Illumina sequencing of clinical samples for virus detection in a public health laboratory

Huang, Bixing; Jennison, Amy V.; Whiley, David M.; McMahon, Jamie; Graham, Rikki; Jong, Amanda De; Warrilow, David

doi:10.1038/s41598-019-41830-w

Cited by 40 publications

(29 citation statements)

References 33 publications

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“…RNA extracted from all six excreta samples collected from groups of experimentally infected mosquitoes were positive for either WNV KUN or RRV by RT-rtPCR, with mean (± standard error of the mean [SEM]) cycle threshold ( C T ) values of 26.3 ± 0.4 and 26.8 ± 1.0, respectively. Based on these results, six libraries were subsequently prepared and next-generation sequenced using the Illumina NextSeq platform ( 21 ). The mean (± SEM) numbers of raw reads obtained from the libraries from mosquitoes exposed to WNV KUN or RRV were 5, 877,227 ± 137,555 and 9,020,173 ± 515,578, respectively.…”

Section: Resultsmentioning

confidence: 99%

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Metagenomic Analysis of the Virome of Mosquito Excreta

Ramírez

Colmant

Warrilow

et al. 2020

mSphere

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Traditional screening for arboviruses in mosquitoes requires a priori knowledge and the utilization of appropriate assays for their detection. Mosquitoes can also provide other valuable information, including unexpected or novel arboviruses, nonarboviral pathogens ingested from hosts they feed on, and their own genetic material. Metagenomic analysis using next-generation sequencing (NGS) is a rapidly advancing technology that allows us to potentially obtain all this information from a mosquito sample without any prior knowledge of virus, host, or vector. Moreover, it has been recently demonstrated that pathogens, including arboviruses and parasites, can be detected in mosquito excreta by molecular methods. In this study, we investigated whether RNA viruses could be detected in mosquito excreta by NGS. Excreta samples were collected from Aedes vigilax and Culex annulirostris experimentally exposed to either Ross River or West Nile viruses and from field mosquitoes collected across Queensland, Australia. Total RNA was extracted from the excreta samples, reverse transcribed to cDNA, and sequenced using the Illumina NextSeq 500 platform. Bioinformatic analyses from the generated reads demonstrate that mosquito excreta provide sufficient RNA for NGS, allowing the assembly of near-full-length viral genomes. We detected Australian Anopheles totivirus, Wuhan insect virus 33, and Hubei odonate virus 5 and identified seven potentially novel viruses closely related to members of the order Picornavirales (2/7) and to previously described, but unclassified, RNA viruses (5/7). Our results suggest that metagenomic analysis of mosquito excreta has great potential for virus discovery and for unbiased arbovirus surveillance in the near future. IMPORTANCE When a mosquito feeds on a host, it ingests not only its blood meal but also an assortment of microorganisms that are present in the blood, thus acting as an environmental sampler. By using specific tests, it is possible to detect arthropod-borne viruses (arboviruses) like dengue and West Nile viruses in mosquito excreta. Here, we explored the use of next-generation sequencing (NGS) for unbiased detection of RNA viruses present in excreta from experimentally infected and field-collected mosquitoes. We have demonstrated that mosquito excreta provide a suitable template for NGS and that it is possible to recover and assemble near-full-length genomes of both arboviruses and insect-borne viruses, including potentially novel ones. These results importantly show the direct practicality of the use of mosquito excreta for NGS, which in the future could be used for virus discovery, environmental virome sampling, and arbovirus surveillance.

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Section: Resultsmentioning

confidence: 99%

“…Libraries were diluted to 1 nM, pooled, denatured and diluted to a final concentration of 1.2 pM. Paired-end sequencing was performed using the NextSeq platform (Illumina) using a NextSeq 500 Mid Output V2 kit (Illumina) (21).…”

Section: Methodsmentioning

confidence: 99%

Metagenomic Analysis of the Virome of Mosquito Excreta

Ramírez

Colmant

Warrilow

et al. 2020

mSphere

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“…A study comparing the diagnostic efficiency of NGS vs. gold standard real time PCR in 89 nasopharyngeal swabs reported a sensitivity of 78% and specificity of 80% for NGS (Thorburn et al, 2015). More recently, a NGS sensitivity of 92% compared to real time PCR was found when testing a range of 52 clinical samples, including 8 of fecal origin (Huang et al, 2019). Target enrichment for sequences of viruses infecting vertebrate organisms, using biotinylated capture probes as a front-end procedure of NGS-based metagenomic sequencing, provides an opportunity for sensitive, broad detection of viruses (Wylie et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

Broad Virus Detection and Variant Discovery in Fecal Samples of Hematopoietic Transplant Recipients Using Targeted Sequence Capture Metagenomics

et al. 2020

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Pediatric allogeneic hematopoietic stem cell transplantation (HSCT) patients often suffer from gastro-intestinal (GI) disease caused by viruses, Graft-versus-Host Disease (GVHD) or a combination of the two. Currently, the GI eukaryotic virome of HSCT recipients remains relatively understudied, which complicates the understanding of its role in GVHD pathogenicity. As decisions regarding immunosuppressive therapy in the treatment of virus infection or GVHD, respectively, can be completely contradicting, it is crucial to better understand the prevalence and relevance of viruses in the GI tract in the HSCT setting. A real time PCR panel for a set of specific viruses widely used to diagnose the most common causes of GI viral gastroenteritis is possibly insufficient to grasp the full extent of viruses present. Therefore, we applied the targeted sequence capture method ViroCap to residual fecal samples of 11 pediatric allogeneic HSCT recipients with GI symptoms and a suspicion of GVHD, to enrich for nucleic acids of viruses that are known to infect vertebrate hosts. After enrichment, NGS was applied to broadly detect viral sequences. Using ViroCap, we were able to detect viruses such as norovirus and adenovirus (ADV), that had been previously detected using clinical diagnostic PCR on the same sample. In addition, multiple, some of which clinically relevant viruses were detected, including ADV, human rhinovirus (HRV) and BK polyomavirus (BKV). Interestingly, in samples in which specific PCR testing for regular viral GI pathogens did not result in a diagnosis, the ViroCap pipeline led to the detection of viral sequences of human herpesvirus (HHV)-7, BKV, HRV, KI polyomavirus and astrovirus. The latter was an only recently described variant and showed extensive sequence mismatches with the applied real time PCR primers and would therefore not have been detected if tested. Our results indicate that target enrichment of viral nucleic acids through ViroCap leads to sensitive and broad possibly clinically relevant virus detection, including the detection of newer variants in clinical HSCT recipient samples. As such, ViroCap could be a useful detection tool clinically, but also in studying the associations between viral presence and GVHD.

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“…Now knowing the potential of DRS for strain level detection of pathogens as determined through this study as well as others [75], we next investigated the analytical sensitivity of PRRSV detection to determine its usefulness for obtaining reliable sequencing results. Previous research examining analytical sensitivity of next-generation sequencing has reported sensitivities that are similar or less sensitive than RT-qPCR [28,76], and the third-generation Oxford Nanopore DRS has previously shown a sensitivity of 1.89 × 10 7 viral copies in an influenza virus study [44]. Our results indicated that samples with a minimum of 10 4 to 10 6 viral copies, depending on the sample type, can be successfully sequenced to accurately identify strains after about 6 hours of sequencing.…”

Section: Discussionmentioning

confidence: 99%

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

Tan

Dvorak

Murtaugh

2019

Viruses

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Prompt detection and effective control of porcine reproductive and respiratory syndrome virus (PRRSV) during outbreaks is important given its immense adverse impact on the swine industry. However, the diagnostic process can be challenging due to the high genetic diversity and high mutation rate of PRRSV. A diagnostic method that can provide more detailed genetic information about pathogens is urgently needed. In this study, we evaluated the ability of Oxford Nanopore MinION direct RNA sequencing to generate a PRRSV whole genome sequence and detect and discriminate virus at the strain-level. A nearly full length PRRSV genome was successfully generated from raw sequence reads, achieving an accuracy of 96% after consensus genome generation. Direct RNA sequencing reliably detected the PRRSV strain present with an accuracy of 99.9% using as few as 5 raw sequencing reads and successfully differentiated multiple co-infecting strains present in a sample. In addition, PRRSV strain information was obtained from clinical samples containing 10 4 to 10 6 viral copies or more within 6 hours of sequencing. Overall, direct viral RNA sequencing followed by bioinformatic analysis proves to be a promising approach for identification of the viral strain or strains involved in clinical infections, allowing for more precise prevention and control strategies during PRRSV outbreaks.2 of 18 against a new introduction from outside the farm indicates a need for enhanced biosecurity, while a re-break of a resident strain suggests better strategies for an elimination or vaccination program are needed. Another big challenge for PRRS control is that PRRSV vaccine is not completely effective at preventing and controlling infection due to the high genetic diversity of the virus, thus outbreaks still occur in vaccinated herds [13][14][15][16]. A diagnostic method which can provide genetic information about the strain causing infection would allow for identification of potential reasons for vaccination failure, such as limited cross-protection due to high genetic divergence from vaccine [17], or in the case of genetic similarity to vaccine, perhaps a reversion to virulence (escape mutation) of the vaccine itself [18]. In addition to the clinical challenges mentioned above, PRRSV has widely divergent genetic lineages and is a rapidly evolving pathogen with novel variants which seem to be more divergent and virulent than those in the past [10,[19][20][21]. The continuous emergence of new virulent strains causes unexpected devastating outbreaks, such as the severe outbreaks of HP-PRRSV in China and MN184 and NADC30 outbreaks in the United States [22][23][24][25]. The increasing incidence of co-infections of multiple strains further complicates PRRS diagnosis and control [26]. Hence, diagnostic tools that can provide more genetic information are extremely important for investigation, prevention, and control strategies for PRRSV outbreaks.Prompt detection of pathogens during an outbreak is essential for efficient disease control. Real-time quantitative...

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Illumina sequencing of clinical samples for virus detection in a public health laboratory

Cited by 40 publications

References 33 publications

Metagenomic Analysis of the Virome of Mosquito Excreta

Metagenomic Analysis of the Virome of Mosquito Excreta

Broad Virus Detection and Variant Discovery in Fecal Samples of Hematopoietic Transplant Recipients Using Targeted Sequence Capture Metagenomics

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

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