FormalPara Key Summary Points

We report an uncommon case of fatal hemorrhagic fever caused by varicella-zoster virus without rash in an inmunocompetent adult.

Varicella zoster virus was fully characterized by whole genome sequencing (WGS) and several relevant mutations affecting essential genes related to viral replication in skin as well as non-coding regions significant in regulating the virus-host interaction were found.

Further studies are needed to relate these mutations to severe disease.

Introduction

Varicella-zoster virus (VZV) is a common human infectious agent worldwide. Acute primary VZV infection causes chickenpox, which is common in unvaccinated childhood populations. VZV disease without rash occurs frequently and is not always neurological. Herpes zoster is due to reactivation and is usually symptomatic with a classical clinical appearance and affects mainly adults [1, 2]. Primary infection in adulthood often causes more severe disease. In Nigeria, primary infection with VZV tends to occur at later stages of life (i.e., adulthood), resulting in a larger population of susceptible adults and potentially a higher proportion of severe cases [3]. Serious complications of chickenpox include pneumonia, encephalitis and hepatitis among adults and children, respectively.

VZV is genetically stable, although up to nine clades with prevailing geographical distributions have been described. Classification is based on seven established clades (1–6 and 9) and two putative clades (VII and VIII) [4, 5]. Genotypes of European origin included Clade 1 and Clade 3; Clade 2 Asian origin (Japanese), Clade 4 (Asia and the Americas), Clade 5 (Asia, Africa, South America) and Mosaic (Clade VI, VII). Clade 5 mostly comprises viruses from Africa and regions with emigrants from Africa [6]. In a study with 327 children with confirmed varicella or zoster, European origin clades were associated with more severe disease [7]. Furthermore, VZV viruses within each clade carry multiple SNPs, and some of them may cause varicella or zoster cases of sufficient severity that the patients seek medical care, mainly immunocompromised or critical patients. However, the consequences of most SNPs on severity and pathogenesis remain unknown. Mutations in ORF68 (gpE) have been associated with a higher virulence of strains [8, 9]. A single nucleotide change within codon 150, GAC to AAC, which in turn led to an amino acid change from aspartic acid to asparagine [10], produced the loss of a B-cell epitope crucial in the immune response to the virus. Furthermore, increased severity of human cases of infection with gE mutant viruses correlates with the data from the severe combine immunodeficient (SCID) mouse model of VZV infection [11].

Recently, the use of human tissue xenografts in murine models of infection demonstrated that deletions and/or mutations in genes such as ORF62 (transcriptional regulator ICP4, IE62 is major viral transactivator; IFN inhibition), ORF63 and 70 (IE63 phosphoprotein; represses IE62; transactivates EF-1α) as well as ORF11 (tegument protein; required for normal levels of IE4, IE62, IE63 and gE) cause impaired virus replication in the human skin [12].

Our study reports a fatal hemorrhagic varicella case in an immunocompetent adult without cutaneous rash and the genome characterization of this VZV strain by mWGS for the investigation of mutations which may confer virulence. All methods were carried out in accordance with relevant guidelines and UE regulations. Written informed consent for the publication of the data included in this case report was obtained from the patient’s wife. Ethical approval was given by Ethics Committee of the “Instituto de Salud Carlos III” (CEI PI 68_2017-v2).

Case Presentation and Clinical Setting

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.

Virological Testing

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.

Shotgun Metagenomic WGS

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).

Table 1 A. Quality of viral consensus genome: Assembly metrics and coverage stats. B. Metagenomics shotgun result
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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).

Table 2 Mutations found on OQ871571 VZV against reference DQ479954.1
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Table 3 VZV protein variants in VZV accession number OQ871571
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Discussion and Conclusions

Overall, the delicate balance between VZV immune evasion and host immune responses enables the virus to replicate and spread to naive individuals in a controlled manner. In its severe form, VZV infection can be fatal, especially in immunocompromised patients. However, a fatal outcome has also been described in immunocompetent individuals [21, 22]. Despite skin involvement, the symptoms of disseminated varicella also include multiple hemorrhages (including intracranial hemorrhage, hemorrhagic gastritis, hemorrhagic pulmonary edema, splenic rupture, adrenal hemorrhage, cystorrhagia and hyphema), encephalitis, pneumonia and abdominal pain [23]. Intense abdominal pain is often an early symptom of dissemination, which reveals that multi-system organs are involved, such as the stomach, bowel and spleen. Thrombocytopenia was observed in this case, which may have led to the occurrence of gastrorrhagia, cystorrhagia and eventually hemorrhagic shock.

Cortisol therapy modified the main events of the infection in some patients [24,25,26,27]. Corticosteroid treatment at the time of virus exposure may have led to hemorrhagic manifestations, acute liver failure and eventually death in the present case.

However, viral genetic features related to virulence could also be involved. Mutations in viral glycoprotein E impairing specific B-cell recognition were involved in some of such fatal outcomes [10] but they were not found in this case. Noticeably, analysis of whole genome revealed several non-synonymous mutations affecting essential genes as well as non-coding regions which recently have been described as significant in regulating the virus-host interaction. These variants included substitutions, insertions, duplications, in-frame deletions and disruptive deletions and insertions. Whether these mutations were related to the uncommon clinical presentation or not needs further study.

Some of these genes have been demonstrated to impair replication of virus in human skin xenografts in the SCID-hu mouse model of infection but have no effect on T cells when they have been deleted (ORF 62) or had mutation of phosphorylation sites (ORF63 and ORF70) [12]. Several mutations were found in gp13 (ORF11) in the present case that may have a high impact on protein functionality if they cause protein truncation, loss of function or triggering nonsense mediated decay. Gp13 is a tegument protein required for normal levels of IE4, IE62, IE63 and gE. Truncation of ORF11 causes impaired replication in skin in animal models of infection [12]. This finding might be associated with VZV disease without rash observed in this patient. Some other mutations are located in non-coding regions of ORF26, ORF28, ORF43, ORF58, ORF61, ORF62 and ORF64. These regions often contain sncRNAs and miRNAs, and some may regulate infection of host cells [28].

When VZV infection is suspected, viral DNA is usually detected by PCR. We applied whole-genome sequencing to further characterize the full genome of the strain. Metagenomic WGS could also be very useful for unbiassed detection and identification of unexpected etiological agents of disease when patients have no skin involvement, which may lead to misdiagnosis and poses diagnostic and therapeutic challenges, especially when some other disease manifestations exist. Testing consecutive sets of viruses by PCR can be time consuming, and, in cases of severe VZV disease, antiviral therapies, which may be effective in reducing disease severity, should be initiated along with supportive treatment as soon as possible to reduce mortality.