Pseudotypes: your flexible friends

Pseudotype viruses: applications and troubleshooting' (EuroSciCon 2013), a 1-day conference held at Cineworld: The O2 (London, UK) on 2 October 2013, focused on the technique of pseudotyping enveloped viruses (for a review of the technique, see [1]). The talks and posters covered the challenges and successes of pseudotyping viruses from a broad range of families (Retroviridae, Flaviviridae, Orthomyxoviridae, Bunyaviridae and Rhabdoviridae) for a variety of applications. The conference was chaired by Nigel Temperton, University of Kent (UK), who placed a strong emphasis on using this event to explore the technical challenges of pseudotyping viruses, especially during the poster and afternoon question and answer sessions

Seroprevalence of Schmallenberg virus in the United Kingdom and Republic of Ireland: 2011-2013

Since its identification in late 2011, Schmallenberg virus (SBV) spread rapidly across Europe. Using archived samples from domestic ruminants collected between October 2011 and June 2013, the seroprevalence in the United Kingdom (UK) and Republic of Ireland (IE) was estimated using a serum neutralisation test. There was no significant difference (P > 0.05) in seroprevalence between sheep and cows suggesting that neither species is significantly more at risk of SBV infection in the UK. A single 2011 sample tested positive; the sample was taken in November from a cow in Wiltshire. There was a steady increase in overall seroprevalence during the first three quarters of 2012, which then more than doubled in quarter 4 (October–December), which may reflect a peak of vector activity. By the end of June 2013, overall seroprevalence was around 72%. However, although seroprevalence was over 50% in Wales and southern and central counties of England, it was below 50% in all other areas of the UK and IE. This suggests that there were still substantial numbers of animals at risk of infection in the latter half of 2013

How have retrovirus pseudotypes contributed to our understanding of viral entry?

Study of virus entry into host cells is important for understanding viral tropism and pathogenesis. Studying the entry of in vitro cultured viruses is not always practicable. Study of highly pathogenic viruses, viruses that do not grow in culture, and viruses that rapidly change phenotype in vitro can all benefit from alternative models of entry. Retrovirus particles can be engineered to display the envelope proteins of heterologous enveloped viruses. This approach, broadly termed ‘pseudotyping’, is an important technique for interrogating virus entry. In this perspective we consider how retrovirus pseudotypes have addressed these challenges and improved our understanding of the entry pathways of diverse virus species, including Ebolavirus, human immunodeficiency virus and hepatitis C virus

How have retrovirus pseudotypes contributed to our understanding of viral entry?

Study of virus entry into host cells is important for understanding viral tropism and pathogenesis. Studying the entry of in vitro cultured viruses is not always practicable. Study of highly pathogenic viruses, viruses that do not grow in culture, and viruses that rapidly change phenotype in vitro can all benefit from alternative models of entry. Retrovirus particles can be engineered to display the envelope proteins of heterologous enveloped viruses. This approach, broadly termed ‘pseudotyping’, is an important technique for interrogating virus entry. In this perspective we consider how retrovirus pseudotypes have addressed these challenges and improved our understanding of the entry pathways of diverse virus species, including Ebolavirus, human immunodeficiency virus and hepatitis C virus

Troubleshooting methods for the generation of novel pseudotyped viruses

A pseudotyped virus (PV) is a virus particle with an envelope protein originating from a different virus. The ability to dictate which envelope proteins are expressed on the surface has made pseudotyping an important tool for basic virological studies such as determining the cellular targets of the envelope protein of the virus as well as identification of potential antiviral compounds and measuring specific antibody responses. In this review, we describe the common methodologies employed to generate PVs, with a focus on approaches to improve the efficacy of PV generation

Troubleshooting methods for the generation of novel pseudotyped viruses

A pseudotyped virus (PV) is a virus particle with an envelope protein originating from a different virus. The ability to dictate which envelope proteins are expressed on the surface has made pseudotyping an important tool for basic virological studies such as determining the cellular targets of the envelope protein of the virus as well as identification of potential antiviral compounds and measuring specific antibody responses. In this review, we describe the common methodologies employed to generate PVs, with a focus on approaches to improve the efficacy of PV generation

Novel functional hepatitis C virus glycoprotein isolates identified using an optimised viral pseudotype entry assay

Retrovirus pseudotypes are a highly tractable model used to study the entry pathways of enveloped viruses. This model has been extensively applied to the study of the hepatitis C virus (HCV) entry pathway, pre-clinical screening of antiviral antibodies and for assessing the phenotype of patient-derived viruses using HCV pseudoparticles (HCVpp) possessing the HCV E1 and E2 glycoproteins. However, not all patient-isolated clones produce particles that are infectious in this model. This study investigated factors that might limit phenotyping of patient-isolated HCV glycoproteins. Genetically related HCV glycoproteins from individual patient quasispecies were discovered to behave very differently in this entry model. Empirical optimisation of the ratio of packaging construct and glycoprotein-encoding plasmid was required for successful HCVpp genesis for different clones. The selection of retroviral packaging construct also influenced the function of HCV pseudoparticles. Some glycoprotein constructs tolerated a wide range of assay parameters, while others were much more sensitive to alterations. Furthermore, glycoproteins previously characterised as unable to mediate entry were found to be functional. These findings were validated using chimeric cell-cultured HCV bearing these glycoproteins. Using the same empirical approach we demonstrated that generation of infectious ebolavirus pseudoviruses (EBOVpv) were also sensitive to the amount, and ratio, of plasmids used, and that protocols for optimal production of these pseudoviruses is dependent on the exact virus glycoprotein construct. These findings demonstrate that it is crucial for studies utilising pseudoviruses to conduct empirical optimisation of pseudotype production for each specific glycoprotein sequence to achieve optimal titres and facilitate accurate phenotyping

Flexible and rapid construction of viral chimeras applied to Hepatitis C Virus

A novel and broadly applicable strategy combining site directed mutagenesis and DNA assembly for constructing seamless viral chimeras is described using Hepatitis C Virus as an exemplar. Full-length HCV genomic cloning cassettes, which contained flexibly situated restriction endonuclease sites, were prepared via a single site-directed mutagenesis reaction and digested to receive PCR amplified virus envelope genes by In-Fusion cloning. Using this method we were able to construct gene-shuttle cassettes for generation of cell culture-infectious JFH-1-based chimeras containing genotype 1-3 E1E2 genes. Importantly, using this method we also show that E1E2 clones that were not able to support cell entry in the HCV pseudoparticle assay did confer entry when shuttled into the chimeric cell culture chimera system. This method can be easily applied to other genes of study and other viruses and, as such, will greatly simplify reverse genetics studies of variable viruses

Expression of human ficolin-2 in hepatocytes confers resistance to infection by diverse hepatotropic viruses

The liver-expressed pattern recognition receptors (PRRs) mannose binding lectin (MBL), ficolin-2 and ficolin-3 contribute to the innate immune response by activating complement. Binding of soluble ficolin-2 to viral pathogens can directly neutralize virus entry. We observed that the human hepatoma cell line HuH7.5, which is routinely used for the study of hepatotropic viruses, is deficient in expression of MBL, ficolin-2 and ficolin-3. We generated a cell line that expressed and secreted ficolin-2. This cell line (HuH7.5 [FCN2]) was more resistant to infection with hepatitis C virus (HCV), ebolavirus (EBOV) and vesicular stomatitis virus (VSV), but surprisingly was more susceptible to infection with rabies virus (RABV). Cell-to-cell spread of HCV was also inhibited in ficolin-2 expressing cells. This illustrates that ficolin-2 expression in hepatocytes contributes to innate resistance to virus infection, but some viruses might utilise ficolin-2 to facilitate entry

Troubleshooting methods for the generation of novel pseudotyped viruses

A pseudotyped virus (PV) is a virus particle with an envelope protein originating from a different virus. The ability to dictate which envelope proteins are expressed on the surface has made pseudotyping an important tool for basic virological studies such as determining the cellular targets of the envelope protein of the virus as well as identification of potential antiviral compounds and measuring specific antibody responses. In this review, we describe the common methodologies employed to generate PVs, with a focus on approaches to improve the efficacy of PV generation