other studies that indicate a minimum of 23 independent introductions into Uruguay, 48 resulting in five major transmission clusters. Our data suggest that most introductions 49 resulting in chains of transmission originate from other South American countries, with 50 the earliest seeding of the virus in late February 2020, weeks before the borders were 51 closed to all non-citizens and a partial lockdown implemented. Genetic analyses suggest 52 a dominance of S and G clades (G, GH, GR) that make up >90% of the viral strains in 53 our study. In our cohort, lethal outcome of SARS-CoV-2 infection significantly 54 correlated with arterial hypertension, kidney failure, and ICU admission (FDR < 0.01), 55 but not with any mutation in a structural or non-structural protein, such as the spike 56 D614G mutation. Our study contributes genetic, phylodynamic, and clinical correlation 57 data about the exceptionally well-curbed SARS-CoV-2 outbreak in Uruguay, which 58 
To amplify the viral genomes in preparation for sequencing, we used the Swift CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 11, 2020 . . https://doi.org/10.1101 /2020 Statistics 159 D'Agostino & Pearson normality tests were performed to assess whether data 160 values followed Gaussian distribution and whether parametric or nonparametric 161 statistical tests were indicated (GraphPad Prism v.8 
To infer our SARS-CoV-2 time-scaled phylogenetic tree, we selected global 181 reference sequences used for the Nextstrain analysis specific for South America as of 182
August 6th, 2020. This dataset consisted of 1747 sequences sampled between December 183 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted October 11, 2020 . . https://doi.org/10.1101 /2020 26th, 2019 and July 17th, 2020, from Africa (40) 
To obtain an estimate of the number of independent introductions of SARS-204
CoV-2 into Uruguay, we performed a preliminary phylogeography analysis using the 205 asymmetric rates discrete diffusion model implemented in BEAST version 1.10.4 [23] , 206 adopting the fixed tree approach [11] . This approach greatly reduces the computational 207 time required to perform the analyses by annotating the phylogeography analyses onto a 208 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 11, 2020. and 59 (24-89) years, respectively. Sixty eight percent of the participants did not require 231 hospitalization, 18% were hospitalized or received in-home care (9% on ventilation and 232 5% on ICU), and 14% were deceased. Whereas the early COVID-19 positive cases in 233 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 11, 2020 . . https://doi.org/10.1101 /2020 March were predominantly clade S infections, clade G viruses subsequently became 234 dominant in April and May 2020. (Figure 1 A-C) . The SARS-CoV-2 genetic variants in our study group are very diverse and 244 comprise sequences from clades S, V, G, GH, and GR ( Figures 1A and 2A , Table S1 ). . In addition, we found three 257 more SNPs to be significantly associated, namely C17470T, C25521T, and C26088T 258 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 11, 2020. . https://doi.org/10.1101/2020.10.08.20208546 doi: medRxiv preprint that all together build a strongly significant, positive correlation cluster composed of 259 five mutations ( Figure S4 ). Interestingly, after April, most sequences belonged to clade 260 G (sublineage B.1), defined by the spike protein's D614G mutation. 57% of our 261 cohort's sequences contain the D614G mutation co-occurring with SNPs C241T, 262 C3037T, and C14408T, the latter causing the AA replacement P323L in the RNA-263 dependent RNA polymerase (RdRP) gene of nsp12 ( Figure S5A ) and the right half of the 280 haplotype network (Figure S5B) , the latter the respective opposite halves, highlighted 281 by green and red arrows for spike D614G and ORF8 L84S key mutations, respectively. 282
The allocation of neighborhood data on the phylogenetic tree indicated regionally 283 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 11, 2020. . https://doi.org/10.1101/2020.10.08.20208546 doi: medRxiv preprint clustered appearances of phylogenetically related viruses, as most evident for Carrasco, 284
Pocitos, Malvin, Reducto (all in Montevideo), and Rivera ( Figure S5A) . 285 286
surrounding South American countries, resulting in five clusters 288
To assess global introductions and the regional spread of SARS-CoV-2 in 289 Uruguay, we performed discrete phylogeographic analysis using BEAST with all 73 290
Uruguayan sequences, complemented with 1737 global reference sequences, based on 291 the global subsampling dataset suggested by Nextstrain (Table S2) The results of the discrete phylogeography analysis we performed that 304 considered seven ancestral state locations further revealed that 18 of the 23 independent 305
introductions are inferred to have originated from other South American countries. The 306 remainder include two independent introductions of GISAID clade GR viruses from 307 Asia, two from North America, and one clade V virus from Oceania (Figure 3) . The 308 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 11, 2020. . https://doi.org/10.1101/2020.10.08.20208546 doi: medRxiv preprint viral sequences from the only two participants in our cohort that reported a possibility 309 of travel-related infection after returning from Japan are inferred to have been imported 310 from Asia and grouped on the tree within separate clades of Asian sequences. The 311 estimated time to the most recent common ancestor (TMRCA), represented by the age 312 of the root node of the entire tree, is in mid-December 2019 (9 th -21 st of December), 313 which is in agreement with the estimated origin of the pandemic in the Hubei Province 314 in China, sometime between October and December, and the first contracted case in 315
China recorded in mid-November [28] [29] [30] [31] . 316
Only five of the 23 independent virus introductions into Uruguay resulted in 317 monophyletic clades with more than two sampled sequences in the country. These 318 clades comprised 21, 18, 5, 4, and 3 sequences, respectively (Figures 3 and 4 , Figure  319 S8), suggesting that these viral outbreaks were maintained by community transmission 320 once introduced into Montevideo. 321
To investigate the timing of the introduction of the viruses that founded these 322 five main clades circulating in Montevideo, we estimated the time of their most recent 323 common ancestor (TMRCA), acknowledging that the actual introduction events likely 324 occurred even before the corresponding TMRCAs. 325
The TMRCA of the first main clade of 21 sampled sequences, highlighted in red 326
in Figure 4 , was estimated to fall between the 2 nd and 5 th of March, 2020 and involved 327 the importation of a GISAID clade S virus. Viruses within this transmission cluster 328 were restricted to Montevideo, where they were distributed among nine of the local 329 neighborhoods, including two Hospitals and one research institute (Figure S8) . A North 330
American sequence from Mexico was positioned basally to this clade on the tree 331 identifying this country as the most plausible source location. 332 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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The TMRCA of the second main clade, which comprised of 18 virus sequences, 333 highlighted in yellow in Figure 4 , was estimated to fall between the 13 th and 17 th March 334 2020 and involved the import of a GISAID clade G virus. Viruses within this 335 transmission cluster were restricted to the city of Montevideo, where they were 336 distributed among nine neighborhoods and included samples from two nursing homes 337 and one hospital ( Figure S8) . The viral sequences in this clade were inferred to have a 338
South American origin. 339
The TMRCA of the third main clade of five sampled sequences, highlighted in 340 blue in Figure 4 and corresponding to a GISAID clade GR, was estimated to fall 341 between March 20 th and May 26th, 2020. This transmission cluster consisted of five 342 viruses from the Hospital de Rivera in Rivera, a small city situated on the border with 343
Brazil (Figure S8 ). This clade was inferred to have originated in South America and 344 groups with a sequence from Brazil on the MCC tree suggesting this was the source 345 location for this viral introduction 346
The TMRCA of the fourth main clade with four sampled sequences, highlighted 347 in purple in Figure 4 , was estimated to fall between March 14 th and May 20 th , 2020 and 348 involved the introduction of a GISAID clade G virus into Montevideo (Figure 4) . This 349 clade comprised four viruses from within two Montevideo neighborhoods ( Figure S8) . 350
The virus responsible for this introduction was also inferred to have originated in South 351
The TMRCA of the fifth main clade of three sequences, highlighted in green in 353 Collectively, sequences from the four main clades described above were sampled 357 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 11, 2020. 
Within these five clades we identified a total of ten sequence clusters (sequences 369 from the same institution that group together on the tree), spread across six of the seven 370 health institutions from which we had more than one sample ( Figure S8) . These 371 included one cluster of five GISAID clade GR sequences in the Hospital de Rivera (red 372 dots), two clusters of two clade G sequences in the Hospital Vilardebó (green dots), 373 three clusters each comprised of two clade S sequences in the Institut Pasteur (brown 374 dots), and two clusters from the Asociación Española Primera en Salud (blue dots) 375 comprising two S and two G clade sequences (Figure S8) . 376
The availability of study participants' clinical and demographic data combined 379 with mutational data of the infecting SARS-CoV-2 viruses enabled us to perform 380 comprehensive correlation analyses (Figures 5 and 6, Figures S9-S11) . Overall, 381
Spearman rank correlation analyses revealed six prominent correlation clusters of 382 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 11, 2020. . https://doi.org/10.1101/2020.10.08.20208546 doi: medRxiv preprint significant positive or mixed correlations (Figure 5, Figure S9) . Four of the highlighted 383 clusters in Figure 5 (clusters 1-4) are dominated by mutations that form mixed clusters 384 with demographic parameters such as sampling location or treating healthcare 385 institution and a few other parameters. Clusters 5 and 6 are different in forming separate 386 clinical correlation clusters together with the parameters "sex" (cluster 5) or "age" 387 (cluster 6) without essential associations to virus mutations. Cluster 5 reveals a 388 significant association of female sex with clinically asymptomatic courses of the disease 389 and a lower risk of developing fever. Cluster 6 indicates a network of positive 390 correlations among age, lethal outcome, and five clinical parameters. In addition to the 391 highlighted clusters, we observed some smaller clusters, mainly composed of mutations. 392
The tight network of positive clinical correlations and a more outspread correlation 393 network of clades with regional appearances and selected demographic parameters are 394 shown in greater detail in Figure 6A . Specifically, statistical analyses of lethal outcome 395 as study parameter revealed significant positive correlations with arterial hypertension 396 (AHT), kidney failure and ICU admission complemented by borderline-significant 397 associations with additional clinical parameters (hospitalization, diabetes mellitus II, 398 and obesity) and age, but no association with any specific mutation ( Figure 6B, Figure  399 S10). Accordingly, the spike D614G mutation and clade G-related viruses, in 400 consequence, are not associated with any clinical parameters, severity or lethality. 401 D614G only correlates with co-occurring/inversely occurring mutations, treating 402 healthcare institutions, and time since sampling started ( Figure 6C, Figure S11 ). 403
Fatality rates among clade G, GR, and S-infected individuals were comparable at 18% 404 (3/17), 14% (1/7), and 12% (2/17), respectively. 405 406 Discussion 407 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 11, 2020 . . https://doi.org/10.1101 /2020 The Uruguayan epidemic is characterized by an early clade S dominance that 408 was subsequently replaced by clade G variants (Figures 1 and 2) , which matches the 409 global trend [27, 32, 33] . Uruguayan clade S viruses are characterized by the key 410 mutations T28144C, causing the AA replacement L84S in ORF8, and C8782T, in 411 agreement with global clade S strains [27] . In our data set, we observed three additional 412 co-occurring SNPs in ORF1b and ORF3 that were present in 15 out of 17 clade S 413 variants, i.e., C17470T, C25521T, and C26088T. These mutations are less common and 414 are presumably characteristic of the regional outbreak. Further studies need to show 415 whether this subclade will be fixed in regional and/or supraregional epidemics, and 416 whether founder effects or functional features accounted for the early clade S 417 dominance over the original clade as well as the subsequent fluctuating prevalence and 418 decline. According to the CoV-GLUE database, ORF8 L84S is the 8th most prevalent 419 AA replacement to date [34] . After a controversial debate about the ancestry of L and S 420 types and the functional impact of L84S [35] [36] [37] [38] , the still limited amount of data 421 indicates that L84S might confer selection advantage and render the virus more virulent 422 based on destabilizing the immuno-and replication-modulatory protein ORF8 and 423 mitigating binding of ORF8 to human complement C3b [35, [37] [38] [39] . 424
End of March/early April 2020, we observed a subsequent switch in dominance 425 from clade S to clade G-variants (G, GR, and GH) (Figures 1 and 2) . It positioned 426
Uruguay somewhere in the middle in the asynchronous transition process from spike 427 D614 to G614 virus predominance, i.e., between the early European and the mostly late 428 Asian countries [32, 33] . Diverse structural and functional assays strongly suggest that 429 the spike D614G mutation renders SARS-CoV-2 more infectious by stabilizing its 430 structure, i.e., through impact on the, compared to SARS-CoV-1, even more fragile 431 metastable SARS-CoV-2 spike protein, thus reducing the shedding of the S1 subunit. 432 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 11, 2020 . . https://doi.org/10.1101 /2020 Furthermore, D614G triggers higher spike numbers on the virion surface and induces a 433 more open, receptor-binding domain (RBD)-up spike conformation toward a receptor-434 binding and fusion-competent state [33, 35, 37, [40] [41] [42] [43] [44] . In D614G spikes, binding to the 435 angiotensin-converting enzyme 2 (ACE2) receptor is not increased, and SARS-CoV-2 436 viruses do not acquire D614G escape mutations in vitro under neutralizing antibody 437 immune pressure [40, 45] . Instead, D614G increases neutralization susceptibility of 438 SARS-CoV-2, which assures high sensitivity to vaccination-induced neutralizing 439 antibodies [46, 47] . 440
Although D614G serves as the clade G-defining mutation with likely effects on 441 virus infectivity/transmissibility, D614G is governed by a very strict co-appearance with 442 C241T, C3037T, and C14408T, both in Uruguayan and global G variants (Figure 5 , 443 Figure S5 ) [27] . Notably, in addition to the D614G-causing A23403G mutation in 444 spike, C14408T is responsible for the AA replacement P323L in the RdRp gene. Based 445 on their central roles in viral entry and replication, their co-evolution is of particular 446 interest, and a mutual contribution to the selective advantage of G-haplotypes is 447 assumed [33] . Interestingly, none of the viruses harboring a single clade G mutation 448 prevailed to achieve epidemiological relevance, e.g., D614G alone or P323L alone have 449 ≤ 0.3 global prevalence, whereas D614G and P323L together have ~70% global 450 prevalence as of August 2020 [33] . P323L, although not located in the active center, 451 possibly influences RdRp fidelity through allosteric effects at the nsp12 interface with 452 the nsp8 cofactor and might increase the viral mutation rate [33, 39, 48 ] . Thus, the 453 strong correlation patterns between key mutations in our Uruguayan data set, mirroring 454 global patterns, allows us hypothesize that coupled mutations, such as D614G in spike 455 and P3233L in RdRp might synergize for the epidemiological success of the virus. It 456 may allow a fine balance between efficient transmission (e.g., by D614G, even in 457 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 11, 2020. . https://doi.org/10.1101/2020.10.08.20208546 doi: medRxiv preprint asymptomatic cases) and limited clinical presentation (e.g., by P323L, decreasing the 458 production of viral RNA) to eventually attenuate an aggressive virus that, as shown for 459 MERS and SARS-CoV-1, is more vulnerable to viral clearance with lack of long-term border with Brazil, has been a main concern for the government and many outbreaks 477 occurred along the border that were rapidly brought under control. In line with our 478 BEAST data that suggested a single transmission from Brazil as the main source of the 479 Rivera infections, the first Rivera outbreak was reported to have begun when a COVID-480 19-positive Uruguayan was diagnosed on May 7 th , 2020. Our BEAST analysis strongly 481 supports the former hypothesis from the Uruguayan Government and Ministry of Health 482 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 11, 2020 . . https://doi.org/10.1101 /2020 (MSP) that a local metallurgic worker that used to travel to Brazil everyday had been 483 infected with SARS-CoV-2 in Brazil and introduced this strain to Rivera [49, 50] . Montevideo neighborhoods, and instead, limited our analysis to describing the spatial 500 relationships among the sampled SARS-CoV-2 sequences. 501
Our discrete phylogeographic analysis provided a new perspective to the 502 believed origin of the Uruguayan SARS-CoV-2 outbreak from overseas by a single 503 traveler returning from Europe [52] [53] [54] . While overall similarities of Uruguayan 504 sequences with European and Asian sequences were observed in mutation patterns and 505 genetic distance-based haplotype networks, our phylogeographic analyses using BEAST 506 indicate that Uruguayan introductions that resulted in outbreaks were mostly restricted 507 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 11, 2020 . . https://doi.org/10.1101 /2020 to neighboring South American countries (Figure 3) , which stands in contrast to the 508 suggestions of a recent preprint article [7] . Instead, our data support the idea that the 509 outbreaks that were seeded after return from overseas travel, were contained 510 successfully through social distancing, mask wearing, rigorous testing, contact tracing, 511 and partial lockdown [2] . 512
Having determined the regional SARS-CoV-2 mutation patterns and 513 phylogeographic spread, the question remained whether and to what extent genomic 514 features are coupled to demographic and/or clinical parameters. Our correlation data 515 revealed significant clusters of co-occurring or mutually excluding mutations with 516 regionally accumulated appearances of clades/mutation clusters (Figures 4 and 5 , 517 Figure S5 ). In contrast, the large bulk of clinical parameters clustered separately 518 without major influence from viral mutations (Figures 5 and 6, Figure S10 ), which is 519 in line with a recent publication that reported no significant impact of genetic variation 520 on clinical outcome [55] . More specifically, we studied pairwise correlations with the 521 spike D614G mutation, which, because of perfectly matching mutation patterns, also 522 represents correlation analyses for the RdRp P323L mutation or G-related clades (G, 523 GR, and GH). In an FDR-adjusted analysis, the presence of D614G mutation was 524 coupled to other viral mutations, treatment of the infected patients in a regional 525 healthcare institution, and late sampling, but not to clinical parameters (Figures 5 and  526 6, Figure S11 ). There have been controversial reports of D614G being associated with 527 higher fatality rates and/or severe illness in a few data sets [56, 57] , whereas more recent 528 data suggests no correlations of D614G with clinical outcome [33], the latter supporting 529 our findings. Our analysis of associations with clinical parameters pointed at significant 530 associations between lethal outcome and arterial hypertension, kidney failure, and ICU 531 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 11, 2020. . https://doi.org/10.1101/2020.10.08.20208546 doi: medRxiv preprint admission, which mirrors clinical studies on associated factors or predictors of disease 532 severity/progression [58, 59] . 533
In sum, our characterization of Uruguayan SARS-CoV-2 phylogenetics, 534 mutation patterns, and their correlation with demographic and clinical parameters did 535 not identify critical viral attenuations or clinical peculiarities that can primarily account 536 for the exceptionally well curbed regional COVID-19 epidemic [2] . It instead suggests 537 that socio-epidemiologic mitigation strategies managed to curtail COVID-19 to 538 restricted regional transmission clusters in Uruguay. 539
We hope that these findings contribute to define the South American COVID-19 540 outbreak better, to optimize and develop efficient, fast, and low-cost mitigation 541 strategies and diagnostic pipelines for Uruguay and other countries, and to assist 542 physicians dealing with strategies for this and future emerging infections. 543 544 Acknowledgments 545
The authors thank all participants who agreed to participate in this study and the 546 healthcare personnel in Uruguay for their dedication to COVID-19 patients' care. We 547 wish to acknowledge the support of New York University's Data Services, Bobst 548 Library, and, in particular, the expertise shared by Christopher Schwarz and Senior 549 Academic Technology Specialist Denis Rubin in software script development. We also 550 acknowledge the support of the NYU Langone Health High-Performance Computing 551 resource and the Laura and Isaac Perlmutter Cancer Center, which partially supports the 552 Genome Technology Center. We thank the scientists across the world who deposited 553 SARS-CoV-2 sequences in GISAID, especially those who deposited 29 Uruguayan 554 sequences that supplemented our 44 sequences. We would also like to thank Flavia 555
Camacho for administrative assistance. 556 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
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The authors declared no potential conflicts of interest with respect to the 559 research, authorship, and/or publication of this article. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
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CoV-GLUE. CoV-GLUE -Amino acid replacements 2020. Available from: 677 http://cov-glue.cvr.gla.ac.uk/#/replacement 678 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
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