Decoded Dog

by Dianne Janczewski

  “Don’t kick yourself! You had no way—”

  “I should have mentioned it to you guys. Frankly I just put it out of my mind because I started to see other cases of CRFS that didn’t seem to have this cat connection.”

  “How many do you think you have?” I asked.

  “Cases with a cat I saw and then a sick dog? I’m checking.”

  Out of the corner of my eye I could see Chris’ face evolve into a look of almost panic. “Anna, can you pull up one or two? What were the cats brought in for?”

  She was laser-focused on her computer. “That’s what I’m looking for.” She clicked to open up two client records, side by side. She scanned them and sat down, deflated, locking knowing eyes with Chris.

  “Someone want to clue me in?” I asked.

  Chris lowered his eyes. Anna murmured, barely audibly, “Vaccinations.”

  “The same vaccination,” Chris said as a statement, not a question.

  “Yes. FeLV,” Anna said.

  I understood then. “And Feline Leukemia Virus vaccination is a mod—”

  “A modified live vaccine, containing a modified version of FeLV,” Anna finished. “Which is shed to the cat’s environment the first twenty-four hours after vaccination.”

  We all sat in dead silence, taking in the gravity of the connection. We could not confirm how vaccinating a cat against a virus that was completely unrelated to anything in the dog could lead to CRFS without further tests, but we all knew this was the smoking gun.

  Chris and I cuddled for a bit while the girls slept. Ania lay regal and aloof next to me, quietly reminding me with her warmth that it was not my job to convince people of the merits of her breed, it was hers.

  She is a standard poodle. An incredible breed loved by those who know it, much maligned and misunderstood by those who can only see a goofy haircut on a pampered white poodle dyed pink, wearing bows in her hair, nails painted, with an owner who insists she demands such attention. “Have you ever actually seen a poodle in that haircut?” I’ll ask. “No,” the response always is. “I’ve only seen them on TV, but so-and-so had one when I was growing up that was really nasty.”

  We poodle people know what you are really thinking.

  It is rather goofy, this caricature of the original hunt cut from the time when a dog’s hair was kept long on the chest and joints to keep it warm in the water, while the hind legs were shaved to help it move through the water, and two patches were left on the hips to keep the kidneys warm. Poodles were bred to be water retrievers, complete with webbed feet and water repellent hair—not fur. But someone who is curious enough to get close at a dog show will see what really goes into crafting these dogs into regal athletes. In addition to careful breeding, there is an artistry to the grooming and a discipline to training that gets lost in the cartoon. And if you are looking for different type of competition, there are also those who participate in grooming completions where they sculpt poodle coats into colorful scenery or to other animals, tis another interesting side of the poodle world.

  I fell in love with the breed by chance, having grown up with a miniature poodle, who was appropriately named for the 1960s Pierre. My college rebellion against everything parental resulted in my owning dogs of two other breeds who shed, smelled odd, and died young of some malady from poor genetics. I loved them each dearly, and anguished over losing them. But by my late twenties I came back home to the poodle, and now I marvel every day at their intelligence and physical ability. They don’t drool, they don’t smell, and they are kickass smart, making them easily corruptible into spoiled and demanding creatures if they are not trained right. They are fiercely loyal, and uniquely intuitive about their surroundings and human companions.

  And they have a great sense of humor. Sofie only needs to find my purse and dig out something to parade around the room to get my attention and make me laugh. Having bred poodles, and watched many reluctant family members and friends become staunch defenders of the breed, I am long past being sensitive to people who don’t get them. And I love their curls!

  In 1998, the first animal genome was sequenced, that of the nematode worm, which consisted of just under one million bases. Next was the Drosophila fruit fly in 2000, with 165 million bases. These early efforts involved a painstaking set of procedures of cloning short pieces of DNA and piecing together the genome from an overlapping puzzle of strands. By the turn of the twenty-first century, a revolution in techniques and laboratory equipment automated the process. It facilitated the sequencing of the human genome in 2003, at over three billion bases, as well as genomes of other species of significance to laboratory research, including the mouse, frog, and dog. While there is controversy over their use, beagles are the most common laboratory dog, with several different lines that have been maintained for decades. Interestingly, the first draft of the dog genome sequence was from a standard poodle named Shadow, because the researcher leading the project wanted to use his own dog. Ultimately, the first complete sequence of a domestic dog, published in 2005, was of a boxer named Tasha. The boxer was chosen because there is very little variation between individuals in the breed, allowing verification of conserved regions of DNA with just a few individuals.

  Ania is tenacious and persistent, and committed to going after what she wants. A ball under the couch has no chance of hiding for long. Neither does a lizard scampering across the rocks. A raptor perched on a wire surveying a field has nothing on Ania’s vantage point from the sunroom. She bolts out the dog door from her ready state and the poor lizard is obliterated. She is relentless in reminding me that my morning coffee is served with a biscuit for her and that the basement door is to be opened in the afternoon to usher in the afternoon ball chase. She is driven like Anna.

  I sat quietly, staring blankly out at the empty campus. Silent, Anna stood at my office door, hugging a small cooler, waiting for me to notice, or maybe simply allowing me a final moment of peace. She looked oddly calm.

  “You’re early,” I said as I hugged her and the box.

  “Actually late, months late, but let’s get to it.”

  We went into the lab and unpacked the box on Megan’s bench. I grabbed markers and labels while Anna laid out spreadsheets and opened her laptop. Everything was already well organized and there was no need for us to rearrange the samples to set up the study, just to put lab-specific accession numbers on them and get started. Megan and Jamie showed up within the hour, excited at the prospect of making a critical connection. Their goal was to pull the modified viruses from the vaccination vials and sequence them the same way they would any sample. I silently hoped to find a match to the promoter sequence we found in the CRFS study dogs.

  A LINE IN THE SAND

  FROM LION to lamb, March in Virginia shifts from frost-covered dawns and frigid nights to mornings perfect for standing on the deck enjoying the warmth of the sun awakening the world. Crocuses poke their purple heads through the softening ground, yellow daffodils open their mouths to sing in the lengthening days. A soft green haze overlays the gray trees. As winter unclenches its stubborn hold, spring quietly takes the stage. By the beginning of April, the season is in full bloom. Spring in Virginia is like a muted fall. Instead of deep oranges, reds, and yellows there are the pale pink feather dusters of the mimosa blooms and cherry blossom petals, faint yellow flowers of tulip poplars, purple redbuds, and white crosses of the dogwoods. A full pastel watercolor palette tints the landscape.

  The dogs too come alive, relentless in their efforts to capitalize on creatures emerging from their underground world. They stand alert in the middle of the yard, heads cocked, listening for the rumble beneath the grass that only their highly attuned ears can hear. They pounce, dig, stop, listen for a change in direction, and repeat. They excavate the tunnels with precision; the moles have no chance. The yard looks like an ant farm. Orange mud oozes from their muzzles, sticks between their toes, and stuffs up their noses. Reluctantly, they make their way to the tub on the command of “shower�
��—but I know they are secretly giggling.

  The origins of CRFS had lain dormant for six months but once it began to germinate, a cascade of discoveries brought it to full bloom. Neil and I had not spoken in the last few weeks—he wanted to keep in the background—though we did communicate through short texts punctuated with key findings.

  I got straight to the point. Found a sequence definitely related.

  His response was immediate. And?

  Sequence associated with the HPA.

  Are you talking about CRFS or Addison’s?

  CRFS. The sequence codes for GR.

  In the pause I could feel him thinking. Are you sure?

  Yes. Think we found the smoking gun.

  What next?

  When can you get here?

  It had taken the better part of two weeks to know, definitively, that we had identified the cause. Neil flew in Saturday night in preparation for an all-day, heavy discussion at our house on Sunday. We told the girls what was going on. They were filled with questions, concerns, and to our delight, genuine interest. As they tried to understand the nuances of our explanation, they were guinea pigs for our eventual presentation to the public. Armed with ninth and eleventh grade educations, they understood most of it and helped us to clarify for the lay public, those concepts that as scientists we took for granted. Genomes, DNA, Addison’s, retrovirus. We needed a glossary of terms. It would be the same if I were to listen to experts in finance or information technology talk their talk.

  We all gathered for cocktails and hors d'oeuvres on the deck consisting of soda water and whatever cheese we had left in the refrigerator. We needed clear minds to work through the night, and worked out our game plan while we waited for dinner to cook. Ania dropped the ball at Neil’s feet. He picked it up and balanced it on his knee for a few seconds, then let it roll down his bent leg and bounce off his toe. She darted after it, pounced, and proudly brought it back to him; again, he let it roll down his leg. “Careful, don’t get her started unless you’re inclined to do that all night,” Chris said.

  “But she’s having such a ball,” Neil retorted.

  “And she looks so fetching doing it, don’t you think?” Chris said without a hint of shame.

  Dinner was calming and rejuvenating. My favorite friends, Anna and Neil, and my family, the loves of my life. The girls were comfortable at a table of adults, entertaining with their syncopated storytelling, and engaging with their fresh perspectives on everything from politics to science to Hollywood gossip. A momentary shrill from the kitchen followed by “mom I dropped my phone in the pie!”, was truncated by Neil’s “Oh! Are we having baked apple?”

  There was a synergy between Anna and I that added hidden meaning to our interchanges, a momentary catch of the eye or subtle knowing smile. The guys competed mercilessly for the role of pun master.

  The girls cleared the table and even did the dishes as the rest of us turned our dining area into a conference room, with stacks of documents and notebooks, and several laptops open and ready for the presentation. Anna, Chris and I planned to walk Neil through all the details of our findings, and tried to anticipate any questions he would have. I knew this was a dress rehearsal as we would be grilled on our data and conclusions many times in the coming weeks. “So you know that Anna had samples from 139 dead dogs,” I began.

  “But I’ve put a stop to that,” she added.

  “And she carefully collected and catalogued all of the data on these animals.”

  “I always liked you,” Neil said, grinning with sarcasm, or maybe not. “What kind of data?”

  “Everything,” Anna said. “All the information leading up to their deaths, blood work, physical profile, vaccinations, treatments, diet. Demographic data like breed, age, information on their owners, other animals in the house.”

  Turning to me he asked, “Okay, other than that she is stunningly beautiful, how does that make her different from all the other researchers with samples of their own or who received samples from the CDC?”

  I looked at her bemused. Neil has never been sexist in the least, and it was an odd time to be flirting. Anna seemed unfazed and answered for me. “You are correct that my good looks separate me from the rest. That and my Addison’s samples. My CRFS sample population includes fifty-two dogs that died which also had Addison’s. I also had stored samples of their blood pre-CRFS, often from different stages of their life and death. But you already know all this. It’s the analyses that are telling.”

  Anna stood, and like a pizza chef in the window of a restaurant, arranged the ingredients of papers and notebooks on the table creating a centerpiece for her story. We all moved to one side of the table to get a better view. I opened and slid to her the notebook that contained all of her spreadsheets: pages of color-coded highlights, dog-eared and scribbled upon. Anna walked Neil through her original findings, that Addison’s dogs lived a bit longer, but eventually succumbed to the disease.

  Seeming to hang on her every word, Neil tipped back a bit and looked at me behind her back. “That’s when you called me.”

  I nodded. He leaned back to the table and Anna talked on. She had called colleagues across the country, and she and I had gone over all of the information that I collected over the holidays. There seemed to be little rhyme or reason to the pockets of outbreaks.

  I walked Neil through our process of elimination as we worked through all the possible laboratory analyses. There was nothing from the toxicology folks, no elevated proteins in the blood, no unusual DNA sequences. Nothing.

  “So from nothing you discovered everything,” Neil said flatly though his rising anticipation was palpable.

  “Well, to be fair,” Chris added, “it wasn’t completely nothing. Claire’s team screened hundreds of blood samples for changes in DNA, and finally noticed a slight barely perceptible, but definite difference not in the DNA, but in a short mRNA sequence that came from transcribing a gene needed to make more of the protein GR.”

  Looking at me Neil said, “Barely perceptible?” He needed proof.

  “Barely,” I said. “In fact, it’s not a matter of a change in the sequence of the mRNA, but instead, in the number of copies.”

  “Well, that’s only part of the change,” Anna inserted.

  “Don’t spoil my presentation,” I said. “I’m going for a big climax.”

  “You were taking too long.”

  “Since when are you the one who is in a hurry? I Usually I have to do the set director’s speed it up sign when you’re telling—

  “You digress,” Neil interrupted. He shook his head, dismissing us, and turned to Chris to rescue him. “So Chris, there is a difference in the number of copies. What else?”

  “Once we’d noted that the number of copies increased, the question became why. Was it in response to CRFS, or was it linked to the cause of CRFS. So the next thing to look at was the sequence itself.”

  “And?” Neil asked.

  “And, while it is true the actual mRNA sequence is unaltered, there is an extra promoter upstream of the normal promoter.”

  “An extra promoter. And that caused . . ?”

  “That caused the increase in production of GR RNA transcripts.”

  “And you see this extra promoter only in CRFS animals.”

  Our three voices answered in unison. “Yup. Yes. Uh-huh”

  “Okay folks,” Neil said. “Let’s hear the punch line because I feel like I have three Cheshire cats hovering over me. What does this extra promoter look like?”

  “Ooh, good analogy,” I said.

  “Yes, purrrrfect,” Anna cooed.

  Neil looked to Chris for relief from their shared purgatory of puns. “It’s a cat virus promoter,” Chris said, spoiling our fun.

  Neil turned and took a few steps away from us towards the window. When he turned back to face us he looked genuinely shocked. “Are you suggesting that there is a cat virus sequence that has jumped to dogs? Do we have a new parvo on our hands?”


  Anna continued. “Yes about the cat sequence, but no on parvo-like. The extra promoter isn’t from a current cat virus like parvo was, but is an ancient one that became part of the cat genome, so it is technically from a cat sequence.”

  “You’ve lost me.”

  I jumped in. “The promoter is from an endogenous retrovirus of the cat. From an ancient virus that long ago got incorporated into the cat’s genome and forever sits there quietly—like a Cheshire cat.”

  The humor was lost on him. “What? Wait, if it is endogenous then it’s inactive. Like you said, forever sitting quietly. How exactly are you proposing that it got turned on then jumped into the dog? That’s a pretty wild assertion.”

  “I didn’t say it did. We only see the promoter part of the sequence, forty-three bases, in front of the GR gene, no other part of the retrovirus,” Anna said. “We also believe the endogenous sequence didn’t just get messed up in the cat and then suddenly jump to the dog.”

  “Then how . . .” Shaking his head, Neil walked back to us and plopped into a chair; slowly the rest of us descended into ours.

  Anna pulled a blank sheet of paper in front of him and began to illustrate the sequence of events. “Here’s what we think happened. A vaccine was created using standard modified live technique.” She drew a vaccine vial and an arrow to a cat. “When it was injected into cats it triggered the typical immune response, which includes shedding the modified virus into the environment through urine, feces or saliva, for maybe twenty-four to forty-eight hours, and the dog then picked it up. Since the vaccine is based on a modified cat virus that doesn’t normally affect dogs, it should have had no effect on the dog, except to wander around the body looking for trouble.

  “But this was not normal.”

  “Correct. The vaccine was contaminated with an extra promoter, so it found trouble in the dog by dropping off the promoter. As it happens, it did so at a location just upstream from the GR gene.” She added a dog to the illustration and a blown-up block with a squiggly double helix and a dot for the GR gene.