key: cord-1042193-22wu0cwj
authors: Pandey, Utsav; Yee, Rebecca; Shen, Lishuang; Judkins, Alexander R; Bootwalla, Moiz; Ryutov, Alex; Maglinte, Dennis T; Ostrow, Dejerianne; Precit, Mimi; Biegel, Jaclyn A; Bender, Jeffrey M; Gai, Xiaowu; Bard, Jennifer Dien
title: High Prevalence of SARS-CoV-2 Genetic Variation and D614G Mutation in Pediatric Patients with COVID-19
date: 2020-11-13
journal: Open Forum Infect Dis
DOI: 10.1093/ofid/ofaa551
sha: da1bbec3e267c92aeea65efe052fea48846c45ff
doc_id: 1042193
cord_uid: 22wu0cwj

BACKGROUND: Full spectrum of disease phenotype and viral genotype of COVID-19 have yet to be thoroughly explored in children. Here, we analyze the relationships between viral genetic variants and clinical characteristics in children. METHODS: Whole genome sequencing was performed on respiratory specimens collected on all SARS-CoV-2 positive children (n=141) between March 13 to June 16, 2020. Viral genetic variations across the SARS-CoV-2 genome were identified and investigated to evaluate genomic correlates of disease severity. RESULTS: Higher viral load was detected in symptomatic patients (p=0.0007) and in children <5 years old (p=0.0004). Genomic analysis revealed a mean pairwise difference of 10.8 SNVs and the majority (55.4%) of SNVs led to an amino-acid change in the viral proteins. The D614G mutation in the spike protein was present in 99.3% of the isolates. The calculated viral mutational rate of 22.2 substitutions/year contrasts the 13.5 substitutions/year observed in California isolates without the D614G mutation. Phylogenetic clade 20C was associated with severe cases of COVID-19 (p=0.0467, OR=6.95). Epidemiological investigation revealed major representation of 3 of 5 major Nextstrain clades (20A, 20B and 20C) consistent with multiple introductions of SARS-CoV-2 in Southern California. CONCLUSIONS: Genomic evaluation demonstrated greater than expected genetic diversity, presence of the D614G mutation, increased mutation rate, and evidence of multiple introductions of SARS-CoV-2 into Southern California. Our findings suggests a possible association of phylogenetic clade 20C with severe disease but small sample size precludes a definitive conclusion. Our study warrants larger and multi-institutional genomic evaluation and has implications for infection control practices.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID- 19) , was first reported in December 2019 in Wuhan, Hubei Province, China [1] . As of September 12, 2020 over 28 million COVID-19 cases and 916,992 deaths have been reported worldwide [2] . The growing significance of COVID-19 in the pediatric population has become more evident in recent months, as cases have grown from a reported 1.7% in April to 7.7% of total reported U.S. cases in August, 2020 [3] . Between July-August 2020 alone, 179,990 new cases were reported in children, a 90% increase [4] . While COVID-19 in children is often reported to be much less severe, analysis of pediatric COVID-19 hospitalization data from 14 states in the US found that 1 in 3 hospitalized children were admitted to an intensive care unit (ICU) [5] . Awareness of the risk of serious long-term sequelae of COVID-19 in children such as Multisystem Inflammatory Syndrome in Children (MIS-C) has increased [6, 7] . Hence, even amongst children with asymptomatic or mild disease manifestations, diagnosis and monitoring of disease course has become a key public health imperative as we seek to safely re-open schools and restart other public activities.

Evaluation of the viral genome during a pandemic can aid in understanding not only the scale and epidemiology of the outbreak, but also provide valuable insights into viral evolution, including potential links to transmissibility and disease severity [8, 9] . Studies investigating SARS-CoV-2 genomes have identified mutations that could potentially alter viral pathogenicity and transmissibility [10, 11] , however few have focused on how this may impact pediatric COVID-19 patients and were limited in scope and sample size. As of September 1, 2020, California has 756,181 confirmed COVID-19 cases and 14,273 deaths [4] . The vast majority of these cases are in Southern California. Los Angeles County has the highest number of confirmed cases and second-highest reported deaths in the US [2] . However, genomic data from these cases are currently limited, with majority of the genomes deposited in GISAID database originating in Northern California [12] .

Using viral whole-genome sequencing (WGS) of clinical specimens we evaluated the associations between disease severity and viral genetic variations in children. This is the largest single institution pediatric COVID-19 case series with integrated clinical and genomic epidemiology reported to date. As such, it represents a significant contribution to the body of knowledge regarding the clinical, genomic, and epidemiological features of COVID-19 in children.

Specimens from 141 COVID-19 patients were obtained from Children's Hospital Los Angeles (CHLA) between March 19 to June 16, 2020. Patients were tested for a variety of reasons including COVID-19 related symptoms, hospital admission unrelated to COVID-19, and asymptomatic pre-procedural screening. Clinical data including age, gender, ethnicity, hospitalization status, coexisting conditions, antimicrobial therapy, modes of oxygen supplementation, history of travel and contacts, clinical signs and symptoms, laboratory test results, and radiographic findings were obtained from the electronic medical record (EMR). Patients were classified based on the following clinical signs, symptoms and radiographic findings using Centers for Disease Control and Prevention's classification: a) Asymptomatic or presymptomatic infections: positive SARS-CoV-2 test in the absence of symptoms; b) Mild illness: signs and symptoms associated with upper respiratory tract M a n u s c r i p t 4 infections (URI) (e.g. fever, cough, malaise, sore throat, headache, vomiting, diarrhea) but without shortness of breath, dyspnea or abnormal chest imaging; c) Moderate illness: evidence of lower respiratory tract infection (LRTI) or radiographic findings and who have saturation of oxygen (SpO2) ≥94% on room air; d) Severe illness: SpO2 <94% on room air, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO2/FiO2) <300 mmHg, respiratory frequency >30 breaths per minute, or lung infiltrates >50%; e) Critical illness: Individuals who have respiratory failure, septic shock, and/or multiple organ dysfunction [13] .

Nasopharyngeal swabs were sent to the Clinical Virology Laboratory at Children's Hospital Los Angeles for testing: 1) Total nucleic acid was extracted using the NucliSENS easyMag (bioMérieux, Durham, NC, USA) followed by real-time reverse transcription-polymerase chain reaction (RT-PCR) using the CDC 2019-Novel Coronavirus Real-Time RT-PCR assay; 2) Total nucleic acid was extracted with the Thermo Fisher KingFisher Flex (Thermo Fisher Scientiftic, Waltham, MA, USA) with the MagMAX Viral and Pathogen Nucleic Acid Isolation Kits followed by RT-PCR using TaqPath COVID-19 Combo Kit. Viral load (copies/mL) were calculated based on a standard curve generated by testing samples with known viral copy numbers. Briefly, viral samples consisting of a gradient of known viral copy numbers (2.0 x 10 7 copies/mL to 2 x 10 3 copies/mL) were processed followed by RT-PCR in triplicates. A standard curve was generated by plotting a linear line in a graph with Ct values plotted on the y-axis and viral load in log (copies/mL) on the x-axis.

WGS of extracted viral RNA was performed as previously described using Paragon Genomics CleanPlex SARS-CoV-2 Research and Surveillance NGS Panel [14] . Libraries were quantified and normalized to approximately 7nM and pooled to a final concentration of 4nM; libraries were denatured and diluted according to Illumina protocols and loaded on the MiSeq at 10pM. Paired-end and dual-indexed 2x150bp sequencing was done using Micro Kit v2 (300 Cycles). Sample performance was selected based on the following metrics: average depth ≥ 1000x, and variant calling at ≥100x.

Nucleotide sequences were aligned with NovoAlign (Novocraft Technologies, Selengor, Malaysia). Coverage profiles, variant calls and consensus genomes were generated using Lightweight Utility tools of Bioinformatics Analysis (LUBA), an in-house proprietary software system [15] . Consensus sequences were built by adjusting the reference genome at high allele frequency (≥ 0.5) SNV and indel loci. Base quality adjusted pileup was generated, and the reference sequence modified accordingly at positions with alternative bases and IN/DELS that accounted for more than 50% of the aligned reads.

Consensus genomes obtained for the 141 CHLA isolates were compared to the Wuhan isolate (NC_045512.2) using SARS-CoV-2 Genome App v1.1 (https://cov2annot.cpmbiodev.net) and CHLA COVID-19 Analysis Research Database (CARD) [16] to identify synonymous, non-synonymous and A c c e p t e d M a n u s c r i p t 5 intergenic variations. CHLA isolates were also compared against the global collection of 77,966 available SARS-CoV-2 sequences in CARD to identify Southern California specific haplotypes.

Phylogenetic analysis and evolutionary rate estimation were performed using packages available through Nextstrain command-line interface (v 2.0.0.) [17] . Consensus sequences for 141 CHLA isolates were combined with full-length SARS-CoV-2 sequences of 436 additional isolates from GISAID that carried the identical, ancestral, or descendent haplotypes as the 141 isolates (Supplementary Table 1 ) to generate a multiple sequence alignment with MAFFT (version 7.460) using speed-oriented option -FFT-NS-i (iterative refinement method, two cycles) optimized for large datasets [18] . A maximum likelihood tree using Bayesian information criteria was generated with IQ-TREE (version 2.0.3) [19] using GTR substitution model. The resulting rate estimation and phylogeny was then time-resolved using TreeTime (version 0.7.6) [20] and visualized using auspice [17] . Phylogenetic clade analysis was performed using Nextclade (version 0.3.7). Similar procedures were also followed for evolutionary rate estimation of 577 SARS-CoV-2 genomes without the D614G mutation, however, analysis was restricted to isolates from California.

Differences of genome copies/mL were compared using Mann-Whitney test. Association between viral genotypes and disease severity was investigated using Fisher's exact test and odds ratio calculation via a 2x2 contingency table. The groupings compared were the investigated phylogenetic clades (e.g. number of patients infected with clade 20C versus number of patients not infected with clade 20C) versus the different categories of disease severity (e.g. number of patients with severe disease versus number of patients without severe disease).

The genomic data and associated meta data necessary for phylogenetic analysis are now part of CHLA CARD [16] . Other de-identified clinical data shown in the manuscript are available upon request from the corresponding author.

Study design conducted at Children's Hospital Los Angeles was approved by the Institutional Review Board under IRB CHLA-16-00429 by which a waiver of consent was approved.

During a 13-week period (March 19 to June 16, 2020) we identified 141 pediatric patients with confirmed COVID-19. All patients resided in Southern California. Demographics and clinical presentation are summarized in Figure 1 and supplementary Table 2 . Most children in our cohort were of Hispanic ethnicity (95/141, 67.4%). Median age of the patients was 8.3 years (range: 10 days to 21 years), 53.2% were male and 13.4% (19) had underlying conditions. Sixteen (11.4 %) patients were admitted to the hospital, of which 14 (87.5%) were admitted due to COVID-19 related symptoms. Of the remaining 125 patients not requiring hospital admission, 48 (38.4%) were screened for pre-operative procedures, 48 (38.4%) presented to the emergency department (ED), M a n u s c r i p t 6 and 29 (23.2%) presented at outpatient clinics. Thirty-three (23.2%) patients had direct contact with individuals positive for COVID-19 or URI symptoms suspicious for COVID-19 suggesting community transmission of the virus plays an important role in pediatric cases. No cases were associated with a positive travel history. Median time to discharge of hospitalized patients was 3.0 days and no deaths were reported in this period.

Extensive chart review was conducted on 88 patients with available inpatient and outpatient EMRs to capture disease severity, comorbidities and clinical course. The majority of patients were symptomatic (62/88, 70.5%), including two patients that were presymptomatic. Whilst the majority (52/62, 83.9%) of symptomatic patients experienced mild infection, 16.1% (10/62) had moderate or severe COVID-19. Three (4.8%) suffered from severe COVID-19 and were admitted to the ICU ( Figure  1 ). Six (4.2%) patients required oxygen supplementation. Six of 16 patients (37.5%) with chestimaging had opacities in the lungs and 2 had massive lung infiltrations ( Figure 1 ). Of the 7 moderate and 3 severe cases, 4 patients total were <1 year old (2 moderate and 2 severe cases). All 3 severe cases were acute with no progression to MIS-C. There were also no critical cases in this cohort.

The median viral load obtained from all positive results was 2.3 x 10 7 copies/mL (range: 81 to 1.0x10 12 copies/mL) with higher viral load detected in symptomatic compared to asymptomatic patients (2.7x10 8 vs 9.9x10 5 copies/mL, p=0.0007) (Figure 2A ). Patients tested within two days of symptom onset also had a higher viral load compared to samples collected >2 days from symptom onset (9.2x10 8 vs 2.4x10 7 copies/mL, p=0.001) ( Figure 2B ). All patients <5 years old were symptomatic and had higher viral loads (2.3x10 8 vs 1.9 x10 6 copies/mL, p=0.0004) ( Figure 2C) . No difference in viral load was observed in association with chronic underlying conditions, gender, or disease severity. Table 3 ). SNVs were located mostly in ORFs, with highest numbers in ORF1ab (n=231) and S (n=42) (Figure 3a, supplementary Table 3 ). The majority (10/15) of the insertion/deletions caused a frame-shift mutation in ORF1ab. Notably, the recently described D614G mutation in the spike protein [10, 22] , caused by nucleotide G-to-A substitution at position 23,403 in the Wuhan reference strain NC_045512.2 (Figure 3b, supplementary Table 3 ), was present in all but one of our isolates (140/141, 99.3%). There were two other mutations that co-exist with the D614G mutation in all isolates located in ORF1ab: F924F (c.2772C>T) is a synonymous mutation while P4715L (c.14144C>T) is a non-synonymous mutation (Figure 3b ). Using CHLA CARD [16] to assess the incidence of these variations globally, we compared our isolates to a global database of 77,966 SARS-CoV-2 isolates worldwide and found 41 variations unique to the isolates present in our cohort, while the remaining 329 variations were present in at least one other global isolate (supplementary Table 3 ). These 41 variants unique to our cohort include 18 missense, 10 synonymous, 10 frameshift, 1 inframe, 1 stop loss and 1 start loss. Interestingly, each of the 10 frameshift mutations was M a n u s c r i p t 7 detected in one isolate. The only other frameshift mutation found in the cohort is the c.361delA mutation in ORF8 that was also found in only one isolate in our cohort but reported 25 times previously according to CHLA CARD. While the clinical significance of these variations remains unclear, they point towards genetic heterogeneity of SARS-CoV-2 isolates circulating in Southern California.

Phylogenetic analysis of the 141 SARS-CoV-2 genomes demonstrated clustering with other isolates from the US and across the world (supplementary Figure 1 ). All but one of the isolates from our cohort belonged to 3 of the 5 phylogenetic clades (20A, 20B, 20C) defined by Nextstrain [17, 23] (  Figure 1, supplementary Figure 1 ). The majority (61.0%, 86/141) of isolates belong to phylogenetic clade 20C, a largely North American clade (Figure 1 ). There was limited evidence of early emerging genotypes of SARS-CoV-2 in this cohort. Only one isolate without the D614G variation in the spike protein belonging to phylogenetic clade 19B was identified and none of the isolates belong to phylogenetic clade 19A, which consists of the very first isolates from Asia ( Figure 1 ).

The estimated evolutionary rate was calculated to be 7.4 × 10 -4 substitutions per site per year yielding, based on the SARS-CoV-2 30kb genome, an overall mutation rate of 22.2 substitutions per year (supplementary Figure 2a) . The inferred time to most recent common ancestor (TMRCA) based on the molecular clock analysis of these isolates was 2019-12-24 (CI: 2019-11-25 -2019-12-26). To further investigate the association between viral isolates with the D614G mutation and the rate of viral evolution, we analyzed 577 viral genomes from California at different time points without the D614G mutation. The mutation rate of these isolates was 13.5 substitutions per year (supplementary Figure 2b ), substantially lower than what was observed for our isolates (supplementary Figure 2a) .

Comparison of viral sequences revealed significant variations across the viral genome, a subset of which are illustrated in Figure 2b . In fact, only 23 of 370 SNVs across the viral genome were present in more than 5% (7/141) of the isolates regardless of the disease phenotype of the patient (supplementary Table 3 ), which made attempts to examine potential associations of individual variants with disease severity significantly underpowered. There was some notable clustering for disease severity and phylogenetic clade, although the overall numbers were small. While the viral isolates detected from asymptomatic patients and patients with mild disease span all three phylogenetic clades (20A, 20B and 20C), LRTI was only seen in only patients with phylogenetic clades 20B and 20C, and more severe clinical course was only encountered in patients with phylogenetic clade 20C (Figure1, 3b) . In fact, 9 of 10 patients with either moderate or severe forms of COVID-19 were infected by virus from phylogenetic clade 20C, which is significantly higher (p = 0.0467, OR=6.95) than the fraction of patients with mild or asymptomatic infections (44/78) (Figure 3b) . No other segregation of clinical and demographic features including age, race/ethnicity, and presence of comorbidities based on viral genotypes was observed (Figure 1 ).

Pediatric patients can present with a broad spectrum of clinical manifestations associated with COVID-19, ranging from asymptomatic infections to severe illness. Over half of our 141 pediatric cohort was symptomatic with a subset requiring hospitalization. Four of 10 cases classified as M a n u s c r i p t 8 moderate to severe cases were in patients < 1 year, consistent with reports of this age group accounting for the highest percentage of hospitalization in pediatric COVID-19 cases [3, 24] . When compared to a recent MMWR summarizing signs and symptoms in 291 pediatric patients, shortness of breath (14.3%) or cough (49.2%) in our patient cohort was comparable but fever was more prevalent in our patients at 71.4% compared to 56% [25] . On the other hand, the hospitalization rate and ICU admission reported in our study was consistent with the estimated ranges reported of 5.7-20% and 0.58-2.0%, respectively [25] . However, only a small number of patients in our study experienced moderate illness with LRTI compared to approximately 65% of 171 children in China with confirmed COVID-19 [25] . These variabilities in disease severities emphasizes the critical need for pediatric specific studies to identify and/or confirm the possible contribution of host factors including age, ethnicity, and immune status.

We observed significantly higher viral loads in symptomatic patients and in children < 5 years of age, corroborating findings from a recent study that demonstrated a significantly higher amount of viral nucleic acid in young children [26, 27] . Association between viral load and days from symptoms onset is consistent with findings from other studies that reported detection of viral RNA as early as one day post symptom onset and highest viral loads in the first two days of symptoms onset [28] [29] [30] [31] . These findings have direct implications for infection control within the community and hospital setting.

Genomic evaluation of SARS-CoV-2 in this cohort demonstrated that nearly all of our isolates carried the D614G mutation in the spike protein. The prevalence of this mutation in our patients earlier in the pandemic (95% in March/April) was particularly striking when compared to other California isolates in the GISAID database, which showed a 10% prevalence in February, 65% in March and not achieving a comparable prevalence of 96% until June. The D614G mutation is shown to have been rapidly fixed in isolates from Europe and North America and has been associated with lower Ct values in vitro and in vivo, but not with disease severity nor case fatality rates [10, 31] . The role of the D614G mutation in SARS-CoV-2 pathogenicity continues to be a focus of active investigation. While some studies suggest this mutation may increase transmissibility, a clear consensus awaits further investigation [32] [33] [34] . In this pediatric cohort the mutation does not appear to be a primary driver of disease severity as it was present in all spectrum of disease, ranging from asymptomatic to severe infection. We also identified the two common mutations (F924F and P4715L) alongside the D614G mutation in our isolates [35] . ORF1ab P4715L is located in Nsp12 which is important for viral replication. A comprehensive analysis of over 12,300 SARS-CoV-2 genome sequences across 28 countries reported that mutation at P4715L and D614G may correlate with higher fatality rates [36] . Nevertheless the functional significance of these mutations still needs to be fully investigated. Confirmation of these observed mutations and better understanding of its potential significance will require sequencing and correlatives studies of larger numbers of pediatric COVID-19 cases.

We also observed an unexpectedly increased mutation rate (22.5 substitutions per year) in this cohort when compared to other SARS-CoV-2 cases from California without the D614G mutation during the same period (13.5 substitutions per year). The degree to which this difference reflects an underlying biologic difference versus the temporal and geographic dynamics of the pandemic in California remains to be determined. However, the effect of this higher than expected rate of mutation is readily apparent in the unexpected diversity of variation across the viral genome as illustrated in Fig. 2B . We postulate that the increased transmissibility caused by D614G may have M a n u s c r i p t 9 also led to this increased mutation rate. Nonetheless, the clinical and biological implications remain to be carefully ascertained.

From a genomic epidemiology perspective, the isolates in our study almost exclusively belonged to the three most recent SARS-CoV-2 phylogenetic clades (20A, 20B, 20C) as defined in Nextstrain [17] . This is consistent with previously described patterns of multiple introduction and community transmission of SARS-CoV-2 lineages in California [37, 38] . Analysis of the genome sequences from the 22 pediatric patients in California not in this study that are currently available for evaluation confirmed the same 3 phylogenetic clades, but with a difference in clade distribution. In these cases phylogenetic clade 20A was most prominent (data not shown). The genetic diversity among the isolates points to multiple potential introductions of the virus in Southern California from across the US and the world. An unexpected finding in our cohort was an apparent association between disease severity and clade 20C. Recognizing the limitations of small case numbers, it is notable that all severe cases and all but one moderately severe case fell into the phylogenetic clade 20C. Two additional mutations (C1059T and G25563T) differentiate phylogenetic clade 20C from 20A and 20B and at least one of these may potentially be associated with increased disease severity [39] . G25563T leads to the Q57H missense mutation ORF3a that is associated with host cell apoptosis and contribute to increased infectivity and virulence [40, 41] . Confirmation of the effect of these mutations, as well as well as further exploration of the genotype-phenotype correlation of phylogenetic clade 20C, warrant further examination in larger scale pediatric populations.

There are some limitations that warrants discussion. First, this is a single-center study on 141 pediatric patients in Southern California with clinical characteristics available on 88 patients. Although we observed interesting associations between viral genotypes and disease severity, this study was not sufficiently powered to establish a statistically significant correlation between individual viral genetic variations and disease severity. Finally, due to the over-representation of Hispanic population (67.3%) in our pediatric cohort, the clinical and genomic findings from this study may not reflect all ethnic groups. Notwithstanding the predominant Hispanic population in this cohort, 6 of the10 moderate or severe cases occurred in non-Hispanic patients. Data from a larger and geographically diverse pediatric population will be required to better characterize these potential associations between disease severity and viral genetic variations.

This study represents the largest single institution pediatric COVID-19 case series with integrated clinical, genomic, and epidemiological characteristics in the US. While we observed limited shared variations between the isolates, our findings demonstrate unique variation in each isolate at the cohort or population level including observation of moderate and severe COVID-19 cases corresponding almost exclusively to phylogenetic clade 20C. Furthermore, the majority of these variants led to amino-acid changes in the viral proteins, possibly indicating an ongoing adaptation of the virus in human population. This genetic diversity of SARS-CoV-2 at the population level has not been well appreciated in previous description of SAR-CoV-2 in children and suggests that substantial collaborative efforts will be required across multiple institutions to confidently establish an association between genetic variations and any specific disease manifestation in pediatric COVID-19 patients. Disease severity: pink = Severe, orange = Moderate, yellow = Mild, green = Asymptomatic. Race/ethnicity: blue = Hispanic, white = unknown, purple = Black, grey = Other, red = Asian, green = White. Location: red = ICU, blue = Inpatient, green = ED, pink = Outpatient. Age: green = ≥ 10 years, grey = ≥ 5 to < 10 years, black = ≥ 1 to < 5 years, blue = < 1 year. Chronic diseases: red = present, green = absent. Days since symptom onset: purple = 1-1.5 days, black = >2 days, white = unknown. Imaging: red = chest imaging with significant findings, green = clear, white = unknown. Figure 2 . Viral load comparison amongst patient groups. Increased viral loads were observed in A, symptomatic individuals, B, patients tested within two days of symptoms, and C, patients that were <5 years old. The line depicts the median viral load. Statistical analysis was performed by Mann Whitney Test (** p < 0.005, *** p < 0.0005) 

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M a n u s c r i p t 10

We would like to acknowledge the staff members of the Clinical Microbiology and Virology laboratory and Center for Personalized Medicine at Children's Hospital Los Angeles for dedication towards providing excellent patient care. We thank Emily Gai for her assistance in editing and formatting of viral sequences. We would like to acknowledge the frontline healthcare workers who remain devoted in the fight against COVID-19. We would also like to acknowledge NCBI, GISAID, and Nextstrain for providing valuable resources for SARS-CoV-2 genomics.

This study was partly supported by the Children's Hospital Los Angeles, The Saban Research Institute.

All authors declare no conflict of interest.A c c e p t e d M a n u s c r i p t 11 A c c e p t e d M a n u s c r i p t