Given the endemic seroprevalence of herpes simplex viruses (HSV), its associated human diseases, and the emergence of acyclovir-resistant strains, there is a continuous need for better antiviral therapies. Towards this aim, identifying mechanistic details of how HSV-1 manipulates infected cells, how it modulates the immune responses, and how it causes diseases are essential. Measuring titers and growth kinetics of clinical isolates and viral mutants are important for a thorough characterization of viral phenotypes in vitro and in vivo. We provide protocols for the preparation as well as titration of HSV-1 stocks, and explain how to perform single-step growth curves to characterize the functions of viral proteins or host factors during infection. In particular, we describe methods to prepare and characterize high-titer HSV-1 stocks with low genome to titer ratios that are required for infection studies in cell culture and animal experiments.
Keywords: Herpes simplex virus type 1, Virus propagation, Plaque assay, Single-step growth curveHerpes simplex virus type 1 (HSV-1) is the prototypic genus of the human pathogenic subfamily Alphaherpesvirinae. Worldwide, it has a seroprevalence of approximately 67% that varies from 30% to more than 90% in different human groups, depending on the age, the socio-economic status, and the geographical region ( Nahmias et al., 1990 ; reviewed in Smith and Robinson, 2002; Looker et al., 2015 ). HSV-1 shares several features with all herpesviruses: (i) a conserved virion architecture, (ii) productive and lytic infection of cells and (iii) establishment of life-long-latent infection in the host (Whitley and Griffiths, 2002; Schleiss, 2009).
During acute infection, HSV-1 causes cutaneous and mucosal herpetic lesions, and in very young or immune-compromised individuals more severe sequelae, such as potentially blinding keratitis and life-threatening disseminated disease or encephalitis (Thompson and Whitley, 2011; Kennedy and Steiner, 2013). Despite a potent antiviral immune response inside the host, the immune system fails to clear the virus. This is achieved by HSV-1-mediated immune evasion mechanisms ( Theodoridis et al., 2011 ; Su et al., 2016 ; Zheng, 2018; Tognarelli et al., 2019 ), and the establishment of life-long latent infection in neurons of peripheral ganglia which are reached via retrograde axonal transport (Smith, 2012; Roizman and Whitley, 2013; Koyuncu et al., 2018 ). During latency, viral antigen expression in the neurons is restricted, but the virus sporadically reactivates upon lowered immune surveillance and systemic stress ( Padgett et al., 1998 ; Huang et al., 2011 ). Recurrent productive infections after reactivation comprise anterograde axonal spread, virus release from the neurons back to mucocutaneous sites, and reinfection of epithelial cells ( Enquist et al., 1998 ; Whitley and Roizman, 2001; Diefenbach et al., 2008 ; Smith, 2012; Koyuncu et al., 2018 ).
Here, we describe protocols to prepare HSV-1 stocks. We generate virus stocks by harvesting post-nuclear supernatants from repeatedly freeze-thawed infected cells, or by pelleting cell-free virions released from infected cells into the conditioned medium. For cell entry and animal experiments, we recommend purifying such preparations further on sedimentation density gradients ( Sathananthan et al., 1997 ; Sodeik et al., 1997 ; Döhner et al., 2006 ; Dai and Zhou, 2014).
Viral stocks will maintain and acquire genomic mutations, if they provide an evolutionary advantage and are not deleterious during virus amplification. To achieve a high genomic homogeneity, a passage 1 preparation from a single viral plaque or a single clone generated by mutagenesis should be generated. Subsequent passage numbers should be kept to a minimum (Harland and Brown, 1998). We characterize virus stocks by measuring the number of plaque-forming units (pfu) by plaque-titration (see Procedure B) and the number of viral genomes by real-time detection PCR ( Döhner et al., 2006 ; Engelmann et al., 2008 ). In addition, the particle to pfu ratio can be determined by electron microscopy (Harland and Brown, 1998; Döhner et al., 2006 ). To this end, virus stocks are mixed with latex beads of known concentration and appropriate heavy metal salts for negative contrast, and analyzed by electron microscopy. Viral particles and latex beads are counted, and using the known concentration of the latex beads as a reference, the number of viral particles can be determined. Low ratios of genome to pfu, total protein to pfu, or particle to pfu are indicative of a high-quality virus stock.
Since HSV-1 causes strong cytopathic effects, we use plaque assays to determine the number of infectious units within a given sample. Due to the high HSV-1 prevalence, it suffices to add a pooled fraction of human IgGs to the cell culture medium to prevent virus spread via the culture medium. Alternatively, an overlay with agarose or methylcellulose could also neutralize extracellular HSV-1 virions; however, such assays require a bit advanced experimental skills. The direct cell-to-cell spread of HSV-1 leads to the formation of macroscopic plaques, which indicates the occurrence of an infectious particle present in this sample.
Titration of virus stocks is not only crucial for subsequent in vitro or in vivo infection experiments, i.e., to use a defined amount of plaque forming units (pfu), but also during the analysis of viral loads in different tissues of infected animals, when comparing phenotypes of different viral mutants, or during the characterization of clinical isolates. Moreover, the impact of perturbing a host function by pharmacological inhibitors, gene silencing, genetic knock-out, or overexpression is determined by performing a time course of virus production.
The growth kinetics of viral mutants lacking essential protein functions are compared in non-complementing versus complementing cells, the latter expressing the ablated protein in trans ( Schipke et al., 2012 ; Sandbaumhüter et al., 2013 ). When analyzing the impact of particular host factors on virus propagation, viral yields on deficient cells obtained by RNAi or CRISPR/Cas9 technology or from knock-out animals are compared to yields on the corresponding wildtype cells ( Döhner et al., 2018 ).
Single-step or multi-step growth curves in susceptible cell lines provide a standardized method to compare different clinical isolates or viral mutants with the respective wildtype strain, or to determine the impact of a particular host factor during infection. In single-step growth curves, a high multiplicity of infection (MOI) of 5 to 20 pfu/cell ensures complete and simultaneous infection. We recommend monitoring virus propagation up to 24 hpi during one replication cycle (Harland and Brown, 1998). In contrast, multi-step growth curves amplify the effect of more subtle phenotypes during several cycles of replication (Harland and Brown, 1998). Thus, cells are infected using a low MOI of 0.01 to 0.1 pfu/cell for 3 days. While both methods involve the same procedures, we focus here on the experimental setup for a synchronous single-step infection.