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coronavirusby Helen Stillwell

Helen is a research associate in David Hafler’s immunobiology lab at Yale University; previously, she worked on poxviruses at Tonix Pharmaceuticals, and she plans to apply to PhD programs in virology this year.

RT-PCR Diagnostics

As cases of COVID-19 continue to rise, diagnostic testing has been a principal topic of discussion. Typically, quantitative (real-time) reverse transcription polymerase chain reaction (RT-PCR) is used for viral diagnostic testing.

RT-PCR requires a nasopharyngeal swab, which is placed into a viral transport media. Viral RNA is isolated via RNA extraction, which requires the use of a specific test kit. Post RNA isolation, reverse transcription – the conversion of RNA into complementary DNA (cDNA) – is facilitated by combining the RNA with deoxyribonucleotides and other reagents and exposing the sample to various temperatures.

Once converted to cDNA, the sample is analyzed via PCR. To do so, the cDNA is mixed with specific probes – short sequences of DNA that are complementary to signature sequences of the viral genome. These probes contain fluorescent markers that are detected by the real-time PCR machine, which provides a read out quantifying the amount of fluorescence detected. This value is called the Ct number, and is inversely related to the amount of DNA. Typically, Ct numbers are analyzed relative to a ‘housekeeping’ gene via the comparative Ct method, which uses the difference in Ct values between these samples to determine whether viral sequences are present.

As such, RT-PCR detects the presence of viral RNA in a sample – it does not necessarily signify the presence of replicating virus.

Delays to Provide Testing in the US

While effective testing paradigms exist, regulatory barriers and equipment shortages have impeded effective scale-up of COVID-19 testing in the US. In mid-February, the CDC shipped test kits to state and local labs; however, these tests proved defective (Baird 2020). On February 29th, in an effort to increase the nation’s testing capacity, the FDA allowed certain laboratories to develop and perform their own diagnostic tests (Baird 2020). Later, on March 12th, the FDA issued Emergency Use Authorizations (EUA) to enable commercial companies to manufacture and issue tests while bypassing certain regulatory measures (Baird 2020).

While these new policies increased the number of laboratories authorized to perform diagnostic testing, material shortages continue to halt efforts to escalate testing. There is a particular need for RNA extraction kits, qPCR reagents, and test swabs (Baird 2020). Some labs, unable to keep up with the influx of samples, have resorted to freezing them until they are able to increase their processing power (Baird 2020).

Diagnostic Innovation for COVID-19

Reduced regulation has invited an influx of proposals for novel COVID-19 testing. Two research groups specializing in CRISPR gene-editing technology, Sherlock Biosciences and Mammoth Biosciences, are working with various collaborators to adapt their CRISPR platforms for COVID-19 diagnosis.

The CRISPR-Cas9 mechanism involves the creation of a small segment of RNA with a “guide” sequence that binds to a specific target of DNA in a genome. The RNA also binds the Cas9 nuclease, which cuts the DNA at the target location. Research on other Cas nucleases has revealed the use of CRISPR to locate rather than cleave certain sequences within a genome (Prabhune 2020). The nucleases Cas12a and Cas13a have been studied for this particular use. Like Cas9, Cas12a/Cas13a nucleases cleave at the site where complementary guide RNA binds to a specific target sequence (Prabhune 2020); however, upon locating the target sequence, these nucleases also cleave other nearby, non-targeted nucleic acid molecules – a mechanism that is appropriated to build reporter systems for a visual diagnostic readout (Prabhune 2020).

Sherlock Biosciences CRISPR-based nucleic acid detection protocol called SHERLOCK (Specific High sensitivity Enzymatic Reporter unLOCKing) targets two SARS-CoV-2 genes – the S gene and Orf1ab. It includes three steps: amplification of synthetic viral RNA and in vitro transcription of amplified DNA into RNA; RNA-detection using the Cas13 nuclease; and a visual readout which captures the cleaved reporter RNA (Prabhune 2020).

Mammoth Biosciences’ platform DETECTR – DNA endonuclease-targeted CRISPR trans reporter (DETECTR) – is a CRISPR-based detection mechanism that targets the N and E viral genes (Prabhune 2020). It includes the following steps: amplification of extracted RNA; detection of DNA using Cas12a; and a visual readout (Prabhune 2020).

Conclusions

As of March 24, 359,161 COVID-19 tests have been performed in the US (The COVID Tracking Project). For comparison, South Korea is testing nearly 20,000 people per day (Khazan 2020). While there are a lot of promising candidates for new testing paradigms, it remains to be seen how and when these new paradigms might be implemented.

References

Baird RP. Why Widespread Coronavirus Testing Isn’t Coming Anytime Soon. New Yorker. (2020).

Cormen VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DKW, Bleicker T, Brunink S, Schneider J, Schmidt ML, Mulders DGJC, Haagmans BL, van der Veer B, van den Brink S, Wijsman L, Goderski G, Romette JL, Ellis J, Zambon M, Peiris M, Goossens H, Reusken C, Koopman MPRG, Drosten C. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. (2020). doi: 10.2807/1560-7917.ES.2020.25.3.2000045.

Khazan O. The 4 Key Reasons the U.S. Is So Behind on Coronavirus Testing. The Atlantic. (2020). https://www.theatlantic.com/health/archive/2020/03/why-coronavirus-testing-us-so-delayed/607954/

Prabhune M. Coronavirus Detection Using CRISPR Diagnostics. Synthego. (2020). https://www.synthego.com/blog/crispr-coronavirus-detection.

The COVID Tracking Project. https://covidtracking.com/us-daily/

I’ve been delinquent in posting recent episodes of This Week in Virology here. They are all focused on SARS-CoV-2 and the disease it causes, COVID-19. Here is a list:

TWiV Special: Coronavirus update with Mark Denison, MD

Pediatric infectious disease physician and coronavirologist Mark Denison joins Vincent for a discussion of COVID-19 and SARS-CoV-2 with an emphasis on antiviral therapeutics.

TWiV 593: Coronavirus update – flatten the curve

Daniel Griffin joins Ori and the TWiV team for an update on the SARS-CoV-2 pandemic, including gastrointestinal illness associated with infection, use of hydroxychloroquine and other antivirals, his experiences treating many patients in the New York area, and much more.

 

TWiV 592: Coronavirus update – dangerous curve ahead

Ori joins TWiV for an update on the SARS-CoV-2 pandemic, including scary modeling, clinical trials for antivirals and vaccines, asymptomatic transmission, and much more inspired by listener email.

 

TWiV Special: SARS-CoV-2 epidemiology with Stephen Morse

Epidemiologist Stephen Morse joins Vincent to discuss epidemiology of SARS-CoV-2 and preparedness for the COVID-19 pandemic.

 

TWiV Special: A medical student perspective on COVID-19

Ori Lieberman joins Vincent to discuss COVID-19, the disease caused by SARS-CoV-2, gleaned from his experience during clinical rotations in medical school.

HydroxychloroquineChloroquine (and a derivative, hydroxychloroquine) has been used for years in the treatment of malaria. The drug is also known to block the entry of many viruses into cells. A small clinical trial has revealed it to be effective in reducing viral loads in COVID-19 patients.

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By David Tuller, DrPh

Like many or most, I don’t know what I’m supposed to be doing right now. I am “sheltering at home” in San Francisco, following the news, making way too many cups of coffee, chatting much more on the phone than usual, checking in with my 90-year-old mom in Manhattan, watching movies I’m not interested in watching, taking walks with Harold (the mutt) around the neighborhood while keeping several feet away from passersby. It’s like living in a Twilight Zone episode, for those old enough to remember that. (Or like living in a Black Mirror episode, for others.)

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By David Tuller, DrPH

I was supposed to be in Bristol, England, last week at the CFS/ME Research Collaborative conference. The conference went on as scheduled, but I decided the weekend before that the situation was getting too dicey to leave home. I didn’t want to get swept up in lockdowns and international travel blockades and not be able to return to the US. Under the circumstances, it seemed wise to change my plans. (It goes without saying that I hope everyone is taking care of themselves in this incredibly stressful moment.)

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coronavirusWhen it comes to safeguarding the health of the US, there is no one I trust more than Dr. Anthony Fauci, head of NIAHD/NIH (whom I was fortunate to interview on TWiV in 2013). So when Dr. Fauci says that 100,000 people could die in the SARS-CoV-2 outbreak just in the US, I take notice. What exactly does he mean? Can we put this in perspective with other outbreaks?

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Ralph Baric joins TWiV to dissect the coronavirus pandemic caused by SARS-CoV-2, including discussion on community spread, asymptomatic infections, origin of the virus, transmission, vaccine development, and much more.

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Download TWiV 591 (75 MB .mp3, 123 min)
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Show notes at microbe.tv

by Helen Stillwell

Helen is a research associate in David Hafler’s immunobiology lab at Yale University; previously, she worked on poxviruses at Tonix Pharmaceuticals, and she plans to apply to PhD programs in virology this year.

Is there a vaccine for SARS-CoV-2? The internet is awash with articles and posts seeking to shed light on this very question.

Currently, there is no approved vaccine against SARS-CoV-2. While several companies have announced vaccine candidates in development, it is still unlikely that a vaccine will play a significant role in the current outbreak.

In the US, vaccine candidates cannot proceed through the appropriate preclinical, clinical, regulatory, and manufacturing pipelines in a mere few weeks. Typically, under non-outbreak circumstances, vaccine development can cost up to $1 billion or more, and it often takes many years to reach approval (Dunn 2020). That being said, certain allowances can be made in the case of an outbreak, and we are getting better at developing platform technologies that allow for the development of viable candidates at increasingly faster rates.

Globally, the therapeutic pipeline for SARS-CoV-2 contains about 15 potential vaccine candidates (Pang et al. 2020). These candidates employ various different technologies, including messenger RNA (mRNA), DNA-based, nanoparticles, synthetic and modified virus-like particles (Pang et al. 2020).

The two principal candidates are those being developed by Inovio Pharmaceuticals and Moderna Therapeutics (in partnership with the NIAID). Both vaccines rely on specific mechanisms that fall under the umbrella term “platform technologies”. That is, both vaccines consist of a primary nucleic construct (in this case RNA or DNA) that is amenable to the insertion of pertinent genetic sequences from a virus of interest so that the construct can be adapted for use against various different viruses. They are ‘platforms’ that allow for more efficient development of vaccine candidates against emerging viruses, such as SARS-CoV-2.

Both candidates are partially funded by the Coalition for Epidemic Preparedness Innovations (CEPI), a global partnership between public, private, philanthropic, and civil society organizations that possesses the resources to fast-track the development of vaccines against emerging infectious disease and enable access to these vaccines during outbreaks. While it will likely take a year or more for the majority of the vaccine candidates to initiate Phase I clinical trials, those funded by CEPI are able to accelerate these timelines (Pang et al. 2020).

The Inovio vaccine is a DNA-based vaccine (Pang et al. 2020). It is in the preclinical stage of development, and phase I testing is projected to occur in the next few months (Pang et al. 2020). Phase I clinical trials for vaccines typically include 20-100 healthy volunteers who are administered a vaccine candidate for the purpose of evaluating safety and determining ideal dose.

The Moderna-NIAID vaccine is a mRNA vaccine. Typically, mRNA vaccines include an open reading frame (ORF) for the target antigen and are flanked by untranslated regions (UTRs) with a terminal poly(A) tail (Zhang et al. 2019). Theoretically, after vaccine delivery, mRNA vaccines are translated to drive transient expression of antigen to promote an immune response (Zhang et al. 2019).

Recently, Moderna escalated development and sent vaccine to NIAID to begin the process of initiating a phase I trial to test the safety and immunogenicity of the vaccine. The trial is projected to begin at the end of April, with preliminary results in July or August (Loftus 2020). The time it took Moderna to develop and prepare the vaccine after learning of the virus’ genetic sequence from Chinese scientists in January is extraordinary. Following the outbreak of SARS-CoV in China in 2002, it took approximately 20 months for NIAID to get a vaccine into the first stage of human testing, according to NIAID Director Dr. Anthony Fauci (Loftus 2020).

Yet, it is still unclear whether Moderna’s vaccine candidate will provoke a sufficient immune response to be effective against SARS-CoV-2. The premise of gene-based platform technologies rests on the ability to target segments of the viral genome that are involved in provoking host immune response. Although we can make well-informed choices on what sequences provoke immunogenicity, we won’t know if the optimal sequence has been selected until human trials are completed (Loftus 2020). So too, there is no precedence for a vaccine of this kind since there are not yet any approved human vaccines that use this gene-based technology (Loftus 2020).

Virologist Dr. Jose Esparza commented the following on Moderna’s vaccine candidate: “The rapid manufacturing of the RNA vaccine is great. But preclinical experiments are important to assess safety before carefully moving ahead with small phase I trials in human volunteers. Special attention should be placed to a potential ‘Antibody Dependent Enhancement of Infectivity’ triggered by the induction of binding but no neutralizing antibodies.”

Indeed, the fast development of a vaccine and imminent phase I testing do not guarantee its efficacy. We will not know until after human trials whether the sequence Moderna and NIAID selected provokes a sufficient immune response to impart protection. And, even if the first studies show encouraging results, the vaccine might not be widely available until 2021 due to the later phase clinical trials and regulatory supervision that will be required to allow for its use in the general public.

To date, the best source of protection against SARS-CoV-2 remains to be the recommendations of the CDC and similar organizations: avoid close contact with those who are sick; avoid touching your eyes, nose and mouth; remain home when you are sick; cover your cough or sneeze with a tissue and discard properly; clean and disinfect frequently touched surfaces; wear a facemask if you are showing symptoms (face masks are not beneficial to those who are well).

References

Dunn A. The Wuhan coronavirus has now claimed more lives than SARS. Top scientists told us it could take years and cost $1 billion to make a vaccine to fight the epidemic. Business Insider. (2020). https://www.businessinsider.com/wuhan-coronavirus-vaccine-could-take-years-timeline-and-cost-2020-2.

Loftus P. Drugmaker Moderna Delivers First Experimental Coronavirus Vaccine for Human Testing. Wall Street Journal. (2020). https://www.wsj.com/articles/drugmaker-moderna-delivers-first-coronavirus-vaccine-for-human-testing-11582579099.

Pang J, Wang MX, Ang IYH, Tan, SHX, Lewis RF, Chen, JI, Gutierrez RA, Gwee SXW, Chua PEY, Yan Q, Ng XY, Yap RKS, Tan HY, Teo YY, Tan CC, Cook AR, Yap JCH, Hsu LY. Potential Rapid Diagnostics, Vaccine Therapeutics for 2019 Novel Coronavirus (2019-nCoV): A Systematic Review. J. Clin. Med. (2020) 9(3). doi: 10.3390/jcm9030623.

Zhang C, Maruggi G, Shan H, Li J. Advances in mRNA Vaccines for Infectious Diseases. Front. Immunol. (2019). doi: 10.3389/fimmu.2019.00594

The TWiV trio continues in-depth coverage of COVID-19 and SARS-CoV-2, including discussion on genome mutation and circulating lineages, handwashing, facemasks, cruise ship outbreaks, the South Korean situation, and much more.

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Show notes at microbe.tv/twiv

Driving the anxiety and uncertainty about the current outbreak of COVID-19 is the case fatality ratio (CFR) being thrown about carelessly by not just the press, but also WHO and other organizations. However the CFR is not a one-size-fits all, and is influenced by many factors.

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