Recovery of yellow fever virus whole genome from an individual with severe vaccine-associated viscerotropic disease
Authors: Harry N Walker, Leon Caly, Ivana Savic, Ammar Aziz, Thomas Tran, Megan Whitley, Ruchir Chavada, Chuan Kok Lim
Journal: The Lancet Microbe (2024)

We read with great interest a recent study on vaccine-strain yellow fever virus (YFV) infection transmitted from a blood donor to a deceased organ donor and then to all four organ recipients.1Gould CV Free RJ Bhatnagar J et al.Transmission of yellow fever vaccine virus through blood transfusion and organ transplantation in the USA in 2021: report of an investigation.Lancet Microbe. 2023; 4: e711-e721Summary Full Text Full Text PDF PubMed Scopus (0) Google Scholar This study highlights some of the diagnostic challenges, including differentiating strains of a single viral species through sequencing from clinical samples with low concentrations of viral RNA. YFV genome sequencing is essential to differentiate the wild-type virus strain from the 17D vaccine strain, particularly when an individual's clinical history is ambiguous. This distinction is of great biosecurity importance given that the mosquito vector is widely endemic in the tropics and subtropics, and wild-type YFV is considered a potential agent of bioterrorism in some countries.2Department of Health and Aged CareAustralian Government, National Health Security (SSBA Standards) Determination.https://www.legislation.gov.au/Details/F2023L01218Date accessed: October 2, 2023Google Scholar Here, we report a case of YFV infection in a recently vaccinated traveller. He presented with fever, hepatitis, thrombocytopenia, and hypotension consistent with yellow fever vaccine-associated viscerotropic disease, which occurs at an incidence of 3–4 per 1 000 000 vaccine doses.3Khromava AY Eidex RB Weld LH et al.Yellow fever vaccine: an updated assessment of advanced age as a risk factor for serious adverse events.Vaccine. 2005; 23: 3256-3263Crossref PubMed Scopus (178) Google Scholar, 4Kitchener S Viscerotropic and neurotropic disease following vaccination with the 17D yellow fever vaccine, ARILVAX.Vaccine. 2004; 22: 2103-2105Crossref PubMed Scopus (0) Google Scholar, 5Lindsey NP Schroeder BA Miller ER et al.Adverse event reports following yellow fever vaccination.Vaccine. 2008; 26: 6077-6082Crossref PubMed Scopus (208) Google Scholar YFV was detected by RT-qPCR in the serum (cycle threshold [Ct] of 36·8) and urine (Ct of 34·4), using previously published primers and probes.6Pang Z Li A Li J et al.Comprehensive multiplex one-step real-time TaqMan qRT-PCR assays for detection and quantification of hemorrhagic fever viruses.PLoS One. 2014; 9e95635Crossref Scopus (54) Google Scholar A hemi-nested, conventional RT-PCR generated a 168-nucleotide amplicon of the YFV NS5 gene.7Russell JS Caly L Kostecki R et al.The first isolation and whole genome sequencing of Murray Valley encephalitis virus from cerebrospinal fluid of a patient with encephalitis.Viruses. 2018; 10: 319Crossref PubMed Scopus (10) Google Scholar Sanger sequencing data of this amplicon were inconclusive in definitely classifying the virus as the 17D vaccine strain. To improve genomic yield, RNA was extracted from urine (5 mL) using an automated magnetic bead system (KingFisher Apex, Thermo Fisher Scientific, Waltham, MA, USA) and eluted into 250 μL of elution buffer. This extraction yielded a concentration of 2·309 copies per μL assessed by digital PCR (from Ct 34·3 to Ct 30·3). The sample RNA was subjected to cDNA generation, amplification, and then hybridisation probe capture using the Comprehensive Viral Research Panel (Twist bioscience, San Francisco, CA, USA). Paired-end, short-read sequencing was performed using an Illumina NextSeq 1000 P1 cartridge (Illumina, San Diego, CA, USA). Reads were aligned to the YFV sequence (NC_002031.1) to create a consensus genome (99·99% coverage, depth >1000x/base position, accession OR700702),8Rice CM Lenches EM Eddy SR et al.Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution.Science. 1985; 229: 726-733Crossref PubMed Scopus (0) Google Scholar with 99·88% nucleotide similarity to the 17D reference strain. This alignment allows the definite exclusion of wild-type YFV as a biothreat organism. Further methodological detail is provided in the appendix. The above approach shows that a large volume of urine is a valuable and under-utilised specimen type for genomic characterisation of flaviviruses. Urine can be obtained non-invasively and concentrated without the issue of PCR inhibition. By using urine as a sample for hybrid probe-capture next-generation sequencing, we showed that a near-whole viral genomic sequence can be derived from specimens with low-level viraemia. This sequencing approach is suitable for the rapid characterisation of flaviviruses during a high-impact public health event. We declare no competing interests. Download .pdf (.19 MB) Help with pdf files Supplementary appendix