A Novel Two-Step, Direct-to-PCR Method for Virus Detection off Swabs using Human Coronavirus 229E

Abstract Background Currently, one of the most reliable methods for viral infection detection are polymerase chain reaction (PCR) based assays. This process is time and resource heavy, requiring multiple steps of lysis, extraction, purification, and amplification procedures. Herein, we have developed a method to detect virus off swabs using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR assay for virus detection, bypassing the substantial reagent and time investments required for extraction and purification steps.Methods Using Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs in vitro for proof-of-concept testing. Swabs were spiked in serial dilutions from 1.2x106 to 1.2x101 copies/mL and then placed in 2mL tubes with viral transport media (VTM) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. After homogenization, 1 µL of lysate was processed using RT-qPCR for amplification of the nucleocapsid (N) gene, qualifying viral detection. Results HCoV-229E in vitro spiked swabs were processed in a novel two-step, direct-to-PCR methodology for viral detection. After running 54 swabs, we confidently determined our limit of detection to be 1.2x103 viral copies/mL with 96.30% sensitivity. Conclusion We have proven that the shaker-mill homogenization-based two-step, direct-to-PCR procedures provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR for the detection of HCoV-229E. This finding allows for reductions in the time and resources required for PCR based virus detection in comparison to the traditional extraction-to-PCR methodology.

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693. Shaking Things Up: Direct-to-PCR Viral Detection off Swabs Using Shaker-Mill Homogenization

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.885 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S399-S399

Author(s):

Zachary P Morehouse ◽

Caleb Proctor ◽

Gabriella Ryan ◽

Rodney J Nash

Keyword(s):

Viral Infections ◽

Limit Of Detection ◽

Genetic Material ◽

N Gene ◽

Viral Detection ◽

Viral Lysis ◽

Specimen Collection ◽

Extraction And Purification ◽

The Cost

Abstract Background As the number of viral diseases are on the rise, it is critical to continue to innovate and advance diagnostic, treatment, and surveillance methods surrounding viral infections. Currently, one of the most reliable methods for viral infection detection are polymerase chain reaction (PCR) based assays. These assays often involve procedures of swabbing a patient, processing the sample to lyse the virus, extract, and purify it’s nucleotides, and then run the purified genetic material via PCR for detection of a gene product needed to confirm the patient’s suspected diagnosis. This process requires time to complete and is dependent on the availability of the reagents and plastics required to complete the lysis, extraction, purification, and amplification procedures. Herein, we have developed a method to detect virus off a swab using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR based assay, bypassing the reagent heavy and time consuming extraction and purification steps. Methods Using Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs with clinically relevant levels of the virus for proof-of-concept testing. Swabs were spiked in serial dilutions from 1.2e7 copies/mL to 1.2e1 copies/mL. The swabs were then placed in 2mL tubes with viral transport media (VTM) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. After homogenization, 1 uL of viral lysate was run in RT-qPCR for amplification of the nucleocapsid (N) gene, qualifying viral detection from the sample. Results HCoV-229E spiked swabs were run through the two-step process of homogenization direct to RT-qPCR for viral detection. After running 54 swabs, we confidently determined our limit of detection to be 1.2e3 viral copies/mL with 96.30% sensitivity in vitro. Conclusion We have successfully proven that shaker-mill homogenization provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR based assays for the detection of virus. This finding allows for decreased run time in traditional PCR based diagnostics and reduces the reagents and plastics required for each sample, ultimately reducing the cost and time of each viral test when compared to traditional PCR based methods. Disclosures Zachary P. Morehouse, MS, OMS-III, Omni International Inc (Consultant) Caleb Proctor, BS, Omni International Inc (Employee) Gabriella Ryan, BS, Omni International Inc (Employee) Rodney J. Nash, PhD, Omni International Inc (Employee)

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Analysis of human coronavirus 229E spike and nucleoprotein genes demonstrates genetic drift between chronologically distinct strains

Journal of General Virology ◽

10.1099/vir.0.81662-0 ◽

2006 ◽

Vol 87 (5) ◽

pp. 1203-1208 ◽

Cited By ~ 32

Author(s):

Doris Chibo ◽

Chris Birch

Keyword(s):

Phylogenetic Analysis ◽

Respiratory Infections ◽

N Gene ◽

Human Coronavirus ◽

S Gene ◽

Limited Variation ◽

Contrast Analysis ◽

Human Coronaviruses ◽

Human Coronavirus 229E ◽

S Genes

Historically, coronaviruses have been recognized as a cause of minor respiratory infections in humans. However, the recent identification of three novel human coronaviruses, one causing severe acute respiratory syndrome (SARS), has prompted further examination of these viruses. Previous studies of geographically and chronologically distinct Human coronavirus 229E (HCoV-229E) isolates have found only limited variation within S gene nucleotide sequences. In contrast, analysis of the S genes of contemporary Human coronavirus OC43 variants identified in Belgium revealed two distinct viruses circulating during 2003 and 2004. Here, the S and N gene sequences of 25 HCoV-229E variants identified in Victoria, Australia, between 1979 and 2004 in patients with symptomatic infections were determined. Phylogenetic analysis showed clustering of the isolates into four groups, with evidence of increasing divergence with time. Evidence of positive selection in the S gene was also established.

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Flavonols and dihydroflavonols inhibit the main protease activity of SARS-CoV-2 and the replication of human coronavirus 229E

10.1101/2021.07.01.450756 ◽

2021 ◽

Author(s):

Yue Zhu ◽

Frank Scholle ◽

Samantha C. Kisthardt ◽

Deyu Xie

Keyword(s):

Docking Simulation ◽

Binding Pocket ◽

Developed Countries ◽

Host Cells ◽

Human Coronavirus ◽

Main Protease ◽

Ic50 Values ◽

In Vitro Inhibition ◽

Human Coronavirus 229E

Since December 2019, the deadly novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the current COVID-19 pandemic. To date, vaccines are available in the developed countries to prevent the infection of this virus, however, medicines are necessary to help control COVID-19. Human coronavirus 229E (HCoV-229E) causes the common cold. The main protease (Mpro) is an essential enzyme required for the multiplication of these two viruses in the host cells, and thus is an appropriate candidate to screen potential medicinal compounds. Flavonols and dihydroflavonols are two groups of plant flavonoids. In this study, we report docking simulation with two Mpro enzymes and five flavonols and three dihydroflavonols, in vitro inhibition of the SARS-CoV-2 Mpro, and in vitro inhibition of the HCoV 229E replication. The docking simulation results predicted that (+)-dihydrokaempferol, (+)-dihydroquercetin, (+)-dihydromyricetin, kaempferol, quercetin, myricentin, isoquercetin, and rutin could bind to at least two subsites (S1, S1', S2, and S4) in the binding pocket and inhibit the activity of SARS-CoV-2 Mpro. Their affinity scores ranged from -8.8 to -7.4. Likewise, these compounds were predicted to bind and inhibit the HCoV-229E Mpro activity with affinity scores ranging from -7.1 to -7.8. In vitro inhibition assays showed that seven available compounds effectively inhibited the SARS-CoV-2 Mpro activity and their IC50 values ranged from 0.125 to 12.9 uM. Five compounds inhibited the replication of HCoV-229E in Huh-7 cells. These findings indicate that these antioxidative flavonols and dihydroflavonols are promising candidates for curbing the two viruses.

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Characteristics of a Long Term in Vitro Persistent Infection with Human Coronavirus 229E

Advances in Experimental Medicine and Biology - Biochemistry and Biology of Coronaviruses ◽

10.1007/978-1-4757-0456-3_25 ◽

1981 ◽

pp. 309-322 ◽

Cited By ~ 4

Author(s):

G. Chaloner-Larsson ◽

C. M. Johnson-Lussenburg

Keyword(s):

Persistent Infection ◽

Human Coronavirus ◽

Human Coronavirus 229E

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Proteolytic Processing at the Amino Terminus of Human Coronavirus 229E Gene 1-Encoded Polyproteins: Identification of a Papain-Like Proteinase and Its Substrate

Journal of Virology ◽

10.1128/jvi.72.2.910-918.1998 ◽

1998 ◽

Vol 72 (2) ◽

pp. 910-918 ◽

Cited By ~ 43

Author(s):

Jens Herold ◽

Alexander E. Gorbalenya ◽

Volker Thiel ◽

Barbara Schelle ◽

Stuart G. Siddell

Keyword(s):

Amino Acids ◽

Cleavage Site ◽

Cysteine Proteinase ◽

Proteolytic Processing ◽

Amino Terminus ◽

Human Coronavirus ◽

Amino Terminal ◽

Cleavage Assay ◽

Human Coronavirus 229E

ABSTRACT Expression of the coronavirus gene 1-encoded polyproteins, pp1a and pp1ab, is linked to a series of proteolytic events involving virus-encoded proteinases. In this study, we used transfection and immunoprecipitation assays to show that the human coronavirus 229E-encoded papain-like cysteine proteinase, PCP1, is responsible for the release of an amino-terminal protein, p9, from the gene 1-encoded polyproteins. The same protein, p9, has also been identified in virus-infected cells. Furthermore, using an in vitrotrans-cleavage assay, we defined the proteolytic cleavage site at the carboxyl terminus of p9 as pp1a-pp1ab amino acids Gly-111 and Asn-112. These results and a comparative sequence analysis suggest that substrate positions P1 and P5 seem to be the major determinants of the PCP1 cleavage site and that the latter can occupy a variable position at the amino terminus of the coronavirus pp1a and pp1ab polyproteins. By combining the trans-cleavage assay with deletion mutagenesis, we were also able to locate the boundaries of the active PCP1 domain between pp1a-pp1ab amino acids Gly-861–Glu-975 and Asn-1209–Gln-1285. Finally, codon mutagenesis was used to show that Cys-1054 and His-1205 are essential for PCP1 proteolytic activity, suggesting that these amino acids most likely have a catalytic function.

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Cytokine Responses in Severe Acute Respiratory Syndrome Coronavirus-Infected Macrophages In Vitro: Possible Relevance to Pathogenesis

Journal of Virology ◽

10.1128/jvi.79.12.7819-7826.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7819-7826 ◽

Cited By ~ 198

Author(s):

Chung Y. Cheung ◽

Leo L. M. Poon ◽

Iris H. Y. Ng ◽

Winsie Luk ◽

Sin-Fun Sia ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Influenza A ◽

Infectious Virus ◽

Expression Profiles ◽

Enzyme Linked Immunosorbent Assay ◽

Viral Gene ◽

Human Coronavirus ◽

Influenza A H1n1 ◽

Human Coronavirus 229E

ABSTRACT The pathogenesis of severe acute respiratory syndrome (SARS) remains unclear. Macrophages are key sentinel cells in the respiratory system, and it is therefore relevant to compare the responses of human macrophages to infections with the SARS coronavirus (SARS-CoV) and other respiratory viruses. Primary human monocyte-derived macrophages were infected with SARS-CoV in vitro. Virus replication was monitored by measuring the levels of positive- and negative-strand RNA, by immunofluorescence detection of the SARS-CoV nucleoprotein, and by titration of the infectious virus. The gene expression profiles of macrophages infected with SARS-CoV, human coronavirus 229E, and influenza A (H1N1) virus were compared by using microarrays and real-time quantitative reverse transcriptase PCR. Secreted cytokines were measured with an enzyme-linked immunosorbent assay. SARS-CoV initiated viral gene transcription and protein synthesis in macrophages, but replication was abortive and no infectious virus was produced. In contrast to the case with human coronavirus 229E and influenza A virus, there was little or no induction of beta interferon (IFN-β) in SARS-CoV-infected macrophages. Furthermore, SARS-CoV induced the expression of chemokines such as CXCL10/IFN-γ-inducible protein 10 and CCL2/monocyte chemotactic protein 1. The poor induction of IFN-β, a key component of innate immunity, and the ability of the virus to induce chemokines could explain aspects of the pathogenesis of SARS.

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Virucidal activity of Nasaleze Cold® & Flu Blocker and Nasaleze® Travel in cell cultures infected with human pathogenic coronavirus 229-E

10.1101/2021.09.23.461483 ◽

2021 ◽

Author(s):

Nigel Hunt ◽

Louise Suleman ◽

Peter D Josling ◽

Todor Popov

Keyword(s):

In Vitro Study ◽

Test Substance ◽

Virucidal Activity ◽

Viral Titre ◽

Human Coronavirus ◽

Test Formulation ◽

Optimal Dosing ◽

Powder Cellulose ◽

Human Coronavirus 229E

This in vitro study determined the anti-viral efficacy of a unique blend of powder cellulose supplemented with powdered garlic extract (PGE) and a signalling agent. The composition, presented as Nasaleze Cold & Flu Blocker/Nasaleze Travel, was assessed against Human Coronavirus 229E, CoV 229E {ATCC VR-740} in an in vitro experiment. The test substance was used at sub-optimal dosing levels to explore its prevention and treatment capabilities. The virucidal activity of this novel formulation was measured at 48, 72 and 112 hour periods after incubation. Results showed strong reductions in viral titre of Coronavirus 229E compared to a control, while no toxicity to human cells from the test formulation was noted. The extract Nasaleze Cold/Travel showed potential to be used as a therapeutic and preventive agent. The data reconfirms the established anti-viral activity of this formulation acting as a barrier preventing the virus from accessing the nasal mucosa and disrupting its replication.

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The MAX dimerization protein MXD1 is differentially expressed and transcriptionally induced in multiple models of coronavirus infection.

10.31219/osf.io/8unvq ◽

2020 ◽

Author(s):

Shahan Mamoor

Keyword(s):

Cellular Response ◽

Differentially Expressed ◽

Human Coronavirus ◽

Lack Of Information ◽

Infection Models ◽

Mammalian Tissues ◽

Coronavirus Infection ◽

Human Coronavirus 229E

The coronavirus COVID19 pandemic is an emerging biosafety threat to the nation and the world (1). There are no treatments approved for coronavirus infection in humans (2) and there is a lack of information available regarding the basic transcriptional behavior of human cells and mammalian tissues following coronavirus infection. We mined independent public (3) and published (4, 5) datasets, containing transcriptome data from infection models of the human coronavirus 229E, severe acute respiratory syndrome (SARS) coronavirus and Middle East (MERS) coronavirus to discover genes that are differentially expressed in coronaviruses and identify potential therapeutic targets and host cell vulnerabilities. We identified MXD1 as differentially expressed genes following coronavirus infection in vivo and in vitro, conserved across HCoV, MERS-CoV, and SARS-CoV. MXD1 may be involved in the cellular response to COVID19 infection.

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