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Figure 1. Major structural proteins of SARS-CoV-2.

The coronaviruses are the largest group in the Nidovirales, which posses some of the largest genomes of all RNA viruses, up to 33.5 kilobases (kb). SARS-CoV-2, like most coronaviruses, is an enveloped RNA virus with a diameter of 120nm. SARS-CoV-2 is a group 2B coronavirus. The name “coronavirus” derives from prominent club-like spike glycoproteins on the surface of the virus that resemble a crown. The single strand of positive-sense RNA that constitutes the viral genome is contained by the coronavirus capsid. The genome encodes 27 viral proteins. Most are transcribed and translated as polymers that must be cleaved before they can mature. Table 1lists the genes and their identities with other two coronaviruses, SARS-CoV and MERS-CoV. The major structural proteins have been studied extensively in the search for potential targets for vaccines and therapies to treat the virus (Figure 1) [1].

The SARS-CoV-2 encoded surface glycoprotein (S protein) includes a receptor binding domain (RBD) on subunit 1 (S1) that is largely unrecognized by immune systems in most human populations that have been examined. This novel RBD accounts for the high rate of susceptibility to the virus in human populations globally. Immune responses to the infection typically include robust production of IgG specific to the surface glycoprotein and memory B cells responsible for the synthesis of these antibodies persist long after serum levels of IgG subside. The S protein encodes a furin cleavage site (PRRAR motif) between the globular receptor-binding domain of S1 and stalk-fusion domain (S2) that is conspicuously absent in other coronaviruses in the 2B and is hypothesized to be a critical factor in the virulence of the SARS-CoV-2 variant. Studies that knocked out this cleavage site saw both increased replication in cell lines and attenuated pathogenesis in vivo. S protein is responsible for cell binding and entry to the host cell [4-6].

SARS-CoV-2 hemagglutinin-esterase dimer (HE protein) synergizes with S protein to promote binding infection as a receptor destroying enzyme and by maintaining the integrity and specificity of S. Researchers found that mutations in HE protein that decrease virion binding efficiency lead to mutations in S protein that compensate for the change in HE. Attachment to host cells is partially mediated by HA specificity to sialic acid receptors [7-9].

Inside the viral particle, the nucleocapsid protein (N protein) of SARS-CoV-2 stabilizes viral RNA by binding the amino-terminal domain (NTD) and the carboxyl-terminal domain (CTD). Once synthesized inside the host cell, the viral N protein possesses RNA interference (RNAi) activity during viral RNA replication in cultured cells. This viral resistance to post-transcriptional gene silencing of host cells likely also occurs in vivo and represents an important mechanism for successful replication. N protein also binds to the viral RNA transcriptase complex and participates in segregating and packaging nascent viral genomes into new viral particles [10, 11].

The envelope small-membrane protein (E protein) of SARS-CoV-2 is also highly conserved between SARS variants. While relatively small as a structural element of the virus, this protein is responsible for interfacing with host cell membranes to promote packaging and intracellular trafficking of new viral particles. The carboxylic-terminal's hydrophobicity is thought to be responsible for initiating host membrane infiltration of the envelope protein and has been modeled as a tetrameric viroporin-like structure. These novel hydrophobic moieties at the C-terminal of the envelope protein are suspected to contribute to some of the amyloidogenic properties of the virus that may serve as targets for research into therapeutics [14-16].

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Figure 2. SARS-CoV-2 Binding to Host Cell. Left panel: The host cell surface protein, Ace2 binds to viral S protein. TMPRSS2 cleaves S1/S2 of the viral S protein. The cleaved section of the S1 subunit is released and the Cend1 site of the S1/S2 is available. Right panel: The host cell surface protein, Nrp1, stabilizes the viral particle as it binds to the host cell.

The SARS-CoV-2 encoded membrane glycoprotein or matrix protein (M protein) shows strong sequence conservation across related coronaviruses. This is one of the more abundant proteins in the viral envelope and has been shown to interact with the S protein, N protein and E proteins during viral assembly and likely cooperates with E protein for viral entry and exit from host cells [15, 17, 18].

Key host factors to date for SARS-CoV-2 interaction with host cells include angiotensin-converting enzyme 2 (Ace2), transmembrane protease serine (TMPRSS2), and neuropilin-1 (Nrp1); they are promising targets for therapeutics (Table 2).

Host-encoded ACE2 initially recognizes and stabilizes the RBD of viral S protein's S1 subunit. Human membrane protein TMPRSS2 is responsible for priming SARS-CoV-2 before the viral particle can enter the host cell. TMPRSS2 cleaves between the “head” S1 and the “binding stalk” S2 of S protein and after that cleavage, most of the S1 domain is released. While this protease is sufficient, it is not necessary for infection by SARS-CoV-2 – research suggests that other proteases can cleave the S1/S2 furin site. Host protein Nrp1 contributes to tissue specificity of SARS-CoV-2 by stabilizing the viral particle on the host cell surface. The binding domain for NRP is the S protein's CendR, revealed by cleavage at the furin site at S1/S2 by TMPRSS2. This protein is abundant in the central nervous system (CNS) as well as the respiratory and olfactory epithelium and has been shown to potentiate SARS-CoV-2 infection by enhancing viral particle binding in tissues where it is abundant (Figure 2) [12, 13, 13, 19-21].

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Figure 3. Life cycle of SARS-CoV-2 in the Host Cell. A. Cell recognition and binding; B. Phagocytosis of viral particle; C. Release of naked capsule. D. Translation of viral genome by host ribosomes, proteolysis of nascent viral polyproteins and maturation of RNA dependent RNA polymerase (RdRp); E. Transcription of viral mRNAs from viral genome; F. Translation of viral structural proteins in the endoplasmic reticulum (ER) and transport to golgi; G. Recruitment of E and N proteins by M protein to package viral genome and assemble viral particle; H. Release of new viral particle.

The genomes of coronaviruses share consistent organization, typically with two overlapping open reading frames. Contained within these two coding regions are suites of structural and non-structural proteins with considerable sequence homology to other coronaviruses of interest from the past 2 decades. The low mutation rate for SARS-CoV-2 is attributed to Nsp14, which is responsible for both methyltransferase activity and 3'-5' exoribonuclease activity that flag nascent RNA and operate as a correction mechanism for mismatch (and incidentally resistance to antivirals like Ribavirin). Within their highly conserved genomes and genomic organization, are many potential targets for therapeutics [22, 23].

Like many of its relatives and as previously mentioned, SARS-CoV-2 must be primed by a host protease before it can interact with host cells. The human protease responsible for priming SARS-CoV-2, TMPRSS2, was previously studied for its role in prostate cancer and infection by influenza; drugs inhibiting its activity are being investigated as potential treatments for SARS-CoV-2. Once the S1 subunit has been cleaved and the S2 subunit has bound to the cell, the SARS-CoV-2 virion sheds its viral envelope and the naked capsid, consisting of N protein and viral genome enters the cell (Figure 3) [24, 25].

The positive-sense RNA genome of the SARS-CoV-2 virus functions as a messenger RNA (mRNA) once inside the cell and includes a 5'-cap and poly-A tail at the 3' end. The two relatively large imbricate ORFs encode all of the structural and non-structural proteins for new viral particles, of which the non-structural proteins are translated first by host ribosomes, including a large positive sense RNA dependent RNA polymerase (RdRp) that is transcribed before most other structural or accessory gene. The first ORF, 5'-. encodes nsp1, nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, and nsp10 and nsp12, nsp13, nsp14, nsp15, and nsp16 and the structural proteins, S, 3a, 3b, E, M, P6, 7a, 7b, 8b and N (9b and orf14) to 3'- in the second, separated by a ribosomal frameshift region [8].

Once inside the host and while creating new viral particles, SARS-CoV-2 evades the immune system in a number of ways. Early studies showed that SARS-CoV-2 does not induce a robust type I interferon (IFN) response; this is typically an early immune response to viral infection by healthy immune systems. Subsequent research has shown that the papain-like protease (PLpro), [26] of SARS-CoV-2 is bifunctional and is at least partly responsible for suppressing IFN response. The primary activity of PLpro is generating mature nonstructural proteins, nsp1, nsp2 and nsp3, by cleaving the viral polyprotein after translation by host ribosomes. The secondary activity of suppressing IFN response is bimodal. PLpro hydrolyzes chains of ubiquitin that would otherwise signal IFN response and also by cleaving modifications made to viral proteins by interferon-stimulated gene 15 (ISG15) which suppresses active and subsequent antiviral responses. Many of the non-structural proteins of SARS-CoV-2 also function as interferon agonists; Nsp1, Nsp3, Nsp12, Nsp13, Nsp14, Orf3, Orf5, Orf6 and M protein are all involved in suppressing interferon-beta generally and specifically in response to Nsp2 and S protein. Nsp1 also interferes with host cell transcription by recruiting 40s and 80s ribosomal subunits to viral RNA while blocking mRNA entry sites for host mRNA – this promotes host translational machinery's activity on viral replication to the exclusion of a local host anti-viral response. These activities that counter host processes are thought to be more recent adaptations; Nsp13 has RNA helicase activity in addition to this immunosuppressive adaptation, for example [27-32].

Newly synthesized structural protein E is involved in the assembly and budding of new viral particles in cooperation with N and M proteins. M protein forms a scaffolding for E and N proteins; the presence of N ensures viral RNA is packaged, stabilized by N and the E protein and the E protein promotes budding of new viral particles. While most of these processes offer opportunities to interfere with the pathogen, S protein has been the focus of most therapeutics and vaccine trials [33].

Despite significant viral genomic material dedicated to evading and countering immune response, a suite of cytokines that promote inflammation including tumor necrosis factor (TNF) α and interleukin (IL) 1β, monokine induced by interferon-gamma (MIG), interferon-inducible protein 10, and others either in concert with the virus itself or on their own contribute to damage at the tissue and organ level. Some treatments focus on tempering the immune response, like with corticosteroids or antibodies developed to sequester IL variants, specifically IL-6, but imprecise attempts to dampen the immune response may lead to more damage by the unimpeded virus [34, 35].

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Figure 4. Physiological Effects of Viral Infection. A. Initial infection by SARS-CoV-2; B. Invasion by SARS-CoV-2 of olfactory nerve after damage to the nasal mucosa and/or increased BBB permeability due to prolonged inflammation and SIRS; C. Anosmia usually precedes more serious symptoms and resolves within weeks, but is usually not attributed to olfactory nerve damage; D. Acute respiratory distress syndrome (ARDS) from prolonged inflammation and severe edema increases hypoxia and is exacerbated by pneumonia; E. Systemic inflammatory response syndrome (SIRS) often termed a “chemokine storm” of inflammatory signal molecules; F. Heart and lung damage may be caused and complicated by a number of factors; G. Lung damage is usually caused by a narrower suite of more direct factors relative to heart and brain damage.

Tissue tropism of SARS-CoV-2 is thought to be largely dictated by Ace2, TMPRSS2 and Nrp1, but there are data to suggest that these factors are merely sufficient and not necessary. For example, gastrointestinal (GI) symptoms are more common in relatives of SARS-CoV-2 like Middle Eastern Respiratory Syndrome (MERS) which binds receptors common to the GI tract, but SARS-CoV-2 cases also occasionally include symptoms of GI involvement. Tests have detected viral particles in human excreta in about 2/3 of patients regardless of the presence of GI symptoms, persisting after nasopharyngeal swabs tested negative for the virus [36-38].

Clinical signs of severe response to SARS-CoV-2 infection include conventional signs of shock, like weak pulse and low body temperature, even when low blood pressure is absent. Coagulation due to increase in blood levels of D-dimer is also a hallmark of infection with SARS-CoV-2 and has resulted in clot related events like clot-induced ischemic events, notably in the heart, lungs and brain, as well as deep vein thrombosis leading to limb amputation in a troubling number of cases. In fact, blood concentrations of blood urea nitrogen (BUN) and D-dimer is a reliable predictor of morbidity and mortality either by themselves or with cytokines that indicate inflammation, like IL6, that also herald poor clinical outcomes and are indicators of systemic immune response syndrome (SIRS). Fortunately, these markers are relatively easy to assay and thromboprophylaxis can be administered, but the risk of persistent coronary artery disease caused by endothelial dysfunction remains. The risk of heart, lung and brain damage are confounded by the ability of the abundance of proinflammatory cytokines that also decreases plaque stability in blood vessels. This decrease in stability increases the risk of plaque rupture, creating another source of stroke and ischemic events in SARS-CoV-2 patients (Figure 4) [39-42].

Persistent CNS involvement after recovering from an active infection with SARS-CoV-2 similar to coronaviruses - SARS and MERS, is a notable quality of this group of viruses. Tissue tropism dictated by the presence of Ace2, TMPRSS1 and Nrp1 suggests that the CNS are potential targets for infection by SARS-CoV-2. Data from 2002-2003 SARS patients showed that SARS viral particles could access the brain, particularly neurons, and post mortem studies of SARS-CoV-2 shows both clinical signs like inflammation and edema as well as the presence of viral particles. Some of the brain injuries seen in SARS-CoV-2 patients may be due to decreased blood oxygen caused by direct damage by the virus to the tissue of the lungs and compounded by immune response and SIRS; this would both increase inflammation and edema in the brain while also increasing the risk that viral particles would cross the blood brain barrier by reducing the structural integrity of the barriers themselves via vasodilation. This process would also recruit materials from both the hematogenic and lymphatic system to the area along with the signal cascade of pro-inflammatory cytokines that accompany them. There are also data that suggest SARS-CoV-2 invades peripheral nerves and travels to the CNS via synaptic transfer and that the virus can enter the CNS through olfactory neurons, despite the conspicuous absence of Ace2 receptors on this particular class of neurons. While direct injury to tissues from infection with SARS-CoV-2, aberrant clotting, viral travel via neuronal pathways (especially through motor transport on dyenin and kinesins) and resulting demyelination, hypoxia, immune response and damage to Ace2 can all cause persistent brain injury via disruption of homeostasis normally dictated by renin–angiotensin–aldosterone system (RAAS) signaling. RAAS signaling perturbed by angiotensin II would promote prothrombotic activity and related ischemic events in the brain. It is likely that most or all of these factors contribute and possibly coact to amplify long term effects of SARS-CoV-2 on the CNS [43-47].

The effect of SARS-CoV-2 on the CNS may be partially to blame for its impact on the respiratory system. Many of the patients with severe SARS-CoV-2 are intubated because they are unable to breathe on their own. The impact of inflammation and edema, mediated both by the immune system at large and locally by microgilal cells, is known to impair the neural aspects of ventilary control. Reduced sensitivity to hypercapnic and hypoxic signals, decreased capacity for rhythm generation and diminished plasticity resulting from intermittent hypoxia and sustained inflammation and edema may leave patients particularly susceptible to erratic and inadequate breathing patterns. This is not to be conflated with damage done directly to the lungs themselves by the virus and inflammation and edema in the lungs and surrounding tissue, but rather a self-reinforcing feedback pattern where reduced lung capacity and reduced efficiency for gas exchange, also caused by inflammation and edema, leads to reduced control for breathing and sensitivity to relevant signals in the brain itself [48, 49].

Another contributor to brain and cardiac injury is RAAS-induced endothelial dysfunction. Proposed models suggest that increased angiotensin secretion in response to cell membrane stress leads to both vasoconstriction and the release of aldosterone which further raises blood volume and blood pressure as sodium is absorbed in the kidneys. It remains unclear as to whether modulating RAAS signaling pharmacologically would decrease the likelihood of neural and cardiac damage or if it would exacerbate the pathogenicity of the virus itself; animal models have yielded enticing results, but data from clinical trials are lacking. The involvement of the Ace2 receptor complicates therapeutic approaches to clinical trials due to the risk that both up- and downregulating the receptor may have deleterious results as one would increase susceptibility to the virus and the other would exacerbate SIRS [50-52].

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Figure 5. Vaccine Design Principle for S protein. 1. Neutralizing antibodies prevent receptor binding at host cell surface by blocking S protein from interacting with ACE2 and other relevant host proteins; 2. Antibody that binds S protein and FcγR (FcR). After binding viral particles on the Fab domain, the antibody then stimulates FcR via interaction with its Fc domain to promote immune response by activating immunoreceptor tyrosine-based activation motifs (ITAMs). This activates Toll-like receptors (TLR), usually TLR3, TLR7 and TLR8 in the endosome, regulates pro-inflammatory and anti-inflammatory cytokines. Note – there is a risk that non-neutralizing antibodies facilitate virus entry to cells, particularly immune cells, resulting in antibody-dependent enhancement of infection.

Early in the SARS-CoV-2 pandemic, loss of taste and smell was reported as an early symptom of infection by the virus in patients a range of seriousness of symptoms as well as in those who were otherwise asymptomatic. This anosmia persists for weeks after the infection has been cleared from the body and patients no longer test positive for the virus. Anosmia resulting from damage directly to olfactory sensory neurons (OSNs) typically resolves on the order of months and OSNs do not express relevant receptor proteins, TMPRSS2 and Ace2, that facilitate infection by SARS-CoV-2. While the specific mode by which SARS-CoV-2 interrupts the ability to smell and taste is yet unknown, researchers have hypothesized that this symptom might result from damage to support neurons for the OSNs. Co-occurrence of TMPRSS2 and Ace2 in sustentacular cells of the olfactory epithelium, and the early secretory, secretory and ciliated cells of the respiratory epithelium suggests that damage to these tissues by SARS-CoV-2 and the associated immune response may be responsible for the anosmia associated with the virus. Still, damage to OSNs themselves from demyelination may be to blame [46, 53].

While we will not fully appreciate the breadth and depth of long term effects of surviving SARS-CoV-2 until conditions allow for resumption of regular visits to health care providers, we can look to other coronaviruses for clues about what to expect in the years following the SARS-CoV-2 pandemic. Follow up studies of SARS patients in Hong Kong suggested that the rate of cognitive and physical effects is alarmingly high four years after contracting the virus, more than a full order of magnitude greater than before contracting the virus. While some of the psychiatric illness may be a result of the mental trauma associated with measures enforced to control the spread of the virus and taken to treat the illness as well as stigma surrounding surviving SARS, the origin of fatigue is less obvious. Just over 40 percent reported chronic fatigue and nearly a third met CDC criteria for chronic fatigue syndrome (CFS). The direct and indirect effects of these comorbidities on productivity and the economy is staggering. Chronic fatigue syndrome has been estimated to reduce an individual's household productivity 37% and contribution to the labor force by 54%. For example, assuming the pre-SARS-CoV-2 rates of CFS conservatively at about half a million Americans and the loss in productivity at $20K per person, this accounts for $10B in lost productivity. Assuming just one in ten of the 20M Americans who have tested positive for SARS-CoV-2 develop CFS, we can anticipate an additional loss of $40B in productivity. Now consider that as of this writing, 90M cases have been reported worldwide [54, 55]. The causes of these health outcomes remains to be seen, but ideally, understanding the relevant pathways will inform our ability to treat survivors of SARS-CoV-2, among them is the viral load which may be a predictor of morbidity and mortality. Microvascular injury is often present in severe SARS-CoV-2 infections, but this cannot account for fatigue, reduced cognitive ability and other common persistent symptoms found in asymptomatic patients or those with mild illness [56-59].

Early studies of SARS-CoV-2 survivors suggest that the persistent effects of SARS-CoV-2 are also common. Italian survivors reported fatigue, difficulty breathing or dyspnea, joint pain and/or chest pain two months after testing negative for the virus. Only 12.6% of participants in the survey were completely free of symptoms. More than half were still experiencing at least three symptoms and most (63%) of those were men. Fatigue and difficulty breathing were the most common persistent symptoms in this cohort at 53 and 43% respectively. Joint pain and chest pain were less common, but still alarmingly high at 27 and 21.7% respectively [60]. One US study submitted to the New England Journal of Medicine surveyed patients who had been hospitalized for SARS-CoV-2 and reported that 94% were still experiencing symptoms including cough, fatigue and dyspnea 2-3 weeks after being cleared of infection [61]. Another suggested that more than half suffered fatigue that persisted a median of 10 weeks after patients had been cleared of the virus and that there was no association between the severity of their infection and the likelihood of this outcome. This study suggested that mental health prior to SARS-CoV infection was correlated with fatigue after recovery [62]. One UK survey of predominantly female participants suggested a lower rate at 10% of symptoms consistent with those observed in Italy and the US. These studies also differ in that most focus on inpatients diagnosed with SARS-CoV-2 and not asymptomatic and outpatients [63]. Another study from the UK examining the cardiovascular, CNS and neuropsychiatric outcomes including acute psychosis without prior history of like psychiatric disorders for SARS-CoV-2 survivors and found that augmented cognition was the second most common long-term effect after fatigue [64]. Studies have also focused on mental health outcomes for patients with SARS-CoV-2 as well as their caregivers. Interestingly, quarantine itself was not associated with worse mental health outcomes, but depression, anxiety and post-traumatic stress disorder were significantly increased in the affected groups and a another study revealed that cognitive behavioral therapy (CBT) may not be sufficient to address the issues associated with living though a pandemic, particularly CFS. Without a meaningful treatment for CFS and for countries who chose to forego masking and contact tracing, vaccination remains the greatest defense against compounding economic woes due to undesirable physical and mental health outcomes for survivors of SARS-CoV-2 [65-68].

Another often mentioned impact of SARS-CoV-2 is its effects on the reproductive capacity of survivors. Studies have shown that motility and concentration of sperm is reduced and that it also impacts the quality of oocytes. The virus can also be transmitted vertically and through breast milk. These data confirm that the CNS and respiratory tract are only the most notable systems affected by infection by SARS-CoV-2 and that other tissues known to be susceptible to the virus will likely present observable impacts long term.

Changes in the strain further complicate our collective efforts to manage the pandemic. Viruses are certainly known for their ability to mutate and adapt in real time and SARS-CoV-2 is no exception. Coronaviruses mutate more slowly than some other viruses due to built-in proofreading that also confers resistance to some antivirals. However, because SARS-CoV-2 has infected so many people, it has had no dearth of opportunities to establish new variants. After the establishment of a reference sequence, a number of variations have been identified [69, 70]. Some variants have been examined as more infectious than others and bioinformatic studies have explored genetic contributions to infectivity of SARS-CoV-2. These data suggested that a variant with a higher R0 was inevitable [71-73]. Virus containing the N501Y variation (lineage B.1.1.7) is estimated to be at least 50% and as much as 80% more infectious than 501N. Children may also be more susceptible to the N501 variant. Another spike protein variant, D614G, also may be more contagious than the wild type, similar to N501Y. Notably, while D614G induces a robust antibody response in animal models, viral titers, another predictor for outcomes, were higher for this strain in hamster specifically in the upper respiratory tract which may account for the heightened virulence [74-78].

While the N501Y variant of SARS-CoV-2 is more virulent than its 501N predecessor, history suggests that current vaccines will continue to provide effective protection, though it is not predicted to endure meaning that regular vaccination against homologous strains may be necessary. Vaccines developed against SARS and MERS using the S protein as an antigen were effective in generating immune responses in animal models and clinical trials and that antibodies were being produced a year later though at greatly reduced levels. Studies observing specific antibody responses to experimental coronavirus infections showed that some patients could be reinfected with the virus despite the presence of specific antibodies. While antibody levels peaked about a week after the challenge, they slowly dissipated thereafter [79-83].

Whatever the end result of this pandemic, the precision of and vigilance surrounding the collective response will have meaningful impacts for decades to come on the individual and global scale.

More than 120 vaccine candidates for SARS-CoV-2 have been announced in 2020 and many are in phase one, two and three testing with promising results and others are available for distribution with emergency use authorization several developed nations. Approaches vary from traditional to novel, but many focus on the S protein of SARS-CoV-2; by creating an immune response that sequesters the S1 subunit, the binding of the virus to host cells and subsequent hijacking of host cell machinery for replication does not occur. Instead the immune system can neutralize viral particles and modulate a more appropriate immune response to a challenge by the virus (Figure 5). Approaches to vaccines include protein-based, RNA and DNA based, live attenuated viruses, replicating and non-replicating viral vectors, and inactivated SARS-CoV-2 [84-86].

Each platform has drawbacks and benefits. Using viral proteins, protein subunits or peptides to promote immune response, for example, traditionally has a good safety record, but researchers must identify the correct antigen to promote an immune response. An adjuvant may also be necessary to promote adequate immune response and all these factors are confounded by a slower rate of manufacture, depending on how the protein is either synthesized or isolated. Numerous protein vaccines for SARS-CoV-2 are currently in development including candidates from GlaxoSmithKline (ClinicalTrials.gov ID: NCT04405908) and a collaborative effort between the University of Queensland and the Coalition for Epidemic Preparedness (CEPI) (ClinicalTrials.gov ID: NCT04495933). Two peptide vaccines have registered for clinical trials, the Vector institute(ClinicalTrials.gov ID: NCT04527575) and Covaxx in partnership with the University of Nebraska Medical Center (ClinicalTrials.gov ID: NCT04545749).

Inactivated virus is a tried and true method for vaccine development and the known drawback is the risk that the vaccine could enhance disease by contributing to SIRS and other immune responses that perturb homeostasis. Still, the potential for rapid production makes this approach appealing. SinoPharm is working on such a vaccine, BBIBP-CorV, in partnership with the Wuhan Institute of Biological Products. Sinovac's “PiCoVacc” (ChiCTR2000031809) consists of inactivated virus and boasts a very low rate of side effects and good seroconversion [87, 88].

Live attenuated and live recombinant virus-based vaccines offer many of the pros of inactivated virus, but they do require careful consideration of the genetics of the virus and even when great care is taken to create an attenuated version that effectively generates the desired immune response, there is always the risk that the attenuated virus will revert to a virulent phenotype. The USA and Austrian collaboration between University of Pittsburgh, Merck, Institut Pasteur, and Themis have registered for clinical trials a live attenuated recombinant measles vector with a modified S protein (ClinicalTrials.gov ID: NCT04497298 and NCT04498247). Similarly, live recombinant viral vectors offer the benefit of rapid production, but may cause potentially serious side effects if the individual receiving the vaccine has pre-existing immunity against the vector. Several non-replicating viral vectors have earned headlines, including the collaboration between AstraZenica and the University of Oxford, the ChAdOx1-s nCoV vaccine (ClinicalTrials.gov ID: NCT04400838). Similarly, CanSino's vaccine candidate (ClinicalTrials.gov ID: NCT04313127) is a type 5 adenovirus vector expressing SARS-CoV-2 S protein [89-91].

Virus-like particles (VLPs) offer the benefits of protein conformations that are more similar to that of the infectious particle which can promote appropriate immunogenic responses, but are limited by slow production. Early data regarding NVX-CoV2373, a candidate from Novavax (ClinicalTrials.gov ID: NCT04533399) based on a trimerized S protein, suggests the candidate effectively generates a substantial immune response to SARS-CoV-2 in recipients, but with moderate to severe side effects. Other such candidates have been registered by the Shenzhen Geno-Immune Medical Institute (ClinicalTrials.gov ID: NCT04299724 and NCT04276896) and Avita Biomedical (ClinicalTrials.gov ID: NCT04386252) [92].

DNA vaccines are appealing because of their low cost and quick production, but are limited by their low immunogenic response. Inovio Pharmaceuticals has partnered with the International Vaccine Institute to study INO-4800a, a plasmid DNA-based vaccine. [93] Small activating RNA (saRNA) may be effective in lower doses at generating an immune response than mRNA. This seems ideal when combined with the relatively low cost and capacity for rapid production, but this class of vaccine is yet successfully tested in humans. The Imperial College in London with VacEquity Global Health (ISRCTN170726920 and Singapore's Arcturus (ClinicalTrials.gov ID: NCT04480957) have begun clinical trials with saRNA vaccine candidates [94].

Like saRNA-based vaccines, mRNA based vaccines offer both the relatively low cost of production along with potential for rapid manufacture. These vaccines are recognized by the body as foreign and therefore in effect self-adjuvant. Pfizer in partnership with German biotech company BioNTech has created the first mRNA based vaccine (ClinicalTrials.gov ID: NCT04368728), revolutionizing the field. Boston based Moderna announced just after Pfizer that their mRNA vaccine was also effective (ClinicalTrials.gov ID: NCT04470427 and NCT04283461). Each reports minimal side-effects, typically mild or moderate pain at the injection site along with swelling and redness in some patients, and high rate of efficacy at ~95%. Some patients reported systemic symptoms after vaccination, fatigue, headache, and chills were the most common. In Pfizer's trial, test patients each received two doses, 21 days apart of vaccine candidate BNT162b1, consisting of a nucleoside-modified mRNA encapsulated in a lipid-nanoparticle coating. The mRNA encodes a trimerized S1 subunit, the receptor-binding domain of SARS-CoV-2 S protein. Participants who tested positive for SARS-CoV-2 achieved 1.9-4.6 fold titers of neutralizing antibodies relative to sera from human convalescent patients. Moderna's candidate, mRNA-1273, similarly encodes SARS-CoV S protein in an mRNA coated with an ionizable lipid, cholesterol, distearoyl phosphatidylcholine, and polyethylene glycol envelope. Participants in Moderna's trial saw similar side effects and rates of immune response to their vaccine. Both vaccines rely on the mRNA being successfully transported into host cells where the target antigen, the S protein of SARS-CoV-2, is synthesized [86, 95].

COVID-19 Rapid Antibody Test Kit from 20/20 BioResponse [96]. Recombinant rabbit monoclonal antibody against SARS-CoV-2 is now available from Sino Biological. Active Motif isolated B-cells from Covid-19 patients and generated multiple recombinant monoclonal antibodies against SARS-CoV 2 spike protein including clones AM004414 and AM006415 that can be used in Western blot and clones AM004414 and AM001414 that can be used for neutralization, as shown in A549 lung epithelial cells expressing the ACE2 receptor with a pseudotyped virus containing the SARS-CoV-2 S1 spike glycoprotein and carrying a luciferase reporter gene. Rockland SARS nucleocapsid protein antibody 200-401-A50 was shown to cross-react with SARS-CoV 2 nucleocapsid protein in immunohistochemistry [97]. Absolute antibody clone CR3022 with many different host/class formats reacts with both SARS-CoV 2 and SARS-CoV S glycoproteins [98].

A Chandrashekar et al detected SARS-CoV-2 viruses from macaque sections with a rabbit anti-SARS-nucleoprotein antibody from NovusBio ( NB100-56576) [99].

S protein from Sino Biological has been used in ELISA assays for detecting S protein antibodies from macaque serum [99]. Sino Biological N protein was used in antibody-dependent cellular phagocytosis, antibody-dependent neutrophil phagocytosis and antibody-dependent complement deposition assays [99].

C Kreer et al detected anti-SARS-Cov 2 IgG antibodies from patients with SARS-CoV-2 detection ELISA kit from Euroimmun [100].

A Grifoni et al indicate their willingness to share the SARS-CoV-2 virus-specific CD4 and CD8 T cell epitope peptide pools in their article [96].

See the detailed instruction here https://www.cdc.gov/coronavirus/SARS-CoV-2/lab/rt-pcr-detection-instructions.html. Primers and probes for Real-time RT-PCR.

• TaqPath™ 1-Step RT-qPCR Master Mix, CG (ThermoFisher; cat # A15299). See Thermo Fisher dedicated webpage for the genetic analysis solutions for SARS-CoV-2 here.
• Acceptable surface decontaminants: DNAZap (Life Technologies, cat. # AM9890); DNA Away (Fisher Scientific; cat. # 21-236-28); RNAse Away (Fisher Scientific; cat. # 21-236-21)
• RNA extraction kits: bioMérieux NucliSens systems, QIAamp Viral RNA Mini [101], QIAamp MinElute Virus Spin Kit [99] or RNeasy Mini Kit (QIAGEN), EZ1 DSP Virus Kit (QIAGEN), Roche MagNA Pure Compact RNA Isolation Kit, Roche MagNA Pure Compact Nucleic Acid Isolation Kit, Roche MagNA Pure 96 DNA and Viral NA Small Volume Kit, and Invitrogen ChargeSwitch Total RNA Cell Kit. Others such RNA-STAT 60 from Tel-test [99] have been also used by researchers.
• PCR machine: AB 7500 Fast DX [99], QIAGEN Rotor-Gene® Q 5-plex real-time PCR cycler [101].
  SARS-CoV-2 isolates

  BEI Resource provides multiple isolates including USA-WA1/2020 , Hong Kong/VM20001061/2020, Italy-INMI1, England/02/2020, Singapore/2/2020, Germany/BavPat1/2020, USA-IL1/2020, USA-CA1/2020, USA-AZ1/2020, USA-WI1/2020, USA-CA3/2020, USA-CA4/2020, USA-CA2/2020, Chile/Santiago_op4d1/2020, New York-PV08410/2020, New York-PV08449/2020, and New York-PV09158/2020. BEI Resources, in addition, provides other SARS-CoV-2 reagents such as inactivated organisms, genomic RNA, ACE2 cell lines, monoclonal antibodies, convalescent Sera, spike and nucleocapsid proteins and their fragments, peptide arrays, and plasmids for various SARS-CoV-2 genes. A Chandrashekar et al inoculated rhesus macaques with USA-WA1/2020 isolate from BEI Resource (NR-52281) [99].

  C-Tan-nCov Wuhan strain 01 and C-F13-nCoV Wuhan strain 02. Contact: ivdcolm@ivdc.chinacdc.cn; chpc@chinacdc.cn

  WHO SARS-CoV-2 detection protocol

  Diagnostic detection of SARS-CoV-2 by real-time RT-PCR. https://www.who.int/docs/default-source/coronaviruse/protocol-v2-1.pdf. An article describing the establishment and evaluation of this protocol has been published [102].

  Diagnostics controls for SARS-CoV-2

  European Virus Archive Global (EVAg), a European Union infrastructure project: https://www.european-virus-archive.com/bundle/diagnostics-controls-wuhan-coronavirus-2019-SARS-CoV-2