The Basic Domain of Herpes Simplex Virus 1 pUS9 Recruits Kinesin-1 To Facilitate Egress from Neurons.

UNLABELLED: The alphaherpesviral envelope protein pUS9 has been shown to play a role in the anterograde axonal transport of herpes simplex virus 1 (HSV-1), yet the molecular mechanism is unknown. To address this, we used an in vitro pulldown assay to define a series of five arginine residues within the conserved pUS9 basic domain that were essential for binding the molecular motor kinesin-1. The mutation of these pUS9 arginine residues to asparagine blocked the binding of both recombinant and native kinesin-1. We next generated HSV-1 with the same pUS9 arginine residues mutated to asparagine (HSV-1pUS9KBDM) and then restored them being to arginine (HSV-1pUS9KBDR). The two mutated viruses were analyzed initially in a zosteriform model of recurrent cutaneous infection. The primary skin lesion scores were identical in severity and kinetics, and there were no differences in viral load at dorsal root ganglionic (DRG) neurons at day 4 postinfection (p.i.) for both viruses. In contrast, HSV-1pUS9KBDM showed a partial reduction in secondary skin lesions at day 8 p.i. compared to the level for HSV-1pUS9KBDR. The use of rat DRG neuronal cultures in a microfluidic chamber system showed both a reduction in anterograde axonal transport and spread from axons to nonneuronal cells for HSV-1pUS9KBDM. Therefore, the basic domain of pUS9 contributes to anterograde axonal transport and spread of HSV-1 from neurons to the skin through recruitment of kinesin-1. IMPORTANCE: Herpes simplex virus 1 and 2 cause genital herpes, blindness, encephalitis, and occasionally neonatal deaths. There is also increasing evidence that sexually transmitted genital herpes increases HIV acquisition, and the reactivation of HSV increases HIV replication and transmission. New antiviral strategies are required to control resistant viruses and to block HSV spread, thereby reducing HIV acquisition and transmission. These aims will be facilitated through understanding how HSV is transported down nerves and into skin. In this study, we have defined how a key viral protein plays a role in both axonal transport and spread of the virus from nerve cells to the skin.

Congenital Cytomegalovirus among Children with Cerebral Palsy.

OBJECTIVES: To determine the proportion of children with cerebral palsy (CP) and cytomegalovirus (CMV) DNA detected retrospectively in their newborn screening cards (NBSC), to compare the proportion of children with CMV DNA in their NBSC across spastic subtypes of CP, and to compare the sex and other characteristics of children with CP and CMV detected on their NSBC with those in whom CMV DNA was not detected. STUDY DESIGN: Retrospective observational study. Data were extracted from patient records on children with CP (birth years 1996-2014) from 2 Australian state CP registers and state-wide paediatric rehabilitation services with consent. NBSCs were retrospectively analyzed for CMV DNA by nested polymerase chain reaction (PCR) using primers against gB. Positive samples were validated using real time PCR for CMV UL83. RESULTS: Of 401 children recruited, 323 (80.5%) had an available NBSC. Of these, 31 (9.6%; 95% CI, 6.8-13.3) tested positive for CMV DNA by nested PCR for CMV gB, of whom 28 (8.7%; 95% CI, 6.1-12.2) also had CMV DNA detected by real-time PCR for CMV UL83. Detection of CMV DNA was significantly associated with epilepsy, but not with clinical or epidemiologic characteristics, including sex and pattern of spasticity. CONCLUSIONS: CMV viremia in the newborn period, indicating congenital CMV infection, is highly prevalent among children with CP. Further research is needed to investigate the mechanisms and contribution of congenital CMV to the causal pathways to CP.

Measurable Residual Disease (MRD) by Flow Cytometry in Adult B-Acute Lymphoblastic Leukaemia (B-ALL) and Acute Myeloid Leukaemia (AML): Correlation with Molecular MRD Testing and Clinical Outcome at One Year.

Measurable residual disease (MRD) detected by flow cytometry (FC) is well established in paediatric B- lymphoblastic leukaemia (B-ALL) and adult chronic lymphocytic leukaemia (CLL), but its utility in adult B-ALL and adult acute myeloid leukaemia (AML) is less clear. In this prospective MRD study, one of the largest in Australia to date, we examined consecutive bone marrow aspirates from adult participants with B-ALL (n = 47) and AML (n = 87) sent for FC-MRD testing at a quaternary referral hospital in Sydney. FC-MRD results were correlated to corresponding Mol-MRD testing where available and clinical outcomes at three-month intervals over 1 year. B-ALL showed a moderate positive correlation (rs = 0.401, p < 0.001), while there was no correlation between FC-MRD and Mol-MRD for AML (rs = 0.13, p = 0.237). Five FC-MRD patterns were identified which had significant associations with relapse (X2(4) = 31.17(4), p > 0.001) and survival (X2(4) = 13.67, p = 0.008) in AML, but not in B-ALL. The three-month MRD results were also strongly associated with survival in AML, while the association in B-ALL was less evident. There was a moderate correlation between FC-MRD and Mol-MRD in B-ALL but not AML. The association of FC-MRD with relapse and survival was stronger in AML than in B-ALL. Overall, these findings suggest divergent utilities of FC-MRD in AML and B-ALL.

Herpes simplex virus infects skin gamma delta T cells before Langerhans cells and impedes migration of infected Langerhans cells by inducing apoptosis and blocking E-cadherin downregulation.

The role individual skin dendritic cell (DC) subsets play in the immune response to HSV remains unclear. We investigated the effect of HSV on DC virus uptake, viability, and migration after cutaneous infection in vitro and in vivo. HSV increased the emigration of skin DCs from whole skin explants over 3 d postinfection (p.i.) compared with mock controls, but the kinetics of emigration was influenced by the skin DC subset. Uninfected (bystander) Langerhans cells (LCs) were the major emigrant DC subset at 24 h p.i., but thereafter, large increases in infected CD103(+)langerin(+) dermal DC (dDC) and uninfected langerin(-) dDC emigration were also observed. LC infection was confirmed by the presence of HSV glycoprotein D (gD) and was associated with impaired migration from cultured skin. Langerin(+) dDC also expressed HSV gD, but infection did not impede migration. We then followed the virus in live MacGreen mice in which LCs express GFP using a fluorescent HSV-1 strain by time-lapse confocal microscopy. We observed a sequential infection of epidermal cells, first in keratinocytes and epidermal gammadelta T cells at 6 h p.i., followed by the occurrence of HSVgD(+) LCs at 24 h p.i. HSV induced CCR7 upregulation on all langerin(+) DC, including infected LCs, and increased production of skin TNF-alpha and IL-1beta. However, a large proportion of infected LCs that remained within the skin was apoptotic and failed to downregulate E-cadherin compared with bystander LCs or mock controls. Thus, HSV infection of LCs is preceded by infection of gammadelta T cells and delays migration.

Murine Skin-resident γδT Cells Impair the Immune Response to HSV in Skin.

BACKGROUND: HSV is an important cause of brain infection. Virus entry is often through breeches in the skin. γδT cells play an immunoprotective role in mice after corneal, genital or footpad (subcutaneous) HSV infection. METHODS: Here we report that the presence of γδT cells in murine skin is associated with increased severity of herpetic disease, reduced protective cytokine responses and increased viral spread from the skin to the sensory ganglia in the zosteriform model. γδT cell-deficient (TCR δ -/-) mice displayed significantly decreased herpetic lesion severity after flank HSV infection compared to WT C57BL/6 controls at both primary and secondary skin infection sites. RESULTS: Viral titer at the primary skin site was similar to WT mice in γδT cell-deficient mice, but was significantly decreased in the ganglia and secondary skin site. γδT cell-deficient mice showed increased Th1 responses by both T cells and non-T cells at the primary site, and decreased T-cell Th17 responses and immune infiltration at the secondary site. CONCLUSION: Cytokine responses of epidermal and dermal γδT cells to HSV also differed in WT mice (Th1 in epidermis, and Th17 in the dermis), suggesting a functional dichotomy between these two subsets. Our data suggest that in contrast to other mouse models of HSV infection, skinresident γδT cells promote the pathogenesis of HSV in skin.

Treg depletion attenuates the severity of skin disease from ganglionic spread after HSV-2 flank infection.

Regulatory T cells (Tregs) attenuate lesion severity and disease after HSV ocular or genital infection, but their role in cutaneous infection remains unclear. Treg depletion (anti-CD25 mAb in C57BL/6 mice or diphtheria toxin (DT) in DEREG mice) prior to tk-deficient HSV-2 flank infection significantly decreased skin lesion severity, granulocyte receptor-1(Gr-1(+)) cell number, and chemokine (KC) expression in the secondary skin, but significantly increased immune effectors and chemokine expression (MCP-1, KC, VEGF-A) in the draining LN, and activated, interferon-γ producing CD8(+)T cells in the ganglia. Treg depletion also significantly reduced HSV-2 DNA in the ganglia. Thus, Tregs increase the severity of recurrent skin lesions, and differentially alter chemokine expression and immune effector homing in the skin and LN after cutaneous infection, and limit CD8(+) T cell responses in the ganglia. Our data suggests that effects of Treg manipulation on recurrent herpes lesions should be considered when developing Treg mediated therapeutics.

Neonatal CD8+ T cells are slow to develop into lytic effectors after HSV infection in vivo.

HSV is an important neonatal pathogen. We defined the kinetics of the primary CTL response to HSV-2 in vivo in neonatal mice. Using a replication-defective HSV-2 virus, we demonstrate that neonates mount a primary HSV-specific CTL effector response in the draining LN, with delayed onset and shortened peak activity, in contrast to the rapid, strong response observed in adult mice. The shortened peak neonatal CTL response is independent of HSV dose and is associated with retarded CD8(+) T cell expansion, reduced expansion of HSV-specific tetramer-positive CD8(+) T cells and a reduced CD8(+) T cell IFN-gamma response. Paradoxically, neonatal CD8(+) T cells display enhanced non-specific early activation that is not sustained. Neonatal HSV-specific TCR-transgenic CD8(+) T cells showed reduced proliferation in vivo when transferred into HSV-infected neonatal mice compared to adult T cell controls. Our data suggest that early events in CD8(+) T cell priming underlie the attenuated newborn CTL response to HSV.

T regulatory cells contribute to the attenuated primary CD8+ and CD4+ T cell responses to herpes simplex virus type 2 in neonatal mice.

The first weeks of life are characterized by immune tolerance and increased susceptibility to intracellular pathogens. The neonatal adaptive response to HSV is attenuated compared with adult control models in humans and mice. T Regulatory cells (Tregs) control autoimmunity and excessive immune responses to infection. We therefore compared Treg responses in the draining lymph nodes (LN) of HSV-infected neonatal and adult C57BL/6 mice with the effect of Treg depletion/inactivation by anti-CD25 (PC61) treatment before infection on Ag-specific T cell effector responses at this site. There was a small, but significant increase in the frequency of CD4(+)Foxp3(+) Tregs at day 3 postinfection (p.i.) in the LN of neonatal and adult mice, compared with age-matched mock-infected controls. Depletion of Tregs before HSV infection significantly enhanced HSV-specific CD8(+) T cell cytotoxicity in vivo, cell number, activation, and granzyme B expression 4 days p.i. only in neonatal mice, and significantly enhanced CD8(+) and CD4(+) T cell IFN-gamma responses in both infected adults and neonates. Treg depletion also reduced the titer of infectious virus in the draining LN and nervous system of infected neonates on days 2 and 3 p.i. Treg suppression of the neonatal CTL response p.i. with HSV was associated with increased expression of TGF-beta in the draining LN at day 4 p.i. compared with uninfected neonates, but IL-10 was increased in infected adults alone. These experiments support the notion that the newborn primary T cell effector responses to HSV are suppressed by Tregs.