1.
LeBlanc, Neil, et al.
(författare)
A novel combination of TaqMan RT-PCR and a suspension microarray assay for the detection and species identification of pestiviruses
2010
Ingår i: Veterinary Microbiology. - : Elsevier BV. - 0378-1135 .- 1873-2542. ; 142:1-2, s. 81-86
Tidskriftsartikel (refereegranskat) abstract
The genus pestivirus contains four recognized species: classical swine fever virus, border disease virus, bovine viral diarrhoea virus types 1 and 2. All are economically important and globally distributed but classical swine fever is the most serious, concerning losses and control measures. It affects both domestic pigs and wild boars. Outbreaks of this disease in domestic pigs call for the most serious measures of disease control, including a stamping out policy in Europe. Since all the members of the pestivirus genus can infect swine, differential diagnosis using traditional methods poses some problems. Antibody tests may lack specificity due to cross-reactions, antigen capture ELISAs may have low sensitivity, and virus isolation may take several days or even longer time to complete. PCR-based tests overcome these problems for the most part, but in general lack the multiplexing capability to detect and differentiate all the pestiviruses simultaneously. The assay platform described here addresses all of these issues by combining the advantages of real-time PCR with the multiplexing capability of microarray technology. The platform includes a TaqMan real-time PCR designed for the universal detection of pestiviruses and a microarray assay that can use the amplicons produced in the real-time PCR to identify the specific pestivirus.
2.
Blomberg, Jonas, et al.
(författare)
Antibodies to Human Herpesviruses in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients
2019
Ingår i: Frontiers in Immunology. - : FRONTIERS MEDIA SA. - 1664-3224. ; 10
Tidskriftsartikel (refereegranskat) abstract
Myalgic encephalomyelitis, also referred to as chronic fatigue syndrome (ME/CFS) is a debilitating disease characterized by myalgia and a sometimes severe limitation of physical activity and cognition. It is exacerbated by physical and mental activity. Its cause is unknown, but frequently starts with an infection. The eliciting infection (commonly infectious mononucleosis or an upper respiratory infection) can be more or less well diagnosed. Among the human herpesviruses (HHV-1 -8), HHV-4 (Epstein-Barr virus; EBV), HHV-6 (including HHV-6A and HHV-6B), and HHV-7, have been implicated in the pathogenesis of ME/CFS. It was therefore logical to search for serological evidence of past herpesvirus infection/reactivation in several cohorts of ME/CFS patients (all diagnosed using the Canada criteria). Control samples were from Swedish blood donors. We used whole purified virus, recombinant proteins, and synthetic peptides as antigens in a suspension multiplex immunoassay (SMIA) for immunoglobulin G (IgG). The study on herpesviral peptides based on antigenicity with human sera yielded novel epitope information. Overall, IgG anti-herpes-viral reactivities of ME/CFS patients and controls did not show significant differences. However, the high precision and internally controlled format allowed us to observe minor relative differences between antibody reactivities of some herpesviral antigens in ME/CFS versus controls. ME/CFS samples reacted somewhat differently from controls with whole virus HHV-1 antigens and recombinant EBV EBNA6 and EA antigens. We conclude that ME/CFS samples had similar levels of IgG reactivity as blood donor samples with HHV-1-7 antigens. The subtle serological differences should not be over-interpreted, but they may indicate that the immune system of some ME/CFS patients interact with the ubiquitous herpesviruses in a way different from that of healthy controls.
3.
Hu, Lijuan, et al.
(författare)
Dynamic and selective HERV RNA expression in neuroblastoma cells subjected to variation in oxygen tension and demethylation
2016
Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - : Wiley. - 0903-4641 .- 1600-0463. ; 124:1-2, s. 140-149
Tidskriftsartikel (refereegranskat) abstract
We studied HERV expression in cell lines after hypoxia, mitogenic stimulation, and demethylation, to better understand if hypoxia may play a role in ERV activation also within the nervous system, as represented by neuroblastoma cell lines. The level of RNA of four human ERV groups (HERVs) (HERVE, I/T, H, and W), and three housekeeping genes, of different cell lines including A549, COS-1, Namalwa, RD-L and Vero-E6, as well as human neuroblastoma cell lines SH-SY5Y, SK-N-DZ, and SK-N-AS were studied using reverse transcription and real-time quantitative PCR (QPCR). During the course of recovery from hypoxia a pronounced and selective activation of RNA expression of HERVW-like sequences, but not of HERVE, I/T, H, and three housekeeping genes, was found in the neuroblastoma cell lines, most pronounced in SK-N-DZ. In the SK-N-DZ cell line, we also tested the expression of HERVs after chemical treatments. HERVW-like sequences were selectively upregulated by 5-azacytidine, a demethylating agent. Some HERVW loci seem especially responsive to hypoxia and demethylation. HERV expression in neuroblastoma cells is selectively and profoundly influenced by some physiological and chemical stimuli.
4.
Jern, Patric, 1976-
(författare)
Genomic Variation and Evolution of HERV-H and other Endogenous Retroviruses (ERVs)
2005
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
An exogenous retrovirus (XRV) that integrates into a germ cell may be inherited as a Mendelian gene; it becomes an endogenous retrovirus (ERV). The human genome consists of up to 8% HERVs. The gammaretroviral (ERV class I) HERV-H, with 926 members, is the largest ERV group. Despite millions of years since integration, it has polymorphic envelope open reading frames in at least three loci. Selections for functional envelopes are indicated on chromosomes 1 and 2. However, envelopes were present only in a fraction of the total HERV-H. Mutated polymerases, indicating old ERVs, contradicted relatively intact long terminal repeats. To explain this, we formulated a “Midwife” element theory where proteins are complemented in trans. A phylogenetic analysis did not support separate HERV-H and -F groups. The new taxonomy included HERV-H like (RGH2-like and RTVLH2-like subgroups) and Adjacent HERV-H like. A bioinformatic reconstruction of a putative ancestral HERV-H exposed novel traits. Two nucleocapsid zinc fingers and a pronounced nucleotide bias for C in the HERV-H like were unique among the gammaretroviruses. Two recently integrated gammaretroviral groups (PtNeo-I[PTERV1] and -II) were found in chimpanzees but not in humans. The PtNeo groups were most similar to baboon ERVs and a macaque sequence, but neither to other chimpanzee nor to any human gammaretroviruses. The pattern was consistent with cross-species transfer via predation. To advance the retroviral taxonomy, we projected structural markers over sequence phylogenetic trees. A number of markers were useful to distinguish between genera and to delineate groups. Basic retroviral knowledge is vital to understand emerging infections. Phylogenetic analyses of taxonomically improved sequences, facilitates the search for common retroviral denominators to target. This thesis provided new insights in retroviral evolution and taxonomy using the ERVs, with special focus on the large gammaretroviral HERV-H group, as an additional source of information next to that of XRVs.
5.
Abdeldaim, Guma M. K., et al.
(författare)
Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction
2009
Ingår i: Diagnostic microbiology and infectious disease. - : Elsevier. - 0732-8893 .- 1879-0070. ; 64:4, s. 366-373
Tidskriftsartikel (refereegranskat) abstract
A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 104 DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.
6.
Abdeldaim, Guma, 1969-, et al.
(författare)
Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis
2010
Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 10, s. 310-
Tidskriftsartikel (refereegranskat) abstract
Background. Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients. Results. The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis) in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 105 genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively. In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both bacteria. Conclusions. The PCR provides increased sensitivity and the multiplex format facilitates diagnosis of S. pneumoniae, H. influenzae and N. meningitidis and the assay enable detection after antibiotic treatment has been installed. Quantification increases the specificity of the etiology for pneumonia.
7.
Abdeldaim, Guma M. K., et al.
(författare)
Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia
2013
Ingår i: Diagnostic microbiology and infectious disease. - : Elsevier. - 0732-8893 .- 1879-0070. ; 76:2, s. 141-146
Tidskriftsartikel (refereegranskat) abstract
A quantitative polymerase chain reaction (PCR) for the fucK gene was developed for specific detection of Haemophilus influenzae. The method was tested on sputum and nasopharyngeal aspirate (NPA) from 78 patients with community-acquired pneumonia (CAP). With a reference standard of sputum culture and/or serology against the patient's own nasopharyngeal isolate, H. influenzae etiology was detected in 20 patients. Compared with the reference standard, fucK PCR (using the detection limit 10(5) DNA copies/mL) on sputum and NPA showed a sensitivity of 95.0% (19/20) in both cases, and specificities of 87.9% (51/58) and 89.5% (52/58), respectively. In a receiver operating characteristic curve analysis, sputum fucK PCR was found to be significantly superior to sputum P6 PCR for detection of H. influenzae CAP. NPA fucK PCR was positive in 3 of 54 adult controls without respiratory symptoms. In conclusion, quantitative fucK real-time PCR provides a sensitive and specific identification of H. influenzae in respiratory secretions.
8.
Nilsson, Anna-Lena, et al.
(författare)
Serological Evaluation of Possible Exposure to Ljungan Virus and Related Parechovirus in Autoimmune (Type 1) Diabetes in Children
2015
Ingår i: Journal of Medical Virology. - : Wiley. - 0146-6615 .- 1096-9071. ; 87:7, s. 1130-1140
Tidskriftsartikel (refereegranskat) abstract
Exposure to Ljungan virus (LV) is implicated in the risk of autoimmune (type 1) diabetes but possible contribution by other parechoviruses is not ruled out. The aim was to compare children diagnosed with type 1 diabetes in 2005-2011 (n=69) with healthy controls (n=294), all from the Jamtland County in Sweden, using an exploratory suspension multiplex immunoassay for IgM and IgG against 26 peptides of LV, human parechoviruses (HPeV), Aichi virus and poliovirus in relation to a radiobinding assay (RBA) for antibodies against LV and InfluenzaA/H1N1pdm09. Islet autoantibodies and HLA-DQ genotypes were also determined. 1) All five LV-peptide antibodies correlated to each other (P<0.001) in the suspension multiplex IgM- and IgG-antibody assay; 2) The LV-VP1_31-60-IgG correlated with insulin autoantibodies alone (P=0.007) and in combination with HLA-DQ8 overall (P=0.022) as well as with HLA-DQ 8/8 and 8/X subjects (P=0.013); 3) RBA detected LV antibodies correlated with young age at diagnosis (P<0.001) and with insulin autoantibodies (P<0.001) especially in young HLA-DQ8 subjects (P=0.004); 4) LV-peptide-VP1_31-60-IgG correlated to RBA LV antibodies (P=0.009); 5) HPeV3-peptide-IgM and -IgG showed inter-peptide correlations (P<0.001) but only HPeV3-VP1_1-30-IgG (P<0.001) and VP1_95-124-IgG (P=0.009) were related to RBA LV antibodies without relation to insulin autoantibody positivity (P=0.072 and P=0.486, respectively). Both exploratory suspension multiplex IgG to LV-peptide VP1_31-60 and RBA detected LV antibodies correlated with insulin autoantibodies and HLA-DQ8 suggesting possible role in type 1 diabetes. It remains to be determined if cross-reactivity or concomitant exposure to LV and HPeV3 contributes to the seroprevalence. J. Med. Virol. 87:1130-1140, 2015.
9.
Herrmann, Björn, et al.
(författare)
Comparison of a duplex quantitative real-time PCR assay and the COBAS Amplicor CMV Monitor test for detection of cytomegalovirus
2004
Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 42:5, s. 1909-14
Tidskriftsartikel (refereegranskat) abstract
A duplex quantitative real-time PCR (qPCR) assay was designed to detect both the polymerase gene (pol) and the glycoprotein gene (gB) of cytomegalovirus (CMV). The detection limit of the qPCR was determined to be 1 to 3 copies/reaction and the linear measure interval was 10(3) to 10(8) copies/ml. The qPCR system was compared to the COBAS Amplicor CMV Monitor test (COBAS) by an analysis of 138 plasma samples. Both systems detected CMV in 71 cases and had negative results for 33 samples. In addition, 34 samples were positive by qPCR and negative by the COBAS assay, but in no case was the COBAS result positive and the qPCR result negative. Thus, qPCR detected 48% more positive cases than the COBAS method. For samples with > or = 10(5) copies/ml by qPCR, a saturation effect was seen in the COBAS assay and quantification required dilution. Copy numbers for pol and gB by qPCR generally agreed. However, the reproducibility of qPCR assays and the need for an international standard are discussed. Discrepant copy numbers for pol and gB by qPCR were found for samples from two patients, and sequence analysis revealed that the corresponding CMV strains were mismatched at four nucleotide positions compared with the gB fragment primer sequences. In conclusion, a duplex qPCR assay in a real-time format facilitates quantitative measurements and minimizes the risk of false-negative results.
10.
Martinez Barrio, Alvaro, 1977-, et al.
(författare)
Data mining of the dog genome reveals novel Canine Endogenous Retroviruses(CfERVs)
Annan publikation (populärvet., debatt m.m.) abstract
Mining the dog genome for canine endogenous retroviruses (CfERV) using the program RetroTector© identified 407 CfERVs (0.15% of the total genome size). Phylogenetic analysis showed that the majority of these CfERVs belong to the gammaretroviridae (n=313) genus. In this group, we found 33 integrated CfERVs with similarity to the human HERV-Fc1. Eighteen of them had conserved open reading frames open and seven of the 18 were recent integrations (≤ 5% LTR divergence). Some of these CfERVs may have potential for active retrotransposition and could actively contribute to the plasticity of canine genomes. Similar to other vertebrates, betaretroviruses (n=28) was the second most common group. In addition, four spuma-like and four gypsy-like CfERVs were identified, the latter group being rare in vertebrate genomes. Moreover, we identified 55 CfERVs that could not be classified unambiguously to any known retroviral genera. The integration landscape shows that all dog chromosomes have CfERV integrations with non-uniform distribution both along and across chromosomes. Some regions were essentially devoid of CfERVs whereas other regions had large numbers. Notably, in a comparison between dog and human genomes, CfERV were approximately one fifth of the amount of HERVs found. Species-specific mechanisms for purging and protection against retroviral infections are suggested to act in the dog genome. The CfERV integration pattern showed that a substantial fraction of annotated genes were found within 100 kb distance from annotated proviruses. The majority of such integrations were placed in antisense orientation relative to the transcriptional direction of the neighboring chromosomal genes. In conclusion, our results from Canis familiaris genome analysis support the notion that different mammals may interact distinctively with endogenous retroviruses.
