Fetal DNA has been detected in maternal plasma by the use of genetic differences between mother and fetus. We explore the possibility of using epigenetic markers for the specific detection of fetal ...DNA in maternal plasma.
A differentially methylated region in the human IGF2-H19 locus and a single-nucleotide polymorphism in this region were chosen for the study. The methylation status in this region is maintained in such a way that the paternal allele is methylated and the maternal allele is unmethylated. The single-nucleotide polymorphism was typed by direct sequencing of PCR products. The methylation status of this region was ascertained by bisulfite conversion and methylation-specific PCR. Differentially methylated fetal alleles were detected in maternal plasma by direct sequencing and a primer-extension assay.
Women in the second (n = 21; 17-21 weeks) and third (n = 18; 37-42 weeks) trimesters of pregnancy were recruited. Among these 39 volunteers, the 16 who were heterozygous for the single-nucleotide polymorphism were chosen for further analysis. In 11 of these 16 cases, paternally inherited methylated fetal alleles were different from the methylated alleles of the respective mothers. Using direct sequencing, we detected paternally inherited methylated fetal DNA in 6 of 11 (55%) cases. In 8 of the 16 heterozygous cases, the fetuses possessed an unmethylated maternally inherited allele that was different from the unmethylated allele of the mother. Using a primer-extension assay, we detected fetal-derived maternally inherited alleles in maternal plasma of four of eight (50%) cases.
These results represent the first use of fetal epigenetic markers in noninvasive prenatal analysis. These data may also have implications for the investigation of other types of chimerism.
Severe acute respiratory syndrome (SARS) is caused by infection of a previously undescribed coronavirus (CoV). L-SIGN, encoded by CLEC4M (also known as CD209L), is a SARS-CoV binding receptor that ...has polymorphism in its extracellular neck region encoded by the tandem repeat domain in exon 4. Our genetic risk association study shows that individuals homozygous for CLEC4M tandem repeats are less susceptible to SARS infection. L-SIGN is expressed in both non-SARS and SARS-CoV-infected lung. Compared with cells heterozygous for L-SIGN, cells homozygous for L-SIGN show higher binding capacity for SARS-CoV, higher proteasome-dependent viral degradation and a lower capacity for trans infection. Thus, homozygosity for L-SIGN plays a protective role during SARS infection.
Background: A novel coronavirus was recently identified as the aetiological agent of Severe Acute Respiratory Syndrome (SARS). Molecular assays currently available for detection of SARS-coronavirus ...(SARS-Cov) have low sensitivity during the early stage of the illness.
Objective: To develop and evaluate a sensitive diagnostic test for SARS by optimizing the viral RNA extraction methods and by applying real-time quantitative RT-PCR technology.
Study design: 50 nasopharyngeal aspirate (NPA) samples collected from days 1–3 of disease onset from SARS patients in whom SARS CoV infections was subsequently serologically confirmed and 30 negative control samples were studied. Samples were tested by: (1) our first generation conventional RT-PCR assay with a routine RNA extraction method (Lancet 361 (2003) 1319), (2) our first generation conventional RT-PCR assay with a modified RNA extraction method, (3) a real-time quantitative RT-PCR assay with a modified RNA extraction method.
Results: Of 50 NPA specimens collected during the first 3 days of illness, 11 (22%) were positive in our first generation RT-PCR assay. With a modification in the RNA extraction protocol, 22 (44%) samples were positive in the conventional RT-PCR assay. By combining the modified RNA extraction method and real-time quantitative PCR technology, 40 (80%) of these samples were positive in the real-time RT-PCR assay. No positive signal was observed in the negative controls.
Conclusion: By optimizing RNA extraction methods and applying quantitative real time RT-PCR technologies, the sensitivity of tests for early diagnosis of SARS can be greatly enhanced.
The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive ...target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5′ single-stranded overhang, indicating a 5′o 3′ polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15–25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT24 binding over 10 times more strongly than dA24 as observed by the apparent Km .
A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RT-PCR) ...assays to detect SARS CoV in clinical specimens.
RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV.
The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar.
NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.
Department of Microbiology, University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong SAR
Correspondence L. L. M. Poon llmpoon{at}hkucc.hku.hk
A novel group 1 coronavirus was previously ...identified in bent-winged bats ( Miniopterus spp.). Here, results are described from our ongoing surveillance of these bats for coronaviruses. These findings show that group 1 coronaviruses are endemic in these bat populations in Hong Kong. Genetic analysis of these viruses indicates that there are at least four different, but closely related, group 1 coronaviruses (bat-CoV 1A, 1B, HKU7 and HKU8) circulating in bent-winged bats. Phylogenetic analysis revealed that these group 1 bat coronaviruses have descended from a common ancestor and that these viruses have been established in these bats for a long period of time. These data provide a better understanding of the emergence and evolution of coronaviruses. Bat-CoV 1A and 1B were detected in apparently healthy Miniopterus magnater and Miniopterus pusillus , respectively, on repeated sampling occasions at a single habitat, suggesting that these viruses have established a persistent infection in these populations.
Published online ahead of print on 14 June 2006 as DOI 10.1099/vir.0.82203-0.
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this study are DQ666337DQ666344.
Supplementary material is available in JGV Online.
► It is essential to define diagnostic performance of rapid influenza detection kits. ► The rapid kits had lower sensitivity for diagnosis of H1pdm09 than seasonal flu. ► Direct immunofluorescence ...test also showed similar finding. ► Lower sensitivity of the rapid kits is related to lower viral load in the patients. ► The detection limit of the rapid kits in clinical specimens was 1×107copies/ml.
Data on analytical sensitivity of rapid diagnostic assays are important for clinical management of influenza, especially during a pandemic. Four rapid antigen detection assays were compared for detection of pandemic influenza A H1N1 2009, seasonal H1N1 and H3N2 in 96 patients with influenza A infection confirmed by real-time RT-PCR. These rapid antigen tests appeared to have lower sensitivity (55.8%) for the diagnosis of pandemic influenza A H1N1 2009 as compared with seasonal H3N2 (71.0%) or H1N1 (69.4%) influenza infections, a difference that was related to a lower viral load in patients infected with the pandemic influenza A H1N1 2009 virus. The detection limit of these antigen detection tests in clinical specimens was an influenza A M gene copy number of average 1.0×107copies/ml. Significant variations between tests in sensitivity for detection of pandemic influenza A H1N1 2009 (43.4–63.3%) were observed. The Directigen EZ Influenza A+B and the Espline Influenza A+B had comparable sensitivity (63%) and were the most sensitive among the four assays evaluated.
The serological response profile of severe acute respiratory syndrome (SARS) coronavirus (CoV) infection was defined by neutralization tests and subclass-specific immunofluorescent (IF) tests using ...serial sera from 20 patients. SARS CoV total immunoglobulin (Ig) (IgG, IgA, and IgM IgGAM) was the first antibody to be detectable. There was no difference in time to seroconversion between the patients who survived (n = 14) and those who died (n = 6). Although SARS CoV IgM was still detectable by IF tests with 8 of 11 patients at 7 months postinfection, the geometric mean titers dropped from 282 at 1 month postinfection to 19 at 7 months (P = 0.001). In contrast, neutralizing antibody and SARS CoV IgGAM and IgG antibody titers remained stable over this period. The SARS CoV antibody response was sometimes associated with an increase in preexisting IF IgG antibody titers for human coronaviruses OC43, 229E, and NL63. There was no change in IF IgG titer for virus capsid antigen from the herpesvirus that was used as an unrelated control, Epstein-Barr virus. In contrast, patients who had OC43 infections, and probably also 229E infections, without prior exposure to SARS CoV had increases of antibodies specific for the infecting virus but not for SARS CoV. There is a need for awareness of cross-reactive antibody responses between coronaviruses when interpreting IF serology.
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