The 21st Annual Meeting of the Rocky Mountain Virology Association.

Nestled within the Rocky Mountain National Forest, 114 scientists and students gathered at Colorado State University's Mountain Campus for this year's 21st annual Rocky Mountain National Virology Association meeting. This 3-day retreat consisted of 31 talks and 30 poster presentations discussing advances in research pertaining to viral and prion diseases. The keynote address provided a timely discussion on zoonotic coronaviruses, lessons learned, and the path forward towards predicting, preparing, and preventing future viral disease outbreaks. Other invited speakers discussed advances in SARS-CoV-2 surveillance, molecular interactions involved in flavivirus genome assembly, evaluation of ethnomedicines for their efficacy against infectious diseases, multi-omic analyses to define risk factors associated with long COVID, the role that interferon lambda plays in control of viral pathogenesis, cell-fusion-dependent pathogenesis of varicella zoster virus, and advances in the development of a vaccine platform against prion diseases. On behalf of the Rocky Mountain Virology Association, this report summarizes select presentations.

Detection of Chronic Wasting Disease Prions in Fetal Tissues of Free-Ranging White-Tailed Deer.

The transmission of chronic wasting disease (CWD) has largely been attributed to contact with infectious prions shed in excretions (saliva, urine, feces, blood) by direct animal-to-animal exposure or indirect contact with the environment. Less-well studied has been the role that mother-to-offspring transmission may play in the facile transmission of CWD, and whether mother-to-offspring transmission before birth may contribute to the extensive spread of CWD. We thereby focused on a population of free-ranging white-tailed deer from West Virginia, USA, in which CWD has been detected. Fetal tissues, ranging from 113 to 158 days of gestation, were harvested from the uteri of CWD+ dams in the asymptomatic phase of infection. Using serial protein misfolding amplification (sPMCA), we detected evidence of prion seeds in 7 of 14 fetuses (50%) from 7 of 9 pregnancies (78%), with the earliest detection at 113 gestational days. This is the first report of CWD detection in free ranging white-tailed deer fetal tissues. Further investigation within cervid populations across North America will help define the role and impact of mother-to-offspring vertical transmission of CWD.

Detection of chronic wasting disease in mule and white-tailed deer by RT-QuIC analysis of outer ear.

Efforts to contain the spread of chronic wasting disease (CWD), a fatal, contagious prion disease of cervids, would be aided by the availability of additional diagnostic tools. RT-QuIC assays allow ultrasensitive detection of prion seeds in a wide variety of cervid tissues, fluids and excreta. The best documented antemortem diagnostic test involving RT-QuIC analysis targets lymphoid tissue in rectal biopsies. Here we have tested a more easily accessed specimen, ear pinna punches, using an improved RT-QuIC assay involving iron oxide magnetic extraction to detect CWD infections in asymptomatic mule and white-tailed deer. Comparison of multiple parts of the ear pinna indicated that a central punch spanning the auricular nerve provided the most consistent detection of CWD infection. When compared to results obtained from gold-standard retropharyngeal lymph node specimens, our RT-QuIC analyses of ear samples provided apparent diagnostic sensitivity (81%) and specificity (91%) that rivaled, or improved upon, those observed in previous analyses of rectal biopsies using RT-QuIC. These results provide evidence that RT-QuIC analysis of ear pinna punches may be a useful approach to detecting CWD infections in cervids.

Cofactor molecules: Essential partners for infectious prions.

The protein-only hypothesis predicts that infectious mammalian prions are composed solely of PrPSc, a misfolded conformer of the normal prion protein, PrPC. However, to date, all wild type protein-only PrPSc preparations lack significant levels of prion infectivity. Using a systemic biochemical approach, our laboratory isolated and identified two different endogenous cofactor molecules, RNA (Deleault et al., 2003 [50]; Deleault et al., 2007 [59]) and phosphatidylethanolamine (Deleault et al., 2012 [61]; Deleault et al., 2012 [18]), which facilitate the formation of prions with high levels of specific infectivity, leading us to propose to the alternative hypothesis that cofactor molecules are required to form wild type infectious prions (Deleault et al., 2007 [59]; Deleault et al., 2012 [18]; Geoghegan et al., 2007 [57]). In addition, we found that purified cofactor molecules restrict the strain properties of chemically defined infectious prions (Deleault et al., 2012 [18]), suggesting a "cofactor selection" model in which natural variation in the distribution of strain-specific cofactor molecules in different parts of the brain may be responsible for strain-dependent patterns of neurotropism (Deleault et al., 2012 [18]; Geoghegan et al., 2007 [57]).

Highly infectious prions are not directly neurotoxic.

Prions are infectious agents which cause rapidly lethal neurodegenerative diseases in humans and animals following long, clinically silent incubation periods. They are composed of multichain assemblies of misfolded cellular prion protein. While it has long been assumed that prions are themselves neurotoxic, recent development of methods to obtain exceptionally pure prions from mouse brain with maintained strain characteristics, and in which defined structures-paired rod-like double helical fibers-can be definitively correlated with infectivity, allowed a direct test of this assertion. Here we report that while brain homogenates from symptomatic prion-infected mice are highly toxic to cultured neurons, exceptionally pure intact high-titer infectious prions are not directly neurotoxic. We further show that treatment of brain homogenates from prion-infected mice with sodium lauroylsarcosine destroys toxicity without diminishing infectivity. This is consistent with models in which prion propagation and toxicity can be mechanistically uncoupled.

A cold water, ultrasonically activated stream efficiently removes proteins and prion-associated amyloid from surgical stainless steel.

BACKGROUND: Sterile service department decontamination procedures for surgical instruments struggle to demonstrate efficient removal of the hardiest infectious contaminants, such as prion proteins. A recently designed novel system, which uses a low pressure ultrasonically activated, cold water stream, has previously demonstrated efficient hard surface cleaning of several biological contaminants. AIM: To test the efficacy of an ultrasonically activated stream for the removal of tissue proteins, including prion-associated amyloid, from surgical stainless steel surfaces. METHODS: Test surfaces were contaminated with 22L, ME7 or 263K prion-infected brain homogenates. The surfaces were treated with the ultrasonically activated water stream for contact times of 5 and 10 s. Residual proteinaceous and amyloid contamination were quantified using sensitive microscopic analysis, and immunoblotting was used to characterize the eluted prion residues before and after treatment with the ultrasonically activated stream. FINDINGS: Efficient removal of the different prion strains from the surgical stainless steel surfaces was observed, and reduced levels of protease-susceptible and -resistant prion protein was detected in recovered supernatant. CONCLUSION: This study demonstrated that an ultrasonically activated stream has the potential to be a cost-effective solution to improve current decontamination practices and has the potential to reduce hospital-acquired infections.

Failure To Detect Prion Infectivity in Ticks following Prion-Infected Blood Meal.

Chronic wasting disease (CWD) is an emerging and fatal contagious prion disease that affects cervids, including mule deer, white-tailed deer, black-tailed deer, red deer reindeer, elk, and moose. CWD prions are widely distributed throughout the bodies of CWD-infected animals and are found in the nervous system, lymphoid tissues, muscle, blood, urine, feces, and antler velvet. The mechanism of CWD transmission in natural settings is unknown. Potential mechanisms of transmission include horizontal, maternal, or environmental routes. Due to the presence of prions in the blood of CWD-infected animals, the potential exists for invertebrates that feed on mammalian blood to contribute to the transmission of CWD. The geographic range of the Rocky Mountain Wood tick, Dermancentor andersoni, overlaps with CWD throughout the northwest United States and southwest Canada, raising the possibility that D. andersoni parasitization of cervids may be involved in CWD transmission. We investigated this possibility by examining the blood meal of D. andersoni that fed upon prion-infected hamsters for the presence of prion infectivity by animal bioassay. None of the hamsters inoculated with a D. andersoni blood meal that had been ingested from prion-infected hamsters developed clinical signs of prion disease or had evidence for a subclinical prion infection. Overall, the data do not demonstrate a role for D. andersoni in the transmission of prion disease.IMPORTANCE Chronic wasting disease (CWD) is an emerging prion disease that affects cervids, including mule deer, white-tailed deer, black-tailed deer, red deer reindeer, elk, and moose. The mechanism of CWD transmission in unknown. Due to the presence of prions in the blood of CWD-infected animals, it is possible for invertebrates that feed on cervid blood to contribute to the transmission of CWD. We examined the blood meal of D. andersoni, a tick with a similar geographic range as cervids, that fed upon prion-infected hamsters for the presence of prion infectivity by animal bioassay. None of the D. andersoni blood meals that had been ingested from prion-infected hamsters yielded evidence of prion infection. Overall, the data do not support a role of D. andersoni in the transmission of prion disease.

Spontaneous generation of prions and transmissible PrP amyloid in a humanised transgenic mouse model of A117V GSS.

Inherited prion diseases are caused by autosomal dominant coding mutations in the human prion protein (PrP) gene (PRNP) and account for about 15% of human prion disease cases worldwide. The proposed mechanism is that the mutation predisposes to conformational change in the expressed protein, leading to the generation of disease-related multichain PrP assemblies that propagate by seeded protein misfolding. Despite considerable experimental support for this hypothesis, to-date spontaneous formation of disease-relevant, transmissible PrP assemblies in transgenic models expressing only mutant human PrP has not been demonstrated. Here, we report findings from transgenic mice that express human PrP 117V on a mouse PrP null background (117VV Tg30 mice), which model the PRNP A117V mutation causing inherited prion disease (IPD) including Gerstmann-Sträussler-Scheinker (GSS) disease phenotypes in humans. By studying brain samples from uninoculated groups of mice, we discovered that some mice (≥475 days old) spontaneously generated abnormal PrP assemblies, which after inoculation into further groups of 117VV Tg30 mice, produced a molecular and neuropathological phenotype congruent with that seen after transmission of brain isolates from IPD A117V patients to the same mice. To the best of our knowledge, the 117VV Tg30 mouse line is the first transgenic model expressing only mutant human PrP to show spontaneous generation of transmissible PrP assemblies that directly mirror those generated in an inherited prion disease in humans.

A new approach for the separation, characterization and testing of potential prionoid protein aggregates through hollow-fiber flow field-flow fractionation and multi-angle light scattering.

Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.

Estimation of prion infectivity in tissues of cattle infected with atypical BSE by real time-quaking induced conversion assay.

Atypical bovine spongiform encephalopathy (BSE), first identified in 2004, poses a threat due to the potential to spread the disease to cattle and other animals, including humans. Here, we estimated prion titers in various tissues of cattle infected with atypical BSE using a real-time quaking-induced conversion assay that detects amyloid seeding activity of a disease-specific prion protein, PrPSc, a major component of prions. PrPSc was detected both in and outside of nerve tissues, and some of the peripheral nerve tissues contained relatively high prion titers. Low titers of prions were also observed in masseter, jejunum, and adrenal glands. Quantitative data on prion infectivity in tissues of atypical BSE-affected cattle is useful to assess the risk of atypical BSE.

Early and Pre-Clinical Detection of Prion Seeding Activity in Cerebrospinal Fluid of Goats using Real-Time Quaking-Induced Conversion Assay.

Since 2005, two cases of natural bovine spongiform encephalopathies (BSE) have been reported in goats. Furthermore, experimental transmissions of classical (C-BSE) and atypical (L-BSE) forms of BSE in goats were also reported. To minimize further spreading of prion diseases in small ruminants the development of a highly sensitive and specific test for ante-mortem detection of infected animals would be of great value. Recent studies reported high diagnostic value of a second generation of cerebrospinal fluid (CSF) Real-Time Quaking-Induced Conversion (RT-QuIC) assay across a wide spectrum of human prions. Here, we applied this improved QuIC (IQ-CSF) for highly efficient detection of TSEs prion protein in goat cerebrospinal fluid. IQ-CSF sensitivity and specificity were evaluated on CSF samples collected at disease endpoint from goats naturally and experimentally infected with scrapie or bovine isolates of C-BSE and L-BSE, respectively. Next, CSF samples collected from L-BSE infected goats during pre-symptomatic stage were also analysed. PrPL-BSE associated seeding activity was detected at early time points after experimental inoculation, with an average time of 439 days before clinical symptoms appeared. Taken together these data are indicative of the great potential of this in vitro prion amplification assay as ante-mortem TSE test for live and asymptomatic small ruminants.

First Detection of Chronic Wasting Disease in a Wild Red Deer (Cervus elaphus) in Europe.

Chronic wasting disease (CWD) is a fatal contagious prion disease naturally occurring in cervids in North America. In 2016, CWD was detected in wild reindeer (Rangifer tarandus) and moose (Alces alces) in Norway. Here, we report the first known naturally infected wild Norwegian red deer (Cervus elaphus).

Kuru, the First Human Prion Disease.

Kuru, the first human prion disease was transmitted to chimpanzees by D. Carleton Gajdusek (1923⁻2008). In this review, we summarize the history of this seminal discovery, its anthropological background, epidemiology, clinical picture, neuropathology, and molecular genetics. We provide descriptions of electron microscopy and confocal microscopy of kuru amyloid plaques retrieved from a paraffin-embedded block of an old kuru case, named Kupenota. The discovery of kuru opened new vistas of human medicine and was pivotal in the subsequent transmission of Creutzfeldt⁻Jakob disease, as well as the relevance that bovine spongiform encephalopathy had for transmission to humans. The transmission of kuru was one of the greatest contributions to biomedical sciences of the 20th century.

Rapid recontamination of a farm building occurs after attempted prion removal.

The transmissible spongiform encephalopathy scrapie of sheep/goats and chronic wasting disease of cervids are associated with environmental reservoirs of infectivity. Preventing environmental prions acting as a source of infectivity to healthy animals is of major concern to farms that have had outbreaks of scrapie and also to the health management of wild and farmed cervids. Here, an efficient scrapie decontamination protocol was applied to a farm with high levels of environmental contamination with the scrapie agent. Post-decontamination, no prion material was detected within samples taken from the farm buildings as determined using a sensitive in vitro replication assay (sPMCA). A bioassay consisting of 25 newborn lambs of highly susceptible prion protein genotype VRQ/VRQ introduced into this decontaminated barn was carried out in addition to sampling and analysis of dust samples that were collected during the bioassay. Twenty-four of the animals examined by immunohistochemical analysis of lymphatic tissues were scrapie-positive during the bioassay, samples of dust collected within the barn were positive by month 3. The data illustrates the difficulty in decontaminating farm buildings from scrapie, and demonstrates the likely contribution of farm dust to the recontamination of these environments to levels that are capable of causing disease.

Synthetic Prion Selection and Adaptation.

Prion pathologies are characterized by the conformational conversion of the cellular prion protein (PrPC) into a pathological infectious isoform, known as PrPSc. The latter acquires different abnormal conformations, which are associated with specific pathological phenotypes. Recent evidence suggests that prions adapt their conformation to changes in the context of replication. This phenomenon is known as either prion selection or adaptation, where distinct conformations of PrPSc with higher propensity to propagate in the new environment prevail over the others. Here, we show that a synthetically generated prion isolate, previously subjected to protein misfolding cyclic amplification (PMCA) and then injected in animals, is able to change its biochemical and biophysical properties according to the context of replication. In particular, in second transmission passage in vivo, two different prion isolates were found: one characterized by a predominance of the monoglycosylated band (PrPSc-M) and the other characterized by a predominance of the diglycosylated one (PrPSc-D). Neuropathological, biochemical, and biophysical assays confirmed that these PrPSc possess distinctive characteristics. Finally, PMCA analysis of PrPSc-M and PrPSc-D generated PrPSc (PrPSc-PMCA) whose biophysical properties were different from those of both inocula, suggesting that PMCA selectively amplified a third PrPSc isolate. Taken together, these results indicate that the context of replication plays a pivotal role in either prion selection or adaptation. By exploiting the ability of PMCA to mimic the process of prion replication in vitro, it might be possible to assess how changes in the replication environment influence the phenomenon of prion selection and adaptation.

In Vitro Prion Amplification Methodology for Inhibitor Screening.

Prion (PrPC) is an endogenous protein found mainly in the nervous system, and its misfolded isoform (PrPSc) is associated with a group of neurodegenerative disorders known as transmissible spongiform encephalopathies, or simply prion diseases. The PrPSc isoform shows an intriguing ability to self-perpetuate, acting as template for PrPC misfolding and consequent aggregation. Aggregation in vitro and in vivo follows a fibrillation processes that is associated with neurodegeneration. Therefore, it is important to investigate and understand the molecular mechanisms involved in this process; such understanding also allows investigation of the action of possible candidate molecules to inhibit this process. Here, we highlight useful in vitro methodologies and analyses that were developed using PrP as a protein model but that, as other amyloid proteins also exhibit the same behavior, may be applied to understand other "prion-like" diseases such as Alzheimer's and Parkinson's disease.

Prion Seeds Distribute throughout the Eyes of Sporadic Creutzfeldt-Jakob Disease Patients.

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common prion disease in humans and has been iatrogenically transmitted through corneal graft transplantation. Approximately 40% of sCJD patients develop visual or oculomotor symptoms and may seek ophthalmological consultation. Here we used the highly sensitive real-time quaking-induced conversion (RT-QuIC) assay to measure postmortem prion seeding activities in cornea, lens, ocular fluid, retina, choroid, sclera, optic nerve, and extraocular muscle in the largest series of sCJD patient eyes studied by any assay to date. We detected prion seeding activity in 100% of sCJD eyes, representing three common sCJD subtypes, with levels varying by up to 4 log-fold among individuals. The retina consistently showed the highest seed levels, which in some cases were only slightly lower than brain. Within the retina, prion deposits were detected by immunohistochemistry (IHC) in the retinal outer plexiform layer in most sCJD cases, and in some eyes the inner plexiform layer, consistent with synaptic prion deposition. Prions were not detected by IHC in any other eye region. With RT-QuIC, prion seed levels generally declined in eye tissues with increased distance from the brain, and yet all corneas had prion seeds detectable. Prion seeds were also present in the optic nerve, extraocular muscle, choroid, lens, vitreous, and sclera. Collectively, these results reveal that sCJD patients accumulate prion seeds throughout the eye, indicating the potential diagnostic utility as well as a possible biohazard.IMPORTANCE Cases of iatrogenic prion disease have been reported from corneal transplants, yet the distribution and levels of prions throughout the eye remain unknown. This study probes the occurrence, level, and distribution of prions in the eyes of patients with sporadic Creutzfeldt-Jakob disease (sCJD). We tested the largest series of prion-infected eyes reported to date using an ultrasensitive technique to establish the prion seed levels in eight regions of the eye. All 11 cases had detectable prion seeds in the eye, and in some cases, the seed levels in the retina approached those in brain. In most cases, prion deposits could also be seen by immunohistochemical staining of retinal tissue; other ocular tissues were negative. Our results have implications for estimating the risk for iatrogenic transmission of sCJD as well as for the development of antemortem diagnostic tests for prion diseases.

Modified Protein Misfolding Cyclic Amplification Overcomes Real-Time Quaking-Induced Conversion Assay Inhibitors in Deer Saliva To Detect Chronic Wasting Disease Prions.

Chronic wasting disease (CWD), a fatal neurodegenerative prion disease of cervids, has spread across North America and has been detected in The Republic of Korea, Finland, and Norway. CWD appears to spread by horizontal transmission, and prions shed in saliva, feces, and urine are thought to contribute. However, studies investigating the rapid spread of CWD have been hampered by assay inhibitors and a lack of consistent and sensitive means to detect the relatively low levels of prions in these samples. Here we show that saliva frequently contains an inhibitor of the real-time quaking-induced conversion assay (RT-QuIC) and that the inhibitor is a member of the mucin family. To circumvent the inhibitor, we developed a modified protein misfolding cyclic amplification (PMCA) method to amplify CWD prions in saliva that were undetectable or ambiguous by RT-QuIC. Our results reinforce the impact of saliva in horizontal CWD transmission and highlight the importance of detection optimization.

Comparative analysis of prions in nervous and lymphoid tissues of chronic wasting disease-infected cervids.

The prevalence, host range and geographical bounds of chronic wasting disease (CWD), the prion disease of cervids, are expanding. Horizontal transmission likely contributes the majority of new CWD cases, but the mechanism by which prions are transmitted among CWD-affected cervids remains unclear. To address the extent to which prion amplification in peripheral tissues contributes to contagious transmission, we assessed the prion levels in central nervous and lymphoreticular system tissues in white-tailed deer (Odocoileus virginianus), red deer (Cervus elaphus elaphus) and elk (Cervus canadensis). Using real-time quaking-induced conversion, cervid prion cell assay and transgenic mouse bioassay, we found that the retropharyngeal lymph nodes of red deer, white-tailed deer and elk contained similar prion titres to brain from the same individuals. We propose that marked lymphotropism is essential for the horizontal transmission of prion diseases and postulate that shed CWD prions are produced in the periphery.