Mapping of genomic segments of influenza B virus strains by an oligonucleotide microarray method

Abstract

Similar to other segmented RNA viruses, influenza viruses can exchange genome segments and form a wide variety of reassortant strains upon coreplication within a host cell. Therefore, the mapping of genome segments of influenza viruses is essential for understanding their phenotypes. In this work, we have developed an oligonucleotide microarray hybridization method for simultaneous genotyping of all genomic segments of two highly homologous strains of influenza B virus. A few strain-specific oligonucleotide probes matching each of the eight segments of the viral genomes of the B/Beijing/184/93 and B/Shangdong/7/97 strains were hybridized with PCR-amplified fluorescently labeled single-stranded DNA. Even though there were a few mismatches among the genomes of the studied virus strains, microarray hybridization showed highly significant and reproducible discrimination ability and allowed us to determine the origins of individual genomic segments in a series of reassortant strains prepared as vaccine candidates. Additionally, we were able to detect the presence of at least 5% of mixed genotypes in virus stocks even when conventional sequencing methods failed, for example, for the NS segment. Thus, the proposed microarray method can be used for (i) rapid and reliable genome mapping of highly homologous influenza B viruses and (ii) extensive monitoring of influenza B virus reassortants and the mixed genotypes. The array can be expanded by adding new oligoprobes and using more quantitative assays to determine the origin of individual genomic segments in series of reassortant strains prepared as vaccine candidates or in mixed virus populations.

FIG. 1.

Influenza B virus array design. The array consists of four identical subarrays. A solid circle indicates a spot containing printed oligoprobe; an open circle indicates a control spot containing no printed oligoprobe to assess background sample binding. Each subarray contained 40 oligoprobes (Table 1). The left part of each subarray contained 20 oligoprobes complementary to the Beijing strain; the right part contained 20 oligoprobes complementary to the Shangdong strain. The pairs of homologous oligoprobes of the Beijing and Shangdong strains were printed symmetrically across the vertical axis of a subarray. Each subarray consisted of four areas. Each area contained oligoprobes complementary to one of the four fluorescently labeled cDNA samples: B1, B2, S1, or S2 (see “Multiplex PCR and synthesis of Cy-5-labeled hybridization samples”). To assist in the visual presentation, the areas of oligoprobes complementary to the B1 and S1 samples are separated from the B2 and S2 samples.

FIG. 2.

Typical hybridization for reference virus strains B/Beijing/184/93 and B/Shangdong/7/97 on a subarray. For purposes of visual presentation, hybridization patterns obtained for multiplex PCR products containing PB2, PB1, PA, and HA segments and NP, NA, M, and NS segments were combined in one image. (A) Hybridization spots for labeled single-stranded DNA samples of Beijing virus homotypically hybridized with 20 Beijing-complementary oligoprobes and heterotypically hybridized with 20 Shangdong-complementary oligoprobes (control). (B) Hybridization spots for labeled single-stranded DNA samples of Shangdong virus heterotypically hybridized with 20 Beijing-complementary oligoprobes (control) and homotypically hybridized with 20 Shangdong-complementary oligoprobes. The hybridization spots in the rows of a subarray represent hybridizations of a Cy-5 fluorescently labeled single-stranded DNA sample with different oligoprobes printed in the row. For purposes of visual presentation and inspection, the vertical line going through the subarray separates three columns containing the Beijing-complementary oligoprobes (on the left side of the subarray) and the three columns containing the Shangdong-complementary oligoprobes (on the right side of the subarray). Each row on a subarray represents oligoprobes of one genome segment of influenza virus. The oligoprobes were printed in a mirror symmetry manner relative to this line (see details in Fig. 1). The identifiers of genome segments are showed on the left in panels A and B. The PB2, PA, NP, and NS genome segments of the Beijing and Shangdong strains are represented by three different oligoprobes and by three observed hybridization spots. The PB1, NA, HA, and M genome segments of these strains are represented by two different oligoprobes and by the two hybridization spots. In the last case, two “empty” spots on the rows of the subarray are observed.

FIG. 3.

Discrimination of hybridization patterns of four different reassortants on a subarray. See details in the text. Microarray elements specific to the prevailing RNA segment are boxed.

FIG. 4.

RFLP of NS segments digested by HgaI. Lane 2, reassortant (the size of the band undigested by HgaI is 1,030 bp, and the sizes of the digested bands are 730 and 300 bp); lane 3, Beijing (the size of the band undigested by HgaI is 1,030 bp); lane 4, Shangdong (the sizes of the bands digested by HgaI are 730 and 300 bp); lanes 1 and 5, DNA ladders.

FIG. 5.

Hybridization of mixed samples with NS segment oligoprobes. The initial Shangdong sample (175 mM) was diluted to final concentrations of 10 and 5% in a Beijing sample (175 mM) and then hybridized with Shangdong-specific oligonucleotides. The heterotypic hybridization signal of the Beijing sample to Shangdong-specific oligoprobes was set as the control. The hybridization was repeated five times. The error bars represent standard deviations.