A NK complex-linked locus restricts the spread of herpes simplex virus type 1 in the brains of C57BL/6 mice.

The most frequent cause of sporadic viral encephalitis in western countries is Herpes simplex virus (HSV). Despite treatment, mortality rates reach 20-30% while survivors often suffer from significant morbidity. In mice, resistance to lethal Herpes simplex encephalitis (HSE) is multifactorial and influenced by mouse and virus strain as well as route of infection. The ability to restrict viral spread in the brain is one factor contributing to resistance. After infection of the oral mucosa with HSV type 1 (HSV-1), virus spreads throughout the brains of susceptible strains but is restricted in resistant C57BL/6 mice. To further investigate restriction of viral spread in the brain, mendelian analysis was combined with studies of congenic, intra-natural killer complex (intra-NKC) recombinant and antibody-depleted mice. Results from mendelian analysis support the restriction of viral spread as a dominant trait and consistent with a single gene effect. In congenic mice, the locus maps to the NKC on chromosome 6 and is provisionally termed Herpes Resistance Locus 2 (Hrl2). In intra-NKC recombinants, the locus is further mapped to the segment Cd69 through D6Wum34; a different location from previously identified loci (Hrl and Rhs1) also associated with HSV-1 infection. Studies with antibody-depleted mice indicate the effect of this locus is mediated by NK1.1(+) expressing cells. This model increases our knowledge of lethal HSE, which may lead to new treatment options.

The effect of mouse strain on herpes simplex virus type 1 (HSV-1) infection of the central nervous system (CNS).

BACKGROUND: Mice infected with HSV-1 can develop lethal encephalitis or virus induced CNS demyelination. Multiple factors affect outcome including route of infection, virus and mouse strain. When infected with a sub-lethal dose of HSV-1 strain 2 via the oral mucosa, susceptible SJL/J, A/J, and PL/J mice develop demyelinating lesions throughout the brain. In contrast, lesions are restricted to the brainstem (BST) in moderately resistant BALB/c mice and are absent in resistant BL/6 mice. The reasons for the strain differences are unknown. METHODS: In this study, we combine histology, immunohistochemistry, and in-situ hybridization to investigate the relationship between virus and the development of lesions during the early stage (< 24 days PI) of demyelination in different strains of mice. RESULTS: Initially, viral DNA and antigen positive cells appear sequentially in non-contiguous areas throughout the brains of BALB/c, SJL/J, A/J, and PL/J mice but are restricted to an area of the BST of BL/6 mice. In SJL/J, A/J, and PL/J mice, this is followed by the development of 'focal' areas of virus infected neuronal and non-neuronal cells throughout the brain. The 'focal' areas follow a hierarchical order and co-localize with developing demyelinating lesions. When antigen is cleared, viral DNA positive cells can remain in areas of demyelination; consistent with a latent infection. In contrast, 'focal' areas are restricted to the BST of BALB/c mice and do not occur in BL/6 mice. CONCLUSIONS: The results of this study indicate that susceptible mouse strains, infected with HSV-1 via the oral mucosa, develop CNS demyelination during the first 24 days PI in several stages. These include: the initial spread of virus and infection of cells in non-contiguous areas throughout the brain, the development of 'focal' areas of virus infected neuronal and non-neuronal cells, the co-localization of 'focal' areas with developing demyelinating lesions, and latent infection in a number of the lesions. In contrast, the limited demyelination that develops in BALB/c and the lack of demyelination in BL/6 mice correlates with the limited or lack of 'focal' areas of virus infected neuronal and non-neuronal cells in these two strains.

Redundancy in the immune system restricts the spread of HSV-1 in the central nervous system (CNS) of C57BL/6 mice.

Resistance to lethal encephalitis in mice infected with HSV-1 via the oral mucosa is mouse strain dependent. In susceptible BALB/c, HSV-1 spreads throughout the CNS but in resistant BL/6 mice, virus is restricted to the brainstem. To examine the contribution of cellular immunity in restricting viral spread, we used a combination of antibody depleted and KO mice. Individually, NK/NKT, iNKT, CD4(+), CD8(+), and gammadelta T-cells do not restrict HSV-1 spread. In contrast, virus spreads throughout the CNS of BL/6 CL I KO mice and BL/6 mice treated with either anti-asialoGM1 Ab or both anti-CD8 and anti-NK1.1 mAbs. The results highlight the importance of redundancy in the immune system in restricting viral spread in the CNS, argue for a role of NK/NKT and CD8(+) T-cells in mediating the restriction, and provide a hierarchical order of the individual elements in controlling virus in BL/6 mice infected with HSV-1 via the oral mucosa.

Real time quantitative PCR as a method to evaluate xenotropic murine leukemia virus removal during pharmaceutical protein purification.

Chinese hamster ovary cells used for pharmaceutical protein production express noninfectious retrovirus-like particles. To assure the safety of pharmaceutical proteins, validation of the ability of manufacturing processes to clear retrovirus-like particles is required for product registration. Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for clearance studies. Traditionally, cell-based infectivity assay has been the standard virus quantification method. In this article, a real time quantitative PCR (Q-PCR) method has been developed for X-MuLV detection/quantification. This method provides accurate and reproducible quantification of X-MuLV particle RNA (pRNA) over a linear dynamic range of at least 100,000-fold with a quantification limit of approximately 1.5 pRNA copies microL(-1). It is about 100-fold more sensitive than the cell-based infectivity assay. High concentrations of protein and cellular DNA present in test samples have been demonstrated to have no impact on X-MuLV quantification. The X-MuLV clearance during chromatography and filtration procedures determined by this method is highly comparable with that determined by the cell-based infectivity assay. X-MuLV clearance measured by both methods showed that anion exchange chromatography (QSFF) and DV50 viral filtration are robust retroviral removal steps. In addition, combination of the two methods was able to distinguish the viral removal from inactivation by the Protein A chromatography, and fully recognize the viral clearance capacity of this step. This new method offers significant advantages over cell-based infectivity assays. It could be used to substitute cell-based infectivity assays for process validation of viral removal procedures, but not inactivation steps. Its availability should greatly facilitate and reduce the cost of viral clearance evaluations for new biologic product development.