Bacterial Toxin Fusion Proteins Elicit Mucosal Immunity against a Foot-and-Mouth Disease Virus Antigen When Administered Intranasally to Guinea Pigs

Peptides corresponding to the foot-and-mouth disease virus VP1 G-H loop are capable of inducing neutralizing antibodies in some species but are considered relatively poor immunogens, especially at mucosal surfaces. However, intranasal administration of antigens along with the appropriate delivery vehicle/adjuvant has been shown to induce mucosal immune responses, and bacterial enterotoxins have long been known to be effective in this regard. In the current study, two different carrier/adjuvant approaches were used to augment mucosal immunity to the FMDV O1 BFS G-H loop epitope, in which the G-H loop was genetically coupled to the E. coli LT-B subunit and coexpressed with the LTA2 fragment (LTA2B-GH), or the nontoxic pseudomonas exotoxin A (ntPE) was fused to LTA2B-GH at LT-A2 to enhance receptor targeting. Only guinea pigs that were inoculated intranasally with ntPE-LTA2B-GH and LTA2B-GH induced significant anti-G-H loop IgA antibodies in nasal washes at weeks 4 and 6 when compared to ovalbumin or G-H loop immunized animals. These were also the only groups that exhibited G-H loop-specific antigen-secreting cells in the nasal mucosa. These data demonstrate that fusion of nonreplicating antigens to LTA2B and ntPE-LTA2B has the potential to be used as carriers/adjuvants to induce mucosal immune responses against infectious diseases

Comparative susceptibility of eastern cottontails and New Zealand white rabbits to classical rabbit haemorrhagic disease virus (RHDV) and RHDV2

Rabbit haemorrhagic disease virus (RHDV) is associated with high morbidity and mortality in the European rabbit (Oryctolagus cuniculus). In 2010, a genetically distinct RHDV named RHDV2 emerged in Europe and spread to many other regions, including North America in 2016. Prior to this study it was unknown if eastern cottontails (ECT(s); Sylvilagus floridanus), one of the most common wild lagomorphs in the United States, were susceptible to RHDV2. In this study, 10 wild-caught ECTs and 10 New Zealand white rabbits (NZWR(s); O. cuniculus) were each inoculated orally with either RHDV (RHDVa/GI.1a; n = 5 per species) or RHDV2 (a recombinant GI.1bP-GI.2; n = 5 per species) and monitored for the development of disease. Three of the five ECTs that were infected with RHDV2 developed disease consistent with RHD and died at 4 and 6 days post-inoculation (DPI). The RHDV major capsid protein/antigen (VP60) was detected in the livers of three ECTs infected with RHDV2, but none was detected in the ECTs infected with RHDV. Additionally, RHD viral RNA was detected in the liver, spleen, intestine and blood of ECTs infected with RHDV2, but not in the ECTs infected with RHDV. RHD viral RNA was detected in urine, oral swabs and rectal swabs in at least two of five ECTs infected with RHDV2. One ECT inoculated with RHDV2 seroconverted and developed a high antibody titre by the end of the experimental period (21 DPI). ECTs inoculated with the classic RHDV did not seroconvert. In comparison, NZWRs inoculated with RHDV2 exhibited high mortality (five of five) at 2 DPI and four of five NZWRs inoculated with RHDV either died or were euthanized at 2 DPI indicating both of these viruses were highly pathogenic to this species. This experiment indicates that ECTs are susceptible to RHDV2 and can shed viral RNA, thereby suggesting this species could be involved in the epidemiology of this virus

The Vehicle, 1965, Vol. 7

Vol. 7 Table of Contents CommentaryElaine Lancepage 3 Lost Island and The Unseen SeaDaun Alan Leggpage 5 ElegyWilliam Mosierpage 6 AwayDavid Dixpage 7 DulceyRoberta Mathewspage 8 Alarum Tuam JonneDavid Walkerpage 11 Little BrotherSteve Gibbspage 13 River RunningDaun Alan Leggpage 15 PortraitRobert D. Thomaspage 16 The RockRoger Lewis Hudsonpage 17 Jarman HospitalElaine Lancepage 18 Of Domes and DiamondsDwight Ashbypage 19 Friday NightRoger J. Barrypage 20 MurderHelen Coxpage 23 Vigil SongDaun Alan Leggpage 24 Had You But Been the OneDavid Helmpage 25 To A Useless WeaponDarlene Brewerpage 25 Out of the NightPat Hartpage 26 La MortAdrian Beardpage 28 Mrs. Milton\u27s LamentBob Millerpage 30 Cockle CoveSusan McCabepage 31 Loss of VirtueJim Rinnertpage 32 The KeepsakeDwight Ashbypage 33 The RuinsRoger Lewis Hudsonpage 35 Ante Major OdysseyDaun Alan Leggpage 38 ReligionAnthony Barrettepage 39 All JoyJim Rinnertpage 40 SesameElaine Lancepage 40 CenterpieceDwight Ashbypage 41 A Great White WaveJohn Rhodespage 42 QueryElaine Lancepage 44 PistachioRita Salyerspage 45 FacadeKathleen McCormackpage 46 Winter Wisp AwaySteve Gibbspage 46 ScenarioDavid Dixpage 47 Damn-GodSteve Gibbspage 48 AccidentElaine Lancepage 48https://thekeep.eiu.edu/vehicle/1013/thumbnail.jp

Quantifying Specific Antibody Concentrations by Enzyme-Linked Immunosorbent Assay Using Slope Correction

Assessing the magnitude of an antibody response is important to many research and clinical endeavors; however, there are considerable differences in the experimental approaches used to achieve this end. Although the time-honored approach of end point titration has merit, the titer can often be misleading due to differences in how it is calculated or when samples contain high concentrations of low-avidity antibodies. One frequently employed alternative is to adapt commercially available enzyme-linked immunosorbent assay kits, designed to measure total antibody concentrations, to estimate antigen-specific antibody concentrations. This is accomplished by coating the specific antigen of interest in place of the capture antibody provided with the kit and then using the kit's standard curve to quantify the specific antibody concentration. This approach introduces considerable imprecision, due primarily to its reliance on a single sample dilution. This “single-point” approach fails to address differences in the slope of the sample titration curve compared to that of the standard curve. Here, we describe a general approach for estimating the effective concentration of specific antibodies, using antisera against foot-and-mouth disease virus VP1 peptide. This was accomplished by initially calculating the slope of the sample titration curve and then mathematically correcting the slope to that of a corresponding standard curve. A significantly higher degree of precision was attained using this approach rather than the single-point method

Real-Time Reverse Transcription PCR Assay for Detection of Senecavirus A in Swine Vesicular Diagnostic Specimens.

Senecavirus A (SV-A), formerly, Seneca Valley virus (SVV), has been detected in swine with vesicular lesions and is thought to be associated with swine idiopathic vesicular disease (SIVD), a vesicular disease syndrome that lacks a defined causative agent. The clinical presentation of SIVD resembles that of other more contagious and economically devastating vesicular diseases, such as foot-and-mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS), that typically require immediate rule out diagnostics to lift restrictions on animal quarantine, movement, and trade. This study presents the development of a sensitive, SYBR Green RT-qPCR assay suitable for detection of SV-A in diagnostic swine specimens. After testing 50 pigs with clinical signs consistent with vesicular disease, 44 (88%) were found to be positive for SV-A by RT-qPCR as compared to none from a negative cohort of 35 animals without vesicular disease, indicating that the assay is able to successfully detect the virus in an endemic population. SV-A RNA was also detectable at a low level in sera from a subset of pigs that presented with (18%) or without (6%) vesicular signs. In 2015, there has been an increase in the occurrence of SV-A in the US, and over 200 specimens submitted to our laboratory for vesicular investigation have tested positive for the virus using this method. SV-A RNA was detectable in all common types of vesicular specimens including swabs and tissue from hoof lesions, oral and snout epithelium, oral swabs, scabs, and internal organ tissues such as liver and lymph node. Genome sequencing analysis from recent virus isolates was performed to confirm target amplicon specificity and was aligned to previous isolates

Development of a Feature and Template-Assisted Assembler and Application to the Analysis of a Foot-and-Mouth Disease Virus Genotyping Microarray.

Several RT-PCR and genome sequencing strategies exist for the resolution of Foot-and-Mouth Disease virus (FMDV). While these approaches are relatively straightforward, they can be vulnerable to failure due to the unpredictable nature of FMDV genome sequence variations. Sequence independent single primer amplification (SISPA) followed by genotyping microarray offers an attractive unbiased approach to FMDV characterization. Here we describe a custom FMDV microarray and a companion feature and template-assisted assembler software (FAT-assembler) capable of resolving virus genome sequence using a moderate number of conserved microarray features. The results demonstrate that this approach may be used to rapidly characterize naturally occurring FMDV as well as an engineered chimeric strain of FMDV. The FAT-assembler, while applied to resolving FMDV genomes, represents a new bioinformatics approach that should be broadly applicable to interpreting microarray genotyping data for other viruses or target organisms

Consensus sequence buildup.

<p>Resolved sequence is selected by alignment of the candidate sequences for each of the thresholds. Sequence assembly is performed through selection of the individual resolved (non-ambiguous) nucleotides at the greatest MFI threshold.</p

Synthetic template scaffold build-up compared to native template selected by the algorithm.

<p>This is the comparison of the components of the synthetic template buildup for a chimeric FMDV, compared to the selected template compared to an individual scaffold template selection.</p