Cytomegalovirus (CMV) is one of the most common human herpesviruses, infecting nearly half of the adult population in the US. Vertical transmission can occur during pregnancy, and congenital CMV infections affect more children each year than all of the common conditions tested for during newborn screening combined. Congenital CMV infection is estimated to occur in 1 out of every 200 births in the US and is the leading cause of non-genetic sensorineural hearing loss and neurodevelopmental abnormalities in infants, and yet public awareness remains low. Though most infants born with congenital CMV infection will appear healthy at birth, about 10% will present with a range of symptoms and complications at birth, and many more will experience delayed onset of sequelae. Early detection of congenital CMV infection is critical for timely clinical intervention, including treatment with antiviral medications, which may lessen the severity of the hearing and developmental impairments associated with this infection. This seminar will shed light on the prevalence, epidemiology, and clinical presentation associated with congenital CMV infections, test methods for diagnosis, and universal versus targeted screening approaches.

Learning Objectives:

1. Summarize the prevalence, disease manifestations, treatment and management of congenital CMV infections.

2. Identify the current clinical guidelines and laboratory methods used to diagnose congenital CMV infections.

3. Discuss universal versus targeted screening approaches and legislation updates.

Alphaviruses are enveloped RNA viruses that cause disease in humans ranging from acute febrile illness with rash and arthritis to lethal encephalitis. Like many other mosquito-borne viruses, they infect vertebrate species and insect vectors separated by hundreds of millions of years of evolutionary history. Viral entry into evolutionarily divergent host cells could be accomplished by recognition of different cellular receptors in different species, or by binding to receptors that are highly conserved across species. Although multiple alphavirus receptors have been described, most are not shared among vertebrate and invertebrate hosts. I will discuss our laboratory’s recent identification of the very low-density lipoprotein receptor (VLDLR) as a receptor for the alphaviruses Semliki Forest virus (SFV), eastern equine encephalitis (EEEV), and Sindbis (SINV). We showed that these viruses interact with the ligand binding domains (LBDs) of VLDLR and apolipoprotein E receptor 2 (ApoER2), a closely related receptor whose expression is mostly restricted to the central nervous system. We further demonstrated that ectopic expression of either VLDLR or ApoER2 facilitated cellular attachment and internalization of virus-like particles (VLPs), that a VLDLR LBD-Fc fusion protein or a ligand binding antagonist can block SFV E2/E1 envelope protein-mediated infection of human and mouse neurons in culture, and that administration of a VLDLR LBD-Fc fusion protein had protective activity against rapidly fatal SFV infection in mouse neonates. Overexpression studies further revealed that invertebrate receptor orthologs as evolutionary distant from vertebrates as mosquitoes and worms could serve as functional alphavirus receptors. We propose that the ability of some alphaviruses to infect a wide range of hosts is a result of their engagement of evolutionarily conserved lipoprotein receptors and contributes to their pathogenesis.

Learning objectives:
1. Clarify the role of alphaviruses as agents that cause acute human febrile illnesses with either arthritis or encephalitis as the outcome of infection.
2. Explain the organization of the alphavirus envelope glycoproteins and their role in mediating viral entry.
3. Summarize the role of LDLR-related receptors as factors that facilitate the cell surface binding and internalization of some alphaviruses.

The evolution of the planetary situation is presently knowing multiple rapid and severe complications impacting both geophysical as well as all the life aspects. Multiple parameters undergo radical changes: climate change, large losses of biodiversity (respectively 70%, 80%, 50% of vertebrates, insects and birds disappeared in less than 40 years, and all the rest being in an accelerated extinction phase). In addition, due to the extraction of fossil resources, mega-fires and dams we see the disappearance of natural habitats (wetlands, mangroves, glaciers, polar ice caps, rivers, lakes, rising of water temperatures), forests. These data are all correlated with an uncontrolled demography growth, mainly since the 20th century, reaching eight billion humans (consumers/polluters) this year as compared with only a single billion humans in 1800. Indeed, due to an accelerated growth of discoveries these last decades and globalization, the humanity has known an easier access to natural resources for a better comfort of life for much less efforts than by the past. Moreover, since the Second World War, the use of antibiotics, the development of multiple vaccines and therapeutic strategies has contributed to an unprecedent demographic growth. This is a paradox, where the medical progress, in a total absence of population control, indirectly contributes to accelerate the planet degradation.

The drastic reduction of wild habitats (previously not-visited by humans) induces a high concentration of hosts of infectious agents (SARS-CoV-2, Monkeypox, Tick-borne agents, etc.), favoring their spread into highly-dense promiscuous new populations. To provide a high-quality public health medicine in the present unfavorable context, we need to rethink several aspects, including the population control and its behavior and two mandatory actions: (i) measures to rapidly reverse the environmental situations mentioned above, but also (ii) set up a more efficient preparedness surveillance using ultra-sensitive and ultra-rapid detection of pathogens to adopt early and optimized counter-measures, to contain epidemics. Here, I will provide integrative ways to think about the crucial Gordian knot question on “human health and health of the planet”, and propose complementary technologies that could meet unmet medical needs.

Gene Synthesis ensures fast access to nearly any DNA sequence, regardless of its origin or complexity. Its reliability, flexibility and fast turnaround time offer a clear advantage over traditional cloning methods. In this webinar we will show you the easiest and most convenient way to get your desired DNA sequence. We will walk you through the design and ordering process, and illustrate the benefits of gene optimization to achieve maximum expression in your final host. Finally, we will discuss the benefits of the gene-to-protein service, including protein structure determination.

Learning Objectives:

1. Discover the benefits of gene synthesis.
2. Find out how gene optimization boosts the expression of your protein.
3. Learn about integrated and reliable services, all the way through to purified proteins.
4. Discover the fastest and easiest way to assemble your DNA construct using Invitrogen GeneArt DNA Fragments and GeneArt Gibson Assembly® cloning kits.

Since its discovery in late 2019 the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a global pandemic of coronavirus disease 2019 (COVID-19).  As of July of 2022, more than 6.5 million SARS-CoV-2 deaths have been reported worldwide to the World Health Organization (WHO). Limited access to testing in underserved communities and incomplete reporting of COVID-19  contribute to official numbers being a fraction of the total infections and deaths from the COVID-19 pandemic. In addition, novel viral variants including of Omicron have higher transmission rates than the original virus and B.1.1.529 omicron even higher than the delta variant. Due to the vaccination efforts to reduce viral replication around the world, there are less severe cases. Outbreak management has been hindered by high transmission, especially among those with mild to no symptoms, and limitations in testing capacity for SARS-CoV-2. To limit spread of SARS-CoV-2, effective public health tools are needed for rapid and early identification of infections. While the current diagnostic standard is quantitative real time polymerase chain reaction (qRT-PCR), its high cost and long turnaround times constrain its utility for widespread surveillance use.  Lack of such surveillance has resulted in the closing of businesses and schools, and severe economic disruption. We present novel ways to apply mathematics, probability theory and statistics using image analysis of COVID-19 rapid antigen tests, and show examples of powerful reporting systems, data and result Interpretation from Real World Data obtained from Chelsea, MA. USA.

Learning Objectives:

1. Describe the situation of antigen tests' high bar approval process in the US market.
2. Describe novel methodologies for image processing and analysis of antigen tests enabling competitiveness and acceptance in diagnostics.
3. Classify the need for rapid and frequent antigen testing as essential for control of the spread of emergent diseases.

The COVID-19 pandemic has underlined the need for an efficient collaboration between scientists, policy makers and the general public to tackle the crisis. Infectious disease experts play a key role in setting the foundations for an acute and accurate communication of the risks of emerging pandemics, however, they often lack the resources and tools to translate their findings into messages that resonate with decision-makers and/or the public. This presentation will introduce the audience to the principles of effective communication of the risk posed by emerging communicable diseases, bridging the fields of infectology, behavior science and communication.

Learning Objectives:

1. List 3 attributes of a risk that make audiences more likely to perceive the risk of an infection to be higher than the actual risk.

2. Name 1 behavioral science framework that helps explain how people behave in the face of health risks.

3. Name 3 characteristics of the target audience that could modify the way the risk communication should be delivered.

Researchers are increasingly turning to RNA sequencing for gene expression analysis from challenging samples. These samples often include fine needle biopsies, micro-dissected tumors or highly fragmented RNA isolated from FFPE curls containing less than 1 ng of total RNA (i.e., low-input samples). One of the biggest bottlenecks that researchers encounter is the ability to prepare complete transcriptome RNA libraries without capturing all of the unwanted ribosomal RNA (rRNA) that makes up over 75% of the sample. Current rRNA removal methods are often designed only for use with full-length RNA or rely on capturing the coding RNA with specific enrichment probes. Both strategies can be time-consuming and involve complex workflows.     

To address these challenges, QIAGEN has developed QIAseq FastSelect – an innovative rRNA removal technology. QIAseq FastSelect works with existing RNA library kits to inhibit cDNA synthesis of specific ribosomal transcripts, effectively removing them from the RNA-seq library. Designed to work after RNA fragmentation, QIAseq FastSelect is suitable for use with both high- and low-quality RNA. In this webinar, we will highlight how QIAseq FastSelect can be paired with various RNA library kits and workflows to decrease the amount of rRNA in your difficult samples. Discover how this powerful rRNA removal solution will increase RNA-seq sensitivity without increasing your sequencing costs.

The global effort to sequence millions of SARS-CoV-2 genomes has provided an unprecedented view of viral evolution. Characterizing how selection acts on SARS-CoV-2 is critical to developing effective, long-lasting vaccines and other treatments, but the scale and complexity of genomic surveillance data make rigorous analysis challenging. To meet this challenge, we develop Bayesian Viral Allele Selection (BVAS), a principled and scalable probabilistic method for inferring the genetic determinants of differential viral fitness and the relative growth rates of viral lineages, including newly emergent lineages. After demonstrating the accuracy and efficacy of our method through simulation, we apply BVAS to 6.9 million SARS-CoV-2 genomes. We identify numerous mutations that increase fitness, including previously identified mutations in the SARS-CoV-2 Spike and Nucleocapsid proteins, as well as mutations in non-structural proteins whose contribution to fitness is less well characterized. In addition, we extend our baseline model to identify mutations whose fitness exhibits strong dependence on vaccination status as well as pairwise interaction effects, i.e. epistasis. Strikingly, both these analyses point to the pivotal role played by the N501 residue in the Spike protein. Our method, which couples Bayesian variable selection with a diffusion approximation in allele frequency space, lays a foundation for identifying fitness-associated mutations under the assumption that most alleles are neutral.

• pre-print: https://www.biorxiv.org/content/10.1101/2022.05.07.490748v2

• github: https://github.com/broadinstitute/bvas

Parasitic infection alters the host in many ways.  Intracellular parasites have extensive needs of their host cells and exploit properties of host cell signaling networks to facilitate their own survival and proliferation. Parasites alter surrounding cells by cell-cell contact and by promoting inflammation, creating differences in and around infected cells.  Some of these changes are required for infection and thus could create a therapeutic index for targeting infection without damaging uninfected cells. Yet, a systematic methodology to identify key infection-associated host targets and translate these targets into interventions is not yet available.  I will present our work towards creating this framework for the malaria-causing parasite Plasmodium.  Plasmodium parasites undergo multiple complex life cycle transitions and interact with a variety of host cell types.  Upon transmission to the mammalian host, Plasmodium parasites are carried through the bloodstream to the liver, where they infect a single hepatocyte.  This represents a first critical stage of infection, as clearance of the parasite during this stage would eliminate all disease and subsequent transmission.  We have also focused on host-targeted interventions that strengthen the blood-brain endothelial barrier, as breakdown of the blood-brain barrier has been implicated in cerebral malaria and adjunctive therapy that restores normal brain function could improve survival rates.  To systematically identify relevant host targets for each of these stages, we utilized existing, and built new, machine-learning driven tools.  These tools include kinase regression and a newly developed tool, Temporally Resolved Kinase Network Generation (TREKING). In each case, we aim to identify critical kinases and/or phosphosignaling networks that drive infection and/or pathogenesis but do not eliminate uninfected cells. These strategies have led us to kinase inhibitors that both eliminate most liver stage parasites and protect the blood brain barrier during inflammation.  While we have taken a malaria-focused approach, our strategies are disease state agnostic and could be used to interrogate multiple diseases either independently or in combination. 

Learning Objectives:

1. Interpret the life cycle of the malaria causing parasite, Plasmodium.

2. Consolidate new approaches to identifying host regulators of infection.

3. Describe advantages and disadvantages to targeting infectious diseases with host targeted interventions.
 

Recent outbreaks of Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have exposed our limited therapeutic options for such diseases and our poor understanding of the cellular mechanisms that block viral infections. Using a transposon-mediated gene-activation screen in human cells, we identify that the major histocompatibility complex (MHC) class II transactivator (CIITA) has antiviral activity against EBOV. CIITA induces resistance by activating expression of the p41 isoform of invariant chain CD74, which inhibits viral entry by blocking cathepsin-mediated processing of the Ebola glycoprotein. We further show that CD74 p41 can block the endosomal entry pathway of coronaviruses, including SARS-CoV-2. However, coronavirus entry mediated by cell surface proteases like TMPRSS2 bypasses inhibition by CD74 p41. These data therefore implicate CIITA and CD74 in host defense against a range of viruses, and they identify an additional function of these proteins beyond their canonical roles in antigen presentation.

Learning Objectives:

1. Analyze about transposon activation tagging and its potential use to rapidly identify virus/host interactions for emerging viral pathogens.

2. Clarify how upregulation of the master regulator of MHC class II (CIITA) and the invariant chain (CD74) leads to cellular restriction of Ebola and SARS-CoV-2 viral entry independent of their role in adaptive immunity.

3. Classify the similarities and differences in the entry pathways of Ebola and SARS-CoV-2.

As obligate intracellular pathogens, viruses require host-derived nucleotides, amino acids, and lipids, among others, and the bioavailability of these metabolites can restrict or promote virus infection. Additionally, viruses alter cellular metabolism to foster replication, by promoting cell cycle, enhancing synthetic enzyme activity, or inhibiting catabolic enzymes. Additionally, large DNA viruses encode entire biosynthetic pathways. In contrast, RNA viruses have limited genomic capacity to encode metabolic enzymes and rely on host metabolism to support replication. Additionally, several antiviral molecules directly or indirectly affect metabolism, including classic molecules like ribavirin, which inhibits IMPDH and de novo guanine synthesis to quell virus replication. Further, cellular metabolic state varies between cell type and even individual cells within a population. We study how RNA viruses interact with cellular metabolism, with particular focus on molecules called polyamines. These polyamines are crucial for virus replication, and we can target their synthesis with small molecule inhibitors. We study how viruses use polyamines, how viruses manipulate polyamines, and how the cell fights back to prevent viruses from obtaining polyamines. Through this work, we discover new ways that viruses infect, identify potential drug targets, and investigate a unique way that RNA viruses interface with cellular metabolism.

Pathogen detection and surveillance have become a high priority in both healthcare and environmental settings for the safety of patients and the general public. The COVID-19 pandemic highlighted the need for strategies to identify and differentiate pathogen variants of concern. Municipalities employ NGS-based wastewater analysis to identify not only SARS-CoV-2 variants but also other viral and bacterial species as early alert systems of community health. In clinical settings where co-infections of two or more pathogens can complicate patient outcomes, such as with SARS-CoV-2 and influenza, microbial surveillance of patient areas may help circumvent disease outbreaks. Thus, there is a widespread need for targeted and sensitive variant detection through whole genome characterization of a wide variety of microbial pathogens.

This webinar will discuss new probe-based NGS enrichment solutions. We’ll introduce our QIAseq xHYB Viral and Bacterial Panels, which use hybrid capture technology to provide high-sensitivity targeted whole genome enrichment for detecting and sequencing:

Respiratory viruses
Sexually transmitted pathogens
Antimicrobial-resistant (AMR) genes from bacteria 
Adventitious agents 
Details of the content of these new panels and their performance across several different applications and sample types will be presented.

In the last decade or so, R&D into the human microbiome-based therapeutics and diagnostics has accelerated the widespread availability of next-generation DNA sequencing technologies that  comprehensively profile complex microbiota through metagenomic approaches. However, measurements generated by different laboratories often display large variability due to protocol-dependent bias introduced at each step in the workflow. Driven by the needs identified by the industry-led Japan Microbiome Consortium (JMBC), we aimed to establish standard operating procedures (SOPs) for metagenomics-based microbiome profiling of human fecal samples. Join this webinar to hear about the steps that help set the SOPs in human fecal microbial profiling:

- The first phase of this study systematically compared a myriad of protocols for DNA extraction and library construction using newly developed mock communities.

- In the second phase, reproducibility and transferability of highly performant protocols were assessed in an industry-based interlaboratory study and as part of the MOSAIC Standards Challenge study.

The widespread uptake of our SOPs, which include the QIAseq FX DNA Library Kit for library construction, and best-practice guidelines, is expected to increase the accuracy of NGS-based metagenome profiling results across the microbiome field.

The lack of preparedness for detecting the highly infectious SARS-CoV-2 pathogen, the pathogen responsible for the COVID-19 disease, has caused enormous harm to public health and the economy. It took ~60 days for the first reverse transcription quantitative polymerase chain reaction (RT-qPCR) tests for SARS-CoV-2 infection developed by the United States Centers for Disease Control (CDC) to be made publicly available. It then took >270 days to deploy 800,000 of these tests at a time when the estimated actual testing needs required over 6 million tests per day. Testing was therefore limited to individuals with symptoms or in close contact with confirmed positive cases. Testing strategies deployed on a population scale at ‘Day Zero’ i.e., at the time of the first reported case, would be of significant value. Next Generation Sequencing (NGS) has such Day Zero capabilities with the potential for broad and large-scale testing. However it has limited detection sensitivity for low copy numbers of pathogens which may be present. Here we demonstrate that by using CRISPR-Cas9 to remove abundant sequences that do not contribute to pathogen detection, NGS detection sensitivity of COVID-19 is equivalent to RT-qPCR. In addition, we show that this assay can be used for variant strain typing, co-infection detection, and individual human host response assessment, all in a single workflow using existing open-source analysis pipelines. This NGS workflow is pathogen agnostic, and therefore has the potential to transform how both large-scale pandemic response and focused clinical infectious disease testing are pursued in the future.

Learning Objectives: 

1. Clarify how Next Generation Sequencing (NGS) as an infectious disease testing strategy for Day Zero capabilities has the potential for broad and large-scale testing.

2. Cite how using CRISPR-Cas9 to remove abundant sequences that do not contribute to pathogen detection, NGS detection sensitivity of COVID-19 is equivalent to RT-qPCR.

3. Express how this all-in-one NGS workflow can be used for variant strain typing, co-infection detection, and individual human host response assessment while using existing open-source analysis pipelines.

Altered enteric microorganisms in concert with host genetics shape inflammatory bowel disease (IBD) phenotypes. However, insight is limited to bacteria and fungi. We found that eukaryotic viruses and bacteriophages (collectively, the virome), enriched from non-IBD, noninflamed human colon resections, actively elicited atypical anti-inflammatory innate immune programs. Conversely, ulcerative colitis or Crohn's disease colon resection viromes provoked inflammation, which was successfully dampened by non-IBD viromes. The IBD colon tissue virome was perturbed, including an increase in the enterovirus B species of eukaryotic picornaviruses, not previously detected in fecal virome studies. Mice humanized with non-IBD colon tissue viromes were protected from intestinal inflammation, whereas IBD virome mice exhibited exacerbated inflammation in a nucleic acid sensing-dependent fashion. Furthermore, there were detrimental consequences for IBD patient-derived intestinal epithelial cells bearing loss-of-function mutations within virus sensor MDA5 when exposed to viromes. Our results demonstrate that innate recognition of IBD or non-IBD human viromes autonomously influences intestinal homeostasis and disease phenotypes. Thus, perturbations in the intestinal virome, or an altered ability to sense the virome due to genetic variation, contribute to the induction of IBD. Harnessing the virome may offer therapeutic and biomarker potential.

Learning Objectives:

1. Classify that prokaryotic and eukaryotic viruses (i.e. the virome) occupy tissue niches of asymptomatic individuals and alterations in this virome associate with many complex immune diseases, including inflammatory bowel disease (IBD).
2. Discuss that the innate immune system senses these viruses which can be protective in health but contribute to inflammation in disease.
3. Explain that Enterovirus B viruses was one eukaryotic virus family to be significantly elevated in IBD patients.

Critical in control of this pandemic is the ability to rapidly identify infectious individuals prior to their ability to infect others. With the introduction of better testing capacity and an understanding of how to use these tests, the current challenge is making sure patients have access to the correct therapies at the right time. A simplified framework for identifying the phases for different therapies and evidence-based application of available therapeutics can greatly improve the chances of a positive outcome.

Learning Objectives:

1. Identify the viral kinetics and impact on testing for patients with COVID-19.
2. Outline available testing options.
3. Be able to apply recognize the right time and the right therapeutic for an individual patient.

Many clinically approved drugs that are prescribed to modulate human physiology are also potent antimicrobials at physiological concentrations. Recent systematic studies indicate that bacterial toxicity is frequent in anti-neoplastic drugs used for treating cancer. Using pooled genetic screens, we systematically mapped all loss-of-function mutations modulating sensitivity against three chemotherapy drugs in Escherichia coli. We further characterized the mechanisms that can confer resistance by evolving bacteria in serial transfer evolution experiments and identifying adaptive mutations by whole-genome-sequencing. Our experiments revealed that changes in multiple and diverse pathways can confer resistance, indicating that resistance can readily emerge over short evolutionary time scales. Interestingly, given that many of pathways underlying resistance can potentially influence drug transport and metabolism, we predicted that bacterial resistance will frequently impact drug availability for host cells neighboring resistant bacteria. Lastly, we examined the impact of bacterial evolved resistance on the host by measuring chemotherapy toxicity in Caenorhabditis elegans while controlling for its bacterial diet. We discovered that nutrient availability during bacterial evolution dictated the dietary effect on the host – only bacteria evolving in nutrient-poor media reduced host drug toxicity. Our work suggests that bacteria can rapidly adapt to tumor-targeting chemotherapies and by doing so may also impact host treatment success.

Learning Objectives:

1. Explain how bacterial drug metabolism by the microbiome can impact treatment of the host.

2. Describe how a genetic loss-of-function screen provide insight on potential resistance mechanism during evolutionary adaption.

3. Clarify why information on the bacterial species identity in the host-microbiome can be insufficient for determining its influence on drug-tumor interaction.

Over 80% of opportunistic infections in humans are caused by microbial biofilms. Traditional methods of study for these infections have focused on single species yet there is a wealth of evidence that these infections are polymicrobial in nature. Polymicrobial infections are often chronic in nature, as is evident for example in diabetic foot ulcers, chronic obstructive pulmonary disease, and cystic fibrosis. These polymicrobial biofilms are complex with various spatial and chemical interactions that can happen at distance or in contact with one another. Recent research has focused on understanding these interactions and how they can be harnessed or inhibited for favourable outcomes.
We have been developing polymicrobial biofilm models to investigate the interactions between 3 cornerstone opportunistic pathogens Pseudomonas aeruginosa, Staphylococcus aureus and the polymorphic fungi Candida albicans. These models have been used to investigate antimicrobial repurposing, adjunctive therapeutics and understanding the quorum sensing interactions that occur between these microbes in models that recapitulate certain aspects of the infection environment.
In summary, we are continuing to develop polymicrobial models that are standardised and can be replicated across multiple labs. These models are designed to better recapitulate the infection environment e.g., nutrient availability and oxygenation status for example, which will lead to improved research translation.
 
Learning Objectives:
1. Clarify the complexity of infections and the importance of polymicrobial biofilms.
2. Explain the importance of clinically relevant biofilm models.
3. Describe the importance of multiple endpoint assessment in polymicrobial biofilms and harnessing new analysis techniques.

New research suggests that the vaginal microbiota (i.e. the communities of bacteria inhabiting the vagina) may play a critical role in women’s reproductive health. “Optimal” vaginal communities dominated by specific Lactobacilli may help protect women from acquisition of sexually transmitted infections and possibly even urinary tract infections. In contrast, vaginal microbiota that are low in Lactobacilli or lack the most protective types of Lactobacilli, may put women at increased risk of these infections. In this talk we will discuss the basic structure of the vaginal microbiota, associations with STIs and UTIs as well as some possible mechanisms by which the vaginal microbiota may affect susceptibility to these infections. Lastly, we will discuss future research-both the promise of and some challenges relating to developing microbiome based therapeutics to improve women’s health.

The vaginal microbiome is a critical component of female health. More than 30% of people with vaginas suffer from imbalances in the microbiome (e.g. bacterial vaginosis, yeast infections, and recurrent UTIs) that drastically affect our quality of life. Additionally, research has uncovered groundbreaking links between the vaginal microbiome and infertility, HIV risk, preterm birth, gynecologic cancers, and more. Evvy’s longitudinal, metagenomic testing is unlocking personalized definitions of health and disease in the vagina — and providing women with education and insights about their vaginal health along the way. This talk will highlight opportunities to leverage the vaginal microbiome to positively impact women’s health outcomes across the board.

Mammalian mucosal niches are colonized by a complex ecosystem of viruses, bacteria and fungi termed the microbiome. Unlike the gut microbiome, the vaginal microbiome is composed of small number of bacterial species and is consistently found in people around the world. The bacterial communities fall into lactobacilli-dominant, low diversity communities and lactobacilli-low high diversity communities. In contrast to other mucosal niches, high-diversity communities are associated with increased acquisition of sexually transmitted infections and poor pregnancy outcomes including preterm birth. Determining the contribution of the microbiome to mucosal immunity is critical to designing new therapeutics and vaccine techniques. We will discuss the prime and pull vaccine technique, which leverages a commonly-used antibiotic increase antiviral immunity at the vaginal mucosa.

Learning Objectives:

1. Describe the vaginal microbiome and bacterial communities.

2. Identify the main differences between the intestinal and vaginal microbiome. 

3. Describe the mechanics of the prime and pull vaccine.

This is a technical talk. Its objective is to provide details of virus sequencing, using SARS-CoV-2 as an example. Knowing the details, strengths, and limitations of virus sequencing will enable the audience to understand and sometimes judge sensationalistic reports of new stains and phylogenetic analyses of the origin of SARS-CoV-2. This talk can be a blueprint to set up a Next Generation Sequencing pipeline for any virus. Background:  McNamara et al. Cell Reports 2020.  
 

Learning Objectives:

1. Clarify principles of targeted virus sequencing using Next Generation Sequencing technology.
2. Clarify principal limitations of Next Generation Sequencing sequencing and the unavoidable existence of gaps in any virus sequence.
3. Appreciate the utility of single-nucleotide variants (SNVs) in deducing virus evolution.

In all organisms, innate immune pathways sense infection and rapidly activate potent immune responses while maintaining a high degree of specificity to prevent inappropriate activation (autoimmunity). In humans, the innate immune receptor cGAS detects viral infection to produce the nucleotide second messenger cGAMP, which initiates STING-dependent antiviral signaling. Bacteria encode predecessors of the cGAS-STING pathway and bacterial cGAS/DncV-like nucleotidyltransferases (CD-NTases) detect bacteriophage infection to produce cGAMP. How bacterial CD-NTase activation is controlled, however, remains unknown. In this presentation, I will describe our work showing that CD-NTase-associated protein 2 (Cap2) primes bacterial CD-NTases for activation through a ubiquitin transferase-like mechanism. A cryoelectron microscopy structure of the Cap2–CD-NTase complex reveals Cap2 as an all-in-one ubiquitin transferase-like protein, with distinct domains resembling the eukaryotic E1 protein ATG7 and the E2 proteins ATG10 and ATG3. The structure captures a reactive-intermediate state with the CD-NTase C-terminus extending into the Cap2 E1 active site and conjugated to AMP. We find that Cap2 ligates the CD-NTase C-terminus to a target molecule in cells, a process that primes the CD-NTase for increased cGAMP production. We further demonstrate that Cap2 activity is balanced by a specific endopeptidase, Cap3, which antagonizes antiviral signaling by deconjugating the CD-NTase. Our data demonstrate that bacteria control immune signaling using an ancient, minimized ubiquitin transferase-like system and provide insight into the evolution of E1 and E2 machinery across domains of life.

Learning Objectives:

1. Describe cGAS-like enzyme regulation by Cap2 and Cap3, enzymes homologous to ubiquitin transferases and deubiquitinases.
2. Discuss about phage biology and phage resistance in bacteria.
3. Appreciate the parallels between human and bacterial immune systems.

Tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis (Mtb) remains one of the leading causes of morbidity and mortality worldwide. This presentation will summarise the epidemiology and pathobiology of TB, examine the evidence that Mtb infection is associated with dissemination into the blood stream and explore why this may be important for understanding the infection and potential implications for developing new tools.

Learning Objectives:

1. Develop basic knowledge and understanding of global TB epidemiology.

2. Classify current concepts in the pathogenesis of human Mtb infection.

3. Develop awareness of existing and emerging evidence supporting dissemination of Mtb away from the lung in both severe and milder forms of TB infection.  

Human intestinal epithelial cells (hIECs) are arranged as a monolayer of cells and provide the first line of defense against invading pathogens. Upon viral infection hIECs upregulate type I and III interferons to control the infection. These interferons lead to the induction of hundreds of interferon stimulated genes (ISGs) that aid in the clearance of the virus and protection of uninfected cells. Upon infection hIECs preferentially upregulate type III interferons to clear the infection. We and others have shown that when added in trans both type I and type III interferons act as key antiviral cytokines, however they achieve their antiviral results using unique patterns of ISGs. Importantly, while hundreds of ISGs have been described using mainly enterocyte models, whether all cell types in the human gastrointestinal tract upregulate the same ISGs following enteric virus infection has yet to be addressed. Using human intestinal mini-gut organoids and both multiplex RNA FISH and single cell RNA sequencing (scRNA-Seq) following virus infection, we have been able to determine the cell type specific response to virus infection. Our analysis showed that each cell type in the human gastrointestinal tract upregulated a unique pattern of ISGs to combat enteric virus infection. Interestingly, we could also show that each cell type in the human ileum also expressed cell-type specific basal ISGs. Together our results illustrate that each individual cell type has a unique ISG pattern that can help us understand virus tropism and virus clearance mechanisms used by hIECs.

Learning Objectives:

1. Develop a basic knowledge of interferon response to viral infection.
2. Describe methods used to evaluate viral response at the single cell level.
3. Describe methods to determine virus infection and propagation in vitro.

NGS library preparation can be a long, multi-step process, but it is essential for proper sample management and reproducible results. Automating this process reduces costs, frees up researchers' valuable time and streamlines laboratory workflows. One area where this automation step has become extremely advantageous is in vaccine development. The emergence of novel infectious diseases, and outbreaks of well-known communicable agents, has underscored the increased demand for vaccine candidates for humans and livestock. Products that can make NGS preparation and analysis more straightforward and effective are a welcome addition to any laboratory.

In this webinar, distinguished presenter, Dr. Graham Freimanis, will compare data from sequencing-ready libraries generated using both manual workflows and the new Tecan MagicPrep™ NGS automated library preparation system. Samples were extracted from several species of economically important livestock inoculated with different vaccine candidates, and were processed in parallel using various prep kits. Dr. Freimanis will discuss the differences between each of the kits, and the quality of the sequencing data. This webinar will provide insights into the benefits of automated library preparation, its impact on the resulting sequence datasets and how it can be integrated into existing workflows.

We live in a revolutionary era during which technology  is leading to amazing advances in medicine.  Parallel and aided by the development of artificial intelligence (AI) are the emergence of the various “Omic” disciplines. These “omics” (genome, microbiome, transcriptome, proteome, metabolome, etc. ) can now be integrated using various bioinformatic platforms that meld nature (the genome) with nurture (the effects of the environment) that illustrate mechanisms of normal physiology and disease. These multiomics combined with the power of AI are opening new avenues for biomarker discovery, disease prevention and therapeutics.
In this lecture review, we will discuss some of the advances that AI is bringing to the field of perinatology and neonatology. We will provide an overview of current capabilities and where we may be headed in the next couple of decades.

Learning Objectives: 

1. Elaborate on the rationale for integration of different “omic” technologies.
2. Discuss several disease entities in the perinatal/neonatal period as well as strategies for personalize nutrition that might be elucidates with the multiomic integration and AI.
3. Contrast the advantages of multiomics versus single omics.

Human respiratory syncytial virus (hRSV) is a significant cause of lower respiratory tract infections in children under the age of 5, the elderly, and immunocompromised individuals, leading to millions of hospitalizations that also contribute to mortality. Prior exposure to hRSV affords little protection for subsequent infections and therefore individuals are susceptible to reinfection. Despite the substantial burden to global human health, there are limited effective and safe prophylactic and therapeutic options available. The hRSV genome encodes for two ORFs at the 3’ end, the multifunctional nonstructural proteins NS1 and NS2. These proteins are implicated in several functions, including immune antagonism.  However, the mechanisms by which NS1 and NS2 proteins modulate host responses remain incompletely defined. We recently solved the X-ray crystal structures of NS1 and NS2. These structures provide the molecular basis for function and have advanced our understanding of nonstructural protein interactions with host components. Consequences of host-viral interactions include direct inhibition of cytosolic pathogen sensors and binding to host nuclear transcriptional machinery to shape the immune response to infection. Insights from these studies also provide a basis to target NS1 and NS2 therapeutically, including opportunities to generate structure-guided mutant viruses as future vaccine candidates with altered host interactions through NS1 and NS2.
 

Macrophages have been implicated in both the resolution and progression of influenza, but the drivers of these outcomes are poorly understood. We probed mouse lung transcriptomic datasets using tissue deconvolution algorithms to predict immune cell subsets that correlated with mild or severe influenza A virus (IAV) infection outcomes. This allowed us to identify a novel lung macrophage population that was transcriptionally similar to small serosal cavity macrophages and was only present in mice that survived IAV infection. Large serosal macrophages have been implicated as a source of macrophages for visceral organs in inflammatory diseases. However, until now, the study of serosal macrophage translocation in the context of infections, and the study of small serosal macrophage translocation in general has been neglected. We used flow cytometry and microscopy to show that fluorescently-labeled pleural macrophages (PMs) migrate from the pleural cavity into the lung after infection with pH1N1 A/California/04/2009 influenza virus. The numbers of translocated PMs peak at day 9, which is during the resolution phase of the disease and after virus has been cleared. Our data show that PM recruitment to the lung parenchyma occurs across the mesothelial layer and not through the vasculature. Translocated PMs phenotypically resemble a MHCIIhi subpopulation of interstitial macrophages (IMs) that rapidly expands in number during influenza virus infection. Depletion of PMs prior to IAV infection led to increased morbidity when compared to the control group. Lack of PMs also resulted in increased pleural cavity inflammation and an influx of neutrophils within the lung. Our results show that both small and large PMs are recruited to the lung during IAV infection and contribute to recovery from influenza. This study expands our knowledge of PM plasticity and provides a new source of IMs independent of monocyte recruitment and local proliferation.

Learning Objectives:

1. Discuss the impact of pleural macrophages on influenza outcomes.
2. Describe how to identify mouse pleural and lung macrophages using flow cytometry.
3. Describe how to track pleural macrophage translocation using in vivo labeling.

HIV-1 replicates in the body as a large viral population. Along with its high error rate during viral DNA synthesis and its short replication time (24 hours), the virus maintains sequence diversity within the viral population. The application of selective pressure results in the differential outgrowth of sequence variants that are more fit in the presence of the selective pressure. The evolution of drug resistance is an example of minor drug resistant variants growing out in the face of a new selective pressure. For HIV-1, the availability of three potent antiviral drugs with different resistance pathways allowed for combination treatment that suppresses viral replication and precludes the evolution of drug resistance. The same three drugs given sequentially would give rise to resistance against all three. Combination therapy results in the rapid collapse of the viral population emphasizing the role population size plays in allowing the evolution of resistance. Antiviral drugs can be evaluated for their potency (the ability to inhibit viral replication) and the size of the genetic barrier to resistance (the number of mutations that must evolve to overcome the potency of the drug). HIV-1 protease inhibitors are unique among anti-HIV-1 drugs in that they have very high potency and they also have a very high genetic barrier to resistance. The viral protease is essential in the virus life cycle. In the absence of protease activity, uncleaved viral precursor proteins form an immature and noninfectious particle. HIV-1 protease inhibitors mimic the protease cleavage site with very tight binding to the protease to block its activity. The current most potent protease inhibitor is darunavir, which reaches levels in the blood that are nearly 1000-fold higher than needed to inhibit virus in cell culture. We will examine the pathway to resistance to darunavir and consider the next generation of protease inhibitors with even greater potency.

Antibiotic treatment failure poses a burden on global public health systems. Bacteria can acquire mutations and extrachromosomal genes that allow them to be resistant to our existing drugs. In addition to developing resistance, some bacteria in genetically clonal populations, coined persisters, can reversibly reprogram their phenotypic responses, enabling them to endure antimicrobial therapy and resume growth once treatment terminates. While the persister phenotype is not heritable, persistence is thought to contribute to infection relapse and accelerate resistance development, which motivates us to elucidate defenses that persisters depend on to survive antibiotic treatment. In this presentation, we focus on antibiotic persistence to DNA-damaging topoisomerase inhibitors. We show that persisters originating from slow-/non-growing cultures suffer DNA damage during topoisomerase inhibitor treatment, and they need to repair their DNA during the post-antibiotic treatment recovery period. We also found that persisters depend on multidrug efflux pumps to survive treatment with certain compounds that inhibit topoisomerases. Finally, we explore metabolic, biochemical, and microbial competition-basedapproaches to sensitize bacterial persisters to our existing and emerging topoisomerase inhibitors.

Learning Objectives:

1. Describe how bacterial phenotypic heterogeneity and antibiotic persistence contribute to antibiotic treatment failure.

2. Discuss the defenses that bacterial persisters depend on the survive treatment with topoisomerase inhibitors.

3. Describe methods of sensitizing bacterial persisters to existing and emerging topoisomerase inhibitors.