B19 inactivation by heat treatment

with epithelial cell lines

Yoshiaki Okada

Kiyoko Umemori

Kazunari Yamaguchi

National Institute of Infectious Disease

Introduction

We made a presentation of B19 infectivity assay with epitherial cell line HTB-41 in last SoGAT meeting. With HTB-41( human salivary gland carcinoma) and NCC(human teratocarcinoma) , we have estimated B19 inactivation by dry heat treatment or pasteurization. B19 was inactivated by pasteurization on KU-812 experiments. The animal parvovirus as used model virus of B19 are resistant to heat treatment and other inactivation treatments.Their assays are estimated by infectivity with epitherial cells. This encouraged us to re-estimate B19 inactivation by heat treatment with human epitherial cell lines.

Preparation of samples

1. Pasteurization

5% and 25% albumin solutions contained B19 positive plasma(10%of total volume) were incubated for 24 hours at 60℃ .

2. Dry heating

5% albumin solutions contained B19 positive plasma(10%of total volume) were dried up and heated for 48 hours and 96 hours at 60℃ .

Detection of spliced RNA of B19 on HTB-41

by semi-nested RT-PCR

-4 -5 -6 -7 -8

Position of spliced sites in genome for

detection of B19 infection

-----AACTAAC AGGTA--------GC AGATGCCC------585 2089

---------AACTAACAGATGCCC---------mRNA

Method

-1day : seed 2X105 NCC or

HTB-41 in 24well plate.

0day : add 200µl sample solution with polybrene (final 5 µg/ml)

Incubate for 2hrs at 37degree.

Add 1 ml fresh medium.

Extraction of RNA and RT-PCR

Culture for 2days

585 2089

Genomic DNA

Spliced RNA

0 h

60 10h℃

0 h

　 60 10℃ ｈ

A

A

B

B

A: 5% albumin B: 25% albumin

10-1 　 10-2 10-3 10-4 10-5 10-6 10-7

10-8

B19 Inactivation by Pasteurization

0 hr

60 48hrs℃

60 96 hrs ℃

10-2 10-3 10-4 10-5 10-6

B19 inactivation by dry heat treatment

conclusions

1.B19 was also inactivated by pasteurization in

albumin product on epitherial cells.

2.B19 was resistant to dry heating (60℃ ,96hrs) in

albumin product on epitherial cells.

Though it was not applied in inactivation process of albumin, efficiency of inactivation may change by inactivation conditions.