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Improving molecular tools for global surveillance of measles virus.

ABSTRACT: The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis.The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels.A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity.A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months.These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures.

SUBMITTER: Bankamp B

PROVIDER: S-EPMC5751696 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Improving molecular tools for global surveillance of measles virus.

Bankamp Bettina B Byrd-Leotis Lauren A LA Lopareva Elena N EN Woo Gibson K S GK Liu Chunyu C Jee Youngmee Y Ahmed Hinda H Lim Wilina W WW Ramamurty Nalini N Mulders Mick N MN Featherstone David D Bellini William J WJ Rota Paul A PA

Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 20130624 1

<h4>Background</h4>The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis.<h4>Objectives</h4>The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a meth ...[more]

PMID: 23806666

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Project description:BACKGROUND: Highly pathogenic avian influenza A(H5N1) viruses are an important health problem in many Asian and African countries. The current increase in human cases demonstrates that influenza A(H5N1) is still a significant global pandemic threat. Many health organizations have recognized the need for new strategies to improve influenza global surveillance. Specifically, the World Health Organization through the global technical consultation for influenza surveillance have called for a detailed picture of the current limitations, especially at the nation level, to evaluate, standardize and strength reporting systems. The main goal of our study is to demonstrate the value of genetic surveillance as part of a strategic surveillance plan. As a proof of concept, we evaluated the current situation of influenza A(H5N1) in Asian and Africa. RESULTS: Our analysis revealed a power-law distribution in the number of sequences of A(H5N1) viruses analyzed and/or reported to influenza surveillance networks. The majority of the Asian and African countries at great risk of A(H5N1) infections have very few (approximately three orders of magnitude) sequenced A(H5N1) viruses (e.g. hemagglutinin genes). This suggests that countries under pandemic alert for avian influenza A(H5N1) have very limited participation (e.g. data generation, genetic analysis and data share) in avian influenza A(H5N1) surveillance. More important, this study demonstrates the usefulness of influenza genetic surveillance to detect emerging pandemic threat viruses. CONCLUSIONS: Our study reveals that some countries suffering from human cases of avian influenza have limited participation (e.g. genetic surveillance or data share) with global surveillance networks. Also, we demonstrate that the implementation of genetic surveillance programs could increase and strengthen worldwide epidemic and pandemic preparedness. We hope that this work promotes new discussions between policy makers and health surveillance organizations to improve current methodologies and regulations.

Project description:Outbreaks of the emerging arbovirus chikungunya virus (CHIKV) affect millions of individuals in Asia, Africa, and Latin America. Vector competence can be changed dramatically by single amino acid exchanges located predominantly within the CHIKV E1 and E2 envelope proteins, which are associated with enhanced transmissibility by anthropophilic Aedes mosquitoes. Commonly used reference assays for molecular surveillance cover only a few adaptive mutations within the envelope domains and have not been validated for all CHIKV genotypes. The recognized landscape of CHIKV adaptive mutations is thus likely incomplete. We designed two nested reverse transcription-PCR (RT-PCR) assays that cover hot spots of viral adaptation to vectors within the E1 and E2 genomic domains. Primers were designed in conserved genomic regions to allow broad usability across CHIKV genotypes. The sensitivity of both assays was at least equivalent to E1- and E2-based reference assays and robust among CHIKV genotypes at 51.4 IU/reaction (E1, 95% confidence interval [CI], 39.8 to 78.9) and 4.0 IU/reaction (E2, 95% CI, 2.0 to 7.4). Upon analysis of the complete known CHIKV genomic diversity, up to 11 nucleotide mismatches with CHIKV variants occurred under oligonucleotide binding sites of reference assays, potentially limiting assay sensitivity, whereas no critical mismatches occurred in the new assays. Specificity testing with nine alphaviruses representing all serocomplexes showed amplification of Mayaro virus and O'nyong-nyong virus by the E1-based assay, but not by the E2-based assay. The high sensitivity and specificity of the new E2-based assay may allow its diagnostic usage in resource-limited settings. The combined new assays allow improved molecular epidemiological surveillance of CHIKV globally.IMPORTANCE The life cycle of arboviruses relies on efficient infection of and transmission by arthropod vectors. Adaptation to new vectors can thus dramatically increase the geographic range of an arbovirus. Several adaptive mutations enhance chikungunya virus (CHIKV) transmissibility by different mosquito species. The appearance of those adaptive mutations has led to large-scale CHIKV outbreaks in Asia, Africa, and Europe. Molecular surveillance of circulating CHIKV strains for adaptive mutations contributes to precise risk assessments and efficient vector control and provides new insight into the evolution of vector adaptation. Existing assays for molecular CHIKV surveillance are limited by poor coverage of known adaptive mutations, low sensitivity, and cost-intensive deep sequencing approaches, preventing universal application. We developed two highly sensitive nested RT-PCR assays that cover hot spots of vector adaptation in CHIKV envelope domains. The new assays allow unprecedented molecular surveillance across all CHIKV genotypes and diagnostic use in resource-limited settings globally.

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Improving molecular tools for global surveillance of measles virus.

Publications

Improving molecular tools for global surveillance of measles virus.

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