High DNA Levels Research Articles

Detection frequencies and viral load distribution of parvovirus B19 DNA in blood and plasma donations in England.

Infections with human parvovirus B19 (B19V) are transmissible by blood components and plasma-derived medicines. The European Pharmacopoeia regulates maximum levels of virus allowed in manufacturers' plasma pools. To evaluate contamination risk prior to re-introduction of UK-sourced plasma for manufacturing, we investigated viraemia frequencies of B19V in plasma samples collected from blood donors before and during COVID-enforced lockdown. Quantitative PCR for B19V DNA was used to screen pools of 96 anonymised plasma samples collected in England from 2017 (n= 29 505), 2020 (n= 3360) and 2021 (n= 43 200). Selected positive pools were resolved into individual samples. Data on donor notifications and related lookback investigations were collected from European countries by on-line survey in 2020. Screening of 76 065 donations identified 80 B19V-positive pools. While most positive samples had low viral loads (<105 IU ml-1 ), primarily from 2017 (77/29 505; 0.3%), two contained high levels of B19V DNA (1.3 × 108 and 6.3 × 106 IU ml-1 ), both likely to contaminate a final manufacturer's pool and lead to discard. The incidence of B19V infection during lockdown was reduced (1/3360 in 2020; 0/43 200 in 2021). Genomic analysis of positive pools resolved to single samples identified B19V genotype 1 in all nine samples. Seroprevalence of anti-B19V IgG antibodies was 75% (143/192). A survey of B19V screening practices in Europe demonstrated considerable variability. Two blood establishments informed infected blood donors of positive B19V results. Information on seroprevalence, incidence and viral loads of B19V viraemia is contributory the evaluation of alternative operational screening strategies for plasma testing.

Prevalence and Evolution Analysis of Porcine Circovirus 3 in China from 2018 to 2022.

Simple SummaryAn increasing number of studies have shown that the PCV3 virus causes signs and symptoms similar to PDNS in pigs, and since first being identified in the United States, it has caused reproductive failure in pigs. Studies have shown that it has spread worldwide, especially in China. However, to date, there are only a few reports of PCV3 detection and sequence variation, and limited information is known about its distribution in China’s major swine-producing regions. This study examined the prevalence of PCV3 in China and its evolutionary relationship. A high level of PCV3 infection has been found in serum samples, and it has been found that PCV3 infection has a widespread distribution among Chinese pig herds. The ORF2 genes of the strains were analyzed and compared with other PCV3 strains, which were downloaded from the NCBI. Our phylogenetic analysis indicated a close relationship with the strains previously described in pigs, and additional analysis revealed that all isolates obtained in this study could be divided into two sub-clades: 3a and 3b. Overall, this study showed that PCV3 prevalence in China is high and there is a lot of genetic divergence among the strains, which may pose a threat to the porcine industry.Porcine circovirus 3 (PCV3) is an emerging virus, causing substantial economic losses in pig populations, that was first detected in 2016. Furthermore, the virus has already been reported in Europe, the Americas, and Asia, including China, indicating that the virus has spread worldwide. However, the molecular epidemiology of PCV3 still needs further study. To investigate PCV3 epidemiological characteristics in China, 2707 serum samples of pigs were randomly collected from 17 provinces in China between September 2018 and March 2022 and analyzed via PCR assays. The study showed that PCV3 infection was prevalent in the overall population with 31.07% (841/2707) and 100.0% (17/17) at sample and province levels, respectively, though the positivity rate of PCV3 varied from 7.41 to 70.0% in different provinces, suggesting that PCV3 infection has a widespread distribution in China. We selected 22 serum samples from different regions that had high levels of viral DNA for amplification and sequenced their ORF2 (Cap) gene. According to the phylogenetic analysis, all isolates in the current study could be grouped into two separate subclades, with 15 strains belonging to clade 3a and 7 strains belonging to clade 3b, indicating that PCV3a and PCV3b were the predominant subtypes in the regions of China studied. Meanwhile, additional analysis revealed that the capsid gene sequences identified in this study displayed 97.46~99.8% nucleotide (nt) and 97.06~100% amino acid (aa) sequence similarity with other PCV3 available reference strains, respectively. In general, our studies provide important insights for understanding the prevalence and evolution of PCV3 in China and will guide future efforts to develop measures for preventing and controlling the disease.

The Role of Autophagy in the Mother-to-Child Transmission of Pregnant Women With a High Level of HBV DNA.

BackgroundMother-to-child transmission (MTCT) is the most common propagation mode of hepatitis B virus (HBV) transmission. Exploring the mechanisms of HBV MTCT is the key to protect infant from infection. In this study, we aim to clarify the important role of autophagy complicated in HBV MTCT.MethodsA total of 169 placental samples were collected in this study, includes 144 HBV positive pregnant women and 25 normal pregnant women. In vitro, JEG-3 cells were treated with serum contained different HBV viral loads. Electron microscope was used to observed the number of autophagosome. RT-qPCR and western blotting were used to measure the expression level of autophagy relative genes and proteins respectively. Immunofluorescence was used to analyzed the expression of LC-3 of the frozen section of placental tissue.ResultsAccording to the number of autophagosomes and the expression level of autophagic genes mRNA and protein, autophagy was increased in HBV maternal placenta. Among the control, low viral load, medium viral load and high viral load groups, autophagy was significantly up-regulated with the increase of HBV viral loads. Also, autophagy was increased in the HBeAg positive pregnant women compared with their HBeAg negative counterparts. Also, autophagy in infant-infected group was up-regulated compared with infant-uninfected group. In vitro, choriocarcinoma JEG-3 cells were treated with the different HBV viral loads or different time incubation, the mRNA and protein of autophagy related genes was maximum expression in the medium viral load or treatment in a short period, but decreased in the high viral load treatment or with long-term HBV exposure.ConclusionOur study determines the high levels of viremia could be the cause of both increase autophagy activities and MTCT. Autophagy was significantly up-regulated in pregnant women with high viral load or HBeAg positive, which plays an important part in the HBV MTCT.

Epstein-Barr Virus Enhances Cancer-Specific Aberrant Splicing of TSG101 Pre-mRNA.

Tumor viruses gain control of cellular functions when they infect and transform host cells. Alternative splicing is one of the cellular processes exploited by tumor viruses to benefit viral replication and support oncogenesis. Epstein-Barr virus (EBV) participates in a number of cancers, as reported mostly in nasopharyngeal carcinoma (NPC) and Burkitt lymphoma (BL). Using RT-nested-PCR and Northern blot analysis in NPC and BL cells, here we demonstrate that EBV promotes specific alternative splicing of TSG101 pre-mRNA, which generates the TSG101∆154-1054 variant though the agency of its viral proteins, such as EBNA-1, Zta and Rta. The level of TSG101∆154-1054 is particularly enhanced upon EBV entry into the lytic cycle, increasing protein stability of TSG101 and causing the cumulative synthesis of EBV late lytic proteins, such as VCA and gp350/220. TSG101∆154-1054-mediated production of VCA and gp350/220 is blocked by the overexpression of a translational mutant of TSG101∆154-1054 or by the depletion of full-length TSG101, which is consistent with the known role of the TSG101∆154-1054 protein in stabilizing the TSG101 protein. NPC patients whose tumor tissues express TSG101∆154-1054 have high serum levels of anti-VCA antibodies and high levels of viral DNA in their tumors. Our findings highlight the functional importance of TSG101∆154-1054 in allowing full completion of the EBV lytic cycle to produce viral particles. We propose that targeting EBV-induced TSG101 alternative splicing has broad potential as a therapeutic to treat EBV-associated malignancies.

Pathogenic Interactions between Macrophomina phaseolina and Magnaporthiopsis maydis in Mutually Infected Cotton Sprouts

The soil fungus Macrophomina phaseolina, the charcoal rot disease agent, poses a major threat to cotton fields. In Israel, highly infected areas are also inhabited by the maize pathogen Magnaporthiopsis maydis. This study reveals the relationships between the two pathogens and their impact on cotton sprouts. Infecting the soil 14 days before sowing (DBS) with each pathogen or with M. phaseolina before M. maydis caused a strong inhibition (up to 50–65%) of the sprouts’ development and survival, accompanied by each pathogen’s high DNA levels in the plants. However, combined or sequence infection with M. maydis first led to two distinct scenarios. This pathogen acted as a beneficial protective endophyte in one experiment, leading to significantly high emergence and growth indices of the plants and a ca. 10-fold reduction in M. phaseolina DNA in the sprouts’ roots. In contrast, M. maydis showed strong virulence potential (with 43–69% growth and survival suppression) in the other experiment, proving its true nature as an opportunist. Interestingly, soil inoculation with M. phaseolina first, 14 DBS (but not at sowing), shielded the plants from M. maydis’ devastating impact. The results suggest that the two pathogens restrict each other, and this equilibrium may lead to a moderate disease burst.

Herpesviruses in patients after renal transplantation

To estimate graft function after kidney transplantation during active herpesviruses or superinfection Materials and methods. The study included 32 patients (men 21, women 11) with end-stage chronic kidney disease. The median age was 43 years. Cytomegalovirus (CMV), EpsteinBarr virus (EBV) and human herpes virus 6 (HHV-6) DNAs were screened by RT-PCR in the donor's transplant biopsy, and recipients peripheral blood and urine after kidney transplantation (KT) on 0, 1, 2, 4, 6, 12 months. Antiviral antibodies (IgM and IgG) were also screened by Enzyme-linked immunoassay analysis (ELISA) along with PCR. The 500 or less copies of viral DNA per 105 nuclear cells or 1 ml of urine was considered as low, more than 1000 copies high. On the first month after KT CMV DNA was detected in 50% of pts., EBV DNA in 40% and HHV-6 DNA in 33%. During first year after KT two or three viruses simultaneously were found in 12 recipients: CMV, EBV, and HHV-6 were detected in 5 recipients; CMV and EBV in 4 patients; CMV and HHV-6 in 2 pts; EBV and HHV-6 in 1 pt. Graft dysfunction was observed in 9 patients with a high concentration of viral DNA of one, two or three viruses simultaneously. An upraise of the concentration of virus DNA (CMV, EBV and HHV 6) was detected primarily in the urine, while in the blood its concentration was less than 500 cop or undetectable. Renal dysfunction was not observed on the background of low concentrations of viral DNA in urine and blood. However, with an increase of DNA concentration, an impaired graft function in 8 of 12 patients appeared. Low viral DNA level proved to be a background for another virus activation or bacterial/fungal superinfection. Graft dysfunction occurs at high viral DNA levels detection during mono-or superinfection. Low viral load can serve as a background for another virus activation and/or bacterial/fungal superinfection.

Technical Note: Lysis and purification methods for increased recovery of degraded DNA from touch deposit swabs

Touch deposits are a routine yet challenging sample type in forensic casework and research. Recent work investigating their contents has indicated corneocytes to be the major cellular constituent while cell-free DNA is present at significant levels. Prolonged incubation including a reducing agent such as DTT has been shown to lyse corneocytes; a plasma cfDNA recovery kit which targets shorter DNA fragments has been demonstrated to improve cfDNA recovery from hand rinses. Herein these methods are combined and tested on mock casework touch deposit swabs from communal surface areas. Both fluorescence- and qPCR-based quantification methods are used and their results compared to query DNA degradation levels. Both proposed lysis and purification methods demonstrate increased recovery of DNA detectable with fluorescence quantification and some additional alleles at short loci, indicating high levels of fragmented DNA in these samples

Dynamics of methylated cell-free DNA in the urine of non-small cell lung cancer patients

ABSTRACT High levels of methylated DNA in urine represent an emerging biomarker for non-small cell lung cancer (NSCLC) detection and are the subject of ongoing research. This study aimed to investigate the circadian variation of urinary cell-free DNA (cfDNA) abundance and methylation levels of cancer-associated genes in NSCLC patients. In this prospective study of 23 metastatic NSCLC patients with active disease, patients were asked to collect six urine samples during the morning, afternoon, and evening of two subsequent days. Urinary cfDNA concentrations and methylation levels of CDO1, SOX17, and TAC1 were measured at each time point. Circadian variation and between- and within-subject variability were assessed using linear mixed models. Variability was estimated using the Intraclass Correlation Coefficient (ICC), representing reproducibility. No clear circadian patterns could be recognized for cfDNA concentrations or methylation levels across the different sampling time points. Significantly lower cfDNA concentrations were found in males (p=0.034). For cfDNA levels, the between- and within-subject variability were comparable, rendering an ICC of 0.49. For the methylation markers, ICCs varied considerably, ranging from 0.14 to 0.74. Test reproducibility could be improved by collecting multiple samples per patient. In conclusion, there is no preferred collection time for NSCLC detection in urine using methylation markers, but single measurements should be interpreted carefully, and serial sampling may increase test performance. This study contributes to the limited understanding of cfDNA dynamics in urine and the continued interest in urine-based liquid biopsies for cancer diagnostics.

HERPES SIMPLEX VIRUS PNEUMONITIS IN AN IMMUNOCOMPETENT, CRITICALLY ILL HOST

TOPIC: Chest Infections TYPE: Medical Student/Resident Case Reports INTRODUCTION: Pneumonitis due to herpes simplex virus is well reported in immunocompromised patients. It is extraordinarily uncommon in immunocompetent patients. CASE PRESENTATION: A 68 year old female with a history of chronic hypoxic respiratory failure on 2 Liters home oxygen presented to our facility with one day of dizziness and profound shortness of breath. At presentation, her oxygen saturation was 75%. A CT of the chest demonstrated significant bilateral diffuse infiltrates of ground glass appearance with consolidation. She was intubated and was transferred to the ICU. She had septic shock requiring vasopressors, and was started on community acquired pneumonia coverage with vancomycin, doxycycline, and ceftriaxone. She was extubated to high flow nasal canula and she was transferred to Step Down. On the floor, she failed to improve and required significant high flow nasal canula and bilevel positive airway pressure (BIPAP) support. A bronchoscopy was performed which showed that the lungs appeared normal. Cytopathology of bronchial washings showed positive staining for HSV-1. Transbronchial biopsy was also performed which demonstrated organizing pneumonia with focal nuclear atypia, findings suggestive of a viral pneumonitis. She was initiated on IV Acyclovir and was evaluated for immunocompromise, however, an extensive workup revealed no cause of compromise. She was noted to have perioral lesions consistent with herpes labialis which she reported started prior to her admission. She improved significantly after initiation of anti-viral therapy. DISCUSSION: Unfortunately, the signs and symptoms of herpes simplex pneumonitis are quite non specific, and in the intensive care setting, diagnosis can be obscured by positive HSV PCR on bronchial specimens in patients without HSV Pneumonitis. . A 2020 retrospective study showed that patients being treated for antibiotic-resistant ventilator associated pneumonia who were also found to have high levels of HSV DNA by PCR experienced a mortality benefit when treated with acyclovir. This finding suggests that HSV may be an underrepresented pathogen in immunocompetent hosts. Despite the vague nature of HSV pneumonitis, there are several features which can be suggestive. It should be considered in patients with disease resistant to antibacterial therapy, difficulty weaning from oxygen, and unexplained leukocytosis. Chest X-ray findings are nonspecific, but CT imaging usually shows ground glass opacities. CONCLUSIONS: Prior case reports have suggested that HSV pneumonitis in immunocompetent hosts occurs late in the course of illness and is associated with ventilator use. This case is significant as it represents HSV as a potential pathogen in immunocompetent patients at the onset of illness prior to initiation of invasive ventilation. REFERENCE #1: 1. Ishihara T, Yanagi H, Ozawa H, Takagi A. Severe herpes simplex virus pneumonia in an elderly, immunocompetent patient. BMJ Case Rep. 2018;2018:bcr2017224022. Published 2018 Jul 18. doi:10.1136/bcr-2017-224022 REFERENCE #2: 2. Schuierer L, Gebhard M, Ruf HG, et al. Impact of acyclovir use on survival of patients with ventilator-associated pneumonia and high load herpes simplex virus replication. Crit Care. 2020;24(1):12. Published 2020 Jan 10. doi:10.1186/s13054-019-2701-5 REFERENCE #3: 3. Pandita A, Dannheim K, Madhuripan N, Ehsan A, Farmakiotis D. Acute Necrotizing Pneumonia from Herpes Simplex Virus. Am J Med. 2021;134(2):e121-e122. doi:10.1016/j.amjmed.2020.06.050 DISCLOSURES: No relevant relationships by Jacob Gelman, source=Web Response No relevant relationships by Lucas Hamrick, source=Web Response No relevant relationships by Allison Lastinger, source=Web Response

DMD – ANIMAL MODELS: EP.90 A novel AAV8-based gene therapy for Duchenne muscular dystrophy: preclinical studies in the mdx mouse

RGX-202, a recombinant adeno-associated virus of serotype 8 (AAV8) with an optimized human microdystrophin transgene under the control of a muscle specific promoter (spc5-12), is a potential gene therapy candidate for the treatment of DMD. The microdystrophin includes 4 rods and 3 hinges and an extended coding region of the carboxyl terminal (CT) domain, known to bind α-dystrobrevin and α-syntrophin. Enhanced association of α-syntrophin may help recruiting neuronal nitric oxide synthase (nNOS) at the sarcolemma, facilitating vasodilation during exercise and protecting muscle against ischemic stress. The protein coding sequence was codon-optimized and depleted of CpG dinucleotides. RGX-202 was administered intravenously to male mdx mice and evaluated over 26 weeks. RGX-202-treated animals exhibited significant increases in muscle strength and normalization of gait as shown by fine kinematic analysis. Using T2 magnetic resonance imaging (MRI) to probe muscle damage characteristic of mdx, dose-dependent reductions of the hyper intense lesions were measured, consistent with an improvement in dystrophic pathology. High levels of RGX-202 vector DNA and microdystrophin protein were detected in the skeletal and cardiac muscles, and membrane assembly of the Dystrophin Associated Protein Complex (DAPC) was observed by immunostaining of muscle sections. Alongside staining for α-syntrophin, dystrobrevin, and ß-dystroglycan, there was also evidence for nNOS recruitment at the sarcolemma. RGX-202 treatment resulted in a reduction of the number of activated Pax7 positive satellite cells, further suggesting an improvement in abnormal muscle regeneration. Our data showed that treatment with RGX-202 resulted in the expression of an active microdystrophin which was able to improve muscle function and reduce dystrophic pathology in mdx mice. We are currently developing RGX-202 as a candidate for microdystrophin gene therapy in DMD patients.

Establishment and validation of a guinea pig model for human congenital toxoplasmosis

BackgroundToxoplasma gondii is an obligate intracellular parasite with a worldwide distribution. Congenital infection in humans and animals may lead to severe symptoms in the offspring, especially in the brain. A suitable animal model for human congenital toxoplasmosis is currently lacking. The aim of this study is to establish and validate the guinea pig as a model for human congenital toxoplasmosis by investigating the impact of the T. gondii infection dose, the duration of infection and the gestational stage at infection on the seroconversion, survival rate of dams, fate of the offspring, T. gondii DNA loads in various offspring tissues and organs and the integrity of the offspring brain.MethodsPregnant guinea pigs were infected with three different doses (10, 100, 500 oocysts) of T. gondii strain ME49 at three different time points during gestation (15, 30, 48 days post-conception). Serum of dams was tested for the presence of T. gondii antibodies using immunoblotting. T. gondii DNA levels in the dam and offspring were determined by qPCR. Offspring brains were examined histologically.ResultsWe found the survival rate of dams and fate of the offspring to be highly dependent on the T. gondii infection dose with an inoculation of 500 oocysts ending lethally for all respective offspring. Moreover, both parameters differ depending on the gestational stage at infection with infection in the first and third trimester of gestation resulting in a high offspring mortality rate. The duration of infection was found to substantially impact the seroconversion rate of dams with the probability of seroconversion exceeding 50% after day 20 post-infection. Furthermore, the infection duration of dams influenced the T. gondii DNA loads in the offspring and the integrity of offspring brain. Highest DNA levels were found in the offspring brain of dams infected for ≥ 34 days.ConclusionThis study contributes to establishing the guinea pig as a suitable model for human congenital toxoplasmosis and thus lays the foundation for using the guinea pig as a suitable animal model to study scientific questions of high topicality and clinical significance, which address the pathogenesis, diagnosis, therapy and prognosis of congenital toxoplasmosis.Graphical abstract

Topical cationic hairy particles targeting cell free DNA in dermis enhance treatment of psoriasis

Abnormal high level of cell free DNA (cfDNA) triggers chronic inflammation to exacerbate psoriasis symptoms. Scavenging cfDNA by topical cationic polymeric nanoparticles has been certified as an effective therapeutic strategy for treating psoriasis. However, cationic cfDNA scavengers have a great potential risk to organs after entering systemic circulation through skin barrier. For better transformation to clinical application, herein a series of poly(2-(dimethylamino)ethyl methacrylate) (PDMA) grafted hairy silica particles (cSPs) with tunable PDMA length and particle size are applied to scavenge cfDNA in dermis. We reveal that the structure of cSPs correlates with the permeation ability across stratum corneum, retention time in dermis, binding affinity to cfDNA, and toxicity tolerance, which in turn affect the therapeutic effect. Especially, the cSPs of 700 nm show more accumulation and longer retention in psoriatic lesions, leading to excellent treatment results. They also outperform the cSPs of 200 nm at a lower administration frequency. Thus, we address the issues of size, cationic content of cSPs to open a potential new avenue to topically treatment of psoriasis by targeting cfDNA in dermis.

Pathogenesis of Erysipelothrix piscisicarius infection in tiger barbs (Puntigrus tetrazona).

Erysipelothrix piscisicarius is an emerging bacterial pathogen and the aetiologic agent of piscine erysipelosis, a recently recognized disease of ornamental fish. However, little is known regarding the dynamics of infection in fish. The purpose of this study was to gain a better understanding of the pathogenesis of piscine erysipelosis in the tiger barb (Puntigrus tetrazona) by investigating tissue tropisms and responses to bacterial dissemination following immersion challenge with a virulent strain recovered from diseased fish. The challenge resulted in 83% mortality by day 16. Erysipelothrix piscisicarius DNA was first detected in the skin using quantitative PCR, and bacteria were visualized in association with microscopic lesions on day 4. By day 8, E.piscisicarius DNA was further detected in intestines, hearts, spleens, gills and skin; parenchymal organs were largely spared. The data suggest a primary cutaneous portal of entry and tropism for collagenous tissues, particularly those within vascular walls. Initial spread occurs directly from the dermis into interstitial areas of skeletal muscle, then centrally to the peritoneum and coelomic cavity following collagenous tissue pathways. Although histopathology revealed widespread bacterial dissemination over time, the severity of skin and muscle lesions with high levels of bacterial DNA identifies these tissues as primary targets of infection.

Abstract 575: Enhancing cell-free DNA mutation detection with novel liquid-phase extraction

Abstract Background: Low yields of extracted cell-free DNA (cfDNA) from plasma limit further development of liquid biopsies for early-stage cancer diagnostics and cancer screening applications. Conventional nucleic acid extraction methods have limitations in capturing low levels of cfDNA and risk capturing high levels of high molecular weight contaminating genomic DNA. We developed a liquid-phase based DNA extraction method (PHASIFY™ ENRICH; Phase Scientific International Ltd., Kwun Tong, Hong Kong) that leverages aqueous two-phase systems (ATPS) to purify, concentrate, and enrich for cfDNA to improve mutant signal detection. Methods: The PHASIFY ATPS consists of unique formulations that are optimized to drive cfDNA into one liquid phase and plasma contaminants into the other, achieving simultaneous target isolation and concentration. In a previous study using the PHASIFY technique, we demonstrated improved cfDNA and mutant copy yield over solid-phase methods. We further developed an additional size-selection feature (PHASIFY ENRICH) to remove high molecular weight genomic DNA (&amp;gt;500 bp) and to reduce signal noise for molecular diagnostics. We compared cfDNA recovery and mutation detection from plasma samples extracted with the industry standard QIAamp Circulating Nucleic Acid kit (QCNA; Qiagen, Hilden, Germany) and PHASIFY ENRICH cfDNA extraction kit. According to the respective kit protocols, the cfDNA was extracted from plasma from patients with advanced cancers, including those known to shed low amounts of cfDNA into circulation (e.g., low grade serious ovarian, appendiceal, thyroid, and pancreatic cancer), with known tissue mutation status. Total DNA recovery was determined with the Quant-iT PicoGreen dsDNA Assay Kit. Mutation status was determined with probe-based Droplet Digital PCR (ddPCR; Bio-Rad, Hercules, CA). In a follow up study, we tested a similar cohort of plasma samples that were previously tested negative for cfDNA mutation status when using QCNA cfDNA extraction, despite known positive mutation status from corresponding tumor tissue genotyping. Results: In the head-to-head study against QCNA, 57 plasma samples from 23 unique patients with diverse advanced cancers with known tissue mutation status were used for cfDNA extraction. The PHASIFY ENRICH method resulted in a 153% increase in median mutant copy recovery compared to QCNA (P = 0.01 Wilcoxon Signed-Rank). In the follow up study, 47 plasma samples from 31 patients with diverse advanced cancers and prior cfDNA negative mutation status with tissue positive mutation status were used for extraction. Of the 47samples extracted by PHASIFY ENRICH, 9 samples resulted in a conversion to positive cfDNA mutation status compared to no mutant signals when previously using QCNA. Conclusion: Our results indicate that PHASIFY ENRICH provides improved cfDNA mutation detection by increasing cfDNA recovery and reducing contaminating genomic DNA background. Citation Format: Filip Janku, Helen Huang, David Y. Pereira, Masae Kobayashi, Steven G. Call, Kristen T. Woodbury, Felix Chao, Daniel R. Marshak, Ricky Y. Chiu. Enhancing cell-free DNA mutation detection with novel liquid-phase extraction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 575.

Abstract 474: Extracellular vesicle - MDM2 as liquid biopsy biomarker for disease identification in retroperitoneal liposarcoma

Abstract Introduction: Extracellular vesicles (EVs) are membrane vesicles containing proteins and nucleic acids that aresecreted by all cells and circulate in the blood. In the tumor microenvironment their contents havebeen shown to carry important pro-carcinogenic molecules. Soft tissue sarcoma of theretroperitoneum of which the most common types are well differentiated/dedifferentiatedliposarcoma (WD/DDLPS) are marked by recurrence rates of &amp;gt;50% even after complete resection.A molecular hallmark of RLS WD/DDLPS are high levels of MDM2 DNA, a finding observed innearly all WD/DDLPS tumors. Indeed, FISH assessment of MDM2 in resected tissues is currentlythe definitive diagnostic methodology for WD/DDLPS. Prognosis in WD/DDLPS is enhanced byearly detection of recurrent lesions. However, current scanning technologies cannot resolverecurrent liposarcoma vs postoperative scarring in many patients (97% sensitivity/51%specificity); the need for easily retrieved and analyzed circulating factors that are diagnostic andprognostic is urgent. Methods: A cohort of 11 WD/DDLPS serum samples were collected at the time of surgery and during followup visits (6-12 months after surgery). Serum from normal healthy people (N=15) were used as acontrol. EVs were isolated from the serum through precipitation technology (ExoQuick) andultracentrifugation. The EVs-DNA was then isolated and the level of MDM2 was measured by Q-PCR. The level of MDM2 DNA in the WD/DDLPS tissues was also measured by Q-PCR andcompared with Normal Adjacent tissues. Results: MDM2 DNA was detected in EVs of the serum of RLS WD/DDLPS patients at significantly higherlevels than normal controls (geometric mean MDM2 DNA number of molecules value=74,434 vs10,146, p=0.0025). This increase was concordant with the level of MDM2 DNA as measured inWD/DDLPS tissues compared to normal adjacent tissues (p= 0.0009). Moreover, serumWD/DDLPS EV MDM2 was dramatically decreased in RLS WD/DDLPS patients after tumorresection (geometric mean MDM2 DNA number of molecules value pre-op=74,434 vs post-op=13,879, p=0.0048), and appears to correlate with CT-scan evidence of recurrent or persistentdisease after surgery. Importantly, the level of MDM2 DNA in EV's post-operatively decreasedreaching a level similar to the level of MDM2 in the controls (difference between post- surgeryand controls: p=0.47). Conclusion: In conclusion RLS WD/DDLPS EVMDM2 may potentially serve as an efficacious RLSWD/DDLPS first-ever prognostic and therapy predictive biomarker for a lethal disease currentlylacking such determinants. Citation Format: Lucia Casadei, Patricia Sarchet, Luciano Cascione, Giovanni Nigita, Fernanda Costas Casal de Faria, Carlo Maria Croce, Valerie Grignol, Raphael E. Pollock. Extracellular vesicle - MDM2 as liquid biopsy biomarker for disease identification in retroperitoneal liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 474.

Abstract 112: Analytical performance of a cfDNA-based targeted methylation multi-cancer early detection test for population-scale screening

Abstract Introduction: cfDNA-based tests have the potential to facilitate earlier detection of cancer and provide greater opportunity for curative intervention. Previously, we demonstrated that a cfDNA-based targeted methylation multi-cancer early detection (MCED) test can detect multiple cancers across all stages with high specificity, and predict the signal origin (SO; ie, tissue of origin) with high accuracy; we also validated the repeatability and reproducibility of its performance. Here, we validate a recent version of this MCED test planned for use as a screening tool. Methods: In the Circulating Cell-free Genome Atlas study (NCT02889978), cfDNA was analyzed using targeted methylation sequencing and machine learning to classify samples as cancer or non-cancer, and predict SO. Dilutions of cfDNA samples from participants with early-stage lung, head and neck, sarcoma, upper gastrointestinal, colorectal, or ovarian cancer into cfDNA from individuals without cancer were analyzed to establish the limit of detection (LOD95%) defined by the lowest variant allele frequency (VAF) at which accurate classification of cancer signals was attained in at least 95% of replicates. Analytical specificity was assessed by the true negative rate in 66 non-cancer cfDNA samples. Classification performance was evaluated as a function of cfDNA input (0.5-100 ng) using cancer samples (liver/bile duct, lung, head and neck, ovarian, breast, bladder/urothelial, uterine). Repeatability within-run and reproducibility between-runs were characterized by pairwise comparisons among 53 cancer samples (anorectal, breast, head and neck, uterine) and among 62 non-cancer samples processed across multiple reagent lots, instruments, and operators. The effects of high levels of potential interferents hemoglobin, bilirubin, triglycerides, and genomic DNA on classification when spiked into 88 plasma samples along with cfDNA from individuals with cancer (liver/bile duct or lung) or when spiked into 87 non-cancer samples were evaluated. Results: LOD95% was 0.11% VAF. No false positives were detected in non-cancer samples (100% analytical specificity). Across all input cfDNA levels tested, in 50/50 (100%) cancer samples, a cancer signal was detected with a correct SO prediction. All sample pairs in within-run (n=110) and between-run (n=696) analyses were concordant with respect to cancer/non-cancer classification and SO prediction (100% repeatability and reproducibility). None of the tested interferents affected cancer signal detection (100% correct) or SO prediction (100% accuracy). Conclusions: These results suggest that the analytical performance of a cfDNA-based targeted methylation MCED screening test is robust for clinical implementation. This MCED test can be a complementary tool to the existing repertoire of cancer screening options currently available for clinical use. Citation Format: Gregory E. Alexander, Byoungsok Jung, Lijuan Ji, Ekaterina Revenkova, Payal Shah, Jacqueline Brooks, Jeremy Carter, Zhao Dong, Lane Eubank, Maryam Hosseini, Xinyi Hou, Hannah Kiarie, Neda Ronaghi, Fabian E. Ortega, Madhuvanthi Ramaiah, Kate Rhodes, Rita Shaknovich, Seyedmehdi Shojaee, Sonya Parpart-Li, Nathan Hunkapiller. Analytical performance of a cfDNA-based targeted methylation multi-cancer early detection test for population-scale screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 112.

Transmission of infectious laryngotracheitis virus vaccine and field strains: the role of degree of contact and transmission by whole blood, plasma and poultry dust

Understanding the mechanisms of transmission of infectious laryngotracheitis virus (ILTV) is critical to proper control as both vaccine and wild-type strains circulate within chicken flocks with potential adverse consequences. The relative efficiency of transmission by direct contact between chickens and airborne transmission has not been investigated. Furthermore, relatively high levels of ILTV DNA have been detected in poultry dust and blood but the infectivity of these is unknown. In this study, comparison of in-contact and airborne transmission of two vaccine and one field strain of ILTV revealed that all transmitted to 100% of in-contact birds by 6 days post-exposure (dpe). Airborne transmission without contact resulted in 100% transmission by 14 and 17 dpe for the wild-type and Serva vaccine virus but only 27% transmission by 21 dpe for the A20 vaccine virus. The infectivity of dust or extracts of dust and blood or plasma from infected chickens at various stages of infection was assessed by inoculation into susceptible chickens. There was no transmission by any of these materials. In conclusion, direct contact facilitated efficient ILTV transmission but the virus was unable to be transmitted by dust from infected chickens suggestive of a limited role in the epidemiology of ILTV.

Replicating minichromosomes as a new tool for plastid genome engineering.

Plant molecular farming, that is, using plants as hosts for production of therapeutic proteins and high-value compounds, has gained substantial interest in recent years. Chloroplasts in particular are an attractive subcellular compartment for expression of foreign genes. Here, we present a new method for transgene introduction and expression in chloroplasts that, unlike classically used approaches, does not require transgene insertion into the chloroplast genome. Instead, the transgene is amplified as a physically independent entity termed a 'minichromosome'. Amplification occurs in the presence of a helper protein that initiates the replication process via recognition of specific sequences flanking the transgene, resulting in accumulation of extremely high levels of transgene DNA. Importantly, we demonstrate that such amplified transgenes serve as a template for foreign protein expression, are maintained stably during plant development and are maternally transmitted to the progeny. These findings indicate that the minichromosome-based approach is an attractive tool for transgene expression in chloroplasts and for organelle genome engineering.

Porcine Circovirus 3a Field Strains in Free-Living Wild Boars in Paraná State, Brazil.

Simple SummaryPorcine circovirus 3 (PCV-3) was first identified in pigs in the USA and was subsequently detected in several other countries, including Brazil. PCV-3 can be associated with diseases in pigs. To date, there are only a few reports of PCV-3 in wild boars worldwide. This study aimed to investigate the presence of PCV-3 in wild boars in Paraná state, Brazil. The results revealed that PCV-3 was present in the serum and lungs of the sampled boars. The complete genome of the PCV-3a strain was determined and compared with other PCV-3 strains around the world. Phylogenetic analysis has shown a close relationship to the strains already described in domestic and wild pigs. At this moment, there is no evidence that PCV-3 causes disease in wild boars. However, the monitoring of circulation of PCV-3 in wild boars is important for pig industry biosecurity because these animals share pathogens with domestic pigs.Porcine circovirus 3 (PCV-3) was identified in domestic pigs worldwide. Although PCV-3 has also been detected in wild boars, information regarding its circulation in this free-living animal species is scarce. To investigate PCV-3 occurrence in free-living wild boars in Brazil, 70 serum samples collected between January 2017 and June 2019 in Paraná state, Brazil were analyzed by PCR assay. Amplicons measuring 330 bp in length were amplified in seven (10.0%) of the serum samples and confirmed to be PCV3-specific by nucleotide (nt) sequencing. As the amplified products from the serum samples yielded only intermediate levels of viral DNA, lung samples from the seven PCR-positive wild boars were also evaluated by PCR. Of these samples, five lung samples were positive and provided high levels of viral DNA. The three lung samples that presented the highest levels of viral DNA were selected for amplification and sequencing of the whole PCV-3 genome. The three full-length sequences obtained were grouped in PCV-3 clade “a”, and the sequences exhibited 100% nucleotide similarity among them. The PCV-3 field strains of this study showed nucleotide and amino acid similarities of 98.5–99.8% and 98.8–100%, respectively, with whole-genome PCV-3 sequences from around the world.

Minimal residual disease (MRD) in patients with resected stage I NSCLC: Results of the prospective adjuvant IFCT-0703 trial.

8526 Background: MRD aims to detect circulating biomarkers of micrometastatic disease and ultimately predict recurrences. The IFCT-0703 randomized phase II trial failed to show a benefit of 6 months adjuvant pazopanib (P) vs. placebo after resection of stage I NSCLC (7th TNM edition). The outcome of pts based on their MRD status has been evaluated. Methods: Blood samples were collected in EDTA tubes (Becton Dickinson Company) after surgery (T0), after 3 months (T3) of P or placebo and at the end of treatment (T6). Plasmas were obtained after double centrifugation of total blood. Total nucleic acid was extracted using the Maxwell RSC LV plasma kit (Promega) according to the manufacturer’s protocol. Samples were quantified using the QuBit dsDNA HS Assay kit on a QuBit 3.0 flurometer (Thermo Fisher Scientific). Molecular analysis was performed by next generation sequencing using the Oncomine Lung cfDNA Assay (ThermoFisher Scientific). Two MRD definitions were tested : 1) high level of DNA in the blood or 2) any mutation detected by the standard bioinformatic pipeline was considered present, whatever the allelic fraction. Results: 143 pts were randomized in 29 centers between March 2009 and August 2012, 71 and 72 in the placebo and P arms respectively. Among the 119 pts with evaluable T0 samples, 27 pts recurred and 14 died. Median DNA concentration ([DNA]) was 6.6 ng/ml and an increase of [DNA] of 10 ng/ml was found prognostic of poor DFS and OS, HR=1.4, 95%CI [1.14-1.72], p=0.0016 and HR=1.62, 95%CI [1.15-2.30], p=0.0057 respectively. In 81 pts with available T0-T6 samples, [DNA] variation had no different impact on DFS and OS, in the P arm and the placebo arm. ctDNA mutations (ctDNA+) were detected in 31/119 pts. ctDNA+ were more frequent in samples with high DNA quantity (p=0.0002). Genes mutated at T0 were TP53 in 16, NRAS in 6, MAP2K1 in 2, KRAS in 1, EGFR in 5, BRAF in 1, ALK in 2. 29 pts had 1 mutation, 2 had 2 mutations. DFS and OS were similar between pts with or without ctDNA+ : HR= 1.038 (95%CI 0.438-2.456, p=0.93) and 1.193 (95% CI 0.367-3.882, p=0.77) respectively. Among 27 pts with ctDNA+ at T0 and available sample at T6, 23 had no more mutations at T6. Two pts had a ctDNA+ only at T6 (not at T3), one of them had a recurrence at 7 months. Conclusions: Post-operative ctDNA mutations are found in 26.0% of the pts but their positivity had no impact on DFS or OS. In contrast, DFS and OS were poorer in pts with increased plasma DNA concentration. ctDNA mutations status do not recapitulate the complexity of MRD characterization. NGS will be performed on matched tissues in order to refine MRD definition. Clinical trial information: NCT00775307.