Lab Matters Fall 2023 | Page 72

APHL 2023 POSTER ABSTRACTS

a model , we demonstrate the effectiveness of target enrichment combined with next generation sequencing . HPV is a family of more than 200 DNA viruses , is the most common sexually transmitted infection , and the etiologic agent of cervical and other anogenital and head / neck cancers . Current assays focus on the 14 high risk types associated with most HPV-related cancers , but identification of the full spectrum of HPV types and variants could provide insights on tropism and disease associations . eWGS was used in this study to explore the genetic diversity and potentially identify novel and unknown sequences of HPV . Methods : Samples ( total nucleic acid [ TNA ] extracts of exfoliated cervical cells ) were indexed and pooled prior to target enrichment . The pools of samples were enriched by hybridizing to RNA bait composed of 25,239 RNA probes targeting 202 HPV types and 12 probes targeting the human beta-globin gene . The Agilent SureSelect protocol ( XT2 ) was used for library preparation , and sequencing was performed on a HiSeq 2500 . An in-house bioinformatic pipeline was used for automated HPV typing and identification of full-length sequences . Results : The bioinformatic pipeline identified 2,376 HPV types ( 2,320 alpha , 9 beta and 45 gamma genomes ) in the cervical samples ( n = 761 ). The prevalence of HPV types in the alpha genomes were HPV16 ( 12.5 %), HPV31 ( 5.8 %), HPV51 ( 4.8 %), HPV66 ( 4.7 %), HPV52 ( 3.8 %), HPV90 ( 2.24 %), HPV39 ( 3.9 %) and HPV18 ( 2.5 %). Of 2,320 alpha genomes , 1,374 genomes ( 59 %) met the definition of full-length sequences (> 1,000 mapped reads with > 80 % genome coverage ). This suggests that up to 41 % of the HPV types could be integrated or have lost segments of their genome . HPV 16 ( 218 / 291 , 75 %) and HPV52 ( 72 / 87 , 82 %) had the highest proportion of intact genomes and HPV39 had the lowest ( 47 / 90 , 52 %). Conclusions : eWGS allows the full spectrum of HPV genetic diversity to be explored in one assay . This approach could be adapted for a wide variety of pathogens of public health importance .

Presenter : Brian Wakeman , mrq9 @ cdc . gov

Genome Sequence of Serodino Virus , a Novel Oliveros-like Clade E Mammarenavirus

E . Shedroff , S . Whitmer , J . D . Klena and J . Montgomery ; US Centers for Disease Control and Prevention

A novel Mammarenavirus genome was identified within a suspect hantavirus sample collected from a rodent in Maciel , Argentina . Metagenomic deep sequencing analysis revealed Mammarenaviruslike reads in addition to those belonging to a species of hantavirus . The genome for the novel Mammarenavirus-like virus was initially built using de novo assembly . BLAST analysis of the partial genome yielded hits within the Mammarenavirus family . A guided de novo assembly was performed to complete the novel Serodino genome . Mammarenaviruses are negative-sense RNA viruses that are strongly associated with rodent species as their typical reservoir and contain a small ( S ) and large ( L ) segment . The S segment encodes the viral nucleoprotein ( NP ) and glycoprotein precursors ( GPC ), while the L segment encodes the viral polymerase ( L ) and RING-finger domain ( Z ). Mammarenaviruses are divided into two major serocomplexes : Old World and New World ; the New World serocomplex is further segregated by Clades A-D . Serodino virus is most closely related to Oliveros virus ; an arenavirus of Argentinian origin , discovered in 1990 . Oliveros virus was first detected in the town of Oliveros , Argentina , located north of the Argentinean

Hemorrhagic Fever ( AHF ) endemic area that was undergoing rodent survey between 1987 and 1990 in response to a Junín virus outbreak . Phylogenetic analysis revealed that the Serodino S segment is 90.1 % identical to Oliveros virus , however , the L segment is 70.4 % identical . At the protein level , the S segment proteins ( NP and GP ) are at or above 99 % similar and greater diversity is observed for the L segment proteins ( L and Z ) at 88.2 % and 83.0 %, respectively . Presence of these segments by RT-PCR has been confirmed and confirmatory sequencing will ensue . The International Committee on Taxonomy of Viruses ( ICTV ) mandates that new viral species may be classified by a specific host species or reservoir , specific geographic location , a cause of human disease , significant differences in antigenic cross-reactivity or a minimum of 12 % amino acid sequence dissimilarity to existing viruses within the genus . Based on the observed L segment diversity and ICTV species-level designations , we propose that Serodino virus is a distinct New World Mammarenavirus and a member of a unique Clade “ E ” species .

Presenter : Elizabeth Shedroff , ubu2 @ cdc . gov

Implementation of a Targeted Nanopore Sequencing Assay for Antimicrobial Resistance Detection in Mycobacterium tuberculosis

S . Murphy , C . Smith , K . Patel , J . Shea , T . Halse , M . Dickinson , V . Escuyer , M . Rowlinson , P . LaPierre , K . Musser ; New York State Department of Health , Wadsworth Center

The time and cost associated with the diagnosis and treatment of Mycobacterium tuberculosis ( MTB ) infections can be considerable , specifically in cases involving multi-drug or extensively-drug resistant ( DR ) strains . Due to the slow growth rate of this bacterium , traditional ( phenotypic ) methods for determining antimicrobial susceptibilities require weeks to months to complete . Meanwhile , patients may be placed on inappropriate therapies leading to treatment failure and further resistance development . To improve turnaround times for resistance detection , a targeted Next Generation Sequencing ( tNGS ) assay was developed at the New York State Department of Health that can profile MTB antimicrobial susceptibilities direct from primary specimens . This assay utilizes multiplex PCR to amplify thirteen loci implicated in resistance to first and second-line antimicrobials . Amplicon libraries are sequenced on an Oxford Nanopore device and analyzed in real-time using a custom bioinformatics pipeline . The assay cost , including reagents and consumables and excluding labor , is approximately $ 75 per sample . tNGS was attempted on 45 specimens previously determined to be MTB-positive . A subset of these samples yielded sufficient DNA for sequencing and aided in the establishment of sample quality criteria for referring samples to tNGS . These criteria include a positive AFB smear and / or a real-time PCR Ct value of ≤34.0 With these parameters met , tNGS accurately provided susceptibility profiles and lineage identification for all samples sequenced , which included 28 pan-susceptible and two multi-DR strains . Comparison of turnaround times showed a mean improvement of 13 days ( and a maximum of 31 days ) for direct specimen tNGS when compared to whole genome sequencing performed on the resulting patient isolate . These results demonstrate the utility of tNGS to offer early comprehensive antimicrobial resistance detection for MTB .

Presenter : Shannon Murphy , shgmurphy @ gmail . com