Sex-related differences in the incidence or severity of infection have been described for multiple viruses. With herpes simplex viruses, the best example is HSV-2 genital infection where women have a higher incidence of infection and can have more severe infections than men. HSV-1 causes several types of infections including skin and mucosal ulcers, keratitis, and encephalitis in humans that do not appear to have a strong biological sex component. Given that mouse strains differ in their MHC loci it is important to determine if sex differences occur in multiple strains of mice. Our goal was to answer two questions: Are virus-related sex differences present in BALB/C mice and does virulence of the viral strain have an effect? We generated a panel of recombinant HSV-1 viruses with differing virulence phenotypes and characterized multiple clinical correlates of ocular infection in BALB/c mice. We found no sex-specific differences in blepharitis, corneal clouding, neurovirulence, and viral titers in eye washes. Sex differences in neovascularization, weight loss, and eyewash titers were observed for some recombinants, but these were not consistent across the phenotypes tested for any recombinant virus. Considering these findings, we conclude that there are no significant sex-specific ocular pathologies in the parameters measured, regardless of the virulence phenotype following ocular infection in BALB/c mice, suggesting that the use of both sexes is not necessary for the bulk of ocular infection studies.

Ethics Statement

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Wisconsin (Protocol Number: M006440). All corneal scarification, disease scoring, tear film collection was performed under isoflurane anesthesia. Sustained release buprenorphine was used for analgesia. Euthanasia was carried out by first anesthetizing the mice with isoflurane and then performing cervical dislocation.

Cells 

Vero cells (CCL-81; ATCC, Manassas, VA, USA) were used for producing viral stocks generating viral DNA. The cells were propagated in Dulbecco’s modified Eagles medium (DMEM), supplemented with 5% serum (1:1 ratio of bovine calf and fetal bovine serum) plus antibiotics and grown at 37^oC with 5% CO₂.

Viruses

The HSV-1 viruses in this study were recombinants generated by mixed corneal infection with two, avirulent, plaque-purified clinical isolates, OD4 and CJ994, in 3- to 4-week-old BALB/c mice (Envigo, Indianapolis, IN, USA). The ocular virulence phenotypes of the parental OD4 and CJ994 strains have been previously described [1]. Corneal scarification was carried out using a 30-gauge needle followed by application of 1x10⁵ PFU of virus (1:1 ratio of OD4 and CJ994) in 5 µL of DMEM with 2% serum. Seven days following infection, the animals were sacrificed, and the trigeminal ganglia were removed, followed by tissue bead homogenization (CK14, Precellys, Rockville, MD, USA). The homogenate was subjected to 3 rounds of freeze-thaw, serially diluted, and plated on confluent Vero cell, 6-well plates for plaque purification.  Each recombinant virus was plaque purified 3 times prior to the preparation of frozen stocks.

Viral DNA Purification and Screening for Recombinants

Briefly, five confluent TC100 plates of Vero cells were infected with recombinant viral stock in DMEM + 2% serum. The infected cells were harvested 24 hours after the monolayer reached 100% cytopathic effect (CPE). The cells were centrifuged at 600 x g for 10 minutes, and the cell pellet with 5 mL of supernatant was subjected to three freeze-thaw cycles. The lysate was then combined with the remaining supernatant and centrifuged at 600 x g for 10 minutes. The supernatant was then layered on a 36% sucrose cushion (in phosphate buffered saline; PBS), and centrifuged for 80 min at 24,000 × g. Following centrifugation, the supernatant was removed, and the pellet was resuspended in 3 mL of TE (10 mM Tris [pH 7.4], 1 mM EDTA) buffer plus 0.15M sodium acetate (pH 5.5). The virus preparation was then incubated with 50 µg/µL of RNase A for 30 minutes at 37°C.  Proteinase K and SDS (50 µg/µL and 0.1% respectively) were then added and the preparation was incubated 30 minutes at 37°C.  The DNA was then purified by phenol/chloroform extraction. The DNA was then precipitated using ice-cold 95% ethanol and desalted with 70% ethanol, followed by resuspension in sterile water. To determine if a strain was a recombinant, BamHI (R6021; Promega, Madison, WI, USA) digested DNA was electrophoresed on a 1% agarose-tris/borate/EDTA (TBE) gel, and the restriction fragment patterns were compared to those of the OD4 and CJ994 parental strains. A strain was determined to be a recombinant if the RFLP mapping revealed patterns which were a combination of the parental strains.  Following confirmation as a recombinant by RFLP analysis, high titer stocks were produced as described previously [1]. 

Genomic Sequencing

Prior to genomic sequencing, the quality of the DNA from each sample was measured using a NanoDrop One (ThermoFisher Scientific, Waltham, MA, USA).  Quantification of the extracted DNA was determined using a Qubit dsDNA High Sensitivity kit (ThermoFisher Scientific). The DNA samples were then diluted and loaded into an Agilent FemtoPulse (Agilent, Santa Clara, CA, USA) electrophoresis system to evaluate DNA size and quality. The samples were subsequently prepared as a Pacific Biosciences Microbial Multiplex library according to PN 101-696-100 v07 instructions.  Modifications included DNA shearing with Covaris gTUBES (Covaris, Woburn, MA, USA). Library quality was assessed using the Agilent Femto Pulse system, followed by library quantification with the Qubit dsDNA High Sensitivity kit. The library was then sequenced on a PacBio Sequel II (PacBio, Menlo Park, CA, USA), using one SMRT cell and the Sequel Polymerase Binding kit 2.2 at the University of Wisconsin-Madison Biotechnology Center DNA Sequencing Facility.

The resulting raw PacBio reads were processed and filtered by CCS calling (CSS 6.2.2; https://github.com/PacificBiosciences/ccs), followed by demultiplexing. The demultiplexed reads were then assembled into contigs using hifiasm (https://hifiasm.readthedocs.io/en/latest/index.html), a de novo PacBio HiFi read assembler. The viral genomes were then manually assembled from contigs using Mega7. Following assembly, the genomes were annotated using VAPiD v1.3.

Multiple Sequence Alignment and Recombination Analysis

A multiple sequence alignment (MSA) for each OD4-CJ994 recombinant, including each parent, was separately generated using MAFFT v7.45, with the FFT-NS-1 option.  Recombination breakpoints for each individual recombinant were detected with RDP4 v4.101, using 1,500 bp sliding window, a step-size of 500 bp, 300 bootstrap replicates, and the Jin and Nei nucleotide substitution model options. A MSA including all 20 recombinants, the OD4 and CJ994 parental, and strain 17 reference sequence was also generated using MAFFT to map each of the breakpoints to strain 17 genome coordinates for data continuity.

Ocular Infection and Disease Scoring

Ocular infection and disease scoring has been described previously [1]. Briefly, to assess the disease phenotype of the 20 novel OD4-CJ994 recombinants,10 females and 10 males (per recombinant), 4-6 week-old BALB/c mice underwent corneal scarification in the right eye with a 30-gauge needle, followed by placement of 1x10⁵ PFU of virus in 5 µL of DMEM in 2% serum on the cornea. The eyes of the infected mice were examined 1, 3, 5, 7, 9, 11 and 13-days post-infection using a Wild-Heerbrugg M8 (Heerbrugg, SG, Switzerland) microscope. The blepharitis, neovascularization, and stromal keratitis ocular disease phenotypes were scored for severity as follows. Blepharitis: 1 +, mild swelling of eyelids; 2+, moderate swelling with some crusting; 3 +, eye swollen 50% shut with severe crusting; 4 +, eye crusted shut. Corneal neovascularization: 1+, less than 25% involvement (vessel ingrowth of the cornea from the limbus); 2+, 25 to 50% involvement; 3+, more than 50% involvement. Stromal keratitis: 1 +, some haziness; iris detail visible; 2+, moderate clouding, iris detail obscured; 3+, cornea totally opaque; 4+, perforated cornea.  For each of the ocular disease phenotypes, a graded score of 0 denotes no observable pathology.

For each viral recombinant, the mean peak disease scores (MPDS) of each of the three ocular disease phenotypes were separately determined according to biological sex by averaging the highest disease score for each animal during the 13-day study. Tear film samples were taken from each mouse by washing the eye with 10 mL DMEM + 2% serum (plus penicillin/streptomycin and amphotericin B), on days 1, 3, 5, and 7 post-infection and subsequently titered using serial dilutions on Vero cell monolayers. A separate average titer for each recombinant was calculated according to sex. Animal weights for each individual mouse were measured at baseline and on days 1, 3, 5, 7, 9, 11, and 13 post-infection. Food intake was not determined. The mean peak weight loss (MPWL) measure (average of the highest weight loss from baseline weight for each mouse over the course of the 13-day study) was calculated for each recombinant according to sex. 

Signs of encephalitis, (neurovirulence) were also scored for each OD4-CJ994 recombinant on days 1, 3, 5, 7, 9, 11, and 13 post-infection. The scoring was binary under the categories of slow respiration rate, tremors/seizures, head tilt, circular gait, ataxia, paralysis, hyperactivity, and coma. A positive score in any of these categories resulted in an infected mouse being scored as positive for neurovirulence. The animal experiments observed the Association for Research in Vision and Ophthalmology and NIH animal welfare guidelines and were approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee (IACUC). Sustained release burprenorphine was used for analgesia and mice with severe neurological signs were humanely euthanized to prevent suffering,

Statistical Analysis

Animal ocular disease phenotypic data graphing, Mann-Whitney U test statistical analyses and regression analyses between phenotypes were performed using SigmaPlot 11 (Systat, Palo Alto, CA, USA). Linear regression comparison was performed using an operator (e.g. MPDS Blepharitis) as the response variable (the expected effect) and the independent variable (e.g. average percent neurovirulence; the experimental cause) interacted with “female” as the independent data.  he P values associated with the female and variable: female coefficients indicate if there is a difference in the relationship between the variable and the operator (R software package v.3.6.1). For all statistical analyses, a P value of 0.001 or less was considered the threshold for significance.

1. Grau DR, Visalli RJ, Brandt CR. Herpes simplex virus stromal keratitis is not titer-dependent and does not correlate with neurovirulence. Invest Ophthalmol Vis Sci. 1989;30(12):2474-80.

Microsoft Excel, Data tables

Microsoft Word, Figure files

Mega, sequence alignments