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. 2017 Jul 7;12(7):e0180497.
doi: 10.1371/journal.pone.0180497. eCollection 2017.

Genotyping assay for differentiation of wild-type and vaccine viruses in subjects immunized with live attenuated influenza vaccine

Victoria Matyushenko  1 , Irina Isakova-Sivak  1 , Tatiana Smolonogina  1 , Irina Dubrovina  1 , Tatiana Tretiak  1 , Larisa Rudenko  1
Affiliations

Genotyping assay for differentiation of wild-type and vaccine viruses in subjects immunized with live attenuated influenza vaccine

Victoria Matyushenko et al. PLoS One. .

Abstract

Live attenuated influenza vaccines (LAIVs) are considered as safe and effective tool to control influenza in different age groups, especially in young children. An important part of the LAIV safety evaluation is the detection of vaccine virus replication in the nasopharynx of the vaccinees, with special attention to a potential virus transmission to the unvaccinated close contacts. Conducting LAIV clinical trials in some geographical regions with year-round circulation of influenza viruses warrants the development of robust and reliable tools for differentiating vaccine viruses from wild-type influenza viruses in nasal pharyngeal wash (NPW) specimens of vaccinated subjects. Here we report the development of genotyping assay for the detection of wild-type and vaccine-type influenza virus genes in NPW specimens of young children immunized with Russian-backbone seasonal trivalent LAIV using Sanger sequencing from newly designed universal primers. The new primer set allowed amplification and sequencing of short fragments of viral genes in NPW specimens and appeared to be more sensitive than conventional real-time RT-PCR protocols routinely used for the detection and typing/subtyping of influenza virus in humans. Furthermore, the new assay is capable of defining the origin of wild-type influenza virus through BLAST search with the generated sequences of viral genes fragments.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. RT-PCR results with the trivalent LAIV using designed universal primers.
The upper panel: influenza A-specific primers. The lower panel: influenza B-specific primers. L–DNA ladder.
Fig 2
Fig 2. RT-PCR results of PB1 gene fragment of wild-type influenza A viruses of different subtypes using the newly designed universal primers.
The strain names can be found in Table 1. NC, negative control.
Fig 3
Fig 3. RT-PCR results of NPW samples with primers specific for PB1 gene of influenza A viruses.
Numbers represent NPW specimens tested. L–DNA ladder. nc–negative control (no RNA added to the RT-PCR mixture).
Fig 4
Fig 4. Evolutionary relationship between HAs of wild-type H1N1 viruses detected in NPW samples of phase III LAIV trial in Bangladesh.
The tree was generated using Archaeopteryx Phylogenetic Tree Viewer of the Influenza Research Database (fludb.org).
See this image and copyright information in PMC

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