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2.
Vaccine ; 37(43): 6550-6557, 2019 10 08.
Article in English | MEDLINE | ID: mdl-31521415

ABSTRACT

BACKGROUND: The impact of annual influenza epidemics and prevailing strains varies worldwide and regional. The majority of vaccines used contained two influenza A strains and only one influenza B strain (trivalent vaccine). AIM: The aim of the study was to compare laboratory confirmed influenza B cases during three consecutive years with respect to vaccination history, clinical symptoms and molecular virology. METHODS: Partial HA gene sequences were analyzed for lineage determination and complete HA sequence in cases with reported vaccination and in fatal cases. Clinical data were retrieved from patient charts. FINDINGS: During the 2015/16 season, 75 influenza B cases were retrieved; 11 in 2016/17, and 274 in 2017/18. The frequency of Yamagata-lineage strains increased from 7.6% to 100%. No difference was detected in the relative frequency of co-morbidities in season 2017/18. 37.7% of the adult patients and 4.5% of pediatric patients were vaccinated against influenza. INTERPRETATION: Phylogenetically, Yamagata strains clustered similarly in 2017/2018 when compared to the previous two influenza seasons. While the relative frequency of influenza B cases differed, the clinical symptoms remained similar. CONCLUSION: World Health Organization recommendations for the use of tetravalent vaccines that contain two influenza B strains (Yamagata and Victoria) in addition to the two influenza A strains (H1N1 and H3N2) should be implemented in national vaccination guidelines. FUNDING: This research was partially supported by the Association of Sponsors and Friends of Leipzig University.


Subject(s)
Influenza B virus/genetics , Influenza B virus/pathogenicity , Influenza, Human/epidemiology , Adolescent , Adult , Age Distribution , Female , Germany/epidemiology , Humans , Influenza Vaccines/therapeutic use , Influenza, Human/etiology , Influenza, Human/prevention & control , Male , Middle Aged , Phylogeny , Seasons , Young Adult
3.
Epidemiol Infect ; 147: e218, 2019 01.
Article in English | MEDLINE | ID: mdl-31364546

ABSTRACT

Childhood morbidity and mortality of diarrhoeal diseases are high, particularly in low-income countries and noroviruses and sapoviruses are among the most frequent causes worldwide. Their epidemiology and diversity remain not well studied in many African countries. To assess the positivity rate and the diversity of sapoviruses and noroviruses in Northwest Ethiopia, during November 2015 and April 2016, a total of 450 faecal samples were collected from outpatient children aged <5 years who presented with diarrhoea. Samples were screened for noroviruses and sapoviruses by real-time RT-PCR. Partial VP1 genes were sequenced, genotyped and phylogenetically analysed. Norovirus and sapovirus stool positivity rate was 13.3% and 10.0%, respectively. Noroviruses included GII.4 (35%), GII.6 (20%), GII.17 (13.3%), GII.10 (10%), GII.2 (6.7%), GII.16 (5%), GII.7 (3.3%), GII.9, GII.13, GII.20 and GI.3 (1.7% each) strains. For sapoviruses, GI.1, GII.1 (20.0% each), GII.6 (13.3%), GI.2 (8.9%), GII.2 (11.1%), GV.1 (8.9%), GIV.1 (6.7%), GI.3 and GII.4 (2.2% each) genotypes were detected. This study demonstrates a high genetic diversity of noroviruses and sapoviruses in Northwest Ethiopia. The positivity rate in stool samples from young children with diarrhoea was high for both caliciviruses. Continued monitoring is recommended to identify trends in genetic diversity and seasonal variations.


Subject(s)
Bacterial Proteins/genetics , Caliciviridae Infections/epidemiology , Gastroenteritis/epidemiology , Norovirus/genetics , Sapovirus/genetics , Caliciviridae Infections/genetics , Child , Child, Preschool , Cohort Studies , Developing Countries , Ethiopia/epidemiology , Feces/virology , Female , Gastroenteritis/genetics , Gastroenteritis/microbiology , Genetic Variation , Genotype , Humans , Incidence , Infant , Male , Norovirus/isolation & purification , Outpatients/statistics & numerical data , Phylogeny , Public Health , Retrospective Studies , Risk Assessment , Sapovirus/isolation & purification , Seasons , Survival Analysis
4.
Clin Microbiol Infect ; 25(1): 102-106, 2019 Jan.
Article in English | MEDLINE | ID: mdl-29649603

ABSTRACT

OBJECTIVES: Rotavirus infections are common causes of infant hospitalization. The present study examined the effectiveness of anti-rotavirus vaccination in preventing rotavirus-related hospitalizations in Germany, following its state and nationwide introductions in 2008 and 2013, respectively. METHODS: During 15 consecutive seasons 9557 stool samples of hospitalized children of 5 years and younger with acute gastroenteritis were screened for rotavirus A. Rotavirus G and P genotypes were assessed after vaccine introduction. Vaccine effectiveness was determined by comparison of rotavirus incidence in pre-vaccine and post-vaccine cohorts. The herd effect was calculated as the difference between the observed reduction of rotavirus-related hospitalizations and the expected direct vaccine effect. RESULTS: The number of rotavirus-related hospitalizations declined after vaccine introduction. Approximately 26% (503/1955) of prevented cases could be attributed to the herd effect. Human rotaviruses of genotypes G3P[8], G1P[8], G9P[8], G4P[8], G2P[4] and G12P[8] were most frequent. Uncommon genotypes remained rare. The direct, indirect, total and overall vaccine effectiveness was 86% (95% confidence interval (CI) 83.2-89.1%), 48% (95% CI 42.8-52.6%), 93% (95% CI 91.3-94.3%) and 69% (95% CI 66.5-72.0%), respectively. There was no significant difference in vaccine-type or in genotype-specific vaccine effectiveness. CONCLUSIONS: Anti-rotavirus vaccination efficiently reduced rotavirus-related hospitalizations in Germany in the past decade. The vaccines analysed in this article provide a broadly heterologous and long-lasting protection. The herd effect substantially contributed to the observed drop in the number of incidences of severe rotavirus infections. Presumably, constant high vaccine coverage will lead to a continued upward trend in the overall vaccine efficiency.


Subject(s)
Gastroenteritis/epidemiology , Gastroenteritis/prevention & control , Hospitalization/statistics & numerical data , Rotavirus Infections/prevention & control , Rotavirus Vaccines/therapeutic use , Child, Preschool , Feces/virology , Female , Genotype , Germany/epidemiology , Humans , Immunity, Herd , Incidence , Infant , Male , Rotavirus , Rotavirus Infections/epidemiology , Rotavirus Vaccines/immunology , Vaccination/statistics & numerical data , Vaccines, Attenuated/immunology , Vaccines, Attenuated/therapeutic use
5.
Arch Virol ; 162(2): 477-486, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27807657

ABSTRACT

Although teratogenic rubella virus (RV) causes a vaccine-preventable disease, it is still endemic in several countries worldwide. Thus, there is a constant risk of RV importation into non-endemic areas. RV monitoring, especially during measles and Zika virus outbreaks, requires reliable diagnostic tools. For this study, a TaqMan-based one-step reverse transcription-quantitative PCR (RT-qPCR) assay, with the p90 gene as a novel and so far unexplored target for detection of clade I and II genotypes, was developed and evaluated. Automated nucleic acid extraction was carried out. Performance characteristics of the TaqMan RT-qPCR assay were determined for a RV plasmid standard and RNA extracted from virus-infected cell culture supernatants representing clade I and II genotypes. Diagnostic specificity and sensitivity were validated against other RNA and DNA viruses, relevant for RV diagnostic approaches and for RV-positive clinical samples, respectively. The assay is specific and highly sensitive with a limit of detection as low as five to one copies per reaction or 200 infectious virus particles per ml. The coefficients of variation (CV) were specified as intra- (within one run) and inter- (between different runs) assay variation, and calculated based on the standard deviations for the obtained Ct values of the respective samples. Intra- and inter-assay CV values were low, with a maximum of 3.4% and 2.4%, respectively. The assay was shown to be suitable and specific for the analysis of clinical samples. With p90 as a novel target, the highly sensitive and specific TaqMan assay outlined in this study is suitable for RV diagnosis worldwide.


Subject(s)
RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Rubella virus/genetics , Rubella/diagnosis , Viral Proteins/genetics , Base Sequence , Gene Expression , Genotype , Humans , Observer Variation , Phylogeny , Real-Time Polymerase Chain Reaction/standards , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/standards , Rubella/virology , Rubella virus/classification , Rubella virus/isolation & purification , Sensitivity and Specificity , Sequence Alignment
6.
Z Gastroenterol ; 54(3): 231-7, 2016 Mar.
Article in English | MEDLINE | ID: mdl-27043886

ABSTRACT

BACKGROUND: Elderly patients are under-represented in hepatitis B and C screening approaches, but may be at increased risk for advanced liver disease. We therefore screened a hospitalized elderly population. MATERIALS AND METHODS: 6011 admissions to the department of internal medicine and neurology within one year were screened for HBsAg and anti-HCV (Elecsys(®)-HBsAg and -anti-HCV). Positive anti-HCV results were confirmed with the INNO-LIA™ assay. HCV-RNA was analyzed by real-time PCR in the case of confirmed positive anti-HCV results, HBV-DNA in the confirmed HBsAg positive individuals. RESULTS: Patient´s mean age (62.4 years) was 19 years above that of the average German population. The confirmed HBsAg prevalence was 0.6 %. 34 % (n = 12/35) of HBsAg positive cases were newly diagnosed, three of them presented with HBV-DNA levels > 2000 IU/mL. The confirmed anti-HCV prevalence was 0.9 %. 14 % (n = 8/56) of anti-HCV positive patients were previously undiagnosed. HCV-RNA was positive in three of them. In newly diagnosed individuals cirrhosis was present in 1/12 of the HBsAg and in 3/8 of the anti-HCV positive individuals. Compared to non-infected controls, the following risk factors were significantly more frequent in infected patients: (i) HBsAg: sexual exposure (20 % vs. 2 %), blood transfusion before 1992 (13 % vs. 6 %), referrals from nursing homes (10 % vs. 1 %). (ii) Anti-HCV: blood transfusion before 1992 (41 % vs. 6 %), IVDU (25 % vs. 0.5 %), organ transplantation (20 % vs. 5 %), hemodialysis (11 % vs. 3 %). CONCLUSIONS: HBsAg and anti-HCV were underdiagnosed in a senescent population, however, only few cases presented with advanced liver disease. Referrals from nursing homes were at increased risk for HBV infection.


Subject(s)
Hepatitis B Surface Antigens/blood , Hepatitis B/epidemiology , Hepatitis C Antibodies/blood , Hepatitis C/epidemiology , Hospitalization/statistics & numerical data , Mass Screening/statistics & numerical data , Age Distribution , Aged , Aged, 80 and over , Biomarkers/blood , Cohort Studies , Comorbidity , Female , Geriatric Assessment/methods , Geriatric Assessment/statistics & numerical data , Germany/epidemiology , Health Services for the Aged/statistics & numerical data , Hepatitis B/blood , Hepatitis B/diagnosis , Hepatitis B Surface Antigens/immunology , Hepatitis C/blood , Hepatitis C/diagnosis , Hepatitis C Antibodies/immunology , Humans , Male , Mass Screening/methods , Middle Aged , Prevalence , Risk Assessment , Sex Distribution , Tertiary Care Centers/statistics & numerical data
7.
Arch Virol ; 159(6): 1267-77, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24343264

ABSTRACT

Mitochondria fulfil several key functions within cellular metabolic and antiviral signalling pathways, including their central role in ATP generation. Viruses, as intracellular parasites, require from their cellular host the building blocks for generation of their viral progeny and the energy that drives viral replication and assembly. While some viruses have adopted ways to manipulate the infected cell such that cellular metabolism supports optimal virus production, other viruses simply exhaust cellular resources. The association of viruses with mitochondria is influenced by several important factors such as speed of the viral replication cycle and viral dependence on cellular enzymes and metabolites. This review will highlight the complex interconnectivity of viral life cycles with the three main mitochondrial metabolic pathways, namely ß-oxidation, the tricarboxylic (TCA) cycle, and oxidative phosphorylation. This interconnectivity has the potential to reveal interesting points for antiviral therapy with either prometabolites or antimetabolites and highlights the importance of the viral association with mitochondrial metabolism.


Subject(s)
Host-Pathogen Interactions , Mitochondria/metabolism , Virus Replication , Viruses/growth & development , Citric Acid Cycle , Energy Metabolism , Oxidation-Reduction , Oxidative Phosphorylation
8.
J Virol ; 87(15): 8481-92, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23720730

ABSTRACT

Mitochondria are important for the viral life cycle, mainly by providing the energy required for viral replication and assembly. A highly complex interaction with mitochondria is exerted by rubella virus (RV), which includes an increase in the mitochondrial membrane potential as a general marker for mitochondrial activity. We aimed in this study to provide a more comprehensive picture of the activity of mitochondrial respiratory chain complexes I to IV. Their activities were compared among three different cell lines. A strong and significant increase in the activity of mitochondrial respiratory enzyme succinate:ubiquinone oxidoreductase (complex II) and a moderate increase of ubiquinol:cytochrome c oxidoreductase (complex III) were detected in all cell lines. In contrast, the activity of mitochondrial respiratory enzyme cytochrome c oxidase (complex IV) was significantly decreased. The effects on mitochondrial functions appear to be RV specific, as they were absent in control infections with measles virus. Additionally, these alterations of the respiratory chain activity were not associated with an elevated transcription of oxidative stress proteins, and reactive oxygen species (ROS) were induced only marginally. Moreover, protein and/or mRNA levels of markers for mitochondrial biogenesis and structure were elevated, such as nuclear respiratory factors (NRFs) and mitofusin 2 (Mfn2). Together, these results establish a novel view on the regulation of mitochondrial functions by viruses.


Subject(s)
Electron Transport Complex III/metabolism , Electron Transport Complex II/metabolism , Electron Transport Complex IV/metabolism , Electron Transport Complex I/metabolism , Oxidative Stress , Rubella virus/physiology , Animals , Gene Expression Profiling , Mitochondria/enzymology , Reactive Oxygen Species/metabolism
9.
J Virol Methods ; 180(1-2): 18-25, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22197190

ABSTRACT

Two truncated sequences (designated P1 and rHA1) of influenza A virus subtype H5 haemagglutinin (HA) were cloned and expressed in yeast Pichia pastoris (P. pastoris). These polypeptides were used in an indirect recombinant ELISA (rELISA) for detection of H5 antibodies in poultry. Serum samples obtained from broiler chickens vaccinated with commercial inactivated vaccine (H5N2) and control negative sera from non-vaccinated chickens against influenza were tested using rP1-ELISA, rHA1-ELISA, whole H5N1-ELISA, Western blot, agar gel immunodiffusion (AGID) and haemagglutination inhibition (HI) tests. The rHA1-ELISA proved to be highly sensitive and specific. To study the validity of rHA1-ELISA, a total of 179 serum samples obtained from commercial broiler chickens vaccinated previously with commercial H5N2 inactivated vaccines, were tested by rHA1-ELISA, commercial ELISA (cELISA) and HI. The relative sensitivity and specificity between rHA1-ELISA, and HI tests were 100% and 70%, respectively, and between cELISA and HI were 100% and 57%, respectively. The agreement ratio between rHA1-ELISA and HI was 84.9% and between cELISA and HI tests was 76.5%. Serum samples obtained from ducks vaccinated with commercial inactivated H5N2 were tested by rHA1-ELISA and the results showed significant reactivity with duck sera. In conclusion, the results demonstrate the potential applicability of the rELISA for the determination of antibodies to H5 influenza virus in chickens and ducks.


Subject(s)
Antibodies, Viral/blood , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H5N1 Subtype/immunology , Influenza in Birds/diagnosis , Peptides/immunology , Pichia/genetics , Recombinant Proteins/immunology , Animals , Chickens/virology , Ducks/virology , Hemagglutination Inhibition Tests , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Influenza A Virus, H5N2 Subtype/immunology , Influenza Vaccines/immunology , Influenza in Birds/virology , Peptides/genetics , Peptides/metabolism , Pichia/metabolism , Poultry Diseases/diagnosis , Poultry Diseases/virology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reproducibility of Results , Sensitivity and Specificity
10.
J Gen Virol ; 93(Pt 3): 516-525, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22113006

ABSTRACT

Rubella virus (RUBV) contains a plus-strand RNA genome with two ORFs, one encoding the non-structural replicase proteins (NS-ORF) and the second encoding the virion structural proteins (SP-ORF). This study describes development and use of a trans-encapsidation system for the assembly of infectious RUBV-like replicon particles (VRPs) containing RUBV replicons (self replicating genomes with the SP-ORF replaced with a reporter gene). First, this system was used to map signals within the RUBV genome that mediate packaging of viral RNA. Mutations within a proposed packaging signal did not significantly affect relative packaging efficiency. The insertion of various fragments derived from the RUBV genome into Sindbis virus replicons revealed that there are several regions within the RUBV genome capable of enhancing encapsidation of heterologous replicon RNAs. Secondly, the trans-encapsidation system was used to analyse the effect of alterations within the capsid protein (CP) on release of VRPs and subsequent initiation of replication in newly infected cells. Deletion of the N-terminal eight amino acids of the CP reduced VRP titre significantly, which could be partially complemented by native CP provided in trans, indicating that this mutation affected an entry or post-entry event in the replication cycle. To test this hypothesis, the trans-encapsidation system was used to demonstrate the rescue of a lethal deletion within P150, one of the virus replicase proteins, by CP contained within the virus particle. This novel finding substantiated the functional role of CP in early post-entry replication.


Subject(s)
Capsid Proteins/metabolism , Rubella virus/physiology , Virus Assembly , Virus Replication , Animals , Capsid Proteins/genetics , Cell Line , Genetic Complementation Test , RNA, Viral/genetics , Replicon , Sequence Deletion , Sindbis Virus
11.
J Virol ; 85(8): 3881-92, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21248045

ABSTRACT

The interaction of the rubella virus (RV) capsid (C) protein and the mitochondrial p32 protein is believed to participate in virus replication. In this study, the physiological significance of the association of RV with mitochondria was investigated by silencing p32 through RNA interference. It was demonstrated that downregulation of p32 interferes with microtubule-directed redistribution of mitochondria in RV-infected cells. However, the association of the viral C protein with mitochondria was not affected. When cell lines either pretreated with respiratory chain inhibitors or cultivated under (mild) hypoxic conditions were infected with RV, viral replication was reduced in a time-dependent fashion. Additionally, RV infection induces increased activity of mitochondrial electron transport chain complex III, which was associated with an increase in the mitochondrial membrane potential. These effects are outstanding among the examples of mitochondrial alterations caused by viruses. In contrast to the preferential localization of p32 to the mitochondrial matrix in most cell lines, RV-permissive cell lines were characterized by an almost exclusive membrane association of p32. Conceivably, this contributes to p32 function(s) during RV replication. The data presented suggest that p32 fulfills an essential function for RV replication in directing trafficking of mitochondria near sites of viral replication to meet the energy demands of the virus.


Subject(s)
Host-Pathogen Interactions , Microtubules/metabolism , Mitochondria/metabolism , Mitochondria/virology , Mitochondrial Proteins/metabolism , Rubella virus/pathogenicity , Viral Core Proteins/metabolism , Animals , Carrier Proteins , Cell Line , Electron Transport , Gene Silencing , Humans , Membrane Potential, Mitochondrial , Mitochondrial Proteins/antagonists & inhibitors , RNA Interference
12.
J Clin Virol ; 50(2): 136-41, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21074491

ABSTRACT

BACKGROUND: Efforts to reduce the impact of group A rotaviruses on human morbidity and mortality rely on oral immunisation with live attenuated or recombinant vaccines. A major challenge in immunisation is the vast inter- and intragenotypic diversity accomplished by circulating rotaviruses. OBJECTIVES: To monitor rotavirus inter- and intragenotypic diversity in hospitalised children. STUDY DESIGN: From January 2008 to December 2009 stool samples from 1994 paediatric in-patients suffering from diarrhoea were screened for rotavirus. Rotavirus G- and P-genotypes were determined by nucleotide sequencing and phylogenetic analysis was performed. RESULTS: Rotavirus A was detected in stool samples of 341 children, comprising G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], as well as uncommon G12P[6] genotypes and mixed infections. Predominant strains shifted from G1P[8] and G9P[8] genotypes in the first season to G3P[8] and G4P[8] genotypes in the second season. The highest intragenotypic diversity was detected in G1 strains and consisted of co-circulating G1-Ic, G1-Id, G1-Ie and G1-II rotaviruses. The G2 analysis revealed different intragenotypic lineages: G2-IIa, G2-IIb and G2-IIc. Interestingly, the circulating G4-Ib rotaviruses were characterised by insertions of 3 or 6 additional coding nucleotides within variable region 4 of VP7. Whereas different G9-III VP7 gene segments were detected G3-Ia sequences were highly homologous. In the VP4 analysis P[8]-III gene segment predominated over P[4]-Vb, P[8]-I, P[8]-IV and P[6]-I. CONCLUSIONS: A remarkable rotavirus heterogeneity was detected in the limited local setting and time span. Continued monitoring and nucleotide sequencing is necessary to document possible effects of rising immunisation levels on intragenotypic rotavirus diversity.


Subject(s)
Antigens, Viral/genetics , Capsid Proteins/genetics , Genetic Variation , Rotavirus Infections/virology , Rotavirus/classification , Rotavirus/genetics , Adolescent , Base Sequence , Child , Child, Preschool , Diarrhea/virology , Feces/virology , Gastroenteritis/virology , Genes, vif , Genotype , Germany , Humans , Polymerase Chain Reaction , Rotavirus/immunology , Sequence Analysis, DNA , Serotyping
13.
Infection ; 38(3): 231-5, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20358246

ABSTRACT

CASE PRESENTATION: We report on the case of a 5 year-old girl who developed fulminant myocarditis due to acute infection with influenza virus type B. Cardiac arrest occurred suddenly, resuscitation efforts were not successful, and the patient died of congestive heart failure 24 h after admission to the hospital. DIAGNOSIS: Lymphocytic infiltration of cardiac tissues and virologic studies confirmed the suspected diagnosis of acute viral myocarditis. CONCLUSION: In conclusion, influenza virus type B is one of the infective agents that can cause rapid and fatal myocarditis in previously healthy children. Early cardiac support may be the only option to prevent fatal outcome.


Subject(s)
Influenza B virus/isolation & purification , Influenza, Human/virology , Myocarditis/virology , Acute Disease , Age Factors , Child, Preschool , Fatal Outcome , Female , Histocytochemistry , Humans , Influenza B virus/genetics , Influenza, Human/cerebrospinal fluid , Influenza, Human/diagnosis , Myocarditis/cerebrospinal fluid , Myocarditis/diagnosis , Radiography, Thoracic , Reverse Transcriptase Polymerase Chain Reaction
14.
J Cell Biochem ; 110(1): 118-28, 2010 May.
Article in English | MEDLINE | ID: mdl-20217898

ABSTRACT

Reference genes are generally employed in real-time quantitative PCR (RT-qPCR) experiments to normalize variability between different samples. The aim of this study was to identify and validate appropriate reference genes as internal controls for RT-qPCR experiments in rubella virus (RV)-infected Vero and MCF-7 cell lines using SYBR green fluorescence. The software programs geNorm and NormFinder and the DeltaDeltaC(t) calculation were used to determine the expression stability and thus reliability of nine suitable reference genes. HPRT1 and HUEL, and HUEL and TBP were identified to be most suitable for RT-qPCR analysis of RV-infected Vero and MCF-7 cells, respectively. These genes were used as normalizers for transcriptional activity of selected cellular genes. The results confirm previously published microarray and Northern blot data, particularly on the transcriptional activity of the cyclin-dependent kinase inhibitor p21 and the nuclear body protein SP100. Furthermore, the mRNA level of the mitochondrial protein p32 is increased in RV-infected cells. The effect on cellular gene transcription by RV-infection seems to be cell line-specific, but genes of central importance for viral life cycle appear to be altered to a similar degree. This study does not only provide an accurate and flexible tool for the quantitative analysis of gene expression patterns in RV-infected cell lines. It also indicates, that the suitability of a reference gene as normalizer of RT-qPCR data and the host-cell response to RV-infection are strictly cell-line specific.


Subject(s)
Gene Expression Profiling/methods , Gene Expression Profiling/standards , Gene Expression Regulation , Reverse Transcriptase Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/standards , Rubella virus/physiology , Rubella/genetics , Animals , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Gene Expression Regulation, Neoplastic , Genome, Viral/genetics , Humans , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reference Standards , Reproducibility of Results , Rubella virus/genetics , Software , Species Specificity
15.
J Med Virol ; 82(2): 335-40, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20029797

ABSTRACT

Immunity to rubella virus (RV) is conventionally determined by measuring specific immunoglobulin G (IgG). However, several individuals may be considered immune despite undetectable antibody levels. In the present study RV-specific interferon-gamma (IFN gamma)-ELISpot and rubella-IgG-ELISA were compared in 75 young adults aged between 20 and 30 years. In a subgroup, not only rubella-like particles (RLP), but also HPV77 rubella vaccine derived antigen was used in IFN gamma-ELISpot. The results from both, ELISA and ELISpot were independent of previous encounter to RV (vaccination, exanthematous disease, or childhood infection). There was no difference between RLP and RV vaccine antigen in IFN gamma-ELISpot response, and there was no correlation between IFN gamma-ELISpot and RV-specific IgG levels. IFN gamma-producing cells were found in 78.7% of all tested persons, and 83.8% of them were positive in ELISA. In almost all individuals seronegative for RV antibody, IFN gamma-producing cells were detected. Considering both humoral and cell-mediated immune responses, a positive RV immune reaction was seen in 98.6%. The results indicate that the IFN gamma-ELISpot can provide valuable additional information in seronegative individuals.


Subject(s)
Immunity, Cellular , Immunoassay/methods , Interferon-gamma/metabolism , Leukocytes, Mononuclear/immunology , Rubella virus/immunology , Adult , Antibodies, Viral/blood , Antigens, Viral , Cells, Cultured , Female , Humans , Immunity, Humoral , Immunoglobulin G/blood , Male , Virosomes , Young Adult
16.
J Clin Microbiol ; 47(11): 3569-76, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19741083

ABSTRACT

A rare G8P[4] rotavirus, designated GER1H-09, was detected in a stool sample from an infant suffering from repeated episodes of emesis for 2 days without diarrhea. Sequencing of all genomic RNA segments was performed, and complete coding sequences were determined. The VP7 amino acid sequence revealed a close phylogenetic relationship to human G8P[6] and G8P[8] isolates from Slovenia and Africa. GER1H-09 shared typical amino acid residues within variable regions VR3 to VR7 with those strains, and their subclassification as lineage G8-II rotaviruses is proposed. The variability in VR3 was identified as the likely reason for the failure in genotyping G8-II rotaviruses by commonly used multiplex PCR. Furthermore, the sequences of associated structural and nonstructural proteins showed high amino acid identities to DS-1-like rotaviruses. The genotype composition of GER1H-09 (G8-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2) suggests the occurrence of reassortment events between G8 genotypes and human DS-1-like G2P[4] rotaviruses.


Subject(s)
Rotavirus Infections/virology , Rotavirus/classification , Rotavirus/genetics , Amino Acid Sequence , Antigens, Viral/genetics , Capsid Proteins/genetics , Cluster Analysis , Feces/virology , Female , Genetic Variation , Genotype , Germany , Humans , Infant , Molecular Sequence Data , Phylogeny , RNA, Viral/genetics , Rotavirus/isolation & purification , Rotavirus Infections/physiopathology , Sequence Analysis, DNA , Sequence Homology, Amino Acid
17.
Rheumatology (Oxford) ; 48(2): 144-8, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19074187

ABSTRACT

OBJECTIVES: To evaluate the influence of low-dose MTX and etanercept treatment on efficacy of measles, mumps and rubella (MMR) revaccination in children with juvenile idiopathic arthritis. METHODS: A prospective nested case-control study was performed to investigate markers of MMR revaccination induced humoral and cell-mediated immunity in 15 patients with juvenile idiopathic arthritis (ages 6-17 yrs), treated with either low-dose MTX therapy alone or in combination with etanercept. The control group consisted of 22 healthy children. Production of IFN-gamma by T memory cells upon in vitro stimulation with measles, mumps and rubella antigens and seroprevalence of virus-specific IgG antibodies were assessed. Medication use, disease activity and patients' comments on side-effects were observed during the period of 6 months before and after revaccination. RESULTS: Low-dose MTX therapy following MMR vaccination proved not to hamper T-cell mediated immunity in vitro. Neither low-dose MTX nor etanercept treatment, given simultaneously with revaccination, markedly interfered with generation of long-lived virus-restricted T cells and protective levels of virus-specific IgG antibodies. No increase in disease activity or medication use was seen within 6 months after MMR revaccination, including JIA patients using etanercept. No overt measles, mumps, rubella or secondary severe infections were noted. CONCLUSIONS: Low-dose MTX and etanercept treatment do not seem to interfere with intended outcome of MMR revaccination in children with JIA.


Subject(s)
Antirheumatic Agents/therapeutic use , Arthritis, Juvenile/drug therapy , Immunoglobulin G/therapeutic use , Measles-Mumps-Rubella Vaccine/administration & dosage , Methotrexate/therapeutic use , Receptors, Tumor Necrosis Factor/therapeutic use , Adolescent , Adult , Antibodies, Viral/immunology , Arthritis, Juvenile/immunology , Case-Control Studies , Child , Child, Preschool , Drug Therapy, Combination , Etanercept , Female , Humans , Immunoglobulin G/immunology , Infant , Interferon-gamma/immunology , Male , Prospective Studies , Statistics, Nonparametric , T-Lymphocytes/immunology , Treatment Outcome , Young Adult
18.
Pneumologie ; 62(5): 297-304, 2008 May.
Article in German | MEDLINE | ID: mdl-18398786

ABSTRACT

Diseases of the lung are one of the main causes of morbidity and mortality in the elderly. The risk of respiratory infections is increased due to structural changes, malnutrition, co-morbidity, and a variety of other factors. Bacterial and viral pathogens cause acute bronchitis and exacerbations of chronic bronchitis (AECB). Community acquired pneumonias (CAP) show a different spectrum of pathogens and clinical course in comparison to nosocomial pneumonias (hospital acquired pneumonia, HAP). Institutionalised patients are at risk of a health care associated pneumonia (HCAP), with often a different spectrum of pathogens in comparison to CAP and HAP. Elderly patients with cerebrovascular disease and impairment of swallowing or cough reflexes often suffer from aspiration pneumonias. The mortality is highest in the elderly, comorbid, and immunocompromised patient with nosocomial pneumonia. Important preventive measures include influenza and pneumococcal vaccination, avoidance of immobility, oral hygiene, and sufficient nutrition.


Subject(s)
Bronchitis/diagnosis , Bronchitis/prevention & control , Geriatric Assessment/methods , Pneumonia/diagnosis , Pneumonia/prevention & control , Aged , Aged, 80 and over , Bronchitis/epidemiology , Female , Humans , Male , Pneumonia/epidemiology
19.
Bone Marrow Transplant ; 39(5): 293-9, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17262060

ABSTRACT

Human cytomegalovirus (CMV) is a major cause of death after transplantation. The frequency of pp65-specific T cells was examined in 38 HLA-A2+ stem cell recipients during the first year after transplantation. Patients were divided into four groups based on donor/recipient serostatus: d+/r+ (n=17), d+/r- (n=7), d-/r+ (n=9) and d-/r- (n=5). Peripheral blood mononuclear cells were stimulated with the CMVpp65 peptide NLVPMVATV, and the specific T-cell frequency was assessed by interferon gamma (IFN-gamma) ELISPOT assay. Responding T cells were characterized by flow cytometry revealing a terminal differentiated effector phenotype. Surveillance of CMV infection was carried out by real-time polymerase chain reaction (n=26) or immunofluorescence (n=12). Infection was present in 7/9 d-/r+ high-risk patients, and CMV disease occurred exclusively in this group with delayed or absent virus-specific T-cell recovery. In contrast, 16/24 intermediate-risk patients showed CMV-specific T cells. Our data suggest that CMV infection and disease rates are elevated in high-risk patients with delayed CMV-specific T-cell immune reconstitution and lower in those with early recovery of T-cell immunity. We recommend preferring CMV seropositive donors for CMV seropositive recipients, as this should lead to durable CMV-specific T-cell responses soon after transplantation with consecutive protection from CMV disease.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cytomegalovirus Infections/immunology , Cytomegalovirus/immunology , Recovery of Function/immunology , Stem Cell Transplantation , Adolescent , Adult , Aged , Female , Follow-Up Studies , HLA-A2 Antigen/immunology , Hematologic Neoplasms/immunology , Hematologic Neoplasms/therapy , Humans , Interferon-gamma/immunology , Male , Middle Aged , Monitoring, Physiologic , Peptides/immunology , Phosphoproteins/immunology , Risk Factors , Transplantation, Homologous , Viral Matrix Proteins/immunology
20.
Pneumologie ; 61(1): 41-5, 2007 Jan.
Article in German | MEDLINE | ID: mdl-17253209

ABSTRACT

New respiratory viruses associated with pneumonia have in the past few years been detected in humans. The sudden appearance of the severe acute respiratory syndrome (SARS) in 2003 demonstrated that an emerging and highly infectious disease caused by a hitherto unknown virus was able to spread rapidly, but could finally be contained by stringent measures. The avian influenza A-H5N1-virus of high pathogenicity has crossed in multiple instances the species barriers between humans, mammals, and birds posing a serious pandemic threat. The application of the so far learnt and the continued development of preventive strategies, efficient vaccines, and antiviral substances are besides worldwide surveillance decisive to rapidly detect the repeated, enforced, or new appearance of viruses like the SARS-CoV, influenza A-H5N1 virus, or of new viruses, to contain their spread, and to defeat them.


Subject(s)
Influenza A Virus, H5N1 Subtype/pathogenicity , Influenza in Birds/diagnosis , Influenza, Human/diagnosis , Severe Acute Respiratory Syndrome/diagnosis , Severe acute respiratory syndrome-related coronavirus/pathogenicity , Animals , Antiviral Agents/therapeutic use , Birds , Humans , Influenza in Birds/drug therapy , Influenza in Birds/transmission , Influenza, Human/drug therapy , Influenza, Human/transmission , Risk Factors , Severe Acute Respiratory Syndrome/drug therapy , Severe Acute Respiratory Syndrome/transmission , Species Specificity , Virulence
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