Molecular epidemiology of respiratory syncytial virus in hospitalised children in Heidelberg, Southern Germany, 2014-2017.

BACKGROUND: Respiratory syncytial virus (RSV) is the leading cause of hopitalisation in young children with respiratory tract infections (RTI). The aim of this research project was to analyse RSV genotypes and the diversification of RSV strains among hospitalised children in Heidelberg, Germany. METHODS: We prospectively analysed nasopharyngeal swabs (NPS) from children who were hospitalised with acute RTI at the University Hospital Heidelberg, Germany, during winter seasons 2014 to 2017. RSV RT-PCR and RSV sequence analysis of the G gene coding for the attachment glycoprotein were performed. Clinical data was obtained using a standardised questionnaire. RESULTS: RSV was detected in 405 out of 946 samples from hospitalised children. Most RSV positive children were below the age of two years (84.4%) and had a lower RTI (78.8%). The majority of RSV positive children was male, significantly younger than RSV negative children with a median age of 0.39 years and with more severe respiratory symptoms. Out of 405 positive samples, 317 RSV strains were successfully sub-grouped into RSV subtypes A (57.4%; 182/317) and B (42.6%; 135/317). Both RSV subtypes cocirculated in all analysed winter seasons. Phylogenetic analysis of 317 isolates revealed that the majority of RSV-A strains (180/182) belonged to the ON1 genotype, most RSV-B strains could be attributed to the BAIX genotype (132/135). ON1 and BAIX strains showed a sub-differentiation into different lineages and we were able to identify new (sub)genotypes. CONCLUSION: Analysis of the molecular epidemiology of RSV from different seasons revealed the cocirculation and diversification of RSV genotypes ON1 and BAIX.

Respiratory syncytial virus A in haematological patients with prolonged shedding: Premature stop codons and deletion of the genotype ON1 72-nucleotide-duplication in the attachment G gene.

BACKGROUND: Respiratory syncytial virus (RSV) can be associated with severe disease and prolonged shedding in immunocompromised patients. OBJECTIVE: To investigate the genetic variability of RSV in consecutive samples of haematological patients with prolonged RSV shedding. STUDY DESIGN: Haematological patients at the University Hospital Heidelberg are routinely screened for respiratory viruses during winter season. In patients with prolonged RSV shedding between 2011 and 2014, Sanger-sequencing of the second hypervariable region of the RSV G gene was performed in consecutive samples. Further, deep-sequencing was performed in representative samples. RESULTS: Patients with prolonged RSV-A shedding were analysed (n=16, mean shedding 90days, 81.2% male). Phylogenetic analysis identified RSV genotypes NA1 (2011/12) or ON1 (2012/13). In most patients (n=12/16), Sanger-sequencing of the G gene showed identical sequences over the course of the shedding period. However, in two patients with particularly long viral shedding (333 and 142days), Sanger-sequencing revealed the presence of mutations leading to premature stop codons (37 and 70 amino acids truncated) in the G gene. In one additional patient, deep-sequencing revealed variants with premature stop codons at different positions. All three patients received repeatedly intravenous immunoglobulins. Interestingly, deep-sequencing revealed also a loss of the characteristic 72-nucleotide-duplication in all analysed ON1 strains. CONCLUSIONS: Long shedding periods and lack of immune selective pressure in the immunocompromised host seems to allow the persistence of viruses stripping a part of the C-terminus of the G glycoprotein. The loss of the characteristic 72-nucleotide-duplication in RSV-A ON1 variant strains is here described for the first time.

Susceptibility of herpes simplex virus type 1 to monoterpenes thymol, carvacrol, p-cymene and essential oils of Sinapis arvensis L., Lallemantia royleana Benth. and Pulicaria vulgaris Gaertn.

In recent years, with increased the prevalence of viral infections and having no specific for  their treatment  and also the continuous appearance of resistant viral strains, the finding of novel antiviral agents is necessary. In this study, monoterpenes of thymol, carvacrol, p-cymene and essential oils from Sinapis arvensis L., Lallemantia royleana Benth. and Pulicaria vulgaris Gaertn. were screened for their inhibitory effect against herpes simplex virus type 1 (HSV-1) in vitro on Vero cell line CCL-81-ATCC using a plaque reduction assay. The antiviral activity of three monoterpenes (thymol, carvacrol and p-cymene) and three essential oils were evaluated by cytotoxicity assay, direct plaque test. In addition, the modes of antiviral action of these compounds were investigated during the viral infection cycle. Results showed that the inhibitory concentrations (IC50) were determined at 0.002%, 0.037%, >0.1%, 0.035%, 0.018% and 0.001% for thymol, carvacrol, p-cymene, S. arvensis oil, L. royleana oil and P. vulgaris oil, respectively. A manifestly dose-dependent virucidal activity against HSV-1 could be exhibited for compounds tested. In order to determine the mode of the inhibitory effect, compounds were added at different stages during the viral infection cycle. At maximum noncytotoxic concentrations of the compounds, plaque formation was significantly reduced by more than 80% when HSV-1 was preincubated with p-cymene. However, no inhibitory effect could be observed when the compounds were added to the cells prior to infection with HSV-1 or after the adsorption period. CONCLUSION: These results indicate that compounds affected HSV-1 mostly before adsorption and might interact with the viral envelope. Thymol exhibited a high selectivity index and seems to be a promising candidate for topical therapeutic application as antiviral agent for treatment of herpetic infections.

Bacterial and viral contamination of breathing circuits after extended use - an aspect of patient safety?

BACKGROUND: In the past, anaesthetic breathing circuits were identified as a source of pathogen transmission. It is still debated, whether breathing circuits combined with breathing system filters can be safely used for more than 1 day. The aim of this study was to evaluate the transmission risk of bacteria and also viruses via breathing circuits after extended use. METHODS: The inner and outer surface of 102 breathing circuits used for 1 day and of 101 circuits used for 7 days were examined for bacteria and viruses. Additionally, 10 and 20 breathing circuits each were examined after use on patients with pulmonary virus infection and with multidrug-resistant organism (MDRO) colonisation/infection respectively. Bacteria were detected by standard microbiological procedures; PCR techniques were applied for herpes simplex virus, cytomegalovirus, influenza, parainfluenza and respiratory syncytial virus. RESULTS: Endoluminal bacterial contamination of breathing circuits remained unchanged after 7-day vs. 1-day use (5.9% vs. 7.8%) [CI95%: -0.0886-0.0506, pnon-inferiority 0.0260]. Only outside surface contamination with bacteria belonging to environmental species or human flora increased (16.8 vs. 6.9%) [CI 95%: 0.0118 - 0.1876, pnon-inferiority 0.8660]. Viruses occurred on the patient side, but not in breathing circuits. No MDRO occurred in the 20 circuits after use on patients harbouring such germs. CONCLUSION: Endoluminal contamination of breathing circuits with bacteria did not increase after extended use. No viruses were detected in the breathing circuits using filters. Based on our results, the extended use of ABC without exceptions appears safe, if a high level of anaesthesia workplace cleaning is secured.

Impact of Everolimus and Low-Dose Cyclosporin on Cytomegalovirus Replication and Disease in Pediatric Renal Transplantation.

In order to investigate the hypothesis that the mammalian target of rapamycin inhibitor everolimus (EVR) shows anticytomegalovirus (CMV) activity in pediatric patients, we analyzed the impact of EVR-based immunosuppressive therapy on CMV replication and disease in a large cohort (n = 301) of pediatric kidney allograft recipients. The EVR cohort (n = 59), who also received low-dose cyclosporin, was compared with a control cohort (n = 242), who was administered standard-dose cyclosporin or tacrolimus and an antimetabolite, mostly mycophenolate mofetil (91.7%). Multivariate analysis revealed an 83% lower risk of CMV replication in the EVR cohort than in the control cohort (p = 0.005). In CMV high-risk (donor+/recipient-) patients (n = 88), the EVR-based regimen was associated with a significantly lower rate of CMV disease (0% vs. 14.3%, p = 0.046) than the standard regimen. In patients who had received chemoprophylaxis with (val-)ganciclovir (n = 63), the CMV-free survival rates at 1 year and 3 years posttransplant (100%) were significantly (p = 0.015) higher in the EVR cohort (n = 15) than in the control cohort (n = 48; 1 year, 75.0%; 3 years, 63.3%). Our data suggest that in pediatric patients at high risk of CMV, an EVR-based immunosuppressive regimen is associated with a lower risk of CMV disease than a standard-dose calcineurin inhibitor-based regimen.

Piroxicam inhibits herpes simplex virus type 1 infection in vitro.

Piroxicam is a potent, nonsteroidal, anti-inflammatory agent (NSAID) which also exhibits antipyretic activity. The antiviral effect of piroxicam against herpes simplex virus type 1 (HSV-1) was examined in vitro on RC-37 monkey kidney cells using a plaque reduction assay. Piroxicam was dissolved in ethanol or dimethylsulfoxide (DMSO) and the 50% inhibitory concentration (IC50) was determined at 4 µg/ml and 75 µg/ml, respectively. The IC50 for the standard antiherpetic drug acyclovir was determined at 1.6 µM. At non-cytotoxic concentrations of these piroxicam solutions, plaque formation was significantly reduced by 62.4% for ethanolic piroxicam and 72.8% for piroxicam in DMSO. The mode of antiviral action of these drugs was assessed by time-on-addition assays. No antiviral effect was observed when cells were incubated with piroxicam prior to infection with HSV-1 or when HSV-1 infected cells were treated with dissolved piroxicam. Herpesvirus infection was, however, significantly inhibited when HSV-1 was incubated with piroxicam prior to the infection of cells. These results indicate that piroxicam affected the virus before adsorption, but not after penetration into the host cell, suggesting that piroxicam exerts a direct antiviral effect on HSV-1. Free herpesvirus was sensitive to piroxicam in a concentration-dependent manner and the inhibition of HSV-1 appears to occur before entering the cell but not after penetration of the virus into the cell. Considering the lipophilic nature of piroxicam, which enables it to penetrate the skin, it might be suitable for topical treatment of herpetic infections.

Pentacyclic triterpenes in birch bark extract inhibit early step of herpes simplex virus type 1 replication.

Antiviral agents frequently applied for treatment of herpesvirus infections include acyclovir and its derivatives. The antiviral effect of a triterpene extract of birch bark and its major pentacyclic triterpenes, i.e. betulin, lupeol and betulinic acid against acyclovir-sensitive and acyclovir-resistant HSV type 1 strains was examined. The cytotoxic effect of a phytochemically defined birch bark triterpene extract (TE) as well as different pentacyclic triterpenes was analyzed in cell culture, and revealed a moderate cytotoxicity on RC-37 cells. TE, betulin, lupeol and betulinic acid exhibited high levels of antiviral activity against HSV-1 in viral suspension tests with IC50 values ranging between 0.2 and 0.5 µg/ml. Infectivity of acyclovir-sensitive and clinical isolates of acyclovir-resistant HSV-1 strains was significantly reduced by all tested compounds and a direct concentration- and time-dependent antiherpetic activity could be demonstrated. In order to determine the mode of antiviral action, TE and the compounds were added at different times during the viral infection cycle. Addition of these drugs to uninfected cells prior to infection or to herpesvirus-infected cells during intracellular replication had low effect on virus multiplication. Minor virucidal activity of triterpenes was observed, however both TE and tested compounds exhibited high anti-herpetic activity when viruses were pretreated with these drugs prior to infection. Pentacyclic triterpenes inhibit acyclovir-sensitive and acyclovir-resistant clinical isolates of HSV-1 in the early phase of infection.

First case report of an acute hepatitis E subgenotype 3c infection during pregnancy in Germany.

Hepatitis E is usually a self-limiting disease and an important cause of acute hepatitis in endemic countries in Asia and Africa. However, the mortality rate for pregnant women infected with hepatitis E virus (HEV) in this area is about 25%. In Germany, sporadic cases of acute hepatitis E infections have been described and the number of autochthonous infections is increasing. Here we report an autochthonous HEV subgenotype 3c infection in a 27-year old pregnant woman. This is the first documented case of a hepatitis E infection during pregnancy in Germany. The patient presented in week 26 of gestation with acute hepatitis and elevated transaminases. During follow-up, she tested positive for anti-HEV antibodies. HEV viral load during the acute hepatitis was 2.3×10(6) copies/ml serum, however viremia declined and cleared rapidly. Sequence analysis revealed a HEV subgenotype 3c closely related to European isolates. The patient had not travelled outside Germany, had regular contact to animals, but the source of infection remains unclear. The newborn was delivered in week 40 of gestation in good health, HEV was not transmitted and liver enzymes were normal. In conclusion, hepatitis E should be considered in differential diagnosis in patients with acute hepatitis especially during pregnancy, even without travel history to countries with high endemicity.

Antimicrobial activity of propolis special extract GH 2002 against multidrug-resistant clinical isolates.

The need to discover and develop alternative therapies to treat multidrug-resistant bacterial infections is timely. The aim of this study was to examine the antimicrobial potential of propolis, as a purified and concentrated special extract GH 2002, against clinical isolates of Streptococcus pyogenes, methicillin-resistent Stapylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE) and Candida. Minimal inhibitory concentrations (MICs) and minimal microbicidial concentrations (MMCs) of propolis against microbial pathogens were evaluated using the broth microdilution method. Propolis extract GH 2002 revealed low MICs in the range of 0.03 to 2 mg/ml against S. pyogenes, S. aureus, E. faecium and Candida. A high bactericidal activity of propolis extract in the range of 0.06 to 1.0 mg/ml was determined for S. pyogenes and S. aureus, however propolis was not bactericidal against E. faecium. Propolis concentrations between 0.6 and 2.4 mg/ml displayed fungicidal activity against different Candida species. Whereas all tested MRSA strains were highly susceptible against propolis, only minor activity was found against VRE strains. Time-kill curves demonstrated a high antimicrobial activity at low MICs already after few hours of incubation against reference strains, clinical antibiotic-susceptible strains, clinical antifungal susceptible strains as well as all tested clinical MRSA strains, but not against VRE strains. In conclusion, clinical drug-sensitive as well as some clinical multidrug-resistant microbial isolates, i.e. MRSA, were susceptible to propolis with different degrees of susceptibility. These results suggest that the special propolis extract GH2002 might be used in the development of alternative products for therapy of microbial infections.

Antiherpetic activity of the traditionally used complex essential oil Olbas.

Essential oils of medicinal plants are increasingly of interest as novel drugs for antiherpetic agents, since herpes simplex virus (HSV) might develop resistance to commonly used antiviral drugs. The antiviral effect of Olbas, a traditionally used complex essential oil, and of cajuput oil, a major constitutent of Olbas, against HSV type 1 was examined. The antiviral activity of these essential oils was tested in vitro on monkey kidney cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of Olbas and cajuput oil for herpes simplex virus plaque formation was determined at 1.8 microg/ml and 7.5 microg/ml, respectively. At noncytotoxic concentrations of these oils, plaque formation was significantly reduced by 99% for Olbas and 66% for cajuput oil. The selectivity index of 150 for Olbas against herpes simplex virus was superior to a rather low selectivity index for cajuput oil. The mode of antiviral action of these essential oils was assessed by time-on-addition assays. Herpesvirus was significantly inhibited by pretreatment with Olbas essential oil prior to infection of cells. These results indicate that Olbas affected the virus before adsorption, but not after penetration into the host cell, thus Olbas exerted a direct antiviral effect on HSV. A clearly time-dependent antiviral activity for Olbas and cajuput oil could be demonstrated. Considering the lipophilic nature of the Olbas complex essential oil mixture, which enables it to penetrate the skin, and a high selectivity index, Olbas might be suitable for topical treatment of herpetic infections.

Risk factors and containment of respiratory syncytial virus outbreak in a hematology and transplant unit.

Respiratory syncytial virus (RSV) usually causes self-limiting upper respiratory tract infections, but can be associated with severe lower respiratory tract infection disease (LRTID) in infants and in patients with hematologic malignancies. We have analyzed the risk factors and the measures for containment within an outbreak of nosocomial RSV infections in a hematology and SCT unit. A total of 56 patients were affected (53 RSV-A and 3 RSV-B) including 32 transplant patients (16 allogeneic and 16 autologous). Forty (71%) of the 56 patients suffered from LRTID and 14 (35%) of the patients with LRTID subsequently died. However, because of concomitant infections with fungal and bacterial pathogens, the impact of RSV on the fatal outcome was difficult to assess. Multivariate analysis showed that low levels of IgG were significantly associated with fatal outcome (P=0.007), treatment with oral ribavirin represented a protective factor (P=0.02). An extremely protracted viral shedding was observed in this cohort of patients (median=30.5 days, range: 1-162 days), especially pronounced in patients after allogeneic transplantation (P=0.002). Implementation of rigorous isolation and barrier measures, although challenged by long-term viral carriers, was effective in containment of the outbreak.

Antiherpetic efficacy of aqueous extracts of the cyanobacterium Arthrospira fusiformis from Chad.

Natural substances offer interesting pharmacological perspectives for antiviral drug development with regard to broad spectrum antiviral properties and novel modes of action. Drugs currently used to treat cutaneous or genital herpetic infections are effective in limiting disease, but the emergence of drug-resistant viruses in immunocompromised individuals can be problematic. A nontoxic cyanobacterium Arthrospira strain from Chad has been characterized by sequence analysis of the intergenic spacer region of the phycocyanin gene. This cyanobacterium was identified as Arthrospira fusiformis by phylogenetic tree analysis. The antiherpetic activity of crude aqueous extracts from the Chad A. fusiformis isolate was determined. Antiviral efficacy against herpes simplex virus of cold water extract, hot water extract and phosphate buffer extract was assessed in plaque reduction assays and their mode of antiherpetic action was analysed. In virus suspension assays, cold water extract, hot water extract and phosphate buffer extract inhibited virus infectivity by 54.9%, 64.6%, and 99.8%, respectively, in a dose-dependent manner. The mode of antiviral action was determined by addition of cyanobacterial extracts separately at different time periods during the viral infection cycle. Extracts of A. fusiformis strain clearly inhibited herpesvirus multiplication before and during virus infection of host cells. The phosphate buffer extract of the A. fusiformis strain affected free herpes simplex virus prior to infection of host cells and inhibited intracellular viral replication. It is concluded, that Arthrospira compounds warrant further investigation to examine their potential role in the treatment of herpetic infections.

Atypical severe Puumala hantavirus infection and virus sequence analysis of the patient and regional reservoir host.

Nephropathia epidemica (NE) caused by Puumala hantavirus (PUUV) is a mild variant of haemorrhagic fever with renal syndrome, which is endemic in Germany. We describe the case of a 67-year-old man initially presenting with acute bilateral angle-closure glaucoma, an atypical clinical presentation of PUUV infection. Subsequently, the patient developed a severe course of disease additionally complicated by profound hepatitis and interstitial nephritis, both phenomena which are rarely described in association with hantavirus infection. Serologic diagnosis was complicated by delayed antibody production until the 10th day of illness; however, PUUV RNA was detectable early in disease. To further analyse this unusual case, sequencing of the PUUV S segment was performed from the patient and regional reservoir host which showed a close relation.

[Efficacy of plant products against herpetic infections]. / Wirksamkeit von Pflanzenprodukten gegen Herpesinfektionen.

Essential oils from various aromatic medicinal plants are highly active against some viral infections, e.g. labial herpes caused by herpes simplex virus type 1. Balm oil, tea tree oil and peppermint oil demonstrate in vitro a significant antiherpetic activity, mainly related to a direct drug-virus particle interaction, some essential oils also act directly virucidal. Interestingly, these essential oils are also highly active against acyclovir-resistant herpes simplex virus strains. In clinical studies, tea tree oil has been shown to possess antiherpetic, anti-inflammatory and pain-relieving properties, as well as to accelerate the healing process of herpes labialis. Applying diluted essential oils three to four times daily for the antiherpetic treatment of affected areas is recommended. Some companies have marketed plant products, e.g. from Melissa, for the treatment of recurrent herpetic infections.

Molecular analysis of linezolid resistance in clinical Enterococcus faecium isolates by polymerase chain reaction and pyrosequencing.

Resistance to linezolid has been associated with a G2576T mutation in the 23 S rRNA gene. Clinical isolates of linezolid-sensitive and linezolid-resistant vancomycin-resistant Enterococcus faecium of a liver transplant patient have been analysed for the G2576T mutation by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), conventional sequencing and pyrosequencing. A clear association between the number of mutated 23 S rRNA genes and the level of linezolid resistance has been demonstrated. Linezolid susceptibility re-emerged after cessation of linezolid therapy; however, the re-initiation of linezolid therapy resulted in the re-emergence of linezolid-resistant strains. Pyrosequencing rapidly detected the number of mutated alleles and is superior to conventional PCR-RFLP for the detection of heterozygous mutations.

Cytomegalovirus infection after liver transplantation incidence, risks, and benefits of prophylaxis.

BACKGROUND: Cytomegalovirus (CMV) infection and related disease is a feared complication after liver transplantation. Antiviral prophylaxis is recommended in clinical practice guidelines depending on the CMV status of both donor and recipient as well as the individual risk profile. METHODS: We retrospectively analyzed 211 liver transplant recipients with respect to the incidence of CMV infection after transplantation, the influence of donor and recipient CMV status, and the effect of antiviral prophylaxis. In addition, the underlying liver disease and immunosuppressive regimen were compared with the incidence of CMV infection. Patients were divided into 4 groups according to CMV donor/recipient (D/R) profile: group A (D-/R-) 28 patients (13.3%), group B (D-/R+) 64 patients (30.3%), group C (D+/R+) 79 patients (37.4%), and group D (D+/R-) 40 patients (19.0%). RESULTS: CMV infection was observed in 17.9%, 29.7%, 24.1%, and 22.5% of the patients, respectively, with no significant difference in infection rates between the groups. CMV infection occurred in 5 patients (17.9%) in the presumed low-risk profile (group A), despite an antiviral prophylaxis in 4 out of these 5 patients. In contrast, CMV infection occurred in only 9/40 patients (22.5%) in the presumed high-risk profile (group D). The most frequent infection rates were found in groups B and C (R+ groups). After successful treatment of CMV infection, no recurrence was detected. Underlying liver disease or immunosuppressive protocol had no influence on CMV infection. CONCLUSION: Approximately one fourth of patients will acquire CMV infection after liver transplantation independent of donor/recipient status. Surprisingly, antiviral prophylaxis does not seem to be sufficient to reduce this proportion of patients, either in presumed high-risk or in presumed low-risk situations.

Molecular authentication and characterization of the antiherpetic activity of the cyanobacterium Arthrospira fusiformis.

In recent years there has been an increasing interest for application of natural products as antiinfectives and concerns about the safety of synthetic compounds have encouraged more detailed studies of natural resources. Two different strains of the nontoxic cyanobacterium Arthrospira from the United States and Egypt have been characterized by sequence analysis of the intergenic spacer region of the phycocyanin gene. Both cyanobacteria were identified as Arthrospira fusiformis by phylogenetic tree analysis. The antiherpetic activity of crude aqueous extracts from the US and the Egyptian A. fusiformis isolates was determined. Antiviral activity against herpes simplex virus of cold water extracts, hot water extracts and phosphate buffer extracts from the American and the Egyptian strains was assessed in plaque reduction assays and their mode of antiherpetic action was analysed. In virus suspension assays, all extracts of the American cyanobacterium and the phosphate buffer extract of the Egyptian cyanobacterium inhibited virus infectivity by > 90% in a dose-dependent manner. Phosphate buffer extract and hot water extract of the US cyanobacterium demonstrated the highest antiviral activity at low extract concentrations with high selectivity indices of 7464 and 542, respectively. The mode of antiviral action has been determined by addition of cyanobacterial extracts separately at different time periods during the viral infection cycle. Two extracts of the US A. fusiformis strain clearly inhibited herpesvirus multiplication before and after virus infection of host cells. In contrast, extracts of the Egyptian A. fusiformis strain affected only free herpes simplex virus prior to infection of host cells by direct inactivation of virus particles. In this study different Arthrospira crude extracts showed a significant antiviral effect and might be applied in recurrent herpetic infections.

Sporadic cases of acute autochthonous hepatitis E virus infection in Southwest Germany.

Hepatitis E infection is usually a self-limiting disease and an important cause of acute hepatitis in tropical and subtropical regions where the virus is endemic. In industrialized countries, sporadic cases of acute hepatitis E virus (HEV) infections have been described and the number of documented autochthonous infections seems to be increasing. We report three sporadic cases of autochthonous hepatitis E infections in Southwestern Germany which presented at our university hospital within two years. All cases were men who presented with acute hepatitis, icterus and elevated liver. In case 1 and case 2, liver biopsy revealed acute hepatitis, both patients were positive for anti-HEV antibodies, case 1 was also positive for HEV RNA with a viral load of 3.0 x 10(3)copies/ml in serum. In case 3, anti-HEV antibodies were detectable and HEV RNA was detected in serum (4.3 x 10(3)copies/ml) and stool (1.4 x 10(6)copies/ml). None of the patients had a recent travel history outside Germany and close contact to animals has been denied. HEV sequence analysis of two patients revealed genotype 3 with homologies to other European isolates and isolates from swine. Thus the source of infection remains unclear. Hepatitis E should be considered in differential diagnosis in patients with unexplained hepatitis and patients with acute hepatitis, whatever their age or travel history might be, should be tested for HEV.

Antiviral activity of Rhus aromatica (fragrant sumac) extract against two types of herpes simplex viruses in cell culture.

We report on the antiviral potency of an aqueous extract of root/stem bark of Rhus aromatica (fragrant sumac extract) against herpes simplex virus type 1 and type 2 in cell culture (RC-37 cells) using a plaque reduction assay. The extract exhibited a high level of anti-HSV activity with IC50-values of 0.0005% for HSV-1 and 0.0043% for HSV-2 as well as high selectivity indices (SI) of 5400 for HSV-1 and 628 for HSV-2. In order to determine the mode of antiviral action, the fragrant sumac extract was added at different times to the cells or viruses during the viral infection cycle. At maximum non-cytotoxic concentration (0.25%), plaque formation was significantly reduced by more than 99% when herpes simplex viruses were pretreated with the plant extract for 1 h prior to cell infection. When the host cells were pretreated with the fragrant sumac extract for 1 h prior to virus infection, the infectivity of viruses was reduced by 50% for HSV-1 but only moderately for HSV-2. No antiviral effect was seen when the plant extract was added to already infected host cells. Based on these findings the plant extract seems to interact not only with the viral envelope but also with the surface of the host cells impairing the ability of herpes simplex viruses to adsorb to and penetrate into the host cells. In conclusion, the aqueous fragrant sumac extract revealed a strong antiviral activity against HSV-1 and HSV-2 in vitro.