No abstract

About 10% of the large (L) protein gene of Strawberry crinkle virus (SCV) was sequenced after amplification with degenerate primers designed to conserved regions of the rhabdovirus L protein. The virus sequence was extended to 1362 nucleotides through rapid amplification of cDNA ends. One pair of degenerate L gene primers amplified a 683-bp fragment from four different isolates of SCV cultured in the experimental host Physalis pubescens ; the nucleotide sequences of these fragments differed by < 1% to 10% indicating the suitability of this region as a diagnostic target. This information enabled the development of a reverse transcription polymerase chain reaction (RT-PCR) detection method for SCV using primers designed to the L gene sequence. SCV was amplified from infected P. pubescens (573 bp fragment) and from infected Chaetosiphon fragaefolii aphids (770 bp fragment). SCV was also detected by RT-PCR in total RNA extracts from three strawberry plants showing symptoms typical of SCV infection but failed when the intensity of the disease symptoms decreased. However, both SCV positive-sense RNA, and negative-sense genomic RNA, were detected by nested PCR in chronically infected strawberry plants sampled in September.

A murine cytomegalovirus (MCMV) temperature-sensitive (ts) mutant, tsm5, of the K181 (Birmingham) strain, showed approximately 10-fold and approximately 10,000-fold reductions in yields at the permissive (33 degrees C) and non-permissive temperature (40 degrees C), respectively. It did not replicate to detectable levels in any tissue of 1-week-old Balb/c mice for up to 21 days following i.p. inoculation with 4 x 10(3) pfu although it did replicate, albeit with considerably delayed kinetics, in SCID mice. tsm5 expressed all kinetic classes of transcript (immediate-early, early and late) both in vitro at the non-permissive temperature and in vivo. To identify mutations contributing to this phenotype, chimaeric viruses produced from overlapping cosmids generated from tsm5 and the Smith strain of MCMV were examined. A virus, Smith/tsm5DGIK, comprising the central conserved region of the tsm5 genome, was not attenuated at 33 or 37 degrees C but was ts at 40 degrees C, although not to the same extent as tsm5. In contrast to tsm5, this chimaeric virus replicated to similar levels as parental viruses in adult BALB/c mice. These results suggested that genes contributing to reduced replication at 33 degrees C and lack of replication in vivo are located at the ends of the tsm5 genome while those contributing to the ts phenotype are located in the central conserved region of the genome. Sequencing of some immune evasion genes known to be located at the 3' or 5' ends of the MCMV genome showed that no mutations were present in ORFs m04, m06, M33, M37, m38.5, m144, m152, or m157 although mutations were found in M27 (A658S) and M36Ex1 (V54I). tsm5 made few capsids at 40 degrees C and these lacked DNA. DNA synthesis was significantly reduced in tsm5-infected cells at 40 degrees C although DNA cleavage occurred with close to wt efficiency. Sequencing of the herpesvirus conserved cis-acting elements, pac1 and pac2, and genes involved in DNA packaging and cleavage located in the central core region of the genome identified few point mutations. Two were identified that alter the encoded protein in tsm5 ORFs M98 (P324S) and M56 (G439R). Furthermore, a point mutation (C890Y) was identified in M70, the primase. Another mutant, tsm30, which is also defective in DNA packaging and processing, has a point mutation in M52 (D494N). Thus, a number of mutations have been identified in tsm5 that suggests that it is defective in genes involved in immune evasion, DNA replication and DNA encapsidation.

The present study found significant risk association for TNF-α -308 (G/A), and TNF-β +252 (A/G) genotypes, alleles and haplotypes, with CAD in a North Indian population.

No abstract