Conducting Research on Highly Pathogenic Viruses Using Virus Pseudotypes: The Influence of Ultrapure Water on Data Quality

Conducting Research on Highly Pathogenic Viruses Using Virus Pseudotypes: The Influence of Ultrapure Water on Data Quality


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  • Source: SARTORIUS

  • Date: 09 Oct,2021

From 2013 to 2016, more than 11,000 people died of Ebola virus infections in West Africa. This most recent epidemic shows how dangerous so-called emerging viruses can be. Virus pseudotypes can be used to easily investigate the entry pathways of such viruses.
Novel viruses, termed emerging viruses, pose a threat to human health of a magnitude that should not be underestimated.

These viruses have acquired the ability to infect humans either as a consequence of interspecies transmission or of naturally occurring changes in the viral genome. Infections caused by emerging viruses often result in serious diseases or even death as the human immune system may not be capable of combatting an unfamiliar virus, especially of zoonotic origin. Various factors promote the occurrence and spread of emerging viruses. These include ecological factors, such as deforestation to gain new land for development, and also our lifestyle involving global mobility, as well as international commerce. Thus, the loss of habitat increases the probability that people, pets and farm animals will come in contact with wildlife species originally inhabiting isolated areas and therefore not previously encountered as natural hosts of emerging viruses.

Furthermore, the surrounding circumstances of a globalised society involving increased travel also enable dissemination of pathogenic viruses around the world, even before the onset of clinical symptoms. Within the last 100 years, multiple introductions of emerging viruses into the human population have occurred, which have led to local epidemics or worldwide outbreaks (pandemics; see Table 1).

In particular, the Ebola virus most recently raised concerns across the globe, as the West African Ebola virus epidemic, which claimed more than 11,000 lives, devastatingly demonstrated the danger posed by emerging viruses. Research involving emerging viruses is often limited to high-containment laboratories of biosafety levels (BSL) 3 and 4 (see Table 1). Working in BSL-4 laboratories is highly laborious, expensive and permitted only at a few locations so that rapid scientific progress in characterizing viral pathogens and developing antiviral drugs is difficult to achieve

Given this situation, virus pseudotypes offer an attractive option for studying the entry of highly pathogenic viruses into cells safely and efficiently.

This is possible as not the entire pathogen is analysed, but rather only its components that mediate host cell entry, the envelope proteins. These represent the key to virus entry into cells.

In the case of virus pseudotypes, envelope proteins of highly pathogenic viruses are incorporated into a carrier virus (pseudotyping), which cannot replicate autonomously, i.e., are replication deficient. Commonly used systems for pseudotyping are based on rhabdoviruses (e.g., vesicular stomatitis virus, VSV; Fig. 1) and retroviruses. The objective of this study was to verify whether the envelope protein-mediated entry of virus pseudotypes reflects host cell entry of intact viruses. In addition, this investigation was designed to determine which influence the level of purity of the reagents used, in this case laboratory water, has on the production of virus pseudotypes

It is known that the Middle East respiratory syndrome coronavirus (MERS-CoV, formerly called human coronavirus, EMC = hCoV-EMC), binds to the cell surface through the interaction between the viral spike glycoprotein (S) and the cellular membrane protein dipeptidyl peptidase 4 (DPP4), thus enabling cell entry [7]. To confirm whether this also applies in the context of VSV pseudotypes, target cells were transfected with an expression vector for DPP4 or an empty expression plasmid (no receptor). As expected, the directed expression of DPP4 led to a significant increase in the host cell entry of VSV pseudotypes if the pseudotypes had MERS-CoV S embedded in their envelope (see Fig. 2A). Influenza A viruses, the causative agents of influenza disease, require terminal sugar structures, so-called sialic acids occurring as natural modifications on cellular membrane glycoproteins and glycolipids, as receptors to mediate entry into target cells (see literature for further reading [8]). To study whether the incorporation of influenza A viral envelope proteins in VSV pseudotypes also results in sialic acid-dependent cell entry, VSV pseudotypes with H1N1 (1918) HA/NA were employed, and the sialic acids were enzymatically removed from the surfaces of target cells. As expected, removal of sialic acids resulted in a dramatic decrease in the host cell entry of VSV pseudotypes that had H1N1 (1918) HA/NA embedded in their envelope (see Fig. 2B). This finding confirms that virus pseudotypes reflect the entry mechanism of authentic influenza A viruses into cells.

The purity level of the laboratory water used influences the quality of VSV pseudotypes

After it could be shown in the previous trials that VSV pseudotypes are suitable models for studying the host cell entry of highly pathogenic viruses, the next issue to be clarified was the particular influence that the purity level of the laboratory water used has on the quality of VSV pseudotypes. During production of VSV pseudotypes, various buffers and solutions are employed that are all prepared with water. However, it must be noted that not just any type of water suffices for preparation of these reagents. Rather, the right choice of purity level must be made for laboratory use. To investigate whether the use of ultrapure water yields higher quality VSV pseudotypes, two batches of VSV pseudotypes were generated in a parallel experiment: One batch was prepared using demineralised water from a central lab water supply source (conductivity of 3.7 – 4.1 μS/cm at 19°C) as the basic solvent for all solutions and buffers, while a further batch utilising solutions and buffers based on Arium® Pro VF ultrapure water (conductivity 0.055 μS/cm compensated to 25°C) was generated.

EBOV GP and MERS-CoV S as envelope proteins were examined. Following parallel production of VSV pseudotypes by maintaining identical incubation conditions, the target cells were inoculated and the envelope protein-mediated entry of VSV pseudotypes was quantified. This experimental setup demonstrated that host entry (as a parameter for the degree of quality) of the VSV pseudotypes produced using Arium® Pro VF ultrapure water as the basic solvent for all buffers and solutions was significantly higher compared with that of pseudotypes for which demineralised water was employed in their production.


In conclusion, it can be stated that virus pseudotypes are important tools for investigating host cell entry of highly pathogenic viruses. As these virus pseudotypes do not restrict research on such highly pathogenic viruses to BLS-3 or BLS-4 laboratories, this enables a larger number of scientific facilities to conduct such research. As a result, the cell entry of emerging viruses can be characterised and suitable detection procedures and antiviral strategies (medications, vaccines) can be developed faster.

The use of pseudotypes also reduces the considerable labour intensity involved in high-containment laboratories requiring whole-body protection suits, as well as the considerable costs and the limitations entailed by such lab work (e.g., no access to equipment that is not directly located inside the high-containment laboratory). Furthermore, compared with authentic, highly pathogenic viruses, virus pseudotypes minimise the risk of infection of lab personnel following unintentional exposure, thus representing a significant safety aspect. Optimisation of the production process, for example, through the use of highly pure reagents, particularly those based on water, can additionally contribute to improving the sensitivity of subsequent test procedures, increasing production quantities and thus further lowering production costs

This application note was first published by Sartorius- click here for the full application note and references.  Find out more on the importance of the quality of ultrapure water, among other factors, for the production of such virus pseudotypes.

Other Covid 19 related resources- Sartorius Covid 19 Resource page


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