Analysis of Neurovirulence in the Mouse Model System Using Deletion Variants of Herpes Simplex Virus Type 2 (HSV-2)

Taha, Mahmoud Younis Mohamed (1990) Analysis of Neurovirulence in the Mouse Model System Using Deletion Variants of Herpes Simplex Virus Type 2 (HSV-2). PhD thesis, University of Glasgow.

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The aim of the work described in this thesis was to identify gene (s) involved in determining the neurovirulence of HSV-2 strain HG52 in the mouse model system using deletion variants. The availability of variants with deletions in specific regions of the genome afforded the unique opportunity to determine the possible role of specific sequences in virulence. The phenotype of the parental wild type virus has also been determined by examining the neurovirulence of individual plaque stocks to identify the baseline from which to evaluate the deletion variants. Twenty well separated plaques were picked from the elite stock of HG52 and passaged twice at 37C. Restriction endonuclease analysis of the DNA from each of the twenty plaque stocks showed no differences in the size of fragments and distribution of the sites. To determine their neurovirulence, ten of the individual plaque stocks were selected randomly for mice inoculation. Following intracranial inoculation of 3 week old BALB/c mice, the plaque stocks segregated into three classes of neurovirulence on the basis of their LD50 values; high (10 3 pfu/mouse), intermediate (10e3-10e4 pfu/mouse) and low virulence (>10 5pfu/mouse). The particle : pfu ratios of the plaque stocks were within the acceptable range for HSV-2. Restriction endonuclease analysis of viruses reisolated from the brains of infected mice showed no apparent difference in their DNA profiles compared to the initial infecting viruses. Two plaque stocks of high, one of intermediate and one of low virulence were selected on the basis of their LD50 values and particle : pfu ratios. These stocks retained their original values compared to the non-plaque purified elite stock of HG52 (10 2pfu/mouse) when retested in mice. Following intraperitoneal inoculation, the selected plaque stocks showed differences in their LD50 values comparable to the differences seen following intracranial inoculation. The selected plaque stocks grew as well as the parental HG52 in one step growth experiments in BHK-21 C13 cells. However, they showed differences in the growth kinetics in vivo in mouse brain where the high virulence stocks showed comparable growth to HG52, while the intermediate and low virulence stocks grew less well. The selected plaque stocks were passaged five times in BHK-21 C13 cells and ten plaques from each were picked and stocks grown. Restriction endonuclease analysis of their DNA showed no differences compared to the wild type HG52. Following intracranial inoculation of the virus stock derived from the fifth passage, those derived from intermediate or low virulence virus remained stably of intermediate or low virulence, while those derived from high virulence virus showed in some cases a shift to intermediate levels of virulence. These results clearly demonstrate virulence heterogeneity within the elite stock of HSV-2 strain HG52. The LD50 value of high virulence virus was chosen as a baseline from which to evaluate the virulence of deletion variants A number of deletion variants of HSV-2 strain HG52 with deletions ranging in size from 1.5 to 9 kb have been tested for virulence following intracranial inoculation of 3 week old BALB/c mice. The variants were segregated into three categories of virulence on the basis of their LD50 values; avirulent (>10e7 pfu/mouse), reduced (10 -10 pfu/mouse) and attenuated (10 pfu/mouse) compared to 10 pfu/mouse for the elite stock of HG52. Analysis of the variants with deletions in U S /TR S indicated that the US4, US10, US11 and US12 genes have a role in neurovirulence. Deletion of one copy of oris and one copy of the IE3 gene has a minor effect on neurovirulence. Analysis of the variants with deletions in IR L/TR L regions of the genome implies that deletion of one copy of the IEl gene and part of LAT transcripts in IR L has a minor effect on neurovirulence. The analysis demonstrated that none of the deleted genes appear to be a unique determinant of neurovirulence with the exception of the DNA sequences between 0-0.02 and 0.81-0 83 m. u. The variant JH2604 whose genome is deleted by 1.5 kb in both copies of the BamHI v fragment between 0-0.02 and 0.81-0.83 m. u. in TR L and IR L respectively was avirulent for mice following intracranial and footpad inoculation with LD 50 values of >10e7 and >10e8 pfu/mouse respectively compared to 10 pfu/mouse for the elite stock of HG52. Therefore, the variant JH2604 is at least 6 logs less neurovirulent than the wild type virus. The variant JH2604 grew as well as the wild type virus in vitro in BHK-21 C13 and 3T6 cells, but it failed to grow in mouse brain in vivo demonstrating that the lack of neurovirulence was due to inefficient replication in mouse brain.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology
Date of Award: 1990
Depositing User: Enlighten Team
Unique ID: glathesis:1990-78020
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:43
Last Modified: 30 Jan 2020 15:43

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