Microarray analysis of evolution of RNA viruses: evidence of circulation of virulent highly divergent vaccine-derived polioviruses

Abstract

Two approaches based on hybridization of viral probes with oligonucleotide microarrays were developed for rapid analysis of genetic variations during microevolution of RNA viruses. Microarray analysis of viral recombination and microarray for resequencing and heterogeneity analysis were able to generate instant genetic maps of vaccine-derived polioviruses (VDPVs) and reveal the degree of their evolutionary divergence. Unlike conventional methods based on cDNA sequencing and restriction fragment length polymorphism, the microarray approaches are better suited for analysis of heterogeneous populations and mixtures of different strains. The microarray hybridization profile is very sensitive to the cumulative presence of small quantities of different mutations, including those that cannot be revealed by sequencing, making this approach useful for characterization of profiles of nucleotide sequence diversity in viral populations. By using these methods, we identified a type-3 VDPV isolated from a healthy person and missed by conventional methods of screening. The mutational profile of the polio strain was consistent with >1 yr of circulation in human population and was highly virulent in transgenic mice, confirming the ability of VDPV to persist in communities despite high levels of immunity. The proposed methods for fine genotyping of heterogeneous viral populations can also have utility for a variety of other applications in studies of genetic changes in viruses, bacteria, and genes of higher organisms.

Fig. 1.

MARSH analysis of strain 11264. (A) Hybridization patterns of reference Sabin 3 (Upper) and test 11264 (Lower) fluorescent RNA samples with MARSH arrays. In each case, only one quadruplicate array is shown. (B) The ratio of signals from two MARSH chips hybridized with Sabin 3 reference is shown with open circles. The ratio of signals obtained with the Sabin 3 reference and strain 11264 is shown by filled circles. x axis, the nucleotide coordinates on poliovirus genome; y axis, ratio values. Diamonds show the location of mutations determined by sequencing.

Fig. 2.

MAVR analysis of OPV and VDPV strains.

Fig. 3.

Mutational profiles of the VP1 coding region of Sabin 1 viral stocks obtained at different passage levels. (A) The ratio of signals obtained from hybridization of MARSH with reference sample (first passage of Sabin 1) and other passages: passage 3, yellow; passage 4, green; passage 6, blue; passage 9, red. x and y axes, see legend to Fig. 1. (B) The raw sequencing data of VP1 genomic regions (nucleotides 2767–2777 and 2970–2980) of Sabin 1 passages 3, 4, 6, and 9. Nucleotides 2772 and 2975 are underlined.