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About viruses and viral disease

reactivation

Why do Some People Develop Long COVID?

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by Gertrud U. Rey

Long COVID is a chronic manifestation of SARS-CoV-2 infection, and it is most commonly characterized by lingering fatigue, brain fog, memory impairment, and confusion. Although it is unclear how the viral infection leads to long COVID, experts speculate that one or more of the following factors may contribute: an inability to successfully clear virus, a reactivation of latent viruses, a disturbance of the gut microbiome, continuing inflammation, and/or autoimmunity.

Yale University researcher Akiko Iwasaki and colleagues recently explored some of these hypotheses in an attempt to identify diagnostic biomarkers associated with long COVID. The study involved four groups of participants, with the experimental group consisting of both vaccinated and unvaccinated individuals with long COVID. The other three groups served as three separate types of controls:

  1. healthy, vaccinated, uninfected individuals;
  2. healthy, unvaccinated, previously infected individuals without long COVID; and
  3. healthy, vaccinated, previously infected individuals without long COVID.

The investigators obtained blood samples from all participants and analyzed the samples for the presence of specific immune cells. They found that compared to control groups, long COVID participants had lower levels of conventional dendritic cells and memory T helper cells. Conventional dendritic cells typically activate cytotoxic T cells, which in turn kill infected cells. Memory T helper cells are a central element of the adaptive immune response, where they help orchestrate downstream immune functions upon recognition of antigen. Long COVID participants also had increased numbers of “exhausted” T cells, which are no longer functional or capable of eliminating virus. These results suggested that people with long COVID may have insufficient numbers of immune cells able to inactivate virus, thus allowing viruses to linger and continue replicating and circulating. Whether this assumption is true, and whether long COVID participants do indeed have persistently circulating virus, is subject to ongoing analysis.

Previous studies have shown that patients with severe COVID-19 can have increased levels of functional antibodies directed against self antigens that circulate outside of cells (i.e., extracellular autoantibodies), suggesting that SARS-CoV-2 infection can cause autoimmune disorders. To identify a possible connection between autoimmunity and long COVID, the authors screened the collected blood samples for autoantibodies using a technique called Rapid Extracellular Antigen Profiling (REAP). Among other applications, REAP allows one to assess antibody reactivity against a panel of extracellular human proteins known to contribute to well-studied autoimmune disorders. Interestingly, long COVID participants did not have increased levels of autoantibodies compared to control groups, suggesting that the autoantibodies detected in patients with severe COVID-19 may only be present during the acute phase of disease. However, REAP only assesses antibodies directed to extracellular self proteins and does not provide any information regarding intracellular or non-protein-specific autoantibodies. Therefore, one cannot rule out a role for autoimmunity in long COVID.  

The authors also used REAP to detect antibodies against various viruses. They found that long COVID participants had antibodies against several other viruses in addition to SARS-CoV-2, including Epstein-Barr virus (EBV), a herpesvirus that is well known for causing infectious mononucleosis (i.e., “mono”), a condition that is often compared to encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID. However, because most adults have antibodies against EBV and there was no significant difference in the percentage of EBV antibody-positive participants between experimental and control groups, it is unlikely that a positive EBV antibody status contributes to the risk of developing long COVID.

The most interesting observation in this study was that compared to control groups, long COVID participants had about 50% lower levels of the steroid hormone cortisol. Because cortisol is a potent anti-inflammatory agent, it is feasible that a shortage of cortisol would lead to persistent inflammation and the long-term tissue damage associated with inflammation. In line with this reasoning, cortisol levels were highest in healthy, vaccinated, uninfected controls (group 1 controls); lower in healthy, unvaccinated, previously infected individuals without persistent symptoms (group 2 controls); and lowest in long COVID participants. Using machine learning methods, the authors determined that cortisol deficiency was the single most significant predictor of a long COVID diagnosis. Low cortisol has also been implicated in ME/CFS, and treatment with hydrocortisone can provide some relief from symptoms. 

The authors are careful to note that the small sample size of 215 participants is a considerable limitation of this study. Nevertheless, the results offer some valuable insight that may apply to other chronic conditions. In contrast to public perception, long-term symptoms following an acute viral infection are not unique to COVID-19. Unexplained chronic syndromes involving similar symptoms to long COVID have also been reported after dengue virus, poliovirus, SARS-CoV, Chikungunya virus, West Nile virus, Ross River virus, Coxsackie virus, and influenza virus infections. Because these syndromes are associated with high public health and economic burdens, more work needs to be done to clarify their underlying mechanisms.  

CRISPR-ing herpes simplex virus

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herpesvirus latencyby Gertrud U. Rey

Herpes simplex viruses establish lifelong persistent infection in sensory neurons of infected individuals, a phenomenon called latency. Latent viral genomes are “dormant” but can sporadically reactivate and begin replicating in a phase called lytic replication, which is often accompanied by shedding of virus particles and the appearance of painful lesions. There is no vaccine to prevent infection with either herpes simplex virus type 1 or 2 (HSV-1 or -2), and currently available therapeutics do not clear latent viruses or prevent their reactivation.

The emergence of CRISPR genome editing tools has inspired renewed efforts for preventing the reactivation of latent viruses by targeting and cleaving their genomes. An exemplary CRISPR editing system consists of the bacterial nuclease Cas9 and a small “guide” RNA molecule. The RNA molecule, which is complementary to the target sequence, guides the nuclease to its destination, where the nuclease cleaves the target DNA. CRISPR/Cas9 complexes can be introduced into cells by various mechanisms. For example, viruses engineered to encode the nuclease and the guide RNA can be transferred into cells using a technique called transduction.

A team at Harvard Medical School recently determined that specifically designed guide RNAs not only inhibit lytic replication of HSV-1, but can also cleave and edit latent HSV-1 genomes, thereby inhibiting their reactivation.

The authors of the study screened 58 potential guide RNAs for their ability to direct the cleavage of HSV-1 target DNAs in vitro. In this assay, they incubated individual nuclease/guide RNA complexes together with different DNA substrates containing various target sequences and measured cleavage efficiency by gel electrophoresis. The guide RNAs that led to the best cleavage efficiency were then further tested for their efficacy in inhibiting HSV-1 lytic replication in human fibroblast cells. The authors transduced the cells with the various nuclease/guide RNA complexes, infected them with HSV-1, and measured viral (lytic) replication by plaque assay. Although several of the guide RNAs significantly reduced viral replication, the guide RNA targeting the UL30 region, which encodes the viral DNA polymerase, reduced viral levels by more than 10,000-fold.

To see whether this editing system could inhibit reactivation of latent HSV-1 genomes, the authors infected cells with a replication-defective HSV-1 strain, thus mimicking latency, and transduced the cells with Cas9 nuclease and various guide RNAs that had been effective in the in vitro cleavage screen. They then reactivated the latent virus by “superinfecting” the latently infected cells with wild type HSV-1 and measured the ability of the individual guide RNAs to inhibit this reactivation. The replication-defective strain encodes a green fluorescent protein, allowing the authors to distinguish between replication of the wild type input HSV-1 and the reactivated virus. When used individually, four of the guide RNAs reduced reactivation of latent viruses by about 100-fold. However, the authors were able to reduce reactivation by an additional 10-fold by targeting two genes simultaneously with two different guide RNAs, suggesting that one can achieve an increased effect by combining several guide RNAs.

Sequencing analyses also showed that some of the CRISPR/Cas9 complexes introduced detrimental mutations into the target sequence, and that the guide RNA targeting the UL30 gene led to mutations in about 40-80% of the latent viral genomes. Although these mutations did not reduce the actual number of latent genomes, they did reduce their ability to reactivate.

During latency, HSV-1 and HSV-2 exist as circular chromosomes wrapped around cellular chromatin components called nucleosomes. This temporary association with nucleosomes implies that portions of the latent viral DNA are tightly folded and inaccessible to guide RNAs. Because the UL30 target site was consistently cleaved so efficiently, the authors speculate that this site may be in an open portion of the viral DNA that is more accessible to guide RNAs than other sites in the viral genome. If this is true, future guide RNA design strategies could include sequencing latent genomes using methods that identify open or accessible DNA.

Previous attempts to eliminate and/or prevent the reactivation of latent HSV virus in infected cells have had limited success. This study provides the first evidence that CRISPR/Cas9 can efficiently edit latent HSV genomes. Other studies are underway to determine whether CRISPR/Cas9 can edit the HSV genome during latent infection in the resting sensory neuron host cell and other in vivo models. Although more work is needed to figure out how to deliver Cas9 and guide RNAs to latently infected sensory or other neurons in vivo, the therapeutic potential of CRISPR/Cas9 in the context of HSV latency is encouraging, particularly when considered in combination with other existing therapies.

TWiV 550: Covering up the shiny parts

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TWiV explains the use of a neuronal cell line to study herpes simplex virus latency and reactivation, and a strategy for creating vaccines that induce antibodies against specific epitopes.

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Show notes at microbe.tv/twiv

TWiV 543: Stoned and senile

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The TWiV team discusses Medusavirus, isolated from a hot spring in Japan, and induction of neurodegeneration by recurrent herpes simplex virus 1 infection of mice.

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A Live-Attenuated Herpes Simplex Virus Vaccine Candidate

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herpesvirusBy Gertrud U. Rey

There is currently no vaccine to prevent infection with herpes simplex virus type 1 or type 2 (HSV-1 or HSV-2). Infection with either of these viruses results in life-long viral latency. Sporadic reactivation and viral shedding may lead to painful oral and genital disease and an increased risk of HIV transmission.

[Read more…] about A Live-Attenuated Herpes Simplex Virus Vaccine Candidate

TWiV 369: Camel runny noses and other JNK

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On the latest episode of the science show This Week in Virology, a swarm of virologists discusses testing of a MERS coronavirus vaccine for camels, and how a neuronal stress pathway reactivates herpes simplex virus.

You can find TWiV #369 at www.microbe.tv/twiv.