Viruses have become a regular part of basic biological research as well as clinical therapy. These biological tools are useful because they’re derived from viruses that can infect people, cells, and animals. Some of these viruses are completely inert, but others can cause diseases. Nervous about handling viruses? That’s okay! In this article we will discuss general viral safety considerations and best practices for handling lab-grown viruses as well as potential risks and how to minimize them.
Built-in safety features
When viruses were first being engineered as biological tools, thought was given on how to keep the replication of virus and its infectivity in check. Engineering controls, safety systems that are built into how something functions, were designed for this purpose. One such control is dividing up the essential components of a virus into different plasmids. For a retroviral system this looks like the genetic cargo in one plasmid (the transfer plasmid), the envelope (Env) gene on a separate plasmid (envelope plasmid), and the polymerase (Pol) for replication and Gag structural protein on yet a third plasmid (packaging plasmid). Viral production then proceeds by transfection of all three plasmids into a producer cell line, such as HEK-293T, production of viral particles, and transduction of a target cell line with those particles. Since the Env, Gag, and Pol genes are not encoded in the transfer plasmid, the component that physically integrates into the host genome, the virus is replication incompetent, meaning it can’t replicate and continue the infection cycle once inside the host. While the virus can still enter (and thus infect) cells, this safety feature prevents the virus from replicating and spreading in non-target cells, including yourself as the researcher.
 |
| Figure 1: Separation of retroviral packaging components onto distinct plasmids for safety. |
Viral cargo-specific safety
After replication competency, the second concern with virus safety is the disease-risk of what is being integrated by the virus. If an oncogene overexpression construct is being delivered, that is a higher risk cargo for a researcher to come into contact with than GFP. Similarly, if the viral vector carries an shRNA to a gene essential for cellular function, this would also be a higher risk vector. While it is unlikely that you, or anyone else in the lab or community would be infected by a lab-grown virus, it's important to understand the risks of the specific virus you’re working with before you work with it. This will help you understand what safety precautions to take, and if something does happen, ensure you can receive appropriate treatment and/or monitoring.
Virus-specific concerns
In nature, some viruses are more dangerous than others, and the same holds true for types of viruses generated in the lab. The four most common types of viruses grown in the lab are gamma retrovirus, lentivirus, adenovirus, and adeno-associated virus (AAV). The least risky of these four viruses is AAV – it is not known to cause any diseases in humans and does not always integrate into the host genome as part of the viral infection process (though it can integrate at chromosome 19q13.4 qtr. (AAVS1)). For these reasons, AAV is usually classified as a BSL-1 safety level. Adenovirus is similar to AAV in that it does not integrate into the host’s genome. However, some adenovirus strains are known to cause respiratory diseases in humans, earning this virus a BSL-2 classification. Lentivirus (a type of retrovirus) and gamma retrovirus require more caution as they are integrating viruses – they physically deposit their cargo into the host’s DNA. These viruses also have a propensity to integrate into proto-oncogenes, which can lead to cancer. These viruses need to be handled in a BSL-2 (or BSL-2+) lab and require additional safety measures.
General viral handling safety
Containment and personal protection are key to lab safety and the same principles apply to viral handling. Gloves are always required and lab coats are either recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus, many organizations require or recommend disposable gowns over your lab coats and double gloving, so that, if needed, a layer can quickly be removed and disposed of without risking exposure. You can request disposable gowns from your lab or health and safety department if they are not currently provided.
Eye protection or face shields are required whenever there’s a splash or spray risk in BSL-1. In BSL-2 labs, eye protection is required any time you have to work with BSL-2 materials, such as lentivirus. Most of this work will need to be done in a biosafety cabinet. Many researchers choose to wear eye protection when they are working in a biosafety cabinet, even if it is not required by their organization. It’s always okay to add another level of protection!
When centrifuging viruses (a necessary step during production), there is a significant risk for aerosol contamination. As most centrifugation has to be done outside of a biosafety cabinet, you’ll need to take extra steps to ensure that particles are not sprayed into the air. For lentivirus and other BSL-2 materials, you’ll need to use aerosol-tight caps, compatible with your centrifugation speed, to cover the centrifuge bins. These have to be added and removed in a biosafety cabinet. For ultracentrifugation, heat sealed tubes can be used for extra security. Finally, when spinning down lentivirus, you’ll need to use centrifuge bin covers, which you should open in the biosafety cabinet and decontaminate after use.
Virus-exposed plastics and other surfaces should be thoroughly decontaminated after use, typically with bleach (plastic) or ethanol (other surfaces). For AAVs, which are resistant to ethanol and isopropyl alcohol, 0.5% peracetic acid, 10% bleach, or 1% iodine are useful. Some organizations or cities may require a higher percentage of the decontaminate to be used for BSL-2 waste, and of course, if you are working with a substance in a biosafety cabinet, it needs to be decontaminated in a biosafety cabinet. When decontaminating liquid waste, remember that the percentage indicates the proportion of the decontaminate in the waste itself, not in the solution that should be added. After decontamination, single-use plastics and other handling tools should then be disposed of following your lab’s biohazard policies.
Administering Virus
Many virus experiments involve needles or scalpels (sharps). Cuts and scrapes from sharp equipment are one of the most common forms of lab accidents, so extra precaution should be used to reduce the risk of exposing yourself via needlestick or cut. Following all sharps procedures for your organization will significantly reduce the risk of an accident (the most common reason for lab accidents is not following safety protocols.) If administering virus to mice, part of your preparation should be doing your best to be well-rested and alert the day of the procedure. Mice move much more quickly than cell cultures do, and tired researchers are accident-prone researchers.
Note that safety policies will vary slightly from organization to organization, so you’ll need to be familiar with your specific organization/department/lab’s policies – basically, you’ll need to get trained in each new place (and pay attention to the trainings!), even if you know guidelines for best practices and have been trained elsewhere. You’ll also need to familiarize yourself with your organization’s reporting mechanisms before you start an experiment, just in case something does happen.
Finally, the best way to be safe is to know and closely follow safety protocols. This is especially true when you’re tired, in a rush, or having an ‘off’ day – you are both more likely to make a mistake and more likely to want to cut a few corners.
For many researchers, viruses might be the most dangerous things they work with, and that can understandably induce some nerves. Following safety procedures and practicing safety culture, however, can let you work with these valuable tools without endangering yourself or others. Happy transducing!
Resources
Resources on Addgene.org
- Addgene viral services
- Addgene Biosafety Guide
- Addgene Viral Plasmids
- Which Type of Virus Should I Use?
Resources on the Addgene blog
Topics: Viral Vectors
Leave a Comment