ABSTRACT
Mutant strains of pseudorabies virus (PRV) of reduced virulence, such as Bartha or BUK-TK900, have been used for vaccination purposes for many years. In contrast to the Bartha strain, BUK-TK900 has not been well characterised at the molecular level. The detailed analysis of this vaccine strain was urged by the fact of the isolation in Poland of field strains which were suspected to originate from BUK-TK900. We characterised changes in the U(S) region of this strain, focusing our attention on gE and gI genes. The only deletion, about 300 bp, found in BamHI 7 fragment (covering most of the U(S) region) was located in the 28 K (US2) gene. BUK-TK 900 produced small plaques on all cell lines tested in our laboratory (SK6, Vero, MDBK, 3T3). The plaque size was restored to about 70% of wild type virus plaque size when growing BUK-TK900 virus on 3T3 complementing cell line expressing PRV gE and up to 100% when cell line producing gE and gI was used. Both gE and gI genes from BUK-TK900 and from some derivative field isolates have been amplified by PCR reaction but no deletions in these genes have been found. Molecular weight of gene products differed from wild type proteins: gE was bigger than wild type gE while gI was smaller. Both proteins were correctly recognised by all tested polyclonal and monoclonal antibodies. Radioimmunoprecipitation study showed that BUK-TK900 gE and gI interact forming a complex. The whole ORF of BUK-TK900 gE was sequenced and only few point mutations were found; only two of them led to changes of amino acids in the polypeptide chain. These were: methionine at position 124 replaced by threonine and glutamine at position 162 replaced by arginine. The introduction of first of these mutations (Met to Thr) to PRV wild type strain NIA-3 resulted in 22% reduction of plaque size. This result confirms the importance of this domain of gE for its function; it was found previously by others that deletion of amino acids 125 and 126 reduced virulence and neurotropism of PRV. More changes were found in BUK-TK900 gI sequence. Over 80% of these changes were located in the terminal 1/3rd of the sequence. Some of these mutations may have significant effect on the secondary structure of gI glycoprotein. The change of the secondary structure may be responsible for the decrease of gI stability and the observed reduction of gI molecular mass.
Subject(s)
Herpesvirus 1, Suid/classification , Herpesvirus 1, Suid/genetics , Point Mutation , Pseudorabies Vaccines , Viral Envelope Proteins/genetics , Animals , Base Sequence , Cell Line , Genetic Complementation Test , Herpesvirus 1, Suid/immunology , Herpesvirus 1, Suid/pathogenicity , Molecular Sequence Data , Polymerase Chain Reaction , Pseudorabies/prevention & control , Restriction Mapping , Sequence Analysis, DNA , Swine , Viral Envelope Proteins/metabolism , Viral Plaque AssayABSTRACT
The paper describes the procedure of efficient elution of glycoproteins from Immobilon membrane after their blotting from polyacrylamide gel. The yield of elution from membrane is 30-70%.
Subject(s)
Glycoproteins/isolation & purification , Animals , Cattle , Electrophoresis, Polyacrylamide Gel , Fibrinogen/isolation & purification , Horseradish Peroxidase/isolation & purification , Humans , Immunoglobulin G/isolation & purification , Ovalbumin/isolation & purification , Ovomucin/isolation & purification , Ribonucleases/isolation & purification , alpha-Fetoproteins/isolation & purificationABSTRACT
The aim of this study was to compare 17 different Aujeszky's disease virus (ADV) isolates from clinical outbreaks of AD by using DNA biotinylated probes. All isolates were collected in Poland between 1984 and 1991. The restriction fragment pattern (RFP) analysis done by hybridization to NIA-3 DNA biotinylated probe indicated that all Polish ADV field strains can be classified as type I of Suid herpesvirus 1. Hybridization with BamHI fragment 7 and gI gene biotinylated probes revealed an unusual heterogeneity of BamHI fragment 7 in almost 50% of strains isolated in Poland. The nature of the molecular changes in this fragment will be discussed.
