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In this week’s coverage of the coronavirus epidemic, the TWiV team discusses the fatality rate, China’s initial reaction to the outbreak, conspiracy theories, how long the virus remains infectious on surfaces, and evidence for virus in the intestinal tract.

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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.

By David Tuller, DrPH

On its website, Mahana Therapeutics has listed sixteen gastroenterology and psychology advisors from prominent academic and medical institutions. Companies often add such names to their rosters as a way of signaling their significance and their access to great minds. Sometimes these people are compensated; sometimes not. In many instances, these advisors play little role in day-to-day company activities.

In the case of Mahana, that means those advisors not directly involved in the development of the web-based cognitive behavior therapy program for treating irritable bowel syndrome might have little knowledge or awareness of the details of the matter. However, that doesn’t mean they have no responsibility to express their thoughts on the company’s promotional strategy–especially because Mahana itself has so far failed to respond to my questions.

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By David Tuller, DrPH

Last week I wrote about the recently announced licensing deal between Mahana Therapeutics and King’s College London. The deal involves a web-based course of cognitive behavior therapy designed to treat irritable bowel syndrome. In a major study, the reported improvements in symptoms among participants in the web-based program were modest at best. Yet Mahana is promoting these improvements as “dramatic” and “potentially game-changing for patients.”

On Monday, I e-mailed Mahana Therapeutics with a few questions. I have not heard back, although I followed up my initial e-mail with two nudges. On Friday, I sent the following e-mail to Professor Rona Moss-Morris, the study’s co-investigator. (Professor Moss-Morris has stock options in the company and has received payment as a consultant.).

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The TWiVerers continue their coverage of the new coronavirus outbreak in China, as the number of cases increase dramatically and the virus begins person-to-person transmission in other countries.

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coronavirusAn unprecedented amount of information is emerging on the new coronavirus, provisionally called 2019-nCoV, that originated in China and is spreading globally. As of this writing there are 8,236 confirmed cases (8,124 in China) with 171 deaths (Click the link for real-time updates).

What was the origin of the virus?

The virus was first isolated by infection of cells in culture with broncho-alveolar wash from a patient in Wuhan with pneumonia. The infected cells showed cytopathic effects, and staining of cells with an antibody to coronavirus NP protein, which is conserved among coronaviruses, revealed intracellular staining.

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By David Tuller, DrPH

As I wrote yesterday, Mahana Therapeutics has recently licensed from King’s College London an “innovative digital therapeutic”—a web-based program delivering a course of cognitive behavioral therapy to patients with irritable bowel syndrome. A page on the Mahana site promoting this web-delivered IBS-CBT program furthers the impression that this deal is steeped mostly in hype.

A January 10thpress release announcing the deal declared the changes in symptoms produced by the web-based program to be “substantial,” which already seemed like an exaggeration. The company’s website has apparently upgraded this observation with its description of the symptom improvements as “dramatic and potentially game-changing for patients.” Wow!

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By David Tuller, DrPH

Yesterday I sent a freedom-of-information request to King’s College London about the recently announced licensing deal it has with Mahana Therapeutics. The deal involves a web-based CBT program for irritable bowel syndrome, which I have written about here and here. This morning I sent a note to the e-mail address for press contact listed on Mahana’s site.

I have posted both of these messages below–first the FOI request, then the letter to Mahana.

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By David Tuller, DrPH

On January 10th, the following information was announced in a press release:

Mahana Therapeutics, a digital therapeutics company reimagining the treatment of chronic diseases, today announced that the Company has entered into a licensing and collaboration agreement with King’s College London, a leading research university and one of the oldest and most prestigious universities in England. Mahana has acquired a worldwide exclusive license to an innovative digital therapeutic for the treatment of irritable bowel syndrome (IBS).

This exciting development in Mahana’s efforts to reimagine the treatment of chronic diseases is related to the IBS study I wrote about last week. In the study, two versions of CBT performed somewhat better than treatment-as-usual (TAU) at 12 and, to a lesser extent, at 24 months.* [*I initially wrote “weeks” rather than “months” in this sentence, although subsequent references to the time frame were correct. I am correcting this shortly after having posted the blog. The effects do diminish but not that quickly!] One group received a course of telephone CBT from a therapist. The second received a web-based course of CBT called “Regul8: A Self-Management Programme for IBS,” supplemented by a minimal amount of therapist contact. After the study ended, those in the TAU arm were offered online access to the Regul8 program [American spelling], but without therapist support.

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Coronavirus expert Ralph Baric joins TWiV to explain the virology and epidemiology of the recent zoonotic outbreak spreading across China and overseas.

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Show notes at microbe.tv/twiv