Here we present a fatal case of VZV infection with hemorrhagic fever in an inmunocompetent adult diagnosed by real-time PCR and subsequent whole-genome sequencing to characterize a putative virulent VZV strain.

A 38-year-old man born in Nigeria was admitted to the emergency unit of Virgen de la Arrixaca Hospital (Murcia, Spain) on January 29, 2012. The patient displayed abdominal pain without fever, regional lymph node enlargement or other significant symptoms. He had been living in Spain for 12 years and had not traveled abroad during the last 6 years but had consumed food brought from his country by his relatives at home. Anamnesis recorded discal herniation at L5–S1, diagnosed in 2008 and under current treatment with non-steroidal anti-inflammatory drugs and analgesics. No recent corticosteroid treatment was noticed at that time. Leukocytosis (12,230 cells/mm³, 66% neutrophils) and abnormal liver enzyme levels (GOT: 382 UI/l; GPT: 364; FA: 39; GGT: 55; LDH: 873) were found, but liver looked normal under CT examination and ascites was absent. Neurological exploration was also normal. The patient was discharged to the Gastroenterology Unit of the hospital a few hours later. Leucocytosis persisted, and platelet count was under the lower limit (120,000/mm³). Cardiac echography performed the next day revealed myopericarditis. Lung endoscopy was planned, but the patient's condition deteriorated quickly, and he was transferred to the Intensive Care Unit. High fever and hemorrhagic features developing in conjunctiva, ears and nose emerged during the next 48 h. VHF was suspected, and he was moved into an isolation room. Multiorgan failure presented on January 31, and the patient died on February 1. Investigation of risk factors for severe VZV primary infection was begun after the laboratory findings and suggested data previously unnoticed: Due to recent lumbar lesions, the patient had self-prescribed corticosteroid treatment at the time of virus exposure. Disseminated VZV infection with hemorrhagic presentation resulting in death without rash was suspected.

Serum markers of hepatitis B and C viruses and HIV infections, and PCR for ebolaviruses RNA, were analyzed on a serum sample taken at presentation. Because of the lack of significant findings, other pathogens were tested by PCR: Plasmodium, Leptospira, Rickettsia, Crimea-Congo hemorrhagic fever virus, dengue viruses, enteroviruses, herpes simplex virus 1 and 2, VZV, human cytomegalovirus, Epstein-Barr virus and human herpesvirus 6, human herpesvirus 7 and human herpesvirus 8. Only VZV tested positive, and VZV DNA was found in serum samples and in liver and spleen tissue samples from autopsy as well. The presence of VZV DNA was examined by using a Multiplex real-time PCR, which included enterovirus, HSV and VZV. In addition, both serum samples available tested negative for VZV-specific IgG and IgM antibody.

Whole-genome sequencing was performed using a metagenomic approach. Briefly, total nucleic acid from a blood sample was extracted using QiAmp Mini Elute Virus Spin Kit (Qiagen) without RNA carrier. Libraries were constructed using NEBNext Ultra II Directional RNA Library Prep kit (New England Biolabs). To enrich VZV genomes, a Twist Capture Panel V2 comprising a wide set of probes against viral pathogens including VZV was used. The sequencing was performed on a Miseq sequencer using MiSeq Reagent Micro Kit, v2 (Illumina).

Sequencing samples were analyzed for viral consensus genome reconstruction and de novo assembly using Viralrecon v2.5 pipeline (https://github.com/nf-core/viralrecon) [13] written in Nextflow (https://www.nextflow.io/) in collaboration between nf-core (https://nf-co.re/) community and the Bioinformatics Unit of the Institute of Health Carlos III (BU-ISCIII) (https://github.com/BU-ISCIII). Trimmed reads were mapped with bowtie2 v.2.4.4 [14] against the reference genome DQ479954.1. Variant calling was carried out using ivar variant v.1.3.1 [15], which calls for low- and high-frequency variants from which variants with an allele frequency > 75% were kept for inclusion in the consensus genome sequence. Finally, Bedtools v2.30.0 [16] was used to obtain the viral genome consensus with the filtered variants and mask genomic regions with coverage values < 10×.

Host reads were removed prior to de novo assembly performing kmer-based mapping of the trimmed reads against the GRCh38 NCBI human genome with Kraken2 v.2.1.2 [17]. For the de novo assembly analysis, the remaining reads after host removal were assembled using SPADES v3.15.4 [18]. A fully ordered genome sequence was generated using ABACAS v1.3.1 [19] based on the Kel strain DQ479954.1 genome because it is a clinical strain of clade 1.

The annotated complete genome was uploaded to GenBank (NCBI) with accession number OQ871571. Previous analysis of seven relevant SNPs from ORF22, ORF 21 and ORF 50 was confirmed by WGS and showed that strain OQ87571 belongs to clade 1, which is predominant in Spain [20].

In addition to Viralrecon v2.5 pipeline, sequencing data were also analyzed on IDseq v3.1 pipeline (https://czid.org/) for de novo assembly and consensus reconstruction against three sequence references. Whole-genome sequencing yielded a 95/95.2/95.5% coverage breadth and 90.7/91.1/91.1% of genome called against clade 1 clinical strain DQ479954.1, Refseq MH709361.1 and Dumas strain NC_001348.1, respectively (Table 1).

Compared to clinical clade 1, Kel strain DQ479954.1 up to 33 genome variants or mutations were found producing changes in amino acid composition (Table 2). These variants affected 14 relevant proteins of VZV (Table 3).