Herpes simplex virus infection causes cellular beta-amyloid accumulation and secretase upregulation.

It is uncertain whether environmental factors contribute to the formation of senile plaques and neurofibrillary tangles, the abnormal features that define the Alzheimer's disease (AD) brain. We previously proposed that herpes simplex virus type 1 (HSV1) is a strong risk factor for AD when it is present in the brains of people who possess the type 4 allele of the apolipoprotein E gene (APOE-epsilon4); however a direct biochemical link between viral infection and the development of the AD pathological features has never previously been examined. Here we show that infection of cultured neuronal and glial cells with HSV1 leads to a dramatic increase in the intracellular levels of beta-amyloid (Abeta) 1-40 and 1-42, whilst levels of amyloid precursor protein (APP) in cells decrease. Similarly, Abeta1-42 deposits are present in mouse brain after HSV1 infection. In the cultured cells the mechanism involves increased Abeta production, rather than merely greater retention of cellular Abeta, as levels of beta-site APP-cleaving enzyme (BACE-1) and of nicastrin, a component of gamma-secretase, both increase in HSV1-infected cells. These novel data show that HSV1 can directly contribute to the development of senile plaques.

Asymptomatic shedding of HSV-1 in patients undergoing oral surgical procedures and attending for noninvasive treatment.

Oral surgery and stress can trigger and/or increase asymptomatic shedding of herpes simplex virus type-1 (HSV-1) into human saliva. In this investigation we examined the frequency of HSV-1 shedding in 32 patients undergoing an oral surgery procedure compared with 40 control patients attending for noninvasive treatment. Control patients comprised 18 migraine patients and 22 patients with temporomandibular (TMD) joint problems. Nested-PCR was carried out on oral rinses collected from each patient prior to treatment and 7 days post-treatment. Fifty-two of sixty-one seropositive patients were positive for HSV-1 DNA in one or both oral rinses. The frequencies of HSV-1 shedding for the oral surgery and control patients were 84.6% and 85.7% respectively. Seropositive patients who started shedding after treatment were significantly higher in oral surgery patients (46.2%) compared to control patients (34.3%). Shedding of HSV-1 in the oral cavity is not only increased by direct surgical trauma, but also appears to be common in migraine and TMD patients attending for general dental treatment. Thus pain or pain-induced stress as well as anxiety associated with dental treatment may also be a risk factor for asymptomatic shedding in specific seropositive patients attending for dental treatment.

Restraint stress facilitates systemic dissemination of Theiler's virus and alters its pathogenecity.

Theiler's murine encephalomyelitis virus (TMEV), a Picornavirus used as a viral model for multiple sclerosis (MS), causes an acute encephalomyelitis and chronic demyelination. The failure to clear the virus, which can result from stress, is a prerequisite for development of the later disease. Similarly, stressful life events have been associated with the development of MS. In the present study, a restraint stress (RS) model was used to investigate the effect of stress on the systemic dissemination of TMEV during the early stage of disease. Experimental data demonstrated that repeated RS remarkably facilitated the spread of virus from the CNS to such systemic organs as the spleen, lymph nodes, thymus, lungs and heart and compromised the ability of viral clearance within those tissues. RS also altered the pathogenecity of TMEV, enabling it to become cardiotropic, resulting in higher myocardial infectivity. These results demonstrate the profound impact that RS has upon both the tissue and organ dissemination of the virus, and the organ tropism of TMEV. An additional finding associated with stress was hepatic necrosis in the restrained animals, regardless of whether or not they were infected.

Epstein-Barr virus shedding by astronauts during space flight.

Patterns of Epstein-Barr virus (EBV) reactivation in 32 astronauts and 18 healthy age-matched control subjects were characterized by quantifying EBV shedding. Saliva samples were collected from astronauts before, during, and after 10 space shuttle missions of 5-14 days duration. At one time point or another, EBV was detected in saliva from each of the astronauts. Of 1398 saliva specimens from 32 astronauts, polymerase chain reaction analysis showed that 314 (23%) were positive for EBV DNA. Examination by flight phase showed that 29% of the saliva specimens collected from 28 astronauts before flight were positive for EBV DNA, as were 16% of those collected from 25 astronauts during flight and 16% of those collected after flight from 23 astronauts. The mean number of EBV copies from samples taken during the flights was 417 per mL, significantly greater (p<.05) than the number of viral copies from the preflight (40) and postflight (44) phases. In contrast, the control subjects shed EBV DNA with a frequency of 3.7% and mean number of EBV copies of 40 per mL of saliva. Ten days before flight and on landing day, titers of antibody to EBV viral capsid antigen were significantly (p<.05) greater than baseline levels. On landing day, urinary levels of cortisol and catecholamines were greater than their preflight values. In a limited study (n=5), plasma levels of substance P and other neuropeptides were also greater on landing day. Increases in the number of viral copies and in the amount of EBV-specific antibody were consistent with EBV reactivation before, during, and after space flight.

Increased expression of 5-HT1B receptor in dorsal raphe nucleus decreases fear-potentiated startle in a stress dependent manner.

5-HT(1B) autoreceptors regulate serotonin release from terminals of dorsal raphe nucleus (DRN) projections. Due to postsynaptic 5-HT(1B) receptors in DRN terminal fields, it has not previously been possible to manipulate 5-HT(1B) autoreceptor activity without also changing 5-HT(1B) heteroreceptor activity. We have developed a viral gene transfer strategy to express epitope-tagged 5-HT(1B) and green fluorescent protein in vivo, allowing us to increase 5-HT(1B) expression in DRN neurons. We have shown that increased 5-HT(1B) autoreceptor expression reduced anxiety in unstressed animals but increased anxiety following inescapable stress. These findings suggest that effects of increased 5-HT(1B) autoreceptor expression are dependent on stress context. To better understand the mechanisms underlying these observations, we have used fear-potentiated startle (FPS). FPS is especially sensitive to the activity of the amygdala, which shares reciprocal connections with DRN. In the absence of an inescapable stressor, increased 5-HT(1B) autoreceptor expression attenuated FPS response compared with animals injected with a virus expressing only green fluorescent protein. Administration of the 5-HT(1B) antagonist SB224289 (5 mg/kg i.p.) before startle testing blocked the effects of increased 5-HT(1B) autoreceptor expression. Since SB224289 had no effect on FPS in the absence of viral gene transfer, these results suggest that the antagonist reversed the behavioral effects of increased 5-HT(1B) autoreceptor expression through blockade of transgenic receptors. When tested 24 h following water-restraint stress, animals with increased 5-HT(1B) autoreceptors demonstrated restoration of robust FPS response. These results extend our previous studies and suggest explanations for the complex relationship between 5-HT(1B) autoreceptor expression, stress, and anxiety behavior.

Suppression of thermotolerance in mumps virus-infected cells is caused by lack of HSP27 induction contributed by STAT-1.

Viral infection modulates the regulation of apoptosis in host cells. Here, we report a novel mechanism by which human cells infected with mumps virus become susceptible to apoptosis caused by extracellular stresses. Mumps virus stimulates proteasome-dependent degradation of STAT-1 by action of viral accessory protein V, resulting in a severe decrease in STAT-1 protein in infected cells. We exposed mumps virus-infected and uninfected cells to heat and chemical stress. The infected cells failed to acquire resistance to apoptotic stimuli (thermotolerance) after exposure to these mild stresses. The induction of HSP27 by stress exposure was dramatically suppressed in the infected cells, but HSP70 induction was not affected. STAT-1 was required for transcriptional activation of the HSP27 gene, but not for the HSP70 gene, and cDNA transfection of STAT-1 in mumps virus-infected cells restored thermotolerance. Phosphorylated heat shock factor-1 (HSF-1) and STAT-1 phosphorylated on neither tyrosine nor serine residues were co-transported to the nucleus in response to stress. Furthermore, overexpression of unphosphorylatable mutants of STAT-1 also restored thermotolerance in mumps virus-infected cells. These lines of evidence indicate that the induction of HSP27 by stress requires STAT-1 in addition to the activated HSF-1. Furthermore, STAT-1 required for the induction of HSP27 worked independent to its phosphorylation. Thus, HSP27-dependent thermotolerance is suppressed by mumps virus infection through the destruction of STAT-1. The lack of thermotolerance should allow the infected cells to be eliminated by apoptosis and might be a host defense against viral infection.

Stress-induced reactivation of Epstein-Barr virus in astronauts.

Herpesviruses are leading causes of infectious blindness and death in immunocompromised individuals. Impaired cellular immunity, which is known to result in increased frequency and severity of herpesvirus infections, has been demonstrated both during and after spaceflight. Therefore, we examined whether Epstein-Barr virus (EBV), a well-characterized latent herpesvirus, undergoes reactivation in astronauts. Sera from Shuttle astronauts, taken before and after spaceflight, were examined for evidence of EBV reactivation. The geometric mean antibody titer to EBV viral capsid antigen (VCA) was significantly increased prior to flight compared to baseline (p = 0. 0001). After spaceflight, evidence of acute lytic replication was found in which 8- to 64-fold increases in EBV early antigen (EA) antibodies occurred without significant increases in antibodies to measles virus. Additionally, stress-induced shifts in circulating leukocytes and elevated levels of urinary cortisol and epinephrine were found. Overall, significant increases in EA or high VCA/EA antibody titers were found in 8 of 23 (35%) male astronauts and 3 of 5 (60%) female astronauts. These results indicate that stress reactivates EBV prior to flight and suggest that acute lytic replication of EBV occurs during spaceflight.

Social isolation may influence responsiveness to infection with bovine herpesvirus 1 in veal calves.

An experiment was performed to develop a model to study the impact of stress on responsiveness to infection with bovine herpesvirus 1 (BHV1) in veal calves. Social isolation after previous group-housing was used as a putatively stressful treatment. Group-housed specific pathogen-free veal calves (n=8) were experimentally infected with BHV1 at the age of 12 weeks. Half of the calves were socially isolated at the time of infection. Clinical, virological and serological responses to BHV1, and adreno-cortical reactivity to exogenous ACTH were examined. In comparison with group-housed calves, calves socially isolated at the time of infection showed a diminished clinical and fever response, and delayed viral excretion after primary infection with BHV1. Four weeks after social isolation, basal cortisol levels before, and the integrated cortisol response after administration of a low dose of ACTH, were significantly depressed in socially isolated calves. The results suggest that social isolation in veal calves influences the response to an experimental BHV1 infection. A possible mechanism is discussed.

A novel link between stress and human cytomegalovirus (HCMV) infection: sympathetic hyperactivity stimulates HCMV activation.

Recently, inflammatory mediators such as TNFalpha were identified as triggering active human cytomegalovirus (HCMV) infection. Here, we demonstrate that a highly stressful event in the absence of systemic inflammation, as observed in patients with acute myocardial infarction, leads to the development of an active HCMV infection in latently infected patients. Elucidating the molecular mechanism of virus activation, we could show that catecholamines directly stimulate the HCMV immediate-early (IE) enhancer/promoter in monocytic cells via beta-2 adrenergic receptors. Subsequent activation of the cAMP/PK-A-signaling pathway results in enhanced synthesis and binding of the transcription factor CREB-1/ATF-1 to the cAMP-responsive elements within the IE enhancer. Epinephrine also enhanced HCMV gene expression in infected THP-1 cells by about 50% in three of four experiments. These data suggest that HCMV, like HSV-1 and VZV, can be (re)activated under stress conditions.

TNFalpha, interferon, and stress response induction as a function of age-related susceptibility to fatal Sindbis virus infection of mice.

The age-related acquisition of resistance to fatal Sindbis virus infection was examined using a molecularly cloned laboratory strain of the AR339 isolate designated TRSB. TRSB caused 100% mortality in mice up to 5 days of age. Resistance to fatal infection developed abruptly between 5 and 9 days of age. Lethal Sindbis virus infection of mice inoculated at 4 days of age was characterized by high levels of virus replication, induction of high levels of interferon-alpha/beta and TNF-alpha and severe thymic involution indicative of a systemic stress response. These changes correlated with predominantly noninflammatory lesions. In contrast, TRSB infection of older mice was characterized by survival, more limited virus replication, reduced cytokine induction, and the development of inflammatory responses leading to encephalitis, myositis, and myocarditis. Previous studies utilized infections of neonatal mice with TRSB and an attenuated mutant of TRSB to compare fatal and nonfatal Sindbis infection (Trgovcich et al., 1996. Virology 224, 73-83). The experiments reported here utilize mouse age at the time of infection to create conditions for examination of fatal and nonfatal TRSB infections. Both experiments suggest that fatal infection is associated with a shock-like syndrome and little or no inflammatory pathology, while survival is correlated with greatly reduced cytokine levels and inflammatory lesions.

Stress-related modulation of central nervous system immunity in a murine model of herpes simplex encephalitis.

This study assesses the immunomodulatory effects of stress on the pathogenesis of herpes simplex encephalitis (HSE) in a mouse model. Physical restraint served as the stressor and HSE developed subsequent to HSV-1 inoculation into the tongues of subject animals. Clinical data showed that stressed mice lost more weight and had greater mortality rates than unrestrained animals during the course of infection. Histologic tissue sections demonstrated a stress-related reduction of the cellular inflammatory response in the central nervous system (CNS). This model may be useful to further investigate the mechanisms of stress-related immunosuppression in the CNS.

Role of the hypothalamic pituitary adrenal axis and IL-6 in stress-induced reactivation of latent herpes simplex virus type 1.

Hyperthermic stress induces reactivation of herpes simplex virus type 1 (HSV-1) in latently infected mice and also stimulates corticosterone release from the adrenals via activation of the hypothalamic pituitary adrenal axis. In the present study, we tested the hypothesis that stress-induced elevation of corticosterone potentiates HSV-1 reactivation in latently infected mice. Because of the putative role of IL-6 in facilitating HSV-1 reactivation in mice, the effect of hyperthermic stress and cyanoketone treatment on IL-6 expression in the trigeminal ganglion was also measured. Preadministration of cyanoketone, a glucocorticoid synthesis inhibitor, blocked the stress-induced elevation of corticosterone in a dose-dependent manner. Furthermore, inhibition of corticosterone synthesis was correlated with reduced levels of HSV-1 reactivation in latently infected mice. Hyperthermic stress elicited a transient rise in IL-6 mRNA levels in the trigeminal ganglion, but not other cytokine transcripts investigated. In addition, there was a significant reduction in MAC-3+, CD8+, and DX5+ (NK cell marker) cells in the trigeminal ganglion of latent HSV-1-infected mice 24 h after stress. Cyanoketone blocked the stress-induced rise in IL-6 mRNA and protein expression in the trigeminal ganglion latently infected with HSV-1. Collectively, the results indicate that the activation of the hypothalamic pituitary adrenal axis plays an important role in stimulating IL-6 expression and HSV-1 reactivation in the trigeminal ganglion following hyperthermic stress of mice.

Herpes simplex virus Us11 protein enhances recovery of protein synthesis and survival in heat shock treated HeLa cells.

One of the herpes simplex virus type 1 (HSV-1) true late gene products, Us11 protein, is brought into the cell by the infecting virion and may play a role in the virally-induced post-transcriptional control of gene expression. Us11 protein forms large oligomers, exhibits RNA binding features, concentrates into the nucleolus and is able to replace Rex protein in post-transcriptional control of human T-cell leukemia/lymphoma virus type I (HTLV-I) expression. As heat shock drastically alters protein synthesis, and because HSV-1 infection stimulates heat shock protein (Hsp) expression, we analyzed the consequence of heat shock in HeLa cells expressing Us11 alone, either transiently or constitutively. No detectable modification of the overall pattern of protein synthesis was observed in cells growing at normal temperatures, including no induction of Hsp expression or accumulation. However, Us11 protein expression induced an enhanced recovery of protein synthesis after heat shock. Moreover, the level of Us11 protein-mediated protection of protein synthesis was similar to that observed for cells made thermotolerant, but only when submitted to a mild heat shock. Finally, Us11 protein expression induced in cells an enhanced survival to heat shock.

Sindbis virus infection of neonatal mice results in a severe stress response.

Neonatal mice were infected with virus derived from a molecular clone of a laboratory strain of Sindbis virus, TRSB. The resulting acute fatal infection was typified by few if any of the classic hallmarks of encephalitis, very high levels of interferon-alpha/beta (IFNalphabeta), and lesions in the thymus and hematopoietic tissues usually associated with a severe stress response. Infection with an attenuated mutant of TRSB, which harbors a single amino acid change in the E2 surface glycoprotein (TRSBr114), was characterized by encephalitis, reduced mortality, low levels of IFNalphabeta, and no thymic pathology (J. Trgovcich, J. F. Aronson, and R. E. Johnston, 1996, Virology 224, 73-83). Here we report that infection of neonatal mice with TRSB, but not TRSBr114, resulted in induction of high levels of tumor necrosis factor-alpha as well as high and sustained levels of adrenalcorticotropin-releasing hormone and corticosterone. This syndrome of potentially toxic cytokine and stress hormone induction correlates with lethal Sindbis virus infection and constitutes a previously unrecognized aspect of Sindbis virus pathogenesis in mice.

Cold stress-induced neuroinvasiveness of attenuated arboviruses is not solely mediated by corticosterone.

In previous studies we have shown that various stress paradigms can induce the penetration of noninvasive, attenuated viruses into the central nervous system (CNS). Since glucocorticoids levels are elevated during stress, we compared the effect of cold stress and corticosterone (CS) injection on neuroinvasiveness of a non-invasive encephalitic virus, WN-25 (West Nile). Exposure of inoculated mice to cold stress or CS resulted in high viremia and a marked increase in mortality when compared to control untreated mice. Exposure of WN-25 inoculated mice to cold treatment or CS injection led to high blood virus levels as compared to nontreated mice (3.2 and 3.1 vs > 1 log 10 PFU/ml). Cold stress or CS (5000 ng/mouse) treatment caused a mortality rate of 70% and 50% of the WN-25 inoculated mice respectively. No mortality was recorded in control inoculated groups (p < 0.05). Passive transfer serum from uninfected cold stressed mice to WN-25 inoculated nonstressed mice, resulted in similar mortality. The levels of CS in passive transferred serum from cold stressed animals was 500 ng/ml, only 2% (100 vs. 5000 ng) of the CS dose required to obtain a similar effect on viral penetration and mortality when CS was injected directly. Therefore, we concluded that CS was not the sole factor responsible for the cold stress effect on the viral infection outcome.