Ocular Immunology and Inflammation
ISSN: 0927-3948 (Print) 1744-5078 (Online) Journal homepage: http://www.tandfonline.com/loi/ioii20
Genotypic Detection of Epstein Barr Virus from Clinically Suspected Viral Retinitis Patients in a Tertiary Eye Care Centre, India Madhuravasal Krishnan Janani MS MLT, Jambulingam Malathi PhD, Jyothirmay Biswas MS, Sudharshan Sridharan DO DNB & Hajib Naraharirao Madhavan MD, PhD To cite this article: Madhuravasal Krishnan Janani MS MLT, Jambulingam Malathi PhD, Jyothirmay Biswas MS, Sudharshan Sridharan DO DNB & Hajib Naraharirao Madhavan MD, PhD (2015) Genotypic Detection of Epstein Barr Virus from Clinically Suspected Viral Retinitis Patients in a Tertiary Eye Care Centre, India, Ocular Immunology and Inflammation, 23:5, 384-391, DOI: 10.3109/09273948.2014.968265 To link to this article: http://dx.doi.org/10.3109/09273948.2014.968265
Published online: 17 Oct 2014.
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Date: 28 October 2015, At: 19:59
Ocular Immunology & Inflammation, 2015; 23(5): 384–391 ! Vision Research Foundation ISSN: 0927-3948 print / 1744-5078 online DOI: 10.3109/09273948.2014.968265
ORIGINAL ARTICLE
Genotypic Detection of Epstein Barr Virus from Clinically Suspected Viral Retinitis Patients in a Tertiary Eye Care Centre, India Madhuravasal Krishnan Janani, MS MLT, Jambulingam Malathi, PhD, Jyothirmay Biswas, MS, Sudharshan Sridharan, DO DNB, and Hajib Naraharirao Madhavan, MD, PhD
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Vision Research Foundation, L&T Microbiology Research Centre, Sankara Nethralaya, Chennai, India
ABSTRACT Purpose: To evaluate the diagnostic value of PCR on aqueous humour for detection and genotyping of Epstein Bar Virus in patients with viral retinitis. Methods: 70 AH samples were collected from 20 HIV positive patients with clinically suspected viral retinitis and 25 patients with serpignous choroiditis and 25 AH from patients undergoing cataract surgery. PCR was performed to screen HHV-1 to HHV-5, Mtb and Toxoplasma gondii. Genotype prevalence was confirmed by phylogenetic analysis targetig EBV. Results: EBV was detected in 17 (37.7%) samples. Genotyping to subtype EBV, revealed the circulation of only one subtype (Type 1). PCR results for other infective agents were negative except for the presence of CMV in 5 (11.1%) AH. Conclusion: The application of PCR to detect genotypes can be used as an epidemiological tool for clinical management. To our knowledge this is the first report on genotyping of EBV performed on intra ocular samples. Keywords: Detection, Epstein-Barr virus, genotyping, PCR, viral retinitis
Viral retinitis is an important vision-threatening infectious disease of the retina which can occur in both immunocompetent and immunocompromised or immunodeficient (acquired immunodeficiency syndrome—AIDS)1 individuals. In immunocompetent patients, acute retinal necrosis (ARN) has been recognized as a vision-threatening inflammation caused primarily by the herpes group of viruses.2,3 Epstein-Barr virus has been described in various ocular inflammatory diseases, including multifocal choroiditis in healthy patients.4,5 In immunocompromised patients, various opportunistic viral infections can occur, the most common being cytomegalovirus (CMV) infection. Other less common viruses causing retinal infections include herpes simplex virus (HSV) and varicella zoster virus (VZV).5 Although viral retinitis can usually be identified by the fundus picture and circumstantial evidence of
immunosuppression (use of immunosuppressive drugs or a diagnosis of AIDS), the precise diagnosis and identification of the virus can be done best only with the help of rapid and sensitive virological studies.6 The etiology of viral retinitis was earlier established by demonstrating the presence of antibodies raised against specific viral agents in the aqueous humor and serum samples. With the advent of the polymerase chain reaction (PCR) technique it was possible to detect the specific infectious agents associated with retinitis as antibodies raised against the herpes group of viruses cross-react due to the antigenic similarity among gene products of herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus.7,8 Epstein-Barr virus is one of the important viral pathogens described in the vision-threatening ocular condition called viral retinitis among
Received 26 April 2014; revised 18 September 2014; accepted 18 September 2014; published online 16 October 2014 Correspondence: Dr. Jambulingam Malathi, Vision Research Foundation, Larsen &Toubro Microbiology Research Centre, No.18, College Road, Sankara Nethralaya, Chennai 600006, India. E-mail: [email protected]
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Detection of Epstein Barr Virus 385 immunocompetent patients.9 In immunocompromised patients human cytomegalovirus (HCMV) has been predominantly reported as the causative agent. With the advent of PCR it was possible to accurately identify the infective agent associated with the clinical condition.10 Though many reports exist on the application of PCR for detection of EBV and other herpes viruses from intraocular fluids of patients,9 no reports are available on associated genotypes. Analyzing the genotype of EBV associated with viral retinitis will aid in understanding the phylogeny of the virus and also whether multiple genotypes are associated. In normal healthy individuals detection of EBV genotype 1 is reported and also in cases of infectious mononucleosis. In malignant conditions EBV2 is reported. So far the genotypic prevalence of EBV among ocular clinical specimens collected from otherwise healthy and immnuocompromised patients has not been studied. Therefore, the current study was undertaken to determine the rate of detection of EBV and also to find the genotypic prevalence. To our knowledge, this is the first report investigating the presence of EBV DNA and genotyping in intraocular fluids of patients with retinitis.
MATERIALS AND METHODS
Positive Controls EBV standard strain type 1: Culture infiltrate of Marmoset cell line infected with EBV B958 (National Eye Institute, Bethesda, USA) EBV standard strain type 2: Culture infiltrate of Ag876 cell line (kind gift from Alan Rickinson, Glasgow University, United Kingdom) DNA extraction: DNA extracted from standard strains, tests, and control samples following the manufacture’s instructions of QIAGEN DNA extraction kit (Hilden, Germany).
Polymerase Chain Reaction for Detection of EBV To confirm the presence of EBV, two PCRs targeting the genes that code for EBV-VCA and EBNA1 were standardized and used on all clinical samples. Primers targeting genes that code for VCA were designed using Primer Premier (Biosoft International, USA) based on the consensus sequence obtained with specific sequences of EBV-specific genes submitted in GenBank. The nucleotide sequences of the primers and the expected respective product size are given in Table 1. All primers and PCR reagents were procured from VBC – Biotech Service (Vienna).
Patients and Clinical Specimens All patients and the control population were recruited from Sankara Nethralaya, Chennai, India. The study protocol was approved by the ethics committee of the institute and informed consent was obtained from each patient and control subject. A total of 70 aqueous humor (AH) samples were collected from January 2012 to January 2013 (20 AH samples from 20 HIVpositive patients with clinical picture [necrosis, blurred vision, AC flare] suggestive of retinitis [blind spot, blurred vision and other vision problems, floaters] and atypical necrotizing retinitis, 25 AH samples from 25 patients with serpignous choroidoitis with no significant abnormality on the chest radiography, and 25 AH samples from patients undergoing cataract surgery, which was considered the negative control). The samples were processed immediately and DNA was extracted and subjected for molecular assays.
Processing of Aqueous Humor AH samples (150–200 mL) were collected aseptically in a tuberculin syringe with a 30-gauge needle under aseptic precautions by the ophthalmologist; the sample was transferred into presterilized microfuge tubes and stored at 20 C for DNA extraction. !
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Polymerase Chain Reaction for Genotyping of EBV Uniplex PCR for detection of EBNA2, EBNA3C, and LMP genes was standardized using the EBV1 and EBV2 standard strains. In brief, 10 mL of DNA elute was subjected to amplification of the EBV-specific genes mentioned. Primers targeting genes that code for EBNA2, EBNA3C, and LMP were designed using Primer Premier based on the consensus sequence obtained with specific sequences of EBV-specific genes submitted in GenBank. The nucleotide sequences of the primers and the expected respective product size are given in Table 2. All primers and PCR reagents were procured from VBC – Biotech Service.
Optimization of PCR All PCRs (VCA, EBNA1, EBNA2, and EBNA3C) were optimized to be carried out in the same thermal profile through the gradient PCR temperature profile. The PCR mixture (50 mL) contained 100 mM of dNTP mixture, 10 PCR buffer with 15 mM MgCl2, 1 mM of each forward and reverse primer, and 3 U/mL Taq DNA polymerase. Ten microliters of extracted DNA was added to the PCR reaction mixture. The thermal
386 M. K. Janani et al. TABLE 1. List of primers used for amplification of genes those codes for VCA and EBNA1 of EBV. Strain number and gene target 1. VCA
2. EBNA1
Primer
Primer sequence
Expected base pair
EBV EBV EBV EBV
50 -TTTGGCGTCTCAGGCTAT-30 50 -CGTGGTCGTGTTCCCTCA-30 50 -CGGTGTAACTACCCGCAATG-30 50 -CGTGGTCGTGTTCCCTCA-30
Round 1:172
50 -GCAGTAACAGGTAATCTCTGG-30 50 -ACCAGAAATAGCTGCAGGACC-30 50 -GATTTGGACCCGAAATCTGA-30 50 -CCTCCCTAGAACTGACAATTGG-30
Round 1: 490
FI PP R PP F PP R
EBV up EBV low EBV up (R) EBV low (R)
Round 2:126
Round 2: 336
TABLE 2. List of primers used for amplification of genes those codes for EBNA2, EBNA3C, and LMP of EBV.
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Strain number and gene target
Primer
Primer sequence
Expected base pair
1. EBNA2
EBNA-2 F EBNA-2R
50 -TTTCACCAATACATGAACC-30 50 -TGGCAAAGTGCTGAGAGCAA-30
Type 1:378 Type 2:483
2. EBNA3C
EBNA3C-F
50 -AGAAGGGGAGCGTGTGTTGT-30
Type 1:153
0
EBNA3C-R
5 -GGCTCGTTTTTGACGTCGGC-3 0
0
Type 2:246 0
160 234
3. LMP1
LMP 1 c F LMP 1c R
5 -AGTGATGAACACCACCACGA-3 50 -GTGCGCCTAGGTTTTGAGAG-30
LMP2
LMP 2b F LMP 2b R
50 -TTCTGGTGATGCTTGTGCTC-30 50 -AAAGGGCTGCACCAAGAGTA-30
TABLE 3. The optimized thermal profile followed to amplify PCRs. Steps
1st round
2nd round
Initial denaturation Denaturation Annealing Extension Final extension No. of cycles
94 C for 1 min 94 C for 30 s 51 C for 1 min 72 C for 30 s 72 C for 5 mins 30 cycles
94 C for 1 min 94 C for 30 s 51 C for 1 min 72 C for 30 s 72 C for 5 min 20 cycles
profile is given in Table 3. Two controls (a reagent control and a reaction control) were included in each PCR run. The PCR results were considered valid only when the reagent controls were negative and the specific amplified product was obtained with the positive controls. To prevent amplicon contamination, DNA extraction, PCR cocktail preparation, amplification, and analysis of results were carried out in separate rooms. Visualization of PCR product was done by subjecting 10 mL of amplified reaction mixture to electrophoresis on a 2% agarose gel incorporated with 5 mg mL1 of ethidium bromide in 1 Tris-borate buffer (pH 8.2–8.6) and documented with the gel documentation system (Vilber Lourmat, France).
Specificity of PCR Primers Specificity of the primers was determined against DNA extracted from herpes simplex virus 1, herpes simplex virus 2, cytomegalo virus, varicella zoster virus, Chlamydia trachomatis, Toxoplasma gondii, human DNA (extracted from whole blood), Eubacteria (Propionibacterium acne), and fungus (Candida albicans). Sensitivity of Primers DNA was extracted from EBV standard strain. Serial tenfold dilutions of the DNA were made from 101 to 1010, i.e. 5 mL of DNA with 45 mL of Milli Q water. Ten microliters was taken from each dilution for PCR reaction. PCR for Detection of Genes that Code for VCA and EBNA1 The standardized PCRs were applied on AH samples collected from patients and control samples. Real-time PCR for Determination of Viral Load in Clinical Samples The viral load was estimated in the DNA extracts from AH samples using the RoboGene Quantification Kit (Hilden, Germany). The amplification reaction was carried out following the manufacturer’s instructions. PCR was carried out at 50 C for 30 min Ocular Immunology & Inflammation
Detection of Epstein Barr Virus 387 followed by initial denaturation at 95 C for 15 min followed by 50 cycles of initial denaturation at 95 C for 30 s, annealing at 50 C for 60 s, and extension at 72 C for 30 s. The viral load was expressed in IU/mL. Samples that were positive for CMV by nested PCR were also subjected to CMV real-time PCR using the ‘‘artus CMV RG PCR kit’’ on a Rotor-Gene 3000 instrument as per manufacture’s instruction.
Phylogenetic Analysis The nucleotide sequences of the EBNA2 and EBNA3C PCR-positive amplified products were analyzed by comparison with EBV standard strains nucleotide sequences. The nucleotide sequences were analyzed using BioEdit software. Evolutionary distances were estimated by constructing a phylogram using the UPGMA algorithm with perform bootstrap analysis (Replicates 100) in CLC Main Workbench6.71 software.
Genotyping
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PCR for Detection of Genes That Code for EBNA2, EBNA3C, and LMP of EBV The samples that were confirmed positive by both conventional and real-time PCR were further subjected to genotyping by PCR-based DNA sequencing by PCR targeting EBNA2 and EBNA3C genes. DNA Sequencing The positive PCR-amplified products were further subjected to DNA sequencing to be compared with the standard strain and to determine the homology percentage. Cycle sequencing of the amplified products was performed in a 10-mL reaction volume, containing 0.5 mL of RR mix, 3.5 mL of sequencing buffer, 2 mL of forward primer (1:100 diluted), 2 mL MilliQ water, and 2 mL of the amplified product. Amplification was carried out in the Perkin-Elmer thermocycler using 25 cycles at 96 C for 10 s, at 50 C for 5 s, and at 60 C for 4 min, with initial denaturation at 96 C for 1 min. The cycle-sequenced products were then purified and sequenced using an ABI Prism 3130 AVANT (Applied Biosystems, USA) genetic analyzer, which works based on the principle of Sanger’s dideoxy termination method. The sequences were then analyzed by BioEdit sequence alignment software, and BLAST analysis (www.ncbi.nlm.nih.gov/ BLAST) was done to confirm the sequenced data with the standard strains and to determine the homology percentage.
RESULTS All the standardized PCRs (2 nPCRs, 3 uniplex genotyping PCR) were specific to detect only EBV DNA and the sensitivity was 2.5 pg of EBV DNA for VCA gene (primer set 1), 2.5 pg for EBNA1 gene (primer set 2), 0.63 fg for EBNA2 gene (primer set 3), 7.23 fg for EBNA3C gene (primer set 4), and 6.2 fg for LMP gene (primer set 5), respectively. Among the 45 test samples, 17 clinical samples (37.7%) tested positive for EBV by both VCA and EBNA1 PCR (Figures 1 and 2). The samples that were positive for nPCR were also found to be positive by real time. The real-time PCR results correlated 100% with the conventional PCR. The mean EBV viral load is 84,310.35 IU/mL. The real-time PCR values signifying the viral load in the samples that are positive are listed in Table 4. Genotyping was performed on samples that tested positive by nPCR and real-time PCR using PCR targeting EBNA2, EBNA3C, and LMP genes. EBV type 1 was detected in all 17 samples that were proved positive by both nPCR and by real-time PCR. EBV type 2 was not found any of the samples processed. The application of genotyping PCR to subtype EBV revealed the circulation of only one subtype (type 1) in the ocular sample. The genotyping PCR electrophoretogram of EBNA2 on clinical samples is shown in Figure 3.
FIGURE 1. PCR for detection of the EBV-VCA gene. Agarose gel electrophoresis showing amplification of the EBV-VCA gene of Epstein-Barr virus in DNA extracted from an AH sample. Analysis products amplified: NC2, negative control for second round; NC2, negative control for first round; PCR, lanes 1–10, DNA extracts from samples; PC, EBV DNA extracted from standard strain of B95-8; MW, molecular weight marker (100- to 1000-bp ladder). !
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388 M. K. Janani et al.
FIGURE 2. PCR for detection of the EBNA1 gene. Agarose gel electrophoresis showing amplification of the EBNA1 gene of EpsteinBarr virus in DNA extracted from AH sample. Analysis products amplified: NC2, negative control for second round; NC2, negative control for first round PCR; lanes 1–11, DNA extracts from samples; PC, EBV DNA extracted from standard strain of B95-8; MW, molecular weight marker (100 - to 1000-bp ladder).
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TABLE 4. The relevant clinical details of the patients and results of PCR tests performed with the aqueous humor for the infectious agents listed. Patient Reference No. Age Sex 1 2 3 4 5 6 7 8 9 10 11 12 *13 *16 *17 *18 *19
33 25 55 14 45 26 20 22 38 57 29 35 26 32 52 36 46
M M F M F M F M F F M M M M F M M
Clinical condition GHPC HAC SC SC AC GHPC AMC GHPC SC GHPC SC SC Healed GHPC NR NR ANR NR
Quantiferon TB Gold test VCA EBNA & CD4 count PCR PCR P N N N N N N N N N P N N 84 22 100 76
P P P P P P P P P P P P P P P P P
P P P P P P P P P P P P P P P P P
EBV RT PCR
CMV PCR
14 427 607 1,693 1,032 123,714 820,955 140,392 5,077 249,000 14,138 1,123 98 16,408 42,216 2,141 14,241
N N N N N N N N N N N N N P N P P
CMV RT HSV1 HSV 2 VZV Mtb TOXO PCR PCR PCR PCR PCR PCR N N N N N N N N N N N N N 7,504 N 76 146
N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N
GHPC, giant helicoid peripapillary choroidopathy: HAC, healed ampiginous choroiditis: SC, serpiginous choroiditis: AC, ampiginous choroiditis: AMC, active multifocal choroiditis H1, Herpes simplex virus1; H2, Herpes simplex virus2; CMV, cytomegalo virus; VZV, varicella zoster virus; Mtb, Mycobacterium tuberculosis; TOXO, Toxoplasma gondii; VCA, Epstein Barr viral capsid antigen; EBNA1, Epstein Barr nuclear antigen; P, positive; N, negative; *, HIV-positive patients; NR, necrotizing retinitis; ANR; atypical necrotizing retinitis.
FIGURE 3. PCR for detection of the EBNA2 gene. Agarose gel electrophoresis showing amplification of the EBNA2 gene of EBV in DNA extracted from AH sample. Analysis products amplified: lanes 1–3, DNA extracts from samples; NC, negative control; PC1, positive control; DNA extracted from type 1 standard strain, B95-8; PC 2, positive control; DNA extracted from type 2 standard strain, Ag876; 100 bp, molecular weight marker (100 - to 1000-bp ladder). EBV type 1-specific band observed in test samples 1 and 2.
The specificity of VCA was further confirmed in 25 AH samples collected from patients who underwent cataract surgery because none of them were positive for these genes. PCRs were performed for other possible causative infectious agents of retinitis, Mycobacterium tuberculosis, Toxoplasma gondii, herpes simplex virus 1, herpes simplex virus 2, cytomegalovirus, and varicella zoster virus as shown in Table 3. PCR results for the other infective agents were negative in all patients except for the presence of CMV DNA in 5 (20%) AH collected from HIV-positive patients with clinically suspected viral retinitis. The results of genotyping PCRs were further confirmed by DNA sequencing using the PCR amplified products. The sequences obtained were submitted to Genbank. The accession numbers are Ocular Immunology & Inflammation
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Detection of Epstein Barr Virus 389
FIGURE 4. Phylogenetic analyses of the EBNA2 PCR-positive samples. The 483 bp of the EBNA2 gene of the representative samples were analyzed by constructing a phylogram using a UPGMA algorithm together with EBV type 1 and EBV type 2 standard strain sequences.
(A)
(C)
(B)
(D)
(E)
FIGURE 5. (A) Fundus of the patient with Epstein-Barr viral retinitis showing the typical chorioretinal lesions with EBV high viral titer and negative for the CMV retinal aspect of viral retinitis. (B) Funduscopy of the left eye at presentation with giant helicoid peripapillary choroidopathy. (C) Funduscopy of the left eye at presentation with ampiginous choroiditis. (D) Funduscopy of the left eye at presentation with multifocal choroiditis. (E) Funduscopy of the left eye at presentation with ampiginous serpiginous choroiditis.
KF651147–KF651163. The nucleotide sequences of the EBNA2-positive amplified products were analyzed by comparison with the nucleotide sequences of EBV standard strains. The sequences were aligned using BioEdit software, and a phylogram was constructed using UPGMA algorithm with perform bootstrap analysis (Replicates 100) in CLC Main Workbench6.71 software. All 17 samples that were confirmed type 1 by genotyping PCR were found to form a unique clade with EBV type 1 standard strain B95_8 strain. The results of phylogeny correlated with !
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genotyping PCR results (Figure 4). The fundus photography of patients with the highest EBV load, showing the typical chorioretinal lesions, is given in Figure 5.
DISCUSSION Herpesviruses represent some of the most successful viruses in humans, infecting over 90% of humans and persisting for the lifetime of the individuals.11,12
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390 M. K. Janani et al. Epstein-Barr virus is a recognized cause of intraocular inflammation and has been implicated as a possible cause of acute referretinal necrosis syndrome ARN. EBV may be directly or indirectly involved in the pathogenesis of a variety of ocular diseases.13–20 EBV was detected in the necrotic retina of patients receiving immunosuppression.21 EBV and CMV cause severe necrotizing retinitis in patients with acquired immune deficiency syndrome (AIDS), and other herpesviruses have been implicated in ARN, seen in both the immunocompetent and the immunosuppressed. Diagnosis of intraocular EBV-induced PTLD can be made from the vitreous using the PCR amplification technique.22,23 In this study, the nested PCR method was used to detect EBV, CMV, VZV, HSV, Mtb, and Toxoplasma gondii in AH samples collected from HIV-positive patients with suspected viral retinitis and patients diagnosed multifocal choroididtis. PCR was used for genotyping process because of the ease of the technique. PCR-based methods are used for strain (EBV type 1 or 2) distinction. In the current study, we showed that intraocular HHV-DNA was detectable over a wide range of HHVassociated uveitis when analysis was performed using the two PCR methods. When positive results were noted, we then used real-time quantitative PCR to examine the viral load using commercially available kits based on TaqMan probe assay. This allowed us to confirm our positive results through the use of two PCR combinations. The finding of high viral loads in the ocular fluids suggests that virus replication takes place in the eye, suggesting a direct pathogenic role in intraocular inflammation. The real-time PCR results correlated 100% with those of conventional PCR. In the current study we have also standardized PCR targeting varying latency genes of EBV for genotyping and applied it to clinical samples that are confirmed EBV PCR positive. All samples that were tested were found to be EBV type 1 by all 3 PCR methods targeting genes that code for EBNA2, EBNA3C, and LMP of EBV. The genotyping PCR results were concordant with all the three sets of primers used for amplification of PCR targeting 3 different latency genes of EBV. Since all three primer sets have given concordant results, any of the primers can be used for the genotyping of the EBV. All the previous studies published have analyzed the viral cause of herpetic retinitis by only PCR and further genotyping has not been done.24,25 To our knowledge, this is the first study involved in both detection and genotyping of EBV in ocular samples. Several studies have shown discrepancies between frequencies of EBV genotypes in blood samples and cultures collected from malignant patients based on the analysis of certain genes, such as EBNA-2, 3A, 3B, and 3C.26 According to Correa et al., applications of PCR on viral DNA from cell culture isolation of clinical samples, in most cases, have revealed only one
genotype, usually EBV1. In contrast, when PCR was applied directly on the clinical samples, co-infection with both EBV genotypes was observed. The discrepancies may be attributed to biological differences between genotypes, in which EBV1 is more efficient than EBV2 in the immortalization of growing B cells in vitro.27 In an attempt to avoid this tendency, the present study used PCR methodology directly in the same sample collected, the type 1 strain being prevalent in developed and developing countries with a greater potential of transforming B cells while type 2 is more prevalent in Africa.28 In this study the application of genotyping PCR to subtype EBV revealed the circulation of only one subtype (type 1) in the ocular sample, in contrast to some studies, which proposed that in HIV-1-infected individuals there is high prevalence of EBV type 2 infection or superinfection with both types 1 and 2.29,30 Our study identified EBV1 in all cases in the study. Use of PCR assay to examine ocular samples in patients with vial retinitis seems to be clinically useful for detecting infectious antigen DNA. Thus, this PCR method is a reliable tool for both diagnosing ocular disorders and further screening of patients for intraocular infections. The application EBV typespecific PCR described here serves to be a rapid, reliable, and cost effective assay to detect EBV subtypes, which can be used as an epidemiological tool for clinical management of this virus disease.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. The research work was carried out with the Indian Council of Medical Research grant, Project No. 5/8/ 7/17/2006-ECD-I. We express our profound gratitude to Ms. Nishi Rani Singha and Ms. Padmapriya for helping us with the collection of clinical specimens. Ethical approval: Approved by institutional ethics subcommittee (IRB) No.48/2004-P.
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Detection of Epstein Barr Virus 391 4. Raymond LA, Wilson CA, Linnemann Jr CC, et al. Punctate outer retinitis in acute Epstein Barr virus infection. Am J Ophthalmol. 1987;104:424–426. 5. Tiedeman JS. Epstein Barr viral antibodies in multifocal choroiditis and panuveitis. Am J Ophthalmol. 1987;103: 659–663. 6. Muccioli C, Belfort Jr R, Lottenberg C, et al. Ophthalmologic findings in AIDS: review of 445 cases seen in a year. Rev Bras Med Assoc (1992). 1994;40:155–158. 7. Balachandran N, Oba DE, Hutt-Fletcher LM. Antigenic cross-reactions among herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus. J Virol. 1987;61: 1125–1135. 8. Lang D, Vornhagen R, Rothe M, et al. Cross-reactivity of Epstein-Barr virus-specific immunoglobulin M antibodies with cytomegalovirus antigens containing glycine homopolymers. Clin Diagn Lab Immunol. 2001;8: 747–756. 9. Holland GN, Tufail A, Jordan MC. Cytomegalovirus diseases. In: Pepose JS, Holland GN, Wilhelmus KR, eds. Ocular Infection and Immunity. 1996;1088–1129. 10. Sugita S, et al. Use of a comprehensive polymerase chain reaction system for diagnosis of ocular infectious diseases. Ophthalmology. 2013;120:1761–1768. 11. Koizumi N, Yamasaki K, Kawasaki S, et al. Cytomegalovirus in aqueous humor from an eye with corneal endotheliitis. Am J Ophthalmol. 2006;141: 564–565. 12. Knox CM, Chandler D, Short GA, Margolis TP. Polymerase chain reaction-based assays of vitreous samples for the diagnosis of viral retinitis: use in diagnostic dilemmas. Ophthalmology. 1998;105:37–44. 13. Jones J, Gardner W, Newman T. Severe optic neuritis in infectious mononucleosis. Ann Emerg Med. 1988;17: 361–364. 14. Davie CJ, Ceballos R, Little SC. Infectious mononucleosis with fatal neuronitis. Arch Neurol. 1963;9:265–272. 15. Grose C, Henle W, Henle G, Feorine PM. Primary EpsteinBarr virus infections in acute neurologic diseases. N Engl J Med. 1975;292:392–395. 16. Raymond LA, Wilson CA, Linnemann CC, et al. Punctate outer retinitis in acute Epstein-Barr virus infection. Am J Ophthalmol. 1987;104:424–426. 17. Kelly SP, Rosenthal AR, Nicholson KG, Woodard CG. Retinochoroiditis in acute Epstein-Barr virus infection. Br J Ophthalmol. 1989;73:1002–1003.
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18. Grossniklaus HE, Aaberg TM, Purnell EW, et al. Retinal necrosis in X-linked lymphoproliferative disease. Ophthalmology. 1994;101:705–709. 19. Wong KW, D’Amico DJ, Hedges TR, et al. Ocular involvement associated with chronic Epstein-Barr virus disease. Arch Ophthalmol. 1987;105:788–792. 20. Tiedeman JS. Epstein-Barr viral antibodies in multifocal choroiditis and panuveitis. Am J Ophthalmol. 1987; 103:659–663. 21. Schaal S, Kagan A, Wang Y, et al. Acute retinal necrosis associated with epstein-barr virus: immunohistopathologic. JAMA Ophthalmol. 2014;132:881–882. 22. Demols PF, Cochaux PM, Velu T, Caspers-Velu L. Chorioretinal post-transplant lymphoproliferative disorder induced by the Epstein-Barr virus. Br J Ophthalmol. 2001;85:93–95. 23. Hershberger VS, Hutchins RK, Witte DP, Schneider S, Harris RE, McGonegle SJ. Epstein-Barr virus-related bilateral acute retinal necrosis in a patient with X-linked 2003;121:1047–1049. 24. Short GA, Margolis TP, Kuppermann BD, et al. A polymerase chain reaction-based assay for diagnosing varicella-zoster virus retinitis in patients with acquired immunodeficiency syndrome. Am J Ophthalmol. 1997;123: 157–164. 25. Correa RM, Fellner MD, Alonio LV, et al. Epstein-Barr virus in healthy carriers: distribution of genotypes and 30bp deletion in latent membrana-1 oncogene. J Med Virol. 2004; 73:583–588. 26. Murray PG, Young LS. Themed issue: the biology and pathology of the Epstein-Barr virus. Mol Pathol. 2003;53: 219–220. 27. Sculley TB, Apolloni A, Hurren L, et al. Coinfection with A- and B-type Epstein-Barr virus in human immunodeficiency virus positive subjects. J Infect Dis. 1990;162:643–648. 28. van Baarle D, Hovemkamp E, Dukers NHTM. High prevalence of Epstein-Barr virus type-2 among homosexual men is caused by sexual transmission. J Infect Dis. 2000; 181:2045–2049. 29. Cohen JI. Epstein-Barr virus infection. N Engl J Med. 2000; 343:481–492. 30. Reeves MB, MacAry PA, Lehner PJ, et al. Latency, chromatin remodeling, and reactivation of human cytomegalovirus in the dendritic cells of healthy carriers. Proc Natl Acad Sci USA. 2005;102:4140–4145.
ISSN: 0927-3948 (Print) 1744-5078 (Online) Journal homepage: http://www.tandfonline.com/loi/ioii20
Genotypic Detection of Epstein Barr Virus from Clinically Suspected Viral Retinitis Patients in a Tertiary Eye Care Centre, India Madhuravasal Krishnan Janani MS MLT, Jambulingam Malathi PhD, Jyothirmay Biswas MS, Sudharshan Sridharan DO DNB & Hajib Naraharirao Madhavan MD, PhD To cite this article: Madhuravasal Krishnan Janani MS MLT, Jambulingam Malathi PhD, Jyothirmay Biswas MS, Sudharshan Sridharan DO DNB & Hajib Naraharirao Madhavan MD, PhD (2015) Genotypic Detection of Epstein Barr Virus from Clinically Suspected Viral Retinitis Patients in a Tertiary Eye Care Centre, India, Ocular Immunology and Inflammation, 23:5, 384-391, DOI: 10.3109/09273948.2014.968265 To link to this article: http://dx.doi.org/10.3109/09273948.2014.968265
Published online: 17 Oct 2014.
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Date: 28 October 2015, At: 19:59
Ocular Immunology & Inflammation, 2015; 23(5): 384–391 ! Vision Research Foundation ISSN: 0927-3948 print / 1744-5078 online DOI: 10.3109/09273948.2014.968265
ORIGINAL ARTICLE
Genotypic Detection of Epstein Barr Virus from Clinically Suspected Viral Retinitis Patients in a Tertiary Eye Care Centre, India Madhuravasal Krishnan Janani, MS MLT, Jambulingam Malathi, PhD, Jyothirmay Biswas, MS, Sudharshan Sridharan, DO DNB, and Hajib Naraharirao Madhavan, MD, PhD
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Vision Research Foundation, L&T Microbiology Research Centre, Sankara Nethralaya, Chennai, India
ABSTRACT Purpose: To evaluate the diagnostic value of PCR on aqueous humour for detection and genotyping of Epstein Bar Virus in patients with viral retinitis. Methods: 70 AH samples were collected from 20 HIV positive patients with clinically suspected viral retinitis and 25 patients with serpignous choroiditis and 25 AH from patients undergoing cataract surgery. PCR was performed to screen HHV-1 to HHV-5, Mtb and Toxoplasma gondii. Genotype prevalence was confirmed by phylogenetic analysis targetig EBV. Results: EBV was detected in 17 (37.7%) samples. Genotyping to subtype EBV, revealed the circulation of only one subtype (Type 1). PCR results for other infective agents were negative except for the presence of CMV in 5 (11.1%) AH. Conclusion: The application of PCR to detect genotypes can be used as an epidemiological tool for clinical management. To our knowledge this is the first report on genotyping of EBV performed on intra ocular samples. Keywords: Detection, Epstein-Barr virus, genotyping, PCR, viral retinitis
Viral retinitis is an important vision-threatening infectious disease of the retina which can occur in both immunocompetent and immunocompromised or immunodeficient (acquired immunodeficiency syndrome—AIDS)1 individuals. In immunocompetent patients, acute retinal necrosis (ARN) has been recognized as a vision-threatening inflammation caused primarily by the herpes group of viruses.2,3 Epstein-Barr virus has been described in various ocular inflammatory diseases, including multifocal choroiditis in healthy patients.4,5 In immunocompromised patients, various opportunistic viral infections can occur, the most common being cytomegalovirus (CMV) infection. Other less common viruses causing retinal infections include herpes simplex virus (HSV) and varicella zoster virus (VZV).5 Although viral retinitis can usually be identified by the fundus picture and circumstantial evidence of
immunosuppression (use of immunosuppressive drugs or a diagnosis of AIDS), the precise diagnosis and identification of the virus can be done best only with the help of rapid and sensitive virological studies.6 The etiology of viral retinitis was earlier established by demonstrating the presence of antibodies raised against specific viral agents in the aqueous humor and serum samples. With the advent of the polymerase chain reaction (PCR) technique it was possible to detect the specific infectious agents associated with retinitis as antibodies raised against the herpes group of viruses cross-react due to the antigenic similarity among gene products of herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus.7,8 Epstein-Barr virus is one of the important viral pathogens described in the vision-threatening ocular condition called viral retinitis among
Received 26 April 2014; revised 18 September 2014; accepted 18 September 2014; published online 16 October 2014 Correspondence: Dr. Jambulingam Malathi, Vision Research Foundation, Larsen &Toubro Microbiology Research Centre, No.18, College Road, Sankara Nethralaya, Chennai 600006, India. E-mail: [email protected]
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Detection of Epstein Barr Virus 385 immunocompetent patients.9 In immunocompromised patients human cytomegalovirus (HCMV) has been predominantly reported as the causative agent. With the advent of PCR it was possible to accurately identify the infective agent associated with the clinical condition.10 Though many reports exist on the application of PCR for detection of EBV and other herpes viruses from intraocular fluids of patients,9 no reports are available on associated genotypes. Analyzing the genotype of EBV associated with viral retinitis will aid in understanding the phylogeny of the virus and also whether multiple genotypes are associated. In normal healthy individuals detection of EBV genotype 1 is reported and also in cases of infectious mononucleosis. In malignant conditions EBV2 is reported. So far the genotypic prevalence of EBV among ocular clinical specimens collected from otherwise healthy and immnuocompromised patients has not been studied. Therefore, the current study was undertaken to determine the rate of detection of EBV and also to find the genotypic prevalence. To our knowledge, this is the first report investigating the presence of EBV DNA and genotyping in intraocular fluids of patients with retinitis.
MATERIALS AND METHODS
Positive Controls EBV standard strain type 1: Culture infiltrate of Marmoset cell line infected with EBV B958 (National Eye Institute, Bethesda, USA) EBV standard strain type 2: Culture infiltrate of Ag876 cell line (kind gift from Alan Rickinson, Glasgow University, United Kingdom) DNA extraction: DNA extracted from standard strains, tests, and control samples following the manufacture’s instructions of QIAGEN DNA extraction kit (Hilden, Germany).
Polymerase Chain Reaction for Detection of EBV To confirm the presence of EBV, two PCRs targeting the genes that code for EBV-VCA and EBNA1 were standardized and used on all clinical samples. Primers targeting genes that code for VCA were designed using Primer Premier (Biosoft International, USA) based on the consensus sequence obtained with specific sequences of EBV-specific genes submitted in GenBank. The nucleotide sequences of the primers and the expected respective product size are given in Table 1. All primers and PCR reagents were procured from VBC – Biotech Service (Vienna).
Patients and Clinical Specimens All patients and the control population were recruited from Sankara Nethralaya, Chennai, India. The study protocol was approved by the ethics committee of the institute and informed consent was obtained from each patient and control subject. A total of 70 aqueous humor (AH) samples were collected from January 2012 to January 2013 (20 AH samples from 20 HIVpositive patients with clinical picture [necrosis, blurred vision, AC flare] suggestive of retinitis [blind spot, blurred vision and other vision problems, floaters] and atypical necrotizing retinitis, 25 AH samples from 25 patients with serpignous choroidoitis with no significant abnormality on the chest radiography, and 25 AH samples from patients undergoing cataract surgery, which was considered the negative control). The samples were processed immediately and DNA was extracted and subjected for molecular assays.
Processing of Aqueous Humor AH samples (150–200 mL) were collected aseptically in a tuberculin syringe with a 30-gauge needle under aseptic precautions by the ophthalmologist; the sample was transferred into presterilized microfuge tubes and stored at 20 C for DNA extraction. !
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Polymerase Chain Reaction for Genotyping of EBV Uniplex PCR for detection of EBNA2, EBNA3C, and LMP genes was standardized using the EBV1 and EBV2 standard strains. In brief, 10 mL of DNA elute was subjected to amplification of the EBV-specific genes mentioned. Primers targeting genes that code for EBNA2, EBNA3C, and LMP were designed using Primer Premier based on the consensus sequence obtained with specific sequences of EBV-specific genes submitted in GenBank. The nucleotide sequences of the primers and the expected respective product size are given in Table 2. All primers and PCR reagents were procured from VBC – Biotech Service.
Optimization of PCR All PCRs (VCA, EBNA1, EBNA2, and EBNA3C) were optimized to be carried out in the same thermal profile through the gradient PCR temperature profile. The PCR mixture (50 mL) contained 100 mM of dNTP mixture, 10 PCR buffer with 15 mM MgCl2, 1 mM of each forward and reverse primer, and 3 U/mL Taq DNA polymerase. Ten microliters of extracted DNA was added to the PCR reaction mixture. The thermal
386 M. K. Janani et al. TABLE 1. List of primers used for amplification of genes those codes for VCA and EBNA1 of EBV. Strain number and gene target 1. VCA
2. EBNA1
Primer
Primer sequence
Expected base pair
EBV EBV EBV EBV
50 -TTTGGCGTCTCAGGCTAT-30 50 -CGTGGTCGTGTTCCCTCA-30 50 -CGGTGTAACTACCCGCAATG-30 50 -CGTGGTCGTGTTCCCTCA-30
Round 1:172
50 -GCAGTAACAGGTAATCTCTGG-30 50 -ACCAGAAATAGCTGCAGGACC-30 50 -GATTTGGACCCGAAATCTGA-30 50 -CCTCCCTAGAACTGACAATTGG-30
Round 1: 490
FI PP R PP F PP R
EBV up EBV low EBV up (R) EBV low (R)
Round 2:126
Round 2: 336
TABLE 2. List of primers used for amplification of genes those codes for EBNA2, EBNA3C, and LMP of EBV.
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Strain number and gene target
Primer
Primer sequence
Expected base pair
1. EBNA2
EBNA-2 F EBNA-2R
50 -TTTCACCAATACATGAACC-30 50 -TGGCAAAGTGCTGAGAGCAA-30
Type 1:378 Type 2:483
2. EBNA3C
EBNA3C-F
50 -AGAAGGGGAGCGTGTGTTGT-30
Type 1:153
0
EBNA3C-R
5 -GGCTCGTTTTTGACGTCGGC-3 0
0
Type 2:246 0
160 234
3. LMP1
LMP 1 c F LMP 1c R
5 -AGTGATGAACACCACCACGA-3 50 -GTGCGCCTAGGTTTTGAGAG-30
LMP2
LMP 2b F LMP 2b R
50 -TTCTGGTGATGCTTGTGCTC-30 50 -AAAGGGCTGCACCAAGAGTA-30
TABLE 3. The optimized thermal profile followed to amplify PCRs. Steps
1st round
2nd round
Initial denaturation Denaturation Annealing Extension Final extension No. of cycles
94 C for 1 min 94 C for 30 s 51 C for 1 min 72 C for 30 s 72 C for 5 mins 30 cycles
94 C for 1 min 94 C for 30 s 51 C for 1 min 72 C for 30 s 72 C for 5 min 20 cycles
profile is given in Table 3. Two controls (a reagent control and a reaction control) were included in each PCR run. The PCR results were considered valid only when the reagent controls were negative and the specific amplified product was obtained with the positive controls. To prevent amplicon contamination, DNA extraction, PCR cocktail preparation, amplification, and analysis of results were carried out in separate rooms. Visualization of PCR product was done by subjecting 10 mL of amplified reaction mixture to electrophoresis on a 2% agarose gel incorporated with 5 mg mL1 of ethidium bromide in 1 Tris-borate buffer (pH 8.2–8.6) and documented with the gel documentation system (Vilber Lourmat, France).
Specificity of PCR Primers Specificity of the primers was determined against DNA extracted from herpes simplex virus 1, herpes simplex virus 2, cytomegalo virus, varicella zoster virus, Chlamydia trachomatis, Toxoplasma gondii, human DNA (extracted from whole blood), Eubacteria (Propionibacterium acne), and fungus (Candida albicans). Sensitivity of Primers DNA was extracted from EBV standard strain. Serial tenfold dilutions of the DNA were made from 101 to 1010, i.e. 5 mL of DNA with 45 mL of Milli Q water. Ten microliters was taken from each dilution for PCR reaction. PCR for Detection of Genes that Code for VCA and EBNA1 The standardized PCRs were applied on AH samples collected from patients and control samples. Real-time PCR for Determination of Viral Load in Clinical Samples The viral load was estimated in the DNA extracts from AH samples using the RoboGene Quantification Kit (Hilden, Germany). The amplification reaction was carried out following the manufacturer’s instructions. PCR was carried out at 50 C for 30 min Ocular Immunology & Inflammation
Detection of Epstein Barr Virus 387 followed by initial denaturation at 95 C for 15 min followed by 50 cycles of initial denaturation at 95 C for 30 s, annealing at 50 C for 60 s, and extension at 72 C for 30 s. The viral load was expressed in IU/mL. Samples that were positive for CMV by nested PCR were also subjected to CMV real-time PCR using the ‘‘artus CMV RG PCR kit’’ on a Rotor-Gene 3000 instrument as per manufacture’s instruction.
Phylogenetic Analysis The nucleotide sequences of the EBNA2 and EBNA3C PCR-positive amplified products were analyzed by comparison with EBV standard strains nucleotide sequences. The nucleotide sequences were analyzed using BioEdit software. Evolutionary distances were estimated by constructing a phylogram using the UPGMA algorithm with perform bootstrap analysis (Replicates 100) in CLC Main Workbench6.71 software.
Genotyping
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PCR for Detection of Genes That Code for EBNA2, EBNA3C, and LMP of EBV The samples that were confirmed positive by both conventional and real-time PCR were further subjected to genotyping by PCR-based DNA sequencing by PCR targeting EBNA2 and EBNA3C genes. DNA Sequencing The positive PCR-amplified products were further subjected to DNA sequencing to be compared with the standard strain and to determine the homology percentage. Cycle sequencing of the amplified products was performed in a 10-mL reaction volume, containing 0.5 mL of RR mix, 3.5 mL of sequencing buffer, 2 mL of forward primer (1:100 diluted), 2 mL MilliQ water, and 2 mL of the amplified product. Amplification was carried out in the Perkin-Elmer thermocycler using 25 cycles at 96 C for 10 s, at 50 C for 5 s, and at 60 C for 4 min, with initial denaturation at 96 C for 1 min. The cycle-sequenced products were then purified and sequenced using an ABI Prism 3130 AVANT (Applied Biosystems, USA) genetic analyzer, which works based on the principle of Sanger’s dideoxy termination method. The sequences were then analyzed by BioEdit sequence alignment software, and BLAST analysis (www.ncbi.nlm.nih.gov/ BLAST) was done to confirm the sequenced data with the standard strains and to determine the homology percentage.
RESULTS All the standardized PCRs (2 nPCRs, 3 uniplex genotyping PCR) were specific to detect only EBV DNA and the sensitivity was 2.5 pg of EBV DNA for VCA gene (primer set 1), 2.5 pg for EBNA1 gene (primer set 2), 0.63 fg for EBNA2 gene (primer set 3), 7.23 fg for EBNA3C gene (primer set 4), and 6.2 fg for LMP gene (primer set 5), respectively. Among the 45 test samples, 17 clinical samples (37.7%) tested positive for EBV by both VCA and EBNA1 PCR (Figures 1 and 2). The samples that were positive for nPCR were also found to be positive by real time. The real-time PCR results correlated 100% with the conventional PCR. The mean EBV viral load is 84,310.35 IU/mL. The real-time PCR values signifying the viral load in the samples that are positive are listed in Table 4. Genotyping was performed on samples that tested positive by nPCR and real-time PCR using PCR targeting EBNA2, EBNA3C, and LMP genes. EBV type 1 was detected in all 17 samples that were proved positive by both nPCR and by real-time PCR. EBV type 2 was not found any of the samples processed. The application of genotyping PCR to subtype EBV revealed the circulation of only one subtype (type 1) in the ocular sample. The genotyping PCR electrophoretogram of EBNA2 on clinical samples is shown in Figure 3.
FIGURE 1. PCR for detection of the EBV-VCA gene. Agarose gel electrophoresis showing amplification of the EBV-VCA gene of Epstein-Barr virus in DNA extracted from an AH sample. Analysis products amplified: NC2, negative control for second round; NC2, negative control for first round; PCR, lanes 1–10, DNA extracts from samples; PC, EBV DNA extracted from standard strain of B95-8; MW, molecular weight marker (100- to 1000-bp ladder). !
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388 M. K. Janani et al.
FIGURE 2. PCR for detection of the EBNA1 gene. Agarose gel electrophoresis showing amplification of the EBNA1 gene of EpsteinBarr virus in DNA extracted from AH sample. Analysis products amplified: NC2, negative control for second round; NC2, negative control for first round PCR; lanes 1–11, DNA extracts from samples; PC, EBV DNA extracted from standard strain of B95-8; MW, molecular weight marker (100 - to 1000-bp ladder).
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TABLE 4. The relevant clinical details of the patients and results of PCR tests performed with the aqueous humor for the infectious agents listed. Patient Reference No. Age Sex 1 2 3 4 5 6 7 8 9 10 11 12 *13 *16 *17 *18 *19
33 25 55 14 45 26 20 22 38 57 29 35 26 32 52 36 46
M M F M F M F M F F M M M M F M M
Clinical condition GHPC HAC SC SC AC GHPC AMC GHPC SC GHPC SC SC Healed GHPC NR NR ANR NR
Quantiferon TB Gold test VCA EBNA & CD4 count PCR PCR P N N N N N N N N N P N N 84 22 100 76
P P P P P P P P P P P P P P P P P
P P P P P P P P P P P P P P P P P
EBV RT PCR
CMV PCR
14 427 607 1,693 1,032 123,714 820,955 140,392 5,077 249,000 14,138 1,123 98 16,408 42,216 2,141 14,241
N N N N N N N N N N N N N P N P P
CMV RT HSV1 HSV 2 VZV Mtb TOXO PCR PCR PCR PCR PCR PCR N N N N N N N N N N N N N 7,504 N 76 146
N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N
GHPC, giant helicoid peripapillary choroidopathy: HAC, healed ampiginous choroiditis: SC, serpiginous choroiditis: AC, ampiginous choroiditis: AMC, active multifocal choroiditis H1, Herpes simplex virus1; H2, Herpes simplex virus2; CMV, cytomegalo virus; VZV, varicella zoster virus; Mtb, Mycobacterium tuberculosis; TOXO, Toxoplasma gondii; VCA, Epstein Barr viral capsid antigen; EBNA1, Epstein Barr nuclear antigen; P, positive; N, negative; *, HIV-positive patients; NR, necrotizing retinitis; ANR; atypical necrotizing retinitis.
FIGURE 3. PCR for detection of the EBNA2 gene. Agarose gel electrophoresis showing amplification of the EBNA2 gene of EBV in DNA extracted from AH sample. Analysis products amplified: lanes 1–3, DNA extracts from samples; NC, negative control; PC1, positive control; DNA extracted from type 1 standard strain, B95-8; PC 2, positive control; DNA extracted from type 2 standard strain, Ag876; 100 bp, molecular weight marker (100 - to 1000-bp ladder). EBV type 1-specific band observed in test samples 1 and 2.
The specificity of VCA was further confirmed in 25 AH samples collected from patients who underwent cataract surgery because none of them were positive for these genes. PCRs were performed for other possible causative infectious agents of retinitis, Mycobacterium tuberculosis, Toxoplasma gondii, herpes simplex virus 1, herpes simplex virus 2, cytomegalovirus, and varicella zoster virus as shown in Table 3. PCR results for the other infective agents were negative in all patients except for the presence of CMV DNA in 5 (20%) AH collected from HIV-positive patients with clinically suspected viral retinitis. The results of genotyping PCRs were further confirmed by DNA sequencing using the PCR amplified products. The sequences obtained were submitted to Genbank. The accession numbers are Ocular Immunology & Inflammation
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Detection of Epstein Barr Virus 389
FIGURE 4. Phylogenetic analyses of the EBNA2 PCR-positive samples. The 483 bp of the EBNA2 gene of the representative samples were analyzed by constructing a phylogram using a UPGMA algorithm together with EBV type 1 and EBV type 2 standard strain sequences.
(A)
(C)
(B)
(D)
(E)
FIGURE 5. (A) Fundus of the patient with Epstein-Barr viral retinitis showing the typical chorioretinal lesions with EBV high viral titer and negative for the CMV retinal aspect of viral retinitis. (B) Funduscopy of the left eye at presentation with giant helicoid peripapillary choroidopathy. (C) Funduscopy of the left eye at presentation with ampiginous choroiditis. (D) Funduscopy of the left eye at presentation with multifocal choroiditis. (E) Funduscopy of the left eye at presentation with ampiginous serpiginous choroiditis.
KF651147–KF651163. The nucleotide sequences of the EBNA2-positive amplified products were analyzed by comparison with the nucleotide sequences of EBV standard strains. The sequences were aligned using BioEdit software, and a phylogram was constructed using UPGMA algorithm with perform bootstrap analysis (Replicates 100) in CLC Main Workbench6.71 software. All 17 samples that were confirmed type 1 by genotyping PCR were found to form a unique clade with EBV type 1 standard strain B95_8 strain. The results of phylogeny correlated with !
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genotyping PCR results (Figure 4). The fundus photography of patients with the highest EBV load, showing the typical chorioretinal lesions, is given in Figure 5.
DISCUSSION Herpesviruses represent some of the most successful viruses in humans, infecting over 90% of humans and persisting for the lifetime of the individuals.11,12
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390 M. K. Janani et al. Epstein-Barr virus is a recognized cause of intraocular inflammation and has been implicated as a possible cause of acute referretinal necrosis syndrome ARN. EBV may be directly or indirectly involved in the pathogenesis of a variety of ocular diseases.13–20 EBV was detected in the necrotic retina of patients receiving immunosuppression.21 EBV and CMV cause severe necrotizing retinitis in patients with acquired immune deficiency syndrome (AIDS), and other herpesviruses have been implicated in ARN, seen in both the immunocompetent and the immunosuppressed. Diagnosis of intraocular EBV-induced PTLD can be made from the vitreous using the PCR amplification technique.22,23 In this study, the nested PCR method was used to detect EBV, CMV, VZV, HSV, Mtb, and Toxoplasma gondii in AH samples collected from HIV-positive patients with suspected viral retinitis and patients diagnosed multifocal choroididtis. PCR was used for genotyping process because of the ease of the technique. PCR-based methods are used for strain (EBV type 1 or 2) distinction. In the current study, we showed that intraocular HHV-DNA was detectable over a wide range of HHVassociated uveitis when analysis was performed using the two PCR methods. When positive results were noted, we then used real-time quantitative PCR to examine the viral load using commercially available kits based on TaqMan probe assay. This allowed us to confirm our positive results through the use of two PCR combinations. The finding of high viral loads in the ocular fluids suggests that virus replication takes place in the eye, suggesting a direct pathogenic role in intraocular inflammation. The real-time PCR results correlated 100% with those of conventional PCR. In the current study we have also standardized PCR targeting varying latency genes of EBV for genotyping and applied it to clinical samples that are confirmed EBV PCR positive. All samples that were tested were found to be EBV type 1 by all 3 PCR methods targeting genes that code for EBNA2, EBNA3C, and LMP of EBV. The genotyping PCR results were concordant with all the three sets of primers used for amplification of PCR targeting 3 different latency genes of EBV. Since all three primer sets have given concordant results, any of the primers can be used for the genotyping of the EBV. All the previous studies published have analyzed the viral cause of herpetic retinitis by only PCR and further genotyping has not been done.24,25 To our knowledge, this is the first study involved in both detection and genotyping of EBV in ocular samples. Several studies have shown discrepancies between frequencies of EBV genotypes in blood samples and cultures collected from malignant patients based on the analysis of certain genes, such as EBNA-2, 3A, 3B, and 3C.26 According to Correa et al., applications of PCR on viral DNA from cell culture isolation of clinical samples, in most cases, have revealed only one
genotype, usually EBV1. In contrast, when PCR was applied directly on the clinical samples, co-infection with both EBV genotypes was observed. The discrepancies may be attributed to biological differences between genotypes, in which EBV1 is more efficient than EBV2 in the immortalization of growing B cells in vitro.27 In an attempt to avoid this tendency, the present study used PCR methodology directly in the same sample collected, the type 1 strain being prevalent in developed and developing countries with a greater potential of transforming B cells while type 2 is more prevalent in Africa.28 In this study the application of genotyping PCR to subtype EBV revealed the circulation of only one subtype (type 1) in the ocular sample, in contrast to some studies, which proposed that in HIV-1-infected individuals there is high prevalence of EBV type 2 infection or superinfection with both types 1 and 2.29,30 Our study identified EBV1 in all cases in the study. Use of PCR assay to examine ocular samples in patients with vial retinitis seems to be clinically useful for detecting infectious antigen DNA. Thus, this PCR method is a reliable tool for both diagnosing ocular disorders and further screening of patients for intraocular infections. The application EBV typespecific PCR described here serves to be a rapid, reliable, and cost effective assay to detect EBV subtypes, which can be used as an epidemiological tool for clinical management of this virus disease.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. The research work was carried out with the Indian Council of Medical Research grant, Project No. 5/8/ 7/17/2006-ECD-I. We express our profound gratitude to Ms. Nishi Rani Singha and Ms. Padmapriya for helping us with the collection of clinical specimens. Ethical approval: Approved by institutional ethics subcommittee (IRB) No.48/2004-P.
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Detection of Epstein Barr Virus 391 4. Raymond LA, Wilson CA, Linnemann Jr CC, et al. Punctate outer retinitis in acute Epstein Barr virus infection. Am J Ophthalmol. 1987;104:424–426. 5. Tiedeman JS. Epstein Barr viral antibodies in multifocal choroiditis and panuveitis. Am J Ophthalmol. 1987;103: 659–663. 6. Muccioli C, Belfort Jr R, Lottenberg C, et al. Ophthalmologic findings in AIDS: review of 445 cases seen in a year. Rev Bras Med Assoc (1992). 1994;40:155–158. 7. Balachandran N, Oba DE, Hutt-Fletcher LM. Antigenic cross-reactions among herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus. J Virol. 1987;61: 1125–1135. 8. Lang D, Vornhagen R, Rothe M, et al. Cross-reactivity of Epstein-Barr virus-specific immunoglobulin M antibodies with cytomegalovirus antigens containing glycine homopolymers. Clin Diagn Lab Immunol. 2001;8: 747–756. 9. Holland GN, Tufail A, Jordan MC. Cytomegalovirus diseases. In: Pepose JS, Holland GN, Wilhelmus KR, eds. Ocular Infection and Immunity. 1996;1088–1129. 10. Sugita S, et al. Use of a comprehensive polymerase chain reaction system for diagnosis of ocular infectious diseases. Ophthalmology. 2013;120:1761–1768. 11. Koizumi N, Yamasaki K, Kawasaki S, et al. Cytomegalovirus in aqueous humor from an eye with corneal endotheliitis. Am J Ophthalmol. 2006;141: 564–565. 12. Knox CM, Chandler D, Short GA, Margolis TP. Polymerase chain reaction-based assays of vitreous samples for the diagnosis of viral retinitis: use in diagnostic dilemmas. Ophthalmology. 1998;105:37–44. 13. Jones J, Gardner W, Newman T. Severe optic neuritis in infectious mononucleosis. Ann Emerg Med. 1988;17: 361–364. 14. Davie CJ, Ceballos R, Little SC. Infectious mononucleosis with fatal neuronitis. Arch Neurol. 1963;9:265–272. 15. Grose C, Henle W, Henle G, Feorine PM. Primary EpsteinBarr virus infections in acute neurologic diseases. N Engl J Med. 1975;292:392–395. 16. Raymond LA, Wilson CA, Linnemann CC, et al. Punctate outer retinitis in acute Epstein-Barr virus infection. Am J Ophthalmol. 1987;104:424–426. 17. Kelly SP, Rosenthal AR, Nicholson KG, Woodard CG. Retinochoroiditis in acute Epstein-Barr virus infection. Br J Ophthalmol. 1989;73:1002–1003.
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