Publications by authors named "Isabelle Dietrich"

26 Publications

  • Page 1 of 1

First report of epidemic dengue fever and malaria co-infections among internally displaced persons in humanitarian camps of North Darfur, Sudan.

Int J Infect Dis 2021 Jul 24;108:513-516. Epub 2021 May 24.

World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, TX, USA; Institute for Human Infections and Immunity and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA.

Objectives: This study aimed to investigate an outbreak of a non-malaria, undifferentiated febrile illness, among internally displaced persons (IDPs) living in humanitarian camps in North Darfur, Sudan, in 2019.

Methods: An investigation team was deployed to North Darfur to identify suspected cases and collect blood samples, and clinical and demographical data. Blood samples were examined microscopically for Plasmodium spp and tested for dengue (DENV) and yellow fever viruses by reverse transcriptase-quantitative polymerase chain reaction.

Results: Between September 7 and December 18, 2019, we clinically identified 18 (24%), 41 (54%), and 17 (22%) cases of dengue fever, dengue with warning signs, and severe dengue, respectively. Blood samples were collected from 22% of patients, and 47% of these tested positive for DENV-1 RNA. We confirmed 32 malaria cases with 5 co-infections with DENV. This outbreak of dengue was the first among IDPs in the humanitarian camps.

Conclusions: Our findings indicate that dengue has become endemic or that there has been a new introduction. Further epidemiological, entomological, and phylogenetic studies are needed to understand disease transmission in the area. An early warning and response system and an effective health policy are crucial for preventing and controlling arboviruses in Sudan.
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http://dx.doi.org/10.1016/j.ijid.2021.05.052DOI Listing
July 2021

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses.

Nat Commun 2021 01 22;12(1):542. Epub 2021 Jan 22.

The Pirbright Institute, Ash Road, Pirbright, GU24 0NF, UK.

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.
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http://dx.doi.org/10.1038/s41467-020-20654-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822889PMC
January 2021

The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins.

PLoS Biol 2020 12 21;18(12):e3001016. Epub 2020 Dec 21.

The Pirbright Institute, Woking, Surrey, United Kingdom.

SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, leading to the Coronavirus Disease 2019 (COVID-19) pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock, and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus, we demonstrate that, in addition to human angiotensin-converting enzyme 2 (ACE2), the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the 22 different hosts we investigated, ACE2 proteins from dog, cat, and cattle were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the 3 genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Comparison of SARS-CoV-2 receptor usage to the related coronaviruses SARS-CoV and RaTG13 identified distinct tropisms, with the 2 human viruses being more closely aligned. Finally, using bioinformatics, structural data, and targeted mutagenesis, we identified amino acid residues within the Spike-ACE2 interface, which may have played a pivotal role in the emergence of SARS-CoV-2 in humans. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock, and wildlife.
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http://dx.doi.org/10.1371/journal.pbio.3001016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751883PMC
December 2020

Unique Outbreak of Rift Valley Fever in Sudan, 2019.

Emerg Infect Dis 2020 12;26(12):3030-3033

We report a unique outbreak of Rift Valley fever in the Eldamar area, Sudan, May-July 2019, that resulted in 1,129 case-patients and 19 (1.7%) deaths. Patients exhibited clinical signs including fever (100%), headache (79%), and bleeding (4%). Most (98%) patients also reported death and abortions among their livestock.
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http://dx.doi.org/10.3201/eid2612.201599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706939PMC
December 2020

Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19.

NPJ Vaccines 2020 27;5(1):69. Epub 2020 Jul 27.

The Pirbright Institute, Ash Road, Pirbright, GU24 0NF UK.

Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.
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http://dx.doi.org/10.1038/s41541-020-00221-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385486PMC
July 2020

Identification and RNAi Profile of a Novel Iflavirus Infecting Senegalese Mosquitoes.

Viruses 2020 04 14;12(4). Epub 2020 Apr 14.

Institute for Parasitology, University of Veterinary Medicine Hannover, 30559 Hannover, Germany.

The inland floodwater mosquito (Meigen, 1830) is a competent vector of numerous arthropod-borne viruses such as Rift Valley fever virus () and Zika virus (). spp. have widespread Afrotropical distribution and are common European cosmopolitan mosquitoes. We examined the virome of samples from Barkédji village, Senegal, with small RNA sequencing, bioinformatic analysis, and RT-PCR screening. We identified a novel 9494 nt iflavirus () designated here as Aedes vexans iflavirus (AvIFV). Annotation of the AvIFV genome reveals a 2782 amino acid polyprotein with iflavirus protein domain architecture and typical iflavirus 5' internal ribosomal entry site and 3' poly-A tail. Aedes vexans iflavirus is most closely related to a partial virus sequence from (a parasitoid wasp) with 56.77% pairwise amino acid identity. Analysis of AvIFV-derived small RNAs suggests that AvIFV is targeted by the exogenous RNA interference pathway but not the PIWI-interacting RNA response, as ~60% of AvIFV reads corresponded to 21 nt Dicer-2 virus-derived small RNAs and the 24-29 nt AvIFV read population did not exhibit a "ping-pong" signature. The RT-PCR screens of archival and current (circa 2011-2020) laboratory samples and wild-caught mosquitoes from Barkédji suggest that AvIFV is ubiquitous in these mosquitoes. Further, we screened wild-caught European samples from Germany, the United Kingdom, Italy, and Sweden, all of which tested negative for AvIFV RNA. This report provides insight into the diversity of commensal viruses and the host RNAi response towards iflaviruses.
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http://dx.doi.org/10.3390/v12040440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232509PMC
April 2020

The Aedes aegypti Domino Ortholog p400 Regulates Antiviral Exogenous Small Interfering RNA Pathway Activity and Expression.

mSphere 2020 04 8;5(2). Epub 2020 Apr 8.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland

Arboviruses are pathogens of humans and animals. A better understanding of the interactions between these pathogens and the arthropod vectors, such as mosquitoes, that transmit them is necessary to develop novel control measures. A major antiviral pathway in the mosquito vector is the exogenous small interfering RNA (exo-siRNA) pathway, which is induced by arbovirus-derived double-stranded RNA in infected cells. Although recent work has shown the key role played by Argonaute-2 (Ago-2) and Dicer-2 (Dcr-2) in this pathway, the regulatory mechanisms that govern these pathways have not been studied in mosquitoes. Here, we show that the Domino ortholog p400 has antiviral activity against the alphavirus Semliki Forest virus () both in -derived cells and Antiviral activity of p400 was also demonstrated against chikungunya virus () and Bunyamwera virus (e) but not Zika virus (). p400 was found to be expressed across mosquito tissues and regulated but not transcript levels in mosquitoes. These findings provide novel insights into the regulation of an important aedine exo-siRNA pathway effector protein, Ago-2, by the Domino ortholog p400. They add functional insights to previous observations of this protein's antiviral and RNA interference regulatory activities in Female mosquitoes are vectors of human-infecting arthropod-borne viruses (arboviruses). In recent decades, the incidence of arthropod-borne viral infections has grown dramatically. Vector competence is influenced by many factors, including the mosquito's antiviral defenses. The exogenous small interfering RNA (siRNA) pathway is a major antiviral response restricting arboviruses in mosquitoes. While the roles of the effectors of this pathway, Argonaute-2 and Dicer-2 are well characterized, nothing is known about its regulation in mosquitoes. In this study, we demonstrate that p400, whose ortholog Domino in is a chromatin-remodeling ATPase member of the Tip60 complex, regulates siRNA pathway activity and controls expression levels. In addition, we found p400 to have antiviral activity against different arboviruses. Therefore, our study provides new insights into the regulation of the antiviral response in mosquitoes.
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http://dx.doi.org/10.1128/mSphere.00081-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142294PMC
April 2020

Risks and Challenges of Arboviral Diseases in Sudan: The Urgent Need for Actions.

Viruses 2020 01 9;12(1). Epub 2020 Jan 9.

Institute for Human Infections and Immunity, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77755, USA.

The risk of emergence and/or re-emergence of arthropod-borne viral (arboviral) infections is rapidly growing worldwide, particularly in Africa. The burden of arboviral infections and diseases is not well scrutinized because of the inefficient surveillance systems in endemic countries. Furthermore, the health systems are fully occupied by the burden of other co-existing febrile illnesses, especially malaria. In this review we summarize the epidemiology and risk factors associated with the major human arboviral diseases and highlight the gap in knowledge, research, and control in Sudan. Published data in English up to March 2019 were reviewed and are discussed to identify the risks and challenges for the control of arboviruses in the country. In addition, the lack of suitable diagnostic tools such as viral genome sequencing, and the urgent need for establishing a genomic database of the circulating viruses and potential sources of entry are discussed. Moreover, the research and healthcare gaps and global health threats are analyzed, and suggestions for developing strategic health policy for the prevention and control of arboviruses with focus on building the local diagnostic and research capacity and establishing an early warning surveillance system for the early detection and containment of arboviral epidemics are offered.
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http://dx.doi.org/10.3390/v12010081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019415PMC
January 2020

Use of an Outbred Rat Hepacivirus Challenge Model for Design and Evaluation of Efficacy of Different Immunization Strategies for Hepatitis C Virus.

Hepatology 2020 03 11;71(3):794-807. Epub 2019 Oct 11.

Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK.

Background And Aims: The lack of immunocompetent small animal models for hepatitis C virus (HCV) has greatly hindered the development of effective vaccines. Using rodent hepacivirus (RHV), a homolog of HCV that shares many characteristics of HCV infection, we report the development and application of an RHV outbred rat model for HCV vaccine development.

Approach And Results: Simian adenovirus (ChAdOx1) encoding a genetic immune enhancer (truncated shark class II invariant chain) fused to the nonstructural (NS) proteins NS3-NS5B from RHV (ChAd-NS) was used to vaccinate Sprague-Dawley rats, resulting in high levels of cluster of differentiation 8-positive (CD8 ) T-cell responses. Following RHV challenge (using 10 or 100 times the minimum infectious dose), 42% of vaccinated rats cleared infection within 6-8 weeks, while all mock vaccinated controls became infected with high-level viremia postchallenge. A single, 7-fold higher dose of ChAd-NS increased efficacy to 67%. Boosting with ChAd-NS or with a plasmid encoding the same NS3-NS5B antigens increased efficacy to 100% and 83%, respectively. A ChAdOx1 vector encoding structural antigens (ChAd-S) was also constructed. ChAd-S alone showed no efficacy. Strikingly, when combined with ChAd-NS, ChAD-S produced 83% efficacy. Protection was associated with a strong CD8 interferon gamma-positive recall response against NS4. Next-generation sequencing of a putative RHV escape mutant in a vaccinated rat identified mutations in both identified immunodominant CD8 T-cell epitopes.

Conclusions: A simian adenovirus vector vaccine strategy is effective at inducing complete protective immunity in the rat RHV model. The RHV Sprague-Dawley rat challenge model enables comparative testing of vaccine platforms and antigens and identification of correlates of protection and thereby provides a small animal experimental framework to guide the development of an effective vaccine for HCV in humans.
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http://dx.doi.org/10.1002/hep.30894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154631PMC
March 2020

The role of ZAP and OAS3/RNAseL pathways in the attenuation of an RNA virus with elevated frequencies of CpG and UpA dinucleotides.

Nucleic Acids Res 2019 09;47(15):8061-8083

Nuffield Department of Medicine, Peter Medawar Building for Pathogen Research, University of Oxford, Oxford OX1 3SY, UK.

Zinc finger antiviral protein (ZAP) is a powerful restriction factor for viruses with elevated CpG dinucleotide frequencies. We report that ZAP similarly mediates antiviral restriction against echovirus 7 (E7) mutants with elevated frequencies of UpA dinucleotides. Attenuation of both CpG- and UpA-high viruses and replicon mutants was reversed in ZAP k/o cell lines, and restored by plasmid-derived reconstitution of expression in k/o cells. In pull-down assays, ZAP bound to viral RNA transcripts with either CpG- and UpA-high sequences inserted in the R2 region. We found no evidence that attenuation of CpG- or UpA-high mutants was mediated through either translation inhibition or accelerated RNA degradation. Reversal of the attenuation of CpG-high, and UpA-high E7 viruses and replicons was also achieved through knockout of RNAseL and oligodenylate synthetase 3 (OAS3), but not OAS1. WT levels of replication of CpG- and UpA-high mutants were observed in OAS3 k/o cells despite abundant expression of ZAP, indicative of synergy or complementation of these hitherto unconnected pathways. The dependence on expression of ZAP, OAS3 and RNAseL for CpG/UpA-mediated attenuation and the variable and often low level expression of these pathway proteins in certain cell types, such as those of the central nervous system, has implications for the use of CpG-elevated mutants as attenuated live vaccines against neurotropic viruses.
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http://dx.doi.org/10.1093/nar/gkz581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735852PMC
September 2019

Spindle-E Acts Antivirally Against Alphaviruses in Mosquito Cells.

Viruses 2018 02 18;10(2). Epub 2018 Feb 18.

Bernhard-Nocht-Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, Hamburg 20359, Germany.

Mosquitoes transmit several human- and animal-pathogenic alphaviruses ( family). In alphavirus-infected mosquito cells two different types of virus-specific small RNAs are produced as part of the RNA interference response: short-interfering (si)RNAs and PIWI-interacting (pi)RNAs. The siRNA pathway is generally thought to be the main antiviral pathway. Although an antiviral activity has been suggested for the piRNA pathway its role in host defences is not clear. Knock down of key proteins of the piRNA pathway (Ago3 and Piwi5) in -derived cells reduced the production of alphavirus chikungunya virus (CHIKV)-specific piRNAs but had no effect on virus replication. In contrast, knock down of the siRNA pathway key protein Ago2 resulted in an increase in virus replication. Similar results were obtained when expression of Piwi4 was silenced. Knock down of the helicase Spindle-E (SpnE), an essential co-factor of the piRNA pathway in , resulted in increased virus replication indicating that SpnE acts as an antiviral against alphaviruses such as CHIKV and the related Semliki Forest virus (SFV). Surprisingly, this effect was found to be independent of the siRNA and piRNA pathways in cells and specific for alphaviruses. This suggests a small RNA-independent antiviral function for this protein in mosquitoes.
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http://dx.doi.org/10.3390/v10020088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850395PMC
February 2018

Mutational analysis of Rift Valley fever phlebovirus nucleocapsid protein indicates novel conserved, functional amino acids.

PLoS Negl Trop Dis 2017 12 21;11(12):e0006155. Epub 2017 Dec 21.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom.

Rift Valley fever phlebovirus (RVFV; Phenuiviridae, Phlebovirus) is an important mosquito-borne pathogen of both humans and ruminants. The RVFV genome is composed of tripartite, single stranded, negative or ambisense RNAs. The small (S) segment encodes both the nucleocapsid protein (N) and the non-structural protein (NSs). The N protein is responsible for the formation of the viral ribonucleoprotein (RNP) complexes, which are essential in the virus life cycle and for the transcription and replication of the viral genome. There is currently limited knowledge surrounding the roles of the RVFV nucleocapsid protein in viral infection other than its key functions: N protein multimerisation, encapsidation of the RNA genome and interactions with the RNA-dependent RNA polymerase, L. By bioinformatic comparison of the N sequences of fourteen phleboviruses, mutational analysis, minigenome assays and packaging assays, we have further characterised the RVFV N protein. Amino acids P11 and F149 in RVFV N play an essential role in the function of RNPs and are neither associated with N protein multimerisation nor known nucleocapsid protein functions and may have additional roles in the virus life cycle. Amino acid Y30 exhibited increased minigenome activity despite reduced RNA binding capacity. Additionally, we have determined that the N-terminal arm of N protein is not involved in N-L interactions. Elucidating the fundamental processes that involve the nucleocapsid protein will add to our understanding of this important viral protein and may influence future studies in the development of novel antiviral strategies.
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http://dx.doi.org/10.1371/journal.pntd.0006155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764413PMC
December 2017

CpG and UpA dinucleotides in both coding and non-coding regions of echovirus 7 inhibit replication initiation post-entry.

Elife 2017 09 29;6. Epub 2017 Sep 29.

Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.

Most vertebrate and plant RNA and small DNA viruses suppress genomic CpG and UpA dinucleotide frequencies, apparently mimicking host mRNA composition. Artificially increasing CpG/UpA dinucleotides attenuates viruses through an entirely unknown mechanism. Using the echovirus 7 (E7) model in several cell types, we show that the restriction in E7 replication in mutants with increased CpG/UpA dinucleotides occurred immediately after viral entry, with incoming virions failing to form replication complexes. Sequences of CpG/UpA-high virus stocks showed no evidence of increased mutational errors that would render them replication defective, these viral RNAs were not differentially sequestered in cytoplasmic stress granules nor did they induce a systemic antiviral state. Importantly, restriction was not mediated through effects on translation efficiency since replicons with high CpG/UpA sequences inserted into a non-coding region were similarly replication defective. Host-cells thus possess intrinsic defence pathways that prevent replication of viruses with increased CpG/UpA frequencies independently of codon usage.
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http://dx.doi.org/10.7554/eLife.29112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5659819PMC
September 2017

RNA Interference Restricts Rift Valley Fever Virus in Multiple Insect Systems.

mSphere 2017 May-Jun;2(3). Epub 2017 May 3.

Bernhard-Nocht-Institut für Tropenmedizin, Hamburg, Germany.

The emerging bunyavirus Rift Valley fever virus (RVFV) is transmitted to humans and livestock by a large number of mosquito species. RNA interference (RNAi) has been characterized as an important innate immune defense mechanism used by mosquitoes to limit replication of positive-sense RNA flaviviruses and togaviruses; however, little is known about its role against negative-strand RNA viruses such as RVFV. We show that virus-specific small RNAs are produced in infected mosquito cells, in cells, and, most importantly, also in RVFV vector mosquitoes. By addressing the production of small RNAs in adult sp. and mosquitoes, we showed the presence of virus-derived Piwi-interacting RNAs (piRNAs) not only in sp. but also in mosquitoes, indicating that antiviral RNA interference in mosquitoes is similar to the described activities of RNAi in sp. mosquitoes. We also show that these have antiviral activity, since silencing of RNAi pathway effectors enhances viral replication. Moreover, our data suggest that RVFV does not encode a suppressor of RNAi. These findings point toward a significant role of RNAi in the control of RVFV in mosquitoes. Rift Valley fever virus (RVFV; , ) is an emerging zoonotic mosquito-borne pathogen of high relevance for human and animal health. Successful strategies of intervention in RVFV transmission by its mosquito vectors and the prevention of human and veterinary disease rely on a better understanding of the mechanisms that govern RVFV-vector interactions. Despite its medical importance, little is known about the factors that govern RVFV replication, dissemination, and transmission in the invertebrate host. Here we studied the role of the antiviral RNA interference immune pathways in the defense against RVFV in natural vector mosquitoes and mosquito cells and draw comparisons to the model insect . We found that RVFV infection induces both the exogenous small interfering RNA (siRNA) and piRNA pathways, which contribute to the control of viral replication in insects. Furthermore, we demonstrate the production of virus-derived piRNAs in mosquitoes. Understanding these pathways and the targets within them offers the potential of the development of novel RVFV control measures in vector-based strategies.
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http://dx.doi.org/10.1128/mSphere.00090-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415632PMC
May 2017

The Antiviral RNAi Response in Vector and Non-vector Cells against Orthobunyaviruses.

PLoS Negl Trop Dis 2017 01 6;11(1):e0005272. Epub 2017 Jan 6.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom.

Background: Vector arthropods control arbovirus replication and spread through antiviral innate immune responses including RNA interference (RNAi) pathways. Arbovirus infections have been shown to induce the exogenous small interfering RNA (siRNA) and Piwi-interacting RNA (piRNA) pathways, but direct antiviral activity by these host responses in mosquito cells has only been demonstrated against a limited number of positive-strand RNA arboviruses. For bunyaviruses in general, the relative contribution of small RNA pathways in antiviral defences is unknown.

Methodology/principal Findings: The genus Orthobunyavirus in the Bunyaviridae family harbours a diverse range of mosquito-, midge- and tick-borne arboviruses. We hypothesized that differences in the antiviral RNAi response in vector versus non-vector cells may exist and that could influence viral host range. Using Aedes aegypti-derived mosquito cells, mosquito-borne orthobunyaviruses and midge-borne orthobunyaviruses we showed that bunyavirus infection commonly induced the production of small RNAs and the effects of the small RNA pathways on individual viruses differ in specific vector-arbovirus interactions.

Conclusions/significance: These findings have important implications for our understanding of antiviral RNAi pathways and orthobunyavirus-vector interactions and tropism.
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http://dx.doi.org/10.1371/journal.pntd.0005272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5245901PMC
January 2017

Dengue in Java, Indonesia: Relevance of Mosquito Indices as Risk Predictors.

PLoS Negl Trop Dis 2016 Mar 11;10(3):e0004500. Epub 2016 Mar 11.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom.

Background: No vaccine is currently available for dengue virus (DENV), therefore control programmes usually focus on managing mosquito vector populations. Entomological surveys provide the most common means of characterising vector populations and predicting the risk of local dengue virus transmission. Despite Indonesia being a country strongly affected by DENV, only limited information is available on the local factors affecting DENV transmission and the suitability of available survey methods for assessing risk.

Methodology/principal Findings: We conducted entomological surveys in the Banyumas Regency (Central Java) where dengue cases occur on an annual basis. Four villages were sampled during the dry and rainy seasons: two villages where dengue was endemic, one where dengue cases occurred sporadically and one which was dengue-free. In addition to data for conventional larvae indices, we collected data on pupae indices, and collected adult mosquitoes for species identification in order to determine mosquito species composition and population density. Traditionally used larval indices (House indices, Container indices and Breteau indices) were found to be inadequate as indicators for DENV transmission risk. In contrast, species composition of adult mosquitoes revealed that competent vector species were dominant in dengue endemic and sporadic villages.

Conclusions/significance: Our data suggested that the utility of traditional larvae indices, which continue to be used in many dengue endemic countries, should be re-evaluated locally. The results highlight the need for validation of risk indicators and control strategies across DENV affected areas here and perhaps elsewhere in SE Asia.
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http://dx.doi.org/10.1371/journal.pntd.0004500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788303PMC
March 2016

Antiviral immunity of Anopheles gambiae is highly compartmentalized, with distinct roles for RNA interference and gut microbiota.

Proc Natl Acad Sci U S A 2015 Jan 29;112(2):E176-85. Epub 2014 Dec 29.

Unit of Genetics and Genomics of Insect Vectors, Department of Parasites and Insect Vectors and CNRS Unit of Hosts, Vectors, and Pathogens, Unité de Recherche Associée 3012, Paris, France; Department of Microbiology, University of Minnesota, Minneapolis, MN 55108;

Arboviruses are transmitted by mosquitoes and other arthropods to humans and animals. The risk associated with these viruses is increasing worldwide, including new emergence in Europe and the Americas. Anopheline mosquitoes are vectors of human malaria but are believed to transmit one known arbovirus, o'nyong-nyong virus, whereas Aedes mosquitoes transmit many. Anopheles interactions with viruses have been little studied, and the initial antiviral response in the midgut has not been examined. Here, we determine the antiviral immune pathways of the Anopheles gambiae midgut, the initial site of viral infection after an infective blood meal. We compare them with the responses of the post-midgut systemic compartment, which is the site of the subsequent disseminated viral infection. Normal viral infection of the midgut requires bacterial flora and is inhibited by the activities of immune deficiency (Imd), JAK/STAT, and Leu-rich repeat immune factors. We show that the exogenous siRNA pathway, thought of as the canonical mosquito antiviral pathway, plays no detectable role in antiviral defense in the midgut but only protects later in the systemic compartment. These results alter the prevailing antiviral paradigm by describing distinct protective mechanisms in different body compartments and infection stages. Importantly, the presence of the midgut bacterial flora is required for full viral infectivity to Anopheles, in contrast to malaria infection, where the presence of the midgut bacterial flora is required for protection against infection. Thus, the enteric flora controls a reciprocal protection tradeoff in the vector for resistance to different human pathogens.
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http://dx.doi.org/10.1073/pnas.1412984112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4299212PMC
January 2015

Understanding the Wolbachia-mediated inhibition of arboviruses in mosquitoes: progress and challenges.

J Gen Virol 2014 Mar 16;95(Pt 3):517-530. Epub 2013 Dec 16.

MRC - University of Glasgow Centre for Virus Research, 8 Church Street, Glasgow G11 5JR, UK.

Arthropod-borne viruses (arboviruses) pose a considerable threat to human and animal health, yet effective control measures have proven difficult to implement, and novel means of controlling their replication in arthropod vectors, such as mosquitoes, are urgently required. One of the most exciting approaches to emerge from research on arthropods is the use of the endosymbiotic intracellular bacterium Wolbachia to control arbovirus transmission from mosquito to vertebrate. These α-proteobacteria propagate through insects, in part through modulation of host reproduction, thus ensuring spread through species and maintenance in nature. Since it was discovered that Wolbachia endosymbiosis inhibits insect virus replication in Drosophila species, these bacteria have also been shown to inhibit arbovirus replication and spread in mosquitoes. Importantly, it is not clear how these antiviral effects are mediated. This review will summarize recent work and discuss determinants of antiviral effectiveness that may differ between individual Wolbachia/vector/arbovirus interactions. We will also discuss the application of this approach to field settings and the associated risks.
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http://dx.doi.org/10.1099/vir.0.057422-0DOI Listing
March 2014

Non-structural proteins of arthropod-borne bunyaviruses: roles and functions.

Viruses 2013 Oct 4;5(10):2447-68. Epub 2013 Oct 4.

Department of Botany and Microbiology, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia.

Viruses within the Bunyaviridae family are tri-segmented, negative-stranded RNA viruses. The family includes several emerging and re-emerging viruses of humans, animals and plants, such as Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, La Crosse virus, Schmallenberg virus and tomato spotted wilt virus. Many bunyaviruses are arthropod-borne, so-called arboviruses. Depending on the genus, bunyaviruses encode, in addition to the RNA-dependent RNA polymerase and the different structural proteins, one or several non-structural proteins. These non-structural proteins are not always essential for virus growth and replication but can play an important role in viral pathogenesis through their interaction with the host innate immune system. In this review, we will summarize current knowledge and understanding of insect-borne bunyavirus non-structural protein function(s) in vertebrate, plant and arthropod.
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http://dx.doi.org/10.3390/v5102447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814597PMC
October 2013

Recent developments in human immunodeficiency virus-1 latency research.

J Gen Virol 2013 May 30;94(Pt 5):917-932. Epub 2013 Jan 30.

MRC-University of Glasgow Centre for Virus Research, Bearsden Road, Glasgow G61 1QH, UK.

Almost 30 years after its initial discovery, infection with the human immunodeficiency virus-1 (HIV-1) remains incurable and the virus persists due to reservoirs of latently infected CD4(+) memory T-cells and sanctuary sites within the infected individual where drug penetration is poor. Reactivating latent viruses has been a key strategy to completely eliminate the virus from the host, but many difficulties and unanswered questions remain. In this review, the latest developments in HIV-persistence and latency research are presented.
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http://dx.doi.org/10.1099/vir.0.049296-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3709588PMC
May 2013

Conditional mutagenesis of a novel choline kinase demonstrates plasticity of phosphatidylcholine biogenesis and gene expression in Toxoplasma gondii.

J Biol Chem 2012 May 26;287(20):16289-99. Epub 2012 Mar 26.

Institute of Biology, Department of Molecular Parasitology, Humboldt University, Philippstrasse 13, House 14, 10115 Berlin, Germany.

The obligate intracellular and promiscuous protozoan parasite Toxoplasma gondii needs an extensive membrane biogenesis that must be satisfied irrespective of its host-cell milieu. We show that the synthesis of the major lipid in T. gondii, phosphatidylcholine (PtdCho), is initiated by a novel choline kinase (TgCK). Full-length (∼70-kDa) TgCK displayed a low affinity for choline (K(m) ∼0.77 mM) and harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. Conditional mutagenesis of the TgCK gene in T. gondii attenuated the protein level by ∼60%, which was abolished in the off state of the mutant (Δtgck(i)). Unexpectedly, the mutant was not impaired in its growth and exhibited a normal PtdCho biogenesis. The parasite compensated for the loss of full-length TgCK by two potential 53- and 44-kDa isoforms expressed through a cryptic promoter identified within exon 1. TgCK-Exon1 alone was sufficient in driving the expression of GFP in E. coli. The presence of a cryptic promoter correlated with the persistent enzyme activity, PtdCho synthesis, and susceptibility of T. gondii to a choline analog, dimethylethanolamine. Quite notably, the mutant displayed a regular growth in the off state despite a 35% decline in PtdCho content and lipid synthesis, suggesting a compositional flexibility in the membranes of the parasite. The observed plasticity of gene expression and membrane biogenesis can ensure a faithful replication and adaptation of T. gondii in disparate host or nutrient environments.
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http://dx.doi.org/10.1074/jbc.M112.347138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3351348PMC
May 2012

Restriction of the felid lentiviruses by a synthetic feline TRIM5-CypA fusion.

Vet Immunol Immunopathol 2011 Oct 12;143(3-4):235-42. Epub 2011 Jun 12.

Centre for Virus Research, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Bearsden Road, Glasgow G61 1QH, United Kingdom.

Gene therapy approaches to the treatment of HIV infection have targeted both viral gene expression and the cellular factors that are essential for virus replication. However, significant concerns have been raised regarding the potential toxic effects of such therapies, the emergence of resistant viral variants and unforeseen biological consequences such as enhanced susceptibility to unrelated pathogens. Novel restriction factors formed by the fusion of the tripartite motif protein (TRIM5) and cyclophilin A (CypA), or "TRIMCyps", offer an effective antiviral defence strategy with a very low potential for toxicity. In order to investigate the potential therapeutic utility of TRIMCyps in gene therapy for AIDS, a synthetic fusion protein between feline TRIM5 and feline CypA was generated and transduced into cells susceptible to infection with feline immunodeficiency virus (FIV). The synthetic feline TRIMCyp was highly efficient at preventing infection with both HIV and FIV and the cells resisted productive infection with FIV from either the domestic cat or the puma. Feline TRIMCyp and FIV infection of the cat offers a unique opportunity to evaluate TRIMCyp-based approaches to genetic therapy for HIV infection and the treatment of AIDS.
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http://dx.doi.org/10.1016/j.vetimm.2011.06.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4261132PMC
October 2011

The role of BST2/tetherin in feline retrovirus infection.

Vet Immunol Immunopathol 2011 Oct 12;143(3-4):255-64. Epub 2011 Jun 12.

Retrovirus Research Laboratory, MRC - University of Glasgow Centre for Virus Research, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Bearsden Road, Glasgow G611QH, United Kingdom.

Pathogenic retroviral infections of mammals have induced the evolution of cellular anti-viral restriction factors and have shaped their biological activities. This intrinsic immunity plays an important role in controlling viral replication and imposes a barrier to viral cross-species transmission. Well-studied examples of such host restriction factors are TRIM5α, an E3 ubiquitin ligase that binds incoming retroviral capsids in the cytoplasm via its C-terminal PRY/SPRY (B30.2) domain and targets them for proteasomal degradation, and APOBEC3 proteins, cytidine deaminases that induce hypermutation and impair viral reverse transcription. Tetherin (BST-2, CD317) is an interferon-inducible transmembrane protein that potently inhibits the release of nascent retrovirus particles in single-cycle replication assays. However, whether the primary biological activity of tetherin in vivo is that of a restriction factor remains uncertain as recent studies on human tetherin suggest that it is unable to prevent spreading infection of human immunodeficiency virus type 1 (HIV-1). The feline tetherin homologue resembles human tetherin in amino acid sequence, protein topology and anti-viral activity. Transiently expressed feline tetherin displays potent inhibition of feline immunodeficiency virus (FIV) and HIV-1 particle release. However, stable ectopic expression of feline tetherin in a range of feline cell lines has no inhibitory effect on the growth of either primary or cell culture-adapted strains of FIV. By comparing and contrasting the activities of the felid and primate tetherins against their respective immunodeficiency-causing lentiviruses we may gain insight into the contribution of tetherins to the control of lentiviral replication and the evolution of lentiviral virulence.
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http://dx.doi.org/10.1016/j.vetimm.2011.06.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4261133PMC
October 2011

Feline tetherin efficiently restricts release of feline immunodeficiency virus but not spreading of infection.

J Virol 2011 Jun 13;85(12):5840-52. Epub 2011 Apr 13.

MRC-University of Glasgow Centre for Virus Research, University of Glasgow, Glasgow, United Kingdom.

Domestic cats endure infections by all three subfamilies of the retroviridae: lentiviruses (feline immunodeficiency virus [FIV]), gammaretroviruses (feline leukemia virus [FeLV]), and spumaretroviruses (feline foamy virus [FFV]). Thus, cats present an insight into the evolution of the host-retrovirus relationship and the development of intrinsic/innate immune mechanisms. Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterize the feline homologue of tetherin and assess its effects on the replication of FIV. Tetherin was expressed in many feline cell lines, and expression was induced by interferons, including alpha interferon (IFN-α), IFN-ω, and IFN-γ. Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release; however, this activity resisted antagonism by either HIV-1 Vpu or the FIV Env and "OrfA" proteins. Further, as overexpression of complete FIV genomes in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV; indeed, syncytium formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Thus, while tetherin may prevent the release of nascent viral particles, cell-to-cell spread remains efficient in the presence of abundant viral receptors and tetherin upregulation may enhance syncytium formation. Accordingly, tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell-to-cell spread.
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http://dx.doi.org/10.1128/JVI.00071-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126296PMC
June 2011

Potent lentiviral restriction by a synthetic feline TRIM5 cyclophilin A fusion.

J Virol 2010 Sep 16;84(17):8980-5. Epub 2010 Jun 16.

Retrovirus Research Laboratory, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, United Kingdom.

A synthetic feline TRIM5-cyclophilin A fusion protein (feTRIMCyp) was generated and transduced into feline cells. feTRIMCyp was highly efficient at preventing infection with human (HIV) and feline (FIV) immunodeficiency virus pseudotypes, and feTRIMCyp-expressing cells resisted productive infection with either FIV-Fca or FIV-Pco. The restriction of FIV infection by feTRIMCyp was reversed by the cyclosporine (Cs) derivatives NIM811 and Debio-025 but less so by Cs itself. FeTRIMCyp and FIV infections of the cat offer a unique opportunity to evaluate TRIMCyp-based approaches to genetic therapy for HIV infection and the treatment of AIDS.
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http://dx.doi.org/10.1128/JVI.00858-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2919025PMC
September 2010
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