Klonierung und Mutagenese des Bovinen Herpesvirus Typ 1 als ein infektiöses künstliches bakterielles Chromosom (bacterial artificial chromosome, BAC)

For the first time the complete BHV-1 Schönböken genome was cloned
as an infectious bacterial artificial chromosome (BAC), by
inserting mini F plasmid sequences into the glycoprotein E (gE)
open reading frame (ORF). DNA of the resulting BAC clone pBHV-1DgE
was transfected into permissive bovine kidney cells and infectious
BHV-1DgE could be recovered. Using RecE/T cloning in Escherichia
coli BHV-1 genomes with a deletion of either glycoprotein G (gG) or
gM or gK in addition to gE (pBHV-1DgE-gG, pBHV-1DgE-gM and
pBHV-1DgE-gK) were generated. The recombinant viruses with a
simultaneous deletion of gE and gG (BHV-1DgE-gG) or gE and gM
(BHV-1DgE-gM), respectively, were reconstituted after transfection
of manipulated BAC DNA into eukaryotic cells. However, no virus
could be recovered after transfection of recombinant pBHV-1DgE-gK
DNA, suggesting that gK is likewise essential for the replication
of BHV-1 as demonstrated for other alphaherpesviruses. Growth
properties of the other BAC derived virus mutants BHV-1DgE,
BHV-1DgE-gG and BHV-1DgE-gM were analysed in vitro. The mutant
viruses exhibited no markedly lowered virus titres compared to wild
type strain Schönböken. However, BHV-1DgE specific virus plaques
were reduced by 45% compared to BHV-1 Schönböken as assessed by
plaque size measurement. Plaques sizes of BHV-1DgE-gG and
BHV-1DgE-gM were reduced by 56% (BHV-1DgE-gG) and 54%
(BHV-1DgE-gM). The observations made here emphasize the influence
of gE, gG and gM on the BHV-1 cell-to-cell spread. Moreover, they
clearly demonstrate that viral cell-to-cell spread is slightly more
inhibited by a simultaneous deletion of gE and gG or gE and gM than
by a single gE deletion. Comparing the plaque sizes of BHV-1DgE,
BHV-1DgE-gG and BHV-1DgG it could be shown that gE and gG function
independently from each other in cell-to-cell spread, because an
additive and no synergistic effect was observed in the gE-gG double
deletion mutant. These studies illustrate, that the propagation and
manipulation of herpesviruses in bacterial systems provides the
advantage of a more rapid and accurate generation and
characterisation of BHV-1 deletion mutants over conventional
cloning procedures. In conclusion the new technique will allow the
fast generation of BHV-1 mutants as well as the investigation for
their suitability as future (marker) vaccines.