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1.  Association Study in a South Indian Population Supports rs1015213 as a Risk Factor for Primary Angle Closure 
Purpose.
Three loci defined by single nucleotide polymorphisms (SNPs) rs11024102 in PLEKHA7, rs3753841 in COL11A1, and rs1015213 between the PCMTD1 and ST18 genes, recently have been associated with primary angle closure glaucoma (PACG). We explored the genetic association of these SNPs with subtypes of primary angle closure in a South Indian population.
Methods.
The study included three case definitions: primary angle closure/primary angle closure glaucoma (PAC/PACG, N = 180); primary angle closure suspect (PACS, N = 171), and a combined any-angle closure group. Controls consisted of 411 individuals from South India. Genotyping for all three SNPs was performed using the TaqMan allelic discrimination assay. Genetic association was estimated using a χ2 test statistics and logistic regression.
Results.
Among the three studied SNPs, significant genetic association was identified for rs1015213 in the PAC/PACG (P = 0.002) and any-angle closure (P = 0.003) analyses. However, no significant genetic association was seen when in PACS subjects (P = 0.052). SNPs rs3753841 and rs11024102 showed no evidence of genetic association with angle-closure phenotypes (P > 0.05) in South Indian participants.
Conclusions.
In our study, rs1015213 (located in the intergenic region between PCMTD1 and ST18) was associated significantly with PAC/PACG, confirming prior reports of an association between this region and angle closure glaucoma. Further work with a larger sample size is necessary to confirm the importance of COL11A1 and PLEKHA7 in the pathogenesis of glaucoma.
Our study confirms the association of a newly identified genetic marker, rs1015213 (PCMTD1-ST18) with subtypes of primary angle closure and fails to support associations with two other markers (rs3753841 and rs11024102) in a South Indian population.
doi:10.1167/iovs.13-12186
PMCID: PMC3747718  PMID: 23847314
PACG; PCMTD1-ST18; South India
2.  Mutational Screening of LCA Genes Emphasizing RPE65 in South Indian Cohort of Patients 
PLoS ONE  2013;8(9):e73172.
Background
Leber congenital amaurosis (LCA) is the most severe form of inherited retinal visual impairment in children. So far, mutations in more than 20 genes have been known to cause LCA and among them, RPE65 is a suitable candidate for gene therapy. The mutational screenings of RPE65 and other LCA genes are requisite in support of emerging gene specific therapy for LCA. Therefore, we have carried out a comprehensive LCA genes screening using a combined approach of direct sequencing and DNA microarray based Asper chip analysis.
Methodology/Principal Findings
Thirty clinically diagnosed index LCA cases from Southern India were screened for coding and flanking intronic regions of RPE65 through direct sequencing. Among thirty, 25 cases excluded from RPE65 mutations were subjected to Asper chip analysis, testing 784 known pathogenic variations in 15 major LCA genes. In RPE65 screening, four different pathogenic variations including two novel (c.361insT & c.939T>A) and two known (c.394G>A & c.361delT) mutations were identified in five index cases. In the chip analysis, seven known pathogenic mutations were identified in six index cases, involving genes GUCY2D, RPGRIP1, AIPL1, CRX and IQCB1. Overall, 11 out of 30 LCA cases (36.6%) revealed pathogenic variations with the involvement of RPE65 (16.6%), GUCY2D (10%), RPGRIP1 (3.3%), AIPL1 (3.3%) and CRX & IQCB1 (3.3%).
Conclusions/Significance
Our study suggests that such combined screening approach is productive and cost-effective for mutation detection and can be applied in Indian LCA cohort for molecular diagnosis and genetic counselling.
doi:10.1371/journal.pone.0073172
PMCID: PMC3774716  PMID: 24066033
3.  Polymorphisms in ARMS2/HTRA1 and Complement Genes and Age-Related Macular Degeneration in India: Findings from the INDEYE Study 
Purpose.
Association between genetic variants in complement factor H (CFH), factor B (CFB), component 2 (C2), and in the ARMS2/HTRA1 region with age-related macular degeneration (AMD) comes mainly from studies of European ancestry and case-control studies of late-stage disease. We investigated associations of both early and late AMD with these variants in a population-based study of people aged 60 years and older in India.
Methods.
Fundus images were graded using the Wisconsin Age-Related Maculopathy Grading System and participants assigned to one of four mutually exclusive stages based on the worse affected eye (0 = no AMD, 1–3 = early AMD, 4 = late AMD). Multinomial logistic regression was used to derive risk ratios (RR) accounting for sampling method and adjusting for age, sex, and study center.
Results.
Of 3569 participants, 53.2% had no signs of AMD, 45.6% had features of early AMD, and 1.2% had late AMD. CFH (rs1061170), C2 (rs547154), or CFB (rs438999) was not associated with early or late AMD. In the ARMS2 locus, rs10490924 was associated with both early (adjusted RR 1.22, 95% confidence interval [CI]: 1.13–1.33, P < 0.0001) and late AMD (adjusted RR 1.81, 95% CI: 1.15–2.86; P = 0.01); rs2672598 was associated only with early AMD (adjusted RR 1.12, 95% CI: 1.02–1.23; P = 0.02); rs10490923 was not associated with early or late AMD.
Conclusions.
Two variants in ARMS2/HTRA1 were associated with increased risk of early AMD, and for one of these, the increased risk was also evident for late AMD. The study provides new insights into the role of these variants in early stages of AMD in India.
We report results from a genetic association study of early AMD in an Indian population. Two variants in the ARMS/HTRA1 region were associated with early AMD but variants in C2, CFH, and CFB were not.
doi:10.1167/iovs.12-10073
PMCID: PMC3490538  PMID: 23060141
4.  Investigation of VSX1 sequence variants in South Indian patients with sporadic cases of keratoconus 
BMC Research Notes  2013;6:103.
Background
The involvement of VSX1 gene for the genetic basis of keratoconus is unclear and controversial. The genetic screening of VSX1 from different ethnic populations can enlighten this subject. The aim of the present study is to investigate the role of VSX1 gene in patients with sporadic cases of keratoconus from South India.
Methods
The VSX1 gene coding regions, including exon-intron boundaries were screened by direct sequencing analysis in 117 sporadic cases of keratoconus. The identified variations were also analyzed in 108 ethnic matched healthy blood donors.
Results
In the VSX1 gene screening, no pathogenic mutation was identified, whereas we could find the presence of four reported single nucleotide polymorphisms; c.546A>G (rs12480307), c.627+23G>A (rs6138482), c.627+84T>A (rs56157240) and c.504-24C>T (IVS3-24C). These variations were observed in similar frequency between cases and controls.
Conclusions
The lack of VSX1 pathogenic variations in a large number of unrelated sporadic keratoconus patients tend to omit its role, and corroborate the involvement of other genetic, environmental or behavioural factors in the development of this complex disorder.
doi:10.1186/1756-0500-6-103
PMCID: PMC3608990  PMID: 23506487
KTCN - Keratoconus; VSX1 - Visual system homeobox 1; SNP - Single nucleotide polymorphism
5.  EPHA2 Polymorphisms and Age-Related Cataract in India 
PLoS ONE  2012;7(3):e33001.
Objective
We investigated whether previously reported single nucleotide polymorphisms (SNPs) of EPHA2 in European studies are associated with cataract in India.
Methods
We carried out a population-based genetic association study. We enumerated randomly sampled villages in two areas of north and south India to identify people aged 40 and over. Participants attended a clinical examination including lens photography and provided a blood sample for genotyping. Lens images were graded by the Lens Opacification Classification System (LOCS III). Cataract was defined as a LOCS III grade of nuclear ≥4, cortical ≥3, posterior sub-capsular (PSC) ≥2, or dense opacities or aphakia/pseudophakia in either eye. We genotyped SNPs rs3754334, rs7543472 and rs11260867 on genomic DNA extracted from peripheral blood leukocytes using TaqMan assays in an ABI 7900 real-time PCR. We used logistic regression with robust standard errors to examine the association between cataract and the EPHA2 SNPs, adjusting for age, sex and location.
Results
7418 participants had data on at least one of the SNPs investigated. Genotype frequencies of controls were in Hardy-Weinberg Equilibrium (p>0.05). There was no association of rs3754334 with cataract or type of cataract. Minor allele homozygous genotypes of rs7543472 and rs11260867 compared to the major homozygote genotype were associated with cortical cataract, Odds ratio (OR) = 1.8, 95% Confidence Interval (CI) (1.1, 3.1) p = 0.03 and 2.9 (1.2, 7.1) p = 0.01 respectively, and with PSC cataract, OR = 1.5 (1.1, 2.2) p = 0.02 and 1.8 (0.9, 3.6) p = 0.07 respectively. There was no consistent association of SNPs with nuclear cataract or a combined variable of any type of cataract including operated cataract.
Conclusions
Our results in the Indian population agree with previous studies of the association of EPHA2 variants with cortical cataracts. We report new findings for the association with PSC which is particularly prevalent in Indians.
doi:10.1371/journal.pone.0033001
PMCID: PMC3297613  PMID: 22412971
6.  Association analysis of nine candidate gene polymorphisms in Indian patients with type 2 diabetic retinopathy 
BMC Medical Genetics  2010;11:158.
Background
Diabetic retinopathy (DR) is classically defined as a microvasculopathy that primarily affects the small blood vessels of the inner retina as a complication of diabetes mellitus (DM).It is a multifactorial disease with a strong genetic component. The aim of this study is to investigate the association of a set of nine candidate genes with the development of diabetic retinopathy in a South Indian cohort who have type 2 diabetes mellitus (T2DM).
Methods
Seven candidate genes (RAGE, PEDF, AKR1B1, EPO, HTRA1, ICAM and HFE) were chosen based on reported association with DR in the literature. Two more, CFH and ARMS2, were chosen based on their roles in biological pathways previously implicated in DR. Fourteen single nucleotide polymorphisms (SNPs) and one dinucleotide repeat polymorphism, previously reported to show association with DR or other related diseases, were genotyped in 345 DR and 356 diabetic patients without retinopathy (DNR). The genes which showed positive association in this screening set were tested further in additional sets of 100 DR and 90 DNR additional patients from the Aravind Eye Hospital. Those which showed association in the secondary screen were subjected to a combined analysis with the 100 DR and 100 DNR subjects previously recruited and genotyped through the Sankara Nethralaya Hospital, India. Genotypes were evaluated using a combination of direct sequencing, TaqMan SNP genotyping, RFLP analysis, and SNaPshot PCR assays. Chi-square and Fisher exact tests were used to analyze the genotype and allele frequencies.
Results
Among the nine loci (15 polymorphisms) screened, SNP rs2070600 (G82S) in the RAGE gene, showed significant association with DR (allelic P = 0.016, dominant model P = 0.012), compared to DNR. SNP rs2070600 further showed significant association with DR in the confirmation cohort (P = 0.035, dominant model P = 0.032). Combining the two cohorts gave an allelic P < 0.003 and dominant P = 0.0013). Combined analysis with the Sankara Nethralaya cohort gave an allelic P = 0.0003 and dominant P = 0.00011 with an OR = 0.49 (0.34 - 0.70) for the minor allele. In HTRA1, rs11200638 (G>A), showed marginal significance with DR (P = 0.055) while rs10490924 in LOC387715 gave a P = 0.07. No statistical significance was observed for SNPs in the other 7 genes studied.
Conclusions
This study confirms significant association of one polymorphism only (rs2070600 in RAGE) with DR in an Indian population which had T2DM.
doi:10.1186/1471-2350-11-158
PMCID: PMC2994838  PMID: 21067572
7.  Genetic analysis of patients with Fuchs endothelial corneal dystrophy in India 
BMC Ophthalmology  2010;10:3.
Background
Mutations in COL8A2 gene which encodes the collagen alpha-2 (VIII) chain have been identified in both familial and sporadic cases of Fuchs endothelial corneal dystrophy (FECD). Heterozygous mutations in the SLC4A11 gene are also known to cause late-onset FECD. Therefore we screened for COL8A2, SLC4A11 gene variants in Indian FECD patients.
Methods
Eighty patients with clinically diagnosed FECD and 100 age matched normal individuals were recruited. Genomic DNA was isolated from peripheral blood leukocytes. Mutations in COL8A2, SLC4A11 coding regions were screened using bi-directional sequencing. Fischer's exact test or Pearson's chi squared test were used to predict the statistical association of genotypes with the phenotype.
Results
Screening of COL8A2 gene revealed 2 novel c.1610G>A, c.1643A>G and 3 reported variations c.112G>A, c.464G>A and c.1485G>A. In SLC4A11 gene, novel c.1659C>T, c.1974C>T and reported c.405G>A, c.481A>C and c.639G>A variants were identified. However all the variations in both the genes were also present in unaffected controls.
Conclusions
This is the first study analysing COL8A2 gene in Indian patients with FECD. No pathogenic mutations were identified in COL8A2. Merely silent changes, which showed statistically insignificant association with FECD, were identified in the screening of SLC4A11 gene. These results suggest that COL8A2, SLC4A11 genes may not be responsible for FECD in patients examined in this study.
doi:10.1186/1471-2415-10-3
PMCID: PMC2834660  PMID: 20144242
8.  Spectrum of candidate gene mutations associated with Indian familial oculocutaneous and ocular albinism 
Molecular Vision  2010;16:1514-1524.
Purpose
Albinism is a group of genetic disorders, showing a broad spectrum of different phenotypes. The purpose of this study was to screen known candidate genes for oculocutaneous albinism (OCA) and ocular albinism (OA) mutations in Indian patients.
Methods
Blood samples were collected from 23 probands and 13 affected family members from 23 genetically unrelated Indian families (22 diagnosed as OCA and 1 diagnosed as OA) and analyzed by bidirectional DNA sequencing of the classic OCA genes— tyrosinase (TYR, or oculocutaneous albinism IA), pink eyed dilution (P; or oculocutaneous albinism II (OCA2]), tyrosinase-related protein 1 (TYRP1), solute carrier family 45, member 2 (SLC45A2; or membrane-associated transporter protein [MATP])—and the OA1 gene, G protein-coupled receptor 143 (GPR143).
Results
Three missense mutations, c. 715 C>T (R239W), c. 896 G>A (R299H), c.1255 G>A (G419R), and one termination c. 832 C>T (R278X), were identified in TYR, as well as one novel mutation, c.1453 G>A (G485R) in P. One novel single nucleotide polymorphism (SNP) was identified in both TYR and P; few reported SNPs were identified. The G>A base substitution caused relatively conservative amino acid changes, which altered glycine to arginine residues within the topological domain. The novel OCA2 mutation was not present in 100 control samples. This study identified two probands carrying mutations alone, 16 probands carrying SNPs alone, 4 probands carrying both mutations and SNPs and only one proband carrying neither mutations nor SNPs.
Conclusions
Although sequence analysis was performed with all five candidate genes, only four (17.39%) of the 23 probands showed mutations in TYR and 2 probands (8.69%) showed an unreported novel mutation in P. Genetic counseling for phenotypical diagnosis and genetic mutation screening of these genes will help to minimize the incidence of OCA and OA in future generations.
PMCID: PMC2925902  PMID: 20806075
9.  Mutations that are a common cause of Leber congenital amaurosis in northern America are rare in Southern India 
Molecular Vision  2009;15:1781-1787.
Purpose
To test patients from southern India for the presence of mutations that most commonly cause Leber congenital amaurosis (LCA) in northern America.
Methods
A review of the literature identified 177 unique LCA causing mutations in eight different genes: aryl hydrocarbon receptor interacting protein-like 1 (AIPL1), crumbs homolog 1 (CRB1), cone-rod homeobox (CRX), guanylate cyclase 2D (GUCY2D), nephronophthisis 6 (NPHP6), retinol dehydrogenase 12 (RDH12), retinal pigment epithelium-specific protein 65 kDa (RPE65), and retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1). Allele-specific ligation assay and bidirectional sequencing were used to test 38 unrelated LCA patients from southern India for 104 of these mutations, which contribute to more than 30% of the LCA cases in a northern American population.
Results
Only one participant was found to harbor one of the 104 mutations in the allele-specific assay (homozygous RPE65 Tyr368His). A mutation that was not part of the assay (homozygous RPE65 Tyr143Asp) was incidentally detected in a second patient when an equivocal signal from one allele on the assay was followed up with automated DNA sequencing.
Conclusions
Mutations that contribute to 30% of the LCA cases in northern America were detected in only 2.6% of LCA cases in our cohort from southern India. There were no instances of IVS26 c.2991+1655 A>G in NPHP6, the most commonly detected mutation in LCA. These data suggest that LCA in India is caused primarily by a different set of mutations in the same genes associated with disease in northern America, or by mutations in other genes that have not yet been discovered. Therefore, mutation-specific assays developed for European and northern American cohorts may not be suited for testing LCA patients from India or other ethnically distinct populations.
PMCID: PMC2742639  PMID: 19753312
10.  Crystallin gene mutations in Indian families with inherited pediatric cataract 
Molecular Vision  2008;14:1157-1170.
Purpose
Pediatric cataract is the most common form of treatable childhood blindness and is both clinically and genetically heterogeneous. Autosomal dominant and recessive forms of cataract have been reported to be caused by mutations in 22 different genes so far. Of the cataract mutations reported to date, about half the mutations occur in crystallins, a quarter of the mutations in connexins, and the remainder is evenly divided between intrinsic membrane proteins, intermediate filament proteins, and transcription factors. This study is aimed at identification of the spectrum and frequency of crystallin gene mutations in cataractous patients in an Indian population.
Methods
Genetic analysis was extended to screen the entire coding region of the CRYAA, CRYAB, CRYBA1, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGC, CRYGD, and CRYGS genes using single stranded conformational polymorphism (SSCP) analysis as a screening technique followed by direct sequencing of all subjects that displayed an electrophoretic shift.
Results
This report describes the first simultaneous mutation analysis of 10 crystallin genes in the same population, represented by 60 south Indian families. The analysis allowed the identification of causative mutations in 10 of the families (three novel and six reported). This includes six missense mutations (CRYAA-R12C, R21W, R54C, CRYAB- A171T, CRYGC-R168W, CRYGS- S39C), two nonsense mutations (CRYBB2- Q155X, CRYGD- R140X), and one splice mutation, which was identified in two families (CRYBA1-IVS3+1G>A).
Conclusions
Crystallin mutations are responsible for 16.6% of the inherited pediatric cataract in this population. As causative mutations have not been found in many of the families analyzed, this study suggests the presence of further novel genes or sequence elements involved in the pathogenesis of cataract in these families.
PMCID: PMC2435160  PMID: 18587492
11.  Investigation of founder effects for the Thr377Met Myocilin mutation in glaucoma families from differing ethnic backgrounds 
Molecular Vision  2007;13:487-492.
Purpose
The aim of this study was to determine if there is a common founder for the Thr377Met myocilin mutation in primary open angle glaucoma (POAG) families with various ethnic backgrounds.
Methods
Genomic DNA of 24 POAG-affected individuals from nine pedigrees with the Thr377Met mutation and 104 unaffected family members was genotyped with six microsatellite markers and four single nucleotide polymorphisms. The families were from Greece, India, Finland, the USA, and Australia. To assess the degree of linkage disequilibrium across MYOC in the general population we also investigated data generated from the HapMap consortium.
Results
Three distinct haplotypes associated with the Thr377Met myocilin mutation were identified. The families from the USA and Greece, as well as the three Australian families originating from Greece and the former Yugoslavian Republic of Macedonia had one common haplotype. Interestingly, however, HapMap data suggest that linkage disequilibrium across MYOC was not strong.
Conclusions
The Thr377Met myocilin mutation has arisen at least three separate times. Evidence for genetic founder effects in this prevalent age-related, yet heterogeneous, disease has important implications for future gene identification strategies.
PMCID: PMC2649311  PMID: 17417609
12.  Genetic variations in the hotspot region of RS1 gene in Indian patients with juvenile X-linked retinoschisis 
Molecular Vision  2007;13:611-617.
Purpose
X-linked juvenile retinoschisis (XLRS) is the leading cause of macular degeneration in males. This condition is caused by mutations in the RS1 gene and is, characterized by schisis within the retina. The purpose of this study was to identify the mutations in the RS1 gene associated with XLRS in an Indian cohort.
Methods
The coding region of RS1 was analyzed for mutations by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and restriction fragment length polymorphism (RFLP) analysis in six unrelated subjects clinically diagnosed as having XLRS and in their available family members. Direct sequencing was performed for all samples that displayed an electrophoretic mobility shift in SSCP gel.
Results
Mutation analysis of RS1 gene revealed five mutations in exon 6 like c.574C>T, c.583A>G, c.608C>T, c.617G>A, and c.637C>T, respectively, among them four missense mutations, one nonsense mutation, and two novel sequence variations. These mutations were found in individuals who exhibited clinical features of bilateral foveal and peripheral retinoschisis consistent with XLRS. The mutations were absent in the 100 age matched control samples analyzed.
Conclusions
This is the first report of mutations in RS1 to be associated with XLRS in the Indian population. The identified genetic variations, phenotype and genotype correlations were consistent with other studies. Identification of the causative mutation in patients with XLRS is helpful in confirming the diagnosis and in counseling of family members.
PMCID: PMC2669507  PMID: 17515881
13.  Identification of novel mutant PAX6 alleles in Indian cases of familial aniridia 
BMC Ophthalmology  2006;6:28.
Background
Haploinsufficiency at the PAX6 locus causes aniridia, a panocular eye condition characterized by iris hypoplasia and a variety of other anterior and posterior eye defects leading to poor vision. This study was performed to identify novel PAX6 mutations that lead to familial aniridia in Indian patients.
Methods
Genomic DNA was isolated from affected individuals (clinically diagnosed aniridia) from nine unrelated aniridic pedigrees, unaffected family members, and unrelated normal controls. The coding regions of PAX6 were amplified and subjected to single strand conformation polymorphism (SSCP) gel analysis, and direct cloning and sequencing.
Results
SSCP band shifts, indicative of DNA base pair mutations, were observed in five of these unrelated families. Four mutations were shown to be previously unreported insertion or deletions in PAX6, leading to frameshifts. These new mutations were c.1174delTG (in exon 10), c.710delC (exon 6), c.406delTT (exon 5) and c.393insTCAGC (exon 5). The other nonsense mutation, a transition (c.1080C>T) in exon 9, has been reported previously as a mutation hotspot for PAX6 in other ethnic pedigrees. All mutant alleles transmitted through aniridic individuals in each family.
Conclusion
These new deletions and an insertion create frameshifts, which are predicted to introduce premature termination codons into the PAX6 reading frame. The genetic alterations carried by affected individuals are predicted to lead to loss-of-function mutations that would segregate in an autosomal dominant manner to subsequent generations. This is the first report of the 'hotspot' c.1080C>T transition from Indian families.
doi:10.1186/1471-2415-6-28
PMCID: PMC1544350  PMID: 16803629
14.  PAX6 gene variations associated with aniridia in south India 
Background
Mutations in the transcription factor gene PAX6 have been shown to be the cause of the aniridia phenotype. The purpose of this study was to analyze patients with aniridia to uncover PAX6 gene mutations in south Indian population.
Methods
Total genomic DNA was isolated from peripheral blood of twenty-eight members of six clinically diagnosed aniridia families and 60 normal healthy controls. The coding exons of the human PAX6 gene were amplified by PCR and allele specific variations were detected by single strand conformation polymorphism (SSCP) followed by automated sequencing.
Results
The sequencing results revealed novel PAX6 mutations in three patients with sporadic aniridia: c.715ins5, [c.1201delA; c.1239A>G] and c.901delA. Two previously reported nonsense mutations were also found: c.482C>A, c.830G>A. A neutral polymorphism was detected (IVS9-12C>T) at the boundary of intron 9 and exon 10. The two nonsense mutations found in the coding region of human PAX6 gene are reported for the first time in the south Indian population.
Conclusion
The genetic analysis confirms that haploinsuffiency of the PAX6 gene causes the classic aniridia phenotype. Most of the point mutations detected in our study results in stop codons. Here we add three novel PAX6 gene mutations in south Indian population to the existing spectrum of mutations, which is not a well-studied ethnic group. Our study supports the hypothesis that a mutation in the PAX6 gene correlates with expression of aniridia.
doi:10.1186/1471-2350-5-9
PMCID: PMC419353  PMID: 15086958
15.  Attenuated, Replication-Competent Herpes Simplex Virus Type 1 Mutant G207: Safety Evaluation in Mice 
Journal of Virology  2000;74(8):3832-3841.
Herpes simplex virus type 1 (HSV-1) mutants that are attenuated for neurovirulence are being used for the treatment of cancer. We have examined the safety of G207, a multimutated replication-competent HSV-1 vector, in mice. BALB/c mice inoculated intracerebrally or intracerebroventricularly with 107 PFU of G207 survived for over 20 weeks with no apparent symptoms of disease. In contrast, over 80% of animals inoculated intracerebrally with 1.5 × 103 PFU of HSV-1 wild-type strain KOS and 50% of animals inoculated intracerebroventricularly with 104 PFU of wild-type strain F died within 10 days. Similarly, after intrahepatic inoculation of G207 (3 × 107 PFU) all animals survived for over 10 weeks, whereas no animals survived for even 1 week after inoculation with 106 PFU of KOS. After intracerebroventricular inoculation, LacZ expression was initially observed in the cells lining the ventricles and subarachnoid space; expression decreased until almost absent within 5 days postinfection, with no apparent loss of ependymal cells. G207 DNA could be detected by PCR in the brains of mice 8 weeks after intracerebral inoculation; however, no infectious virus could be detected after 2 days. As a model for latent HSV in the brain, we used survivors of an intracerebral inoculation of HSV-1 KOS at the 50% lethal dose. Inoculation of a high dose of G207 at the same stereotactic coordinates did not result in reactivation of detectable infectious virus or symptoms of disease. We conclude that G207 is safe at or above doses that were efficacious in mouse tumor studies.
PMCID: PMC111891  PMID: 10729157
16.  In Vivo Immune Evasion Mediated by the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor 
Journal of Virology  1998;72(7):5351-5359.
Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE339, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE339 expresses gE and gI, is FcγR−, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE339 causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR− mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE339-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.
PMCID: PMC110157  PMID: 9620988

Results 1-16 (16)