RNA Copy Number Research Articles

Real-world outcome analysis of patients with advanced gastric and gastroesophageal adenocarcinoma with HER2-low expression treated with first-line therapy.

326 Background: HER2-low tumors, defined as a score of 1+ on immunohistochemical (IHC) analysis or as an IHC score of 2+ and negative results on in situ hybridization (ISH), are not well characterized among gastric or gastroesophageal junction adenocarcinomas. More importantly, this patient population is currently treated as HER2-negative (HER2-low and HER2-zero [IHC score of 0]) without anti-HER-2 directed therapy. Herein, we describe a real world outcome analysis of this distinct genomically defined underrepresented population. Methods: De-identified, multimodal real-world data (RWD) of 429 advanced gastric, esophageal, and GEJ adenocarcinomas from the Tempus Lens database was analyzed. Inclusion criteria was a diagnosis of gastric, esophageal, or GEJ adenocarcinoma between January 2017 to May 2021 and treatment with first-line therapy. Median overall survival (mOS) was estimated using Kaplan-Meier methods. Results: HER2-low was identified in 24.9% (107/429) of the total cohort. Histologies within HER2 low group were 83.2% (89/107) adenocarcinoma (NOS), 7.5% (8/107) signet ring cell carcinoma, 5.6% (6/107) diffuse-type, and 3.7% (4/107) mucinous-type. PDL-1 TPS status was available in only 50.5% (54/107) of the HER2-low group. TPS distribution was: 74.1% (40/54) for TPS <1, 22.2% (12/54) for TPS >=1-<10, and TPS >= 10: 3.7% (2/54). ERBB2 amplification or focal gain was detected in 1.1% (1/88) of patients in the HER-2 low group that had undergone tumor genomic sequencing, and 0% (0/28) of patients that had circulating tumor DNA (ctDNA) sequenced. Median followup time was 20.7 (8.5, 35.6) months. Median OS (mOS, months) was 9.8 (9.0, 12.7) for HER2-low, 13.2 (8.9, 27.0) for HER2(-), and 14.1 (11.0, 19.2) for HER2(+). Survival differences were also compared with variations across ERBB2 RNA expression and copy number variation (CNV). HER2 groups were defined on RNA expression and copy number estimates based on thresholds determined from the proportion of samples in each of the HER2 groups from IHC/ISH data. For RNA expression analysis, the mOS was 10.8 (8.8, 13.5) months for HER2-low, 10.9 (7.2, 17.4) months for HER2(-), and 13.0 (11.4, 20.5) months for HER2(+). For CNV data, the mOS(months) was: HER2-low: 10.9 (8.5, 14.1), HER2(-): 10.2 (7.0, 15.7), HER2(+): 14.2 (11.5, 21.4). Conclusions: Our real world, biomarker outcome analysis suggests that advanced gastric and GEJ patients with HER2-low expression identified by IHC and RNA expression have a poor prognosis with shortest survival. Furthermore, our data suggest that there is a heterogeneity of PDL-1 enrichment within this group. Hence, targeting expression of low levels of HER2 similar to HER2+ group alone or in combination with immunotherapy should be evaluated in future clinical trials to improve efficacy outcomes.

Monitoring COVID-19 spread in selected Prague's schools based on the presence of SARS-CoV-2 RNA in wastewater

The COVID-19 pandemic has demanded a broad range of techniques to better monitor its extent. Owing to its consistency, non-invasiveness, and cost effectiveness, wastewater-based epidemiology has emerged as a relevant approach to monitor the pandemic's course. In this work, we analyzed the extent of the COVID-19 pandemic in five primary schools in Prague, the Czech Republic, and how different preventive measures impact the presence of SARS-CoV-2 RNA copy numbers in wastewaters. Copy numbers were measured by reverse transcription–multiplex quantitative real-time PCR. These copy numbers were compared to the number of infected individuals in each school identified through regular clinical tests. Each school had a different monitoring regime and subsequent application of preventive measures to thwart the spread of COVID-19. The schools that constantly identified and swiftly quarantined infected individuals exhibited persistently low amounts of SARS-CoV-2 RNA copies in their wastewaters. In one school, a consistent monitoring of infected individuals, coupled with a delayed action to quarantine, allowed for the estimation of a linear model to predict the number of infected individuals based on the presence of SARS-CoV-2 RNA in the wastewater. The results show the importance of case detection and quarantining to stop the spread of the pandemic and its impact on the presence of SARS-CoV-2 RNA in wastewaters. This work also shows that wastewater-based epidemiological models can be reliably used even in small water catchments, but difficulties arise to fit models due to the nonconstant input of viral particles into the wastewater systems.

Hybrids of Ixodes ricinus and Ixodes persulcatus ticks effectively acquire and transmit tick-borne encephalitis virus.

Ixodes rici nus and Ixodes persulcatus ticks are the main vectors of tick-borne encephalitis virus (TBEV), which has three main subtypes connected with certain tick species: the European subtype, associated with I. ricinus, and the Siberian and Far-Eastern subtypes, associated with I. persulcatus. Distribution ranges of these species overlap and form large sympatric areas in the East European Plain and Baltic countries. It has previously been shown that crossing of I. ricinus and I. persulcatus is possible, with the appearance of sterile hybrids. Hybridization of ticks can affect not only the spread of ticks but also the properties of natural foci of arbovirus infections, in particular TBEV. In the present study, we analyzed the effectiveness of virus transmission from infected mice to larvae and nymphs and trans-stadial transmission (from larvae to nymph and adult) in I. ricinus, I. persulcatus, and hybrids. For this purpose, we bred a hybrid generation from the crossing of I. persulcatus females and I. ricinus males, and we used the Siberian and European subtypes of TBEV. We showed that after feeding on infected mice, virus prevalence in engorged ticks decreased over time, and after molting, the opposite was true. In hybrids we observed the highest acquisition effectiveness and RNA copy numbers during Siberian TBEV subtype transmission. The efficiency of trans-stadial transmission of both TBEV subtypes was similar in hybrids and parental species. After the second trans-stadial TBEV transmission, a significant increase in ticks' infection rates was observed only in specific subtype-tick combination. Our data demonstrate the possible features of TBEV circulation in the I. ricinus and I. persulcatus sympatry area.

Limited Capacity for Aedes aegypti to Mechanically Transmit Chikungunya Virus and Dengue Virus.

Mechanical transmission is an understudied mode of arbovirus transmission that occurs when a biting insect transmits virus among hosts by the direct transfer of virus particles contaminating its mouthparts. Multiple arboviruses have been shown to be capable of utilizing this transmission route, but most studies were conducted 40 to 70 years ago using dated methodologies. To gain a better understanding of this phenomenon, we used molecular techniques to evaluate the efficiency of mechanical transmission by Aedes aegypti mosquitoes for two evolutionarily divergent arboviruses, chikungunya virus (CHIKV) and dengue virus (DENV). Viral RNA and/or infectious DENV could be detected on 13.8% of mosquito proboscises sampled immediately after an infectious bloodmeal, but positivity rates declined within hours. CHIKV RNA and/or infectious virus was detected on 38.8% of proboscises immediately after feeding but positivity rates dropped to 2.5% within 4 hours. RNA copy numbers were low for both viruses, and we were unable to demonstrate mechanical transmission of CHIKV using an established animal model, suggesting that this mode of transmission is unlikely under natural conditions.

Brazilin from Caesalpinia sappan inhibits viral infection against PRRSV via CD163ΔSRCR5 MARC-145 cells: an in silico and in vitro studies

This research aimed to identify bioactive compounds from Caesalpinia sappan extract that function as novel porcine reproductive and respiratory syndrome virus (PRRSV) infection inhibitors by computational molecular screening. We obtained a set of small-molecule compounds predicted to target the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163. In addition, the functions of positive hits were assessed and verified utilizing an in vitro antiviral activity assay with PRRSV-infected MARC-145 cells. Combining molecular docking with the results of binding affinity and ligand conformation, it was found that brazilin had the highest binding energy with the SRCR5 receptor compared to catechin and epicatechin (− 5.8, − 5.5, and − 5.1 kcal/mol, respectively). In terms of molecular mechanics, the binding free energy between the SRCR5 receptor was − 15.71 kcal/mol based on the Poisson-Boltzmann surface area of brazilin. In addition, PRRSV infection in MARC-145 cells was significantly inhibited by brazilin compared to the control (virus titer, 4.10 vs. 9.25 TCID50/mL, respectively). Moreover, brazilin successfully limited the number of PRRSV RNA copies in MARC-145 cells as determined by RT-qPCR. By inhibiting the PRRSV-CD163 interaction with brazilin from Caesalpinia sappan, it may be possible to prevent PRRSV infection in pigs, as suggested by this research.

Using T cell lymphokines of hyperimmunized chickens with Salmonella pullorum to enhance immune response of layer hens against avian influenza

The current study used immune lymphokines from chickens that had been hyperimmunized with Salmonella pullorum to improve the resistance of layer hens at the production stage to AIV-type (H5N1) infected hens (SP). Two groups (each consisting of 25 pullets) were treated; the first group received three doses of SP vaccination at 12, 14, and 16 weeks; the second group received no vaccination and was used as a control group. At 18 weeks, the T cells of the first group released immune lymphokines (S-ILK), whereas the T cells of the second group produced non-immune lymphokines (N-ILK). The following procedures were then used on a total of 100-layer hens (ISSA brown), divided into four groups of 25 each. G1: S-ILK treatment and H5N1 challenge. G2: N-ILK treatment and H5N1 challenge. G3: Untreated and challenged to (H5N1). G4: untreated or unchallenged. Blood samples were obtained at 31, 32, and 33 weeks of age to assess the (IgG, IFN-γ). Additionally, lung and tracheal tissues were obtained to assess the viral load of influenza RNA copies at (7 and 14) days following the challenge. The experiment's findings indicated that the first group produced the highest mean (IgG and IFN-γ) titers and had the lowest mortality of the other groups. The findings of the viral load test showed that G2 and G3 had the highest number of influenza RNA copies and had significantly lower egg production than G1, which had the lowest viral load and kept egg production at a normal level. The conclusion of the current study shows that using SILK gives layer hens a higher level of homogeneous protection against AIV without any dangers during production.

Evaluation of Rapid Lateral-Flow Tests Directed against the SARS-CoV-2 Nucleoprotein Using Viral Suspensions Belonging to Different Lineages of SARS-CoV-2

Within the successive waves that occurred during the SARS-CoV-2 pandemic, recommendations arose to test symptomatic and contact subjects by using rapid antigen devices directed against the viral nucleocapsid protein with the aim to isolate contagious patients without delay. The objective of this study was to evaluate the ability of four rapid lateral-flow tests (RLFT) that were commercially available on the French market in 2022 to recognize various strains of SARS-CoV-2. Series of five-fold dilutions of seven viral suspensions belonging to different lineages of SARS-CoV-2 (19A, 20A, Alpha, Beta, Gamma, Delta and Omicron) were used to evaluate the analytical sensitivity of four commercially available RLFTs (manufacturers: Abbott, AAZ, Becton-Dickinson and Biospeedia). Cell culture and quantitative RT-PCR were used as references. Excellent correlations were observed for each lineage strain between the viral titer obtained via cell culture and the number of RNA copies measured by quantitative RT-PCR. Although the four tests were able to recognize all the tested variants, significant differences in terms of sensitivity were observed between the four RLFTs. Despite the limitation represented by the small number of devices and clinical isolates that were tested, this study contributed by rapidly comparing the sensitivity of SARS-CoV-2 RLFTs in the Omicron era.

246 (PB-070) Poster - One-step nucleic acid amplification assay in palpable and non-palpable breast tumours

Background: The one-step nucleic acid amplification (OSNA) assay quantifies the cytokeratin 19 (CK19) messenger RNA copy number, which is currently being used for assessment of axillary sentinel lymph node (SLN) status in breast cancer. The total tumour load (TTL), defined as the total amount of CK19 mRNA copies in all positive SLNs, may help predicting additional metastatic axillary involvement besides SLN. There is evidence suggesting that palpable and non-palpable breast tumours manifest distinct pathological features, making tumour palpability a putative predictive factor of axillary lymph node involvement.

A triple-RBD-based mucosal vaccine provides broad protection against SARS-CoV-2 variants of concern.

The rapid mutation and spread of SARS-CoV-2 variants urge the development of effective mucosal vaccines to provide broad-spectrum protection against the initial infection and thereby curb the transmission potential. Here, we designed a chimeric triple-RBD immunogen, 3Ro-NC, harboring one Delta RBD and two Omicron RBDs within a novel protein scaffold. 3Ro-NC elicits potent and broad RBD-specific neutralizing immunity against SARS-CoV-2 variants of concern. Notably, intranasal immunization with 3Ro-NC plus the mucosal adjuvant KFD (3Ro-NC + KFDi.n) elicits coordinated mucosal IgA and higher neutralizing antibody specificity (closer antigenic distance) against the Omicron variant. In Omicron-challenged human ACE2 transgenic mice, 3Ro-NC + KFDi.n immunization significantly reduces the tissue pathology in the lung and lowers the viral RNA copy numbers in both the lung (85.7-fold) and the nasal turbinate (13.6-fold). Nasal virologic control is highly correlated with RBD-specific secretory IgA antibodies. Our data show that 3Ro-NC plus KFD is a promising mucosal vaccine candidate for protection against SARS-CoV-2 Omicron infection, pathology and transmission potential.

Association of SARS-CoV-2 RNA Copy Number with the COVID-19 Mortality Rate and Its Effect on the Predictive Performance of Mortality in Severe Cases.

Factors associated with mortality are important in the treatment of coronavirus disease 2019 (COVID-19). Polymerase chain reaction (PCR) is the gold standard for diagnosing COVID-19, which reflects the viral load in the upper respiratory tract. In total, 523 patients were enrolled in this study; of them, 441 and 75 patients underwent PCR testing of nasopharyngeal swabs and sputum samples, respectively, within 20 days from onset of COVID-19. We investigated the association between RNA copy number and the COVID-19 severity and mortality rate and its effect on the predictive performance for severity and mortality. RNA copy numbers in nasopharyngeal swabs were higher in the non-survivor group than in the survivor group. Multivariate logistic regression analysis identified that the high RNA copy number (≥9 log10 /swab) in nasopharyngeal swabs was a factor associated with mortality (odds ratio, 4.50; 95% confidence interval, 1.510-13.100; P = 0.008). Furthermore, adding RNA copy number (≥9 log10 /swab) in severe cases, adjusted by duration from onset to PCR, improved mortality predictive performance based on known factors. The RNA copy number is a factor associated with the mortality of patients with COVID-19 and can improve the predictive performance of mortality in severe cases.

A collaborative study to establish the national standard for SARS-CoV-2 RNA nucleic acid amplification techniques (NAAT) in Taiwan

The conventional PCR remains a valuable method to detect the newly emergent coronavirus rapidly and accurately. Our investigation aimed to establish the standard materials of SARS-CoV-2 for NAAT detection. We provided formalin-inactivated SARS-CoV-2 and confirmed RNA copy numbers. In addition, the virus genome was confirmed with whole-genome sequencing and identified as Wuhan/WI04/2019. Seven laboratories were invited for this collaborative study, according to the reporting data, we determined the SARS-CoV-2 with the unit of 6.35 Log10 copies/mL as the national standard. The availability of the national standard (NS) of SARS-CoV-2 will facilitate the standardization and harmonization of SARS-CoV-2 NAAT assays.

Bacteriophage-Derived Double-Stranded RNA Exerts Anti-SARS-CoV-2 Activity In Vitro and in Golden Syrian Hamsters In Vivo.

Bacteriophage-derived dsRNA, known as Larifan, is a nationally well-known broad-spectrum antiviral medication. This study aimed to ascertain the antiviral activity of Larifan against the novel SARS-CoV-2 virus. Larifan’s effect against SARS-CoV-2 in vitro was measured in human lung adenocarcinoma (Calu3) and primary human small airway epithelial cells (HSAEC), and in vivo in the SARS-CoV-2 infection model in golden Syrian hamsters. Larifan inhibited SARS-CoV-2 replication both in vitro and in vivo. Viral RNA copy numbers and titer of infectious virus in the supernatant of Calu3 cells dropped significantly: p = 0.0296 and p = 0.0286, respectively. A reduction in viral RNA copy number was also observed in HSAEC, especially when Larifan was added before infection (p = 0.0218). Larifan markedly reduced virus numbers in infected hamsters’ lungs post-infection, with a more pronounced effect after intranasal administration (p = 0.0032). The administration of Larifan also reduced the amount of infections virus titer in the lungs (p = 0.0039). Improvements in the infection-induced pathological lesion severity in the lungs of animals treated with Larifan were also demonstrated. The inhibition of SARS-CoV-2 replication in vitro and the reduction in the viral load in the lungs of infected hamsters treated with Larifan alongside the improved lung histopathology suggests a potential use of Larifan in also controlling the COVID-19 disease in humans.

Crystal digital RT-PCR for the detection and quantification of norovirus and hepatitis A virus RNA in frozen raspberries

Berries are important vehicles for norovirus (NoV) and hepatitis A virus (HAV) foodborne outbreaks. Sensitive and quantitative detection of these viruses in food samples currently relies on RT-qPCR, but remains challenging due to their low concentration and the presence of RT-qPCR inhibitors. Moreover, quantification requires a standard curve. In this study, crystal digital RT-PCR (RT-cdPCR) assays were adapted from RT-qPCR sets of primers and probe currently used in our diagnostic laboratory for the detection and precise quantification of norovirus genogroups I and II (NoV GI, GII) and hepatitis A virus (HAV) RNA in frozen raspberry samples. We selected assay conditions based on optimal separation of positive and negative droplets, and peak resolution. Using virus-specific in vitro RNA transcripts diluted in raspberry RNA extracts, we showed that all three RT-cdPCR assays were sensitive, and we estimated the 95 % detection limit at 9 copies per RT-cdPCR reaction for NoV GI, 3 for NoV GII, and 14 for HAV. Serial dilutions of the RNA transcripts showed excellent linearity over a range of four orders of magnitude. We achieved precise quantification (CV ≤ 35 %) of the RNA transcripts between runs down to 15–145 copies per reaction for NoV GI, <20 for NoV GII, and < 15 for HAV. The three RT-cdPCR assays also proved to be tolerant to inhibitors from frozen raspberries, although not as tolerant as the RT-qPCR assays in the case of NoV GI and HAV. We further evaluated the assays with inoculated frozen raspberry samples and compared their performance to that of the RT-qPCR assays. As compared to the corresponding RT-qPCR assays, the NoV GI and HAV RT-cdPCR assays showed a decreased qualitative sensitivity, while the NoV GII RT-cdPCR assay had an increased sensitivity. As for quantification, the NoV GI and NoV GII RT-cdPCR assays produced similar estimates of RNA copy number than their respective RT-qPCR assays, whereas for HAV, the RT-cdPCR assay produced lower estimates than the RT-qPCR assay. However, all the RT-cdPCR assays provided more precise quantitative measurements at low levels of contamination than the RT-qPCR assays. In conclusion, the potential of the RT-cdPCR assays in this study to detect viral RNA from frozen raspberries varied according to assay, but these RT-cdPCR assays should be considered for precise absolute quantification in difficult matrices such as frozen raspberries.

A ribavirin-induced ORF2 single-nucleotide variant produces defective hepatitis E virus particles with immune decoy function

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and is the leading cause of enterically transmitted viral hepatitis worldwide. Ribavirin (RBV) is currently the only treatment option for many patients; however, cases of treatment failures or posttreatment relapses have been frequently reported. RBV therapy was shown to be associated with an increase in HEV genome heterogeneity and the emergence of distinct HEV variants. In this study, we analyzed the impact of eight patient-derived open reading frame 2 (ORF2) single-nucleotide variants (SNVs), which occurred under RBV treatment, on the replication cycle and pathogenesis of HEV. The parental HEV strain and seven ORF2 variants showed comparable levels of RNA replication in human hepatoma cells and primary human hepatocytes. However, a P79S ORF2 variant demonstrated reduced RNA copy numbers released in the supernatant and an impairment in the production of infectious particles. Biophysical and biochemical characterization revealed that this SNV caused defective, smaller HEV particles with a loss of infectiousness. Furthermore, the P79S variant displayed an altered subcellular distribution of the ORF2 protein and was able to interfere with antibody-mediated neutralization of HEV in a competition assay. In conclusion, an SNV in the HEV ORF2 could be identified that resulted in altered virus particles that were noninfectious invitro and invivo, but could potentially serve as immune decoys. These findings provide insights in understanding the biology of circulating HEV variants and may guide development of personalized antiviral strategies in the future.

Accurate quantification of SARS-CoV-2 RNA by isotope dilution mass spectrometry and providing a correction of reverse transcription efficiency in droplet digital PCR.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 505 million confirmed cases, including over 6 million deaths. Reference materials (RMs) of SARS-CoV-2 RNA played a crucial role in performance evaluation and quality control of testing laboratories. As the potential primary characterization method of RMs, reverse transcription digital PCR (RT-dPCR) measures the copy number of RNA, but the accuracy of reverse transcription (RT) efficiency has yet to be confirmed. This study established a method of enzymatic digestion followed by isotope dilution mass spectrometry (IDMS), which does not require an RT reaction, to quantify in vitro–transcribed SARS-CoV-2 RNA. RNA was digested to nucleotide monophosphate (NMP) within 15 min and analyzed by IDMS within 5 min. The consistency among the results of four different NMPs demonstrated the reliability of the proposed method. Compared to IDMS, the quantitative result of RT-dPCR turned out to be about 10% lower, possibly attributed to the incompleteness of the reverse transcription process. Therefore, the proposed approach could be valuable and reliable for quantifying RNA molecules and evaluating the RT efficiency of RT-based methods.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-022-04238-6.

Knockdown of FLT4, Nup98, and Nup205 Cellular Genes Effectively Suppresses the Reproduction of Influenza Virus Strain A/WSN/1933 (H1N1) In vitro.

Influenza is one of the most common infectious diseases, which affects the lower respiratory tract, and can lead to serious complications, including death. It is known that currently available therapeutic agents and vaccines do not provide 100% protection against influenza viruses. The development of drugs based on the RNA interference mechanism in the context of this problem is a promising area. This paper aims to assess the effect of FLT4, Nup98, and Nup205 cellular gene knockdown on the reproduction of the influenza A virus in human lung cell culture. Influenza virus strain A/WSN/1933 (St. Jude's Children's Research Hospital, USA) was used in this work as well as A549 cell culture (human lung adenocarcinoma, ATCC® CCL- 185, USA) and MDCK cell culture (dog kidney cells, Institut Pasteur, France). Small interfering RNAs (siRNAs) (Syntol, Russia) were synthesized for targeting the FLT4, Nup98, and Nup205 genes. Lipofectamin 2000 (Invitrogen, USA) was used for transfection. After 4 hours, the transfected cells were infected with the influenza virus at MOI = 0.1. Virus-containing fluid was collected within three days from the moment of transfection, and the intensity of viral reproduction was assessed by CPE titration and hemagglutination reactions. Viral RNA concentration was determined by RT-PCR. Mann- Whitney U test was used for statistical analysis. In cells treated with siRNA for FLT4, Nup98, and Nup205 genes, there was a significant decrease in the expression of target genes and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) at MOI = 0.1, although the cell survival rate did not decrease significantly. On the first day, the viral titer in cells treated with declared siRNA was lower, on average, by 1 Lg, and on the second and third days, by 2.2-2.3 Lg, compared to cells treated with nonspecific siRNA. During RT-PCR, a significant decrease in the concentration of viral RNA with Nup98.1 and Nup205 siRNA was detected: up to 190 times and 30 times on the first day, 26 and 29 times on the second day, and 6 and 30 times on the third day, respectively. For FLT4.2 siRNA, the number of viral RNA copies has been decreased by 23, 18, and 16 times on the first, second, and third days. Similar results were obtained while determining the hemagglutinating activity of the virus. The hemagglutinating activity decreased mostly (by 16 times) in cells treated with Nup205 and FLT4.2 siRNAs on the third day. In cells treated with FLT4.1, Nup98.1, and Nup98.2 siRNAs, the hemagglutinating activity decreased by 8 times. We identified a number of genes, such as FLT4, Nup98, and Nup205, whose expression can efficiently suppress viral reproduction when their expression is decreased. The original siRNA sequences were also obtained. These results are important for the creation of therapeutic and prophylactic agents, whose action is based on the RNA interference mechanism.

COVID-19 in Children: Correlation Between Epidemiologic, Clinical Characteristics, and RT-qPCR Cycle Threshold Values.

Background:Initially, the impact of SARS-CoV-2 infection on children was unknown. Standard COVID-19 diagnosis is confirmed using real-time qPCR. Cycle threshold (Ct) values of RT-qPCR are inversely proportional to viral load and the test indirectly quantifies viral RNA copy numbers. The objective of this study was to determine the correlation between epidemiology, clinical characteristics, severity of confirmed COVID-19 cases, and Ct values.Methods:An observational, analytical, cross-sectional study. All children with COVID-19 under 18 years old admitted to the Ricardo Gutiérrez Children’s Hospital between March 1, 2020, and February 28, 2021, were included. SARS-CoV-2 infection was confirmed using RT-qPCR.Results:Median age of patients was 7 years. Ct values were estimated in 419 cases, median Ct value was 23.5 [interquartile range (IQR): 18.9–30.9]. Levels were significantly lower in symptomatic than asymptomatic patients (Ct: 22.1; IQR: 18.4–22.1), in children <2 years of age (Ct: 20.6; IQR: 17.3–27.3) and when sample collection was <4 days after symptom onset (Ct: 21.1; IQR: 18.1–27.5). In children >2 years of age, Ct values were significantly lower in symptomatic (Ct: 22.6; IQR: 18.7–29.3) than asymptomatic (Ct: 31.2; IQR: 24.5–33.3) patients.Conclusions:Children younger than 2 years with COVID-19 have lower values of Ct—as a proxy for higher viral load—than older children. Symptomatic children over 2 years of age had lower Ct values compared with asymptomatic children.

Survival and Replication of Zika Virus in Diapause Eggs of Aedes Albopictus From Beijing, China

Zika virus (ZIKV) has emerged as a globally important arbovirus. The virus is primarily transmitted to humans through the bite of an infective Aedes albopictus in temperate area. Vertical transmission of ZIKV by Ae. albopictus is determined and has been suggested to be a means by which the virus could persist in nature. Ae. albopictus undergoes a well-characterized photoperiodic diapause. Viruses are harbored by overwintering mosquitoes in diapause that contributes to the resurgence of vertebrate diseases in the following spring, yet little is known about the impact of diapause on the regulation of viral replication and survival. The purpose of this study is to determine that Ae. albopictus in Beijing are highly susceptible to ZIKV (92.3%), and viable virus is passed to their organs of progeny via vertical transmission. Moreover, diapause eggs (diapause incidence 97.8%) had significantly lower minimum infection rates and filial infection rates of the first gonotrophic cycle than those of the second gonotrophic cycle in the short-day photoperiod group. Regarding the development of diapause eggs, the minimum infection rates and ZIKV RNA copy number increased significantly, suggesting that virus RNA replication occurred in the diapause eggs. Meanwhile, eggs from the ZIKV-infected mosquitoes had a significantly lower hatching rate compared with uninfected mosquitoes, implying an intriguing interaction between diapause eggs and virus. The findings here suggest that vertical transmission of ZIKV from diapause eggs to progeny may have a critical epidemiological role in the dissemination and maintenance of ZIKV circulating in the vector.

Abstract 770: Multiregional imaging and genomic analysis in high-grade IDH-mutant astrocytoma identifies intratumor subclones with aggressive features

Abstract High-grade gliomas represent the most common type of primary adult malignant brain tumor historically diagnosed and graded from histologic criteria alone. Gliomas harboring isocitrate dehydrogenase (IDH) 1 or 2 mutations, which are present in more than 80% of World Health Organization (WHO) grade 2 or 3 tumors, portend a favorable prognosis as compared to IDH-wildtype tumors. However, recent molecular profiling has identified the presence of additional alterations in these lower grade tumors that transform the biology toward an aggressive high-grade phenotype clinically that are independent of histologic grading and confer a worse clinical prognosis. Of these, the loss of CDKN2A/B is the strongest implicated alteration and is sufficient to define a grade 4 tumor in IDH-mutant astrocytomas regardless of histologic appearance. However, there remain no effective therapies targeted at molecular subgroups in aggressive gliomas to date. Here we have profiled the intratumoral heterogeneity via whole exome and RNA sequencing and copy number alteration analysis within a cohort of de-identified IDH-mutant astrocytoma patient tumors collected through the Mayo Clinic and Barrow Neurological Institute (BNI) biorepositories to determine the spatial evolution of aggressive molecular features. Within this cohort we have collected 42 image-localized intratumoral biopsy specimens representing 13 IDH-mutant patient tumors. We identified 11 biopsies from 3 tumors that harbored loss of CDKN2A/B which included 1 tumor that displayed intratumoral heterogeneity of CDKN2A/B expression across subclones. We performed an unsupervised cluster analysis to profile the transcriptional signatures in IDH-mutant astrocytomas across both differential CDKN2A/B status and tumor geographic location comparing contrast enhancing versus non-enhancing regions, and we analyzed the MRI features in IDH-mutant tumors across CDKN2A/B status. We found that tumors with CDKN2A/B alteration demonstrated lower signal intensity on delayed post-contrast T2*W images, which could represent lower pre-contrast T2*, unique contrast agent kinetics, or dissimilar diffusion properties related to increased cellular density and/or greater heterogeneity of cell size. Interestingly, we uncovered spatial heterogeneity of additional molecular drivers of high-grade glioma including KRAS, MYCN, and PDGFRA. While EGFR alterations are rarely reported in IDH-mutant tumors, we identified one treatment naïve patient tumor that harbored EGFR alteration only in the contrast non-enhancing region. Based on this work we propose there is early spatial evolution of aggressive tumor subclones prior to primary tumor diagnosis, and multiregional molecular profiling may better inform tumor biology and predict therapeutic strategies in IDH-mutant astrocytomas. Citation Format: Shannon P. Fortin Ensign, Fulvio D'Angelo, Francesca P. Caruso, Christopher Sereduk, Kyle Singleton, Jennifer Eschbacher, Gina Mazza, Maciej Mrugala, Alyx Porter, Bernard Bendok, Richard Zimmerman, Kris Smith, Peter Nakaji, Kamala Swanson, Junwen Wang, Nathenael Semmineh, Christopher Quarles, Anna Lasorella, Kristin Swanson, Michele Ceccarelli, Antonio Iavarone, Leland Hu, Nhan L. Tran. Multiregional imaging and genomic analysis in high-grade IDH-mutant astrocytoma identifies intratumor subclones with aggressive features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 770.

#73 Single Cell RNA Sequencing of Zika Infection in Neural Tissues Reveals an Astrocyte-Driven Innate Immune Response Governed by Interferon Beta.

Abstract Background Congenital infection with Zika virus (ZIKV) has been linked to severe of fetal brain disruption and other developmental anomalies referred to collectively as congenital Zika syndrome (CZS). Long-term follow up of ZIKV-exposed infants has shown that even those without overt findings of CZS can have significant cognitive and developmental difficulties. However, the pathogenesis of ZIKV-related microcephaly and cognitive dysfunction remain poorly understood. To begin answering these questions, we sought to define the consequences of ZIKV infection on specific cell types in the developing brain. Method We have used primary human fetal brain cultures and human induced pluripotent stem cell-derived neural progenitor cell lines to probe innate immune signaling to ZIVK infection in vitro. We used single-cell RNA sequencing (10x Genomics) to identify the cell type-specific transcriptional changes induced by ZIKV infection. Results We found that neural progenitor cells (NPCs) support higher ZIKV RNA copy number than do differentiated neural cells. Moreover, while IFN-β is the predominant cytokine released by differentiated cells in response to ZIKV infection, NPCs do not produce IFN-β. By additionally characterizing the transcriptional response to IFN-β treatment, we have been able to distinguish virus-stimulated genes (VSGs) from interferon-stimulated genes (ISGs) across cell types. We find extensive heterogeneity in cell type-specific VSG and ISG profiles. Astrocytes undergo the largest transcriptional changes in response to infection and are the main producers of IFN-β. Our findings also identify genes whose expression is altered during viral infection specifically in NPCs and neurons, providing candidate pathways that may underlie neural dysfunction. Conclusion Across cell types, the response to Zika infection closely matches the response to IFN-β, arguing that astrocyte-derived IFN-β is the main driver of the innate immune response to ZIKV in the developing brain. Our findings provide insight into the pathogenesis of CZS and offer novel targets for improving developmental outcomes after fetal ZIKV infection.