genome structure ,replication,entry,corona virus test and treatment

2. TOPIC: PHENOTYPE AND
GENOTYPE OF COVID 19
COURSE TEACHER-
Dr.ANITA PETER
PROFFESOR
DEPT OF PLANT BIOTECHNOLOGY
PRESENTED BY-
BHARATI.G.S.
I.D. NO- PALB9299
DEPT. OF PLANT BIOTECHNOLGY
UAS ,GKVK,BANGALORE.
2

3. Coronaviruses are a group of
related RNA viruses that cause
diseases in mammals and birds.
 In human viruses cause respiratory
tract infections that can range from
mild to lethal.
Mild illnesses include some cases of
the common cold (which is caused
also by certain other viruses,
predominantly rhinoviruses), while
more lethal varieties
include SARS, MERS, and COVID-19.

4. Genera:: Betacoronavirus.
Corona : prefix comes from the
latin word for crown.
 subfamily: orthocoronavirinae
family - Coronaviridae
order - Nidovirales
realm - riboviria
Genera- Alphacoronavirus,
Betacoronavirus,
Gammacoronavirus
Deltacoronavirus
Human corona viruses are alpha
and Beta coronaviruses.
Genome size :26-30kb

5. HISTORY

7. Severe Acute Respiratory Sysndrome(SARS)-2002/2003
Guangdong province of china
Transmitted from bats to civets to humans
Began in February 2003 and outbreak lasted until July 2003
More than 8000 total cases ,774 deaths.
Middle East Respiratory Syndrome(MERS)-2012
Saudi Arabia
Betacorna virus
Transmitted from camels to humans
More than 2400 cases ,858 deaths.
2019 Novel corona virus (2019-nCoV)
Wuhan in china
Transmitted from snake or bats.

9. Corona viruses are large,
roughly spherical, particles
with bulbous surface
projections.
 The average diameter of
the virus particles is around
125 nm (.125 μm).
 The diameter of the
envelope is 85 nm and
the spikes are 20 nm long.
structure

10. Coronaviruses are large, roughly spherical, particles with
bulbous surface projections.
 The average diameter of the virus particles is around
125 nm (.125 μm).
 The diameter of the envelope is 85 nm and the spikes are
20 nm long.
The envelope of the virus in electron micrographs appears as
a distinct pair of electron-dense shells (shells that are
relatively opaque to the electron beam used to scan the virus
particle).
Cross sectional model of corona virus

11. The viral envelope consists of a lipid bilayer, in which the (M), (E)
and (S) structural proteins are anchored.
 The ratio of E:S:M in the lipid bilayer is approximately 1:20:300.
 On average a corona virus particle has 74 surface spikes.
A subset of corona viruses (specifically the members
of betacoronavirus subgroup A) also have a shorter spike-like
surface protein called hem agglutinin esterase (HE).
 The coronavirus surface spikes are homotrimers of the S protein,
which is composed of an S1 and S2 subunit.

12. The homotrimeric S protein is a class I fusion protein which
mediates the receptor binding and membrane fusion between
the virus and host cell.
The S1 subunit forms the head of the spike and has the
receptor binding domain (RBD).
The S2 subunit forms the stem which anchors the spike in the
viral envelope and on protease activation enables fusion with
the host cell.

13. The E and M protein are important in forming the viral
envelope and maintaining its structural shape.
Inside the envelope, there is the nucleocapsid, which is formed
from multiple copies of the nucleocapsid (N) protein, which are
bound to the positive-sense single-stranded RNA genome in a
continuous beads-on-a-string type conformation.
 The lipid bilayer envelope, membrane proteins, and
nucleocapsid protect the virus when it is outside the host cell.

14. The most abundant structural protein is the membrane (M) glycoprotein;
it spans the membrane bilayer three times, leaving a short NH2-terminal
domain outside the virus and a long COOH terminus (cytoplasmic domain)
inside the virion.
The spike protein (S) as a type I membrane glycoprotein constitutes the
peplomers. In fact, the main inducer of neutralizing antibodies is S protein.
M plays a predominant role in the intracellular formation of virus particles
without requiring S.
In the presence of tunicamycin coronavirus grows and produces spikeless,
noninfectious virions that contain M but devoid of S.

15. Coronaviruses contain a positive-sense, single-stranded
RNA genome.
 The genome size for corona viruses ranges from 26.4 to
31.7 kilobases. With G+C content varying from 32-43%
The genome has a 5′ methylated cap and a 3′ polyadenylated tail.
The genome organization for a coronavirus is 5′-leader-UTR-
replicase(ORF1ab)-spike (S)-envelope (E)-membrane (M)-
nucleocapsid (N)-3′UTR-poly (A) tail.
A typical CoV contains at least six ORFs in its genome. Four main
structural proteins are encoded by ORFs 10, 11 on the one-third of
the genome near the 3 prime –terminus
Genome

16. The open reading frames 1a and 1b, which occupy
the first two-thirds of the genome, encode the
replicase-transcriptase polyprotein (pp1ab).
 The replicase-polyprotein self cleaves to form
16 nonstructural proteins (nsp1–nsp16).

18. This figure is modified from the sequence comparison and genomic
organization of 2019-nCoV, 2020.9
The differences in the arrangement of the envelope (E), membrane (M), and
nucleoprotein (N) among COVID-19, SARS-CoV, and MERS-CoV are shown
at 3’ end.

19. Entry
The life cycle of a coronavirus Infection begins when the viral
spike protein attaches to its complementary host cell receptor.
After attachment, a protease of the host cell cleaves and
activates the receptor-attached spike protein.
. Depending on the host cell protease available, cleavage and
activation allows the virus to enter the host cell
by endocytosis or direct fusion of the viral envelop with
the host membrane.
Replication cycle

20. In order for the virus to
complete entry into the cell
following initial process .
the sike protein has to be
primed by an enzyme called a
protease ,similar to SAESCoV
,SARSCoV2 use protease called
TMPRSS2 to complete this
process.
In order to attach virus
receptor (spike protein)to its
cellular ligand(ACE2)activation
by TMPRSS2 as a protease is
needed.
Figure 4. The attachment protein “spike” of the new coronavirus COVID-19 andSARS-CoV
use the same cellular attachment factor (ACE2) and the cellular protease TMPRSS2 for
their activation. Existing, clinically approved drugs directed against TMPRSS2 inhibit
SARS-CoV-2 infection of lung cells.

21. The replicase complex is believed to be comprised of up to 16
viral subunits and a number of cellular proteins.
Besides RNAdependent RNA polymerase, RNA helicase, and
protease activities, which are common to RNA viruses, the corona
virus replicase was recently predicted to employ a variety of RNA
processing enzymes that are not (or extremely rarely) found in
other RNA viruses and include putative sequence-specific
endoribonuclease, 30-to-50 exoribonuclease, 20-O-ribose
methyltransferase, ADP ribose 10-phosphatase and, in a subset of
group 2 coronaviruses, cyclic Phosphodiesterase

22. On entry into the host cell, the virus particle is uncoated, and
its genome enters the cell cytoplasm.
The coronavirus RNA genome has a 5′ methylated cap and
a 3′ polyadenylated tail, which allows it to act like a messennger
RNA and be directly translated by the host cells ribosomes.
 The host ribosome translates the initial overlapping open
reading frames ORF1a and ORF 1b of the virus genome into
two large overlapping polyproteins pp1a and pp1ab
The larger polyprotein pp1ab is a result of a -1 ribosomal
frameshift caused by a slippery sequence (UUUAAAC) and a
downstream RNA pseudoknot at the end of open reading frame
GENOME TRANSLATION

23. The ribosomal frameshift allows for the continuous translation of ORF1a
followed by ORF1b.
The polyproteins have their own proteases, PLpro and 3CLpro, which cleave
the polyproteins at different specific sites.
The cleavage of polyprotein pp1ab yields 16 nonstructural proteins (nsp1 to
nsp16). Product proteins include various replication proteins such as RNA-
dependent RNA polymerase (nsp12), RNA
helicase (nsp13),and exoribonuclease (nsp14)

26. A number of the nonstructural proteins coalesce to form
a multi-protein replicase-transcriptase complex (RTC).
The main replicase-transcriptase protein is the RNA-
dependent RNA polymerase (RdRp).
 It is directly involved in the replication and transcription of
RNA from an RNA strand. The other nonstructural proteins in
the complex assist in the replication and transcription
process.
The exoribonuclease nonstructural protein, for instance,
provides extra fidelity to replication by providing
a proofreading function which the RNA-dependent RNA
polymerase lacks.
Replicase-transcriptase complex

27. Replication –
One of the main functions of the complex is to replicate the viral
genome.
 RdRp directly mediates the synthesis of negative-sense
genomic RNA from the positive-sense genomic RNA. This is
followed by the replication of positive-sense genomic RNA from
the negative-sense genomic RNA.

28. Transcription – The other important function of the complex is to
transcribe the viral genome.
RdRp directly mediates the synthesis of negative-sense subgenomic RNA
molecules from the positive-sense genomic RNA.
This process is followed by the transcription of these negative-sense
subgenomic RNA molecules to their corresponding positive-sense mRNAs.
The subgenomic mRNAs form a "nested set" which have a common 5'-
head and partially duplicate 3'-end.

29. Recombination –
The replicase-transcriptase complex is also capable of genetic
recombination when at least two viral genomes are present in the same
infected cell.
RNA recombination appears to be a major driving force in determining
genetic variability within a coronavirus species, the capability of a
coronavirus species to jump from one host to another and, infrequently, in
determining the emergence of novel coronaviruses.
]The exact mechanism of recombination in coronaviruses is unclear, but
likely involves template switching during genome replication.

30. The replicated positive-sense genomic RNA becomes the genome
of the progeny viruses.
The mRNAs are gene transcripts of the last third of the virus
genome after the initial overlapping reading frame.
These mRNAs are translated by the host's ribosomes into the
structural proteins and a number of accessory proteins.
RNA translation occurs inside the endoplasmic reticulum.
Release

31. The viral structural proteins S, E, and M move along the
secretory pathway into the Golgi intermediate compartment.
There, the M proteins direct most protein-protein
interactions required for assembly of viruses following its
binding to the nucleocapsid.
 Progeny viruses are then released from the host cell
by exocytosis through secretory vesicles. Once released the
viruses can infect other host cells.

32. CUTANEOUS MANIFESTATIONS
1. COVID toes : erythematous or purpuric macules on toes ,
lateral aspect of feet , fingers , elbows Pernio like lesions of acral
surfaces Pathogenesis

33. MORBILIFORM RASH : this rash involves trunk
Most common manifestation
Noted after recovery

34. Transmission

36. DETECTION OF CORONA VIRUS
RT PCR
Nosal
parengil/NP/SP
Swab
Require more
time and costly
RAPID
BLOOD TEST
blood
Require less
time 10-15 mins

37. Sample is collected from nose swab
The sample is treated with several chemical solutions that
remove substances, such as proteins and fats, and extracts
only the RNA present in the sample.
 This extracted RNA is a mix of a person’s own genetic
material and, if present, the coronavirus’ RNA.
RNA is reverse transcribed to Ds cDNA
This ds DNA is amplified with the help of the florescent
probe which probe contain known sequence of corona virus
genome
This specific sequence was provided by china in march
After 35 cycle if amplification curve shows S shaped curve
then the person should +ve
RT PCR

39.  RADIOLOGICAL
 CHEST XRAY :

41. If there is a line in IgM
region with control line
then we are infected with
corona virus first time.
If there is a line in IgG
region with control line
then we are infect corona
virus second time
If there is line in both
IgG and IgM region we
infected corona virus
earlier now we are in
recovery stage.

42. 1253 STUDIES ARE ONGOING FOR
MANAGEMENT OF COVID 19 .
Includes
hydroxychloroquine .
 Plasma based therapy
 Lopinavir/ Ritonavir
 Azithromycin
 Remdesivir
 Vaccine
 Tocilizumab
Favipiravir
 Sarilumab
 Anakinra
 Interferon therapy
 Umifenovir
 Corticosteroids
 Steam cell therapy

43. REMDESIVIR
•Used against ss RNA
•Developed in 2009 to treat hepC,
EBOLA
• It is administered via injection into a
vein
•It is an adenosine nucleotide prodrug
Competes for incorporation into RNA
chains
•Delayed chain termination during viral
RNA replication .
• DOSING : I.V. 200 mg on day 1
Followed by 100 mg OD for 5 or 10
days based on severity .

44. HYDROXYCHLOROQUINE / CHLOROQUINE
Anti malarial drug using for covid 19
 Changes pH at cell membrane surface
Inhibits viral fusion Inhibits nucleic acid replication , viral
assembly and release .
 DOSE: as per FDA
1. 800 mg PO on day 1
2. 400 mg PO OD for 4-7 days .
Baseline : ECG , RFT , electrolytes , LFT to be done .
Repeat ECG 2-4 hours , 48 hours and 96 hours after 1st dose

45. Plasma which the portion of blood which
is responsible for maintaing blood pH level
and it also contain many specific proteins
mainly antibodies.
When virus enters into body our immune
system produces IgM and IgG which are
specific against covid 19
If the infected person have high immune
response he produce these antibodies and
recovered from corona virus by killing it
The plasma from recovered patient taken
by seperating blood cells and plasma and
transported to reciepient.
PLASMA THERAPY