Nomenclature- and Database-Compatible Names for the Two Ebola Virus Variants that Emerged in Guinea and the Democratic Republic of the Congo in 2014

In 2014, Ebola virus (EBOV) was identified as the etiological agent of a large and still expanding outbreak of Ebola virus disease (EVD) in West Africa and a much more confined EVD outbreak in Middle Africa. Epidemiological and evolutionary analyses confirmed that all cases of both outbreaks are connected to a single introduction each of EBOV into human populations and that both outbreaks are not directly connected. Coding-complete genomic sequence analyses of isolates revealed that the two outbreaks were caused by two novel EBOV variants, and initial clinical observations suggest that neither of them should be considered strains. Here we present consensus decisions on naming for both variants (West Africa: “Makona”, Middle Africa: “Lomela”) and provide database-compatible full, shortened, and abbreviated names that are in line with recently established filovirus sub-species nomenclatures.     

Challenges,Progress, and Opportunities: Proceedings of the Filovirus Medical Countermeasures Workshop

On August 22–23, 2013, agencies within the United States Department of Defense (DoD) and the Department of Health and Human Services (HHS) sponsored the Filovirus Medical Countermeasures (MCMs) Workshop as an extension of the activities of the Filovirus Animal Non-clinical Group (FANG). The FANG is a federally-recognized multi-Agency group established in 2011 to coordinate and facilitate U.S. government (USG) efforts to develop filovirus MCMs. The workshop brought together government, academic and industry experts to consider the needs for filovirus MCMs and evaluate the status of the product development pipeline. This report summarizes speaker presentations and highlights progress and challenges remaining in the field.     

Niemann-Pick C1 (NPC1)/NPC1-like1 Chimeras Define Sequences Critical for NPC1’s Function as a Filovirus Entry Receptor

We recently demonstrated that Niemann-Pick C1 (NPC1), a ubiquitous 13-pass cellular membrane protein involved in lysosomal cholesterol transport, is a critical entry receptor for filoviruses. Here we show that Niemann-Pick C1-like1 (NPC1L1), an NPC1 paralog and hepatitis C virus entry factor, lacks filovirus receptor activity. We exploited the structural similarity between NPC1 and NPC1L1 to construct and analyze a panel of chimeras in which NPC1L1 sequences were replaced with cognate sequences from NPC1. Only one chimera, NPC1L1 containing the second luminal domain (C) of NPC1 in place of its own, bound to the viral glycoprotein, GP. This engineered protein mediated authentic filovirus infection nearly as well as wild-type NPC1, and more efficiently than did a minimal NPC1 domain C-based receptor recently described by us. A reciprocal chimera, NPC1 containing NPC1L1’s domain C, was completely inactive. Remarkably, an intra-domain NPC1L1-NPC1 chimera bearing only a ~130-amino acid N–terminal region of NPC1 domain C could confer substantial viral receptor activity on NPC1L1. Taken together, these findings account for the failure of NPC1L1 to serve as a filovirus receptor, highlight the central role of the luminal domain C of NPC1 in filovirus entry, and reveal the direct involvement of N–terminal domain C sequences in NPC1’s function as a filovirus receptor.     

Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies

The filovirus plaque assay serves as the assay of choice to measure infectious virus in a cell culture, blood, or homogenized tissue sample. It has been in use for more than 30 years and is the generally accepted assay used to titrate virus in samples from animals treated with a potential antiviral therapeutic or vaccine. As these animal studies are required for the development of vaccines and therapeutics under the FDA Animal Rule, it is essential to have a standardized assay to compare their efficacies against the various filoviruses. Here, we present an evaluation of the conditions under which the filovirus plaque assay performs best for the Ebola virus Kikwit variant and the Angola variant of Marburg virus. The indicator cell type and source, inoculum volumes, length of incubation and general features of filovirus biology as visualized in the assay are addressed in terms of the impact on the sample viral titer calculations. These optimization studies have resulted in a plaque assay protocol which can be used for preclinical studies, and as a standardized protocol for use across institutions, to aid in data comparison. This protocol will be validated for use in GLP studies supporting advanced development of filovirus therapeutics and vaccines.     

Genetic Characterization of the Tick-Borne Orbiviruses

The International Committee for Taxonomy of Viruses (ICTV) recognizes four species of tick-borne orbiviruses (TBOs): Chenuda virus, Chobar Gorge virus, Wad Medani virus and Great Island virus (genus Orbivirus, family Reoviridae). Nucleotide (nt) and amino acid (aa) sequence comparisons provide a basis for orbivirus detection and classification, however full genome sequence data were only available for the Great Island virus species. We report representative genome-sequences for the three other TBO species (virus isolates: Chenuda virus (CNUV); Chobar Gorge virus (CGV) and Wad Medani virus (WMV)). Phylogenetic comparisons show that TBOs cluster separately from insect-borne orbiviruses (IBOs). CNUV, CGV, WMV and GIV share low level aa/nt identities with other orbiviruses, in ‘conserved’ Pol, T2 and T13 proteins/genes, identifying them as four distinct virus-species. The TBO genome segment encoding cell attachment, outer capsid protein 1 (OC1), is approximately half the size of the equivalent segment from insect-borne orbiviruses, helping to explain why tick-borne orbiviruses have a ~1 kb smaller genome.     

An Interagency Collaboration to Facilitate Development of Filovirus Medical Countermeasures

The Filovirus Animal Non-Clinical Group (FANG) is a US interdepartmental and interagency group established to support and facilitate the advanced development of filovirus Medical Countermeasures (MCM), both vaccines and therapeutics. It is co-led by one representative from the Department of Defense (DoD), the first author, and one from the Department of Health and Human Services (HHS), the second author. The FANG membership includes operational level program staff and Subject Matter Experts (SME) from performing organizations as well as scientific staff and program managers from DoD and HHS funding and regulatory agencies. Focus areas include animal models, assays, reagents, product manufacture and characterization, and other interagency product development issues that will support Food and Drug Administration (FDA) licensure of safe and effective filovirus MCMs. The FANG continues to develop strategies to address broadly applicable and interagency product development challenges relevant to filovirus MCM development. This paper summarizes FANG structure and accomplishments and is meant to heighten community awareness of this government-led collaborative effort.     

A Limited Structural Modification Results in a Significantly More Efficacious Diazachrysene-Based Filovirus Inhibitor

Ebola (EBOV) and Marburg (MARV) filoviruses are highly infectious pathogens causing deadly hemorrhagic fever in humans and non-human primates. Promising vaccine candidates providing immunity against filoviruses have been reported. However, the sporadic nature and swift progression of filovirus disease underlines the need for the development of small molecule therapeutics providing immediate antiviral effects. Herein we describe a brief structural exploration of two previously reported diazachrysene (DAAC)-based EBOV inhibitors. Specifically, three analogs were prepared to examine how slight substituent modifications would affect inhibitory efficacy and inhibitor-mediated toxicity during not only EBOV, but also MARV cellular infection. Of the three analogs, one was highly efficacious, providing IC₅₀ values of 0.696 µM ± 0.13 µM and 2.76 µM ± 0.21 µM against EBOV and MARV infection, respectively, with little or no associated cellular toxicity. Overall, the structure-activity and structure-toxicity results from this study provide a framework for the future development of DAAC-based filovirus inhibitors that will be both active and non-toxic in vivo.     

Antimicrobial activity of bismuth subsalicylate on Clostridium difficile,Escherichia coli O157:H7, norovirus,and other common enteric pathogens

Previous studies have shown bismuth subsalicylate (BSS) has antimicrobial properties, but few studies have addressed the mechanism of action. Furthermore, following BSS ingestion other bismuth salts form throughout the gastrointestinal tract including bismuth oxychloride (BiOCl) that also act upon enteric pathogens. To further understand the antimicrobial activity of bismuth in infectious diarrhea, the antimicrobial effect of BSS and BiOCl on Clostridium difficile, Salmonella, Shigella, Shiga toxin-producing Escherichia coli strains and norovirus (NoV) were measured. Bacterial enteric pathogens in pure culture or in human fecal material were exposed to 35mg/ml BSS or BiOCl with or without a vehicle suspension. BSS and BiOCl treated samples were quantified and visualized by transmission electron microscopy. To measure the effect on NoV, reduction of infectious murine NoV (MNV), a surrogate for human NoV, and Norwalk virus RNA levels were measured by viral plaque assay and RT-qPCR, respectively. BSS and BiOCl reduced bacterial growth by 3–9 logs in all strains with majority resulting in populations of <10 cfu/ml within 24 h. Similar results were found when fecal material was included. Microscopy images detected bismuth on bacterial membranes and within the bacterial organisms at 30 min post-treatment. At 8.8mg/ml BSS and BiOCl reduced infectivity of MNV significantly by 2.7 and 2.0 log after 24 h of exposure. In addition, both BSS and BiOCl slightly reduced the level of Norwalk replicon-bearing cells suggesting that bismuth may inhibit NoV in vivo. Collectively, our results confirm and build on existing data that BSS has antimicrobial properties against a wide-range of diarrhea-causing pathogens.     

Increased activity of Diaphanous homolog 3 (DIAPH3)/diaphanous causes hearing defects in humans with auditory neuropathy and in Drosophila

Auditory neuropathy is a rare form of deafness characterized by an absent or abnormal auditory brainstem response with preservation of outer hair cell function. We have identified Diaphanous homolog 3 (DIAPH3) as the gene responsible for autosomal dominant nonsyndromic auditory neuropathy (AUNA1), which we previously mapped to chromosome 13q21-q24. Genotyping of additional family members narrowed the interval to an 11-Mb, 3.28-cM gene-poor region containing only four genes, including DIAPH3. DNA sequencing of DIAPH3 revealed a c.-172G > A, g. 48G > A mutation in a highly conserved region of the 5′ UTR. The c.-172G > A mutation occurs within a GC box sequence element and was not found in 379 controls. Using genome-wide expression arrays and quantitative RT-PCR, we demonstrate a 2- to 3-fold overexpression of DIAPH3 mRNA in lymphoblastoid cell lines from affected individuals. Likewise, a significant increase (≈1.5-fold) in DIAPH3 protein was found by quantitative immunoblotting of lysates from lymphoblastoid cell lines derived from affected individuals in comparison with controls. In addition, the c.-172G > A mutation is sufficient to drive overexpression of a luciferase reporter. Finally, the expression of a constitutively active form of diaphanous protein in the auditory organ of Drosophila melanogaster recapitulates the phenotype of impaired response to sound. To date, only two genes, the otoferlin gene OTOF and the pejvakin gene PJVK, are known to underlie nonsyndromic auditory neuropathy. Genetic testing for DIAPH3 may be useful for individuals with recessive as well as dominant inheritance of nonsyndromic auditory neuropathy.     

Connexin 32 and 43 promoter methylation in Helicobacter pylori-associated gastric tumorigenesis

AIM: To explore the mechanism of abnormal Connexin (Cx) 32 and Cx43 expression in the gastric mucosa after Helicobacter pylori (H. pylori) infection.METHODS: Biopsy specimens of gastric mucosa in different gastric carcinogenesis stages with H. pylori infection, that is, non-atrophic gastritis (NAG; n = 24), chronic atrophic gastritis (CAG; n = 25), intestinal metaplasia (IM; n = 28), dysplasia (DYS; n = 24), and gastric cancer (GC; n = 30), as well as specimens of normal gastric mucosa without H. pylori infection (NGM; n = 25), were confirmed by endoscopy and pathological examination. Cx32 and Cx43 mRNA expression was detected by real-time polymerase chain reaction (PCR). Cx32 and Cx43 promoter CpG island methylation status was determined by methylation-specific PCR (MSP), bisulfite PCR sequencing (BSP) and MassArray methods.RESULTS: The relative mRNA expression levels in the gastric mucosa of patients with NGM, NAG, CAG, IM, DYS and GC were 0.146 ± 0.011, 0.133 ± 0.026, 0.107 ± 0.035, 0.039 ± 0.032, 0.037 ± 0.01 and 0.03 ± 0.011 for Cx32; and 0.667 ± 0.057, 0.644 ± 0.051, 0.624 ± 0.049, 0.555 ± 0.067, 0.536 ± 0.058 and 0.245 ± 0.121 for Cx43, respectively, which were gradually decreasing and significantly different (GC vs NGM: P < 0.001 for Cx32, P < 0.001 for Cx43). The promoter methylation levels in the gastric mucosa from NGM to GC stages by MSP were 38.8% ± 9.0%, 43.1% ± 9.4%, 56.5% ± 3.1%, 64.4% ± 9.7%, 72.5% ± 4.2% and 79.6% ± 6.8% for Cx32; and 49.0% ± 3.9%, 58.1% ± 5.0%, 66.5% ± 7.9%, 74.0% ± 8.8%, 78.3% ± 3.6% and 88.7% ± 6.2% for Cx43, respectively, which were gradually increasing and significantly different (P = 0.039, P = 0.019). The promoter methylation levels by BSP and MassArray exhibited similar trends. Cx32 and Cx43 mRNA expression was negatively correlated with promoter methylation status and gastric carcinogenesis stages (P < 0.001, P = 0.016).CONCLUSION: Cx32 and Cx43 mRNA expression decreased gradually during H. pylori infection-associated gastric carcinogenesis, and it is associated with hypermethylation of these genes’ promoter.     

Filoviruses: One of These Things is (not) Like the Other

The family Filoviridae contains several of the most deadly pathogens known to date and the current Ebola virus disease (EVD) outbreak in Western Africa, due to Ebola virus (EBOV) infection, highlights the need for active and broad research into filovirus pathogenesis. However, in comparison, the seven other known filovirus family members are significantly understudied. Many of these, including Marburgviruses and Ebolaviruses other than EBOV, are also highly virulent and fully capable of causing widespread epidemics. This review places the focus on these non-EBOV filoviruses, including known immunological and pathological data. The available animal models, research tools and currently available therapeutics will also be discussed along with an emphasis in the large number of current gaps in knowledge of these less highlighted filoviruses. It is evident that much research is yet to be done in order to bring the non-EBOV filovirus field to the forefront of current research and, importantly, to the development of more effective vaccines and therapeutics to combat potential future outbreaks.     

A Call to Action to Enhance Filovirus Disease Outbreak Preparedness and Response

The frequency and magnitude of recognized and declared filovirus-disease outbreaks have increased in recent years, while pathogenic filoviruses are potentially ubiquitous throughout sub-Saharan Africa. Meanwhile, the efficiency and effectiveness of filovirus-disease outbreak preparedness and response efforts are currently limited by inherent challenges and persistent shortcomings. This paper delineates some of these challenges and shortcomings and provides a proposal for enhancing future filovirus-disease outbreak preparedness and response. The proposal serves as a call for prompt action by the organizations that comprise filovirus-disease outbreak response teams, namely, Ministries of Health of outbreak-prone countries, the World Health Organization, Médecins Sans Frontières, the Centers for Disease Control and Prevention—Atlanta, and others.     

Phylogenetic profiles reveal evolutionary relationships within the "twilight zone" of sequence similarity

Inferring evolutionary relationships among highly divergent protein sequences is a daunting task. In particular, when pairwise sequence alignments between protein sequences fall <25% identity, the phylogenetic relationships among sequences cannot be estimated with statistical certainty. Here, we show that phylogenetic profiles generated with the Gestalt Domain Detection Algorithm–Basic Local Alignment Tool (GDDA-BLAST) are capable of deriving, ab initio, phylogenetic relationships for highly divergent proteins in a quantifiable and robust manner. Notably, the results from our computational case study of the highly divergent family of retroelements accord with previous estimates of their evolutionary relationships. Taken together, these data demonstrate that GDDA-BLAST provides an independent and powerful measure of evolutionary relationships that does not rely on potentially subjective sequence alignment. We demonstrate that evolutionary relationships can be measured with phylogenetic profiles, and therefore propose that these measurements can provide key insights into relationships among distantly related and/or rapidly evolving proteins.     

Comparative study of mutations in SNP loci of K-RAS,hMLH1 and hMSH2 genes in neoplastic intestinal polyps and colorectal cancer

AIM: To clarify the molecular mechanism involved in pathogenesis of colorectal cancer as well as clinical significance of genetic analysis of histological samples.METHODS: A total of 480 blood and tissue specimens were collected in our hospital from January 2011 to October 2012. In the observation group, there were 120 blood specimens and 120 intestinal tract tissue specimens collected from patients with neoplastic intestinal polyps. In the control group I there were 80 blood specimens and 80 intestinal tract tissue specimens collected from patients with colorectal cancer. In the control group II there were 40 blood specimens and 40 intestinal tract tissue specimens collected from healthy individuals. The gene segments were amplified using PCR and DNA gel electrophoresis along with DNA sequence analysis were employed for the detection of the following single nucleotide polymorphisms (SNPs): K-RAS codons 12 and 13; hMLH1 (human mutS homolog 1) gene missense mutation at Va1384Asp; hMSH2 (human mutS homolog 2) gene missense mutation at 2783C/A.RESULTS: The mutation rate of the SNP at Va1384Asp locus of the hMLH1 gene from blood and tissue specimens in the observation group showed no statistical difference from those in the control group I. The mutation rates of SNPs in codons 12 and 13 of K-RAS and at 2783C/A locus of the hMSH2 gene were significantly lower in the observation group than in the control group I (χ² = 15.476, 29.670, 10.811, 16.618, 33.538, 7.898, P < 0.05). The mutation rate of SNP at Va1384Asp locus of the hMLH1 gene was significantly higher in the observation group when compared to the control group II (χ² = 10.486, 4.876, P < 0.05). The mutation rates of SNPs in codons 12 and 13 of K-RAS and at 2783C/A locus of the hMSH2 gene did not show any statistical difference from those in the control group II.CONCLUSION: There may be important clinical significance and relevance between neoplastic intestinal polyps and colorectal cancer in terms of the mechanisms involved in the pathogenesis.     

Reciprocal impact of host factors and Helicobacter pylori genotypes on gastric diseases

AIM: To assess the impact of Helicobacter pylori (H. pylori) genotypes and patient age and sex on the development of gastric diseases.METHODS: H. pylori-infected patients (n = 233) referred to the endoscopy unit at Tehran University of Medical Sciences (Tehran, Iran) were diagnosed with chronic gastritis (CG), gastric ulcer (GU), or duodenal ulcer (DU). Brucella blood agar was used for biopsy cultures and H. pylori isolation under microaerobic conditions. H. pylori isolates were confirmed with biochemical tests and through amplification of the 16S rRNA gene. DNA was extracted from fresh cultures of the H. pylori isolates and used for amplification of vacA alleles and the cagA gene. Statistical analysis was performed to determine the association between H. pylori genotypes, age (< 40 years vs > 40 years) and sex of the patient, and gastric diseases.RESULTS: CG was the most prevalent gastric disease (113/233; 48.5%), compared to GU (64/233; 27.5%) and DU (56/233; 24%). More patients were male, and gastric diseases were more frequent in patients > 40 years (P < 0.05). The percentage of CG and GU patients that were male and female did not show a significant difference; however DU was more common in males (P < 0.05). Interestingly, a diagnosis of CG in patients > 40 years was more common in females (18.5%) than males (11.6%) (P = 0.05), whereas a diagnosis of GU or DU in patients > 40 years was more frequent in males (14.6% vs 10.7% and 12.4% vs 4.3%, respectively). Overall, genotyping of the H. pylori isolates revealed that the vacA s1 (82%), vacA m2 (70%), and cagA⁺ (72.5%) alleles were more frequent than vacA s2 (18%), vacA m1 (29.2%), and cagA^- (all P < 0.05). The vacA s1m2cagA⁺ genotype was the most prevalent within the three disease groups. vacA s1m2 frequency was 56.2% with a similar occurrence in all diagnoses, while vacA s1m1 appeared more often in DU patients (33.9%). A genotype of vacA s2m2 occurred in 15% of isolates and was more common in CG patients (21.2%); vacA s2m1 was the least common genotype (3%). The vacA s1 allele was found to be a risk factor for DU, vacA s2 for CG, and vacA s1 and vacA s2 for GU (all P < 0.05). The vacA s2m2 genotype was associated with the development of CG and GU compared to DU (P < 0.05). No correlation was found between vacA m or cagA and gastric diseases.CONCLUSION: The outcome of H. pylori infection is the result of interaction between bacterial genotypes and the age and sex of infected individuals.