The document discusses mumps virus, which causes mumps disease. It is classified in the Paramyxoviridae family. Mumps virus has a non-segmented, negative-stranded RNA genome containing 7 transcription units that encode 8 proteins. The study analyzed the lipid and protein profiles of mumps virus grown in two cell lines, CEF and Vero, using mass spectrometry. In CEF cells, 4 viral proteins were identified (HN, NP, M, P), while in Vero cells 6 were found (HN, NP, M, P, L, V). The lipid profile depended on the host cell line, with more information obtained from Vero cells. Common proteins in both cell lines were

1. DANIEL MADRID
MARTHA FLÓREZ

2. INTRODUCTION

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

4. 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.

5. VIRUS:
Structure
The genomic RNA have
15384 nucleotides
contains 7 transcription
units that encode open
reading frames for:
Nucleop
rotein
NP
Small
hydrophobic
protein - SH
V
protein
Phosphop
rotein
P
Hemagglutinin
neuraminidase
protein - HN
I
protein
Fusion
protein
F
Large protein
L
Matrix
protein
M

6. VIRUS:
Structure

7. VIRUS:
Structure.

8. MASS SPECTROMETRY - MS
MS is a powerful tool in the field of biochemistry and virology.

9. MASS SPECTROMETRY - MS
utility
• 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.

10. 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.

11. 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.

12. MATERIALES Y METODOS

13. LINEAS CELULARES Y VIRUS

14. 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.

15. ANALISIS DE LIPIDOS
Extractos
obtenidos.
Secar, separar. Y
detectar con luz
UV 365nm.
MALDI
Calibración de
masas.

16. ANALISIS DE LIPIDOS:
MALDI MS
matriz asistida por láser de desorción / ionización
Ionizacion
Analisis de
biomoleculas
Fragilidad
Fragmentacion por
métodos
convencionales
Acoplada a un
analizador
TOF

17. ANALISIS DE LIPIDOS:
HPLC
Separa macro
moleculas
Fase móvil
liquida
Fase
estacionaria
activa

18. RESULTADOS

19. 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

20. CEF (Chick embryo fibroblasts)
 Usadas generalmente para la cultivación de virus
 Utilizadas en este experimento con la cepa L- Zagreb

21. 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

22. 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

23. 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

24. 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

25. Fig 3

26. Fig 3

27. Fig 4

28. Fig 5

29. Fig 5

30. DISCUSION

31. 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 isolation
procedure and ability to clearly
clearly discern contaminating
contaminating
from interacting or constituting
constituting host cell proteins in
proteins in virions
has been recognized as
controversial”

32. 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 incorporated
into virions simply by being
being fortuitously present at the
at the
site of budding or preferentially
preferentially incorporated into
incorporated into virions
thereby influencing viral biology
biology and pathogenesis”

33. 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 Nterminal
caspase cleavage of influenza
influenza NP correlates
with the host origin of the virus
virus and is speculated to be
be
a molecular determinant for
for host range”

34. AUTOR LO QUE DIJO SI O NO
Vigerust DJ, Sheperd VL, Stehle
T, Khan ZM, et al.
“The importance of
glycosylation
in virulence and immune
interactions on the level of
of glycoproteins
is quite well known”

35. CONCLUTIONS

36.  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.

37.  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.

38. Daniel Madrid

39. Martha
Flórez