[Show abstract][Hide abstract] ABSTRACT: Background:
HCV replication in persistently infected cell culture remains resistant to IFN-α/RBV combination treatment, whereas IFN-λ1 induces viral clearance. The antiviral mechanisms by which IFN-λ1 induces sustained HCV clearance have not been determined.
To investigate the mechanisms by which IFN-λ clears HCV replication in an HCV cell culture model.
IFN-α sensitive (S3-GFP) and resistant (R4-GFP) cells were treated with equivalent concentrations of either IFN-α or IFN-λ. The relative antiviral effects of IFN-α and IFN-λ1 were compared by measuring the HCV replication, quantification of HCV-GFP expression by flow cytometry, and viral RNA levels by real time RT-PCR. Activation of Jak-Stat signaling, interferon stimulated gene (ISG) expression, and miRNA-122 transcription in S3-GFP and R4-GFP cells were examined.
We have shown that IFN-λ1 induces HCV clearance in IFN-α resistant and sensitive replicon cell lines in a dose dependent manner through Jak-Stat signaling, and induces STAT 1 and STAT 2 activation, ISRE-luciferase promoter activation and ISG expression. Stat 3 activation is also involved in IFN-λ1 induced antiviral activity in HCV cell culture. IFN-λ1 induced Stat 3 phosphorylation reduces the expression of hepatocyte nuclear factor 4 alpha (HNF4α) through miR-24 in R4-GFP cells. Reduced expression of HNF4α is associated with decreased expression of miR-122 resulting in an anti-HCV effect. Northern blot analysis confirms that IFN-λ1 reduces miR-122 levels in R4-GFP cells. Our results indicate that IFN-λ1 activates the Stat 3-HNF4α feedback inflammatory loop to inhibit miR-122 transcription in HCV cell culture.
In addition to the classical Jak-Stat antiviral signaling pathway, IFN-λ1 inhibits HCV replication through the suppression of miRNA-122 transcription via an inflammatory Stat 3-HNF4α feedback loop. Inflammatory feedback circuits activated by IFNs during chronic inflammation expose non-responders to the risk of hepatocellular carcinoma.
[Show abstract][Hide abstract] ABSTRACT: The 1000 Genomes Project set out to provide a comprehensive description of common human genetic variation by applying whole-genome sequencing to a diverse set of individuals from multiple populations. Here we report completion of the project, having reconstructed the genomes of 2,504 individuals from 26 populations using a combination of low-coverage whole-genome sequencing, deep exome sequencing, and dense microarray genotyping. We characterized a broad spectrum of genetic variation, in total over 88 million variants (84.7 million single nucleotide polymorphisms (SNPs), 3.6 million short insertions/deletions (indels), and 60,000 structural variants), all phased onto high-quality haplotypes. This resource includes >99% of SNP variants with a frequency of >1% for a variety of ancestries. We describe the distribution of genetic variation across the global sample, and discuss the implications for common disease studies.
[Show abstract][Hide abstract] ABSTRACT: Background:
Throughout the 2014-2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples.
Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development.
Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus-specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone.
The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins.
Full-text · Article · Jul 2015 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: dence for import or export of EBOV across national borders after its initial introduction. Using high-depth
The 2013–2015 Ebola virus disease (EVD) epidemic is caused by the Makona variant of Ebola virus (EBOV). Early in the epidemic, genome sequencing provided insights into virus evolution and transmission and offered important information for outbreak response. Here, we analyze sequences from 232 patients sampled over 7 months in Sierra Leone, along with 86 previously released genomes from earlier in the epidemic. We confirm sustained human-to-human transmission within Sierra Leone and find no evidence for import or export of EBOV across national borders after its initial introduction. Using high-depth replicate sequencing, we observe both host-to-host transmission and recurrent emergence of intrahost genetic variants. We trace the increasing impact of purifying selection in suppressing the accumulation of nonsynonymous mutations over time. Finally, we note changes in the mucin-like domain of EBOV glycoprotein that merit further investigation. These findings clarify the movement of EBOV within the re- gion and describe viral evolution during prolonged human-to-human transmission.
[Show abstract][Hide abstract] ABSTRACT: Lassa fever (LF) is a potentially fatal disease that affects an estimated 300,000-500,000 people in endemic areas of west Africa each year. Though past studies have identified fatality rates of 5-20% in patients suspected to have contracted Lassa virus (LASV), new studies using more precise clinical diagnoses and modern diagnostic assays show fatalities rates above 60% in acutely ill patients from endemic regions. Currently, there are no approved vaccines or therapeutics, and only one Comformité Européenne (CE) marked rapid immunodiagnostic for acute LASV infection. Therefore, preventing LASV transmission is the primary goal in endemic regions. Development of rapid immunodiagnostics and research into the efficacy of current treatment options continues toward saving lives in west Africa as well as creating a line of defense against the nefarious use of LASV in bioterrorism settings.
[Show abstract][Hide abstract] ABSTRACT: Next-generation sequencing (NGS) has the potential to transform the discovery of viruses causing unexplained acute febrile illness (UAFI) because it does not depend on culturing the pathogen or a priori knowledge of the pathogen's nucleic acid sequence. More generally, it has the potential to elucidate the complete human virome, including viruses that cause no overt symptoms of disease, but may have unrecognized immunological or developmental consequences. We have used NGS to identify RNA viruses in the blood of 195 patients with UAFI and compared them with those found in 328 apparently healthy (i.e., no overt signs of illness) control individuals, all from communities in southeastern Nigeria. Among UAFI patients, we identified the presence of nucleic acids from several well-characterized pathogenic viruses, such as HIV-1, hepatitis, and Lassa virus. In our cohort of healthy individuals, however, we detected the nucleic acids of two novel rhabdoviruses. These viruses, which we call Ekpoma virus-1 (EKV-1) and Ekpoma virus-2 (EKV-2), are highly divergent, with little identity to each other or other known viruses. The most closely related rhabdoviruses are members of the genus Tibrovirus and Bas-Congo virus (BASV), which was recently identified in an individual with symptoms resembling hemorrhagic fever. Furthermore, by conducting a serosurvey of our study cohort, we find evidence for remarkably high exposure rates to the identified rhabdoviruses. The recent discoveries of novel rhabdoviruses by multiple research groups suggest that human infection with rhabdoviruses might be common. While the prevalence and clinical significance of these viruses are currently unknown, these viruses could have previously unrecognized impacts on human health; further research to understand the immunological and developmental impact of these viruses should be explored. More generally, the identification of similar novel viruses in individuals with and without overt symptoms of disease highlights the need for a broader understanding of the human virome as efforts for viral detection and discovery advance.
[Show abstract][Hide abstract] ABSTRACT: Filoviruses, such as Ebola and Marburg viruses, cause severe outbreaks of human infection, including the extensive epidemic of Ebola virus disease (EVD) in West Africa in 2014. In the course of examining mutations in the glycoprotein gene associated with 2014 Ebola virus (EBOV) sequences, a differential level of conservation was noted between the soluble form of glycoprotein (sGP) and the full length glycoprotein (GP), which are both encoded by the GP gene via RNA editing. In the region of the proteins encoded after the RNA editing site sGP was more conserved than the overlapping region of GP when compared to a distant outlier species, Tai Forest ebolavirus. Half of the amino acids comprising the "delta peptide", a 40 amino acid carboxy-terminal fragment of sGP, were identical between otherwise widely divergent species. A lysine-rich amphipathic peptide motif was noted at the carboxyl terminus of delta peptide with high structural relatedness to the cytolytic peptide of the non-structural protein 4 (NSP4) of rotavirus. EBOV delta peptide is a candidate viroporin, a cationic pore-forming peptide, and may contribute to EBOV pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: Background
Zoonotic infections, which transmit from animals to humans, form the majority of new human pathogens. Following zoonotic transmission, the pathogen may already have, or may acquire, the ability to transmit from human to human. With infections such as Lassa fever (LF), an often fatal, rodent-borne, hemorrhagic fever common in areas of West Africa, rodent-to-rodent, rodent-to-human, human-to-human and even human-to-rodent transmission patterns are possible. Indeed, large hospital-related outbreaks have been reported. Estimating the proportion of transmission due to human-to-human routes and related patterns (e.g. existence of super-spreaders), in these scenarios is challenging, but essential for planned interventions.
Here, we make use of an innovative modeling approach to analyze data from published outbreaks and the number of LF hospitalized patients to Kenema Government Hospital in Sierra Leone to estimate the likely contribution of human-to-human transmission. The analyses show that almost of the cases at KGH are secondary cases arising from human-to-human transmission. However, we found much of this transmission is associated with a disproportionally large impact of a few individuals (‘super-spreaders’), as we found only of human cases result in an effective reproduction number (i.e. the average number of secondary cases per infectious case) , with a maximum value up to .
This work explains the discrepancy between the sizes of reported LF outbreaks and a clinical perception that human-to-human transmission is low. Future assessment of risks of LF and infection control guidelines should take into account the potentially large impact of super-spreaders in human-to-human transmission. Our work highlights several neglected topics in LF research, the occurrence and nature of super-spreading events and aspects of social behavior in transmission and detection.
[Show abstract][Hide abstract] ABSTRACT: Abstract Lassa fever (LF) is a severe viral hemorrhagic fever caused by Lassa virus (LASV). The LF program at the Kenema Government Hospital (KGH) in Eastern Sierra Leone currently provides diagnostic services and clinical care for more than 500 suspected LF cases per year. Nearly two-thirds of suspected LF patients presenting to the LF Ward test negative for either LASV antigen or anti-LASV immunoglobulin M (IgM), and therefore are considered to have a non-Lassa febrile illness (NLFI). The NLFI patients in this study were generally severely ill, which accounts for their high case fatality rate of 36%. The current studies were aimed at determining possible causes of severe febrile illnesses in non-LF cases presenting to the KGH, including possible involvement of filoviruses. A seroprevalence survey employing commercial enzyme-linked immunosorbent assay tests revealed significant IgM and IgG reactivity against dengue virus, chikungunya virus, West Nile virus (WNV), Leptospira, and typhus. A polymerase chain reaction-based survey using sera from subjects with acute LF, evidence of prior LASV exposure, or NLFI revealed widespread infection with Plasmodium falciparum malaria in febrile patients. WNV RNA was detected in a subset of patients, and a 419 nt amplicon specific to filoviral L segment RNA was detected at low levels in a single patient. However, 22% of the patients presenting at the KGH between 2011 and 2014 who were included in this survey registered anti-Ebola virus (EBOV) IgG or IgM, suggesting prior exposure to this agent. The 2014 Ebola virus disease (EVD) outbreak is already the deadliest and most widely dispersed outbreak of its kind on record. Serological evidence reported here for possible human exposure to filoviruses in Sierra Leone prior to the current EVD outbreak supports genetic analysis that EBOV may have been present in West Africa for some time prior to the 2014 outbreak.
No preview · Article · Dec 2014 · Viral Immunology
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0519-7) contains supplementary material, which is available to authorized users.