Recovery Of RNA Research Articles

A comparative study of RNA yields from museum specimens, including an optimized protocol for extracting RNA from formalin-fixed specimens

Animal specimens in natural history collections are invaluable resources in examining the historical context of pathogen dynamics in wildlife and spillovers to humans. For example, natural history specimens may reveal new associations between bat species and coronaviruses. However, RNA viruses are difficult to study in historical specimens because protocols for extracting RNA from these specimens have not been optimized. Advances have been made in our ability to recover nucleic acids from formalin-fixed paraffin-embedded samples (FFPE) commonly used in human clinical studies, yet other types of formalin preserved samples have received less attention. Here, we optimize the recovery of RNA from formalin-fixed ethanol-preserved museum specimens in order to improve the usability of these specimens in surveys for zoonotic diseases. We provide RNA quality and quantity measures for replicate tissues subsamples of 22 bat specimens from five bat genera (Rhinolophus, Hipposideros, Megareops, Cynopterus, and Nyctalus) collected in China and Myanmar from 1886 to 2003. As tissues from a single bat specimen were preserved in a variety of ways, including formalin-fixed (8 bats), ethanol-preserved and frozen (13 bats), and flash frozen (2 bats), we were able to compare RNA quality and yield across different preservation methods. RNA extracted from historical museum specimens is highly fragmented, but usable for short-read sequencing and targeted amplification. Incubation of formalin-fixed samples with Proteinase-K following thorough homogenization improves RNA yield. This optimized protocol extends the types of data that can be derived from existing museum specimens and facilitates future examinations of host and pathogen RNA from specimens.

Multifaceted Assessment of Wastewater-Based Epidemiology for SARS-CoV-2 in Selected Urban Communities in Davao City, Philippines: A Pilot Study.

Over 60 countries have integrated wastewater-based epidemiology (WBE) in their COVID-19 surveillance programs, focusing on wastewater treatment plants (WWTP). In this paper, we piloted the assessment of SARS-CoV-2 WBE as a complementary public health surveillance method in susceptible communities in a highly urbanized city without WWTP in the Philippines by exploring the extraction and detection methods, evaluating the contribution of physico-chemical–anthropogenic factors, and attempting whole-genome sequencing (WGS). Weekly wastewater samples were collected from sewer pipes or creeks in six communities with moderate-to-high risk of COVID-19 transmission, as categorized by the City Government of Davao from November to December 2020. Physico-chemical properties of the wastewater and anthropogenic conditions of the sites were noted. Samples were concentrated using a PEG-NaCl precipitation method and analyzed by RT-PCR to detect the SARS-CoV-2 N, RdRP, and E genes. A subset of nine samples were subjected to WGS using the Minion sequencing platform. SARS-CoV-2 RNA was detected in twenty-two samples (91.7%) regardless of the presence of new cases. Cycle threshold values correlated with RNA concentration and attack rate. The lack of a sewershed map in the sampled areas highlights the need to integrate this in the WBE planning. A combined analysis of wastewater physico-chemical parameters such as flow rate, surface water temperature, salinity, dissolved oxygen, and total dissolved solids provided insights on the ideal sampling location, time, and method for WBE, and their impact on RNA recovery. The contribution of fecal matter in the wastewater may also be assessed through the coliform count and in the context of anthropogenic conditions in the area. Finally, our attempt on WGS detected single-nucleotide polymorphisms (SNPs) in wastewater which included clinically reported and newly identified mutations in the Philippines. This exploratory report provides a contextualized framework for applying WBE surveillance in low-sanitation areas.

Storage media and RNA extraction approaches substantially influence the recovery and integrity of livestock fecal microbial RNA.

BackgroundThere is growing interest in understanding gut microbiome dynamics, to increase the sustainability of livestock production systems and to better understand the dynamics that regulate antibiotic resistance genes (i.e., the resistome). High-throughput sequencing of RNA transcripts (RNA-seq) from microbial communities (metatranscriptome) allows an unprecedented opportunity to analyze the functional and taxonomical dynamics of the expressed microbiome and emerges as a highly informative approach. However, the isolation and preservation of high-quality RNA from livestock fecal samples remains highly challenging. This study aimed to determine the impact of the various sample storage and RNA extraction strategies on the recovery and integrity of microbial RNA extracted from selected livestock (chicken and pig) fecal samples.MethodsFecal samples from pigs and chicken were collected from conventional slaughterhouses. Two different storage buffers were used at two different storage temperatures. The extraction of total RNA was done using four different commercially available kits and RNA integrity/quality and concentration were measured using a Bioanalyzer 2100 system with RNA 6000 Nano kit (Agilent, Santa Clara, CA, USA). In addition, RT-qPCR was used to assess bacterial RNA quality and the level of host RNA contamination.ResultsThe quantity and quality of RNA differed by sample type (i.e., either pig or chicken) and most significantly by the extraction kit, with differences in the extraction method resulting in the least variability in pig feces samples and the most variability in chicken feces. Considering a tradeoff between the RNA yield and the RNA integrity and at the same time minimizing the amount of host RNA in the sample, a combination of storing the fecal samples in RNALater at either 4 °C (for 24 h) or −80 °C (up to 2 weeks) with extraction with PM kit (RNEasy Power Microbiome Kit) had the best performance for both chicken and pig samples.ConclusionOur findings provided a further emphasis on using a consistent methodology for sample storage, duration as well as a compatible RNA extraction approach. This is crucial as the impact of these technical steps can be potentially large compared with the real biological variability to be explained in microbiome and resistome studies.

Inactivation and Recovery of High Quality RNA From Positive SARS-CoV-2 Rapid Antigen Tests Suitable for Whole Virus Genome Sequencing.

The diagnostic protocol currently used globally to identify Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is RT-qPCR. The spread of these infections and the epidemiological imperative to describe variation across the virus genome have highlighted the importance of sequencing. SARS-CoV-2 rapid antigen diagnostic tests (RADTs) are designed to detect viral nucleocapsid protein with positive results suggestive of the presence of replicating virus and potential infectivity. In this study, we developed a protocol for recovering SARS-CoV-2 RNA from “spent” RADT devices of sufficient quality that can be used directly for whole virus genome sequencing. The experimental protocol included the spiking of RADTs at different concentrations with viable SARS-CoV-2 variant Alpha (lineage B.1.1.7), lysis for direct use or storage. The lysed suspensions were used for RNA extraction and RT-qPCR. In parallel, we also tested the stability of the viral RNA in the RADTs and the RNA extracted from the RADTs was used as a template for tiling-PCR and whole virus genome sequencing. RNA recovered from RADTs spiked with SARS-CoV-2 was detected through RT-qPCR with Ct values suitable for sequencing and the recovery from RADTs was confirmed after 7 days of storage at both 4 and 20°C. The genomic sequences obtained at each time-point aligned to the strain used for the spiking, demonstrating that sufficient SARS-CoV-2 viral genome can be readily recovered from positive-RADT devices in which the virus has been safely inactivated and genomically conserved. This protocol was applied to obtain whole virus genome sequence from RADTs ran in the field where the omicron variant was detected. The study demonstrated that viral particles of SARS-CoV-2 suitable for whole virus genome sequencing can be recovered from positive spent RADTs, extending their diagnostic utility, as a risk management tool and for epidemiology studies. In large deployment of the RADTs, positive devices could be safely stored and used as a template for sequencing allowing the rapid identification of circulating variants and to trace the source and spread of outbreaks within communities and guaranteeing public health.

Implementing a SARS-CoV-2 Early Warning System in Valencia (Spain) and its Correlation with Epidemiological Indicators

PurposeTo implement a SARS-CoV-2 wastewater surveillance system in Valencia and study the correlation between SARS-CoV-2 concentration levels and epidemiological indicators.Methods & MaterialsThe working strategy developed in the municipality of Valencia consisted of monitoring wastewater effluents collected by 790,000 people. The city was divided into 24 different hydraulic sectors. 3,023 samples of untreated wastewater were collected and analysed between May 2020 and May 2021. RNA extraction from sewage material was carried out using the NucleoSpin RNA virus Kit. SARS-CoV-2 RNA detection was performed by RT-qPCR using One-Step PrimeScript™ RT-PCR Kit (Perfect Real Time), targeting the nucleoprotein (N), N1 and N2 fragments, and envelope protein (E) gene. Mengovirus RNA recovery rates were used as quality assurance parameters according to ISO 15216-1:2017.ResultsA total of 2,169 samples were positive for SARS-CoV-2 RNA material. SARS-CoV-2 variations were detected throughout the entire study evidencing trends during the first, second and third wave. As the National State of Emergency ended (June 2020), SARS-CoV-2 values began to increase reaching the first and most significant concentration spike of the study (20th-26th of July 2020), with a weekly average aggregate concentration of 34,5M GC/L (an increase of 2 units in the log scale). This translated in the worsening of epidemiological indicators (number of cases, hospitalizations, deaths and cumulative incidence (CI)), which maintained a stable increase until a second spike was detected during an important bank holiday in October (9th-12th), where the concentration changed from 28M GC/L to 345M GC/L in a 3-day period while the city was registering 1,100 new weekly cases and a CI of 200 cases/100,000 residents. Finally, a third wave placed Valencia with the worst historical epidemiological data (6,545 new cases; 1,000 new hospitalizations; CI= 1,318,04 cases/100,000 inhabitants) with a weekly SARS-CoV-2 average aggregate concentration of 967M GC/L, leading health officials to implement new restrictive measures.ConclusionWastewater surveillance could be used as a complimentary tool to estimate the presence and prevalence of COVID-19 in communities and can be used for preventive purposes, as an increasing SARS-CoV-2 trend in wastewater could be a signal of the possible re-emergence of the pandemic.

Development of an efficient wastewater testing protocol for high-throughput country-wide SARS-CoV-2 monitoring

Wastewater-based surveillance has been widely used as a non-intrusive tool to monitor population-level transmission of COVID-19. Although various approaches are available to concentrate viruses from wastewater samples, scalable methods remain limited. Here, we sought to identify and evaluate SARS-CoV-2 virus concentration protocols for high-throughput wastewater testing. A total of twelve protocols for polyethylene glycol (PEG) precipitation and four protocols for ultrafiltration-based approaches were evaluated across two phases. The first phase entailed an initial evaluation using a small sample set, while the second phase further evaluated five protocols using wastewater samples of varying SARS-CoV-2 concentrations. Permutations in the pre-concentration, virus concentration and RNA extraction steps were evaluated. Among PEG-based methods, SARS-CoV-2 virus recovery was optimal with 1) the removal of debris prior to processing, 2) 2 h to 24 h incubation with 8% PEG at 4 °C, 3) 4000 xg or 14,000 xg centrifugation, and 4) a column-based RNA extraction method, yielding virus recovery of 42.4–52.5%. Similarly, the optimal protocol for ultrafiltration included 1) the removal of debris prior to processing, 2) ultrafiltration, and 3) a column-based RNA extraction method, yielding a recovery of 38.2%. This study also revealed that SARS-CoV-2 RNA recovery for samples with higher virus concentration were less sensitive to changes in the PEG method, but permutations in the PEG protocol could significantly impact virus yields when wastewater samples with lower SARS-CoV-2 RNA were used. Although both PEG precipitation and ultrafiltration methods resulted in similar SARS-CoV-2 RNA recoveries, the former method is more cost-effective while the latter method provided operational efficiency as it required a shorter turn-around-time (PEG precipitation, 9–23 h; Ultrafiltration, 5 h). The decision on which method to adopt will thus depend on the use-case for wastewater testing, and the need for cost-effectiveness, sensitivity, operational feasibility and scalability.

SARS-CoV-2 RNA Recovery from Air Sampled on Quartz Fiber Filters: A Matter of Sample Preservation?

The airborne route of transmission of SARS-CoV-2 was confirmed by the World Health Organization in April 2021. There is an urge to establish standardized protocols for assessing the concentration of SARS-CoV-2 RNA in air samples to support risk assessment, especially in indoor environments. Debates on the airborne transmission route of SARS-CoV-2 have been complicated because, among the studies testing the presence of the virus in the air, the percentage of positive samples has often been very low. In the present study, we report preliminary results on a study for the evaluation of parameters that can influence SARS-CoV-2 RNA recovery from quartz fiber filters spotted either by standard single-stranded SARS-CoV-2 RNA or by inactivated SARS-CoV-2 virions. The analytes were spiked on filters and underwent an active or passive sampling; then, they were preserved at −80 °C for different numbers of days (0 to 54) before extraction and analysis. We found a mean recovery of 2.43%, except for the sample not preserved (0 days) that showed a recovery of 13.51%. We found a relationship between the number of days and the recovery percentage. The results presented show a possible issue that relates to the quartz matrix and SARS-CoV-2 RNA recovery. The results are in accordance with the already published studies that described similar methods for SARS-CoV-2 RNA field sampling and that reported non-detectable concentrations of RNA. These outcomes could be false negatives due to sample preservation conditions. Thus, until further investigation, we suggest, as possible alternatives, to keep the filters: (i) in a sealed container for preservation at 4 °C; and (ii) in a viral transport medium for preservation at a temperature below 0 °C.

Automated DNA, RNA, and Protein Extraction from Urine for Biobanking.

DNA, RNA, and proteins are unavoidable human biomarkers. Today, blood remains the commonly used source of biomarkers despite numerous limitations. Therefore, other sources of biomarkers such as urine could be more appropriate for research in the field of bladder cancer. The aim of this study was to set up a new automated procedure for urinary DNA, RNA, and protein extraction and to evaluate their quality and quantity. This study was conducted in the setting of the COBLAnCE cohort. Urinary DNA and RNA were extracted using the Maxwell 16 system, and urinary proteins were isolated by precipitation from the supernatant and the cell pellet. The concentration and purity of nucleic acids were determined by spectrophotometry. RNA integrity was determined by the Agilent Bioanalyzer. PCR assays were also used to ensure the quality of DNA and RNA samples. The quality of protein samples obtained was determined by Western blot analysis. PCR experiments performed highlighted that it is possible to use the DNA and RNA samples for amplification, gene expression, or genotyping. However, DNA and RNA recovery from urine was highly variable among patients, with a significant impact of the patient's gender. The samples were highly degraded. Finally, our protocol of protein isolation was effective in extracting urinary supernatant proteins as well as pellet proteins. Therefore, urine samples could constitute valuable resources for subsequent investigations in bladder cancer. These samples will allow identifying new easy-access biomarkers for the early detection of cancer, monitoring cancer progression, and assessing response to therapy.

Evaluation of micro-RNA in extracellular vesicles from blood of patients with prostate cancer.

Extracellular vesicles (EVs) contain various types of molecules including micro-RNAs, so isolating EVs can be an effective way to analyze and diagnose diseases. A lot of micro-RNAs have been known in relation to prostate cancer (PCa), and we evaluate miR-21, miR-141, and miR-221 in EVs and compare them with prostate-specific antigen (PSA). EVs were isolated from plasma of 38 patients with prostate cancer and 8 patients with benign prostatic hyperplasia (BPH), using a method that showed the highest recovery of RNA. Isolation of EVs concentrated micro-RNAs, reducing the cycle threshold (Ct) value of RT-qPCR amplification of micro-RNA such as miR-16 by 5.12 and miR-191 by 4.65, compared to the values before EV isolation. Normalization of target micro-RNAs was done using miR-191. For miR-221, the mean expression level of patients with localized PCa was significantly higher than that of the control group, having 33.45 times higher expression than the control group (p < 0.01). Area under curve (AUC) between BPH and PCa for miR-221 was 0.98 (p < 0.0001), which was better than AUC for prostate-specific antigen (PSA) level in serum for the same patients. The levels of miR-21 and miR-141 in EVs did not show significant changes in patients with PCa compared to the control group in this study. This study suggests isolating EVs can be a helpful approach in analyzing micro-RNAs with regard to disease.

Optimization of RNA Aptamer SELEX Methods: Improved Aptamer Transcript 3'-End Homogeneity, PAGE Purification Yield, and Target-Bound Aptamer RNA Recovery.

Since its inception in the early 1990s, SELEX remains the gold standard for discovering RNA aptamers specific for proteins and small molecules. The SELEX process has undergone countless modifications and now encompasses a breadth of innovative selection schemes to pare an aptamer library toward target-specific aptamers. Common to all these RNA aptamer SELEX processes are the steps for the preparation of DNA template and in vitro transcription of aptamer RNA. These steps have remained mostly unchanged over the past three decades and would benefit from optimization. We focused on three key areas: improving the homogeneity of in vitro transcribed aptamer RNA, increasing the efficiency of in vitro transcribed aptamer RNA purification by PAGE, and improving the quality of target-bound aptamer RNA recovered during SELEX. Together, these optimizations contribute toward a more efficient SELEX process and are applicable to both protein-based and cell-based RNA aptamer selections.

Direct detection of polioviruses using a recombinant poliovirus receptor.

Polioviruses are positive-sense, single-stranded RNA picornaviruses and the principal cause of poliomyelitis. Global poliovirus surveillance has relied on poliovirus isolation in cells, which may take a minimum of 10 days, involves maintaining two cell lines, and propagates virus in high titers. With eradication underway, a major objective of the Global Polio Eradication Initiative (GPEI) is to develop culture-independent detection of polioviruses as an alternative method to complement the current virus isolation technique. A culture-independent method on poliovirus-positive stool suspensions was assessed with commercially available recombinant soluble poliovirus receptor (PVR) coupled to Histidine (His) tags. Viral RNA was screened by quantitative real-time reverse transcription PCR using the poliovirus intratypic differentiation kit. Poliovirus recovery was optimized with PVR-His–tagged protein and buffers supplemented with polyethylene glycol. To validate the poliovirus-PVR–His tag purification assay, 182 poliovirus-positive stools of programmatic importance were parallel tested against the GPLN-accepted virus isolation method. The PVR-His tag enrichment method detected poliovirus in 164 of 171 poliovirus-positive stools, whereas the virus isolation method misidentified 38 stools as poliovirus-negative (McNemar χ2 p<0.0001). Using this method in combination with RNA extraction, viral RNA recovery increased and showed similar (WPV1) or higher (Sabin 1) sensitivity than the World Health Organization accredited variation of the virus isolation method. The PVR-His enrichment method could be a viable addition to poliovirus surveillance; similar methods have the potential to capture other human pathogens such as EV71 using an appropriate soluble His tag receptor.

Mengovirus as Process Control Virus in the Monitoring of Genomic RNA and Infectivity of Enteric Viruses in Water Matrices

The present study, developed in the scope of a survey to monitor enteric viruses in natural surface water and drinking water sources, addressed the suitability of mengovirus to assess viral recovery rates at two steps of the water sampling process. In a pilot campaign comprising two samples from each type of water source, when mengovirus was added after the filtration/primary concentration step, the recovery rates of viral RNA were higher than 18% and identical for both water matrices. In a one-year sampling campaign, where mengovirus was present along the whole sample processing (addition in the filtration/primary concentration step), significantly different recovery rates were observed between water matrices: usually higher than 1% in drinking water and under 1% in surface water. The results suggest the first stage of the water sampling process and the type of water matrix are the most influential factors for viral RNA recovery. This study also addressed and evidenced mengovirus replication and titration in Vero E6 cultures and showed infectious mengovirus to be recovered from samples of both types of water matrix. These results anticipate a more comprehensive applicability of mengovirus as a process control virus in the monitoring of viruses in water, extended to viral infectivity.

Deciphering the molecular signal from past and alive bacterial communities in aquatic sedimentary archives.

Lake sediments accumulate information on biological communities thus acting as natural archives. Traditionally paleolimnology has focussed on fossilized remains of organisms, however, many organisms do not leave fossil evidence, meaning major ecosystem components are missing from environmental reconstructions. Many paleolimnology studies now incorporate molecular methods, including investigating microbial communities using environmental DNA (eDNA), but there is uncertainty about the contribution of living organisms to molecular inventories. In the present study, we obtained DNA and RNA inventories from sediment spanning 700years to investigate the contribution of past and active communities to the molecular signal from sedimentary archives. Additionally, a droplet digital PCR (ddPCR) targeting the 16S ribosomal RNA (16S rRNA) gene of the photosynthetic cyanobacterial genera Microcystis was used to explore if RNA signals were from legacy RNA. We posit that the RNA signal is a mixture of legacy RNA, dormant cells, living bacteria and modern-day trace level contaminants that were introduced during sampling and preferentially amplified. The presence of legacy RNA was confirmed by the detection of Microcystis in sediments aged to ~200years ago. Recent comparisons between 16S rRNA gene metabarcoding and traditional paleo proxies showed that past changes in bacterial communities can be reconstructed from sedimentary archives. The recovery of RNA in the present study has provided new insights into the origin of these signals. However, caution is required during analysis and interpretation of 16S rRNA gene metabarcoding data especially in recent sediments were there are potentially active bacteria.

Aerosol SARS-CoV-2 in hospitals and long-term care homes during the COVID-19 pandemic.

BackgroundFew studies have quantified aerosol concentrations of SARS-CoV-2 in hospitals and long-term care homes, and fewer still have examined samples for viability. This information is needed to clarify transmission risks beyond close contact.MethodsWe deployed particulate air samplers in rooms with COVID-19 positive patients in hospital ward and ICU rooms, rooms in long-term care homes experiencing outbreaks, and a correctional facility experiencing an outbreak. Samplers were placed between 2 and 3 meters from the patient. Aerosol (small liquid particles suspended in air) samples were collected onto gelatin filters by Ultrasonic Personal Air Samplers (UPAS) fitted with <2.5μm (micrometer) and <10 μm size-selective inlets operated for 16 hours (total 1.92m3), and with a Coriolis Biosampler over 10 minutes (total 1.5m3). Samples were assayed for viable SARS-CoV-2 virus and for the viral genome by multiplex PCR using the E and N protein target sequences. We validated the sampling methods by inoculating gelatin filters with viable vesicular stomatitis virus (VSV), and with three concentrations of viable SARS-CoV-2, operating personal samplers for 16hrs, and quantifying viable virus recovery by TCID50 assay.ResultsIn total, 138 samples were collected from 99 rooms. RNA samples were positive in 9.1% (6/66) of samples obtained with the UPAS 2.5μm samplers, 13.5% (7/52) with the UPAS 10μm samplers, and 10.0% (2/20) samples obtained with the Coriolis samplers. Culturable virus was not recovered in any samples. Viral RNA was detected in 15.1% of the rooms sampled. There was no significant difference in viral RNA recovery between the different room locations or samplers. Method development experiments indicated minimal loss of SARS-CoV-2 viability via the personal air sampler operation.

Microglial FACS for Robust RNA Recovery for Next-Generation Sequencing.

Microglia are immune cells of the central nervous system (CNS), which play an instrumental role in eliminating invading pathogens and help regulate localized inflammation. They also assist in maintaining homeostasis of the brain microenvironment. Microglia isolation from primary brain tissue can be difficult with poor yields from tissue dissociation which precludes many downstream assays from being efficiently conducted. Recovery of intact microglia for single-cell or next-generation RNA sequencing (NGS, RNAseq) can be a difficult process. The recovery of intact RNA transcripts inside viable cells has its challenges. Here we describe a method to enrich CD11b+microglial cells from brain tissue followed by FACS, for a reliable and reproducible method for the recovery of high-quality RNA from sorted microglia for downstream sequencing.

Factors influencing recovery of SARS-CoV-2 RNA in raw sewage and wastewater sludge using polyethylene glycol-based concentration method.

Wastewater surveillance for monitoring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important epidemiologic tool for the assessment of population-wide coronavirus disease 2019 (COVID-19). This tool can be successfully implemented only if SARS-CoV-2 RNA in wastewater can be accurately recovered and quantified. The lack of standardized procedure for wastewater virus analysis has resulted in varying SARS-CoV-2 concentrations for the same sample. This study reports the effect of 4 key factors-sample volume, percentage polyethylene glycol (PEG)-NaCl, incubation period, and storage duration at 4°C-on the recovery of spiked noninfectious SARS-CoV-2 RNA in raw sewage and sludge samples. N1 and N2 genes of SARS-CoV-2 were quantified using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and digital droplet PCR (RT-ddPCR) techniques. Results indicate that 1) for raw sewage, 50-ml sample volume, 30% PEG-NaCl addition, 6-h incubation, and sample analysis within 24 h of collection can result in much better RNA recovery (RT-qPCR: 72% for N1 and 82% for N2; RT-ddPCR: 55% for N1 and 85% for N2) when compared with commonly used PEG-based method; 2) for sludge, the sample analysis using raw sewage protocol and all other variations of each factor mostly resulted in false negatives for both N1 and N2. The absence of N1 and N2 suggests that sludge samples probably need a pretreatment step that releases RNA entrapped in sludge solids back into bulk solution. In conclusion, our modified PEG-based concentration method can cut down the analysis time at least by half, which in turn helps to implement early detection system for SARS-CoV-2 in wastewater.

Autochthonous dengue outbreak in Italy 2020: clinical, virological and entomological findings.

BackgroundIn August 2020, in the context of COVID-19 pandemics, an autochthonous dengue outbreak was identified for the first time in Italy.MethodsFollowing the reporting of the index case of autochthonous dengue, epidemiological investigation, vector control and substances of human origin safety measures were immediately activated, according to the national arbovirus surveillance plan. Dengue cases were followed-up with weekly visits and laboratory tests until recovery and clearance of viral RNA from blood.ResultsThe primary dengue case was identified in a young woman, who developed fever after returning from Indonesia to northern Italy, on 27 July 2020. She spent the mandatory quarantine for COVID-19 at home with relatives, six of whom developed dengue within two weeks. Epidemiological investigation identified further five autochthonous dengue cases among people who lived or stayed near the residence of the primary case. The last case of the outbreak developed fever on 29 September 2020. Dengue cases had a mild febrile illness, except one with persistent asthenia and myalgia. DENV-1 RNA was detected in blood and/or urine in all autochthonous cases, up to 35 days after fever onset. All cases developed IgM and IgG antibodies which cross-reacted with West Nile virus (WNV) and other flaviviruses. Sequencing of the full viral genome from blood samples showed over 99% nucleotide identity with DENV-1 strains isolated in China in 2014–2015; phylogenetic analysis classified the virus within Genotype I. Entomological site inspection identified a high density of Aedes albopictus mosquitoes, which conceivably sustained local DENV-1 transmission. Aedes koreicus mosquitoes were also collected in the site.ConclusionsAreas in Europe with high density of Aedes mosquitoes should be considered at risk for dengue transmission. The presence of endemic flaviviruses, such as WNV, might pose problems in the laboratory diagnosis.

Impact of sampling and storage stress on the recovery of airborne SARS-CoV-2 virus surrogate captured by filtration

Environmental air sampling of the SARS-CoV-2 virus in occupational and community settings is pertinent to reduce and monitor the spread of the COVID pandemic. However, there is a general lack of standardized procedures for airborne virus sampling and limited knowledge of how sampling and storage stress impact the recovery of captured airborne viruses. Since filtration is one of the commonly used methods to capture airborne viruses, this study analyzed the effect of sampling and storage stress on SARS-CoV-2 surrogate virus (human coronavirus OC43, or HCoV-OC43) captured by filters. HCoV-OC43, a simulant of the SARS-CoV-2, was aerosolized and captured by PTFE-laminated filters. The impact of sampling stress was evaluated by comparing the RNA yields recovered when sampled at 3 L/min and 10 L/min and for 10 min and 60 min; in one set of experiments, additional stress was added by passing clean air through filters with the virus for 1, 5, and 15 hr. The impact of storage stress was designed to examine RNA recovery from filters at room temperature (25 °C) and refrigerated conditions (4 °C) for up to 1 week of storage. To our knowledge, this is the first report on using HCoV-OC43 aerosol in air sampling experiments, and the mode diameter of the virus aerosolized from the growth medium was 40–60 nm as determined by SMPS + CPC system (TSI Inc.) and MiniWRAS (Grimm Inc.) measurements. No significant difference was found in virus recovery between the two sampling flow rates and different sampling times (p > 0.05). However, storage at room temperature (25 °C) yielded ∼2x less RNA than immediate processing and storage at refrigerated conditions (4 °C). Therefore, it is recommended to store filter samples with viruses at 4 °C up to 1 week if the immediate analysis is not feasible. Although the laminated PTFE filter used in this work purposefully does not include a non-PTFE backing, the general recommendations for handling and storing filter samples with viral particles are likely to apply to other filter types.

Comparative analysis of rapid concentration methods for the recovery of SARS-CoV-2 and quantification of human enteric viruses and a sewage-associated marker gene in untreated wastewater

To support public-health-related disease surveillance and monitoring, it is crucial to concentrate both enveloped and non-enveloped viruses from domestic wastewater. To date, most concentration methods were developed for non-enveloped viruses, and limited studies have directly compared the recovery efficiency of both types of viruses. In this study, the effectiveness of two different concentration methods (Concentrating pipette (CP) method and an adsorption-extraction (AE) method amended with MgCl2) were evaluated for untreated wastewater matrices using three different viruses (SARS-CoV-2 (seeded), human adenovirus 40/41 (HAdV 40/41), and enterovirus (EV)) and a wastewater-associated bacterial marker gene targeting Lachnospiraceae (Lachno3). For SARS-CoV-2, the estimated mean recovery efficiencies were significantly greater by as much as 5.46 times, using the CP method than the AE method amended with MgCl2. SARS-CoV-2 RNA recovery was greater for samples with higher titer seeds regardless of the method, and the estimated mean recovery efficiencies using the CP method were 25.1 ± 11% across ten WWTPs when wastewater samples were seeded with 5 × 104 gene copies (GC) of SARS-CoV-2. Meanwhile, the AE method yielded significantly greater concentrations of indigenous HAdV 40/41 and Lachno3 from wastewater compared to the CP method. Finally, no significant differences in indigenous EV concentrations were identified in comparing the AE and CP methods. These data indicate that the most effective concentration method varies by microbial analyte and that the priorities of the surveillance or monitoring program should be considered when choosing the concentration method.

Abstract 2232: Optimizing Performance of Whole Transcriptome RNA-Seq Reference

Abstract Purpose: Whole transcriptome RNA-sequencing (RNA-Seq) has emerged as one of the most effective methods for detection of genomic rearrangements in cancer. Enrichment for poly(A) + RNA as part of library preparation is a standard method to select for mRNAs, but ribosomal RNA depletion and exon capture methods may be preferred for samples of suboptimal RNA quality, such as FFPE. Regardless of the selection and library preparation procedure, RNA-Seq for clinical use requires well characterized reference materials for assay quality control and fusion detection verification. However, reference materials developed for targeted NGS panels performed sub-optimally on RNA-Seq. This study was undertaken to design, optimize and improve RNA-Seq performance with a novel transcriptome-scale RNA Fusion reference material. Procedure: Seraseq® Fusion RNA Mix is highly multiplexed with 18 clinically relevant fusion RNAs and is designed for targeted NGS panels. The biosynthetic fusions are targeted to 60 copies per nanogram of total RNA from GM24385 human reference cell line and contain poly-A tail of ~37 templated A's. We tested whether increasing the length of the poly-A tail would improve the efficiency of binding to an oligo dT column and improve performance in total RNA-Seq NGS assays that use poly(A) + selection. E. coli Poly(A) Polymerase was used to extend tails, and efficiency of RNA recovery from oligo-dT columns was assessed visually by gel analysis. The fusion RNAs (with standard or lengthened poly-A tails) were mixed with total RNA from GM24385 human reference cell line. Since GM24385 is a lymphoid cell line, highly abundant immune transcripts may reduce the prevalence of the fusion RNAs in the selected RNAs. Therefore, mixtures were made at the normal concentration (~60 copies/ng, or 1X) as well as at 3-fold and 10-fold concentrations. Fusion detection was performed using Illumina TruSeq Stranded mRNA(polyA) kit and Illumina HiSeq, run in 2 × 150bp mode. Results: The proportional recovery was improved with the longer (&amp;gt;100 A's) tail length. This translated to improvement in NGS detection with longer poly-A tail length giving from 10% to 50% more fusion reads. Increasing the Fusion RNA concentration led to a proportion increase in fusion reads per million total reads. The increase from &amp;lt;1 fusion reads/million total reads to &amp;gt;5 fusion reads/million resulted in more robust and consistent fusion detection. Conclusions: These studies indicate that reference materials similar to the Seraseq Fusion RNA mix reference material, but with greater concentration of Fusion RNAs (3 - 10 fold higher concentration) and with longer polyA tail length will be applicable quality control materials for whole transcriptome RNA-Seq NGS assays. These novel reference materials may aid clinical labs in developing sensitive new RNA-Seq Fusion detection assays and tuning their bioinformatics pipeline to more precise selection of therapeutic strategies for patients. Citation Format: Catherine Huang, Matthew G. Butler, Yves Konigshofer, Jessica Dickens, Rajeswari Vemula, Omoshile Clement, Bharathi Anekella. Optimizing Performance of Whole Transcriptome RNA-Seq Reference [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 2232.