Did a small mu­ta­tion fuel virus’s con­quest?

Sci­en­tists race to solve a ge­netic mys­tery: What makes it so in­fec­tious

The Washington Post - - FRONT PAGE - BY SARAH KA­PLAN AND JOEL ACHEN­BACH

When the first coro­n­avirus cases in Chicago ap­peared in Jan­uary, they bore the same ge­netic sig­na­tures as a germ that emerged in China weeks be­fore.

But as Egon Ozer, an in­fec­tious-dis­ease spe­cial­ist at the North­west­ern Univer­sity Fein­berg School of Medicine, ex­am­ined the ge­netic struc­ture of virus sam­ples from lo­cal pa­tients, he no­ticed some­thing dif­fer­ent.

A change in the virus was ap­pear­ing again and again. This mu­ta­tion, as­so­ci­ated with out­breaks in Europe and New York, even­tu­ally took over the city. By May, it was found in 95 per­cent of all the genomes Ozer se­quenced.

At a glance, the mu­ta­tion seemed triv­ial. About 1,300 amino acids serve as build­ing blocks

for a pro­tein on the sur­face of the virus. In the mu­tant virus, the ge­netic in­struc­tions for just one of those amino acids — num­ber 614 — switched in the new vari­ant from a “D” (short­hand for as­par­tic acid) to a “G” (short for glycine).

But the lo­ca­tion was sig­nif­i­cant, be­cause the switch oc­curred in the part of the genome that codes for the all-im­por­tant “spike pro­tein” — the pro­trud­ing struc­ture that gives the coro­n­avirus its crown­like pro­file and al­lows it to en­ter hu­man cells the way a bur­glar picks a lock.

And its ubiq­uity is un­de­ni­able. Of the ap­prox­i­mately 50,000 genomes of the new virus that re­searchers worldwide have up­loaded to a shared data­base, about 70 per­cent carry the mu­ta­tion, of­fi­cially des­ig­nated D614G but known more fa­mil­iarly to sci­en­tists as “G.”

“G” hasn’t just dom­i­nated the out­break in Chicago — it has taken over the world. Now sci­en­tists are racing to fig­ure out what it means.

At least four lab­o­ra­tory ex­per­i­ments sug­gest that the mu­ta­tion makes the virus more in­fec­tious, al­though none of that work has been peer-re­viewed. An­other un­pub­lished study led by sci­en­tists at Los Alamos Na­tional Lab­o­ra­tory as­serts that pa­tients with the G vari­ant ac­tu­ally have more virus in their bod­ies, mak­ing them more likely to spread it to oth­ers.

The mu­ta­tion doesn’t ap­pear to make peo­ple sicker, but a grow­ing num­ber of sci­en­tists worry that it has made the virus more con­ta­gious.

“The epi­demi­o­log­i­cal study and our data to­gether re­ally ex­plain why the [ G vari­ant’s] spread in Europe and the U.S. was re­ally fast,” said Hy­eryun Choe, a vi­rol­o­gist at Scripps Re­search and lead au­thor of an un­pub­lished study on the G vari­ant’s en­hanced in­fec­tious­ness in lab­o­ra­tory cell cul­tures. “This is not just ac­ci­den­tal.”

But there may be other ex­pla­na­tions for the G vari­ant’s dom­i­nance: bi­ases in where ge­netic data are be­ing col­lected, quirks of tim­ing that gave the mu­tated virus an early foothold in sus­cep­ti­ble pop­u­la­tions.

“The bot­tom line is, we haven’t seen any­thing de­fin­i­tive yet,” said Jeremy Luban, a vi­rol­o­gist at the Univer­sity of Mas­sachusetts Med­i­cal School.

The scram­ble to un­ravel this mu­ta­tion mys­tery em­bod­ies the chal­lenges of science dur­ing the coro­n­avirus pan­demic. With mil­lions of peo­ple in­fected and thou­sands dy­ing ev­ery day around the world, re­searchers must strike a high-stakes bal­ance be­tween get­ting in­for­ma­tion out quickly and mak­ing sure that it’s right.

A bet­ter lock pick

SARS- COV-2, the novel coro­n­avirus that causes the dis­ease covid-19, can be thought of as an ex­tremely de­struc­tive bur­glar. Un­able to live or re­pro­duce on its own, it breaks into hu­man cells and co-opts their bi­o­log­i­cal ma­chin­ery to make thou­sands of copies of it­self. That leaves a trail of dam­aged tis­sue and trig­gers an im­mune sys­tem re­sponse that for some peo­ple can be dis­as­trous.

This repli­ca­tion process is messy. Even though it has a “proof­read­ing” mech­a­nism for copy­ing its genome, the coro­n­avirus fre­quently makes mis­takes, or mu­ta­tions. The vast ma­jor­ity of mu­ta­tions have no ef­fect on the be­hav­ior of the virus.

But since the virus’s genome was first se­quenced in Jan­uary, sci­en­tists have been on the look­out for changes that are mean­ing­ful. And few ge­netic mu­ta­tions could be more sig­nif­i­cant than ones that af­fect the spike pro­tein — the virus’s most pow­er­ful tool.

This pro­tein at­taches to a re­cep­tor on res­pi­ra­tory cells called ACE2, which opens the cell and lets the virus slip in­side. The more ef­fec­tive the spike pro­tein, the more eas­ily the virus can break into the bod­ies of its hosts. Even when the orig­i­nal vari­ant of the virus emerged in Wuhan, China, it was ob­vi­ous that the spike pro­tein on SARS- COV-2 was al­ready quite ef­fec­tive.

But it could have been even bet­ter, said Choe, who has stud­ied spike pro­teins and the way they bind to the ACE2 re­cep­tor since the se­vere acute res­pi­ra­tory syn­drome out­break in 2003.

The spike pro­tein for SARSCOV-2 has two parts that don’t al­ways hold to­gether well. In the ver­sion of the virus that arose in China, Choe said, the outer part — which the virus needs to at­tach to a hu­man re­cep­tor — fre­quently broke off. Equipped with this faulty lock pick, the virus had a harder time in­vad­ing host cells.

“I think this mu­ta­tion hap­pened to com­pen­sate,” Choe said.

Study­ing both ver­sions of the gene us­ing a proxy virus in a petri dish of hu­man cells, Choe and her col­leagues found that viruses with the G vari­ant had more spike pro­teins, and the outer parts of those pro­teins were less likely to break off. This made the virus ap­prox­i­mately 10 times more in­fec­tious in the lab ex­per­i­ment.

The mu­ta­tion does not seem to lead to worse out­comes in pa­tients. Nor did it al­ter the virus’s re­sponse to an­ti­bod­ies from pa­tients who had the D vari­ant, Choe said, sug­gest­ing that vac­cines be­ing de­vel­oped based on the orig­i­nal ver­sion of the virus will be ef­fec­tive against the new strain.

Choe has up­loaded a man­u­script de­scrib­ing this study to the web­site biorxiv, where sci­en­tists can post “pre­print” re­search that has not yet been peer-re­viewed. She has also sub­mit­ted the pa­per to an aca­demic jour­nal, which has not yet pub­lished it.

The dis­tinc­tive in­fec­tious­ness of the G strain is so strong that sci­en­tists have been drawn to the mu­ta­tion even when they weren’t look­ing for it.

Neville San­jana, a ge­neti­cist at the New York Genome Cen­ter and New York Univer­sity, was try­ing to fig­ure out which genes en­able SARS- COV-2 to in­fil­trate hu­man cells. But in ex­per­i­ments based on a gene se­quence taken from an early case of the virus in Wuhan, he strug­gled to get that form of the virus to in­fect cells. Then the team switched to a model virus based on the G vari­ant.

“We were shocked,” San­jana said. “Voilà! It was just this huge in­crease in vi­ral trans­duc­tion.” They re­peated the ex­per­i­ment in many types of cells, and ev­ery time the vari­ant was many times more in­fec­tious.

Their find­ings, pub­lished as a pre­print on biorxiv, gen­er­ally matched what Choe and other lab­o­ra­tory sci­en­tists were see­ing.

But the New York team of­fers a dif­fer­ent ex­pla­na­tion as to why the vari­ant is so in­fec­tious. Whereas Choe’s study pro­poses that the mu­ta­tion made the spike pro­tein more sta­ble, San­jana said ex­per­i­ments in the past two weeks, not yet made pub­lic, sug­gest that the im­prove­ment is ac­tu­ally in the in­fec­tion process. He hy­poth­e­sized that the G vari­ant is more ef­fi­cient at be­gin­ning the process of in­vad­ing the hu­man cell and tak­ing over its re­pro­duc­tive ma­chin­ery.

Luban, who has also been ex­per­i­ment­ing with the D614G mu­ta­tion, has been drawn to a third pos­si­bil­ity: His ex­per­i­ments sug­gest that the mu­ta­tion al­lows the spike pro­tein to change shape as it at­taches to the ACE2 re­cep­tor, im­prov­ing its abil­ity to fuse to the host cell.

Dif­fer­ent ap­proaches to mak­ing their model virus might ex­plain th­ese dis­crep­an­cies, Luban said. “But it’s quite clear that some­thing is go­ing on.”

Unan­swered ques­tions

Al­though th­ese ex­per­i­ments are com­pelling, they’re not con­clu­sive, said Kristian An­der­sen, a Scripps vi­rol­o­gist not in­volved in any of the stud­ies. The sci­en­tists need to fig­ure out why they’ve iden­ti­fied dif­fer­ent mech­a­nisms for the same ef­fect. All the stud­ies still have to pass peer re­view, and they have to be re­pro­duced us­ing the real ver­sion of the virus.

Even then, An­der­sen said, it will be too soon to say that the G vari­ant trans­mits faster among peo­ple.

Cell cul­ture ex­per­i­ments have been wrong be­fore, noted An­der­son Brito, a com­pu­ta­tional bi­ol­o­gist at Yale Univer­sity. Early ex­per­i­ments with hy­drox­y­chloro­quine, a malaria drug, hinted that it was ef­fec­tive at fight­ing the coro­n­avirus in a petri dish. The drug was touted by Pres­i­dent Trump, and the Food and Drug Ad­min­is­tra­tion au­tho­rized it for emer­gency use in hos­pi­tal­ized covid-19 pa­tients. But that au­tho­riza­tion was with­drawn this month after ev­i­dence showed that the drug was “un­likely to be ef­fec­tive” against the virus and posed po­ten­tial safety risks.

So far, the big­gest study of trans­mis­sion has come from Bette Kor­ber, a com­pu­ta­tional bi­ol­o­gist at Los Alamos Na­tional Lab­o­ra­tory who helped build one of the world’s big­gest vi­ral genome data­bases for track­ing HIV. In late April, she and col­leagues at Duke Univer­sity and the Univer­sity of Sh­effield in Bri­tain re­leased a draft of their work ar­gu­ing that the mu­ta­tion boosts trans­mis­sion of the virus.

An­a­lyz­ing se­quences from more than two dozen re­gions across the world, they found that most places where the orig­i­nal virus was dom­i­nant be­fore March were even­tu­ally taken over by the mu­tated ver­sion. This switch was es­pe­cially ap­par­ent in the United States: Ninety-six per­cent of early se­quences here be­longed to the D vari­ant, but by the end of March, al­most 70 per­cent of se­quences car­ried the G amino acid in­stead.

The Bri­tish re­searchers also found ev­i­dence that peo­ple with the G vari­ant had more vi­ral par­ti­cles in their bod­ies. Al­though this higher vi­ral load didn’t seem to make peo­ple sicker, it might ex­plain the G vari­ant’s rapid spread, the sci­en­tists wrote. Peo­ple with more virus to shed are more likely to in­fect oth­ers.

The Los Alamos draft drew in­tense scrutiny when it was re­leased in the spring, and many re­searchers re­main skep­ti­cal of its con­clu­sions.

“There are so many bi­ases in the data set here that you can’t con­trol for and you might not know ex­ist,” An­der­sen said. At a time when as many as 90 per­cent of U.S. in­fec­tions are still un­de­tected and coun­tries with lim­ited pub­lic health in­fra­struc­ture are strug­gling to keep up with surg­ing cases, a short­age of data means “we can’t answer all the ques­tions we want to answer.”

Pardis Sa­beti, a com­pu­ta­tional bi­ol­o­gist at Har­vard Univer­sity and the Broad In­sti­tute, noted that the vast ma­jor­ity of se­quenced genomes come from Europe, where the G vari­ant first emerged, and the United States, where in­fec­tions thought to have been in­tro­duced by trav­el­ers from Europe spread un­de­tected for weeks be­fore the coun­try shut down. This could at least partly ex­plain why it ap­pears so dom­i­nant.

The mu­ta­tion’s suc­cess might also be a “founder ef­fect,” she said. Ar­riv­ing in a place like North­ern Italy — where the vast ma­jor­ity of se­quenced in­fec­tions are caused by the G vari­ant — it found easy pur­chase in an older and largely un­pre­pared pop­u­la­tion, which then un­wit­tingly spread it far and wide.

Sci­en­tists may be able to rule out th­ese al­ter­na­tive ex­pla­na­tions with more rig­or­ous sta­tis­ti­cal analy­ses or a con­trolled ex­per­i­ment in an an­i­mal pop­u­la­tion. And as stud­ies on the D614G mu­ta­tion ac­cu­mu­late, re­searchers are start­ing to be con­vinced of its sig­nif­i­cance.

“I think that slowly we’re be­gin­ning to come to a con­sen­sus,” said Judd Hultquist, a vi­rol­o­gist at North­west­ern Univer­sity.

Solv­ing the mys­tery of the D614G mu­ta­tion won’t make much of a dif­fer­ence in the short term, An­der­sen said. “We were un­able to deal with D,” he said. “If G trans­mits even bet­ter, we’re go­ing to be un­able to deal with that one.”

But it’s still es­sen­tial to un­der­stand how the genome in­flu­ences the be­hav­ior of the virus, sci­en­tists say. Iden­ti­fy­ing emerg­ing mu­ta­tions al­lows re­searchers to track their spread. Know­ing what genes af­fect how the virus trans­mits en­ables pub­lic health of­fi­cials to tai­lor their ef­forts to con­tain it. Once ther­a­peu­tics and vac­cines are dis­trib­uted on a large scale, hav­ing a base­line un­der­stand­ing of the genome will help pin­point when drug re­sis­tance starts to evolve.

“Un­der­stand­ing how trans­mis­sions are hap­pen­ing won’t be a magic bul­let, but it will help us re­spond bet­ter,” Sa­beti said. “This is a race against time.”

OC­TAVIO JONES/GETTY IMAGES

Health-care work­ers ad­min­is­ter coro­n­avirus tests in Tampa on Thurs­day. Sci­en­tists have dis­cov­ered a mu­ta­tion they sus­pect has made the novel coro­n­avirus more con­ta­gious and al­lowed it to spread faster.

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