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

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The impact of within-herd genetic variation upon inferred transmission trees for foot-and-mouth disease virus

Full-genome sequences have been used to monitor the fine-scale dynamics of epidemics caused by RNA viruses. However, the ability of this approach to confidently reconstruct transmission trees is limited by the knowledge of the genetic diversity of viruses that exist within different epidemiological units. In order to address this question, this study investigated the variability of 45 foot-and-mouth disease virus (FMDV) genome sequences (from 33 animals) that were collected during 2007 from eight premises (10 different herds) in the United Kingdom. Bayesian and statistical parsimony analysis demonstrated that these sequences exhibited clustering which was consistent with a transmission scenario describing herd-to-herd spread of the virus. As an alternative to analysing all of the available samples in future epidemics, the impact of randomly selecting one sequence from each of these herds was used to assess cost-effective methods that might be used to infer transmission trees during FMD outbreaks. Using these approaches, 85% and 91% of the resulting topologies were either identical or differed by only one edge from a reference tree comprising all of the sequences generated within the outbreak. The sequence distances that accrued during sequential transmission events between epidemiological units was estimated to be 4.6 nucleotides, although the genetic variability between viruses recovered from chronic carrier animals was higher than between viruses from animals with acute-stage infection: an observation which poses challenges for the use of simple approaches to infer transmission trees. This study helps to develop strategies for sampling during FMD outbreaks, and provides data that will guide the development of further models to support control policies in the event of virus incursions into FMD free countries

Receptor for Fc on the surfaces of schistosomes

Schistosoma mansoni masks its surface with adsorbed host proteins including erythrocyte antigens, immunoglobulins, major histocompatibility complex class I, and beta (2)-microglobulin (beta (2)m), presumably as a means of avoiding host immune responses, How this is accomplished has not been explained. To identify surface receptors for host proteins, we biotinylated the tegument of live S, mansoni adults and mechanically transformed schistosomula and then removed the parasite surface with detergent, Incubation of biotinylated schistosome surface extracts witt l human immunoglobulin G (IgG) Fc-Sepharose resulted in purification of a 97-kDa protein that was subsequently identified as paramyosin (Pmy), using antiserum specific for recombinant Pmy, Fc also bound recombinant S. mansoni Pmy and native S. japonicum Pmy, Antiserum to Pmy decreased the binding of Pmy to Fc-Sepharose, and no proteins bound after removal of Pmy from extracts. Fluoresceinated human Fe bound to the surface, vestigial penetration glands, and nascent oral cavity of mechanically transformed schistosomula, and rabbit anti-Pmy Fab fragments ablated the binding of Fc to the schistosome surface, Pmy coprecipitated with host IgG from parasite surface extracts, indicating that complexes formed on the parasite surface as well as in vitro. Binding of Pmy to Fe was not inhibited by soluble protein A, suggesting that Pmy does not bind to the region between the CH2 and CH3 domains used by many other Fc-binding proteins. beta (2)m did not bind to the schistosome Fc receptor (Pmy), a finding that contradicts reports from earlier workers but did bind to a heteromultimer of labeled schistosomula surface proteins, This is the first report of the molecular identity of a schistosome Fc receptor; moreover it demonstrates an additional aspect of the unusual and multifunctional properties of Pmy from schistosomes and other parasitic flatworms

Recovery of viral RNA and infectious foot-and-mouth disease virus from positive lateral-flow devices

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories

DetectiV: visualization, normalization and significance testing for pathogen-detection microarray data

DetectiV is a tool for analyzing pathogen-detection microarray datasets that allows simple visualisation, normalisation and significance testing

Foot-and-mouth disease surveillance using pooled milk on a large-scale dairy farm in an endemic setting

Pooled milk is used for the surveillance of several diseases of livestock. Previous studies demonstrated the detection of foot-and-mouth disease virus (FMDV) in the milk of infected animals at high dilutions, and consequently, the collection of pooled milk samples could be used to enhance FMD surveillance. This study evaluated pooled milk for FMDV surveillance on a large-scale dairy farm that experienced two FMD outbreaks caused by the A/ASIA/G-VII and O/ME-SA/Ind-2001d lineages, despite regular vaccination and strict biosecurity practices. FMDV RNA was detected in 42 (5.7%) of the 732 pooled milk samples, and typing information was concordant with diagnostic reports of clinical disease. The FMDV positive milk samples were temporally clustered around reports of new clinical cases, but with a wider distribution. For further investigation, a model was established to predict real-time RT-PCR (rRT-PCR) C values using individual cattle movement data, clinical disease records and virus excretion data from previous experimental studies. The model explained some of the instances where there were positive results by rRT-PCR, but no new clinical cases and suggested that subclinical infection occurred during the study period. Further studies are required to investigate the effect of vaccination on FMDV excretion in milk, and to evaluate more representative sampling methods. However, the results from this pilot study indicate that testing pooled milk by rRT-PCR may be valuable for FMD surveillance and has provided evidence of subclinical virus infection in vaccinated herds that could be important in the epidemiology of FMD in endemic countries where vaccination is used. [Abstract copyright: Copyright © 2020 Armson, Gubbins, Mioulet, Qasim, King and Lyons.

GoPrime: development of an in silico framework to predict the performance of real-time PCR primers and probes using foot-and-mouth disease virus as a model

Real-time PCR (rPCR) is a widely accepted diagnostic tool for the detection and quantification of nucleic acid targets. In order for these assays to achieve high sensitivity and specificity, primer and probe-template complementarity is essential; however, mismatches are often unavoidable and can result in false-negative results and errors in quantifying target sequences. Primer and probe sequences therefore require continual evaluation to ensure they remain fit for purpose. This paper describes the development of a linear model and associated computational tool (GoPrime) designed to predict the performance of rPCR primers and probes across multiple sequence data. Empirical data were generated using DNA oligonucleotides (n = 90) that systematically introduced variation in the primer and probe target regions of a diagnostic assay routinely used to detect foot-and-mouth disease virus (FMDV); an animal virus that exhibits a high degree of sequence variability. These assays revealed consistent impacts of patterns of substitutions in primer and probe-sites on rPCR cycle threshold (CT) and limit of detection (LOD). These data were used to populate GoPrime, which was subsequently used to predict rPCR results for DNA templates (n = 7) representing the natural sequence variability within FMDV. GoPrime was also applicable to other areas of the FMDV genome, with predictions for the likely targets of a FMDV-typing assay consistent with published experimental data. Although further work is required to improve these tools, including assessing the impact of primer-template mismatches in the reverse transcription step and the broader impact of mismatches for other assays, these data support the use of mathematical models for rapidly predicting the performance of rPCR primers and probes in silico

Interfering ribonucleic acids that suppress expression of multiple unrelated genes

<p>Abstract</p> <p>Background</p> <p>Short interfering RNAs (siRNAs) have become the research tool of choice for gene suppression, with human clinical trials ongoing. The emphasis so far in siRNA therapeutics has been the design of one siRNA with complete complementarity to the intended target. However, there is a need for multi-targeting interfering RNA in diseases in which multiple gene products are of importance. We have investigated the possibility of using a single short synthetic duplex RNA to suppress the expression of <it>VEGF-A </it>and <it>ICAM-1</it>; genes implicated in the progression of ocular neovascular diseases such as diabetic retinopathy.</p> <p>Results</p> <p>Duplex RNA were designed to have incomplete complementarity with the 3'UTR sequences of both target genes. One such duplex, CODEMIR-1, was found to suppress VEGF and ICAM-1 by 90 and 60%, respectively in ARPE-19 cells at a transfected concentration of 40 ng/mL. Use of a cyan fusion reporter with target sites constructed in its 3'UTR demonstrated that the repression of VEGF and ICAM-1 by CODEMIR-1 was indeed due to interaction with the target sequence. An exhaustive analysis of sequence variants of CODEMIR-1 demonstrated a clear positive correlation between activity against VEGF (but not ICAM-1) and the length of the contiguous complementary region (from the 5' end of the guide strand). Various strategies, including the use of inosine bases at the sites of divergence of the target sequences were investigated.</p> <p>Conclusion</p> <p>Our work demonstrates the possibility of designing multitargeting dsRNA to suppress more than one disease-altering gene. This warrants further investigation as a possible therapeutic approach.</p