Bo Svennerholm

HIV-1 Viral Escape in Cerebrospinal Fluid of Subjects on Suppressive Antiretroviral Treatment

BACKGROUND Occasional cases of viral escape in cerebrospinal fluid (CSF) despite suppression of plasma human immunodeficiency virus type 1 (HIV-1) RNA have been reported. We investigated CSF viral escape in subjects treated with commonly used antiretroviral therapy regimens in relation to intrathecal immune activation and central nervous system penetration effectiveness (CPE) rank. METHODS Sixty-nine neurologically asymptomatic subjects treated with antiretroviral therapy >6 months and plasma HIV-1 RNA <50 copies/mL were cross-sectionally included in the analysis. Antiretroviral therapy regimens included efavirenz, lopinavir/ritonavir or atazanavir/ritonavir combined with tenofovir, abacavir, or zidovudine and emtricitabine or lamivudine. HIV-1 RNA was analyzed with real-time polymerase chain reaction assays. Neopterin was analyzed by enzyme-linked immunosorbent assay. RESULTS Seven (10%) of the 69 subjects had detectable CSF HIV-1 RNA, in median 121 copies/mL (interquartile range, 54-213 copies/mL). Subjects with detectable CSF virus had significantly higher CSF neopterin and longer duration of treatment. Previous treatment interruptions were more common in subjects with CSF escape. Central nervous system penetration effectiveness rank was not a significant predictor of detectable CSF virus or CSF neopterin levels. CONCLUSIONS Viral escape in CSF is more common than previously reported, suggesting that low-grade central nervous system infection may continue in treated patients. Although these findings need extension in longitudinal studies, they suggest the utility of monitoring CSF responses, as new treatment combinations and strategies modify clinical practice.

Uptake and Transport of Herpes Simplex Virus in Neurites of Rat Dorsal Root Ganglia Cells in Culture

Attachment and neuritic transport of herpes simplex virus (HSV) type 1 (McIntyre) were studied in a cell culture system with dissociated cells of rat dorsal root ganglia. The two-chamber cell culture system containing a diffusion barrier penetrated by neurites of cultured sensory neurons permitted infection of neurites extending outside the diffusion barrier without exposure of the neuronal cell soma. HSV adsorbed to neuritic extensions and reached the neuronal soma within 1.5 h post-inoculation. Neuritic uptake and transport of HSV were inhibited in the presence of cytochalasin B. Internalization of virus in neurites was preceded by attachment of virus to the neurite plasma membrane. Neurites transported viral nucleocapsids (NC) through the diffusion barrier of the cultures. Destruction of the neuritic extensions before or shortly after peripheral virus inoculation blocked spread of infection to the cell soma. No infection was established when neuritic extensions were exposed to viral NC and NC were then not observed inside the neurite plasma membrane. Virus produced in neurons, when HSV was inoculated into the inner culture chamber containing the neuronal cell bodies, was transported as enveloped virus in cytoplasmic vesicles from the neuronal cell body towards the periphery. Schwann cells were infected by viropexis. Shortly after infection virions were observed in vacuoles of the cytoplasm.

Herpes simplex virus infection of the human sensory neuron An electron microscopy study

SummaryHerpes simplex virus (HSV) type 1 was used to infect cultures of human embryonic dorsal root ganglion cells. Infected cultured were studied by electron microscopy. Viral nucleocapsids were observed to be internalized into neuronal cells bodies and neuritic extensions by fusion of the viral envelope and the plasma membrane. No signs of internalization by endocytosis were noted. Nucleocapsids were transported in neurites and were within 2 hrs post-infection found located near the microtubules and close to the nuclear pores in the perikaryon.A primary envelopment of nucleocapsids occurred at the inner lamina of the nuclear membrane and virions appeared between the two laminae. Presence of non-enveloped nucleocapsids outside the nuclear membrane and in close contact with the endoplasmic reticulum suggested that nucleocapsids could pass to the cytoplasmic side probably by de-envelopment at the outer nuclear membrane. A secondary envelopment occurred at the endoplasmic reticulum where the virions also became enclosed in transport vesicles. Enveloped virus appearing in the cytoplasm of neurons and neuritic extensions was always found only inside these transport vesicles.During their passage through the cytoplasm the virion-transport vesicle complexes were surrounded by smaller lysosome-like vesicles possibly derived from the Golgi apparatus. Fusion reactions between vesicles with virions and the smaller vesicles seemed to occur. We discuss if in this way the virion-transport vesicle complexes might be provided with glycosyl transferases and substrates necessary for maturation and completion of glycosylation of the viral envelope glycoproteins. The transport vesicles seemed essential for egress of virions from the infected cell by releasing virus when fusing with the plasma membrane.

Binding of herpes simplex virus to cellular heparan sulphate, an initial step in the adsorption process

It has been suggested that heparan sulphate has a receptor function in the initial phase of the attachment of herpes simplex virus (HSV) to cells. We have studied the influence of glycosaminoglycans on cell adsorption of, and plaque formation by, HSV-1 and HSV-2, with regard to the role of saccharide structure, chain length and charge density. Heparin and highly-sulphated heparan sulphate (1.5 sulphate groups/disaccharide unit), but neither chondroitin sulphate nor dermatan sulphate, were found to compete with the cellular receptor for attachment of HSV. Heparan sulphate preparations of low sulphate content (0.5 and 0.7 sulphate groups/disaccharide unit) failed to show any significant interaction with HSV. Oligosaccharides generated by partial deaminative cleavage of heparin were used to determine the minimum molecular size required for the binding of virus; the smallest oligosaccharide which reacted with HSV was composed of 10 monosaccharide units. The importance of charge density was demonstrated more directly by subfractionation of the heparin dodecasaccharide fraction by anion-exchange HPLC. The virus-binding capacities of the four resulting dodecasaccharide subfractions increased from the least sulphated to the most heavily sulphated fraction. The results reported are discussed in relation to virus-receptor interactions involved in the attachment of HSV, including the reported binding of HSV to the fibroblast growth factor receptor.

Neuritic Transport of Herpes Simplex Virus in Rat Sensory Neurons in vitro. Effects of Substances Interacting with Microtubular Function and Axonal Flow (Nocodazole, Taxol and Erythro-9-3-(2-hydroxynonyl)adenine)

Herpes simplex virus type 1 and a fluorescein-labelled lectin (wheat germ agglutinin) were selectively transported to nerve cell bodies located in the inner compartment of a two-chamber tissue culture system after the application of virus or lectin to the neuritic processes in the outer culture compartment. Taxol, which stabilizes and alters intracellular arrangements of microtubules, and nocodazole, which disrupts microtubules, both inhibited this retrograde axonal transport of viral particles and lectin. The transport was also inhibited by erythro-9-3-(2-hydroxynonyl)adenine (EHNA), which blocks ATPases. However, EHNA was also an effective inhibitor of infection with the virus in non-neuronal cells (GMK AH-1). The nature of the action(s) of EHNA on neuritic transport of the virus is therefore less clear.

Differential roles of B cells and IFN-gamma-secreting CD4(+) T cells in innate and adaptive immune control of genital herpes simplex virus type 2 infection in mice.

The role of B, CD4(+) T and CD8(+) T cells in both primary genital infection with attenuated herpes simplex virus type 2 (HSV-2) and development of protective immunity to a later challenge with virulent HSV-2 using lymphocyte-deficient mice has been elucidated. Following primary inoculation with attenuated thymidine kinase-deficient (TK(-)) HSV-2, B cell-deficient (microMT) mice developed a local viraemia and transient genital inflammation, suggesting a role for B cells in the innate control of local infection and inflammation. Natural antibodies are implicated in this process, as passive transfer of normal serum into microMT mice significantly reduced HSV-2 TK(-) shedding in the vaginal lumen, although it did not affect subsequent inflammation. Protection against lethal HSV-2 challenge was noted in HSV-2-vaccinated wild-type, CD8(+) T cell-deficient and microMT mice and was characterized by strong virus-specific IFN-gamma responses in vitro and delayed type hypersensitivity (DTH) responses in vivo. In contrast, CD4(+) T cell-deficient (CD4(-/-)) mice had impaired HSV-2-specific IFN-gamma production and DTH responses and succumbed rapidly to genital HSV-2 challenge. However, protective responses to HSV-2 could be induced in HSV-2-vaccinated CD4(-/-) mice by treatment with recombinant IFN-gamma. Taken together, these results suggest that CD4(+) T cells secreting IFN-gamma are critical for immune protection against lethal genital HSV-2 re-infection, whereas B cells/natural antibodies have anti-viral and -inflammatory effects in the innate control of a primary infection.

Acyclovir treatment of relapsing-remitting multiple sclerosis. A randomized, placebo-controlled, double-blind study.

Acyclovir treatment was used in a randomized, double-blind, placebo-controlled clinical trial with parallel groups to test the hypothesis that herpes virus infections are involved in the pathogenesis of multiple sclerosis (MS). Sixty patients with the relapsing-remitting form of MS were randomized to either oral treatment with 800 mg acyclovir or placebo tablets three times daily for 2 years. The clinical effect was investigated by an extensive test battery consisting of neurological examinations, neuro-ophthalmological and neuropsychological tests, and evoked potentials. Results were based on “intent-to-treat” data and the primary outcome measure was the exacerbation rate. In the acyclovir group (n = 30), 62 exacerbations were recorded during the treatment period, yielding an annual exacerbation rate of 1.03. The placebo group (n = 30) had 94 exacerbations and an annual exacerbation rate of 1.57. Thus, 34% fewer exacerbations were encountered during acyclovir treatment. This difference in exacerbation rate between the treatment groups was not significant (P = 0.083). However, this trend to a lower disease activity in acyclovir-treated patients was supported in subsequent data analysis. If the patients were grouped according to exacerbation frequencies, i.e. into low (0–2), medium (3–5) and high (6–8) rate groups, the difference between acyclovir and placebo treatment was significant (P = 0.017). Moreover, in a subgroup of the population with a duration of the disease of at least 2 years providing an exacerbation rate base-line before entry, individual differences in exacerbation rates were compared between the 2-year pre-study period and the study period in acyclovir-treated (n = 19) and placebo (n = 20) patients and acyclovir-treated patients showed a significant reduction of exacerbations (P = 0.024). Otherwise, neurological parameters were essentially unaffected by acyclovir treatment and there were no convincing signs of reduced neurological deterioration in the acyclovir group. This study indicates that acyclovir treatment might inhibit the triggering of MS exacerbations and thus suggests that acyclovir-susceptible viruses might be involved in the pathogenesis of MS. This possibility warrants further investigation.

Herpes Simplex Virus Types 1 and 2 Differ in Their Interaction with Heparan Sulfate

ABSTRACT Cell surface heparan sulfate (HS) serves as an initial receptor for many different viruses, including herpes simplex virus types 1 and 2 (HSV-1 and 2, respectively). Glycoproteins C and B (gC and gB) are the major components of the viral envelope that mediate binding to HS. In this study, purified gB and gC homologous proteins as well as purified HSV-1 and HSV-2 virions were compared for the ability to bind isolated HS receptor molecules. HSV-1 gC and HSV-2 gC bound comparable amounts of HS. Similarly, HSV-1 gB and its HSV-2 counterpart showed no difference in the HS-binding capabilities. Despite the similar HS-binding potentials of gB and gC homologs, HSV-1 virions bound more HS than HSV-2 particles. Purified gC and gB proteins differed with respect to sensitivity of their interaction with HS to increased concentrations of sodium chloride in the order gB-2 > gB-1 > gC-1 > gC-2. The corresponding pattern for binding of whole HSV virions to cells in the presence of increased ionic strength of the medium was HSV-2 gC-neg1 > HSV-1 gC−39 > HSV-1 KOS 321 > HSV-2 333. These results relate the HS-binding activities of individual glycoproteins with the cell-binding abilities of whole virus particles. In addition, these data suggest a greater contribution of electrostatic forces for binding of gB proteins and gC-negative mutants compared with binding of gC homologs and wild-type HSV strains. Binding of wild-type HSV-2 virions was the least sensitive to increased ionic strength of the medium, suggesting that the less extensive binding of HS molecules by HSV-2 than by HSV-1 can be compensated for by a relatively weak contribution of electrostatic forces to the binding. Furthermore, gB and gC homologs exhibited different patterns of sensitivity of binding to cells to inhibition with selectively N-, 2-O-, and 6-O-desulfated heparin compounds. The O-sulfate groups of heparin were found to be more important for interaction with gB-1 than gB-2. These results indicate that HSV-1 and HSV-2 differ in their interaction with HS.

Interleukin-12 (IL-12) and IL-18 Are Important in Innate Defense against Genital Herpes Simplex Virus Type 2 Infection in Mice but Are Not Required for the Development of Acquired Gamma Interferon-Mediated Protective Immunity

ABSTRACT Using a combination of gene-targeted mice and neutralizing antibodies, we showed that interleukin-12 (IL-12) and IL-18 are important in the innate control of genital herpes simplex virus type 2 infection but were not found to be critical, either singly or in combination, for the development of a protective gamma interferon-mediated immune response.

Localization of a functional site on herpes simplex virus type 1 glycoprotein C involved in binding to cell surface heparan sulphate

The amino acid residues critical for interaction between herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) and cell surface heparan sulphate (HS) were localized to two separate regions within antigenic site II of this glycoprotein. These amino acids were Arg-143, Arg-145, Arg-147 and Thr-150 in one region and Gly-247 in the other. This conclusion is based on the following observations. (i) Monoclonal antibodies defining gC-1 antigenic site II, and not those reactive with antigenic site I, inhibited HSV-1-induced haemagglutination and virus binding to susceptible cells. (ii) A number of HSV-1 mar mutants, altered at these critical residues, were impaired in attachment to cells. (iii) Synthetic peptides, corresponding to these two regions inhibited virus attachment to cells and infectivity. In addition these peptides were found to agglutinate red blood cells. This agglutination was inhibited by soluble HS, and was prevented by the pretreatment of red blood cells with heparitinase suggesting that cell surface HS was a site of peptide binding. The same was observed with the polycationic substances neomycin and poly-L-lysine. In conclusion, we propose that the regions of gC-1 represented by the HS-binding peptides may form a functional site of a polycationic nature, active in attachment to the polyanionic glycosaminoglycan chain of cell surface HS.