Milestones in coronavirus discovery and research

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Coronaviruses

• Coronaviruses are

enveloped ssRNA(+) viruses,

belonging to replication

class IV, which infect

mammals and birds. The

name derives from

latin corona (crown),

referring to the shape of

proteins around the virion

as observed in electron

micrographs. These viruses

have the largest RNA

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Milestones in coronavirus discovery and research

Coronaviruses (CoV) were identified as human pathogens in the 1960s. Most coronaviruses infect animals, (i.e., bats, birds and mammals), which act as an intermediate host reservoir. Sometimes they change host and infect humans.

There are currently seven coronaviruses known to infect humans. Four of them cause mild to moderate disease. More specifically, HCoV-OC43, HCoV-HKU1 and HCoV-229E cause common colds. The other three cause more severe and even fatal disease and have emerged more recently (in the last 20 years): SARS-CoV responsible for the Severe Acute Respiratory Syndrome (SARS) in 2002, MERS-CoV the Middle East Respiratory Syndrome (MERS) in 2012 and SARS-CoV-2, identified with a cluster of pneumonia cases in Wuhan, China in late 2019.

Illness in humans mostly affects the respiratory tract, with symptoms ranging from those of a common cold to very severe lower respiratory infections

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SARS-CoV-2 genome

PLp; papain- like protease

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Coronavirus non-structural proteins and their functions

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Coronavirus replication and discontinuous transcription

.

Full- length positive- sense genomic RNA is used as a template to produce both full- length

negative- sense copies for genome replication and subgenomic negative- sense RNAs (–sgRNA) to produce the subgenomic mRNAs (sg mRNA). The negative strand RNA synthesis involving a

template switch from a body transcription regulatory sequence (TRS-B) to the leader TRS (TRS- L) is illustrated to produce one sg mRNA. This process can take place at any TRS- B and will

collectively result in the production of the characteristic nested set of coronaviral mRNAs.

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Subgenomic RNAs (sgRNAs) are created by discontinous transcription. During transcription of minus strand RNA, the polymerase have chances to pause on transcription-regulating

sequences (TRS) and jump to leader TRS, thereby creating a major deletion. This creates

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Coronavirus subgenomig mRNA products

Subgenomic mRNA are translated into four structural proteins: S, E, M and nucleocapsid (N) proteins and accessory proteins.

S (spike glycoprotein) is responsible for host cell receptor recognition and binding, and for fusion of virion envelope with endosomal membrane

E proteins are small integral membrane proteins with roles in virus morphogenesis, assembly and budding. In the absence of E proteins, virus release is inhibited

completely (in the case of transmissible gastroenteritis virus (TGEV)) or partially (in the case of SARS-CoV and MHV). The E protein also possesses ion channel activity, which is required for optimal virus replication.

M protein is the most abundant protein in the coronavirus virion. It is a multipass transmembrane protein. Homotypic interaction between M protein provides the scaffold for virion assembly, while heterotypic interaction recruits other structural protein and genomic RNA to the assembly site.

N protein is important for encapsidation of viral RNA and acts as an interferon (IFN) antagonist.

Accessory proteins are not required for virus replication in cultured cells. However, they are conserved in virus species isolated at different times and locales (for

example, for SARS-CoV), which suggests that these proteins have an important role in replication in the natural host. Several accessory proteins are virion-associated,

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The coronavirus

life cycle

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The coronavirus virion and life cycle. a | The coronavirus virion consists of structural proteins, namely

spike (S), envelope (E), membrane (M), nucleocapsid (N) and, for some betacoronaviruses,

haemagglutinin- esterase (not shown). The positive- sense, single- stranded RNA genome (+ssRNA) is encapsidated by N, whereas M and E ensure its incorporation in the viral particle during the assembly process. S trimers protrude from the host- derived viral envelope and provide specificity for cellular entry receptors. b | Corona virus particles bind to cellular attachment factors and specific S interactions with the cellular receptors (such as angiotensin- converting enzyme 2 (ACE2)), together with host

factors (such as the cell surface serine protease TMPRSS2), promote viral uptake and fusion at the cellular or endosomal membrane. Following entry, the release and uncoating of the incoming genomic RNA subject it to the immediate translation of two large open reading frames, ORF1a and ORF1b. The resulting polyproteins pp1a and pp1ab are co- translationally and post- translationally processed into the individual non- structural proteins (nsps) that form the viral replication and transcription complex. Concordant with the expression of nsps, the biogenesis of viral replication organelles consisting of characteristic perinuclear double- membrane vesicles (DMVs), convoluted membranes (CMs) and small open double- membrane spherules (DMSs) create a protective microenvironment

for viral genomic RNA replication and transcription of subgenomic mRNAs (sg mRNAs) comprising the characteristic nested set of coronavirus mRNAs. Translated structural proteins translocate into

endoplasmic reticulum (ER) membranes and transit through the ER- to- Golgi intermediate

compartment (ERGIC), where interaction with N- encapsidated, newly produced genomic RNA results in budding into the lumen of secretory vesicular compartments. Finally, virions are secreted from the

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Animal origins of human coronaviruses.

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Animal origins of human coronaviruses

Severe acute respiratory syndrome coronavirus (SARS- CoV) is a new coronavirus that emerged through recombination of bat SARS- related coronaviruses (SARSr- CoVs). The recombined virus infected civets and humans and adapted to these hosts before causing the SARS epidemic. Middle East respiratory syndrome coronavirus

(MERS-CoV) likely spilled over from bats to dromedary camels at least 30 years ago and since then has been prevalent in dromedary camels. HCoV-229E and HCoV- NL63 usually cause mild infections in immunocompetent humans. Progenitors of these viruses have recently been found in African bats, and the camelids are likely intermediate hosts of HCoV-229E. HCoV- OC43 and HKU1, both of which are also mostly harmless in humans, likely originated in rodents. Recently, swine acute diarrhoea syndrome (SADS) emerged in piglets. This disease is caused by a novel strain of Rhinolophus bat coronavirus HKU2, named SADS coronavirus (SADS- CoV); there is no evidence of

infection in humans.

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Cross-species transmission of coronaviruses

a | Severe acute respiratory syndrome (SARS)-like bat coronavirus (BtCoV) spread and adapted to wild animals such as the Himalayan palm civet that was sold as food in Chinese wet markets. The virus frequently spread to animal handlers in these markets, but caused minimal or no disease. Further adaptation resulted in strains that replicated efficiently in the human host, caused disease and could spread from person to person.

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SARS-CoV-2

SARS- CoV-2 shared 79.6% nucleotide identity with SARS- CoV and close relations to severe acute respiratory syndrome- related coronaviruses (SARSr- CoVs) ZC45 and ZXC21 from Rhinolophus sinicus, whereas RaTG13 from Rhinolophus affinis showed the highest nucleotide similarity of 96.2%

The intermediate host

The example of pangolin CoV MP789, which shared five essential amino acids for ACE2 binding in the S with SARS- CoV-2 highlights the existence of a variety of unidentified betacoronaviruses in wildlife animals and their roles as possible intermediate hosts

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The Potential Intermediate Hosts for SARS-CoV-2

The potential transmission of SARS-CoV-2 between hosts and humans. SARS-CoV-2, originated from bat-nCoV, infected wild animals and gradually evolved in the intermediate host after mutation and recombination. Wildlife business give chance for SARS-CoV-2 to infect humans and domestic

animals.

SARS-CoV-2 induce a pandemic in human population by respiratory droplet transmission and close contact transmission.

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19/11/20

Biologia, zoonosi e filogenesi dei Betacoronavirus umani: SARS- CoV,

MERS-CoV e SARS-CoV-2. _{Pagina 20}

SARS-CoV

2002

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Comparison with SARS-2003

There are major differences between SARS-CoV-2 and SARS-CoV 2003 time course. The most important is that SARS 2003 viral shedding starts three to four days after symptom onset, whereas in COVID-19 people can propagate the virus two days before symptom onset. Of note, SARS-CoV-2 replicates more efficiently at temperatures

encountered in the upper respiratory tract than SARS 2003

The early spreading of SARS-CoV-2 contributed to the failure of control measures taken to prevent the pandemic. Isolating symptomatic patients and their relative had proven effective for SARS-2003, but SARS-CoV-2 patients spread the virus much earlier.

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NATURE REVIEWS | MICROBIOLOGY Ottobre 2020

Targets for

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Treatment of COVID-19

SARS-CoV and SARS-CoV-2 cell recognition/interaction

Coronavirus entry into host cells is mediated by the transmembrane spike (S)

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SARS-CoV-2 Spike protein

S comprises two functional subunits responsible for binding to the host cell receptor (S1 subunit) and fusion of the viral and cellular membranes (S2

subunit). For many CoVs, S is cleaved at the boundary between the S1 and S2 subunits, which remain non-covalently bound in the prefusion conformation. The distal S1 subunit comprises the receptor-binding domain and contributes to stabilization of the prefusion state of the membrane-anchored S2 subunit that contains the fusion machinery. For all CoVs, S is further cleaved by host proteases at the so-called S2’ site located immediately upstream of the fusion peptide. This cleavage has been proposed to activate the protein for

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SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2

and Is Blocked by a Clinically Proven Protease Inhibitor

Highlights

• SARS-CoV-2 uses the SARS-CoV receptor ACE2 for host cell entry

• The spike protein of SARS-CoV-2 is primed by TMPRSS2

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SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2

and Is Blocked by a Clinically Proven Protease Inhibitor

Schematic illustration of SARS-S including functional domains (RBD, receptor binding domain; RBM, receptor binding motif; TD, transmembrane domain) and proteolytic cleavage sites (S1/S2, S2ʹ). Amino acid sequences around the two protease recognition sites (red) are indicated for SARS-S and SARS-2-S (asterisks indicate conserved residues). Arrow heads indicate the cleavage site. SARS-CoV-2 uses the serine protease TMPRSS2 for S protein priming at S’ site and is independent from the endosomal cysteine

proteases cathepsin B (CatB) and CatL. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option.

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•Letter

•Published: 03 December 2020

Therapeutically administered ribonucleoside analogue MK-4482/EIDD-2801 blocks SARS-CoV-2 transmission in ferrets

•Robert M. Cox, Josef D. Wolf & Richard K. Plemper

Nature Microbiology (2020)

Approved antiviral treatments such as remdesivir and reconvalescent serum cannot be delivered orally2,3_{, making them poorly suitable for transmission control. We previously} reported the development of an orally efficacious ribonucleoside analogue inhibitor of

influenza viruses, MK-4482/EIDD-2801 (refs. 4,5_{), that was repurposed for use against severe} acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is currently in phase II/III

clinical trials (NCT04405570 and NCT04405739). Here, we explored the efficacy of

therapeutically administered MK-4482/EIDD-2801 to mitigate SARS-CoV-2 infection and block transmission in the ferret model, given that ferrets and related members of the

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Diagnostic testing and screening for SARS-CoV-2

qRTPCR

N gene (Pan Corona virus - screening) E gene (2019-nCoV specific target gene)

RdRP/S gene (2019-nCoV specific target gene)

Antigen tests detect the presence of a viral antigen, typically part of a surface protein.