Reduction of Thymoglobuline from 7.5 mg/kg to 6 mg/kg in conditioning regimen extended time to the first cytomegalovirus detection after allogenic haematopoietic stem cell transplantation.

INTRODUCTION: The optimal dosage of anti-thymocyte globulin (ATG) may influence the outcome of patients after allogenic haematopoietic stem cell transplantation (HSCT). The aim of our study was to analyse human cytomegalovirus (CMV) infection data, incidence of graft-versus-host disease and other clinical endpoints comparing two patients cohorts that were administered two different Thymoglobuline Genzyme doses as part of the HSCT conditioning regimen. MATERIALS AND METHODS: Total of 65 adult patients received ATG (7.5 mg/kg or 6 mg/kg) as a part of the fludarabine/busulfan/ATG conditioning regimen. CMV DNAemia was monitored after HSCT using quantitative real-time PCR and preemptive treatment was started for viral loads above 1000 cp/ml. RESULTS: The mild ATG dose reduction extended the time to the first CMV detection after transplantation (28 days for 7.5 mg/kg dose vs. 40 days for 6 mg/kg dose, p = 0.04). But it did not reduce the incidence or influence first anti-CMV treatment onset, the initial viral load, peak viral load in whole blood or the antiviral therapy parameters (all p 0.18). No impact of ATG dose reduction on incidence of graft-versus-host-disease, relapse of underlying disease or mortality within first year after transplantation (all p 0.32) were observed. CONCLUSIONS: The reduced ATG dosages can allow lower toxicity of conditioning regimen while keeping the performance.

Locally advanced colorectal cancer in pregnancy.

OBJECTIVE: To highlight the issue of diagnostics and treatment management in colorectal cancer of pregnant women. DESIGN: Case report. SETTING: Department of Obstetrics and Gynaecology, Pardubice Hospital. CASE REPORT: We present a case of a 24-year-old secundigravida, diagnosed as colorectal cancer in week 34 of pregnancy, manifesting as a diarrhoea and a stomach ache. The diagnosis was made in an advanced stage due to underestimation of clinical symptoms. CONCLUSION: The diagnosis of the colorectal cancer in pregnancy can be difficult because the presenting symptoms are easily attributable to pregnancy. Therefore the advanced disease is diagnosed more frequently in pregnant patients than in the general population. Colorectal cancer should be managed by multidisciplinary team with regard to gestation.

[Spontaneous rupture of the uterine artery in connection with pregnancy]. / Spontánní ruptura uterinní arterie v souvislosti s tehotenstvím.

OBJECTIVE: To present two cases of spontaneous uterine artery rupture in connection with pregnancy, accompanied by life endangering bleeding. In the first case we talk about a thirty-three years old primigravida nullipara in the 20th week of pregnancy. In the second case there is a woman, who gave spontaneous birth in the 36th week of pregnancy. DESIGN: Case report. SETTING: Department of Obstetrics and Gynecology, Pardubice Hospital. METHODS: Own observation. CONCLUSION: Spontaneous rupture of the uterine artery in connection with pregnancy is a rare complication that can be fatal for mother and fetus. Clinical symptoms are often nonspecific, diagnostics is difficult, and diagnosis is often made during the surgery. It is necessary to include this rare complication in the differential diagnosis in pregnant women with abdominal pain and haemoperitoneum of unknown etiology and in postpartal women with an unknown source of bleeding.

Primary malignant melanoma of the vagina, case report and review of the literature.

OBJECTIVE: Presentation of primary malignant melanoma of the vagina. Summary of clinical findings and management. DESIGN: Case report. SETTING: Nemocnice Pardubického kraje a.s., Porodnicko-gynekologická klinika, Pardubice. OBSERVATION: We report a case of primary malignat melanoma in a 44 year old woman, with symptoms of abnormal vaginal bleeding and painful intercourse. CONCLUSION: Primary malignant melanoma of the vagina is rare disease. Goal of therapy is complete removal of primary tumor.

[Suburethral sling removal due to chronic pain]. / Exstirpace suburetrální pásky z duvodu chronických bolestí.

OBJECTIVE: To point out one possible complication after suburethral tape insertion. To present methods of treatment, especially partial or total removal of the tape. Few successful cases of treatment are shown in case reports. DESIGN: Case report. SETTING: Department of obstetrics and gynecology, Pardubice hospital. OBSERVATION: Chronic pain after insertion of suburethral tape is a rare complication. There are several possible causes, such as tape erosion with chronic inflamation, post-colposuspension syndrome, myo-fascial syndrome, nerve damage during tape implantation or due to overproduction of fibrous tissue. Partial or total tape removal is needed when the conservative treatment fails. Patients may remain continent or a new tape is placed. Pain relief is usually complete but there are times when additional treatment is needed. CONCLUSION: Sling removal is a successful method of treating chronic pain after suburethral tape insertion. Patients describe plain relief and improvement of quality of life.

[Ganciclovir treatment failure in adult allogeneic hematopoietic stem cell transplant recipients with cytomegalovirus infection--a single centre experience]. / Klinická rezistence lidského cytomegaloviru pri lécbe gancyklovirem u pacientu po alogenní transplantaci hematopoetických bunek--zkusenosti jednoho centra.

OBJECTIVE: To determine the incidence of infection with ganciclovir-resistant cytomegalovirus (CMV) in adult allogeneic hematopoietic stem cell transplant (HSCT) recipients. Clinical resistance or treatment failure was defined as persistent DNAemia or increasing viral load in peripheral blood after 2 weeks of virostatic treatment. The association between the treatment failure and viral resistance was analysed. The presence of ganciclovir-resistant CMV strains was confirmed by genotypic testing able to detect mutations conferring resistance. METHODS: In 2012 and 2014, 40 patients who underwent allogeneic HSCT for hematologic malignancies and were treated for human CMV reactivation/disease were followed up prospectively. In patients with treatment failure, CMV DNA was isolated and analysed by nucleotide sequence analysis of the UL 97 and UL 54 genes conferring resistance to the virostatic agent. RESULTS: The treatment failure occurred in seven patients, but ganciclovir resistance conferring mutations were only detected in two of them (mutations L595F and M460I in the UL 97 gene). Another mutation in the UL 97 gene (N510S) was found in a patient with recurrent CMV replication who needed to be retreated but did not meet the criteria for treatment failure. CONCLUSION: The low incidence of genetically confirmed ganciclovir-resistant CMV isolates in HSCT recipients with relatively common clinical treatment failure suggests that the mechanism underlying slower viral clearance is often other than mutations conferring ganciclovir resistance to the virus.

[Lipid and lipoprotein levels in normal and complicated pregnancies]. / Zmeny lipidu a lipoproteinu provázející fyziologická a riziková tehotenství

OBJECTIVE: Lipid and lipoprotein levels during pregnancy and postpartum were compared in healthy pregnant women and in high risk pregnancy groups including women with pre-existing metabolic syndrome. The second half of a normal pregnancy is accompanied by hyperlipidaemia and glucose intolerance. Physiological response to pregnancy represents a transient excursion into a metabolic syndrome. DESIGN: Clinical study. SETTING: Department of Obstetrics and Gynaecology, Regional Hospital Pardubice, Faculty of Health Studies, University of Pardubice. METHODS: Fasting plasma lipid and lipoprotein levels were studied in 54 women during pregnancy and postpartum at the Regional Hospital Pardubice. The group of patients consisted of 25 healthy pregnant women,11 women with gestational diabetes, 9 women with diabetes type 1 and 9 women with pre-existing metabolic syndrome. The results were compared to measurements in 30 non-pregnant healthy controls. RESULTS: Total triglyceride levels in late gestation were at least but significantly (p < 0.001) elevated above those in non-pregnant controls in the group of patients with pre-existing metabolic syndrome. In the same group, in contrast with other groups, no changes in plasma cholesterol and LDL-cholesterol were found in late pregnancy. However, significant decline in HDL-cholesterol, when compared to non-pregnant controls, was observed 6 and 24 months postpartum only in the group of women with pre-existing metabolic syndrome (p < 0.01, p < 0.001 respectively). CONCLUSION: The gynaecologist may be the first physician that a woman of reproductive age and with a metabolic syndrome encounters, it is important to suspect the presence of this syndrome and inform the woman about the most notable clinical implications.

[Hyperlipidaemia in pregnancy]. / Hyperlipidémie v tehotenství

OBJECTIVE: We investigated the degree of pregnancy - induced hyperlipidaemia in normal and complicated pregnancies. Under the "stress of pregnancy" supraphysiologic increase in lipid and lipoprotein levels may delevop (similar to changes in carbohydrate metabolism). DESIGN: Clinical study. SETTING: Department of Obstetrics and Gynaecology, Regional Hospital Pardubice, Faculty of Health Studies, University of Pardubice. METHODS: Fasting plasma lipid and lipoprotein levels were studied in 26 healthy pregnant women, in 30 women with high risk pregnancy and in 30 nonpregnant healthy controls. The results were compared to reference values given in percentiles to find those exceeding the 95th percentile of the reference values for the third trimester. RESULTS: Two subjects (3.6%) with supraphysiologic increase in plasma lipids in late pregnancy were identified among the 56 women studied. The details are reviewed in the study as well as the rare forms of extreme hyperlipoproteinaemia during pregnancy in women with pre-existing disorder of lipid metabolism. CONCLUSION: The measurement of lipid and lipoprotein levels cannot be recommended in all pregnant women in late pregnancy. Hovewer, in subjects with family history of dyslipidaemia or premature atherosclerosis in the first - degree relatives and without hyperlipidaemia outside pregnancy, supraphysiologic increase in lipid and lipoprotein levels developed under the "stress of pregnancy" might indentify women at risk of dyslipidaemia in future life.

[3D MRI-based brachytherapy in patients with locally advanced cervical carcinoma - early clinical results]. / 3D MR-based brachyterapie v lécbe lokálne pokrocilého karcinomu delozního hrdla - casné klinické výsledky.

OBJECTIVE: To demonstrate the clinical benefit of MRI-based brachytherapy using CT and MRI data fusion. DESIGN: Clinical trial. SETTING: Oncology Centre, Multiscan and Pardubice Regional Hospital, Pardubice. METHODS: Thirty six patients with locally advanced cervical cancer were treated with MRI-based brachytherapy (MRI-based preplanning 15 patients, MRI approximation 21 patients). All patients were continuously followed during and after treatment. Tumor control and toxicity were evaluated at each visit. Late gastrointestinal and genitourinary symptoms were recorded, using Fox Chase (FC) modification of the Radiation Therapy Oncology Group (RTOG) and Late Effects Normal Tissue Task Force (LENT) toxicity criteria. RESULTS: We observed no complications during the entire MRI procedure at the radiology department. The cumulative incidence of grade 2 late genitourinary and gastrointestinal toxicity at median follow up of 30.3 months was 4.8% and 9.5% for MRI approximation, respectively 0% and 13.3% for MRI-based preplanning. Local control was 86.7% for MRI-based preplanning and 76.2% for MRI approximation. CONCLUSION: 3D MRI-based brachytherapy with consecutive CT/MRI data fusion yields excellent local control with minor toxicity.

BAL fluid analysis in the identification of infectious agents in patients with hematological malignancies and pulmonary infiltrates.

The present study aims to evaluate the diagnostic yield of bronchoalveolar lavage (BAL) fluid in patients with hematological malignancies and describe the most common pathogens detected in BAL fluid (BALF.) An analysis of 480 BALF samples was performed in patients with hematological malignancies over a period of 7 years. The results of culture methods, PCR, and immunoenzymatic sandwich microplate assays for Aspergillus galactomannan (GM) in BALF were analyzed. Further, the diagnostic thresholds for Aspergillus GM and Pneumocystis jiroveci were also calculated. Microbiological findings were present in 87% of BALF samples. Possible infectious pathogens were detected in 55% of cases; 32% were classified as colonizing. No significant difference in diagnostic yield or pathogen spectrum was found between non-neutropenic and neutropenic patients. There was one significant difference in BALF findings among intensive care units (ICU) versus non-ICU patients for Aspergillus spp. (22% versus 9%, p = 0.03). The most common pathogens were Aspergillus spp. (n = 86, 33% of BAL with causative pathogens) and Streptococcus pneumoniae (n = 46, 18%); polymicrobial etiology was documented in 20% of cases. A quantitative PCR value of > 1860 cp/mL for Pneumocystis jirovecii was set as a diagnostic threshold for pneumocystis pneumonia. The absorbance index of GM in BALF of 0.5 was set as a diagnostic threshold for aspergillosis. The examination of BAL fluid revealed the presence of pathogen in more than 50% of cases and is, therefore, highly useful in this regard when concerning pulmonary infiltrates.

Prediction of long-term prognosis of age-related macular degeneration treated by hemorheologic therapy using baseline laboratory indicators - Experimental-clinical model.

BACKGROUND + OBJECTIVE:Age-related macular degeneration (AMD) is the most common cause of practical blindness in people over 60 years of age in industrialised countries. We formulated a hypothesis that a group of initial laboratory parameters would be suitable for prediction of prognosis of AMD, allowing for individual modifications in treatment intensity. PATIENTS AND METHODS: 66 patients with dry form of AMD were treated using rheohaemapheresis with an individual follow-up period of more than 5 years. The patients' initial laboratory data was split in two subgroups based on treatment success and analysed using discriminant analysis (analysis of the linear and quadratic models using the automated and interactive step-wise approach) by means of the Systat 13 software. RESULTS: Prediction of prognosis based on the initial laboratory parameters was correct in 79% of unsuccessfully treated patients, allowing for early detection of high-risk patients. With the use of a quadratic model, the prediction was correct in 100% of unsuccessfully treated patients and in 75% of successfully treated patients. CONCLUSION: Implementation of discriminant analysis is a promising method for prediction of prognosis, especially when the patient is at risk of AMD progression, which allows for early and more intensive monitoring and treatment.

Toxic epidermal necrolysis data from the CELESTE multinational registry. Part II: Specific systemic and local risk factors for the development of infectious complications.

The aim of the study was to identify the most important systemic and local risk factors for the development of infectious complications in patients with toxic epidermal necrolysis (TEN). MATERIAL AND METHODOLOGY: This is a multicentric study that included all patients with TEN who were hospitalized between 2000-2015 in specialized centres in the Czech Republic and Slovakia. The total catchment area included a population of over 12.5 million inhabitants. The actual implementation of the project was carried out using data obtained from the CELESTE (Central European LyEll Syndrome: Therapeutic Evaluation) registry, wherein specific parameters related to epidemiological indicators and infectious complications in patients with TEN were evaluated as a retrospective analysis. RESULTS: A total of 38 patients (97%) of the group were treated with corticosteroids. The comparison of patients with different doses of corticosteroids did not exhibit a statistically significant effect of corticosteroid administration on the development of infectious complications (p=0.421). There was no effect of the extent of the exfoliated area on the development of infectious complications in this area. The average extent of the exfoliated area was 66% TBSA (total body surface area) in patients with reported infectious complications and 71% TBSA (p=0.675) in patients without infectious complications. In the case of the development of an infectious complication in the bloodstream (BSI), the increasing effect of the SCORTEN (SCORe of Toxic Epidermal Necrosis) value was monitored during hospitalization. Within 5days from the beginning of the hospitalization, the average SCORTEN value was 2.7 in 6 patients with BSI and 3.0 in 32 patients without BSI (p=0.588). In the period after the 15th day of hospitalization, 7 patients with BSI had an average SCORTEN value of 3.4, and 16 patients without BSI had an average SCORTEN value of 2.5 (p=0.079). In the case of low respiratory tract infection (LRTI), the effects of the necessity for artificial pulmonary ventilation and the presence of tracheostomy were monitored. The statistically significant effect of mechanical ventilation on the development of LRTI occurred only during the period of 11-15days from the beginning of the hospitalization (p=0.016). The effect of the tracheostomy on the development of LRTI was proven to be more significant. CONCLUSION: We did not find any statistically significant correlation between the nature of immunosuppressive therapy and the risk of developing infectious complications. We failed to identify statistically significant risk factors for the development of BSI. Mechanical ventilation and tracheostomy increase the likelihood of developing LRTIs in patients with TEN.

Toxic epidermal necrolysis data from the CELESTE multinational registry. Part I: Epidemiology and general microbiological characteristics.

INTRODUCTION: Toxic epidermal necrolysis (TEN) is a rare, life-threatening autoimmune disease predominantly manifested in the skin and mucous membranes. Today, infectious complications have the dominant share in mortality of TEN patients. Due to the nature of the therapy and administration of immunosuppressive medications, a wide range of potentially pathogenic microorganisms, which cause infectious complications in different compartments in these patients, is not surprising. MATERIAL AND METHODOLOGY: This is a multicentric study, which included all patients with TEN hospitalized between 2000-2015 in specialized centres in the Czech Republic and Slovakia. The total catchment area was over 12.5 million inhabitants. The actual implementation of the project was carried out using data obtained from the registry CELESTE (Central European LyEll Syndrome: Therapeutic Evaluation), when specific parameters relating to epidemiological indicators and infectious complications in patients with TEN were evaluated in the form of a retrospective analysis. RESULTS: In total, 39 patients with TEN were included in the study (12 patients died, mortality was 31%), who were hospitalized in the monitored period. The median age of patients in the group was 63 years (the range was 4-83 years, the mean was 51 years), the median of the exfoliated area was 70% TBSA (total body surface area) (range 30-100%, mean 67%). SCORTEN was calculated for 38 patients on the day of admission. Its median in all patients was 3 (range 1-6; mean 3). Any kind of infectious complication in the study group was recorded in 33 patients in total (85%). In total, 30 patients (77%) were infected with gram-positive cocci, 27 patients (69%) with gram-negative rods, and yeast cells or fibrous sponge were cultivated in 12 patients (31%). A total of 32 patients (82%) were found to have infectious complications in the exfoliated area, 15 patients (39%) had lower respiratory tract infections, 18 patients (46%) urinary tract infections and 15 patients (39%) an infection in the bloodstream. The most common potentially pathogenic microorganism isolated in our study group was coagulase neg. Staphylococcus, which caused infectious complications in 24 patients. Enterococcus faecalis/faecium (19 patients), Pseudomonas aeruginosa (17 patients), Staphylococcus aureus (11 patients) and Escherichia coli (11 patients) were other most frequently isolated micro-organisms. CONCLUSION: The published data were obtained from the unique registry of TEN patients in Central Europe. In the first part, we have succeeded in defining the basic epidemiological indicators in the group of patients anonymously included in this registry. The study clearly confirms that infectious complications currently play an essential role in TEN patients, often limiting the chances of survival. The study also shows a high prevalence of these complications in the period after 15days from the start of hospitalization, when most patients already have completely regenerated skin cover.

Antireticulin antibodies in sera of children with insulin-dependent diabetes mellitus.

Sera from 125 children (mean age 9.5 +/- 3.9; range 0.5-18 years) with newly diagnosed insulin-dependent diabetes mellitus were examined for the presence of antireticulin antibodies (ARA). Fifty-four of these children were followed up over a period of 150-400 days after the onset of the disease with respect to their serum ARA. The indirect immunofluorescence method on human and rat tissue was used to detect autoantibodies. In each serum, the level of islet cell antibodies (ICA) was determined. The prevalence of ARA in our diabetic children (16%) was significantly higher than in normal population (P less than 0.05). In sera of newly diagnosed ICA-negative children, ARA were more frequent than in ICA-positive patients (P less than 0.025). The difference in ARA prevalence was even higher when patients were divided into two groups one with less and one with more than 30 JDFu (P less than 0.005). On the contrary, sera sampled 150-400 days after the manifestation of IDDM revealed neither a positive nor a negative association between ICA and ARA. Thus, the negative association of ARA with ICA in the early stages of IDDM may suggest the role of an autoimmune response to reticulin in part of the IDDM patients, and gives further evidence to the heterogeneity of IDDM.

Troponin T levels in the cord blood of healthy term neonates.

Troponin T (TnT) is recently being considered to be an important diagnostic marker of myocardial damage in adults, but this marker has not yet been used in neonates. The present study was designed to determine the normal level of cardiac TnT in the cord blood of healthy term neonates. Cardiac troponin T concentration in cord blood was measured in 15 healthy term neonates using commercial kit (Enzymun-Test System, Boehringer, Mannheim). TnT serum concentration was 0.05 +/- 0.04 micrograms/l in 10 of 15 babies whereas in the remaining 5 haemolysed samples its concentration was elevated (mean 0.19 +/- 0.07 micrograms/l). It is important to consider that incidental haemolysis of blood samples can mimic pathological elevation of TnT by interfering with the assay.

Regions US6 and US7 of herpes simplex virus type 1 DNA encoding glycoproteins D and I may influence neuroinvasivity.

Recombinants were prepared by replacing a 1931 bp region of the BamHI J fragment (0.906-0.920) of the pathogenic ANGpath DNA-coding for glycoprotein D (gD) and a part of glycoprotein I (gI)--by the corresponding sequence of nonpathogenic KOS DNA (Kaerner et al., 1991) and tested in DBA/2 mice. The strain ANGpath and the control recombinant ANGpath/gD-gIpath, prepared by back transfer of the given ANGpath DNA fragment into ANGpath/gD-gIdellacZ+ DNA, were pathogenic after intraperitoneal inoculation. In contrast, mice infected with the strain KOS and the low-pathogenic recombinant ANGpath/gD-gIKOS survived peripheral virus administration. Both the strain KOS and the low-pathogenic recombinant ANDpath/gD-gIKOS spread by bloodstream to spleen, liver and adrenal glands but did not multiply in spinal cord. Nevertheless, the antigen of low-pathogenic recombinant ANGpath/gD-gIKOS was found in retroperitoneal vegetative nerves and ganglia. On the other hand, the strain ANGpath and the pathogenic recombinant ANGpath/gD-gIpath multiplied in cerebrospinal nerves and spinal cord causing typical hind leg paralysis.

The glycoprotein B gene and its syn3 locus of herpes simplex virus type 1 are involved in the synthesis of virus-associated growth factor (HSGF-1).

A putative growth factor (HSGF-1) associated with herpes simplex virus type 1 (HSV-1), which is similar to PRGF associated with pseudorabies virus, and/or HSGF-2 associated with HSV-2, was described. Experiments with four syncytial (syn) and four nonsyncytial (syn+) HSV-1 strains showed that the ability of this virus to produce HSGF-1 in infected cells is associated with the syn+ phenotype. Double infection of cells with syn+ and syn strain resulted either in enhancement or complete inhibition of HSGF-1 production, depending on the chosen pair of syn+ and syn strains. The studies with the recombinants between the syn+ strain KOS and syn strain ANGpath in the gene for glycoprotein B (gB) and syn3 locus revealed that the gB gene and its syn3 locus play a role in the HSGF-1 synthesis.

Pathogenicity and latency competence for rabbits of the herpes simplex virus type 1 ANGpath gC and gE defective mutants.

A total of 139 rabbits was infected to the right scarified cornea with HSV type 1 strains Kupka, ANG, ANGpath and their gE defective (ANGpathI2-4), gC defective (ANGpathgC18), gC/gE negative (ANGpathCI-8) and gC/ICP4 deletion (ANGpathY1) mutants. Strains ANG, ANGpath, ANGpathgC18 and ANGpathY1 were, in contrast to the two gE negative mutants, highly lethal, but 79% of rabbits infected with the non-encephalitogenic Kupka strain survived. Strain Kupka and strain ANGpath gE-negative mutants I2-4 and gCI-8 were tested for their latency competence. While Kupka established latency in the homolateral trigeminal ganglia from 80% of infected rabbits, I2-4 did so in one of 10 animals only, and the gC/gE mutant gCI-8 was not harboured in any of infected animals in an inducible form. Significant correlation was found between shedding into the culture fluid of reactivated virus from the explanted ganglion and brain stem fragments at one hand and the presence of the viral DNA in these organs on the other hand as judged by spot blot hybridization with the HSV-1 strain 17 Kpn I fragments h and i to DNA extracts prepared from these organs. Hybridizations were predominantly negative with the DNA from the corresponding non-cultured organs, except in a few cases of non-cultured ganglion and brain stem from rabbits previously infected with the gE deletion mutants which displayed positive hybridization, although no virus reactivation could be observed in corresponding explants.

Replacement of glycoprotein B gene in the herpes simplex virus type 1 strain ANGpath DNA by that originating from nonpathogenic strain KOS reduces the pathogenicity of recombinant virus.

Herpes simplex virus type-1 (HSV-1) strain ANGpath and its recombinants, in which the 8.1 kbp BamHI G restriction fragment (0.345-0.399) containing the glycoprotein B (gBpath) gene (UL27) or its subfragments-coding either for cytoplasmic or surface domains of gB-had been replaced with the corresponding fragments from nonpathogenic KOS virus DNA (gBKOS), were tested for their pathogenicity for DBA/2 mice and rabbits. The recombinant ANGpath/B6KOS prepared by transferring the 2.7 kbp SstI-SstI subfragment (0.351-0.368) of the BamHI GKOS fragment still had the original sequence of ANGpath DNA coding for the syn3 marker in the cytoplasmic domain of gB and was pathogenic for mice as well as for rabbits. Virological and immunohistological studies in DBA/2 mice infected with the latter pathogenic recombinant and with ANGpath showed the presence of infectious virus and viral antigen at inoculation site (epidermis, subcutaneous connective tissue and striated muscle in the area of right lip), in homolateral trigeminal nerve and ganglion, brain stem, midbrain, thalamic and hypothalamic nuclei. In contrast, nonpathogenic recombinants ANGpath/syn+B6KOS (prepared by transferring the whole BamHI GKOS fragment) and ANGpath/syn+KOS (prepared by transferring the 0.8 kbp BamHI-SstI subfragment of the BamHI GKOS fragment) showed limited haematogenous and neural spread, but no evidence of replication in CNS; thus, their behaviour resembled that of the wild type strain KOS. The recombinant ANGpath/syn+KOS, which was not pathogenic for mice, still remained pathogenic for rabbits, a phenomenon indicating the presence of an additional locus in the gB molecule participating on virulence. Sequencing the 1478 bp SstI-SstI subfragment of the BamHI G(path) fragment (nt 53,348-54,826 of UL segment) showed the presence of at least 3 mutations as compared to the KOS sequence, from which the change of cytosine to thymine at nt 54,251 altered the codon for arginine to that for histidine (amino acid 515) in the gB polypeptide chain.

Latency competence of herpes simplex virus strains ANG, ANGpath and its gC and gE minus mutans.

The latency competence of herpes simplex virus type 1 (HSV-1) strains SC16, KOS, ANG, ANGpath and its mutants ANGpathgC18 (gC minus, spontaneous point mutation), KOSgC39 (gC minus deletion), ANGpathI2-4 (gE minus deletion), and ANGpathgCI-8 (gE and gC minus double mutan) was compared and DBA/2 mice. While the latent SC16 and KOS reactivated spontaneously in explanted homolateral trigeminal ganglion fragments coming from Velaz DBA/2 mice, methylation inhibitor 5-azacytidine (5-AzaC) was required to achieve reactivation of SC16 in the ganglion explants from Hannover DBA/2 mice. Reactivation of ANGpath in the cultured trigeminal ganglia from both lines of DBA/2 mice occurred only in the presence of the drug. The compound also enhanced the reactivation incidence in the ganglion explants from ANG-infected Hannover DBA/2 mice but not from Velaz DBA/2 mice: in the latter it remained low even in the presence of the inducer. Both gE- mutants failed to establish latency as judged by the failure of reactivation either in the presence or the absence of 5-AzaC. This seemed in accordance with the absence of neural (quick axonal) spread of these mutans in mice (Rajcáni et al., 1990). In contrast, both gC- mutans established latency: ANGpathgC18 at an unchanged rate and KOSgC39 at a lower frequency than the parent strain.