Median Tissue Culture Infectious Dose 50 (TCID50) Assay to Determine Infectivity of Cytopathic Viruses.

The emergence of zoonotic viruses like severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 have significantly impacted global health and economy. The discovery of other viruses in wildlife reservoir species present a threat for future emergence in humans and animals. Therefore, assays that are less reliant on virus-specific information, such as neutralization assays, are crucial to rapidly develop diagnostics, understand virus replication and pathogenicity, and assess the efficacy of therapeutics against newly emerging viruses. Here, we describe the discontinuous median tissue culture infectious dose 50 (TCID50) assay to quantitatively determine the titer of any virus that can produce a visible cytopathic effect in infected cells.

Applications of Quantitative PCR (qPCR) in Studies of Virus-Host Interactions.

Emerging viruses pose significant threats to human health and the global economy. In the past two decades, three different coronaviruses have emerged to cause worldwide public health concerns. The advent of high throughput genomic and transcriptomic technologies facilitated the study of virus-host interactions, accelerating the development of diagnostics, vaccines, and therapeutics. Here, we describe quantitative PCR (qPCR) in studies of virus-host interactions to dissect host responses and viral kinetics and how these relate to one another.

Establishing Air-Liquid Interface (ALI) Airway Culture Models for Infectious Disease Research.

Air-liquid interface (ALI) airway culture models serve as a powerful tool to emulate the characteristic features of the respiratory tract in vitro. These models are particularly valuable for studying emerging respiratory viral and bacterial infections. Here, we describe an optimized protocol to obtain the ALI airway culture models using normal human bronchial epithelial cells (NHBECs). The protocol outlined below enables the generation of differentiated mucociliary airway epithelial cultures by day 28 following exposure to air.

Mycobacterium tuberculosis and SARS-CoV-2 co-infections: The knowns and unknowns.

Health impacts of Mycobacterium tuberculosis (Mtb) and SARS-CoV-2 co-infections are not fully understood. Both pathogens modulate host responses and induce immunopathology with extensive lung damage. With a quarter of the world's population harboring latent TB, exploring the relationship between SARS-CoV-2 infection and its effect on the transition of Mtb from latent to active form is paramount to control this pathogen. The effects of active Mtb infection on establishment and severity of COVID-19 are also unknown, despite the ability of TB to orchestrate profound long-lasting immunopathologies in the lungs. Absence of mechanistic studies and co-infection models hinder the development of effective interventions to reduce the health impacts of SARS-CoV-2 and Mtb co-infection. Here, we highlight dysregulated immune responses induced by SARS-CoV-2 and Mtb, their potential interplay, and implications for co-infection in the lungs. As both pathogens master immunomodulation, we discuss relevant converging and diverging immune-related pathways underlying SARS-CoV-2 and Mtb co-infections.

Lytic Cell Death Mechanisms in Human Respiratory Syncytial Virus-Infected Macrophages: Roles of Pyroptosis and Necroptosis.

Human respiratory syncytial virus (RSV) is the most common cause of viral bronchiolitis and pneumonia in infants and children worldwide. Inflammation induced by RSV infection is responsible for its hallmark manifestation of bronchiolitis and pneumonia. The cellular debris created through lytic cell death of infected cells is a potent initiator of this inflammation. Macrophages are known to play a pivotal role in the early innate immune and inflammatory response to viral pathogens. However, the lytic cell death mechanisms associated with RSV infection in macrophages remains unknown. Two distinct mechanisms involved in lytic cell death are pyroptosis and necroptosis. Our studies revealed that RSV induces lytic cell death in macrophages via both of these mechanisms, specifically through the ASC (Apoptosis-associated speck like protein containing a caspase recruitment domain)-NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome activation of both caspase-1 dependent pyroptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), as well as a mixed lineage kinase domain like pseudokinase (MLKL)-dependent necroptosis. In addition, we demonstrated an important role of reactive oxygen species (ROS) during lytic cell death of RSV-infected macrophages.