Insights from a workplace SARS-CoV-2 specimen collection program, with genomes placed into global sequence phylogeny.

In 2020, the Department of Energy established the National Virtual Biotechnology Laboratory (NVBL) to address key challenges associated with COVID-19. As part of that effort, Pacific Northwest National Laboratory (PNNL) established a capability to collect and analyze specimens from employees who self-reported symptoms consistent with the disease. During the spring and fall of 2021, 688 specimens were screened for SARS-CoV-2, with 64 (9.3%) testing positive using reverse-transcriptase quantitative PCR (RT-qPCR). Of these, 36 samples were released for research. All 36 positive samples released for research were sequenced and genotyped. Here, the relationship between patient age and viral load as measured by Ct values was measured and determined to be only weakly significant. Consensus sequences for each sample were placed into a global phylogeny and transmission dynamics were investigated, revealing that the closest relative for many samples was from outside of Washington state, indicating mixing of viral pools within geographic regions.

COVID19 Disease Map, a computational knowledge repository of virus-host interaction mechanisms.

We need to effectively combine the knowledge from surging literature with complex datasets to propose mechanistic models of SARS-CoV-2 infection, improving data interpretation and predicting key targets of intervention. Here, we describe a large-scale community effort to build an open access, interoperable and computable repository of COVID-19 molecular mechanisms. The COVID-19 Disease Map (C19DMap) is a graphical, interactive representation of disease-relevant molecular mechanisms linking many knowledge sources. Notably, it is a computational resource for graph-based analyses and disease modelling. To this end, we established a framework of tools, platforms and guidelines necessary for a multifaceted community of biocurators, domain experts, bioinformaticians and computational biologists. The diagrams of the C19DMap, curated from the literature, are integrated with relevant interaction and text mining databases. We demonstrate the application of network analysis and modelling approaches by concrete examples to highlight new testable hypotheses. This framework helps to find signatures of SARS-CoV-2 predisposition, treatment response or prioritisation of drug candidates. Such an approach may help deal with new waves of COVID-19 or similar pandemics in the long-term perspective.

Leveraging Structured Biological Knowledge for Counterfactual Inference: A Case Study of Viral Pathogenesis.

Counterfactual inference is a useful tool for comparing outcomes of interventions on complex systems. It requires us to represent the system in form of a structural causal model, complete with a causal diagram, probabilistic assumptions on exogenous variables, and functional assignments. Specifying such models can be extremely difficult in practice. The process requires substantial domain expertise, and does not scale easily to large systems, multiple systems, or novel system modifications. At the same time, many application domains, such as molecular biology, are rich in structured causal knowledge that is qualitative in nature. This article proposes a general approach for querying a causal biological knowledge graph, and converting the qualitative result into a quantitative structural causal model that can learn from data to answer the question. We demonstrate the feasibility, accuracy and versatility of this approach using two case studies in systems biology. The first demonstrates the appropriateness of the underlying assumptions and the accuracy of the results. The second demonstrates the versatility of the approach by querying a knowledge base for the molecular determinants of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced cytokine storm, and performing counterfactual inference to estimate the causal effect of medical countermeasures for severely ill patients.

Polysaccharide-based liquid storage and transport media for non-refrigerated preservation of bacterial pathogens.

The preservation of biological samples for an extended time period of days to weeks after initial collection is important for the identification, screening, and characterization of bacterial pathogens. Traditionally, preservation relies on cold-chain infrastructure; however, in many situations this is impractical or not possible. Thus, our goal was to develop alternative bacterial sample preservation and transport media that are effective without refrigeration or external instrumentation. The viability, nucleic acid stability, and protein stability of Bacillus anthracis Sterne 34F2, Francisella novicida U112, Staphylococcus aureus ATCC 43300, and Yersinia pestis KIM D27 (pgm-) was assessed for up to 28 days. Xanthan gum (XG) prepared in PBS with L-cysteine maintained more viable F. novicida U112 cells at elevated temperature (40°C) compared to commercial reagents and buffers. Viability was maintained for all four bacteria in XG with 0.9 mM L-cysteine across a temperature range of 22-40°C. Interestingly, increasing the concentration to 9 mM L-cysteine resulted in the rapid death of S. aureus. This could be advantageous when collecting samples in the built environment where there is the potential for Staphylococcus collection and stabilization rather than other organisms of interest. F. novicida and S. aureus DNA were stable for up to 45 days upon storage at 22°C or 40°C, and direct analysis by real-time qPCR, without DNA extraction, was possible in the XG formulations. XG was not compatible with proteomic analysis via LC-MS/MS due to the high amount of residual Xanthomonas campestris proteins present in XG. Our results demonstrate that polysaccharide-based formulations, specifically XG with L-cysteine, maintain bacterial viability and nucleic acid integrity for an array of both Gram-negative and Gram-positive bacteria across ambient and elevated temperatures.

Unified feature association networks through integration of transcriptomic and proteomic data.

High-throughput multi-omics studies and corresponding network analyses of multi-omic data have rapidly expanded their impact over the last 10 years. As biological features of different types (e.g. transcripts, proteins, metabolites) interact within cellular systems, the greatest amount of knowledge can be gained from networks that incorporate multiple types of -omic data. However, biological and technical sources of variation diminish the ability to detect cross-type associations, yielding networks dominated by communities comprised of nodes of the same type. We describe here network building methods that can maximize edges between nodes of different data types leading to integrated networks, networks that have a large number of edges that link nodes of different-omic types (transcripts, proteins, lipids etc). We systematically rank several network inference methods and demonstrate that, in many cases, using a random forest method, GENIE3, produces the most integrated networks. This increase in integration does not come at the cost of accuracy as GENIE3 produces networks of approximately the same quality as the other network inference methods tested here. Using GENIE3, we also infer networks representing antibody-mediated Dengue virus cell invasion and receptor-mediated Dengue virus invasion. A number of functional pathways showed centrality differences between the two networks including genes responding to both GM-CSF and IL-4, which had a higher centrality value in an antibody-mediated vs. receptor-mediated Dengue network. Because a biological system involves the interplay of many different types of molecules, incorporating multiple data types into networks will improve their use as models of biological systems. The methods explored here are some of the first to specifically highlight and address the challenges associated with how such multi-omic networks can be assembled and how the greatest number of interactions can be inferred from different data types. The resulting networks can lead to the discovery of new host response patterns and interactions during viral infection, generate new hypotheses of pathogenic mechanisms and confirm mechanisms of disease.

The landscape of viral proteomics and its potential to impact human health.

INTRODUCTION: Advances in mass spectrometry-based proteomic technologies are enhancing studies of viral pathogenesis. Identification and quantification of host and viral proteins and modifications in cells and extracellular fluids during infection provides useful information about pathogenesis, and will be critical for directing clinical interventions and diagnostics. AREAS COVERED: Herein we review and discuss a broad range of global proteomic studies conducted during viral infection, including those of cellular responses, protein modifications, virion packaging, and serum proteomics. We focus on viruses that impact human health and focus on experimental designs that reveal disease processes and surrogate markers. Expert commentary: Global proteomics is an important component of systems-level studies that aim to define how the interaction of humans and viruses leads to disease. Viral-community resource centers and strategies from other fields (e.g., cancer) will facilitate data sharing and platform-integration for systems-level analyses, and should provide recommended standards and assays for experimental designs and validation.

The interplay between immune maturation, age, chronic viral infection and environment.

BACKGROUND: The worldwide increase in life expectancy has been associated with an increase in age-related morbidities. The underlying mechanisms resulting in immunosenescence are only incompletely understood. Chronic viral infections, in particular infection with human cytomegalovirus (HCMV), have been suggested as a main driver in immunosenescence. Here, we propose that rhesus macaques could serve as a relevant model to define the impact of chronic viral infections on host immunity in the aging host. We evaluated whether chronic rhesus CMV (RhCMV) infection, similar to HCMV infection in humans, would modulate normal immunological changes in the aging individual by taking advantage of the unique resource of rhesus macaques that were bred and raised to be Specific Pathogen Free (SPF-2) for distinct viruses. RESULTS: Our results demonstrate that normal age-related immunological changes in frequencies, activation, maturation, and function of peripheral blood cell lymphocytes in humans occur in a similar manner over the lifespan of rhesus macaques. The comparative analysis of age-matched SPF-2 and non-SPF macaques that were housed under identical conditions revealed distinct differences in certain immune parameters suggesting that chronic pathogen exposure modulated host immune responses. All non-SPF macaques were infected with RhCMV, suggesting that chronic RhCMV infection was a major contributor to altered immune function in non-SPF macaques, although a causative relationship was not established and outside the scope of these studies. Further, we showed that immunological differences between SPF-2 and non-SPF macaques were already apparent in adolescent macaques, potentially predisposing RhCMV-infected animals to age-related pathologies. CONCLUSIONS: Our data validate rhesus macaques as a relevant animal model to study how chronic viral infections modulate host immunity and impact immunosenescence. Comparative studies in SPF-2 and non-SPF macaques could identify important mechanisms associated with inflammaging and thereby lead to new therapies promoting healthy aging in humans.

Human herpes simplex virus type 1 in confiscated gorilla.

In 2007, we detected human herpes simplex virus type 1, which caused stomatitis, in a juvenile confiscated eastern lowland gorilla (Gorilla beringei graueri) that had a high degree of direct contact with human caretakers. Our findings confirm that pathogens can transfer between nonhuman primate hosts and humans.

Patterns of acute rhesus cytomegalovirus (RhCMV) infection predict long-term RhCMV infection.

We previously reported that long-term rhesus cytomegalovirus (RhCMV) excretion in infected macaques was related to UL/b' coding content. Acute biopsy specimens of the inoculation sites from the previous study have now been analyzed to determine whether there were acute phenotypic predictors of long-term RhCMV infection. Only in animals displaying acute endothelial tropism and neutrophilic inflammation was RhCMV excretion detected. The results imply that vaccinating against these early viral determinants would significantly impede long-term RhCMV infection.

Immunogenicity of a vaccine formulated with the Chlamydia trachomatis serovar F, native major outer membrane protein in a nonhuman primate model.

To determine the ability of a vaccine formulated with the genital Chlamydia trachomatis, serovar F, native major outer membrane protein (Ct-F-nMOMP), to induce systemic and mucosal immune responses, rhesus macaques (Macaca mulatta) were immunized three times by the intramuscular (i.m.) and subcutaneous (s.c.) routes using CpG-2395 and Montanide ISA 720 VG, as adjuvants. As controls, another group of M. mulatta was immunized with ovalbumin instead of Ct-F-nMOMP using the same formulation and routes. High levels of Chlamydia-specific IgG and IgA antibodies were detected in plasma, vaginal washes, tears, saliva, and stools from the Ct-F-nMOMP immunized animals. Also, high neutralizing antibody titers were detected in the plasma from these animals. Monkeys immunized with ovalbumin had no detectable Chlamydia-specific antibodies. Furthermore, as measured by a lymphoproliferative assay, significant Chlamydia-specific cell-mediated immune responses were detected in the peripheral blood mononuclear cells (PBMC) from the rhesus macaques vaccinated with Ct-F-nMOMP when compared with the animals immunized with ovalbumin. In addition, the levels of two Th1 cytokines, IFN-γ and TNF-α, were significantly higher in the animals immunized with Ct-F-nMOMP when compared with those from the monkeys immunized with ovalbumin. To our knowledge, this is the first time that mucosal and systemic immune responses have been investigated in a nonhuman primate model using a subunit vaccine from a human genital C. trachomatis serovar.

Open reading frames carried on UL/b' are implicated in shedding and horizontal transmission of rhesus cytomegalovirus in rhesus monkeys.

Implicit with the use of animal models to test human cytomegalovirus (HCMV) vaccines is the assumption that the viral challenge of vaccinated animals reflects the anticipated virus-host interactions following exposure of vaccinated humans to HCMV. Variables of animal vaccine studies include the route of exposure to and the titer of challenge virus, as well as the genomic coding content of the challenge virus. This study was initiated to provide a better context for conducting vaccine trials with nonhuman primates by determining whether the in vivo phenotype of culture-passaged strains of rhesus cytomegalovirus (RhCMV) is comparable to that of wild-type RhCMV (RhCMV-WT), particularly in relation to the shedding of virus into bodily fluids and the potential for horizontal transmission. Results of this study demonstrate that two strains containing a full-length UL/b' region of the RhCMV genome, which encodes proteins involved in epithelial tropism and immune evasion, were persistently shed in large amounts in bodily fluids and horizontally transmitted, whereas a strain lacking a complete UL/b' region was not shed or transmitted to cagemates. Shedding patterns exhibited by strains encoding a complete UL/b' region were consistent with patterns observed in naturally infected monkeys, the majority of whom persistently shed high levels of virus in saliva for extended periods of time after seroconversion. Frequent viral shedding contributed to a high rate of infection, with RhCMV-infected monkeys transmitting virus to one naïve animal every 7 weeks after introduction of RhCMV-WT into an uninfected cohort. These results demonstrate that the RhCMV model can be designed to rigorously reflect the challenges facing HCMV vaccine trials, particularly those related to horizontal transmission.

Vaccine-induced control of viral shedding following rhesus cytomegalovirus challenge in rhesus macaques.

The use of animal models of human cytomegalovirus (HCMV) infection is critical to refine HCMV vaccine candidates. Previous reports have demonstrated that immunization of rhesus monkeys against rhesus cytomegalovirus (RhCMV) can reduce both local and systemic replication of RhCMV following experimental RhCMV challenge. These studies used prime/boost combinations of DNA expression plasmids alone or DNA priming and boosting with either inactivated virion particles or modified vaccinia virus Ankara (MVA) expressing the same antigens. Viral outcomes included reduced RhCMV replication at the site of subcutaneous inoculation and RhCMV viremia following intravenous inoculation. Since shedding of cytomegalovirus from mucosal surfaces is critical for horizontal transmission of the virus, DNA priming/MVA boosting was evaluated for the ability to reduce oral shedding of RhCMV following subcutaneous challenge. Of six rhesus monkeys vaccinated exclusively against RhCMV glycoprotein B (gB), phosphoprotein 65 (pp65), and immediate-early 1 (IE1), half showed viral loads in saliva that were lower than those of control monkeys by 1 to 3 orders of magnitude. Further, there was a strong association of memory pp65 T cell responses postchallenge in animals exhibiting the greatest reduction in oral shedding. These results highlight the fact that a DNA/MVA vaccination regimen can achieve a notable reduction in a critical parameter of viral replication postchallenge. The recently completed clinical trial of a gB subunit vaccine in which the rate of HCMV infection was reduced by 50% in the individuals receiving the vaccine is consistent with the results of this study suggesting that additional immunogens are likely essential for maximum protection in an outbred human population.

Protein coding content of the UL)b' region of wild-type rhesus cytomegalovirus.

A recent comparison of two rhesus cytomegalovirus (RhCMV) genomes revealed that the region at the right end of the U(L) genome component (U(L)b') undergoes genetic alterations similar to those observed in serially passaged human cytomegalovirus (HCMV). To determine the coding content of authentic wild-type RhCMV in this region, the U(L)b' sequence was amplified from virus obtained from naturally infected rhesus macaques without passage in vitro. A total of 24 open reading frames (ORFs) potentially encoding >99 amino acid residues were identified, 10 of which are related to HCMV ORFs and 15 to previously listed RhCMV ORFs. In addition, the analysis revealed a cluster of three novel alpha chemokine-like ORFs, bringing the number of predicted alpha chemokine genes in this region to six. Three of these six genes exhibit a high level of sequence diversity, as has been observed for the HCMV alpha chemokine gene UL146.