Issue #113: The persistence of SAR-CoV-2 and Long COVID, 1 - Looking for infectious virus

Written by Nobel Laureate Professor Peter Doherty

A key question with Long COVID (LC) is, of course, whether the SARS-CoV-2 virus, or some components of this virus, are persisting in us. Does Long COVID (LC) reflect a continuing immune response, with all the deleterious consequences for ‘feeling unwell’ that the constant production of ‘immune effector molecules’ (like cytokines and chemokines) that spill over into the blood and circulate to the brain can entail (#110)?

 Over the past two weeks (#111, #112) we’ve looked at this question for the human herpesviruses (HHVs) that normally establish latency and/or ‘grumbling’ low-level infections in us. Though the HHVs can be transiently reactivated during COVID-19, there’s no obvious signal that this is a cause of LC. Furthermore, the type of immunosuppression (resulting from cancer therapy or monoclonal antibody treatment, #75-77) that can flush an HHV out of hiding to cause lethal disease does not reveal any ‘cryptic’ reservoir of SARS-CoV-1 or SARS-CoV-2 in those who recovered from SARS or COVID-19.

Similarly, we discussed the RNA retroviruses that carry a reverse transcriptase (RT) and integrase (INT) to copy their genetic information back into our genomes. But the CoVs do not share this RT/INT strategy. Unlike the situation for HIV and HTLV1 (#112), there should be no ‘hidden code’ to make new CoV virions if immune control is withdrawn.

So, is SARS-CoV-2 persisting in us, either as infectious virus (like the JCV DNA virus that continues to grow in uroepithelium, #112), or as individual viral components? If it is persisting, how does that work, both from the aspect of molecular strategy and a cause of LC symptoms? That’s where we’ll go over the next few weeks. This discussion will be developed systematically in the context of the types of approaches that scientists use to probe the question of possible virus persistence. The most dramatic demonstration would, of course, be the isolation of infectious virus from this or that anatomical site.

The benchmark test for infectious virus is to take, say, mucosal fluid from the nose or throat of a person with symptoms of COVID-19 and overlay that material on a monolayer of susceptible cells (VERO cell line) growing on the plastic ‘floor’ of a tissue culture ‘well’ (they come as individual plates with 6 to 96 wells).  The aim is to see if there is any virus present that can infect, multiply and cause a ‘cytopathic effect’(CPE) in the underlying VERO cells. The surrounding tissue culture fluid (‘medium’) contains antibiotics to limit bacterial contamination, and the sample may first have been centrifuged to remove some of the ‘gunk’. Using a syringe, it might also have been pushed through a tiny filter to get rid of any larger bacteria. This was ‘state of the art’ virology more than 50 years ago!

Virus isolation approaches may also use ground up (or dissociated) tissue samples, which would require a biopsy or be obtained at post-mortem in humans. One problem here is that a given individual may be simultaneously producing both the infectious virus, and antibodies (immunoglobulins, Igs) to that virus. If both are present in our sample and the two get together, then the CoV may be neutralised by Igs blocking the receptor binding domain (RBD) of the SARS-CoV-2 spike (#22). That would give us a false negative. A way around this is to take, say, epithelial cells obtained from a ‘vigorously acquired’ nasal swab - or a dissociated (with trypsin enzyme) tissue sample - then, ‘drop’ the (possibly) infected cells onto our uninfected VERO monolayers. This ‘co-cultivation’ can allow viruses to go from cell-to-cell without any exposure to Igs in the surrounding fluid phase.  Potentially, this could also be a way of maintaining an active infection within an otherwise immune individual. Normally, though, we would expect that our emerging ‘killer T cells (#33, #34) should recognize and eliminate any such infected cells in our bodies.

Traditionally, virologists screened by eye for any virus-induced CPE using an ‘inverted microscope’ where the objective lens is below the tissue culture plate and the observer looks up at the infected cells. Now, though, a more automated strategy could be to do sequential PCRs on small samples of the overlying tissue culture medium.  A day-to-day increase in the amount of viral RNA detected is likely to be reflective of virus production. Another approach is to take the tissue culture fluid, spin it down in a high-speed centrifuge, and look for virus particles in the deposited material using an electron microscope. Using these techniques, infectious virus has been found as late as 37 days after the initial development of symptoms in an otherwise recovered person.

This type of work is time consuming, expensive and is not done routinely in any survey or diagnostic situation. The top reference laboratories will make the effort to isolate any new virus variant (determined by gene sequencing) that comes along, so that it is available for virus neutralisation tests in cell culture, or animal challenge experiments for vaccine or drug development. Thus, as we continue to survey published data on the persistence of SARS-CoV-2 in people, we’ll be looking mainly at information generated from RNA and/or protein detection methods.

To be continued…