Optimisation of methodology for whole genome sequencing of Measles Virus directly from patient specimens

Schulz, Helene; Hiebert, Joanne; Frost, Jasmine Rae; McLachlan, Elizabeth; Severini, Alberto

doi:10.1016/j.jviromet.2021.114348

Cited by 9 publications

(4 citation statements)

References 26 publications

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“…[44] With this approach, coverage can be reduced by lower sample quality, lower viral load, and higher point mutations. [45, 46] In addition, our primers were designed using 2016 consensus sequences in the genome database, and might, therefore, be susceptible to viral evolution leading to amplicon dropouts. [44, 47] This underscores the need for continual refinement of tiled primers for optimal amplification of circulating virus strains, as documented during the COVID pandemic by the need to update ARTIC tiled primers for sequencing SARSCoV-2 genomes.…”

Section: Discussionmentioning

confidence: 99%

Dengue Virus Surveillance in Nepal Yields the First On-Site Whole Genome Sequences of Isolates from the 2022 Outbreak

Napit,

Ngono,

Mihindukulasuriya

et al. 2024

Preprint

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BackgroundThe 4 serotypes of dengue virus (DENV1-4) can each cause potentially deadly dengue disease, and are spreading globally from tropical and subtropical areas to more temperate ones. Nepal provides a microcosm of this global phenomenon, having met each of these grim benchmarks. To better understand DENV transmission dynamics and spread into new areas, we chose to study dengue in Nepal and, in so doing, to build the onsite infrastructure needed to manage future, larger studies.Methods and ResultsDuring the 2022 dengue season, we enrolled 384 patients presenting at a hospital in Kathmandu with dengue-like symptoms; 79% of the study participants had active or recent DENV infection (NS1 antigen and IgM). To identify circulating serotypes, we screened serum from 50 of the NS1+participants by RT-PCR and identified DENV1, 2, and 3 – with DENV1 and 3 codominant. We also performed whole-genome sequencing of DENV, for the first time in Nepal, using our new on-site capacity. Sequencing analysis demonstrated the DENV1 and 3 genomes clustered with sequences reported from India in 2019, and the DENV2 genome clustered with a sequence reported from China in 2018.ConclusionThese findings highlight DENV’s geographic expansion from neighboring countries, identify China and India as the likely origin of the 2022 DENV cases in Nepal, and demonstrate the feasibility of building onsite capacity for more rapid genomic surveillance of circulating DENV. These ongoing efforts promise to protect populations in Nepal and beyond by informing the development and deployment of DENV drugs and vaccines in real time.

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Section: Discussionmentioning

confidence: 99%

Dengue Virus Surveillance in Nepal Yields the First On-Site Whole Genome Sequences of Isolates from the 2022 Outbreak

Napit,

Ngono,

Mihindukulasuriya

et al. 2024

Preprint

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“…For the whole N and HA sequences we have approximately 99.93% coverage and a depth of 10×, thus minimizing any technology bias. 13 Partial N (450 bp) and HA (1854 bp) consensus sequences (AC# OR224894 to OR224917) were used for genotyping using MEGA 7 14 according to CDC and WHO recommendations. 15,16…”

Section: Methodsmentioning

confidence: 99%

“…These samples were thus representative of the 12 weeks of the epidemic follow‐up and presented with a Ct ≤ 30 that allowed a clean sequencing with the MinION. For the whole N and HA sequences we have approximately 99.93% coverage and a depth of 10×, thus minimizing any technology bias 13 . Partial N (450 bp) and HA (1854 bp) consensus sequences (AC# OR224894 to OR224917) were used for genotyping using MEGA 7 14 according to CDC and WHO recommendations 15,16…”

Section: Methodsmentioning

confidence: 99%

Viral etiology of measles‐like rash in Guinean children during the COVID epidemic in 2022

Anguinze,

Touré,

Cissé

et al. 2024

Journal of Medical Virology

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Covid‐19 in West Africa masked outbreaks of vaccine‐preventable diseases such as the measles epidemic in children in Guinea in 2021–2022 characterized by a lack of confirmation of suspected clinical cases. During weeks 13–22 of 2022, saliva samples were collected from 213 children (3–60 months old) with measles‐like symptoms within the St Gabriel dispensary in Conakry. Samples were processed in Virus Transport Medium (VTM) and tested on the same day by triplex reverse transcriptase ‐real‐time polymerase chain reaction for Measles, Rubella and RNaseP. Samples were also tested for HHV6 and Parvovirus B19, viruses causing clinical signs similar to measles. We confirmed 146 (68.5%) measles cases, 27 (12.7%) rubella, 5 (2.3%) double‐positive measles‐rubella, 35 (16.4%) HHV‐6 and 8 (3.75%) Parvovirus B19. To test the assay's robustness, 27 samples were kept at 26–30°C. Measles and rubella were still detected after 7 days at 26–30°C, and after 21 days measles and rubella were still detectable in all samples but one. Sequencing indicated the circulation of the B3 measles genotype, as expected in West Africa. This study highlights the robustness of the measles/rubella diagnostic test on saliva samples stored in VTM. The high level of rubella detection questioned the single valence measles vaccination strategy.

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“…Total nucleic acids from clinical specimens were extracted on the MagNA Pure 96 using the DNA and Viral NA large volume kit (catalog 06374891001, Roche), and for the isolate, the QIAamp Viral RNA kit (Qiagen catalog 52906). All next-generation sequencing (NGS) libraries were prepared as previously described and used a probe enrichment modification of the TruSeq DNA Nano LP kit (catalog 20016328 Illumina) ( 4 ). Libraries were paired-end sequenced on an Illumina MiSeq with a v3 reagent kit (catalog MS-102–3003).…”

Section: Announcementmentioning

confidence: 99%

Genome sequence of a measles virus strain with a novel loss of stop codon mutation in the phosphoprotein gene

Zubach,

Schulz,

Kim

et al. 2024

Microbiol Resour Announc

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Optimisation of methodology for whole genome sequencing of Measles Virus directly from patient specimens

Cited by 9 publications

References 26 publications

Dengue Virus Surveillance in Nepal Yields the First On-Site Whole Genome Sequences of Isolates from the 2022 Outbreak

Dengue Virus Surveillance in Nepal Yields the First On-Site Whole Genome Sequences of Isolates from the 2022 Outbreak

Viral etiology of measles‐like rash in Guinean children during the COVID epidemic in 2022

Genome sequence of a measles virus strain with a novel loss of stop codon mutation in the phosphoprotein gene

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