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

The development of two field‐ready reverse transcription loop‐mediated isothermal amplification assays for the rapid detection of Seneca Valley virus 1

Seneca Valley virus 1 (SVV‐1) has been associated with vesicular disease in swine, with clinical signs indistinguishable from those of other notifiable vesicular diseases such as foot‐and‐mouth disease. Rapid and accurate detection of SVV‐1 is central to confirm the disease causing agent, and to initiate the implementation of control processes. The development of rapid, cost‐effective diagnostic assays that can be used at the point of sample collection has been identified as a gap in preparedness for the control of SVV‐1. This study describes the development and bench validation of two reverse transcription loop‐mediated amplification (RT‐LAMP) assays targeting the 5′‐untranslated region (5′‐UTR) and the VP3‐1 region for the detection of SVV‐1 that may be performed at the point of sample collection. Both assays were able to demonstrate amplification of all neat samples diluted 1/100 in negative pig epithelium tissue suspension within 8 min, when RNA was extracted prior to the RT‐LAMP assay, and no amplification was observed for the other viruses tested. Simple sample preparation methods using lyophilized reagents were investigated, to negate the requirement for RNA extraction. Only a small delay in the time to amplification was observed for these lyophilized reagents, with a time from sample receipt to amplification achieved within 12 min. Although diagnostic validation is recommended, these RT‐LAMP assays are highly sensitive and specific, with the potential to be a useful tool in the rapid diagnosis of SVV‐1 in the field

NsrR from Streptomyces coelicolor is a nitric oxide-sensing [4Fe-4S] cluster protein with a specialized regulatory function

The Rrf2 family transcription factor NsrR controls expression of genes in a wide range of bacteria in response to nitric oxide (NO). The precise form of the NO-sensing module of NsrR is the subject of controversy because NsrR proteins containing either [2Fe-2S] or [4Fe-4S] clusters have been observed previously. Optical, Mössbauer, resonance Raman spectroscopies and native mass spectrometry demonstrate that Streptomyces coelicolor NsrR (ScNsrR), previously reported to contain a [2Fe-2S] cluster, can be isolated containing a [4Fe-4S] cluster. ChIP-seq experiments indicated that the ScNsrR regulon is small, consisting of only hmpA1, hmpA2, and nsrR itself. The hmpA genes encode NO-detoxifying flavohemoglobins, indicating that ScNsrR has a specialized regulatory function focused on NO detoxification and is not a global regulator like some NsrR orthologues. EMSAs and DNase I footprinting showed that the [4Fe-4S] form of ScNsrR binds specifically and tightly to an 11-bp inverted repeat sequence in the promoter regions of the identified target genes and that DNA binding is abolished following reaction with NO. Resonance Raman data were consistent with cluster coordination by three Cys residues and one oxygen-containing residue, and analysis of ScNsrR variants suggested that highly conserved Glu-85 may be the fourth ligand. Finally, we demonstrate that some low molecular weight thiols, but importantly not physiologically relevant thiols, such as cysteine and an analogue of mycothiol, bind weakly to the [4Fe-4S] cluster, and exposure of this bound form to O2 results in cluster conversion to the [2Fe-2S] form, which does not bind to DNA. These data help to account for the observation of [2Fe-2S] forms of NsrR

Pandemic Strain of Foot-and-Mouth Disease Virus Serotype O

The PanAsia strain is spreading explosively in Asia and extending to parts of Africa and Europe

Near-complete genome sequencing of swine vesicular disease virus using the Roche GS FLX sequencing platform

Swine vesicular disease virus (SVDV) is an enterovirus that is both genetically and antigenically closely related to human coxsackievirus B5 within the Picornaviridae family. SVDV is the causative agent of a highly contagious (though rarely fatal) vesicular disease in pigs. We report a rapid method that is suitable for sequencing the complete protein-encoding sequences of SVDV isolates in which the RNA is relatively intact. The approach couples a single PCR amplification reaction, using only a single PCR primer set to amplify the near-complete SVDV genome, with deep-sequencing using a small fraction of the capacity of a Roche GS FLX sequencing platform. Sequences were initially verified through one of two criteria; either a match between a de novo assembly and a reference mapping, or a match between all of five different reference mappings performed against a fixed set of starting reference genomes with significant genetic distances within the same species of viruses. All reference mappings used an iterative method to avoid bias. Further verification was achieved through phylogenetic analysis against published SVDV genomes and additional Enterovirus B sequences. This approach allows high confidence in the obtained consensus sequences, as well as provides sufficiently high and evenly dispersed sequence coverage to allow future studies of intra-host variation

Proposals for the classification of human rhinovirus species A, B and C into genotypically assigned types

Human rhinoviruses (HRVs) frequently cause mild upper respiratory tract infections and more severe disease manifestations such as bronchiolitis and asthma exacerbations. HRV is classified into three species within the genus Enterovirus of the family Picornaviridae. HRV species A and B contain 75 and 25 serotypes identified by cross-neutralization assays, although the use of such assays for routine HRV typing is hampered by the large number of serotypes, replacement of virus isolation by molecular methods in HRV diagnosis and the poor or absent replication of HRV species C in cell culture. To address these problems, we propose an alternative, genotypic classification of HRV-based genetic relatedness analogous to that used for enteroviruses. Nucleotide distances between 384 complete VP1 sequences of currently assigned HRV (sero)types identified divergence thresholds of 13, 12 and 13 % for species A, B and C, respectively, that divided inter- and intra-type comparisons. These were paralleled by 10, 9.5 and 10 % thresholds in the larger dataset of >3800 VP4 region sequences. Assignments based on VP1 sequences led to minor revisions of existing type designations (such as the reclassification of serotype pairs, e.g. A8/A95 and A29/A44, as single serotypes) and the designation of new HRV types A101–106, B101–103 and C34–C51. A protocol for assignment and numbering of new HRV types using VP1 sequences and the restriction of VP4 sequence comparisons to type identification and provisional type assignments is proposed. Genotypic assignment and identification of HRV types will be of considerable value in the future investigation of type-associated differences in disease outcomes, transmission and epidemiology

Viral meningitis epidemics and a single, recent, recombinant and anthroponotic origin of swine vesicular disease virus

Background and objectives: Swine vesicular disease virus (SVDV) is a close relative of the human Enterovirus B serotype, coxsackievirus B5. As the etiological agent of a significant emergent veterinary disease, several studies have attempted to explain its origin. However, several key questions remain, including the full biological ancestry of the virus, and its geographical and temporal origin. Methodology: We sequenced near-complete genomes of 27 SVDV and 13 coxsackievirus B5 samples, all originally isolated between 1966 and 2006, and analysed these in conjunction with existing sequences and historical information. Results: While analyses incorporating 24 additional near-complete SVDV genomic sequences indicate clear signs of within-SVDV recombination, all 51 SVDV isolates remain monophyletic. This supports a hypothesis of a single anthroponotic transfer origin. Analysis of individual coding and non-coding regions supports that SVDV has a recombinant origin between coxsackievirus B5 and another Enterovirus B serotype, most likely coxsackievirus A9. Extensive Bayesian sequence-based analysis of the time of the most recent common ancestor of all analysed sequences places this within a few years around 1961. Epidemiological evidence points to China as an origin, but there are no available samples to test this conclusively. Conclusions and implications: Historical investigation and the clinical aspects of the involved Enterovirus B serotypes, makes the current results consistent with a hypothesis stating that SVDV originated through co-infection, recombination, and a single anthroponotic event, during large viral meningitis epidemics around 1960/1961 involving the ancestral serotypes. The exact geographical origin of SVDV may remain untestable due to historical aspects