Mutations in Coding and Non-Coding Regions in Varicella-Zoster Virus Causing Fatal Hemorrhagic Fever Without Rash in an Immunocompetent Patient: Case Report

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).

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.

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 [24‐27]. 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.