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Development of real-time RT-PCR for detection of human metapneumovirus and genetic analysis of circulating strains (2009-2011) in Pune, India.

ABSTRACT: Human metapneumovirus (HMPV) is an important respiratory virus implicated in respiratory infections. The purpose of this study was to develop a one-step real-time RT-PCR assay that can detect all four lineages of HMPV and to identify the HMPV lineages circulating in Pune, India. Conserved regions of the nucleoprotein gene were used to design real-time primers and a probe. A total of 224 clinical samples that were positive for different respiratory viruses (including 51 samples that were positive for HMPV) were tested using the real time RT-PCR assay, and the specificity of the assay was observed to be 100 %. Using in vitro-synthesized RNA, the sensitivity of the assay was ascertained to be 100 copies of the target gene per reaction. Phylogenetic analysis of the nucleoprotein (N) and attachment glycoprotein (G) genes confirmed that this assay detected all lineages of HMPV. A2, B1 and B2 strains were observed during the study period. Our assay is highly sensitive and specific for all known lineages of HMPV, making it a valuable tool for rapid detection of the virus. A2 and B2 were the predominant subtypes circulating in Pune, Western India.

SUBMITTER: Choudhary ML

PROVIDER: S-EPMC7087245 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Development of real-time RT-PCR for detection of human metapneumovirus and genetic analysis of circulating strains (2009-2011) in Pune, India.

Choudhary Manohar Lal ML Anand Siddharth P SP Sonawane Nupoor S NS Chadha Mandeep S MS

Archives of virology 20130809 2

Human metapneumovirus (HMPV) is an important respiratory virus implicated in respiratory infections. The purpose of this study was to develop a one-step real-time RT-PCR assay that can detect all four lineages of HMPV and to identify the HMPV lineages circulating in Pune, India. Conserved regions of the nucleoprotein gene were used to design real-time primers and a probe. A total of 224 clinical samples that were positive for different respiratory viruses (including 51 samples that were positive ...[more]

PMID: 23929232

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Project description:Prior experience and the persisting threat of influenza pandemic indicate the need for global and local preparedness and public health response capacity. The pandemic of 2009 highlighted the importance of such planning and the value of prior efforts at all levels. Our review of the public health response to this pandemic in Pune, India, considers the challenges of integrating global and national strategies in local programmes and lessons learned for influenza pandemic preparedness.Global, national and local pandemic preparedness and response plans have been reviewed. In-depth interviews were undertaken with district health policy-makers and administrators who coordinated the pandemic response in Pune.In the absence of a comprehensive district-level pandemic preparedness plan, the response had to be improvised. Media reporting of the influenza pandemic and inaccurate information that was reported at times contributed to anxiety in the general public and to widespread fear and panic. Additional challenges included inadequate public health services and reluctance of private healthcare providers to treat people with flu-like symptoms. Policy-makers developed a response strategy that they referred to as the Pune plan, which relied on powers sanctioned by the Epidemic Act of 1897 and resources made available by the union health ministry, state health department and a government diagnostic laboratory in Pune.The World Health Organization's (WHO's) global strategy for pandemic control focuses on national planning, but state-level and local experience in a large nation like India shows how national planning may be adapted and implemented. The priority of local experience and requirements does not negate the need for higher level planning. It does, however, indicate the importance of local adaptability as an essential feature of the planning process. Experience and the implicit Pune plan that emerged are relevant for pandemic preparedness and other public health emergencies.

Project description:BackgroundBoth respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are important viral pathogens causing respiratory tract infection (RTI) in the pediatric population. However, the clinical manifestations of RSV and hMPV infections are similar. Therefore, a reliable and rapid diagnostic tool is needed for diagnostic performance.MethodsIn order to optimize diagnosis efficiency of RTI, the aim of this study is to establish a rapid and advanced method for simultaneous detecting RSV and hMPV in nasopharyngeal aspirates specimens from patients. We designed a one-step triplex real-time RT-PCR (qRT-PCR) protocol using TaqMan probes for detecting RSV and hMPV. The plasmid clones containing RSV nucleoprotein gene and hMPV fusion gene were established as reference standards. We used virus culture supernatants from 86 known pediatric RTI patient to test the specificity and sensitivity of our assay. Then we used total 222 nasopharyngeal aspirates specimens from pediatric patients hospitalized with respiratory symptoms to evaluate our assay.ResultsOur one-step triplex qRT-PCR assay showed 100% sensitivity and specificity in testing RSV and hMPV in 86 known virus culture supernatants, with excellent linearity (R2 > 0.99) and reliable reproducibility (CV lower than 1.04%). This assay has a wide dynamic range 102-109copies/reaction (limit of detection; LOD = 100 copies/reaction). A total of 222 patients hospitalized with respiratory symptoms were enrolled for clinical evaluation. In these samples, our qRT-PCR assay detected 68 RSV positive and 18 hMPV positive cases. However, standard virus culture only detected 8 RSV positive cases and 0 hMPV cases. Based on this improved triplex qRT-PCR assay, we found that RSV infection was associated with severe inflammation by chest X-ray and occurrence of pneumonia which were not observed previously.ConclusionsIn summary, we have developed a highly specific and sensitive one-step triplex qRT-PCR assay to detect hMPV and RSV simultaneously. This assay offers a valuable tool for routine diagnosis.

Dataset Information

Development of real-time RT-PCR for detection of human metapneumovirus and genetic analysis of circulating strains (2009-2011) in Pune, India.

Publications

Development of real-time RT-PCR for detection of human metapneumovirus and genetic analysis of circulating strains (2009-2011) in Pune, India.

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