Determination of HSV type was done by PCR specific for the HSV pol gene using a common forward primer and type-specific reverse primers as performed by Abraham, et. al and Kimura, et al. DNA was extracted (Invitrogen PureLink viral DNA/RNA mini kit) from purified virus of HSV-1 (McIntyre strain), HSV-2 (Strain 333), and from the Y3369 isolate. PCR products were then analyzed on a 1% agarose gel, which revealed that clinical isolate Y3369 contains the pol gene of an HSV-1 virus. To confirm the analysis, DNA was then extracted from the gel (QIAquick gel extraction kit, Qiagen) and sequenced (Parallab 350, ABI 3730xl). DNA sequencing confirmed Y3369 specimen was a strain of HSV-1 with the sequenced amplicon having 100% identity when compared to the published HSV-1 pol gene sequence (GenBank accession #X04771) and only 85% homology with the HSV-2 sequence. Confirmation of the isolate as an HSV-1 strain was done by successful PCR amplification of HSV-1 genes UL1, UL10, UL22, glycoprotein D, and glycoprotein G.
Glycoprotein G was PCR amplified (see supplementary table) and sequenced. Examination of the sequences showed that the probable cause for the non-reactivity of the mAb assay was the presence of a valine residue in glycoprotein G at amino acid (AA) 111. This valine is near the immunodominant region of antibody binding during normal immune response. Sequencing results were deposited [GenBank:HQ833203], and compared to other isolates on GenBank. Sequencing revealed that the clinical isolate Y3369 contains an amino acid sequence consistent with a common HSV-1 sequence found in many parts of the world.
A meta analysis of three population studies which have sequenced this region of the HSV-1 US4 gene was conducted to determine the prevalence of valine at position 111, as was identified in our sample. Included were isolates from individuals from China, Japan, Kenya, South Korea, Sweden, and the United Kingdom. We discovered the valine at position 111 to be present in all HSV-1 isolates (100%, N = 141) taken from human populations from Asia and Africa. The other populations, from the UK and Sweden, contained the valine at position 111 in 36% (N = 185) of isolates. This valine at position 111 is located within the binding site for a commonly used typing mAb, which recognizes the epitope AFPL. The phenylalanine is replaced to form the sequence AVPL in this variant.
Sequences for the middle region encoding AA 110 to 164 of glycoprotein G were analyzed and a phylogenetic tree created. Phylogenetic analysis groups our isolate Y3369 as an HSV-1 with sequence V (representing valine at 111) which contains the sequence AVPL instead of AFPL, as well as other common nucleotides. All isolates from populations from Africa and Asia, as well as 36% of the European population contained the sequence AVPL, which would not be recognized by the mAb which tests for the AFPL epitope. Another study found that all isolates with a valine residue at position 111 of glycoprotein G were untypable when assaying viral antigens. This specific test would not be likely to function diagnostically in these African or Asian populations.