- SARS-CoV-2 viral infection was found to not significantly trigger IFN expression and only activated 5 of 13 pro-inflammatory mediators, which play important roles in the body's innate immune response. The virus possesses an gene identified as ORF3b that produces an IFN-antagonizing protein which inhibits synthesis and signaling and leads to unchecked viral replication.

- Virus entry triggers the generation of pro-inflammatory cytokines (IL-6, IP-10, macrophage inflammatory protein 1α (MIP1α), MIP1β and MCP1) which recruit monocytes, macrophages, and T-cells to the sites of infections. This promotes further inflammation and creation of a feedback loop leading to a cytokine storm.

Manipulation of Innate Immune and Pro-inflammatory Response

1. Entry through the naval cavity: because intranasal drug delivery bypasses the blood brain barrier to delivery therapeutic agents directly to the CNS, it is thought that SARS-CoV-2 can likewise infect the brain via olfactory nerves.
  
2. Entry through the lungs: previous speculation that SARS-CoV could infect the , brainstem via chemoreceptors and mechanoreceptors in the respiratory system lend possibility for a neurotropic mechanism for SARS-CoV-2.

These hypotheses have yet to be validated.  
Figure below shows coronavirus infection mechanisms (Wu et. al).

Hypothesized Mechanisms for SARS-CoV-2 Direct Invasion of the CNS 

- Transcription works through the replication-transcription complex (RCT) organized in double-membrane vesicles via synthesis of sgRNA sequences.
- Different CoVs present special structural and accessory proteins translated by dedicated sgRNAs. 

Diagram of SARS-CoV-2 Replication in Host Cell

Upon receptor recognition, the viral envelope will fuse with the host cell membrane or by receptor mediated endocytosis by the spike protein. Once inside the host cytoplasm, the viral RNA is released with ORF1a and ORF1ab translated to make 2 polyproteins: PP1a and PP1ab. In order to make 2 distinct polyproteins, the viral RNA has a slippery sequence (5’-UUUAAAC-3’) and a RNA pseudoknot secondary structure that will cause frameshift activity in the ribosomes, analogous to attenuation in bacterial Tryptophan operon. When the ribosome fails to unwind the psuedoknot in time, it will pause at the slippery sequence and move back by one nucleotide, causing a -1 frameshift before resolving the secondary structure. This upstream frameshift is what gives rise to the second polyprotein PP1ab. Interestingly, this delay in psuedoknot resolution occurs 25% of the time to create a stoichiometric 3 to 1 ratio of PP1a and PP1ab. This ratio might be the ideal stoichiometric balance for efficient viral replication and assembly (Baranov et al. 2005).

After translation of PP1a and PP1ab, these precursors are then cleaved by proteases to form 16 nsps that will associate into the RNA Replicase-transcriptase complex localized in bilayer vesicles derived from the ER to produce the genomic (–) RNAs. The (¬–) RNAs are transcribed and replicated discontinuously to give a diverse pool of subgenomic (+) mRNAs/RNAs. During this process, there are many cis-regulatory elements that strictly regulate different stages of RNA synthesis. For instance, the 5’UTR of the genome consists of 7 stem-loop structures that invade into the replicase 1a gene (Raman et al., 2003). Meanwhile, the 3’UTR end consists of the bulged stem-loop and the pseudoknot sequences that overlap. In other words, both secondary structures cannot form simultaneously, implying that different structures are formed in a time-dependent manner to modulate different stages of replication and translation (Liu et al., 2001).


Infection Pathway & Mechanism

- Manipulation of Innate Immune and Pro-inflammatory Response

University of California, Davis

University of Michigan - Ann Arbor

Smith College

- Animal Studies on SARS-CoV-2 Virology
- Antigen Detection
- Classification
- Evolution and Mutations
- Methods of Host Cell Infection and Exploitation
- Stability in different environmental conditions
- Structure
- Vaccine Candidates, Immunogenicity, and Adaptive Immunity
- Reinfection

Focuses of Virology of COVID-19

SARS-CoV-2 is comprised of four main structural components: the spike glycoprotein (S), the small envelope glycoprotein (E), the membrane glycoprotein (M), and the nucleocapsid protein (N). There are also several accesory proteins. 

Structure of SARS-CoV-2

Methods of Host Cell Infection and Exploitation

A study found that SARS-CoV-2 can be highly sensitive to heat. The virus was still detectable after being left out for 14 days in a 4°C room, while the virus inactive 5 minutes after being left in a 70°C. The study also found a difference in stability on different surfaces, such as paper, plastic, and metal.  At room temperature, the virus is highly stable in a range of pH3 - pH10. However, the standard disinfectant method was still able to affect the virus, with the researchers finding no active infection in different disinfectants after an incubation of 5 minutes (except for hand soap).

Stability of SARS-CoV-2 in different environmental conditions

Investigations are still ongoing in determining the current evolution and mutating nature of SARS-CoV-2, which will aid scientists in understanding more about this virus and in developing effective treatments and vaccines against the disease. 

Evolution and Mutations of SARS-CoV-2

- Application of Rhesus Macaques for Studying SARS-CoV-2 Virology
- Application of Syrian Hamsters for Studying SARS-CoV-2 Virology
- Application of Mosquitoes for Studying SARS-CoV-2 Virology
- Application of African Green Monkeys for Studying SARS-CoV-2 Virology

Animal Studies on SARS-CoV-2 Virology

So far, vaccines consist of either inactivated or weakened versions of antigens or virions. Many pharmaceutical companies like Pfizer and Moderna are developing inactivated antigens in the form of mRNA, DNA, and proteins from the viral envelope or capsid that are often fragmented. 

Vaccine Candidates, Immunogenicity, and Adaptive Immunity

Until vaccine has passed all preclinical and clinical trials as well as FDA approval, the best approach to containing the pathogenicity and infectivity of COVID-19 will involve testing. The Safra center at Harvard advised to have up to 5 million tests being conducted within a day. Testing will not only slow down the spread, but also allow reopening of businesses and schools. So far, many testing methods involve viral RNAs or antigens. For detecting viral RNAs, most involve using RT-qPCR conjugated with Next Generation Sequencing to identify specific unique viral sequences. Recently, SHERLOCK detection technique utilizing CRISPR-Cas13 was tested in Broad Institute using a catalytically dead Cas13 to bind to specific viral sequences. On the other hand, detection of viral antigens involve either using ELISA with selected antibodies against viral antigens or using lateral flow assay.

Antigen Detection

Until August 28, 2020, there has been several cases of reinfections around the world (Hong Kong, Belgium, the Netherlands, and the US). 

Reinfection of SARS-CoV-2

The coronaviruses belong to the broad viral order Nidovirales within the family, Coronaviridae, notoriously known to be infecting mammals and humans in the recent years. In general, they can be commonly distinguished by their unique morphological structures of spherical or disk-like envelopes embedded with glycoproteins (Burrell et al., 2017). In terms of its genetic composition, it has a large positive sense RNA (33.5 kb) belonging to Group VI in the Baltimore classification. Other shared similarities include highly conserved genomes, Replicase gene upstream of structural and accessory genes as well as the translation of many nonstructural mRNAs from ribosomal frameshift. These viruses are very diverse and can be further subdivided into 4 subfamilies: alpha-, beta-, delta-, and gamma-coronaviruses based on serology (Fehr et al., 2015). 

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