Hop Latent Viroid

A 256 base pair piece of RNA is a Billion dollar hit to the Cannabis Industry.

The COVID pandemic has a lot of people obsessed over viruses. Let’s take a tour through one of their evolutionary ancestors (viroids) that runs roughshod over plants that produce our flavor in beer and weed.

Viroids are viruses that don’t have protein coats nor do they require translation of proteins to replicate. They are ‘living’ fossils that predate viruses and usually exhibit Ribozyme activity (catalytic RNA). They are not known to infect humans but they sure do a number on plants.

Some rough estimates imply $4B in annual damages to the Cannabis industry from Hop Latent Viroid. This viroid was first detected in Cannabis sativa’s nearest cousin, Hop or Humulus lupulus. As prohibition was lifted, molecular tools began finding their way into the cannabis industry and next generation sequencing confirmed the existence of this viroid in Cannabis. This is a 256 base pair piece of semi-circular RNA that has no protein coat and does not code for any proteins. Compare this to SARs-CoV-2 which is ~29,900 base pair linear RNA that codes for over 28 proteins including spike, envelope and nucleocapsid proteins.

How does it do this?

The viroid has figured out how to high jack Rolling Circle Amplification (RCA) and RNA ligase in the host plant. When RNA or DNA molecules are circular, they can be replicated like a wheel that becomes and infinite template. This creates long concatemers of the viroid genome. These concatemers are then sliced and diced into 256 base segments with the Ribozyme activity of the original double stranded viroid. Once the genome is diced, plant RNA ligases seal it back into many circles and the process can repeat itself.

What does it look like in Cannabis?

This month, Medicinal Genomics pal, Collin Palmer became concerned over some of his plants that he has cloned successfully for over 20 years. A new addition to his library began showing bizarre phenotypes. The most symptomatic plant was just introduced from a nursery. Cannabis nurseries handle diverse cannabis genetics and clone them for their customers. Cloning is one way way to cheat Mendel. Cannabis can have a SNP every 50 bases so sexually derived seeds are a chemotypic lottery machine and cloning helps to produce consistent THC or CBD levels. The industry is highly regulated on chemotype so cloning is more popular for indoor THC positive plants.

As a result, the nurseries play a critical but challenging role as a nexus of many different plants. Just like Hospitals and Nursing homes with SARs-CoV-2, they can be sources of nosocomial infections and super spreaders if sterile technique ever slips.

We initially tested Collin’s imported symptomatic plant against 10 different viruses/viroids using RT-qPCR as it had the classic look of a Mosaic virus (looks are always deceiving). This is achieved with multiplex qPCR where 3 viruses are detected in a single reaction with the addition of a 4th assay targeting the cannabis genome so viral/viroid loads can be properly counted.

Viruses initially tested:

1. Hop Latent Viroid (HLVd)

2. Lettuce Chlorosis Virus (LCV)

3. Cannabis Cryptic Virus (CCV)

4. Hop Mosaic Virus

5. Cucumber Mosaic Virus

6. Arabis Mosaic Virus

7. Tobacco Streak Virus

8. Tobacco Mosaic Virus

9. Tobacco RingSpot Virus

10. Beet Curly Top Virus

This Cannabis Internal Control (Green line) is critical as the extraction of the Virus/Viroid from the plant tissue can vary sample to sample and without adjusting for the yield of cannabis DNA recovered, you can’t compare samples to understand viroid load. In the case above, the viroid load is 13+ CT’s (Red, Blue and Purple) earlier than the cannabis genomic DNA. This is 10,000 fold more viroid/virus RNA than the Cannabis genomic DNA.

This is something the COVID Testing space got horribly wrong. In many jurisdictions they were using tests that didn’t have this internal control while speaking about viral loads (some assays did have them in the form of RNaseP assays). This is something known in the field as Delta Delta qPCR. It requires 2 CT values to calculate viral loads (The host and the pathogen) and the COVID testing space was often just running off of one (the pathogen). So when you hear people discussing the viral load in C19, they are often referring to a singular viral CT score which technically is a numerator with no denominator.

Back to the plant…

Only the HLVd qPCR came back positive and it wasn’t constrained to the symptomatic plant. 7 other (7/12) asymptomatic plants were also infected.

In retrospect one could find leaves on the asymptomatic plants that had signs of inter-veinal chlorosis (yellowing between the veins of the leaf) but this is a non-unique symptom that is found in many Calcium or Magnesium deficient plants. Brittle petioles were noticed retrospectively.

Why the variable phenotype or pleiotropy with HLVd infections? What is the mechanism of disease? How can such a small viroid reek so much havoc to plant yield? Why is it asymptomatic in so many plants only to emerge later to cause problems? Does it always exit latency and is the yield loss a guarantee?

These are all questions that have parallels to SARs-CoV-2.

The precise pathology of Viroids is still in debate. There are a few papers suggesting these viroids may induce RNA silencing in plants and other that suggest they activate specific kinases that may interfere with other metabolic processes in the plant. My personal theory is grounded in the qPCR data we continue to see. In most infected plants the CT for the virus is 500-1000 times higher than the cannabis genomic DNA. Rolling circle amplification of RNA consumes truckloads of ATP as ATP is literally the source of the Adenosine written into the RNA code. As a result, symptoms may emerge when the plant is metabolically challenged…like during flowering. These hypotheses are not mutually exclusive. The RNA interference hypothesis is worth investigating as well. There is a lot more literature support for the RNAi pathway and recently we found some sequence homology of HpLVd to the Cannabis genome.

Like SARs-CoV-2, there can be asymptomatic transmission. This is exceedingly rare in SARs-CoV-2 so the analogy is a bit of a stretch….And the viroid transmission requires a mechanical vector for transmission. Scissors/shears should be bleached before use between plants (EtOH/Isopropanol doesn’t destroy viroids). Insects are another suspected vector well documented in the transfer of LCV between cannabis plants. Even mechanical touch by gardeners is known to transmit the viroid.

This begs the question of the bio-distribution of the viroids. To address this we screened many clones in hydroponic cloning system and tested the leaves, roots and water. The cloner water was clean of detectable RNA signal but the roots had similar viroid loads as the leaves. This was a bit surprising.

After many screens of dozens of plants, we have a good sense of which plants require isolation, which ones (Strawberry) are worthy of an attempted Tissue culture rescue and which ones are free of disease. We were able to isolate positive from negative plants and get a view to how much this viroid load varied with multiple samplings and tissue types. Collin recognized some of the asymptomatically infected plants shared tents or cloners emphasizing the importance of tracking and tracing these lineages. This wasn’t a won and done day trip to the lab. After initial screening with a panel of viruses/viroids, HLVd was zeroed in on and repeat tested two more times roughly a week apart.

Unlike SARs-CoV-2, we don’t yet know if plants can build an immune response to such a small 37nm viroid? For viroids, Mother Nature has left us a few hints, as these rarely transmit via seeds. The process of making pollen, up-regulates a few plant based nucleases that lower viroid level such that they don’t transmit very effectively to the next generation. It is these very genes that might lead to an understanding of why 25% of the plants in this study were never infected. Whole genome sequencing of these dozen plants are underway.

In summary, size can be deceiving. Even 256 base pair pieces of RNA can have dramatic effects on reprogramming the biology of a 900 Million base pair genome like Cannabis. The Hop genome is 2-3X the size of the Cannabis genome.

The very high attack rate of HLVd is a concern (75% in this study or 8/12 mother plants). Simply burning every plant down that tests positive may lead to a massive genetic bottleneck in cannabis diversity. Tissue culture rescue of these plants is time consuming (3-9 months) and doesn’t alway deliver viroid free plants but will be explored for valuable heirloom cultivars.

The market is impatient. Cannabis cultivars are like the fashion industry. They come and go like fads every 2-3 years such that few grows want to rescue heirloom strains and usually opt to just bring in new genetics and start over with the latest in vogue cultivars.

Given these viroids are benign to humans, experiments that lower viroid loads such that the host remains asymptomatic and yield competent are worthwhile. qPCR will be a valuable tool in guiding these programs.

This resonates with what we are learning about the COVID pandemic. Rarely does a silver bullet exist but treatments that reduce viral load are our best defense against mutagenic viruses. It is important to slow the replication cycles of the virus for both the patient and the herd. Treatments that increase viral load are a dangerous trade off. Complete elimination of the viroid may be unrealistic and making the assumption there is no potential symbiosis for certain genotypes. Heterozygous sickle-cell humans are resistant to malaria. An important hubris solvent in the complex world of biology.