DANIEL MADRIDMARTHA FLÓREZ

INTRODUCTION

VIRUS

Mumps virus is a nonsegmented, negative stranded RNA virus that causes mumps disease.

VIRUS: general characteristics

• Family: Paramyxoviridae.• Subfamily: Paramyxovirinae.

• Genus: Rubulavirus.• Pleomorphic particles ranging from 100 to

800 nm in size.• Helical ribonucleocapsid core sorrounded

by a host cell derived lipid envelope.

VIRUS: Structure

The genomic RNA have 15384

nucleotides contains 7 transcription units

that encode open reading frames for:

NucleoproteinNP

Small hydrophobic protein - SH

V prote

in

Phosphoprot

ein P

Hemagglutinin neuraminidase protein - HN

Fusion protei

nF

Large proteinL

VIRUS: Structure

VIRUS:Structure.

MASS SPECTROMETRY - MS

MS is a powerful tool in the field of biochemistry and virology.

MASS SPECTROMETRY - MSutility

• Measuring nanoparticle size.• Looking for toxins.

• Looking for pesticides.• Determine the elements and isotopes found in the solar wind.• To identify the structures of complex biological molecules.

• To measure the metabolic gas Exchange.• To locate oil deposits by measuring petroleum precursors in

rock.

RELATIONSHIP MS / VIRUS.

• Combination of MS and HPTLC has been shown as a very convenient method for lipid analysis.

• Virus proteome and lipidome care expressed virus features that to some extent depend on the host cell and these must have an impact on virus characterists such as infectivity and

stability.• Methods characterizing both virus proteins and lipids could be a tool in raising the level of viral vaccines quality control.

GENERAL OBJECTIVE

The aim of this research was to analyse lipid and protein pattern of the mumps virus derived from two cell lines by

means of mass spectrometry.

MATERIALES Y METODOS

LINEAS CELULARES Y VIRUS

JL5

L-Zg

CEF

JL5

L-Zg

CEF

JL5

L-Zg

CEF

Crec

imien

to,

elim

inació

n.

ANALISIS DE PROTEINAS:Western Blot

Es una técnica analítica usada para detector proteinas especificas en una muestra determinada, mediante una

electrophoresis en gel se separan las proteínas.

Sirve para confirmer un resultado positive de una prueba, con base en la producción de anticuerpos contra un virus.

ANALISIS DE LIPIDOS

Extractos obtenidos.

Secar, separar. Y detectar con luz UV 365nm.

ANALISIS DE LIPIDOS:MALDI MS

matriz asistida por láser de desorción / ionización

Ionizacion

Analisis de biomolecul

as

Fragilidad

Fragmentacion por métodos

convencionales

Acoplada a un

analizador

TOF

ANALISIS DE LIPIDOS:HPLC

Separa macro

moleculas

Fase móvil liquida

Fase estacionaria activa

RESULTADOS

RESULTADOS

7 unidades de transcripción que codifican los marcos de lectura abiertos para: NP (Nucleoprotein) P phosphoprotein V protein I protein M matrix protein F fusion protein SH small hydrophobic protein L large protein HN Hemagglutinin Neuraminidase protein

CEF (Chick embryo fibroblasts)

Usadas generalmente para la cultivación de virus Utilizadas en este experimento con la cepa L- Zagreb

Células Vero

“Riñón verde” Utilizado en cultivos celulares Aislado a partir de células epiteliales del riñón de mono verde africano Puede replicarse a través de muchos ciclos sin envejecer Permite producción de vacunas contra enfermedades virales

RESULTADOS

De las 9 proteínas solo 4 fueron identificadas en virus de paperas cultivados en =

CEF: HN, NP, M y P Vero: HN, NP, M, P, L y V

Fig. 1

- Izquierda: Mediante condiciones no reductoras: Proteínas identificadas con MS

- Derecha: Condiciones reductoras, pesos moleculares teóricos del virus de las paperas

- 3 Bandas de NP, 61, 55, 48 kDa respectivamente

Fig 2

- Izquierda: Proteínas teóricas del virus de las paperas y sus pesos moleculares calculados.

- Derecha: Proteínas identificadas mediante MS, NP como la proteína más abundante en virus de las paperas.

Proteínas no detectadas: - F: Puede deberse a baja expresión génica, nivel de

glicosilación, extracción poco efectiva.

- SH: No se ha detectado aún en viriones de paperas

- I: No se esperaba su aparición al no ser proteína de membrana

Fig 3

Fig 3

Fig 4

Fig 5

Fig 5

DISCUSION

AUTOR LO QUE DIJO SI O NO

Varnum SM, Streblow DN, Monroe ME, Smith P, Auberry KJ, Paša-Tolić LJ,et al.

“Problem of the virus isolationprocedure and ability to clearly discern contaminatingfrom interacting or constituting host cell proteins in virionshas been recognized as controversial”

AUTOR LO QUE DIJO SI O NO

Chertova E, Chertov O, Coren LV, Roser JD, Trubey CM, Bess JW, et al.

“However, host cell proteins can be incorporatedinto virions simply by being fortuitously present at thesite of budding or preferentially incorporated into virionsthereby influencing viral biology and pathogenesis”

AUTOR LO QUE DIJO SI O NO

Lipatov AS, Yen H-L, Salomon R, Ozaki H, Hoffman E, Webster RG, et al

“The role of Nterminalcaspase cleavage of influenza NP correlateswith the host origin of the virus and is speculated to bea molecular determinant for host range”

AUTOR LO QUE DIJO SI O NO

Vigerust DJ, Sheperd VL, Stehle T, Khan ZM, et al.

“The importance of glycosylationin virulence and immune interactions on the level of glycoproteinsis quite well known”

CONCLUTIONS

Knowing a virus proteome can be really helpfull for the development of vaccines.

Cells Vero and CEF are both useful as cell culture and for virus studying

Is important to do more studies about the function of glycolipids in the pathogenesis and development of the virus inside the cell.

L-Zagreb strain is also used for the development of vaccines against mumps, is relatively straightforward to work with them.

The obtained data indicate that lipid profile of mumps virus depends on the host cell line.

The results derived from Vero cells contained more information about the lipids .

Although HN and F proteins are similar, because they are glycosylated by the host cell, the F protein was not detected.

The proteins in common in both cells were NP, M and P, because the damage or alterations in these proteins determines the disease.

Daniel Madrid

Martha Flórez