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Viral Pathogen Research Cheat Sheet (DRAFT) by

This is a draft cheat sheet. It is a work in progress and is not finished yet.

One Minute Pitch

RNAscope ISH detects viral RNA, provides context, is highly sensitive and strand­-sp­ecific; therefore delivers where the actual virus is and what viral state it is in (resting, replic­ating). Assay can be applied to any sequenced virus, already applied to >100 different viruses.

Pain Points & Our Solutions

Viral Location
Context provides viral locali­zation which answers where virus entered, replicate, cause disease, shed
No Antibody
Any Gene of any sequenced virus, or for any gene in a virus.
Antibody Surrogate
Direct measur­ement of the actual virus not a protein that is produced as a host response
Low viremia
High sensit­ivity to detect single RNA molecules, proven for early infections and HIV/SIV reservoirs
Emerging Viruses
Rapid design and assay enable resear­chers to respond to emerging viral invest­iga­tions
Viral State
Strand­-sp­ecific probes discern viral stages; hybridize to sense (+) or anti-sense (-) strand to detect replic­ation, resting stages
Viral Causality
Locali­zation & viral state are needed to determine whether virus is just present or actually causing the disease
Uncommon species
Any species with a sequence. We have probes for viruses that infects raccoon, seal, fox
RNA/DNA
RNAscope and/or DNAscope to visualize viral states.

Solution for DNA & Retrov­iru­ses­--D­NAscope ISH

DNAscope ISH is a customer proven protocol. It can be used to detect viral DNA using a non-st­andard, modified protocol. It can be combined with RNA ISH so you see both viral RNA & DNA
Defining HIV and SIV Reservoirs in Lymphoid Tissues.
Non-st­andard, can be cumber­some, recommend with caution

Key Tools / References

URLs https:­//a­cdb­io.c­om­/zika https:­//a­cdb­io.c­om/HIV
https:­//a­cdb­io.c­om­/in­fec­tio­usd­iseases
DNAscope Inform­ation
2 Spotlight Interviews Pesavento & Getu and Smits (Showpad)
Presen­tation Viral Pathogen Compre­hensive PPT (Showpad)
Data Loads of public­ations & data images in PPT mentioned above
Who McCune Lab at UCSF for HIV, US CDC for Zika,

Research Goals

Viral pathog­enesis how biological viruses cause diseases in their target hosts, Pathog­enesis is a process in which an initial infection becomes a disease
Viral immunity correlates of immunity to infection and the develo­pment of novel approaches to the diagnosis, prevention and treatment
Viral emergence mechanisms that underpin host switching, examine same virus in multiple host species
Viral spread examine viral spread between livestock and wildlife. Examine resevoirs of infection and routes of transm­ission

Virus and Hosts

What is the viral type & viral group? Are there closely related species?
What is the host species and is there more than one? Is the parthe­nog­enesis specific to the host?
  

Qualifying Questions

Is your goal to locate the virus, find what state it is in or both?
RNAscope ISH can provide location and viral state info
Which specific viral replic­ation state do you want to detect? Attach­men­t/P­ene­tra­tion/ Uncoat­ing­/Re­pli­cat­ion­/As­sem­bly­/Re­lease?
For that specific viral state is it + /- strand­ed-­-probes designed comple­mentary to that strand.
Is the antibody available for viral protein or is it a surrogate marker?
RNAscope hybridize to the actual viral RNA and is strand specific, so it measures the actual virus in viral state.
Is low viremia a concer­n--­early infection or viral reserv­oirs?
Single molecule detection is proven with RNAscope. Several RNAscope HIV/SIV resevoir papers available.
Do you need to link a viral stage to a disease? Do you want to detect a viral RNA and a disease marker?
Multip­lexing options available, so one probe can be design for the virus and another for the disease marker
Is host switching a concern?
If the virus is the same in both hosts, then we can use one probe. But if the virus evolves from host to host, our probes are specific and be designed to the sequence

Backgr­ound-- Compatible with all 7 Viral Groups

Public­ations using RNAscope ISH can be found for 6/7 viral groups

Backgr­oun­d--­Con­cepts & Vocabulary

Tropism
viral specif­icity for host tissue­/cell (viral surface structures & host surface receptors)
Dynamics
speed of viral progre­ssion within host
Load/ Burden
quantity of virus in a given volume
Latency
ability of a path­oge­nic­ vi­rus to lie do­rmant within a cell
Reservoir
cell type or anatomical site where (resti­ng/­latent) virus can hide, stay stable for future replic­ation. May also be used to describe species that harbour the virus without causing disease.
Virions
virus while not inside an infected cell or in the process of infecting a cell
3 viral types
DNA, RNA (most common) & Retrovirus
7 viral groups
see chart
Sense (+) anti-sense virus
Sense virus hybridize with anti-sense probes, anti-sense virus hybridize with sense probes
Ambi-sense strand virus
single stranded genome with both +- strands requiring both sense & anti-sense probes
Host switching
species jumping, cross species transm­iss­ion--a virus infects a new host species and may have changed at molecular level...new strain (SARS, Ebola, HIV)