Amplification and sequencing of varicella-zoster virus (VZV) gene 4: point mutation in a VZV strain causing chickenpox during pregnancy.
The varicella-zoster virus (VZV) causes chickenpox (varicella) as the primary disease and shingles (zoster) as a recurrent manifestation of infection, both being generally benign and self-limiting. While these infections may be severe in adults and even life-threatening in immunosuppressed individuals, they may be amenable to effective antiviral drugs or varicella-zoster immune globulin, provided the treatment is administered early. The prompt diagnosis of VZV infections may be accelerated by rapid, sensitive and specific molecular techniques such as amplification by polymerase chain reaction (PCR) compared with slower and more cumbersome tissue culture and serological procedures. Based on the VZV gene 4 which encodes a transcriptional activator, primers were designed for use in PCR to amplify a target fragment of 381 bp. Distinct diagnostic bands were observed by agarose gel electrophoresis of PCR products of VZV strains isolated from 11 varicella and 7 zoster patients in Singapore, as well as of the Japanese vaccine Oka strain. The detection sensitivity of this PCR assay was determined to be 1 pg of purified VZV DNA equivalent to about 7,000 viral DNA copies. No target bands were amplified from negative control templates from five related human herpes-viruses and from human DNA. The specificity of the PCR products was ensured by direct cycle DNA sequencing, which revealed complete identity of the 18 VZV isolates with the published European Dumas strain. The strong sequence conservation of the target fragment renders this PCR assay highly reliable for detecting the VZV sequence. Only one VZV strain isolated from a patient with varicella during pregnancy exhibited a GGA to GAA point mutation at codon 46 of gene 4, culminating in the non-conservative substitution of Ser with Phe. The predicted secondary structure of the mutant polypeptide portrayed a radical alteration, which may influence its function in transcriptional activation.
Chow
V T
VT
Department of Microbiology, Faculty of Medicine, National University of Singapore, Republic of Singapore.
Lim
K P
KP
D001345:Autoradiography; D001483:Base Sequence; D002644:Chickenpox / Q000175:diagnosis* / Q000821:virology; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D005260:Female; D005814:Genes, Viral / Q000235:genetics*; D014645:Herpesvirus 3, Human / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011251:Pregnancy Complications, Infectious / Q000175:diagnosis* / Q000821:virology; D017433:Protein Structure, Secondary; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012846:Singapore; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics
D017931:DNA Primers; D015534:Trans-Activators; D014764:Viral Proteins