Segment reassortment is an essential feature of many viral pathogens, including influenza viruses. More than one type of reassortant virus can be present simultaneously in natural populations, further complicating their analysis. Detailed genome segment mapping of virus isolates is needed to elucidate the mechanisms leading to the emergence and evolution of viral pathogens and to facilitate the creation of new protective vaccines.
The rational design of vaccines against influenza includes the generation of reassortants between a recent field isolate and a reference strain adapted for efficient growth in the substrate used for vaccine production. After a variety of reassortant strains is generated in vitro by growing two parental strains together, a prospective vaccine strain is selected by genotyping all relevant genomic segments to identify the right combination of segments coding for immunogenic proteins and those determining high-growth characteristics. Traditionally, this time-critical task of selection of prospective vaccine strains has been accomplished by RFLP, sequencing of cDNA, or other approaches (1
). The recently developed high-throughput microarray analysis method appears to be ideally suited for one-step mapping of segmented viruses.
Microarray hybridization was previously used to genotype individual segments of influenza A virus (16
). In our work, we have developed and used an improved oligonucleotide microarray method to discriminate viruses with high levels of genomic homology through identification of minor genetic differences, including single-nucleotide variations, simultaneously in all segments of influenza B viruses. We observed unambiguous discrimination between the B/Beijing/184/93 and B/Shangdong/7/97 influenza strains. These strains were discriminated with statistical certainty by using different oligoprobes, including PA-1979, PA-2034, NP-188, and M-1004, differing at only 1 nucleotide and representing the highly homologous segments PA, NP, and M of both viruses. The high level of nucleotide homology between the PA, NP, and M segments of B/Shangdong/7/97 and B/Beijing/184/93 has been an obstacle to the genotyping of reassortants derived from the two strains by RT-PCR and RFLP because of the lack of suitable restriction sites (23
Microarray hybridization, coupled with statistical interpretation of the results, allowed us to estimate the effects of cross-hybridization and to account for nonspecific sample binding (background). Our statistical analysis demonstrated high levels of sensitivity and reproducibility for all individual oligoprobes to all influenza B virus segments; P values for specific hybridization signals for all of the 40 selected oligoprobes were highly significant (P < 0.01).
The statistical planning of the number of hybridization experiments developed in this work can be used in the future in the design of similar oligonucleotide microchip experiments to assess the discriminating power of individual oligoprobes and to select only those that produce the clearest results. Identification of the origin of each segment based on hybridization with two or more specific oligoprobes significantly increased the reliability of identification. To this end, we estimated the maximum probability of false-negative identification of each segment based on multiple results for individual oligoprobes in 56 hybridization experiments. The P values for segments of different origin were calculated by multiplication of P values for homotypic hybridization with oligoprobes specific to each segment. They were statistically significant and demonstrated high overall discriminating power of the microarray for all eight segments of influenza B virus.
Note that our approaches for estimating the minimum number of replicate experiments and for estimating the false-negative error rate for a given gene segment seem novel for microarray genotyping. However, depending on the specific designs of microarray experiments, other statistical approached can also be useful (22a
Interestingly, the influenza B virus microarray can detect the presence in mixed viral stocks of as little as 5% of a second closely related influenza B virus strain (Fig. ). The greater ability to detect mixed viral populations is an important advantage of the method over conventional methods based on nucleotide sequencing, since natural isolates often contain more than one genotype. Although the restriction pattern of DNA shown in Fig. indicates that the NS segments of both the Beijing and Shangdong strains were present, similar analyses of other segments and other reassortant strains prepared as vaccine candidates need to be studied in the future. Our preliminary experiments presented here showed that the method could not be used to quantify the components of a mixture without further refinement. The ability to detect as little as 5% of a second strain in a mixture on one hand and the lack of statistically significant hybridization between the values obtained for 5% and increasing spike concentrations on the other suggest that the dose-response curve may be nonlinear. There may a number of reasons for this, including saturation of immobilized probes during hybridization and nonlinear response of the fluorescence detector. Further experiments are needed to make this method more quantitative and to accurately assess the detection limit for mixtures.
The data presented in this paper suggest that the microarray method can be used for high-throughput screening of influenza B virus reassortants, substantially reducing the time needed for vaccine development. The influenza B virus array described in this paper contains only a few oligoprobes specific to each segment of two influenza B virus strains, Beijing and Shangdong. The microarray method can be expanded by adding new oligoprobes, including those specific to other strains, possibly covering the entire repertoire of currently circulating influenza B virus strains. Such extension of the array might allow us to use a more quantitative assay to determine the origins of individual genomic segments in a series of reassortant strains prepared as vaccine candidates or mixed virus populations.
Our preliminary analysis of the repertoire of vaccine reassortants suggests a highly biased (nonrandom) generation of certain segment combinations. Extensive studies of a large number of reassortant strains may help to reveal the preferred combination of segments and to examine the reason for this nonrandomness of the reassortment process.
It is known that rates of homologous recombination in negative-sense RNA viruses, including influenza virus, are substantially lower than their point mutation rates (24
). However, patterns of sequence variations compatible with the action of recombination were recently observed in several RNA viruses, including influenza A virus and influenza B virus (3
). A search for the evidence of within-segment recombination in influenza viruses could be done by a microarray method similar to the one developed to analyze recombinants in poliovirus (5
The ability to perform rapid and comprehensive genomic analysis of multiple field isolates can help in efforts to recognize and respond to the emergence of new pandemic strains of influenza virus by providing a better understanding of the processes of viral evolution in populations of susceptible hosts. Similar approaches can also be developed for analysis of influenza A viruses and for other viruses with segmented genomes and can substantially advance our understanding of their evolution and help to create new effective vaccines.