Microarray assay for detection and discrimination of Orthopoxvirus species

 

Dr. Alexander N. Sinyakov, Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, State Research Center of Virology and Biotechnology "Vector,"

A microarray method was developed for simultaneous detection and identification of six species of Orthopoxvirus (OPV) including Variola, Monkeypox, Cowpox, Camelpox, Vaccinia, and Ectromelia viruses. The method allowed us to discriminate OPV species from varicella-zoster virus (VZV), Herpes Simplex 1 virus (HSV-1), and Herpes Simplex 2 virus (HSV-2) that cause infections with clinical manifestations similar to OPV infections. The nucleotide sequences of the C23L/B29R and the B19R genes identified for 86 and 72 different OPV strains, respectively, were used to design species-specific microarray oligonucleotide probes (oligoprobes). The microarray also contained several oligoprobes selected from the ORF31, US4, and US5 genes of VZV, HSV-1 and HSV-2, respectively.

 

The results obtained in this study demonstrated that this new microarray method is a valuable tool for the rapid and accurate detection and differentiation of these important viral pathogens.

 

Introduction

The genus Orthopoxvirus contains several species of viruses causing infections in different vertebrate species. Among these, Variola virus (VARV), Monkeypox virus (MPXV), Cowpox virus (CPXV), and Vaccinia virus (VACV) can potentially infect humans and cause illnesses of varying severity. VARV is a strictly anthroponotic agent associated with devastating endemic infections, known as smallpox. The two forms of smallpox, variola major and variola minor, cause 30% and 1% of case-fatality rates, respectively.   Vaccines for smallpox have been available for more than 200 years. Smallpox vaccination program, coordinated by World Health Organization (WHO) included active immunization campaigns in all endemic and neighboring countries. These efforts resulted in significant reduction of spontaneous smallpox cases during the period from 1966 to 1976; the last detected case of smallpox was reported in Somalia in 1977. Three years later, in 1980 the WHO declared that the eradication of smallpox infection was complete, and mass vaccination against smallpox was discontinued in many countries. As a result, the vast majority of human population does not have protective immunity against poxvirus infection. Current susceptibility of human population to smallpox infection raises a serious concern about the possibility of potential use of Variola virus as a weapon of bioterrorism. Thus, development of rapid and sensitive methods for detection and discrimination of Orthopoxviruses from other infections producing similar symptoms (VZV, etc.) is an important public health and biodefense objective.

Traditional methods used for Orthopoxvirus detection are based on electron microscopy, characteristic growth of Poxviruses on chorioallantoic membrane of chick embryos and serological methods. These methods produce reliable results but are very time consuming.

Microarrays consisting of multiple individual short oligoprobes are likely to be the most effective tool for accurate detection and analysis of human pathogens. The main advantage of microarray technology is the ability to simultaneously analyze several genetic markers using multiple oligoprobes for each marker. This redundancy ensures significant improvement of reliability of pathogen identification, despite their variability and rapid changes.

 

 

 

Results

PCR primers for individual or multiplex PCR amplification of the two OPV marker genes C23L/B29R and B19R, were designed on the basis of conserved sequences found within these genes. Using different OPV DNA samples available from the collection of the State Research Center of Virology and Biotechnology ‘‘Vector’’ (Kol’tsovo, Russia), we demonstrated that designed primers efficiently amplified any OPV species from the collection. We also designed separate sets of PCR primers that successfully amplified target regions within the ORF31, US4, and US5 genes used for specific detection of VZV, HSV-1 and HSV-2, respectively.

To develop a microarray-based assay for detection and identification of orthopoxviruses, we used two previously characterized OPV genes, C23L/B29R and B19R, conserved among all orthopoxviruses. The C23L/B29R gene encodes the viral CC-chemokine binding protein, while B19R gene encodes the a/b-IFN binding protein.  Herpesviruses VZV, HSV-1, and HSV-2 were also included in this study due to the ability of these viruses to cause the diseases with clinical symptoms similar to smallpox. The conserved regions within the ORF31, US4 and US5 genes were used to design PCR primers and microarray probes for identifying VZV, HSV-1, and HSV-2 viruses, respectively. The design of PCR primers as well as   OPV species and strain-specific microarray oligoprobes was done by analysis of the C23L/B29R gene sequences available for 86 OPV strains and the B19R gene sequences for 72 OPV strains using OligoScan computer software. All these OPV strains were isolated at different time periods and in different geographic areas of the world. Oligoprobes were selected based on the sequences in these two genes that were conserved for all virus strains of each of six OPV species including VARV, MPXV, CPXV, VACV, Camelpox (CMLV), and Ectromelia (ECTV) viruses. 

Using the nucleotide differences in the C23L/B29R gene of OPV, we were able to design oligoprobes for accurate differentiation of four OPV species, VARV, MPXV, ECTV and CMLV.  However, high intra-species divergence observed for CPXVs and VACV did not allow to find a suitable oligoprobes for theirs specific detection using the microarray method.  To increase the specificity of microarray for detection of CPXV strains, we included  more oligoprobes

The use of the B19R gene allowed us to design oligoprobes for specific detection of six OPV species i.e. VARV, MPXV, ECTV, CMLV, VACV, and CPXV.  However, high genetic heterogeneity of the cowpox viruses was a serious hindrance for universal CPXV microarray oligoprobes based on B19R gene. To embrace all genetic variations in the B19R gene, we divided all CPXV species into three subgroups A, B, and C.

 

Evaluation of OPV microarray

The oligoprobes designed for detection and discrimination of OPV species and for their differentiation from herpesviruses were immobilized on silylated glass slides. Fluorescently labeled DNAs samples for microarray hybridization were prepared from 52 OPV strains representing six OPV species, two VZV, two HSV-1 and from three HSV-2 isolates, and were used for evaluation of the assay specificity. The results of this study showed that the oligonucleotide microarray could unambiguously detect and discriminate all tested OPV species. The OPV identification was based on hybridization signals from OPV genetic markers, C23L/B29R and B19R. For microarray evaluation purposes, we used separate PCR to prepare gene-specific amplicons. We found that oligoprobes that contained two and more mismatches with heterologous DNA samples demonstrated a significantly lower cross-hybridization in comparison with oligoprobes with only one mismatch.

Discrimination ability of the microarray was estimated as a mean square of normalized fluorescent signal intensities (Y^m) using the formula:

Y^m = ()/N,

Where m is a group of spots discriminating specific OPV species (or HSVs), - normalized signal from each spot belonging to m, and N is the number of specific oligoprobes for detection the specific OPV species. The signal quantification by using the mean square of normalized fluorescent signals (Y^m) was found to be useful for accurate and reliable OPV speciation. All herpes virus-specific oligoprobes showed high specificity and revealed no interspecies cross-hybridization.

Detection of OPV species by using concurrent microarray analysis of the B19R and C23L/B29R genes

To increase the reliability of microarray-based assay for identification of OPV species, we concurrently analyzed both OPV genetic markers in one experiment. DNA samples of OPVs were subjected to multiplex PCR in the presence of primers for simultaneous amplification of the C23L/B29R and B19R genes. As a model, we used two OPV strains, M/LIB and S/SOK that belonged to MPXV and VARV OPV species, respectively.  ssDNAs prepared by asymmetric PCR in the presence of primers for amplification of both genes was fluorescently labeled by chemical DNA staining. The results of this study demonstrated that microarray hybridization unambiguously and specifically detected and identified M/LIB strain as MPXV and S/SOK strain as VARV.

 

Identification of previously uncharacterized variola strains

To test the ability of the new microarray to identify a diverse spectrum of VARV strains, we analyzed three unrelated and previously genetically uncharacterized VARV strains; Ind 4A (Isolated in India, 1967 year), M-Gavr-60 (Isolated in Russia, 1960 year), and 13/62 (Isolated in Tanzania, 1962 year). The sequences of the B19R and C23/B29R genes of these strains were not previously determined. Single and multiplex PCR were used for the microarray analysis of these strains.  These particular VARV strains were correctly identified on the basis of the hybridization profile specific to species Variola virus.

Incorporation of herpesvirus-specific oligoprobes into the same microchip allowed us to quickly discriminate chickenpox virus (VZV) from Orthopoxviruses, and also to differentiate VZV from human Herpes viruses 1 and 2.

Our results demonstrate the exceptional power of microarrays to rapidly detect and identify single or multiple viral species and strains of potential bioterrorism agents on a small glass slide. Inclusion of oligoprobes from multiple genes increases robustness of the method and expands the repertoire of strains that can be reliably discriminated. It also opens the possibility to identify recombinant strains that carry genes originating from different viruses.