Inactivation of Plasmodium falciparum in whole blood by riboflavin plus irradiation.

BACKGROUND: Malaria parasites are frequently transmitted by unscreened blood transfusions in Africa. Pathogen reduction methods in whole blood would thus greatly improve blood safety. We aimed to determine the efficacy of riboflavin plus irradiation for treatment of whole blood infected with Plasmodium falciparum. STUDY DESIGN AND METHODS: Blood was inoculated with 10(4) or 10(5) parasites/mL and riboflavin treated with or without ultraviolet (UV) irradiation (40-160 J/mL red blood cells [mL(RBCs)]). Parasite genome integrity was assessed by quantitative amplification inhibition assays, and P. falciparum viability was monitored in vitro. RESULTS: Riboflavin alone did not affect parasite genome integrity or parasite viability. Application of UV after riboflavin treatment disrupted parasite genome integrity, reducing polymerase-dependent amplification by up to 2 logs (99%). At 80 J/mL(RBCs), riboflavin plus irradiation prevented recovery of viable parasites in vitro for 2 weeks, whereas untreated controls typically recovered to approximately 2% parasitemia after 4 days of in vitro culture. Exposure of blood to 160 J/mL(RBCs) was not associated with significant hemolysis. CONCLUSIONS: Riboflavin plus irradiation treatment of whole blood damages parasite genomes and drastically reduces P. falciparum viability in vitro. In the absence of suitable malaria screening assays, parasite inactivation should be investigated for prevention of transfusion-transmitted malaria in highly endemic areas

Investigating intra-host and intra-herd sequence diversity of foot-and-mouth disease virus

Due to the poor-fidelity of the enzymes involved in RNA genome replication, foot-and-mouth disease (FMD) virus samples comprise of unique polymorphic populations. In this study, deep sequencing was utilised to characterise the diversity of FMD virus (FMDV) populations in 6 infected cattle present on a single farm during the series of outbreaks in the UK in 2007. A novel RT–PCR method was developed to amplify a 7.6 kb nucleotide fragment encompassing the polyprotein coding region of the FMDV genome. Illumina sequencing of each sample identified the fine polymorphic structures at each nucleotide position, from consensus level changes to variants present at a 0.24% frequency. These data were used to investigate population dynamics of FMDV at both herd and host levels, evaluate the impact of host on the viral swarm structure and to identify transmission links with viruses recovered from other farms in the same series of outbreaks. In 7 samples, from 6 different animals, a total of 5 consensus level variants were identified, in addition to 104 sub-consensus variants of which 22 were shared between 2 or more animals. Further analysis revealed differences in swarm structures from samples derived from the same animal suggesting the presence of distinct viral populations evolving independently at different lesion sites within the same infected animal

The detection and role of human endogenous retrovirus K (HML-2) in rheumatoid arthritis

Human endogenous retroviruses are the remnants of ancient retroviral infections present within our genome. These molecular fossils show similarities with present day exogenous retroviruses but act as typical Mendelian elements that are passed vertically between generations. Despite being repeatedly linked to a number of autoimmune diseases and disorders, no conclusive proof has been identified. Rheumatoid arthritis (RA) is one such disease which has been associated with an increase in HERV expression, compared to controls. In order to elucidate a clear role for HERVs in RA pathogenesis, autoantigens implicated in disease pathogenesis were scanned for sequence homology to retroviral genes. Such epitopes would induce antibodies cross reactive with host proteins, resulting in disease. Short peptides mimicking these regions were synthesised and the prevalence of anti-HERV antibodies was determined in RA patients and disease controls. Additionally, a novel real-time Polymerase Chain Reaction (PCR) assay was developed to accurately quantify levels of HERV-K (HML-2) gag expression, relative to normalised levels of housekeeping gene expression. Both serological and molecular assays showed significant increases in HERV-K (HML-2) activity in RA patients compared to disease controls with CD4+ lymphocytes harbouring the highest activity. The real-time assay was also used to determine whether factors within the synovium could modulate HERVs, resulting in their upregulation. Exogenous viral protein expression and pro-inflammatory cytokines were shown to exert a significant modulatory effect over HERV-K (HML-2) transcription. From this data, it is clear that RA patients have increased levels of HERV-K (HML-2) gag activity compared to controls. Despite this it is likely that factors within the synovium such as exogenous viral expression and pro-inflammatory cytokines also influence HERV-K (HML-2) transcription possibly contributing to a role of bystander activation, i.e. being influenced by external factors, rather than actively contributing to disease processes. The exact role of HERVs in RA pathology remains elusive; however this research proposes several mechanisms by which HERV-K (HML-2) may contribute to disease.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Attenuation of infectious bronchitis virus in eggs results in different patterns of genomic variation across multiple replicates

The gammacoronavirus infectious bronchitis virus (IBV) causes an acute, highly contagious respiratory disease of poultry. Live attenuated vaccines are traditionally generated by serial passage of a virulent strain in embryonated chicken eggs, however the molecular mechanism of attenuation is unknown. The virulent lab adapted strain of IBV, M41-CK, was egg-passaged over one hundred times in four parallel independent replicates. All four final egg-passaged viruses were attenuated and exhibited similar growth phenotypes in adult chicken kidney cells and tracheal organ cultures. The virus populations were sequenced by 454 pyrosequencing at the end of passaging, showing that overall sequence diversity in the IBV population increased but the four replicates only had between 11 and 17 consensus-level single nucleotide polymorphisms (SNPs). Although hotspots of variation were identified in spike and nucleocapsid structural proteins as well as the 3' untranslated region, each attenuated virus possessed a different pattern of genomic variation. Overall, only a small number of consensus-level SNPs were acquired during egg passage, leaving a potentially short route back to virulence. These results highlight the unpredictable nature of attenuation by serial egg passage and the need to develop mechanisms to rationally attenuate IBV for the next generation of effective vaccines. Infectious Bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines are currently developed by serial passage of a virulent strain on embryonated hen's eggs until attenuation, however little is known about the evolution of the viral population during the process of attenuation. High throughput sequencing of four replicates of a serially egg-passaged IBV revealed a different pattern of genomic variation in each attenuated replicate and few consensus-level SNPs. This raises concerns that only a small number of genomic mutations are required to revert to a virulent phenotype, which may result in vaccine breakdown in the field. The observed hotspots of variation in the attenuated viruses has the potential to be used in the rational attenuation of virulent IBV for next generation vaccine design

Multiple novel non-canonically transcribed sub-genomic mRNAs produced by avian coronavirus infectious bronchitis virus

Funding: This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/L003988/1 and 1645891, and strategic funding to The Pirbright Institute, BBS/E/I/00007035, BBS/E/I/00007034, BBS/E/I/00007037 and BBS/E/I/00007039.Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5' untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3'-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified.Publisher PDFPeer reviewe

Sequencing and Analysis of Lumpy Skin Disease Virus Whole Genomes Reveals a New Viral Subgroup in West and Central Africa

Lumpy skin disease virus (LSDV) is a member of the capripoxvirus (CPPV) genus of the Poxviridae family. LSDV is a rapidly emerging, high-consequence pathogen of cattle, recently spreading from Africa and the Middle East into Europe and Asia. We have sequenced the whole genome of historical LSDV isolates from the Pirbright Institute virus archive, and field isolates from recent disease outbreaks in Sri Lanka, Mongolia, Nigeria and Ethiopia. These genome sequences were compared to published genomes and classified into different subgroups. Two subgroups contained vaccine or vaccine-like samples ("Neethling-like" clade 1.1 and "Kenya-like" subgroup, clade 1.2.2). One subgroup was associated with outbreaks of LSD in the Middle East/Europe (clade 1.2.1) and a previously unreported subgroup originated from cases of LSD in west and central Africa (clade 1.2.3). Isolates were also identified that contained a mix of genes from both wildtype and vaccine samples (vaccine-like recombinants, grouped in clade 2). Whole genome sequencing and analysis of LSDV strains isolated from different regions of Africa, Europe and Asia have provided new knowledge of the drivers of LSDV emergence, and will inform future disease control strategies.</p

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

The Detection and Role of Human Endogenous Retrovirus K (HML-2) In Rheumatoid Arthritis

A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of PhilosophyHuman endogenous retroviruses are the remnants of ancient retroviral infections present within our genome. These molecular fossils show similarities with present day exogenous retroviruses but act as typical Mendelian elements that are passed vertically between generations. Despite being repeatedly linked to a number of autoimmune diseases and disorders, no conclusive proof has been identified. Rheumatoid arthritis (RA) is one such disease which has been associated with an increase in HERV expression, compared to controls. In order to elucidate a clear role for HERVs in RA pathogenesis, autoantigens implicated in disease pathogenesis were scanned for sequence homology to retroviral genes. Such epitopes would induce antibodies cross reactive with host proteins, resulting in disease. Short peptides mimicking these regions were synthesised and the prevalence of anti-HERV antibodies was determined in RA patients and disease controls. Additionally, a novel real-time Polymerase Chain Reaction (PCR) assay was developed to accurately quantify levels of HERV-K (HML-2) gag expression, relative to normalised levels of housekeeping gene expression. Both serological and molecular assays showed significant increases in HERV-K (HML-2) activity in RA patients compared to disease controls with CD4+ lymphocytes harbouring the highest activity. The real-time assay was also used to determine whether factors within the synovium could modulate HERVs, resulting in their upregulation. Exogenous viral protein expression and pro-inflammatory cytokines were shown to exert a significant modulatory effect over HERV-K (HML-2) transcription. From this data, it is clear that RA patients have increased levels of HERV-K (HML-2) gag activity compared to controls. Despite this it is likely that factors within the synovium such as exogenous viral expression and pro-inflammatory cytokines also influence HERV-K (HML-2) transcription possibly contributing to a role of bystander activation, i.e. being influenced by external factors, rather than actively contributing to disease processes. The exact role of HERVs in RA pathology remains elusive; however this research proposes several mechanisms by which HERV-K (HML-2) may contribute to disease

Comparison of Antigenic Regions Identified on IgG1Fc Using Bioinformatics vs Pepscan Analysis

Clinical Medicine: Arthritis and Musculoskeletal Disorders is an international, open access, peer reviewed journal.Epitope mapping allowed the location of antigenic determinants on a protein macromolecule to be identified. In particular, pepscan techniques that utilize a series of overlapping peptides, help detect key amino acid residues that are important in antibody recognition and binding. In a previous study, we employed 15-mer peptides spanning the entire length of IgG1Fc to ascertain successfully the target epitopes of isotypic/allotypic monoclonal reagents. As an extension to this work we have used these peptides to evaluate the location of epitope targets of five IgM rheumatoid factor antibodies (RFAbs). Overall, 2 antibodies, RFAb TS2 and TS1, detected a similar epitope within the CH3 domain (360-KNQVSLTCLVKGFYP-374), whilst 1 (RFAb SJ1) recognised an epitope in the CH2 domain (294- EQYNSTYRVVSVLTV-308). In contrast, 2 RFAbs, PRSJ2 and PRTS1 detected four and five epitopes respectively within the Fc region. RFAb PRSJ2 recognised epitopes detected by RFAB TS2 and TS1 but also further epitopes in the CH2 domain (256-TPEVTCVVVDVSHED-270) and CH3 domain (418-QQGNVFSCSVMHEAL-432). Similarly, RFAb PRTS1 detected all four epitopes plus a fifth in the CH3 domain (382-ESNGQPENNYKTTPP-396). In essence there was a consensus of target epitopes identified by these rheumatoid factor antibodies. Interestingly, two epitopes (256–270, CH2 domain and 360–374, CH3 domain) were novel in that they had not been identified in previous pepscan studies. The other epitopes recognised, either overlapped or were immediately adjacent to previous epitopes detected by poly/monoclonal rheumatoid factor antibodies. Molecular modelling (PCImdad) of IgG1Fc showed that all five epitopes were exposed and surface accessible for antibody interaction. In addition, a bioinformatics analysis of the Fc region using ExPASy was employed to identify key antigenic determinants. This ‘in silico’ approach may provide a means of determining key regions without the need to develop overlapping peptides spanning the entire length of a macromolecule