Propagation and Titration of Influenza Viruses.

Influenza viruses are constantly circulating among humans, in which they cause seasonal epidemics of severe respiratory disease. Additionally, these zoonotic viruses infect different mammals and birds, from which new antigenic variants are occasionally transmitted to humans leading to devastating global pandemics. Surveillance programs, in which viruses from the main reservoir (waterfowl), intermediate hosts (like pigs and other farm animals), and other affected species are isolated and characterized, are crucial for the global influenza prevention strategy. This chapter gives an overview of the most commonly used methods for the propagation and titration of influenza viruses, which are key steps in surveillance procedures, as well as in vaccine development and basic research. Depending on the host and the viral strain, primary isolates are obtained from biological samples of different origin and subsequently amplified in embryonated chicken eggs or cell cultures. These propagation procedures are the focus of the first part of this chapter. Once the initial isolates have been amplified, virus titration methods based on particular characteristics of influenza viruses, such as their ability to agglutinate red blood cells (RBCs) or to induce cytopathic effects (CPE) in cell monolayers, are used to estimate the amount of viral particles. Such approaches, like the hemagglutination assay (HA assay), 50% tissue culture infectious dose (TCID50), or plaque assay, are included in the second part of this chapter. Although they are simple and cost-effective, some of these techniques have been partially replaced by faster and more sensitive methods based on the quantification of viral genomes, such as the quantitative real-time reverse transcription PCR (RT-qPCR), which is presented at the end of this section. The different protocols are explained in detail in order to facilitate the preparation and quantification of infectious virus stocks.

MxB is an interferon-induced restriction factor of human herpesviruses.

The type I interferon (IFN) system plays an important role in controlling herpesvirus infections, but it is unclear which IFN-mediated effectors interfere with herpesvirus replication. Here we report that human myxovirus resistance protein B (MxB, also designated Mx2) is a potent human herpesvirus restriction factor in the context of IFN. We demonstrate that ectopic MxB expression restricts a range of herpesviruses from the Alphaherpesvirinae and Gammaherpesvirinae, including herpes simplex virus 1 and 2 (HSV-1 and HSV-2), and Kaposi's sarcoma-associated herpesvirus (KSHV). MxB restriction of HSV-1 and HSV-2 requires GTPase function, in contrast to restriction of lentiviruses. MxB inhibits the delivery of incoming HSV-1 DNA to the nucleus and the appearance of empty capsids, but not the capsid delivery to the cytoplasm or tegument dissociation from the capsid. Our study identifies MxB as a potent pan-herpesvirus restriction factor which blocks the uncoating of viral DNA from the incoming viral capsid.