Critical in control of this pandemic is the ability to rapidly identify infectious individuals prior to their ability to infect others.  The original testing approaches for COVID-19 diagnosis through detection of SARS-CoV-2 RNA focused on high sensitivity and are not being used to discriminate between the infected and the infectious.  Many diagnostic tests are expensive and suffer from significant resulting delays. The introduction of tests that may be less sensitive at lower RNA copy number and use alternative technology such as antigen detection are potentially of use and may be better at rapidly identifying infectious individuals prior to transmission.  We have a growing body of evidence that patients with low levels of RNA and negative antigen tests are very low risk for spread of disease. This information along with timing in disease course can allow for a cut off for infectious levels of RNA and can help guide development and use of different testing modalities as well as pooling decisions.

Learning Objectives:

1. Understand the viral kinetics and impact on testing for patients with COVID-19.

2. Understand available testing options.

3. Be able to determine positive and negative predictive values based on pre-test probabilities.

Over the past decade, the availability and feasibility for clinical laboratories of all sizes to implement molecular platforms has grown exponentially.  No longer does a laboratory need to be a large research or teaching facility to be able to perform all manner of detection of various molecular targets.

This presentation will show how a small, county hospital laboratory has been able to expand its molecular capabilities and rethink the use of multiple platforms for redundancy and rapid testing modifications based on experiences and challenges presented by the COVID-19 Pandemic.

The Microbiology/Molecular section of the Riverview Heath Laboratory supports testing for a 156- county hospital, a 16-bed critical access hospital, three ER/Urgent care centers, two dozen physician practices, 14 long term care facilities, and two non-affiliated client hospital.

Prior to the Pandemic, Riverview Health was utilizing four different molecular platforms for a variety of testing.  Influenza A&B and RSV detection were the only assays that could be performed on more than one platform.  During the Pandemic, Riverview added two ARIES® instruments, two MAGPIX® instruments (including extraction and thermocycling instrumentation) and five rapid antigen instruments. COVID testing was also brought on to one existing platform and evaluated on two other existing platforms.

Discussion topics for this presentation will include factors that drove a major change in testing philosophy, challenges with implementing these new platforms, positive outcomes from expanding capabilities, and future planning for utilization of multiple platforms for testing.

Learning Objectives:

1. Learn from Riverview’s approach for adapting to the pandemic and their testing needs.

2. Learn to evaluate the capabilities of your lab, instruments, and storage capacity.

3. Learn how to develop or update a lab-specific Pandemic Preparedness Plan.

Varicella zoster virus (VZV) causes two clinically distinct forms of disease, varicella and herpes zoster. These forms can each present with multiple manifestations ranging from lesions including primary chickenpox or shingles to a diverse spectrum of neurologic complications including aseptic meningitis and encephalitis. This workshop will discuss the clinical spectrum of VZV infection as well as laboratory testing for VZV.

Learning Objectives:

1. Describe the general biology of human herpesviruses.

2. Summarize the transmission, epidemiology, and diverse disease manifestations of VZV infections.

3. Explore the laboratory methods used to diagnose the various stages of VZV infection.

Over the last two decades, DNA synthesis has established itself as a standard tool propelling life science research in a wide range of fields. In this session, we will highlight how DNA synthesis tools can support mRNA therapeutics development.

One key benefit of synthetic DNA is that it enables flexible sequence design independent of any naturally occurring template. Methods for optimization of expression in a target organism will be discussed, focusing on how to achieve best results in mammalian systems. The ability to design sequences also enables incorporation of additional layers of information into the DNA. To this end, a method for “molecular watermarking” will be described, which can enable tracking and tracing of a molecule throughout the application.

mRNA Therapeutics have a wide range of applications ranging from personalized approaches in cancer immunotherapy to global applications in the infectious disease landscape. Depending on the application, the needs for DNA input differ widely. These needs will be discussed using specific examples of how Thermo Fisher GeneArt DNA synthesis technologies can address a variety of needs across myriad applications in the life science industry.

In summary, this presentation will highlight how different Synthetic Biology technologies can support emerging needs in mRNA therapeutics development and commercialization.

Learning Objectives:

1. Discover Synthetic Biology technologies to support emerging mRNA therapeutics needs

2. Learn about the importance of DNA input for Infectious Disease applications

3. Understand the different methods for optimization of DNA expression in target organisms

Existing methods for antibiotic selection are based on outdated and inaccurate microbiological principles, causing millions of inappropriate antibiotic selections and hundreds of thousands of preventable deaths, particularly in patients with respiratory infections. These tests take days and fail to select effective antibiotics against multidrug resistant pathogens because they do not account for critical characteristics of “real-life” infections. AtbFinder is a novel rapid culture-based diagnostics test system, employing a novel paradigm of antibiotic selection.  It mimics conditions of antibiotics  interactions  with bacteria within a host by remodeling polymicrobial biofilms from the site of infection in which interbacterial interactions that affect bacterial sensitivity to antibiotics are modulated, and testing antibiotics taken at concentrations that can be achieved in affected tissue or organ. AtbFinder selects antibiotics that treat the bacterial community at the site of infection as a whole and  enables: (i)The selection of antibiotics effective against extensively drug-resistant polymicrobial biofilm challenged infections (ii)The reporting of results in <4 hours, saving over 44 hours compared to conventional methods.

Here, we, for the first time, present findings from a 3 year long clinical study reporting the performance of antibiotic therapy selected based on recommendations of AtbFinder for patients with multidrugresistant bacteria in patients with cystic fibrosis.

Results: Antibiotic optimization aided by the AtbFinder led to: Eradicated drug-resistant P.aeruginosa Infections in 73% of cases; FEV1% increased up to 27%; Decreased the incidence of hospitalizations by 100%; Normalized WBC and CRP inflammatory markers, that correlate with lung injury; Number of systemic antibiotic courses was reduced from 355 to 178.

Conclusion: Antibiotic optimization with AtbFinder results in progressive eradication of P.aeruginosa from airways of patients with CF.

Learning objectives:

1. Selection of antibiotics that treat the bacterial community at the site of infection as a whole is a better option for patients.

2. Mult-idrug resistant bacteria can be treated with antibiotics active against bacteria that support their growth 

Early detection is critical for prompt and effective treatment of acute infectious diseases. For tick-borne diseases (TBD), the lack of accurate early diagnosis can result in delayed treatment, augment morbidity, and increase the likelihood of developing persistent symptoms. For diagnosis of Lyme disease, the low sensitivity of molecular assays in the majority of clinical presentations has prevented their extensive implementation as an effective diagnostic tool. Serology remains the primary means of diagnosis for Lyme disease and the majority of TBD. TBDCapSeq is a sequencing assay that uses hybridization capture probes that cover the complete genomes of the eleven most common tick-borne agents found in the United States. We evaluated the performance of TBDCapSeq to surveil samples that included human whole blood, mouse tissues, and field-collected ticks. For Borrelia burgdorferi and Babesia microti, the sensitivity of TBDCapSeq was comparable and occasionally exceeded the performance of agent-specific quantitative Polymerase Chain Reaction (PCR) and resulted in 25 to > 10,000-fold increase in pathogen reads when compared to standard unbiased sequencing.
 
Current virus diagnostic assays that are commonly based on PCR assays targeting one or a few specific agents are limited in scope, may fail to detect virus variants and provide only limited genotypic information. Our assay VirCapSeq-VERT addresses many of the current challenges of PCR and sequencing for diagnostics and virome analysis. The sensitivity and specificity of the VirCapSeq-VERT are comparable to those of agent-specific real-time PCR. 
 

Learning Objectives:

1. Limitations of microbial diagnostic molecular assays for genomic and pathogenesis studies of tick-borne and viral agents.

2. Offer valuable new insights into our understanding of tick-borne and viral diseases by facilitating analysis of previously challenging specimens using standard sequencing.

Microbiome sequencing data are known to be biased; the measured taxa relative abundances can be systematically distorted from their true values at every step in the experimental/analysis workflow. If this bias is not accounted for, it can lead to spurious discoveries and invalid conclusions. Unfortunately, in order to measure bias it is necessary to have samples for which the true relative abundances are known, such as model or mock community samples.  In this chapter, we propose a log-linear model for the biases observed when analyzing model communities data.  Our model expands the recent work from McLaren, Willis and Callahan (MWC) [eLife, 8:e46923, 2019] that proposed a multiplicative bias structure for microbiome data. Our extension of the MWC model is general enough to allow testing of complex hypotheses, and readily handles situations in which samples have different number of bacteria present by design. An F-test with permutation-based hypothesis testing is proposed to assess statistical significance. We conduct simulations to show the validity and the power of our method, and also demonstrate the utility of our method through an analysis of a complex model communities dat=aset that allows us to directly test  the multiplicative bias assumption of the MWC model. An R package implementing the proposed work is publicly available at https://github.com/zhaoni153/MicroBias

Learning Objectives:

1. Understanding the multiplicative bias-generation procedure in microbiome sequencing.

2. Understanding how we can measure the sequencing bias as ratios of abundances.

Ribonucleic acid (RNA) has a breathtaking variety of biological functions, which far exceeds its classical role as a carrier of genetic information. The repertoire of this versatile molecule is further diversified by covalent post-transcriptional modifications (PTMs) or chemical marks that decorate all four canonical nucleotides in mRNAs and noncoding RNAs. Although, methylation of the N6-position of adenosine (m6A) was recently shown to be a key regulator of viral gene expression and modulate nuclear export, expression, stability and packaging of viral genomic RNA, the role of the more than 170 other RNA modifications during viral infection is largely unknown. To investigate these other RNA modifications we employed an unbiased and comprehensive analytical approach based on mass spectroscopy to characterize the changes in the landscape of RNA modifications in the cell during virus infection as well as identify the epitranscriptomic marks on the RNA genomes of Zika virus. Zika virus is a re-emerging mosquito-borne flavivirus that during the 2016 outbreak in the Americas was associated with developmental and neurological defects in newborns. Our data show that the RNA modification landscapes change significantly during viral infection. Moreover, during infection of mammalian cells we observed the appearance of two dimethylcytosine modifications namely 5,2’-O-dimethylcytosine (m5Cm) and N4,N4-dimethylcytosine (m44C). Our studies with other RNA viruses show that this dimethylcytosine mark also appears following infection with human immunodeficiency virus 1 (HIV-1), poliovirus, hepatitis C virus and Dengue virus. Remarkably when we analyzed the viral RNA, we identified not just m6A but more than 30 different epitranscriptomic marks on Zika virus genomes. To investigate whether epitranscriptomic profile would similarly change following Zika virus infection in mosquitoes, we fed Aedes aegypti mosquitoes a bloodmeal without and with Zika virus. Mosquitoes were collected 14 days post-infection for analysis. We found that particular dimethyl modifications were more prevalent among infected mosquitoes and may impart a role during the mosquito immune response to infection. While the localization of specific RNA modifications, the function and putative modifying enzymes remains to be identified, our research demonstrates a way in which RNA viruses interface with cellular RNA pathways that may present new targets for bioassays and antiviral treatments.

Learning Objectives:

1.    To be able to define what an RNA modification is and the function of these chemical marks.

2.    To be able to describe one experimental approach to identifying RNA modifications.

Health economists are great at calculating a value-based price for diagnostic tests. Given the unexpected supply and demand for SARS-CoV-2 tests during the pandemic, efficient allocation of tests and money is important to maximize public health benefits and economic recovery. This talk will help you think like an economist. What is the decision the test result is intended to inform? What is the most efficient and effective cadence of frequent testing for surveillance? We will define the dimensions of diagnostic testing value, including time-to-result, accuracy, health benefits, and access. We will discuss which tests and protocols were chosen to reopen universities, sports leagues, retail stores, and Broadway theaters. Examples will highlight self-collected saliva, pooling specimens, and sequencing. Lastly, we will use the testing value framework to categorize existing technologies on the market and highlight opportunities for innovation. 

Learning Objectives:

1.    Think like an economist

2.    Define the dimensions of value for diagnostic testing

3.    Identify product-market fit

Saliva serves as a reservoir for the upper aerodigestive tract (UADT). Saliva collection is non-invasive, convenient, and amenable to self-collection. Thus, saliva is an ideal resource for liquid biopsy of cancers or infectious diseases derived from UADT. Cancers arising from UADT include head and neck, esophagus, and lung cancers, which, in combination, have the highest rate of incidence and mortality among all cancers. Detection of respiratory infection, particularly viral infection, is the most common lab test in clinic. In this talk, we will focus on our proprietary inventions of saliva-based tests for head and neck cancer, i.e., HNKlearTM, and SARS-CoV-2, i.e., CovidFASTTM, two best examples to demonstrate the value of saliva diagnostics for cancers and infectious diseases from both clinical development and real-world experience.

Learning Objectives:

1. Advantages of using saliva in clinical tests for cancers and infectious diseases derived from the upper aerodigestive tract.

2. HNKlear, a saliva-based test for head and neck cancer, from invention to clinical application.

3. CovidFAST, an RNA extraction-free method for SARS-CoV-2 using either saliva (SalivaFAST) or swab (SwabFAST), serves as a powerhouse for COVID Check Colorado.

We currently do not have effective vaccines or antiviral drugs for most of the viral diseases that afflict humans.  Antiviral therapies that enable long-term control over human immunodeficiency virus (HIV) infection and cure chronic hepatitis C virus (HCV) have been landmark successes in the treatment of viral infections.  These therapies work through the use of multiple direct-acting antiviral (DAA) drugs that act by potently inhibiting the function of viral proteins, generally enzymes such as viral polymerases and proteases. Narrow spectrum activity (i.e., the “one bug, one drug” problem) and drug resistance are two major challenges for the antivirals field.  This is especially true against viruses with RNA genomes because limited or no proofreading function of the viral polymerase generates tremendous genetic diversity.  There is thus a need for antiviral strategies that make use of alternative targets, mechanisms, and modalities to combat viral diseases and to avoid antiviral resistance.  My group has focused on the discovery and validation of new antiviral targets and strategies. Here I hope to provide to examples of our proof-of-concept work establishing targeted protein degradation as an alternative antiviral strategy.

Learning objectives:

1. Understand the mechanism of what a degrader (also known as PROTAC, “degronimid”) is.

2.  Understand how one experimentally determines if a small molecule has a “degrader” mechanism.

3. Explain potential advantages of degraders versus conventional inhibitors as direct-acting antivirals.

Analysis of community wastewater has been shown to be an effective method for tracking the prevalence of SARS-COV-2.  Our group has developed techniques for sequencing a portion of the SARS-COV-2 genome to determine what viral viriants are present in wastewater samples.  In addition to tracking known lineages, a subset of sewersheds were found to contain viral lineages that are distinct from lineages that have been seen in patients.  These novel lineages all have mutations in the receptor binding domain (RBD) of the Spike protein in positions known to affect viral tropism.   Spike proteins generated with these additional mutations were found to have expanded tropism and resistance to some neutralizing antibodies.  While the source of these novel lineages is currently not known, it is suspected that they are derived from non-human hosts.

Learning Objectives:

1.    What are the different techniques for identifying SARS-COV-2 variants from wastewater.

2.    What important SARS-COV-2 properties can be elucidated by analyzing the viral sequence.

In this webinar Prof. Andersson introduces HMGB1 and its function in the innate immune system to further elucidate all hints and scientific findings explaining HMGB1’s role in COVID-19 disease aetiology.

Learning Objectives:

1. What is HMGB1 and which function does it play in the immune system.

2. What role does HMGB1 play in Sars-CoV2 infections.

The COVID-19 pandemic has brought awareness to the dangers of emerging pathogens to global human health and welfare. Sensitivity and flexibility are important features for methods used to detect emerging pathogens. While PCR testing is rapid and sensitive, a significant advantage next generation sequencing (NGS) approaches have over PCR-based analyses is the ability to detect previously undiscovered pathogens while also providing genomic information that can detect SARS-CoV-2 genome sequence, identify source of co-infection, and the host transcriptional response in a single workflow. The critical component enabling this approach is Jumpcode CRISPRclean technology which removes abundant human and bacterial ribosomal RNA sequences from NGS libraries. Metatranscriptomics powered by CRISPR-mediated depletion of rRNA offers a robust methodology to acquire viral genomic data, microbiome composition, co-infection information, and the transcriptional status of the host immune response in a single workflow. This sequencing-based approach can be available on the first day of the next viral outbreak and should be considered as a first-line test for novel zoonotic virus detection.

Learning Objectives:

1.    Learn about metagenomic sample prep.

2.    Learn about metagenomic sequencing.

3.    Learn about metagenomic analysis.

Flow cytometry is a powerful technology for investigating questions related to the immune response and its sustenance, as well as detailed mechanisms of COVID-19. Simplified cell-based and biomarker-based strategies can provide rapid insights into these aspects of the disease, and the evaluation of anti-SARS-CoV-2 antibodies and their isotypes has become a crucial aspect of COVID-19 research, supporting the development and understanding of vaccine-based strategies. In this webinar, we discuss the results from a new flow cytometry-based assay—the Guava® SARS-CoV-2 Multi-Antigen Antibody Assay. This multiplex, bead-based assay detects the presence of IgG, IgM, and IgA antibodies against multiple SARS-CoV-2 antigens, including the nucleocapsid protein, the receptor-binding domain of the spike protein, and the S1 subunit of the spike protein, with high detection sensitivity. Although the assay was optimized on Luminex’s Guava® Platforms, it can be used on any flow cytometer equipped with either a 488 nm or 532 nm laser. We also review data from longitudinal studies, as well as data from vaccinated donors and serological samples from natural infection taken at different durations post-symptom onset. Finally, the capabilities of flow cytometry platforms to provide important immune cell information will also be discussed.
For Research Use Only. Not for use on diagnostic procedures.
 

Learning Objectives:

1. Value of multi-antigen approaches for detection of SARS-CoV-2 antibodies.

2. Importance of the study of SARS-CoV-2 antibody isotypes in SARS-CoV-2 research.

3. Continued importance of serology in natural infection and vaccine research.

Highly efficacious vaccines against COVD-19 have been generated in a record time, and allowed a great impact in preventing severe disease due to SARS-CoV-2 infection in vaccinated individuals. Unfortunately, the pace in vaccination in developing countries continues to be very slow, mainly due to the lack of vaccine supply. The development of affordable COVID-19 vaccines that can be produced locally to satisfy the needs of developing countries is highly desirable to speed up the world COVID-19 vaccination. We have generated a  COVID-19 vaccine based on a Newcastle disease virus (NDV) vector expressing a stabilized form of the spike (S) protein of SARS-CoV-2.  This vaccine can be manufactured using the same technology in palce for the generation of the annual inactivated influenza virus vaccines,. The NDV-S  vaccine provides high level of protection against SARS-CoV-2 challenge in multiple animal models. Phase 1 clinical trial are being conducted in multiple countries to determine the safety profile and immunogenicity of this novel COVID-19 vaccine.

Learning Objectives:

1. To understand viral vector vaccines.

2. To gain knowledge on Newcastle disease virus-based vaccine vectors.

3. To gain knowledge on vaccine development steps

4. To understand the current limitations of COVID-19 vaccines.

Microbes are recently recognized as driving the energy and nutrient transformations that fuel Earth’s ecosystems in soils, oceans and humans. Where studied, viruses appear to modulate these microbial impacts in ways ranging from mortality and nutrient recycling to extensive metabolic reprogramming during infection. As environmental virology strives to get a handle on the global virosphere (the diversity of viruses in nature), we face challenges to organize this ‘sequence space’ (create a sequence-based viral taxonomy), link these viruses to their natural hosts (who infects whom), and establish how virus populations are structured (ecological drivers) and impact natural ecosystems (their impacts). Here I will share current thinking on how to study viruses in complex communities and how these efforts are revealing new biology in the oceans, soils and the clinic that will help enable a new generation of eco-systems biology and medical treatments.

Learning Objectives:

1. Why study viruses, particularly of microbes, in complex systems?

2. What challenges have been overcome to enable their study? 

3. What have we learned across biomes?
 

Self-amplifying RNA replicons are promising candidates for next generation vaccines against human coronaviruses with pandemic potential. Self-amplification of RNAs in host cells generates more potent immune
responses with significantly reduced amounts of RNA delivered. Their defects in essential functions for viral dissemination make them highly safe as vaccines. We have shown that the deletion of the envelope (E) gene from Middle East respiratory syndrome coronavirus (MERS-CoV) led to a replication-competent propagation-defective RNA replicon (MERS-CoV-ΔE) that does not produce infectious progeny and behaves as a safe single-cycle virus in cell cultures and in vivo. The combined deletion of E gene with accessory proteins 3, 4a, 4b and 5, which are involved in the interference with the innate immune response, further attenuated the propagation-defective RNA replicon in vivo, in a mouse model of infection. 

Inside the cells, in the absence of E protein, MERS RNA replicons formed highly immunogenic polymeric structures. Intranasal administration of MERS-CoV RNA replicons to mice provided 100% protection after challenge with a lethal dose of virulent MERS-CoV. Interestingly, they induced sterilizing immunity, as no infectious virus was detected in the lungs of challenged mice. These results represent a proof of concept for the development of safe and effective RNA replicon vaccines against deadly human CoVs. We are currently developing replication-competent propagation-defective RNA replicons derived from SARS-CoV-2 as safe and more efficient vaccine candidates, since they simultaneously express multiple viral antigens.

LEARNING OBJECTIVES

1.    Define the potential of self-amplifying RNA replicons derived from coronaviruses as vaccine candidates.
2.    Explain current approaches to the development of RNA-replicon vaccine candidates against SARS-CoV-2.

Influenza viruses represent a significant burden to human health.  In order to limit respiratory viral disease, new approaches that can prevent viral infection and spread are needed.  We have performed a number of genetic screens to identify host factors that can either directly restrict infection or modulate the immune response to better control the virus.  Those data and thoughts on the future development of host-directed antiviral therapeutics will be discussed in this presentation.

Learning Objectives:
1. Understand how host proteins can be targeted to prevent viral disease.
2. Explain theoretical advantages and disadvantages for host-directed therapeutics.
 

The gut microbiota has been proposed to affect the risk and prognosis of various health outcomes. Related to pregnancy, existing studies suggest that pregnant women have a distinct microbiota profile compared to non-pregnant women. Further, there are also differences within pregnancy, with distinct profile between women from the first and third trimester. The microbiota profile in the third trimester has been noted to be similar to the second trimester while the first trimester samples are closer to non-pregnant women. However, data is lacking in diverse resource-limited settings that have a unique baseline microbiota profile, as well as whether there are differences in the temporal profile by disease status (e.g. HIV status); whether the gut microbiota profile impacts maternal-infant health outcomes is also not clear. To address these gaps, we conducted a cohort study of pregnant women with and without HIV from India. Preliminary data suggests no differences in α- and β-diversity between women with and without HIV. At the genus level, we observed lower levels of Prevotella and higher levels of Megasphaera in women with HIV compared to women without HIV. Ongoing analysis will i) characterize the temporal dynamics of the gut microbiota during pregnancy, ii) study the relationship of maternal gut microbiome with diet and inflammation, and iii) determine the relationship of maternal gut microbiome with a) infant gut microbiome and b) infant health outcomes (birth outcomes and growth).

Learning Objectives:

1. Understand the temporal dynamics of gut microbiota during pregnancy and infancy.

2. Explain the role of maternal HIV infection on maternal-infant gut microbiota.

Genetic Molecular Surveillance is a key component of a modern public health toolkit. This talk will detail the molecular microbial make up of urban environments. It will discuss how these environments have changed with the advent of the COVID-19 pandemic and how variant identification can be used to accurately identify SARS-CoV-2 strains in a population.

Learning Objectives:

1. Understand the urban microbiome and it’s potential impact on health.

2. Understand how molecular assays can be used to make fine grained public health decisions.

Computational or mathematical modeling generally refers to a mechanism-based formalism that help us test hypotheses, expand our understanding of a system, or make mechanism-aware predictions. These models often vary in their level of granularity. For example, metabolic models focus on biochemical interactions on the level of enzymes and molecules while population models such as Lotka-Volterra focus on species-species interactions. What these models share in common is that for any moderately sized microbial community, they become exceedingly complex in terms of number of interactions and free parameters to evaluate. These modeling fields continue to advance rapidly to meet these challenges. In this talk, I will highlight the potential of different machine learning approaches to augment mechanistic modeling efforts and synergize with large data and modeling efforts in the microbiome sciences.

Learning Objectives:

1. What is a complex system?

2. Examples of modeling approaches for microbial populations.

3. Machine learning approaches for understanding higher-order concepts.

For centuries, humans have become infected with endemic circulating Coronaviruses (hCoV), typically repeatedly throughout life. To understand how these respiratory infections establish CD4 T cells memory and whether this memory has the potential to be recruited into the immune response to SARS-CoV-2, we studies human peripheral blood samples collected prior to 2019. Our studies revealed highly variable reactivity to the human coronavirus proteins, related both to the age of subjects and with regard to the functional potential, including cytotoxic activityl. Particularly striking was the diminished human CD4 memory to hCoV associated with age, in contrast to reactivity to influenza epitopes, studied in parallel. We also provide evidence that CD4 T cells reactive with the alpha hCoV can be cross boosted by the two lineages, based on accumulation of CD4 T cells specific for the highly conserved S2 domains of the spike proteins derived from these endemic viruses. Finally, we found that the human CD4 T cells elicited by the endemic hCoV are able to recognize SARS-CoV-2 epitopes and display functional potential linked to the specific viral antigen examined, particularly notable in the cytotoxic potential of the SARS CoV-2 reactive CD4 T cells.
 
Learning Objectives
 
1.     Understand the epidemiology of human Coronaviruses.
2.     Learn the types of human CD4 T cells elicited by human Coronaviruses.
3.     Understand the basis of cross-reactive T cell recognition of related viruses.

The ongoing COVID-19 pandemic has increased awareness about sex-specific differences in immunity and outcomes following respiratory virus infections. Strong evidence of a male bias in COVID-19 disease severity will be presented based on clinical data and preclinical animals models, which illustrate sex differential immune responses against SARS-CoV-2. Prior to the pandemic, data from other viral infections, including influenza viruses, showed profound sex differences in virus-specific immunity, including locally in the respiratory tract. We  have used influenza A viruses to interrogate sex-specific immunity to infection and vaccination. Although males are more susceptible to most viral infections, females possess immunological features that contribute to greater vulnerability to immune-mediated pathology but also better protection following vaccination. Both sex chromosome complement and related X-linked genes (e.g., TLR7) as well as sex steroids, including estrogens and androgens, play important roles in mediating the development of sex differences in immunity to respiratory viral infections and vaccination.

Learning objectives:

1.    Definition of biological sex and distinction from gender, with consideration of diverse respiratory viruses.

2.    Characterization of sex differences in respiratory virus pathogenesis in humans and animal models, with emphasis on influenza A viruses and SARS-CoV-2.

3.    Insights into the genetic and hormonal mechanisms that underlie sex differences in immunity to respiratory virus infection and vaccination.

The Human Microbiome Project was conceived almost 15 years ago, as an extension of the Human Genome Project, to explore the diversity of human-associated microorganisms at multiple body sites (skin, mouth, nose, colon, and vagina) and their impact on human health.  Pioneering studies highlighted the extent of intra- and inter-individual microbiome variations, and how these variations played key roles in nutrition, health, disease, and immunity. Different models were proposed to describe the human microbiome, likening it, for example, to a missing organ or a second genome. The origin and definition of the term microbiome is discussed, and different views of illustrating or modeling the microbiome are debated, including a proposed cloud model. Subsequently, the major obstacles/bottlenecks related to microbiome analysis are listed, and examples of analysis strategies that address them are presented. Moreover, recent research results are presented on microbiome alterations in hepatitis C and conjunctivitis patients, as well as the interplay between the human and surrounding environmental microbes among nurses and drug factory workers. Finally, the nascent field of pharmacomicrobiomics, or drug–microbiome interactions, is introduced and discussed in the light of ‘the microbiome cloud model’. Key examples of drugs that are dramatically affected by gut and vaginal microbes are presented. Finally, recently developed web resources and big data analysis tools are demonstrated and applied to accelerate drug-microbiome interactions in the hope of customizing therapeutic intervention, minimizing drug toxicity, and improving the therapeutic outcomes of available medicines.

Learning Objectives:

1. List and compare the different definitions of, and images associated with, the terms microbiome and human microbiome.

2. Define pharmacomicrobiomics, and differentiate the term from the associated subfields of pharmacometagenomics, pharmacometabonomics, pharmacometabonomics

3. List the major bottleneck in the microbiome analysis pipeline, and mention at least one strategy to address each of them

4. List potential applications of pharmacomicrobiomics in precision medicine (therapeutics and diagnostics).

Innate and adaptive immunity mechanisms provide antiviral protection in mammals. In this talk, I shall present recent findings from my lab and collaborators revealing a distinct form of mammalian antiviral response directed by the RNA interference (RNAi) pathway. In the first part of the presentation, I discuss the data published in a joint paper with Dr. Kate Jeffrey reporting the first evidence for a human virus to induce and suppress antiviral RNAi in cultured mammalian cells. In the second part, I present results of our functional and mechanistic studies on antiviral RNAi from a mouse model of infection by Nodamura virus (NoV), a positive-strand RNA virus transmissible by mosquitoes. We show that NoV RNA replication triggers Dicer-dependent production of predominantly 22-nt virus-derived siRNAs, loaded in RNA-induced silencing complex (RISC) to guide specific RNA cleavages by Argonaute-2. We demonstrate that NoV encodes a dsRNA-binding B2 protein as a viral suppressor of RNAi (VSR) essential for infection by inhibiting both Dicer processing and siRNA loading into RISC by sequestering long dsRNA and siRNA duplexes, respectively. Notably, VSR-B2 enhances virus accumulation exclusively by suppressing the RNAi response since it becomes inactive in RNAi-defective mouse fibroblasts. Moreover, we found that expression of a functional VSR-B2 is essential for NoV infection and virulence not only in suckling mice, but also in adult mice either intact or completely defective in the signaling of type I, II and III interferons (IFN). Together, our findings illustrate that the RNAi response confers in vivo antiviral protection in the presence or absence of IFN-regulated antiviral responses and that specific viral suppression of the RNAi response is essential for in vivo infection and virulence. After a brief summary of data published by others on this topic, I discuss the main properties of mammalian RNAi response to virus infection as compared to the known innate and adaptive immunity mechanisms.

 
Learning objectives:

1. Define the main components of the mammalian RNAi response to virus infection

2. Explain the RNA-based specificity mechanism of the RNAi antiviral response.

The host antiviral innate immune response involves activation of multiple signaling pathways that result in the production of type I interferons (IFN-I) and inflammatory cytokines, which together control virus infections. However, excessive inflammation and cytokine production can promote exacerbated disease. To achieve the right balance, these signaling pathways are regulated by different molecular processes, including the host ubiquitin system. This system is best known for its function in marking unnecessary proteins for degradation, but can also be used as a weapon against invading pathogens. However, viruses have developed mechanisms to hijack this system for its own use. For example, Zika virus hijacks a host antiviral factor, the E3-ubiquitin ligase TRIM7, which puts an ubiquitin marker on the viral envelope. This ubiquitin on the surface of the virus allows the virus to attach better to receptors expressed in specific cell types, driving the tissue specificity of Zika virus. Therefore, pathogenic viruses can hijack antiviral factors of the ubiquitin system, turning the antiviral functions of ubiquitin into ‘pro-viral’ pathogenic factors. This is a constant battle between the defensive response of the host cell and viruses, resulting in virus evolution and adaptation. In this talk, we will discuss novel molecular mechanism by which E3-ubiquitin ligases of the TRIM family regulate host antiviral pathways, and how different pathogenic viruses hijack the ubiquitin system to enhance virus replication. 

Learning objectives:

1.    Learn mechanisms of regulation of innate antiviral immune signaling that lead to protection against virus infection.

2.    Strategies used by viruses to hijack host antiviral factors to enhance virus replication and pathology

Polymerase errors are the major source of variation in virus and viroid genomes, and thus polymerase error rates are critical determinants of adaptation potential.  Potato spindle tuber viroid (PSTVd) has a circular non-coding RNA genome of 359 nucleotides that replicates and spreads systemically in host plants.  Consequently, all functions needed to establish a productive infection are mediated by sequence and structure elements within the genomic RNA.  During replication, PSTVd hijacks host transcription machinery, and specifically DNA-dependent RNA polymerase II (Pol II), to copy its genome.  We sought to determine the Pol II error rate when transcribing PSTVd RNA, a non-native template.  However, estimation of in vivo error/mutation rates is complicated by selection, which leads to loss or proliferation of certain mutations.  To circumvent this issue, we sequenced minus-strand PSTVd dimers from concatemeric replication intermediates.  The underlying rationale is that mutations found in only one of the monomers were likely generated de novo during Pol II transcription of the circular plus-strand RNA genome.  This approach yielded an apparent error rate ~100-fold greater than Pol II transcription on DNA templates.  Nevertheless, the error spectrum was similar to that observed during mRNA transcription by Pol II.  Remarkably, however, de novo mutations were rare in the most highly conserved, replication-critical genome regions, suggesting these sequences are selected for both important function and enhanced transcription fidelity.  Such biased fidelity reveals a novel strategy to ensure population survival while maximizing adaptation potential.  Comparison of mutations identified by minus-strand dimer sequencing with mutations observed in progeny variants derived from wild type PSTVd showed that most de novo mutations are lost through selection.

Learning Objectives: 

1.  Identify viroids as infectious non-coding RNAs

2.  Understand polymerase errors as drivers of virus/viroid evolution

Flaviviruses are pathogens of global public health concern. They include dengue virus (DENV), West Nile virus (WNV), and zika virus (ZIKV). There are no approved US Food and Drug Administration antivirals for any flavivirus. The type I interferon (IFN-I) system is the first line of defense against viral infection. Activation of IFN-I signaling leads to the induction of hundreds of IFN stimulated genes (ISGs), the products of many serve to limit viral replication. One of these ISGs, Cytidine monophosphate kinase (CMPK2), is a potential anti-flavivirus drug target. CMPK2 is an interferon inducible enzyme with a mitochondrial localization sequence (MLS) at its N-terminus that localizes it to the mitochondria where it plays an important role in maintenance of cellular nucleic acid synthesis. The N-terminal domain (NTD) of human CMPK2 contains no known sequence homology to any other species and the C-terminal domain (CTD) is highly conserved across species and is responsible for its kinase activity. CMPK2 was reported to inhibit human immunodeficiency virus (HIV) and DENV replication. However, the molecular mechanism by which CMPK2 restricts these viruses is unclear. Here we show that expression of CMPK2 inhibits the replication of multiple flaviviruses including ZIKV.  We show that the mitochondrial localization of CMPK2 is required for its antiviral activity. Moreover, we show that the NTD of CMPK2 is sufficient for its antiviral activity thus the kinase activity is not required for CMPK2 antiviral function. Future studies will focus on the molecular mechanisms by which CMPK2 inhibits flavivirus replication. This knowledge will aid in the design of novel therapeutic antiviral strategies.

Learning objectives:
1. Identify CMPK2 as a host restriction factor for multiple flaviviruses.
2. To explain that the mitochondrial localization of CMPK2 is required for its antiviral function, and its kinase function is dispensable. 

Infection with human immunodeficiency virus (HIV)-1 compromises the immune system and causes in about half of the affected individuals neurological and neurocognitive complication categorized as NeuroHIV and HIV-associated neurocognitive disorders (HAND). The pathological mechanisms leading to HAND remain incompletely understood but the available data suggest an important role for the innate immune system. Transgenic mice expressing HIV envelope protein gp120 in the central nervous system (HIVgp120tg) display key features features observed in NeuroHIV patients, including loss of neuronal dendrites and synapses, astrocytosis and increased numbers of microglia. We have recently shown that the acute phase protein lipocalin-2 (LCN2) is one of the most upregulated genes in this transgenic mouse model of NeuroHIV. Furthermore, we observed that LCN2 is significantly upregulated in brains of HIV+ patients when compared to non-infected controls. Higher levels of LCN2 were present in HIV+ patients with brain pathology than in patients without brain injury, suggesting a role for LCN2 in HIV-associated brain injury. In order to explore the biological role of LCN2 we cross-bred HIVgp120tg with LCN2-deficient mice (LCN2ko). LCN2 deficiency prevented behavioral deficits, and largely abrogated synaptic and dendritic damage indicating almost complete neuroprotection and providing evidence for a role of LCN2 in HIV-associated brain injury. We also observed that HIVgp120tg brains express genes induced by interferon-β (IFNβ), indicating another component of the innate immune response to the neurotoxicity inducing viral protein. In separate experiments we demonstrated that intranasal treatment with exogenous IFNβ prevented neuronal damage triggered by HIVgp120. Altogether, our studies strongly suggest that components of innate immunity can promote and prevent HIV-associated neurodegeneration.

Learning Objectives:

1. Explain how viral infections can spread from the periphery to the central nervous system.

2. Define the dual role of innate immunity in injury and protection of the brain from viral infection.

3. Identify challenges and opportunities to treat viral infections and neurodegeneration.
 

Flaviviruses are pathogens of global public health concern. They include dengue virus (DENV), West Nile virus (WNV), and zika virus (ZIKV). There are no approved US Food and Drug Administration antivirals for any flavivirus. The type I interferon (IFN-I) system is the first line of defense against viral infection. Activation of IFN-I signaling leads to the induction of hundreds of IFN stimulated genes (ISGs), the products of many serve to limit viral replication. One of these ISGs, Cytidine monophosphate kinase (CMPK2), is a potential anti-flavivirus drug target. CMPK2 is an interferon inducible enzyme with a mitochondrial localization sequence (MLS) at its N-terminus that localizes it to the mitochondria where it plays an important role in maintenance of cellular nucleic acid synthesis. The N-terminal domain (NTD) of human CMPK2 contains no known sequence homology to any other species and the C-terminal domain (CTD) is highly conserved across species and is responsible for its kinase activity. CMPK2 was reported to inhibit human immunodeficiency virus (HIV) and DENV replication. However, the molecular mechanism by which CMPK2 restricts these viruses is unclear. Here we show that expression of CMPK2 inhibits the replication of multiple flaviviruses including ZIKV.  We show that the mitochondrial localization of CMPK2 is required for its antiviral activity. Moreover, we show that the NTD of CMPK2 is sufficient for its antiviral activity thus the kinase activity is not required for CMPK2 antiviral function. Future studies will focus on the molecular mechanisms by which CMPK2 inhibits flavivirus replication. This knowledge will aid in the design of novel therapeutic antiviral strategies.

Learning objectives:
1. Identify CMPK2 as a host restriction factor for multiple flaviviruses.
2. To explain that the mitochondrial localization of CMPK2 is required for its antiviral function, and its kinase function is dispensable. 

Plant-infecting viruses cause significant losses to agriculture, including to staple crops such as wheat, maize, and rice. Preventing and managing endemic and emerging plant diseases is an ongoing challenge. Maize lethal necrosis, or MLN, has emerged globally since 2010-2011 in East Africa, Asia, and South America. MLN is caused by synergistic co-infection of two viruses: maize chlorotic mottle virus (MCMV) and any of several maize-infecting potyviruses that are endemic worldwide--sugarcane mosaic virus (SCMV), maize dwarf mosaic virus (MDMV), Johnsongrass mosaic virus (JGMV), or wheat streak mosaic virus (WSMV). Although the route(s) of introduction and emergence of MLN are not fully elucidated, considerations for preventing plant pathogen introduction and managing disease once present, including diagnostics in global trade and genetic resistance in plants, will be discussed. In agricultural and uncultivated settings, mixed infections of viruses frequently occur with various possible outcomes. In the mixed infection of MCMV and a potyvirus, titer of MCMV increases and disease severity is strongly enhanced, with tissue and often plant death (lethal necrosis) that does not occur in single infections with either virus alone. Other viruses also coexist and frequently co-infect plants in field settings where MLN is emergent, including a recently discovered maize-infecting polerovirus, variously named maize yellow mosaic virus RMV2 (MYDV RMV2), maize yellow mosaic virus (MaYMV), and MYDV-like. Outcomes of mixed infections of MCMV, SCMV, and MaYMV show interactions of each virus but synergistic lethal necrosis only for the MCMV + SCMV combination.

Learning Objectives:

1.    Identify two or more ways in which plant viruses can be introduced to new locations.
2.    Describe commonly-employed approaches to limit plant virus introduction and/or minimize losses caused by plant disease.
3.    Describe two or more possible outcomes of virus mixed infections in plants.

Plant viruses are obligate parasites that highjack the resources of the host to facilitate their infection cycle.  Plants defend themselves using a variety of mechanisms, including antiviral RNA silencing.  Viruses counteract these defense responses, by encoding specialized proteins such as suppressors of silencing.  In some plant-virus interactions, notably in plants infected with nepoviruses, symptom recovery is observed.  In this complex phenotype, plants are initially diseased but later recover from infection and become asymptomatic.  Symptom recovery can be considered as a form of inducible tolerance in which the virus persists but does not cause significant disease.  Using Nicotiana benthamiana plants infected with tomato ringspot virus (ToRSV) as a model system, we have shown that symptom recovery is dependent on environmental conditions and on the specific ToRSV strain.  In this pathosystem, the plant antiviral RNA silencing is activated to reduce but not eliminate virus infection.  Temperature-dependent symptom recovery against a severe strain of ToRSV was shown to be associated with specific repression of the translation of viral RNAs, preventing the formation of viral proteins.  This process was dependent on the plant ARGONAUTE 1 (AGO1), a key enzyme of the antiviral RNA silencing mechanism.  A suppressor of silencing encoded by ToRSV was shown to counteract the AGO1-dependent translation repression mechanism although inefficiently.  In contrast, symptom recovery against a mild strain of ToRSV at a lower temperature did not depend on AGO1.  AGO2 restricted virus accumulation under these conditions but was not absolutely required for symptom recovering, suggesting that other AGOs are also likely to be involved.  The results highlight the complexity and diversity of molecular mechanisms associated with symptom recovery and illustrate how the delicate balance between plant defense responses and virus counter-defenses determines the outcome from infection. 

Learning Objectives:

1. Understand the diversity and complexity of plant antiviral defense mechanisms, in particular antiviral RNA silencing

2. Understand how the balance between plant defense responses and viral counter-defenses determine the outcome from infection from susceptibility to tolerance to resistance

3. Understand that redundant and overlapping defense mechanisms can simultaneously operate in virus-infected plants

4. Understand the impact of environmental factors on plant virus-induced disease
 

The Coronavirus disease 2019 (COVID-19) pandemic poses an unprecedented challenge for public health, which is caused by infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is one of Coronaviruses. These viruses are the known RNA viruses with the largest RNA genome, ∼30 kb-long. Its replication is completed by a complex of non-structural proteins (NSP), composed of a core factor, NSP12 and two other small proteins, NSP7 and NSP8. All three factors are assembled highly dynamically in solution and NSP7 and 8 play a critical role in regulating the RNA dependent polymerase (RdRp) activity of NSP12. However, the underlying mechanism remains unknown. Here, we are using both biophysical and biochemical analysis on the interaction between NSP7 and NSP8. We obtained a structural information for the NSP7-NSP8 complex. Formation of the NSP7-NSP8 complex is mediated by two distinct oligomer interfaces, with interface I responsible for heterodimeric NSP7-NSP8 assembly, and interface II mediating the heterotetrameric interaction between the two NSP7-NSP8 dimers. Conformation guided mutation, combined with biochemical analysis, reveals a structural coupling between the two oligomer interfaces and the importance of these interfaces for the RdRp activity. We identified one NSP7 mutation that differentially affects the stability of the NSP7-NSP8 and NSP7-NSP8-NSP12 complexes, resulting in the compromised RdRP activity. This information shed light on the critical regulating role of NSP7-NSP8 on replication of SARS-CoV-2 genome, which has implications for antiviral drug development.

Learning Objectives:

1. Regulators for RNA dependent polymerase of SARS-CoV-2

2. RNA dependent polymerase of SARS-CoV-2

3. Antiviral development for SARS-CoV-2

Recent years have seen an explosion in our knowledge on the molecular interplay between viruses and their hosts. Plus-stranded RNA viruses, such as Sars-CoV-2 or Dengue virus, recruit cellular vesicles and co-opt cellular proteins involved in metabolism and lipid biosynthesis to build viral replicase complexes within the large viral replication organelles. We use tombusviruses (TBSV), which are small (+)RNA viruses, as model plant viruses to study virus replication, recombination, and virus - host interactions using yeast (Saccharomyces cerevisiae) surrogate host. Several systematic genome-wide screens and global proteomics and lipidomics approaches have led to the identification of ~500 host proteins/genes that are implicated in TBSV replication. We characterized the role of ~100 co-opted host proteins, sterols and phospholipids in tombusvirus replication organelle biogenesis. We will present an integrated view how tombusviruses built the replication organelle through co-opting cellular proteins and subverting cellular pathways. Finally, we show evidence that TBSV uses the replication organelle to protect the viral RNA in a unique microenvironment from degradation by the host antiviral machinery. Altogether, hijacking of cellular proteins and networks is key for TBSV to exert supremacy over the host cell leading to robust replication of the viral RNA. 

 
Learning Objectives:

1. Virus-host interaction network

2. Understanding viral replication

3. Broad-spectrum antiviral approaches