Classical BSE dismissed as the cause of CWD in Norwegian red deer despite strain similarities between both prion agents.

The first case of CWD in a Norwegian red deer was detected by a routine ELISA test and confirmed by western blotting and immunohistochemistry in the brain stem of the animal. Two different western blotting tests were conducted independently in two different laboratories, showing that the red deer glycoprofile was different from the Norwegian CWD reindeer and CWD moose and from North American CWD. The isolate showed nevertheless features similar to the classical BSE (BSE-C) strain. Furthermore, BSE-C could not be excluded based on the PrPSc immunohistochemistry staining in the brainstem and the absence of detectable PrPSc in the lymphoid tissues. Because of the known ability of BSE-C to cross species barriers as well as its zoonotic potential, the CWD red deer isolate was submitted to the EURL Strain Typing Expert Group (STEG) as a BSE-C suspect for further investigation. In addition, different strain typing in vivo and in vitro strategies aiming at identifying the BSE-C strain in the red deer isolate were performed independently in three research groups and BSE-C was not found in it. These results suggest that the Norwegian CWD red deer case was infected with a previously unknown CWD type and further investigation is needed to determine the characteristics of this potential new CWD strain.

Routinized Syphilis Screening Among Men Living With Human Immunodeficiency Virus: A Stepped Wedge Cluster Randomized Controlled Trial.

BACKGROUND: We implemented an opt-out clinic-based intervention pairing syphilis tests with routine human immunodeficiency virus (HIV) viral load testing. The primary objective was to determine the degree to which this intervention increased the detection of early syphilis. METHODS: The Enhanced Syphilis Screening Among HIV-Positive Men (ESSAHM) Trial was a stepped wedge cluster-randomized controlled trial involving 4 urban HIV clinics in Ontario, Canada, from 2015 to 2017. The population was HIV-positive adult males. The intervention was standing orders for syphilis serological testing with viral loads, and control was usual practice. We obtained test results via linkage with the centralized provincial laboratory and defined cases using a standardized clinical worksheet and medical record review. We employed a generalized linear mixed model with a logit link to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of the intervention. RESULTS: A total of 3895 men were followed over 7471 person-years. The mean number of syphilis tests increased from 0.53 to 2.02 tests per person per year. There were 217 new diagnoses of syphilis (control, 81; intervention, 136), for which 147 (68%) were cases of early syphilis (control, 61 [75%]; intervention, 86 [63%]). The annualized proportion with newly detected early syphilis increased from 0.009 to 0.032 with implementation of the intervention; the corresponding time-adjusted OR was 1.25 (95% CI, .71-2.20). CONCLUSIONS: The implementation of standing orders for syphilis testing with HIV viral loads was feasible and increased testing, yet produced less-than-expected increases in case detection compared to past uncontrolled pre-post trials. CLINICAL TRIALS REGISTRATION: NCT02019043.

Utility and Impact of the Implementation of Same-Day, Self-administered Electronic Patient-Reported Outcomes Assessments in Routine HIV Care in two North American Clinics.

The PROgress study assessed the value and feasibility of implementing web-based patient-reported outcomes assessments (PROs) within routine HIV care at two North American outpatient clinics. People with HIV (PWH) completed PROs on a tablet computer in clinic before their routine care visit. Data collection included PROs from 1632 unique PWH, 596 chart reviews, 200 patient questionnaires, and 16 provider/staff questionnaires. During an initial setup phase involving 200 patients, PRO results were not delivered to providers; for all subsequent patients, providers received PRO results before the consultation. Chart review demonstrated that delivery of PRO results to providers improved patient-provider communication and increased the number of complex health and behavioral issues identified, recorded, and acted on, including suicidal ideation (88% with vs 38% without PRO feedback) and anxiety (54% with vs 24% without PRO feedback). In post-visit questionnaires, PWH (82%) and providers (82%) indicated that the PRO added value to the visit.

Discovery of peptide ligands targeting a specific ubiquitin-like domain-binding site in the deubiquitinase USP11.

Ubiquitin-specific proteases (USPs) reverse ubiquitination and regulate virtually all cellular processes. Defined noncatalytic domains in USP4 and USP15 are known to interact with E3 ligases and substrate recruitment factors. No such interactions have been reported for these domains in the paralog USP11, a key regulator of DNA double-strand break repair by homologous recombination. We hypothesized that USP11 domains adjacent to its protease domain harbor unique peptide-binding sites. Here, using a next-generation phage display (NGPD) strategy, combining phage display library screening with next-generation sequencing, we discovered unique USP11-interacting peptide motifs. Isothermal titration calorimetry disclosed that the highest affinity peptides (KD of ∼10 µm) exhibit exclusive selectivity for USP11 over USP4 and USP15 in vitro Furthermore, a crystal structure of a USP11-peptide complex revealed a previously unknown binding site in USP11's noncatalytic ubiquitin-like (UBL) region. This site interacted with a helical motif and is absent in USP4 and USP15. Reporter assays using USP11-WT versus a binding pocket-deficient double mutant disclosed that this binding site modulates USP11's function in homologous recombination-mediated DNA repair. The highest affinity USP11 peptide binder fused to a cellular delivery sequence induced significant nuclear localization and cell cycle arrest in S phase, affecting the viability of different mammalian cell lines. The USP11 peptide ligands and the paralog-specific functional site in USP11 identified here provide a framework for the development of new biochemical tools and therapeutic agents. We propose that an NGPD-based strategy for identifying interacting peptides may be applied also to other cellular targets.

BSE infectivity survives burial for five years with only limited spread.

The carcasses of animals infected with bovine spongiform encephalopathy (BSE), scrapie or chronic wasting disease (CWD) that remain in the environment (exposed or buried) may continue to act as reservoirs of infectivity. We conducted two experiments under near-field conditions to investigate the survival and dissemination of BSE infectivity after burial in a clay or sandy soil. BSE infectivity was either contained within a bovine skull or buried as an uncontained bolus of BSE-infected brain. Throughout the five-year period of the experiment, BSE infectivity was recovered in similar amounts from heads exhumed annually from both types of soil. Very low levels of infectivity were detected in the soil immediately surrounding the heads, but not in samples remote from them. Similarly, there was no evidence of significant lateral movement of infectivity from the buried bolus over 4 years although there was a little vertical movement in both directions. However, bioassay analysis of limited numbers of samples of rain water that had drained through the bolus clay lysimeter indicated that infectivity was present in filtrates. sPMCA analysis also detected low levels of PrPSc in the filtrates up to 25 months following burial, raising the concern that leakage of infectivity into ground water could occur. We conclude that transmissible spongiform encephalopathy infectivity is likely to survive burial for long periods of time, but not to migrate far from the site of burial unless a vector or rain water drainage transports it. Risk assessments of contaminated sites should take these findings into account.

A Trematode Parasite Derived Growth Factor Binds and Exerts Influences on Host Immune Functions via Host Cytokine Receptor Complexes.

The trematode Fasciola hepatica is responsible for chronic zoonotic infection globally. Despite causing a potent T-helper 2 response, it is believed that potent immunomodulation is responsible for rendering this host reactive non-protective host response thereby allowing the parasite to remain long-lived. We have previously identified a growth factor, FhTLM, belonging to the TGF superfamily can have developmental effects on the parasite. Herein we demonstrate that FhTLM can exert influence over host immune functions in a host receptor specific fashion. FhTLM can bind to receptor members of the Transforming Growth Factor (TGF) superfamily, with a greater affinity for TGF-ß RII. Upon ligation FhTLM initiates the Smad2/3 pathway resulting in phenotypic changes in both fibroblasts and macrophages. The formation of fibroblast CFUs is reduced when cells are cultured with FhTLM, as a result of TGF-ß RI kinase activity. In parallel the wound closure response of fibroblasts is also delayed in the presence of FhTLM. When stimulated with FhTLM blood monocyte derived macrophages adopt an alternative or regulatory phenotype. They express high levels interleukin (IL)-10 and arginase-1 while displaying low levels of IL-12 and nitric oxide. Moreover they also undergo significant upregulation of the inhibitory receptor PD-L1 and the mannose receptor. Use of RNAi demonstrates that this effect is dependent on TGF-ß RII and mRNA knock-down leads to a loss of IL-10 and PD-L1. Finally, we demonstrate that FhTLM aids newly excysted juveniles (NEJs) in their evasion of antibody-dependent cell cytotoxicity (ADCC) by reducing the NO response of macrophages-again dependent on TGF-ß RI kinase. FhTLM displays restricted expression to the F. hepatica gut resident NEJ stages. The altered fibroblast responses would suggest a role for dampened tissue repair responses in facilitating parasite migration. Furthermore, the adoption of a regulatory macrophage phenotype would allow for a reduced effector response targeting juvenile parasites which we demonstrate extends to an abrogation of the ADCC response. Thus suggesting that FhTLM is a stage specific evasion molecule that utilises host cytokine receptors. These findings are the first to clearly demonstrate the interaction of a helminth cytokine with a host receptor complex resulting in immune modifications that facilitate the non-protective chronic immune response which is characteristic of F. hepatica infection.

Expression of four S. pneumoniae type 2 polysaccharide biosynthetic enzymes utilising the endogenous Kex2 protease activity in tobacco.

In order to express multisubunit proteins, or to manipulate metabolic pathways in plants it is essential to be able to efficiently express multiple proteins within the same plant cell. To increase the efficiency of multi-protein expression, we demonstrate the use of the Golgi localized Kex2 protease activity in tobacco to process a large polyprotein precursor consisting of four individual protein domains into its individual protein constituents. Four genes encoding enzymes involved in the biosynthesis of S. pneumoniae type 2 polysaccharide were assembled into a single expression cassette as a large polyprotein driven by a single cauliflower mosaic virus (CaMV) 35S promoter. Each of the individual protein domains were separated by three sequential Kex2 protease digestion sites. At the N-terminus a Pr1b signal peptide was incorporated for efficient targeting of the polyprotein to the apoplast. Each individual protein domain was tagged with its own immuno-tag. The construct was used for the transformation of Nicotiana tabacum and stable lines were selected. All four processed proteins could be immunologically detected in protein extracts using Western blotting indicating correct expression and Kex2 processing. Utilisation of the Kex2 protease system represents an efficient way of expressing multiple proteins in the same plant. This method simplifies the transformation procedures, and presents a method for expression of multiple proteins within the same plant.

Evidence for a novel Kit adhesion domain mediating human mast cell adhesion to structural airway cells.

BACKGROUND: Human lung mast cells (HLMCs) infiltrate the airway epithelium and airway smooth muscle (ASM) in asthmatic airways. The mechanism of HLMC adhesion to both cell types is only partly defined, and adhesion is not inhibited by function-blocking anti-Kit and anti-stem cell factor (SCF) antibodies. Our aim was to identify adhesion molecules expressed by human mast cells that mediate adhesion to human ASM cells (HASMCs) and human airway epithelial cells. METHODS: We used phage-display to isolate single chain Fv (scFv) antibodies with adhesion-blocking properties from rabbits immunised with HLMC and HMC-1 membrane proteins. RESULTS: Post-immune rabbit serum labelled HLMCs in flow cytometry and inhibited their adhesion to human BEAS-2B epithelial cells. Mast cell-specific scFvs were identified which labelled mast cells but not Jurkat cells by flow cytometry. Of these, one scFv (A1) consistently inhibited mast cell adhesion to HASMCs and BEAS-2B epithelial cells by about 30 %. A1 immunoprecipitated Kit (CD117) from HMC-1 lysates and bound to a human Kit-expressing mouse mast cell line, but did not interfere with SCF-dependent Kit signalling. CONCLUSION: Kit contributes to human mast cell adhesion to human airway epithelial cells and HASMCs, but may utilise a previously unidentified adhesion domain that lies outside the SCF binding site. Targeting this adhesion pathway might offer a novel approach for the inhibition of mast cell interactions with structural airway cells, without detrimental effects on Kit signalling in other tissues.

Circulation of prions within dust on a scrapie affected farm.

Prion diseases are fatal neurological disorders that affect humans and animals. Scrapie of sheep/goats and Chronic Wasting Disease (CWD) of deer/elk are contagious prion diseases where environmental reservoirs have a direct link to the transmission of disease. Using protein misfolding cyclic amplification we demonstrate that scrapie PrP(Sc) can be detected within circulating dusts that are present on a farm that is naturally contaminated with sheep scrapie. The presence of infectious scrapie within airborne dusts may represent a possible route of infection and illustrates the difficulties that may be associated with the effective decontamination of such scrapie affected premises.

Incubation of ovine scrapie with environmental matrix results in biological and biochemical changes of PrP(Sc) over time.

Ovine scrapie can be transmitted via environmental reservoirs. A pool of ovine scrapie isolates were incubated on soil for one day or thirteen months and eluted prion was used to challenge tg338 mice transgenic for ovine PrP. After one-day incubation on soil, two PrP(Sc) phenotypes were present: G338 or Apl338ii. Thirteen months later some divergent PrP(Sc) phenotypes were seen: a mixture of Apl338ii with either G338 or P338, and a completely novel PrP(Sc) deposition, designated Cag338. The data show that prolonged ageing of scrapie prions within an environmental matrix may result in changes in the dominant PrP(Sc) biological/biochemical properties.

Comparison of Schmallenberg virus antibody levels detected in milk and serum from individual cows.

BACKGROUND: Schmallenberg virus (SBV) is a recently emerged virus of ruminants in Europe. Enzyme-linked immunosorbent assays (ELISA) are commonly used to detect SBV-specific antibodies in bulk tank milk samples to monitor herd exposure to infection. However, it has previously been shown that a bulk tank milk sample can test positive even though the majority of cows within the herd are seronegative for SBV antibodies. Development of a pen-side test to detect antibodies in individual milk samples would potentially provide a cheaper test (for which samples are obtained non-invasively) than testing individual serum samples by ELISA. Therefore, the aim of this study was to investigate the agreement between antibody levels measured in milk and serum. RESULTS: Corresponding milk and serum samples from 88 cows in two dairy herds in the UK were tested for presence of immunoglobulin G antibodies to SBV using a commercially-available indirect ELISA. A serum neutralisation test (NT) was also performed as a gold standard assay. The ELISA values obtained for the bulk tank milk samples corresponded with the mean values for individual milk samples from each herd (bulk tank milk values were 58% and 73% and mean individual milk values 50% and 63% for herds A and B, respectively). Of the 88 serum samples tested in the NT, 82 (93%) were positive. Although at higher antibody levels, the ELISA values tended to be higher for the individual milk samples than for the corresponding serum samples, the positive predictive value for milk samples was 98% and for serum samples 94%. The serum ELISA was more likely to give false positive results around the lower cut-off value of the assay. CONCLUSIONS: The results indicate that testing of individual milk samples for antibodies against SBV by ELISA could be used to inform decisions in the management of dairy herds such as which, if any, animals to vaccinate.

Highly sensitive detection of small ruminant bovine spongiform encephalopathy within transmissible spongiform encephalopathy mixes by serial protein misfolding cyclic amplification.

It is assumed that sheep and goats consumed the same bovine spongiform encephalopathy (BSE)-contaminated meat and bone meal that was fed to cattle and precipitated the BSE epidemic in the United Kingdom that peaked more than 20 years ago. Despite intensive surveillance for cases of BSE within the small ruminant populations of the United Kingdom and European Union, no instances of BSE have been detected in sheep, and in only two instances has BSE been discovered in goats. If BSE is present within the small ruminant populations, it may be at subclinical levels, may manifest as scrapie, or may be masked by coinfection with scrapie. To determine whether BSE is potentially circulating at low levels within the European small ruminant populations, highly sensitive assays that can specifically detect BSE, even within the presence of scrapie prion protein, are required. Here, we present a novel assay based on the specific amplification of BSE PrP(Sc) using the serial protein misfolding cyclic amplification assay (sPMCA), which specifically amplified small amounts of ovine and caprine BSE agent which had been mixed into a range of scrapie-positive brain homogenates. We detected the BSE prion protein within a large excess of classical, atypical, and CH1641 scrapie isolates. In a blind trial, this sPMCA-based assay specifically amplified BSE PrP(Sc) within brain mixes with 100% specificity and 97% sensitivity when BSE agent was diluted into scrapie-infected brain homogenates at 1% (vol/vol).

Longitudinal Analysis of Mpox Virus DNA Detectability From Multiple Specimen Types During Acute Illness: A Cohort Study.

Background: Longitudinal data on the detectability of monkeypox virus (MPXV) genetic material in different specimen types are scarce. Methods: We describe MPXV-specific polymerase chain reaction (PCR) results from adults with confirmed mpox infection from Toronto, Canada, including a cohort undergoing weekly collection of specimens from multiple anatomic sites until 1 week after skin lesions had fully healed. We quantified the time from symptom onset to resolution of detectable viral DNA (computed tomography [Ct] ≥ 35) by modeling exponential decay in Ct value as a function of illness day for each site, censoring at the time of tecovirimat initiation. Results: Among 64 men who have sex with men, the median (interquartile range [IQR]) age was 39 (32.75-45.25) years, and 49% had HIV. Twenty received tecovirimat. Viral DNA was detectable (Ct < 35) at baseline in 74% of genital/buttock/perianal skin swabs, 56% of other skin swabs, 44% of rectal swabs, 37% of throat swabs, 27% of urine, 26% of nasopharyngeal swabs, and 8% of semen samples. The median time to resolution of detectable DNA (IQR) was longest for genital/buttock/perianal skin and other skin swabs at 30.0 (23.0-47.9) and 22.4 (16.6-29.4) days, respectively, and shortest for nasopharyngeal swabs and semen at 0 (0-12.1) and 0 (0-0) days, respectively. We did not observe an effect of tecovirimat on the rate of decay in viral DNA detectability in any specimen type (all P > .05). Conclusions: MPXV DNA detectability varies by specimen type and persists for over 3-4 weeks in skin specimens. The rate of decay did not differ by tecovirimat use in this nonrandomized study.

The oral secretion of infectious scrapie prions occurs in preclinical sheep with a range of PRNP genotypes.

Preclinical sheep with the highly scrapie-susceptible VRQ/VRQ PRNP genotype secrete prions from the oral cavity. In order to further understand the significance of orally available prions, buccal swabs were taken from sheep with a range of PRNP genotypes and analyzed by serial protein misfolding cyclic amplification (sPMCA). Prions were detected in buccal swabs from scrapie-exposed sheep of genotypes linked to high (VRQ/VRQ and ARQ/VRQ) and low (ARR/VRQ and AHQ/VRQ) lymphoreticular system involvement in scrapie pathogenesis. For both groups, the level of prion detection was significantly higher than that for scrapie-resistant ARR/ARR sheep which were kept in the same farm environment and acted as sentinel controls for prions derived from the environment which might contaminate the oral cavity. In addition, sheep with no exposure to the scrapie agent did not contain any measurable prions within the oral cavity. Furthermore, prions were detected in sheep over a wide age range representing various stages of preclinical disease. These data demonstrate that orally available scrapie prions may be a common feature in sheep incubating scrapie, regardless of the PRNP genotype and any associated high-level accumulation of PrP(Sc) within lymphoreticular tissues. PrP(Sc) was present in buccal swabs from a large proportion of sheep with PRNP genotypes associated with relatively low disease penetrance, indicating that subclinical scrapie infection is likely to be a common occurrence. The significance of positive sPMCA reactions was confirmed by the transmission of infectivity in buccal swab extracts to Tg338 mice, illustrating the likely importance of orally available prions in the horizontal transmission of scrapie.

Factors affecting antiretroviral pharmacokinetics in HIV-infected women with virologic suppression on combination antiretroviral therapy: a cross-sectional study.

BACKGROUND: Although some studies show higher antiretroviral concentrations in women compared to men, data are limited. We conducted a cross-sectional study of HIV-positive women to determine if protease inhibitor (PI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) C(min) and Cmax values were significantly different than historical general population (predominantly male) averages and to evaluate correlates of higher concentrations. METHODS: HIV-positive women with virologic suppression (viral load < 50copies/mL) on their first antiretroviral regimen were enrolled. Timed blood samples for C(min) and Cmax were drawn weekly for 3 weeks. The ratio of each individual's median C(min) and Cmax to the published population mean values for their PI or NNRTI was calculated and assessed using Wilcoxon sign-rank. Intra- and inter-patient variability of antiretroviral drug levels was assessed using coefficient of variation and intra-class correlation. Linear regression was used to identify correlates of the square root-transformed C(min) and Cmax ratios. RESULTS: Data from 82 women were analyzed. Their median age was 41 years (IQR=36-48) and duration of antiretrovirals was 20 months (IQR=9-45). Median antiretroviral C(min) and Cmax ratios were 1.21 (IQR=0.72-1.89, p=0.003) (highest ratios for nevirapine and lopinavir) and 0.82 (IQR=0.59-1.14, p=0.004), respectively. Nevirapine and efavirenz showed the least and unboosted atazanavir showed the most intra- and inter-patient variability. Higher CD4+ count correlated with higher C(min). No significant correlates for Cmax were found. CONCLUSIONS: Compared to historical control data, C(min) in the women enrolled was significantly higher whereas Cmax was significantly lower. Antiretroviral C(min) ratios were highly variable within and between participants. There were no clinically relevant correlates of drug concentrations. TRIAL REGISTRATION: NCT00433979.