Development Of An Indirect Immunofluorescence Assay For Detection Of Human Metapneumovirus And Comparision Of Different Methods Used In Detection Of Human Metapneumovirus

PARTⅠ:Development and application of an indirect immunofluorescence assay for detection of Human MetapneumovirusObjective Human metapneumovirus (hMPV) is a newly recognized viral pathogen causing upper and lower respiratory infection in all age groups in particular young children. This study was to develop an indirect immunofluorescence assay(IFA) for detection of human Metapneumovirus.Method Vero-E6 cells were infected by recombinant hMPV strain NL/1/00 and NL/1/99. When maximum CPE was observed, cells were used to prepare cell antigen smear for IFA detection. The assay was validated by using serially diluted human Metapneumovirus infected cells and common respiratory virus (RSV, parainfluenza, adenovirus and influenza viruses) positive anigens. The hMPV IFA was then performed to detect hMPV in clinical specimens.Result The hMPV IFA did not recognize common respiratory viral antigens but hMPV antigen from both subgroup, indicating good specificity and reproducibility. The analytical detection limit of this IFA was 102.2TCID50/ml. A total of 523 nasopharygeal secretions specimens were screened for the presence of hMPV by IFA and 48 specimens were positive.Conclusion An indirect immunofluorescence assay for detection of hMPV has been successfully developed, which could be utilized in clinical diagnosis and epidemiological studies for hMPV。PARTⅡ:Comparison of different methods used in detection of human MetapneumovirusObjective Compared the sensitivity and specificity of conventional RT-PCR,Real-time PCR and IFA in detecting hMPV.And compared the virus detection rate in nasopharyngeal secretions by Real-time PCR with IFA .Method (1)Tenfold serial dilutions of recombinate hMPV whose titer was 105.2TCID50/ml were inoculated onto Vero-E6 cells in six-well plate.The CPE was observed in cells inoculated with 105 times dilutions virus appeared at 6 post inoculation.Subsequenfly,experiments were undertaken to assess diagnostic criteria such as specificity,sensitivity and reproducibility of RT-PCR,Real-time PCR and IFA respectively. (2)Tenfold serial dilutions of the recombinate hMPV were amplified by Real-time PCR and RT-PCR respectively. (3) The hMPV IFA and Real-time PCR were then performed to detected hMPV in clinical specimens.Result (1)The hMPV IFA recognized the antigen of 104 times dilution of hMPV , RT-PCR detected the nucleic acid of 105 times dilution of hMPV ,The Real-time PCR detected the nucleic acid of 107 times dilution of hMPV . (2)The analytical detection limit of RT-PCR and Real-time PCR were 103TCID50/ml and 1TCID50/ml respectively .(3) A total of 609 clinical specimens from patients with acute respiratory tract infections were collected.86 of them had no enough cells for IFA. A total of 523 nasopharygeal secretions specimens were screened for the presence of hMPV by IFA and 48 specimens were positive.When 500copies/ul was the reference standard, both of Real-time PCR and IFA were positive for 33 of the 165 Real-time PCR-positive children.Conclusion The RT-PCR was more sensitive than IFA but less than Real-time PCR . The quantitate analytical detection limit of RT-PCR was lower than Real-time PCR. The Real-time PCR was proved to be more sensitive than IFA when they were used to detect clinical specimens . The results taken together indicate that Real-time PCR is an efficient method for detecting hMPV .