Lab Matters Fall 2024 | Page 111

APHL 2024 POSTER ABSTRACTS

Validation of Influenza Sequencing on the Illumina and Oxford Nanopore Platforms

C . Barnes , B . Schwem , S . Delaney , M . Pugliese , D . Woell , R . Siderits , T . Kirn , New Jersey Public Health and Environmental Laboratories

National and global surveillance of Influenza A and B viruses is incredibly important , as its genome can accumulate small mutations over time or even have a major reassortment events resulting in a different hemagglutinin ( HA ) and / or neuraminidase ( NA ) genes . Next generation sequencing has become a pivotal tool to monitor these genomic changes over time , which in turn helps all public health laboratories and the Centers of Disease Control and Prevention ( CDC ) make important public health decisions for the upcoming Influenza season . To strengthen Influenza surveillance in New Jersey , the following project aims to validate and implement the CDC ’ s Multi-Segment Reverse Transcription PCR ( MRT-PCR ) sequencing of Influenza A and B viruses on the Illumina and Oxford Nanopore Technologies ( ONT ) platforms . Generally , RNA extracted from a confirmed Influenza positive patient sample is used for MRT-PCR , which is a one-step protocol for cDNA synthesis and amplification of the Influenza virus . Subsequently , the cleaned PCR product is used for library preparation using the Illumina DNA Prep Kit and ONT Rapid Barcoding Kit for sequencing on the iSeq / MiSeq instrument or MinION Mk1B device . The resulting sequencing reads are processed and analyzed using the Terra bioinformatic platform and NextClade , which provides a determined type / subtype , HA / NA clade and percent coverage of the virus for each sample . Experiments were conducted to assess the accuracy , precision , analytical specificity and sensitivity on both platforms . The accuracy experiment compared the type / subtype results between the sequencing and in-house qPCR assay results , as well as the HA and NA clade determination against CDC ’ s results for the samples . It was determined that both Illumina and ONT showed over 98 % accuracy . We were also able to demonstrate 100 % precision between operators , day and instruments for both platforms . The analytical specificity study revealed that Illumina primers is more specific than the ONT primers , but the reference coverage would not meet the quality requirements set by the CDC for submission . Lastly , the results of the analytical sensitivity experiment revealed the Illumina platform would have a higher Ct cutoff value when compared to the ONT platform , meaning samples with a higher Ct value were able to be sequenced and reach the required quality metrics . In conclusion , we were able to successfully validate MRT-PCR Influenza sequencing on the Illumina and ONT platforms with high accuracy and precision . Although there were some differences seen in the specificity and sensitivity between the two platforms , we find that Illumina and ONT are viable platforms for sequencing of Influenza to help bolster the surveillance of Influenza in New Jersey .

Presenter : Cherrelle Barnes , cherrelle . barnes @ doh . nj . gov

Whole Genome Sequencing of an Mpox Outbreak Cluster in Chicago , IL — March – September 2023

H . Barbian 1 , A . Kittner 2 , D . Foulkes 2 , S . Green 1 , K . Kunstman 1 , F . Araujo-Perez 1 , G . Balangue 1 , C . Chau 1 , K . Anderson 2 , E . Faherty 3 , M . Pacilli 2 , Rush University Medical Center 1 , Chicago Department of Public Health 2 , CDC Epidemic Intelligence Service 3

Introduction : Prior to the 2022 pandemic , mpox was a rare zoonotic disease primarily involving animal to human transmission .

While cases in 2023 were much lower than in 2022 , the mpox virus ( MPXV ) was circulating on-and-off and in limited locations in 2023 . The Chicago Department of Public Health ( CDPH ) began tracking a cluster of mpox illness in the spring and early summer of 2023 , a time during which case numbers were very low elsewhere in the country . CDPH sequenced MPXV samples to determine ( i ) the presence of specific mutations which could explain the illness resurgence and ( ii ) possible epidemiologic links based on genomic relatedness of the MPXV in patients .

Methods : Following a lab confirmed diagnosis of MPXV Clade II , specimens were routed to the Regional Innovative Public Health Laboratory ( RIPHL ), a public health / academic partnership between CDPH and Rush University Medical Center for advanced molecular detection . At RIPHL , whole genome sequence libraries were prepared using the PrimalSeq amplicon scheme and and deeply sequenced using Illumina chemistry . Consensus sequences were generated using reference MT903345 . A phylogenetic tree including cluster patients and contextual isolates was prepared using Nextstrain .

Results : Isolates were obtained for whole genome sequencing for 60 patient specimens and 55 had adequate genome coverage for analysis (> 89 %). Fifty-four of 55 sequences were assigned to lineage B . 1.20 and fell within a single phylogenetic clade . The single non-clustering sequence belonged to the B . 1.13 lineage and caused a prolonged infection with initial onset of illness in December 2022 . Among the 54 B . 1.20 isolates , 21 were identical , 29 were closely related ( i . e ., clustering with and 1-6 nucleotide mutations diverged from the identical specimens ) and four were less closely related and did not cluster with the identical specimens ( i . e ., lacked 1-2 mutations shared by the identical sequences and contained 4-6 other unique mutations ). Using public sequencing data of US cases , the Chicago isolates were found to diverge by three nucleotides from two genomes ( undisclosed state ) in March 2023 and diverged by four nucleotides ( Texas ) in 2022 . There was little diversification within this clade : identical genomes were obtained from specimens collected up to 10 weeks apart . No genetic differences were observed between MPXV sequences from vaccinated and unvaccinated patients and sequences obtained from vaccinated patients were interspersed with sequences from unvaccinated patients . A few amino acid mutations were inferred from the genomes of viruses within the B . 1.20 Chicago cluster and none occurred within the gene for the MPXV protein VP37 , associated with tecovirimat resistance .

Conclusion : Analysis revealed that 50 of 54 ( 93 %) B . 1.20 specimens clustered into a single closely related group , indicating a limited number of introductions to Chicago and close transmission linkages . Multiple MPXV lineages from national and international samples in 2023 clustered closely with Chicago sequences , indicating that this lineage was not limited to Chicago . During the 2023 illness cluster , MPXV sequences were analyzed to understand the progression of the epidemic outbreak in the city . Sequencing revealed that MPXV circulating in the outbreak cluster had not acquired concerning mutations associated with vaccine escape or increased disease severity .

Presenter : Alyse Kittner , alyse . kittner @ cityofchicago . org