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Prevalence of herpes simplex virus type 1 glycoprotein G (gG) and gI genotypes in patients with herpetic keratitis
  1. R Duan1,2,
  2. J M van Dun1,2,
  3. L Remeijer2,
  4. M Siemerink2,
  5. P G H Mulder3,
  6. P Norberg4,
  7. A D M E Osterhaus1,
  8. G M G M Verjans1
  1. 1
    Departments of Virology, Erasmus Medical Center, Rotterdam, The Netherlands
  2. 2
    Rotterdam Eye Hospital, Rotterdam, The Netherlands
  3. 3
    Departments of Epidemiology & Biostatistics, Erasmus Medical Center, Rotterdam, The Netherlands
  4. 4
    Department of Virology, University of Göteborg, Göteborg, Sweden
  1. Dr G M G M Verjans, Department of Virology, Erasmus Medical Center, ‘s-Gravendijkwal’ 230, 3015 CE Rotterdam, The Netherlands; g.verjans{at}erasmusmc.nl

Abstract

Aim: Recent phylogenetic analyses on the herpes simplex virus type 1 (HSV-1) genes US4, encoding glycoprotein G (gG) and US7, encoding gI, of clinical HSV-1 isolates have led to the classification of HSV-1 into three genotypes, arbitrarily designated as A, B and C. The prevalence of the HSV-1 gG and gI genotypes and their potential disease association was determined in a large cohort of patients with herpetic keratitis (HK).

Methods: Primary corneal HSV-1 isolates of 178 HK patients were genotyped by a PCR-based restriction fragment length polymorphism method targeting the viral genes US4 and US7.

Results: Genotype B was more frequently expressed by the corneal HSV-1 isolates compared with genotypes A and C. Fifty-five of 178 corneal isolates (31%) had different genotypes in both loci. No clinically relevant associations were observed between the HSV-1 genotypes and disease outcome in the HK patients studied.

Conclusions: The data presented demonstrate a high frequency of recombinant corneal HSV-1 isolates and suggest that clinical outcome of HSV-1-induced keratitis is independent of a gG or gI genotype.

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Herpes simplex virus type 1 (HSV-1) infection is a leading cause of visual morbidity in developed countries. The prevalence of ocular HSV-1 disease, mainly affecting the anterior segment of the eye, has been estimated at 149 cases per 100 000 US citizens.1 Corneal HSV-1 infections cause a spectrum of clinical manifestations ranging from blepharitis, acute infectious epithelial keratitis (IEK) to the potentially blinding chronic inflammatory disease herpetic stromal keratitis (HSK). Patients who have had a corneal HSV-1 infection risk recurrent corneal disease throughout life. Particularly prolonged or recurrent episodes of herpetic keratitis (HK) can result in the development of HSK.2

The pathogenesis of ocular HSV-1 disease is dependent on a number of factors, including the genetic constitution of the virus and host, and the host’s immune system. Current knowledge on the pathogenesis of HK is largely based on studies on experimental mouse models, which have provided important insights into the role of host factors involved in both the prevention and immunopathology of HK.3 4 Differences among inbred mouse strains have a strong influence on the reactivation of HSV-1 and the disease phenotype.5 6 In humans, specific HLA alleles, polymorphisms in the genes encoding interleukin 10, apolipoprotein E and toll-like receptors, and a gene located within a region on the long arm of chromosome 21 have been described to affect HSV-1 disease.712 By comparison, only a few studies have addressed the role of the viral genotype in the disease process.13 14 Studies in experimental animal models have shown that different clinical patterns of herpetic ocular disease may be attributed at least in part to the differing biological behaviour of specific HSV-1 strains.1519

Recently, Norberg and colleagues have described that clinical HSV-1 isolates can be divided into three distinct genotypes, arbitrarily designated as genotype A, B and C.20 The classification was based on DNA sequencing of the viral genes, located in the unique short (US) region of the HSV-1 genome, encoding glycoprotein G (gG; gene US4), gI (US7) and gE (US8). The gE and gI proteins have been shown to form a complex involved in cell-to-cell spread and the binding of IgG to its Fc receptor. The gG protein is essential for virus entry through the apical surfaces of polarised cells.21 Genotype-specific synonymous and non-synonymous nucleotide substitutions have been identified; the latter mutations may modify the biological activity of the encoding proteins.20 21 The contribution of the viral genotype to the clinical entity or outcome of HSV-1-induced diseases is still unknown.

The objective of the current study was twofold. First, we determined the prevalence of the US4 and US7 HSV-1 genotypes in a large cohort of 178 unrelated HSV-1 isolates recovered from affected corneas of HK patients. Second, we explored the possible association between the viral genotypes and the clinical outcome of HSV-1-induced keratitis.

PATIENTS AND METHODS

Patients and clinical specimens

Corneal swabs (n = 178) were obtained for diagnostic purposes at the Rotterdam Eye Hospital (Rotterdam, The Netherlands) between 1981 and 2002 from otherwise healthy patients with suspected herpetic corneal lesions. The classification of HK was defined on clinical criteria.2 The corneal swabs were inoculated on human embryonic lung fibroblasts. The virus was harvested when approximately 75% of the monolayer showed a viral cytopathic effect, typed for HSV-1 or -2 by immunocytology and PCR, and subsequently culture supernatant frozen in aliquots as described previously.22 The clinical items scored retrospectively were previous history of ocular disease, gender, age, number of recurrences, regimen and response to therapy (steroid and/or antiviral), cornea transplantation(s), glaucoma, anatomic location of the lesion and clinical presentation of HK at time of virus culture and at the end of the follow-up period (6 (SD 2.9) years). The study was performed according to the tenets of the Declaration of Helsinki, approved by the local Ethical Committee, and written informed consent was obtained from all patients.

HSV-1 genotyping

Total DNA was isolated from the virus culture supernatants using the MagNA Pure LC total nucleic acid isolation kit and the MagNAPure LC isolation station according to the manufacturer’s instructions (Roche diagnostics, Almere, The Netherlands). Classification of the corneal HSV-1 isolates into the three distinct genotypes A, B and C was based on a PCR-based restriction fragment length polymorphism (RFLP) method as described in detail previously.23 In brief, the PCR was carried out in a 50 μl volume containing 2.5 U Taq-polymerase with the corresponding buffer containing 15 mM MgCl2, 0.5 μM of each of the primers, 200 μM dNTP (all from Roche diagnostics) and 10 μl of the purified DNA sample on a Peltier Thermal Cycler 200 DNA Engine (Bio-Rad, Veenendaal, The Netherlands). The gG gene (US4) was amplified using the following primers: forward 5′-GACTCTCCCACCGCCATCAG-3′ (HSV-1 genome sequence nucleotide positions 136912–136932; GenBank accession number NC_001806), reverse 5′-TGTCTTCGGGCGACTGGTCT-3′ (nucleotide positions 137162–137181). For the gI gene (US7) the following primers were used: forward 5′-CCTGCTTATTCTCGGGGAGCTTC-3′ (nucleotide positions 139934–139957) and reverse 5′-AGCAGTTTCGGGTCGCAGGA-3′ (nucleotide positions 140325–140344). Genotype classification was done by restriction enzyme (RE) analysis. Ten microlitres of the unpurified PCR product was mixed with PflM I and Dde I (US4 gene) or Sac I and Ple I (US7 gene), supplemented with the correct buffer, bovine serum albumin and water to a final volume of 20 μl (all from New England Biolabs, Leusden, The Netherlands). After incubation for 3 h at 37°C, the samples were run on 3% Metaphor agarose gel (BioWhittaker) along with a 50 bp DNA marker (Invitrogen, Breda, The Netherlands). Differential RE cleavage patterns of the amplicons, based on genotype-specific point mutations within the RE sites, enabled accurate classification of the HSV-1 isolate analysed.20 23

Statistics

Comparisons of categorical or numeric variables within the group of patients and the US4 and US7 viral genotypes were done using the chi-square test, Kruskal–Wallis test or one-way analysis of variance with the Bonferroni multiple comparison test. In total, 13 clinical variables were explored in this way.

RESULTS

Recently, Norberg and colleagues described a PCR-RFLP assay for rapid and accurate genotyping of clinical HSV-1 isolates.23 This assay is based on genotype-specific mutations within RE sites of discrete regions within US4 and US7.20 This assay was applied to determine the genotype prevalence among unrelated corneal HSV-1 isolates from 178 HK patients. The HK patient group consisted of 108 men and 70 women (mean age, 50 (22) years). The HK clinical entities included IEK (n = 63), HSK (n = 81), blepharitis (n = 24) and keratouveitis (n = 10). Based on the genotyping target US4, 46 isolates were classified as genotype A, 104 isolates were genotype B, and 28 isolates were genotype C. Based on the US7 gene, 52 isolates were classified as genotype A, 86 isolates were genotype B, and 40 isolates were genotype C (table 1). Interestingly, 55 out of 178 isolates (31%) had different genotype identities in the two genotyping targets, suggesting that these isolates are intergenic recombinants. The most prevalent combinations between US4 and US7 found were A/B (n = 14), B/A (n = 21) and B/C (n = 14). Contrastingly, the US4/US7 combinations A/C (n = 2), C/A (n = 1) and C/B (n = 3) were less common (table 1).

Table 1 Results of cornea HSV-1 isolate genotyping based on HSV-1 US4 and US7 polymorphisms

Next, we linked the genotype data to the patient’s clinical records to look for a possible association between the patient’s corneal HSV-1 genotype and outcome of disease. Of the 13 clinical variables considered, four variables were identified with at least one or both p values for either US4 or US7 smaller than 0.05. The outcomes of the analyses are shown in table 2 and fig 1A. The relative frequencies of genotypes A and C were significantly lower and higher in the right and left eye of the patient for both the US4 (p = 0.046) and US7 gene (p = 0.028), respectively. In case of US7, a lower proportion of female patients than male HK patients were infected with HSV-1 genotype C (p = 0.035). None of the HK patients who developed HSV-1-induced blepharitis were infected with an US4-based genotype C virus, while this particular genotype was identified among 28 of 154 (18%) of the patients with blepharitis (p = 0.029). The US7-based genotype B was significantly associated with reduced sensitivity of the diseased cornea (p = 0.024; table 2).

Figure 1 Comparison of the corneal herpes simple× virus type 1 (HSV-1) US4 and US7 genotypes, and the combinations theveof, with the age and number of recurrences of the herpetic keratitis patients studied. (A) Corneal HSV-1 isolates of 178 herpetic keratitis patients were genotyped on the HSV-1 US4 and US7 locus and the three alleles of each locus, arbitrarily defined as A, B or C, analysed for the possible association with the age (upper panel) and number of recurrences until end of follow-up (lower panel). (B) US4 and US7 genotypes combined and analysed as in (A). Data were analysed by one-way analysis of variance with Bonferroni multiple comparison testing. Bars represent mean values.
Table 2 Comparison of the corneal herpes simplex virus type 1 US4 and US7 genotypes and clinical parameters of the herpetic keratitis patients studied

All other clinical parameters of interest, either before or at the end of follow-up, including the presence of inflammatory cells in the aqueous chamber (AC), glaucoma, clinical presentation of herpetic keratitis, transplantation of the affected cornea, outcome of therapy, patient’s age and number of recurrences did not show a significant association with either genotype (table 2, fig 1A).

About one-third of the corneal HSV-1 isolates had different genotypes in both loci, raising the possibility that a specific US4 and US7 genotype combination may affect the clinical presentation and outcome of disease. The clinical parameters were evaluated for their possible association with either US4 or US7 genotype combination (n = 9) (table 3 and fig 1B). Except for the anatomic location of the lesion (left or right eye) and blepharitis, no significant association with either US4 or US7 genotype combination was detected (table 3). In comparison, lesions of patients with corneal HSV-1 isolates expressing the genotype combination US4A/US7A were more often located in the left eye (p = 0.038), and none of the 24 patients with an US4C/US7C genotype combination had blepharitis (p = 0.013) (table 3). Patients with an US4B/US7C genotype combination were predominantly males with lesions in their right eye. However, the number of patients expressing this genotype combination is too low to draw conclusions and warrants re-evaluation in a larger group of HK patients.

Table 3 Comparison of the corneal herpes simplex virus type 1 US4 and US7 genotype combinations and clinical parameters of the herpetic keratitis patients studied

DISCUSSION

Herpetic keratitis caused by HSV-1 is the most common infective cause of corneal blindness in developed countries, largely because of its recurrent nature.2 The clinical manifestations of HK vary extensively. The most common presentations are blepharitis and conjunctivitis, with the least common being HSK.1 Despite the development of new therapeutic regimens targeting both the virus and host immune response involved, HSK remains the most devastating manifestation of herpetic keratitis.2 Whereas more than 80% of the adult population have acquired HSV-1, only 0.15% will develop herpetic keratitis. Successful treatment of the disease will benefit from the elucidation of factors, of virus or host origin, predisposing to the development and severity of HK in the population. The recent division of HSV-1 into three distinct genotypes has raised the question of whether either viral genotype may be associated with clinical disease patterns.20

In the present study, we first determined the frequency of the three viral genotypes within a large panel of 178 unrelated corneal HSV-1 isolates of HK patients. The genotype of the isolates was defined by a validated PCR-RFLP method targeting the polymorphic locations within the viral genes US4 and US7.23 The genotype frequencies were not equally distributed. Genotype B was more frequently expressed by the corneal HSV-1 isolates compared with genotypes A and C. The genotype frequencies among HK patients resemble those described recently on 28 Swedish clinical isolates obtained from patients with various diseases including genital and oral HSV-1 infections.20 23 Although these data suggest that neither genotype predisposes to the development of HK, a larger cohort of patients with other HSV-1 diseases, including patients from other geographical locations in the world, are warranted to validate this assumption.

Whereas the majority of the corneal HSV-1 isolates displayed a similar genotype for both the US4 and US7 gene, the genotypes of about one-third of the isolates were disparate. The data suggest that the latter isolates are intergenic recombinants with recombination points located between the respective genes. A similar high frequency of intergenic recombinants has been reported previously, suggesting the relative instability of the HSV-1 genome between both genes.20 23 Among the intergenic recombinant corneal HSV-1 isolates, combinations of genotypes A and B were over-represented, whereas genotype A and C combinations were only rarely identified. These differences may be due in part to the genetic distance between genotype A and B compared with C.20 Alternatively, genotypes A and B may have been introduced in The Netherlands earlier than genotype C, facilitating genotypes A and B strains to recombine over a longer time frame.

As for other viral infections, the clinical outcome of HK depends on the interaction between virus and host.2 4 Based on previous studies in experimental HK mouse models, the genetic make-up of the virus may affect the pathology of the disease.1417 Consequently, the second objective of the present study was to determine the possible association between the viral genotypes identified and the patients’ clinical parameters. Among the set of clinical data analysed, several clinical parameters appeared to be associated to some extent with a specific genotype. However, the clinical relevance of the associations found is questionable. Particularly, the statistically significant associations between genotype C and US4AUS7A and disease in the right eye and, only in the case of US7, its high prevalence in male patients than in female HK patients are inexplicable. Additionally, two other disease-related parameters including blepharitis and reduced corneal sensitivity of the diseased eye tended to be more or less associated with specific viral genotypes. This is an explorative study in order to look for clinically relevant associations or clinically relevant combinations of associations. For that reason, no correction for multiple testing was applied. A formal multiple testing correction for 13 variables and two tests per variable would have led to such a small alpha value per test that relevant (combinations of) associations would have certainly been overlooked as non-significant. Of course, the price to be paid here is that one should afterwards critically judge the resulting (combinations of) associations for clinical relevance, as they could have quite easily arisen by chance.

Hitherto, the data do not provide conclusive evidence that the US4 and US7 genotypes predispose to the clinical outcome of HK. The data presented leave us with the question as to which viral genotypes determine the outcome of disease in HK patients and how to identify these candidate markers. The extensively studied HSK mouse model will be of value to screen potential virulence genes before in-depth analyses on HSK patients. The disease closely mimics the immunopathology of human HSK, and the application of inbred mouse strains enables the plethora of host factors involved in the disease process to be standardised.1 2 Furthermore, experiments in the HSK mouse model have shown HSV-1 strain differences in reactivation and pathogenesis. For instance, the HSV-1 reference strains McKrae and 17syn+ reactivate with similar frequency in mouse and rabbit models of disease, whereas strain KOS does not reactivate in these animals.2426 Moreover, corneal infection of A/J mice with the HSV-1 strains KOS and RE induces HSK, which is orchestrated by different effector cells. The disease in KOS and RE infected A/J mice is dependent on CD8 and CD4 T cells, respectively.27 Thus, virulence genes encoded by HSV-1 truly affect the severity of corneal disease in mice. Among the 80 HSV-1 genes, only a few have been formally tested in mice for their role in HSV keratitis. Polymorphisms within the HSV-1 genes UL9, UL33, UL36, UL41, UL42 and US1 have been show to affect corneal virulence in mice and are of interest for inclusion in human studies.14 However, it should be kept in mind that the genetics of HSV-1 virulence is complicated by the constellation of genes encoded by each strain. Both the interactions between viral proteins and viral-host protein interactions determine the outcome of disease. Future studies are mandatory to identify and determine the role of HSV-1 virulence genes involved in HSV-1-induced keratitis. This may eventually provide new diagnostic means to identify and treat patients more effectively that are at risk of developing severe herpetic keratitis.

REFERENCES

Footnotes

  • Funding: The study was financially supported by grants from Stichting Wetenschappelijk Onderzoek Oogziekenhuis, Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, and Hoornvlies Stichting Nederland, Stichting OOG and Stichting voor Ooglijders (RD, JMvD).

  • Competing interests: None.

  • Ethics approval: The study was performed according to the tenets of the Declaration of Helsinki, approved by the local ethical committee.

  • Patient consent: Obtained.