Molecular techniques for the genomic viral RNA detection of West Nile, Dengue, Zika and Chikungunya arboviruses: a narrative review

2021 ◽  
Author(s):  
Antonio Mori ◽  
Elena Pomari ◽  
Michela Deiana ◽  
Francesca Perandin ◽  
Sara Caldrer ◽  
...  
Keyword(s):  
Molecular Techniques ◽  
Narrative Review ◽  
Viral Rna ◽  
West Nile ◽  
Rna Detection

scholarly journals Do I Have HIV or Not? Lack of RNA Detection and the Case for Sensitive DNA Testing

2020 ◽  
Vol 7 (11) ◽  
Author(s):  
Sandra A Springer ◽  
Silvina Masciotra ◽  
Jeffrey A Johnson ◽  
Sheldon Campbell
Keyword(s):  
Antiretroviral Therapy ◽  
Hiv Infection ◽  
Viral Rna ◽  
Infection Status ◽  
Test Results ◽  
Dna Testing ◽  
Rna Detection ◽  
Hiv Test

Abstract We present a case of a 20-year-old male who had ambiguous HIV test results after entering new provider care and whose status was later complicated by undetectable viral RNA off antiretroviral therapy (ART). Verifying HIV infection status may occasionally require sensitive DNA testing that might need to be considered in diagnostic guidelines to resolve diagnosis and ensure appropriate ART management.

scholarly journals Direct Viral RNA Detection of SARS-CoV-2 and DENV in Inactivated Samples by Real-Time RT-qPCR: Implications for Diagnosis in Resource Limited Settings with Flavivirus Co-Circulation

Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1558
Author(s):  
Zhan Qiu Mao ◽  
Mizuki Fukuta ◽  
Jean Claude Balingit ◽  
Thi Thanh Ngan Nguyen ◽  
Co Thach Nguyen ◽  
...  
Keyword(s):  
Real Time ◽  
Early Stage ◽  
Rna Extraction ◽  
Viral Rna ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Resource Limited Settings ◽  
Rna Detection ◽  
Resource Limited ◽  
Starting Point

The RT-qPCR method remains the gold standard and first-line diagnostic method for the detection of SARS-CoV-2 and flaviviruses, especially in the early stage of viral infection. Rapid and accurate viral detection is a starting point in the containment of the COVID-19 pandemic and flavivirus outbreaks. However, the shortage of diagnostic reagents and supplies, especially in resource-limited countries that experience co-circulation of SARS-CoV-2 and flaviviruses, are limitations that may result in lesser availability of RT-qPCR-based diagnostic tests. In this study, the utility of RNA-free extraction methods was assessed for the direct detection of SARS-CoV-2 and DENV-2 in heat-inactivated or chemical-inactivated samples. The findings demonstrate that direct real-time RT-qPCR is a feasible option in comparison to conventional real-time RT-qPCR based on viral genome extraction-based methods. The utility of heat-inactivation and direct real-time RT-qPCR for SARS-CoV-2, DENV-2 viral RNA detection was demonstrated by using clinical samples of SARS-CoV-2 and DENV-2 and spiked cell culture samples of SARS-CoV-2 and DENV-2. This study provides a simple alternative workflow for flavivirus and SARS-CoV-2 detection that includes heat inactivation and viral RNA extraction-free protocols, with aims to reduce the risk of exposure during processing of SARS-CoV-2 biological specimens and to overcome the supply-chain bottleneck, particularly in resource limited settings with flavivirus co-circulation.

scholarly journals SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification

2020 ◽  
Vol 117 (39) ◽  
pp. 24450-24458 ◽  
Author(s):  
Brian A. Rabe ◽  
Constance Cepko
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Reverse Transcription ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Viral Rna ◽  
Rna Detection ◽  
Sample Stability ◽  
Rapid Inactivation ◽  
Diagnostic Capabilities ◽  
Purification Protocol

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.

scholarly journals SARS-CoV-2 Isolation From Ocular Secretions of a Patient With COVID-19 in Italy With Prolonged Viral RNA Detection

2020 ◽  
Vol 173 (3) ◽  
pp. 242-243 ◽  
Author(s):  
Francesca Colavita ◽  
Daniele Lapa ◽  
Fabrizio Carletti ◽  
Eleonora Lalle ◽  
Licia Bordi ◽  
...  
Keyword(s):  
Viral Rna ◽  
Rna Detection

scholarly journals West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis

2007 ◽  
Vol 88 (4) ◽  
pp. 1163-1168 ◽  
Author(s):  
Jason M. Mackenzie ◽  
Mark T. Kenney ◽  
Edwin G. Westaway
Keyword(s):  
West Nile Virus ◽  
Rna Polymerase ◽  
Virus Strain ◽  
Viral Rna ◽  
West Nile ◽  
Replication Complex ◽  
Virus Rna ◽  
West Nile Virus Strain ◽  
First Time ◽  
Cytoplasmic Location

Using West Nile virus strain Kunjin virus (WNVKUN) as a model system for flavivirus replication, we showed that the virus replication complex (RC) is associated with the dsRNA template located in induced membranes only in the cytoplasm. In this report we established for the first time that the RNA-dependent RNA polymerase NS5 is located in flavivirus-induced membranes, including the site of viral RNA replication. We found no evidence for nuclear localization of the essential RC components NS5 and its dsRNA template for WNVKUN or the closely related WNV strain Sarafend, by immuno-electron microscopy or by immunofluorescence. Metabolic radiolabelling with [32P]orthophosphate revealed that WNVKUN NS5 was phosphorylated and this was confirmed by Western blotting with antibodies specific for phosphorylated serine and threonine only. These observations of a cytoplasmic location for the WNV polymerase and its phosphorylation state correspond to the characteristics of the hepatitis C virus RNA polymerase NS5B.

DEVELOPMENT AND ASSESSMENT OF A REAGENT KIT FOR RNA DETECTION OF CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS WITH USING REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION METHOD

2019 ◽  
Vol 64 (9) ◽  
pp. 571-577
Author(s):  
V. A. Ternovoi ◽  
Yu. V. Kononova ◽  
A. V. Zaykovskaya ◽  
E. V. Chub ◽  
A. S. Volynkina ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
High Speed ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Isothermal Amplification ◽  
Viral Rna ◽  
Rna Detection ◽  
Crimean Congo Hemorrhagic Fever ◽  
Loop Mediated Isothermal Amplification ◽  
Hemorrhagic Fever Virus

This study presents the results of laboratory trials of the reagent kit for the rapid detection of RNA of the Crimean-Congo hemorrhagic fever virus (CCHFV) using loop-mediated isothermal amplification with reverse transcription (RT-LAMP). The developed RT-LAMP reagent kit was used to detect the CCHFV and showed a sensitivity of 103 GE/ml of viral RNA, which is sufficient for detection of the CCHFV in the early stage of human infections. The kit showed high specificity and no cross-reactivity with viral panel from the State collection of viruses of the FBRI SRC VB «Vector» (arboviruses and hemorrhagic fever viruses). Laboratory trials of the RT-LAMP kit are showed a high analytical and diagnostic sensitivity and specificity for RNA detection of the CCHFV and high speed of the analysis (60-70 min with sample preparation) compared to real-time PCR. Approbation of the kit field version has showed the possibility of setting the RT-LAMP reaction and viral RNA detection without the using of analytical equipments.

Viral RNA detection by RIG-I-like receptors

2015 ◽  
Vol 32 ◽  
pp. 48-53 ◽  
Author(s):  
Mitsutoshi Yoneyama ◽  
Koji Onomoto ◽  
Michihiko Jogi ◽  
Teppei Akaboshi ◽  
Takashi Fujita
Keyword(s):  
Viral Rna ◽  
Rna Detection

scholarly journals Cell Proteins TIA-1 and TIAR Interact with the 3′ Stem-Loop of the West Nile Virus Complementary Minus-Strand RNA and Facilitate Virus Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 11989-12000 ◽  
Author(s):  
W. Li ◽  
Y. Li ◽  
N. Kedersha ◽  
P. Anderson ◽  
M. Emara ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Rna Binding ◽  
Peptide Sequencing ◽  
Viral Rna ◽  
Cell Protein ◽  
Affinity Column ◽  
Rna Recognition Motif ◽  
The West ◽  
West Nile ◽  
Stem Loop

ABSTRACT It was reported previously that four baby hamster kidney (BHK) proteins with molecular masses of 108, 60, 50, and 42 kDa bind specifically to the 3′-terminal stem-loop of the West Nile virus minus-stand RNA [WNV 3′(−) SL RNA] (P. Y. Shi, W. Li, and M. A. Brinton, J. Virol. 70:6278-6287, 1996). In this study, p42 was purified using an RNA affinity column and identified as TIAR by peptide sequencing. A 42-kDa UV-cross-linked viral RNA-cell protein complex formed in BHK cytoplasmic extracts incubated with the WNV 3′(−) SL RNA was immunoprecipitated by anti-TIAR antibody. Both TIAR and the closely related protein TIA-1 are members of the RNA recognition motif (RRM) family of RNA binding proteins. TIA-1 also binds to the WNV 3′(−) SL RNA. The specificity of these viral RNA-cell protein interactions was demonstrated using recombinant proteins in competition gel mobility shift assays. The binding site for the WNV 3′(−) SL RNA was mapped to RRM2 on both TIAR and TIA-1. However, the dissociation constant (Kd ) for the interaction between TIAR RRM2 and the WNV 3′(−) SL RNA was 1.5 × 10−8, while that for TIA-1 RRM2 was 1.12 × 10−7. WNV growth was less efficient in murine TIAR knockout cell lines than in control cells. This effect was not observed for two other types of RNA viruses or two types of DNA viruses. Reconstitution of the TIAR knockout cells with TIAR increased the efficiency of WNV growth, but neither the level of TIAR nor WNV replication was as high as in control cells. These data suggest a functional role for TIAR and possibly also for TIA-1 during WNV replication.

scholarly journals THE INFORMATIVE VALUE OF IMMUNOLOGICAL AND GENETIC TESTS IN THE DIAGNOSIS WEST NILE FEVER

2020 ◽  
Vol 76 (4) ◽  
pp. 42-45
Author(s):  
Т.V. Zamarina ◽  
◽  
N.P. Khrapova ◽  
I.A. Barkova ◽  
E.V. Pimenova ◽  
...  
Keyword(s):  
Viral Infection ◽  
Viral Rna ◽  
Unknown Etiology ◽  
West Nile Fever ◽  
West Nile ◽  
Respiratory Viral Infection ◽  
Blood Samples ◽  
Monitoring Centre ◽  
Laboratory Confirmation ◽  
Acute Respiratory Viral Infection

We used ELISA and PCR for laboratory verification of West Nile fever (MUC 4.2.3009-12). We analyzed serum and whole blood samples which had been sent to West Nile fever reference monitoring centre in 2018–2019. A total of 270 blood samples obtained from patients with presumed viral encephalitis of unknown etiology, acute respiratory viral infection, acute respiratory viral infection, meningitis, acute gastroenteritis were analyzed. Antibodies against WNV were detected in 193 (71,4 %) blood samples, while 146 samples were found to be capable of developing an immune response with viral RNA not being detected in them. Both WNV antibodies and viral RNA were detected in 47 (17,4 %) samples tested. WNV RNA was detected in only 14 (5,1 %) cases. Thus, laboratory confirmation of WNF was obtained in 76,6 % of cases (207 out of 270).

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