Entry receptor bias in evolutionarily distant HSV-1 clinical strains drives divergent ocular and nervous system pathologies

Abstract

Purpose

Herpes simplex virus-1 (HSV-1) infection leads to varying pathologies including the development of ocular lesions, stromal keratitis and encephalitis. While the role for host immunity in disease progression is well understood, the contribution of genetic variances in generating preferential viral entry receptor usage and resulting immunopathogenesis in humans are not known.

Methods

Ocular cultures were obtained from patients presenting distinct pathologies of herpes simplex keratitis (HSK). Next-generation sequencing and subsequent analysis characterized genetic variances among the strains and estimated evolutionary divergence. Murine model of ocular infection was used to assess phenotypic contributions of strain variances on damage to the ocular surface and propagation of innate immunity. Flow cytometry of eye tissue identified differential recruitment of immune cell populations, cytokine array probed for programming of local immune response in the draining lymph node and histology was used to assess inflammation of the trigeminal ganglion (TG). Ex-vivo corneal cultures and in-vitro studies elucidated the role of genetic variances in altering host-pathogen interactions, leading to divergent host responses.

Results

Phylogenetic analysis of the clinical isolates suggests evolutionary divergence among currently circulating HSV-1 strains. Mutations causing alterations in functional host interactions were identified, particularly in viral entry glycoproteins which generated a receptor bias to herpesvirus entry mediator, an immune modulator involved in immunopathogenic diseases like HSK, leading to exacerbated ocular surface pathologies and heightened viral burden in the TG and brainstem.

Conclusions

Our data suggests receptor bias resulting from genetic variances in clinical strains may dictate disease severity and treatment outcome.

Introduction

Herpes simplex virus 1 (HSV-1) is a well-adapted, highly conserved human pathogen with high worldwide prevalence (1). HSV-1 is a double-stranded DNA virus belonging to the Alphaherpesvirinae subfamily of Herpesviridae which consists of HSV-1, HSV-2 and varicella zoster virus (VZV). HSV-1 is particularly associated with ocular pathologies and manifests as keratoconjunctivitis that may be unilateral or, in rare cases, bilateral. Progressive HSV infection results in scarring of the ocular surface and keratitis or branching dendritic ulcers, a major cause of blindness worldwide (1–3). In addition, HSV-1 can progress to cause rare but life-threatening encephalitis (4). HSV-1 establishes latency in sensory ganglia neurons, where the virus remains dormant awaiting opportunities of lytic reactivation causing epithelial lesions. Nucleoside analogs are commonly prescribed to block active viral replication and subside symptoms, however, once a latent reservoir is established, therapy options are limited leading to lifelong infection (5). There is a growing need for novel therapeutics particularly in the case of drug resistant HSV strains and in the case of patients experiencing severe disease like neurotrophic keratitis (6–8).

Patients presenting HSV-1 ocular infection exhibit varying degrees of pathologies (9–11). The clinical course depends on multiple factors involving the innate resistance, host immune response and genetic makeup of the virus (12). HSV-1 strains have geographically evolved, forming clusters in at least three major clades with differences of 2 to 4% in the viral genomes (13, 14). Different virulence patterns are observed among these strains as well as ranges in the severity of keratitis. In murine studies, different strains of HSV-1 demonstrate varying pathologies from no apparent disease to severe corneal surface damage or even lethal encephalitis (15, 16). However, the differential contributions caused by viral strain-dependent genetic variations especially in regard to viral entry are poorly characterized. Recently, high-throughput sequencing has allowed for genome-wide evolutionary studies in the viral population being transmitted between different hosts (14, 17–19). However, only a limited analysis of global HSV-1 sequence diversity has been performed and studies on the genetic correlation of varying clinical manifestations are not well supported. Furthermore, how emergence of new viral strains drives antigenic evolution and influences infectivity, transmissibility and host reactivity to these variants remains unclear (14). Lack of such knowledge has negatively impacted development of vaccines or more effective therapies against the virus. Therefore, studies on pathogen genomics and the impact of viral genetic variants are critical for therapeutic development as well as predicting prognosis of herpes simplex virus disease in patients.

In this study we provide whole genome sequence analysis of two new clinical isolates of ocular HSV-1 from human patients and demonstrate the large degree of coding sequence variability that lead to divergent host responses and ultimately disease outcome. Particularly, we found mutations in the glycoproteins that generated an acquired receptor usage bias, leading to significant differences in entry receptor-dependent phenomena such entry kinetics, cell-to-cell spread, viral plaque formation, and immune signaling. Sequence analysis demonstrated that glycoprotein D of one particular strain yields a favorable interaction with Herpesvirus Entry Mediator (HVEM), a member of the tumor necrosis family that has been shown to mediate ocular immunopathogenesis through the induction of T cell response that promotes HSK. Ocular murine infection with this clinical isolate displayed unique properties of heightened virulence and neuroinvasiveness, characterized by a robust T cell response, accompanied by other immune cell infiltrates leading to loss of corneal integrity and dissemination to the central nervous system, causing eventual mortality. While receptor bias has not been previously shown to have clinical relevance, we show that ocular HSV-1 isolates encompass genetic variations leading to preferential entry receptor interactions and ultimately influencing disease outcome and immunopathogenesis.

Results

Isolation of clinical strains from ocular material of patients presenting either unilateral or bilateral herpetic keratitis.

Bilateral herpetic keratitis is rare and only present in about 2-12% of ocular HSV infections (20). A patient, 61-year-old male, initially presented in clinic with a pseudomonas ulcer in the right eye (Fig. 1A) and after two days of treatment with antibiotics, a geographic ulcer indicative of HSV keratitis developed in the left eye (Fig. 1B). Prior to the visit, this patient was diagnosed with graft-versus-host disease (GVHD) and taking multiple immunosuppressive medications including Vemlidy, preniSONE, Methylprednisone, Levothyroxine and hydrocortisone. Ophthalmic examination conducted after initial symptoms began developing included fluorescein staining and slit-lamp images which clearly demonstrate presence of HSV through the observed geographic ulcer, which is indicative of damage to the ocular surface as a result of replicating virus. Both eyes began responding to treatment with Valacyclovir and continued to heal after 14 days (Fig 1C, D). Ocular material from both the right (BL.R strain) and left (BL.L strain) eyes of this patient with bilateral infection was collected and cultured. In another case, ocular wash was collected from a female patient with persistent unilateral infection (UL strain) in the right eye that presented extreme light sensitivity, increased ocular discomfort and severe tear deficiency. Prior to visit, this patient was diagnosed with GVHD and taking medications including prednisone, spironolactone and tacrolimus. During ophthalmic examination, fluorescein staining and slit-lamp images show developed geographic ulcer, consistent with herpes keratitis (Fig. 1E). The ocular surface disease index (OSDI) was measured to be 87.5. The patient was treated with Valacyclovir and the infection was resolved after 10 days (Fig. 1F). Both patients were diagnosed with GVHD and were prescribed immunosuppressive medications; however, history regarding recurrence of ocular HSV is unknown. While both patients exhibited similar medical conditions, each presented different manifestations of ocular HSV with varying symptoms and responses to therapy.

Figure 1. Infection with ocular HSV-1 clinical isolates leads to extreme divergence in pathogenesis in murine model of ocular infection.

Ocular image of (A) pseudomonas ulcer in the right eye and (B) geographic ulcer in the left eye shown by slit-lamp and fluorescein staining of the patient with bilateral infection. (C-D) Images of the right and left eye, respectively, after administration of topical therapeutic. (E) Ocular images unilaterally infected patient with prominent geographic ulcers. (F) Resolution of infection after therapy. C57BL/6 mice were infected on the right eye with 5x105 PFU/mL of ULS or BLS. (G) Representative photographs from each group were taken. (H) Fluorescein staining of the right eye at 4 d.p.i shows at both 10 and 20X magnification. (I) Secreted virus titers assessed from swabs of the right eye at 2 and 4 d.p.i and the left eye at 2, 4 and 7 d.p.i. Significance between BLS and ULS infected mice was determined by one-way ANOVA with Dunnett’s correction test *p<0.0001. Each dot in the bar graph represents a single mouse (n=5) (J) Ocular disease scores (0 to 5, 5 being severe) in mice (n=5). Significance was determined by two-way ANOVA with Dunnett’s multiple comparisons test. (K) Images of right and left mouse eyes showing levels of corneal opacity.

HSV clinical isolates from immunocompromised patients seen in the same clinic generate divergent pathologies in an ocular murine model of infection.

Our initial curiosity stemmed from the observation that the patient with active infection in both the right and left eye contained live replicating virus that we were able to culture and grow in the lab. We sought to determine whether this isolated viral strain, due to its genetic composition, could cause a bilateral infection in mice. We infected C57BL/6 mice with 5x10⁵ plaque forming units (PFU) of either UL, BL.R or BL.L strains in the right eye and monitored several parameters including viral shedding and disease progression in both the right and left eyes. Infection with BL.R and BL.L resulted in very similar pathogenesis, suggesting high genomic similarity between the strains (Fig. 1G, Fig. S1A–C). Mice infected with either BL strains (BLS) showed minor signs of inflammation at 4 and 6 d.p.i. in the right eye and seemed to fully recover by 8 d.p.i (Fig. 1G). In contrast, mice infected with UL strain (ULS) developed significantly higher clinical signs of acute ocular herpes infection as well as heightened levels of inflammation in the periorbital region (Fig. 1G, J). Viral shedding in the ocular tear film was similar between UL and BL-infected mice at 2 d.p.i. However, at 4 d.p.i., significantly increased viral titers in tear samples collected from mice infected with UL, compared to BL, was seen, as well as increased HSV-1 viral transcripts from that eye (Fig. 1H–I, Fig. S1E). Fluorescein staining was used to assess damage of the corneal epithelium as a result of infection. At 4 d.p.i., mice infected with ULS developed dendritic ulcers, which indicates the presence of actively replicating virus. Fluorescein staining was also performed at 2 and 7 d.p.i., however staining of the corneal epithelium was not observed in either group. To determine whether a bilateral infection was developed in either group, we assayed secreted virus titers from the tears of the left eyes on 2, 4 and 7 d.p.i. Unexpectedly, mice infected with ULS developed severe bilateral infection while mice infected with BLS did not secrete any infectious particles from the left eyes (Fig. 1I, Fig. S1F, G). This suggests that, since the strain isolated from a patient with bilateral infection did not cause a bilateral infection in murine ocular studies, bilateral infections observed in patients may not be a result of infection or exposure to a certain strain, but rather the individual’s host response to the infection or other contributing exogenous factors.

Often times, optical transparency is compromised as a result of inflammatory immune cell infiltration in response to ocular infection (2). To determine whether these strains are capable of, or even differ in, generating such a response we visually assessed corneal opacification of the right and left eye. Corneal opacification was significantly more prominent in the right and left eyes of mice infected with ULS, compared to mice infected with BLS (Fig. 1K, Fig. S1D). In contrast, mice infected with BLS quickly suppressed virus replication and recovered without generating disease phenotypes like corneal scarring or opacification (Fig. 1G–K). Our data demonstrates that viruses collected from immunocompromised patients with varying disease outcomes are capable of generating host responses that lead to different pathologies. To further investigate the differential virus-host interaction between the strains and identify mechanisms causing the different phenotypes in vivo, we examined the strain-dependent regulation of heparanase (HPSE), a host enzyme that participates in extracellular matrix remodeling through heparan sulfate (HS) degradation and intracellular processes like gene transcription. Our lab has shown that virus-induced upregulation of HPSE in drives key processes in herpes pathogenesis, including the generation of pro-inflammatory factors and disruption of immune tolerance in the human cornea (21). Interestingly, we found that while both strains upregulate HPSE expression during the initial hours post infection (h.p.i.) in human corneal epithelial (HCE) cells, only BLS infection causes a sustained gradual increase at 24 and 32 h.p.i (Fig. S2A). We also found that BLS, but not ULS, significantly upregulated HPSE expression in ocular tissue at 4 d.p.i relative to mock-infected mice (Fig. S2B). Furthermore, in the absence of HPSE, ULS and BLS exhibit significantly different replication kinetics marked by different viral protein expression over the course of infection (Fig. S2C). ULS also generated a more cytotoxic and destructive cellular environment in the absence of HPSE than BLS (Fig. S2D).

ULS, but not BLS, infection generates a robust CD8⁺ T lymphocyte response characterized by inflammatory infiltrate consisting of proinflammatory cells and cytokines.

After observing clear differences in the corneal opacity of mice infected with either UL or BL clinical strains, we sought to examine the local immune response generated as a result of infection with either strains and identify factors contributing to the apparent pathological differences. Due to the importance of T cells in perpetuating inflammatory diseases like stromal keratitis, we assessed the amount of CD4⁺ and CD8⁺ T cells in the whole eye (22). At early d.p.i., we examined the expression of key chemokines, CXCL10 and CCL5, involved in T lymphocyte and leukocyte recruitment and found that these two chemokines were significantly upregulated upon ULS infection in the eye at 4 d.p.i. (Fig. 2A) (23–25). Flow cytometry was then used to quantify immune cell populations in the eyes collected at 8 d.p.i. from mice infected with either BL or UL strains. In accordance with the elevated chemokine levels, significantly higher number of CD3CD8⁺ T cell infiltrates were found in the eyes of mice infected with UL compared to BL strain (Fig. 2B, Fig. S3). This was accompanied by increased levels of ocular-infiltrating macrophages and plasmacytoid dendritic cells in ULS infected mice (Fig. 2G–H, Fig. S3). While the population of CD4⁺ T cells were similar among both groups of mice, mice infected with ULS exhibited slightly higher number of CD4⁺ T cells (Fig. S3C). In addition, the cytokine profile elicited in response to infection varies, suggesting potential differential naive CD4⁺ T cell differentiation in each microenvironment. For this reason, we examined the cytokine profile at the primary site of infection. At 4 d.p.i., infection with ULS generated significantly higher proinflammatory cytokines; namely IL-1β, IL-6 and TNF-α in the right eyes (Fig. 2C–E). These proinflammatory mediators have been shown to cause pathological inflammatory disorder and much of the tissue damage observed in herpes keratitis (26, 27). While both BLS and ULS resulted in an increase of IL-10, it is one of the few cytokines upregulated in mice infected with BLS and therefore suggests playing a protective role and driving anti-inflammatory signals that control the disease (Fig. 2F) (28).

Figure 2. ULS, but not BLS, infection generates a robust CD8⁺ T lymphocyte response characterized by inflammatory infiltrate consisting of proinflammatory cells and cytokines.

(A) Quantification of local chemokine expression in mock, ULS and BLS infected mice with 5x10⁵ PFU/mL. Tissue was collected at 4 d.p.i. and subjected to qRT-PCR. Copy numbers relative to GAPDH are shown. Mouse eyes collected at 8 d.p.i with either ULS or BLS were collected and subjected to flow cytometry analysis of immune cell populations. (B) Representative flow cytometry plots of anti-CD8 vs.anti-CD3 with indicated number of double positive cells. The whole eye for each mouse was suspended in 500μL and diluted 4x from the stock cell suspension before performing the flow cytometry read. (C-F) Quantification of pro-inflammatory cytokine transcripts in eye tissue at 4 d.p.i with 5x10⁵ PFU/mL mock, ULS or BLS. Representative flow cytometry plots of (G) APC anti-GR-1 vs. FITC anti-CD45 and (H) anti-CD317 vs. anti-CD11c with indicated percentages of double positive cells from eyes collected 8 d.p.i. Statistical significance was measured in by (B, G, H) student T test and (A, C-F) One-way ANOVA with multiple comparisons. *P<0.05, **P<0.01. Each dot in the bar graphs represents data from a single mouse (n=5).

To further validate that these strains are capable of generating different immune responses, we infected ex vivo human cornea cultures with either UL or BL strains. We found that ULS was capable of replicating and producing a greater number of infectious particles in culture accompanied by increased expression of viral transcripts and proinflammatory cytokines in response to ULS infection (Fig. S4).

UL and BL clinical isolates are capable of generating unique programming of local immune response.

To map out the trajectory of the immune responses generated following exposure to either viral strain, we evaluated the initiation of the adaptive immune response in the draining lymph nodes (dLNs). Since cytokines are systemic effectors of lymphatic function, we began by examining the cytokine profile (29). We found that the cytokine profile in the lymph node at 4 d.p.i. was comparable between both ULS and BLS groups of infection, suggesting similar initial immune responses (Fig. 3A). Notably, CCL5, CXCL10, IL-10, IL-17, IL-6 and IL-1b were all significantly upregulated in both mouse groups relative to mock-infected mice. However, at 8 d.p.i., the draining lymph node size of mice infected with UL strain was significantly larger than those of mice infected with BLS (Fig. 3B, Fig. S3A). This suggested sustained infection resulting in lymphatic inflammation which in turn generates the observed disease phenotypes in ULS infected mice, but not in BLS infected mice. Analyzing the cytokine protein levels, we observe significant upregulation of key cytokines including IL-17A, MIP3a/CCL20, IL-1β and IL-5 protein expression in the lymph nodes of mice infected with ULS (Fig. 3C). In contrast, IL-2 was significantly upregulated in the lymph node of mice infected with BLS, along with modest increase in TNF-α and IL-21 protein levels (Fig. 3D).

Figure 3. Differential local immune response as a result of infection with divergent clinical isolates.

Mice were infected in the right eye with 5x10⁵ PFU/mL of either ULS or BLS. Draining lymph node (dLN) tissue collected from mice sacrificed at (A) 4 d.p.i and (B-D) 8 d.p.i. (A) dLN tissue was subjected to cytokine expression analysis by qRT-PCR. (B) Length of lymph nodes collected was measured and graphed. (C) Heatmap of the cytokine protein array in the dLN of each mouse in either the UL or BL infected group represented in the columns and the cytokine analytes in the rows. The expression heat map was generated through Heatmapper where relative abundance is represented by color (blue is lower abundance and red is higher abundance). Both rows and columns are clustered using Kendall′s Tau distance measurement and average linkage. (D) Protein quantification of select cytokines from (C). Each dot in the bar graphs represents data from a single mouse (n=5). Statistical significance was measured by student T test. *P<0.05, **P<0.01. ***P<0.001, ****P<0.0001

Ocular infection with ULS HSV-1 strain leads to heightened viral burden in the trigeminal ganglion and brainstem.

Ocular infection with UL strain caused 0% survival by 8 d.p.i, compared to 100% survival and no apparent weight loss in mice infected with BLS (Fig. 4A, Fig. S1H). To further assess and determine the cause of this phenotype, we examined the ability of each strain to infect and replicate in the central nervous system. After primary ocular infection, the virus travels through retrograde transport to the trigeminal ganglion (TG), where it briefly replications and establishes latency (30). We found that mice infected with ULS harbored more virus in the TG causing a robust and destructive inflammatory response indicated by mononuclear infiltrate in the TG in mice infected with ULS with mild infiltrates seen in BLS infected mice (Fig. 4B–D, Fig. S5). Dissemination of the virus to the brainstem was detected in mice infected with ULS indicating development of encephalitis, the likely cause of the observed weight loss and mortality (Fig. 4). Furthermore, since we reported that infection of the right eye with ULS caused bilateral viral shedding, we assessed whether virus could be detected in the left TG from either mouse group. We detected immediate early and late viral transcripts in the left TG only in the group of mice infected with ULS (Fig. S1G).

Figure 4. Sequence variability found in the clinical isolates in proteins known to contribute to cell-to-cell spread and neurovirulence may be the cause of divergence in CNS viral burden and mortality rate.

(A) Mice were inoculated with 5x10⁵ PFU/mL of either BLS or ULS (n=5) following ocular scarring of the right eye and their weights were monitored over the course of infection and used to calculate percent of original body weight. (B) Representative images of hematoxylin and eosin staining of the right trigeminal ganglion of mock. BLS and ULS infected mice at 8 dpi. Arrow indicates focal mononuclear inflammatory infiltrates. qRT-PCR of (C) trigeminal ganglion and (D) brainstem collected from mice 4 days following ocular infection with 5x10⁵ PFU/mL of mock, ULS or BLS showing gene expression of HSV-1 viral proteins ICP4 and gD. Each dot in the bar graphs represents data from a single mouse (n=5). (E) Comparison of nonsynonymous to synonymous nucleotide differences (dN/dS), between UL and BL strains isolated and cultured in the lab, of viral proteins known to contribute in cell-to-cell spread and neurovirulence.

With the drastic differences observed in the host control of neuronal infection between the two strains, we compared the sequence variations of key viral proteins known to contribute to cell spread and neurovirulence. A comparison of the dN/dS ratios between the two strains revealed interhost coding diversity at the consensus level in UL23, US3, UL38, UL42, UL19, US6, UL24, US2, UL27, UL8 and particularly substantial differences UL22 and UL49 (Fig. 4E, Table 1) (31–40).

Table 1. List of proteins known to contribute to cell spread and neurovirulence.

The number of single nucleotide polymorphisms (SNPs) and amino acid changes in ULS relative to BLS is reported. The percent amino acid difference is relative to the protein length.

Gene	Protein	# of SNPs	# of AA changes	ORF length	protein length	%AA diff	dN/dS
UL49	tegument protein VP22	7	5	276	300	1.67	2.50
UL8	DNA helicase/primase complex-associated protein	19	6	2253	750	0.80	0.46
UL24	Nuclear protein UL24	8	4	810	269	1.49	1.00
US2	Virion protein US2	7	3	876	291	1.03	0.75
UL27	Glycoprotein B	23	8	2715	904	0.88	0.53
US6	Glycoprotein D	1	1	1185	394	0.25	1.00
UL19	Major capsid protein	21	5	4125	1374	0.363	0.31
UL42	DNA polymerase processivity subunit	6	3	1467	488	0.61	1.00
UL38	Capsid triplex subunit 1	6	1	1398	465	0.21	0.20
US3	Serine/threonine protein kinase US3	4	2	1446	481	0.42	1.00
UL22	Glycoprotein H	9	8	2517	838	0.95	8.00
UL23	Thymidine kinase	9	5	1131	376	1.33	1.25

Genomic sequencing and assembly of ocular HSV-1 clinical isolates show evolutionary divergence and interstrain diversity.

We isolated HSV-1 DNA from sucrose gradient purified virus preparations and subjected them to next generation sequencing followed by reference-assisted genome assembly (Fig. S6). The virus collected from the right and left eye of the patient with bilateral infection showed identical consensus sequences. However, the UL strain (ULS) BL strain (BLS) encompassed roughly 800 single nucleotide polymorphisms across the genome (Fig. 5A).

Figure 5. Circulating ocular HSV-1 clinical isolates are genetically divergent from one another.

(A) Diagram of the HSV-1 genome structure. Coverage of sequenced and reference mapped genomes using KOS strain shown in gray. Mutations across the genomes are indicated. (B) Whole genome phylogenetic analysis of multiple HSV-1 strains, including the ocular clinical isolates by neighbor network based alignment using SplitsTrees. (C) Percent of genes in the entire genome that encompass either no mutations, missense or gene-level mutations in ULS relative to BLS. (D) Number of mutations across the genomes that result in amino acid variations, single nucleotide polymorphisms (SNPs), deletions or indels. (E) Percent amino acid variable for unique short (U_s) and unique long (U_L) proteins. This is calculated relative to the total protein length. The bars shown in the lighter shade of gray represent glycoproteins while the darker shade represents the remaining proteins.

To assess the overall degrees of relatedness between these viral genomes and how geographic origins may be influencing the observed phenotype patterns, phylogenetic analysis using a sequence-based network was performed. The consensus sequences of the clinical isolates and 38 HSV-1 whole genomes, along with 1 outgroup HSV-2 genome, from Genbank were compared using neighbor network-based alignment to construct the phylogenetic tree (Fig. 5B). Three major phylogenetic groups with distinct geographical distribution were identified in accordance with previously published papers (13, 14, 41). The first phylogroup, highlighted in green in Figure 5, contains strains isolated in Europe and North America. The second phylogroup, highlighted in blue, is comprised of strains mostly from Asia and the third group, highlighted in orange, consists of strains from Africa. The clinical strains segregated into different groupings and proved to be evolutionarily distant. UL strain grouped into phylogroup I and exhibited highest sequence homology to 17 and McKrae while BL strain grouped into phylogroup II and exhibited highest sequence homology to KOS strain. Furthermore, comparative analysis of the coding sequences revealed that 72% of the genome encompassed nonsense mutations, 24% consisted of missense mutations and only 4% of the genome encompassed genes conserved at both the nucleic acid and amino acid level, relative to each other (Fig. 5C, D). Moreover, only 19 viral proteins were conserved at the amino acid level between these two strains (Table S1), with the majority encompassing 0.5-3% amino acid variations (Fig. 5E).

Nonsynonymous mutations in HSV glycoproteins contribute to decreased viral entry and replication in the BL strain.

Further phenotypic characterization was then pursued in order to understand the influence of genomic variations on viral growth kinetics. Most distinctly, infection of HCE cells with McKrae, KOS, UL and BL resulted in different morphology and plaque size (Fig. 6A, B). Plaques formed as a result of infection with BL were significantly larger than plaques generated by UL. Although BL and KOS are genetically similar with only 16 sequence mutations relative to each other, they still produced plaque sizes that are significantly different from each other suggesting that these 16 SNPs play a role in cell-to-cell spread (Table S2). Unique growth kinetics of each clinical and laboratory strains was also observed (Fig. 6D, E). Given these findings, we sought to determine whether these were caused by alterations in entry kinetics among the strains using HCE cells. HCE cells express HSV-1 entry receptors nectin-1, herpesvirus entry mediator (HVEM), and paired immunoglobulin-like 2 receptor alpha (PILR-a) (42). We found that BL strain exhibits decreased cellular entry compared to UL, KOS or McKrae (Fig. 6H, Fig. S7A). HSV-1 entry is governed by the interaction of multiple viral envelope glycoproteins (gC, gB, gD, gH/gL) with cell surface components (43). Looking closely at the sequence variability in the viral glycoproteins between UL and BL, we found that all glycoproteins, and particularly those involved in entry, exhibited synonymous and/or nonsynonymous mutations that may be contributing to the clear differences in rate of cellular entry (Fig. 6F). Structural studies have reported the interaction of carbonyl group of gD amino acid L25 with the hydroxyl group of Y23 in HVEM, an entry receptor (44). L25P substitution has been reported to affect this interaction leading to reduced efficiency of viral entry into cells expressing HVEM (44–47). We show that the gD sequence of BL strain exhibits the L25P substitution which may contribute to the significant differences observed in viral gD (Fig. 6G, Fig. 6A). To verify this, we transfected Chinese hamster ovary (CHO) cells, nonpermissive cells for HSV-1 infection due to the lack of specific entry receptors, with HVEM or nectin-1 and performed an entry assay with 5 MOI of ULS or BLS (48). We found that ULS was able to entry at higher rates in all transfection conditions and even more so in co-transfection of both receptors, relative to BLS (Fig. 6I).

Figure 6. Coding sequence variations in glycoproteins between the clinical isolates are associated with their varying capacity of viral entry, replication and spread.

(A) HCE cells infected at 1 MOI with clinical isolates (BLS and ULS) and laboratory strains (KOS and McKrae). Brightfield images taken at 12 and 24 hpi. (B) Representative images of plaques produced by each strain are shown on Vero cell monolayer are shown. (C) Area of 20 plaques from each sample was taken and graphed in pixels. HCE cells were infected at 0.1 MOI with lab and clinical isolates to measure the (D) intracellular and (E) extracellular viral growth curve. (F) Profile of SNP and amino acid mutations in HSV-1 glycoproteins. (G) Causative mutation in the receptor binding domain of glycoprotein D (H) HCE cells or (I) CHO cells transfected with HVEM, nectin-1 or both were subjected to an entry assay. Representative immunoblot of VP16. Significance between the groups was determined by Student T test. *p<0.05

Discussion

Most clinical isolates of HSV contain significant genetic variation, however the relative contributions of these genetic variations on clinical manifestation and disease outcome are rarely characterized. In this study, we report an in-depth phenotypic and genotypic characterization of two ocular HSV-1 isolates from immunocompromised patients with similar immune backgrounds but different clinical manifestations of herpetic infection. Although comparative analysis of two clinical isolates is limiting, the associations identified here provide key insights into the potential impact of viral variability on clinical outcomes and serve as guidance for further mechanistic studies. We found that two viral strains (UL and BL) are capable of generating divergent host responses in murine model of ocular as well as nervous system infection, ex vivo human corneal cultures and in vitro cell culture. This includes differential immune programming and tissue-specific tropism leading to divergent disease outcomes and mortality rate. High-throughput genome sequencing of divergent virus strains enables unbiased and comprehensive mapping of viral genotype to differences in phenotypes. Using comprehensive comparative genomics, we reveal viral genetic mutations involved in altering functional interactions with entry receptors leading to unique growth characteristics, cell-to-cell spread and early entry kinetics between the two strains. Furthermore, we found high sequence variability in proteins known to contribute to cell-to-cell spread and/or neurovirulence in mouse models of CNS disease. While further studies are required to determine the impact of these variations, here we show that intrastrain variations across the viral genomes particularly in viral gD affecting receptor usage play a critical role in determining the disease outcome.

Despite the high prevalence of HSV-1 worldwide, it remains unclear why certain individuals develop pathologies of recurrent herpetic disease while others remain asymptomatic. It is important to understand whether genetic diversity among HSV strains results in the expression of viral proteins that differentially modulate host immune responses, leading to disparate disease outcomes. It is apparent that both host and viral factors influence susceptibility to herpetic disease; however, the specific virus-host interactions that promote differences in the initiation of the adaptive immune response after exposure to different HSV-1 strains are currently unknown (49). While the mechanism is unclear, previous findings suggest that degree of corneal damage depends on the virus strain that infects the cornea. Moreover, studies have shown strain dependent influence on the preferential recruitment and activation of CD4 and CD8⁺ T cells, leading to pathogenesis of or protection against stromal keratitis (12, 22). In this study, we show that inflammatory cellular infiltrates induced by these two clinical viral strains were markedly different. More specifically, differential induction of CXCL10 and CCL5, likely by infected epithelial cells or resident cell types like DCs, was observed leading to different amounts of CD8⁺ T cell recruitment to the eye. The activation of DCs, acquisition of viral antigens, and migration into the dLN are crucial for initiating antiviral T cell responses, and our data indicate that these events are differentially regulated between the two HSV-1 isolates. UL strain elicited robust macrophage and plasmacytoid dendritic cell infiltration, causing loss of corneal transparency and integrity. In contrast, a more robust and efficient control of BLS infection was observed and likely dependent on elevated IL-10 levels in the cornea, which has been shown to suppress production of certain cytokines and minimized ocular inflammation. A better understanding of the viral and host factors contributing to tissue destruction may facilitate development of immunology-based therapy for this blinding disease.

Heparanase (HPSE) is an endoglycosidase capable of degrading cell surface heparan sulfate and has been implicated as a driver of pathogenesis and inflammation in multiple disease models including HSV-1 infection (45, 50–52). We have previously reported that host encoded HPSE is upregulated upon infection by herpesviruses and that this in turn facilitates release of viral progeny from parent cells (53). We also show that HSPE is capable of translocating to the nucleus and regulating transcription of key host molecules that drive the pathological and inflammatory conditions seen with HSV-1 infection (21). Most recently, we show that cells lacking HPSE are intrinsically resistant to HSV-1 infection and the absence of HPSE in vivo protects against proinflammatory cellular infiltrate in the cornea (54). The strain dependent differences in HPSE regulation and potential role in varying clinical manifestations of HSV-1 is not known. In this study, differential dependence on HPSE activity is observed between genetically distinct viral strains however how this leads to alterations in disease outcomes in not well understood. HPSE is a multifunctional protein that is also involved in cell survival and communication in stress induced environments (55, 56). It may be that in one case HPSE is promoting a proinflammatory environment leading to tissue destruction and damage while in another case HPSE is promoting cellular survival and proliferation. Both strains investigated in this study upregulate HPSE after infection however this is prolonged past 24hpi in BL but not UL. Furthermore, the ability of ULS to infect cells deficient in HPSE suggests the strains lack of dependence on HPSE for viral replication and progression of infection. It is evident that there is differential dependence on the host factor HPSE between the two strains and further investigating this may give a mechanistic insight to diverse immune responses and clinical outcomes observed.

While we were anticipating that the strains isolated from the patient with bilateral infection would give us some insight as to the genetic contribution of this phenotype, we found that, at least in murine ocular infection, there was no association. Interestingly, we found that the viruses from this patient preserved its genetic and phenotypic characteristics during bilateral infection. This may suggest that bilateral infections are a result of exogenous factors like contamination from rubbing eyes. Infection with UL strain caused development of corneal dendritic ulcers, heightened levels of viral shedding from both the right and left eyes as well as severe weight loss and lethality. In contrast, infection with BL strain was quickly controlled by the host and viral shedding was suppressed by 4 d.p.i. Our murine studies indicate that early host-pathogen interaction drive diverse responses and differences in the ability of the host to control viral replication and dissemination. This suggests that upon diagnosis of herpes infection, identifying the strain present may influence the course of treatment and determine whether aggressive action must be taken such as the administration of anti-inflammatory agents.

HSV-1 strain-dependent variation in neural spread influences the virus’s ability to cause acute neurological disease. Viral genetic factors have been reported to contribute to neuroinvasive disease potential. Previous studies have reported increased virulence and neuroinvasiveness of HSV-1 strain McKrae compared to other wild-type strains like KOS (15). This data suggests that the viral genetic origin may be responsible for determining the cellular and clinical outcomes of ocular HSV-1 infection. While we do report apparent neuroinvasive properties of UL, but not BL, strain, further studies are needed to fully elucidate the mechanism behind this. In vitro studies also indicate ULS’s capability of generating small plaque sizes, which has previously been a key characteristic of cytopathic and neurovirulent strains. One possibility for these differences is the mechanism and kinetics of T cell dependent HSV control in the nervous system, as well as other host antiviral mechanisms like autophagy, are differentially triggered as a result of varying genetic factors. Examining the interstrain coding sequence variability between UL and BL, we found genes involved in neurovirulence to be highly variable and likely contributing to the observed differences. Mutations or sequence variability in these viral proteins have been shown to be capable of altering the ability of the virus to replicate in peripheral sites, neural entry and spread as well as evasion from immunological surveillance. This is particularly interesting and relevant to other DNA and RNA viruses that exhibit strain dependent neurotropism. For example, recent studies show SARS-CoV-2, an RNA virus that primarily causes disease in respiratory systems, is capable of intracranial invasion and neurological manifestations (57). At present, it remains unclear whether certain variants of SARS-CoV-2, or Zika virus, exhibit enhanced neurotropism and increased susceptibility to causing neurological disease (58, 59). With the emergence of new SARS-CoV-2 strains, this highlights the importance of monitoring molecular evolution as a result of host adaptation.

Viral glycoproteins are involved in multiple host interactions including viral entry, cell-to-cell spread, cellular tropism, immune recognition/evasion and play a definite role in pathogenesis (60). Four HSV glycoproteins (gB, gD, gH and gL) are essential for viral entry and interact with cell surface receptors. Entry receptors for HSV include herpes virus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family, two members of the immunoglobulin superfamily, nectin-1 and nectin-2, as well as specific sites on heparan sulfate generated by 3-O-sulffotransferases (43, 61, 62). In vitro, we found that ULS exhibited enhanced entry capabilities relative to BLS. Comparative analysis of ULS and BLS demonstrated that all of the glycoproteins involved in entry exhibit amino acid variations potentially altering receptor usage or interaction. Particularly, a single amino acid substitution was found in gD which has been observed to alter the physical and functional interaction with herpes entry receptors expressed on cell surfaces. This was mainly driven through a leucine to proline substitution found in residue 25 in the N-terminus HVEM-binding region of gD in BLS. L25P has been reported to reduce HVEM-mediated virus infection as well as abrogate interactions with 3-O-sulfated heparan sulfate (3-O-S HS) (45). Glycoprotein sequence variability and change of receptor tropism may also pose immunological implications with pathological consequences after HSV-1 infection. Furthermore, receptor bias of ULS may be a factor for the resulting bilateral infection and mediates enhanced entry and replication in neuronal cells. While it is likely that multiple host-pathogen interactions influence the aggressive infectious patterns of ULS, our study suggests that early entry kinetics contributes to differences in disease outcomes. Together, our data suggests viral genomic variations are capable of generating an acquired entry receptor bias usage leading to disparate immunopathogenic responses and ultimately disease outcomes.

Materials and Methods

Virus stock.

UL and BL strains were isolated from ocular material of patients who showed various clinical features of HSV infection including herpetic stomatitis, herpes labialis and kerato-conjunctivitis. The isolates obtained by the authors were passaged three times in Vero cells at relatively low multiplicities in order to prepare a seed virus of high titer. Other laboratory virus strains used in these studies were HSV-1 KOS and McKrae which were provided by Dr. Patricia G. Spear (Northwestern University, Chicago, IL).

Nucleocapsid DNA preparation.

Infected Vero cells were harvested 48 h post-infection, subjected to three freeze-thaw cycles, and cellular debris was removed by low-speed centrifugation. Viral particles were concentrated by two sucrose cushions and treated with DNase I to eliminate cellular DNA. Lysates were banded by equilibrium centrifugation in 57% CsCl for 72 h at 40,000 rpm in a Beckman 6OTi rotor. After centrifugation, the viral DNA band was dialyzed against 1x TE (0.01 M Tris, 0.001 M EDTA [trisodium salt], pH 7.4). The viral pellet was resuspended in 5 mL of TE buffer (10 mM Tris [pH 7.4], 1 mM EDTA) with 0.15 M sodium acetate and 50 g/mL RNase A, and incubated for 30 minutes at 37°C. Proteinase K and SDS (50 g/mL and 0.1%, respectively) were then added, and the solution was incubated for 30 minutes at 37°C. The viral DNA was then purified by phenol:chloroform extraction and ethanol precipitation, resuspended in deionized water, and stored at 20°C

Next-generation sequencing

Library preparation was performed using Celero DNA-seq (NuGEN Technologies) and following manufacturer’s protocol. Libraries passing quality control were subjected to Illumina NextSeq500. Illumina reads were trimmed using a Q30 setting, and sequences with ambiguous nucleotides were removed. Reads shorter than 50 bases were removed, as were broken paired end reads and passed through FastQC for verification.

Genome Assembly

After trimming, reference-guided assembly with the complete genome sequence of HSV-1 strain KOS (JQ780693.1) using BWA-MEM was performed. Resulting alignment was visualized with the Integrated Genome Viewer (IGV) v2.4.60 (52). SAMtools mpileup was used on BAM-formatted mapped reads to generate information on match, mismatch, indel, strand, and mapping quality per reference genomic position. To reduce the impact of false positives on variant calling, only high-quality mapped reads and reads with base quality and mapping quality of ≥20 were utilized in the pile-up. SNP variants were filtered and reported in variant calling format (VCF) and used to generate the consensus sequence for each strain.

Phylogenetic Analysis

FASTA files generated for each strain, along with 35 strains on NCBI GenBank, were used to generate a neighbor network based whole-genome alignment using SplitsTrees (v4.14) with 1,000 bootstraps (Table S3) (53).

Murine ocular infection model

All animal care and procedures were performed in accordance with the institutional and NIH guidelines, and approved by the Animal Care Committee at University of Illinois at Chicago (ACC protocol 20-065). Male and female C57/B6 mice (6-8 week old) were used for all experiments. Anaesthetized mouse corneas were scarified in a 3x3 grid using a 30-gauge needle and infected with 5x10⁵ PFU/mL of HSV-1, as previously described (15). Images of the corneal surface to monitor disease was acquired with SteREO Discovery.V20 stereoscope (Zeiss, Germany). Humane endpoint criteria was set at 15% or more weight loss, large ocular/periorbital lesions or corneal perforation.

Cell lines

HCE cell line was obtained from Kozaburo Hayashi (National Eye Institute, Bethesda, MD) and the cells were cultured in Minimum Essential Media, MEM (Thermo Fisher Scientific) supplemented with 1% penicillin/streptomycin (P/S) (Thermo Fisher Scientific) and 10% fetal bovine serum (FBS, Sigma-Aldrich). African green monkey fetal kidney epithelial (Vero) cells were provided from P. Spear (Northwestern University) and cultured in DMEM (Life Technologies) with 10% FBS and 1% P/S. Wildtype and heparanase-knockout mouse embryonic fibroblasts were provided by Dr. Israel Vlodavsky (Rappaport Institute, Haifa, Israel). All cells were maintained in a Heracell VIOS 160i CO2 incubator (Thermo Scientific) and have been confirmed negative for mycoplasma contamination. Patricia G. Spear (Northwestern University, Chicago, IL) provided wildtype Chinese hamster ovary (CHO-K1) cells and cultured in Ham’s F12 (Invitrogen) supplemented with 10% fetal bovine serum (FBS).

Plasmids encoding HSV entry receptors included pcDNA3-based constructs as follows: pBEC10 expressing human HVEM and pBG38 expressing human nectin-1 (42, 54). Expression vector pcDNA3 was used as an empty vector control.

Viral Entry Assay

HCE cells were plated in a 12-well plate (ca. 5x10⁵ cells per well). The next day, cells were incubated with 5 MOI of HSV-1 McKrae, KOS, ULS or BLS viruses for 15 minutes at 20°C, followed by a 30-minute incubation in 37°C with 5% CO2. Non-internalized virions were removed by washing cells with low pH treatment (50 mM sodium citrate buffer pH 3). The cells were then collected and subjected to subsequent analysis. CHO cells were plated in 12-well plates (ca. 4x10⁵ cells per well). The next day, the cells were transfected with either pcDNA3 empty vector, HVEM, nectin-1 or both HVEM and nectin-1 plasmids at 0.5 ug/well. 24 hours after transfection, the entry assay was performed as previously described.

Plaque Assay

Plaque assay was performed with tear samples from infected mice as well as with cell supernatant from in vitro experiments. Vero cell monolayers were inoculated for 2 h with virus containing sample and incubated at sample at 37°C, 5% CO2. Viral suspension was aspirated and replaced with complete DMEM containing 0.5% methyl cellulose (Fisher Scientific) for 48 to 72 h. Cells were then fixed with 100% methanol and finally stained with crystal violet solution to visualize plaques.

Quantitative real time-polymerase chain reaction

RNA was extracted from cultured cells using TRIzol (Thermo Scientific, 15596018), following the manufacturer’s protocol, and complementary DNA was produced using High Capacity cDNA Reverse Transcription kit (Life Technologies). For in vivo experiments, mouse tissue was extracted and incubated in 50 μL of 2 mg/mL collagenase D (Sigma C0130) in PBS for 1 h at 37°C. Tissues were then triturated with a pipet tip, resuspended in TRIzol and extraction of RNA and cDNA were performed as above. Real-time quantitative polymerase chain reaction (qPCR) was performed using Fast SYBR Green Master Mix (Life Technologies) on QuantStudio 7 Flex system (Life Technologies). The following primers were used:

• HSV-1 gD Fwd 5’-GTGTGACACTATCGTCCATAC-3’, Rev 5’-ATGACCGAACAACTCCCTAAC-3’

• HSV-1 ICP0 Fwd 5’-ACAGACCCCCAACACCTACA-3’, Rev 5’-GGGCGTGTCTCTGTGTATGA-3’

Flow cytometry

Corneas were extracted from mice after euthanasia and treated with 2 mg/mL collagenase D (Sigma C0130) for 1 h at 37°C and triturated with a pipet tip. Cell suspensions were filtered through a 70 μm mesh and resuspended in FACS buffer (PBS + 5% FBS). Cells were then aliquoted into U-bottom 96-well plates for subsequent staining. The following fluorophore-conjugated primary antibodies from Biolegend were used for cell surface staining: FITC anti-mouse CD45 (103107) and APC anti-mouse Ly-6G/Ly-6C (Gr-1) (108411). Cells were immunolabeled, washed, and analyzed with Accuri C6 Plus flow cytometer (BD Biosciences). BD Accuri C6 Plus software and Treestar FlowJo v10.0.7 were used for all flow cytometry data analysis.

Western blot

Cellular proteins were extracted using radioimmunoprecipitation (RIPA, Sigma) buffer and Halt Protease Inhibitor Cocktail (Thermo Scientific). Lysis was performed on ice with agitation for 1 hour, followed by 10 min centrifugation at 13,000 rpm. After removing cellular debris, lysates were then denatured at 95°C for 8 min in the presence of 4X LDS sample loading buffer (Life Technologies) and 5% beta-mercaptoethanol (Bio-Rad, Hercules, CA) and electrophoresed by SDS-PAGE with NuPAGE 4-12% Bis-Tris 1.5 mm 15-well gels (Thermo Scientific). Proteins were then transferred to nitrocellulose using iBlot2 system (Thermo Scientific) and membranes were blocked in 5% milk/TBS-T for 1 h at room temperature, followed by incubation with primary antibody overnight at 4°C. After washes with TBS-T and incubation with respective horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, protein bands were visualized using SuperSignal West Femto substrate (Thermo Scientific) with Image-Quant LAS 4000 biomolecular imager (GE Healthcare Life Sciences, Pittsburgh, PA). The housekeeping gene GAPDH was used for normalization, as a loading control. Antibodies used in this study are GAPDH (Proteintech), 10494 at dilution 1:1000, gB (Abcam, ab6505) at dilution 1:10,000, VP16 (Abcam, ab110226) at dilution 1:100, gD (Abcam, ab6507) at dilution 1:10,000, HVEM (Santa Cruz Biotechnology, sc-365971) at dilution 1:100 and Nectin-1 (Santa Cruz Biotechnology, sc-271063) at dilution 1:100.

Cytokine Multiplex Assay

Mouse lymph nodes were collected at 8 d.p.i and, using liquid nitrogen, they were flash frozen and stored in −80°C. Processing to cytokine analysis was performed following manufacturer’s protocol and reagents (Millipore Sigma).

Hematoxylin and eosin staining

Collected mouse tissue was first fixed with 4% PFA for 24 hours then incubated in 70% ethanol. Paraffin embedding and sectioning using Microm HM340 was performed by the UIC core facilities. Subsequent H&E staining was also performed by the core facility.

Data acquisition and statistical analysis.

Graphpad Prism Software was used for statistical analysis with bar heights representing the mean and error bars representing standard deviation. Statistical analysis in each figure is described in the legend, as well as explanations of dot plot representation.

Study approval.

This study was performed with approval by the University of Illinois, Chicago IRB Committee (2011-0975). Consent was obtained from participants prior to inclusion in this study

Supplementary Material

1

Supplemental Figure 1. Infection with ocular HSV-1 clinical isolates leads to extreme divergence in pathogenesis in murine model of ocular infection. (A-C) C57BL/6 mice (n=5) infected in the right eye with 5x10⁵ PFU/mL of BL strain collected from the right eye of the patient. (A) Representative photographs taken 4, 6 and 8 dpi. (B) Fluorescein staining of the right eye at 4 dpi shown in 10X and 20X magnification. (C) Images of the right and left eyes at 9 dpi. (D) Quantification of corneal opacity at 9 days following infection with either ULS or BLS (related to figure 1K). (E) Viral transcripts of immediate early expressed infected cell protein 4 (ICP4) and HSV-1 glycoprotein B relative to GAPDH measured by quantitative PCR from eyes of mice collected 4 days post infection in the right eye with 5x10⁵ PFU/mL of either mock, ULS or BLS. (F) Left eyes were also collected and HSV-1 glycoprotein D was measured by qPCR. Each dot in the bar graph represents data from a single mouse (n=5). Statistical significance was measured by one-way ANOVA with Tukey’s multiple comparison test. *P<0.01, **P<0.01. (G) ICP4 and gD were measured in the left trigeminal ganglion tissue following 9 days of murine infection with 5x10⁵ PFU/mL of either mock, ULS or BLS in the right cornea (n=5). (H) C57BL/6 mice (n=5) infected in the right eye with 5x10⁵ PFU/mL of either ULS or BLS and monitored over the course of 10 days. Survival was determined by the occurance of death or a humane end-point criteria like >15% loss of body weight or large ocular/periorbital lesions.

Supplemental Figure 2. In vitro and in vivo strain dependent differential regulation of the host enzyme HPSE. (A) Quantification of HPSE expression by qRT-PCR following infection time course of HCE cells with 0.1 MOI of either ULS or BLS. Data from three biological replicates are shown (B) Quantification of HPSE expression in murine eyes following infection with 5x10⁵ PFU/mL of either ULS or BLS. Ocular tissue was collected at 4 dpi and HPSE expression was measured by qRT-PCR. Each dot in the bar graphs represents data from a single mouse (n=5). (C) Representative western blot analysis of cell lysates from wildtype and HPSE−/− MEFs following infection time course with 0.1 MOI of ULS or BLS. HSV-1 glycoprotein B (gB) and D (gD) are HSV-1 late viral genes. (D) Representative images of wildtype and HPSE−/− MEFs infected with 0.1 MOI of ULS or BLS at 24 and 48 hours. Statistical significance was measured in (A) by student T test and in (B) by one-way ANOVA with Tukey’s multiple comparison test. *P<0.05, **P<0.01.

Supplemental Figure 3. ULS, but not BLS, infection generates a robust CD8+ T lymphocyte response following ocular infection. C57BL/6 mice (n=5) infected in the right eye with 5x10⁵ PFU/mL of either ULS or BLS. (A) Images of the draining lymph nodes 8 dpi. Quantification of sizes shown in Figure 3B. (B) Mouse eyes were collected at 8 dpi and subjected to flow cytometry analysis of immune cell populations. Number of (B) CD3+ and (C) CD3+CD4+ T cells are graphed. Each dot in the bar graph represents data from a single mouse. Statistical significance was measured using Mann-Whitney test. *P<0.05. (D-F) Flow cytometry data plots for each mouse in the study is shown. Experimental details are described in Figure 2.

Supplemental Figure 4. ULS and BLS exhibit different replication capabilities and induction of proinflammatory cytokines in ex vivo human cornea cultures. (A) Ex vivo human cornea cultures were infected with 1x106 PFU/mL of McKrae, KOS, ULS or BLS. Infectious particles secreted were assessed by plaque assay at 2 and 3 dpi. Sidak’s multiple comparisons was performed and the statistical analysis for BLS vs ULS is shown (*P<0.05). Quantification of (B) viral glycoprotein D and (C-E) proinflammatory cytokines measured at 3 d.p.i. by qRT-PCR. Gene expression is shown relative to GAPDH. Statistical significance was measured by ordinary one-way ANOVA (*P<0.05). Each dot in the bar graphs represents data from a single mouse (n=5).

Supplemental Figure 5. ULS infection results in increased CNS viral burden and inflammation. Histology sections of trigeminal ganglion from each mouse shown following ocular infection with either (A) ULS or (BLS). Representative images and experimental details are provided in Figure 4B. (C) Quantification of ImageJ Software was used to quantitatively analyze the number of positive cells per image shown for all five mice in each group (including images from the mice shown in Figure 4B). Positive cells were selected for based on color, size and roundness to ensure exclusion of Schwann cells and neuronal cell bodies. Statistical analysis was measured by student T-test. ***P>0.0005.

Supplemental Figure 6. Circulating ocular HSV-1 clinical isolates are genetically divergent from one another. (A) Coverage depth of aligned reads (range 138,375-138,415) generated using IGV. Reads from BLS sequencing library aligned to ULS reference genome. The sequence shown at the bottom is that of the reference sequence ULS. The blue band indicates a T to C substitution at position 138,395 of the genome and amino acid 25 of glycoprotein D. Mapping summary report of (B) distribution of read length and (C) distribution of mapped read length.

Supplemental Figure 7. ULS and BLS exhibit varying capacity of viral entry. (A) ULS exhibits enhanced viral entry relative to other clinical and laboratory strains. Quantification of western blot shown in Figure 6H and other biological replicates (n=4) using ImageJ software. (B) HCE cells were infected with 0.1 MOI for 24 hours. Lysates were then collected and prepared for western blot analysis and blotted for infected cell protein 0 (ICP0), glycoproteins D and B (gD and gB, respectively) and tegument protein VP16.

2

Table S1. List of proteins conserved at the amino acid level in ULS relative to BLS.

3

Table S2. List of genes with sing nucleotide polymorphisms in BLS relative to KOS.

4

Table S3. List of accession numbers and strain names used from GenBank to generate the phylogenetic tree.