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Doctoral thesis, 2002

Porcine cytomegalovirus : studies on the viral genome and development of novel diagnostic techniques

Widén, Frederik

Abstract

Porcine cytomegalovirus (PCMV) is a recognised pathogen in pigs causing reproductive failure, piglet mortality, rhinitis and possibly retarded growth. The virus is highly prevalent in pig populations worldwide. The introduction of PCMV in nonimmune herds can cause substantial losses. The current interest in transplantation of tissues from pigs to humans (xenotransplantation) and the related risks for cross species virus infections and interactions constitute an important incentive for research on PCMV. In order to eliminate the risk of xenogeneic PCMV infections and recombinations, effective diagnostic techniques are needed. PCMV is a poorly characterised virus. It has previously been assigned to the subfamily Betaherpesvirinae based on its biological properties but no information regarding molecular characteristics was available. Very few studies have been performed on this highly prevalent virus. The main reason for this being the lack of practically usable and effective diagnostic techniques. This in turn is due to the biological properties of PCMV and the difficulties in finding a usable in vitro cultivation system.

In order to develop efficient diagnostic techniques for direct and indirect detection of PCMV and gain more information on the evolutionary relationship and taxonomical position of PCMV it was necessary to perform molecular characterisation. As a first step a PCR assay for PCMV was developed and a short fragment ofthe DNA polymerase (DPOL) gene was sequenced. This provided initial information regarding the evolutionary relationship to other members of the Herpesviridae family and served as a basis for further characterisation ofthe DPOL gene.

The complete sequencing of the DPOL gene provided information regarding the genetic stability of PCMV and identified conserved and variable regions thereby enabling the development of sensitive and reliable PCR assays. It also confirmed previous results regarding evolutionary relationship of PCMV to other viruses. The results demonstrated that PCMV is a betaherpesvirus and indicated that it belongs to the genus Roseolovirus. Also, the DPOL sequence served as a basis for further acquisition of genetic information. Thus, it was possible to sequence the complete glycoprotein B (gB) gene, one complete and two partial flanking genes, together with the DPOL gene comprising 7200 base pairs. In this manner it was possible to show that in PCMV exist a block of five genes that is conserved amongst betaherpesviruses. The sequencing of the gB gene provided further information regarding genetic stability of PCMV and a basis for the development of a serological assay.

Further characterisation of the gB gene and the deduced gB protein identified immunogenic regions. The cloning of a fragment encoding such a region and its expression in E. coli provided a highly immunogenic truncated protein. With the use of this truncated protein it was possible to perform initial studies of an ELISA detecting antibodies to PCMV. The ELISA was used to analyse 198 Swedish pig serum samples. 54% of the samples were classified as positive 37% as negative and 7% as inconclusive. The results should however be interpreted with caution since the assay has not yet been fully validated.

Keywords

PCMV; PCR; ELISA; DNA polymerase; Glycoprotein B; genome; diagnostic techniques

Published in

Acta Universitatis Agriculturae Sueciae. Veterinaria
2002, number: 134
ISBN: 91-576-6388-2
Publisher: Swedish University of Agricultural Sciences

Authors' information

Swedish University of Agricultural Sciences, Department of Veterinary Microbiology

UKÄ Subject classification

Pathobiology

URI (permanent link to this page)

https://res.slu.se/id/publ/117502