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 (tester) as a recurrent manifestation of infection, both bring generally benign and self-limiting. While these infections may be severe in adults and even life-threatening in immuno suppressed individuals, they may be amenable to effective antiviral dr ugs or varicella-zoster immune globulin, provided the treatment is adm inistered early. The prompt diagnosis of VZV infections may be acceler ated by rapid, sensitive and specific molecular techniques such as amp lification by polymerase chain reaction (PCR) compared with slower and more cumbersome tissue culture and serological procedures. Based on t he VZV gene 4 which encodes a transcriptional activator, primers were designed for use in PCR to amplify a target fragment of 381 bp. Distin ct diagnostic bands were observed by agarose gel electrophoresis of PC R products of VZV strains isolated from 11 varicella and 7 tester pati ents 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 p urified VZV DNA equivalent to about 7,000 viral DNA copies. No target bands were amplified from negative control templates from five related human herpesviruses and from human DNA. The specificity of the PCR pr oducts was ensured by direct cycle DNA sequencing, which revealed comp lete identity of the 18 VZV isolates with the published European Dumas strain. The strong sequence conservation of the target fragment rende rs this PCR assay highly reliable for detecting the VZV sequence. Only one VZV strain isolated from a patient with varicella during pregnanc y exhibited a GGA to GAA point mutation at codon 46 of gene 4, culmina ting in the non-conservative substitution of Ser with Phe. The predict ed secondary structure of the mutant polypeptide portrayed a radical a lteration, which may influence its function in transcriptional activat ion.