Evidence of varicella zoster virus (VZV) reactivation in children with arterial ischemic stroke: Results of the VIPS II Study.

Background: Varicella zoster virus (VZV) has been associated with focal cerebral arteriopathy (FCA) and arterial ischemic stroke (AIS) in childhood. The Vascular effects of Infection in Pediatric Stroke (VIPS) II study aimed to examine this relationship in the modern era when most children in North America and Australia receive VZV vaccination with live, attenuated virus. Methods: This 22-center prospective cohort study enrolled 205 children (28 days-18 years) with AIS (2017-2022), collected baseline [hyperacute (≤72 hours; n=194) and acute (4-6 days; n=181)] and convalescent (1-6 weeks; n=74) serum samples. Sites enrolled 95 stroke-free controls with single serum samples. A virology research laboratory measured VZV IgM and IgG titers by an in-house enzyme-linked immunosorbent assay (ELISA). Baseline IgG seropositivity indicated prior exposure (vaccination/infection) and elevated IgM titers indicated recent reactivation. Results: Median (IQR) age was 11.6 (5.5-15.6) years for cases and 11.8 (6.8-15.3) years for controls. Baseline serologies indicated prior VZV exposure in 198 cases (97%) and all controls. Parents of cases reported VZV vaccination in 160 (78%) and remote chicken pox in three (1.4%). Twenty cases (9.8%) and three controls (3.1%) had serologic evidence of recent VZV reactivation (p=0.06); all had remote VZV exposure (vaccination in 19 cases and all controls) and all were asymptomatic. Recent VZV reactivation was seen in similar proportions in arteriopathic, cardioembolic, and idiopathic stroke. Of 32 cases of FCA, 4 (12.5%) had recent VZV reactivation, versus no cases of arterial dissection (n=10) or moyamoya (n=16). Conclusions: Serologic evidence of recent VZV reactivation (≈1-6 weeks prior to stroke) was present in one in 10 cases of childhood AIS, including those without arteriopathy. Clinically silent VZV reactivation may be a childhood stroke trigger despite widespread vaccination. These cases could represent waning immunity with reactivation of either vaccine virus or wild-type virus after an unrecognized secondary VZV infection.

Serious neurological adverse events in immunocompetent children and adolescents caused by viral reactivation in the years following varicella vaccination.

Serious adverse events following vaccination include medical complications that require hospitalisation. The live varicella vaccine that was approved by the Food and Drug Administration in the United States in 1995 has an excellent safety record. Since the vaccine is a live virus, adverse events are more common in immunocompromised children who are vaccinated inadvertently. This review includes only serious adverse events in children considered to be immunocompetent. The serious adverse event called varicella vaccine meningitis was first reported in a hospitalised immunocompetent child in 2008. When we carried out a literature search, we found 15 cases of immunocompetent children and adolescents with varicella vaccine meningitis; the median age was 11 years. Eight of the children had received two varicella vaccinations. Most of the children also had a concomitant herpes zoster rash, although three did not. The children lived in the United States, Greece, Germany, Switzerland, and Japan. During our literature search, we found five additional cases of serious neurological events in immunocompetent children; these included 4 cases of progressive herpes zoster and one case of acute retinitis. Pulses of enteral corticosteroids as well as a lack of herpes simplex virus antibody may be risk factors for reactivation in immunocompetent children. All 20 children with adverse events were treated with acyclovir and recovered; 19 were hospitalised and one child was managed as an outpatient. Even though the number of neurological adverse events remains exceedingly low following varicella vaccination, we recommend documentation of those caused by the vaccine virus.

Common Features Between Stroke Following Varicella in Children and Stroke Following Herpes Zoster in Adults : Varicella-Zoster Virus in Trigeminal Ganglion.

The cerebral arteries are innervated by afferent fibers from the trigeminal ganglia. Varicella-zoster virus (VZV) frequently resides in the trigeminal ganglion. Reports of arterial ischemic stroke due to VZV cerebral vasculopathy in adults after herpes zoster have been described for decades. Reports of arterial ischemic stroke due to post-varicella cerebral arteriopathy in children have also been described for decades. One rationale for this review has been post-licensure studies that have shown an apparent protective effect from stroke in both adults who have received live zoster vaccine and children who have received live varicella vaccine. In this review, we define common features between stroke following varicella in children and stroke following herpes zoster in adults. The trigeminal ganglion and to a lesser extent the superior cervical ganglion are central to the stroke pathogenesis pathway because afferent fibers from these two ganglia provide the circuitry by which the virus can travel to the anterior and posterior circulations of the brain. Based on studies in pseudorabies virus (PRV) models, it is likely that VZV is carried to the cerebral arteries on a kinesin motor via gE, gI and the homolog of PRV US9. The gE product is an essential VZV protein.

The Role of Infection and Inflammation in the Pathogenesis of Pediatric Arterial Ischemic Stroke.

Infections play an important role in the pathogenesis of acute ischemic stroke (AIS) in neonates and children. In neonates, chorioamnionitis or intrauterine inflammation has been implicated as a common risk factor for AIS. In infants and children, recent investigations demonstrated that even minor childhood infections are associated with subsequent increased risk for AIS. Post-infectious inflammatory mechanisms following infections with herpesviruses may lead to focal cerebral arteriopathy (FCA), one of the most common causes of AIS in a previously healthy child. Other agents such as parvovirus B19, dengue virus, and SARS-CoV-2 have recently been implicated as other potential triggers. Infections are compelling treatable stroke risk factors, with available therapies for both pathogens and downstream inflammatory effects. However, infections are common in childhood, while stroke is uncommon. The ongoing VIPS II (Vascular effects of Infection in Pediatric Stroke) study aims to identify the array of pathogens that may lead to childhood AIS and whether either unusual strains or unusual combinations of pathogens explain this paradox. Immune modulation with corticosteroids for FCA is another active area of research, with European and U.S. trials launching soon. The results of these new pediatric stroke studies combined with findings emerging from the larger field of immune-mediated post-infectious diseases will likely lead to new approaches to the prevention and treatment of pediatric stroke. This review highlights recent developments from both clinical and animal model research enhancing our understanding of this relationship between infection, inflammation, and stroke in neonates and children.

Reassessment of Evidence about Coinfection of Chickenpox and Monkeypox (Mpox) in African Children.

In west and central Africa, monkeypox occurs mainly in older children, adolescents and young adults. In two large epidemiology studies of monkeypox outbreaks, the investigators observed a sizable number of coinfections of chickenpox (varicella) and monkeypox. Based on a review of the literature, we propose that chickenpox (human herpesvirus-3 infection) is a risk factor for acquisition of monkeypox infection. Our hypothesis states that the chickenpox skin lesion provides an entry site for the monkeypox virus, which is harbored on a fomite in the environment of the patient. The fact that monkeypox can enter via a scratch or abrasion is a known mechanism of spread for three other poxviruses, including mousepox (ectromelia), orf and molluscum contagiosum. There are many similarities in pathogenesis between certain poxviruses and chickenpox, including a viremia with a cellular stress response leading to high levels of the IL-6 cytokine. One very revealing observation in the two epidemiology studies was that the number of pox as well as the severity of disease in children with chickenpox and monkeypox coinfection was not greater than found in children with monkeypox alone. Based on the above observations, we conclude that, when chickenpox precedes monkeypox, priming of the immune system by the earlier chickenpox infection moderates the severity of the secondary infection with monkeypox. This conclusion also has important public health implications about chickenpox surveillance.

The Enduring Legacy of Randall Cohrs: A Meeting of the Minds in the Rocky Mountains.

Randall Cohrs established the Colorado Alphaherpesvirus Latency Society (CALS) in 2011 [...].

Meningitis Caused by the Live Varicella Vaccine Virus: Metagenomic Next Generation Sequencing, Immunology Exome Sequencing and Cytokine Multiplex Profiling.

Varicella vaccine meningitis is an uncommon delayed adverse event of vaccination. Varicella vaccine meningitis has been diagnosed in 12 children, of whom 3 were immunocompromised. We now report two additional cases of vaccine meningitis in twice-immunized immunocompetent children and we perform further testing on a prior third case. We used three methods to diagnose or investigate cases of varicella vaccine meningitis, none of which have been used previously on this disease. These include metagenomic next-generation sequencing and cytokine multiplex profiling of cerebrospinal fluid and immunology exome analysis of white blood cells. In one new case, the diagnosis was confirmed by metagenomic next-generation sequencing of cerebrospinal fluid. Both varicella vaccine virus and human herpesvirus 7 DNA were detected. We performed cytokine multiplex profiling on the cerebrospinal fluid of two cases and found ten elevated biomarkers: interferon gamma, interleukins IL-1RA, IL-6, IL-8, IL-10, IL-17F, chemokines CXCL-9, CXCL-10, CCL-2, and G-CSF. In a second new case, we performed immunology exome sequencing on a panel of 356 genes, but no errors were found. After a review of all 14 cases, we concluded that (i) there is no common explanation for this adverse event, but (ii) ingestion of an oral corticosteroid burst 3-4 weeks before onset of vaccine meningitis may be a risk factor in some cases.

Corticosteroids Contribute to Serious Adverse Events Following Live Attenuated Varicella Vaccination and Live Attenuated Zoster Vaccination.

Corticosteroids, when given in high dosages, have long been recognized as a risk factor for severe infection with wild-type varicella-zoster virus in both children and adults. The goal of this review is to assess the degree to which both low-dosage and high-dosage corticosteroids contribute to serious adverse events (SAEs) following live varicella vaccination and live zoster vaccination. To this end, we examined multiple published reports of SAEs following varicella vaccination (VarivaxTM) and zoster vaccination (ZostavaxTM). We observed that five of eight viral SAEs following varicella vaccination, including two deaths, occurred in children receiving corticosteroids, while one of three fatal viral SAEs following live zoster vaccination occurred in an adult being treated with low-dosage prednisone. The latter death after live zoster vaccination occurred in a 70 year-old man with rheumatoid arthritis, being treated with prednisone 10 mg daily. Thus, corticosteroids contributed to more severe infectious complications in subjects immunized with each of the two live virus vaccines. Further, when we surveyed the rheumatology literature as well as individual case reports, we documented examples where daily dosages of 7.5-20 mg prednisone were associated with increased rates of severe wild-type varicella-zoster virus infections in children and adults.

Twelve Children with Varicella Vaccine Meningitis: Neuropathogenesis of Reactivated Live Attenuated Varicella Vaccine Virus.

Varicella vaccine is a live attenuated varicella-zoster virus (VZV). Like its parental strain called VZV pOka, the vaccine virus vOka retains some neurotropic properties. To better understand vOka neuropathogenesis, we reassessed 12 published cases of vOka meningitis that occurred in once-immunized and twice-immunized children, all of whom had bouts of herpes zoster preceding the central nervous system infection. Eight of the 12 meningitis cases occurred in children who had received only one immunization. There was no pattern to the time interval between varicella vaccination and the onset of herpes zoster with meningitis. Four of the meningitis cases occurred in children who had received two immunizations. Since all four children were 14 years old when meningitis was diagnosed, there was a strong pattern to the interval between the first vaccination at age 1 year and onset of meningitis, namely, 13 years. Knowledge of pathogenesis requires knowledge of the location of herpes zoster; the majority of dermatomal rashes occurred at sites of primary immunization on the arm or thigh, while herpes zoster ophthalmicus was uncommon. Based on this literature review, currently there is no consensus as to the cause of varicella vaccine meningitis in twice-immunized children.

Varicella Vaccine Meningitis as a Complication of Herpes Zoster in Twice-Immunized Immunocompetent Adolescents.

Varicella-zoster virus vaccination is recommended for virtually all young children in the United States, Canada, and several other countries. Varicella vaccine is a live attenuated virus that retains some of its neurotropic properties. Herpes zoster caused by vaccine virus still occurs in immunized children, although the rate is much lower than in children who had wild-type varicella. It was commonly thought that 2 varicella vaccinations would protect children against the most serious complication of meningitis following herpes zoster; however, 2 meningitis cases have already been published. We now report a third case of varicella vaccine meningitis and define risk factors shared by all 3 immunized adolescents. The diagnosis in cerebrospinal fluid in this third case was verified by amplifying and sequencing portions of the viral genome, to document fixed alleles found only in the vaccine strain. Viral antibody was also detected in the cerebrospinal fluid by confocal microscopy. When compared with the other 2 cases, remarkably all 3 were 14 years old when meningitis occurred. All 3 were treated with intravenous acyclovir, with complete recovery. The adolescent in our case report also had recurrent asthma, which was treated with both prednisone tablets and beclomethasone inhaler before onset of meningitis. When the 3 cases were considered together, they suggested that immunity to varicella-zoster virus may be waning sufficiently in some twice-immunized adolescents to make them vulnerable to varicella vaccine virus reactivation and subsequent meningitis. This complication rarely happens in children after wild-type varicella.

Exocytosis of Progeny Infectious Varicella-Zoster Virus Particles via a Mannose-6-Phosphate Receptor Pathway without Xenophagy following Secondary Envelopment.

The literature on the egress of different herpesviruses after secondary envelopment is contradictory. In this report, we investigated varicella-zoster virus (VZV) egress in a cell line from a child with Pompe disease, a glycogen storage disease caused by a defect in the enzyme required for glycogen digestion. In Pompe cells, both the late autophagy pathway and the mannose-6-phosphate receptor (M6PR) pathway are interrupted. We have postulated that intact autophagic flux is required for higher recoveries of VZV infectivity. To test that hypothesis, we infected Pompe cells and then assessed the VZV infectious cycle. We discovered that the infectious cycle in Pompe cells was remarkably different from that of either fibroblasts or melanoma cells. No large late endosomes filled with VZV particles were observed in Pompe cells; only individual viral particles in small vacuoles were seen. The distribution of the M6PR pathway (trans-Golgi network to late endosomes) was constrained in infected Pompe cells. When cells were analyzed with two different anti-M6PR antibodies, extensive colocalization of the major VZV glycoprotein gE (known to contain M6P residues) and the M6P receptor (M6PR) was documented in the viral highways at the surfaces of non-Pompe cells after maximum-intensity projection of confocal z-stacks, but neither gE nor the M6PR was seen in abundance at the surfaces of infected Pompe cells. Taken together, our results suggested that (i) Pompe cells lack a VZV trafficking pathway within M6PR-positive large endosomes and (ii) most infectious VZV particles in conventional cell substrates are transported via large M6PR-positive vacuoles without degradative xenophagy to the plasma membrane.IMPORTANCE The long-term goal of this research has been to determine why VZV, when grown in cultured cells, invariably is more cell associated and has a lower titer than other alphaherpesviruses, such as herpes simplex virus 1 (HSV1) or pseudorabies virus (PRV). Data from both HSV1 and PRV laboratories have identified a Rab6 secretory pathway for the transport of single enveloped viral particles from the trans-Golgi network within small vacuoles to the plasma membrane. In contrast, after secondary envelopment in fibroblasts or melanoma cells, multiple infectious VZV particles accumulated within large M6PR-positive late endosomes that were not degraded en route to the plasma membrane. We propose that this M6PR pathway is most utilized in VZV infection and least utilized in HSV1 infection, with PRV's usage being closer to HSV1's usage. Supportive data from other VZV, PRV, and HSV1 laboratories about evidence for two egress pathways are included.

Personalized viral genomic investigation of herpes simplex virus 1 perinatal viremic transmission with dual fatality.

Here we present a personalized viral genomics approach to investigating a rare case of perinatal herpes simplex virus 1 (HSV-1) transmission that ended in death of both mother and neonate. We sought to determine whether the virus involved in this rare case had any unusual features that may have contributed to the dire patient outcome. A pregnant woman with negative HerpeSelect antibody test underwent cesarean section at 30 wk gestation and died the same day. The premature newborn died 5 d later. Both individuals were found postmortem to have positive blood HSV-1 PCR tests. Using oligonucleotide enrichment and deep sequencing, we determined that viral transmission from mother to infant was nearly perfect at the consensus genome level. At the virus population level, 77% of minor variants (MVs) in the mother's blood also appeared on the neonate's skin, of which more than half were disseminated into the neonate's blood. We also detected nonmaternal MVs that arose de novo in the neonate's viral populations. Of note, one de novo MV in the neonate's skin virus induced a nonsynonymous mutation in the UL6 protein, which is a component of the portal that allows DNA entry into new progeny capsids. This case suggests that perinatal viremic HSV-1 transmission includes the majority of genetic diversity from the maternal virus population and that new, nonsynonymous mutations can occur after relatively few rounds of replication. This report expands our understanding of viral transmission in humans and may lead to improved diagnostic strategies for neonatal HSV-1 acquisition.

Progeny Varicella-Zoster Virus Capsids Exit the Nucleus but Never Undergo Secondary Envelopment during Autophagic Flux Inhibition by Bafilomycin A1.

Varicella-zoster virus (VZV) is an alphaherpesvirus that lacks the herpesviral neurovirulence protein ICP34.5. The underlying hypothesis of this project was that inhibitors of autophagy reduce VZV infectivity. We selected the vacuolar proton ATPase inhibitor bafilomycin A1 for analysis because of its well-known antiautophagy property of impeding acidification during the late stage of autophagic flux. We documented that bafilomycin treatment from 48 to 72 h postinfection lowered VZV titers substantially (P ≤ 0.008). Because we were unable to define the site of the block in the infectious cycle by confocal microscopy, we turned to electron microscopy. Capsids were observed in the nucleus, in the perinuclear space, and in the cytoplasm adjacent to Golgi apparatus vesicles. Many of the capsids had an aberrant appearance, as has been observed previously in infections not treated with bafilomycin. In contrast to prior untreated infections, however, secondary envelopment of capsids was not seen in the trans-Golgi network, nor were prototypical enveloped particles with capsids (virions) seen in cytoplasmic vesicles after bafilomycin treatment. Instead, multiple particles with varying diameters without capsids (light particles) were seen in large virus assembly compartments near the disorganized Golgi apparatus. Bafilomycin treatment also led to increased numbers of multivesicular bodies in the cytoplasm, some of which contained remnants of the Golgi apparatus. In summary, we have defined a previously unrecognized property of bafilomycin whereby it disrupted the site of secondary envelopment of VZV capsids by altering the pH of the trans-Golgi network and thereby preventing the correct formation of virus assembly compartments.IMPORTANCE This study of VZV assembly in the presence of bafilomycin A1 emphasizes the importance of the Golgi apparatus/trans-Golgi network as a platform in the alphaherpesvirus life cycle. We have previously shown that VZV induces levels of autophagy far above the basal levels of autophagy in human skin, a major site of VZV assembly. The current study documented that bafilomycin treatment led to impaired assembly of VZV capsids after primary envelopment/de-envelopment but before secondary reenvelopment. This VZV study also complemented prior herpes simplex virus 1 and pseudorabies virus studies investigating two other inhibitors of endoplasmic reticulum (ER)/Golgi apparatus function: brefeldin A and monensin. Studies with porcine herpesvirus demonstrated that primary enveloped particles accumulated in the perinuclear space in the presence of brefeldin A, while studies with herpes simplex virus 1 documented an impaired secondary assembly of enveloped viral particles in the presence of monensin.