Download Preparation of plasmids containing HBV-full genome of

Preparation of plasmids containing HBV-full
genome of genotype A to H and trial of HBV
inactivation method
Yoshiaki Okada
Kiyoko
Umemori
Kazunari Yamaguchi
National Institute of Infectious Disease
Background and Objects
genotype B and C of HBV are dominant in
Japan.Recently detection rate of genotype A is
increasing in Tokyo. The preparations of HBV
genotype panel are required to keep blood safety.
Genotypes except A, B and C are difficult to
collect in Japan.
We have prepared cloned HBV-full genome by PCR.
1.Evaluation of NAT sensitivity among HBV genotypes.
2.Evaluation of HBs-Ag Diagnostic kits among HBV
genotypes.
Design of Primers for HBV Full Genome Amplification
(J.Virol.vol.69.p.5437-5444)
Panels of Plasmids containing HBV-full genome
genotype
cloning
recombinant HBs-Ag sequence
A
B
done
done
+
+
done
done
C
D
done
done
+
+
done
done
E
done
+
done*
F
done
+
done
G
done
+
done
Ｈ
done
+
done
*several bases are deleted.
Amplification efficiency among HBV genotypes
Genotype
A.B,C,D,and G
genotype E and F
Evaluation of HBs-Ag diagnostic kits with recombinant
HBs-Ag which were produced by prepared plasmids
35
Genot ype
A
30
B
25
C
C.O.I.
20
D
15
E
F
10
G
5
H
0
1
2
3
4
5
6
7
8
9
10
Kit No.
J Virological methods 136,254-256,2006
Development of estimation method of HBV inactivation
HBV does not replicate in cell culture.
Recently Owada reported that the treated HBV with neuraminidase
enhance the binding to the cells which express asialoglycoprotein
receptor .(J.Viral hepat.vol.13.11-18.2006)
Neuraminidase treatment enhance HBV infectivity ?
HBV (+)
neuraminidase ( - )
HBV (+)
neuraminidase (+)
Planning to quantitative assay
Conclusion
1.We have prepared plasmids containing HBV-full
genome of genotype A to H respectively.
2.Prepared plasmid panel is useful to evaluate NAT
and HBs-Ag detection kits.
appendix
Establishment of cell culture method of PrPres from BSE
BSE (-)
(+)
I will report next SoGAT
meeting
1 2
3 4
5 6 7
1.Neg.
2.X 1
3.X 102
Infected cells secreted
infectious PrPres into the
culture supernatant.
4.X 103
5.X 104
6.X 105
7.X 106