using next generation sequencing analysis of five sequential in vitro passages. The results show that genomic entropy of WT ZIKV steadily increases during in vitro passage, whereas that of LAVs also increased by passage number five but was variable throughout passaging. Additionally, clusters of single nucleotide variants (SNVs) were found to be present in the pre-membrane/membrane (prM), envelope (E), nonstructural protein NS1 (NS1), and other nonstructural protein genes, depending on the specific deletion, whereas in the parent WT ZIKV, they are more abundant in prM and NS1. Ultimately, both the parental WT and LAV derivatives increase in genetic diversity, with evidence of adaptation following passage.
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http://dx.doi.org/10.3390/vaccines8020161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349499PMC
April 2020

Characterization of Port Bolivar Virus, a Novel Entomobirnavirus ) Isolated from Mosquitoes Collected in East Texas, USA.

Viruses 2020 03 31;12(4). Epub 2020 Mar 31.

Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555-0609, USA.

This report describes and characterizes a novel entomobirnavirus, designated Port Bolivar virus (PTBV), that was isolated from a pool of mosquitoes collected in a saltwater marsh in East Texas, USA. Full genome sequencing and phylogenetic analyses indicate that PTBV is distinct but genetically related to Drosophila X virus and mosquito X virus, which are assigned to species in the genus , family . PTBV produced cytopathic effect (CPE) in cultures of mosquito (C6/36) cells, but not in Vero cell cultures. Ultrastructural studies of PTBV in infected C6/36 cells demonstrated unenveloped virus particles about 55 nm in diameter.
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http://dx.doi.org/10.3390/v12040390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232177PMC
March 2020

Severe Acute Respiratory Syndrome Coronavirus 2 from Patient with Coronavirus Disease, United States.

Emerg Infect Dis 2020 06 17;26(6):1266-1273. Epub 2020 Jun 17.

The etiologic agent of an outbreak of pneumonia in Wuhan, China, was identified as severe acute respiratory syndrome coronavirus 2 in January 2020. A patient in the United States was given a diagnosis of infection with this virus by the state of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens from this patient and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into 2 virus repositories, making it broadly available to the public health and research communities. We hope that open access to this reagent will expedite development of medical countermeasures.
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http://dx.doi.org/10.3201/eid2606.200516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7258473PMC
June 2020

Ebola Virus Produces Discrete Small Noncoding RNAs Independently of the Host MicroRNA Pathway Which Lack RNA Interference Activity in Bat and Human Cells.

J Virol 2020 02 28;94(6). Epub 2020 Feb 28.

Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, USA

The question as to whether RNA viruses produce microRNAs (miRNAs) during infection has been the focus of intense research and debate. Recently, several groups using computational prediction methods have independently reported possible miRNA candidates produced by Ebola virus (EBOV). Additionally, efforts to detect these predicted RNA products in samples from infected animals and humans have produced positive results. However, these studies and their conclusions are predicated on the assumption that these RNA products are actually processed through, and function within, the miRNA pathway. In the present study, we performed the first rigorous assessment of the ability of filoviruses to produce miRNA products during infection of both human and bat cells. Using next-generation sequencing, we detected several candidate miRNAs from both EBOV and the closely related Marburg virus (MARV). Focusing our validation efforts on EBOV, we found evidence contrary to the idea that these small RNA products function as miRNAs. The results of our study are important because they highlight the potential pitfalls of relying on computational methods alone for virus miRNA discovery. Here, we report the discovery, via deep sequencing, of numerous noncoding RNAs (ncRNAs) derived from both EBOV and MARV during infection of both bat and human cell lines. In addition to identifying several novel ncRNAs from both viruses, we identified two EBOV ncRNAs in our sequencing data that were near-matches to computationally predicted viral miRNAs reported in the literature. Using molecular and immunological techniques, we assessed the potential of EBOV ncRNAs to function as viral miRNAs. Importantly, we found little evidence supporting this hypothesis. Our work is significant because it represents the first rigorous assessment of the potential for EBOV to encode viral miRNAs and provides evidence contrary to the existing paradigm regarding the biological role of computationally predicted EBOV ncRNAs. Moreover, our work highlights further avenues of research regarding the nature and function of EBOV ncRNAs.
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http://dx.doi.org/10.1128/JVI.01441-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7158719PMC
February 2020

Japanese encephalitis vaccine-specific envelope protein E138K mutation does not attenuate virulence of West Nile virus.

NPJ Vaccines 2019 5;4:50. Epub 2019 Dec 5.

1Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555 USA.

West Nile (WNV) and Japanese encephalitis viruses (JEV) are closely related, mosquito-borne neurotropic flaviviruses. Although there are no licensed human vaccines for WNV, JEV has multiple human vaccines, including the live, attenuated vaccine SA14-14-2. Investigations into determinants of attenuation of JE SA14-14-2 demonstrated that envelope (E) protein mutation E138K was crucial to the attenuation of mouse virulence. As WNV is closely related to JEV, we investigated whether or not the E-E138K mutation would be beneficial to be included in a candidate live attenuated WNV vaccine. Rather than conferring a mouse attenuated phenotype, the WNV E-E138K mutant reverted and retained a wild-type mouse virulence phenotype. Next-generation sequencing analysis demonstrated that, although the consensus sequence of the mutant had the E-E138K mutation, there was increased variation in the E protein, including a single-nucleotide variant (SNV) revertant to the wild-type glutamic acid residue. Modeling of the E protein and analysis of SNVs showed that reversion was likely due to the inability of critical E-protein residues to be compatible electrostatically. Therefore, this mutation may not be reliable for inclusion in candidate live attenuated vaccines in related flaviviruses, such as WNV, and care must be taken in translation of attenuating mutations from one virus to another virus, even if they are closely related.
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http://dx.doi.org/10.1038/s41541-019-0146-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895119PMC
December 2019

Genes of the cGMP-PKG-Ca signaling pathway are alternatively spliced in cardiomyopathy: Role of RBFOX2.

Biochim Biophys Acta Mol Basis Dis 2020 03 25;1866(3):165620. Epub 2019 Nov 25.

Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston, 77555-1070, TX, United States of America; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, 77555, TX, United States of America. Electronic address:

Aberrations in the cGMP-PKG-Ca pathway are implicated in cardiovascular complications of diverse etiologies, though involved molecular mechanisms are not understood. We performed RNA-Seq analysis to profile global changes in gene expression and exon splicing in Chagas disease (ChD) murine myocardium. Ingenuity-Pathway-Analysis of transcriptome dataset identified 26 differentially expressed genes associated with increased mobilization and cellular levels of Ca in ChD hearts. Mixture-of-isoforms and Enrichr KEGG pathway analyses of the RNA-Seq datasets from ChD (this study) and diabetic (previous study) murine hearts identified alternative splicing (AS) in eleven genes (Arhgef10, Atp2b1, Atp2a3, Cacna1c, Itpr1, Mef2a, Mef2d, Pde2a, Plcb1, Plcb4, and Ppp1r12a) of the cGMP-PKG-Ca pathway in diseased hearts. AS of these genes was validated by an exon exclusion-inclusion assay. Further, Arhgef10, Atp2b1, Mef2a, Mef2d, Plcb1, and Ppp1r12a genes consisted RBFOX2 (RNA-binding protein) binding-site clusters, determined by analyzing the RBFOX2 CLIP-Seq dataset. H9c2 rat heart cells transfected with Rbfox2 (vs. scrambled) siRNA confirmed that expression of Rbfox2 is essential for proper exon splicing of genes of the cGMP-PKG-Ca pathway. We conclude that changes in gene expression may influence the Ca mobilization pathway in ChD, and AS impacts the genes involved in cGMP/PKG/Ca signaling pathway in ChD and diabetes. Our findings suggest that ChD patients with diabetes may be at increased risk of cardiomyopathy and heart failure and provide novel ways to restore cGMP-PKG regulated signaling networks via correcting splicing patterns of key factors using oligonucleotide-based therapies for the treatment of cardiovascular complications.
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http://dx.doi.org/10.1016/j.bbadis.2019.165620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954967PMC
March 2020

Dianke virus: A new mesonivirus species isolated from mosquitoes in Eastern Senegal.

Virus Res 2020 01 4;275:197802. Epub 2019 Nov 4.

Virology Department, Arbovirus and Hemorrhagic Fever Viruses Unit, Institut Pasteur de Dakar, Dakar, Senegal.

An increasing number of insect-specific viruses are found around the world. Very recently, a new group of insect-specific viruses, the Mesoniviridae family, was discovered in Africa, Asia, North America and Australia. Here we report the first detection and isolation of a new virus belonging to Mesonivirus genus in Senegal, West Africa. The so-called Dianke virus was detected in 21 species of arthropods trapped in the eastern part of the country. Male individuals were also infected, supporting vertical transmission assertion of insect specific viruses. As described for other mesoniviruses, no viral replication was observed after inoculation of mammalian cells. Viral replication in mosquito cells was blocked at a temperature of 37 °C, highlighting the importance of thermal conditions in Mesonivirus host restriction. Similar to our study, where a diverse range of arthropod vectors were found infected by the new virus, several studies have detected mesonivirus infection in mosquitoes with concerns for human health. It has been shown that dual infections in mosquito can alter viral infectivity. Due to their extensive geographic distribution and host range, as well as their use as potential disease control agents in vector populations, more studies should be done for a better knowledge of arthropod-restricted viruses prevalence and diversity.
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http://dx.doi.org/10.1016/j.virusres.2019.197802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075714PMC
January 2020

Characterization of Three Novel Viruses from the Families , and , Isolated from Dead Birds Collected during West Nile Virus Surveillance in Harris County, Texas.

Viruses 2019 10 10;11(10). Epub 2019 Oct 10.

Department of Pathology, University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555, USA.

This report describes and characterizes three novel RNA viruses isolated from dead birds collected during West Nile virus surveillance in Harris County, TX, USA (the Houston metropolitan area). The novel viruses are identified as members of the families , , and and have been designated as San Jacinto virus, Mason Creek virus, and Buffalo Bayou virus, respectively. Their potential public health and/or veterinary importance are still unknown.
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http://dx.doi.org/10.3390/v11100927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832935PMC
October 2019

Attenuation of Live-Attenuated Yellow Fever 17D Vaccine Virus Is Localized to a High-Fidelity Replication Complex.

mBio 2019 10 22;10(5). Epub 2019 Oct 22.

Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, USA

The molecular basis of attenuation for live-attenuated vaccines is poorly understood. The yellow fever (YF) 17D vaccine virus was derived from the wild-type, parental strain Asibi virus by serial passage in chicken tissue and has proven to be a very safe and efficacious vaccine. We have previously shown that wild-type Asibi is a typical RNA virus with high genetic diversity, while the 17D vaccine virus has very little genetic diversity. To investigate this further, we treated Asibi and 17D viruses with ribavirin, a GTP analog with strong antiviral activity that increases levels of mutations in the viral genome. As expected, ribavirin treatment introduced mutations into the Asibi virus genome at a very high frequency and decreased viral infectivity while, in contrast, the 17D vaccine virus was resistant to ribavirin, as treatment with the antiviral introduced very few mutations into the genome, and viral infectivity was not lost. The results were confirmed for another YF wild-type parental and vaccine pair, a wild-type French viscerotropic virus and French neurotropic vaccine. Using recombinant Asibi and 17D viruses, ribavirin sensitivity was located to viral nonstructural genes. Thus, two live-attenuated YF vaccine viruses are genetically stable even under intense mutagenic pressure, suggesting that attenuation of live-attenuated YF vaccines is due, at least in part, to fidelity of the replication complex resulting in high genetic stability. Live-attenuated viral vaccines are highly safe and efficacious but represent complex and often multigenic attenuation mechanisms. Most of these vaccines have been generated empirically by serial passaging of a wild-type (WT) virus in cell culture. One of the safest and most effective live-attenuated vaccines is yellow fever (YF) virus strain 17D, which has been used for over 80 years to control YF disease. The availability of the WT parental strain of 17D, Asibi virus, and large quantities of clinical data showing the effectiveness of the 17D vaccine make this WT parent/vaccine pair an excellent model for investigating RNA virus attenuation. Here, we investigate a mechanism of 17D attenuation and show that the vaccine virus is resistant to the antiviral compound ribavirin. The findings suggest that attenuation is in part due to a low probability of reversion or mutation of the vaccine virus genome to WT, thus maintaining a stable genotype despite external pressures.
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http://dx.doi.org/10.1128/mBio.02294-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805994PMC
October 2019

Genotypic and phenotypic characterization of West Nile virus NS5 methyltransferase mutants.

Vaccine 2019 11 11;37(48):7155-7164. Epub 2019 Oct 11.

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, United States; Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, United States; Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX 77555, United States. Electronic address:

Although West Nile virus (WNV) causes annual cases of neurological disease and deaths in humans, a vaccine has not been licensed for human use. Several WNV genes have been targeted for mutagenesis in attempts to generate live attenuated vaccine candidates, including the non-structural protein NS5. Specifically, mutation of WNV NS5-K61A or NS5-E218A in the catalytic tetrad of the methyltransferase decreases enzyme activity of the NS5 protein and correspondingly attenuates the virus in mice. In this report, NS5-K61A, NS5-E218A, and a double mutant encoding both mutations (NS5-K61A/E218A) were compared both in vitro and in vivo. Each single mutant was strongly attenuated in highly susceptible outbred mice, whereas the double mutant unexpectedly was not attenuated. Sequencing analysis demonstrated that the double mutant was capable of reversion at both residues NS5-61 and NS5-218, whereas the genotype of the single mutants did not show evidence of reversion. Overall, either NS5-K61A or NS5-E218A methyltransferase mutations could be potential mutations to include in a candidate live WNV vaccine; however, multiple mutations in the catalytic tetrad of the methyltransferase are not tolerated.
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http://dx.doi.org/10.1016/j.vaccine.2019.09.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996054PMC
November 2019

Whole-Genome Sequence of Rickettsia parkeri Strain Atlantic Rainforest, Isolated from a Colombian Tick.

Microbiol Resour Announc 2019 Sep 26;8(39). Epub 2019 Sep 26.

Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA.

is classified as a member of the alphaproteobacterial microorganisms, genus Here, we report the complete genome sequence of strain Atlantic Rainforest, which was isolated from an tick collected in the municipality of Necoclí, Colombia.
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http://dx.doi.org/10.1128/MRA.00684-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763642PMC
September 2019

Ixodes scapularis salivary gland microRNAs are differentially expressed during Powassan virus transmission.

Sci Rep 2019 09 11;9(1):13110. Epub 2019 Sep 11.

SUNY Center for Environmental Health and Medicine, SUNY Upstate Medical University, Syracuse, NY, United States.

Successful tick feeding is facilitated by an assortment of pharmacologically-active factors in tick saliva that create an immunologically privileged micro-environment in the host's skin. Through a process known as saliva-assisted transmission, bioactive tick salivary factors modulate the host environment, promoting transmission and establishment of a tick-borne pathogen. This phenomenon was previously demonstrated for Powassan virus (POWV), a North American tick-borne flavivirus that is the causative agent of a severe neuroinvasive disease in humans. Here, we sought to characterize the Ixodes scapularis salivary gland microRNAs (miRNAs) expressed during the earliest period of POWV transmission to a mammalian host. POWV-infected and uninfected I. scapularis females were fed on naïve mice for 1, 3, and 6 hours, and Illumina next generation sequencing was used to characterize the salivary gland miRNA expression profiles of POWV-infected versus uninfected ticks. 379 salivary miRNAs were detected, of which 338 are reported here as putative novel I. scapularis miRNAs. 35 salivary gland miRNAs were significantly up-regulated and 17 miRNAs were significantly down-regulated in response to POWV infection. To investigate the potential role of salivary gland miRNAs in POWV replication in-vitro, we transfected miRNA inhibitors into VeroE6 cells to profile temporal POWV replication in mammalian cells. Together, the small RNA sequencing data and the in vitro miRNA inhibition assay suggest that the differentially expressed tick salivary miRNAs could act in regulating POWV replication in host tissues.
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http://dx.doi.org/10.1038/s41598-019-49572-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739385PMC
September 2019

Attenuation of Zika Virus by Passage in Human HeLa Cells.

Vaccines (Basel) 2019 08 20;7(3). Epub 2019 Aug 20.

Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USA.

Zika virus (ZIKV) is a mosquito-borne Flavivirus. Previous studies have shown that mosquito-transmitted flaviviruses, including yellow fever, Japanese encephalitis, and West Nile viruses, could be attenuated by serial passaging in human HeLa cells. Therefore, it was hypothesized that wild-type ZIKV would also be attenuated after HeLa cell passaging. A human isolate from the recent ZIKV epidemic was subjected to serial HeLa cell passaging, resulting in attenuated in vitro replication in both Vero and A549 cells. Additionally, infection of AG129 mice with 10 plaque forming units (pfu) of wild-type ZIKV led to viremia and mortality at 12 days, whereas infection with 10 pfu of HeLa-passage 6 (P6) ZIKV led to lower viremia, significant delay in mortality (median survival: 23 days), and increased cytokine and chemokine responses. Genomic sequencing of HeLa-passaged virus identified two amino acid substitutions as early as HeLa-P3: pre-membrane E87K and nonstructural protein 1 R103K. Furthermore, both substitutions were present in virus harvested from HeLa-P6-infected animal tissue. Together, these data show that, similarly to other mosquito-borne flaviviruses, ZIKV is attenuated following passaging in HeLa cells. This strategy can be used to improve understanding of substitutions that contribute to attenuation of ZIKV and be applied to vaccine development across multiple platforms.
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http://dx.doi.org/10.3390/vaccines7030093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789458PMC
August 2019

Type II Epithelial-Mesenchymal Transition Upregulates Protein N-Glycosylation To Maintain Proteostasis and Extracellular Matrix Production.

J Proteome Res 2019 09 28;18(9):3447-3460. Epub 2019 Aug 28.

Institute for Clinical and Translational Research , University of Wisconsin-Madison School of Medicine and Public Health , Madison , Wisconsin 53705 , United States.

Type II epithelial-mesenchymal transition (EMT) plays a vital role in airway injury, repair, and remodeling. Triggered by growth factors, such as transforming growth factor beta (TGFβ), EMT induced a biological process that converts epithelial cells into secretory mesenchymal cells with a substantially increased production of extracellular matrix (ECM) proteins. Epithelial cells are not professional secretory cells and produce few ECM proteins under normal conditions. The molecular mechanism underlying the transformation of the protein factory and secretory machinery during EMT is significant because ECM secretion is central to the pathogenesis of airway remodeling. Here we report that type II EMT upregulates the protein N-glycosylation of ECMs. The mechanism study reveals that the substantial increase in synthesis of ECM proteins in EMT activates the inositol-requiring protein 1 (IRE1α)-X-box-binding protein 1 (XBP1) axis of the unfolded protein response (UPR) coupled to the hexosamine biosynthesis pathway (HBP). These two pathways coordinately up-regulate the protein N-glycosylation of ECM proteins and increase ER folding capacity and ER-associated degradation (ERAD), which improve ER protein homeostasis and protect transitioned cells from proteotoxicity. Inhibition of the alternative splicing of XBP1 or protein N-glycosylation blocks ECM protein secretion, indicating the XBP1-HBP plays a prominent role in regulating the secretion of ECM proteins in the mesenchymal transition. Our data suggest that the activation of XBP1-HBP pathways and elevation of protein N-glycosylation is an adaptive response to maintain protein quality control and facilitate the secretion of ECM proteins during the mesenchymal transition. The components of the XBP1-HBP pathways may be therapeutic targets to prevent airway remodeling.
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http://dx.doi.org/10.1021/acs.jproteome.9b00342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195216PMC
September 2019

Inter- and intra-lineage genetic diversity of wild-type Zika viruses reveals both common and distinctive nucleotide variants and clusters of genomic diversity.

Emerg Microbes Infect 2019 ;8(1):1126-1138

c Department of Pathology, University of Texas Medical Branch , Galveston , USA.

Zika virus (ZIKV) strains belong to the East African, West African, and Asian/American phylogenetic lineages. RNA viruses, like ZIKV, exist as populations of genetically-related sequences whose heterogeneity may impact viral fitness, evolution, and virulence. Genetic diversity of representative ZIKVs from each lineage was examined using next generation sequencing (NGS) paired with downstream entropy and single nucleotide variant (SNV) analysis. Comparisons showed that inter-lineage diversity was statistically supported, while intra-lineage diversity. Intra-lineage diversity was significant for East but not West Africa strains. Furthermore, intra-lineage diversity for the Asian/American lineage was not supported for human serum isolates; however, a placenta isolate differed significantly. Relative in the pre-membrane/membrane (prM/M) gene of several ZIKV strains. Additionally, the East African lineage contained a greater number of synonymous SNVs, while a greater number of non-synonymous SNVs were identified for American strains. Further, inter-lineage SNVs were dispersed throughout the genome, whereas intra-lineage non-synonymous SNVs for Asian/American strains clustered within prM/M and NS1 gene. This comprehensive analysis of ZIKV genetic diversity provides a repository of SNV positions across lineages. We posit that increased non-synonymous SNV populations and increased relative genetic diversity of the prM/M and NS1 proteins provides more evidence for their role in ZIKV virulence and fitness.
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http://dx.doi.org/10.1080/22221751.2019.1645572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6711133PMC
December 2019

Hippocampal stem cells promotes synaptic resistance to the dysfunctional impact of amyloid beta oligomers via secreted exosomes.

Mol Neurodegener 2019 06 14;14(1):25. Epub 2019 Jun 14.

Mitchell Center for Neurodegenerative Diseases, Department of Neurology, University of Texas Medical Branch, Galveston, TX, 77555, USA.

Background: Adult hippocampal neurogenesis plays an important role in synaptic plasticity and cogntive function. We reported that higher numbers of neural stem cells (NSC) in the hippocampus of cognitively-intact individuals with high Alzheimer's disease (AD) pathology (plaques and tangles) is associated with decreased synaptic amyloid beta oligomers (Aβο), an event linked to onset of dementia in AD. While these findings suggest a link between NSC and synaptic resistance to Aβο, the involved mechanism remains to be determined. With this goal in mind, here we investigated the ability of exosomes secreted from hippocampal NSC to promote synaptic resilience to Aβo.

Methods: Exosomes isolated from media of hippocampus NSC (NSC-exo) or mature hippocampal neuronal (MN-exo) cultures were delivered intracerebroventricularly (ICV) to mice before assessment of Aβο-induced suppression of hippocampal long-term potentiation (LTP) and memory deficits. Aβο binding to synapses was assessed in cultured hippocampal neurons and on synaptosomes isolated from hippocampal slices from wild type mice and from an inducible mouse model of NSC ablation (Nestin-δ-HSV-TK mice) treated with exosomes. Expression of CaMKII and of AMPA and NMDA glutamate receptor subunits in synaptosomes was measured by western blot. Small RNA Deep sequencing was performed to identify microRNAs enriched in NSC-exo as compared to MN-exo. Mimics of select miRNAs were injected ICV.

Results: NSC-exo, but not MN-exo, abolished Aβo-induced suppression of LTP and subsequent memory deficits. Furthermore, in hippocampal slices and cultured neurons, NSC-exo significantly decreased Aβo binding to the synapse. Similarly, transgenic ablation of endogenous NSC increased synaptic Aβo binding, which was reversed by exogenous NSC-exo. Phosphorylation of synaptic CaMKII was increased by NSC-exo, while AMPA and NMDA receptors were not affected. Lastly, we identified a set of miRNAs enriched in NSC-exo that, when injected ICV, protected the synapses from Aβo-binding and Aβo-induced LTP inhibition.

Conclusions: These results identify a novel mechanism linking NSC-exo and synaptic susceptibility to Aβo that may underscore cognitive resilience of certain individuals with increased neurogenesis in spite of AD neuropathology and unmask a novel target for the development of a new treatment concept for AD centered on promoting synaptic resilience to toxic amyloid proteins.
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http://dx.doi.org/10.1186/s13024-019-0322-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570890PMC
June 2019

Development of a Novel Nasal Epithelial Cell Model Supporting Colonization With Human Nasal Microbiota.

Front Cell Infect Microbiol 2019 21;9:165. Epub 2019 May 21.

School of Medicine, University of Texas Medical Branch, Galveston, TX, United States.

The nasal mucosa provides first line defense against inhaled pathogens while creating a unique microenvironment for bacterial communities. Studying the impact of microbiota in the nasal cavity has been difficult due to limitations with current models including explant cultures, primary cells, or neoplastic cell lines. Most notably, none have been shown to support reproducible colonization by bacterial communities from human donors. Therefore, to conduct controlled studies of the human nasal ecosystem, we have developed a novel mucosal model that supports bacterial colonization of a cultured host mucosa created by immortalized human nasal epithelial cells (NEC). For this model, immortalized NEC established from 5 male and 5 female donors were cultured with an air-interfaced, apical surface on a porous transwell membrane. NEC were grown from nasal turbinate tissues harvested from willed bodies or from discarded tissue collected during sinonasal procedures. Immortalized cells were evaluated through molecular verification of cell type, histological confirmation of tissue differentiation including formation of tight junctions, NEC multilayer viability, metabolism, physiology and imaging of the luminal surface by scanning electron microscopy. Results showed proper differentiation and multilayer formation at seven to 10 days after air interface that was maintained for up to 3 weeks. The optimized mucosal cultures created an environment necessary to sustain colonization by nasal microbiomes (NMBs) that were collected from healthy volunteers, cryogenically preserved and characterized with customized quantitative polymerase chain reaction (qPCR) arrays. Polymicrobial communities of nasal bacteria associated with healthy and inflamed states were consistently reproduced in matured NEC co-cultures by transplant of NMBs from multiple community types. The cultured NMBs were stable after an initial period of bacterial replication and equilibration. This novel culture system is the first model that supports controlled cultivation of NMBs, allowing for lab-based causation studies and further experimentation to explore the complexities of host-microbe and microbe-microbe interactions.
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http://dx.doi.org/10.3389/fcimb.2019.00165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536665PMC
January 2020

Genome Sequence of Chiqui Virus, a Novel Reovirus Isolated from Mosquitoes Collected in Colombia.

Microbiol Resour Announc 2018 Sep 27;7(12). Epub 2018 Sep 27.

Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas, USA.

We report here the complete genome sequence of a novel reovirus, designated Chiqui virus (CHQV) strain CoB38d, that was isolated from a pool of unidentified mosquitoes collected in northern Colombia in 2013. CHQV has nine double-stranded DNA (dsRNA) genome segments and has similarity to viruses belonging to the family , subfamily .
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http://dx.doi.org/10.1128/MRA.00881-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256685PMC
September 2018

Genomic characterisation of Cuiaba and Charleville viruses: arboviruses (family Rhabdoviridae, genus Sripuvirus) infecting reptiles and amphibians.

Virus Genes 2019 Feb 3;55(1):87-94. Epub 2018 Dec 3.

School of Biological Sciences, The University of Queensland, St Lucia, QLD, Australia.

Viruses in the family Rhabdoviridae are ecologically very diverse, infecting mammals, birds, reptiles, fish, plants and a wide range of other terrestrial and aquatic invertebrates. The genus Sripuvirus currently comprises five viruses that appear to circulate in transmission cycles involving reptiles and sandflies. Here, we report an analysis of the complete coding sequences of Cuiaba virus (CUIV), isolated from an amphibian (Bufo marinus) collected in Brazil, and Charleville virus (CHVV), isolated from sandflies (Phlebotomus sp.) and lizards (Gehyra australis), collected in Australia. CUIV and CHVV cluster phylogenetically with the sripuviruses in maximum likelihood trees generated from complete L protein (RdRp) sequences. They also share with sripuviruses unique features in genome organisation, including an additional gene (U1) between the matrix protein (M) gene and glycoprotein (G) gene, and an alternative long open reading frame near the start of the G ORF that encodes a predicted transmembrane protein. In view of their phylogenetic relationships, similar genome organisations and similar ecological characteristics, we propose the assignment of CUIV and CHVV as novel members of the genus Sripuvirus.
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http://dx.doi.org/10.1007/s11262-018-1620-xDOI Listing
February 2019

Comparative Transcriptomics in Ebola Makona-Infected Ferrets, Nonhuman Primates, and Humans.

J Infect Dis 2018 11;218(suppl_5):S486-S495

Departments of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston.

The domestic ferret is a uniformly lethal model of infection for 3 species of Ebolavirus known to be pathogenic in humans. Reagents to systematically analyze the ferret host response to infection are lacking; however, the recent publication of a draft ferret genome has opened the potential for transcriptional analysis of ferret models of disease. In this work, we present comparative analysis of longitudinally sampled blood taken from ferrets and nonhuman primates infected with lethal doses of the Makona variant of Zaire ebolavirus. Strong induction of proinflammatory and prothrombotic signaling programs were present in both ferrets and nonhuman primates, and both transcriptomes were similar to previously published datasets of fatal cases of human Ebola virus infection.
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http://dx.doi.org/10.1093/infdis/jiy455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249602PMC
November 2018

Terrestrial Bird Migration and West Nile Virus Circulation, United States.

Emerg Infect Dis 2018 12;24(12):2184-2194

Host migration and emerging pathogens are strongly associated, especially with regard to zoonotic diseases. West Nile virus (WNV), a mosquitoborne pathogen capable of causing severe, sometimes fatal, neuroinvasive disease in humans, is maintained in highly mobile avian hosts. Using phylogeographic approaches, we investigated the relationship between WNV circulation in the United States and the flight paths of terrestrial birds. We demonstrated southward migration of WNV in the eastern flyway and northward migration in the central flyway, which is consistent with the looped flight paths of many terrestrial birds. We also identified 3 optimal locations for targeted WNV surveillance campaigns in the United States-Illinois, New York, and Texas. These results illustrate the value of multidisciplinary approaches to surveillance of infectious diseases, especially zoonotic diseases.
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http://dx.doi.org/10.3201/eid2412.180382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256381PMC
December 2018

Structural and Nonstructural Genes Contribute to the Genetic Diversity of RNA Viruses.

mBio 2018 10 30;9(5). Epub 2018 Oct 30.

Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston, Texas, USA

One paradigm to explain the complexity of viral RNA populations is that the low fidelity of the RNA-dependent RNA polymerase (RdRp) drives high mutation rates and consequently genetic diversity. Like most RNA viruses, wild-type yellow fever virus (YFV) replication is error-prone due to the lack of proofreading by the virus-encoded RdRp. However, there is evidence that replication of the live attenuated YF vaccine virus 17D, derived from wild-type strain Asibi, is less error-prone than wild-type RNA viruses. Recent studies comparing the genetic diversity of wild-type Asibi and 17D vaccine virus found that wild-type Asibi has the typical heterogeneous population of an RNA virus, while there is limited intra- and interpopulation variability of 17D vaccine virus. Utilizing chimeric and mutant infectious clone-derived viruses, we show that high and low genetic diversity profiles of wild-type Asibi virus and vaccine virus 17D, respectively, are multigenic. Introduction of either structural (pre-membrane and envelope) genes or NS2B or NS4B substitutions into the Asibi and 17D backbone resulted in altered variant population, nucleotide diversity, and mutation frequency compared to the parental viruses. Additionally, changes in genetic diversity of the chimeric and mutant viruses correlated with the phenotype of multiplication kinetics in human alveolar A549 cells. Overall, the paradigm that only the error-prone RdRp controls genetic diversity needs to be expanded to address the role of other genes in genetic diversity, and we hypothesize that it is the replication complex as a whole and not the RdRp alone that controls genetic diversity. With the advent of advanced sequencing technology, studies of RNA viruses have shown that genetic diversity can contribute to both attenuation and virulence and the paradigm is that this is controlled by the error-prone RNA-dependent RNA polymerase (RdRp). Since wild-type yellow fever virus (YFV) strain Asibi has genetic diversity typical of a wild-type RNA virus, while 17D virus vaccine has limited diversity, it provides a unique opportunity to investigate RNA population theory in the context of a well-characterized live attenuated vaccine. Utilizing infectious clone-derived viruses, we show that genetic diversity of RNA viruses is complex and that multiple genes, including structural genes and NS2B and NS4B genes also contribute to genetic diversity. We suggest that the replication complex as a whole, rather than only RdRp, drives genetic diversity, at least for YFV.
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http://dx.doi.org/10.1128/mBio.01871-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6212827PMC
October 2018

Analysis By Deep Sequencing of Discontinued Neurotropic Yellow Fever Vaccine Strains.

Sci Rep 2018 09 7;8(1):13408. Epub 2018 Sep 7.

Department of Pathology, Galveston, TX, 77555, USA.

Deep sequencing of live-attenuated viral vaccines has focused on vaccines in current use. Here we report characterization of a discontinued live yellow fever (YF) vaccine associated with severe adverse events. The French neurotropic vaccine (FNV) strain of YF virus was derived empirically in 1930 by 260 passages of wild-type French viscerotropic virus (FVV) in mouse brain. The vaccine was administered extensively in French-speaking Africa until discontinuation in 1982, due to high rates of post-vaccination encephalitis in children. Using rare archive strains of FNV, viral RNAs were sequenced and analyzed by massively parallel, in silico methods. Diversity and specific population structures were compared in reference to the wild-type parental strain FVV, and between the vaccine strains themselves. Lower abundance of polymorphism content was observed for FNV strains relative to FVV. Although the vaccines were of lower diversity than FVV, heterogeneity between the vaccines was observed. Reversion to wild-type identity was variably observed in the FNV strains. Specific population structures were recovered from vaccines with neurotropic properties; loss of neurotropism in mice was associated with abundance of wild-type RNA populations. The analysis provides novel sequence evidence that FNV is genetically unstable, and that adaptation of FNV contributed to the neurotropic adverse phenotype.
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http://dx.doi.org/10.1038/s41598-018-31085-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128858PMC
September 2018