Neonatal herpes simplex virus (HSV) infection is a devastating disease with high mortality, particularly when disseminated. Studies in adults and children suggest that susceptibility to herpes simplex encephalitis (HSE) may represent phenotypes for inborn errors in toll-like receptor 3 (TLR3) signaling. However, the genetic basis of susceptibility to neonatal HSV including disseminated disease remains unknown. To test the hypothesis that variants in known HSE-susceptible genes as well as genes mediating HSV immunity will be identified in neonatal HSV, we performed an unbiased exome sequencing study in 10 newborns with disseminated, HSE, and skin, eyes, and mouth disease. Determination of potential impact on function was determined by following American College of Medical Genetics and Genomics guidelines. We identified deleterious and potentially deleterious, rare variants in known HSE-related genes including a stop IRF3 variant (disseminated), nonsynonymous variants in TLR3 and TRAF3 (HSE), STAT1 (skin, eyes, and mouth), and DBR1 (disseminated) in our cohort. Novel and rare variants in other immunodeficiency genes or HSV-related immune genes GRB2, RAG2, PRF1, C6, C7, and MSR1 were found in 4 infants. The variant in GRB2, essential for T-lymphocyte cell responses to HSV, is a novel stop variant not found in public databases. In this pilot study, we identified deleterious or potentially deleterious variants in TLR3 pathway and genes that regulate anti-HSV immunity in neonates with HSV including disseminated disease. Larger, definitive studies incorporating functional analysis of genetic variants are required to validate these data and determine the role of immune genetic variants in neonatal HSV susceptibility.

Neonatal herpes simplex virus (HSV) infection presents in the first 28 days of life as 1 of 3 subtypes: skin, eyes, and mouth (SEM), central nervous system (CNS), and disseminated disease. Disseminated HSV, defined as involvement of multiple organ systems, is the most devastating form. If left untreated, the mortality rate of disseminated HSV is as high as 80% to 90%.1  Despite implementation of risk factor–based screening, mortality rates have increased between 2004 and 2013, as compared to the previous 2 decades.2  This mandates newer approaches to identify infants susceptible to severe disease.

A major component of the host’s immune response to HSV relies on a family of innate immune receptors called toll-like receptors. By recognizing double-stranded RNA produced during HSV replication, toll-like receptor 3 (TLR3) signaling activates interferon regulating factor 3 (IRF3)– and interferon regulating factor 7–dependent type-1 interferon response, necessary for the host immune response.35  Enrichment of TLR3 in several resident cell types in the CNS ensures that brain HSV invasion is prevented by intact TLR3 signaling.3,4  Loss of function variants in the TLR3 signaling pathway genes, including TLR3, TRIF, TRAF3, TBK1, UNC93B, STAT1, and IRF3, have been identified in children and adults with herpes simplex encephalitis (HSE).4,69  Whether deleterious variants in TLR3 pathway genes or other known immunodeficiency genes underpin susceptibility to neonatal HSV phenotypes remains unknown. In addition, the genetic basis of disseminated HSV susceptibility has not been investigated. This is particularly important because neonates are immunocompromised hosts, and genetic variants are likely to be more penetrant, resulting in systemic phenotypes.10  Herein, we undertook an unbiased exome sequencing approach to test the hypothesis that novel or rare TLR3 pathway genetic variants will be identified in infants with neonatal HSV infections.

For detailed methods, please see the Supplemental Information.

Among the 10 patients enrolled in our study, the male/female ratio was 7:3, with gestational age ranging from 30 to 40 weeks (Table 1). There was no documented history of maternal HSV infection or suppressive therapy with valacyclovir during pregnancy in any of the infants. With the exception of one, all patients were diagnosed with HSV within the first 3 weeks of life. Our cohort included the following HSV phenotypes: 4 SEM, 1 intrauterine infection with CNS and SEM manifestations after birth, 1 CNS, and 4 disseminated disease. Of those 4 infants with disseminated disease, 2 had confirmed CNS involvement. Two of the 10 infants died, an infant died secondary to multiorgan failure related to disseminated HSV, and the family of an infant with HSE acquired in utero chose to redirect care because of severe brain anomalies. All 10 participants were treated with intravenous acyclovir at 20 mg/kg every 8 hours.

TABLE 1

Patient Characteristics

InfantSexRaceGADiagnosisDOL at DiagnosisHSV SerotypeHSV Blood PCR, ±HSV CSF PCR, ±HSV Surface Culture, ±Skin Lesion, ±Patient OutcomeaHSV History or Suppressive Therapy, ±Comments
Male AA 37 + 2/7 Disseminated N/A − Living − LP was attempted; no CSF was obtained. 
Male White 39 + 2/7 Disseminated − Living − — 
Male White 39 + 5/7 SEM 18 − − Living b Healing skin lesions were present. 
Male Biracial 39 SEM 56 + (86 × 108 copies per mL) − Living b SEM HSV with transient viremia was noted; no other signs of dissemination were noted. Skin lesions were present at 3 wk of age; infant was undiagnosed until DOL 56. 
Male White 40 + 2/7 Disseminated − Living − — 
Female White 38 SEM 10 N/A − Living b HSV PCR blood was not obtained; no other signs of dissemination were noted. 
Female AA 30 + 4/7 Disseminated N/A Deceased − HSV PCR blood was not obtained; evidence of dissemination was noted: DIC, respiratory failure, transaminitis, CNS involvement, and thrombocytopenia. 
Male AA 34 SEM − Living − HSV PCR blood result was positive. No other signs of dissemination were noted. 
Male White 31 + 4/7 Intrauterine (CNS and SEM) N/A Deceased − Infant died on DOL 10; redirection of care occurred. No LP was conducted per parental wishes. 
10 Female White 40 + 2/7 CNS N/A − Living b Patient with fever, elevated CRP, but no transaminitis or coagulopathy. No HSV PCR of blood was documented. 
InfantSexRaceGADiagnosisDOL at DiagnosisHSV SerotypeHSV Blood PCR, ±HSV CSF PCR, ±HSV Surface Culture, ±Skin Lesion, ±Patient OutcomeaHSV History or Suppressive Therapy, ±Comments
Male AA 37 + 2/7 Disseminated N/A − Living − LP was attempted; no CSF was obtained. 
Male White 39 + 2/7 Disseminated − Living − — 
Male White 39 + 5/7 SEM 18 − − Living b Healing skin lesions were present. 
Male Biracial 39 SEM 56 + (86 × 108 copies per mL) − Living b SEM HSV with transient viremia was noted; no other signs of dissemination were noted. Skin lesions were present at 3 wk of age; infant was undiagnosed until DOL 56. 
Male White 40 + 2/7 Disseminated − Living − — 
Female White 38 SEM 10 N/A − Living b HSV PCR blood was not obtained; no other signs of dissemination were noted. 
Female AA 30 + 4/7 Disseminated N/A Deceased − HSV PCR blood was not obtained; evidence of dissemination was noted: DIC, respiratory failure, transaminitis, CNS involvement, and thrombocytopenia. 
Male AA 34 SEM − Living − HSV PCR blood result was positive. No other signs of dissemination were noted. 
Male White 31 + 4/7 Intrauterine (CNS and SEM) N/A Deceased − Infant died on DOL 10; redirection of care occurred. No LP was conducted per parental wishes. 
10 Female White 40 + 2/7 CNS N/A − Living b Patient with fever, elevated CRP, but no transaminitis or coagulopathy. No HSV PCR of blood was documented. 

AA, African American; CRP, C-reactive protein; DIC, disseminated intravascular coagulation; DOL, day of life; GA, gestational age; LP, lumbar puncture; N/A, not available.

a

Outcomes at the time samples were obtained.

b

Distant history of cold sores in mother or father.

We identified several deleterious rare variants with a minor allele frequency (MAF) <1% within our cohort, both in genes previously associated with HSV and in other immune- or immunodeficiency genes (Table 2). Functional annotation of the identified variants using commonly used prediction software is shown in Supplemental Table 4.

TABLE 2

Variants in Viral-Sensing Immunodeficiency Genes

CaseDiagnosisGeneVariantImpactProtein ChangePopulation MAF,a %ACMG Class
Disseminated IRF3b rs149842990 Premature stop R411Ter 0.03 
 — MSR1 rs72552387 NSV D174Y 0.22 
SEM PRF1 rs12161733 NSV R4C 0.11 
SEM STAT1b rs148775168 NSV 1265V 0.07 
Disseminated DBR1 rs146690949 NSV D349G 0.0003 
Disseminated GRB2 rs759793650 Premature stop R112Ter Novel 
 — RAG2 rs34629171 NSV F386L 0.97 
Intrauterine (CNS and SEM manifestations) TLR3b rs112077022 NSV D280N 0.15 
 — TRAF3b rs567455701 Deletion — 0.17 
10 CNS C6 rs76202909 Splice donor — 0.23 
 — C7 rs121964920 NSV R521S 0.25 
CaseDiagnosisGeneVariantImpactProtein ChangePopulation MAF,a %ACMG Class
Disseminated IRF3b rs149842990 Premature stop R411Ter 0.03 
 — MSR1 rs72552387 NSV D174Y 0.22 
SEM PRF1 rs12161733 NSV R4C 0.11 
SEM STAT1b rs148775168 NSV 1265V 0.07 
Disseminated DBR1 rs146690949 NSV D349G 0.0003 
Disseminated GRB2 rs759793650 Premature stop R112Ter Novel 
 — RAG2 rs34629171 NSV F386L 0.97 
Intrauterine (CNS and SEM manifestations) TLR3b rs112077022 NSV D280N 0.15 
 — TRAF3b rs567455701 Deletion — 0.17 
10 CNS C6 rs76202909 Splice donor — 0.23 
 — C7 rs121964920 NSV R521S 0.25 

ACMG, American College of Medical Genetics and Genomics; C6, complement 6; C7, complement 7; —, not applicable.

a

From gnomAD.

b

Genes that have been previously implicated in cutaneous or HSE in children and adults.

Infant 1, with disseminated HSV-2, was heterozygous for a stop variant in IRF3, which truncates the protein. IRF3 is a key transcription factor, which regulates protective immunity against HSV by inducing a type-1 interferon response.11  In 2 previous studies, authors have reported single-nucleotide polymorphisms in IRF3 that impair TLR3-induced IRF3 function and are inherited in an autosomal dominant manner.8,11  Thus, the heterozygous IRF3 variant identified in our study could impair functional responses and contribute to HSV vulnerability. In addition, this patient had a rare variant in the MSR1 gene, which encodes macrophage scavenger receptor 1 (MSR1). MSR1 recognizes extracellular double-stranded RNA such as HSV-1 and mediates its endocytosis. It then engages with TLR3 and triggers the TLR3 signaling pathway. Suzuki et al12  found that MSR1 knock-out variant mice had increased susceptibility to HSV infection.13 

Infant 7 with disseminated HSV disease who succumbed to end-organ failure had a novel (not found in Genome Aggregation Database [gnomAD]), premature stop variant in GRB2 that truncates the protein at amino acid 112. Growth factor receptor bound protein 2 (GRB2) is important for lymphocyte antiviral immunity.14  Strunk et al15  found that the HSV tegument protein virion phosphoprotein 11/12 contains tyrosine-based motifs that bind to a specific domain of GRB2 at position 633. A variant at Y633 abolishes the interaction between virion phosphoprotein 11/12 and GRB2, required for downstream antiviral signaling.15 Our patient had truncation of the protein upstream of Y633, indicating the potential for poor recognition of the virus in this patient. Interestingly, this infant also had a variant in RAG2, a gene implicated in autosomal-recessive severe combined immunodeficiency and essential to T-cell function, which is necessary to combat infection from HSV.16 

In infant 5, also with disseminated HSV, we identified a rare nonsynonymous variant (NSV) in DBR1, a gene that encodes an RNA lariat debranching enzyme. Zhang et al17  reported 2 children with HSE found to have hypomorphic variants (partial loss of function) in DBR1. Through functional studies, Zhang et al17  confirmed mutations in DBR1 as a genetic etiology of brainstem viral encephalitis. Although (debranching RNA lariat 1) DBR1 is expressed most strongly in the brainstem and spinal cord, its expression is ubiquitous and therefore could possibly increase susceptibility to disseminated HSV. The HSV polymerase chain reaction (PCR) result of the cerebrospinal fluid (CSF) was negative in infant 5 indicating the absence of encephalitis despite disseminated infection. Whether mutations in DBR1 can cause disseminated disease in immunocompromised neonates needs to be examined in larger studies. In the remaining infant with disseminated HSV, infant 2, we did not identify any pathogenic variants.

In the infants with SEM disease (n = 4), infant 3 had a missense variant in PRF1, a gene implicated in familial hemophagocytic lymphohistiocytosis type 2 and essential for antiviral cytotoxic responses. Interestingly, 30% of infants with a familial hemophagocytic lymphohistiocytosis diagnosis in the neonatal period present with HSV infection.18  Infant 4, also with SEM disease, had a rare single-nucleotide variant in STAT1. STAT1 is a major regulator of innate and adaptive immunity to intracellular bacteria and viruses, and heterozygous gain of function mutations in STAT1 is associated with cutaneous HSV.9  Although a causal role for the STAT1 I126V mutation in our proband with SEM cannot be established without further genetic evidence, our data are consistent with previous studies implying a role for STAT1 in immunodeficiency and HSV.9  Infants 6 and 8 with SEM disease did not have any pathogenic variants identified.

Among infants with CNS disease, infant 9 was born at 31 + 4/7 weeks’ gestational age with a prenatal diagnosis of lissencephaly. A postnatal MRI confirmed diffuse cystic encephalomalacia presumably related to an in utero vascular or infectious event. On day of life 4, he developed a vesicular rash, and the result from the HSV PCR obtained from the lesions and blood was positive for HSV-2. A lumbar puncture was not performed because of his critical status and parents’ wish for comfort care. The patient’s extensive encephalomalacia and presence of seizure activity were consistent with in utero HSV infection. The vesicular lesions manifested after birth are also within the clinical spectrum of in utero HSV infection.19  This infant had 2 rare variants in TLR3 and TRAF3, which are both genes previously implicated in susceptibility to childhood HSE.4,7  In a review, Mielcarska et al4  highlight several variants identified in the TLR3 gene, with both autosomal-recessive and dominant patterns of inheritance identified in patients diagnosed with childhood HSE. Given the increased susceptibility to developing HSE with TLR3 variants, it is likely that this infant was infected in utero, resulting in his severe presentation. TRAF3 is a key adapter downstream of TLR3 required for optimal anti-HSV immunity, and autosomal dominant, hypomorphic mutations are implicated in childhood HSE.20  Infant 10, also with CNS disease, had heterozygous variants in complement genes C6 and C7. Although these genes are typically implicated in susceptibility to meningococcal disease, a role for the complement system in anti-HSV immunity has been reported previously in a single case report from 1 family, as well as in animal studies.21,22 

In the first hypothesis-generating pilot studies, we report the presence of deleterious gene variants in the TLR3 pathway, as well as MSR1, DBR1, and other viral-sensing genes, in neonatal disseminated and other HSV phenotypes. Although there were no identical variants in our cohort, 5 of the genes identified (IRF3, TLR3, TRAF3, STAT1, DBR1) have been linked to HSV in childhood and adults. Importantly, TLR3 pathway variants have only been reported in the context of CNS or SEM disease in children and adults but not in disseminated disease. We identified 2 stop variants in separate infants both with disseminated HSV: a rare stop variant in the IRF3 gene and an interesting novel stop variant in GRB2, a gene essential for T-cell responses to HSV. Our study has limitations, including a small cohort of 10 neonates. Given the low prevalence of neonatal HSV disease, a longitudinal multicenter study would be required for further investigation. The addition of parental and familial genotyping would have been helpful in classifying variants as inherited versus de novo. In addition, completion of functional studies in cell lines and analysis of peripheral blood mononuclear cell responses to TLR3 ligands would have strengthened our conclusions. Although these data are hypothesis generating, we speculate that disseminated neonatal HSV may be a phenotype for immunodeficiency arising from variants in the TLR3 pathway and other viral-sensing immune genes. In contrast to adults, the immunocompromised infant may be much more susceptible to disseminated disease as opposed to CNS disease in the presence of deleterious genetic variants. Identification of candidate genes for neonatal HSV susceptibility may allow screening-based approaches for disease prevention in the future. Whether infants with neonatal HSV should be evaluated for mutations in immunodeficiency genes is a pressing question that needs further research.

We thank Heather Menden, MS, and Wei Yu, PhD, for their time and effort with the DNA extraction. Dr Cummings also thanks Joshua Petrikin, MD, and Abedayo Oshodi, MD, as members of her scholarship oversight committee, for their support in my project. Without your help and guidance, this would not be possible. Lastly, we thank our patients and their parents for their participation.

Dr Cummings conceptualized and designed the study, designed the data collection instruments, collected data, conducted the initial analyses and interpretation, drafted the initial manuscript, and reviewed and revised the manuscript; Dr Tucker collected data and reviewed and revised the manuscript; Ms Gibson and Mr Johnston collected data, conducted the initial analyses and interpretation, drafted the initial manuscript, and reviewed and revised the manuscript; Drs Myers and Farrow conducted the initial analyses and interpretation, drafted the initial manuscript, and reviewed and revised the manuscript; Dr Pastinen provided oversight for the genetic analysis and reviewed the data and final manuscript; Dr Sampath conceptualized and designed the study, coordinated and supervised data collection, drafted the initial manuscript, and critically reviewed and revised the manuscript for important intellectual content; and all authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

FUNDING: Supported by institutional funds awarded to Dr Sampath.

CNS

central nervous system

CSF

cerebrospinal fluid

DBR1

debranching RNA lariat 1

gnomAD

Genome Aggregation Database

GRB2

growth factor receptor bound protein 2

HSE

herpes simplex encephalitis

HSV

herpes simplex virus

IRF3

interferon regulatory factor 3

MAF

minor allele frequency

MSR1

macrophage scavenger receptor 1

NSV

nonsynonymous variant

PCR

polymerase chain reaction

SEM

skin, eyes, and mouth

TLR3

toll-like receptor 3

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Competing Interests

POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.

FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose.

Supplementary data