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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Acknowledgments
 ~  References
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  Table of Contents  
Year : 2014  |  Volume : 32  |  Issue : 3  |  Page : 261-269

Significance of Epstein-Barr virus (HHV-4) and CMV (HHV-5) infection among subtype-C human immunodeficiency virus-infected individuals

1 Department of Clinical Virology, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Dermatology, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Neurovirology, National Institute of Mental Health and Neurosciences, Bangalore, Karnataka, India
4 Department of Internal Medicine, Christian Medical College, Vellore, Tamil Nadu, India
5 Department of Biostatistics, Christian Medical College, Vellore, Tamil Nadu, India
6 Sri Sakthi Amma Biomedical Research Institute, Sri Narayani Hospital and Research Center,Vellore, Tamil Nadu, India

Date of Submission10-Apr-2013
Date of Acceptance26-Nov-2013
Date of Web Publication10-Jul-2014

Correspondence Address:
R Kannangai
Department of Clinical Virology, Christian Medical College, Vellore, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.136558

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 ~ Abstract 

Purpose: Opportunistic viral infections are one of the major causes of morbidity and mortality in HIV infection and their molecular detection in the whole blood could be a useful diagnostic tool. Objective: The frequency of opportunistic DNA virus infections among HIV-1-infected individuals using multiplex real-time PCR assays was studied. Materials and Methods: The subjects were in two groups; group 1: Having CD4 counts <100 cells/µl (n = 118) and the group 2: counts >350 cells/µl (n = 173). Individuals were classified by WHO clinical staging system. Samples from 70 healthy individuals were tested as controls. In-house qualitative multiplex real-time PCR was standardised and whole blood samples from 291 were tested, followed by quantitative real-time PCR for positives. In a proportion of samples genotypes of Epstein-Barr virus (EBV) and CMV were determined. Results: The two major viral infections observed were EBV and CMV. The univariate analysis of CMV load showed significant association with cryptococcal meningitis, oral hairy leukoplakia (OHL), CMV retinitis, CD4 counts and WHO staging (P < 0.05) while the multivariate analysis showed an association with OHL (P = 0.02) and WHO staging (P = 0.05). Univariate analysis showed an association of EBV load with CD4 counts and WHO staging (P < 0.05) and multivariate analysis had association only with CD4 counts. The CMV load was significantly associated with elevated SGPT and SGOT level (P < 0.05) while the EBV had only with SGOT. Conclusion: This study showed an association of EBV and CMV load with CD4+ T cell counts, WHO staging and elevated liver enzymes. These viral infections can accelerate HIV disease and multiplex real-time PCR can be used for the early detection. Genotype 1 and 2 of EBV and genotype gB1 and gB2 of CMV were the prevalent in the HIV-1 subtype C-infected south Indians.

Keywords: Cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, India, opportunistic infection

How to cite this article:
Sachithanandham J, Kannangai R, Pulimood S A, Desai A, Abraham A M, Abraham O C, Ravi V, Samuel P, Sridharan G. Significance of Epstein-Barr virus (HHV-4) and CMV (HHV-5) infection among subtype-C human immunodeficiency virus-infected individuals. Indian J Med Microbiol 2014;32:261-9

How to cite this URL:
Sachithanandham J, Kannangai R, Pulimood S A, Desai A, Abraham A M, Abraham O C, Ravi V, Samuel P, Sridharan G. Significance of Epstein-Barr virus (HHV-4) and CMV (HHV-5) infection among subtype-C human immunodeficiency virus-infected individuals. Indian J Med Microbiol [serial online] 2014 [cited 2021 Feb 24];32:261-9. Available from:

 ~ Introduction Top

In human immunodeficiency virus (HIV)-infected individuals herpes viruses often cause a wide spectrum of opportunistic infections. [1] Herpes simplex virus-1 (HSV-1) is associated with extensive skin manifestations and CNS infections. Majority of the adults get sero-converted during late childhood. [2],[3] Latent VZV reactivates in the host and causes zoster-associated dermatological, ophthalmic and neurological complications in HIV-infected individuals. [4] Epstein-Barr virus (EBV) infects more than 90% of world's population and known to have a unique association with several malignancies. [5],[6] It is also associated with CNS infection and hepatitis. The prevalence of EBV-associated lymphomas has been increasing in the developing countries like India. [7]

In HIV-infected individuals, CMV causes a various life-threatening infections like retinitis, gastrointestinal and neurological disease. CMV viraemia will hasten the HIV disease progression. [8] There are many reports showing the co-infection of CMV in HIV-infected individuals. The severity of CMV disease is associated with CD4+ T cells below <100 cells/µl in HIV. [9] CMV Retinitis is the most common manifestation seen in HIV-infected individuals with low CD4+ T cell counts. [10] The early diagnosis of CMV in HIV-infected individuals is very important for the management of CMV-related disease and death. [11] Polyomaviruses like JCV causes fatal demyelinating disease, while BKV causes nephropathy or hemorrhagic cystitis in HIV-infected individuals. [12],[13]

The screening and detection of these opportunistic viral infections with proper treatment are very crucial in the management of HIV-infected individuals. Sensitive molecular assays like multiplex real- time PCR could be useful in screening for opportunistic viruses in HIV-infected individuals. [14] The diagnostic utility of multiplex real-time PCR is multi-factorial such as of saving time, cost and also found to be very useful in diagnosing co-infections. The aim of this study was to evaluate the frequency of opportunistic viruses like, HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-8, JCV, BKV and Adenovirus among HIV-1-infected individuals using multiplex real-time PCR in whole blood samples and to investigate the viral load level variation with WHO clinical staging and CD4+ T cell counts.

 ~ Materials and Methods Top

The study was carried out at the department of Clinical Virology of a tertiary care hospital in south India with the approval from its Institutional Review Board. Whole blood samples were collected from HIV-1-infected individuals who are treatment-naοve for antiretroviral therapy. The study participants were categorised into two groups while recruitment based on CD4 counts; group-1 with CD4 + T cell count < 100 cells/µl) and group 2 with CD4+ T cell count >350 cells/µl. This was done to get an obvious demarcation in the immunodeficiency status of the comparable group. All the study participants categorised based on the CD4 counts were treatment-naοve for antiretroviral therapy. All the clinical and other laboratory data were collected from the individuals' hospital chart and based on the clinical features one experienced HIV physician classified the individuals in to different WHO clinical staging. To establish the baseline findings whole blood samples from 70 healthy individuals were also tested as controls. All participants were enrolled for the study only after informed consent for HIV-related testing by a UNAIDS-approved HIV antibody test.

Sample collection

Whole blood was collected in a sterile K2 EDTA vacutainer (Beckton Dickinson, NJ, USA) for CD4+ T cell estimation and for real-time PCR. DNA was extracted using QIA amp DNA blood minikit (Qiagen, Hilden, Germany). The manufacturer's instructions were strictly followed at all steps.

CD4+ T cell counts for all these patients were performed using the standard procedures by Guava Technologies with Easy CD4™ and cytosoft™ software version 2.2 (Guava Technologies, CA, USA) or BD FACS Count system with FACS Count CD4/CD4 SW, Version 1.0.

Real-time multiplex PCR

A qualitative real-time multiplex PCR assay was carried out on all samples for of all 10 viruses, in four cocktail mixes as shown: Cocktail-1: CMV, EBV and HSV-1, Cocktail-2: VZV and HHV-6, Cocktail-3: JC and HSV-2. Cocktail-4: HHV-8, BK and Adenovirus. The primer and probe sequences and the gene targeted used for the study were taken from the published literature. [15],[16],[17] The uniplex real-time PCR was initially standardised. For HSV-2, CMV, HHV-6 and HHV-8 probes had FAM dye as their fluorophore while, EBV and BKV probes had ROX dye as their fluorophore and HSV-1, VZV, JCV and adenovirus had cy5 dye as their fluorophore. The combination of viruses was selected for the multiplex real-time PCR based on their respective fluorophore with available fluorescent channels. Cell culture supernatants were used as the sources of positive controls for HSV-1, HSV-2, EBV, CMV, HHV-6 and Adenovirus, for VZV OKA vaccine strain and plasmids for JCV, BKV and HHV-8. Sterile Milli-Q water control was included after every 5th place to check the PCR cross contamination.

Multiplex Real-time PCR was carried out (Rotor gene RG-3000 and RG-6000 Corbett Research, Sydney Australia) using 10 μL of extracted DNA in 25 μL reaction mix containing 12.5 µl of QuantiTect Multiplex PCR NoROX master mix (Hamburg, Germany) and 0.075 µl (7.5 picomoles) of forward and reverse of each viruses in the cocktail primers, 0.05 µl (5 picomoles) of respective TaqMan probes. The thermal cycling conditions used were 95°C for 15 min, 95°C for 45 sec and 60°C for 75 sec for 50 cycles. An in-house ERV-3 (endogenous retrovirus) qualitative real-time PCR was attempted to check the DNA integrity for all PCR negative samples. The assay based on TaqMan chemistry was standardised using the same PCR protocol described above and the primers and probe were taken from the published literature. [18]

In-house real-time Quantitation PCR

Samples positive in qualitative multiplex real-time PCR was tested by in-house real-time quantitation PCR. The standards were prepared using plasmids of respective targets cloned using Topo TA Clone/TOP10 kit, (Invitrogen, Carisbad, USA). The plasmids were quantitated using the DNA spectrophotometry (BioTek Instruments, Vermont, USA) and then diluted serially in tenfold dilutions of MilliQ water to obtain 10 6 /10 5 to 10 2 copies/ µl. Amplification of viral DNA was carried out using the same protocol as qualitative PCR with respective primers and probes. The thermal cycling conditions used were same as the qualitative PCR.

Amplification of DNA for genotyping

Genotyping was carried out for a proportion of samples positive for EBV and CMV to determine the prevalent genotypes circulating among HIV-infected individuals. A total of 27 samples for EBV-positive samples with viral load of >1000 copies/ml were attempted for amplification and for CMV a total of 21 samples were attempted for amplification from which 16 samples were amplified. EBV genotyping: A nested PCR target 447 bp of LMP-2A gene, outer primers were designed using Primer 3 tool software and inner primers sequences from the published literature. [19] CMV genotyping: 410-bp gB region was amplified as reported earlier. [20] The amplification was performed using 5 µL of DNA input to a PCR mix containing 2.5 U of Hot star taq master mix (Qiagen, Hilden, Germany) and 25 picomoles of forward and reverse primers in a total reaction volume of 50 µL. The PCR was carried out in thermal cycler PTC-100 (MJ research, CA, USA) with following cycling conditions; 95°C for 15 min followed by 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min for 40 cycles with final extension cycle at 72°C for 10 min.

DNA sequencing

The amplified products were purified using Millipore sequencing clean up reagents (Montage, MA, USA) and then sequenced directly using the same amplification primers of respective targets by the Big Dye Terminator Cycle Sequencing assay (Applied Biosystems, Foster City, CA). The bidirectional alignment sequences for EBV and CMV was made using the Finch TV software (version 1.4.0) and Bio Edit sequence alignment editor software version The phylogenetic analyses was carried out by the neighbour joining method using 1000 bootstrap replicates in MEGA4 software.

HIV Subtyping

Among the 291 HIV-positive individuals, samples from 82 (28%) individuals were subjected to HIV-1 subtyping based on the sequencing of PCR amplified products targeting pol region using Stanford data base (

Statistical analysis

Chi Square test was used to see the difference in proportion of EBV and CMV-positive status among 2 groups of HIV-1-infected individuals and controls. The two-tailed t test was used to analyze the difference in EBV and CMV viral load among two groups of HIV-1-infected individuals and controls. The percentile value, Chi Square and the two-tailed t test analysis were done with the Medcal software version ( The univariate and multivariate analysis of all the clinical and laboratory data were performed using STATA 10.0 (Stata Corp, College Station, TX).

 ~ Results Top

All the 82 sequences submitted to the Stanford data base for subtyping was found to be HIV-1 subtype C with 93- 96% similarity. The EBV DNA was found to be positive in 97 (82%) individuals with viral load range of 100-194900 copies/ml among group 1 and 127 (73%) with viral load range of 100-543300 copies/ml in group 2. The CMV-DNA was found to be positive in 35 (29.6%) with viral load range of 100-17615100 copies/ml among individuals of group-1 and 5 (2.8%) with viral load range of 100-1100 copies/ml among group-2 individuals. One each sample was positive for VZV, HHV-6 and adenovirus while 2 each were positive for JCV and BKV. All the study samples were negative for HSV-1, HSV-2 and HHV-8 DNA. Among the 70 healthy individuals who were negative for HIV, 9 (12.8%) samples were positive for EBV, 1 each were positive for HHV-6 (1.4%) and adenovirus (1.4%). The real-time PCR-positive samples with viral load level among the 291 study participants were categorised based on WHO clinical staging and are shown in [Table 1].
Table 1: The number of real - time PCR - positive samples and their virus load profile for the different opportunistic viruses in the HIV-infected individuals who were categorised based on WHO clinical staging

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Different clinical and laboratory findings obtained from the patients' hospital charts were analyzed to identify associations between the EBV and CMV load level using univariate and multivariate analysis. The univariate and multivariate analysis for EBV and CMV load level was analyzed based on the percentile viral load values. The viral load level which gave the best outcome of the univariate and multivariate analysis was chosen and it was 2.87 log copies/ml (median/50th percentile) for CMV and 3.35 copies/ml (60th percentile) for EBV. These data and the statistical significance are shown in the [Table 2] and [Table 3]. The univariate analysis of CMV viral load outcome showed significant association with Cryptococcal meningitis, oral hairy leukoplakia (OHL), CMV retinitis, CD4+ T cell counts and WHO clinical staging (P value range from 0.000 to 0.039). The variables, which were significant in the univariate analysis only, were taken for multivariate analysis. CMV multivariate analysis showed a significant association with OHL (P = 0.02) and WHO staging (P = 0.05). The univariate analysis of EBV viral load showed significant association with CD4 counts and WHO clinical staging (P value 0.01) with multivariate analysis showed only significance with CD4 counts (P = 0.02). The different clinical presentations observed in the four WHO clinical stages of the study group individuals are shown in [Table 4].
Table 2: Univariate and multivariate analysis of different clinical presentation, CD4 T cell count and WHO staging with median CMV virus load (2.87 log10 copies/ml) as outcome

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Table 3: Univariate and multivariate analysis of different clinical presentation, CD4+T cell count and WHO staging with EBV virus load (log10 3.35 copies/ml ( 60th percentile)) as outcome

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Table 4: Different clinical presentation of the study participants based on the WHO staging

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Among the laboratory profile the median level of SGOT in the study group (n = 174) was 32 U/L (range 11-601 U\L), the median level of SGPT (n = 174) was 25 U/L (range 8-224 U/L) and the median level of alkaline phosphates (n = 171) was 80 U/L (range 17-981 U/L). The univariate analysis showed a significant association between the liver enzyme levels and the EBV/CMV viral load when the cutoff the enzymes were fixed at the double the level of upper limit. The CMV load showed significant association with SGPT and SGOT (P value range from 0.002-0.005). The EBV load had a significance association with only SGOT (P = 0.02). Both EBV and CMV not showed any significant association with alkaline phosphatase.

The difference in the mean EBV viral load detected between the group 1 and group 2 based on the CD4 + T cells of HIV-infected individuals fell just short of significance (two-tailed probability for EBV P = 0.063). The EBV viral load level between healthy controls and HIV-1-infected individuals were found to be significant (two-tailed probability P = 0.012). The difference in the CMV viral load level among the group 1 and group 2 were significant (Two-tailed probability P = 0.009). The comparison of EBV and CMV viral load in whole blood samples among the two groups of HIV-1-infected individuals and healthy controls using the box and whisker plot were shown in [Figure 1]. Among the 40 CMV-positive individuals 35 were coinfected with EBV. The HIV-1 viral load data was only available for few study samples so it was excluded from the statistical analysis.
Figure 1: Comparison of viral load level among the study groups. (a) EBV viral load among the two groups of HIV-1-infected individuals (b) EBV load among HIV infected and controls (c) Comparison of CMV viral load among the two groups of HIV-1-infected individuals

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All the primers and probes used for testing clinical samples were specific to their respective targets. The lower detection limits for all the viruses were previously reported [21] except for adenovirus the lower detection of limit was 15 plasmids/10 µl input (1500 copies/ml).

EBV and CMV genotyping

Out of 27 EBV-positive samples genotyped 19 (70%) were found to be EBV genotype 2 and the remaining 8 (30%) were genotype 1. Among the 16 CMV-positive samples genotyped 9 (56%) strains were found to be gB1 genotype and 7 (44%) were gB2 genotype. The phylogenetic tree analysis determining the genotypes of EBV and CMV are shown in [Figure 2] and [Figure 3]. The EBV and CMV sequences used for genotyping were submitted to Gen Bank data base and obtained accession numbers JX 237780-JX 237787, JX 237789-JX 237800, JX 237802- JX 237807, JX 237809 for EBV and JX 287268-JX287283 for CMV.
Figure 2:

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Figure 3:

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Quality control

DNA from 26 whole blood samples (every tenth) was taken to Neurovirology Department of NIMHANS, Bangalore, a collaborating institute. The quality control testing of 26 samples showed concordant results between the testing at two centres. Our EBV and CMV in-house quantitation assays used in the study were validated with samples received through UK NEQAS (National External Quality Assessment Service for Microbiology) program. During the last 2 years, we received eight samples for EBV and six samples for CMV. All the samples we received for CMV and EBV viral load were within the expected results.

 ~ Discussion Top

The early detection of opportunistic viruses like, EBV and CMV and intervention is useful in HIV-infected individuals for delaying HIV disease progression. These opportunistic viruses are a major hurdle in the management of the HIV/AIDS-infected individuals, since they are one of the most frequent causes of morbidity and mortality in infected individuals.

In our earlier study based on the conventional multiplex PCR assay we obtained a 72% EBV DNA-positive status and 7% CMV DNA-positive status among the HIV-infected individuals. [22] Compston et al. (2009) looked at the prevalence of persistent and latent viruses in treatment-naοve HIV-1-infected individuals ( both asymptomatic and symptomatic) and HIV-negative blood donors from Ghana in which the investigators used multiplex PCR for the detection of viruses like, Parvovirus B19, HBV, GB virus-C, CMV, EBV, HHV-8 and VZV. EBV viraemia was significantly higher in asymptomatic (44.6%) and symptomatic (14.6%) HIV-infected individuals compared to blood donors (8.3%). CMV viraemia was significantly increased in symptomatic (7.6%) compared to asymptomatic HIV-infected individuals (1.8%) and blood donors (0.8%) P < 0.001. [23] Another real-time PCR-based study had reported a high prevalence of HSV-1 (16%), EBV (90%) and CMV (31%) in saliva of HIV-infected individuals compared to controls; HSV-1 (2%), EBV (48%) and CMV (2%). [24] Similar to the above mentioned data we observed significant associations between the EBV and CMV load with the CD4+ T cell count and WHO clinical staging. When we analyzed the percentage positive of EBV in the two groups of individuals based on CD4+ T cell count, higher EBV% positives (82%) was observed among the group 1 than the group 2 (73%). Compared to healthy controls (13%) again the proportion of positive individuals was significantly (P = 0.012) higher in HIV-1-infected individuals. The CMV percentage positive was also significantly (P = 0.009) higher in group 1 (29.6%) compared to group 2 (2.9%). We observed only very few cases in which other herpes viral DNA like HHV-6 and VZV were positive. Only a few individuals were positive for the JCV and BKV. A study form our center reported JCV DNA in the CSF (62% of samples) and brain tissue samples (42% of the samples tested) from HIV-infected individuals. [21] One notable finding in our study is that all the 291 tested samples were also found negative for HHV-8 DNA. [25]

There was a significant association with elevated SGPT/SGOT serum level with high EBV and CMV loads. CMV showed significant association with SGPT and SGOT (P value range from 0.02 to 0.04). The EBV viral load had a significance association with SGOT (P = 0.03). This association of the increased viral load and elevated liver enzymes may be due to mild hepatitis. The increase in the levels of SGOT/SGPT compared to the level of alkaline phosphatase indicates a typical hepatocellular damage rather than a cholestatic or infiltrative damage. In EBV and CMV infection also there may be only hepatocellular damage and that may be the reason for this non-association of alkaline phosphatase level and viral load. [26] The CMV viraemia was also significantly associated with a few clinical conditions like cryptococcal meningitis, OHL and retinitis. The 15 individuals who presented with cryptococcosis in this study, 12 were on antifungal drugs like liposomal amphotericin B or fluconazole and for the other three information was not available. The aetiology of retinitis in these cases could be CMV but the other two conditions may co-existed when the CD4+ T cell count was less then < 100 cells/µl. Although EBV association with OHL seen in HIV-infected individuals our study not showed any significance of EBV with OHL.

Robaina et al. (2008) had analyzed PCR based EBV genotypes among HIV-1 sero-positive individuals. Among their study group they observed 58% of EBV genotype-1, 23% with EBVgenotype-2 and the remaining 19% having mixed infections with EBV-1 and 2 genotypes. [27] Another study from Australia looked at EBV genotyping based on the DNA sequence analysis of EBNA-2 region and found EBVgenotype-1 (98.2%) was predominantly seen. The study also reported non-association of EBV genotypes with EBV-associated disease. [28] In contrast we observed a higher percentage (70%) of EBV-2 genotype compared to EBV-1 in our study populations. In our previous study, we reported EBV and CMV genotypes from HIV-1-infected individuals with CNS-related opportunistic infections. We reported EBV genotype-2 in 8 (57%) and EBV genotype-1 in 6 (43%) samples. In case of CMV 8 (89%) samples were genotype gB1 and 1 (11%) sample showed genotype gB2 in organ-specific samples like CSF and brain tissue samples. [21] Currently there are no reports on EBV genotypes among HIV-infected individuals from India other than our study. A recent study reported from New Delhi, India had studied the glycoprotein B genotypes in blood and urine of babies with symptomatic congenital/perinatal CMV infection and found gB 1, 2 and 3 genotypes in the population. The gB 3 was the most prevalent genotype which showed an association with hepatosplenomegaly and gB2 was more commonly associated with long-term sequelae of congenital CMV infection. [29] An RFLP-based genotyping study from India among non-HIV-infected individuals reported with CMV disease found only CMV gB2 and gB3 in (53.8 and 46.1%, respectively) of the patients' studied. [30] A study from Brazil reported the frequency of CMV genotypes from patients with AIDS and observed genotype gB2 (45.16%) followed by gB3 (35.48%). They also reported that presence of gB2 genotype could be a predictor of patient's poor prognosis. [31] Roubalovα et al. (2009) had looked for the prevalence of CMV gB genotypes in various risk groups and gB2 (55%) was most dominantly seen in HIV patients. [32] In our study though gB1 genotype of CMV was predominant, we could not find any significant association of a particular genotype with HIV infection.

This study clearly showed an association of increased EBV and CMV loads with CD4 + T cell counts, WHO clinical staging and an elevated liver enzymes. The care and management of HIV-infected individuals partly depends on prompt diagnosis of these opportunistic viruses. This study had utilised multiplex real-time PCR assays successfully for the demonstration of different herpes viruses. Based on our findings, it is ideal to introduce screening for CMV as a baseline test among HIV-infected individuals. This information will help the clinician to see whether the individuals subsequently develop CMV-related diseases. It is also important to do a prospective study to see individuals with CMV DNAemia when they started on antiretroviral therapy will become negative for CMV DNAemia subsequently without any CMV-specific treatment. Genotype 1 and 2 of EBV and genotype gB1 and gB2 of CMV were the prevalent in the HIV-1 subtype C-infected south Indians.

 ~ Acknowledgments Top

We gratefully acknowledge the funding received from Department of Biotechnology (Govt. of India) for the opportunistic infection study and Indian council for Medical Research for HIV-1 subtype study. Data presented in this manuscript forms part of the PhD thesis of Mr. Jaiprasath Sachithanandham.

 ~ References Top

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3], [Table 4]


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2004 - Indian Journal of Medical Microbiology
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