Simultaneous identification of orthopoxviruses and alphaviruses by oligonucleotide macroarray with special emphasis on detection of variola and Venezuelan equine encephalitis viruses.

The researchers have developed a versatile, straightforward method using a macroarray format to identify specific types of viruses that are a concern for biodefense. This method can detect or exclude their presence in environmental samples, making it useful for biodefense applications.

Methodology

• The study revolves around the development of a method to identify alphaviruses and orthopoxviruses, which are of biodefense concern, using a macroarray format. This method uses capture oligonucleotides – short DNA fragments – that are designed to bind to generic members of the orthopox- or alphavirus families.
• Apart from the generic DNA fragments, the researchers also used a collection of additional oligonucleotides specifically designed to bind with the nucleic acids of five individual alphaviruses, including the Venezuelan equine encephalitis virus, and DNA from each of four orthopoxviruses, including the variola virus.
• These oligonucleotides were deposited onto nylon membranes. The technique involves hybridization – the process of joining two complementary strands of DNA – of digoxigenin-labeled PCR products to the macroarray, creating results that can be observed with the naked eye.

Results

• Using the developed method, researchers noticed that multiplex RT-PCR (a method used to amplify specific regions of a DNA strand), using both orthopox- and alphavirus-generic primers, led to the amplification of DNA of the expected sizes of the orthopoxvirus and alphavirus fragments.
• The samples, when hybridized to capture oligonucleotides in the macroarray membranes, correctly identified generic sequences of both orthopoxviruses and alphaviruses.
• The hybridizations were also able to correctly identify each of the three alphaviruses and two orthopoxviruses tested in the study. Cross-hybridization was observed only once among two alphaviruses but was less intense than the spots created by correct hybridization.

Conclusion

• The macroarray test developed in this study is easy to execute, budget-friendly, relatively fast, and simple to interpret. The results can easily be seen and interpreted without the need for extra lab equipment. This characteristic can be of acute importance in resource-limited settings.
• This nucleic acid hybridization assay, performed on nylon membranes in a macroarray format, holds promise for detecting or excluding the presence of threat viruses in environmental samples. This could have significant implications for biodefense applications, such as early warning systems for potential viral outbreaks or as part of routine surveillance.