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Quantitative real-time PCR (qPCR) is a fast and reliable method to quantify viral genomes as a surrogate to titering on monolayers of cells for measuring virus replication. Whether it be for determining the number of virions released, the total number of genomes produced during infection, or the number of virions bound to a cell, qPCR assays can be adapted to quickly enumerate total viral genomes in a broad range of experiments comparing virus replication under different conditions. In addition, qPCR offers several advantages compared to plaque assays including time, linearity over 9 logs, and scalability from tens-to-hundreds of samples, depending on the qPCR machine. Here we describe a qPCR assay for quantifying vaccinia virus' dsDNA genome that can be used to determine the total number of virions produced. Furthermore, we describe a straightforward protocol for a cell-binding assay that is sensitive enough to use with small concentrations of inoculating virions. This protocol is suitable for measuring the cell-binding ability of mutations that affect virus production and infectivity., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Poxviruses represent evolutionary successful infectious agents. As a family, poxviruses can infect a wide variety of species including humans, fish, and insects. While many other viruses are species-specific, an individual poxvirus species is often capable of infecting diverse hosts and cell types. For example, the prototypical poxvirus, vaccinia, is well known to infect numerous human cell types but can also infect cells from divergent hosts like frog neurons. Notably, poxvirus infections result in both detrimental human and animal diseases. The most infamous disease linked to a poxvirus is smallpox caused by variola virus. Poxviruses are large double-stranded DNA viruses, which uniquely replicate in the cytoplasm of cells. The model poxvirus genome encodes ~200 nonoverlapping protein-coding open reading frames (ORFs). Poxvirus gene products impact various biological processes like the production of virus particles, the host range of infectivity, and disease pathogenesis. In addition, poxviruses and their gene products have biomedical application with several species commonly engineered for use as vaccines and oncolytic virotherapy. Nevertheless, we still have an incomplete understanding of the functions associated with many poxvirus genes. In this chapter, we outline evolutionary insights that can complement ongoing studies of poxvirus gene functions and biology, which may serve to elucidate new molecular activities linked to this biomedically relevant class of viruses., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A modernized version of an old saying goes that "If a picture is worth a thousand words, then a video is worth a million." Although made with reference to "YouTube", the quotation also has relevance for microbiologists when one considers how modern microscopes can be used to track biological fluorophores for hours without bleaching or phototoxicity. Confocal fluorescence microscopy provides a powerful tool for capturing dynamic processes within a cellular context that are better understood when viewed using time-lapse videos. In our laboratory we have long been interested in the links between poxvirus DNA replication and recombination and, since these are cytoplasmic viruses, such DNA-dependent processes are easily imaged throughout the virus life cycle without interference from signals coming from nuclear DNA. In this chapter we outline methods that can be used to follow the movement and replication of vaccinia virus DNA, and to also detect the products of poxvirus-catalyzed recombination reactions. We describe how to use the bacteriophage lambda DNA-binding protein, cro, as a way of labeling DNA within a cell when it is conjugated to fluorescent proteins. When used in conjunction with other fluorescent reagents, new labeling technologies, and tagged reporter constructs, these approaches can generate visually appealing and highly informative insights into diverse aspects of vaccinia virus biology.

Poxviruses cause many diseases in humans and animals worldwide, and there is a need for vaccines with improved safety and good efficacy. In addition, poxvirus vectors are widely used as recombinant vaccines for various infectious diseases and as recombinant and oncolytic vaccines for cancer. One concern with poxvirus vaccine vectors is that some poxviruses can infect a developing fetus and cause fetal loss or congenital disease. This can be an issue both for patients receiving a vaccine and for pregnant health care providers, including doctors, nurses, and veterinarians, who might receive accidental exposure to the poxvirus by injection or during patient care. We describe here a method for analyzing the safety of virus exposure in pregnant mammals using a mouse model testing vaccinia, canarypox, and raccoonpox virus vectors.

Antigen presentation to T lymphocytes is the seminal triggering event of the specific immune response, and poxviruses encode immunomodulatory genes that disrupt this process. Discovery of viral proteins that interfere with steps in the antigen presentation process requires a robust, easily manipulated antigen-presenting and T lymphocyte response system. Use of fresh primary antigen-presenting cells (APC) is preferable because cell lines that can present antigen in vitro are often not representative of APC in vivo and are typically weak stimulators. To study immunomodulatory poxvirus genes, we have used infected primary rat macrophages to present a model antigen, the myelin basic protein peptide, to a cognate CD4+ RsL11 T cell clone. Using this system, viruses can be assessed for difference in immunomodulation, and viral gene functions may also be assayed by comparing effects of wild type virus and mutant viruses (e.g., a deletion in the putative immunomodulatory gene). While antigen presentation can be thought of as a single event, it can also be considered as a larger process comprising multiple steps including: antigen acquisition, antigen processing, peptide loading onto MHC molecules, transport to the surface, MHC binding to T cell receptor, interaction of costimulatory molecules, cell signaling, cytokine synthesis by both cells, and proliferation of antigen specific T lymphocytes. This system allows for the initial determination of whether there is a phenotype and then also allows the stepwise deconstruction of the system to analyze this process at several points to focus in on the mechanism of immunomodulation. We have used this model system to elucidate the function of a highly conserved but previously uncharacterized poxvirus gene that we showed was important for virulence in rodents. The experimental system developed should be broadly applicable to analyzing viral effects on immunity.

Viral vector is the most effective means of gene transfer to modify specific cell type or tissue and can be manipulated to express therapeutic genes. Several virus types are currently being investigated for use to deliver genes to cells to provide either transient or permanent transgene expression. These include adenoviruses (Ads), retroviruses (γ-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses. The choice of virus for routine clinical use will depend on the efficiency of transgene expression, ease of production, safety, toxicity, and stability. This chapter provides an introductory overview of the general characteristics of viral vectors commonly used in gene transfer and their advantages and disadvantages for gene therapy use.

Poxviruses including canarypox (ALVAC) and vaccinia viruses have, in recent years, received considerable attention as live vectors for the development of vaccines against infectious diseases such as AIDS, malaria, and tuberculosis. However, the cellular targets for viral infection within the human immune system and the consequences of infection for cells involved in the generation of immune responses have not been clearly delineated. Using recombinant enhanced green fluorescence protein (EGFP)-expressing ALVAC and vaccinia viruses, we have focused here on a side-by-side comparison of ALVAC and vaccinia virus tropism for cells from human peripheral blood and bone marrow. Both ALVAC and vaccinia viruses showed a strong bias toward monocyte infection. ALVAC minimally infected CD19(+) B cells and was unable to infect ex vivo NK cells and T lymphocytes, whereas vaccinia virus could infect B lymphocytes and NK cell populations. Vaccinia virus was also able to infect T lymphocytes at low but detectable levels which could be enhanced upon their activation. Both ALVAC and vaccinia viruses could infect immature monocyte-derived dendritic cells (MDDCs), but only ALVAC infection induced their subsequent maturation. Infection in human bone marrow cells showed that ALVAC infection was restricted to a myelomonocytoid cell-specific CD33(+) cell population, while vaccinia virus showed a strong, but not exclusive, preference for these cells.

Cytoplasmic replication of poxviruses dictates the encoding of most, if not all, of the trans-acting factors required for faithful genome duplication. Several of these proteins have been identified through genetic and biochemical evaluation, including the catalytic DNA polymerase (E9), an essential and stoichiometric component of the processive polymerase (A20), a single-strand DNA-binding protein (I3), a type I topoisomerase (H6), the uracil DNA glycosylase (D4), a nucleic acid-independent nucleoside triphosphatase (D5), a serine/threonine protein kinase (B1), and a Holliday Junction resolvase (A22). All of these factors work in concert to faithfully duplicate the viral genome. Although a replication origin has not been defined for the poxviruses, cis-acting sequences found within the telomeric 200 bp have been implicated as necessary and sufficient for minichromosome replication. Replication occurs within cytoplasmic foci from approx 3 to 12 h postinfection. This chapter includes several methodologies to assay and quantitate replication in vivo, visualize replication foci microscopically, and test the integrity of central replication enzymes in vitro.

Poxviruses are cell-associated viruses that can be grown in adherent- or suspension-cell cultures or chorioallantoic membranes of embryonated hen's eggs. The main principle of isolating the virus is to mechanically lyse infected cells. The virus can then be semipurified by centrifugation through a sucrose cushion. Further purification can be achieved with a sucrose gradient. This chapter describes methods for the preparation of large-scale growth and purification of the virus.