Rapid and highly fieldable viral diagnostic
The present invention relates to a rapid, highly fieldable, nearly reagentless diagnostic to identify active RNA viral replication in a live, infected cells, and more particularly in leukocytes and tissue samples (including biopsies and nasal swabs) using an array of a plurality of vertically-aligned nanostructures that impale the cells and introduce a DNA reporter construct that is expressed and amplified in the presence of active viral replication.
1. A method for rapid diagnosis of infection by a RNA virus in a sample which comprises
(a) adsorbing either
(i) a sample of blood onto a porous filter to entrap leukocytes but permit gravimetric flow through of platelets and red blood cells, thereby obtaining a sample of leukocytes,
(ii) a tissue sample onto a filter or other surface,
(iii) a biological fluid sample onto a filter or other surface, or
(iv) a sample of leukocytes onto a filter or other surface;
(b) impaling cells in said sample on the filter or surface onto an array of a plurality of vertically aligned nanostructures carrying at least one DNA reporter construct thereon, wherein the at least one DNA reporter construct encodes an mRNA viral replicon, wherein said viral replicon comprises cis-acting RNA viral replication elements and any other adjacent regions necessary for viral replication of said RNA virus flanking and operably linked to a reporter gene or operon, wherein said viral replicon lacks a nucleic acid encoding at least one factor required for viral replication and provided in trans by cells infected by said RNA virus, and wherein the viral replicon is amplified in cells infected by said RNA virus but not in cells not infected by said virus; and
(c) detecting expression of said reporter gene or operon, wherein the expression of said reporter gene is indicative of a RNA virus infection.
2. The method of claim 1 , wherein expression of said reporter gene or operon occurs by virally-mediated replication and amplification of RNA encoding said reporter gene or operon.
3. The method of claim 1 , wherein detecting expression is by detecting amplification of a signal from said reporter gene or operon when virally-infected cells are present.
4. The method of claim 3 , wherein a non-amplifiable reporter DNA construct with a distinct spectral response is co-delivered with said at least one DNA reporter construct to allow ratiometric comparison of amplified and unamplified signals to account for cell- or tissue-specific expression dynamics and variation in stoichiometries of DNA delivered to each cell.
5. The method of claim 4 , wherein said ratiometric comparison is conducted temporally.
6. The method of claim 1 , wherein said sample is washed with buffer prior to step (b).
7. The method of claim 1 , wherein said blood sample is from about 10 to about 100 microliters.
8. The method of claim 1 wherein said porous filter is Leukosorb.
9. The method of claim 1 , wherein said sample of leuokocytes is prepared by a conventional leukocyte enrichment protocol.
10. The method of claim 1 , wherein said array of a plurality of vertically aligned nanostructures comprise nanostructures selected from the group consisting of nanospikes, nanotubes, micro-machined needles and microneedles.
11. The method of claim 10 , wherein said array is spatially indexed.
12. The method of claim 1 , wherein said cis-acting RNA viral replication elements are selected from a human or animal pathogenic RNA virus.
13. The method of claim 12 , wherein said human pathogenic RNA virus is a Hanta, Dengue, Ebola, Marburg, Lassa, Hepatitis A, Hepatitis C, Yellow Fever or West Nile virus or other human encephalitis virus.
14. The method of claim 12 , wherein said animal pathogenic RNA virus is foot and mouth disease virus, Rift Valley fever virus, common swine fever virus, or Japanese encephalitis virus.
15. The method of claim 1 , wherein said cis-acting replication elements operably linked to a reporter gene or operon comprise 5′ and 3′ RNA virus untranslated regions.
16. The method of claim 1 , wherein said reporter gene or operon encodes a fluorescent reporter protein or protein complex, or a bioluminescent reporter protein or protein complex.
17. The method of claim 16 , wherein said fluorescent reporter protein is selected from the group consisting of GFP, YFP, BFP or RFP.
18. The method of claim 16 , wherein said bioluminescent reporter protein complex is LuxCDABE.
19. The method of claim 1 , wherein expression is detected by fluorescence or luminescence.
20. The method of claim 19 , wherein fluorescence is detected with a battery-powered unit providing laser diode excitation and channel-photomultiplier detection.
21. The method of claim 19 , wherein fluorescence is detected by microscopy.
22. The method of claim 1 , wherein the at least one viral replication factor is a viral replicase specific to said RNA virus.
23. The method of claim 22 , wherein the replicase is an NS5 replicase.
24. The method of claim 22 , wherein the virus is a Dengue virus.