Last data update: Apr 22, 2024. (Total: 46599 publications since 2009)
Records 1-19 (of 19 Records) |
Query Trace: Marine RL[original query] |
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Serological and metagenomic interrogation of cerebrospinal fluid implicates enteroviruses in pediatric acute flaccid myelitis (preprint)
Schubert RD , Hawes IA , Ramachandran PS , Ramesh A , Crawford ED , Pak JE , Wu W , Cheung CK , O'Donovan BD , Tato CM , Lyden A , Tan M , Sit R , Sowa GA , Sample HA , Zorn KC , Banerji D , Khan LM , Bove R , Hauser SL , Gelfand AA , Johnson-Kerner BL , Nash K , Krishnamoorthy KS , Chitnis T , Ding JZ , McMillan HJ , Chiu CY , Briggs B , Glaser CA , Yen C , Chu V , Wadford DA , Dominguez SR , Ng TFF , Marine RL , Lopez AS , Nix WA , Soldatos A , Gorman MP , Benson L , Messacar K , Konopka-Anstadt JL , Oberste MS , DeRisi JL , Wilson MR . bioRxiv 2019 666230 Background Since 2014, the United States has experienced a biennial spike in pediatric acute flaccid myelitis (AFM). Epidemiologic evidence suggests non-polio enteroviruses (EVs) are a potential etiology, yet EV RNA is rarely detected in cerebrospinal fluid (CSF) and only inconsistently identified from the respiratory tract, serum, or stool.Methods We interrogated CSF from children with AFM (n=42) and pediatric controls with other neurologic diseases (OND) (n=58). Samples were incubated with T7 bacteriophage expressing 481,966 sixty-two amino acid peptides with a fourteen amino acid overlap tiled across all known vertebrate virus and arbovirus genomes, an adaption of the VirScan method. Antibody-bound phage were deep sequenced to quantify enriched peptides with normalized counts expressed as reads per hundred thousand (rpK). EV antibody findings were confirmed with ELISA using whole viral protein 1 (VP1) from contemporary enterovirus (EV) A71 and D68 strains. Separately, metagenomic next-generation sequencing (mNGS) of CSF RNA, both unbiased and with targeted enrichment for EVs, was performed.Results The most significantly enriched viral family by VirScan of CSF in AFM versus OND controls was Picornaviridae (mean rpK 11,266 versus mean rpK 950, p-adjusted < 0.001, Wilcoxon signed-rank test with Bonferroni adjustment). Enriched Picornaviridae peptides belonged almost entirely to the genus Enterovirus. The mean EV VP1 ELISA signal in AFM (mean OD 0.51) was significantly higher than OND controls (mean OD 0.08, p-value < 0.001, Mann-Whitney test). mNGS did not detect additional enterovirus RNA in CSF.Conclusion Despite the rare detection of EV RNA in the CNS of patients with AFM, a pan-viral serologic assay identified high levels of CSF EV antibodies in AFM CSF compared to CSF from OND controls. These results provide further evidence for a causal role of non-polio enteroviruses in AFM. |
The effect of variant interference on de novo assembly for viral deep sequencing (preprint)
Castro CJ , Marine RL , Ramos E , Ng TFF . bioRxiv 2019 815480 Viruses have high mutation rates and generally exist as a mixture of variants in biological samples. Next-generation sequencing (NGS) approach has surpassed Sanger for generating long viral sequences, yet how variants affect NGS de novo assembly remains largely unexplored. Our results from >15,000 simulated experiments showed that presence of variants can turn an assembly of one genome into tens to thousands of contigs. This “variant interference” (VI) is highly consistent and reproducible by ten most used de novo assemblers, and occurs independent of genome length, read length, and GC content. The main driver of VI is pairwise identities between viral variants. These findings were further supported by in silico simulations, where selective removal of minor variant reads from clinical datasets allow the “rescue” of full viral genomes from fragmented contigs. These results call for careful interpretation of contigs and contig numbers from de novo assembly in viral deep sequencing. |
Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses (preprint)
Marine RL , Magana LC , Castro CJ , Zhao K , Montmayeur AM , Schmidt A , Diez-Valcarce M , Fan Ng TF , Vinje J , Burns CC , Allan Nix W , Rota PA , Oberste MS . bioRxiv 2019 705632 Next-generation sequencing is a powerful tool for virological surveillance. While Illumina® and Ion Torrent® sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. We evaluated the Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of assembled viral genomes, and an estimation of cost per sample to generate the data was calculated. The choice of library preparation kit and sequencing platform was found to impact the breadth of genome coverage and accuracy of consensus viral genomes. The Ion Torrent S5 outperformed the older Ion Torrent PGM platform in data quality and cost, and generated the highest proportion of reads for enterovirus D68 samples. However, indels at homopolymer regions impacted the accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., Ion Torrent 510 or Illumina MiSeq Nano V2), the cost per sample was lower on the MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 or Illumina MiSeq V2) the cost per sample was comparable. These findings suggest that the Ion Torrent S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of RNA viruses, each with specific advantages and tradeoffs. |
Nearly Complete Genome Sequences of Type 2 Sabin-Like Polioviruses from Northern Nigerian Poliovirus Surveillance, 2016 to 2018.
Zhao K , Schmidt A , Tang K , Castro CJ , Liu H , Pang H , Chen Q , Baba M , Soji OB , Bukbuk D , Akinola M , Adeniji JA , Marine RL , Ng TFF , Jorba J , Burns CC . Microbiol Resour Announc 2022 12 (1) e0073522 We sequenced 109 type 2 Sabin-like poliovirus isolates that had been collected from acute flaccid paralysis patients or healthy children in Nigeria. Understanding the genetic makeup of these viruses may contribute to polio eradication efforts. |
VPipe: an Automated Bioinformatics Platform for Assembly and Management of Viral Next-Generation Sequencing Data.
Wagner DD , Marine RL , Ramos E , Ng TFF , Castro CJ , Okomo-Adhiambo M , Harvey K , Doho G , Kelly R , Jain Y , Tatusov RL , Silva H , Rota PA , Khan AN , Oberste MS . Microbiol Spectr 2022 10 (2) e0256421 Next-generation sequencing (NGS) is a powerful tool for detecting and investigating viral pathogens; however, analysis and management of the enormous amounts of data generated from these technologies remains a challenge. Here, we present VPipe (the Viral NGS Analysis Pipeline and Data Management System), an automated bioinformatics pipeline optimized for whole-genome assembly of viral sequences and identification of diverse species. VPipe automates the data quality control, assembly, and contig identification steps typically performed when analyzing NGS data. Users access the pipeline through a secure web-based portal, which provides an easy-to-use interface with advanced search capabilities for reviewing results. In addition, VPipe provides a centralized system for storing and analyzing NGS data, eliminating common bottlenecks in bioinformatics analyses for public health laboratories with limited on-site computational infrastructure. The performance of VPipe was validated through the analysis of publicly available NGS data sets for viral pathogens, generating high-quality assemblies for 12 data sets. VPipe also generated assemblies with greater contiguity than similar pipelines for 41 human respiratory syncytial virus isolates and 23 SARS-CoV-2 specimens. IMPORTANCE Computational infrastructure and bioinformatics analysis are bottlenecks in the application of NGS to viral pathogens. As of September 2021, VPipe has been used by the U.S. Centers for Disease Control and Prevention (CDC) and 12 state public health laboratories to characterize >17,500 and 1,500 clinical specimens and isolates, respectively. VPipe automates genome assembly for a wide range of viruses, including high-consequence pathogens such as SARS-CoV-2. Such automated functionality expedites public health responses to viral outbreaks and pathogen surveillance. |
Next-Generation Sequencing of Human Respiratory Syncytial Virus Subgroups A and B Genomes.
Wang L , Ng TFF , Castro CJ , Marine RL , Magaña LC , Esona M , Peret TCT , Thornburg NJ . J Virol Methods 2021 299 114335 Human respiratory syncytial virus (HRSV) is a leading cause of acute respiratory illness in young children worldwide. Whole genome sequencing of HRSV offers enhanced resolution of strain variability for epidemiological surveillance and provides genomic information essential for antiviral and vaccine development. A 10-amplicon one-step RT-PCR assay and a 20-amplicon nested RT-PCR assay with enhanced sensitivity were developed to amplify whole HRSV genomes from samples containing high and low viral loads, respectively. Ninety-six HRSV-positive samples comprised of 58 clinical specimens and 38 virus isolates with C(t) values ≤ 24 were amplified successfully using the 10-amplicon one-step RT-PCR method and multiplexed in a single MiSeq run. Genome coverage exceeded 99.3% for all 96 samples. The 20-amplicon nested RT-PCR NGS method was used to generate >99.6% HRSV full-length genome for 72 clinical specimens with C(t) values ranging from 24 to 33. Phylogenetic analysis of the genome sequences obtained from the 130 clinical specimens revealed a wide diversity of HRSV genotypes demonstrating methodologic robustness. |
The effect of variant interference on de novo assembly for viral deep sequencing.
Castro CJ , Marine RL , Ramos E , Ng TFF . BMC Genomics 2020 21 (1) 421 BACKGROUND: Viruses have high mutation rates and generally exist as a mixture of variants in biological samples. Next-generation sequencing (NGS) approaches have surpassed Sanger for generating long viral sequences, yet how variants affect NGS de novo assembly remains largely unexplored. RESULTS: Our results from > 15,000 simulated experiments showed that presence of variants can turn an assembly of one genome into tens to thousands of contigs. This "variant interference" (VI) is highly consistent and reproducible by ten commonly-used de novo assemblers, and occurs over a range of genome length, read length, and GC content. The main driver of VI is pairwise identities between viral variants. These findings were further supported by in silico simulations, where selective removal of minor variant reads from clinical datasets allow the "rescue" of full viral genomes from fragmented contigs. CONCLUSIONS: These results call for careful interpretation of contigs and contig numbers from de novo assembly in viral deep sequencing. |
Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses.
Marine RL , Magana LC , Castro CJ , Zhao K , Montmayeur AM , Schmidt A , Diez-Valcarce M , Fan Ng TF , Vinje J , Burns CC , Allan Nix W , Rota PA , Oberste MS . J Virol Methods 2020 280 113865 Next-generation sequencing is a powerful tool for virological surveillance. While Illumina(R) and Ion Torrent(R) sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. The Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms were evaluated using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of assembled viral genomes, and an estimation of cost per sample to generate the data was calculated. The choice of library preparation kit and sequencing platform was found to impact the breadth of genome coverage and accuracy of consensus viral genomes. The Ion Torrent S5 510 chip runs produced more reads at a lower cost per sample than the highest output Ion Torrent PGM 318 chip run, and generated the highest proportion of reads for enterovirus D68 samples. However, indels at homopolymer regions impacted the accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., Ion Torrent 510 and Illumina MiSeq Nano V2), the cost per sample was lower on the MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 and Illumina MiSeq V2) there is less of a difference in the cost per sample between the two sequencing platforms ($5.47-$10.25 more per sample for an Ion Torrent 530 chip run when multiplexing 24 samples). These findings suggest that the Ion Torrent S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of RNA viruses, each with specific advantages and tradeoffs. |
Nearly Complete Genome Sequence of an Echovirus 30 Strain from a Cluster of Aseptic Meningitis Cases in California, September 2017.
Pan CY , Huynh T , Padilla T , Chen A , Ng TFF , Marine RL , Castro CJ , Nix WA , Wadford DA . Microbiol Resour Announc 2019 8 (44) We report the nearly complete genome sequence of a human enterovirus, a strain of echovirus 30, obtained from a cerebrospinal fluid specimen from a teenaged patient with aseptic meningitis in September 2017. |
Pan-viral serology implicates enteroviruses in acute flaccid myelitis.
Schubert RD , Hawes IA , Ramachandran PS , Ramesh A , Crawford ED , Pak JE , Wu W , Cheung CK , O'Donovan BD , Tato CM , Lyden A , Tan M , Sit R , Sowa GA , Sample HA , Zorn KC , Banerji D , Khan LM , Bove R , Hauser SL , Gelfand AA , Johnson-Kerner BL , Nash K , Krishnamoorthy KS , Chitnis T , Ding JZ , McMillan HJ , Chiu CY , Briggs B , Glaser CA , Yen C , Chu V , Wadford DA , Dominguez SR , Ng TFF , Marine RL , Lopez AS , Nix WA , Soldatos A , Gorman MP , Benson L , Messacar K , Konopka-Anstadt JL , Oberste MS , DeRisi JL , Wilson MR . Nat Med 2019 25 (11) 1748-1752 Since 2012, the United States of America has experienced a biennial spike in pediatric acute flaccid myelitis (AFM)(1-6). Epidemiologic evidence suggests non-polio enteroviruses (EVs) are a potential etiology, yet EV RNA is rarely detected in cerebrospinal fluid (CSF)(2). CSF from children with AFM (n = 42) and other pediatric neurologic disease controls (n = 58) were investigated for intrathecal antiviral antibodies, using a phage display library expressing 481,966 overlapping peptides derived from all known vertebrate and arboviruses (VirScan). Metagenomic next-generation sequencing (mNGS) of AFM CSF RNA (n = 20 cases) was also performed, both unbiased sequencing and with targeted enrichment for EVs. Using VirScan, the viral family significantly enriched by the CSF of AFM cases relative to controls was Picornaviridae, with the most enriched Picornaviridae peptides belonging to the genus Enterovirus (n = 29/42 cases versus 4/58 controls). EV VP1 ELISA confirmed this finding (n = 22/26 cases versus 7/50 controls). mNGS did not detect additional EV RNA. Despite rare detection of EV RNA, pan-viral serology frequently identified high levels of CSF EV-specific antibodies in AFM compared with controls, providing further evidence for a causal role of non-polio EVs in AFM. |
Antibodies to Enteroviruses in Cerebrospinal Fluid of Patients with Acute Flaccid Myelitis.
Mishra N , Ng TFF , Marine RL , Jain K , Ng J , Thakkar R , Caciula A , Price A , Garcia JA , Burns JC , Thakur KT , Hetzler KL , Routh JA , Konopka-Anstadt JL , Nix WA , Tokarz R , Briese T , Oberste MS , Lipkin WI . mBio 2019 10 (4) Acute flaccid myelitis (AFM) has caused motor paralysis in >560 children in the United States since 2014. The temporal association of enterovirus (EV) outbreaks with increases in AFM cases and reports of fever, respiratory, or gastrointestinal illness prior to AFM in >90% of cases suggest a role for infectious agents. Cerebrospinal fluid (CSF) from 14 AFM and 5 non-AFM patients with central nervous system (CNS) diseases in 2018 were investigated by viral-capture high-throughput sequencing (VirCapSeq-VERT system). These CSF and serum samples, as well as multiple controls, were tested for antibodies to human EVs using peptide microarrays. EV RNA was confirmed in CSF from only 1 adult AFM case and 1 non-AFM case. In contrast, antibodies to EV peptides were present in CSF of 11 of 14 AFM patients (79%), significantly higher than controls, including non-AFM patients (1/5 [20%]), children with Kawasaki disease (0/10), and adults with non-AFM CNS diseases (2/11 [18%]) (P = 0.023, 0.0001, and 0.0028, respectively). Six of 14 CSF samples (43%) and 8 of 11 sera (73%) from AFM patients were immunoreactive to an EV-D68-specific peptide, whereas the three control groups were not immunoreactive in either CSF (0/5, 0/10, and 0/11; P = 0.008, 0.0003, and 0.035, respectively) or sera (0/2, 0/8, and 0/5; P = 0.139, 0.002, and 0.009, respectively).IMPORTANCE The presence in cerebrospinal fluid of antibodies to EV peptides at higher levels than non-AFM controls supports the plausibility of a link between EV infection and AFM that warrants further investigation and has the potential to lead to strategies for diagnosis and prevention of disease. |
Complete Genome Sequences of Human Astrovirus Prototype Strains (Types 1 to 8).
Castro CJ , Reynolds E , Monroe SS , Marine RL , Vinje J . Microbiol Resour Announc 2019 8 (7) We report the complete genome sequences of the eight human astrovirus Oxford prototype strains. These sequences share 94.9% to 99.9% nucleotide identity with open reading frame 2 (ORF2) genes of astrovirus genomes previously deposited in GenBank and include the first complete genome of human astrovirus type 7. |
Strengthening laboratory surveillance of viral pathogens: Experiences and lessons learned building next-generation sequencing capacity in Ghana.
Marine RL , Ntim NAA , Castro CJ , Attiku KO , Pratt D , Duker E , Agbosu E , Ng TFF , Gatei W , Obodai E , Odoom JK , Walker CL , Rota PA , Oberste MS , Ampofo WK , Balajee SA . Int J Infect Dis 2019 81 231-234 OBJECTIVES: To demonstrate the feasibility of applying next-generation sequencing (NGS) in medium-resource reference laboratories in Africa to enhance global disease surveillance. METHODS: A training program was developed to support implementation of NGS at Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana. The program was divided into two training stages, first at the Centers for Disease Control and Prevention (CDC) in Atlanta, GA, followed by on-site training at NMIMR for a larger cohort of scientists. RESULTS: Self-assessment scores for topics covered during the NGS training program were higher post-training relative to pre-training. During the NGS Training II session at NMIMR, six enterovirus isolates from acute flaccid paralysis cases in Ghana were successfully sequenced by trainees, including two echovirus 6, two echovirus 11 and one echovirus 13. Another genome was an uncommon type (EV-B84), which has not been reported in Africa since its initial discovery from a Cote d'Ivoire specimen in 2003. CONCLUSIONS: The success at NMIMR provides an example of how to approach transferring of NGS methods to international laboratories. There is great opportunity for collaboration between institutes that have genomics expertise to ensure effectiveness and long-term success of global NGS capacity building programs. |
Genome Sequences of Rhinovirus Genotype C56 Detected in Three Patients with Acute Respiratory Illness, California, 2016 to 2017.
Pan CY , Yagi S , Padilla T , Fei Fan Ng T , Marine RL , Nix WA , Wadford DA . Microbiol Resour Announc 2018 7 (7) We report here two genome sequences of a newly designated rhinovirus genotype, RV-C56, which were obtained from respiratory specimens of three patients with acute respiratory illness in 2016 and 2017. To our knowledge, these sequences represent the first near-complete genomes for RV-C56 strains. |
Genetic diversity of human sapovirus across the Americas.
Diez-Valcarce M , Castro CJ , Marine RL , Halasa N , Mayta H , Saito M , Tsaknaridis L , Pan CY , Bucardo F , Becker-Dreps S , Lopez MR , Magana LC , Ng TFF , Vinje J . J Clin Virol 2018 104 65-72 BACKGROUND: Sapoviruses are responsible for sporadic and epidemic acute gastroenteritis worldwide. Sapovirus typing protocols have a success rate as low as 43% and relatively few complete sapovirus genome sequences are available to improve current typing protocols. OBJECTIVE/STUDY DESIGN: To increase the number of complete sapovirus genomes to better understand the molecular epidemiology of human sapovirus and to improve the success rate of current sapovirus typing methods, we used deep metagenomics shotgun sequencing to obtain the complete genomes of 68 sapovirus samples from four different countries across the Americas (Guatemala, Nicaragua, Peru and the US). RESULTS: VP1 genotyping showed that all sapovirus sequences could be grouped in the four established genogroups (GI (n=13), GII (n=30), GIV (n=23), GV (n=2)) that infect humans. They include the near-complete genome of a GI.6 virus and a recently reported novel GII.8 virus. Sequences of the complete RNA-dependent RNA polymerase gene could be grouped into three major genetic clusters or polymerase (P) types (GI.P, GII.P and GV.P) with all GIV viruses harboring a GII polymerase. One (GII.P-GII.4) of the new 68 sequences was a recombinant virus with the hotspot between the NS7 and VP1 regions. CONCLUSIONS: Analyses of this expanded database of near-complete sapovirus sequences showed several mismatches in the genotyping primers, suggesting opportunities to revisit and update current sapovirus typing methods. |
Whole-Genome Sequence of Human Rhinovirus C47, Isolated from an Adult Respiratory Illness Outbreak in Butte County, California, 2017.
Pan CY , Padilla T , Yagi S , Lewis LS , Ng TFF , Marine RL , Nix WA , Wadford DA . Genome Announc 2018 6 (5) Here, we report the full coding sequence of rhinovirus C47 (RV-C47), obtained from a patient respiratory sample collected during an acute respiratory illness investigation in Butte County, California, in January 2017. This is the first whole-genome sequence of RV-C47 to be reported. |
Near-Complete Genome Sequences of Several New Norovirus Genogroup II Genotypes.
Chhabra P , Aswath K , Collins N , Ahmed T , Olortegui MP , Kosek M , Cebelinski E , Cooper PJ , Bucardo F , Lopez MR , Castro CJ , Marine RL , Ng TFF , Vinje J . Genome Announc 2018 6 (6) We report here the near-complete genome sequences of 13 norovirus strains detected in stool samples from patients with acute gastroenteritis from Bangladesh, Ecuador, Guatemala, Peru, Nicaragua, and the United States that are classified into one existing (genotype II.22 [GII.22]), 3 novel (GII.23, GII.24 and GII.25), and 3 tentative novel (GII.NA1, GII.NA2, and GII.NA3) genotypes. |
Complete Genome Sequences of Mumps and Measles Virus Isolates from Three States in the United States.
Magana LC , Espinosa A , Marine RL , Ng TFF , Castro CJ , Montmayeur AM , Hacker JK , Scott S , Whyte T , Bankamp B , Oberste MS , Rota PA . Genome Announc 2017 5 (33) We report here the full coding sequence of nine paramyxovirus genomes, including two full-length mumps virus genomes (genotypes G and H) and seven measles virus genomes (genotypes B3 and D4, D8, and D9), from respiratory samples of patients from California, Virginia, and Alabama obtained between 2010 and 2014. |
Genetic and Epidemiologic Trends of Norovirus Outbreaks in the US Demonstrated Emergence of Novel GII.4 Recombinant Viruses, 2013-2016.
Cannon JL , Barclay L , Collins NR , Wikswo ME , Castro CJ , Magana LC , Gregoricus N , Marine RL , Chhabra P , Vinje J . J Clin Microbiol 2017 55 (7) 2208-2221 Noroviruses are the most frequent cause of epidemic acute gastroenteritis in the United States (US). Between September 2013 and August 2016, 2,715 genotyped norovirus outbreaks were submitted to CaliciNet. GII.4 Sydney viruses caused 58% of outbreaks during these years. A GII.4 Sydney variant with a novel GII.P16 polymerase emerged in November 2015, causing 60% of all GII.4 outbreaks in the 2015-2016 season. Multiple polymerase types were found associated with GII.2 (3), GII.3 (3), GII.4 Sydney (3), GII.13 (2) and GII.17 (2) genotypes, 4 of which included GII.P16 variants. GII.P16 polymerase sequences associated with GII.2 and GII.4 Sydney strains were nearly identical, suggesting common ancestry. Other common genotypes, each causing 5-17% of outbreaks in a season, included GI.3, GI.5, GII.2, GII.3, GII.6, GII.13 and GII.17 Kawasaki. Acquisition of alternative RNA polymerases by recombination is an important mechanism for norovirus evolution and a phenomenon that was shown to occur more frequently than previously recognized in the US. Continued molecular surveillance of norovirus strains, including typing of both polymerase and capsid genes, is important for monitoring emerging strains in our continued efforts to reduce the overall burden of norovirus disease. |
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