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IntroductionThe SARS-CoV-2 pandemic represented a formidable scientific and technological challenge to public health due to its rapid spread and evolution. To meet these challenges and to characterize the virus over time, the State of California established the California SARS-CoV-2 Whole Genome Sequencing (WGS) Initiative, or “California COVIDNet”. This initiative constituted an unprecedented multi-sector collaborative effort to achieve large-scale genomic surveillance of SARS-CoV-2 across California to monitor the spread of variants within the state, to detect new and emerging variants, and to characterize outbreaks in congregate, workplace, and other settings.MethodsCalifornia COVIDNet consists of 50 laboratory partners that include public health laboratories, private clinical diagnostic laboratories, and academic sequencing facilities as well as expert advisors, scientists, consultants, and contractors. Data management, sample sourcing and processing, and computational infrastructure were major challenges that had to be resolved in the midst of the pandemic chaos in order to conduct SARS-CoV-2 genomic surveillance. Data management, storage, and analytics needs were addressed with both conventional database applications and newer cloud-based data solutions, which also fulfilled computational requirements.ResultsRepresentative and randomly selected samples were sourced from state-sponsored community testing sites. Since March of 2021, California COVIDNet partners have contributed more than 450,000 SARS-CoV-2 genomes sequenced from remnant samples from both molecular and antigen tests. Combined with genomes from CDC-contracted WGS labs, there are currently nearly 800,000 genomes from all 61 local health jurisdictions (LHJs) in California in the COVIDNet sequence database. More than 5% of all reported positive tests in the state have been sequenced, with similar rates of sequencing across 5 major geographic regions in the state.DiscussionImplementation of California COVIDNet revealed challenges and limitations in the public health system. These were overcome by engaging in novel partnerships that established a successful genomic surveillance program which provided valuable data to inform the COVID-19 public health response in California. Significantly, California COVIDNet has provided a foundational data framework and computational infrastructure needed to respond to future public health crises.

Remdesivir is the first FDA-approved drug for treating severe SARS-CoV-2 infection and targets RNA-dependent RNA polymerase (RdRp) that is required for viral replication. To monitor for the development of mutations that may result in remdesivir resistance during prolonged treatment, we sequenced SARS-CoV-2 specimens collected at different treatment time points in two transplant patients with severe COVID-19. In the first patient, an allogeneic hematopoietic stem cell transplant recipient, a transient RdRp catalytic subunit mutation (nsp12:A449V) was observed that has not previously been associated with remdesivir resistance. As no in vitro study had been conducted to elucidate the phenotypic effect of nsp12:A449V, its clinical significance is unclear. In the second patient, two other transient RdRp mutations were detected: one in the catalytic subunit (nsp12:V166A) and the other in an accessory subunit important for processivity (nsp7:D67N). This is the first case report for a potential link between the nsp12:V166A mutation and remdesivir resistance in vivo, which had only been previously described by in vitro studies. The nsp7:D67N mutation has not previously been associated with remdesivir resistance, and whether it has a phenotypic effect is unknown. Our study revealed SARS-CoV-2 genetic dynamics during remdesivir treatment in transplant recipients that involved mutations in the RdRp complex (nsp7 and nsp12), which may be the result of selective pressure. These results suggest that close monitoring for potential resistance during the course of remdesivir treatment in highly vulnerable patient populations may be beneficial. Development and utilization of diagnostic RdRp genotyping tests may be a future direction for improving the management of chronic COVID-19.

Heat Shock Factor 1 (HSF-1) is a key regulator of the heat shock response (HSR). Upon heat shock, HSF-1 binds well-conserved motifs, called Heat Shock Elements (HSEs), and drives expression of genes important for cellular protection during this stress. Remarkably, we found that substantial numbers of HSEs in multiple Caenorhabditis species reside within Helitrons, a type of DNA transposon. Consistent with Helitron-embedded HSEs being functional, upon heat shock they display increased HSF-1 and RNA polymerase II occupancy and up-regulation of nearby genes in C. elegans. Interestingly, we found that different genes appear to be incorporated into the HSR by species-specific Helitron insertions in C. elegans and C. briggsae and by strain-specific insertions among different wild isolates of C. elegans. Our studies uncover previously unidentified targets of HSF-1 and show that Helitron insertions are responsible for rewiring and diversifying the Caenorhabditis HSR.

The DREAM (Dp/Retinoblastoma(Rb)-like/E2F/MuvB) transcriptional repressor complex acts as a gatekeeper of the mammalian cell cycle by establishing and maintaining cellular quiescence. How DREAM's three functional components, the E2F-DP heterodimer, the Rb-like pocket protein, and the MuvB subcomplex, form and function at target gene promoters remains unknown. The current model invokes that the pocket protein links E2F-DP and MuvB and is essential for gene repression. We tested this model by assessing how the conserved yet less redundant DREAM system in Caenorhabditis elegans is affected by absence of the sole C. elegans pocket protein LIN-35. Using a LIN-35 protein null mutant, we analyzed the assembly of E2F-DP and MuvB at promoters that are bound by DREAM and the level of expression of those "DREAM target genes" in embryos. We report that LIN-35 indeed mediates the association of E2F-DP and MuvB, a function that stabilizes DREAM subunit occupancy at target genes. In the absence of LIN-35, the occupancy of E2F-DP and MuvB at most DREAM target genes decreases dramatically and many of those genes become upregulated. The retention of E2F-DP and MuvB at some target gene promoters in lin-35 null embryos allowed us to test their contribution to DREAM target gene repression. Depletion of MuvB, but not E2F-DP, in the sensitized lin-35 null background caused further upregulation of DREAM target genes. We conclude that the pocket protein functions primarily to support MuvB-mediated repression of DREAM targets and that transcriptional repression is the innate function of the evolutionarily conserved MuvB complex. Our findings provide important insights into how mammalian DREAM assembly and disassembly may regulate gene expression and the cell cycle.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.