ABSTRACT
Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.
Subject(s)
Animals , Genetic Variation , Herpesviridae Infections/veterinary , Herpesvirus 1, Equid/classification , Herpesvirus 1, Equid/genetics , Horse Diseases/virology , Brazil , Cluster Analysis , Conserved Sequence , DNA, Viral/chemistry , DNA, Viral/genetics , Genotype , Horses , Herpesviridae Infections/virology , Molecular Sequence Data , Open Reading Frames , Phylogeny , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
Objective To establish a fluorescent quantitative polymerase chain reaction method for quantifying the ICP4 gene expression of herpes simplex virus type 2(HSV2).Methods According to the HSV2 ICP4 gene sequence,we designed and synthesized PCR primer.The purified PCR product was sequenced after connecting with pMD-18 T plasmid.According to the sequence assay results,the primer and probe of fluorescent quantitative PCR was designed and synthesized.Standard recombinant plasmid extracted from the positive bacteriumclone was used as standard substance.The plasmid as standard substance was diluted for 10 times,then PCR reaction proceeded.The sensitivity and dependability of the real-time fluorescent quantitative PCR were analyzed.Results The sequence result indicated that there was non-sense mutation of A-G and T-C.And the detection sensitivity was 101 copy.The Ct value were 14.275土0.137,17.988?0.162,22.081土0.259,25.957土0.345,29.565?0.203,33.269土0.287,37.737?0.698,respectively with 107~ 101 copies/?l.The coefficient of variability were 0.965%,0.902%,1.174%,1.329%,0.686%,0.862% and1.851%,respectively.There was a good linear function in statistics between the Ct value and the concentration gradient of standard plasmid DNA specimen.The coefficient of regression was 0.998.Conclusion The method of quantification of ICP4 gene of HSV2 with real-time fluorescent quantitative PCR is successfully established,and the method has good sensitivity and dependability,which can be used to quantitative detecting HSV2 ICP4.