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. 1999 May;73(5):3843-53.

Intrastrain Variants of Herpes Simplex Virus Type 1 Isolated From a Neonate With Fatal Disseminated Infection Differ in the ICP34.5 Gene, Glycoprotein Processing, and Neuroinvasiveness

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Intrastrain Variants of Herpes Simplex Virus Type 1 Isolated From a Neonate With Fatal Disseminated Infection Differ in the ICP34.5 Gene, Glycoprotein Processing, and Neuroinvasiveness

J R Bower et al. J Virol. .
Free PMC article

Abstract

Two intrastrain variants of herpes simplex virus type 1 (HSV-1) were isolated from a newborn with fatal disseminated infection. A small-plaque-producing variant (SP7) was the predominant virus (>99%) in the brain, and a large-plaque-producing variant (LP5) was the predominant virus (>99%) in the lung and gastrointestinal tract. EcoRI and BamHI restriction fragment patterns indicated that SP7 and LP5 are related strains. The large-plaque variants produced plaques similar in size to those produced by HSV-1 KOS. Unlike LP5 or KOS, SP7 was highly cell associated and processing of glycoprotein C and glycoprotein D was limited to precursor forms in infected Vero cells. The large-plaque phenotype from KOS could be transferred into SP7 by cotransfection of plasmids containing the EK or JK EcoRI fragment or a 3-kb plasmid with the UL34.5 gene of HSV-1 KOS together with SP7 DNA. PCR analysis using primers from within the ICP34.5 gene indicated differences for SP7, LP5, and KOS. Sequencing data indicated two sets of deletions in the UL34.5 gene that distinguish SP7 from LP5. Both SP7 and LP5 variants were neurovirulent (lethal following intracranial inoculation of young BALB/c mice); however, the LP5 variant was much less able to cause lethal neuroinvasive disease (footpad inoculation) whereas KOS caused no disease. Passage of SP7 selected for viruses (SLP-5 and SLP-10) which were attenuated for lethal neuroinvasive disease, were not cell-associated, and differed in the UL34.5 gene. UL34.5 from SLP-5 or SLP-10 resembled that of KOS. These findings support a role for UL34.5 in promoting virus egress and for neuroinvasive disease.

Figures

FIG. 1
FIG. 1
Plaque morphology of the small-plaque (SP7) and large-plaque (LP5) variants grown in Vero cells. The cells were fixed and stained with crystal violet in ethanol.
FIG. 2
FIG. 2
HSV-1 glycoprotein differences associated with large-plaque and small-plaque variants. Whole-cell detergent extracts from Vero cells infected with large- or small-plaque variants from the CSF, kidney, brain, lung, or GI tract were examined by SDS-PAGE and Western blot analysis using either polyclonal anti-gC (A) or anti-gD (B). The laboratory strain KOS was included as a control, and molecular weights of the major glycoforms (kilodaltons) are indicated.
FIG. 3
FIG. 3
RFLP of large- and small-plaque variants isolated from different tissues. DNA was digested with BamHI (lanes 1 to 4) or EcoRI (lanes 5 to 8) and electrophoresed in 0.8% agarose gels containing ethidium bromide. Lanes 1 and 5, large-plaque variant from the kidney; lanes 2 and 6, small-plaque variant from the kidney; lanes 3 and 7, large-plaque variant from the GI tract; lanes 4 and 8, small-plaque variant from the brain. Bracket indicates fragments exhibiting RFLP.
FIG. 4
FIG. 4
Evaluation of initial isolates from cotransfection of genomic DNA from HSV-1 SP7 and plasmid DNA containing EcoRI fragments of HSV-1 KOS. (A) Plaque size. Vero cells were cotransfected with HSV-1 and plasmid DNA by using lipofection. The cells were maintained in growth medium for 4 days, and large and small plaques were counted. (B) Extracellular-to-intracellular virus ratios. Virus obtained from cell cultures described in the legend to panel A was applied to Vero cells and allowed to infect cells for 20 h. Virus obtained from the medium (0.5 volumes) and virus from the cells and the remaining medium were quantitated by plaque assay.
FIG. 5
FIG. 5
Comparison of glycoforms of gC from cotransfectants of HSV-1 SP7 and HSV-1 KOS EcoRI JK and EK fragments. Virus obtained from the cotransfection experiment described in the legend to Fig. 4 was triple plaque purified. Infected Vero cell detergent extracts were prepared and analyzed by SDS-PAGE and Western blotting. Images were scanned into the computer.
FIG. 6
FIG. 6
PCR amplification products of UL34.5 sequences. DNA purified from Vero cells infected with the indicated virus was amplified by PCR using unique primers from the termini of the UL34.5 gene and evaluated by electrophoresis on 1.5% agarose gels. (A) Individual PCR products from HSV-1 KOS, LP5, and SP7 DNA and a mixture of the PCR product of LP5 and SP7 to illustrate differences in mobility. (B) Lanes 1 to 8, PCR products from different plaque-picked isolates from suspected recombinants of SP7/JK; lane 9, SP7/EK plaque isolate; lane 10, is another SP7/JK isolate. Std, size standards, positions of which are indicated in base pairs.
FIG. 7
FIG. 7
Comparisons of UL34.5 DNA sequences. DNA obtained by PCR amplification of the UL34.5 gene, as described for Fig. 6, were submitted for sequencing. The sequences were aligned by using the MAP alignment program. Differences as well as deletions in the sequences are indicated.
FIG. 8
FIG. 8
Comparisons of the predicted peptide sequences of UL34.5 from KOS, SP7, and LP5. Dashes denote lack of corresponding sequence; shading denotes differences in sequence; hatched boxes enclose HSV-1 conserved sequence with similarity to the murine MyD116 myeloid differentiation primary response protein. KOS 70 and KOS 321 (marked with asterisks) have the same sequence.
FIG. 9
FIG. 9
Neuroinvasive progression of SP7 and LP5 variants in the mouse. BALB/c mice were inoculated in the right rear footpad with 5 × 105 PFU of either the SP7 or LP5 variant. Mice were euthanized (three mice per time point) on 0, 1, 3, 5, 7, and 9 days p.i. The foot, sciatic nerve, DRG, spinal cord, and brain were removed, virus was extracted into 1 ml of medium, and viral titers were determined. Virus titers are representative of the amount of virus in the total tissue sample.
FIG. 10
FIG. 10
HSV-1 gC profiles from SP7, SLP7, LP5, and KOS. Whole-cell detergent extracts from infected Vero cells were examined by SDS-PAGE and Western blot analysis using polyclonal anti-gC. Lanes: 1, SP7; 2, SLP5; 3, LP5; 4, KOS.
FIG. 11
FIG. 11
Electrophoretic mobilities of UL34.5 PCR products from SLP, LP5, SP7, and KOS strains. (A) PCR products from KOS 321, SP7, and SLP5. (B) PCR products from SP7 and KOS 321 (mixed to show differences) and from SP7 virus obtained after passages 1, 4, 5, 8, 9, and 10. Virus from passages 9 and 10 showed phenotypic differences from the parent and are therefore designated SL (small to large plaque) and SLP9 and SLP10 in the text. Positions of size standards (Std) are indicated in base pairs.

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