Cryptosporidium uses multiple distinct secretory organelles to interact with and modify its host cell.

Cryptosporidium is a leading cause of diarrheal disease in children and an important contributor to early childhood mortality. The parasite invades and extensively remodels intestinal epithelial cells, building an elaborate interface structure. How this occurs at the molecular level and the contributing parasite factors are largely unknown. Here, we generated a whole-cell spatial proteome of the Cryptosporidium sporozoite and used genetic and cell biological experimentation to discover the Cryptosporidium-secreted effector proteome. These findings reveal multiple organelles, including an original secretory organelle, and generate numerous compartment markers by tagging native gene loci. We show that secreted proteins are delivered to the parasite-host interface, where they assemble into different structures including a ring that anchors the parasite into its unique epicellular niche. Cryptosporidium thus uses a complex set of secretion systems during and following invasion that act in concert to subjugate its host cell.

Design, Implementation, and Outcomes of a Volunteer-Staffed Case Investigation and Contact Tracing Initiative at an Urban Academic Medical Center.

Importance: The COVID-19 pandemic has claimed nearly 6 million lives globally as of February 2022. While pandemic control efforts, including contact tracing, have traditionally been the purview of state and local health departments, the COVID-19 pandemic outpaced health department capacity, necessitating actions by private health systems to investigate and control outbreaks, mitigate transmission, and support patients and communities. Objective: To investigate the process of designing and implementing a volunteer-staffed contact tracing program at a large academic health system from April 2020 to May 2021, including program structure, lessons learned through implementation, results of case investigation and contact tracing efforts, and reflections on how constrained resources may be best allocated in the current pandemic or future public health emergencies. Design, Setting, and Participants: This case series study was conducted among patients at the University of Pennsylvania Health System and in partnership with the Philadelphia Department of Public Health. Patients who tested positive for COVID-19 were contacted to counsel them regarding safe isolation practices, identify and support quarantine of their close contacts, and provide resources, such as food and medicine, needed during isolation or quarantine. Results: Of 5470 individuals who tested positive for COVID-19 and received calls from a volunteer, 2982 individuals (54.5%; median [range] age, 42 [18-97] years; 1628 [59.4%] women among 2741 cases with sex data) were interviewed; among 2683 cases with race data, there were 110 Asian individuals (3.9%), 1476 Black individuals (52.7%), and 817 White individuals (29.2%), and among 2667 cases with ethnicity data, there were 366 Hispanic individuals (13.1%) and 2301 individuals who were not Hispanic (82.6%). Most individuals lived in a household with 2 to 5 people (2125 of 2904 individuals with household data [71.6%]). Of 3222 unique contacts, 1780 close contacts (55.2%; median [range] age, 40 [18-97] years; 866 [55.3%] women among 1565 contacts with sex data) were interviewed; among 1523 contacts with race data, there were 69 Asian individuals (4.2%), 705 Black individuals (43.2%), and 573 White individuals (35.1%), and among 1514 contacts with ethnicity data, there were 202 Hispanic individuals (12.8%) and 1312 individuals (83.4%) who were not Hispanic. Most contacts lived in a household with 2 to 5 people (1123 of 1418 individuals with household data [79.2%]). Of 3324 cases and contacts who completed a questionnaire on unmet social needs, 907 (27.3%) experienced material hardships that would make it difficult for them to isolate or quarantine safely. Such hardship was significantly less common among White compared with Black participants (odds ratio, 0.20; 95% CI, 0.16-0.25). Conclusions and Relevance: These findings demonstrate the feasibility and challenges of implementing a case investigation and contact tracing program at an academic health system. In addition to successfully engaging most assigned COVID-19 cases and close contacts, contact tracers shared health information and material resources to support isolation and quarantine, thus filling local public health system gaps and supporting local pandemic control.

Multiple Introductions of SARS-CoV-2 Alpha and Delta Variants into White-Tailed Deer in Pennsylvania.

The SARS-CoV-2 pandemic began by viral spillover from animals to humans; today multiple animal species are known to be susceptible to infection. White-tailed deer, Odocoileus virginianus, are infected in North America at substantial levels, and genomic data suggests that a variant in deer may have spilled back to humans. Here, we characterize SARS-CoV-2 in deer from Pennsylvania (PA) sampled during fall and winter 2021. Of 123 nasal swab samples analyzed by RT-qPCR, 20 (16.3%) were positive for SARS-CoV-2. Seven whole genome sequences were obtained, together with six more partial spike gene sequences. These annotated as alpha and delta variants, the first reported observations of these lineages in deer, documenting multiple new jumps from humans to deer. The alpha lineage persisted in deer after its displacement by delta in humans, and deer-derived alpha variants diverged significantly from those in humans, consistent with a distinctive evolutionary trajectory in deer. IMPORTANCE Coronaviruses have been documented to replicate in numerous species of vertebrates, and multiple spillovers of coronaviruses from animals into humans have founded human epidemics. The COVID-19 epidemic likely derived from a spillover of SARS-CoV-2 from bats into humans, possibly via an intermediate host. There are now several examples of SARS-CoV-2 jumping from humans into other mammals, including mink and deer, creating the potential for new animal reservoirs from which spillback into humans could occur. For this reason, data on formation of new animal reservoirs is of great importance for understanding possible sources of future infection. Here, we identify extensive infection in white-tailed deer in Pennsylvania, including what appear to be multiple independent transmissions. Data further suggests possible transmission among deer. These data thus help identify a potential new animal reservoir and provide background information relevant to its management.

Evaluation of a Contact Tracing Training Program and Field Experience.

Introduction: The study objective was to evaluate a contact tracing training program and the role of contact tracing on volunteers' professional development. Methods: A COVID-19 contact tracing program was conducted at an urban academic medical center, in collaboration with the local health department, between March 2020 and May 2021. Contact tracers, most of whom were health professions students, completed pretraining and post-training surveys to assess knowledge and self-efficacy to conduct contact tracing, plus an 18-month follow-up survey regarding career impacts. Results: We observed statistically significant post-training increases in knowledge and self-efficacy to conduct contact tracing. Contact tracers described benefiting from training regarding cultural humility, empathy, and trauma-informed interviewing. They also expressed a deeper understanding of COVID-19 inequities and their structural causes and reported that the work was emotionally demanding. Conclusions: Key to pandemic preparedness is having a trained and supported workforce. This study showed how contact tracing training and field experience strengthened students' education in the health professions by sharpening interpersonal skills and structural competency and by generating insights regarding current gaps in both public health infrastructure and support for vulnerable populations.

SARS-CoV-2 variants associated with vaccine breakthrough in the Delaware Valley through summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed three-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of 1.25-3.18). This study thus provides a detailed picture of viral evolution in the Delaware Valley and a geographically matched analysis of vaccine breakthroughs; it also introduces a rigorous statistical approach to interrogating enrichment of viral variants.

SARS-CoV-2 Variants Associated with Vaccine Breakthrough in the Delaware Valley through Summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here, we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed 3-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of1.25-3.18). This study thus overviews viral evolution and vaccine breakthroughs in the Delaware Valley and introduces a rigorous statistical approach to interrogating enrichment of breakthrough variants against a changing background. IMPORTANCE SARS-CoV-2 vaccination is highly effective at reducing viral infection, hospitalization and death. However, vaccine breakthrough infections have been widely observed, raising the question of whether particular viral variants or viral mutations are associated with breakthrough. Here, we report analysis of 2621 surveillance isolates from people diagnosed with COVID-19 in the Delaware Valley in southeastern Pennsylvania, allowing rigorous comparison to 159 vaccine breakthrough case specimens. Our best estimate is a 3-fold enrichment for some lineages of delta among breakthroughs, and enrichment of a notable spike substitution, N501Y. We introduce statistical methods that should be widely useful for evaluating vaccine breakthroughs and other viral phenotypes.

MIST1 Links Secretion and Stress as both Target and Regulator of the Unfolded Protein Response.

Transcriptional networks that govern secretory cell specialization, including instructing cells to develop a unique cytoarchitecture, amass extensive protein synthesis machinery, and be embodied to respond to endoplasmic reticulum (ER) stress, remain largely uncharacterized. In this study, we discovered that the secretory cell transcription factor MIST1 (Bhlha15), previously shown to be essential for cytoskeletal organization and secretory activity, also functions as a potent ER stress-inducible transcriptional regulator. Genome-wide DNA binding studies, coupled with genetic mouse models, revealed MIST1 gene targets that function along the entire breadth of the protein synthesis, processing, transport, and exocytosis networks. Additionally, key MIST1 targets are essential for alleviating ER stress in these highly specialized cells. Indeed, MIST1 functions as a coregulator of the unfolded protein response (UPR) master transcription factor XBP1 for a portion of target genes that contain adjacent MIST1 and XBP1 binding sites. Interestingly, Mist1 gene expression is induced during ER stress by XBP1, but as ER stress subsides, MIST1 serves as a feedback inhibitor, directly binding the Xbp1 promoter and repressing Xbp1 transcript production. Together, our findings provide a new paradigm for XBP1-dependent UPR regulation and position MIST1 as a potential biotherapeutic for numerous human diseases.