Enhancing the traceability of infectious clones via the incorporation of sequence signatures in order to reduce biosafety risk

Seamless cloning leaves no trace of ligation, and is often used for constructing infectious clones (ICs), which are used to express synthetic viruses. The resulting lack of a signature of sequence modification inhibits the tracing of IC viruses generated using seamless cloning, in the event of escape of the virus. This has significant biosafety costs as it ameliorates corrective measures and fails to disincentivize future escapes. Here, it is discussed whether seamless cloning is necessary for the construction of ICs. A key argument used to justify its use is the need to preserve the biological properties of the virus by maintaining synonymous sites. However, it is argued that the alteration of individual synonymous sites is unlikely to lead to a significant reduction in translational optimization, and that disruption of RNA structure, while a possibility, is low. However, avoiding the use of seamless cloning would not necessarily enhance traceability, given that naturally occurring restriction sites can often be used to construct an IC instead, also avoiding sequence modification. Given these considerations, it is proposed that a short non-disruptive signature sequence be incorporated into the IC virus genome, to ensure traceability. The sequence may be cryptographically signed allowing attribution to the originating lab. Experimental validation of candidate insertion sites, such as upstream of the poly(A) tail in RNA viruses, would address concerns over potential alteration of the virus biology resulting from the insert. While the stability of the insert is unlikely to be problematic given low indel frequencies in RNA and DNA viruses, it can be characterized empirically using serial passaging and evolutionary modelling. Consequently, the proposal represents a cheap and straightforward method of enhancing the biosafety of ICs.