scholarly journals Enrichment approach for unbiased sequencing of respiratory syncytial virus directly from clinical samples

2021 ◽  
Vol 6 ◽  
pp. 99
Author(s):  
Jacqueline Wahura Waweru ◽  
Zaydah de Laurent ◽  
Everlyn Kamau ◽  
Khadija Said ◽  
Elijah Gicheru ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Bacterial Contamination ◽  
Random Sequence ◽  
Statistical Significance ◽  
Rna Extraction ◽  
N Gene ◽  
Clinical Samples ◽  
Rank Test ◽  
Dnase Treatment ◽  
Syncytial Virus

Background: Nasopharyngeal samples contain higher quantities of bacterial and host nucleic acids relative to viruses; presenting challenges during virus metagenomics sequencing, which underpins agnostic sequencing protocols. We aimed to develop a viral enrichment protocol for unbiased whole-genome sequencing of respiratory syncytial virus (RSV) from nasopharyngeal samples using the Oxford Nanopore Technology (ONT) MinION platform. Methods: We assessed two protocols using RSV positive samples. Protocol 1 involved physical pre-treatment of samples by centrifugal processing before RNA extraction, while Protocol 2 entailed direct RNA extraction without prior enrichment. Concentrates from Protocol 1 and RNA extracts from Protocol 2 were each divided into two fractions; one was DNase treated while the other was not. RNA was then extracted from both concentrate fractions per sample and RNA from both protocols converted to cDNA, which was then amplified using the tagged Endoh primers through Sequence-Independent Single-Primer Amplification (SISPA) approach, a library prepared, and sequencing done. Statistical significance during analysis was tested using the Wilcoxon signed-rank test. Results: DNase-treated fractions from both protocols recorded significantly reduced host and bacterial contamination unlike the untreated fractions (in each protocol p<0.01). Additionally, DNase treatment after RNA extraction (Protocol 2) enhanced host and bacterial read reduction compared to when done before (Protocol 1). However, neither protocol yielded whole RSV genomes. Sequenced reads mapped to parts of the nucleoprotein (N gene) and polymerase complex (L gene) from Protocol 1 and 2, respectively. Conclusions: DNase treatment was most effective in reducing host and bacterial contamination, but its effectiveness improved if done after RNA extraction than before. We attribute the incomplete genome segments to amplification biases resulting from the use of short length random sequence (6 bases) in tagged Endoh primers. Increasing the length of the random nucleotides from six hexamers to nine or 12 in future studies may reduce the coverage biases.

scholarly journals RS virus diagnosis: comparison of isolation, immunofluorescence and enzyme immunoassay

1986 ◽  
Vol 81 (2) ◽  
pp. 225-232 ◽  
Author(s):  
Marilda M. Siqueira ◽  
Vanja Ferreira ◽  
Jussara P. Nascimento
Keyword(s):  
Tissue Culture ◽  
Respiratory Syncytial Virus ◽  
Enzyme Immunoassay ◽  
Bacterial Contamination ◽  
Virus Isolation ◽  
Rapid Diagnosis ◽  
Tissue Cultures ◽  
Virus Diagnosis ◽  
Under Five ◽  
Syncytial Virus

Two techniques for rapid diagnosis, immunofluorescence (IFAT) and enzyme immunoassay (EIA), have been compared with virus isolaion in tissue culture for the detection of respiratory syncytial virus (RSV) in specimens of nasopharyngeal secretions. The specimens were obtained from children under five years of age suffering from acute respiratory iliness, during a period of six months from January to June 1982. Of 471 specimens examined 54 (11.5%) were positive by virus isolation and 180 (38.2%) were positive by immunofluorescence. The bacterial contamination of inoculated tissue cultures unfortunately prevented the isolation of virus from many samples. Specimens from 216 children were tested to compare enzyme immunoassay and immunofluorescence. Of these 60 (27%) were positive by EIA and 121 (56%) were positive by IFAT. Our results suggest that the EIA technique although highly specific is rather insensitive. This may be because by the time these tests were done the originl nasopharyngeal secretions were considerably diluted and contained more mucus fragments than the call suspension used for IFAT. Of the three techniques, IFAT gives the best results although EIA may be useful where IFAT is not possible.

scholarly journals Circulation of Respiratory Viruses in Hospitalized Adults before and during the COVID-19 Pandemic in Brescia, Italy: A Retrospective Study

2021 ◽  
Vol 18 (18) ◽  
pp. 9525
Author(s):  
Maria Antonia De Francesco ◽  
Caterina Pollara ◽  
Franco Gargiulo ◽  
Mauro Giacomelli ◽  
Arnaldo Caruso
Keyword(s):  
Respiratory Syncytial Virus ◽  
Influenza A ◽  
Statistical Significance ◽  
Respiratory Viruses ◽  
Influenza Viruses ◽  
Contact Tracing ◽  
Nucleic Acid Detection ◽  
Respiratory Pathogens ◽  
Syncytial Virus ◽  
Human Coronaviruses

Different preventive public health measures were adopted globally to limit the spread of SARS-CoV-2, such as hand hygiene and the use of masks, travel restrictions, social distance actions such as the closure of schools and workplaces, case and contact tracing, quarantine and lockdown. These measures, in particular physical distancing and the use of masks, might have contributed to containing the spread of other respiratory viruses that occurs principally by contact and droplet routes. The aim of this study was to evaluate the prevalence of different respiratory viruses (influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses 1, 2, 3 and 4, rhinovirus, adenovirus, metapneumovirus and human coronaviruses) after one year of the pandemic. Furthermore, another aim was to evaluate the possible impact of these non-pharmaceutical measures on the circulation of seasonal respiratory viruses. This single center study was conducted between January 2017–February 2020 (pre-pandemic period) and March 2020–May 2021 (pandemic period). All adults >18 years with respiratory symptoms and tested for respiratory pathogens were included in the study. Nucleic acid detection of all respiratory viruses was performed by multiplex real time PCR. Our results show that the test positivity for influenza A and B, metapneumovirus, parainfluenza virus, respiratory syncytial virus and human coronaviruses decreased with statistical significance during the pandemic. Contrary to this, for adenovirus the decrease was not statistically significant. Conversely, a statistically significant increase was detected for rhinovirus. Coinfections between different respiratory viruses were observed during the pre-pandemic period, while the only coinfection detected during pandemic was between SARS-CoV-2 and rhinovirus. To understand how the preventive strategies against SARS-CoV-2 might alter the transmission dynamics and epidemic patterns of respiratory viruses is fundamental to guide future preventive recommendations.

scholarly journals Human Respiratory Syncytial Virus-Induced Immune Signature of Infection Revealed by Transcriptome Analysis of Clinical Pediatric Nasopharyngeal Swab Samples

2020 ◽  
Author(s):  
Claire Nicolas De Lamballerie ◽  
Andrés Pizzorno ◽  
Julia Dubois ◽  
Blandine Padey ◽  
Thomas Julien ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Transcriptome Analysis ◽  
Real Life ◽  
Human Respiratory Syncytial Virus ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Immune Signature ◽  
Real Life Setting ◽  
Syncytial Virus ◽  
New Diagnosis

Abstract Human respiratory syncytial virus (HRSV) constitutes one the main causes of respiratory infection in neonates and infants worldwide. Transcriptome analysis of clinical samples using high-throughput technologies remains an important tool to better understand virus-host complex interactions in the real-life setting but also to identify new diagnosis/prognosis markers or therapeutics targets. A major challenge when exploiting clinical samples such as nasal swabs, washes, or bronchoalveolar lavages is the poor quantity and integrity of nucleic acids. In this study, we applied a tailored transcriptomics workflow to exploit nasal wash samples from children who tested positive for HRSV. Our analysis revealed a characteristic immune signature as a direct reflection of HRSV pathogenesis and highlighted putative biomarkers of interest such as IP-10, TMEM190, MCEMP1, and TIMM23.

scholarly journals Genotyping of Type A Human Respiratory Syncytial Virus Based on Direct F Gene Sequencing

Medicina ◽  
2019 ◽  
Vol 55 (5) ◽  
pp. 169 ◽  
Author(s):  
Daifullah Al Aboud ◽  
Nora M. Al Aboud ◽  
Mater I. R. Al-Malky ◽  
Ahmed S. Abdel-Moneim
Keyword(s):  
Respiratory Syncytial Virus ◽  
Mutational Analysis ◽  
Gene Sequencing ◽  
Human Respiratory Syncytial Virus ◽  
Clinical Samples ◽  
Escape Mutant ◽  
Respiratory Pathogens ◽  
Virus Genotype ◽  
F Gene ◽  
Syncytial Virus

Background and objectives: The human respiratory syncytial virus (hRSV) is among the important respiratory pathogens affecting children. Genotype-specific attachment (G) gene sequencing is usually used to determine the virus genotype. The reliability of the fusion (F) gene vs. G gene genotype-specific sequencing was screened. Materials and Methods: Archival RNA from Saudi children who tested positive for hRSV-A were used. Samples were subjected to a conventional one-step RT-PCR for both F and G genes and direct gene sequencing of the amplicons using the same primer sets. Phylogeny and mutational analysis of the obtained sequences were conducted. Results: The generic primer set succeeded to amplify target gene sequences. The phylogenetic tree based on partial F gene sequencing resulted in an efficient genotyping of hRSV-A strains equivalent to the partial G gene genotyping method. NA1, ON1, and GA5 genotypes were detected in the clinical samples. The latter was detected for the first time in Saudi Arabia. Different mutations in both conserved and escape-mutant domains were detected in both F and G. Conclusion: It was concluded that a partial F gene sequence can be used efficiently for hRSV-A genotyping.

scholarly journals A quite sensitive fluorescent loop-mediated isothermal amplification for rapid detection of respiratory syncytial virus

2019 ◽  
Vol 13 (12) ◽  
pp. 1135-1141 ◽  
Author(s):  
Yihong Hu ◽  
Zhenzhou Wan ◽  
Yonglin Mu ◽  
Yi Zhou ◽  
Jia Liu ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
High Morbidity ◽  
Rt Pcr ◽  
Loop Mediated Isothermal Amplification ◽  
Syncytial Virus ◽  
Syto 9

Introduction: Human respiratory syncytial virus (hRSV) is a common respiratory virus closely related to respiratory tract infection (RTI). Rapid and accurate detection of hRSV is urgently needed to reduce the high morbidity and mortality due to hRSV infection. Methodology: Here, we established a highly sensitive and specific reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of A and B group hRSV simultaneously. The specific primer sets for hRSV A and B groups were designed in the M and M2-2 gene, respectively. SYTO 9 was used as the fluorescent dye for real-time monitoring of the amplification of hRSV RNA without cross reaction between hRSV A and B. Results: The limit of detection (LOD) of our new method was 281.17 50% tissue culture infective doses (TCID50)/mL for hRSV A and 1.58 TCID50/mL for hRSV B. Using 90 clinical samples, a comparison to traditional RT-PCR was performed to validate this assay. The positivity rate of RT-LAMP and RT-PCR were 67.8% and 55.6%, respectively, and the positivity rate of RT-LAMP was significantly higher than RT-PCR (χ2 test, P < 0.01). Conclusions: Compared with traditional RT-PCR method, the newly developed fluorescent RT-LAMP combined with well-designed primers and SYTO 9 is quite sensitive, specific, rapid and well applicable to hRSV clinical diagnosis.

scholarly journals Obtaining and Characterization of the Monoclonal Antibodies Against G-Protein of the Respiratory Syncytial Virus

2020 ◽  
pp. 7-14
Author(s):  
N. A. Demidova ◽  
R. R. Klimova ◽  
A. A. Kushch ◽  
E. I. Lesnova ◽  
O. V. Masalova ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Respiratory Syncytial Virus ◽  
G Protein ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rsv Infection ◽  
Hybridoma Technology ◽  
Infected Cells ◽  
Syncytial Virus

The aim of this study was to obtain hybridomas producing monoclonal antibodies (Mabs) to the G-protein of the respiratory syncytial virus (RSV), and to evaluate their immunological characteristics and virus-neutralizing activity.Material and methods. Mouse Mabs were obtained using hybridoma technology. The properties of Mabs were studied by enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining (IF) of infected cells, as well as by biological neutralization test in vitro (NT). To identify epitopes recognized by the Mabs on G protein ELISA additivity test was used.Results. Hybridization of splenocytes with Sp2/0 myeloma cells and primary screening showed that 75 hybridomas produce antibodies interacting with purified virus, 17 of them also react with the recombinant G-protein in ELISA. In NT 4, hybridomas suppressed in vitro RSV infection by more than 50%. Cloning of these hybridomas revealed 4 monoclones producing the most active Mabs. Mab 1C11 was IgG2a, 3 others (5D4, 5G11 and 6H4) were IgM. Three IgM Mabs actively reacted with both RSV A2 and Long, and with G-protein; Mab 1C11 was less reactive with all antigens tested. All Mabs suppressed RSV infection, while Mab 5D4 supressed it almost completely (98%). IF analysis showed that all Mabs detected RSV G-protein in the cell cytoplasm, the largest number of infected cells was detected using Mab 5D4 (80%). It was shown that the isolated Mabs were directed to two non-overlapping epitopes on the RSV G-protein.Conclusion. The isolated Mabs can be used to detect RSV in clinical samples by ELISA and IF. The isolated Mabs can be used for humanized recombinant antibodies construction and for the treatment of RSV infection in future.

scholarly journals Human Respiratory Syncytial Virus-induced immune signature of infection revealed by transcriptome analysis of clinical pediatric nasopharyngeal swab samples

2020 ◽  
Author(s):  
Claire Nicolas De Lamballerie ◽  
Andrés Pizzorno ◽  
Julia Dubois ◽  
Blandine Padey ◽  
Thomas Julien ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Transcriptome Analysis ◽  
Real Life ◽  
Human Respiratory Syncytial Virus ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Immune Signature ◽  
Real Life Setting ◽  
Syncytial Virus ◽  
New Diagnosis

AbstractHuman Respiratory Syncytial Virus (HRSV) constitutes one the main causes of respiratory infection in neonates and infants worldwide. Transcriptome analysis of clinical samples using high-throughput technologies remains an important tool to better understand virus-host complex interactions in the real-life setting but also to identify new diagnosis/prognosis markers or therapeutics targets. A major challenge when exploiting clinical samples such as nasal swabs, washes or bronchoalveolar lavages is the poor quantity and integrity of nucleic acids. In this study, we applied a tailored transcriptomics workflow to exploit nasal wash samples from children who tested positive for HRSV. Our analysis revealed a characteristic immune signature as a direct reflection of HRSV pathogenesis and highlighted putative biomarkers of interest.

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