Cumulative cortisol concentrations in hair of patients with atopy are lower than in healthy subjects and are not related to their perceived stress experience.

Patients with atopy were found to exhibit blunted cortisol responses to acute stress stimuli. The aim of this study was to test the hypothesis that cumulative cortisol concentrations in the hair of patients with atopy are lower than in healthy subjects when related to their perceived stress experience. The sample consisted of 31 participants. The most proximal 3 cm of hair (as close to the scalp as possible), reflecting the cumulative cortisol secretion during the previous 3 months, was used for the analysis. Only in 20 subjects (9 patients with atopy and 11 healthy controls), there was a sufficient amount of hair for precise analysis using a new methodology. The results showed lower hair cortisol concentrations in patients with atopy compared to those in controls. The perceived stress scores in patients with atopy and healthy controls were not statistically different. The cortisol concentration/perceived stress score ratios were lower in patients with atopy compared to those in controls. No statistically significant correlation between hair cortisol and long-term experienced stress assessed via perceived stress scale was observed. In conclusion, the cumulative cortisol secretion in the hair of atopic patients is lower than would be expected according to their subjective scores of perceived stress. Most importantly, the previously lower stress hormone increase found in acute stress situations and in children now was confirmed in adult patients with chronic stress load.

Coronaviruses.

The members of coronavirus family are facultative pathogens of birds and mammals, including men. From their first isolation 60 years ago, they caused smaller or larger epidemics mainly originating from China. The most recent pandemic quickly spreading worldwide has affected over 2,000,000 people. Keywords: coronavirus; epidemic; single strand vRNA.

Patients with atopy exhibit reduced cortisol awakening response but not cortisol concentrations during the rest of the day.

It has been documented that cortisol release in response to acute stressors is reduced in patients with atopic dermatitis, allergic rhinitis, and other atopic diseases compared to that in healthy subjects. We aimed to test the hypothesis that atopic patients exert reduced salivary cortisol awakening response (CAR) in comparison with healthy subjects. The hypothesis was tested on a stressful and a relax day selected subjectively. Moreover, we evaluated the impact of trait anxiety. The sample consisted of 60 subjects, out of which 28 were patients with atopy and 32 healthy volunteers of both sexes. Saliva samples were collected in the morning to evaluate CAR as well as in the early afternoon and evening to look at cortisol concentrations during the rest of the day. The results showed reduced CAR in atopic patients compared to that in healthy subjects. This effect was modulated by sex with a significant difference observed in males. While CAR was reduced, atopic patients had unchanged cortisol concentrations throughout the day. The evening cortisol was even higher in atopic patients. If the subjects were stratified according to the trait anxiety, no significant differences in CAR between high and low anxiety were observed. No differences in cortisol variables including CAR were observed between the stressful and relax day. In conclusion, this study presents evidence on reduced CAR suggesting an insufficient HPA axis reactivity in atopy. Furthermore, the data in atopic patients demonstrate that reduced HPA axis reactivity does not necessarily mean lower cortisol concentrations throughout the day. This might be of relevance to immune system function and the course of the disease.

Attenuated cortisol reactivity to psychosocial stress is associated with greater visual dependency in postural control.

Despite known anatomical links between the hypothalamic-pituitary-adrenal (HPA) axis and the vestibular system, there are no studies on the relationship between postural control and HPA axis function. Visual dependence in postural control, often measured by increased postural sway on exposure to visual motion, is an indication of altered visual-vestibular integration with greater weighting towards visual cues for balance. Visual dependence is more common in older age and a range of vestibular and non-vestibular health conditions. The relationship between visual dependence in postural control was investigated in relation to cortisol reactivity to psychosocial stress (using the Trier Social Stress Test for groups: TSST-G), as an index of HPA axis function, in healthy young females. In those who exhibited a cortisol response (>2 nmol/l), a negative relationship between stress-induced cortisol reactivity and visual dependence in postural control was observed, since those with the largest cortisol response showed less visual motion induced postural sway (measured by force platform). This finding in healthy females indicates that subtle non-clinical differences in vestibular function are associated with dysregulated HPA axis activity as indicated by lower cortisol reactivity to psychosocial stress. It adds to the growing body of evidence linking blunted cortisol reactivity to stress to poor homeostatic regulation and potential negative health and behavioural outcomes.

Allergy and high trait anxiety are related to increases in heart rate variability: results of naturalistic long-term design study.

Summary: A number of studies report heart rate variability (HRV) changes in allergic as well as high trait anxious people, and associations between allergic inflammation and trait anxiety. This study investigated HRV of 20 low anxious allergic, 19 healthy high trait anxious and 18 healthy low anxious, in naturalistic setting. On arranged research days, subjects performed measurements using portable ECG device and subjective self-assessment of perceived stress. Five repeated measurements data from each subject have shown increases in overall HRV, as well as HRV on respiratory frequencies in both allergy and high trait anxiety. Subject's sex was an important factor, because HRV increases in allergy were only apparent in women. Data from self-assessment show no differences in experienced stress attributable to allergy, only to trait anxiety.

Immunohistochemical characterization of urothelial carcinoma.

From the archive of BB Biocyt company, 32 urinary bladder carcinomas (urothelium carcinomas, UC) and 7 cases of chronic cystitis were selected and examined in semiserial sections for the following antigens: 1) cell proliferation marker Ki-67 (expressed in the nuclei), 2) cell cycle regulator p16/INK4a polypeptide (expressed in the cytoplasm and nuclei), 3) urothelium marker p63 (expressed in the nuclei), 4) cytokeratin 7 (CK7). 5) cytokeratin 20 (CK20) and 6) high molecular weight cytokeratin (HMWCK). Invasive urothelium carcinomas showing a high grade dysplasia (invasive HG UC) comprised over the half (20 out of 32) of the investigated tumours. Microinvasion to lamina propria (seen in three HG papillary carcinomas) was regarded as an early infiltration even when the position of muscular layer could not be determined. Classical invasion across the urinary bladder wall and/or to surrounding tissues was found in 17 cases of low-differentiated HG UCs. The rest (9 out of 32 neoplasms) were either non-invasive papillary carcinomas of high (non-invasive HG UC, 5 cases) or low malignant potential (noninvasive LG UC, 4 cases). Finally, 3 cases were papillary urothelium neoplasms of low malignant potential (PUNLMP). HMWCK was present in all invasive tumours, whereas the frequency of other urothelium markers ranged from 65 to 88 %. Nevertheless, at least two markers were expressed in each invasive tumour. Staining for Ki-67 antigen was positive in over 50 % of the nuclei of HG UCs, while in the LG UCs, the frequency of positive Ki-67 staining did not exceed 25 %. In PUNLMP, the positive rate of Ki-67 stained dysplastic cells was below 10 %. The staining for p16 antigen did not correlate with the degree of dysplasia within urothelium tumours. For routine diagnostic, we recommend to combine the Ki-67 staining with detection of HMWCK. In cases of chronic cystitis, which developed urothelial hyperplasia and/or squamous metaplasia, the presence of p63 antigen was a relevant marker confirming the urothelial origin of the altered transitional cells (Tab. 6, Fig. 4, Ref. 69).

Correlations of survivin expression with clinicomorphological parameters and hormonal receptor status in breast ductal carcinoma.

The antiapoptotic protein survivin is widely expressed in most human cancers, including carcinomas of the breast. It is rarely detected in corresponding normal adult tissues. Therefore, survivin comes into the limelight as a promising diagnostic biomarker and prognostic parameter. Immunohistochemically, we examined the expression of this protein in 126 cases of ductal breast carcinoma to determine the association with clinicomorphological parameters such as age of patients, grade, stage and size of the primary tumor, lymph node metastasis, vascular invasion as well as estrogen and progesterone status. In each section, the subcellular location of survivin antigen, the intensity of staining and the percentage of labeled cells were assessed. Overall, survivin was expressed in 111 cases (88.1%). The statistical analysis revealed a significant correlation between the nuclear location of survivin and tumor grade 3. Furthermore, a significant relation was also found between vascular invasion and nuclear and combined nuclear and cytoplasmic survivin expression, together with a higher intensity of immunoreaction. However, no significant correlations were shown with other clinicomorphological parameters, such as stage and size of the tumor, lymph node metastasis, estrogen and progesterone receptors and age. Our findings revealed that survivin was frequently overexpressed in carcinoma cells, where it was present in different subcellular compartments. The nuclear positivity of survivin or combined nuclear and cytoplasmic expression was shown to be a poor prognostic parameter in ductal breast carcinoma.

Herpesvirus-associated lymphomas: Investigations in humans and animal models.

Lymphomas are solid tumors consisting of lymphoid cells; they form a heterogeneous group of less or more malignant disorders. A portion of lymphomas develop due to latent herpesvirus infections established in B and/or T-lymphocytes. The basis for latency is a lifelong presence of the circularized covalently linked viral genome within nuclei of carrier lymphocytes. In certain cases, however, the essential event leading to tumor formation is the integration of a portion(s) of viral DNA into the host cell DNA. This leads to rearrangements within the host cell genome on one hand, and, on other hand, to unregulated expression of oncoproteins encoded by the integrated fragment. Our review deals with mechanisms of lymphoma formation regarding to the role of non-structural herpesvirus oncoproteins interfering with the regulation of cell division and/or exerting anti-apoptotic effects. In addition, the authors wish to highlight the common procedures, which allowed isolation and/or identification of lymphoma-associated viruses in cell cultures derived from tumors and/or proliferating lymphatic tissues.

Expression pattern of anti-apoptotic protein survivin in dysplastic nevi.

UNLABELLED: The anti-apoptotic protein survivin was detected in a panel of 27 dysplastic nevi. From each representative paraffin block 4 mm sections were cut and stained with anti-survivin antibody (DAKO, Clone 12C4). In each section, the labeling intensity, the subcellular location of survivin antigen, the percentage of labeled cells and the degree of dysplasia were assessed. Survivin was present in 23 out of 27 cases (85.2%), but absent in 4/27 cases (14.8%). Positive staining was confined to the cytoplasm (C) of nevus cells only in 18 cases (66.7%), while cytoplasmic as well as nuclear positivity (NC) was found in 5 cases (18.5%). In no case solely nuclear staining could be seen. Furthermore, in 4 out of 5 cases (80%) with NC staining, severe dysplasia was found. Our data point at usefulness of survivin staining, otherwise rarely performed in dysplastic nevi. We confirm the importance of nuclear location of the survivin antigen, which may be helpful for assessing the possible progression to melanoma. KEYWORDS: survivin, immunohistochemistry, nevi, dysplasia, melanoma.

Multiorgan distribution of human influenza A virus strains observed in a mouse model.

Multiorgan spread and pathogenesis of influenza infection with three human influenza A viruses was studied in mice. Mouse-adapted viruses A/Dunedin/4/73(H3N2), A/Mississippi/1/85(H3N2), and A/PR/8/34(H1N1) differed considerably in virulence (p.f.u./LD(50)): 79,000 p.f.u. for Dunedin, 5,000 p.f.u. for Mississippi, and 65 p.f.u. for PR/8, which qualified Dunedin as low virulent, Mississippi as intermediate, and PR/8 as highly virulent. All three viruses were detected in lungs, heart, and thymus by cultivation and RT-PCR. Moreover, vRNA of all viruses was found in liver and spleen, of Dunedin and PR/8 also in kidneys and that of Dunedin and Mississippi in blood. Only vRNA of Dunedin was demonstrated in brain. Lung damage accompanied by histopathological changes and thymus reduction were most extensive after infection with the highly virulent virus PR/8. We assume that the ability to spread to multiple organs may be a more common property of influenza viruses in mammalian hosts than previously believed.

[Detection of regulatory protein p16/INK4A in the dysplastic cervical squamous cell epithelium is a diagnostic tool for carcinoma prevention]. / Detekcia regulacného proteínu p16/INK4A v dysplastickom dlazdicovom epiteli krcka maternice ako diagnostický nástroj prevencie karcinómu.

Parallel sections from 423 randomly selected blocks representing biopsies of 178 women with the diagnosis of cervical dysplasia and/or erosion were stained for p16 polypeptide. The p16/INK4A (inhibitory kinase 4) protein is a cellular division regulator, expression of which increases in the presence of oncoprotein E7, encoded by human papillomavirus (HPV). Expression of p16 protein was seen in the nuclei and cytoplasm of dysplastic squamous epithelium cells as well as in carcinoma cells. In 16.6% of erosion cases, the p16 antigen was present in the basal and suprabasal layer of the surrounding squamous epithelium revealing features of CIN I/LSIL. In CIN I/LSIL as classified by HE staining, the p16 antigen was found in 65 out of 80 (81%) cases. The p16 protein was typically seen in dysplastic basal and suprabasal cells encompassing a confluent layer in the lowest third segment of stratified epithelium. In CIN II and CIN III grouped as HSIL, the positive rate of p16 antigen presence was 95% (in 45 cases out of 47) and/or 100% (in each of 27 cases), respectively. The typical sign of p16 antigen distribution in HSIL was its staining over two thirds and/or throughout the whole dysplastic epithelium. Extensive staining for p16 antigen was registered within nuclei as well as cytoplasm of neoplastic cells in all 6 cervical squamous cell carcinomas, which were examined in many sections when being used as positive controls. Based on our experience, we consider the p16 antigen staining a helpful tool indicating dysplastic cells and estimating their extent.

Immune response and cytokine production following immunization with experimental herpes simplex virus 1 (HSV-1) vaccines.

Balb/c mice were immunized with the recombinant fusion protein gD1/313 (FpgD1/313 representing the ectodomain of HSV-1 gD), with the non-pathogenic ANGpath gE-del virus, with the plasmid pcDNA3.1-gD expressing full-length gD1 and with the recombinant immediate early (IE) HSV-1 protein ICP27. Specific antibodies against these antigens (as detected by ELISA) reached high titers with the exception of the DNA vaccine. High-grade protection against challenge with the virulent strain SC16 was found following immunization with the pcDNA3.1-gD plasmid and with the gE-del virus. Medium grade, but satisfactory protection developed after immunization with the FpgD1/313 and minimum grade protection was seen upon immunization with the IE/ICP27 polypeptide. A considerable response of peripheral blood cells (PBL) and splenocytes in the lymphocyte transformation test (LTT) was found in mice immunized with FpgD1/313, with the pcDNA3.1-gD plasmid and with the live ANGpathgE-del virus. For lymphocyte stimulation in vitro, the FpgD1/313 antigen was less effective than the purified gD1/313 polypeptide (cleaved off from the fusion protein); both proteins elicited higher proliferation at the 5 microg per 0.1 mL dose than at the 1 microg per 0.1 mL dose. The secretion of Th type 1 (TNF, IFN-gamma and IL-2) and Th type 2 (IL-4 and IL-6) cytokines was tested in the medium fluid of purified PBL and splenocyte cultures; their absolute values were expressed in relative indexes. The PBL from FpgD1/313 immunized mice showed increased secretion of both T(H)1 (TNF) as well as T(H)2 (IL-4) cytokines (7-10-fold, respectively). Splenocytes from FpgD1/313 immunized mice showed a significant (23-fold) increase in IL-4 production.

Reactivation of latent DNA viruses during immunosuppression: basic contemplation.

Immunosuppression (ISP) affects the outcome of acute virus primoinfection as well as the course of virus latency. ISP may result from viral infection of the immune cells. This infection can be either transient, as in measles virus infection, or progressive as in Human immunodeficiency virus (HIV-1, 2) infections, which proceed to acquired immunodeficiency syndrome (AIDS). Innate ISP occurs due to various congenital defects of cells, participating within immune response. In the majority of cases, ISP may be initiated by physical causes (e.g. irradiation) or by chemical substances including drugs used for post-transplantation therapeutic regimens. The hallmark of ISP is the impairment of B or T lymphocyte functions. Helper T lymphocytes produce a great variety of cytokines providing B lymphocyte proliferation and cytotoxic T cell maturation. The mature and/or activated memory cytotoxic T lymphocytes destroy the target cells, which synthesize virus-specific proteins. Since many ISP drugs hamper or downregulate the proliferation of B and T lymphocytes, their administration creates favorable conditions for the replication of reactivated DNA viruses otherwise produced in low amounts. Thus, ISP causes an extensive increase in the low-grade production of persisting virus, which might regularly occur during latency. In addition to reactivation of virus latency, ISP potentiates the replication and spread of any intercurrent infectious agent, whether bacterial, parasitic, yeast or fungal origin.

Developments in herpes simplex virus vaccines: old problems and new challenges.

Vaccination has remained the best method for preventing virus spread. The herpes simplex virus (HSV) candidate vaccines tested till now were mostly purified subunit vaccines and/or recombinant envelope glycoproteins (such as gB and gD). In many experiments performed in mice, guinea pigs and rabbits, clear-cut protection against acute virus challenge was demonstrated along with the reduction of the extent of latency, when established in the immunized host. The immunotherapeutic effect of herpes vaccines seems less convincing. However, introduction of new adjuvants, which shift the cytokine production of helper T-cells toward stimulation of cytotoxic T-cells (TH1 type cytokine response), reveals a promising development. Mathematical analysis proved that overall prophylactic vaccination of seronegative women, even when eliciting 40-60 % antibody response only, would reduce the frequency of genital herpes within the vaccinated population. Even when partially effective, immunotherapeutic vaccination might represent a suitable alternative of chronic chemotherapy in recurrent labial and genital herpes.

Efficacy of recombinant herpes simplex virus 1 glycoprotein D candidate vaccines in mice.

To compare the immunogenity of the herpes simplex virus 1 (HSV-1/HHV-1) recombinant glycoprotein D (gD1), as a potential protective vaccine, Balb/c mice were immunized with either gD1/313 (the ectodomain of the gD1 fusion protein consisting of 313 amino acid residues), or the plasmid pcDNA3.1-gD (coding for a full length gD1 protein, FLgD1). A live attenuated HSV-1 (deleted in the gE gene), and a HSV-1 (strain HSZP)-infected cell extract served as positive controls, and three non-structural recombinant HSV-1 fusion proteins (ICP27, UL9/OBP and thymidine kinase--TK) were used as presumed non-protective (negative) controls. Protection tests showed that the LD50 value of the challenging infectious virus increased 90-fold in mice immunized with ICP27, but remained unchanged in other control mice immunized with TK and OBP polypeptides. A significant protection (the LD50 value of challenging virus increased 800-fold) was noted following immunization with gD1/313, while immunization with the gE-del virus and/or the gD1 DNA vaccine resulted in a more than 4,000-fold increase of the challenging virus dose killing 50% of the animals. Using ELISA, elevated antibody titers were detected following immunizations with gD1/313, gE-del virus, and/or HSV-1-infected-cell extract. In addition, all of the three non-structural proteins elicited a good humoral response (with titres ranging from 1:16,000 to 1:128,000). The lowest IgG response (1:8,000) was noted after immunization with the gD1 DNA vaccine. Peripheral blood leukocytes (PBLs) as well as splenocytes from mice immunized with gD1/313, gE-del virus, and gD1-plasmid responded in lymphocyte transformation test (LTT) to the presence of purified gD1/313 antigen. For PBLs, the most significant stimulation of thymidine incorporation was registered at a gD1/313 concentration of 5 microg/100 microl, while the splenocytes from DNA vaccine-immunized mice responded already at a concentration of 1 microg/100 microl.

Murine herpesvirus pathogenesis: a model for the analysis of molecular mechanisms of human gamma herpesvirus infections.

Murine herpes virus (MHV), a natural pathogen originally isolated from free-living rodents, constitutes the most amenable animal model for human gamma herpesviruses. Based on DNA sequence homology, this virus was classified as Murid Herpesvirus 4 to subfamily Gammaherpesvirinae. Pilot studies in our laboratory, using mice inoculated by the intranasal route, showed that MHV infects macrophages, B lymphocytes, lung alveolar as well as endothelial cells. From the lungs the virus spreads via the bloodstream to spleen and bone marrow and via the lymphatics to the mediastinal lymph nodes. Similarly to other gamma herpesviruses, MHV established life-long latency maintained in host B lymphocytes and macrophages. An IM-like syndrome (per analogy to EBV) may develop during acute MHV infection, in which the atypical T/CD8+ lymphocytes eliminate viral DNA carrying B cells expressing the M2 latency associated protein. During latency, the MHV LANA (a KSHV LANA homologue) maintains the latent viral genome, assuring its copying and partition to new carrier cells in the course of division of the maternal cell. The nonproductive latency is turned onto virus replication by means of Rta protein. The chronic lymphoproliferative syndrome of unclear pathogenesis, which occurs in a certain part of latent MHV carriers, is related to the expression of gamma herpesvirus common latency-associated genes such as v-cyclin and/or to that of a virus-specific (M11/bcl-2) gene. This review attempts to summarize our knowledge concerning the function of MHV genes (either gamma herpesvirus common or MHV specific) related to immune evasion, latency and lymphoproliferation when highlighting the unsolved problems and/or controversial opinions.

Current developments in viral DNA vaccines: shall they solve the unsolved?

This review describes the mechanisms of immune response following DNA vaccination. The efficacy of DNA vaccines in animal models is highlighted, especially in viral diseases against which no widely accepted vaccination is currently available. Emphasis is given to possible therapeutic vaccination in chronic infections due to persisting virus genomes, such as recurrent herpes (HSV-1 and HSV-2), pre-AIDS (HIV-1) and/or chronic hepatitis B (HBV). In these, the problem of introducing foreign viral DNA may not be of crucial importance, since the immunised subject is already a viral DNA (or provirus) carrier. The DNA-based immunisation strategies may overcome several problems of classical viral vaccines. Novel DNA vaccines could induce immunity against multiple viral epitopes including the conservative type common ones, which do not undergo antigenic drifts. Within the immunised host, they mimic the effect of live attenuated viral vaccines when continuously expressing the polypeptide in question. For this reason they directly stimulate the antigen-presenting cells, especially dendritic cells. The antigen encoded by plasmid elicits T helper cell activity (Th1 and Th2 type responses), primes the cytotoxic T cell memory and may induce a satisfactory humoral response. The efficacy of DNA vaccines can be improved by adding plasmids encoding immunomodulatory cytokines and/or their co-receptors.

Expression of herpes simplex virus 1 glycoprotein D in prokaryotic and eukaryotic cells.

Recombinant plasmids encoding either the full-length glycoprotein D (FLgD) or truncated gDs were constructed. The recombinant plasmids were expressed in Escherichia coli and BHK-21 cells. The strongest expression was obtained with the recombinant plasmid encoding a truncated gD which corresponded to the gD ectodomain. The cells transformed with this plasmid showed good exponential growth ensuring satisfactory yields of the expressed polypeptide in the form of the fusion protein. The fusion protein was biotinylated and efficiently purified. The shortest truncated gD, which contained the main continuous antigenic locus VII binding neutralization antibody and additional continuous antibody binding epitopes, still reacted with specific antibody as proven by immunoblot analysis. In addition, a shuttle vector for expression of FLgD in mammalian cells was constructed. This vector-transfected BHK-21 cells expressed gD for 40 days during 9 consecutive passages. The expression of gD began on day 2 and culminated at day 9 post transfection (p.t.).

Leukemia-like syndrome in Balb/c mice infected with the lymphotropic gamma herpesvirus MHV-Sumava: an analogy to EBV infection.

Based on our previous observation that primary infection with the murine gamma herpesvirus (MHV) isolate Sumava (MHV-SU) undergoes a lymphoproliferative phase resembling to Epstein-Barr virus (EBV) induced infectious mononucleosis (IM), we evaluated white blood cell (WBC) counts at late stages following MHV-SU infection. In consequence of intranasal inoculation with MHV-SU a leukemia-like syndrome in Balb/c mice developed. The syndrome in question was accompanied with significant splenomegaly; in the peripheral blood leukocytosis (from 8 x 10(4) to 5 x 10(5) leukocytes/microl) and a high percentage of atypical lymphocytes (60-80%) was found. Presented results are bringing further evidence for lymphoproliferative effect of MHV and point at analogic course of MHV-SU and EBV infections.

Detection of immediate early protein ICP27/IE63 and thymidine kinase in the course of reactivation of latent herpes simplex virus 1 infection.

We followed the kinetics of reactivation of latent Herpes simplex virus 1 (HSV-1) infection established in rabbits by corneal route. The corresponding trigeminal ganglia (TG) were cultured and the culture medium was examined at daily intervals for release of infectious virus. Sections from the cultured TG fragments were stained with antisera against non-structural proteins such as the immediate early (IE) protein ICP27 and the early (E) proteins thymidine kinase (TK), the large subunit of ribonucleotide reductase (RR1), the ori-binding protein OBP and with a human serum obtained from volunteers immunized with an experimental subunit HSV-1 envelope (env) vaccine containing late structural proteins gB1, gC1, gD1 and gG1 (env antiserum). By indirect immunofluorescence (IF) test, ICP27 was detected in a few neurons from day 1 post explantation (p.e.), while TK was observed in neurons from day 2 p.e. Fluorescence with the human env antiserum was seen at day 3 p.e. The RR1 and OBP antisera stained productively infected Vero cells from 3 and 4 hrs post inoculation (p.i.), respectively. However, these sera showed no IF in cultured ganglion fragments at any interval examined. Our results showed the same cascade of HSV-1 IE and E protein expression during productive infection and reactivation in vitro.