Development of a real-time RT-PCR assay for detection and quantitation of parainfluenza virus 3.

A TaqMan-based real-time RT-PCR assay was developed to detect and quantify human parainfluenza virus 3 (PIV3). Two sets of primer-probe pairs were designed based on the nucleotide (nt) sequence of the nucleocapsid (N) gene. The primer-probe pairs were derived from the 3' end of the N gene (set 1) and the 5' region of the gene (set 2), respectively. Using real-time RT-PCR, the sensitivity of set 1 was determined to be about 9 copies of PIV3 genome, while the sensitivity of set 2 was about 93 copies of PIV3 genome. Set 1 was chosen for subsequent experiments. This primer-probe pair detected PIV3, but not any of several other respiratory viruses, indicating that the assay is PIV3 specific. For clinical evaluation, the assay was employed to test 80 nasopharyngeal aspirates from children with respiratory symptoms. The results confirmed the presence of PIV3 in 12 specimens previously identified as positive by culture confirmation, and showed all of which contained more than 100 copies of PIV3 genome. In addition, the method also detected PIV3 genomes in specimens found negative by culture confirmation, indicating the value of this RT-PCR assay. These data thus demonstrate the application of the real-time RT-PCR assay for the detection and quantification of PIV3 in clinical specimens.

Detection and characterization of new influenza B virus variants in 2002.

One-hundred five influenza B-positive specimens obtained from southeast Asia in 2002 were categorized on the basis of DNA sequencing of HA1 gene as well as real-time PCR analysis of the NA gene. Phylogenetic analysis of the HA1 gene sequences showed that the majority of the viruses (96.2%) belonged to the B/Victoria/2/87 lineage, while a smaller percentage of the viruses (3.8%) belonged to the B/Yamagata/16/88 lineage. The B/Yamagata/16/88 viruses displayed significant antigenic drift in the deduced amino acid sequences of the HA1 protein, and the B/Victoria/2/87-like viruses consisted of B/Hong Kong/1351/02-like (72.3%) and B/Hong Kong/330/01-like (27.7%) viruses. The B/Hong Kong/1351/02-like viruses were reassortants with the HA gene belonging to the B/Victoria/2/87 lineage and the NA gene belonging to the B/Yamagata/16/88 lineage, whereas both the HA and NA genes of B/Hong Kong/330/01 virus belonged to the B/Victoria/2/87 lineage. In this study, however, all the B/Hong Kong/330/01-like isolates exhibited the B/Yamagata/16/88-like NA gene, which likely resulted from reassortment of B/Hong Kong/330/01 and B/Hong Kong/1351/02 viruses during coinfection. Additional molecular characterization of the six internal genes showed that the M, NS, PA, and PB2 genes of the new variants were B/Hong Kong/1351/02 in origin, whereas the NP and PA genes retained the B/Hong Kong/330/01 origin. Interestingly, these new variants all appeared late in the year 2002. These results support the notion that influenza B viruses continued to evolve through antigenic drift and shift.

[Clinical characteristics of bronchiolitis caused by human metapneumovirus in infants].

OBJECTIVE: The fact that the acute lower respiratory infections (ALRI) are associated with a newly discovered virus, human metapneumovirus (hMPV), has been shown in several studies. The authors conducted this study to understand the etiological and clinical characteristics of bronchiolitis, one of the most common ALRI in infants, caused by hMPV. METHODS: Nasopharyngeal aspirate specimens from 54 out of 126 infants with bronchiolitis admitted to the Children's Hospital Affiliated to Capital Institute of Pediatrics, Beijing from November 2002 to February 2003 were examined for hMPV gene fragments by reverse transcription-polymerase chain reaction (RT-PCR). Prior to the detection, the specimens were confirmed as negative for the common respiratory pathogens including RSV, influenza A and B, parainfluenza I, II, III, adenovirus, Mycoplasma pneumoniae by indirect immunofluorescence test, virus isolation and ELISA test. The clinical data of the patients diagnosed etiologically as hMPV infection analyzed included the infants' age, sex, the degree of fever, the severity of wheezing and clinical Lowell score, the findings of chest examination and chest X-ray, the white blood cell count and blood gas analysis, the course of the disease, the major treatments and the outcome of the disease. RESULTS: Twenty-one specimens showed the predicted 213 bp PCR products in agarose gel and the positive rate was 16.7% of all patients (21/126) and 39% of the patients with negative results for common respiratory pathogens detections (21/54). The range of patients' age was 2 - 15 months and the young infants with hMPV bronchiolitis (1 - 6 month of age) accounted for 62% and the male:female ratio was 3.2:1. The patients presented a low-medium grade fever (T < 39 degrees C) accounted for 86%; 81.0% of patients had a white blood cell count lower than 10.0 x 10(9)/L. The radiological findings were patchey opacity in both lungs (68%) and(or) hyperinflation (62%). Assessed by the Lowell score system, 5 out of 21 cases were considered as severe cases. The major clinical findings of hMPV bronchiolitis had no significant difference compared with that of subgroup A hRSV bronchiolitis, and showed longer course of disease than that of subgroup B hRSV bronchiolitis (P < 0.01). CONCLUSIONS: Of the infants with bronchiolitis hospitalized in our hospital from November of 2002 through February of 2003, 16.7% were caused by hMPV infection. These data showed that the major clinical characteristics and the outcome of treatment of hMPV bronchiolitis had no statistically significant difference compared to the cases with either subgroup A or subgroup B hRSV infection.

Detection of influenza B in clinical specimens: comparison of high throughput RT-PCR and culture confirmation.

Influenza virus is one of the major causes of worldwide respiratory tract infections during the winter season. Here we describe a high throughput (HTP) protocol for rapid diagnosis of influenza B that combines automated viral RNA extraction with detection and quantification by TaqMan-based PCR. Using this methodology, we tested 4176 nasal swabs collected from children enrolled in a European influenza vaccine trial during the winter of 2000 to compare our HTP PCR method to culture confirmation for detection of influenza B. Among these, 37 were positive by culture and 169 were positive by PCR irrespective of virus copy number. However, when specimens with fewer than 20 copies of the viral genome were disregarded, a good correlation between two methods was observed. At this cut-off, 34 specimens were positive and 4106 were negative by both methods. Statistical analysis of the data using culture confirmation as the standard indicated that the sensitivity of HTP RT-PCR for influenza B was 92% (95% CI, 0.83-1), and the specificity was 99% (95% CI, 0.99-1). In summary, HTP RT-PCR was proved to be more rapid and sensitive than culture confirmation, and it correlated significantly with culture confirmation for specimens containing more than 20 copies of the viral genome.

Simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus A and B by real-time PCR.

Timely diagnosis of respiratory syncytial virus (RSV) infection is critical for appropriate treatment of lower respiratory infection in young children. To facilitate diagnosis, we developed a rapid, specific, and sensitive TaqMan PCR method for detection of RSV A and RSV B. Two sets of primer-probe pairs were selected from the nucleotide sequences encoding the nucleocapsid protein--one targeting RSV A and the other targeting RSV B. The specificity of the TaqMan reverse transcription-PCR assay was evaluated by testing each primer-probe pair against various viruses derived from laboratory virus stocks, as well as clinical respiratory specimens. Fluorescent signals were observed only in the presence of RSV A and/or RSV B. The sensitivity of our quantitative PCR assay was determined on the basis of PFU and virus particle counts. The resulting assay sensitivity was found to be 0.023 PFU, or two copies of viral RNA, for RSV A and 0.018 PFU, or nine copies of viral RNA, for RSV B. This quantitative TaqMan PCR assay was utilized to diagnose 175 nasopharyngeal aspirates obtained from children in Hong Kong with respiratory symptoms during the winter of 2000 and 2001. Among these specimens, TaqMan PCR detected 36 RSV-positive samples, 10 of which were identified as RSV A and 26 of which were identified as RSV B, whereas culture confirmation identified 21 RSV-positive specimens and immunofluorescence identified 32 RSV-positive specimens, all of which were among those identified by PCR. The results confirmed the accuracy of our TaqMan PCR assay and demonstrated its improved sensitivity versus classical methods.

[Human metapneumovirus may associate with acute respiratory infections in hospitalized pediatric patients in Beijing, China].

OBJECTIVE: A new respiratory virus, human metapneumovirus (HMPV) was recently identified by scientists in the Netherlands first and then in a few other countries. To investigate if this newly discovered virus is associated with the acute respiratory infections in pediatric patients in Beijing, tests were developed to detect HPMV gene fragments from nasopharyngeal aspirates collected from infants and young children hospitalized for acute respiratory infections from November 2002 to March 2003. METHODS: The HMPV was screened by reverse transcription-polymerase chain reaction (RT-PCR). RNAs were extracted by Trizol from 247 specimens which had been determined as negative for conventional respiratory viruses including RSV, influenza A and B, parainfluenza I, II, III and adenovirus by indirect immunofluorescence test as well as virus isolation. The HMPV RNAs were detected by reverse transcription tests using random primer and M-MLV reverse transcriptase followed by PCR using the primers designed from the published sequence of the N protein-encoding gene from the first HMPV identified in the Netherlands. PCR products were visualized by 1.2% agarose gel electrophoresis. Selected positive PCR products were sequenced and the sequences of the nucleotides and deduced amino acids were compared with those in the GenBank. RESULTS: Among those 247 specimens negative for common respiratory viruses, 74 (30.0%) showed the predicted 213 bp PCR products in agarose gel. Most of clinical diagnoses for these 58 patients were pneumonia (36, 48.6%), bronchiolitis (21, 28.4%), and bronchitis and asthma in some patients. Nearly 90 percent of positive specimens were from patients under 2 years of age. Ten out of 74 amplicons were randomly selected for sequence analysis. When compared with the sequences in the GenBank, the nucleotide sequences of these 10 amplicons shared high homology only with those of HMPVs. The nucleotide sequence identities of these 10 samples with those from the Netherlands and Canada were 87% - 99%. When compared with the nucleotide sequence from the first reported strain by Van den Hoogen (strain HMPV 00-1), the sequence identities of these 10 fragments ranged from 88.7% to 99.1%. Among the 10 amplicons from the specimens, the nucleotide identities were 87.3% - 100%. One of the 10 amplicons (No. 1816) shared lower identity with others (87.3% - 89.7%), whereas the other 9 shared higher identities (95.8% - 100%) with each other. The comparison of amino acids showed that these 10 amplicons showed high homology (95.8% - 100%). Again, amplicon No.1816 shared lower homology (95.8% - 97.2%) with others, whereas the other 9 shared higher homology (98.6% - 100%). The amino acid homology between No.1816 and HMPV 00-1 was 95.8%, whereas that of the other 9 with HMPV 00-1 was 98.6% - 100%. CONCLUSION: These data suggested that some of acute respiratory infections in pediatric patients in Beijing area are related to the newly identified human metapneumovirus. The HMPV circulating in Beijing may have different genotypes.