Genotypic Characterization of African Swine Fever

Virus Isolated from Outbreaks in Kenya between

2011 and 2013

Cynthia Onzere

MSc. International Animal Health

University Of Edinburgh

18th October 2013

Background & Significance

The study explores four genetic markers in addition to the B646L gene to identify genotypes and

determine variations within and between genotypes. The study also intends to eventually evaluate the

effects of the variations on the pathogenicity of the virus. These markers include:

The complete E183L gene that encodes the p54 ASFV protein

essential in the recruitment of envelope precursors to the

assembly site (Rodriguez et al., 2004).

The variable 3′-end of the B646L gene that encodes the

major capsid protein p72

Inner membrane

Matrix

The B602L gene that encodes the central variable region (CVR)

where repeated amino acid tetramers that vary in number and

type among ASFV isolates are located. This variation is

important in identifying and grouping the ASFV isolates.

Red Blood cell

Infected leucocyte

The EP402R gene encodes

the CD2v protein that is

responsible for erythrocytes

haemadsorption around

ASFV infected cells (Borca et

al.,1998).

The CP204L gene that encodes

the p30 protein that Modifies

the subcellular distribution of

heterogeneous nuclear

ribonucleoprotein K and may

contribute to modulate

HNRNPK functions related to

processing and export of

mRNAs during ASFV infection.

Conceptual or theoretical framework

Materials and Methods

ASFV diagnosis andverification usingconventional PCR,UPL and TCORPCR and selectionof ASFV positivesamples.

Collection of ASFV naïve blood for PBMC isolation. Culture of the leucocytes using RPMI medium and autologous serum and infection of the resultant macrophages with the ASFV isolates.

Monitoring haemadsorption (HAD)

Harvesting and extraction of DNA from HAD positive cultures.

Genotyping and sequencing of thepartial and full length VP72, VP54,and CVR markers.

Nucleotide and molecular evolutionary analyses using CLC workbench, MEGA version 5.2, Mobyle and Bioedit

Blood, tissues and serum samples are obtained from the ASF cross sectional survey, longitudinal survey and suspected outbreak areas

Purification

P72

CVR

ken12/bus.sig

ug13.231

ken12/burumba.F1

P73

P72

P69

ken12/kia.p59

P43

ken12/nrb.p39

ken12/nrb.p38

ken12/nrb.P36

ken12/kia.p32

ken13/kiri.P31

ken13/kia.P30

ken12/athi.p26

ken12/athi.p24

P21

P09

ken13/bus.obai

Ken13/bus.1033

ken13/bus.564

ken13/bus.125

ken11/kisauni.P52

ken11/Kisauni.p13

Ug12.Kabale1

Ug10.Adjumani

Ug11.Mpigi

Ug10.Moyo2

Ug12.Lira

Ug12.Kampala3

Ug13.Busia2

UGA/1/95

UG07.Wak4

Ken11/Bus1.2

Ken11/Kia2.1

Ken11/ThikP06

Ken11/KakSP

Ken10/KAKFA1

Ken10/Kis027

Ken06.Bus

Ken06.Kis

Ken07.Eld2

Ken07.Kia

Ken07.Nak

Ken08WH/8

Ken05/Tk1

Ken05/Tk6

Ken05/Tk10

Ken05.DPN2

Ken09Tk.15/6

Ken08Tk.2/1

Ken09Tk.15/4

Ken09Tk.19/7

57

62

61

69

98

0.002

P54

IX

X

Dendrograms generated using three marker loci

ken12/nrb.p39

ken12/kia.p59

ken12/nrb.p38

ken12/nrb.P36

ken12/kia.p32

ken13/kiri.P31

ken13/kia.P30

ken12/athi.p26

ken12/athi.p24

ken13/bus.125

ken12/burumba.F1

ken11/kisauni.P52

ken11/Kisauni.p13

Ug10.Kumi

Ug13.Busia2

Ug12.Kyenjojo

Ug03P.6

Ken06.Kis

Ken07.Nak

Ken08WH/4

UG07.Wak4

Ken10/Kis027

Ken11/Bus1.2

Ken11/ThikP06

Ug12.Lira

Ug11.Mpigi

Ug10.Adjumani

Ug12.Kabale1

Uga 95/1

86

64

0.005

ug13.231

ken12/athi.p26

ken12/kia.p59

ken12/nrb.p38

ken12/kia.p32

ken12/nrb.P36

ken13/bus.125

ken11/kisauni.P52

ken11/Kisauni.p13

Ug12.Kabale1

Ug10.Kumi

Ug10.Adjumani

Ug11.Mpigi

Ug12.Lira

Ug13.Busia2

UG07.Wak4

Ken11/Bus1.2

Ken11/Kia2.1

Ken11/ThikP06

Ken10/KAKFA1

Ken10/Kis027

Ken08WH/4

Ken06.Kis

Ken07.Nak

ken12/athi.p24

UGA/95/1

0.002

THANK YOU