RSV notes, out of office notice

A weekend offline.

I will be away from my workstation until Monday.

Below is a highlight of a paper about Alpha and a cross-posted bit of RSV and polio history.

On my way out, I would also like to break glass on the Level 10 Recommended Viewing Alert button for Maria Gutschi’s powerful overview of the flaws and follies of the mRNA vaccine logistical infrastructure, and associated regulatory delinquency:

RTE Roundtable Talk #19: Pseudovaccine Quality Inconsistency

Alpha Was As Anomalous as Omicron

Once back in office, I hope to put together something to show for my argument that there have been multiple releases of SARS-CoV-2. As a preview, a wholistic breakdown of why “Alpha” cannot be explained by any other acceptable-in-polite-company origin except “chronic infection” was quietly uploaded this March by man-of-mystery Andrew Rambaut and collaborators.

Hill, V. et al. The original biorxiv.org upload makes for a somewhat more digestible presentation.

“Chronic infection,” of course, might as well be a cop-out to “act of God.” It’s a fig leaf over what published science is not allowed to say, namely that humans synthesize viruses intentionally.

At any rate, the best part is the discussion section.

Any proposed origin of B.1.1.7 must explain three observations: first, the long branch leading to the B.1.1.7 clade with at most one intermediate sequence, despite high genomic surveillance; second, an increased evolutionary rate along this branch; and third, a single geographical and evolutionary origin of B.1.1.7 (Kraemer et al. 2021).

In a country with an extensive virus genomic surveillance programme like the UK, which includes random and relatively dense sampling (an average of 7.9% of weekly reported cases in Kent and Medway between 24th April 2020 and 19th September 2020 were sequenced), it is unlikely that a precursor lineage was circulating in Kent over the summer of 2020 and was not detected. It is worth noting also that B.1.1.7 was captured by this surveillance programme within at most two days of its origin - the MRCA of the clade is the 19th September 2020 (see above), and the first sample was taken on 20th of the same month [meaning, no sequences ever appeared later to offer genetic evidence of the Alpha’s presence in the world before September 20, 2020]

Alpha vs. background. Note how “pure Alpha” sequences continue to be recorded through December (the blue dots that do not gain distance from root). The supplemental materials contain provocative plots for the other VOCs in which evolution seems to travel in reverse; I believe these are artifacts of the same stasis but in lower-surveillance countries where the VOCs are not first identified until later in the winter.

My offhand reply to the chronic infection origin theory is that chronic infections result in higher “mutation rates” because of the loss of consensus and genetic decay that results from allowing the virus to mutate in a permissive environment, which removes stabilizing selection. Essentially you get a lot of inbred virus. So a chronic infection origin theory has to suppose that removing stabilizing selection somehow tricks the virus into crossing a fitness valley, akin to inbreeding resulting in a king who suddenly sprouts huge muscles and a gorgeous, giant chin.

Nope.

Not how Alpha was made.

Comment cross-post: RSV and Polio history.

In another follow-up to the blogger who goes by “the Naked Emperor,” I offered some context on the early days of discovering viruses in cell culture and how that might limit the nefarious implications of certain coincidences, over at yesterday’s post. I more or less did this for my own benefit, as I am always seeking to understand this era better as well as to convert the research in Oshinsky’s Polio to a more manageable format [in fact in my original comment I failed to parse the correct identity of the generic, reader-friendly “monkey” Oshinsky usually refers to].

I am also thinking of declaring December “Polio Month” for Unglossed, as a gimmick to force myself to publish follow-ups to my Decoding Polio post.

---

Me on the “isolation ceiling.”

Cell culturing of viruses wasn't a thing before penicillin and streptomycin, so it doesn't really get a foothold until 1949 when Enders makes it work for polio virus. This also facilitates the realization that chimpanzees and cynomolgous monkeys fed orally are a better infection model than rhesus macaques injected with the MV strain, and that there are at least three very different serotypes, which is Bodian's contribution.

And so you have the typing project go from 1949 to 51 and a major ramp-up of the monkey supply chain, with Indian rhesus monkeys becoming the favored model and 17,000 eventually sacrificed. Chimps are being used a lot too, even after Salk's vaccine is licensed because Sabin is using them for his work. And "monkey kidney" is used for growing vaccine starting in 1951 but Oshinsky doesn't specify which kind, I would guess Indian rhesus again [see excerpt below].

But in parallel, cell culture makes it possible to start isolating viruses that aren't easily transmitted to rabbits, mice, ferrets or eggs. In 1946 the MRC launched a common cold center where human volunteers tried to passage cold viruses to each other for three years, to give you an idea of how difficult things were. Essentially there was no ability to find new viruses before 1949 because of the "isolation ceiling." After that is a bold new journey of virus finding. So RSV's discovery is around the same time as discovering viruses with cell culture becomes possible to begin with.

The RSV as a lab escape theory would be pretty easy to test just by seeing if adults were already seropositive in the 50s or 60s, implying the virus was already in the roster of human childhood encounters. Off hand I would say that if it could make children ill, it should have killed a lot of adults if it was actually a novel virus.

Addendum: After writing this comment, I traveled to the original paper reporting on the virus that was eventually described as RSV. Antibodies were already immediately found in teens and young adults, suggesting the virus was already in humanity before first being cultured from chimps with colds:

However, a number of human beings, particularly adolescents and young adults, have antibodies in their sera directed against the coryza agent suggesting that these individuals have experienced infection with the new agent or one closely related to it.

Excerpts from Oshinsky’s Polio: An American Story on cell culturing and monkey-business:

The cell-cultural revolution

It wasn’t for lack of effort. In 1907 an obscure Yale biologist named Ross Harrison made what some have hailed as “one of the ten most important discoveries in Western medicine.” It was the concept of tissue culture: the ability to grow and nurture living cells in vitro, outside the hosts—plant, animal, and human—from which they came. “Harrison’s discovery,” wrote two distinguished researchers, “has made possible the study of living organisms at the cellular and even the molecular level, and the development of modern vaccines, including those for poliomyelitis, measles, mumps and rabies…. Indeed, because of tissue culture more has been learned about the basic mechanism of disease in the past fifty years than in the previous five thousand.”

Tissue culture seemed perfectly suited for the study of polio, which, like other viruses, can exist only in living cells. Yet success did not come quickly. In 1936 Sabin and Peter Olitsky of the Rockefeller Institute had shown that poliovirus could, indeed, be grown in test tube cultures. That was the good news. The bad news was that it would only grow in nervous tissue.

There was a logical explanation for this, though no one knew it at the time. The poliovirus in vogue at the institute was Simon Flexner’s “MV,” a highly neurotropic strain, unable to grow in anything except nervous tissue. And by using it in their experiments, Sabin and Olitsky had confirmed the mistaken belief that poliovirus could not survive anywhere else. This presented a dilemma for researchers, since the nervous tissue of monkeys was known to cause encephalomyelitis, an inflammation of the brain and spinal cord, when injected into human beings. If Sabin and Olitsky were correct—if poliovirus would only grow in dangerous nerve tissue—then how did one go about harvesting it for use in a vaccine? In the matter of polio, at least, the promise of growing a safe virus in tissue culture had reached a dead end. And there the matter stood. It was not easy to challenge the wisdom of Simon Flexner and two leading lights from the nation’s most respected research institute. “That work was so meticulously done that I believed it was absolutely correct,” Tom Rivers recalled. “Hell … every working virologist that I know believed it, with the possible exception of John Enders at Harvard.”

[…]

By 1948, the art of in vitro cultivation was rapidly advancing. The introduction of antibiotics such as penicillin and streptomycin made it simpler to maintain sterile cultures by cutting down on bacterial contamination. New techniques were being employed to gently roll the test tubes, exposing the tissue inside to the proper amounts of fluid and air. And Tom Weller discovered that the tissue would survive longer if the nutrient medium was changed at regular intervals, about every four days.

The great breakthrough in Enders’s lab that year came largely through scientific intuition. “One day, when Tom and I were preparing a new set of cultures,” said Fred Robbins. “Dr. Enders suggested that since we had some poliovirus stored in the freezer, we might inoculate some of the cultures with this material, which we did.” The cultures contained both nerve and non-nerve embryonic tissue. Four were injected with chickenpox virus, four with Lansing Type II poliovirus, and four were left as controls.

The typing project: Monkeys, Salk-insults, etc.

Most researchers favored the cynomolgous monkey from the Philippines because it appeared to closely mimic the polio experience of humans, with the virus entering through the mouth and replicating in the gastrointestinal tract. But these animals were scarce and delicate, so American labs looked to the Indian rhesus monkey, a more resilient and plentiful source. The Indian government was happy to oblige. Exporting rhesus monkeys was good for the economy, providing both foreign currency and local employment. Workers were needed to capture the animals and carry them “on shoulder poles to the nearest railway station and from there to New Delhi,” where they were put on airplanes for the four-thousand-mile trip to London, and then on to New York. Furthermore, a thinning of the monkey population appealed to government officials, because monkeys were responsible for destroying ten percent of India’s crops.

There was a problem, however. Because monkeys are sacred in the Hindu religion, concerns were raised about their mistreatment by non-Hindus during their capture, their travel, and their time in the laboratories. One such mishap, in which 390 monkeys died of suffocation at a London airport, almost caused Indian officials to ban future exports. In response, the National Foundation agreed to monitor this process, and to promise that the monkeys would be well cared for and used only for polio research.

The bargain made sense. In 1949, the foundation established a special facility known as Okatie Farms in rural South Carolina to process the monkeys arriving from abroad. Veterinarians screened them for disease, and nutritionists supervised their diet. Once in shape, they were trucked to foundation grantees throughout North America. (“We should like to have 50 conditioned cynomolgous monkeys, three to five pounds, delivered monthly,” said Tommy Francis in a typical request.) The cost was about $26 per animal, including transportation. “Okatie,” said an observer, “was in its way a little Warm Springs for monkeys.”

More than 17,000 of them would be sacrificed in the typing project alone. The procedure went like this: fecal samples from human polio victims were injected into the brains of monkeys, who were exercised daily to look for telltale signs of polio. When paralysis appeared, the animals were destroyed so their brains and spinal cords could be harvested for poliovirus. Tissue-serum mixtures of the virus were then injected into the brains of healthy monkeys. Those that received a known Type I strain and recovered were considered to have immunity to all other Type I strains. “They now would be inoculated with virus of an unknown type. If they proved susceptible to infection, it would mean that the unknown strain belonged to Type II or Type III…. The tests then would have to be conducted all over again, using the same unknown virus to challenge monkeys immune to Type II or Type III.”

While the typing program certainly expanded Salk’s reach within the foundation, it also marked his lowly position in the calcified pecking order of polio research. Salk got an early whiff of this at a meeting of the typing committee, when he posed what he thought was a modest query about procedure. “Albert Sabin … turned to me and said, ‘Now Dr. Salk, you should know better than to ask a question like that.’” It was, Salk recalled, “like being kicked in the teeth. I could feel the resistance and the hostility and the disapproval. I never attended a single one of those meetings afterward without that same feeling.”

Salk bit his lip and played the role of junior partner. He visited Sabin’s lab in Cincinnati, offered the appropriate compliments, and even spent the night at Sabin’s home. He thanked Sabin profusely for sending him reprints of articles and new strains of virus to be typed. It counted for little. Sabin didn’t think much of Salk, and never would. He viewed him as an errand boy for the National Foundation, ambitious but mediocre, who would do whatever Harry Weaver wanted in order to advance. In an early letter to Salk, reviewing a draft paper on the typing project, Sabin dropped a heavy hint of the trouble that lay ahead. “You have made a number of references of extraordinary praise, etc., to the National Foundation,” he wrote. “My own reaction is that this is perhaps in bad taste…. It is quite obvious that this entire project was sponsored by the National Foundation, and I can see no need for expressions regarding the help that was given by [them].”

The final results of the typing project were reassuring. David Bodian’s prediction had held up well. The 196 tested strains of poliovirus all fit neatly into three distinct types. The poliovirus “family” was remarkably, conveniently, small.

Growing vax: When all you have is a monkey, every problem looks like a kidney

By 1951, with the typing program now behind him, Salk moved seamlessly to the next plateau. [...]

“After some intense work,” Youngner added, “I realized that monkey kidney was the answer.” A single such organ, properly handled, could produce enough raw material for 6,000 shots of polio vaccine. The process was exacting, to say the least. Though all the monkeys came from the foundation’s Okatie Farms in South Carolina, Salk demanded that each one be given a “physical” by Jim Lewis and his staff. This done, the animal was anesthetized and its kidneys removed. The cortex (outer layer) was then separated, chopped into tiny fragments, and “rinsed several times with salt solution to remove blood and debris.”

Oshinksy, David. Polio: An American Story. Oxford University Press.

If you derived value from this post, please drop a few coins in your fact-barista’s tip jar.

click button or visit https://ko-fi.com/unglossed