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SARS Severe Acute Respiratory Syndrome

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Severe Acute Respiratory Syndrome

What We Know About SARS
• Viral infection – Coronavirus is suspected as the cause • Affects all age groups, highest number of deaths have been among people with pre-existing chronic conditions. • Suspected to have originated in Guandong, China. • Causes atypical pneumonia in infected patients.

Cell Culture
--- Vero E6 cells (fibroblast cell line from primate) inoculated with Oropharyngeal specimens

Cytopathic effect (40X)

Fluorescence antibody assay (400X)

Atypical Pneumonia

Typical Pneumonia

Atypical Pneumonia fuzzy shadow

without clear boundaries

Temporal Clinical Profiles
(75 patients)

• Attenuation is a phenomenon seen in some members of the coronavirus family, where the virulence decreases when it jumps from person to person. • The SARS virus seems exhibit this phenomenon

Group IV: (+)sense RNA Viruses
Order Nidovirales - "Nested" Viruses
Family Genus Type Species Hosts

Arteriviridae Coronaviridae

Arterivirus Coronavirus Torovirus

Equine arteritis virus Infectious bronchitis virus Equine totovirus

Vertebrates Vertebrates Vertebrates

In 1937, Coronaviruses were first isolated from chickens. In 1965, Tyrrell and Bynoe used cultures of human ciliated embryonal trachea to propagate the first human coronavirus (HCoV) in vitro. > 15 species in this family

• “Coronavirus” refers to the protein molecules surrounding the virus, making it look like a crown. • Irregularly-shaped, ~60-220nm in diameter. • Cause mild to moderate respiratory illnesses such as common cold. • Able to survive in dry air for up to 3 hours. • Can be killed by exposure to ultra-violet light. • Mutate easily

• • • • Non-segmented, single-stranded, (+)sense RNA 27-31 kb - the longest of any RNA virus The genome has a 5' methylated cap and 3' poly-A Functions directly as mRNA (unlike (-)sense RNA viruses, no polymerase in particles!)

Human Coronaviruses
229E-like and OC43-like Differ in antigenic determinants and culturing requirements Little antigenic cross-reaction
HCoV-229E Persistent infection in the cell lines (oligodendrocytes and glial cells) S glycoprotein binds with the receptor (aminopeptidase N) and the two fuse. The virion is taken up by endocytosis --- Be isolated in embryonic fibroblast cultures HCoV-OC43 --- Induce apoptosis and persistent infection in diploid fetal lung cells --- Be isolated, or adapted to growth, in suckling mouse brain.

SARS Coronavirus (SARS-CoV)
The envelope carries three proteins • S - Spike glycoprotein: receptor binding, cell fusion, major antigen

• E - Envelope protein: small, envelope-associated protein
• M - Membrane protein: transmembrane - budding & envelope formation The genome is associated with a basic phosphoprotein, N.

Homology of SARS-CoV Genome Sequence (SIN2500) to Other Coronaviruses

Sequence Alignment
• 405 Nucleotides of the Coronavirus Polymerase Gene ORF 1b

Genome Organization

Genome Structure

NSP=Non-structural proteins. S=spike protein. E=small envelop protein. N=nucleocapsid. M=Membrane protein. MHV=murine hepatitis virus. MD=metal binding. 3CL-PRO=3C-like proteinase. The SARS genome is predicted to encode 23 putative mature proteins (blue bars).

mRNA Mapping

Lane 1, SARS-CoV mRNA

Lane 2, Vero E6 cell mRNA Lane 3, molecular mass marker (sizes in kilobases)

Nested-set Transcription
•Intergenic sequence

Regulation of mRNA transcription
DI (defective interfering)

Models for transcription attenuation


nsp13 Domain of SARS-CV pp1ab Polyprotein

• Based on cap-1 methyltransferase

(the reovirus λ2 protein)
• Conserved residues ~ (K-D-K-E) • AdoMet cofactor • Efficient replication

HCoV Main Proteinase

C plot of SARS-CoV Mpro
• Model-built on the basis of the crystal structures of HCoV 229E Mpro and TGEV Mpro.

Inhibitor of Main protein

(A) Inhibitor is shown in red, protein in gray. Cys144 is yellow. (B) HCoV Mpro (blue), SARS-CoV Mpro (gray), TGEV Mpro (green) hexapeptidyl CMK inhibitor (red). The covalent bond between the inhibitor and Cys144 of TGEV Mpro is in

Inhibitor of Main protein

TGEV Mpro (marine) the hexapeptidyl CMK inhibitor (red) HRV2 3Cpro (green) the inhibitor AG7088 (yellow)

antirhinoviral drug AG70

Diagnostic Test
• Serological testing
Indirect fluorescent antibody testing and enzyme-linked immunosorbent assays (ELISA) which detect antibodies against the virus produced in response to infection. (Negative coronavirus antibody tests is possible only for specimens obtained >21 days after onset of fever)
• Molecular testing Reverse transcriptase-polymerase chain reaction (RTPCR) tests specific for the RNA from this novel coronavirus. This can detect infection within the first 10 days after the onset of fever in some SARS patients, but the duration of detectable viraemia and virus shedding is unknown, so RT-PCR tests performed too late could give negative results.

Melanin Inhibition

Skin – the body’s largest organ Functions - Thermoregulation - Protection from physical and chemical & invasion by microorganisms injury - Sensation - Excretion - Immunity (Langerhans cells) - Manufactures essential nutrient (Vit. D) - Blood reservoir (1/10)


Distribution of Skin Colour

Skin colour distribution – an association with environmental factors varying with latitude
Ultraviolet radiation (the quantity of UV rays striking the surface of the earth from the sun) & temperature

Human Complexions
Six skin types

I – light skinned, burns easily, never tans II – light skinned, burns easily, tans some III – light skinned, burns occasionally, tans well IV – light skinned, tans well, rarely burns V – brown skinned (Asian, Indo-Asian, Chinese, Japanese), tans well, burns rarely, can sunburn after prolonged exposure to UVR VI – black skinned (Afro-Caribbean), deeply pigmented, can burn after prolonged exposure to UVR

Biological Determinants of Skin Colour
The pigments Carotene, Haemoglobin, and Melanin are involved in skin colour

Carotene The least common skin pigment results in a yellowing of skin – Results primarily from the over-consumption of carotene containing foods (like carrots)

– This pigment is significant almost exclusively in pathological or abnormal skin coloration

The complex molecule responsible for transport of oxygen throughout our bodies

– The primary protein constituent of Red Blood Cells
Oxygenated haemoglobin has a reddish hue (produces a pinkish tint to lightly pigmented skin) Deoxygenated haemoglobin has a purplish colour (produces the bluish tint to lightly pigmented skin that is characteristic of oxygen deprivation and suffocation)

Melanin The primary determinant of variability in human skin colour is the amount, density, and distribution of the pigment melanin
– Has a dark brown/purple/black colour that is intensified by denser compaction of the melanin granules in the cells of the upper layers of the skin

Melanin Metabolism

Melanin Synthesis
The metabolic pathway to melanin is extremely complicated, involving several intermediate steps Starts with the amino acid tyrosine oxidized by the copper-containing enzyme tyrosinase to dihydroxyphenylalanine (dopa) and then to dopaquinone

Dopaquinone undergoes a series of nonenzymatic reactions and rearrangements forming the different molecules that are co-polymerized to make up one of the types of melanin
Eumelanin is the dark brown/purple/black compound found in skin and hair Phaeomelanin is the yellow-to-reddish-brown pigment which is present in red hair

Both forms of melanin combine with other proteins to form the melanosome that is distributed from the melanocyte to surrounding cells

Melanin Synthesis

Structure of the Epidermis

Structure of the Epidermis

Regulation of Melanin Synthesis
Schematic illustration of the possible approaches to interfere with melanogenesis pathway. Tyr, tyrosinase; M, melanosomes; ROS, reactive oxygen species.

Skin Lightening / Depigmenting Agents
An important industrial chemical, readily available in cosmetic and nonprescription forms for skin lightening Competitive inhibitor of tyrosinase OTC in the USA up to 2% May damage melanocytes at 5% or more Prolonged use of high concentrations may result in ochronosis Other side-effects : mutagenic, skin irritation, nail discoloration, post-inflammatory hyperpigmentation Currently banned in Japan and, as a cosmetic, in Europe
Ochronosis is the bluish-black discoloration of certain tissues, such as the ear cartilage and ocular tissue, seen with alkaptonuria, a metabolic disorder. Additionally, ochronosis can occur occasionally from exposure to noxious substances, such as phenol, trinitrophenol, benzene and hydroquinone.

Monobenzyl ether of hydroquinone (MBEH)
Phenol/catechol class of chemical agents Always causes nearly irreversible depigmentation of skin In dermatology, MBEH is only used to eliminate residual areas of normally pigmented skin in patients with refractory and generalized vitiligo Selective melanocytic destruction through free radical formation and competitive inhibition of tyrosinase enzyme system

Azelaic acid
Saturated dicarboxylic acid originally isolated from Pityrosporum ovale (yeast) Weak competitive inhibitor of tyrosinase Used as a 20% cream Often used in combinations

Magnesium Ascorbyl Phosphate (MAP)
Stable derivative of ascorbic acid (vitamin C) Used as a 10% cream Reducing agent on melanin intermediates Blocks the oxidation chain reaction at various points from tyrosine / DOPA to melanin Protective effect against skin damage induced by UV-B irradiation (conversion of MAP to ascorbic acid)

Kojic acid (5-hydroxy-4-pyran-4-one-2-methyl)
Fungal metabolic product Tyrosinase inhibitor Suppresses melanin synthesis Dipalmitate has enhanced stability and efficacy

Arbutin (hydroquinone-beta-D-glucopyranoside)
Glycosidase HQ found at high concentration in certain plants (Plant Glycoside) Surviving extreme and sustained dehydration Tyrosinase inihibitor

Paper mulberry
Isolated from a plant herbal extract (Korea) Contains Prenylflavones Inhibits melanogenesis

Arctostaphylos patula and Arctostaphylos viscida
Their leaves are potent inhibitors of tyrosinase Inhibit the production of melanin from dopachrome Exhibited superoxide dismutase-like activity

Secreted by the pineal gland in response to sunlight, which is responsible for lightening the colours of amphibians Inhibition of melanogenesis occurs at the post-tyrosinase step in the melanin biosynthetic pathway Inhibit adenosine 3’,5’-cyclic phosphate (cAMP) driven processes in pigment cells

Possible Multi-Step Protocol for the Evaluation of Putative Depigmenting Compounds

Example Project : Development of Cosmeceutical
and Phytopharmaceutical Products
1. Evaluation of cosmeceutical properties of extracts or phytochemicals obtained from temulawak and lempoyang - skin whitening/anti-hyper pigmentation effect (in vitro, tyrosinase inhibition assay) - anti-inflammatory(in vitro, serine protease inhibitory activity - quenching of free radicals(reduction of DPPH) - UV protection (measurement of UVB, UVA absorbance)
2. Preparation of aqueous extracts for biological activity study and for product formulation - cytotoxicity tests (agar overlay, neutral rate uptake), analysis of heavy metals content and preservative study.

3. Product Formulation

- Formulation of skin whitening products such as cleanser, toner and moisturizer for all skin types, oily skin and dry skin. - Formulation will be based on aqueous extracts or phytochemicals derived from the two plants

4. Product safety and efficacy study
- Cytotoxicity tests, dermal and ocular irritation tests and photosensitization test will be conducted on formulated products. - Product efficacy study will be carried out through in vitro and in vivo methods (animals/human volunteers). - The study on human volunteers will be conducted according to COLIPA protocol.

Isolation of active constituents

Chemical Modification


Bioactive Derivatives


Showing potential
Skin whitening assay


Fractionation VLC

Highest potential

Skin whitening assay
Anti-microbial assay

Active Fractions

Cell-Based Tyrosinase Assay


+ Test Sample

Harvest & Pellet Cells Cell Lysis – 30 min incubation to release tyrosinase from melanosome membrane

+ Lysis Buffer

96-well Plate

ELISA Plate Reader Measure absorbance at 405 nm (10 min interval for 1 hr at 37oC)

Enzymatic Assay of Tyrosinase Activity
L-Tyrosine + Test Sample + Tyrosinase 96-well Plate

Incubation at 37oC for 30 min

ELISA Plate Reader Measure absorbance at 475 nm

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