Amount Of Viral RNA Research Articles

T cells protect against hepatitis A virus infection and limit infection-induced liver injury

Hepatitis A virus (HAV) is a common cause of enterically transmitted viral hepatitis. In non-immune individuals, infection results in typically transient but occasionally fulminant and fatal inflammatory liver injury. Virus-specific T cell frequencies peak when liver damage is at its zenith, leading to the prevalent notion that T cells exacerbate liver disease, as suspected for other hepatotropic virus infections. However, the overall contribution of T cells to the control of HAV and the pathogenesis of hepatitis A is unclear and has been impeded by a historic lack of small animal models. Ifnar1-/- mice are highly permissive for HAV and develop pathogenesis that recapitulates many features of hepatitis A. Using this model, we identified HAV-specific CD8+ and CD4+ T cells by epitope mapping, and then used tetramers andfunctional assays to quantify T cells in the liver at multiple times after infection. We assessed the relationships between HAV-specific T cell frequency, viral RNA amounts, and liver pathogenesis. A large population of virus-specific T cells accumulated within the livers of Ifnar1-/- mice during the first 1-2 weeks of infection and persisted over time. HAV replication was enhanced and liver disease exacerbated when mice were depleted of T cells. Conversely, immunization with a peptide vaccine increased virus-specific CD8+ T cell frequencies in the liver, reduced viral RNA abundance, and lessened liver injury. These data show that T cells protect against HAV-mediated liver injury and can be targeted to improve liver health. Hepatitis A virus is a leading cause of acute viral hepatitis worldwide. T cells were thought to contribute to liver injury during acute infection. We now show that virus-specific T cells protect against infection and limit liver injury.

COVID-19 rapid antigen testing strategies require careful evaluation

COVID-19 rapid antigen testing strategies require careful evaluation

Novel Multiplexed Amplicon-Based Sequencing to Quantify SARS-CoV-2 RNA from Wastewater.

The application of wastewater-based epidemiology (WBE) to support the global response to the COVID-19 pandemic has shown encouraging outcomes. The accurate, sensitive, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in municipal wastewater is critical for WBE. Here, we present a novel approach based on multiplexed amplicon-based sequencing, namely the ATOPlex platform, for detecting SARS-CoV-2. The ATOPlex platform is capable of quantifying SARS-CoV-2 RNA at concentrations that are at least 1 order of magnitude lower than the detection limit of reverse transcription quantitative polymerase chain reaction (RT-qPCR). Robust and accurate phylogenetic placement can be done at viral concentrations 4 times lower than the detection limit of RT-qPCR. We further found that the solid fraction in wastewater harbors a considerable amount of viral RNA, highlighting the need to extract viral RNA from the solid and liquid fractions of wastewater. This study delivers a highly sensitive, phylogenetically informative, and high-throughput analytical workflow that facilitates the application of WBE.

Municipal wastewater viral pollution in Saudi Arabia: effect of hot climate on COVID-19 disease spreading.

The viral RNA of SARS-Coronavirus-2 is known to be contaminating municipal wastewater. We aimed to assess if COVID-19 disease is spreading through wastewater. We studied the amount of viral RNA in raw sewage and the efficiency of the sewage treatment to remove the virus. Sewage water was collected before and after the activated sludge process three times during summer 2020 from three different sewage treatment plants. The sewage treatment was efficient in removing SARS-CoV-2 viral RNA. Each sewage treatment plant gathered wastewater from one hospital, of which COVID-19 admissions were used to describe the level of disease occurrence in the area. The presence of SARS-CoV-2 viral RNA–specific target genes (N1, N2, and E) was confirmed using RT-qPCR analysis. However, hospital admission did not correlate significantly with viral RNA. Moreover, viral RNA loads were relatively low, suggesting that sewage might preserve viral RNA in a hot climate only for a short time.

Potent priming by inactivated whole influenza virus particle vaccines is linked to viral RNA uptake into antigen presenting cells

Current detergent or ether-disrupted split vaccines (SVs) for influenza do not always induce adequate immune responses, especially in young children. This contrasts with the whole virus particle vaccines (WPVs) originally used against influenza that were immunogenic in both adults and children but were replaced by SV in the 1970s due to concerns with reactogenicity. In this study, we re-evaluated the immunogenicity of WPV and SV, prepared from the same batch of purified influenza virus, in cynomolgus macaques and confirmed that WPV is superior to SV in priming potency. In addition, we compared the ability of WPV and SV to induce innate immune responses, including the maturation of dendritic cells (DCs) in vitro. WPV stimulated greater production of inflammatory cytokines and type-I interferon in immune cells from mice and macaques compared to SV. Since these innate responses are likely triggered by the activation of pattern recognition receptors (PRRs) by viral RNA, the quantity and quality of viral RNA in each vaccine were assessed. Although the quantity of viral RNA was similar in the two vaccines, the amount of viral RNA of a length that can be recognized by PRRs was over 100-fold greater in WPV than in SV. More importantly, 1000-fold more viral RNA was delivered to DCs by WPV than by SV when exposed to preparations containing the same amount of HA protein. Furthermore, WPV induced up-regulation of the DC maturation marker CD86 on murine DCs, while SV did not. The present results suggest that the activation of antigen-presenting DCs, by PRR-recognizable viral RNA contained in WPV is responsible for the effective priming potency of WPV observed in naïve mice and macaques. WPV is thus recommended as an alternative option for seasonal influenza vaccines, especially for children.

Detection of SARS-CoV-2 genome and whole transcriptome sequencing in frontal cortex of COVID-19 patients

SARS-Cov-2 infection is frequently associated with Nervous System manifestations. However, it is not clear how SARS-CoV-2 can cause neurological dysfunctions and which molecular processes are affected in the brain. In this work, we examined the frontal cortex tissue of patients who died of COVID-19 for the presence of SARS-CoV-2, comparing qRT-PCR with ddPCR. We also investigated the transcriptomic profile of frontal cortex from COVID-19 patients and matched controls by RNA-seq analysis to characterize the transcriptional signature.Our data showed that SARS-CoV-2 could be detected by ddPCR in 8 (88%) of 9 examined samples while by qRT-PCR in one case only (11%). Transcriptomic analysis revealed that 11 genes (10 mRNAs and 1 lncRNA) were differential expressed when frontal cortex of COVID-19 patients were compared to controls. These genes fall into categories including hypoxia, hemoglobin-stabilizing protein, hydrogen peroxide processes. This work demonstrated that the quantity of viral RNA in frontal cortex is minimal and it can be detected only with a very sensitive method (ddPCR). Thus, it is likely that SARS-CoV-2 does not actively infect and replicate in the brain; its topography within encephalic structures remains uncertain. Moreover, COVID-19 may have a role on brain gene expression, since we observed an important downregulation of genes associated to hypoxia inducting factor system (HIF) that may inhibit the capacity of defense system during infection and oxigen deprivation, showing that hypoxia, well known multi organ condition associated to COVID-19, also marked the brain.

Field performance evaluation of the PanBio rapid SARS-CoV-2 antigen assay in an epidemic driven by the B.1.351 variant in the Eastern Cape, South Africa

BackgroundSouth Africa was the African country with the most recorded cases of SARS-CoV-2 during 2020, experiencing 2 waves of infection. During the first wave, diagnostics were largely based on reverse transcription-linked PCR (RT-PCR). The Abbott PanBio antigen test was deployed during the 2nd wave which may have been driven by emergence of the B.1.351 variant. At the time of evaluation in mid-November 2020, B.1.351 was the dominant circulating virus in Nelson Mandela Bay, in the Eastern Cape Province. MethodsUsed PanBio antigen swabs (collected from patients with genetically characterised virus) were first validated as suitable for PCR. A prospective study was then undertaken to evaluate assay performance in the field. Testing was conducted at mobile community testing centres on 677 ambulant patients. Used swabs were kept and tested by RT-PCR. ResultsDuring initial validation, used swabs in proprietary lysis buffer were found to be suitable for PCR and secondly, the PB assay reliably detected patients infected with B.1.351. In the field study, of 146 RT-PCR positive individuals, 101 were RTD positive in the clinic. The RTD had a sensitivity of 69.2% (95%CI 61.4, 75.8) and specificity of 99.0% (95%CI 98.8, 99.3). Sensitivity was dependent on the amount of viral RNA in clinical samples, as reflected by the PCR cycle threshold (CT) value. ConclusionsThe assay reliably detected B.1.351 infections in ambulatory ill patients during initial validation and in field testing. In the field, assay sensitivity was >90% in patients with high viral loads who are expected to be most infectious. Negative and positive predictive values were also >90%.

Which factors influence the extent of indoor transmission of SARS-CoV-2? A rapid evidence review.

BackgroundThis rapid evidence review identifies and integrates evidence from epidemiology, microbiology and fluid dynamics on the transmission of SARS-CoV-2 in indoor environments.MethodsSearches were conducted in May 2020 in PubMed, medRxiv, arXiv, Scopus, WHO COVID-19 database, Compendex & Inspec. We included studies reporting data on any indoor setting except schools, any indoor activities and any potential means of transmission. Articles were screened by a single reviewer, with rejections assessed by a second reviewer. We used Joanna Briggs Institute and Critical Appraisal Skills Programme tools for evaluating epidemiological studies and developed bespoke tools for the evaluation of study types not covered by these instruments. Data extraction and quality assessment were conducted by a single reviewer. We conducted a meta-analysis of secondary attack rates in household transmission. Otherwise, data were synthesised narratively.ResultsWe identified 1573 unique articles. After screening and quality assessment, fifty-eight articles were retained for analysis. Experimental evidence from fluid mechanics and microbiological studies demonstrates that aerosolised transmission is theoretically possible; however, we found no conclusive epidemiological evidence of this occurring. The evidence suggests that ventilation systems have the potential to decrease virus transmission near the source through dilution but to increase transmission further away from the source through dispersal. We found no evidence for faecal-oral transmission. Laboratory studies suggest that the virus survives for longer on smooth surfaces and at lower temperatures. Environmental sampling studies have recovered small amounts of viral RNA from a wide range of frequently touched objects and surfaces; however, epidemiological studies are inconclusive on the extent of fomite transmission. We found many examples of transmission in settings characterised by close and prolonged indoor contact. We estimate a pooled secondary attack rate within households of 11% (95% confidence interval (CI) = 9, 13). There were insufficient data to evaluate the transmission risks associated with specific activities. Workplace challenges related to poverty warrant further investigation as potential risk factors for workplace transmission. Fluid mechanics evidence on the physical properties of droplets generated by coughing, speaking and breathing reinforce the importance of maintaining 2 m social distance to reduce droplet transmission.ConclusionsThis review provides a snap-shot of evidence on the transmission of SARS-CoV-2 in indoor environments from the early months of the pandemic. The overall quality of the evidence was low. As the quality and quantity of available evidence grows, it will be possible to reach firmer conclusions on the risk factors for and mechanisms of indoor transmission.

Characterizing and Quantifying Arbovirus Transmission by Aedes aegypti Using Forced Salivation and Analysis of Bloodmeals.

Simple SummaryMosquitoes transmit hundreds of arboviruses. The ability to estimate the titers of viruses transmitted from infectious mosquitoes to a host is critical. In this study, we evaluated forced salivation techniques to estimate and compare titers of Zika virus and chikungunya virus transmitted by the mosquitoes. We demonstrated that performing forced salivation on mosquitoes after blood feeding might be an efficient way to estimate the virus transmitted during blood feeding. Additionally, by comparing titers of bloodmeals and saliva post-feeding, we showed that mosquitoes re-ingest much of their saliva during artificial blood feeding. The results from this study add new information to understanding and quantifying the transmission of arboviruses.Arbovirus transmission studies are dependent on the ability to estimate the titer of virus transmitted from infectious mosquitoes to a host. There are several methods for estimating virus titer in mosquito saliva, including (1) using forced salivation (FS) whereby the infectious mosquito’s proboscis is forced into a capillary tube containing media to collect and test their saliva for virus, and (2) by quantifying virus expectorated into host tissues or into the blood contained in an artificial feeder immediately after blood feeding. We studied FS and bloodmeals to estimate and compare titers of Zika virus and chikungunya virus transmitted by the mosquito vector Aedes aegypti. Infectious virus and viral genomes of both viruses were detected more often from individual mosquitoes using immersion oil for the FS media compared to fetal bovine serum (FBS) plus glycerol, but the FS media had no influence on virus quantification from positive samples. FS virus titers were equivalent when comparing individuals or groups of mosquitoes that never received a blood meal compared to those that were blood fed immediately prior, showing that blood feeding does not influence FS. This suggested that performing FS on mosquitoes after blood feeding might be an efficient way to estimate virus transmitted during blood feeding. However, detecting virus from the blood remaining in an artificial feeder post-blood feeding was mostly unsuccessful relative to quantifying virus from FS of the post-blood fed mosquitoes. In contrast, immunocompromised mice always became infected after being fed on by Zika-infected mosquitoes, even when no infectious virus was detected in their saliva by FS post-blood feed. Due to this discrepancy, we tested the ingested bloodmeals of individual mosquitoes that fed on artificial blood feeders for virus, and compared these to virus in their saliva harvested from FS and to virus in their bodies. These experiments revealed ~50–100 times higher virus titers in the dissected bloodmeals compared to those detected in the same mosquitoes’ saliva, demonstrating how mosquitoes re-ingest much of their saliva during artificial blood feeding, and highlighting a large increase in virus transmission during Aedes aegypti blood feeding. Both FS and the dissected bloodmeals of artificially blood-fed mosquitoes showed that the quantity of viral RNA expectorated by mosquitoes was 2–5 logs more than the quantity of infectious virus. The results from this study add critical information to understanding and quantifying the transmission of Aedes aegypti arboviruses.

Revealing Tissue-Specific SARS-CoV-2 Infection and Host Responses using Human Stem Cell-Derived Lung and Cerebral Organoids.

SummaryCOVID-19 is a transmissible respiratory disease caused by a novel coronavirus, SARS-CoV-2, and has become a global health emergency. There is an urgent need for robust and practical in vitro model systems to investigate viral pathogenesis. Here, we generated human induced pluripotent stem cell (iPSC)-derived lung organoids (LORGs), cerebral organoids (CORGs), neural progenitor cells (NPCs), neurons, and astrocytes. LORGs containing epithelial cells, alveolar types 1 and 2, highly express ACE2 and TMPRSS2 and are permissive to SARS-CoV-2 infection. SARS-CoV-2 infection induces interferons, cytokines, and chemokines and activates critical inflammasome pathway genes. Spike protein inhibitor, EK1 peptide, and TMPRSS2 inhibitors (camostat/nafamostat) block viral entry in LORGs. Conversely, CORGs, NPCs, astrocytes, and neurons express low levels of ACE2 and TMPRSS2 and correspondingly are not highly permissive to SARS-CoV-2 infection. Infection in neuronal cells activates TLR3/7, OAS2, complement system, and apoptotic genes. These findings will aid in understanding COVID-19 pathogenesis and facilitate drug discovery.

Single-cell analysis of arthritogenic alphavirus-infected human synovial fibroblasts links low abundance of viral RNA to induction of innate immunity and arthralgia-associated gene expression

ABSTRACT Infection by (re-)emerging RNA arboviruses including Chikungunya virus (CHIKV) and Mayaro virus primarily cause acute febrile disease and transient polyarthralgia. However, in a significant subset of infected individuals, debilitating arthralgia persists for weeks over months up to years. The underlying immunopathogenesis of chronification of arthralgia upon primary RNA-viral infection remains unclear. Here, we analysed cell-intrinsic responses to ex vivo arthritogenic alphaviral infection of primary human synovial fibroblasts isolated from knee joints, one the most affected joint types during acute and chronic CHIKV disease. Synovial fibroblasts were susceptible and permissive to alphaviral infection. Base-line and exogenously added type I interferon (IFN) partially and potently restricted infection, respectively. RNA-seq revealed a CHIKV infection-induced transcriptional profile that comprised upregulation of expression of several hundred IFN-stimulated and arthralgia-mediating genes. Single-cell virus-inclusive RNA-seq uncovered a fine-tuned switch from induction to repression of cell-intrinsic immune responses depending on the abundance of viral RNA in an individual cell. Specifically, responses were most pronounced in cells displaying low-to-intermediate amounts of viral RNA and absence of virus-encoded, fluorescent reporter protein expression, arguing for efficient counteraction of innate immunity in cells expressing viral antagonists at sufficient quantities. In summary, cell-intrinsic sensing of viral RNA that potentially persists or replicates at low levels in synovial fibroblasts and other target cell types in vivo may contribute to the chronic arthralgia induced by alphaviral infections. Our findings might advance our understanding of the immunopathophysiology of long-term pathogenesis of RNA-viral infections.

Severe acute respiratory syndrome Corona virus and intraocular fluid sampling

Dear Editor, We congratulate the authors for the excellent documentation of their cases.[1] We would like to share our experiences dealing with similar cases and would also like to seek clarifications from the authors. We have also been routinely doing reverse transcriptase polymerase chain reaction (RT-PCR) for severe acute respiratory syndrome Corona virus (SARS-CoV-2) in our patients with retinal vascular occlusion, endogenous endophthalmitis, panophthalmitis (unpublished data), and with posterior uveitis.[23] The authors statement “To the best of our knowledge, no study to date evaluated the intraocular fluids for the presence of SARS-CoV-2 in live humans is not correct.” Bilgic et al.[4] have isolated the SARS-CoV-2 in patients with endophthalmitis from the vitreous sample. In an another study by Koo et al.[5], six (19.4%) patients demonstrated detectable SARS-CoV-2 RNA in the aqueous sample, and fortunately, all these individuals did not have any systemic symptoms. We were not able to isolate the SARS-CoV-2 virus in either aqueous or vitreous sample. We have been adopting the following procedure for processing the samples. Our samples are collected in a 1 mL tubeculin syringe and capped, transported in an viral transport medium available from Bhat Bio-Scan™ to the laboratory maintaining a temperature between 2 and 8°C. TaqPath™ Corona Virus Disease-19 (COVID-19) Multiplex Diagnostic Solution from Thermo Fisher Scientific™ is used for multiplex real-time RT-PCR test. This helps in qualitative detection of nucleic acid from SARS-CoV-2. Three regions of the SARS-CoV-2 single-stranded RNA genome – The Orf1ab, N gene, and S gene – are amplified and detected with the help of primer and the probe. It is of importance as SARS-CoV-2 is either not detected or detected in a very low percentage, of ocular samples in patients who have COVID-19.[6] We would also like to suggest that a positive control may be useful by adding a known quantity of viral RNA to ocular fluid to rule out the presence of polymerase chain reaction inhibitors. It would also be useful if the authors could share if SARS-CoV-2 antibodies and other inflammatory markers were raised in their seven patients. Our patients who were not vaccinated had raised levels of antibodies in addition to raised levels of D-dimer, serum ferritin, and lactate dehydrogenase. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Comparison of Digital PCR and Quantitative PCR with Various SARS-CoV-2 Primer-Probe Sets.

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.

Juice of Citrullus lanatus var. citroides (wild watermelon) inhibits the entry and propagation of influenza viruses in vitro and in vivo.

Vaccines and various anti‐influenza drugs are clinically used to prevent and treat influenza infections. However, with the antigenic mismatch of vaccines and the emergence of drug‐resistant viral strains, new approaches for treating influenza are warranted. This study focused on natural foods as potential candidates for the development of new treatment options for influenza infections. The screening of plants from the Cucurbitaceae family revealed that the juice of Citrullus lanatus var. citroides (wild watermelon) had the strongest ability to inhibit the replication of influenza virus in Madin–Darby canine kidney cells. The results of a time‐of‐addition assay indicated that wild watermelon juice (WWMJ) inhibits the adsorption and late stages of viral replication, suggesting that WWMJ contains multiple constituents with effective anti‐influenza activity. A viral adsorption analysis showed that WWMJ reduces the amount of viral RNA in the cells at 37°C but not at 4°C, confirming that WWMJ inhibits viral entry into the host cells at 37°C. These results suggest that a mechanism other than the inhibition of viral attachment is involved in the anti‐influenza action of WWMJ, which is perhaps responsible for a reduction in internalization of the virus. Administration of WWMJ into the nasal mucosa of BALB/c mice infected with the A/PR/8/34 mouse‐adapted influenza virus was seen to significantly improve the survival rate. The findings of this study, therefore, demonstrate the anti‐influenza potential of WWMJ in vitro and in vivo, thereby suggesting the candidature of WWMJ as a functional food product that can be used to develop anti‐influenza agents and drugs.

SARS‐CoV‐2 evades immune detection in alveolar macrophages

Respiratory infections, like the current COVID‐19 pandemic, target epithelial cells in the respiratory tract. Alveolar macrophages (AMs) are tissue‐resident macrophages located within the lung. They play a key role in the early phases of an immune response to respiratory viruses. AMs are likely the first immune cells to encounter SARS‐CoV‐2 during an infection, and their reaction to the virus will have a profound impact on the outcome of the infection. Interferons (IFNs) are antiviral cytokines and among the first cytokines produced upon viral infection. In this study, AMs from non‐infectious donors are challenged with SARS‐CoV‐2. We demonstrate that challenged AMs are incapable of sensing SARS‐CoV‐2 and of producing an IFN response in contrast to other respiratory viruses, like influenza A virus and Sendai virus, which trigger a robust IFN response. The absence of IFN production in AMs upon challenge with SARS‐CoV‐2 could explain the initial asymptotic phase observed during COVID‐19 and argues against AMs being the sources of pro‐inflammatory cytokines later during infection.

Detection of SARS-COV N2 Gene: Very low amounts of viral RNA or false positive?

BackgroundThe detection of a low amount of viral RNA is crucial to identify a SARS-CoV-2 positive individual harboring a low level of virus, especially during the convalescent period. However, the detection of one gene at high Cycle threshold (Ct) has to be interpreted with caution.In this study we address this specific issue and report our real-life experience.Study designA total of 1639 nasopharyngeal swabs (NPS) were analyzed with Xpert® Xpress SARS-CoV-2. Positive samples showing high Ct values (Ct>35) were concentrated by centrifugation and re-tested with Cepheid or other methods (RealStar SARS-CoV2 RT-PCR, Altona Diagnostics; GeneFinder COVID-19 Plus RealAmp Kit, Elitech).Results1599 (97.5%) negative samples, 36 (2.3%) positive samples and 4 (0.2%) presumptive positive samples were detected. In 17 out of 36 positive patients, very low viral RNA copies were suspected since positivity was detected at high Ct. We confirmed positivity for patients who showed both E and N genes detected and for patients with only N detected but with Ct <39. On the contrary, samples with only gene N detected with Ct values >39 were found negative. NPS taken 24 hours after the first collection confirmed the negativity of the 12 samples. Clinical data sustained these results since only 2 of these 12 patients showed COVID-19-like symptoms.ConclusionsThese data support our consideration that detection of the N2 gene at high Ct needs to be interpreted with caution, suggesting that collaboration between virologists and clinicians is important for better understanding of results.

Evaluation of U.S. Food and Drug Administration Enteric Viruses Microarray for Detection of Hepatitis A Virus and Norovirus in Inoculated Tomatoes, Green Onions, and Celery

Foodborne viral contamination of fresh produce has been associated with numerous outbreaks. Detection of such contaminated foods is important in protecting public health. Here, we demonstrate for the first time the capability of the U.S. Food and Drug Administration Enteric Viruses tiling microarray (FDA-EVIR) to perform rapid molecular identification of hepatitis A virus (HAV) and human norovirus extracted from artificially inoculated fresh produce. Two published viral extraction strategies, total RNA extraction or virus particle isolation, were used to prepare the viral targets. The total RNA extraction method was used on material eluted from tomatoes, using an alkaline Tris-glycine-beef extract (TGBE) buffer. Optimization procedures including DNase treatment and poly(A)-RNA enrichment were adopted to improve microarray sensitivity. For green onions or celery, material was eluted using either glycine buffer or TGBE buffer supplemented with pectinase, respectively, and then virus particles were concentrated by ultracentrifugation. We also assessed the amount of viral RNA extracted from celery using three commercially available kits and how well that RNA performed on FDA-EVIR. Our results confirm that FDA-EVIR can identify common enteric viruses isolated from fresh produce when present as either a single or mixed species of viruses. Using total RNA extraction from tomatoes yielded a limit of detection of 1.0 × 105 genome equivalents (ge) of HAV per array input. The limit of detection for viral RNA obtained using ultracentrifugation was 1.2 × 105 ge of HAV from green onions and 1.0 × 103 ge of norovirus from celery per array input. Extending microarray methods to other food matrices should provide important support to surveillance and outbreak investigations.

Multi-route transmission potential of SARS-CoV-2 in healthcare facilities

Understanding the transmission mechanism of SARS-CoV-2 is a prerequisite to effective control measures. To investigate the potential modes of SARS-CoV-2 transmission, 21 COVID-19 patients from 12–47 days after symptom onset were recruited. We monitored the release of SARS-CoV-2 from the patients’ exhaled breath and systematically investigated environmental contamination of air, public surfaces, personal necessities, and the drainage system. SARS-CoV-2 RNA was detected in 0 of 9 exhaled breath samples, 2 of 8 exhaled breath condensate samples, 1 of 12 bedside air samples, 4 of 132 samples from private surfaces, 0 of 70 samples from frequently touched public surfaces in isolation rooms, and 7 of 23 feces-related air/surface/water samples. The maximum viral RNA concentrations were 1857 copies/m3 in the air, 38 copies/cm2 in sampled surfaces and 3092 copies/mL in sewage/wastewater samples. Our results suggest that nosocomial transmission of SARS-CoV-2 can occur via multiple routes. However, the low detection frequency and limited quantity of viral RNA from the breath and environmental specimens may be related to the reduced viral load of the COVID-19 patients on later days after symptom onset. These findings suggest that the transmission dynamics of SARS-CoV-2 differ from those of SARS-CoV in healthcare settings.

Stability of MERS-CoV RNA on spin columns of RNA extraction kit at room temperature

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus causing a highly fatal respiratory disease in humans. Confirmation of MERS-CoV infection and molecular study on the virus may require transportation of samples to specialized laboratories. While freezing at −80 °C is the gold standard method for RNA preservation, maintaining the integrity of viral RNA during transport will require additional precautions and, as a result, increase transport costs. We aimed at testing the stability of MERS-CoV RNA on spin columns of RNA extraction kit at room temperature for 16 weeks. Respiratory samples spiked with stock culture of MERS-CoV were extracted and loaded on QIAamp Viral RNA Mini Kit spin columns and preserved at room temperature. Amount of viral RNA was evaluated periodically by real-time quantitative reverse-transcription polymerase chain reaction. Minimal changes in cycle threshold values over the study period were noted, suggesting stability of viral RNA by this preservation method.

Severe Acute Respiratory Syndrome Coronavirus 2 and Coronavirus Disease 2019: A Clinical Overview and Primer.

Following its emergence in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused what rapidly became a global pandemic. The precise origin and subsequent path of transmission have not yet been established-but like the other novel coronaviruses that it closely resembles, it appears to have evolved naturally in a bat host. The disease caused by SARS-CoV-2 infection, designated as coronavirus disease 2019 (COVID-19), ranges from asymptomatic, to mild self-limited illness, to progressive pneumonia, respiratory compromise, multiorgan failure, and death. In addition, a hyperinflammatory disease state occurs in a subset of patients, and may be seen either during acute infection or following recovery. The search for effective pharmacological management of COVID-19 continues, but several promising candidates have been identified, including the viral nucleoside analog remdesivir. However, despite the existence of literally thousands of clinical trials, the management of COVID-19 remains challenging, and the development of an optimal, evidence-based therapeutic approach is ongoing. The impact of SARS-CoV-2 and COVID-19 on the biobanking world is evolving and profound-in particular, it is likely that many of mysteries surrounding COVID-19 will be solved via the availability of high-quality, large-scale collection, storage, and analysis of patient specimens. The purpose of this review article is therefore to provide a rapid, comprehensive, and relevant overview and primer on SARS-CoV-2 and COVID-19, with attention to the epidemiology, virology, transmission, clinical features, and major therapeutic options currently existent.