- 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

Research in the virology of COVID-19 encompasses several primary areas of study. These include understanding the physical structure of SARS-CoV-2, its classification among coronaviruses, and its continuous evolution and mutations. Key focuses also involve the mechanisms of host cell infection, the stability of the virus in various environmental conditions, and the potential for patient reinfection. Furthermore, applied research areas include antigen detection methods, animal studies on the virus, and the development of vaccine candidates to build adaptive immunity.

Areas of Study in COVID-19 Virology

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

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

This node surveys animal studies conducted to investigate SARS-CoV-2 virology and pathogenesis, including: - 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 - Study of the Application of Green African Monkeys

Animal Studies on SARS-CoV-2 Virology

Cases of SARS-CoV-2 reinfection have been documented globally, demonstrating that prior infection does not guarantee permanent immunity. Confirmed instances, verified through whole genome sequencing, have shown that individuals can be reinfected by phylogenetically distinct strains of the virus.

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). 

Coronaviruses Classification

SARS-CoV-2 antigen detection involves identifying viral proteins to diagnose a COVID-19 infection. Unlike nucleic acid tests that detect viral RNA, antigen detection primarily utilizes methods such as Enzyme-Linked Immunosorbent Assay (ELISA) with antibodies selected against specific viral antigens, or lateral flow assays. These rapid testing methods are a critical approach to containing the pathogenicity and spread of the virus.

SARS-CoV-2 Antigen Detection

SARS-CoV-2 vaccine candidates use several platforms. mRNA and DNA constructs encode viral antigens, adenoviral vectors express the spike protein, and protein-based candidates deliver spike-derived antigen directly. These candidates are evaluated for their ability to elicit adaptive immune responses, including neutralizing antibodies and T-cell responses.

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