Microbiology Virtual Week 2020

While global demand for food is expected to increase significantly (70% by 2050), the agricultural industry is suffering from a decline in soil fertility, the adverse effects of climate change and the high cost of agrochemicals. Therefore food security is in danger. A need arises for sustainable agriculture methods that complement contemporary farming. Harnessing natural resources – particularly, plant and soil microbiomes – has the potential to enable the agricultural industry to remain profitable while ensuring food security and environmental sustainability. However, technical, translational and policy challenges must first be addressed. The presentation will identify these challenges and will propose a framework to solve them,  highlighting the critical role of the Global Initiative of Crop Microbiome as well as sustainable agriculture to bring together global expertise to address key challenges and to deliver on the United Nation's Sustainable Development Goals.

Learning Objectives:

1. Understand the challenges to respond to an increase in global demand for food and fiber

2. Identify the need to harness natural resources to combine food security and sustainability

3. Discuss the critical role of the Global Crop Microbiome initiative

While live cell imaging offers advantages over traditional static imaging, this approach has been challenging for studying microbes due to the difficulty in tracking very small cells in a single focal plane during long term culture. Together with the CellASIC® ONIX2 Microfluidic System for live cell imaging, the CellASIC® Trap Plates offer unique architecture that positions nonadherent live cells in a focal plane to enable continuous imaging and simpler generational analysis of progeny over time.  In addition, these plates offer special features for monitoring biofilm growth and its response to environmental perturbation. Here, we report additional capability enabled by our next generation trap plate with a proprietary design that alleviates drawbacks previously imposed by cellular overgrowth. When combined with the ONIX2 system, the new plate offers an unprecedented turnkey solution to the mechanistic investigation of the impact of environmental conditions on microbes.

Learning Objectives:

In Pacific Northwest watersheds several species of Pacific salmonid fishes are hosts for the rhabdovirus infectious hematopoietic necrosis virus (IHNV).  In this multi-host ecosystem specialist genogroups of IHNV have evolved with high fitness and high virulence in sockeye salmon, steelhead trout, or Chinook salmon. A generalist genogroup of IHNV also occurs with high frequency in both Chinook salmon and steelhead trout, but it exhibits low virulence in the field. In controlled wet laboratory infection studies we are investigating the specialist and generalist phenotypes of the four IHNV subgroups in all three salmonid fish host species.  Results of virulence trials mimic the specialist and generalist trends observed in the field, confirming that for IHNV specialization leads to high virulence. We find that all viruses are able to infect all host species but there are differences in early replication and they vary dramatically in ability to persist. This suggests that the basis of host specificity involves ability to avoid the strong innate immune response of the host, rather than differing ability to gain entry.  In addition, a new project is investigating the evolution of virulence after a historic host jump of IHNV from sockeye salmon to rainbow trout in the 1970s, leading to evolution of a major pathogen impacting rainbow trout farming world-wide. These controlled infection studies comprise tractable model systems for investigating the natural evolution of a generalist from a specialist ancestor, empirical testing of specialist-generalist theory predictions in vivo, and tracking of virulence evolution after a vertebrate virus host jump. In combination with genetic surveillance in the field and landscape virus transmission modeling of IHNV in salmonid fish, this system provides a broad understanding of evolution of virulence for an aquatic pathogen in natural and anthropogenically impacted environments.

Learning Objectives:

1. Describe different possible "lifestyles" for viruses evolving in a multi-host ecosystem

2. Understand the differences between specialist, generalist, and non-adapted virus:host relationships

3. Consider different possible outcomes for the evolution of virulence after a virus jumps into a new host

In this webinar, we will present QIAGEN CLC Genomics Workbench and its utility for bacterial isolate identification, strain discrimination using core genome multi-locus typing (cgMLST) and detailed genomic analysis of factors contributing to multi-drug resistance.

Learning Objectives:

1. Demonstrate bioinformatics analysis workflows built using CLC Genomics ProSuite for routine food testing and surveillance purposes

2.  Demonstrate methods using the Microbial Insights database to CLC users seeking to identify the genetic determinants of AMR in bacterial pathogens

The influence of the human microbiome on health is an important area of ongoing investigation for medicine and diagnostics. For these studies, it is crucial to have a reliable, reproducible extraction method which is able to extract DNA of all microorganisms with comparable efficiency, while simultaneously depleting inhibitory substances which might disturb subsequent downstream analysis. We present the results of DNA extraction and microbiome analysis from stool, saliva, and a variety of swabs (buccal, vaginal, rectal, skin) using the DNeasy PowerSoil Pro/QIAamp PowerFecal Pro kits, including automation options and process handling improvements. Materials/methods: Human microbiome samples (stool, saliva, and buccal, vaginal, rectal and skin swabs) were homogenized and microbial cells (including gram-negative and gram-positive bacteria, fungi, and archaea) were rapidly and efficiently lysed by bead beating. Inhibitory substances were removed through the use of Inhibitor Removal Technology (IRT). Thereafter, inhibitor-free DNA was purified and used for 16S rDNA sequencing experiments. Within these experiments, the effect of using inoculating loops for stool sampling was investigated, as well as the impact of automating the procedure. Results: The DNeasy PowerSoil Pro/QIAamp PowerFecal Pro Kits were able to efficiently extract microbial DNA with up to 20-fold higher yields compared to other methods.  The extracted samples showed no inhibition in PCR. The use of inoculation loops for stool sampling was shown to be feasible and accurate. 16S rDNA sequencing revealed distinct and complex communities within the different sample types. Conclusion: The DNeasy PowerSoil Pro and QIAamp PowerFecal Pro kits are an optimal method for extracting microbial DNA from human samples for microbiome analysis and can be used for a wide variety of sample types in addition to stool.

Learning Objectives:

1. Understand the challenges of extracting DNA from human samples for microbiome analysis; learn about the best technologies for accomplishing this

2. Learn about process optimization for DNA extraction

3. Learn about applications of the standard stool kits for the processing of different human sample types

Enigmatic and often vilified, viruses are now known to play important and possibly indispensable roles in the biology and ecology of cellular organisms. Evidence of viral impacts are everywhere. Animal and plant genomes are littered with genes of viral origin. Ecosystems contain large numbers of viruses, an estimated global population of 10e31 individuals, dwarfing co-occurring microbial abundances. Most viruses observed within ecosystems infect microbes. During infection, viruses can alter the phenome of host cells in ways that change the population biology of microbial communities and the flow of nutrients and energy within ecosystems. While we have a high-level view of viral impacts, the details are largely a mystery. These details matter as viral impacts on ecosystems are the net result of hundreds or thousands of interacting populations of viruses and microbial hosts. The mechanistic underpinnings of viral effects on ecosystems can only be fully appreciated through a greater understanding of the phenomic features of viral-host interaction networks. Genomic data have shown that viral genetic diversity is vast and largely unknown. Often less than half of the genes within a dsDNA viral genome can be assigned a function based on homology. This situation is worse for viral metagenomes (viromes). Predicting important phenomic features of an unknown virus based on genomic information alone is not currently possible.  However, we have identified three genes — DNA polymerase A, ribonucleotide reductase (RNRs), and chaperonins — which appear to demonstrate particularly strong links to the phenomic characteristics of viruses of microbes (VoMs).  Mutations within PolA appear to be predictive of whether a virus has a lytic or lysogenic life cycle and polA genes seem to be critical in determining the broader suite of genes involved in viral genome replication.  The class and sub-group of RNR genes carried by a virus appear to predict the environmental conditions most favorable to lytic viral production.  The propensity of a virus to carry chaperonin genes may predict its genome size and its capability for altering, more generally, the protein folding machinery within infected cells.  Viral chaperonins within viromes have also demonstrated the existence of unknown viruses infecting marine archaeal populations.  Ultimately, uncovering genome to phenome links within ecologically-important VoM-host systems will improve the predictive utility of viral genomic and metagenomic data for advancing scientific understanding on the role of viruses within ecosystems.

Learning Objectives:

1. Give examples of how viral infection influences ecosystem processes

2. How might the biochemistry of a replication enzyme inform us about the biology of a virus

3. Why are metagenome data critical to building genome to phenome hypotheses for viruses

Congenital CMV is the most frequent infectious cause of neonatal malformation in developed nations. It is more prevalent than other neonatal conditions such as spina bifida and Down syndrome. However, this disease has been under-recognized by many health authorities around the world, as well as largely unknown among pregnant women and their partners. Congenital CMV can lead to hearing loss, neurological impairment and even death, however appropriate and timely treatment can minimize the severity and reduce the burden of this disease. This presentation will address the fundamentals of congenital CMV in prevention, diagnosis and management. We will touch on some recent research, and how we can move forward to continue reducing the impact of congenital CMV globally.

Learning Objectives:

1. Describe the clinical presentation of congenital CMV

2. Understand diagnosis and indications for testing and possibilities of screening

3. Recognize the opportunities for prevention, management and therapeutic strategies

4. Understand research strategies in development to reduce the global impact of congenital CMV

The selective pressure placed on the resident microbiota by local changes in the host environment – DNA damage, chronic inflammation, metabolic shifts, barrier damage, reduced immunosurveillance – might make tumor microenvironment a perfect niche for certain bacterial clades. While bacteria have long been known to play a role in carcinogenesis, only a few have been identified as initiators. In this work we uncover a unique microbiome-gene interaction in lung cancer, between the tumor suppressor gene TP53 and a consortia of bacteria. One taxa in particular, Acidovorax, we discovered was highly abundant in smokers, the primary risk factor for lung cancer. Knowing that mutations in TP53 are prevalent in 70-80% of squamous cell carcinoma (SCC), and confer reduced barrier function, we found that mutations in TP53 were associated with higher Acidovorax in SCC tumors. Further, preliminary data show that Acidovorax temperans administered by nasal instillation accelerates lung tumorigenesis and is detrimental to the survival of a mouse model of adenocarcinoma driven by AdCre-activated Kras and mutant Trp53. Importantly, A. temperans, has the ability to degrade hydrocarbons, such as those found in tobacco smoke, which strongly suggests that microbial metabolism impacts host cellular metabolism and pharmacokinetics. In conducting this research, many lessons were learned, especially in regards to collecting and analyzing low biomass samples. These lessons will be conveyed as critical concepts in human microbiome research and guidelines will be presented for future research studies.

Learning Objectives:

1. Define the main confounders in low-biomass microbiome studies

2. List the key controls you need in human low-biomass studies

3. Name at least two additional methods required to demonstrate ‘proof of life’ beyond sequencing in human microbiome studies

Sepsis is a life-threatening condition that is caused by the immune system’s inability to respond appropriately to an infection. How sepsis can change the gut microbiome in ways that alter the brain and behavior is not well understood. One project in my laboratory is to identify ways to prevent or mitigate the neurological impact of sepsis by understanding how gut microbial communities change in response to sepsis. To investigate this, we compared changes in the gut microbiome between wild type (WT) and Alzheimer’s disease transgenic (CVN-AD) mice who received a sepsis injury. WT and CVN-AD mice were subjected to the cecal ligation and puncture model of sepsis or a sham surgery. Fecal samples were collected from each mouse at baseline (day 0) and 6 days post-sepsis. Each fecal sample was processed in triplicate using Shoreline Biome’s StrainID kit, which features a 16S-23S amplicon product that includes the highly variable internal transcribed spacer (ITS) region. Pooled amplicons (~2500 bp) were sequenced on the PacBio Sequel platform, and reads were demultiplexed using SBAnalyzer software and mapped to the novel Athena database. Dramatic differences in gut microbiome profiles between day 0 and day 6 post-sepsis were observed in both WT and CVN-AD mice. First, a dramatic decrease of the commensal Muribaculum was observed in all septic mice. Second, a unique cluster of septic samples displayed a large increase in Clostridium with a proportional decrease in Muribaculum on day 6. Third, samples with a higher percentage of Lactobacillus at day 0 had a lower decrease in Muribaculum and increase in Clostridium on day 6. WT and CVN-AD mice showed the greatest differences in their abundance of the Lactobacillus strains: L. murinus L. johnsonii, and L. reuteri. These results show that the combination of the StrainID kit, PacBio Sequel, SBAnalyzer software, and Athena database provide an optimal strategy to uncover species- and strain-differences in microbial communities.

Learning Objectives:

1. Explain how sepsis can change the gut microbiome in ways that affect human health and behavior

2. Identify ways that Alzheimer’s disease-like pathology can modify gut microbiome responses during sepsis

3. Describe advantages of 16S-ITS-23S rRNA amplicon sequencing over standard short-read 16S rRNA sequencing

Focusing on the urgent clinical problem of increasing carbapenem resistance in Enterobacteriaceae we have been evaluating detection methods in clinical microbiology and molecular transmission of carbapenemase genes for the last twelve years. Molecular characterization has included analysis of mobile resistance mechanisms with evaluation of plasmid evolution and mobility across species with next generation sequencing paired with more traditional techniques.  During the talk a real world setting in which a plasmid and transposon mediated antimicrobial resistance gene transfer has occurred will act as a backdrop for understanding the different molecular approaches. We will compare and contrast the different molecular and next generation sequencing techniques for evaluating gene exchange in this context.  Through this real world example of ongoing hospital transmission involving the hospital environment and patients we can compare the advantages and disadvantages of different sequencing approaches to understand the challenges of molecular epidemiology of antimicrobial resistance transmission when horizontal gene transfer occurs.

Learning Objectives:

1. Understand the primary mechanisms of horizontal gene transfer between strains and species

2. Describe why horizontal gene transfer can more quickly disseminate antimicrobial resistance

3. Understand how long read and short read sequencing may be needed to describe the genetic context of horizontal gene transfer stting in which a plasmid mediated transmission can be seen

The host response to infection is a critical determinant of virus pathogenicity. Emerging viruses require the host cellular machinery to replicate and successfully infect new hosts, and must subvert host immunity to effectively hijack host cells. The interplay between pathogen and host, and the subsequent responses induced by infection, is the battleground that determines disease phenotype and clinical outcome. Viruses induce myriad host responses, including both antiviral responses that protect the host and aberrant responses that cause damage and enhance pathogenicity. My talk focuses on my approach to understand the complexity of virus-host interactions by combining classical virology with modern systems biology approaches. A key component to this is using reliable experimental models such as the Collaborative Cross mouse systems genetics resource. We used this panel of genetically diverse mice to characterize host responses to infection, elucidate mechanisms of pathogenesis, predict disease outcome, and identify translational opportunities for therapeutic intervention in Ebola virus disease. This model is now being applied to studying SARS-CoV-2.

Learning Objectives:

1. Understanding how systems approaches can be used to investigate the role of the host response in virus pathogenesis

2. Explain the advantages and disadvantages to using different animal models to study human viral disease

3. Understand how the Collaborative Cross mouse model is useful for investigating the host response to Ebola virus infection and its role in disease pathogenesis

The transfer of antimicrobial resistance genes (ARG) to pathogenic microbes is a major concern in modern medicine. Antibiotic therapies are often rendered ineffective by horizontal acquisition of these genes by previously susceptible microbes; however, the vectors of ARGs in the environment are difficult to identify. We have characterized the mechanisms for how mobile DNA that contains ARGs can enter other potential host bacterial cells, but we still have difficulty in identifying all of the potential hosts for these genomic islands in the surrounding environment. Using the cattle rumen microbiome as a model, I will show how improvements in DNA sequencing and chromatin conformation capture (Hi-C) technologies can be used to identify candidate host bacteria for viral and plasmid DNA that could carry ARGs to new bacterial hosts. Use of long-read DNA sequence data uncovered 10-fold more ARG alleles than discovered by short-read sequencing, suggesting that the true number and diversity of alleles were underestimated previously. Application of Hi-C links enabled the detection of living microbes likely to carry these ARG alleles in the community and would therefore serve as vectors for their transmission to other species or strains. This methodology and network analysis algorithm has been released to the community, and has already been used in a similar study looking at ARG allele distribution in human gut microbes. As we apply new technologies and methods to metagenomics data, we are slowly uncovering the true extent to which ARGs are transferred between microbial species in the same environments. These findings impact future considerations for antibiotic usage in clinical medicine and agriculture.

Learning Objectives:

1. Define several different classes of mobile DNA

2. Identify the challenges in identifying antimicrobial resistance genes in the microbiome

3. Discuss the implications of horizontal transfer of resistance genes for antibiotic use.

Microbial biofilms form on all aquatic surfaces and can harbor pathogenic bacteria. In the aquaculture industry, Flavobacteria species can cause serious diseases and lead to high mortality. These disease outbreaks can reoccur on potential reservoir of these pathogens are biofilms. We characterized the biofilms in a commercial trout farm by filtering water entering and leaving the raceways and collecting surface swabs from walls and baffles. DNA was extracted and either the V4 region of the 16S rRNA gene was amplified and sequenced on an Illumina MiSeq or the V1 to V9 regions were amplified and sequenced on a PacBio Sequel II. During the course of this study the raceways walls were not cleaned after stocking the fish, so the age of the fish reflects the age of the biofilm. The data analysis revealed the biofilms in raceways harboring the youngest fish had a simple community and in raceways that harbored older fish had more complex communities. We also cultured hundreds of bacteria from this site. 570 isolates were grown up in broth, their DNA extracted and the V1-V9 region of the 16S rRNA gene amplified using the Shoreline Biome DNA V1-V9 kit. Six different extraction plates were used and six SMRTbells with different barcodes were used to create libraries that were sequenced on a PacBio Sequel II. The data was analyzed using the SBanalyzer, DADA2 and Qiime2. This allowed the identification of the isolates. We identified bacteria that belonged to 22 families and 48 genera. In summary, PacBio, full-length 16S rRNA gene sequences provides high resolution insights into microbial communities and allows the rapid identification of bacteria.

Learning Objectives:

1. Gain an understanding of the changes in composition and complexity of microbial biofilms over time

2. Learn a novel approach for identifying hundreds of bacterial strains by sequencing the full-length 16S rRNA gene using a PacBio Sequel2

The microbiome has emerged as a major contributor to human health and disease. Numerous sources implicate shifts in the gut microbiome as potentially pathologic for a variety of autoimmune diseases. For many of these diseases, changes in the microbiome are subtle and descriptive in nature. Further, limitations in 16S rRNA sequencing make inter-study comparisons and identification of causative microbes difficult. This heterogeneity can be traced to the evolution of sequencing technology and variability in sampling methodology. For example, in multiple sclerosis, numerous research groups have described shifts in gut microbes among patients, but identified taxa have been inconsistent across studies. To address these challenges, we have implemented long-amplicon sequencing of the 16S-ITS-23S rRNA operon, which, when combined with machine learning-based denoising, allows reliable taxonomic resolution down to the species and strain levels, facilitating more definitive comparison between clinical cohorts and possible identification of causative microbes. Using these technologies, we set out to study the microbiomes of new-onset treatment-naïve relapsing-remitting multiple sclerosis at baseline and post B cell depletion therapy, with healthy donors as a control group. We identify Bacteroides emerge as being enriched in the gut microbiota of MS patients. This trend reverses upon initiation of effective immunomodulatory therapy for MS. Manipulation of the gut microbiome is a putative mechanism of action by which immunomodulatory therapies may impact MS and other immune-medicated diseases. Further exploration of these phenomena may open new avenues for understanding and treating MS.

Learning Objectives:

1. Understand the pros/cons of generating and processing long-read microbiome data

2. Identify differences between the microbiome of healthy subjects and MS patients at baseline

3. Understand how immunotherapy may influence the microbiome of MS patients

Lessons around leveraging high-complexity next-generation sequencing tests for precision infectious disease discovery to guide patient treatment and improve health outcomes.

Learning Objectives:

1. Reviewing current available technology and limitations for microbial profiling

2. What is NGS and metagenomics

3. Applications of NGS in urine, stool, and respiratory samples and case studies

Viruses are the causative agents of approximately 12% of human cancers. The most recently discovered herpesvirus, Kaposi’s sarcoma herpesvirus (KSHV) is known to cause three human cancers. Effective antiviral therapeutics are needed for the treatment of KSHV. Viral DNA replication and the KSHV DNA replication proteins are essential to successful viral replication and, thus, appealing therapeutic targets. Our focus is characterizing the functions and molecular interactions of the core KSHV DNA replication proteins in order to develop potential therapeutic strategies against KSHV infection. Herpesviruses encode their own DNA replication machinery. KSHV expresses six DNA replication proteins: DNA polymerase (ORF9), polymerase accessory factor (ORF59), helicase (ORF44), primase (ORF56), primase associated factor (ORF40/41), and single-stranded DNA binding protein (ORF6). The binding of the KSHV polymerase processivity factor, ORF59 (PF-8), to the viral polymerase, ORF9 (Pol-8) promotes continual synthesis of DNA. We visualized ORF59 using negative staining with electron microscopy. We predict head to tail dimers assemble into higher order oligomeric rings composed of four or six monomers, which may function as a sliding clamp during DNA replication. To investigate ORF50 binding in the context of viral DNA replication, we performed in vitro binding assays with purified ORF50 and isolated lytic origin DNA. We visualized purified ORF50 binding to the 2.4kB lytic origin of replication (OriLyt-R) using tungsten metal shadow casting with electron microscopy. We mapped ORF50 binding to OriLyt-R to three distinct regions. Two regions represent novel binding locations. We measured the average area of origin bound ORF50. We predict ORF50 dimerization may stabilize protein-DNA interaction as well as facilitate the binding of additional KSHV DNA replication proteins. Our preliminary finding provides novel insights into the activities of KSHV proteins. Our in-depth molecular study of the core DNA replication proteins will advance the general understanding of KSHV biology.

Learning Objectives:

1. Define the phases of herpesvirus infection

2. Explain high resolution electron microscopy-based approaches to study protein-protein and protein-DNA interactions

3. Identify novel activities of KSHV DNA viral replication proteins.

Asthma is an increasing health concern affecting more than 25 million individuals in the United States and more than 300 million individuals worldwide. In some cases, sensitization or exposure to specific microbes can exacerbate asthma. In 2006, a new asthma phenotype termed “severe asthma with fungal sensitization” (SAFS) was described for individuals whose asthma was poorly-controlled and who were sensitized to fungi. We have previously reported that asthmatics sensitized to fungi demonstrate worse lung function, higher serum IgE, blood eosinophil and exhaled nitric oxide levels. A growing area of interest is the identification of factors that contribute to immunopathogenesis in allergic asthma, including those sensitized to fungi, and determining whether these could be viable therapeutic targets. Indeed, biologics targeting the type 2 cytokines IL-4, IL-5 and IL-13 have been successful in treating individuals with severe asthma. To this end, we have reported that human asthmatics sensitized to fungi have increased levels of multiple cytokines, chemokines and growth factors in bronchoalveolar lavage fluid and sputum, some of which correlate with lung function or atopic measurements. This presentation will provide an overview of these findings and how some of these mediators mechanistically contribute to disease severity using an experimental animal model of fungal-associated allergic airway inflammation.

Learning Objectives:

1. To understand the clinical significance of allergic asthma as it relates to fungal sensitization

2. To understand experimental animal models as it relates to determining immunoprotective vs. immunopathogenic responses during fungal asthma

Epidemics are occurring at an increasing pace and scale. Our laboratory group has developed platform technologies for discovery of broad and potent neutralizing antibodies for many emerging infectious diseases. Studying diverse pathogens, we have discovered general principles about the genetic, molecular, and structural basis for virus neutralization by human antibodies. I will discuss mechanisms of neutralization and review specific case studies of antibiotic discovery programs for emerging infections leading to therapeutic drug candidates now in the clinic.

Learning Objectives:

1. Explain the concepts of sites of vulnerability for antibody-mediated neutralization

2. Define 3 common mechanisms of virus neutralization

3. Explain the rationale for rational selection of antibody combinations

HIV currently infects almost 40 million people worldwide. The virus is responsible for ~2 million new infections per year and ~1 million deaths. Like all retroviruses, HIV integrates a viral DNA copy of its RNA genome into host chromatin, thereby establishing a permanent and irreversible infection in the target cell. Integration depends on the obligate formation of a large oligomeric nucleoprotein complex containing the viral integrase enzyme assembled on viral DNA ends, commonly referred to as an intasome. Intasomes are also targeted by the latest generation antiretrovirals, integrase strand transfer inhibitors (INSTIs). Cryo-EM methods are beginning to shed light on the structures of HIV intasomes and how INSTIs bind, revealing how small changes in the integrase active site can have significant implications for drug binding. This work has implications for expanding effective treatments available for HIV-infected individuals. In this webcast, Dr. Dmitry Lyumkis will describe how advances in cryo-EM are facilitating structure-based drug design targeting HIV integration.

Learning Objectives:

1. The basics of single-particle cryo-EM, including the latest technical advances that are facilitating high-resolution structural studies

2. The biology of HIV integration, and why this step of the viral lifecycle is vulnerable to inhibition

3. How single-particle cryo-EM is becoming applicable to drug design and helping to combat the global HIV epidemic

My group is addressing fundamental questions in evolutionary biology, using both genome- and phenotype-first approaches. A few years ago, we discovered that Arabidopsis thaliana is a great model for the study of hybrid necrosis. This widespread syndrome of hybrid failure in plants is caused by plant paranoia – regardless of the presence of enemies, plants “think” they are being attacked by pathogens. Over the past decade, we have studied in detail the underlying genetics, finding that often one or two loci encoding NLR immune receptors are causal. NLRs make up the most variable gene family in plants, and it is not surprising that they are often involved in genome-genome conflicts. Hybrid necrosis results when NLR genes meet that have not been co-adapted. Our goal for the next decade is to understand the genomic and geographic patterns for immune system diversity. In 2018, we initiated a project, Pathodopsis, in which we aim to describe genetic diversity in the host A. thaliana and two of its important pathogens, the generalist Pseudomonas sp. and the specialist Hyaloperonospora arabidopsidis. The long-term vision is to produce maps of resistance alleles in the host, and of effector alleles in the pathogens, in order to learn when the pathogens win in a wild plant pathosystem – and when the hosts prevail. Additional information about our work can be found on our websites, http://weigelworld.org and http://pathodopsis.org.

Learning Objectives:

1. Understand drivers of plant immune receptor diversity

2. Understand limits of plant immune receptor diversity

Human chromosome 19q13.4 contains genes encoding killer-cell immunoglobulin-like receptors (KIR). The region has certain properties such as single nucleotide variation, structural variation, homology, and repetitive elements that make it hard to align accurately beyond single gene alleles. The 68 reference haplotypes in the human genome reference range in length from 67 to 269 kilobases and contain 4 to 18 genes. We leveraged these references and tools from our long-read KIR haplotype assembly algorithm to define and align KIR haplotypes at <5 kb resolution on average. We then used a standard alignment algorithm to refine that alignment down to single base resolution. This processing demonstrated that the high-level alignment recapitulates human-curated annotation of the human haplotypes as well as a chimpanzee haplotype. Further, assignments and alignments of gene alleles were consistent with their human curation in haplotype and allele databases. These results define KIR haplotypes as 14 loci containing 9 genes. The multiple sequence alignments have been applied in two computational workflows as in silico gene markers and in vitro capture probes. The first workflow is an efficient computational approach for in silico KIR probe interpretation (KPI) to accurately interpret individual’s KIR genes and haplotype-pairs from KIR sequencing reads. The second workflow efficiently captures, sequences, and assembles diploid human KIR haplotypes from PacBio HiFi reads.

Learning Objectives:

1. Identify the types of variation in the KIR region

2. Explain how this variation affects sequence interpretation

3. Define the difference in the output between the two WGS interpretation algorithms

Prokaryotic DNA contains three types of methylation: N6-methyladenine, N4-methylcytosine and 5-methylcytosine. The lack of tools to analyse the frequency and distribution of methylated residues in bacterial genomes has prevented a full understanding of their functions. Now, advances in DNA sequencing technology, including single-molecule, real-time sequencing and nanopore-based sequencing, have provided new opportunities for systematic detection of all three forms of methylated DNA at a genome-wide scale and offer unprecedented opportunities for achieving a more complete understanding of bacterial epigenomes. Indeed, as the number of mapped bacterial methylomes approaches 2,000, increasing evidence supports roles for methylation in regulation of gene expression, virulence and pathogen–host interactions.

Learning Objectives:

1. What are the state of the art methods for mapping bacterial DNA methylation?

2. How can we exploit bacterial DNA methylation to better understand bacteria and microbiome?

Traditionally, virology has been focused in studying the pathogenic effect of viruses. In the recent years, however, this perception is changing and viruses are being studied as mutualistic components of ecosystems. Even pathogenic viruses can be beneficial for its host under certain circumstances: e.g., virus-infected plants showed a higher drought tolerance than the non-infected ones. We are studying the evolutionary dynamics of a pathosystem under drought stress conditions. To do so, we evolved turnip mosaic potyvirus (TuMV) in the model plant Arabidopsis thaliana. Viruses have been subjected to five evolutionary passages in four ecotypes of A. thaliana that were previously shown to have different transcriptomic responses to potyviral infection. In each accession, three independent viral lineages were evolved in normal conditions (SD) and another three were evolved in water-deficient conditions (DT). After evolution, DT-evolved viruses showed a higher infectivity than SD-evolved viruses. All infections increased the drought tolerance of the host, although DT-evolved viruses induced a significantly higher tolerance to water deficit than SD-evolved viruses. DT-evolved viruses provided its host with higher survival rates when facing water withdrawal. As TuMV is a sterilizing virus, plants infected with SD-evolved viruses barely produced seeds. However, plants infected with DT-evolved viruses produced larger amount of seeds than plants infected by SD-evolved viruses, a direct measure of increased plant fitness. Interestingly, the magnitude of this beneficial effects is ecotype-specific. The genetic and physiological causes of these differential host responses to both viruses are being characterized with targeted metabolomic and transcriptomic approaches. Results are consistent with a virus-induced activation of salicylic acid signaling pathway, which is also involved in drought tolerance. This research shows how environmental conditions shape the evolutionary dynamics in a plant-virus pathosystem, modifying the relationships between the virus and the host. Effects are variable among natural ecotypes but in all cases DT-evolved viruses were more virulent but less pathogenic for the host, providing the host an increased tolerance to drought.

Learning Objectives:

1. The triangle of pathology: disease is the outcome of an interplay between host and virus genotypes and environment

2. Host-virus interactions can evolve from parasitic to mutualistic as a consequence of environmental stress

Over the last several decades, antibodies (Abs) have become a valuable weapon in the fight against viral infections, with several studies demonstrating the importance of both neutralizing and non-neutralizing Ab responses in preventing or controlling viral infections in-vivo. Fc receptor mediated non-neutralizing effector functions such as, Ab-dependent Cellular Cytotoxicity (ADCC), phagocytosis and cytokine expression, have all been shown to contribute to effective immune responses in-vivo. However, in the case of some viral infections, such as dengue, Ab/Fc receptor mediated interactions enhance the severity of dengue hemorrhagic fever via Antibody Dependent Enhancement (ADE), which is hypothesized to primarily occur via Fc gamma receptor (FcγR) interactions. Importantly, in the context of the current SARS CoV-2 pandemic, researchers have observed that disease severity correlates with anti-SARS CoV-2 spike protein levels. Further, previous studies on a SARS1 vaccine suggest that ADE may contribute to mortality in animal models. These findings have important implications for anti-spike based SARS CoV-2 therapeutics and vaccine approaches. Therefore, our laboratory has begun to profile the levels of FcγR activation in a cohort of SARS CoV-2 convalescent patients versus severe hospitalized patients, to begin to understand whether ADE is a primary driver of severity and mortality during COVID-19 disease.

Learning objectives:

1. To learn how non-neutralizing Ab responses contribute to immune regulation during viral infections

2. To learn how in the majority of viral infections, non-neutralizing antibodies help to potently control infections in-vivo

3. To learn that in the context of certain viral infections, such as dengue, non-neutralizing Ab functions can be detrimental to the host and enhance severity of disease

The statement by Dimitri Ivanovsky in 1882 that "the sap of leaves infected with tobacco mosaic disease retains its infectious properties even after filtration through Chamberland filter candles" gave birth to a new field of science; Virology. Since that time, and well-beyond the essential goal of crop protection, virology studies conducted in plants have made seminal contributions to science with such landmark demonstrations as the infectious nature of nucleic acids, RNA silencing, and identification of host factors with roles in viral replication and recombination.  This engaging presentation considers how plant virology can inform the most pressing epidemiological concern of our time under the hypothesis that; if plant virology can inform zoonotic virus emergence, then the two should share in common: “outbreak” phenomena (sudden appearance), “spillover” phenomena (transfer from wild to domesticated areas), common genetics (similar types of viruses), reliance on ”vectors” for viral spread, and maintenance of viruses in “reservoir” species.

Learning Objectives:

1. Identify three plant viruses that are genetically similar to viruses that infect humans

2. Identify common vectors and reservoir species for plant viruses

3. Explain how ecology and population biology studies of plant viruses parallel those for zoonotic viruses for informing risk of virus emergence

As a response to various inflammatory stimuli, neutrophils and macrophages expel a mixture of their nuclear and granular elements in the form of extracellular traps (ETs). These web-like substances represent an evolutionarily conserved defense mechanism across humans, animals and even plants. ETs are composed of DNA, histones, and granular enzymes, and are capable of the entrapment of invading pathogens. Besides their protective function, ETs have been detected in a variety of pathological scenarios (from infections and autoimmune conditions to thrombosis) with detrimental effects, therefore they have been often referred to as ‘double-edged swords of immunity’. Most recently, neutrophil ETs have been identified as major contributors to the widespread immune-thrombosis in COVID-19 induced acute respiratory distress syndrome.

Learning Objectives:

1. Understand the basic types of ETs and the route of their formation

2. Name three pathological conditions where ETs play a role

3. Outline the suspected steps of ET contribution to (COVID-19 triggered) thrombosis

The regions of our genome responsible for encoding the genes that regulate our immune response are some of the most complex and polymorphic known. This complexity encompasses multiple types of genetic variation, from enrichment of single nucleotide variants through large structural differences. In several instances this variation has been shown to contribute to differential, individual susceptibility to disease. Investigating these regions is incredibly difficult using traditional short read approaches, as these reads often do not span entire genes or structural variants. Highly accurate long reads, such as those generated by single-molecule, real time (SMRT) sequencing, provide resolution of full-length immune genes without imputation and assembly of large, complex immune loci in a haplotype-specific manner. This presentation will use two examples, the human leukocyte antigen (HLA) locus, which is critically involved in effective T cell immunity, as well as the immunoglobulin heavy chain (IGH) locus, which encodes antibody responses, to demonstrate the power of SMRT sequencing for unparalleled resolution of these loci. In both cases, end-to-end genotyping pipelines have been developed and are available to leverage the power of long read sequencing to provide HLA and IGH genotyping data for most samples of interest.

Learning Objectives:

1. Identify mechanisms of genomic variation in immune loci that provide the extreme diversity of B and T cell responses

2. Understand the benefits of long read (vs. short read) sequencing approaches for resolving immune loci

3. Design an experiment utilizing HLA and/or IGH genotyping to understand the impact of immune loci variation in your specific disease model

Influenza severity is determined by the interplay between the virus and the host response. Previously, we identified a three-pronged lung gene expression signature that predicted severe influenza outcomes in mice. We hypothesized that this signature resulted from differential activation or infiltration of immune cells in the lungs during mild versus severe influenza infection. In order to build a comprehensive model of lung immune dynamics in response to influenza virus infections, we employed tissue deconvolution and linear regression to model the lung immune cell changes that occurred during mild or severe influenza virus infections. Modeling accurately predicted known macrophage dynamics that occur during influenza infection in vivo, and further predicted a novel population that transcriptionally resembled small serosal macrophages. This macrophage population was predicted to be present in the lungs of animals that recovered from influenza infection and absent from the lungs of those that succumbed. Overall, we provide a multidimensional analysis that delineates how immune cell responses relate to immunopathological consequences of influenza infection. We also demonstrate the utility of bulk tissue gene expression data in generating hypotheses about immune cell dynamics.

Learning Objectives:

1. Understand how different lung immune cell populations impact influenza severity

2. Understand how gene expression data can be used to generate hypotheses about immune cell dynamics

To establish productive infection, plant viruses need to be able to efficiently invade and spread within a plant. Most viruses are introduced into a plant via the epidermal or mesophyll cells where they undergo disassembly, replication, translation of their proteins, and assemble new virions. Viruses then move to the adjacent cells until they reach the phloem, which serves as the highway by which viruses spread throughout their hosts. It was shown that the phloem has evolved additional protection against these molecular intruders. Virus entry into, translocation through, and exit from the phloem involve additional viral factors and complex virus-host interactions. Although the majority of plant viruses are capable of infecting most types of cells within their hosts, there are several groups of plant viruses whose tissue tropism is limited to the phloem. These viruses are typically introduced directly into the phloem by vectoring phloem-feeding insects. Interestingly, the movement systems of such viruses are complex and involve multiple viral proteins. The factors and processes that control phloem restriction of these viruses are not well understood. We are working with Citrus tristeza virus (CTV), the largest non-segmented positive-sense RNA virus of plants, which belongs to the family Closteroviridae. In our recent study, we showed that the p33 protein of CTV is a viral effector, which affects viral pathogenicity by modulating a host immune response. Upon CTV infection, p33 triggers the accumulation of reactive oxygen species (ROS) and plant cell death. Deletion of the p33 gene from the CTV genome resulted in a significant decrease in the ROS production during virus invasion of the host plants and an increase in the virus accumulation and spread.  Remarkably, the p33 deletion mutant was able to spread beyond the phloem and enter the immature xylem cells where it perturbed the vascular tissue differentiation leading to the enhancement of the stem pitting syndrome. We hypothesize that the plant recognizes p33 and activates the host immune response to restrict CTV into the phloem tissue and minimize the disease.

Learning Objectives:

1. Learn how plant viruses move cell-to-cell and long-distance in their hosts

2. Understand how plant viruses modulate phloem to their advantage

3. Get familiar with mechanisms plants have developed to perceive and defeat viruses

4. Learn what factors mediate phloem restriction of certain plant viruses.

Background: Haemophilus influenzae is the causative agent of multiple human disease conditions among multiple sites in the human body. Underlying genetic mechanisms are elusive, particularly in species with diverse ecological niches in the human body.  Our lab and others have sequenced the whole genomes of over 1,600 genomes of Non-typeable Haemophilus influenzae (NTHi).  These strains were isolated from human patients with various disease states (as well as colonizing without disease symptoms) from multiple locations within and on subject’s bodies.

Methods: 1,618 genomes were assembled using sequencer-appropriate assembly software. Automatic gene annotation was performed using Prokka, and pan-genome gene cluster analysis was performed with Roary. Gene presence/absence matrix of 4,207 gene clusters were used as gene ‘features’ to predict isolates from A) Body Site of isolation, and B) Disease State of patient. Additionally, ‘core’ genes (genes present in all NTHi strains) were converted to numeric vectors and used as a separate feature set. Three algorithms used for class prediction were explored, among the two feature sets.

Results: Imbalance in the number of classes within the dataset proved challenging for the machine learning (ML) algorithm predictions. All algorithms performed significantly better than ‘No Information Criteria’ in predicting either body site and disease state, though in all cases predicting fewer, and more balanced classes was correlated with higher accuracy.

Conclusion: Both gene presence/absence, and core gene genetic composition information in NTHi strains can successfully be used to predict both ecological niche and disease state of origin. Future work is warranted, specifically increasing the number of genomes in classes with low representation and exploring additional methods and feature selection techniques.

Learning Objectives:

1. Define horizontal gene transfer in naturally competent bacterial species

2. Identify importance of gene possession to phenotype

3. Explain current approaches to use gene sets to predict clinical provenance

Over the past five years there has been an explosion of research the deep learning field. Companies like Google, Facebook, and OpenAI have created neural networks that are significantly more accurate and more versatile than their counterparts of a decade ago. However, biological applications of these techniques have lagged behind. In this talk we will explore how modern neural network architectures can be applied to a diverse set of biological problems. As a use-case we will explore the HIV protein Tat, the transactivator of transcription. Utilizing techniques from natural language processing and computer visions we will predict its biological functions, interacting partners, and potential future sequences. We will also discuss how to use techniques like transfer learning to take advantage of small datasets and how to use our biological knowledge to avoid common pitfalls.

Learning Objectives:

1. Recognize the components of an RCNN-based predictor

2. Understand how to use transfer learning to take advantage of mixed data

3. Recognize the components of a GAN

4. Understand how GANs can be used to generate sequences with particular properties

The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor, and is a major determinant of host range and a dominant target of neutralizing antibodies. Here we experimentally measure how all amino-acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBD’s surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial number of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. Our data will inform vaccine design and functional annotation of mutations observed during viral surveillance, and motivate future directions in molecular evolution and immunology of SARS-related coronaviruses.

Learning Objectives:

1. Define deep mutational scanning and its use in understanding protein function and evolution

2. Interpret patterns of mutational tolerance in light of SARS-CoV-2 functional and antigenic evolution

3. Describe patterns of circulating genetic variation in SARS-CoV-2

While viral fusion proteins are highly desirable for subunit vaccine generation, their inherent metastable nature complicates implementation and development.  We have harnessed the structural rigidity that dityrosine bonding imparts as a means of surmounting these difficulties.  Dityrosine bonds are naturally occurring and are formed through multiple reaction mechanisms including photo-activation, chemical, and enzymatic catalyzation.  While most in vitro reaction mechanisms are too harsh for the maintenance of protein conformation, we have modulated the enzymatic reaction in order to target crosslinks to specific regions of fully-folded protein immunogens in order to maintain potently neutralizing conformational epitopes or preserve conformational integrity in otherwise unstable molecules.    By focusing Ab responses toward desired epitopes, dityrosine-stabilized immunogens elicit focused and more potently neutralizing Ab responses, and thus improve their performance as vaccine immunogens.

Learning Objectives:
1. Describe the inherent stability issues for viral fusion proteins that make them difficult to use as subunit vaccines

2. Describe the characteristics of dityrosine crosslinking in terms of bond length, length of reaction, bond characteristics, and when the bond is introduced

3. Describe the attributes that dityrosine bonding imparts on the RSV F protein

4. Describe the advantage of formulation of DT-preF on AdvaxSM

5. Describe the characteristics of Headless HA that make it well suited as a dityrosine bond substrate.

Most currently used conventional influenza vaccines are based on 1940s technology. Advances in immunogen design and vaccine delivery emerging over the last decade open novel opportunities for improving influenza vaccines that are previously inaccessible. These next-generation vaccine design tools are particularly promising in their potential to provide solutions to challenging targets for which conventional approaches have proven ineffective—for example, a universal influenza vaccine. Among the new vaccine technologies, nanoparticle-based vaccines have unique properties that are highly designable and programable. I will discuss our efforts on the development of next-generation nanoparticle-based influenza vaccines to elicit broad and potent protective immunity to influenza virus. A few of such designed nanoparticle-based influenza vaccine candidates are currently evaluated in human clinical trials.

Learning Objectives:

1. Nanoparticle-based immunogen design to elicit broad and potent protective immunity to influenza virus

2. How immunofocusing to the viral sites of vulnerability can be achieved through immunogen design

The recent outbreak of SARS-CoV-2 underscores the need for understanding the evolutionary processes that drive the emergence and adaptation of zoonotic viruses in humans. Here, we show that recombination in betacoronaviruses, including human-infecting viruses like SARS-CoV and MERS-CoV, frequently encompasses the Receptor Binding Domain (RBD) in the Spike gene. We find that SARS-CoV and SARS-CoV-2 are involved in an ancestral recombination event affecting the RBD. As a result of this recombination event, SARS-CoV and SARS-CoV-2 share a similar genotype in RBD, including two insertions (positions 432-436 and 460-472), and alleles 427N and 436Y which belong to a helix that interacts with the human ACE2 receptor. We perform reconstruction of ancestral states and protein-binding affinity analyses in order to understand what role this ancestral recombination involving SARS-CoV and SARS-CoV-2 might have played in the emergence of the new pandemic virus. We also analyze the ACE2-binding affinity of lineage-specific mutations in the SARS-CoV-2 RBD.

Learning Objectives:

1. Discuss the major drivers of viral evolution

2. Identify hotspots of recombination in coronaviruses

3. Present in-silico predictions of virus binding affinity to the host receptor, and explain what these predictions show about the emergence of SARS-CoV-2

While the frequency of pandemic threats seems to be increasing, we fortunately have new tools and technologies to make vaccines with more precision and speed and that support a more proactive approach to pandemic preparedness and response. There are ~25 virus families associated with human infection from which the next pandemic threat will likely arise. Within each relevant virus family, a database of information with accompanying reagents, assays, and animal models could be developed for prototypic viruses based on properties of tropism, transmission routes, and other distinguishing features of pathogenesis. Candidate vaccine approaches could be designed based on virus structure, transmission dynamics, entry requirements, and replication strategy. The prototype pathogen approach for pandemic preparedness has been applied to MERS CoV over the last 7 years. It was informed by structure-based immunogen-design concepts established for RSV F subunit vaccines, and focused on solving coronavirus spike structures, defining mechanisms of CoV neutralization, and evaluating MERS CoV vaccine candidates in collaboration with a commercial mRNA manufacturer. Prior spike protein engineering experience resulted in rapid sequence selection and using the mRNA manufacturing platform provided rapid GMP production a COVID-19 mRNA vaccine in record time. This candidate was tested in mice in ~25 days and humans in ~65 days from the time sequence was released. The product has been shown to be immunogenic and protective in mice, hamsters, and NHP and no safety signals have been noted. Phase 1 and 2 clinical trials demonstrated that mRNA-1273 is well tolerated and immunogenic. These data supported initiation of a 30,000 subject Phase 3 trial on July 27th, 198 days from sequence availability. The proactive preparation not only facilitated rapid vaccine development and evaluation but provided stabilized spike protein reagents that were the basis for developing serological assays and isolating potent human neutralizing mAbs being clinically evaluated for prevention and therapy.

Learning Objectives:

1. Understand the prototype pathogen approach for pandemic preparedness and role of new technologies particularly structure-based vaccine design

2. Know which types of COVID-19 vaccines have advanced into clinical vaccine trials

Influenza infections are initiated by just a handful of virions infecting a handful of cells, so it is important to understand what happens in single infected cells. I will describe work that characterizes how the mutations in individual virions affect the outcome of infection in single cells.

Learning Objectives:

1. Explain what is measured during single cell transcriptomic experiments

2. Explain how mutations affect innate immune recognition of influenza infection

Seasonal and pandemic influenza virus infections can cause significant disease worldwide. Current vaccines only provide limited, short-lived protection, and antigenic drift/shift in the hemagglutinin (HA) surface glycoprotein necessitates their annual reformulation and re-administration. To overcome these limitations, universal influenza virus vaccine strategies aim at eliciting broadly protective antibodies to conserved epitopes of the HA. We have developed ‘chimeric’ HA constructs which retain the conserved stalk domain of the HA and have exotic HA heads. Vaccination and boosting with such constructs successfully redirects the immune system in animals and in humans towards the conserved immune sub-dominant domains of the HA stalks resulting in an antigenic silencing of the HA heads and a protective immune response facilitated by the conserved HA stalks. In mice and ferrets such a strategy protects the animals against challenge with different influenza A strains as well as against different influenza B variants. Although phase I trials have been successfully conducted, further extensive trials will be necessary in the future in order to make this ‘chimeric’ HA approach an FDA-approved vaccine.

Learning Objectives:

1. Why is antigenic drift/shift responsible for the occasional very low efficacy of influenza virus vaccines and why is the composition of influenza virus vaccines adjusted on an annual basis?

2. Why are other vaccines not altered on an annual basis and why can they be used effectively over many decades? What is the principle behind the approach to develop a universal influenza virus vaccine?

3. Why is the technology of reverse genetics instrumental in making safe and protective universal influenza virus vaccines?

This webinar covers various fundamental aspects of performing a successful digital PCR (dPCR) assay on the QIAcuity instruments. An essential parameter to consider in designing a dPCR assay is the choice of templates and their concentrations. Here, we describe the various templates used in digital PCR and focus on their quantitative and qualitative aspects. Further, an insight into a few example applications of the technology, including absolute quantification, will be provided, and underlying calculations and statistics involved in the analysis will be explained. Lastly, we will walk you through the QIAcuity Software Suite, explaining the different views and interpretations of sample data.