The “Wuhan Virus” Outbreak—Molecular Aspects of Coronaviruses (CoV)

  •  The “Wuhan Virus,” a Novel CoV Designated as 2019-nCoV, has Been Declared a Public Health Emergency of International Concern by the World Health Organization
  • Genomic Sequencing and qRT-PCR Detection of 2019-nCoV Has Already Been Achieved    
  • Existing Antiviral Drugs Are Being Evaluated and an mRNA Vaccine is Being Investigated

Over the past two months, there has been extensive media coverage on the “Wuhan virus” outbreak. According to an official health advisory, the U.S. Centers for Disease Control and Prevention (CDC) “continues to closely monitor an outbreak of a 2019 novel CoV (2019-nCoV) in Wuhan City,” China that started in December 2019. On January 30th, 2020, the World Health Organization (WHO) declared that “the outbreak now meets the criteria for a Public Health Emergency of International Concern.” CoVs are a large family of viruses, and they get their name from the surface proteins on virions visualized by electron microscopy, which are reminiscent of a solar corona, as seen here.

Coronavirus electron micrograph. Credit: CDC/Dr. Fred Murphy. This image is in the public domain and free of any copyright restrictions.

In keeping with the Zone’s focus on what’s trending in nucleic acid research, this blog will provide information about CoV genome structure and function, as well as 2019-nCoV-specifc aspects for sequencing, qRT-PCR detection, nucleic acid-based drug candidates, and an mRNA vaccine. Before doing that, I will provide some historical background about coronaviruses in the next section. Readers interested in obtaining updated information from the CDC regarding the 2019-nCoV outbreak can use this link.   

Background

According to a 2012 review by Geller et al., human coronaviruses (HCoVs) were first identified in the 1960s, but their severity was underappreciated until the worldwide epidemic of Severe Acute Respiratory Syndrome (SARS) in 2002–2003. Based on the sequencing of RT-PCR amplicons, it was determined that SARS originated in China due to the emergence of a new coronavirus, the SARS-CoV. The emergence of Middle East Respiratory Syndrome coronavirus, MERS-CoV, in 2012 rekindled memories of this SARS outbreak. Moreover, say Geller et al., these viruses show an environmental resistance that increases their probability of transfer between contaminated hosts via surfaces, hands, etc. Because of this resistance, suitably strong yet safe disinfectants are needed, as no treatment or vaccines are currently available to cure HCoVs infections.

For example, Geller et al. note that SARS-CoV has been shown to survive after drying on different kinds of materials or when diluted in water, revealing a decreased infectivity only after 72 to 96 hours, depending on the conditions. Thus, SARS-CoV was found on different environmental samples, such as chairs, elevators, and a computer mouse. Studies using surrogate viruses, which required 17 to 22 days for 99% reduction in water at room temperature, have also implicated water and sewage in the transmission of SARS-CoV.

Coronavirus Genome Structure and Function

Diagram of coronavirus virion structure. Taken from Belouzard et. al. via wikipedia.org and free to use under the Creative Commons Attribution 3.0 Unported license.

CoV is an enveloped virus that carries a ∼30-kb positive-sense RNA genome, which encodes multiple different polyproteins in multiple open reading frames (ORFs), as depicted in detail elsewhere by Wada et al. in 2018. CoV particles carry a helical nucleocapsid, which is a complex of the viral genomic RNA. The nucleocapsid protein is enclosed in an envelope composed of viral envelope proteins known as spike, membrane protein (M), and envelope protein (E), as diagramed here. 

After infection, the genomic RNA is released into the cytoplasm and translated to produce two large polyproteins encoded in gene 1, which occupies the 5′ two-thirds of the genome with two partially overlapping ORFs, as depicted in detail elsewhere. The two polyproteins are processed by viral proteases to generate 15 or 16 nonstructural proteins, most of which are required for viral RNA synthesis, according to Wada et al. In addition to mRNA 1 (the intracellular form of genomic RNA), several subgenomic mRNAs are synthesized in infected cells. These subgenomic mRNAs encode viral structural proteins and accessory proteins, the latter of which are not essential for virus replication in cell culture but play a role in viral pathogenicity, as detailed elsewhere by Menachery et al., who also discuss involvement of interferon, depicted here, and NF-κB-mediated inflammation. 

3D molecular model of interferon-alpha, a protein produced by leukocytes and involved in the innate immune response against viral infections.

Sequencing Coronavirus, Including 2019-nCoV

Taken from Workman et al. in bioRxiv, posted online November 9, 2018, and free to use. The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

According to a 2020 report by Viehweger et al., short-read second-generation sequencing technologies—such as IonTorrent and Illumina—are restricted by read length (200-400 nucleotides). For example, the use of highly fragmented viral RNAs considerably complicates the investigation of haplotypes. Consequently, short-read data can usually not be unambiguously assigned to specific subgenomic RNA species of CoV. Viehweger et al. therefore performed direct RNA sequencing (DRS) of human CoV-229E on an array of nanopores developed by Oxford Nanopore Technologies (ONT), using the workflow depicted here.

Using DRS, these researchers were able to map the longest (∼26kb) contiguous read to the viral reference genome. By combining Illumina and nanopore sequencing, they constructed a highly accurate consensus sequence of the human CoV-229E genome (27.3kb). Furthermore, using long reads that did not require an assembly step, Viehweger et al. were able to identify diverse and novel human CoV-229E subgenomic RNAs in infected cells. These remain to be characterized. The DRS approach also allowed for direct detection of 5-methylcytosine (5mC) sites in human CoV-229E RNAs and, by extension, 5mC sites in RNA viruses in general. On January 31, 2020, it was reported that ONT has shipped 200 MinION sequencers and related consumables to China to be used to support the ongoing surveillance of the current CoV outbreak.

5-Methylcytosine. Taken from commons.wikimedia.org and free to use.

The following two links to genomes for samples of 2019-nCoV have been posted in GenBank, the NIH genetic sequence database. The first item was uploaded on January 24, 2020, by Chan et al. in China, and is currently in press for publication in Lancet titled A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. The second item provided by Queen et al. from the CDC is unpublished and titled Full genome sequence of first U.S. case of nCoV-2019. In each case, a Graphics link provides access to the assembled genome structure.

1. Accession: MN975262.1 29,891 bp linear RNA      

2. Accession: MN985325.1 29,882 bp linear RNA    

On January 31, 2020, it was reported that the Institut Pasteur in France has sequenced the genome of 2019-nCoV samples from French patients.

The horseshoe bat (Rhinolophus spp.) is associated with SARS-CoV, according to Banerjee et al.

According to a post by the CDC, the 2019-nCoV is a betacoronavirus, like MERS and SARS, all of which have originated from bats. The CDC added that “[t>

he sequences from U.S. patients are similar to the one that China initially posted, suggesting a likely single, recent emergence of this virus from an animal reservoir”. For more information, interested readers can explore Bats and Coronaviruses by Banerjee et al. in Viruses (2019) 11(1): 41.

qRT-PCR Detection of Coronavirus, Including 2019-nCoV 

In response to the aforementioned SARS-CoV outbreak, Vijgen et al. published a very sensitive and specific TaqMan-based, real-time, quantitative reverse-transcription PCR (qRT-PCR) for the rapid detection and quantitation of human CoVs in 2005. Absolute viral load measurement in clinical samples was achieved through the construction of human CoV OC43 and 229E RNA standards for the generation of a standard curve. The CoV OC43 assay allowed quantitation over a range of 20 to 2 X 108 RNA copies per reaction mixture, which when extrapolated to clinical samples, corresponds to a detection range of 103 to 1010 viral genome equivalents per mL. By using the CoV 229E qRT-PCR assay, viral RNA copies ranging from 20 to 2 X 109 per reaction mixture were detected, which corresponds to 104 to 1011 viral genome equivalents per mL.

Now, in response to the 2019-nCoV outbreak, the CDC has developed a qRT-PCR assay for this new human CoV that was posted on January 24, 2020. One downloadable provides a list of the sequences, dye-labeling, and working concentrations of the required panel of primers and probes. Another downloadable document provides detailed instructions for this assay, and includes sections on reagents, supplies, and equipment; nucleic acid extraction; quality control; qRT-PCR assys, interpreting the results, assay limitations, and contact information. Considering the relatively short amount of elapsed time between initial recognition of this outbreak and the posting of full documentation on this assay, the CDC response has been remarkably quick.

Nucleic Acid-Based Drug Candidates for Coronavirus, Including 2019-nCoV  

Taken from commons.wikimedia.org and free to use.

The previously mentioned SARS-CoV outbreak also prompted investigations of ASOs as possible therapeutic agents against this virus and, by extension, phylogenetically related viruses. For example, Burrer et al. used several strains of murine hepatitis virus (MHV)—a close phylogenetic relative of SARS-CoV—in cell culture and in vivo in mouse models to investigate the antiviral characteristics of peptide-conjugated antisense phosphorodiamidate morpholino oligomers (P-PMOs). 

Ten P-PMOs directed against various target sites in the viral genome were tested in cell culture, and one of these (5TERM), which was complementary to the 5’ terminus of the genomic RNA, was effective against six strains of MHV. Further studies were carried out with various arginine-rich peptides (P) conjugated to the 5TERM PMO sequence in order to evaluate efficacy and toxicity and thereby select candidates for in vivo testing. In uninfected control mice, prolonged treatment with the P-PMO conjugate did not result in weight loss or detectable histopathologic changes, whereas treatment of infected mice reduced viral titers in target organs and protected mice against virus-induced tissue damage. It was concluded that “the strong antiviral effect observed suggests that with further development, P-PMO may provide an effective therapeutic approach against a broad range of coronavirus infections”.

At about the same time, Li et al. reported a study in which potent siRNA inhibitors of SARS-CoV in vitro were further evaluated for efficacy and safety in a rhesus macaque (pictured here) SARS model using clinically viable delivery, while comparing three dosing regimens. Observations of SARS-like symptoms, measurements of SARS-CoV RNA presence, and lung histopathology and immunohistochemistry consistently showed siRNA-mediated anti-SARS efficacy by either prophylactic or therapeutic treatment regimes. 

In response to the current CoV outbreak, Gilead Sciences is considering repositioning its nucleotide prodrug remdesivir (GS-5734), depicted here, as a treatment for 2019-nCoV, according to a January 24, 2020 news report. CoV susceptibility to remdesivir was previously shown by Agostini et al. to be mediated by the viral polymerase and the proofreading exoribonuclease, using SARS-CoV and MERS-CoV. Remdesivir was developed by Gilead Sciences as a treatment for Ebola virus disease and Marburg virus infections. In related news, on January 27, 2020, AbbVie and Johnson & Johnson began shipping drugs approved to treat HIV to Chinese health authorities to assess whether the medicines could help contain 2019-nCoV. 

An mRNA Vaccine for 2019-nCoV 

An official statement by the CDC regarding the 2019-nCoV reads as follows: “There are currently no vaccines available to protect you against human coronavirus infection”. Traditional approaches to development of vaccines are relatively slow, as evidenced by the situation for a vaccine for MERS-CoV, which evolved in seriousness beginning in 2013-2014. About six years later, in October 2019, an article in Lancet Infectious Disease states that “[t>

he phase 1, open-label, single-arm, first-in-human evaluation of the Middle East respiratory syndrome (MERS) coronavirus DNA vaccine by Kayvon Modjarrad and colleagues is an important step forward for achieving one of the WHO R&D Blueprint for MERS aims, which calls for development of two types of human MERS vaccines”. Given the need for additional pivotal clinical trials prior to possible regulatory approvals, such a vaccine seems to be far in the future. 

Fortunately, the advent of “RNA vaccines” has led to the growing opinion among experts that much faster timelines for vaccine development are possible. In a nutshell, mRNA vaccines are composed of biosynthetic messenger RNA (mRNA), which encode antigen genes of an infectious agent. When administered to host cells, the mRNAs are translated into protein antigens that elicit protective immunity against the infectious agent. Synthesizing mRNA—usually with chemically modified bases—can be achieved relatively quickly, as discussed elsewhere. In addition, use of mRNA not only eliminates the need to work with an infectious organism per se, but also avoids relatively old-style, cell culture-based manufacturing methods.

The crisis-like situation for 2019-nCoV has prompted action by the Coalition for Epidemic Preparedness Innovations (CEPI), the mission of which is “to stimulate and accelerate the development of vaccines against emerging infectious diseases and enable access to these vaccines for people during outbreaks”. CEPI was founded in Davos by the governments of Norway and India, the Bill & Melinda Gates Foundation, the Wellcome Trust, and the World Economic Forum. CEPI has secured $750 million toward its $1billion funding target, with multi-year funding from Norway, Germany, Japan, Canada, Australia, the Bill & Melinda Gates Foundation, and Wellcome.

On January 23, 2020, Moderna, Inc., a clinical stage biotechnology company pioneering mRNA therapeutics and vaccines, and CEPI announced a new collaboration to develop an mRNA vaccine against 2019-nCoV. Under the terms of the agreement, Moderna will manufacture an mRNA vaccine against 2019-nCoV, which will be funded by CEPI. The Vaccine Research Center (VRC) of the National Institute of Allergy and Infectious Diseases (NIAID), part of NIH, collaborated with Moderna to design the vaccine. NIAID will conduct IND-enabling studies and a Phase 1 clinical study in the U.S. Information about two other CoV vaccine approaches funded by CEPI is available in this link to Science magazine.

Concluding Comments

Since December 31, 2019 and as of February 13, 2020, there have been 60,330 laboratory-confirmed cases of 2019-nCoV infection and 1,369 deaths, according to a post by the European Centre for Disease Prevention and Control, which is updated daily. The vast majority (59,805) of these cases and deaths have been reported in China. 

A National Institutes of Health press release stated that “the cluster ‘marks the third time in 20 years that a member of the large family of coronaviruses (CoVs) has jumped from animals to humans and sparked an outbreak,’ and officials are making an effort to share with the public information about current research that relates to the virus, including in a JAMA essay published last week.”

Only time will tell whether the current measures being taken globally to stem the outbreak will be effective. In the meantime, we can only hope for the best, and that use of the existing antiviral agents mentioned in this blog will be found helpful in the near term. Beyond that, we must remain hopeful that the push for a vaccine against 2019-nCoV will be successful.

Your comments are welcomed, as usual.

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