COVID-19

Sequencing and analyzing an RNA virus genome (continued)

Isolate fluid that contains virus (and other stuff) --> Extract RNA --> Deplete host ribosomal RNA --> Reverse transcribe (remaining RNA into cDNA) --> Illumina sequencing: 150bp reads --> 56,565,928 reads --> Assemble into contigs --> filter out human sequences --> 23,712,657 --> Compare to known sequences --> Longest contig had high abundance and best alignment with coronavirus genomes --> Further analysis and confirmation with PCR and sequencing

Types of Coronavirus

- SARS (CoV1), SARS-CoV2

- MERS (Middle East Respiratory Syndrome Coronavirus)

Viral Replication Strategies

- Plus Strand RNA Virus Families

- Cause many serious diseases of animals and man

- Foot and mouth virus first animal virus described (1898).

SARS-CoV2

- Causes COVID-19 disease

- First identified late Dec 2019 in Wuhan, China on patients with pneumonia

- First genome sequence made publicly available Jan 10, 2020

- Metagenomic RNA sequencing of a spale of Bronchoalveolar lavage fluid patient

Covid-19 is a....

Betacoronavirus

- Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses Likely "zoonosis" from a bat

SARS CoV-1, MERS, and SARS CoV-2 likely all emerged from bats and were transferred to humans via..

- Intermediate host

- More than 500 CoVs have been identified in bats in China, with estimates of unknown CoV diversity reaching >5,000

SARS: 8,098 cases, 774 deaths (epidemic ended)

MERS: 2,521 cases, 866 total deaths (infections still occuring)

We were able to control CoV-1/SARS within one year but not CoV2/COVID19. Why?

How does SARS-CoV2 enter host cells?

- Genome sequence indicated the receptor-binding domain (RBDD) of SARS-CoV2 spike protein should interact with same receptor (angiotensin converting enzyme receptor ACE2) as SARS-CoV

Overall Structure of SARS-CoV1

- CoV-2 entry is driven via binding between Spike protein and Angiotensin-converting enzyme 2 (ACE2)

- Then, protease (TMPRSS2) cleavage (multiple) allows entry

How does SARS-CoV2 enter host cells?

- Genome sequence indicated the receptor-binding domain (RBD) of SARS-CoV2 spike protein should interact with same receptor (angiotensin converting enzyme receptor ACE2) as SARS-CoV

- Experimental support of this model just published

How do viruses enter host cells?

- Bind to cell surface receptors on host cells

- e.g. CCR5 gene and HIV/AIDS

How might ACE2R mutations affect human susceptibility to SARS-CoV2?

- Loss of function mutations would cause resistance to infection

Viral Life Cycle

1. Enter

2. Replicate

3. Exit

The 2019-nCoV genome was annotated to posses 14 ORFs enconding 27 proteins

One large RNA to translate two polyproteins by RNA structure, called Pseudoknot

- Programed ribosomal frameshifting generates two polyproteins coding for genes involved in replication

- A stop codon at the end of ORF1a is no longer read as a stop codon because of the slippery heptanucleotide

RNA Polymerase Gymnastic Allows generation of the CoV-2 structural and accessory proteins

- long Range RNA-RNA interaction

- Once polymerase codes cCS-B via long range RNA-RNA interaction

- JUMP JUMP

then..., (-) subgenomic mRNA templates have to become (+) sub genomic mRNA

- discontinuous transcription

- A very high recombination rates (up to 25%) due to RNA pol jumping

- Unique to SARS CoV-2 compare to other RNA viruses

Flavivirus Envelope Protein Translation and Processing

- A transmembrane insertion occurs at the ER during viral protein translation

- The capsid protein has a hydrophobic signal sequence at its C-terminus that translocates the prM (membrane precursor protein) into the ER lumen

- The capsid protein associates with viral RNA and assembles at the ER surface as an immature virion

- Double membrane vesicle

- SARS replication-transcription complexes (RTC)

Expression and Cleavage of Middle East REspiratory Syndrome Coronavirus nsp3-4 Polyprotein...

Induce the Formation of Double-Membrane Vesicles That Mimic Those Associated with Coronaviral RNA Replication